A comparative study of IL-12 (cytotoxic lymphocyte maturation factor)-, IL-2-, and IL-7-induced effects on immunomagnetically purified CD56+ NK cells.

IL-12, or cytotoxic lymphocyte maturation factor, is a recently cloned cytokine shown to influence lymphokine-activated killer cells activity in heterogeneous lymphocyte populations, proliferative activity as a costimulus in PBMC/PBL populations and IFN-gamma production in PBL. We have investigated the effects of IL-12 on immunomagnetically highly purified CD56+ lymphocytes, and compared the effects with those of IL-7 and IL-2. Our results show that IL-12 directly generated high lymphokine-activated killer cell activity in CD56+ NK cells, without the need for accessory cells. The IL-12-induced lymphokine-activated killer cell activity reached 50% of what was obtained with IL-2. In contrast, only low proliferative activity was induced by IL-12, as 10% of the IL-2-induced- and approximately 50% of the IL-7-induced proliferative activity was detected with IL-12. The CD56+ cells expressed high levels of IL-2R alpha and 75-kDa TNFR in response to IL-12, comparable to what was registered with IL-2 and IL-7. Furthermore, an extensive up-regulation of the CD56 Ag, to the level obtained with IL-2, was detected in the CD56+ NK cells in the presence of IL-12. Stimulation with IL-7 resulted in a more limited CD56 up-regulation in the CD56+ NK cells. Low concentrations of TNF-alpha were produced in response to both IL-12 and IL-7, with little or no TNF-beta production. Time course of the IL-2-induced TNF production revealed an initial TNF-alpha production, whereas significant levels of TNF-beta were detected after 72 h. The effects of both IL-12 and IL-7 on the CD56+ NK cells were inhibited by an anti-TNF-alpha mAb. Thus, IL-12 can directly influence NK cell activities in purified CD56+ cells, and endogenously produced TNF-alpha is involved in mediating the effects of both IL-12 and IL-7.


A mechanism of action for anaphylatoxin C3a stimulation of mast cells.

Incubation of either C3a, C3ades Arg, or synthetic analogues of the C-terminal sequence of C3a with purified rat peritoneal mast cells resulted in a rapid and dose-dependent histamine release. The natural factors C3a and C3ades Arg were the most active of the factors tested exhibiting EC50 values of 3.3 and 2.2 microM, respectively. The corresponding 21- and 22-residue C-terminal analogues of C3a (Y21R and Y21) were less potent than intact factor exhibiting EC50 values of 10.9 and 25.1 microM, respectively. Histamine was released in a nonlytic manner and the mast cell stimulation by both natural and synthetic factors was sensitive to pertussis toxin, neuraminidase, benzalkonium chloride, and to an excess of calcium. C3a stimulated the generation of inositol polyphosphates that was inhibited by either pertussis toxin or benzalkonium chloride. The C3a anaphylatoxin also directly stimulates purified G proteins (i.e., GTPase activity) in a dose-dependent manner. The evident correlation between efficiency of C3a and C3a analogues to stimulate purified G proteins and their capacity to induce cellular histamine release led us to conclude that C3a fails to activate mast cells via a mechanism involving specific receptors on the cell. Instead, we propose that C3a either causes direct activation of G proteins of the Gi subtype, with a subsequent activation of phospholipase C, or interacts with a binding site of the cell surface specific for cationic molecules that is coupled to the G protein cascade.


In vivo binding and clearance of circulating antigen by bispecific heteropolymer-mediated binding to primate erythrocyte complement receptor.

We have used the avidin/biotin system to construct soluble, cross-linked bispecific heteropolymers containing mAb to both the primate E C receptor and the DNP group. These heteropolymers facilitate in vitro binding of DNP-bovine gamma-globulin (DNP-BGG) to both human and squirrel monkey E. Intravenous injection in squirrel monkeys of DNP-BGG followed by heteropolymer leads to E binding and clearance from the circulation of a significant fraction of both heteropolymer and DNP-BGG, without lysis or clearance of the E. This methodology may potentially be used to treat a variety of infectious diseases and other syndromes associated with blood-borne pathogens.


Preliminary identification and role of phosphodiesterase isozymes in human basophils.

We attempted to identify and establish the role of cyclic nucleotide phosphodiesterase (PDE) isozymes in human basophils by using standard biochemical techniques as well as describing the effects of isozyme-selective and nonselective inhibitors of PDE. The nonselective PDE inhibitors, theophylline and 3-isobutyl-1-methylxanthine, inhibited anti-IgE-induced release of histamine and leukotriene C4 (LTC4) from basophils. This inhibition was accompanied by elevations in cAMP levels. Rolipram, an inhibitor of the low Km cAMP-specific PDE (PDE IV), inhibited the release of both histamine and LTC4 from activated basophils and increased cAMP levels in these cells. In contrast, mediator release from basophils was not inhibited by either siguazodan or SK&F 95654, inhibitors of the cGMP-inhibited PDE (PDE III) or zaprinast, an inhibitor of the cGMP-specific PDE (PDE V). SK&F 95654 failed to elevate basophil cAMP in these experiments whereas zaprinast induced significant increases in cAMP content. The inhibitory effect of rolipram on mediator release was potentiated by siguazodan or SK&F 95654, but not by zaprinast. SK&F 95654 also enhanced the ability of rolipram to increase cAMP content. Forskolin, a direct activator of adenylate cyclase, inhibited IgE-dependent release of mediators from basophils and increased cAMP levels in these cells. These effects were enhanced by rolipram, but not by SK&F 95654 or zaprinast. The cell permeant analog of cAMP, dibutyryl cAMP, inhibited mediator release from these cells, a property not shared by either dibutyryl-cGMP or sodium nitroprusside, an activator of soluble guanylate cyclase. The presence of both PDE III and PDE IV was confirmed by partially purifying and characterizing PDE activity in broken cell preparations. Overall, these data lend support to the hypothesis that cAMP inhibits mediator release from basophils and suggest that the major PDE isozyme responsible for regulating cyclic AMP content in these cells is PDE IV, with a minor contribution from PDE III. However, the finding that zaprinast caused increases in cAMP without inhibiting mediator release indicates that cAMP accumulation is not invariably linked to an inhibition of basophil activation.


The antigen-processing mutant T2 suggests a role for MHC-linked genes in class II antigen presentation.

.174xCEM.T2 (T2) is a human cell hybrid that has a large homozygous deletion within the MHC, including all of the functional class II genes. We have generated stable HLA-DR3 and H-2 I-Ak transfectants of T2 that express parental levels of class II molecules at the cell surface. T2.Ak transfectants fail to stimulate a hen egg lysozyme (HEL)-specific, I-Ak-restricted T cell when incubated with intact HEL. However, stimulation occurs if the appropriate HEL peptide is provided. The T2 cell line therefore has a defect in class II-restricted Ag processing. Biosynthetic studies demonstrate that the kinetics of I-Ak transport in T2.Ak are similar to the parental rates of transport, although the percentage of I-Ak molecules transported appears somewhat lower. I-Ak glycoproteins in T2.Ak associate normally with the I-chain, which appears to be proteolytically cleaved after transport through the Golgi apparatus in a similar fashion to that in the parent cell line, .174xCEM.T1 (T1). The DR alpha beta heterodimers in T2 differ from the parental phenotype in two ways. First, HLA-DR3 expressed in T2 does not have the epitope recognized by the DR3-specific mAb 16.23, although DR3 expressed in the parent does have the epitope. Second, the alpha beta subunits in the parent remain associated when exposed to SDS at room temperature, although those in T2 dissociate.


Age-dependent changes in the oligosaccharide structure of the major myelin glycoprotein, P0.

The single oligosaccharide moiety of the major myelin glycoprotein, P0, resides in an immunoglobulin-like domain that appears to participate in homophilic binding. The studies presented here indicate that the structure of the P0 oligosaccharide from rat nerve changes as a function of Schwann cell age. Examination of 5-day-old nerve revealed that P0 contained predominantly endo-beta-N-acetylglucosaminidase H (endo H)-resistant, complex-type oligosaccharide. In contrast, P0 from adult rats had mostly endo H-sensitive carbohydrate, indicating the presence of appreciable high-mannose and/or hybrid-type oligosaccharide on the glycoprotein. The endo H-sensitive and -resistant P0 of adult nerve could be readily phosphorylated by protein kinase C, as could the complex-type P0 from 5-day-old nerve. This suggests that the glycoprotein progresses to the plasma membrane and myelin regardless of the type of oligosaccharide chain. Analysis of 35SO4(2-)-labeled P0 showed that the sulfate group was found on both endo H-sensitive and -resistant oligosaccharide. The endo H-sensitive P0 carbohydrate from adult nerve appears to be primarily of the hybrid type, as evidenced by (a) the elution profile of [3H]mannose-labeled P0 glycopeptides from adult nerve during concanavalin A chromatography and (b) the inability of P0 from adult nerve to interact with Galanthus nivalis agglutinin. The observed age-dependent changes of P0 oligosaccharide may modify the binding properties of this myelin glycoprotein.


Purification and partial structural and functional characterization of mouse myelin/oligodendrocyte glycoprotein.

The myelin/oligodendrocyte glycoprotein (MOG) is found exclusively in the CNS, where it is localized on the surface of myelin and oligodendrocyte cytoplasmic membranes. The monoclonal antibody 8-18C5 identifies MOG. Several studies have shown that anti-MOG antibodies can induce demyelination, thus inferring an important role in myelin stability. In this study, we demonstrate that MOG consists of two polypeptides, with molecular masses of 26 and 28 kDa. This doublet becomes a single 25-kDa band after deglycosylation with trifluoromethanesulfonic acid or peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase, indicating that there are no or few O-linked sugars and that the doublet band represents differential glycosylation. Partial trypsin cleavage, which also gave a doublet band of lower molecular weight, confirmed this idea. MOG was purified by polyacrylamide gel electrophoresis, followed by electroelution. Three N-terminal sequences of eight to 26 amino acids were obtained. By western blot analysis, no binding was found between MOG and cerebellar soluble lectin. MOG does not seem to belong to the signal-transducing GTP-binding proteins. Reduced MOG concentrations were observed in jimpy and quaking dysmyelinating mutant mice, giving further support to its localization in compact myelin of the CNS.


On the use of multiple probe insertions at the same site for repeated intracerebral microdialysis experiments in the nigrostriatal dopamine system of rats.

The effects of implantation of a dialysis probe into the striatum of awake rats on indices of dopamine (DA) and serotonin neurotransmission were assessed, first over 24 h following initial insertion of a probe, and then again following reinsertion of a probe at the same site 1 week later. It was found that the basal concentration of DA in dialysate stabilized within 20-40 min after probe implantation, although DA showed a modest decline 24 h later. There was, however, no significant difference in basal DA between two test sessions separated by 1 week. On the other hand, the basal concentrations of the DA metabolites, 3,4-dihydroxyphenylacetic acid and homovanillic acid, progressively increased for 2-3 h after probe implantation and decreased markedly by 24 h later. Furthermore, in contrast to DA, the DA metabolites decreased even further after the second probe insertion. Amphetamine-stimulated DA release was also greatly attenuated following the second probe insertion, relative to the first probe insertion. Two probe insertions had only modest effects on the concentration of 5-hydroxyindoleacetic acid in dialysate, relative to the DA metabolites. It is suggested the effects of two probe insertions on DA metabolism and amphetamine-stimulated DA release described here are indicative of probe-induced damage to the nigrostriatal DA system. If this is the case, multiple probe insertions may not provide a feasible strategy for within-subjects design dialysis experiments over extended periods of time, at least in the DA system of small animals. It is suggested further that a stable basal concentration of DA in dialysate may be an especially poor indicator of the integrity of the dopaminergic input to the striatum.


Properties of muscarinic-stimulated adenylate cyclase activity in rat olfactory bulb.

The muscarinic stimulation of adenylate cyclase activity in rat olfactory bulb was characterized, with the aim of elucidating the nature of the molecular mechanism involved. Carbachol (CCh) stimulated the enzyme activity in either crude or purified cell membrane preparations and increased cyclic AMP accumulation in miniprisms of olfactory bulb. The CCh stimulation of adenylate cyclase activity displayed a fast onset and was rapidly reversed by addition of atropine. The stimulation was associated with an increase in the apparent Vmax of the enzyme, with no change in the Km for Mg-ATP. The affinity of the enzyme for Mg2+ was enhanced by CCh. The muscarinic effect required GTP at concentrations higher than those needed for enzyme stimulation with either l-isoproterenol or vasoactive intestinal peptide. Moreover, contrary to the beta-adrenergic stimulation, the muscarinic effect disappeared when guanosine 5'-O-(3'-thiotriphosphate) was substituted for GTP. In vivo treatment of olfactory bulbs with pertussis toxin completely prevented the muscarinic stimulation of adenylate cyclase, whereas cholera toxin was without effect. These results indicate that in rat olfactory bulb muscarinic receptors increase adenylate cyclase activity by interacting with a pertussis toxin-sensitive GTP-binding protein different from the stimulatory GTP-binding protein.


A rabbit autoantibody specific for the 46-kDa form of 2',3'-cyclic nucleotide 3'-phosphodiesterase.

An autoantibody occurring in the serum of an apparently normal rabbit that immunocytochemically stains myelin sheaths and oligodendrocytes in rat brain was shown to react specifically with the 46-kDa isoform of 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNP) (EC 3.1.4.37) in a number of species. Identification of the shorter isoform of the enzyme (CNP1) as the antigen was achieved by comparing the immunostaining of Western blots by the autoantibody with that of a well-characterized anti-CNP antiserum. The 46-kDa antigen reacting with the autoantibody exhibited the same Mr and pI as the small isoform of CNP on two-dimensional gels and showed a similar enrichment in purified CNS myelin. The autoantibody has very high affinity for CNP1 and is capable of detecting the very low amounts of this enzyme in peripheral nerve, spleen, adrenal gland, pancreas, testis, and intestine. Testing the reactivity of the autoantibody with synthetic peptides by enzyme-linked immunosorbent assay revealed that it reacted with the N-acetylated decapeptide corresponding to the N-terminus of CNP1, but did not react if the peptide was not acetylated or if the acetyl group was replaced with a palmityl group. The lack of reactivity with CNP2, which differs from CNP1 by a 20-amino acid extension at the N-terminus of the protein as a result of alternative splicing, may be due to the absence of the N-acetyl moiety that is part of the epitope and/or blocking of antibody binding to the decapeptide by extension of the polypeptide chain.(ABSTRACT TRUNCATED AT 250 WORDS)


Decreased density of presynaptic alpha 2-adrenoceptors in postmortem brains of patients with Alzheimer's disease.

The full agonist [3H]bromoxidine (UK 14304) was used to quantitate alpha 2-adrenoceptors in postmortem brains of patients with Alzheimer's disease. The effects of aging and human serum Cohn fraction IV on [3H]bromoxidine binding were also assessed. In patients with Alzheimer's disease, the binding capacity (Bmax) of [3H]bromoxidine was lower in the frontal cortex (37%), hypothalamus (33%), and cerebellum (52%) than in matched controls. In the hippocampus, amygdala, and head of caudate, the binding capacities (Bmax) were unchanged. Quantitative autoradiographic analyses with [3H]bromoxidine confirmed the existence of a marked reduction (55-60%) in alpha 2A-adrenoceptor density in the frontal cortex (layers I and III). In patients with dementia who did not meet neuropathological criteria for Alzheimer's disease, the density of alpha 2-adrenoceptors was unchanged. In control subjects, the density of alpha 2A-adrenoceptors in the frontal cortex showed a significant negative correlation with age at death. The inhibitory effect of human serum Cohn fraction IV on [3H]bromoxidine was very similar in control subjects and patients with Alzheimer's disease. The observed decrease in the density of brain alpha 2-adrenoceptors in Alzheimer's disease may represent direct biochemical evidence of a presynaptic location of this receptor on noradrenergic nerve terminals in the human CNS.


Estrogen induction of cytochrome c oxidase subunit III in rat hippocampus.

Differential screening of a cDNA library prepared from mRNA of the hippocampus of estrogen-stimulated ovariectomized female rats led to the identification of a single estrogen-induced clone. Analysis of the sequence identified this cDNA as the gene coding for subunit III of the enzyme cytochrome c oxidase. Cytochrome c oxidase subunit III mRNA levels significantly increased as early as 3 h following the administration of a single dose of hormone. This effect was visible in the hippocampus and in the hypothalamus, but not in the other brain areas examined. Because subunit III of the cytochrome c oxidase is of mitochondrial origin, the mechanism involved in the estrogenic effect is still unknown. The observation that the activity of cytochrome c oxidase can also be induced by estrogens in the hippocampus indicates that this induction may be secondary to the increased expression of the other subunits of cytochrome c oxidase or to the general increase of neuronal activity.


Detergent-insoluble cortical Lewy body fibrils share epitopes with neurofilament and tau.

Lewy bodies are cytoskeletal inclusions associated with neuronal injury and death in idiopathic Parkinson's disease and other neurodegenerative disorders. The chemical composition of the 8-10-nm fibrils of the Lewy body is unknown, although they are related to both normal cytoskeletal elements and paired helical filaments of Alzheimer neurofibrillary tangles. From the Lewy body-rich cerebral cortex of patients with diffuse Lewy body disease we have isolated intact Lewy bodies using a high salt buffer/nonionic detergent gradient centrifugation procedure and extracted the constitutive fibrils with urea and sodium dodecyl sulfate. Urea/detergent-resistant Lewy body fibrils were solubilized with formic acid and found to contain a single protein band of 68 kDa, which was not found in identically prepared normal brain homogenates. The Lewy body derived-polypeptide was recognized on immunoblots by a polyclonal antibody that reacted with both the 68-kDa neurofilament subunit and the microtubule-associated protein tau. The 68-kDa Lewy body protein was not labeled by the monoclonal antibody tau-1 despite prior in vitro enzymatic dephosphorylation. We conclude that the detergent-insoluble component of the cortical Lewy body fibril shares epitopes with neurofilament and tau and may be a posttranslationally modified derivative of either neurofilament or tau with substantially altered biochemical and immunologic properties.


Localization of actin in the retina of the crayfish Procambarus clarkii.

The distribution of actin in the retina of the crayfish was investigated at the LM level using FITC-phalloidin. Fluorescent staining was associated with the main rhabdom and eighth cell rhabdom, the zonula adherens junctions between retinula cells, and the basement membrane of the retina. EM and S1 decoration were used to confirm the presence of actin and identify its structural relationships. Phalloidin staining of the rhabdom and S1 decoration of actin filaments in the rhabdom microvilli confirmed earlier findings that actin is a component of the microvillus cytoskeleton in the crayfish. At the zonula adherens junctions, actin filaments, identified by S1 decoration, run longitudinally within the plaque of the junction. At the extreme proximal end of the rhabdom, actin filaments associated with the junctions fill each small area of retinula cell cytoplasm. In the basement membrane, EM and S1 decoration show that basilar cells contain large bundles of actin filaments which are associated with cell-matrix adherens junctions. Foot cells which lie immediately below the rhabdom also contain similar junctions and actin is tentatively identified in these cells. The functional role of actin at these various locations is discussed in relation to retinal organization in the crayfish and other invertebrates.


BUdR as an S-phase marker for quantitative studies of cytokinetic behaviour in the murine cerebral ventricular zone.

BUdR incorporation into replicating DNA, detected immunohistochemically, is used as an S-phase marker in the proliferative cell populations of the cerebral wall of the mouse embryo on the 14th gestational day (E14). The analysis initiates a series of studies concerned with the cytokinetic behaviour and cell output of proliferative populations involved in neocortical histogenesis. On E14 there are two periventricular proliferative zones in the cerebral wall. These are the ventricular and subventricular zones. The ventricular zone is a pseudostratified epithelium. DNA replication occurs with the cell nucleus in the outer zone of the epithelium and mitoses at the ventricular surface. Prior applications of BUdR for studies of cytogenesis in the CNS have been extended in two principal ways: (1) basic fuchsin was used as counterstain for BUdR-negative nuclei and (2) labelling indices were determined separately in strata or bins, 10 microns in height, through the full depth of the ventricular zone and overlying cerebral wall. It was established that a single injection of 50 micrograms g-1 into the pregnant dam was associated with labelling of 100% of nuclei in S-phase over an interval extending from 15 min to at least 2.0 h after injection. The zone where nuclei are undergoing S-phase (S-phase zone) extends through the outer four bins of the ventricular zone. The method has high quantitative reproducibility with an SE for labelling indices in bins within the S-phase zone less than 10% of the average values. Evidence is provided that BUdR incorporation is initiated with the nucleus in the outer aspect of the S-phase zone. The efficiency of incorporation of the marker is reduced as nuclei near the end of DNA replication and move to the inner aspect of the S-phase zone.


Phosphorylation-dependent neurofilament epitopes are reduced at the node of Ranvier.

Neurofilaments in axons are highly phosphorylated at multiple sites on the 200 kDa neurofilament (neurofilament-H) and 160 kDa (neurofilament-M) subunit peptides. We used a panel of monoclonal and polyclonal antibodies against distinct neurofilament epitopes to study the distribution of these epitopes along the axons of large myelinated fibres in rat sciatic nerve using quantitative electron microscopic immunocytochemistry with colloidal gold. Antibodies specific for phosphorylated epitopes on neurofilament-H showed a 60% reduction in density of immunoreactivity at the node of Ranvier, compared to the internodal axon. Antibodies directed against neurofilament-M, which recognized phosphorylated epitopes preferentially, showed a 40% reduction in density of immunoreactivity at the node. Following dephosphorylation of the neurofilaments in tissue sections by alkaline phosphatase treatment, antibodies which recognized dephosphorylated forms of neurofilament-H showed no reduction in density of immunoreactivity at the node. Similarly, an antibody directed against the 70 kDa subunit (neurofilament-L), showed no reduction in density of immunoreactivity at the node. An antibody against tubulin also showed no decrease in the density of immunoreactivity at the node of Ranvier. Tubulin immunoreactivity was similar in myelinated and unmyelinated fibres. In contrast to phosphorylated neurofilament epitopes, immunoreactivity was much greater in myelinated than unmyelinated fibres. These results suggest that the degree of phosphorylation of neurofilament-H and neurofilament-M subunits is reduced at the node of Ranvier, in comparison to internodal neurofilaments, and imply that a post-translational modification of neurofilaments must occur along the length of the axon at the node.


A search for Pneumocystis carinii in post-mortem lungs by DNA amplification.

DNA amplification of specific sequences and subsequent oligonucleotide hybridization were used to search for Pneumocystis carinii in post-mortem lung samplings from non-immunosuppressed individuals ranging from 15 to 70 years of age. No P. carinii-specific DNA was detected in 45 DNA amplification reactions from 15 lungs.


Rapid acinar to ductal transdifferentiation in cultured human exocrine pancreas.

Experiments have been performed to define conditions for the primary culture of human exocrine pancreas, as a first step towards molecular reconstruction experiments of pancreatic neoplasia. Normal human exocrine pancreas was digested using collagenase and dispase and the resulting cellular aggregates were cultured in vitro. The phenotype of the digested pancreatic cells was almost exclusively acinar (amylase-positive, keratin 19 and mucin antigens-negative), yet within 4 days of culture the cells had taken on a ductal phenotype (amylase-negative, keratin 19 and mucin antigens-positive). The kinetics of these observations exclude the possibility of overgrowth of the acinar population by a ductal sub-population, and selective adherence is excluded by examination of those cells that do not adhere, which are representative of the initiating population. We interpret these data as indicating that, under the conditions of culture, the acinar cell phenotype is not stable and can transdifferentiate to a ductal phenotype. Taken together with recent data from transgenic animals, this in vitro observation has possible implications for our view of the pathogenesis of pancreatic neoplasia.


Role of the gamma-aminobutyric acid receptor/chloride channel complex in tolerance to ethanol and cross-tolerance to diazepam and pentobarbital.

The role of the gamma-aminobutyric acid (GABA) receptor/chloride channel complex in the development of tolerance to ethanol and cross-tolerance to diazepam and pentobarbital was assessed. Rats given a low (1.8 g/kg) dose of ethanol before daily practice on the moving belt test of motor incoordination, and those given a high daily dose (3.6 g/kg) not paired with practice, showed tolerance to ethanol and cross-tolerance to diazepam and pentobarbital, whereas rats receiving 1.8 g/kg of ethanol after practice did not. Control rats were trained on the moving belt, but received no ethanol treatment. No differences were seen among the treatment groups in the abilities of GABA or ethanol to increase 36Cl uptake into cerebral cortical microsacs. However, diazepam potentiation of GABA-mediated chloride flux was significantly lower in rats receiving daily intoxicated practice, but only if they received an i.p. injection of ethanol 1 hr before sacrifice. The degree of pentobarbital potentiation of the effect of GABA did not correlate with the behavioral cross-tolerance observed. The results indicate that behaviorally augmented cross-tolerance from ethanol to diazepam correlates incompletely with changes on the biochemical level.


Discrimination of agonist-antagonist opioids in humans trained on a two-choice saline-hydromorphone discrimination.

The stimulus properties of four opioid agonist-antagonists were assessed in postaddict volunteers trained in a two-choice drug discrimination procedure to discriminate between the effects of i.m. saline and hydromorphone (3 mg/70 kg). Behavioral, subjective and physiological measures were concurrently collected. After training, generalization curves were determined for hydromorphone, pentazocine, butorphanol, nalbuphine and buprenorphine. In generalization testing, hydromorphone produced dose-related increases in hydromorphone-appropriate responses and in characteristic opioid agonist-like subjective effects measures. In general, each of the study drugs produced a profile of subjective and physiological effects similar to that reported in other human studies. These subjective indices showed the study drugs to be heterogeneous. However, in this two-choice drug discrimination procedure, they were discriminated as homogeneous (i.e., all were discriminated as hydromorphone-like). The present study also found that all test drugs were subjectively identified as being opiates, and all were subjectively rated as similar to the hydromorphone training condition. A previous three-choice discrimination was sensitive to the heterogeneity among these drugs on both the discrimination measures and subjective ratings of similarity to the hydromorphone training condition. Thus, the specific procedures of drug discrimination studies may have an effect on some subjective indices, although this interaction is not apparent for the majority of subjective effect measures. Also, the characterization of opioids in drug discrimination testing appears to depend upon the specific training subjects receive; this is most clear with discrimination measures, and to a more limited extent, with subjective measures.


Synthesis, characterization, and anti-human immunodeficiency virus activity of water-soluble salts of polyoxotungstate anions with covalently attached organic groups.

The cesium and tetramethylammonium (TMA) salts of polyoxotungstate anions with covalently attached organosilyl groups of formula [(RSi)2O]SiW11O39(4-), where R = CH2CH2COCH3, (CH2)3CN, and CH==CH2 (1-R, cesium salt, unless otherwise noted) have been prepared, purified, and spectroscopically characterized. The water solubility (25 degrees C) of these 10 new compounds ranges from 0.14 mM to 2.16 mM. All appear to be stable in aqueous media over a period of several hours as assessed by 1H NMR. The activities (EC50) of the new compounds against human immunodeficiency virus in primary human lymphocytes range from 3.3 microM to 39.0 microM. Their toxicities (IC50) are all greater than 100 microM. The inhibition constants of the new compounds against purified virion-derived HIV-1 reverse transcriptase are in the 1-10 microM range.


Synthesis and substance P antagonist activity of naphthimidazolium derivatives.

The synthesis of unsymmetrical naphth[2,3-d]imidazolium and bridged naphth[2,3-d]imidazolium derivatives and their substance P (SP) antagonist activity are described. All compounds were evaluated for their ability to displace SP from neurokinin-1 (NK-1) receptor sites using standard receptor binding methodology (rat forebrain membrane). 1,3-Diethyl-2-[3-(1,3-dihydro-1,3,3-timethyl-2H-indol-2-ylidene) -1-propenyl]-1H-naphth[2,3-d]imidazolium chloride (7a), a representative compound in this series, was further evaluated for SP antagonist activity in a guinea pig ileum contractility assay. In vivo SP antagonist activity of 7a was demonstrated using SP-induced salivation and paw edema models performed in rats.


Amplification of 16S rRNA sequences to detect Mycobacterium paratuberculosis.

A probe based on 16S ribosomal RNA (rRNA) sequences was developed to detect Mycobacterium paratuberculosis, the causative agent of Johne's disease in cattle. Three universal primers were used to sequence the amplified fragments of the 16S rRNA gene of various species of mycobacteria. When the nucleotide sequences were analysed, a deletion was detected in the sequence of the fast-growing species. An oligonucleotide probe (P) directed to this region was synthesised and hybridised directly with total RNA of various mycobacterial strains in a dot-spot assay. The probe detected M. paratuberculosis, some other slow-growing mycobacteria of the M. avium-intracellulare (MAI) complex, and one atypical strain, M. gordonae. To increase the sensitivity of the probe, a 413-bp fragment of the 16S rRNA gene of M. paratuberculosis between P and a second oligonucleotide primer was amplified and hybridised with a M. paratuberculosis/M. avium-specific probe. When faecal samples of cattle were tested, all culture-positive samples were positive in the PCR assay.


Entero-adherent Escherichia coli is an important diarrhoeagenic agent in infants aged below 6 months in Calcutta, India.

Escherichia coli adherent to HEp-2 and HeLa cells were isolated from the faeces of 43 (19.7%) of 218 hospitalised infants aged below 6 months with acute diarrhoea. No conventional virulence factors, including enterotoxin production--heat-labile (LT) or heat-stable (ST), the verotoxin (VT) or shiga-like toxin (SLT)--or the invasive phenotype (determined by the Sereny test) could be detected among these isolates. Out of the 43 isolates, 16 (37.2%) were of the known enteropathogenic O:K serogroups--enteropathogenic E. coli (EPEC). The remaining 27 (62.8%) isolates showed different types of adherence to HEp-2 and HeLa cells which was diffuse (40.7%), localised (37.0%), or both (22.3%); they were identified as entero-adherent E. coli (EAEC). The EAEC isolates adhered to HEp-2 and HeLa cells in the presence of mannose, lactose, fucose, galactose, and fetuin, indicating that adhesion was not specific for these sugars or glycoprotein. Haemagglutination and the salt aggregation test (SAT) did not correlate with patterns of adherence. The results of this study indicate that LA-EAEC is an important aetiological agent of acute diarrhoea in infants aged below 6 months in Calcutta.


Nucleotides of tRNA governing the specificity of Escherichia coli methionyl-tRNA(fMet) formyltransferase.

In Escherichia coli, the free amino group of the aminoacyl moiety of methionyl-tRNA(fMet) is specifically modified by a transformylation reaction. To identify the nucleotides governing the recognition of the tRNA substrate by the formylase, initiator tRNA(fMet) was changed into an elongator tRNA with the help of an in vivo selection method. All the mutations isolated were in the tRNA acceptor arm, at positions 72 and 73. The major role of the acceptor arm was further established by the demonstration of the full formylability of a chimaeric tRNA(Met) containing the acceptor stem of tRNA(fMet) and the remaining of the structure of tRNA(mMet). In addition, more than 30 variants of the genes encoding tRNA(mMet) or tRNA(fMet) have been constructed, the corresponding mutant tRNA products purified and the parameters of the formylation reaction measured. tRNA(mMet) became formylatable by the only change of the G1.C72 base-pair into C1-A72. It was possible to render tRNA(mMet) as good a substrate as tRNA(fMet) for the formylase by the introduction of a limited number of additional changes in the acceptor stem. In conclusion, A73, G2.C71, C3.G70 and G4.C69 are positive determinants for the specific processing of methionyl-tRNA(fMet) by the formylase while the occurrence of a G.C or C.G base-pair between positions 1 and 72 acts as a major negative determinant. This pattern appears to account fully for the specificity of the formylase and the lack of formylation of any aminoacylated tRNA, excepting the methionyl-tRNA(fMet).


Application of staining of nucleolar organizer regions in cytological smears of the bronchus.

An argyrophil technique for the staining nucleolar organizer regions (AgNOR) was applied to cytological preparations obtained from bronchoscopic brushing materials. The number of AgNOR has been thought to be related to cellular activation. To differentiate malignant cells from non-malignant atypical cells, this study was carried out in 20 cases of adenocarcinoma of the lung (mean AgNOR: 18.34), 12 cases of pulmonary inflammatory diseases (mean AgNOR: 6.54) and 10 normal bronchial epithelial specimens for control (mean AgNOR: 2.07). On the basis of AgNOR number, we could differentiate between the three groups. The differences observed were statistically highly significant (p less than 0.0001). Moreover, the nucleolar organizer regions (NOR) in cancer cells were found the more irregularly distributed and more variable in sized than those in atypical and normal bronchial columnar cells. We suggest that the AgNOR technique will find increasing application as a complementary test in diagnostic cytopathology.


Target epitope in the Tax protein of human T-cell leukemia virus type I recognized by class I major histocompatibility complex-restricted cytotoxic T cells.

A trans-acting regulatory gene product p40tax (Tax) of human T-cell leukemia virus type I (HTLV-I) is one of the main target antigens recognized by cytotoxic T lymphocytes (CTL) specific for HTLV-I. A CTL epitope within the Tax protein was identified in this report. HTLV-I-specific CD8+ CTL lines established from two HTLV-I carriers with HTLV-I-associated myelopathy or Sjögren syndrome were previously demonstrated to kill predominantly the target cells expressing HTLV-I Tax. The CTL from two patients showed significant levels of cytotoxicity to autologous target cells pulsed with a synthetic peptide of 24 amino acids corresponding to the amino-terminal sequences of the Tax protein. Allogeneic target cells were also sensitized for CTL by this peptide when the target cells have HLA-A2. Tax-specific cytotoxicity, detected as cytolysis of the target cells infected with vaccinia virus-HTLV-I recombinant expressing Tax protein, was almost completely inhibited by competitor cells pulsed with the synthetic peptide. This indicates that a major CTL epitope is present in this peptide. Further analysis using shorter peptides revealed that the core sequence of the CTL epitope was LLFGYPVYV at positions 11 through 19. This sequence can be aligned with the HLA-A2-specific motifs reported recently.


Cytotoxicity of a replication-defective mutant of herpes simplex virus type 1.

Replication-defective mutants of herpes simplex virus type 1 (HSV-1) may prove useful as vectors for gene transfer, particularly to nondividing cells. Cgal delta 3 is an immediate-early gene 3 (IE 3) deletion mutant of HSV-1 that expresses the lacZ gene of Escherichia coli from the human cytomegalovirus immediate-early control region but does not express viral early or late genes. This vector was able to efficiently infect and express lacZ in cells refractory to traditional methods of gene transfer. However, 1 to 3 days postinfection, Cgal delta 3 induced cytopathic effects (CPE) in many cell types, including neurons. In human primary fibroblasts Cgal delta 3 induced chromosomal aberrations and host cell DNA fragmentation. Other HSV-1 strains that caused CPE, tested under conditions of viral replication-inhibition, included mutants of the early gene UL42, the virion host shutoff function, single mutants of IE 1, IE 2, and IE 3, and double mutants of IE 3 and 4 and IE 3 and 5. Inhibition of viral gene expression by UV irradiation of virus stocks or by preexposure of cells to interferon markedly reduced the CPE. We conclude from these studies that HSV-1 IE gene expression is sufficient for the induction of CPE, although none of the five IE gene products appear to be solely responsible. After infection of human fibroblasts with Cgal delta 3 at a low multiplicity of infection, we were able to recover up to 6% of the input virus 2 weeks later by a superinfection-rescue procedure, even though the virally transduced human cytomegalovirus-lacZ transgene was not expressed at this time. It is therefore likely that inhibition or inactivation of viral IE gene expression, either for establishing latency or for the long-term transduction of foreign genes by HSV-1 vectors, is essential to avoid the death of infected cells.


Ternary complex formation by vaccinia virus RNA polymerase at an early viral promoter: analysis by native gel electrophoresis.

We have resolved, by native gel electrophoresis, two intermediates in the transcription of a vaccinia virus early gene by the virus-encoded RNA polymerase. Polymerase holoenzyme containing the vaccinia virus early transcription factor (VETF) forms a complex of VETF bound to the promoter as the first step in a pathway leading to establishment of a committed ternary elongation complex. Formation of the VETF-DNA complex is stimulated by magnesium but is uninfluenced by nucleoside triphosphates. A stable binary complex of RNA polymerase bound to DNA is not detected. Assembly of a gel-stable polymerase-DNA complex depends on conditions permissive for RNA synthesis. Nucleotide omission experiments suggest that at least a tetrameric RNA must be made before a ternary complex is stabilized. RNA analysis indicates that complexes containing nascent transcripts 20 nucleotides long are stable and active. Ternary complex formation requires hydrolyzable ATP. This is consistent with an essential role for the ATPase activity of VETF at a step subsequent to DNA binding, as proposed by Broyles (S. S. Broyles, J. Biol. Chem. 266:15545-15548, 1991). The ternary complex, once formed, is resistant to dissociation by competitor DNA, as well as by salt, Sarkosyl, and heparin. The effects of these inhibitory agents on transcription complex formation suggest that they target different steps in the assembly pathway.


Identification of T-helper epitopes in the VP1 capsid protein of poliovirus.

Poliovirus-specific T lymphocytes were isolated from virus-immunized mice of different H-2 haplotypes. Immunological characterization of this population indicates that the effector population involved in the observed poliovirus-specific proliferative response was that of CD4-positive T-helper cells. Proliferative responses also were induced within these T-lymphocyte populations upon stimulation with either purified VP1 capsid protein or VP1 synthetic peptides. By using these synthetic peptides, several T-helper epitopes were identified. Generally, proliferative responses were observed in three regions of VP1. Two regions spanning VP1 residues 86 to 120 and 201 to 241 were recognized by T lymphocytes from BALB/c (H-2d), C57BL/6 (H-2b), and C3H/HeJ (H-2k) backgrounds. Analyses using synthetic peptides of nonoverlapping sequences indicated that the region spanning residues 201 to 241 may contain several T epitopes and may account for the strong proliferative response observed. In addition, for two of the three haplotypes examined, T epitopes were observed within residues 7 to 24 of VP1. Additional epitopes which appeared to be restricted to specific H-2 backgrounds were identified. T epitopes within VP1 that are common between different strains of mice appeared to lie within previously identified neutralizing antigenic sites in poliovirus.


5-Azacytidine and RNA secondary structure increase the retrovirus mutation rate.

A broad spectrum of mutations occurs at a high rate during a single round of retrovirus replication (V.K. Pathak and H. M. Temin, Proc. Natl. Acad. Sci. USA 87:6019-6023, 1990). We have now determined that this high rate of spontaneous mutation can be further increased by 5-azacytidine (AZC) treatment or by regions of potential RNA secondary structure. We found a 13-fold increase in the mutation rate after AZC treatment of retrovirus-producing cells and target cells. The AZC-induced substitutions were located at the same target sites as previously identified spontaneous substitutions. The concordance of the AZC-induced and spontaneous substitutions indicates the presence of reverse transcription "pause sites," where the growing point is error prone. An analysis of nucleotides that neighbored substitutions revealed that transversions occur primarily by transient template misalignment, whereas transitions occur primarily by misincorporation. We also introduced a 34-bp potential stem-loop structure as an in-frame insertion within a lacZ alpha gene that was inserted in the long terminal repeat (LTR) U3 region and determined whether this potential secondary structure increased the rate of retrovirus mutations. We found a threefold increase in the retrovirus mutation rate. Fifty-seven of 96 mutations were deletions associated with the potential stem-loop. We also determined that these deletion mutations occurred primarily during minus-strand DNA synthesis by comparing the frequencies of mutations in recovered provirus plasmids containing both LTRs and in provirus plasmids containing only one LTR.


The pathogenesis of infection with minute virus of mice depends on expression of the small nonstructural protein NS2 and on the genotype of the allotropic determinants VP1 and VP2.

Neonatal C3H/He mice were oronasally inoculated with similar doses of four genotypes of minute virus of mice (MVM). MVMp, a fibroblast-specific variant, caused an asymptomatic infection. MVM(1035), a chimera which had the allotropic determinant of virulent MVMi inserted onto an MVMp background, caused a lethal infection and renal papillary infarcts, the hallmark of MVMi infection. MVMi(NS2-1990), the virulent lymphocyte-specific variant mutated to eliminate NS2 synthesis, was infectious but caused an asymptomatic infection. Sequential virus titration, histology, in situ hybridization with a full-length MVMi genomic probe, and immunohistochemistry for viral capsid antigen were used to compare the pathogenesis of infection with the four MVM genotypes. Infectious virus was recovered from multiple organs of mice infected with MVMi, MVMp, and MVM(1035) but not from mice infected with MVMi(NS2-1990). MVMp titers were lower than MVMi titers in all organs except the intestine. MVM(1035) titers were higher than MVMi titers in all organs except the blood. MVMp was localized to connective tissue elements of the intestine, to cells in mesenteric lymph nodes, and rarely to cells in other organs. MVM(1035) was localized to multiple organs and shared the same target cells, endothelium, lymphoid cells, and hematopoietic cells, as MVMi. MVM(1035) also replicated in external germinal cells of the cerebellum and smooth muscle cells of the stomach and colon, which were not targets of MVMi or MVMp infection. MVMi(NS2-1990) replicated to a limited degree in some MVMi target organs.


Mapping genetic determinants for human immunodeficiency virus type 1 resistance to soluble CD4.

Neutralization of human immunodeficiency virus type 1 (HIV-1) infection with soluble CD4 (sCD4) can be achieved over a broad range of concentrations for different virus strains. Laboratory virus strains passaged in transformed T-cell lines are typically sensitive to sCD4 neutralization, whereas primary virus isolates require over 100-fold-higher sCD4 concentrations. Using recombinant viruses generated from a laboratory strain, HIV-1NL4-3, and a primary macrophagetropic strain, HIV-1JR-FL, we mapped a region of gp120 important for determining sensitivity to sCD4 neutralization. This same region has previously been defined as important for macrophage and transformed T-cell line tropism and includes the V3 neutralization domain but does not include regions of gp120 that have been shown to be most important for CD4 binding.


Mutation of human immunodeficiency virus type 1 at amino acid 585 on gp41 results in loss of killing by CD8+ A24-restricted cytotoxic T lymphocytes.

A human leukocyte antigen A24-restricted CD8+ cytotoxic T-cell clone specific for gp41 of human immunodeficiency virus type 1 was isolated from an infected individual. The epitope was localized to amino acids 584 to 591 (YLKDQQLL, NL43 env sequence) of gp41 by using a panel of recombinant vaccinia viruses that contain truncated env genes and synthetic peptides. The clone killed autologous B-lymphoblastoid cell lines pulsed with a synthetic peptide reflecting the sequence of the IIIB and MN strains. This clone, however, failed to kill target cells pulsed with the peptides that have a mutation from Lys to Arg or Gln at amino acid 585 which is present in some prototype human immunodeficiency virus type 1 strains, e.g., ADA, JFL, SC, ALA1, BAL1, SF2, VRF, SF33, and WMJ2. This finding that a mutation at amino acid 585 on gp41 results in nonrecognition by human leukocyte antigen A24-restricted CD8+ cytotoxic T lymphocytes suggests that antigenic variation at T-cell epitopes contributes to the failure of immune control of human immunodeficiency virus type 1 infections.


Poliovirus chimeras expressing sequences from the principal neutralization domain of human immunodeficiency virus type 1.

Sequences from the principal neutralization domain of human immunodeficiency virus type 1 (HIV-1) strain LAI or RF have been expressed in antigenic site 1 of the capsid of the Sabin strain of poliovirus type 1. A number of the resulting chimeras were viable. Viable variants bearing mutations within the insertion site spontaneously arose from several nonviable chimeras. In general, these mutations result in a decrease in positive charge in the substituted antigenic site 1. Two of the chimeras were genetically stable and have been further characterized. Both chimeras were neutralized by various HIV-1 neutralizing antibodies. In rabbits, both chimeras produced high levels of antibodies which react with HIV-1 gp120/160 in immunoprecipitation and enzyme-linked immunosorbent assays. One of the chimeras (HIV-1LAI) produced a significant but weak HIV-1 neutralizing response.


Reverse transcriptase of human immunodeficiency virus type 1: functionality of subunits of the heterodimer in DNA synthesis.

From an in vitro analysis of the DNA-synthesizing abilities of certain specifically mutated forms of the heterodimeric reverse transcriptase of human immunodeficiency virus type 1, we can conclude that in a heterodimer, the functionality of p66 is necessary while the functionality of the p51 subunit is not needed. Conversely, p51 is not able to catalyze DNA synthesis when associated with p66, and yet when the p66 protein is absent, p51 can function. These conclusions applied to DNA synthesis on heteropolymeric RNA and DNA templates.


[Anesthetic management of a hemophilia A carrier for palliative operation and radical repair of a cardiac anomaly].

We experienced the anesthetic management of a hemophilia A carrier for two palliative operations (left and right unifocalization) and radical repair of cardiac anomaly. The patient was a ten-year-old girl and her father had hemophilia A. She was diagnosed as tetralogy of Fallot, major aortopulmonary collateral arteries and pulmonary atresia. Laboratory findings showed prolonged bleeding time and activated partial thromboplastin time, and decreased levels of factor VIII activity of 26%. When she was eight and nine years old, she underwent unifocalization. At both occasions, excessive bleeding tendency continued into postoperative period. This time she underwent radical repair in which her bleeding tendency was successfully controlled by use of a usual dose of concentrated factor VIII for twice.


[Effect of ursodeoxycholate on pancreatic exocrine secretion in vitro and in vivo study].

In the present study, we examined the effect of ursodeoxycholate (UDCA) and it's taurine conjugate (TUDC) on rat pancreatic exocrine secretion using dispersed pancreatic acini (in vitro) and conscious rats (in vivo). In in vitro study 300 microM UDCA significantly increased 10(-12)-10(-9) M CCK-8 stimulated amylase release and change of intracellular Ca2+ concentration, but TUDC did not have these effects. In in vivo study intraduodenal infusion of UDCA but not TUDC stimulated pancreatic exocrine secretion. Intravenous infusion of secretin antibody decreased bicarbonate output, however, this increase was not prevented by CCK antagonist. Thus, it was suggested that UDCA has direct action on pancreatic acini and UDCA infused intraduodenally stimulates pancreatic secretion, possibly via the release of a secretin-like substance. The taurine conjugate has weak bioactivity on pancreatic exocrine secretion in both in vitro and in vivo.


A block in release of progeny virus and a high particle-to-infectious unit ratio contribute to poor growth of enteric adenovirus types 40 and 41 in cell culture.

The fastidious enteric adenovirus (FEAd) types 40 (Ad40) and 41 (Ad41) are found in stool specimens of infants and young children in association with gastroenteritis. Although they can be isolated routinely from clinical specimens by using 293 cells, they are propagated with variable success in cell lines which support the replication of other adenovirus serotypes. HeLa cells are generally considered to be nonpermissive for the replication of FEAds, but in this study, Ad40 and Ad41 grew to comparable titers in individual 293 and HeLa cells. However, virus was not efficiently released from infected HeLa cells and thus did not undergo multiple cycles of infection in HeLa cell cultures. The block in virus release was not overcome in KB18 cells which, like 293 cells, constitutively express proteins encoded by the E1B region of a subgroup C adenovirus (in this case Ad2). Moreover, it was apparent from these studies that Ad40 and Ad41 have particle-to-infectious unit ratios several orders of magnitude greater than that for Ad5, even in 293 cells which express the E1A and E1B proteins of Ad5 and are considered to be permissive for replication of the FEAds. Neither the block in release of progeny virus nor the high particle-to-infectious unit ratio is explained solely by the defect in expression of the E1B 55K protein identified by Mautner et al. (V. Mautner, N. MacKay, and V. Steinthorsdottir, Virology 171:619-622, 1989; V. Mautner, N. MacKay, and K. Morris, Virology 179:129-138, 1990).


On the biochemical modulation of 6-mercaptopurine by methotrexate in murine WEHI-3b leukemia cells in vitro.

The chemicals 6-mercaptopurine (6-MP) and methotrexate (MTX) are the cornerstones in the maintenance treatment of acute lymphoblastic leukemia. The intracellular metabolism of 6-MP to 6-thioguanosine nucleotides (TGN) via 6-thioinosine 5'-monophosphate (TIMP) is crucial for its cytotoxic effect. MTX inhibits purine de novo synthesis and thereby increases the intracellular PRPP being a substrate for the phosphoribosylation of 6-MP to TIMP. Hypoxanthine has been shown to inhibit the uptake of 6-MP over the cell membrane and the phosphoribosylation of 6-MP to TIMP. We have previously shown that the conversion of TIMP to TGN decreases at 6-MP concentrations above 5 microM in vitro. The aim of the present study was therefore to investigate the effect of MTX increasing the PRPP and TIMP concentrations and of hypoxanthine decreasing the TIMP concentration on the formation of TGN from TIMP. Murine myelomonocytic leukemia cells (WEHI-3b) were treated with 6-MP in vitro. The drug concentration was kept constant by continuous addition of 6-MP during the experiment. With this technique, the concentration of TGN begins to decrease already at 6-MP concentrations above 2 microM. The addition of 0.2 microM MTX 6 h before 6-MP strongly inhibited the purine de novo synthesis, decreased the ATP, and increased the PRPP concentration 4-fold. The intracellular concentrations of TIMP and to a lesser extent TXMP also increased. The concentrations of the TGN were, however, basically unaffected by the preincubation with MTX. Simultaneous addition of 20-50 microM hypoxanthine and 6-MP decreased the accumulation of all cellular 6-MP metabolites. It is concluded that the synergistic cytotoxic effect of the combination of 6-MP and MTX is not based on biochemical modulation of the 6-MP metabolism by MTX.


[Wilson's disease. A histological review of 7 patients and the value of histological copper positivity in relation to other hepatopathies].

Wilson's disease is an infrequent entity. Biopsy is essential for establishing its presence and degree of hepatopathy but it lacks diagnostic specificity. It is necessary to know the significance of positivity or negativity of histochemically demonstrated copper when faced with the diagnosis of Wilson's disease and cholestatic diseases. Nine hepatic sample from 7 patients with clinical-biochemical Wilson's disease were reviewed. The rodanine histochemical technique was used for demonstration of copper in the samples aforementioned and in another 3 kinds of liver diseases: 16 biopsies of primary biliary cirrhosis, 86 biopsies of cholestatic hepatopathies (serum bilirubin greater than 68 mumol/l) and control biopsies with serum bilirubin less than 42.5 mumol/l. Among the 7 patients with Wilson's disease, 6 were diagnosed with cirrhosis and one chronic active hepatitis. The incidence of erosive necrosis (86%), Mallory hyaline (86%), macrovacuolar steatosis (71%), glucogenated nuclei (71%) and giant mitochondria (29%) was evaluated. Histologic copper deposit with Shikata orcein was objectified in 3 cases (43%) and with rodanine in 5 (83%). Positivity with rodanine was 69% among the series of primary biliary cirrhosis, 20% among the cholestatic hepatopathies and 0 in the control series. Histology of Wilson's disease remains without definitive diagnostic criteria. The disease is diagnosed in a late phase in Spain. The sensitivity of the histochemical demonstration of copper with rodanine is high (6/7) but its specificity is low, being positive in 68% of primary biliary cirrhosis and 19% of other cholestatic hepatopathies.


Cytophotometric analysis of magnocellular azure B-RNA and Feulgen-DNA following chronic GABA infusion into the nucleus basalis of rats.

This investigation was undertaken to examine possible cytopathic effects of GABA infusion on nucleus basalis (NBM) magnocellular neurons. Sixty-three male Long-Evans rats received unilateral, intra-NBM infusions of either GABA100 (100 micrograms/microliters/h), GABA10 (10 micrograms/microliters/h), or ultrafiltered saline (1 microliter/h) for a period of 24 hours. Rats from each of these groups were sacrificed at either 24 hours, 48 hours or 8 days following initiation of infusions. The sham operated hemisphere of each rat served as a control for the infused hemisphere. After stoichiometric azure B-RNA and Feulgen-DNA staining of brain sections, scanning-integrating microdensitometry was used to quantify GABA-induced alterations in these well established indices of neuronal toxicity. These results provide evidence that the neurotoxic effects of 24 hours of 100 micrograms/microliters-h GABA infusion are manifested within 48 hours post-initiation of infusions. Although 24 hours of 10 micrograms/microliters-h GABA infusion suppressed NBM neuronal metabolism, the lower magnitude and duration of this effect signified an impending recovery. GABA infusion resulted in little if any NBM neuronal chromatin template impairment (i.e., reduced Feulgen-DNA reactivity), irrespective of the dosage employed and the delay prior to sacrifice.


Porins and specific channels of bacterial outer membranes.

Porins and specific channels both produce water-filled pores that allow the transmembrane diffusion of small solutes, but the latter contain specific ligand-binding sites within the channels. Recent structural studies show that many or most of these proteins exist as beta-barrels with the beta-strands traversing the thickness of the outer membrane. The channels often have diameters in the range of 1 nm, and thus the penetration rates of solutes through porin channels are likely to be affected strongly by what appear to be minor differences in the size, shape, hydrophobicity or charge of the solute molecule. With the specific channels, the presence of binding sites can accelerate very significantly the diffusion of some ligands when they are present at low concentrations. Thus these simple channels can sometimes achieve a surprising degree of real or apparent specificity. Recent data tend to favour the idea that these proteins are first exported into the periplasm, and then inserted into the outer membrane. Although lipopolysaccharides seem to play a significant role in the final assembly of the trimeric porins, the details of the targeting process still remain to be elucidated.


Chemical purity and mutagenicity: case study of a drug in development.

During a routine Ames assay of a potential antipsychotic drug candidate, the compound appeared to be a frameshift mutagen in Salmonella typhimurium strains TA98 and TA1538. Additional testing indicated the mutagenic activity was due to one or more contaminants incurred during synthesis. While the compound was initially shown to be greater than 98% pure by high-performance liquid chromatography, the presence of small amounts (0.01-0.1%) of a highly mutagenic impurity produced positive mutagenicity results. The need to assess for chemical purity before discontinuing development of drug candidates found positive in the Ames assay is discussed.


Mutagenicity of the reaction products of dibenzo-p-dioxin with nitrogen oxides.

Dibenzo-p-dioxin (DD) was made to react with various concentrations of nitrogen oxides in the dark. The mutagenicities of the reaction products were tested using Salmonella typhimurium strains TA98, TA100, TA98NR and TA98/1,8-DNP6 in the presence or absence of a mammalian metabolic activation system (S9 mix). DD-NOx (molar ratios 1:3, 1:6 and 1:18) reaction products exhibited mutagenic potency in strains TA98 and TA98/1,8-DNP6 without S9 mix. In a gas chromatography/mass spectrometry study, 2-nitrodibenzo-p-dioxin (NDD) was identified with authentic sample in the mutagenic reaction products. DD-NOx (1:18) reaction products were reduced by sodium hydrogen sulfide and the reduction mixture was analyzed by HPLC. 2,7-Dinitrodibenzo-p-dioxin (DNDD) and 2,8-DNDD were identified as corresponding diamino-DDs in the reduction mixture. 2-NDD, 2,7-DNDD and 2,8-DNDD were also mutagenic in strains TA98 and TA98/1,8-DNP6 without S9 mix and the mutagenicity of DD-NOx reaction products was largely accounted for by the nitro-DDs.


Genotoxic activity of methyl mercury chloride and dimethyl mercury in human lymphocytes.

The genotoxicity of methyl mercury chloride (MMC, 0-25 x 10(-6) M) and dimethyl mercury (DMM, 0-434 x 10(-6) M) was evaluated by chromosome metaphase analysis in human lymphocytes treated in vitro for 24 h. Structural (CA) and numerical (AN) chromosomal aberrations were scored for the assessment of induced genotoxic effects, while the variation in mitotic index (MI) was considered a monitor for induced cellular toxicity. MMC induced CA and AN in a dose-related manner at doses exceeding 0.6 x 10(-6) M, and the proportion of cells with CA was constantly and significantly higher than that of cells with AN. DMM was able to induce both effects as well, although to a lesser extent than MMC, CA and AN being induced at doses exceeding 43.4 x 10(-6) M and 1.73 x 10(-6) M, respectively. MMC was 6-fold more effective in inducing CA than DMM at equivalent toxic doses. On the other hand, no significant difference was observed between the two compounds in inducing AN. Therefore MMC was much more clastogenic than DMM, whereas mitotic spindle disturbances appeared to be almost equally induced by both compounds.


Published data on mutagenesis by ionizing radiation of plasmids in solution probably reflect in part the specificity of adventitious transition metal ions complexed to the DNA.

A number of recent papers show that single base changes induced by mutagenesis with ionizing radiation of genes on plasmids in solution, followed by transfection into mammalian or bacterial cells for assay, are mostly at G:C base pairs, with mutagenic hot spots. Genes irradiated in mammalian or bacterial cells, on the other hand, have comparable numbers of base changes at all sites, with no evidence for hot spots. The differences are ascribed to induction of many base change mutations in vitro by reactions catalyzed by adventitious transition metal ions complexed to the DNA. Reasons are given why this process should play a much smaller role in vivo.


Antagonists to cholinergic receptors increase the frequency of binuclear V79 Chinese hamster cells. A mechanism for induction of aneuploidy.

V79 Chinese hamster cells were found to produce significant amounts of acetylcholine. Asynchronously growing V79 cells were treated with five different antagonists to cholinergic receptors: atropine and scopolamine, which are inhibitors of muscarinic receptors, and mecamylamine, d-tubocurarine and alpha-bungarotoxin, which are inhibitors of nicotinic receptors. All compounds caused a slight but significant increase of the frequency of binuclear interphase cells and also of the frequency of cells in late telophase and early G1 that had not completed cleavage. In addition, hemicholinium-3, a specific choline uptake antagonist, inhibited cleavage. Taken together, it seems reasonable to hypothesize that acetylcholine and its receptors take part in the regulation of cleavage in these cells. As binuclear cells are prone to aberrant spindle functions in following mitoses, inhibition of cleavage may constitute a risk for generation of cells with highly aberrant chromosome numbers.


Effects of benzyladenine on prokaryotic and eukaryotic cells.

Comparative studies of the effect of benzyladenine (BA) on the yeast Saccharomyces cerevisiae, the bacterium Salmonella typhimurium, the shallot Allium ascalonicum and Chinese hamster fibroblast cells were performed. The tested substance had no mutagenic activity on yeast, bacteria and cultured fibroblast cells. Changes in mitotic activity and cell division abnormalities were observed after BA treatment in shallot root-tip cells.


The effect of tequila in the synaptonemal complex structure of mouse spermatocytes.

The effect of tequila in the synaptonemal complex (SC) of mouse spermatocytes was determined. We tested 3 dosages (2.1, 4.2 and 8.4 g/kg) administered in a single intraperitoneal inoculation. The frequency of SC alterations was established in pachytenic nuclei 5 days after the administration using a silver impregnation technique. Three types of alterations were observed (desynapses, breaks and multiaxials) and the rate of each alteration was compared with that obtained with appropriate controls, including cyclophosphamide (CP) (150 mg/kg). The results showed a significant increase induced by tequila only in the frequency of desynapses. This damage began at the second highest dose (4.2 g/kg). The other SC alterations were in the control range. CP, however, induced a significant increase in all 3 types of SC alterations.


Kinetochore-staining of spermatid micronuclei: studies of mice treated with X-radiation or acrylamide.

The rodent spermatid micronucleus (MN) assay was used in conjunction with immunofluorescent techniques to distinguish kinetochores in MN following exposure of mice to X-radiation or acrylamide. After either treatment, modest increases in kinetochore-positive MN were observed. Spermatids which had been exposed during meiotic prophase to X-rays (400 cGy) had approximately 10-fold increases in MN compared to controls; up to 15% of the MN observed were kinetochore-positive. Following acrylamide treatment of meiotic prophase cells, there was a doubling of spermatid MN over baseline levels, approximately one-third of which were kinetochore-positive.


Protein kinase C reduces Mg2+ block of NMDA-receptor channels as a mechanism of modulation.

The roles of N-methyl-D-aspartate (NMDA) receptors and protein kinase C (PKC) are critical in generating and maintaining a variety of sustained neuronal responses. In the nociceptive (pain-sensing) system, tissue injury or repetitive stimulation of small-diameter afferent fibres triggers a dramatic increase in discharge (wind-up) or prolonged depolarization of spinal cord neurons. This central sensitization can neither be induced nor maintained when NMDA receptor channels are blocked. In the trigeminal subnucleus caudalis (a centre for processing nociceptive information from the orofacial areas), a mu-opioid receptor agonist causes a sustained increase in NMDA-activated currents by activating intracellular PKC. There is also evidence that PKC enhances NMDA-receptor-mediated glutamate responses and regulates long-term potentiation of synaptic transmission. Despite the importance of NMDA-receptors and PKC, the mechanism by which PKC alters the NMDA response has remained unclear. Here we examine the actions of intracellularly applied PKC on NMDA-activated currents in isolated trigeminal neurons. We find that PKC potentiates the NMDA response by increasing the probability of channel openings and by reducing the voltage-dependent Mg2+ block of NMDA-receptor channels.


Normal development and behaviour of mice lacking the neuronal cell-surface PrP protein.

PrPC is a host protein anchored to the outer surface of neurons and to a lesser extent of lymphocytes and other cells. The transmissible agent (prion) responsible for scrapie is believed to be a modified form of PrPC. Mice homozygous for disrupted PrP genes have been generated. Surprisingly, they develop and behave normally for at least seven months, and no immunological defects are apparent. It is now feasible to determine whether mice devoid of PrPC can propagate prions and are susceptible to scrapie pathogenesis.


[Somatostatin in the prevention of postoperative increase of pancreatic enzyme after pancreatic surgery].

The prophylactic effect of perioperative use of somatostatin on postoperative increase of pancreatic digestive enzymes was investigated in this double blind, randomized study. Thirty three patients undergoing pancreatic surgery because of chronic pancreatitis were divided randomly into two groups. Fifteen patients received somatostatin- (dose 125 micrograms/hour), 18 placebo-infusion, pre- and postoperatively for a total time of 48 hours. The level of serum amylase, lipase, gammaGT, calcium, creatinine and blood glucose was determined every 12 hours. In the placebo group the serum lipase and amylase increased significantly (p less than 0.001), while the calcium decreased. In the somatostatin treated patients only the lipase level increased significantly (p less than 0.01), while the amylase and calcium showed no significant changes compared to their initial values. The postoperative increase in serum enzyme levels is interpreted as being an indicator of pancreatic injury. These results suggest that the perioperative use of somatostatin has beneficial effect for the prevention of pancreatic enzymes increases, associated with pancreatic surgery or its complications in patients with chronic pancreatitis.


Decreased granulocyte-macrophage colony-stimulating factor production by human neonatal blood mononuclear cells and T cells.

Impaired production and delivery of neutrophils to the site of infection have been implicated in the increased susceptibility of the neonate to infection. Because granulocyte-macrophage colony-stimulating factor (GM-CSF) and granulocyte colony-stimulating factor (G-CSF) play critical roles in the production of neutrophils from marrow precursors, we assessed the ability of leukocytes from neonates and adults to produce GM-CSF, G-CSF, and, for comparison, macrophage colony-stimulating factor (M-CSF) after stimulation with concanavalin A +/- phorbol myristate acetate [blood mononuclear cells (MC) and T lymphocytes] or lipopolysaccharide (monocytes). MC and monocytes from adult and neonatal subjects produced mRNA for GM-CSF, G-CSF, and M-CSF, whereas T cells produced only GM-CSF mRNA. Neonatal MC and T cells accumulated only approximately 30% as much GM-CSF mRNA as did adult MC and T cells. In contrast, the accumulation of GM-CSF mRNA by neonatal and adult monocytes was similar. Neonatal MC also accumulated similar amounts of G-CSF mRNA and somewhat more M-CSF mRNA than did adult MC; results with monocytes were similar to those with MC. Results of colony-stimulating activity bioassays on supernatants from neonatal and adult MC stimulated with concanavalin A paralleled the mRNA results.(ABSTRACT TRUNCATED AT 250 WORDS)


Hepatic protein synthesis in suckling rats: effects of stage of development and fasting.

We studied the developmental changes in hepatic protein synthesis in suckling rats between postpartum d 1 and 28 and investigated the effect of fasting for 10 or 18 h on hepatic protein turnover at postpartum d 5, 10, 16, and 28. Fractional protein synthesis rates (KS, %/d) were measured in vivo using a flooding dose of L-[4-3H]phenylalanine. Although hepatic KS and translation efficiency (protein synthesis/unit RNA) were significantly higher at postpartum d 28 than d 1, the pattern of change was biphasic: KS and translational efficiency were higher at d 10 and 28 than at d 5 and 16. The largest increase in KS and translational efficiency occurred during the period normally associated with weaning (between postpartum d 16 and 28). At all stages of development, the KS and translational efficiency in fasted rats were significantly lower than those in control (fed) rats, although the relative decline in both measurements was largest at postpartum d 10. The absolute rates of hepatic protein synthesis declined to similar levels on d 5, 10, and 16 after 10 h of fasting and changed little after 18 h of fasting; this level was significantly higher at postpartum d 28. Our results suggest that postnatal development in suckling rats was marked by a biphasic pattern in the rates of hepatic protein synthesis, which increased during the neonatal and weaning periods. The relative changes in the synthesis and loss of hepatic protein in response to fasting were greater during the neonatal than during the late suckling and weaning periods.


Synthesis and base-pairing properties of the nuclease-resistant alpha-anomeric dodecaribonucleotide alpha-[r(UCUUAACCCACA)].

The non natural oligoribonucleotide alpha-[r(UCUUAACCCACA)] consisting exclusively of alpha-anomeric ribonucleoside units was synthesized according to the phosphoramidite methodology and the solid support technology. For this purpose, the base-protected alpha-ribonucleosides were synthesized and converted into their O-methylphosphoramidites. Assembling was carried out on a DNA synthesizer with an average efficiency of 97% per step. Base composition of this nuclease-resistant alpha-RNA strand was ascertained after chemical and enzymatic hydrolysis and HPLC analysis of the hydrolysate. Whereas no spectroscopic evidence of base pairing was found above 0 degrees C between alpha-[r(UCUUAACCCACA)] and beta-[d(TGTGGGTTAAGA)], a clear UV absorbance transition (Tm 25.5 degrees C) was observed during the hybridization of the same alpha-RNA strand with beta-[d(AGAATTGGGTGT)]. In this latter case, the mixing curve titration suggests formation at low temperature of a triplex involving two alpha-RNA and one beta-DNA strands. Moreover, this alpha-decaribonucleotide complementary in parallel orientation of the splice receptor of HIV-1 tat mRNA was found to inhibit (10 microM less than ED50 less than 20 microM), with apparent lack of sequence specificity, the de novo HIV-1 infection in cultured cells.


Mutations which alter splicing in the human hypoxanthine-guanine phosphoribosyltransferase gene.

A large proportion of mutations at the human hprt locus result in aberrant splicing of the hprt mRNA. We have been able to relate the mutation to the splicing abnormality in 30 of these mutants. Mutations at the splice acceptor sites of introns 4, 6 and 7 result in splicing out of the whole of the downstream exons, whereas in introns 1, 7 or 8 a cryptic site in the downstream exon can be used. Mutations in the donor site of introns 1 and 5 result in the utilisation of cryptic sites further downstream, whereas in the other introns, the upstream exons are spliced out. Our most unexpected findings were mutations in the middle of exons 3 and 8 which resulted in splicing out of these exons in part of the mRNA populations. Our results have enabled us to assess current models of mRNA splicing. They emphasize the importance of the polypyrimidine tract in splice acceptor sites, they support the role of the exon as the unit of assembly for splicing, and they are consistent with a model proposing a stem-loop structure for exon 8 in the hprt mRNA.


Chemical synthesis of RNA using fast oligonucleotide deprotection chemistry.

The exocyclic amine protecting groups in oligonucleotide synthesis which require 8-16 hours at 55 degrees C for deprotection in ammonia have been replaced with more labile base protecting groups (dimethylformamidine for adenine and guanine and isobutyryl for cytosine). Using these fast oligonucleotide deprotecting groups which require 2-3 hours at 55 degrees C for complete deprotection, a new set of cyanoethyl phosphoramidite ribonucleoside monomers and supports has been developed. Ribozymes and substrate RNAs which were synthesized with these phosphoramidites were assayed and were found to have full catalytic (biological) activity.


A pyrimidine-guanine sequence-specific ribonuclease from Rana catesbeiana (bullfrog) oocytes.

A pyrimidine-guanine sequence-specific ribonuclease (RC-RNase) was purified from Rana catesbeiana (bullfrog) oocytes by sequential phosphocellulose, Sephadex G75, heparin Sepharose CL 6B and CM-Sepharose CL 6B column chromatography. The purified enzyme with molecular weight of 13,000 daltons gave a single band on SDS-polyacrylamide gel. One CNBr-cleaved fragment has a sequence of NVLSTTRFQLNT/TRTSITPR, which is identical to residues 59-79 of a sialic acid binding lectin from R. catesbeiana eggs, and is 71% homologous to residues 60-80 of an RNase from R. catesbeaina liver. The RC-RNase preferentially cleaved RNA at pyrimidine residues with a 3' flanking guanine under various conditions. The sequence specificity of RC-RNase was further confirmed with dinucleotide as substrates, which were analyzed by thin layer chromatography after enzyme digestion. The values of kcat/km for pCpG, pUpG and pUpU were 2.66 x 10(7) M-1s-1, 2.50 x 10(7) M-1s-1 and 2.44 x 10(6) M-1s-1 respectively, however, those for other phosphorylated dinucleotides were less than 2% of pCpG and pUpG. As compared to single strand RNA, double strand RNA was relatively resistant to RC-RNase. Besides poly (A) and poly (G), most of synthetic homo- and heteropolynucleotides were also susceptible to RC-RNase. The RC-RNase was stable in the acidic (pH 2) and alkaline (pH 12) condition, but could be inactivated by heating to 80 degrees C for 15 min. No divalent cation was required for its activity. Furthermore, the enzyme activity could be enhanced by 2 M urea, and inhibited to 50% by 0.12 M NaCl or 0.02% SDS.


Photodynamic inactivation of an ion channel: gramicidin A.

The ion channel formed by the peptide gramicidin A in planar lipid membranes is inactivated by visible light in the presence of the photosensitizer Rose Bengal. This is concluded from the strong decrease of the membrane conductance by more than two orders of magnitude. Experiments performed at different oxygen concentrations, in the presence of the singlet oxygen quenchers beta-carotene or alpha-tocopherol indicate, that presumably a type I process between the dye Rose Bengal and the tryptophan residues of the gramicidin channel with a subsequent oxidation of the tryptophans is responsible for the loss of the conductance properties of the channel.


Malignant microvasculature in abdominal tumors in children: detection with Doppler US.

Malignant tumors produce a network of microscopic, thin-walled vessels that invade the host and provide the blood supply essential for growth. This network, situated at the periphery of malignant tumors, gives rise to characteristic, high-velocity Doppler shifts at ultrasound (US) scanning and was sought in 54 children with abdominal masses. Characteristic high-frequency Doppler signals (greater than 2.5 kHz) were found in 27 of 35 malignant tumors. These signals disappeared during successful chemotherapy in three children with neuroblastoma in whom the signals had been documented initially. The signals were absent in three other children, who had undergone clinically successful therapy, and were present in two children with rapidly progressing tumors despite chemotherapy. In 14 children, none of the benign tumors showed high-frequency Doppler shifts. The search for malignant neovasculature with Doppler US scanning may provide insight into tumor behavior. The Doppler US examination, a noninvasive adjunct to routine US, may become useful at both the time of diagnosis of a mass and afterward to assess the effect of chemotherapy.


[Transposition of the great arteries. A follow-up of patients operated on with atrial correction].

The late results of 146 patients with transposition of the great arteries (simple: 119, complex: 27) surviving to physiological correction (Mustard: 33, Senning: 113) are analyzed. The mean age at operation was 27 months (1-120), and the mean follow-up was 78 months (6-187). The mean P wave voltage significantly decreased (from 0.3 mV preoperatively to 0.16 mV postoperatively). Frontal mean P wave axis varied from 63.9 degrees to 71.3 degrees. Mean heart rate were significantly lower than those for age-matched normal children. Previous atrioseptectomy had been performed in 36 patients and five of them had arrhythmias before operation. 78 (55%) out of the remaining 141 showed arrhythmias in the surface ECG: sinus node disfunction in 51, tachyarrhythmias in eight, atrioventricular block in eight and several association of arrhythmias in 11. Actuarial survival free of arrhythmias is 81% at the first year, 49% at 5 years and 22% at 15 years. The incidence of arrhythmias in the group of 92 patients with Holter monitoring was higher, with 73 cases having arrhythmias (79%): sinus node disfunction in 47, tachyarrhythmias in ten, atrioventricular block in four and associated in 12. Survival free of arrhythmias by both methods (ECG and Holter) shows a 78% at the first year, 28% at 5 years and 5.6% at 15 years. No significant correlation was found between the incidence of arrhythmias and the type of TGA (simple or complex) or the surgical technique (Mustard or Senning). Permanent pacemakers were implanted in seven to a mean follow-up of 7 years. Five patients were reoperated to a mean follow-up of 46 months. There were 7 late deaths to a mean follow-up of 13 months, four of them being sudden. The actuarial survival rate for the whole group was 93.5% at 15 years. No significant correlation was found between the late mortality and the type of TGA or operation. There was also no correlation with the incidence of arrhythmias. Residual lesions were found in 46 patients, hemodynamically significant in ten. Functional status was class I (NYHA) in 123, and class II in 16. There was correlation between the functional class and the residual lesions.


[The expansion of NK CD56+ cells with reduced cytotoxicity in a female patient with systemic sclerosis].

An absolute and relative increase of circulating CD56+ (NKH1, Leu19) natural killer cells has been found in a patient with systemic sclerosis. The expanded natural killer cells exhibited reduced natural killer activity and antibody-dependent cell-mediated cytotoxicity. Furthermore, the limiting dilution analysis of spontaneous in vitro immunoglobulin synthesis demonstrated that the precursor frequency of immunoglobulin-secreting cells and the "single-hit" kinetics of the titration curve were similar to healthy controls, but strongly different from previously reported patients in whom CD16+NK cell subset was expanded. Thus, our findings might suggest that expanded CD56+ natural killer cells exhibit unique regulatory properties on B cell function in systemic sclerosis.


Rhabdomyosarcoma in a congenital pigmented nevus.

A 7-month-old boy had a giant pigmented lesion involving the trunk and thighs that exhibited many hyperpigmented hairy and verrucous nevi. One of the nevi ulcerated and on histological examination consisted of pleomorphic rhabdomyosarcoma cells that stained for muscle-specific actin (HHF-35), desmin, and myoglobin. Around the tumor, in the dermis, benign pigmented nevus cells were observed. The occurrence of malignant tumors, other than malignant melanoma, in pigmented nevi is rarely described.


Acute cytotoxic effect of cyclosporin A on pancreatic acinar cells in rats. Protective effect of the synthetic protease inhibitor E3123.

This study was designed to evaluate the acute toxic effects of cyclosporin A on the exocrine pancreas and the protective effects of a new potent protease inhibitor, 4-(2-succinimidoethylthio) phenyl 4-guanidinobenzoate methanesulfonate, E3123. Cyclosporin A in a dose of 5 mg/kg.h caused a significant increase in serum amylase levels, pancreatic amylase content, and interstitial edema, suggesting that it induces exocrine pancreatic injury. Cyclosporin A also caused redistribution of cathepsin B from the lysosomal fraction to the zymogen fraction, indicating colocalization of lysosomal enzymes with pancreatic digestive enzymes. E3123 in a dose of 2 mg/kg.h prevented almost completely these changes caused by cyclosporin A. These results indicate that exocrine pancreatic injury is induced by cyclosporin A and that lysosomal enzymes play important roles in the pathogenesis of this injury and also suggest that E3123 might protect the exocrine pancreas of patients receiving cyclosporin A after organ transplantation.


The roles of ionic processes in muscular fatigue during intense exercise.

Muscular fatigue is manifested by a decline in force- or power-generating capacity and may be prominent in both submaximal and maximal contractions. Disturbances in muscle electrolytes play an important role in the development of muscular fatigue. Intense muscular contraction is accompanied by an increased muscle water content, distributed in both intracellular and extracellular spaces. This water influx will modify ionic changes in both compartments. Changes in muscle intracellular electrolyte concentrations with intense contraction may be summarised as including decreases in potassium (6 to 20%) and in creatine phosphate (up to 70 to 100%) and increases in lactate (more than 10-fold), sodium (2-fold) and small, variable increases in chloride. The net result of these intracellular ionic concentration changes with exercise will be a reduction in the intracellular strong ion difference, with a consequent marked rise in intracellular hydrogen ion concentration. This intracellular acidosis has been linked with fatigue via impairment of regulatory and contractile protein function, calcium regulation and metabolism. Potassium efflux from the contracting muscle cell dramatically decreases the intracellular to extracellular potassium ratio, leading to depolarisation of sarcolemmal and t-tubular membranes. Surprisingly little research has investigated the effects of intense exercise training on electrolyte regulation and fatigue.


[Approaches to neuropsychiatry as a transition in the separation of neurology and psychiatry].

The focus of the article is on the separation of neurology and psychiatry and their interconnection by means of the discipline of neuropsychiatry. First, the oppositions of "Structure versus Functions" and "Localization versus Holism" are worked out as the main reasons for the separation of neurology and psychiatry. This is followed by the demonstration of the recent developments of "biological psychiatry", "behavioral neurology" and "neuropsychology" which try to bridge the gulf between neurology and psychiatry. Their inadequacies with regard to a real bridge between the two disciplines, which takes account for both sides, is shown. Consecutively, a specific neuropsychiatric conception as a functional dynamic one, is suggested, which can be distinguished from the approaches in "biological psychiatry" and "behavioral neurology". This neuropsychiatric conception is applied, in a last step, to the oppositions of "Structure versus Function" and "Localization versus Holism", by means of which these oppositions, and the one between neurology and psychiatry, can be bridged--this is exemplified by means of the Parkinson-Syndrome with its neurologic and psychiatric symptoms.


[Electrodermal hypoactivity in depression: psychobiological marker or differential psychophysiologic disposition?].

Research about the dominating hypoactivity in electrodermal system in affective disorders led to the assumption that electrodermal activity (EDA) could be an important biopsychological trait in the etiology of depression. However, experimental results are contradictory. Some studies find nosological validity, other studies an influence of psychomotor status on EDA or correlations to clinical improvement, etc. Most of these results are based on laboratory conditions (habituation experiment with tones). The person-situation-environment interaction and their dynamic process regulation on electrodermal activity are neglected. Also lacking is a comparable description of nosology, psychomotor status and experimental design. After discussing the neuropsychological background of the habituation paradigm which led to the "traditional" assumptions of the correlation between electrodermal activity and depression, results of different samples are shown tested over years using always the same classification and experimental methods. None of our assumptions could be verified. Although low levels in SCL dominate, we could not find results of high sensitivity and specificity in contrast to controls. However we found differences in other, more complex experiments containing personality traits and reactions to emotional words. In a multi-level process-oriented stress paradigm of the success/failure type we found a delay in regeneration of psychological and electrodermal measures after failure. This results demonstrate that investigation in electrodermal activity needs more complex and process-oriented experimental designs. EDA probably has not the quality of a biological marker, but seen in the context of person and situation factors it differentiates depressed states.


[Continuity and discontinuity of psychopathology: a study of patients examined as children and as adults. I. Antecedents of adult schizophrenic disorders].

The demographical stability of the Canton of Geneva made it possible for us to do a prospective study of all the children who had consulted the Child Psychiatry Service between 1963 and 1967 and who later consulted the public adult psychiatry services. On the basis of the adult diagnoses, wo looked for specific clinical "clusters" in childhood which would allow us to differentiate statistically the groups of children. We present here the results of the group of children who developed schizophrenia in adulthood. We found a specific childhood "cluster" which clearly distinguishes these children from the others. It includes: serious relational disorders, neurodevelopmental impairment, attention deficit and school learning disorders.


[Dimensions of depressive behavior].

This study investigates the significance of a schedule of modes of behavior by comparing depressive and non-depressive persons. Using factor analytical methods it can be shown that behavioral modes of depressives form a heterogeneous group. Furthermore, it seems that two diametrically opposed mechanisms underlie depressive behavior: increase or decrease of behavioral mode frequency.


Methylbromide intoxication: a case report.

This work focuses on the neurophysiological features in a patient with action myoclonus and mental deterioration following methylbromide intoxication. The patient is a 28-year-old man, without respiratory distress or exposure to other toxics. Myoclonus improved with polytherapy (clonazepam, 5-HT, carbidopa, GABA). The neurophysiological and neuropsychological evidence in this patient suggests a possible double site of action of methylbromide at cortical and subcortical levels.


[Polyneuropathy in hyperthyroidism--a clinical neurophysiologic study].

In this prospective study, 27 patients with hyperthyroidism were evaluated neurophysiologically. There were 24 women and 3 men: the mean age was 56.4 years (range 21 to 84 years). Neurologic complaints of the patients were muscle weakness (59.3%), muscle cramps (18.5%) and sensory symptoms (11.1%). Tendon reflexes were increased in 18.5% and decreased in 14.8%, respectively. While the motor nerve conduction velocities (NCV) of the tibial and the peroneal nerve revealed normal results in the majority of patients, there were pathological findings of the sensory NCV of the sural nerve in 29.6% of patients. In another 22.2%, borderline findings were obtained. Six patients presented myopathic changings in electromyography of the m.vastus lateralis. Neither neuropathic nor myopathic findings correlated with the serum levels of TT3, TT4 or the T4/TBG-quotient. A polyneuropathy in hyperthyroidism seems not to be uncommon and is probably caused by hypermetabolism in thyroid hyperfunction.


T2- and diffusion-weighted magnetic resonance imaging of a focal ischemic lesion in rat brain.

We sought to evaluate the application of T2-weighted and diffusion-weighted magnetic resonance imaging techniques in the study of a focal ischemic lesion in the rat brain. Unilateral cortical infarcts were induced using the photosensitive dye rose bengal and 560 nm light irradiation. Magnetic resonance images were recorded from a total of 11 rats at selected intervals from 1.5 hours to several days after induction of the lesion. Parallel experiments were performed in which Evans blue dye was injected into the lesioned animals either immediately after lesion induction (n = 11) or 1 hour before the animals were killed (n = 11). The second procedure was designed to show regions of blood-brain barrier permeability to plasma proteins at the time of sacrifice, whereas the first procedure showed the accumulation and subsequent dispersion of plasma protein following disruption of the blood-brain barrier. Regions of the cortex highlighted by the T2-weighted images corresponded well to the pattern of dye staining seen from the first procedure while the diffusion-weighted images showed visual correspondence with the staining pattern obtained using the second procedure. These results illustrate the complementary use of T2-weighted and diffusion-weighted magnetic resonance imaging in discerning the pathophysiology of developing lesions.


[Clodronate in the palliative therapy of bone-metastasized prostatic carcinoma].

Activity and side-effects of clodronate (Ostac), an inhibitor of osteoclastic bone resorption, were recorded in an open prospective uncontrolled study on 35 patients with metastatic prostatic cancer. All patients had progressive symptomatic bone metastases despite prior hormone therapy. Clodronate was initially administered i.v. for 8 days with 300 mg/day. This was followed by a daily oral administration of 1600 mg. The analgesic effect was evaluated by using a visual analogue scale and by recording the daily consumption of analgesic drugs. Karnofsky index and routine blood examinations, including PSA, were assessed. Repeated bone scans and radiological evaluations were performed. An improvement in pain was observed in 71% of the patients. The mean duration of improvement was 4 weeks. Average survival time was 12 weeks. There were no side-effects after i.v. administration. Slight gastrointestinal discomfort was observed in 3 patients after oral administration. No effect was observed on the extent or biology of the metastases. Clodronate is an effective drug for palliative treatment of symptomatic bone metastases of prostatic carcinoma. It causes fewer and less pronounced side effects than other palliative drug therapies.


Specificity of affinity-purified Trichinella spiralis antigens.

An affinity-purified fraction (APF) was obtained by passing crude somatic antigens of Trichinella spiralis muscle larvae through an Affi-Gel 10 column coupled with anti-Trichuris suis IgG. The fraction contained seven antigens with molecular weights ranging from 28 to 55 kDa. When tested with antiserum against other common nematodes of pigs from China, the APF was found to be markedly more specific than S3 antigens (prepared by a combination of cell fractionation and differential centrifugation according to Despommier and Lacetti, 1981) and fractions produced by Sephacryl S-200 gel filtration (F1 to F12). When the APF was used in an indirect IgG-enzyme-linked immunosorbent assay (IgG-ELISA) to screen serum samples from 2000 pigs imported from China, a positive rate of 7.5% was obtained. Similar screenings using the crude somatic antigens F1 and S3 gave a large number of cross-reactions and false positive reactions. Positive rates of 48%, 39% and 59.5% respectively were obtained for the three antigens.


Prevalence of anti-hepatitis C virus antibodies in positive FTA-ABS non-drug abusing female prostitutes in Spain.

The prevalence of antibodies against Hepatitis C Virus (anti-HCV antibodies) and its association with the presence of treponemal antibodies (FTA-ABS) was studied in 227 non-drug abusing female prostitutes. The overall anti-HCV antibody prevalence was 8.8%, and a significantly higher prevalence of anti-HCV antibodies was found in prostitutes testing positive for FTA-ABS (19.4%) than in prostitutes testing negative for FTA-ABS (3.9%) (PR = 5.023, P less than 0.001). No statistical association was found between patient age or the duration of prostitution and the presence of anti-HCV antibodies.


Macromolecular self-association of a synthetic peptide derived from the hepatitis B surface antigen: construction of a quaternary epitope.

A major impediment to the development of peptide vaccines has been the inability accurately to mimic conformationally constrained epitopes on the envelope proteins of pathogens. This limitation is further compounded by the fact that several viral envelope proteins exist either as covalently or non-covalently associated homo-oligomers in the native state. Evidence is now accumulating to indicate that, at least in some instances, these homo-oligomers display antigenic determinants that are not present in the dissociated monomer units. Clearly this problem will have to be addressed if peptide-based vaccines are ever to become feasible alternatives. In this report we demonstrate that an oligomerized synthetic peptide that was derived from the hepatitis B surface antigen aggregates in solution to form macromolecular structures. These aggregates appear to represent a 'native' form of the group-specific determinant presented by the hepatitis B surface antigen.