Enhancement of the effect of low-dose cyclosporin A by sulphasalazine in prevention of cardiac allograft rejection in the rat.

Sulphasalazine (SASP) is an immunomodulatory compound with disease-modifying activity in ulcerative colitis and in other autoimmune disorders. SASP was previously shown to prolong the survival of heart allografts in rats treated with cyclosporin A (CyA) for 9 days after transplantation. We have now evaluated whether SASP also exerts a beneficial effect under continuous treatment with CyA, when CyA is discontinued after 14 days, or alone if given 10 days prior to transplantation. Cardiac grafts were transplanted from PVG donors to Wistar/Kyoto recipients using an accessory cervical heart transplantation technique. Rejection was defined as the absence of palpable contractions and occurred in the control group in a very reproducible manner on day 8 or 9. SASP alone was given orally (100 mg/kg body weight) starting 10 days before transplantation and resulted in a minor prolongation of graft survival. When SASP was given in addition to oral CyA (1 mg/kg or 2 mg/kg from day 0 to rejection) there was a significant prolongation in graft survival [from medians of 8 (range 6-11) and 9 (range 8-11) days, respectively, to medians of 10 (range 8-15) and 12 (range 11-15) days, respectively]. When SASP was given from day 0 to rejection, in addition to a schedule of oral CyA (10 mg/kg) for 15 days, there was no prolongation of graft survival [median of 30 (range 26-42) days vs median of 32 (26-38) days]. The data show that SASP acts as a weak immunosuppressive agent which enhances the effect of CyA given at a low dose.(ABSTRACT TRUNCATED AT 250 WORDS)


Prolyl endopeptidase and dipeptidyl peptidase IV are distantly related members of the same family of serine proteases.

Prolyl endopeptidase and dipeptidyl peptidase IV are serine proteases which cleave the peptide bonds at the carboxy group of proline residues. They do not show amino acid sequence homology with the known serine enzymes, but a possible relationship between them has not yet been examined. We have compared the amino acid sequences, and this revealed a distant evolutionary relationship between the two enzymes. Conserved segments were found mainly in the C-terminal region which also contained the catalytic residues. It is concluded that the C-terminal region is a protease domain which is attached to some noncatalytic structures.


The infectivity of spongiform encephalopathies: does a modified membrane hypothesis account for lack of immune response?

Scrapie, the prototype of a group of diseases which have the unique property of being both hereditary and infectious, is also exceptional in that it fails to evoke an immune response. Purification of crude scrapie preparations revealed a strong association of infectivity with a membrane protein ('PrPsc'); but a protein with the same amino acid sequence ('PrPc') was subsequently also found in normal mammalian nervous tissue. It is postulated by some investigators that 'PrPsc' is itself the infectious agent, or the most important part thereof, but in papers making that proposal immunological aspects have not been addressed. Experimental evidence supporting the hypothesis of a membrane fragment as agent has likewise lately not been taken into account. A modified form of the membrane hypothesis could account for immunological as well as genetic aspects of these diseases.


Activation pathways and human immunodeficiency virus type 1 replication are not altered in CD4+ T cells expressing the nef protein.

While recent studies in Rhesus monkeys have pointed out the importance of an intact nef gene for the development of acquired immunodeficiency syndrome (AIDS), no biological function has been so far unambiguously attributed to its product. Since Nef has been described to possess GTP-binding properties and to down-regulate CD4 cell surface expression, we looked for evidences of Nef interfering with the transduction of activating signals in human CD4+ T cells. We used a murine leukemia retroviral vector to express the HIV-1BRU nef gene in two permanent tumoral T-cell lines (CEM and Jurkat) and in two nonimmortalized, interleukin-2 (IL2)-dependent, T-cell clones. The single copy recombinant provirus integrated in the genome of these cells directed the synthesis of a 27-kD protein with a half-life greater than 5 h. The levels of expression of cell surface molecules involved in T-cell functions (CD4, CD3, CD28, CD29, IL-2 receptor) were not modified in cell populations expressing Nef. In immunocompetent T-cell clones, cell proliferation and lymphokine production in response to activating stimuli (IL-2, alloantigens, phorbol esters, or antibodies directed against CD2, CD3, CD4, CD28) remained unmodified. Moreover, the presence of Nef did not change the kinetics of human immunodeficiency virus (HIV) infection.


Prediction of doxorubicin resistance in gastrointestinal cancer by P-glycoprotein staining.

Feasibility of immunohistochemical staining of P-glycoprotein for the prediction of doxorubicin resistance in gastrointestinal cancers was examined. Among 10 cancer cell lines which consist of two gastric cancer cell lines and eight colon cancer cell lines, seven cell lines were stained positively by the monoclonal antibody to P-glycoprotein, C219. In consequence of the evaluation on the effect of doxorubicin on these tumour cells by means of succinic dehydrogenase inhibition test (SDI test), zero out of seven cell lines stained positively by C219 was sensitive to doxorubicin, but two out of three cell lines stained negatively were sensitive. Among 23 fresh surgical specimens of gastrointestinal cancers which consisted of 15 gastric cancers and eight colon cancers, seven tumour tissues were stained positively by C219. All P-glycoprotein positive tumours were resistant to doxorubicin. On the other hand, four of 16 P-glycoprotein tumours were sensitive to doxorubicin. These data indicate that positively stained cancer cells by C219 are resistant to doxorubicin.


[Comparative study of the efficacy and tolerance of cetirizine verses astemizole in patients with allergic rhinitis].

Thirty four patients with allergic rhinitis were included in two randomized groups and treated with Cetirizine and Astemizole respectively, 10 mg/day in oral administration during 15 days. This study was designed in order to compare the efficacy and tolerance of Cetirizine versus Astemizole, with special reference on the collateral effects on the central nervous system. Although both drugs showed good results. Cetirizine had a statistically significant better outcome in the evolution of symptoms, above all at the second day of treatment. Also it showed superior results, based on a subjective approach, related to the rapidity, power and efficacy of its action.


Inhibition of gastrin-stimulated growth of gastrointestinal tumour cells by octreotide and the gastrin/cholecystokinin receptor antagonists, proglumide and lorglumide.

The rat pancreatic cell line, AR42J possessed high-affinity gastrin and somatostatin receptors and its growth was stimulated by physiological gastrin-17 concentrations between 5 x 10(-11) mol/l and 10(-9) mol/l as measured by [75Se]selenomethionine uptake. The somatostatin analogue, octreotide (2 x 10(-7) to 2 x 10(-11) mol/l), reduced this stimulated growth. Gastrin-stimulated AR42J growth was also inhibited by proglumide (3 x 10(-4) mol/l) and lorglumide (3 x 10(-5) mol/l) at maximal G17 concentrations of 5 x 10(-11) and 10(-10) mol/l, respectively, and the analogues competed with [125I] gastrin-17 (5 x 10(-10) mol/l) for binding to gastrin receptors on AR42J (50% inhibitory concentrations, less than or equal to 10(-3) mol/l and 4 x 10(-6) mol/l, respectively. Octreotide reduced the basal growth of the human gastric cell line, MKN45G, (which is associated with intracellular gastrin immunoreactivity) in serum-free medium to 73% of control at a concentration of 2 x 10(-8) mol/l, which was reversed by gastrin-17 (10(-10) mol/l). Lorglumide (3 x 10(-5) mol/l) also reduced the basal growth to 30% of control, which was reversed to 78% by 10(-5) mol/l gastrin. Proglumide had no effect on the basal growth of MKN45G.


Plasma triglyceride and high density lipoprotein cholesterol as predictors of ischaemic heart disease in British men. The Caerphilly and Speedwell Collaborative Heart Disease Studies.

To assess the roles of plasma triglyceride and high density lipoprotein (HDL) cholesterol concentrations in predicting ischaemic heart disease. Two prospective cohort studies with common core protocols. Both cohorts are 100% samples of middle aged men. In Caerphilly the 2512 men were living within a defined area. In Speedwell the 2348 men were registered with local general practitioners. Fasting blood samples were taken at initial examination and plasma lipid concentrations were measured. Major ischaemic heart disease events were assessed from hospital notes, death certificates, and electrocardiograms. At first follow up, after an average of 5.1 years in Caerphilly and 3.2 years in Speedwell, 251 major ischaemic heart disease events had occurred. Men with triglyceride concentrations in the top 20% of the distribution had a relative odds value for ischaemic heart disease of 2.3 (95% confidence interval (95% CI) 1.3 to 4.1) compared with men in the bottom 20%, after adjusting for both plasma total and HDL cholesterol, and non-lipid risk factors. Men in the lowest 20% of the distribution of HDL cholesterol concentration had a relative odds value of 1.7 (95% CI 1.0 to 2.8) compared with the top 20%, after adjustment was made for total cholesterol and triglyceride concentrations, and non-lipid risk factors. These relations were not caused by beta blockers, which were being taken by 5% of the men. Plasma triglyceride concentration predicts major ischaemic events after allowance is made for total and HDL cholesterol concentrations and other risk factors. In these populations, triglyceride is a more important predictor than total cholesterol concentration.


Population genetics and forensic efficiency data of 4 AMPFLP's.

Family studies were carried out in a population sample from north west Germany using 4 amplifiable VNTR polymorphic systems D1S80 (MCT118), ApoB, D17S30 (YNZ22) and COL2A1. Separation was carried out in polyacrylamide gels and visualised using silver staining. In family studies (n = 30) no evidence of new mutations was found. The population study of unrelated individuals (mothers and putative fathers) showed that all 4 systems were highly polymorphic and similar to other population studies. The combined exclusion chance was calculated to be approximately 99% and the combined discrimination index 1.5.10(-4). The Hardy-Weinberg equilibrium was checked by forming groups of alleles and no significant deviations could be found in all systems.


Functional interaction between the two zinc finger domains of the v-erb A oncoprotein.

The v-erb A oncogene of avian erythroblastosis virus is a mutated and virally transduced copy of a host cell gene encoding a thyroid hormone receptor. The protein expressed by the v-erb A oncogene binds to DNA and acts as a dominant negative inhibitor of both the thyroid hormone receptor and the closely related retinoic acid receptor. The v-erb A protein has sustained two amino acid alterations within its DNA-binding domain relative to that of c-erb A, one of which, at serine 61, is known to be important for v-erb A function in the neoplastic cell. We report here that the second alteration, at threonine 78, also plays an important, although more indirect, role: alteration of the sequence at threonine 78 such that it resembles that of c-erb A can act as an intragenic suppressor and can partially restore function to a v-erb A protein rendered defective due to a mutation at position 61. Threonine 78 lies within the D-box of the v-erb A protein, a region thought to mediate receptor-receptor dimerizations, and is not in physical proximity to the serine at position 61. It therefore appears that an indirect interaction occurs between these two sites and that this interaction is crucial for v-erb A function.


The volume of the mediodorsal thalamic nucleus in treated and untreated schizophrenics.

Reductions of 40% in total cell number and 25% in volume of the mediodorsal thalamic nucleus were recently reported in an unbiased neurostereological study of neuroleptic-treated schizophrenic patients. In order to investigate whether these results might be secondary to many years of treatment with neuroleptic drugs, eight brains from schizophrenics never treated with neuroleptics and eight controls were studied using the unbiased Cavalieri volume estimator. To compare left-right differences in this region, twelve neuroleptic-treated schizophrenics and eleven control cases were compared. The brains used for the left-right comparison study and five of 20 used for comparison of treated and untreated brain volumes have been used in an earlier study. The mediodorsal thalamus volume was reduced by 31% in untreated schizophrenics and by 22% in neuroleptic-treated schizophrenics. No differences were found in mean total volume of the left and right mediodorsal thalamus in brains from controls nor from schizophrenics. A major difference exists with respect to time of fixation in controls (12 years) and untreated schizophrenics (39 years) that makes shrinkage differences a possible confounding variable. The results suggest that the consistent reduction in number of neurons in the mediodorsal thalamic nucleus are not secondary to prolonged treatment with neuroleptic drugs and that asymmetry in this specific brain region is not a feature of the schizophrenia-afflicted brain.


Cooperative DNA binding of the highly conserved human Hox 2.1 homeodomain gene product.

The human homeobox-containing gene Hox 2.1 (hHox 2.1) and its murine cognate mHox 2.1 are part of evolutionarily conserved gene clusters, encode an identical Antennapedia-type homeodomain, and are expressed in a similar pattern in the developing embryo. We have isolated cDNA clones of hHox 2.1 and found that the human/murine Hox 2.1 gene structure is strikingly conserved, with only 3 out of 269 amino acid differences in the entire predicted protein sequence. We show that purified hHox 2.1 protein is a sequence-specific DNA binding protein capable of binding to a variety of DNA sequences, including multiple sites in the promoter of the hHox 2.1 gene. In a footprint titration assay, the apparent affinity of the hHox 2.1 protein for a consensus binding site (LP) increases when the site is present in tandem copies. Quantitative footprint challenge experiments revealed that the in vitro half-life of the protein-DNA complex is less than 30 s for a single LP binding site (kd greater than 1.4 min-1), but 126 min for proteins bound to two tandem LP binding sites (kd = 5.5 x 10(-3) min-1). A domain distinct from the homeodomain is necessary for cooperative DNA binding, because a 61-amino-acid peptide containing only the Hox 2.1 homeodomain can specifically bind to LP sites, but exhibits no cooperativity. A different full-length human homeodomain protein, hHox 1.3, was also found to show cooperative DNA binding quantitatively similar to hHox 2.1. Therefore, cooperative DNA binding to adjacent sites may be a crucial component in the overall affinity of mammalian Antennapedia-type homeodomain proteins for their DNA target sites.


The amino-terminal peptide of HIV-1 glycoprotein 41 lyses human erythrocytes and CD4+ lymphocytes.

Functional studies assessed the cytolytic activity of the amino terminal peptide (FP-I; 23 residues 519-541) of the glycoprotein 41,000 (gp41) of the Human Immunodeficiency Virus Type-1 (HIV-1). Synthetically prepared FP-I efficiently hemolyzed human red blood cells at 37 degrees C, with 40% lysis at 32 microM. Kinetic studies indicated that FP-I induced maximal hemolysis in 30 min, probably through tight binding of the peptide with the red cell membrane. The Phe-Leu-Gly-Phe-Leu-Gly (residues 526-531) motif in FP-I apparently plays a critical role in lysis of red cells, since no hemolytic activity was observed for an amino-acid-substituted FP-I in which the unique Phe-Leu-Gly-Phe-Leu-Gly was converted to Ala-Leu-Gly-Ala-Leu-Gly. As neither smaller constituent peptides (e.g., residues 519-524 and residues 526-536) nor a N-terminal flanking peptide (e.g., residues 512-523) induced red cell hemolysis, the entire 23-residue (519-541) sequence of FP-I may be required for hemolytic activity. FP-I was also cytolytic with CD4(+)-bearing Hut-78 cells, with 40% lysis at approx. 150 microM. These results are consistent with an earlier hypothesis that the N-terminal peptide of gp41 may partially contribute to the in vivo cytopathic actions of HIV-1 infection (Gallaher, W.R. (1987) Cell 50, 327-328).


The amino-terminal peptide of HIV-1 glycoprotein 41 interacts with human erythrocyte membranes: peptide conformation, orientation and aggregation.

Structural studies assessed interactions between the amino-terminal peptide (FP-I; 23 residues 519-541) of the glycoprotein 41,000 (gp41) of Human Immunodeficiency Virus Type-1 (HIV-1) and human erythrocyte membranes and simulated membrane environments. Peptide binding was examined at sub-hemolytic (approx. less than 5 microM) and hemolytic (greater than or equal to 5 microM) doses (Mobley et al. (1992) Biochem. Biophys. Acta 1139, 251-256), using circular dichroism (CD) and Fourier-transform infrared (FTIR) measurements with FP-I, and electron spin resonance (ESR) studies employing FP-I spin-labeled at either the amino-terminal alanine (FP-II; residue 519) or methionine (FP-III; position 537). In the sub-lytic regime, FP-I binds to both erythrocyte lipids and dispersions of SDS with high alpha-helicity. Further, ESR spectra of FP-II labeled erythrocyte ghosts indicated peptide binding to both lipid and protein. In ghost lipids, FP-II was monomeric and exhibited low polarity and rapid, anisotropic motion about its long molecular axis (i.e., alpha-helical axis), with restricted motion away from this axis. The spin-label at the amino-terminal residue (Ala-519) is insensitive to the aqueous broadening agent chromium oxalate and buried within the hydrophobic core of the membrane; the angle that the alpha-helix (residues 519-536) makes to the normal of the bilayer plane is either 0 degree or 40 degrees. Contrarily, ESR spectra of ghost lipids labeled with sub-lytic doses of FP-III indicated high mobility and polarity for the reporter group (Met-537) at the aqueous-membrane interface, as well as extreme sensitivity to chromium oxalate. At lytic FP-I doses, CD and FTIR showed both alpha-helix and beta-structure for peptide in ghost lipids or detergent, while ESR spectra of high-loaded FP-II in ghost membranes indicated peptide aggregates. Membrane aggregates of FP-I may be involved in hemolysis, and models are suggested for N-terminal gp41 peptide participation in HIV-induced fusion and cytolysis.


A single-base-pair substitution abolishes D-amino-acid oxidase activity in the mouse.

Mutant ddY/DAO- mice lacking D-amino-acid oxidase (DAO) activity were examined for the cause of their lack of enzyme activity. Total RNA was extracted from the kidney of the ddY/DAO- mice and cDNA was synthesized. After cDNA encoding DAO was amplified by the polymerase chain reaction it was cloned into a plasmid and sequenced. Comparison of the DAO cDNA sequence with that of normal BALB/c mice revealed the presence of a single-base substitution (G----A) which causes a Gly-181----Arg substitution in the middle of the enzyme molecule. The mutant DAO cDNA was inserted into an expression vector and was expressed in transfected COS-1 cells. The transfected cells synthesized the DAO mutant protein, but they did not show DAO activity. In contrast, when cells were transfected with an expression vector carrying wild-type DAO cDNA, where the substituted base-pair was replaced by a normal base-pair, they showed DAO activity. These results indicate that the single base-pair substitution is the cause of the loss of DAO activity in the ddY/DAO- mice.


Receptor systems affecting force of contraction in the human heart and their alterations in chronic heart failure.

Catecholamines acting through beta 1- and beta 2-adrenergic receptors cause positive inotropic and chronotropic effects in the human heart. However, recent evidence suggests that in the human heart other receptor systems can also affect heart rate and contractility. Positive inotropic effects can be mediated by receptor systems acting through accumulation of intracellular cyclic adenosine monophosphate (cAMP; Gs-protein-coupled receptors such as 5-hydroxytryptamine(5-HT)4-like, histamine H2, and vasoactive intestinal peptide) or by receptor systems acting independently of cAMP, possibly through the phospholipase C/diacylglycerol/inositol-1,4,5-trisphophate pathway (such as alpha 1-adrenergic, angiotensin II, and endothelin). In the nonfailing human heart, activation of all these receptor systems induces only submaximal positive inotropic effects compared with those caused by beta-adrenergic receptor stimulation, indicating that in humans the cardiac beta-adrenergic receptor/Gs-protein/adenylate cyclase pathway is the most powerful mechanism to increase heart rate and contractility. However, the human heart contains only a few spare receptors for beta-adrenergic receptor-mediated positive inotropic effects and nearly all beta-adrenergic receptors are needed to cause maximal inotropic effects. Thus any decrease in the number of beta-adrenergic receptors will automatically lead to a reduction in functional responsiveness of beta-adrenergic receptors. In chronic heart failure the number and responsiveness of cardiac beta-adrenergic receptors are reduced, presumably because of the enhanced sympathetic drive to the heart and hence endogenous down-regulation by an elevated release of (cardiac-derived) norepinephrine, and this loss in cardiac beta-adrenergic receptor function is strongly related to the severity of the disease. However, beta 1- and beta 2-adrenergic receptors are differentially changed in different forms of heart failure. In dilated cardiomyopathy and possibly in aortic valve disease the number of cardiac beta 1-adrenergic receptors is selectively reduced without alteration in the number of beta 2-adrenergic receptors (although beta 2-adrenergic receptors become somewhat uncoupled). In ischemic cardiomyopathy, mitral valve disease, and possibly tetralogy of Fallot, the number of both beta 1- and beta 2-adrenergic receptors is concomitantly decreased. Because of the lack of a substantial receptor reserve, such a decrease in the number of beta-adrenergic receptors is accompanied by reduced inotropic and chronotropic responses to beta-adrenergic receptor stimulation in vitro and in vivo.(ABSTRACT TRUNCATED AT 400 WORDS)


GYKI 52466 blocks the increase in extracellular glutamate induced by ischaemia.

In vivo microdialysis has been used to study the effect of pre- or post-ischaemic administration of the non-NMDA antagonist 1-(4-amino-phenyl)-4-methyl-7, 8-methyl-endioxyl-5H-2,3- benzodiazepine hydrochloride (GYKI 52466), on the increases in extracellular glutamate levels induced by 20 minutes of four vessel occlusion in rats. In control rats, ischaemia resulted in transient increases in glutamate (4 fold), aspartate (6 fold) and gamma-aminobutyric acid (GABA) (15 fold) and decreases in glutamine (0.5 fold). Intravenous administration of GYKI 52466 (10 mg kg-1 bolus followed by 10 mg kg-1 h-1 infusion) beginning 20 minutes prior to the induction of ischaemia abolished ischaemia-induced glutamate release without affecting the increases in aspartate and GABA and the decrease in glutamine. Administration of GYKI 52466 immediately post-ischaemia resulted in a more rapid return of glutamate levels to basal values.


Transplantation of human neuroblastoma cells, catecholaminergic and non-catecholaminergic: effects on rotational behavior in Parkinson's rat model.

Cultured human catecholaminergic and non-catecholaminergic donor cells were used in neural transplantation experiments in a rat model of Parkinson's disease. Using two different human catecholaminergic neuroblastoma cell lines, one control non-catecholaminergic neuroblastoma cell line, and one sham control (tissue culture medium), transplants were made into the striatum using a modified Ungerstedt hemiparkinsonian rat model. Significant decreases in apomorphine-induced rotational behavior were produced by two of three catecholaminergic cell lines. Grafted cells staining positively for tyrosine hydroxylase (TH) and catecholamine fluorescence indicated viable catecholamine activity in the two cell lines which produced reductions in rotational behavior. Catecholamine fluorescence was not detected in either of the two controls. These data suggest a link between catecholamine secretion by transplanted cells and motor improvement using a rat rotational behavior model.


Implication of non-NMDA and NMDA receptors in cochlear ischemia.

We have investigated the hypothesis that the acute ischemic swelling of the radial dendrites connected to the inner hair cells (IHCs) is mediated by glutamatergic receptors. In control cochleas, after 20 min ischemia all the dendrites were dramatically swollen. Conversely, after a perfusion of 50 microM 6-7-dinitroquinoxaline-2,3-dione (DNQX) before ischemia, most dendrites were protected although those contacting the IHCs on their modiolar side frequently swelled. After 50 microM of D-2-amino-5-phosphonopentanoate (D-AP5), no dendrite protection could be obtained. Finally, after DNQX and D-AP5, no dendrite swelling occurred. These results suggest that, in the cochlea, the acute ischemic swelling of dendrites primarily occurs via non-NMDA receptors. However, in radial dendrites contacting the IHCs on their modiolar side, NMDA receptors may contribute to excitotoxicity.


Effects of adrenal medulla and sciatic nerve co-grafts in rats with unilateral substantia nigra lesions.

Major limitations of adrenal medulla transplantation in animal models of Parkinson's disease have been the relatively small behavioral effects and the poor or inconsistent graft survival. Transplantation of fragments of sural nerve in combination with adrenal medulla has been reported to increase the survival of chromaffin cells in adrenal medulla grafts in primates. In the present study, the possibility was tested that peripheral nerve co-grafts would increase the functional effects of adrenal medulla grafts in a 6-hydroxydopamine-lesioned rat model. Animals received unilateral substantia nigra lesions, and subsequently received intraventricular grafts of adrenal medulla, sciatic nerve, adrenal medulla plus sciatic nerve, or sham grafts consisting of medium only. Functional effects of the grafts were tested using apomorphine-induced rotational behavior. The sciatic nerve co-grafts did not increase the survival of TH-immunoreactive chromaffin cells. The co-grafting treatment also did not augment the overall effect of adrenal medulla grafts on rotational behavior. In the animals with substantial numbers of surviving chromaffin cells, however, the animals with sciatic nerve co-grafts showed greater decreases in rotational behavior as compared to the animals with adrenal medulla grafts alone, even though the number of surviving cells was not increased.


Characterization of the Ac/Ds behaviour in transgenic tomato plants using plasmid rescue.

We describe the use of plasmid rescue to facilitate studies on the behaviour of Ds and Ac elements in transgenic tomato plants. The rescue of Ds elements relies on the presence of a plasmid origin of replication and a marker gene selective in Escherichia coli within the element. The position within the genome of modified Ds elements, rescued both before and after transposition, is assigned to the RFLP map of tomato. Alternatively to the rescue of Ds elements equipped with plasmid sequences, Ac elements are rescued by virtue of plasmid sequences flanking the element. In this way, the consequences of the presence of an (active) Ac element on the DNA structure at the original site can be studied in detail. Analysis of a library of Ac elements, rescued from the genome of a primary transformant, shows that Ac elements are, infrequently, involved in the formation of deletions. In one case the deletion refers to a 174 bp genomic DNA sequence immediately flanking Ac. In another case, a 1878 bp internal Ac sequence is deleted.


The type 1 (EGFR-related) family of growth factor receptors and their ligands.

This review considers the biology of the type 1 growth factor receptor family which is increasingly recognised as important in the control of normal cell proliferation and in the pathogenesis of human cancer. The family currently comprises three closely related members: the epidermal growth factor (EGF) receptor, c-erbB-2 and c-erbB-3, all of which show abnormalities of expression in various human tumours. The family of factors related to EGF has also expanded recently and now includes transforming growth factor alpha, heparin-binding EGF, amphiregulin, cripto and heregulin, as well as several other potential ligands for the c-erbB2-2 receptor. The involvement of these receptors and growth factors in human cancer has implications for the design of novel forms of therapy for cancer, and we review recent advances and future avenues for investigation.


Detection of human T-cell leukemia/lymphoma virus, type II, in a patient with large granular lymphocyte leukemia.

We studied a patient with large granular lymphocyte (LGL) leukemia for evidence of human T-cell leukemia/lymphoma virus (HTLV) infection. Serum from this patient was positive for HTLV-I/II antibodies by enzyme-linked immunosorbent assay (ELISA) and was confirmed positive in Western blot and radioimmunoprecipitation assays. Results of a synthetic peptide-based ELISA showed that the seropositivity was caused by HTLV-II and not HTLV-I infection. Analyses of enzymatic amplification of DNA from bone marrow sections using the polymerase chain reaction (PCR) were positive for HTLV-II specific gag, pol, env, and pX gene sequences. Cloning and sequencing of amplified products showed that the HTLV-II pol and pX sequences in patient DNA differed from the sequences of 17 other HTLV-II isolates examined in our laboratory. HTLV infection may have a role in some patients in the pathogenesis of LGL leukemia.


Contact-induced neutrophil activation by platelets in human cell suspensions and whole blood.

Platelet-dependent activation of polymorphonuclear neutrophils (PMNL) was investigated with a lumi-aggregometer in heparinized whole blood and platelet-PMNL suspensions. The lumi-aggregometer allowed us to simultaneously monitor increases in impedance or light transmission as consequences of platelet aggregation and luminol-enhanced chemiluminescence (CL) as a measure of the oxidative burst in PMNL. Aggregation and platelet-PMNL contacts were also checked by light and electron microscopy. In whole blood, adenosine diphosphate (ADP) and the thromboxane A2 mimetic U 46619 induced the aggregation (increase in impedance) and the CL, which were both suppressed by EDTA, arginyl-glycyl-aspartyl-serine (RGDS) peptide, and the absence of stirring. In contrast, FMLP caused only CL that was unaffected by EDTA, RGDS peptide, and nonstirring. Similar observations were obtained with mixed suspensions containing washed platelets and PMNL at their physiologic concentrations. ADP, U 46619, and thrombin induced both aggregation (increase in light transmission) and CL, whereas FMLP caused CL but only very weak aggregation. Exogenous fibrinogen strongly enhanced the effects of ADP and U 46619. Iloprost, EDTA, RGDS peptide, red blood cell (RBC) ghosts, and nonstirring inhibited the effects induced by the platelet agonists, but were ineffective on the CL induced by FMLP. Treatment of platelets with aspirin did not affect the CL of PMNL induced by platelets. Microscopic examination, the requirements of stirring, Ca2+, and fibrinogen, and the inhibitory effects of RGDS peptide and RBC ghosts show that stimulated platelets activate PMNL in a contact-dependent manner that depends on fibrinogen binding. This was confirmed by the immunochemical demonstration of fibrinogen (but not of fibronectin) in the contact spaces between activated platelets and PMNL. Because supernatants and lysates of resting or thrombin-stimulated platelets did not induce the CL of PMNL, soluble agonists did not appear to be involved. Nonstimulated washed platelets also caused CL of PMNL that required stirring and Ca2+ and was inhibited by RBC ghosts. No CL occurred in unstimulated stirred whole blood, suggesting that a preactivation of platelets during the preparation may be responsible for the effects of unstimulated washed platelets. The results show that platelets provide a strong stimulus for PMNL that requires intercellular contact. Fibrinogen exposure on the platelet surface seems to be necessary for the activation of PMNL by stimulated platelets.


Inflammatory bowel disease: an uncommon problem in Singapore.

Fifty patients with inflammatory bowel disease (ulcerative colitis, 40; Crohn's disease, seven; indeterminate colitis, three) treated in one gastroenterology unit in Singapore over a 10 year period were reviewed. Clinical features were similar to those described in Western patients. Of the three main races of Singapore it was found that Indians are more susceptible to these diseases than Chinese or Malays. A survey of all gastroenterologists in Singapore indicated a possible prevalence of 8.6 per 100,000 people for ulcerative colitis and 1.3 per 100,000 people for Crohn's disease. These prevalence rates are much lower than those reported for Western populations.


Growth inhibition of estrogen independent MXT mouse mammary carcinomas in mice treated with an agonist or antagonist of LH-RH, an analog of somatostatin, or a combination.

Female BDF1 mice inoculated with MXT (3.2) estrogen independent mouse mammary carcinoma were treated for three weeks with microcapsules of the luteinizing hormone-releasing hormone (LH-RH) agonist [D-Trp6]LH-RH, the antagonist SB-75, the somatostatin analog RC-160, or combinations. The lack of estrogen dependence of the tumor was proved by bilateral surgical ovariectomy, which had no effect. In two experiments, treatment with 25 micrograms/day doses of each analog alone resulted in a significant inhibition of tumor growth as shown by a 40-53% inhibition of tumor volumes, 38-43% decrease in tumor weights, and histological signs of tumor regression. However, the combination of SB-75 or [D-Trp6]LH-RH with somatostatin analog RC-160 caused greater reduction of tumor volume (68 and 61%) or tumor weights (59 and 56%), than single analogs, and histologically the occurrence of apoptosis and decrease in AgNOR numbers was more pronounced in the groups receiving combination therapy. Specific binding sites for [D-Trp6]LH-RH, EGF, and IGF-I were demonstrated in the tumor membranes. The binding capacity of LH-RH receptors was decreased by treatment with the analogs, the greatest down-regulation being caused by combination therapy. A significant decrease in EGF binding capacity was observed after treatment with the LH-RH analogs, alone or especially in combination with somatostatin analog RC-160. The combination of these analogs also caused a reduction in IGF-I receptors. The finding that LH-RH agonists and antagonists and somatostatin analogs inhibit the growth of estrogen independent mammary tumors, and that combinations are more effective than single analogs, might be of practical importance in human breast cancer therapy.


Amino acid substitutions in the Dictyostelium G alpha subunit G alpha 2 produce dominant negative phenotypes and inhibit the activation of adenylyl cyclase, guanylyl cyclase, and phospholipase C.

Previous studies have demonstrated that the Dictyostelium G alpha subunit G alpha 2 is essential for the cAMP-activation of adenylyl cyclase and guanylyl cyclase and that g alpha 2 null mutants do not aggregate. In this manuscript, we extend the analysis of the function of G alpha 2 in regulating downstream effectors by examining the in vivo developmental and physiological phenotypes of both wild-type and g alpha 2 null cells carrying a series of mutant G alpha 2 subunits expressed from the cloned G alpha 2 promoter. Our results show that wild-type cells expressing G alpha 2 subunits carrying mutations G40V and Q208L in the highly conserved GAGESG (residues 38-43) and GGQRS (residues 206-210) domains, which are expected to reduce the intrinsic GTPase activity, are blocked in multicellular development. Analysis of down-stream effector pathways essential for mediating aggregation indicates that cAMP-mediated activation of guanylyl cyclase and phosphatidylinositol-phospholipase C (PI-PLC) is almost completely inhibited and that there is a substantial reduction of cAMP-mediated activation of adenylyl cyclase. Moreover, neither mutant G alpha 2 subunit can complement g alpha 2 null mutants. Expression of G alpha 2(G43V) and G alpha 2(G207V) have little or no effect on the effector pathways and can partially complement g alpha 2 null cells. Our results suggest a model in which the dominant negative phenotypes resulting from the expression of G alpha 2(G40V) and G alpha 2(Q208L) are due to a constitutive adaptation of the effectors through a G alpha 2-mediated pathway. Analysis of PI-PLC in g alpha 2 null mutants and in cell lines expressing mutant G alpha 2 proteins also strongly suggests that G alpha 2 is the G alpha subunit that directly activates PI-PLC during aggregation. Moreover, overexpression of wild-type G alpha 2 results in the ability to precociously activate guanylyl cyclase by cAMP in vegetative cells, suggesting that G alpha 2 may be rate limiting in the developmental regulation of guanylyl cyclase activation. In agreement with previous results, the activation of adenylyl cyclase, while requiring G alpha 2 function in vivo, does not appear to be directly carried out by the G alpha 2 subunit. Our data are consistent with adenylyl cyclase being directly activated by either another G alpha subunit or by beta gamma subunits released on activation of the G protein containing G alpha 2.


Neuropharmacology of pain.

Recent research on the site of action of morphine, its distribution following systemic administration and activity in a model of neuropathic pain is reviewed. Neuropeptides and pain is discussed in relation to tachykinins and their antagonists, cholecystokinin (CCK) and its antagonists and somatostatin.


Effects of omeprazole on the number of immunoreactive gastrin- and somatostatin-cells in the rat gastric mucosa.

The effects of omeprazole--an inhibitor of gastric acid secretion--on gastrin (G)- and somatostatin (D)-cell density in the gastric antral mucosa epithelium in rats were examined, following a 5-day treatment. It was found that omeprazole increased the density of G-cells, whereas it decreased the density of D-cells. That effect was probably independent of hypergastrinaemia, since it could not be blocked by a simultaneous treatment with proglumide--a gastrin receptor blocker. It is concluded that the observed phenomenon is a direct result of a lower gastric acidity, as a consequence of omeprazole treatment.


[Interferon-gamma suppresses expression of the HER-2 oncogene in ovarian cancer cells].

The overexpression of the proto-oncogene HER-2 (c-erbB-2/neu) in ovarian and mammary carcinoma is an important indicator for a bad prognosis. In this study we demonstrate that in 7 out of 8 ovarian carcinoma cell lines there is an interferon-gamma-mediated reduction in HER-2 specific protein, and this effect was found to correlate with the antiproliferative action. It is interesting to note that there is no relationship between the absolute amount of HER-2 protein expressed and the sensitivity of the ovarian carcinoma cells for an antiproliferative activity of interferon-gamma. Other chemotherapeutic agents did not affect HER-2 expression although they inhibited the proliferation. The oncogene expression was lowered only in the ovarian carcinoma cell lines and not in 3 interferon-gamma sensitive human breast cancer cell lines. Expression of the oncogene HER-2 is the leading prognostic factor in ovarian cancer. Its modulation might represent a mechanism by which interferon-gamma inhibits cell proliferation.


A mathematical model of the P-glycoprotein pump as a mediator of multidrug resistance.

Cells displaying the classic multidrug resistant (MDR) phenotype possess a transmembrane protein (p170 or P-glycoprotein) which can actively extrude cytotoxic agents from the cytoplasm. A mathematical model of this drug efflux pump has been developed. Outward transport is modeled as a facilitated diffusion process. Since energy-dependent efflux of cytotoxic agents requires that ATP also bind to p170, the model includes a dynamic calculation for efflux rate which considers Michaelis-Menten kinetics for both the substrate agent and ATP. The final system consists of one partial differential equation (PDE) for the facilitated diffusion of substrate agents out of the cell, a 2 x 2 ordinary differential equation (ODE) system for the dynamic calculation of the ATP-ADP pool, and a dynamic algebraic calculation of the efflux rate given substrate levels at the interior cell membrane interface and ATP levels in the cell. A stability analysis of the ATP-ADP pool distribution and a simplistic closed form solution of the linearized PDE are included. Numerical simulations are also provided.


Effect of methylxanthine derivatives on T cell activation.

In a Phase I-II trial examining the effect of prophylactic administration of pentoxifylline (PTX) on bone marrow transplant-associated morbidity, there was an apparent reduction in the incidence and severity of acute graft-versus-host disease. To determine if PTX might be directly immunosuppressive, its effects on T cell activation and proliferation were examined. PTX and several cogeners were found to directly suppress T cell proliferation in response to phytohemagglutinin, to allogeneic cells in a mixed leukocyte reaction, and to cross-linking the CD3 complex. The effects were dose-related and associated with suppression of secretion of tumor necrosis factor-alpha (TNF alpha). However, the inhibition of proliferation was not solely due to this effect since adding excess recombinant TNF alpha did not restore the proliferative response. These data suggest that PTX may be clinically useful in suppressing allogeneic reactions in bone marrow transplantation.


Microinjection of L-glutamate into the nucleus tractus solitarii increases arterial pressure in conscious rats.

Microinjection of L-glutamate into the nucleus tractus solitarii (NTS) of anesthetized rats produces a fall in mean arterial pressure (MAP) similar to that observed during activation of baroreceptor afferents. In the present study we examined the effect of bilateral microinjections of L-glutamate through chronically implanted cannulae in the NTS of conscious freely moving rats. Group I (n = 6) was studied under conscious conditions and 24 h later the rats were anesthetized with urethane and the effects of L-glutamate re-examined. In conscious rats, L-glutamate (30 pmol to 5 nmol/100 nl) produced dose-dependent increases in MAP (+37 +/- 7 mmHg, 5 nmol), whereas under urethane anesthesia falls in MAP were observed (-11 +/- 3 mmHg, 5 nmol). Group II (n = 7) was studied under conscious conditions and 1 h later the rats were anesthetized with chloralose and the effects of L-glutamate re-examined. In this group of conscious rats L-glutamate (300 pmol to 5 nmol/100 nl) also produced dose-dependent increases in MAP (+37 +/- 5 mmHg, 5 nmol), whereas under chloralose anesthesia a dose-dependent depressor response was observed (-33 +/- 6 mmHg, 5 nmol). Saline microinjections into the NTS of conscious and anesthetized rats produced negligible effects. These data demonstrate that microinjection of L-glutamate into the NTS of rats produces a pressor response in conscious animals in contrast to depressor responses in animals anesthetized with chloralose or urethane.(ABSTRACT TRUNCATED AT 250 WORDS)


The learning capacity of high or low performance rats is related to the hippocampus NMDA receptors.

The hippocampal synaptic plasticity of rats with an inborn high (HP) or low (LP) learning capacity to perform in a shuttle box is closely related to their percentage of conditioned responses (Crs). HP rats show less sensitivity to the blocking effect of 2-aminophosphonopentanoic acid (AP5) on the generation of long-term potentiation (LTP) than do LP rats. Results described in the present report are indicative of an increased density of N-methyl-D-aspartate (NMDA) receptors in HP rats compared to control and LP rats. We postulate that the differential pharmacological sensitivity of LTP in these rats is a reflection of this biochemical difference. Also, from these results we suggest that the learning capacity may be related to the density of glutamate NMDA receptors of HP, LP and control rats.


Stimulation of alpha 2-adrenergic receptors in nucleus tractus solitarius is required for the baroreceptor reflex.

Bilateral injection into the nucleus tractus solitarius (NTS) of the alpha 2-adrenergic receptor antagonist yohimbine produced a dose-related (10-500 pmol) increase in arterial pressure, with a maximal response of approximately 60 mm Hg. Idazoxan, also an alpha 2-adrenergic receptor antagonist, produced a similar response although idazoxan was less potent than yohimbine. The pressor response elicited by these drugs was attenuated by stimulation of adrenergic receptors in the NTS by local administration of either clonidine or tyramine. Doses of yohimbine (200 pmol) or idazoxan (5 nmol) that maximally increased arterial pressure also completely inhibited the depressor and bradycardic responses to electrical stimulation of the aortic depressor nerve. These results indicate that tonic stimulation of alpha 2-adrenergic receptors in the NTS is required for baroreceptor reflex function.


Glutamate release and presynaptic action of AP4 during inspiratory drive to phrenic motoneurons.

High-performance liquid chromatography (HPLC) was used to detect the presence of excitatory amino acids released from bulbospinal axon terminals projecting to cervical spinal respiratory motoneurons during transmission of inspiratory drive in an in vitro neonatal rat brainstem-spinal cord preparation. Measurements were then repeated under paradigms where transmitter release was decreased by either depression of bulbospinal respiratory drive, or by adding DL-2-amino-4-phosphonobutyrate (AP4) to the solution bathing the spinal cord. The amounts of glutamate, but not aspartate, released decreased significantly with depressed brainstem inspiratory drive or the activation of AP4-sensitive receptors within the cervical (C) spinal cord.


Acetylcholine release in the hippocampus: regulation by monoaminergic afferents as assessed by in vivo microdialysis.

The role of monoamines in the functional regulation of the septo-hippocampal cholinergic system was studied using in vivo microdialysis of acetylcholine (ACh) release in the hippocampus of awake unrestrained rats. Systemic administration of the dopamine receptor agonist apomorphine (2.0 mg/kg) resulted in a 170% increase in hippocampal ACh overflow. Similarly the catecholamine-releasing agent amphetamine (2.5 mg/kg) produced a 400% increase in ACh overflow. The effect induced by amphetamine, but not that of apomorphine, was blocked in animals pretreated with the tyrosine hydroxylase inhibitor alpha-methyl-p-tyrosine (AMPT). The effect of amphetamine on ACh release was reduced by 75% after a 6-hydroxydopamine (6-OHDA) lesion of the ventral tegmental area (VTA) but was not affected by 6-OHDA lesions of the noradrenergic dorsal and ventral bundles. However, baseline ACh overflow was increased by 130% by the dorsal and ventral bundle lesions. The serotonin-releasing agent p-chloroamphetamine (2.5 mg/kg) produced a 160% increase in hippocampal ACh release, and this effect was enhanced after a 5,7-dihydroxytryptamine (5,7-DHT) lesion of the serotonin projection system. The results show that surgical or pharmacological manipulations of the ascending brainstem monoaminergic systems, which innervate wide areas of the forebrain, including the septum and the hippocampal formation, have pronounced effects on septo-hippocampal cholinergic activity. Thus, the present data provide support for the view that information regarding behavioral state and arousal is conveyed to the septo-hippocampal system via ascending monoaminergic systems.


Age-related alterations of NMDA-receptor properties in the mouse forebrain: partial restoration by chronic phosphatidylserine treatment.

The effect of aging on the properties of N-methyl-D-aspartate (NMDA) receptors in the forebrain of female NMRI mice was investigated using the antagonist [3H]MK-801 as radioligand. Compared to young (3 months) mice, aged (20 months) mice showed changes of the properties of the NMDA receptor at three different levels: (1) the density was reduced by about 35%; (2) the efficacy of L-glutamate and glycine for stimulating specific [3H]MK-801 binding was enhanced, probably because more NMDA receptor-associated ion channels are closed under baseline conditions in the aged brain; (3) the affinity of L-glutamate and glycine to its binding sites at the NMDA receptor complex was also enhanced. Chronic treatment of aged mice with phosphatidylserine (20 mg/kg, i.p., once daily) for three weeks completely normalized enhanced efficacy and affinity of L-glutamate and glycine and elevated NMDA receptor density by approximately 25%. These findings are consistent with the assumptions that deficits of the NMDA receptor are one of the mechanisms of age-related cognitive impairment and that the beneficial effects of phosphatidylserine treatment on cognitive deficits of aged individuals might be partially due to the effects of this drug on age-related NMDA receptor deficits.


The effect of morphine tolerance dependence and abstinence on immunoreactive dynorphin (1-13) levels in discrete brain regions, spinal cord, pituitary gland and peripheral tissues of the rat.

The effect of morphine tolerance dependence and protracted abstinence on the levels of dynorphin (1-13) in discrete brain regions, spinal cord, pituitary gland and peripheral tissues was determined in male Sprague-Dawley rats. Of all the tissues examined, the highest level of dynorphin (1-13) was found to be in the pituitary gland. Among the brain regions and spinal cord examined, the levels of dynorphin (1-13) in descending order were: hypothalamus, spinal cord, midbrain, pons and medulla, hippocampus, cortex, amygdala and striatum. The descending order for the levels of dynorphin (1-13) in peripheral tissues was: adrenals, heart and kidneys. In morphine tolerant rats, the levels of dynorphin (1-13) increased in amygdala but were decreased in pons and medulla. In morphine abstinent rats, the levels of dynorphin (1-13) were increased in amygdala, hypothalamus and hippocampus. The levels of dynorphin (1-13) were increased in pituitary but decreased in spinal cord and remained so even during protracted abstinence. The levels of dynorphin (1-13) in the peripheral tissues of morphine tolerant rats were unaffected. However, in the heart and kidneys of morphine abstinent rats, the levels of dynorphin (1-13) were increased significantly. It is concluded that both morphine tolerance and abstinence modify the levels of dynorphin (1-13) in pituitary, central and peripheral tissues. Morphine abstinence differed from non-abstinence process in that there were additional changes (increases) in the levels of dynorphin (1-13) in brain regions (hypothalamus and hippocampus) and peripheral tissues (heart and kidneys) and may contribute to the symptoms of the morphine abstinence syndrome.(ABSTRACT TRUNCATED AT 250 WORDS)


Tyrosine-hydroxylase-containing neurons in the primate basal forebrain magnocellular complex.

Immunocytochemistry and in situ hybridization for tyrosine hydroxylase (TH) were used to study the distribution of putative catecholaminergic neurons in the basal forebrain magnocellular complex (BFMC) of monkeys and humans. Magnocellular TH-expressing neurons in the primate BFMC are distributed along a rostrocaudal gradient, with the largest proportion of these cells located in the medial septal nucleus and nucleus of the diagonal band of Broca; smaller TH-containing neurons generally follow the same distribution. These findings suggest that, within rostromedial segments of the BFMC, there is a distinct subpopulation of neurons that express catecholamine-synthesizing enzymes. Further research is necessary to establish whether these neurons utilize one or more catecholamines as neurotransmitters.


Comparison of the expression, transcription and genomic organization of D2 dopamine receptors in outbred and inbred strains of rat.

Three outbred (Sprague-Dawley, Wistar and Long-Evans) and five inbred (Brown-Norway, Buffalo, DA, Fisher and Lewis) strains of rat were used to investigate the extent of genetic variation in the expression and organization of the rat D2 receptor locus. Radioligand binding studies were performed using 125I-iodobenzamide ([125I]IBZM), a high-affinity antagonist for D2 dopamine receptors, to determine the extent of variation in the expression of D2 receptors in these strains of rat. A comparison of the affinities (Kd = 0.26-0.38 nM) and densities (450-580 fmol/mg of protein) of binding sites for [125I]IBZM in the striatum of the eight strains of rat did not reveal statistically significant differences. Solution hybridization using 32P-labeled riboprobes complementary to the coding region of the third intracellular loop of the D2 receptor was used to investigate the extent of variation in transcription of the long (D2L) and short (D2S) isoforms of D2 receptor mRNA in rat striatal tissue. The level of expression of these two mRNA isoforms was found to be invariant in the strains of rats that were examined. The genomic organization of the D2 receptor locus for each strain of rat was compared using Southern blot hybridization. Southern blots were hybridized with a DNA probe that codes for the D2L receptor isoform. Restriction fragment lengths were conserved between each rat strain for genomic DNA digested with BamHI, EcoRI, HindIII, PstI and TaqI. Restriction fragment length polymorphisms (RFLPs) were identified when genomic DNA was digested with XbaI or MspI. The XbaI polymorphism was mapped to within 2 kb of the exon coding for the third intracellular loop of the D2 receptor. Both RFLPs differentiated Sprague-Dawley and Brown-Norway rats from Wistar, Long-Evans, Buffalo, DA, Lewis and Fisher rats. The RFLPs for the rat D2 receptor locus provide genetic markers that can be used with classic genetic studies to determine whether strain differences in behavior and/or in the response to pharmacologic intervention are determined by genetic elements linked to the D2 receptor locus.


Dopamine and melatonin interactions in the intact chicken eye. Electrooculographic and biochemical study.

Electrophysiological and biochemical techniques were used to investigate the interactions between dopamine (DA) and melatonin (MEL) in the intact chicken eye. Endogenous DA depletion induced by intraocular administration of alpha-methyl-para-tyrosine (alpha-MPT), a selective tyrosine hydroxylase inhibitor, decreases the transepithelial potential (TEP) of the retinal pigment epithelium and reduces the light peak (LP) recorded by an indirect electro-oculographic (EOG) method. An intraocular injection of MEL also reduces the TEP but does not reduce the LP. Retinal MEL is increased after endogenous DA depletion and a tight inverse correlation between DA and MEL contents was found. The present data, together with other findings support the hypothesis (1) that in the intact chicken eye, DA and MEL play respectively a role of light and dark signals on the TEP, and (2) that a balance between these two neurohormones may be responsible for the regulation of RPE events which are dependent on light-dark conditions.


Analgesia from the periaqueductal gray in the developing rat: focal injections of morphine or glutamate and effects of intrathecal injection of methysergide or phentolamine.

The aim of these experiments was to examine the changes in antinociception elicited by morphine or glutamate stimulation of the periaqueductal gray of the midbrain (PAG) during the postnatal development of the rat. Pups, aged 3, 10, and 14 days, were implanted with cannulas aimed at either the dorsal or the ventral aspect of the PAG, and glutamate (vehicle, 60 mM or 180 mM) or morphine (vehicle, 2 micrograms or 6 micrograms) was microinjected into one of those two sites. Pups were tested for analgesia against noxious thermal and mechanical stimuli. Morphine produced analgesia at 3 and 10 days of age only when administered to the ventral part of the PAG and the thermal noxious stimulus was tested. Conversely, analgesia induced by glutamate was seen at 3 and 10 days of age only when glutamate was given to the dorsal aspect of the PAG and the mechanical stimulus was used. In 14-day-old pups, both drugs produced analgesia against both types of noxious stimuli regardless of their site of administration within the PAG. Systemically administered naloxone attenuated the analgesic effects of both drugs when they were administered to the ventral PAG, but did not consistently attenuate the analgesic effect of either compound given to the dorsal aspect of the PAG. When either morphine or glutamate was injected into the ventral PAG, intrathecal injections of methysergide attenuated analgesia against the thermal stimulus to a significantly greater degree than the mechanical stimulus and intraspinal injection of phentolamine attenuated analgesia against the mechanical stimulus more potently. When glutamate was given to the dorsal PAG, analgesia against both stimulus types was significantly attenuated. These results indicate that the morphine- and glutamate-induced analgesia mediated by the PAG are developmentally differentiated. These ontogenetic differences most likely reflect differences in the mechanism of action by which these drugs produce analgesia when administered to the PAG, as well as neuroanatomical differences within the dorsal and the ventral regions of the PAG.


Demonstration of proteases in basal cell carcinomas. A histochemical study using amino acid-4-methoxy-2-naphthylamides as chromogenic substrates.

Proteases are reported to play an essential part in the proliferative, invasive, and metastasizing behavior of malignant tumors. The aim of the current study was to determine the activity and localization of proteases in basal cell carcinomas (BCC) histochemically. Various proteases were identified histochemically in frozen sections of BCC. The following amino acid-4-methoxy-2-naphthylamides (MNA) were used as chromogenic substrates:alanine-MNA for the detection of aminopeptidase M (APM), glycyl-proline-MNA for dipeptidyl peptidase IV (DPP IV), lysyl-proline-MNA and lysyl-alanine-MNA for dipeptidyl peptidase II (DPP II), glycyl-arginine-MNA for dipeptidyl peptidase I (DPP I), and carbobenzoxy (CBZ)-arginyl-arginine-MNA for cathepsin B. APM activity was high in the peritumorous connective tissue, whereas the tumor epithelium and epidermis had negative results. DPP IV showed a highly positive reaction in both tumor epithelium and surrounding connective tissue. Cathepsin B and DPP I reacted strongly in the tumor epithelium but not in the peritumorous connective tissue. The marked activity of APM, DPP IV, DPP I, and cathepsin B may be related to the proliferation and invasive growth of BCC. The distribution of the activity of APM and DPP IV indicates dynamic interactions between the tumor epithelium and the adjacent connective tissue in the neoplastic process.


The expression of proliferating cell nuclear antigen in paraffin sections of peripheral, node-negative non-small cell lung cancer.

Cell proliferation of 40 peripheral, node-negative non-small cell lung cancers (NSCLC) treated with surgery alone was investigated by immunohistochemical analysis with the monoclonal antibody (MoAb) PC10, which recognizes a proliferating cell nuclear antigen (PCNA) in formalin-fixed and paraffin-embedded material. Results were correlated with DNA ploidy and S-phase fraction (SPF) analyzed by DNA flow cytometric study. Mitotic count (MC) was analyzed by light microscopic study and histopathologic features. PCNA immunoreactivity was seen in all samples and confined to the nuclei of cancer, but not to the surrounding, tumor-negative cells; its frequency ranged from 0-70% (median, 15%), and tumors expressed either a low (0-25%, n = 25) or intermediate (26-75%, n = 15) proliferative activity. There was no relationship between PCNA immunoreactivity and tumor stage or among size, histologic type, and mitotic count (MC). Tumors with intratumoral blood vessel invasion (BVI) showed a significantly higher (P less than 0.005) PCNA immunoreactivity than BVI-negative tumors. PCNA scores were significantly higher (P less than 0.005) in DNA aneuploid (n = 22) than in DNA diploid (n = 18) tumors and correlated significantly with the SPF of DNA aneuploid tumors (r = 0.825, P less than 0.0001), but not with diploid tumors (r = 0.002, P = 0.9). Intermediate proliferating tumors had a significantly higher (P less than 0.01) MC than their counterparts. In univariate analysis, significant predictors of survival were tumor classification (T1 versus T2), tumor size (less than or equal to 2.6 cm versus more than 2.6 cm), BVI (BVI-negative versus BVI-positive), MC (less than or equal to 8 versus more than 8), and PCNA immunoreactivity (low versus intermediate). DNA ploidy and SPF did not influence survival significantly. Only PCNA immunoreactivity retained its independent level of significance (P = 0.02) by multivariate analysis. It was concluded that PCNA immunostaining is a simple and clinically useful method for estimating cell proliferation in formalin-fixed, paraffin-embedded tissue of resected peripheral, node-negative NSCLC.


Prognostic significance of the PC10 index as a prospective assay for cervical cancer treated with radiation therapy alone.

The monoclonal antibody PC10 recognizes proliferating cell nuclear antigen (PCNA) in conventionally fixed and processed histologic materials. Formaldehyde-fixed and paraffin-embedded specimens taken from 194 patients with Stage III squamous cell carcinoma of the cervix treated with radiation therapy alone were investigated for PC10 positivity using immunohistochemical methods. A few squamous epithelial cells in the basal layer and the deepest zone of the prickle cell layer were positive for PC10. Cancer cells also were positive for PC10, showing a diffuse or granular nuclear staining pattern. The multiple logistic regression model analysis and the Kaplan-Meier method indicate that a strong correlation exists between the PC10 index and prognosis (P less than 0.001). Thus, the PC10 index may be a predictive indicator for the prognosis of patients with squamous cell carcinoma of the cervix treated with radiation therapy alone.


Genetic markers as prognostic indicators in breast cancer.

Identifying markers that have the potential to predict tumor behavior is important in breast cancer because of the variability in clinical disease progression. Genetic alterations in tumors may appear as changes in total DNA content, individual chromosomes, single genes, or gene expression. Alteration in DNA content is an imprecise but accessible measurement of the genome. Diploid tumors have been associated with a better clinical outcome, and increased ploidy correlates with other indicators of poor prognosis. Concurrent analysis of DNA content with markers of genetic expression is feasible (e.g., myc oncogene) and may increase its prognostic power. Chromosomal studies could provide a more precise tool for localizing genetic damage, but there is little cytogenetic information about primary breast cancers, no convincing evidence has emerged to target locations in the karyotype that appear specifically altered, and many primary and cultured breast cancers contain cells that appear chromosomally normal. Attempts to define molecular markers have used probes of different chromosomal sites, some chosen because of logical associations with hormonal activity, known oncogenes, or tumor-suppressor genes, and some by chance. Currently, to the authors' knowledge, none has shown uniform changes by mutation, loss, or overexpression in all breast cancers, although a remarkable number of loci are altered to some extent. These lesions must be associated with particular disease subsets or, retrospectively, with differential survival if they are to have prognostic value. The authors examined several loci (ERBB2, INT2, MUC1) for gene amplification or loss of heterozygosity by Southern blotting and for gene expression by immunohistochemistry in breast tumors from patient groups selected by survival. A retrospective series showed gene amplification at the erbB2 locus in 22% of rapidly recurrent (RR) tumors and 13% of tumors from long-term tumor-free survivors (LTS), but the difference was not statistically significant (P = 0.18). The erbB2 product was displayed histochemically with equal frequency between those with RR tumors and LTS patients. Moreover, the correlation was poor between different analytic measures on the same tumors. This result was tested using a prospective study of erbB2 to correlate DNA analysis with western blot findings and frozen and fixed histochemical results. Another oncogene, int2, showed significant correlation between amplification and recurrence; 16% of RR tumors showing genetic amplification (P = 0.02). Loci on other chromosomes, 1 (muc1) and 17 (cmm86), also are being investigated in groups selected for differences in survival.


Prognosis and treatment decisions in patients with breast cancer without axillary node involvement.

Every month, treatment decisions must be made for more than 6000 patients with breast cancer without axillary node involvement in the United States. Approximately 70% of these patients will survive more than 10 years after surgery and/or radiation treatment without additional systemic adjuvant therapy. If we had good methods to identify patients who are destined to have a recurrence of their disease, only those patients should receive adjuvant therapy. The authors reviewed the literature supporting the use of currently available prognostic factors for patients with node-negative breast cancer, and formulated a framework on which prognostic factor information can be based to help make these treatment decisions. The steps involved in making treatment decisions are: use prognostic factors to determine the recurrence probability; calculate the expected treatment benefit; and weigh the expected benefits against the potential risks. Prognostic factors can be used to help make treatment decisions for patients with breast cancer without axillary node involvement. However, the final treatment decision must take into account all aspects of the patient and her disease, and the physician must help the patient evaluate her prognostic factors, arrive at an understanding of her particular risk of recurrence, and weigh the potential benefits and risks of adjuvant therapy.


Genetic aspects of multidrug resistance.

Mammalian cells exposed to a single cytotoxic natural product drug, such as vincristine or dactinomycin, can develop resistance to the selective agent and cross-resistance to a broad spectrum of structurally and functionally distinct antibiotics and alkaloids. This phenomenon, termed multidrug resistance (MDR), has been widely studied experimentally. The most consistent feature of cells with high-level MDR is amplification and overexpression of genes encoding an integral plasma membrane protein known as P-glycoprotein. The MDR genes belong to a small family (two members in humans and three members in mouse and Chinese hamster). Based on several lines of evidence, P-glycoprotein is thought to act as an adenosine triphosphate-dependent efflux pump that decreases accumulation of drugs and increases resistance to their effects. The normal function of P-glycoprotein, apart from its role in MDR, is not known. Proposed roles in detoxification and steroid transport systems are speculative but suggest that the membrane protein may have distinct functions in normal tissues and in tumor cells with acquired MDR. Although possible endogenous substrates for P-glycoprotein have not been identified, insight into normal function may be gained from tissue distribution studies. For example, studies using molecular probes to P-glycoprotein messenger RNA and monoclonal antibodies to different epitopes of the molecule have shown that P-glycoprotein is expressed at high levels in the more differentiated or specialized cells of the colon or kidney. Amplification of MDR genes in vivo has not been observed. Whether intrinsic or acquired MDR plays a causal and potentially modifiable role in clinical nonresponsiveness to cancer chemotherapeutic agents is a topic of current interest. Prospective studies and serial determinations during the course of disease are needed to clarify the importance of this membrane protein in clinical drug resistance.


Oral cancer progression and c-erbB-2/neu proto-oncogene expression.

Monoclonal antibody PAb3 to c-erbB-2/neu protein was utilized in the immunoperoxidase staining of 86 human specimens from oral mucosa. These tissue specimens represented a spectrum from 7 normal to 9 simple hyperplasia, 15 mild dysplasia, 14 moderate dysplasia, 20 severe dysplasia and 21 squamous cell carcinoma. Our study indicated that as the cells acquire a more malignant phenotype, there was a progressive increase in neu expression. It also suggested that neu may be involved in the development of oral cancers and that its evaluation in the early stages may assist in the diagnosis and management of oral cancers.


Increase in gamma-glutamylcysteine synthetase activity and steady-state messenger RNA levels in melphalan-resistant DU-145 human prostate carcinoma cells expressing elevated glutathione levels.

The biochemical and molecular basis for the elevation of glutathione (GSH) levels commonly detected in many drug-resistant cells has not been elucidated. In a series of L-phenylalanine mustard-resistant human prostate carcinoma cell lines (DU-145), resistance was associated with elevated GSH levels, increased activity of gamma-glutamylcysteine synthetase (GCS), the rate-limiting enzyme in GSH biosynthesis, and a marked increase in the steady-state levels of GCS-specific transcripts (4.0 and 3.2 kilobases). Loss of the resistant phenotype was accompanied by a reduction in GSH and a return of GCS activity and transcript levels to values comparable to those detected in the drug-sensitive parent cells. These data strongly implicate up-regulation of GCS activity as an important mechanism in the evolution of drug resistance associated with increased levels of intracellular GSH. The results further suggest that the ability to synthesize GSH may be more indicative of resistance than steady-state GSH levels per se.


Weak antiparkinsonian activity of the D1 agonist C-APB (SKF 82958) and lack of synergism with a D2 agonist in primates.

The role of D1 receptors in motor control is poorly understood. In parkinsonian squirrel monkeys, the full D1 agonist C-APB (SKF 82958; 0.1-0.4 mg/kg s.c.) caused weak stimulation of locomotor activity. However, the motor stimulant effects of the D2 agonist (+)-PHNO (0.001 mg/kg s.c.) were not potentiated by C-APB (0.3 mg/kg). Differences between these observations and expectations from experiments using rodents are discussed.


Respiratory dyskinesia: a variety of clinical forms differentially diagnosed by using a spirograph.

Four cases of respiratory dyskinesia were investigated by using a spirograph before and after biperiden injection. The abnormal respiratory patterns in four cases appeared to be in two types and these abnormalities were abolished after biperiden injection. The present study showed that respiratory dyskinesia could be defined more clearly by using a spirograph and these results are useful for the diagnosis of patients with respiratory discomfort while undergoing neuroleptic drug treatment.


The synaptic vesicle protein SV2 is a novel type of transmembrane transporter.

The primary function of synaptic vesicles is to store and release neurotransmitter. Synaptic vesicles are locally recycled following exocytosis and rapidly refilled with neurotransmitter from the cytoplasm by a process that depends on the electrochemical gradient generated by a proton pump. Little is known about the molecules that import neurotransmitter into synaptic vesicles. We report here that the sequence of the synaptic vesicle protein SV2 identifies this protein as a novel type of transmembrane transporter. The deduced amino acid sequence of SV2 contains two sets of six predicted transmembrane domains: the six most N-terminal transmembrane domains are highly homologous to a subfamily of transporters that includes the human glucose transporter, while the six most C-terminal domains are homologous to the plasma membrane transporters for neurotransmitters. We propose that SV2 mediates transport of neurotransmitters into synaptic vesicles.


Leukotriene B4 mediates shear rate-dependent leukocyte adhesion in mesenteric venules.

Previous studies have demonstrated that low shear rates promote leukocyte adherence to microvascular endothelium in postcapillary venules. The objective of this study was to determine whether an accumulation of inflammatory mediators such as platelet activating factor and leukotriene B4 is responsible for shear rate-dependent leukocyte-endothelial cell adhesion. Postcapillary venules (25-39 microns in diameter) in cat mesentery were studied by intravital microscopy. Venular wall shear rate was varied over a wide range by graded occlusion of the mesenteric artery. Red blood cell velocity, vessel diameter, leukocyte rolling velocity, and the numbers of rolling and adherent leukocytes were measured at each shear rate. In one series of experiments, shear rate-dependent leukocyte adherence was monitored at different superfusion rates (1.0 and 2.5 ml/min). At the lower superfusion rate, the number of adherent leukocytes was significantly higher at any given shear rate when compared with results obtained at the higher superfusion rate. This suggests that reduced washout of inflammatory mediators contributes to shear rate-dependent leukocyte adhesion. Pretreatment with different platelet activating factor receptor antagonists (WEB 2086 or WEB 2170) had no effect on the number of adherent leukocytes normally observed at lower shear rates, suggesting that platelet activating factor does not play a major role in this process. However, shear rate-dependent leukocyte adhesion was largely prevented by pretreatment with either a leukotriene B4 receptor antagonist (SC-41930) or a leukotriene synthesis inhibitor (L663,536). The results of this study indicate that a reduced washout of leukotriene B4 is responsible for the enhanced leukocyte adherence that occurs at low venular wall shear rates.


Report of the Conference on Low Blood Cholesterol: Mortality Associations.

A National Heart, Lung, and Blood Institute (NHLBI) Conference was held October 9-10, 1990, to review and discuss existing data on U-shaped relations found between mortality rates and blood total cholesterol levels (TC) in some but not other studies. Presentations were given from 19 cohort studies from the United States, Europe, Israel, and Japan. A representative of each study presented its findings and also submitted tables of proportional hazards regression coefficients for entry TC levels in regard to death, and these were incorporated into a formal statistical overview adjusted for age, diastolic blood pressure, cigarette smoking, body mass index, and alcohol intake, as available. The U-shape for total mortality in men and the flat relation in women resulted largely from a positive relation of TC with coronary heart disease death and an inverse relation with deaths caused by some cancers (e.g., lung but not colon), respiratory disease, digestive disease, trauma, and residual deaths. Risk for combined noncardiovascular, noncancer causes of death decreased steadily across the range of TC. The conference considered possible explanations for the statistical associations found between low TC levels or active TC lowering and certain causes of death. One is that TC is lowered by some disease conditions themselves, such as wasting in chronic pulmonary disease or reduced production and secretion of cholesterol-bearing lipoproteins with liver disease. In this sort of situation, the TC:mortality association found in observational studies may be due to preexisting disease. This was addressed by excluding early deaths from the analysis, which did not change the results. The conference considered as well the biological function of cholesterol, which, if seriously deranged, might hypothetically cause a wide variety of diseases and dysfunction. The conference also considered the biological functions that might provide plausible mechanisms for the associations found. Definitive interpretation of the associations observed was not possible, although most participants considered it likely that many of the statistical associations of low or lowered TC level are explainable by confounding in one form or another. The conference focused on the apparent existence and nature of these associations and on the need to understand their source rather than on any pertinence of the findings for public health policy. Further research is recommended to explain the observed associations of low TC levels (and TC lowering) with certain noncardiovascular diseases. This includes studies of the time course of TC change in disease, the relation of TC to morbidity, further studies of possible epidemiological confounding, monitoring of population trends in TC and mortality, further studies of the relations in women, auditing of noncardiovascular events in trials, studies of cell membrane, genetic and molecular links to cholesterol metabolism, TC level and disease, studies of disease manifestations in specific lipid disorders, and further study of the proposed causal mechanisms linking low TC and hemorrhagic stroke.


Coronary endothelial and cardiac protective effects of a monoclonal antibody to intercellular adhesion molecule-1 in myocardial ischemia and reperfusion.

Intercellular adhesion molecule-1 (ICAM-1) is a major ligand on endothelial cells for adherence of activated polymorphonuclear leukocytes (PMNs). The major purpose of this study was to study the effects of RR1/1, a monoclonal antibody against ICAM-1 (i.e., MAb RR1/1), on myocardial injury and endothelial dysfunction associated with myocardial ischemia and reperfusion. Either MAb RR1/1 (2 mg/kg, n = 7), an antibody that was found to bind selectively to endothelial cells in the cat, or MAb R3.1 (2 mg/kg, n = 7), a nonbinding control antibody, was given as an intravenous bolus 10 minutes before reperfusion. Two hundred eighty minutes later, hearts were excised. The left ventricle area-at-risk (AAR) was similar in MAb RR1/1 (29 +/- 2%) and MAb R3.1 (30 +/- 3%) groups. In MAb R3.1-treated cats, 90 minutes of myocardial ischemia plus 4.5 hours of reperfusion induced a significant myocardial injury (necrotic tissue/AAR, 28 +/- 2%), high myeloperoxidase activity (0.65 +/- 0.16 units/100 mg ischemic tissue), and a marked decrease in endothelium-dependent vasorelaxation in isolated left anterior descending coronary arteries (vasorelaxation to acetylcholine, 29 +/- 3%) with no change in endothelium-independent vasorelaxation (relaxation to NaNO2, 91 +/- 3%). However, cats treated with MAb RR1/1 developed significantly less myocardial necrosis (10 +/- 2% of the AAR, p less than 0.01), lower myeloperoxidase activity in ischemic myocardial tissue (0.2 +/- 0.03 units/100 mg ischemic tissue, p less than 0.01), and enhanced vasorelaxant responses to endothelial-dependent relaxation to acetylcholine (53 +/- 5%) compared with ischemic/reperfused cats treated with Mab R3.1. Furthermore, addition of MAb RR1/1 in vitro significantly inhibited unstimulated PMN adherence to ischemic-reperfused coronary artery endothelium. These results suggest that ICAM-1-dependent PMN adherence plays an important role in reperfusion injury, and that PMN adherence and infiltration contribute significantly to coronary endothelial dysfunction.


Mechanisms of interaction between the sulfhydryl precursor L-methionine and glyceryl trinitrate.

L-Methionine potentiates systemic hemodynamic effects of intravenous glyceryl trinitrate (GTN) in tolerant and nontolerant patients to a similar extent as N-acetylcysteine (NAC). This potentiation of GTN action by L-methionine has been attributed to enhanced intracellular formation of nitrosothiols, known to be potent stimulators of soluble guanylyl cyclase. This study was performed to analyze directly the effects of L-methionine on GTN-induced dilation of large epicardial arteries and the venous capacitance system of the dog in the tolerant and nontolerant states. Cultured rat aortic vascular smooth muscle cells and purified guanylyl cyclase were used to study potential intracellular and extracellular mechanisms responsible for this interaction. In awake nontolerant dogs, L-methionine (100 mg/kg) potentiated the tachycardic response to GTN (5.0 and 15 micrograms/kg/min) and enhanced the hypotensive action of GTN (1.5 and 5.0 micrograms/kg/min) in anesthetized, nonreflexic dogs. In nontolerant and tolerant dogs, however, L-methionine did not alter the dose-response of large epicardial artery dilation to intravenous GTN challenges and did not modify nitrate tolerance of the low pressure system of the dog. The infusion of L-methionine (100 mg/kg) significantly increased plasma methionine levels (from 52 +/- 12 to 1,141 +/- 239 microM), cystine levels (from 12 +/- 4 to 26 +/- 7 microM), but not homocystine levels. In vitro, the L-methionine conversion product L-cysteine (0.1-1.0 mM) but not homocysteine significantly enhanced the augmentation of purified guanylyl cyclase activity by GTN (100 microM). Incubation of cultured rat aortic smooth muscle cells with L-methionine (10 microM or 1 mM) did not result in a significant increase of free intracellular sulfhydryl group content. The L-methionine conversion product L-cysteine mediates tolerance independent the potentiation of GTN action. This may result from an L-cysteine-induced formation of a vasoactive metabolite of GTN (nitric oxide) or nitrosothiol. This effect occurs primarily in the resistance vessel circulation, not in large epicardial arteries and veins. The lack of effect of L-methionine on sulfhydryl group content in large conductance vessels indicates that hepatic L-methionine metabolism constitutes the significant source of L-cysteine. These findings strongly suggest that administration of sulfhydryl-group precursor L-methionine does not represent a therapeutic alternative to a nitrate-free interval to restore nitrate sensitivity in tolerant large epicardial arteries and veins.


A fragile X family with high penetrance in females: risk heterogeneity?

A fragile X family is described which shows two interesting features. In a sibship of seven, the two males and four of the five females are affected with mental retardation. Since the only normal daughter is not a carrier, the penetrance of the fragile X mutation in carrier daughters is 100%. Nevertheless, the penetrance of this syndrome in affected daughters of normal mothers has been estimated at a third. Also, DNA typing analysis of flanking RFLP markers revealed a higher than expected number of crossing-overs. We also include the molecular study of the mutation in the (CGG)n repeat of the FMR-1 gene.


Amyloidogenic and non-amyloidogenic transthyretin Asn 90 variants.

Recently, a new transthyretin (TTR) variant was described in the normal Portuguese and German populations. The same substitution was found associated with familial amyloidotic polyneuropathy (FAP) in an American family of Italian origin. Comparative isoelectric focusing studies showed a difference in the mobility pattern between the non-pathogenic and pathogenic variants. However, comparative DNA sequencing between them did not reveal any additional mutation. Comparative isoelectric focusing between the variants and TTR Asn 90 produced by recombinant techniques indicated that the non-pathogenic variant has the electrophoretic behaviour expected for the mutation. We suggest that an as yet unknown post-translational modification may have occurred in the FAP-associated Asn 90 variant, turning it into an amyloidogenic molecule.


Variable dystrophin expression in different muscles of a Duchenne muscular dystrophy carrier.

The majority of Duchenne muscular dystrophy (DMD) female carriers show dystrophin immunostaining abnormalities, although a significant proportion of clinically non-manifesting carriers are normal following this analysis. We had the opportunity to study dystrophin immunostaining in two different muscles, the vastus lateralis and the rectus abdominis of a possible DMD carrier. While the vastus showed normal dystrophin immunostaining, pathological staining was detected in her rectus abdominis. These findings seem to indicate that dystrophin expression can vary in different muscle groups of a DMD carrier. The implications of these findings in DMD carrier detection and possible dystrophin function are discussed.


Adrenergic agents: clinical trials and experiences.

Beta-adrenergic blocking agents constitute first-line therapy for hypertension in many countries of the world. Comparative trials have been extensive in duration and have included large numbers of patients. Still, the desired cardioprotective effect of beta blockers has yet to be established. Their antiatherosclerotic effect has been noted in several animal experiments. Confirming evidence is needed before the clinical relevance of this effect can be evaluated, but these studies point to a potential therapeutic effect that may be immensely important in the future. Beta blockers can reverse left ventricular hypertrophy secondary to hypertension, but it remains to be shown that regression of left ventricular hypertrophy will reduce the associated risks.


Disorders of excessive sleepiness. Treatment improves ability to stay awake but does not reduce sleepiness.

A total of 47 patients with sleep disorder (36 male and 11 female) with a mean age of 47.5 +/- 15 years were evaluated for daytime symptoms with a Multiple Sleep Latency Test (MSLT) and a Maintenance of Wakefulness Test (MWT) given on the same day--once at the time of their diagnostic evaluation and again after one to six months of treatment. The MSLT and MWT data are consistent with the notion that sleep tendency, as measured by the MSLT and ability to remain awake, as measured by the MWT, represent different physiologic processes. Data show a marked treatment-related improvement in ability to stay awake as measured by the MWT and no treatment-related improvement in sleepiness as measured by the MSLT. We conclude that there is a heterogeneous subpopulation of patients with sleep disorders whose symptoms of daytime sleepiness will show no treatment-related improvement in daytime symptoms if they are evaluated only by the MSLT. We suggest that, since ability to stay awake (and not ability to fall asleep) is a requisite for all job-related duties, an objective, physiologically based test such as the MWT should be used to assess the impact of sleep disorders in cases where there is a clinical concern about fitness to drive or work.


Expression of alveolar macrophage adhesion molecules in pulmonary sarcoidosis.

Beta-2-integrins belong to a family of leukocyte surface glycoproteins that are essential for immune functions of bronchoalveolar cells. The expression of three alpha chains designed as CD11a, CD11b, CD11c, a common beta chain CD18, and of a ligand for several integrins CD54 (ICAM-1) was studied on alveolar macrophages of patients with active and inactive sarcoidosis and in control subjects. The percentage of macrophages expressing CD11b (CR3) was significantly increased in patients with active sarcoidosis compared with patients who had inactive disease and control subjects. The adhesion molecule CD54 (ICAM-1) was detected on a higher percentage of alveolar macrophages in patients with active rather than inactive sarcoidosis and in control subjects. Since integrin-mediated adhesion seems to be important in macrophage-lymphocyte interactions during the immune response, higher expression of both CD11b and CD54 on sarcoid alveolar macrophages may be related to several immune abnormalities reported in pulmonary sarcoidosis.


Molecular mechanisms of pattern formation in the vertebrate hindbrain.

During early stages of neural development a series of repeated bulges, termed rhombomeres, form in the vertebrate hindbrain. Studies in the chick have shown that rhombomeres are segments that underlie the patterning of nerves in the hindbrain, and this raises the question of the molecular basis of segment development. Several genes have been found with expression patterns consistent with roles in the formation or differentiation of rhombomeres. The zinc finger gene Krox-20 is expressed in two alternating rhombomeres, r3 and r5, in the mouse hindbrain; these stripes of gene expression are established prior to the morphological appearance of segments. Krox-20 is also expressed in this pattern in the chick and Xenopus, suggesting that it has a conserved role, possibly in the formation of rhombomeres. Four members of the Hox-2 homeobox gene cluster have limits of expression at rhombomere boundaries. Three genes, Hox-2.6, -2.7 and -2.8 have progressively more anterior limits of expression at two-segment intervals, whereas expression of Hox-2.9 is restricted to one rhombomere, r4. The Hox-2 genes are expressed in spatially restricted patterns in early neural crest cells. These findings suggest that the Hox genes have roles in specifying the identity of rhombomeres and of neural crest.


Effect of cetirizine, ketotifen and chlorpheniramine on the dynamics of the cutaneous hypersensitivity reaction: a comparative study.

Allergic cutaneous challenge causes mast cell and basophil mediator release which recruit inflammatory cells to the site of antigen administration. This secondary cell infiltration and mediator release is responsible for the changes seen during the late phase of allergic diseases. In this randomised, double-blind, cross-over, placebo controlled study, it was demonstrated that, at steady-state drug concentrations, chlorpheniramine reduced the wheal-and-flare reaction by about 50% compared to the 75% reduction, on average, by cetirizine and ketotifen. Cetirizine significantly reduced eosinophil vacuolisation at all observation periods, i.e. 2,6,10 and 24 h, and also inhibited basophil accumulation significantly at 10 h (75% reduction), while chlorpheniramine had a negligible effect on these variables. These changes would indicate that the late phase reaction was modified, especially as eosinophil vacuolisation is known to correlate with late phase intensity, T-lymphocyte infiltration and subsequent tissue damage. It further supports previous speculation that cetirizine inhibit late histamine release by acting on basophils. The extent of induration in the late phase reaction did not differ significantly among the three treatments. Cetirizine and ketotifen, noticeably although not significantly, reduced eosinophil and lymphocyte recruitment. As these two antihistamines differ structurally and in regard to receptor specificity, it is possible that they exert their actions on other, unspecified, receptors.


Effects of loratadine and cetirizine on actual driving and psychometric test performance, and EEG during driving.

Sixteen healthy male and female volunteers took part in a 6-way, double-blind cross-over trial to compare the effects of single doses of cetirizine 10 mg, loratadine 10 mg and placebo, with and without alcohol (0.72 g.kg-1, lean body mass). Performance was measured in two repetitions of a psychometric test battery, and a standard, over-the-road driving test. EEG was also measured during driving. Alcohol significantly affected almost every performance measure and altered the EEG energy spectrum during driving whilst the blood concentrations declined from 0.37 to 0.20 mg.ml-1. The effects of cetirizine of on driving performance resembled those of alcohol. It caused the subjects to operate with significantly greater variability in speed and lateral position ('weaving' motion). The effects of alcohol and cetirizine appeared to be additive. Certain cetirizine-placebo differences in subjective feelings and test battery performance were also significant. Loratadine had no significant effect on any performance parameter. It was concluded that cetirizine, but not loratadine, generally caused mild impairment of performance after a single 10 mg dose.


Interaction of talinolol and sulfasalazine in the human gastrointestinal tract.

The absorption of talinolol (TA) 50 mg was investigated without and together with the co-administration of sulfasalazine (SASP) 4 g in 11 healthy young volunteers, in order to clarify gastrointestinal transit of TA. Without SASP, the tmax of TA was 2.8 h, Cmax was 112 ng.ml-1 and the half life was 12 h; the AUCo-t was 958 ng.ml-1.h. In the case of concomitant administration of SASP, TA was found only in serum from 3 individuals, with a Cmax of 23 ng.ml-1 and a mean AUCo-t of 84 ng.ml-1.h. TA was not detectable in 5 subjects and it was at the limit of detection (2 ng.ml-1) in 3 subjects. Pharmacokinetic analysis was not possible in any of those individuals. The reason for the interaction appears to be the adsorption of TA by SASP. An interval of 2-3 h should elapse between giving SASP and other drugs.


Mechanism of platelet activating factor-induced vascular leakage in the rat trachea.

Platelet activating factor (PAF) is a phospholipid mediator of inflammation and vascular leakage that may be important in the etiology of asthma. We and others have demonstrated that PAF causes vascular leakage in the rat trachea. In the present study, we attempted to determine how PAF mediates this effect. Vascular leakage was quantitated by measuring the amount of intravascular Evans blue dye extravasated into tracheal tissue. Intravenously administered PAF increased vascular leakage, although Lyso-PAF and Enantio-PAF had no effect. PAF-induced vascular leakage was inhibited in a dose-dependent fashion by the PAF receptor blocker WEB 2086. However, PAF-induced vascular leakage was not inhibited by blockade of cyclooxygenase/lipoxygenase, calmodulin, calcium channels, protein kinase C, histamine receptors, or by destruction of peptidergic sensory nerves. We conclude that PAF causes vascular leakage in the rat trachea by a stereospecific receptor-mediated mechanism that does not depend on arachidonic acid metabolites, calcium, protein kinase C, histamine, or peptidergic sensory nerves.


Heterogeneous susceptibility of human melanoma clones to monocyte cytotoxicity: role of ICAM-1 defined by antibody blocking and gene transfer.

Five clones derived from the same human malignant melanoma lesion were studied for their susceptibility to killing by human monocytes activated by exposure to interferon (IFN)-gamma and lipopolysaccharide. Melanoma clones were heterogeneous in their susceptibility to human monocyte cytotoxicity, with one clone (2/21) exhibiting extremely low levels of lysis. The different levels of susceptibility to monocyte cytotoxicity were not accounted for by susceptibility or resistance to monokines [tumor necrosis factor (TNF), interleukin (IL)-1 and IL-6] because: (a) these effector molecules had little (TNF) or no (IL-1 and IL-6) cytolytic activity under these conditions; and (b) anti-TNF antibodies had marginal effects on cytotoxicity. Monocytes bound less to resistant than to susceptible melanoma cells. Monocyte-resistant 2/21 melanoma cells expressed substantially lower levels of ICAM-1 and VLA-4 than susceptible cells. Anti-CD18 and, to a lesser extent, anti-ICAM-1 mAb inhibited binding and cytotoxicity of human monocytes on malignant melanoma whereas anti-VLA-4 had no inhibitory action. Transfection of the ICAM-1 gene under the control of a constitutive promotor resulted in high levels of expression of ICAM-1 in 2/21 melanoma cells and, concomitantly, in augmented susceptibility to activated monocyte cytotoxicity. The augmented killing of ICAM-1 transfected 2/21 cells was inhibited by anti-ICAM-1 mAb. These results demonstrate that the CD18-ICAM-1 adhesion pathway can play an important role in the expression of human monocyte cytotoxicity on melanoma target cells and that heterogeneity in expression of ICAM-1 can underlie differences in susceptibility to tumoricidal activity.