Enhanced myocardial adenylate cyclase activity in spontaneously hypertensive rats.

This study was designed to clarify the state of beta-adrenergic signal transduction and the disordered level of its transduction in hypertensive hearts, using myocardium from spontaneously hypertensive rats (SHR) as a generic model of essential hypertension. Beta-adrenergic receptor binding sites and dissociation constants in the extracted membranes of adult (70-100 days of age) SHR heart were not significantly different from those of Wistar-Kyoto (WKY) rats, the non-hypertensive control. The adenylate cyclase activities stimulated by isoproterenol with GTP, NaF and forskolin were significantly higher in SHR compared to those in WKY. To determine whether differences in signal transduction are natural or are a result of hypertension, we evaluated chronotropic responses in cultured cells of fetal hearts which had not been exposed to hypertension. Fetal cardiac muscle cells of SHR were more sensitive than WKY to isoproterenol stimulation over a wide concentration range. However, there were no statistically significant differences between these two strains with respect to the density of binding sites. These results suggest that in the transduction of adrenergic signals, alterations distal to the beta-receptors are present in the adult hearts of hypertensive rats, and, that the adrenergic signal transduction is already exaggerated in the pre-hypertensive fetal stage.


[Prostaglandin E1 infusion fails to inhibit the increase of serum granulocyte elastase and myeloperoxidase and the decrease of plasma angiotensin converting enzyme in patients undergoing open-heart surgery].

We studied whether prostaglandin E1 (PGE1) could inhibit the increase of serum granulocyte elastase (GEL) and myeloperoxidase (MPO), and the decrease of plasma angiotensin converting enzyme (ACE) induced by oxygenator in 19 patients undergoing open-heart surgery. The patients were randomly allocated into 2 groups: one group (PGE1 group, n = 9) received a continuous infusion of PGE1 at a rate of 30 ng.kg-1.min-1 during cardiopulmonary bypass (CPB), and the other group (control group, n = 10) received saline infusion. GEL, MPO and ACE were measured serially at 8 points: before induction of anesthesia (as baseline), immediately before initiation of CPB, 10 min after initiation, 60 min after initiation, immediately after the end of CPB, 60 min after CPB, 120 min after CPB, and on the first postoperative day. Serum levels of GEL and MPO during 120 min after the end of CPB in both groups increased significantly compared with the baseline values. There was no significant difference between the two groups. Plasma levels of ACE in both groups decreased significantly immediately after the end of CPB compared with values taken 10 min after the initiation of CPB. There was no significant difference between the groups. We conclude that the infusion of PGE1 30 ng.kg-1.min-1 failed to inhibit the increase of GEL as well as MPO, and the decrease of ACE.


Regulation of Na+ channels in the cortical collecting duct by AVP and mineralocorticoids.

A variety of experimental approaches have shown that AVP and mineralocorticoids stimulate Na+ transport through their effects on the number and kinetic properties of amiloride-sensitive Na+ channels in the apical membrane. The different mechanisms by which AVP and mineralocorticoid act on the Na+ channel provide a basis for synergism in their actions, perhaps by a scheme such as that proposed in Figure 5. However, the details of this interaction will require a better understanding of the molecular details involved in activating quiescent channels, increasing their open probability, and reorientating or inserting channels to an operational position in the apical membrane. Electrophysiological and biochemical approaches have gone a long way toward elucidating some of these molecular details. But the latter approach in particular has indicated that the Na+ channel may have multiple regulatory subunits and thus be a target for several intracellular second messengers and autacoids other than those involved in the actions of AVP and aldosterone. The challenges for future research in this area are multiple. It seems likely that the primary amino acid sequence of the channel subunits will soon become available from cloning and sequencing approaches, but the application of this knowledge to understanding how the subunits are integrated into the complete protein and mediate regulatory signals will be a formidable task. It will be important to determine the normal extracellular signals (other than aldosterone and AVP) and the associated intracellular second messengers that alter channel activity. It will also be important to understand how some species such as the rabbit may "turn off" the stimulatory effect of AVP on Na+ reabsorption in the CCD, and how this regulatory process is altered when these cells are cultured. At the whole animal level, it will also be important to investigate whether changes in one or more of the normal regulatory pathways that impinge on the Na+ channel might be involved in a diminished ability to excrete a salt load, as is observed in some models of hypertension. All of these issues need to be understood at the molecular level, and it seems likely they will provide exciting physiological insights at all levels.


Glomerular cell proliferation and PDGF expression precede glomerulosclerosis in the remnant kidney model.

Increasing evidence supports a role of glomerular cell proliferation in the development of focal or diffuse glomerulosclerosis. This study investigates the chronology and sequence of cellular events that precede glomerulosclerosis in 5/6 nephrectomized rats. Within three days of renal ablation, a phenotypic switch occurred in which some mesangial cells expressed alpha-smooth muscle actin. This was followed by proliferation of mesangial cells, and to a lesser degree endothelial cells from day 5 to week 4 as detected by immunostaining for the proliferating cell nuclear antigen (PCNA). Glomerular cell proliferation was accompanied by increased immunohistochemical expression of PDGF B-chain. In situ hybridization showed no glomerular PDGF B-chain mRNA expression at the induction of proliferation (day 5), and a marked increase between week 1 and 4 in operated rats. In parallel, increased expression of PDGF receptor beta-subunit protein and mRNA was demonstrated by immunohistochemistry and Northern analysis of total glomerular RNA. The onset of glomerular cell proliferation was also associated with mild glomerular platelet accumulation (as defined by 111In-labelled platelet studies) as well as with fibrinogen deposition. Proteinuria, glomerular sclerotic changes, and leukocyte infiltration all followed cell proliferation. The glomerular leukocyte infiltrate consisted of monocytes/macrophages and increased markedly at week 10 in rats with renal ablation. Thus, our results suggest that in the remnant kidney model: 1) proliferation of intrinsic glomerular cells precedes glomerulosclerosis; 2) proliferation may be initiated by degranulating platelets and sustained by PDGF released from intrinsic glomerular cells; and 3) glomerular monocyte/macrophage infiltration occurs after the proliferation, and may possibly contribute to the development of glomerular sclerotic changes.


Regulation of endothelin-1 production in cultured rat mesangial cells.

We investigated the regulatory mechanisms of endothelin (ET)-1 production in cultured rat mesangial cells (MC), with a special focus on the roles of protein kinase A (PKA)- and protein kinase C (PKC)-mediated signaling systems. Vasoactive agents and growth promoting factors, including platelet-derived growth factor, vasopressin and thrombin, which act through receptors coupled to the phospholipase C-mediated signaling system, as well as phorbol ester and fetal calf serum stimulated ET-1 production. This effect was attenuated in PKC-depleted or H-7 (a PKC inhibitor) treated MC. On the other hand, an increase in intracellular cyclic AMP by forskolin or beta-adrenergic agonist, isoproterenol, which act as anti-mitogenic agents, inhibited serum-stimulated ET-1 production. In addition this effect was mimicked by the addition of 8-bromo-cyclic AMP to the medium. The effect of isoproterenol was abolished by propranolol. H-8, a PKA inhibitor, attenuated the inhibitory effect of forskolin. These findings suggest that ET-1 production in MC is regulated by interaction of both positive and negative signals mediated by PKC- and PKA-dependent mechanisms.


Anti-myeloperoxidase antibodies stimulate neutrophils to damage human endothelial cells.

Anti-myeloperoxidase autoantibodies are found in association with idiopathic necrotizing glomerulonephritis and systemic vasculitis. It is not known if their presence is an epiphenomen or an integral part of the pathogenic process. To further delineate their hypothesized pathogenicity, we studied their ability to stimulate neutrophils to damage human umbilical vein endothelial cells in vitro. Anti-myeloperoxidase antibodies from human, rabbit and mouse sources were utilized. These antibodies stimulated neutrophils to damage endothelial cells as determined by 51Cr release. The effect was dependent on priming the neutrophils with tumor necrosis factor-alpha, and further enhanced with the addition of endotoxin. The amount of endothelial cell damage was dependent on the dose of anti-myeloperoxidase, the source of the neutrophils, the concentration of TNF, and the presence of endotoxin. Under identical conditions, control antibodies did not stimulate neutrophils to damage endothelial cells. The effect was confirmed by labeling the endothelial cells with 3H-adenine which yielded the same results. These results provide further in vitro evidence that anti-myeloperoxidase autoantibodies may play a significant role in the pathogenesis of idiopathic pauci-immune glomerulonephritis and vasculitis.


Detection of Epstein-Barr virus genome in natural-killer-like cell line, YT.

YT cells, originally reported as a natural-killer-like (NK-like) lymphoid cell line, were investigated for Epstein-Barr virus (EBV) genome, gene rearrangement for T-cell receptor (TCR), phenotype and function. The YT cells of the original batch (YT-0) and two subclones (YT2C2 and YTC3) expressed EBV-associated nuclear antigen, and the BamHI-digested DNA showed the 3.4 kb hybridizing band with the BamHIW probe of EBV DNA in Southern blot analysis. When tested with latent-infection membrane protein probe, an identical hybridizing band was shared, indicating that all three sources of YT cells were of monoclonal derivation in terms of the terminal repeat junctional structure of EBV DNA, and that the original YT cells had been infected with EBV before the isolation of the two subclones. The cell-surface antigen analysis revealed the expression of CD7, CD28, CD30, CD45R0, TLiSA and S6F1 antigen besides the originally recorded CD25, CD56 and HLA-DR antigen. Gene rearrangement analysis showed the germ-line genotype, including TCR gamma and delta as well as beta chain. The Northern blot study using the CD3 epsilon and CD3 delta chain gene probes revealed CD3 epsilon, but not CD3 delta RNA. The YT-0 cells exhibited NK and antibody-dependent cellular cytotoxicity activity, but the YT2C2 and YTC3 cells did not. It was not resolved whether the fresh neoplastic NK-like cells of the YT-cell donor carry EBV genome, but YT cells, the first lymphoid cell line found to have EBV genome and non-B lymphoid properties, are valuable for investigating the relationship between EBV and human non-B lymphoid hematopoietic cells.


[Acute hepatitis C. A clinical and epidemiological study].

The discovery of the hepatitis C virus (HCV) and the possibility of determining anti-HCV serum antibodies has permitted greater etiological, clinical and epidemiological knowledge of non A non B hepatitis (NANB). The clinical and epidemiological characteristics of 47 patients with acute hepatitis C were studied. Diagnosis was based on compatible clinical and analytical picture as well as positivity of the anti-HCV antibodies. Thirty-eight percent of the patients were intravenous drug abusers (IVDA). Thirty-four percent had transfusion antecedents and the remaining were sporadic forms of which 3 cases corresponded to health care workers. The study of other viral markers demonstrated that only in the IVDA collective did infections by hepatitis B and delta (HBV and HDV) and by the human immunodeficiency virus (HIV) coexist. Anti-HCV antibodies were detected between 15 days and 8 months (mean 38 +/- 74 days) of clinic initiation; with no differences being found regarding the form of contagion. The incubation period of transfused patients was 50 +/- 15 days. In 2 cases the disease followed a biphasic course, in 4 patients a cholestatic profile was observed, and in 12 the hepatitis was anicteric. In 28% of the cases controlled over 6 months the disease autolimited itself and 72% developed active chronic hepatitis. Most cases of acute hepatitis C are of intravenous origin with a prolonged period of anti-HCV positivization. No differences exist in the clinical picture of the disease regarding the form of contagion and the great tendency to chronicity of hepatitis C is confirmed, regardless of the mechanisms of acquiring the disease.


Synthesis and binding characteristics of the highly delta-specific new tritiated opioid peptide, [3H]deltorphin II.

A radiolabelled form of deltorphin II was synthesized by catalytic tritiation using [p-IPhe3]-deltorphin II as a precursor. The ligand labels rat brain membranes with a Kd value of 1.9 nM, and the Bmax was found to be 92 fmol/mg protein. This new tritiated ligand exhibits high affinity for the delta opioid binding site, whereas its binding to the mu type is weak and extremely low for the kappa type. Mu/delta and kappa/delta selectivity ratios were about 900 and 10,000, respectively. The highly delta selective binding properties of this new radioligand suggest that it could serve as an excellent tool for investigating the delta opioid receptors in various species.


Regulation of [3H]nitrendipine binding by phospholipases A2 and C through direct and GTP-sensitive mechanisms.

We compared the effects of Phospholipases A2, C, B and D on [3H]nitrendipine binding to hamster cardiac membranes, in the absence and presence of ATP or GTP. Phospholipase A2, competitively inhibited [3H]nitrendipine binding to hamster cardiac membranes unchanged by ATP or GTP (Ki = 5 ng/ml); as evidenced by complete and reversible displacement of [3H]nitrendipine binding and increase in KD on Scatchard analyses. Phospholipase C also completely inhibited [3H]nitrendipine binding to hamster cardiac membranes (Ki = 5 micrograms/ml) with a decrease in Bmax and no change in KD on Scatchard analyses. The addition of GTP alone inhibited the PLC effect in EGTA-treated membranes. The addition of GTP with either CaCl2 or ATP or both resulted in an equal and opposite enhancement of the PLC effect. Phospholipases B and D had no effect on [3H]nitrendipine binding. These data support: (1) Direct effect of PLA2 on dihydropyridine binding. (2) Indirect regulation of dihydropyridine binding by Phospholipase C through a GTP and ATP-sensitive mechanism.


Characterization of [3H]naltrindole binding to delta opioid receptors in rat brain.

[3H]Naltrindole binding characteristics were determined using homogenized rat brain tissue. Saturation binding studies at 25 degrees C measured an equilibrium dissociation constant (Kd) value of 37.0 +/- 3.0 pM and a receptor density (Bmax) value of 63.4 +/- 2.0 fmol/mg protein. Association binding studies showed that equilibrium was reached within 90 min at a radioligand concentration of 30 pM. Naltrindole, as well as the ligands selective for delta (delta) opioid receptors, such as pCI-DPDPE and Deltorphin II inhibited [3H]naltrindole binding with nanomolar IC50 values. Ligands selective for mu (mu) and kappa (kappa) opioid receptors were only effective in inhibiting [3H]naltrindole binding at micromolar concentrations. From these data, we conclude that [3H]naltrindole is a high affinity, selective radioligand for delta opioid receptors.


Sigma (sigma) antagonist BMY 14802 prevents methamphetamine-induced sensitization.

We have demonstrated for the first time that the sigma antagonist BMY 14802 prevents the development of behavioral sensitization induced by repeated administration of methamphetamine. Rats received an intraperitoneal injection of 15 or 30 mg/kg BMY 14802 followed by 2 mg/kg methamphetamine 30 min later. Unlike dopamine antagonists, BMY 14802 did not induce major changes in the acute motor effects of 2 mg/kg methamphetamine. Repeated administration of methamphetamine induced progressive augmentation of stereotyped behaviors and resulted in behavioral sensitization. However, repeated administration of methamphetamine in combination with BMY 14802 at either dose produced no increase in the intensity of stereotypy when compared with the first treatment. After a 7-day abstinence period, a challenge test with methamphetamine alone revealed supersensitivity of methamphetamine-sensitized rats to subsequent methamphetamine, whereas rats pretreated with repeated methamphetamine in combination with BMY 14802 exhibited no difference in the intensity of stereotypy from rats pretreated with repeated saline. These results suggest that sigma receptors play a crucial role in the induction of methamphetamine-induced sensitization.


Chronic morphine treatment increases the number, but decreases the affinity of [3H]-U69,593 binding sites in the rat heart.

In the present study, the effects of chronic morphine treatment on the binding properties of tritiated U69,593, a specific kappa-ligand, in the rat heart were determined. Adult rats were given morphine through osmotic pumps at a rate of 80 micrograms/hour supplemented by daily injection of morphine at increasing doses for 9 days. The increase in colonic temperature to morphine, used as an indicator of development of tolerance in the rat, was measured daily. It was shown that, on day 7 following morphine treatment, the rats developed tolerance to morphine as indicated by an attenuated hyperthermic response to morphine. The [3H]-U69,593 specific binding properties were determined by direct receptor binding assay. The binding sites increased gradually and reached a significantly higher level at day 10. Scatchard analysis showed that both Bmax and Kd increased at day 10 following morphine treatment, indicating an increase in number of sites and a reduction in affinity to the kappa-ligand. Acute morphine injection at a dose of 10 mg/kg did not cause any significant alteration of [3H]-U69,593 binding sites. Two days after withdrawal of morphine, the [3H]-U69,593 binding sites returned to the original level. The finding of the present study indicates that there is an up-regulation of number, but a reduction in affinity of kappa-binding sites following chronic morphine treatment.


An opioid growth factor regulates the replication of microorganisms.

An opioid growth factor (OGF), [Met5]-enkephalin, interacts with the zeta (zeta) opioid receptor to modulate development of eukaryotes. We have found that [Met5]-enkephalin, an endogenous opioid peptide serves to inhibit the growth of S. aureus. This effect on growth involves cell proliferative events and is under tonic control, since potent opioid antagonists accelerate cell replication. Both the OGF and zeta opioid receptor were associated with these microorganisms. Other opioid receptors (mu, delta and kappa) were not detected. OGF also controlled the growth of other bacteria: P. aeruginosa and S. marcesans. These results indicate that OGF and its receptor, known to be important in the regulation of mammalian development, also function in the growth of simple unicellular organisms. We suggest that the endogenous opioid system related to growth originated billions of years ago.


Gestational modulation of myometrial proteins in the timed-pregnant Sprague-Dawley rat.

These studies sought to test the hypothesis that the expression of myometrial proteins is modulated as the onset of parturition approaches. Myometrial proteins from timed-pregnant rats were analyzed utilizing sodium dodecylsulfate polyacrylamide gel and 2-dimensional electrophoresis, and Western blot techniques. SDS-PAGE gels demonstrated increased expression of at least 10 protein bands from 17 to 200+ KD. 2-dimensional gels confirmed the presence of at least five groups of gestationally modulated proteins. Western blots for phosphoinositide-specific phospholipase C demonstrated significant modulation of the expression of three isozymes. These studies have confirmed differential expression of myometrial proteins near term in the timed-pregnant rat; some of which play an important role in intracellular signal transduction in response to hormones and pharmacologic agents.


Mitochondrial DNA phylogeny of the Old-World monkey tribe Papionini.

The evolution of the Old World monkey tribe Papionini, composed of macaques, baboons, mandrills, drills, and mangabeys, was examined using mitochondrial DNA (mtDNA) sequence data on the cytochrome oxidase subunit II gene. When analyzed cladistically, these data support a baboon clade of savannah (Papio) plus gelada (Theropithecus) baboons, as well as a clade containing drill (Mandrillus) plus mangabey (Cerocebus) genera. This result stands in opposition to most morphological phylogenies, which break up the baboon clade by placing Papio and Mandrillus as sister taxa and Theropithecus as a more distantly related lineage. Analyses of COII gene sequences also suggest that the papionin ancestral stock divided into two lineages, one leading to macaques and the other to the purely African genera. From a molecular evolutionary perspective, the papionin COII gene sequences reveal a pattern of amino acid replacements concentrated in the regions spanning the mitochondrial membrane.


SecB-binding does not maintain the translocation-competent state of prePhoE.

The role of SecB protein in the export of the precursor of outer membrane protein PhoE and mutant forms of this precursor was studied in vitro. When synthesized in the absence of SecB, translocation-competent prePhoE was observed post-translationally, but addition of SecB was required for efficient translocation into inner membrane vesicles. The translocation competency of in vitro synthesized prePhoE diminished with a similar half-life during incubations in the presence or absence of SecB. The loss of translocation competency of prePhoE, synthesized in the presence of SecB, was not due to dissociation of prePhoE-SecB complexes as could be demonstrated in co-immunoprecipitation experiments with anti-SecB serum. Apparently, SecB does not maintain the translocation-competent conformation of prePhoE, but is mainly required for efficient targeting of this precursor to the export apparatus.


Cloning, sequencing and deletion from the chromosome of the gene encoding subunit I of the aa3-type cytochrome c oxidase of Rhodobacter sphaeroides.

The ctaD gene encoding subunit I of the aa3-type cytochrome c oxidase from Rhodobacter sphaeroides has been cloned. The gene encodes a polypeptide of 565 residues which is highly homologous to the sequences of subunit I from other prokaryotic and eukaryotic sources, e.g. 51% identity with that from bovine, and 75% identity with that from Paracoccus denitrificans. The ctaD gene was deleted from the chromosome of R. sphaeroides, resulting in a strain that spectroscopically lacks cytochrome a. This strain maintains about 50% of the cytochrome c oxidase activity of the wild-type strain owing to the presence of an alternate o-type cytochrome c oxidase. The aa3-type oxidase was restored by complementing the chromosomal deletion with a plasmid-borne copy of the ctaD gene. This system is well suited for site-directed mutagenesis probing of the structure and function of cytochrome c oxidase.


[Clinical evaluation of the measurement of hepatosplenic volume ratio by computed tomography].

The liver and spleen volume ratio (S/L ratio) was estimated with X-ray computed tomography. Clinical usefulness of S/L ratio was evaluated by comparison with other liver functions (retention rate of ICG, total bilirubin, serum albumin and cholinesterase activity) in 42 hepatocellular carcinoma patients with liver cirrhosis. The correlation between S/L ratio and retention rate of ICG, total bilirubin, serum albumin or cholinesterase activity was good (r = 0.870, r = 0.719, r = -0.691, or r = -0.606, respectively p less than 0.001). Positive correlation was observed between S/L ratio and retention rate of ICG or total bilirubin. Negative correlation was observed between S/L ratio and serum albumin or cholinesterase activity. In conclusion, the measurement of S/L ratio on computed tomography was considered to be useful as an evaluation for the degree of severity in liver cirrhosis by considering both effective hepatic blood flow and portal hypertension.


The promoter region of the Escherichia coli pepD gene: deletion analysis and control by phosphate concentration.

A series of deletions removing progressively larger parts of the 5' flanking region of the Escherichia coli pepD gene was constructed. After fusing the resulting promoter fragments to the chromosomal malPQ operon, their activities were determined by assaying for amylomaltase, the product of the malQ gene. Transcription from the pepD promoter region in exponentially growing cells was estimated to be about 5 times less efficient than transcription from the induced lac promoter. Approximately 115 bp preceding the translation start site of the pepD gene are important for regular promoter functioning, whereas the more distal sequences could be deleted without any significant effects. In bacterial cultures containing limiting amounts of inorganic phosphate, the rate of de novo synthesis of peptidase D, simultaneously with the derepression of alkaline phosphatase, increased about fivefold as a consequence of phosphate starvation. This regulation was shown to occur at the transcriptional level by the use of chromosomal pepD promoter-malPQ fusions. The inducibility by phosphate limitation was conserved in all of the deletion clones in which the pepD promoter region was still functional. As demonstrated by the use of phoB, R, and M mutants, the modulation of pepD expression is independent of the genetic system controlling the pho regulon.


Fot1, a new family of fungal transposable elements.

We report here the discovery of a family of transposable elements, which we refer to as Fot1 elements, in the fungal plant pathogen Fusarium oxysporum. The first element was identified as an insertion in the gene encoding nitrate reductase. It is 1928 bp long, has 44 bp inverted terminal repeats, contains a large open reading frame and is flanked by a 2 bp (TA) target site duplication. This element shares significant structural similarities with a class of transposons that includes Tc1 from Caenorhabditis elegans and therefore represents a new class of transposable elements in fungi.


The pogo transposable element family of Drosophila melanogaster.

A 190 bp insertion is associated with the white-eosin mutation in Drosophila melanogaster. This insertion is a member of a family of transposable elements, pogo elements, which is of the same class as the P and hobo elements of D. melanogaster. Strains typically have many copies of a 190 bp element, 10-15 elements 1.1-1.5 kb in size and several copies of a 2.1 kb element. The smaller elements all appear to be derived from the largest by single internal deletions so that all elements share terminal sequences. They either always insert at the dinucleotide TA and have perfect 21 bp terminal inverse repeats, or have 22 bp inverse repeats and produce no duplication upon insertion. Analysis by DNA blotting of their distribution and occupancy of insertion sites in different strains suggests that they may be less mobile than P or hobo. The DNA sequence of the largest element has two long open reading frames on one strand which are joined by splicing as indicated by cDNA analysis. RNAs of this strand are made, whose sizes are similar to the major size classes of elements. A protein predicted by the DNA sequence has significant homology with a human centrosomal-associated protein, CENP-B. Homologous sequences were not detected in other Drosophila species, suggesting that this transposable element family may be restricted to D. melanogaster.


Tissue-specific position effects on alcohol dehydrogenase expression in Drosophila melanogaster.

Twenty transformed lines have been isolated as a result of the germ line insertion of a 3.2 kb alcohol dehydrogenase (Adh) gene fragment into an Adh negative strain of Drosophila melanogaster by P element-mediated transformation. More than half of these lines exhibited abnormal ADH expression. The level of ADH expression ranges from zero in some lines to near normal levels in others, and the pattern of ADH expression in the larval gut is also abnormal in many of these lines. Each of the abnormal tissue-specific patterns is stable and characterized by the absence or reduction of ADH expression in certain tissues. High levels of ectopic expression were not observed. In two of these lines, the pattern of ADH staining is highly restricted: it is limited to the medial midgut in line MM-50, and to the gastric caecae and the proventriculus in line GC-1. In heterozygotes between these two lines ADH is expressed in both of these tissues. To test the hypothesis that this abnormal expression is due to position effects, inserts were mobilized to new locations. The mobilized inserts exhibited new patterns of tissue-specific expression associated with new cytological insert locations, showing that the abnormal expression in lines MM-50 and GC-1 is due to tissue-specific position effects and not to mutations. The results are discussed in the context of chromatin structure as a possible cause of these position effects.


Sensitivity to plating of Escherichia coli cells expressing the cryA gene from Bacillus thuringiensis var. israelensis.

The gene (cytA) coding for the 27 kDa polypeptide of the Bacillus thuringiensis var. israelensis mosquito larvicidal delta-endotoxin, was cloned into a plasmid containing the T7 bacteriophage promoter. The plasmid was used to transform an Escherichia coli strain containing the T7 RNA polymerase gene 1, under the control of lacP. Loss of colony-forming ability without substantial lysis, associated with immediate inhibition of DNA synthesis, was observed after induction of transformed cells. The cytA gene product may kill E. coli cells by disrupting their chromosome replicating apparatus.


Extra sequences found at P element excision sites in Drosophila melanogaster.

We have previously established a transgenic Drosophila line with a highly transposable P element insertion. Using this strain we analyzed transposition and excision of the P element at the molecular level. We examined sequences flanking the new insertion sites and those of the remnants after excision. Our results on mobilization of the P element demonstrate that target-site duplication at the original insertion site does not play a role in forward excision and transposition. After P element excision an 8 bp target-site duplication and part of the 31 bp terminal inverted repeat (5-18 bp) remained in all the strains examined. Moreover, in 11 out of 28 strains, extra sequences were found between the two remaining inverted repeats. The double-strand gap repair model does not explain the origin of these extra sequences. The mechanism creating them may be similar to the hairpin model proposed for the transposon Tam in Antirrhinum majus.


Reconstitution of an episomal mouse aprt gene as a consequence of recombination.

When a functional murine adenine phosphoribosyltransferase (aprt) gene linked to bovine papilloma virus (BPV) DNA is transfected into Aprt- L cells, the cells are rendered Aprt+ and the aprt gene persists as an episome. Cotransfection with two BPV vectors, one containing the 5' half of the aprt gene and the other the 3' half of the gene, that share about 300 bp of common sequence in intron 2, produces Aprt+ cells with functional aprt as an episome. Southern blot analysis of low molecular weight DNA derived from Hirt extracts revealed the regeneration of a diagnostic SmaI fragment, consistent with establishment of an episome with functional aprt that was reconstituted as a consequence of recombination. To establish cells with an episomal target for recombination, BPV vectors containing a G418 resistance marker and either the 5' half or 3' half of aprt were transfected into Aprt- L cells. Stably transfected cells, selected by their growth in G418, were in turn transfected with DNA containing the other half of the aprt gene. Following selection of Aprt+ cells, Southern blot and polymerase chain reaction (PCR) analysis of low molecular weight DNA confirmed the presence of a complete episomal aprt gene. The region of DNA shared by the episomal aprt fragment and the transfected aprt half was sequenced after PCR amplification of the reconstituted, episomal gene and was found to be wild type. The region of overlap that serves as the substrate for recombination lies entirely within an intron and can, therefore, tolerate nucleotide substitutions and deletions. The absence of such errors in the sequences examined is consistent with recombination events that are not error prone.


New mammalian chloride channel identified by expression cloning.

Ion channels selectively permeable to chloride ions regulate cell functions as diverse as excitability and control of cell volume. Using expression cloning techniques, a complementary DNA from an epithelial cell line has been isolated, sequenced and its putative structure examined by site-directed mutagenesis. This cDNA, encoding a 235-amino-acid protein, gave rise to a chloride-selective outward current when expressed in Xenopus oocytes. The expressed, outwardly rectifying chloride current was calcium-insensitive and was blocked by nucleotides applied to the cell surface. Mutation of a putative nucleotide-binding site resulted in loss of nucleotide block but incurred dependence on extracellular calcium concentration. The unusual sequence of this putative channel protein suggests a new class of ion channels not related to other previously cloned chloride channels.


Glycosyl-phosphatidylinositol linkage as a mechanism for cell-surface expression of immunoglobulin D.

The B-cell antigen receptor of the IgM and IgD class is a multimeric complex consisting of the membrane-bound form of the immunoglobulin molecule and two other proteins, Ig-alpha and Ig-beta. The Ig-alpha and Ig-beta proteins form a disulphide-linked alpha/beta heterodimer and are encoded by the mb-1 (ref 9, 10) and B29 genes, respectively. Surface expression of the membrane-bound IgM molecule requires assembly with the alpha/beta heterodimer. The IgD molecule, however, can be expressed on the cell surface in an alpha/beta-dependent and -independent form. We show here that in the alpha/beta-independent form the IgD molecule is anchored in the plasma membrane through a glycosyl-phosphatidylinositol linker. In the presence of the alpha/beta heterodimer, most of the otherwise glycosyl-phosphatidylinositol-linked IgD molecule is expressed on the cell surface as transmembrane proteins.


Cardioviral internal ribosomal entry site is functional in a genetically engineered dicistronic poliovirus.

High mutation rates have driven RNA viruses to shorten their genomes to the minimum possible size. Mammalian (+)-strand RNA viruses and retroviruses have responded by reducing the number of cis-acting regulatory elements, a constraint that has led to the emergence of the polyprotein. Poliovirus is a (+)-stranded picornavirus whose polyprotein, encoded by an open reading frame spanning most of the viral RNA, is processed by virus-encoded proteinases. Despite their genetic austerity, picornaviruses have retained long 5' untranslated regions, which harbour cis-acting elements that promote initiation of translation independently of the uncapped 5' end of the viral messenger RNA. These elements are termed 'internal ribosomal entry sites' and are formed from highly structured RNA segments of at least 400 nucleotides. How these elements function is not known, but special RNA-binding proteins may be involved. The ribosome or its 40S subunit probably binds at or near a YnXmAUG motif (where Y is a pyrimidine and X is a purine) at the 3' border of the internal ribosomal entry site, which either provides the initiating codon or enables the ribosome to translocate to one downstream (E.W. et al., submitted). Initiation from most eukaryotic messenger RNAs usually occurs by ribosomal recognition of the 5' and subsequent scanning to the AUG codon. Here we describe a genetic strategy for the dissection of polyproteins which proves that an internal ribosomal entry site element can initiate translation independently of the 5' end.


Hepatitis C virus in chronic hemodialysis patients with non-A, non-B hepatitis.

Sixteen of 110 hemodialysis (HD) patients fulfilling criteria of non-A, non B hepatitis (NANBH), i.e. alanine aminotransferase (ALT) greater than 50 U/ml in the absence of both serologic markers for acute HBV and HAV infections and clinical evidence of another cause of hepatitis, were tested for the presence of antibodies against hepatitis C virus (anti-HCV) by enzyme immunoassay (Ortho, Diagnostics). All (100%) were anti-HCV-positive. There were 5 patients with a monophasic (M) rise pattern (1 or 2 ALT rises), and 11 cases demonstrated a polyphasic (P) rise elevation pattern (more than 2). The mean ALT value of the M group was 202.3 +/- 209 U/ml and that of the P group was 116.6 +/- 39.1 U/ml. The patients received a mean of 19.1 +/- 16.2 units of packed red cells during the follow-up period (69.9 months). Only 1 patient received no blood transfusion. Six patients had a past HBV infection and 3 became HIV-infected in the course of this study. The high rate of infection of hemodialysis patients with hepatitis C virus in our setting points to the need for improved control measures.


The effect of benzodiazepines on the binding of [3H]SCH 23390 in vivo.

The effect of flunitrazepam upon the binding of [3H]SCH 23390 in vivo was investigated. Acute treatment with flunitrazepam decreased the binding of [3H]SCH 23390 in the striatum in a dose-dependent manner. The time course of radioactivity in the striatum, cerebral cortex and cerebellum in controls and flunitrazepam (1 mg/kg)-treated mice was measured after intravenous injection of [3H]SCH 23390. The binding kinetics were calculated, using the cerebellum as a reference region for the estimation of the amount of free ligand in the brain. Flunitrazepam significantly decreased the input rate constant to the receptor compartment and the dissociation rate constant in vivo. An in vivo displacement study, using carrier SCH 23390, also showed significant reduction in the dissociation rate constant of [3H]SCH 23390 in vivo. The drug Ro 15-1788 reversed the effect of flunitrazepam, suggesting that this reduction in binding of [3H]SCH 23390 was mediated by benzodiazepine receptors. To evaluate the relationship between the reduction in binding of [3H]SCH 23390 in vivo and in vivo occupancy of benzodiazepine receptors, in vivo occupancy of benzodiazepine receptors was measured using [3H]Ro 15-1788. A non-linear relationship was found between the reduction in dopamine D1 receptor binding in vivo and the occupancy of benzodiazepine receptors in vivo, indicating that benzodiazepines exerted the maximum change in dopamine receptor binding at a low fractional occupancy of receptors.


Down-regulation of central receptors for thyrotropin-releasing hormone in kappa opiate agonist-induced abstinence in the rat.

The effect of U-50,488H, a selective kappa opiate agonist, on tolerance-dependence and abstinence on the TRH receptors of the spinal cord and discrete regions of the brain of male Sprague-Dawley rats was determined. Rats were injected intraperitoneally twice daily with 25 mg/kg of U-50,488H for 4 days. Rats serving as controls were injected with the vehicle. On day 5, rats which were labeled as tolerant to U-50,488H were injected with U-50,488H (25 mg/kg) and sacrificed 1 hr later, whereas those labeled as abstinent were sacrificed without any injection. The above procedure has been previously shown to produce a high degree of tolerance to the analgesic and hypothermic effects of U-50,488H. The spinal cord and regions of the brain (hippocampus, cortex, midbrain, hypothalamus, corpus striatum, pons and medulla, and amygdala) were isolated for binding studies. The ligand [3H]MeTRH was used for TRH receptors. The binding constants, Bmax and Kd values, of [3H]MeTRH to bind to membranes prepared from various regions of the brain and spinal cord of rats tolerant-dependent on U-50,488H were unaffected. However, in rats abstinent to U-50,488H, the binding of [3H]MeTRH to membranes of the hypothalamus, and pons and medulla, was decreased. The decreased binding of [3H]MeTRH to hypothalamic membranes was due to changes in Bmax value, while in pons and medulla it was due to an increase in the Kd value.(ABSTRACT TRUNCATED AT 250 WORDS)


Effects of prolonged administration of milnacipran, a new antidepressant, on receptors and monoamine uptake in the brain of the rat.

Most antidepressants produce changes in monoamine receptors in brain after chronic administration in animals. The most commonly described alterations are a decreased density and function of beta-adrenergic receptors and have been postulated to be the mechanisms by which antidepressants exert their therapeutic effect. Milnacipran (previous name midalcipran) is a new, clinically-effective antidepressant, which inhibits the uptake of both serotonin and noradrenaline but has no affinity for any pre- or postsynaptic receptor studied. When given either orally at 7.5 mg/kg twice daily for 3 days, at 30 mg/kg once daily for 3 weeks, by osmotic mini-pump at 30 mg/kg/day for 27 days, or in drinking water at approximately 15 mg/kg/day for 6 weeks and after a washout period of 24 hr, milnacipran produced no down-regulation of beta-adrenoceptors. In addition, there were no alterations of alpha 1- or alpha 2-adrenoceptors, 5-HT1, 5-HT2 receptors or benzodiazepine binding sites. Moreover, uptake and accumulation of serotonin and noradrenaline were unmodified. In addition, the potency for milnacipran to inhibit monoamine uptake in vitro in the cortex was not altered in treated rats, compared to control animals. Thus, in spite of its action on both the uptake of serotonin and noradrenaline, milnacipran appears to be without long-term action on beta-adrenoceptors or the other receptors studied, suggesting that, at least for this antidepressant, these modifications are not essential for clinical activity.


Prior chronic exposure to cocaine inhibits the serotonergic stimulation of ACTH and secretion of corticosterone.

The effect of long-term pretreatment with cocaine on serotonergic regulation of ACTH (adrenocorticotropic hormone; corticotropin) and secretion of corticosterone in rats was investigated. The following observations were made: (1) Pretreatment with cocaine had no significant effect on basal levels of ACTH and corticosterone in plasma. However, cocaine caused a reduction in the ability of the 5-HT (5-hydroxytryptamine, serotonin) releaser p-chloroamphetamine (PCA) to increase corticosterone in plasma, 42 hr after the last injection of cocaine. (2) Exposure to cocaine for 7 days was sufficient to produce a maximal inhibition of the PCA-induced increase in ACTH in plasma. (3) The inhibitory effect of cocaine on PCA-induced release of ACTH was more marked than on corticosterone. (4) Conversely, the dose-dependent stimulatory effect of two 5-HT1 agonists, RU 24969 (5-methoxy-3-(1,2,3,4-tetrahydro-4-pyridinyl)-1H-indole) and m-CPP (m-chlorophenylpiperazine), on ACTH and corticosterone was not reduced by 7 days of exposure to cocaine. Taken together, these findings indicate that pretreatment with cocaine reduced the function of serotonergic nerve-terminals but not postsynaptic receptors, that stimulate ACTH and secretion of corticosterone.


Peripheral cerebral aneurysm associated with a glioma.

This 42-year-old man with acute subarachnoid, intraventricular and parenchymal hemorrhage from an angiographically and surgically confirmed successfully clipped, right splenial artery aneurysm, subsequently manifested a grade 3 astrocytoma at the site of the aneurysm. Intracranial aneurysms are recognized in association with metastases from cardiac myxoma and choriocarcinoma, but are rarely seen with primary brain tumors. In patients with nontraumatic peripheral aneurysms it would seem prudent to biopsy the aneurysm and/or surrounding necrotic tissue at the time of surgical clipping.


Selective intra-arterial chemotherapy with BCNU in recurrent malignant gliomas.

Study of intra-arterial BCNU in 12 patients with recurrent glioblastoma multiforme and 5 patients with recurrent anaplastic astrocytoma already treated by surgery and radiotherapy indicates that the risk of BCNU toxicity can be reduced by the use of relatively low doses (160-180 mg/m2) of BCNU, the use of dextrose as solvent, selective injections of BCNU, and fast "bolus" injections of drug. No certain toxic effects of BCNU were observed in the 17 patients treated this way. Anaplastic astrocytomas, particularly in young patients, seem to respond relatively well to this treatment.


[Prognostic implications of the proliferation index (H3-TdR-labeling index) and ploidy in neuroectodermal tumors (astrocytomas and glioblastomas)].

Kinetic cellular methods were used in 53 cases of various sizes of astrocytomas and glioblastomas to obtain reliable prognostic information. Kinetic analysis revealed a wide variation of proliferative activity, with a gradual increase of the mean value from the most differentiated astrocytomas to glioblastomas. DNA content was significantly correlated with the degree of histological differentiation. Chromosomic patterns, which were detected in a few cases, were generally aspecific. The sole recurrent alteration in the karyotype was a small metacentric marker. The comparison of results obtained with the follow-up of patients showed that the labeling index offers a more accurate estimate of the proliferative potential of individual tumours, and thus of the probability of survival, than histological diagnosis and the evaluation of other biological parameters such as ploidy and karyotype.


[Chronic use of glucomannan in the dietary treatment of severe obesity].

Two groups of 25 severely obese patients underwent 3 months of hypocaloric diet therapy either alone or associated with a glucomannan-based fibrous diet supplement (approx. 4 g/die in 3 doses). The comparative analysis of the results obtained in both groups showed that the diet + glucomannan group had a more significant weight loss in relation to the fatty mass alone, an overall improvement in lipid status and carbohydrate tolerance, and a greater adherence to the diet in the absence of any relevant side effects. Due to the marked ability to satiate patients and the positive metabolic effects, glucomannan diet supplements have been found to be particularly efficacious and well tolerated even in the long-term treatment of severe obesity.


Delta 9-tetrahydrocannabinol discrimination in rats as a model for cannabis intoxication.

Studies of the discriminative stimulus effects of delta 9-THC are reviewed. The results of generalization studies in rats trained to discriminate delta 9-THC from nondrug provide evidence for the pharmacological specificity of the delta 9-THC stimulus. Only cannabinoid analogs of delta 9-THC substitute fully for delta 9-THC. The ability of cannabinoids to produce delta 9-THC-like discriminative stimulus effects appears to predict delta 9-THC- or marihuana-like effects in humans. Of the 11 cannabinoids tested in rats and humans, the results with 9 are in complete concordance, and results with the remaining two are inconclusive. This concordance provides evidence for the validity of delta 9-THC discrimination in rats as an animal model of cannabis intoxication. A number of natural and synthetic cannabinoids have delta 9-THC-like discriminative stimulus effects. They represent a 5,600-fold range of relative potencies. Micromolar potency estimates and relative potencies to delta 9-THC for these compounds are provided. Correlations between these values and potencies for cellular actions of cannabinoids can be used to establish the neural substrates of cannabis intoxication.


Enoxaparin prophylaxis for deep vein thrombosis in orthopaedic surgery.

Seventeen patients undergoing elective total hip or knee joint replacement were given enoxaparin prophylaxis against deep vein thrombosis. Four patients developed clinical evidence of deep vein thrombosis, confirmed by venography. Two patients developed disseminated intravascular coagulation.


Differential short-term desensitization to vasopressin, isoproterenol, glucagon, parathyroid hormone and calcitonin in the thick ascending limb of rat kidney.

Short-term desensitization to hormone-induced cAMP accumulation was investigated in the medullary (MTAL) and the cortical (CTAL) thick ascending limbs of Henle's loop isolated by microdissection from the rat kidney. The following agonists were studied: vasopressin, glucagon and human calcitonin in the MTAL, and vasopressin, glucagon, human calcitonin, parathyroid hormone (PTH) and the beta-adrenergic agonist isoproterenol in the CTAL. Isolated tubules were preincubated in vitro for 60 min in the presence or absence of a maximal concentration of one of the five agonists (vasopressin 10 nM, glucagon 10 nM, calcitonin 100 nM, PTH 10 nM, isoproterenol 1 microM). Desensitization induced by each agent to its own action was then quantified by measuring the amount of cAMP accumulating in the presence of the phosphodiesterase inhibitor isobutylmethylxanthine and the same agonist concentration as that used during preincubation. In the MTAL, as previously reported, preincubation with vasopressin led to a marked (80%-85%) desensitization to this hormone. A significant hormone self-induced desensitization of about 45% was also obtained with glucagon, but not with calcitonin. In the CTAL, the following order of potency to elicit desensitization was observed: vasopressin (80%) greater than isoproterenol (50%) greater than glucagon (30%) greater than PTH (20%, NS) greater than calcitonin (10%, NS). Thus, the magnitude of desensitization varied greatly from one hormone to another, but for a given hormone, was of roughly similar extent in both MTAL and CTAL.(ABSTRACT TRUNCATED AT 250 WORDS)


Cyclic AMP-dependent phosphorylation modifies the gating properties of L-type Ca2+ channels in bovine adrenal chromaffin cells.

We investigated the effects of cAMP-dependent phosphorylation on the voltage- and time-dependent gating properties of Ca2+ channel currents recorded from bovine adrenal chromaffin cells under whole-cell voltage clamp. Extracellular perfusion with the membrane-permeant activator of cAMP-dependent protein kinase, 8-bromo(8-Br)-cAMP (1 mM), caused a 49%, 29%, and 21% increase in Ca2+ current (ICa) amplitudes evoked by voltage steps to 0, +10, and +20 mV respectively (mean values from eight cells, p less than or equal to 0.05). Analysis of voltage-dependent steady-state activation (m infinity) curves revealed a 0.70 +/- 0.27 charge increase in the activation-gate valency (zm) following 8-Br-cAMP perfusion. Similar responses were observed when Ba2+ was the charge carrier, where zm was increased by 1.33 +/- 0.34 charges (n = 8). The membrane potential for half activation (V1/2) was also significantly shifted 6 mV more negative for IBa (mean, n = 8). The time course for IBa (and ICa) activation was well described by second-order m2 kinetics. The derived time constant for activation (tau m) was voltage-dependent, and the tau m/V relation shifted negatively after 8-Br-cAMP treatment. Ca2+ channel gating rates were derived from the tau m and m infinity 2 values according to a Hodgkin-Huxley type m2 activation process. The forward rate (alpha m) for channel activation was increased by 8-Br-cAMP at membrane potentials greater than or equal to 0 mV, and the backward rate (beta m) decreased at potentials less than or equal to + 10 mV. Time-dependent inactivation of ICa consisted of a slowly decaying component (tau h approximately 300 ms) and a "non-inactivating" steady-state component. The currents contributed by the two inactivation processes displayed different voltage dependences, the effects of 8-Br-cAMP being exclusively on the slowly inactivating L-type component.


Inhibition of L-type calcium channels by internal GTP [gamma S] in mouse pancreatic beta cells.

Pretreatment of pancreatic beta cells with pertussis toxin resulted in a 30% increase in peak whole-cell Ca2+ currents recorded in the absence of exogenous intracellular guanine nucleotides. Intracellular application of 90 microM GTP[gamma S], by liberation from a caged precursor, resulted in 40% reduction of the peak Ca2+ current irrespective of whether the current was carried by Ca2+ or Ba2+. Effects on the delayed outward K+ current were small and restricted to a transient Ca(2+)-dependent K+ current component. Inhibition by GTP[gamma S] of the Ca2+ current was not mimicked by standard GTP and could not be prevented either by pretreatment with pertussis toxin or by inclusion of GDP[beta S] or cyclic AMP in the intracellular medium. The inhibitory effect of GTP[gamma S] could be counteracted by a prepulse to a large depolarizing voltage. A similar effect of a depolarizing prepulse was observed in control cells with no exogenous guanine nucleotides. These observations indicate that inhibition of beta cell Ca2+ current by G protein activation results from direct interaction with the channel and does not involve second-messenger systems. Our findings also suggest that the beta cell Ca2+ current is subject to resting inhibition by G proteins.


Proton transport mechanism in the cell membrane of Xenopus laevis oocytes.

Mechanisms of H+ transport across the plasma cell membrane of prophase-arrested oocytes of Xenopus laevis were investigated by testing the effect of ion substitutions and inhibitors on cytoplasmic pH (pHi), membrane potential (Vm) and membrane resistance (Rm). During superfusion with control solution of pH = 7.4, pHi was 7.49 +/- 0.12 (n = 15), Vm was -61.9 +/- 7.8 mV (n = 34) (cytoplasm negative), and Rm was 2.9 +/- 1.5 M omega (n = 19). These data confirm that H+ ions are not distributed at electrochemical equilibrium. By following pHi during recovery of the oocytes from an acid load (20 mmol/l NH4Cl) in the presence and absence of extracellular Na+ or amiloride (1 mmol/l), a Na/H exchanger was identified. On the basis of the known Na+ gradient across the cell membrane, this transporter could suffice to generate the observed H+ disequilibrium distribution. Utilizing blockers or ion-concentration-step experiments no evidence was obtained for an ATP-driven H+ pump or for passive acid/base transporters such as H+ conductances or Na+ (HCO3-)3 cotransport. The membrane depolarization observed in response to extracellular acidification appeared to result from a pH-dependent, Ba(2+)-inhibitable K+ conductance.


High-resolution measurements of gap-junctional conductance during perfusion with anti-connexin antibodies in pairs of cultured mammalian cells.

Antibodies against the main proteins in hepatic gap junctions--connexin-26 and connexin-32--have been used in conjunction with high-resolution patch-clamp techniques to investigate whether a relation exists between connexin type and conductance of single gap-junctional channels. Two different cell lines, BRL cells, derived from rat liver, and FL cells a human amniotic cell line exhibited the same single-channel conductances in double whole-cell recordings, but reacted differently upon dialysis with antibodies. Preliminary results indicate that both cell lines express mainly connexin-43. Thus, in spite of the inhibitory action of anti-cx26 and anti-cx32 antibodies observed, the data question the reliability of these antibodies for the functional characterization of gap-junction proteins in electrophysiological experiments.


Effects of serotonin and cAMP on calcium currents in different neurones of Helix pomatia.

Effects of application of serotonin (5-HT) and intracellular administration of cyclic adenosine monophosphate (cAMP) on voltage-gated calcium current (ICa) were studied in isolated, intracellularly perfused Helix pomatia neurones. Two types of the effects of 5-HT (1-10 microM) were observed in different neurones: reversible inhibition (by about 20%) or reversible potentiation (up to 50%) of the current amplitude. Some cells did not respond to 5-HT application. In cells with the potentiating effect of 5-HT, ICa could also be increased by intracellular introduction of cAMP (100 microM), but not the guanosine analogue, cGMP (50-100 microM). These effects were not additive and could be potentiated by theophylline (5 mM) and 3-isobutyl-1-methylxanthine (IBMX, 100-500 microM); they could be mimicked by forskolin (10-50 microM) and abolished by tolbutamide (1-5 mM) or protein kinase inhibitor (500 micrograms/ml), indicating that cAMP-dependent phosphorylation mediates the potentiating action of 5-HT on ICa. In neurones showing inhibitory effect of 5-HT, neither cAMP nor forskolin increased ICa. Methiothepin (10-50 microM), a 5-HT1,2 receptor antagonist, irreversibly inhibited the potentiating effect of 5-HT, while antagonists of 5-HT2 receptors cyproheptadine (10-50 microM) or ketanserine (10-60 microM) and of 5-HT3 receptors ISC 205-930 (10-50 microM) or cocaine (5-25 microM) had no effect on ICa and its enhancement by 5-HT. It is suggested that in certain snail neurones the possibility of cAMP-dependent up-regulation of ICa correlates with the presence of 5-HT1-like receptors in the neuronal membrane.


Pharmacokinetic evaluation of drug interactions with anti-human immunotrophic virus (HIV) Drugs. III. 2',3'-Dideoxycytidine (ddC) and zidovudine in monkeys.

A pharmacokinetic evaluation of a potential drug interaction between zidovudine (AZT) and dideoxycytidine (ddC) was conducted in monkeys. Each of six animals received 20 mg/kg of AZT intragastrically in the absence and presence of an intravenous steady-state dosage regimen of ddC. The regimen was designed to produce steady-state ddC plasma concentration of 1.77 micrograms/ml for 30 min. Plasma and urine samples were analyzed for AZT, its major glucuronide metabolite, GAZT, and ddC by HPLC techniques. Pharmacokinetic parameters for AZT and GAZT were calculated by non-compartmental methods. The mean apparent clearance of AZT was 1.40 and 1.78 L/hr/kg in the absence and presence of ddC, respectively. The mean AUC for GAZT was 36.39 micrograms-hr/ml in the absence of ddC and 28.81 micrograms-hr/ml in the presence of ddC. No statistical differences were found in these and other pharmacokinetic parameters in the absence and presence of ddC. The absence of an effect on AZT's pharmacokinetics by ddC is attributed to the primary metabolic and renal elimination pathways for AZT and ddC, respectively. The results of this study provide a rational basis to design combined AZT-ddC treatment regimens in AIDS patients.


Pancreatic exocrine carcinoma producing adrenocorticotropic hormone.

A rare case of pancreatic exocrine carcinoma with an endocrine component secreting adrenocorticotropic hormone (ACTH) is reported and discussed in relation to other cases previously published. The patient initially presented with a severe form of diabetes, which was treated accordingly. Persistence of hyperglycemia, along with other metabolic alterations and marked hypokalemia, led to the suggestion of abnormal ACTH secretion. In this patient, however, a florid Cushing's syndrome was not observed. The patient also developed hematological alterations, mainly leukopenia and thrombocytopenia, whose origins were unclear. At autopsy, a poorly-defined mass was discovered between the body and tail of the pancreas. Standard histology showed a moderately-differentiated adenocarcinoma. Immunohistochemical analysis of the tumor specimen demonstrated the presence of some neoplastic cells immunoreactive for chromogranin A, neuron-specific enolase and ACTH. These findings are consistent with the existence of an endocrine component within the exocrine carcinoma with ACTH differentiation.


CLIX: a search algorithm for finding novel ligands capable of binding proteins of known three-dimensional structure.

A computer algorithm, CLIX, capable of searching a crystallographic data-base of small molecules for candidates which have both steric and chemical likelihood of binding a protein of known three-dimensional structure is presented. The algorithm is a significant advance over previous strategies which consider solely steric or chemical requirements for binding. The algorithm is shown to be capable of predicting the correct binding geometry of sialic acid to a mutant influenza-virus hemagglutinin and of proposing a number of potential new ligands to this protein.


Metal ion stabilization of the conformation of a recombinant 19-kDa catalytic fragment of human fibroblast collagenase.

A recombinant 19-kDa human fibroblast collagenase catalytic fragment modeled on a naturally occurring proteolytic product was purified from E. coli inclusion bodies. Following renaturation in the presence of zinc and calcium, the fragment demonstrated catalytic activity with the same primary sequence specificity against small synthetic substrates as the full-length collagenase. Unlike the parent enzyme, it rapidly cleaved casein and gelatin but not native type I collagen. Intrinsic fluorescence of the three tryptophan residues was used to monitor the conformational state of the enzyme, which underwent a 24-nm red shift in emission upon denaturation accompanied by quenching of the fluorescence and loss of catalytic activity. Low concentrations of denaturant unfolded the fragment while the full-length enzyme displayed a shallow extended denaturation curve. Calcium remarkably stabilized the 19-kDa fragment, zinc less so, while together they were synergistically stabilizing. Among divalent cations, calcium was the most effective stabilizer, EC50 approximately 60 microM, and similar amounts were required for substrate hydrolysis. Catalytic activity was more sensitive to denaturation than was tryptophan fluorescence. Least sensitive was the polypeptide backbone secondary structure assessed by CD. These observations suggest that the folding of the 19-kDa collagenase fragment is a multistep process stabilized by calcium.


3H-norepinephrine release in caudal artery of spontaneously hypertensive and Wistar-Kyoto rats: effects of altered salt diets.

The potassium-induced release of 3H-norepinephrine (3H-NE) was examined in the rat caudal artery. A simple and reliable method was developed to examine stimulated neurotransmitter release from this highly innervated resistance vessel. The potassium-induced release of 3H-NE was dose- and calcium-dependent. Stimulated 3H-NE release was elevated in the spontaneously hypertensive (SHR) compared to age-matched Wistar-Kyoto (WKY) animals (p less than 0.05). However, in animals at 5-6 weeks of age, where the blood pressure was not different between the SHR and WKY, the stimulation-induced release of 3H-NE was also not significantly different. Low salt diets for 7 days significantly decreased the potassium-induced release of 3H-NE from both 10- to 11-week-old SHR and WKY animals. High salt diets did not significantly effect 3H-NE release, even though the high salt diets for 7 days significantly increased the blood pressure in the SHR. The results suggest that the caudal artery is a good model to study stimulation-induced transmitter release and that elevated 3H-NE release plays a role in the development of hypertension in the SHR. Low salt diets, which did not lower blood pressure, were shown to decrease 3H-NE release. High salt diets elevated the blood pressure in the SHR but had no significant effect on stimulated 3H-NE release.


Failure of adrenocorticotrophic hormone to release serum amyloid A in cattle.

The objective of this study was to determine whether adrenocorticotrophic hormone plays a direct role in the secretion of serum amyloid A (SAA) in cattle. Seven lactating Holstein-Friesian cows were given either saline or 25 iu adrenocorticotrophic hormone (ACTH) intravenously at 09.00 in a two factor crossover design. Plasma cortisol concentrations had increased significantly by one hour after the injection of ACTH, whereas the SAA level had not increased by 24 hours after injection. It is concluded that in cattle adrenocorticotrophic hormone has no direct influence on the release of SAA by the hepatocyte.


Detection of TNF alpha receptors on ovine leucocytes by flow cytofluorimetry.

Tumour necrosis factor receptor activity was detected on sheep leucocytes using flow cytofluorimetry and commercially available fluorescent human tumour necrosis factor. The density of receptors per cell and the proportion of cells expressing TNF receptor activity was found to be in the following rank order: monocytes greater than granulocytes greater than lymphocytes.


Dietary fibre in the diet of the growing pig: effects on apparent digestibility and resultant implications for its use in reducing the incidence of oesophagogastric parakeratosis.

The effects on apparent digestibility of increasing dietary fibre concentrations in barley-based diets by substituting for barley either bran, oatmeal by-product, guar gum or pectin were studied using 72 pigs. Increasing substitutions of bran up to a maximum of 300 g kg-1 gave progressive decreases in apparent digestibility of dry matter, ether extract and gross energy fractions and in digestible and metabolisable energy contents, progressive increases in apparent digestibility of modified acid detergent fibre but had no consistent effect on either nitrogen or extract free of nitrogen fractions. Increasing substitution of oatmeal by-product up to the same maximum gave significant linear decreases in apparent digestibility of dry matter, extract free of nitrogen and gross energy, significant depressions in apparent digestibility of modified acid detergent fibre and significant increases in ether extract. Guar gum and pectin, both substituted to a maximum of 50 g kg-1 had no significant effects on apparent digestibility. The results are discussed in the context of the concentrations of dietary fibre needed to reduce the incidence of oesophagogastric parakeratosis.


Effects of vagotomy on ventilatory responses to CO2 in alligators.

Reptiles increase ventilation during hypercapnia at a constant temperature. In this study, the contributions of vagal vs non-vagal receptors to CO2 ventilatory responses were investigated in 16 sedated Alligator mississippiensis (25 mg/kg pentobarbital; 3 days prior to data collection). Four animals served as controls to assess the effects of time and/or anesthetic drift on ventilation and blood gases; significant ventilatory drift was not detected during the observation period. The effects of bilateral vagotomy on CO2 ventilatory responses were determined during spontaneous breathing (n = 6) and unidirectional ventilation (UDV; n = 6) at two body temperatures (Tb = 30 and 20 degrees C). Resting PaCO2, minute ventilation (VI), tidal volume (VT) and breathing frequency (f) were elevated at 30 degrees C relative to 20 degrees C in spontaneously breathing alligators. Increasing inspired CO2 to 5% increased PaCO2, f, VT and VI at both levels of Tb. Ventilatory sensitivity to CO2 (S = delta VI/delta PaCO2) was higher at 30 degrees C with a temperature coefficient (Q10) of 2.3. Vagotomy increased PaCO2 and VT, decreased f and had no effect on VI at either Tb. After vagotomy, hypercapnia had no effects on ventilation. When CO2 feedback loops were opened by UDV at a high flow rate (greater than 2 L/min), Tb had no effects on ventilatory efforts at constant PCO2, but hypercapnia significantly increased f, VT and VI. S was variable with a Q10 of 2.1. After vagotomy, a significant CO2-ventilatory response remained during UDV, but S was unaffected by Tb (Q10 = 0.8). The results indicate that non-vagal chemoreceptors contribute to CO2 ventilatory responses in alligators, although their contribution following vagotomy is evident only during unidirectional ventilation. Although tentative, the data also suggest that CO2-sensitive vagal receptors may be necessary for the temperature dependency of S.


Primary liver cancer and occupational exposure.

In a dynamic population-based case-referent study on primary liver cancer and occupational exposure, the work histories of 344 cases and 861 referents, derived from the follow-up of the whole Finnish population in 1976-1978 and 1981, were analyzed by industry, occupation, and agent. After adjustment for alcohol consumption, elevated odds ratios were found for the categories other agricultural workers (mainly milkmaids), clerical workers, persons exposed to welding fumes and those exposed to other inorganic dusts (mainly silica). One possible explanation for the excess among milkmaids was exposure to dust from cattle feed contaminated with aflatoxins. The excess among clerical workers was probably due to nonoccupational factors or chance because occupational exposure was generally rare. The excesses for welding fumes and inorganic dusts, although compatible with occupational etiology, contradict the results of many previous studies carried out among workers exposed to silica dust and welders.


Requirements for phosphorylation of MAP kinase during meiosis in Xenopus oocytes.

Mitogen-activated protein (MAP) kinases are activated in response to a variety of extracellular stimuli by phosphorylation on tyrosine and threonine residues. Xp42 is a Xenopus laevis MAP kinase that is activated during oocyte maturation. Modified forms of Xp42 that lacked enzymatic activity or either of the phosphorylation sites were expressed in Xenopus oocytes. When meiotic maturation was induced with progesterone, each mutant Xp42 was phosphorylated, indicating that at least one kinase was activated that can phosphorylate Xp42 on tyrosine and threonine. Phosphorylation of one residue is not strictly dependent on phosphorylation of the other.


Intercellular communication and cell-cell adhesion.

In developmental biology, binary cell-cell interactions often determine the fate of one or both cell partners. The two cells must adhere to one another to allow chemical signals to be transmitted in one or both directions across the regions of cell-cell contact. The molecular mechanisms of cell-cell adhesion and intercellular communication, even if they are mediated by different cell surface components, may be functionally integrated in several different ways. Studies of helper T cells with antigen-presenting B cells in culture have illuminated such binary interactions. The possible application of similar mechanisms to other binary developmental systems is briefly explored.


Cloning of a second type of activin receptor and functional characterization in Xenopus embryos.

A complementary DNA coding for a second type of activin receptor (ActRIIB) has been cloned from Xenopus laevis that fulfills the structural criteria of a transmembrane protein serine kinase. Ectodermal explants from embryos injected with activin receptor RNA show increased sensitivity to activin, as measured by the induction of muscle actin RNA. In addition, injected embryos display developmental defects characterized by inappropriate formation of dorsal mesodermal tissue. These results demonstrate that this receptor is involved in signal transduction and are consistent with the proposed role of activin in the induction and patterning of mesoderm in Xenopus embryos.


Tumor necrosis factor-alpha activates the sphingomyelin signal transduction pathway in a cell-free system.

The mechanism of tumor necrosis factor (TNF)-alpha signaling is unknown. TNF-alpha signaling may involve sphingomyelin hydrolysis to ceramide by a sphingomyelinase and stimulation of a ceramide-activated protein kinase. In a cell-free system, TNF-alpha induced a rapid reduction in membrane sphingomyelin content and a quantitative elevation in ceramide concentrations. Ceramide-activated protein kinase activity also increased. Kinase activation was mimicked by addition of sphingomyelinase but not by phospholipases A2, C, or D. Reconstitution of this cascade in a cell-free system demonstrates tight coupling to the receptor, suggesting this is a signal transduction pathway for TNF-alpha.


Toxicity of treatment of germ cell tumors.

Great progress has been made in the management of testicular cancer in the past 15 years, with the attainment of the expectation of cure for most patients. In all of the efforts to reduce the toxicity of treatment, one governing principle should be retained: we must not reduce the chance of successful treatment in our attempts to avoid toxicity. Continued progress will only be achieved by the meticulous implementation of carefully structured trials with appropriate follow-up schedules. If future studies are effected in an unstructured and uncontrolled fashion, physicians could become the most common cause of death of patients with testicular cancer.


Mechanisms of tumor necrosis factor action.

Tumor necrosis factor (TNF) is able to induce a great diversity of cellular responses via modulating the expression of a number of different genes. The multitude of TNF activities may be explained by both structural and functional heterogeneity in TNF receptors as well as by a diversification of postreceptor signal transduction pathways. Purification of TNF receptors has revealed two major, distinct binding proteins (TR60 and TR80). TR60 seems to be an essential component for TNF signaling; the functional role of TR80 remains to be elucidated. The pathway of postreceptor signal transduction involves phospholipase A2, a phosphatidylcholine-specific phospholipase C, protein kinase C, and other serine/threonine and tyrosine-specific protein kinases with as yet unknown function. At the receiving end of TNF signaling, induction of gene expression is mediated through activation of nuclear transcription factors, such as NFkB, AP-1, IRF-1, and NF-GMa.


The effect of 6-nitro-1,3,8-trichlorodibenzofuran as a partial estrogen in the female rat uterus.

Administration of 6-nitro-1,3,8-trichlorodibenzofuran (6-NCDF) caused a dose- and time-dependent increase in uterine wet weight and cytosolic and nuclear estrogen receptor (ER) and progesterone receptor (PR) levels in immature female Sprague-Dawley rats. These estrogenic effects persisted for up to 96 or 144 hr after initial administration of 6-NCDF and could be observed at a dose as low as 2 mumol/kg. In contrast, 6-NCDF (25 mumol/kg) did not increase rat uterine peroxidase activity or epidermal growth factor (EGF) receptor binding activity. 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD), which exhibits a broad spectrum of antiestrogenic effects in the female rat uterus, inhibited the 17 beta-estradiol-induced increase in uterine wet weights, cytosolic and nuclear ER and PR levels, peroxidase activity, and EGF receptor binding activity. In contrast, 2,3,7,8-TCDD inhibited the uterotropic effects caused by 6-NCDF but did not affect the 6-NCDF-induced uterine ER and PR levels. 6-NCDF is a weak inducer of hepatic microsomal ethoxyresorufin O-deethylase activity and competitively binds to the aryl hydrocarbon (Ah) receptor but not the PR or ER. Thus both 6-NCDF and 2,3,7,8-TCDD, two ligands which bind to the Ah receptor, exhibit both partial estrogenic and antiestrogenic properties and serve as useful models for delineating the complex biochemical interactions between the ER and Ah receptor signal transduction pathways.


[Nerve sparing retroperitoneal lymphadenectomy with intraoperative electrostimulation in patients with nonseminomatous testicular tumors].

Thirteen patients with nonseminomatous testicular germ cell tumours underwent nerve-sparing retroperitoneal lymphadenectomy (RLA) with prospective isolation and preservation of postganglionic sympathetic nerve fibres. In all ten patients with pathological stage I in whom a unilateral nerve-sparing RLA was performed, ejaculation was preserved. In two of three patients with pathological stage IIa who underwent a bilateral RLA with a nerve-sparing technique on one side, ejaculation was also preserved. In nine patients intraoperative electrostimulation of isolated sympathetic nerve fibres was performed employing Brindley's stimulation device. The result was an immediate and reproducible ejaculation in seven patients. Thus electrostimulation of sympathetic nerves may be a useful intraoperative test for the identification of the nerve fibres that need to be protected for ejaculation. Nerve-sparing RLA seems to be superior to modified (unilateral) RLA with regard to preservation of ejaculation.


A simple and rapid technique to process formalin-fixed, paraffin-embedded tissues for the detection of viruses by the polymerase chain reaction.

The use of chelating resin in a simple, rapid and efficient pre-treatment protocol to process formalin-fixed, paraffin-embedded specimens for the polymerase chain reaction (PCR) is described and compared to other pre-treatment techniques. With this modified PCR protocol, a variety of human autopsy and biopsy specimens were investigated for presence of DNA of human papilomaviruses, cytomegalovirus or Epstein-Barr virus. These viruses were detected in a productive or non-/low-productive state. Amplimers generated ranged from 88 to 450 base pairs length. Under the specified technical conditions, a considerable range of DNA molecules can be amplified from paraffin-embedded material, some older than 10 years.


Immunohistochemical localization of platelet-derived growth factor receptors in the porcine uterus during the oestrous cycle and pregnancy.

The distribution of alpha- and beta-type receptors for PDGF was studied immunohistochemically in the uterus of cycling and pregnant pigs with well-documented hormonal status in order to determine a possible steroid hormone-dependence of its expression. Peroxidase-antiperoxidase (PAP) labelling at light microscopical level was done with rabbit polyclonal antisera recognizing either both alpha- and beta-type receptors (PDGFR-1), or selectively beta-type (PDGFR-3). Immunoreactivity to the PDGF-receptors of alpha- and beta-type (PDGFR-1) was present in all endometrial samples examined, being located principally on subepithelial stromal and endothelial cells, the wall of endometrial blood vessels and in the fetal placenta (trophoblast and adjoining blood capillaries). In the myometrium, the major reactive cells were the non-vascular smooth muscle cells, and to some extent, also those of the tunica media of major uterine vessels. No dramatic variations in the intensity of the reaction were noticed during the oestrous cycle. A very intense immunoreactivity was present at early pregnancy and at term, i.e. under major oestrogenic influence. The immunoreactivity to the PDGF-receptor antiserum R-3 (against beta-type) was similarly localized as that showed for R-1 antiserum, while of a weaker intensity. The results indicate the presence of PDGF receptors (alpha- and beta-type) in the endo- and myometrium of cycling and pregnant pigs, and that their expression seems to be accentuated under oestrogenic influence.


Synovial sarcoma in a dog.

Synovial sarcoma is an uncommon malignant tumour of mesenchymal origin, occurring mainly in the vicinity of a joint. Subsequent invasion of the joint and adjacent bones may occur. Less frequently the tumour may be localized in soft tissues remote from joints. Synovial sarcoma usually affects male middle-aged dogs of large breeds. The rate of tumour growth can vary from very slow to very rapid. The tumour is usually poorly defined and infiltrates the deeper structures. Metastasis and recurrence are common. Wide-field surgical excision of the tumour or amputation of the affected limb are the most frequently used forms of treatment. In the present case the dog, a 7-year-old male Alsatian, had a slowly growing tumour in the vicinity of the hip joint, bilaterally. There was no involvement of the bones surrounding the tumour. Surgical resection was performed and one year later the dog was reported to be normal.


[Protective effect of dextromethorphan on the ischemic retinal damage in rabbit].

To investigate the effect of dextromethorphan (DEX), N-methyl-D-aspartate (NMDA) receptor antagonist, on the retinal ischemia, 0.4%DEX hydrobromide was intravenously given to rabbits before, during and after retinal ischemia. Retinal function was monitored by electroretinogram (ERG). Retinal ischemia was induced by increasing intraocular pressure to 130 mmHg for 90 or 120 min. Amplitudes of ERG.b-waves recorded after the 90 min ischemia recovered to 72.5 +/- 9.0% in the DEX group and 38.5 +/- 8.5% in the control group which was given normal saline. The maximal recovery rates of b-wave amplitudes after the 120 min ischemia were 44.0 +/- 7.9% in the DEX group and 21.0 +/- 1.3% in the control group. The recovery rates of the b-wave amplitudes in DEX group were significantly higher than in the control group (p less than 0.01). It was found that the effective dose of DEX was 0.1-0.4%.


On the mechanism of action of antiarrhythmic agents.

Cardiac arrhythmias arise from disturbances in the functioning of the specific ion channels that normally control excitation or from the functional expression of previously latent channels. Antiarrhythmic agents act by blocking the membrane sodium, potassium, and calcium channels, but no agent has exclusive action on a given type of channel. Arrhythmias resulting from reentry form the largest group of clinically significant arrhythmias. Most arrhythmias result from depressed sodium channel function. The local anesthetic class of sodium channel blockers (class I agents) acts by slowing conduction and converting regions of unidirectional block to bidirectional block. Class III agents act by prolonging the action potential duration. Because potassium currents are normally responsible for repolarization of the cardiac action potentials, these agents are generally assumed to be potassium channel blockers. Class IV antiarrhythmics--calcium channel blockers--are used when a group of reentrant arrhythmias arises in regions in which conduction is primarily sustained by increases in permeability to calcium ions. The mechanisms of action of antiarrhythmic agents are discussed with respect to the basic cellular mechanisms of cardiac arrhythmias.


Mechanisms of pharmacologic intervention at the level of the calcium channel.

Calcium channels are large, complex membrane proteins that mediate transmembrane calcium currents. At least 2 kinds of calcium channels are found in heart muscle--the transient type and the long-lasting type. Calcium currents are modulated by diverse endogenous and exogenous factors including hormones, catecholamines, and calcium antagonists. Calcium antagonists act preferentially on vascular smooth muscle and have relatively less effect on the calcium channels of heart muscle. Compared with heart muscle, vascular smooth muscle is relatively depolarized, suggesting that vascular smooth muscle cells have predominantly the long-lasting type of calcium currents. The differential binding to different types of calcium channels underlies the clinical efficacy of the calcium antagonists. A drug such as bepridil, which acts preferentially on the coronary vasculature rather than on the peripheral vasculature, dilates the coronary vessels without depressing cardiac contraction, a putative clinical advantage.


Phase II trial of combination chemotherapy and irradiation in non-small-cell lung cancer, Radiation Therapy Oncology Group 88-04.

Encouraging results of several clinical trials utilizing combination chemotherapy and irradiation in unresectable non-small-cell lung cancer have been reported. A recent report from a cooperative group study suggested that preirradiation vinblastine and cisplatin improved survival over irradiation alone. In an attempt to enhance the possible effectiveness of combination chemotherapy and irradiation, the Radiation Therapy Oncology Group embarked on a Phase II trial utilizing preirradiation vinblastine (5 mg/m2 weekly x 5) and cisplatin (100 mg/m2) on days 1 and 29 prior to irradiation and on days 50, 71, and 92 during irradiation. The irradiation began on day 50 and consisted of 6300 cGy in 7 weeks. Between May 20, 1988 and May 1, 1989, 30 patients were entered on study. Seventy-two percent of patients had Karnofsky status greater than 90, and 76% had weight loss less than 5%. Forty-eight percent of the patients were younger than 60 years of age. Forty-five percent of the patients had Stage IIIA disease. Eighty-three percent of the patients received at least four courses of vinblastine, and 59% received at least four courses of cisplatin. Seventy-eight percent of the patients received at least 95% of the prescribed irradiation. The major toxicity was hematologic, and there were two fatal complications in the study group. The preliminary survival figures are encouraging. This combination of chemotherapy and irradiation appears to be tolerable and may merit further investigation.


Fibrous histiocytoma of the parotid gland.

The light microscopic, electron microscopic, and immunocytochemical characteristics of a case of a fibrous histiocytoma arising in the parotid gland is presented. This neoplasm is very rare in this site and must be distinguished from other spindle cell tumors of the parotid gland, particularly those of epithelial and myoepithelial origins. Histologic characteristics similar to those displayed by dermatofibromas and dermatofibrosarcoma protuberans help to differentiate this tumor from other spindle cell tumors. The absence of cytochemical epithelial markers and the electron microscopic demonstration of fibroblasts and histiocytes are useful in establishing the diagnosis. This tumor, which was confined completely to the parotid gland, appears to have arisen from mesenchymal elements within the gland.


A rapid, specific assay for superoxide release from phagocytes in small volumes of whole blood.

A rapid, specific assay was developed to measure the release of superoxide anion from stimulated phagocytic cells in small volumes of whole blood. The assay is based on the chemiluminescence that results when lucigenin (bis-N-methyl acridinium nitrate) is reduced by superoxide. Heparinized whole blood, at volumes from 0.2 mL to a single drop from a 20-gauge needle, was mixed with lucigenin and either a soluble or particulate stimulus (phorbol myristate acetate or opsonized zymosan particles, respectively) in a standard volume of 1 mL. The chemiluminescence was measured at 3-minute intervals for a 30-minute period in a luminometer capable of automated operation. Characteristic plots of chemiluminescence versus time were obtained. This assay is rapid and simple, obviates the need to isolate leukocytes from whole blood, is specific for superoxide, can be performed with both soluble and particulate stimuli, and is sensitive enough to detect characteristic responses with as little as a single drop of blood. Patients with chronic granulomatous disease were readily identified using this assay. The ability to use very small blood volumes makes the assay suitable for use in prenatal diagnosis. The main disadvantage of this assay compared to the often-employed slide nitroblue tetrazolium test, with respect to testing for chronic granulomatous disease, is that carriers of x-linked chronic granulomatous disease are not readily detected with this chemiluminescence assay, as they are with the nitroblue tetrazolium test.


Clinical significance of serum and urinary neopterin levels in patients with various liver diseases.

Serum and urinary neopterin levels were measured by radioimmunoassay in 120 healthy controls, 16 asymptomatic HBsAg carriers, 12 patients with acute hepatitis, 13 with chronic inactive hepatitis, 35 with chronic active hepatitis, 46 with liver cirrhosis, 18 with hepatocellular carcinoma, and 6 with alcoholic liver disease. Serum and urinary neopterin levels were significantly higher in almost all patients than in normal subjects. Neopterin levels were highest in acute hepatitis and correlated with the results of liver function tests, but did not show this correlation in chronic liver disease. In chronic liver disease, the levels of serum neopterin in non-A non-B viral patients was significantly increased, compared with those in B viral and alcoholic patients. The rate of abnormal urinary neopterin levels in chronic liver disease was higher than the rate of abnormal serum neopterin levels, but no difference was observed between the rates of abnormal serum and urinary levels in acute hepatitis and asymptomatic HBsAg carriers. These results indicate that serum and urinary neopterin levels may be useful markers for cell-mediated immunity in liver disease, and that the immune system response in chronic liver disease may be different for different pathogens.


DNA methylation patterns of the gamma delta beta-globin genes in human fetal and adult erythroid tissues.

An investigation of the correlation between the gamma----beta-globin switch and DNA methylation was carried out. The restriction patterns obtained with methylation-sensitive and -insensitive enzymes indicated hypomethylation in the promoter region of the gamma-globin genes in fetal liver DNA but high methylation of the same region in all other samples (except in the presence of an elevated erythroblast count or leukemia). All samples appeared to be partially hypomethylated at the 5' end of the delta-globin gene and hypomethylated at the 3' region of the beta-globin gene. Although consistent with a role for DNA methylation in globin gene regulation, the results also suggest that other factors besides methylation may be required for regulation of the level of expression, and switching of the globin genes.


Time-weighted averages, peaks, and other indices of exposure in occupational epidemiology.

Dose surrogates commonly used in occupational epidemiology are exposure intensity, exposure duration, and cumulative exposure. The appropriateness of any of these measures as dose indicators depends on the nature of the induction process for the disease under consideration. Peak exposure intensity is often associated with acute health outcomes, whereas cumulative exposure is generally more relevant for diseases with long induction times, i.e., "chronic" diseases. However, there may be situations where peak exposure is etiologically relevant in chronic disease induction, such as might occur with nonlinear rates of damage during brief intervals of very high exposure. An approach is described for evaluating the effect of peak exposures in which peaks may be defined on a relative basis for each worker, or with respect to an absolute value, such as the permissible occupational exposure limit. The analytic strategy is illustrated with data from a case-control study of silicosis in relation to quantitative estimates of silica exposure. In this example, relative peak exposures and average non-peak exposures appear to be better predictors of silicosis risk than cumulative exposure.


Partial gonadal dysgenesis in a patient with a marker Y chromosome.

We evaluated a patient with partial gonadal dysgenesis including a right dysgenetic testis and a left streak gonad with rudimentary fallopian tube and uterus. She had ambiguous external genitalia and was raised female. Although her height is normal (25th centile at age 12 years), she has some findings of Ullrich-Turner syndrome. Her karyotype was reported to be 46,X,+marker; subsequent molecular investigations showed the marker to be the short arm of the Y chromosome. Genomic DNA, isolated from leukocytes of the patient and her father, was digested with a variety of restriction endonucleases and subjected to Southern blot analysis. A positive hybridization signal was obtained with probes for the short arm of the Y chromosome (pRsY0.55, SRY, ZFY, 47Z, pY-190, and YC-2) in DNA from the patient, indicating the presence of most if not all of the short arm, while long arm probes (HinfA and pY3.4) indicated that at least 75% of the long arm of the Y chromosome was missing. The gene responsible for testicular determination (TDF) is on the distal portion of the short arm of the Y chromosome; Yq has no known influence on sex determination. Hence, the deletion of the long arm of the Y chromosome cannot explain the gonadal dysgenesis in this patient. One explanation for the gonadal dysgenesis and Ullrich-Turner phenotype in the patient could be undetected 45,X/46,X,+marY mosaicism but no such mosaicism was observed in peripheral lymphocytes. Several investigators have suggested the presence of an "anti-Turner" gene near TDF. Hence it is possible that the clinical phenotype in our patient results from a Y chromosomal defect in sequences flanking TDF, which reduces the function of both TDF and the "anti-Turner" genes.


Effects of halothane on electrophysiologic properties and cyclic adenosine 3',5'-monophosphate content in isolated guinea pig hearts.

We studied the effects of halothane on the electrophysiologic and biochemical properties of both Langendorff perfused hearts and single ventricular myocytes isolated from guinea pigs. Isometric contractions of left ventricles in perfused hearts, elicited by atrial pacing, decreased to 14% of control after exposure to 2% halothane-equilibrated perfusate. Subsequently the slow inward Ca2+ current (ICa) was recorded in isolated myocytes with a whole cell voltage clamp technique. ICa, recorded in response to 100-ms depolarizations from -40 mV to 0 mV, was decreased by 2% halothane to 28.4% of control. Halothane-induced ICa depression did not exhibit use dependency. To define a possible site at which halothane acts, we measured the cyclic adenosine 3',5'-monophosphate (cAMP) content of single ventricular myocytes using a radioimmunoassay. Two percent halothane decreased myocardial cAMP content to 68.9% of control. Further addition of dibutyryl cAMP (10(-3) mol/L) partially reversed the depressed contractility during 2% halothane administration in perfused hearts. In conclusion, the present study demonstrated that the decrease of myocardial cAMP by halothane was due to a direct action, at least partly, and not to other factors such as catecholamines, and suggested that the decreases in contractility and ICa were induced possibly through the decrease in cellular cAMP.


Surfactant replacement therapy improves pulmonary mechanics in end-stage influenza A pneumonia in mice.

Surfactant replacement therapy may be a promising approach for treatment of respiratory failure caused by viral pneumonia. This study in mice demonstrates that during the development of lethal influenza A pneumonia, thorax-lung compliance (Ctl/kg) and lung volume at 5 cm H2O PEEP (V5/kg) significantly decrease (28 and 54%, respectively), whereas lung water content significantly increases (25%). Surfactant replacement therapy during the end stage of pneumonia significantly increases Ctl/kg (31%) and V5/kg (21%). Instillation of the vehicle for surfactant in control animals does not significantly affect Ctl/kg (5% decrease), but it significantly decreases V5/kg (25% decrease). Further, a new method for postmortem measurement of lung volumes in small laboratory animals based on Archimedes' principle is presented.


Detection of Epstein-Barr virus in lymphocytic interstitial pneumonia by in situ hybridization.

The involvement of Epstein-Barr virus (EBV) in the pathogenesis of lymphocytic interstitial pneumonia (LIP) was investigated using an in situ hybridization technique. Archival lung tissue samples from 14 patients (six men and eight women with a mean age of 58 +/- 3 yr) in whom a diagnosis of LIP had been previously established were retrospectively examined and compared with samples from 10 patients (six men and four women with a mean age of 58 +/- 3 yr) with idiopathic pulmonary fibrosis (IPF) who served as control subjects. In patients with LIP, the immunophenotype of the lymphocytic infiltrate was determined by using monoclonal antibodies to both pan-B-cell and pan-T-cell markers. In situ hybridization studies were performed by using the BamHI-W region of the EBV genome as a probe and a colorimetric detection method. The immunophenotyping studies showed that the interstitial infiltrate in LIP was primarily made up of B-lymphocytes, particularly within the lymphoid aggregates, whereas T-lymphocytes were sparsely distributed along the alveolar septa. The in situ hybridization studies showed the presence of cells bearing the EBV genome in nine cases of LIP and in two cases of IPF (p less than 0.05, Fisher's exact test). In LIP, the EBV-positive cells were observed in both enlarged and normal septa and occasionally within the lymphoid aggregates. We conclude that EBV may promote the proliferation of B-lymphocytes in a substantial number of patients with LIP.


A combination of human cytomegalovirus (HCMV)-specific murine monoclonal antibodies exhibits synergistic antiviral activity in vitro.

A combination of HCMV-specific monoclonal antibodies (MAbs) reactive with glycoproteins in gcI complexes which exhibit synergistic antiviral activity in vitro is described. MAbs directed against different structural and biological properties of HCMV have been selected to increase the antiviral activity against all possible strains, and to reduce the likelihood that resistant strains will emerge with prolonged exposure. Furthermore, in vitro analysis demonstrates that certain of the MAbs in the combination augment the virus-neutralizing activity of other component antibodies, thereby decreasing the amount of total antibody protein required to inhibit HCMV infection. Certain MAbs have been selected to inactivate extracellular virus during the early phase of HCMV infection, whereas others have been selected to prevent its spread once cells have been infected. These data suggest that a MAb cocktail may be useful for prophylaxis and treatment of patients at risk of life-threatening HCMV infections.