The DNA sequence of the sulfate activation locus from Escherichia coli K-12. The DNA sequence of the sulfate activation locus from Escherichia coli K-12 has been determined. The sequence includes the structural genes encoding the enzymes ATP sulfurylase (cysD and cysN) and APS kinase (cysC) which catalyze the synthesis of activated sulfate. These are the only genes known to reside in the sulfate activation operon. Consensus elements of the operon promoter were identified, and the start codons and open reading frames of the Cys polypeptides were determined. During this work, another gene, iap, was partially sequenced and mapped. The activity of ATP sulfurylase is stimulated by an intrinsic GTPase. Comparison of the primary sequences of CysN and Ef-Tu revealed that CysN has conserved many of the residues integral to the three-dimensional structure important for guanine nucleotide binding in Ef-Tu and RAS. nodP and nodQ, from Rhizobium meliloti, are essential for nodulation in leguminous plants. The Cys and Nod proteins are remarkably similar. NodP appears to be the smaller subunit of ATP sulfurylase. NodQ encodes homologues of both CysN and CysC; thus, these enzymes may be covalently associated in R. meliloti. The consensus GTP-binding sequences of NodQ and CysN are identical suggesting that NodQ encodes a regulatory GTPase.


NhaR, a protein homologous to a family of bacterial regulatory proteins (LysR), regulates nhaA, the sodium proton antiporter gene in Escherichia coli. On the basis of protein homology, nhaR has previously been shown to belong to a large family of regulatory proteins, the LysR family (Henikoff, S., Haughn, G.W., Calvo, J.M., and Wallace, J.C. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 6602-6606). In this work we show that nhaR is a regulator of nhaA, a gene encoding a Na+/H+ antiporter in Escherichia coli. Multicopy plasmid bearing nhaR enhances the Na(+)-dependent induction of a chromosomal nhaA'-'lacZ fusion. Extracts derived from cells overexpressing nhaR exhibit specific DNA binding capacity to the upstream sequences of nhaA. Construction of an nhaR deletion mutant (OR100) shows that nhaR is required in addition to nhaA to tolerate the extreme conditions under which nhaA is indispensable. Whereas OR100 grows like the wild type at neutral pH even at high Na+ concentrations (700 mM), it becomes much more sensitive to Na+ (greater than 300 mM) at pH 8.5; furthermore, OR100 is more sensitive to Li+ (100 mM) than the wild type. Nevertheless, the phenotype of OR100, which is more resistant to Na+, Li+, and alkaline pH than a delta nhaA strain (NM81), implies that the regulation exerted by nhaR is not complete and that some expression of nhaA exists in OR100. Accordingly, the effect of nhaR in cells is dependent on the level of nhaA. OR200, a nhaA and nhaR deletion mutant, has the same phenotype as NM81. Multicopy plasmid bearing nhaR does not change the phenotype of either OR200 or NM81. On the other hand, multicopy nhaA renders the cells Li(+)- and and Na(+)-resistant even without nhaR.


Growth factor stimulation of phospholipase C-gamma 1 activity. Comparative properties of control and activated enzymes. We demonstrated previously tyrosine phosphorylation-dependent modulation of phospholipase C-gamma 1 (PLC-gamma 1) catalytic activity (Nishibe, S., Wahl, M. I., Hernandez-Sotomayor, S. M. T., Tonks, N. K., Rhee, S. G., and Carpenter, G. (1990) Science 250, 1253-1256). The increase in PLC-gamma 1 catalytic activity in A-431 cells occurs rapidly, with maximal activation 5 min after epidermal growth factor (EGF) stimulation. Certain other growth factors (fibroblast growth factor, platelet-derived growth factor) also stimulate PLC-gamma 1 catalytic activity, whereas insulin does not. A similar increase in PLC-gamma 1 specific activity (2-3-fold) was observed in both soluble (cytosol) and particulate (membrane) preparations from EGF-treated cells. Tyrosine-phosphorylated PLC-gamma 1 was detected in both cytosol and membrane fractions in lysates from EGF-treated A-431 cells, but the proportion of tyrosine-phosphorylated PLC-gamma 1 was higher in the cytosol (approximately 50%) than in the membrane (approximately 20%). Because a micellar concentration of the non-ionic detergent Triton X-100 allows detection of the tyrosine phosphorylation-dependent increase in PLC-gamma 1 catalytic activity in this assay, we evaluated the kinetic properties of PLC-gamma 1, immunoprecipitated from cytosol of control or EGF-treated cells, using substrate, phosphatidylinositol 4,5-bisphosphate (PtdIns 4,5-P2), solubilized in Triton X-100 at various molar ratios. The behavior of the control enzyme differed from the EGF-activated enzyme with respect to both Ks and Km. The control enzyme has a 7.5-fold higher Ks value than the activated enzyme (1.5 mM as compared with 0.22 mM). Activation by EGF is also a positive allosteric modifier of PLC-gamma 1-catalyzed PtdIns 4,5-P2 hydrolysis, i.e. the activated enzyme displayed apparent Michalis-Menton kinetics, with a Km of 0.6 mol fraction PtdIns 4,5-P2, whereas the control enzyme displayed sigmoidal kinetics with respect to PtdIns 4,5-P2 hydrolysis. At low substrate mol fractions (e.g. 0.07), the reaction velocity of the control enzyme was 4-fold lower than the activated enzyme. However, at a high substrate mol fraction (e.g. 0.33), the estimated maximal reaction velocities (Vmax) for both forms of PLC-gamma 1 were equivalent. PLC-gamma 1 activity from both control and EGF-treated cells was stimulated by increasing nanomolar Ca2+ concentrations. Although the catalytic activity of PLC-gamma 1 from EGF-treated cells was greater than control PLC-gamma 1 at every Ca2+ concentration tested, the relative stimulation of activity was markedly greater at Ca2+ concentrations above approximately 300 nM.


The interaction of RNA polymerase II with the adenovirus-2 major late promoter is precluded by phosphorylation of the C-terminal domain of subunit IIa. Mammalian RNA polymerase II contains at the C terminus of its largest subunit an unusual domain consisting of 52 tandem repeats of the consensus sequence Tyr-Ser-Pro-Thr-Ser-Pro-Ser. The phosphorylation of this domain is thought to play an important role in the transition of RNA polymerase II from a preinitiation complex to an elongating complex. The unphosphorylated form of RNA polymerase II is designated IIA, whereas the phosphorylated form is designated IIO. In an effort to determine the consequence of C-terminal domain phosphorylation on complex formation, 32P-labeled RNA polymerases IIA and IIO were prepared and examined for their ability to form a stable preinitiation complex on the adenovirus-2 major late promoter in the presence of a reconstituted HeLa cell transcription extract. Preinitiation complexes were formed in the absence of ATP and purified from free RNA polymerase II by chromatography on Sepharose CL-4B. The state of phosphorylation of the largest subunit was monitored by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the transcriptional activity was determined by assaying specific transcript formation upon the addition of nucleotides and a competing DNA template. RNA polymerase IIA was recovered in transcriptionally active complexes in reactions in which the input enzyme was RNA polymerase IIA. In reactions with RNA polymerase IIO as the input enzyme, no IIO was recovered in excluded fractions that normally contain preinitiation complex. In reactions with equimolar amounts of RNA polymerases IIO and IIA, purified preinitiation complexes contained almost exclusively RNA polymerase HA. These results support the idea that RNA polymerase II containing an unphosphorylated C-terminal domain preferentially associates with the adenovirus-2 major late promoter. The state of phosphorylation of the C-terminal domain can, therefore, directly influence preinitiation complex formation. We also report here the presence of an activity in HeLa cell extracts that catalyzes dephosphorylation of the C-terminal domain, thereby converting RNA polymerase IIO to IIA. This C-terminal domain phosphatase is specific in that it does not catalyze the dephosphorylation of a serine residue phosphorylated by casein kinase II. The presence of a C-terminal domain phosphatase in in vitro transcription reactions containing RNA polymerase IIO results in the formation of RNA polymerase IIA. This RNA polymerase IIA associates preferentially with preinitiation complexes.


Endothelin inhibits adenylate cyclase and stimulates phosphoinositide hydrolysis in adult cardiac myocytes. We have assessed the effects of endothelin-1 (ET-1) on transmembrane signaling in adult rat ventricular myocytes. ET-1 stimulates phosphoinositide hydrolysis with an EC50 of 0.3-0.8 nM. This stimulation is linear for up to 30 min in the presence of a protease inhibitor, is additive with the effects of other stimulators of phosphoinositide hydrolysis, is not inhibited by the Ca2+ entry blocker, nifedipine, and is insensitive to pertussis toxin. ET-1 also reduces cyclic AMP production in myocytes in response to isoproterenol and forskolin (EC50, 1 nM). This cyclic AMP-lowering effect of ET-1 is sensitive to pertussis toxin, can be demonstrated directly in assays of adenylate cyclase activity of myocyte membranes, and seems to be mediated by Gi. These data indicate that the effects of endothelin on adult cardiac myocytes involve multiple signaling pathways, including enhanced activity of the inositol phosphate pathway and a decrease in cyclic AMP-mediated responses, neither of which seems likely to account for the positive contractile effects of endothelin.


Expression of antioxidant enzymes in rat lungs after inhalation of asbestos or silica. Several studies indicate that active oxygen species play an important role in the development of pulmonary disease (asbestosis and silicosis) after exposure to mineral dust. The present study was conducted to determine if inhaled fibrogenic minerals induced changes in gene expression and activities of antioxidant enzymes (AOE) in rat lung. Two different fibrogenic minerals were compared, crocidolite, an amphibole asbestos fiber, and cristobalite, a crystalline silicon dioxide particle. Steady-state mRNA levels, immunoreactive protein, and activities of selected AOE were measured in lungs 1-10 days after initiation of exposure and at 14 days after cessation of a 10-day exposure period. Exposure to asbestos resulted in significant increases in steady-state mRNA levels of manganese-containing superoxide dismutase (MnSOD) at 3 and 9 days and of glutathione peroxidase at 6 and 9 days. An increase in steady-state mRNA levels of copper, zinc-containing superoxide dismutase (CuZnSOD), was observed at 6 days. Exposure to asbestos also resulted in overall increased enzyme activities of catalase, glutathione peroxidase and total superoxide dismutase in lung. In contrast, silica caused a dramatic increase in steady-state levels of MnSOD mRNA at all time periods and an increase in glutathione peroxidase mRNA levels at 9 days. Activities of AOE remained unchanged in silica-exposed lungs. In both models, increases in gene expression of MnSOD correlated with increased amounts of MnSOD immunoreactive protein in lung and the pattern and extent of inflammation. These data indicate that the profiles of AOE are dissimilar during the development of experimental asbestosis or silicosis and suggest different mechanisms of lung defense in response to these minerals.


Different sorting of Lys-Asp-Glu-Leu proteins in rat liver. Most of the resident soluble proteins of the endoplasmic reticulum (ER) seem to be sorted into this compartment via their COOH-terminal tetrapeptide Lys-Asp-Glu-Leu (KDEL). This sorting is supposed to occur in a post-ER compartment. Three resident soluble ER glycoproteins belonging to the KDEL family are CaBP1, CaBP2, CaBP3 (= calreticulin), and CaBP4 (= grp94) (Nguyen Van, P., Peter, F., and Söling, H.-D. (1989) J. Biol. Chem. 264, 17494-17501). In rat liver, calreticulin possesses a carbohydrate moiety of the complex hybrid type with terminal galactoses (Nguyen Van, P., Peter, F., and Söling, H.-D. (1989) J. Biol. Chem. 264, 17494-17501). We can show now that practically all calreticulin molecules (and not only a fraction) possess terminal galactoses as well as the COOH-terminal KDEL sequence. This as well as pulse-chase experiments performed at 37 and 15 degrees C indicate that calreticulin must have passed through the trans-Golgi. Subcellular fractionations of post-mitochondrial supernatants from isolated rat hepatocytes by sucrose-Nycodenz gradient centrifugation revealed that calreticulin is confined mainly to the rough ER, grp94 mainly to the smooth ER. CaBP1, a member of the thioredoxin family, was recovered in fractions which most likely represent the intermediate compartment. This indicates that KDEL is a sorting signal which leads to the retention of these proteins in the pre-Golgi compartments. However, additional factors, most likely residing within the specific KDEL protein itself, determine the final location of the protein within the pre-Golgi compartments. This is underlined by experiments in which the density dependent distribution of total KDEL proteins was studied using a COOH-terminal KDEL-specific antibody.


Antipeptide antibody to the insulin-like growth factor-I receptor sequence 1232-1246 inhibits the receptor kinase activity. To approach the question of why insulin-like growth factor-I (IGF-I) and insulin have different physiological actions, we developed antibodies directed against cytoplasmic regions of the IGF-I receptor exhibiting a low degree of homology with the corresponding sequences of the insulin receptor. We found that an antipeptide antibody directed against the beta-subunit carboxyl-terminal sequence (1232-1246) of the IGF-I receptor significantly reduced the in vitro receptor autophosphorylation. The ability of the synthetic peptide corresponding to the IGF-I receptor sequence 1232-1246 to abolish this inhibitory effect reflects the specific nature of the antibody interaction with the targeted domain in the receptor. Antipeptide antibody to IGF-I receptor sequence 1232-1246 also decreased receptor phosphorylation activity toward the exogenous substrate poly(Glu/Tyr). The reduction in poly(Glu/Tyr) phosphorylation was seen even when the antibody was incubated with a receptor previously activated and phosphorylated. Therefore, the inhibitory action on substrate phosphorylation is likely to be unrelated to the antibody reduction of receptor autophosphorylation but rather results from a global decrease in receptor enzymatic activity. The effect of the antipeptide antibody on receptor tyrosine kinase cannot be accounted for by a lowering of the receptor Km for ATP or of its affinity for the substrate poly(Glu/Tyr). Moreover, the interaction of the antibody with the receptor had no repercussion on the ligand binding site as shown by the unaltered IGF-I binding. Taken together our data suggest that the beta-subunit carboxyl-terminal domain of the IGF-I receptor plays a key role in regulating its kinase activity and that the particular sequence recognized by our antipeptide antibody could be involved in negative regulation of receptor functioning.


n-butyrate reduces the expression of beta-galactoside alpha 2,6-sialyltransferase in Hep G2 cells. n-Butyrate, a short chain fatty acid that is produced by colonic bacterial fermentation, is detectable in portal blood and induces differentiation in various human neoplastic cell lines. Earlier reports indicated approximately 20-fold induction in vitro by n-butyrate of the sialyltransferase that catalyzes terminal glycosylation of GM3 ganglioside in HeLa and colon cancer cells. We previously isolated a 1.3-kilobase cDNA for a human beta-galactoside alpha 2,6-sialyltransferase, for which N-linked glycoproteins are the acceptors. We report here that treatment of Hep G2 cells with 5 mM n-butyrate for 24 h reduced beta-galactoside alpha 2,6-sialyltransferase mRNA levels by approximately 90%. Reductions in mRNA level were followed by approximately 75 and approximately 90% reductions, respectively, in specific beta-galactoside alpha 2,6-sialyltransferase enzyme activity after treatment for 24 and 36 h with 5 mM n-butyrate. However, in contrast with earlier reports of enhanced ganglioside synthesis in response to n-butyrate treatment, incubation of Hep G2 cells with n-butyrate did not alter the ganglioside pattern as assessed by thin layer chromatography of lipids extracted from treated cells. Nuclear run-on reactions indicated that the rate of transcription of beta-galactoside, alpha 2,6-sialyltransferase was not altered by treatment with 5 mM n-butyrate for 24 h, but the effects of this treatment on cytoplasmic levels of beta-galactoside alpha 2,6-sialyltransferase mRNA were largely negated by co-treatment with actinomycin D or cycloheximide. Therefore, our results show that n-butyrate reduces expression of mature beta-galactoside alpha 2,6-sialyltransferase mRNA by post-transcriptional mechanisms.


Insulin-like growth factor I receptor gene structure. The insulin-like growth factor I (IGF I) receptor is a tyrosine kinase-containing transmembrane protein that plays an important role in cell growth control. We have isolated and characterized human genomic DNA clones containing the entire coding sequence of the IGF I receptor. Results of restriction analysis and sequencing of multiple overlapping clones were consistent with the existence of a single IGF I receptor gene. The complete receptor coding sequence is contained in 21 exons. There is striking homology with the insulin receptor gene in overall size (approximately 100 kilobases) and in the number and size of individual exons. An exon analogous to the alternatively spliced exon 11 of the insulin receptor gene could not be detected. An alternative internal splice site corresponding to a known alternatively spliced mRNA transcript was shown to be located at the 5' end of exon 14. Knowledge of the structure of the IGF I receptor gene should facilitate further studies on the structural determinants of receptor function, the relationships between insulin and IGF I receptors, and the molecular basis for multiple IGF I receptor species.


Inhibition of myogenesis by okadaic acid, an inhibitor of protein phosphatases, 1 and 2A, correlates with the induction of AP1. Recently, we demonstrated that okadaic acid, an inhibitor of protein phosphatases 1 and 2A, inhibits myogenesis by extinguishing the expression of MyoD1 and inducing the expression of Id. Since it has been reported that transformation by c-fos also inhibits myogenesis through inhibition of MyoD1 expression, we examined the effects of okadaic acid on the activation of the c-fos and jun family of proto-oncogenes in an attempt to understand the mechanism by which okadaic acid inhibits the myogenic differentiation. Treatment of C2C12 cells in growth medium with okadaic acid increased expression of the mRNAs for the c-fos family continuously and for the jun family to a lesser extent. In contrast, in differentiation medium, the induction of c-fos, c-jun, and fos B mRNAs by okadaic acid was transient, whereas fra-1, jun D, and jun B mRNAs were induced continuously, suggesting that okadaic acid regulates the expression of the c-fos and jun family through complex regulatory mechanisms depending on the state of differentiation of the cells. Transfection of c-jun and c-fos promoter-chloramphenicol acetyltransferase constructs demonstrated that the effects of okadaic acid on the induction of c-fos and c-jun are mediated through the activation of promoter elements. These results suggest that some of the targets of protein phosphatases 1 and 2A may include transcription factors capable of forming AP1 complexes and that these factors may play an important role during myogenic differentiation.


Positive and negative regulatory DNA elements including a CCArGG box are involved in the cell type-specific expression of the human muscle dystrophin gene. The muscle-specific promoter of the dystrophin gene is active in skeletal, cardiac, and smooth muscles and is specifically stimulated during differentiation of myoblasts into multinucleated myotubes. An 850-base pair (bp) DNA fragment upstream from the cap site is able to confer a partial muscle specificity to a reporter gene. The region between -850 and -140 bp includes nonspecific negative and positive regulatory sequences. A continuous stretch of 140 bp upstream from the cap site exhibits a striking conservation between rodents and human (93% homology) and still retains muscle preference of expression. It contains two putative binding sites for factors involved in regulation of other muscle-specific genes, a CCArGG box and an E box. This latter element, however, is unable to confer the ability to be transactivated by MyoD1 to the dystrophin promoter. The -140-bp promoter fragment exhibits antagonist effects contributed by one inhibiting sequence (nucleotide -140/-96), active in all cell types, and one activating region, from nucleotide -96 to the cap site, sufficient to confer a muscle preference of expression, in which the CCArGG box seems to play a major role.


Ligand-receptor interactions in affinity cell partitioning. Studies with transferrin covalently linked to monomethoxypoly(ethylene glycol) and rat reticulocytes. The partitioning of rat reticulocytes in poly(ethylene glycol) (PEG)-dextran two-phase systems increases into the PEG-rich top phase when the cells are incubated with transferrin covalently modified with monomethoxy-PEG (MPEG-transferrin) prior to partitioning. Two observations support the suggestion that such an increase in top-phase partitioning is due to the specific interaction of the MPEG-transferrin conjugate with the transferrin receptor on the surface of the reticulocyte: first, the MPEG-transferrin conjugate competes with [125I]transferrin for the transferrin receptor on reticulocytes (Ka = 6.28 x 10(6) l mol-1); and second, the MPEG-modified transferrin is unable to change the partitioning of rat erythrocytes, cells lacking the transferrin receptor. This example illustrates the feasibility of manipulating the partitioning of a selected cell population when ligand-receptor interactions are exploited. The increase in the partitioning of the reticulocytes takes place within a narrow range of MPEG-transferrin bound per cell, viz., 10.2-11.3 fg per cell. The latter range corresponds to ca. 80,000-89,000 molecules of MPEG-transferrin bound per cell.


Comparative auto-controlled study between swim-up and Percoll preparation of fresh semen samples for in-vitro fertilization. This study compared swim-up and Percoll preparation of fresh semen samples for in-vitro fertilization. Sixty trials of in-vitro fertilization (IVF), 38 with normal semen and 22 with abnormal semen, comprising 734 oocytes were included in the study. Each semen sample was prepared by both a swim-up technique and a simplified discontinuous (50%, 70%, 90%) Percoll gradient. The oocytes for each trial were distributed at random between the two sperm preparations and incubated with the same number of motile spermatozoa. Percoll gradient preparation produced a significantly higher final concentration of spermatozoa than swim-up preparation (mean +/- SEM: 6.6 +/- 1.5 x 10(6)/ml versus 1.9 +/- 0.2 x 10(6)/ml; P less than 0.01) but a significantly lower sperm motility (69 +/- 2% versus 94 +/- 1%; P less than 0.001) and a lower number of normal forms (55 +/- 2% versus 64 +/- 2%; P less than 0.01). The ability of the Percoll gradient method to extract motile spermatozoa was higher than that of the swim-up technique (20 +/- 15.6% versus 0.8 +/- 13.6%). Nevertheless, the rates of fertilization (61%), fertilization failure (18%) and polyspermia (9%), embryo quality evaluated by mean embryo scores (3.8 +/- 0.3) and the mean number of spare embryos frozen per trial (1.4 +/- 0.3) were strictly identical in both groups. The 24 pregnancies (including three from frozen--thawed embryos) obtained in these 60 trials (40% per oocyte retrieval) could not be separated according to the sperm preparation method, as embryos from both groups were replaced together.(ABSTRACT TRUNCATED AT 250 WORDS)


Ultrastructural immunogold localization of prostaglandin endoperoxide synthase (cyclooxygenase) to non-membrane-bound cytoplasmic lipid bodies in human lung mast cells, alveolar macrophages, type II pneumocytes, and neutrophils. Lipid bodies are non-membrane-bound, lipid-rich cytoplasmic inclusions that occur in many mammalian cell types. Because lipid bodies are more prominent in cells associated with inflammation and are repositories of arachidonyl-phospholipids, a role for lipid bodies in the oxidative metabolism of arachidonic acid to form eicosanoids has been suggested. To evaluate further whether lipid bodies, in addition to serving as non-membranous sources of substrate arachidonate, are involved in eicosanoid formation, we used cells isolated from human lung to investigate the intracellular localization of prostaglandin endoperoxide (PGH) synthase (cyclooxygenase), the key initial, rate-limiting enzyme in the formation of prostaglandins and thromboxanes. Isolated lung cells containing a mixture of mast cells, alveolar macrophages, Type II alveolar pneumocytes, and neutrophils from short-term cultures were fixed in suspension in a dilute aldehyde mixture, post-fixed in osmium tetroxide, stained en bloc with uranyl acetate, dehydrated in a graded series of alcohols, and embedded in Epon. A post-embedding immunogold procedure was used with a primary PGH synthase monoclonal antibody and 20-nm gold-conjugated secondary antibody to demonstrate enzyme locations. Specificity controls were also done. We found PGH synthase in lipid bodies of human lung mast cells, alveolar macrophages, Type II alveolar pneumocytes, and neutrophils. Specific secretory and lysosomal granules and plasma membranes did not express PGH synthase. Specificity controls, including omission of the primary antibody or substitution with an irrelevant antibody, were negative. Absorption of the specific PGH synthase antibody with purified solid-phase PGH synthase resulted in a marked reduction of label in lipid bodies of all four cell types. These findings establish the presence of PGH synthase in lipid bodies of human lung mast cells, alveolar macrophages, Type II alveolar pneumocytes, and neutrophils and, in concert with previous studies, suggest that these cytoplasmic lipid-rich organelles may be non-membrane sites of eicosanoid formation.


Involvement of the same region of the T cell antigen receptor in thymic selection and foreign peptide recognition. The Ag receptor (TCR) on T lymphocytes has been shown to be specific for foreign antigenic peptides bound to MHC-encoded molecules. During T cell differentiation in the thymus this same TCR mediates the recognition of MHC molecules in the absence of foreign Ag, a process termed positive selection. To analyze the structural relationship between MHC-restricted Ag recognition and positive selection, we characterized two different transgenic lines of mice bearing TCR specific for pigeon cytochrome c and the Ek class II MHC molecule. The two TCR expressed in these animals differed by only one amino acid in the V-J junction of the alpha-chain. In vitro, we find that this TCR difference alters Ag fine specificity. Analysis of transgenic animals demonstrates that this change in the putative third complementarity determining region of the TCR also alters the specificity of positive selection in the thymus. These results suggest that the diversity of a TCR region that can be shown to affect the specificity of foreign Ag recognition may be influenced by selection in the thymus. The findings presented here are discussed in relation to the possible role of self-peptides in positive selection.


Selective decrease in cell surface expression and mRNA level of the 55-kDa tumor necrosis factor receptor during differentiation of HL-60 cells into macrophage-like but not granulocyte-like cells. Expression of the two known receptors for TNF was studied in the promyelocytic leukemia cell line HL-60 before and after differentiation of the cells along the granulocyte lineage (induced by incubation with retinoic acid), or along the macrophage lineage (induced by incubation with the phorbol diester, PMA). The extent of inhibition of TNF binding by receptor-specific antisera, as well as the size of the complexes formed after cross-linking TNF to its receptors on intact cells, indicated that both receptor species were expressed on the surface of the undifferentiated HL60 cells. Differentiation into granulocyte-like cells resulted in some increase in TNF binding. The increase was apparently due to enhanced expression of the 75-kDa TNF-R, whereas the amounts of the 55-kDa TNF-R did not change significantly. In contrast, in HL-60 cells induced to differentiate into macrophage-like cells, expression of the 55-kDa TNF-R species was completely abolished. The pattern of TNF-R expression in the differentiated HL-60 cells was similar to that observed in leukocytes isolated from peripheral blood: on granulocytes, there were about equal amounts of both receptor species, whereas on monocytes the 75-kDa receptor was predominant. The loss of 55-kDa receptors during differentiation of HL-60 cells into macrophage-like cells was accompanied by a pronounced decrease in the level of the mRNA for that receptor, suggesting that at least part of the change in TNF-R expression is due to mechanisms that control the amounts of receptor mRNA. Although little is yet known regarding the functional differences between the two receptor species, marked changes in the pattern of their expression, as observed during HL-60 cell differentiation, are likely to alter the kind of response of the cells to TNF and may therefore play an important role in the coordination of TNF effects in the organism.


Functional nerve growth factor receptors on human B lymphocytes. Interaction with IL-2. In addition to its neurotrophic activity, nerve growth factor (NGF) has been shown to interact with cells of the immune system. We have characterized the effects of NGF on human B cell proliferation and the regulation of NGF receptor expression on these cells. Nerve growth factor receptors were expressed on all tonsillar and peripheral blood B cells and this expression was increased upon activation of the cells. NGF augmented the mitogenic effect of the T-independent B cell mitogen, Staphylococcus aureus Cowan I strain, and provided a progression signal to competent B cells. The proliferative response was augmented when the progression signal provided by NGF was combined with that provided by IL-2 but not with IL-4. One effect of the interaction between NGF and IL-2 appears to occur at the receptor level, because each of these ligands increased the expression of the receptor for the other ligand, whereas IL-4 was without effect. These results demonstrate the expression of functional receptors on human B lymphocytes, the involvement of NGF in immuno-regulation, and indicate that NGF may act as a B cell growth factor.


Differential responsiveness of human neutrophils to the autocrine actions of 1-O-alkyl-homologs and 1-acyl analogs of platelet-activating factor. The phlogistic actions of six molecular species of platelet-activating factor (PAF) (1-O-alkyl-PAF homologs, 16:0-, 18:0- and 18:1-alkyl-PAF, 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine (AGEPC) and their respective 1-acyl-PAF analog counterparts, 16:0-, 18:0- and 18:1-acyl-PAF, 1-acyl-2-acetyl-sn-glycero-3-phosphocholine (AGPC)) were assessed relative to five human neutrophilic polymorphonuclear leukocyte (PMN) functional responses: 1) lysosomal enzyme secretion; 2) specific desensitization to 16:0-AGEPC-induced lysosomal enzyme secretion; 3) O2- production; 4) chemotaxis; and 5) priming for enhanced O2- production. With respect to inducing lysozyme secretion, 18:0-AGEPC was 30- and 75-fold less potent than 16:0-AGEPC and 18:1-AGEPC, respectively, and was 25- and 40-fold less potent for inducing beta-glucuronidase secretion. 18:0-AGEPC was also 10-fold less active than 18:1- and 16:0-AGEPC for inducing O2- production. Thus, the rank order of potency of the alkyl-PAF homologs for inducing both lysosomal enzyme secretion and O2- production was 18:1- greater than or equal to 16:0- much greater than 18:0-AGEPC. In contrast, these three alkyl-PAF homologs had the same potency for desensitizing PMN to subsequent 16:0-AGEPC-induced lysosomal enzyme secretion and for priming PMN for augmented O2- production in response to FMLP or human recombinant C5a. Paradoxically, however, the rank order of potency of the alkyl-PAF homologs for effecting PMN chemotaxis was 18:0- greater than 18:1- much greater than 16:0-AGEPC. At concentrations as high as 1.0 microM, the acyl-PAF analogs did not initiate PMN lysosomal enzyme secretion, O2- production, or chemotaxis. However, the acyl-PAF analogs induced partial PMN desensitization to 16:0-AGEPC. A novel finding of potential (patho)-physiologic significance was the ability of acyl-PAF at nM concentrations to prime PMN for significantly enhanced O2- production after stimulation with FMLP or human recombinant C5a. The priming action of acyl-PAF was due to an increase in the rate as opposed to a prolongation of O2- production. The differing rank orders of potency of the alkyl-PAF homologs and acyl-PAF analogs for stimulating several physiologic responses of the same target cell, the human PMN, support the premise that there may be more than one PAF receptor subtype on the PMN and/or that differences in the biophysical properties of the various molecular species of PAF modulate their interaction with PAF receptor(s) linked to stimulus-response coupling.


In vitro, Candida albicans releases the immune modulator adenosine and a second, high-molecular weight agent that blocks neutrophil killing. We previously described a lyophilized supernatant from germinated Candida albicans that blocks human neutrophil (PMN) O2- production and degranulation stimulated by several PMN agonists but does not block stimulation by PMA. In studies to further characterize this Candida hyphal inhibitory product (CHIP), we noted several physicochemical parallels with the purine nucleoside adenosine (Ado). A Sephadex G-10 semipurified fraction of CHIP had an absorption peak near 260 nm, an apparent m.w. of less than 400, and was resistant to boiling and proteases. Maximally effective doses of CHIP (100 micrograms/ml) and Ado (100 microM) blocked 0.1 microM FMLP-stimulated O2- production by 76.8 +/- 4.1 and 81.7 +/- 4.8%, respectively. Ado deaminase, known to inactivate Ado, reversed inhibition by both Ado and CHIP. Results were comparable for the effect of CHIP and Ado on FMLP-stimulated beta-glucuronidase and lactoferrin release. Activation of the respiratory burst by opsonized C. albicans yeast was also inhibited by CHIP and Ado, but the extent of inhibition was less than for FMLP. At yeast:PMN ratios of 4:1, 10:1, and 40:1, CHIP inhibited O2- by -3.8%, 14.3%, and 12.8%, respectively; Ado blocked production by 32.9%, 24.2%, and 11.5%, respectively. The effect of CHIP and Ado on Candida killing by PMN was compared using two viability assays in each of four experiments. Ado (100 microM) had no effect on killing, although CHIP (100 micrograms/ml) inhibited killing in the MTT assay at 15 and 45 min by 81.6 +/- 6.3 and 24.7 +/- 6.2%, respectively; as assayed by CFU, CHIP inhibited killing by 34.1 +/- 6.2 and 10.3 +/- 2.5%, respectively. The ability of CHIP to inhibit killing was not affected by adding Ado deaminase, providing additional evidence that an Ado-like effect by CHIP is not essential for killing inhibition. Killing of opsonized Streptococcus pneumoniae was also inhibited in a concentration-dependent manner. Reverse-phase HPLC of the semipurified fraction revealed a peak, eluting identically to authentic Ado, which was eliminated by adding Ado deaminase. Ado content of the G-10 fraction was sufficient to account fully for the FMLP-inhibitory activity. The antikilling activity was resistant to boiling and proteases but was eliminated by mild periodation. Fractions eluting from a Sephadex CL6B column between 0.8 and 2.0 x 10(6) m.w. had increased sp. act. for killing inhibition. Sp. act. increased as carbohydrate content increased, but killing inhibition by various Candida cell wall constituents was absent to modest compared to inhibition induced by CHIP.(ABSTRACT TRUNCATED AT 400 WORDS)


Asymmetric selection of T cell antigen receptor alpha- and beta-chains in HLA-B27 alloreactivity. Endogenous peptides constitutively bind to class I MHC Ag and are thought to be integral parts of allospecific T cell epitopes. However, allospecific TCR can recognize structural features of the alloantigen as foreign. To define some crucial parameters determining HLA-B27 allorecognition, the structure of TCR alpha- and beta-chains from HLA-B27-specific CTL was analyzed. A strategy, based on V alpha and V beta family-specific oligonucleotides, was used for specific amplification and direct sequencing of TCR-alpha and -beta cDNA. We observed nonrandom usage of V beta segments and recurrent structural motifs within beta-chain junctional regions. In contrast, no structural restrictions were apparent among alpha-chains, even from CTL clones of related fine specificity. These results indicate an asymmetric contribution of TCR alpha- and beta-chains to HLA-B27 allospecificity among the CTL clones analyzed. They suggest recognition of multiple peptides and involvement of beta-chain junctional regions in recognizing shared motifs among some of these peptides.


cDNA for Mo3, a monocyte activation antigen, encodes the human receptor for urokinase plasminogen activator. We have cloned the cDNA for Mo3, an activation Ag expressed by human monocytes and myelomonocytic cell lines after stimulation by PMA, LPS, muramyl dipeptide, certain cytokines, and cAMP agonists. We have previously shown that Mo3 expression in vivo is associated predominantly with macrophages in inflammatory sites. Mo3 is a highly glycosylated protein of about 50 kDa in monocytes and U-937 cells and is anchored to the plasma membrane by glycosyl-phosphatidylinositol linkage. We purified Mo3 protein by cleavage from the U-937 cell surface with phosphatidylinositol-specific phospholipase C, followed by affinity chromatography using a mAb. An internal peptide sequence was determined and used to design oligonucleotide probes for screening an expression cDNA library. Nucleotide sequencing indicated that the complete coding sequence encodes 335 amino acids, including a predicted signal peptide of 22 residues and a hydrophobic C-terminal portion that is probably cleaved during formation of the GPI linkage. The resulting mature protein of about 290 amino acids is consistent with the 29-kDa molecular mass of deglycosylated Mo3. A Northern blot of RNA from U-937 cells revealed a 1.5-kb band that was induced by PMA treatment. Mo3 cDNA was transfected into Cos cells and surface expression of Mo3 was detected by ELISA using various anti-Mo3 mAb. We performed a computer search of the National Biomedical Research Foundation database and found that Mo3 is identical to the human receptor for the urokinase plasminogen activator (uPA-R). Purified soluble Mo3, as well as anti-Mo3 antibodies, were able to block uPA binding to its receptor on U-937 cells, indicating that Mo3 is indeed uPA-R. The use of these anti-Mo3 antibodies may be helpful in assessing the role of uPA-R in processes such as inflammation and tumor invasion.


Relationship between increased cyclic AMP-phosphodiesterase activity and abnormal adenylyl cyclase regulation in leukocytes from patients with atopic dermatitis. Atopic dermatitis (AD) is characterized by a variety of abnormal physiologic and pharmacologic responses in the skin. Leukocyte abnormalities of the cyclic nucleotide system include increased cAMP phosphodiesterase (PDE) and adenylyl cyclase activities. We have evaluated the possibility that a defect of the inhibitory GTP-binding protein (Gi) might cause inadequate modulation of adenylyl cyclase activity in AD leukocytes. We carried out a series of studies assessing adenylyl cyclase and Gi subunits in monocyte membranes. Using both pertussis toxin ribosylation and direct monoclonal antibody labeling of Gi proteins, we have shown evidence for a decrease or possible absence of one of the Gi proteins in atopic monocyte membranes. A genetic defect or toxin-mediated abnormality in leukocyte membrane Gi could account for these findings. Increased cAMP degradation by PDE may be a compensatory mechanism for increased cAMP synthesis that is regulated by GTP-binding proteins. But this increased PDE activity also rendered AD leukocytes hypo-responsive to immunofunction regulatory signals mediated by cAMP.


Structure, functions, and mechanisms of substance P receptor action. Substance P is a member of a family of structurally related peptides, called tachykinins, that are involved in the regulation of many biologic processes. Diversity in the generation of multiple tachykinin peptides arises due to multiple genes encoding these peptides as well as by mechanisms of alternative RNA processing and differential posttranslational processing. The multiple peptides are neurotransmitters and/or neuromodulator substances, and they bring about their actions mainly by activating three primary types of receptors, NK-1, NK-2, and NK-3. The pharmacology and tissue locations of these receptor sites are discussed, as is their involvement in certain biologic responses. These three receptor sites have been molecularly characterized by cDNA cloning and functional expression, and all are members of the superfamily of receptors coupled to G-regulatory proteins. Second messenger systems established to be activated by tachykinin receptor stimulation include the hydrolysis of inositol containing phospholipids by a phospholipase C mechanism. The role of substance P in neurogenic inflammation and plasma extravasation is briefly discussed. The generation of new research tools recently in the tachykinin field should allow for a detailed examination of the mechanisms of peptide action, including a focus on receptor structure-function relations and regulation of receptor sensitivity.


Receptor-initiated activation of cells and their oncogenes by herpes-family viruses. The interaction of human cytomegalovirus (HCMV) with the cell membrane has been shown to initiate a cascade of physiologic and biochemical responses that result in the transcriptional activation of specific cellular proto-oncogenes. The cell-activation responses initiated by the virus membrane interaction appear to be important for efficient HCMV replication, as pharmacologic inhibition of cell activation responses significantly reduces the expression of immediate early viral genes and the production of infectious progeny virus. Cellular receptor proteins for other viruses have been shown to be molecules with physiologic activities. Binding of virus to these receptors may trigger the cell to initiate changes that are important for efficient viral replication. These viruses may also trigger inappropriate physiologic responses in the absence of viral replication, thereby causing more covert manifestations of viral pathology.


Retinoic acid receptors and binding proteins in human skin. Nuclear retinoic acid receptors (RAR) are likely to mediate many of the pleiotypic cutaneous actions of retinoids by acting as ligand-dependent enhancer factors. The presence of nuclear RAR in skin was confirmed by identification of a 45-kDa nuclear RA binding activity by fast protein liquid chromatography (FPLC). Analysis of RNA extracted from skin specimens demonstrated expression of RAR-alpha and RAR-gamma transcripts, as well as expression of the homologous low-affinity receptor, RXR-alpha. Both isoforms of RAR-gamma RAR-gamma 1 and RAR-gamma 2 were detectable, with RAR-gamma 1 being the more strongly expressed. FPLC analysis also demonstrated a 15-kDa peak of specific RA binding activity, consistent with the presence of cellular retinoic acid binding protein (CRABP). Of the two known forms of CRABP, CRABP-II was much more strongly expressed than CRABP-I at the level of steady-state mRNA. CRABP-II was also expressed in keratinocytes and fibroblasts in vitro. CRABP-II was up-regulated by agents that induce keratinocyte differentiation, and inhibited by prolonged exposure to high concentrations of RA. In contrast, CRABP-II was consistently induced by RA in dermal, but not in lung fibroblasts. CRABP-I was expressed at low to undetectable levels under all these conditions. The presence of tissue-specific and differentiation-related regulation of CRABP-II suggests that it may be an important regulator of RA action in human skin.


Excess insulin binding to insulin-like growth factor receptors: proposed mechanism for acanthosis nigricans. Clinical and epidemiologic evidence has shown acanthosis nigricans to be closely related to defective tissue utilization of insulin in a number of previously recognized (e.g., obesity, lipodystrophy, and leprechaunism) as well as recently characterized (e.g., type A and type B syndromes) disorders. This article reviews the relationship of acanthosis nigricans to these insulin-resistant states. It also focuses attention on the possibility that interaction between excessive amounts of circulating insulin with insulin-like growth factor receptors on keratinocytes and dermal fibroblasts leads to the development of acanthosis nigricans.


The genetics of encapsulation in Haemophilus influenzae. In Haemophilus influenzae type b (Hib) strains the cap locus with very few exceptions contains an unstable direct repeat of approximately 17 kb of DNA flanking an approximately 1-kb bridge region containing the gene bexA. Each repeat contains genes necessary for polysaccharide synthesis, export, and surface expression, with BexA a critical component of the polysaccharide exporter. Only rare Hib strains have been identified in which cap lacks a direct repeat, though this is the norm for non-b serotypes. Examination of the ends of this single-copy locus shows that cap has the structure of a compound transposon: Copies of the insertion element IS1016 flank the gene cluster. This gives strains the capacity to amplify genes at cap by unequal homologous recombination. The cap duplication in Hib strains--subserving augmented production of polysaccharide--has apparently arisen in this way and become fixed in the population through deletion of one copy of bexA.


Serum angiotensin-1 converting enzyme activity processes a human immunodeficiency virus 1 gp160 peptide for presentation by major histocompatibility complex class I molecules. T cell stimulation by the human immunodeficiency virus 1 gp160-derived peptide p18 presented by H-2Dd class I major histocompatibility complex molecules in a cell-free system was found to require proteolytic cleavage. This extracellular processing was mediated by peptidases present in fetal calf serum. In vitro processing of p18 resulted in a distinct reverse phase high performance liquid chromatography profile, from which a biologically active product was isolated and sequenced. This peptide processing can be specifically blocked by the angiotensin-1 converting enzyme (ACE) inhibitor captopril, and can occur by exposing p18 to purified ACE. The ability of naturally occurring extracellular proteases to convert inactive peptides to T cell antigens has important implications for understanding cytotoxic T lymphocyte responses in vivo, and for rational peptide vaccine design.


Mls-1-like superantigen in the MA/MyJ mouse is encoded by a new mammary tumor provirus that is distinct from Mtv-7. Mls-1 is an endogenous superantigen that leads to in vivo deletion and in vitro stimulation of T cell receptor (TCR) V beta 6-, 7-, 8.1-, and 9-expressing cells. The MA/MyJ mouse deletes the identical set of TCR from its mature T cell repertoire; however, it does not contain Mtv-7, the murine mammary tumor provirus (MMTV), whose sag gene encodes Mls-1. Interestingly, the superantigen activity of this mouse strain segregates with a new mammary tumor provirus, Mtv-43, not seen in other inbred strains. The predicted amino acid sequence of the sag gene of Mtv-43 was compared with that of Mtv-7. Strikingly, the COOH terminus of the two molecules is very similar, while all other MMTV-encoded superantigens differ 100% in this segment.


An exogenous mouse mammary tumor virus with properties of Mls-1a (Mtv-7). The classical minor lymphocyte stimulating (Mls) antigens, which induce a strong primary T cell response in vitro, are closely linked to endogenous copies of mouse mammary tumor viruses (MMTV). Expression of Mls genes leads to clonal deletion of T cell subsets expressing specific T cell receptor (TCR) V beta chains. We describe the isolation and characterization of a new exogenous (infectious) MMTV with biological properties similar to the Mls antigen Mls-1a. In vivo administration of either Mls-1a-expressing B cells or the infectious MMTV (SW) led to an increase of T cells expressing V beta 6 followed by their deletion. Surprisingly, different kinetics of deletion were observed with the exogenous virus depending upon the route of infection. Infection through the mucosa led to a slow deletion of V beta 6+ T cells, whereas deletion was rapid after subcutaneous infection. Sequence analysis of the open reading frames in the 3' long terminal repeat of both this exogenous MMTV (SW) and of Mtv-7 (which is closely linked to Mls-1a) revealed striking similarities, particularly in the COOH terminus, which has been implicated in TCR V beta recognition. The identification of an infectious MMTV with the properties of a strong Mls antigen provides a new, powerful tool to study immunity and tolerance in vivo.


Interallelic V(D)J trans-rearrangement within the beta T cell receptor gene is infrequent and occurs preferentially during attempted D beta to J beta joining. Previous work has demonstrated that intergenic V(D)J rearrangement, a process referred to as trans-rearrangement, occurs at an unexpectedly high frequency. These rearrangements generate novel V(D)J combinations which could conceivably have some role in the normal immune system, and since they probably arise through chromosomal rearrangements akin to those associated with lymphoid neoplasia, they may also serve as a model for investigating recombinational events which underlie oncogenesis. In view of the existence of a mechanism that permits relatively frequent intergenic trans-rearrangements, it seems reasonable that interallelic trans-rearrangements involving segments belonging to each of the two alleles of a single antigen receptor gene might also occur. To determine the frequency of such rearrangements, we examined thymocytes of F1 progeny of a cross between SWR mice, which have a deletion spanning 10 of the known V beta segments, and NZW mice, which have a deletion involving all J beta 2 segments. Rearranged TCR-beta genes containing V beta segments from the NZW chromosome and J beta segments from the SWR chromosome were amplified from the DNA of F1 thymocytes with the polymerase chain reaction. Using this approach, we found that such rearrangements are relatively uncommon, being present in about 1 in 10(5) thymocytes, a frequency lower than that of V gamma/J beta intergenic trans-rearrangements. The ratio of conventional cis-rearrangement to interallelic trans-rearrangement for any particular V beta segment appears to be about 10(4):1. The structure of the junctions in all trans-rearrangements analyzed closely resembles conventional cis-rearrangements, indicating involvement of V(D)J recombinase in the ultimate joining event. However, in contrast to cis-rearrangements, a strong bias for inclusion of D beta 1 segments over D beta 2 segments was noted, suggesting that interallelic trans-rearrangement may occur preferentially during attempted D-J joining. J beta 2 segment usage in trans-rearrangements also appeared to differ from that expected from previously studied cis-rearrangements. The results have implications with respect to the events and timing of conventional cis-rearrangement during thymocyte differentiation, and the prevalence of various types of trans-rearrangements.


Transition from interleukin 1 beta (IL-1 beta) to IL-1 alpha production during maturation of inflammatory macrophages in vivo. In situ production of interleukin 1 alpha (IL-1 alpha) and IL-1 beta was investigated in Peyer's patches (PP) of mice undergoing an acute bacterial infection with Yersinia enterocolitica O8. Synthesis of IL-1 beta, as determined by immunohistochemistry, was found primarily in monocytes migrating into the inflamed PP. In comparison, synthesis of IL-1 alpha was temporarily delayed by at least 24 h and was only found in mature macrophages, which did not produce detectable levels of IL-1 beta. This indicates a transition from IL-1 beta to IL-1 alpha production during maturation of monocytes into inflammatory macrophages, and further emphasizes a dichotomy between IL-1 alpha and IL-1 beta.


Two monoclonal antibodies generated against human hsp60 show reactivity with synovial membranes of patients with juvenile chronic arthritis. Heat-shock proteins have been shown to be critical antigens in a number of autoimmune diseases. In human arthritis and in experimentally induced arthritis in animals, disease development was seen to coincide with development of immune reactivity directed against not only bacterial hsp60, but also against its mammalian homologue. We have developed murine monoclonal antibodies after immunization with recombinant human hsp60. Antibodies with unique specificity for mammalian hsp60, not crossreactive with the bacterial counterpart (LK1), and antibodies recognizing both human and bacterial hsp60 (LK2) were selected. Both antibodies recognize epitopes located between amino acid positions 383 and 447 of human hsp60. In immunogold electron microscopy, the mitochondrial localization of hsp60 in HepG2 cells was shown. Furthermore, both LK1 and LK2 showed a raised level of staining in light microscopy immunohistochemistry of synovial membranes in patients with juvenile chronic arthritis. The increased staining for LK1, with a unique specificity for mammalian hsp60, thus unequivocally demonstrates that this is due to a raised level of expression of endogenously produced host hsp60 and not to deposition of bacterial antigens.


Calcium currents in the A7r5 smooth muscle-derived cell line. An allosteric model for calcium channel activation and dihydropyridine agonist action. We have investigated the gating kinetics of calcium channels in the A7r5 cell line at the level of single channels and whole cell currents, in the absence and presence of dihydropyridine (DHP) calcium channel agonists. Although latencies to first opening and macroscopic currents are strongly voltage dependent, analysis of amplitude histograms indicates that the primary open-closed transition is voltage independent. This suggests that the molecular mechanisms for voltage sensing and channel opening are distinct, but coupled. We propose a modified Monod-Wyman-Changeux (MWC) model for channel activation, where movement of a voltage sensor is analogous to ligand binding, and the closed and open channels correspond to inactive (T) and active (R) states. This model can account for the activation kinetics of the calcium channel, and is consistent with the existence of four homologous domains in the main subunit of the calcium channel protein. DHP agonists slow deactivation kinetics, shift the activation curve to more negative potentials with an increase in slope, induce intermingled fast and slow channel openings, and reduce the latency to first opening. These effects are predicted by the MWC model if we make the simple assumption that DHP agonists act as allosteric effectors to stabilize the open states of the channel.


Analysis of the mutant proBA operon from a proline-producing strain of Serratia marcescens. The nucleotide sequence of the proBA operon from a proline-hyperproducing mutant of Serratia marcescens was determined. Two base substitutions were found: one in the proB structural gene, coding for gamma-glutamyl kinase (GK), and a second one in the promoter region of the operon. The former base substitution led to a change of the predicted amino acid at position 117 from an alanine to a valine in GK. This mutation rendered GK 700-fold less sensitive to proline-mediated feedback inhibition than the wild-type enzyme. The other base substitution, a transversion from a G-C to an A-T, was located in the spacer region between the '-35' and '-10' sequences of the promoter, and it increased the transcriptional activity of this operon fourfold. Both these two base substitutions, which were acquired at the step of selecting mutants resistant to a toxic proline analogue, 3,4-dehydroproline, confer upon cells a high proline productivity and an increased osmotolerance.


Molecular cloning and expression of a spike protein of neurovirulent murine coronavirus JHMV variant cl-2. A cDNA encoding the spike (S) protein of the neurovirulent murine coronavirus JHMV variant cl-2 was isolated and sequenced. Analysis of the cDNA revealed that the S protein consists of 1376 amino acids, as does the S protein of mouse hepatitis virus 4. We inserted the cDNA into the genome of vaccinia virus to obtain a recombinant vaccinia virus (rVV). The S protein expressed in RK13 cells infected by the rVV was shown to be electrophoretically and immunologically indistinguishable from the S protein produced in DBT cells infected with cl-2 virus. RVV infection of rats and mice induced S protein-specific antibody production detectable by immunofluorescence and neutralization. Moreover, the S protein expressed by the rVV induced syncytium formation not only in mouse DBT and L cells, which are susceptible to cl-2 virus infection, but also in rabbit RK13 cells, which are not susceptible to cl-2 virus infection. This result suggests the possibility that RK13 cells have binding sites for the cl-2 virus S protein.


Analysis of a new hepatitis C virus type and its phylogenetic relationship to existing variants. Sequences obtained in the 5' non-coding region (5'NCR) of hepatitis C virus (HCV) were obtained from Scottish blood donors and compared with previously published HCV sequences. Phylogenetic analysis revealed the existence of three distinct groups of sequences; two of these corresponded to the recently described HCV types 1 and 2 variants, while viral sequences detected in around a third of the blood donors formed a separate phylogenetic group that probably represents infection with a novel virus species. Nucleotide sequences of this latter group differed from all previously published 5'NCR sequence variants by at least 9%. This new virus type also differed considerably from previously published variants in other regions of the viral genome (core, NS-3 and NS-5), with corrected nucleotide distances of 15, 43 and 49% respectively from the prototype HCV-1 sequence. Formal phylogenetic analysis of each of the coding regions confirmed that HCV type 1 variants could be clearly differentiated into regional variants (Far East and U.S.A./European), in contrast to the clearly overlapping geographical distributions of the main HCV types in U.K. blood donors. We discuss the evidence for and against the hypothesis that the three main phylogenetic groups identified in this study represent separate species of HCV.


Simian varicella virus: characterization of virion and infected cell polypeptides and the antigenic cross-reactivity with varicella-zoster virus. Simian varicella virus (SVV) causes a varicella-like disease in non-human primates. In this study, SVV virions were purified from SVV-infected BSC-1 cells by zonal and differential gradient centrifugation and the virion polypeptide composition was analysed by SDS-PAGE. SVV virions had a buoyant density of 1.21 g/ml, identical to the value obtained for varicella-zoster virus (VZV) virions purified by the same method. Electron microscopy of the concentrated SVV virions revealed characteristic herpesvirus morphology. SVV virions consisted of at least 30 polypeptide species ranging from 16K to greater than 200K. The electrophoretic profiles of radiolabelled SVV and VZV virion polypeptides were very similar. Immunoprecipitations of solubilized SVV-infected cell preparations using SVV immune sera revealed at least 18 viral polypeptides with an Mr range of 12K to 142K and six glycoproteins ranging from 46K to 115K. In addition, extensive cross-reactivity between SVV and VZV proteins and glycoproteins was demonstrated by immunoprecipitation with heterologous immune sera. The high degree of antigenic relatedness between SVV and VZV provides further support for simian varicella as a model for VZV infections.


Molecular cloning and physical mapping of the genome of simian herpes B virus and comparison of genome organization with that of herpes simplex virus type 1. The molecular structure of the genome of simian herpes B virus (SHBV) was determined by restriction endonuclease mapping studies. Genomic DNA was cleaved with restriction endonucleases BamHI and SalI into 41 and 58 fragments, respectively. Most of these fragments were cloned into the plasmid vector pACYC184; uncloned fragments were identified following isolation from agarose gels. Terminal fragments were identified by exonuclease digestion and radioactive end-labelling, and linkage of fragments was deduced by a combination of single and double digest experiments and cross-blot hybridizations. The genome is larger than that of herpes simplex virus type 1 (HSV-1), being approximately 165 kilobase pairs. Like that of HSV-1, the SHBV genome is composed of a long and a short unique region each flanked by inverted repeat sequences, which allow the unique regions to invert relative to one another, resulting in four possible isomeric arrangements of the molecule. Genome locations of several SHBV genes were compared with their HSV-1 homologues.


Identification of the equine herpesvirus type 1 glycoprotein 17/18 as a homologue of herpes simplex virus glycoprotein D. The DNA sequence of the equine herpesvirus type 1 (EHV-1) gD gene homologue has been determined for the strain Ab1 and compared with previously published sequences. A portion of the gene has been located to a region of the genome which also encodes homologues of the herpes simplex virus type 1 genes for gE and gI and is known to encode an epitope of the virion protein gp17/18. Analysis of the EHV-1 strain Kentucky A (KyA) by DNA hybridization showed the presence of a gD gene homologue and established the absence of genes for gI and gE. Western blot analysis, however, showed that KyA virus particles contain gp17/18, thus indicating that this protein is encoded by the gD gene homologue. The KyA gp17/18 was found to be smaller than that detected in other strains and this is accounted for by a frameshift mutation in the KyA sequence relative to Ab1. The mutation in the KyA strain results in an altered C-terminal sequence and could explain the apparent structural differences suggested by the reactivities with monoclonal antibodies (MAbs). We have also expressed part of the Ab1 gD gene as a fusion protein with glutathione S-transferase in Escherichia coli and shown that this reacts with the MAb 5H6 originally used to map gp17/18. These experiments establish that gp17/18 is encoded by the gD gene homologue.


Detection of human serum antibodies that neutralize infectious human papillomavirus type 11 virions. A selection of human sera were tested for the presence of antibodies that neutralized infectious human papillomavirus (HPV) type 11. Neutralizing antibodies were detected by prevention of HPV-11-induced condylomatous transformation of human foreskin chips transplanted subrenally into athymic mice. Test sera were obtained from 21 female patients with genital condylomas and eight patients with laryngeal papillomas. Control patients consisted of 57 adult random blood donors and five asymptomatic children. ELISAs demonstrated that all sera from patients with genital papillomas were strongly reactive to disrupted papillomavirus (PV) antigens of HPV-11, bovine PV type 1 and cottontail rabbit PV, but only two were weakly reactive to intact HPV-11. None of the eight sera from the laryngeal papilloma bearers reacted significantly to disrupted PV antigens, but four of the eight showed strong specific responses to intact HPV-11 only. The majority of the sera that were reactive to intact HPV-11 by ELISA neutralized HPV-11 infectivity in the athymic mouse xenograft system. The data indicated that ELISA reactivity to intact HPV-11 virions was a good predictor for the presence of HPV-11 neutralizing antibodies.


T cell responses to the human papillomavirus type 16 E7 protein in mice of different haplotypes. The response of murine T cells to the E7 molecule of human papillomavirus type 16 (HPV-16) was studied using eight different mouse strains of six distinct H-2 haplotypes. HPV-16 E7 protein was prepared as a fusion protein with glutathione-S-transferase, purified by affinity chromatography and used for immunization. Cells from the lymph nodes were cultured with whole fusion protein, glutathione-S-transferase or HPV-16 E7 protein synthetic peptides. All the mouse strains tested, with the exception of BALB/c, recognized the E7 molecule, as evidenced by a proliferative response to at least two of the peptides. The profile of responses to peptides varied between and within a strain, but five distinct immunodominant regions could be identified. These regions were defined on the basis of a reaction to one or more peptides in a given part of the E7 molecule by at least four strains. The five regions were encompassed by amino acid residues 1 to 9, 17 to 32, 42 to 59, 62 to 77 and 87 to 98. The findings suggest that in an outbred population, such as man, the E7 molecule of HPV-16 would be recognized by a large proportion of the population. However, the poor response of two mouse strains [B10.RIII (71NS) and BALB/c] could also have a corollary in man.


Mutagenesis of human papillomavirus types 6 and 16 E7 open reading frames alters the electrophoretic mobility of the expressed proteins. The E7 open reading frames of human papillomavirus type 6 (HPV-6) and HPV-16 encode proteins consisting of 98 amino acids that are quite similar in sequence yet different in electrophoretic mobility. Moreover, these proteins vary strikingly in oncogenicity. To investigate the molecular basis of the differences in structure and function, site-directed mutagenesis was used to exchange non-conserved amino acid residues between the two proteins. The mutated coding regions were expressed as fusion proteins in Escherichia coli and identified by Western blotting. Comparative analysis of the affinity-purified mutated E7 fusion proteins in polyacrylamide slab mini-gels in the presence of SDS and 2-mercaptoethanol revealed altered electrophoretic mobilities. This analysis suggests that the aspartic acid at residue 4 (Asp 4) contributes to the characteristic aberrant migration of the HPV-16 E7 protein in SDS-polyacrylamide gels.


Synchronous appearance of antigen-positive and latently infected neurons in spinal ganglia of mice infected with a virulent strain of herpes simplex virus. Studies with replication-defective mutants of herpes simplex virus (HSV) have defined the minimum requirements for establishment of latency, but their behaviour may not reflect the course of events following infection by wild-type HSV, in which ability to express viral genes has not been precluded by a genetic lesion. To address this issue we devised a strategy for studying establishment of latency by a virulent strain of HSV, based on the distinctive molecular characteristics of latently infected neurons. By combining in situ hybridization for detection of latency-associated transcripts with immunohistochemical analysis of viral proteins we demonstrate here that antigen-positive and latently infected neurons appear synchronously in spinal ganglia during the earliest stages of acute ganglionic infection. This is consistent with early divergence of the molecular pathways leading to productive and latent infection, supporting and extending the results obtained with viral mutants.


Two patterns of persistence of herpes simplex virus DNA sequences in the nervous systems of latently infected mice. The number of herpes simplex virus (HSV) genome equivalents recovered from latently infected mouse spinal ganglia was compared with the proportion of neurons containing latency-associated transcripts (LATs). Two distinct patterns of HSV persistence were observed, depending on the anatomical location of ganglia with respect to the site of cutaneous inoculation. The location of the bulk of latent viral DNA did not correspond with the highest prevalence of LAT+ neurons. Viral DNA was most abundant in spinal ganglia directly innervating the inoculation site and the amount recovered, which was similar to that found previously in human trigeminal ganglia, suggested that LAT+ neurons each contain hundreds of copies of HSV DNA. In stark contrast, although LAT+ neurons were most abundant in neighbouring ganglia, viral DNA was scarce (approx. 20 copies/LAT+ cell). These data indicate that amplification of HSV DNA sequences is greatest in ganglia previously shown to be associated with viral antigen expression during the productive phase of primary infection.


Identification and characterization of glycoprotein gp1 of bovine herpesvirus type 4. Three major bovine herpesvirus type 4 (BHV-4) glycoproteins have been described previously. By using monoclonal antibodies produced against BHV-4 envelope proteins from which the three major antigens had been removed by immunoaffinity, a fourth glycoprotein was identified. This protein (gp1) has a high Mr (greater than 300K), is detected about 8 h post-inoculation of infected cells and is strictly expressed as a gamma protein. Moreover, gp1 was identified by a polyclonal antiserum from an infected animal, indicating that this glycoprotein is an antigen recognized by the immune system of infected animals.


Spectroscopic properties of the nitric oxide derivative of ferrous man, horse, and ruminant hemoglobins: a comparative study. The spectroscopic (EPR and absorbance) properties of the nitric oxide derivative of ferrous man, horse, buffalo, deer, mouflon, musk ox, ox, and reindeer hemoglobin (HbNO) have been investigated in the absence of any allosteric effector at pH 6.5 (in 0.1 M 2-[N-morpholino]ethanesulphonic acid/NaOH chloride-free buffer system), as well as at 100 K and/or 20 degrees C. Man and horse HbNO show spectroscopic properties that are generally taken as typical of the high affinity state of ferrous tetrameric Hb's; on the other hand, the spectroscopic properties of ruminant (i.e., buffalo, deer, mouflon, musk ox, ox, and reindeer) HbNO are characteristic of the low affinity conformation. These results are in keeping with the functional properties of the mammalian Hb's considered and have been related to the peculiar low oxygen affinity of ruminant Hb's.


Thermodynamic parameters of the interaction between Co(II) bovine carbonic anhydrase and anionic inhibitors. The pH dependence of the apparent affinity constants of perchlorate for cobalt(II)bovine carbonic anhydrase II has been measured by electronic absorption spectroscopy. The obtained data have been analyzed in terms of the ionization of two acidic groups of CoBCAII, and the affinity of perchlorate for the two water-containing species of the enzyme have been estimated. Furthermore, the affinity constants of nitrate, perchlorate, and azide for CoBCAII in the temperature range 5 degrees C-30 degrees C have been determined by spectrophotometric titrations at pH 7. The affinity constants for these ligands decrease with increasing temperatures. The temperature dependence of binding was used to estimate the enthalpy and entropy parameters for the formation of the corresponding 1:1 adducts. The obtained results indicate that binding of these anions to the cobalt enzyme is an enthalpy driven process which is opposed by a moderate entropy change.


Does thoracic irradiation improve survival and local control in limited-stage small-cell carcinoma of the lung? A meta-analysis. Our main purpose was to determine whether the addition of thoracic radiation therapy to systemic chemotherapy improves 2-year survival, improves local (intrathoracic) tumor control, and affects treatment-related mortality in patients with limited-stage small-cell carcinoma of the lung. Eleven randomized trials addressing this issue were identified using a computerized literature search (Medline and Cancerline) and by polling senior investigators in the field. A meta-analysis was then performed and the results of the trials were analyzed in two ways, the odds ratio (OR) (Peto) method and the risk difference method (Dersimonian and Laird). The overall OR for benefit of thoracic radiation on 2-year survival (ie, the odds of surviving 2 years among patients allocated to radiation compared with the odds of surviving 2 years among patients allocated to control) is 1.53 (95% confidence interval [CI], 1.30 to 1.76; chi 2 = 12.76; P less than .001). The risk difference method showed that radiation therapy improved 2-year survival by 5.4% (95% CI, 1.1% to 9.7%). Local control results were available for only nine studies, the OR for treatment benefit is 3.02 (95% CI, 2.80 to 3.24; chi 2 = 101.48; P less than .0001), and intrathoracic tumor control was improved by 25.3% (95% CI, 16.5% to 34.1%). The OR for excess treatment-related deaths in the thoracic radiation-treated patients was 2.54 (95% CI, 1.90 to 3.18; chi 2 = 8.24; P less than .01). The risk difference for treatment-related deaths was 1.2% (95% CI, -0.6% to 3.0%). This meta-analysis shows a small but significant improvement in survival and a major improvement in tumor control in the thorax in patients receiving thoracic radiation therapy. However, this is achieved at the cost of a small increase in treatment-related mortality.


A comprehensive multiattribute system for classifying the health status of survivors of childhood cancer. A multiattribute health status classification system was devised to describe comprehensively the health status of survivors of childhood cancer. The system consists of seven attributes: sensation, mobility, emotion, cognition, self-care, pain, and fertility. Three to five levels of functioning are defined for each attribute. Any specific combination of seven attribute levels constitutes a health state. In the first survey, the system was used to classify the health status of 20 children currently undergoing therapy for high-risk acute lymphoblastic leukemia (ALL), Wilms' tumor, or neuroblastoma, and eight who had completed treatment. A second survey consisted of 13 children with brain tumors on active treatment. In general, independent ratings by clinicians were in agreement, and consensus was readily achieved in 1 to 2 minutes per patient. Children on therapy experienced a higher burden of morbidity than those off treatment. Brain tumor patients experienced more morbidity than patients in the first survey. The multiattribute system provides a compact but comprehensive tool for long-term follow-up of survivors of childhood cancer. It captures both multiple sequelae and varying levels of severity. By using a mathematical utility function, a single summary score of health-related quality of life may be assigned to each health state. Additional studies to establish reproducibility, validity, responsiveness, and generalizability are indicated.


Hypoxia-induced functional alterations in adult rat neocortex. 1. Brief periods of hypoxia (2-7 min) were induced in rat neocortical slices maintained in an interface-type recording chamber at 34-35 degrees C by changing the aerating gas from 95% O2-5% CO2 to 95% N2-5% CO2. Field potential (FP) and intracellular recordings were obtained in layers II/III of primary somatosensory cortex. Intracellular injection of biocytin revealed the characteristic morphology of supragranular spiny pyramidal neurons. 2. Excitatory synaptic transmission reversibly decreased by 45% as estimated from FP responses to orthodromic stimulation of the underlying white matter/layer VI. Excitatory postsynaptic potentials (EPSPs) were suppressed by 36% in amplitude and recovered within 2-3 min after reoxygenation. During the recovery period, EPSPs showed a reversible increase in duration by 72%. 3. Inhibitory synaptic transmission was completely blocked as determined in FP responses with a paired-pulse inhibition protocol. The fast inhibitory postsynaptic potential (IPSP) declined by 58% during hypoxia. The long-lasting IPSP was suppressed by 75% and showed incomplete recovery. During hypoxia, the amplitude of both IPSPs was significantly more strongly suppressed than the EPSP. 4. In 40% of the cells, hypoxia induced an early anoxic hyperpolarization with a reversal potential of E = -80.8 mV, followed by a postanoxic hyperpolarization (E = -89.4 mV). In a second group of cells (37%), a gradual anoxic depolarization with E = -57.5 mV was observed instead of an early hyperpolarization. In both groups of cells, the anoxic response was associated with a marked decrease in input resistance, by 42 and 31%, respectively. 5. The spike discharge frequency was reversibly suppressed by 71% during hypoxia. A transient hyperexcitability accompanied with a rise in input resistance and discharge rate was observed in 38% of the cells on reoxygenation. 6. The reversal potential of the anoxic hyperpolarization was unaffected by tetrodotoxin (TTX) but was significantly altered by application of the ATP-sensitive K+ channel (KATP) blocker gliquidone. Application of gliquidone additionally resulted in a significantly smaller hypoxia-induced decline in paired-pulse inhibition. 7. Increases in tissue high-energy phosphates induced by preincubating the slices in 25 mM creatine for greater than 2 h had a pronounced protective effect on excitatory and inhibitory synaptic transmission. 8. These data suggest a selective vulnerability of the neocortical inhibitory system during hypoxia. Our results further indicate that hypoxia activates a pre- and postsynaptic KATP conductance because of the decline in intracellular ATP.


Cortical oscillations and temporal interactions in a computer simulation of piriform cortex. 1. A large-scale computer model of the piriform cortex was constructed on the basis of the known anatomic and physiological organization of this region. 2. The oscillatory field potential and electroencephalographic (EEG) activity generated by the model was compared with actual physiological results. The model was able to produce patterns of activity similar to those recorded physiologically in response to both weak and strong electrical shocks to the afferent input. The model also generated activity patterns similar to EEGs recorded in behaving animals. 3. In addition to replicating known physiological responses, it has been possible to use the simulations to explore the interactions of network components that might underlie these responses. This analysis suggests that the physiological properties of the cortex are dependent on the complex interaction of both network and cellular properties. In particular, we have found that the relationship between conduction velocities in intrinsic cortical fiber systems and the time constants of excitatory and inhibitory effects are critical for replicating physiological results. 4. Analysis of the model also suggests a correspondence between the 40-Hz oscillatory patterns of activity induced by low levels of odor-like stimulation and oscillatory patterns seen in lightly anesthetized cortex in response to weak electrical shocks to the afferent fiber system. 5. The specific relationships we have found between the different components of the model also support several speculations on their functional significance. The simulations suggest that during each 40-Hz cycle of EEG activity there is a convergence in rostral cortex of afferent information from the olfactory bulb and recurrent association fiber information from caudal cortex. This convergence could underlie an iterative process central to the recognition of complex olfactory stimuli.


Endocrinological evaluation of ACTH-secreting pituitary microadenomas: their location and alpha-melanocyte stimulating hormone immunoreactivity. It has been hypothesized by Lamberts and coworkers in their analysis of 15 cases that adrenocorticotropic hormone (ACTH)-secreting pituitary adenomas may be derived from either the anterior lobe or the intermediate lobe. The intermediate lobe type of Cushing's disease is thought to be controlled through a hypothalamic pathway and is characterized by hyperprolactinemia; suppressibility of cortisol with bromocriptine, and lower sensitivity to dexamethasone. The authors investigated the validity of this hypothesis in 125 cases of ACTH-secreting pituitary microadenomas by analyzing the endocrine findings, the locations of the microadenomas, and alpha-melanocyte stimulating hormone (alpha-MSH) immunoreactivity in the adenoma cells. No significant differences in the basal hormone levels, cortisol suppressibility with bromocriptine, sensitivity to dexamethasone, and recurrence rate were observed between patients with the microadenoma adjacent to the posterior lobe (considered typical of the intermediate lobe-derived tumor) or those with the microadenoma located in the anterior lobe. The locations of the microadenoma were not correlated with alpha-MSH immunoreactivity in the adenoma cells. No significant differences in endocrine findings were noticed between adenomas positive or negative for alpha-MSH. Thus, Cushing's disease cannot be simply divided into either the anterior lobe type or the intermediate lobe type by endocrinological evaluation as described by Lamberts, et al.


Dietary fat influences Ia antigen expression and immune cell populations in the murine peritoneum and spleen. Peritoneal cells (PEC) and splenocytes were obtained from Listeria monocytogene (LM)-infected or noninfected mice fed a 20% fat diet rich in either (n-3) polyunsaturated fatty acids [(n-3) PUFA diet], linoleate [(n-6) PUFA diet], oleate (MONO diet), or saturated fatty acids (SAT diet) for 6 wk and were assessed for T cells, B cells, macrophages and Ia expression by flow cytometric analysis. In the peritoneum of noninfected mice, dietary fat did not affect total cell yield or the percentage of B cells, macrophages or Ia+ cells, but the (n-3) PUFA-fed group had a greater percentage of T cells than did the other groups. Among the LM-infected mice, the (n-3) PUFA-fed group generally had the highest percentage of B cells and the lowest percentages of T cells, macrophages and Ia+ cells in the peritoneum. Listeria monocytogene infection elevated peritoneal T cell numbers in all mice except the (n-3) PUFA-fed group. The density of Ia molecules on PEC was 40% lower in mice fed the (n-3) PUFA diet. In the spleen, dietary fat also influenced the immune cell populations and Ia+ cells. Two-color staining of spleen cells revealed that Ia+ splenocytes were predominately B cells. These data demonstrate that dietary fats influence Ia expression and immune cell populations and that the effects observed in one immune tissue or cell type may not be readily extrapolated to others.


Zinc deficiency alters barrier function of cultured porcine endothelial cells. Zinc is necessary for normal membrane function and stability. We postulated that Zn deficiency may disrupt the integrity of the vascular endothelium by decreasing its barrier function. To test this hypothesis, endothelial cells were cultured on polycarbonate filters and exposed to media enriched with either 1% fetal bovine serum (FBS) (low FBS; total Zn, 1.07 mumols/L medium) or 5% FBS (control; total Zn, 2.29 mumols/L) or low FBS plus two supplemental levels of Zn, 3.36 and 5.66 mumols total zinc/L. Endothelial cell barrier function, expressed as albumin transfer across cultured endothelial monolayers, was significantly lower in cultures exposed to low FBS compared with control medium. Supplementation with 5.66 mumols total Zn/L completely restored endothelial barrier function. A divalent cation chelator, 1,10-orthophenanthroline, was used to induce Zn deficiency in vitro. Compared with control cultures, the presence of 1,10-orthophenanthroline in the culture medium resulted in markedly lower endothelial barrier function that was increased by the addition of Zn but not calcium or magnesium. Activity of the membrane-bound zinc-dependent angiotensin-converting enzyme (ACE) was depressed by low zinc medium, whereas membrane-bound Ca(2+)-ATPase and total ATPase were not depressed. Furthermore, cells cultivated in low zinc medium did not have greater cytosolic release of adenine, indicating no increase in cell injury or death. These data suggest that Zn is vital to endothelial cell integrity and that Zn may play an important role in vascular endothelial barrier function.


Cholesterol-lowering effects and utilization of protein, lipid, fiber and energy in rats fed unprocessed and baked oat bran. The effects of the addition of 15% or 30% non-heated oat bran or 30% baked oat bran to a purified diet on apparent digestibility of dry matter, protein, lipid and fiber were measured in balance experiments with male Wistar rats. The effects of oat bran on dietary metabolizable energy, fecal bulking capacity and transit time of the ingested food were also studied. Heat processing of oat bran occurred in conditions of moisture and temperature similar to those of bread baking. Compared with the unprocessed oat bran, the baked product had a higher content of insoluble fiber, mainly due to higher Klason lignin content and a shift from soluble to insoluble beta-glucans. Relative to the fiber-free control diet, feeding the oat bran diets increased wet and dry fecal weight and decreased the transit time of the food. Fecal bulking capacity increased proportionally with oat fiber intake. Metabolizable energy of the diets, as well as apparent digestibility of dry matter and protein decreased with oat bran supplementation; on the other hand, lipid digestibility was not significantly changed. Baking of oat brain resulted in no statistically significant effects on dietary metabolizable energy or apparent digestibility of dry matter, protein, lipid and fiber. The measured metabolizable energy of oat bran ranged between 12.7 and 13.2 MJ/kg. Total plasma cholesterol concentration diminished with oat brain intake; non-heated and baked oat bran had comparable effects on plasma cholesterol. An inverse linear relationship (r = -0.80, P less than 0.1) was found between plasma cholesterol concentration and fecal excretion of bile acids.


ZDV delays but does not prevent the transmission of MAIDS by LP-BM5 MuLV-infected macrophage-monocytes. LP-BM5 MuLV infection of monocyte-macrophages (MM cells) and the ability of MM cells infected with this murine oncornavirus complex to transmit murine acquired immune deficiency syndrome (MAIDS) were assessed. Adherent cells expressing Mac-1 antigen (Mac-1+) were isolated from the peritoneum of infected C57BL/6 mice at weekly intervals postinoculation. A small percentage of MM cells was infected by 7 days after inoculation with LP-BM5 MuLV and virus production could be detected in MM cells throughout the course of disease. MAIDS could be induced in naive C57BL/6 mice by i.p. injection of 0.5-3 x 10(5) MM cells derived from infected mice as early as 8 weeks postinfection. When 3 x 10(5) cells (300 infectious centers) were injected, the progression of disease was similar to that seen after inoculation with a known virus pool (log10 5.78 XC pfu/ml). Treatment with zidovudine (ZDV) at 1 mg/ml in drinking water delayed disease progression if started 24 h prior to inoculation of MM cells and given continuously.


Efficacy of continuous zidovudine infusion at early stages of retroviral infection in mice. We studied the pharmacokinetics of zidovudine (ZDV) in mice after twice-daily s.c. bolus injections and during continuous infusion with s.c. ALZET mini-osmotic pumps. We also compared the antiretroviral efficacy of these two modes of administration against Friend leukemia virus (FLV) infection. Mice were infected by retro-orbital inoculation of about 50 focus-forming units (ffu) of FLV, and treatment was started 1 or 4 h later with ZDV at 40 mg/kg/day for 5 days. Efficacy was evaluated in terms of spleen weight and/or virus titer (spleen focus assay) on day 21 in comparison with untreated infected mice. In a separate experiment, survival time after infection was also monitored over a 140-day period. Plasma concentrations of ZDV were determined by means of high-performance liquid chromatography. Following bolus administration, the peak plasma ZDV concentration (30.5 mg/ml) was reached within 10 min, and elimination was rapid (mean half-life, 0.7 h). During the continuous infusion, the mean concentration was constant at about 1.2 mg/ml. After 5 days of treatment, continuous ZDV infusion consistently inhibited virus-induced splenomegaly by more than 97%; bolus injections were less effective with inhibition ranging from 13 to 98%. These results suggest that moderate constant levels of ZDV have greater antiretroviral efficacy than intermittent high concentrations.


Cytomegalovirus and Candida esophagitis in patients with AIDS. We define the clinical importance of cytomegalovirus (CMV) in the natural history of patients with concomitant candida and CMV infection of the esophagus. Prospective evaluation was made of patients with Candida and CMV esophagitis enrolled in a trial of antifungal therapy for Candida esophagitis. Retrospective review was also made of the course of patients who had been found to have both Candida and CMV infection during a previous prospective endoscopic study investigating the etiology of esophageal symptoms in HIV infection. Ten (21%) of 48 patients with Candida esophagitis in the prospective study had evidence of esophageal CMV (nine by culture, one by histology). One died after 4 weeks of therapy, with minimal retrosternal pain. None of the remaining nine had any symptoms or gross CMV esophagitis after antifungal therapy. Thirteen other patients with CMV and Candida were included in the retrospective review (mean follow-up of 8 months). Eight patients received antifungal therapy alone: six (CMV determined by histology in three and by culture in three) had symptomatic resolution; one (CMV by culture) had ongoing symptoms, and a second endoscopy showed an esophageal ulcer due to CMV (histology and culture); and one had ongoing symptoms but a negative repeat endoscopy. Two died without receiving treatment, and three were treated with antifungal and anti-CMV therapy together because of concurrent CMV retinitis (esophageal symptoms resolved in all three). Thus, CMV was of clinical importance in the esophagus in only one of 18 patients with CMV and Candida who received antifungal therapy alone.(ABSTRACT TRUNCATED AT 250 WORDS)


Molecular studies of viral pathogenesis in the central nervous system. The Linacre Lecture 1991. What is the molecular biological basis of viral pathogenesis in the central nervous system (CNS), ie by what molecular mechanisms do different viruses produce particular patterns of neurological disease in man and animal models, and can one use molecular techniques to ascertain the viral aetiology of certain neurological conditions? This complex subject can be approached in three different but interrelated ways. First, one may relate molecular techniques to specific biological properties such as viral spread to the CNS, to neurotropism, ie the affinity of the virus for particular neural regions and cells, and to neurovirulence, which refers to the actual ability to cause neurological disease. Second, the reverse approach can be adopted by considering these different aspects of virus-host relationships and then how the techniques have contributed to their understanding. Third, one can select specific neurotropic viruses, such as polio or herpes viruses, and then relate these to both particular techniques and pathogenetic mechanisms [1]. The second component of this paper will deal with the immunopathological mechanisms seen in three specific CNS viral infections, all of which have been the focus of study in the author's laboratory over the past six years.


Synthesis and antiviral activity of 1-cyclobutyl-5-(2-bromovinyl)uracil nucleoside analogues and related compounds. A series of racemic (1 alpha (E), 2 beta, 3 alpha)-1-[2,3-bis(hydroxymethyl)cyclobutyl]-5-(2-halovinyl)uracils was synthesized and evaluated in cell culture. The bromovinyl, iodovinyl, and chlorovinyl analogues, 13, 15, and 16, respectively, are all potent inhibitors of varicella zoster virus (VZV), but are less inhibitory to the replication of human cytomegalovirus (HCMV) and herpes simplex viruses 1 and 2 (HSV-1, HSV-2). The excellent anti-VZV activities of 13, 15, and 16 coupled with their virtual inability to inhibit WI-38 cell growth indicate high in vitro therapeutic indices. VZV thymidine kinase readily converts these compounds to their respective monophosphates but not to their corresponding diphosphates. Compound 13a, the (1'R) enantiomer of the bromovinyl analogue 13, was also synthesized, and its potency is comparable to that of the racemate. A lower homologue 14, (1 alpha (E),2 beta, 3 alpha)-1-[2-hydroxy-3-(hydroxymethyl)cyclobutyl]-5- (2-bromovinyl)uracil, was found to be inactive against VZV, HCMV, HSV-1, and HSV-2.


Leukotriene B4 receptor antagonists: the LY255283 series of hydroxyacetophenones. A series of hydroxyacetophenones was prepared for evaluation as leukotriene B4 (LTB4) receptor antagonists, culminating in 1-[5-ethyl-2-hydroxy-4-[[6-methyl-6-(1H-tetrazol-5- yl)heptyl]oxy]phenyl]ethanone (compound 35, LY255283). Using an assay for inhibition of specific [3H]LTB4 binding to human PMN, we found that substitution of a nonpolar substituent in the 5-position was required for activity. Best activity was realized with hydrogen in the 3-position, hydroxyl in the 2-position, short chain alkyl ketone in the 1-position, and a six- or eight-carbon chain linking the oxygen in the 4-position with an unsaturated terminal function. Compound 35, having an IC50 of 87 nM in the binding assay, was chosen for further preclinical evaluation.


Reversible inhibitors of the gastric (H+/K+)-ATPase. 2. 1-Arylpyrrolo[3,2-c]quinolines: effect of the 4-substituent. Further work on compounds 1 has identified the 4-position as a site where substantial modifications are tolerated, leading to analogues which are more potent and less toxic than those described previously. The best compound in the series is 13a (SK&F 96356), which is a potent inhibitor of gastric acid secretion in both the pentagastrin-stimulated rat and the histamine-stimulated dog. This compound shows reversible, K(+)-competitive binding to the enzyme. Because of its fluorescent properties, it is also proving useful in vitro as a probe of the structure and function of the (H+/K+)-ATPase.


Alanine series of ovine corticotropin releasing factor (oCRF): a structure-activity relationship study. Previous structure-activity relationship studies of CRF have shown that residues 1-4 were not necessary for receptor binding or transduction, that residues 4-8 were important for activation, and that residues 12-41 were mostly responsible for binding. Finally it was proposed that CRF assumed an alpha-helical structure when interacting with its receptor. By systematic substitution of each residue (except residues 1-4) in ovine CRF (oCRF) by Ala, we have investigated the role played by individual side chains in receptor recognition and activation. Out of 33 analogues (synthesized using SPPS on an MBHA resin, purified by RPHPLC and characterized by amino acid and mass spectral analyses), a significant loss of biological potency (less than 1% potency of native) was observed for 6 analogues ([Ala6], [Ala8], [Ala10], [Ala12], [Ala14], and [Ala38]); 12 analogues had biological potencies ranging from 1% to 60% and ranked as follows: [Ala35] less than [Ala16] less than [Ala9] less than [Ala19] less than [Ala15] less than [Ala13] less than [Ala7] less than [Ala23] less than [Ala11] less than or equal to [Ala21] less than [Ala27] less than or equal to [Ala18]; 8 analogues were found to be equipotent (greater than 60% and less than 150%) ([Ala5], [Ala17], [Ala26], [Ala29], [Ala30], [Ala34], [Ala36], and [Ala37]; and 7 analogues were found to be approximately 2-5 times more potent than native oCRF ([Ala25] = [Ala40] less than or equal to [Ala39] less than or equal to [Ala33] less than [Ala20] less than [Ala22] less than [Ala32], in an in vitro pituitary cell culture assay. In summary, the Ala substitutions which showed the greatest loss of potency (less than 1% of native oCRF) were those replacing hydrophobic residues while those showing the greatest increase in potency were replacing hydrophilic residues. Of the 22 Ala-containing analogues in the C-terminal half of the molecule, 17 analogues have equal or greater potencies than native oCRF. Substitution of Ala in the N-terminal region (residues 5-19) on the other hand is generally detrimental to biological activity. These results suggest that the side chains of residues 5-19 are very important for receptor binding and activation while, in the C-terminal region, the amino acid side chains may be more responsible for structural conservation than for functional expression.


Antibacterial activity of a 1,8-naphthyridine quinolone, PD 131628. A new 1,8-naphthyridine quinolone antimicrobial, PD131628, was found to be as, or more active than ciprofloxacin or ofloxacin against Escherichia coli and considerably more active than the two 4-quinolone agents against staphylococci in terms of both MIC and OBC (the optimum bactericidal concentration, at which the rate of kill is greatest). In common with ciprofloxacin and ofloxacin, the rate of kill of PD131628 against Enterococcus faecalis was considerably slower than against the other three species tested. Bacterial protein synthesis, RNA synthesis and cell division were not required for the bactericidal activity of PD131628 against E. coli or the staphylococci, although the lethality of PD131628 in the absence of these activities was reduced.