Mapping the binding site on ankyrin for the voltage-dependent sodium channel from brain. Erythrocyte ankyrin is a member of a family of proteins that mediate the linkage between membrane proteins and the underlying spectrin-actin-based cytoskeleton. Ankyrin has been shown to interact with a variety of integral membrane proteins such as the anion exchanger, the Na+K(+)-ATPase, and the voltage-dependent sodium channel (NaCh) in brain. To understand how ankyrin interacts with these proteins and maintains its specificity and high affinity for the voltage-dependent NaCh, we have mapped the binding site on ankyrin for the NaCh by examining the binding of purified ankyrin subfragments, prepared by proteolytic cleavage, to the purified rat brain NaCh incorporated into liposomes. 125I-Labeled ankyrin and the radiolabeled 89- and 43-kDa fragments of ankyrin bind to the NaCh with high affinities and with Kd values of 34, 22, and 63 nM, respectively, and have stoichiometries of approximately 1 mol/mol NaCh. The 72-kDa spectrin binding domain is inactive and does not bind to the NaCh. Dissection of ankyrin reveals that the 43-kDa domain retains all the binding properties of native ankyrin to the NaCh. Analysis of the primary structure reveals that the NaCh binding site is confined to a domain of ankyrin consisting entirely of the 11 terminal 33-amino acid repeats and is distinct from the ankyrin domains that interact with spectrin and the Na+K(+)-ATPase.


Metal-tetracycline/H+ antiporter of Escherichia coli encoded by transposon Tn10. Roles of the aspartyl residues located in the putative transmembrane helices. Three conserved aspartyl residues located in the putative transmembrane helices in the Tn10-encoded metal-tetracycline/H+ antiporter were replaced by Asn, Lys, or Glu with oligonucleotide-directed site-specific mutagenesis. Replacement of Asp84 or Asp15 by Asn or Lys caused a severe defect in tetracycline transport activity, however, the Glu84 and Glu15 mutants retained 150 and 40% of the wild type activity, respectively, indicating the critical role of the negative charge. The increase in the activity of the Glu84 mutant was due to an increase in the affinity for the substrate. H+/tetracycline coupling was intact in these mutants, including Asn and Lys mutants. On the other hand, all of the Asp285-substitution mutants showed a severe defect in tetracycline transport activity and a complete lack of tetracycline-coupled H+ transport. However, since in vivo tests showed the tetracycline resistance for the Glu285 mutant, a negative charge in position 285 plays some role in maintaining the possible down-hill and/or low affinity efflux of accumulated tetracycline from intact cells. Similar work was done for Asp365, and here the Asn and Glu mutants showed decreased but high activity, while the Lys mutant was only marginally active (5%), indicating that a negative charge is not so demanding in position 365, possibly because it is not in the membrane.


Stabilization of human neutrophil NADPH oxidase activated in a cell-free system by cytosolic proteins and by 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide. The superoxide-generating respiratory burst oxidase (NADPH oxidase) from human neutrophils can be activated in a cell-free system consisting of plasma membrane and cytosol by anionic amphiphiles such as sodium dodecyl sulfate and arachidonate (McPhail, L. C., Shirley, P. S., Clayton, C. C., and Snyderman, R. (1985) J. Clin. Invest. 75, 1735-1739; Curnutte, J. T. (1985) J. Clin. Invest. 75, 1740-1743; Bromberg, Y., and Pick, E. (1984) Cell. Immunol. 88, 213-221). Herein, the activity thus obtained is shown to be very labile at 37 degrees C. The rate of inactivation varied inversely with cytosol concentration. The stabilizing factor(s) was destroyed by heat and trypsin, indicating that it is protein in nature. Whereas cytosol from normal cells and from a chronic granulomatous disease patient lacking p67phox stabilized the oxidase activity, that from a chronic granulomatous disease patient lacking p47phox did not. Also, dialdehyde NADPH-treated cytosol showed no stabilizing effect, indicating that p47phox and a putative NADPH-binding component both participate in stabilization. The mechanism of inactivation was further explored by examining the stabilizing effect of agents that can act as chemical cross-linkers. Of several tested, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) was the most effective, but others that utilize different chemical mechanisms were also partially effective. EDC extended the half-life at 37 degrees C from 2 to 120 min, protected against the inactivating effects of Triton X-100 and high salt, and did not affect the Km for NADPH. Stabilization required prior activation in the presence of both cytosol and membrane; and EDC treatment of cytosol, membrane, or a mixture of the two prior to the addition of sodium dodecyl sulfate failed to induce stabilization. EDC eliminated the requirement for the continuous presence of cytosol and activator. Dialysis did not cause a loss in activity, whereas control activity was diminished with dialysis and was largely restored with added sodium dodecyl sulfate. In the absence of EDC, the separation of cytosol from the membrane fraction resulted in a significant loss of activity, which was largely restored by the addition of cytosol. However, EDC treatment allowed the isolation of a nearly fully active oxidase in the membrane fraction, the activity of which was not influenced by added cytosol. These results support a model in which the active NADPH oxidase consists of a dissociable complex among membrane and cytosolic components and indicate that the longevity of the activated state requires continuous association of these components.


DNA-induced dimerization of the Escherichia coli rep helicase. Allosteric effects of single-stranded and duplex DNA. The Escherichia coli Rep helicase is a stable monomer (Mr = 72,802) in the absence of DNA; however, binding of single-stranded (ss) or duplex (ds) DNA induces Rep monomers to dimerize. Furthermore, a chemically cross-linked Rep dimer retains both its DNA-dependent ATPase and helicase activities, suggesting that the functionally active Rep helicase is a dimer (Chao, K., and Lohman, T. M. (1991) J. Mol. Biol. 221, 1165-1181). Using a modified "double-filter" nitrocellulose filter binding assay, we have examined quantitatively the equilibrium binding of Rep to a series of ss-oligodeoxynucleotides, d(pN)n (8 less than or equal to n less than or equal to 20) and two 16-base pair duplex oligodeoxynucleotides, which are short enough so that only a single Rep monomer can bind to each oligonucleotide. This strategy has enabled us to examine the linkage between DNA binding and dimerization. We also present a statistical thermodynamic model to describe the DNA-induced Rep dimerization in the presence of ss- and/or ds-oligodeoxynucleotides. We observe quantitative agreement between this model and the experimental binding isotherms and have analyzed these isotherms to obtain the seven independent interaction constants that describe Rep-DNA binding and Rep dimerization. We find that Rep monomers (P) can bind either ss-DNA (S) or ds-DNA (D) to form PS or PD, respectively, which can then dimerize to form P2S or P2D. Furthermore, both protomers of the DNA-induced Rep dimer can bind DNA to form either P2S2, P2D2 or the mixed dimer species P2SD and ss- and ds-DNA compete for the same sites on the Rep protein. When bound to DNA, the Rep dimerization constants are approximately 1-2 x 10(8) M-1 (6 mM NaCl, pH 7.5, 4 degrees C), which are greater than the dimerization constant for free Rep monomers by at least 10(4)-fold. The Rep-ss-DNA interaction constants are independent of base composition and sequence, consistent with its role as a nonspecific DNA-binding protein. Allosteric effects are associated with ss- and ds-DNA binding to the half-saturated Rep dimers, i.e. the affinity of either ss- or ds-DNA to the free promoter of a half-saturated Rep dimer is clearly influenced by the conformation of DNA bound to the first protomer. These allosteric effects further support the proposal that the Rep dimer is functionally important and that the Rep-DNA species P2S2 and P2SD may serve as useful models for intermediates that occur during DNA unwinding.(ABSTRACT TRUNCATED AT 400 WORDS)


The murine gene for cellular retinoic acid-binding protein type II. Genomic organization, chromosomal localization, and post-transcriptional regulation by retinoic acid. The cellular retinoic acid-binding protein type II (CRABP-II) is a member of the serum and cytoplasmic retinoid-binding protein family. It is expressed during embryonic development and in adult skin and is upregulated by retinoic acid (RA) in F9 teratocarcinoma cells. We have determined the genomic organization of the murine CRABP-II gene and performed a detailed analysis of its transcriptional unit. The CRABP-II gene, located on mouse chromosome 2, is approximately 4.6 kilobases long and divided into four exons in a structure common to other members of the family of serum and cellular retinoid-binding proteins. Primer extension analysis and S1 nuclease protection assay were used to identify the transcription initiation site which is located 27 base pairs downstream of a typical TATAA box. Sequence analysis of the promoter also revealed a GC-rich region with overlapping putative SP1-binding sites at nucleotides -61 and AP-1 and AP-2-binding sites at nucleotides -518 and -544, respectively. The 3'-untranslated region contains two copies of the pentanucleotide AUUUA shown to be involved in messenger RNA destabilization. Consensus sequence for retinoic acid response elements were not detected in the promoter region of the CRABP-II gene. Results of nuclear run on experiments show that the CRABP-II gene is not transcriptionally activated by RA in F9 teratocarcinoma cells. These results suggest that the up-regulation of CRABP-II mRNA levels by RA is mainly controlled by a post-transcriptional mechanism.


Substrate specificity of the Rad3 ATPase/DNA helicase of Saccharomyces cerevisiae and binding of Rad3 protein to nucleic acids. Rad3 protein from the yeast Saccharomyces cerevisiae is a single-stranded DNA-dependent ATPase which catalyzes the unwinding of DNA.DNA duplexes. In the present studies we have demonstrated that the purified enzyme additionally catalyzes the displacement of RNA fragments annealed to complementary DNA. Quantitative comparisons using otherwise identical partially duplex DNA.DNA and DNA.RNA substrates indicate a significant preference for the latter. Competition for ATPase or DNA helicase activity by various homopolymers suggests that Rad3 protein does not discriminate between ribonucleotide and deoxyribonucleotide homopolymers with respect to binding. However, neither single-stranded RNA nor various ribonucleotide homopolymers supported the hydrolysis of nucleoside 5'-triphosphates. Additionally, Rad3 protein was unable to catalyze the displacement of oligo(dA) annealed to poly(U), suggesting that the catalytic domain of the enzyme is exquisitely sensitive to chemical and/or or conformational differences between DNA and RNA. Hence, it appears that Rad3 protein is not an RNA helicase.


Activation of RNA polymerase II transcription by the specific DNA-binding protein LSF. Increased rate of binding of the basal promoter factor TFIIB. While the components of the initiation complex at an RNA polymerase II basal promoter have been well characterized, few mechanistic studies have focused on how upstream DNA-binding, transcriptional activators influence protein assembly at the initiation site. Our analysis of basal transcription on both the simian virus 40 and adenovirus major late promoters demonstrates that two slow steps in initiation of transcription are the assembly of the general transcription factors TFIID and TFIIB onto the template DNA. On the simian virus 40 major late promoter, the rate of initiation complex formation is dramatically increased in the presence of the cellular transcriptional activator LSF. Direct analysis by band mobility shift assays demonstrates that LSF has no effect on the rate of binding, or the stability of TFIID on the promoter, predicting that LSF would not affect the template commitment step. Rather, kinetic analyses demonstrate that LSF reduces the lag in the rate of initiation complex formation attributable to the slow addition of TFIIB and suggest that LSF increases the rate of association of TFIIB with the committed template. In addition, LSF increases the total number of transcription complexes in long term assays, which is also consistent with LSF increasing the rate of association of TFIIB, where TFIIB is not saturating. These results indicate a mechanism for the activation of the initiation of RNA polymerase II transcription by one upstream activating protein, LSF. This mechanism may also be applicable to other activators that function in cases where limiting concentrations of TFIIB in the cell dictate slow binding of TFIIB.


Mutational analysis of active site residues in pig heart aconitase. A cDNA encoding mature porcine heart aconitase was over-expressed in Escherichia coli under the control of a phage T7 promoter. Recombinant aconitase purified from E. coli was identical to the enzyme from pig and beef heart in size, [3Fe-4S] and [4Fe-4S] cluster structure and enzymatic activity. Nine amino acid residues in close proximity to the Fe-S cluster and bound substrate (Lauble, H., Kennedy, M.C., Beinert, H., and Stout, C.D. (1992) Biochemistry, in press) were replaced by site-directed mutagenesis. Fe-S cluster environment as indicated by the EPR spectrum, tight binding of substrate, and enzymatic activity were compared for the mutant and wild type enzymes. Significant perturbations were detected for all of the mutant enzymes. Replacements for Asp100, His101, Asp165, Arg580, and Ser642 result in a 10(3)-10(5)-fold drop in activity, which suggests that these residues are involved in critical aspects of the reaction. Arg580 appears to be a key residue for substrate binding, as shown by a 30-fold increased Km and loss of tight substrate binding. Results of mutagenesis support the interpretation of the x-ray model, namely that Asp100 and His101 form an ion pair for elimination of the substrate hydroxyl and Ser642 may function as a general base for proton abstraction from citrate or isocitrate in the dehydration step and protonation of cis-aconitate in the hydration step. Asp165 appears to play a critical role in the interaction of Fea with substrate.


Specific reduction of delta-opioid receptor binding in transfected NG108-15 cells. We have recently identified and sequenced the cDNA for an opioid-binding protein with homologies to cell adhesion molecules (OBCAM) (Schofield, P. R., McFarlard, K. C., Hayflick, J. S., Wilcox, J. N., Cho, T. M., Roy, S., Lee, N. M., Loh, H. H., and Seeburg, P. H. (1989) EMBO J. 8, 489-495). Several lines of evidence using antibodies suggest that OBCAM may play a functional role in NG108-15 neuroblastoma x glioma cells, a useful model system that contains a homogeneous population of delta-opioid receptors. A logical extension of this research is to further test this hypothesis. As part of this study, NG108-15 cells were stably transfected with either sense or antisense sequences of a portion of pROM, the rat cDNA for OBCAM. [3H] Diprenorphine binding was greatly reduced in antisense-transfected cells relative to non-transfected cells. Binding to alpha 2-adrenergic, muscarinic, and insulin receptors was unaffected. These results further support the notion that OBCAM or its analogue is part (or a subunit) of an opioid receptor. Furthermore, our observation of an apparently specific reduction in opioid binding in these transfected cells suggests that they may provide a novel genetic approach for studying regulation of the opioid receptor in this defined cell line.


Proteins are secreted by both constitutive and regulated secretory pathways in lactating mouse mammary epithelial cells. Lactating mammary epithelial cells secrete high levels of caseins and other milk proteins. The extent to which protein secretion from these cells occurs in a regulated fashion was examined in experiments on secretory acini isolated from the mammary glands of lactating mice at 10 d postpartum. Protein synthesis and secretion were assayed by following the incorporation or release, respectively, of [35S]methionine-labeled TCA-precipitable protein. The isolated cells incorporated [35S]methionine into protein linearly for at least 5 h with no discernible lag period. In contrast, protein secretion was only detectable after a lag of approximately 1 h, consistent with exocytotic secretion of proteins immediately after passage through the secretory pathway and package into secretory vesicles. The extent of protein secretion was unaffected by the phorbol ester PMA, 8-bromo-cAMP, or 8-bromo-cGMP but was doubled by the Ca2+ ionophore ionomycin. In a pulse-label protocol in which proteins were prelabeled for 1 h before a chase period, constitutive secretion was unaffected by depletion of cytosolic Ca2+ but ionomycin was found to give a twofold stimulation of the secretion of presynthesized protein in a Ca(2+)-dependent manner. Ionomycin was still able to stimulate protein secretion after constitutive secretion had terminated. These results suggest that lactating mammary cells possess both a Ca(2+)-independent constitutive pathway and a Ca(2+)-activated regulatory pathway for protein secretion. The same proteins were secreted by both pathways. No ultrastructural evidence for apocrine secretion was seen in response to ionomycin and so it appears that regulated casein release involves exocytosis. Ionomycin was unlikely to be acting by disassembling the cortical actin network since cytochalasin D did not mimic its effects on secretion. The regulated pathway may be controlled by Ca2+ acting at a late step such as exocytotic membrane fusion.


Nerve growth factor nonresponsive pheochromocytoma cells: altered internalization results in signaling dysfunction. Variant rat pheochromocytoma (PC12) cells which fail to respond to nerve growth factor (NGF) (PC12nnr5) (Green, S. H., R. E. Rydel, J. L. Connoly, and L. A. Greene. 1986. J. Cell Biol. 102:830-843) bind NGF at both high and low affinity sites. Although still undefined at the molecular level, these have been referred to as type I (high) and type II (low) receptors. They are apparently composed of two membrane-bound proteins, p75 and the protooncogene trk, both of which bind NGF, and apparently contribute singularly or in concert to the two observed affinities, and to the promotion of the NGF effects. In native PC12 cells, only the high affinity receptors are apparently capable of mediating internalization and degradation. PC12nnr5 cells also display type I binding, but the subsequent internalization is not the same fashion as in the parental cell line, nor is it subjected to lysosomal degradation. Rather it is initially sequestered during the first 15 min, and is eventually released intact into the medium. In contrast, EGF is bound, internalized, and degraded by PC12nnr5 cells, albeit less efficiently than in the parent cells. These observations argue that the defect(s) preventing the PC12nnr5 variants from responding to NGF prevents competent internalization, which in the case of NGF, may be required for the full expression of activity. The absence of trk, as one alteration in PC12nnr5 cells (Loeb, D. M., J. Maragos, D. Martin-Zanca, M. V. Chao, L. F. Parada, and L. A. Greene. 1991. Cell. 66:961-966), is consistent with this conclusion.


Astrocyte process growth induction by actin breakdown. cAMP analogues such as dibutyryl cAMP (dBcAMP) have been shown to induce the formation of processes in cultured primary astrocytes. We observe that the processes form by elongation as well as the previously reported retraction of cytoplasm around cytoskeletal elements. The most prominent cytoskeletal change that occurs in response to dBcAMP is a rearrangement of actin filaments characterized by a loss of cortical F-actin staining and the appearance of actin filament staining at the tips of the processes. If cortical actin filaments are disrupted with dihydrocytochalasin B, processes form that are similar to those induced by dBcAMP suggesting that the disruption of the cortical actin network is the pivotal step in process formation. Reorganization of the actin filament network in response to cAMP is accompanied by a decrease in phosphate incorporation into the regulatory light chain of myosin (MLC). Two selective inhibitors of MLC kinase (MLCK), ML-9 and KT5926, as well as a calmodulin antagonist (W7), which would also inhibit MLCK activation, all induce astrocytic process growth implicating MLCK as a control point in process initiation. We also found that dBcAMP and ML-9 both cause a decrease in the phosphate content of actin depolymerizing factor, suggesting that this protein and myosin light chain are the effectors of actin cytoskeleton reorganization and process growth.


A mechanism generating heterogeneity in thyroid epithelial cells: suppression of the thyrotropin/cAMP-dependent mitogenic pathway after cell division induced by cAMP-independent factors. The mechanisms that generate the intercellular heterogeneity of functional and proliferation responses in a tissue are generally unknown. In the thyroid gland, this heterogeneity is peculiarly marked and it has been proposed that it could result from the coexistence of genetically different subpopulations of thyrocytes. To evaluate the heterogeneity of proliferative responses in primary culture of dog thyrocytes, we asked whether the progeny of cells having incorporated 3H thymidine in a first period of the culture could have a distinct proliferative fate during a second labeling period (incorporation of bromodeoxyuridine revealed by immunofluorescence staining combined with autoradiography of 3H thymidine). No growth-prone subpopulations were detected and the great majority of cells were found to response to either EGF or thyrotropin (TSH) through cAMP. However, only a fraction of cells replicated DNA at one given period and a clustered distribution of labeled cells within the monolayer, which was different for thymidine- or bromodeoxyuridine-labeled cells, indicates some local and temporal synchrony of neighboring cells. The TSH/cAMP-dependent division of thyrocytes preserved their responsiveness to both TSH and EGF mitogenic pathways. By contrast, cells that had divided during a momentary treatment with EGF lost the mitogenic sensitivity to TSH and cAMP (forskolin) but retained the sensitivity to EGF. Since cells that had not divided kept responsiveness to both TSH and EGF, this generated two subpopulations differing in mitogen responsiveness. The extinction of the TSH/cAMP-dependent mitogenic pathway was delayed (1-2 d) but stable. Cell fusion experiments suggest it was due to the induction of a diffusible intracellular inhibitor of the cAMP-dependent growth pathway. These findings provide a useful model of the generation of a qualitative heterogeneity in the cell sensitivity to various mitogens, which presents analogies with other epigenetic processes, such as differentiation and senescence. They shed a new light on the significance of the coexistence of different modes of cell cycle controls in thyroid epithelial cells.


Release of cell surface-associated basic fibroblast growth factor by glycosylphosphatidylinositol-specific phospholipase C. Heparan sulfate proteoglycans (HSPG) are ubiquitous constituents of mammalian cell surfaces and most extracellular matrices. A portion of the cell surface HSPG is anchored via a covalently linked glycosyl-phosphatidylinositol (Pl) residue, which can be released by treatment with a glycosyl-Pl specific phospholipase C (Pl-PLC). We report that exposure of bovine aortic endothelial and smooth muscle cells to Pl-PLC resulted in release of cell surface-associated, growth-promoting activity that was neutralized by antibasic fibroblast growth factor (bFGF) antibodies. Active bFGF was also released by treating the cells with bacterial heparitinase. Under the same conditions there was no release of mitogenic activity from cells (BHK-21, NIH/3T3, PF-HR9) that expressed little or no bFGF, as opposed to Pl-PLC-mediated release of active bFGF from the same cells transfected with the bFGF gene. The released bFGF competed with recombinant bFGF in a radioreceptor assay. Addition of Pl-PLC to sparsely seeded vascular endothelial cells resulted in a marked stimulation of cell proliferation, but there was no mitogenic effect of Pl-PLC on 3T3 fibroblasts. Studies with exogenously added 125I-bFGF revealed that about 6.5% and 20% of the cell surface-bound bFGF were released by treatment with Pl-PLC and heparitinase, respectively. Both enzymes also released sulfate-labeled heparan sulfate from metabolically labeled 3T3 fibroblasts. Pl-PLC failed to release 125I-bFGF from the subendothelial extracellular matrix (ECM), as compared to release of 60% of the ECM-bound bFGF by heparitinase. Our results indicate that 3-8% of the total cellular content of bFGF is associated with glycosyl-Pl anchored cell surface HSPG. This FGF may exert both autocrine and paracrine effects, provided that it is released by Pl-PLC and adequately presented to high affinity bFGF cell surface receptor sites.


Stimulatory and inhibitory effects of catecholamines on DNA synthesis in primary rat hepatocyte cultures: role of alpha 1- and beta-adrenergic mechanisms. Previous studies suggest that catecholamines may be involved in the regulation of liver growth. Considerable evidence implicates alpha 1-adrenergic mechanisms in the initiation of hepatocyte proliferation, while the role of beta-adrenoceptors is less clear. We have examined further the adrenergic regulation of hepatocyte DNA synthesis, using primary monolayer cultures. In hepatocytes that were also treated with epidermal growth factor and insulin, epinephrine or norepinephrine added early after the seeding strongly accelerated the rate of S phase entry. The beta-adrenergic agonist isoproterenol and the alpha-adrenergic agonist phenylephrine also stimulated the DNA synthesis, but were less efficient than epinephrine and norepinephrine. Experiments with the alpha 1-receptor blocker prazosine and the beta-receptor blocker timolol showed that the stimulatory effect of norepinephrine consisted of both an alpha 1- and a beta-adrenergic component. The alpha 1-component was most prominent in terms of maximal response at high concentrations of the agonist, but the beta-component contributed significantly and predominated at low concentrations (less than 0.1 microM) of norepinephrine. At later stages (about 40 h) of culturing norepinephrine strongly but reversibly inhibited the cells, acting at a point late in the G1 phase. This inhibition was mimicked by isoproterenol and abolished by timolol but was unaffected by prazosine, suggesting a beta-adrenoceptor-mediated effect. The results confirm the alpha 1-adrenoceptor-mediated stimulatory effect, but also show that beta-adrenoceptors may contribute to the growth stimulation by catecholamines. Furthermore, catecholamines, via beta-adrenoceptors and cyclic AMP, inhibit the G1-S transition, and may thus play a role in the termination of hepatic proliferation.


Changes in pyridine and adenine nucleotide levels in Friend erythroleukaemia cells during growth and differentiation. Pyridine and adenine nucleotide levels were measured in Friend erythroleukaemia cells (FELC) stimulated to growth and induced to differentiate by hexamethylene bisacetamide (HMBA) and N'-methylnicotinamide (N'-MNAM). A three- to fourfold increase in the NADP(H) was found to parallel cell growth stimulation in both the presence and absence of differentiation inducers. NAD(H) increased about twofold in control and to a minor extent in HMBA-treated FELC but did not vary significantly in N'-MNAM-treated cells. ATP was significantly higher in control cells stimulated to growth than in resting ones, but it did not vary in inducer-treated cells. These data confirm the relationship between high NADP(H) levels and cell resumption to growth; moreover they show that NAD(H) pool reduction and NAD/NADH ratio rise are associated with the process of FELC differentiation. The activities of NAD pyrophosphorylase and NAD kinase are much more enhanced in growth-stimulated FELC than in resting ones. On the other hand transition from the quiescent to the proliferative state was accompanied by a decrease in the activity of poly(ADP-ribose) polymerase. A decrease in poly(ADP-ribose) polymerase activity was also found in differentiated cells in contrast to controls.


Changes in luminal flow rate modulate basal and bradykinin-stimulated cell [Ca2+] in aortic endothelium. Hemodynamic forces influence many endothelial cell functions. The coupling between hemodynamic forces and cell function could be mediated by mechano-sensitive ion channels present in the plasma membrane of endothelial cells. Because one of these channels is permeable to Ca2+, we tested whether hemodynamic forces influence endothelial cell Ca2+ ([Ca2+]i). Bovine aortic endothelial cells were grown inside cylindrical glass tubes, loaded with fura-2, and perfused at different pressures and flow rates on the stage of a fluorescence microscope. Decreasing flow from 110 to 2 ml.min-1 raised [Ca2+]i from 57 +/- 11 to 186 +/- 29 nM (mean +/- SEM, p less than 0.01) by increasing the entry of extracellular Ca2+ into the cytoplasm. Increasing flow from 2 to 110 ml.min-1 transiently decreased [Ca2+]i from 62 +/- 3 to 33 +/- 5 nM (p less than 0.01) apparently due to reduced Ca2+ entry and concomitant extrusion by the plasma membrane Ca(2+)-ATPase. The rise in [Ca2+]i induced by bradykinin was magnified during a decrease in flow; in control cells, 10(-7) M bradykinin increased [Ca2+]i by 162 +/- 26 nM, whereas [Ca2+]i increased 350 +/- 67 nM (p less than 0.05) in cells previously exposed to 110 ml.min-1. These observations suggest that flow-induced changes in [Ca2+]i might be a signal-transduction mechanism for endothelial functions responsive to hemodynamic forces and may also modulate the magnitude of hormonally mediated increases in [Ca2+]i.


Mechanisms of platelet adhesion and aggregation: an update. A hemostatic plug is initially composed of an adherent layer of platelets, but it is subsequent platelet aggregation that stems the flow of leaking blood. Unlike platelet adhesion--which is a passive process--platelet aggregation is active, requiring ongoing platelet metabolism and stimulation by one or more specific agonists.


Detection of FMDV RNA amplified by the polymerase chain reaction (PCR). Molecular detection of foot-and-mouth disease virus (FMDV) using the polymerase chain reaction (PCR) is a rapid and accurate method. In this study we present PCR for the detection of FMDV RNA in infected BHK cells. Using PCR and two primers selected from the RNA polymerase gene, a conserved sequence in all types and subtypes of FMDV, we were able to detect FMDV RNA present in RNA extracted from the FMDV-infected cells. RNA from uninfected BHK cells gave negative results. Another set of primers selected from the nucleotide sequence of the variable VP1 gene permitted the demonstration of variations among different FMDV Israeli isolates by PCR. Two 01 type FMDV isolates out of a total of 6 FMDV field isolates (including 01 Geshur) gave a positive PCR while two other 01 isolates and two ASIA isolates were detected with the RNA polymerase gene primers but not with the VP1 primers. Serial dilutions of the RNA used in each reaction showed that a very small amount of RNA may be detected by PCR. The PCR products from the RNA polymerase and the VP1 genes were sequenced and the nucleotide sequences obtained were compared with a known nucleotide sequence of the FMDV 01 genome.


Detection of feline immunodeficiency virus by a nested polymerase chain reaction. A specific and sensitive polymerase chain reaction (PCR) procedure for the detection of feline immunodeficiency virus (FIV) in peripheral blood mononuclear cells (PBMC) was developed. PBMC from both blood samples and cultures were digested by proteinase K in a lysis buffer, and after heat inactivation of the proteinase, the resultant material was used in a two step amplification protocol using nested sets of primers. Two independent amplifications, from the gag and pol genes respectively, were performed in each tube. The PCR was positive for six of 14 samples from FIV seropositive adult cats, while all 36 samples from seronegative cats were negative. In comparison with an antigen-capturing ELISA procedure, the PCR detected FIV infection in PBMC cultures on average two days earlier.


Indirect ELISA for the detection of HSV-2 specific IgG and IgM antibodies with glycoprotein G (gG-2). The glycoprotein G (gG-2) purified from HSV-2 infected cells has been reported to be useful for determination of HSV-2 type-specific antibodies using conventional ELISA formats. This study further confirmed the specificity of gG-2 and demonstrated the feasibility of a specific IgM assay. The gG-2 ELISA was developed to detect HSV-2 specific IgG and IgM antibodies in human sera with high levels of sensitivity and specificity. Of 45 patients with culture-proven recurrent HSV-2 genital infection 44 were reactive for gG-2 IgG. Of 30 sera from patients with culture-proven recent initial HSV-2 genital infection 29 were positive for gG-2 IgM. Three patients with primary HSV-2 genital infection showed gG-2 IgM in the convalescent but not in the acute sera. The IgG- and IgM-gG-2 ELISA showed high specificity. None of 40 sera from children were reactive by either assay. Only one of 94 sera from patients with antibody to herpesviruses other than HSV reacted in the IgG assay but none reacted in the IgM assay. There was no cross-reaction with sera from patients with proven HSV-1 infection with the gG-2 antigen. The results suggest that the IgG assay can be used for demonstration of past HSV-2 infection and the IgM assay for the diagnosis of HSV-2 in neonatal herpes and primary genital herpes, when cultures or rapid diagnostic techniques are unavailable.


Vaginal infection of mice with HSV type 2 variant ER-: a new animal model for human primary genital HSV type 2 infections. Studying the pathogenesis of vaginal infections in mice with two variants of Herpes simplex virus type 2 (HSV-2) strain ER we observed that both variants ER+ and ER- caused severe vaginitis but only ER+ invaded the CNS leading to lethal neurological disease. In contrast, mice infected with ER- cleared the virus from the vagina and recovered from infection. ER+ and ER- expressed equal levels of thymidine kinase (TK) indicating a TK-independent difference in neurovirulence. Using the non-neurovirulent variant ER-, we were able to investigate humoral immune responses later after infection. Vaginal infection with ER- suppressed serum antibody formation after a secondary systemic HSV-1 infection. Fresh isolates of HSV-1 and HSV-2 caused uniformly a lethal neurological disease after vaginal inoculation of mice. However, some animals survived an intraperitoneal infection with these isolates. Infection with HSV-1 isolates stimulated a strong antibody production, whereas infection with HSV-2 isolates suppressed antibody formation, thus supporting earlier results from our group obtained with laboratory strains. Since suppression of antibody formation could be demonstrated with clinical HSV-2 isolates and likewise after vaginal infection with HSV-2 variant ER- we consider this phenomenon to be of relevance in human genital HSV-2 infections. Vaginal infection of mice with variant ER- represents a new model for primary genital HSV-2 infections; this model could be useful for histopathological, virological, immunological and drug testing studies.


Establishment of a genomic bank of bovine herpesvirus 1 using a novel positive selection plasmid vector. A small positive selection cloning vector, designated pSiig1, suitable for the construction of genomic banks in E. coli is described and used for the establishment of a bank of the bovine herpesvirus 1 genome. Hybrid transformants are directly selected on agar plates containing ampicillin. The vector is based on the replicon of R1 and has a lambda PR promotor inserted upstream of the replication control genes. The vector has an uncontrolled (runaway) replication and is lethal to the host cell unless the PR promotor is brought under trans-acting control of the lambda cI repressor or runaway replication is blocked by an insertion between the PR promotor and the replicon. The vector contains a unique Bg/II site between PR and the replicon which is suitable for insertion of genomic DNA.


Sympathetic nervous system modulation of tumor metastases and host defense mechanisms. The sympathetic nervous system can signal cells of the immune system through release of norepinephrine (NE), and may thus modulate several aspects of immune reactivity. We have examined the consequences of chemical denervation using 6-hydroxydopamine (6-OHDA) on the response of BALB/c mice to tumor cell challenge. In this study, chemical axotomy prior to the intravenous (i.v.) injection of the alveolar carcinoma line 1 significantly increased the number of pulmonary metastases. In contrast, axotomy performed after i.v. injection of tumor cells had no effect on the number of lung metastases. Line 1 tumor cells have been reported to be susceptible to lysis by natural killer (NK) cells. To examine possible mechanisms through which prior axotomy leads to increased lung metastases, we tested the effects of axotomy on in vitro and in vivo NK cell activity. No differences in NK cell activity were found between 6-OHDA- and vehicle-treated mice. Line 1 tumor cell growth in vitro was unaffected by both 6-OHDA and NE, and the tumor cells do not express beta-adrenergic receptors. Priming mice with lethally irradiated line 1 cells significantly reduced the number of lung metastases following challenge with live tumor cells; axotomy did not alter this decrease in metastases associated with priming. In summary, chemical axotomy of mice prior to injection of alveolar carcinoma cells resulted in an increased number of pulmonary metastases that was not correlated with alterations in either NK cell cytotoxicity or the putative immunological consequences of in vivo priming.


Transforming growth factor beta (TGF-beta) in inflammation: a cause and a cure. As we continue to explore the biology of TGF-beta in the network of cells and mediators contributing to host defense, the mechanisms controlling whether the pro- or antiinflammatory effects of this peptide prevail will be unraveled. Understanding these basic mechanisms may offer new approaches for identifying agonists and/or antagonists and in which circumstances their use might be appropriate. The striking differences between local and systemic administration of this cytokine reaffirm that the functional consequences of any biologic mediator must be considered in context (9) and, furthermore, suggest avenues of therapeutic application (Table III). In summary, the central role of TGF-beta in normal and aberrant host defense has become indisputable.


Stress-induced changes in muscarinic and beta-adrenergic binding sites on rat thymocytes and lymphocytes. This study examined the effect of immobilization stress on the expression of muscarinic and beta-adrenergic receptors on thymocytes and lymphocytes obtained from 5-month-old L-E male rats. After 2 h immobilization (acute stress) there was a significant increase in specific binding of [3H]-DHA to beta-adrenergic receptors on thymocytes and on lymphocytes from the blood but not from the spleen, whereas [3H]-QNB binding to muscarinic receptors in those cells was not altered in comparison with the undisturbed control. Chronic immobilization stress (5 days, for 2 h) decreased the [3H]-QNB binding to lymphocytes collected from the spleen and blood but not from thymus; it caused neither a significant change in the 3H-DHA binding to thymocytes nor lymphocytes obtained from the blood and spleen.


Color and conventional image-directed Doppler ultrasonography: accuracy and sources of error in quantitative blood flow measurements. Accuracy of two systems--conventional (DRF 400, Diasonics) and color-coded (Angiodynograph, Quantum/Phillips) image-directed Doppler ultrasonography--was investigated using an in vitro model that generated both monophasic and triphasic pulsatile flow patterns. Estimated and actual blood volume flow rates showed good correlations, but the sampling with a hand-held transducer led to wide variations in measurement error for the conventional (-69.2% to 50%) and the color-coded (-79.3% to 265.7%) systems. By performing multiple measurements, one could improve accuracy considering only the maximal values of a series instead of the mean values. Accuracy was impaired by interposed muscular or fatty tissue due to false low time-average velocity measurements caused by a loss of Doppler signal. Comparison of both systems revealed significant differences between pulsatility index values (p less than 0.001), blood flow velocities (p less than 0.001), and blood volume flow rates (p less than 0.05 for program flow, p less than 0.001 for manual and automatic flow program of the color-coded system).


A review of fetal organ measurements obtained with ultrasound: normal growth. This review article presents fetal organ measurements obtained with ultrasound. Data derived from measurements of the brain, heart, lung, thymus, liver, spleen, pancreas, stomach, gallbladder, kidney, adrenal glands, intestine, and bladder are summarized, compared and evaluated critically. The selection of optimal parameters, development of valid measurement procedures, and use of mathematical modeling and descriptive statistics are necessary for quantitative studies by ultrasound of fetal organ growth.


An assessment of hysterosalpingosonography (HSSG) as a diagnostic tool for uterine cavity defects and tubal patency. The value of hysterosalpingosonography (HSSG) as a diagnostic tool was evaluated in 76 patients and compared to hysteroscopic, laparoscopic, and/or hysterosalpingographic (HSG) findings. Saline solution and Dextran 60 were used as distension media. Patients were divided in three groups: group A (n = 22), patients submitted for control post-tubal electrocoagulation. Group B (n = 38), patients with a history of pathological metrorrhagias, and group C (n = 16) infertile women with possible tubal pathology. Comparison between the different diagnostic techniques for the evaluation of the uterine cavity and tubes was carried out. Our results indicated that HSSG had more sensitivity but less specificity than hysteroscopy or HSG in the diagnosis of uterine cavity pathology. Hysteroscopy seems to be the best technique for the diagnosis of endometrial pathology, and HSSG seems to be the most effective in the study of the myometrium. HSSG cannot be considered a reliable and accurate method for the diagnosis of tubal patency.


Efficiency of cleaning agents for an inorganic membrane after milk ultrafiltration. Cleaning of inorganic membranes after ultrafiltration (UF) of skim milk has been assessed using hydraulic, physicochemical and spectroscopic (i.r. and X-ray photoelectron spectroscopy) measurements. A cleaning sequence using hypochlorite alone or hypochlorite followed by HNO3 restored the membrane hydraulic resistance, in contrast to cleaning with HNO3 alone. When using NaOH, addition of Ca complexants (EDTA, gluconate, tripolyphosphate) and surfactants was required to obtain similar results. Three types of criteria (hydraulic, kinetic, chemical) are available to assess the effect of the sequestrant and surfactant types. In all the cases studied, traces of protein and Ca were detected on and within the membrane after cleaning. Nevertheless, it was concluded that it is possible to develop a single-step alkaline product to clean inorganic milk UF membranes if suitable surfactants and Ca sequestrants are included in its formula.


Effect of canola fat on ruminal and total tract digestion, plasma hormones, and metabolites in lactating dairy cows. The effects of canola fat on digestion and metabolism were investigated by incorporating 0, 4.5, 9, 13.2, or 17.4% Jet-Sploded canola seed into a diet containing a 60:40 (DM) concentrate:forage ratio. The diets contained 16.5% CP, 30% alfalfa silage, and 10% whole-crop oat silage on a DM basis and were fed for ad libitum consumption as TMR to 10 ruminally cannulated Holstein cows in early lactation. Jet-Sploded canola seed supplementation did not change ruminal pH or NH3 N concentrations, but VFA concentrations declined with increasing level of inclusion. Apparent digestibilities of DM, OM, CP, NDF, and ADF were unaffected by level of inclusion of Jet-Sploded canola seed, but ether extract digestibility declined linearly, which resulted in similar ether extract absorption across the three diets supplemented with canola fat. Based on in sacco data, the percentages of ruminal digestion of OM and CP declined with increasing inclusion of Jet-Sploded canola seed. Plasma glucose and FFA concentrations tended to respond in a quadratic fashion, plasma insulin concentration declined linearly, and plasma glucagon and somatotropin concentrations were unaffected by dietary treatment. The results indicate that a positive productive response may be expected from dietary inclusion of about 5% Jet-Sploded canola seed, but the benefits of increased energy density associated with higher inclusion levels may be offset by reduced availability of energy in the rumen and decreased fat digestibility postruminally. The substantial effects of time postfeeding on ruminal fermentation and on concentrations of plasma hormone and metabolites in animals fed TMR demonstrate that infrequent sampling can result in misleading results and, thus, invalid interpretation of the influence of dietary fat on these parameters.


Influence of rumen fermentable neutral detergent fiber levels on feed intake and milk production of dairy cows. Sixteen dairy cows in midlactation were fed four isonitrogenous (mean 2.46% N) mixed rations containing 46.4% DM and 40.7% NDF in a 16-wk Latin square design experiment. Diets were 42% barley-based concentrate, on a DM basis; forage was provided by combinations of two timothy silages of either slow (poor timothy) or rapid (good timothy) rumen fermentability and an alfalfa silage. Differences between the timothy forages caused the 24-h in sacco mixed diet NDF fermentability, determined in dry cows fed hay, to be 53.8, 56.0, 60.2, and 62.1% in the four diets formulated to rise from 0% [as a percentage of total timothy DM] to 33, 66, and 100% good timothy. Intake of DM and NDF were not influenced by forage source. Rumen pool sizes of OM and NDF were similarly unaffected by treatment. Milk production increased linearly as the proportion of rapidly fermentable NDF in the diet increased, although the increases were small and not statistically significant, whereas BW gain declined (nonsignificantly). As a result, total net energy output was not influenced by diet. Although cows appeared able to extract more energy than expected from the slower fermenting forage, increasing levels in the diet resulted in a shift in energy output from milk products to BW gain, supported by a shift in rumen fermentation end products from propionic to acetic acid. Results do not appear to support the concept that NDF quality influences and can be used to predict voluntary feed intake. There is no indication that cows responded to more rapidly fermentable dietary NDF by increasing feed intake or extent of NDF fermentation in the rumen.


Optimal EPR detection of weak nitroxide spin adduct and ascorbyl free radical signals. We have investigated the optimal nominal power settings for the electron paramagnetic resonance detection of typical nitroxides, nitroxide spin adducts, and the ascorbyl free radical. In room temperature aqueous solution, we find that, for all the nitroxides examined, saturation effects begin at approx. 25 mW nominal power with maximum signal intensity achieved at approx. 100 mW power when using a TM110 cavity. For the ascorbyl free radical, we find that saturation effects begin at approx. 16 mW nominal power and that maximum peak-to-peak signal amplitude is obtained at approx. 40 mW microwave power. For the ascorbyl free radical, we find that a modulation amplitude of approx. 0.65 G yields the maximum signal height for the doublet signal. This information will help researchers maximize the EPR signal height of minimally detectable free radicals such as encountered in biological systems.


Isolated adrenocorticotropic hormone deficiency secondary to hypothalamic deficit of corticotropin releasing hormone. A 42-year-old man and a 51-year-old woman with a positive history of weakness and gastrointestinal complaints were shown to have low basal plasma cortisol and ACTH levels, and low daily urinary excretion of free cortisol. An empty sella was found in patient no. 1, while patient no. 2 was hypothyroid. Both patients showed a normal plasma cortisol response to ACTH and an increment in plasma ACTH and lipotropin levels after ovine CRH (oCRH), lysine vasopressin (LVP) and oCRH-LVP stimulation tests. These studies clearly report an isolated idiopathic ACTH deficiency due to a deficit in CRH in two adult subjects.


Critical role of corneal Langerhans cells in the CD4- but not CD8-mediated immunopathology in herpes simplex virus-1-infected mouse corneas. Previous studies have revealed that the RE strain of HSV type 1 (HSV-1) induces a tissue-destructive inflammatory response in the mouse cornea that is mediated by CD4 T lymphocytes, whereas the KOS strain of HSV-1 preferentially activates CD8 T lymphocytes in the cornea. Langerhans cells (LC) normally reside only at the periphery of the cornea but can migrate centripetally after HSV-1 infection. We studied the relative contribution of LC to the corneal inflammation induced by the KOS and RE strains of HSV-1. Ten days after infection, the central one-third of RE HSV-1-infected corneas contained an average of 5.7 LC/high-power field compared with 0.6 LC/high-power field in KOS-infected corneas. We hypothesized that the increased density of LC in RE HSV-1-infected corneas at the time of T lymphocyte infiltration contributed to the preferential activation of CD4 T lymphocytes in these corneas. To test this hypothesis, we gave mice a low dose of UV-B corneal irradiation (150 mJ/cm2) 1 day before infection with HSV-1. UV-B irradiation effectively prevented the migration of LC into the central cornea when measured 10 or 21 days after corneal infection with either HSV-1 strain. UV-B corneal irradiation had no effect on the CTL response to HSV-1 Ag in the regional lymph nodes after corneal infection with KOS or RE HSV-1. The delayed-type hypersensitivity response induced by both strains of virus, when measured 8 and 14 days after corneal infection, was significantly reduced by UV-B irradiation. UV-B irradiation significantly reduced the incidence (p = 0.0023) and severity (p = 0.0008) of corneal stromal disease induced by RE HSV-1 but did not significantly affect the stromal disease induced by KOS HSV-1. To distinguish between the effect of UV-B treatment on the afferent and efferent arms of the Ir in mice, we administered UV-B treatment to one eye, followed 24 h later by RE HSV-1 infection of both eyes. These mice developed a normal delayed-type hypersensitivity response, and stromal inflammation developed normally in the untreated eye. However, stromal inflammation was significantly reduced in the treated eye. Our findings suggest that LC play a critical role in the activation of HSV-reactive CD4 T lymphocytes in the cornea. Moreover, the type of corneal inflammation induced by different strains of HSV-1 may reflect their differential capacity to induce LC migration into the central cornea.


Identification and molecular cloning of tactile. A novel human T cell activation antigen that is a member of the Ig gene superfamily. We have identified and cloned cDNA for a novel cell-surface protein that we have named Tactile for T cell activation, increased late expression. It is expressed on normal T cell lines and clones, and some transformed T cells, but no other cultured cell lines tested. It is expressed at low levels on peripheral T cells and is strongly up-regulated after activation, peaking 6 to 9 days after the activating stimulus. It is also up-regulated on NK cells activated in allogeneic cultures. It is not found on peripheral B cells but is expressed at very low levels on activated B cells. Tactile-specific mAb immunoprecipitates a band of 160 kDa when reduced and bands of 240, 180, and 160 kDa nonreduced. Using an antiserum produced with affinity-purified Tactile protein to screen a lambda gt11 library, we have identified Tactile cDNA. Northern blot analysis shows an expression pattern similar to that of the protein and transfection of COS cells with the full-length 5.2-kb cDNA results in cell-surface expression. Comparison with the sequence databanks show that Tactile is a member of the immunoglobulin gene superfamily, with similarity to Drosophila amalgam, the melanoma Ag MUC-18, members of the carcinoembryonic Ag family, the poliovirus receptor, and the neural cell adhesion molecule. The deduced primary sequence encodes a protein with three Ig domains, a long serine/threonine/proline-rich region typical of an extensively O-glycosylated domain, a transmembrane domain, and a 45 residue cytoplasmic domain. These data suggest that Tactile may be involved in adhesive interactions of activated T and NK cells during the late phase of the immune response.


Induction of cytotoxic T lymphocytes specific for a syngeneic tumor expressing the E6 oncoprotein of human papillomavirus type 16. Human papillomavirus (HPV) type 16 has been implicated in the etiology of cervical carcinomas, but it is unknown whether HPV-specific immunity can function in controlling the growth of HPV-associated carcinomas. We previously demonstrated that CD8+ T lymphocytes can inhibit the in vivo outgrowth of murine tumor cells transfected with the HPV-16 E7 gene and have now established a murine model to study the CTL responses to the E6 oncoprotein of HPV-16. Immunization of C3H/HeN mice with syngeneic fibroblasts expressing a transfected HPV-16 E6 gene induced regression of transplanted tumors expressing this gene. Populations of CTL isolated from the spleens of mice whose E6+ tumors had regressed were shown to specifically lyse E6+ target cells. The cytolytic activity was mediated by CD8+ CTL in a MHC restricted pattern. These data and our previous findings with transfected tumor cells expressing the E7 gene, support the conclusion that tumor cells associated with HPV-16 can be inhibited by CTL specific for molecules encoded by the HPV-16 E6 and E7 genes.


Biochemical, physiological, and pathological aspects of the peripheral benzodiazepine receptor. The PBR is a mitochondrial protein composed of at least two subunits, an approximately 30-kDa subunit that contains the site for BZs and an approximately 18-kDa subunit that binds isoquinoline carboxamide derivatives. Porphyrins and diazepam binding inhibitor are putative endogenous ligands for these receptors, which are under neural and hormonal control. Alterations in the density of PBR seem to be a sensitive indicator of stress: up-regulation after acute stress and down-regulation induced by repeated stress. PBR-specific ligands are involved in the control of cell proliferation and differentiation, and their binding is increased in some cancer tumors. Numerous studies in various endocrine organs have revealed that PBR are located in specific regions or tissues in the organs. Furthermore, PBR densities in various organs subject to hormonal control are regulated by organotropic hormones. At least in some cases, BZ ligands do not exert a specific effect in an organ, but rather modulate the well-documented effects of that particular hormone. To the best of our knowledge, BZ ligand action in peripheral tissues is dependent on recognition of PBR, which may suggest a receptor-mediated action.


Depression of mesolimbic dopamine transmission and sensitization to morphine during opiate abstinence. To investigate the role of mesolimbic dopamine (DA) in the mechanism of drug dependence, extracellular DA was monitored by transcerebral dialysis in the caudal nucleus accumbens under basal conditions and after challenge with morphine (5 mg/kg s.c.) in control rats and in rats made dependent on and then deprived of morphine. Withdrawal from morphine resulted in a marked reduction of extracellular DA concentrations from control values at 1, 2, 3, and 5 days of withdrawal. After 7 days of withdrawal, DA output was less, but still significantly, reduced. Challenge with morphine resulted in stimulation of DA output in controls (maximum, 35%), no effect on the first day of withdrawal, and stimulation similar to controls' on days 2 and 7 of withdrawal. On day 5 and, particularly, on day 3 of withdrawal, morphine-induced stimulation of DA output was markedly potentiated (maximum, 100 and 160%, respectively). Changes in the sensitivity of DA transmission to morphine challenge were associated with changes in the behavioral stimulant effects of morphine, with tolerance on day 1 and marked sensitization on days 3 and 5 but also on day 7, when morphine-induced stimulation of transmission was no longer potentiated. The results indicate that repeated morphine administration induces a state of dependence in DA neurons and a short-lasting tolerance followed by an increased sensitivity to its stimulant effects on DA transmission. These changes might play an important role in the development of opiate addiction and in the maintenance of opiate self-administration in dependent subjects.


L-fucose is a potent inhibitor of myo-inositol transport and metabolism in cultured neuroblastoma cells. It has been proposed that abnormal myo-inositol metabolism may be a factor in the development of diabetic complications. Studies with animal models of diabetes and cultured cells have suggested that hyperglycemia by an unknown mechanism may alter myo-inositol metabolism and content. Recently, we have shown that L-fucose, a 6-deoxy sugar whose content has been reported to be increased in diabetes, is a potent inhibitor of myo-inositol transport. To examine the effect of L-fucose on myo-inositol metabolism, neuroblastoma cells were cultured in medium supplemented with L-fucose. L-Fucose is a competitive inhibitor of Na(+)-dependent, high-affinity myo-inositol transport. The Ki for inhibition of myo-inositol transport by L-fucose is about 3 mM. L-Fucose is taken up and accumulates in neuroblastoma cells. The uptake of L-fucose is inhibited by Na+ depletion, D-glucose, glucose analogues, phloridzin, and cytochalasin B. In contrast, neither myo-inositol nor L-glucose inhibits L-fucose uptake. Chronic exposure of neuroblastoma cells to 1-30 mM L-fucose causes a decrease in myo-inositol accumulation and incorporation into inositol phospholipids, intracellular free myo-inositol content, and phosphatidylinositol levels. Na+,K(+)-ATPase transport activity is decreased by about 15% by acute or chronic exposure of neuroblastoma cells to L-fucose. Similar defects occur when neuroblastoma cells are exposed chronically to 30 mM glucose. Cell myo-inositol metabolism and Na+/K(+)-pump activity are maintained when 250 microM myo-inositol is added to the L-fucose-supplemented medium. Unlike the effect of chronic exposure of neuroblastoma cells to medium containing 30 mM glucose, the resting membrane potential of neuroblastoma cells is not altered by chronic exposure of the cells to 30 mM L-fucose. The effect of L-fucose on cultured neuroblastoma cell properties occurs at concentrations of L-fucose which may exist in the diabetic milieu. These data suggest that increased concentrations of L-fucose may have a role in myo-inositol-related defects in mammalian cells.


Distinct cellular expression of calcineurin A alpha and A beta in rat brain. Specific polyclonal antibodies that distinguish the two distinct isoforms of the catalytic subunit of calmodulin-dependent protein phosphatase, calcineurin A alpha and A beta, were prepared, and the distribution of calcineurin A alpha and A beta in rat brain was studied using immunochemical and immunocytochemical techniques. Immunochemical measurement revealed that the regional distributions of the two isoforms differed and that A alpha was more abundant than A beta in the rat brain. The subcellular distribution patterns of both isoforms were similar. Both isoforms were highly enriched in cytosolic fractions, including the synaptosomal cytosol. Immunocytochemical analysis revealed that both A alpha and A beta immunoreactivities differed in regional and cellular localizations. These different patterns of expression suggest that the two isoforms of calcineurin A may each have specific functions in modulating neuronal activity in particular cell types.


Ischemia-induced translocation of Ca2+/calmodulin-dependent protein kinase II: potential role in neuronal damage. The activities of Ca2+/calmodulin (CaM)-dependent, Ca2+/phospholipid-dependent, and cyclic AMP-dependent protein kinases (CaM-KII, PKC, and PKA, respectively) were determined in rat brains after global ischemia. Both CaM-KII and PKC activities were significantly depressed in both hippocampal and cerebral cortical regions of ischemic animals, whereas no change was detected in PKA activity. The loss of CaM-KII activity was more dramatic and more sustained than the loss of PKC activity and correlated with the duration of ischemia. These decreases in enzyme activity were found in both supernatant and pellet fractions from crude homogenates. When the supernatant and pellet were analyzed for the amount of CaM-KII 50-kDa protein, a significant decrease was detected in supernatant fractions that paralleled a gain in the amount of CaM-KII in the pellet. Thus, the loss of CaM-KII activity in the supernatant can be explained by translocation of the enzyme to the pellet. Whether inactivation of CaM-KII occurs during or after the enzyme translocates from the supernatant to the pellet is unknown. Our results indicate that loss in CaM-KII activity parallels neuronal damage associated with ischemia; down-regulation of CaM-KII activity coincided with translocation of the enzyme to the particulate fraction, and it is proposed that this may be, in fact, a mechanism for controlling excessive CaM-KII phosphorylation.


Calcium independence of phosphoinositide hydrolysis-induced increase in cyclic AMP accumulation in SK-N-SH human neuroblastoma cells. Previous work has shown that stimulation of muscarinic receptors in various cell lines increases intracellular cyclic AMP (cAMP) levels. This unusual response has been hypothesized to be mediated by stimulation of calcium/calmodulin-sensitive adenylate cyclase, secondary to inositol trisphosphate (IP3)-mediated calcium mobilization. To test this hypothesis, we stimulated muscarinic receptors in SK-N-SH human neuroblastoma cells while blocking the IP3-mediated rise in intracellular calcium concentration using two different methods. Loading cells with the intracellular calcium chelator 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) abolished the carbachol-mediated intracellular calcium release without abolishing the carbachol-mediated increase in cAMP level. Similarly, in cells preexposed to carbachol, the agonist-induced change in intracellular calcium level was blocked, but the cAMP response was not. Thus, both of these methods failed to block the muscarinic receptor-mediated increase in cAMP level, thereby demonstrating that this cAMP level increase is not mediated by a detectable rise in intracellular calcium concentration.


Regional and temporal alterations in Ca2+/calmodulin-dependent protein kinase II and calcineurin in the hippocampus of rat brain after transient forebrain ischemia. We have investigated regional and temporal alterations in Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) and calcineurin (Ca2+/calmodulin-dependent protein phosphatase) after transient forebrain ischemia. Immunoreactivity and enzyme activity of CaM kinase II decreased in regions CA1 and CA3, and in the dentate gyrus, of the hippocampus early (6-12 h) after ischemia, but the decrease in immunoreactivity gradually recovered over time, except in the CA1 region. Furthermore, the increase in Ca2+/calmodulin-independent activity was detected up to 3 days after ischemia in all regions tested, suggesting that the concentration of intracellular Ca2+ increased. In contrast to CaM kinase II, as immunohistochemistry and regional immunoblot analysis revealed, calcineurin was preserved in the CA1 region until 1.5 days and then lost with the increase in morphological degeneration of neurons. Immunoblot analysis confirmed the findings of the immunohistochemistry. These results suggest that there is a difference between CaM kinase II and calcineurin in regional and temporal loss after ischemia and that imbalance of Ca2+/calmodulin-dependent protein phosphorylation-dephosphorylation may occur.


Receptor-linked hydrolysis of phosphoinositides and production of prostacyclin in cerebral endothelial cells. The receptor agonist-mediated hydrolysis of phosphoinositides and production of prostacyclin were studied in murine cerebral endothelial cells (MCEC). Of 11 neurotransmitters and neuromodulators examined, carbachol, noradrenaline (NE), bradykinin, and thrombin significantly increased 3H-inositol phosphate accumulation in the presence of LiCl (20 mM). The maximal stimulation of [3H]inositol monophosphate ([3H]IP1) reached approximately 11, 11, seven, and four times the basal levels for carbachol, NE, bradykinin, and thrombin, respectively. The EC50 values of IP1 accumulation for carbachol and NE were 34 and 0.16 microM, respectively. The muscarinic antagonists, atropine and pirenzepine, blocked the carbachol-induced IP1 accumulation with Ki values of 0.3 and 30 nM, respectively. The adrenergic antagonist, prazosin, blocked NE-induced IP1 accumulation with a Ki of 0.1 nM. The calcium ionophore A23187, histamine, glutamate, vasopressin, serotonin, platelet activating factor, and substance P did not stimulate IP1 accumulation. A23187, bradykinin, and thrombin stimulated prostacyclin release to approximately four, four, and two times the basal levels, respectively, whereas carbachol and NE had little effect upon prostacyclin release. These results suggest that the activation of phospholipase C and of phospholipase A2 in MCEC are regulated separately.


Angiotensin-induced cyclic GMP production is mediated by multiple receptor subtypes and nitric oxide in N1E-115 neuroblastoma cells. Angiotensin II (AngII) elicited a rapid and dose-related production of intracellular cyclic GMP (cGMP) in murine neuroblastoma N1E-115 cells. The agonist-induced rise in cGMP levels was blocked in a monophasic fashion by the AT1-selective antagonist DuP 753 or the nonselective antagonist [Sarc1,Ile8]-AngII, and both antagonists produced complete inhibition of the cGMP response elicited by submaximal concentrations of AngII. In contrast, the AT2-selective antagonist CGP 42112A inhibited the cGMP response biphasically. At lower antagonist concentrations, agonist-induced cGMP production was only partially inhibited, whereas complete inhibition was observed only when the concentration of CGP 42112A was increased sufficiently to interact with both AT1 and AT2 receptor subtypes. AngII also increased inositol trisphosphate (InsP3) levels in N1E-115 cells. However, the InsP3 response was mediated exclusively by the AT1 receptor subtype because it was inhibited by lower, AT1-selective concentrations of DuP 753, whereas only higher, nonselective concentrations of CGP 42112A were effective. Finally, the stimulatory effects of AngII on cGMP production appeared to be mediated by the intracellular formation of nitric oxide in that they were attenuated by the nitric oxide synthase inhibitor, N-monomethyl-L-arginine. Collectively, these results suggest that the AngII-elicited rise in cGMP levels may require an interaction between AT1-mediated mobilization of intracellular Ca2+, as well as some partial role of AT2 receptors.


Inhibition of forskolin-stimulated cyclic AMP formation by 1-aminocyclopentane-trans-1,3-dicarboxylate in guinea-pig cerebral cortical slices. The effects of the selective metabotropic glutamate receptor agonist 1-aminocyclopentane-trans-1,3-dicarboxylate (t-ACPD) on forskolin-stimulated cyclic AMP formation in guinea-pig cerebral cortex slices were determined. t-ACPD inhibited the accumulation of [3H]cyclic AMP by approximately 80%, with an IC50 value of 35 +/- 4 microM. The effect was reversible and stereoselective, with the 1S,3R isomer being approximately 400-fold more potent than the 1R,3S isomer. L-Glutamate (over a restricted concentration range) also partially inhibited the response to forskolin, but quisqualate, alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA), and N-methyl-D-aspartate (NMDA) were ineffective. The effect of t-ACPD was not blocked by antagonists of the phospholipase C-linked metabotropic glutamate receptor, the AMPA ionotropic glutamate receptor, or the NMDA receptor. In summary, our results indicate the presence of a glutamate receptor in guinea-pig brain that is activated selectively by t-ACPD and that is negatively linked to adenylyl cyclase.


Evidence for distinct neuronal localization of gamma and delta subunits of Ca2+/calmodulin-dependent protein kinase II in the rat brain. Localization of the gamma and delta types of mRNAs for Ca2+/calmodulin-dependent protein kinase II (CaM-kinase II) was determined in the rat brain, making use of in situ hybridization histochemistry. The gamma and delta mRNAs as well as the alpha and beta mRNAs for CaM-kinase II were heterogeneously and distinctly distributed. In the Purkinje cell layer of the cerebellum, alpha, beta, and gamma mRNAs but not delta mRNA were present, whereas beta, gamma, and delta mRNAs were present in the locus coeruleus. These findings provide evidence that CaM-kinase II exists in a variety of forms in different cells composed of a variable number and type of subunits.


Changes in nerve growth factor receptor-like immunoreactivity in the spinal cord after ventral funiculus lesion in adult cats. Spinal motoneurons have a capability to regenerate CNS-type axons after intramedullary lesions in the adult cat. Regrowing axons have been traced through CNS-type scar tissue in the ventral funiculus of the spinal cord and into adjacent ventral root fascicles. This scar tissue, which appears to support and sustain regenerating axons, has been shown to have a persistent defect in the blood-brain barrier. It has been suggested that the blood-brain barrier may play a vital role in CNS regeneration by regulating the access of blood-borne trophic factors to the lesion area. In the present study, the binding of antibodies to the human nerve growth factor receptor in the cat spinal cord was examined with immunohistochemical methods 2 days to 8 weeks after a ventral funiculus lesion. The results show that, while no neurons in the ventral horn of the control material contained nerve growth factor receptor-like immunoreactivity as revealed by fluorescence microscopy, affected motoneurons expressed nerve growth factor receptor after ventral funiculus lesion. Nerve growth factor receptor-like immunoreactivity associated to both capillaries and interstitium was present in the scar tissue. Electron microscopic examination of sections labelled with the immunogold-silver method showed that perivascular nerve growth factor receptor-like immunoreactivity was located exclusively to non-pericytic perivascular cells. These cells were abundant in the expanded capillary perivascular spaces adjacent to the traumatic lesion. Similar cells, with or without relation to blood vessels, were observed in the scar tissue and in the pia mater. In a separate set of specimens it was observed that a ventral funiculus lesion combined with ventral root avulsion, which removes denervated PNS tissue, resulted in an expression of nerve growth factor receptor-like immunoreactivity which was similar to the one observed after ventral funiculus lesion only. The results of the present study show that affected motoneurons and cells in the scar tissue express nerve growth factor receptor after ventral funiculus lesion which implies that neurotrophic factors related to nerve growth factor may be of importance for the regenerative response.


Efficacy of '8-drugs-in-one-day' combination in treatment of recurrent GBM patients. Thirty-five adult recurrent GBM patients, divided randomly in two groups of 19 and 16 cases, had been treated with two regimens of chemotherapy: a) 'eight-drugs-in-one-day'; b) procarbazine + CCNU + vincristine (PCV) respectively. Chemotherapy was planned at the tumour relapse and delivered as long as tolerated without irreversible sequelae or until the CT scan showed tumor progression. Multiple agents are used simultaneously in the therapeutic approach using 'eight-in-one' to kill as many heterogeneous cells of malignant glial tumor as possible and minimize the emergence of cellular resistance to chemotherapy. Rate response to chemotherapy and the median adjunctive survival time (6.5 and 6 months, respectively) are not significantly different in the two arms of this study. Our experience with such an aggressive multi-drugs combination 'eight-in-one-day' was disappointing if compared with less toxic, better tolerated and easy delivered (PCV) regimen.


[Sensitivity test to chemotherapeutic agents and cytotoxicic test against immuno-effecter cells of established malignant fibrous histiocytoma cell line]. A cell line was established from a patient with malignant fibrous histiocytoma, which had originated in the maxillary sinus. Using this cell line, sensitivity to chemotherapeutic agents and cytotoxicity against various immunoeffecter cells were tested. This MFH cell line was sensitive in some degree to adriamycin, 5-fluorouracil, cisplatin, peplomycin, and methotrexate at high doses, but insensitive to mitomycin-C, vincristine and cyclophosphamide. Furthermore, this cell line showed no sensitivity to cytolytic cytokines, such as tumor necrosis factor-alpha and interferon-gamma. Lymphokine activated killer (LAK) cells and lymphokine activated tumor infiltrating lymphocytes (LA-TIL), induced by culture of peripheral blood lymphocytes and TIL respectively with rIL-2 showed high NK and LK activities and remarkable anti-autologous tumor ability.


Immunohistological detection of human parvovirus B19 in formalin-fixed, paraffin-embedded tissues. Human parvovirus B19 is a cause of aplastic crises in patients with haemolytic anaemias, prolonged bone marrow failure in the immunosuppressed, and fetal death secondary to non-immune hydrops. The immunohistological detection of parvovirus B19 in formalin-fixed, paraffin-embedded tissues has not previously been reported, and definitive diagnosis of infection in such specimens has relied on the use of specialized DNA hybridization and amplification techniques. A new monoclonal antibody to B19 capsid proteins, R92F6, was found to be capable of labelling infected cells in paraffin-embedded tissues from all 19 cases of parvovirus-related fetal hydrops tested, and in bone marrow from a child with congenital immunodeficiency and chronic parvovirus infection. Viral antigen was detected both in cytoplasmic and in nuclear distributions using the alkaline phosphatase anti-alkaline phosphatase (APAAP) technique without preceding proteolytic digestion. The viral epitope recognized appears to be highly conserved, as specimens were obtained over a 13-year period from widely spaced locations in the U.K. Antibody R92F6 should facilitate rapid diagnosis of parvovirus B19 infection in routinely processed and archival specimens.


Detection of Epstein-Barr virus in lymphoid tissues of patients with infectious mononucleosis by in situ hybridization. Although the immunological response during infectious mononucleosis (IMN) has been studied in detail, little is known about the spread of Epstein-Barr virus (EBV) in lymphoid organs or the topographical distribution of the infected cells. In this study, EBV was detected in 11 lymph nodes, 4 tonsils, and 1 spleen of 16 patients with IMN. The predominant cell type positive for the EBV genome was identified as small lymphocytes localized chiefly within typical T areas, preferentially in perifollicular and interfollicular regions of the lymph node. A few endothelia of epithelioid venules were also found to be positive. Furthermore, a small number of sinus lining cells of lymph nodes exhibited labelling. Altogether, only a small number of cells, not exceeding 1 per cent of all cells, were infected with EBV. Our results show that only a small number of lymphocytes carry the EBV and that besides B lymphocytes, other cell constituents of lymphatic tissues are infected by EBV during IMN.


Serum cholesterol, cholesteryl ester, and high-density lipoprotein development in newborn infants: response to formulas supplemented with cholesterol and gamma-linolenic acid. Healthy newborn infants were either breast-fed or randomly designated to receive a standard formula, formula plus cholesterol, or formula plus gamma-linolenic acid at birth. At 0, 7, and 30 days of life, the following variables were measured: cholesteryl esters (cholesteryl arachidonate, cholesteryl oleate, cholesteryl palmitate, and cholesteryl linoleate), high-density lipoprotein (HDL) cholesterol, apoproteins (A-I, A-II, B, C-II, C-III, and E), and the cholesterol and apoprotein A-I content of the HDL subfractions HDL-2b, HDL-(2a + 3a), and HDL-(3b + 3c). Breast-fed infants had higher serum levels of cholesterol, cholesteryl oleate, cholesteryl palmitate, cholesteryl arachidonate, and HDL-2b than had formula-fed infants. Addition of gamma-linolenic acid to formula raised cholesteryl-arachidonate levels, and cholesterol and gamma-linolenic acid raised serum HDL-2b levels compared with those produced by unsupplemented formula. Our data suggest that both exogenous cholesterol and gamma-linolenic acid contribute to the maturation of HDL particles associated with human milk consumption in newborn infants. They may also promote adequate delivery of cholesterol and arachidonic acid to the developing brain.


HIV-1 and HIV-2 infection in children in Abidjan, Côte d'Ivoire. We conducted a study of 1,003 well and hospitalized children, birth to 5 years old, in Abidjan, Côte d'Ivoire, to determine the prevalence of HIV-1 and HIV-2 infection, evaluate risk factors for infection, and describe associated clinical characteristics. The overall seroprevalence was significantly higher for children in the hospital (10.8%) than for those attending the clinic (3.6%). HIV-1 was the predominant virus in both populations, comprising 87% (hospital) and 77% (clinic) of the seroreactive blood specimens. Ninety-two percent of seroreactive children of all ages had a mother who was HIV positive; 77% of seroreactive children greater than or equal to 15 months old had an HIV-infected mother. The remaining seropositive children had a history of receiving blood transfusions. Hospitalized children who were HIV-1 positive or dually seroreactive were more likely to have HIV-related clinical signs and symptoms than HIV-negative children. These findings suggest that HIV infection is an important cause of morbidity for children in Abidjan and that maternal infection is the primary risk factor for both HIV-1 and HIV-2 infection in children. Further evaluation and attention should be given to transmission, clinical characteristics, and the impact of HIV infection in children in West Africa, where both HIV-1 and HIV-2 are present.


Marijuana smoking: effect of varying delta 9-tetrahydrocannabinol content and number of puffs. The purpose of this study was to determine marijuana dose-effects on subjective and performance measures over a wider dosage range than previously reported using technology which allowed for specification of both the volume and delta 9-tetrahydrocannabinol (THC) content of smoke delivered, and to relate these effects to plasma THC levels. Seven male community volunteers, who were moderate users of marijuana, smoked 4, 10 or 25 puffs from cigarettes containing either 1.75 or 3.55% THC on 6 separate days. Postsmoking plasma THC levels were systematically related to both number of puffs and cigarette THC content. Maximal THC levels occurred immediately after smoking and ranged from 57 to 268 ng/ml. These plasma levels provided a measure of systemic delivery when a known volume and THC content of marijuana smoke was inhaled. Orderly dose-related increases were also observed for heart rate, expired air carbon monoxide and subjective report of drug effects. The 25-puff, 3.55%-THC condition produced greater plasma THC levels than previously reported and reliably impaired performance on a battery of psychomotor and cognitive tasks with substantial individual differences noted in the degree of performance impairment. Puff number/THC content combinations producing comparable plasma THC levels resulted in similar subjective effects and performance impairment. This study provided a comprehensive assessment of the pharmacological effects of smoked marijuana over a wider and more precisely controlled dosage range than has been accomplished previously.(ABSTRACT TRUNCATED AT 250 WORDS)


Predominant role for nitric oxide in the relaxation induced by acetylcholine in cat cerebral arteries. The possible role of nitric oxide (NO) and other endothelial relaxant factors in the vasodilation induced by acetylcholine (ACh) in isolated segments of cat cerebral arteries was analyzed by using the following treatments: 1) the blockers of cyclo- and lipoxygenase, indomethacin and 5,8,11,14-eicosatetraynoic acid; 2) the NO inactivators, phenidone, hydroquinone and oxyhemoglobin; 3) the inhibitors of NO synthesis, NG-nitro-L-arginine methyl ester and NG-monomethyl-L-arginine; 4) the blockers of sodium pump activity, ouabain and K(+)-free medium; and 5) the antagonist of K+ channels, 4-aminopyridine (4-AP). A comparative study between the relaxant actions of exogenous NO and ACh was also performed. The most relevant results obtained were: 1) cat cerebral arteries are very sensitive to the endothelial effects of ACh, as well as to the exogenous NO; 2) ACh-induced endothelium-dependent dilatation is not affected by indomethacin and 4-AP, partially inhibited by 5,8,11,14-eicosatetraynoic acid, phenidone, hydroquinone, oxyhemoglobin and NG-nitro-L-arginine methyl ester and abolished by NG-monomethyl-L-arginine; 3) this latter effect is selectively antagonized by L-arginine, suggesting that the inhibition of NO synthase may be enough to abolish relaxation to ACh; and 4) sodium pump blockade abolished endothelial but not exogenous NO effects. From these results, we conclude that ACh-induced relaxation in these vessels can be entirely mediated by the release of endothelial NO. Although other endothelial factors cannot be discarded, their possible contribution to ACh-evoked relaxation is likely negligible.


Effect of newly developed tachykinin agonist and antagonists on the guinea pig isolated gallbladder. The present study was undertaken to assess the activity of the neurokinin-2 (NK2) tachykinin receptor-selective antagonists MEN 10,376, L 659,877 and R 396 along with the NK2 receptor-selective ligand, MDL 28,564 on the isolated guinea pig gallbladder. None of the antagonists tested inhibited the response to the peptide CCK-8, although they competitively antagonized the contractile effect produced by NKA in the above tissue. The following rank order of potency was observed: MEN 10,376 (pKB = 6.77) greater than L 659,877 (pKB = 6.29) greater than R396 (pKB = 5.26). MEN 10,376 also antagonized the response to the NK3-selective agonist [MePhe7].NkB (10 microM), showing that the agonist activity of this peptide is due to NK2 receptor stimulation. On the other hand, MDL 28,564 had an agonist effect in the isolated gallbladder, its maximal effect approaching 58% of that to NKA. The results obtained using the above NK2 receptor-selective drugs in the guinea pig gallbladder was similar to that observed in other mammalian tissues bearing the NK2A receptor subtype, such as the endothelium-denuded rabbit pulmonary artery, the guinea pig bronchus and the circular muscle of the human colon and ileum. Therefore, we conclude that in the isolated guinea pig gallbladder tachykinins exert their contractile effect by activating NK2 receptors, which seem to belong to the NK2A receptor subtype.


Highly potent beta-2 sympathomimetics convert to less potent partial agonists as relaxants of guinea pig tracheae maximally contracted by carbachol. Comparison of relaxation with receptor binding and adenylate cyclase stimulation. Beta-adrenergic agonists are among to the most potent dilators of airway smooth muscle available and act as functional antagonists of a variety of contractile stimuli. In order to elucidate their loss of relaxant potency in dependence on antagonistic stimuli tracheal relaxation by the beta-2 sympathomimetics (+/-)-salbutamol (+/-)-fenoterol, and (+/-)-formoterol was compared to (-)-isoprenaline in guinea pig tracheae partially and maximally precontracted by carbachol. In partially precontracted tracheae, salbutamol, fenoterol and formoterol exerted maximum relaxation with low EC50 of 20, 5.6 and 0.29 nmol/l, respectively. In maximally precontracted tracheae, however, salbutamol, fenoterol and formoterol were only partial agonists for relaxation with different intrinsic activities (0.62, 0.62 and 0.77, respectively) and increased EC50 (120, 50 and 3.6 nmol/l, respectively). A reduction of relaxant potency by increased muscarinic stimuli was also observed for beta-1 adrenoceptors stimulated by (-)-noradrenaline after blockade of beta-2 adrenoceptors. In order to investigate if the reduced relaxant potency of beta-2 sympathomimetics was caused by a reduced spare receptor capacity or a reduced intrinsic activity for stimulation of adenylate cyclase (AC), we performed experiments in membranes from lung and tracheal cells. In radioligand binding, beta-2 sympathomimetics recognized the high-affinity state (57%) of the beta-2 adrenoceptor with a lower effectiveness than (-)-isoprenaline, which exhibited a 100-fold higher affinity for high over low-affinity states. Dissociation constants for the low-affinity state matched EC50 for AC stimulation. Intrinsic activities (%) for AC stimulation were significantly lower for salbutamol (67%), fenoterol (67%) and formoterol (89%) than for (-)-isoprenaline (100%), indicating that the reduced relaxation potency of the beta-2 sympathomimetics of maximally precontracted tracheae is caused by a reduced intrinsic activity for AC stimulation. It might be speculated that formoterol could improve drug therapy of asthma due to its high binding affinity and its high intrinsic activity for relaxation.


Desensitization and down-regulation of the 5-hydroxytryptamine1B receptor in the opossum kidney cell line. In the opossum kidney cell line the 5-hydroxytryptamine (serotonin; 5-HT)1B receptor is negatively coupled to adenylyl cyclase via a Gi protein. Preincubation of opossum kidney cell line cell monolayers with 5-HT resulted in 5-HT1B receptor-mediated desensitization expressed as a 4-fold rightward shift of the dose-response curve and a 10 to 29% decrease of maximal inhibition of forskolin-stimulated cyclic AMP production. These moderate decreases in potency and efficacy were concentration- and time-dependent. Maximal desensitization occurred with 3 hr of 5-HT preincubation. Preincubation with 5-HT caused no change in the potency or efficacy of alpha-2 adrenergic agonist-mediated inhibition of forskolin-stimulated cyclic AMP production. Therefore, the desensitization caused by 5-HT preincubation appears to be homologous. Down-regulation of the 5-HT1B receptor, assessed with the high affinity radioligand [125I]iodocyanopindolol, also occurred, and was concentration- and time-dependent. Maximum down-regulation of 40% occurred after 20 hr of exposure to 10 microM 5-HT. These results demonstrate that, like other receptors coupled to the inhibition of adenylyl cyclase, exposure of 5-HT1B receptors to an agonist causes desensitization of the functional response followed by down-regulation of the receptor.


Differential influence of N-dealkylation on the stimulus properties of some opioid agonists. The capacity of several opioid agonists and their N-dealkylated derivatives (normetabolites) to substitute for the discriminative and reinforcing stimulus properties of codeine was evaluated in rhesus monkeys, and the affinity of these compounds in binding to mu receptors in rhesus monkey brain membranes was determined. Heroin (0.1 mg/kg), 6-acetylmorphine (0.1 mg/kg), methadone (0.6 mg/kg), 3-acetylmorphine (1 mg/kg), morphine (1 mg/kg) and codeine (1.8 mg/kg) substituted for the codeine cue, but the normetabolites of heroin, 6-acetylmorphine, morphine and codeine did not (up to 10 mg/kg). l-alpha-Acetylmethadol (3 mg/kg) and its mono (0.1 mg/kg) and double (0.6 mg/kg) N-dealkylated derivatives all substituted. In self administration, subjects responded for morphine (0.1 mg/kg/injection) and codeine (0.3 mg/kg/injection), but not for norcodeine (up to 1 mg/kg/injection) or normophine (up to 3 mg/kg/injection). l-alpha-Acetylmethadol (up to 0.3 mg/kg/injection) did not maintain responding, but its mono (0.1 mg/kg/injection) and double (0.1 mg/kg/injection) normetabolites did. In receptor binding, the normetabolites of morphine and 6-acetylmorphine were less potent than their parent agonists in displacing [3H]Tyr-D-Ala-Gly-(Me)Phe-Gly-ol, but the normetabolites of l-alpha-acetylmethadol had greater affinity than their parent. Codeine and norcodeine were inactive in binding. If l-alpha-acetylmethadol is converted only slowly to its active normetabolites, this may explain the lack of efficacy found with this compound in the self administration procedure.


Chronic benzodiazepine administration. X. Concurrent administration of the peripheral-type benzodiazepine ligand PK11195 attenuates chronic effects of lorazepam. Chronic administration of benzodiazepine active at the tau-aminobutyric acidA receptor ("central" benzodiazepine sites) is associated with behavioral tolerance and receptor downregulation. Recent reports indicate possible interactions between central sites and benzodiazepines active at "peripheral-type" sites located primarily on non-neuronal cells. To evaluate these interactions during chronic administration, we treated mice with lorazepam for 1 to 14 days alone or in combination with the peripheral-type site ligand PK11195 [N-methyl-N-(methyl-1-propyl)chloro-2-phenyl-1-isoquinoline-3-carboxamid e]. Lorazepam was associated with tolerance at 7 days, but tolerance was not observed during concurrent administration of PK11195. Lorazepam was also associated with benzodiazepine receptor down-regulation in cortex and hippocampus at 7 days. With concurrent administration of PK11195, this effect remained in cortex but was absent in hippocampus. tau-Aminobutyric acid-dependent chloride uptake was reduced in both cortex and hippocampus with lorazepam, but not with concurrent lorazepam and PK11195. PK11195 administration alone did not affect behavior or neurochemical parameters, or did it alter brain lorazepam concentrations. These data indicate that concurrent PK11195 administration attenuates behavioral and neurochemical effects of chronic lorazepam administration.


Pyrazole, an alcohol dehydrogenase inhibitor, has dual effects on N-methyl-D-aspartate receptors of hippocampal pyramidal cells: agonist and noncompetitive antagonist. Electrophysiological and biochemical studies demonstrated that pyrazole, an inhibitor of alcohol dehydrogenase and a proposed therapeutic agent for treatment of alcoholic intoxication, activated and blocked the N-methyl-D-aspartate (NMDA) receptor and did not interact significantly with the end-plate nicotinic acetylcholine receptor (AChR). Pyrazole, at concentrations as low as 0.5 microM, applied to outside-out patches excised from the membrane of cultured rat hippocampal neurons, elicited single-channel currents of 48 pS which were blocked by DL-2-amino-5-phosphorovaleric acid, a competitive antagonist of NMDA. In addition, binding studies showed that pyrazole displaced 1-(cis-2-carboxypiperidine-4-yl)methyl-1-phosphoric acid from the agonist recognition site of the NMDA receptor in a concentration-dependent manner and enhanced the binding of (+)-5-methyl-10,11-dihydro-5H- dibenzo[a,d]cyclohepten-5,10-imine to this complex. These data indicate that pyrazole is an agonist at NMDA receptors. However, at higher concentrations, open and burst times as well as the frequency of single-channel currents activated by pyrazole were reduced significantly, a finding which suggests that this compound is also an open channel blocker. In agreement with these results, it was shown biochemically that pyrazole was able to stimulate influx of Ca++ into rat brain microsomes via NMDA receptors and on the other hand to block the influx of Ca++ induced by NMDA. Pyrazole was unable to affect the neuromuscular transmission of frog sartorius muscle-sciatic nerve preparations. Additionally, pyrazole did not interact either with the agonist recognition site or with noncompetitive sites of the AChR. However, this drug had a very weak agonist-like action on the AChR of the Torpedo electric organ, most likely via binding sites different from those described previously for acetylcholine. Therefore, the therapeutic efficacy of pyrazole may be related at least in part to its effects on the NMDA receptor. Furthermore, this compound, because of the small size and rigidity of its molecular structure, becomes a promising drug for the study of the NMDA receptor. Indeed its use may allow a better understanding of the physiological and pathological processes involving this receptor.


Abnormal platelet 5-hydroxytryptamine uptake and imipramine binding in postnatal dysphoria. Platelet 14C-5-hydroxytryptamine (14C-5-HT) uptake, 3H-imipramine binding and monoamine oxidase (MAO) activity were measured in women 5 days postpartum and compared with depression scores (Edinburgh Postnatal Depression Scale) at that time and 6 weeks later. Mean Km of 14C-5-HT uptake was significantly reduced in the group showing dysphoria at 5 days (p less than 0.01). Mean Kd of 3H-imipramine binding was significantly increased in the group who later went on to become depressed at 6 weeks postpartum (p less than 0.03). Vmax for 14C-5-HT uptake, Bmax for 3H-imipramine binding and MAO activity did not differ between depressed and non-depressed patients on either occasion. Although the observed changes manifested in a system known to be disturbed in other forms of depression, they were in affinity rather than Bmax or Vmax. Even though probably not of direct physiological significance, such results, if confirmed, together with other pointers in the literature, suggest biochemical abnormalities specific to the puerperal period.


Irritable bowel syndrome: assessment of psychological disturbance and its influence on the response to fibre supplementation. This study describes the effect of fibre supplementation on the gastrointestinal symptoms and general wellbeing of patients with constipated irritable bowel syndrome. In a single centre, double blind, placebo controlled trial of 3 months duration, a daily supplement of 4.1 g fibre produced no greater change in gastrointestinal symptoms than placebo. Pretreatment constipation was related to baseline fibre intake. Overall outcome was the same in treated and control groups; a considerable placebo response was evident. This level of fibre supplementation is not a useful treatment; improving neither constipation nor other symptoms. At the outset pain severity correlated with depression score on psychometric testing. Those who felt better at the end of the study scored significantly lower for depression at outset than those who felt no better. In irritable bowel syndrome a depressive emotional state profile is a powerful determinant of outcome, shaping the response to treatment, which includes a considerable placebo element.


Development of a series of phenyltetrazole leukotriene D4 (LTD4) receptor antagonists. A hypothetical model for receptor binding of leukotriene D4 (LTD4) was deduced from conformational analysis of LTD4 and from the structure-activity relationships (SAR) of known LTD4 receptor antagonists. A new structural series of LTD4 receptor antagonists exemplified by 5-[4-(4-phenylbutoxy)phenyl]-2-[4-(tetrazol-5-yl)butyl]-2H-t etrazole was designed in which a phenyltetrazole moiety was incorporated as a receptor binding equivalent of the triene unit of LTD4. A number of these phenyltetrazoles were prepared and found to possess LTD4 receptor antagonist activity. The structure-activity relationship (SAR) of this series is described.


Optimization of the quinoline and substituted benzyl moieties of a series of phenyltetrazole leukotriene D4 receptor antagonists. This report describes the development of a series of highly potent quinoline-based leukotriene D4 (LTD4) receptor antagonists containing an N-benzyl-substituted phenyltetrazole moiety. They were designed to provide both the correct positioning of the acidic function and secondary lipophilic domain required for strong receptor binding. Members of this series possess high activity in blocking LTD4-induced contractions of isolated guinea pig ileum. Compound 32, LY287192 (2-[[5-[3-[2-(7-chloroquinolin-2- yl)ethenyl]phenyl]-2H-tetrazol-2-yl]methyl]-5-fluorobenzoic acid sodium salt), blocked contraction with a pKB value of 9.1 +/- 0.3. Qualitative structure-activity studies have demonstrated specific requirements for the best activity. In particular, ortho substitution of the benzyl group with an acidic function was crucial for maximum potency. In cases similar to 32, where the benzyl group possesses an ortho carboxylate, the N-2-substituted tetrazole isomer showed 100-fold greater activity relative to the corresponding N-1 isomer. This pattern was reversed when the acid was substituted at the para position. The quinoline unit may be replaced by other nitrogen-containing heterocycles.