Regulation of receptor-mediated shape change in astroglial cells. Activation of adenylate cyclase in astroglial cells in culture results in a rapid change in cell shape that appears to occur by the active movement of cytoplasm from peripheral cell regions to the perinuclear space with processes being formed along regions that remain extended. Three series of experiments were designed to determine how shape change occurred. First, the Ca(2+)-dependency of shape change was determined by reducing intracellular Ca2+ concentrations to less than or equal to 50 nM or increasing intracellular Ca2+ concentrations to greater than or equal to 1 microM. Neither of these changes significantly affected the rate of receptor-mediated shape change. Second the role that longer-lived, acetylated microtubules play in receptor-mediated shape change was assessed by visualizing microtubules using a polyclonal antibody to brain 6S tubulin or a monoclonal antibody to oligomers of tubulin to monitor total tubulin distribution and a monoclonal antibody to acetylated tubulin to describe the distribution of these microtubules. Three-dimensional distribution of microtubules was observed by optical sectioning of cultures using a laser scanning confocal imaging system. The distribution of acetylated tubules in control cells was similar to that observed with the antibodies to tubulin. Following treatment with 100 nM isoproterenol to stimulate shape change, there was a dramatic redistribution of microtubules; however, the distribution of acetylated tubules was again similar to the total microtubules. Analysis of the optical sections recorded using the confocal attachment revealed that while control cells were relatively flat (cell height = 4 microns), the perinuclear region of isoproterenol-treated cells extended much higher above the substrate (cell height = 13 microns). Third, the role of microtubule assembly and disassembly were assessed using colchicine and taxol. Results from these experiments suggest that microtubule reassembly is necessary for receptor-mediated shape change. Control experiments indicated that colchicine or taxol treatment did not inhibit either cAMP synthesis or another cAMP-dependent process, receptor-mediated taurine release. Together these results indicate that receptor-mediated shape change in astroglial cells occurs by a Ca(2+)-independent mechanism that results in active movement of cytoplasm to the perinuclear region. This process is dependent on microtubule reassembly suggesting that shape change may occur by active movement of material along microtubules or by microtubule redistribution.


Co-cultures of microglia and astrocytes from kainic acid-lesioned adult rat hippocampus: effects of glutamate. The long-standing question concerning the direct actions of glutamate on the membrane potential of astroglial cells in the central nervous system was addressed using the in vitro kainic acid-lesioned hippocampal slice preparation and primary cell co-cultures of astrocytes and microglia derived from such lesions. The ultrastructure of the lesioned hippocampus was examined to aid in the identification of the cells appearing in culture. In culture, microglia appeared as flat cells, less than 1 micron in thickness at the edge of the cell, but thicker (about 5 microns) near the nucleus. The cytoplasm was packed with granular inclusions. Microglia appeared in two morphological forms, amoeboid and ramified. The amoeboid form was characterized by a cell body with a single process, and was always observed 1 day after starting the cell culture. Such cells became less frequent after 1 week in culture. The ramified form appeared as a rounded cell, devoid of processes, and were frequently observed in older cultures (greater than 1 week). Microglia did not round up after exposure to dibutyrylcyclic adenosine monophosphate (cAMP), and did not stain for glial fibrillary acidic protein (GFAP). An ultrastructural examination of the lesion demonstrated that microglia were present and that they contained many cytoplasmic granules similar to lipofuscin-containing granules. No filaments were observed in the cytoplasm of microglia. By contrast, the cytoplasm of astrocytes in culture had far fewer granules, rounded up to dibutyryl-cAMP, exhibited multiple processes, and stained for GFAP. In slices, astrocytes had no lipofuscin-containing granules, but numerous cytoplasmic filaments were present.(ABSTRACT TRUNCATED AT 250 WORDS)


Serotonin promotes region-specific glial influences on cultured serotonin and dopamine neurons. To test the hypothesis that glia mediate interactions between embryonic serotonergic (5-HT) neurons and dopamine neurons, we studied the effects of 5-HT in co-cultures of E14 raphe neurons of mesencephalic dopamine neurons and radial glia/astrocytes derived from the same (homotypic) or opposite (heterotypic) brain region using a dose (10(-5) M) that would produce 5-HT uptake into glial cells as well as activate 5-HT receptors. Morphometric analysis of 5-HT and tyrosine hydroxylase (TH) immunoreactive neurons revealed regional differences in the effects of 5-HT (and nialamide) on survival, cell soma size, and dendrite-like neurite outgrowth in neuronal-glial co-cultures. In general, 5-HT had more significant effects on both types of monoamine neuron when they were cultured with mesencephalic glia (GSN). Stimulatory effects of 5-HT on growth of TH neurons in GSN cultures suggest that developing raphe axons, which reach the mesencephalon during the early differentiation of these neurons, may enhance the influence of local glial-derived trophic factors. Likewise, the promotion of 5-HT neuronal survival in these cultures suggests that glial factors in the mesencephalon may contribute to the support of 5-HT neurons in addition to the influences of raphe glia. The inhibitory effects of 5-HT on neurite outgrowth by raphe neurons in GSN co-cultures indicates enhanced sensitivity of these neurons to the inhibitory effects of 5-HT in the presence of mesencephalic glia. The region-specific effects of 5-HT and nialamide in glial co-cultures suggest that raphe and mesencephalic glia may express different capacities for 5-HT uptake, receptors, and/or monoamine oxidase (MAO) activities. These characteristics could be important for the specificity of growth-regulatory influences of glial cells on the development of brain monoamine neurons.


Aggregative adherence fimbriae I of enteroaggregative Escherichia coli mediate adherence to HEp-2 cells and hemagglutination of human erythrocytes. Strains of enteroaggregative Escherichia coli (EAggEC) have been implicated in several studies as important agents of persistent diarrhea among infants in the developing world. We have previously shown that the aggregative adherence (AA) property of EAggEC is associated with the presence of a 60-MDa plasmid which confers AA when introduced into E. coli HB101. Here, we report the cloning of the AA determinant from EAggEC strain 17-2 into the 21.5-kb cosmid vector pCVD301. TnphoA mutagenesis of the AA cosmid clone pJPN31 implicated an AA region of approximately 12 kb. Transmission electron microscopy of HB101 (pJPN31) revealed the presence of bundle-forming fimbriae, which were absent in AA- TnphoA insertion mutants. The presence of these fimbriae, AA, and hemagglutination (HA) of human erythrocytes were all concurrently lost by single-insertion mutations. A 14-kDa protein was seen on polyacrylamide gel electrophoresis and Western blotting (immunoblotting) of surface shear preparations from fimbriated clones. Twelve of nineteen volunteers fed EAggEC 17-2 developed rises in antibodies to the 14-kDa protein as determined by Western blot. We have termed the cloned bundle-forming fimbriae aggregative adherence fimbriae I (AAF/I); positivity with a previously described EAggEC probe and human erythrocyte HA appear to correlate with the presence of AAF/I.


Characterization of the heat shock response in Brucella abortus and isolation of the genes encoding the GroE heat shock proteins. In an effort to define the heat shock response in the bovine intracellular pathogen Brucella abortus, a rough variant lacking extensive lipopolysaccharide was pulse-labeled with [35S]methionine following exposure to elevated temperatures. The major heat shock proteins observed following sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography migrate at 70, 62, 18, and 10 kDa. The maximum response was observed between 42 and 46 degrees C and within 2 to 3 h of the shif in temperature and varied slightly for the different proteins. Accumulation of the 62-kDa heat shock protein (62-kDa Hsp) was observed to continue for up to 5 h following the shift in temperature. In an effort to better define the heat shock response and its potential relationship with protective immunity, genes encoding the major heat shock proteins were isolated from recombinant libraries constructed from B. abortus S19 and S2308 and sequenced. The 62-kDa Hsp shares more than 60% amino acid homology with members of the GroEL family and is immunoprecipitated with polyclonal antibodies to Escherichia coli GroEL and monoclonal antibodies to mycobacterial Hsp 65. Western blot (immunoblot) analysis with pooled sera from vaccinated and infected cattle revealed that the 62-kDa Hsp is a predominantly recognized antigen. The roles of these gene products during environmental stress and in protective immunity against brucellosis are under investigation.


Functional expression of heterologous fimbrial subunits mediated by the F41, K88, and CS31A determinants of Escherichia coli. F41, K88, and CS31A are fimbrial adhesins associated with enterotoxigenic Escherichia coli. These adhesins are distinct from one another in the composition of their structural subunits and the adherence characteristics associated with their expression. Despite these differences, extensive homology exists between the genetic determinants mediating the expression of these adhesins, extending throughout the region of each determinant encoding the accessory proteins involved in adhesin biogenesis. This suggests that the regulatory and assembly systems mediating expression of these adhesins may be functionally interchangeable. In the present study we demonstrated that the accessory systems of the F41, K88, and CS31A determinants are able to mediate the functional expression of heterologous fimbrial subunit proteins. Plasmid constructs containing the isolated fimbrial subunit gene of the F41 or CS31A determinant were prepared and introduced into E. coli harboring the F41, K88, and CS31A accessory genes contained on compatible plasmid vectors. The ability of each of the three accessory systems to mediate stable expression of the F41 or CS31A fimbrial subunit peptide was demonstrated by Western blot (immunoblot) analysis. Functional expression of the F41 or CS31A subunit on the bacterial cell surface was demonstrated by the ability of these proteins to confer mannose-resistant hemagglutination of human erythrocytes or in vitro adherence to epithelial cells, respectively. The accessory system of an unrelated adhesin determinant, F1845, did not mediate functional expression of F41 adherence. Taken together, these data indicate that the genetic determinants mediating expression of the F41, K88, and CS31A adhesins are members of a closely related family and suggest that a mechanism exists in the family for the more rapid divergence of genes encoding antigenic and adhesive determinants.


Neurotransmitters as neurotrophic factors: a new set of functions. At the start of this review, factors were deemed trophic if they stimulated mitosis, permitted neural cell survival, promoted neurite sprouting and growth cone motility, or turned on a specific neuronal phenotype. The in vitro evidence from cell cultures is overwhelming that both neurotransmitters and neuropeptides can have such actions. Furthermore, the same chemical can exert several of these effects, either on the same or on different cell populations. Perhaps the most striking example is that of VIP, which can stimulate not only mitosis, but also survival and neurite sprouting of sympathetic ganglion neuroblasts (Pincus et al., 1990a,b). The in vivo data to support the in vitro experiments are starting to appear. A role for VIP in neurodevelopment is supported by in vivo studies that show behavioral deficits produced in neonatal rats by treatment with a VIP antagonist (Hill et al., 1991). The work of Shatz' laboratory (Chun et al., 1987; Ghosh et al., 1990) suggests that neuropeptide-containing neurons, transiently present, serve as guideposts for thalamocortical axons coming in to innervate specific cortical areas. Along similar lines, Wolff et al. (1979) demonstrated gamma-aminobutyric acid-accumulating glia in embryonic cortex that appeared to form axoglial synapses and suggested the possibility that gamma-aminobutyric acid released from the glia might play a role in synaptogenesis by increasing the number of postsynaptic thickenings. Meshul et al. (1987) have provided evidence that astrocytes can regulate synaptic density in the developing cerebellum. The work of Zagon and McLaughlin (1986a,b, 1987) has shown that naltrexone, an antagonist of the endogenous opioid peptides, affects both cell number and neuronal sprouting. Lauder's laboratory (Lauder et al., 1982) has shown a role for 5-HT in regulation of the proliferation of numerous cell types. These studies illustrate another important point, that neurotransmitters and neuropeptides function in communication not only between neurons, but also between neurons and glial cells, and between glial cells. Given that astrocytes can express virtually all of the neural receptors and can produce at least some of the neurotransmitters and neuropeptides, they must now be considered equal partners in the processes of intercellular communication in the nervous system, including the trophic responses. The actions of neurotransmitters and neuropeptides have to be considered in terms of a broad spectrum of actions that range from the trophic actions described in this review, to the classic transmitter actions, to potential roles in neurotoxicity.(ABSTRACT TRUNCATED AT 400 WORDS)


The retinoblastoma gene product suppresses neu oncogene-induced transformation via transcriptional repression of neu. The retinoblastoma susceptibility gene (Rb) is a tumor suppressor gene involved in the etiology of many types of human cancers. However, the molecular mechanisms involved in tumor suppression by Rb are largely unknown. The neu gene is a dominant transforming oncogene and a member of the growth factor receptor tyrosine kinase gene family. Both inactivation of the Rb gene and overexpression of the neu gene are involved in human breast and lung cancers. Therefore, it is of interest and importance to investigate the potential interactions between Rb and neu. Here we show that Rb suppresses neu-induced transformation by focus formation assays. This transformation suppression by Rb was further shown to be due to transcriptional repression of neu using Rb expressing effector plasmid and neu promoter-chloramphenicol acetyltransferase reporter gene. The cis-acting element conferring Rb-mediated repression was mapped to a recently identified novel enhancer in the neu promoter. The data indicate that the growth factor receptor neu is a target for the Rb gene product and transcriptional repression of a dominant oncogene expression may be one of the molecular mechanisms of Rb-mediated tumor suppression.


The physical and enzymatic properties of Escherichia coli recA protein display anion-specific inhibition. The enzymatic activities of Escherichia coli recA protein are sensitive to ionic composition. Here we report that sodium glutamate (NaGlu) is much less inhibitory to the DNA strand exchange, DNA-dependent ATPase, and DNA binding activities of the recA protein than is NaCl. Both joint molecule formation and complete exchange of DNA strands occur (albeit at reduced rates) at NaGlu concentrations as high as 0.5 M whereas concentrations of NaCl greater than 0.2 M are sufficient for complete inhibition. The single-stranded DNA (ssDNA)-dependent ATPase activity is even less sensitive to inhibition by NaGlu; ATP hydrolysis stimulated by M13 ssDNA is unaffected by 0.5 M NaGlu and is further stimulated by E. coli ssDNA binding protein approximately 2-fold. Finally, NaGlu has essentially no effect on the stability of recA protein-epsilon M13 DNA complexes, with concentrations of NaGlu as high as 1.5 M failing to dissociate the complexes. Surprisingly, NaGlu also has little effect on the concentration of NaCl required to disrupt the recA protein-epsilon M13 DNA complex, demonstrating that destabilization is dependent on both the concentration and type of anionic rather than cationic species. Quantitative analysis of DNA binding isotherms establishes that the intrinsic binding affinity of recA protein is affected by the anionic species present and that the cooperativity parameter is relatively unaffected. Consequently, the sensitivity of recA protein-ssDNA complexes to disruption by NaCl does not result from the competitive effects associated with cation displacement from the ssDNA upon protein binding but rather results from anion displacement upon complex formation. The magnitude of this anion-specific effect on ssDNA binding is large relative to that of other nucleic acid binding proteins.


Glutamate-malate metabolism in liver mitochondria. A model constructed on the basis of mitochondrial levels of enzymes, specificity, dissociation constants, and stoichiometry of hetero-enzyme complexes. The level of aspartate aminotransferase in liver mitochondria was found to be approximately 140 microM, or 2-3 orders of magnitude higher than its dissociation constant in complexes with the inner mitochondrial membrane and the high molecular weight enzymes (M(r) = 1.6 x 10(5) to 2.7 x 10(6)) carbamyl-phosphate synthase I, glutamate dehydrogenase, and the alpha-ketoglutarate dehydrogenase complex. The total concentration of aminotransferase-binding sites on these structures in liver mitochondria was more than sufficient to accommodate all of the aminotransferase. Therefore, in liver mitochondria, the aminotransferase could be associated with the inner mitochondrial membrane and/or these high molecular weight enzymes. The aminotransferase in these hetero-enzyme complexes could be supplied with oxalacetate because binding of aminotransferase to the high molecular weight enzymes can enhance binding of malate dehydrogenase, and binding of both malate dehydrogenase and the aminotransferase facilitated binding of fumarase. The level of malate dehydrogenase was found to be so high (140 microM) in liver mitochondria, compared with that of citrate synthase (25 microM) and the pyruvate dehydrogenase complex (0.3 microM), that there would also be a sufficient supply of oxalacetate to citrate synthase-pyruvate dehydrogenase.


Energy-dependent accumulation of daunorubicin into subcellular compartments of human leukemia cells and cytoplasts. Anthracycline accumulation was evaluated by flow cytometry or radiolabeled drug assays in cells and cytoplasts (enucleated cells) prepared from parental and multidrug-resistant human K562 leukemia cells. Treatment with energy inhibitors, such as dinitrophenol (DNP) or sodium azide/deoxyglucose, led to a marked decrease in daunorubicin accumulation in parental cells and cytoplasts. Another ionophore, monensin, also caused a significant decrease in daunorubicin accumulation; however, ATPase inhibitors ouabain, vanadate, and N-ethylamaleimide had little or no effect. The lysosomatropic agents chloroquine and methylamine caused a moderate decrease in anthracycline accumulation. Fluorescence microscopy showed that the DNP-sensitive daunorubicin uptake occurred in a nonnuclear subcellular compartment. Studies using increasing daunorubicin concentrations demonstrated fluorescence quenching that occurred in the nonnuclear, DNP-sensitive compartment. The effect of inhibitors on the accumulation of rhodamine 123 and acridine orange strongly implicated lysosomes as the principal compartment of this inhibitable daunorubicin accumulation. Cytoplasts from P-glycoprotein containing multidrug-resistant K562 cells demonstrated a verapamil-reversible, decreased daunorubicin accumulation that was observed in resistant whole cells. Verapamil pretreatment of cytoplasts from resistant cells revealed the subcellular DNP-sensitive uptake present in parental cytoplasts. These studies demonstrate that cytoplasts are an effective means to study drug transport in mammalian cells without nuclear drug binding. Parental K562 cells and cytoplasts exhibit an energy-dependent accumulation of daunorubicin into cytoplasmic organelles that is also present in resistant cells and cytoplasts when P-glycoprotein mediated efflux is inhibited.


The p88 molecular chaperone is identical to the endoplasmic reticulum membrane protein, calnexin. We previously described a novel molecular chaperone (designated p88) that participates in the assembly of murine class I histocompatibility molecules (Degen, E., and Williams, D. B. (1991) J. Cell Biol. 112, 1099-1115). Our findings suggest that p88 may either promote proper assembly of class I molecules or retain them, probably within the endoplasmic reticulum (ER), until assembly of the ternary complex of heavy chain, beta 2-microglobulin, and peptide ligand is complete. In this report, we compare p88 to calnexin, a calcium-binding 90-kDa phosphoprotein of the ER membrane (Wada, I., Rindress, D., Cameron, P. H., Ou, W.-J., Doherty, J.-J., II, Louvard, D., Bell, A.W., Dignard, D., Thomas, D. Y., and Bergeron, J. J. M. (1991) J. Biol. Chem. 266, 19599-19610). We show that p88 and calnexin share antigenic epitopes defined by a polyclonal anti-calnexin antiserum. Furthermore, both proteins were immunoprecipitated in association with an intracellularly retained variant of the class I H-2Kb molecule. Since p88 and calnexin were also indistinguishable by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis, were resistant to digestion with endoglycosidase H, and exhibited virtually identical patterns of peptide fragments following digestion with either V8 protease or trypsin, we conclude that p88 and calnexin represent the same protein. The identification of the p88 chaperone as a phosphorylated, calcium-binding protein of the ER membrane suggests possible means whereby its interaction with class I molecules may be regulated.


Dietary regulation of stearoyl-CoA desaturase 1 gene expression in mouse liver. Expression of the murine liver stearoyl-CoA desaturase gene (SCD1) is induced upon feeding fasted mice a fat-free, high carbohydrate diet (Ntambi, J. M., Buhrow, S. A., Kaestner, K. H., Christy, R. J., Sibley, E., Kelly, T. J., and Lane, M.D. (1988) J. Biol. Chem. 263, 17291-17300). In the present study, SCD1-specific RNA riboprobes and cDNA probes were used to study the mechanism for the induction of SCD1 mRNA. Based on the time course of induction, the SCD1 mRNA increased from 2-fold within 6 h to 45-fold within 36 h. Nuclear run-on transcription studies showed that the accumulation of SCD1 mRNA after refeeding starved mice a fat-free, high carbohydrate diet was a consequence of the transcriptional activation of the SCD1 gene. The SCD1 mRNA level decreased rapidly (t1/2 = approximately 4 h) within 24 h when mice fed the fat-free, high carbohydrate diet were switched to a regular chow diet. Furthermore, when the fat-free diet was supplemented with triacylglycerides containing polyunsaturated fatty acids, the transcription of the SCD1 gene and the induction of the SCD1 mRNA were significantly blunted. Triacylglycerides containing saturated and monounsaturated fatty acids were without dramatic effects. In contrast, synthesis of liver albumin mRNA was little affected by any of these dietary variations, indicating that the observed changes in the transcription of the SCD1 gene and mRNA levels were specific. These data demonstrate that both dietary carbohydrates and polyunsaturated fatty acids or their metabolites directly or indirectly regulate the expression of the SCD1 gene in mouse liver.


Effect of xamoterol in a patient with idiopathic autonomic failure. We report the effect of xamoterol on autonomic, humoral and haemodynamic function in a patient with idiopathic autonomic neuropathy. Xamoterol produced marked symptomatic improvement, slightly increased heart rates in the sitting, supine and upright tilt positions and higher blood pressures while seated and on recovery from upright tilt. There were, however, no differences in blood pressure responses following a meal or during head-up tilt. We suggest that before ascribing symptomatic benefit to xamoterol in this condition, objective haemodynamic improvement should be recorded. Further studies of treatment in idiopathic autonomic failure require to be placebo controlled.


Effects of tannic acid on antigenicity and membrane contrast in ultrastructural immunocytochemistry. We have modified our previous method for immunogold staining of unosmicated, plastic-embedded tissue by addition of tannic acid as a post-fixative to increase membrane contrast. Overall cell ultrastructure and organelle membranes, in particular, appeared well preserved after this treatment. We evaluated quantitatively the effect of tannic acid on the antigenicity of several membrane proteins in rat liver and intestine. For all antigens tested, significant antigenicity was retained on both intracellular and plasma membranes. However, the level of antigenicity decreased with increased concentrations of tannic acid. This effect was most apparent on the apical and basolateral membranes of hepatocytes and on the apical membrane of enterocytes, surfaces that had been in direct contact with the tannic acid fixative. The results indicate that when low concentrations of tannic acid are employed, this method yields greatly enhanced membrane contrast while preserving sufficient antigenicity to facilitate the ultrastructural localization of many membrane and other antigens.


Cold stress response in patients with severe burns after beta-blockade. Adrenergic receptor blockade has been shown to be of benefit in the treatment of adverse cardiovascular changes in patients with burns during the hypermetabolic phase. This article examines the stress response to cold exposure in adults and children with 33% to 95% total body surface area burns with and without beta-blockade. Resting energy expenditures were measured by indirect calorimetry; the test subjects were exposed to mean temperatures of 27.5 degrees C (room temperature) or 24.6 degrees C (cold). The mean resting energy expenditure at ambient room temperature in patients with burns without beta-blockade was 1411 +/- 70 kcal/m2/day (mean +/- SEM). This value was 142% of that predicted for normal control subjects without burns. When placed in a cold temperature, patients with burns significantly increased their resting energy expenditures by 160% of predicted values, whereas patients with similar burns and beta-blockade increased their resting energy expenditures by 156%. Adults with septic burns had a mean resting energy expenditure 198% of the predicted value. These patients did not significantly increase their resting energy expenditures when they were exposed to cold either with or without beta-blockade. Data suggest that patients with septic burns already have a maximal metabolic response and that cold stress does not further increase this response. Males, ages 17 to 54 years, were found to increase their resting energy expenditures by 11.4 kcal/m2/day for each percent total body surface area burn. We conclude that beta-blockade with propranolol in therapeutic doses may be used in patients with burns without adversely affecting the cold stress response.


T-deficient transmembrane signaling in CD4+ T cells of retroviral-induced immune-deficient mice. The defective virus found in the LP-BM5 mixture of murine leukemia viruses induces a severe immune deficiency disease in C57BL/6 mice that is characterized by the activation and expansion of T and B cells that become unresponsive to normal immune stimuli. The nature of the biochemical lesion in these defective lymphocyte populations remains unknown. Flow cytometric analysis of the T cell population in infected animals has demonstrated expansion of both CD4+ and CD8+ subsets. Despite chronic expansion in vivo, CD4+ T cells by wk 4 postinfection failed to up-regulate cell surface IL-2R expression, produced IL-2, or proliferate in vitro in response to either Con A, Staphylococcal enterotoxin super-antigens, or anti-CD3 stimulation. Exogenous IL-2 did not restore the proliferative response and also failed to up-regulate IL-R expression on CD4+ T cells from infected mice, even though basal IL-2R expression was initially elevated compared to normals. In contrast, CD4+ T cells from infected mice could be induced to proliferate by stimulation with PMA and ionomycin resulting in IL-2R up-regulation, IL-2 production, and proliferation. Moreover, proliferation could also be induced by anti-CD3 plus PMA, although anti-CD3 plus ionomycin was without effect. These studies suggest that chronic expansion of CD4+ T cells in infected mice is probably not maintained by normal TCR signaling, which appears defective in these cells. In addition, the lesion in biochemical signaling appears to result in defective activation of protein kinase C, which can be overcome by direct activation with PMA.


Glutamate differentially inhibits the expression of class II MHC antigens on astrocytes and microglia. MHC molecules are required for Ag recognition by T cells. Inasmuch as cells in the central nervous system do not express MHC constitutively, appearance of MHC, in inflammatory and degenerative diseases of the brain, may indicate local Ag presentation and subsequent immune response. Although both astrocytes and microglia are capable of class II MHC expression in vitro, in vivo studies failed to show the presence of significant amounts of class II on astrocytes compared to microglia. Our study is designed to clarify possible regulatory mechanisms that can explain the differences in inducibility of class II MHC between astrocytes and microglia in vivo. Using dissociated rat brain cell cultures, we have found that glutamate, an excitatory neurotransmitter, exerted a profound inhibitory effect on IFN-gamma-induced expression of class II on astrocytes, but not on microglia. Both glutamate and norepinephrine, a neurotransmitter previously reported to down-regulate class II on astrocytes, inhibited the induction of class II on astrocytes by eliminating accumulation of class II MHC mRNA. The kinetics of class II mRNA induction by IFN-gamma in the presence of glutamate suggested that glutamate may act as a transcriptional inhibitor. It is likely that class II induction on astrocytes in vivo may be selectively down-regulated by neurotransmitters such as glutamate and norepinephrine.


Cloning and expression of murine IL-12.

Cloning and expression of murine IL-12. Human IL-12 (NK cell stimulatory factor, cytotoxic lymphocyte maturation factor) is a heterodimeric cytokine that can act as a growth factor for activated human T and NK cells, enhance the lytic activity of human NK/lymphokine-activated killer cells, and stimulate the production of IFN-gamma by resting human PBMC. Because in our hands, human IL-12 did not elicit similar responses in murine lymphocytes, we have cloned and expressed the murine IL-12 subunit cDNA in order to obtain recombinant protein for murine studies. Comparison of the predicted amino acid sequences of the murine subunits with their human counterparts revealed that the p40 subunits are more highly conserved than the p35 subunits (70% vs 60% identity, respectively). The sizes of the p35 and p40 subunit mRNA were estimated to be 1.5 kb and 2.6 kb, respectively. RNA blot analysis showed that p35 mRNA was expressed in lymphoid tissues (spleen, thymus) and nonlymphoid tissues (lung, brain), whereas p40 mRNA expression was only detected in lymphoid cells. Incubation of splenocytes with pokeweed mitogen did not significantly affect p35 mRNA levels, however, it resulted in a decrease of p40 mRNA. Coexpression of the murine p35 and p40 cDNA clones in COS cells resulted in the secretion of IL-12, which was active in human and mouse T cell proliferation, murine NK cell activation, and murine IFN-gamma induction assays. Transfection of each subunit cDNA alone did not result in measurable secreted IL-12 activity. A hybrid heterodimer consisting of murine p35 and human p40 subunits retained bioactivity on murine cells; however, the combination of human p35 and murine p40 was completely inactive on murine cells. These results indicate that the observed inability of human IL-12 to act on murine cells is largely determined by the p35 subunit.


Regulation of tumor necrosis factor production by adrenaline and beta-adrenergic agonists. The effects of adrenaline and isoproterenol, a specific beta-adrenergic agonist, on TNF production were investigated. Both agents inhibited the production of TNF by human blood and THP-1 cells stimulated by LPS. The effect of adrenaline was prevented by a beta-receptor antagonist, but not by an alpha-receptor antagonist. Levels of TNF mRNA were not reduced by adrenaline. Inhibition of TNF production was observed only if cells were first exposed to adrenaline or isoproterenol at about the same time as to LPS; incubation of THP-1 cells with isoproterenol for 24 h before LPS stimulation dramatically increased response, and prevented suppression of TNF production by a second dose of isoproterenol. Intracellular cAMP levels were increased by adrenaline and isoproterenol, at concentrations that inhibited TNF production. However, prolonged incubation of THP-1 cells with isoproterenol resulted in depression of cAMP concentrations to below basal levels. These data suggest that TNF production can be regulated by beta-receptor stimulation, that such regulation is mediated by changes in intracellular cAMP concentrations and is exerted at a posttranscriptional level. Adrenaline may be an important endogenous regulator of TNF production in sepsis.


Parallel regulation of IL-4 and IL-5 in human helminth infections. To investigate the relationship between cytokine production and the increased levels of serum IgE and peripheral eosinophilia commonly accompanying human helminth infections, we studied the ability of PBMC of normal (N1) (n = 18) and eosinophilic individuals with helminth infections (H1) (n = 9) to produce IL-3, IL-4, IL-5, granulocyte-macrophage-CSF, and IFN-gamma in vitro after stimulation with PMA (50 ng/ml) and ionomycin (1 microgram/ml). The two groups differed in both the levels of serum IgE and eosinophilia. For mitogen-induced production of granulocyte-macrophage-CSF and IFN-gamma, there was no difference in cytokine production between the two groups. In marked contrast, supernatants from PBMC of infected individuals had significantly higher levels of IL-4 (mean = 213 pg/ml for N1 and 944 pg/ml for H1, p less than 0.02), IL-5 (mean = 180 pg/ml for N1 and 1118 pg/ml for HL, p less than 0.001), and IL-3 (mean = 13900 pg/ml for N1, 28029 pg/ml for H1, p less than 0.05). In addition, helminth-infected patients had approximately 5-fold greater numbers of T cells capable of producing IL-5 and 2.5-fold greater frequency of IL-4-secreting cells than did normal individuals; GM-CSF- and IFN-gamma-producing T cell numbers were not significantly different in the two groups. IL-3-producing cell frequencies could not be evaluated by this method. There was a direct correlation between IL-4 production and IL-5 production at the level of both protein production and frequency of T cells capable of producing these cytokines. These data indicate that individuals with reactive eosinophilia and elevated serum IgE have an expanded population of lymphocytes producing IL-4 and IL-5 and the association of the two suggests that the regulation of IL-4 and IL-5 may be linked.


Fibroblast stimulation in schistosomiasis. XII. Identification of CD4+ lymphocytes within schistosomal egg granulomas as a source of an apparently novel fibroblast growth factor (FsF-1). Granulomas that form around Schistosoma mansoni eggs deposited in the liver secrete a variety of fibrogenic factors that may provide a molecular link between chronic inflammation and hepatic fibrogenesis in schistosomiasis. We recently isolated from conditioned medium of egg granuloma cultures a approximately equal to 60-kDa heparin-binding growth factor for fibroblasts. Because this protein is distinct from other defined heparin-binding growth factors, we designated it "fibroblast stimulating factor-1" (FsF-1). We now report that FsF-1 is a lymphokine. We prepared IgG antibody against purified FsF-1 and determined that it did not cross-react with a variety of growth factors or recombinant interleukins. Using two-color flow cytometry of dissociated granuloma cell suspensions, we observed that approximately 20% to 25% of granuloma CD4+ lymphocytes express surface FsF-1. We isolated CD4+ granuloma lymphocytes by FACS and observed that these cells spontaneously secrete into culture supernatant a fibroblast mitogen that is neutralized by anti-FsF-1 antibody. Furthermore, anti-FsF-1 can specifically immunoprecipitate a metabolically labeled protein produced by the granuloma CD4+ lymphocytes. The labeled protein has the same apparent molecular mass (approximately equal to 60 kDa) as FsF-1 purified from granuloma culture supernatants. These findings define CD4+ lymphocytes as a source of FsF-1. Because FsF-1 has biologic and chemical features distinct from most other defined lymphokines and from other heparin-binding growth factors, FsF-1 appears to be a novel lymphokine.


Structure of the mouse IL-10 gene and chromosomal localization of the mouse and human genes. The nucleotide sequence of a 7.2-kb segment containing the mouse IL-10 (mIL-10) gene was determined. Comparison to the mIL-10 cDNA sequence (Moore, K. W., et al. 1990. Science 248:1230; 250:494) revealed the presence of five exons that span approximately 5.1 kb of genomic DNA. The noncoding regions of the mIL-10 gene contain sequences that have been associated with transcriptional regulation of several cytokine genes. The mIL-10 gene was mapped to mouse chromosome 1 and the human IL-10 gene was also mapped to human chromosome 1.


Apparent ineffectiveness of natural killer cells vis-à-vis retrovirus-infected targets. The role of NK cells in the defense against retroviral infections is ill defined. The discovery of the pathogenic human retroviruses and their epidemic spread have made more urgent a better understanding of how such infections may be naturally controlled. Therefore, a systematic study was undertaken to determine whether NK cells obtained from healthy individuals are able to recognize and lyse target cells that have been infected with HTLV-I, HTLV-II, or HIV. The studies demonstrated that NK cells can recognize retrovirus-infected cells as evidenced by rapid conjugation, but that neither freshly isolated, nor IL-2 stimulated cells cause lysis of such targets. As has been reported for NK-resistant tumor cells, removal of sialic acid residues rendered the retrovirus-infected target cells vulnerable to NK cell attack. Although these data do not suggest that boosting natural immunity would be a useful treatment modality for patients with AIDS or HTLV-related diseases, the observations may help to explain why the small number of cells that harbor retroviruses in patients with subclinical infection are not eliminated.


Human intercellular adhesion molecule-1 gene and its expression in the skin. Cell adhesion molecules are cell-surface proteins that allow specific cell-cell interactions among leukocytes, as well as between leukocytes and other cells. Recent studies have shown that the differential expression of selected cell-adhesion molecules plays a critical role in cutaneous inflammation, immunologic responses, and wound repair. Intercellular adhesion molecule-1 (ICAM-1) is a cell-adhesion molecule that is constitutively expressed on human dermal microvascular endothelial cells (HDMEC) and is inducible on human keratinocytes (HK). Its regulated expression is vital to the initiation and evolution of localized inflammatory processes in the skin. ICAM-1 serves as a specific ligand for lymphocyte function-associated antigen-1 (LFA-1), a cell-surface protein expressed on all leukocytes. The regulated expression of ICAM-1 allows leukocytes to bind to endothelial cells at sites of inflammation and, after exiting into the tissue, to interact with specific target cells, such as HK. Furthermore, specific cytokines are capable of differentially regulating ICAM-1 expression on HDMEC, HK, and other cells. The biologic relevance of ICAM-1 expression in cutaneous inflammation is further supported by functional studies demonstrating the critical role of ICAM-1/LFA-1 interactions in mediating the binding of peripheral blood leukocytes to HDMEC and to HK--cells known to be participants and targets in specific cutaneous immunologic responses. Thus, the delineation of precise molecular mechanisms that regulate the tissue-specific and cytokine-specific expression if ICAM-1 is important to both our understanding of the biology of localized inflammation and to the development of directed anti-inflammatory therapeutic strategies. Current evidence indicates that ICAM-1 expression is regulated at the level of gene transcription. Recently our laboratory has isolated and characterized a human genomic clone that contains the 5' regulatory region of the ICAM-1 gene. In the current studies, we further describe the genomic ICAM-1 clones isolated to date and demonstrate the presence of consensus regulatory elements located within the 5' flanking region of the ICAM-1 gene that are potentially involved in regulating ICAM-1 gene transcription.


Fimbria-mediated adherence and hemagglutination of Haemophilus influenzae. The gram-negative bacterium Haemophilus influenzae expresses morphologically and functionally distinct types of fimbriae, of which the LKP fimbriae mediate hemagglutination and adherence to human epithelial cells but hamper mucosal invasion. Therefore, the both in vivo and in vitro observed fimbrial phase variation may contribute to the pathogenesis of the infection. The existence of greater than 14 LKP serotypes hampers vaccine development based on fimbriae, since a monovalent fimbria vaccine confers protection against only the homologous strain. Cloning of the fimbrial genes in Escherichia coli results in the expression of morphologically intact fimbriae. Analysis of the cloned DNA indicates that a fimbrial gene cluster is necessary for formation of complete fimbriae and for fimbria-mediated adherence. The gene encoding the subunit is highly conserved among H. influenzae and belongs to the family of E. coli fimbriae. The phase variation is transcriptionally regulated by variation of the length of the reiterated sequence that forms the promoter region of the subunit gene.


A variable number of tandem repeats locus within the human complement C2 gene is associated with a retroposon derived from a human endogenous retrovirus. We have previously described multiallelic restriction fragment length polymorphisms of the C2 gene, suggesting the presence of a variable number of tandem repeats (VNTR) locus. We report here the cloning and sequencing of the polymorphic fragments from the two most common alleles of the gene, a and b. The results confirm the presence of a VNTR locus consisting of a nucleotide sequence, 41 bp in average length, repeated tandemly 23 and 17 times in alleles a and b, respectively. The difference in the number of repeats between the two alleles is due to the deletion/insertion of two noncontiguous segments, 143 and 118 bp long, of allele a, and of a 40-bp segment of allele b. The VNTR region is associated with a SINE (short interspersed sequence)-type retroposon, SINE-R.C2, located within the third intron of the C2 gene. SINE-R.C2 is a member of a previously described large retroposon family of the human genome, apparently derived from the human endogenous retrovirus, (HERV) K10, which is homologous to the mouse mammary tumor virus.


Tumour necrosis factor and interleukin 6 production during interaction between activated CD4+ lymphocytes and simian immunodeficiency virus-infected macrophages. The mechanism for the gradual loss of CD4+ T lymphocytes and the development of the slowly progressive inflammatory/degenerative lesions that accompany human immunodeficiency virus infection are poorly understood. Using the Simian immunodeficiency virus (SIVmac) macaque model of AIDS, we found that persistently infected primary macrophages fuse with primary activated CD4+ lymphocytes and that this interaction results in production of tumour necrosis factor-alpha (TNF alpha) and interleukin 6 (IL-6). An earlier report had shown that SIV-infected macaque macrophages fuse with CEM174 cells (a human CD4+ cell line) and cause their lysis. In the present report, we have shown that TNF-alpha and IL-6 are also produced during the early stages of this interaction. Data from cocultivation of infected macrophages with several CD4+ T cell lines, including CEM174, suggested that the cytokines are produced by the T cells, and that cytokine production is restricted to those cells which not only express CD4, but are also capable of fusing with the infected macrophages. These data suggest that infected macrophages in vivo could fuse with and eliminate activated CD4+ lymphocytes and, during this interaction, release cytokines, which would contribute to the degenerative and inflammatory lesions characteristic of this disease.


Potentiation of mossy fiber-evoked EPSPs in turtle cerebellar Purkinje cells by the metabotropic glutamate receptor agonist 1S,3R-ACPD. 1. The effects of the metabotropic glutamate receptor (mGluR) agonist 1S,3R-ACPD on excitatory postsynaptic potentials (EPSPs) evoked by stimulation of mossy fibers (MF) and parallel fibers (PF) were examined in turtle cerebellar Purkinje cells. 2. 1S,3R-ACPD (1-25 microM) reversibly potentiated the amplitude of the MF-evoked EPSPs and revealed a late, slow EPSP component, but was without effect on PF-evoked EPSPs. The potentiation of both components of MF-evoked EPSPs was dose dependent, with an ED50 of approximately 3 microM. At higher doses (15-25 microM) 1S,3R-ACPD produced a direct depolarization of Purkinje cells in 57% of cells examined. 3. The enhancement of MF EPSPs by 1S,3R-ACPD was blocked by the N-methyl-D-aspartate (NMDA) receptor antagonist D-2-amino-5-phosphonovalerate (AP-5), but not by the mGluR antagonist L-2-amino-3-phosphonopionic acid (L-AP3; 1 mM), or the 1R,3S isomer of ACPD (25-500 microM). 4. The results demonstrate that mGluR activation by 1S,3R-ACPD produces a potent, stereospecific facilitation of NMDA receptor-mediated transmission at the MF-granule cell synapse.


Isolated NMDA receptor-mediated synaptic responses express both LTP and LTD. 1. The possibility of use-dependent, long-lasting modifications of pharmacologically isolated N-methyl-D-aspartate (NMDA) receptor-mediated synaptic transmission was examined by intracellular recordings from granule cells of the hippocampal dentate gyrus in vitro. In the presence of the non-NMDA receptor antagonist 6-cyano-7-nitroquinaxaline-2,3-dione (CNQX, 10 microM) robust, long-term potentiation (LTP) of NMDA receptor-mediated synaptic potentials was induced by brief, high (50 Hz) and lower (10 Hz) frequency tetanic stimuli of glutamatergic afferents (60 +/- 6%, n = 8, P less than 0.001 and 43 +/- 12%, n = 3, P less than 0.05, respectively). 2. Hyperpolarization of granule cell membrane potential to -100 mV during 50-Hz tetanic stimuli reversibly blocked the induction of LTP (-6 +/- 2%, n = 6, P greater than 0.05) indicating that simultaneous activation of pre- and postsynaptic elements is a prerequisite for potentiation of NMDA receptor-mediated synaptic transmission. In contrast, hyperpolarization of the granule cell membrane potential to -100 mV during 10-Hz tetanic stimuli resulted in long-term depression (LTD) of NMDA receptor-mediated synaptic potentials (-34 +/- 8%, n = 8, P less than 0.01). 3. We also studied the role of [Ca2+]i in the induction of LTP and LTD of NMDA receptor-mediated synaptic responses. Before tetanization, [Ca2+]i was buffered by iontophoretic injections of bis-(o-aminophenoxy)-N,N,N',N'-tetraacetic acid (BAPTA). BAPTA completely blocked the induction of LTP (3 +/- 5%, n = 13) and partially blocked LTD (-14.8 +/- 6%, n = 10).(ABSTRACT TRUNCATED AT 250 WORDS)


Modulation of cortical evoked potentials by stimulation of nucleus raphe magnus in rats. 1. In pentobarbital sodium-anesthetized rats, we evaluated changes in cortical evoked potentials (EPs) associated with electrical and chemical stimulation of nucleus raphe magnus (NRM). A condition-test (C-T) paradigm was used. Cortical EPs were produced by test stimuli delivered to a hindpaw or the thalamic ventral posterior lateral nucleus (VPL; electrical stimulation), or by photic stimulation of the eyes or electrical stimulation of contralateral homotopical cortex (transcallosal EPs). These test stimuli were then preceded by electrical or chemical conditioning stimulation (CS) delivered to NRM through a stereotaxically implanted electrode or injection cannula, respectively. Effects of CS on EPs produced by the test stimuli were characterized. 2. Electrical CS preceding a test stimulus delivered to the foot reduced the amplitude of EPs at thresholds as low as 10-25 microA. The magnitude of EP reduction was dependent on CS intensity, frequency, and the C-T interval. Optimal parameters were trains of 10 pulses (400 Hz) delivered at a C-T interval of 5-10 ms. Injection of glutamate and lidocaine into NRM demonstrated that these effects were due to activation of NRM neurons and not to current spread to medial lemniscus (ML). NRM CS also reduced cortical EPs produced by test stimulation in VPL but did not alter EPs from visual stimulation or from electrical stimulation of contralateral homotopical cortex. 3. These findings suggest that NRM CS attenuates EPs by inhibiting thalamic or thalamocortical afferent activity. Because NRM CS affected all components of the cortical EPs, the effect appears to involve alteration of general sensory activity and is not nociception specific.(ABSTRACT TRUNCATED AT 250 WORDS)


Long-term potentiation in slices of kitten visual cortex and the effects of NMDA receptor blockade. 1. A slice preparation was used to study layer III field potentials (FPs) evoked by electrical stimulation of the white matter-layer VI border and their potentiation by patterned stimuli. 2. The dependence of the FP on recording position was investigated. The maximum field was recorded in layer III at a position radial to the site of stimulation. Because this negative FP reflects an excitatory synaptic current sink, this site was chosen for all subsequent experiments. 3. Under normal recording conditions, components of the layer III FP with latencies greater than 3 ms were completely abolished by kynurenate but unaffected by 2-amino-5-phosphonovalerate (AP5), indicating that this potential reflects the activation of non-NMDA excitatory amino acid receptors. 4. Addition of the gamma-aminobutyric acid (GABA)A receptor antagonist bicuculline methiodide (BMI) broadened the field potential and revealed an AP5-sensitive component. By filling the recording pipette with BMI, it was possible to substantially reduce inhibition locally around the recording site while avoiding stimulus-driven and spontaneous epileptiform activity. 5. Tetanic stimulation elicited a long-term potentiation (LTP) of the FP in 14 of 17 experiments when the BMI-filled pipette method was used. 6. Addition of 100 microM D,L-AP5 significantly reduced the average probability and magnitude of LTP. Nonetheless, in 2 of 8 experiments, significant LTP was observed after a tetanus in the presence of AP5. Control experiments confirmed that this concentration of AP5 was sufficient to maximally block cortical NMDA receptors. 7. We conclude that LTP of layer III field potentials can be reliably elicited, provided that GABAA-receptor mediated inhibition is blocked locally at the site of recording and that NMDA receptors are recruited during the conditioning stimulation. However, activation of NMDA receptors is apparently not an obligatory step for the induction of use-dependent increases in synaptic strength in the kitten striate cortex.


Selective beta 3-adrenergic agonists of brown adipose tissue and thermogenesis. 1. [4-[2-[(2-Hydroxy-3-phenoxypropyl)amino]ethoxy]phenoxy]acetates. The ester methyl [4-[2-[(2-hydroxy-3-phenoxypropyl)amino]ethoxy]phenoxy]acetate (8) has been identified as the most interesting member of a series of selective beta 3-adrenergic agonists of brown adipose tissue and thermogenesis in the rat. In vivo it acts mainly via the related acid 10. Potency was generally markedly reduced by placing substituents on the phenyl ring of the phenoxypropanolamine unit of 8; only the 2-fluoro analogue 16 had comparable potency to 8. Other structure-activity relationships are discussed. Further testing of 8 (ICI 198157) has shown that in the rat it stimulates the beta 3-adrenergic receptor in brown adipose tissue at doses lower than those at which it affects beta 1 and beta 2 adrenergic receptors in other tissues. It increases metabolic rate, as judged by an increase in oxygen consumption, and in the genetically obese Zucker rat it causes a reduced rate of weight gain. This class of compound may be useful in the treatment of obesity in man.


Selective beta 3-adrenergic agonists of brown adipose tissue and thermogenesis. 2. [4-[2-[(2-Hydroxy-3-phenoxypropyl)amino]ethoxy]phenoxy]acetamides. The ester methyl [4-[2-[(2-hydroxy-3-phenoxypropyl)amino]ethoxy]phenoxy]acetate (1) (R1 = OMe) had previously been identified as the most interesting member of a series of selective beta 3-adrenergic agonists of brown adipose tissue and thermogenesis in the rat. In vivo it acts mainly via the related acid 1 (R1 = OH). Amides have been examined to determine whether they have advantages over the ester. In particular, in the rat and dog the half-lives of amides of appropriate potency were no longer than those of the ester. The amide (S)-4-[2-[(2-hydroxy-3-phenoxypropyl)amino]ethoxy]-N-(2- methoxyethyl)phenoxyacetamide [S-27, ICI D7114] was selected as having properties consistent with a sustained-release formulation should that prove necessary. Unlike the ester it is resistant to hydrolysis in the gut lumen. Further testing of ICI D7114 has shown that in the rat, cat, and dog it stimulates the beta 3-adrenergic receptor in brown adipose tissue at doses lower than those at which it affects beta 1- and beta 2-adrenergic receptors in other tissues. Slimming effects were observed in the dog. ICI D7114 may be a selective thermogenic agent in man and may be useful in the treatment of obesity and diabetes.


Neurotransmitter-mediated mechanisms of traumatic brain injury: acetylcholine and excitatory amino acids. Research into traumatic brain injury (TBI), focusing on changes in energy metabolism, cerebrovascular dysfunction, and brain parenchymal morphology, has not produced complete descriptions of mechanisms mediating the pathophysiology of TBI. New studies indicate that neurochemical alterations mediate important components of brain physiology associated with TBI, and these alterations may be responsive to pharmacologic therapy. We discuss rodent models of TBI, review current experimental evidence of muscarinic cholinergic and excitatory amino acid (EAA) receptor involvement in its pathophysiology, and address issues relevant to the interpretation of these data.


Adaptive regulation in skeletal muscle glutamine metabolism in endotoxin-treated rats. The effects of a single dose of endotoxin (7.5 mg/kg BW) on skeletal muscle glutamine metabolism were studied in vivo in rats to gain further understanding of the altered glutamine metabolism that characterizes sepsis and other catabolic diseases. In endotoxin-treated animals the arterial glutamine concentration fell early initially and then increased compared with control values. Twelve hours after treatment, the arteriovenous concentration difference for glutamine across the hindquarter doubled, resulting in a significant increase in net muscle glutamine release in endotoxin-treated rats. As a consequence, the muscle glutamine concentration fell in the endotoxin-treated animals by 25%-40%, an event that was apparent as early as two hours after endotoxin treatment. Skeletal muscle glutaminase activity, the major enzyme of glutamine breakdown, was unchanged by endotoxemia, but expression of glutamine synthetase mRNA and glutamine synthetase specific activity increased in a time-dependent fashion. The glutamine depletion that develops in skeletal muscle during endotoxemia is caused by accelerated muscle glutamine release rather than an increase in intracellular degradation or a fall in intracellular biosynthesis. The adaptive increase in glutamine synthetase expression that occurs requires de novo RNA and protein synthesis and may be designed to prevent complete depletion of the intracellular glutamine pool.


Isolation and characterization of the major vegetative RNA polymerase of Streptomyces coelicolor A3(2); renaturation of a sigma subunit using GroEL. The promoter region of the agarase gene (dagA) of Streptomyces coelicolor A3(2) is complex; it consists of four distinct promoters with different -10 and -35 regions. We report the isolation of a form of RNA polymerase that mediates transcription in vitro from the dagAp4 promoter. The core components of this RNA polymerase are associated with a polypeptide of c. 66 kDa; holoenzyme reconstitution experiments show that the 66 kDa polypeptide functions as a sigma factor that directs transcription from the dagAp4 and Bacillus subtilis veg promoters in vitro. Alignment of the DNA sequences of these two promoters shows that they have bases in common in the -10 and -35 regions and that these sequences are similar to those observed for the major RNA polymerases of other bacteria. N-terminal amino acid sequence analysis of the 66 kDa polypeptide revealed it to be the product of the hrdB gene. Previous experiments showed that the predicted amino acid sequence of the hrdB gene product is very similar to the major sigma factors of other bacteria and suggested that disruption of the hrdB gene is lethal. These observations together lead to the conclusion that we have isolated the major RNA polymerase of Streptomyces coelicolor A3(2). We have developed an improved protocol for the renaturation of sigma factors that have been isolated by preparative sodium dodecyl sulphate/polyacrylamide gel electrophoresis (SDS-PAGE). This method involves renaturing the polypeptide in the presence of the bacterial chaperonin GroEL. We expect this protocol to find general application for renaturation of other polypeptides that have been subjected to SDS-PAGE.


High-frequency rearrangements in the chromosome of Mycoplasma pulmonis correlate with phenotypic switching. Mycoplasma pulmonis is a murine pathogen that causes chronic respiratory disease in laboratory rats and mice. Several examples of high-frequency phenotypic switching have been reported for M. pulmonis, the molecular basis of which is unknown. We report here that during growth the M. pulmonis chromosome undergoes DNA rearrangements at a high frequency. Some of the rearrangements we examined correlated with changes in the susceptibility of the cells to mycoplasma virus P1, an example of phenotypic switching involving changes in surface antigen structure. Other rearrangements, unrelated to phenotypic switching, involved a DNA element present in the chromosome in multiple copies. The high level of DNA recombination that occurred in M. pulmonis indicates that this may be one of the most variable genomes studied to date. High levels of DNA recombination may contribute to the unusually high rate of evolution that mycoplasmas are thought to be undergoing. Understanding the molecular basis for this phenomenon may provide an insight into the chronic nature of many mycoplasmal infections.


Effects of adrenergic blockers on central nervous system-mediated hyperglycemia in fed rats. We studied the effect of adrenergic blockade on hepatic venous hyperglycemia and the activation of a hepatic glycogenolytic enzyme, phosphorylase-a, in response to cerebral cholinergic activation. Neostigmine was injected into the third cerebral ventricle of bilaterally adrenodemedullectomized (ADMX) rats, while somatostatin and insulin were administered intravenously. Hepatic venous plasma glucose concentrations and hepatic phosphorylase-a activity were measured. Intracerebroventricular injection of neostigmine (5 x 10(-8) mol) caused increases in hepatic venous glucose concentrations and hepatic phosphorylase-a activity. Both of these changes were prevented by intraperitoneal (IB) pretreatment with phentolamine (5 x 10(-7), 1 x 10(-6) mol) without the intervention of insulin secretion, but not by pretreatment with the alpha-adrenoreceptor antagonist phenoxybenzamine (1 x 10(-6) mol), the beta-adrenoreceptor antagonist propranolol (1 x 10(-6) mol), the alpha 1-antagonists prazosin or bunazosin (1 x 10(-6) mol), the alpha 2-antagonist yohimbine (1 x 10(-6) mol), or prazosin (5 x 10(-7) mol) plus yohimbine (5 x 10(-7) mol). These results suggest that phentolamine prevented brain-mediated hepatic glycogenolysis by a mechanism that may not be classified pharmacologically as involving either alpha 1- or alpha 2-receptors.


Drug overdose--reducing the load.

Drug overdose--reducing the load. To review available information about various methods for reducing gastrointestinal absorption of a poison or drug. Articles on overdose and accidental poisoning generated by the Australian Medlars Service and concentrating on the period between 1985 and 1990 were surveyed. Earlier studies were included if relevant. English language articles with an emphasis on studies using objective methods to measure individual and comparative efficacy of gastrointestinal decontamination techniques were selected. A total of 65 articles were reviewed. Gastric emptying procedures (gastric lavage or emesis caused by syrup of ipecac) are only effective if performed within one hour of drug ingestion. Gastric lavage is superior to syrup of ipecac. Oral administration of activated charcoal is more effective than either gastric emptying procedure, and is recommended for most cases of poisoning. Cathartics (sorbitol) can be used with activated charcoal. Whole bowel lavage with polyethylene glycol is indicated in selected cases of potentially lethal overdose where the toxic substance cannot be absorbed by charcoal and has passed the pylorus. Children--syrup of ipecac can be given at home to children older than 12 months. Most children who reach hospital can be treated by charcoal alone. ADULTS--Most patients are managed with supportive care and, in the absence of contraindications, a single dose of activated charcoal if seen within four hours of ingestion of the poison or drug. Gastric lavage is used if the patient presents within one hour of ingestion and has clinical features of toxicity.


Guidelines for the performance of CD4+ T-cell determinations in persons with human immunodeficiency virus infection. This document has been developed by CDC to give guidance to laboratories performing lymphocyte immunophenotyping assays in human immunodeficiency virus-infected persons. The recommendations in this document reflect current technology in a field that is rapidly changing. The recommendations apply to laboratory safety, specimen collection, specimen transport, maintenance of specimen integrity, specimen processing, flow cytometer quality control, sample analyses, data analysis, data storage, data reporting, and quality assurance.


Chronic selegiline administration transiently decreases tyrosine hydroxylase activity and mRNA in the rat nigrostriatal pathway. Selegiline, a selective monoamine oxidase type B inhibitor, is beneficial in the treatment of Parkinson's disease. However, this beneficial effect is only transient, and patients must ultimately resort to treatment with standard levodopa therapy. We studied the effects of chronic selegiline treatment on the rat nigrostriatal pathway, to elucidate a neurochemical correlate for this adaptive clinical response. Selegiline treatment for 3, 7, 14, or 21 days decreased tyrosine hydroxylase (the enzyme that catalyzes the rate-limiting step in catecholamine biosynthesis) activity in the cell body regions (substantia nigra) of the nigrostriatal pathway. However, tyrosine hydroxylase activity measurements in the major terminal field region (corpus striatum) of the pathway did not correspond to those in the substantia nigra; in the corpus striatum, tyrosine hydroxylase activity was decreased at 3 and 7 days of treatment and recovered by 14 days. We tested whether the decrease in tyrosine hydroxylase activity was mediated by a decrease in tyrosine hydroxylase mRNA. Northern blot and RNA dot blot analyses (using a tyrosine hydroxylase-specific cDNA probe) of substantia nigra homogenates revealed a significant decrease in tyrosine hydroxylase mRNA at 3, 7, and 14 days of selegiline treatment, compared with controls. Conversely, after 21 days of selegiline, tyrosine hydroxylase mRNA levels were significantly higher (3-fold) than controls; this finding was not reflected in substantia nigra tyrosine hydroxylase activity. The 21-day increase in mRNA may be associated with the rebound in tyrosine hydroxylase activity observed in the corpus striatum. Thus, it is possible that the recovery in tyrosine hydroxylase activity in the corpus striatum is mediated through an increase in tyrosine hydroxylase protein transport from the substantia nigra to the corpus striatum and/or that the tyrosine hydroxylase enzyme exists in a more stabilized state during this period of time. These results demonstrate that monoamine oxidase type B-selective inhibitory doses of selegiline are capable of inducing transient decreases in tyrosine hydroxylase activity and tyrosine hydroxylase mRNA levels. Furthermore, these reversible effects may represent adaptive responses associated with pharmacological tolerance and the transient beneficial actions of this drug in Parkinson's disease.


Beta 1- and beta 2-adrenergic receptors display subtype-selective coupling to Gs. beta-Adrenergic receptor (beta AR) subtypes differ in their affinities for some agonists and antagonists and thus may potentially impart different cellular effects based on this ligand-binding specificity. However, the possibility that there may be subtype-specific events subsequent to ligand binding has not been evaluated extensively. In particular, although beta ARs stimulate adenylyl cyclase by coupling to the guanine nucleotide-binding protein Gs, no studies have directly assessed the coupling efficiencies among isolated beta AR subtypes. We, therefore, permanently transfected the mammalian fibroblast cell line CHW-1102 with beta 1- or beta 2AR cDNAs and studied the coupling characteristics of these two receptor subtypes, each expressed at approximately 335 fmol/mg of protein. Both receptors mediated equivalent maximal increases in adenylyl cyclase activities (6.63 +/- 1.85-fold for beta 1AR versus 6.10 +/- 0.53-fold for beta 2AR; p = not significant). However, the isoproterenol dose-response curves for the beta 2AR were shifted to the left, compared with those for the beta 1AR (EC50 of 52.3 +/- 2.87 nM and 191 +/- 10.5 nM, respectively; p less than 0.05), resulting in an approximately 4-fold greater potency for the beta 2AR versus the beta 1AR. Thus, at the submaximal isoproterenol concentration of 30 nM, the beta 2AR stimulated adenylyl cyclase approximately 50% more than did the beta 1AR. This finding was not due to a difference in the affinities of isoproterenol for these receptors, which were found to be the same, as determined by competition binding studies with 125I-cyanopindolol in the presence of GTP. The ability of beta 1- and beta 2ARs to form the high affinity ternary complex was assessed in agonist competition studies without guanine nucleotide. We found that, whereas the proportion of receptors in the high affinity state was equivalent between the two receptor subtypes, the affinity of this state for isoproterenol was approximately 5-fold greater for the beta 2AR, compared with the beta 1AR (KH for beta 2AR, 11.8 +/- 3.1 nM; KH for beta 1AR, 61.7 +/- 18.3 nM; p less than 0.05). In addition, we examined physical and functional coupling of beta 1- and beta 2ARs to Gs using the agonist epinephrine, which also has equal binding affinity for both receptor subtypes. As with isoproterenol, epinephrine was more potent in stimulating adenylyl cyclase and promoted a higher affinity ternary complex for the beta 2AR. Thus, a greater degree of both physical and functional agonist-promoted coupling occurs between Gs and beta 2AR, compared with beta 1AR. We conclude that coupling to Gs by beta 1- and beta 2ARs is subtype selective and is a potentially important distinguishing feature among these members of the beta AR family.


Conservation of the kinaselike regulatory domain is essential for activation of the natriuretic peptide receptor guanylyl cyclases. The natriuretic peptide receptors, NPR-A and NPR-B, are two members of the newly described class of receptor guanylyl cyclases. The kinaselike domain of these proteins is an important regulator of the guanylyl cyclase activity. To begin to understand the molecular nature of this type of regulation, we made complete and partial deletions of the kinase domain in NPR-A and NPR-B. We also made chimeric proteins in which the kinase domains of NPR-A and NPR-B were exchanged or replaced with kinase domains from structurally similar proteins. Complete deletion of the kinase homology domain in NPR-A and NPR-B resulted in constitutive activation of the guanylyl cyclase. Various partial deletions of this region produced proteins that had no ability to activate the enzyme with or without hormone stimulation. The kinase homology domain can be exchanged between the two subtypes with no effect on regulation. However, structurally similar kinaselike domains, such as from the epidermal growth factor receptor or from the heat-stable enterotoxin receptor, another member of the receptor guanylyl cyclase family, were not able to regulate the guanylyl cyclase activity correctly. These findings suggest that the kinaselike domain of NPR-A and NPR-B requires strict sequence conservation to maintain proper regulation of their guanylyl cyclase activity.


X rays induce interallelic homologous recombination at the human thymidine kinase gene. We have developed a human lymphoblast cell line for the study of interchromosomal homologous recombination at the endogenous thymidine kinase (tk) gene on chromosome 17 (M. B. Benjamin, H. Potter, D. W. Yandell, and J. B. Little, Proc. Natl. Acad. Sci. USA 88:6652-6656, 1991). This cell line (designated 6:86) carries unique heterozygous frameshift mutations in exons 4 and 7 of its endogenous tk alleles and can revert to TK+ by frame-restoring mutations, gene conversion, or reciprocal recombination. Line 6:86 reverts spontaneously to TK+ at a frequency of 10(-7) to 10(-8), and exposures to X-irradiation or the frameshift mutagen ICR-191 induce increased reversion frequencies in a dose-dependent manner. Another cell line (designated 4:2) carries a homozygous exon 7 frameshift and is not expected to revert through mechanisms other than frame-restoring mutation. Line 4:2 reverts to TK+ at a lower spontaneous frequency than does 6:86 but can be induced with similar kinetics by ICR-191. In contrast to line 6:86, however, X rays did not induce detectable reversion of line 4:2. We have characterized a number of 6:86-derived revertants by means of restriction fragment length polymorphism analysis at tk and linked loci, single-strand conformation polymorphisms, and direct transcript sequencing. For X rays, most revertants retain both original mutations in the genomic DNA, and a subset of these frameshift-retaining revertants produce frameshift-free message, indicating that reversion is the result of reciprocal recombination within the tk gene. Frame-restoring point mutations, restoration of original sequences, and phenocopy reversion by acquisition of aminopterin resistance were also found among X-ray-induced revertants, whereas the ICR-191-induced revertants examined show only loss of the exon 7 frameshift.


Negative autoregulation of the neu gene is mediated by a novel enhancer. In an attempt to study potential feedback regulation of the neu oncogene, we have found that the neu oncogene product specifically represses its own promoter activity. Deletion analysis indicated a 140-bp region (nucleotides -312 to -173 relative to the ATG initiation codon) in the rat neu promoter responsible for neu autorepression. Gel shift assays and methylation interference analysis further demonstrated that a GGTGGGGGGG sequence (nucleotides -243 to -234 relative to the ATG initiation codon) in this 140-bp region interacts with specific protein complexes. The GGTGGGGGGG sequence (GTG element), which functions as an enhancer, is sufficient to cause neu-mediated repression in a heterologous promoter. Furthermore, it produces different gel shift patterns with nuclear extracts from neu-transformed cell lines and their parental lines, suggesting that a transcriptional factor(s) interacting with this enhancer element has been perturbed by the introduction of neu. Taken together, the data presented in this report show that (i) the neu oncogene product autorepresses its own promoter, (ii) the neu promoter contains a novel enhancer, and (iii) neu autorepression is mediated through this enhancer, likely by inhibition of the enhancer activity.


Multidrug resistance in Leishmania donovani is conferred by amplification of a gene homologous to the mammalian mdr1 gene. Drug resistance is a major impediment to the effective treatment of parasitic diseases. The role of multidrug resistance (mdr) genes and their products in this drug resistance phenomenon, however, remains controversial. In order to determine whether mdr gene amplification and overexpression can be connected to a multidrug resistance phenotype in parasitic protozoa, a mutant strain of Leishmania donovani was generated by virtue of its ability to proliferate in medium containing increasing concentrations of vinblastine. The vinblastine-resistant strain, VINB1000, displayed a cross-resistance to puromycin and the anthracyclines, a growth phenotype that could be attributed to an impaired ability to accumulate the toxic drugs. By using the polymerase chain reaction, two different DNA fragments, LEMDR06 and LEMDRF2, were amplified from leishmanial genomic DNA, and each amplified fragment encoded a product that was significantly homologous to parts of the mammalian P-glycoprotein. In the VINB1000 strain, the mdr gene recognized by the LEMDR06 probe was amplified approximately 50-fold in copy number, whereas the mdr genes that hybridized to LEMDRF2 or to a fragment of the previously characterized ltpgpA gene were not amplified. Moreover, the VINB1000 cell line expressed a LEMDR06 gene transcript of 12.5 kb in size that was not detected in the parental wild-type strain. To furnish a functional test for mdr gene amplification and expression in L. donovani, the L. donovani gene recognized by the LEMDR06 polymerase chain reaction product, ldmdr1, was isolated from a genomic library, transfected into wild-type cells, and amplified over 500-fold by selection in 0.5 mg of G418 per ml. The resulting transfectants were resistant to all drugs to which VINB1000 cells were resistant and sensitive to all drugs to which VINB1000 cells were sensitive. These studies demonstrate that amplification of the ldmdr1 gene either by direct selection or subsequent to transfection can confer a drug-resistant phenotype in parasitic protozoa similar to that observed for MDR mammalian cells.


Effect of polymorphism of an MHC-linked transporter on the peptides assembled in a class I molecule. Short antigenic peptides bound in the groove of class I major histocompatibility complex molecules enable T cells to detect intracellular pathogens. It has been assumed that structural features of the class I molecule alone select which peptides are bound. It is now demonstrated that a complex polymorphism in one of the major histocompatibility complex-encoded putative peptide-transporter genes is associated with an altered spectrum of bound peptides.


The spread of Na+ spikes determines the pattern of dendritic Ca2+ entry into hippocampal neurons. The dendrites of many types of neurons contain voltage-dependent Na+ and Ca2+ conductances that generate action potentials (see ref. 1 for review). The function of these spikes is not well understood, but the Ca2+ entry stimulated by spikes probably affects Ca(2+)-dependent processes in dendrites. These include synaptic plasticity, cytotoxicity and exocytosis. Several lines of evidence suggest that dendritic spikes occur within subregions of the dendrites. To study the mechanism that govern the spread of spikes in the dendrites of hippocampal pyramidal cells, we imaged Ca2+ entry with Fura-2 (ref. 9) and Na+ entry with a newly developed Na(+)-sensitive dye. Our results indicate that Ca2+ entry into dendrites is triggered by Na+ spikes that actively invade the dendrites. The restricted spatial distribution of Ca2+ entry seems to depend on the spread of Na+ spikes in the dendrites, rather than on a limited distribution of Ca2+ channels. In addition, we have observed an activity-dependent process that modulates the invasion of spikes into the dendrites and progressively restricts Ca2+ entry to more proximal dendritic regions.


Changing role of even-skipped during the evolution of insect pattern formation. The development of Drosophila is typical of the so-called long germband mode of insect development, in which the pattern of segments is established by the end of the blastoderm stage. Short germband insects, such as the grasshopper Schistocerca americana, by contrast, generate all or most of their metameric pattern after the blastoderm stage by the sequential addition of segments during caudal elongation. This difference is discernible at the molecular level in the pattern of initiation of the segment polarity gene engrailed, and the homeotic gene abdominal-A (ref. 5). For example, in both types of insects, engrailed is expressed by the highly conserved germband stage in a pattern of regularly spaced stripes, one stripe per segment. In Drosophila, the complete pattern is visible by the end of the blastoderm stage, although engrailed appears initially in alternate segments in a pair-rule pattern that reflects its known control by pair-rule genes such as even-skipped. In contrast, in the grasshopper, the engrailed stripes appear one at a time after the blastoderm stage as the embryo elongates. To address the molecular basis for this difference, we have cloned the grasshopper homologue of the Drosophila pair-rule gene even-skipped and show that it does not serve a pair-rule function in early development, although it does have a similar function in both insects during neurogenesis later in development.


[Molecular genetic studies in alpha-thalassemia]. A group of 5,000 patients, suspected of haemolytic anaemia, were investigated with molecular genetic methods for deletion types of alpha-thalassemia. In 776 (15.6%) patients a deletion of one or more alpha-globin genes was found. The same group of patients was also investigated for abnormal haemoglobins and beta-thalassaemia. In about 30% of the patients either an alpha-thalassaemia, an abnormal haemoglobin, a beta-thalassaemia, or a combination was diagnosed. In a group of patients with a haemoglobinopathy, the frequency of alpha-thalassaemia was much higher (i.e. 33%) than in individuals without haemoglobinopathy. Preselection of the patients based on the presence of microcytic erythrocytes and/or a decreased ADW0.5 of the erythrocytes gave a high incidence of false-negative and false-positive results. Therefore, haemoglobin examination should not be restricted to protein chemistry, but should include molecular genetic investigations for deletion types of alpha-thalassaemia.


[Vecuronium in pediatric anesthesia. Repeated bolus vs continuous infusion]. Vecuronium bromide was employed in 26 pediatric patients; 18 received vecuronium infusion and 11 intermittent bolus administration. The aim of the study was to evaluate the pharmacodynamics of the drug, the lack of cumulative properties, the best infusion rate, the possible advantage of the infusion technique. Neuromuscular blockade monitoring was performed in all patients. In the results, vecuronium shows lack of cumulative properties. The best infusion rate was 0.13 +/- 0.02 mg/kg/h. The continuous infusion allows the employment of lesser doses of the drug than the intermittent administration.


[Analgesia-sedation during extracorporeal lithotripsy (ESWL)]. Personal experience of analgesia-sedation in ESWL is reported, stress being laid on the importance of anxiety treatment by means of premedication with benzodiazepine. In personal experience, this preventive treatment proved very useful in reducing the consumption of opioids and thus in rendering the pain experience of patients undergoing treatment less unpleasant. At the same time it is reported that in some patients it was necessary to administer benzodiazepines with a more marked hypnotic effect. In these cases, the possibility of countering the hypnotic effect with a specific antagonist made it possible to resort to these drugs in patients who were subjected to ESWL in a day-hospital regime.


Synaptic inputs to GABAA and GABAB receptors originate from discrete afferent neurons. gamma-Aminobutyric acid (GABA) inhibits neurons by acting at GABAA and GABAB receptors but it is not known whether the two receptors are associated with discretely separate afferent inputs or whether GABA released from a single presynaptic neuron activates both receptors. Intracellular recordings were used to show that, in the lateral amygdala and ventral tegmental area of the rat, distinct sets of GABA-containing neurons provide the synaptic input to GABAA and GABAB receptors. Synaptic potentials resulting from GABAA receptor activation (blocked by bicuculline) and from GABAB receptor activation (blocked by 2-hydroxysaclofen) occurred spontaneously but as unrelated events. Furthermore, the two components of evoked synaptic potentials were differentially inhibited by agonists acting presynaptically (muscarine and 5-hydroxytryptamine). The finding that GABA acting at GABAA and GABAB receptors originates from distinct sets of presynaptic fibers suggests that two groups of GABA-containing neurons might be generally distinguishable in the mammalian nervous system.


Extracellular adenosine 5'-triphosphate-evoked glutamate release in cultured hippocampal neurons. Characteristics of extracellular ATP-evoked electrical responses in rat hippocampal neurons were investigated. Extracellular ATP (100 microM) induced a rapid depolarization followed by repetitive firings of spikes in these cells under whole-cell current-clamp. In whole-cell voltage-clamp experiments, ATP activated 2 types of inward currents that were inhibited by P2-purinoceptor blocker suramin (300 microM). One is a small (about -20 pA) sustained current which is insensitive to tetrodotoxin (TTX), and the other is a large (-100 to -300 pA) transient current which abolished in the presence of 3 microM TTX. The ATP-induced transient current was blocked by 6-cyano-7-nitro-quinoxaline-2,3-dione (CNQX; 30 microM), a non-N-methyl-D-aspartate (non-NMDA) receptor antagonist. ATP failed to induce the transient current in the cell which showed the desensitization to quisqualic acid (QA; 10 microM), a non-NMDA receptor agonist. These findings suggest that ATP directly activates small sustained currents, and indirectly induces the transient currents by evoking glutamate release.


NMDA, non-NMDA and glycine receptors mediate binaural interaction in the lateral superior olive: physiological evidence from mouse brain slice. Binaural interaction was investigated in a 400 microns brain slice taken through the mouse lateral superior olive (LSO). Ipsilateral excitatory and contralateral inhibitory inputs to LSO neurons were examined by recording physiological responses to electrical stimulation of the trapezoid body. Bath application of non-N-methyl-D-aspartate (non-NMDA) antagonists blocked ipsilateral excitation and strychnine blocked contralateral inhibition. N-methyl-D-aspartate (NMDA) had little effect on ipsilateral responses but completely blocked contralateral inhibition. These results suggest that ipsilateral excitation is mediated by non-NMDA receptors and contralateral inhibition by strychnine dependent glycine receptors. NMDA receptors may play a role by modulating contralateral inhibition in LSO.


Reversal by pertussis toxin and N-ethylmaleimide of the facilitation of baroreceptor reflex response by somatostatin in the rat. We evaluated the transmembrane signaling mechanism that may underlie the facilitatory action of somatostatin (SOM) on baroreceptor reflex (BRR), using adult, male, Sprague-Dawley rats anesthetized with pentobarbital sodium (40 mg/kg, i.p.). Intracerebroventricular (i.c.v.) application of SOM (2 nmol) promoted a significant elevation in BRR response, induced by phenylephrine (5 micrograms/kg, i.v.). This potentiatory action of the tetradecapeptide was significantly reversed after pretreating animals with bilateral microinjection of pertussis toxin (25 ng) or N-ethylmaleimide (2 nmol) into the nucleus tractus solitarius (NTS), the terminal site for baroreceptor afferents. These results suggest that a pertussis toxin-sensitive GTP-binding regulatory protein, possibly Gi, may be involved in the modulation of the BRR by SOM at the NTS.


Hypnosis or cognitive behavioral training for the reduction of pain and nausea during cancer treatment: a controlled clinical trial. Few controlled clinical trials have tested the efficacy of psychological techniques for reducing cancer pain or post-chemotherapy nausea and emesis. In this study, 67 bone marrow transplant patients with hematological malignancies were randomly assigned to one of four groups prior to beginning transplantation conditioning: (1) hypnosis training (HYP); (2) cognitive behavioral coping skills training (CB); (3) therapist contact control (TC); or (4) treatment as usual (TAU; no treatment control). Patients completed measures of physical functioning (Sickness Impact Profile; SIP) and psychological functioning (Brief Symptom Inventory; BSI), which were used as covariates in the analyses. Biodemographic variables included gender, age and a risk variable based on diagnosis and number of remissions or relapses. Patients in the HYP, CB and TC groups met with a clinical psychologist for two pre-transplant training sessions and ten in-hospital "booster" sessions during the course of transplantation. Forty-five patients completed the study and provided all covariate data, and 80% of the time series outcome data. Analyses of the principal study variables indicated that hypnosis was effective in reducing reported oral pain for patients undergoing marrow transplantation. Risk, SIP, and BSI pre-transplant were found to be effective predictors of inpatient physical symptoms. Nausea, emesis and opioid use did not differ significantly between the treatment groups. The cognitive behavioral intervention, as applied in this study, was not effective in reducing the symptoms measured.


Does opiate premedication influence postoperative analgesia? A prospective study. The influence of opiate premedication on analgesic requirements postoperatively was investigated. Out of 98 patients with a lumbar disc prolapse 50 were premedicated with flunitrazepam orally, 48 with pethidine and triflupromazine intramuscularly. The operations were performed under inhalational anaesthesia. The average time up to the first demand for an analgesic was longer following opiate premedication (351 vs. 219 min). Only 45.8% of the patients treated with opiates demanded analgesics postoperatively, compared to 80.0% of those who had a benzodiazepine premedication (P less than 0.01). These clinical data confirm the experimental evidence that pretreatment with opiates diminishes the sustained hyperexcitation of the central nervous system caused by peripheral lesions.


Enhancement of pentazocine analgesia by clonidine. Opiate-adrenergic interactions were investigated by studying the effect of the selective alpha 2-adrenergic agonist, clonidine, on the analgesia produced by intravenous placebo and by the predominantly kappa-opiate agonist, pentazocine, in patients with dental postoperative pain. Clonidine did not affect the pain level when administered with intravenous placebo. When administered with pentazocine, clonidine caused a statistically significant increase in pentazocine analgesia. Comparison is made to other opiate-adrenergic interactions and possible mechanisms are discussed.


Is Asacol as effective as sulphasalazine in maintaining remission of Crohn's disease and ulcerative colitis? To compare the efficacy of Asacol (mesalazine coated with Eudraget-S) as a maintenance therapy with that of sulphasalazine, relapse rates of patients with ulcerative colitis and Crohn's disease, treated with sulphasalazine or Asacol were assessed in a retrospective study. A total of 164 patients were investigated, 127 on sulphasalazine and 37 on Asacol. None of the patients on Asacol was changed from sulphasalazine because of lack of efficacy to sulphasalazine. Relapse rates were measured over a 4 year period. In ulcerative colitis these were sulphasalazine 10/77 (13.0%), Asacol 5/20 (25.0%), NS; in all Crohn's disease patients, sulphasalazine 12/50 (24.0%), Asacol 11/17 (64.7%); P less than 0.0025. In patients with Crohn's disease with ileal involvement, relapse rates were sulphasalazine 9/28 (32.1%), Asacol 9/11 (81.6%), P less than 0.0125; without ileal involvement, sulphasalazine 3/22 (13.6%), Asacol 2/6 (33.4%), NS. This study suggests that Asacol is as effective as sulphasalazine in maintaining remission in ulcerative colitis and in patients with Crohn's disease without ileal involvement. Sulphasalazine seems to be more effective than Asacol in maintaining remission in patients with Crohn's disease with terminal ileal involvement.


Difficulties in localization and treatment of insulinomas in type 1 multiple endocrine adenomatosis (MEA). A 15 year old girl with a family history of type 1 multiple endocrine adenomatosis presented with reversible neurological disturbances, hypoglycaemia and hyperinsulinaemia. Initial radiology was normal, but portal venous sampling suggested an insulinoma in the tail of the pancreas which was removed with conservation of the spleen. Hypoglycaemia persisted despite high doses of diazoxide and intravenous dextrose. A second laparotomy revealed a pancreatic endocrine tumour and sub-total pancreatectomy was performed. Histology revealed islet cell microadenomatosis. Hypoglycaemia persisted despite treatment with somatostatin analogues and 40% intravenous dextrose was required to maintain normoglycaemia. A possible lesion near the splenic hilum on computed tomographic scan was reported as a splenunculus although further peripheral, hepatic and portal venous sampling suggested hepatic or systemic lesions. A positron emission scan and selective visceral angiography suggested a lesion in the left upper quadrant. Acute lactic acidosis, rhabdomyolysis and renal failure supervened. Post mortem revealed the putative 'splenunculus' to be a residual insulinoma, whilst the splenic vein was thrombosed, accounting in part for discrepant venous sampling data. Hyperinsulinaemia in type 1 multiple endocrine adenomatosis may require more aggressive surgical and hormonal intervention than when dealing with solitary insulinomas. Insulinomas may mimic developmental abnormalities on computed tomographic scanning.


Immunocytochemical localisation of an FMRFamide-like peptide in the filarial nematodes Dirofilaria immitis and Brugia pahangi. Immunocytochemical techniques were used to detect FMRFamide-like immunoreactivity in adults of the filarial nematodes Dirofilaria immitis and Brugia pahangi. An FMRFamide-like peptide was also located in third- and fourth-stage larvae of D. immitis. Positive immunoreactivity was observed in all parasites examined, irrespective of developmental stage. The major areas of positive immunoreactivity were located in the anterior nerve ring, lateral/dorso-ventral nerves, cephalic papillary ganglia and lateral ganglia. No staining was seen in the intestine or gonads of any parasite. These results indicate that filarial worms possess a peptidergic component in their nervous system. The possible role of an FMRFamide-like peptide in the control of certain physiological events is discussed.


Acute ethanol intoxication may not alter alpha 2-adrenoceptors in the human brain. The specific binding of the alpha 2-adrenoceptor agonists [3H]clonidine and [3H]bromoxidine (UK 14304) was measured in the postmortem brain of ethanol intoxicated nonalcoholic subjects (blood ethanol concentration: 1.37 +/- 0.30 g/l) and matched controls. In the frontal cortex, the density of binding sites for [3H]clonidine (Bmax = 58 +/- 7 fmol/mg protein) and [3H]bromoxidine (UK 14304) (Bmax = 49 +/- 7 fmol/mg protein) in ethanol intoxicated subjects did not differ from those in the control groups (Bmax = 68 +/- 4 and 56 +/- 8 fmol/mg protein for the respective radioligand). The dissociation constants (KD) were also similar in both groups. The binding capacities (Bmax) and KD values for both radioligands also were found unchanged in the hypothalamus, amygdala, head of caudate, hippocampus and cerebellum. The results demonstrate that, contrary to the beta-adrenoceptor, the presence of ethanol in the human brain does not alter the high-affinity state of the alpha 2-adrenoceptor in the frontal cortex and possibly also in other brain regions.


Effects of blockade of 5-HT2 receptors and activation of 5-HT1A receptors on the exploratory activity of rats in the elevated plus-maze. Acute administration of gepirone, a 5-HT1A agonist, caused a dose dependent (1-10 mg/kg, IP) reduction in the locomotor activity (open and closed arms) of rats tested in the elevated plus-maze. However, rats housed in individual cages and submitted to chronic treatment with gepirone (10 mg/kg PO) showed a marked increase in the percentages of number and time spent in the open arms as compared to controls. These results are compatible with the idea that the antiaversive effect due to long-term treatment with 5-HT1A agonists is the result of a progressive desensitization of the somatodendritic 5-HT autoreceptor with the consequent recovery of firing rate of 5-HT neurons along with an activation of normosensitive postsynaptic 5-HT neurons. Ketanserin caused a biphasic effects on the exploratory behavior of rats in the plus-maze. The lower dose (0.5 mg/kg) decreased the aversion to the open arms and the higher dose (1.0 mg/kg) caused an unspecific decrease in the overall activity of the animals. Ketanserin is supposed to have antagonistic action on 5-HT2 and on alpha-adrenergic receptors. As prazosin (0.5-1.0 mg/kg), an alpha-adrenergic receptor blocker, did not present any significant effect in the present work it is suggested that the effects of the lower dose of ketanserin was due to its high antagonistic action on 5-HT2 receptors.


Effects of acute dopamine depletion on responsiveness to D1 and D2 receptor agonists in infant and weanling rat pups. The behavioral responses to separate and combined administration of the D1 agonist SKF-38393 and the D2 agonist quinpirole following acute dopamine (DA) depletion via alpha-methyl-p-tyrosine (AMPT) or AMPT/reserpine were examined in infant (10-day-old) and weanling (21-day-old) rat pups. At both ages, AMPT pretreatment generally had little impact on D1- or D2-agonist-induced responding, whereas the greater DA depletion observed following AMPT/reserpine pretreatment was generally associated with suppression of both D1 and D2-agonist-typical responding. Thus, whereas in adult animals some degree of D1 receptor activation by endogenous dopamine appears to be necessary for D2 responding but not vice versa (e.g. White et al. 1988), in young animals there appears to be a reciprocal co-dependence of these two receptor subtypes, with extensive DA depletion suppressing responding to both agonists when administered separately. At 10 days of age, some D1 and D2 agonist-induced behaviors that were previously blocked by AMPT/reserpine were reinstated following combined administration of both agonists. In contrast, no clear evidence for reinstatement was seen following administration of the combined agonists to AMPT/reserpine-pretreated weanlings, perhaps due to the induction of potential competing behaviors. Whereas DA depletion blocked many D1- and D2-induced behaviors, such depletion conversely promoted the expression in agonist-treated animals of a number of behaviors that were not normally induced by the agonists in non-depleted animals. These behaviors typically involved an oral component and included grooming and mouthing following SKF-38393 in depleted 10-day-old pups, mouthing following administration of either agonist to depleted weanlings, and probing and intense self-mutilation (forepaw and tongue biting) following the combined agonists in depleted weanlings. This rapid induction of potentiated agonist responsiveness following acute DA depletion early in life may have significant implications with regard to animal models for the developmental disorder of Lesch-Nyhan syndrome.