Neurochemical effects of 5-HT1 receptor ligands in pigeons. Pigeon cerebrospinal fluid was assayed for 5-HT (5-hydroxytryptamine) and catecholamine metabolites after systemic drug injection. The 5-HT1-like receptor agonists 8-hydroxy-(di-n-propylamino)tetralin (8-OH-DPAT), 5-methoxy-3(1,2,3,6-tetrahydropyridin-4-yl)1H indole (RU 24969), 1-(m-trifluoromethylphenyl)piperazine (TFMPP), and 1-(3-chlorphenyl)piperazine (mCPP) decreased levels of the 5-HT metabolite 5-hydroxyindoleacetic acid (5-HIAA) without altering other metabolites. 5-HIAA decreases occurred at doses of 8-OH-DPAT and RU 24969 that have anti-conflict effects in pigeons, whereas TFMPP and mCPP decreased 5-HIAA only at behaviorally disruptive doses. The novel compound 1-(2-methoxyphenyl)-1-(4-(2-phthalimido)butyl)piperazine (NAN-190), a putative 5-HT1A receptor antagonist, did not affect 5-HIAA, but attenuated the decreases produced by the agonists. NAN-190 and the alpha 1-adrenoceptor antagonist prazosin increased levels of the norepinephrine metabolite 3-methoxy-4-hydroxyphenylethylene glycol and had additive effects when co-administered. The rank order of potency in inhibiting [3H]8-OH-DPAT binding in pigeon cerebrum was 8-OH-DPAT = RU 24969 > NAN-190 >> mCPP > TFMPP. The results support suggestions that decreased 5-HT neurotransmission underlies the anxiolytic-like effects of 5-HT1A receptor agonists in pigeons.


Endothelium-derived relaxing factor inhibits platelet adhesion to cultured porcine endocardial endothelium. By using a simple platelet binding assay, we investigated whether endothelium-derived relaxing factor (EDRF) released from endocardial endothelium influences the adhesion of unstimulated platelets to these cells. Under basal conditions 8.0 +/- 0.32% of total platelets added adhered. The nitric oxide (NO) synthase inhibitor, i.e. NG-nitro L-arginine methyl ester (L-NAME), and the EDRF inhibitor haemoglobin (Hb) increased this adhesion, but another NO synthase inhibitor, NG-monomethyl L-arginine (L-NMMA), did not. The EDRF releasing agent substance P (SP) decreased adhesion, L-NMMA reversed this inhibition, whereas L-NAME and Hb did so only partially. Superoxide dismutase (SOD) caused a marked decrease in adhesion which was fully reversed by L-NMMA, L-NAME and Hb. SOD and SP together showed a cumulative effect on platelet adhesion; this inhibition was significantly reversed by all the EDRF inhibitors, although the levels of adhesion did not return to those seen under basal conditions. These results indicate that EDRF release can inhibit the adhesion of unstimulated platelets to cultured porcine endocardium and that NO synthase inhibitors have differential effects on basal and stimulated EDRF release by these cells.


Inhibition of islet nitric oxide synthase increases arginine-induced insulin release. NG-Nitro-L-arginine, an inhibitor of nitric oxide (NO) synthase, markedly (+50%) increased the L-arginine-induced insulin release from isolated mouse islets but did not itself influence insulin secretion. An abundance of mouse islet cells were positively stained for the enzyme NADPH diaphorase, which reportedly is a marker for NO synthase. The data suggest that the NO synthase activity in mouse islet tissue may inhibit insulin secreting processes and that L-arginine has a dual action on insulin release.


Neurotensin, substance P, gastrin/cholecystokinin, and bombesin in the intestine of the tilapia (Oreochromis mossambicus) and the goldfish (Carassius auratus): immunochemical detection and effects on electrophysiological characteristics. The distribution of neurotensin-, substance P-, gastrin/cholecystokinin/carerulein- and bombesin-like immunoreactivities has been studied in the gut of the tilapia (Oreochromis mossambicus) and the goldfish (Carassius auratus) using immunohistochemistry and radioimmunoassay; the electrophysiological effects of these peptides on the intestinal epithelium were also examined with the Ussing-type chamber technique. Neurotensin- and gastrin/cholecystokinin/caerulein-like immunoreactivities were present in endocrine cells in both species. Substance P- and bombesin-like immunoreactive endocrine cells were present in the intestine of the tilapia. Neurotensin-like immunoreactivity was observed in varicose fibers and nerve cell bodies in the muscle layers and myenteric plexus of both species, whereas nerve fibers showing substance P-like immunoreactivity were found in the goldfish only. Using radioimmunoassays, neurotensin- and gastrin/cholecystokinin/caerulein-like immunoreactive materials were detected in intestinal extracts of both species. The amounts of substance P- and bombesin-like material were below detection level. The ion selectivity of the intestinal epithelium of both species was modulated by exogenously applied neurotensin. This effect was blocked by tetrodotoxin in the tilapia but not in the goldfish. In the tilapia, neurotensin may act via stimulation of a cAMP-dependent increase of the Cl- conductance of the tight junctions, whereas in the goldfish, neurotensin induced, via an unknown messenger, a transient decrease of the cation selectivity without a decrease in the resistance. Substance P, cholecystokinin, and bombesin were without effect on the electrophysiological characteristics of the epithelium.


Occurrence of alginate gene sequences among members of the pseudomonad rRNA homology groups I-IV. Total genomic DNA of 13 pseudomonads representing rRNA homology groups I-IV were screened for sequences homologous to four Pseudomonas aeruginosa alginate (alg) genes by Southern hybridization. Biotinylated probes for three structural genes (algA, algC and algD) and one regulatory gene (algR1) were prepared. Genomic DNA of strains representing group I (P. syringae pv. glycinea, P. viridiflava and P. corrugata) hybridized with all four gene probes. Hybridizing fragments were of differing sizes, indicating that evolutionary divergence among group I members has occurred. P. corrugata has not been reported to synthesize alginate. Genomic DNA from representatives of groups II-IV gave no or very weak hybridization with the probes except for algC. This study indicates that the ability to produce alginic acid as an exopolysaccharide among the pseudomonads is restricted to members of rRNA homology group I in agreement with earlier physiological studies.


Structure of a neutral glycan from the lipopolysaccharides of reference strains for Serratia marcescens serogroups O2 and O3. Serogroups O2 and O3 of Serratia marcescens are differentiated by acidic glycans present in the aqueous phase when lipopolysaccharides are extracted from the reference strains by the aqueous-phenol method. The phenolic phases of these extracts from both strains also contain lipopolysaccharides, from which the same neutral glycan is released on milk acid hydrolysis. The neutral glycan has the disaccharide repeating-unit shown, and accounts for the cross-reactions between the two serogroups and also with serogroup O21: --> 4)-alpha-D-GlcpNAc-(1-->4)-beta-D-ManpNAc-(1--.


Atopic dermatitis and food hypersensitivity in children. Atopic dermatitis is a common condition of early childhood. The association of atopic dermatitis and allergy has been studied for several years. Recent work has shown that food hypersensitivity does play a role as an exacerbating factor in some children with atopic dermatitis. This hypersensitivity will be limited typically to one or two food antigens. Children placed on appropriate elimination diets can be expected to have significant improvement in their clinical course.


Comparative effect of galanin and pyridostigmine on the growth hormone response to growth hormone-releasing hormone in normal aged subjects. The aim of our study was to investigate the effects of aging on the growth hormone (GH) response to growth hormone-releasing hormone (GHRH) alone and in combination with either the neuropeptide galanin or the acetylcholinesterase inhibitor pyridostigmine (PD) in normal subjects. In protocol 1 (GHRH/galanin), 9 old healthy volunteers, ranging in age from 68 to 97 years, and 6 young subjects, ranging in age from 25 to 31 years, received: (a) human GHRH (1-29)NH2, 100 micrograms in 1 ml saline, as an intravenous bolus, and (b) porcine galanin, 500 micrograms in 100 ml saline, as an intravenous infusion from -10 to 30 min combined with GHRH, 100 micrograms i.v. at time 0. In protocol 2 (GHRH/PD), 14 old healthy volunteers, ranging in age from 65 to 91 years, and 11 young subjects, ranging in age from 19 to 34 years, received: (a) GHRH (1-29)NH2, 100 micrograms in 1 ml saline, as an intravenous bolus, and (b) PD, 120 mg administered per os 60 min before GHRH, 100 micrograms as an intravenous bolus. Blood samples for GH were drawn at -75, -60 (time of PD administration), -45, -30, -15, -10 (time of beginning of galanin infusion), 0 (time of GHRH injection), 15, 30, 45, 60, 90, and 120 min. The GH response to GHRH was significantly (< 0.05) enhanced either by galanin or PD pretreatment both in young and old subjects. However, the GH response to GHRH alone or combined with either galanin or PD was significantly greater in the young subjects as compared to the old subjects.(ABSTRACT TRUNCATED AT 250 WORDS)


New World plants; New World drugs.

New World plants; New World drugs. The "discovery" and eventual colonization and exploitation of the New World by Europeans created the opportunity for the development of medicines from numerous plants native to the Western Hemisphere. Many of these plants had been employed by native cultures for centuries or millenia. The plants and, eventually, isolated drugs derived from them were incorporated into the materia media of the Europeans both in Europe and in the new colonies. Many became official in the United States Pharmacopeia (USP); a few still remain today. In all, 30 plants and/or their derivatives are briefly profiled. The subjects of plant-based medicines is becoming more timely as millions of Europeans and Americans begin to seek "natural" remedies for self-medication. Unfortunately, lack of patentability and high new-drug approval costs keep many traditional plant medicines from obtaining proper recognition.


Endogenous interferon plasma levels and antipyrine pharmacokinetics in patients with viral infections. We investigated the pharmacokinetics of antipyrine and the endogenous plasma levels of interferon alpha (IFN alpha), interferon beta (IFN beta) and interferon gamma (IFN gamma) in 10 otherwise healthy volunteers before outbreak (baseline) and in the symptomatic interval of an acute viral respiratory infection, and also in HIV-1 infected homosexual patients in stadium WR 2-3 (n = 11) and WR 5-6 (n = 11) before and one day after application of the antiretroviral agent zidovudine 800 mg day-1 for 14 days. Interferons were measured by RIA or ELISA, respectively. The concentrations of antipyrine and its metabolites in serum and urine were measured by HPLC. Antipyrine is a pharmacokinetic model substance to estimate the cytochrome P 450 enzyme activity. The plasma levels of IFN alpha and IFN gamma in the symptomatic interval of an acute viral respiratory infection were elevated from 4.7 U ml-1 to 12.6 U ml-1 and from 0.3 U ml-1 to 3.4 U ml-1, respectively. The antipyrine clearance showed a significant decrease from 57.9 ml min-1 to 45.0 ml min-1. In the HIV-1 infected patients in stadium WR 2-3 no alterations in plasma levels of interferons or in the pharmacokinetics of antipyrine were observed after treatment with zidovudine. In contrast to these results, in patients in stadium WR 5-6 we found a significant decrease of IFN alpha and an elevation of the clearance and the clearances to metabolite of antipyrine by about 20 percent.


European animal diseases brought to the New World. Beginning with Columbus' second voyage to the New World in 1493, domesticated animals have been imported to establish herds and flocks for the development of animal agriculture and as mounts for the early explorers. Some of these imports harbored agents of disease that were transmitted to healthy animals during their ocean voyage or to their offspring born in the New World. Some of these diseases were eradicated, others have persisted to the present.


The effect of the new immunosuppressive drug FK506 on the formation of secondary metabolites of cyclosporin A. Interactions of FK506 with the cyclosporin A (CsA) metabolism are described. These interactions were not differentiated between the primary and secondary part of metabolism. The combination-therapy with cyclosporin A and diltiazem has shown, that not only the blood levels of CsA were increased, but also the blood levels of the primary CsA-metabolite M17. In the presented in in-vitro-investigations 1.7 microM tritium-labelled CsA was incubated for 90 min with human liver microsomes. The inhibitory effect of FK506 (6 microM) was observed with coincubation under the same conditions. The metabolites were quantified by detection of radioactivity of the elution-fractions after HPLC. The results showed strong inhibition on the formation of both, the primary and secondary CsA-metabolites by FK506. With the same concentration diltiazem and erythromycin exhibited only an inhibition of the formation of secondary CsA-metabolites. In clinical investigations with FK506 in combination with CsA it is necessary to control blood levels of CsA and also its primary metabolites.


Calcium localization in the shell-forming tissue of the freshwater snail, Biomphalaria glabrata: a comparative study of various methods for localizing calcium. The routes calcium might take across the mantle to the shell have been investigated with various electron-microscopical techniques in the freshwater snail Biomphalaria glabrata (Planorbidae, Basommatophora). In chemically-fixed tissue, calcium was precipitated with a tannic acid-antimonate technique in predominantly the intercellular spaces of the outer mantle epithelium and the interstitium below it. Some vacuoles of the outer mantle epithelium and one type of mucus cell in the inner mantle epithelium also contained precipitate. The presence of calcium in the precipitates was proved by electron energy loss spectroscopy combined with electron spectroscopic imaging. Incubation with lead acetate and uranyl acetate revealed binding-sites for calcium in the intercellular spaces of the epithelia interstitium and the mucus cells of the inner mantle epithelium. Precipitates were also seen after all incubations in the calcium spherites of the connective tissue. The concentrations of calcium and other elements were analysed in freeze-dried ultrathin sections of cryofixed mantle tissue by means of energy-dispersive X-ray microanalysis. Only in mitochondria of the musculature could high amounts of calcium and phosphorous be detected.


Relationship between interphase AgNOR distribution and nucleolar size in cancer cells. We have studied the relationship between interphase nucleolar organizer region (NOR) distribution and nucleolar size in cancer cells at light-microscopical level. Thirteen cases of formalin-fixed bladder cancer and fifteen cases of methacarn-fixed tumours of different origin were used. Nucleoli of the former cases were stained by Phloxine B and of the latter by Toluidine Blue. Selective visualization of interphase NORs was obtained by carrying out the one-step silver staining reaction for AgNOR proteins (Ploton et al., 1986). The area occupied by Phloxine B- or Toluidine Blue-stained nucleoli and interphase silver-stained NORs was measured by means of an automated image analyser. Both in bladder cancers and in the other tumour lesions nucleolar and interphase AgNOR areas were linearly related (r = 0.95 and r = 0.96, respectively, P < 0.001). The close relationship between the area of nucleoli and that of silver-stained nucleolar structures was maintained even if the silver-staining procedure was prolonged beyond the optimal time length for selective interphase NOR staining. In the latter case, however, single interphase AgNORs were no longer visible within the nucleolar body which was, in fact, homogeneously stained. These data indicate that evaluation of the interphase AgNOR area has the same relevance, in tumour pathology, as whole nucleolar size measurement.


Inhibitory effects of organotin compounds on histamine release from rat serosal mast cells. Tributyltin inhibited compound 48/80-induced histamine release from rat serosal mast cells in a dose-related manner. Triphenyltin and tripropyltin also showed a strong inhibitory effect on the histamine release and the degree of the inhibition was the same as that of tributyltin. The inhibitory effects of other organometals were much smaller than those of the organotins. Tributyltin and triphenyltin also showed a tendency to inhibit histamine release induced by concanavalin A + phosphatidylserine and calcium ionophore A 23187. The present results suggest that certain triorganotin compounds may have a specific inhibitory effect on histamine release by acting on the same process in each of the three stimulus responses.


Effect of polysaccharide interactions on antibiotic susceptibility of Pseudomonas aeruginosa. The relative viscosity of Pseudomonas aeruginosa alginate was shown to increase markedly when combined with mucin, Ca2+ ions and the exopolysaccharide from Pseudomonas cepacia. The presence of such a heterodisperse polysaccharide solution significantly reduced the diffusion and hence antimicrobial activity of tobramycin and to a lesser extent ciprofloxacin against Ps. aeruginosa by factors of 90 and 2.5-fold respectively over a 5 h incubation period. The clinical implications of these results are discussed in relation to cystic fibrosis.


Preparation and properties of monoclonal antibodies against lipopolysaccharide-sensitive serine protease zymogen, factor C, from horseshoe crab (Tachypleus tridentatus) hemocytes. Seventeen murine monoclonal antibodies (mAbs) against horseshoe crab clotting factor, factor C, were prepared and characterized. When the binding sites of these mAbs were analyzed by immunoblotting, ten mAbs recognized nonreduced factor C, five mAbs were directed against the heavy chain, and two mAbs were directed against the B chain. Three mAbs, 1H4, 2C12, and 2A7, one selected from each group, were used for further study. The mAb 1H4, which recognized only nonreduced factor C molecule, inhibited the factor C activity in a dose-dependent manner. It also inhibited lipopolysaccharide (LPS)- and alpha-chymotrypsin-mediated activations of the zymogen factor C, suggesting that 1H4 binds close to the active site and/or the substrate-binding site located in the serine protease domain (B chain) of factor C. On the other hand, 2C12 and 2A7 recognized, respectively, an epitope located in the heavy and the B chains, and inhibited LPS-mediated activation of factor C, but not alpha-chymotrypsin-mediated activation of factor C or factor C activity. Both F(ab')2 and Fab' fragments derived from 2C12 inhibited LPS-mediated activation in the same manner. These three mAbs did not bind with LPS, although a factor C-mAb complex was able to bind LPS, suggesting that the LPS-mediated activation of the zymogen factor C was induced through intermolecular interaction between the LPS-bound factor C molecules. The dissociation constants (Kd) for 1H4, 2C12, and 2A7 binding to factor C were determined as 1.9 x 10(-9), 0.6 x 10(-10), and 1.8 x 10(-10) M, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)


Engineering of artificial cell adhesion proteins by grafting the Arg-Gly-Asp cell adhesive signal to a calpastatin segment. A new artificial cell adhesive protein was engineered by grafting the Arg-Gly-Asp (RGD) sequence, the minimal recognition signal of fibronectin for interaction with integrins, to a calpastatin segment by in vitro mutagenesis. The mutagenized protein showed cell adhesive activity in addition to calpain inhibitory activity. The RGD signal grafted to the calpastatin segment was recognized by the vitronectin receptor but not by the fibronectin receptor.


Microbore liquid chromatography with dual electrochemical detection for the determination of serotonin and 5-hydroxyindoleacetic acid in rat brain dialysates. A microbore liquid chromatographic assay with dual electrochemical detection is described for the determination of serotonin and its metabolite 5-hydroxyindoleacetic acid in rat brain dialysates. The concentration of serotonin in these samples is usually in the low nanomolar range (fmol per 20 microliters range). To optimize separation and detection, several adaptations were made to the system with respect to the injection valve, flow-rate of the pump, connections between injector, column and detector, and cell volume of the detector. These aspects are discussed, as well as the procedure developed for optimal peak identification of serotonin and correct estimation of 5-hydroxyindoleacetic acid. The assay allows the measurement of basal serotonin release without the use of a re-uptake inhibitor added to the perfusion fluid.


Ultrastructure of sarcoballs on the surface of skinned amphibian skeletal muscle fibres. The formation of sarcoballs on the surface of skinned fibres from semitendinosus muscles of Xenopus laevis, and the sarcoplasmic reticulum content of the structures, have been studied using conventional electron microscopic techniques and immunoelectron microscopy. Examination of the fibres showed many membrane-bound blebs projecting from the surface in areas where vesicles of internal membranes (including sarcoplasmic reticulum, T-tubules and mitochondria) were clustered in interfilament spaces. The blebs varied in size from 1 micron to 150 microns and those with diameters > 10 microns are referred to as sarcoballs. Small blebs were often seen in close association with each other and might have fused during sarcoball formation. The interior of the sarcoball was filled with foam-like material made up of vesicles with diameters of 100 nm to 1.0 microns. The sarcoplasmic reticulum membrane content of the sarcoballs was evaluated using two monoclonal antibodies, one to the Ca2+ ATPase of the sarcoplasmic reticulum and the second to ryanodine receptor calcium release channels in the junctional-face membrane. The antibodies bound to some components of the surface and interior of the sarcoball, but not to mitochondrial-like structures and tubular vesicles. The results show that a large component of the sarcoball and its surface is derived from sarcoplasmic reticulum and suggest that mitochondria and T-tubules might also contribute membranes to the structures. Our hypothesis is that (a) blebs bud out from the surface of the skinned fibre following fusion of internal vesicles that are extruded along interfilament channels during unrestrained contractures, (b) blebs grow into sarcoballs by additional fusion of internal membrane vesicles and fusion of adjacent blebs, and (c) the sarcoball is a foam-like structure composed of bathing medium and membrane lipid (containing membrane proteins).


The clinical expression in anticentromere antibody-positive patients is not specified by the epitope recognition of CENP-B antigen. Centromere protein B (CENP-B), which is an alphoid DNA binding protein, is the target antigen in autoimmune disease patients (often with scleroderma). From our previous analysis of the reactivity of anticentromere sera, four independent epitopes were identified on recombinant CENP-B. The anticentromere sera displayed heterogeneity in their patterns of reactivity to the four epitopes. We have investigated to what extent this heterogeneity of the target autoepitope on CENP-B accounts for the clinical diversity of anticentromere antibody (ACA)-positive patients. A major autoepitope, epitope I, was recognized by all 40 ACA-positive sera; however, the other three epitopes were recognized differently from case to case. We could not find any significant correlation between the reactivity to CENP-B autoepitopes and the clinical presentation of ACA-positive patients. There was considerable clinical diversity, even among the nine patients showing specificity for the single major autoepitope. In conclusion, we found that, although ACA-positive patients were both clinically and immunologically heterogeneous, in most respects the clinical expression appeared to be independent of the reactivity to the CENP-B autoepitope, a finding which suggests that identification of the target epitope of CENP-B is unlikely to assist in the clinical classification of the disease in ACA-positive patients. The identification of multiple B cell epitopes on CENP-B is consistent with the concept that the self-antigen drives the antibody response. However, factors other than CENP-B autoepitope specificity must determine the clinical expression of ACA responses.


[Fetal growth retardation: prevention. Review of the literature]. The principle underlying preventive treatment of fetal intrauterine growth retardation can only be considered after defining various groups that are at risk and early screening based on the obstetric history, biological tests, and recently using flow rates with Doppler techniques. The use of aspirin in preventive treatment of growth retardation originates in the pharmacological properties of its molecule which allow the re-establishment of a balance of the prostoglandins. Numerous authors have studied clinical effects, its harmlessness as well as the true indications for its use. As there have been no control studies the theoretical possibilities of using Beta-Mimetic drugs has not been tested. The use of ultrasound has made it possible to try to see whether there is an improvement in the fetal state of well-being when oxygen therapy is used by the mother as it is in certain extremes to see what effect it has on placental function. Its use as an effective prophylactic has not yet been demonstrated. Promising lines of research could be assessing the effects of immunological treatments that have already been suggested (especially gammaglobulin transfusions). They have been used prophylactically with success but the series are small for well defined immunological indications.


Assessment of airborne bacterial contamination of clean wounds: results in a tissue model. Ovine skeletal muscle was used as a model wound and inoculated with airborne bacteria collected from a busy communal room. A specialized counting technique involving agar overlay and post-incubation tetrazolium staining was developed to allow accurate counting of small numbers of bacteria on the surfaces of muscle and membrane filters coated with substantial quantities of muscle and fat debris. Two techniques of recovering the inoculated airborne bacteria from the model wound were compared. Pulsed jet lavage with membrane filtration of the recovered fluid showed substantially better recovery, less variability and correlated more closely with controls than a tetrazolium stained 5 microns membrane filter imprint technique. Pulsed jet lavage with membrane filtration is likely to be the more appropriate technique in the assessment of contamination of wounds created in ultraclean air.


Ki-67 antibody immunostaining in benign and malignant human prostatic disease. Indices of mitotic potential may improve prognostic discrimination in patients with malignant disease. Ki-67 is a monoclonal antibody directed against an unknown proliferation antigen which has been shown to be a measure of mitotic potential. Sixty-four benign and eighty malignant prostatic biopsies were stained with the Ki-67 antibody. Nuclear and cytoplasmic staining was identified in benign and malignant biopsies using immunoalkaline phosphatase and immunoperoxidase staining reactions. Nuclear staining was identified in 14 benign and 44 malignant biopsies. Nuclear staining for Ki-67 was seen in 36% of biopsies with Gleason histological score (GHS) 2-4, 71% with GHS 5-7 and 62% with GHS 8-10. Nuclear staining was associated with advanced local disease stage, but not with metastatic disease stage. Clinical follow-up is required to establish the value of Ki-67 immunostaining as a prognostic determinant in prostatic cancer.


Complex modulation of cation channels in the tonoplast and plasma membrane of Saccharomyces cerevisiae: single-channel studies. Detailed patch-clamp studies have been made of ion channels in the plasma membrane and tonoplast of the yeast Saccharomyces cerevisiae. The predominant tonoplast channel is a high-conductance cation-selective inward rectifier (passing ions easily into the cytoplasm from the vacuole), with its open probability (Po) peaking at about -80 mV (cytoplasm negative) and falling to near zero at +80 mV. It has a maximal slope conductance of approximately 150 pS in 100 mmol l-1 KCl, and conducts Na+, K+ and Ca2+. Elevated cytoplasmic Ca2+ concentration, alkaline pH and reducing agents can activate the channel, its likely physiological function being to adjust cytoplasmic Ca2+ concentration from the vacuolar reservoir. The predominant plasma-membrane channel is a strongly outward rectifying K+ channel (passing K+ easily out of the cytoplasm to the extracellular medium), which is activated by positive-going membrane voltages as well as by elevated cytoplasmic Ca2+ concentration and alkaline pH. Interaction between membrane voltage and [Ca2+]cyt is complex and defines three parallel closed states for the channel: a Ca(2+)-independent brief closure (I), a calcium-inhibited long closure (G) and, at large positive voltages, a calcium-induced brief blockade (B). This channel is likely to function in steady-state turgor regulation and in charge balancing during proton-coupled substrate uptake.


Therapy of Morbus Parkinson and radical-induced neurotoxicity in the rat--in vivo voltammetric studies. In vivo pulse voltammetry was used to study the effect of anti-parkinsonian drugs and of neurotoxins in rat striatum. It could be demonstrated that besides the well-known changes of total dopamine (DA) concentrations the applied drugs affected also the extraneuronal DA metabolism in the living rat. In addition, neurotoxin-treated rats can be used as a model to study some aspects of the Parkinson syndrome.


Blockade of ionotropic quisqualate receptor desensitization by wheat germ agglutinin in cultured postnatal rat hippocampal neurons. 1. The effects of the lectin wheat germ agglutinin (WGA) on quisqualate-gated currents were examined in postnatal rat hippocampal neurons using recordings from whole cells and outside-out membrane patches. 2. Rapid applications of quisqualate to whole cells and outside-out patches evoked a current that desensitized to a steady-state level. WGA blocked desensitization by increasing the steady-state current amplitude without altering the current-voltage (I-V) relationship or pharmacology of the current. 3. In outside-out patches quisqualate (2.5-1,000 microM) elicited bursts of channel openings having conductances of 2.7, 6.3, and 13 pS. The mean burst length for all conductances was 8.6 +/- 0.6 ms (mean +/- SE) and exhibited little voltage (-110 to +80 mV) or concentration (2.5-1,000 microM) dependence. Treating patches with 580 nM WGA produced no change in conductance, but the mean burst length for 100 microM quisqualate increased from 9.0 +/- 1.1 to 16 +/- 3.2 ms. 4. Fluctuation analysis of whole cell currents evoked by 1 microM quisqualate at -80 mV revealed an increase in the time constant from 8.7 +/- 0.5 to 13 +/- 1.0 ms after treatment with 580 nM WGA. This treatment produced no change in the estimated single-channel conductance. 5. These findings suggest that an increase in channel burst length, rather than an increase in single-channel conductance, contributes to the WGA-induced augmentation of the steady-state quisqualate current.


Wheat germ agglutinin enhances EPSCs in cultured postnatal rat hippocampal neurons by blocking ionotropic quisqualate receptor desensitization. 1. The effect of the lectin wheat germ agglutinin (WGA), an inhibitor of ionotropic quisqualate receptor desensitization, on both evoked and spontaneous fast excitatory postsynaptic events was examined in cultured postnatal rat hippocampal neurons with the use of whole cell recordings. 2. WGA, at 580 nM, potentiated evoked fast excitatory postsynaptic currents (EPSCs) by increasing the amplitudes by 100 +/- 27% (mean +/- SE) and the time constant of decay from 5.8 +/- 0.6 to 7.9 +/- 0.5 ms. The increases in these parameters were not accompanied by changes in the current-voltage (I-V) relationship or pharmacological profile of the fast EPSCs. 3. WGA did not alter the amplitude or time course of decay of inhibitory postsynaptic currents (IPSCs), and it did not alter neuronal input resistance or action potentials. 4. WGA increased the amplitude of spontaneous fast miniature EPSCs (MEPSCs), defined as spontaneous EPSCs recorded in the presence of tetrodotoxin, by 53 +/- 11% and increased the time required to decay to 50% of the peak amplitude by 48 +/- 23%. These changes were not associated with a change in the rate of MEPSC occurrence. 5. These results suggest that WGA augments hippocampal excitatory postsynaptic events via a postsynaptic mechanism. The results further imply that ionotropic quisqualate receptor desensitization can modulate the amplitude and time course of decay of fast excitatory synaptic events. Thus desensitization may be one factor that regulates fast excitatory synaptic transmission.


A computer-based model for realistic simulations of neural networks. II. The segmental network generating locomotor rhythmicity in the lamprey. 1. To analyze the function of the spinal interneuronal network generating locomotion in the lamprey CNS, a vertebrate model system, we performed computer simulations with realistic model neurons possessing the essential properties of their biological counterparts. 2. The segmental network has been simulated by modeling experimentally established types of neurons with their specific membrane properties and synaptic interconnections. Fictive locomotor activity, which can be experimentally induced by elevating the background excitability by bath application of excitatory amino acids, was simulated by opening membrane conductances for kainate/alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) or N-methyl-D-aspartate (NMDA) receptors. Kainate/AMPA receptor activation induced a rhythm in the middle and upper part of the physiological burst frequency range, whereas NMDA receptor activation evoked bursting in the lower part of the range, which corresponds well to earlier experimental findings. 3. Several factors contributing to the termination of the burst were studied and their interaction was assessed in simulations of the network. 1) The summation of spike afterhyperpolarizations (late AHPs), leading to adaptation of the discharge, acts as a primary burst-terminating factor at lower rates of kainate/AMPA-induced bursting, and it also interacts with the NMDA-induced oscillatory membrane properties during slow rhythmicity. 2) The termination of the depolarized NMDA plateau is another important factor during NMDA-evoked rhythmicity. 3) The synaptic inhibition from lateral interneurons to the interneurons mediating reciprocal inhibition is important at higher rates of kainate/AMPA-induced bursting. 4. The mechanism of action of 5-hydroxytryptamine (5-HT) on the lamprey segmental network was further investigated by simulation. 5-HT is known to lower the burst frequency during fictive locomotion and also to decrease the conductance through the Ca(2+)-dependent K+ channels, and thereby the size of the late AHP that follows the action potential. Decreasing this conductance in the network simulations resulted in a lesser amount of AHP summation and thereby less frequency adaptation during the burst, longer bursts, and a lower locomotor frequency. Thus the selective action of 5-HT on the Ca(2+)-dependent K+ channels, and hence on the AHP, can account for the modulatory effect on the fictive locomotor rhythm seen experimentally. 5. The results demonstrate that the present simulation of the segmental network can account for essential features of the motor pattern seen experimentally during lamprey locomotion.(ABSTRACT TRUNCATED AT 400 WORDS)


Calcium-activated chloride channels in cultured embryonic Xenopus spinal neurons. 1. Single-channel currents were recorded from Xenopus spinal neurons developing in vitro using the patch-clamp technique, to identify the channels underlying the large and small macroscopic Ca(2+)-activated Cl- currents (ICl(Ca)) present in these cells. 2. Channels of large (maxi-channels; 310 pS) and smaller conductance (mini-channels; 50-60 pS) are activated by elevation of cytoplasmic Ca2+ concentration. Channel activity is not altered by subsequent removal of Ca2+ from the bath, arguing against a direct ligand-type Ca2+ dependence. The much higher incidence of channel activation in cell-attached patches from cells permeabilized with the Ca2+ ionophore A23187 than in excised patches also suggests the involvement of some unidentified intracellular factor. 3. The reversal potential of maxi-Cl- channels is not altered by changes in Na+ concentration, but is shifted in the negative direction by the substitution of Cl- by methanesulfonate on the intracellular side of the patch, indicating their anionic selectivity. 4. Maxi-Cl- channels exhibited the presence of multiple probable subconductance states and showed marked voltage-dependent inactivation above and below +/- 20 mV. 5. Examination of maxi-Cl- channels at early times in culture (6-9 h) and 24 h later did not reveal any developmental change in the characteristics described above. However, the mean open duration of the channel was found to increase twofold during this period of time. 6. The simultaneous presence of maxi- and mini-Cl- channels prevented detailed characterization of the latter. The anionic selectivity of mini-Cl- channels is suggested by their reversal potential that lies close to the Cl- equilibrium potential.(ABSTRACT TRUNCATED AT 250 WORDS)


[An immunohistological study of normal parotid gland]. Cytoskeletal proteins from each epithelial component of the parotid gland were studied immunohistologically using monospecific monoclonal antibodies, and we obtained several interesting results: 1) The expression of vimentin, S-100 protein and GFAP were essentially unrecognizable. 2) The expression of alpha-actin was recognized only in myoepithelial cells, and was negative in other tissues. 3) The expression of CK-3, CK-6, CK-7, CK-8 and CK-18 was commonly recognized in both acinal and ductal cells. 4) The expression of CK-13 and CK-19 was recognized in ductal cells, but was negative in acinal cells. 5) We propose that the characteristic cell be referred to as a "duct satellite cell", as it comprises part of the intercalated duct and is sporadically localized around striated and layer ducts. 6) These duct satellite cells express all subclasses of cytokeratin observed in myoepithelial, acinal and ductal cells. 7) The duct satellite cells observed in this study are most likely to correspond to the undifferentiated reserve cells described in "bicellular theory", and we speculate that all epithelial tissues of the parotid gland originate from these cells.


Abdominal pain as a cause of acute admission to hospital. Between 1 January 1988 and 31 December 1989, 525 patients were admitted to one hospital with a diagnosis of acute abdominal pain. Of these, 182 (34.7%) underwent an emergency operation and 14 (7.7%) of these patients subsequently died within 30 days. Death was due to intestinal obstruction in 69%, and there was a 28% mortality rate for emergency colonic resection. Non-specific abdominal pain (NSAP) was the most common diagnosis (36.0%), followed by appendicitis (14.9%) and urological causes (12.8%). There was an unnecessary appendicectomy rate of 25.0%. Admission with pain because of urological causes was over twice that of previous reports. Duration of stay increased greatly with age. Results from this study confirm the high mortality rate in the elderly from emergency colonic resection. Greater care in diagnosis and a conservative approach to appendicitis, with laparoscopy in females of reproductive age, may produce a lower unnecessary appendicectomy rate without an increase in morbidity. If the diagnosis of NSAP could be made earlier and patients discharged sooner, a large saving in resources would result. This early diagnosis is not yet possible.


Preoperative shaving: patient and surgeon preferences and complications for the Gillies incision. A modified technique for the Gillies temporal approach without previous hair removal is described. A randomized prospective study of two groups of patients requiring elevation of fractures of the zygomaticomalar complex via this approach with and without hair removal was performed with respect to patients' attitudes and surgeons' assessment.


Antiphospholipid antibodies: new perspectives on antigenic specificity. Antiphospholipid antibodies (aPL) are found in up to 60% of patients with systemic lupus erythematosus (SLE), and appear to predispose to a number of complications of the disease. We purified IgG from patients with SLE and the antiphospholipid antibody syndrome (APS) who have high levels of aPL and used this IgG in a modified ELISA where we excluded any source of cofactor in the assay. We showed that for 10 of the 11 patients, IgG bound strongly to cardiolipin only in the presence of the cofactor, which was present in fetal calf serum, normal human serum (NHS), and in the infranatant lipoprotein fractions obtained by flotation ultracentrifugation of NHS. We then purified beta 2 glycoprotein I (beta 2GPI) from NHS and showed that for 9 of the 11 samples of IgG, beta 2GPI was as effective, or more effective, than NHS as a source of cofactor. When beta 2GPI was coated on ELISA plate and used as an antigen we found little or no reactivity of patient sera or IgG, suggesting that beta 2GPI alone is not antigenic in our system. Three possible explanations could be put forward to explain the behavior of cardiolipin and beta 2GPI, neither of which is antigenic alone.


Apical and basolateral Na/H exchange in cultured murine proximal tubule cells (MCT): effect of parathyroid hormone (PTH). Kidney proximal tubule Na/H exchange is inhibited by PTH. To analyze further the cellular mechanisms involved in this regulation we have used MCT cells (a culture of SV-40 immortalized mouse cortical tubule cells) grown on permeant filter supports. Na/H exchange was measured using single cell fluorescence microscopy (BCECF) and phosphate transport (measured for comparisons) by tracer techniques. MCT cells express apical and basolateral Na/H exchangers which respond differently to inhibition by ethylisopropylamiloride and by dimethylamiloride, the basolateral membrane transporter being more sensitive. Apical membrane Na/H exchange was inhibited by PTH (10(-8) M; by an average of 25%); similar degrees of inhibition were observed when cells were exposed either to forskolin, 8-bromo-cAMP or phorbol ester. Basolateral membrane Na/H exchange was stimulated either by incubation with PTH (to 129% above control levels) or by addition of phorbol ester (to 120% above control levels); it was inhibited after exposure to either forskolin or 8-bromo-cAMP. The above effects of PTH and phorbol ester (apical and basolateral) were prevented by preincubation of cells with protein kinase C antagonists, staurosporine and calphostin C; both compounds did not affect forskolin or 8-bromo-cAMP induced effects. PTH also inhibited apical Na-dependent phosphate influx (29% inhibition at 10(-8) M); it had no effect on basolateral phosphate fluxes (Na-dependent and Na-independent). Incubation with PTH (10(-8) M) resulted in a rapid and transient increase in [Ca2+]i (measured with the fluorescent indicator, fura-2), due to stimulation of a Ca2+ release from intracellular stores. Exposure of MCT cells to PTH did not elevate cellular levels of cAMP. Taken together, these results suggest that PTH utilizes in MCT cells the phospholipase C/protein kinase C pathway to differently control Na/H exchangers (apical vs. basolateral) and to inhibit apical Na/Pi cotransport.


Anion and cation permeability of a large conductance anion channel in the T84 human colonic cell line. A large conductance multi-state channel was identified and characterized in single channel recordings from cell-attached and excised patches of the human colonic tumor cell line, T84. The channel activity was dependent on the presence of both permeable cations and anions. In Na(+)-free symmetrical Cl- solutions or Cl(-)-free symmetrical Na+ solutions the channel was inactive. Addition of 5 mM NaCl (NaI or KCl) induced channel activity. The selectivity sequence obtained from the shift in reversal potential was I- (1.9) > Cl- (1) > Na+ (0.5) > K+ (0.3). SO2-(4), SCN- (thiocyanate) and NMDG+ were impermeant. Multiple subconductance states were identified at all voltages explored (+/- 90 mV). The minimum conductance encountered in symmetrical 100 mM NaCl was a 15 pS substrate, the maximum, 210 pS. The channel appeared to be composed of multiples of the 15 pS subunits which were reversibly blocked by the loop diuretic bumetanide (5 microM).


Sonographic scoring index for prenatal detection of chromosomal abnormalities. Current indications for cytogenetic evaluation leave the majority of Down syndrome fetuses undetected. Using advanced maternal age and low maternal serum alpha-fetoprotein (AFP) levels as criteria, only 40% of fetuses with Down syndrome (trisomy 21) are identified (positive predictive value, 0.4% to 1%). We evaluate the sonographically detectable physical features of second trimester fetuses to determine whether these features are more sensitive and specific than maternal age for detecting fetuses with abnormal karyotypes. From March 1, 1990, to September 1, 1991, more than 5,000 fetuses between 14 and 20 weeks of development were referred for genetic amniocentesis because of advanced maternal age or abnormal AFP levels. Forty-three of these 5,000 fetuses were later found to have autosomal trisomies by karyotype (32 with trisomy 21, nine with trisomy 18, and two with trisomy 13). A sample of 588 consecutive normal fetuses from the total of more than 5,000 amniocenteses performed during this period of time was used as our control group for statistical analysis. The sonographic features of these 588 normal second trimester fetuses and the 43 trisomic fetuses recorded prospectively prior to knowledge of the karyotype were evaluated statistically. The femur and humerus lengths, nuchal fold, renal pelvic dimension, and major structural defects were compared in the normal and trisomic fetuses. On the basis of our results, a weighted sonographic score was developed to optimize the detection of fetuses at risk for aneuploidy. Using our previously published formulas and criteria for a short femur and humerus, 17/32 (53%) fetuses with Down syndrome and 23/588 (3.9%) of the normal fetuses were identified. Twenty two of 32 Down syndrome fetuses (69%) and 2/588 (0.34%) of normals had a nuchal fold > or = 6 mm, and 11 of 32 Down syndrome fetuses and all those with trisomies 18 and 13 had a major anomaly detected sonographically. The following scoring system was developed for the detection of aneuploidy: nuchal fold = 2, major structural defect = 2, and short femur, short humerus, and pyelectasis = 1 each. Selecting fetuses with a score of > or = 2 would identify 26/32 (81%) Down syndrome fetuses, and 9/9 (100%) and 2/2 (100%) fetuses with trisomies 18 and 13 respectively, but only 26/588 (4.4%) of the normal fetuses. Using the sonographic score of 2 results in a positive predictive value for a 1/250 risk group of 6.87% for identifying Down syndrome fetuses and 7.25% for all three trisomies.(ABSTRACT TRUNCATED AT 400 WORDS)


[Application of PCR and RT-PCR method to molecular biology study in nephrology]. Recent advance in molecular biology and genetic engineering has made a profound influence on basic and clinical medicine. Development of PCR (polymerase chain reaction) has transformed our trend of thought in molecular biology. Applications of PCR method has extended to nephrology study. RT (reverse transcription)-PCR method was elaborated to identify mRNA even in a small number of cells. It is also important and advantageous that PCR facilitates the utilization of a non-radioisotopic detection system. Newly developed chemiluminescent procedure yields as high sensitivity just as radioactive probes. Further applications are now being investigated including in situ PCR. PCR and RT-PCR methods are of great value for studying a molecular biological background of intricate kidney functions and diverse renal disorders.


[Endothelin, EDRF, CGRP].

[Endothelin, EDRF, CGRP]. Endothelin-1 is now known to synthesized in the kidney and influence the renal function. ET-1 mRNA was detected in glomerulus and inner medullary collecting ducts using RT-PCR technique. ETA receptor mRNA was detected only in glomerulus, vasa recta bundle, and arcuate artery. ETB receptor mRNA distributed mainly in glomerulus and collecting ducts. Endothelium derived relaxing factor (EDRF) was believed to be nitric oxide, was synthesized by nitric oxide (NO) synthase from L-arginine. NO stimulates soluble guanylate cyclase and increases cGMP level. NO synthase mRNA was detected in glomerulus and inner medulla. Soluble guanylate cyclase mRNA distributed widely along the nephron segments. NO and cGMP system seems to play some roles in modulating renal functions.


[Ion channels in the kidney: molecular biological aspects]. Ion channels in renal tubules are involved in maintenance of the volume and ion composition of the body. In the kidney these channels are expressed at the apical and/or basolateral cell membranes and are responsible for vectorial ion transport. Structure and function of the renal ion channels differs from those of previous channels which were cloned in excitable tissues. In this review I outline the various types of ion channels in renal and non-renal cells and give examples that emphasize the physiological roles of these channels in the nephron.


[DNA typing of HLA-class II genes in idiopathic nephropathy]. The association between idiopathic nephropathy and HLA-class II antigen has been reported in many ethnic groups. We attempted to ascertain the HLA regions more specifically, associated with Japanese idiopathic membranous nephropathy (IMN), IgA nephropathy (IgAN) and minimal change nephrotic syndrome (MCNS), by examining HLA-class II genes. DNA typing of HLA-class II genes showed that IMN was associated with HLA-DRB1*1501-DRB5*0101-DQA1*0102-DQB1*0602, IgA with HLA-DQA1*0301, and MCNS with DQB1*0302. We also found a common epitope of HLA-class II (at 38th amino acid position of HLA-DR beta in IMN, at 55th of HLA-DQ beta in MCNS) in Japanese and Caucasian patients. These particular epitopes seem to be important for the susceptibility to IMN or MCNS.


[The molecular genetic analysis of polycystic kidney disease]. Autosomal dominant polycystic kidney disease (ADPKD) is one of common single gene disorders. The development of molecular genetic techniques has shown that mutant PKD1 gene assigned to ADPKD was closely linked to alpha-globin on the short arm of chromosome 16. This location was established when genetic linkage was found between ADPKD and a highly polymorphic region at the 3' end of the alpha-globin cluster (3' HVR). The discover of genetic linkage markers such as 3' HVR probe has provided a diagnostic test in presymptomatic stage. We performed this diagnostic test using DNA probes in 3 patients with ADPKD of one Japanese family. They also showed PKD1 gene linkage as previously described by Reeders et al. Linkage analysis of the PKD1 gene might be available to diagnostic test of ADPKD. DNA diagnosis of ADPKD however has to be performed carefully because of an ethical standpoint.


Histamine-induced depolarization and the cyclic AMP--protein kinase A system in isolated guinea pig adipocytes. The relationship between histamine (Hi)-induced depolarization and the cyclic AMP system in adipocytes was studied in guinea pigs, which seem to be more sensitive than rats to Hi. Hi caused a dose-dependent depolarization in guinea pig mesenterial and epididymal adipocytes with EC50 values of 1.69 x 10(-7) M and 1.19 x 10(-7) M, respectively. Guinea pig adipocytes were 280-750 times more sensitive than rat adipocytes to Hi. Isoproterenol, forskolin and 3-isobutyl-1-methylxanthine (IBMX) also caused a depolarization, and the slopes of the concentration response lines for these drugs were almost the same as that for Hi. Furthermore, pretreatment with these drugs resulted in a potentiation of Hi-induced depolarization at lower concentrations which are not effective when each drug is used alone. In addition, Hi-induced depolarization was inhibited by pretreatment with prostaglandin E1 (PGE1) and insulin dose-dependently. The content of cyclic AMP in adipocytes was increased by Hi (10(-7) M) in association with a decrease in membrane potential. KT5720, a protein kinase A inhibitor, which provides no significant effect even at a concentration of 10(-6) M, showed an antagonistic effect on Hi-induced depolarization.


Significant role of peptide leukotrienes (p-LTs) in the antigen-induced contractions of human and guinea pig lung parenchymas and bronchi or tracheas in vitro. Chemical mediators responsible for the antigen-induced contractions of isolated, passively sensitized human and guinea pig lung parenchymas and bronchi or tracheas were evaluated by several antagonists and enzyme inhibitors, with emphasis on the effects of the potent and selective peptide leukotriene (p-LT) antagonist MCI-826. All of these preparations showed long-lasting contractions in response to an antigen challenge which lasted for more than 60 min. In either the human lung parenchyma and brochus or guinea pig lung parenchyma, pretreatment with 10(6) g/ml (2.4 x 10)-6) M) MCI-826 significantly inhibited the late phase at 10 to 60 min after the challenge of the contraction following slight suppression of the early phase. The early phase contractions of these preparations were moderately antagonized by 10(-6) g/ml mepyramine, but the late phases were not influenced or even rather enhanced. The combination treatment of MCI-826 with mepyramine additionally and markedly inhibited both phases of these preparations. On the other hand, although mepyramine apparently inhibited the early phase of the guinea pig tracheal contraction but not the late phase, no synergistic inhibitions of the contraction were observed when it was combined with MCI-826. The p-LT antagonist FPL 55712, atropine and indomethacin at 10(-6) g/ml either slightly inhibited or enhanced the contractions of human lung parenchymas, guinea pig tracheas and lung parenchymas, but the effects were not significant. From these results, it should be emphasized that p-LTs largely contribute to induction of the anaphylactic contractions of human lung parenchymas as well as human bronchi and guinea pig lung parenchymas but not guinea pig tracheas.


Calcium channel blocker-like action of reserpine in smooth muscle. The effect of reserpine on vascular and intestinal smooth muscles was examined. In these muscles, reserpine inhibited the high K(+)-induced contraction, and this inhibitory effect was antagonized by the increase in external Ca2+ concentration and also by a Ca2+ channel activator, Bay k8644. In rabbit aorta, increases in cytosolic Ca2+ level and muscle tension due to high K+ were inhibited in parallel by reserpine. These results suggest that reserpine inhibits L-type Ca2+ channels to inhibit smooth muscle contraction.


Effects of nitric oxide synthase inhibitors on gastric alkaline secretion in rats. The effects of NG-nitro-L-arginine methyl ester (L-NAME), the nitric oxide (NO) synthase inhibitor, on gastric HCO3- secretion were examined in anesthetized rats. Intravenous administration of L-NAME (1, 2.5, 5 mg/kg) increased HCO3- secretion in a dose-related manner. This effect of L-NAME was mimicked by NG-mono-methyl-L-arginine (50 mg/kg, i.v.) and was antagonized significantly by concurrent administration of L-arginine but not D-arginine (200 mg/kg, i.v.). These results indicate that gastric HCO3- secretion is stimulated by inhibition of NO biosynthesis.


End-organ disease in hypertension: what have we learned? The major target organs that suffer from sustained hypertension are the heart, kidneys, and brain. Cardiac adaptation to arterial hypertension consists of left ventricular hypertrophy (LVH) of the concentric type, that is, an increase in wall thickness at the expense of chamber volume. However, LVH can no longer be considered a simple adaptive myocardial process serving to compensate for the increase in afterload and bring left ventricular wall stress back to normal. Data from the Framingham cohort have shown that the occurrence of LVH drastically increases the risk of sudden death and other cardiovascular morbidity and mortality irrespective of the levels of arterial pressure. Renal adaptation to arterial hypertension consists of a decrease in renal blood flow with elevations in filtration fraction and renal vascular resistance. With progressive hypertensive cardiovascular disease, glomerular filtration rate will fall as well. Recent data in patients with mild-to-moderate hypertension demonstrate that despite "efficacious" antihypertensive therapy, one-third to one-half of hypertensive patients may experience a significant decline in renal function. Cerebrovascular adaptation to hypertension consists of micro- and macrovascular disease leading to vascular dementia, or ischemic or hemorrhagic stroke. Cerebrovascular autoregulation, the mechanism by which cerebral blood flow is maintained, despite changes in arterial pressure, may be altered in hypertension.


Kinetics of angiotensin-converting enzyme inhibitors in renal failure. Although there is impressive documentation linking severe hypertension to renal insufficiency, corresponding data for mild-to-moderate hypertension are only now starting to emerge. As a result, it is only now becoming evident that a much larger portion of the hypertensive population could be susceptible to drug accumulation owing to renal insufficiency. Angiotensin-converting enzyme (ACE) inhibitor therapy routinely requires dosage adjustment in the instance of renal insufficiency, as all currently marketed ACE inhibitors are renally eliminated. Such dosage adjustments are usually considered a way to minimize side effects and to limit the duration of any induced hypotension. Dosage adjustment is usually considered at creatinine clearance levels between 30 and 60 ml/min. This is somewhat problematic, as physicians generally rely on serum creatinine determinations to assess renal function, and serum creatinine values are notoriously poor predictors of actual creatinine clearance. This is particularly true in the elderly population, where a greater disparity between the serum creatinine and creatinine clearance commonly exists, with moderate renal insufficiency frequently going unrecognized. Thus, the development of other ACE inhibitors eliminated via renal/hepatic routes may prove to be advantageous in that dosage adjustments might not be required in the setting of declining renal function, whether age-related or not. Fosinopril, a new phosphorus-containing ACE inhibitor, is administered as a prodrug and is hydrolyzed to the pharmacologically active diacid, fosinoprilat.(ABSTRACT TRUNCATED AT 250 WORDS)


Left ventricular hypertrophy and performance: therapeutic options among the angiotensin-converting enzyme inhibitors. The goal of antihypertensive therapy is the reduction in morbidity and mortality associated with high blood pressure. Despite our ability to reduce blood pressure, "standard" antihypertensive therapy has not produced a general decrease in coronary heart disease. This failure might be related to the adverse metabolic consequences of diuretics and beta-adrenergic receptor-blocking agents used in most clinical trials. In the hypertensive patient population, however, the principal physiologic abnormality is increased systemic vascular resistance. This increase in vascular tone leads to compensatory changes in cardiac function that result in left ventricular hypertrophy and diastolic filling abnormalities. Diastolic ventricular dysfunction is present in approximately 50% of asymptomatic hypertensive patients and might be a precursor of the syndrome of congestive heart failure with normal systolic ventricular function. In view of the prevalence of diastolic filling abnormalities in the hypertensive patient population, one should consider the effect of an antihypertensive drug on left ventricular function. In a comparison of the angiotensin-converting enzyme (ACE) inhibitors, captopril, lisinopril, and fosinopril, only fosinopril increased stroke volume, peak ejection rate, and peak filling rate, and decreased time to peak ejection rate. These favorable inotropic and lusitropic responses to fosinopril may reflect an effect on the myocardial renin-angiotensin cascade which is dependent upon the unique chemical structure of the fosinopril molecule.


Treatment of hypertension: the place of angiotensin-converting enzyme inhibitors in the nineties. The past decade has seen a shift in the strategy for hypertension treatment from stepped therapy--a highly structured monolithic series of steps--to recommendations for a more individualized selection of treatment. Severe hypertension is a clear indicator to bypass traditional steps. Demographic factors, such as age, gender, and race, are often cited, but have proved to be less helpful. Concomitant medical conditions and problems are very common and are more often the crucial determinants in the selection of antihypertensive therapy. Coronary artery disease, diabetes mellitus, heart failure, azotemia, asthma, and chronic obstructive pulmonary artery disease, anxiety, and depression are all common, and each has implications for the selection of antihypertensive therapy. Blood pressure reduction is a surrogate for reduction of cardiovascular risk, and therefore, consideration of concomitant medical problems has extended to left ventricular hypertrophy, obesity, mild hyperlipidemia, and insulin resistance, as additional risk factors in hypertension. Consideration of all these factors makes it possible to individualize antihypertensive therapy in most patients today.


Fosinopril: a new generation of angiotensin-converting enzyme inhibitors. Fosinopril, the first agent in a new chemical class of phosphorus-containing angiotensin-converting enzyme (ACE) inhibitors, has unique pharmacologic properties. Fosinopril administration leads to complete inhibition of plasma ACE activity for 12-24 h. In patients with normal renal function, approximately equal amounts of the drug are eliminated via the hepatic and renal routes. With worsening renal function, increasing amounts of fosinopril are eliminated hepatically. This dual elimination allows for the administration of fosinopril using the same starting dosage to any patient, regardless of renal function. Fosinopril may provide end-organ protection against the effects of hypertension and antihypertensive therapy; this drug potentially protects the kidney by increasing renal functional reserve, while maintaining cardiac left ventricular performance. Moreover, despite marked blood reductions, cerebral blood flow is maintained. Controlled trials show that fosinopril in single daily doses of 10-40 mg is efficacious and safe for the long-term treatment of hypertension. At these doses, favorable responses were seen in up to 80% of patients. Fosinopril is equally effective in elderly as well as younger patients and works in black as well as in nonblack patients. The incidence of adverse events does not differ significantly between fosinopril- and placebo-treated patients. Fosinopril represents a clinically useful agent for the treatment of hypertension.


Quadruple labeling of brain-stem neurons: a multiple retrograde fluorescent tracer study of axonal collateralization. Four different fluorochromes were injected into adjacent cervical spinal cord segments, 1 unique tracer per segment. Each tracer, Fluoro-Gold, Fast Blue, Diamidino Yellow dihydrochloride and Propidium Iodide, was taken up by axonal terminals and transported intra-axonally in a retrograde direction to the cell bodies. Some, though by no means all, of these axons were stem axons with terminals in 2, 3 or 4 of the injected spinal segments. Hence as many as 4 different fluorescent tracers could be discerned simultaneously within individual neuronal somata of origin using fluorescent microscopy. These results extend the possibilities for multiple interconnection determinations within the central nervous system. Specifically, the potential for individual neurons of a nucleus to project collateral branches of a stem axon to as many as 4 different central nervous system nuclei now can be studied simultaneously using these 4 fluorescent tracers.


Notes on a light and electron microscopic double-labeling method combining anterograde tracing with Phaseolus vulgaris leucoagglutinin and retrograde tracing with cholera toxin subunit B. Investigations of monosynaptic connections in the central nervous system have been hindered by the lack of compatible markers that can be used at both light and electron microscopic levels. In attempts to determine synaptic contacts between fibers originating in the substantia nigra and neurons projecting to the spinal cord, we have developed a double immunolabeling technique using anterograde transport of Phaseolus vulgaris leucoagglutinin (PHA-L) and retrograde transport of unconjugated cholera toxin subunit B (CTB). In this report, we describe technical modifications which consistently produced superior labeling together with adequate ultrastructural preservation of the tissue and discuss the advantages of the two tracers.


Biotin-dextran: a sensitive anterograde tracer for neuroanatomic studies in rat and monkey. The properties of a new anterograde tracer, biotin-dextran (BD), are reported. Iontophoretic or pressure injections of BD in the spinal cord of rats and monkeys revealed terminal-like BD label in many regions of the brain. The BD label was visualized by an avidin-biotin reaction combined with diaminobenzidine, with and without nickel enhancement. This reaction resulted in permanent label and revealed the fine morphology of terminal boutons and en-passant endings. Biotin-dextran is an excellent anterograde tracer that can also be visualized at the electron microscopic level, used in combination with other retrograde and anterograde tracers and with immunohistochemical labeling of neurotransmitters.


Ca2+ currents of fast blue-labeled superior cervical ganglion neurons. To investigate if Ca2+ currents of acutely isolated superior cervical ganglion (SCG) neurons are altered after dye labeling or ultraviolet (UV) exposure, SCG neurons were labeled by multiple injections of 2% fast blue (FB) (5-microliters total) into the submaxillary gland. Ca2+ currents of both labeled and unlabeled neurons were significantly depressed by 2-min exposure to UV, compared to labeled and unlabeled neurons not exposed to UV. The I-V curve of labeled neurons was shifted hyperpolarized after UV exposure. Tail-current activation curves of both labeled and unlabeled neurons were fitted to a double Boltzmann equation. In labeled neurons, 2-min exposure to UV produced a significant reduction of the amplitude of the more depolarized component as well as a hyperpolarizing shift of the half activation potential of both components of the activation curve. However, the short time (< 10 s) required to identify a target-specific neuron had no significant effect on the biophysical properties of Ca2+ currents of FB-labeled SCG neurons. Thus, use of this label in combination with the patch-clamp technique comprises a powerful approach to study membrane currents of target-specific neurons.


Intra-axonal Neurobiotin injection rapidly stains the long-range projections of identified trigeminal primary afferents in vivo: comparisons with HRP and PHA-L. Currently available methods for studying the morphology of physiologically characterized primary afferents are limited by difficulties inherent in impaling thin fibers and by the limited distances over which conventional tracers move during the course of a recording session. We have encountered an alternative method that overcomes these limitations. Neurobiotin (NB; Vector) injections into rat trigeminal (V) primary afferents in the brain stem or V ganglion provided rapid, long-range staining with recording and electrophoretic parameters that are commonly used to eject horseradish peroxidase (HRP) or Phaseolus vulgaris leucoagglutinin (PHA-L). When NB was injected into brain stem fibers responsive to vibrissal deflection with A-beta conduction velocities, collaterals were darkly stained in each of the 4 V subnuclei, as well as the cervical dorsal horn. Labeled fibers were also seen in the V root and peripherally in the infra-orbital nerve for a distance up to 15 mm from the injection site (30 mm total). Cell bodies in the ganglion were never labeled. When NB was injected into V ganglion cells with low- or high-threshold receptive fields and A-beta or A-delta conduction velocities, parent axons were stained in the V spinal tract to the level of the obex, and collaterals were visible in each of the 4 V subnuclei. Such long-range staining occurred within 4 h of tracer injection. HRP never stained brain stem fibers following ganglion cell injections and, when injected centrally with the same survival intervals used with NB, dark staining was limited to within 4 mm of the injection site. Unlike NB or HRP, PHA-L injections rarely produced useful data, either because of the high mortality accompanying attempts to achieve a 1-2 week survival period or because injected neurons were not recovered. Due to its rapid and robust transport, NB is a more convenient and reliable tracer than PHA-L for producing long-range staining of the projections of identified ganglion cells. Intracellular injection of NB also produces rapid Golgi-like staining of fibers over much greater distances than HRP under equivalent staining parameters.


Comparison of Borrelia burgdorferi isolated from humans and ixodid ticks in Hokkaido, Japan. Four spirochetal isolates (JEM1 to JEM4) were obtained from cutaneous lesions of patients manifesting erythema chronicum migrans in Hokkaido, Japan. In the protein profiles by SDS-PAGE and the reactivities with monoclonal antibodies (H5332 and H9724) by immunoblotting, all the human isolates were identical with the tick isolates from Ixodes persulcatus. These data indicate that I. persulcatus is an important vector of Lyme disease for humans in Japan.


Electron microscopic analysis of fluorescent neuronal labeling after photoconversion. Ultrastructural visualization of non-electron-dense fluorescent retrograde neuronal labeling was attempted by means of photo-oxidation. This procedure was used to convert the fluorescence of neurons labeled by the tracers propidium iodide, rhodamine latex microspheres and fluorogold into a stable diaminobenzidine reaction product. The ultrastructural study revealed an accumulation of electron-dense material in these cells both within lysosomes and scattered in the cytoplasmic matrix. Comparison with several different sets of control samples indicated that this material, on the basis of its amount, electron density and appearance, specifically represents the photoconversion reaction product. The effects of the intensity of the fluorescent labeling and of a prolonged photoconversion on the fine structural features of the reaction product are also described and discussed. The present findings indicate that photoconversion can be effectively applied to ultrastructural study of fluorescent retrogradely labeled neurons. The specificity of the photoconversion reaction product should be tested routinely for each fluorochrome and tissue sample.


Serological cross-reaction between intact and chemically modified lipopolysaccharides of O1 Vibrio cholerae Inaba and non-O1 V. cholerae bio-serogroup Hakata. Serological cross-reaction of intact as well as chemically modified LPS from O1 Vibrio cholerae 569B (Inaba) with non-O1 V. cholerae Hakata LPS, which contain alpha(1-->2)-linked N-acetyl perosamine-homopolymer constituting their O polysaccharide chain, was studied by passive hemolysis test by using their LPS as antigen for sensitizing sheep red blood cells (SRBC). The N-deacylation of the alpha(1-->2)-linked linear 3-deoxy-tetronyl perosamine-homopolymer constituting the O polysaccharide chain in 569B LPS resulted in virtual elimination of their serological reactivity with both homologous Inaba and heterologous Hakata antisera. Furthermore, when the resultant NH2 groups of the N-deacylated perosamine-homopolymers in 569B LPS were N-acylated with acetyl, propionyl or butanoyl groups, they markedly recovered the serological reactivity to a marked extent, in particular, their pronounced cross-serological reactivity with heterologous Hakata antiserum. These results are believed to be compatible with the interpretation that the Inaba antigen factor C possessed by the two bacteria studied is related to the common occurrence of the N-acyl groups, regardless of what the acyl groups are, residing in the perosamine residues of the perosamine-homopolymers constituting the O polysaccharide chain of their LPS.


Staining myelin and myelin-like degradation products in the spinal cords of chronic experimental allergic encephalomyelitis (Cr-EAE) rats using Sudan black B staining of glycol methacrylate-embedded material. A high-resolution light-microscopical (HRLM) technique is described to visualize myelin, and macrophages containing degradation products of myelin, in the spinal cords of chronic relapsing experimental allergic encephalomyelitis (Cr-EAE) rats. This HRLM technique was developed to optimalize the correlation between nuclear magnetic resonance (NMR) characteristics and histopathological images in this well-established animal model for multiple sclerosis (MS). Spinal cords were fixed by perfusion with a combination of cacodylate-buffered glutaraldehyde and formaldehyde, post-fixed in Dalton's fixative (containing osmium tetroxide), rinsed in water, processed in ethanol, acetone, and embedded in glycol methacrylate resin (Technovit 7100/HistoResin). Semi-thin sections were stained with Sudan Black B and counterstained with Cresyl Fast Violet, resulting in black staining of myelin and its degradation products, with blue/violet staining of demyelinated axons and other tissue elements. These dyes were selected with the aid of a numerical model of staining, which took both access and lipophilicity into account. The staining procedure is simple and highly reproducible. The resulting images are contrast rich, and combine excellent morphology with a high degree of lipid retention.


Hepatitis C: an update.

Hepatitis C: an update. The identification of the Hepatitis C virus using molecular cloning techniques, besides making the term Non-A Non-B Hepatitis obsolete, enables the development of specific assays for the detection of antibodies in HCV-infected individuals, thus making it possible to obtain sero-epidemiological data of the disease. The carriage of Hepatitis C antibody varies worldwide. The disease is most prevalent in intravenous drug abusers or haemophiliacs. Parenteral transmission is the most important route of transmission. Sexual, intra-familial and perinatal transmissions are uncommon. About 40% could be community-acquired (sporadic). Diagnostic tests include enzyme-linked immunosorbant (ELISA) anti-HCV assay, recombinant immunoblot assay, HCV-RNA by polymerase chain reaction and HCV-Ag. More than 50% of acute cases becomes chronic and runs a benign and indolent course. About 20% progress to cirrhosis and some of these develop hepatocellular carcinoma. Several published trials have consistently shown that treatment with interferon in some patients is useful. There is however a relapse rate of 50%. Further trials with interferon and other anti-viral agents like ribavirin are awaited for more effective treatment.


Retinoic acid receptor-beta: immunodetection and phosphorylation on tyrosine residues. Polyclonal (RP) and monoclonal (Ab) antibodies were raised against synthetic peptides (or fusion proteins) corresponding to amino acid sequences unique to human and mouse retinoic acid receptor-beta (RAR beta) isoforms. Antibodies directed against the A2 region [Ab6 beta 2(A2), Ab7 beta 2(A2), and RP beta 2(A2)], the D2 region [RP beta(D2)], or the F region [Ab8 beta(F)2, RP beta(F)1, and RP beta(F)2] were selected. The monoclonal and polyclonal antibodies directed against the D2 and F regions specifically immunoprecipitated and recognized by Western blotting all human and mouse RAR beta isoforms (mRAR beta 1, -beta 2, -beta 3, and -beta 4), produced in COS-1 cells transfected with expression vectors containing the corresponding RAR beta cDNA. Furthermore, in gel retardation assays, the monoclonal antibodies supershifted RAR beta protein-RA response element oligonucleotide complexes. Antibodies directed against the A2 region were specific for the RAR beta 2 isoform. The above antibodies allowed us to detect the presence of mRAR beta 2 proteins in mouse embryos and to show that their presence in embryonal carcinoma cells (F9 and P19 cell lines) is dependent upon RA treatment. The antibodies were also used to demonstrate that RAR beta proteins produced by transfection in COS-1 cells are phosphorylated. RAR beta 2 phosphorylation was not affected by RA treatment, whereas the phosphorylation of RAR beta 1 and RAR beta 3 isoforms was greatly enhanced by RA. We also show that, in contrast to RAR alpha 1 and RAR gamma 1, RAR beta 2 proteins contain phosphotyrosine residues and are only weakly phosphorylated in vitro by cAMP-dependent protein kinase. These results support our previous proposal that the various receptors have distinct functions in the RA-signaling pathway.


Insulin rapidly decreases insulin-like growth factor-binding protein-1 gene transcription in streptozotocin-diabetic rats. Insulin-like growth factor-binding protein-1 (IGFBP-1) can inhibit or potentiate IGF action. The biological activity of IGFBP-1 is determined by many factors, including its abundance in tissues and plasma, posttranslational modifications, and localization. IGFBP-1 levels in human plasma are highly regulated. They are increased after acute fasting and in diabetes, and are rapidly reversed by refeeding and insulin treatment, respectively. Similarly, IGFBP-1 mRNA is increased in the liver of severely diabetic and ketotic rats and decreased after 4 days of insulin treatment. Insulin rapidly decreases IGFBP-1 mRNA and IGFBP-1 transcription in rat hepatoma cells. The present study asks whether the increase in IGFBP-1 mRNA in diabetic rat liver reflects increased gene transcription, whether insulin decreases IGFBP-1 mRNA through a transcriptional or posttranscriptional mechanism, and whether this decrease is sufficiently rapid to account for the dynamic fluctuations in plasma IGFBP-1. Rats were injected ip with 100 mg/kg streptozotocin and used 7 days later when they were hyperglycemic and failed to gain weight, but were not ketotic. Hepatic IGFBP-1 mRNA levels were 13.6 +/- 5.3-fold greater in diabetic than control liver and decreased to the low levels in nondiabetic controls within 1 h after insulin treatment. In run-on transcription assays, IGFBP-1 transcription was 12.6 +/- 1.5-fold greater in nuclei from diabetic than control liver and decreased to low control levels by 1 h after insulin injection. Normalization of hepatic IGFBP-1 mRNA in insulin-treated diabetic animals did not require restoration of euglycemia. IGFBP-1 mRNA and IGFBP-1 gene transcription also were increased in the kidney of diabetic ketotic rats. We propose that the dynamic regulation of IGFBP-1 gene transcription in diabetes and after insulin treatment, by determining the availability of IGFBP-1 in tissues and plasma, may be a critical factor in the modulation of IGF action.


Monoclonal antibodies to two adhesive cell surface antigens (CD43 and CD59) with different distribution on hematopoietic and non-hematopoietic tumor cell lines. Two new murine monoclonal antibodies were prepared by hybridoma technique after immunization with the immature pluripotent leukemia cell line K562. The monoclonal antibody Bra10G (IgG2b) reacted in a non-lineage pattern with all examined hematopoietic neoplastic cell lines and peripheral blood cells (granulocytes, lymphocytes, erythrocytes) of healthy donors, with the exception of monoblastoid cell line U-937 and B lymphoma cell line Daudi. This monoclonal antibody immunoprecipitated an 18-20 kDa cell surface protein expressed also on the cell surface of examined non-hematopoietic (malignant glioma, melanoma and breast carcinoma) cell lines. These properties and the efficient inhibition of Bra10G binding to the cell surface of K562 cells by the reference CD59 monoclonal antibody (MEM-43) indicated that Bra10G belongs to the CD59 cluster of monoclonal antibodies which identify the human protectin molecule. The monoclonal antibody Bra7G (IgM) reacted with a 95 kDa cell surface protein expressed on hematopoietic cells (with the exception of erythrocytes) and was absent on the examined non-hematopoietic neoplastic cell lines. These data together with a partial inhibition of Bra7G binding by the reference CD-43 monoclonal antibody suggested the CD43 (leukosialin, sialophorin) specificity of this monoclonal antibody.


Monitoring of patients with non-seminomatous germ cell tumors of the testis by determination of alpha-fetoprotein and beta-human chorionic gonadotropin levels and by computed tomography. The results of a 7-year monitoring of 230 patients with non-seminomatous testicular tumors are reported with respect to the employment of radioimmunoanalysis of alpha-fetoprotein and beta-human chorionic gonadotropin levels and CT examinations of retroperitoneum and lungs. Prior to orchiectomy, elevated levels of at least one of these markers were found in 79% of patients. After orchiectomy, tumor marker levels were in 70.4% of patients in agreement with the results of CT examinations. After the completion of chemotherapy, in more than a half of patients normal tumor marker levels and positive CT findings were observed. These results were most often due to the presence of mature teratoma. In Stage I patients the advantages of tumor marker determinations and CT examinations in the early detection of tumor progression have fully been confirmed.


[Lactic acidosis, hyperamylasemia, and phenformin]. Phenformin is still used in the therapy of non-insulin dependent diabetes in association with sulphonylureas. The controindications to the use of this drug often are not kept in due consideration. It is therefore often possible to observe cases of irreversible lactic acidosis with lethal outcome. In this paper, three cases of lactic acidosis from phenformin administration are reported. Some characteristics of this clinical picture are underscored: accompanying ketoacidosis, hyperamylasemia. On the basis of their observation, the Authors conclude that those diabetic patients who can be treated with diet alone or with diet and sulphonylureas must not be treated with phenformin. Metformin is then proposed as a more flexible and safer drug.


Prevalence of antibodies to hepatitis C in dialysis patients and transplant recipients with possible routes of transmission. The prevalence of hepatitis C infection and possible predisposing factors was assessed in a renal unit. Of 343 patients at our renal dialysis centre, 37 (10.8%) were anti-HCV positive by a 1st-generation assay (ELISA, Ortho/Chiron) and confirmed positive in 35 (10.2%) with a 2nd-generation test (UBI, New York). Anti-HCV positivity was significantly associated with: duration of renal replacement therapy (P < 0.0001); quantity of blood transfused (P < 0.002); duration of hospital haemodialysis (P = 0.0001); duration with a functional renal transplant (P = 0.039); and aspartate aminotransferase (P < 0.0001). Logistic regression determined the following variables to be independent risk factors: duration of renal replacement therapy with a relative risk of 34.3 for 5-9 years and 87.4 when the duration was in excess of 10 years; renal transplant for less than 1 year (relative risk of 5.0); transfusion in excess of 50 units of blood (relative risk of 11.6). Clinical assessment of anti-HCV-positive patients revealed peripheral signs of chronic liver disease in 40%, hepatomegaly in 34%, and splenomegaly in 9%. This prevalence of hepatitis C infection is similar to other European and North American centres, but contrasts with low prevalence rates reported from dialysis populations in the UK. It adds further support for routine screening of blood and possibly organ donors and implementation of further infection control measures in dialysis centres.


Reduced GAP-43 message levels are associated with increased neurofibrillary tangle density in the frontal association cortex (area 9) in Alzheimer's disease. We previously suggested the hypothesis that defective neuronal plasticity is a major neurobiological deficit causing the dementia of Alzheimer's disease (AD). We used message levels of the growth-associated protein, GAP-43, as a marker of axonal plasticity to examine the hypothesis of defective neuronal plasticity in AD. When all AD cases are combined, the average level of GAP-43 message in area 9 of the AD frontal association cortex was not significantly different from the level in the comparably aged control cortex. Differentiation of AD cases on the basis of neurofibrillary tangle (NFT) density revealed that in AD cases with high tangle density average GAP-43 message level was reduced fivefold relative to levels in AD cases with low NFT density. AD cases with low neurofibrillary tangle density had levels of GAP-43 message that were not significantly different from the levels of normal controls. Differentiation of AD cases on the basis of neuritic plaque density did not indicate as strong a relationship to GAP-43 message level. The association between neurofibrillary tangle density and GAP-43 message level suggests the hypothesis that neurofibrillary tangles may reduce GAP-43 expression. Data of others show a relationship between high NFT density and reduced levels of synaptophysin-like immunoreactivity and reduced cerebral glucose metabolism. These data combine to suggest a set of AD cases with high NFT density, reduced axonal plasticity, reduced synaptic density, and reduced cerebral glucose metabolism--all variables that may be directly related to the functioning of the brain.(ABSTRACT TRUNCATED AT 250 WORDS)


Localization of preprogalanin mRNA in the monkey hippocampal formation. The existence of neurons expressing preprogalanin mRNA in the monkey hippocampal formation was demonstrated using in situ hybridization of a radio-labelled cRNA probe transcribed from human preprogalanin cDNA. Specific hybridization occurred in neurons of the hilus of the dentate gyrus, fields CA1-3 in Ammon's horn, subiculum, presubiculum, parasubiculum and occasionally in neurons of the entorhinal cortex. These findings suggest that galanin is synthesized by neurons intrinsic to the monkey hippocampal formation.


Phase shifting of the circadian rhythm in the eye of Bulla by inhibition of chloride conductance. Inhibition of Cl- conductance has been previously found to shorten the free-running period of the circadian eye rhythm of the marine snail Bulla gouldiana. In this study, we describe a phase-response curve for 6-h pulses of Cl(-)-free seawater, consisting only of phase advances with the largest in the late subjective night (1.5 h) and smaller phase shifts in the late subjective day. Intracellular recordings revealed that at both circadian times retinal pacemaker neurons hyperpolarized in response to the removal of extracellular chloride. Since previous studies indicate that membrane potential generates both phase advances and delays it seems unlikely that the action of chloride removal is mediated exclusively by the change in membrane potential.


Activation of a metabotropic excitatory amino acid receptor potentiates A2b adenosine receptor-stimulated cyclic AMP accumulation. Incubation of guinea-pig cerebral cortical slices with the excitatory amino acid agonists DL-alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) or N-methyl-D-aspartate (NMDA) failed to significantly alter either basal or 5'-N-ethylcarboxamidoadenosine- (NECA-)stimulated cAMP levels. Quisqualate, L-glutamate and DL-1-aminocyclopentane-trans-1,3- dicarboxylate (t-ACPD) were also without effect on basal levels of cAMP but increased NECA-stimulated levels of cAMP to approximately 190, 220 and 290% of control levels, respectively. Analysis of t-ACPD concentration-response data for the potentiation of NECA-stimulated cAMP elevations and the stimulation of phosphoinositide turnover gave EC50 values of 51 and 107 microM, respectively. The enhancing effect of t-ACPD on cAMP levels was maintained for up to 40 minutes as was the stimulation of phosphoinositide turnover. We conclude that the cAMP response to A2b adenosine receptor stimulation is augmented by the rigid glutamate analogue t-ACPD, possibly through the action of products of phosphoinositide turnover.


m4 muscarinic receptor subtype activates an inwardly rectifying potassium conductance in AtT20 cells. The modulation of an inwardly rectifying potassium conductance by muscarinic receptor stimulation was studied in the AtT-20 pituitary cell line, using the whole-cell patch-clamp technique. Only m4 mRNA was detected in these cells, thus, it is assumed that the actions of muscarinic receptor stimulation are mediated by the m4 receptor. AtT-20 cells express a slowly activating inwardly rectifying potassium conductance. Application of acetylcholine (ACh), resulted in an atropine sensitive, reversible increase in inwardly rectifying current. The ACh-induced current differed from the current recorded in control, in that it was fast activating, while the control current was slowly activating. Inclusion of GTP gamma S in the patch pipette activated an inward current with characteristics similar to the ACh-induced current, and the ACh-induced current response could be inhibited by pre-incubation with pertussis toxin (PTX). It is concluded that the m4 muscarinic receptor is coupled to an inwardly rectifying potassium conductance via a PTX sensitive G-protein.


Suppression of a calcium current by CNQX and kynurenate. During our investigation of a sustained high voltage-activated (HVA) calcium current in retinal horizontal cells, we found that the glutamate receptor antagonists CNQX and kynurenate but not AP7 reversibly reduced the peak amplitude of the HVA current. Changes in the HVA current kinetics or activation voltage were not apparent; there was only a reduction in the peak current. The novel effects of these antagonists on HVA calcium currents reported here could have an impact on many studies involving glutamatergic synaptic transmission.


Suppression of a ligand operated membrane current by the epileptogenic agent pentylenetetrazol in oocytes of Xenopus laevis after injection of rat brain RNA. The effects of the epileptogenic substance pentylenetetrazol (PTZ) on ligand operated membrane channels were studied. For this purpose serotonin (5-HT) sensitive channels were expressed in RNA injected oocytes of Xenopus laevis. With simultaneous application of both substances, the response to 5-HT was reduced and eventually abolished by PTZ with increasing concentrations (5-100 mM). A reduction of the 5-HT response also appeared when PTZ was applied in various intervals (15 and 240 s) before 5-HT. It may be assumed that PTZ produces a component of its epileptogenic effect by acting on ligand operated membrane channels.


Tunicamycin-induced inhibition of functional expression of glutamate receptors in Xenopus oocytes. The effects of tunicamycin, a specific inhibitor of N-linked glycosylation, on functional expression of glutamate receptor subtypes were investigated in RNA-injected oocytes. In the presence of tunicamycin the expression of ligand-operated receptors sensitive to kainate, alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) and quisqualate were completely blocked. The inhibitory effect was reversible after removal of tunicamycin from the culture medium.


Aminooxyacetic acid causes selective neuronal loss in layer III of the rat medial entorhinal cortex. Aminooxyacetic acid (AOAA) was used to produce a selective lesion in the rat entorhinal cortex (EC). As assessed 7 days following the injection of AOAA (75 micrograms/0.75 microliter) into the EC, neuronal loss in layer III of the medial EC, particularly in its ventral portion, was consistently observed in Nissl-stained horizontal sections. This selective neurodegeneration was seen even when AOAA was injected laterally or in deeper layers. Behavioral seizures occurred between 2 and 4 h after the AOAA injection. AOAA-induced EC lesions may provide experimental models for the study of human diseases in which the EC, particularly layer III neurons, is involved.


1,10-Diaminodecane and 1,12-diaminododecane block NMDA receptor currents by an open channel mechanism. In whole-cell recordings from cultured rat hippocampal neurons (VH = -60 mV), 1,10-diaminodecane (DA10) and 1,12-diaminododecane (DA12) produced a concentration-dependent block of NMDA-induced current (IC50 = 30 and 7 microM, resp.). In contrast, the diamines failed to affect AMPA and kainate currents. The inhibition of NMDA currents was highly voltage-dependent and was substantially relieved at positive holding potentials. In outside-out patches, DA10 and DA12 produced a voltage-dependent flickery block of NMDA-activated single-channel currents. These results indicate that DA10 and DA12 antagonize NMDA responses via an open channel mechanism. DA10 and DA12 have been proposed to be inverse agonists at the spermine facilitatory site on the NMDA receptor. However, the channel blocking effects of the diamines complicate the interpretation of their actions at this site.


Nitric oxide synthase immunoreactivity in rat spinal cord. Immunoreactivity to nitric oxide synthase (NOS-IR) and choline acetyltransferase (ChAT-IR) was detected in the adult rat spinal cord using the avidin-biotin-peroxidase technique. Intensely stained NOS-positive neurons with cell processes were observed in the intermediolateral cell column of the thoracic and sacral segments and around the central canal of all segments. These areas also contained ChAT-IR neurons. A number of small- to medium-sized NOS-IR cells were noted in the superficial and deeper laminae throughout the entire cord. NOS-IR was not detected in the ventral horn motoneurons, which were, however, ChAT-IR. The results indicate that NOS-IR is present in autonomic preganglionic neurons and in selected neurons in the dorsal horn and lamina X, but appears to be absent in motoneurons.


Evidence that CHP100 neuroblastoma cell death induced by N-methyl-D-aspartate involves L-arginine-nitric oxide pathway activation. Evidence suggests that nitric oxide (NO) may mediate, at least in part, excitotoxic effects of excessive N-methyl-D-aspartate (NMDA) receptor activation both in vivo and in vitro. In the present experiments, NMDA-induced excitotoxicity has been studied in CHP100 neuroblastoma cell cultures. Application of NMDA (0.25-1.5 mM) produced concentration-dependent cell death. These effects were antagonized by co-application of dizocilpine (MK801), a selective and non-competitive NMDA receptor complex antagonist. Protection from NMDA-induced lethal effects was also afforded by N omega-nitro-L-arginine methyl ester, a potent NO-synthase inhibitor, and by hemoglobin, a NO-trapping agent. In addition, substitution of L-arginine, normally present in the exposure solution with its D-isomer, abolished the cell death induced by the excitotoxin. In conclusion, the present experiments support the suggestion that excitotoxic effects induced by NMDA receptor stimulation involve L-arginine-NO pathway activation.


Distribution of NADPH-diaphorase-positive nerves in the uterine cervix and neurons in dorsal root and paracervical ganglia of the female rat. Nitric oxide synthase (NOS) was selectively stained in nerve fibers of the uterine cervix and neurons of the paracervical (PG) and dorsal root ganglia (DRG) by NADPH diaphorase histochemistry. In the cervix, numerous NADPH-diaphorase-positive nerve fibers were observed in the myometrium, endometrium and around arteries. In addition, a subpopulation of neurons within ganglia that innervate the cervix, i.e., the PG and DRG, were NADPH-diaphorase positive; thus the fibers in the cervix could be sensory and/or autonomic. NADPH-diaphorase/NOS localization identifies sites where nitric oxide (NO) can be synthesized. Since NO relaxes vascular and nonvascular smooth muscle, the prevalence and anatomical localization of NADPH-diaphorase-positive fibers suggest that they could influence functions of the uterine cervix.


Neonatal capsaicin treatment prevents the development of the thermal hyperalgesia produced in a model of neuropathic pain in the rat. Loose ligation of the sciatic nerve with 4-0 chromic gut sutures in rats produces behavioral evidence of neuropathic pain. In the present experiments we examined the involvement of capsaicin-sensitive afferents in mediating the thermal hyperalgesia produced by this model. Male Sprague-Dawley rats, treated as neonates (within 48 h of birth) with capsaicin (50 mg/kg, s.c.) or vehicle, were used at 16-18 weeks of age. Chromic gut sutures (4-0) were tied around the left sciatic nerve and withdrawal latencies of both hind paws to radiant heat were determined on postoperative days 3, 5, 10 and 20. Whereas there was a pronounced thermal hyperalgesia which lasted for up to 20 days in vehicle-treated rats, there was no evidence of thermal hyperalgesia in capsaicin-treated rats. There was no difference in baseline (pre-surgery) withdrawal latencies between the two groups. Radioimmunoassay revealed that there was a significant depletion of substance P (43.8%) and calcitonin-gene-related peptide (72.6%) in the lumbar spinal cord of neonatal capsaicin-treated rats compared to vehicle-treated rats. These results demonstrate that the chromic gut-induced thermal hyperalgesia is mediated by capsaicin-sensitive afferents and suggest that central mechanisms which process and control the reflex response to heat are different than mechanisms involved in thermal hyperalgesia.