Effects of granulocyte colony-stimulating factor, interleukin-1 alpha, and interleukin-6 on prolonged myelosuppression induced by nimustine hydrochloride in rats. The myelorestorative effects of granulocyte colony-stimulating factor (G-CSF), interleukin-1 alpha (IL-1 alpha) and interleukin-6 (IL-6) were studied in F-344 rats which had been treated with cyclophosphamide (CY), carboplatin (CBDCA), or nimustine hydrochloride (ACNU). In CY- or CBDCA-pretreated rats, significantly higher peripheral white blood cell (WBC) count was observed in animals treated with G-CSF and IL-1 alpha, while the platelet (PLT) count was elevated by IL-6 treatment. All of the cytokines had little effect on the hemoglobin (HB) value. Animals treated with ACNU had prolonged myelosuppression. Treatment of these animals with G-CSF and IL-1 alpha significantly enhanced the recovery of HB value as well as WBC count. Higher PLT counts were observed in treated groups, but a statistical difference was not evident. Combination therapy with G-CSF and IL-1 alpha, G-CSF and IL-6, or IL-1 alpha and IL-6 did not have any significant beneficial effects on the peripheral blood cell count in ACNU-pretreated rats over single agent therapy. Conversely, the combination of IL-6 and G-CSF had an unfavorable effect on HB and PLT levels. In rats which received multiple doses of ACNU, G-CSF treatment exhibited a beneficial effect on WBC, HB, and PLT levels, the most prominent on the HB value. These findings suggest that treatment with hematopoietic cytokines may be most beneficial when combined with anticancer drugs which are known to cause prolonged myelosuppression.


Protein kinase C stimulates dense tubular Ca2+ uptake in the intact human platelet by increasing the Vm of the Ca(2+)-ATPase pump: stimulation by phorbol ester, inhibition by calphostin C. The effects of protein kinase C (PKC) on Ca2+ transport were investigated in human intact platelets. The indicator quin2 was used to measure the free cytoplasmic Ca2+ concentration ([Ca2+]cyt) and to search for possible PKC effects on the Ca(2+)-ATPase extrusion pump located in the plasma membrane. The Ca2+ indicator chlorotetracycline (CTC) was used to study PKC effects on the dense tubular Ca(2+)-ATPase uptake pump. The activity of PKC was stimulated by phorbol 12-myristate 13-acetate (PMA) and was inhibited with calphostin C. Neither PKC activation nor inhibition had any effect on [Ca2+]cyt or the Ca2+ extrusion pump. Substantial activation of the dense tubular pump was observed with PMA. In resting platelets bathed in 2 mM external Ca2+ giving [Ca2+]cyt = 102-106 nM, activation of PKC by PMA (100 nM) increases the rate and extent of dense tubular Ca2+ uptake to 1.62 +/- 0.35 and 1.25 +/- 0.3 times control value (respectively). The Vm of the dense tubular pump was measured by using ionomycin to manipulate [Ca2+]cyt. It is shown that PMA increases the Vm by a factor of 1.7 +/- 0.4 but has no effect on the Km value (= 180 nM). An unexpected finding was that PKC activity supports a portion of the basal activity of the dense tubular Ca2+ pump in resting platelets. Preincubation with the inhibitor calphostin C (100 nM) decreases the rate and extent of dense tubular Ca2+ uptake in resting platelets by 38 +/- 5% and 29 +/- 21% (respectively). This is due to a 28 +/- 9% decrease in the Vm of the dense tubular pump. This suggests that there is a low level of stimulation of dense tubular Ca2+ pump mediated by PKC in resting platelets.


The role of anion channels in the mechanism of acrosome reaction in bull spermatozoa. The involvement of anion channels in the mechanism of the acrosome reaction (AR) was investigated. The AR was induced by Ca2+ or by addition of the Ca2+ ionophore A23187. The occurrence of AR was determined by following the release of acrosin from the cells. In order to investigate the role of anion channels in the AR, several anion-channel inhibitors were tested, mainly DIDS (4,4'-diisothiocyanostilbene-2,2'-disulfonic acid). Other blockers, like SITS (4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid), furosemide, probenecid and pyridoxal 5-phosphate, were also tested. We found that DIDS binds covalently to sperm plasma membrane in a time- and concentration-dependent manner. Maximal binding occurs after 2 h with 0.3 mM DIDS. DIDS and SITS inhibit AR in a concentration-dependent manner. The IC50 of DIDS and SITS in the presence of A23187 is 0.15 and 0.22 mM, respectively. Tributyltin chloride (TBTC), an Cl-/OH- exchanger, partially overcomes DIDS inhibition of the AR. HCO3- is required for a maximal acrosin release and Ca(2+)-uptake, in the presence or absence of A23187. It is known that HCO3- activates adenylate cyclase and therefore, increases the intracellular level of cAMP. The inhibition of the AR by DIDS decreases from 95 to 50% when (dibutyryl cyclic AMP (dbcAMP) was added, i.e., HCO3- is no longer required while elevating the level of cAMP in an alternative way. Moreover, we show that the stimulatory effect of HCO3- on Ca(2+)-uptake is completely inhibited by DIDS. We conclude that DIDS inhibits AR by blocking anion channels, including those that transport HCO3- into the cell.


Sterol specific inactivation of gramicidin A induced membrane cation permeability. Channel inactivation, a time-dependent decrease of the high-cationic permeability induced by gramicidin A, has been found both in cholesterol containing red blood cell membranes and lipid bilayers (Schagina et al., (1989) Biochim. Biophys. Acta 978, 145-150). The rate of channel inactivation strongly depends on the phospholipid to cholesterol molar ratio of the membrane. The channel inactivation is suggested to be the result of an interaction between gramicidin and cholesterol in a stoichiometry of 1:5. Cholesterol dependent inactivation is shown also for gramicidin A analogs: tryptophan-N-formylated gramicidin A, o-pyromellitilgramicidin and malonylbisdesformylgramicidin. When cholesterol in the membrane is substituted by sitosterol, the inactivation of gramicidin-induced cation permeability is preserved, while in the presence of either ergosterol or 7-dehydrocholesterol no indication of the channel inactivation is observed. Thus, the structure of the 'B', ring, not the apolar tail of the sterol molecule, appears to be important in the inactivation process.


Proteolytic modification of raw-starch-digesting amylase from Bacillus circulans F-2 with subtilisin: separation of the substrate-hydrolytic domain and the raw substrate-adsorbable domain. Raw starch-digesting amylase (BF-2A, 93,000 Da) from Bacillus circulans F-2 was converted into two components during digestion with subtilisin. The two components were separated and designated BF-2A' (63 kDa) and BF-2B (30 kDa), respectively. BF-2A' exhibited the same hydrolysis curve for soluble starch as the original amylase (BF-2A). Moreover, the catalytic activities of original and modified enzymes were indistinguishable in Km, Vmax and in their specific activity for soluble starch hydrolysis. However, its absorbability and digestibility on raw starch was greatly decreased. Furthermore, the enzymatic action pattern on soluble starch was differed greatly from that of BF-2A. The stability of the enzymes decreased below pH 5.5 and at 50 degrees C, while it was quite stable even at pH 12. On the other hand, the smaller peptide (BF-2B) could be adsorbed onto raw starch. From these results, it is suggested that the larger peptide (BF-2A') has a region responsible for the expression of the enzyme activity to hydrolyze soluble substrate, and the smaller peptide (BF-2B) plays a role on raw starch adsorption and also contributes to the original enzyme-to-enzyme stabilization. A proposed model of the raw-starch-digesting enzyme from this strain is extensively discussed.


Interaction of catalytic-site mutants of Bacillus subtilis alpha-amylase with substrates and acarbose. The interactions of the three catalytic-site mutants of Bacillus subtilis alpha-amylase/(DN176 [Asp-176----Asn], EQ208 [Glu-208----Gln] and DN269 [Asp-269----Asn]) with substrates and a pseudo-oligosaccharide inhibitor, acarbose, have been studied by means of difference absorption spectroscopy and affinity chromatography. The addition of maltopentaose or soluble starch to the inactive mutant enzymes mostly resulted in difference spectra characteristic of tryptophan perturbation, enabling determination of the dissociation constants. The results show that conversion of Glu-208 to Gln greatly enhanced substrate binding, implying that Glu-208 interacts unfavorably with the substrate's ground state, preventing its optimal fit to the active site. The affinity for acarbose was greatly reduced in DN269 and EQ208, but less so in DN176, suggesting that Asp-269 and Glu-208 are more important than Asp-176 in stabilizing the transition state. These results are consistent with Glu-208 and Asp-269 being the key catalytic residues, as proposed for Taka-amylase A.


Effect of benzodiazepine ligands on Ca2+ channel currents in Xenopus oocytes injected with rat heart RNA. Voltage-dependent Ca2+ channel (VDCC) currents have been measured in Xenopus oocytes injected with heart RNA purified from 7 day old rats, using voltage clamp technique. The currents were evoked by depolarizing voltage steps, using Ba2+ as charge carrier. Electrophysiological analysis of the current revealed two components: a slow, L-type, dihydropyridine (DHP)-sensitive current and a transient, DHP-insensitive, current. The benzodiazepine (BZ) ligands diazepam and Ro5-4864 decreased the current with micromolar affinity, with potency order of Ro5-4864 greater than diazepam greater than clonazepam. The central antagonist Ro15-1788 did not interfere with the effect of these drugs, thus excluding the possible involvement of the "central type" receptor. The slow current that was increased about 3 fold in the presence of 0.5 microM Bay K 8644, was less potentiated when previously treated with Ro5-4864 or diazepam. Current-voltage relation of the peak inward current and the steady state activation curve showed a small shift towards negative potentials in the presence of 50 microM diazepam. It is concluded that the benzodiazepine ligands block DHP-sensitive voltage dependent Ca2+ channels with a very marginal effect on the transient, DHP-insensitive current. Also it is emphasized that Xenopus oocytes can serve as a useful model system to study the pharmacology of these important drugs on cardiac Ca2+ channels.


Endothelin increases [Ca2+]i in rat pancreatic acinar cells by intracellular release but fails to increase amylase secretion. In individual fura-2 loaded cells of rat pancreatic acini endothelin-1 (ET-1) (10-50 nM) induced sustained oscillations in [Ca2+]i. At higher concentrations a larger, but transient increase in [Ca2+]i was observed, which was largely unaffected by removal of extracellular Ca2+. ET-1 induced the release of Ca2+i from the same store as cholecystokinin (CCK), but with less potency. At concentrations of endothelin which transiently increased Ca2+, ET-1 increased the accumulation of inositol phosphates. Specific binding sites for 125I-endothelin were demonstrated on rat pancreatic acini. A single class of binding sites was identified with an apparent Kd 108 +/- 12 pM and Bmax of 171 +/- 17 fmol/mg for ET-1. The relative potency order for displacing [125I]ET was ET-1 greater than ET-2 greater than ET-3. In contrast to CCK and the non-phorbol ester tumour promoter Thapsigargin (TG) which induce both transient and sustained components of [Ca2+]i elevation, ET-1 failed to increase amylase release over the range 100 pM-1 microM.


Signal mechanisms in the activation of basophils and mast cells. While several important biochemical steps have been observed to occur in basophils and mast cells, there remain some very important links to be established. Notably, it is important to understand the exact role of the IgE receptor and the need for its aggregation. The description of the GTP-binding protein involved in IgE-mediated signal transduction is necessary, as is a description of the proteins involved in calcium movement. Control mechanisms remain unclear, and the length of the signal transduction pathway has yet to be defined. Much of the data points to multiple requisite pathways, and yet only calcium elevations have a relatively well-demonstrated pro-degranulatory role. Until some of these events are understood in detail, the means by which cell sensitivity is regulated may remain a mystery. Finally, it will be important to determine if mast cells and basophils use distinctive IgE-mediated signal transduction pathways.


Hemopoietic growth factors regulate basophil function and viability. Table 1 summarizes the activities of hemopoietins on immature and mature basophils. IL-3, GM-CSF, and IL-5 enhanced basophil histamine release and in-vitro survival, while G-CSF, M-CSF, and IL-4 had no enhancing activities at all. In addition, IL-3 and GM-CSF induced basophil chemotaxis. Although IL-3 was proved to have proliferative and differentiation-inducing activities on basophils in vitro and in vivo, the other two factors (GM-CSF and IL-5), which are also capable of regulating basophil functions and viability, might potentially participate in proliferation or differentiation of human basophils, but this hypothesis remains without sure foundation. Studies on the roles of both factors in human basophilopoiesis need extension. The fact that IL-3, GM-CSF, and IL-5 regulate basophil function and viability in vitro demonstrates possible mechanisms for the regulation of basophil function and viability in IgE-mediated reactions (especially in late-phase reactions) in vivo by these factors. Since these factors could be produced by antigen-stimulated T-cells or epithelial cells through the inflammatory process, these factors could participate in exacerbation and prolongation of hypersensitivity reactions by inducing the migration and enhancing functions and the lifespan of basophils.


Augmented secretion of lysosomal enzyme into pancreatic juice after short term obstruction of the pancreatic duct in rats. To find out if and when lysosomal enzymes are excreted into pancreatic juice in physiological and pathological conditions, the changes in the secretion of cathepsin B into pancreatic juice were investigated in 66 Wistar rats with cannulation of common pancreatic-biliary duct and common bile duct, and infusions of caerulein and secretin. In a separate experiment ducts were cannulated and secretin infused as before, but in one group the ducts were "obstructed" and in another they were allowed to remain patent. Obstruction of the pancreatic duct for three hours caused a moderate significant rise in serum amylase activity. Cathepsin B activity in the pancreatic subcellular fractions was redistributed, and the amount of cathepsin B increased. In rats with obstructed ducts the secretion of cathepsin B and other lysosomal enzymes that were stimulated by caerulein was significantly greater than in the animals in which the ducts remained patent. Lysosomal enzymes associated with zymogen granules are secreted into pancreatic juice together with digestive enzymes after stimulation by gut hormones, and they may have pathophysiological roles in pancreatic juice.


Techniques for clearing ovules for studies of megagametophyte and early postfertilization development in Rhododendron. Using ovule clearing, more than 33,600 ovules of Rhododendron nuttallii T. W. Booth (Ericaceae) were examined for megagametophyte and early postfertilization stages at daily intervals from anthesis until 3 weeks after pollination. Pretreatments with amyloglucosidase to digest integumentary and gametophyte starch and Stockwell's bleach to remove tannins from the integumentary epidermis were necessary. Ovules were cleared by a combination or modifications of Herr's four-and-a-half or Stelly's hemalum-methyl salicylate techniques and were observed using differential interference contrast optics. The method proved suitable for large-scale quantitative studies of ovule development and fertilization. A general protocol is suggested as a starting point for ovule clearing studies.


A mixture of paraphenylenediamine and imidazole: its effect on the extraction of lipid droplets during electron microscopy staining. To prevent extraction of lipids during a double staining procedure for electron microscopy, the tissue slices, double fixed with glutaraldehyde and osmium tetroxide to preserve microvesicular lipid droplets in the cytoplasm, were immersed for 2 hr in veronal buffer (pH 9.0) containing 0.5% p-phenylenediamine and 0.5% imidazole immediately after postfixation. The stained sections of the immersed tissue slice showed blackened, well circumscribed lipid droplets similar to those in corresponding unstained sections. Moreover, highly contrasting features of the cellular architecture could be visualized with the double stained, as well as routinely prepared sections.


An update on protein stains: amido black, coomassie blue G, and coomassie blue R. Samples of amido black, Coomassie blue G, and Coomassie blue R obtained over a number of years were tested for dye content, impurities, and effectiveness for staining proteins after polyacrylamide gel electrophoresis and for protein dye-binding assays. Some impurities produced reactions resembling metachromasia with specific proteins, although instances of true metachromatic staining are also reported. Several simple assays are given for determining dye content and relative levels of impurities. Recommendations are made for selecting batches of commercial dyes which are most likely to perform satisfactorily.


A comparison of substrates for human umbilical vein endothelial cell culture. We have studied culture conditions which facilitate the growth of stable, non-proliferating, human umbilical vein endothelial cell (HUVEC) monolayers. Gelatin and fibronectin coatings, with or without glutaraldehyde cross-linking, on both plastic and glass were investigated for initial attachment of HUVEC and growth characteristics. The presence during culture of intercellular (IC) junctions demonstrated by silver staining, expression of platelet endothelial cell adhesion molecule-1 (PECAM-1) and maintenance of a cobblestone appearance of HUVEC monolayers were assessed over time. Glutaraldehyde cross-linked fibronectin and gelatin coatings on glass and glutaraldehyde cross-linked gelatin or untreated fibronectin coatings on plastic served as good substrates for short term culture. Long term (20 days) cultures of HUVEC which maintained silver and PECAM-1 staining of IC junctions and a cobblestone appearance could be achieved if glutaraldehyde cross-linked gelatin coatings on glass were used as substrates.


Epitope mapping by photobleaching fluorescence resonance energy transfer measurements using a laser scanning microscope system. The donor photobleaching method (T. M. Jovin and D. J. Arndt-Jovin. 1989. Annu. Rev. Biophys. Biophys. Chem. 18:271-308.) has been adapted to an ACAS 570 (laser scanning microscope) system to measure fluorescence resonance energy transfer (FRET) on individual human peripheral blood T cells. Photobleaching was completed in approximately 100 ms in our case and it followed double-exponential kinetics. The energy transfer efficiency (E) was approximately 20% between the CD4 epitopes OKT4-FITC and Leu-3a-PE as well as between OKT4E-FITC and OKT4-PE. E was approximately 8% between OKT4-FITC and Leu-4-PE (alpha CD3) and barely detectable (approximately 4%) from OKT4-FITC to Leu-5b-PE (alpha CD2). The E values obtained by the photobleaching method were highly reproducible both in repeated measurement of identical samples and in experiments with different batches of cells and were in agreement with the flow cytometric donor quenching measurements. As expected, E measured between primary and secondary layers of antibodies increased (from approximately 14% to approximately 28%) when F(ab')2 fragments were substituted for whole antibody molecules as the donor. On a T cell line we mapped the distance between the idiotypic determinant of the T cell receptor (TcR) and the Leu-4 epitope of CD3 as proximal as E = 28%, as compared to E = 4% between a framework TcR epitope and Leu-4. In the latter case, however, approximately 40% less Leu-4 was bound suggesting that the antigen binding site of TcR is in close proximity with one of the two CD3 epsilon chains, which hence are not equivalent.


Constant helical pitch of the gramicidin channel in phospholipid bilayers. X-ray diffraction has been applied in measuring the helical pitch of the gramicidin channel in oriented bilayers of dilauroylphosphatidylcholine (DLPC) and dimyristoylphosphatidylcholine (DMPC) at a polypeptide concentration of 9.1 mol %. The diffraction data show the helical pitch of gramicidin to be 4.7 +/- 0.2 A in both gel and liquid-crystalline phase bilayers, with and without monovalent cations. In addition, the width of the reflection due to the pitch of the helical gramicidin channel is consistent with a five turn helix.


Evidence that endothelium-dependent relaxation to histamine in the rat common carotid artery is mediated by EDRF. The mechanism of histamine-induced relaxation in the isolated rat common carotid artery was analysed. Histamine (3 x 10(-7)-10(-4) mol/l) caused a concentration-dependent relaxation of the artery. Indomethacin, a cyclo-oxygenase inhibitor, and diethylcarbamazine, a lipoxygenase inhibitor, did not affect the relaxant response of the artery to histamine. After removal of the vascular endothelium the histamine-induced relaxation was strongly reduced. Moreover, hemoglobin and methylene blue, inhibitors of endothelium-derived relaxing factor (EDRF), prevented or reversed the relaxant effect of histamine. These findings confirm that the histamine-induced relaxation is to a greater extent endothelium dependent. It is concluded that the endothelium-dependent component in the relaxant response of the rat common carotid artery to histamine results from the release of EDRF.


Phosphoamino acid phosphatases in normal and cancerous tissues of the human uterus, cervix and ovary. The activities of phosphoamino acid phosphatases were measured in human myometrium and fibroma and normal and cancerous tissues of the cervix and ovary. Phosphoserine and phosphothreonine phosphatases were detected only in myometrium and fibroma and the values were relatively low. Phosphotyrosine phosphatase activities in myometrium and fibroma fell within a similar range; this was also the case for ovary and ovarian carcinoma, whereas values for cervical tumours were significantly higher than for normal cervix. Activities of phosphotyrosine phosphatase in serum from patients with cervical or other tumours were, in most cases, within the range of values obtained for normal serum.


[Heart volume overload in the rabbit during growth via chronic arteriovenous fistula. Evaluation of an original model]. A heart volume overload model was developed in male New Zealand rabbit nine weeks old, using femorofemoral or jugulo-carotid fistulae, producing 50% increase of the mean cardiac output. An evaluation was performed after 2 and 11 weeks. It was evaluated by dimensional echocardiogram and doppler system flow measurement at the aorta ring, angiography and right cardiac pressure recording. This hemodynamic analysis was completed by a heart and fistulae histopathological study. In some volume overload cases, an increasing volume index parameter was described. Two weeks later an increase of the cardiac frequency and a dilatation of the aorta ring were observed but the stroke volume remained unchanged. 11 old weeks fistula animals presented similar frequency and stroke volume. So, the heart compensatory mechanisms varied with the age of the rabbit. The right catheter and cardiac angiography were not useful for ventricular volume overload estimation. Histopathological study showed parenchymal changes and alveolo capillary congestion. An increase of the heart weight/body weight ratio was described. When analysed by ultrasound and blood flow measurement, data were similar to those obtained by invasive pressure recordings or by dilution system. An experimental model of overload volume in rabbit with non invasive recording methods (bidimensional ultrasound and doppler system) can be proposed.


Characterization of chick serum lipoproteins isolated by density gradient ultracentrifugation. Serum lipoproteins from 12h fasted male chicks (15-day-old) were separated into 20 fractions by isopycnic density gradient ultracentrifugation. A new procedure was described by collecting the different fractions from the bottom of tube instead of by aspiration from the meniscus of each tube. Analyses of chemical composition of serum lipoproteins have permitted to reevaluate the density limits of major classes: VHDL, d greater than 1.132 g/ml; HDL, d 1.132-1.084 g/ml; LDL, d 1.084-1.038; IDL, d 1.038-1.022; and VLDL d less than 1.022. HDL fractions clearly predominated (approx. 77% of total lipoproteins) while IDL and VLDL were present at low percentage. LDL was the fraction richest in cholesterol; triacylglycerol content clearly increased from HDL to VLDL, while protein content decreased. All the chemical components of chick serum lipoproteins were accumulated in HDL, although triacylglycerol was relatively distributed in all the lipoprotein classes.


Bound and purified alkaline phosphatase from rat duodenal and jejunal brush-border membranes: kinetics and magnesium stimulation. Brush-border membranes from rat duodenal and jejunal mucosa were prepared by differential Ca(2+)-precipitation. Kinetical properties and Mg(2+)-stimulation of alkaline phosphatase were studied for the enzyme either bound to these membranes or purified from these membranes by liquid chromatography. With p-nitrophenylphosphate as substrate, the alkaline phosphatase apparent Km was lower in jejunum (90 microM) as compared with duodenum (160 microM), and lower for the purified enzyme (jejunum: 55 microM; duodenum: 97 microM) as compared to the bound one. In the presence of 5 mM MgCl2, the substrate affinity was in all cases decreased. For the bound enzyme Vmax was 10 times greater in duodenum compared to jejunum. 5 mM MgCl2 tripled the Vmax of the duodenal bound enzyme and increased it by 50% for the jejunal one, but a seven-fold increase was recorded for the purified enzyme at both levels of intestine. The apparent affinity for Mg2+ was similar for the bound and the free enzyme, for duodenum and for jejunum (Mg0.5: +/- 40 microM).


The digestive and metabolic utilization of the dietary protein: effect of clenbuterol and protein level. The influence of a beta-agonist, clenbuterol, added to the diet at 0, 30 and 50 mg/Kg was studied on the digestive and metabolic use of the dietary protein in Wistar rats of two different weights (70 and 200 g). The rats had been fed diets containing different protein levels (4, 10 and 15%). Treatment with clenbuterol did not have any effects on the absorption of the dietary protein in any of the studies released. The beta-agonist did not alter fecal nitrogen excretion nor did it change the digestion coefficient of the protein at the doses used. Clenbuterol significantly increased (P less than 0.01) urinary nitrogen excretion in younger animals fed diets containing low levels of protein (4%). On the other hand, in animals of greater age/weight that had been fed diets with a sufficient protein content (15%), the beta-agonist significantly reduced urinary nitrogen excretion (P less than 0.001). From the results of this study, it can be concluded that in order that clenbuterol can significantly increase nitrogen balance and protein retention, the animals must be given a sufficient exogenous protein intake and they must not be at a stage of maximum growth potential.


Are the unusual morphological and physiological features of the leatherback turtle (Dermochelys coriacea) paralleled at the molecular level? A study on A4 (muscle-type) isozyme of its lactate dehydrogenase. A4 (muscle-type) Lactate Dehydrogenase was purified to homogeneity from Dermochelys coriacea. The steady-state kinetic features of the enzyme show remarkable similarities with those displayed by many other heterothermal LDH's from cold-blooded vertebrates.


Biochemical changes in progressive muscular dystrophy, XVI. Effect of glutamic acid, aspartic acid and glycine on the amino acid content of skeletal muscle of dystrophic mice. The effect of exogenous administration of glutamic acid (GL), aspartic acid (A) and glycine (G) on individual amino acids in the free amino acid pool was studied in skeletal muscles of 60- to 70-day-old normal (N) and dystrophic (D) mice. Both N and D mice received either 0.25 ml of saline (S) or 250 mg/kg weight of GL, A or G in 0.25 ml S subcutaneously for 13 days. GL, A, G or S did not cause any significant changes in the body and skeletal muscle weights of either group. Most of the individual amino acids were increased in skeletal muscles of GL-treated mice and were decreased in A- or G-treated animals compared to S administration in the N group. The picture was more dramatic in the D group: GL-induced amino acid elevations were more pronounced than the values of N- or S-treated D controls. A and G elicited amino acid increases in D mice compared to their S-treated counterparts. Most of the individual amino acids in skeletal of the D group were decreased relative to N mice after S, GL or A administration. This was evident when the D/N ratio was calculated for S, GL and A. The situation was very different after G administration since of the individual amino acids were augmented in the skeletal muscle of D mice compared to N animals.(ABSTRACT TRUNCATED AT 250 WORDS)


Effect of diet on folates levels and distribution in selected tissues of the rat. A technique for the extraction, purification and concentration of folates, followed by high performance liquid chromatography assay with fluorometric detection of the three principal derivatives (tetrahydrofolic, 5-methyl-tetrahydrofolic and formyl-tetrahydrofolic acids) has been applied to the determination of tissue folates in rats. The levels found are compared to those of the microbiological assay using Lactobacillus casei. When rats were fed a diet containing 1 mg of folic acid per kg of food, levels in the intestinal mucosae, liver, whole blood and brain were 0.59, 14.87, 0.28 and 0.83 nmol/g of tissue. An exogenous supply of 200 mg of folic acid/kg of food significantly increased folate levels in all tissues studied, except for the brain: 1.51, 28.93, 0.52 and 0.99 nmol/g, respectively in the above four tissues. The separation of the various derivatives and a variable supply of folic acid have enabled the conversions of these metabolites to be studied.


Inhibition of the cholinergic twitch response in guinea-pig common bile duct: an evaluation of the effects of noradrenaline, clonidine and fentanyl. Presynaptic inhibitory effects of noradrenaline, clonidine and fentanyl on twitch contractions evoked by transmural electrical stimulation were investigated in guinea-pig common bile duct (CBD). Antagonism of the response to fentanyl by naloxone and to noradrenaline and clonidine by yohimbine confirmed the involvement of presynaptic opiate and alpha 2-adrenoceptors respectively. Clonidine caused desensitization of presynaptic alpha 2-adrenoceptors. No cross desensitization between clonidine and fentanyl was observed. Furthermore, partial agonistic activity of clonidine at presynaptic alpha 2-adrenoceptors was also observed by the antagonism of noradrenaline response in this preparation.


Management of non-Hodgkin's lymphoma.

Management of non-Hodgkin's lymphoma. This brief review of non-Hodgkin's lymphomas emphasizes aspects of clinical research which have led to important improvements in the management of patients, and examines research questions with relevance for the future. In the intermediate- and high-grade lymphomas, strategies to intensify drug regimens have proved successful in raising rates of curative outcome. With the introduction of growth factors and peripheral stem cell transfusion to support nadir cytopenias it becomes possible to further develop dose-intense regimens. Identification and selection of poor prognostic subsets is key to the further application of these approaches, since the therapeutic ratio may be narrow and up to 50% of patients may be cured with less toxic regimens. In the low-grade lymphomas where a curative outcome is less certain, the application of intensive therapy has proved more controversial and results remain preliminary. Nevertheless, durable remissions appear achievable in advanced-stage CR patients utilizing low-dose consolidative irradiation or autologous transplantation. In contrast to intermediate- and high-grade lymphomas where intensive therapy is often reserved for responding poor-risk presentations, in low-grade lymphoma intensive therapy and consolidation is most successfully applied to good-risk patients.


Modification of electrophysiological properties of rat heart with age. The authors have determined the serum thyroid hormone levels [total (TT3) and free (FT3) triiodothyronine], the heart weight/body weight ratio and the heart rate of differently aged male rats. The variations of these parameters show a modification of thyroid state as a function of ageing. The authors have also recorded, at about 26 degrees C, resting and action potentials from single cells of papillary muscles isolated from the same groups of rats. The animals in the higher thyroid state exhibited a repolarization speed higher than the other animals. The thyroidectomy, performed on 50 day old rats, and T3 treatment of the thyroidectomized rats give rise to modifications of repolarization speed and then of action potential duration analogous to ones obtained in previous study for animals thyroidectomized at 30 days of age. These data demonstrate that the modifications of heart electrophysiological properties with age, are due fundamentally to thyroid state modifications. The results suggest also that the cardiac chronotropism modifications which the rat undergoes as a function of ageing are due to the changes of levels of thyroid hormone which might exert its effect by modifying the ion channel kinetics as well as the cardiac receptors.


Direct chemical ionization mass spectrometry of short-chain polysaccharides. Underivatized short-chain polysaccharides such as two inulins from different plants containing up to 35-40 monosaccharide units with molecular weights of up to 6500 Da and dextran T 1.5 containing up to 16-20 monosaccharide units with molecular weights of up to 3200 Da have been investigated by direct chemical ionization. Under soft ionization conditions such as ammonia chemical ionization and reduced ion-source temperature, it was possible to obtain spectra of the native polysaccharides showing dominant ion series corresponding to ammonia adduct ions of oligosaccharides, and also ion series corresponding to ammonia adduct ions of anhydro-oligosaccharides.


A new method for the measurement of tremor at rest. This paper establishes a standard method for measuring human tremor. The electronic instrument described is an application of this method. It solves the need for an effective and simple tremor-measuring instrument fit for wide distribution. This instrument consists of a piezoelectric accelerometer connected to an electronic circuit and to an LCD display. The signal is also analysed by a computer after accelerometer analogic/digital conversion in order to test the method. The tremor of 1079 healthy subjects was studied. Spectral analysis showed frequency peaks between 5.85 and 8.80 Hz. Chronic cigarette-smoking and coffee drinking did not modify the tremor as compared with controls. Relaxation session decreased tremor significantly in healthy subjects (P less than 0.01). This new tremor-measuring method opens new horizons in the understanding of physiological and pathological tremor, stress, anxiety and in the means to avoid or compensate them.


[Involvement of 3-dehydroecdysteroids in the ecdysone biosynthetic pathway in Locusta migratoria, in vitro]. Prothoracic glands of the locus, Locusta migratoria, incubated in vitro converted tritiated 3-dehydrocetodiol (22,23,24,25 3H4-14 alpha-hydroxy-5 beta-cholest-7-en-3,6-dione) into ecdysteroids and 3-dehydroecdysteroids as far as the final products of the two series, ecdysone and 3-dehydroecdysone. In the two series, the different compounds are formed in the same quantities, except for 2,22-desoxy-products, the nature of which could not have been determined. Converted 3-dehydroecdysone issued from 3-dehydrocetodiol is transformed into ecdysone after several hours incubation with Locusta last instar larvae hemolymph. Till now is has been impossible to determine if the reduction of 3-dehydroecdysteroids took place into the prothoracic glands or in the incubation medium. In no case is 3-dehydrocetodiol converted into cetodiol. Conversion rates of the different compounds, either issued from cetodiol or from 3-dehydrocetodiol as precursors, are of same importance, so that a weak specificity of the hydroxylation enzymes must be considered.


Prophenoloxidase activation in the hemolymph of American cockroach, Periplanata americana. Prophenoloxidase has been successfully extracted in a stable condition from the Hemocytes of Periplaneta americana using cane sugar saline solution. Other media used to extract prophenoloxidase were found not suitable. Among the activators used (trypsin, chymotrypsin, sodium oleate and SDS), trypsin activated the proenzyme maximum. Activation during electrophoresis resulted in the dissociation of proenzyme in to two sub-units. The enzyme has also been localised by the agarose gel electrophoresis. The proenzyme was stable up to 50 degrees C and precipitated at 60 degrees C and above. The activated enzyme showed maximum activity at pH 7.0. Preliminary attempt has also been made to precipitate the enzyme protein against the antibody raised in rabbit.


An arginine regulated gamma-guanidobutyrate ureahydrolase from tench liver (Tinca tinca L.). A gamma-guanidobutyrate ureahydrolase isolated from tench liver has been characterized. Some of its physicochemical properties like pH effect and thermal stability resemble those of arginases, however it shows some peculiarities that makes it different from arginases and other amidino hydrolases. Thus cation requirement is not as strong as in arginases, and the Km value for gamma-guanido-butyric acid (230 +/- 25 mM) is shifted to a lower value (45 +/- 5 mM) by 5 mM arginine. The possible regulatory role of arginine on gamma-guanidobutyrate ureahydrolase activity is discussed.


Circadian rhythms in glutathione and glutathione-S transferase activity of rat liver. The experiments were conducted to examine the existence of circadian rhythms in glutathione concentration and glutathione S-transferase activity in the liver of the rat. In animals synchronized to a 12:12 h light-dark cycle and fasted at 6 different time points to allow exactly 24 h of fasting, both, glutathione concentration and glutathione S-transferase activity show diurnal variation with a maximum during the light period and a minimum at night. On the other hand the hepatic protein level was maximal during the light period and decreased to its lowest level during the dark period. The implications of such oscillations in the circadian rhythms of toxicological or therapeutical effects of many xenobiotic agents are clear.


Chromaffinity, uranaffinity and argentaffinity of small granule-containing (SGC) cells in rat superior cervical ganglia. A systemic examination on the small granule-containing (SGC) cells in rat superior cervical ganglia was conducted by conventional and cytochemical electron microscopy including chromaffin, argentaffin and uranaffin reactions. According to the fine structure of dense cored vesicles (DCVs) in the cytoplasm, three types of small granule-containing (SGC) cells were revealed--Type I: 90-160 nm vesicles with cores of moderate or low electron density; Type II: 130-330 nm vesicles, polymorphic with highly electron dense cores; Type III: elongated vesicles (170 nm x 60 nm) with cores of moderate to low electron density. The majority of SGC cells were the Type I cells (78%) and Type II and III cells made up 13% and 9% of SGC cell population, respectively. Cytochemical results demonstrated that only the Type II cells displayed a positive chromaffin reaction and all three types of SGC cells showed argentaffinity and uranaffinity. The present study is the first to demonstrate the argentaffin reaction at ultrastructural level in SGC cells of sympathetic ganglia. Based on the results of the present study we also concluded that (1) the DCVs of Type II SGC cells contained noradrenaline and (2) biogenic amines and nucleotides (ATPs) coexisted in the DCVs of all three types of SGC cells.


Bleeding stress and metabolic changes in the crab Scylla tranquebarica (Fabricius). In the crab scylla tranquebarica, bleeding stress is characterized by changes in proteins, lactic acid and water content of muscles, hepatopancreas and haemolymph. Proteins seemed to be involved in energy expenditure and internal osmotic balance. Lactate levels increased, suggesting a shift in aerobic metabolism. Water content fell.


In vitro electro-mechanical activity of the human colon. Simultaneous recording of the electrical patterns of the two muscle layers. Electrical and mechanical activity on longitudinal and circular layers of the human sigmoid colon were simultaneously studied. Recordings were obtained from two electrode sites spaced 3 cm apart in a piece of colon which had been resected surgically and perfused in an organ bath. Spontaneous electrical activity of the colon showed slow waves and spikes. Slow waves were present for only 24.5% and 12% of the recording time on the longitudinal and circular layers, respectively, and they appeared as localized activity which was irregular in amplitude and varying in frequency. Electrical coupling between the two muscle layers was rarely seen and slow waves were not associated with pressure changes. Spiking activity were recorded as short and long spike bursts on both muscle layers. Short spike bursts were localized activity superimposed on slow waves. The associated mechanical activity, which consisted of single weak pressure changes or prolonged contractions with summation, was determined by slow wave frequency. Long spike bursts were seen at irregular intervals and were either propagated or not propagated activity associated with electrical oscillations ranging from 24 to 46 cpm. Mechanical activity consisted of sustained tonic contractions propagated or not propagated in the same way as the electrical pattern. Coordinated electrical activity of the two muscle layers seldom occurred when spontaneous activity was being recorded. Electrical activity on both muscle layers was very sensitive to stretching and could be initiated or modulated by pharmacological agents. In particular, our findings showed that stimulation induced coordinated spiking activity on the two muscle layers and caused mechanical activity, propagated orally or aborally, which consisted of long lasting, high amplitude contractions.(ABSTRACT TRUNCATED AT 250 WORDS)


Survival rate of patients with glomerulonephritis. This is a fourteen-year follow-up study of patients with primary and secondary glomerulonephritis, diagnosed by percutaneous renal biopsy. The survival curves of 271 patients were determined, applying the method described by Cutler 1958. The term "kidney death" was used for the patients who had died or developed end stage renal failure. The five-year survival rates were: 32 for diffuse proliferative glomerulonephritis (chronic), 22 for focal glomerulosclerosis, 30 for membranoproliferative glomerulonephritis, 62 for membranous nephropathy, 90 for focal and mesangioproliferative glomerulonephritis, and 94 for IgA nephropathy. Two diseases of the secondary glomerulonephritis group were analyzed: renal amyloidosis with a survival rate of 8 and lupus nephritis 51.


Phagocytic function in patients with recurrent urinary tract infections. Phagocytes play an essential role in the host's defence against uropathogenic bacteria which are mostly extracellular pathogens. The functional capacity of peripheral blood phagocytes (predominantly polymorphonuclears, PMN) was investigated in 47 patients (urethrocystitis, acute or chronic pyelonephritis) and in healthy persons. The random mobility of phagocytes was determined by measuring their spontaneous migration from a capillary tube. Using radioactively labelled sheep erythrocytes as targets and the phagocytes as effector cells, ingestion, digestion and extracellular cytotoxicity were determined. All the four phagocytic functions in patients were significantly lower than in healthy controls, especially in patients with chronic pyelonephritis. These results link reduced phagocytosis by blood phagocytes with recurrent urinary tract infections (UTI). Whether the defect is primary or secondary to infection (and only transient) should be the object of further studies.


Electrocardiographic and clinical anticipation of the left ventricular systolic function, systemic vascular resistance, and myocardial hypertrophy--normal values and validation of method in patients with arterial hypertension. In order to study the hemodynamic effects of the antihypertensive drugs in patients with essential hypertension, systolic time intervals from ECG data were extrapolated. The study was performed in 36 healthy individuals and 38 patients with essential hypertension without a drug therapy and/or in the wash out period more than three weeks and after a treatment. The stroke volume (SV) was determined as a product of the ejection time (ET), the pulse pressure (PP) and the flow coefficient (Kf). The Kf was extrapolated from Doppler-cardiographic parameters and it significantly correlated with the normal diastolic blood pressure (TA(d)) if the QT is not prolonged and the electrical systole/mechanical systole ratio (QS2/MS) not disturbed: Kf = 9.356 e-0.008 TA(d); r = -0.9. In arterial hypertension the correlation was also curvilinear: kf = 3.962 e-0.001 TA(d); r = -0.99. The arteriolar stiffness was defined as the PP/SV ratio. The cardiac output (CO) and systemic vascular resistance (SVR) were determined according to conventional formulas by known clinical and extrapolated ECG data. The cardiac contractile or muscle performance was defined as corrected changes of the ejection function for the given afterload according to the formula: I (-0.004 TA(d) + PEP/ET) -0.014 x 100; the normal 95% confidence limits for the laboratory used are -6.6% to +6%. Systolic time intervals were extrapolated from ECG data in the consecutive manner: QS2 = kQS2 x QT, ET = kET x JT, and MS = kMS x ET. The correlation of kQS2 with QT is curvilinear: kQS2 = 2.760 e-2.732 QT.(ABSTRACT TRUNCATED AT 250 WORDS)


Partial laryngectomy in transglottic carcinoma. At the ENT Department of Zagreb University from 1973 to 1982 119 cases of transglottic carcinoma were treated. In the first group of 20 cases total laryngectomy was done. Five years later, ten of the patients treated had survived. Primary and secondary neck metastases were present in 6 cases, i.e. 30%. In the second group of 99 cases partial laryngectomy (vertical and combined) was done. Five years later 70 cases had survived, i.e. 70%. Primary and secondary neck metastases were present in 19 cases, i.e. 19%. In the partial laryngectomy the reconstruction was done by fascia sternohyoidea and partial resection of the cricoid and totally of the arythenoid in the cases where there was the indication. Postoperative irradiation was given in cases with neck metastases and where resection margin was inadequate.


Mentally ill offenders and their medical treatment according to the criminal law of Croatia. The authors have analyzed psychopathological and criminal characteristics of the 75 patients, regarding the census on 31. 12. 1985. Group 1 (N = 55) was committed by court for inpatient treatment and group 2 (N = 20) was committed by court for outpatient treatment. The analysis was based on the data in psychiatric expertises and medical files containing the relevant facts for the study. 46 patients (92%) in the first group are diagnosed as psychotic disturbances, regarding the ICD-9, while in the second group there were 15 patients (75%) of the same diagnostic category. The other patients were diagnosed as mentally ill (including the mentally retarded, epileptic etc. patients). The age distribution shows that a majority of the patients of the first group are between 31-50 years (73%) and for the second group the rate is 50%. According to the criminal acts the first group mainly consists of patients who had committed one murder or more (36 + 3), while in the second group the patients offended against property and committed body violations (8 + 6). The average institutional treatment, which is not legally limited, is over 10 years, and for outpatient treatment group it is 13 months, legally limited up to 2 years. The main criterion for psychiatric and legal distribution of the patients into two groups is the estimation of their social danger. The given data prove that the estimation of the social danger is mainly defined by the psychopathological characteristics of the subjects and the type of the criminal act they committed, while the other parameters are less influential for the estimation.(ABSTRACT TRUNCATED AT 250 WORDS)


Serum gastrin concentration in chronic renal failure. On the basis of the existing experimental and clinical studies about the factors affecting the appearance of hypergastrinemia in renal failure, it can be concluded that the kidney plays an important, but not the only, role in the degradation of endogenous gastrin in humans. In this process the key role is played by the blood flow through the kidney, the preservation of the peritubular capillary system, and the functional kidney mass. Glomerular filtration has no particular importance in the extraction of gastrin from the circulation, while through the urine only a small amount of gastrin is excreted. In the decomposition of a part or at least some molecular gastrin forms, an important role is played by the capillary systems of extra-renal tissue. One further conclusion is that hypergastrinemia in patients with renal failure is the result of the combined effects of the reduced catabolism of gastrin in the kidney and its increased synthesis which is for the most part connected with hypochlohydria and secondary hyperparathyroidism. In patients with renal failure there exists the inhibition of the gastrin acid secretion which is the cause of the weakening of the mechanism of the feedback connection between HCl and gastrin, while because of a permanent stimulation of G-cells, the hyperplasia of these cells develops, as well as the increased secretory activity, and hypergastrinemia. Parietal cells become less sensitive to a permanently increased serum gastrin concentration but still capable of reacting to the maximal stimulus. In patients with renal failure, especially those with extreme hypergastrinemia, there develops the increased concentration of large, mainly biologically inactive (big big gastrin, component I) molecular forms of gastrin.


Stereological analysis of the female breast alveolar ductulus epithelial cells in premenopause and postmenopause. Stereological characteristics of the ductular parenchyma epithelial cells were analysed ultrastructurally in 18 cases of normal breast tissue. Different physiological states were compared in the premenopause and postmenopause. The volume density (VV), surface density (SV) and specific surface density (SV/VV) of nuclei and the epithelial cell cytoplasm were stereologically examined and compared. After a menopause a slight decrease of the volume density (VVj), surface density (SVj) and specific surface density (SVj/VVj) of epithelial cell nuclei was noted, while the volume density (VVc), surface density (SVc) and the specific surface density (SVc/VVc) of the epithelial cell cytoplasm were slightly increased.


Risks of retrobulbar application of anesthetics and drugs. In spite of hundreds of harmless retrobulbar applications of anesthetics and drugs, a certain risk still exists. In the past ten years in our Department with 7200 retrobulbar procedures performed in local anesthesia and 12500 retrobulbar applications of the drugs (corticosteroids, antibiotics, vasodilatators) 14 incidents were registered. Palpebral chemosis and retrobulbar hematoma or eyeball protrusion were transient in 11 patients. Acute disturbance of the chorioretinal circulation occurred in three patients. In one of them vascular occlusion was total with permanent loss of vision, while in the other two recuperation of vision was the sign that vascular spasm was transient.


Cor triatriatum dextrum.

Cor triatriatum dextrum. Cor triatriatum dextrum was an incidental echocardiographic finding in a 5 year old boy and a 9 year old girl, who were evaluated for the presence of a heart murmur. Apart from slightly enlarged right atria, they had otherwise normal hearts and were symptomless. Most of the patients reported until then had been symptomatic due either to the persistent right sinus venosus valve or to commonly associated structural heart anomalies. Therefore, the clinical significance of asymptomatic cor triatriatum dextrum remains coniectural, but might lie in the possibility of development of arrhythmia, progressively worsening interference with the systemic venous return and thrombus formation.


Prognostic value of some parameters of cellular immunity in breast cancer patients. In 45 histologically verified breast cancer patients the cell-mediated immune responses were tested prior to surgical therapy. The cell immunity was assessed by the percentage of T and active T-lymphocytes and by measuring the lymphocyte reactivity to phytohemagglutin (PHA) and concanavalin A (Con A). Disease-free interval was longer and the mortality after 3, 5 and 8-year follow-up significantly lower in the group of patients with the normal number of T and active T-lymphocytes than in the group of patients with the lower number of those cells. The proliferative response of lymphocytes to Con A and PHA assessed in allogeneic and autologous serum also seemed to be a useful prognostic parameter. The survival was significantly longer in the group of patients with a normal lymphocyte reactivity to those mitogens than in the non-reactive group.


Influence of phenobarbital and triiodothyronine on the development of hepatic eosine transport in rats. In male Wistar rats aged 20 and 60 days the influence of a 5-day-treatment with phenobarbital (PB, 75 mg/kg b.m. i.p.), triiodothyronine (T3, 100 micrograms/kg b.m. s.c.) and both agents simultaneously (PB+T3) on eosine accumulation of liver slices in vitro and biliary excretion of eosine in vivo was investigated. PB enhanced bile acid independent bile flow. T3 accelerated eosine accumulation in liver slices. The combined treatment with PB+T3 had no additional effects. The simultaneous action of PB+T3 could not induce the maturation of the rate limiting excretion process for organic anions in 20-day-old rats.


Influence of stepwise uncoupling and temporary anoxia on pancreatic enzyme secretion by isolated rat acini. For special studies on pancreatic diseases a parameter is needed to record alterations of the cellular energy metabolism. In the in vitro model of isolated pancreatic acini, we investigated whether or not at standardized cholecystokinin stimulation the energy-consuming process of enzyme secretion can be used to monitor changes of the energy-supplying capacity. Rat pancreatic acini were isolated via collagenase digestion and characterized by basal and stimulated release of amylase and trypsin, oxygen uptake under resting and maximally uncoupled conditions and by their ability to accumulate actively rhodamine-6G, as a measure of the mitochondrial membrane potential. The stimulation of enzyme release did not find a measurable reflection in rhodamine-6G accumulation and in the respiratory rat. Stepwise uncoupling of oxidative phosphorylation by 2,4-dinitrophenol (DNP) and temporary anoxia were used to simulate disturbances of the pancreatic energy metabolism in vitro. With increasing DNP concentration the enzyme release was significantly reduced. While after 30 min anoxia the enzyme release still exceeded that of unstimulated control, after 60 min anoxia there was no further response to hormonal stimulation. At standardized stimulation and after suitable calibration the enzyme release by acini may be used to monitor alterations of the pancreatic energy metabolism in vitro.


[A study of infant and child health care "follow up clinic"]. Health screening examinations of children. The 1st. health screening forms a major part of the public health services in community health, but it still encounters many problems. In the present study, the health screening programs and the infant and child health care follow-up clinic (2nd health screening) conducted by the Yoshikawa health center in Saitama-pref., as well as by each city and town, were investigated and analyzed. The findings are as follows: 1. In the first, health screening they pointed out some developmental disorders of 21.2%. About 80% of them are "to continue with follow-up observation". And 20% are "to go to the hospital and are to have an examination at once". Almost all of the above problems are related to psychomotor developmental disorders and physical growth delay. 2. The number of the children who made use of public expense was only 7 in 1st health screening. 3. The major complaints of the 2nd health screening are as follows: 1) speech and language delay 130 (36.4%); 2) motor behavior delay 117 (32.8%); 3) physical growth retardation 51 (14.3%); 4) emotional disturbances 12 (4.8%). The results of medical diagnosis for 2 years (1988-1990) were as follows: 1) speech and language disorders 55 (27.5%); 2) mental retardation 29 (14.5); 3) motor disturbances 28 (14.0%). The number of children without any problems is 18 (9.0%). 5. After they took the 2nd health screening, 138 (38.7%) children consulted with this clinic, and still keep consulting. 107 (30.0%) children had a medical examination, and 44 other children (12.3%) were introduced to other related facilities. As matters stand, there are not enough nurseries or training facilities for borderline children, and high-risk children. We don't have a complete system for border and high-risk children. The facilities for border and high-risk children do not give any specific details as to the various special services available. In the future, we forecast that the number of children with speech disorders and children with emotional disorders including infantile autism will increase, we should analyze the system of border and high-risk infants and children in connection with the 2nd health screening and discuss how to serve high-risk children effectively.


Use of gasoline exposure data and assessment. A summary report of work group III. The overall consensus of the Work Group was that the workshop provided an excellent opportunity for discussion of the scientific issues pertaining to non-occupational exposures to gasoline as it related to public health officials, legal and regulatory agencies, and industrial (workplace) concerns. It was enlightening to discuss the implications of new and existing data and methods for determining the public's exposure to gasoline. The workshop resulted in a list of data needs and a vision of the future research that will be required to aid future users of exposure data.


Identification of inhibitors of nitric oxide synthase that do not interact with the endothelial cell L-arginine transporter. The effects of inhibitors of nitric oxide (NO) synthase and other cationic amino acids on unidirectional L-arginine transport were studied in porcine aortic endothelial cells cultured in microwell plates or perfused in microcarrier columns. L-Homoarginine, L-lysine and L-ornithine inhibited transport of L-arginine. The NO synthase inhibitors NG-monomethyl-L-arginine and NG-iminoethyl-L-ornithine also reduced L-arginine uptake, whereas NG-nitro-L-arginine and its methyl-ester had no inhibitory effect. The ability to modulate selectively endothelial cell L-arginine transport or NO synthase activity will allow further characterization of the arginine transporter and its role in regulating NO biosynthesis.


Individual and population exposures to gasoline. Gasoline is a complex mixture of many constituents in varying proportions. Not only does the composition of whole gasoline vary from company to company and season to season, but it changes over time. The composition of gasoline vapors is dominated by volatile compounds, while "gasoline" in groundwater consists mainly of water-soluble constituents. Hydrocarbons, including alkanes, alkenes, and aromatics, make up the large majority of gasoline, but other substances, such as alcohols, ethers, and additives, may also be present. Given this inability to define "gasoline,h' exposures to individual chemicals or groups of chemicals must be defined in a meaningful exposure assessment. An estimated 111 million people are currently exposed to gasoline constituents in the course of refueling at self-service gasoline stations. Refueling requires only a few minutes per week, accruing to about 100 min per year. During that time, concentrations in air of total hydrocarbons typically fall in the range 20-200 parts per million by volume (ppmV). Concentrations of the aromatic compounds benzene, toluene, and xylene rarely exceed 1 ppmV. Some liquid gasoline is also released, generally as drops less than 0.1 g each, but with enough larger spills to raise the average loss per gallon dispensed to 0.23 g for stations with conventional nozzles and 0.14 g per refueling for stations with vapor recovery nozzles (Stage II controls). Some skin exposure may occur from these spills but the exposure has not been quantified. Two major types of vehicular emissions have been studied. Evaporative emissions include emissions while the vehicle is driven (running losses), emissions after the engine has been shut off but is still warm (hot soak), and emissions during other standing periods (diurnal) emissions. These evaporative emissions are dominated by the more volatile gasoline components. Tailpipe emissions include some unreacted gasoline constituents as well as products of combustion (including chemicals identical to some of the original constituents of the gasoline) and a variety of hydrocarbons and related compounds. Running losses are reported to fall in the range of 0.2 to 2.8 g of total hydrocarbons per mile driven, while benzene evaporative emissions range from 0.002 to 0.007 g/mile. Benzene levels inside travelling vehicles have been reported to average about 13 ppbV in Los Angeles. Tailpipe emissions amount to 0.3 to 1.0 g/mile of total hydrocarbons; emissions of benzene, polycylic aromatic hydrocarbons, and 1,3-butadiene have been reported to range from 0.015 to 0.04 g/mile, 0.00025 to 0.00046 g/mile, and 0.001 to 0.005 g/mile, respectively.(ABSTRACT TRUNCATED AT 400 WORDS)


Exposure to emissions from gasoline within automobile cabins. Gasoline is emitted from automobiles as uncombusted fuel and via evaporation. Volatile organic compounds (VOC) from gasoline are at higher levels in roadway air than in the surrounding ambient atmosphere and penetrate into automobile cabins, thereby exposing commuters to higher levels than they would experience in other microenvironments. Measurements of VOC concentrations and carbon monoxide were made within automobiles during idling, while driving on a suburban route in New Jersey, and on a commute to New York City. Concentrations of VOC from gasoline were determined to be elevated above the ambient background levels in all microenvironments while VOC without a gasoline source were not. The variability of VOC concentrations with location within the automobile was determined to be smaller than inter-day variability during idling studies. VOC and carbon monoxide levels within the automobile cabin differed among the different routes examined. The levels were related to traffic density and were inversely related to driving speed and wind speed. Overall, daily VOC exposure for gasoline-derived compounds during winter commuting in New Jersey was estimated to range between 5 and 20% and constituted between 15 and 40% of an individual's daily exposure based on comparison to urban and suburban settings, respectively. VOC exposure during commuting in Southern California was estimated to range between 15 and 60%.


Tachykininergic transmission to the circular muscle of the guinea-pig ileum: evidence for the involvement of NK2 receptors. 1. The effect of newly developed, receptor-selective tachykinin antagonists (GR 71,251 for NK1 receptors, MEN 10,376 and L 659,877 for NK2 receptors) on noncholinergic transmission to the circular muscle of the guinea-pig ileum has been investigated. 2. In circular muscle strips of the ileum, electrical field stimulation in the presence of atropine (2 microM) and apamin (0.1 microM) evoked a complex motor response. The tonic primary contraction in this response was reduced by GR 71,251 (10 microM) and MEN 10,376 (3-10 microM) but not by L 659,877 (up to 10 microM). The presence of apamin was necessary in this experimental arrangement to unmask an atropine-resistant primary contraction, sensitive to tachykinin antagonists. The motor response was abolished by tetrodotoxin. 3. In circular strips of the ileum GR 71,251 (10 microM) inhibited the tonic contraction produced by [Sar9] substance P sulphone, a selective NK1 receptor agonist but not that produced by [beta Ala8] neurokinin A (4-10), a selective NK2 receptor agonist. By contrast, MEN 10,376 antagonized the effect of the NK2 agonist while leaving the response to the NK1 agonist unaffected. 4. In whole segments of the ileum, distension of the gut wall by an intraluminal balloon placed at about 1 cm from the point of recording of mechanical activity of the circular muscle produced atropine-sensitive phasic contractions (ascending enteric reflex). In the presence of atropine (2 microM), a noncholinergic response was elicited, which required larger volumes of distension that the cholinergic one. The atropine-resistant ascending enteric reflex was enhanced by apamin (0.1 microM) and abolished by tetrodotoxin, either in the presence or absence of apamin.5. MEN 10,376 (3-lOmicroM) inhibited the atropine-resistant ascending enteric reflex in the presence of apamin while GR 71,251 or L 659,877 (10 microM each) were ineffective. MEN 10,376 inhibited the atropine-resistant ascending enteric reflex to a larger extent in the absence than in the presence of apamin and also slightly inhibited the ascending enteric reflex in the absence of atropine.6. These findings provide evidence for an involvement of NK2 tachykinin receptors in excitatory transmission to the circular muscle of the guinea-pig ileum. NK2 receptors are also involved in the physiological-like circular muscle activation produced by stimulation of intramural neuronal pathways which subserve the atropine-resistant ascending enteric reflex.


Characterization of the receptor mediating relaxation to substance P in canine middle cerebral artery: no evidence for involvement of substance P in neurogenically mediated relaxation. 1. The aim of this study was to characterize the neurokinin receptor which mediates relaxation of dog isolated middle cerebral artery by the use of selective agonists and antagonists and to establish whether substance P is involved in the neurogenically mediated relaxant response in this vessel. 2. Substance P caused concentration-related, endothelium-dependent relaxations of dog isolated middle cerebral artery, contracted with prostaglandin F2 alpha. The selective NK1 receptor agonists, GR73632 and substance P methyl ester (SPOMe), also caused relaxation with similar maximum effects to those of substance P. GR73632 and SPOMe were approximately 20 times and 6 times less potent respectively than substance P. The selective NK2 and NK3 receptor agonists, GR64349 and senktide, were only weakly active in causing relaxation being at least 425 times and 245 times less potent respectively than substance P. 3. The selective NK1 receptor antagonist, GR82334, was a potent, specific, competitive antagonist of the relaxant effects of substance P. In contrast, the selective NK2 receptor antagonist, R396 (10 microM) had no effect on the response to substance P. 4. Electrical field stimulation of dog isolated middle cerebral artery, contracted with prostaglandin F2 alpha, caused neurogenically mediated, non-adrenergic non-cholinergic (NANC) relaxations. These NANC relaxations were unaffected by endothelium removal, GR82334 (10 microM) or by capsaicin (10 microM) treatment. However, the nitric oxide synthesis inhibitor, L-NG-monomethyl arginine methyl ester (L-NMMA) (100 microM) markedly attenuated the response to electrical stimulation. 5. These results suggest that substance P causes relaxation of dog isolated middle cerebral artery via activation of NK1 receptors. However, substance P does not appear to be involved in NANC neurotransmission. In contrast, the marked inhibitory effect of L-NMMA on NANC relaxations implicates nitric oxide in NANC neurotransmission in this vessel.


Differences in neurokinin receptor pharmacology between rat and guinea-pig superior cervical ganglia. 1. The depolarizations elicited by seven neurokinin receptor agonists were examined in both rat and guinea-pig superior cervical ganglia by use of grease-gap methodology in the presence of tetrodotoxin (0.1 microM). Responses were normalised with respect to 1 microM eledoisin. 2. The rank order of agonist potency in the rat ganglia was senktide greater than substance P greater than substance P methyl ester = eleidosin = Sar-Met-substance P greater than neurokinin B greater than neurokinin A, whereas in guinea-pig superior cervical ganglion (SCG) the rank order was senktide greater than Sar-Met-substance P greater than neurokinin B = eledoisin = substance P methyl ester. The concentration-effect curves for substance P and neurokinin A in guinea-pig ganglia were biphasic which precluded the determination of meaningful potency values. 3. The maximal depolarization achieved by subtype selective ligands was different between these two species. On rat and guinea-pig SCG, the NK3-selective ligand, senktide, produced a maximal depolarization of 27% and 274% respectively, whereas the NK1-selective ligand, substance P methyl ester, produced depolarizations of 77% and 64% respectively. 4. The depolarizations induced by substance P methyl ester and senktide in either species were unaffected by atropine (1 microM), suggesting a lack of involvement of presynaptic neurokinin receptors in the generation of the response. 5. The potency of substance P methyl ester, senktide, and neurokinin A were unaffected by pretreating ganglia with the peptidase inhibitors bacitracin (40 micrograms ml-1), leupeptin (4 micrograms ml-1), and chymostatin (2 micrograms ml-1). Similarly, these peptidase inhibitors had no effect on the maximal depolarizations achieved by any of these agonists.6. It is evident that rat and guinea-pig superior cervical ganglia possess both NK, and NK3 receptors, but that their net contribution to depolarizations are different between the two species. The depolarizations in guinea-pig SCG are mediated predominantly by an NK3 subtype and in rat SCG by an NK, receptor subtype.


Bronchodilatation by tachykinins and capsaicin in the mouse main bronchus. 1. The effect of sensory neuropeptides and capsaicin on basal and stimulated tone of mouse bronchial smooth muscle has been evaluated. 2. In basal conditions neither sensory neuropeptides (substance P, neurokinin A or calcitonin gene-related peptide (CGRP) nor capsaicin exerted any contractile effects. However, when a tonic contraction was induced with carbachol (1 microM) a prompt relaxation was induced by substance P (1- 100 nM) and by neurokinin A (1- 100 nM), with substance P being more potent. A second application of substance P was without effect. CGRP (10 nM) produced only a very small and erratic relaxation. Relaxation was also induced by capsaicin (1 microM), and this response could be evoked only once in each preparation. In 4 out of 6 preparations a cross-desensitization between substance P and capsaicin was observed. 3. The selective NK1 tachykinin agonist, [Pro9]-SP sulphone (1 microM), exerted potent bronchodilator actions on carbachol-contracted mouse bronchial preparations. In contrast, neither [beta Ala8]-NKA (4-10) nor [MePhe7]-NKB (both at a concentration of 1 microM), selective synthetic agonists for NK2 and NK3 receptors, exerted significant relaxant effects. Furthermore, the selective NK1 tachykinin antagonist, (+/-)-CP 96,345 (1 microM), abolished substance P (1 nM)- but not isoprenaline (0.1 microM)-induced relaxations. 4. Application of electrical field stimulation (EFS) (20 Hz, supramaximal voltage, 0.5 ms for 10 s) to carbachol-contracted preparations evoked a transient contraction followed by a relaxation. The tetrodotoxin-sensitive slow component of this relaxation was reduced following capsaicin desensitization. 5. In the presence of indomethacin (5 microM) the relaxation induced by substance P, capsaicin or EFS was suppressed.6. In conclusion, the mouse main bronchus appears to be a monoreceptorial tissue containing only NK, receptors which subserve bronchodilator functions. Such receptors could be activated by exogenous or endogenously (capsaicin or EFS) released tachykinins and the consequent relaxation is probably mediated by the generation of prostanoids.


Chloride channels and anion fluxes in a human colonic epithelium (HCA-7). 1. Colonic epithelial cells, derived from a human adenocarcinoma (HCA-7), were examined by the patch clamp technique. 2. Outwardly rectifying anion (Cl-) channels were identified in the apical membrane. The conductance was g(in) approximately 26 pS, g(out) approximately 40 pS. The open state probability of the channels increased with depolarization and the selectivity for Cl- over K+ (PCl/PK) was approximately 7.5. 3. The channels were sensitive to intracellular adenosine 3':5'-cyclic monophosphate (cyclic AMP, 0.1 mM), but not to Ca2+ (at concentrations up to 1 mM). At depolarized potentials the channels were blocked by pirentanide (1-5 microM) applied intracellularly. 4. HCA-7 monolayers loaded with 125I- (as a marker for Cl-) were used to measure I- efflux and converted to instantaneous rate constants. 5. The rate constant for I- efflux was increased by forskolin and lysylbradykinin (LBK). The effects of forskolin were not effected by BAPTA (an intracellular calcium chelator). The effects of LBK were inhibited by BAPTA and by Ba2+, indicating that LBK raised intracellular Ca2+ (Cai) which activates Ca(2+)-sensitive K-channels, the latter being blocked by Ba2+. 6. Although it cannot be conclusively proved that the outwardly rectifying chloride channels described here are solely or partially responsible for the increased anion efflux or transepithelial chloride secretion, the channels are likely to be more relevant for cyclic AMP-requiring rather than Ca(2+)-requiring secretagogues.


FR 113680: a novel tripeptide substance P antagonist with NK1 receptor selectivity. 1. We have discovered a novel tripeptide substance P (SP) antagonist, FR 113680 [N alpha-[N alpha-(N alpha-acetyl-L-threonyl)-N'-formyl-D- tryptophyl]-N-methyl-N-phenylmethyl-L-phenylalaninamide]. In binding experiments, FR 113680 inhibited [3H]-SP binding to guinea-pig lung membranes (NK1) in a competitive manner but had not effect on [3H]-SP binding to rat cerebral cortical membranes (NK1), [3H]-neurokinin A ([3H]-NKA) binding to rat duodenum smooth muscle membranes (NK2) and [3H]-eledoisin (Ele) binding to rat cerebral cortical membranes (NK3). 2. In bioassay experiments, FR 113680 dose-dependently inhibited SP-induced guinea-pig ileum contraction (NK1), but did not inhibit either NKA-induced rat vas deferens contraction (NK2) or neurokinin B (NKB)-induced contraction of rat portal vein (NK3). According to Schild plot analysis, the inhibitory effect of FR 113680 on SP-induced guinea-pig ileum contraction is competitive and the pA2 value is 7.53. 3. The inactivity of FR 113680 on NK1 receptors in rat compared to guinea-pig may represent species-specific forms of the NK1 receptor. 4. These findings suggest that FR 113680 interacts selectively with the NK1 neurokinin receptor.


Endothelium-dependent relaxation to acetylcholine in the rabbit basilar artery: importance of membrane hyperpolarization. 1. Muscarinic stimulation of isolated, preconstricted segments of the basilar artery, with either acetylcholine or carbachol, was followed by endothelium-dependent smooth muscle relaxation and membrane hyperpolarization. 2. Smooth muscle relaxation to acetylcholine was stimulated in the presence of lower concentrations than the associated hyperpolarization (EC50 values 3.2 microM and 31.6 microM, respectively), and was sustained during agonist application, while the hyperpolarization was relatively transient. 3. Repeated exposure to acetylcholine was associated with loss of membrane hyperpolarization, while smooth muscle relaxation was unaltered. Following a second exposure to 100 microM acetylcholine, mean hyperpolarization was markedly depressed from 8.5 to 2 mV, and subsequent exposures failed to induce any hyperpolarization. Relaxations with a similar amplitude and rate of development, were recorded with each subsequent addition of acetylcholine. 4. The competitive substrate inhibitors for nitric oxide synthase, L-NG-monomethyl arginine (100 microM L-NMMA) or L-NG-nitro arginine methyl ester (100 microM L-NAME), modified the form and amplitude of both the relaxation and the hyperpolarization to acetylcholine. In the majority of experiments, both the hyperpolarization and the relaxation were almost totally abolished. 5. Neither nitric oxide, applied directly in physiological salt solution, nor sodium nitroprusside, produced smooth muscle hyperpolarization except in high concentrations. Reproducible, small amplitude (around 2 mV) hyperpolarization followed the application of either NO gas (15 microM) or sodium nitroprusside (100 microM), both of which induced almost maximal smooth muscle relaxation. 6. These data show that muscarinic stimulation of endothelial cells in the rabbit basilar artery is followed by both smooth muscle hyperpolarization and relaxation. They indicate that nitric oxide is involved in both of these responses, but that the smooth muscle hyperpolarization is not an essential component of the relaxation.


Kinetic modulation of guinea-pig cardiac L-type calcium channels by fendiline and reversal of the effects of Bay K 8644. 1. The modulation of L-type calcium channel current (ICa) by fendiline, a diphenylalkylamine type of calcium channel blocker was investigated on guinea-pig ventricular myocytes by use of the whole-cell patch-clamp technique. 2. Fendiline-induced block of ICa is accompanied by modulation of the channel kinetics in a complex manner. The time course of ICa inactivation is significantly faster and the channel availability (f infinity) curve is shifted considerably to more negative potentials by fendiline. These findings can be interpreted qualitatively in terms of a modulated receptor. 3. When the 1,4-dihydropyridine agonist (4R, 4S)-Bay K 8644 was added in presence of 30 microM fendiline a further reduction of ICa instead of the expected stimulatory effect was observed. 4. A similar 'paradoxical' inhibition of ICa was produced by the pure agonist enantiomer (4S)-Bay K 8644. Thus this novel effect of Bay K 8644 cannot be attributed to changes in affinity of the 1,4-dihydropyridine receptor site for (4R)-Bay K 8644 during fendiline action. 5. The IC50 for fendiline was reduced to 3.0 +/- 0.1 microM (control value: 17.0 +/- 2.4 microM) and the Hill slope in its presence was increased to 1.90 +/- 0.1 (control value: 1.39 +/- 0.23) by 1 microM (4R, 4S)-Bay K 8644. 6. (4R,4S)-Bay K 8644 caused the expected stimulation of ICa in the presence of verapamil, diltiazem and nifedipine, overcoming the inhibitory effect of these calcium channel blockers. 7. The 'paradoxical' inhibitory effect of the agonist Bay K 8644 can be explained in terms of an allosteric interaction between fendiline and the dihydropyridine agonist.


The effect of monoclonal antibodies to calcitonin gene-related peptide (CGRP) on CGRP-induced vasodilatation in pig coronary artery rings. 1. The modification of the vasodilator effect of calcitonin gene-related peptide (CGRP) by a panel of monoclonal antibodies (MAbs), which map to discrete epitopes on the CGRP molecule, was investigated in pig coronary artery rings (PCA). The preparations were pre-constricted with acetylcholine (3 x 10(-7) M) and concentration-response curves to CGRP (2 x 10(-10)-2.56 x 10(-8) M) were obtained in the presence or absence of each MAb. 2. CGRP caused a concentration-dependent relaxation of PCAs which reached a maximum (98.2 +/- 4.8%, n = 25) at 1.28 x 10(-8) M and gave an EC50 of 3.8 +/- 0.8 x 10(-9) M. 3. Two MAbs which map to the N-terminal, CN1 and CRA3, did not affect the CGRP response whilst a third, CRA5, significantly inhibited its effect. 4. The C-terminal MAb, CRA2, did not modify the CGRP response whilst, in contrast, CB3 (C-terminal) potentiated its effect. A similar augmentation of the CGRP-induced vasodilatation was seen in the presence of the middle-region MAb, CRA8. 5. These results suggest that regional specific MAbs can modify the vasodilator effect of CGRP causing either inhibition (CRA5, N-terminal) or potentiation (CB3, C-terminal; CRA8, middle region).


Actions of arginine polyamine on voltage and ligand-activated whole cell currents recorded from cultured neurones. 1. Toxins from invertebrates have proved useful tools for investigation of the properties of ion channels. In this study we describe the actions of arginine polyamine which is believed to be a close analogue of FTX, a polyamine isolated from the American funnel web spider, Agelenopsis aperta. 2. Voltage-activated Ca2+ currents and Ca(2+)-dependent Cl- currents recorded from rat cultured dorsal root ganglion neurones were reversibly inhibited by arginine polyamine (AP; 0.001 to 100 microM). Low voltage-activated T-type Ca2+ currents were significantly more sensitive to AP than high voltage-activated Ca2+ currents. The IC50 values for the actions of AP on low and high voltage-activated Ca2+ currents were 10 nM and 3 microM respectively. AP was equally effective in inhibiting high voltage-activated currents carried by Ba2+, Sr2+ or Ca2+. However, AP-induced inhibition of Ca2+ currents was attenuated by increasing the extracellular Ca2+ concentration from 2 mM to 10 mM. 3. The actions of AP on a Ca(2+)-independent K+ current were more complex, 1 microM AP enhanced this current but 10 microM AP had a dual action, initially enhancing but then inhibiting the K+ current. 4. gamma-Aminobutyric acid-activated Cl- currents were also reversibly inhibited by 1 to 10 microM AP. In contrast N-methyl-D-aspartate currents recorded from rat cultured cerebellar neurones were greatly enhanced by 10 microM AP. 5. We conclude that at a concentration of 10 nM, AP is a selective inhibitor of low threshold T-type voltage-activated Ca2+ currents. However, at higher concentrations 1-10 microM AP interacts with ion channels or other membrane constituents to produce a variety of actions on both voltage and ligand gated ion channels.


Adenosine modulation of calcium currents in postganglionic neurones of avian cultured ciliary ganglia. 1. Calcium currents in postganglionic neurones of cultured 7- to 10-day embryonic avian ciliary ganglia were analyzed under whole-cell voltage-clamp and their modulation by 2-chloroadenosine determined. 2. In the presence of tetrodotoxin (200 nM) in the medium to block the Na+ current and CsCl (105 mM) in the patch-clamp electrode to block the K+ current, two different components of the calcium currents (transient and sustained) were identified on the basis of their voltage-dependent kinetics as well as their sensitivity to the dihydropyridine agonist Bay K 8644 and antagonist nifedipine. 3. The sustained current inactivated very slowly (tau greater than 1000 ms; for test potentials from -20 mV to +40 mV) but was reactivated at a holding potential (Vh) of -40 mV. The current was increased on average over 50% by 1 microM of Bay K 8644 at a test potential of 0 mV and decreased over 35% by 1 microM of nifedipine. 4. The transient current inactivated slowly (tau less than 200 ms; for test potentials from -20 mV to +40 mV), and could be completely reactivated at a Vh of -80 mV. This current was unaffected by Bay K 8644 (1 microM) but reduced on average by 8% with nifedipine (1 microM). 5. The sustained and transient currents were decreased more than 70% by 5 microM of omega-conotoxin and decreased more than 50% by 250 microM verapamil. 6. 2-Chloroadenosine (1 microM) decreased the transient current by over 50% and the sustained current by less than 10%. In the presence of nifedipine (1 microM), 2-chloroadenosine decreased the transient current by over 30% and the remaining sustained current by 35%.In the presence of 8-phenyltheophylline (10 microM), 2-chloroadenosine no longer decreased either the transient or sustained currents but did have a slight potentiating effect on both the transient and sustained currents.7. These observations of the effects of 2-chloroadenosine on the transient and sustained currents are discussed in relation to the different calcium channel types at preganglionic nerve terminals.


Stimulation of noradrenaline release in human cerebral cortex mediated by N-methyl-D-aspartate (NMDA) and non-NMDA receptors. 1. Human brain cortical slices from patients undergoing neurosurgery for treatment of epilepsy resistant to antiepileptic drugs were used to identify and characterize N-methyl-D-aspartate (NMDA) and non-NMDA receptors mediating stimulation of noradrenaline release. The slices preincubated with [3H]-noradrenaline were superfused with Krebs-Henseleit solution with or without Mg2+ (1.2 mmol l-1) and were stimulated by 2-min exposure to NMDA, kainic acid or (RS)-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA). 2. In slices superfused without Mg2+, NMDA induced a concentration-dependent tritium overflow. 3. The NMDA-evoked tritium overflow was almost abolished by tetrodotoxin (TTX), Mg2+ or by omission of Ca2+ from the superfusion fluid. 2-Amino-5-phosphonopentanoic acid (AP5; a competitive NMDA receptor antagonist) or dizocilpine (formerly MK-801; an antagonist at the phencyclidine receptor within the NMDA-gated ion channel) inhibited the NMDA-evoked tritium overflow. The stimulatory effect of NMDA was not significantly enhanced by glycine added to the superfusion fluid but was reduced by 7-chlorokynurenic acid (an antagonist at the glycine site coupled to the NMDA receptor). 4. In slices superfused with solution containing Mg2+, kainic acid or AMPA induced a concentration-dependent tritium overflow which was susceptible to blockade by TTX. 5. The kainic acid-evoked tritium overflow was not affected by DL-(E)-2-amino-4-methyl-5-phosphono-3-pentanoic acid (CGP37849; a competitive NMDA receptor antagonist), but was inhibited by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; an antagonist at glutamate receptors of the non-NMDA type). 6. The AMPA-evoked tritium overflow was also inhibited by CNQX.2ń


Penicillin-induced potentiation of glycine receptor-operated chloride current in rat ventro-medial hypothalamic neurones. 1. Effects of penicillin G (PCN) on glycine (Gly)-evoked Cl- current (IGly) were investigated in acutely dissociated rat ventro-medial hypothalamic (VMH) neurones by the whole cell mode of patch clamp technique. 2. When PCN was applied simultaneously with Gly, PCN depressed IGly like a Cl- channel blocker. 3. The PCN-induced blocking action was clearly observed at a low PCN concentration (30 u), while the maximal blockade was achieved by 600 u (units per 10 ml) PCN. 4. When tested solution containing both PCN and Gly was quickly substituted with one containing Gly only, a new rebound-like transient current (I(T)) which also passed through Cl- channel, was elicited. 5. The peak amplitude of I(T) induced by PCN at concentrations higher than 100 u was greater than that induced by glycine alone. We termed this phenomenon PCN-induced potentiation of IGly. In all cells tested, PCN potentiated IGly. 6. At a lower PCN concentration below 30 u, I(T) generation was not clear in the presence of 10(-5) M gamma-aminobutyric acid. With PCN a higher concentration than 300 u, I(T) amplitude was greater than that of the original peak IGly. This was observed in 18 neurones out of 21. The maximal amplitude of the I(T) was achieved with 600 u PCN.


Comparison of the contractile effects of endothelin-1 and sarafotoxin S6b in goat isolated cerebral arteries. 1. The effects of endothelium-derived endothelin-1 and snake venom-derived sarafotoxin S6b, peptides with striking structural and functional similarities, were examined and compared in isolated middle cerebral arteries of goats. 2. Endothelin-1 and sarafotoxin S6b contracted cerebral arteries in a concentration-dependent manner. The potency of endothelin-1 (EC50 = 4.9 (3.9-6.2) x 10(-10) M) was about ten times higher than that of sarafotoxin S6b (EC50 = 5.5 (4.4-6.9) x 10(-9) M). The tension returned to basal values after repeated washings and contraction with endothelin-1 could be reproduced. Endothelin-1 and sarafotoxin S6b induced further contraction in arteries precontracted with prostaglandin F2 alpha (10(-5) M). 3. Mechanical removal of the endothelium or incubation with indomethacin (10(-5) M) displaced the concentration-response curves to endothelin-1 and, more pronouncedly, to sarafotoxin S6b to the left. The maximum response to sarafotoxin S6b was also increased by either of these two treatments. 4. Incubation in 'nominally' Ca(2+)-free medium attenuated the vasoconstrictor response to endothelin-1 but not to sarafotoxin S6b, which was inhibited after incubation in Ca(2+)-free medium to which EGTA (10(-4) M) had been added. Pretreatment with caffeine (2 x 10(-2) M) in Ca(2+)-free medium abolished responses to endothelin-1 and sarafotoxin S6b. 5. Bay K 8644 (10(-10) M, 10(-8) M) enhanced and nicardipine (10(-10) M, 10(-8) M) inhibited in a concentration-dependent manner vasoconstrictor response to endothelin-1. Response to sarafotoxin S6b was only affected by 10(-8) M Bay K 8644 or nicardipine.6. It is concluded that endothelin-1 and sarafotoxin S6b are potent vasoconstrictors of goat cerebral arteries, having direct effects on smooth muscle which are counteracted by the endothelium through the release of a vasodilatator substance, probably prostacyclin. Both endothelin-l and sarafotoxin S6b depend on extracellular Ca2+ and on intracellular, caffeine-sensitive Ca2+ stores to develop vasoconstriction.However, endothelin-l depends to a larger extent than sarafotoxin S6b on free extracellular Ca2+.


Kinetics of nicotinic acetylcholine ion channels in the presence of intravenous anaesthetics and induction agents. 1. Single channel currents activated by 250 nM acetylcholine were recorded from cell-attached patches of BC3H1 mouse tumour cells grown in culture. Channels were recorded in the absence and presence of alphaxalone, diazepam, etomidate, fentanyl, ketamine, meperidine, or propofol. 2. All of the anaesthetics tested shortened channel open time but did not alter single channel current amplitude. Drug concentrations calculated to reduce the time constant of open-time distributions by 50% were 99 microM alphaxalone, 66 microM diazepam, 57 microM etomidate, 26 microM fentanyl, 15 microM ketamine, 16 microM meperidine, or 81 microM propofol. 3. Ketamine, meperidine, and propofol reduced channel open time at concentrations comparable to plasma levels attained during therapeutic use of these agents, while alphaxalone, diazepam, etomidate, and fentanyl reduced channel open time only at levels higher than those encountered clinically. 4. The potency of these drugs in decreasing channel open time appears to be directly correlated with their octanol/buffer partition coefficients. In contrast to expectations, however, agents with higher partition coefficients were less potent in altering channel open time. 5. Ketamine and meperidine produced a prominent third component in closed-time distributions, which were otherwise well described by the sum of two exponential components. Alphaxalone, diazepam, and etomidate also produced a small third component, while no additional component was seen with propofol or fentanyl. These additional components probably arise from creation of an additional closed state of the channel. 6. We conclude that these agents are not altering channel properties merely by exerting non-specific effects via the lipid bilayer and that they are probably not all acting by similar mechanisms.


Effect of human recombinant interleukin-5 on in vitro responsiveness to PAF of lung from actively sensitized guinea-pigs. 1. The intra-tracheal (i.t.) administration of human recombinant interleukin-5 (rhIL-5; 100 or 300 ng) to isolated perfused lungs from guinea-pigs actively sensitized to ovalbumin induced an increased bronchoconstriction and release of thromboxane A2 (TXA2) and histamine into the lung effluent following the subsequent (10 min) intra-arterial injection of platelet-activating factor (PAF). Lung responses to 5-hydroxytryptamine were unaffected by rhIL-5. 2. Hyperresponsiveness to PAF was observed when the lungs were obtained from guinea-pigs used 2 or 7 days after a booster injection of the antigen and, to a lower extent, when they were from animals sensitized by a single antigen administration. By contrast, rhIL-5 did not modify the responses to PAF of lungs from passively sensitized or from adjuvant-treated guinea-pigs, suggesting that immunological stimulation is required to allow the expression of synergism between rhIL-5 and PAF. 3. Guinea-pigs killed 2 and 7 days after the booster injection of the antigen exhibited a marked increase in the number of eosinophils in the bronchoalveolar lavage fluid (BAL), as compared to non-sensitized animals. 4. Our results demonstrate that rhIL-5 and PAF act synergistically to induce enhanced bronchoconstriction and mediator release exclusively when lungs are obtained from guinea-pigs sensitized once to ovalbumin and then boosted. Since recruitment of eosinophils into the airways and the development of hyperresponsiveness to PAF are concomitant, it is suggested that eosinophils are the target cells for interaction between rhIL-5 and PAF.


Calcitonin gene-related peptide increases blood flow and potentiates plasma protein extravasation in the rat knee joint. 1. The effects of calcitonin gene-related peptide (CGRP) and other vasoactive mediators of inflammation on blood flow in the synovial vessels and plasma protein extravasation into the knee (femoro-tibial) joint of the pentobarbitone-anaesthetized rat were measured. 2. Changes in synovial blood flow were estimated by 133xenon clearance from the synovial cavity. CGRP (0.1 pmol and 10 pmol) and prostaglandin E1 (PGE1; 3 pmol and 300 pmol) significantly increased clearance from the knee joint measured 5 min after intra-articular injection. Substance P (10 pmol) had no effect on synovial blood flow. 3. Intra-articular perfusion of the rat knee with CGRP at concentrations up to 0.1 mM, or PGE1 at concentrations up to 10 microM, did not increase plasma extravasation into the synovial cavity measured by accumulation of intravenously injected 125I-albumin in the perfusate. 4. Plasma extravasation into the knee was significantly increased by infusion of bradykinin (0.1 microM), 5-hydroxytryptamine (1 microM) and histamine (0.1 mM), compared with the contralateral joints in the same animals which were perfused with Tyrode solution. 5. Perfusion of the knee joint with substance P did not specifically induce 125I-labelled albumin accumulation in the synovial cavity even at doses that had systemic effects as observed by marked plasma extravasation into other tissues. 6. The increase in plasma extravasation induced by histamine (0.1 mM) was potentiated by co-infusion with CGRP (0.1 microM) and PGE1 (3 microM). However the response to a submaximal dose (0.1 microM) of bradykinin, which induced similar plasma extravasation to histamine (0.1 mM), was not increased by co-infusion with CGRP or PGE1.7. These results show that CGRP is a potent vasodilator in the rat knee. CGRP released from sensory nerves may act synergistically with mediators of increased vascular permeability to modify the inflammatory response in this site.


Long-term potentiation is associated with increased [3H]AMPA binding in rat hippocampus. The location and nature of the changes underlying long-term potentiation (LTP) remain controversial issues. In this study, we tested the possibility that changes in binding properties of the alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionate (AMPA/quisqualate and N-methyl-D-aspartate (NMDA) subtype of glutamate receptors are associated with LTP. LTP was elicited in vivo by stimulation of the perforant pathway in anesthetized rats. One hour following stimulation the animals were sacrificed. We performed quantitative ligand binding autoradiography on frozen brain sections using [3H]AMPA and [3H]N-(1-(2-thienyl)cyclohexyl)-3,4-piperidine ([3H]TCP) to label the AMPA/quisqualate and the NMDA receptors, respectively. No changes in [3H]TCP binding were detected in any of the treatment groups. However, increases in [3H]AMPA binding were observed only in animals that exhibited LTP. These increases were bilateral and present in several subfields of the hippocampus and cortical areas. Administration of the NMDA receptor antagonist, ketamine, prior to tetanic stimulation prevented both the increase in binding and the induction of LTP. These results suggest that changes in the characteristics of AMPA/quisqualate receptors are a biochemical correlate of LTP.