First Identification of the MHC-DPA1 Alleles in Tibetan Macaques (Macaca thibetana).
Ten novel MHC-DPA1 alleles were identified in Tibetan macaques (Macaca thibetana).
Animals↗
Biomedical subjects
H Chen
Publications and source records attributed to H Chen.
Mutational analysis of the adenovirus 2 IVa2 initiator and downstream elements.
The initiator element of the adenovirus type 2 IVa2 promoter is sufficient to direct accurate initiation by RNA polymerase II. Analysis of the effects of substitution of specific base pairs on initiator activity in in vitro transcription systems indicated that specific sequences between positions -4 and +5 were essential for initiator activity. Mutations that impaired or eliminated initiator activity altered both base pairs that are conserved in sequence-related initiators and nonconserved sequences. Neither the downstream TA-rich sequence of the IVa2 promoter, nor the adenovirus 2 major late TATA element placed at the same downstream site could overcome the severe inhibitory effects of initiator mutations, indicating that the initiator is the primary determinant of the specificity and direction of IVa2 transcription. By contrast, when the ML TATA element was placed 31 nucleotides upstream of the IVa2 initiator, the precise specificity, but neither the efficiency nor direction of transcription, depended on the presence of a functional initiator. Activity of the IVa2 promoter was relatively insensitive to changes in the orientation or nature of the TA-rich sequence. Furthermore, only a promoter containing the ML TA-TAAAA sequence downstream of the IVa2 initiator was competent to direct both IVa2 transcription and transcription from the opposite strand. The implications of this functional difference for recognition of the downstream element are discussed.
Adenoviruses, Human↗
Sphingomyelin content of intestinal cell membranes regulates cholesterol absorption. Evidence for pancreatic and intestinal cell sphingomyelinase activity.
Micellar cholesterol uptake and secretion were investigated in the human intestinal cell line CaCo-2 following depletion of apical membrane sphingomyelin. The addition of exogenous sphingomyelinase, which hydrolysed 60% of prelabelled sphingomyelin, resulted in a 50% decrease in the uptake of cholesterol from bile salt micelles. The flux of membrane cholesterol into the cell by the hydrolysis of membrane sphingomyelin decreased the rate of cholesterol synthesis by 43% and inhibited hydroxymethylglutaryl-CoA reductase activity by 54%. Moreover, the rate of cholesterol esterification was increased 4-fold. Total cellular cholesterol mass was unchanged by the addition of sphingomyelinase; however, cholesteryl esters increased by 50% and the amount of unesterified cholesterol decreased significantly. The basolateral secretion of cholesterol mass was also decreased following sphingomyelin hydrolysis. Human pancreatic juice was found to contain neutral sphingomyelinase activity which required taurocholate for full expression. The presence of neutral sphingomyelinase activity was also documented in membranes prepared from CaCo-2 cells and in whole homogenates from human duodenal biopsies. The data suggest that the amount of sphingomyelin present in the apical membrane of the intestinal absorptive cell regulates cholesterol uptake from bile salt micelles. Sphingomyelinase activity within intestinal cells and in pancreatic juice could alter the sphingomyelin content of brush-border membranes of small intestinal absorptive cells and thus regulate the amount of cholesterol absorbed by the gut.
Cells, Cultured↗
Derivation of a biologically contained replication system for human immunodeficiency virus type 1.
Human immunodeficiency virus type 1 (HIV-1) proviral mutants that lack viral regulatory genes are unable to replicate unless rescued by complementation in trans. Structurally intact virus can be produced by infecting recombinant cell lines expressing the deficient genes. A HIV-1 mutant functionally defective in tat and rev (vIIIB delta Tat/Rev), which replicates only in a recombinant T-cell line expressing tat and rev (CEMTART), is described in this report. Infection of the CEMTART cell line with vIIIB delta Tat/Rev permits the complete HIV-1 life cycle, including cytopathology, decreased expression of CD4, and production of viral structural proteins, to be biologically contained. Culture supernatants from infected CEMTART contain virus that is able to replicate only in uninfected CEMTART. No reversion of vIIIB delta Tat/Rev to wild-type HIV-1 was observed as measured either by sequencing proviral vIIIB delta Tat/Rev or by detecting the ability of vIIIB delta Tat/Rev to replicate in CEM or activated CD4-bearing T lymphocytes. Defective HIV-1 mutants produced by trans complementation of essential genes permit infection and analysis of defined genotypes on cellular function and phenotype. Authentic HIV-1 structural proteins and infected cells can be prepared in mass, and agents that interfere with the HIV-1 life cycle can be studied on a large scale with minimum risk of exposing workers to virulent HIV-1.
3T3 Cells↗
Quantitation of phenacyl esters of retinal fatty acids by high-performance liquid chromatography.
A high-performance liquid chromatographic (HPLC) method for the separation and quantitation of retinal fatty acids containing long-chain polyunsaturated fatty acids is described. Fatty acids from frog retinal lipids were converted to the corresponding phenacyl derivatives which were separated on a C18 reversed-phase column and detected at 242 nm. Molar absorptivities (peak area units/nmol) of up to seventeen fatty acid phenacyl derivatives were determined and used for quantitation of fatty acids separated by HPLC. Compared with gas chromatography, the HPLC method gave a similar molar percent distribution of the fatty acids and was twenty to fifty times more sensitive. This HPLC method provides a useful means for the study of chemistry and metabolism of long-chain polyunsaturated fatty acids in retina and other tissues where amounts of material may be limited or recovery of individual components desirable.
Acetophenones↗
Effects of somatostatin on potassium currents in bullfrog sympathetic ganglion neurones: possible role of receptor subtypes.
The effects of whole somatostatin (wSS; somatostatin-28) and cyclic somatostatin (cSS; somatostatin-14) were examined on patch-clamped bullfrog sympathetic ganglion neurones. In the C-cells, where muscarine produces hyperpolarization, wSS was also inhibitory and activated an inwardly-rectifying K+ current; cSS was ineffective. By contrast, in the B-cells, where muscarine produces excitatory effects, cSS was also excitatory and was more effective than wSS in suppressing a voltage-dependent, non-inactivating K(+)-current (IM). These results are consistant with the idea that excitatory and inhibitory effects of somatostatin-derived peptides may be mediated via different receptor subtypes.
Animals↗
Purification and characterization of two extracellular beta-glucosidases from Trichoderma reesei.
A major beta-glucosidase I and a minor beta-glucosidase II were purified from culture filtrates of the fungus Trichoderma reesei grown on wheat straw. The enzymes were purified using CM-Sepharose CL-6B cation-exchange and DEAE Bio-Gel A anion-exchange chromatography steps, followed by Sephadex G-75 gel filtration. The isolated enzymes were homogeneous in SDS-polyacrylamide gel electrophoresis and isoelectric focusing. beta-Glucosidase I (71 kDa) was isoelectric at pH 8.7 and contained 0.12% carbohydrate; beta-glucosidase II (114 kDa) was isoelectric at pH 4.8 and contained 9.0% carbohydrate. Both enzymes catalyzed the hydrolysis of cellobiose and p-nitrophenyl-beta-D-glucoside (pNPG). The Km and kcat/Km values for cellobiose were 2.10 mM, 2.45.10(4) s-1 M-1 (beta-glucosidase I) and 11.1 mM, 1.68.10(3) s-1 M-1 (beta-glucosidase II). With pNPG as substrate the Km and kcat/Km values were 182 microM, 7.93.10(5) s-1 M-1 (beta-glucosidase I) and 135 microM, 1.02.10(6) s-1 M-1 (beta-glucosidase II). The temperature optimum was 65-70 degrees C for beta-glucosidase I and 60 degrees C for beta-glucosidase II, the pH optimum was 4.6 and 4.0, respectively. Several inhibitors were tested for their action on both enzymes. beta-Glucosidase I and II were competitively inhibited by desoxynojirimycin, gluconolactone and glucose.
Cations, Divalent↗
Calmodulin increases Ca-dependent inhibition of the Na,K-ATPase in human red blood cells.
Proteins in human red cell hemolysate were purified to determine which of them increase inhibition of the Na,K-ATPase in the presence of 2 microM free Ca. Samples purified 600,000-fold inhibited the Na,K-ATPase of human red cells in a Ca-dependent manner and stimulated the (Ca+Mg)-ATPase. These samples contained two proteins as analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE): calmodulin (18,000 Mr), which comprised most (greater than 90%) of the total protein, and an unidentified protein of approximately 13,000 Mr. Both proteins were a distinctive light yellow when stained with silver. Calmodulin from bovine testes also inhibited the Na,K-ATPase and stimulated the (Ca+Mg)-ATPase. This preparation also contained two proteins as analyzed by SDS-PAGE: calmodulin (95 to 99% of the total protein) and another protein of approximately 13,000 Mr (1 to 5% of the total protein). Both were light yellow when stained with silver. Since the amount of red cell protein was limited, the remainder of the study was carried out with the bovine testes preparation. Heating the testes preparation decreased, but did not abolish, inhibition of the Na,K-ATPase and reduced stimulation of the (Ca+Mg)-ATPase. When corrected for denatured calmodulin, both heated and unheated proteins increased inhibition of the Na,K-ATPase to the same extent. The Na,K-ATPase was inhibited at 2 microM free Ca in a dose-dependent manner over a range of 15 to 100 nM calmodulin. To establish if the inhibition was due to the calmodulin or the 13,000 Mr protein, both were electroeluted after SDS-PAGE. Electroeluted calmodulin stimulated the (Ca+Mg)-ATPase and increased Ca inhibition of the Na,K-ATPase. Electroeluted amounts of the smaller Mr protein slightly stimulated the (Ca+Mg)-ATPase, but had no effect on the Na,K-ATPase. This protein was digested with cyanogen bromide, partially sequenced, and thereby identified as a fragment of calmodulin. We conclude that intact calmodulin increases inhibition of the Na,K-ATPase at 2 microM free Ca. We suggest that calmodulin is part of a mechanism mediating the effects of physiological free Ca on the Na,K-ATPase.
Amino Acid Sequence↗
Genomic analysis using a yeast artificial chromosome library with mouse DNA inserts.
A yeast artificial chromosome library with mouse genomic DNA inserts has been constructed. The library encompasses a 2.5-fold coverage of the mouse genome, with an average insert size of 250 kilobases. The screening strategy uses the polymerase chain reaction on pooled DNAs prepared from individually stored clones. The usefulness of the library for chromosome walking was illustrated by constructing a 600-kilobase-long contig of DNA surrounding Hba-ps4, a DNA marker that is tightly linked to the fused (Fu) locus on chromosome 17.
Animals↗
Changes in lipid content and composition during the development of N-nitrosodiethylamine induced hepatocarcinoma.
Alterations in lipid content and composition in the N-nitrosodiethylamine-induced hepatocarcinoma were investigated. Rats were administered with N-nitrosodiethylamine in the drinking water for 12 weeks followed by normal tap water for another 6 weeks. The cholesterol content in the liver was increased shortly after the administration of N-nitrosodiethylamine and remained elevated after the removal of the nitrosoamine from the water. The phosphatidylethanolamine level was elevated during N-nitrosodiethylamine administration with a concomitant reduction in phosphatidylcholine level. Lysophosphatidylcholine and sphingomyelin levels were increased during the last four weeks of the study. The level of phosphatidylinositol was substantially reduced after eight weeks of N-nitrosodiethylamine treatment, and remained low during the post-treatment period. We postulate that changes in lysophosphatidylcholine and sphingomyelin may be a compensatory mechanism for maintaining the asymmetrical distribution of choline-containing lipids in the outer leaflet of the membrane. The elevated level of cholesterol may be a useful indicator for the early detection of N-nitrosodiethylamine-induced hepatocarcinoma.
Animals↗
Absence of antibodies to human herpesvirus-6 in patients with slowly-progressive human immunodeficiency virus type 1 infection.
To evaluate a possible role for Human Herpesvirus-type 6 (HHV-6) coinfection as a co-factor in the progression of HIV-1 disease, we investigated the prevalence of seropositivity for HHV-6 in a cohort of HIV-1 infected patients. These patients were retrospectively divided into two groups according to the decline of CD4+ T cells during the follow up: 11 were classified as rapid decliners (less than 400 CD4+/cmm within 1 year), and 38 as slow decliners (greater than 400 CD4+/cmm after at least 4 years' follow up). HHV-6 antibodies were detected by a commercial immunofluorescence assay and by a Western blotting assay developed in our laboratory. Our results show that Western blot appears to provide results satisfactorily free of false positivities. We found that the frequency of HHV-6 seropositivity was significantly lower in the group of slow decliners, compared both to rapid decliners and to the general population. These data suggest a role for HHV-6 co-infection in the progression of HIV-1 disease.
Adolescent↗
Neuronal damage, glial response and cerebral metabolism after hypothermic forebrain ischemia in the rat.
We investigated the effect of 30 degrees C whole body hypothermia on neuronal injury, astroglial reactivity and intracellular pH in rats subjected to 15 min of forebrain ischemia. Experimental groups included: (1) normothermic ischemia (n = 8), ischemia induced under 37 degrees C body temperature, (2) hypothermic ischemia (n = 6), ischemia induced under 30 degrees C body temperature. Cerebral intracellular pH was measured using in vivo 31P NMR spectroscopy over 7 days. Neuronal injury and astrocytic reactivity were evaluated using hematoxylin and eosin staining, and immunoreactivity to glial fibrillary acidic protein, respectively. Normothermic animals revealed significant alkalosis (P less than 0.01) at 48 h after ischemia compared to the pre-ischemic value. No significant intracellular pH change was detected after ischemia in the hypothermic group. Ischemic neuronal injury was prevented in the hypothermic animals, compared to the severe neuronal injury found in the normothermic animals (P less than 0.01). The marked astrocytosis of normothermic animals was significantly inhibited in the hypothermic animals (P less than 0.01). Our data indicate, that hypothermia significantly inhibits neuronal injury as well as post-ischemic alkaloids and astrocytosis, induced by 15 min of forebrain ischemia in the rat.
Animals↗
Pharmacokinetics of haloperidol and reduced haloperidol in Chinese schizophrenic patients after intravenous and oral administration of haloperidol.
The pharmacokinetics of haloperidol were studied in eight Chinese schizophrenic patients after intravenous administration and in six of the patients who also received oral haloperidol. After intravenous dosing, haloperidol disposition was best characterized by a three compartment model. The mean elimination half-life of 54.8 h determined by model dependent analysis was similar to the mean elimination half-life of 59.9 h determined by model independent analysis. The mean plasma clearance was 21.71/h and the mean volume of distribution during the distribution phase was 1754.3 1. After oral dosing, bioavailability of haloperidol was 35 +/- 8%, suggesting extensive first pass metabolism. Determination of reduced haloperidol concentration confirmed a previous finding of significant variability in haloperidol reductive capacity in individual patients. Comparison of area under the plasma concentration-time curve of reduced haloperidol after intravenous and oral administration of haloperidol suggests that reduction of haloperidol may only account for a small portion of first pass metabolism of haloperidol. However, conversion of reduced haloperidol back to haloperidol necessitates the monitoring of both haloperidol and reduced haloperidol concentrations in clinical practice.
Administration, Oral↗
The inhibitory effect of water on the Co2+ and Cu2+ catalyzed decomposition of methyl linoleate hydroperoxides.
The inhibitory effect of water on the decomposition of methyl linoleate hydroperoxides (MLHP) catalyzed by Co2+ and Cu2+ was studied in a model system using proton nuclear magnetic resonance (NMR) spectroscopy. MLHP were prepared by photoxidation and purified by chromatographic methods. Proton NMR spectroscopy was used to measure reaction rates by monitoring changes in the intensity of the OOH signal. The rate constant of the reaction was obtained by plotting the natural logarithm of MLHP concentration vs time. In the first part of the study, no transition metals were added to the model system, so that the effect of water could be attributed to the interaction between water and MLHP only. The rate constant of the reaction (K) was found inversely proportional to the concentration of water. There was a downfield chemical shift of both hydroperoxide and water peaks in the NMR spectra when water was added. As temperature increased to 40 degrees C, the difference in K between the systems with 0% and 2% water disappeared. It is proposed that the hydroperoxides were solvated with water which retarded their decomposition. When Co2+ was added to the model system, K decreased as the concentration of water increased from 0% to 1.5%. As temperature increased from 18 degrees C to 40 degrees C, differences between the K for 0% and 2% water disappeared. A similar phenomenon was observed in reactions catalyzed with Cu2+. These findings would support a mechanism in which the protective effect of water involves both the solvation of OOH and hydration of the metal catalyst.
Acetone↗
Docosahexaenoic acid increases in frog retinal pigment epithelium following rod photoreceptor shedding.
The vertebrate retina conserves docosahexaenoic acid (22:6n-3) during n-3 fatty acid deficiency. The mechanism of conservation is not known, although recycling of this fatty acid between the retinal pigment epithelium (RPE) and retina is one possibility. We examined the role of the RPE in conservation of 22:6n-3 by quantitating the fatty acids and phospholipid molecular species (PLMS) in frog RPE before and after light-stimulated shedding of rod outer segments (ROS). RPE cells were dissociated with brush agitation and purified by a discontinuous ficoll density gradient. One hour after the light-induced shedding of ROS, the phagocytosed ROS tip and opsin content of RPE had increased. Simultaneously, the levels of 22:6n-3 and 22:6(n-3)-containing PLMS were increased in the RPE. Within 8 hr following the shedding event, 22:6n-3 in the RPE had returned to the dark level. These findings indicate that the phagocytosed ROS tips contain 22:6n-3 and that the RPE metabolizes these ROS tips and eliminates 22: 6n-3 from the cell. Thus, the RPE is intimately involved in the metabolism of 22: 6n-3 in the retina. The recycling of 22: 6n-3 from the RPE to the retina is a possible means of conserving this important fatty acid in the retina.
Animals↗
Decreased docosahexaenoic acid levels in retina and pigment epithelium of frogs fed crickets.
Whole retina, rod outer segments, and retinal pigment epithelium of frogs (Rana pipiens) fed crickets for more than 1 year had significantly lower levels of docosahexaenoic acid (22: 6n-3) than the same tissues of frogs fed crickets for less than 1 month. Decreases in 22:6n-3 levels in these tissues were compensated for by increases in the n-6 polyunsaturated fatty acids (PUFAs), primarily 22:5n-6. There were no changes in the levels of saturated, monoenoic, or dienoic acids. Analysis of diacyl phospholipid molecular species (PLMS) revealed decreases in both the 22:6(n-3)-containing dipolyenoic molecular species in phosphatidylethanolamine and phosphatidylserine, and the monopolyenoic molecular species in phosphatidylcholine. These PLMS were replaced by species containing 22:5n-6 or other n-6 PUFAs. Examination of fatty acid methyl esters of total lipids extracted from crickets revealed that less than 1 mol% fatty acids were of the n-3 family, while more than 30 mol% were of the n-6 family. Thus, frogs raised on an n-3-deficient diet have reduced levels of n-3 PUFA in their retinas, rod outer segments, and retinal pigment epithelium. Although such changes have been reported for mammals, this is the first report of the effects of n-3 deficiency on the lipids of amphibians.
Animals↗
The effects of post-ischemic hypothermia on the neuronal injury and brain metabolism after forebrain ischemia in the rat.
We investigated the effect of moderate post-ischemic hypothermia on neuropathological outcome and cerebral high energy phosphate metabolism, intracellular pH and Mg2+ concentration in the rat. Three groups of animals were investigated: (1) Wistar rats subjected to 12 min of forebrain ischemia under normothermic conditions (n = 17), (2) rats subjected to the identical procedure of ischemia, except that 30 degrees C hypothermia was induced post-ischemia and maintained for 2 h of reperfusion (n = 6), and (3) control hypothermic rats not subjected to ischemia (n = 4). In vivo 31P NMR spectroscopy was performed prior to ischemia, and at intervals up to 168 h after ischemia. Histological analysis of brain tissues was performed 7 days after ischemia. No significant differences in cortical and hippocampal neuronal damage was detected between the two experimental groups. Significantly lower pH values were detected in the hypothermic ischemic animals at 24 h (P = 0.0001) and 48 h (P = 0.018) post-ischemia compared to the normothermic ischemic animals. Normothermic ischemic animals exhibited significantly lower [Mg2+] at 72 h (P less than 0.006) compared to the pre-ischemia level. Our data indicate that post-ischemic hypothermia modifies the profiles of post-ischemic brain tissue pH and Mg2+ concentration, and this modification is not associated with histopathological outcome 7 days after ischemia.
Adenosine Triphosphate↗
Effects of rapamycin on human HLA-unrestricted cell killing.
Rapamycin (RAPA) is a potent immunosuppressant and can effectively prevent allograft rejection at a dosage 10- to 100-fold lower than that of cyclosporin A. RAPA strongly inhibits proliferation and function of T and B cells. In this study, we investigated the effect of RAPA on human HLA-unrestricted cell killing. It was shown that in vitro RAPA inhibited the cytolytic effect of natural killer cells and lymphokine-activated killer cells (LAK) and inhibited antibody-dependent cell-mediated cytotoxicity. The effective concentration of RAPA was 10- to 100-fold higher than that required for inhibiting T cell proliferation. These results suggest that there could be a therapeutic dose window at which RAPA inhibits T cell activity while it leaves HLA-unrestricted cell killing unaffected. We also demonstrated that while IL4 inhibited LAK activity, and RAPA inhibited IL4-promoted T cell proliferation, RAPA was not able to antagonize IL4's inhibitory effect on LAK. This indicates that the mechanism of interaction between RAPA and IL4 on LAK is different from that on T cells.
Antibody-Dependent Cell Cytotoxicity↗