Effect of Exogenous Gangliosides on Amino Acid Uptake and Na+,K+-ATPase Activity in Superior Cervical and Nodose Ganglia of Rats

The effects of some gangliosides on active uptake of nonmetabolizable alpha-aminoisobutyric acid (AIB) and Na+, K+-ATPase and Ca2+, Mg2+-ATPase activities in superior cervical ganglia (SCG) and nodose ganglia (NG) excised from adult rats were examined during aerobic incubation at 37 degrees C for 2 h. In NG, amino acid uptake was greatly accelerated with the addition of galactosyl-N-acetylgalactosaminyl-[N-acetylneuraminyl]-galactosylgluc osyl ceramide (GM1) (85%) and also with N-acetylgalactosaminyl-[N-acetylneuraminyl]-galactosylglucosyl ceramide (GM2) or [N-acetylneuraminyl]-galactosyl-N-acetylgalactosaminyl-[N-acetyl- neuraminyl]-galactosylglucosyl ceramide (GD1a) (43% each) compared with a nonaddition control at a 5 nM concentration. Under identical conditions, Na+, K+-ATPase activity was strongly stimulated with GM1 (180%) and GD1a (93%), whereas Ca2+, Mg2+-ATPase activity showed no change. In SCG, on the other hand, AIB uptake was apparently inhibited (-27%) by addition of GM1, with a slight decrease in Na+, K+-ATPase but no change in Ca2+, Mg2+-ATPase activity in the tissue. Both asialo-GM1, in which N-acetylneuraminic acid is deficient, and Forssman glycolipid, which is not present in nervous tissue, failed to produce any significant increase in both SCG and NG not only in amino acid uptake, but also in Na+, K+-ATPase activity. A kinetic study of active AIB uptake showed that GM1 ganglioside produced an increase in Km with no change in Vmax in SCG, whereas it caused a decrease in Km with a slight increase in Vmax in NG. Treatment of NG and SCG with neuraminidase from Vibrio cholerae, an enzyme that split off sialic acid from polysialoganglioside, leaving GM1 intact, caused little inhibition of the amino acid uptake.(ABSTRACT TRUNCATED AT 250 WORDS)     

Selective potentiation of 5-methyl-2-(1-methylcyclohexyl)-4-oxazoleacetic acid (AD-4610) on glucose-induced insulin secretion

In the perfused pancreas from normal SD rats, AD-4610 (0.01-0.1 mM) potentiated biphasic insulin secretion induced by 7.5 mM of glucose. The concentration-response curve of insulin secretion to glucose was shifted leftwards with AD-4610 (0.1 mM) without altering either the threshold concentration of glucose to induce insulin secretion or the maximal insulin response to glucose, indicating increased sensitivity of the pancreatic B-cells to glucose. On the other hand, AD-4610 was 10-fold less effective in altering insulin secretion induced by arginine and glyceraldehyde. The effect of AD-4610 on insulin secretion and glucose metabolism was compared with that of tolbutamide in vivo. AD-4610 (100 mg/kg) potentiated insulin secretion induced by an intravenous glucose load, and also accelerated glucose metabolism without altering basal insulin secretion in normal rats. On the other hand, tolbutamide (20 mg/kg) increased basal insulin secretion, but slightly decreased glucose-induced insulin secretion. In yellow KK mice with hyperglycemia, AD-4610 (10-100 mg/kg) had a dose-dependent hypoglycemic action, but tolbutamide did not. Thus, AD-4610 stimulated insulin secretion in a glucose-dependent fashion and enhanced glucose metabolism in vivo. These results suggest that AD-4610 selectively potentiates glucose-induced insulin secretion by increasing the sensitivity of pancreatic B-cells to glucose and may be useful for treating human NIDDM through a different mechanism than that of tolbutamide.     

Transplacental action of sodium nitrite on embryonic cells of Syrian golden hamster

Hamster embryos were treated with various doses of NaNO2 in utero, by its oral administration to the mothers, and then the embryonic cells were examined for micronucleus formation, chromosomal aberrations, morphological or malignant transformation and drug-resistant mutations. For induction of resistant mutations, the cells were cultured in normal medium for 72 h, and then selected in media containing 8-azaguanine (10 or 20 microgram/ml) or 1 mM ouabain. This treatment with NaNO2 caused marked dose-dependent induction of 8-azaguanine- and ouabain-resistant mutations. Cultured embryonic fibroblasts in the resting state also showed a marked dose-dependent increase in micronucleus formation but not an increase in chromosomal aberrations. This treatment also caused morphological and neoplastic transformation of the cells. Transplacental oral treatment with DMN, as a positive control, caused changes of similar extent in biological effects of embryonic fibroblasts, and in addition it caused chromosomal aberrations in metaphase plates. On the contrary, transplacental oral application of NaNO2 did not induce any biological change in cultured embryonic fibroblasts.     

Effects of an inhibitor of glucosylceramide synthase on glycosphingolipid synthesis and neurite outgrowth in murine neuroblastoma cell lines.

1-Phenyl-2-decanolyamino-3-morpholino-1-propanol (PDMP), an effective inhibitor of UDP-glucose:ceramide glucosyltransferase, caused inhibition of cell growth in murine neuroblastoma cell lines. Metabolic labeling of glycosphingolipids with [14C]galactose in NS-20Y, Neuro2a, and N1E-115 cells showed reduced incorporation of radioactivity into gangliosides and neutral glycosphingolipids when threo-PDMP was present in the medium. Treatment of NS-20Y cells with threo-PDMP resulted in a time-dependent decrease in mass levels of gangliosides and neutral glycosphingolipids. After 24 h in the presence of 50 microM threo-PDMP, neutral glycosphingolipid mass was reduced to 32%, where glucosylceramide was the most affected (90% decrease). The ganglioside mass was reduced to 57% of the original content. Neurite outgrowth from neuroblastoma cells in serum-free medium was significantly inhibited by threo-PDMP in a dose-dependent manner. Threo-PDMP also caused retraction of neurites which had been induced to extend in serum-free medium. Pretreatment of cells with GM1 partially restored the ability of NS-20Y cells for neurite outgrowth in the medium containing threo-PDMP. These results suggest a possible role for glycosphingolipids in neurite outgrowth of murine neuroblastoma cells.     

Studies on protein folding, unfolding and fluctuations by computer simulation. I. The effect of specific amino acid sequence represented by specific inter-unit interactions.

A lattice model of proteins is introduced. "A protein molecule" is a chain of nown-intersecting units of a given length on the two-dimensional square lattice. The copolymeric character of protein molecules is incorporated into the model in the form of specificities of inter-unit interactions. This model proved most effective for studying the statistical mechanical characteristics of protein folding, unfolding and fluctuations. The specificities of inter-unit interactions are shown to be the primary factors responsible for the all-or-none type transition from native to denatured states of globular proteins. The model has been studied by the Monte Carlo method of Metropolis et al., which is now shown applied to approximately simulating a kinetic process. In the strong limit of the specificity of the inter-unit interaction the native conformation was reached in this method by starting from an extended conformation. The possible generalization and application of this method for finding the native conformation of proteins form their amino sequence are discussed.     

Cholesterol transport between red blood cells and lipoproteins contributes to cholesterol metabolism in blood

Lipoproteins play a key role in transport of cholesterol to and from tissues. Recent studies have also demonstrated that red blood cells (RBCs), which carry large quantities of free cholesterol in their membrane, play an important role in reverse cholesterol transport. However, the exact role of RBCs in systemic cholesterol metabolism is poorly understood. RBCs were incubated with autologous plasma or isolated lipoproteins resulting in a significant net amount of cholesterol moved from RBCs to HDL, while cholesterol from LDL moved in the opposite direction. Furthermore, the bi-directional cholesterol transport between RBCs and plasma lipoproteins was saturable and temperature-, energy-, and time-dependent, consistent with an active process. We did not find LDLR, ABCG1, or scavenger receptor class B type 1 in RBCs but found a substantial amount of ABCA1 mRNA and protein. However, specific cholesterol efflux from RBCs to isolated apoA-I was negligible, and ABCA1 silencing with siRNA or inhibition with vanadate and Probucol did not inhibit the efflux to apoA-I, HDL, or plasma. Cholesterol efflux from and cholesterol uptake by RBCs from Abca1^+/+ and Abca1^−/− mice were similar, arguing against the role of ABCA1 in cholesterol flux between RBCs and lipoproteins. Bioinformatics analysis identified ABCA7, ABCG5, lipoprotein lipase, and mitochondrial translocator protein as possible candidates that may mediate the cholesterol flux. Together, these results suggest that RBCs actively participate in cholesterol transport in the blood, but the role of cholesterol transporters in RBCs remains uncertain.     

Genetic mapping of ossification of the posterior longitudinal ligament of the spine.

Ossification of the posterior longitudinal ligament of the spine (OPLL) is recognized as a common disorder among Japanese and throughout Asia. Estimates of its prevalence are in the range of 1. 9%-4.3%. Although its etiology is thought to involve a multiplicity of factors, epidemiological and family studies strongly implicate genetic susceptibility in the pathogenesis of OPLL. In this study we report an identification of a predisposing locus for OPLL, on chromosome 6p, close to the HLA complex. The evidence for this localization is provided by a genetic-linkage study of 91 affected sib pairs from 53 Japanese families. In this sib-pair study, D6S276, a marker lying close to the HLA complex, gives evidence for strongly significant linkage (P = .000006) to the OPLL locus. A candidate gene in the region, that for collagen 11A2, was analyzed for the presence of molecular variants in affected probands. Of 19 distinct variants identified, 4 showed strong statistical associations with OPLL (highest P = .0004). These observations of linkage and association, taken together, show that a genetic locus for OPLL lies close to the HLA region, on chromosome 6p.     

Pharmacophore Modeling for Anti-Chagas Drug Design Using the Fragment Molecular Orbital Method

BackgroundChagas disease, caused by the parasite Trypanosoma cruzi, is a neglected tropical disease that causes severe human health problems. To develop a new chemotherapeutic agent for the treatment of Chagas disease, we predicted a pharmacophore model for T. cruzi dihydroorotate dehydrogenase (TcDHODH) by fragment molecular orbital (FMO) calculation for orotate, oxonate, and 43 orotate derivatives.Conclusions/SignificanceFMO-based interaction energy analyses revealed a pharmacophore model for TcDHODH inhibitor. Hydrogen bond acceptor pharmacophores correspond to Lys43 and Lys214, hydrogen bond donor and acceptor pharmacophores correspond to Asn67 and Asn194, and the aromatic ring pharmacophore corresponds to FMN, which shows important characteristics of compounds that inhibit TcDHODH. In addition, the Lys214 residue is not conserved between TcDHODH and human DHODH. Our analysis suggests that these orotate derivatives should preferentially bind to TcDHODH, increasing their selectivity. Our results obtained by pharmacophore modeling provides insight into the structural requirements for the design of TcDHODH inhibitors and their development as new anti-Chagas drugs.     

Peripheral tolerance and the qualitative characteristics of autoreactive T cell clones in primary biliary cirrhosis

Primary biliary cirrhosis is characterized by autoreactive T cells specific for the mitochondrial Ag PDC-E2(163-176). We studied the ability of eight T cell clones (TCC) specific for PDC-E2(163-176) to proliferate or become anergic in the presence of costimulation signals. TCC were stimulated with either human PDC-E2(163-176), an Escherichia coli 2-oxoglutarate dehydrogenase mimic (OGDC-E2(34-47)), or analogs with amino acid substitutions using HLA-matched allogeneic PBMC or mouse L-DR53 fibroblasts as APC. Based on their differential responses to these peptides (human PDC-E2(163-176), E. coli OGDC-E2(34-47)) in the different APC systems, TCC were classified as costimulation dependent or independent. Only costimulation-dependent TCC could become anergic. TCC with costimulation-dependent responses to OGDC-E2 become anergic to PDC-E2 when preincubated with mimic, even if costimulation is independent for PDC-E2(163-176). Anergic TCC produced IL-10. One selected TCC could not become anergic after preincubation with PDC-E2(163-176)-pulsed L-DR53 but became anergic using L-DR53 pulsed with PDC-E2 peptide analogs with a substitution at a critical TCR binding site. TCC that only respond to peptide-pulsed PBMC, but not L-DR53, proliferate with peptide-pulsed CD80/CD86-transfected L-DR53; however, anergy was not induced with peptide-pulsed L-DR53 transfected with only CD80 or CD86. These data highlight that costimulation plays a dominant role in maintaining peripheral tolerance to PBC-specific Ags. They further suggest that, under specific circumstances, molecular mimicry of an autoantigen may restore rather than break peripheral tolerance.