Glycosaminoglycan polysulfate-induced stimulation of hyaluronic acid synthesis in rabbit knee synovial membrane: involvement of binding protein and calcium ion

We have previously reported that naturally occurring sulfated glycosaminoglycans having a chondroitin-type structure and glycosaminoglycan polysulfate (GAGPS, a persulfated derivative of chondroitin sulfate) caused a specific stimulation of hyaluronic acid synthesis in rabbit knee synovial membranes [H. Nishikawa et al. (1985) Arch. Biochem. Biophys. 240, 146-153]. In the present study, the interaction of [3H]GAGPS and the surface of the rabbit knee synovial membranes and the relationship between this interaction and the stimulatory effect of GAGPS on the hyaluronic acid synthesis were examined in order to define the stimulatory mechanism of hyaluronic acid synthesis by GAGPS. A part of the [3H]GAGPS taken up by the synovial membranes was released from the membranes by trypsin treatment. The interaction of [3H]GAGPS and the surface of the isolated synovial membranes was diminished by pretreatment of the membranes with proteases or chelating reagents. Pretreatment of synovial membranes with trypsin or ethylene glycol bis(beta-aminoethyl ether) N,N'-tetraacetic acid had little effect on the basal hyaluronic acid synthesis but caused the loss of GAGPS-induced stimulation of hyaluronic acid synthesis accompanied by significant decrease (20% P less than 0.05-P less than 0.01) in the interaction between GAGPS and the surface of the synovial membranes. Dermatan sulfate having a chondroitin-type structure also stimulated hyaluronic acid synthesis but this effect was not additive to the stimulation of hyaluronic acid synthesis by GAGPS. Heparin had no effect on either the basal hyaluronic acid synthesis or the GAGPS-induced stimulation of hyaluronic acid synthesis. These results indicate that binding of GAGPS to certain distinct protein components on the surface of synovial membranes is involved in the stimulatory mechanism of hyaluronic acid synthesis by GAGPS, and that the binding may be mediated by Ca2+ ion. The binding was also found to be specific for sulfated glycosaminoglycans having a chondroitin-type structure.     

Participation of cytochrome P450 in mutagenic activation of the carcinogen 3'-hydroxymethyl-N,N-dimethyl-4-aminoazobenzene and its N-demethylated compounds by rat liver

Mutagenicity of the hepatocarcinogen 3'-hydroxymethyl-N, N-dimethyl-4-aminoazobenzene (3'-CH2OH-DAB) and its N-demethylated compounds was examined. Rat-liver 9000 X g supernatant (S9) fraction was used together with Salmonella typhimurium TA98 or TA100 as a tester strain. The expression of mutagenicity of 3'-CH2OH-DAB, 3'-hydroxymethyl-N-methyl-4-aminoazobenzene (3'-CH2OH-MAB) and 3'-hydroxymethyl-4-aminoazobenzene (3'-CH2OH-AB) required the presence of both microsomes and cytosol as sources of enzymes as well as NADPH as a cofactor. 3'-CH2OH-AB showed positive mutagenicity on both strains in the presence of liver S9 from untreated rats whereas 3'-CH2OH-DAB and 3'-CH2OH-MAB were negative. The treatment of rats with polychlorinated biphenyls (PCB) or 3-methylcholanthrene (3-MC) resulted in a marked increase in the ability of S9 to activate these three compounds, whereas phenobarbital (PB) induction was not effective, except for the activation of 3'-CH2OH-AB. The mutagenic activities of the three compounds in strain TA98 were considerably decreased by adding cytochrome c to the S9 mixture, but the activation reactions were insensitive to 1-(1-naphthyl)-2-thiourea (NTU) and methimazole, high-affinity flavin-containing monooxygenase (FMO) substrates. Metyrapone and 2-diethylaminoethyl-2,2-diphenylvalerate hydrochloride (SKF-525A, potent inhibitors of cytochrome P450, had no inhibitory effect on the activation of these compounds by S9 from PCB-treated rat livers. In contrast, 7,8-benzoflavone (BF), a specific inhibitor of cytochrome P448, decreased the activities of 3'-CH2OH-DAB and 3'-CH2OH-MAB by 88 and 78%, respectively, but the inhibition was negligible for 3'-CH2OH-AB.     

Zymograms and histochemistry of non-specific esterase in the salivary glands

Summary Zymogramic analysis of esterase in the mouse, rat and guinea-pig salivary glands was undertaken and demonstrated species and organ specificities of esterase with electrophoretic method. Salivary glands esterase was classified into A, B and C types based on the electrophoretic mobility. Mouse submandibular gland had the most complicated pattern, while guinea-pig showed the simpliest patterns which was devoid of B type of esterase. Rat salivary glands exhibited rather regular patterns. Similar zymogram patterns were obtained with many kinds of ester compounds, that is simple and substituted naphthol esters and indoxyl derivatives. The tests of inhibition and activation for esterase activity was obtained. Histochemical properties applied to inhibitor test in the esterase zymogram patterns showed no marked differences between ducts and acini.     

Post-ischemic administration of the acetylcholinesterase inhibitor ENA-713 prevents delayed neuronal death in the gerbil hippocampus

We examined by morphological methodology the effect of (S)-N-ethyl-3-[(1-dimethyl-amino)ethyl]-N-methyl-phenylcarbamate hydrogentartrate (ENA-713), an acetylcholinesterase (AChE) inhibitor, on ischemia-induced neuronal death in the gerbil hippocampus due to a 5-min ligation of bilateral common carotid arteries after light ether anesthesia. Pyramidal cells had been decreased to 27% of sham-operated controls and the number of hypertrophic astrocytes expressing glial fibrillary acidic protein (GFAP) markedly increased in the hippocampal CA1 subfield 14 days after ischemia. However, post-ischemic administration of ENA-713 (three times 0.2 mg/kg, i.p.) significantly ameliorated this ischemia-induced decrease in the number of pyramidal cells by 47% of sham-operated controls, furthermore, it reduced the ischemia-induced accumulation of GFAP-positive astrocyte in the CA1 region. Together with previous results showing that ENA-713 protected against the ischemia-induced cholinergic abnormalities in the gerbil brain and improved cholinergic dysfunctions in the senescent rat brain, our present findings suggest that ENA-713 prove to be useful for treatment with senile dementia such as cerebrovascular dementia.     

A direct evidence for defect in glucose-6-phosphate transport system in hepatic microsomal membrane of glycogen storage disease type IB

Uptake of glucose-6-phosphate by microsomes of hepatocyte in rats, human controls and patients with glycogen storage disease type Ia and Ib was studied. In rat the uptake of glucose-6-phosphate increased rapidly and reached to a plateau, but mannose-6-phosphate was not accumulated. These findings indicate that a glucose-6-phosphate specific transport system exists in the microsomal membrane. In human controls and patients with glycogen storage disease type Ia the uptake of glucose-6-phosphate was clearly observed. On the other hand, no accumulation of it was detected in a patient with glycogen storage disease type Ib. These data provide a direct evidence of the defect in the glucose-6-phosphate transport system of hepatic microsomal membrane in glycogen storage disease type Ib.     

Loose association of ribulose 1,5-bisphosphate carboxylase/oxygenase with chloroplast thylakoid membranes

The intra-chloroplastic distribution of ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO) between thylakoid membranes and stroma was studied by determining the enzyme activities in the two fractions, obtained by the rapid centrifugation of hypotonically disrupted chloroplast preparations of spinach and pea leaf tissues. The membrane-associated form of RuBisCO was found to increase in proportion to the concentration of MgCl₂ in the disrupting medium; with 20 mM MgCl₂ approximately 20% of the total RuBisCO of spinach chloroplasts and 10% of that of pea chloroplasts became associated with thylakoid membranes. Once released from membranes in the absence of MgCl₂, addition of MgCl₂ did not cause reassociation of the enzyme. The inclusion of KCl in the hypotonic disruption buffer also caused the association of RuBisCO with membranes; however, up to 30 mM KCl, only minimal enzyme activities could be detected in the membranes, whereas above 40 mM KCl there was a sharp increase in the membrane-associated form of the enzyme.Higher concentrations of chloroplasts during the hypotonic disruption, as well as addition of purified preparations of RuBisCO to the hypotonic buffer, resulted in an increase of membrane-associated activity. Therefore, the association of the enzyme with thylakoid membranes appears to be dependent on the concentration of RuBisCO. P-glycerate kinase and aldolase also associated to the thylakoid membranes but NADP-linked glyceraldehyde-3-P dehydrogenase did not. The optimal conditions for enzyme association with the thylakoid membranes were examined; maximal association occurred at pH 8.0. The association was temperature-insensitive in the range of 4° to 25° C. RuBisCO associated with the thylakoid membranes could be gradually liberated to the soluble form upon shaking in a Vortex mixer at maximal speed, indicating that the association is loose.Abbreviations DTT dithiothreitol - RuBP ribulose 1,5-bisphosphate - RuBisCO ribulose 1,5-bisphosphate carboxylase/oxygenase - MES 2-(N-morpholino) ethane sulfonic acid