We isolated and characterized heparan sulfate rich proteoglycan (HSPG) present in the basement membrane from 7 M urea insoluble materials of the human placenta by CsCl isopycnic centrifugation, anion-exchange chromatography and gel filtration. The molecular mass of the glycosaminoglycan chain from pronase digestion of HSPG was estimated to be 30-100 kDa. The reported glycosaminoglycan moiety of proteoglycans from the lens capsule, glomerular basement membrane and fibroblast consists only of the heparan sulfate chain. However, the glycosaminoglycan of the proteoglycan isolated from human placenta was composed of heparan sulfate (82%) and other glycosaminoglycans (18%). An affinity-purified rat antibody against HSPG specifically reacted with HSPG, but it did not immunoreact with basement membrane components such as laminin and type IV collagen. Indirect immunohistochemical observations of tissue HSPG revealed staining mostly along with basement membrane (basal layer). From these results, isolated HSPG from human placenta appeared to be a constituent of the basement membrane, together with other basement membrane components such as type IV collagen, laminin and nidogen.
A fungus of Pestalotiopsis species produced an extracellular, water-soluble polysaccharide (PS-N). PS-N exhibited significant hypoglycemic activity in streptozotocin-induced diabetic mice following intraperitoneal administration and had an effect on oral glucose tolerance in normal mice following oral administration. PS-N ([α]D+34.5° in water) was homogeneous on gel chromatography, it is composed of galactose and mannose in a molar ratio of 1 : 9, and its molecular weight was estimated by gel chromatography to be about 24000. Its structure was investigated by a combination of chemical and spectroscopic methods. The results indicated that PS-N, a highly branched galactomannan, is composed of β-(1→3)-linked D-galactofuranosyl and non-reducing terminal β-D-galactofuranosyl residues, in addition to α-D-mannopyranosyl residues of a yeast mannan type.
Most digested food 2 h after overnight feeding in rat remained in the stomach, duodenum, upper small intestine, lower small intestine, cecum and colon, all of which indicated pH between 4 and 7 and had glycyrrhizin (GL) hydrolyzing activity. This enzyme activity was highest in the cecal and colonic contents among all gastrointestinal contents. Also, 3α-hydroxyglycyrrhetic acid (3α-hydroxyGA) and 3β-hydroxyglycyrrhetic acid (3β-hydroxyGA) oxidizing enzymes were localized in the same cecal content. Namely, rat gastrointestinal bacteria had the ability to hydrolyze GL to 3β-hydroxyGA by glycyrrizin β-D-glucuronidase and to oxide 3β-hydroxyGA and 3α-hydroxyGA to 3-oxoGA by 3β-hydroxyglycyrrhetinate dehydrogenase and 3α-hydroxyglycyrrhetinate dehydrogenase, respectively. In medium of pH 1 to pH 10, metabolites 3β-hydroxyGA, 3-oxoGA and 3α-hydroxyGA obtained from the metabolism of GL were the highest in pH 8. The intestinal contents of pH 6 or pH 7 were able to produce metabolites 3β-hydroxyGA in the metabolism of GL. However, the stomach content at pH 4.2 was lowest in metabolite 3β-hydroxyGA. It is unknown whether or not GL is metabolized to 3β-hydroxyGA by the stomach content in vivo.
Human lactoferrin (LF) specifically binds to human monocytic leukemia cell line THP-1 cells differentiated into macrophages, and it has been suggested that the poly-N-acetyllactosaminyl saccharide chains of LF are involved. We partially purified and characterized LF-binding proteins with affinity for polylactosamines from THP-1 cells. LF-binding activity was solubilized by nonionic detergent Triton X-100 from THP-1 cell membrane, and subjected to affinity chromatography using an LF-Sepharose column. LF-binding activity, detected by ligand blotting assay, was eluted and further fractionated by affinity chromatography using a Sepharose column coupled with band 3, a polylactosaminyl chain-containing glycoprotein of human erythrocyte membrane. LF-binding activity was separated into three fractions (frs. B1, B2, and B3). These fractions exhibited band 3-binding activity as detected by ligand blotting assay. Dodecylsulfate-polyacrylamide gel electrophoresis of frs. B1, B2, and B3, followed by detection of LF-binding activity on Western blots, indicated that frs. B1, B2, and B3 contained LF-binding proteins with a molecular mass of 35, 50 and 80, and 35-37 kDa, respectively. Binding of LF to each of the fractions on the dot blots was partially inhibited by LF oligosaccharides, band 3 oligosaccharides and lacto-N-neotetraose, each containing di-N-acetyllactosaminyl or analogous structure, Galβ1→4GlcNAcβ1→3Galβ1→4GlcNAc (or Glc). These results suggest that the 35, 50 and/or 80, and 35-37 kDa proteins on THP-1 cells are LF-binding proteins with polylactosamine-binding ability.
In BIO14.6 cardiomyopathic hamster heart, the dystrophin-glycoprotein complex is disrupted and sarcoglycans are greatly reduced in abundance. We examined whether the gene expression of β- and γ-sarcoglycans is indeed defective in this hamster. We found that mRNA expression for these proteins and the cDNA sequences of their coding regions are identical in both normal and myopathic hamster cardiomyocytes. The results strongly suggest that defect in a currently unknown sarcoglycan-associated protein(s) is responsible for the deficiency of the sarcoglycan complex that leads to muscle cell necrosis in the myopathic hamster.
Astrocytes play an important role in supporting nerve regeneration after brain injuries. In this study, the effects of novel neurotropic synthesized pyrimidine compounds on astrocytic morphological differentiation were examined. Treatment of protoplasmic cultured astrocytes with 2-piperidino-5, 6-dihydro-7-methyl-6-oxo(7H)pyrrolo[2, 3-d]pyrimidine maleate (MS-430) and the related compounds caused astrocytic process formation in 60 min. The morphology of MS-430-treated cells was similar to that of dibutyryl cAMP (DBcAMP)-treated cells. The astrocytic process formation by MS-430 was observed within 60 min and the maximum effect was obtained at the drug concentration of 0.5-1.0 mM. Rhodamine-phalloidin staining showed that astrocytic cytoskeletal actin was reorganized by MS-430 and DBcAMP. MS-430 did not increase cAMP accumulation in cultured astrocytes. These results suggest that the neurotropic pyrimidines induced astrocytic morphological differentiation through a cAMP-independent mechanism.
We investigated the characteristics of β-adrenoceptors (β-ARs) in rat white adipose tissues (WAT) with a radioligand receptor binding assay using (-)-[3H]-CGP12177. Scatchard analysis revealed that there are high- and low-affinity sites for (-)-[3H]-CGP12177 in WAT. The (-)-[3H]-CGP12177 bound to a high-affinity site was displaced by 1 μM propranolol. The rank of pKi values of catecholamines for the site was isoproterenol>epinephrine>norepinephrine. By contrast, BRL37344A, BRL35135A and SR59230A, β3-selective agonists had high affinity for the low-affinity site of (-)-[3H]-CGP12177, whereas (-)-[3H]-CGP12177 bound to a low-affinity site was completely displaced by 100 μM bupranolol but not 1 μM propranolol. The pKi values of the catecholamines (isoproterenol, norepinephrine, epinephrine) for this site were very low. In addition, the correlation between the pKi values of various β-agonists for the low-affinity site of rat WAT and those obtained from rat cloned β3-ARs was significant, but those of human cloned β3-ARs were not. Consequently, the results suggested that the high- and low-affinity sites were β2-ARs and β3-ARs in rat WAT, respectively.
The present work shows that gangliosides prevent glutamate cytotoxicity in PC12 cells. Similarly, ouabain (1 and 5 μg/ml) protected cells against glutamate-induced toxicity in a dose-dependent manner. The cytoprotection observed with ouabain was not maintained after its association with gangliosides and glutamate. Also, the effects of gangliosides in blocking glutamate cytotoxicity did not persist in the presence of arachidonic acid. The results indicate that a higher degree of sodium pump inhibition is necessary for ouabain cytotoxicity to be manifested, and that a partial enzyme inhibition may actually be protective to glutamate and/or arachidonic acid-induced toxicity in PC12 cells.
The uptake of 45Ca by inside-out erythrocyte membrane vesicles (IOV) was dose-dependently inhibited by spermine. A cationic substance, Mg2+, did not affect this inhibitory activity of spermine. Ca2+-ATPase activity, however, was not inhibited by spermine at a concentration that could inhibit the uptake of 45Ca by the IOV. The release of 45Ca from IOV was significantly accelerated by spermine. Spermine bound to the surface of the IOV and was removed by treatment with acetic acid. These results suggest that spermine stimulates the release of Ca2+ from the IOV (or uptake of Ca2+ by erythrocytes) via an effect on the outer surface of the IOV (or the inner surface of erythrocytes).
The uptake of the Cd-metallothionein complex (CdMT) into LLC-PK1 cells was investigated and compared with that of CdCl2. The cells were incubated at 37°C for up to 45 min with 1 μM Cd, as either CdMT or CdCl2 at pH 5.5, 6.4, or 7.4. Under all the experimental conditions described below, the accumulation of Cd from CdMT was markedly lower than that from CdCl2. Cd accumulation at pH 7.4 from CdMT increased linearly with the time of incubation, whereas Cd accumulation from CdCl2 was saturable. Metabolic inhibitors, 2, 4-dinitrophenol(DNP) and carbonylcyanide p-(trifluoromethoxy)-phenylhydrazone (FCCP), and incubation at low temperature significantly decreased Cd accumulation from both Cd compounds. Coincubation with 30 μM ZnCl2, an antagonist of CdCl2 uptake, slightly decreased Cd accumulation from CdMT, but it markedly decreased that from CdCl2. Cd accumulation from CdMT at pH 5.5 was significantly higher than at pH 6.4 or 7.4, whereas Cd accumulation from CdCl2 at pH 5.5 and 6.4 was significantly lower than at pH 7.4. Although Cd accumulation from CdCl2 at pH 7.4 was significantly decreased by coincubation with 100 μM cysteine or glutathione (GSH), this decrease was not observed at pH 5.5 or 6.4. A small amount of Cd was removed, by the chelating agent EGTA, from the cell membranes after incubation with CdMT at pH 6.4 and 7.4, whereas a considerable amount of Cd was removed by EGTA after incubation with CdMT at pH 5.5 and with CdCl2 at three pHs. It appears that the CdMT complex is taken up into LLC-PK1 cells partially via an energy-dependent process, and the increase in Cd accumulation at low pH is due to the liberation of Cd. High stability and molecular size of the CdMT complex explains why it is not taken up readily into LLC-PK1 cells.
DNA single-strand breaks due to the inhibition of repair polymerization in cultured human pulmonary epithelial (L-132) cells after exposure to dimethylarsinic acid (DMAA), a main metabolite of inorganic arsenics in mammals, were examined. The strand breaks were detected by an alkaline elution method with the use of inhibitors of DNA polymerase, aphidicolin (aph) and 2', 3'-dideoxythymidine (ddT); the former inhibits DNA polymerases α, δ and ε, and the latter inhibits DNA polymerase β. Generally, DNA polymerases δ and ε are thought to be associated with necleotide excision (long patch) repair and polymerase β with base excision (short patch) repair. After exposure of the L-132 cells to 10 mM DMAA, the breaks occurred in a time-dependent manner during incubation for 1-6 h under the inhibition of aph-sensitive polymerases with 50 μg/ml aph plus 10 mM hydroxyurea (HU) for the last 1 h of the DMAA exposure. Also, when DNA polymerase β was inhibited with 10 mM ddT plus 1 μM methotrexate (MTX), the exposure of L-132 cells to 10 mM DMAA for 6 h significantly induced DNA single-strand breaks. An experiment of the co-treatment with both aph and ddT suggested that in the DNA repair process, aph-sensitive polymerases, probably polymerases δ and/or ε, and polymerase β, functioned independently on different lesions induced after exposure to DMAA.
Alkyl viologens showed cytotoxicity when incubated with cultured murine leukemia L1210 cells for 48 h, whereas they were not cytotoxic when briefly incubated for 10 min. Under the permeabilizing conditions achieved by vortex-stirring with a high molecular weight polyacrylic acid (A-119), appreciable cytotoxicity was shown even after a 10-min exposure to any of the alkyl and amino viologens examined. Acetylamino viologen showed no cytotoxicity regardless of the presence or absence of A-119. This permeabilizing procedure was demonstrated to be applicable to internalize water-soluble positively charged viologen molecules into the cell.
Arenastatin A, isolated from the Okinawan marine sponge Dysidea arenaria, is an antimitotic depsipeptide containing a 16-membered ring. Interaction of the compound with tubulin was investigated by the use of [3H]arenastatin A and other microtubule disruptors. Scatchard analysis indicated the presence of one binding site for arenastatin A per tubulin heterodimer with a dissociation constant (Kd) of 1.8×10-6 M. Rhizoxin was a competitive inhibitor of arenastatin A binding, and vinblastine also inhibited arenastatin A binding in a partially competitive manner. Arenastatin A had no inhibitory effect on colchicine binding to tubulin.
In a series of compounds containing (2S, 3S)-3-amino-2-hydroxy-4-phenylbutanoic acid (AHPBA), a transition-state mimetic, R-87366 : (2S, 3S)-3-[N-(quinoxaline-2-carbonyl)-L-asparaginyl]amino-2-hydroxy-4-phenylbutanoyl-L-proline tert-butylamide, was found to be a potent human immunodeficiency virus protease inhibitor (Ki value was 11 nM) and anti-HIV agent (IC90 value was 0.5 μM for HIV-1IIIB acutely infected cells) with moderate water-solubility (4.2 mg/ml at 25°C). The compound was also active in chronically infected Molt-4/HIV-1IIIB cells, and inhibited the proteolytic processing of p55 into p17, suggesting that its anti-HIV activity was derived from HIV protease inhibition.The compound showed more potent activity (IC90 value was 0.03-0.25 μM) against clinial isolates of HIV in 5 out of 6 patients examined with varying clinical status in an ex vivo assay. One isolate, however, from the sixth patient, was less sensitive to R-87366 (IC90 value was 0.5 μM). In experiments with this strain, R-87366 showed comparatively low efficacy in acutely infected peripheral blood mononuclear cell (PBMC). This result suggests that the diversity of sensitivity shown in the ex vivo assay could be caused by the viral property itself. As a result of the determination of nucleic acid sequences in the clinical isolates, some amino acids were found to be substituted in the protease region, in contrast to the HIV-1 clade B consensus sequence, and some of them have been reported to contribute to the susceptibility of HIV protease inhibitors.
The biodisposition of fluorescein-labeled dextrans (FDs) with different molecular weights (MW=4-500 kDa) was systematically examined in mice. After intravenous injection of FDs at a dose of 120 mg/kg, the levels of FDs in the blood circulation and in the various organs were measured fluorometrically. FDs with a molecular weight lower than 20 kDa showed poor hepatic distribution (2.1-3.7% of dose/g tissue) due to their rapid elimination from the blood circulation. FDs with higher molecular weights were appreciably distributed in the liver (18.9-24.0% of dose/g tissue) and accumulated there over a long period, whereas the FD levels in the other organs were almost negligible a few days after injection. The hepatic mean residence time of FDs ranged from 22.5 to 28.1 d. Partial depolymerization of FDs which accumulated in the liver was observed within 10 d after administration. The hepatic uptake clearance of FDs was decreased with an increase in molecular weight. A marked molecular weight dependency was also seen in the urinary and fecal excretions of FDs. An appreciable dose-dependency was demonstrated in the hepatic uptake of FDs (MW=40 kDa), as well. The amount of hepatic uptake as a function of dose showed saturation kinetics and was analyzed by a Michaelis-Menten type equation. The apparent values of Km (dose) and Vmax (hepatic level) estimated were 116±5 mg/kg and 1.10±0.05 mg/g tissue, respectively.
Pharmacokinetics of metyrapone and metyrapol enantiomers was studied in the rat to determine the stereoselective reductive metabolism of metyrapone. The HPLC method using a chiral column was developed for the stereoselective analysis of metyrapol enantiomers in rat plasma. The AUC ratio of (-)- and (+)-metyrapol appeared in rat plasma after i.v. administration of metyrapone was about 3 : 1. The interconversion of (-)- or (+)-metyrapol to its antipode was negligible, and the reverse reaction from metyrapol to metyrapone was insignificant. There were similar kinetic parameters of (-)-metyrapol to those of (+)-metyrapol after i.v. administration of racemic metyrapol. These results indicate metyrapone displays product-stereoselective reductive metabolism in the rat. The inhibition of steroid 11β-hydroxylase by metyrapone, racemic metyrapol, (-)-metyrapol or (+)-metyrapol was analyzed in rat adrenal homogenates. Metyrapol was equally as potent as metyrapone in the inhibition of steroid 11β-hydroxylase and each enantiomer of metyrapol showed similar inhibitory activity on the rat adrenal steroid 11β-hydroxylase. These results indicate there is an insignificant difference in the inhibitory effects on steroid 11β-hydroxylase of metyrapol enantiomers, and that the inhibitory effects of metyrapol may be involved in the pharmacological activity of metyrapone in vivo.
The ability of Bordetella heat-labile toxin (HLT) to induce ischemic lesions after intracutaneous injection to guinea pig skin was lost following incubation at 37°C with long-chain saturated acyl-CoA and acyl-carnitine compounds. Short-chain unsaturated acyl-CoA compounds, however, were less potent in inhibiting the induction of HLT activity. Long-chain saturated acyl-CoA and acyl-carnitine compounds, potently inhibited the induction of this activity. On incubation with HLT at 0°C, a long-chain saturated acyl-CoA compound, palmitoyl-CoA, did not inhibit HLT activity. When first mixed with bovine serum albumin or dimethyl-β-cyclodextrin, palmitoyl-CoA lost the ability to inhibit HLT activity. Binding of 14C-palmitoyl-CoA to HLT was measured by Scatchard analysis. The Bmax values of HLT (2.75 mol/mol of protein) were higher than that of acyl-CoA-binding protein from bovine liver (0.95 mol/mol of protein). Neither acyl-CoA hydrolase nor acyl-CoA ligase was detected in the HLT preparation. These results suggest that the acyl-CoA and acyl-carnitine compounds bind directly to HLT and produce a critical change in conformation required for HLT activity.
Spasmolytic effects of efonidipine hydrochloride (efonidipine) on high K+-, U46619- and 3, 4-diaminopyridine (3, 4-DAP)-induced contractions were evaluated in isolated canine coronary artery, and were compared with the effects of nifedipine and nisoldipine. Efonidipine (0.3-30 nM), nifedipine (1-300 nM) and nisoldipine (0.1-100 nM) each relaxed the contractions induced by high K+ and U46619. However, relaxation produced by efonidipine was slower than that produced by nifedipine or nisoldipine. The rank order of potency of these drugs for U46619-induced contraction was efonidipine ≥ nisoldipine > nifedipine, whereas in high K+-induced contraction, it was nisoldipine > efonidipine > nifedipine. Thus, the relaxing effect of efonidipine on U46619-induced contraction appeared to be more potent than its effect on high K+-induced contractions, when compared with the effects of nifedipine and nisoldipine. These three drugs also suppressed 3, 4-DAP-induced rhythmic contractions. However, a marked time-dependent increase in potency was only observed for efonidipine, and was similar to its time-dependent effect on high K+- and U46619-induced contractions. Efonidipine did not change the contraction cycle length whilst suppressing the peak contractions. On the other hand, lower concentration of nifedipine at 3 nM and nisoldipine at 1 nM significantly shortened the cycle length. These results suggest that efonidipine may be an effective agent for the treatment of angina pectoris. The high potency of efonidipine for U46619-induced contractions will provide some advantages in the clinical use of this compound on thromboxane A2-mediated coronary vasoconstriction.
A plasmid (5.4 kbp) from Salmonella Typhi D4 has been identified as encoding a restriction and modification (R-M) system. DNA fragments (2537 bp) that carried the genes for restriction endonuclease and methyltransferase encoded on the plasmid were sequenced. Two divergently arranged open reading frames of 957 bp for the restriction endonuclease consisting of 318 aa (amino acids) and 1140 bp for the DNA methyltransferase consisting of 379 aa were identified. These sequences were similar to the sequences of the SsoII R-M system, including the interspace between the two genes.
The acid salts of aminoalkanecarboxylic acid esters of d-α-tocopherol were in a previous in vitro study identified as prodrug candidates for a parenteral form of d-α-tocopherol. The disposition of d-α-tocopheryl N, N-dimethylaminoacetate hydrochloride (TDMA), the most potential candidate for the prodrug, after a single intravenous administration was investigated and compared with that of the d-α-tocopheryl acetate (TA) and dl-α-tocopherol, solubilized with HCO-60, in order to establish the utility as a prodrug for i.v. administration. The preventive effect of the prodrug against endotoxin (lipopolysaccharide (LPS))-induced liver lipid peroxidation was also investigated in mice. The plasma and liver levels of α-tocopherol (Toc) were increased rapidly after i.v. administration of the prodrug. The distribution of Toc and TDMA in the plasma and the liver at 1 h was as follows; 2.1±0.2 (plasma, Toc), 2.0±0.2 (plasma, TDMA), 32.8±2.9 (liver, Toc), and 35.3±6.5% of dose (liver, TDMA). The rapid and liver-selective uptake and liver-esterase specific regeneration characteristics of the prodrug enhance the delivery of Toc to liver. The liver availability of Toc after i.v. administration of TDMA, TA and Toc were 116, 50 and 100%, respectively. The elevation of liver lipid peroxide induced with LPS was significantly suppressed to a normal range by a single i.v. postadministration of TDMA (over 10 mg/kg equivalent for Toc). These results indicated that the water-soluble and liver-esterase hydrolyzable derivative of Toc was a potential candidate for a parenteral prodrug which can thus achieve the systemic liver-specific delivery of Toc. Such effective and selective delivery of Toc into the liver can therefore lead to enhanced pharmacological efficacy against liver oxidative injury associated with free radicals.