Our previous work has shown that the amino acid residues around 62-67 located in the connecting loop between helix I and II of human growth hormone (hGH) are important in eliciting the differentiation of preadipose 3T3-F442A cells to adipocytes. In this study, we evaluated the role of the charged residues around 62-67 in receptor binding and biological activity. Eight artificial mutant variants of hGH were prepared in Escherichia coli by site-directed mutagenesis. Replacement of Arg64 with Tyr (R64Y variant) resulted in a significant loss of binding to the somatogenic receptors on 3T3-F442A cells, but retained full adipose conversion activity on these cells. Replacement of Arg64 with Glu (R64E) produced a considerable loss in receptor binding and a significant loss in biological activity. hGH variants in which either Glu65 or Glu66 was replaced with Asp (E65D and E66D) and with Gln (E65Q and E66Q) showed a slight loss in binding activity and retained almost a full adipogenic activity. An E65P variant (replacement of Glu65 with Pro) possessed the same binding activity as hGH, although it failed to induce full biological activity. The insertion of Ala between Asn63 and Arg64 (63NAR) caused a marked loss in both activities. These results indicate that the positively charged Arg64 is important for receptor binding and thereby in eliciting the biological activity of hGH, while negatively charged Glu65 and Glu66 are less important. In addition, our findings confirm that the conformation and size of the loop region around Arg64 is important for the adipose conversion activity of hGH.
The uptake of oxidized low density lipoprotein (ox-LDL) by macrophages and the resulting accumulation of low density lipoprotein (LDL) lipids within the cells has been implicated in the pathogenesis of atherosclerosis. The effect of fibronectin (FN) on the binding and uptake of ox-LDL by macrophages was investigated using thioglycollate-induced mouse peritoneal macrophages. The ability of the macrophages to bind ox-LDL was assessed by the binding of mouse red blood cells (RBC) pre-coated with ox-LDL (ox-LDL-RBC) prepared in vitro to macrophages at 37°C. The binding of ox-LDL-RBC to macrophages was significantly enhanced when the macrophages were plated on a FN-coated substrate. Similar enhancement was observed when the macrophages were plated on a substrate pre-coated with Gly-Arg-Gly-Asp-Ser-Pro (GRGDSP) peptide, an adhesive sequence of FN involved in binding to the cells, but not with the control Gly-Arg-Gly-Glu-Ser-Pro (GRGESP) peptide. The effect of FN was inhibited when GRGDSP, but not GRGESP, was present during the macrophage attachment to the FN-coated substrate, suggesting that the specific interaction of this sequence and the FN-receptor is responsible for the effect of FN. The addition of FN or GRGDSP in solution to the macrophage layers on an uncoated substrate was ineffective. Thus, attachment to a substrate is necessary for FN to be effective on the macrophages. The FN-coated substrate also enhanced the uptake of 125I-labeled ox-LDL by the macrophages, and this enhancement was found to be due both to an increased binding of 125I-labeled ox-LDL to the cells and to increased internalization of the surface-bound 125I-labeled ox-LDL into the cells. Consistently, enhanced accumulation of lipid droplets and cholesterol within the cells was observed when the macrophages were plated on the FN-coated substrate. These results indicate that substrate-bound FN (solid-phase FN) enhances the activity of the macrophage receptors for ox-LDL (scavenger receptors) to bind and take up ox-LDL, and thus causes enhanced accumulation of ox-LDL lipids. It is, therefore, possible that insoluble FN present in an extracellular matrix and on other cell surfaces in the artery wall plays a promoting role in atherogenesis.
A nicked form of human chorionic gonadotropin (nicked hCG), in which only one peptide bond between residues 47 and 48 (-Gly-Val-) of β-subunit is cleaved, has been found in the urine and blood of pregnant women. In this study, we investigated the occurrence of nicked hCG and the localization of the nicking enzyme for hCG. First, to determine what type of protease nicks hCG, an in vitro proteolytic study using various proteases was performed. Amino-terminal amino acid sequence analysis of the β-subunit purified from protease-treated hCG indicated that thermolysin actively nicks hCG. Secondly, to determine which tissues are related to the formation of nicked hCG, the distribution of radioactivity in various tissues after i. v. administration of radiolabeled hCG to female rats was examined. The radioactivity accumulated predominantly in the kidney (17%), liver (9.3%) and ovary (0.9%) after 30 min of injection. Analysis of molecular species of β-radiolabeled hCG in various tissues and body fluids, using sodium dodecyl sulfate polyacrylamide gel electrophoresis followed by autoradiography, indicated that a nicked hCG-like molecule was found in the kidney, predominantly, as well as in the serum and urine. To examine the role of the kidney in producing nicked hCG, hCG was incubated with rat kidney particulate fraction (KPF). Immunoblot analysis of KPF-treated hCG indicated that KPF produced a nicked hCG-like molecule. Furthermore, the possibility that placental trophoblast cells produce nicked hCG was also examined using the choriocarcinoma cell BeWo. It was revealed that 1 to 5% of the hCG secreted from BeWo cells to the medium was assessed to be the nicked form by immunoblot analysis of the medium. Taken together, these results suggested that the nicking of hCG may be caused by thermolytic protease in trophoblast cells before or concomitantly on secretion, as well as in the kidney cortex after secretion. In the kidney proximal tubular cells, neutral endopeptidase EC 3. 4. 24. 11 (endopeptidase 24. 11) is localized, and its substrate specificity is very similar to that of thermolysin. Thus, the possibility is raised that endopeptidase 24. 11 might be a nicking enzyme for hCG. However, we have been unable to demonstrate that purified endopeptidase 24. 11 forms nicked hCG from hCG, indicating that the hCG-nicking enzyme may be an endoprotease unidentified so far.
We report the effects of indenestrol A, a metabolite of diethylstilbestrol, and its analogue, indenestrol B, on the relative plating efficiency and cellular microtubular architecture of Chinese hamster V79 cells. In this study, the effects of the monomethyl ethers of indenestrols A and B on these biological activities and their affinity for estrogen receptors in cytosol from mouse uteri were investigated. The results indicate that among eight optically active and four racemic methyl ethers, the 4'-methyl ether of [(-)-3S] indenestrol B exhibits both the strongest cytotoxicity in, and greatest disruption of, the cellular microtubular architecture of Chinese hamster V79 cells in culture. For the 6-and 4'-monomethyl ethers of optically active indenestrols A and B, some correlation was found between cytotoxicity and the effect on the distribution of cellular microtubular networks in Chinese hamster V79 cells. Estrogen receptor competitive binding studies revealed that stereochemistry and the position (6 or 4') of the methyl ether group contributed greatly to their binding affinity. However, no correlation was observed between the affinity for estrogen receptors and the cytotoxicity of the monomethyl ethers tested. This suggests that the important causes of cytotoxicity in this series of compounds involve the inhibitory activity on cellular microtubule networks and not the affinity for estrogen receptors.
To investigate the intracellular transport and maturation of lysosomal cathepsin D, we carried out an in vivo pulse-chase analysis with [35S] methionine in the primary cultures of rat hepatocytes. Cathepsin D was initially synthesized as a proenzyme of 45 kDa. The proenzyme was subsequently processed, becoming a mature enzyme of 43 kDa. The proenzyme and mature enzyme showed complete susceptibility to endoglycosidase H treatment, suggesting the presence of high-mannose type oligosaccharide chains. The effects of tunicamycin and chloroquine were also investigated. In the presence of tunicamycin, the 42.5-kDa unglycosylated precursor polypeptide appeared in the cell, and this protein was exclusively secreted from the cells without undergoing proteolytic processing. These results support the notion that the oligosaccharide moieties are of importance in addressing the lysosomal hydrolases to the lysosomes. However, in the presence of chloroquine, proteolytic processing of the proenzyme was prevented, and the enhanced release of proenzyme from the cells was observed. These results indicate that the processing of proenzyme to mature enzyme would take place in the lysosomes.
Lysosomal cysteine proteinases of cathepsins B and H were isolated to a homogeneous state from rat liver by employing Sephadex G-75, DEAE-Sephacel, CM-Sephadex, and Mono S column chromatography. Each of the purified cathepsins B and H was demonstrated to be composed of a mixture of a single-chain form and the processed two-chain form upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). To investigate the proteolytic maturation of lysosomal cathepsins B and H, turnover kinetics of these enzymes were studied by comparing the specific radioactivities of the incorporated [3H] leucine into either the single-chain form or two-chain form in vivo. The specific radioactivity derived from each protein band of lysosomal cathepsin H in SDS-PAGE at 1, 3, 6, 12, 24 and 48h after the injection of a radiolabel showed that the peak of specific radioactivity of the single-chain form of cathepsin H appeared at 6h and that after 6h, the radiolabel was sequentially incorporated into the two-chain form, while the radiolabel in the single-chain form started to gradually decrease, suggesting that the single-chain form was processed to generate the mature enzyme after the enzyme was incorporated into the lysosomes. In contrast, in the case of cathepsin B, the appearance of a radiolabel in the single-chain form or in the two-chain form was observed almost concomitantly without time lag, indicating that the processing of cathepsin B occurred very rapidly in the lysosomes.
PK-120 is a novel substrate of plasma kallikrein isolated from human plasma. Western blotting using anti-human PK-120 polyclonal antibodies demonstrated the presence of a PK-120-like protein in guinea pig, rat and mouse plasma. We purified the immunoreactive-120 kDa protein from guinea pig plasma by polyethylene glycol fractionation followed by ion-exchange chromatography using Q-Sepharose, heparin-Sepharose, Mono-Q and gel filtration with TSK G3000. The 120 kDa protein thus isolated was similar to human PK-120 with respect to limited proteolysis by kallikrein, amino acid composition, and the N-terminal amino acid sequence, indicating that the immunoreactive 120 kDa protein was guinea pig PK-120.
The main interest of the present study was to determine possible alterations in fatty acid composition in rat plasma and liver phospholipids (PL) caused by chronic ethanol consumption, diazepam treatment and chronic consumption of alcohol and diazepam together. Chronic ethanol consumption (11g/kg/d) elevated the proportion of plasma saturated (SFA) and monounsaturated fatty acids (MUFA), and decreased the most important polyunsaturated fatty acids (PUFA) in plasma and liver PL. The alterations in fatty acid composition in liver PL indicate that ethanol can change the composition of cell membrane lipids. Diazepam treatment (10mg/kg/d) elevated the contents of SFA and MUFA in plasma PL. On the other hand, diazepam produced a drastic decrease in 22 : 6n-3 docosahexaenoic acid (DHA) in plasma and liver PL. These changes in plasma PL fatty acid composition indicated a disturbance of fatty acid metabolism. The changes in fatty acid contents of plasma and liver PL were the greatest in rats treated with a combination of ethanol and diazepam, in which there was a summation of the effects of ethanol or diazepam alone, and the effects were intensified by an ethanol-diazepam interaction.
Shosaiko-to (Xiao-chai-hu-tang, SHO), which is a Kampo medicine prepared by decocting a prescription of 7 kinds of medical plants, has been used mainly to treat chronic hepatitis in Japan. Previously, we reported that an oral administration of SHO augmented natural killer (NK) activity in the peripheral blood. To characterize its active substance, SHO was fractionated. The high molecular weight fraction showed the ability to augment NK activity by oral administration, but the low molecular weight fraction did not. Furthermore, we obtained an active acidic polysaccharide from the high molecular weight fraction. This polysaccharide fraction, with a molecular weight of approximately 1.2×105, is probably responsible for the effect of the original Shosaiko-to. It contained no protein. The sugar moiety was composed of rhamnose, arabinose, mannose, galactose, glucose and galacturonic acid in molar ratios of 1 : 17 : 3 : 21 : 100 : 87.
To investigate the mechanism of the anti-urease action of ecabet sodium (ecabet) observed in Helicobacter pylori in vitro, the effects of ecabet on purified urease from jack bean were studied in comparison with the effects of the specific urease inhibitor benzohydroxamic acid (BHA). After incubation of the enzyme with the test drug for a period of time, urease activity was measured. Ecabet depressed the activity below pH 5, and the lower the pH, the greater the degree of depression. The degree of depression by ecabet increased gradually during incubation and reached a plateau in 20 min, whereas that by BHA attained a maximum rapidly. The IC50 values of ecabet and BHA were 2.1 mg/ml and 0.5 μg/ml, respectively. When the incubation mixture of urease with an inhibitor was diluted and further incubated, the depressed activity by BHA reverted gradually, but that by ecabet did not. When the incubation mixture of urease with ecabet was centrifuged, the urease activity of the mixture decreased in parallel with the reduction in protein concentration of the supernatant. When the incubation mixture of urease and 14C-ecabet was ultrafiltered to remove the drug, the radioactivity in the retentate remained in parallel with the degree of reduction of urease activity of jack bean, and suggest that the depression is caused by irreversible binding of ecabet to urease followed by denaturation of the enzyme protein.
Oral tolerance is thought to have a role in preventing allergic responses and immune-mediated diseases. Modulation of this tolerance by drugs and chemicals can cause or suppress them. An improved model of oral tolerance to ovalbumin (OVA) in mice was developed to detect modulators of the tolerance and to apply it to selected immunomodulating substances, cyclophosphamide (CP), Escherichia coli lipopolysaccharide (LPS) and cadmium chloride (Cd). Male C3H/HeN mice given an oral administration of 20 mg OVA were immunized 7d later with an i. p. injection of 0.1 mg OVA in complete Freund's adjuvant. Effects of oral OVA and agents on systemic immunity were assessed by enzyme-linked immunosorbent assay (ELISA) of immunoglobulin (Ig) levels in serum collected 7 or 14d after immunization. Oral tolerance was adequately induced on day 7 after immunization and was more effective in C3H/HeN mice than in BALB/c mice. It was primarily associated with the decreased serum levels of anti-OVA IgG (including both IgG1 and IgG2a subclasses regulated differently by T-helper subpopulations, Th2 and Th1 cells, respectively). The C3H model of oral tolerance was further examined to detect modulators of the tolerance. An i. p. injection of CP prior to oral OVA, or 5 consecutive daily oral administrations of LPS after oral OVA elevated or reduced serum levels of anti-OVA IgG in C3H mice hyposensitized by the oral OVA, respectively. Concerning IgG subclasses, CP restored anti-OVA IgG2a but not IgG1 levels, while LPS caused greater suppression of both anti-OVA IgG1 and IgG2a levels. Oral administrations of Cd for 5d after oral OVA also suppressed anti-OVA IgG1 levels further. These findings indicate that the C3H model is useful for detecting modulators of oral tolerance, and suggest that the differential modulation of oral tolerance induced by CP, LPS and Cd reflects different responses of T-helper subpopulations.
We examined the interaction among secretagogues that stimulate pepsinogen secretion through different pathways in vivo and in vitro. In in vitro study, a combined administration of secretin and carbachol or cholecystokinin octapeptide (CCK-8) to the culture medium of chief cells potentiated pepsinogen secretion. Moreover, the response induced by carbachol or CCK-8 with forskolin was greater than that with secretin. We examined the interaction among receptor-related second mediators, and found that carbachol-or CCK-8-induced intracellular Ca2+ concentration ([Ca2+]i) increase was not affected by secretin or forskolin. Both these substances, however, significantly reduced secretin-induced cAMP production. On the contrary, CCK-8 significantly increased forskolin-induced cAMP production, while carbachol increased it slightly. Calcium ionophore, A23187, or protein kinase C activator, phorbol 12-myristate 13-acetate (PMA), did not alter secretin-or forskolin-induced cellular cAMP production ; and the reductive effect of carbachol or CCK-8 on secretin-induced cAMP production was restored by their competitive antagonists, atropine or lorglumide. EC50 of those antagonists was almost the same value as IC50 on pepsinogen secretion and [Ca2+] i increase. These results indicate that secretin-induced cAMP production is interfered with by receptor related agonists like CCK-8 and carbachol. It may be suggested that there is a kind of "cross-talk, " between the adenylate cyclase system, that is, the secretin receptor, and carbachol or CCK-8 receptor. The interactions between secretin and other secretagogues (carbachol, CCK-8, tetragastrin and histamine) were examined using the perfused rat stomach. The infusion of CCK-8 induced high pepsin secretion initially, which gradually decreased ; with a simultaneous injection of secretin, however, high pepsin secretion was maintained. High pepsin secretion induced by an infusion of carbachol was obtained when secretin was injected simultaneously. Although tetragastrin or histamine did not stimulate significant pepsin secretion, pepsinogen secretion was stimulated if secretin injected simultaneously. These results suggest that pepsinogen secretion in the rat is not controlled only by pepsinogen secretagogues but by its combination with acid secretagogues.
Oxidized low density lipoprotein (LDL) has a variety of hazardous influences on biological systems. Oxidative modification of LDL by diesel exhaust particles (DEP) was studied in vitro to assess its in vivo health effects. DEP suspensions (1, 10 and 100 μg/ml) were incubated for 1h with LDL (1mg protein/ml) at 37°C. Conjugated diene formation and negative charges in LDL were increased by DEP-treatment in a dose-dependent manner. When native LDL and DEP-treated LDL (DEP-LDL) were incubated for 18h with macrophage, J774A. 1 cell at 37°C, significantly more DEP-LDL was taken up into cells than native LDL. Accumulation of cholesterol ester in cells incubated with DEP-LDL was 4 to 8 times higher than that with native LDL while there was no significant difference between them in free cholesterol content. Incubation (18h) of J774A. 1 with DEP-LDL caused an increase in leakage of lactate dehydrogenase from cells in a DEP-concentration dependent manner, but the incubation with native LDL or DEP per se did not increase the leakage except at the highest concentration of DEP. These results suggest that DEP oxidatively modified LDL giving it cytotoxic, inflammatory and atherogenic properties characteristic of so-called oxidized LDL ; these initial modifications of LDL may be one of the underlying mechanisms for diseases associated with DEP.
To obtain further knowledge of the metabolic fate of carcinogenic 4-aminobiphenyl (ABP) and 4-acetylaminobiphenyl (AABP) in intact guinea pig liver, we performed in situ liver perfusion using a recirculation method. Following a biexponential disappearance of ABP from the perfusate, not only AABP but also its secondary metabolites 4'-hydroxy AABP (4'-OH AABP) and 4-glycolylaminobiphenyl (GABP) appeared as the major metabolites. 4'-Hydroxy ABP (4'-OH ABP) was also detected in the perfusate as another predominant metabolite, while N-hydroxy ABP (N-OH ABP) as a minor one. Most of these metabolites, except for AABP and GABP, found in both perfusate and bile were conjugated with glucuronic acid. In addition, considerable amounts of all the metabolites were also detected in either unconjugated or conjugated form in a homogenate of the liver tissue after perfusion. When AABP was infused, an almost similar metabolic profile to that of ABP was obtained, but the amount of ABP was quite small and N-OH AABP rather than N-OH ABP was found. These results demonstrate that both ABP and AABP are rapidly metabolized by a combination of N-acetylation, C-and N-hydroxylations as well as glucuronidation in guinea pig liver.
Roxithromycin (RXM), a new macrolide antibiotic, has a 14-member macrocycline ring structure which is similar to that of erythromycin. We investigated the effects of RXM on the proliferation of peripheral blood mononuclear cells (PBMCs) and the production of interleukin 1β (IL-1β) and tumor necrosis factor α (TNF-α) by PBMCs stimulated with lipopolysaccharide (LPS). At concentrations greater than 25.0 μg/ml, RXM suppressed the proliferation of PBMCs stimulated with phytohemagglutinin, probably due to cytotoxicity. When the PBMCs were incubated with RXM for 7d, the number of adherent cells (monocyte/macrophages) increased. Incubation with RXM at a concentration of 25.0 μg/ml induced the greatest increase (p<0.05). IL-1β and TNF-α were present 3 h after LPS-stimulation, and IL-1β production reached a peak at 12 h and TNF-α production at between 6 and 12 h, and then their production declined. RXM (25 μg/ml) suppressed the production of IL-1β and TNF-α slightly during the entire course of the incubation. This suppression was dose-dependent. Anti-human granulocyte-macrophage colony-stimulating factor and anti-human macrophage colony stimulating factor antibodies had no effect on the RXM-induced proliferation of adherent cells. Suppression of the production of IL-1β and TNF-α by RXM suggested that this drug might have anti-inflammatory and immunosuppressive effects.
When dissolution is the rate-limiting step in absorption of a drug, both it and gastrointestinal transit can play dominant roles in determining absorption. Therefore, a pharmacokinetic model including the process of dissolution, gastrointestinal transit rate and gastrointestinal pH profile was proposed to explain the behavior of drug formulations in vivo. Clarithromycin (CAM), which exhibits pH-dependent solubility and has high partition coefficients, was used as a drug to test the model, and the effect of a meal on serum concentrations of CAM after a single oral administration to healthy volunteers was evaluated. The results of pharmacokinetic analyses of serum concentration-time data for a single oral administration of CAM to humans in the fasting and postprandial states were well fitted by the new pharmacokinetic model. By substituting the pharmacokinetic parameters obtained into the model equations, the in vivo rate of dissolution could be determined. Pharmacokinetic parameters affecting drug bioavailability were also examined using the model. Factors influencing the behavior of a drug formulation in the gastrointestinal tract were determined. These findings indicate that the model we have proposed can be used to evaluate the behavior of drug formulations in vivo.
Soybean-derived sterylglucoside mixture (SG) is a potentially effective absorption enhancer for the nasal absorption of insulin. Insulin in a peanut oil suspension dosage form and in a powder dosage form with SG were administered into the rabbit nasal cavity. After 0.5 or 1h, nasal mucosa was taken from the nasal cavity and side effects were examined by an optiphoto light microscope. The insulin in the peanut oil suspension produced a histological change in the nasal epithelium mucosae, as well as mucodermal phlogistic cellular infiltration. The peanut oil suspension with SG showed side effects slightly stronger than without SG. SG alone and the insulin powder dosage form with SG produced no signs of inflammation, erosion or squamous metaplasia. The results from this study indicate that SG can be considered safe.
The objective of this study was to determine the stability of insulin in rat lung homogenate and to confirm the effectiveness of various protease inhibitors for insulin delivery from the lung. The stability of insulin in rat lung homogenate was compared with that in the small intestine. Insulin was rapidly degraded in lung homogenate similarly to degradation in the small intestinal homogenate. The effects of various protease inhibitors on the degradation of insulin were studied in the lung. Protease inhibitors such as Na-glycocholate (Na-GC ; 10mм), aprotinin (10mg/ml), soybean trypsin inhibitor (STI ; 10mg/ml) and bacitracin (20mм) effectively reduced insulin degradation in lung homogenate. The rank order of effectiveness for decreasing the insulin hydrolysis in lung homogenate was bacitracin>aprotinin>STI>Na-GC. In addition, a slight correlation was observed between the in situ pulmonary absorption of insulin and its stability in vitro in the lung homogenate in the presence of various protease inhibitors. These findings suggest that the coadministration of protease inhibitors would be useful for improving the stability of insulin in the lung.
Several derivatives of K-252a, a protein kinase inhibitor isolated from Nocardiopsis sp., were investigated for their effects on the replication of vesicular stomatitis virus (VSV) in BHK-21 cell cultures. Among those we tested, KT5926, which preferentially inhibits the myosin light chain kinase (MLCK), suppressed the viral replication by 95-99% at 15μм. K-252a, which inhibits a broad spectrum of cellular protein kinase, similarly affected the viral replication. Other derivatives, KT5720 and KT5823, that are known to inhibit the cAMP-dependent protein kinase (PKA) and cGMP-dependent protein kinase (PKG), respectively, did not suppress VSV replication even at a high concentration as 15μм. None of these inhibitors affected the Sindbis virus replication in BHK-21 cells under similar assay conditions as used for VSV. KT5926 and K-252a seemed to affect the VSV replication at the step (s) after the viral invasion, resulting in decreased viral RNA synthesis. Neither substance inhibited cellular casein kinase (CK) II which is known to be involved in phosphorylation of the nonstructural (NS) protein, a non-catalytic subunit of the viral RNA polymerase. These results suggest that the inhibition of VSV replication by KT5926 and K-252a is not a secondary effect due to generalized suppression of host cell activities, and that the VSV replication requires the KT5926-sensitive function (s) in the cell which would be performed by an enzyme (s) other than CK II.
A sensitive and specific double-antibody enzyme immunoassay (EIA) for detecting an elcatonin-like immunoreactive substance (ECT-IS) in human plasma has been developed. In competitive reactions, the ECT antibody was incubated with a plasma sample (or ECT standard) and β-D-galactosidase-linked synthetic ECT. Free and antibody-bound enzymes were separated using an anti-rabbit IgG-coated immunoplate. Enzyme activity on the plate was determined by fluorescence analysis. This immunoassay allows the detection of 20 to 300 fmol/ml (67 to 1000 pg/ml) of ECT. The EIA was applied to determine the pharmacokinetic behavior of ECT after a single intramuscular administration (20 IU). The maximum level was achieved 30 min after administration, at approximately 30 pg ECT/ml of plasma.
Possible involvement of the peripheral-type benzodiazepine receptor (PBR) in hemin/protoporphyrin-induced erythroid differentiation of human leukemia K562 cells was investigated by the use of the ligands, diazepam and PK11195. Diazepam itself exhibited differentiation-inducing activity on K562 cells. The PBR-specific antagonist, PK11195, dose-dependently inhibited both diazepam-induced and hemin/protoporphyrin-induced K562 cell differentiation. The results imply that PBR is involved in the erythroid differentiation of K562 cells.
Na, K-ATPase receptor density has been shown to be down-regulated with decreasing ejection fraction in patients with chronic heart failure. It was the aim of the present study to determine whether down-regulation is detected also at the mRNA level. Six donor hearts and six explanted hearts due to dilated cardiomyopathy (ejection fraction 23±5%) were analyzed. RNA was extracted. Quantitative Na, K-ATPase receptor catalytic subunit α1, α2 and α3 mRNA expression was determined by solution hybridization. No cross-reactivity occurred between the three probes. α1 mRNA was expressed at about 5 and 10 times higher (p<0.001) concentrations than α2 and α3 mRNA, respectively, and α2 mRNA higher (p<0.001) than α3. There were no differences between right and left ventricles and between donor hearts and patients with dilated cardiomyopathy. In conclusion, Na, K-ATPase α1 mRNA is the predominant subunit expressed in human myocardium. Depressed ejection fraction in dilated cardiomyopathy is not associated with changed mRNA subunit expression. Documented downregulation of Na, K-ATPase activity, therefore, may be associated with the structural and membrane-related β subunit or posttransciptional modification of the catalytic subunits.
The effects of alkaloid extract from the stem bark of Hunteria zeylanica GARD. (H. zeylanica) on the activities of cyclooxygenase and 5-lipoxygenase in A23187-stimulated rat mast cells were investigated. H. zeylanica alkaloid extract (0.3-300μg/ml) inhibited leukotriene C4 (LTC4) production by 5-lipoxygenase in a concentration-dependent manner and it blocked the production by 50% at 300 μg/ml. On the other hand, the extract had no effect on prostaglandin D2 (PGD2) production by cyclooxygenase. Neither (-)-eburnamine nor pleiomutinine, major constituents of H. zeylanica alkaloid extract, inhibited the production of PGD2 and LTC4 in the A23187-stimulated mast cells. The inhibition of arachidonic acid metabolism via 5-lipoxygenase pathway may be due to minute amounts of other components as stated in the Discussion.
Antioxidative inhibition by protoporphyrin (PP) of peroxidative damage in lysosomes, mitochondria and microsomes of rat liver was investigated at 24h after an intravenous administration of PP. Using a lysosome-containing (3500×g) fraction, the release of lysosomal marker enzymes, acid phosphatase and aryl sulfatase, from lysosome which had been stimulated by L-ascorbic acid (AsA), was decreased dose-dependently, as was the inhibition of lipid peroxidation by PP in the fraction. Swelling of mitochondria induced by Fe2+ and AsA was also inhibited in the PP-injected rat. In microsomes, lipid peroxidation stimulated by AsA caused a decrease in activity of a microsomal marker enzyme, glucose 6-phosphatase, and in P450 content. The extent of the decrease by AsA, both in activity and content, was diminished in PP-administered rat liver microsomes. These results indicate that PP protects those subcellular fractions from deterioration by lipid peroxidation.
In many inflammatory conditions including burns, the plasma sialic acid level rises as an acute responder. Sunburn is a kind of burn. In this study, sunburn was experimentally caused in mice by UV-B irradiation and their plasma sialic acid levels were measured. The levels increased, and reached the maximum 3d after irradiation. This level was maintained for about 2d, then it returned to normal within about one week. The increase in the level correlated with the UV dose and the severity of inflammation resulting in sunburn. This assay system was applied to assess the virtue of UV-cut cream in experimental sunburn.
The cell-to-cell transmission of human immunodeficiency virus type 1 (HIV-1) was studied using MOLT-4 cells chronically infected with a variant strain of HIV-1SF-2 (MOLT-4/HIV-1SF-2H) and CD4+ human lymphoid MT-4 cells. MOLT-4/HIV-1SF-2H cells produced less than 1 TCID50 infectious particles per 105 cells per day as determined by the cytopathogenicity in MT-4 cells. However, the expression of envelope glycoproteins gp120 and gp41 on the MOLT-4/HIV-1SF-2H cell membrane was satisfactory for syncytium formation with the uninfected MOLT-4 cells. When MOLT-4/HIV-1SF-2H and MT-4 cells were co-cultured, severe cytopathogenicity was observed in MT-4 cells without being accompanied by the formation of multi-nucleated cells. Thus, the system consisting of MOLT-4/HIV-1SF-2H and MT-4 cells is convenient for exclusive study of the mechanism of cell-to-cell transmission of HIV-1. Using various compounds, it was confirmed that cell-to-cell transmission required both gp120/gp41-CD4 binding and de novo DNA synthesis.
A nickel (II) complex containing N-glycosides derived from the reaction of D-glucosamine (D-GlcN) and ethylenediamine (en) [Ni (D-GlcN-en)2] Cl2·H2O showed effective antifungal activity against pathogenic yeast, Candida albicans, where the MIC (minimal concentration of inhibition) value of the complex is 0.25 mM.
In sera of 29 patients under the chronic administration of carbamazepine (CBZ, dose : 10.9±4.3 mg/kg/day), a ring-contracted metabolite of CBZ was detected at a relatively higher concentration of 0.57±0.30 μg/ml (mean±S.D.) by high-performance liquid chromatogaphic assay. The structure of the metabolite was identified as 9-hydroxymethyl-10-carbamoylacridan (HMCA) by the liquid chromatography-mass spectral examination, and also by comparison with the authentic sample. Although HMCA was already reported in a few papers as a minor metabolite of CBZ in human urine, any reliable report for the determination of HMCA in human blood has never been published. Our results show that HMCA is one of the major metabolites of CBZ
The metabolism of insulin and calcitonin by microorganisms was examined in rat cecal contents. Both insulin and calcitonin were markedly degraded. Calcitonin was more susceptible to proteolysis in rat cecal contents than insulin. Calcitonin was also rapidly degraded in supernatant, while we found few degradation products of insulin. These findings suggest that care should be taken to metabolize the peptide drugs by microorganisms when they are administered to the large intestine for colon-specific drug delivery.