Cytoprotective effect of TRK-100, a prostacyclin analogue, against chemical injuries in cultured human vascular endothelial cells

We studied the cytoprotective effect of TRK-100, a chemically stable analogue of prostacyclin (PGI2), in the cultured human endothelial cells from umbilical vein. TRK-100 (10 and 100 nM) stimulated significantly proliferation of endothelial cells but did not affect PGI2 production in endothelial cells. Exposure of cultured endothelial cells to homocysteine (2.5 mM) or glucose (50 mM) caused concentration-dependent cytotoxicity, as evidenced by a decrease in number of viable cells. When endothelial cells were treated with TRK-100 simultaneously or prior to, but not after, exposure to injury substances, decreases in viable cell were significantly suppressed. The protective effect of TRK-100 against homocysteine-induced cytotoxicity also appeared in endothelial cells treated with acetylsalicylic acid, suggesting that endogenous PGI2 did not involve in the protective effect of TRK-100.     

Intergeneric transfer of cytoplasmic male sterility between Raphanus sativus (cms line) and Brassica napus through cytoplast-protoplast fusion

Summary Cytoplasts isolated from hypocotyl protoplasts of Raphanus sativus cv Kosena (cms line) by ultracentrifugation through Percoll/mannitol discontinuous gradient were fused with iodoacetamide(IOA)-treated protoplasts of Brassica napus cv Westar. Seventeen randomly selected regenerated plants were characterized for morphology and chromosome numbers. All of the regenerated plants had morphology identical to B. napus and 10 of them possessed the diploid chromosome number of B. napus. The remaining plants had chimeric or aneuploid chromosome numbers. The mitochondrial genomes in the 10 fusion products possessing the diploid chromosome numbers of B. napus were examined by Southern hybridization analysis. Four of the 10 plants contained mitochondrial DNA showing novel hybridization patterns. Of these 4 plants, 1 was male sterile, and 3 were male fertile. The remaining plants showed mitochondrial DNA patterns identical to B. napus and were male fertile.     

Mutational analysis of simian immunodeficiency virus from African green monkeys and human immunodeficiency virus type 2

We constructed ten mutants of simian immunodeficiency virus isolated from African green monkey (SIVAGM), and nine mutants of human immunodeficiency virus type 2 (HIV-2) in vitro. Their infectivity, cytopathogenicity, transactivation potential, virus RNA, and protein synthesis were examined by transfection and infection experiments. Mutations in three structural (gag, pol, env) and two regulator (tat, rev) genes abolished the infectivity of both viruses, but vpx, vpr (HIV-2), and nef were dispensable and mutant viruses were indistinguishable phenotypically from wild type virus. A vif mutant of HIV-2 showed poor infectivity in cell-free condition, whereas SIVAGM mutants grew equally well with wild type virus. In transient transfection assays, rev mutants derived from both viruses produced mainly small mRNA species and no detectable virus proteins and particles. Transactivation potential of tat mutants originated from both viruses was about three- to ten-fold less than that of respective wild type DNAs, generating small amounts of virus.     

Molecular cloning, sequence analysis and expression of a cDNA encoding human type-1 angiotensin II receptor.

We isolated a cDNA encoding type-1 angiotensin II receptor from a human liver cDNA library. The cDNA had an open reading frame encoding a protein of 359 amino acid residues with a relative Mr of 41,060. The deduced amino acid sequence of the human angiotensin II (Ang II) receptor was 95.3% and 94.2% identical to those of bovine and rat type-1 Ang II receptors, respectively, and had a significant similarity with the G protein-coupled receptor. The rank order of the binding to the receptor expressed in COS-7 cells was Ang II greater than Ang III greater than Ang I. The expression of the Ang II receptor mRNA was detected in human liver, lung, adrenal and adrenocortical adenomas but not in adrenomedullary tumor, pheochromocytoma, by Northern blot analysis.     

Differential oxidase activity of hepatic and pulmonary microsomal cytochrome P-450 isozymes after treatment with cytochrome P-450 inducers.

Phenobarbital, 3-methylcholanthrene, acetone and pyrazole were used as inducers of cytochrome P450 and the NADPH-dependent oxidase activity (O-2 production) of pulmonary and hepatic microsomes was determined. Oxidase activity of microsomes from 3-methylcholanthrene-treated rats was significantly decreased as compared to that of controls when expressed on the basis of cytochrome P450 content (30% decrease for liver, 60% decrease for lung). The oxidase activity of liver microsomes from pyrazole-treated rats showed a significant increase, whereas phenobarbital treated microsomes had average superoxide-generating activity. The contribution of cytochromes CYP 1A, CYP 2B and CYP 2E1 to superoxide-generating activity was investigated using monoclonal antibodies. Monoclonal antibody 1-91-3 against CYP 2E1 inhibited superoxide generation by 58% in liver microsomes from pyrazole-treated rats. Monoclonal antibodies 1-7-1 and 2-66-3 against CYP 1A and CYP2B, respectively, had no effect on superoxide generation. These results indicate that different cytochrome P450 isoforms are mainly responsible for differential superoxide generating activities of microsomes and complement the reconstitution study of Morehouse and Aust. Furthermore, our study indicates that CYP 1A1, induced by 3-MC, demonstrates an unusually low oxidase activity.     

Circular dichroic evidence for regulation of enzymatic activity by nonsubstrate hydrophobic ligand on glutathione S-transferase P.

1-Anilinonaphthalene-8-sulfonic acid (ANS) noncompetitively inhibited enzyme activity of glutathione S-transferase P for both glutathione and 1-chloro-2,4-dinitrobenzene (Ki = 30 microM). Dissociation constant for ANS.GST-P complex calculated from the binding study was 15 microM. From the similar values of the inhibition constant and the dissociation constant, it was concluded that specific ANS binding caused the loss of enzyme activity. In the protein structural analysis by circular dichroism, the secondary structures remarkably changed by ANS binding in accordance with the decrease of enzymatic activities. The conformational change of the protein and the decrease in enzymatic activity were reversed by dissociation of ANS. This fact strongly suggested that the enzymatic activity was regulated by a nonsubstrate hydrophobic ligand.     

The existence of activin A/erythroid differentiation factor and its inhibitor in human serum: comparison of normal and chronic renal failure sera.

Activin A/EDF, initially found as a differentiation inducer of murine Friend erythroleukemia, also has a stimulatory effect on erythropoiesis in vitro and in vivo. Here we proved activin A/EDF activity in human serum. The activin A/EDF level in 18 normal human serum samples was measured by a specific bioassay and was found to be 8.3 +/- 4.6 ng/ml, indicating that there exists sufficient activity to affect erythropoiesis in normal serum. In contrast, activin A/EDF activity was reduced in the chronic renal failure patients and 23/26 serum samples examined showed levels below 1.2 ng/ml. Further analysis using HPLC revealed that chronic renal failure serum actually contained as much activin A/EDF as normal serum, and that the difference between normal and patient serum existed in the content of a specific inhibitor of activin A/EDF. This observation suggests the possibility that the inhibitor is participating in the regulation of activin A/EDF activity in vivo in chronic renal failure patients and also the possibility of activin A/EDF could be utilized in the therapy of the anemia of such patients.     

Gene expression of metalloproteinase and its inhibitor in mesangial cells exposed to high glucose.

To clarify the roles of metalloproteinases and their inhibitor (TIMP) in diabetic glomerulopathy, we studied the effect of a high glucose concentration on the gene expression of metalloproteinase transin and TIMP as well as collagen type IV and laminin in cultured rat mesangial cells (MCs). In the high glucose group, collagen type IV, laminin, and TIMP mRNA levels were all elevated in a concentration-dependent manner, whereas transin expression was suppressed. Osmotic control of high glucose with mannitol selectively stimulated TIMP expression. We hypothesize that high glucose decreases matrix-degrading activity as well as increases matrix productivity in MCs.     

Metabolic activities in germinated ancient lotus seeds

Seeds of Taizi lotus (Nelumbo nucifera Gaertn cv. Taizi) wereunearthed in a western suburb of Beijing in 1984, and determinedto be 58070-years-old by radiocarbon dating. Pretreatment withconcentrated sulphuric acid for 6 h promoted 75% of the seedstested to germinate. Over 3 weeks of incubation after the additionof water, storage starch, total soluble sugar and globulin inthe cotyledons decreased whereas albumin and soluble -aminonitrogen increased. These changes in cotyledonary componentswere similar to the changes seen in four present-day Indianlotus varieties, although the content of reducing sugar in Taizilotus was significantly lower. The polypeptide compositionsin extracts from the cotyledons and embryonic axis of Taiziseeds, as analysed by SDS-PAGE, were similar to those of present-dayJiangxi seeds. Taizi lotus also showed the ability to incorporateradioactive leucine into the trichloroacetic acid (TCA)-insolublefraction of a cotyledonary extract during the 24 h post-imbibitionperiod. The incorporation was inhibited when seeds of Taizilotus were allowed to rehydrate for 24 h in the presence of-amanitin or cycloheximide. Key words: Ancient lotus, Nelumbo nucifera, protein synthesis, seed germination, seed viability