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Biomedical subjects

J Dai

Publications and source records attributed to J Dai.

At least 199 records · Page 11Linked to original sources
PubMed1993

Combination of DNA single strand synthesis with PCR to construct mung bean trypsin inhibitor gene.

This paper using the synthesis of mung bean trypsin inhibitor gene as an example, presented a new method for gene synthesis. The principle of the method was based on the combination of the single strand strategy and the PCR technique. The synthesis was very simple, convenient and rapid. The mung bean trypsin inhibitor is a protein composed of 72 amino acid residues. Its amino acid sequence has been determined, but the DNA sequence of gene still unknown. The synthetic mung bean trypsin inhibitor gene was 248 bp in length. It contains the encoded sequence, the start and stop codons, the restriction sites of EcoRI and BamHI at both ends, The codon selection of the synthetic gene was carried out according to the codon usage of other plant protease inhibitor gene or plant gene. The synthetic double-stranded DNA was digested with EcoRI and BamHI or PstI first, then cloned into plasmid pUC19. The synthetic gene was proved to be correct by the restriction map and the sequence analysis using the dideoxy-mediated chain termination method.

Amino Acid Sequence↗
PubMed1993

[Change in calcium transport as a factor in causing dysfunction of myocardial mitochondria during hypoxia].

During normoxic incubation of isolated myocardial mitochondria, there were marked increases in mitochondrial calcium when free calcium concentration in the incubation medium was enhanced. The increases were concomitant with rises increase in the rate of state 4 respiration. Moreover, there is a positive correlation between the mitochondrial calcium and the rate of state 4 respiration. During hypoxic incubation, when the calcium concentration of the incubation medium was enhanced, there was no significant increase in mitochondrial calcium and the rises in the rate of state 4 respiration were much lower than the rises during normoxia. Hypoxic incubation in medium of low calcium concentration (pCa 8.0) resulted in slight rises in rate of state 4 respiration. The above results suggest that in myocytes, the mitochondrial dysfunction under hypoxia may be caused indirectly by changes in cytoplasm rather than directly by hypoxia. It seems that the change of cellular free calcium concentration is an important factor in the mitochondrial dysfunction.

Animals↗
PubMed1992

Molecular cloning of the acid-labile subunit of the rat insulin-like growth factor binding protein complex.

The insulin-like growth factors, IGF-I and IGF-II, circulate in both humans and rats as part of a 125-150 kDa complex comprising IGFs, the IGF binding protein IGFBP-3, and an acid-labile subunit. Clones encoding rat acid-labile subunit have been isolated from a rat liver cDNA library probed with a human acid-labile subunit cDNA. Two overlapping clones encode a leucine-rich protein of 576 amino acids preceded by a 27-residue signal sequence, with 78% homology to the human acid-labile subunit. Northern analysis of mRNA from adult rat brain, kidney, heart, lung, spleen, muscle and liver shows a major species of about 4.4 kb and minor bands of about 2 kb, 1.4 kb and 1 kb. The tissue distribution of this protein may therefore be wider than previously recognized.

Amino Acid Sequence↗
PubMed1992

Computer system for the acquisition and analysis of vascular contractility. Application to a bioassay of endothelial cell function.

A system for the digital acquisition and subsequent analysis of the tension developed by isolated blood vessels in response to an endothelial cell superfusate is reported. Tension of the isolated rat aortic rings was measured by strain gauge. Strain-gauge output was then amplified, and the analog signal was digitized on a 16-channel A/D board. Lab tech Notebook software was used to display and store the data. The sampling rate was 0.1 Hz, and the data was written concurrently to hard disk and printer. Both disk and printer output were accompanied by a time stamp for subsequent ease of retrieval. The endothelial cell bioassay system allowed measurement of changes in vascular tension after the release of endothelium-dependent relaxing factor, nitric oxide (EDRF-NO) from cultured cells. Cells were cultured on microcarrier beads, formed into columns, and perfused with physiological salt solution. Significant (p < 0.05) relaxant responses occurred after agonist stimulation with bradykinin (10(-8) M; Emax -31.0% +/- 8.2%), acetylcholine (10(-8) M; Emax -33.2% +/- 5.0%), and calcium ionophore A 23187 (10(-6) M; Emax -55.7% +/- 15.4%). These responses were dependent on EDRF-NO, as shown by both the lack of relaxation in the absence of endothelial cells, and that relaxation to A 23187 was overcome by hemoglobin (3 x 10(-6) M). Results were manipulated graphically to allow the superimposition of data and thereby provide a mean and standard error of the mean for the entire time course of each response. Thus, a system was produced where fidelity of data expression was not dependent on measurements made at single points, but on the sampling frequency of the acquisition system.

Animals↗
PubMed1992

Depression of cardiac sarcolemmal phospholipase D activity by oxidant-induced thiol modification.

Myocardial phospholipase D (PLD) is primarily localized at the sarcolemmal level and selectively hydrolyzes phosphatidylcholine to form phosphatidic acid as part of the signal transduction mechanisms for regulating Ca2+ movements in the heart. Since the myocardial cell damage induced by oxidative stress is associated with abnormalities in Ca2+ homeostasis and thiol status, we examined the thiol group dependence and the effects of oxidant species on this enzyme. Sarcolemmal membranes isolated from rat heart were exposed to several types of thiol group modifiers. Alkylation with N-ethylmaleimide or methyl methanethiosulfonate, mercaptide formation with p-chloromercuriphenylsulfonic acid, and thiol-disulfide exchange with 5,5'-dithio-bis(2-nitrobenzoate) depressed sarcolemmal PLD activity; in all cases the depression was prevented by dithiothreitol. At different concentrations of N-ethylmaleimide the PLD depression correlated well (r = 0.98) with the decrease in total thiol group content of the membrane. The enzyme activity was not affected by xanthine-xanthine oxidase, a superoxide anion-generating system, but was depressed by hydrogen peroxide (H2O2) in a concentration-dependent manner. This inhibitory effect was prevented by catalase as well as by dithiothreitol, but not by D-mannitol. The effect of a hydroxyl radical-generating system (Fenton reaction) could not be assessed because of an interfering direct inhibition by Fe2+. Dithiothreitol was also able to restore PLD activity in H2O2-pretreated membranes and to prevent a severe deactivation of the enzyme by hypochlorous acid (HOCI). Protection by glutathione and inhibition by its oxidized form were also observed.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

Structure and functional expression of the acid-labile subunit of the insulin-like growth factor-binding protein complex.

Nearly all of the insulin-like growth factor (IGF) in the circulation is bound in a heterotrimeric complex composed of IGF, IGF-binding protein-3, and the acid-labile subunit (ALS). Full-length clones encoding ALS have been isolated from human liver cDNA libraries by using probes based on amino acid sequence data from the purified protein. These clones encode a mature protein of 578 amino acids preceded by a 27-amino acid hydrophobic sequence indicative of a secretion signal. Expression of the cDNA clones in mammalian tissue culture cells results in the secretion into the culture medium of ALS activity that can form the expected complex with IGF-I and IGF-binding protein-3. The amino acid sequence of ALS is largely composed of 18-20 leucine-rich repeats of 24 amino acids. These repeats are found in a number of diverse proteins that, like ALS, participate in protein-protein interactions.

Amino Acid Sequence↗
PubMed1992

[Construction and identification of vectors containing maize plasmid-like DNA S1 and reporter genes].

The reporter Gene GUS with CaMV 35S promoter was inserted into plasmid pBS[1] in reversal direction of integration, and two kinds of recombinant plasmids were obtained. These results were verified by restriction endonuclease analysis and southern hybridization. A piece of 10.8kb fragment containing NPT II reporter gene from digested pBI121.1 by BamHI and EcoRI was ligated together with 3.9 kb fragment of plasmid-like DNA S1. Then generate a recombinant plasmid pBIS5 was generated. These three vectors can be used to transform maize protoplasts. High frequency of transformation may be expected due to homology between plasmid-like DNA S1 and maize nuclear DNA.

DNA↗
PubMed1991

Phospholipase D activity in subcellular membranes of rat ventricular myocardium.

The phospholipase D (PL D), which catalyzes the formation of phosphatidic acid (PA), was studied in rat myocardium using 14C-labelled phosphatidylcholine (PC) as an exogenous substrate. Subcellular distribution experiments indicated the presence of PL D in particulate fractions only. Different procedures for the isolation of purified cardiac subcellular organelles showed the presence of PL D in sarcolemma (SL), sarcoplasmic reticulum (SR) and mitochondria with 14-, 11- and 5-fold enrichment when compared to the homogenate value, respectively. The activity of SL PL D was observed over a narrow acid pH range with an optimum at 6.5, and it showed a high specificity for PC while phosphatidylethanolamine and phosphatidylinositol showed a low rate of hydrolysis. Under optimal conditions, PA formation was linear for a 90-min period of incubation and the reaction rate was constant for 10 to 100 micrograms SL protein in the assay medium. The SR PL D displayed properties similar to those seen with the SL PL D. In membrane fractions PL D was also found to catalyze a transphosphatidylation reaction for the synthesis of phosphatidylglycerol. Assessment of the intramembranal levels of radioactive 1,2-diacylglycerol (DAG) in the absence or presence of KF suggested the presence of an active PA phosphohydrolase activity. This study indicates that a PC-specific PL D activity is localized in different membrane systems of the myocardium and may be associated with PA phosphohydrolase to act in a coordinated manner. The functional significance of PL D-dependent formation of PA in cardiac membranes is discussed.

Animals↗
PubMed1991

Role of sulfhydryl groups in phospholipid methylation reactions of cardiac sarcolemma.

The effect of reagents that modify sulfur-containing amino acid residues in the phosphatidylethanolamine N-methyltransferase was studied in the isolated rat cardiac sarcolemma by employing S-adenosyl-L-[methyl-3H]methionine as a methyl donor. Dithiothreitol protected the sulfhydryl groups in the membrane and caused a concentration- and time-dependent increase of phospholipid N-methylation at three different catalytic sites. This stimulation was highest (9-fold) in the presence of 1 mM MgCl2 and 0.1 microM S-adenosyl-L-[methyl-3H]methionine at pH 8.0 (catalytic site I), and was associated with an enhancement of Vmax without changes in Km for the methyl donor. Thiol glutathione was less stimulatory than dithiothreitol; glutathione disulfide inhibited the phosphatidylethanolamine N-methylation by 50%. The alkylating reagents, N-ethylmaleimide and methylmethanethiosulfonate, inhibited the N-methylation with IC50 of 6.9 and 14.1 microM, respectively; this inhibition was prevented by 1 mM dithiothreitol. These results indicate a critical role of sulfhydryl groups for the activity of the cardiac sarcolemmal phosphatidylethanolamine N-methyltransferase and suggest that this enzyme system in cardiac sarcolemma may be controlled by the glutathione/glutathione disulfide redox state in the cell.

Animals↗
PubMed1991

[Morphological and functional features of endodermal cells in rat yolk sac, with special reference to the fetal macrophage differentiation].

Morphological and functional features of the yolk sac endodermal cells with special reference to the fetal macrophage differentiation were investigated morphologically under the light and electron microscopes and immunologically with the antigen phenotypic analysis and the phagocytic activity-test, using the syngeneic DA rat-embryos from 8 to 16 days of gestation. Based on the staining property with toluidin blue and the ultrastructural features, the endodermal layer from day 8 to 16-yolk sacs has been known to consist of two kinds of cell type; 10% "clear" cells with clear cytoplasm and 90% "dark" cells with dark cytoplasm. Numerous primary lysosomes, phagolysosomes, lipid droplets and coated vesicles distributed preferentially in the supranuclear portion of endodermal cells. A broad intercellular space was found between "clear" cells and "dark" cells, indicating the loose intercellular binding. It was often found that "clear" cells tend to migrate from the endodermal layer into the mesenchymal layer, where the poor development of basement membrane was seen between them. Cells phagocytosing red blood cells, that resemble morphologically "clear" cells, were also observed in the fetal liver. At ten hours after latex-injection into the yolk sac cavity of 14 days embryos, some cells which phagocytosed latex beads in their cytoplasm were found in the endodermal layer, and also in the liver tissue and loose connective tissue of fetus. These cells were stained positively with monoclonal antibody Mar3 which recognizes preferentially rat-mononuclear phagocyte system. In vitro-latex uptake of separated endodermal cells was also demonstrated by the culture-study of endodermal cell suspension. The present findings indicate that the yolk sac-endodermal layer derived from the proximal endoderm consists of at least two kinds of cell-population with a great similarity to tissue macrophages in morphological and functional senses, and support the concept that some cell-populations of endodermal layer may migrate into fetal tissue and are closely related to the differentiation of fetal macrophages and their precursors.

Animals↗
PubMed1990

Alteration of lipid methylation by oleic acid in rat heart sarcolemma.

Incubation of rat heart sarcolemma with the methyl donor S-adenosyl-L-[methyl-3H] methionine resulted in N-methylation of phosphatidylethanolamine and methylation of a heterogenous fraction of nonpolar lipids in the membrane. Oleic acid reduced the synthesis of N-methylated phospholipids and stimulated the methyl group incorporation into nonpolar lipids in a concentration-dependent manner. Both methylation reactions were not affected when oleic acid was substituted by methyl ester of oleic acid or by the detergents sodium deoxycholate or Triton X-100. This study suggests that the enzymatic biosynthesis of the N-methylated phospholipids may be altered by free fatty acids.

Animals↗
PubMed1990

[Studies on ultraweak luminescence of bacteria].

Ultraweak luminescence of Escherichia coli, Bacillus subtilis and Brevibacterium ammoniagenes was measured with high sensitive single photon counting equipment (Made in China). The results obtained from ultraweak luminescence of as above three bacterial strains were as follows: spectral distribution curves, photon emission kinetic curves or emission intensity and its qualitative relationship between intensity and bacterial counts.

Bacillus subtilis↗
PubMedDate not supplied

Interactions between cyclic AMP-dependent protein phosphorylation and lipid transmethylation reactions in isolated porcine cardiac sarcolemma.

Premethylation of purified porcine cardiac sarcolemma (SL) in the presence of 0.15, 10 and 150 microM S-adenosyl-L-methionine (AdoMet) did not change the phosphorylation of SL proteins catalyzed either by intrinsic cyclic AMP-dependent protein kinase (cAK) or by added catalytic (C) subunit of this enzyme. On the other hand, membrane exhibited increased lipid methyltransferase activity after preincubation with MgATP and C subunit. Prephosphorylation of membranes stimulated the total [3H]-methyl incorporation into SL lipids assayed at 0.15 microM [3H]AdoMet due to an enhancement of Vmax and without changes in the Km value for AdoMet. Analysis of the methylated lipid products revealed an increased methyl group incorporation into a nonpolar lipid fraction whereas phosphatidylethanolamine-N-methylation was not affected by phosphorylation. The results suggest that the cyclic AMP-mediated signal transduction at the level of cardiac SL is not affected by methylation-induced modifications of the membrane lipid microdomains. On the other hand, an intrinsic SL lipid methyltransferase activity is apparently not related to the N-methylation of phospholipids, is modulated by cyclic AMP-dependent protein phosphorylation.

Adenosine Triphosphate↗
PubMedDate not supplied

Kinetics of myocardial phospholipase D.

Myocardial phospholipase D (PLD) is located in different subcellular membranes, including sarcolemma (SL) and sarcoplasmic reticulum (SR). In this study, the kinetics of PLD-dependent hydrolytic and transphosphatidylation activities were examined in SL and SR fractions isolated from rat heart by measuring the formation of phosphatidic acid and phosphatidylethanol, respectively. The results showed that, compared to SR PLD, SL PLD had a higher Vmax, i.e. 373 vs. 70 nmol/mg protein/h for the hydrolytic activity and 415 vs. 60 nmol/mg protein/h for the transphosphatidylation activity. In comparison with the SR enzyme, SL PLD had a lower Km value for the hydrolytic activity (0.46 vs. 0.65 mM), buy a higher Km for the transphosphatidylation activity (225 vs. 179 mM). These distinctive kinetic parameters suggest that SL PLD and SR PLD may be isoforms of the enzyme and/or have different membrane domain. Therefore, SL- and SR-localized PLD activities may be under independent control mechanism(s) and play distinct roles in normal conditions and pathological processes.

Animals↗