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Biomedical subjects

J Zhao

Publications and source records attributed to J Zhao.

At least 595 records · Page 33Linked to original sources
PubMed1996

Effect of verapamil on Ca2+ influx and CVB3-RNA replication in cultured neonatal rat heart cells infected with CVB3.

The effect of verapamil on Ca2+ influx across the myocardial plasma membrane and coxsackie virus B3 (CVB3) -RNA replication in cultured neonatal rat heart cells infected with CVB3 was investigated. It was found that the Ca2+ influx could be inhibited significantly (P < 0.01) by verapamil (1 mumol/L) after infection of heart cells for 48 h. However, when the cultured heart cells infected with CVB3 and treated with verapamil (1 mumol/L and 10 nmol/L) at the same time for 48 h, the amounts of CVB3-RNA in myocytes were significantly higher than that in infected control group (P < 0.05). These phenomena suggest that the increase of Ca2+ influx of cultured heart cells infected with CVB3 could be inhibited by some calcium antagonists, e.g. verapamil at the early stage. On the other hand, verapamil might accelerate viral replication in myocardium. Thus, although verapamil could be beneficial for decreasing the secondary Ca2+ damages and improve the myocardial electric activity, it isn't a sensible choice for therapy in early stage of virus infection with cardiac symptoms.

Animals↗
PubMed1996

[Expression of myelin basic protein gene in the developing human brain].

The expression of myelin basic protein (MBP) gene in the developing human brain was studied by using Northern dot blot and in situ hybridization with 32P- and biotin-labeled rat MBP cDNA and Diglabeled human MBP cRNA probes so as to explore the relation of temporal and spatial expression of human MBP gene to myelinogenesis and myelination. The results of dot blot analysis showed that the expression of MBP mRNA was near 2.0 A590/microgram of RNA in the fetal brain at 16 weeks of conceptional age, and it increased progressively with development to have 9-fold increment (18 A590/microgram of RNA) in the adult brain. The insitu hybridization was performed with the sections of fetal spinal cord and medulla oblongata at 8-25 weeks of conceptional age, and of pons, midbrain, thalamus, cerebellum and cerebrum at 20, 22 and 25 weeks. The results indicated that the expression of MBP mRNA was found as early as 8-9 weeks and localized within the perinuclear cytoplasm of the scattered cells in the ventral horn of spinal cord and near the basis of the fourth ventricle of medulla oblongata. Whether the above-mentioned labeled cells are neurons or precursors of glial cell remains to be identified because they are larger in size and have more abundant cytoplasm than oligodendrocytes. The labeling of MBP mRNA abruptly increased and spread with the processes of oligodendrocytes to form a network in spinal cord and medulla at 20 weeks; meanwhile, labeling was obvious in pons and scattering in the other brain regions examined.

Adult↗
PubMed1995

Phosphorylation of both serine residues in cardiac troponin I is required to decrease the Ca2+ affinity of cardiac troponin C.

The phosphorylation of cardiac muscle troponin I (CTnI) at two adjacent N-terminal serine residues by cAMP-dependent protein kinase (PKA) has been implicated in the inotropic response of the heart to beta-agonists. Phosphorylation of these residues has been shown to reduce the Ca2+ affinity of the single Ca(2+)-specific regulatory site of cardiac troponin C (CTnC) and to increase the rate of Ca2+ dissociation from this site (Robertson, S. P., Johnson, J. D., Holroyde, M. J., Kranias, E. G., Potter, J. D., and Solaro, R. J. (1982) J. Biol. Chem. 257, 260-263). Recent studies (Zhang, R., Zhao, J., and Potter, J. D. (1995) Circ. Res. 76, 1028-1035) have correlated this increase in Ca2+ dissociation with a reduced Ca2+ sensitivity of force development and a faster rate of cardiac muscle relaxation in a PKA phosphorylated skinned cardiac muscle preparation. To further determine the role of the two PKA phosphorylation sites in mouse CTnI (serine 22 and 23), serine 22 or 23, or both were mutated to alanine. The wild type and the mutated CTnIs were expressed in Escherichia coli and purified. Using these mutants, it was found that serine 23 was phosphorylated more rapidly than serine 22 and that both serines are required to be phosphorylated in order to observe the characteristic reduction in the Ca2+ sensitivity of force development seen in a skinned cardiac muscle preparation. The latter result confirms that PKA phosphorylation of CTnI, and not other proteins, is responsible for this change in Ca2+ sensitivity. The results also suggest that one of the serines (23) may be constitutively phosphorylated and that serine 22 may be functionally more important.

Amino Acid Sequence↗
PubMed1995

Regulation of transforming growth factor beta receptors in H-ras oncogene-transformed rat intestinal epithelial cells.

We have reported that over expression of the H-ras oncogene causes resistance to growth inhibition by transforming growth factor beta 1 (TGF-beta 1) and a time-dependant switch of type II to type I TGF-beta receptor expression in the rat intestinal epithelial cell line IEC-18 (J. Filmus, J. Zhao, and R. N. Buick, Oncogene, 7: 521-526, 1992). Here, we investigate the possible mechanisms involved in H-ras-mediated regulation of TGF-beta receptors in an IEC-18 cell clone expressing H-ras, conditional on the activity of a dexamethasone-sensitive promoter. The switch from type II to type I receptor expression in response to H-ras expression has a requirement for de novo RNA synthesis. In addition, accumulation of TGF-beta receptor type II mRNA is approximately 5-fold lower in ras-expressing cells compared to control cells. Nuclear run-on experiments suggest that the down-regulation of type II receptor mRNA by H-ras oncogene is based, at least in part, on reduced transcription. We have also analyzed the consequences of H-ras expression on the properties of the TGF-beta receptors. Type I and II in IEC-18 cells and type I receptors in ras-transformed cells have similar characteristics in terms of binding affinities for TGF-beta 1 (or TGF-beta 2) turnover rates and glycosylation states. Notably, the type I receptors in ras-transformed cells are not capable of ligand-induced internalization. Although H-ras expression in IEC-18 cells causes resistance to TGF-beta-mediated growth inhibition, the cells remain responsive to TGF-beta 1 stimulation of fibronectin expression. These results are discussed in the context of the knowledge of TGF-beta receptor complexity and signal transduction, and with reference to the potential role for loss of TGF-beta-mediated negative growth regulation in malignant transformation.

Animals↗
PubMed1995

The synthesis of blocked triplet-phosphoramidites and their use in mutagenesis.

A general approach for the synthesis of oligonucleotide-triplet phosphoramidites and the synthesis of four such blocks are described. A strategy was devised to minimize the number of dimer precursors needed for synthesis of a complete set of triplet-amidite blocks encoding all 20 amino acids. Whereas synthesis of 20 triplet-amidite blocks consisting of codon sequences requires 16 dimer blocks, just seven dimer blocks are required to synthesize all required antisense sequences. The antisense sequences are then converted to codons in template mediated replication. Using a mixture of four triplet-amidites and conventional automated solid-phase DNA synthesis, short (6mer) and medium length (30mer) oligonucleotide mixtures were synthesized and analyzed. The latter was replicated in vitro and used as a mutagenic cassette to produce four mutants of Asp 221 in the enzyme thymidylate synthase. The method establishes the direction and utility for the production and use of triplet-amidite blocks in DNA synthesis.

Amides↗
PubMed1995

Modified ligands to FA and FB in photosystem I. I. Structural constraints for the formation of iron-sulfur clusters in free and rebound PsaC.

Cysteines 14, 21, 34, 51, or 58 in PsaC of photosystem I (PS I) were replaced with aspartic acid (C21D and C58D), serine (C14S, C34S, and C51S), and alanine (C14A, C34A, and C51A). When free in solution, the C34S and C34A holoproteins contained two S = 1/2 ground state [4Fe-4S] clusters; all other mutant proteins contained [3Fe-4S] clusters and [4Fe-4S] clusters; in addition, there was evidence in C14S, C51S, C14A, and C51A for high spin (S = 3/2) [4Fe-4S] clusters, presumably in the modified site. These findings are consistent with the assignment of C14, C21, C51, and C58, but not C34, as ligands to FA and FB. The [4Fe-4S] clusters in the unmodified sites in C14S, C51S, C14A, and C51A remained highly electronegative, with Em values ranging from -495 to -575 mV. The [3Fe-4S] clusters in the modified sites were driven 400 to 450 mV more oxidizing than the native [4Fe-4S] clusters, with Em values ranging from -98 mV to -171 mV. A C14D/C51D double mutant contains [3Fe-4S] and S = 1/2 [4Fe-4S] clusters, showing that the 3Cys.1Asp motif is also able to accommodate a low spin cubane. When C34S, C34A, C14S, C51S, C14A, and C51A were rebound to P700-FX cores, electron transfer to FA/FB was regained, but functional reconstitution has not yet been achieved for C21D, C58D, or C14D/C51D. These data imply that PsaC requires two iron-sulfur clusters to refold, one of which must be a cubane. Since two [4Fe-4S] clusters are found in all reconstituted PS I complexes, the presence of two cubanes in free PsaC may be a necessary precondition for binding to P700-FX cores.

Amino Acid Sequence↗
PubMed1995

Arabidopsis thaliana tryptophan synthase alpha: gene cloning, expression, and subunit interaction.

The tryptophan synthase alpha subunit catalyzes the conversion of indole-3-glycerolphosphate to indole, the penultimate reaction in the biosynthesis of the essential amino acid tryptophan. A cDNA encoding Arabidopsis thaliana tryptophan synthase alpha(TSA1) was isolated by complementation of an Escherichia coli delta trpA mutation and by polymerase chain reaction amplification from a cDNA library using degenerate primers. A TSA1 genomic clone was also isolated and 5 kb of the DNA sequence determined. A single sequence in the Arabidopsis genome with homology to the TSA1 cDNA was detected by high-stringency genomic Southern blot hybridization. In contrast under hybridization conditions of reduced stringency, one or two additional homologous sequences were observed. A 1.4 kb transcript was detected in wild-type RNA with the TSA1 cDNA as a probe. Several lines of evidence, including immunoaffinity chromatography, suggest that the active A. thaliana tryptophan synthase enzyme consists of a heterosubunit complex, presumably analogous to the prokaryotic alpha 2 beta 2 complex. Immunoblot analysis indicated that the plant alpha and beta subunits are present throughout development.

Amino Acid Sequence↗
PubMed1995

The complex of phosphatidylinositol 4,5-bisphosphate and calcium ions is not responsible for Ca2+-induced loss of phospholipid asymmetry in the human erythrocyte: a study in Scott syndrome, a disorder of calcium-induced phospholipid scrambling.

Elevation of cytoplasmic Ca2+ levels in human erythrocytes induces a progressive loss of membrane phospholipid asymmetry, a process that is impaired in erythrocytes from a patient with Scott syndrome. We show here that porcine erythrocytes are similarly incapable of Ca2+-induced redistribution of membrane phospholipids. Because a complex of phosphatidylinositol 4,5-bisphosphate (PIP2) and Ca2+ has been proposed as the mediator of enhanced transbilayer movement of lipids (J Biol Chem 269:6347,1994), these cell systems offer a unique opportunity for testing this mechanism. Analysis of both total PIP2 content and the metabolic-resistant pool of PIP2 that remains after incubation with Ca2+ ionophore showed no appreciable differences between normal and Scott erythrocytes. Moreover, porcine erythrocytes were found to have slightly higher levels of both total and metabolic-resistant PIP2 in comparison with normal human erythrocytes. Although loading of normal erythrocytes with exogenously added PIP2 gave rise to a Ca2+-induced increase in prothrombinase activity and apparent transbilayer movement of nitrobenzoxadiazolyl (NBD)-phospholipids, these PIP2-loaded cells were also found to undergo progressive Ca2+-dependent cell lysis, which seriously hampers interpretation of these data. Moreover, loading Scott cells with PIP2 did not abolish their impaired lipid scrambling, even in the presence of a Ca2+-ionophore. Finally, artificial lipid vesicles containing no PIP2 or 1 mole percent of PIP2 were indistinguishable with respect to transbilayer movement of NBD-phosphatidylcholine in the presence of Ca2+. Our findings suggest that Ca2+-induced redistribution of membrane phospholipids cannot simply be attributed to the steady-state concentration of PIP2, and imply that such lipid movement is regulated by other cellular processes.

4-Chloro-7-nitrobenzofurazan↗
PubMed1995

Effect of anthrax toxin's lethal factor on ion channels formed by the protective antigen.

Protective antigen (PA), a component of anthrax toxin, mediates translocation of the toxin's lethal and edema factors (LF and EF, respectively) to the cytoplasm, via a pathway involving their release from an acidic intracellular compartment. PA63, a 63-kDa proteolytic fragment of PA, can be induced to form ionconductive channels in the plasma membrane of mammalian cells by acidification of the medium. These channels are believed to be comprised of dodecyl sulfate-resistant oligomers (heptameric rings) of PA63 seen by electron microscopy of the purified protein. Here we report that the PA63-mediated efflux of 86Rb+ from preloaded CHO-K1 cells under acidic conditions is strongly inhibited (> or = 70%) by LF or LFN, a PA-binding fragment of LF. Control proteins caused no inhibition. Evidence is presented that the inhibition involves partial blockage of ion conductance by the PA63 channel. Also, oligomer formation is slowed somewhat by LF at pH values near the pH threshold of channel formation (pH approximately 5.3), suggesting that channel formation may also be retarded under these conditions. The relevance of these results to the location of the LF-binding site on PA63 and the mechanism of LF and EF translocation is discussed.

Animals↗
PubMed1995

Characterization of an unbalanced de novo rearrangement by microsatellite polymorphism typing and by fluorescent in situ hybridization.

Unbalanced de novo rearrangements, difficult to characterize by conventional cytogenetic techniques, may be elucidated by molecular approaches. By dinucleotide repeat polymorphism typing and fluorescence in situ hybridization (FISH), we have defined the composition of an unbalanced de novo translocation (46,XX,15p+) in a child with multiple congenital anomalies. Use of a microsatellite repeat D5S208 (localized to 5p15) and polymerase chain reaction (PCR) analysis confirmed that the extra segment originated from the short arm of chromosome 5. Amplification of the patient's DNA with primers for dinucleotide repeats D5S350 and D5S118 showed that the entire 5p (from 5pter to 5q11) was present in 3 copies. FISH confirmed the trisomic status of 5p, and further revealed the presence of centromeres of both chromosomes 5 and 15 on the rearranged chromosome thus delineating its dicentric nature. This information allowed us to redefine the de novo rearrangement in this patient as 46,XX,dic der(15)t(5;15)(q11;p11).

Base Sequence↗
PubMed1995

Expression of Rb, E2F1, cdc2, and D, and B cyclins in developing spinal cord.

The spatial and temporal distribution of transcripts for the tumor suppressor gene Rb, transcription factor E2F1, cdc2 kinase, cyclins D1, D2, B1 and B2 during neurogenesis of the spinal cord was determined by in situ hybridization. The Rb and E2F1 transcripts were detectable in proliferating and differentiating cells. By contrast, cdc2, cyclins D1, B1 and B2 are expressed in the ventricular zone where proliferating cells are localized. Cyclin D2 mRNA was detectable only in the marginal zone of the developing neural tube. Electrophoretic mobility shift analyses demonstrated a changing pattern of DNA/protein complexes that bind to E2F binding site. These observations suggest that Rb and E2F1 may be involved in the early stages of neuronal differentiation in addition to the cell cycle regulation.

Animals↗
PubMed1995

Out-of-equilibrium CO2/HCO3- solutions and their use in characterizing a new K/HCO3 cotransporter.

In typical physiological solutions, CO2 is in equilibrium with HCO3- and H+ (CO2 + H2O<==>HCO3- +H+). Because one cannot independently alter CO2 and HCO3- concentrations and pH, it is impossible to distinguish between the effects of CO2 and HCO3- on physiological processes. Here we describe a continuous-flow, rapid-mixing approach for generating out-of-equilibrium CO2/HCO3- solutions with a physiological pH and CO2 (but little HCO3-), or pH and HCO3- (but little CO2). We have exploited these out-of-equilibrium solutions to introduce HCO3- exclusively to either the outside or inside of a squid giant axon, and verify the presence of a new K/HCO3 cotransporter. The out-of-equilibrium approach could be useful in a variety of applications for independently controlling CO2 and HCO3- concentrations and pH.

Animals↗
PubMed1995

Quantitation of matrix Gla protein mRNA by competitive polymerase chain reaction using glyceraldehyde-3-phosphate dehydrogenase as an internal control.

Matrix Gla (gamma-carboxyglutamic acid) protein (MGP) is a vitamin-K-dependent extracellular matrix protein. A method was developed to quantitate MGP mRNA based on competitive polymerase chain reaction following reverse transcription (competitive RT-PCR). The MGP cDNA was coamplified with a mutant MGP cDNA (competitor). The ratio of MGP to competitor after the PCR reaction was compared to standards to determine the amount of MGP mRNA in RT samples. MGP mRNA in as little as 3.125 ng total RNA was accurately quantitated and was far more sensitive than RNA hybridization methods. To control for variations due to sample preparation, a second competitive RT-PCR was developed to measure the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA from the same sample as an internal control. Thus, the amount of MGP is normalized to the amount of the housekeeping gene GAPDH. The accuracy, sensitivity and ease of this new method enables rapid mRNA quantitation without blotting, hybridization or autoradiography. The method is particularly advantageous for MGP mRNA measurement from a small amount of sample. Using this assay, we established that MGP mRNA increases approx. fivefold with co-treatment of retinoic and ascorbic acids.

Animals↗
PubMed1995

Immunological characterization and chloroplast localization of the tryptophan biosynthetic enzymes of the flowering plant Arabidopsis thaliana.

In order to study the tryptophan biosynthetic enzymes of the plant Arabidopsis thaliana, polyclonal antibodies were raised against five of the tryptophan biosynthetic pathway proteins: anthranilate synthase alpha subunit, phosphoribosylanthranilate transferase, phosphoribosylanthranilate isomerase, and the tryptophan synthase alpha and beta subunits. Immunoblot analysis of Arabidopsis leaf protein extracts revealed that the antibodies identify the corresponding proteins that are enriched in Arabidopsis chloroplast fractions. Precursors of phosphoribosylanthranilate isomerase and tryptophan synthase alpha subunit were synthesized by in vitro translation. The precursors were efficiently imported and processed by isolated spinach chloroplasts, and the cleavage sites within the precursors were determined. These results provide the first direct evidence that the tryptophan biosynthetic enzymes from Arabidopsis are synthesized as higher molecular weight precursors and then imported into chloroplasts and processed into their mature forms.

Aldose-Ketose Isomerases↗