Biomedical subjects

A Zhang

Publications and source records attributed to A Zhang.

At least 73 records · Page 4Linked to original sources

PubMed1999

Expression and activity of vitamin D-metabolizing cytochrome P450s (CYP1alpha and CYP24) in human nonsmall cell lung carcinomas.

Extrarenal 25-hydroxyvitamin D3-1alpha-hydroxylase is believed to play a major role in the pathogenesis of hypercalcemia associated with various types of granulomatous and lymphoproliferative diseases and certain solid tumors. In this paper, we describe the cloning of the cytochrome P450 component of the extrarenal enzyme from a human nonsmall cell lung carcinoma, SW 900. The cytochrome P450 for the extrarenal 1alpha-hydroxylase has an amino acid sequence identical to that of the cytochrome P450 component of the CYP1alpha, the renal form of the enzyme, and appears to be a product of the same gene. CYP1alpha messenger RNA (mRNA) and 1alpha-hydroxylase enzyme activity were detected in two (SW 900, SK-Luci-6) of a series of five nonsmall cell lung carcinoma cell lines. All five lung cell lines were cultured with the same medium under the same conditions, but only two of the five expressed 1alpha-hydroxylase enzyme; two others (WT-E, Calu-1) expressed high levels of the reciprocally regulated enzyme, 25-hydroxyvitamin D3-24-hydroxylase, with its specific cytochrome P450 component, CYP24. Although under basal conditions the lung cell line SW 900 expressed only CYP1alpha and showed 1alpha-hydroxylase enzyme activity, when treated with small concentrations of 1alpha,25-dihydroxyvitamin D3 or high concentrations of 25-hydroxyvitamin D3, it began to express CYP24 and exhibit 24-hydroxylase enzyme activity. Somewhat surprisingly, SW 900 cells still had detectable CYP1alpha mRNA some 24 h after vitamin D treatment despite the fact that 1alpha-hydroxylase enzyme activity was unmeasurable. These data are consistent with the emerging hypothesis that vitamin D through its active form does not directly turn off CYP1alpha mRNA production but, rather, strongly stimulates CYP24, thereby masking CYP1alpha activity. The factor(s) responsible for the basal expression of CYP1alpha in SW 900 and SK-Luci-6 is currently unknown.

25-Hydroxyvitamin D3 1-alpha-Hydroxylase↗

PubMed1999

A disulfide-bound HIV-1 V3 loop sequence on the surface of human rhinovirus 14 induces neutralizing responses against HIV-1.

An immunogenic sequence from the V3 loop of the MN isolate of human immunodeficiency virus type 1 (HIV-1), His-Ile-Gly-Pro-Gly-Arg-Ala-Phe, was transplanted onto a surface loop of the VP2 capsid protein of human rhinovirus 14. To optimize for virus viability and immunogenicity of the transplanted sequence, the HIV sequence was flanked by (1) a cysteine residue that could form a disulfide bond and (2) randomized amino acids (in either of two arrangements) to generate numerous presentations of the Cys-Cys loop. The location for engineering in VP2 was chosen by searching the geometries of disulfide-bound loops in known protein structures. A model for the structure of the transplanted V3 loop sequence was developed using molecular dynamics and energy minimization calculations. Proteolytic digestion with and without reducing agent demonstrated the presence of the disulfide bond in the chimeric virus examined. Monoclonal and polyclonal antibodies directed against the V3 region of the HIV-1MN strain potently neutralized two chimeric viruses. Guinea pig antisera against two chimeric viruses were able to neutralize HIV-1MN and HIV-1ALA-1 in cell culture. The ability of chimeric viruses to elicit antibodies capable of neutralizing the source of the transplanted sequence could be favorable for vaccine development.

Animals↗

PubMed1999

[A study on expressions of P53 protein and proliferating cell nuclear antigen in malignant eyelid tumors].

OBJECTIVE: To observe the relationship between P53 gene mutation and malignant eyelid tumor. METHODS: The expressions of P53 protein and proliferating cell nuclear antigen (PCNA) in 102 cases with malignant eyelid tumors were examined by labelled streptravidin biotin (LSAB) immunohistochemical technique. RESULTS: Although overexpression of P53 protein was not detected in normal eyelid cutaneous epithelium, meibomian gland and mild epithelial dysplasia, the positive rate of P53 increased in degree in moderate and severe dysplasia, carcinoma in situ and invasive tumors, and PCNA labeling index (PI) progressively increased too. Overexpression of P53 protein was detected in 30/53 cases of basal cell carcinoma (BCC), 16/32 cases of squamous cell carcinoma (SCC), 10/17 cases of meibomian gland carcinoma, and the intensity of P53 protein immunostaining increased with the lowering in the degree of histological differentiation. The mean PCNA labeling index was 38.1 +/- 31.1, 38.3 +/- 30.1, 42.6 +/- 32.0 respectively in BCC, SCC and meibomian gland carcinoma. The PI value in tumor tissue with positive P53 protein expression was higher than that in tissues with its negative expression (P < 0.05). CONCLUSIONS: The gene mutation of P53 protein might play an important role in the pathogenesis of malignant eyelid tumors, it may be one of the factors that can lead to abnormal hyperactivity of cell proliferation and it possibly can be a malignant sign in the progression of a tumor.

Aged↗

PubMed1999

[Anti-Toxoplasma effect of activated mouse macrophages induced by interferon-gamma combined with TNF-alpha].

AIM: To compare the inhibitory effect of the macrophages activated by IFN-gamma combined with TNF-alpha against RH strain and Fukaya strain. METHODS: The average parasite proliferation rates of the two strains within the cytokine-activated M phi s were calculated at different times post-challenge, the nitric oxide (NO) levels in the medium supernatant were simultaneously determined. RESULTS: In the macrophages activated by 100 U each of IFN-gamma and TNF-alpha, the invaded tachyzoites of RH strain were completely killed, while the invaded tachyzoites of Fukaya strain remained slow proliferation with significantly lower levels of NO detected at 24 h post challenge. CONCLUSION: The difference in the anti-Toxoplasma effect of the activated macrophages against RH and Fukaya strains might be attributed to the different amount of NO produced by the macrophages.

Animals↗

PubMed1999

[Simultaneous determination of molybdenum and chromium by artificial neural networks spectrophotometry].

In this paper, a three-layer artificial neural networks were applied to simultaneous determination of molybdenum and chromium by spectrophotometry. The apparent molar absorptivities of the complex of molybdenum and chromium are 1.4 x 10(5) L x mol(-1) x cm(-1) and 3.7 x 10(4) L x mol(-1) x cm(-1), respectively. A method has been developed for simultaneous determination of molybdenum and chromium in steel with satisfactory results. The paralysis in the procedure of training about ANN has been avoided with the improved back propagation algorithms. The simple transformation method for target vectors and criterion function convenient for selection of the parameters about the networks has been put foward.

English Abstract↗

PubMed1999

[Assay of diclofenac sodium and codeine phosphate in tablet by multiwavelength K-factor spectrophotometry].

Assay of diclofenac sodium and codeine phosphate in tablet is reported in this paper. The calibration curves were linear over the range of 11.18-25.40 microg/mL of diclofenac sodium and over the range of 6.785-15.42 microg/mL of codeine phosphate. The average recoveries and relative standard deviations of diclofenac sodium and codeine phosphate were 100.1%, 0.36% and 100.7%, 0.80%, respectively. The method is simple and rapid, and the results are satisfactory.

Codeine↗

PubMed1999

[Determination of the content of aminopyrine in antongding injection by orthogonal function spectrophotometry].

Determination of the content of aminopyrine in the antongding injection by orthogonal function spectrophotometry was reported in this paper. The six wavelengths for the determination are 257, 261, 265, 269, 273 and 277 nm. The concentration of aminopyrine in the range of 5-25 microg x mL(-1) is in a good linear relation to P2, and the regression equation is P2 = + 0.018 - 0.032 9c, r = 0.999 9. The average recovery and relative standard deviation are 100.3% and 0.40%, respectively. On the basis of the data observed it is concluded that this method shows no obvious difference as compared with Hebei province pharmacy regulation.

Aminopyrine↗

PubMed1998

Mg2+ modulates membrane sphingolipid and lipid second messenger levels in vascular smooth muscle cells.

In vitro studies with smooth muscle cells from rat aorta and dog cerebral blood vessels indicate that variation in free Mg2+, within the pathophysiological range of Mg2+ concentrations, found in human serum, causes sustained changes in membrane phospholipids and lipid second messengers. Incorporation of [3H]palmitic acid into phosphatidylcholine (PC) and sphingomyelin (SM) was altered within 15-30 min after modifying the extracellular Mg2+ ion level ([Mg2+]o). Decreased Mg2+ produced a fall in both [3H]SM and [3H]PC over the first 2 h. After an 18-h incubation, the [3H]PC/[3H]SM ratio changed from about 20:1 to about 50:1. Increased [Mg2+]o resulted in a 2- to 3-fold increase in [3H]SM compared to only a small increase in [3H]PC over the same period. There was a reciprocal relationship between [3H]ceramide and [3H]1,2-DAG levels with highest [3H]ceramide and lowest [3H]-1,2-DAG levels seen at lowest [Mg2+]o. The results indicate that a fall in extracellular ionized Mg2+ concentration produces a rapid and sustained decrease in membrane sphingomyelin and a moderate rise in intracellular ceramide. A major effect of lowering [Mg2+]o appears to be a down-regulation of SM synthase. The increased membrane SM content and a concomitant decrease in cell ceramide, in the presence of elevated [Mg2+]o, may be relevant to the apparent protective role of adequate Mg intake on vascular function in humans.

Animals↗

PubMed1998

Utilizing the C-terminal cleavage activity of a protein splicing element to purify recombinant proteins in a single chromatographic step.

A conventional affinity protein purification system often requires a separate protease to separate the target protein from the affinity tag. This paper describes a unique protein purification system in which the target protein is fused to the C-terminus of a modified protein splicing element (intein). A small affinity tag is inserted in a loop region of the endonuclease domain of the intein to allow affinity purification. Specific mutations at the C-terminal splice junction of the intein allow controllable C-terminal peptide bond cleavage. The cleavage is triggered by addition of thiols such as dithiothreitol or free cysteine, resulting in elution of the target protein while the affinity-tagged intein remains immobilized on the affinity column. This system eliminates the need for a separate protease and allows purification of a target protein without the N-terminal methionine. We have constructed general cloning vectors and demonstrated single-column purification of several proteins. In addition, we discuss several factors that may affect the C-terminal peptide bond cleavage activity.

Affinity Labels↗

PubMed1998

The OxyS regulatory RNA represses rpoS translation and binds the Hfq (HF-I) protein.

The OxyS regulatory RNA integrates the adaptive response to hydrogen peroxide with other cellular stress responses and protects against DNA damage. Among the OxyS targets is the rpoS-encoded sigma(s) subunit of RNA polymerase. Sigma(s) is a central regulator of genes induced by osmotic stress, starvation and entry into stationary phase. We examined the mechanism whereby OxyS represses rpoS expression and found that the OxyS RNA inhibits translation of the rpoS message. This repression is dependent on the hfq-encoded RNA-binding protein (also denoted host factor I, HF-I). Co-immunoprecipitation and gel mobility shift experiments revealed that the OxyS RNA binds Hfq, suggesting that OxyS represses rpoS translation by altering Hfq activity.

Bacterial Proteins↗

PubMed1998

The Escherichia coli OxyS regulatory RNA represses fhlA translation by blocking ribosome binding.

OxyS is a small untranslated RNA which is induced in response to oxidative stress in Escherichia coli. This novel RNA acts as a global regulator to activate or repress the expression of as many as 40 genes, including the fhlA-encoded transcriptional activator and the rpoS-encoded sigma(s) subunit of RNA polymerase. Deletion analysis of OxyS showed that different domains of the small RNA are required for the regulation of fhlA and rpoS. We examined the mechanism of OxyS repression of fhlA and found that the OxyS RNA inhibits fhlA translation by pairing with a short sequence overlapping the Shine-Dalgarno sequence, thereby blocking ribosome binding/translation.

Base Sequence↗

PubMed1998

[PCR in combination with silver as a method for the determination of apolipoprotein E alleles].

OBJECTIVE: To present a rapid, sensitive and safe procedure for the determination of human apolipoprotein E alleles. METHODS: Genomic DNA was extracted from 0.5ml whole blood by a non-phenol protocol. After PCR and restriction enzyme digestion,short DNA fragments were detected by a simplified silver staining method. This technique was used to assay apolipoprotein E alleles in 19 patients with sporadic Alzheimer's disease and 41 normal aged persons. RESULTS: The short DNA fragments were visualized clearly on polyacrylamide gel processed by silver staining. It took 24 hours from DNA extraction to the final result. The A4 allele frequency in patients with sporadic Alzheimer's disease was 0.29, much higher than that in control group (0.02, P<0.001). CONCLUSION: PCR in combination with silver staining can satisfy the need for high-resolution and high-efficiency in the determination of apolipoprotein E alleles and can be used as a routine procedure in clinical and epidemiological investigations.

Alleles↗

PubMed1998

Inhibition of respiratory and bioenergetic mechanisms by hydrogen sulfide in mammalian brain.

The biochemical effects of hydrogen sulfide were investigated by treating enzyme homogenates and synaptosomes prepared from mammalian brain with sodium sulfide. Brain cytochrome c oxidase activity was highly sensitive to inhibition by sodium sulfide, as demonstrated by an IC50 of 0.13 microM. Sodium sulfide was also found to inhibit carbonic anhydrase activity in cerebellum, frontal cortex, and hippocampus. Synaptosomal oxygen consumption was significantly reduced as the concentration of sodium sulfide was increased from 20 to 100 microM; this was accompanied by a concentration-dependent depolarization of the synaptosomal mitochondrial membrane in situ and a reduction in synaptosomal ATP concentration. In other experiments using synaptosomes, sodium sulfide caused a significant calcium-independent increase in the extracellular accumulation of L-glutamate, inhibited Na+-dependent uptake of [3H]glutamate, but was unable to influence intrasynaptosomal free ionic Ca2+. Parallel studies conducted in vivo showed that rats exposed over a 5-d period to hydrogen sulfide (100 ppm for 3 h/d) had significantly higher concentrations of L-glutamate in the hippocampus compared to control animals. In summary, our results indicate that sulfide causes extensive disruption to respiratory and related mitochondrial functions in mammalian brain in vitro. The reduced capacity of nerve endings to take up L-glutamate may contribute to the raised L-glutamate levels observed in vivo.

Adenosine Triphosphate↗

PubMed1998

Regulation of the p70 S6 kinase by phosphorylation in vivo. Analysis using site-specific anti-phosphopeptide antibodies.

The p70 S6 kinase is activated by diverse stimuli through a multisite phosphorylation directed at three separate domains as follows: a cluster of (Ser/Thr) Pro sites in an autoinhibitory segment in the noncatalytic carboxyl-terminal tail; Thr-252 in the activation loop of the catalytic domain; and Ser-394 and Thr-412 in a segment immediately carboxyl-terminal to the catalytic domain. Phosphorylation of Thr-252 in vitro by the enzyme phosphatidylinositol 3-phosphate-dependent kinase-1 or mutation of Thr-412 --> Glu has each been shown previously to engender some activation of the p70 S6 kinase, whereas both modifications together produce 20-30-fold more activity than either alone. We employed phospho-specific anti-peptide antibodies to examine the relative phosphorylation at several of these sites in wild type and various p70 mutants, in serum-deprived cells, and in response to activators and inhibitors of p70 S6 kinase activity. Substantial phosphorylation of p70 Thr-252 and Ser-434 was present in serum-deprived cells, whereas Thr-412 and Thr-444/Ser-447 were essentially devoid of phospho-specific immunoreactivity. Activation of p70 by insulin was accompanied by a coordinate increase in phosphorylation at all sites examined, together with a slowing in mobility on SDS-PAGE of a portion of p70 polypeptides. Upon addition of rapamycin or wortmannin to insulin-treated cells, the decrease in activity of p70 was closely correlated with the disappearance of anti-Thr-412(P) immunoreactivity and the most slowly migrating p70 polypeptides, whereas considerable phosphorylation at Ser-434 and Thr-252 persisted after the disappearance of 40 S kinase activity. The central role of Thr-412 phosphorylation in the regulation of kinase activity was further demonstrated by the close correlation of the effects of various deletions and point mutations on p70 activity and Thr-412 phosphorylation. In conclusion, although p70 activity depends on a disinhibition from the carboxyl-terminal tail and the simultaneous phosphorylation at both Thr-252 and Thr-412, p70 activity in vivo is most closely related to the state of phosphorylation at Thr-412.

Androstadienes↗

PubMed1998

Alpha-tocopherol prevents ethanol-induced elevation of [Ca2+]i in cultured canine cerebral vascular smooth muscle cells.

Exposure of cultured canine cerebral vascular smooth muscle cells to ethanol (10-400 mM) for 1-5 days results in concentration-dependent elevation in resting intracellular free calcium ([Ca2+]i) levels. Preincubation of these cultured vascular cells with alpha-tocopherol (20 microM), alone, did not produce any apparent changes from control resting levels of [Ca2+]i. However, after concomitant addition of alpha-tocopherol (20 microM) and ethanol (10-400 mM), the rises of [Ca2+]i induced by ethanol were attenuated markedly. These results suggest that alcohol-induced lipid peroxidation of cerebral vascular muscle cell membranes triggers membrane entry of extracellular Ca2+, which could play an important role in ethanol-induced cerebrovasospasm, brain ischemia and stroke. Moreover, these new results support the concept recently advanced to suggest that alpha-tocopherol-induced amelioration of membrane lipid alterations of cerebral vascular cells can prevent ethanol-induced excessive accumulation of [Ca2+]i.

Animals↗

PubMed1998

Mechanisms of hydrogen peroxide-induced contraction of rat aorta.

It has been suggested that reactive oxygen species may be involved in the regulation of vascular tone. However, the underlying mechanisms remain to be elucidated. The present studies were designed to investigate the contractile effects of hydrogen peroxide (H2O2), one of the reactive oxygen species, on isolated ring segments of rat aorta with and without endothelium. H2O2 induced an endothelium-independent contraction in isolated rat aorta ring segments in a concentration-dependent manner at concentrations from 5 x 10(-6) to 5 x 10(-3) M. H2O2-induced contractions of denuded rat aorta rings were stronger than those on intact rat aorta segments. The contractile effects of H2O2 were inhibited completely by 1200 u/ml catalase. The presence of 1.0 microM Fe2+ or 10 microM proadifen, a cytochrome P450 monooxygenase inhibitor, potentiated the contractile effect of H2O2 on isolated rat aorta segments. 1 mM deferoxamine (a Fe2+ chelator) or 100 microM dimethyl sulfoxide (a hydroxyl radical scavenger) significantly attenuated the vessel contractions induced by hydrogen peroxide plus Fe2+ or hydrogen peroxide itself. Removal of extracellular Ca2+ ([Ca2+]0), addition of 5 microM verapamil, administration of a protein kinase C inhibitor (staurosporine), treatment with an inhibitor of protein tyrosine phosphorylation (genistein) or employment of 5.0 microM indomethacin resulted in a significant attenuation of the contractile responses of the vessels to H2O2. Pharmacological antagonists (e.g. a muscarinic acetylcholine receptor antagonist (atropine), an antagonist of histamine H1 receptors (diphenhydramine), an antagonist of histamine H2 receptors (cimetidine), an alpha-adrenoceptor antagonist (phentolamine), a beta-adrenoceptor antagonist (propranolol) and an antagonist of serotonin receptor (methysergide)) did not inhibit or attenuate the contractions induced by H2O2. Exposure of primary aortic smooth muscle cells to H2O2 (5 x 10(-6) to 5 x 10(-3) M) produced significant rises of intracellular Ca2+ ([Ca2+]i) within 20 s. Employment of 1.0 microM Fe2+ markedly enhanced the increment in [Ca2+]i in the smooth muscle cells. 10 microM proadifen treatment failed to alter the hydrogen peroxide-induced increment in [Ca2+]i of the smooth muscle cells. However, the presence of 5 microM indomethacin significantly attenuated the rise in [Ca2+]i in smooth muscle cells. The present results suggest that H2O2 can induce contractions of rat aorta segments, at pathophysiological concentrations, which are Ca2+-dependent. Hydroxyl radicals (.OH), cyclooxygenase products, protein kinase C and products of protein tyrosine phosphorylation appear to play some role in hydrogen peroxide-induced contractions. Metabolites catalyzed by cytochrome P450-dependent enzymes (upon treatment with hydrogen peroxide) appear to exert a vasodilator effect on rat aorta segments. Lastly, some unidentified mediators, produced by a cytochrome P450 inhibitor (proadifen), during hydrogen peroxide treatment, appear to play some role in contraction of vascular smooth muscle of rat aorta segments in vitro.

Animals↗

PubMed1998

Molecular cytogenetic analysis of patients with holoprosencephaly and structural rearrangements of 7q.

The holoprosencephaly (HPE) sequence is a malformation complex with abnormal midline cleavage of the embryonic forebrain. HPE is genetically heterogeneous with at least 6 different chromosome regions containing genes involved in the expression of the phenotype. HPE3, recently identified as the human Sonic hedgehog gene, is localized to 7q36. We have used fluorescence in situ hybridization (FISH) and polymerase chain reaction (PCR) amplification in 5 cell lines from patients with HPE (3 cases), HPE and sacral agenesis (1 case), and microcephaly (1 case) to further define the structural rearrangements of the long arm of chromosome 7 in each case. All cell lines demonstrated loss of material in the critical region of HPE3 at band 7q36, which includes the Sonic hedgehog gene. We report here the analysis of these patient cell lines.

Cell Line↗

PubMed1998

Staurosporine and H7 attenuate ethanol-induced elevation in [Ca2+]i in cultured canine cerebral vascular smooth muscle cells.

Chronic exposure of cultured canine cerebral vascular smooth muscle cells to ethanol (10-400 mM) for 1-5 days resulted in significant concentration-dependent elevation in resting intracellular free calcium ([Ca2+]i) levels. Preincubation of these cultured vascular cells with inhibitors of protein kinase C (PKC), staurosporine and H7, induced no apparent changes from the control resting levels of [Ca2+]i. However, the increases of [Ca2+]i due to ethanol treatment were attenuated markedly by staurosporine and H7. Our data suggest that activation of PKC plays an important role in ethanol's action in producing a sustained rise in [Ca2+]i in cerebral vascular smooth muscle cells. Activation of PKC could thus play a crucial role in the pathogenesis of alcohol-induced cerebral ischemia and stroke.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗