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Biomedical subjects

S Ren

Publications and source records attributed to S Ren.

At least 19 recordsLinked to original sources
PubMed2005

Detection of the steroidogenic acute regulatory protein, StAR, in human liver cells.

Overexpressing StAR (a mitochondrial cholesterol transporter) increases (>5-fold) the rate of 27-hydroxylation of cholesterol and the rates of bile acid synthesis in primary rat hepatocytes; suggesting that the transport of cholesterol into mitochondria is rate-limiting for bile acid biosynthesis via the CYP27A1 initiated 'acidic' pathway. Our objective was to determine the level of StAR expression in human liver and whether changes in StAR would correlate with changes in CYP27A1 activity/bile acid synthesis rates in human liver tissues. StAR mRNA and protein were detected in primary human hepatocytes and HepG2 cells by RT-PCR/Northern analysis and by Western analysis, respectively. In immunocompetition assays, liver StAR was competed away with the addition of purified human adrenal StAR. Overexpressing CYP27A1 in both cell types led to >2-fold increases in liver StAR concentration. StAR protein levels also increased approximately 2-fold with the addition of 27-hydroxycholesterol to HepG2 cell culture medium. Overexpressing StAR increased the rates of 27-hydroxylation of cholesterol/bile acid synthesis in both cell lines and increased intracellular levels of 27-hydroxycholesterol. In conclusion, human liver cells contain regulable StAR protein whose level of expression appears capable of regulating cellular cholesterol homeostasis, representing a potential therapeutic target in the management of hyperlipidemia.

Bile Acids and Salts↗
PubMed2005

Mitochondrial cholesterol transport: a possible target in the management of hyperlipidemia.

Sterol 27-hydroxylase (CYP27A1) may defend cells against accumulation of excess cholesterol, making this enzyme a possible target in the management of hyperlipidemia. The study objective was to analyze cholesterol homeostatic responses to increases in CYP27A1 activity in HepG2 cells and primary human hepatocytes. Increasing CYP27A1 activity by increasing enzyme expression led to significant increases in bile acid synthesis with compensatory increases in HMG-CoA reductase (HMGR) activity/protein, LDL receptor (LDLR) mRNA, and LDLR-mediated cholesterol uptake. Under these conditions, only a small increase in cellular 27-hydroxycholesterol (27OH-Chol) concentration was observed. No changes were detected in mature sterol regulatory element-binding proteins (SREBP) 1 or 2. Increasing CYP27A1 activity by increasing mitochondrial cholesterol transport (i.e., substrate availability) led to greater increases in bile acid synthesis with significant increases in cellular 27OH-Chol concentration. Mature SREBP 2 protein decreased significantly with compensatory decreases in HMGR protein. No change was detected in mature SREBP 1 protein. Despite increasing 27OH-Chol and lowering SREBP 2 protein concentrations, LDLR mRNA increased significantly, suggesting alternative mechanisms of LDLR transcriptional regulation. These findings suggest that regulation of liver mitochondrial cholesterol transport represents a potential therapeutic strategy in the treatment of hyperlipidemia and atherosclerosis.

Adenoviridae↗
PubMed2004

Effect of a combination of clevudine and emtricitabine with adenovirus-mediated delivery of gamma interferon in the woodchuck model of hepatitis B virus infection.

Our aim was to evaluate the antiviral effect of a combination of two nucleoside reverse transcriptase inhibitors, emtricitabine (FTC) and clevudine (L-FMAU), with the addition of an adenovirus-driven delivery of recombinant gamma interferon (IFN-gamma) in the woodchuck model of hepatitis B virus infection. Six woodchuck hepatitis virus (WHV)-infected woodchucks received L-FMAU (10 mg/kg) plus FTC (30 mg/kg) intraperitoneally for 8 weeks; six other animals received in addition an intravenous injection of a recombinant adenovirus vector expressing woodchuck IFN-gamma (Ad-IFN) at weeks 4 and 8. In the control group, two animals received Ad-IFN alone, two received adenovirus vector expressing the green fluorescent protein reporter gene, and one remained untreated. In less than 2 weeks, all woodchucks that received L-FMAU plus FTC showed a rapid and marked inhibition of viral replication, with a 4-log(10) drop in serum WHV DNA. In two animals, viremia remained suppressed for several months after the end of treatment. Similarly, a dramatic decrease in intrahepatic replicative intermediates of viral DNA was observed in the L-FMAU/FTC-treated groups. The additional administration of Ad-IFN led to increased inflammation in the liver but did not enhance the antiviral effect of the L-FMAU/FTC combination. In conclusion, therapies combining L-FMAU and FTC in WHV-infected woodchucks resulted in a potent and sustained antihepadnaviral effect both in the liver and in the blood circulation. However, no extra benefit of adding IFN-gamma gene transduction to the L-FMAU/FTC combination could be detected.

Adenoviridae↗
PubMed2002

Determining the mechanisms of toxic action of phenols to Tetrahymena pyriformis.

Phenols are environmentally important compounds that are widely used in agriculture and industry. Thus, phenols are of interest to environmental toxicologists, which has led to the development of several quantitative structure-activity relationship (QSAR) models. The most successful QSARs have been established for compounds having a common mechanism of toxic action (MOA). However, correctly determining the MOA of a compound is not easy. Discriminant analysis was used in this study to separate phenols by their MOAs. The working hypothesis of this study was that phenols with different MOAs lie in different regions in the response surface described by the ionization-corrected hydrophobicity descriptor, log D, the electrophilicity descriptor, ELUMO, and the interaction between hydrophobicity and electrophilicity, D * ELUMO. Results of discriminant analysis showed a total error rate of 15%. In addition, it was difficult to separate phenols that had a polar narcosis MOA from phenols that had a proelectrophile MOA.

Animals↗
PubMed2002

Classifying class I and class II compounds by hydrophobicity and hydrogen bonding descriptors.

The successful development of quantitative structure-activity relationships (QSARs) and the prediction of toxicity based on QSARs depend on the correct classification of the mechanism of toxic action of chemical compounds. The toxicity mechanism of a compound can be determined by a few existing methods, such as studying the chemical structure of the compound for certain substructures and assigning a mechanism accordingly. However, these methods are less reliable for compounds with complex structures and are usually complicated. A novel descriptor-based method employing a multivariate statistical technique--discriminant analysis--was developed in this study for the classification of class I and class II compounds. The discriminating variables used in discriminant analysis were the hydrophobicity descriptor log(K(ow)) and the hydrogen bonding descriptors E(LUMO), E(HOMO), Q(+), and Q(-). Because only numerical values of these descriptors are required for this method, which can be calculated, no additional experimental work was required for the toxicity mechanism classification of new compounds. A nonlinear discriminant function was generated that could not be expressed explicitly. Assessing the predicting ability of the discriminant function by the cross-validation method showed that a low total error rate, 4.2% was achieved.

Environmental Pollutants↗
PubMed2001

Synthesis and in vitro antitumor activity of oligonucleotide-tethered and related platinum complexes.

Three classes of hydroxy-tethered platinum(II) complexes have been synthesized from K(2)PtCl(4) and appropriate amino alcohols. A sequence of selective oxidation and hydrolysis has been developed to prepare hydroxy-tethered platinum(IV) complexes. A novel procedure for the synthesis of amminetrichloroplatinate(II) anion has been generated and used to synthesize a number of monohydroxy-tethered nonchelating platinum complexes. These tethered platinum complexes, including hydroxy-tethered, phosphoramidite-tethered, and monodeoxyribonucleotide-tethered platinum(II) and -(IV) complexes, have been examined in vitro for antitumor activity in both leukemia and ovarian cancer cell lines. Activity of some of these complexes was similar to cis-platin, and most of them showed much better potency than carboplatin. We observed an interesting structure-activity correlation for platinum(II) complexes for both PA-1 and SK-OV-3 ovarian cancer cell lines. However, platinum(IV) complexes showed much more diversified response among cancer cell lines studied. We observed enhanced selectivity among different cancer cell lines for some agents. The most promising is the monodeoxyribonucleotide-tethered platinum(IV) complex, which is the first analogue of the conjugates between a platinum fragment and monodeoxyribonucleotides, showing antitumor activity and selectivity among the cell lines. Finally, the p53 status of the cells appears to contribute to the effectiveness of these agents in that cells harboring wild-type p53 appear to be more sensitive to these agents.

Animals↗
PubMed2001

Caspase-dependent cytosolic release of cytochrome c and membrane translocation of Bax in p53-induced apoptosis.

Activation of p53 induces apoptosis in various cell types. However, the mechanism by which p53 induces apoptosis is still unclear. We reported previously that the activation of a temperature-sensitive mutant p53 (p53(138Val)) induced activation of caspase 3 and apoptosis in Jurkat cells. To elucidate the pathway linking p53 and downstream caspases, we examined the activation of caspases 8 and 9 in apoptotic cells. The results showed that both caspases were activated during apoptosis as judged by the appearance of cleavage products from procaspases and the caspase activities to cleave specific fluorogenic substrates. The significant inhibition of apoptosis by a tetrapeptide inhibitor of caspase 8 and caspase 9 suggested that both caspases are required for apoptosis induction. In addition, the membrane translocation of Bax and cytosolic release of cytochrome c, but not loss of mitochondrial membrane potential, were detected at an early stage of apoptosis. Moreover, Bax translocation, cytochrome c release, and caspase 9 activation were blocked by the broad-spectrum caspase inhibitor, Z-VAD-fmk and the caspase 8-preferential inhibitor, Ac-IETD-CHO, suggesting that the mitochondria might participate in apoptosis by amplifying the upstream death signals. In conclusion, our results indicated that activation of caspase 8 or other caspase(s) by p53 triggered the membrane translocation of Bax and cytosolic release of cytochrome c, which might amplify the apoptotic signal by activating caspase 9 and its downstream caspases.

Apoptosis↗
PubMed2001

Neurological abnormalities in a knock-in mouse model of Huntington's disease.

Mice representing precise genetic replicas of Huntington's disease (HD) were made using gene targeting to replace the short CAG repeat of the mouse Huntington's disease gene homolog (HDH:) with CAG repeats within the length range found to cause HD in humans. Mice with alleles of approximately 150 units in length exhibit late-onset behavioral and neuroanatomic abnormalities consistent with HD. These symptoms include a motor task deficit, gait abnormalities, reactive gliosis and the formation of neuronal intranuclear inclusions predominating in the striatum. This model differs from previously described HDH: knock-ins by its method of construction, longer repeat length and more severe phenotype. To our knowledge, this is the first knock-in mouse model of HD to show increased glial fibrillary acidic protein immunoreactivity in the striatum, suggesting that these mice have neuronal injury similar to that found early in the course of HD. These mice will serve as useful reagents in experiments designed to reveal the molecular nature of neuronal dysfunction underlying HD.

Alleles↗
PubMed2001

Purine analogs as CDK enzyme inhibitory agents: a survey and QSAR analysis.

Characterization of the cell cycle has introduced CDKs and other proteins as possible targets for inhibition of cell proliferation, such as, CDK1 and CDK2, whose inhibition may be useful in the treatment of proliferative disorders. Structure-activity analyses have been instrumental in the design and discovery of potent CDK inhibitors, such as purine analogs, which have increased in potency from the micromolar to the nanomolar level. X-ray crystallography and molecular modeling have provided evidence that these compounds act on the CDK target enzyme. Selected CDK inhibitors have successfully entered clinical trials. Further characterization of the cell cycle to identify molecular targets to inhibit cell proliferation, QSAR and SAR studies, and clinical trials may expedite the development of CDK inhibitors for therapeutic use. The ultimate goal of these studies is to determine whether specific CDKs, CDK1 or CDK2, are enzymes essential to cell proliferation that can be targeted for treatment of proliferative disorders. CDK1 and CDK2 are viable molecular targets for cancer therapies based on isolated-enzyme inhibition by CDK inhibitors, successful clinical trials of CDK1 and CDK2 inhibitors, and x-ray crystallographic confirmation of CDK inhibitors binding to the putative target enzyme active site. It is now reported that CDK1 inhibitory activities of purine analogs correlate with the physiochemical parameters of purine analogs. Enzyme inhibition [1-5], clinical trials (see Tab. 1), x-ray crystallographic [4, 6, 7] and QSAR correlation studies are evidence that specific CDK1 and/or CDK2 inhibitors are potentially useful agents for various cell-proliferation disorders. A brief overview of the cell cycle precedes a literature review of clinical applications of CDK inhibitors, followed by a new QSAR study, and a SAR and molecular modeling discussion.

Animals↗
PubMed2001

The simultaneous determination of diazepam and its three metabolites in dog plasma by high-performance liquid chromatography with mass spectroscopy detection.

A fast, sensitive and specific LC/MS/MS method for the simultaneous determination of diazepam and its three metabolites, oxazepam, temazepam and desmethyldiazepam, in dog plasma is described. The method consists of an automated 96-well solid phase extraction procedure and electrospray LC/MS/MS analysis. D(5)-Diazepam is used as the internal standard for all the compounds. Intra-day and inter-day assay coefficients of variations are less than 12.7%. The lower limit of quantitation (LLOQ) is 1 nM for each analyte, based on 0.1 ml aliquots of dog plasma. The analytical run time was 5 min. Linearity is observed over the range of 1--500 nM. This method has been used to support the discovery of pharmacokinetic studies.

Animals↗
PubMed2001

Disialoganglioside GD3 is selectively expressed by developing and mature human mast cells.

BACKGROUND: Disialoganglioside GD3 is expressed on the surface of selected cell types. Anti-GD3 mAb administered to human subjects with malignant melanoma produces signs and symptoms of immediate hypersensitivity reactions. OBJECTIVE: The expression of GD3 by human mast cells was assessed during mast cell development in vitro and in samples of lung and skin. METHODS: GD3 on tissue- and in vitro-derived mast cells was analyzed after double labeling of cells for tryptase (G3 mAb) or Kit (YB5.B8 mAb) and GD3 (R24 mAb). Glycolipids in extracts of fetal liver-derived mast cells were examined by using high-performance thin-layer chromatography. RESULTS: Flow cytometry showed that the percentage of GD3+ cells increased in parallel to Kit+ cells during the recombinant human stem cell factor-dependent development of fetal liver-derived mast cells. Double-labeling experiments showed that GD3+ cells were also surface Kit+ and granule tryptase positive, identifying them as mast cells in preparations of lung-, skin-, fetal liver-, and cord blood-derived cells. The major acidic glycolipid detected was NeuAcalpha2-8NeuAcalpha2-3Galbeta1-4Glcbeta1-1'Cer (GD3). Among peripheral blood leukocytes, only basophils and about 10% of the T cells were labeled with anti-GD3 mAb. Anti-GD3 mAb-conjugated magnetic beads were used to purify mast cells to greater than 90% purity from dispersed skin cells enriched to approximately 12% purity by means of density-dependent sedimentation but were less proficient for dispersed human lung mast cells, most likely because of other cell types that express GD3. CONCLUSION: GD3 is expressed on the surface of developing human mast cells in parallel to tryptase in secretory granules and, like Kit, can serve as a target for their enrichment by immunoaffinity techniques.

Cells, Cultured↗
PubMed2001

Biotransformation of D-limonene to (+) trans-carveol by toluene-grown Rhodococcus opacus PWD4 cells.

The toluene-degrading strain Rhodococcus opacus PWD4 was found to hydroxylate D-limonene exclusively in the 6-position, yielding enantiomerically pure (+) trans-carveol and traces of (+) carvone. This biotransformation was studied using cells cultivated in chemostat culture with toluene as a carbon and energy source. The maximal specific activity of (+) trans-carveol formation was 14.7 U (g of cells [dry weight])(-1), and the final yield was 94 to 97%. Toluene was found to be a strong competitive inhibitor of the D-limonene conversion. Glucose-grown cells did not form any trans-carveol from D-limonene. These results suggest that one of the enzymes involved in toluene degradation is responsible for this allylic monohydroxylation. Another toluene degrader (Rhodococcus globerulus PWD8) had a lower specific activity but was found to oxidize most of the formed trans-carveol to (+) carvone, allowing for the biocatalytic production of this flavor compound.

Biotransformation↗
PubMed2001

Killing effect and antitoxic activity of the Leptospira interrogans toxin-antitoxin system in Escherichia coli.

We report the first evidence of a chromosome-encoded toxin-antitoxin locus in spirochetes. This locus has been found in the pathogenic spirochete Leptospira interrogans and exhibits homologies with the pem/chp loci. The L. interrogans chp locus consists of two genes: chpK (for "killer protein") and its upstream partner chpI (for "inhibitory protein"). Expression of ChpK in Escherichia coli results in the inhibition of bacterial growth. The coexpression of ChpI neutralizes ChpK toxicity. By Southern blot analysis, chp homologs were found in all representative pathogenic strains of L. interrogans.

Amino Acid Sequence↗
PubMed2001

Hepatitis B virus (HBV) virion and covalently closed circular DNA formation in primary tupaia hepatocytes and human hepatoma cell lines upon HBV genome transduction with replication-defective adenovirus vectors.

Hepatitis B virus (HBV), the causative agent of B-type hepatitis in humans, is a hepatotropic DNA-containing virus that replicates via reverse transcription. Because of its narrow host range, there is as yet no practical small-animal system for HBV infection. The hosts of the few related animal viruses, including woodchuck hepatitis B virus and duck hepatitis B virus, are either difficult to keep or only distantly related to humans. Some evidence suggests that tree shrews (tupaias) may be susceptible to infection with human HBV, albeit with low efficiency. Infection efficiency depends on interactions of the virus with factors on the surface and inside the host cell. To bypass restrictions during the initial entry phase, we used recombinant replication-defective adenovirus vectors, either with or without a green fluorescent protein marker gene, to deliver complete HBV genomes into primary tupaia hepatocytes. Here we show that these cells, like the human hepatoma cell lines HepG2 and Huh7, are efficiently transduced by the vectors and produce all HBV gene products required to generate the secretory antigens HBsAg and HBeAg, replication-competent nucleocapsids, and enveloped virions. We further demonstrate that covalently closed circular HBV DNA is formed. Therefore, primary tupaia hepatocytes support all steps of HBV replication following deposition of the genome in the nucleus, including the intracellular amplification cycle. These data provide a rational basis for in vivo experiments aimed at developing tupaias into a useful experimental animal system for HBV infection.

Adenoviridae↗
PubMed2001

A simplified method to prepare PCR template DNA for screening of transgenic and knockout mice.

Polymerase chain reaction (PCR) amplification of DNA is the most widely used technique for screening of large numbers of genetically engineered transgenic or knockout mice (Mus musculus). In this report, we present a new DNA preparation procedure for running diagnostic PCR. In this procedure, mouse ear tissue was used directly for PCR after the tissue underwent brief digestion in a solution containing only proteinase K. Using this method, we have successfully screened several lines of single, double, and triple transgenic and knockout mice. The results are reliable and reproducible. The advantage of this new method is that DNA purification by organic extraction or isolation kit was omitted. DNA purification is the limiting factor in terms of time and money when screening transgenic and knockout mice by PCR. In addition, using ear instead of tail tissue can reduce distress of animals because the samples can be obtained when the mice are labeled by ear punch.

Animal Identification Systems↗
PubMed2001

[Construction of luciferase reporter plasmid which is under the control of dioxin-responsive enhancers].

To improve the bio-detection of dioxin-type chemicals, we constructed a plasmid containing reporter under the control of dioxin-responsive enhancer. After connected the enhancer from the plasmid pHAV with the MMTV promoter of the plasmid pCatM, the connected segment was linked with the luciferase vector. Then the recombinant plasmid was transfected to the HepG2 human hepatoma cell line, and the luciferase activity was induced by 2,3,7,8-TCDD. The result suggested that expression of the recombinant luciferase was controlled by the dioxin-responsive enhancer, and there were a linear relationship between the activity of luciferase and certain range of 2,3,7,8-TCDD concentration. It is promising that the transfected cell line could be used for rapid screen and semi quantitation of dioxin-type chemicals, and a standard and routine method for the detection of dioxin-type chemicals by this recombinant plasmid is being under way.

Enhancer Elements, Genetic↗
PubMed2001

Development of HIV protease inhibitors: a survey.

In the treatment of infections caused by rapidly mutating viruses like human immunodeficiency virus (HIV), combination therapy with multiple drugs acting by different mechanisms offers several advantages over monotherapy. It may provide: synergistic effect, possible reduction of dosages and side-effects, and reduction of the chance of drug resistance. In the past few years, hundreds of HIV protease inhibitors have been synthesized and tested in order to overcome the limitations of reverse transcriptase inhibitors like zidovudine and others. In this review, emphasis is placed on the development of HIV protease inhibitors as antiviral agents against HIV, and structure-activity relationship analysis of saquinavir and related compounds. Limitations of some protease inhibitors and ways to overcome the shortcomings are presented. Among these many protease inhibitors five have been marketed during 1995-1999. They are saquinavir, ritonavir, indinavir, nelfinavir and amprenavir. Their different structural features, important physicochemical, pharmacokinetic and clinical profiles are presented in a table form for easy comparison. It is hoped that in the future new drugs based on additional mechanisms can be developed for the treatment of AIDS.

Drug Design↗