Biomedical subjects
Publications and source records attributed to W Xia.
Based on the comprehensive survey, the status and countermeasures of systematic development for the resources of traditional Chinese drugs and herbs in the rural exploitative emigration and eco-agricultural economy in the Three Gorges Reservoir Areas are put forth constructively.
The HER-2/neu proto-oncogene is frequently amplified or overexpressed in many different types of human cancers, a phenomenon that has been shown to correlate with shorter survival time and lower survival rate in ovarian cancer patients. We previously reported that increased HER-2/neu expression led to more severe malignancy and increased metastatic potential in animal models and that the adenovirus 5 E1A gene repressed HER-2/neu gene expression at transcriptional level and was able to suppress tumor growth when stably transfected into human ovarian cancer SKOV-3 cells which overexpress HER-2/neu. To investigate whether the E1A gene may be used as a therapeutic agent for HER-2/neu-overexpressing human cancers in living hosts, we first developed tumor-bearing mice by injecting SKOV-3 cells that overexpress HER-2/neu intraperitonealy into female nu/nu mice. Five days later, we used cationic liposomes to directly deliver the E1A gene into adenocarcinomas that developed in the peritoneal cavity and on the mesentery of the mice that received the SKOV-3 cell injection. We found that liposome-mediated E1A gene transfer significantly inhibited growth and dissemination of ovarian cancer cells that overexpress HER-2/neu in the treated mice; about 70% of these mice survived at least 365 days, whereas all the control mice that did not receive the gene therapy developed severe tumor symptoms and died within 160 days. The results suggest that liposome-mediated E1A gene transfer may serve as an effective therapy for human ovarian cancers that overexpress HER-2/neu by directly targeting the HER-2/neu oncogene.
Overexpression of HER-2/neu has been demonstrated in human ovarian cancer and correlated with poor prognosis. We previously found that the adenovirus type 5 early region 1A (E1A) gene product can repress overexpression and suppress the tumorigenic potential of the HER-2/neu-overexpressing cancer cells. To develop an efficient HER-2/neu-targeting gene therapy with E1A, a replication-deficient adenovirus containing the E1A gene, Ad.E1A(+), was used to transduce the HER-2/neu-overexpressing human ovarian cancer cell line SK-OV3.ip1. Tumor cell growth in vitro and colony formation in soft agarose were greatly inhibited by Ad.E1A(+) transduction. To test therapeutic efficacy in vivo, tumor-bearing mice were established by i.p. injection with ovarian cancer cells and treated by i.p. injection of 10(8) PFU viral solution containing either replication-deficient Ad.E1A(+); control virus Ad.E1A(-) which is the same adenovirus as Ad.E1A(+) except for E1A deletion, or just PBS. Ad.E1A(+) significantly prolonged survival in treated mice for 1 year (80%) whereas in control groups, all mice died of cancer within 4.5 months. Immunohistochemistry analysis indicated that Ad.E1A protein was expressed in tumor tissue and expression of HER-2/neu p185 protein was suppressed in vivo. As a control, another ovarian cancer cell line 2774, in which HER-2/neu is expressed at a basal level, was also inoculated i.p. following the same therapeutic procedure. Ad.E1A(+) could not prolong survival of 2774 cells, indicating that Ad.E1A(+) specifically targeted HER-2/neu-overexpressing tumor cells and functioned as an antitumor agent.
Dendritic cells (DC) are widely distributed in the lung where they are distinguished by their morphology and class II major histocompatibility complex (Ia) antigen expression. Although a role for DC as pulmonary antigen-presenting cell (APC) has been suggested, little is currently known concerning how these cells respond to inhaled antigens in vivo. Hen-egg lysozyme (HEL) was injected intratracheally into Lewis rats; DC were subsequently purified from the lung and regional lymph nodes (LN) at intervals of up to 14 d and examined for their ability to stimulate the proliferation of HEL-immune T cells in vitro in the absence of added HEL. Pulmonary DC displayed APC activities at 3 h and for up to 7 d after the injection of antigen. Dendritic cells in the draining hilar LN showed APC activities that appeared at 24 h, peaked at day 3, and then diminished progressively. After the primary sensitization, HEL-immune T cells were detected in hilar LN but not in the lung. A second airway challenge with HEL at day 14 yielded an antigen-specific pulmonary immune response, characterized histologically by the accumulation of mononuclear cells around lung venules. We conclude that APC activities shift from lung to lymph node during the response to inhaled antigen.
In vitro, anionic phospholipids can reactivate inactivated DnaA protein, which is essential for initiation of DNA replication at the oriC site of Escherichia coli [Sekimizu, K. & Kornberg, A. (1988) J. Biol. Chem. 263, 7131-7135]. Mutations in the pgsA gene (encoding phosphatidylglycerophosphate synthase) limit the synthesis of the major anionic phospholipids and lead to arrest of cell growth. We report herein that a mutation in the rnhA gene (encoding RNase H) that bypasses the need for the DnaA protein through induction of constitutive stable DNA replication [Kogoma, T. & von Meyenburg, K. (1983) EMBO J. 2, 463-468] also suppressed the growth arrest phenotype of a pgsA mutant. The maintenance of plasmids dependent on an oriC site for replication, and therefore DnaA protein, was also compromised under conditions of limiting anionic phospholipid synthesis. These results provide support for the involvement of anionic phospholipids in normal initiation of DNA replication at oriC in vivo by the DnaA protein.
Escherichia coli cells are dependent on the anionic phospholipid phosphatidylglycerol for cell growth. Introduction into cells of the ability to make phosphatidylinositol did not suppress the need for naturally occurring phosphatidylglycerol. Therefore, the requirement for phosphatidylglycerol must be more than maintenance of the proper membrane surface charge.
Chlamydiae possess an intracellular developmental cycle defined by the orderly interconversion of infectious, metabolically inactive elementary bodies and noninfectious, dividing reticulate bodies. Only a few stage-specific genes have been cloned and sequenced, including the late-stage cysteine-rich protein operon and two late-stage genes encoding histone-like proteins. The aims of this study were to identify additional late-stage genes of Chlamydia trachomatis, analyze the upstream DNA sequence of late genes, and determine the sigma factor requirement of late genes. Stage-specific RNA, made by chlamydiae isolated from host cells, was used to probe C. trachomatis genomic libraries. Two new late genes, designated ltuA and ltuB, were identified, cloned, and sequenced. The predicted peptides encoded by ltuA and ltuB do not bear strong homology to known proteins, and the function of the new late genes is not known. The 5' ends of the transcripts of ltuA, ltuB, the cysteine-rich protein operon, and the two histone-like genes (hctA and hctB) were mapped, and a consensus -10 promoter region of TATAAT was derived from their upstream DNA sequences. In vitro transcription from templates encoding the promoter regions of ltuA, ltuB, and hctA cloned into the transcription assay vector pUC19-spf was found to be strongly stimulated by the addition of recombinant chlamydial sigma 66, while transcription from the putative hctB promoter region cloned in pUC19-spf was not detected in either the presence or absence of added sigma 66. These results suggest that the transcription of at least some chlamydial late-stage genes is dependent on sigma 66, which is homologous to the major sigma factors of other eubacteria.
cls and nov mutants have similar increased sensitivities to novobiocin and reduced levels of cardiolipin, both of which can be corrected by plasmid-borne copies of either wild-type gene. A comparison of the DNA sequences of both genes further verifies their identity.
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Dihydrodiol dehydrogenase (DD; EC 1.3.1.20) catalyzes a novel pathway of polycyclic aromatic hydrocarbon (PAH) metabolism in which trans-dihydrodiols (proximate carcinogens) are oxidized to reactive o-quinones which are cytotoxic and genotoxic. In this study, the complementary DNA for rat liver DD was used to examine the structure and regulation of the DD gene. Southern analysis of rat genomic DNA confirmed that DD is a member of the multigene aldo-keto reductase superfamily. Conservative estimates indicate that the rat DD gene is at least 20-25 kilobases in length. Northern analysis showed that the rat liver transcript was 2.4 kilobases whereas the complementary DNA contains an open-reading frame of 966 nucleotides. Primer extension of male and female polyadenylated RNA indicated that the major transcription start sites are only 53 and 54 base pairs upstream from the translation start site, confirming that the RNA has a very long 3'-untranslated region. In male and female tissues, 2.4 kilobase transcripts predominate in liver, small intestine, and lung, which is consistent with a role for the enzyme in PAH metabolism. Transcripts were also detected in male (prostate)- and female (ovary, mammary gland, and uterus)-specific tissues. In the ovary, two transcripts were observed of 2.4 and 1.4 kilobases in length. Using benzenedihydrodiol as a model substrate for PAH trans-dihydrodiols, highest levels of DD activity were observed in the liver and small intestine of both sexes. Enzyme activity is 2.5-fold higher in the female liver versus the male liver. This sexual dimorphism can be explained by increases in the DD mRNA and enzyme protein as measured by dot-blot and immunotitration analyses, respectively. The latter measurements indicate that DD represents 1.0% of the soluble protein in female liver but is only 0.5% of the soluble protein in male liver. Hormonal ablation (ovariectomy and hypophysectomy) abolishes the sexual dimorphism observed in levels of DD mRNA, enzyme protein, and enzyme activity. Administration of estrogens to males is sufficient to establish the female pattern of gene expression. These data indicate that DD gene expression is hormonally regulated, that estrogens exert their effect at the level of the mRNA, and that aldo-keto reductases involved in PAH metabolism may have their expression regulated by female sex hormones.
Connective tissue dendritic accessory cells (DC) accumulate at sites of extracellular matrix (ECM) deposition. The adherence of DC to purified ECM proteins was examined. Splenic and pulmonary DC were purified from Lewis rats and incubated on slides coated with fibronectin (FN), laminin (LN), or collagen (COLL) types I, III and IV. In other experiments, DC were pre-incubated with selected proinflammatory cytokines (IL-1, IL-2, IFN-gamma, TNF-alpha) in order to determine their abilities to modulate cell adherence to ECM. Both splenic and pulmonary DC showed dose-dependent binding to FN that was inhibited by the synthetic peptide arg-gly-asp-ser. There was minimal DC binding to LN or COLL. Pretreating DC with IFN-gamma or TNF-alpha enhanced DC binding to FN but did not increase binding to LN or COLL. IL-1 and IL-2 had no demonstrable effect on DC binding to ECM. Our results suggest that FN is a critical ligand for DC in their binding to the ECM. IFN-gamma and TNF-alpha increase adherence of DC to FN and may promote their accumulation in the lung during inflammation.
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A substantial body of evidence indicates that the primary sensitization of naive T cells to inhaled antigens occurs in the regional lymph nodes, whereas secondary responses may be generated directly within lung tissue. Ia+ pulmonary dendritic cells are widely distributed within the rat lung where they can participate in the induction of the immune response to inhaled antigens. Recently, two subsets of Ia+ pulmonary dendritic cells have been distinguished based on their expression of Fc receptors (FcR), but little is known concerning their abilities to support the responses of naive or sensitized T cells. In order to address this question, pulmonary FcR+/- dendritic cells have been purified from enzymatic digests of Lewis rat lungs, based on their differential binding to heat-aggregated immunoglobulin. The FcR+/- dendritic cell subsets differed with respect to their light microscopic appearance and in their expression of non-specific esterase. Only the FcR+ subset was able to phagocytize latex beads and showed intracellular phagolysosomes by electron microscopy. Both of the FcR+/- subsets rapidly formed clusters with naive (OX-22+) and sensitized (OX-22-) T cells. However, the clusters yielded by the FcR+ subset were substantially smaller, possibly reflecting their diminished surface membrane expression of the intercellular adhesion molecule-1. The FcR+/- subsets were capable of presenting soluble and particulate antigens to OX-22- T cells. FcR+ cells were less effective than FcR- cells in promoting the proliferative response of OX-22+ T cells to concanavalin A and in the primary mixed leukocyte reaction. We conclude that the FcR+/- pulmonary dendritic cells differ in their abilities to support the responses of naive and sensitized T lymphocytes. This observation may have significance for how primary and secondary pulmonary cell-mediated immune responses are generated.
The Tradescantia-micronucleus (Trad-MCN) bioassay is an efficient short-term test for genotoxicity of pollutants. In order to increase the efficiency and to standardize the micronucleus (MCN) scoring process, an automated scoring system was developed using the principle of image analysis in computer science. This assemblage is called the Tradescantia-micronucleus image analysis (Trad-MCNIA) system. The MCN frequencies scored by this system were compared with those scored by human observation for its proficiency. A set of low MCN frequency (around 5 MCN/100 tetrads) slides prepared from a control group, a set of medium MCN frequency (around 20 MCN/100 tetrads) slides prepared from sodium azide treated plant cuttings and a set of high MCN frequency (around 50 MCN/100 tetrads) slides prepared from X-ray treated materials were used for this study. In the low MCN frequency slides, the Trad-MCNIA system scored about the same value as human observation. In the medium and high frequency slides, MCN frequencies scored by the system were lower than those scored by human observers. This discrepancy was corrected by increasing the power of the objective of the microscope in the system. The MCN frequencies scored by the system attained 90% congruity with those scored by human observers after the correction. The scoring speed of the system was about 3.5 times as fast as that by human observers, and the data could be statistically analyzed immediately after the data scores were recorded. Further improvements can be made by upgrading the video camera and the computer speed.
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