Gene expression profile in immunologically injured liver cell of mice

To study the gene expression profiles between immunologically injured liver cell and normal liver cell of mice and to screen on a large scale the differentially expressed genes associated with the formation of liver injury, the experimental mice were randomly divided into the normal group for controlling and the immunologically liver-injured group induced by BCG and LPS. The liver mRNA of the two groups were extracted respectively and reversely-transcribed to cDNA with the incorporation of different fluorescence (Cy3, Cy5) labeled dUTP as the hybridization probes. The mixed probes were hybridized to the cDNA microarray chips. The fluorescent signal results were acquired by scanner ScanArray 4000 and analyzed with software GenePix Pro 3.0. Among the 14112 target genes, 293 genes were found to be significantly differentially expressed, in which 188 genes were up-regulated and 105 genes were down-regulated. Based on the analysis of biological functions of those differentially expressed genes, it was indicated that the occurrence and development of mouse liver damage induced by BCG and LPS were highly correlated with the processes of immune reactions, cell synthesis, metabolism, apoptosis and transportation in liver cell, which might be quite important for elucidating the regulatory network of gene expression associated with the liver damage, also important for finally discovering the pathogenic mechanisms of immunological liver damage.     

Host recognition in the pupal parasitoid Trichopria drosophilae: a morpho-functional approach

The diapriid wasp Trichopria drosophilae Perkins (Hymenoptera: Diapriidae) attacks and develops in puparia of the common fruit fly, Drosophila melanogaster Meigen (Diptera: Drosophilidae). Host recognition of T. drosophilae was studied using both a morphological and behavioural approach. Scanning and electron microscopical observations of female parasitoid antennae showed the presence of two types of sensilla, which we named MGS1 and MGS2. The former are present on the ventral side of both the apical (A11) and sub-apical (A12) antennomeres, while the latter occur only on A12. Ultrastructural features suggest a gustatory function for these sensilla. Arena bioassays using intact or antennaectomised females and intact host puparia showed that MGS2 are necessary for achieving host acceptance. Further bioassays, where the host's anterior spiracles were covered with wax, led to a very low level of host acceptance. We suggest that the secretion produced by glands associated with the anterior spiracles act as a contact kairomone, which has to be perceived by MGS2 in order to elicit host recognition. The removal of both the female apical antennomeres (A12) led to the failure of the parasitoid to recognize its host.     

Use of Galactosyltransferase to Assess the Biological Function of O-linked N-Acetyl- -Glucosamine: A Potential Role for O-GlcNAc during Cell Division

Many cytosolic and nuclear proteins are modified by monomeric O-linked N-acetyl- -glucosamine (O-GlcNAc). The biological functions of this form of glycosylation are unclear but evidence suggests that it heightens regulation of protein function. To assess the biological function of O-GlcNAc addition, we examined the biological effects of galactosyltransferase (GalT) microinjected into the cytoplasm of Xenopus ovarian oocytes. GalT, which catalyzes β1-4-galactose addition to O-GlcNAc, should inhibit deglycosylation and lectin-like interactions requiring unmodified O-GlcNAc residues. Although GalT injection into diplotene-arrested oocytes has no detectable effects on cell viability, it is toxic to oocytes entering meiosis. Cell-cycle-specific toxicity is recapitulated in vitro as GalT inhibits formation of nuclei and microtubule asters from cell-free extracts of ovulated frog eggs. These observations suggest that regulation of O-GlcNAc is important for cell cycle progression and may be important in diseases in which O-GlcNAc metabolism is abnormal. The methods described here outline a viable experimental scheme for ascribing a biological function to this form of glycosylation.     

Effect of FeSO4 treatment on glucose metabolism in diabetic rats

Iron, the prosthetic group of haemoglobin, was found to lower serum glucose levels of diabetic rats. Its regulative mechanism and effects on enzymatic activities of glucose metabolism are still unknown. In this study, the correlation between iron supply and enzymatic activities of glucose metabolism and respiratory chain were evaluated in liver and kidney tissues of alloxan induced-diabetic rats. After FeSO₄ and metformin administration, serum samples were collected for serum glucose and fructosamine level measurements. Kidney and liver tissues were excised at the end of the study for assaying enzymatic activities of isocitrate dehydrogenase, succinate dehydrogenase, malate dehydrogenase, NADH-dehydrogenase and cytochrome-c-oxidase. Results showed significantly decreased serum glucose and fructosamine levels in treatment groups and enhanced enzymatic activities of several proteins as compared with the diabetic control group. Therefore, these data suggested that FeSO₄ administration could increase the supply of oxygen, enhance enzymatic activities of glucose metabolism and the respiratory chain, accelerate glucose metabolism and consequently decrease serum glucose levels.     

Synonymous codon usage bias and overexpression of a synthetic gene encoding Interferon α2b in yeast

To achieve higher level expression of Interferon α2b (IFN-α2b) in methylotrophic yeast (Pichia pastoris), a cDNA fragment coding for the mature IFN-α2b was designed and synthesized based on the synonymous codon bias of P. pastoris and optimized G+C content. The synthetic IFN-α2b was inserted into the secreted expression vector pPICZαA, and then integrated into P. pastoris GS115 genome by electroporation. Multi-copy integrants in the Mut⁺ recombinant P. pastoris strain were screened by high concentrations of Zeocin. 120 hours culturing allowed expression of the IFN-α2b transformant up to 810 mg/L as detected by SDS-PAGE and quantitative methods. In addition, Western blot analysis showed that the recombinant proteins had immunogenicity. The significant antiviral activity of the recombinant IFN-α2b protein was verified by WISH/ VSV system, which was 3.3×10⁵ IU/mL. Foundation items: The National ‘973’ Basic Research Program (2002CB111302); The National Natural Science Foundation of China (30370807)     

Catalase and estradiol inhibit mitochondrial protein S-glutathionylation

Regulation and downstream effects of mitochondrial protein S-glutathionylation in response to oxidative stress are poorly understood. The study aim was to determine whether anti-oxidants such as catalase and estradiol alter mitochondrial protein S-glutathionylation and in turn affect apoptosis following ultraviolet B (UV-B) light irradiation. HeLa cells were transduced with increasing amounts of adenovirus encoding catalase (Ad-Cat) and β-galactosidase (Ad-Lac Z) or pre-incubated with estradiol before induction of apoptosis by UV-B light exposure. Inhibition of mitochondrial protein S-glutathionylation was assessed using autoantibodies specific for the non-S-glutathionylated form of PDC-E2. The percentage of apoptotic cells following UV-B irradiation were not significantly different between mock cells (cells with no virus infection) and Ad-Cat and Ad-Lac Z infected cells at all viral doses (all p > 0.050). Autoantibody staining of non-S-glutathionylated PDC-E2 in apoptotic cells was three times greater in only Ad-Cat infected cells compared to only Ad-Lac Z infected cells (81.3 ± 16.7 vs 26 ± 7.2 %, respectively, p = 0.030). Similarly estradiol treatment (33 and 100 nM) also significantly increased PDC-E2 staining in apoptotic cells compared to non-treated cells (both p < 0.010). The percentage of apoptotic cells was not significantly different with any of the estradiol concentrations (all p > 0.100). The observed procaspase 12 cleavage following UV-B irradiation suggests that a mitochondrial-independent apoptotic pathway was activated. In conclusion, following an apoptotic stimulus, estradiol may inhibit mitochondrial protein S-glutathionylation without inhibiting apoptosis. This effect may play a role in ninefold greater prevalence of autoantibodies against PDC-E2 in women with primary biliary cirrhosis.     

Studies on the interaction mechanism between hexakis(imidazole) manganese(II) terephthalate and DNA and preparation of DNA electrochemical sensor

The interaction between hexakis(imidazole) manganese(II) terephthalate ([Mn(Im)(6)](teph).4H(2)O) and salmon sperm DNA in 0.2M pH 2.30 Britton-Robinson buffer solution was studied by fluorescence spectroscopy and cyclic voltammetry. Increasing fluorescence was observed for [Mn(Im)(6)](2+) with DNA addition, while quenching fluorescence phenomenon appeared for EB-DNA system when [Mn(Im)(6)](2+) was added. There were a couple quasi-reversible redox peaks of [Mn(Im)(6)](2+) from the cyclic voltammogram on the glassy carbon electrode. The peak current of [Mn(Im)(6)](2+) decreased with positive shift of the formal potential in the presence of DNA compared with that in the absence of DNA. All the experimental results indicate that [Mn(Im)(6)](2+) can bind to DNA mainly by intercalative binding mode. The binding ratio of the DNA-[Mn(Im)(6)](2+) association complex is calculated to be 1:1 and the binding constant is 4.44x10(3) M(-1). By using [Mn(Im)(6)](teph).4H(2)O as the electrochemical hybridization indicator, the DNA electrochemical sensor was prepared by covalent interaction and the selectivity of ssDNA modified electrode were described. The results demonstrate the use of electrochemical DNA biosensor in the determination of complementary ssDNA.     

ORAI-mediated calcium influx in T cell proliferation, apoptosis and tolerance

Ca²⁺ homeostasis controls a diversity of cellular processes including proliferation and apoptosis. A very important aspect of Ca²⁺ signaling is how different Ca²⁺ signals are translated into specific cell functions. In T cells, Ca²⁺ signals are induced following the recognition of antigen by the T cell receptor and depend mainly on Ca²⁺ influx through store-operated CRAC channels, which are mediated by ORAI proteins following their activation by STIM proteins. The complete absence of Ca²⁺ influx caused by mutations in Stim1 and Orai1 leads to severe immunodeficiency. Here we summarize how Ca²⁺ signals are tuned to regulate important T cell functions as proliferation, apoptosis and tolerance, the latter one being a special state of immune cells in which they can no longer respond properly to an otherwise activating stimulus. Perturbations of Ca²⁺ signaling may be linked to immune suppressive diseases and autoimmune diseases.