Standardizing global gene expression analysis between laboratories and across platforms

To facilitate collaborative research efforts between multi-investigator teams using DNA microarrays, we identified sources of error and data variability between laboratories and across microarray platforms, and methods to accommodate this variability. RNA expression data were generated in seven laboratories, which compared two standard RNA samples using 12 microarray platforms. At least two standard microarray types (one spotted, one commercial) were used by all laboratories. Reproducibility for most platforms within any laboratory was typically good, but reproducibility between platforms and across laboratories was generally poor. Reproducibility between laboratories increased markedly when standardized protocols were implemented for RNA labeling, hybridization, microarray processing, data acquisition and data normalization. Reproducibility was highest when analysis was based on biological themes defined by enriched Gene Ontology (GO) categories. These findings indicate that microarray results can be comparable across multiple laboratories, especially when a common platform and set of procedures are used.     

Novel soluble epoxide hydrolase inhibitor protects mitochondrial function following stress

Epoxyeicosatrienoic acids (EETs) are active metabolites of arachidonic acid that are inactivated by soluble epoxide hydrolase enzyme (sEH) to dihydroxyeicosatrienoic acid. EETs are known to render cardioprotection against ischemia reperfusion (IR) injury by maintaining mitochondrial function. We investigated the effect of a novel sEH inhibitor (sEHi) in limiting IR injury. Mouse hearts were perfused in Langendorff mode for 40 min and subjected to 20 min of global no-flow ischemia followed by 40 min of reperfusion. Hearts were perfused with 0.0, 0.1, 1.0 and 10.0 μmol·L(-1) of the sEHi N-(2-chloro-4-methanesulfonyl-benzyl)-6-(2,2,2-trifluoro-ethoxy)-nicotinamide (BI00611953). Inhibition of sEH by BI00611953 significantly improved postischemic left-ventricular-developed pressure and reduced infarct size following IR compared with control hearts, and similar to hearts perfused with 11,12-EETs (1 μmol·L(-1)) and sEH(-/-) mice. Perfusion with the putative EET receptor antagonist 14,15-epoxyeicosa-5(Z)-enoic acid (14,15-EEZE, 10 μmol·L(-1)), or the plasma membrane K(ATP) channels (pmK(ATP)) inhibitor (glibenclamide, 10 μmol·L(-1)) abolished the improved recovery by BI00611953 (1 μmol·L(-1)). Mechanistic studies in H9c2 cells demonstrated that BI0611953 decreased ROS generation, caspase-3 activity, proteasome activity, increased HIF-1∝ DNA binding, and delayed the loss of mitochondrial membrane potential (ΔΨ(m)) caused by anoxia-reoxygenation. Together, our data demonstrate that the novel sEHi BI00611953, a nicotinamide-based compound, provides significant cardioprotection against ischemia reperfusion injury.     

Evaluation of immunostimulatory effect of the arrowroot (<Emphasis Type="Italic">Maranta arundinacea</Emphasis>. L) in vitro and in vivo

Arrowroot (Maranta arundinacea. L) is an underutilized local crop potentially to be developed as carbohydrate source and functional food in Indonesia. The objectives of this research are to evaluate the immunostimulatory effects of arrowroot extracts in vitro by using animal cell culture techniques, and in vivo by using BALB/c mice. The arrowroot tuber extracts were prepared by heat-treatment at 121 °C for 20 min in distilled water. The IgM production stimulatory activity of arrowroot tuber extracts against human hybridoma HB4C5 cells and mouse splenocytes was assessed. The result indicated that the arrowroot tuber extract stimulated IgM production by HB4C5 cells and immunoglobulin (IgG, IgA and IgM) production by splenocytes in vitro. In addition, the arrowroot tuber extracts strongly enhanced interferon γ production by splenocytes. In vivo study indicated that the diet containing arrowroot extracts increased the serum IgG, IgA and IgM levels in mice. These results revealed that the arrowroot tuber extracts have immunostimulatory effects in vivo as well as in vitro.     

Anti‐hepatitis C virus compounds obtained from Glycyrrhiza uralensis and other Glycyrrhiza species

Development of complementary and/or alternative drugs for treatment of hepatitis C virus (HCV) infection is still much needed from clinical and economic points of view. Antiviral substances obtained from medicinal plants are potentially good targets to study. Glycyrrhiza uralensis and G. glabra have been commonly used in both traditional and modern medicine. In this study, extracts of G. uralensis roots and their components were examined for anti‐HCV activity using an HCV cell culture system. It was found that a methanol extract of G. uralensis roots and its chloroform fraction possess anti‐HCV activity with 50%‐inhibitory concentrations (IC₅₀) of 20.0 and 8.0 μg/mL, respectively. Through bioactivity‐guided purification and structural analysis, glycycoumarin, glycyrin, glycyrol and liquiritigenin were isolated and identified as anti‐HCV compounds, their IC₅₀ being 8.8, 7.2, 4.6 and 16.4 μg/mL, respectively. However, glycyrrhizin, the major constituent of G. uralensis, and its monoammonium salt, showed only marginal anti‐HCV activity. It was also found that licochalcone A and glabridin, known to be exclusive constituents of G. inflata and G. glabra, respectively, did have anti‐HCV activity, their IC₅₀ being 2.5 and 6.2 μg/mL, respectively. Another chalcone, isoliquiritigenin, also showed anti‐HCV activity, with an IC₅₀ of 3.7 μg/mL. Time‐of‐addition analysis revealed that all Glycyrrhiza‐derived anti‐HCV compounds tested in this study act at the post‐entry step. In conclusion, the present results suggest that glycycoumarin, glycyrin, glycyrol and liquiritigenin isolated from G. uralensis, as well as isoliquiritigenin, licochalcone A and glabridin, would be good candidates for seed compounds to develop antivirals against HCV.     

Directed evolution of P-glycoprotein cysteines reveals site-specific,non-conservative substitutions that preserve multidrug resistance

Pgp (P-glycoprotein) is a prototype ABC (ATP-binding-cassette) transporter involved in multidrug resistance of cancer. We used directed evolution to replace six cytoplasmic Cys (cysteine) residues in Pgp with all 20 standard amino acids and selected for active mutants. From a pool of 75000 transformants for each block of three Cys, we identified multiple mutants that preserved drug resistance and yeast mating activity. The most frequent substitutions were glycine and serine for Cys⁴²⁷ (24 and 20%, respectively) and Cys¹⁰⁷⁰ (37 and 25%) of the Walker A motifs in the NBDs (nucleotide-binding domains), Cys¹²²³ in NBD2 (25 and 8%) and Cys⁶³⁸ in the linker region (24 and 16%), whereas close-by Cys⁶⁶⁹ tolerated glycine (16%) and alanine (14%), but not serine (absent). Cys¹¹²¹ in NBD2 showed a clear preference for positively charged arginine (38%) suggesting a salt bridge with Glu²⁶⁹ in the ICL2 (intracellular loop 2) may stabilize domain interactions. In contrast, three Cys residues in transmembrane α-helices could be successfully replaced by alanine. The resulting CL (Cys-less) Pgp was fully active in yeast cells, and purified proteins displayed drug-stimulated ATPase activities indistinguishable from WT (wild-type) Pgp. Overall, directed evolution identified site-specific, non-conservative Cys substitutions that allowed building of a robust CL Pgp, an invaluable new tool for future functional and structural studies, and that may guide the construction of other CL proteins where alanine and serine have proven unsuccessful.     

Expression of hyaluronan synthase 1 and distribution of hyaluronan during follicular atresia in pig ovaries

CD44 on macrophages is recognized as a phagocytic receptor involved in the phagocytosis of apoptotic cells. Recently, we detected CD44 on macrophages in atretic follicles during atresia. In this study, we evaluated the distribution of the principal CD44 ligand hyaluronan (HA) and the expressions of HA synthases (HAS: HAS1, HAS2, and HAS3) during atresia in pig ovaries. We determined the 2139-bp sequence of Sus scrofa HAS1 and raised an anti-HAS1 polyclonal antibody. The S. scrofa HAS1 sequence contained six putative HA-binding motifs and conserved amino acid residues crucial for GlcNac transferase activity. HAS1 mRNA expression was upregulated during atresia; however, HAS2 and HAS3 mRNA expression levels were low and very low to undetectable, respectively. Western blotting showed that HAS1 was markedly upregulated during atresia. Immunohistochemical analyses revealed HAS1 distribution in theca cells of healthy and early atretic (stages I and II) follicles and in progressing atretic (stage III) follicles. Hyaluronan was visualized with the HA-binding protein; it accumulated in the theca layer during all stages and in stage III follicles. Hyaluronan assay showed a significantly increased HA concentration in follicular fluid at stage III. Flow cytometry showed HAS1 expression in 55.7% of SIRPA-positive macrophages in stage III follicles. Our results suggest that the HA concentration in follicular fluids increased during atresia and that HAS1 may be the dominant HAS protein in theca cells to produce HA in pig ovaries.     

Virus-encoded b7-2 costimulation molecules enhance the protective capacity of a replication-defective herpes simplex virus type 2 vaccine in immunocompetent mice

Herpes simplex virus 2 (HSV-2) and, to a lesser extent, HSV-1 cause the majority of sexually transmitted genital ulcerative disease. No effective prophylactic vaccine is currently available. Replication-defective HSV stimulates immune responses in animals but produces no progeny virus, making it potentially useful as a safe form of live vaccine against HSV. Because it does not replicate and spread in the host, however, replication-defective virus may have relatively limited capacity to solicit professional antigen presentation. We previously demonstrated that in mice devoid of B7-1 and B7-2 costimulation molecules, replication-defective HSV-2 encoding B7-1 or B7-2 induces stronger immune responses and protection against HSV-2 challenge than immunization with replication-defective virus alone. Here, we vaccinated wild-type mice fully competent to express endogenous B7 costimulation molecules with replication-defective HSV-2 or replication-defective virus encoding B7-2 and compared their capacities to protect against vaginal HSV-2 infection and disease. Replication-defective virus encoding B7-2 induced more IFN-γ-producing CD4 T cells than did replication-defective virus alone. Immunization with B7-2-expressing virus decreased challenge virus replication in the vaginal mucosa, genital and neurological disease, and mortality more effectively than did immunization with the parental replication-defective virus. Prior immunization with B7-expressing, replication-defective virus also effectively suppressed infection of the nervous system compared to immunization with the parental virus. Thus, B7 costimulation molecules expressed at the site of HSV infection can enhance vaccine efficacy even in a fully immunocompetent host.     

Comparative structural analysis of a novel glutathioneS-transferase (ATU5508) from Agrobacterium tumefaciens at 2.0 A resolution

Glutathione S-transferases (GSTs) comprise a diverse superfamily of enzymes found in organisms from all kingdoms of life. GSTs are involved in diverse processes, notably small-molecule biosynthesis or detoxification, and are frequently also used in protein engineering studies or as biotechnology tools. Here, we report the high-resolution X-ray structure of Atu5508 from the pathogenic soil bacterium Agrobacterium tumefaciens (atGST1). Through use of comparative sequence and structural analysis of the GST superfamily, we identified local sequence and structural signatures, which allowed us to distinguish between different GST classes. This approach enables GST classification based on structure, without requiring additional biochemical or immunological data. Consequently, analysis of the atGST1 crystal structure suggests a new GST class, distinct from previously characterized GSTs, which would make it an attractive target for further biochemical studies.