Ralstonia eutropha H16 encodes two and possibly three intracellular Poly[D-(-)-3-hydroxybutyrate] depolymerase genes

Intracellular poly[D-(-)-3-hydroxybutyrate] (PHB) depolymerases degrade PHB granules to oligomers and monomers of 3-hydroxybutyric acid. Recently an intracellular PHB depolymerase gene (phaZ1) from Ralstonia eutropha was identified. We now report identification of candidate PHB depolymerase genes from R. eutropha, namely, phaZ2 and phaZ3, and their characterization in vivo. phaZ1 was used to identify two candidate depolymerase genes in the genome of Ralstonia metallidurans. phaZ1 and these genes were then used to design degenerate primers. These primers and PCR methods on the R. eutropha genome were used to identify two new candidate depolymerase genes in R. eutropha: phaZ2 and phaZ3. Inverse PCR methods were used to obtain the complete sequence of phaZ3, and library screening was used to obtain the complete sequence of phaZ2. PhaZ1, PhaZ2, and PhaZ3 share approximately 30% sequence identity. The function of PhaZ2 and PhaZ3 was examined by generating R. eutropha H16 deletion strains (Delta phaZ1, Delta phaZ2, Delta phaZ3, Delta phaZ1 Delta phaZ2, Delta phaZ1 Delta phaZ3, Delta phaZ2 Delta phaZ3, and Delta phaZ1 Delta phaZ2 Delta phaZ3). These strains were analyzed for PHB production and utilization under two sets of conditions. When cells were grown in rich medium, PhaZ1 was sufficient to account for intracellular PHB degradation. When cells that had accumulated approximately 80% (cell dry weight) PHB were subjected to PHB utilization conditions, PhaZ1 and PhaZ2 were sufficient to account for PHB degradation. PhaZ2 is thus suggested to be an intracellular depolymerase. The role of PhaZ3 remains to be established.     

Synthesis of 2H-benzo[b][1,4]oxazin-3(4H)-one derivatives as platelet aggregation inhibitors

Novel 2H-benzo[b][1,4]oxazin-3(4H)-ones have been synthesized by condensation, reduction, O-alkylation and Smiles rearrangement using 3-bromo-4-hydroxy benzaldehyde, anilines, and chloroacetyl chloride as starting materials. All the synthesized compounds have been characterized by (1)H NMR, (13)C NMR, and HRMS, and tested for the inhibitory ability on platelet aggregation. The results have shown that the ADP (adenosine 5'-diphosphate)-induced platelet aggregation was inhibited by 7a-g with the IC(50) value at 10.14-18.83 μmol/L. Compound 7a exhibited the most potent inhibitory effect (IC(50)=10.14 μmol/L) among all the compounds, but less potent than the control drug ticlopidine (3.18 μmol/L) and aspirin (6.07 μmol/L). The preliminary structure-activity relationship (SAR) was initially investigated in the study.     

Use of Granulocyte Colony-Stimulating Factor for the Treatment of Thin Endometrium in Experimental Rats

Granulocyte colony-stimulating factor (G-CSF) induces stem cells to mobilize to the injury site, which have beneficial effect on tissue repair. The aim of this study was to investigate the effect of G-CSF on the thin endometrium in rat models. In the present study, rats with thin endometrium were divided into 4 groups (experimental group I: administrated with G-CSF (40 µg/kg/d) 4–6 hours post-modeling; control group I: administrated with saline 4–6 hours post-modeling; experimental group II: administrated with G-CSF (40 µg/kg/d) 12 days post-modeling; control group II: administrated with saline 12 days post-modeling. The agentia was given once daily and last for 5 days. Endometrial morphology was analyzed by Hematoxylin-Eosin staining, and the regeneration of endometrial cells was evaluated by immunohistochemistry and western-blot with cytokeratin and vimentin. We found that endometrial thickness and morphology presented a significant difference between experimental groups and control groups. No matter when we start with G-CSF, there was a significantly thicker endometrium and stronger expression of cytokeratin/vimintin in the experimental groups compared with the control groups (P<0.01). There were significant thicker endometrial lining and stronger expression of cytokeratin/vimintin in experimental group I than that of experimental group II (P<0.05), but there was no difference in the endometrial lining and the expression of cytokeratin/vimintin between the two control groups (P>0.05). In conclusion, G-CSF can promote the regeneration of endometrial cells in animal research, especially when the G-CSF was administrated earlier.     

Regulation of progesterone during follicular development by FSH and LH in sheep

Progesterone (P4) can participate in the development of female mammalian antral follicles through nuclear receptor (PGR). In this experiment, the differences of P4 synthesis and PGR expression in different developmental stages of sheep antral follicles (large > 5mm, medium 2-5mm, small < 2mm) were detected by enzyme-linked immunosorbent assay, immunohistochemistry, qRT-PCR and Western blotting. Secondly, sheep follicular granulosa cells were cultured in vitro. The effects of different concentrations of FSH and LH on P4 synthesis and PGR expression were studied. The results showed that acute steroid regulatory protein (StAR), cholesterol side chain lyase (P450scc) and 3β Hydroxysteroid dehydrogenase (3β-HSD) and PGR were expressed in antral follicles, and with the development of antral follicles in sheep, StAR, P450scc and the expression of 3β-HSD and PGR increased significantly. In vitro experiments showed that FSH and LH alone or together treatment could regulate P4 secretion and PGR expression in sheep follicular granulosa cells to varying degrees, hint P4 and PGR by FSH and LH, and LH was the main factor. Our results supplement the effects of FSH and LH on the regulation of P4 synthesis during follicular development, which provides new data for further study of steroid synthesis and function in follicular development.     

An inducible transgenic Cre mouse line for the study of hippocampal development and adult neurogenesis

The hippocampus is crucial for higher brain functions, such as learning, memory, and emotion. Many diseases like epilepsy and Down's syndrome are associated with abnormalities in early hippocampal development. In addition, adult dentate neurogenesis is thought to be defective in several classes of psychiatric disorders. However, the mechanisms regulating hippocampal development and adult neurogenesis remain unclear. One of the limitations to studying these processes is the scarcity of available specific mouse tools. Here, we report an inducible transgenic Cre mouse line, Frizzled 9‐CreER?, in which tamoxifen administration induces Cre recombinant. Our data show that Cre is expressed in the developing hippocampal primordium, confined to the granule cell layer at P20 and further limited to the subgranular zone in the adult dentate gyrus. Cre recombinase shows very high activity in all of these regions. Thus, this transgenic line will be a powerful tool in understanding the mechanisms of hippocampal development, adult neurogenesis, and associated diseases. genesis 49:919–926, 2011. © 2011 Wiley Periodicals, Inc.     

Ataxin-10 is involved in Golgi membrane dynamics

<正>Cytokinesis is the final stage of cell division that generates two daughter cells(Fededa and Gerlich,2012).The textbook version di-vides the plant and animal cell cytokinesis into two categories.Plant cells form a mid-zone phragmoplast via vesicle delivering and fusion,and cell wall materials are thus deposited.Animal cells form actomyosin contractile rings,which are the sole force that drives abscission.However,recent evidence has been mounting and pinpointing a pivotal role of membrane transport and subse-     

Retrospective Analysis of 234 Nasopharyngeal Carcinoma Patients with Distant Metastasis at Initial Diagnosis: Therapeutic Approaches and Prognostic Factors

Purpose

The purpose of this retrospective study was to identify the independent prognostic factors and optimize the treatment for nasopharyngeal carcinoma (NPC) patients with distant metastasis at initial diagnosis.

Methods

A total of 234 patients referred between January 2001 and December 2010 were retrospectively analyzed. Among the 234 patients, 94 patients received chemotherapy alone (CT), and 140 patients received chemoradiotherapy (CRT). Clinical features, laboratory parameters and treatment modality were examined with univariate and multivariate analyses.

Results

The median overall survival (OS) time was 22 months (range, 2-125 months), and the 1-year, 2-year, 3-year overall survival rates were 82.2%, 51.3% and 34.1%. The overall response and disease control rates of metastatic lesions after chemotherapy were 56.0% and 89.8%. The factors associated with poor response were karnofsky performance score (KPS) <80, liver metastasis, lactate dehydrogenase (LDH)>245 IU/L, and number of chemotherapy cycles <4. The 3-year OS of patients receiving CRT was higher than those receiving CT alone (48.2% vs. 12.4%, p<0.001). Subgroup analysis showed that significantly improved survival was also achieved by radiotherapy of the primary tumor in patients who achieved complete remission (CR)/partial remission (PR) or stable disease (SD) of metastatic lesions after chemotherapy. Significant independent prognostic factors of OS were KPS, liver metastasis, levels of LDH, and multiple metastases. Treatment modality, response to chemotherapy and chemotherapy cycles were also associated with OS.

Conclusion

A combination of radiotherapy and chemotherapy seems to have survival benefits for selected patients with distant metastases at initial diagnosis. Clinical and laboratory characteristics can help to guide treatment selection. Prospective randomized studies are needed to confirm the result.     

Asynchronous accumulation of lettuce infectious yellows virus RNAs 1 and 2 and identification of an RNA 1 trans enhancer of RNA 2 accumulation

Time course and mutational analyses were used to examine the accumulation in protoplasts of progeny RNAs of the bipartite Crinivirus, Lettuce infectious yellow virus (LIYV; family Closteroviridae). Hybridization analyses showed that simultaneous inoculation of LIYV RNAs 1 and 2 resulted in asynchronous accumulation of progeny LIYV RNAs. LIYV RNA 1 progeny genomic and subgenomic RNAs could be detected in protoplasts as early as 12 h postinoculation (p.i.) and accumulated to high levels by 24 h p.i. The LIYV RNA 1 open reading frame 2 (ORF 2) subgenomic RNA was the most abundant of all LIYV RNAs detected. In contrast, RNA 2 progeny were not readily detected until ca. 36 h p.i. Mutational analyses showed that in-frame stop codons introduced into five of seven RNA 2 ORFs did not affect accumulation of progeny LIYV RNA 1 or RNA 2, confirming that RNA 2 does not encode proteins necessary for LIYV RNA replication. Mutational analyses also supported that LIYV RNA 1 encodes proteins necessary for replication of LIYV RNAs 1 and 2. A mutation introduced into the LIYV RNA 1 region encoding the overlapping ORF 1B and ORF 2 was lethal. However, mutations introduced into only LIYV RNA 1 ORF 2 resulted in accumulation of progeny RNA 1 near or equal to wild-type RNA 1. In contrast, the RNA 1 ORF 2 mutants did not efficiently support the trans accumulation of LIYV RNA 2. Three distinct RNA 1 ORF 2 mutants were analyzed and all exhibited a similar phenotype for progeny LIYV RNA accumulation. These data suggest that the LIYV RNA 1 ORF 2 encodes a trans enhancer for RNA 2 accumulation.     

Pacer Is a Mediator of mTORC1 and GSK3-TIP60 Signaling in Regulation of Autophagosome Maturation and Lipid Metabolism

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