Cloning and characterization of an mRNA encoding a novel G protein alpha-subunit abundant in mantle and gill of pearl oyster Pinctada fucata

Nacre formation is an ideal model to study biomineralization processes. Although much has been done about biomineralization mechanism of nacre, little is known as to how cellular signaling regulates this process. We are interested in whether G protein signaling plays a role in mineralization. Degenerate primers against conserved amino acid regions of G proteins were employed to amplify cDNA from the pearl oyster Pinctada fucata. As a result, the cDNA encoding a novel G(s)alpha (pfG(s)alpha) from the pearl oyster was isolated. The G(s)alpha cDNA encodes a polypeptide of 377 amino acid residues, which shares high similarity to the octopus (Octopus vulgaris) G(s)alpha. The well-conserved A, C, G (switch I), switch II functional domains and the carboxyl terminus that is a critical site for interaction with receptors are completely identical to those from other mollusks. However, pfG(s)alpha has a unique amino acid sequence, which encodes switch III and interaction sites of adenylyl cyclase respectively. In situ hybridization and Northern blotting analysis revealed that the oyster G(s)alpha mRNA is widely expressed in a variety of tissues, with highest levels in the outer fold of mantle and epithelia of gill, the regions essential for biomineralization. We also show that overexpression of the pfG(s)alpha in mammalian MC3T3-E1 cells resulted in increased cAMP levels. Mutant pfG(s)alpha that has impaired CTX substrate diminished its ability to induce cAMP production. Furthermore, the alkaline phosphatase (ALP) activity, an indicator for mineralization, is induced by the G(s)alpha in MC3T3-E1 cells. These results indicated that G(s)alpha may be involved in regulation of physiological function, particularly in biological biomineralization.     

Selection of TAR RNA-binding chameleon peptides by using a retroviral replication system

The interaction between the arginine-rich motif (ARM) of the human immunodeficiency virus (HIV) Tat protein and TAR RNA is essential for Tat activation and viral replication. Two related lentiviruses, bovine immunodeficiency virus (BIV) and Jembrana disease virus (JDV), also require Tat ARM-TAR interactions to mediate activation, but the viruses have evolved different RNA-binding strategies. Interestingly, the JDV ARM can act as a "chameleon," adopting both the HIV and BIV TAR binding modes. To examine how RNA-protein interactions may evolve in a viral context and possibly to identify peptides that recognize HIV TAR in novel ways, we devised a retroviral system based on HIV replication to amplify and select for RNA binders. We constructed a combinatorial peptide library based on the BIV Tat ARM and identified peptides that, like the JDV Tat ARM, also function through HIV TAR, revealing unexpected sequence characteristics of an RNA-binding chameleon. The results suggest that a retroviral screening approach may help identify high-affinity TAR binders and may provide new insights into the evolution of RNA-protein interactions.     

Two extracellular proteins with alkaline peroxidase activity, a novel cytochrome c and a catalase-peroxidase, from Bacillus sp. No.13

A novel cytochrome c and a catalase-peroxidase with alkaline peroxidase activity were purified from the culture supernatant of Bacillus sp. No.13 and characterized. The cytochrome c exhibited absorption maxima at 408 nm (Soret band) in its oxidized state, and 550 (alpha-band), 521 (beta-band), and 415 (Soret band) nm in its reduced state. The native cytochrome c with a relative molecular mass of 15,000 was composed of two identical subunits. The cytochrome c showed over 50 times higher peroxidase activity than those of known c-type cytochromes from various sources. The optimum pH and temperature of the peroxidase activity were about 10.0 and 70 degrees C, respectively. The peroxidase activity is stable in the pH range of 6.0 to 10.8 (30 degrees C, 1-h treatment), and at temperatures up to 80 degrees C (pH 8.5, 20-min treatment). The heme content was determined to be 1 heme per subunit. The amino acid sequence of the cytochrome c showed high homology with those of the c-type cytochromes from Bacillus subtilis and Bacillus sp. PS3. The catalase-peroxidase showed high catalase activity and considerable peroxidase activity, the specific activities being 55,000 and 0.94 micromol/min/mg, respectively. The optimum pH and temperature of the peroxidase activity were in the range of 6.4 to 10.1 and 60 degrees C, respectively. The catalase-peroxidase showed a lower K(m) value (0.67 mM) as to H(2)O(2) than known catalase-peroxidases.     

Suppression of androgen receptor-mediated transactivation and cell growth by the glycogen synthase kinase 3 beta in prostate cells

Androgens play important roles in the growth of normal prostate and prostate cancer via binding to the androgen receptor (AR). In addition to androgens, AR activity can also be modulated by selective growth factors and/or kinases. Here we report a new kinase signaling pathway by showing that AR transactivation was repressed by wild type glycogen synthase kinase 3beta (GSK3 beta) or constitutively active S9A-GSK3 beta in a dose-dependent manner. In contrast, the catalytically inactive kinase mutant GSK3 beta showed little effect on the AR transactivation. The suppression of AR transactivation by GSK3 beta was abolished by the GSK3 beta inhibitor lithium chloride. The in vitro kinase assay showed that GSK3 beta prefers to phosphorylate the amino terminus of AR that may lead to the suppression of activation function 1 activity located in the NH(2)-terminal region of AR. GSK3 beta interrupted the interaction between the NH(2) and COOH termini of AR, and overexpression of the constitutively active form of GSK3 beta, S9A-GSK3 beta, reduced the androgen-induced prostate cancer cell growth in stably transfected CWR22R cells. Together, our data demonstrated that GSK3 beta may function as a repressor to suppress AR-mediated transactivation and cell growth, which may provide a new strategy to modulate the AR-mediated prostate cancer growth.     

Developing an <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>-mediated genetic transformation for a selenium-hyperaccumulator <Emphasis Type="Italic">Astragalus racemosus</Emphasis>

Agrobacterium tumefaciens strain LBA4404 containing the plasmid pBI121, carrying the reporter gene uidA and the kanamycin resistance gene nptII, was used for gene transfer experiments in selenium (Se)-hyperaccumulator Astragalus racemosus. The effects of kanamycin on cell growth and division and acetosyringone on transformation efficiency were evaluated. The optimal concentration of kanamycin that could effectively inhibit cell growth and division in non-transgenic tissues was 50 mg l⁻¹ and thus all putative transgenic plants were obtained on induction medium containing 50 mg l⁻¹ kanamycin. The verification of transformants was achieved by both histochemical GUS assay and PCR amplification of nptII gene. Southern blot analysis was performed to further confirm that transgene nptII was stably integrated into the A. racemosus genome. A transformation frequency of approximately 10% was achieved using this protocol, but no beneficial effect from the addition of acetosyringone (50 μM) was observed. This transformation system will be a useful tool for future studies of genes responsible for Se-accumulation in A. racemosus.     

A platform to standardize,store, and visualize proteomics experimental data

With the development of functional genomics research, large-scale proteomics studies are now widespread, presenting significant challenges for data storage, exchange, and analysis. Here we present the Integrated Proteomics Exploring Database （IPED） as a platform for managing proteomics experimental data （both process and result data）. IPED is based on the schema of the Proteome Experimental Data Repository （PEDRo）, and complies with the General Proteomics Standard （GPS） drafted by the Proteomics Standards Committee of the Human Proteome Organization. In our work, we developed three components for the IPED platform： the IPED client editor, IPED server software, and IPED web interface. The client editor collects experimental data and generates an extensible markup language （XML） data file compliant with PEDRo and GPS; the server software parses the XML data file and loads information into a core database; and the web interface displays experimental results, to provide a convenient graphic representation of data. Given software convenience and data abundance, IPED is a powerful platform for data exchange and presents an important resource for the proteomics community. In its current release, IPED is available at http：//www. biosino.org/iped2.     

Improvement and Optimization of Standards for a Preclinical Animal Test Model of Laser Induced Choroidal Neovascularization

Background

As the murine model of laser-induced choroidal neovascularization (CNV) is becoming the most established and commonly utilized model worldwide for studying the pathogenesis of CNV and its response to treatment, specific operating standards are yet to be clarified. The purpose of this study is to compare the lesion size of CNV in mice with different ages, sex, durations of CNV process, and treated positions of laser spots, to make recommendations that may improve and optimize the quality of the model.

Methods and Results

C57/BL6 mice of different ages were treated with diode laser photocoagulation per eye and perfused with PBS containing fluorescein-labeled dextran at different time of observation. Choroid flat mounts, were then examined by fluorescence microscopy for the measurement of CNV area. Messenger-RNA expression levels of several angiogenic cytokines in eye cups of male and female C57BL/6 mice at 5–8 and 16–20 week-old were analyzed by real-time RT-PCR assay. The results showed significantly more CNV area in eyes of female mice compared to male mice with the expression level of several angiogenic cytokines elevated. 16–20-week-old female mice developed the biggest area of CNV. The mean area of CNV increased significantly at the 14^th day after photocoagulation. Laser spots delivered 1PD away from the optic disc induced the biggest area of CNV compared to those 2PD or 3PD away. Interaction of NV was observed in laser spots delivered less than 1PD away from each other.

Conclusion

The current results suggest that 16–20-week-old female C57BL/6 mice developed the most distinct CNV lesion size with laser spots delivered 1PD away from the optic disc. The best time to observe and analyze is the 14^th day after photocoagulation.     

A Modified Delta-Shaped Gastroduodenostomy in Totally Laparoscopic Distal Gastrectomy for Gastric Cancer: A Safe and Feasible Technique

Background

The present study introduced a modified delta-shaped gastroduodenostomy (DSG) technique and assessed the safety, feasibility and clinical results of this procedure in patients undergoing totally laparoscopic distal gastrectomy (TLDG) for gastric cancer (GC).

Materials and Methods

A total of 102 patients with distal GC undergoing TLDG with modified DSG between January 2013 and December 2013 were enrolled. A retrospective study was performed using a prospectively maintained comprehensive database to evaluate the results of the procedure. Univariate and multivariate analyses were performed to estimate the predictive factors for postoperative morbidity.

Results

The mean operation time was 150.6±30.2 min, the mean anastomosis time was 12.2±4.2 min, the mean blood loss was 48.2±33.2 ml, and the mean times to first flatus, fluid diet, soft diet and postoperative hospital stay were 3.8±1.3 days, 5.0±1.0 days, 7.4±2.1 days and 12.0±6.5 days, respectively. Two patients with minor anastomotic leakage after surgery were managed conservatively; no patient experienced any complications around the anastomosis, such as anastomotic stricture or anastomotic hemorrhage. Univariate analysis showed that age, gastric cancer with hemorrhage and cardiovascular disease combined were significant factors that affected postoperative morbidity (P<0.05). Multivariate analysis found that gastric cancer with hemorrhage was the independent risk factor for the postoperative morbidity (P = 0.042). At a median follow-up of 7 months, no patients had died or experienced recurrent or metastatic disease.

Conclusions

The modified DSG was technically safe and feasible, with acceptable surgical outcomes, in patients undergoing TLDG for GC, and this procedure may be promising in these patients.     

酵母菌相关实验的一些改进

高中生物学教学中,与酵母菌相关的实验有多个,如“探究酵母菌的呼吸方式”、“酵母菌的酒精发酵”等。下面就针对这些实验的可操作性,对实验装置进行一些改进,以利于探究实验的顺利进行。