Identification and cloning of xp95, a putative signal transduction protein in Xenopus oocytes

A 95-kDa protein in Xenopus oocytes, Xp95, was shown to be phosphorylated from the first through the second meiotic divisions during progesterone-induced oocyte maturation. Xp95 was purified and cloned. The Xp95 protein sequence exhibited homology to mouse Rhophilin, budding yeast Bro1, and Aspergillus PalA, all of which are implicated in signal transduction. It also contained three conserved features including seven conserved tyrosines, a phosphorylation consensus sequence for the Src family of tyrosine kinases, and a proline-rich domain near the C terminus that contains multiple SH3 domain-binding motifs. We showed the following: 1) that both Xp95 isolated from Xenopus oocytes and a synthetic peptide containing the Src phosphorylation consensus sequence of Xp95 were phosphorylated in vitro by Src kinase and to a lesser extent by Fyn kinase; 2) Xp95 from Xenopus oocytes or eggs was recognized by an anti-phosphotyrosine antibody, and the relative abundance of tyrosine-phosphorylated Xp95 increased during oocyte maturation; and 3) microinjection of deregulated Src mRNA into Xenopus oocytes increased the abundance of tyrosine-phosphorylated Xp95. These results suggest that Xp95 is an element in a tyrosine kinase signaling pathway that may be involved in progesterone-induced Xenopus oocyte maturation.     

月季愈伤组织的诱导及植株再生

以可在黑龙江地区露地越冬的5个现代月季（Rosa chinensis）品种为实验材料,分别以其无菌苗的叶片和茎段为外植体,研究了愈伤组织诱导及植株再生方法。实验结果表明：5个寒地月季品种的叶片和茎段均可诱导出愈伤组织,2,4-D诱导愈伤组织的效果较好,高浓度的细胞分裂素不适合用于月季叶片和茎段愈伤组织的诱导;TDZ在月季愈伤组织分化培养过程中具有重要作用,光照培养可促进月季愈伤组织的分化,愈伤组织的分化能力随着继代次数的增加呈下降趋势。该实验成功地从2004-8和2004-9（2个月季品种）愈伤组织中诱导出再生植株,其愈伤组织的分化率分别为45%和38%。     

Non-genetic individuality in Escherichia coli motor switching

By analyzing 30 min, high-resolution recordings of single Escherichia coli flagellar motors in the physiological regime, we show that two main properties of motor switching-the mean clockwise and mean counter-clockwise interval durations-vary significantly. When we represent these quantities on a two-dimensional plot for several cells, the data do not fall on a one-dimensional curve, as expected with a single control parameter, but instead spread in two dimensions, pointing to motor individuality. The largest variations are in the mean counter-clockwise interval, and are attributable to variations in the concentration of the internal signaling molecule CheY-P. In contrast, variations in the mean clockwise interval are interpreted in terms of motor individuality. We argue that the sensitivity of the mean counter-clockwise interval to fluctuations in CheY-P is consistent with an optimal strategy of run and tumble. The concomittent variability in mean run length may allow populations of cells to better survive in rapidly changing environments by 'hedging their bets'.     

ORF66 protein kinase function is required for T-cell tropism of varicella-zoster virus in vivo

Several functions have been attributed to the serine/threonine protein kinase encoded by open reading frame 66 (ORF66) of varicella-zoster virus (VZV), including modulation of the apoptosis and interferon pathways, down-regulation of major histocompatibility complex class I cell surface expression, and regulation of IE62 localization. The amino acid sequence of the ORF66 protein contains a recognizable conserved kinase domain. Point mutations were introduced into conserved protein kinase motifs to evaluate their importance to ORF66 protein functions. Two substitution mutants were generated, including a G102A substitution, which blocked autophosphorylation and altered IE62 localization, and an S250P substitution, which had no effect on either autophosphorylation or IE62 localization. Both kinase domain mutants grew to titers equivalent to recombinant parent Oka (pOka) in vitro. pOka66G102A had slightly reduced growth in skin, which was comparable to the reduction observed when ORF66 translation was prevented by stop codon insertions in pOka66S. In contrast, infection of T-cell xenografts with pOka66G102A was associated with a significant decrease in infectious virus production equivalent to the impaired T-cell tropism found with pOka66S infection of T-cell xenografts in vivo. Disrupting kinase activity with the G102A mutation did not alter IE62 cytoplasmic localization in VZV-infected T cells, suggesting that decreased T-cell tropism is due to other ORF66 protein functions. The G102A mutation reduced the antiapoptotic effects of VZV infection of T cells. These experiments indicate that the T-cell tropism of VZV depends upon intact ORF66 protein kinase function.     

Peony Glycosides Protect Against Corticosterone-Induced Neurotoxicity in PC12 Cells

Preclinical and clinical investigations have shown the involvement of dysregulation of hypothalamic–pituitary–adrenal (HPA) axis in the pathogenesis of depression. Hypercortisolemia and the associated hippocampal atrophy were observed in patients with depression, which could be ameliorated by the treatment with antidepressants. Therefore, neuroprotection has been proposed to be one of the acting mechanisms of antidepressant. Previous studies in our laboratory have demonstrated the antidepressant-like activity of total glycosides of peony (TGP) in mice. This study aimed to examine the effect of TGP treatment on corticosterone-induced neurotoxicity in cultured rat pheochromocytoma (PC12) cells. Treating the cells with corticosterone at 200 μM for 48 h caused apoptotic cell death. The cytotoxicity was associated with the activation of caspase-3 activity and the decrease in the mRNA ratio of bcl-2 to bax. TPG treatment at increasing doses (1–10 mg/l) protected against the corticosterone-induced toxicity in PC12 cells in a dose-dependent manner. The cytoprotection afforded by TGP treatment was associated with the inhibition of caspase-3 activity and the up-regulation of bcl-2/bax mRNA ratio. The anti-apoptotic effect of TGP is therefore likely mediated by the suppression of the mitochondrial pathway leading to apoptosis.     

The effects of simulated rainfall on immature population dynamics of Aedes albopictus and female oviposition

Larvae of Aedes albopictus Skuse typically inhabit natural and artificial containers. Since these larval habitats are replenished by rainfall, Ae. albopictus may experience increased loss of immature stages in areas with high levels of rainfall. In this study, we investigated the effects of rainfall and container water level on population density, and oviposition activity of Ae. albopictus. In field and laboratory experiments, we found that rainfall resulted in the flushing of breeding habitats. Excess rain negatively impacted larval and pupal retention, especially in small habitats. When filled with water to overflowing, container habitats were significantly repellent to ovipositing females. Taken together, these data suggest that rainfall triggers population loss of Ae. albopictus and related species through a direct detrimental effect (flushing out) and an indirect effect (ovipositional repellency).     

Comparative Characteristics of Porous Bioceramics for an Osteogenic Response In Vitro and In Vivo

Porous calcium phosphate ceramics are used in orthopedic and craniofacial applications to treat bone loss, or in dental applications to replace missing teeth. The implantation of these materials, however, does not induce stem cell differentiation, so suitable additional materials such as porous calcium phosphate discs are needed to influence physicochemical responses or structural changes. Rabbit adipose-derived stem cells (ADSC) and mouse osteoblastic cells (MC3T3-E1) were evaluated in vitro by the MTT assay, semi-quantitative RT-PCR, and immunoblotting using cells cultured in medium supplemented with extracts from bioceramics, including calcium metaphosphate (CMP), hydroxyapatite (HA) and collagen-grafted HA (HA-col). In vivo evaluation of the bone forming capacity of these bioceramics in rat models using femur defects and intramuscular implants for 12 weeks was performed. Histological analysis showed that newly formed stromal-rich tissues were observed in all the implanted regions and that the implants showed positive immunoreaction against type I collagen and alkaline phosphatase (ALP). The intramuscular implant region, in particular, showed strong positive immunoreactivity for both type I collagen and ALP, which was further confirmed by mRNA expression and immunoblotting results, indicating that each bioceramic material enhanced osteogenesis stimulation. These results support our hypothesis that smart bioceramics can induce osteoconduction and osteoinduction in vivo, although mature bone formation, including lacunae, osteocytes, and mineralization, was not prominent until 12 weeks after implantation.     

Ubc9 acetylation modulates distinct SUMO target modification and hypoxia response

While numerous small ubiquitin‐like modifier (SUMO) conjugated substrates have been identified, very little is known about the cellular signalling mechanisms that differentially regulate substrate sumoylation. Here, we show that acetylation of SUMO E2 conjugase Ubc9 selectively downregulates the sumoylation of substrates with negatively charged amino acid‐dependent sumoylation motif (NDSM) consisting of clustered acidic residues located downstream from the core ψ‐K‐X‐E/D consensus motif, such as CBP and Elk‐1, but not substrates with core ψ‐K‐X‐E/D motif alone or SUMO‐interacting motif. Ubc9 is acetylated at residue K65 and K65 acetylation attenuates Ubc9 binding to NDSM substrates, causing a reduction in NDSM substrate sumoylation. Furthermore, Ubc9 K65 acetylation can be downregulated by hypoxia via SIRT1, and is correlated with hypoxia‐elicited modulation of sumoylation and target gene expression of CBP and Elk‐1 and cell survival. Our data suggest that Ubc9 acetylation/deacetylation serves as a dynamic switch for NDSM substrate sumoylation and we report a previously undescribed SIRT1/Ubc9 regulatory axis in the modulation of protein sumoylation and the hypoxia response.     

Effect of chitosan‐alginate nanoparticles and ultrasound on the efficiency of gene transfection of human cancer cells

Background

Gene therapy has been used to treat a variety of health problems, but transfection inefficiency and the lack of safe vectors have limited clinical progress. Fabrication of a vector that is safe and has high transfection efficiency is crucial for the development of successful gene therapy. The present study aimed to synthesize chitosan‐alginate nanoparticles that can be used as carriers of the pAcGFP1‐C1 plasmid and to use these nanoparticles with an ultrasound protocol to achieve high efficiency gene transfection.

Methods

Chitosan was complexed with alginate and the pAcGFP1‐C1 plasmid at different charge ratios to create chitosan‐alginate‐DNA nanoparticles (CADNs). The average particle size and loading efficiency were measured. Plasmid DNA retardation and integrity were analysed on 1% agarose gels. The effect of CADNs and ultrasound on the efficiency of transfection of cells and subcutaneous tumors was evaluated.

Results

In the CADNs, the average size of incorporated plasmid DNA was 600–650 nm and the loading efficiency was greater than 90%. On the basis of the results of the plasmid DNA protection test, CADNs could protect the transgene from DNase I degradation. The transgene product expression could be enhanced efficiently if cells or tumor tissues were first given CADNs and then treated with ultrasound.

Conclusions

The use of CADNs combined with an ultrasound regimen is a promising method for safe and effective gene therapy. Copyright © 2009 John Wiley & Sons, Ltd.

     

BRCA1基因在人食管癌中的表达

目的本研究利用组织芯片检测BRCA1基因在人食管癌中的表达与食管癌的生长、分化和转移等临床特征的关系,期望找到BRCA1与食管癌发生、发展的关系。方法收集48例食管癌患者标本,分别取其肿瘤组织、癌前病变组织及正常组织制成组织微阵列,免疫组织化学SP法检测BRCA1蛋白的表达,分析BRCA1在各种组织的表达特点及其与肿瘤的关系。选择其中10例患者的上述组织的新鲜标本,采用Western blot检测BRCA1蛋白的表达。结果免疫组织化学结果显示,BRCA1在肿瘤组织阳性占70.50%,癌前组织阳性占43.10%,正常组织阳性占39.00%,食管癌组织与癌前病变组织、食管癌组织与正常组织相比BRCA1的表达均存在显著差异（P〈0.01）。BRCA1的表达与食管癌的病理分化有统计学上的差异（P〈0.05）。Western blot显示,BRCA1在食管癌肿瘤组织、癌前病变组织及正常组织中表达量依次降低,差异具有统计学意义（P〈0.05）;BRCA1在高、中、低分化程度的食管癌组织中表达量依次降低,差异具有统计学意义（P〈0.05）。结论BRCA1与食管癌发生发展有关,BRCA1的表达与食管癌的分化呈正相关。