Identification and expression of a novel member of Ly-6 superfamily in zebrafish <Emphasis Type="Italic">Denio rerio</Emphasis>

Ly-6 superfamily members are present in many metazoans and are divided into two groups: secreted proteins and glycosylphosphatidyl inositol (GPI)-anchored membrane proteins. They both contain one or more conserved domain identified as Ly-6/uPAR (LU) domain and play key roles in cellular adhesion and signaling. Here, we identify a novel member, lymphocyte antigen-6 epidermis (lye), of Ly-6 superfamily in zebrafish. In silico analyses revealed that lye codes for a predicted GPI-anchored membrane protein containing a conserved LU domain and 10 position-specific conserved cysteines typical of known Ly-6 proteins. Whole mount in situ hybridization showed that lye is predominantly expressed in epidermis. We thus named the gene lye, highlighting it is expressed in epidermis. Lye exhibits a dynamic expression pattern during development, which is initially expressed in enveloping layer at gastrula stage, then expressed in epidermis at later stages. It is also expressed in olfactory placode at 24 h post-fertilization. Subsequently, epidermal expression of lye becomes weaker gradually, whereas the expression in pharyngeal arch and pectoral fin increases at 2 and 3 days post-fertilization. Our study lays a foundation for further investigation of lye roles in early developmental stages.     

WNT signaling increases proliferation and impairs differentiation of stem cells in the developing cerebellum

The WNT pathway plays multiple roles in neural development and is crucial for establishment of the embryonic cerebellum. In addition, WNT pathway mutations are associated with medulloblastoma, the most common malignant brain tumor in children. However, the cell types within the cerebellum that are responsive to WNT signaling remain unknown. Here we investigate the effects of canonical WNT signaling on two important classes of progenitors in the developing cerebellum: multipotent neural stem cells (NSCs) and granule neuron precursors (GNPs). We show that WNT pathway activation in vitro promotes proliferation of NSCs but not GNPs. Moreover, mice that express activated β-catenin in the cerebellar ventricular zone exhibit increased proliferation of NSCs in that region, whereas expression of the same protein in GNPs impairs proliferation. Although β-catenin-expressing NSCs proliferate they do not undergo prolonged expansion or neoplastic growth; rather, WNT signaling markedly interferes with their capacity for self-renewal and differentiation. At a molecular level, mutant NSCs exhibit increased expression of c-Myc, which might account for their transient proliferation, but also express high levels of bone morphogenetic proteins and the cyclin-dependent kinase inhibitor p21, which might contribute to their altered self-renewal and differentiation. These studies suggest that the WNT pathway is a potent regulator of cerebellar stem cell growth and differentiation.     

A SNX10/V-ATPase pathway regulates ciliogenesis in vitro and in vivo

Sorting nexins (SNXs) are phosphoinositide-binding proteins implicated in the sorting of various membrane proteins in vitro, but the in vivo functions of them remain largely unknown. We reported previously that SNX10 is a unique member of the SNX family genes in that it has vacuolation activity in cells. We investigate the biological function of SNX10 by loss-of-function assay in this study and demonstrate that SNX10 is required for the formation of primary cilia in cultured cells. In zebrafish, SNX10 is involved in ciliogenesis in the Kupffer's vesicle and essential for left-right patterning of visceral organs. Mechanistically, SNX10 interacts with V-ATPase complex and targets it to the centrosome where ciliogenesis is initiated. Like SNX10, V-ATPase regulates ciliogenesis in vitro and in vivo and does so synergistically with SNX10. We further discover that SNX10 and V-ATPase regulate the ciliary trafficking of Rab8a, which is a critical regulator of ciliary membrane extension. These results identify an SNX10/V-ATPase-regulated vesicular trafficking pathway that is crucial for ciliogenesis, and reveal that SNX10/V-ATPase, through the regulation of cilia formation in various organs, play an essential role during early embryonic development.     

In vivo,ex vivo,and in vitro studies on apelin's effect on myocardial glucose uptake

Apelin is an endogenous peptide hormone recently implicated in glucose homeostasis. However, whether apelin affects glucose uptake in myocardial tissue remains undetermined. In this study, we utilized in vivo, ex vivo and in vitro methods to study apelin's effect on myocardial glucose uptake. Pyroglutamated apelin-13 (2 mg/kg/day) was administered to C57BL6/J mice for 7 days. In vivo myocardial glucose uptake was measured by FDG-PET scanning, and GLUT4 translocation was assessed by immunofluorescence imaging. For in vitro studies, differentiated H9C2 cardiomyoblasts were exposed to pyroglutamated apelin-13 (100 nM) for 2 h. To test their involvement in apelin-stimulated myocardial glucose uptake, the energy sensing protein kinase AMPK were inhibited by pharmacologic inhibition (compound C) and RNA interference. IRS-1 phosphorylation was assessed by western blotting using an antibody directed against IRS-1 Ser-789-phosphorylated form. We found that apelin increased myocardial glucose uptake and GLUT4 membrane translocation in C57BL6/J mice. Apelin was also sufficient to increase glucose uptake in H9C2 cells. Apelin-mediated glucose uptake was significantly decreased by AMPK inhibition. Finally, apelin increased IRS-1 Ser-789 phosphorylation in an AMPK-dependent manner. The results of our study demonstrated that apelin increases myocardial glucose uptake through a pathway involving AMPK. Apelin also facilitates IRS-1 Ser-789 phosphorylation, suggesting a novel mechanism for its effects on glucose uptake.     

非人灵长类位置行为的辐射适应与研究进展

野生动物在长期进化中形成特定的行为模式以适应特定的生存环境。灵长类的位置行为研究对于理解灵长类进化、适应性的多样化以及生态学和解剖学十分关键。该文对已有文献进行了综述,针对位置行为的分类、季节性、以及具体地点和年龄性别差异性进行了总结,分析了灵长类位置行为的发展、特征以及研究现状,以期对今后非人灵长类位置行为的研究提供借鉴和参考,从而促进我国灵长类行为生态学理论体系的发展。     

Use of nucleofection to efficiently transfect primary rabbit lacrimal gland acinar cells

Lacrimal gland acinar cells are an important cell type to study due to their role in production and release of tear proteins, a function essential for ocular surface integrity and normal visual acuity. However, mechanistic studies are often limited by problems with transfection using either plasmid DNA or siRNA. Although various gene delivery methods are available, many have been unproductive due to consistently low transfection efficiencies. We have developed a method using nucleofection that can result in 50% transfection efficiency and 60% knockdown efficiency for plasmid DNA and siRNA, respectively. These results are vastly improved relative to previous studies, demonstrating that nucleofection offers an efficient transfection technique for primary lacrimal gland acinar cells.     

替米沙坦对UUO小鼠肾脏HIF-1α表达的影响及其与巨噬细胞的关系

目的 研究单侧输尿管结扎小鼠肾脏缺氧诱导因子-1α(Hypoxia-inducible factor-1 alpha,HIF-1α)的表达及替米沙坦对其表达的影响和机制.方法 采用单侧输尿管结扎(UUO)建立肾间质纤维化小鼠模型.30只雄性CD1小鼠随机分为假手术组(0d)、单侧输尿管结扎7天组(7d)及14天组(14d)、单侧输尿管结扎7天及14天并替米沙坦(10mg·kg-1·d-1)治疗组(7dT、14dT).MASSON染色观察肾脏病理改变;免疫组织化学检测各组α平滑肌动蛋白(α-SMA)、HIF-1α、巨噬细胞(F4/80)在肾脏组织中的表达及分布、连续切片检测HIF-1α与F4/80的共定位;Western-blot检测α-SMA、HIF-1α的变化.结果 随时间进展,输尿管结扎小鼠(7d、14d)肾脏病理改变逐渐加重,α-SMA、HIF-1α、F4/80表达均增加,与假手术组相比有统计学意义(P＜0.05);HIF-1α主要分布于肾间质细胞中,HIF-1α与F4/80存在明显的共定位现象;给予替米沙坦治疗后,它们的表达均下降(均P＜0.05).结论 替米沙坦可抑制巨噬细胞的浸润,降低UUO小鼠HIF-1α的表达,减轻UUO小鼠肾脏纤维化.     

Siah1 interacts with the scaffold protein POSH to promote JNK activation and apoptosis

Siah proteins are ubiquitin-protein isopeptide ligases (E3) that have been implicated in a variety of cellular actions, including promotion of apoptotic death. Here, we show that Siah1 is a binding partner for POSH (plenty of SH3s), a scaffold component of the apoptotic JNK pathway, and that Siah contributes to death of neurons and other cell types by activating the JNK pathway. Such proapoptotic activity requires the E3 ligase activity of Siah1. Moreover, apoptotic stimuli markedly elevate cellular Siah1 levels by a mechanism reliant on Siah1 protein stabilization. This stabilization requires JNK pathway activation and interaction with POSH and is enhanced by phosphorylation of SIAH1 at tyrosines 100 and 126. Depletion of intracellular Siah proteins via small interference RNA partially protects cells from death evoked by apoptotic stimuli such as trophic factor deprivation and DNA damage. These findings thus reveal a "loop" mechanism in which the JNK pathway promotes SIAH1 stabilization and in which SIAH1 in turn activates the JNK pathway and, ultimately, contributes to cell death.     

SNAP-25/syntaxin 1A complex functionally modulates neurotransmitter gamma-aminobutyric acid reuptake

Neurotransmitter gamma-aminobutyric acid (GABA) release to the synaptic clefts is mediated by the formation of a soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex, which includes two target SNAREs syntaxin 1A and SNAP-25 and one vesicle SNARE VAMP-2. The target SNAREs syntaxin 1A and SNAP-25 form a heterodimer, the putative intermediate of the SNARE complex. Neurotransmitter GABA clearance from synaptic clefts is carried out by the reuptake function of its transporters to terminate the postsynaptic signaling. Syntaxin 1A directly binds to the neuronal GABA transporter GAT-1 and inhibits its reuptake function. However, whether other SNARE proteins or SNARE complex regulates GABA reuptake remains unknown. Here we demonstrate that SNAP-25 efficiently inhibits GAT-1 reuptake function in the presence of syntaxin 1A. This inhibition depends on SNAP-25/syntaxin 1A complex formation. The H3 domain of syntaxin 1A is identified as the binding sites for both SNAP-25 and GAT-1. SNAP-25 binding to syntaxin 1A greatly potentiates the physical interaction of syntaxin 1A with GAT-1 and significantly enhances the syntaxin 1A-mediated inhibition of GAT-1 reuptake function. Furthermore, nitric oxide, which promotes SNAP-25 binding to syntaxin 1A to form the SNARE complex, also potentiates the interaction of syntaxin 1A with GAT-1 and suppresses GABA reuptake by GAT-1. Thus our findings delineate a further molecular mechanism for the regulation of GABA reuptake by a target SNARE complex and suggest a direct coordination between GABA release and reuptake.