The PDZ-GEF Gef26 regulates synapse development and function via FasII and Rap1 at the Drosophila neuromuscular junction

Guanine nucleotide exchange factors (GEFs) are essential for small G proteins to activate their downstream signaling pathways, which are involved in morphogenesis, cell adhesion, and migration. Mutants of Gef26, a PDZ-GEF (PDZ domain-containing guanine nucleotide exchange factor) in Drosophila, exhibit strong defects in wings, eyes, and the reproductive and nervous systems. However, the precise roles of Gef26 in development remain unclear. In the present study, we analyzed the role of Gef26 in synaptic development and function. We identified significant decreases in bouton number and branch length at larval neuromuscular junctions (NMJs) in Gef26 mutants, and these defects were fully rescued by restoring Gef26 expression, indicating that Gef26 plays an important role in NMJ morphogenesis. In addition to the observed defects in NMJ morphology, electrophysiological analyses revealed functional defects at NMJs, and locomotor deficiency appeared in Gef26 mutant larvae. Furthermore, Gef26 regulated NMJ morphogenesis by regulating the level of synaptic Fasciclin II (FasII), a well-studied cell adhesion molecule that functions in NMJ development and remodeling. Finally, our data demonstrate that Gef26-specific small G protein Rap1 worked downstream of Gef26 to regulate the level of FasII at NMJs, possibly through a βPS integrin-mediated signaling pathway. Taken together, our findings define a novel role of Gef26 in regulating NMJ development and function.     

Numerical simulation of two-phase non-Newtonian blood flow with fluid-structure interaction in aortic dissection

The behavior of blood cells and vessel compliance significantly influence hemodynamic parameters, which are closely related to the development of aortic dissection. Here the two-phase non-Newtonian model and the fluid-structure interaction (FSI) method are coupled to simulate blood flow in a patient-specific dissected aorta. Moreover, three-element Windkessel model is applied to reproduce physiological pressure waves. Important hemodynamic indicators, such as the spatial distribution of red blood cells (RBCs) and vessel wall displacement, which greatly influence the hemodynamic characteristics are analyzed. Results show that the proximal false lumen near the entry tear appears to be a vortex zone with a relatively lower volume fraction of RBCs, a low time-averaged wall shear stress (TAWSS) and a high oscillatory shear index (OSI), providing a suitable physical environment for the formation of atherosclerosis. The highest TAWSS is located in the narrow area of the distal true lumen which might cause further dilation. TAWSS distributions in the FSI model and the rigid wall model show similar trend, while there is a significant difference for the OSI distributions. We suggest that an integrated model is essential to simulate blood flow in a more realistic physiological environment with the ultimate aim of guiding clinical treatment.     

ATP25, a new nuclear gene of Saccharomyces cerevisiae required for expression and assembly of the Atp9p subunit of mitochondrial ATPase

We report a new nuclear gene, designated ATP25 (reading frame YMR098C on chromosome XIII), required for expression of Atp9p (subunit 9) of the Saccharomyces cerevisiae mitochondrial proton translocating ATPase. Mutations in ATP25 elicit a deficit of ATP9 mRNA and of its translation product, thereby preventing assembly of functional F(0). Unlike Atp9p, the other mitochondrial gene products, including ATPase subunits Atp6p and Atp8p, are synthesized normally in atp25 mutants. Northern analysis of mitochondrial RNAs in an atp25 temperature-sensitive mutant confirmed that Atp25p is required for stability of the ATP9 mRNA. Atp25p is a mitochondrial inner membrane protein with a predicted mass of 70 kDa. The primary translation product of ATP25 is cleaved in vivo after residue 292 to yield a 35-kDa C-terminal polypeptide. The C-terminal half of Atp25p is sufficient to stabilize the ATP9 mRNA and restore synthesis of Atp9p. Growth on respiratory substrates, however, depends on both halves of Atp25p, indicating that the N-terminal half has another function, which we propose to be oligomerization of Atp9p into a proper size ring structure.     

Role of protein kinase A in GABAA receptor dysfunction in CA1 pyramidal cells following chronic benzodiazepine treatment

One-week treatment with the benzodiazepine (BZ) flurazepam (FZP), results in anticonvulsant tolerance, associated with reduced GABAA receptor (GABAR) subunit protein and miniature inhibitory post-synaptic current (mIPSC) amplitude in CA1 neurons of rat hippocampus. Because protein kinase A (PKA) has been shown to modulate GABAR function in CA1 pyramidal cells, the present study assessed whether GABAR dysfunction is associated with changes in PKA activity. Two days after 1-week FZP treatment, there were significant decreases in basal (- 30%) and total (- 25%) PKA activity, and a 40% reduction in PKA RIIbeta protein in the insoluble fraction of CA1 hippocampus. The soluble component of CA1 showed a significant increase in basal (100%) but not total PKA activity. Whole-cell recording in vitro showed a 50% reduction in mIPSC amplitude in CA1 pyramidal cells, with altered sensitivity to PKA modulators. Neurons from FZP-treated rats responded to 8-bromo-cAMP with a significant increase (31%) in mIPSC amplitude. Likewise, vasoactive intestinal polypeptide (VIP), an endogenous PKA activator, caused a significant 36% increase in mIPSC amplitude in FZP-treated cells. Neither agent had a significant effect on mIPSC amplitude in control cells. This study supports a role for PKA in GABAR dysfunction after chronic FZP treatment.     

人红细胞生成素单克隆抗体的制备、鉴定及应用研究

用rhEPo作为抗原,免疫BALB／c小鼠,取其脾细胞与x63Ag8．653小鼠骨髓瘤细胞融合,再碱性PAGE方法进一步分离并纯化的rhEpo,包被Pvc板,对杂交瘤用ELlSA方法进行筛选,获得两株稳定分泌抗hEPO单抗的杂交瘤细胞株。经鉴定分别属于IgG1、IgG2b,轻链均为k链,Kd分别为5．53×10^-10mol／L和1．34×1O^-10mol／L．用western blot方法证明两者对hEPO具有高度韵专一性．能特异地识别rhEPO和尿源hEPO。所制备单抗可作为亲和层析的配体,用于再生障碍性贫血病人尿中EPO及哺乳类工程细胞所表达的hEPO的分离、纯化,并可用于hEPO的定量检测．     

The N-Terminal HSDCIF Motif Is Required for Cell Surface Trafficking and Dimerization of Family B G Protein Coupled Receptor PAC1

PAC1 is PACAP (pituitary adenylate cyclase-activating polypeptide) preferring receptor belonging to class B G protein coupled receptor (GPCR) mediating the most effects of PACAP. The important role of G protein coupled receptor homo/heteromerization in receptor folding, maturation, trafficking, and cell surface expression has become increasingly evident. The bimolecular fluorescence complementation (BiFC) and bioluminescence resonance energy transfer (BRET) assay were used in this research to confirm the dimerization of PAC1 for the first time. The structure-activity relationship focused on the N-terminal HSDCIF motif, which locates behind the signal sequence and has high homology with PACAP (1–6), was assayed using a receptor mutant with the deletion of the HSDCIF motif. The fluorescence confocal microscope observation showed that the deletion of the HSDCIF motif impaired the cell delivery of PAC1. The results of BiFC, BRET and westernblot indicated that the deletion of HSDCIF motif and the replacement of the Cys residue with Ala in HSDCIF motif resulted in the disruption of receptor dimerization. And the exogenous chemically synthesized oligopeptide HSDCIF (100 nmol/L) not only down-regulated the dimerization of PAC1, induced the internalization of PAC1, but also inhibited the proliferation of CHO cells expressing PAC1 stably and decreased the activity of PACAP on the cell viability. All these data suggested that the N-terminal HSDCIF motif played key role in the trafficking and the dimerization of PAC1, and the exogenous oligopeptide HSDCIF had effects on the cell signaling, trafficking and the dimerization of PAC1.     

Association analysis between feed efficiency studies and expression of hypothalamic neuropeptide genes in laying ducks

Residual feed intake (RFI) is now considered a more reasonable metric to evaluate animal feed efficiency. In this study, the correlation between RFI and other feed efficiency traits was investigated and gene expression within the hypothalamus was determined in low RFI (LRFI) and high RFI (HRFI) ducks. Further, several hypothalamic neuropeptide genes were measured using quantitative real‐time PCR. The mean feed intake value was 160 g/day, whereas the egg mass laid (EML) and body weight were approximately 62.4 g/day and 1.46 kg respectively. Estimates for heritability of RFI, feed conversion ratio (FCR) and feed intake were 0.26, 0.18 and 0.23 respectively. RFI is phenotypically positively correlated with feed intake and FCR (P < 0.01). The expression of neuropeptide Y (NPY) and neuropeptide Y receptor Y5 (NPY5R) mRNA was higher in HRFI ducks compared with LRFI ducks (P < 0.05), whereas that of proopiomelanocortin (POMC), melanocortin 4 receptor (MC4R) and cholecystokinin (CCK) was lower (P < 0.05). The mRNA expression of gonadotropin‐releasing hormone 1 (luteinizing‐releasing hormone) (GNRH1) and prolactin receptor (PRLR) was unchanged between LRFI and HRFI ducks. The results indicate that selection for LRFI could reduce feed intake without significant changes in EML, whereas selection on FCR will increase EML.