Rab3A and calmodulin regulate acrosomal exocytosis by mechanisms that do not require a direct interaction

The interaction between Rab3A and calmodulin is necessary for the inhibitory effect of Rab3A in neuroendocrine cells. Contrastingly, Rab3A triggers the exocytosis known as acrosome reaction in permeabilized spermatozoa. Here we show that a Rab3A mutant that cannot bind calmodulin was fully capable of triggering acrosomal exocytosis. Additionally, calmodulin by itself abrogated the exocytosis triggered by Rab3A. The effect was observed with both the wild type protein and the calmodulin binding deficient mutant. Our results indicate that the inhibitory and stimulatory effects of Rab3A in different exocytic processes are mediated by different effectors.     

MiR-203 Suppresses ZNF217 Upregulation in Colorectal Cancer and Its Oncogenicity

Zinc finger protein 217 (ZNF217) is essential for cell proliferation and has been implicated in tumorigenesis. However, its expression and exact roles in colorectal cancer (CRC) remain unclear. In this study, we demonstrated that ZNF217 expression was aberrantly upregulated in CRC tissues and associated with poor overall survival of CRC patients. In addition, we found that ZNF217 was a putative target of microRNA (miR)-203 using bioinformatics analysis and confirmed that using luciferase reporter assay. Moreover, in vitro knockdown of ZNF217 or enforced expression of miR-203 attenuated CRC cell proliferation, invasion and migration. Furthermore, combined treatment of ZNF217 siRNA and miR-203 exhibited synergistic inhibitory effects. Taken together, our results provide new evidences that ZNF217 has an oncogenic role in CRC and is regulated by miR-203, and open up the possibility of ZNF217- and miR-203-targeted therapy for CRC.     

Dynamic analysis of differential scanning calorimetry data

The apparent heat capacity function measured by high-sensitivity differential scanning calorimetry contains dynamic components of two different origins: (1) an intrinsic component arising from the finite instrument time response; and (2) a sample component arising from the kinetics of the thermal transition under study. The intrinsic instrumental component is always present and its effect on the shape of the experimental curve depends on the magnitude of the calorimeter response time. Usually, high-sensitivity instruments exhibit characteristic time constants varying from 10 to 100 s. This slow response introduces distortions in the shape of the heat capacity function especially at fast scanning rates. In addition to this instrumental component, dynamic effects due to sample relaxation processes also contribute to the shape of the experimental heat capacity profile. Since the nature and magnitude of these effects are a function of the kinetic parameters of the transition, they can be used to obtain kinetic information. This communication presents a dynamic deconvolution technique directed to remove artificial distortions in the shape of the heat capacity function measured at any scanning rate, and to obtain a kinetic characterization of a thermally induced transition. The kinetic characterization obtained by this method allows the researcher to obtain transition relaxation times as a continuous function of temperature. This technique has been applied to the thermal unfolding of ribonuclease A and the pretransition of dipalmitoylphosphatidylcholine (DPPC). In both systems the transition relaxation times are temperature dependent. For the protein system the relaxation time is very slow below the transition temperature (approximately 30 s) and very fast above Tm (less than 1 s) in agreement with direct kinetic measurements. For the pretransition of DPPC, the relaxation time is maximal at the transition midpoint and of the order of approx. 40 s.     

Serum miR-210 Contributes to Tumor Detection,Stage Prediction and Dynamic Surveillance in Patients with Bladder Cancer

MiR-210 is the master hypoxamir that generally exhibits oncogenic properties in most human solid tumors including bladder cancer (BC). However, it remains unknown about the clinical significance of circulating miR-210 levels in BC. In this study, we found that serum miR-210 was up-regulated in patients with BC, and serum levels of miR-210 increased with advancing stage and grade. Moreover, serum miR-210 expression was found to be significantly reduced in paired post-operative samples and elevated in most patients with relapsed BC. Taken together, our data suggest that serum miR-210 could be a potential noninvasive biomarker for screening, predicting and monitoring BC.     

禄丰蜥龙动物群的组成及初步分析

本文在对禄丰蜥龙动物群的组成进行分析的同时,修正了部分化石产出层位.分层列出了迄今报道过的采自禄丰盆地下禄丰组的脊椎动物化石名单;并以此为依据,倾向于下禄丰组深红层,更确切地说是表4的第"8"层中所含化石,代表里阿斯期.     

四川侏罗纪三列齿类头后骨骼

本文记述了最晚期的三列齿类——似卞氏兽 (Bienotheroides) 的头后骨骼,并和其他三列齿类以及原始哺乳动物作了对比.肩胛骨上雏型岗上窝的出现,证明三列齿类与原始哺乳类的关系要比以往想象的更为密切.     

Small interference ITGA6 gene targeting in the human thymic epithelium differentially regulates the expression of immunological synapse-related genes

The thymus supports differentiation of T cell precursors. This process requires relocation of developing thymocytes throughout multiple microenvironments of the organ, mainly with thymic epithelial cells (TEC), which control intrathymic T cell differentiation influencing the formation and maintenance of the immunological synapse. In addition to the proteins of the major histocompatibility complex (MHC), this structure is supported by several adhesion molecules. During the process of thymopoiesis, we previously showed that laminin-mediated interactions are involved in the entrance of T-cell precursors into the thymus, as well as migration of differentiating thymocytes within the organ. Using small interference RNA strategy, we knocked-down the ITGA6 gene (which encodes the CD49f integrin α-chain) in cultured human TEC, generating a decrease in the expression of the corresponding CD49f subunit, in addition to modulation in several other genes related to cell adhesion and migration. Thymocyte adhesion to TEC was significantly impaired, comprising both immature and mature thymocyte subsets. Moreover, we found a modulation of the MHC, with a decrease in membrane expression of HLA-ABC, in contrast with increase in the expression of HLA-DR. Interestingly, the knockdown of the B2M gene (encoding the β-2 microglobulin of the HLA-ABC complex) increased CD49f expression levels, thus unraveling the existence of a cross-talk event in the reciprocal control of CD49f and HLA-ABC. Our data suggest that the expression levels of CD49f may be relevant in the general control of MHC expression by TEC and consequently the corresponding synapse with developing thymocytes mediated by the T-cell receptor.     

小煤炱科子囊壳超微结构研究

用扫描电镜对小煤炱科Meliolaceae的美座附丝壳Appendiculella calotroma(Desm.)Hoehnel和莫勒针壳炱Irenopsis molleriana(Wint.)Stev.的子囊壳进行观察的结果,发现它们的子囊壳都有一个小孔口,孔口周围有2～4层小圆柱形至椭圆形细胞;它们的子囊壳表面细胞呈疣状或乳突状,具纵向沟纹。其中美座附丝壳还有具横条纹的蠕虫状附属物,莫勒针壳炱有光滑的子囊壳刚毛。子囊壳表面孔口的发现为小煤炱目Meliolales的确立提供了直接的证据。     

Epac Activates the Small G Proteins Rap1 and Rab3A to Achieve Exocytosis

Exocytosis of the acrosome (the acrosome reaction) relies on cAMP production, assembly of a proteinaceous fusion machinery, calcium influx from the extracellular medium, and mobilization from inositol 1,4,5-trisphosphate-sensitive intracellular stores. Addition of cAMP to human sperm suspensions bypasses some of these requirements and elicits exocytosis in a protein kinase A- and extracellular calcium-independent manner. The relevant cAMP target is Epac, a guanine nucleotide exchange factor for the small GTPase Rap. We show here that a soluble adenylyl cyclase synthesizes the cAMP required for the acrosome reaction. Epac stimulates the exchange of GDP for GTP on Rap1, upstream of a phospholipase C. The Epac-selective cAMP analogue 8-pCPT-2′-O-Me-cAMP induces a phospholipase C-dependent calcium mobilization in human sperm suspensions. In addition, our studies identify a novel connection between cAMP and Rab3A, a secretory granule-associated protein, revealing that the latter functions downstream of soluble adenylyl cyclase/cAMP/Epac but not of Rap1. Challenging sperm with calcium or 8-pCPT-2′-O-Me-cAMP boosts the exchange of GDP for GTP on Rab3A. Recombinant Epac does not release GDP from Rab3A in vitro, suggesting that the Rab3A-GEF activation by cAMP/Epac in vivo is indirect. We propose that Epac sits at a critical point during the exocytotic cascade after which the pathway splits into two limbs, one that assembles the fusion machinery into place and another that elicits intracellular calcium release.During fertilization in eutherian mammals, the spermatozoon must penetrate the zona pellucida to reach the oolema. Only sperm that have completed the acrosome reaction (AR)⁴ can successfully accomplish this task (1). The AR is a regulated exocytosis where the membrane of the acrosome, the single dense core secretory granule in sperm, fuses to the plasma membrane surrounding the anterior portion of the head. This process releases hydrolytic enzymes stored in the granule. These enzymes, together with the physical thrust derived from strong flagellar beating, enable sperm to penetrate the zona pellucida (1, 2). Physiological agonists accomplish the AR by inducing an influx of calcium from the extracellular medium and the assembly of a conserved proteinaceous fusion machinery that includes Rab3A, α-SNAP/NSF, synaptotagmin, complexin, and neurotoxin-sensitive SNAREs; the AR also requires an efflux of calcium from inside the acrosome through IP₃-sensitive channels (reviewed in Refs. 3, 4).In certain neurons, neuroendocrine and exocrine acinar cells, cAMP potentiates calcium-dependent exocytosis. Either cAMP-dependent protein kinase (PKA) or the exchange protein directly activated by cAMP (Epac) can be the targets of cAMP in the cAMP-regulated exocytosis. On the other hand, cAMP is the principal trigger of regulated secretion in various non-neuronal cells (5–7). Likewise, an elevation of cAMP alone is sufficient to trigger exocytosis in human sperm. Moreover, calcium relies on endogenous cAMP to accomplish acrosomal release, and it does so through a PKA-insensitive pathway involving Epac. The stimulation of endogenous Epac by the selective cAMP analogue 8-(p-chlorophenylthio)-2′-O-methyladenosine-3′,5′-cyclic monophosphate (8-pCPT-2′-O-Me-cAMP) is sufficient to trigger the AR even in the absence of extracellular calcium. Furthermore, when Epac is sequestered with specific antibodies, cAMP, calcium (8), and recombinant Rab3A (this study) are unable to elicit exocytosis.Epac1 and Epac2 are multidomain proteins that consist of an N-terminal regulatory region and a C-terminal catalytic region (9–11). The regulatory domain harbors the cAMP-binding site, which auto-inhibits the catalytic activity in the absence of cAMP (12–15). The catalytic portion bears a guanine-nucleotide exchange factor (GEF) activity specific for Rap1 and Rap2 (16, 17). Like all small G proteins, Raps cycle between an inactive GDP-bound and an active GTP-bound conformation. The GDP-GTP cycle is regulated by GEFs that induce the release of the bound GDP to be replaced by the more abundant GTP and by GTPase-activating proteins that coax the intrinsic GTPase activity to rapidly hydrolyze bound GTP, returning the G proteins to the inactive GDP-bound state (18, 19). Most small G proteins are linked to biological membranes via lipid modifications at their C terminus; for instance, Rap2A is farnesylated, and Rap1A/B, Rap2B, and Rabs are geranylgeranylated (20, 21). Guanine nucleotide dissociation inhibitors (GDIs) remove Rabs from membranes by sequestration of their lipid tails (22).Extracellular stimuli often result in the activation of cellular adenylate cyclases and an increase in cAMP levels. By serving as a cAMP-binding protein with intrinsic GEF activity, Epac couples cAMP production to a variety of Rap-mediated processes such as the control of cell adhesion and cell-cell junction formation, water resorption, cell differentiation, inflammatory processes, etc. (9–11). Many are the effectors of Epac and Epac-Rap signaling. Of particular interest to us is the observation that Epac stimulates phospholipase Cϵ (PLCϵ) through the activation of Rap1 and -2, resulting in IP₃-mediated release of calcium from internal stores (23, 24). PLCϵ is an unusual enzyme with two catalytic activities as follows: the typical phosphatidylinositol 4,5-bisphosphate hydrolyzing PLC activity plus a Rap-GEF activity. Thus, PLCϵ acts both downstream and upstream of Ras-like GTPases, perhaps to guarantee sustained Rap signaling (25).During membrane fusion, Rab proteins direct the recognition and physical attachments of the compartments that are going to fuse (26, 27). This association, or tethering, represents one of the earliest known events in membrane fusion and is accomplished through the recruitment of tethering factors. Rab3A localizes to vesicles and secretory granules and is one of the isoforms directly implicated in regulated exocytosis of neurotransmitters and hormones (28). Rab3A interacts in a GTP-dependent manner with at least two effector proteins, rabphilin and Rim (29–31). Rab3A is present in the acrosomal region of human (32), rat (33), and mouse sperm (34). Rab3A (full-length recombinant protein or a synthetic peptide corresponding to the effector domain) stimulates human (32, 35) and ram (36) and inhibits rat sperm AR (33). Rab3A is required for the AR triggered by calcium (37, 38) and cAMP (8).Epac is a multifunctional protein in which cAMP exerts its effects not only by promoting the exchange of GDP for GTP on Rap but also by allosterically regulating other molecules (10). In exocytosis for instance, a number of Rap-independent, Epac-linked signaling pathways have been described. They include the interaction of Epac2 with Rim2 (39) and the Rim2-related protein Piccolo (40). Epac2 also stimulates exocytosis by interacting with SUR1 (41). Finally, Epac2 controls ryanodine-sensitive calcium channels that are involved in calcium-induced calcium release (CICR) from internal stores in insulin-secreting cells (42).In this study, we piece together the analysis of two phenomena as follows: calcium mobilization and protein-protein interactions preceding exocytosis. To the best of our knowledge, this constitutes the first integrated molecular model that includes both the assembly of the fusion and intravesicular calcium release protein machineries during regulated exocytosis. By enquiring further into the signaling pathways operating during sperm exocytosis, we have found more players than previously suspected, and we discovered that the key components of these cascades are not arranged in a linear sequence. Epac sits at a central point of the signaling cascade after which the exocytotic pathway splits into two limbs as follows: one that assembles the fusion machinery into place, and another that elicits the release of calcium from the acrosome; both need to act in concert to achieve exocytosis. Our results identify Rab3A for the first time as a downstream target for Epac and place this small GTPase as an early component of the “fusion machinery” branch of the pathway. They also show that Epac stimulates the exchange of GDP for GTP on Rap1 and that this protein, as well as a PLC, drives intracellular calcium mobilization. Finally, our data reveal that a soluble adenylyl cyclase (sAC) (43, 44) synthesizes the cAMP that activates Epac. Again, we believe that this is the first report linking sAC to an exocytotic event.