Porcine zona pellucida glycoprotein ZP4 is responsible for the sperm-binding activity of the ZP3/ZP4 complex

Summary The zona pellucida (ZP) is a transparent envelope that surrounds the mammalian oocyte and mediates species-selective sperm-egg interactions. Porcine and bovine ZPs consist of glycoproteins ZP2, ZP3, and ZP4. In both pig and bovine a heterocomplex consisting of ZP3 and ZP4 binds to sperm, however it is not clarified whether ZP3 or ZP4 in the complex is responsible for the sperm binding. Previously, we have established a baculovirus-Sf9 cell expression system for porcine ZP glycoproteins. A mixture of recombinant ZP3 (rZP3) and rZP4 displayed sperm-binding activity toward bovine sperm but not porcine sperm, probably due to differences in carbohydrate structure between the native and recombinant ZP glycoproteins. In this study, a mixture of porcine rZP3 and native ZP4 (nZP4) inhibited the binding of porcine sperm to the ZP. In contrast, a mixture of porcine nZP3 and rZP4 did not inhibit the binding of porcine sperm, although the mixture inhibited the binding of bovine sperm. The porcine rZP3/nZP4 mixture bound to the acrosomal region of porcine sperm, in a manner similar to that of the nZP3/nZP4 mixture. nZP3 was precipitated with rZP4, and nZP4 was precipitated with rZP3 by utilising the N-terminal tags on the recombinant proteins. These results indicated that nZP4, but not rZP4, is necessary for binding activity of porcine ZP3/ZP4 complex towards porcine sperm and further suggested that the carbohydrate structures of ZP4 in the porcine ZP3/ZP4 complex are responsible for porcine sperm-binding activity of the complex.     

Direct evidence that ventral forebrain cells migrate to the cortex and contribute to the generation of cortical myelinating oligodendrocytes

Cortical neuroepithelial cells generate neurons, astrocytes, and oligodendrocytes (OLs) in vitro. However, whether cortical OLs are derived from the cortical neuroepithelium or migrate from the ventral forebrain is under severe debate yet. This is due to the fact that OL progenitor cells (OPCs), as marked by the expression of PDGFRalpha or NG2, are generated at around embryonic day (E) 11 or 12 in the mouse ganglionic eminences, but the myelinating OLs appear during the second week postnatally in the cortex. There has been no labeling method for long-term glial cell-lineage tracing. Thus, we developed a new strategy: plasmid DNA encoding Cre recombinase was introduced into the Cre/loxP reporter forebrain in ventral- or dorsal-specific manner by in utero DNA electroporation. The reporter gfp gene is expressed permanently owing to the chromosomal DNA recombination. The GFP-labeled myelinating OLs were detected in the adult cortex when electroporation was targeted to the ventral neuroepithelium, demonstrating at least some of the myelinating OLs are derived from the ventral forebrain. However, when electroporation was targeted to the dorsal, we could not find GFP-labeled myelinating OLs. This suggests that the progenitors of cortical OPCs are absent or located at restricted regions in the dorsal forebrain (cortex) at E12.     

Role of transient receptor potential vanilloid 2 in LPS-induced cytokine production in macrophages

There is considerable evidence indicating that intracellular Ca²⁺ participates as a second messenger in TLR4-dependent signaling. However, how intracellular free Ca²⁺ concentrations ([Ca²⁺]_i) is increased in response to LPS and how they affect cytokine production are poorly understood. Here we examined the role of transient receptor potential (TRP), a major Ca²⁺ permeation pathway in non-excitable cells, in the LPS-induced cytokine production in macrophages. Pharmacologic experiments suggested that TRPV family members, but neither TRPC nor TRPM family members, are involved in the LPS-induced TNFα and IL-6 production in RAW264 macrophages. RT-PCR and immunoblot analyses showed that TRPV2 is the sole member of TRPV family expressed in macrophages. ShRNA against TRPV2 inhibited the LPS-induced TNFα and IL-6 production as well as IκBα degradation. Experiments using BAPTA/AM and EGTA, and Ca²⁺ imaging suggested that the LPS-induced increase in [Ca²⁺]_i involves both the TRPV2-mediated intracellular and extracellular Ca²⁺ mobilizations. BAPTA/AM abolished LPS-induced TNFα and IL-6 production, while EGTA only partially suppressed LPS-induced IL-6 production, but not TNFα production. These data indicate that TRPV2 is involved in the LPS-induced Ca²⁺ mobilization from intracellular Ca²⁺ store and extracellular Ca²⁺. In addition to Ca²⁺ mobilization through the IP₃-receptor, TRPV2-mediated intracellular Ca²⁺ mobilization is involved in NFκB-dependent TNFα and IL-6 expression, while extracellular Ca²⁺ entry is involved in NFκB-independent IL-6 production.     

Wnt3a Promotes Hippocampal Neurogenesis by Shortening Cell Cycle Duration of Neural Progenitor Cells

The effects of Wnt signaling on neural progenitor cells have been controversial. Activation of the canonical Wnt signaling pathway either promotes neural progenitor cell proliferation or accelerates their differentiation into postmitotic neurons. This study demonstrates that activation of the Wnt signaling pathway by itself induces neural progenitor cell proliferation but does not directly affect neuronal differentiation processes. To investigate whether Wnt signaling promotes expansion and/or differentiation of neural progenitor cells in the developing hippocampus, we prepared primary mouse hippocampal progenitors and treated them with Wnt3a in a chemically defined culture medium. Wnt3a increased the total number of cells, including the numbers of Ki67⁺ proliferating cells and Tuj1⁺ differentiated neurons. This result verified that Wnt3a promoted neural progenitor cell proliferation. Meanwhile, Wnt3a did not appear to actively enhance the neuronal differentiation process itself, because (1) the ratio of Tuj1⁺ cells to the total cells, and (2) the ratio of BrdU⁺ Tuj1⁺ cells to the total BrdU⁺ cells, were both comparable between cultures with or without Wnt3a. Indeed, Wnt3a caused no significant change in either cell survival or the proportion of symmetric and asymmetric cell divisions that directly affected neuron production. We finally demonstrated that the Wnt3a treatment simply shortened cell cycle duration of neural progenitor cells by 2.9 h. The accelerated cell cycle progression without affecting the ratio of symmetric/asymmetric cell divisions explains how Wnt signaling per se leads to the expansion of both proliferative cell population and differentiated neuronal cell population.     

Acute Effects of Sex Steroid Hormones on Susceptibility to Cardiac Arrhythmias: A Simulation Study

Acute effects of sex steroid hormones likely contribute to the observation that post-pubescent males have shorter QT intervals than females. However, the specific role for hormones in modulating cardiac electrophysiological parameters and arrhythmia vulnerability is unclear. Here we use a computational modeling approach to incorporate experimentally measured effects of physiological concentrations of testosterone, estrogen and progesterone on cardiac ion channel targets. We then study the hormone effects on ventricular cell and tissue dynamics comprised of Faber-Rudy computational models. The “female” model predicts changes in action potential duration (APD) at different stages of the menstrual cycle that are consistent with clinically observed QT interval fluctuations. The “male” model predicts shortening of APD and QT interval at physiological testosterone concentrations. The model suggests increased susceptibility to drug-induced arrhythmia when estradiol levels are high, while testosterone and progesterone are apparently protective. Simulations predict the effects of sex steroid hormones on clinically observed QT intervals and reveal mechanisms of estrogen-mediated susceptibility to prolongation of QT interval. The simulations also indicate that acute effects of estrogen are not alone sufficient to cause arrhythmia triggers and explain the increased risk of females to Torsades de Pointes. Our results suggest that acute effects of sex steroid hormones on cardiac ion channels are sufficient to account for some aspects of gender specific susceptibility to long-QT linked arrhythmias.     

Nitrogen Fixation by Termites in Tropical Forests, Thailand

Nitrogen (N) fixed by termites was evaluated as a N input to decomposition processes in two tropical forests, a dry deciduous forest (DDF) and the neighboring dry evergreen forest (DEF), Thailand. A diverse group of termite species were assayed by acetylene reduction method and only the wood/litter-feeding termites were found to fix N. More intensive samplings of two abundant species, Microcerotermes crassus and Globitermes sulphureus, were done across several seasons, suggesting N fixation rates of 0.21 and 0.28 kg ha⁻¹ y⁻¹ by termites in the DDF and DEF, respectively. Also, estimates of asymbiotic N fixation rates were 0.75 and 3.95 kg ha⁻¹ y⁻¹. N fixed by termites and by asymbiotic fixers is directly supplied to decomposers breaking down dead plant material and could be a major source of their N. N fixed by termites was 7–22% of that fixed by termites and asymbiotic fixers. Although N fixed by termites is a small input compared to other inputs, this N is likely important for decomposition processes.     

Aquaporin-2 trafficking is regulated by PDZ-domain containing protein SPA-1

Targeted positioning of water channel aquaporin-2 (AQP2) strictly regulates body water homeostasis. Trafficking of AQP2 to the apical membrane is critical to the reabsorption of water in renal collecting ducts. Controlled apical positioning of AQP2 suggests the existence of proteins that interact with AQP2. A biochemical search for AQP2-interacting proteins led to the identification of PDZ-domain containing protein, signal-induced proliferation-associated gene-1 (SPA-1) which is a GTPase-activating protein (GAP) for Rap1. The distribution of SPA-1 coincided with that of AQP2 in renal collecting ducts. The site of colocalization was concomitantly relocated by hydration status. AQP2 trafficking to the apical membrane was inhibited by the SPA-1 mutant lacking Rap1GAP activity and by the constitutively active mutant of Rap1. AQP2 trafficking was impaired in SPA-1-deficient mice. Our results show that SPA-1 directly binds to AQP2 and regulates at least in part AQP2 trafficking.     

Subcellular localization of herpes simplex virus type 1 UL51 protein and role of palmitoylation in Golgi apparatus targeting

The herpes simplex virus type 1 (HSV-1) UL51 gene products are virion-associated phosphoproteins with apparent molecular masses of 27, 29, and 30 kDa in HSV-1-infected cells. In this study, we have investigated the intracellular localization and distribution of UL51 protein both in infected cells and in transfected cells expressing only UL51. We found that this protein colocalized closely with Golgi marker proteins such as the Golgi-58K protein and GM130 in transfected cells expressing only UL51. However, in infected cells, the UL51 protein localized to the juxtanuclear region but only partially colocalized with the Golgi maker proteins. Mutant protein analysis revealed that the N-terminal 15 amino acid residues of the UL51 protein sufficed for this Golgi localization property. The UL51 protein redistributed on addition of brefeldin A. This was prevented by pretreatment with 2-deoxyglucose and sodium azide, which results in ATP depletion, but not by pretreatment with NaF and AlCl(3), which activates heterotrimeric G proteins. Moreover, we found that palmitoylation of the UL51 protein through the N-terminal cysteine at position 9 was necessary for its Golgi localization. Protease digestion analysis suggested that the UL51 protein localized on the cytoplasmic face of the membrane in UL51-transfected cells, while in infected cells it localized mainly to the inside of cytoplasmic vesicles and/or the viral envelope. Transmission immunoelectron microscopy revealed an association of UL51 protein-specific labeling with cytoplasmic virions and also with some membranous structure. We infer from these observations that internalization of UL51 protein into the cytoplasmic vesicle and/or virion may occur in association with viral envelopment in HSV-infected cells.