A comparative study of the characteristics of eIF-2 and eIF-2-ancillary factor activities from yeast Saccharomyces cerevisiae and rabbit reticulocytes

The characteristics of yeast eukaryotic initiation factor 2 (eIF-2) and Co-eIF-2A have been studied and compared with those of the corresponding factors from rabbit reticulocytes. 1) Unlike eIF-2r, purified eIF-2y did not contain bound GDP. 2) Purified eIF-2y preparation contained GTPase activity and dephosphorylated GTP to GDP. 3) An anti-eIF-2r preparation which predominantly precipitated the gamma-subunit (Mr 54,000) of eIF-2r also precipitated the larger subunit (Mr 54,000) of eIF-2y. 4) Unlike eIF-2r, ternary complex formation by eIF-2y was not inhibited by Mg2+. 5) Both Co-eIF-2A20y and Co-eIF-2r significantly enhanced Met-tRNAf binding to eIF-2y and, again, Mg2+ did not have any effect on this stimulated Met-tRNAf binding to eIF-2y. 6) Both Co-eIF-2A20y and Co-eIF-2r were similarly effective in stimulating Met-tRNAf binding to eIF-2r in the absence of Mg2+. However, in the presence of Mg2+, Co-eIF-2A20y was significantly less effective than Co-eIF-2r as Co-eIF-2A20y did not promote displacement of GDP from eIF-2r X GDP. 7) eIF-2y bound [3H]GDP and this binding was significantly enhanced in the presence of Mg2+. Also, [3H]GDP in the preformed eIF-2y X [3H]GDP complex was rapidly exchanged with exogenously added unlabeled GDP in the presence of Mg2+. Co-eIF-2A20y had no effect on GDP binding to eIF-2y nor on GDP exchange reactions. 8) Reticulocyte heme-regulated protein synthesis inhibitor, which phosphorylated almost completely (in excess of 80%) the alpha-subunit (Mr 38,000) of eIF-2r, also phosphorylated similarly the smaller subunit (Mr 36,000) of eIF-2y. However, such phosphorylation had no significant effect on ternary complex formation, GDP binding, and GDP exchange reactions.     

Alteration of domain architecture of chicken erythrocyte chromatin

The higher-order organisation of chromatin in chicken erythrocyte nuclei as a function of the ionic strength of the nuclear suspension buffer and also of the time of incubation in this buffer prior to nuclease digestion has been investigated. This organisation is described in terms of a physical parameter called the domain length. The 45-kbp-long domains of control nuclei were unravelled to give rise to domains of length 150 kbp on overnight equilibration at 0 degree C of the nuclei in standard isolation buffer containing 0.135 M NaCl prior to nuclease digestion. However, transition to the equilibrium state was preceded by a metastable and irregular domain architecture when the nuclei were incubated for only 1 h. In contrast, the domain length remained unchanged when nuclei were incubated in the isolation buffer alone for identical periods of time. The proteins dissociated at the higher ionic strength were characterised and their role in stabilising the domain structure is discussed.     

Dynamic Redox Regulation of IL-4 Signaling

Quantifying the magnitude and dynamics of protein oxidation during cell signaling is technically challenging. Computational modeling provides tractable, quantitative methods to test hypotheses of redox mechanisms that may be simultaneously operative during signal transduction. The interleukin-4 (IL-4) pathway, which has previously been reported to induce reactive oxygen species and oxidation of PTP1B, may be controlled by several other putative mechanisms of redox regulation; widespread proteomic thiol oxidation observed via 2D redox differential gel electrophoresis upon IL-4 treatment suggests more than one redox-sensitive protein implicated in this pathway. Through computational modeling and a model selection strategy that relied on characteristic STAT6 phosphorylation dynamics of IL-4 signaling, we identified reversible protein tyrosine phosphatase (PTP) oxidation as the primary redox regulatory mechanism in the pathway. A systems-level model of IL-4 signaling was developed that integrates synchronous pan-PTP oxidation with ROS-independent mechanisms. The model quantitatively predicts the dynamics of IL-4 signaling over a broad range of new redox conditions, offers novel hypotheses about regulation of JAK/STAT signaling, and provides a framework for interrogating putative mechanisms involving receptor-initiated oxidation.     

Myocardial protection by Protykin,a novel extract of trans-resveratrol and emodin

Protykin is an all-natural, high potency standardized extract of trans-resveratrol (20%) and emodin (10%) derived from the dried rhizome of Polygonum cuspidatum. Previous studies have demonstrated free radical scavenging and anti-inflammatory activities of resveratrol. Since free radicals play a crucial role in the pathogenesis of myocardial ischemia/reperfusion injury, we examined whether Protykin could preserve the heart during ischemic arrest. Sprague—Dawley rats were divided into two groups: experimental group was gavaged Protykin (100 mg/kg body wt) dissolved in corn oil for three weeks, while the control group was gavaged corn oil alone. After three weeks, rats were sacrificed, isolated hearts perfused via working mode, were made globally ischemic for 30 min followed by 2 h of reperfusion. Left ventricular functions were continuously monitored and malonaldehyde (MDA) (presumptive marker for oxidative stress) formation were estimated. At the end of each experiment, myocardial infarct size was measured by TTC staining method. Peroxyl radical scavenging activity of Protykin was determined by examining its ability to remove peroxyl radical generated by 2,2′-azobis (2-amidinopropane) dihydrochloride, while hydroxy radical scavenging activity was tested with its ability to reduce 7-OH^·-coumarin-3-carboxylic acid. The results of our study demonstrated that the Protykin group provided cardioprotection as evidenced by improved post-ischemic left ventricular functions (dp, dp/dt_max) and aortic flow as compared to control group. This was further supported by the reduced infarct size in the Protykin group. Formation of MDA was also reduced by Protykin treatment. In vitro studies demonstrated that Protykin possessed potent peroxyl and hydroxyl radical scavenging activities. The results of this study indicate that Protykin can provide cardioprotection, presumably by virtue of its potent free radical scavenging activity.     

A Non-enveloped Virus Hijacks Host Disaggregation Machinery to Translocate across the Endoplasmic Reticulum Membrane

Mammalian cytosolic Hsp110 family, in concert with the Hsc70:J-protein complex, functions as a disaggregation machinery to rectify protein misfolding problems. Here we uncover a novel role of this machinery in driving membrane translocation during viral entry. The non-enveloped virus SV40 penetrates the endoplasmic reticulum (ER) membrane to reach the cytosol, a critical infection step. Combining biochemical, cell-based, and imaging approaches, we find that the Hsp110 family member Hsp105 associates with the ER membrane J-protein B14. Here Hsp105 cooperates with Hsc70 and extracts the membrane-penetrating SV40 into the cytosol, potentially by disassembling the membrane-embedded virus. Hence the energy provided by the Hsc70-dependent Hsp105 disaggregation machinery can be harnessed to catalyze a membrane translocation event.     

Rac1 Regulates Endometrial Secretory Function to Control Placental Development

During placenta development, a succession of complex molecular and cellular interactions between the maternal endometrium and the developing embryo ensures reproductive success. The precise mechanisms regulating this maternal-fetal crosstalk remain unknown. Our study revealed that the expression of Rac1, a member of the Rho family of GTPases, is markedly elevated in mouse decidua on days 7 and 8 of gestation. To investigate its function in the uterus, we created mice bearing a conditional deletion of the Rac1 gene in uterine stromal cells. Ablation of Rac1 did not affect the formation of the decidua but led to fetal loss in mid gestation accompanied by extensive hemorrhage. To gain insights into the molecular pathways affected by the loss of Rac1, we performed gene expression profiling which revealed that Rac1 signaling regulates the expression of Rab27b, another GTPase that plays a key role in targeting vesicular trafficking. Consequently, the Rac1-null decidual cells failed to secrete vascular endothelial growth factor A, which is a critical regulator of decidual angiogenesis, and insulin-like growth factor binding protein 4, which regulates the bioavailability of insulin-like growth factors that promote proliferation and differentiation of trophoblast cell lineages in the ectoplacental cone. The lack of secretion of these key factors by Rac1-null decidua gave rise to impaired angiogenesis and dysregulated proliferation of trophoblast cells, which in turn results in overexpansion of the trophoblast giant cell lineage and disorganized placenta development. Further experiments revealed that RAC1, the human ortholog of Rac1, regulates the secretory activity of human endometrial stromal cells during decidualization, supporting the concept that this signaling G protein plays a central and conserved role in controlling endometrial secretory function. This study provides unique insights into the molecular mechanisms regulating endometrial secretions that mediate stromal-endothelial and stromal-trophoblast crosstalk critical for placenta development and establishment of pregnancy.