Control of cell polarity and motility by the PtdIns(3,4,5)P3 phosphatase SHIP1

Proper neutrophil migration into inflammatory sites ensures host defense without tissue damage. Phosphoinositide 3-kinase (PI(3)K) and its lipid product phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P(3)) regulate cell migration, but the role of PtdIns(3,4,5)P(3)-degrading enzymes in this process is poorly understood. Here, we show that Src homology 2 (SH2) domain-containing inositol-5-phosphatase 1 (SHIP1), a PtdIns(3,4,5)P(3) phosphatase, is a key regulator of neutrophil migration. Genetic inactivation of SHIP1 led to severe defects in neutrophil polarization and motility. In contrast, loss of the PtdIns(3,4,5)P(3) phosphatase PTEN had no impact on neutrophil chemotaxis. To study PtdIns(3,4,5)P(3) metabolism in living primary cells, we generated a novel transgenic mouse (AktPH-GFP Tg) expressing a bioprobe for PtdIns(3,4,5)P(3.) Time-lapse footage showed rapid, localized binding of AktPH-GFP to the leading edge membrane of chemotaxing ship1(+/+)AktPH-GFP Tg neutrophils, but only diffuse localization in ship1(-/-)AktPH-GFP Tg neutrophils. By directing where PtdIns(3,4,5)P(3) accumulates, SHIP1 governs the formation of the leading edge and polarization required for chemotaxis.     

Expression and Localization of Type II Diacylglycerol Kinase Isozymes δ and η in the Developing Mouse Brain

The functions of type II diacylglycerol kinase (DGK) δ and -η in the brain are still unclear. As a first step, we investigated the spatial and temporal expression of DGKδ and -η in the brains of mice. DGKδ2, but not DGKδ1, was highly expressed in layers II–VI of the cerebral cortex; CA–CA3 regions and dentate gyrus of hippocampus; mitral cell, glomerular and granule cell layers of the olfactory bulb; and the granule cell layer in the cerebellum in 1- to 32-week-old mice. DGKδ2 was expressed just after birth, and its expression levels dramatically increased from weeks 1 to 4. A substantial amount of DGKη (η1/η2) was detected in layers II–VI of the cerebral cortex, CA1 and CA2 regions and dentate gyrus of the hippocampus, mitral cell and glomerular layers of the olfactory bulb, and Purkinje cells in the cerebellum of 1- to 32-week-old mice. DGKη2 expression reached maximum levels at P5 and decreased by 4 weeks, whereas DGKη1 increased over the same time frame. These results indicate that the expression patterns of DGK isozymes differ from each other and also from other isozymes, and this suggests that DGKδ and -η play distinct and specific roles in the brain.     

Substrate specificity of an esterase from the archaeon Sulfolobus tokodaii bearing a GGG(A)X motif

A GGG(A)X-type esterase (Est0071) from an archaeon catalyzes asymmetric hydrolysis of prochiral bulky malonic diesters in good enantioselectivity. The selectivity of Est0071 was for the opposite enantiomer to that previously shown for pig liver esterase, and the resulting enantiomeric excess of the products was higher. Est0071 could also catalyze the hydrolysis of various acetates of secondary alcohols, and showed moderate enantioselectivity in these reactions.     

A deletion in a cis element of Foxe3 causes cataracts and microphthalmia in rct mice

The Rinshoken cataract (rct) mutation, which causes congenital cataracts, is a recessive mutation found in SJL/J mice. All mutants present with opacity in the lens by 2?months of age. The rct locus was mapped to a 1.6-Mb region in Chr 4 that contains the Foxe3 gene. This gene is responsible for cataracts in humans and mice, and it plays a crucial role in the development of the lens. Furthermore, mutation of Foxe3 causes various ocular defects. We sequenced the genomic region of Foxe3, including the coding exons and UTRs; however, no mutations were discovered in these regions. Because there were no differences in Foxe3 sequences between the rct/rct and wild-type mice, we inferred that a mutation was located in the regulatory regions of the Foxe3 gene. To test this possibility, we sequenced a 5' noncoding region that is highly conserved among vertebrates and is predicted to be the major enhancer of Foxe3. This analysis revealed a deletion of 22-bp located approximately 3.2-kb upstream of the start codon of Foxe3 in rct mice. Moreover, we demonstrated by RT-PCR and in situ hybridization that the rct mutant has reduced expression of Foxe3 in the lens during development. We therefore suggest that cataracts in rct mice are caused by reduced Foxe3 expression in the lens and that this decreased expression is a result of a deletion in a cis-acting regulatory element.     

Methotrexate chronotherapy is effective against rheumatoid arthritis

Methotrexate (MTX) is the most important drug for treating rheumatoid arthritis (RA). It has been stated that cytokines play an important role in the pathogenesis of RA, and that cytokine levels increase and show 24-h rhythms in RA patients. Previously, we found that arthritis was relieved after the administration of MTX at specific times in synchronization with the 24-h rhythm of tumor necrosis factor (TNF)-α in collagen-induced arthritis (CIA) animals. Based on our findings in an earlier study of the dosing time-dependent effects of MTX in MRL/lpr mice, which develop autoimmune disorders that share similarities with human RA, we examined here the utility of MTX chronotherapy in Japanese RA patients. In an initial animal modeling study, we collected blood from MRL/lpr mice at different times (2, 6, 10, 14, 18, or 22 hours after the light was turned on [HALO]), and we measured TNF-α mRNA expression in leukocytes. MTX was administered to the mice at two different dosing times (6 or 18 HALO), and various blood parameters were measured to estimate arthritis activity. TNF-α mRNA levels showed a clear 24-h rhythm with a peak at 22 HALO and a trough at 18 HALO after RA had developed. In these MRL/lpr mice, inflammation and TNF-α were markedly reduced when the MTX dosing time was matched to the time (18 HALO) when the TNF-α level began to increase. We then applied these findings to Japanese RA patients by switching them from the standard MTX three times/wk (day 1: after breakfast and supper; day 2: after breakfast schedule), to chronotherapy, in which the dose and number of doses/wk were not changed but MTX was administered once-a-day at bedtime. Disease Activity Score (DAS)28, modified health assessment questionnaire (MHAQ), and adverse effects were assessed. With MTX chronotherapy, DAS28, which is commonly used to quantitatively assess RA symptoms, was significantly improved at all follow-up clinical visit times compared with the baseline (vs. 1 mo: p?=?.0197, 2 mos: p?=?.0107, 3 mos: p?=?.0087). Significant symptom recovery was observed in 41.2% of patients, and 23.5% of patients achieved clinical remission during the 3 mos of follow-up. Functional capacity of RA patients, as indicated by the MHAQ, was markedly improved by chronotherapy. There were no severe adverse effects. Thus, we demonstrated (i) inflammation and plasma TNF-α concentrations were significantly reduced in MRL/lpr mice treated with MTX at 18 HALO, the time when TNF-α mRNA level began to increase; and (ii) MTX bedtime chronotherapy was safe, markedly reduced disease activity, and improved the functional capacity of RA patients. The findings on RA patients show that bedtime MTX chronotherapy can improve RA symptoms compared to the current standard dosing methods.     

The ESCRT system is required for hepatitis C virus production

Background

Recently, lipid droplets have been found to be involved in an important cytoplasmic organelle for hepatitis C virus (HCV) production. However, the mechanisms of HCV assembly, budding, and release remain poorly understood. Retroviruses and some other enveloped viruses require an endosomal sorting complex required for transport (ESCRT) components and their associated proteins for their budding process.

Methodology/Principal Findings

To determine whether or not the ESCRT system is needed for HCV production, we examined the infectivity of HCV or the Core levels in culture supernatants as well as HCV RNA levels in HuH-7-derived RSc cells, in which HCV-JFH1 can infect and efficiently replicate, expressing short hairpin RNA or siRNA targeted to tumor susceptibility gene 101 (TSG101), apoptosis-linked gene 2 interacting protein X (Alix), Vps4B, charged multivesicular body protein 4b (CHMP4b), or Brox, all of which are components of the ESCRT system. We found that the infectivity of HCV in the supernatants was significantly suppressed in these knockdown cells. Consequently, the release of the HCV Core into the culture supernatants was significantly suppressed in these knockdown cells after HCV-JFH1 infection, while the intracellular infectivity and the RNA replication of HCV-JFH1 were not significantly affected. Furthermore, the HCV Core mostly colocalized with CHMP4b, a component of ESCRT-III. In this context, HCV Core could bind to CHMP4b. Nevertheless, we failed to find the conserved viral late domain motif, which is required for interaction with the ESCRT component, in the HCV-JFH1 Core, suggesting that HCV Core has a novel motif required for HCV production.

Conclusions/Significance

These results suggest that the ESCRT system is required for infectious HCV production.     

Distinct characteristics of circulating vascular endothelial growth factor-a and C levels in human subjects

The mechanisms that lead from obesity to atherosclerotic disease are not fully understood. Obesity involves angiogenesis in which vascular endothelial growth factor-A (VEGF-A) plays a key role. On the other hand, vascular endothelial growth factor-C (VEGF-C) plays a pivotal role in lymphangiogenesis. Circulating levels of VEGF-A and VEGF-C are elevated in sera from obese subjects. However, relationships of VEGF-C with atherosclerotic risk factors and atherosclerosis are unknown. We determined circulating levels of VEGF-A and VEGF-C in 423 consecutive subjects not receiving any drugs at the Health Evaluation Center. After adjusting for age and gender, VEGF-A levels were significantly and more strongly correlated with the body mass index (BMI) and waist circumference than VEGF-C. Conversely, VEGF-C levels were significantly and more closely correlated with metabolic (e.g., fasting plasma glucose, hemoglobin A1c, immunoreactive insulin, and the homeostasis model assessment of insulin resistance) and lipid parameters (e.g., triglycerides, total cholesterol (TC), low-density-lipoprotein cholesterol (LDL-C), and non-high-density-lipoprotein cholesterol (non-HDL-C)) than VEGF-A. Stepwise regression analyses revealed that independent determinants of VEGF-A were the BMI and age, whereas strong independent determinants of VEGF-C were age, triglycerides, and non-HDL-C. In apolipoprotein E-deficient mice fed a high-fat-diet (HFD) or normal chow (NC) for 16 weeks, levels of VEGF-A were not significantly different between the two groups. However, levels of VEGF-C were significantly higher in HFD mice with advanced atherosclerosis and marked hypercholesterolemia than NC mice. Furthermore, immunohistochemistry revealed that the expression of VEGF-C in atheromatous plaque of the aortic sinus was significantly intensified by feeding HFD compared to NC, while that of VEGF-A was not. In conclusion, these findings demonstrate that VEGF-C, rather than VEGF-A, is closely related to dyslipidemia and atherosclerosis.     

In vivo effects of a recombinant molt-inhibiting hormone on molt interval and hemolymph ecdysteroid level in the kuruma prawn, Marsupenaeus japonicus

In order to determine the function of molt-inhibiting hormone (MIH) in vivo, we examined the effects of injecting of a recombinant MIH on the molt interval and hemolymph ecdysteroid level in the kuruma prawn, Marsupenaeus japonicus. The injection of recombinant MIH significantly prolonged the molt interval (9.0 +/-0.4 days in the control group, 9.5+/-0.5 days in the 2500 ng/g-body weight/injection-group, mean+/-SD), and significantly decreased the hemolymph ecdysteroid level (ratio of levels between after and before injection: 1.94+/-1.09 in the control and 1.28+/-0.39 in the 3000 ng/g-body weight/injection-group, mean+/-SD). These results conclusively show the inhibitory effects of MIH on molting in vivo.     

Endothelial cell Ca2+ increases are independent of membrane potential in pressurized rat mesenteric arteries

In rat mesenteric arteries, the ability of ACh to evoke hyperpolarization of smooth muscle cells and consummate dilatation relies on an increase in endothelial cell cytosolic free [Ca2+] and activation of Ca2+-activated K+ channels (KCa). The time course of average and spatially organized rises in endothelial cell [Ca2+]i and concomitant effects on membrane potential were investigated in individual cells of pressurized arteries and isolated sheets of native cells stimulated with ACh. In both cases, ACh stimulated a sustained and oscillating rise in endothelial cell [Ca2+]i. Overall, the oscillations remained asynchronous between cells, yet occasionally localized intercellular coordination became evident. In pressurized arteries, repetitive waves of Ca2+ moved longitudinally across endothelial cells, and depended on Ca2+-store refilling. The rise in endothelial cell Ca2+ was associated with sustained hyperpolarization of endothelial cells in both preparations. This hyperpolarization was also evident when recording from smooth muscle cells in pressurized arteries, and from resting membrane potential, selective inhibition of small-conductance K Ca (SK Ca) with apamin (50 nM) was sufficient to inhibit this response. In the presence of phenylephrine-tone, both apamin and the selective inhibitor of intermediate conductance K Ca (IK Ca) TRAM-34 (1 microM) were required to inhibit the non-nitric oxide-mediated dilatation to ACh. When hyperpolarization of endothelial cells was fully prevented either with inhibitors of K Ca or in KCl (35 mM)-depolarized cells, both the time course and frequency of oscillations in endothelial cell [Ca2+]i to ACh were unaffected. Together, these data show that although a rise in endothelial cell [Ca2+]i stimulates hyperpolarization, depletion of intracellular stores with ACh stimulates Ca2+-influx which is not significantly influenced by the increase in cellular electrochemical gradient for Ca2+ caused by that hyperpolarization.