Crystallization and MAD data collection of high-molecular weight cytochrome c from Desulfovibrio vulgaris Miyazaki F

Hexadecaheme high molecular weight cytochrome c from a sulfate-reducing bacterium, Desulfovibrio vulgaris Miyazaki F has been successfully purified and crystallized. X-ray diffraction data have been collected by the multiple wavelength anomalous dispersion method. The crystal belongs to the space group P2(1)2(1)2(1) with unit-cell parameters a=60.42, b=84.29 and c=144.16 A and contains one molecule per asymmetric unit.     

The Lipoprotein LpqW Is Essential for the Mannosylation of Periplasmic Glycolipids in Corynebacteria

Phosphatidylinositol mannosides (PIM), lipomannan (LM), and lipoarabinomannan (LAM) are essential components of the cell wall and plasma membrane of mycobacteria, including the human pathogen Mycobacterium tuberculosis, as well as the related Corynebacterineae. We have previously shown that the lipoprotein, LpqW, regulates PIM and LM/LAM biosynthesis in mycobacteria. Here, we provide direct evidence that LpqW regulates the activity of key mannosyltransferases in the periplasmic leaflet of the cell membrane. Inactivation of the Corynebacterium glutamicum lpqW ortholog, NCgl1054, resulted in a slow growth phenotype and a global defect in lipoglycan biosynthesis. The NCgl1054 mutant lacked LAMs and was defective in the elongation of the major PIM species, AcPIM2, as well as a second glycolipid, termed Gl-X (mannose-α1–4-glucuronic acid-α1-diacylglycerol), which function as membrane anchors for LM-A and LM-B, respectively. Elongation of AcPIM2 and Gl-X was found to be dependent on expression of polyprenol phosphomannose (ppMan) synthase. However, the ΔNCgl1054 mutant synthesized normal levels of ppMan, indicating that LpqW is not required for synthesis of this donor. A spontaneous suppressor strain was isolated in which lipoglycan synthesis in the ΔNCgl1054 mutant was partially restored. Genome-wide sequencing indicated that a single amino acid substitution within the ppMan-dependent mannosyltransferase MptB could bypass the need for LpqW. Further evidence of an interaction is provided by the observation that MptB activity in cell-free extracts was significantly reduced in the absence of LpqW. Collectively, our results suggest that LpqW may directly activate MptB, highlighting the role of lipoproteins in regulating key cell wall biosynthetic pathways in these bacteria.     

Development of human T-cell leukemia virus type 1-transformed tumors in rats following suppression of T-cell immunity by CD80 and CD86 blockade

Host immunity influences clinical manifestations of human T-cell leukemia virus type 1 (HTLV-1) infection. In this study, we demonstrated that HTLV-1-transformed tumors could develop in immunocompetent rats by blocking a costimulatory signal for T-cell immune responses. Four-week-old WKA/HKm rats were treated with monoclonal antibodies (MAbs) to CD80 and CD86 and subcutaneously inoculated with syngeneic HTLV-1-infected TARS-1 cells. During MAb treatment for 14 days, TARS-1 inoculation resulted in the development of solid tumors at the site of inoculation, which metastasized to the lungs. In contrast, rats not treated with MAbs promptly rejected tumor cells. Splenic T cells from MAb-treated rats indicated impairment of proliferative and cytotoxic T-lymphocyte responses against TARS-1 in vitro compared to untreated rats. However, tumors grown in MAb-treated rats regressed following withdrawal of MAb therapy. Recovery of TARS-1-specific T-cell immune responses was associated with tumor regression in these rats. Our results suggest that HTLV-1-specific cell-mediated immunity plays a critical role in immunosurveillance against HTLV-1-transformed tumor development in vivo.     

Mouse embryonic stem cell culture for generation of three-dimensional retinal and cortical tissues

Generation of compound tissues with complex structures is a major challenge in cell biology. In this article, we describe a protocol for mouse embryonic stem cell (ESC) culture for in vitro generation of three-dimensional retinal tissue, comparing it with the culture protocol for cortical tissue generation. Dissociated ESCs are reaggregated in a 96-well plate with reduced cell-plate adhesion and cultured as floating aggregates. Retinal epithelium is efficiently generated when ESC aggregates are cultured in serum-free medium containing extracellular matrix proteins, spontaneously forming hemispherical vesicles and then progressively transforming into a shape reminiscent of the embryonic optic cup in 9-10 d. In long-term culture, the ESC-derived optic cup generates a fully stratified retinal tissue consisting of all major neural retinal components. In contrast, the cortical differentiation culture can be started without exogenous extracellular matrix proteins, and it generates stratified cortical epithelia consisting of four distinct layers in 13 d.     

Proprotein convertases regulate activity of prostate epithelial cell differentiation markers and are modulated in human prostate cancer cells

Prostate derived factor (PDF) is a member of transforming growth factor-beta (TGF-beta) superfamily proteins involved in differentiation of the prostate epithelium. Proprotein convertases (PCs) such as furin are thought to mediate the processing of TGF-beta superfamily. In the present study, we demonstrated for the first time that human prostate cancer cell lines differentially synthesize and secret prostate derived factor (PDF), and that PDF secreted by LNCaP is processed by PCs. Exposure of LNCaP cells to the decanoyl-Arg-Val-Lys-Arg-chloromethylketone (CMK), a synthetic furin-like protease inhibitor, inhibited PDF processing and resulted in the loss of luminal cell phenotype and induction of basal cell phenotype in LNCaP cells as demonstrated by alternations in the expression of cytokeratins 8, 14, 18, and 19, markers of prostate epithelial cell differentiation. These results suggest that proprotein convertases may be involved in the regulation of prostate epithelial cell differentiation, and may be an important target of prostate cancer therapy.     

Two Forms of Glucoamylase from Mucor rouxianus 1. Purification and Crystallization

Mucor rouxianus produced two forms (isoenzymes) of glucoamylase which could be separated from each other by polyacrylamide gel electrophoresis or by chromatography on SP-Sephadex C-50, and they were designated glucoamylase I and glucoamylase II. Glucoamylases I and II were isolated in crystalline form, and were homogeneous in poly acrylamide gel electrophoresis and in ultracentrifugation, respectively. The sedimentation coefficient () molecular weight of glucoamylase I were 4.39 S and 59,000, and those of glucoamylase II were 4.29 S and 49,000, respectively.     

Differential regulation of the low affinity Fc receptor for IgE (Fc epsilon R2/CD23) and the IL-2 receptor (Tac/p55) on eosinophilic leukemia cell line (EoL-1 and EoL-3)

Two types of activation Ag, low affinity FcR for IgE (Fc epsilon R2)/CD23 and IL-2R (Tac/p55), were expressed and differently regulated on human eosinophilic leukemia cell lines (EoL-1 and EoL-3). Because the binding of IgE on EoL-3 cells was completely inhibited by H107 (anti-Fc epsilon R2/CD23 mAb) but not by irrelevant mAb, essentially all the low affinity Fc epsilon R2 on EoL-3 seemed to be the Fc epsilon R2/CD23 molecules. Both IL-4 and IFN-gamma enhanced the surface expression of Fc epsilon R2, whereas IL-1, IL-2, and IL-5 showed no effects, as determined by surface staining with anti-Fc epsilon R2 antibody (H107). In contrast to Fc epsilon R2 up-regulation, IL-4 and IFN-gamma showed a differential effect on the regulation of IL-2R (Tac/p55). Whereas IFN-gamma up-regulated the receptor expression of IL-2R/Tac, IL-4 did not. The result suggests that these lymphokines are involved in the different aspects of the activation pathway of the eosinophils. The possible role of Fc epsilon R2 and IL-2R on the function of eosinophils in allergic reaction is discussed.     

Trend and Risk Factors of Diverticulosis in Japan: Age,Gender, and Lifestyle/Metabolic-Related Factors May Cooperatively Affect on the Colorectal Diverticula Formation

Background

Despite the marked increase of diverticulosis, its risk factors have not been adequately elucidated. We therefore aim to identify significantly associated factors with diverticulosis. We also aim to investigate the present state of diverticulosis in Japan.

Methods

We reviewed the medical records from 1990 to 2010 that included the data of consecutive 62,503 asymptomatic colonoscopy examinees from the general population in Japan. Most recent 3,327 examinees were analyzed with 16 background factors.

Results

Among the 62,503 subjects (47,325 men and 15,178 women; 52.1 ± 9.2 years old), diverticulosis was detected in 11,771 subjects (18.8%; 10,023 men and 1,748 women). The incidences of diverticulosis in 1990-2000 and 2001-2010 were respectively 13.0% (3,771 of 29,071) and 23.9% (8,000 of 33,432): the latter was much higher than the former in all age groups and for both genders. Considering the anatomical locations of colorectal diverticula, left-sided ones have markedly increased with age but not significantly changed with times. Univariate analyses of the 3,327 subjects showed significant association of diverticulosis with four basic factors (age, sex, body mass index, blood pressure), three life style-related factor (smoking, drinking, severe weight increase in adulthood), and two blood test values (triglyceride, HbA1c). The multiple logistic analysis calculating standardized coefficients (β) and odds ratio (OR) demonstrated that age (β = 0.217-0.674, OR = 1.24-1.96), male gender (β = 0.185, OR = 1.20), smoking (β = 0.142-0.200, OR = 1.15-1.22), severe weight increase in adulthood (β = 0.153, OR = 1.17), HbA1c (β = 0.136, OR = 1.15), drinking (β = 0.109, OR = 1.11), and serum triglyceride (β = 0.098, OR = 1.10) showed significantly positive association with diverticulosis whereas body mass index and blood pressure did not.

Conclusions

The large-scale data of asymptomatic colonoscopy examinees from the general population from 1990 to 2010 indicated that the prevalence of diverticulosis is still increasing in Japan. Age, male gender, smoking, severe weight increase in adulthood, serum HbA1c, drinking, and serum triglyceride showed significant positive association with diverticulosis.     

Cryopreservation of zebrafish (Danio rerio) primordial germ cells by vitrification of yolk-intact and yolk-depleted embryos using various cryoprotectant solutions

The aim of this study was to examine the effects of partial removal of yolk and cryoprotectant mixtures on the viability of cryopreserved primordial germ cells (PGCs) and elucidated the differentiation ability of cryopreserved PGCs in zebrafish. First, dechorionated yolk-intact and yolk-depleted (partially yolk removed) embryos, PGCs of which were labeled with green fluorescence protein (GFP), were vitrified after serial exposures to pretreatment solution (PS) and vitrification solution (VS) that contained ethylene glycol (EG), dimethyl sulfoxide (Me₂SO) or propylene glycol at 3 and 5 M, respectively. Although partial removal of yolk improved the viability of cryopreserved PGCs, numbers of PGCs with pseudopodial movement were limited (0–2.6 cells/embryo). Next, yolk-depleted embryos were cryopreserved using mixtures of two types of cryoprotectants. The maximum survival rate of PGCs (81%; 9.6 cells/embryo) was obtained from the yolk-depleted embryos vitrified using PS containing 2 M EG + 1 M Me₂SO and VS containing 3 M EG + 2 M Me₂SO and 56% (5.3 cells/embryo) of PGCs showed pseudopodial movement. Finally, PGCs recovered from yolk-depleted embryos (wild-type) that were vitrified under the optimum condition were transplanted individually into 236 sterilized recipient blastulae (recessive light-colored). Seven recipients matured and generated progeny with characteristics inherited from the PGC donor. In conclusion, the authors confirmed the beneficial effects of partial removal of yolk on the viability of cryopreserved PGCs and that the viability of the PGCs was improved by using PS and VS that contained two types of cryoprotectants, especially PS containing 2 M EG + 1 M Me₂SO and VS containing 3 M EG + 2 M Me₂SO, and that recovered PGCs retained ability to differentiate into functional gametes.