Structural basis of sugar-recognizing ubiquitin ligase

SCF(Fbs1) is a ubiquitin ligase that functions in the endoplasmic reticulum (ER)-associated degradation pathway. Fbs1/Fbx2, a member of the F-box proteins, recognizes high-mannose oligosaccharides. Efficient binding to an N-glycan requires di-N-acetylchitobiose (chitobiose). Here we report the crystal structures of the sugar-binding domain (SBD) of Fbs1 alone and in complex with chitobiose. The SBD is composed of a ten-stranded antiparallel beta-sandwich. The structure of the SBD-chitobiose complex includes hydrogen bonds between Fbs1 and chitobiose and insertion of the methyl group of chitobiose into a small hydrophobic pocket of Fbs1. Moreover, NMR spectroscopy has demonstrated that the amino acid residues adjoining the chitobiose-binding site interact with the outer branches of the carbohydrate moiety. Considering that the innermost chitobiose moieties in N-glycans are usually involved in intramolecular interactions with the polypeptide moieties, we propose that Fbs1 interacts with the chitobiose in unfolded N-glycoprotein, pointing the protein moiety toward E2 for ubiquitination.     

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.     

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.     

Production of transgenic miniature pigs by pronuclear microinjection

Miniature pig is an attractive animal for a wide range of research fields, such as medicine and pharmacology, because of its small size, the possibility of breeding it under minimum environmental controls and the physiology that is potentially similar to that of human. Although transgenic technology is useful for the analysis of gene function and for the development of model animals for various diseases, there have not yet been any reports on producing transgenic miniature pig. This study is the first successful report concerning the production of transgenic miniature pig by pronuclear microinjection. The huntingtin gene cloned from miniature pig, which is a homologue of candidate gene for Huntington's disease, connected with rat neuron-specific enolase promoter region, was injected into a pronucleus of fertilized eggs with micromanipulator. The eggs were transferred into the oviduct of recipient miniature pigs, whose estrus cycles were previously synchronized with a progesterone analogue. A total of 402 injected eggs from 171 donors were transferred to 23 synchronized recipients. Sixteen of them maintained pregnancy and delivered 65 young, and one resulted in abortion. Five of the 68 offspring (three of which were aborted) were determined to have transgene by PCR and Southern analysis. The overall rate of transgenic production was 1.24% (transgenic/injected eggs). This study provides the first success and useful information regarding production of transgenic miniature pig for biomedical research.     

Interleukin-12 genetic administration suppressed metastatic liver tumor unsusceptible to CTL

A cytokine gene therapy approach was conducted against metastatic lesions of cytotoxic T lymphocyte (CTL)-unsusceptible tumor in mice. The EBV-based and conventional plasmid vectors that encode murine interleukin-12 (IL-12) gene (pGEG.mIL-12 and pG.mIL-12, respectively) were intravenously transfected into the mice that had received a subcutaneous inoculation of M5076 sarcoma cells. The pGEG.mIL-12 transfection drastically suppressed the subcutaneous as well as hepatic metastatic tumors, resulting in significant prolongation of survival period of the animals. Although single administration with pG.mIL-12 was not effective, repetitive transfection with the plasmid significantly prolonged the longevity of the mice-bearing the metastatic liver tumors. Multiple transfection with either pGEG.mIL-12 or pG.mIL-12 also suppressed peritoneal carcinomatosis in mice that had been injected with M5076 cells into the peritoneal cavity. It was suggested that a high level IL-12 production elicited by the intravenous delivery of the cytokine gene may be quite effective in inhibiting metastatic and CTL-unsusceptible neoplasms.     

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.     

诱导型一氧化氮合酶对内毒素休克小肠微循环的影响

采用静脉注射脂多糖(1ipopolysaccharide,LPS)的方法建立小鼠内毒素休克模型,探讨内毒素休克时小肠微循环的变化以及诱导型一氧化氮合酶(iNOS)对小肠微循环的影响。实验过程中连续监测小鼠平均动脉血压(mean afterial pressure,MAP)变化情况。利用FTTC标记红细胞和活体显微镜方法直接观察并计算小鼠小肠绒毛尖端小动脉和毛细血管内红细胞的流速和流量,并观察敲除小鼠iNOS基因和选择性iNOS抑制剂S-methylthiourea sulfate(SMT)对实验过程中小肠微循环的影响。结果显示,对于野生型小鼠,应用SMT处理和敲除iNOS基因对基线的MAP、小肠绒毛尖端小动脉和毛细血管的红细胞流速和流量没有显著性差别。给予LPS后,小鼠的MAP进行性下降。给予LPS前,应用SMT和敲除小鼠iNOS基因可以显著提高MAP：给予LPS后,小鼠小肠绒毛尖端小动脉和毛细血管内红细胞流速和流量显著下降。给予LPS前,应用SMT和敲除小鼠iNOS基因可以显著提高小肠绒毛尖端小动脉和毛细血管的红细胞流速和流量。结果表明,iNOS在内毒素休克小肠微循环衰竭的过程中发挥重要作用。一能性     

Phylogenetic analysis of long-chain hydrocarbon-degrading bacteria and evaluation of their hydrocarbon-degradation by the 2,6-DCPIP assay

Thirty-six bacteria that degraded long-chain hydrocarbons were isolated from natural environments using long-chain hydrocarbons (waste car engine oil, base oil or the c-alkane fraction of base oil) as the sole carbon and energy source. A phylogenetic tree of the isolates constructed using their 16S rDNA sequences revealed that the isolates were divided into six genera plus one family (Acinetobacter, Rhodococcus, Gordonia, Pseudomonas, Ralstonia, Bacillus and Alcaligenaceae, respectively). Furthermore, most of the isolates (27 of 36) were classified into the genera Acinetobacter, Rhodococcus or Gordonia. The hydrocarbon-degradation similarity in each strain was confirmed by the 2,6-dichlorophenol indophenol (2,6-DCPIP) assay. Isolates belonging to the genus Acinetobacter degraded long-chain normal alkanes (n-alkanes) but did not degrade short-chain n-alkanes or cyclic alkanes (c-alkanes), while isolates belonging to the genera Rhodococcus and Gordonia degraded both long-chain n-alkanes and c-alkanes.