Conserved Loop Cysteines of Vitamin K Epoxide Reductase Complex Subunit 1-like 1 (VKORC1L1) Are Involved in Its Active Site Regeneration

Vitamin K epoxide reductase complex subunit 1 (VKORC1) reduces vitamin K epoxide in the vitamin K cycle for post-translational modification of proteins that are involved in a variety of biological functions. However, the physiological function of VKORC1-like 1 (VKORC1L1), a paralogous enzyme sharing about 50% protein identity with VKORC1, is unknown. Here we determined the structural and functional differences of these two enzymes using fluorescence protease protection (FPP) assay and an in vivo cell-based activity assay. We show that in vivo VKORC1L1 reduces vitamin K epoxide to support vitamin K-dependent carboxylation as efficiently as does VKORC1. However, FPP assays show that unlike VKORC1, VKORC1L1 is a four-transmembrane domain protein with both its termini located in the cytoplasm. Moreover, the conserved loop cysteines, which are not required for VKORC1 activity, are essential for VKORC1L1''s active site regeneration. Results from domain exchanges between VKORC1L1 and VKORC1 suggest that it is VKORC1L1''s overall structure that uniquely allows for active site regeneration by the conserved loop cysteines. Intermediate disulfide trapping results confirmed an intra-molecular electron transfer pathway for VKORC1L1''s active site reduction. Our results allow us to propose a concerted action of the four conserved cysteines of VKORC1L1 for active site regeneration; the second loop cysteine, Cys-58, attacks the active site disulfide, forming an intermediate disulfide with Cys-139; the first loop cysteine, Cys-50, attacks the intermediate disulfide resulting in active site reduction. The different membrane topologies and reaction mechanisms between VKORC1L1 and VKORC1 suggest that these two proteins might have different physiological functions.     

麦角固醇高产菌株的构建及其培养优化条件的研究

通过初筛、单倍体分离、诱变及酵母菌种间原生质体融合技术构建了2株麦角固醇产量明显提高的优良菌株YEF-21和YEF-29。并对YEF-21的培养优化条件进行了研究。结果表明在优化的实验条件下YEF-21的生物量及麦角固醇含量的综合值分别为原始亲株YE227和YE180的1.54和1.55倍。经遗传稳定性分析,没有发现标记基因的分离现象,从而证明获得的融合菌株是遗传稳定的,是具有一定实际应用价值的麦角固醇高产菌株。     

High-Glucose Inhibits Human Fibroblast Cell Migration in Wound Healing via Repression of bFGF-Regulating JNK Phosphorylation

One of the major symptoms of diabetes mellitus (DM) is delayed wound healing, which affects large populations of patients worldwide. However, the underlying mechanism behind this illness remains elusive. Skin wound healing requires a series of coordinated processes, including fibroblast cell proliferation and migration. Here, we simulate DM by application of high glucose (HG) in human foreskin primary fibroblast cells to analyze the molecular mechanism of DM effects on wound healing. The results indicate that HG, at a concentration of 30 mM, delay cell migration, but not cell proliferation. bFGF is known to promote cell migration that partially rescues HG effects on cell migration. Molecular and cell biology studies demonstrated that HG enhanced ROS production and repressed JNK phosphorylation, but did not affect Rac1 activity. JNK and Rac1 activation were known to be important for bFGF regulated cell migration. To further confirm DM effects on skin repair, a type 1 diabetic rat model was established, and we observed the efficacy of bFGF on both normal and diabetic rat skin repair. Furthermore, proteomic studies identified an increase of Annexin A2 protein nitration in HG-stressed fibroblasts and the nitration was protected by activation of bFGF signaling. Treatment with FGFR1 and JNK inhibitors delayed cell migration and increased Annexin A2 nitration levels, indicating that Annexin A2 nitration is modulated by bFGF signaling via activation of JNK. Together with these results, our data suggests that the HG-mediated delay of cell migration is linked to the inhibition of bFGF signaling, specifically through JNK suppression.     

The Systematic Classification of Gallbladder Stones

Background

To develop a method for systematic classification of gallbladder stones, analyze the clinical characteristics of each type of stone and provide a theoretical basis for the study of the formation mechanism of different types of gallbladder stones.

Methodology

A total of 807 consecutive patients with gallbladder stones were enrolled and their gallstones were studied. The material composition of gallbladder stones was analyzed using Fourier Transform Infrared spectroscopy and the distribution and microstructure of material components was observed with Scanning Electron Microscopy. The composition and distribution of elements were analyzed by an X-ray energy spectrometer. Gallbladder stones were classified accordingly, and then, gender, age, medical history and BMI of patients with each type of stone were analyzed.

Principal Findings

Gallbladder stones were classified into 8 types and more than ten subtypes, including cholesterol stones (297), pigment stones (217), calcium carbonate stones (139), phosphate stones (12), calcium stearate stones (9), protein stones (3), cystine stones (1) and mixed stones (129). Mixed stones were those stones with two or more than two kinds of material components and the content of each component was similar. A total of 11 subtypes of mixed stones were found in this study. Patients with cholesterol stones were mainly female between the ages of 30 and 50, with higher BMI and shorter medical history than patients with pigment stones (P<0.05), however, patients with pigment, calcium carbonate, phosphate stones were mainly male between the ages of 40 and 60.

Conclusion

The systematic classification of gallbladder stones indicates that different types of stones have different characteristics in terms of the microstructure, elemental composition and distribution, providing an important basis for the mechanistic study of gallbladder stones.     

α-Domain of human metallothionein IA can bind to metals in transgenic tobacco plants

We have introduced a genetically marked Dissociation transposable element (Ds ^HPT ) into tomato (Lycopersicon esculentum) by Agrobacterium tumefaciens-mediated transformation. Probes for the flanking regions of the T-DNA and transposed Ds ^HPT elements were obtained with the inverse polymerase chain reaction (IPCR) technique and used in RFLP linkage analyses. The RFLP map location of 11 T-DNAs carrying Ds ^HPT was determined. The T-DNAs are distributed on 7 of the 12 tomato chromosomes. To explore the feasibility of gene tagging strategies in tomato using Ds ^HPT , we examined the genomic distribution of Ds ^HPT receptor sites relative to the location of two different, but very closely linked, T-DNA insertion sites. After crosses with plants expressing Ac transposase, the hygromycin phosphotransferase (HPT) marker on the Ds element and the excision markers β-glucuronidase (GUS) and Basta resistance (BAR) facilitated the identification of plants bearing germinally transposed Ds ^HPT elements. RFLP mapping of 21 transposed Ds ^HPT elements originating from the two different T-DNA insertions revealed distinct patterns of reintegration sites.     

BRG1 variant rs1122608 on chromosome 19p13.2 confers protection against stroke and regulates expression of pre-mRNA-splicing factor SFRS3

A single nucleotide polymorphism (SNP) rs1122608 on chromosome 19p13.2 and in the BRG1/SMARCA4 gene was previously associated with coronary artery disease (CAD). CAD and ischemic stroke are both associated with atherosclerosis. Thus, we tested the hypothesis that rs1122608 is associated with ischemic stroke. Further studies were used to identify the most likely mechanism by which rs1122608 regulates atherosclerosis. For case–control association studies, two independent Chinese Han GeneID cohorts were used, including a Central cohort with 1,075 cases and 2,685 controls and the Northern cohort with 1,208 cases and 824 controls. eQTL and real-time RT-PCR analyses were used to identify the potential candidate gene(s) affected by rs1122608. The minor allele T of SNP rs1122608 showed significant association with a decreased risk of ischemic stroke in the Central GeneID cohort (adjusted P _adj = 2.1 × 10^?4, OR 0.61). The association was replicated in an independent Northern GeneID cohort (P _adj = 6.00 × 10^?3, OR 0.69). The association became more significant in the combined population (P _adj = 7.86 × 10^?5, OR 0.73). Allele T of SNP rs1122608 also showed significant association with a decreased total cholesterol level (P _adj = 0.013). Allele T of rs1122608 was associated with an increased expression level of SFRS3 encoding an mRNA splicing regulator, but not with the expression of BRG1/SMARCA4 or LDLR (located 36 kb from rs1122608). Increased expression of SFSR3 may decrease IL-1β expression and secretion, resulting in reduced risk of atherosclerosis and stroke. This is the first study that demonstrates that rs1122608 confers protection against ischemic stroke and implicates splicing factor SFSR3 in the disease process.     

LeETR1反义基因对番茄的遗传转化

从番茄果实中提取总RNA,根据GeneBank中LeETR1序列,设计合成特异性引物,利用RT-PCR及技术克隆了LeETR1基因3’端非编码区313bp的cDNA,经酶切图谱和序列分析鉴定无误后,反向插入到植物表达载体pPZP11A中,构建了表达LeETR1反义RNA的双元载体。经农杆菌途径转化番茄品种B1后,通过PCR检测从抗卡那霉素再生植株中筛选到13株阳性植株,Southern blot杂交确证反义基因已经整合到番茄染色体中。对果实乙烯释放的测定结果表明,转基因番茄乙烯释放高峰的出现比对照果实推迟10天,番茄红素的合成受到显抑制,果实不能形成正常的红色。推测LeETR1和番茄的成熟有着密切的关系。