Isolation of two saxitoxin-sensitive sodium channel subtypes from rat brain with distinct biochemical and functional properties

Summary Two different³H-saxitoxin-binding proteins, with distinct biochemical and functional properties, were isolated from rat brain using a combination of anion exchange and lectin affinity chromatography as well as high resolution size exclusion and anion exchange HPLC. The alpha subunits of the binding proteins had different apparent molecular weights on SDS-PAGE (Type A: 235,000; Type B: 260,000). When reconstituted into planar lipid bilayers, the two saxitoxin-binding proteins formed sodium channels with different apparent single-channel conductances in the presence of batrachotoxin (Type A: 22 pS; Type B: 12 pS) and veratridine (Type A: 9 pS; Type B: 5 pS). The subtypes were further distinguished by scorpion (Leiurus quinquestriatus) venom which had different effects on single-channel conductance and gating of veratridine-activated Type A and Type B channels. Scorpion venom caused a 19% increase in single-channel conductance of Type A channels and a 35-mV hyperpolarizing shift in activation. Scropion venom double the single-channel conductance of Type B channels and shifted activation by at least 85 mV.     

Endogenously generated DNA nucleobase modifications source,and significance as possible biomarkers of malignant transformation risk,and role in anticancer therapy

The DNA of all living cells undergoes continuous structural and chemical alteration, which may be derived from exogenous sources, or endogenous, metabolic pathways, such as cellular respiration, replication and DNA demethylation. It has been estimated that approximately 70,000 DNA lesions may be generated per day in a single cell, and this has been linked to a wide variety of diseases, including cancer. However, it is puzzling why potentially mutagenic DNA modifications, occurring at a similar level in different organs/tissue, may lead to organ/tissue specific cancers, or indeed non-malignant disease – what is the basis for this differential response? We suggest that it is perhaps the precise location of damage, within the genome, that is a key factor. Finally, we draw attention to the requirement for reliable methods for identification and quantification of DNA adducts/modifications, and stress the need for these assays to be fully validated. Once these prerequisites are satisfied, measurement of DNA modifications may be helpful as a clinical parameter for treatment monitoring, risk group identification and development of prevention strategies.     

Interaction between ZBP-89 and p53 mutants and its contribution to effects of HDACi on hepatocellular carcinoma

Normal hematopoiesis is suppressed during the development of leukemia. In the T-ALL leukemia mouse model described in our recent study (Hu X, et al. Blood 2009), the impacts of leukemic environment on normal hematopoietic stem cells (HSCs) and hematopoietic progenitor cells (HPCs) were distinct, in that normal HSCs were preserved in part because of increased mitotic quiescence of HSCs and resulting exhaustion of HPCs proliferation. Stem cell factor (SCF) secreted by leukemic cells in Nalm6 B-ALL model was previously suggested to force normal HSCs/HPCs out of their bone marrow niches and allow leukemic cells to occupy the niches (Colmone A, et al. Science 2008). Here we found that stem cell factor (SCF) expression in PB and BM of T-ALL model was increased, but SCF mRNA and protein levels in normal hematopoietic cells were higher than those in leukemia cells, which suggested that upregulated SCF was mainly contributed by non-leukemic cells in response to the leukemia development. To further elucidate the molecular mechanisms, microarray analysis was conducted on normal HSCs in this model and verified by real-time RT-PCR. The expression of Hes1 and its downstream target p21 were elevated in normal HSCs, whereas their expression showed no significant alteration in HPCs. Interestingly, although overexpression of Hes1 by retroviral infection inhibited the in vitro colony formation of normal hematopoietic cells, in vivo results demonstrated that normal Lin^- cells and HSPCs were better preserved when normal Lin^- cells with Hes1 overexpression were co-transplanted with T-ALL leukemia cells. Our results suggested that the differential expression of Hes1 between HSCs and HPCs resulted in the distinct responses of these cells to the leukemic condition, and that overexpression of Hes1 could enhance normal HSPCs in the leukemic environment.     

Flt3配体的同源二聚体构建及其生物学特性

Flt3配体 (FL)是一种早期造血生长因子 ,与相应受体结合后 ,驱动受体二聚化 ,从而启动细胞内信号转导。体内 ,可溶性的FL主要以单体形式存在。将 2个相同的编码可溶性FL的基因片段 ,通过连接序列以首尾相连的方式构建成FL FL基因 ,并在毕氏酵母 (Pichiapastoris)中表达。SDS PAGE试验表明 ,FL FL融合蛋白 (rFL FL)分子量约为 4 3kD ,与预期相符 ,并为Western印迹试验所证实。骨髓祖细胞体外集落形成试验提示 ,rFL FL具有更强的生物学活性 ,约是单体FL(rhFL)的 10倍之多。另外 ,FL FL融合蛋白还能促进和延长饥饿的Raji细胞存活 ,与rhFL相比 ,也表现出较强的生物学功能。     

Reversible binding of zinc in Plasmodium falciparum Sir2: Structure and activity of the apoenzyme

Reversible zinc chelation via thiol groups of cysteines leading to modulation of activity in redox regulated proteins forms a basis for switching on–off of various biochemical processes. Silent information regulator 2 (Sir2), a NAD⁺ dependent deacetylase, contains a non-catalytic zinc ion coordinated by thiol groups of cysteines. Using Plasmodium falciparum Sir2 (PfSir2), we have examined the effect of zinc removal on the structure and activity of this enzyme. Our studies show that the enzyme with high affinity for zinc exhibits partial collapse of structure upon removal of the metal ion. Zinc reconstitution of apo PfSir2 led to recovery of both structure and activity highlighting the reversibility of the process.     

A proper transmembrane Ca2+ gradient is essential for the stimulation of adenylate cyclase activity by stimulatory GTP-binding protein

Stimulatory GTP-binding Protein (Gs) and adenylate cyclase prepared from bovine brain cortices were co-reconstituted into asolectin vesicles with or without 1000-fold transmembrane Ca²⁺ gradient. The results showed that both basal activity and Gs-stimulated activity of adenylate cyclase were highest in proteoliposomes with a transmembrane Ca²⁺ gradient similar to physiological condition (1 M Ca²⁺ outside and 1 mM Ca²⁺ inside) and lowest when the transmembrane Ca²⁺ gradient was in the inverse direction. Such a difference could be diminished following dissipation of the transmembrane Ca²⁺ gradient by A23187. Comparable conformational changes of Gs in proteoliposomes were also observed when Gs was labeled with the fluorescence probe, acrylodan. These results may indicate that a proper transmembrane Ca²⁺ gradient is essential not only for higher adenylate cyclase activity but also for its stimulation by Gs.     

Cloning of three A-type cytochromes P450, CYP71E1, CYP98, and CYP99 from Sorghum bicolor (L.) Moench by a PCR approach and identification by expression in Escherichia coli of CYP71E1 as a multifunctional cytochrome P450 in the biosynthesis of the cyanogenic glucoside dhurrin

A cDNA encoding the multifunctional cytochrome P450, CYP71E1, involved in the biosynthesis of the cyanogenic glucoside dhurrin from Sorghum bicolor (L.) Moench was isolated. A PCR approach based on three consensus sequences of A-type cytochromes P450 – (V/I)KEX(L/F)R, FXPERF, and PFGXGRRXCXG – was applied. Three novel cytochromes P450 (CYP71E1, CYP98, and CYP99) in addition to a PCR fragment encoding sorghum cinnamic acid 4-hydroxylase were obtained.Reconstitution experiments with recombinant CYP71E1 heterologously expressed in Escherichia coli and sorghum NADPH–cytochrome P450–reductase in L--dilaurylphosphatidyl choline micelles identified CYP71E1 as the cytochrome P450 that catalyses the conversion of p-hydroxyphenylacetaldoxime to p-hydroxymandelonitrile in dhurrin biosynthesis. In accordance to the proposed pathway for dhurrin biosynthesis CYP71E1 catalyses the dehydration of the oxime to the corresponding nitrile, followed by a C-hydroxylation of the nitrile to produce p-hydroxymandelonitrile. In vivo administration of oxime to E. coli cells results in the accumulation of the nitrile, which indicates that the flavodoxin/flavodoxin reductase system in E. coli is only able to support CYP71E1 in the dehydration reaction, and not in the subsequent C-hydroxylation reaction.CYP79 catalyses the conversion of tyrosine to p-hydroxyphenylacetaldoxime, the first committed step in the biosynthesis of the cyanogenic glucoside dhurrin. Reconstitution of both CYP79 and CYP71E1 in combination with sorghum NADPH-cytochrome P450–reductase resulted in the conversion of tyrosine to p-hydroxymandelonitrile, i.e. the membranous part of the biosynthetic pathway of the cyanogenic glucoside dhurrin. Isolation of the cDNA for CYP71E1 together with the previously isolated cDNA for CYP79 provide important tools necessary for tissue-specific regulation of cyanogenic glucoside levels in plants to optimize food safety and pest resistance.     

转录因子与造血调控

血细胞生成是一个极其复杂的过程,转录因子在这个过程中起到了重要的调控作用。而转录因子的表达具有阶段和细胞系特异性。在造血干细胞的增殖和分化、髓系和淋巴系细胞等的成熟过程中,众多转录因子既相互作用又表现出各自的特异性。转录因子数目较多,该文仅就一些与造血细胞分化、成熟相关的转录因子近年来的研究进展作一介绍。