Chemical dissection of the link between streptozotocin, O-GlcNAc, and pancreatic cell death

Streptozotocin is a natural product that selectively kills insulin-secreting beta cells, and is widely used to generate mouse models of diabetes or treat pancreatic tumors. Several studies suggest that streptozotocin toxicity stems from its N-nitrosourea moiety releasing nitric oxide and possessing DNA alkylating activity. However, it has also been proposed that streptozotocin induces apoptosis by inhibiting O-GlcNAcase, an enzyme that, together with O-GlcNAc transferase, is important for dynamic intracellular protein O-glycosylation. We have used galacto-streptozotocin to chemically dissect the link between O-GlcNAcase inhibition and apoptosis. Using X-ray crystallography, enzymology, and cell biological studies on an insulinoma cell line, we show that, whereas streptozotocin competitively inhibits O-GlcNAcase and induces apoptosis, its galacto-configured derivative no longer inhibits O-GlcNAcase, yet still induces apoptosis. This supports a general chemical poison mode of action for streptozotocin, suggesting the need for using more specific inhibitors to study protein O-GlcNAcylation.     

Structural and molecular evolutionary analysis of Agouti and Agouti-related proteins

Agouti (ASIP) and Agouti-related protein (AgRP) are endogenous antagonists of melanocortin receptors that play critical roles in the regulation of pigmentation and energy balance, respectively, and which arose from a common ancestral gene early in vertebrate evolution. The N-terminal domain of ASIP facilitates antagonism by binding to an accessory receptor, but here we show that the N-terminal domain of AgRP has the opposite effect and acts as a prodomain that negatively regulates antagonist function. Computational analysis reveals similar patterns of evolutionary constraint in the ASIP and AgRP C-terminal domains, but fundamental differences between the N-terminal domains. These studies shed light on the relationships between regulation of pigmentation and body weight, and they illustrate how evolutionary structure function analysis can reveal both unique and common mechanisms of action for paralogous gene products.     

Cloning, expression, and biochemical characterization of Streptomyces rubellomurinus genes required for biosynthesis of antimalarial compound FR900098

The antibiotics fosmidomycin and FR900098 are members of a unique class of phosphonic acid natural products that inhibit the nonmevalonate pathway for isoprenoid biosynthesis. Both are potent antibacterial and antimalarial compounds, but despite their efficacy, little is known regarding their biosynthesis. Here we report the identification of the Streptomyces rubellomurinus genes required for the biosynthesis of FR900098. Expression of these genes in Streptomyces lividans results in production of FR900098, demonstrating their role in synthesis of the antibiotic. Analysis of the putative gene products suggests that FR900098 is synthesized by metabolic reactions analogous to portions of the tricarboxylic acid cycle. These data greatly expand our knowledge of phosphonate biosynthesis and enable efforts to overproduce this highly useful therapeutic agent.     

Analysis of the ambruticin and jerangolid gene clusters of Sorangium cellulosum reveals unusual mechanisms of polyketide biosynthesis

Ambruticins and jerangolids are structurally related antifungal polyketides produced by Sorangium cellulosum strains. Comparative analysis of the gene clusters and characterization of compounds produced by gene knockout strains suggested hypothetical schemes for biosynthesis of these compounds. Polyketide synthase (PKS) architecture suggests that the pyran ring structure common to ambruticins and jerangolids forms by an intramolecular reaction on a PKS-bound intermediate. Disrupting ambM, encoding a discrete enzyme homologous to PKS C-methyltransferase domains, gave 15-desmethylambruticins. Thus, AmbM is required for C-methylation, but not pyran ring formation. Several steps in the post-PKS modification of ambruticin involve new enzymology. Remarkably, the methylcyclopropane ring and putative carbon atom excision during ambruticin biosynthesis apparently occur on the PKS assembly line. The mechanism probably involves a Favorskii rearrangement, but further work is required to elucidate these complex events.     

Covalent inhibitors of human monoacylglycerol lipase: ligand-assisted characterization of the catalytic site by mass spectrometry and mutational analysis

The active site of recombinant hexa-histidine-tagged human monoacylglycerol lipase (hMGL) is characterized by mass spectrometry using the inhibitors 5-((biphenyl-4-yl)methyl)-N,N-dimethyl-2H-tetrazole-2-carboxamide (AM6701), and N-arachidonylmaleimide (NAM) as probes. Carbamylation of Ser(129) by AM6701 in the putative hMGL catalytic triad demonstrates this residue's essential role in catalysis. Partial NAM alkylation of hMGL cysteine residues 215 and/or 249 was sufficient to achieve approximately 80% enzyme inhibition. Although Cys(215) and/or Cys(249) mutations to alanine(s) did not affect hMGL hydrolytic activity as compared with nonmutated hMGL, the C215A displayed heightened NAM sensitivity, whereas the C249A evidenced reduced NAM sensitivity. These data conclusively demonstrate a sulfhydryl-based mechanism for NAM inhibition of hMGL in which Cys(249) is of paramount importance. Identification of amino acids critical to the catalytic activity and pharmacological modulation of hMGL informs the design of selective MGL inhibitors as potential drugs.     

Synthesis of a hairpin pyrrole-imidazole polyamide conjugate containing a quinone methide precursor and vinyl linking group

Standard procedures for elaborating a quinone methide precursor for conjugation to a DNA ligand was not compatible with the presence of a vinyl group. Instead, an acrylate linker was attached by Heck coupling subsequent to o-substitution of the phenolic precursor. This transformation required protection of the phenolic group and use of ethyl acrylate rather than acrylic acid. The presence of the vinyl group also rendered the quinone methide precursor more labile to alkaline conditions than its equivalent saturated derivative and required mild conditions for coupling to the pyrrole-imidazole polyamide.     

Structural basis for discriminative regulation of gene expression by adenine- and guanine-sensing mRNAs

Metabolite-sensing mRNAs, or "riboswitches," specifically interact with small ligands and direct expression of the genes involved in their metabolism. Riboswitches contain sensing "aptamer" modules, capable of ligand-induced structural changes, and downstream regions, harboring expression-controlling elements. We report the crystal structures of the add A-riboswitch and xpt G-riboswitch aptamer modules that distinguish between bound adenine and guanine with exquisite specificity and modulate expression of two different sets of genes. The riboswitches form tuning fork-like architectures, in which the prongs are held in parallel through hairpin loop interactions, and the internal bubble zippers up to form the purine binding pocket. The bound purines are held by hydrogen bonding interactions involving conserved nucleotides along their entire periphery. Recognition specificity is associated with Watson-Crick pairing of the encapsulated adenine and guanine ligands with uridine and cytosine, respectively.     

Development of an efficient endothelial cell specific vector using promoter and 5' untranslated sequences from the human preproendothelin-1 gene

We report here, that a vector constructed based on ppET-1 gene promoter and 5' untranslated region induced a high level of gene expression in endothelial cells and the specificity is even further enhanced under hypoxia-mimic conditions due to a natural hypoxia responsive element within the promoter region. A naked DNA vector that confers endothelial cell specific gene expression as well as efficient levels of gene expression was constructed with an endothelial cell specific naked DNA vector, pETlong, by using the full length promoter of the preproendothelin-1 gene and the entire 5' untranslated region upstream from the start codon. Inclusion of the entire 5' untranslated region in pETlong increased gene expression 2.96 fold as compared with that from pETshort, which contains only the promoter sequences. Reporter gene expression from pETlong was 7.9 fold higher as compared with that from CMV-driven promoter based vector in calf pulmonary endothelial cells. However, in nonendothelial COS cells, luciferase activity from pETlong was only 0.3 fold as compared with that of CMV-based vector. Similar results were observed in other nonendothelial cells. These results demonstrate that the pETlong drives gene expression in endothelial cells with high efficacy and specificity. We have examined hypoxia responsiveness of pETlong as the promoter region of the preproendothelin-1 gene contains hypoxia responsive elements. The activity of the pETlong vector was increased 1.6 fold under hypoxia-mimic conditions using cobalt chloride. The high levels of hypoxia-inducible expression in endothelial cells relative to the low levels of background expression in other cells shows that pETlong could be a useful tool for vascular targeting of vascular disease and cancer gene therapy.     

A microfluidic processor for gene expression profiling of single human embryonic stem cells

The gene expression of human embryonic stem cells (hESC) is a critical aspect for understanding the normal and pathological development of human cells and tissues. Current bulk gene expression assays rely on RNA extracted from cell and tissue samples with various degree of cellular heterogeneity. These 'cell population averaging' data are difficult to interpret, especially for the purpose of understanding the regulatory relationship of genes in the earliest phases of development and differentiation of individual cells. Here, we report a microfluidic approach that can extract total mRNA from individual single-cells and synthesize cDNA on the same device with high mRNA-to-cDNA efficiency. This feature makes large-scale single-cell gene expression profiling possible. Using this microfluidic device, we measured the absolute numbers of mRNA molecules of three genes (B2M, Nodal and Fzd4) in a single hESC. Our results indicate that gene expression data measured from cDNA of a cell population is not a good representation of the expression levels in individual single cells. Within the G0/G1 phase pluripotent hESC population, some individual cells did not express all of the 3 interrogated genes in detectable levels. Consequently, the relative expression levels, which are broadly used in gene expression studies, are very different between measurements from population cDNA and single-cell cDNA. The results underscore the importance of discrete single-cell analysis, and the advantages of a microfluidic approach in stem cell gene expression studies.     

LEA proteins in higher plants: structure, function, gene expression and regulation

Late embryogenesis abundant (LEA) proteins are mainly low molecular weight (10–30 kDa) proteins, which are involved in protecting higher plants from damage caused by environmental stresses, especially drought (dehydration). These findings and the fact that the breeding of drought tolerant varieties would be of great value in agriculture, form the basis of search for anti-drought inducible genes and their characterization. LEA proteins are generally classified into six groups (families) according to their amino acid sequence and corresponding mRNA homology, which are basically localized in cytoplasm and nuclear region. LEA protein synthesis, expression and biological activities are regulated by many factors (e.g. developmental stages, hormones, ion change and dehydration), signal transduction pathways and lea genes. No tissue-specific lea gene expression has been considered as one main regulatory mechanism on the basis of extensive studies with the model plant, Arabidopsis thaliana. The study of the regulatory mechanism of lea gene expression is an important feature of modern plant molecular biology.     

人α-防御素基因克隆及其毕赤酵母表达的研究

为探讨人α-防御素(HNP2)酵母表达,根据HNP2氨基酸序列和巴斯德毕赤酵母密码偏爱性,设计HNP2基因序列片段,构建pGAPZαA-HNP2酵母表达载体,转化E.coliJM109,扩增重组子,经限制性核酸内切酶BlnI线性化,转毕赤酵母.PCR扩增挑选阳性转化子,SDS-PAGE分析酵母表达,利用阳离子交换树脂纯化HNP2,开展抗菌试验.结果显示转入巴斯德毕赤酵母HNP2基因得到表达,表达的HNP2具有显著的抗菌作用.     

Molecular cloning and transgenic expression of a synthetic human erythropoietin gene in tobacco

Erythropoietin (EPO) is a hormone belonging to a group of hematopoietic growth factors that control the proliferation and differentiation of bone marrow cells. It induces the production of erythrocytes, thereby increasing the amount of circulating hemoglobin and oxygen. Previous attempts to transgenically express human EPO in plants failed to succeed because the plants exhibited abnormal morphology and infertility. In the present work, we describe the generation of fertile transgenic tobacco plants able to express a synthetic version of human EPO. A 582-bp fragment of the human EPO gene was synthesized using a PCR-based method and ligated into pCR-Blunt. After sequencing, the human EPO fragment was transferred to pWUbi.tm1 and the expression cassette was then transferred to the binary vector pWBVec4a. After Agrobacterium-mediated transformation of Nicotiana tabacum SR1 plants, integration of the transgene into T₀ and T₁ plant genomes was confirmed by PCR. The human EPO gene was found to be expressed in tobacco leaves at the mRNA and protein levels. Self-crossing allowed us to obtain T₁ plants exhibiting Mendelian segregation of the transgene. None of the plants presented any kind of malformation or deformity.     

Development of a sensitive and specific liquid chromatography/mass spectrometry method for the determination of tenofovir in human plasma

A sensitive and specific method for the quantitation of tenofovir (TFV) in human plasma by liquid chromatography/electrospray ionization mass spectrometry was developed and validated. Plasma samples were prepared by solid-phase extraction performed on Waters Oasis cation-exchange cartridges (30 mg). Chromatographic separation was performed isocratically on a reversed-phase Waters Atlantis dC18 column (2.0x100 mm, 3 microm). The mobile phase consisted of a hydroxylamine/acetic acid buffer (pH 6.75) and methanol (93:7, v/v). The acquisition was performed in selected ion monitoring mode for the protonated molecular ions [M+H]+ of m/z 288.2 for TFV and 212.2 for the internal standard, zalcitibine. The method was fully validated to determine its specificity, recovery, linearity and sensitivity, accuracy and precision. The analytical range was set at 1-750 ng/mL using a 200 microL plasma sample, with a mean coefficient of determination (r2) of 0.9969. The mean accuracies for the calibration standards ranged from -5.0 to 4.3%, while the precisions were within 1.2 and 6.4%. Intra-assay and inter-assay mean accuracies for three quality control concentrations (2, 60, and 600 ng/mL) ranged from -6.1 to 10.7%, while the precisions were within 1.3 and 9.1%. TFV was shown to be stable under normal storage and assay conditions; no degradation was seen when stored at -20 degrees C or -80 degrees C for up to 6 months, and after 16 h at room temperature in the injection matrix. The present method provides an accurate, precise, and sensitive tool for TFV quantitation and was successfully applied to an external proficiency-testing program and pharmacokinetic analysis.     

Development and characterization of new polyclonal antibodies specific for three polychlorinated biphenyls

Three polychlorinated biphenyls(PCBs)congeners and their corresponding haptens bearing four carbon length carboxylic groups that can be linked to a protein for raising antibodies were synthesized.The three resultant immunogens were fabricated and used to stimulate immune responses in rabbits to survey the characteristics of the haptens.Three of the resultant polyclonal antibodies(Pabs)were obtained.The antiserum exhibited relatively high antibody titres(1:32-64)in double agar diffusion.◎2008 Hui Sheng Zhuang.Published by Elsevier B.V.on behalf of Chinese Chemical Society.All rights reserved.     

The electrical properties of the nuclear envelope,and their possible role in the regulation of eukaryotic gene expression

The regulation of eukaryotic gene expression is discussed from a bioelectrochemical perspective. The ways in which the nuclear envelope/membranes can regulate ion fluxes and generate electrical potentials and fields at the nuclear periphery are considered. Evidence for close association of DNA and proteins with the nuclear envelope is also presented. Possible functional consequences of this association are outlined in view of the polyelectrolyte nature of DNA and proteins, and the behavior of such biopolymers in the presence of the strong electric fields and ion microenvironments which can be generated at the perimeter of the nucleoplasm.     

Sensitive and specific LC-MS assay for quantification of digoxin in human plasma and urine

Digoxin, a commonly prescribed cardiac glycoside with a narrow therapeutic window, is routinely used in pharmacokinetic studies to assess the in vivo activity of the drug efflux pump P-glycoprotein. To minimize adverse events, a sub-therapeutic dose of digoxin is usually administered, producing low plasma concentrations requiring a sensitive detection technique. Commonly available immunoassay techniques do not provide the required sensitivity to measure these low plasma concentrations and are potentially non-specific in certain subject populations. Previously published mass spectrometric techniques require either large plasma volumes or a tandem mass spectrometer. To overcome these challenges we have developed a sensitive and specific LC-MS method for the quantification of digoxin in small volumes of human plasma and urine. Plasma (1 mL) was extracted with methyl t-butyl ether under basic conditions followed by LC-MS detection of the sodium adducts of digoxin (803.4 m/z) and digitoxin (787.4 m/z, internal standard). Linearity and accuracy were demonstrated across a wide range of digoxin plasma concentration (0.05-1.5 ng/mL). This specific, sensitive, validated digoxin LC-MS assay can be used to quantify sub-therapeutic digoxin plasma concentrations in men and women (pregnant and non-pregnant).     

Development of a drinking water regulation for perchlorate in California

Perchlorate is an environmental contaminant often associated with military installations and rocket propellant manufacture and testing facilities across the U.S. Highly water soluble, perchlorate has been found by federal and state agencies at almost 400 sites within the U.S. in groundwater, surface water, soil or public drinking water. There is no federal drinking water standard for perchlorate, but it is on the drinking water Contaminant Candidate List, and falls under the Unregulated Contaminant Monitoring Rule (UCMR) for which monitoring is required. The recent National Academy of Science (NAS) report on the potential health effects of perchlorate recommended a perchlorate reference dose of 0.0007 mg/kg of body weight which would be equivalent to a drinking water concentration of 24.5 μg/L.In California, approximately 395 wells in 96 water systems have been shown to contain perchlorate, and about 90% of these are located in Southern California. Water taken from the Colorado River, a major surface water supply to Southern California, has had reported detections of perchlorate ranging from non-detect to 9 μg/L. California has established a Public Health Goal (PHG) of 6 μg/L for perchlorate, and a proposed drinking water regulation is imminent. This review details the regulatory process involved with particular attention given to the occurrence of perchlorate in California drinking water sources and analytical methodology utilized.