Expression of a novel gene, <Emphasis Type="Italic">gluP</Emphasis>, is essential for normal <Emphasis Type="Italic">Bacillus subtilis</Emphasis> cell division and contributes to glucose export

Background  

The Bacillus subtilis glucokinase operon was predicted to be comprised of the genes, yqgP (now named gluP), yqgQ, and glcK. We have previously established a role for glcK in glucose metabolism. In the absence of enzymes that phosphorylate glucose, such as GlcK and/or enzyme II^Glc, accumulated cytoplasmic glucose can be transported out of the cell. Genes within the glucokinase operon were not previously known to play a role in glucose transport. Here we describe the expression of gluP and its function in glucose transport.     

Discovery of antichagasic inhibitors by high-throughput screening with Trypanosoma cruzi glucokinase

A high-throughput screening (HTS) campaign was carried out for Trypanosoma cruzi glucokinase (TcGlcK), a potential drug-target of the pathogenic protozoan parasite. Glycolysis and the pentose phosphate pathway (PPP) are important metabolic pathways for T. cruzi and the inhibition of the glucose kinases (i.e. glucokinase and hexokinase) may be a strategic approach for drug discovery. Glucose kinases phosphorylate d-glucose with co-substrate ATP to yield G6P, and moreover, the produced G6P enters both pathways for catabolism. The TcGlcK – HTS campaign revealed 25 novel enzyme inhibitors that were distributed in nine chemical classes and were discovered from a primary screen of 13,040 compounds. Thirteen of these compounds were found to have low micromolar IC₅₀ enzyme – inhibition values; strikingly, four of those compounds exhibited low toxicity towards NIH-3T3 murine host cells and notable in vitro trypanocidal activity. These compounds were of three chemical classes: (a) the 3-nitro-2-phenyl-2H-chromene scaffold, (b) the N-phenyl-benzenesulfonamide scaffold, and (c) the gossypol scaffold. Two compounds from the 3-nitro-2-phenyl-2H-chromene scaffold were determined to be hit-to-lead candidates that can proceed further down the early-stage drug discovery process.     

<Emphasis Type="Italic">PGM2</Emphasis> overexpression improves anaerobic galactose fermentation in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

Background  

In Saccharomyces cerevisiae galactose is initially metabolized through the Leloir pathway after which glucose 6-phosphate enters glycolysis. Galactose is controlled both by glucose repression and by galactose induction. The gene PGM2 encodes the last enzyme of the Leloir pathway, phosphoglucomutase 2 (Pgm2p), which catalyses the reversible conversion of glucose 1-phosphate to glucose 6-phosphate. Overexpression of PGM2 has previously been shown to enhance aerobic growth of S. cerevisiae in galactose medium.     

Effect of <Emphasis Type="Italic">HXT</Emphasis> 1 and <Emphasis Type="Italic">HXT</Emphasis> 7 hexose transporter overexpression on wild-type and lactic acid producing <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> cells

Background  

Since about three decades, Saccharomyces cerevisiae can be engineered to efficiently produce proteins and metabolites. Even recognizing that in baker's yeast one determining step for the glucose consumption rate is the sugar uptake, this fact has never been conceived to improve the metabolite(s) productivity.     

Mining metadata from unidentified ITS sequences in GenBank: A case study in <Emphasis Type="Italic">Inocybe</Emphasis> (<Emphasis Type="Italic">Basidiomycota</Emphasis>)

Background  

The lack of reference sequences from well-identified mycorrhizal fungi often poses a challenge to the inference of taxonomic affiliation of sequences from environmental samples, and many environmental sequences are thus left unidentified. Such unidentified sequences belonging to the widely distributed ectomycorrhizal fungal genus Inocybe (Basidiomycota) were retrieved from GenBank and divided into species that were identified in a phylogenetic context using a reference dataset from an ongoing study of the genus. The sequence metadata of the unidentified Inocybe sequences stored in GenBank, as well as data from the corresponding original papers, were compiled and used to explore the ecology and distribution of the genus. In addition, the relative occurrence of Inocybe was contrasted to that of other mycorrhizal genera.     

Phylogenetic analysis of bacterial and archaeal <Emphasis Type="Italic">arsC</Emphasis> gene sequences suggests an ancient,common origin for arsenate reductase

Background  

The ars gene system provides arsenic resistance for a variety of microorganisms and can be chromosomal or plasmid-borne. The arsC gene, which codes for an arsenate reductase is essential for arsenate resistance and transforms arsenate into arsenite, which is extruded from the cell. A survey of GenBank shows that arsC appears to be phylogenetically widespread both in organisms with known arsenic resistance and those organisms that have been sequenced as part of whole genome projects.     

Hepatic glucokinase promoter polymorphism is associated with hepatic insulin resistance in Asian Indians.

Background  

The role of glucokinase (GCK) in the pathogenesis of maturity-onset diabetes of the young is well established. However, its role in the common form of type 2 diabetes is far from convincing. We investigated the role of the G-to-A polymorphism in the hepatic GCK promoter on insulin sensitivity and beta cell function in 63 normotensive Asian Indians with normal glucose tolerance. As proposed by Matsuda and DeFronzo, hepatic insulin sensitivity (ISI_H) and total body insulin sensitivity (ISI_M) were estimated from the oral glucose tolerance test. Beta cell function was estimated using %B from the Homeostasis Model Assessment and insulingenic index (dI/dG).     

Glycolysis and the regulation of glucose transport in <Emphasis Type="Italic">Lactococcus lactis</Emphasis> spp. <Emphasis Type="Italic">lactis</Emphasis> in batch and fed-batch culture

Background  

Despite the fact that many reports deal with glycolysis in Lactococcus lactis, there is not much information on the regulation of uptake of glucose itself. The aim of the present work was to investigate the effect of the glucose level on its specific uptake rate.     

Heterologous expression of glucose oxidase in the yeast <Emphasis Type="Italic">Kluyveromyces marxianus</Emphasis>

Background  

In spite of its advantageous physiological properties for bioprocess applications, the use of the yeast Kluyveromyces marxianus as a host for heterologous protein production has been very limited, in constrast to its close relative Kluyveromyces lactis. In the present work, the model protein glucose oxidase (GOX) from Aspergillus niger was cloned into K. marxianus CBS 6556 and into K. lactis CBS 2359 using three different expression systems. We aimed at verifying how each expression system would affect protein expression, secretion/localization, post-translational modification, and biochemical properties.     

Improved vanillin production in baker's yeast through <Emphasis Type="Italic">in silico</Emphasis> design

Background  

Vanillin is one of the most widely used flavouring agents, originally obtained from cured seed pods of the vanilla orchid Vanilla planifolia. Currently vanillin is mostly produced via chemical synthesis. A de novo synthetic pathway for heterologous vanillin production from glucose has recently been implemented in baker's yeast, Saccharamyces cerevisiae. In this study we aimed at engineering this vanillin cell factory towards improved productivity and thereby at developing an attractive alternative to chemical synthesis.     

Glucose-free fructose production from Jerusalem artichoke using a recombinant inulinase-secreting <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> strain

Purpose of work  

Using inulin (polyfructose) obtained from Jerusalen artichokes, we have produced fructose free of residual glucose using a recombinant inulinase-secreting strain of Saccharomyces cerevisiae in a one-step fermentation of Jerusalem artichoke tubers.     

The VirusBanker database uses a Java program to allow flexible searching through <Emphasis Type="Italic">Bunyaviridae</Emphasis> sequences

Background  

Viruses of the Bunyaviridae have segmented negative-stranded RNA genomes and several of them cause significant disease. Many partial sequences have been obtained from the segments so that GenBank searches give complex results. Sequence databases usually use HTML pages to mediate remote sorting, but this approach can be limiting and may discourage a user from exploring a database.     

Molecular cloning,genomic characterization and over-expression of a novel gene, <Emphasis Type="Italic">XRRA1</Emphasis>, identified from human colorectal cancer cell HCT116<Superscript>Clone2_XRR</Superscript> and macaque testis

Background  

As part of our investigation into the genetic basis of tumor cell radioresponse, we have isolated several clones with a wide range of responses to X-radiation (XR) from an unirradiated human colorectal tumor cell line, HCT116. Using human cDNA microarrays, we recently identified a novel gene that was down-regulated by two-fold in an XR-resistant cell clone, HCT116^Clone2_XRR. We have named this gene as X-ray radiation resistance associated 1 (XRRA1) (GenBank BK000541). Here, we present the first report on the molecular cloning, genomic characterization and over-expression of the XRRA1 gene.     

Bisphenol A Impairs Hepatic Glucose Sensing in C57BL/6 Male Mice

Aims/Hypothesis

Glucose sensing (eg. glucokinase activity) becomes impaired in the development of type 2 diabetes, the etiology of which is unclear. Estrogen can stimulate glucokinase activity, whereas the pervasive environmental pollutant bisphenol A (BPA) can inhibit estrogen action, hence we aimed to determine the effect of BPA on glucokinase activity directly.

Methods

To evaluate a potential acute effect on hepatic glucokinase activity, BPA in water (n = 5) vs. water alone (n = 5) was administered at the EPA’s purported “safe dose” (50 µg/kg) by gavage to lean 6-month old male C57BL/6 mice. Two hours later, animals were euthanized and hepatic glucokinase activity measured over glucose levels from 1–20 mmol/l in liver homogenate. To determine the effect of chronic BPA exposure on hepatic glucokinase activity, lean 6-month old male C57BL/6 mice were provided with water (n = 15) or water with 1.75 mM BPA (∼50 µg/kg/day; n = 14) for 2 weeks. Following the 2-week exposure, animals were euthanized and glucokinase activity measured as above.

Results

Hepatic glucokinase activity was signficantly suppressed after 2 hours in animals given an oral BPA bolus compared to those who received only water (p = 0.002–0.029 at glucose 5–20 mmol/l; overall treatment effect p<0.001). Exposure to BPA over 2 weeks also suppressed hepatic glucokinase activity in exposed vs. unexposed mice (overall treatment effect, p = 0.003). In both experiments, the Hill coefficient was higher and Vmax lower in mice treated with BPA.

Conclusions/Interpretation

Both acute and chronic exposure to BPA significantly impair hepatic glucokinase activity and function. These findings identify a potential mechanism for how BPA may increase risk for diabetes.     

A SSR-based composite genetic linkage map for the cultivated peanut (<Emphasis Type="Italic">Arachis hypogaea</Emphasis> L.) genome

Background  

The construction of genetic linkage maps for cultivated peanut (Arachis hypogaea L.) has and continues to be an important research goal to facilitate quantitative trait locus (QTL) analysis and gene tagging for use in a marker-assisted selection in breeding. Even though a few maps have been developed, they were constructed using diploid or interspecific tetraploid populations. The most recently published intra-specific map was constructed from the cross of cultivated peanuts, in which only 135 simple sequence repeat (SSR) markers were sparsely populated in 22 linkage groups. The more detailed linkage map with sufficient markers is necessary to be feasible for QTL identification and marker-assisted selection. The objective of this study was to construct a genetic linkage map of cultivated peanut using simple sequence repeat (SSR) markers derived primarily from peanut genomic sequences, expressed sequence tags (ESTs), and by "data mining" sequences released in GenBank.     

Combinatorial modulation of <Emphasis Type="Italic">galP</Emphasis> and <Emphasis Type="Italic">glk</Emphasis> gene expression for improved alternative glucose utilization

Phosphoenolpyruvate (PEP) is an important precursor for anaerobic production of succinate and malate. Although inactivating PEP/carbohydrate phosphotransferase systems (PTS) could increase PEP supply, the resulting strain had a low glucose utilization rate. In order to improve anaerobic glucose utilization rate for efficient production of succinate and malate, combinatorial modulation of galactose permease (galP) and glucokinase (glk) gene expression was carried out in chromosome of an Escherichia coli strain with inactivated PTS. Libraries of artificial regulatory parts, including promoter and messenger RNA stabilizing region (mRS), were firstly constructed in front of β-galactosidase gene (lacZ) in E. coli chromosome through λ-Red recombination. Most regulatory parts selected from mRS library had constitutive strengths under different cultivation conditions. A convenient one-step recombination method was then used to modulate galP and glk gene expression with different regulatory parts. Glucose utilization rates of strains modulated with either galP or glk all increased, and the rates had a positive relation with expression strength of both genes. Combinatorial modulation had a synergistic effect on glucose utilization rate. The highest rate (1.64 g/L h) was tenfold higher than PTS⁻ strain and 39% higher than the wild-type E. coli. These modulated strains could be used for efficient anaerobic production of succinate and malate.     

Cholesterol‐α‐glucosyltransferase gene is present in most Helicobacter species including gastric non‐Helicobacter pylori helicobacters obtained from Japanese patients

Background

Non‐Helicobacter pylori helicobacters (NHPHs) besides H. pylori infect human stomachs and cause chronic gastritis and mucosa‐associated lymphoid tissue lymphoma. Cholesteryl‐α‐glucosides have been identified as unique glycolipids present in H. pylori and some Helicobacter species. Cholesterol‐α‐glucosyltransferase (αCgT), a key enzyme for the biosynthesis of cholesteryl‐α‐glucosides, plays crucial roles in the pathogenicity of H. pylori. Therefore, it is important to examine αCgTs of NHPHs.

Materials and Methods

Six gastric NHPHs were isolated from Japanese patients and maintained in mouse stomachs. The αCgT genes were amplified by PCR and inverse PCR. We retrieved the αCgT genes of other Helicobacter species by BLAST searches in GenBank.

Results

αCgT genes were present in most Helicobacter species and in all Japanese isolates examined. However, we could find no candidate gene for αCgT in the whole genome of Helicobacter cinaedi and several enterohepatic species. Phylogenic analysis demonstrated that the αCgT genes of all Japanese isolates show high similarities to that of a zoonotic group of gastric NHPHs including Helicobacter suis, Helicobacter heilmannii, and Helicobacter ailurogastricus. Of 6 Japanese isolates, the αCgT genes of 4 isolates were identical to that of H. suis, and that of another 2 isolates were similar to that of H. heilmannii and H. ailurogastricus.

Conclusions

All gastric NHPHs examined showed presence of αCgT genes, indicating that αCgT may be beneficial for these helicobacters to infect human and possibly animal stomachs. Our study indicated that NHPHs could be classified into 2 groups, NHPHs with αCgT genes and NHPHs without αCgT genes.     

A novel hydroxyfuroic acid compound as an insulin receptor activator - structure and activity relationship of a prenylindole moiety to insulin receptor activation

Background  

Diabetes Mellitus is a chronic disease and many patients of which require frequent subcutaneous insulin injection to maintain proper blood glucose levels. Due to the inconvenience of insulin administration, an orally active insulin replacement has long been a prime target for many pharmaceutical companies. Demethylasterriquinone (DMAQ) B1, extracted from tropical fungus, Pseudomassaria sp., has been reported to be an orally effective agent at lowering circulating glucose levels in diabetic (db/db) mice; however, the cytotoxicity associated with the quinone moiety has not been addressed thus far.     

Metabolic engineering for the production of shikimic acid in an evolved <Emphasis Type="Italic">Escherichia coli</Emphasis> strain lacking the phosphoenolpyruvate: carbohydrate phosphotransferase system

Background  

Shikimic acid (SA) is utilized in the synthesis of oseltamivir-phosphate, an anti-influenza drug. In this work, metabolic engineering approaches were employed to produce SA in Escherichia coli strains derived from an evolved strain (PB12) lacking the phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS^-) but with capacity to grow on glucose. Derivatives of PB12 strain were constructed to determine the effects of inactivating aroK, aroL, pykF or pykA and the expression of plasmid-coded genes aroG ^fbr, tktA, aroB and aroE, on SA synthesis.