The first crystal structure of gluconolactonase important in the glucose secondary metabolic pathways

The first gluconolactonase crystal structure from bacteria has been determined to a resolution of 1.61 Å using X-ray crystallography. It belongs to the senescence marker protein 30/gluconolaconase superfamily but exhibits substrate specificity mainly toward d-glucono-δ-lactone. It forms a novel disulfide-bonded clamshell dimer comprising two doughnut-shaped six-bladed β-propeller domains, yet with an exceptionally long N-terminal subdomain forming an extra helix and four additional β-strands to enclose half of the outermost β-strands of each propeller. Extensive interactions, including H-bonds, salt bridges, disulfide bonds, and coordination bonds, along with numerous bridging water molecules, are present in the interface to institute the “top-to-top” clamshell-type dimer. Three calcium ions per subunit were observed. Two are present in the central water-filled channel, with the top one coordinated to four highly conserved amino acids and is possibly involved in substrate hydrolysis, while the bottom one is coordinated to the backbone oxygen atoms, which is possibly for stabilizing the propeller domain. One calcium ion is situated in the interface also to stabilize the dimer form. Since gluconolactonase is essential in the glucose secondary metabolic pathways leading to the synthesis of pentose, vitamin C, or “antiaging” factors, determination of its tertiary structure should help understand these important biochemical processes.     

Baicalein induces proliferation inhibition in B16F10 melanoma cells by generating reactive oxygen species via 12-lipoxygenase

In a previous study, we demonstrated that baicalein induces hydroxyl radical formation in human platelets but the mechanisms are unclear. Herein, we show, using an electron spin resonance technique, that baicalein also induces hydroxyl radical formation in B16F10 melanoma cells in a dose-dependent manner. Baicalein produced superoxide anions in the presence of an iron chelator and superoxide dismutase (SOD) inhibitor. We suggest that superoxide anions produced by baicalein were promptly converted to hydroxyl radicals through SOD and the Fenton reaction in B16F10 melanoma cells. According to Western blotting results, the 12-LOX protein was expressed in B16F10 melanoma cells, but baicalein had no effect on 12-LOX expression. Decreases in 12-LOX protein expression and hydroxyl radical signals occurred in a 12-LOX small interfering RNA knockdown protein group compared with the baicalein control. In the MTT assay, we also found that baicalein caused a reduction in cellular viability, which was reversed by the addition of ROS scavengers. On the basis of these data, we conclude that ROS formation catalyzed by 12-LOX is one possible mechanism of growth inhibition by baicalein in B16F10 melanoma cells.     

Shorter GT repeat polymorphism in the heme oxygenase-1 gene promoter has protective effect on ischemic stroke in dyslipidemia patients

Background  

The microsatellite polymorphism of heme oxygenase (HO)-1 gene promoter has been shown to be associated with the susceptibility to ischemic event, including coronary artery disease (CAD), myocardial infarction, and peripheral vascular disease. We aimed to examine whether the length of (GT)_n repeats in HO-1 gene promoter is associated with ischemic stroke in people with CAD risk factors, especially low level of HDL.     

Anchoring 9,371 maize expressed sequence tagged unigenes to the bacterial artificial chromosome contig map by two-dimensional overgo hybridization

Our goal is to construct a robust physical map for maize (Zea mays) comprehensively integrated with the genetic map. We have used a two-dimensional 24 x 24 overgo pooling strategy to anchor maize expressed sequence tagged (EST) unigenes to 165,888 bacterial artificial chromosomes (BACs) on high-density filters. A set of 70,716 public maize ESTs seeded derivation of 10,723 EST unigene assemblies. From these assemblies, 10,642 overgo sequences of 40 bp were applied as hybridization probes. BAC addresses were obtained for 9,371 overgo probes, representing an 88% success rate. More than 96% of the successful overgo probes identified two or more BACs, while 5% identified more than 50 BACs. The majority of BACs identified (79%) were hybridized with one or two overgos. A small number of BACs hybridized with eight or more overgos, suggesting that these BACs must be gene rich. Approximately 5,670 overgos identified BACs assembled within one contig, indicating that these probes are highly locus specific. A total of 1,795 megabases (Mb; 87%) of the total 2,050 Mb in BAC contigs were associated with one or more overgos, which are serving as sequence-tagged sites for single nucleotide polymorphism development. Overgo density ranged from less than one overgo per megabase to greater than 20 overgos per megabase. The majority of contigs (52%) hit by overgos contained three to nine overgos per megabase. Analysis of approximately 1,022 Mb of genetically anchored BAC contigs indicates that 9,003 of the total 13,900 overgo-contig sites are genetically anchored. Our results indicate overgos are a powerful approach for generating gene-specific hybridization probes that are facilitating the assembly of an integrated genetic and physical map for maize.     

Dual stable point model of muscle activation and deactivation

Two dynamic models of muscle activation and deactivation based on the concepts of ion transport, reaction rates, and muscle mechanics are proposed. Storage release and uptake of calcium by the sarcoplasmic reticulum, and a two-step chemical reaction of calcium and troponin are included in the first model. This is a concise version of the complex chemical reactions of muscle activation and deactivation in sarcoplasm. The second model is similar to the first, but calcium-troponin reactions are simplified into two nonlinear rates functions. Due to these nonlinear dynamics, the second model can explain the catch-like enhancement of isometric force response. Simulation results which match experimental data are shown. Also, two new phenomena which need further experiment to verify are predicted by the second model.     

Biogeochemistry and microbial community composition in sea ice and underlying seawater off East Antarctica during early spring

Pack ice, brines and seawaters were sampled in October 2003 in the East Antarctic sector to investigate the structure of the microbial communities (algae, bacteria and protozoa) in relation to the associated physico-chemical conditions (ice structure, temperature, salinity, inorganic nutrients, chlorophyll a and organic matter). Ice cover ranged between 0.3 and 0.8 m, composed of granular and columnar ice. The brine volume fractions sharply increased above −4°C in the bottom ice, coinciding with an important increase of algal biomass (up to 3.9 mg C l⁻¹), suggesting a control of the algae growth by the space availability at that period of time. Large accumulation of NH₄ ⁺ and PO₄ ³⁻ was observed in the bottom ice. The high pool of organic matter, especially of transparent exopolymeric particles, likely led to nutrients retention and limitation of the protozoa grazing pressure, inducing therefore an algal accumulation. In contrast, the heterotrophs dominated in the underlying seawaters.     

One-step refolding and purification of disulfide-containing proteins with a C-terminal MESNA thioester

Background  

Expression systems based on self-cleavable intein domains allow the generation of recombinant proteins with a C-terminal thioester. This uniquely reactive C-terminus can be used in native chemical ligation reactions to introduce synthetic groups or to immobilize proteins on surfaces and nanoparticles. Unfortunately, common refolding procedures for recombinant proteins that contain disulfide bonds do not preserve the thioester functionality and therefore novel refolding procedures need to be developed.     

MemBrain: improving the accuracy of predicting transmembrane helices

Prediction of transmembrane helices (TMH) in alpha helical membrane proteins provides valuable information about the protein topology when the high resolution structures are not available. Many predictors have been developed based on either amino acid hydrophobicity scale or pure statistical approaches. While these predictors perform reasonably well in identifying the number of TMHs in a protein, they are generally inaccurate in predicting the ends of TMHs, or TMHs of unusual length. To improve the accuracy of TMH detection, we developed a machine-learning based predictor, MemBrain, which integrates a number of modern bioinformatics approaches including sequence representation by multiple sequence alignment matrix, the optimized evidence-theoretic K-nearest neighbor prediction algorithm, fusion of multiple prediction window sizes, and classification by dynamic threshold. MemBrain demonstrates an overall improvement of about 20% in prediction accuracy, particularly, in predicting the ends of TMHs and TMHs that are shorter than 15 residues. It also has the capability to detect N-terminal signal peptides. The MemBrain predictor is a useful sequence-based analysis tool for functional and structural characterization of helical membrane proteins; it is freely available at http://chou.med.harvard.edu/bioinf/MemBrain/.     

EGF upregulates Na+/H+ exchanger NHE1 by post-translational regulation that is important for cervical cancer cell invasiveness

Na+/H+ exchanger 1 (NHE1) is involved in cell migration but little is known about the signal pathways that regulate NHE1 activity and that are associated with tumor cell invasiveness. This study is to investigate the mechanisms by which epidermal growth factor (EGF) regulates NHE1 expression to promote cervical cancer cell invasiveness and the clinical significance in early-stage cervical cancer. NHE1 protein was scanty in normal or noncancerous cervical tissues of all surgical specimens examined (n = 92). Tumor tissues clearly expressed NHE1 protein with different amounts. The differential expression level of NHE1 is associated with the clinical outcome. NHE1 protein was also differentially expressed between normal cervical epithelial cells and two cervical cancer cell lines. Cervical cancer cells benefit some enhanced cellular functions from NHE1 abundance, such as cell volume regulation, migration, and invasion. Interestingly, NHE1 colocalized with EGF in cervical cancer tissues. Studies in cell culture systems indicated that EGF-stimulated NHE1 abundance in a time-dependent manner by post-translational regulation. This implies a likely autocrine or paracrine EGF stimulation of NHE1 production in vivo. In addition, the phosphoinositide 3-kinase pathway is the dominant signal controlling EGF-stimulated NHE1 abundance. Pharmacological inhibition of NHE1 activity markedly inhibited the basal and EGF-stimulated cervical cancer cell migration. Image studies and immunoprecipitaion experiments suggest that EGF-induced NHE1 translocation to the leading-edge lamellipodia, where NHE1 interacted with actin-associated protein Ezrin, thereby remodeling cytoskeleton and stimulating cervical cancer cell migration. In conclusion, EGF upregulates NHE1 by post-translational regulation that is important for cervical cancer cell invasiveness.     

Overexpression, one-step purification, and biochemical characterization of a recombinant gamma-glutamyltranspeptidase from Bacillus licheniformis

A truncated gene from Bacillus lichenifromis ATCC 27811 encoding a recombinant γ-glutamyltranspeptidase (BLrGGT) was cloned into pQE-30 to generate pQE-BLGGT, and the overexpressed enzyme was purified from the crude extract of IPTG-induced E. coli M15 (pQE-BLGGT) to homogeneity by nickel-chelate chromatography. This protocol yielded over 25 mg of purified BLrGGT per liter of growth culture under optimum conditions. The molecular masses of the subunits of the purified enzyme were determined to be 41 and 22 kDa, respectively, by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The optimum pH and temperature for the recombinant enzyme were 6–8 and 40 °C, respectively. The chloride salt of metal ions Mg²⁺, K⁺, and Na⁺ can activate BLrGGT, whereas that of Pb²⁺ dramatically inhibited it. The substrate specificity study showed that l-γ-glutamyl-p-nitroanilide (l-γ-Glu-p-NA) is a preference for the enzyme. Steady-state kinetic study revealed that BLrGGT has a k _cat of 105 s⁻¹ and a K _m of 21 μM when using l-γ-Glu-p-NA as the substrate. With this overexpression and purification system, BLrGGT can now be obtained in quantities necessary for structural characterization and synthesis of commercially important γ-glutamyl compounds.