Identification and characterization of proteins isolated from microvesicles derived from human lung cancer pleural effusions

Microvesicles (MVs, also known as exosomes, ectosomes, microparticles) are released by various cancer cells, including lung, colorectal, and prostate carcinoma cells. MVs released from tumor cells and other sources accumulate in the circulation and in pleural effusion. Although recent studies have shown that MVs play multiple roles in tumor progression, the potential pathological roles of MV in pleural effusion, and their protein composition, are still unknown. In this study, we report the first global proteomic analysis of highly purified MVs derived from human nonsmall cell lung cancer (NSCLC) pleural effusion. Using nano‐LC–MS/MS following 1D SDS‐PAGE separation, we identified a total of 912 MV proteins with high confidence. Three independent experiments on three patients showed that MV proteins from PE were distinct from MV obtained from other malignancies. Bioinformatics analyses of the MS data identified pathologically relevant proteins and potential diagnostic makers for NSCLC, including lung‐enriched surface antigens and proteins related to epidermal growth factor receptor signaling. These findings provide new insight into the diverse functions of MVs in cancer progression and will aid in the development of novel diagnostic tools for NSCLC.     

Sex steroid changes during temperature‐induced gonadal differentiation in Paralichthys olivaceus (Temminck & Schegel, 1846)

Sex steroid changes during temperature‐induced gonadal differentiation were evaluated in the olive flounder, Paralichthys olivaceus. Larvae were reared at 21 ± 0.5°C, 24 ± 0.5°C and 28 ± 0.5°C from day 40 post‐hatching (dph) to 90 dph. The proportion of males was 61.1, 76.7, 87.8 and 47.8% in 21°C, 24°C, 28°C and in control groups, respectively. Gonadal differentiation was circa 65 dph, when fishes were a mean 39 mm total length (TL). The gonads developed faster when fishes were reared in higher temperatures. Radioimmunoassay (RIA) analyses indicated that the level of estradiol‐17β (E2) changed during the period of gonadal differentiation and peaked at an onset of ovarian differentiation in all groups. Compared with fish in control groups, the levels of E2 were lower in thermal‐treated groups, especially in the highest temperature groups. The present results indicate that E2 plays a major role in the process of ovarian differentiation, and suggest that temperature‐induced masculinization in P. olivaceus is mainly due to a decrease in the E2 level during the period of ovarian differentiation.     

Improving heterologous polyketide production in <Emphasis Type="Italic">Escherichia coli</Emphasis> by overexpression of an <Emphasis Type="Italic">S</Emphasis>-adenosylmethionine synthetase gene

An S-adenosylmethionine synthetase gene (metK) from Streptomyces spectabilis was cloned into an expression plasmid under the control of an inducible T7 promoter and introduced into a strain of Escherichia coli (BAP1(pBP130/pBP144)) capable of producing the polyketide product 6-deoxyerythronolide B (6-dEB). The metK coexpression in BAP1(pBP130/pBP144) improved the specific production of 6-dEB from 10.86 to 20.08 mg l⁻¹ . In an effort to probe the reason for this improvement, a series of gene deletion and expression experiments were conducted based on a metK metabolic pathway that branches between propionyl-CoA (a 6-dEB precursor) and autoinducer compounds. The deletion and expression studies suggested that the autoinducer pathway had a larger impact on improved 6-dEB biosynthesis. Supporting these results were experiments demonstrating the positive effect conditioned media (the suspected location of the autoinducer compounds) had on 6-dEB production. Taken together, the results of this study show an increase in heterologous 6-dEB production concomitant with heterologous metK gene expression and suggest that the mechanism for this improvement is linked to native autoinducer compounds.     

Chromium picolinate and conjugated linoleic acid do not synergistically influence diet- and exercise-induced changes in body composition and health indexes in overweight women

This study assessed the effects of combined chromium picolinate (CP) and conjugated linoleic acid (CLA) supplementation on energy restriction and exercise-induced changes in body composition, glucose metabolism, lipid lipoprotein profile and blood pressure in overweight, premenopausal women. For 12 weeks, 35 women [age 36+/-1 years (mean+/-S.E.M.); BMI 28.0+/-0.5 kg/m2] were counseled to consume a 2092 kJ/day (500 kcal/day) energy deficit diet and performed 30 min of moderate-intensity walking or jogging 5 days/week. The women were randomly assigned to ingest either CP-CLA [400 mug chromium (Cr), 1.8 g CLA in 2.4 g tonalin oil, n=19] or placebo (<0.1 microg Cr, 2.4 g canola oil, n=16). Compared to baseline, urinary Cr excretion increased 22-fold, plasma CLA isomer 18:2 (c9,t11) content increased 79% and plasma CLA isomer 18:2 (t10,c12) became detectable in CP-CLA and were unchanged in Placebo. Over time, body weight decreased 3.5+/-0.5% (CP-CLA -2.6+/-0.5; placebo -2.5+/-0.5 kg) and fat mass decreased 8.9+/-1.3% (CP-CLA -2.7+/-0.5, placebo -2.4+/-0.5 kg), with no differences in responses between groups. Fasting blood hemoglobin A1c, plasma glucose and insulin, a homeostatic assessment of insulin resistance, serum total cholesterol (CHOL), high-density lipoprotein cholesterol (HDL), low-density lipoprotein cholesterol, triacylglycerol (TG), CHOL/HDL ratio, TG/HDL ratio and sitting systolic and diastolic blood pressures were not changed over time or influenced by CP-CLA. The use of a combined CP and CLA supplement for 3 months does not affect diet- and exercise-induced changes in weight and body composition or improve indexes of metabolic and cardiovascular health in young overweight women.     

A nicardipine-sensitive Ca2+ entry contributes to the hypotonicity-induced increase in [Ca2+]i of principal cells in rat cortical collecting duct

We examined the mechanisms involved in the [Ca(2+)](i) response to the extracellular hypotonicity in the principal cells of freshly isolated rat cortical collecting duct (CCD), using Fura-2/AM fluorescence imaging. Reduction of extracellular osmolality from 305 (control) to 195 mosmol/kgH(2)O (hypotonic) evoked transient increase in [Ca(2+)](i) of principal cells of rat CCDs. The [Ca(2+)](i) increase was markedly attenuated by the removal of extracellular Ca(2+)(.) The application of a P(2) purinoceptor antagonist, suramin failed to inhibit the hypotonicity-induced [Ca(2+)](i) increase. The [Ca(2+)](i) increase in response to extracellular hypotonicity was not influenced by application of Gd(3+) and ruthenium red. On the other hand, a voltage-gated Ca(2+) channel inhibitor, nicardipine, significantly reduced the peak amplitude of [Ca(2+)](i) increase in the principal cells. In order to assess Ca(2+) entry during the hypotonic stimulation, we measured the quenching of Fura-2 fluorescence intensity by Mn(2+). The hypotonic stimulation enhanced quenching of Fura-2 fluorescence by Mn(2+), indicating that a Ca(2+)-permeable pathway was activated by the hypotonicity. The hypotonicity-mediated enhancement of Mn(2+) quenching was significantly inhibited by nicardipine. These results strongly suggested that a nicardipine-sensitive Ca(2+) entry pathway would contribute to the mechanisms underlying the hypotonicity-induced [Ca(2+)](i) elevation of principal cells in rat CCD.     

Syntheses, crystal structures, and magnetic properties of Ni(mnt)2-based molecular solids containing substituted isoquinolinium

Two novel ion-pair complexes, [RBzIQl]⁺[Ni(mnt)₂]⁻ (mnt²⁻ = maleonitriledithiolate, [RBzIQl]⁺ = 4-R-benzylisoquinolinium; R = H (1), Cl (2)) have been characterized structurally and magnetically. The anions and [BzIQl]⁺ cations of 1 form 1D column of alternating between cations and anions via π?π stacking interaction between Ni(mnt)₂ plane and isoquinoline ring, and the Ni(mnt)₂ anions between adjacent columns exist C?N, C?N, and N?N interaction. The anions and cations of 2 stack into well-segregated columns in the solid state; and the Ni(III) ions form a 1D zigzag chain in a Ni(mnt)₂ column through intermolecular Ni?S, S?S, Ni?Ni or π?π interactions. The chain is uniform in 2 with the Ni?Ni distances of 3.784 Å. Magnetic susceptibility measurements for these complexes in the temperature range 1.8-300 K show that 1 exhibits antiferromagnetic coupling behavior, and 2 exhibits unusual magnetic phase transitions around 45 K. The overall magnetic behavior for 2 indicates the presence of antiferromagnetic interaction in the high-temperature phase (HT) and spin gap in the low-temperature phase (LT).     

Incorporation of quantum dots on virus in polycationic solution

Developing methods to label viruses with fluorescent moieties has its merits in elucidating viral infection mechanisms and exploring novel antiviral therapeutics. Fluorescent quantum dots (QDs), an emerging probe for biological imaging and medical diagnostics, were employed in this study to tag retrovirus encoding enhanced green fluorescent protein (EGFP) genes. Electrostatic repulsion forces generated from both negatively charged retrovirus and QDs were neutralized by cationic Polybrene, forming colloidal complexes of QDs-virus. By examining the level of EGFP expression in 3T3 fibroblast cells treated with QDs-tagged retroviruses for 24 hours, the infectivity of retrovirus incorporated with QDs was shown to be only slightly decreased. Moreover, the imaging of QDs can be detected in the cellular milieu. In summary, the mild method developed here makes QDs-tagged virus a potential imaging probe for direct tracking the infection process and monitoring distribution of viral particles in infected cells.     

Effects of a HP0859 (rfaD) knockout mutation on lipopolysaccharide structure of Helicobacter pylori 26695 and the bacterial adhesion on AGS cells

Lipopolysaccharide (LPS) is considered as an important virulence factor of Helicobacter pylori, and contributes to infection persistence and disease severity. ADP-l-glycero-d-manno-heptose-6-epimerase is an enzyme essential for LPS synthesis and understanding of its biochemistry is critical for drug development. We cloned one putative ortholog of Escherichia colirfaD, HP0859, from H. pylori 26695. Determination of the native molecular weight of the recombinant HP0859 protein suggests that the protein is likely a hexamer. NADP⁺, instead of NAD⁺, was proved to be the physiological cofactor for HP0859 protein. Circular dichroism spectrum analysis demonstrated that the secondary structure of this protein is significantly altered when the cofactor is removed. We also constructed an HP0859 knockout mutant and examined its phenotypic properties. The HP0859 knockout mutant exhibited a severe truncation of LPS, a decreased growth rate, and a higher susceptibility to novobiocin. Disruption of HP0859 also reduced the adhesive capacity of H. pylori to AGS cells, and the infected cells failed to display the classic hummingbird phenotype. Complementation of the HP0859 knockout mutation restored these phenotypes completely. In conclusion, we demonstrate that HP0859 codes for a protein essential for the LPS inner core biosynthesis in H. pylori and an intact LPS structure contributes to the adherence ability of this bacterium.