Influence of heme and heme oxygenase-1 transfection of pulmonary microvascular endothelium on oxidant generation and cGMP

Heme is a co-factor required for the stimulation of soluble guanylate cyclase (sGC) by nitric oxide (NO) and carbon monoxide, and sGC activation by these agents is inhibited by superoxide. Because heme promotes oxidant generation, we examined the influence of rat pulmonary microvascular endothelial cells (PMECs) with a stable human heme oxygenase-1 (HO-1) transfection and heme on oxidant generation and cGMP. Culture of PMEC with low serum heme decreased cGMP and the detection of peroxide with 10 microM 2',7'-dichlorofluorescin diacetate and increased HO-1 further decreased cGMP without altering the peroxide detection under these conditions. Under conditions where heme (30 microM) has been shown to stimulate cGMP production in PMECsby mechanisms involving NO and CO, heme increased the detection of peroxide in a PMEC-dependent manner and HO-1 transfection did not markedly alter the effects heme on peroxide detection. The addition of 1 microM catalase markedly inhibited the effects of heme on peroxide detection whereas increasing (0.1 mM ebselen) or decreasing (depleting glutathione with 7 mM diethylmaleate) rates of intracellular peroxide metabolism or inhibiting the biosynthesis of oxidants (with 10 microM diphenyliodonium or 0.1 mM nitro-L-arginine) had only modest effects. The detection of superoxide by 10 microM dihydroethidium from PMECs was not increased by exposure to heme. These actions of oxidant probes suggest that intracellular oxidants have a minimal influence on the response to heme. Thus, exposure of PMECs to heme causes a complex response involving an extracellular generation of peroxide-derived oxidant species, which do not appear to originate from increases in intracellular superoxide or peroxide. This enables heme and HO to regulate sGC through mechanisms involving NO and CO, which are normally inhibited by superoxide.     

Methylotrophic yeast Pichia pastoris as a host for production of ATP-diphosphohydrolase (apyrase) from potato tubers (Solanum tuberosum)

ATP-diphosphohydrolase (apyrase) catalyzes the hydrolysis of phosphoanhydride bonds of nucleoside tri- and di-phosphates in the presence of divalent cations. This enzyme has broad substrate specificity for nucleotides, which makes it an ideal enzyme for different biotechnical applications, such as DNA sequencing and platelet-aggregation inhibition. The only commercially available apyrase is isolated from potato tubers. To avoid batch-to-batch variations in activity and quality, we decided to produce a recombinant enzyme. The methylotrophic yeast Pichia pastoris was chosen as an eukaryotic expression host. The coding sequence of potato apyrase, without the signal peptide, was cloned into the YpDC541 vector to create a fusion with the alpha-mating secretion signal of Saccharomyces cerevisiae. The gene was placed under the control of the methanol-inducible alcohol oxidase promoter. The YpDC541-apyrase construct was integrated into P. pastoris strain SMD1168. Methanol induction resulted in secretion of apyrase to a level of 1mg/L. The biologically active recombinant apyrase was purified by hydrophobic interaction and ion exchange chromatography. According to SDS-PAGE and Western blot analysis, the purified enzyme showed to be hyperglycosylated. By enzymatic removal of N-glycans, a single band corresponding to a molecular mass of 48kDa was detected. The recombinant apyrase was found to function well when it was used in combination with the Pyrosequencing technology.     

STRP Screening Sets for the human genome at 5 cM density

Background  

Short tandem repeat polymorphisms (STRPs) are powerful tools for gene mapping and other applications. A STRP genome scan of 10 cM is usually adequate for mapping single gene disorders. However mapping studies involving genetically complex disorders and especially association (linkage disequilibrium) often require higher STRP density.     

Functional organization of adult motor cortex is dependent upon continued protein synthesis

The functional organization of adult cerebral cortex is characterized by the presence of highly ordered sensory and motor maps. Despite their archetypical organization, the maps maintain the capacity to rapidly reorganize, suggesting that the neural circuitry underlying cortical representations is inherently plastic. Here we show that the circuitry supporting motor maps is dependent upon continued protein synthesis. Injections of two different protein synthesis inhibitors into adult rat forelimb motor cortex caused an immediate and enduring loss of movement representations. The disappearance of the motor map was accompanied by a significant reduction in synapse number, synapse size, and cortical field potentials and caused skilled forelimb movement impairments. Further, motor skill training led to a reappearance of movement representations. We propose that the circuitry of adult motor cortex is perpetually labile and requires continued protein synthesis in order to maintain its functional organization.     

A novel heparan sulphate with high degree of N-sulphation and high heparin cofactor-II activity from the brine shrimp Artemia franciscana

With the aid of heparinase and heparitinases from Flavobacterium heparinum and 13C and IH NMR spectroscopy it was shown that the heparan sulphate isolated from the brine shrimp Artemia franciscana exhibits structural features intermediate between those of mammalian heparins and heparan sulphates. These include an unusually high degree of N-sulphation (with corresponding very low degree of N-acetylation), a relatively high content of iduronic acid residues (both unsulphated and 2-O-sulphated) and a relatively low degree of 6-O-sulphation of the glucosamine residues. The major sequences (glucuronic acid-->N-sulphated glucosamine and glucuronic acid-->N, 6-disulphated glucosamine) are most probably arranged in blocks. Although exhibiting negligible anticlotting activity in the APTT and anti-factor Xa assays the A. franciscana heparan sulphate has a high heparin cofactor-II activity (about 1/3 that of heparin).     

Autocrine secretion of TGF-β1 and TGF-β2 by pre-adipocytes and adipocytes: A potent negative regulator of adipocyte differentiation and proliferation of mammary carcinoma cells

Summary We have developed an in vitro system to examine the influence of adipocytes, a major mammary stromal cell type, on the growth of a murine mammary carcinoma, SP1. Previously, we have shown that 3T3-L1 adipocytes release a mitogenic factor, hepatocyte growth factor, which strongly stimulates proliferation of SP1 cells. We now show that 3T3-L1 pre-adipocytes secrete active inhibitory molecules which inhibit DNA synthesis in SP1 cells. In addition, latent inhibitory activity is present in conditioned media (CM) from both pre-adipocytes and adipocytes, and is activated following acid treatment. CM also inhibited DNA synthesis in Mv1Lu wild type epithelial cells, but not DR27 mutant epithelial cells which lack TGF-β type II receptor. Inhibitory activity of CMs was partially abrogated by neutralizing anti-TGF-β1 and anti-TGF-β2 antibodies, and was removed following ultrafiltration through membranes of 10 000 M_r but not 30 000 M_r pore size. These results show that the inhibitory effect on DNA synthesis is mediated by TGF-β1-like and TGF-β2-like molecules. In addition, acid-treated CM as well as purified TGF-β inhibited differentiation of pre-adipocytes. Untreated pre-adipocyte CM, but not mature adipocyte CM, spontaneously inhibited adipocyte differentiation. Together, these findings indicate that pre-adipocytes spontaneously activate their own secreted TGF-β, whereas mature adipocytes do not, and suggest that activation of TGF-β has a potent negative regulatory effect on adipocyte differentiation and tumor growth. Thus, TGF-β may be an important modulator of tumor growth and adipocyte differentiation via both paracrine and autocrine mechanisms. These findings emphasize the importance of adipocyte-tumor interactions in the regulation of tumor microenvironment.     

Growth of Tomato Plants in Soil Contaminated with Kuwait Crude Oil

This laboratory study measured growth of one plant species, Lycopersicon esculentum Big Girl (tomato), that is sensitive to the presence of soil contamination, in Kuwait soil amended with crude-oil-contaminated soil. Germinated tomato seeds were placed in containers with soil containing 0, 0.12, 0.24, 0.36, 0.48, 0.60, 1.2, and 2.4% crude oil and were grown in an indoor growth chamber. Plants grew in Kuwait soil containing up to 0.36% crude oil; however, growth and fruit production were compromised at crude oil concentrations greater than 0.12% when compared with control plants. Plants did not grow in Kuwait soil amended with 0.48% crude oil or higher.     

Serum Biomarkers in Patients with Relapsing Eosinophilic Granulomatosis with Polyangiitis (Churg-Strauss)

Introduction

Previous studies suggest a role for eotaxin-3, TARC/CCL17 and IgG4 in newly- diagnosed patients with eosinophilic granulomatosis with polyangiitis (EGPA, Churg-Strauss) with highly active disease. The role of these biomarkers in relapsing disease is unclear.

Methods

Serum levels of TARC/CCL17, eotaxin-3, IgG4, and IgG4/IgG ratio were determined in serum samples from a longitudinal cohort of patients with EGPA (105 visits of 25 patients). Epidemiological, clinical and laboratory data were available for all visits.

Results

At the first visit, 80% of patients were using glucocorticoids and 68% additional immunosuppressive drugs. Disease flares were seen at 18 visits. The median BVAS and BVAS/WG scores at time of relapse were 4 and 2, respectively. None of the biomarkers tested were useful to discriminate between active disease and remission. Patients treated with prednisone had lower eotaxin-3 and eosinophil levels compared to patients not taking glucocorticoids irrespective of disease activity. Use of immunosuppressive agents was not associated with biomarker levels.

Conclusions

Serum levels of TARC/CCL17, eotaxin-3, IgG4, and IgG4/IgG ratio do not clearly differentiate active and inactive disease in established EGPA. Defining biomarkers in EGPA remains a challenge especially during times of glucocorticoid use.     

Impact of Sn/F Pre-Treatments on the Durability of Protective Coatings against Dentine Erosion/Abrasion

For preventing erosive wear in dentine, coating with adhesives has been suggested as an alternative to fluoridation. However, clinical studies have revealed limited efficacy. As there is first evidence that Sn²⁺ increases bond strength of the adhesive Clearfil SE (Kuraray), the aim of the present study was to investigate whether pre-treatment with different Sn²⁺/F⁻ solutions improves the durability of Clearfil SE coatings. Dentine samples (eight groups, n=16/group) were freed of smear layer (0.5% citric acid, 10 s), treated (15 s) either with no solution (control), aminefluoride (AmF, 500 ppm F⁻, pH 4.5), SnCl₂ (800/1600 ppm Sn²⁺; pH 1.5), SnCl₂/AmF (500 ppm F⁻, 800 ppm Sn²⁺, pH 1.5/3.0/4.5), or Elmex Erosion Protection Rinse (EP, 500 ppm F⁻, 800 ppm Sn²⁺, pH 4.5; GABA International), then rinsed with water (15 s) and individually covered with Clearfil SE. Subsequently the specimens were subjected to an erosion/abrasion protocol consisting of 1320 cycles of immersion in 0.5% citric acid (5°C/55°C; 2 min) and automated brushing (15 s, 200 g, NaF-toothpaste, RDA 80). As the coatings proved stable up to 1320 cycles, 60 modified cycles (brushing time 30 min/cycle) were added. Wear was measured profilometrically. After SnCl₂/AmF, pH 4.5 or EP pre-treatment all except one coating survived. In the other groups, almost all coatings were lost and there was no significant difference to the control group. Pre-treatment with a Sn²⁺/F⁻ solution at pH 4.5 seems able to improve the durability of adhesive coatings, rendering these an attractive option in preventing erosive wear in dentine.