Diversity of halophilic archaea from six hypersaline environments in Turkey

The diversity of archaeal strains from six hypersaline environments in Turkey was analyzed by comparing their phenotypic characteristics and 16S rDNA sequences. Thirty-three isolates were characterized in terms of their phenotypic properties including morphological and biochemical characteristics, susceptibility to different antibiotics, and total lipid and plasmid contents, and finally compared by 16S rDNA gene sequences. The results showed that all isolates belong to the family Halobacteriaceae. Phylogenetic analyses using approximately 1,388 bp comparisions of 16S rDNA sequences demonstrated that all isolates clustered closely to species belonging to 9 genera, namely Halorubrum (8 isolates), Natrinema (5 isolates), Haloarcula (4 isolates), Natronococcus (4 isolates), Natrialba (4 isolates), Haloferax (3 isolates), Haloterrigena (3 isolates), Halalkalicoccus (1 isolate), and Halomicrobium (1 isolate). The results revealed a high diversity among the isolated halophilic strains and indicated that some of these strains constitute new taxa of extremely halophilic archaea.     

Characterization of thermostable α-glucosidases from newly isolated <Emphasis Type="Italic">Geobacillus</Emphasis> sp. A333 and thermophilic bacterium A343

We have partially purified and characterized two new thermostable exo-α-1,4-glucosidases (E.C.3.2.1.20) isolated from Geobacillus sp. A333 and thermophilic bacterium A343 strains. A333 α-glucosidase showed optimum activity at 60°C, pH 6.8 and had a value of 1.38 K _m for the pNPG substrate, whereas these results were found to be 65°C, 7.0 and 0.85, respectively for A343 enzyme. Specificity for 20 different substrates and thin layer chromatography studies demonstrated that the A333 enzyme had high transglycosylation activity, and A343 had wide substrate specificity. The substrate specificity of A333 α-glucosidase was determined as maltose, dextrin, turanose, maltotriose, maltopentaose, meltotetraose, maltohexaose and phenyl-α-d-glycopyranoside. On the other hand, the A343 α-glucosidase mostly hydrolyzed dextrin, turanose, maltose, phenyl-α-d-glucopyranoside, maltotriose, maltotetraose, maltopentaose, isomaltose, saccharose and kojibiose by acting α-1,2, α-1,3, α-1,4 and α-1,6 bonds of these substrates. The relative activites of A333 and A343 enzymes were determined to be 83 and 92% when incubated at 60°C for 5 h whereas, the pH of 50% inactivation at 60°C for 15 h were determined to be pH 4.5/10.0 and pH 5.0/10.0, respectively. In addition, the results not only showed that both of the α-glucosidases were stable in a wide range of pH and temperatures, but were also found to be resistant to most of the denaturing agents, inhibitors and metal ions tested. With this study, thermostable exo-α-1,4-glucosidases produced by two new thermophilic strains were characterized as having biotechnological potential in transglycosylation reactions and starch hydrolysis processes.     

Influence of pH and temperature on soluble substrate generation with primary sludge fermentation

The study was undertaken to evaluate the effect of pH and temperature control on the generation of soluble fermentation products from primary sludge. The effect was tested by running parallel experiments under pH and temperature controlled and uncontrolled conditions. In fermentation experiments conducted at 20 degrees C without pH control, the average soluble COD release was 14 mg per liter of wastewater treated, representing a potential increase of 5% in the biodegradable COD content of the primary sedimentation effluent. The corresponding average VFA generation was 9.2mg COD l(-1). The nutrient release was practically negligible and stayed at 0.4 mg l(-1) for nitrogen and 0.1mg l(-1) for phosphorus. Acetic acid accounted more than 45% of the generated VFA in all experimental runs. The acetic acid content of the VFA decreased with increasing initial VSS concentrations and higher pH levels. VFA generation by fermentation was significantly affected with temperature and pH control. Temperature change between 10 and 24 degrees C induced a five-fold increase in VFA generation, from 610 mg l(-1) at 10 degrees C to 2950 mg l(-1) at 24 degrees C.     

Miscarriage, and TNF-α and osteopontin relationship in women patients with Hashimoto's thyroiditis

Objective: Infertility and reproductive impairment can be compromised by abnormalities in both endocrine and immune system. TNF-α promotes apoptotic cell death in fetal membrane tissues and pro-inflammatory, proapoptotic, and procoagulant properties of TNF-α probably contribute to widely accepted abortogenic profile of this cytokine. The aim of this study was to assess the alteration in the levels of TSH, FT3, FT4, TNF-α, osteopontin in pregnant and controls. Methods: Study subjects were 28 pregnant women, 28 non-pregnant women, and 28 healthy controls. All subjects underwent venous blood drawing for levels of TNF-α, osteopontin, and also hormonal assays including the levels of anti-TPO, anti-TG antibodies, TSH, FT3, FT4. Results: Both patient and control groups are similar in terms of age. Pregnancy age in conceived patients is 23.64 ± 2.040. No statistically meaningful relation was found in correlation analysis between TNF-α and osteopontin among the groups (p = 0.963). Anti-thyroglobuline antibody and anti-microsomal antibody levels were found to be higher in patients with non-pregnant patients with Hashimoto thyroiditis than the control group (p < 0.001). No statistically meaningful relation was found in terms of TNF-α (p = 0.66) and osteopontin serum levels (p = 0.50) in patient groups with or without miscarriage history. Conclusions: In our study, no statistically meaningful relation was found in terms of TNF-α and osteopontin serum levels in patient groups with and without miscarriage history.     

The expression of URGCP gene in prostate cancer cell lines: correlation with rapamycin

Molecular targets in prostate cancer are continually being explored, for which there are currently few therapeutic options. Rapamycin (RPM) is an antifungal macrolide antibiotic isolated from Streptomyces hygroscopicus which can inhibit the G1 to S transition. URGCP (upregulator of cell proliferation) is a novel gene located on chromosome 7p13. We aimed to investigate the role of URGCP gene expression changes in PC3, DU145, and LNCAP cell lines with/out RPM. Average cell viability and cytotoxic effect of rapamycin were investigated at 24?h intervals for three days by using Trypan blue dye exclusion test and XTT assay. Cytotoxic effects of rapamycin in DU145, PC3 and LNCAP cells were detected in time and dose dependent manner with the IC₅₀ doses within the range of 1–100?nM. As the results were evaluated, IC₅₀ doses in the DU145, PC3, and LNCaP cells were detected as 10, 25, and 50?nM, respectively. The mean relative ratios of URGCP gene expression in DU145, LNCAP and PC3 cells were found as ?1.48, 6.59 and ?13.00, respectively, when compared to rapamycin-free cells. The False Discovery Rate adjusted p value in DU145, LNCAP and PC3 were 1.25?×?10^?5, 2.20?×?10^?8 and 6.20?×?10^?9, respectively. When the URGCP gene expression level is compared between the dose and control group, we found that URGCP gene expression was significantly decreased in dose groups of DU145 and PC3 cells.     

Dog liver glucose-6-phosphate dehydrogenase: purification and kinetic properties

Glucose-6-phosphate dehydrogenase (G6PD) catalyses the first step of the pentose phosphate pathway which generates NADPH for anabolic pathways and protection systems in liver. G6PD was purified from dog liver with a specific activity of 130 U x mg(-1) and a yield of 18%. PAGE showed two bands on protein staining; only the slower moving band had G6PD activity. The observation of one band on SDS/PAGE with M(r) of 52.5 kDa suggested the faster moving band on native protein staining was the monomeric form of the enzyme.Dog liver G6PD had a pH optimum of 7.8. The activation energy, activation enthalpy, and Q10, for the enzymatic reaction were calculated to be 8.96, 8.34 kcal x mol(-1), and 1.62, respectively.The enzyme obeyed "Rapid Equilibrium Random Bi Bi" kinetic model with Km values of 122 +/- 18 microM for glucose-6-phosphate (G6P) and 10 +/- 1 microM for NADP. G6P and 2-deoxyglucose-6-phosphate were used with catalytic efficiencies (kcat/Km) of 1.86 x 10(6) and 5.55 x 10(6) M(-1) x s(-1), respectively. The intrinsic Km value for 2-deoxyglucose-6-phosphate was 24 +/- 4mM. Deamino-NADP (d-NADP) could replace NADP as coenzyme. With G6P as cosubstrate, Km d-ANADP was 23 +/- 3mM; Km for G6P remained the same as with NADP as coenzyme (122 +/- 18 microM). The catalytic efficiencies of NADP and d-ANADP (G6P as substrate) were 2.28 x 10(7) and 6.76 x 10(6) M(-1) x s(-1), respectively. Dog liver G6PD was inhibited competitively by NADPH (K(i)=12.0 +/- 7.0 microM). Low K(i) indicates tight enzyme:NADPH binding and the importance of NADPH in the regulation of the pentose phosphate pathway.     

Characterization of extracellular esterase and lipase activities from five halophilic archaeal strains

A total of 118 halophilic archaeal collection of strains were screened for lipolytic activity and 18 of them were found positive on Rhodamine agar plates. The selected five isolates were further characterized to determine their optimum esterase and lipase activities at various ranges of salt, temperature and pH. The esterase and lipase activities were determined by the hydrolysis of pNPB and pNPP, respectively. The maximum hydrolytic activities were found in the supernatants of the isolates grown at complex medium with 25% NaCl and 1% gum Arabic. The highest esterase activity was obtained at pH 8-8.5, temperature 60-65 degrees C and NaCl 3-4.5 M. The same parameters for the highest lipase activities were found to be pH 8, temperature 45-65 degrees C and NaCl 3.5-4 M. These results indicate the presence of salt-dependent and temperature-tolerant lipolytic enzymes from halophilic archaeal strains. Kinetic parameters were determined according to Lineweaver-Burk plot. The KM and V (max) values were lower for pNPP hydrolysis than those for pNPB hydrolysis. The results point that the isolates have higher esterase activity comparing to lipase activity.     

Treatment of Cells with Detergent Activates Caspases and Induces Apoptotic Cell Death

Due to their amphiphilic properties, detergents readily disrupt cellular membranes and cause rapid cytolysis. In this study we demonstrate that treatment of cells with sublytic concentrations of detergents such as Triton X-100, Nonidet P-40, n-octylglucoside and the bile salt sodium deoxycholate induce typical signs of apoptosis including DNA fragmentation and cleavage of poly(ADP-ribose) polymerase molecules. The detergent concentration required for apoptosis was below the critical micellar concentration. Induction of apoptosis was not restricted to human cells but similarly occurred in a variety of other vertebrate cell lines. Unstimulated peripheral blood mononuclear cells were susceptible to apoptosis induction by detergent suggesting that apoptosis in this circumstance is not mediated by CD95. Cell death was not due to influx of calcium from the medium. Apoptosis was blocked and cytolysis prevented by treatment with peptide inhibitors of caspases. These findings suggest a process of apoptosis that is initiated upon nonspecific alterations at the cell membrane level. Physiologic correlates of this process still have to be defined. Received: 12 November 1999/Revised: 6 March 2000