Diversity and abundance of sulfate-reducing microorganisms in the sulfate and methane zones of a marine sediment, Black Sea

The Black Sea, with its highly sulfidic water column, is the largest anoxic basin in the world. Within its sediments, the mineralization of organic matter occurs essentially through sulfate reduction and methanogenesis. In this study, the sulfate-reducing community was investigated in order to understand how these microorganisms are distributed relative to the chemical zonation: in the upper sulfate zone, at the sulfate-methane transition zone, and deeply within the methane zone. Total bacteria were quantified by real-time PCR of 16S rRNA genes whereas sulfate-reducing microorganisms (SRM) were quantified by targeting their metabolic key gene, the dissimilatory (bi)sulfite reductase (dsrA). Sulfate-reducing microorganisms were predominant in the sulfate zone but occurred also in the methane zone, relative proportion was maximal around the sulfate-methane transition, c. 30%, and equally high in the sulfate and methane zones, 5-10%. The dsrAB clone library from the sulfate-methane transition zone, showed mostly sequences affiliated with the Desulfobacteraceae. While, the dsrAB clone libraries from the upper, sulfate-rich zone and the deep, sulfate-poor zone were dominated by similar, novel deeply branching sequences which might represent Gram-positive spore-forming sulfate- and/or sulfite-reducing microorganisms. We thus hypothesize that terminal carbon mineralization in surface sediments of the Black Sea is largely due to the sulfate reduction activity of previously hidden SRM. Although these novel SRM were also abundant in sulfate-poor, methanogenic areas of the Black Sea sediment, their activities and possibly very versatile metabolic capabilities remain subject of further study.     

Phosphomannose isomerase: An efficient selectable marker for plant transformation

Summary Phosphomannose isomerase (PMI) catalyzes the reversible interconversion of mannose 6-phosphate and fructose 6-phosphate. Plant cells lacking this enzyme are incapable of surviving on synthetic medium containing mannose as a carbon source. Maize, wheat and barley plants, genetically modified to express the Escherichia coli manA gene (pmi) under the control of a plant promoter, were able to survive selection on mannose-containing medium. Transformation frequencies averaged 45% for maize transformation via Biolistics^™ 35% for maize Agrobacterium-mediated transformation, 20% for wheat, 3% for barley, and 2% for watermelon transformation. Moreover, the frequencies exceeded those obtained for maize and wheat using the pat or bar gene with Basa^? selection. A preliminary safety assessment has been conducted for PMI. Purified PMI protein demonstrates no adverse, effects in an acute mouse toxicity test. Purified PMI protein was readily digested in simulated mammalian gastric and intestinal fluids. Plants derived from surgar beet and corn cells that had been genetically modified to express the E. coli manA gene were evaluated for biochemical changes in mannose-associated pathways. No detectable changes in glycoprotein profiles were detected in PMI-transformed plants as compared to nontransgenic controls. The yield and nutritional composition of grain from PMI-transformed corn plants compared to their non-transformed isogenic counterparts were also determined and no statistically significant differences were found. The inherent safety of a system based on simple sugar metabolism coupled with high transformation frequencies for monocots make pmi and ideal selectable marker for plant transformation.     

Inhibition of the helicase activity of HCV NTPase/helicase by 1-beta-D-ribofuranosyl-1,2,4-triazole-3-carboxamide-5 '-triphosphate (ribavirin-TP)

In the presented study the ribavirin-TP--an established inhibitor of the NTPase activity of the superfamily NTPase/helicases II--was investigated as an inhibitor of the unwinding activity of the hepatitis C virus (HCV) NTPase/helicase. The kinetics of the reaction revealed that ribavirin-TP reduces the turnover number of the helicase reaction by a mechanism that does not correspond to that of the inhibition of the NTPase activity. Our results suggest that derivatives of ribavirin-TP with enhanced stability towards hydrolytic attack may be effective inhibitors of the enzyme.     

Functional abnormalities in protein tyrosine phosphatase epsilon-deficient macrophages

Protein tyrosine phosphatase epsilon (PTP epsilon)-deficient mice were generated by targeted deletion of exons 3, 4, and 5 of the Ptpre gene. Mice homozygous for this deletion (Ptpre(Delta3-5)) were fertile, bred and developed normally and exhibited no overt phenotype. However, closer examination of the function of macrophages from these mice revealed a defect in the regulation of the respiratory burst. While bacterial lipopolysaccharide (LPS) or tumour necrosis factor alpha (TNFalpha) were able to prime bone marrow-derived macrophages (BMM) from wild type (Ptpre(+)) macrophages for an enhanced respiratory burst, they were unable to do so in macrophages from PTP epsilon-deficient mice. PTP epsilon-deficient BMM also had abnormalities in cytokine production with a reduced ability to produce TNFalpha and enhanced IL-10 production in response to challenge with LPS. These findings suggest an important role for PTP epsilon in the control of macrophage function.     

ATP-binding domain of NTPase/helicase as a target for hepatitis C antiviral therapy

To enhance the inhibitory potential of 1-beta-D-ribofuranosyl-1,2,4-triazole-3-carboxamide (ribavirin) vs hepatitis C virus (HCV) NTPase/helicase, ribavirin-5'-triphosphate (ribavirin-TP) was synthesized and investigated. Ribavirin-TP was prepared with the use of modified Yoshikawa-Ludwig-Mishra-Broom procedure (cf. Mishra & Broom, 1991, J. Chem. Soc., Chem. Commun, 1276-1277) involving phosphorylation of unprotected nucleoside. Kinetic analysis revealed enhanced inhibitory potential of ribavirin-TP (IC50=40 microM) as compared to ribavirin (IC50 > 500 microM). Analysis of the inhibition type by means of graphical methods showed a competitive type of inhibition with respect to ATP. In view of the relatively low specificity towards nucleoside-5'-triphosphates (NTP) of the viral NTPase/helicases, it could not be ruled out that the investigated enzyme hydrolyzed the ribavirin-TP to less potent products. Investigations on non- hydrolysable analogs of ribavirin-TP or ribavirin-5'-diphosphate (ribavirin-DP) are currently under way.     

Blood flow in ductus venosus in early uncomplicated pregnancy

The ductus venosus is the very important part of fetal venous circulation. It plays a central role in return of venous blood from the placenta. This unique shunt carries well-oxygenated blood from the umbilical vein through the inferior atrial inlet on its way across the foramen ovale. Using Doppler ultrasound, it is possible to assess the blood flow in fetal vessels including ductus venosus. It is observed, in animal and human studies, that the typical waveform for blood flow in ductus venosus in early pregnancy can be different depending on numerous conditions e.g. fetal karyotype. This study is performed to assess the physiologic parameters of blood flow in ductus venosus in uncomplicated early pregnancy. Material and Methods: 404 women were examined between 11+0 and 13+6 weeks (+ days) of gestation by ultrasound. Fetal crown-rump length (CRL) was measured to assess the gestational age. The assessment of risk of fetal abnormalities was based on nuchal translucency (NT) measurement. The ductus venosus blood flow with color and spectral Doppler was obtained in all patients. The following features were assessed: pulsatility index (PI), and direction of flow (positive/negative) during atrial contraction (wave A). All cases were followed up to 22 weeks of gestation when the control scan was performed. Results and conclusions: 30 cases were excluded from the uncomplicated group due to: high risk of fetal abnormalities, fetal loss, confirmed fetal abnormalities and utero-placental pathology. 374 women were considered as uncomplicated pregnancy. In both uncomplicated and complicated groups the mean values for pulsatility index (PI) were established. The mean PI value in uncomplicated pregnancies was: 0.91 (SD +/- 0.32). No significant differences between groups were noticed. In 370 cases of uncomplicated pregnancy the A wave direction was positive but in 1.1% of cases the reverse flow in atrial contraction was observed.     

Experiences with the dual digestion of municipal sewage sludge

The dual digestion process was investigated using sludge samples collected at the WWTP of Tomaszow Mazowiecki (Poland). Mixed sludge was treated in a laboratory setup under batch and semi-continuous conditions. Dual digestion with a 1d SRT aerobic thermophilic pretreatment followed by an anaerobic step with 20 d of SRT turned out to be optimal, since a 44-46% VS reduction and a biogas yield of 480 dm(3)/kg VS fed were achieved. In the course of the process, the concentration of nitrogen in supernatant increased over 5 times and its major portion was converted into ammonia. Phosphorus also entered the supernatant, reaching over 200 g/m(3). The dual digestion noticeably deteriorated the sludge dewaterability. Following completion of the process, capillary suction time measurements averaged 64 s for the raw sludge, 400 s for aerobically pretreated sludge and 310-360 for the anaerobically digested sludge. Aerobic pretreatment consistently reduced Enterobacteriaceae content to below detectable limits.