Identification of anaerobic selenate-respiring bacteria from aquatic sediments

The diversity population of microorganisms with the capability to use selenate as a terminal electron acceptor, reducing it to selenite and elemental selenium by the process known as dissimilatory selenate reduction, is largely unknown. The overall objective of this study was to gain an in-depth understanding of anaerobic biotransformation of selenium in the environment, particularly anaerobic respiration, and to characterize the microorganisms catalyzing this process. Here, we demonstrate the isolation and characterization of four novel anaerobic dissimilatory selenate-respiring bacteria enriched from a variety of sources, including sediments from three different water bodies in Chennai, India, and a tidal estuary in New Jersey. Strains S5 and S7 from India, strain KM from the Meadowlands, NJ, and strain pn1, categorized as a laboratory contaminant, were all phylogenetically distinct, belonging to various phyla in the bacterial domain. The 16S rRNA gene sequence shows that strain S5 constitutes a new genus belonging to Chrysiogenetes, while strain S7 belongs to the Deferribacteres, with greater than 98% 16S rRNA gene similarity to Geovibrio ferrireducens. Strain KM is related to Malonomonas rubra, Pelobacter acidigallici, and Desulfuromusa spp., with 96 to 97% 16S rRNA gene similarity. Strain pn1 is 99% similar to Pseudomonas stutzeri. Strains S5, S7, and KM are obligately anaerobic selenate-respiring microorganisms, while strain pn1 is facultatively anaerobic. Besides respiring selenate, all these strains also respire nitrate.     

Reduced creatine-stimulated respiration in doxorubicin challenged mitochondria: particular sensitivity of the heart

Doxorubicin (DXR) belongs to the most efficient anticancer drugs. However, its use is limited by a risk of cardiotoxicity, which is not completely understood. Recently, we have shown that DXR impairs essential properties of purified mitochondrial creatine kinase (MtCK), with cardiac isoenzyme (sMtCK) being particularly sensitive. In this study we assessed the effects of DXR on respiration of isolated structurally and functionally intact heart mitochondria, containing sMtCK, in the presence and absence of externally added creatine (Cr), and compared these effects with the response of brain mitochondria expressing uMtCK, the ubiquitous, non-muscle MtCK isoenzyme. DXR impaired respiration of isolated heart mitochondria already after short-term exposure (minutes), affecting both ADP- and Cr-stimulated respiration. During a first short time span (minutes to 1 h), detachment of MtCK from membranes occurred, while a decrease of MtCK activity related to oxidative damage was only observed after longer exposure (several hours). The early inhibition of Cr-stimulated respiration, in addition to impairment of components of the respiratory chain involves a partial disturbance of functional coupling between MtCK and ANT, likely due to interaction of DXR with cardiolipin leading to competitive inhibition of MtCK/membrane binding. The relevance of these findings for the regulation of mitochondrial energy production in the heart, as well as the obvious differences of DXR action in the heart as compared to brain tissue, is discussed.     

Solution structure of Domains IVa and V of the tau subunit of Escherichia coli DNA polymerase III and interaction with the alpha subunit

The solution structure of the C-terminal Domain V of the τ subunit of E. coli DNA polymerase III was determined by nuclear magnetic resonance (NMR) spectroscopy. The fold is unique to τ subunits. Amino acid sequence conservation is pronounced for hydrophobic residues that form the structural core of the protein, indicating that the fold is representative for τ subunits from a wide range of different bacteria. The interaction between the polymerase subunits τ and α was studied by NMR experiments where α was incubated with full-length C-terminal domain (τ_C16), and domains shortened at the C-terminus by 11 and 18 residues, respectively. The only interacting residues were found in the C-terminal 30-residue segment of τ, most of which is structurally disordered in free τ_C16. Since the N- and C-termini of the structured core of τ_C16 are located close to each other, this limits the possible distance between α and the pentameric δτ₂γδ′ clamp–loader complex and, hence, between the two α subunits involved in leading- and lagging-strand DNA synthesis. Analysis of an N-terminally extended construct (τ_C22) showed that τ_C14 presents the only part of Domains IVa and V of τ which comprises a globular fold in the absence of other interaction partners.     

Nonenzymatic chromophore attachment in biliproteins: conformational control by the detergent Triton X-100

While chromophore attachment to alpha-subunits of cyanobacterial biliproteins has been studied in some detail, little is known about this process in beta-subunits. The ones of phycoerythrocyanin and C-phycocyanin each carry two phycocyanobilin (PCB) chromophores covalently attached to cysteins beta84 and beta155. The differential nonenzymatic reconstitution of PCB to the apoproteins, PecA, PecB, CpcA and CpcB, as well as to mutant proteins of the beta-subunits lacking either one of the two binding cysteins, was studied using overexpression of the respective genes. PCB adds selectively to Cys-84 of CpcA, CpcB, PecA, and PecB, but the bound chromophore has a nonnative configuration, and in the case of CpcA, is partly oxidized to mesobiliverdin (MBV). The oxidation is independent of thiols but can be suppressed by ascorbate. The addition to Cys-beta84 is suppressed in the presence of detergents like Triton X-100, in favor of an addition to Cys-beta155 yielding the correctly bound chromophore. Triton X-100 also inhibits oxidation of the chromophore during addition to CpcA. The effect of Triton X-100 was studied on the isolated components of the reconstitution system. Absorption, fluorescence and circular dichroism spectra indicate a major conformational change of the chromophore upon addition of the detergent, which probably controls the site selectivity of the addition reaction, and inhibits the oxidation of PCB to MBV.     

Sensitivity of Leishmania braziliensis promastigotes to meglumine antimoniate (glucantime) is higher than that of other Leishmania species and correlates with response to therapy in American tegumentary leishmaniasis

The first line drugs for the treatment of leishmaniasis are antimonial derivatives. Poor clinical response may be credited to factors linked to the host, the drug, or the parasite. We determined the sensitivity of Leishmania sp. promastigotes and amastigotes by counting parasites exposed to increasing concentrations of meglumine antimoniate (Glucantime). Leishmania braziliensis promastigotes were significantly more sensitive than those belonging to other species. The sensitivity of L. braziliensis isolates from patients with unfavorable clinical outcome, such as therapeutic failure or relapse, was significantly lower than those from patients who had clinical cure. Poor clinical response to therapy (therapeutic failure or relapse) was also associated with inadequate antimonial therapy. We also found a significant and positive correlation between promastigotes and intracellular amastigotes with regard to their in vitro susceptibilities to meglumine antimoniate. Our data provide evidence for an association between the sensitivity of promastigotes to antimonials in vitro and clinical response to therapy in American tegumentary leishmaniasis. The high sensitivity of the local L. braziliensis to meglumine antimoniate in vitro provides an explanation for the good clinical response of cutaneous leishmaniasis in the municipality of Rio de Janeiro, Brazil, even when low-dose regimens are employed.     

Trifolium pratense isoflavones in the treatment of menopausal hot flushes: A systematic review and meta-analysis

OBJECTIVE: To critically assess the evidence of supplements containing Trifolium pratense (red clover) isoflavones in the reduction of hot flush frequency in menopausal women. DATA SOURCES: Systematic literature searches were performed in (Medline (1951 - April 2006), Embase (1974 - April 2006), CINAHL (1982 - April 2006), Amed (1985 - April 2006) and The Cochrane Library (Issue 2, 2006). Reference lists located were checked for further relevant publications. Experts in the field and manufacturers of identified products were contacted for unpublished material. No language restrictions were imposed. REVIEW METHODS: Studies were selected according to predefined inclusion and exclusion criteria. All randomized clinical trials of monopreparations containing T. pratense isoflavones for treating hot flushes were included. Study selection, data extraction and validation were performed by at least two reviewers with disagreements being settled by discussion. Weighted means and 95% confidence intervals were calculated and sensitivity analyses were performed. RESULTS: Seventeen potentially relevant articles were retrieved for further evaluation. Five were suitable for inclusion in the meta-analysis. The meta-analysis indicates a reduction in hot flush frequency in the active treatment group (40-82 mg daily) compared with the placebo group (weighted mean difference -1.5 hot flushes daily; 95% CI -2.94 to 0.03; p=0.05). CONCLUSION: There is evidence of a marginally significant effect of T. pratense isoflavones for treating hot flushes in menopausal women. Whether the size of this effect can be considered clinically relevant is unclear. Whereas there is no apparent evidence of adverse events during short-term use, there are no available data on the safety of long-term administration.     

The Formation of Endoderm-Derived Taste Sensory Organs Requires a Pax9-Dependent Expansion of Embryonic Taste Bud Progenitor Cells

In mammals, taste buds develop in different regions of the oral cavity. Small epithelial protrusions form fungiform papillae on the ectoderm-derived dorsum of the tongue and contain one or few taste buds, while taste buds in the soft palate develop without distinct papilla structures. In contrast, the endoderm-derived circumvallate and foliate papillae located at the back of the tongue contain a large number of taste buds. These taste buds cluster in deep epithelial trenches, which are generated by intercalating a period of epithelial growth between initial placode formation and conversion of epithelial cells into sensory cells. How epithelial trench formation is genetically regulated during development is largely unknown. Here we show that Pax9 acts upstream of Pax1 and Sox9 in the expanding taste progenitor field of the mouse circumvallate papilla. While a reduced number of taste buds develop in a growth-retarded circumvallate papilla of Pax1 mutant mice, its development arrests completely in Pax9-deficient mice. In addition, the Pax9 mutant circumvallate papilla trenches lack expression of K8 and Prox1 in the taste bud progenitor cells, and gradually differentiate into an epidermal-like epithelium. We also demonstrate that taste placodes of the soft palate develop through a Pax9-dependent induction. Unexpectedly, Pax9 is dispensable for patterning, morphogenesis and maintenance of taste buds that develop in ectoderm-derived fungiform papillae. Collectively, our data reveal an endoderm-specific developmental program for the formation of taste buds and their associated papilla structures. In this pathway, Pax9 is essential to generate a pool of taste bud progenitors and to maintain their competence towards prosensory cell fate induction.     

Colostrum from Cows Immunized with a Vaccine Associated with Bovine Neonatal Pancytopenia Contains Allo-Antibodies that Cross-React with Human MHC-I Molecules

In 2006, a new haemorrhagic syndrome affecting newborn calves, Bovine Neonatal Pancytopenia (BNP), was reported in southern Germany. It is characterized by severe bleeding, destruction of the red bone marrow, and a high case fatality rate. The syndrome is caused by alloreactive, maternal antibodies that are ingested by the calf with colostrum and result from a dam vaccination with one particular vaccine against Bovine-Viral-Diarrhoea-Virus. Because bovine colostrum is increasingly gaining interest as a dietary supplement for human consumption, the current study was initiated to elucidate whether BNP alloantibodies from BNP dams (i.e. animals that gave birth to a BNP-affected calf) cross-react with human cells, which could pose a health hazard for human consumers of colostral products. The present study clearly demonstrates that BNP alloantibodies cross-react with human lymphocytes in vitro. In agreement with previous reports on BNP, the cross-reactive antibodies are specific for MHC-I molecules, and sensitize opsonised human cells for in vitro complement lysis. Cross-reactive antibodies are present in serum and colostrum of individual BNP dams. They can be traced in commercial colostrum powder manufactured from cows immunized with the vaccine associated with BNP, but are absent from commercial powder manufactured from colostrum excluding such vaccinated cows. In humans alloreactive, MHC-I specific antibodies are generally not believed to cause severe symptoms. However, to minimize any theoretical risk for human consumers, manufacturers of bovine colostrum for human consumption should consider using only colostrum from animals that have not been exposed to the vaccine associated with BNP.