Molecular Insights into the Coding Region Determinant-binding Protein-RNA Interaction through Site-directed Mutagenesis in the Heterogeneous Nuclear Ribonucleoprotein-K-homology Domains

The ability of its four heterogeneous nuclear RNP-K-homology (KH) domains to physically associate with oncogenic mRNAs is a major criterion for the function of the coding region determinant-binding protein (CRD-BP). However, the particular RNA-binding role of each of the KH domains remains largely unresolved. Here, we mutated the first glycine to an aspartate in the universally conserved GXXG motif of the KH domain as an approach to investigate their role. Our results show that mutation of a single GXXG motif generally had no effect on binding, but the mutation in any two KH domains, with the exception of the combination of KH3 and KH4 domains, completely abrogated RNA binding in vitro and significantly retarded granule formation in zebrafish embryos, suggesting that any combination of at least two KH domains cooperate in tandem to bind RNA efficiently. Interestingly, we found that any single point mutation in one of the four KH domains significantly impacted CRD-BP binding to mRNAs in HeLa cells, suggesting that the dynamics of the CRD-BP-mRNA interaction vary over time in vivo. Furthermore, our results suggest that different mRNAs bind preferentially to distinct CRD-BP KH domains. The novel insights revealed in this study have important implications on the understanding of the oncogenic mechanism of CRD-BP as well as in the future design of inhibitors against CRD-BP function.     

Production-related petroleum microbiology: progress and prospects

Microbial activity in oil reservoirs is common. Methanogenic consortia hydrolyze low molecular weight components to methane and CO2, transforming light oil to heavy oil to bitumen. The presence of sulfate in injection water causes sulfate-reducing bacteria to produce sulfide. This souring can be reversed by nitrate, stimulating nitrate-reducing bacteria. Removing biogenic sulfide is important, because it contributes to pitting corrosion and resulting pipeline failures. Increased water production eventually makes oil production uneconomic. Microbial fermentation products can lower oil viscosity or interfacial tension and produced biomass can block undesired flow paths to produce more oil. These biotechnologies benefit from increased understanding of reservoir microbial ecology through new sequence technologies and help to decrease the environmental impact of oil production.     

Dosimetry in molecular nuclear therapy

Advanced personalized dosimetry for molecular nuclear therapy has been shown to be feasible in clinical practice. At the same time instrumentation and dosimetric software are still evolving at a high pace. Procedures developed so far differ in approach and sophistication, and standard operating procedures necessary for accurate patient specific dosimetry do not yet exist. For this reason we restricted ourselves to reviewing the literature and highlighting relevant developments.     

Hyperacidification of trans-Golgi network and endo/lysosomes in melanocytes by glucosylceramide-dependent V-ATPase activity

Sphingolipids are considered to play a key role in protein sorting and membrane trafficking. In melanocytic cells, sorting of lysosomal and melanosomal proteins requires the sphingolipid glucosylceramide (GlcCer). This sorting information is located in the lumenal domain of melanosomal proteins. We found that two processes dependent on lumenal pH, protein sialylation and lysosomal acid lipase (LAL) activity were aberrant in GM95 melanocyte cells, which do not produce glycosphingolipids. Using fluorescence lifetime imaging microscopy (FLIM), we found that the lumenal pH in the trans-Golgi network and lysosomes of wild-type melanocyte MEB4 cells are >1 pH unit lower than GM95 cells and fibroblasts. In addition to the lower pH found in vivo, the in vitro activity of the proton pump, the vacuolar-type H(+) -translocating ATPase (V-ATPase), was twofold higher in MEB4 compared to GM95 cells. The apparent K(i) for inhibition of the V-ATPase by concanamycin A and archazolid A, which share a common binding site on the c-ring, was lower in glycosphingolipid-deficient GM95 cells. No difference between the MEB4 and GM95 cells was found for the V-ATPase inhibitors apicularen A and salicylihalimide. We conclude that hyperacidification in MEB4 cells requires glycosphingolipids and propose that low pH is necessary for protein sorting and melanosome biogenesis. Furthermore, we suggest that glycosphingolipids are indirectly involved in protein sorting and melanosome biogenesis by stimulating the proton pump, possibly through binding of GlcCer. These experiments establish, for the first time, a link between pH, glycosphingolipids and melanosome biogenesis in melanocytic MEB4 cells, to suggest a role for glycosphingolipids in hyperacidification in melanocytes.     

The overlooked role of a biotin precursor for marine bacteria - desthiobiotin as an escape route for biotin auxotrophy

Biotin (vitamin B₇) is involved in a wide range of essential biochemical reactions and a crucial micronutrient that is vital for many pro- and eukaryotic organisms. The few biotin measurements in the world’s oceans show that availability is subject to strong fluctuations. Numerous marine microorganisms exhibit biotin auxotrophy and therefore rely on supply by other organisms. Desthiobiotin is the primary precursor of biotin and has recently been detected at concentrations similar to biotin in seawater. The last enzymatic reaction in the biotin biosynthetic pathway converts desthiobiotin to biotin via the biotin synthase (BioB). The role of desthiobiotin as a precursor of biotin synthesis in microbial systems, however, is largely unknown. Here we demonstrate experimentally that bacteria can overcome biotin auxotrophy if they retain the bioB gene and desthiobiotin is available. A genomic search of 1068 bacteria predicts that the biotin biosynthetic potential varies greatly among different phylogenetic groups and that 20% encode solely bioB and thus can potentially overcome biotin auxotrophy. Many Actino- and Alphaproteobacteria cannot synthesize biotin de novo, but some possess solely bioB, whereas the vast majority of Gammaproteobacteria and Flavobacteriia exhibit the last four crucial biotin synthesis genes. We detected high intra- and extracellular concentrations of the precursor relative to biotin in the prototrophic bacterium, Vibrio campbellii, with extracellular desthiobiotin reaching up to 1.09 ± 0.15*10⁶ molecules per cell during exponential growth. Our results provide evidence for the ecological role of desthiobiotin as an escape route to overcome biotin auxotrophy for bacteria in the ocean and presumably in other ecosystems.Subject terms: Water microbiology, Ecosystem ecology, Marine microbiology     

Mortality and Potential Years of Life Lost Attributable to Alcohol Consumption by Race and Sex in the United States in 2005

Background

Alcohol has been linked to health disparities between races in the US; however, race-specific alcohol-attributable mortality has never been estimated. The objective of this article is to estimate premature mortality attributable to alcohol in the US in 2005, differentiated by race, age and sex for people 15 to 64 years of age.

Methods and Findings

Mortality attributable to alcohol was estimated based on alcohol-attributable fractions using indicators of exposure from the National Epidemiologic Survey on Alcohol and Related Conditions and risk relations from the Comparative Risk Assessment study. Consumption data were corrected for undercoverage (the observed underreporting of alcohol consumption when using survey as compared to sales data) using adult per capita consumption from WHO databases. Mortality data by cause of death were obtained from the US Department of Health and Human Services. For people 15 to 64 years of age in the US in 2005, alcohol was responsible for 55,974 deaths (46,461 for men; 9,513 for women) representing 9.0% of all deaths, and 1,288,700 PYLL (1,087,280 for men; 201,420 for women) representing 10.7% of all PYLL. Per 100,000 people, this represents 29 deaths (29 for White; 40 for Black; 82 for Native Americans; 6 for Asian/Pacific Islander) and 670 PYLL (673 for White; 808 for Black; 1,808 for Native American; 158 for Asian/Pacific Islander). Sensitivity analyses showed a lower but still substantial burden without adjusting for undercoverage.

Conclusions

The burden of mortality attributable to alcohol in the US is unequal among people of different races and between men and women. Racial differences in alcohol consumption and the resulting harms explain in part the observed disparities in the premature mortality burden between races, suggesting the need for interventions for specific subgroups of the population such as Native Americans.     

Biosynthesis and genetic control of isovitexin 2″-O-arabinoside in petals of Silene dioica

In petals of Silene dioica plants, an enzyme has been demonstrated which catalyses the transfer of the arabinose moiety of UDP-arabinose to the hydroxyl group on the 2″-position of the carbon-carbon bound glucose of isovitexin. The presence of this arabinosyltransferase activity is controlled by the dominant allele gl^A. Maximal activity takes place at pH 7.2–7.5; the reaction is stimulated by the divalent metal ions Mg and Mn. For optimal solubilization of the enzyme, Triton X-100 is necessary. Substrate specificity and kinetic behaviour have been investigated.     

Flavour formation by lactic acid bacteria and biochemical flavour profiling of cheese products

Flavour development in dairy fermentations, most notably cheeses, results from a series of (bio)chemical processes in which the starter cultures provide the enzymes. Particularly the enzymatic degradation of proteins (caseins) leads to the formation of key-flavour components, which contribute to the sensory perception of dairy products. More specifically, caseins are degraded into peptides and amino acids and the latter are major precursors for volatile aroma compounds. In particular, the conversion of methionine, the aromatic and the branched-chain amino acids are crucial. A lot of research has focused on the degradation of caseins into peptides and free amino acids, and more recently, enzymes involved in the conversion of amino acids were identified. Most data are generated on Lactococcus lactis, which is the predominant organism in starter cultures used for cheese-making, but also Lactobacillus, Streptococcus, Propionibacterium and species used for surface ripening of cheeses are characterised in their flavour-forming capacity. In this paper, various enzymes and pathways involved in flavour formation will be highlighted and the impact of these findings for the development of industrial starter cultures will be discussed.