Mercury methylation independent of the acetyl-coenzyme A pathway in sulfate-reducing bacteria

Sulfate-reducing bacteria (SRB) in anoxic waters and sediments are the major producers of methylmercury in aquatic systems. Although a considerable amount of work has addressed the environmental factors that control methylmercury formation and the conditions that control bioavailability of inorganic mercury to SRB, little work has been undertaken analyzing the biochemical mechanism of methylmercury production. The acetyl-coenzyme A (CoA) pathway has been implicated as being key to mercury methylation in one SRB strain, Desulfovibrio desulfuricans LS, but this result has not been extended to other SRB species. To probe whether the acetyl-CoA pathway is the controlling biochemical process for methylmercury production in SRB, five incomplete-oxidizing SRB strains and two Desulfobacter strains that do not use the acetyl-CoA pathway for major carbon metabolism were assayed for methylmercury formation and acetyl-CoA pathway enzyme activities. Three of the SRB strains were also incubated with chloroform to inhibit the acetyl-CoA pathway. So far, all species that have been found to have acetyl-CoA activity are complete oxidizers that require the acetyl-CoA pathway for basic metabolism, as well as methylate mercury. Chloroform inhibits Hg methylation in these species either by blocking the methylating enzyme or by indirect effects on metabolism and growth. However, we have identified four incomplete-oxidizing strains that clearly do not utilize the acetyl-CoA pathway either for metabolism or mercury methylation (as confirmed by the absence of chloroform inhibition). Hg methylation is thus independent of the acetyl-CoA pathway and may not require vitamin B(12) in some and perhaps many incomplete-oxidizing SRB strains.     

The cAMP signaling pathway has opposing effects on Rac and Rho in B16F10 cells: implications for dendrite formation in melanocytic cells

A hallmark of melanocytic cells is their ability to form dendrites in response to growth factors and to ultraviolet irradiation. It is known that the cyclic adenosine monophosphate (cAMP) second messenger pathway stimulates melanocyte dendrite formation because agents that increase cAMP such as forskolin and dibutyrl cAMP induce dendrite formation in normal human and murine melanocytes and melanoma cell lines. The Rho family of guanosine triphosphate (GTP)-binding proteins regulates cytoskeletal reorganization in all cells tested and Rac and Rho have both been shown to regulate melanocyte dendrite formation. In this report, we analyzed the effect of cAMP on the activation of Rac and Rho and show that elevation of cAMP stimulates Rac and inhibits Rho in B16F10 cells. The Rho GTP-binding proteins have also been shown to either cross-activate or inhibit each other and in this report we show that Rac activates Rho in B16F10 cells. Microinjection of C3 botulinum exoenzyme toxin, an agent that specifically inactivates Rho or microinjection of constitutively active mutant Rac protein-induced dendricity in human melanocytes and in B16F10 and B16F1 murine melanoma cell lines. We conclude that cAMP-mediated dendrite formation in melanocytic cells is mediated through upregulation of Rac activity and downregulation of Rho activity.     

Differences in Elasticity of Vinculin-Deficient F9 Cells Measured by Magnetometry and Atomic Force Microscopy

We have investigated a mouse F9 embryonic carcinoma cell line, in which both vinculin genes were inactivated by homologous recombination, that exhibits defective adhesion and spreading [Collet al.(1995)Proc. Natl. Acad. Sci. USA92, 9161–9165]. Using a magnetometer and RGD-coated magnetic microbeads, we measured the local effect of loss and replacement of vinculin on mechanical force transfer across integrins. Vinculin-deficient F9Vin(−/−) cells showed a 21% difference in relative stiffness compared to wild-type cells. This was restored to near wild-type levels after transfection and constitutive expression of increasing amounts of vinculin into F9Vin(−/−) cells. In contrast, the transfection of vinculin constructs deficient in amino acids 1–288 (containing the talin- and α-actinin-binding site) or substituting tyrosine for phenylalanine (phosphorylation site, amino acid 822) in F9Vin(−/−) cells resulted in partial restoration of stiffness. Using atomic force microscopy to map the relative elasticity of entire F9 cells by 128 × 128 (n= 16,384) force scans, we observed a correlation with magnetometer measurements. These findings suggest that vinculin may promote cell adhesion and spreading by stabilizing focal adhesions and transferring mechanical stresses that drive cytoskeletal remodeling, thereby affecting the elastic properties of the cell.     

Investigating the Role of Gene-Gene Interactions in TB Susceptibility

Tuberculosis (TB) is the second leading cause of mortality from infectious disease worldwide. One of the factors involved in developing disease is the genetics of the host, yet the field of TB susceptibility genetics has not yielded the answers that were expected. A commonly posited explanation for the missing heritability of complex disease is gene-gene interactions, also referred to as epistasis. In this study we investigate the role of gene-gene interactions in genetic susceptibility to TB using a cohort recruited from a high TB incidence community from Cape Town, South Africa. Our discovery data set incorporates genotypes from a large a number of candidate gene studies as well as genome-wide data. After limiting our search space to pairs of putative TB susceptibility genes, as well as pairs of genes that have been curated in online databases as potential interactors, we use statistical modelling to identify pairs of interacting SNPs. We attempt to validate the top models identified in our discovery data set using an independent genome-wide TB case-control data set from The Gambia. A number of models were successfully validated, indicating that interplay between the NRG1 - NRG3, GRIK1 - GRIK3 and IL23R - ATG4C gene pairs may modify susceptibility to TB. Gene pairs involved in the NF-κB pathway were also identified in the discovery data set (SFTPD - NOD2, ISG15 - TLR8 and NLRC5 - IL12RB1), but could not be tested in the Gambian study group due to lack of overlapping data.     

Gene-gene interaction between tuberculosis candidate genes in a South African population

In a complex disease such as tuberculosis (TB) it is increasingly evident that gene-gene interactions play a far more important role in an individual??s susceptibility to develop the disease than single polymorphisms on their own, as one gene can enhance or hinder the expression of another gene. Gene-gene interaction analysis is a new approach to elucidate susceptibility to TB. The possibility of gene-gene interactions was assessed, focusing on 11 polymorphisms in nine genes (DC-SIGN, IFN-??, IFNGR1, IL-8, IL-1Ra, MBL, NRAMP1, RANTES, and SP-D) that have been associated with TB, some repeatedly. An optimal model, which best describes and predicts TB case?Ccontrol status, was constructed. Significant interactions were detected between eight pairs of variants. The models fitted the observed data extremely well, with p?<?0.0001 for all eight models. A highly significant interaction was detected between INFGR1 and NRAMP1, which is not surprising because macrophage activation is greatly enhanced by IFN-?? and IFN-?? response elements that are present in the human NRAMP1 promoter region, providing further evidence for their interaction. This study enabled us to test the theory that disease outcome may be due to interaction of several gene effects. With eight instances of statistically significant gene-gene interactions, the importance of epistasis is clearly identifiable in this study. Methods for studying gene-gene interactions are based on a multilocus and multigene approach, consistent with the nature of complex-trait diseases, and may provide the paradigm for future genetic studies of TB.     

Exercise Improves Body Fat,Lean Mass,and Bone Mass in Breast Cancer Survivors

Given the negative effects of a breast cancer diagnosis and its treatments on body weight and bone mass, we investigated the effects of a 6‐month randomized controlled aerobic exercise intervention vs. usual care on body composition in breast cancer survivors. Secondary aims were to examine the effects stratified by important prognostic and physiologic variables. Seventy‐five physically inactive postmenopausal breast cancer survivors were recruited through the Yale–New Haven Hospital Tumor Registry and randomly assigned to an exercise (n = 37) or usual care (n = 38) group. The exercise group participated in 150 min/week of supervised gym‐ and home‐based moderate‐intensity aerobic exercise. The usual care group was instructed to maintain their current physical activity level. Body composition was assessed at baseline and 6‐months through dual‐energy X‐ray absorptiometry (DXA) by one radiologist blinded to the intervention group of the participants. On an average, exercisers increased moderate‐intensity aerobic exercise by 129 min/week over and above baseline levels compared with 45 min/week among usual care participants (P < 0.001). Exercisers experienced decreases in percent body fat (P = 0.0022) and increases in lean mass (P = 0.047) compared with increases in body fat and decreases in lean mass in usual care participants. Bone mineral density (BMD) was also maintained among exercisers compared with a loss among usual care participants (P = 0.043). In summary, moderate‐intensity aerobic exercise, such as brisk walking, produces favorable changes in body composition that may improve breast cancer prognosis.