An Intertwined Evolutionary History of Methanogenic Archaea and Sulfate Reduction

Hydrogenotrophic methanogenesis and dissimilatory sulfate reduction, two of the oldest energy conserving respiratory systems on Earth, apparently could not have evolved in the same host, as sulfite, an intermediate of sulfate reduction, inhibits methanogenesis. However, certain methanogenic archaea metabolize sulfite employing a deazaflavin cofactor (F₄₂₀)-dependent sulfite reductase (Fsr) where N- and C-terminal halves (Fsr-N and Fsr-C) are homologs of F₄₂₀H₂ dehydrogenase and dissimilatory sulfite reductase (Dsr), respectively. From genome analysis we found that Fsr was likely assembled from freestanding Fsr-N homologs and Dsr-like proteins (Dsr-LP), both being abundant in methanogens. Dsr-LPs fell into two groups defined by following sequence features: Group I (simplest), carrying a coupled siroheme-[Fe₄-S₄] cluster and sulfite-binding Arg/Lys residues; Group III (most complex), with group I features, a Dsr-type peripheral [Fe₄-S₄] cluster and an additional [Fe₄-S₄] cluster. Group II Dsr-LPs with group I features and a Dsr-type peripheral [Fe₄-S₄] cluster were proposed as evolutionary intermediates. Group III is the precursor of Fsr-C. The freestanding Fsr-N homologs serve as F₄₂₀H₂ dehydrogenase unit of a putative novel glutamate synthase, previously described membrane-bound electron transport system in methanogens and of assimilatory type sulfite reductases in certain haloarchaea. Among archaea, only methanogens carried Dsr-LPs. They also possessed homologs of sulfate activation and reduction enzymes. This suggested a shared evolutionary history for methanogenesis and sulfate reduction, and Dsr-LPs could have been the source of the oldest (3.47-Gyr ago) biologically produced sulfide deposit.     

Lactate is a mediator of metabolic cooperation between stromal carcinoma associated fibroblasts and glycolytic tumor cells in the tumor microenvironment

Human mesenchymal stem cells (hMSCs) are bone marrow-derived stromal cells, which play a role in tumor progression. We have shown earlier that breast cancer cells secrete higher levels of interleukin-6 (IL-6) under hypoxia, leading to the recruitment of hMSCs towards hypoxic tumor cells. We found that (i) MDA-MB-231 cells secrete significantly higher levels of lactate (3-fold more) under hypoxia (1% O(2)) than under 20% O(2) and (ii) lactate recruits hMSCs towards tumor cells by activating signaling pathways to enhance migration. The mRNA and protein expression of functional MCT1 in hMSCs is increased in response to lactate exposure. Thus, we hypothesized that hMSCs and stromal carcinoma associated fibroblasts (CAFs) in the tumor microenvironment have the capacity to take up lactate expelled from tumor cells and use it as a source of energy. Our (13)C NMR spectroscopic measurements indicate that (13)C-lactate is converted to (13)C-alpha ketoglutarate in hMSCs and CAFs supporting this hypothesis. To our knowledge this is the first in vitro model system demonstrating that hMSCs and CAFs can utilize lactate produced by tumor cells.     

Identification of Bacillus anthracis Spore Component Antigens Conserved across Diverse Bacillus cereus sensu lato Strains

We sought to identify proteins in the Bacillus anthracis spore, conserved in other strains of the closely related Bacillus cereus group, that elicit an immune response in mammals. Two high throughput approaches were used. First, an in silico screening identified 200 conserved putative B. anthracis spore components. A total of 192 of those candidate genes were expressed and purified in vitro, 75 of which reacted with the rabbit immune sera generated against B. anthracis spores. The second approach was to screen for cross-reacting antigens in the spore proteome of 10 diverse B. cereus group strains. Two-dimensional electrophoresis resolved more than 200 protein spots in each spore preparation. About 72% of the protein spots were found in all the strains. 18 of these conserved proteins reacted against anti-B. anthracis spore rabbit immune sera, two of which (alanine racemase, Dal-1 and the methionine transporter, MetN) overlapped the set of proteins identified using the in silico screen. A conserved repeat domain protein (Crd) was the most immunoreactive protein found broadly across B. cereus sensu lato strains. We have established an approach for finding conserved targets across a species using population genomics and proteomics. The results of these screens suggest the possibility of a multiepitope antigen for broad host range diagnostics or therapeutics against Bacillus spore infection.The anthrax causing bacterium Bacillus anthracis is a member of the Bacillus cereus sensu lato (s.l.)¹ group, a term given to the polyphyletic species consisting of Bacillus thuringiensis, Bacillus cereus, Bacillus mycoides, Bacillus weihenstephanensis, and Bacillus pseudomycoides (1). Genomics studies of B. cereus s.l. strains have shown a similar chromosomal gene composition within this group (2–7). Many phenotypes that distinguish B. cereus s.l. members, such as crystalline toxin production (8), emesis in humans (9), and anthrax virulence (10), are encoded by genes on large plasmids. Experimental conjugative transfer of plasmids between B. cereus s.l. strains has been demonstrated in vitro, in complex media, and in vector species (11–13). Therefore there is a concern about transfer of virulence genes between genetic backgrounds creating new pathogen lineages. In this regard, there is an emerging evidence of natural dissemination of the pXO1 and pXO2 plasmids that encode the anthrax lethal toxin and capsule, respectively. For example, B. cereus G9241 carries a pXO1 plasmid and lethal toxin genes almost identical to those in B. anthracis (6), and a B. cereus strain, which causes anthrax-like illness in African great apes, apparently contains both pXO1 and pXO2 plasmids (14).The infectious agent of most if not all human B. cereus s.l. diseases is the spore. The spore is a dormant, environmentally resistant structure that persists in nutrient- or water-limiting conditions. Anthrax infection occurs after introduction of the B. anthracis spore into a skin abrasion or via inhalation or ingestion (10). The spore germinates inside host cells, and the resulting vegetative bacteria express toxins and capsules that elicit an immune response (10, 15, 16). Formation of the B. cereus spore involves asymmetric cell division during which a copy of the genome is partitioned into each of the sister cells. The smaller cell (prespore) develops into mature endospore, and the larger cell (mother cell) contributes to the differentiation process but undergoes autolysis following its completion to release the endospore into the surrounding medium. Synthesis of cortex, coat, and exosporium are a function mainly of the mother cell. The cortex and coat layers are in close proximity to one another, whereas the exosporium tends to appear as an irregularly shaped, loosely attached, balloon-like layer (17–20). The coat and the exosporium contribute to the remarkable resistance of spores to extreme physical and chemical stresses including the exposure to extraterrestrial conditions (21, 22). Recent work on the structure, composition, assembly, and function of the spore coat and exosporium of pathogenic organisms like B. anthracis and B. cereus have highlighted the crucial link that exists between the origin of these layers (19, 23). There are differences in the appearance and thickness of the coat layers among the spores of various strains and species. In some B. thuringiensis strains, the inner coat is laminated but consists of a patchwork of striated packets, appearing either stacked or comblike, and the outer coat is granular (24), whereas in B. anthracis and other B. cereus s.l. isolates the coat appears compact (25–27). The coat layers comprise about 30% of the total proteins present in the spore (19, 28). Intraspecies variation in the structure and composition of the spore surface layers may reflect the environmental conditions under which these spores are formed (29–31).Because the spore is crucial to infection and persistence of B. anthracis and its close relatives, we undertook an investigation of its protein profile variability across the B. cereus s.l. group. Our goal in this study was to identify conserved antigenic spore proteins that may be transitioned in the future as candidates for immunodiagnostics, therapeutics, or vaccines. We used two high throughput approaches: genome-based bioinformatics analysis and comparative proteomics analysis of spores of B. cereus s.l. to select conserved targets. Our analysis revealed a list of conserved spore proteins within B. cereus but relatively few cross-reacting antigens. Two of these spore conserved antigens (Crd and MetN) have not been described previously for B. anthracis.     

Directed evolution of a yeast arsenate reductase into a protein-tyrosine phosphatase

Arsenic, which is ubiquitous in the environment and comes from both geochemical and anthropogenic sources, has become a worldwide public health problem. Every organism studied has intrinsic or acquired mechanisms for arsenic detoxification. In Saccharomyces cerevisiae arsenate is detoxified by Acr2p, an arsenate reductase. Acr2p is not a phosphatase but is a homologue of CDC25 phosphatases. It has the HCX5R phosphatase motif but not the glycine-rich phosphate binding motif (GXGXXG) that is found in protein-tyrosine phosphatases. Here we show that creation of a phosphate binding motif through the introduction of glycines at positions 79, 81, and 84 in Acr2p resulted in a gain of phosphotyrosine phosphatase activity and a loss of arsenate reductase activity. Arsenate likely achieved geochemical abundance only after the atmosphere became oxidizing, creating pressure for the evolution of an arsenate reductase from a protein-tyrosine phosphatase. The ease by which an arsenate reductase can be converted into a protein-tyrosine phosphatase supports this hypothesis.     

Bidirectional silencing and DNA methylation-sensitive methylation-spreading properties of the Kcnq1 imprinting control region map to the same regions

The mechanisms underlying the phenomenon of genomic imprinting are poorly understood. Accumulating evidence suggests that imprinting control regions (ICR) associated with the imprinted genes play an important role in creation of imprinted expression domains by propagating parent-of-origin-specific epigenetic modifications. We have recently documented that the Kcnq1 ICR unidirectionally blocks enhancer-promoter communications in a methylation-dependent manner in Hep-3B and Jurkat cell lines. In this report we show that the Kcnq1 ICR harbors bidirectional silencing and methylation-sensitive methylation-spreading properties in a lineage-specific manner. We fine map both of these functions to two critical regions, and loss of one these regions results in loss of silencing as well as methylation spreading. The cell type-specific functions of the Kcnq1 ICR suggest binding of cell type-specific factors to various cis elements within the ICR. Fine mapping of the silencing and methylation-spreading functions to the same regions explains the fact that the silencing factors associated with this region primarily repress the neighboring genes and that methylation occurs as a consequence of silencing.     

Regulation of vascular endothelial growth factor production by Leydig cells in vitro: the role of protein kinase A and mitogen-activated protein kinase cascade

We previously reported the presence of vascular endothelial growth factor (VEGF) in testicular cells, and high concentrations of VEGF have been measured in semen, although its role in male reproduction remains obscure. In the present study we focus on understanding the mechanism of VEGF production by mouse Leydig cells cultured in vitro. Production of VEGF protein in medium by testicular cells was markedly increased by the addition of hCG in a time- and dose-dependent manner. Gonadotropin-stimulated VEGF production was mediated by cAMP-dependent protein kinase A (PKA), as evidenced by the effect of hCG being mimicked by 8Br-cAMP and being abolished in the presence of a PKA-specific inhibitor, H-89. Protein kinase C was not involved, as evidenced by phorbol 12-myristate 13-acetate having no influence on VEGF production by Leydig cells. In addition to hCG, atrial natriuretic peptide was also able to stimulate VEGF production, suggesting that cGMP is able to cross-activate PKA. A specific Src kinase inhibitor, PP2, could completely block the stimulatory effects of both gonadotropin and 8Br-cAMP on VEGF production by Leydig cells, implying an involvement of the Src kinase pathway. Furthermore, addition of U0126, an inhibitor of MEK 1/2, abolished the increase in VEGF production stimulated by both hCG and 8Br-cAMP. A similar inhibitory effect was observed by the addition of SB203580, a p38 mitogen-activated protein kinase inhibitor. Thus, in conclusion, Leydig cells are able to produce VEGF by a process under gonadotropic control, and PKA plays a key role in this process. Downstream of PKA, it appears that both MEK 1/2 and Src kinase-dependent pathways are involved, although further research will be necessary to determine the precise link between PKA and other kinases involved.     

A simple one-pot method for the synthesis of partially protected mono- and disaccharide building blocks using an orthoesterification-benzylation-orthoester rearrangement approach

A simple one-pot method is reported for making partially protected glycosyl acceptors from O-methyl or S-alkyl/aryl glycosides of D-glucose, D-galactose, D-arabinose, L-rhamnose, L-fucose and lactose via orthoester formation, benzylation and selective hydrolysis.     

Recent advances in molecular genetics of glaucoma

Glaucoma represents a heterogeneous group of optic neuropathies, with different genetic bases. It can affect all ages generally with a rise in intra-ocular pressure. Three major types of glaucoma have been reported: primary open angle glaucoma (POAG), primary acute closed angle glaucoma (PACG) and primary congenital glaucoma (PCG), as well as a few others associated with developmental abnormalities. In recent years impressive progress has been made in the molecular genetic studies of POAG and PCG. These include the discovery of three genes – Myocilin, Optineurin and CYP1B1 – defects in which results in Mendelian transmission of glaucoma. Identification of single nucleotide polymorphisms in multiple other genes that are associated with glaucoma and alteration of drug sensitivity are enriching our knowledge regarding the complex nature of the disease. This review attempts to present the recent progress made in the molecular genetics of glaucoma.     

Visualization of membrane protein domains by cryo-electron microscopy of dengue virus

Improved technology for reconstructing cryo-electron microscopy (cryo-EM) images has now made it possible to determine secondary structural features of membrane proteins in enveloped viruses. The structure of mature dengue virus particles was determined to a resolution of 9.5 A by cryo-EM and image reconstruction techniques, establishing the secondary structural disposition of the 180 envelope (E) and 180 membrane (M) proteins in the lipid envelope. The alpha-helical 'stem' regions of the E molecules, as well as part of the N-terminal section of the M proteins, are buried in the outer leaflet of the viral membrane. The 'anchor' regions of E and the M proteins each form antiparallel E-E and M-M transmembrane alpha-helices, leaving their C termini on the exterior of the viral membrane, consistent with the predicted topology of the unprocessed polyprotein. This is one of only a few determinations of the disposition of transmembrane proteins in situ and shows that the nucleocapsid core and envelope proteins do not have a direct interaction in the mature virus.