YbeA is the m3Psi methyltransferase RlmH that targets nucleotide 1915 in 23S rRNA

Pseudouridines in the stable RNAs of Bacteria are seldom subjected to further modification. There are 11 pseudouridine (Ψ) sites in Escherichia coli rRNA, and further modification is found only at Ψ1915 in 23S rRNA, where the N-3 position of the base becomes methylated. Here, we report the identity of the E. coli methyltransferase that specifically catalyzes methyl group addition to form m³Ψ1915. Analyses of E. coli rRNAs using MALDI mass spectrometry showed that inactivation of the ybeA gene leads to loss of methylation at nucleotide Ψ1915. Methylation is restored by complementing the knockout strain with a plasmid-encoded copy of ybeA. Homologs of the ybeA gene, and thus presumably the ensuing methylation at nucleotide m³Ψ1915, are present in most bacterial lineages but are essentially absent in the Archaea and Eukaryota. Loss of ybeA function in E. coli causes a slight slowing of the growth rate. Phylogenetically, ybeA and its homologs are grouped with other putative S-adenosylmethionine-dependent, SPOUT methyltransferase genes in the Cluster of Orthologous Genes COG1576; ybeA is the first member to be functionally characterized. The YbeA methyltransferase is active as a homodimer and docks comfortably into the ribosomal A site without encroaching into the P site. YbeA makes extensive interface contacts with both the 30S and 50S subunits to align its active site cofactor adjacent to nucleotide Ψ1915. Methylation by YbeA (redesignated RlmH for rRNA large subunit methyltransferase H) possibly functions as a stamp of approval signifying that the 50S subunit has engaged in translational initiation.     

Genotoxicity of alpha-asarone analogues

The role of cholesterol in the formation of atherosclerotic lesions during hypercholesterolemia has been confirmed. alpha-Asarone is a substance of a potent hypolipidemic activity which is isolated from plants. We previously described the synthesis of several alpha-asarone analogues exhibiting hypolipidemic and antiplatelet activity. Genotoxic activity of four selected alpha-asarone analogues was theoretically evaluated based on quantum-mechanical method for calculation of enthalpy of carbocations formation (DeltaH(R)) according to the Testa's method. In the present paper, we evaluated the mutagenic and genotoxic activity of alpha-asarone isomers 2-5 based on the reference Ames test and micronucleus test. Results obtained in the study show that tested isomers were non-mutagenic, however, they exhibited growing cytotoxic activity. Relationship between the heat of formation of their putative metabolic intermediates and mutagenic/genotoxic activity was not confirmed.     

Used of oral antidiabetic agents in pediatric patients - own observations

Our present investigation demonstrates that in adolescents with various impaired glucose homeostasis oral antidiabetic agents can be used to improve glucose metabolism. Metformin is widely used in pediatric patients and is considered to be the most effective oral agent. Metformin is beneficial in improving glucose tolerance and insulin sensitivity, in lowering insulinemia, and in reducing elevated androgen levels. Addition of metformin to insulin in pediatric patients with type 1 diabetes mellitus improves metabolic control. Metformin acts by promoting glucose utilization and reducing hepatic glucose production. In many patients with type 2 diabetes, hyperglycemia can be reduced with appropriate changes in diet and exercise, however, some patients with type 2 diabetes and insulin resistance syndromes need pharmacological therapy to improve their metabolic control. The first oral agent concerned to use should be metformin. More severe pancreatic b-cell dysfunction in the group of children requires insulin therapy. Some forms of monogenic diabetes can be successfully managed by sulphonylurea agents. Metformin should be considered a first-line agent in girls with PCOS.     

Sequence-structure-function analysis of the bifunctional enzyme MnmC that catalyses the last two steps in the biosynthesis of hypermodified nucleoside mnm5s2U in tRNA

MnmC catalyses the last two steps in the biosynthesis of 5-methylaminomethyl-2-thiouridine (mnm(5)s(2)U) in tRNA. Previously, we reported that this bifunctional enzyme is encoded by the yfcK open reading frame in the Escherichia coli K12 genome. However, the mechanism of its activity, in particular the potential structural and functional dependence of the domains responsible for catalyzing the two modification reactions, remains unknown. With the aid of the protein fold-recognition method, we constructed a structural model of MnmC in complex with the ligands and target nucleosides and studied the role of individual amino acids and entire domains by site-directed and deletion mutagenesis, respectively. We found out that the N-terminal domain contains residues responsible for binding of the S-adenosylmethionine cofactor and catalyzing the methylation of nm(5)s(2)U to form mnm(5)s(2)U, while the C-terminal domain contains residues responsible for binding of the FAD cofactor. Further, point mutants with compromised activity of either domain can complement each other to restore a fully functional enzyme. Thus, in the conserved fusion protein MnmC, the individual domains retain independence as enzymes. Interestingly, the N-terminal domain is capable of independent folding, while the isolated C-terminal domain is incapable of folding on its own, a situation similar to the one reported recently for the rRNA modification enzyme RsmC.     

Mycobacterium tuberculosis Is Able To Accumulate and Utilize Cholesterol

It is expected that the obligatory human pathogen Mycobacterium tuberculosis must adapt metabolically to the various nutrients available during its cycle of infection, persistence, and reactivation. Cholesterol, which is an important part of the mammalian cytoplasmic membrane, is a potential energy source. Here, we show that M. tuberculosis grown in medium containing a carbon source other than cholesterol is able to accumulate cholesterol in the free-lipid zone of its cell wall. This cholesterol accumulation decreases the permeability of the cell wall for the primary antituberculosis drug, rifampin, and partially masks the mycobacterial surface antigens. Furthermore, M. tuberculosis was able to grow on mineral medium supplemented with cholesterol as the sole carbon source. Targeted disruption of the Rv3537 (kstD) gene inhibited growth due to inactivation of the cholesterol degradation pathway, as evidenced by accumulation of the intermediate, 9-hydroxy-4-androstene-3,17-dione. Our findings that M. tuberculosis is able to accumulate cholesterol in the presence of alternative nutrients and use it when cholesterol is the sole carbon source in vitro may facilitate future studies into the pathophysiology of this important deadly pathogen.Mycobacterium tuberculosis, the causative agent of tuberculosis, is a very successful pathogen that infects one-third of the human population (21). Only 10% of primary infected individuals develop active disease during their lifetimes. Tubercle bacilli are able to persist in a dormant state, from which they may reactivate and induce the contagious disease state (13). In asymptomatic hosts, M. tuberculosis exists in reservoirs called granulomas, which are cellular aggregates that restrict bacterial spreading (40). Granulomas are organized collections of mature macrophages that exhibit a certain typical morphology and that arise in response to persistent intracellular pathogens (1, 4). Pathogenic mycobacteria can induce the formation of foamy macrophages filled with lipid-containing bodies; these have been postulated to act as a secure, nutrient-rich reservoir for tubercle bacilli (31). Moreover, M. tuberculosis DNA has been detected in fatty tissues surrounding the kidneys, as well as those of the stomach, lymph nodes, heart, and skin. Tubercle bacilli are able to enter adipocytes, where they accumulate within intracytoplasmic lipid inclusions and survive in a nonreplicating state (26). In vivo, it is expected that M. tuberculosis adapts metabolically to nutrient-poor conditions characterized by glucose deficiency and an abundance of fatty acids (25, 26). The presence of a complex repertoire of lipid metabolism genes in the genome of M. tuberculosis suggests that lipids, including steroids, are important alternative carbon and energy sources for this pathogen (7).One attractive potential alternative nutrient that is readily available in the mammalian host is cholesterol, a major sterol of the plasma membrane. The presence of cholesterol in lipid rafts is required in order for microorganisms to enter the intracellular compartment (14). Studies have shown that cholesterol is essential for the uptake of mycobacteria by macrophages, and it has been found to accumulate at the site of M. tuberculosis entry (2, 12, 30). Moreover, cholesterol depletion overcomes the phagosome maturation block experienced by Mycobacterium avium-infected macrophages (10).It is well known that cholesterol can be utilized by fast-growing, nonpathogenic mycobacteria (5, 20, 22), but it was previously thought that pathogenic mycobacteria might not be able to use cholesterol as a carbon and energy source (3). Recently, however, bioinformatic analysis identified a cassette of cholesterol catabolism genes in actinomycetes, including the M. tuberculosis complex (41). Microarray analysis of Rhodococcus sp. grown in the presence of cholesterol revealed the upregulation of 572 genes, most of which fell within six clearly discernible clusters (41). Most of the identified genes had significant homology to known steroid degradation genes from other organisms and were distributed within a single 51-gene cluster that appears to be very similar to a cluster present in the genome of M. tuberculosis (41). Many of the cholesterol-induced genes had been previously selected by transposon site hybridization analysis of genes that are essential for survival of tubercle bacilli (33) and/or are upregulated in gamma interferon-activated macrophages (37, 42). It was also demonstrated that the M. tuberculosis complex can grow on mineral medium with cholesterol as a primary source of carbon (27, 41). Moreover, the growth of tubercle bacilli on cholesterol was significantly affected by knockout of the mce4 gene, which encodes an ABC transporter responsible for cholesterol uptake (24, 27). Earlier studies had shown that disruption of mce4 attenuated bacterial growth in the spleens of infected animals that had developed adaptive immunity (17, 35).In the present study, we demonstrate for the first time that M. tuberculosis utilizes cholesterol via the 4-androstene-3,17-dione/1,4-androstadiene-3,17-dione pathway (AD/ADD) and that this process requires production of an intact KstD enzyme. We also show that tubercle bacilli growing in medium containing an alternative carbon source can accumulate cholesterol in the free-lipid zone of their cell walls, and this accumulation affects cell wall permeability.     

Mutations in CNNM4 Cause Recessive Cone-Rod Dystrophy with Amelogenesis Imperfecta

Cone-rod dystrophies are inherited dystrophies of the retina characterized by the accumulation of deposits mainly localized to the cone-rich macular region of the eye. Dystrophy can be limited to the retina or be part of a syndrome. Unlike nonsyndromic cone-rod dystrophies, syndromic cone-rod dystrophies are genetically heterogeneous with mutations in genes encoding structural, cell-adhesion, and transporter proteins. Using a genome-wide single-nucleotide polymorphism (SNP) haplotype analysis to fine map the locus and a gene-candidate approach, we identified homozygous mutations in the ancient conserved domain protein 4 gene (CNNM4) that either generate a truncated protein or occur in highly conserved regions of the protein. Given that CNNM4 is implicated in metal ion transport, cone-rod dystrophy and amelogenesis imperfecta may originate from abnormal ion homeostasis.     

The immunomodulatory sphingosine 1-phosphate analog FTY720 reduces lesion size and improves neurological outcome in a mouse model of cerebral ischemia

Cerebral ischemia is accompanied by fulminant cellular and humoral inflammatory changes in the brain which contribute to lesion development after stroke. A tight interplay between the brain and the peripheral immune system leads to a biphasic immune response to stroke consisting of an early activation of peripheral immune cells with massive production of proinflammatory cytokines followed by a systemic immunosuppression within days of cerebral ischemia that is characterized by massive immune cell loss in spleen and thymus. Recent work has documented the importance of T lymphocytes in the early exacerbation of ischemic injury. The lipid signaling mediator sphingosine 1-phosphate-derived stable analog FTY720 (fingolimod) acts as an immunosuppressant and induces lymphopenia by preventing the egress of lymphocytes, especially T cells, from lymph nodes. We found that treatment with FTY720 (1 mg/kg) reduced lesion size and improved neurological function after experimental stroke in mice, decreased the numbers of infiltrating neutrophils, activated microglia/macrophages in the ischemic lesion and reduced immunohistochemical features of apoptotic cell death in the lesion.     

Identification of conserved modes of expression profiles during hippocampal development and neuronal differentiation in vitro

Gene expression profiles can be regarded as sums of simpler modes, analogous to the modes of a vibrating violin string. Decomposition of temporal gene expression profiles into modes by singular value decomposition (SVD) was reported before, but the question as to what degree the SVD modes can be interpreted in terms of biology remains open. We report and compare the results of SVD of published datasets from hippocampal development, neuronal differentiation in vitro, and a control time-series hippocampal dataset. We demonstrate that the first SVD mode reflects the magnitude of expression, interpretable on the Affymetrix platform. In the datasets from gene profiling of hippocampal development and neuronal differentiation, the second mode reflects a monotonous change in expression, either up- or down-regulation, in the time course of experiment. We demonstrate that the top two SVD modes are conserved between datasets and therefore, likely reflect properties of the underlying system (gene expression in hippocampus) rather than of a particular experiment or dataset. Our results also indicate that the magnitude of expression, and the direction of change in expression during hippocampal development, are uncorrelated, suggesting that they are regulated by largely independent mechanisms.