Conditional silencing of topoisomerase I gene of Mycobacterium tuberculosis validates its essentiality for cell survival

Topoisomerases are an important class of enzymes for regulating the DNA transaction processes. Mycobacterium tuberculosis (Mtb) is one of the most formidable pathogens also posing serious challenges for therapeutic interventions. The organism contains only one type IA topoisomerase (Rv3646c), offering an opportunity to test its potential as a candidate drug target. To validate the essentiality of M.tuberculosis topoisomerase I (TopoI(Mt)) for bacterial growth and survival, we have generated a conditionally regulated strain of topoI in Mtb. The conditional knockdown mutant exhibited delayed growth on agar plate. In liquid culture, the growth was drastically impaired when TopoI expression was suppressed. Additionally, novobiocin and isoniazid showed enhanced inhibitory potential against the conditional mutant. Analysis of the nucleoid revealed its altered architecture upon TopoI depletion. These studies establish the essentiality of TopoI for the M.tuberculosis growth and open up new avenues for targeting the enzyme.

GTP Cyclohydrolase I Gene Transfer Augments Intracellular Tetrahydrobiopterin in Human Endothelial Cells: Effects on Nitric Oxide Synthase Activity and Dimerisation.

Objectives: Tetrahydrobiopterin (BH4) is an essential cofactor for endothelial nitric oxide synthase (eNOS) activity. BH4 levels are regulated by de novo biosynthesis; the rate-limiting enzyme is GTP cyclohydrolase I (GTPCH). BH4 activates and promotes homodimerisation of purified eNOS protein, but the intracellular mechanisms underlying BH4-mediated eNOS regulation in endothelial cells remain less clear. We aimed to investigate the role of BH4 levels in intracellular eNOS regulation, by targeting the BH4 synthetic pathway as a novel strategy to modulate intracellular BH4 levels. Methods: We constructed a recombinant adenovirus, AdGCH, encoding human GTPCH. We infected human endothelial cells with AdGCH, investigated the changes in intracellular biopterin levels, and determined the effects on eNOS enzymatic activity, protein levels and dimerisation. Results: GTPCH gene transfer in EAhy926 endothelial cells increased BH4 >10-fold compared with controls (cells alone or control adenovirus infection), and greatly enhanced NO production in a dose-dependent, eNOS-specific manner. We found that eNOS was principally monomeric in control cells, whereas GTPCH gene transfer resulted in a striking increase in eNOS homodimerisation. Furthermore, the total amounts of both native eNOS protein and a recombinant eNOS–GFP fusion protein were significantly increased following GTPCH gene transfer. Conclusions: These findings suggest that GTPCH gene transfer is a valid approach to increase BH4 levels in human endothelial cells, and provide new evidence for the relative importance of different mechanisms underlying BH4-mediated eNOS regulation in intact human endothelial cells. Additionally, these observations suggest that GTPCH may be a rational target to augment endothelial BH4 and normalise eNOS activity in endothelial dysfunction states.

Molecular Medicine and Molecular Pathology for Haematologists:I. Gene Speak made Easy: An Overview of Genes and Gene Expression

Molecular Medicine and Molecular Pathology are integral parts of Haematology as we enter the new millennium. Their origins can be linked to fundamental developments in the basic sciences, particularly genetics, chemistry and biochemistry. The structure of DNA and the genetic code that it encrypts are the critical starting points to our understanding of these new disciplines. The genetic alphabet is a simple one, consisting of just 4 letters, buts its influence is crucial to human development and differentiation. The concept of a gene is not a new one but the Human Genome Project (a joint world-wide effort to characterise our entire genetic make-up) is providing an invaluable understanding of how genes function in normal cellular processes and pinpointing how disruption of these processes can lead to disease. Transcription and translation are the key events by which our genotype is converted to our phenotype (via a messenger RNA intermediate), producing the myriad proteins and enzymes which populate the cellular factory of our body. Unlike the bacterial or prokaryotic genome, the human genome contains a large amount of non coding DNA (less than 1% of our genome codes for proteins), and our genes are interrupted, with the coding regions or exons separated by non coding introns. Precise removal of the intronic material after transcription (though a process called splicing) is critical for efficient translation to occur. Incorrect splicing can lead to the generation of mutant proteins, which can have a dilaterious effect on the phenotype of the individual. Thus the 100,000-200,000 genes which are present in each cell in our body have a defined control mechanism permitting efficient and appropriate expression of proteins and enzymes and yet a single base change in just one of those genes can lead to diseases such as haemophilia or fanconis anaemia.

Conservation of the ST6Gal I gene and its expression in the mammary gland

Milk sialoglycoconjugates can protect the gastrointestinal tract of the suckling neonate by competitively binding to invading pathogens and promoting growth of beneficial flora, and their potential role in postnatal brain development is of particular interest in human infant nutrition. Although the concentration and the distribution of sialoglycoconjugates have been extensively studied in the milk of various species, the investigation of sialyltransferase gene expression in the mammary gland, in the context of lactation, has been limited. The sialyltransferase enzyme ST6Gal I transfers sialic acid from CMP-sialic acid to type 2 (Galβ1,4GlcNAc) free disaccharides or the termini of N- or O-linked oligosaccharides using an α2,6-linkage. Expression of the ST6Gal I gene is primarily regulated at the level of transcription through the use of several cell and development- specific promoters, producing transcripts with divergent 5′ untranslated regions (UTR). In the mouse mammary gland, the novel 5′UTR exon (L) appears to be associated with a drastic increase in ST6Gal I gene expression during lactation. We find that rats also possess an exon (L), suggesting conservation of this regulatory mechanism in rodents. In contrast, an exon (L)-containing transcript was not detected in the lactating bovine or human mammary gland. We also observed a trend of increasing ST6Gal I gene expression in the bovine mammary gland, culminating in involution. This is in contrast to species such as mice where the greatest change in ST6Gal I gene expression occurs between pregnancy and lactation, suggesting different roles in rodents vs. other mammals for α2,6-sialylated oligosaccharides present in milk.

Growth and carcass traits associated with GH1/Alu I and POU1F1/Hinf I gene polymorphisms in Zebu and crossbred beef cattle

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Effects of cyclosporin, phenytoin, and nifedipine on the synthesis and degradation of gingival collagen in tufted capuchin monkeys (Cebus apella): Histochemical and MMP-1 and -2 and collagen I gene expression analyses

Background: The purpose of this experimental study was to evaluate the collagen fiber distribution histologically after phenytoin, cyclosporin, or nifedipine therapy and to correlate it with collagen I and matrix metalloproteinase (MMP)-1 and -2 gene expression levels.Methods: Gingival samples from the canine area were obtained from 12 male monkeys (Cebus apella). The mesial part of each sample was assessed by reverse transcription-polymerase chain reaction, whereas the distal part was processed histologically for picrosirius red and hematoxylin and eosin stainings, as well as for collagen IV immunostaining. One week after the first biopsy, the animals were assigned to three groups that received daily oral dosages of cyclosporin, phenytoin, or nifedipine for 120 days. Additional gingival samples were obtained on days 52 and 120 of treatment from two animals from each group on the opposite sides from the first biopsies.Results: Picrosirius red staining showed a predominance of mature collagen fibers in the control group. Conversely, there was an enlargement of areas occupied by immature collagen fibers in all groups at days 52 and 120, which was not uniform over each section. There was a general trend to lower levels of MMP-1 gene expression on day 52 and increased levels on day 120. Phenytoin led to increased levels of MMP-2 and collagen I gene expression on day 120, whereas the opposite was observed in the nifedipine group.Conclusion: Cyclosporin, phenytoin, and nifedipine led to phased and drug-related gene expression patterns, resulting in impaired collagen metabolism, despite the lack of prominent clinical signs.

Analysis of Bothrops jararacussu venomous gland transcriptome focusing on structural and functional aspects: I - gene expression profile of highly expressed phospholipases A(2)

Snake venom glands are a rich source of bioactive molecules such as peptides, proteins and enzymes that show important pharmacological activity leading to in local and systemic effects as pain, edema, bleeding and muscle necrosis. Most studies on pharmacologically active peptides and proteins from snake venoms have been concerned with isolation and structure elucidation through methods of classical biochemistry. As an attempt to examine the transcripts expressed in the venom gland of Bothrops jararacussu and to unveil the toxicological and pharmacological potential of its products at the molecular level, we generated 549 expressed sequence tags (ESTs) from a directional cDNA library. Sequences obtained from single-pass sequencing of randomly selected cDNA clones could be identified by similarities searches on existing databases, resulting in 197 sequences with significant similarity to phospholipase A(2) (PLA(2)), of which 83.2% were Lys49-PLA(2) homologs (BOJU-1), 0.1% were basic Asp49-PLA(2)s (BOJU-II) and 0.6% were acidic Asp49-PLA(2)s (BOJU-III). Adjoining this very abundant class of proteins we found 88 transcripts codifying for putative sequences of metalloproteases, which after clustering and assembling resulted in three full-length sequences: BOJUMET-I, BOJUMET-II and BOJUMET-III; as well as 25 transcripts related to C-type lectin like protein including a full-length cDNA of a putative galactose binding C-type lectin and a cluster of eight serine-proteases transcripts including a full-length cDNA of a putative serine protease. Among the full-length sequenced clones we identified a nerve growth factor (Bj-NGF) with 92% identity with a human NGF (NGHUBM) and an acidic phospholipase A2 (BthA-I-PLA(2)) displaying 85-93% identity with other snake venom toxins. Genetic distance among PLA(2)s from Bothrops species were evaluated by phylogenetic analysis. Furthermore, analysis of full-length putative Lys49-PLA(2) through molecular modeling showed conserved structural domains, allowing the characterization of those proteins as group II PLA(2)s. The constructed cDNA library provides molecular clones harboring sequences that can be used to probe directly the genetic material from gland venom of other snake species. Expression of complete cDNAs or their modified derivatives will be useful for elucidation of the structure-function relationships of these toxins and peptides of biotechnological interest. (C) 2004 Elsevier SAS. All rights reserved.

Slow skeletal troponin I gene transfer, expression, and myofilament incorporation enhances adult cardiac myocyte contractile function

The functional significance of the developmental transition from slow skeletal troponin I (ssTnI) to cardiac TnI (cTnI) isoform expression in cardiac myocytes remains unclear. We show here the effects of adenovirus-mediated ssTnI gene transfer on myofilament structure and function in adult cardiac myocytes in primary culture. Gene transfer resulted in the rapid, uniform, and nearly complete replacement of endogenous cTnI with the ssTnI isoform with no detected changes in sarcomeric ultrastructure, or in the isoforms and stoichiometry of other myofilament proteins compared with control myocytes over 7 days in primary culture. In functional studies on permeabilized single cardiac myocytes, the threshold for Ca2+-activated contraction was significantly lowered in adult cardiac myocytes expressing ssTnI relative to control values. The tension–Ca2+ relationship was unchanged from controls in primary cultures of cardiac myocytes treated with adenovirus containing the adult cardiac troponin T (TnT) or cTnI cDNAs. These results indicate that changes in Ca2+ activation of tension in ssTnI-expressing cardiac myocytes were isoform-specific, and not due to nonspecific functional changes resulting from overexpression of a myofilament protein. Further, Ca2+-activated tension development was enhanced in cardiac myocytes expressing ssTnI compared with control values under conditions mimicking the acidosis found during myocardial ischemia. These results show that ssTnI enhances contractile sensitivity to Ca2+ activation under physiological and acidic pH conditions in adult rat cardiac myocytes, and demonstrate the utility of adenovirus vectors for rapid and efficient genetic modification of the cardiac myofilament for structure/function studies in cardiac myocytes.

Cell stress-regulated human major histocompatibility complex class I gene expressed in gastrointestinal epithelium.

Conventional major histocompatibility complex (MHC) class I genes encode molecules that present intracellular peptide antigens to T cells. They are ubiquitously expressed and regulated by interferon gamma. Two highly divergent human MHC class I genes, MICA and MICB, are regulated by promoter heat shock elements similar to those of HSP70 genes. MICA encodes a cell surface glycoprotein, which is not associated with beta 2-microglobulin, is conformationally stable independent of conventional class I peptide ligands, and almost exclusively expressed in gastrointestinal epithelium. Thus, this MHC class I molecule may function as an indicator of cell stress and may be recognized by a subset of gut mucosal T cells in an unusual interaction.

Induction of a Cryphonectria parasitica cellobiohydrolase I gene is suppressed by hypovirus infection and regulated by a GTP-binding-protein-linked signaling pathway involved in fungal pathogenesis.

Extracellular cellulase activity is readily induced when the chestnut blight fungus Cryphonectria parasitica is grown on cellulose substrate as the sole carbon source. However, an isogenic C. parasitica strain rendered hypovirulent due to hypovirus infection failed to secrete detectable cellulase activity when grown under parallel conditions. Efforts to identify C. parasitica cellulase-encoding genes resulted in the cloning of a cellobiohydrolase (exoglucanase, EC 3.2.1.91) gene designated chb-1. Northern blot analysis revealed an increase in cbh-1 transcript accumulation in a virus-free virulent C. parasitica strain concomitant with the induction of extracellular cellulase activity. In contrast, induction of cbh-1 transcript accumulation was suppressed in an isogenic hypovirus-infected strain. Significantly, virus-free C. parasitica strains rendered hypovirulent by transgenic cosuppression of a GTP-binding protein alpha subunit were also found to be deficient in the induction of cbh-1 transcript accumulation.

Scrambling of the actin I gene in two Oxytricha species.

The DNA in a germ-line nucleus (a micronucleus) undergoes extensive processing when it develops into a somatic nucleus (a macronucleus) after cell mating in hypotrichous ciliates. Processing includes destruction of a large amount of spacer DNA between genes and excision of gene-sized molecules from chromosomes. Before processing, micronuclear genes are interrupted by numerous noncoding segments called internal eliminated sequences (IESs). The IESs are excised and destroyed, and the retained macro-nuclear-destined sequences (MDSs) are spliced. MDSs in some micronuclear genes are not in proper order and must be reordered during processing to create functional gene-sized molecules for the macronucleus. Here we report that the micronuclear actin I gene in Oxytricha trifallax WR consists of 10 MDSs and 9 IESs compared to the previously reported 9 MDSs and 8 IESs in the micronuclear actin I gene of Oxytricha nova. The MDSs in the actin I gene are scrambled in a similar pattern in the two species, but the positions of MDS-IES junctions are shifted by up to 14 bp for scrambled and 138 bp for the nonscrambled MDSs. The shifts in MDS-IES junctions create differences in the repeat sequences that are believed to guide MDS splicing. Also, the sizes and sequences of IESs in the micronuclear actin I genes are different in the two Oxytricha species. These observations give insight about the possible origins of IES insertion and MDS scrambling in evolution and show the extraordinary malleability of the germ-line DNA in hypotrichs.