In vitro splicing of erythropoietin by the Mycobacterium tuberculosis RecA intein without substituting amino acids at the splice junctions

2003 ◽  
Vol 1619 (2) ◽  
pp. 193-200 ◽  
Author(s):  
Jaya Pal Gangopadhyay ◽  
Shu-qin Jiang ◽  
Patrick van Berkel ◽  
Henry Paulus
Keyword(s):  
Amino Acids ◽  
Mycobacterium Tuberculosis ◽  
In Vitro Splicing ◽  
Splice Junctions

scholarly journals A mixed nitrogen diet and compartmentalized utilization for Mycobacterium tuberculosis replicating in host cells: results of a systems-based analysis

2019 ◽  
Author(s):  
Khushboo Borah ◽  
Martin Beyss ◽  
Axel Theorell ◽  
Huihai Wu ◽  
Piyali Basu ◽  
...  
Keyword(s):  
Amino Acids ◽  
Mycobacterium Tuberculosis ◽  
Nitrogen Source ◽  
Nitrogen Metabolism ◽  
Nitrogen Sources ◽  
Spectral Ratio ◽  
Host Cells ◽  
Ratio Analysis ◽  
Nitrogen Donor

Nitrogen metabolism of Mycobacterium tuberculosis (Mtb) is crucial for the survival and virulence of this pathogen inside host macrophages but little is known about the nitrogen sources acquired from the host or their route of assimilation. Here we performed a systems-based analysis of nitrogen metabolism in intracellullar Mtb and developed 15N-Flux Spectral Ratio Analysis (FSRA) to probe the metabolic cross-talk between the host cell and Mtb. We demonstrate that intracellular Mtb acquires nitrogen from multiple amino acids in the macrophage including glutamate, glutamine, aspartate, alanine, glycine and valine, with glutamine being the predominant nitrogen donor. Each nitrogen source is uniquely assimilated into specific intracellular pools indicating compartmentalised metabolism. This was not observed for in vitro-grown Mtb indicating that there is a switch in nitrogen metabolism when the pathogen enters the intracellular environment. These results provide clues about the potential metabolic targets for development of innovative anti-tuberculosis therapies.

U1 small nuclear ribonucleoproteins are required early during spliceosome assembly

1987 ◽  
Vol 7 (8) ◽  
pp. 2877-2883
Author(s):  
M Zillmann ◽  
S D Rose ◽  
S M Berget
Keyword(s):  
Gel Electrophoresis ◽  
Early Recognition ◽  
Splice Junction ◽  
Specific Antibodies ◽  
U1 Snrnp ◽  
In Vitro Splicing ◽  
Precursor Rna ◽  
Splice Junctions ◽  
Small Nuclear Ribonucleoproteins

U1 small nuclear ribonucleoproteins (snRNPs) are required for in vitro splicing of pre-mRNA. Sequences within U1 RNA hybridize to, and thus recognize, 5' splice junctions. We have investigated the mechanism of association of U1 snRNPs with the spliceosome. U1-specific antibodies detected U1 association with precursor RNA early during assembly. Removal of the 5' terminal sequences of U1 RNA by oligo-directed cleavage or removal of U1 snRNPs by immunoprecipitation prior to the addition of precursor RNA depressed the association of all snRNPs with precursor RNA as detected by immunoprecipitation of splicing complexes by either Sm or U1-specific antibodies. Assembly of the spliceosome as monitored by gel electrophoresis was also depressed after cleavage of U1 RNA. The dependency of Sm precipitability of precursor RNA upon the presence of U1 snRNPs suggests that U1 snRNPs participate in the early recognition of substrate RNAs by U2 to U6 snRNPs. Although removal of the 5'-terminal sequences of U1 depressed U1 snRNP association with precursor RNA, it did not eliminate it, suggesting semistable association of U1 snRNPs with the assembling spliceosome in the absence of U1 RNA hybridization. This association was not dependent upon 5' splice junction sequences but was dependent upon 3' intronic sequences, indicating that U1 snRNPs interact with factors recognizing 3' intronic sequences. Mutual dependence of 5' and 3' recognition factors suggests significant snRNP-snRNP communication during early assembly.

scholarly journals Inactivation of the ilvB1 gene in Mycobacterium tuberculosis leads to branched-chain amino acid auxotrophy and attenuation of virulence in mice

Microbiology ◽  
2009 ◽  
Vol 155 (9) ◽  
pp. 2978-2987 ◽  
Author(s):  
Disha Awasthy ◽  
Sheshagiri Gaonkar ◽  
R. K. Shandil ◽  
Reena Yadav ◽  
Sowmya Bharath ◽  
...  
Keyword(s):  
Amino Acids ◽  
Mycobacterium Tuberculosis ◽  
Amino Acid ◽  
Mutant Strain ◽  
Branched Chain Amino Acids ◽  
Branched Chain Amino Acid ◽  
Branched Chain ◽  
Kinetics Of

Acetohydroxyacid synthase (AHAS) is the first enzyme in the branched-chain amino acid biosynthesis pathway in bacteria. Bioinformatics analysis revealed that the Mycobacterium tuberculosis genome contains four genes (ilvB1, ilvB2, ilvG and ilvX) coding for the large catalytic subunit of AHAS, whereas only one gene (ilvN or ilvH) coding for the smaller regulatory subunit of this enzyme was found. In order to understand the physiological role of AHAS in survival of the organism in vitro and in vivo, we inactivated the ilvB1 gene of M. tuberculosis. The mutant strain was found to be auxotrophic for all of the three branched-chain amino acids (isoleucine, leucine and valine), when grown with either C6 or C2 carbon sources, suggesting that the ilvB1 gene product is the major AHAS in M. tuberculosis. Depletion of these branched chain amino acids in the medium led to loss of viability of the ΔilvB1 strain in vitro, resulting in a 4-log reduction in colony-forming units after 10 days. Survival kinetics of the mutant strain cultured in macrophages maintained with sub-optimal concentrations of the branched-chain amino acids did not show any loss of viability, indicating either that the intracellular environment was rich in these amino acids or that the other AHAS catalytic subunits were functional under these conditions. Furthermore, the growth kinetics of the ΔilvB1 strain in mice indicated that although this mutant strain showed defective growth in vivo, it could persist in the infected mice for a long time, and therefore could be a potential vaccine candidate.

scholarly journals U1 small nuclear ribonucleoproteins are required early during spliceosome assembly.

1987 ◽  
Vol 7 (8) ◽  
pp. 2877-2883 ◽  
Author(s):  
M Zillmann ◽  
S D Rose ◽  
S M Berget
Keyword(s):  
Gel Electrophoresis ◽  
Early Recognition ◽  
Splice Junction ◽  
Specific Antibodies ◽  
U1 Snrnp ◽  
In Vitro Splicing ◽  
Precursor Rna ◽  
Splice Junctions ◽  
Small Nuclear Ribonucleoproteins

U1 small nuclear ribonucleoproteins (snRNPs) are required for in vitro splicing of pre-mRNA. Sequences within U1 RNA hybridize to, and thus recognize, 5' splice junctions. We have investigated the mechanism of association of U1 snRNPs with the spliceosome. U1-specific antibodies detected U1 association with precursor RNA early during assembly. Removal of the 5' terminal sequences of U1 RNA by oligo-directed cleavage or removal of U1 snRNPs by immunoprecipitation prior to the addition of precursor RNA depressed the association of all snRNPs with precursor RNA as detected by immunoprecipitation of splicing complexes by either Sm or U1-specific antibodies. Assembly of the spliceosome as monitored by gel electrophoresis was also depressed after cleavage of U1 RNA. The dependency of Sm precipitability of precursor RNA upon the presence of U1 snRNPs suggests that U1 snRNPs participate in the early recognition of substrate RNAs by U2 to U6 snRNPs. Although removal of the 5'-terminal sequences of U1 depressed U1 snRNP association with precursor RNA, it did not eliminate it, suggesting semistable association of U1 snRNPs with the assembling spliceosome in the absence of U1 RNA hybridization. This association was not dependent upon 5' splice junction sequences but was dependent upon 3' intronic sequences, indicating that U1 snRNPs interact with factors recognizing 3' intronic sequences. Mutual dependence of 5' and 3' recognition factors suggests significant snRNP-snRNP communication during early assembly.

Tuberkulose - Screening

2011 ◽  
Vol 68 (7) ◽  
pp. 381-387
Author(s):  
Otto Schoch
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Interferon Gamma ◽  
Wird Eine ◽  
Interferon Gamma Release Assays

Das primäre Ziel der Aktivitäten zur bevölkerungsbezogenen Tuberkulosekontrolle ist die Identifizierung von Patienten mit sputummikroskopisch positiver Lungentuberkulose. Wenn diese Patienten umgehend therapiert werden, haben sie nicht nur eine optimale Heilungschance, sondern übertragen auch den Krankheitserreger nicht weiter auf andere Personen. Das Screening, die systematische Suche nach Tuberkulose, erfolgt in der Regel radiologisch bei der Suche nach Erkrankten, während immunologische Teste bei der Suche nach einer Infektion mit Mycobacterium tuberculosis zur Anwendung kommen. Diese Infektion, die ein erhöhtes Risiko für die Entwicklung einer Tuberkulose-Erkrankung mit sich bringt, wird im Rahmen der Umgebungsuntersuchungen oder bei Hochrisikogruppen gesucht. Neben dem traditionellen in vivo Mantoux Hauttest stehen heute die neueren in vitro Blutteste, die sogenannten Interferon Gamma Release Assays (IGRA) zur Verfügung, die unter anderem den Vorteil einer höheren Spezifität mit sich bringen, weil die verwendeten Antigene der Mykobakterien-Wand beim Impfstamm Bacille Calmitte Guerin (BCG) und bei den meisten atypischen Mykobakterien nicht vorhanden sind. Zudem kann bei Immunsupprimierten dank einer mitgeführten Positivkontrolle eine Aussage über die Wahrscheinlichkeit eines falsch negativen Testresultates gemacht werden. Bei neu diagnostizierter Infektion mit Mycobacterium tuberculosis wird eine präventive Chemotherapie mit Isoniazid während 9 Monaten durchgeführt.

Characterization of the Original Christmas Disease Mutation (Cysteine 206 → Serine): From Clinical Recognition to Molecular Pathogenesis

1992 ◽  
Vol 67 (01) ◽  
pp. 063-065 ◽  
Author(s):  
Sherryl A M Taylor ◽  
Jacalyn Duffin ◽  
Cherie Cameron ◽  
Jerome Teitel ◽  
Bernadette Garvey ◽  
...  
Keyword(s):  
Nucleotide Substitution ◽  
Novel Mutation ◽  
Factor Ix ◽  
Causative Mutation ◽  
Coding Region ◽  
Body Of Knowledge ◽  
Polymerase Chain ◽  
Splice Junctions

SummaryChristmas disease was first reported as a distinct clinical entity in two manuscripts published in 1952 (1, 2). The eponym associated with this disorder, is the surname of the first patient examined in detail and reported by Biggs and colleagues in a paper describing the clinical and laboratory features of seven affected individuals (3). This patient has severe factor IX coagulant deficiency (less than 0.01 units/ml) and no detectable circulating factor IX antigen (less than 0.01 units/ml). Coding sequence and splice junctions of the factor IX gene from this patient have been amplified in vitro through the polymerase chain reaction (PCR). One nucleotide substitution was identified at nucleotide 30,070 where a guanine was replaced by a cytosine. This mutation alters the amino acid encoded at position 206 in the factor IX protein from cysteine to serine. The non conservative nature of this substitution, the absence of this change in more than 200 previously sequenced factor IX genes and the fact that the remainder of the coding region of this gene was normal, all provide strong circumstantial evidence in favour of this change being the causative mutation in this patient. The molecular characterization of this novel mutation in the index case of Christmas disease, contributes to the rapidly expanding body of knowledge pertaining to Christmas disease pathogenesis.

Antimyotoxic Activity of Synthetic Peptides Derived from Bothrops atrox Snake Gamma Phospholipase A2 Inhibitor Selected by Virtual Screening

2019 ◽  
Vol 19 (22) ◽  
pp. 1952-1961 ◽  
Author(s):  
J.C. Sobrinho ◽  
A.F. Francisco ◽  
R. Simões-Silva ◽  
A.M. Kayano ◽  
J.J. Alfonso Ruiz Diaz ◽  
...  
Keyword(s):  
Amino Acids ◽  
Molecular Docking ◽  
Synthetic Peptides ◽  
Cell Damage ◽  
Neutralization Assay ◽  
Phospholipase Activity ◽  
Phospholipases A2 ◽  
Catalytically Active ◽  
Bothrops Atrox

Background: Several studies have aimed to identify molecules that inhibit the toxic actions of snake venom phospholipases A2 (PLA2s). Studies carried out with PLA2 inhibitors (PLIs) have been shown to be efficient in this assignment. Objective: This work aimed to analyze the interaction of peptides derived from Bothrops atrox PLIγ (atPLIγ) with a PLA2 and to evaluate the ability of these peptides to reduce phospholipase and myotoxic activities. Methods: Peptides were subjected to molecular docking with a homologous Lys49 PLA2 from B. atrox venom modeled by homology. Phospholipase activity neutralization assay was performed with BthTX-II and different ratios of the peptides. A catalytically active and an inactive PLA2 were purified from the B. atrox venom and used together in the in vitro myotoxic activity neutralization experiments with the peptides. Results: The peptides interacted with amino acids near the PLA2 hydrophobic channel and the loop that would be bound to calcium in Asp49 PLA2. They were able to reduce phospholipase activity and peptides DFCHNV and ATHEE reached the highest reduction levels, being these two peptides the best that also interacted in the in silico experiments. The peptides reduced the myotubes cell damage with a highlight for the DFCHNV peptide, which reduced by about 65%. It has been suggested that myotoxic activity reduction is related to the sites occupied in the PLA2 structure, which could corroborate the results observed in molecular docking. Conclusion: This study should contribute to the investigation of the potential of PLIs to inhibit the toxic effects of PLA2s.

Sign in / Sign up

Export Citation Format

Share Document