Antihypertensive and Antioxidant Action of Amlodipine and Vitamin C in Patients of Essential Hypertension

The etiology of essential hypertension includes increased oxidative stress. The role of antihypertensive drug amlodipine as an antioxidant and the benefit of addition of vitamin C, an antioxidant to antihypertensive therapy were studied. Forty male patients of essential hypertension were randomly divided into two groups and treated with 5 mg amlodipine. In addition one group also received 1000 mg vitamin C (as two 500 mg tablets) once daily for three months. Although blood pressure decreased in both groups, the systolic blood pressure in patients given vitamin C was less (126.4 ± 7.47) compared to the other group (130.9 ± 7.27). A decrease in malondialdehyde, an increase in erythrocyte sodium-potassium adenosine triphosphatase (Na+ K+ ATPase) and an increase in the superoxide dismutase levels were observed in both groups. The increase in SOD was statistically more in the patients given vitamin C in addition to amlodipine (0.1717 ± 0.0150 compared to 0.152 ± 0.0219 units/100 ml assay). In spite of the known antihypertensive, antioxidant activity, similarity in correcting endothelial dysfunction independently, giving the two drugs together and early introduction of vitamin C perhaps decreases oxidative stress and augments the antioxidant status. This may prevent further vascular damage due to oxidative stress, leading to a better prognosis in essential hypertension patients

Conditional Silencing by CRISPRi Reveals the Role of DNA Gyrase in Formation of Drug-Tolerant Persister Population in Mycobacterium tuberculosis

Drug tolerance in mycobacterial pathogens is a global concern. Fluoroquinolone (FQ) treatment is widely used for induction of persisters in bacteria. Although FQs that target DNA gyrase are currently used as second-line anti-tuberculosis (TB) drugs, little is known about their impact on Mycobacterium tuberculosis (Mtb) persister formation. Here we explored the CRISPRi-based genetic repression for better understanding the effect of DNA gyrase depletion on Mtb physiology and response to anti-TB drugs. We find that suppression of DNA gyrase drastically affects intra- and extracellular growth of Mtb. Interestingly, gyrase depletion in Mtb leads to activation of RecA/LexA-mediated SOS response and drug tolerance via induction of persister subpopulation. Chemical inhibition of RecA in gyrase-depleted bacteria results in reversion of persister phenotype and better killing by antibiotics. This study provides evidence that inhibition of SOS response can be advantageous in improving the efficacy of anti-TB drugs and shortening the duration of current TB treatment

Mycobacterial transcriptional signals: requirements for recognition by RNA polymerase and optimal transcriptional activity

Majority of the promoter elements of mycobacteria do not function well in other eubacterial systems and analysis of their sequences has established the presence of only single conserved sequence located at the −10 position. Additional sequences for the appropriate functioning of these promoters have been proposed but not characterized, probably due to the absence of sufficient number of strong mycobacterial promoters. In the current study, we have isolated functional promoter-like sequences of mycobacteria from the pool of random DNA sequences. Based on the promoter activity in Mycobacterium smegmatis and score assigned by neural network promoter prediction program, we selected one of these promoter sequences, namely A(37) for characterization in order to understand the structure of housekeeping promoters of mycobacteria. A(37)–RNAP complexes were subjected to DNase I footprinting and subsequent mutagenesis. Our results demonstrate that in addition to −10 sequences, DNA sequence at −35 site can also influence the activity of mycobacterial promoters by modulating the promoter recognition by RNA polymerase and subsequent formation of open complex. We also provide evidence that despite exhibiting similarities in −10 and −35 sequences, promoter regions of mycobacteria and Escherichia coli differ from each other due to differences in their requirement of spacer sequences between the two positions

Successful Shortening of Tuberculosis Treatment Using Adjuvant Host-Directed Therapy with FDA-Approved Phosphodiesterase Inhibitors in the Mouse Model

Global control of tuberculosis (TB), an infectious disease that claims nearly 2 million lives annually, is hindered by the long duration of chemotherapy required for curative treatment. Lack of adherence to this intense treatment regimen leads to poor patient outcomes, development of new or additional drug resistance, and continued spread of M.tb. within communities. Hence, shortening the duration of TB therapy could increase drug adherence and cure in TB patients. Here, we report that addition of the United Stated Food and Drug Administration-approved phosphodiesterase inhibitors (PDE-Is) cilostazol and sildenafil to the standard TB treatment regimen reduces tissue pathology, leads to faster bacterial clearance and shortens the time to lung sterilization by one month, compared to standard treatment alone, in a murine model of TB. Our data suggest that these PDE-Is could be repurposed for use as adjunctive drugs to shorten TB treatment in humans

Functional Characterization of EngAMS, a P-Loop GTPase of Mycobacterium smegmatis

Bacterial P-loop GTPases belong to a family of proteins that selectively hydrolyze a small molecule guanosine tri-phosphate (GTP) to guanosine di-phosphate (GDP) and inorganic phosphate, and regulate several essential cellular activities such as cell division, chromosomal segregation and ribosomal assembly. A comparative genome sequence analysis of different mycobacterial species indicates the presence of multiple P-loop GTPases that exhibit highly conserved motifs. However, an exact function of most of these GTPases in mycobacteria remains elusive. In the present study we characterized the function of a P-loop GTPase in mycobacteria by employing an EngA homologue from Mycobacterium smegmatis, encoded by an open reading frame, designated as MSMEG_3738. Amino acid sequence alignment and phylogenetic analysis suggest that MSMEG_3738 (termed as EngAMS) is highly conserved in mycobacteria. Homology modeling of EngAMS reveals a cloverleaf structure comprising of α/β fold typical to EngA family of GTPases. Recombinant EngAMS purified from E. coli exhibits a GTP hydrolysis activity which is inhibited by the presence of GDP. Interestingly, the EngAMS protein is co-eluted with 16S and 23S ribosomal RNA during purification and exhibits association with 30S, 50S and 70S ribosomal subunits. Further studies demonstrate that GTP is essential for interaction of EngAMS with 50S subunit of ribosome and specifically C-terminal domains of EngAMS are required to facilitate this interaction. Moreover, EngAMS devoid of N-terminal region interacts well with 50S even in the absence of GTP, indicating a regulatory role of the N-terminal domain in EngAMS-50S interaction

Role of 5'-TGN-3' motif in the interaction of mycobacterial RNA polymerase with a promoter of 'extended -10' class

In a systematic approach to understand the transcriptional machinery of mycobacteria, we had previously isolated and characterized mycobacterial promoter regions. In this study, we have investigated molecular interactions between mycobacterial RNA polymerase holoenzyme, reconstituted with different sigma subunits and the promoter element of the Mycobacterium tuberculosis gene pknH (Rv1266c), a representative of promoters belonging to the 'extended -10' class. In vitro transcription assays using the pknH promoter and reconstituted RNA polymerase holoenzyme demonstrated that transcription from the pknH promoter is specifically initiated by σ<SUP>A</SUP>, the principal sigma factor of mycobacteria. DNase I protection assay and deletion studies with the pknH promoter revealed that the minimal region required for optimal transcription carries the sequence from position -37 to position +6. Moreover, mutation in the TGN motif of the pknH promoter resulted in the loss of &gt;75% of its activity. Binding of RNA polymerase with wild-type promoter as well as its TG<SUP>-</SUP> mutant revealed that the TGN motif is required for the transition from a close complex into an open complex. Further, it was observed that the presence of the TGN motif reduces the thermal energy required for the conversion of a close complex into an open complex, necessary for initiation of transcription

Synthesis and antimicrobial activity of 1-aminomethyl-3-[4′-(4″-nitrobenzyloxy)-benzohydrazono]isatins

330-333A new series of 1-aminomethyl-3-[4′-(4″-nitrobenzyloxy)-benzo­hydrazono]isatins (Mannich bases) have been synthesized and screened for their antimicrobial potential against human pathogenic bacteria and fungi. The structures of the compounds have been established by means of elemental analysis and spectral data (IR and 1H NMR)