Biomedical subjects

N K Verma

Publications and source records attributed to N K Verma.

At least 19 recordsLinked to original sources

PubMed2006

The anti-leishmanial drug miltefosine causes insulin resistance in skeletal muscle cells in vitro.

AIMS/HYPOTHESIS: Miltefosine, the first oral anti-leishmanial drug, is reported to inhibit phosphatidylinositol 3-kinase (PI3K)/Akt activity in carcinoma cell lines. Inhibition of the PI3K/Akt pathway is known to result in insulin resistance. Therefore, we investigated whether miltefosine has any deleterious effect(s) on insulin sensitivity in L6E9 skeletal muscle cells. MATERIALS AND METHODS: L6E9 myotubes were treated with miltefosine and its effect was observed on insulin-signalling proteins such as Akt, PI3K, insulin receptor-beta, IRS-1, c-Jun N-terminal kinase, p38 and glycogen synthase kinase beta, as well as on glucose uptake. RESULTS: Miltefosine caused skeletal muscle insulin resistance in vitro by interfering with the insulin-signalling pathway and inhibiting insulin-stimulated glucose uptake. CONCLUSIONS/INTERPRETATION: Miltefosine may contribute to the risk of type 2 diabetes and needs further clinical exploration.

Animals↗

PubMed2004

RNA-mediated gene silencing: mechanisms and its therapeutic applications.

RNA interference, part of a complicated network of interconnected pathways for cellular defence, RNA surveillance and development, has become a powerful tool for the experimental manipulation of gene expression. It is the process by which double-stranded (dsRNA) silences specific gene expression through homology-dependent degradation of cognate mRNA. The dsRNA is converted into 21nt small interfering RNAs (siRNAs), which directs a complex ribonuclease system to substrate mRNA targets. The degradation of the target mRNA is initiated with the cleavage at a position corresponding to the centre of the siRNA. Dissecting individual cellular pathways to reveal the function of numerous proteins is an approach to drug discovery. Interfering RNA (RNAi) serves as a rapid and convenient tool, which works in various organisms. RNAi technology has the potential to facilitate our understanding of biological processes and potentially lead to exciting new drugs. Here we review various experimental approaches adopted with RNAi and possible therapeutic applications.

Drug Design↗

PubMed2000

A study of drug utilisation and cost of treatment in patients hospitalised with unstable angina.

OBJECTIVE: The current study was designed to investigate drug utilisation in the management of unstable angina in India and to calculate the costs incurred by patients in the treatment of a single episode of unstable angina. METHODS: We conducted a prescription survey to examine the use of antianginal drugs in patients with unstable angina in a tertiary care Indian hospital. The use of concurrent medications such as antidiabetic, antihypertensive and lipid-lowering agents was also examined. Data on the cost of treatment, investigations, income, and family size were collected from the case histories or direct interviews with the patients/relatives. RESULTS: A total of 336 consecutive prescriptions were evaluated. Aspirin was the most frequently prescribed drug (98%) followed by nitroglycerin infusion (90%) and enoxaparin (52%). One of the heparins was used by 89% of all patients and beta-blockers by up to 62% of the patients. Besides antianginals, antihypertensive (49%) and antidiabetic (16%) drugs were commonly coadministered. The mean (+/- SD) cost of treatment of a single episode of unstable angina in the hospital was US $494 (+/- 271) against an annual per capita income of US $245. The mean (+/- SD) cost incurred by the patients due to drugs alone during the hospital stay was US $70 (+/- 18) and enoxaparin accounted for 60% of the expenditure due to drugs. CONCLUSIONS: The results of our study show that low-molecular-weight heparin, enoxaparin, is replacing unfractionated heparin in the treatment of unstable angina. In view of the use of costly new drugs, there is an urgent need for carrying out pharmacoeconomic analysis in developing countries as the treatment of a single episode of unstable angina imparts a considerable economic burden on the patient.

Aged↗

PubMed2000

Serotype-converting bacteriophages and O-antigen modification in Shigella flexneri.

O-antigen modification (serotype conversion) in Shigella flexneri, which is an important virulence determinant, is conferred by temperate bacteriophages. Several serotype-converting phages have been isolated and preliminary characterization has identified the genes involved in O-antigen modification, and has also provided insight into the molecular biology of these phages.

Acetylation↗

PubMed1999

Immune response to rotavirus VP4 expressed in an attenuated strain of Shigella flexneri.

An attenuated strain of Shigella flexneri was utilised to express viral protein (VP) 4 of rotavirus and the immunogenicity of the recombinant constructs was studied in BALB/c mice. VP4 was expressed as a fusion with maltose binding protein (MBP) in both the cytoplasm and periplasm, with a much higher level of expression occurring in the former. While all constructs induced a Shigella-specific response in mice, only the construct expressing MBP-VP4 in the cytoplasm of Shigella stimulated an immune response specific to rotavirus. This study demonstrates that Shigella can be used to deliver rotavirus antigens and induces an immune response directed towards both rotavirus and Shigella.

Animals↗

PubMed1999

Antigen-specific systemic and reproductive tract antibodies in foxes immunized with Salmonella typhimurium expressing bacterial and sperm proteins.

Attenuated Salmonella typhimurium strains are potential 'safe' delivery vectors of an oral immunocontraceptive vaccine for the European red fox (Vulpes vulpes). In the present study, model bacterial (Escherichia coli heat-labile enterotoxin B subunit, LTB) and fox sperm (fSP10) antigens were expressed in S. typhimurium SL3261 (delta aroA) under the control of the trc promoter. Adult female foxes were given three oral immunizations with SL3261 containing either LTB (SL3261/pLTB), fSP10 (SL3261/pFSP10) or a control plasmid (pKK233-2 or pTrc99A). All foxes raised serum (IgG) and vaginal (IgG and IgA) antibodies against S. typhimurium lipopolysaccharide (LPS). Each fox that received SL3261/pLTB raised high titre LTB-specific serum and vaginal IgG antibodies. However, only one of four foxes immunized with SL3261/pFSP10 raised an anti-fSP10 immune response, in the form of low titre serum and vaginal IgG antibodies. No vaginal IgA antibodies were raised against either LTB or fSP10 in these experiments. The immune responses against recombinant LTB and fSP10 resulted chiefly from the initial dose of antigen in the inocula and were minimally influenced by continued in vivo antigen expression. This study demonstrates for the first time in the female red fox that oral Salmonella can elicit specific systemic and reproductive tract antibodies against heterologous, recombinant proteins.

Acrosome↗

PubMed1999

Serotype 1a O-antigen modification: molecular characterization of the genes involved and their novel organization in the Shigella flexneri chromosome.

The factors responsible for serotype 1a O-antigen modification in Shigella flexneri were localized to a 5.8-kb chromosomal HindIII fragment of serotype 1a strain Y53. The entire 5.8-kb fragment and regions up- and downstream of it (10.6-kb total) were sequenced. A putative three-gene operon, which showed homology with other serotype conversion genes, was identified and shown to confer serotype 1a O-antigen modification. The serotype conversion genes were flanked on either side by phage DNA. Multiple insertion sequence (IS) elements were located within and upstream of the phage DNA in a composite transposon-like structure. Host DNA homologous to the dsdC and the thrW proA genes was located upstream of the IS elements and downstream of the phage DNA, respectively. The sequence analysis indicates that the organization of the 10.6-kb region of the Y53 chromosome is unique and suggests that the serotype conversion genes were originally brought into the host by a bacteriophage. Several features of this region are also characteristic of pathogenicity islands.

Chromosomes, Bacterial↗

PubMed1998

Serotype conversion of a Shigella flexneri candidate vaccine strain via a novel site-specific chromosome-integration system.

Shigella flexneri SFL124 (serotype Y) is a promising live oral vaccine candidate, which has been shown to be safe and immunogenic in human volunteers. To change the serotype of this vaccine strain, we inserted a serotype conversion gene cluster into the chromosome of SFL124 by using a bacteriophage-based site-specific integration system. By cloning an integrase gene (int), an attachment site (attP) and a glucosyl transfer gene cluster from bacteriophage SfX into a suicide vector, and subsequently introducing this construct into S. flexneri SFL124, we obtained a S. flexneri strain (designated SFL1213) expressing the serotype X somatic antigen specificity. The strain retained other characteristics of the parent strain, such as colony shape, growth rate, and Congo red binding property. Stability test showed that the serotype X O-antigen specificity in SFL1213 was 100% stable after being cultured approximately 72 successive hours under non-selective condition. In a mouse pulmonary model, the recombinant strain elicited a significant level of humoral antibodies which recognized the lipopolysaccharide (LPS) of a wild-type S. flexneri serotype X strain. The site-specific insertion system will be useful when stable expression of a cloned single copy gene is desired in the chromosome of S. flexneri vaccine candidate, SFL124.

Animals↗

PubMed1997

Cloning and analysis of the glucosyl transferase gene encoding type I antigen in Shigella flexneri.

The O-antigen of most Shigella flexneri serotypes contains an identical tetrasaccharide repeating unit. Apart from serotype Y, the O-antigen is modified by addition of a glucosyl and/or O-acetyl residue to a specific position in the O-unit. In this study the glucosyl transferase gene from a serotype 1 a has been cloned and identified. The bacteriophage SfV integrase (int) gene was used to probe a S. flexneri Y53 (serotype 1 a) cosmid library and 18 unique clones were identified. Southern hybridisation of these clones indicated two unlinked regions of the chromosome contained the int homologue. When expressed in a live candidate vaccine strain of S. flexneri serotype Y (SFL124), clones with one region produced type I antigen, whereas clones containing the other region produced mainly type Y antigen. One of the cosmid clones positive for type I antigen by agglutination and Western blotting was selected for further study. Genes involved in O-antigen glucosyl modification were mapped on a 5.8 kb fragment and subclones were produced which fully or partially expressed the type I antigen, depending on the extent of the clone. Fully and partially expressing clones may be useful vaccine candidate strains for protection against disease caused by two serotypes of S. flexneri.

Antigens, Bacterial↗

PubMed1997

Environmental auditing programme in India.

The concept of environmental auditing in industrial units in India was formally introduced in March 1992 with the overall objective minimising consumption of resources and promoting use of clean technologies in industrial production to minimise generation of wastes. A series of discussions were held among the concerned regulatory agencies before and after making environmental auditing and submission of Annual Environmental Statements, a mandatory requirement for the industries. This followed organisation of a number of workshops, seminars and training programmers all over the country by different agencies dealing with environment protection, conducting of audit studies in 125 selected polluting industries in the country by the Central Pollution Control Board (CPCB), Delhi, and a number of other activities. The present paper gives the details of the activities before and after the introduction of this requirement in the country. The aspects covered include (i) the evolution of the concept, (ii) the history of the introduction of the scheme in the country and the organisational structure, (iii) the mandatory requirement and its applicability, (iv) the implementing agencies and their responsibilities, (v) summary of the training programmes/workshops organised in the country, (vi) case studies conducted by CPCB in selected polluting industries and the major findings, (vii) development of guidelines for conducting environmental audit in different categories of polluting industries, and (viii) the current status of the scheme and plans for its effective implementation.

Air Pollution↗

PubMed1997

Molecular characterization of the genes involved in O-antigen modification, attachment, integration and excision in Shigella flexneri bacteriophage SfV.

Bacteriophage SfV is a temperate phage of Shigella flexneri responsible for converting serotype Y (3,4) to serotype 5a (V; 3,4) through its glucosyl transferase gene. The glucosyl transferase (gtr) gene of SfV has been cloned and shown to partially convert S. flexneri serotype Y to serotype 5a. In this study, we found that the serotype-converting region of SfV was approximately 2.5 kb in length containing three continuous ORFs. The recombinant strain carrying the three complete ORFs expressed the type V and group antigen 3,4, both indistinguishable from that of S. flexneri 5a wild-type strain. The interruption of orf5 or orf6 gave partial conversion in the S. flexneri recombinant strain indicated by the incomplete replacement of group antigen 3,4. The region adjacent to the serotype-conversion genes was found to be identical to the attP-int-xis region of phage P22. Altogether, an approximately 2.2-kb sequence covering a portion of the serotype-conversion (approximately 500 nt)-attP-int-xis regions of SfV was remarkably similar to that of P22.

Amino Acid Sequence↗

PubMed1997

Shigella flexneri type-specific antigen V: cloning, sequencing and characterization of the glucosyl transferase gene of temperate bacteriophage SfV.

With lysogeny by bacteriophage SfV, Shigella flexneri serotype Y is converted to serotype 5a. The glucosyl transferase gene (gtr) from bacteriophage SfV of S. flexneri, involved in serotype-specific conversion, was cloned and characterized. The DNA sequence of a 3.7 kb EcoRI-BamHI fragment of bacteriophage SfV which includes the gtr gene was determined. This gene, encoding a polypeptide of 417 aa with 47.67 kDa molecular mass, caused partial serotype conversion of S. flexneri from serotype Y to type V antigen as demonstrated by Western blotting and the sensitivity of the hybrid strain to phage Sf6. The deduced protein of the partially sequenced open reading frame upstream of the gtr showed similarity to various glycosyl transferases of other bacteria. Orf3, separated from the gtr by a non-coding region and transcribed convergently, codes for a 167 aa (18.8 kDa) protein found to have homology with tail fibre genes of phage lambda and P2.

Amino Acid Sequence↗

PubMed1997

The immune response to a B-cell epitope delivered by Salmonella is enhanced by prior immunological experience.

Attenuated, heterologous strains of Salmonella have shown potential as live, recombinant vaccines against foreign pathogens. Studies in animal models have demonstrated that immunization with these heterologous vaccines is an effective way to induce both cellular and humoral immune responses against Salmonella and the foreign antigen. We studied the consequence of priming mice with Salmonella dublin 3-6 months before intraperitoneal administration with the same strain carrying a model B-cell epitope. Mice primed with the carrier strain demonstrated enhanced serum Ig titres against the foreign antigen. This immune enhancement was observed up to approximately 6 months after priming. These findings suggest that previous immunological experience with Salmonella does not limit the immune response to a foreign antigen carried by the same organism. In fact, prior exposure to Salmonella appears to enhance the response to the foreign antigen.

Animals↗

PubMed1997

Enhancement of the specific mucosal IgA response in vivo by interleukin-5 expressed by an attenuated strain of Salmonella serotype Dublin.

It has been shown that cytokines have potential as therapeutic adjuvants in vaccination. Interleukin-5 (IL-5) is a cytokine that regulates antibody production, in particular enhancing IgA production by activated mucosal B cells. This study examined the expression of a cloned cytokine gene encoding murine IL-5 (mIL-5) by an attenuated aroA strain (SL5631) of Salmonella serotype Dublin. The resulting strain, SL5631(pTRXFUS-mIL-5), expressed mIL-5 as a fusion with thioredoxin as demonstrated by immunological and biological assays. When strain SL5631(pTRXFUS-mIL-5) was used as a live vaccine in BALB/c mice, it colonised and multiplied at higher levels in spleens and livers than the strain carrying the empty plasmid. A reduction in invasiveness of SL5631(pTRXFUS-mIL-5) was observed in in-vitro invasion assays. Enhanced IgA response against salmonella LPS in mucosal secretions and enhanced IgA and IgG responses were detected by ELISA and ELISPOT methods in sera of mice immunised with the strain expressing mIL-5. Results with IL-5-deficient mice showed that the enhanced IgA response was due to Salmonella-expressed mIL-5 rather than endogenous mIL-5.

Animals↗

PubMed1997

Immune response to a Murray Valley encephalitis virus epitope expressed in the flagellin of an attenuated strain of Salmonella.

Recent developments in vaccine construction include the use of attenuated, avirulent strains of Salmonella as carriers of foreign antigens. These recombinant strains can elicit a heterologous immune response when injected into animals, demonstrating potential for their use in the construction of many vaccines. In the present study, a B-cell epitope of Murray Valley encephalitis virus (MVE) was identified and expressed in a Salmonella strain to evaluate its potential to induce a specific immune response to MVE. A synthetic oligonucleotide encoding the B-cell epitope (residues E201-224) of the envelope protein of MVE was inserted into the cloned flagellin gene of the Salmonella strain. The construct was sequenced to ensure correct orientation of the epitope. Expression of the epitope was demonstrated by Western blot analysis and immunogold electron microscopy with monoclonal antibody specific to the epitope. Electron microscopy analysis revealed multiple copies of the epitope along the flagella. The recombinant Salmonella carrying the hybrid flagellin gene elicited an immune response to the MVE epitope in a mouse model. The MVE-specific antibodies partially neutralised the virus in vitro. The significance of this system for engineering vaccines for other medically important flaviviruses is discussed.

Amino Acid Sequence↗

PubMed1997

Cloning and characterisation of the aroA and aroD genes of Shigella dysenteriae type 1.

The aroA and aroD genes from Shigella dysenteriae type 1, encoding 5-enolpyruvylshikimate 3-phosphate synthase and 3-dehydroquinase, respectively, were cloned by polymerase chain reaction (PCR). Their nucleotide sequences were determined and predicted to code for 46 kDa and 27.5 kDa proteins, respectively. Protein expressed from these genes using the minicell system, corresponded to the size of the predicted protein products. The cloned genes were shown to be functional by complementation of Escherichia coli aroA- and aroD- mutants. The predicted amino acid sequences of the cloned aroA (427 amino acids) and aroD (252 amino acids) genes of S. dysenteriae type 1 were found to be highly homologous to the corresponding genes in other bacterial species, indicating the high conservation of these housekeeping genes. The use of the cloned aroA and aroD genes in the development of a vaccine strain against S. dysenteriae is discussed.

3-Phosphoshikimate 1-Carboxyvinyltransferase↗

PubMed1995

Delivery of class I and class II MHC-restricted T-cell epitopes of listeriolysin of Listeria monocytogenes by attenuated Salmonella.

Using a Salmonella vaccine-Listeria infection model of intracellular infection, we studied the capacity of an attenuated strain of Salmonella carrying T-cell epitopes of listeriolysin (LLO) of L. monocytogenes to elicit epitope-specific T-cell responses. Class II (LLO 215-226) or class I (LLO 91-99) MHC-restricted T-cell epitopes of LLO were inserted within a central, hypervariable domain of the flagellin protein of an attenuated delta aroA Salmonella dublin strain. T cells from Listeria-immunized mice were activated by lysates or heat-killed preparations of Salmonella construct expressing the LLO 215-226 epitope, indicating that LLO 215-226 is processed and presented to T cells when offered to antigen-presenting cells as part of a flagellin-epitope fusion protein. The chimeric flagellin genes were integrated into the chromosome of the flagellin-negative S. dublin strain to obtain stable expression of the epitopes. Immunization with the living, chromosomally integrated Salmonella construct carrying LLO 215-226 epitope as part of the flagellin protein generated T cells reactive with the corresponding LLO peptide, indicating that this chimera can stimulate a class-specific immune response in vitro. The effect of flanking residues on the processing and presentation of MHC class I LLO 91-99 epitope was studied using Salmonella vaccine strains that express chimeric flagellins containing one of three LLO 91-99 inserts: 91-99 (normal flagellin amino acids as flanking residues); KK91-99KK (Lys-Lys flanking residues); and AAA91-99AAA (Ala-Ala-Ala flanking residues).(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed1995

Induction of a cellular immune response to a defined T-cell epitope as an insert in the flagellin of a live vaccine strain of Salmonella.

Attenuated strains of Salmonella have been used as vaccines to deliver heterologous antigens mainly to generate a humoral immune response. However, little is known about their ability to induce a cell-mediated immune response to the T-cell epitopes of another infectious agent or how optimally to deliver these epitopes to the host immune system. In order to study this question, a well defined MHC class II-restricted epitope (residues 88-103) from moth cytochrome C (MCC) was inserted into the central hypervariable domain of the flagellin of an attenuated strain of Salmonella dublin. The resulting flagellin was exported to the bacterial surface and polymerized into flagellar filaments that contained multiple copies of the MCC epitope. When flanked by Lys-Lys cathepsin B cleavage sites to facilitate its proteolytic release within the endosomal compartment of antigen-presenting cells, the MCC-chimeric flagellin epitope was efficiently processed in vitro by mouse peritoneal macrophages and presented to 2B4 T-hybridoma cells (specific for the MCC epitope 88-103). Stable expression of the epitope and a higher immune response was obtained in H-2k mice by integrating the chimeric flagellin gene into the chromosome of the vaccine strain. Bacteria with MCC-chimeric flagellins that were expressed from a stable chromosomal locus and flanked by cathepsin B cleavage sites were cleared more rapidly from the livers and spleens of transgenic mice with T-cell receptor (TCR) alpha and beta chains specific for the MCC epitope than were bacteria lacking the epitope. Antigen processing and presentation of class II-restricted epitopes expressed as chimeric proteins by attenuated bacterial vaccine vectors may be facilitated by the presence of endosomal protease cleavage sites on each side of the epitope and by chromosomal integration of the coding sequence.

Amino Acid Sequence↗