Biomedical subjects

Vivek Kapur

Publications and source records attributed to Vivek Kapur.

At least 19 recordsLinked to original sources

PubMed2006

Rapid expression of Mycobacterium avium subsp. paratuberculosis recombinant proteins for antigen discovery.

Mycobacterium avium subsp. paratuberculosis is the causative agent of Johne's disease, a chronic granulomatous enteritis of ruminants and other species. Detection of infection in animals is hampered by the lack of sensitive and specific diagnostic assays. We describe here an approach that utilizes translationally active PCR fragments for the rapid in vitro transcription and translation of recombinant proteins for antigen discovery in M. avium subsp. paratuberculosis. The investigations showed that the MAP1272c protein selectively reacts with sera from Johne's disease-positive cattle and represents an antigen of potential utility in M. avium subsp. paratuberculosis immunodiagnostics.

Animals↗

PubMed2006

PBP3 inhibition elicits adaptive responses in Pseudomonas aeruginosa.

Adaptive evolution depends on both the genetic variability in a population of organisms and the selection of the better adapted genotypes. However, for the fittest variants to be selected they must survive over a sufficient period under the new conditions. Bacteria are often exposed to different types of stress in nature, including antibiotics. We analysed the global expression profiles of the opportunistic pathogen Pseudomonas aeruginosa in response to ceftazidime, a PBP3 inhibitor, at different concentrations and times. PBP3 inhibition exerts a global impact on the transcription of a large number of genes. From an adaptive perspective, it is noteworthy the induction of several SOS genes, as well as adaptation, protection and antibiotic resistance genes. Intriguingly, transcription of pyocin genes, previously described as SOS-regulated, was repressed upon PBP3 inhibition. Ciprofloxacin, an SOS inducer, produced transcriptional induction of pyocins. Our results indicate that: (i) the SOS responses resulting from treatments with these two antibiotics cause only partially overlapping transcription profiles; (ii) PBP3 and DNA-gyrase inhibition produce opposite effects on transcription of pyocin genes. Consequently, ceftazidime decreases ciprofloxacin toxicity; (iii) error-prone DNA-polymerase DinB is induced by PBP3 inhibition but not by DNA-gyrase inhibition; (iv) PBP3 inhibition causes induced mutagenesis; (v) ceftazidime upregulates several antibiotic-resistance and adaptation genes; and (vi) ceftazidime concentrations thought previously to be lethal are not, as most cells treated with ceftazidime remain alive and recover their capacity to form colonies. Thus, transcriptional changes demonstrated in this work are likely to be adaptively relevant to cells that survive.

Adaptation, Physiological↗

PubMed2006

Differentiation of embryonic stem cells conditionally expressing neurogenin 3.

Expression of the proendocrine gene neurogenin 3 (Ngn3) is required for the development of pancreatic islets. To better characterize the molecular events regulated by Ngn3 during development, we have determined the expression profiles of murine embryonic stem cells (mESCs) uniformly induced to overexpress Ngn3. An mESC line was created in order to induce Ngn3 by adding doxycycline to the culture medium. Genome-wide microarray analysis was performed to identify genes regulated by Ngn3 in a variety of contexts, including undifferentiated ESCs and differentiating embryoid bodies (EBs). Genes regulated by Ngn3 in a context-independent manner were identified and analyzed using systematic gene ontology tools. This analysis revealed Notch signaling as the most significantly regulated signaling pathway (p = .009). This result is consistent with the hypothesis that Ngn3 expression makes the cell competent for Notch signaling to be activated and, conversely, more sensitive to Notch signaling inhibition. Indeed, EBs induced to express Ngn3 were significantly more sensitive to gamma-secretase inhibitor-mediated Notch signaling inhibition (p < .0001) when compared with uninduced EBs. Moreover, we find that Ngn3 induction in differentiating ESCs results in significant increases in insulin, glucagon, and somatostatin expression.

Animals↗

PubMed2006

Cytokine responses of bovine macrophages to diverse clinical Mycobacterium avium subspecies paratuberculosis strains.

BACKGROUND: Mycobacterium avium subsp. paratuberculosis (MAP), the causative agent of Johne's disease (JD) persistently infects and survives within the host macrophages. While it is established that substantial genotypic variation exists among MAP, evidence for the correlates that associate specific MAP genotypes with clinical or sub-clinical disease phenotypes is presently unknown. Thus we studied strain differences in intracellular MAP survival and host responses in a bovine monocyte derived macrophage (MDM) system. RESULTS: Intracellular survival studies showed that a bovine MAP isolate (B1018) and a human MAP isolate (Hu6) persisted in relatively higher numbers when compared with a sheep MAP isolate (S7565) at 24-hr, 48-hr and 96-hr post infection (PI). MDMs stimulated with B1018 up-regulated IL-10 at the transcript level and down-regulated TNFalpha at the protein and transcript levels compared with stimulations by the S7565 and Hu6. MDMs infected with Hu6 showed a down regulatory pattern of IL-10 and TNFalpha compared to stimulations by S7565. Cells stimulated with B1018 and Hu6 had low levels of matrix metalloprotease-3 (MMP3) and high levels of tissue inhibitor of metalloprotease-1 (TIMP1) at 96-hr PI relative to MDMs stimulated by S7565. CONCLUSION: Taken together, results suggest that the bovine (B1018) and the human (Hu6) MAP isolates lead to anti-inflammatory and anti-invasive pathways in the macrophage environment whereas the sheep (S7565) MAP isolate induces a pro-inflammatory pathway. Thus the infecting strain genotype may play a role in polarizing the host immune responses and dictate the clinicopathological outcomes in this economically important disease.

Animals↗

PubMed2006

Current understanding of the genetic diversity of Mycobacterium avium subsp. paratuberculosis.

Mycobacterium avium subsp. paratuberculosis (MAP) is the etiological agent of Johne's disease (or paratuberculosis). Paratuberculosis is a chronic gastroenteritis mainly affecting cattle, sheep and other ruminants. MAP is also of concern due to the heretofore unresolved issue of its possible role in Crohn's disease in humans. We present here a review of MAP (i) mobile genetic elements; (ii) repetitive elements; (iii) single nucleotide polymorphisms; and (iv) whole-genome comparisons to study the molecular epidemiology of MAP. A summary of the findings to date is presented, and the discriminatory power, advantage and disadvantages of each of the methods are compared and discussed.

Animals↗

PubMed2006

Comparative transcriptional analysis of human macrophages exposed to animal and human isolates of Mycobacterium avium subspecies paratuberculosis with diverse genotypes.

Mycobacterium avium subsp. paratuberculosis is the causative agent of Johne's disease in animals and has been hypothesized to be associated with Crohn's disease in humans. Recently, M. avium subsp. paratuberculosis isolates recovered from Crohn's disease patients were shown to have limited diversity, implying the existence of human disease-associated genotypes and strain sharing with animals (A. H. Ghadiali et al., J. Clin. Microbiol. 42:5345-5348, 2004). To explore whether these genotypic differences or similarities among human and animal isolates translated to functionally significant attributes such as variance in host preference and/or difference in magnitude of infections, we performed a global scale analysis of M. avium subsp. paratuberculosis isolates that were representative of different genotypes and host species using DNA microarrays. Genome-wide characterization of the transcriptional changes was carried out using a human monocytic cell line (THP-1 cells) in response to different genotypes of M. avium subsp. paratuberculosis isolates recovered from various hosts. We identified several differentially expressed genes during early intracellular infection, including those involved in common canonical pathways such as NF-kappaB, interleukin-6 (IL-6), mitogen-activated protein kinase/extracellular signal-regulated kinase, and Jun N-terminal protein kinase signaling, as well as genes involved in T helper type 1 (Th1) responses (such as CCL5 ligand) and those that encode several proinflammatory cytokines and chemokine receptors. The cattle and human isolates of M. avium subsp. paratuberculosis, regardless of their short sequence repeat (SSR) genotype, induced similar global gene expression patterns in THP-1 cells. They differentially regulated genes necessary for cell survival without causing major alterations in proinflammatory genes. In contrast, the sheep isolates representing diverse SSR genotypes closely resembled the global gene expression pattern of an M. avium subsp. avium isolate, and they significantly up-regulated proinflammatory genes related to IL-6, T-cell receptor, B-cell receptor, and death receptor signaling within THP-1 cells. Additionally, we demonstrated consistency among infecting genotypes of M. avium subsp. paratuberculosis isolated from diverse hosts [cattle (n=2), human (n=3), sheep (n=2), and bison (n=1)] in quantitative reverse transcription-PCR analysis of seven differentially expressed genes. While the levels of expression induced by the bison isolate were different compared with cattle or human isolates, they followed the common anti-inflammatory, antiapoptotic trend. Our data suggest that the macrophage responses to M. avium subsp. paratuberculosis isolates from cattle and human sources, regardless of genotype, follow a common theme of anti-inflammatory responses, an attribute likely associated with successful infection and persistence. However, these expression patterns differ significantly from those in THP-1 cells infected with sheep isolates of M. avium subsp. paratuberculosis or the M. avium subsp. avium isolate. These data provide a transcriptional basis for a variety of pathophysiological changes observed during early stages of infection by different strains of M. avium subsp. paratuberculosis, a first step in understanding trait-allele association in this economically important disease.

Animals↗

PubMed2006

Genomic comparison of Mycobacterium avium subsp. paratuberculosis sheep and cattle strains by microarray hybridization.

Microarray-based comparisons of three Mycobacterium avium subsp. paratuberculosis isolates, including one sheep strain and two cattle strains, identified three large genomic deletions in the sheep strain, totaling 29,208 bp and involving 24 open reading frames. These deletions may help explain some of the differences in pathogenicity and host specificity observed between the cattle and sheep strains of Mycobacterium avium subsp. paratuberculosis.

Animals↗

PubMed2006

Short-sequence-repeat analysis of Mycobacterium avium subsp. paratuberculosis and Mycobacterium avium subsp. avium isolates collected from animals throughout the United States reveals both stability of loci and extensive diversity.

We analyzed the multilocus short sequence repeats (SSRs) of 211 and 56 isolates of Mycobacterium avium subsp. paratuberculosis and M. avium subsp. avium, respectively. The M. avium subsp. paratuberculosis isolates could be differentiated into 61 genotypes. The M. avium subsp. avium isolates showed limited diversity. These SSRs are stable and suitable for studying the molecular epidemiology of M. avium subsp. paratuberculosis.

Animals↗

PubMed2006

TimeView: for comparative gene expression analysis.

UNLABELLED: TimeView is a MATLAB program that compares multiple temporal datasets from microarray experiments under two or more conditions, for example, temporal variation of cellular response upon exposure to different drugs. The current paucity of programs designed to efficiently compare and visualise gene expression profiles in such datasets led us to design TimeView, which also enhances data visualisation by plotting the expression profiles of a large number of genes on a single screen. AVAILABILITY: TimeView is available free of charge to all users at http://hugroup.cems.umn.edu/Research/Genomics/Timeview/timeview.htm. To use TimeView, users will require access to the commercial software MATLAB (version 6.5). A help document is available on the TimeView website.

Algorithms↗

PubMed2005

Avian metapneumovirus phosphoprotein targeted RNA interference silences the expression of viral proteins and inhibits virus replication.

Avian metapneumovirus (aMPV) is one of the major causes of serious respiratory infections of poultry and leads to considerable economic losses to food animal production worldwide. Here, we show that double stranded short interfering RNA (siRNA) molecules corresponding to aMPV phosphoprotein (P) gene silence P RNA and protein expression. These siRNAs broadly reduced the expression of other viral proteins in addition to P, but did not have a discernable effect on cellular protein expression. The exposure of cells to P-specific siRNAs also led to inhibition of virus replication as evidenced by marked reduction in the progeny virion titers. Taken together, the findings suggest that exogenous P silencing siRNAs can inhibit aMPV replication with potential implications in the design of novel siRNA based prophylactics.

Animals↗

PubMed2005

Mycobacterium tuberculosis-induced cytokine and chemokine expression by human microglia and astrocytes: effects of dexamethasone.

Although corticosteroids are recommended as adjunctive therapy for tuberculous meningitis, the mechanism underlying their beneficial effect is poorly understood. In this study, human microglia and astrocytes were infected with Mycobacterium tuberculosis H37Rv, and cytokine and chemokine expression was examined with and without dexamethasone treatment. Microglia were the principal cells infected by tubercle bacilli, which elicited robust amounts of several cytokines and chemokines. Treatment with dexamethasone markedly suppressed production of these mediators. The results of this study support the concept that microglia play an important role in neuropathogenesis of tuberculosis and that dexamethasone could operate via modulation of the production of proinflammatory cytokines and chemokines by these brain macrophages.

Animals↗

PubMed2005

The complete genome sequence of Mycobacterium avium subspecies paratuberculosis.

We describe here the complete genome sequence of a common clone of Mycobacterium avium subspecies paratuberculosis (Map) strain K-10, the causative agent of Johne's disease in cattle and other ruminants. The K-10 genome is a single circular chromosome of 4,829,781 base pairs and encodes 4,350 predicted ORFs, 45 tRNAs, and one rRNA operon. In silico analysis identified >3,000 genes with homologs to the human pathogen, M. tuberculosis (Mtb), and 161 unique genomic regions that encode 39 previously unknown Map genes. Analysis of nucleotide substitution rates with Mtb homologs suggest overall strong selection for a vast majority of these shared mycobacterial genes, with only 68 ORFs with a synonymous to nonsynonymous substitution ratio of >2. Comparative sequence analysis reveals several noteworthy features of the K-10 genome including: a relative paucity of the PE/PPE family of sequences that are implicated as virulence factors and known to be immunostimulatory during Mtb infection; truncation in the EntE domain of a salicyl-AMP ligase (MbtA), the first gene in the mycobactin biosynthesis gene cluster, providing a possible explanation for mycobactin dependence of Map; and Map-specific sequences that are likely to serve as potential targets for sensitive and specific molecular and immunologic diagnostic tests. Taken together, the availability of the complete genome sequence offers a foundation for the study of the genetic basis for virulence and physiology in Map and enables the development of new generations of diagnostic tests for bovine Johne's disease.

Animals↗

PubMed2005

An analysis of the use of genomic DNA as a universal reference in two channel DNA microarrays.

BACKGROUND: DNA microarray is an invaluable tool for gene expression explorations. In the two-dye microarray, fluorescence intensities of two samples, each labeled with a different dye, are compared after hybridization. To compare a large number of samples, the 'reference design' is widely used, in which all RNA samples are hybridized to a common reference. Genomic DNA is an attractive candidate for use as a universal reference, especially for bacterial systems with a low percentage of non-coding sequences. However, genomic DNA, comprising of both the sense and anti-sense strands, is unlike the single stranded cDNA usually used in microarray hybridizations. The presence of the antisense strand in the 'reference' leads to reactions between complementary labeled strands in solution and may cause the assay result to deviate from true values. RESULTS: We have developed a mathematical model to predict the validity of using genomic DNA as a reference in the microarray assay. The model predicts that the assay can accurately estimate relative concentrations for a wide range of initial cDNA concentrations. Experimental results of DNA microarray assay using genomic DNA as a reference correlated well to those obtained by a direct hybridization between two cDNA samples. The model predicts that the initial concentrations of labeled genomic DNA strands and immobilized strands, and the hybridization time do not significantly affect the assay performance. At low values of the rate constant for hybridization between immobilized and mobile strands, the assay performance varies with the hybridization time and initial cDNA concentrations. For the case where a microarray with immobilized single strands is used, results from hybridizations using genomic DNA as a reference will correspond to true ratios under all conditions. CONCLUSION: Simulation using the mathematical model, and the experimental study presented here show the potential utility of microarray assays using genomic DNA as a reference. We conclude that the use of genomic DNA as reference DNA should greatly facilitate comparative transcriptome analysis.

DNA↗

PubMed2005

Transcriptional response of avian cells to infection with Newcastle disease virus.

Newcastle disease virus (NDV) causes widespread disease in poultry and wild-birds throughout the world. cDNA microarray analysis was used to examine the effect of NDV infection on host cell transcription. The results show that NDV infection causes an apparent suppression of the interferon response genes during the early stages of infection. In addition, the results reveal transcriptional silencing of cytoskeletal proteins such as the alpha, beta, and gamma types of actin, and a downregulation of the thioredoxin gene, a likely mediator of apoptosis with possible implications in NDV pathogenesis. Comparative analyses show that a majority of genes that were transcriptionally regulated during infection with another common respiratory pathogen of poultry, the avian pneumovirus, remained unaltered during NDV infection, suggesting that even phylogenetically related viruses elicit unique or "signature" patterns of host transcriptional profiles during infection of host cells.

Actins↗

PubMed2005

Transcriptional analysis of avian embryonic tissues following infection with avian infectious bronchitis virus.

Avian infectious bronchitis virus (IBV) infection is one of the major viral respiratory diseases of chickens. Better understanding of the molecular basis of viral pathogenesis should contribute significantly towards the development of improved prophylactic, therapeutic and diagnostic reagents to control infections. In the present investigation, transcriptional profiles were analyzed by using RNA recovered from the lung tissue of IBV infected 18-day-old chicken embryos at 6, 24, 48 and 72 h post IBV infection. This microarray analysis was completed using avian cDNA arrays comprised of fragments of 1191 unique chicken and turkey gene transcripts. These arrays were generated from normalized cDNA subtraction libraries that were derived from avian pneumovirus (APV) infected chicken embryo fibroblast (CEF) cultures and tissues obtained from APV infected turkeys subtracted with their respective uninfected cultures and tissues. Of the 1191 unique genes represented on the array, the expression of a total of 327 genes (27% of total) were altered by two-fold or more from 6 through 72 h post-infection. A comparative analysis of IBV regulated genes with genes previously reported to change in expression following infection with other avian respiratory viruses revealed both conserved and unique changes. Real-time qRT-PCR was used to confirm the regulated expression of genes related to several functional classes including kinases, interferon induced genes, chemokines and adhesion molecules, vesicular trafficking and fusion protein genes, extracellular matrix protein genes, cell cycle, metabolism, cell physiology and development, translation, RNA binding, lysosomal, protein degradation and ubiquitination related genes. Microarray analysis served as an efficient tool in facilitating a comparative analysis of avian respiratory viral infections and provided insight into host transcriptional changes that were conserved as well as those which were unique to individual pathogens.

Animals↗

PubMed2005

Transcriptional response of Enterococcus faecalis V583 to erythromycin.

A transcriptional profile of Enterococcus faecalis V583 (V583) treated with erythromycin is presented. This is the first study describing a complete transcriptional profile of Enterococcus. E. faecalis is a common and nonvirulent bacterium in many natural environments, but also an important cause of nosocomial infections. We have used a genome-wide microarray based on the genome sequence of V583 to study gene expression in cells exposed to erythromycin. V583 is resistant to relatively high concentrations of erythromycin, but growth is retarded by the treatment. The effect of erythromycin treatment on V583 was studied by a time course experiment; samples were extracted at five time points over a period of 90 min. A drastic change in gene transcription was seen with the erythromycin-treated cells compared to the untreated cells. Altogether, 260 genes were down-regulated at one or more time points, while 340 genes were up-regulated. Genes encoding hypothetical proteins and genes encoding transport and binding proteins were the two most dominating groups of differentially expressed genes. The gene encoding ermB (EFA0007) was expressed, but not differentially, which indicated that other genes are important for the survival and growth maintenance of V583 treated with erythromycin. One of these genes is a putative MsrC-like protein, which was up-regulated at all time points studied. Other specific genes that were found to be up-regulated were genes encoding ABC transporters and two-component regulatory systems, and these may be genes that are important for the specific response of V583 to erythromycin.

Anti-Bacterial Agents↗

PubMed2005

Characterization of the pheromone response of the Enterococcus faecalis conjugative plasmid pCF10: complete sequence and comparative analysis of the transcriptional and phenotypic responses of pCF10-containing cells to pheromone induction.

The sex pheromone plasmids in Enterococcus faecalis are one of the most efficient conjugative plasmid transfer systems known in bacteria. Plasmid transfer rates can reach or exceed 10(-1) transconjugants per donor in vivo and under laboratory conditions. We report the completion of the DNA sequence of plasmid pCF10 and the analysis of the transcription profile of plasmid genes, relative to conjugative transfer ability following pheromone induction. These experiments employed a mini-microarray containing all 57 open reading frames of pCF10 and a set of selected chromosomal genes. A clear peak of transcription activity was observed 30 to 60 min after pheromone addition, with transcription subsiding 2 h after pheromone induction. The transcript activity correlated with the ability of donor cells to transfer pCF10 to recipient cells. Remarkably, aggregation substance (Asc10, encoded by the prgB gene) was present on the cell surface for a long period of time after pheromone-induced transcription of prgB and plasmid transfer ability had ceased. This observation could have relevance for the virulence of E. faecalis.

Chromosome Mapping↗

PubMed2005

Comparative genomic hybridizations reveal genetic regions within the Mycobacterium avium complex that are divergent from Mycobacterium avium subsp. paratuberculosis isolates.

Mycobacterium avium subsp. paratuberculosis is genetically similar to other members of the Mycobacterium avium complex (MAC), some of which are nonpathogenic and widespread in the environment. We have utilized an M. avium subsp. paratuberculosis whole-genome microarray representing over 95% of the predicted coding sequences to examine the genetic conservation among 10 M. avium subsp. paratuberculosis isolates, two isolates each of Mycobacterium avium subsp. silvaticum and Mycobacterium avium subsp. avium, and a single isolate each of both Mycobacterium intracellulare and Mycobacterium smegmatis. Genomic DNA from each isolate was competitively hybridized with DNA from M. avium subsp. paratuberculosis K10, and open reading frames (ORFs) were classified as present, divergent, or intermediate. None of the M. avium subsp. paratuberculosis isolates had ORFs classified as divergent. The two M. avium subsp. avium isolates had 210 and 135 divergent ORFs, while the two M. avium subsp. silvaticum isolates examined had 77 and 103 divergent ORFs. Similarly, 130 divergent ORFs were identified in M. intracellulare. A set of 97 ORFs were classified as divergent or intermediate in all of the nonparatuberculosis MAC isolates tested. Many of these ORFs are clustered together on the genome in regions with relatively low average GC content compared with the entire genome and contain mobile genetic elements. One of these regions of sequence divergence contained genes homologous to a mammalian cell entry (mce) operon. Our results indicate that closely related MAC mycobacteria can be distinguished from M. avium subsp. paratuberculosis by multiple clusters of divergent ORFs.

Conserved Sequence↗