Biomedical subjects

Jagjit S Yadav

Publications and source records attributed to Jagjit S Yadav.

13 recordsLinked to original sources

PubMed2006

Global gene expression changes underlying Stachybotrys chartarum toxin-induced apoptosis in murine alveolar macrophages: evidence of multiple signal transduction pathways.

The overall mechanism(s) underlying macrophage apoptosis caused by the toxins of the indoor mold Stachybotrys chartarum (SC) are not yet understood. In this direction, we report a microarray-based global gene expression profiling on the murine alveolar macrophage cell line (MH-S) treated with SC toxins for short (2 h) and long (24 h) periods, coinciding with the pre-apoptotic (<3 h) and progressed apoptotic stages of the treated cells, respectively. Microarray results on differential expression were validated by real-time RT-PCR analysis using representative gene targets. The toxin-regulated genes corresponded to multiple cellular processes, including cell growth, proliferation and death, inflammatory/immune response, genotoxic stress and oxidative stress, and to the underlying multiple signal transduction pathways involving MAPK-, NF-kB-, TNF-, and p53-mediated signaling. Transcription factor NF-kB showed dynamic temporal changes, characterized by an initial activation and a subsequent inhibition. Up-regulation of a battery of DNA damage-responsive and DNA repair genes in the early stage of the treatment suggested a possible role of genotoxic stress in the initiation of apoptosis. Simultaneous expression changes in both pro-survival genes and pro-apoptotic genes indicated the role of a critical balance between the two processes in SC toxin-induced apoptosis. Taken together, the results imply that multiple signaling pathways underlie the SC toxin-induced apoptosis in alveolar macrophages.

Animals↗

PubMed2006

DNA damage, redox changes, and associated stress-inducible signaling events underlying the apoptosis and cytotoxicity in murine alveolar macrophage cell line MH-S by methanol-extracted Stachybotrys chartarum toxins.

Spore-extracted toxins of the indoor mold Stachybotrys chartarum (SC) caused cytotoxicity (release of lactate dehydrogenase), inhibition of cell proliferation, and cell death in murine alveolar macrophage cell line MH-S in a dose- and time-dependent manner. Apoptotic cell death, confirmed based on morphological changes, DNA ladder formation, and caspase 3/7 activation, was detectable as early as at 3 h during treatment with a toxin concentration of 1 spore equivalent/macrophage and was preceded by DNA damage beginning at 15 min, as evidenced by DNA comet formation in single cell gel electrophoresis assay. The apoptotic dose of SC toxins did not induce detectable nitric oxide and pro-inflammatory cytokines (IL-1beta, IL-6, and TNF-alpha) but showed exacerbated cytotoxicity in presence of a non-apoptotic dose of the known pro-inflammatory agent LPS (10 ng/ml). Intracellular reduced glutathione (GSH) level showed a significant decrease beginning at 9 h of the toxin treatment whereas oxidized glutathione (GSSG) showed a corresponding significant increase, indicating a delayed onset of oxidative stress in the apoptosis process. The toxin-treated macrophages accumulated p53, an indicator of DNA damage response, and showed activation of the stress-inducible MAP kinases, JNK, and p38, in a time-dependent manner. Chemical blocking of either p38 or p53 inhibited in part the SC toxin-induced apoptosis whereas blocking of JNK did not show any such effect. This study constitutes the first report on induction of DNA damage and associated p53 activation by SC toxins, and demonstrates the involvement of p38- and p53-mediated signaling events in SC toxin-induced apoptosis of alveolar macrophages.

Animals↗

PubMed2005

Occurrence and characterization of multiple novel genotypes of Mycobacterium immunogenum and Mycobacterium chelonae in metalworking fluids.

Rapidly growing mycobacteria colonize metalworking fluids, leading to contamination of occupational environments and exposure-related respiratory illnesses in machine workers. Lately, it has been emphasized that these fluids are colonizable by a single genotype of a rapidly growing mycobacterium species, Mycobacterium immunogenum. Here, we report on the genotypic diversity of mycobacteria in these fluids, including isolation and characterization of multiple novel genotypes of two distinct species, Mycobacterium chelonae and M. immunogenum. Using agar culturing and Mycobacterium-specific PCR, 13 mycobacterial isolates were recovered from 100 geographically diverse in-use metalworking fluid samples. Based on restriction fragment length polymorphism of PCR products, DNA sequencing (hsp65 gene segment), and phylogenetic analysis of 16S-23S rDNA internal transcribed spacer (ITS) sequences, six isolates were identified as M. immunogenum and seven as M. chelonae; an additional isolate from metalworking fluid diluent water was identified as M. diernhoferi. Genomic DNA macro-restriction fragment pattern analysis, using pulsed-field gel electrophoresis with XbaI and SpeI restriction digestions, showed intraspecies variation among the isolates of M. immunogenum and M. chelonae. Visual and computer-assisted dendrogram analysis of the XbaI macro-restriction patterns revealed three novel genotypes of M. immunogenum and two of M. chelonae, whereas SpeI macro-restriction patterns revealed only two genotypes for each isolate. None of the identified genotypes matched the reportedly dominant one of M. immunogenum from metalworking fluids. Both mycobacterial species are prevalent in metalworking fluids and there is a considerable strain-level genetic diversity within them.

Bacterial Proteins↗

PubMed2005

Genome-wide structural and evolutionary analysis of the P450 monooxygenase genes (P450ome) in the white rot fungus Phanerochaete chrysosporium: evidence for gene duplications and extensive gene clustering.

BACKGROUND: Phanerochaete chrysosporium, the model white rot basidiomycetous fungus, has the extraordinary ability to mineralize (to CO2) lignin and detoxify a variety of chemical pollutants. Its cytochrome P450 monooxygenases have recently been implied in several of these biotransformations. Our initial P450 cloning efforts in P. chrysosporium and its subsequent whole genome sequencing have revealed an extraordinary P450 repertoire ("P450ome") containing at least 150 P450 genes with yet unknown function. In order to understand the functional diversity and the evolutionary mechanisms and significance of these hemeproteins, here we report a genome-wide structural and evolutionary analysis of the P450ome of this fungus. RESULTS: Our analysis showed that P. chrysosporium P450ome could be classified into 12 families and 23 sub-families and is characterized by the presence of multigene families. A genome-level structural analysis revealed 16 organizationally homogeneous and heterogeneous clusters of tandem P450 genes. Analysis of our cloned cDNAs revealed structurally conserved characteristics (intron numbers and locations, and functional domains) among members of the two representative multigene P450 families CYP63 and CYP505 (P450foxy). Considering the unusually complex structural features of the P450 genes in this genome, including microexons (2-10 aa) and frequent small introns (45-55 bp), alternative splicing, as experimentally observed for CYP63, may be a more widespread event in the P450ome of this fungus. Clan-level phylogenetic comparison revealed that P. chrysosporium P450 families fall under 11 fungal clans and the majority of these multigene families appear to have evolved locally in this genome from their respective progenitor genes, as a result of extensive gene duplications and rearrangements. CONCLUSION: P. chrysosporium P450ome, the largest known to date among fungi, is characterized by tandem gene clusters and multigene families. This enormous P450 gene diversity has evolved by extensive gene duplications and intragenomic recombinations of the progenitor genes presumably to meet the exceptionally high metabolic demand of this biodegradative group of basidiomycetous fungi in ecological niches. In this context, alternative splicing appears to further contribute to the evolution of functional diversity of the P450ome in this fungus. The evolved P450 diversity is consistent with the known vast biotransformation potential of P. chrysosporium. The presented analysis will help design future P450 functional studies to understand the underlying mechanisms of secondary metabolism and oxidative biotransformation pathways in this model white rot fungus.

Alternative Splicing↗

PubMed2005

Physiological regulation, xenobiotic induction, and heterologous expression of P450 monooxygenase gene pc-3 (CYP63A3), a new member of the CYP63 gene cluster in the white-rot fungus Phanerochaete chrysosporium.

In order to characterize the functional diversity in CYP63 cluster of tandemly linked P450 genes (pc-1, pc-2, and pc-3) in Phanerochaete chrysosporium, here we report the functional characterization of pc-3 (CYP63A3), a newly cloned member of this group. pc-3 expression was favored in nutrient-limited versus nutrient-rich media in 3-6-day-old cultures and was upregulated by starch as a carbon source or by oxygenation of cultures. pc-3 was induced by various xenobiotics in defined nutrient-limited (3-9-fold) and nutrient-rich (2-5-fold) cultures. Particularly, a range of unsubstituted and substituted aliphatic hydrocarbons (alkanes and fatty acids) induced the expression under the two nutrient conditions albeit in a differential manner. Interestingly, pc-3 was also inducible by certain oxygenated mono aromatics (nitrophenol, benzoate, and resorcinol), lower molecular weight (2 to 4 ring size) polycyclic aromatic hydrocarbons (PAHs) and alkali-treated lignin derivatives in nutrient-rich malt extract cultures. The study further establishes that the three CYP63 genes (CYP63A1, A2, and A3) are independently regulated despite being members of the tandem gene cluster with high gene structural similarity (13-14 introns) and protein sequence homology (59-85%). The pc-3 cDNA (1,812 bp) was expressed in E. coli as a His-tagged protein (approximately 74 kDa). This constitutes the first report on heterologous expression of a P450 monooxygenase enzyme from this model white-rot fungus.

Adaptation, Physiological↗

PubMed2005

Biocidal activity of formaldehyde and nonformaldehyde biocides toward Mycobacterium immunogenum and Pseudomonas fluorescens in pure and mixed suspensions in synthetic metalworking fluid and saline.

The microbicidal activity of four different biocides was studied in synthetic metalworking fluid (MWF) against Mycobacterium immunogenum, a suspected causative agent for hypersensitivity pneumonitis, and Pseudomonas fluorescens, a representative for the predominant gram-negative bacterial contaminants of MWF. The results indicated that M. immunogenum is more resistant than P. fluorescens to the tested formaldehyde-releasing biocides (Grotan and Bioban), isothiazolone (Kathon), and phenolic biocide (Preventol). Kathon was effective against mycobacteria at lower concentrations than the other three test biocides in MWF. In general, there was a marked increase in biocidal resistance of both the test organisms when present in MWF matrix compared to saline. Increased resistance of the two test organisms to biocides was observed when they were in a mixed suspension (1:1 ratio). The results indicate the protective effect of the MWF matrix against the action of commonly used biocides on the MWF-colonizing microbial species of occupational health significance, including mycobacteria.

Alveolitis, Extrinsic Allergic↗

PubMed2005

Method for rapid identification and differentiation of the species of the Mycobacterium chelonae complex based on 16S-23S rRNA gene internal transcribed spacer PCR-restriction analysis.

Members of the Mycobacterium chelonae complex (MCC), including M. immunogenum, M. chelonae, and M. abscessus, have been associated with nosocomial infections and occupational hypersensitivity pneumonitis due to metalworking fluid (MWF) exposures. In order to minimize these health hazards, an effective and rapid assay for detection of MCC species and differentiation of MCC species from other species of rapidly growing mycobacteria (RGM) and from one another is warranted. Here we report such a method, based on the variable 16S-23S rRNA gene internal transcribed spacer (ITS) region. Mycobacterium genus-specific primers derived from highly conserved sequences in the ITS region and the flanking 16S rRNA gene were used. Specificity of the primers was verified using the MCC member species, 11 non-MCC RGM species, 3 slow-growing mycobacterial (SGM) species (two strains each), and 19 field isolates, including 18 MCC isolates (from in-use MWF) and one non-MCC isolate (from reverse osmosis water). The ITS amplicon size of M. immunogenum varied from those of M. chelonae and M. abscessus. Sequencing of the approximately 250-bp-long ITS amplicons of the three MCC member species showed differences in 24 to 34 bases, thereby yielding variable deduced restriction maps. ITS PCR-restriction analysis using the in silico-selected restriction enzyme MaeII or HphI differentiated the three MCC members from one another and from other RGM and SGM species without sequencing. The enzyme MaeII discriminated all three member species; however, HphI could only differentiate M. immunogenum from M. chelonae and M. abscessus. Use of an optimized rapid DNA template preparation step based on direct cell lysis in the PCR tube added to the simplicity and adaptability of the developed assay.

Bacterial Typing Techniques↗

PubMed2004

A new method for species identification and differentiation of Mycobacterium chelonae complex based on amplified hsp65 restriction analysis (AHSPRA).

Members of the Mycobacterium chelonae complex (MCC), namely M. chelonae, Mycobacterium abscessus and Mycobacterium immunogenum, have been implicated in nosocomial infections and occupational respiratory illnesses like hypersensitivity pneumonitis (HP) associated with contaminated metalworking fluid (MWF) exposures. Close relationship among these member species makes their differentiation cumbersome using the existing methods. Here we report a simple and rapid method for unambiguous identification and differentiation of the three-member species of the MCC group with PCR-restriction analysis targeting a 667-bp segment of a variable region of the 65-kDa-heat shock protein (hsp65) gene. This assay, described as Amplified hsp65 Restriction Analysis (AHSPRA), can discriminate all the three individual species using a one-step restriction digestion using either BbvI or Eco0109I. The enzyme NarI can differentiate M. immunogenum from the other two MCC species (M. chelonae and M. abscessus). The developed method was validated using several non-MCC reference species of other rapidly growing mycobacteria (RGM) and MCC field isolates from MWF samples. Direct cell-lysis was used instead of the conventional DNA template preparation, which improved the rapidity, simplicity and adaptability of the developed method. The results suggest that the developed method can unambiguously differentiate species of the M. chelonae complex from other RGM species and from one another.

Bacterial Proteins↗

PubMed2004

Differential regulation and xenobiotic induction of tandem P450 monooxygenase genes pc-1 (CYP63A1) and pc-2 (CYP63A2) in the white-rot fungus Phanerochaete chrysosporium.

The two tandem P450 monooxygenase genes (pc-1 and pc-2) reported by us earlier in Phanerochaete chrysosporium were investigated for their regulation under nutrient-limited and nutrient-rich culture conditions. Transcript analysis based on real-time quantitative RT-PCR showed higher expression of pc-1 in defined low-nitrogen and pc-2 in defined high-nitrogen media, with maximum expression on day 4, indicating that the two genes, though tandemly linked, are regulated in a non-coordinate manner. Transcript levels of pc-1 and pc-2 were differentially influenced by the type of carbon source, incubation temperature, and oxygenation. Both genes were inducible by organic xenobiotic chemicals. Of the 42 xenobiotics tested in nutrient-rich cultures, pc-1 transcription was induced 2.12(+/-0.40)-fold to 6.27(+/-0.48)-fold in the presence of 19 compounds and pc-2 transcription was induced 2.00(+/-0.73)-fold to 29.39(+/-9.40)-fold in the presence of 22 compounds. Particularly, it is significant that both pc-1 and pc-2 are induced by polycyclic aromatic hydrocarbons (PAHs) of varying ring size, including naphthalene (4.35+/-0.09, 6.02+/-1.39), phenanthrene (2.82+/-0.12, 2.14+/-0.61), pyrene (3.93+/-0.01, 1.0+/-0.12), benzanthracene (1.67+/-0.03, 6.08+/-1.50), and benzo(a)pyrene (1.55+/-0.01, 5.54+/-2.75) respectively. This study constitutes the first report on the identification of P450 genes in a fungus that are responsive to environmentally significant pollutant chemicals (PAHs, DDT, long-chain alkyl phenols) and lignin derivatives.

Adaptation, Physiological↗

PubMed2004

Real-time PCR assays for genus-specific detection and quantification of culturable and non-culturable mycobacteria and pseudomonads in metalworking fluids.

Genus-specific real-time PCR assays were developed and optimized for the direct culture-independent detection and quantification of Mycobacteria and Pseudomonads in contaminated metalworking fluids (MWF) and the results were compared with conventional culturing using selective media. It included optimization of the direct DNA isolation from the fluid matrix and the amplification conditions using genus-specific primers. Mycobacterium-specific primers based on 65-kDa heat shock protein (hsp) gene, and Pseudomonas-specific primers based on 16S rRNA gene were used. A standard curve was developed each for the two model bacterial species Mycobacterium immunogenum and Pseudomonas fluorescens, representing two important genera frequently isolated from MWF. A minimum quantification limit of 10 cells/ml was achieved although as low as 1 cell/ml yielded a detectable amplicon signal. Of the twenty MWF field samples contaminated with mixed microflora, only two samples yielded putative colonies of Mycobacteria and Pseudomonads by culturing method, while seven samples responded to the genus-specific real-time PCR detection and quantification for each genus. In contrast to the low culturable counts, the real-time PCR based cell counts ranged from 1.3 x 10(2) to 5.5 x 10(5)cells/ml and 5.2 x 10(2) to 7.0 x 10(5)cells/ml for Mycobacteria and Pseudomonads, respectively, indicating a significant non-culturable fraction in the fluids, for the two genera. This is the first application of real-time PCR protocol to MWF samples for detection and quantification of total (culturable and non-culturable) Mycobacteria and Pseudomonads without culturing.

Bacterial Proteins↗

PubMed2004

Development of a single-tube, cell lysis-based, genus-specific PCR method for rapid identification of mycobacteria: optimization of cell lysis, PCR primers and conditions, and restriction pattern analysis.

A single-tube PCR method was developed for efficient identification of nontuberculous mycobacteria (NTM) and their environmental isolates in about 3 h without conventional DNA isolation. The following three steps were optimized or developed: (i). a simple, 6-min direct cell lysis protocol as a PCR prestep for generation of DNA-template, (ii). an improved Mycobacterium-specific PCR amplification protocol with a broader species specificity using newly designed primers targeting a 228-bp region of the 65-kDa heat shock protein (hsp) gene and optimal PCR amplification conditions, and (iii). a genus-specific restriction analysis of the PCR product for conclusive identification of the unknown NTM isolates.

Bacteriolysis↗

PubMed2003

Tandem cytochrome P450 monooxygenase genes and splice variants in the white rot fungus Phanerochaete chrysosporium: cloning, sequence analysis, and regulation of differential expression.

Phanerochaete chrysosporium, the model white rot fungus, uses P450 monooxygenation for oxidation of xenobiotic chemicals. To characterize its P450 systems, we have isolated two tandemly linked full-length P450 monooxygenase genes, pc-1 (2510bp) and pc-2 (2503bp). cDNA sequence and splice site analysis showed multiple small putative introns (35-82bp), 17 in pc-1 and 16 in pc-2. The deduced P450 proteins PC-1 (531 aa, MW 60,070) and PC-2 (538 aa, MW 61,765) showed 55% aa identity with each other and low relatedness (25-36%) with P450 gene families CYP52, CYP86, CYP4, and CYP3 decreasing in that order. Alternate splicing was identified for pc-1 transcription. Real-time quantitative RT-PCR analysis on 4-day cultures showed an elevated level of expression ( approximately 8-fold) of pc-1 in nutrient-limited (ligninolytic) as compared to nutrient-rich (non-ligninolytic) conditions. Interestingly, the pc-1 gene is expressible in both primary and secondary phases of growth with peak expression occurring with secondary metabolism.

Alternative Splicing↗

PubMed2003

DNA-based methodologies for rapid detection, quantification, and species- or strain-level identification of respiratory pathogens (Mycobacteria and Pseudomonads) in metalworking fluids.

Mycobacteria and pseudomonads occurring in modern metalworking fluids (MWF) have been implicated in occupational health hazards as causal agents for hypersensitivity pneumonitis (HP) and other respiratory illnesses in machine workers exposed to these fluids and their aerosols. Unlike the conventional cultural and biochemical methods, which are often slow and ambiguous and detect only culturable cells, DNA-based methods offer a time-saving alternative for reliable detection and identification of both culturable and nonculturable bacteria in MWF and for selective quantification of individual genera of pathogens of interest in these fluids. This is the first report on DNA-based direct detection of mycobacteria and pseudomonads in MWF without culturing. Genus-specific PCR approach was successfully applied for screening of field MWF samples originating from different industrial users for detection of mycobacteria or pseudomonads including both culturable and nonculturable cells. PCR in combination with amplicon DNA sequencing led to the identification of Mycobacterium chelonae, Pseudomonas nitroreducens, and an undefined Pseudomonas species from these fluids. Genome fingerprinting by pulsed-field gel electrophoresis (PFGE) on Mycobacterium isolates further showed that the isolates represented three strains of M. chelonae although the possibility of one of the strains being clonal with M. immunogenum cannot be excluded. In parallel efforts, a quantitative competitive PCR method developed based on the Pseudomonas-specific PCR was applied to quantify total P. fluorescens cells in contaminated metalworking fluid and MWF aerosol without culturing. The DNA-based protocols developed in this study will allow rapid screening of field MWF samples for the presence of both culturable and nonculturable cells and thus facilitate effective fluid management and timely exposure assessment.

DNA Fingerprinting↗