Biomedical subjects

S K Arora

Publications and source records attributed to S K Arora.

At least 19 recordsLinked to original sources

PubMed2004

In vitro pharmacological investigations of Sapindus trifoliatus in various migraine targets.

Phytotherapies have offered alternative sources of therapy for migraine and gained much importance in prophylactic treatment. The aqueous extract of pericarp of fruits of Sapindus trifoliatus Linn (ST), family Sapindaceae was evaluated for its affinity for 5-HT(1B/1D) receptors in rabbit saphenous vein, alpha-adrenoceptors in rabbit aorta, GABA receptors in guinea pig ileum, 5-HT(2B) receptors in rat fundus and vanilloid receptors in guinea pig trachea. The calcium blocking effect was studied in rabbit aorta while the modulatory role of ST on platelet serotonin release was evaluated in human platelets. The aqueous extract of Sapindus trifoliatus exhibited significant 5-HT(2B) receptor inhibition and moderate platelet serotonin release inhibition.

Adult↗

PubMed2004

Structural and genetic characterization of glycosylation of type a flagellin in Pseudomonas aeruginosa.

Type a flagellins from two strains of Pseudomonas aeruginosa, strains PAK and JJ692, were found to be glycosylated with unique glycan structures. In both cases, two sites of O-linked glycosylation were identified on each monomer, and these sites were localized to the central, surface-exposed domain of the monomer in the assembled filament. The PAK flagellin was modified with a heterogeneous glycan comprising up to 11 monosaccharide units that were O linked through a rhamnose residue to the protein backbone. The flagellin of JJ692 was less complex and had a single rhamnose substitution at each site. The role of the glycosylation island gene cluster in the production of each of these glycosyl moieties was investigated. These studies revealed that the orfA and orfN genes were required for attachment of the heterologous glycan and the proximal rhamnose residue, respectively.

Amino Acid Sequence↗

PubMed2004

Investigations into the antinociceptive activity of Sapindus trifoliatus in various pain models.

The effect of the aqueous extract of Sapindus trifoliatus (ST) on chemical, thermal-induced pain, nitroglycerin-induced hyperalgesia and pain on inflamed tissue was investigated. The extract (20 and 100 mg x kg(-1), i.p.) significantly inhibited acetic-acid-induced abdominal constrictions, formalin-induced pain licking and hotplate-induced pain in mice. Furthermore, the extract significantly increased the response latencies of nitroglycerin-induced hyperalgesia by the tail-flick method and mechanical pain on carrageenan-induced inflamed paw in rats. The data suggest that ST has an inhibitory activity on both peripheral and central pain mechanisms and has a modulatory role in NO-mediated nociceptive transmission.

Analgesics↗

PubMed2003

Stroke: a rare presentation of cardiac hydatidosis.

A 13-year-old boy presented with acute stroke leading to right-sided hemiparesis. A contrast CT scan of the brain showed a hemorrhagic infarct in the left basal ganglia region with surrounding edema. Echocardiography showed a hydatid cyst in the right atrial chamber extending into the left atrium. A single hepatic hydatid was also seen.

Adolescent↗

PubMed2002

Identification and functional characterization of flgM, a gene encoding the anti-sigma 28 factor in Pseudomonas aeruginosa.

We describe here the functional characterization of the putative flgM gene of Pseudomonas aeruginosa. FlgM of P. aeruginosa is most similar to FlgM of Vibrio parahaemolyticus. A conserved region is present in the C-terminal half of the FlgM of P. aeruginosa and in FlgM homologues of other organisms that includes the sigma(28) binding domain. A role for the flgM gene of P. aeruginosa in motility was demonstrated by its inactivation. The beta-galactosidase activity of a transcriptional fusion of the fliC promoter to lacZ was upregulated in the flgM mutant, suggesting that the activity of FliA, the sigma factor that regulates fliC, was increased. Consistent with these results, an increased amount of flagellin was demonstrated in the flgM mutant of P. aeruginosa strain PAK by Western blot, suggesting that FlgM negatively regulates transcription of fliC by inhibiting the activity of FliA. Direct interaction of the P. aeruginosa FlgM with the alternative sigma factor sigma(28) was demonstrated by utilizing the yeast two-hybrid system. Three putative consensus sigma(54) recognition sites and one sigma(28) site were found in the flgM upstream region. However, analysis of the transcriptional fusion of the flgM promoter to lacZ in different mutant backgrounds showed that the flgM promoter was not entirely dependent on either sigma(28) or sigma(54). A transcript was detected by primer extension that was 8 bp downstream of the consensus sigma(28)-binding site. Thus, a system for the control of flagellin synthesis by FlgM exists in P. aeruginosa that is different from that in the enteric bacteria and seems to be most similar to that of V. cholerae where both sigma(28)-dependent and -independent mechanisms of transcription exist.

Amino Acid Sequence↗

PubMed2001

A genomic island in Pseudomonas aeruginosa carries the determinants of flagellin glycosylation.

Protein glycosylation has been long recognized as an important posttranslational modification process in eukaryotic cells. Glycoproteins, predominantly secreted or surface localized, have also been identified in bacteria. We have identified a cluster of 14 genes, encoding the determinants of the flagellin glycosylation machinery in Pseudomonas aeruginosa PAK, which we called the flagellin glycosylation island. Flagellin glycosylation can be detected only in bacteria expressing the a-type flagellin sequence variants, and the survey of 30 P. aeruginosa isolates revealed coinheritance of the a-type flagellin genes with at least one of the flagellin glycosylation island genes. Expression of the b-type flagellin in PAK, an a-type strain carrying the glycosylation island, did not lead to glycosylation of the b-type flagellin of PAO1, suggesting that flagellins expressed by b-type bacteria not only lack the glycosylation island, they cannot serve as substrates for glycosylation. Providing the entire glycosylation island of PAK, including its a-type flagellin in a flagellin mutant of a b-type strain, results in glycosylation of the heterologous flagellin. These results suggest that some or all of the 14 genes on the glycosylation island are the genes that are missing from strain PAO1 to allow glycosylation of an appropriate flagellin. Inactivation of either one of the two flanking genes present on this island abolished flagellin glycosylation. Based on the limited homologies of these gene products with enzymes involved in glycosylation, we propose that the island encodes similar proteins involved in synthesis, activation, or polymerization of sugars that are necessary for flagellin glycosylation.

Chromosomes, Bacterial↗

PubMed2001

Recognition of mucin components by Pseudomonas aeruginosa.

Pseudomonas aeruginosa remains one of the most important bacterial pathogens in lung diseases and especially in Cystic fibrosis. This unusual predilection is best explained by the existence of defects in host defense mechanisms as resulting from the genetic lesion and the presence of a specific colonization niche within the lungs. The niche has been identified as the mucus layer wherein mucin glycoproteins provide a substrate for binding and allows the persistence of this organism in this milieu by a number of possible mechanisms. While this organism is capable of binding to non CF mucins, it is perhaps a combination of factors e.g. increased binding and decreased mucociliary clearance that is responsible for this marked state of colonization in CF. The organism uses chiefly proteins of its flagellar apparatus to initiate this binding and recognizes a variety of oligosaccharides that have been identified in mucins. Among these are both, neutral oligosaccharides and several forms of acidic oligosaccharides derived from the Lewis antigens. There are more than likely a larger repertoire of receptors than those identified and certainly more adhesins present than those currently known. However, the information gathered to date provides an excellent example of the specificity of bacterial interactions with mucins that will certainly be expanded as we study more pulmonary pathogens.

Bacterial Adhesion↗

PubMed2001

FlhA, a component of the flagellum assembly apparatus of Pseudomonas aeruginosa, plays a role in internalization by corneal epithelial cells.

Pseudomonas aeruginosa invades various epithelial cell types in vitro and in vivo. The P. aeruginosa genome possesses a gene (flhA) which encodes a protein that is believed to be part of the export apparatus for flagellum assembly and which is homologous to invA of Salmonella spp. Because invA is required for invasion of Salmonella spp., a role for flhA in P. aeruginosa invasion was explored using cultured rabbit corneal epithelial cells. An flhA mutant of P. aeruginosa strain PAO1 was constructed and was shown to be nonmotile. Complementation with flhA in trans restored motility. Corneal cells were infected for 3 h with the wild type (PAO1), the flhA mutant, the flhA mutant complemented with flhA in trans, an flhA mutant containing the plasmid vector control, or an fliC mutant (nonmotile mutant control). Invasion was quantified by amikacin exclusion assays. Both the flhA and the fliC mutants invaded at a lower level than the wild-type strain did, suggesting that both fliC and flhA played roles in invasion. However, loss of motility was not sufficient to explain the reduced invasion by flhA mutants, since centrifugation of bacteria onto cells did not restore invasion to wild-type levels. Unexpectedly, the flhA mutant adhered significantly better to corneal epithelial cells than wild-type bacteria or the fliC mutant did. The percentage of adherent bacteria that invaded was reduced by approximately 80% for the flhA mutant and approximately 50% for the fliC mutant, showing that only part of the role of flhA in invasion involves fliC. Invasion was restored by complementing the flhA mutant with flhA in trans but not by the plasmid vector control. Intracellular survival assays, in which intracellular bacteria were enumerated after continued incubation in the presence of antibiotics, showed that although flhA and fliC mutants had a reduced capacity for epithelial cell entry, they were not defective in their ability to survive within those cells after entry. These results suggest that the flagellum assembly type III secretion system plays a role in P. aeruginosa invasion of epithelial cells. Since the flhA mutants were not defective in their ability to adhere to corneal epithelial cells, to retain viability at the cell surface, or to survive inside epithelial cells after entry, the role of flhA in invasion of epithelial cells is likely to occur during the process of bacterial internalization.

Animals↗

PubMed2001

Recognition of Lewis x derivatives present on mucins by flagellar components of Pseudomonas aeruginosa.

Pseudomonas aeruginosa binds to human respiratory mucins by mechanisms involving flagellar component-receptor interactions. The adhesion of P. aeruginosa strain PAK is mediated by the flagellar cap protein, FliD, without the involvement of flagellin. Two distinct types of FliD proteins have been identified in P. aeruginosa: A type, found in strain PAK, and B type, found in strain PAO1. In the present work, studies performed with the P. aeruginosa B-type strain PAO1 indicate that both the FliD protein and the flagellin of this strain are involved in the binding to respiratory mucins. Using polyacrylamide-based fluorescent glycoconjugates in a flow cytometry assay, it was previously demonstrated that P. aeruginosa recognizes Le(x) (or Lewis x) derivatives found at the periphery of human respiratory mucins. The aim of the present work was therefore to determine whether these carbohydrate epitopes (or glycotopes) are receptors for FliD proteins and flagellin. The results obtained by both flow cytometry and a microplate adhesion assay indicate that the FliD protein of strain PAO1 is involved in the binding of glycoconjugates bearing Le(x) or sialyl-Le(x) determinants, while the binding of flagellin is restricted to the glycoconjugate bearing Le(x) glycotope. In contrast, the type A cap protein of P. aeruginosa strain PAK is not involved in the binding to glycoconjugates bearing Le(x), sialyl-Le(x), or sulfosialyl-Le(x) glycotopes. This study demonstrates a clear association between a specific Pseudomonas adhesin and a specific mucin glycotope and demonstrates that fine specificities exist in mucin recognition by P. aeruginosa.

Bacterial Adhesion↗

PubMed2001

Evaluation of polymerase chain reaction for detection of Mycobacterium tuberculosis in pleural fluid.

OBJECTIVES: Tuberculosis, a reemergent killer, is threatening to assume serious proportions all over the world, particularly in view of the AIDS pandemic. The detection of mycobacterial DNA by polymerase chain reaction (PCR) in clinical samples is a promising approach for the rapid diagnosis of tuberculous infections. The aims of this study were to evaluate PCR for detection of Mycobacterium tuberculosis in pleural fluids and to correlate the results with adenosine deaminase activity (ADA) estimation and acid-fast bacilli (AFB) screening. METHODS: The sensitivity and specificity of PCR in detection of mycobacterial DNA in 20 samples of tuberculous pleural effusion were evaluated using 40 samples of nontubercular pleural effusion as controls. The results were correlated with the ADA in all 60 pleural fluids. In addition, AFB detection by Ziehl-Neelsen staining on cytospin smears of all pleural fluids was also compared. RESULTS: Of the 20 samples of tuberculous pleural effusion, mycobacterium could be detected by AFB staining in 4 samples. Fourteen samples were PCR positive. None of the samples from the control group were AFB or PCR positive. The sensitivity of PCR, therefore, was 70.0% with specificity of 100% (positive predictive value, 100%; negative predictive value, 86.95%). The sensitivity of AFB screening was at best 20%. The mean of ADA values in tubercular pleural effusions was 63.21 U/L (SD, 33.01), and the mean in the control samples was 51.1 U/L (SD, 29.71). Taking a cut-off value of 50 U/L, both the sensitivity and specificity of ADA estimation in diagnosing tuberculosis were only 55%. CONCLUSION: PCR represents a rapid and sensitive method for the detection of mycobacterial DNA in tuberculous pleural effusions. AFB screening has low sensitivity, and ADA estimation has both low sensitivity and specificity. Therefore, when the clinical suspicion is high and smear result is negative, but the signs and symptoms of M tuberculosis are apparent, PCR is the method of choice for identifying the infection.

Adenosine Deaminase↗

PubMed2000

Development of a polymerase chain reaction dot-blotting system for detecting cutaneous tuberculosis.

For a definitive diagnosis of cutaneous tuberculosis the demonstration of mycobacteria is essential, but this is generally not possible in skin lesions. Routinely available techniques have poor sensitivity and are time consuming, therefore, delaying the institution of timely therapy. The high sensitivity and speed of polymerase chain reaction (PCR) for the detection of infectious agents has prompted investigators to use this technique for the detection of Mycobacterium tuberculosis in body fluids such as cerebrospinal fluid or pleural fluid. In the present study, PCR was used to examine punch biopsy specimens from the affected skin of 10 patients with clinical diagnoses of tuberculosis verrucosa cutis, lupus vulgaris, scrofuloderma, papulonecrotic tuberculide and erythema induratum. A control group of 20 patients included individuals having skin manifestations with definite clinical diagnoses other than cutaneous tuberculosis, such as leprosy, fungal mycetoma, chronic bullous disease of childhood and pemphigus vulgaris. The PCR amplified products were dot hybridized with a probe which was random prime labelled with 32P. The results were compared with routine microbiological and histological findings. Among the test group, six of 10 (60%) were positive for M. tuberculosis by PCR, although their histopathology showed non-specific chronic inflammation with no definite diagnosis. Microbiological investigations, including acid-fast bacillus smear and culture, were positive in a single case of scrofuloderma. All patients in the control group were negative by PCR for M. tuberculosis. The data indicate that the combination of dot hybridization with PCR markedly increased the sensitivity and specificity of PCR. This may be a useful tool in the diagnosis of tuberculosis when conventional methods fail.

Humans↗

PubMed2000

Identification of two distinct types of flagellar cap proteins, FliD, in Pseudomonas aeruginosa.

Binding of Pseudomonas aeruginosa strain PAK to mucin has been shown to be mediated by the flagellar cap protein, product of the fliD gene. Since the flagellar cap is very likely an exposed structure, the FliD polypeptide should be recognized by the host immune system, analogous to the recognition of dominant epitopes located in the exposed parts of the flagellin polypeptide within the assembled flagellum. In P. aeruginosa, a number of distinct flagellin variants are made, and these variable sequences presumably allow the newly infected P. aeruginosa to escape recognition by the antibody induced during a previous infection. Since similar mechanisms may direct the selection of FliD variants, we examined the extent of sequence heterogeneity among various FliD sequences among a selected group of P. aeruginosa. The results of PCR and nucleotide sequencing of the fliD region of eight different P. aeruginosa strains (laboratory strains PAK, PAO1, and PA103; clinical strains 1244, CS2, and CS32; cystic fibrosis strains CS29 and MDR) suggested that there were two distinct types of FliD in P. aeruginosa, which we named A type and B type. The results of Western blotting using the polyclonal antibodies raised against the purified FliD of A type (PAK) or B type (PAO1) further confirmed the existence of two distinct antigenic types of FliD proteins, with no cross-reactivity between the two serotypes. Further Western immunoblot analysis of the same strains using polyclonal FliC antibody showed that the strains with A-type FliD possessed a-type FliC and those with B-type FliD had b-type FliC. Similar Western blot analyses of 50 more P. aeruginosa strains obtained from varied sources revealed that all strains contained either A-type or B-type FliD, suggesting the existence of only two types of FliD in P. aeruginosa and indicating that fliC and fliD were coinherited. This limited diversity of FliC and FliD serotypes seems to be a unique feature of flagellar proteins. A chromosomal mutant having an insertion in the fliD gene of P. aeruginosa PAO1 was constructed. The motility defect of this mutant and a previously constructed PAK fliD mutant was better complemented with the fliD gene of the homologous types.

Amino Acid Sequence↗

PubMed2000

fleN, a gene that regulates flagellar number in Pseudomonas aeruginosa.

The single polar flagellum of Pseudomonas aeruginosa plays an important role in the pathogenesis of infection by this organism. However, regulation of the assembly of this organelle has not been delineated. In analyzing the sequence available at the Pseudomonas genome database, an open reading frame (ORF), flanked by flagellar genes flhF and fliA, that coded for a protein (280 amino acids) with an ATP-binding motif at its N terminus was found. The ORF was inactivated by inserting a gentamicin cassette in P. aeruginosa PAK and PAO1. The resulting mutants were nonmotile on motility agar plates, but under a light microscope they exhibited random movement and tumbling behavior. Electron microscopic studies of the wild-type and mutant strains revealed that the mutants were multiflagellate, with three to six polar flagella per bacterium as rather than one as in the wild type, indicating that this ORF was involved in regulating the number of flagella and chemotactic motility in P. aeruginosa. The ORF was named fleN. An intact copy of fleN on a plasmid complemented the mutant by restoring motility and monoflagellate status. The beta-galactosidase activities of eight flagellar operon or gene promoters in the wild-type and fleN mutant strains revealed a direct correlation between six promoters that were upregulated in the fleN mutant (fliLMNOPQ, flgBCDE, fliEFG, fliDS orf126, fleSR, and fliC) and positive regulation by FleQ, an NtrC-like transcriptional regulator for flagellar genes. Based on these results, we propose a model where FleN influences FleQ activity (directly or indirectly) in regulating flagellar number in P. aeruginosa.

Amino Acid Sequence↗

PubMed1999

Synthesis and antitumor activity of goniofufurone analogues.

Synthesis and antitumor activity of goniofufurone analogues 15, 16, 17, 33, and 46 is reported. Key step in the synthesis is Pd (II) mediated oxidative cyclisation of vinyl-(hydroxy) furans 18, 19 to the corresponding lactols 32, 43. Cytotoxicities of 15, 16, 17, 33, and 46 tested against six human cancer cell lines are reported. Change of stereochemistry at C-5, C-6 and C-7 position of goniofufurone (1) did not enhance the cytotoxicities significantly.

Antineoplastic Agents↗

PubMed1999

Diagnostic efficacy of polymerase chain reaction in granulomatous uveitis.

SETTING: The granulomatous uveitis, multifocal choroiditis and periphlebitis have been suspected to be of tubercular origin but no definitive reports about detection of etiological agents have been documented in the literature. Conventional bacteriological methods are not generally helpful in diagnosing ocular tuberculosis due to difficulty with potential morbidity associated with obtaining the biopsy material from the eye. Thus, the diagnosis of ocular tuberculosis is most often presumptive. OBJECTIVE: We evaluated the role of polymerase chain reaction (PCR) for detection of Mycobacterium tuberculosis in the aqueous humor samples obtained from eyes with active uveitis. METHODS: Aqueous samples from 53 patients having cellular reaction in the anterior chamber along with any one or more of the following: 1) active vasculitis; 2) anterior vitreous cells; 3) snowball opacities; 4) snow banking in the pars plana; 5) retinochoroiditis were withdrawn by anterior chamber paracentesis and subjected to PCR. Seventeen samples from patients with definite clinical diagnoses other than tuberculosis formed a disease control group. Fifteen aqueous samples obtained from healthy subjects undergoing routine cataract surgery served as healthy controls. PCR was performed using primers capable of amplifying a 150 b.p. segment from a conserved repetitive sequence in the genome of M. tuberculosis. RESULTS: Twenty out of the 53 samples (37.7%) in the study group were positive where as only one sample out of 17 in the disease control group (5.7%) showed a weakly positive band. No sample from the healthy control group showed a positive PCR. CONCLUSION: Our study shows that PCR can be effectively used for the diagnosis of intraocular tuberculosis in the presence of uveitis.

Aqueous Humor↗

PubMed1998

Genetic polymorphism of Leishmania species using kinetoplast DNA restriction fragment length polymorphism and cDNA probe of Leishmania donovani.

Leishmaniasis represents a group of diseases that range from simple cutaneous lesions through metastasizing diffused cutaneous to severe systemic infection depending upon the taxon to which the causative parasite belongs. Therefore, it is important to identify the infecting Leishmania. Methods presently being used, including immunology, biochemistry and molecular biology have one or the other limitations, leaving scope for the search for newer probes. This study reports the characterization of leishmania isolates both by restriction fragment length polymorphism of kinetoplast DNA (kDNA) and genomic DNA. The genomic DNA was probed with a cDNA probe B2a1. Using a kDNA restriction pattern technique, different isolates of Leishmania donovani could be differentiated from the UR6 strain of L. tropica, but it was not possible to differentiate between newer local isolates of L. donovani with most of the restriction enzymes except AluI. However, the B2a1 cDNA probe was able to differentiate these isolates effectively. Both of these techniques could differentiate newer local isolates of L. donovani from the older isolates of L. donovani from India, i.e., DD8, RMRI and SS. The Indian isolates of L. donovani could also be differentiated from isolates of L. donovani from Jeddah and Germany using both techniques. The present study indicates that the cDNA probe B2a1 can be used as an important adjunct to kDNA restriction analysis for the characterization of Leishmania species.

Animals↗

PubMed1998

Heterogeneity in heat shock protein genes in Leishmania isolates.

Leishmaniasis, a group of visceral and cutaneous diseases, is caused by parasites belonging to one genus comprising approximately 13 different species. Many methods including serological, biochemical and molecular biological techniques have been used by various workers to characterize these different species and isolates of Leishmania, yet there is no single generally accepted criterion. We have identified certain cDNA clones from a library generated from the promastigotes of S1 strain of Leishmania donovani and used them as probes for identification of various isolates of L. donovani and Leishmania tropica. Two of the probes used, E2b (2.0 kb) and E1a (1.3 kb), sequence characterized to be hsp70 of Leishmania, were able to distinguish various isolates of L. donovani from different geographical origins as well as strains of L. donovani from those of L. tropica. Thus, by using recombinant hsp70 cDNA probes, the data indicated that there is a considerable degree of heterogeneity in the heat-shock genes of Leishmania.

Animals↗

PubMed1998

The Pseudomonas aeruginosa flagellar cap protein, FliD, is responsible for mucin adhesion.

Mucin-specific adhesion of Pseudomonas aeruginosa plays an important role in the initial colonization of this organism in the airways of cystic fibrosis patients. We report here that the flagellar cap protein, FliD, participates in this adhesion process. A polar chromosomal insertional mutation in the P. aeruginosa fliD gene made this organism nonadhesive to mucin in an in vitro mucin adhesion assay. The adhesive phenotype was restored by providing the fliD gene alone on a multicopy plasmid, suggesting involvement of this gene in mucin adhesion of P. aeruginosa. Further supporting this observation, the in vitro competition experiments demonstrated that purified FliD protein inhibited the mucin adhesion of nonpiliated P. aeruginosa PAK-NP, while the same concentrations of PilA and FlaG proteins of P. aeruginosa were ineffective in this function. The regulation of the fliD gene was studied and was found to be unique in that the transcription of the fliD gene was independent of the flagellar sigma factor sigma28. Consistent with this finding, no sigma28 binding sequence could be identified in the fliD promoter region. The results of the beta-galactosidase assays suggest that the fliD gene in P. aeruginosa is regulated by the newly described transcriptional regulator FleQ and the alternate sigma factor sigma54 (RpoN).

Amino Acid Sequence↗