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Biomedical subjects

M Singh

Publications and source records attributed to M Singh.

At least 253 records · Page 14Linked to original sources
PubMed2000

Blastocystis hominis: A simplified, high-efficiency method for clonal growth on solid agar.

Colony growth of protozoan parasites in agar can be useful for axenization, cloning, and viability studies. This is usually achieved with the pour plate method, for which the parasite colonies are situated within the agar. This technique has been described for Giardia intestinalis, Trichomonas vaginalis, and Entamoeba and Blastocystis species. Extracting such colonies can be laborious. It would be especially useful if parasites could be grown on agar as colonies. These colonies, being exposed on the agar surface, could be conveniently isolated for further investigation. In this study, we report the successful culture of B. hominis cells as colonies on solid agar. Colonies were enumerated and the efficiency of plating was determined. It was observed that B. hominis could be easily cultured on agar as clones. The colonies were dome-shaped and mucoid and could grow to 3 mm in diameter. Flow cytometric analyses revealed that parasite colonies remained viable for up to 2 weeks. Viable colonies were conveniently expanded in liquid or solid media. Scanning electron microscopy revealed that each colony consists of two regions; a dome-shaped, central core region and a flattened, peripheral region. Older colonies possessed numerous strand-like surface coat projections. This study provides the first report of clonal growth of B. hominis on agar and a simple, effective method for cloning and expansion of B. hominis cells.

Agar↗
PubMed2000

Electronic Spectrum of the Antimony Monoxide Radical.

The 0-1 band (4271.3 Å) of the B(2)Sigma(+)-X(2)(2)Pi(3/2) transition, the 0-6 band (4275 Å) of the H(2)Pi(3/2)-X(2)(2)Pi(3/2) transition, the 0-0 band (3776.2 Å) of the B(2)Sigma(+)-X(1)(2)Pi(1/2) transition, and the 2-0 (3251.3 Å) as well as the 3-0 (3194.2 Å) bands of the C(2)Sigma(-)-X(1)(2)Pi(1/2) transition of SbO have been photographed at high resolution using the single isotope of antimony (121)Sb. An unambiguous analysis of the rotational structure of all these bands has been carried out for the first time. The magnitude and sign of the Lambda doubling constant p(0) of the X(1)(2)Pi(1/2) state have been determined and as a result of that, the symmetry of the C state has been uniquely identified as C(2)Sigma(-). Accurate rotational constants of all the states involved have been determined by a global linear least-squares fit. Copyright 2000 Academic Press.

Journal Article↗
PubMed2000

Possible mechanism of cardioprotective effect of ischaemic preconditioning in isolated rat heart.

The present study is designed to investigate the mechanism of the cardioprotective effect of ischaemic preconditioning. Isolated perfused rat heart was subjected to global ischaemia for 30 min followed by reperfusion for 120 min. Coronary effluent was analysed for LDH and CK release to assess the degree of cardiac injury. Myocardial infarct size was estimated macroscopically using TTC staining. Four episodes of ischaemic preconditioning markedly reduced LDH and CK release in the coronary effluent and decreased myocardial infarct size. Administration of prazosin (alpha(1)adrenoceptor antagonist) before global ischaemia reduced the extent of ischaemia-reperfusion induced myocardial injury. The cardioprotective effect of ischaemic preconditioning was abolished by prazosin and colchicine (microtubule disaggregator). On the basis of these results, it may be concluded that the cardioprotective effects of ischaemic preconditioning may be mediated through stimulation of alpha(1)adrenoceptors and translocation of PKC.

Animals↗
PubMed2000

Oral ketamine for radiotherapy in children with cancer.

Children coming for radiotherapy under sedation usually get repeated injections, which cause distress to both the child and the parents. A prospective study was conducted to evaluate the efficacy of oral ketamine for sedation for radiotherapy (RT) in children with cancer. Ten children who received 49 sittings of RT were given 8-15 mg/kg body weight of oral ketamine. The onset time, recovery time, efficacy of sedation and incidence of abnormal movements were compared with another group of 8 children, who received intramuscular ketamine in the dose of 6 mg/kg for a total of 28 sittings of RT. Onset time and recovery time were significantly longer in oral ketamine group as compared to the intramuscular group (p < 0.001). Limb movements in patients receiving oral ketamine necessitated further supplement of sedation and interruption of RT. These drawbacks discourage use of oral ketamine as a good sedative for radiotherapy treatment in paediatric oncology patients.

Administration, Oral↗
PubMed2000

His-tagged tryparedoxin peroxidase of Trypanosoma cruzi as a tool for drug screening.

Tryparedoxin peroxidase has recently been identified as a constituent of the complex peroxidase system in the trypanosomatid Crithidia fasciculata [Nogoceke E, Gommel DU, Kiess M, Kalisz HM, Flohe L (1997) Biol Chem 378: 827-836]. In trypanosomatids, hydroperoxides are reduced at the expense of NADPH by means of a cascade of three oxidoreductases: the flavoprotein trypanothione reductase, tryparedoxin and tryparedoxin peroxidase. Inhibitors of these enzymes are presumed to be trypanocidal drugs. Here, we present the heterologous expression of a putative tryparedoxin peroxidase gene of Trypanosoma cruzi (accession no AJ012101) as an N-terminally His-tagged protein (TcH6TXNPx). The product was purified with a high yield (8.75 mg from 11 fermentation broth of A(600)2.1) from the cytosolic fraction of sonified Escherichia coli BL21(DE3)[pET22b( + )/TcH6TXNPx] by metal-chelating chromatography. TcH6TXNPx proved to be fully active when tested with heterologous tryparedoxins of C. fasciculata (His-tagged TXN1H6 and TXN2H6). TcH6TXNPx displayed ping-pong kinetics with a k(cat) of 1.7 s(-1) and limiting Km values of 51.8 microM and 1.7 microM for t-butyl hydroperoxide and CfTXN2H6, respectively.

Amino Acid Sequence↗
PubMed2000

Duplex RT-PCR: reagent concentrations at reverse transcription stage affect the PCR performance.

Test conditions for the simultaneous detection of potato leafroll virus (PLRV) and potato virus Y (PVY) in dormant tubers and leaves by reverse transcription-polymerase chain reaction (RT-PCR) were optimized. Various factors optimized at the reverse transcription (RT) stage rather than at the amplification (PCR) stage affected the outcome. In the simplex RT-PCR a onefold dNTPs concentration (0.5 mM) was sufficient in yielding a PLRV or PVY band. In contrast, the duplex RT-PCR required a minimum twofold dNTPs concentration (1.0 mM) during RT to produce distinct bands in PCR. Similarly, various proportions of antisense primers of PLRV and PVY used during RT affected subsequent duplex RT-PCR. Optimal amplification of both viruses were obtained at a ratio of 0.90:0.49 microM of PLRV:PVY antisense primers. An interaction of dNTPs and RNA template concentration was observed. A higher concentration of RNA was required at onefold dNTPs concentration than at twofold dNTPs. Dilutions down to 1:300 of RNA template yielded distinct bands of both viruses at twofold dNTPs concentration. At optimized conditions of the duplex RT-PCR both viruses were reliably detected in composite samples at a ratio of one part infected sap mixed with 399 parts of sap from healthy tubers. Application of optimized conditions to singly- and doubly-infected tubers detected both viruses from naturally infected field-grown tubers. A nearly perfect correlation (r(2)=0.99) was observed between visible plant symptoms and the virus detection from leaves and tubers by the duplex RT-PCR.

DNA Primers↗
PubMed2000

Improving outcome over time of percutaneous coronary interventions in unstable angina.

OBJECTIVE: This study was performed to evaluate the recent changes in the outcome of coronary interventions in patients with unstable angina (UA). BACKGROUND: An early invasive strategy has not been shown to be superior to conservative treatment in patients with UA. Earlier studies had utilized older technology. Interventional approaches have changed in the recent past, but to our knowledge, no large studies have addressed the impact of these changes on the outcome of coronary interventions. METHODS: We analyzed the in-hospital and intermediate-term outcome in 7,632 patients with UA who underwent coronary interventions in the last two decades. The study population was divided into three groups: group 1, n = 2,209 who had coronary intervention from 1979 to 1989; group 2, n = 2,212 with interventions from 1990 to 1993; and group 3, n = 3,211 treated from 1994 to 1998. RESULTS: Group 2 and 3 patients were older and sicker compared with group 1 patients. The clinical success improved significantly in group 3 (94.1%) compared with group 2 (87%) and group 1 (76.5%) (p < 0.001). There was a significant reduction in in-hospital mortality, Q-wave myocardial infarction and need for emergency bypass surgery in group 3 compared with the earlier groups. One-year event-free survival was also significantly higher in the recent group compared with the earlier groups: 77% in group 3, 70% in group 2 and 74% in group 1 (p < 0.001). With the use of multivariate models to adjust for clinical and angiographic variables, treatment during the most recent era was found to be independently associated with improved in-hospital and intermediate-term outcomes. CONCLUSIONS: There has been significant improvement in the in-hospital and intermediate-term outcome of coronary interventions in patients with UA in recent years; newer trials comparing conservative and invasive strategies are therefore needed.

Aged↗
PubMed2000

Connective eccentricity index: a novel topological descriptor for predicting biological activity.

A simple, adjacency-cum-path length based, topological descriptor termed the connective eccentricity index has been conceptualized and its discriminating power investigated with regard to antihypertensive activity. A data set consisting of 81 derivatives of N-benzylimidazole was selected for the present investigation. These derivatives are potent, competitive, and nonpeptide angiotensin II receptor antagonists. The value of connective eccentricity index for each derivative was computed and active range identified. Subsequently, each derivative was assigned a biological activity that was compared with the reported antihypertensive activity. The results obtained using connective eccentricity index were better than the corresponding values obtained using Balaban's mean square distance index. The accuracy of prediction was found to be about 80% in the active range using connective eccentricity index.

Angiotensin Receptor Antagonists↗
PubMed2000

Blastocystis elongation factor-1alpha: genomic organization, taxonomy and phylogenetic relationships.

The elongation factor-1 alpha (EF-1alpha) is a highly conserved ubiquitous protein that is involved in translation and is desirable for use in phylogenetic studies on Blastocystis, an enigmatic intestinal parasite with a contentious taxonomic position. In the present study, a PCR product (BEalpha) that codes for a major part of the coding region of the EF-lalpha protein was amplified. Genome walking experiments together with cloning were implemented to elucidate the 5' and 3' ends of the EF-1alpha gene of the human isolate, Blastocystis hominis C. The genomic organization and the potential transcription factor binding sites of the 5' end of B. hominis C EF-1alpha were identified. A comparative study on the deduced amino acid sequences of BEalpha of 13 Blastocystis isolates from various hosts was done to evaluate the phylogenetic relationship among the species. A phylogenetic reconstruction analysis with other eukaryotic EF-1alpha sequences was carried out to trace the phylogenetic position of Blastocystis among eukaryotic organisms.

Amino Acid Sequence↗
PubMed2000

Effect of ethylisopropyl amiloride, a Na+ - H+ exchange inhibitor, on cardioprotective effect of ischaemic and angiotensin preconditioning.

The present study is designed to investigate the role of Na+ -H+ exchanger in the cardioprotective effect of ischaemic and angiotensin (Ang II) preconditioning. Isolated perfused rat heart was subjected to global ischaemia for 30 min followed by reperfusion for 120 min. Coronary effluent was analysed for LDH and CK release to assess the degree of cardiac injury. Myocardial infarct size was estimated macroscopically using TTC staining. Left ventricular developed pressure (LVDP) and dp/dt were recorded to evaluate myocardial contractility. Four episodes of ischaemic or Ang II preconditioning markedly reduced LDH and CK release in coronary effluent and decreased myocardial infarct size. 5-(N-ethyl-N-isopropyl)amiloride (EIPA), a Na+ - H+ exchange inhibitor, produced no marked effect on ischaemic preconditioning and Ang II preconditioning induced cardioprotection. On the other hand, EIPA administration prior to global ischaemia produced a similar reduction in myocardial injury as was noted with ischaemic preconditioning or Ang II preconditioning. On the basis of these results, it may be concluded that inhibition of Na+ - H+ exchanger protects against ischaemia-reperfusion induced myocardial injury whereas activation of Na+ - H+ exchanger may not mediate the cardioprotective effect of ischaemic and Ang II preconditioning.

Amiloride↗
PubMed2000

Rat models of premalignant breast disease.

While a number of agents have been shown to induce mammary carcinogenesis in the rat, premalignant stages of the disease have been best characterized in chemically-induced models, specifically those initiated by either 7,12 dimethylbenz[alpha]anthracene (DMBA) or 1-methyl-1-nitrosourea (MNU). In general, it appears that epithelial cells in mammary terminal end buds or terminal ductules are the targets of carcinogenic initiation, and that a series of morphologically identifiable steps are involved in the development of mammary carcinoma. The premalignant steps include ductal hyperplasia of the usual type and carcinoma in situ of the cribriform or comedo type; atypical ductal hyperplasia has not been reported. Thus the histogenesis of lesions occurring in chemically induced mammary carcinogenesis in the rat is similar to that observed in the human; although, the spectrum of lesions observed in the rat is limited. Opportunities to investigate the biological and molecular characteristics of premalignant breast disease in the rat are presented.

9,10-Dimethyl-1,2-benzanthracene↗
PubMed2000

Cytokine analysis at the single cell level and lymphoproliferative responses to mycobacterial antigens in HIV-1 patients with successful virologic response to potent antiretrovirals.

Immunologic parameters, known to be grossly abnormal in HIV-1-infected subjects, were analyzed in 22 patients with sustained viral load suppression (<200 copies/ml) following long-term highly active antiretroviral therapy (HAART). Responses were compared with those from 18 HIV-seronegative healthy controls. Persistent phenotypic alterations in patients' blood mononuclear cells were minimal, though the percentages of lymphocytes that could be activated to produce interleukin-2 (IL-2) remained severely depressed. Using lymphoproliferative assays, a striking deficit in the capacity of patients to respond to the common mycobacterial antigens and particularly to recombinant heat-shock proteins paralleled the absence of responses to virus p24 antigen. In view of the important immunoregulatory role of stress proteins, these findings reveal profound functional deficiencies and persistent immune dysregulation in HIV-1 patients, despite successful HAART and a considerable recovery of CD4+ lymphocyte numbers. Rational immunotherapeutic approaches should be aimed to correct the characterized immune abnormalities.

Antigens, Bacterial↗
PubMed2000

Classification of premalignant and malignant lesions developing in the rat mammary gland after injection of sexually immature rats with 1-methyl-1-nitrosourea.

Premalignant and malignant stages of mammary carcinogenesis can be rapidly induced by injecting female rats i.p. with 1-methyl-1-nitrosourea (MNU)3 at 21 days of age. In this paper, the characteristics of this model are briefly reviewed and the histology of the lesions induced is presented and compared to those that occur in humans. Malignant mammary lesions induced in rats injected with MNU at 21 days of age are compared with the lesions that develop when MNU is administered to 50-day-old female rats.

Adenocarcinoma↗
PubMed2000

A comparison of the histopathology of premalignant and malignant mammary gland lesions induced in sexually immature rats with those occurring in the human.

The injection of sexually immature female rats with 1-methyl-1-nitrosourea results in a rapid induction of premalignant and malignant mammary gland lesions within 35 days of carcinogen administration. This model affords the opportunity for investigators to study the process of mammary carcinogenesis over a very short latency and to investigate early events in this process. We have recently published on various aspects of this system including the histology of the lesions induced, the time frame of their occurrence, and their dependence on ovarian hormones for their maintenance and growth. In this report we present evidence that many aspects of the histopathology of mammary lesions in this model system are similar to those occurring in humans. We also discuss aspects of the human disease, which are not recapitulated in this model.

Animals↗
PubMed2000

Plasmid DNA adsorbed onto cationic microparticles mediates target gene expression and antigen presentation by dendritic cells.

Dendritic cells (DC) play a key role in antigen presentation and activation of specific immunity. Much current research focuses on harnessing the potency of DC for vaccines, gene therapy, and cancer immunotherapy applications. However, DC are not readily transfected in vitro by traditional nonviral techniques. A novel DNA vaccine formulation was used to determine if DC are transfected in vitro. The formulation consists of plasmid DNA adsorbed on to cationic microparticles composed of the biodegradable polymer polylactide-co-glycolide (PLG) and the cationic surfactant, cetyltrimethylammonium bromide (CTAB). Using preparations of fluorescent-labeled plasmid DNA formulated on PLG-CTAB microparticles to study internalization by macrophages and dendritic cells in vitro and in vivo, we found that most, but not all, of the fluorescence was concentrated in endosomal compartments. Furthermore, uptake of plasmid DNA encoding HIV p55 gag adsorbed to PLG-CTAB microparticles by murine bone marrow-derived dendritic cells resulted in target gene expression, as detected by RT-PCR. The antigen was subsequently processed and presented, resulting in stimulation of an H-2kd-restricted, gag-specific T cell hybridoma. Activation of the hybridoma, detected by IL-2 production, was dose-dependent in the range of 0.1-20 microg DNA (10-2000 microg PLG) and was sustained up to 5 days after transfection. Thus, adsorption of plasmid DNA on PLG-CTAB microparticles provides a potentially useful nonviral approach for in vitro transfection of dendritic cells. Gene Therapy (2000) 7, 2105-2112.

Adsorption↗
PubMed2000

Change of Th0 to Th1 cell-cytokine profile following tuberculosis chemotherapy.

T cells mediate protection against tuberculosis, but little is known about their role during chemotherapy of patients with active disease. Here we examined the cytokine profile of CD4 T cells before and after four months of chemotherapy in six initial skin test anergic cases. Purified protein derivative (PPD) and 16-kDa antigen-reactive CD4 T-cell clones prior to therapy resided mostly in disease-associated body fluids and were of the Th0 (interferon (IFN)-gamma + interleukin (IL)-4) secreting profile. In contrast, the majority of postchemotherapy CD4 T-cell clones originated from blood and were of the IFN-gamma secreting Th1 type. However, the recognition of several peptides derived from the 16-kDa antigen was not significantly different between the Th1 and Th0 clones. We conclude that chemotherapy shifts CD4 T cells from the affected body fluids to the blood circulation, accompanied by a change from Th0 to Th1 cytokine profile.

CD4-Positive T-Lymphocytes↗