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Biomedical subjects

M Joshi

Publications and source records attributed to M Joshi.

At least 55 records · Page 3Linked to original sources
PubMed1998

Hemoglobin biosynthesis in Vitreoscilla stercoraria DW: cloning, expression, and characterization of a new homolog of a bacterial globin gene.

In the strictly aerobic, gram-negative bacterium Vitreoscilla strain C1, oxygen-limited growth conditions create a more than 50-fold increase in the expression of a homodimeric heme protein which was recognized as the first bacterial hemoglobin (Hb). The recently determined crystal structure of Vitreoscilla Hb has indicated that the heme pocket of microbial globins differs from that of eukaryotic Hbs. In an attempt to understand the diverse functions of Hb-like proteins in prokaryotes, we have cloned and characterized the gene (vgb) encoding an Hb-like protein from another strain of Vitreoscilla, V. stercoraria DW. Several silent changes were observed within the coding region of the V. stercoraria vgb gene. Apart from that, V. stercoraria Hb exhibited interesting differences between the A and E helices. Compared to its Hb counterpart from Vitreoscilla strain C1, the purified preparation of V. stercoraria Hb displays a slower autooxidation rate. The differences between Vitreoscilla Hb and V. stercoraria Hb were mapped onto the three-dimensional structure of Vitreoscilla Hb, which indicated that the four changes, namely, Ile7Val, Ile9Thr, Ile10Ser, and Leu62Val, present within the V. stercoraria Hb fall in the region where the A and E helices contact each other. Therefore, alteration in the relative orientation of the A and E helices and the corresponding conformational change in the heme binding pocket of V. stercoraria Hb can be correlated to its slower autooxidation rate. In sharp contrast to the oxygen-regulated biosynthesis of Hb in Vitreoscilla strain C1, production of Hb in V. stercoraria has been found to be low and independent of oxygen control, which is supported by the absence of a fumarate and nitrate reductase regulator box within the V. stercoraria vgb promoter region. Thus, the regulation mechanisms of the Hb-encoding gene appear to be quite different in the two closely related species of Vitreoscilla. The relatively slower autooxidation rate of V. stercoraria Hb, lack of oxygen sensitivity, and constitutive production of Hb suggest that it may have some other function(s) in the cellular physiology of V. stercoraria DW, together with facilitated oxygen transport, predicted for earlier reported Vitreoscilla Hb.

Amino Acid Sequence↗
PubMed1997

The importance of L-arginine metabolism in melanoma: an hypothesis for the role of nitric oxide and polyamines in tumor angiogenesis.

There is a worldwide increase in the incidence of melanoma. Without treatment, melanomas can progress to metastatic disease and result in death. It is now accepted that for a tumor to grow, non-tumorous host tissue must form blood vessels in and around the tumor. Tumor cells and blood vessels must form a highly regulated system whereby endothelial cells can be switched from a resting state to one of rapid growth. Tumor cells have been shown to produce diffusible angiogenic regulatory molecules. Nitric oxide [NO] and polyamines [PA] have been implicated in the angiogenic process. This paper hypothesizes that NO and PA regulate melanoma angiogenesis differently. During early stages of malignant melanoma an increase in PA synthesis is expected to promote endothelial cell proliferation and therefore angiogenesis. NO is expected to be maintained at low levels. During the vascular stage of malignant melanoma, NO synthesis is hypothesized to be elevated which will decrease endothelial cell proliferation and maintain a vasodilator tone in and around the tumor. PA concentrations are expected to be lower. A regulatory link between NO and PA may be involved in the maintenance of tumor homeostasis. The regulation of L-arginine metabolism in tumor angiogenesis requires investigation as it may lead to novel selective therapeutic interventions in cancer therapy.

Arginine↗
PubMed1997

Osteonecrosis following meningococcemia and disseminated intravascular coagulation in an adult: case report and review.

Meningococcemia and disseminated intravascular coagulation (DIC) have a known association, and they have been identified as a rare cause of osteonecrosis in children. To our knowledge, we report only the second case of an adult with DIC and Neisseria meningitidis infection whose condition was subsequently diagnosed as osteonecrosis. We also review the world medical literature that pertains to osteonecrosis as a sequelae of meningococcal infection associated with DIC.

Adult↗
PubMed1997

Improving the quality of the NCQA (National Committee for Quality Assurance) Annual Member Health Care Survey Version 1.0.

The National Committee for Quality Assurance (NCQA) developed a standardized survey instrument in 1995 designed to measure enrollee satisfaction with the care and services received from health plans across the United States. After the survey was administered for a large number of health plans and thousands of responses were received, some areas for survey improvement have emerged. The objective of this research was to evaluate the NCQA Annual Member Health Care Survey Version 1.0 (the standard form) relative to an alternate survey form created in cooperation with the HMO Group, Maritz Marketing Research, Inc., and Healthcare Research Systems, Ltd. The alternate form of the NCQA survey was constructed to test several theories of measurement improvement via rewording of items, reordering of items, deletion of items, and addition of items. The most important findings of the research project are reported herein. Ten geographically dispersed member health maintenance organizations (HMOs) of The HMO Group took part in the project. A split-half design was used to test the standard and alternate survey forms concurrently. Surveys were administered by using the NCQA-recommended mail methodology of survey and cover letter, reminder card, and second survey. Assuming a 50% response rate, a target of 400 responses (200 for each survey form) per HMO was planned. The window for responding was allowed to remain open 4 weeks beyond the mailing of the second survey to achieve the desired response rate. A total of 4,056 responses were collected (2,022 for the standard form and 2,034 for the alternate form). It was found that the addition of "No Experience" and "No Opinion" response options to the majority of satisfaction items reduced the random error associated with informed responses and produced statistically significant higher correlations with the global satisfaction items relative to the standard form items. Only four of the eight Short Form 12 summary scales, General Health, Reported Health Transition, Mental Health, and Social Functioning, were useful for adjusting data (covariation). The entire set of comorbidities (chronic disease checklist) could be eliminated without losing significant data adjustment capability. The multiple linear regression models generated by using the global satisfaction items on the alternate form had higher adjusted R2 values than the standard-form models. The alternate-form item Overall Value correlated highly with cost items and general satisfaction item, making it a useful global satisfaction variable for predictive modeling.

Health Care Surveys↗
PubMed1996

Isolation and characterization of Leishmania donovani calreticulin gene and its conservation of the RNA binding activity.

Calreticulin has been implicated in multiple cell functions. Recently, we have shown that both human and simian calreticulin are RNA binding proteins and that their binding activity is due to phosphorylation. To demonstrate that the RNA binding property of calreticulin is an intrinsic part of this multi-functional molecule and is evolutionarily conserved, we isolated and characterized the calreticulin gene from the unicellular parasite, Leishmania donovani. Amino acid sequence homology between human and Leishmania calreticulin (L. d. cal) is limited, but like the human homologue, L. d. cal binds Ca+2, can be phosphorylated in vitro and binds certain RNA sequences in a phosphorylation-dependent manner. Unlike human calreticulin, L. d. cal is glycosylated and its binding to endogenous Leishmania RNA is phosphorylation-independent. The binding of L. d. cal to Leishmania RNA suggests that the RNA binding activity of calreticulin has remained evolutionarily conserved.

Amino Acid Sequence↗
PubMed1996

Conservation of low-copy gene loci in Old World leishmanias identifies mechanisms of parasite evolution and diagnostic markers.

Genome plasticity has been hypothesized to be a driving force behind parasite speciation. We have evaluated divergence in single and low-copy genes in terms of locus organization, chromosomal localization and gene expression in Leishmania infantum, L. major, L. tropica and three widely divergent geographic isolates of L. donovani. Seventeen genes of low to moderate copy number (1-4 copies/haploid genome) were analyzed to identify restriction fragment length polymorphisms (RFLPs) providing heritable markers distinguishing Old World (OW) leishmanias. These RFLP markers were conserved in parasite isolates from primary infections demonstrating their utility as diagnostic tools. The species designations established by RFLP analysis of field isolates was confirmed by use of monoclonal antibodies. All 17 genes were present in each OW leishmania analyzed except LSIP (A45), which was absent from L. infantum. The 17 genes were found to be distributed among 9 distinct chromosomes. However, in spite of variations in chromosome karyotypes among the various OW leishmanias, individual gene probes localized to a similar sized chromosome from each isolate. These observations coupled with a molecular tree derived from RFLP data suggest that the OW leishmanias comprise a monophyletic lineage, with species associated with cutaneous disease exhibiting the greatest level of divergence. Data from this study supports previous observations that species causing cutaneous and visceral disease have diverged primarily by nucleotide substitutions. Such nucleotide divergence may not only lead to changes in protein function and antigenicity, but may also alter gene regulation programs as exemplified by the finding that the LdI-9-5 and LdE-6-1 genes were expressed only in visceralizing leishmanias.

Animals↗
PubMed1996

Nitric oxide synthase activity is up-regulated in melanoma cell lines: a potential mechanism for metastases formation.

Nitric oxide (NO) may be an important mediator of tumour angiogenesis and metastasis formation. Tumour cell derived NO may be important in the regulation of angiogenesis and vasodilatation of the blood vessels surrounding a tumour. The aims of the present study were, firstly, to determine whether malignant melanoma cells and normal melanocytes had nitric oxide synthase (NOS) activity (measured by the conversion of L-arginine to L-citrulline) and, secondly, to determine whether there was a difference in NOS activity between malignant and normal cell types. This paper assays NOS activity directly in lysates from normal human melanocyte and malignant melanoma cell lines. The enzyme activity was not inducible with bacterial lipopolysaccharide and could be heat denatured. The activity of NOS was demonstrated to be both NADPH- and calcium-dependent and it was inhibitable in a dose-dependent manner by the NOS inhibitor Nw-nitro-L-arginine methyl ester. We conclude that melanoma and melanocyte cells express a constitutive form of NOS. Finally, nitric oxide synthase activity in melanoma cell lines was found to be significantly greater than in normal melanocytes. These findings suggest that NO synthesis is elevated in malignant melanoma. An elevated NO concentration in melanoma is expected to promote metastases by maintaining a vasodilator tone in the blood vessels in and around the melanoma.

Cells, Cultured↗
PubMed1996

Role of the accident and emergency department in the non-heart-beating donor programme in Leicester.

OBJECTIVE: To describe the development of a non-heart-beating donor (NHBD) programme in an accident and emergency (A&E) department over a three year period. BACKGROUND AND METHODS: The A&E department at the Leicester Royal Infirmary at present deals with approximately 200 prehospital cardiopulmonary arrests per year. A programme of kidney retrieval from non-heart-beating donors was started in April 1992. Strict criteria for admission to the programme, appropriate consent procedures, facilities, lines of communication, and feedback were developed to enhance its success. RESULTS: Of 66 patients referred to the NHBD programme over a three year period from 1 April 1992, 51 sets of relatives were available to be asked for possible organ donation, and 34 sets (66%) gave their consent. Twenty five patients had successful in situ perfusion of the kidneys. Forty seven organs were retrieved and 34 went on to be transplanted. To date, 27 kidneys are still working. As a result, 23.8% of kidneys transplanted in Leicester over this time period have been from the NHBD programme. CONCLUSIONS: The NHBD programme in Leicester has proved very successful, requiring organisation of resources and personnel both from the transplant service and the A&E department. The programme has provided such a significant boost to the renal transplant rate in Leicester that other hospitals with large A&E departments should consider setting up similar programmes.

Emergency Service, Hospital↗
PubMed1995

Investigation of the role of reactive oxygen species in bilirubin metabolism in the Gunn rat.

It has been previously established that the attenuation of hepatic lipid peroxidation by a fat-free diet is accompanied by a marked rise in plasma bilirubin in Gunn rats. Present in vitro studies confirmed that microsomal lipid peroxidation caused the concurrent degradation of added bilirubin but failed to show that microsomal superoxide, hydroxyl radical or hydrogen peroxide would degrade bilirubin. Moreover, although injection of vitamin E completely inhibited microsomal lipid peroxidation and bilirubin degradation it had no effect on plasma bilirubin. No evidence has therefore been obtained that in Gunn rats, in the absence of bilirubin glucuronidation, that reactive oxygen species provide a significant physiological pathway of bilirubin disposal.

Animals↗
PubMed1995

Proteolysis in cultured liver epithelial cells during oxidative stress. Role of the multicatalytic proteinase complex, proteasome.

Exposure to various forms of mild oxidative stress significantly increased the intracellular degradation of both "short-lived" and "long-lived," metabolically radiolabeled, cell proteins in cultures of Clone 9 liver cells (normal liver epithelia). The oxidative stresses employed were bolus H2O2 addition; continuous H2O2 flux; the redox cycling quinones, menadione and paraquat; and the aldehydic products of lipid peroxidation, 4-hydroxynonenal, malonyldialdehyde, and hexenal. In general, exposure to more severe oxidative stress produced a concentration-dependent decline in intracellular proteolysis, in some cases to below baseline levels. Oxidatively modified "foreign" proteins (superoxide dismutase and hemoglobin) were also selectively degraded, in comparison with untreated foreign proteins, when added to lysates of Clone 9 liver cells. As with intracellular proteolysis, the degradation of foreign proteins added to cell lysates was greatly increased by mild oxidative modification, but depressed by more severe oxidative modification. The proteinase activity was recovered in > 300-kDa cell fractions, and inhibitor profiles and immunoprecipitation studies indicated that the multicatalytic proteinase complex, proteasome, was responsible for most of the selective degradation observed with mild oxidative stress; up to approximately 95% for intracellular proteolysis and 65-80% for degradation of foreign modified proteins. Seven days of daily treatment with an antisense oligodeoxynucleotide, directed against the initiation codon region of the proteasome C2 subunit gene, severely depressed the intracellular levels of several proteasome subunit polypeptides (by Western blot analysis), and also depressed the H2O2 induced increase in intracellular proteolysis by approximately 95%, without significantly affecting baseline proteolytic rates. Extensive studies revealed only small or no increases in the overall capacity of oxidatively stressed cells to degrade oxidatively modified protein substrates; a finding supported by both Western blot and Northern blot analyses which revealed no significant increase in the levels of proteasome subunit polypeptides or mRNA transcripts. We conclude that mild oxidative stress increases intracellular proteolysis by modifying cellular proteins, thus increasing their proteolytic susceptibility. In contrast, severe oxidative stress diminishes intracellular proteolysis, probably by generating severely damaged cell proteins that cannot be easily degraded (e.g. cross-linked/aggregated proteins), and by damaging proteolytic enzymes. We further conclude that the multicatalytic proteinase complex proteasome is responsible for most of the recognition and selective degradation of oxidatively modified proteins in Clone 9 liver cells.

Amino Acid Sequence↗
PubMed1995

Mitral dilatation with the Multi-Track system: an alternative approach.

We developed a simple and versatile new technique (Multi-Track) for percutaneous mitral valvotomy using two two separate balloon catheters positioned on a single guidewire. The first catheter, with only a distal guidewire lumen and a proximal balloon, is introduced over the guidewire into the vein and then advanced into the mitral valve orifice. Subsequently, a normal balloon catheter running on the same guidewire is inserted and lined up with the first catheter so the two are positioned side by side. The balloons are then inflated simultaneously. The technique was applied in 12 patients between 10 and 44 years of age (mean, 27.1) and weighing 24-80 kg (mean, 50.3). Valve area increased from 0.66 cm2 (range, 0.3-0.9 cm2) to 1.97 cm2 (range, 1.3-3.1 cm2) and mean left atrial pressure dropped from 31 mmHg (range, 18-52 mmHg) to 12 mmHg (range, 5-22 mmHg). Mitral dilatation with the Multi-Track system gives results comparable to those with previously described techniques and uses simpler and less costly catheters.

Adolescent↗
PubMed1994

Oxygen dependent regulation of Vitreoscilla globin gene: evidence for positive regulation by FNR.

Vitreoscilla globin (vgb) gene, encoding for Vitreoscilla haemoglobin (VtHb) has been cloned and functionally expressed in heterologous bacterial hosts. Analysis of vgb gene expression and the study on vgb-xylE transcriptional fusion revealed that vgb promoter is preferentially activated in response to oxygen limitation in Vitreoscilla and other heterologous bacterial hosts. Microaerobic mode of induction in various hosts, provided evidence for a common regulatory factor involved in activation of vgb promoter under hypoxic condition. Primary structure analysis of vgb upstream regulatory region indicated the presence of a possible binding site for the transcriptional activator, FNR. Further, the E.coli mutant lacking fnr gene product was not able to activate vgb promoter under microaerobic condition, suggesting the involvement of FNR or FNR-like proteins in modulating its activity. The possibility of a second level of control by CRP is also indicated. Oxygen responsive nature and regulatory characteristics of vgb promoter offers a novel system for the expression of gene in heterologous bacterial hosts in an oxygen dependent manner.

Azotobacter vinelandii↗
PubMed1994

Monocyte procoagulant activity: development of a microtitre plate chromogenic assay.

A monocyte procoagulant assay was developed based on the original method of Surprenant and Zuckerman, which quantitates a factor Xa-specific chromogenic substrate (at 405 nm) activated via the extrinsic coagulation pathway. Normal tissue factor initiation of the pathway is replaced by tissue factor generated from monocytes, stimulated by various agents including bacterial lipopolysaccharide, and antibody/antigen complexes. Hydrolysis of the chromogenic substrate is therefore directly proportional to the degree of monocyte activation. Using a chromogenic substrate as an end-point the assay was performed in a standard microtitre plate.

Blood Coagulation Tests↗
PubMed1993

Cloning and characterization of differentially expressed genes from in vitro-grown 'amastigotes' of Leishmania donovani.

Leishmanial parasites routinely undergo cyclic differentiation from promastigotes to amastigotes during their life cycle. This process involves both morphological and macromolecular changes. To study such changes, we used a axenic culture system which permits the continuous generation and cycling of Leishmania donovani from promastigotes to 'amastigotes' in vitro. cDNA libraries were constructed from poly(A)+ RNA isolated from both the pro- and amastigote forms. Using differential cDNA hybridization techniques, 3 unique cDNAs clones (P17, A41 and A45) were isolated from the amastigote library. To assess whether these clones were differentially expressed by the pro-or 'amastigotes' forms, they were hybridized to RNA isolated from each of these parasite forms in Northern and slot-blots. Results of these analyses showed that 'amastigotes' had approx. 2-fold higher levels of the A41 and A45 RNAs compared to the promastigotes. Conversely, promastigotes showed approx. 2-fold higher levels of the P17 RNA than 'amastigotes'. Nucleotide sequence analysis and comparison with those in Gene bank, revealed that the 3 cDNAs represent unique leishmanial genes. Comparison of the deduced amino acid sequences revealed that P17 open reading frame (ORF) had significant similarity with a soybean ribosomal protein S11; A41 ORF with a Bacillus subtilis spore germination gene (gerC) and A45 ORF with yeast stress-inducible protein (STI1). It is of interest to note that, of the 3 cDNAs identified, the A45-encoded protein was recognized by sera from patients with clinically active visceral leishmaniasis and was encoded by a single copy gene.

Amino Acid Sequence↗
PubMed1993

Microbiological study of mycetoma patients from Bombay with special reference to actinomyces immunofluorescence.

17 of the 21 clinically diagnosed mycetoma and actinomycosis cases studied yielded positive cultures. Foot, leg, inguinal region, chest wall, jaw and scalp were the affected sites. 15 of the patients were from Bombay, 13 of them had infection due to Nocardia species and 2 had Actinomyces israelii infection. The other two patients from South India had Madurella mycetomi infection. N.brasiliensis was commonest isolate (8). Other isolates were N.asteroides (2), N.pelletieri (2), N.caviae (1) and A.israelii (2). A direct fluorescent antibody test to detect actinomyces was used in selected cases (4). Two of whom gave positive immunofluorescence with A.israelii FITC labeled globulin, in primary smears as well as in cultures.

Actinomyces↗
PubMed1992

Pulmonary abnormalities in Klippel-Trenaunay syndrome. A histologic, ultrastructural, and immunocytochemical study.

Klippel-Trenaunay (KT) syndrome is a rare, sporadic, congenital vascular disease of unknown etiology. We describe pulmonary findings in an 18-year-old male patient followed up since birth with the KT syndrome. The patient developed pleural and pericardial serous effusions that led to an open lung biopsy. Previous pulmonary findings have been limited to thromboembolic phenomena and pulmonary vein varicosities. On the other hand, reports of lymphatic hyperplasia, aplasia, and hypoplasia in KT have been limited to the extremities. For the first time, we describe lymphatic involvement of the lung in KT. The plexiform hyperplasia of the lymphatic channels with smooth muscle hyperplasia leading to lymphatic obstruction, pleural and pericardial effusions are new findings. The lymphatic nature of the plexiform channels was confirmed by immunohistochemistry. Von Willebrand factor and QD-END/10 monoclonal antibodies either did not react or reacted poorly with lymphatic endothelium, features used to distinguish lymphatic and venous endothelium. Ultrastructurally, the absence of basement membrane continuity further substantiated the lymphatic nature of the channels. From our findings, the lymphatic abnormality in the syndrome appears to be more generalized than previously thought. This entity should be distinguished from lymphangioleiomyomatosis to which it bears a superficial morphologic appearance.

Adolescent↗