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Biomedical subjects

V M Katoch

Publications and source records attributed to V M Katoch.

108 records · Page 6Linked to original sources
PubMedDate not supplied

Metabolic studies on mycobacteria. IV. Assay of isocitrate lyase and malate synthase activity in M. leprae.

Cell free extracts from M. tuberculosis H37 Rv, M. smegmatis armadillo derived M. leprae and normal armadillo liver homogenates were assayed for the presence of isocitrate lyase and malate synthase activity. It was observed that significant amount of isocitrate lyase and malate synthase activity was present in M. tuberculosis H37 Rv, M. smegmatis and armadillo derived M. leprae. No such activity was demonstrable in cell free extracts of normal armadillo liver. It is concluded that M. leprae like other mycobacteria has the capability to metabolise via glyoxylate bypass of TCA cycle. These findings may be relevant for understanding the energy metabolism of M. leprae under stress conditions and possibly the 'persister' stage.

Animals↗
PubMedDate not supplied

Metabolic studies on mycobacteria. III. Demonstration of key enzymes of TCA cycle in M. leprae.

Cell free extracts of armadillo derived M. leprae, M. phlei, M. smegmatis and normal armadillo liver were analysed for the two key enzymes of TCA cycle. Aconitase activity was assayed in the presence of inhibitor fluorocitrate and it was observed that cell free extracts from cultivable mycobacteria as well as aramadillo derived M. leprae had this enzyme activity and 66-82% of this activity was inhibited by 0.1 mM fluorocitrate. 74% of M. leprae derived enzyme activity was inhibited by fluorocitrate in contrast with armadillo derived enzyme which was only 29% inhibited by fluorocitrate. PAGE separation of cell free extracts and staining for Isocitrate dehydrogenase (ICD) activity showed that an additional bond of ICD activity was demonstrable in the cell free extracts of armadillo derived M. leprae and this was NADP dependent. The mobility (ef) of this band of activity was in the same range as ICD from cultivable mycobacteria and much lower than ICD from normal armadillo liver. From this study and from the previously reported work, it is concluded that like other mycobacteria TCA cycle is operative in M. leprae.

Aconitate Hydratase↗
PubMedDate not supplied

Metabolic studies on mycobacteria. V. A preliminary report on the ATP synthesis by mycobacteria including M. leprae by using different substrates.

By deletion and addition of various substrates in Sauton's and Dubos media, an experimental system has been standardised in which the role of various nutrients in the energy synthesis of mycobacteria can be determined. By using this system with cultivable mycobacteria it was observed that glycerol and asparagine are the important ingredients for ATP synthesis by mycobacteria. Glucose further enhanced the ATP synthesis and growth of these mycobacteria. In the media containing asparagine or glycerol, there was marginal increase in the ATP in the M. leprae suspensions initially but this was not sustained and there was no progressive increase in biomass or multiplication. When M. leprae was incubated in the media from which both these substrates were deleted, there was progressive decline in ATP levels right from the beginning. From these preliminary results, it appears that asparagine and glycerol may be useful as substrates for ATP synthesis by M. leprae and need to be investigated further. In depth studies are necessary to find out the factors which results in the inability of M. leprae to utilise these and other substrates in a substrained manner for its multiplication and growth in artificial media.

Adenosine Triphosphate↗
PubMedDate not supplied

Metabolic studies on mycobacteria-I. Demonstration of key enzymes of glycolysis and tricarboxylic acid cycle on polyacrylamide gels.

Polyacrylamide gel electrophoresis (PAGE) technique was standardised to demonstrate some key enzymes of glycolysis, hexose mono phosphate (HMP) pathway and tricarboxylic acid cycle in slow growing mycobacteria (M. avium. M. gastri) as well as in fast growing mycobacteria (M. vaccae, M. phlei). The enzymes studied were lactate dehydrogenase (LDH) glucose-6-phosphate dehydrogenase (G6PD), aconitase, isocitrate dehydrogenase (ICD), succinic dehydrogenase (SDH), fumerase and malate dehydrogenase (MDH). All the three pathways were found to be operative in slow as well as fast growing mycobacteria. Using this technique M. leprae specific MDH activity was demonstrated in the cell free extract of M. leprae. It's (MDH) electrophoretic mobility on gels lies in the range shown by other mycobacterial species studied and was distinct from that of host MDH. It appears that PAGE offers a useful tool for metabolic characterization of M. leprae using infected tissues.

Citric Acid Cycle↗
PubMedDate not supplied

Metabolic studies on mycobacteria--II. Glyoxylate by-pass (TCA cycle) enzymes of slow and fast growing mycobacteria.

Glyoxylate by-pass of tricarboxylic acid cycle (TCA) comes into prominence during survival of microorganisms under oxygen limitations and study of these enzymes may contribute to understanding of physiology of 'persisters' in various mycobacterial diseases. The enzymes of glyoxylate by-pass have been assayed in the extracts of various mycobacterial species, namely, M. tuberculosis H37Rv, M. tuberculosis H37Ra, M. flavescens, M. vaccae, M. smegmatis and Mycobacteria strain w (M.w.). M.w. has been included because of its close antigenic resemblance to M. leprae. It has been found that all of the above investigated species possess isocitrate lyase and malate synthetase, the key enzymes of glyoxylate by-pass. The presence of the enzymes is being reported for the first time in M. flavescens, M. vaccae and M.w. whereas these were earlier shown to be present in M. tuberculosis and M. smegmatis. It was also demonstrated in M.w. where acetate alone could not serve as sole source of carbon, but in the presence of glycerol stimulates the activity of glyoxylate pathway enzymes. The importance of these findings has been discussed.

Citric Acid Cycle↗
PubMedDate not supplied

Isoenzymes of mycobacteria. II. Relevance of LDH zymograms in taxonomy and identification.

The cell free extracts of mycobacteria namely M. kansasii M. avium, M. tuberculosis, BCG (Glaxo), M. gastri, M. phlei, M. smegmatis, M. vaccae, M. strain w., M. scrofulaceum, M. gordonae, M. nonchromogenicum E. coli, Staph, aureus, and M. leprae infected skin have been electrophoresed and stained for LDH activity. Normal skin tissue was also taken as control. It was found that all the organisms tested showed distinct species specific LDH isoenzyme patterns. There was no extra band but an aberrant zone of LDH activity was seen in M. leprae infected human skin in comparison to LDH isoenzymes from normal skin. No strain variations was found among the different strains of species investigated. Results described in the present paper indicate that LDH isoenzyme patterns of mycobacteria could be of identification value at species level.

Humans↗
PubMedDate not supplied

Isoenzyme patterns of mycobacteria. I. Factors influencing LDH isoenzymes of mycobacteria.

Cell free extracts of a fast growing mycobacterium (M. phlei) and a slow growing mycobacterium (M. tuberculosis H37Ra) were analysed for lactate dehydrogenase (LDH) isoenzymes under different experimental conditions. It was observed that growth of M. phlei when taken from Lowenstein Jensen (LJ) as well as Sauton's medium showed identical band but for (M. tuberculosis H37Ra the number of bands observed were less when grown on LJ-medium. There was no difference in LDH isoenzyme patterns when the mycobacteria were incubated at 30 degrees C and 37 degrees C and under different pH conditions (6.2-8.2). Actively growing cultures of both the species showed distinct LDH isoenzyme patterns whereas the activity and bands became indistinct in old cultures. The LDH bands from lyophilized growth studied resembled to those of fresh growth. The treatment of growth with 1M NaOH for one hour resulted in marked diminution of LDH activity. Sonication with wet growth weight of 0.5 gm per ml of distilled water was found to give clearer bands as compared to phosphate buffer. No loss of LDH isoenzymes activity was noticed after storing the extracts at -80 degrees C for one month, treating to 58 degrees C for one hour or freezing and thawing for 2 times whereas these isoenzymes were quite unstable at other storage temperatures. Increasing the staining time was found helpful in getting clearer bands when activity was low. It is concluded that the factors studied have important bearing on LDH isoenzyme patterns of mycobacteria and must be kept in mind while studying the LDH zymograms for any taxonomic identification of mycobacteria or for studying the metabolic role. These are important both for sensitivity and reproducibility of LDH zymograms.

Culture Media↗
PubMedDate not supplied

Chest infection due to M. fortuitum in a case of lepromatous leprosy--a case report.

Lepromatous leprosy cases may be immunocompromised due to the extensive disease and also because of steroid therapy for repeated reactions. Such patients are likely to be at higher risk for getting opportunistic infection due to various environmental microbes. This paper reports a case of lepromatous leprosy with repeated lepra reaction who was found to have chest infection due to M. fortuitum. It is suggested that mycobacterial culture and sensitivity should be recommended in cases who are immunocompromised and whose pathological specimens contain acid fast bacilli. Species identification and sensitivity can be very helpful in proper management of such cases who will otherwise pass off as tuberculosis.

Drug Resistance, Microbial↗
PubMedDate not supplied

Alanine dehydrogenase in mycobacteria--a preliminary report.

Various mycobacterial species namely M. phlei, M. vaccae, M. scrofulaceum, M. avium and M. tuberculosis have been investigated for the presence of enzyme alanine dehydrogenase which could be important for utilization of alanine by TCA cycle. It was found that alanine dehydrogenase was present in all species of mycobacteria tested irrespective of the fact whether they are rapid or slow growers. Electrophoretic mobilities of alanine dehydrogenase from different species of mycobacteria were not found to be significant for taxonomical differentiation of rapid and slow growers.

Alanine Dehydrogenase↗
PubMedDate not supplied

Serological approaches for the characterization of catalase in tissue-derived mycobacteria.

Cell-free extracts of Mycobacterium lepraemurium from mouse liver and M. leprae from armadillo liver were analysed for the presence of any mycobacterial catalase by using the specific inhibitor 3-amino-1,2,4-triazole and seroprecipitation titrations. These studies clearly demonstrated the presence of a "T" type of mycobacterial catalase in M. lepraemurium and placed it, in terms of immunological distance, in a position between M. tuberculosis and M. avium. The results did not reveal any detectable "T" catalase activity in the M. leprae preparations. The "M" type catalase activity which was observed did not bind to antisera against "M" catalase of M. kansasii, but was bound to the extent of 80% to antisera against normal armadillo liver catalase. The significance of the component of the "M" catalase in M. leprae preparations which did not react against antibodies to normal liver remains to be determined.

Amitrole↗
PubMedDate not supplied

Comparative assessment of viability of M.leprae by mouse foot-pad and fluorescent staining techniques.

Morphological characteristics have been used as a parameter to assess the viability of M.leprae in leprosy patients. However, with the advent of the mouse foot-pad technique, viability of M.leprae is determined by growing the bacilli in the mouse foot-pad. In recent years, a fluorescent staining technique using fluorescent diacetate-ethidium bromide (FDA-EB) has been used to assess the viability of cultivable mycobacteria as well as M.leprae. The purpose of this study was to compare the viability of M.leprae by both mouse foot-pad and fluorescent staining techniques. M.leprae strains from both untreated and treated patients as well as mouse passaged strains of M.leprae were used for the comparison. Percentage of green-stained bacilli in the inoculum was compared with that of multiplication of M.leprae in the mouse foot-pad. It was observed that there was no correlation between the estimates of viable M.leprae by fluorescent staining and by mouse foot-pad inoculation. FDA-EB staining appears to reflect only trends as absence of green staining cells had overall general correlation with loss of infectivity to mouse foot-pad but, the converse was not found to be true.

Animals↗
PubMedDate not supplied

Studies on microbial aerobic flora of skin in leprosy patients.

This study reports the isolation and identification of aerobic organisms from biopsies/slit-skin smears/scrapings from 129 leprosy patients and 50 healthy controls. These include 56 paucibacillary (PB) and 73 multibacillary (MB) cases. Thirty-six isolates from the specimens from 21 patients and 15 healthy controls were grown. The non-mycobacterial isolates from clinically PB leprosy (TT/BT/I) patients were: (1) Gram-positive cocci: Staphylococcus aureus(1), Staphylococcus albus(1); (b) Gram-positive bacilli: Bacillus subtilis(1), Corynebacterium xerosis(1); (c) Gram-negative bacilli: Escherichia coli(1), Proteus mirabilis(2), Klebsiella pneumoniae(1) and Pseudomonas aeruginosa(1). The isolates from clinically MB leprosy (BB/BL/LL) patients were: (a) Gram-positive cocci: Micrococci(1), Staphylococcus aureus(1) and Staphylococcus albus(1); (b) Gram-positive bacilli: Corynebacterium xerosis(1); Corynebacterium hofmanni(1) and Bacillus cereus(1). (c) Gram-negative bacilli: Escherichia coli(2), Klebsiella pneumoniae(1) and Proteus mirabilis(2). The specimens from healthy controls yielded similar organisms. These were (a) Gram-positive cocci: Staphylococcus albus(2), Staphylococcus aureus(2) and Micrococci(2); (b) Gram-positive bacilli: Corynebacterium xerosis(1), Bacillus subtilis(2), Corynebacterium hofmanni(1) and Bacillus cereus(1); (c) Gram-negative bacilli: Escherichia coli(3), Proteus vulgaris(1) and Proteus mirabilis(1). While these results show no significant differences in the species types of non-mycobacterial aerobic organisms isolated from healthy skin and PB/MB types of leprosy, these isolates need to be characterized by immunological/molecular methods to find out subtypes if any.

Bacteria, Aerobic↗
PubMedDate not supplied

Isolation and characterization of cultivable mycobacteria from leprosy skin.

Attempts were made to isolate cultivable mycobacteria from 129 biopsies/slit-skin scrapings from the skin of leprosy patients (73 multibacillary-BB/BL/LL and 56 paucibacillary-TT/BT/I) as well as 50 healthy controls. Among the 19 isolates obtained, 17 were from specimens from leprosy cases whereas two were from healthy controls. 14 of the 17 isolates were from multibacillary cases and three were from paucibacillary patients. The mycobacteria isolated were: M.scrofulaceum (4 = all LL cases); M.avium (3 = 2 from LL cases and 1 from healthy control); M.avium-intracellulare complex (1 LL); M.gordonae (2 = 1 from BT and BB each); M.flavescens (1 BL); M.smegmatis (2 = both LL); M.phlei (4 = 1 LL, 1 BL, 1 BT and 1 healthy control); M.fortuitum (1 BL); and M.chelonei (1 BT relapse). The results of this study suggest a preferential colonization of skin of lepromatous leprosy cases by M.scrofulaceum and M.avium. As such isolates have been reported by the investigators from other parts of the world, independent confirmation of such trends in Indian patients is significant and casual relationship (if any) between such colonization and development of lepromatous disease merits further investigation.

Biopsy↗
PubMedDate not supplied

Understanding the phenomenon of persistence in mycobacterial infections.

Persistence of live organisms despite chemotherapy for long periods is a significant problem in both leprosy and tuberculosis. The consequence of this persistence is varying rates of relapses which undermine the success of treatment. The mechanisms of the dormancy are ill-understood, and as explanation a switch over to alternate modes of metabolism such as glyoxylate bypass and other shunts has been suggested. This presentation reviews the information available on this aspect. In-depth studies by designing and investigating model system(s) using molecular genetic approaches may help in gaining better understanding of the mechanisms of dormancy and persistence in mycobacterial infections and devising appropriate strategies and tools for the better management of these complications.

Animals↗