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A M Dhople

Publications and source records attributed to A M Dhople.

At least 55 records · Page 3Linked to original sources
PubMed1983

Taxonomic studies on Mycobacterium leprae.

M. leprae harvested from livers and spleens of infected armadillos showed close similarity in biochemical characteristics with M. vaccae than with any other cultivable mycobacteria. Further more. M. lepraemurium was least similar to M. leprae. Thus, M. leprae can be linked with fast-growing groups of acid-fast bacilli, and perhaps most closely with the M. vaccae complex.

Animals↗
PubMed1983

Effect of lysozyme on Mycobacterium leprae.

Because large amounts of lysozyme are present in phagocytes, it was selected, as one of the bactericidal substances, to study the in vitro effects on M. leprae. The results obtained led the author to conclude that M. leprae is not susceptible to lysozyme as many other atypical as well as attenuated pathogenic mycobacteria. This resistance of M. leprae to lysozyme was seen up to a level of 100-500 micrograms per ml, while levels of 1000 micrograms and more had very significant effect; the ATP content of such treated M. leprae declined to 30% of the original levels in 5 days of incubation at 37 degrees C, with optimum pH of 6.0, and these M. leprae after such exposure to lysozyme failed to multiply in the foot pads of mice. The interpretation of these results, though not definitive, have been discussed.

Adenosine Triphosphate↗
PubMed1982

Effect of freezing Mycobacterium leprae in tissues.

The effects of freezing tissues from M. leprae-infected armadillos as well as biopsy specimens from leprosy patients have been studied. No loss of viability of M. leprae was observed using mouse foot pad technique. Similarly, using bioluminescent determinations of bacterial ATP, it was shown that these bacilli retain all their metabolic activity during such freezing and thawing process. The practical implications of these findings have been discussed.

Adenosine Triphosphate↗
PubMed1982

Adenosine triphosphate content of Mycobacterium leprae: effect of purification procedures.

Various procedures to decontaminate and purify M leprae free of host tissue material resulted in total retention of their intracellular ATP and also infectiousness. The ATP content of one million M. leprae cells, isolated from either livers, spleens, or lymph nodes of infected armadillos, or a nude mouse foot pad or a human biopsy specimen, was in the range of 1.17 to 1.40 picograms. Suspensions could be decontaminated with 4% NaOH and all non-bacterial ATP could be eliminated by the combined action of trypsin, chymotrypsin, and collagenase initially, followed by Triton X-100 plus ATPase. These findings further assure that M. leprae are different from M. lepraemurium in that they can withstand even the severest purification procedures that are necessary in order for them to be used for sophisticated biochemical and metabolic studies.

Adenosine Triphosphate↗
PubMed1981

Role of sulfhydryls in in vitro growth of Mycobacterium lepraemurium.

In an attempt to evaluate various factors that influence the growth of Mycobacterium lepraemurium in NC-5 medium, the effects of sulfur and --SH compounds were investigated. Cysteine could be replaced by equimolar concentrations of other --SH compounds containing carboxyl group, and at lower concentrations by nonpolar sulfhydryl compounds. The oxidized form of sulfhydryls, as well as certain organic and inorganic reducing agents, did not support growth. The results suggest that the function of sulfhydryl compounds is to maintain low reducing potential in the medium and also to participate in metabolic or biosynthetic pathways or both. A combination of dithiothreitol and thioglycolate gave better results than when these compounds were incorporated individually in the medium. This suggests the protective action of dithiothreitol in preventing oxidation of monothiols.

Adenosine Triphosphate↗
PubMed1980

Pedigreed stocks of Mycobacterium lepraemurium for cultivation and metabolic studies.

Experience has shown that a prime requirement for investigations with Mycobacterium lepraemurium is pedigreed stocks of cells which provide constant baselines over a long period of time. Biochemical indicator, ATP, was employed to devise a method for preservation of metabolic pools of M. lepraemurium during storage. ATP assays were made by the luciferin-luciferase bioluminescent method. Several cryoprotective agents were compared at -76 and 4 degrees C. The essential steps have been found to be (i) for prolonged storage and constant supply of material, to freeze the intracellular bacteria within infected cells containing 28% proteins, i.e., to freeze the infected tissue; and (ii) when a large number of diverse experiments are to be undertaken on a single suspension, to stabilize working stocks of refrigerated cells for 12-16 weeks by using bovine serum albumin, fraction V, and Difco yeast supplement B to compensate for the leaching of intracellular cofactors, metabolites, nucleotides, etc.

Adenosine Triphosphate↗
PubMed1980

Microscopic counts carried out on Mycobacterium leprae and M. tuberculosis suspensions. A comparison of three staining procedures.

Standard smears of heat-killed Mycobacterium leprae and M. tuberculosis H37Rv were counted microscopically following staining by the Ziehl-Neelsen, auramine, and silver-methenamine methods. The numbers of stained bacillary bodies were consistently higher in the silver-methenamine stained smears compared to the Ziehl-Neelsen and auramine stained smears. The auramine smears were examined under ultraviolet illumination and permitted the enumeration of the brightly fluorescent bacilli against a black background. The auramine counts were not as high as those obtained using the silver-methenamine stained preparations but were consistently higher than those obtained with the Ziehl-Neelsen preparations. Both the auramine and silver-methenamine stained preparations clearly outlined the cell walls of the bacilli in the smears and this greatly facilitated the counting process, especially if the cell suspension was badly clumped.

Mycobacterium leprae↗
PubMed1977

In vitro growth of Mycobacterium lepraemurium, an obligate intracellular microbe.

By using an ultrasensitive technique to measure adenosine triphosphate in terms of functional biomass, we have confirmed that Mycobacterium lepraemurium (the agent of rat leprosy and a classical obligate intracellular microbe) grows in vitro in the Nakamura system. By using a sulfhydryl-containing medium that ocupies 65 to 75% of the culture tube volume, together with the five supplements recommended by Nakamura, we have obtained growth rates some eight times above the original. The new physicochemical environment and the use of adenosine triphosphate as an index of energy status in the presence and absence of growth provide a basis for investigating the physiology and growth of other noncultivated microbes.

Adenosine Triphosphate↗
PubMed1973

Quantitative extraction of adenosine triphosphate from cultivable and host-grown microbes: calculation of adenosine triphosphate pools.

EXISTING DATA ON ADENOSINE TRIPHOSPHATE (ATP) POOLS IN MICROBES ARE DEFICIENT FOR TWO REASONS: (i) incomplete extractions of ATP, and (ii) the failure to take into account that the adverse effects of extracting procedures on standard ATP exert analogous effects on the ATP released from bacterial cells. Methods for correcting observed yields and calculating ATP pools have been demonstrated. Three bacterial species were used in the studies on extraction of ATP: Escherichia coli, Mycobacterium phlei, and Mycobacterium lepraemurium. Perchloric acid and n-butanol were disqualified because of their failure to extract total bacterial ATP even from E. coli and because of inconvenient procedures. The new extraction procedure had minimal effects on standard ATP, liberated 100% of the ATP pools from the three representative species of microbes, and caused no ionic imbalance or quenching of bioluminescence. This method involves vortexing of cell suspensions for 10 s with 23% chloroform (vol/vol), heating at 98 C for the required time (E. coli, 3 min; M. phlei, 5 min; M. lepraemurium, 10 min) and then 1 min at 98 C with vacuum to dry the samples. Heat or chloroform alone may suffice for some microbes and release total ATP from plant and animal cells.

Adenosine Triphosphate↗
PubMed1973

Energetics (adenosine 5'-triphosphate) of Mycobacterium lepraremurium in diffusion chambers incubated in vitro and in mice.

Adenosine 5'-triphosphate (ATP) measurements and the processing of samples have been refined to a point where the energetics and growth potential of microscopic samples of unwashed host-grown, host-dependent microbes can be investigated. Mycobacterium lepraemurium, the noncultivated agent of murine leprosy, was employed to examine three reports of the slow microscopic growth of this organism in the absence of host cells. A few million bacterial cells were enclosed in Rightsel- and Ito-type diffusion chambers, which were incubated in vitro and in the peritoneal cavities of mice. In the in vitro experiments, a complex medium containing bovine serum and mouse brain extracts, renewed three times a week, did not sustain the energetics of the bacilli. The microscopic counts declined to 72% and the ATP per culture to 9% of the original values. Very different results were obtained from chambers incubated in the peritoneal cavities of mice. The bacterial biomass increased 2.7-fold and the ATP per culture increased 2.5-fold. Because the ATP per cell was 93% of the original, this system is regarded as the first to permit the extracellular growth of a so-called "obligate intracellular microbe." The results obtained with only 1 x 10(6) host-grown cells per assay demonstrate a significant biochemical tool for investigating the growth potential of host-grown microbes during the progression, regression, and therapy of disease.

Adenosine Triphosphate↗
PubMedDate not supplied

Limited in vitro multiplication of Mycobacterium leprae.

The inability to cultivate Mycobacterium leprae in vitro has been a major bottleneck in leprosy research. There have been numerous reports on successful in vitro cultivation of this organism, but these reports could not be confirmed by others in the field. Hence, in vitro multiplication of M. leprae was evaluated in various culture media. Only 2 media supported limited multiplication of M. leprae. One medium was used previously by one of the authors (AMD) for in vitro growth of M. lepraemurium and the other was a conditioned medium used for growth of mouse dorsal root ganglion. Growth was evaluated by 3 biochemical parameters: bacterial ATP, DNA and 3H-thymidine uptake. All 3 measurements revealed a 4-6-fold increase in cell biomass after 16 weeks of incubation at 34 degrees C. The harvested bacilli demonstrated a few of the important properties of M. leprae, including growth in mouse footpads. However, subcultures of these in-vitro-grown cells in the respective media could not be achieved. By the end of 12 weeks, the bacilli lost all intracellular ATP and the ability to incorporate 3H-thymidine; they also failed to multiply in mouse footpads.

Adenosine Triphosphate↗
PubMedDate not supplied

Search for newer antileprosy drugs.

In 1991 World Health Organization proclaimed the goal of global elimination of leprosy as a public health problem by year 2000 by implementing multidrug therapy (MDT). Since then the prevalence rate has declined by 85%. However, during the same period the incidence rate of leprosy has remained constant or even has been increasing. This suggests that it will take a long time for the eradication of leprosy and that without in-vitro cultivation of M. leprae, eradication of leprosy is not likely to be achieved. While in-vitro cultivation is a long-term goal, as an immediate measure, there is an urgent need for the development of newer drugs and newer multidrug therapy regimens. Using the in-vitro system for screening potential antileprosy drugs and also using the mouse foot-pad system we have evaluated several compounds in four classes of drugs--dihydrofolate reductase inhibitors, fluoroquinolones, rifampicin analogues and phenazines--and identified at least two compounds that appear to be more potent than dapsone, rifampicin and clofazimine. Newer combinations of rifampicin analogues and fluoroquinolones have also been identified that seem to be better than the combination of rifampicin and ofloxacin.

Animals↗
PubMedDate not supplied

Utility of beige mouse in leprosy research.

Dissemination of M. leprae to visceral organs is seen by four months onwards only in beige (C57BL/6/bgj) but not BALB/c mice following intravenous or intraperitoneal infections. Inoculation of the beige mouse derived M. leprae showed all the characteristics of M. leprae, including growth pattern in the foot-pads of BALB/c mice. M. leprae inoculated into foot-pads of beige mice multiplied faster than those in the foot-pads of BALB/c mice. The possibility of using beige mouse in chemotherapeutic studies in leprosy is discussed.

Animals↗