Five alternative methods were used to explore in vitro the effects of normal and activated murine macrophages on the metabolic well-being of intracellular Mycobacterium leprae: fluorescein diacetate-ethidium bromide staining, ATP content, synthesis of phenolic glycolipid 1, and two techniques to quantitate oxidation of palmitic acid. In relatively short-term experiments (7 to 10 days), each of these procedures provided strong evidence that activated macrophages exerted a deleterious effect on the leprosy bacillus. These findings appear to confirm the contention that activated macrophages underlie host resistance to clinical leprosy and limitation of M. leprae growth in paucibacillary leprosy. Clinical leprosy is characterized by a broad spectrum of host response, with great variability in clinical course of infection, histopathology, humoral antibody response, and cell-mediated immunity. As an obligate intracellular microorganism, Mycobacterium leprae is capable of prolific growth in macrophages (MO) in vivo. However, little is known about the immune-mediated activation of M+, and their ability to kill and digest M. leprae is an issue of central importance to understanding the spectrum of host resistance characteristic of leprosy (12). Unfortunately, the direct * Corresponding author. t Present address:
Mycobacterium leprae synthesizes large quantities of a specific phthiocerol-containing phenolic glycolipid in vivo. We had shown earlier that viable M. leprae readily incorporates radiolabeled palmitic acid into phenolic glycolipid I when residing in cultured macrophages in vitro and that this process is inhibited by the antileprosy drug rifampin. In the present paper we report the application of this observation to the rapid evaluation of over 25 antimicrobial agents for potential antileprosy activity in vitro. All the known antileprosy drugs rifampin, dapsone, clofazimine, and ethionamide inhibited phenolic glycolipid I synthesis. Rifabutin, a spiropiperidyl derivative of rifamycin, also reported to be active in the mouse model, was very effective. Interestingly, the macrolides erythromycin, clarithromycin, and roxithromycin were also found to be active in this system, while D-cycloserine and other cell wall synthesis inhibitors showed no effect. Many of the compounds found to be active in this system have been reported to be effective in vivo in mice. This correlation lends support to the feasibility of using phenolic glycolipid I synthesis for the rapid evaluation of new drugs against leprosy.The increased emergence of drug-resistant strains of Mycobacterium leprae in recent years has emphasized the need to identify new compounds effective in the treatment of leprosy. Since M. leprae does not multiply in vitro, conventional microbiological assays cannot be used for primary evaluations of new drugs for antileprosy activity. Prior to 1960, antileprosy drugs were selected primarily on the basis of empirical results of clinical trials with leprosy patients. The mouse footpad technique developed in 1960 by Shepard (38) proved to be an invaluable tool in the primary selection of new drugs by facilitating comparison of the bactericidal potency of different compounds and by making it possible to differentiate bactericidal activity from bacteriostatic effects. The activity of a drug in the mouse model is strongly correlated with its ultimate efficacy in humans. However, primary evaluation of new drugs by this approach has severe practical limitations since the assay is time-consuming (requires 4 to 8 months) and laborious, requires at least 4 to 5 g of a test compound for a test screening, and is heavily dependent on the compound's having favorable pharmacokinetics in the mouse. The development of a rapid and reliable in vitro drug-screening system would allow primary screening and selection of more compounds with potential antileprosy activity, the activity of which could then be tested in mice prior to clinical trials.A number of in vitro metabolic assay systems have been reported in recent years, and these have been extensively reviewed (22,47,48). These assays are based on the observation that M. leprae, although incapable of proliferation in vitro, retains many of its metabolic functions for a limited period of time outside the host either in tissue culture or in cell-free systems. The metabolism of the organism...
The incorporation of "4C-labeled palmitic acid ([U-14C]PA) into the phenolic glycolipid-I (PGL-I) fraction of Mycobacterium keprae was studied in a murine macrophage system in vitro. Peritoneal macrophages from Swiss Webster mice were infected with fresh viable or Formalin-killed M. leprae harvested from infected foo,pads of nulnu mice, and [U-"'C]PA was added to the culture medium. Labeled glycolipid synthesized by live M. leprae was fractionated on a Florisil-silicic acid column and identified as PGL-I by using thin-layer chromatography and localization on a polysulfone membrane with an anti-PGL-I monoclonal antibody. Increased incorporation of (U-"'C]PA into the PGL-I fraction was observed in macrophages infected with only live M. leprae. Treatment of the infected macrophages with rifampin caused a significant reduction in the incorporation of palmitic acid into PGL-I. These preliminary studies suggest that PGL-I synthesis can be used to quantitate the metabolism of M. keprae in macrophages in vitro.The inability to rapidly quantitate the viability of Mycobacterium leprae has vastly impeded progress in leprosy research. To monitor the viability of the noncultivable leprosy bacillus, tedious indirect titrations employing the mouse footpad technique developed by Shepard (21) are required, often taking a year or more to complete (13). Clearly, to more easily screen antileprosy chemotherapeutic regimens or to study host resistance factors in leprosy there is a need to develop alternate and more rapid techniques for determining the viability of M. leprae.M. leprae is a natural intracellular parasite that depends on the internal milieu of the host cell for survival. Within the host cell, M. leprae synthesizes large amounts of a characteristic phenolic glycolipid (PGL-I) which has been shown by Brennan and colleagues to be biochemically unique and immunologically specific for M. leprae (1,9,10). Since mononuclear phagocytes have been shown to serve as important host cells for M. leprae, the present study explores the biosynthesis of PGL-I by the leprosy bacillus in tissue cultures of mouse peritoneal macrophages. The in vitro biosynthesis of PGL-I from 14C-acetate by M. leprae within schwannoma cell lines has been reported by Mukherjee et al. (16). In our studies PGL-I synthesis was monitored in vitro by measuring the incorporation of radiolabeled palmitic acid (PA) into the extractable PGL-I fraction of M. leprae in infected macrophages. The present work describes the methodology developed and reports our preliminary findings on the effects of rifampin on PGL-I synthesis by M. leprae in vitro. MATERIALS AND METHODSMacrophages. Peritoneal cells (PC) were harvested from normal unstimulated Swiss Webster mice by using methods described previously (12). Briefly, PC harvested in heparinized Hanks balanced salt solution containing 10 U of heparin per ml were washed by centrifugation (150 x g for 10 min) and suspended in medium RPMI 1640 (GIBCO Laboratories, Chagrin Falls, Ohio) supplemented with 20% heatinactivated fetal ...
Two isoenzymes of amyloglucosidase, designated as AG‐I and AG‐II, elaborated exocellularly by a strain of Aspergillus niger van Tieghem, were separated and purified to homogeneity. The enzymes, produced in a selective medium, were separated and purified on a column of DEAE‐Sephadex A‐50. The molecular weights of AG‐I and AG‐II were found to be 69, 810 and 89,130 respectively. The two enzymes were glycoproteins and differed in their carbohydrate contents, pH and temperature stabilities and optima for activity. Their activation energies and Km values also varied. AG‐II, had a higher molecular weight, carbohydrate content, increased acid tolerance and was synthesized earlier to AG‐I (when the pH of the medium was acidic). Hg and Ag salts caused partial inhibition of their activities.
The exocellular α‐amylase of a strain of Aspergillus niger van Tieghem (elaborating both dextrifying and saccharifying thermophilic amylases) was purified to homogeneity. Purification was achieved by providing cultural conditions for the organism to preferentially synthesize α‐amylase and fractionation of the culture filtrate by DEAE‐Sephadex chromatography. Its purity was established by gel electrophoresis and confirmed by sedimentation studies. The molecular weight of the enzyme was 56,230. Its Km values on different starches, temperature and pH optima for activity and energy of activation were established. Compared to literature values for other fungal α‐amylases, this enzyme exhibited a lower energy of activation, increased tolerance to lower pH and enhanced affinity to starch, highlighting its potential industrial application. While Ag+, Pb2+, Hg+, Al3+ and EDTA inhibited the activity of the enzyme, Ca2+ enhanced its activity, apart from conferring thermal stability and lowered activation energy. The product of its action on starch were maltooligosaccharides, maltose and glucose.
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