The enzyme fraction A, a constituent enzyme of the three-fraction enzyme mycobacillin synthetase, independently and sequentially activated five amino acids starting from L-proline, producing the pentapeptide Pro(Asp1,Glu1,Tyr1)Asp. The fractions B and C were unable to function independently. However, the fraction B synthesized the nonapeptide Pro(Asp3,Glu1,Tyr2,Ser1)Leu, sequentially activating the pentapeptide and next four amino acids, whereas the fraction C synthesized mycobacillin by the sequential activation of the nonapeptide and the remaining four amino acids. The pH optima of the above enzymes are almost identical (pH 7.8), but their Km values are a little different.
Thirteen mycobacillin-negative (My-) mutants of Bacillus subtilis B3 were isolated from an auxotrophically tagged mycobacillin producer organism. The wild-type producer, three feeble producers and three strictly My- mutants did not accumulate any ninhydrin-positive peptide in the culture medium while the remaining seven My- mutants did accumulate ten such peptides whose amino acid composition indicated that there might be only three different peptides. The N-terminal and C-terminal amino acid residues implicated one of these peptides as a pentapeptide intermediate in mycobacillin synthesis; this was further confirmed by its molecular weight and sequence. Studies on cell-free synthesis showed that only the enzyme system from the wild-type strain synthesized mycobacillin while the defective ones from all the My- mutants synthesized one and the same pentapeptide as found in the culture broth of some of the mutants. Further studies in which the enzymes responsible for mycobacillin synthesis by cell-free extracts were separated into three fractions, A, B and C, showed that seven of the mutants were defective in fraction B whereas the three other mutants had defects in both fractions B and C. Thus the pentapeptide Pro----Asp----Glu----Tyr----Asp appears to be implicated in mycobacillin biosynthesis.
The growth of Mycobacterium microti was inhibited within J774A.1 macrophage cells activated with either interferon-gamma or tumor necrosis factor-alpha. Activation with interferon-gamma or tumor necrosis factor-alpha alone did not stimulate the production of nitrite in J774A.1 cells. Interferon-gamma but not tumor necrosis factor-alpha increased the production of hydrogen peroxide in a concentration dependent manner but scavengers of reactive oxygen species did not influence the growth inhibiting effect of interferon-gamma within J774A.1 cells. Both interferon-gamma and tumor necrosis factor-alpha enhanced the fusion of M. microti containing phagosomes with lysosomes and the ultimate degradation of bacteria. Our results showed that growth inhibition of M. microti within interferon-gamma or tumor necrosis factor-alpha stimulated J774A.1 cells was independent of reactive oxygen intermediate and reactive nitrogen intermediate production.
The effect of mouse recombinant interleukin-1 alpha on the intracellular growth of Mycobacterium microti in a murine macrophage cell line J774A.1 was investigated. Interleukin-1 alpha added after infection to the M. microti-infected macrophage monolayers enhanced the growth of M. microti in a concentration-dependent manner and this growth enhancement was abrogated by neutralization of interleukin-1 alpha with anti-interleukin-1 alpha antibody. Cyclic adenosine monophosphate level in J774A.1 cells was increased by the addition of interleukin-1 alpha. Addition of dibutyryl cyclic adenosine monophosphate to infected J774A.1 cells increased the number of intracellular bacteria in a concentration-dependent manner. These results suggest that interleukin-1 alpha acts as a growth enhancer for intracellular M. microti and the growth enhancing effect of interleukin-1 alpha may be due to enhanced cellular cyclic adenosine monophosphate level.
The growth of Mycobacterium microti was inhibited within J774A. 1 macrophage cells activated with either interferon‐γ or tumor necrosis factor‐α. Activation with interferon‐γ or tumor necrosis factor‐α alone did not stimulate the production of nitrite in J774A. 1 cells. Interferon‐γ but not tumor necrosis factor‐a increased the production of hydrogen peroxide in a concentration dependent manner but scavengers of reactive oxygen species did not influence the growth inhibiting effect of interferon‐γ within J774A.1 cells. Both interferon‐γ and tumor necrosis factor‐α enhanced the fusion of M. microti containing phagosomes with lysosomes and the ultimate degradation of bacteria. Our results showed that growth inhibition of M. microti within interferon‐γ or tumor necrosis factor‐a stimulated J774A. 1 cells was independent of reactive oxygen intermediate and reactive nitrogen intermediate production.
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