Pathological investigation of armadillos infected with Mycobacterium leprae.

An infection experiment with M. leprae was carried out using 20 nine-banded armadillos. As a result, the development of leprous lesions and a marked multiplication of AFB were confirmed in a high rate of 13 out of 15 cases (86.8%) in the inoculated groups. These changes were found to be progressing at post mortem of one case even with the shortest life period for 7.5 months and were very serious in one case with the longest life period for 33 months, suggesting the continuation of symptoms, though it is an expression neglecting the individual difference in susceptibility to leprosy. Among infected viscera with AFB, the most conspicuous lesions were found in the liver and spleen. The developed lesions were found in the lung, stomach and kidney which had been never seen in HD in human cases, and so, which may characterize armadillos' leprosy. The change in the peripheral nerve was not so severe when compared with that in HD in human cases. This difference will remain as a future pathological problem to be solved.

Inhibition of the multiplication of Mycobacterium leprae in nude mice by intermittent administration of a new rifamycin derivative, 3'-hydroxy-5'-(4-isobutyl-1-piperazinyl)benzoxazinorifamycin (KRM-1648) combined with sparfloxacin.

Inhibition of the multiplication of Mycobacterium leprae in the footpads of nude mice by the oral administration of sparfloxacin, a new quinolone, and 3'-hydroxy-5'-(4-isobutyl-1-piperazinyl)benzoxazinorifamycin (KRM-1648), selected from a series of newly synthesized benzoxazinorifamycins, was studied. When the 2 drugs were administered alternately at intervals of 3 or 4 days, (i.e., each drug was administered once weekly), or simultaneously once weekly, between 3 and 5 months after inoculation of nude mice with M. leprae, 10 mg sparfloxacin and 0.6 mg KRM-1648 per kg bodyweight were sufficient to prevent multiplication of the organisms. Only partial inhibition of multiplication was achieved by alternate administration of 5 mg sparfloxacin and 0.3 mg KRM-1648 per kg, as was the case for 20 mg sparfloxacin per kg or 1 mg KRM-1648, each drug administered alone once weekly. The addition to these 2 drugs of dapsone, administered in the diet in a concentration of 0.001 g per 100 g, enhanced their effect. The potential usefulness of multidrug regimens including these compounds is considered.

[Phospholipid-deacylating activities of the mouse peritoneal macrophages during phagocytosis].

In the macrophages (M phi) obtained from mouse peritoneal exudates, five kinds of phospholipid-deacylating activities were detected using phosphatidylethanolamine (PE) and phosphatidylcholine (PC) labeled with [1-14C]oleic acid either in 1- or 2- position and 1- [1-14C]oleoyl-lysoPE, as substrates. Two types of phospholipipase A1 with pH optima of 4 to 6 and 8, respectively, and two types of phospholipase A2 activities with pH optima of 4 to 5 and 8, respectively, were identified. A detected lysophospholipase activity exhibited a broad pH optimum between 4 and 8. Both types of the phospholipase A1 and A2 of M phi hydrolyzed PE more than PC. Exogenously added Ca2+ did not increase the enzymatic activities. A comparison was made of three kinds of the M phi-phospholipid deacylating activities at pH8, after challenging the M phi with Mycobacterium lepraemurium, Escherichia coli, zymosan, or latex beads for 17 hours at 37 degrees C. The bacteria used to the phagocytosis were autoclaved. When the M phi were challenged with M. lepraemurium, the phospholipase A1, A2 and lysophospholipase activities were stimulated by about 160%, 150% and 140%, respectively. However, when challenged with E. coli, the phospholipase A1 activity remarkably decreased by about a third, although the phospholipase A2 activity was stimulated by about 150% that is similar to the challenge with M. lepraemurium. An inflammatory substance, zymosan seemed an effective inducer of the phospholipase A2, the enzymatic activity was remarkably stimulated by 260%, when challenged with 200 micrograms of zymosan. The increase in phospholipase A2 activity of the M phi pretreated with the bacteria or zymosan seems to result in an increase in the hydrolysis of arachidonic acid from the M phi-phospholipids to synthesize its inflammatory oxygenated metabolites. The lysophospholipase activity was not stimulated by the substances used to challenge the M phi, except for M. lepraemurium. No significant increase in three kinds of phospholipid-deacylating activities was observed after challenging the M phi with latex beads. It was considered from the above results that the M phi-phospholipid-deacylating activities at pH8 might be affected by sort of the ingested substances.

Phospholipid deacylating activities in murine leprosy bacilli.

1) The particulate fraction of cultivated murine leprosy bacilli (Mycobacterium lepraemurium, rough colonies of the Hawaiian-Ogawa strain) contained phospholipid deacylating activities with acidic pH optima. It hydrolyzed phosphatidylcholine and phosphatidylethanolamine at similar rates, and phosphatidylinositol oligomannosides more slowly. It also hydrolyzed 1-acyl- and 2-acyl-GPCs (sn-glycerol 3-phosphocholine) more rapidly than phosphatidylcholine. 2) Ca2+ did not stimulate either diacyl- or monoacyl-hydrolase activity. Triton X-100 and Emulgen 913 had little influence on the hydrolysis of phosphatidylcholine, but at rather high concentrations inhibited the hydrolyses of 1-acyl- and 2-acyl-GPCs. Iron ions strongly inhibited the hydrolysis of phosphatidylcholine, but caused little or no inhibition of the deacylations of 1-acyl- and 2-acyl-GPCs. 3) With 1-[stearoyl-14C]phosphatidylcholine and 2-[oleoyl-14C]phosphatidylcholine as substrates, both labeled fatty acid and lysophosphatidylcholine were produced. Labeled fatty acid appeared more rapidly from 2-[oleoyl-14C]phosphatidylcholine than labeled lysophosphatidylcholine, while labeled lysophosphatidylcholine was produced more than labeled fatty acid from 1-[stearoyl-14C]phosphatidylcholine in the early stage of incubation.

Metabolism of triacylglycerol in Mycobacterium smegmatis.

Mycobacterium smegmatis cells incorporated [1-14C]oleic acid into triacylglycerols (TG) from the medium more rapidly than shorter chain fatty acids, caprilic and butyric acids. This incorporation was inhibited more strongly by 10(-3) M N-ethylmaleimide than by 10(-3) M KCN. [14C]TG in the bacterial cells was utilized when the cells were in poor nutritional conditions, such as phosphate buffer (pH 7.0) containing oleic acid. Accumulation of TG was observed in the cells at late stages of growth. Diglyceride acyltransferase [EC 2.3.1.20] activity was detected in a cell-free extract from this bacterium. The pH optimum of this enzyme was between pH 7 and 9. F- and Tween 20 showed remarkable enhancing and inhibitory effects, respectively.

Effect of metal ions in the culture medium on the stearoyl-coenzyme A desaturase activity of Mycobacterium phlei.

A particulate fraction prepared from Mycobacterium phlei grown in a metal-deficient medium exhibited a greatly reduced activity of stearoyl-CoA desaturase compared to that from normally grown cells. Metal deficiency, however, had no effect on the FAD-dependent NADPH-cytochrome C reductase activity, which has been suggested to participate in the desaturation process. When the cells were grown in the deficient medium supplemented with both Fe2+ and Mg2+, the desaturase activity was restored to the normal level. Supplementation with Mg2+ alone promoted growth but did not restore the desaturase activity, whereas Fe2+ alone did cause a significant restoration. Among the various metal ions tested, only Fe2+ and Fe3+ enhanced the formation of desaturase activity in the deficient medium. When added to the assay medium in vitro, Fe2+ and Fe3+ did not stimulate the desaturase activity of the particulate fraction from the deficient cells. Cultivation in the metal-deficient medium had essentially no effect on the levels of cytochromes in the particulate fraction, but dramatically decreased the non-heme iron content and the amount of a high-spin ferric species exhibiting an ESR signal at g=4.3. No labile sulfur could be detected in the normal or metal-deficient particulate fractions. It is concluded that the presence of iron ions in the culture medium is necessary for the synthesis and/or assembly of the terminal portion of the desaturase system.