Search PubMed⌕ Search

Biomedical subjects

L Kato

Publications and source records attributed to L Kato.

69 records · Page 4Linked to original sources
PubMedDate not supplied

Cholesterol, a factor which is required for growth of mycobacteria from leprous tissues.

In a yeast extract, glycerol and sheep serum containing medium, slow but abundant growth of mycobacteria occurred when media were inoculated with M. leprae isolated from leprous tissues of armadillos (Dasypus novemcinctus Linn.). The lipid fraction of the serum was the essential factor for growth. Cholesterol not only replaced, but surpassed the growth promoting effect of the lipid fraction. However, growth of mycobacteria was observed only when media were enriched with serum. The relationship of the obtained strains of mycobacteria to leprosy is not yet clear. The following cholesterol medium, stabilized with lecithin, is proposed for primary cultivation of mycobacteria from leprous tissues: KH2PO4-8.2 gm, Na2HPO4-0.5 gm, yeast extract (Difco)-4 gm, and glycerol 30 gm, dissolved to make one liter basal medium in distilled water. Cholesterol (200 mg) dissolved in 4 ml warm acetone is injected with a syringe into the basal medium. The solution is autoclaved for ten minutes to evaporate the acetone. Lecithin, 200 mg dissolved in 20 ml of the basal medium is mixed to the medium cooled to room temperature. Nine milliliter aliquots are distributed into each of a series of 50 ml screw cap tubes and autoclaved for 25 minutes. One milliliter of filter sterilized sheep serum is added to each of the tubes containing 9 ml of the cholesterol-lecithin medium. Semisolid media are prepared the same way but 1.5% agar w/v is added to the cholesterol-lecithin medium before autoclaving. When cooled to 56 degrees C, 10% w/v sheep serum is mixed to the liquid. The medium is distributed into screw cap tubes and agar slants are poured and allowed to solidify in the inclined tubes at room temperature. Macrophages contain considerable amounts of cholesterol. Cholesterol is proposed as a possible growth factor for host grown M. leprae in the macrophages of the susceptible host and the same sterol as a growth factor for primary cultivation of mycobacteria from leprous tissues.

Animals↗
PubMedDate not supplied

In vitro cultivation of mycobacteria in cholesterol lecithin media from lepromas of rats infected with Mycobacterium lepraemurium.

In vivo grown M. lepraemurium suspensions were inoculated into a basal medium containing cholesterol and lecithin. Slow growing strains of mycobacteria were cultured regularly in these media. The presence of free cholesterol or cholesterol in serum or cholesterol in trypsin-digested egg yolk was essential for growth. The primary cultures were difficult to obtain, but the strains were easily subcultured. A heavy inoculum was necessary to obtain primary cultures in the liquid media, no growth occurred on semisolid agar slants. Similarly slow-growing primary cultures were obtained on Ogawa egg yolk media. Growth developed in a considerably shorter time if Ogawa's medium was enriched with 0.4% yeast extract (Difco). The cultures obtained on Ogawa egg yolk media were successfully subcultered in liquid cholesterol-lecithin media. The relation of the cultured strains of mycobacteria to the pathology of murine leprosy is not yet clear. The dynamics of cholesterol metabolism in the macrophages related to murine leprosy is discussed.

Animals↗
PubMedDate not supplied

Absence of mycobactin in Mycobacterium leprae; probably a microbe dependent microorganism implications.

Ferric mycobactins were prepared from Mycobacterium phlei. Mycobacterium avium--intracellulare A and H, isolated respectively from armadillo and human leprosy specimens. Attempts were made to extract mycobactin from host grown M. leprae cells. The crude ferric mycobactin extracts were tested for growth supporting effect on the mycobactin dependent M. paratuberculosis strain ATCC 19698. Mycobactins prepared from M. phlei and the two M. avium--intracellulare strains had growth promoting effect on M. paratuberculosis. The same test organism did not grow in media supplemented with the extract prepared from M. leprae. Results indicate the absence of mycobactin from host grown M. leprae. Since M. leprae cells contain cytochrome c and since mycobactin is essential to growth of all mycobacteria, M. leprae might be considered as a microbe dependent microbe. It is proposed that secondary mycobacteria present in M. leprae infected humans and armadillos might provide mycobactin for in vivo multiplication of M. leprae.

Iron↗
PubMedDate not supplied

A culture medium for cultivation of mycobacteria, probably Mycobacterium leprae from Mycobacterium leprae infected tissues.

Mycobacterium leprae suspensions were prepared from infected armadillos. The M. leprae cells were inoculated into culture media containing KH2PO4 4.7. g. Na2HPO4 2 g, sodium thioglycolate 1 g, (NH4)2SO4 2 g, MgSO4 0.1 g, ferric ammonium citrate 0.05 g, and lipoic acid (thioctic acid) 0.1 g in one liter distilled water. The solution was enriched with heat killed, sonicated leprosy derived Mycobacterium X or crude mycobactin extract from M. phlei to contain + 0.2 micrograms mycobactin per 1 ml in the final medium. Twenty ml media was distributed into each of 25 ml screw cap tubes and autoclaved for 30 minutes. Positive growth was obtained from seven out of ten specimens when incubated at 34 degrees C. The cultures developed as a sediment in the liquid media, suggesting preference for microaerophylic conditions. No growth was seen on the surface of the semi-solid agar media containing the same ingredients. Latency period of growth was estimated as 10-16 days and time of division as 6 days. Subcultures were obtained. Cells were long, acid fast, arranged side by side or end to end, with a tendency to form long spiral cords or clumps when sedimented on siliconized slides. Pyridine extraction eliminated acid fastness, but not gram positivity. Cultures did not grow on Dubos, Lowenstein or 7H10 media. They produce the disease in the foot pads of mice characteristic of M. leprae. Subcultures remain dependent on the heat killed sonicated mycobacteria, or crude mycobactin extract, and reduced oxygen tension in the media. Results suggest that cultures might be identical to M. leprae.

Ammonium Sulfate↗
PubMedDate not supplied

[A model for growing mycobacteria from leprous tissues placed in aliphatic hydrocarbons, tetradecane, a preliminary report].

Strains of Mycobacteria were regularly cultured from human and armadillo lepromata in liquid media, supplemented with tetradecane and dimethylsulfoxide. These substances had to be emulsified with heavy inoculum in order to obtain positive subcultures in the tetradecane-DMSO media. The use of nonpurified suspension of host-grown cells was advantageous for the primary cultivation. Subcultures were grown in the inorganic salt-tetradecane-dimethyl-sulfoxide liquid media. Strains did not grown on Löwenstein or Dubos medium. There is indication that the obtained cultures might be identical to M. leprae since the strongly acid-fast strains were obtained only in dimethylsulfoxide media. They were cultivated from a high proportion of human and armadillo lepromata collected from distant geographical locations. Cultures did not grow on any other media used for the cultivation of mycobacteria.

Alkanes↗
PubMedDate not supplied

A simplified hyaluronic acid based culture medium for mycobacteria isolated from human lepromata.

Acid-fast bacilli multiplied in liquid culture media containing hyaluronic acid when inoculated with mycobacteria from a lepromatous leprosy nodule. The culture was readily subcultured at ten day intervals in the homologue media, but failed to grow in the Dubos, Middlebrook and Lowenstein media. These findings confirm the results of Skinsnes et al (1975). Identification of this culture is not yet available, however it gives positive immunofluorescence with authentic anti-M. leprae serum. The obtained culture also grows as a chromogenic culture at 34 degrees C on a simple medium prepared from trypsin digested human umbilical cord, yeast extract powder and glycerol. This medium can be sterilized in an autoclave, but filter sterilized sheep, bovine or horse serum must be added aseptically as an essential ingredient. The medium does not differ considerably from the hyaluronic acid medium proposed by Skinsnes et al, but it is easier to prepare, it is inexpensive and permits a logarithmic growth within seven days of the so far unidentified culture isolated from leprotic nodules.

Cell Division↗
PubMedDate not supplied

Oxidation of 3,4-dihydroxyphenylalanine by connective tissue constituents. Identification of Mycobacterium leprae not related to phenolase activity.

The oxidation of 3,4-dihydroxyphenylalanine (DOPA) was studied by spectrophotometric methods at pH 6.8. In the presence of L- or D-DOPA, a color development occurred in the presence of the following substances as measured by increase in absorption both at 540 nm and 480 nm: hyaluronic acid, trypsinized human skin and umbilical cord extract, trypsin treated rat tissue from subcutaneous rat leproma, trypsin treated M. lepraemurium isolated from rat lepromata, and trypsinized M. leprae isolated from non-treated lepromatous leprosy cases. Normal human skin and connective tissue extract and nontrypsinized connective tissue of rat leprosy granuloma did not oxidize DOPA. While the trypsin-treated partially purified M. leprae suspension oxidized DOPA at both wave-lengths, the hyaluronidase-treated same suspension of M. leprae failed to oxidize these phenolic compounds. Mushroom tyrosinase oxidized D-DOPA, L-DOPA, epinephrine and norepinephrine at 480 nm. Hyaluronic acid also oxidized epinephrine and norepinephrine at both wave-lengths. Since it is known that M. leprae in the human host is closely associated with the presence of the acid mucopolysaccharides of the skin, and since acid mucopolysaccharides and skin constituents strongly oxidized DOPA, and since the hyaluronidase treated M. leprae failed to oxidize DOPA, it became evident that hyaluronic acid and not M. leprae is responsible for DOPA oxidation, and phenolase activity is not associated with the metabolism of M. leprae. Evidence is presented that DOPA is not a unique characteristic of the human leprosy bacillus. For instance, trypsin-treated murine leprosy bacilli from the rat strongly oxidized DOPA. The reaction of DOPA oxidation, therefore, must be rejected as a test for the identification of M. leprae. The obtained results confirmed the pertinent findings of Skinsnes and his co-workers.

Animals↗