Search PubMed⌕ Search

Biomedical subjects

D H Howard

Publications and source records attributed to D H Howard.

At least 73 records · Page 4Linked to original sources
PubMed1973

Fate of Histoplasma capsulatum in guinea pig polymorphonuclear leukocytes.

Guinea pig polymorphonuclear leukocytes (PMN) were parasitized in vitro with yeast cells of Histoplasma capsulatum. Preparations stained after 3 h at 37 C with May Greenwald-Giemsa revealed that 87% of the yeasts were tinctorially altered. Such alterations corresponded to those displayed by fungus cells intentionally killed by heat or other means and thus the altered yeast were presumed to be dead. A combination of 10(-5) M H(2)O(2), 10(-5) M KI, and horseradish peroxidase killed H. capsulatum. Death was assessed by the eosin-y dye exclusion test. All of the listed components were required for death of the fungus. A granule lysate preparation derived from guinea pig PMN leukocytes could replace the horseradish peroxidase in the fungicidal system. The granule lysates behaved in keeping with the attributes of a myeloperoxidase. Thus, PMN leukocytes and certain extracellular peroxidase systems kill the yeast cell phase of the dimorphic fungus H. capsulatum.

Animals↗
PubMed1973

Further studies on the inhibition of Histoplasma capsulatum within macrophages from immunized animals.

Mononuclear phagocytes freshly harvested from immunized animals restrict the intracellular growth of Histoplasma capsulatum. The fate of the inhibited fungus is the subject of this report. Macrophages harvested from mice immunized by sublethal infection were parasitized in vitro with yeast cells of H. capsulatum. The subsequent fate of the fungus was assessed by staining characteristics, viability on subculture, and radioisotopic techniques. No tinctorial distinction could be made between yeasts within normal or immune phagocytes stained by the May Greenwald-Giemsa or by the Gridley techniques. Yeast cells recovered after 24 h of residence within the cytoplasm of immune monocytes excluded the dye eosin-Y, germinated on Casamino Acid agar incubated at 30 C, and initiated growth at a normal rate within phagocytes from normal animals. Thus even though the growth of H. capsulatum was inhibited within macrophages from immunized animals, the fungus was not killed by the encounter. However, autoradiographic studies established that protein synthesis by the fungus in situ was impaired.

Animals↗
PubMed1973

Uptake and utilization of glutamic acid by Cryptococcus albidus.

Cryptococcus albidus utilizes glutamate as a sole carbon source. The kinetics of uptake of this amino acid were studied. l-Glutamic acid was taken up by two saturable systems: a high affinity system with a Michaelis constant (K(m)) of 1.15 x 10(-5) M and a V(max) of 0.049 mumol per mg per h and a low affinity system with a K(m) of 2.5 x 10(-3) M and a V(max) of 3.61 mumol per mg per h. Both systems possessed characteristics of active transport which were dependent on temperature and pH and which required metabolic energy. Uptake was inhibited at 37 C but the temperature-sensitive step was reversible. Chemical fractionation of cells with 5% trichloroacetic acid showed that glutamic acid initially entered a soluble pool which decreased after 1 h as the amino acid was incorporated into the protein and nucleic acid fractions of the yeast. Some of the glutamate was completely oxidized and could be recovered as (14)CO(2). Therefore, the amino acid was also used as an energy source.

Amino Acids↗
PubMed1973

Metabolism, macromolecular synthesis, and nuclear behavior of Cryptococcus albidus at 37 C.

The temperature-sensitive events which prevent Cryptococcus albidus from growing at 37 C were investigated. Cultures incubated at 37 C immediately after inoculation did not increase in optical density nor in cell numbers, and by 24 h 90% of cells in such cultures were deformed and dead. When cultures in log phase were shifted from 23 to 37 C the optical density increased but the cell numbers did not. Morphological observations revealed that the increase in turbidity at 37 C represented enlargement and distortion of cells without appreciable replication. Uptake and incorporation of (14)C-leucine were similar at 23 and 37 C. There was no difference in (14)CO(2) evolution from cells at either temperature. Uptake and incorporation of adenine-8-(14)C into RNA was slightly lower in cells incubated at 37 C. There was, however, a 60% reduction in incorporation of adenine-8-(14)C into DNA after 3 hr at 37 C. Nuclear staining revealed that nuclear migration did not occur in cells incubated at 37 C. Thus the data indicate that both adenine incorporation into DNA and nuclear migration prior to nuclear division by C. albidus are temperature sensitive.

Adenine↗
PubMed1971

Characterization of antigens from the yeast phase of Histoplasma capsulatum.

Preliminary studies established methods for obtaining maximum yields of viable cells. Liquid shaken cultures of Histoplasma capsulatum provided a maximum of 4 x 10(8) to 5 x 10(8) cells/ml regardless of the inoculum size. Under optimum conditions, cells were viable (90 to 95%) for 168 to 240 hr. Generation times ranged from 7.52 to 8.36 hr. Immunodiffusion, immunoelectrophoresis, and ultracentrifugation studies on phenol and ethylenediamine extracts of intact cells and cell walls revealed the presence of two components in the ethylenediamine extracts and three in the phenol preparations. The ethylenediamine extracts from intact cells and cell walls seemed to be identical although one of the components was more abundant in cell walls. Mice injected intraperitoneally with intact cells or cell walls were protected against intravenous challenge with H. capsulatum. Among the extracts, the water-soluble ethylenediamine extract from cell walls was most immunogenic. The other extracts gave only a light protection or none at all. Intact cells and cell walls were slightly toxic to mice. Two of the extracts were toxic when incorporated into Freund's complete adjuvant.

Animals↗
PubMed1971

Lymphocyte-mediated cellular immunity in histoplasmosis.

Mononuclear phagocytes freshly harvested from immunized animals restrict the intracellular growth of Histoplasma capsulatum, whereas the same cells maintained in culture for 48 hr do not. Experiments established that within the mixed cell population of the mouse peritoneal cavity the lymphocyte was the most likely mediator of the intracellular growth restriction observed. Partially purified lymphocytes, from the peritoneal cavity of mice immunized by sublethal infection, mediated the suppression of intracellular growth of the fungus in normal mouse macrophages in cell culture.

Animals↗
PubMed1971

Comparative physiologial studies of the yeast and mycelial forms of Histoplasma capsulaum: uptake and incorporation of L-leucine.

l-Leucine entered the cells of both morphological forms of Histoplasma capsulatum by a permease-like system at low external concentrations of substrate. However, at levels greater than 5 x 10(-5)m l-leucine, the amino acid entered the cells both through a simple diffusion-like process and the permease-like system. The rate of the amino acid diffusion into yeast and mycelial forms appeared to be the same, whereas the initial rate of accumulation through the permease-like system was 5 to 10 times faster in the mycelial phase than it was in the yeast phase. The Michaelis constants were 2.2 x 10(-5)m in yeast phase and 2 x 10(-5)m in mycelial phase cells. Transport of l-leucine at an external concentration of 10(-5)m showed all of the characteristics of a system of active transport, which was dependent on temperature and pH. Displacement or removal of the alpha-amino group, or modification of the alpha-carboxyl group abolished amino acid uptake. The process was competitively inhibited by neutral aliphatic side-chain amino acids (inhibition constants ranged from 1.5 x 10(-5) to 6.2 x 10(-5)m). Neutral aromatic side-chain amino acids and the d-isomers of leucine and valine did not inhibit l-leucine uptake. These data were interpreted to mean that the l-leucine transport system is stereospecific and is highly specific for neutral aliphatic side-chain amino acids. Incorporation of l-leucine into macromolecules occurred at almost the same rate in both morphological forms of the fungus. The mycelial phase but not the yeast phase showed a slight initial lag in incorporation. In both morphological forms the intracellular pool of l-leucine was of limited capacity, and the total uptake of the amino acid was a function of intracellular pool size. The initial rate of l-leucine uptake was independent of the level of intracellular pool. Both morphological forms deaminated and degraded only a minor fraction of the accumulated leucine.

Amino Acids↗
PubMed1970

Uptake of Cystine by the Yeast Phase of Histoplasma capsulatum.

This report deals with factors affecting the uptake of cystine by the yeast phase of Histoplasma capsulatum. The kinetics of uptake showed a saturation at 70 muM and an average K(m) value of 3 x 10(-5)m. The optimal pH and temperature for transport of cystine were 6.5 and 37 C, respectively. The energy of activation was 14.1 kcal/mole, and the temperature coefficient value was 2.1. A requirement for energy supplied by metabolic activity was demonstrated by the inhibition of incorporation of the amino acid by cells preincubated with either 2,4-dinitrophenol or sodium azide. Although uptake was not inhibited by any single amino acid, a combination of amino acids did cause a decrease in uptake. Thus, the data show that the uptake of cystine by yeast cells of H. capsulatum has the characteristics of a system of transport that requires the expenditure of energy by the cells.

Journal Article↗
PubMed1970

Incorporation of Cystine by Yeast Cells of Histoplasma capsulatum.

This report deals with the incorporation of cystine into macromolecules by yeast cells of Histoplasma capsulatum. The results show that at saturating concentrations of cystine in a rich medium, total uptake of the cystine occurs in 10 hr, whereas the amount of label in the cold trichloroacetic acid-soluble material reaches a maximum at 3 to 4 hr and remains at this value. The amount of label in the cold acid-insoluble material accumulates linearly for 4 to 5 hr and reaches a plateau at 7 to 9 hr. A chemical fractionation of labeled cells shows that 25% of the cystine taken up remains as low-molecular-weight components, of which cystine comprises 60 to 75%. Approximately 30% of the total label incorporated is ethanol-soluble, and polyacrylamide gel electrophoresis of this material shows a rather uniform incorporation of the amino acid into many proteins. The combined hot KOH fractions account for 40% of the total label incorporated. The amount of hot KOH-insoluble material almost doubles in a 10-hr pulse, whereas there is an increase in hot KOH-soluble material. Hence, the greatest amount of label from cells pulsed with (14)C-cystine is recovered from cell wall material.

Journal Article↗
PubMed1967

Effect of temperature on the intracellular growth of Histoplasma capsulatum.

The rate and form of growth of Histoplasma capsulatum within histiocytes derived from homothermic and poikilothermic animals, and incubated at 25, 30, and 37 C, are described. The generation time of the fungus in mouse cells incubated at 37 and 25 C was 11 and 24 hr, respectively. Blastospore formation was progressively retarded in cells at 25 C, and this retardation was accompanied by germination of some of the blastospores. The generation time of the fungus in mouse cells incubated at 30 C was the same as it was at 37 C. Germ tube formation was not a prominent feature of intracellular growth at 30 C. The rate of growth of H. capsulatum within frog histiocytes at 30 and 25 C was slower than it was in mouse cells at the same temperatures. Some loss of frog histiocytes in cultures incubated at 37 C prevented accurate estimation of the rate of growth of the fungus at this temperature. Growth of H. capsulatum in frog histiocytes kept at 25 C was progressively retarded, and the retardation was accompanied by germination of the yeasts. Yeast-phase growth predominated in fish histiocytes incubated at 30 C, whereas germ tubes were formed within such cells incubated at 25 C. Cell survival of fish histiocytes was relatively poor in culture, and no estimates of rate of growth of the fungus within these cells were made.

Animals↗
PubMed1967

Carbon catabolism and synthesis of macromolecules during spore germination of Microsporum gypseum.

Either d- or l-leucine (10(-3)m) and unsaturated long-chain fatty acids such as oleic, linoleic, and arachidonic (10(-4)m) significantly stimulated macroconidia germination of Microsporum gypseum. Saturated long-chain fatty acids did not affect germination, whereas saturated short-chain fatty acids such as caprylic, hexanoic, and butyric were completely inhibitory. Germination was followed by an increase in endogenous respiration and a decrease in dry weight of approximately 5% at 4 hr. Endogenous fatty acids and soluble carbohydrates were utilized significantly during germination. Tritiated leucine, uridine, and thymidine were incorporated respectively into protein, ribonucleic acid (RNA), and deoxyribonucleic acid (DNA) fractions within the first 5 min of germination. Incorporation of oleic-1-C(14) into RNA and protein was significantly increased after germ tube development. Net synthesis of RNA and protein started prior to germ tube protrusion. Increase in DNA could be detected only later. A significant increase in RNA and protein during the 4th hr of germination was correlated with vegetative development. Inhibition of respiration and incorporation of leucine-H(3) and uridine-H(3) into corresponding macromolecules by dl-fluorophenylalanine and phenethyl alcohol started before germ tube appearance. Griseofulvin significantly inhibited incorporation of uridine-H(3) and thymidine-H(3), but not of leucine-H(3). This inhibition occurred only after initial vegetative development. In contrast to the two other inhibitors, which substantially inhibited germination, griseofulvin only slightly retarded the period of germination and did not affect respiration.

Alcohols↗