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G S Kobayashi

Publications and source records attributed to G S Kobayashi.

At least 19 recordsLinked to original sources
PubMed1992

4-oxatetradecanoic acid is fungicidal for Cryptococcus neoformans and inhibits replication of human immunodeficiency virus I.

Candida albicans and Cryptococcus neoformans are major causes of systemic fungal infections, particularly in patients with acquired immunodeficiency syndrome. Metabolic labeling studies revealed that these organisms synthesize a small number of N-myristoylproteins, the most prominent being 20-kDa ADP-ribosylation factors (Arfs). C. albicans Arf has approximately 80% identity with the essential Arf1 and Arf2 proteins of Saccharomyces cerevisiae. [3H]Myristic acid analogs with oxygen for -CH2- substitutions at C4, C6, C11, and C13 are incorporated into cellular N-myristoylproteins, phospholipids, and neutral lipids produced by these three yeasts during exponential growth at 30 degrees C in complex media. Analog- and organism-specific differences in the efficiency of labeling of proteins and lipid classes were observed. The effects of oxatetradecanoic acids with oxygen for -CH2- substitutions at C3-C13 on C. neoformans, C. albicans, and S. cerevisiae were assessed during mid-log phase growth at 30 degrees C. A single dose of 3-oxa-, 4-oxa-, 5-oxa- or 6-oxatetradecanoic acid (O3-O6, final concentration = 300 microM) was able to inhibit growth of C. neoformans in the order O4 greater than O5 greater than O3 approximately O6. The other compounds were inactive. 4-Oxatetradecanoic acid was fungicidal, producing a 10,000-fold reduction in viable cell number 1 h after administration and continued suppression of cell growth for 7 h. A clear dose response was observed over a concentration range of 100-300 microM. 4-Oxatridecanoic acid was 100-fold less potent in reducing cell viability than 4-oxatetradecanoic acid but more potent than 5-oxatridecanoic acid. O4 produced approximately 10-100-fold reductions in the viability of C. albicans and S. cerevisiae at 300-500 microM, respectively, whereas O5 and O6 were less active. Since N-myristoylation of the Pr55gag polyprotein precursor produced by human immunodeficiency virus I (HIV-I) is essential for its assembly, we also assessed the antiviral effects of 4-oxatetradecanoic acid. O4 is able to produce a 50% reduction in the replication of HIV-I in acutely infected human T-lymphocyte cell lines at a concentration of 18 microM. Together, these data suggest that (i) the position of the oxygen for methylene substitution is a critical determinant of the fungicidal activity of O4 and (ii) NMT may be an attractive therapeutic target for treating opportunistic fungal infections in patients infected with HIV-I.

ADP-Ribosylation Factor 1↗
PubMed1992

Isolation, characterization, and sequencing of Candida albicans repetitive element 2.

A 1059-bp Sau3A fragment, designated Candida albicans repetitive element 2 (CARE-2), was isolated from the genome of the pathogenic yeast, C. albicans. CARE-2 DNA was detected on several C. albicans chromosomes separated by transverse alternating-field electrophoresis. A high degree of interstrain variation in the pattern of hybridizing bands were observed by Southern blot analysis, with a minimum of 10-14 copies of CARE-2 per strain. A low frequency of new CARE-2 polymorphisms was observed over time for three strains grown at 25 degrees C or 37 degrees C. No new CARE-2 polymorphisms were observed from two naturally occurring switch phenotypes. To localize repeated DNA, oligodeoxyribonucleotide probes, each representing a different region of CARE-2, were hybridized to genomic blots. A lower number of copies were observed 5' and 3' to a 600-bp region of CARE-2. Nucleotide (nt) sequence analysis of CARE-2 DNA shows the element is characterized by six perfect direct repeats 6 bp in length and shows no significant DNA similarity with any known nt sequence.

Base Sequence↗
PubMed1992

Standardized susceptibility testing of fluconazole: an international collaborative study.

An international collaborative study of broth dilution (MIC) and disk diffusion susceptibility testing of fluconazole was conducted by using a chemically defined medium (High-Resolution Antifungal Assay Medium; Oxoid Ltd., Basingstoke, United Kingdom) and standard test methods performed in eight reference laboratories. Ten yeast isolates were tested by each test method in duplicate on each of 3 separate days. The intralaboratory reproducibility of the MIC test was excellent; 95.7% of the replicate tests (n = 220) were within 2 doubling dilutions of the other values in the set for the eight laboratories. The intralaboratory reproducibility of the disk test was also good, with 91% of the replicate tests (n = 234) agreeing with each other within an arbitrarily chosen value of 4 mm. Interlaboratory agreement of MIC test results was acceptable, with 84% of the MICs agreeing within 2 doubling dilutions. In contrast, the interlaboratory agreement of the disk test was not good, with only 59% of test results agreeing within 4 mm. Comparison of the rank order of MICs obtained in each laboratory with the reference rank order gave an agreement of 70 to 80% (median, 80%) with the MIC test and 70 to 90% (median, 80%) with the disk test. These preliminary results are encouraging for the development of standardized testing methods for testing fluconazole.

Culture Media↗
PubMed1992

Typing of Histoplasma capsulatum by restriction fragment length polymorphisms in a nuclear gene.

We previously described yps-3, a Histoplasma-specific nuclear gene probe useful in the identification of Histoplasma capsulatum. By using restriction fragment length polymorphisms (RFLPs) of DNA detected by the yps-3 gene and mitochondrial DNA, 76 clinical and soil isolates of H. capsulatum were classified. The majority of North American isolates obtained from endemic regions of the midwestern United States were members of the previously characterized class 2, although four clinical isolates from different patients with AIDS from that region were grouped in class 1 with the temperature-sensitive Downs strain. A Florida soil isolate (FLS1) was placed in class 4 on the basis of RFLP with both probes. Two American Type Culture Collection strains (G184B and G186B) from Panama were grouped into class 3 by this analysis. A group of five H. capsulatum isolates obtained from patients with AIDS in New York City were typed into a new class 5 on the basis of yps-3 polymorphisms; those organisms fell into two broad mitochondrial DNA patterns, designated 5b and 5c. Two new isolates from Panama were also members of this broad yps-3 class 5 group, but they exhibited a distinct mitochondrial DNA profile (class 5a). A sixth class was detected in DNA obtained from a patient with AIDS from Panama; that DNA had unique RFLP profiles with respect to both probes. These observations suggest that the Histoplasma-specific yps-3 gene probe is a sensitive tool for typing H. capsulatum in clinical specimens. Additionally, these studies provide molecular support for the hypothesis that AIDS-associated histoplasmosis in nonendemic areas is due to reactivation of a previously acquired infection.

Acquired Immunodeficiency Syndrome↗
PubMed1991

Characterization of CARE-1: Candida albicans repetitive element-1.

A middle repetitive DNA element, Candida albicans repetitive element-1 (CARE-1) has been isolated from the pathogenic yeast C. albicans. CARE-1 appears to be species-specific and constitutes approx. 0.045% of total C. albicans DNA, or a reiteration frequency of about two to twelve copies per haploid genome. The CARE-1 element has been detected on several C. albicans chromosomes separated by field-inversion gel electrophoresis, suggesting that the element is dispersed. Interstrain variation was observed in the number and distribution of hybridizing bands. The element is well conserved, since no nucleotide (nt) heterogeneity was observed when the sequences of two CARE-1 family members isolated from two different chromosomes (A and B) of C. albicans were compared. CARE-1 possesses 467 bp and is characterized by several stretches of A's and T's, short direct repeats and shows no significant homology to any known nt sequence.

Base Sequence↗
PubMed1991

Systemic fungal infections: an overview.

Increasingly more common, these infections pose diagnostic and therapeutic problems. The distinction between primary infection in healthy hosts and opportunistic infection in the immunocompromised remains clinically useful. Three major diseases in each category are discussed. A promising therapeutic advance is the introduction of orally administered azoles.

Antifungal Agents↗
PubMed1991

Structure-activity study of inhibition of amphotericin B (Fungizone) binding to sterols, toxicity to cells, and lethality to mice by esters of sucrose.

The effects of four monoesters of sucrose with different acyl chain lengths (palmitate, C16; myristate, C14; laurate, C12; and caprate, C10) on the aggregation state of amphotericin B (AmB), its binding to cholesterol and ergosterol, its toxicity to cells, and its lethality to mice were determined. In solution, all four of these esters inhibited AmB binding to cholesterol more than to ergosterol; this effect correlated with the ester-induced shift from the mainly aggregated form of AmB to the mainly monomeric form. In experiments with cells, the esters inhibited the toxicity of AmB to mouse erythrocytes and cultured mouse fibroblast L-929 cells more than its toxicity to Candida albicans cells. When injected intravenously with AmB, these esters decreased AmB lethality to mice. In all of these assays, the ester with the shortest chain length (caprate) was much less potent than the other three esters. Our results indicate a correlation between in vitro and in vivo assays and suggest that the in vitro and in vivo selectivity of AmB may be enhanced by surface-active agents which modulate the aggregation state of AmB.

Amphotericin B↗
PubMed1990

Temperature-sensitive variants of Histoplasma capsulatum isolated from patients with acquired immunodeficiency syndrome.

Histoplasma capsulatum isolates from three St. Louis area AIDS patients with disseminated histoplasmosis were found to be closely related to the temperature-sensitive, previously unique, Downs strain based on growth phenotype and restriction fragment length polymorphisms (RFLP) involving mitochondrial DNA, ribosomal DNA, and the yps-3 gene. H. capsulatum isolates from five non-AIDS patients in the St. Louis area with disseminated histoplasmosis or chronic pulmonary histoplasmosis had the growth phenotype and RFLP pattern characteristic of most strains isolated from other regions of the USA.

Acquired Immunodeficiency Syndrome↗
PubMed1990

In vitro and in vivo activities of Sch 39304, fluconazole, and amphotericin B against Histoplasma capsulatum.

The antifungal activities of amphotericin B and two triazoles, Sch 39304 and fluconazole, were tested against Histoplasma capsulatum. In this study Sch 39304 compared favorably with amphotericin B in treating histoplasmosis in normal and leukopenic mice, whereas fluconazole was much less active. The differences in the efficacies of the triazoles appeared to be due to differences in their pharmacokinetics and the dosage schedule that was used. For amphotericin B there was a good correlation between in vitro and in vivo efficacy, but this was not true of the triazole derivatives. These results further demonstrate that, with the methods used in this study, in vitro susceptibility testing of triazoles may not be predictive of in vivo activity against isolates of H. capsulatum.

Amphotericin B↗
PubMed1989

Comparison of the separation of Candida albicans chromosome-sized DNA by pulsed-field gel electrophoresis techniques.

Pulsed-field gel electrophoresis techniques were used to study chromosome-sized DNA molecules of C. albicans. Chromosome-sized DNA of two strains of Candida albicans has been resolved into 8 bands by orthogonal-field-alternation gel electrophoresis (OFAGE). Six bands were observed in chromosomal preparations of C. albicans using field-inversion gel electrophoresis (FIGE). Differences in the electrophoretic mobilities of bands of the strains of C. albicans examined suggests that chromosome-length polymorphisms exist and make it difficult to correlate the banding patterns among strains. These correlations were facilitated, however, by assignment of C. albicans chromosomes by hybridization using a collection of cloned DNA probes specific for each of the 8 observed bands. Southern blotting showed that the 6 FIGE bands consisted of 4 singlets and 2 comigrating doublets, accounting for the 8 bands observed by OFAGE analysis. The agreement between OFAGE and FIGE analysis suggests that the C. albicans haploid genome contains a minimum of 8 chromosomes.

Candida albicans↗
PubMed1989

Effects of ascorbic acid on the antifungal action of amphotericin B.

Ascorbic acid enhanced the lethal but not the permeabilizing effects of amphotericin B on Candida albicans and Cryptococcus neoformans cells. Two other ene-diol acids, D-erythorbate and dihydroxyfumarate, also enhanced the lethal action of amphotericin B on Can. albicans. Maleic acid and gulanolactone, compounds structurally related to ascorbic acid but not containing the ene-diol group, had no such effect. It is assumed that ascorbic acid and the two other ene-diol acids acting as pro-oxidants augmented the oxidation-dependent killing of fungal cells induced by amphotericin B.

Amphotericin B↗
PubMed1989

Effects of Histoplasma capsulatum on murine macrophage functions: inhibition of macrophage priming, oxidative burst, and antifungal activities.

Histoplasma capsulatum yeast cells fail to trigger an oxidative burst response in normal murine macrophages. The results of this study, in which an in vitro assay of macrophage antifungal effects was used, extend these findings. During 18 h of incubation, unprimed elicited murine macrophages inhibited H. capsulatum growth only when macrophages were present in great excess. Gamma interferon (IFN-gamma)-primed macrophages showed enhanced fungal growth inhibition but a similar requirement for an excess of phagocytes. Macrophages containing heat-killed H. capsulatum exhibited diminished antifungal effects toward viable H. capsulatum and Saccharomyces cerevisiae cells. Parallel experiments showed no comparable effect of ingested latex particles on macrophage antifungal activity. Using chemiluminescence as a measure of the oxidative burst, we found that macrophages primed in vitro with IFN-gamma alone failed to exhibit a significant response to triggering by H. capsulatum yeast cells unless a second priming agent (tumor necrosis factor alpha or bacterial lipopolysaccharide) was added to IFN-gamma. Furthermore, macrophage priming with single agents was blocked by the prior ingestion of heat-killed H. capsulatum. These studies provide evidence that ingestion of H. capsulatum yeast cells can induce a prompt and enduring deactivation of murine macrophages.

Animals↗
PubMed1989

Variable expression of a yeast-phase-specific gene in Histoplasma capsulatum strains differing in thermotolerance and virulence.

By means of differential hybridization techniques, several yeast-phase-specific DNA sequences were identified in the dimorphic pathogenic fungus Histoplasma capsulatum. A 1.85-kilobase (kb) HindIII fragment from one genomic clone, yps-3, hybridized to at least three distinct yeast poly(A)+ RNAs of 1.3, 1.05, and 0.95 kb from the virulent strain G217B. These mRNAs were not detected in mycelia. When mycelia from G217B were induced to become yeast by transfer from 25 to 37 degrees C, a process requiring approximately 9 days, expression of yps-3 was detected within 24 h, although not in the initial 2 h following the temperature shift. In contrast, a low-virulence strain (Downs) which completes the transition in approximately 2 weeks failed to express the yps-3 gene during phase transitions. A third isolate, G186B, intermediate in its virulence properties and in the time required for the transition (11 days), expressed a single 1.25-kb mRNA but only at low levels in the yeast phase and only after 3 days following the 25-to-37 degrees C temperature shift. Although yps-3 expression does not appear to be essential for the transformation to the yeast phase, it may facilitate the early adaptive processes which permit the mycelium-to-yeast transition and survival of the yeast phase of H. capsulatum at elevated host temperatures. The phase-specific yps-3 nuclear gene is carried on highly polymorphic restriction fragments in all three strains, suggesting that this probe may provide a sensitive diagnostic tool for the classification of H. capsulatum isolates.

Blotting, Northern↗