Induction of N-acetyl-D-glucosamine catabolic enzymes and germinative response in Candida albicans

The regulation of N-acetylglucosamine catabolic enzymes was studied in both yeast and germ tube forms of the dimorphic fungus Candida albicans. The induction pattern of these enzymes was the same for yeast cells incubated at 28 degrees C and in cells incubated at 37 degrees C which formed germ tubes. However, the level of activity of these enzymes in germ tube stage is lower as compared to yeast phase cells. A strain of C. albicans that did not form germ tubes was endowed with a pronounced ability for induction of N-acetylglucosamine catabolic enzymes. This result suggests that germ tube formation and N-acetylglucosamine metabolism are mutually exclusive events.     

Comparative study of a new Chrysosporium species with Histoplasma capsulatum

The Chrysosporium state of a new ascomycete, Renispora flavissima (Gymnoascaceae), resembles Histoplasma capsulatum in its macroconidial morphology. It was discovered growing in bat guano, from which it was readily isolated by direct plating of diluted soil, but only rarely from mice inoculated with soil suspensions. The fungus was consistently reisolated from tissues of mice inoculated intravenously and intraperitoneally with conidial and mycelial suspensions from cultures of the fungus. Nevertheless, there is no evidence that this species is pathogenic. Cultures grew at 37 degrees C, but did not convert to a yeast form on agar media or within cultured mouse peritoneal macrophages. Although the hyphae and conidia of this fungus fluoresce when stained with H. capsulatum fluorescent antibodies, exoantigens of the fungus produce neither H nor M precipitin bands, thus differentiating it from H. capsulatum. Both H. capsulatum and the new Chrysosporium sp. demonstrate isozyme polymorphism, and isozymic differences have been discussed between the two species.     

Involvement of calcium, calmodulin and protein phosphorylation in morphogenesis of Candida albicans

N-Acetyl-D-glucosamine-induced germ tube formation in Candida albicans at 37 degrees C was accompanied by an increase in the rate of protein phosphorylation. The calmodulin antagonist trifluoperazine and the Ca2+ ionophore A23187, which inhibited germ tube formation, also reduced the rate of phosphorylation. The rate of phosphorylation was also reduced when cells were incubated at 25 degrees C, which favoured yeast-phase growth. Two-dimensional SDS-PAGE analysis of phosphoproteins from germ-tube-forming and yeast cells revealed two germ-tube-specific and three yeast-specific phosphoproteins. Germ tubes and hyphae had more calmodulin activity than yeast cells, irrespective of the germ-tube-inducing condition used. As a first step towards understanding the inhibitory effect of trifluoperazine on germ tube formation, calmodulin from C. albicans was purified to homogeneity. It was heat stable, and displayed a pronounced Ca2(+)-induced shift in electrophoretic mobility.     

Histoplasma capsulatum heat-shock 60 orchestrates the adaptation of the fungus to temperature stress

Heat shock proteins (Hsps) are among the most widely distributed and evolutionary conserved proteins. Hsps are essential regulators of diverse constitutive metabolic processes and are markedly upregulated during stress. A 62 kDa Hsp (Hsp60) of Histoplasma capsulatum (Hc) is an immunodominant antigen and the major surface ligand to CR3 receptors on macrophages. However little is known about the function of this protein within the fungus. We characterized Hc Hsp60-protein interactions under different temperature to gain insights of its additional functions oncell wall dynamism, heat stress and pathogenesis. We conducted co-immunoprecipitations with antibodies to Hc Hsp60 using cytoplasmic and cell wall extracts. Interacting proteins were identified by shotgun proteomics. For the cell wall, 84 common interactions were identified among the 3 growth conditions, including proteins involved in heat-shock response, sugar and amino acid/protein metabolism and cell signaling. Unique interactions were found at each temperature [30°C (81 proteins), 37°C (14) and 37/40°C (47)]. There were fewer unique interactions in cytoplasm [30°C (6), 37°C (25) and 37/40°C (39)] and four common interactions, including additional Hsps and other known virulence factors. These results show the complexity of Hsp60 function and provide insights into Hc biology, which may lead to new avenues for the management of histoplasmosis.     

The requirements for bicarbonate and metabolism of the inducer during germ tube formation by Candida albicans

Factors affecting germ tube formation in Candida albicans at suboptimal temperatures were investigated. Candida albicans formed germ tubes between 22 and 30 degrees C in solution when incubated without shaking, in the presence of bicarbonate (2 mg mL-1). Other conditions depended on the inducer used. Proline could induce germ tube formation optimally only when its concentration was between 200 and 400 mM. A concentration of 0.05 mM N-acetylglucosamine was sufficient to induce germ tube formation. N-Acetylglucosamine could induce germ tube formation at 30 but not at 25 degrees C. N-Acetylglucosamine induced germ tube formation was most reproducible when the cells were first starved by incubation in water for 16-24 h at 20 degrees C. Germ tubes induced by proline could be formed at pH values between 3.8 and 9.0 at 30 degrees C, but only between 7.0 and 7.5 at 25 degrees C. The addition of 0.05 to 5 mM glucose to a 5 mM proline induction solution allowed germ tube formation at 30 but not at 25 degrees C. Glucose (400 mM) did not suppress germ tube formation at 30 degrees C but only 5 mM was sufficient to cause a 65% suppression at 25 degrees C. The results show the importance of CO2 and (or) bicarbonate to the induction of germ tube formation and are consistent with the metabolism of the inducer.     

Recovery from potentially lethal damage of irradiated L5178Y-R and L5178Y-S cells: the effect of temperature and benzamide

Mouse lymphoma L5178 Y-S and Y-R cells differing in radiosensitivity by 1.5 times were treated with benzamide, an inhibitor of poly(ADP-ribosylation), for 24 h before and 18 h after X-irradiation, and incubated after irradiation at 25 degrees C and 37 degrees C. Clonogenic capacity of LY-S cells incubated at 25 degrees C exceeded that of the same cells incubated at 37 degrees C; the clonogenic capacity of LY-R cells did not vary with the postirradiation incubation temperature. Benzamide increased equally the radiosensitivity of LY-R cells incubated at both temperatures, whereas that of LY-S cells was only increased at 37 degrees C. Repair of potentially lethal damages to LY-S cells incubated at 25 degrees C was independent of the effectiveness of poly(ADP-ribosylation).     

Lipid synthesis during reinitiation of growth from stationary phase cultures of Candida albicans

Lipid synthesis has been studied in the dimorphic fungus Candida albicans. ¹⁴C-acetate incorporation into lipid material was used to measure new lipid synthesis in two cultures in which either yeast or mycelial growth was initiated from stationary phase yeast cells. When resuspended in fresh medium at 37 °C, cells resume growth and change morphology while at 30 °C cells resume budding growth. When resuspended at the appropriate temperature, both yeast and germ tube cultures immediately incorporated ¹⁴C-acetate into lipid material. The labeled lipid was more or less evenly divided between neutral and phospholipid. Phosphatidyl choline was the major phospholipid fraction and along with phosphatidyl ethanolamine accounted for 60–65 % of the total phospholipid. Lipid synthesis during growth initiation of either morphology showed a similar pattern, with no significant differences observed in neutral or phospholipid or phospholipid components between yeast and mycelial forms.     

Use of gradient plates to study combined effects of temperature, pH, and NaCl concentration on growth of Monascus ruber van Tieghem, an Ascomycetes fungus isolated from green table olives

The effect of temperature, pH, and sodium chloride concentration on the growth of the Ascomycetes fungus Monascus ruber van Tieghem, the main spoilage microorganism during storage of table olives, was studied by using the gradient plate technique. Gradients of NaCl (3 to 9%, wt/vol) at right angles to gradients of pH (2 to 6.8) were prepared for the plates, which were incubated at 25, 30, and 35 degrees C. Visible fungal growth, expressed in optical density units, was recorded by image analysis and graphically presented in the form of three-dimensional grids. Results obtained from the plates indicated that the fungus was salt and acid tolerant, being able to grow at NaCl concentrations of up to 9% (wt/vol) and pH values of as low as 2.2, depending on the incubation temperature. The inhibitory effect of NaCl increased as the pH decreased progressively at 25 and 30 degrees C but not at 35 degrees C. Growth was better at 30 and 25 degrees C as judged by the larger extent of the plates covered by mycelium compared with that at 35 degrees C, where no growth was observed at pHs below 3.7. Differentiation between vegetative (imperfect-stage) and reproductive (perfect-stage) growth was evident on all plates, providing useful information about the effect of environmental conditions on the form of fungal growth. When the growth/no-growth surface model was obtained by applying linear logistic regression, it was found that all factors (pH, NaCl, and temperature) and their interactions were significant. Plots of growth/no-growth interfaces for P values of 0.1, 0.5, and 0.9 described the results satisfactorily at 25 and 35 degrees C, whereas at 35 degrees C the model predicted lower minimum pH values for growth in the range of 7 to 10% NaCl than those observed on the plates. Overall, it is suggested that the fungus cannot be inhibited by any combination of pH and NaCl within the limits of the brine environment, so further processing is required to ensure product stability in the market.     

Methodological studies on growth of mouse granulocyte/macrophage colonies in methylcellulose-containing media

We studied the influence of various physicochemical parameters on colony development and total cell numbers in 7-day methylcellulose cultures of mouse bone marrow cells. Colony growth was markedly retarded by an increase of PO2 from approximately 6.7 kPa towards that in ambient air and by a decrease of incubator temperature from 37 degrees C to 33 degrees C. Medium osmolality above approximately 340 mosm/kg inhibited formation of granulocytes (in cultures containing growth factors from pokeweed mitogen-stimulated spleen cells), but not macrophages (L cell-produced growth factors). At most, colony growth was affected slightly by moderate changes in pH (7.17-7.47) or concentration of methylcellulose (0.77-1.02%), or by the presence of 2-mercaptoethanol (50 mumol/1), Hepes buffer (25 mmol/1), or erythropoietin (0.1-1 units/ml). The culture trays could be stored for one day at 4 degrees C in the incubation boxes before colonies or cells were counted.     

Control of microbial contamination of Franz diffusion cell receptor phase in the development of transcutaneous breast cancer therapeutics

Aims:  To determine the micro-organism contamination of excised porcine (pig) ear, and evaluate the use of Cyclopore track-etched membranes (CTEM) for preventing ingress into Franz-type diffusion cells.
Methods:  Swabs were taken from four locations and used to inoculate Tryptone Soya Agar (TSA) and Sabouraud Dextrose Agar (SDA) plates. Diffusion cells were assembled to include porcine skin with and without CTEM, and the receptor phase sampled periodically and spread onto plates.
Results:  Five distinct colony types were isolated after incubation of all swabs on TSA plates at 37°C; on SDA plates, one fungal colony was found at 30°C and one at 37°C. The SDA agar plate incubated at 30°C resulted in the growth of a large diffused white fungal colony. No regional differences were observed. Without the CTEM, the receptor phase became contaminated within 6 h. With the CTEM present, microbial ingress was substantially retarded with visible presumptive fungal growth occurring at 24 h and detectable contamination on both microbiological media at 48 h.
Conclusions:  As expected, the native porcine ears were considerably contaminated. The ingress of contamination into the diffusion cell receptor phases can be largely, but not entirely, eliminated using CTEM. The addition of antimicrobial agents was necessary to eliminate micro-organisms that were observed at later time points.
Significance and Impact of the Study:  This article, while highlighting the presence of a high number of micro-organisms on native porcine skin, presents a practical means to reduce the risk of microbial contamination in transdermal/transcutaneous permeation studies, particularly in the study of cell cultures grown within Franz diffusion cell receptor compartments.     

Effects of deferoxamine methanesulfonate on Trichophyton mentagrophytes.

Deferoxamine methanesulfonate (Desferal), an iron chelator, inhibited germ tube formation and growth of Trichophyton mentagrophytes in a microculture assay. A 50% reduction of germ tube formation required Desferal at 5 mg/ml and a 50% reduction of growth required 1.5 mg/ml. Growth was almost completely inhibited with 50 and 100 mg/ml. Also, Desferal at 100 mg/ml inhibited further elongation when added to short hyphae (II and 21 micrometer), but showed less inhibitory effects when added to long hyphae (64 micrometer). Iron (133 microgram/ml) reversed the inhibition of growth produced by incubating spores with Desferal at 5 mg/ml, providing iron was added before 72 h incubation. Desferal at 100 mg/ml decreased viability of activated spores incubated for 3 days at 30 degrees C, but did not decrease viability of spores incubated for 3 days at 4 degrees C. The growth inhibitory effect of Desferal and transferrin were compared. Transferrin was inhibitory at low molarities (0.001 to 1.0 mM), while Desferal was inhibitory only at higher molarities (greater than 1 mM). Desferal (0.05 mM) also reversed the inhibition expected with 0.05 mM transferrin. These findings indicate that Desferal and transferrin deprive T. mentagrophytes of nutritional iron and thus inhibit growth of the fungus. Low concentrations of Desferal can also promote growth in the presence of transferrin.     

Adhesion of Histoplasma capsulatum to pneumocytes and biofilm formation on an abiotic surface

The pathogenic fungus, Histoplasma capsulatum, causes the respiratory and systemic disease 'histoplasmosis'. This disease is primarily acquired via inhalation of aerosolized microconidia or hyphal fragments of H. capsulatum. Evolution of this respiratory disease depends on the ability of H. capsulatum yeasts to survive and replicate within alveolar macrophages. It is known that adhesion to host cells is the first step in colonization and biofilm formation. Some microorganisms become attached to biological and non-biological surfaces due to the formation of biofilms. Based on the importance of biofilms and their persistence on host tissues and cell surfaces, the present study was designed to investigate biofilm formation by H. capsulatum yeasts, as well as their ability to adhere to pneumocyte cells. H. capsulatum biofilm assays were performed in vitro using two different clinical strains of the fungus and biofilms were characterized using scanning electron microscopy. The biofilms were measured using a 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl]-2H-tetrazolium-hydroxide (XTT) reduction assay. The results showed that both the H. capsulatum strains tested were very efficient at adhering to host cells and forming biofilm. Therefore, this is a possible survival strategy adopted by this fungus.     

Electron microscopy of yeastlike cell development from the microconidium of Histoplasma capsulatum.

Fine details of the sequential morphological events occurring during transition of microconidia (spores less than 5 micrometer in diameter) to the yeastlike phase of Histoplasma capsulatum as seen in ultrathin section are described and illustrated by electron micrographs. Masses of microconidia were obtained when the fungas was grown on a garden soil extract medium. Spores were incubated under in vitro environmental conditions conducive for phase transition (an enriched medium at 37 degrees C). Within 48 h of incubation, the microconidia either germinated to give rise to a short mycelium or the germ tube process became a yeast mother cell without further extension. The wall of the yeast mother cell was thin and smooth, and its cytoplasmic content was ultrastructurally complex, consisting of numerous lipid bodies, vacuoles, glycogen-like deposits, and membrane systems. Within 96 h, the mother cell underwent multipolar budding to form simultaneously linear hyphal and/or ovate yeastlike daughter cells. During the transition, new cell wall materials of the germ tube, the mother cell, and yeastlike daughter cells arose by blastic action from the innermost layer(s) of the wall of the precursor form. Lomasome-like vesicles were often seen in association with areas of new cell wall formation. After organellar migration into and septation of the daughter cells, the yeast mother cell's cytoplasmic content underwent marked degenerative changes.     

A monoclonal antibody specific for Candida albicans Als4 demonstrates overlapping localization of Als family proteins on the fungal cell surface and highlights differences between Als localization in vitro and in vivo

The Candida albicans agglutinin-like sequence (ALS) family encodes large cell surface glycoproteins that function in adhesion of the fungus to host and abiotic surfaces. Monoclonal antibodies (mAbs) specific for each Als protein were developed to study Als localization on the C. albicans surface. An anti-Als4 mAb demonstrated that Als4 covers the surface of yeast cells, with a greater abundance of Als4 on cells grown at 30 °C compared to 37 °C. On germ tubes, Als4 is localized in a restricted area proximal to the mother yeast. Immunolabeling with several anti-Als mAbs showed overlapping localization of Als1 and Als4 on yeast cells and Als1, Als3 and Als4 on germ tubes. Overlapping localization of Als proteins was also observed on yeast and hyphae recovered from mouse models of disseminated and oral candidiasis. Differences between Als localization in vivo and in vitro suggested changes in regulation of Als production in the host compared to the culture flask. Characterization with the anti-Als mAbs reveals the simultaneous presence and differences in relative abundance of Als proteins, creating an accurate image of Als representation and localization that can be used to guide conclusions regarding individual and collective Als protein function.     

Characterization of a Thermolabile Sulfite Reductase from Salmonella pullorum

The biochemical basis for sulfite accumulation by sulfate-using revertants of Salmonella pullorum was determined. All of the sulfate-using mutants isolated from wild-type S. pullorum accumulated sulfite when grown at 37 but not at 25 C. The specific activity of reduced nicotinamide adenine dinucleotide (NADPH)-dependent sulfite reductase (H ₂S-NADP oxidoreductase, EC 1.8.1.2) and of reduced methyl viologen (MVH)-dependent sulfite reductase (H ₂S-MV oxidoreductase), in extracts prepared from cells incubated at 37 C, declined as the incubation period lengthened. However, the specific activity of both reductases from cells incubated at 25 C did not decline. Thermolability of cell-free NADPH-dependent sulfite reductase from cells of S. pullorum incubated at 37 C was greater than the lability of this enzyme either from cells of S. typhimurium incubated at 37 C or from cells of S. pullorum incubated at 25 C. Cells cultured at 37 C continued to accumulate sulfite when the incubation temperature was shifted to 25 C; cells cultured at 25 C and shifted to 37 C accumulated no sulfite, whereas these cells shifted to 41 C accumulated sulfite. It was concluded that the configuration of the sulfite reductase of S. pullorum strain 6–18 is a function of the incubation temperature at which synthesis occurs.     

Effect of hypothermia on cell kinetics and response to hyperthermia and X rays

Hyperthermia is a potent radio enhancer. Studies using hypothermia in combination with irradiation have given confusing results due to lack of uniformity in experimental design. This report shows that hypothermia might have potential significance in the treatment of malignant cells with both thermo- and radiotherapy. Reuber H35 hepatoma cells, clone KRC-7 were used to study the effect of hypothermia on cell kinetics and subsequent response to hyperthermia and/or X rays. Cells were incubated at 8.5 degrees C or between 25 and 37 degrees C for 24 hr prior to hyperthermia or irradiation. Hypothermia caused sensitization to both hyperthermia and X rays. Maximum sensitization was observed between 25 and 30 degrees C and no sensitization was found at 8.5 degrees C. At 25 degrees C maximum sensitization was achieved in approximately 24 hr, cell proliferation was almost completely blocked, and cells gradually accumulated in the G2 phase of the cell cycle. In contrast to the effect of hypothermia on either hyperthermia or X rays alone, thermal radiosensitization was decreased in hypothermically pretreated cells (24 hr at 25 degrees C) compared to control cells (37 degrees C). The expression of thermotolerance and the rate of development at 37 degrees C after an initial heating at 42.5 degrees C were not influenced after preincubation at 25 degrees C for 24 hr. The expression of thermotolerance for heat or heat plus X rays during incubation at 41 degrees C occurred in a significantly smaller number of cells after 24 hr preincubation at 25 degrees C. The enhanced thermo- and radiosensitivity in hypothermically treated cells disappeared in approximately 6 hr after return to 37 degrees C.     

Hirsutella thompsonii, a fungal pathogen of mites. I. Biology of the fungus in vitro

Hirsutella thompsonii, a moniliaceous fungus pathogenic to mites, grew and sporulated on sterilised wheat bran. The effects of environmental factors were studied on the fungus grown on potato-dextrose-agar (PDA). The fungus was mesothermophilic. Growth, sporulation and conidial germination were best at 25^o-30 ^oC. Conidia kept at 37 ^oC for 5 days on PDA died, but those held at 5 ^oC germinated upon a subsequent removal to 25 ^oC. Almost all conidial germ tubes survived an 8 h exposure to 3–5% r.h. and to 60% r.h., but subsequently the former grew poorly at 100% r.h. H. thompsonii sporulated equally well in continuous darkness or light, and produced typical chlorinous to light olive-green mycelium and conidia under all conditions. A 2 h exposure of naked mycelium and conidia (which have melanised walls) to u.v. irradiation failed to kill the fungus.     

In vitro culture of mouse primordial germ cells

Germ cells were isolated from mouse fetal gonads 11 1/2-16 1/2 days post coitum (dpc), and exposed to various methods of in vitro culture. From 13 1/2 dpc onwards, both male and female germ cells survived well at 37 degrees C for several days. During the culture period the proportion of female germ cells in meiosis increased and later stages of meiotic prophase were seen. The gonadal environment is therefore not essential for the progress of meiosis. Male germ cells in vitro did not enter meiosis. Germ cells isolated from gonads 11 1/2 or 12 1/2 dpc did not survive at 37 degrees C in any of the three culture systems used (Petri dishes, microtest plate wells, drops under oil); cell density, substrate and culture medium were varied, and several additives tested, but no improvement in viability was detected. Below 30 degrees C, on the other hand, 11 1/2 and 12 1/2 day germ cells survived in vitro for at least a week. They did not enter meiosis in culture, but continued to undergo mitotic proliferation.     

Requirement of newly synthesized protein for the priming activity of interferon.

Pretreatment of mouse L cells with interferon (IF) enhanced IF production in response to polyinosinic-polycytidylic acid (poly I-poly C). Post-treatment of cells with IF caused no significant enhancement of IF production. The enhancing effect of IF pretreatment (priming) reached a maximum after incubation with IF (10 or 100 units/ml) for 4-6 hr at 37 C, but this effect was absent when the incubation was done at 4 C. Cells which were incubated for additional several hours at 37 C after IF pretreatment at 4 C did not develop the primed state nor the antiviral state. The presence of protein synthesis inhibitors during the IF pretreatment depressed, though not completely, the development of the primed state. The residual priming effect was lost when the cells were incubated with the inhibitors at 37 C for 2 hr before they were exposed to poly I-poly C. There was no significant difference in the binding rate of poly I-poly C to cells between IF-treated and untreated cells. The degradation rate of cell-bound poly I-poly C and its sensitivity to exogenous pancreatic ribonuclease in the pretreated cells were also similar to those in the untreated cells.