The presumptive identification of Candida albicans with germ tube induced by high temperature

For direct identification of Candida albicans from other Candida species, the chlamydospore formation and the mycelial transition induced by high temperature and by sera were examined in 198 Candida isolates. The germ tubes of C. albicans developed early at 30 min in high temperature-induction, but at 60 min in serum-induction. C. albicans generated germ tubes well at concentrations lower than 2 x 10(7) cells/ml, but the germ tube formation was markedly restrained at concentrations higher than 4 x 10(7) cells/ml. In a serum-free, yeast extract-peptone-dextrose (YEPD) medium, C. albicans grew as a yeast form at 30 degrees C and as a mycelial form at 35-42 degrees C. Mycelial development was maximal at 37 degrees C in serum and at 39 degrees C in YEPD. Germ tubes were formed within 30 min in YEPD at 39 degrees C, but after 60 min in serum at 37 degrees C. Our examination showed that the 39 degrees C-induced germ tube formation tests were very reliable (sensitivity 100%, specificity 100%) at discerning C. albicans from other Candida species. These results suggest that the high temperature-induced germ tube formation testing could be a useful identification method of C. albicans in clinical laboratories.     

Melaleuca alternifolia (tea tree) oil inhibits germ tube formation by Candida albicans.

The effect of tea tree oil (TTO) on the formation of germ tubes by Candida albicans was examined. Two isolates were tested for germ tube formation (GTF) in the presence of TTO concentrations (% v/v) ranging from 0.25% (1/2 minimum inhibitory concentration [MIC]) to 0.004% (1/128 MIC). GTF at 4 h in the presence of 0.004 and 0.008% (both isolates) and 0.016% (one isolate) TTO did not differ significantly (P > 0.05) from controls. At all other concentrations at 4 h, GTF differed significantly from controls (P < 0.01). A further eight isolates were tested for GTF in the presence of 0.031% TTO, and at 4h the mean GTF for all 10 isolates ranged 10.0-68.5%. Two isolates were examined for their ability to form germ tubes after 1 h of pre-exposure to several concentrations of TTO, prior to induction of germ tubes in horse serum. Cells pre-exposed to 0.125 and 0.25% TTO formed significantly fewer germ tubes than control cells at 1 h (P < 0.05), but only those cells pre-exposed to 0.25% differed significantly from control cells at later time points (P < 0.01). GTF by C. albicans is affected by the presence of, or pre-exposure to, sub-inhibitory concentrations of TTO. This may have therapeutic implications.     

A simple technique for observing germ tube formation in Candida albicans.

Candida albicans is identified in most laboratories by the rapid formation of germ tubes when it is grown in human serum (Taschdjian et al, 1960). Demonstration of the production of germ tubes on solid media, 1% bactopeptone in 2% agar (Joshi and Gavin, 1974), and the appearance of other yeasts on a variety of carbohydrate media (Joshi et al, 1975) has led to the development of a simple technique for observing germ tube production.     

Nucleoid segregation pattern during branching in Streptomyces granaticolor mycelia

A survey of GIEMSA-stained S. granaticolor mycelia grown in liquid medium revealed that branches developed only in subapical older regions of main hyphae for which large nucleoids were characteristic. The branches themselves contained only small nucleoids, as did the apical (about 30 μm long) region of the main hyphae. From the distribution pattern of the nucleoids at nascent branching areas it was concluded that during branch outgrowth one large nucleoid segregation into two small ones, both of which in most cases entered the branch. In order to determine in vivo the number of genome equivalents provided from main hyphae for a single branch, the kinetics of outgrowth of branches was measured and compared to that of germ tubes. The analysis of series of micrographs revealed that at the beginning of outgrowth, a branch received two genome equivalents from the main hypha while a germ tube got only one (from the spore). The subsequently expressed exponential elongation of branches as well as germ tubes ruled out that further nucleoids were supplied thereafter.     

Blockade of nitric oxide formation down-regulates cyclooxygenase-2 and decreases PGE2 biosynthesis in macrophages.

Elevated levels of nitric oxide (NO*) produced by expression of inducible nitric oxide synthase (iNOS/NOS type 2) and high levels of prostaglandins (PGs) generated by expression of inducible cyclooxygenase (COX-2/PGH2 synthase-2) are important mediators of immune and inflammatory responses. Previous studies have shown that endogenous levels of NO* can influence the formation of PGs. We examined the mechanism by which NO* regulates PG biosynthesis in macrophages. Treatment of a murine macrophage cell line (ANA-1) with lipopolysaccharide (LPS, 10 ng/mL) and interferon-gamma (IFN-gamma, 10 U/mL) for 20 h elicited high levels of nitrite (NO2-) and prostaglandin E2 (PGE2) that were inhibited in a dose-dependent fashion by the NOS inhibitor, aminoguanidine (AG), with IC50 values of 15.06 and 0.38 microM for NO2- and PGE2, respectively. Stimulation of cultures with LPS and IFN-gamma for 20 h induced de novo iNOS protein expression that was not altered by the addition of AG (0.1, 10, or 1000 microM). In contrast, treatment of cultures with LPS and IFN-gamma for 20 h promoted COX-2 mRNA and protein expression that were decreased in a dose-dependent fashion by AG (P < 0.05 with 10 and 1000 microM). LPS and IFN-gamma-induced COX-2 protein expression was not decreased in cultures treated with AG for 2 h, illustrating that AG does not inhibit the formation of COX-2 protein. Analysis of partially purified enzyme extracts demonstrated that AG did not directly inhibit the enzymatic activity of COX. Additional experiments revealed that NO* donors (S-nitroso-N-aceytl-D-L-pencillamine, SNAP, at 0.1, 10, and 1000 microM) did not induce de novo COX-2 protein expression or potentiate COX-2 expression in cells treated with LPS and/or IFN-gamma. Our results suggest that, while endogenous NO* is not required for de novo COX-2 mRNA and protein expression, NO* is necessary for maintaining prolonged COX-2 gene expression.     

Trehalose hydrolysis is not required for human serum-induced dimorphic transition in Candida albicans: evidence from a tps1/tps1 mutant deficient in trehalose synthesis

Exponential yeast-like cells of a Candida albicans wild-type strain exhibited strong capacity for germ tube formation in a glucose-containing medium (YPD) after induction with human serum at 37 degrees C, whereas the isogenic double disruptant tps1/tps1 mutant, which is deficient in trehalose synthesis, failed to produce germ tubes. In a medium without glucose (YP), the morphological transition fraction was roughly equivalent in both strains. Substitution of glucose by galactose or glycerol increased the number of wild-type proliferating cells able to enter the dimorphic program with no noticeable change in their trehalose content, while stationary cells, which accumulate a large amount of trehalose, did not form germ tubes. When fresh medium was added, a high proportion of these resting cells recovered their ability to carry out dimorphic transition. The tps1/tps1 mutant followed the same pattern of hyphae formation, despite the fact that it was unable to accumulate trehalose either during dimorphism induction or after several stress challenges. Furthermore, trehalose-6-phosphate synthase activity was barely detectable in the mutant. These results strongly suggest that serum-induced dimorphic transition does not require trehalose mobilization; they also support the idea that TPS1 is the only activity involved in trehalose biosynthesis in C. albicans.     

Inhibition of fast sodium current in rabbit ventricular myocytes by protein tyrosine kinase inhibitors

The present study investigated the effects of protein tyrosine kinase inhibitors on the fast sodium current ( I(Na)) in rabbit ventricular myocytes. Single rabbit ventricular myocytes were isolated enzymatically using Langendorff perfusion. I(Na) was recorded using the whole-cell patch-clamp technique at room temperature. The protein tyrosine kinase inhibitors genistein, AG957, ST638, and PP2 reversibly inhibited I(Na) in a concentration-dependent manner. At a test pulse potential of -30 mV, genistein (n=7) inhibited I(Na) by 37.7+/-3.2%, 53.4+/-2.5%, and 71.8+/-2.7% at concentrations of 15, 50, and 100 microM, respectively, without changing the voltage dependence of activation, while 100 microM AG957, 100 microM ST638, and 30 microM PP2 inhibited I(Na) by 38.7+/-2.4, 35.8+/-3.4, and 21.1+/-3.9%, respectively. Genistein (100 microM) and AG957 (100 microM) shifted the voltage for half-maximal inactivation of I(Na) from -76.7+/-2.0 mV (n=10) in control to -88.37+/-2.6 mV (n=6, P<0.05), and -82.9+/-1.7 (n=4, P<0.05), respectively, without changing the slope factor. Genistein and AG957 also significantly prolonged the time course of I(Na) recovery from inactivation. Daidzein and PP3, inactive analogs of genistein and PP2, respectively, did not inhibit I(Na) significantly. We conclude that protein tyrosine kinase signaling pathways may play an important role in regulation of I(Na) in cardiac myocytes.     

Inductions of germ tube and hyphal formations are controlled by mRNA synthesis inhibitor in Candida albicans.

Candida albicans is a pathogenic dimorphic fungus. When yeast cells were pre-incubated in YPD medium at 25degreesC and released into HFM7 medium containing 4% serum at 37degreesC, germ tubes emerged within 0.5 h. To determine whether mRNA or protein synthesis was necessary for germ tube formation, we examined the effects of mRNA and protein syntheses inhibitors on this formation. In the presence of cycloheximide, cells were unbudded and no germ tube was observed. However, in the presence of actinomycin D, germ tube formation was observed while budding growth and true hyphae elongation were blocked. Next, we measured mRNA or protein accumulation during induction of germ tube formation in the presence of the inhibitors. In the presence of cycloheximide, protein was not synthesized, while in the presence of actinomycin D, mRNA synthesis decreased to 6.3% and protein synthesis to 37.7%. The condition we found which allows only germination but not budding or filamentation might be convenient to use in screening genes involved in the initial stage of morphological change in C. albicans.     

Studies on the early stages of infection of Alternaria alternata on groundnut (Arachis hypogaea L.)

Studies were conducted on the early stages of infection of groundnut leaves by Alternaria alternata. Inoculated leaves were periodically sampled, checked for germination of spores, growth of germ tubes, appressorium formation and method of sporulation. Evidence was observed for the entry of the germ tubes through the stomates (with or without the formation of appressorium-like structures) or through the host surface by the formation of appressoria.     

Modulation of cell surface-associated mannoprotein antigen expression in experimental candidal vaginitis.

The monoclonal antibody (MAb) AF1 recognizes an oligosaccharide epitope present on highly immunogenic and immunomodulatory mannoproteins (MP) of Candida albicans. The expression of this epitope (AF1-MP) during experimental candidal vaginitis was studied in two strains of C. albicans (3153 and CA-2) which were equally vaginopathic but differed in the mode of hypha formation in the vagina. In both strains, immunofluorescence of vaginal samples, taken 1 h after challenge, revealed an intense, MAb AF1-specific labelling of the yeast cells. This labelling was very scarce in fungal cells taken at 24 h and on subsequent days during the development of filamentous forms. Electron-microscopic gold immunolabelling observations showed that molecules carrying AF1-MP spanned the entire cell wall in the initial yeast cells but were absent on the cell surface and in the outermost, capsular layer of the cell wall of the germ tubes and filamentous forms. In both strains, at any time and for any form of intravaginal growth, AF1-MP was clearly expressed in the cytoplasm and cytoplasmic vesicles, and was fully incorporated into the inner layers of the cell wall. As seen by immunofluorescence, the vaginal fluid from C. albicans-infected rats did not hinder the expression of AF1-MP on the yeast cells surface in vitro. In electron-microscopic gold immunolabelling, a hypha-specific MAb (3D9) labelled the surface of the hyphal but not of the yeast cells of C. albicans harvested from rat vagina. Overall, these data strongly suggest that cell surface expression of MP antigen is modulated during intravaginal growth and morphogenesis of C. albicans.     

Mikroskopische Untersuchungen zur Wachstumskinetik von Streptomyces hygroscopicus

Growth kinetics, branch formation, and cytological properties of mycelial growth of Streptomyces hygroscopicus on solid media were investigated by phase-contrast microscopy using a microculture method. Measurements were made on taken photographs of the growing hyphae from the beginning of spore germination up to maximal 18 hours. The specific growth rate of the germ tube was much higher than the specific growth rate of the mycelium. The doubling time of the total length of the mycelial hyphae and the doubling time of branch formation was quite the same for the period investigated. After a short time of outgrowth each individual hypha grows at a constant rate, i.e. the length increases linearly, but the growth kinetics of the whole mycelium becomes exponentially by branching. It seems, that nucleoids only divide in that part of a hypha between the tip and the nearest branch. A cell unit (1.4–1.9 μm on different media) could be calculated by the length of a hypha and the number of nucleoids. A hypha is growing with the cell unit at the tip or with the polar cap only. No interkalary growth in length could be found. Branches were formed only up to about 100 μm from the tip on complex medium and up to about 50 μm from the tip on mineral salt medium. A simple model of mycelial growth has been developed. Some properties showing the connection between Streptomycetes and prokaryotic organisms on one hand and hyphal growing fungi on the other hand are discussed.     

Effect of pH, carbon source and K+ on the Na+-inhibited germ tube formation of Candida albicans.

The effect of pH, carbon source and K+ on the Na+ -inhibited germ tube formation of the pathogenic fungus Candida albicans was examined in the arginine-phosphate modified (APM) medium. All C. albicans cells formed germ tubes in APM medium at pH 5.0-9.0. Na+ inhibited germ tube formation in a concentration dependent manner ranging from 0.2 to 1.0 M, and was further influenced by the pH of the medium. The inhibitory effect of Na+ was lowest at pH 8.0, and germ tube formation ceased at 1.0 M Na+ for any pH (4.0-9.0). At pH > or = 6.0, non-germ tube-forming cells did not show yeast growth; whereas at pH < or = 5.0, Na+ inhibited only germ tube formation but did not inhibit yeast growth. The inhibitory effect of Na+ was stronger in glucose medium than in galactose medium as carbon source. K+, at 0-0.8 M, had almost no effect on germ tube formation. However, in the presence of Na+, a very low concentration of K+ (0.5 mM) was able to release the cells from Na+ arrest and produced an increase in the rate as well as the percentage of germ tube formation. Intracellular Na+/K+ ratios increased with the increase in extracellular Na+ concentration, whereas the ratios decreased and remained within nontoxic levels when the extracellular K+ concentration was increased.     

Germ-tube formation by oral strains of Candida tropicalis.

Candida species isolated from the mouths of healthy children and of patients with denture stomatitis included strains of Candida tropicalis that formed germ tubes when incubated in serum. Twenty-six germ-tube-forming strains of C. albicans and of C. tropicalis were subcultured weekly for 9 wk on blood agar and on Sabouraud's agar and the ability of each subculture to form germ tubes was measured. All the strains of C. albicans formed almost as many germ tubes after nine weekly subcultures as they did when first isolated. By contrast, although all 26 strains of C. tropicalis formed germ tubes when first isolated, all had lost the ability to do so after six serial weekly subcultures. Germ-tube formation should not be the sole criterion for the identification of oral C. albicans strains.     

Characterization of antigens specific to the surface of germ tubes of Candida albicans by immunofluorescence

To characterize germ tube-specific antigens of Candida albicans, rabbit antiserum prepared to Formalin-treated yeast possessing germ tubes was adsorbed with stationary-phase blastospores. By immunofluorescence and enzyme-linked immunosorbent assay, this antibody did not react with blastospores but detected germ tube-specific antigens in hyphal forms. Germ tube-specific antigens appeared 30 min after placing blastospores in appropriate conditions for germ tube formation. Hyphae, formed by allowing yeast to germinate for 24 h, still retained germ tube-specific antigens, but blastospores budding off these hyphae were unstained, as were log-phase blastospores. Germ tube-specific antigens were sensitive to heat, sodium metaperiodate oxidation, dithiothreitol reduction, and proteolysis with pronase, trypsin, or chymotrypsin, whereas antigens common to blastospores and germ tubes were stable to boiling, treatment with proteolytic enzymes, and dithiothreitol reduction. Thus, surfaces of germ tubes can be distinguished from those of blastospores not only immunologically, but also by the sensitivity of germ tube-specific antigens to proteolytic treatments.     

In vitro effect of fibrinogen on Candida albicans germ tube formation

Pseudohyphae formation by Candida albicans blastoconidia, as seen in vaginal smears, is a phenotypical change commonly assumed to mean fungal invasiveness, i.e. not mere colonization. C. albicans forms germ tubes in vitro in the presence of serum. In our search for inhibitory components of germ tube formation, we decided to study fibrinogen. The inhibition of germ tube formation by clinical isolates of C. albicans was evaluated in the presence of serial concentrations of fraction I, type IV and fraction I, type Is of fibrinogen from bovine plasma. Fibrinogen showed a dose-dependent, pH-independent inhibitory effect on the germ tube formation by C. albicans.     

Further characterization of human salivary anticandidal activities in a human immunodeficiency virus-positive cohort by use of microassays.

Salivary anticandidal activities play an important role in oral candidal infection. R. P. Santarpia et al. (Oral Microbiol. Immunol. 7:38-43, 1992) developed in vitro anticandidal assays to measure the ability of saliva to inhibit the viability of Candida albicans blastoconidia and the formation of germ tubes by C. albicans. In this report, we describe modifications of these assays for use with small volumes of saliva (50 to 100 microl). For healthy subjects, there is strong inhibition of blastoconidial viability in stimulated parotid (75%), submandibular-sublingual (74%), and whole (97%) saliva, as well as strong inhibition of germ tube formation (>80%) for all three saliva types. The susceptibility of several Candida isolates to inhibition of viability by saliva collected from healthy subjects is independent of body source of Candida isolation (blood, oral cavity, or vagina) or the susceptibility of the isolate to the antifungal drug fluconazole. Salivary anticandidal activities in human immunodeficiency virus (HIV)-infected patients were significantly lower than those in healthy controls for inhibition of blastoconidial viability (P < 0.05) and germ tube formation (P < 0. 001). Stimulated whole-saliva flow rates were also significantly lower (P < 0.05) for HIV-infected patients. These results show that saliva of healthy individuals has anticandidal activity and that this activity is reduced in the saliva of HIV-infected patients. These findings may help explain the greater incidence of oral candidal infections for individuals with AIDS.     

Antigenic differences between mannoproteins of germ tubes and blastospores of Candida albicans.

To determine the nature of germ tube-specific antigens of Candida albicans, procedures for intrinsically labeling cell wall antigens metabolically were developed. Blastospores or germ tubes labeled either in their proteins with L-[35S]methionine or in mannose-containing carbohydrates with D[2-3H]mannose contained surface components similar to those found previously with 125I-labeled organisms. Germ tube-specific determinants, were found on a 200-kilodalton protein in digests from germ tubes, whereas a component of similar molecular size in blastospore extracts reacted weakly or not at all with germ tube-specific antibody. In addition, a glycan fraction prepared from germ tubes reacted with the unadsorbed anti-C. albicans polyvalent antibody but not with the germ tube-specific antibody, suggesting that the germ tube-specific determinants are not carbohydrates.     

Relationships between chemical composition, physical properties and transfection efficiency of polysaccharide-spermine conjugates

Biodegradable water-soluble polysaccharide-spermine (SPM) polycation conjugates for nucleic acid delivery were synthesized by oxidizing polysaccharides using potassium periodate, followed by SPM conjugation. The polycations differ in their polysaccharide type, arabinogalactan (AG) or dextran (D), and/or in the IO(4)- /saccharide mole ratio used for polysaccharide oxidation (1:1, 1:3, or 1:5), resulting in either D(1:1)-SPM, AG(1:1)-SPM, D(1:3)-SPM, AG(1:3)-SPM, or AG(1:5)-SPM. Chemical structure of the conjugates was characterized for total nitrogen and primary amino groups. Surface pH and electrical surface potential were determined by means of spectral changes of covalently attached 7-hydroxycoumarin (HC, a pH- and electrical surface potential-sensitive fluorophore). The binding and the electrostatic neutralization of the polycations by plasmid DNA, as well as the relationship between chemical structure, physical parameters, and transfection of NIH3T3 cells, were also studied. D(1:1)-SPM, the only polycation that showed efficient cell transfection in culture, was shown to have: (1) high SPM content (2000 nmol/mg); (2) high levels of cross-linked SPM (39-51%); (3) at DNA P-/NH3+ ratio of 2.0, a plateau in neutralization of cationic groups (+48 mV, as determined by HC-labeled D(1:1)-SPM titration with DNA), and a drop in zeta-potential from +42 mV for the polymer alone to 0 mV for the polyplex, suggesting that some of the charges are hidden from the DNA; (4) pH(surface) value of 9.2, suggesting that at physiological bulk pH the polymer is only partially ionized, and therefore can act as a "proton sponge" in the endosome; and (5) high sensitivity to serum-rich growth medium. An oleyl derivative, N-oleyl-dextran-spermine (ODS), was synthesized and demonstrated improved transfection efficiency in serum-rich medium.     

IL-1β通过JAK2-STAT3促进大鼠脊髓损伤后胶质瘢痕形成

目的探讨IL-1β促进脊髓损伤后胶质瘢痕形成的机制。方法将大鼠随机分为模型组(采用钳夹脊髓的方法建立SCI模型)、假手术组(sham group)、IL-1β特异性抑制剂组(IL-1RA)、IL-1β组(IL-1β)及IL-1β+JAK2-STAT3特异性抑制剂组(IL-1β+AG490)。假手术组只打开椎板,不作其他处理。在术后相应时间点(术后8及12 h和1、3、7及14 d)进行大鼠后肢BBB评分,用Western blot、免疫荧光和免疫组化技术检测GFAP、vimentin、p-STAT3的表达变化。结果 p-STAT3(术后第8 h和第12 h)及GFAP、vimentin(术后第7和第14天)表达趋势:模型组显著高于假手术组(P0.01),IL-1RA组明显低于模型组(P0.05),但仍高于假手术组(P0.05);IL-1β+AG490组明显低于模型组(P0.05),但仍高于假手术组(P0.05);IL-1β组均显著高于模型组(P0.05)。术后第14天,BBB评分模型组显著低于假手术组(P0.01),IL-1RA组显著高于模型组(P0.05),但仍低于假手术组(P0.01);IL-1β组显著低于模型组(P0.05)。结论 IL-1β可通过JAK2-STAT3促进脊髓损伤后胶质瘢痕形成,抑制IL-1β或JAK2-STAT3可减弱胶质瘢痕形成,促进脊髓神经功能恢复。     

Evidence for two mechanisms of amino acid osmolyte release from hippocampal slices

A 30% decrease in osmolarity stimulated 3H-taurine, 3H-GABA and glutamate (followed as 3H-D-aspartate) efflux from rat hippocampal slices. 3H-taurine efflux was activated rapidly but inactivated slowly. It was decreased markedly by 100 microM 5-nitro-(3-phenylpropylamino)benzoic acid (NPPB) and 600 microM niflumic acid and inhibited strongly by tyrphostins AG18, AG879 and AG112 (25-100 microM), suggesting a tyrosine kinase-mediated mechanism. Hyposmolarity activated the mitogen-activated protein kinases (MAPK) extracellular-signal-related kinase-1/2 (ERK1/ERK2) and p38, but blockade of this reaction did not affect 3H-taurine efflux. Hyposmosis also activated phosphatidylinositide 3-kinase (PI3K) and its prevention by wortmannin (100 nM) essentially abolished 3H-taurine efflux. 3H-taurine efflux was insensitive to the protein kinase C (PKC) blocker chelerythrine (2.5 microM) or to cytochalasin E (3 microM). The release of 3H-GABA and 3H-D-aspartate occurred by a different mechanism, characterized by rapid activation and inactivation, insensitivity to NPPB, niflumic acid, tyrphostins or wortmannin. 3H-GABA and 3H-D-aspartate efflux was not due to external [NaCl] decrease, cytosolic Ca2+ increase or depolarization, or to reverse operation of the carrier. This novel mechanism of amino acid release may be mediated by Ca2+-independent exocytosis and modulated by PKC and actin cytoskeleton disruption, as suggested by its inhibition by chelerythrine and potentiation by 100 nM phorbol-12-myristate-13 acetate (PMA) and cytochalasin E. GABA and glutamate osmosensitive efflux may explain the hyposmolarity-elicited increase in amplitude of inhibitory and excitatory postsynaptic potentials in hippocampal slices as well as the hyperexcitability associated with hyponatraemia.