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1.
The mechanisms used by Paracoccidioides brasiliensis(Pb 18) to survive into monocytes are not clear. Cellular iron metabolism is of critical importance to the growth of several intracellular pathogens, including P. brasiliensis, whose capacity to multiply in mononuclear phagocytes is dependent on the availability of intracellular iron. Chloroquine, by virtue of its basic properties, has been shown to prevent release of iron from holotransferrin by raising endocytic and lysosomal pH, and thereby interfering with normal iron metabolism. Then, in view of this, we have studied the effects of CHLOR on P. brasiliensis multiplication in human monocytes and its effect on the murine paracoccidioidomycosis. CHLOR induced human monocytes to kill P. brasiliensis. The effect of CHLOR was reversed by FeNTA, an iron compound that is soluble at neutral to alkaline pH, but not by holotransferrin, which releases iron only in an acidic environment. CHLOR treatment of Pb 18-infected BALB/c mice significantly reduced the viable fungi recovery from lungs, during three different periods of evaluation, in a dose-dependent manner. This study demonstrates that iron is of critical importance to the survival of P. brasiliensis yeasts within human monocytes and the CHLOR treatment in vitro induces Pb 18 yeast-killing by monocytes by restricting the availability of intracellular iron. Besides, the CHLOR treatment in vivo significantly reduces the number of organisms in the lungs of Pb-infected mice protecting them from several infections. Thus, CHLOR was effective in the treatment of murine paracoccidioidomycosis, suggesting the potential use of this drug in patients' treatment. 相似文献
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Roslia Santos Amorim Jesuino Renata de Bastos Asceno Soares Silvia Maria Salem-Izacc Maristela Pereira Maria Sueli Soares Felipe Clia Maria de Almeida Soares 《Microbiology and immunology》1997,41(2):93-99
The in vitro effects of amphotericin B deoxycholate suspension (fungizone) on Paracoccidioides brasiliensis growth, cell viability and transformation were investigated. We also analyzed the protein synthesis patterns of both cellular forms, yeast and mycelium in the presence of AmB. This drug, at 30 μg/ml, highly inhibited yeast growth, which could be recovered depending on treatment time, where the most effective reversion was observed after 6 hr of incubation. The yeast cell viability, that had been partially affected by the drug, could also be efficiently recovered after AmB was removed. The effect of AmB on the cellular dimorphism process showed a strong reduction in the mycelium to yeast transformation (80% inhibition compared to the control without the drug). On the other hand, the transformation from yeast to mycelium in the presence of AmB was 50% affected, relative to the control. In contrast to the growth and cell viability experiments, the reversion effects on dimorphism were partial when the drug was removed, even with only 6 hr treatment. The two-dimensional gels of 35S-labeled proteins revealed a strong reduction in the three species of 80, 71 and 56 kDa in yeast and mycelium when treated with AmB. 相似文献
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Pereira Crott Luciana Simon Lucisano-Valim Yara Maria Silva Celio Lopes Barbosa José Elpidio 《Mycopathologia》1997,140(1):19-28
The aim of our study was to investigate differences that might exist in the activation of the human complement system by F1
fractions from four different isolates of P. brasiliensis. Isolates HC and 18 (virulent), 265 (low virulence), and 9 (intermediate
virulence, attenuated) were used; before the experiments, the virulence of isolates HC and 18 was recovered by in vivo passage
in guinea pigs. The four isolates of the fungus were processed for purification of F1 fractions and the activation of the
human complement system was studied by a kinetic method of hemolytic activity measurement. The incubation of F1 fractions
in normal human serum resulted in different degrees of inhibition of the classical and alternative pathways. The F1 fraction
from the low virulence isolate was more efficient than the F1 fraction from the virulent isolates (HC and 18). Previous absorption
of sera with F1 fractions completely abolished classical pathway activation. Using zymosan, instead of F1, in the absorption
process caused the same phenomenon, suggesting that natural or nonspecific antibodies are responsible for the classical pathway
activation. The alternative pathway activation did not depend on these antibodies, but was enhanced by their presence. On
the other hand, F1 fractions from virulent isolates were more active in the stimulation of neutrophil chemiluminescence compared
with the F1 fraction from the low virulence isolate. Whole P. brasiliensis yeast cells (WYC) from two distinct strains, 18
and 265, showed the same patterns of response of those observed with the F1 fractions in the functions tested. These differences
in the behavior of the F1 fractions as well as WYC in relation to human complement activation and consequently to neutrophil
stimulation may correlate with the virulence of individual isolates and may contribute to the understanding of the inflammatory
response generation and maintenance processes in paracoccidioidomycosis.
This revised version was published online in June 2006 with corrections to the Cover Date. 相似文献
7.
Sano Ayako Tanaka Reiko Yokoyama Koji Franco Marcello Bagagli Eduardo Montenegro Mario Rubens Mikami Yuzuru Miyaji Makoto Nishimura Kazuko 《Mycopathologia》1998,143(3):165-169
Sixty-three Paracoccidioides brasiliensis isolates obtained from three nine-banded armadillos ( Dasypus novemcinctus), one
Amazonian armadillo's and 19 clinical isolates were compared by random amplified polymorphic DNA analysis with the primer
OPG-19. The isolates were divided into three major clusters, I, II and III. Coincidences between human and armadillo isolates
were observed in clusters I and II. Cluster III consisted only of armadillos' isolates. The results suggested that (I) humans
may acquire P. brasiliensis infection by contact with armadillo's environment, (II) there may be P. brasiliensis genotypes
peculiar to the animal, and (III) individual armadillos may be infected with P. brasiliensis cells with different genotypes.
This revised version was published online in June 2006 with corrections to the Cover Date. 相似文献
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Julhiany de Fátima da Silva Juliana Vicentim Haroldo Cesar de Oliveira Caroline Maria Marcos Patricia Akemi Assato Patrícia Ferrari Andreotti Juliana Leal Monteiro da Silva Christiane Pienna Soares Gil Benard Ana Marisa Fusco Almeida Maria José Soares Mendes-Giannini 《Memórias do Instituto Oswaldo Cruz》2015,110(4):476-484
The fungal strain Paracoccidioides brasiliensis remains viable
inside of epithelial cells and can induce apoptosis in this population. However,
until now, the molecules that participate in this process remained unknown. Thus,
this study evaluated the contribution of two P. brasiliensis
molecules, the 14-3-3 and glycoprotein of 43 kDa proteins, which had been previously
described as extracellular matrix adhesins and apoptosis inductors in human
pneumocytes. Accordingly, epithelial cells were treated with these molecules for
different periods of time and the expression of the apoptosis regulating-proteins
Bak, Bax, Bcl-2, p53 and caspases were evaluated by terminal deoxynucleotidyl
transferase dUTP nick end labelling, flow cytometry and real-time polymerase chain
reaction analysis. Our results demonstrated that treatment with these molecules
induces apoptosis signalling in pulmonary epithelial cells, showing the same pattern
of programmed cell-death as that observed during infection with P.
brasiliensis. Thus, we could conclude that P.
brasiliensis uses these molecules as virulence factors that participate
not only in the fungal adhesion process to host cells, but also in other important
cellular mechanisms such as apoptosis. 相似文献
9.
Detection of gp43 of Paracoccidioides brasiliensis by the loop-mediated isothermal amplification (LAMP) method 总被引:8,自引:0,他引:8
Endo S Komori T Ricci G Sano A Yokoyama K Ohori A Kamei K Franco M Miyaji M Nishimura K 《FEMS microbiology letters》2004,234(1):93-97
Paracoccidioidomycosis is a deep mycosis caused by the thermo-dependent dimorphic fungus Paracoccidioides brasiliensis and is prevalent in Latin American countries. We detected the species specific gp43 gene of P. brasiliensis by loop-mediated isothermal amplification (LAMP) in 22 clinical and seven armadillo-derived isolates. The amplified DNA appeared as a ladder with a specific banding pattern. The advantage of the LAMP method is speed; only 3 h were necessary for identification of the organism and diagnosis of the disease. We were also able to obtain positive results from DNA extracted from a paraffin-embedded tissue sample of paracoccidioidomycosis, suggesting that this method may achieve clinical application in the near future. 相似文献
10.
Nobuyuki Kurita Kiyoshi Terao Elmer Brummer Emiko Ito Kazuko Nishimura Makoto Miyaji 《Mycopathologia》1991,115(3):207-213
The basis for resistance of yeast form of Histoplasma capsulatum to antifungal activity of human neutrophils was studied. In limiting dilution assays and short term coculture assays human neutrophils were ineffective in killing H. capsulatum whereas Candida albicans was readily killed. By contrast, in a cell free hydrogen peroxide-peroxidase-halide system H. capsulatum was as sensitive to killing as C. albicans. Moreover, lysate of human neutrophils effectively substituted for horse-radish peroxidase in a cell free system for killing H. capsulatum. H. capsulatum elicited significant products of the oxidative burst in human neutrophils as detected by luminol-enhanced chemiluminescence. However, the response was two-fold less (p<0.05) than that induced by C. albicans. Transmission electron microscopy studies showed that phagosome-lysosome fusion took place when neutrophils phagocytosed C. albicans or H. capsulatum. Taken together, these findings indicate that, even though H. capsulatum elicits an oxidative burst and phagosome-lysosome fusion within the phagosome, it is capable of evading damage in short term assays.Abbreviations CFU
colony forming units
- PMN
polymorphonuclear neutrophil
- CTCM
complete tissue culture medium
- CL
chemiluminescence
- HPO
horseradish peroxidase
- P-L
lysosomal peroxidase positive material 相似文献
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U. Reineke R. Sabat H. D. Volk J. Schneider-Mergener 《Protein science : a publication of the Protein Society》1998,7(4):951-960
The discontinuous interleukin-10(IL-10)/interleukin-10 receptor (IL-10R) combining site was mapped using sets of overlapping peptides derived from both binding partners bound to continuous cellulose membranes. Low affinity binding of single regions of the discontinuous contact sites on IL-10 and IL-10R could be identified due to (1) high peptide density on the membrane support, (2) incubation with high protein concentrations, (3) indirect immunodetection of the ligates after electrotransfer onto polyvinylene difluoride membranes, and (4) use of highly overlapping peptide scans of different length (6-mers and 15-mers). The single binding regions identified for each protein species are separated in the protein sequences, but form continuous areas on the surface of IL-10 (X-ray structure) and IL-10R (computer model). Furthermore, four epitopes of neutralizing anti-IL-10 and anti-IL-10R antibodies were mapped and overlap with these binding regions. Soluble peptides (15- to 19-mers) each spanning one of the three identified IL-10-derived receptor binding regions displayed no significant affinity to IL-10R as expected, whereas a peptide (35-mer) comprising two of these regions had considerably higher binding activity. The data are consistent with a previously published computer model of the IL-10/IL-10R complex. This approach should be generally applicable for the mapping of non-linear protein-protein contact sites. 相似文献
12.
de Carvalho MJ Amorim Jesuino RS Daher BS Silva-Pereira I de Freitas SM Soares CM Felipe MS 《Fungal genetics and biology : FG & B》2003,39(3):204-210
Calmodulin (CaM) modulates intracellular calcium signalling and acts on several metabolic pathways and gene expression regulation in many eukaryotic organisms including human fungal pathogens, such as Candida albicans and Histoplasma capsulatum. The temperature-dependent dimorphic fungus Paracoccidioides brasiliensis is the aetiological agent of paracoccidioidomycosis (PCM). The mycelium (M) to yeast (Y) transition has been shown to be essential for establishment of the infection, although the precise molecular mechanisms of dimorphism in P. brasiliensis are still unknown. In this work, several inhibitory drugs of the Ca(2+)/calmodulin signalling pathway were tested to verify the role of this pathway in the cellular differentiation process of P. brasiliensis. EGTA and the drugs calmidazolium (R24571), trifluoperazine (TFP), and W7 were able to inhibit the M-Y transition. We have cloned and characterized the calmodulin gene from P. brasiliensis, which comprises 924 nucleotides and five introns that are in a conserved position among calmodulin genes. 相似文献
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Nadya da Silva Castro Kelly Pacheco de Castro Ivan Orlandi Luciano dos Santos Feitosa Lívia Kmetzsch Rosa e Silva Marilene Henning Vainstein Sônia Nair Báo Marina Vai & Célia Maria de Almeida Soares 《FEMS yeast research》2009,9(1):103-114
The fungus Paracoccidioides brasiliensis causes paracoccidioidomycosis, a systemic granulomatous mycosis prevalent in Latin America. In an effort to elucidate the molecular mechanisms involved in fungus cell wall assembly and morphogenesis, β-1,3-glucanosyltransferase 3 ( Pb Gel3p) is presented here. Pb Gel3p presented functional similarity to the glucan-elongating/glycophospholipid-anchored surface/pH-regulated /essential for pseudohyphal development protein families, which are involved in fungal cell wall biosynthesis and morphogenesis. The full-length cDNA and gene were obtained. Southern blot and in silico analysis suggested that there is one copy of the gene in P. brasiliensis . The recombinant Pb Gel3p was overexpressed in Escherichia coli , and a polyclonal antibody was obtained. The PbGEL3 mRNA, as well as the protein, was detected at the highest level in the mycelium phase. The protein was immunolocalized at the surface in both the mycelium and the yeast phases. We addressed the potential role of Pb Gel3p in cell wall biosynthesis and morphogenesis by assessing its ability to rescue the phenotype of the Saccharomyces cerevisiae gas1 Δ mutant. The results indicated that Pb Gel3p is a cell wall-associated protein that probably works as a β-1,3-glucan elongase capable of mediating fungal cell wall integrity. 相似文献
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白细胞介素—10对血管一氧化氮/一氧化氮合酶系统的影响 总被引:4,自引:1,他引:4
为探讨抗炎因子--白细胞介素-10(IL-10)对大鼠主动脉一氧化氮(NO)/一氧化氮合酶(NOS)系统的影响,应用Griess试剂、^3H-瓜氨酸生成及蛋白免疫印迹杂交等方法,测定IL-10孵育对血管NO释放、NOS活性及表达的影响。结果发现细菌脂多糖(LPS)呈浓度领带性地激活诱导型NOS(iNOS),促进NO生成。IL-10(10^-10-10^-8g/ml)呈浓度依赖性地上调内皮型NOS(eNOS)蛋白表达及其活性,但对iNOS活性及表达无明显影响,IL-10(10^-9-10^-8g/ml)显著抑制10μg/ml LPS诱导的NO生成和iNOS激活;而高浓度IL-10(10^-7g/ml)则上调iNOS的活性,对eNOS蛋白的表达知活性无明显影响。因此IL-10对NO/NOS系统具有双重影响,一方面可抑制炎症介质诱发的作为炎性物质的iNOS的表达及激活,另一方面可上调内皮源扩血管物质NO的释放。 相似文献
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Ayako Sano Makoto Miyaji Kazuko Nishimura Marcello Fabiano de Franco 《Mycopathologia》1991,114(2):93-101
We performed comparative studies of the pathogenicity of six strains of Paracoccidioides brasiliensis (Bt-9, Bt-4, Pb-9, Pb-18, Bt-7 and B-1183) for young adult male ddY mice and the growth rate of each strain under different oxygen atmospheres (aerobic, micro-aerobic and anaerobic atmospheres) at 37 °C. 106 units of yeast cells were intravenously injected into each mouse. The pathogenicity of each isolate was determined by a scoring system based on organ culture and histopathological findings. The growth rates under different oxygen atmospheres were determined by a scoring system in which 300 fungal units per strain were counted. The strain Bt-9 showed the greatest pathogenicity, followed by Bt-4. Pb-9 and Pb-18 had on intermediate rank of pathogenicity. Bt-7 and B-1183 were the least pathogenic of the strains tested. Except for strain Bt-7 all strains showed an excellent growth under an aerobic atmosphere. Bt-4 and Bt-9 also showed excellent growth under a micro-aerobic atmosphere, followed by Pb-9, whereas the growth of Pb-18, Bt-7 and B-1183 was limited. There was a correlation between the growth rate under a micro-aerobic atmosphere and the pathogenicity of a strain. The growth rate of P. brasiliensis under a micro-aerobic atmosphere strongly correlated to its pathogenicity. 相似文献
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用凝胶阻滞分析的方法, 发现鼠T淋巴细胞系CTLL-2在白细胞介素-2(IL-2)刺激下可活化一个DNA结合因子, 它与γ-干扰素活化序列(GAS)专一性结合, 命名这个DNA结合因子为白细胞介素-2活化核因子(IL-2-NAF).IL-2-NAF的活化非常迅速, 不需要新的蛋白质合成, 并且它的活化程度随着IL-2刺激细胞的时间的不同而发生相应的变化. 进一步研究表明, IL-2-NAF的活化过程是通过酪氨酸激酶的信号传递途径, 并且它本身的酪氨酸残基也被磷酸化, 酪氨酸残基的磷酸化为其结合DNA所必需. IL-4、γ-IFN刺激CTLL-2细胞不活化与GAS专一性结合的因子. 而在Hut-102细胞中, IL-2、IL-4均可活化GAS结合因子, 但活化程度较弱. 相似文献
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Thomas K. Hughes Patrick Cadet Peter L. Rady Stephen K. Tyring Robert Chin Eric M. Smith 《Cellular and molecular neurobiology》1994,14(1):59-69
Summary 1. Interleukin-10 (IL-10) has a wide range of activities in the immune system such as modulation of interferon-gamma (IFN-) and antibody production. The neuropeptide hormone corticotropin (ACTH) has similar activities, suggesting that a bidirectional communication mechanism operates between the immune and the neuroendocrine system involving these two substances.2. Murine pituitary tumor cells (AtT-20) were found to produce up to 3 ng/ml of IL-10.3. Pituitary cell corticotropin production was enhanced by IL-10 treatment.4. IL-10 induced the production of ACTH in mouse splenocytes.5. Authenticity of pituitary-derived IL-10 was shown by the demonstration of identical neucleic acid sequences of reverse-transcribed, polymerase chain reaction amplified fragments of cDNA obtained from murine splenocytes, a murine pituitary tumor cell line, and freshly isolated murine pituitaries. 相似文献
19.
对急性分离的人中性粒细胞采用4℃预处理是进行膜片钳实验前经常采取的步骤,但这一步骤对电生理记录结果有何影响尚无文献报道。本实验探讨这一步骤对电生理记录过程和实验结果的影响。结果显示,4℃预处理可以显著提高细胞的封接率,有利于对中性粒细胞进行电生理记录;封接率提高的原因与4℃预处理降低细胞的极性活动有关,但记录到的电压依赖性钾通道全细胞电流和大电导Ca^2+依赖性K^+单通道电流动力学没有显著的变化。这些结果表明,4℃预处理可能影响到细胞膜上与极性有关的脂膜变化,但对细胞膜上蛋白的功能影响较少。 相似文献
20.
Darawan Rinchai Donporn Riyapa Surachat Buddhisa Kusumawadee Utispan Richard W Titball Mark P Stevens Joanne M Stevens Michinaga Ogawa Isei Tanida Masato Koike Yasuo Uchiyama Manabu Ato Ganjana Lertmemongkolchai 《Autophagy》2015,11(5):748-755
Neutrophils play a key role in the control of Burkholderia pseudomallei, the pathogen that causes melioidosis. Here, we show that survival of intracellular B. pseudomallei was significantly increased in the presence of 3-methyladenine or lysosomal cathepsin inhibitors. The LC3-flux was increased in B. pseudomallei-infected neutrophils. Concordant with this result, confocal microscopy analyses using anti-LC3 antibodies revealed that B. pseudomallei-containing phagosomes partially overlapped with LC3-positive signal at 3 and 6 h postinfection. Electron microscopic analyses of B. pseudomallei-infected neutrophils at 3 h revealed B. pseudomallei-containing phagosomes that occasionally fused with phagophores or autophagosomes. Following infection with a B. pseudomallei mutant lacking the Burkholderia secretion apparatus Bsa Type III secretion system, neither this characteristic structure nor bacterial escape into the cytosol were observed. These findings indicate that human neutrophils are able to recruit autophagic machinery adjacent to B. pseudomallei-containing phagosomes in a Type III secretion system-dependent manner. 相似文献