Paracocci

Am J Trop Med Hyg, 2004 Dec, 71(6), 828 - 30
SHORT REPORT: INHIBITION BY TUMOR NECROSIS FACTOR-{alpha}-ACTIVATED MACROPHAGES OF THE TRANSITION OF PARACOCCIDIOIDES BRASILIENSIS CONIDIA TO YEAST CELLS THROUGH A MECHANISM INDEPENDENT OF NITRIC OXIDE; Gonzalez A et al.; It is known that peritoneal murine macrophages activated with interferon-gamma exert a fungicidal effect against Paracoccidioides brasiliensis conidia by a nitric oxide (NO)-mediated mechanism . This NO-mediated effect can also be induced by other cytokines such as tumor necrosis factor-alpha (TNF-alpha) . The aim of this study was to determine if TNF-alpha-activated peritoneal murine macrophages infected with P . brasiliensis were able to show fungistatic/fungicidal effects mediated by NO . The results indicated that although macrophage activation with TNF-alpha did not result in NO production, these cells played an important role in inhibiting the conidia from becoming yeast cells . In vivo, the NO-independent inhibitory effect would prove of importance for the establishment of P . brasiliensis in host tissues.

J Clin Microbiol, 2005 Jan, 43(1), 491 - 3
Variable gp43 secretion by Paracoccidioides brasiliensis clones obtained by two different culture methods; Berzaghi R et al.; The main objectives of this study were to obtain clones of Paracoccidioides brasiliensis by two methods (micromanipulation and plating assay) and to determine if the secretion of the 43-kDa glycoprotein (gp43) is dependent on the clonal culture . The results show that the secretion of gp43 is not dependent on clonal cultures . Clones that originally were secretors of this molecule, after subculturing, lost this characteristic; on the other hand, clones that originally did not secrete gp43 began to secrete gp43 after subculturing.

Surg Neurol, 2005 Jan, 63(Suppl 1), S13 - 21
Central nervous system paracoccidioidomycosis: diagnosis and treatment; Elias J Jr et al.; BACKGROUND: Paracoccidioidomycosis (PCM) is a systemic mycosis caused by Paracoccidioides brasiliensis . The involvement of the central nervous system (CNS) in paracoccidioidomycosis is higher than previously thought and 2 clinical presentations have been reported, meningitis and pseudotumoral . METHODS: Twenty medical records of patients with CNS paracoccidioidomycosis treated from 1986 to 2003 were analyzed . The follow-up ranged from 1 to 18 years (mean = 8.9 +/- 4.2) . RESULTS: Besides CNS paracoccidioidomycosis, all patients but one had the chronic systemic form and the pseudotumoral clinical presentation was the most frequent . Based on computed tomography scan findings, 4 image patterns were identified: low-density lesion with ring enhancement, lesion with calcification and ring enhancement, multiloculated low-density lesion with ring enhancement, and diffuse subarachnoid enhancement . The magnetic resonance imaging was performed in 3 patients and showed subarachnoid enhancement in 1 patient and heterogeneous lesion with ring enhancement in 2 patients . Eleven patients were submitted to medical treatment and 9 needed neurosurgical treatment; ventriculoperitoneal shunts in 4 patients, brain lesions resection in 3 patients, and partial resection of spinal cord lesions in 2 patients . Eleven patients had excellent outcome, 4 patients died, 3 are in good clinical condition with residual pulmonary dysfunction, and 1 patient was lost to follow-up . CONCLUSIONS: The diagnosis of paracoccidioidomycosis with involvement of the CNS is difficult and clinical suspicion is a key point to achieve the correct diagnosis . Patients with early diagnosis have a favorable outcome with clinical or surgical treatment.

Clin Infect Dis, 2005 Jan 1, 40(1), e1 - 4 Epub 2004 Dec 06.
Contribution to the natural history of paracoccidioidomycosis: identification of the primary pulmonary infection in the severe acute form of the disease--a case report; Benard G et al.; Several aspects of the pathogenesis of paracoccidioidomycosis (PCM) have not yet been fully clarified . We describe a patient with an overwhelmingly acute form of PCM who presented with clinically apparent pulmonary infection that spontaneously subsided while yeast cells disseminated systemically . This case may help to explain the paradox of the absence of pulmonary involvement in the acute disseminated form of PCM.

Eur J Neurol, 2005 Jan, 12(1), 73 - 4
Focal status epilepticus as the first manifestation of paracoccidioidomycosis; Franca MC Jr et al.; We report a 56-year old man with prolonged focal motor status epilepticus as the first clinical manifestation of paracoccidioidomycosis (PCM) and discuss this unusual presentation . We emphasize the need for a comprehensive work-up and increased awareness for central nervous system involvement in PCM, particularly in endemic areas.

Eur J Clin Microbiol Infect Dis, 2004 Dec, 23(12), 916 - 9
Chronic paracoccidioidomycosis in a female patient in Austria; Mayr A et al.; Presented here is a case of chronic paracoccidioidomycosis that occurred in a Cuban female living in Austria and was first misdiagnosed as tuberculosis . The clinical picture was one of progressive pulmonary insufficiency with fever, weight loss and productive cough . Since antituberculous therapy was started but did not achieve a long-term clinical response, an intensive diagnostic work-up was performed . Paracoccidioides brasiliensis was then diagnosed by histopathology, serology, microbiology and molecular identification . Antifungal therapy was commenced immediately with amphotericin B (1 mg/kg/day) for 10 days, followed by voriconazole (200 mg/day po) for at least 3 months, and the lesions disappeared almost completely . This report presents the first published case of imported paracoccidioidomycosis in a female patient in Austria.

Clin Immunol, 2005 Jan, 114(1), 86 - 94
The role of interleukin-10 in the differential expression of interleukin-12p70 and its beta2 receptor on patients with active or treated paracoccidioidomycosis and healthy infected subjects; Romano CC et al.; Paracoccidioidomycosis patients present an antigen-specific Th1 immunosuppression . To better understand this phenomenon, we evaluated the interleukin (IL)-12 pathway by measuring IL-12p70 production and CD3(+) T cell expression of the IL-12 receptor (IL-12R)beta1/beta2 chains, induced with the main fungus antigen (gp43) and a control antigen, from Candida albicans (CMA) . We showed that gp43-induced IL-12p70 production and IL-12Rbeta2 expression were significantly decreased in acute and chronic patients as compared to healthy subjects cured from PCM or healthy infected subjects from endemic areas . Interestingly, the healthy infected subjects had higher gp43-induced IL-12p70 production and beta2 expression than the cured subjects . The addition of a neutralizing anti-IL-10 antibody to the cultures increased IL-12p70 levels and beta2 expression in acute and chronic patients to levels observed in cured subjects . Conversely, addition of the cytokine IL-10 strongly inhibited both parameters in the latter group . In conclusion, we have shown that paracoccidioidomycosis-related Th1 immunosuppression is associated with down-modulation of the IL-12 pathway, that IL-10 may participate in this process, and that patients cured from paracoccidioidomycosis may not fully recover their immune responsiveness.

Fungal Genet Biol, 2005 Jan, 42(1), 51 - 60
Paracoccidioides brasiliensis presents two different cDNAs encoding homologues of the fructose 1,6-biphosphate aldolase: protein isolation, cloning of the cDNAs and genes, structural, phylogenetic, and expression analysis; Carneiro LC et al.; A proteomic approach was used to identify a 39kDa antigen of Paracoccidioides brasiliensis . Amino acid sequences of the N-terminal and of endoproteinase Lys-C digested peptides revealed the protein to be a fructose 1,6-biphosphate aldolase (FBA) Class II of P . brasiliensis . Two cDNA homologues, Pbfba1 and Pbfba2, were cloned and characterized . Pbfba1 encoded a predicted polypeptide of 360 amino acids that was highly homologous in the primary structure to the same enzyme from fungi and bacteria . The other DNA, Pbfba2, encoded a polypeptide predicted to be 363 amino acids . The sequence of Pbfba2 differed significantly from Pbfba1 . Phylogenetic and molecular analysis supports the concept of gene duplication for FBAs in P . brasiliensis, constituting a two-member family . Expression analysis demonstrated differential expression for both fbas genes in P . brasiliensis cells.

Biochemistry, 2004 Nov 23, 43(46), 14696 - 703
Sulfide dehydrogenase activity of the monomeric flavoprotein SoxF of Paracoccus pantotrophus; Quentmeier A et al.; Flavocytochrome c-sulfide dehydrogenases (FCSDs) are complexes of a flavoprotein with a c-type cytochrome performing hydrogen sulfide-dependent cytochrome c reduction in vitro . The amino acid sequence analysis revealed that the phylogenetic relationship of different flavoproteins reflected the relationship of sulfur-oxidizing bacteria . The flavoprotein SoxF of Paracoccus pantotrophus is 29-67% identical to the flavoprotein subunit of FCSD of phototrophic sulfur-oxidizing bacteria . Purification of SoxF yielded a homogeneous emerald-green monomeric protein of 42 797 Da . SoxF catalyzed sulfide-dependent horse heart cytochrome c reduction at the optimum pH of 6.0 with a k(cat) of 3.9 s(-1), a K(m) of 2.3 microM for sulfide, and a K(m) of 116 microM for cytochrome c, as determined by nonlinear regression analysis . The yield of 1.9 mol of cytochrome c reduced per mole of sulfide suggests sulfur or polysulfide as the product . Sulfide dehydrogenase activity of SoxF was inhibited by sulfur (K(i) = 1.3 microM) and inactivated by sulfite . Cyanide (1 mM) inhibited SoxF activity at pH 6.0 by 25% and at pH 8.0 by 92% . Redox titrations in the infrared spectral range from 1800 to 1200 cm(-1) and in the visible spectral range from 400 to 700 nm both yielded a midpoint potential for SoxF of -555 +/- 10 mV versus Ag/AgCl at pH 7.5 and -440 +/- 20 mV versus Ag/AgCl at pH 6.0 (-232 mV versus SHE') and a transfer of 1.9 electrons . Electrochemically induced FTIR difference spectra of SoxF as compared to those of free flavin in solution suggested a strong cofactor interaction with the apoprotein . Furthermore, an activation/variation of SoxF during the redox cycles is observed . This is the first report of a monomeric flavoprotein with sulfide dehydrogenase activity.

Biochemistry, 2004 Nov 23, 43(46), 14566 - 76
A copper protein and a cytochrome bind at the same site on bacterial cytochrome c peroxidase; Pauleta SR et al.; Pseudoazurin binds at a single site on cytochrome c peroxidase from Paracoccus pantotrophus with a K(d) of 16.4 microM at 25 degrees C, pH 6.0, in an endothermic reaction that is driven by a large entropy change . Sedimentation velocity experiments confirmed the presence of a single site, although results at higher pseudoazurin concentrations are complicated by the dimerization of the protein . Microcalorimetry, ultracentrifugation, and (1)H NMR spectroscopy studies in which cytochrome c550, pseudoazurin, and cytochrome c peroxidase were all present could be modeled using a competitive binding algorithm . Molecular docking simulation of the binding of pseudoazurin to the peroxidase in combination with the chemical shift perturbation pattern for pseudoazurin in the presence of the peroxidase revealed a group of solutions that were situated close to the electron-transferring heme with Cu-Fe distances of about 14 A . This is consistent with the results of (1)H NMR spectroscopy, which showed that pseudoazurin binds closely enough to the electron-transferring heme of the peroxidase to perturb its set of heme methyl resonances . We conclude that cytochrome c550 and pseudoazurin bind at the same site on the cytochrome c peroxidase and that the pair of electrons required to restore the enzyme to its active state after turnover are delivered one-by-one to the electron-transferring heme.

Scand J Immunol, 2004 Nov, 60(5), 500 - 5
Modulation of CD28 and CD86 expression in patients with paracoccidioidomycosis in different periods of treatment; Bozzi A et al.; Paracoccidioides brasiliensis, a thermo-dimorphic fungus, is the ethiologic agent of paracoccidioidomycosis (PCM) . The recidive is the greatest obstacle of this disease, because the yeast usually returns after the long treatment period . In the present work, we have investigated the cellular immune response of cells from peripheral blood drawn from patients with different duration of PCM . The classification of patients ranged from nontreated to those with long-standing disease over 5 years . Unstimulated as well as cells stimulated with phytohemaglutinin or two different antigen preparations, secreted (MEXO) or somatic (PbAg) of P . brasiliensis, were characterized . We found that cells from patients with disease proliferate considerably upon stimulation with the antigen preparations and that cells from patients with disease of long duration does not proliferate that vigorously as from patients with more recent diagnosis . Both interferon (IFN)-gamma and interleukin (IL)-4 appear to be increased in patients, but IFN-gamma tended to increase upon treatment while IL-4-secretion decreased . With respect to CD28 and CD86, we found that the subset of CD28 positive CD8 cells are decreased in all stages of the disease as compared to control individuals . A subset of CD86 positive CD19 cells appeared to be considerably increased compared to the controls . Indeed, our results demonstrated that the treatment of PCM patients promoted a regulation of IFN-gamma, IL-4 levels and CD28, CD86 expression bringing new insight to the cellular immune response in PCM.

J Clin Microbiol, 2004 Nov, 42(11), 5007 - 14
Detection and selection of microsatellites in the genome of Paracoccidioides brasiliensis as molecular markers for clinical and epidemiological studies; Nascimento E et al.; Paracoccidioides brasiliensis, a thermodimorphic fungus, is the causative agent of the prevalent systemic mycosis in Latin America, paracoccidioidomycosis (PCM) . Here, we describe the microsatellite patterns observed in a collection of P . brasiliensis random sequence tags . We identified 1,117 microsatellite patterns in about 3.8 Mb of unique sequences (0.47% of the total DNA used in the analysis) . The majority of these microsatellites (87.5%) are found in noncoding sequences . We used two polymorphic microsatellites located on noncoding and coding sequences, as well as two microsatellites located on introns, as molecular markers to discriminate P . brasiliensis isolates, to look for relationships between the genetic background of the strains and the types of human disease they cause . We did not observe any correlation between the clinical form of human PCM and four simple sequence repeat patterns analyzed.

Rev Inst Med Trop Sao Paulo, 2004 Sep-Oct, 46(5), 279 - 81 Epub 2004 Oct 22.
Paracoccidioidomycosis in organ transplant recipient: case report; Zavascki AP et al.; Paracoccidioidomycosis is a common disease in Latin America but it is rare in organ transplant recipient patients . We report on a case of such mycosis in a renal transplant recipient . The patient presented with a large lung cavity on the left lower lobe, a rare radiological presentation of paracoccidioidomycosis . Unusual clinical and radiological manifestations of Paracoccidioides brasiliensis infection can occur in immunocompromised patients.

Mycopathologia, 2004 Jul, 158(1), 57 - 9
Paracoccidioidomycosis in a woman with idiopathic hirsutism; dos Santos RP et al.; Paracoccidioidomycosis, especially the chronic pulmonary form of the disease, is not commonly described in females . Data from in vitro and vivo studies support the hypothesis that estrogens might influence the pathogenesis of paracoccidioidomycosis in humans by inhibition of transition of conidia or mycelia to yeast form of Paracoccidioides brasiliensis . The authors describe a chronic progressive pulmonary form of paracoccidioidomycosis in a woman with idiopathic hirsutism . In addition to estrogens, the present report suggests that other hormonal factors might play an important role in the pathogenesis of paracoccidioidomycosis, including the increased production of 5alpha-dehydrotestosterone frequently described in individuals with idiopathic hirsutism.

Rev Iberoam Micol, 1997 Mar, 14(1), 14 - 22
{Oral mycoses in the AIDS era.}; Delgado W et al.; The present advancement and generalitation of oral mycosis is probably due to different factors associated to the progress and development of the countries and the presence of medically compromised patients and immunosupressor therapies . From all these mycoses, oral candidosis is the commonest, but histoplasmosis and paracoccidioidomycosis are also important . The main clinicopatho-logic and therapeutic features of those oral diseases are reviewed in this paper.

Ultrasound Q, 2004 Mar, 20(1), 12 - 8
Ultrasound of tropical and infectious diseases that affect the scrotum; de Cassio Saito O et al.; Ultrasonography of the scrotum permits assessment of testicular and extratesticular masses with high sensitivity . It can differentiate a variety of conditions involving the scrotum, testicles, and epididymis with similar clinical manifestations, including infectious and tropical diseases . The authors performed conventional and color Doppler ultrasonographic examinations in 76 patients who presented with scrotal pain, swelling, and/or tenderness . Their diagnoses included sexually transmitted disease (eg, gonorrhea, syphilis, chlamydial infection), tuberculosis, mumps, and various tropical diseases (eg, filariasis, leishmaniasis, schistosomiasis, paracoccidioidomycosis) . The most common imaging findings were enlarged hypoechoic testes, hypervascularity, small hydroceles, and cutaneous edema . This report reviews these and other possible presentations of tropical and infectious diseases affecting the scrotum, emphasizing ultrasound findings that facilitate diagnosis.

Med Mycol, 2004 Aug, 42(4), 379 - 83
Canine paracoccidioidomycosis; Ricci G et al.; Paracoccidioidomycosis (PCM) is a severe disease caused by the dimorphic fungus Paracoccidioides brasiliensis, which is characterized by granulomatous pulmonary and systemic lesions, affecting mainly men between 20 and 60 years of age . Reports of PCM disease in animals are rare, but the disease has been described in armadillos . On the other hand, PCM infection of domestic and wild animals detected by serological or cutaneous tests in the absence of apparent disease has been frequently reported . We present here the case of a female adult Doberman that developed cervical lymphadenomegaly . Histopathological examination of a cervical biopsy specimen revealed active PCM, with an epithelioid, granulomatous inflammation containing numerous yeast-like, multiple budding fungal forms . The diagnosis of PCM was confirmed by immunohistochemistry using a specific antibody anti-gp43 and by nested PCR using primers for the amplification of the gp43 gene region . This is the first report of PCM disease occurring in a dog, an animal that has been shown to play an important role in the natural history of North American blastomycosis.

Med Mycol, 2004 Aug, 42(4), 363 - 8
IgM and IgG antibody response to Paracoccidioides brasiliensis in naturally infected wild armadillos (Dasypus novemcinctus); Fernandes GF et al.; We studied the extent to which wild nine-banded armadillos, Dasypus novemcinctus, produce immune humoral responses specifically directed against characteristic Paracoccidioides brasiliensis antigens . Such antibody production might reflect direct contact with the ecological microniche of P . brasiliensis, or might merely reflect inhalation of widely distributed airborne propagules . An enzyme-linked immunosorbent assay (ELISA) was designed containing purified glycoprotein gp43 and gp70 antigens from P . brasiliensis as well as cross-reactive antisera originally targeted against human IgM (mu chain) and armadillo anti-IgG (gamma-chain) . It was used to detect and classify IgM and IgG antibodies to P . brasiliensis in the armadillo . In a serological survey of 47 wild armadillos, IgM antibodies to gp43 were detected in seven animals (14.8%), and IgG antibodies were detected in 20 (42.5%) . IgM antibodies to gp70 were detected in 10 (21.3%) animals and IgG antibodies were detected in 18 (38.3%) . These results, showing a pattern consistent with infection, suggest that P . brasiliensis is enzootic in armadillos . How the animals became exposed could not be determined.

Med Mycol, 2004 Aug, 42(4), 349 - 53
Separation of Paracoccidioides brasiliensis conidia through percoll gradients; del P Jimenez M et al.; The conidia of Paracoccidioides brasiliensis are the structures most likely to serve as the infectious propagules of this fungus . This study describes our attempts to purify conidia by eliminating mycelial fragments . Purification was attempted using discontinuous 95% and 60% Percoll gradients with densities of 1.167 and 1.107, respectively, prepared either in 0.15 mol/L PBS or 0.25 mol/L sucrose . The best results were observed with the 95% and 90% gradients in sucrose; with the former, conidial purity ranged from 70.6 to 100%, with a mean of 82.3% and a coefficient of variation (VC) of 11.7 . With 90% gradients, purity was achieved between 70.4 and 92.5% . The mean in this case was 80.6% and the VC was 9.2% . The use of two consecutive 95% Percoll gradients in sucrose was tested . The recovery efficiency per plate, which averaged 2.5 x 10(6) conidia per plate with one gradient, increased to 5.1 +/- 1.3 x 10(6) conidia with two gradients . The use of Percoll did not affect the viability of the conidia, which was always > or = 90% . This method allows the preparation of a conidial sample almost free from contamination with mycelial fragments, thus facilitating quantitative determination of cause and effect in in-vivo interactions between P brasiliensis and its hosts.

J Clin Microbiol, 2004 Oct, 42(10), 4480 - 6
Detection of Paracoccidioides brasiliensis gp70 circulating antigen and follow-up of patients undergoing antimycotic therapy; da Silva SH et al.; Paracoccidioidomycosis (PCM), one of the most important systemic mycoses in Central and South America, is caused by the dimorphic fungus Paracoccidioides brasiliensis and has a high prevalence in Brazil . Glycoproteins of 43 and 70 kDa are the main antigenic compounds of P . brasiliensis and are recognized by Western blotting by 100 and 96% of PCM patient sera, respectively . In the present study, an inhibition enzyme-linked immunosorbent assay (ELISA) was used to detect gp70 in different biological samples from patients with PCM . gp70 was detected in 98.76% of 81 serum samples, with an average concentration of 8.19 microg/ml . The test was positive for 100% of the patients with the acute and chronic unifocal forms of PCM and 98.43% of the patients with the multifocal chronic form, with average concentrations of 11.86, 4.83, and 7.87 microg/ml, respectively . Bronchoalveolar lavage fluid from 23 patients with pulmonary unifocal PCM and 14 samples of cerebrospinal fluid from patients with neurological PCM were also tested for gp70 detection, with the test showing 100% sensitivity and 100% specificity, with mean gp70 concentrations of 7.5 and 6.78 microg/ml, respectively . To investigate the potential of gp70 detection by inhibition ELISA for the follow-up of PCM patients during antimycotic therapy with itraconazole (ITZ), the sera of 23 patients presenting with the chronic multifocal form of PCM were monitored at regular intervals of 1 month for 12 months . The results showed a decrease in circulating gp70 levels during treatment which paralleled the reduction in anti-P . brasiliensis antibody levels . The detection of P . brasiliensis gp70 from the biological fluids of patients suspected of having PCM proved to be a promising method for diagnosing infection and evaluating the efficacy of ITZ treatment.

Rev Argent Microbiol, 2004 Apr-Jun, 36(2), 68 - 74
{Immunodiagnosis of endemic mycoses and bronchopulmonary aspergillosis: a multicenter study in Argentina}; Canteros CE et al.; In order to contribute to the knowledge of the relative frequency of chronic fungal diseases and assess the performance of diagnostic laboratories in Argentina, a multicenter study was performed with the participation of 25 medical centers located in 12 different provinces and Buenos Aires City . Between 04-01-2000 and 03-30-2001, 965 serum specimens from patients clinically suspected of having histoplasmosis (HP), paracoccidioidomycosis (PCM), coccidioidomycosis (CM) or aspergilosis were analyzed . Agar immunodiffusion tests (IDD) were done locally . All positive and 35% of negative sera were retested in the reference center . Results of laboratories of origin showed 98.8% concordance with those of reference center . Antibodies against any of the etiological agents were detected in 120 specimens from 98 patients . Endemic mycoses (HP, PCM and CM) were diagnosed in 70 patients (71.4%) and aspergilosis in 28 (28.6%) . The frequencies of the different mycoses in decreasing order were PCM 47 patients (47.9%), aspergilosis 28 patients (28.6%), HP 13 patients (13.3%) and CM 10 patients (10.2%) . The study was carried out on a voluntary basis and some areas of the country were not represented . However, the frequencies were in range with the expected rates in the population under study.

Invest Clin, 2004 Sep, 45(3), 277 - 88
Central nervous system paracoccidioidomycosis: case report and review; Tristano AG et al.; Paracoccidioidomycosis is a systemic infection caused by a dimorphic fungus (Paracoccidioides brasiliensis) . The most common lesions frequently occur in the bucopharinx mucosa . Other lesions occur in the adrenal glands, liver, bone, gastrointestinal tract, lungs and nervous system . We report here a case of neuroparacoccidioidomycosis . The patient was a 49 year-old male, who consulted due to neurological symptoms (cephalalgia, speech difficulty and one tonic clonic seizure with urinary incontinence) of eight months duration . Upon physical examination it was observed an emaciated male with nail clubbing, a skin ulcer with raised edges and a crusted bottom of 4 x 2 cm in diameter located in the right supraclavicular region and an ulcerated lesion in the left tonsil with edema . The rest of the physical examination reveled a discrete left side hemiparesis and pulmonary rales in the left hemitorax . The fungus was identified through direct examination of cerebrospinal fluid (CSF) . The histopathology of suprarenal, lungs, brain and skin showed multiple paracoccidioidal granulomas . To the best of our knowledge, this is the third case reported in the literature . We review the literature on the pathogenesis and prevalence of neuroparacoccidioidomycosis.

J Antimicrob Chemother, 2004 Nov, 54(5), 956 - 8 Epub 2004 Sep 24.
Therapeutic activity of a killer peptide against experimental paracoccidioidomycosis; Travassos LR et al.; OBJECTIVES: To evaluate whether an engineered synthetic decapeptide (KP) derived from the sequence of a recombinant anti-idiotypic antibody, that represents the internal image of a Pichia anomala killer toxin, could be fungicidal in vitro and therapeutic in vivo against Paracoccidioides brasiliensis and paracoccidioidomycosis (PCM) . METHODS: Fungicidal activity of KP was assessed in vitro and in vivo by inhibition of colony forming units and by histological examination, 8 days after infection, of organs from mice intravenously injected with a virulent strain of P . brasiliensis (3 x 10(6) yeast cells) and intraperitoneally treated with KP (3.3 mug/g body weight, three doses), in comparison with control animals equally administered with a scrambled decapeptide (SP) . RESULTS: KP but not SP was fungicidal in vitro at 39 ng/multiply-budding yeast cell and less efficiently in its D-isomeric form (0.31 mug/multiply-budding yeast cell) . It was also able to markedly reduce the fungal load in organs (liver, lung, spleen) of infected animals . CONCLUSIONS: The therapeutic effect observed opens the way for using the antifungal peptide as an alternative control of PCM in association with conventional antifungal drugs.

Curr Microbiol, 2004 Sep, 49(3), 145 - 51
A novel exopolymer-producing bacterium, Paracoccus zeaxanthinifaciens subsp . payriae, isolated from a "kopara" mat located in Rangiroa, an atoll of French Polynesia; Raguenes G et al.; An aerobic, mesophilic and heterotrophic marine bacterium designated RA19, able to produce two different exocellular polymers and zeaxanthin, was isolated from a French polynesian bacterial mat (localy named "kopara") situated in the atoll of Rangiroa . This microorganism, on the basis of its phenotypical features and the genotypic investigations, can be clearly assigned to the Parococcus zeaxanthinifaciens species and the name Parococcus zeaxanthinifaciens subsp . payriae is proposed . Optimal growth occurs between 30 degrees C and 35 degrees C, at pH between 6.5 and 7.5 and at ionic strength between 20 and 40 g/L of NaCl . The guanine-plus-cytosine content of DNA was 65.6% . This bacterium excreted, under laboratory conditions, two different polymers: a water-soluble exopolysaccharide (EPSI) consisting of 5 different sugars and a non-water-soluble macromolecule assumed to be of a glycoproteinic nature . The high sulfate content of the EPS1 and preliminary biological tests clearly showed that applications could be found in the very near future for both polymers in the cosmetic area . Their contribution to the viscous laminated microbial mat locally called "kopara" can be also mentioned.

Acta Dermatovenerol Croat, 2004, 12(3), 173 - 80
Facial and oral aspects of some venereal and tropical diseases; Ramos-E-Silva M; Diseases of the tropical areas include some venereal diseases, and they are still very prevalent in some countries; Brazil is one of them . Very few cases are originated in large cities, as Rio de Janeiro, but at the University Hospital of the Federal University of Rio de Janeiro we also see those patients who come from the interior of the State of Rio de Janeiro or from other states to seek medical care at better equipped hospitals for this type of investigation and therapy . Venereal and tropical dermatoses have many different cutaneous manifestations and may affect skin in several locations . The face is one of the affected areas especially when the disease has a predilection for cartilage, oral and/or nasal mucosa . Alterations observed on the skin of the face and on the mucosa of the mouth of some tropical diseases, such as leprosy, leishmaniasis, paracoccidioidomycosis, donovanosis, and syphilis, as they are observed in Brazil, are presented and discussed in this article.

Biochemistry, 2004 Sep 7, 43(35), 11214 - 25
Paracoccus pantotrophus pseudoazurin is an electron donor to cytochrome c peroxidase; Pauleta SR et al.; The gene for pseudoazurin was isolated from Paracoccus pantotrophus LMD 52.44 and expressed in a heterologous system with a yield of 54.3 mg of pure protein per liter of culture . The gene and protein were shown to be identical to those from P . pantotrophus LMD 82.5 . The extinction coefficient of the protein was re-evaluated and was found to be 3.00 mM(-1) cm(-1) at 590 nm . It was confirmed that the oxidized protein is in a weak monomer/dimer equilibrium that is ionic-strength-dependent . The pseudoazurin was shown to be a highly active electron donor to cytochrome c peroxidase, and activity showed an ionic strength dependence consistent with an electrostatic interaction . The pseudoazurin has a very large dipole moment, the vector of which is positioned at the putative electron-transfer site, His81, and is conserved in this position across a wide range of blue copper proteins . Binding of the peroxidase to pseudoazurin causes perturbation of a set of NMR resonances associated with residues on the His81 face, including a ring of lysine residues . These lysines are associated with acidic residues just back from the rim, the resonances of which are also affected by binding to the peroxidase . We propose that these acidic residues moderate the electrostatic influence of the lysines and so ensure that specific charge interactions do not form across the interface with the peroxidase.

J Neuroimaging, 2004 Oct, 14(4), 377 - 9
Proton magnetic resonance spectroscopy and magnetic resonance imaging findings in a patient with central nervous system paracoccidioidomycosis; Faria AV et al.; The authors describe a patient with brain paracoccidioidomycosis whose magnetic resonance image (MRI) showed multiple hypointense lesions on T2-weighted images with peripheral enhancement after gadolinium injection . Single-voxel proton magnetic resonance spectroscopy (1H-MRS) of one of the lesions showed 2 peaks at 0.9 and 1.32 ppm, corresponding to lipid signals, indicating intense necrosis . The other characteristic peaks of 1H-MRS were undifferentiated from baseline . These findings, although not specific, may help to differentiate fungal abscess from tumoral lesions and other types of abscess.

Nihon Kokyuki Gakkai Zasshi, 2004 Jul, 42(7), 629 - 33
{A case of chronic pulmonary paracoccidioidomycosis}; Igarashi T et al.; In a 43-year-old Japanese Brazilian who came to Japan in 2001, since subjective symptoms such as cough, sputum, and dyspnea on exertion had become severe, he was referred to our hospital because of suspicion of pulmonary tuberculosis in chest radiography and CT findings . A chest radiograph of initial examination showed interstitial shadows in both lungs with nodular, infiltrative or cavitary changes . No Mycobacterium tuberculosis was found . The mycetocyte with multipolar budding resembling the steerage of a ship, which was characteristic of Paracoccidioides was observed in sputum and transbronchial lung biopsy specimens . We cultured a fungus to show dimorphism of temperature dependency, and a diagnosis of chronic lung paracoccidioidomycosis was arrived at . By administration of ITCZ 200 mg/day, the chest radiography findings and clinical manifestations were improved . This case seems to be worthy of reporting in Japan since the affected site or organ was limited to the two lungs with multiple cavitary lesions and fibrotic changes on radiographic examination, and final diagnosis was made by cytology of sputum and pathology of TBLB specimens.

Arkh Patol, 2003 Mar-Apr, 65(2), 47 - 9
{Mycotic lesions in the brain in chronic opium-ephedrone addiction}; Bogomolov DV et al.; Histological examination of the brain of a opium-ephedrone addict has detected large spherical cells resembling Coccidioides or Paracoccidioides spherules . Roundish and oval structures were found in subependymal space of the fourth ventricle . These structures may be spores of many fungi . Areas of normal structure and these with productive inflammation were observed in the surrounding nervous tissue.

Microbes Infect, 2004 Aug, 6(10), 892 - 900
Proteomic identification, nucleotide sequence, heterologous expression and immunological reactivity of the triosephosphate isomerase of Paracoccidioides brasiliensis; Pereira LA et al.; An antigen of Paracoccidioides brasiliensis (Pb) was gel isolated and characterized . Endoproteinase Lys-C-digested peptides of the purified protein, which presented a molecular mass of 29 kDa and pI of 5.8, were subjected to sequence analysis of their amino acids . Searches at databases comparing the sequence of amino acids from the three peptides of the native protein revealed strong homology to triosephosphate isomerase (TPI: E.C . 5.3.1.1) from several sources . The complete cDNA and gene encoding PbTPI were obtained and both contained an open reading frame predicted to encode a 249-amino acid protein that presented all the peptides characterized in the native PbTPI . The Pbtpi gene contained six exons interrupted by five introns . Analysis performed with the deduced PbTPI suggested its usefulness in providing phylogenetic relatedness, as well as evidencing the correlation between the phylogeny provided by the deduced protein and intron positions in the cognate genes . The immunological reactivity of PbTPI was examined . The complete coding cDNA of PbTPI was overexpressed in an Escherichia coli host to produce high levels of recombinant fusion protein with glutathione S-transferase (GST) that had been purified by affinity chromatography . The purified recombinant TPI was recognized by sera of patients with confirmed paracoccidioidomycosis and not by sera of healthy individuals . Thus, recombinant PbTPI can be a valuable addition to the still small arsenal of P . brasiliensis immunoreactive proteins, which could be tested for incorporation into assays for serodiagnosis of the disease.

Microbes Infect, 2004 Aug, 6(10), 882 - 91
Invasion of epithelial mammalian cells by Paracoccidioides brasiliensis leads to cytoskeletal rearrangement and apoptosis of the host cell; Mendes-Giannini MJ et al.; Paracoccidioides brasiliensis (Pb) yeast cells can enter mammalian cells and probably manipulate the host cell environment to favor their own growth and survival . We studied the uptake of strain Pb 18 into A549 lung and Vero epithelial cells, with an emphasis on the repercussions in the cytoskeleton and the apoptosis of host cells . Cytoskeleton components of the host cells, such as actin and tubulin, were involved in the P . brasiliensis invasion process . Cytochalasin D and colchicine treatment substantially reduced invasion, indicating the functional participation of microfilaments (MFs) and microtubules (MTs) in this mechanism . Cytokeratin could also play a role in the P . brasiliensis interaction with the host . Gp43 was recognized by anti-actin and anti-cytokeratin antibodies, but not by anti-tubulin . The apoptosis induced by this fungus in infected epithelial cells was demonstrated by various techniques: TUNEL, DNA fragmentation and Bak and Bcl-2 immunocytochemical expression . DNA fragmentation was observed in infected cells but not in uninfected ones, by both TUNEL and gel electrophoresis methods . Moreover, Bcl-2 and Bak did not show any differences until 24 h after infection of cells, suggesting a competitive mechanism that allows persistence of infection . Overexpression of Bak was observed after 48 h, indicating the loss of competition between death and survival signals . In conclusion, the mechanisms of invasion of host cells, persistence within them, and the subsequent induction of apoptosis of such cells may explain the efficient dissemination of P . brasiliensis.

Microsc Microanal, 2004 Apr, 10(2), 215 - 23
A morphological and cytochemical study of the interaction between Paracoccidiodes brasiliensis and neutrophils; Dias MF et al.; Paracoccidioidomycosis is a systemic granulomatous disease caused by the dimorphic fungus Paracoccidioides brasiliensis . It is the most prevalent systemic mycosis of Latin America and 80% of the reported cases are from Brazil . Because of the great number of neutrophils found in the P . brasiliensis granuloma, studies have been done to evaluate the role of these cells during the development of the infection . Scanning and transmission electron microscopy of thin sections showed that the neutrophils ingest yeast cells through a typical phagocytic process with the formation of pseudopodes . The pseudopodes even disrupt the connection established between the mother and the bud cells . Neutrophils also associate to each other, forming a kind of extracellular vacuole where large yeast cells are encapsulated . Cytochemical studies showed that once P . brasiliensis attaches to the neutrophil surface, it triggers a respiratory burst with release of oxygen-derived products . Attachment also triggers neutrophils degranulation, with release of endogenous peroxidase localized in cytoplasmic granules . Together, these processes lead to killing of both ingested and extracellular P . brasiliensis.

Rev Inst Med Trop Sao Paulo, 2004 May-Jun, 46(3), 127 - 31 Epub 2004 Jul 20.
Paracoccidioidomycosis in children: clinical presentation, follow-up and outcome; Pereira RM et al.; From February, 1981 to May, 2001, 63 children under 15 y old (ages 2 - 15 y, median = 8 y, mean +/- 1 SD = 8 +/- 3 y) presenting 70 episodes of Paracoccidioidomycosis were admitted . The main clinical manifestations and laboratory features observed upon admission were: lymph node enlargement (87.1%), fever (75.7%), weakness (48.6%), pallor (41.4%), hepatomegaly (40%), splenomegaly (35.7%), anemia (90%), hypergammaglobulinemia (88.5%), eosinophilia (75.5%) and hypoalbuminemia (72.5%) . Moderate to severe malnutrition was detected in 35.7% of the episodes (Gomez's criterion) . Radiographic and technetium studies showed bone lesions in 20 of the episodes, most of them being multiple lytic lesions, involving both long (70%) and plain bones (30%) . First line treatment consisted of an association of sulfametoxazole-trimethoprin, which was used, exclusively, in 50 episodes . Follow-up of hemoglobin levels, number of eosinophils in the peripheral blood, albumin and gammaglobulin serum levels revealed significant sequential improvement one and six months after hospital admission, being quite useful to evaluate treatment effectiveness . Six patients died (9.3%) and four developed sequelae (6.3%) . In conclusion, the juvenile and disseminated forms can be observed in about 70% of the episodes of PCM occurring in children younger than 15 y old, most of them presenting with a febrile lymphoproliferative syndrome associated to anemia, eosinophilia and hypergammaglobulinemia.

Can J Microbiol, 2004 Jun, 50(6), 445 - 9
Evaluation of the in vitro and in vivo dimorphism of Sporothrix schenckii, Blastomyces dermatitidis, and Paracoccidioides brasiliensis isolates after preservation in mineral oil; Lima RF et al.; Morphological differentiation has commanded attention for its putative impact on the pathogenesis of invasive fungal infections . We evaluated in vitro and in vivo the dimorphism from mycelial to yeast-phase of Sporothrix schenckii, Blastomyces dermatitidis and Paracoccidioides brasiliensis isolates, two strains for each species, preserved in mineral oil . S . schenckii strains showed typical micromorphology at 25 degrees C but one strain was unable to complete the dimorphic process in vitro . After in vivo passage through mice the strains had the ability to turn into yeast-like cells and to form colonies on brain-heart infusion medium at 36 degrees C . B . dermatitidis strains grew as dirty white to brownish membranous colonies at 25 degrees C and their micromorphology showed thin filaments with single hyaline conidia . At 36 degrees C the colonies did not differ from those grown at 25 degrees C, but produced a transitional micromorphology . P . brasiliensis strains grew as cream-colored cerebriform colonies at 25 degrees C showing a transitional morphology . B . dermatitidis and P . brasiliensis strains did not turn into yeast-like cells in vivo . The present results demonstrate that B . dermatitidis and P . brasiliensis strains were unable to complete the dimorphic process even after in vivo passage, in contrast to the S . schenckii strain.

Med Mycol, 2004 Jun, 42(3), 247 - 53
Molecular cloning and characterization of a cDNA encoding the N-acetyl-beta-D-glucosaminidase homologue of Paracoccidioides brasiliensis; Santos MO et al.; A cDNA encoding the N-acetyl-beta-D-glucosaminidase (NAG) protein of Paracoccidioides brasiliensis, Pb NAG1, was cloned and characterized . The 2663-nucleotide sequence of the cDNA consisted of a single open reading frame encoding a protein with a predicted molecular mass of 64.73 kDa and an isoeletric point of 6.35 . The predicted protein includes a putative 30-amino-acid signal peptide . The protein as a whole shares considerable sequence similarity with 'classic' NAG . The primary sequence of Pb NAG1 was used to infer phylogenetic relationships . The amino acid sequence of Pb NAG1 has 45, 31 and 30% identity, respectively, with homologous sequences from Trichoderma harzianum, Aspergillus nidulans and Candida albicans . In particular, striking homology was observed with the active site regions of the glycosyl hydrolase group of proteins (family 20) . The expected active site consensus motif G X D E and catalytic Asp and Glu residues at positions 373 and 374 were found, reinforcing that Pb NAG1 belongs to glycosyl hydrolase family 20 . The nucleotide sequence of Pb nag1 and its flanking regions have been deposited, along with the amino acid sequence of the deduced protein, in GenBank under accession number AF419158.

Med Mycol, 2004 Jun, 42(3), 217 - 21
Molecular cloning and characterization of a cDNA encoding the Paracoccidioides brasiliensis 135 ribosomal protein; Jesuino RS et al.; A 630 bp cDNA encoding an L35 ribosomal protein of Paracoccidioides brasiliensis, designated as Pbl35, was cloned from a yeast expression library . Pbl35 encodes a polypeptide of 125 amino acids, with a predicted molecular mass of 14.5 kDa and a pI of 11.0 . The deduced PbL35 shows significant conservation in respect to other described ribosomal L35 proteins from eukaryotes and prokaryotes . Motifs of ribosomal proteins are present in PbL35, including a bipartite nuclear localization signal (NLS) that could be related to the protein addressing to the nucleolus for the ribosomal assembly . The mRNA for PbL35, about 700 nucleotides in length, is expressed at a high level in P . brasiliensis . The PbL35 and the deduced amino acid sequence constitute the first description of a ribosomal protein in P . brasiliensis . The cDNA was deposited in GenBank under accession number AF416509.

Fungal Genet Biol, 2004 Jul, 41(7), 667 - 75
The glyceraldehyde-3-phosphate dehydrogenase homologue is differentially regulated in phases of Paracoccidioides brasiliensis: molecular and phylogenetic analysis; Barbosa MS et al.; Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) plays important roles in various cellular processes . Here we report the sequence and analysis of a novel developmentally regulated gene and cDNA (Pbgadph), encoding a GAPDH homologue (PbGAPDH), of the pathogenic dimorphic fungus Paracoccidioides brasiliensis . We have analyzed the protein, the cDNA and genomic sequences to provide insights into the structure, function, and potential regulation of PbGAPDH . That Pbgapdh encodes PbGAPDH was demonstrated by micro-sequencing of the native protein homologue isolated from the fungus proteome . The deduced amino acid sequence of Pbgapdh showed identity to those of from other species (88-76%) . Phylogenetic analysis indicated that GAPDH could be useful for the determination of evolutionary relationships . Expression of the Pbgapdh gene and the cognate protein were developmentally regulated in phases of P . brasiliensis, with a higher expression in the yeast parasitic phase and was induced during the transition from mycelium to yeast and decreased during the reverse process, transition from yeast to mycelium.

Dalton Trans, 2004 Apr 7, (7), 996 - 1002 Epub 2004 Mar 05.
Characterisation of {Cu4S}, the catalytic site in nitrous oxide reductase, by EPR spectroscopy; Oganesyan VS et al.; The enzyme nitrous oxide reductase (N(2)OR) has a unique tetranuclear copper centre {Cu(4)S}, called Cu(Z), at the catalytic site for the two-electron reduction of N(2)O to N(2) . The X- and Q-band EPR spectra have been recorded from two forms of the catalytic site of the enzyme N(2)OR from Paracoccus pantotrophus, namely, a form prepared anaerobically, Cu(Z), that undergoes a one-electron redox cycle and Cu(Z)*, prepared aerobically, which cannot be redox cycled . The spectra of both species are axial with that of Cu(Z) showing a rich hyperfine splitting in the g||-region at X-band . DFT calculations were performed to gain insight into the electronic configuration and ground-state properties of Cu(Z) and to calculate EPR parameters . The results for the oxidation state {Cu(+1)(3)Cu(+2)(1)S}(3+) are in good agreement with values obtained from the fitting of experimental spectra, confirming the absolute oxidation state of Cu(Z) . The unpaired spin density in this configuration is delocalised over four copper ions, thus, Cu(I) 20.1%, Cu(II) 9.5%, Cu(III) 4.8% and Cu(IV) 9.2%, the mu(4)-sulfide ion and oxygen ligand . The three copper ions carrying the highest spin density plus the sulfide ion lie approximately in the same plane while the fourth copper ion is perpendicular to this plane and carries only 4.8% spin density . It is suggested that the atoms in this plane represent the catalytic core of Cu(Z), allowing electron redistribution within the plane during interaction with the substrate, N(2)O.

J Clin Microbiol, 2004 Jul, 42(7), 3366 - 8
First comprehensively documented case of Paracoccus yeei infection in a human; Funke G et al.; Paracoccus yeei was isolated in pure culture from an aerobic blood culture and bulla fluid from a 67-year-old male . The biochemical identification scheme for this recently described species is outlined . Because of its reaction pattern it is not unlikely that P . yeei is underdiagnosed.

Am J Trop Med Hyg, 2004 Jul, 71(1), 82 - 86
PARACOCCIDIOIDOMYCOSIS: AN EPIDEMIOLOGIC SURVEY IN A PEDIATRIC POPULATION FROM THE BRAZILIAN AMAZON USING SKIN TESTS; Kalmar EM et al.; Since Paracoccidioides brasiliensis and Histoplasma capsulatum are known to be present in similar environments, there have been many epidemiologic investigations regarding the prevalences of these two organisms . However, cross-reactivity can occur in paracoccidioidin and histoplasmin skin tests, and this usually results in the overestimation of the prevalence of P . brasiliensis.The prevalence of infection with P . brasiliensis was evaluated in a cross-sectional study of 298 asymptomatic school children in the Brazilian Amazon region (Mato Grosso State) . In this investigation, the reactivity of children to two different P . brasiliensis antigen preparations, paracoccidioidin and a purified 43-kD glycoprotein (gp43), was compared with or without the co-administration of histoplasmin . In the group of individuals receiving paracoccidioidin who had a positive histoplasmin skin test result, the prevalence of exposure to P . brasiliensis was 44% (16 of 36) . This reactivity to P . brasiliensis was significantly higher than that observed in other groups, which ranged from 4% to 6% (P < 5 x 10(-4) for each) . Overall prevalence was 4.6% (95% confidence interval = 2.5-7.7%) . These data suggest that gp43 provides a better estimate of exposure to P . brasiliensis when the co-administration of histoplasmin is desired.

Immunol Lett, 2004 Jun 15, 94(1-2), 107 - 14
Down-regulation of dendritic cell activation induced by Paracoccidioides brasiliensis; Ferreira KS et al.; Paracoccidioidomycosis (PCM) endemic in Latin America, is a progressive systemic mycosis caused by Paracoccidioides brasiliensis (Pb) . The infection can evolve to different clinical forms that are associated to various degrees of suppressed cell-mediated immunity . With the recognition that DCs are able to initiate response in naive T cells and that they also participate in Th cell education, the present study was undertaken to check whether DCs interact with P . brasiliensis, as well as to elucidate possible mechanisms and consequences of this interaction . Our results indicate that P . brasiliensis infection and purified gp43, its main antigenic component, lead to down-regulation of MHC-II and adhesion properties of immature DCs . The down-regulation was also observed in LPS-induced DC maturation . In addition, an inhibition of IL-12 and TNF-alpha production by both P . brasileinsis or gp43, was observed in LPS-induced DC maturation . These results suggest that protein, released in great amounts by the fungus, might be used, to reduce the effectiveness of the immune response.

Infect Immun, 2004 Jul, 72(7), 3932 - 40
Dual role of interleukin-4 (IL-4) in pulmonary paracoccidioidomycosis: endogenous IL-4 can induce protection or exacerbation of disease depending on the host genetic pattern; Arruda C et al.; Resistance to paracoccidioidomycosis, the most important endemic mycosis in Latin America, is thought to be primarily mediated by cellular immunity and the production of gamma interferon . To assess the role of interleukin-4 (IL-4), a Th2 cytokine, pulmonary paracoccidioidomycosis in IL-4-depleted susceptible (B10.A) and intermediate (C57BL/6) mice was studied . Two different protocols were used to neutralize endogenous IL-4 in B10.A mice: 1 mg of anti-IL-4 monoclonal antibody (MAb)/week and 8 mg 1 day before intratracheal infection with 10(6) Paracoccidioides brasiliensis yeast cells . Unexpectedly, both protocols enhanced pulmonary infection but did not alter the levels of pulmonary cytokines and specific antibodies . Since in a previous work it was verified that C57BL/6 mice genetically deficient in IL-4 were more resistant to P . brasiliensis infection, we also investigated the effect of IL-4 depletion in this mouse strain . Treatment with the MAb at 1 mg/week led to less severe pulmonary disease associated with impaired synthesis of Th2 cytokines in the lungs and liver of control C57BL/6 mice . Conversely, in IL-4-depleted C57BL/6 mice, increased levels of tumor necrosis factor alpha and IL-12 were found in the lungs and liver, respectively . In addition, higher levels of immunoglobulin G2a (IgG2a) and lower levels of IgG1 antibodies were produced by IL-4-depleted mice than by control mice . Lung pathologic findings were equivalent in IL-4-depleted and untreated B10.A mice . In IL-4-depleted C57BL/6 mice, however, smaller and well-organized granulomas replaced the more extensive lesions that developed in untreated mice . These results clearly showed that IL-4 can have a protective or a disease-promoting effect in pulmonary paracoccidioidomycosis depending on the genetic background of the host.

Mol Med, 2003 Sep-Dec, 9(9-12), 209 - 19
In silico prediction of peptides binding to multiple HLA-DR molecules accurately identifies immunodominant epitopes from gp43 of Paracoccidioides brasiliensis frequently recognized in primary peripheral blood mononuclear cell responses from sensitized individuals; Iwai LK et al.; One of the major drawbacks limiting the use of synthetic peptide vaccines in genetically distinct populations is the fact that different epitopes are recognized by T cells from individuals displaying distinct major histocompatibility complex molecules . Immunization of mice with peptide (181-195) from the immunodominant 43 kDa glycoprotein of Paracoccidioides brasiliensis (gp43), the causative agent of Paracoccidioidomycosis (PCM), conferred protection against infectious challenge by the fungus . To identify immunodominant and potentially protective human T-cell epitopes in gp43, we used the TEPITOPE algorithm to select peptide sequences that would most likely bind multiple HLA-DR molecules and tested their recognition by T cells from sensitized individuals . The 5 most promiscuous peptides were selected from the gp43 sequence and the actual promiscuity of HLA binding was assessed by direct binding assays to 9 prevalent HLA-DR molecules . Synthetic peptides were tested in proliferation assays with peripheral blood mononuclear cells (PBMC) from PCM patients after chemotherapy and healthy controls . PBMC from 14 of 19 patients recognized at least one of the promiscuous peptides, whereas none of the healthy controls recognized the gp43 promiscuous peptides . Peptide gp43(180-194) was recognized by 53% of patients, whereas the other promiscuous gp43 peptides were recognized by 32% to 47% of patients . The frequency of peptide binding and peptide recognition correlated with the promiscuity of HLA-DR binding, as determined by TEPITOPE analysis . In silico prediction of promiscuous epitopes led to the identification of naturally immunodominant epitopes recognized by PBMC from a significant proportion of a genetically heterogeneous patient population exposed to P . brasiliensis . The combination of several such epitopes may increase the frequency of positive responses and allow the immunization of genetically distinct populations.

Clin Exp Immunol, 2004 Jul, 137(1), 123 - 8
B and T cell responses elicited by monoclonal anti-idiotypic antibody (Ab2beta) mimicking gp43 from Paracoccidioides brasiliensis; Souza EB et al.; Paracoccidioidomycosis is a systemic mycosis endemic in Latin America, with a high prevalence in Brazil, Argentina, Colombia and Venezuela . The aetiological agent of disease is the thermal dimorphic fungus, Paracoccidioides brasiliensis . A glycoprotein of 43 kD (gp43) is the major antigen of P . brasiliensis . Antibodies directed to this antigen are detected in the sera of all patients with paracoccidioidomycosis (PCM) . Recently, it has been shown that mice immunized with anti-gp43 monoclonal antibodies (MAbs) (Ab1), induce the idiotypic cascade in the gp43 system, which produced both, anti-Id antibodies (Ab2) and anti-anti-Id antibodies (Ab3) . To further characterize the idiotypic cascade modulation in mice immunized with anti-gp43 MAb 17c, hybridomas were produced . Ab2 MAbs named 7.B12 inhibited (>95%) the binding of gp43 to MAb 17c (Ab1), suggesting that this anti-Id MAb bind to the idiotope, thus fulfilling the internal image criteria . To elucidate whether Ab2 MAb could act as antigen in serological assays, instead of gp43, sera from PCM patients were tested . Using an ELISA test, it was observed that antibodies from patients and not normal serum bound to Ab2 . However, the ELISA test using Ab2 bound to the solid phase made possible to serologically monitor the patients after antifungal therapy, showing an equivalent curve when compared with ELISA test employing purified gp43 . Our results also showed that, when mice were immunized with Ab2beta and their cells were exposed to gp43 in vitro, a T cell proliferation response was observed.

J Clin Microbiol, 2004 Jun, 42(6), 2419 - 24
Monitoring gp43 antigenemia in Paracoccidioidomycosis patients during therapy; Marques da Silva SH et al.; Paracoccidioidomycosis (PCM) is a systemic fungal disease that is particularly important among individuals living and working in rural areas of endemicity in Latin America . Detection of anti-Paracoccidioides brasiliensis antibodies is of limited value due to false-negative results . Detection of P . brasiliensis-gp43 circulating antigen is a practical approach for a specific diagnosis of the disease . In a previous study we described an inhibition enzyme-linked immunosorbent assay able to detect the 43-kDa P . brasiliensis antigen in sera of 100% of patients with the acute form of PCM and in 95.31 and 100% of patients with the chronic multifocal and unifocal forms of PCM . To investigate its potential application for the follow-up of PCM patients during treatment, antigen levels were monitored at regular intervals for up 8 to 12 months in serum samples from 23 patients . The results showed that treatment with itraconazole resulted in decreasing levels of circulating gp43 that were correlated with the reduction of anti-gp43 antibodies . It was also observed that by the end of 12 months of treatment gp43 levels were <5 microg/ml in all patients.

J Clin Microbiol, 2004 Jun, 42(6), 2411 - 8
Development of a species-specific probe for Pythium insidiosum and the diagnosis of pythiosis; Schurko AM et al.; Pythium insidiosum, the only species in the genus that infects mammals, is the etiological agent of pythiosis, a granulomatous disease characterized by cutaneous and subcutaneous lesions and vascular diseases . Accurate diagnosis of pythiosis and identification of its causal agent are often inconsistent with current immunological diagnostic methods . A species-specific DNA probe was constructed by using a 530-bp HinfI fragment from the ribosomal DNA intergenic spacer of P . insidiosum . When the probe was incubated with dot blots of genomic DNA from 104 Pythium species, it hybridized only to the DNA of P . insidiosum and P . destruens-two species that have been considered conspecific . The probe also hybridized to DNA from 22 P . insidiosum isolates in this study, regardless of their geographic origin or animal host . When tested against genomic DNA from other pathogenic organisms (Aspergillus fumigatus, Basidiobolus ranarum, Conidiobolus coronatus, Lagenidium giganteum, Paracoccidioides brasiliensis, and Prototheca wickerhamii), no cross-hybridization of the probe was detected . The specificity of the probe to hybridize to genomic DNA from all isolates of P . insidiosum and not cross-react with DNA from other Pythium species or pathogens that cause symptoms similar to pythiosis in their hosts makes it a powerful tool for the accurate diagnosis of pythiosis . In addition, the probe has the potential for pathological and environmental diagnostic applications.

Curr Microbiol, 2004 Jun, 48(6), 452 - 8
Phylogenetically diverse new sulfur chemolithotrophs of alpha-proteobacteria isolated from Indian soils; Deb C et al.; Five facultative sulfur chemolithotrophs were isolated from soils to study the diversity of sulfur lithotrophy . Phenotypic characteristics, including sulfur lithotrophic properties and chemotaxonomic features of the isolates, were similar to those of the members of the colorless sulfur bacteria . 16S rDNA sequence analyses rendered placing the isolates to three distinct phylogenetic clusters of alpha-proteobacteria . Three isolates, AS001, AS002, and KCT002, were identified as members of the genus Paracoccus . The strains AS001 and AS002, having identical 16S-rDNA sequence, showed significant 16S rDNA sequence similarity (99.1%) to Paracoccus versutus . The strain KCT002 showed highest (98%) 16S rDNA sequence similarity to P . alcaliphilus and 96% similarity to the pair AS001 and AS002 . Isolate KCT001 appeared to be closely related to Pseudaminobacter salicylatoxidans, although sulfur lithotrophy of P . salicylotoxidans is not known . The other isolate, TCK, showed almost identical 16S rDNA (99.9%) sequence with two recently described unclassified chemolithoautotrophic arsenite oxidizing strains . Physiological and chemotaxonomic characteristics and phylogenetic analyses of the five new strains emphasize the need of polyphasic bacterial taxonomy of sulfur lithotrophs.

Ophthal Plast Reconstr Surg, 2004 May, 20(3), 212 - 6
Eyelid involvement in paracoccidioidomycosis; Cruz AA et al.; PURPOSE: To describe a series of patients with eyelid lesions caused by paracoccidioidomycosis and to estimate the prevalence of eyelid involvement in this disease METHODS: The medical records of 439 patients with paracoccidioidomycosis admitted to our hospital from 1992 to 2002 were reviewed . Age, sex, and clinical forms of the disease were recorded . All patients with eyelid involvement had a skin biopsy positive for paracoccidioidomycosis and were examined by an ophthalmologist with oculoplastic training . RESULTS: Of 439 patients with acute, subacute, or chronic paracoccidioidomycosis, 11 (2.5%) had eyelid involvement . Active lesions ranged from erythematous patches of madarosis to frank destructive ulcers indistinguishable from malignancies . Healed lesions were characterized by a high degree of fibrosis . Cicatricial changes induced eyelid malpositions (entropion or ectropion) and fusion of eyelid tissues to the globe . Madarosis was a constant finding in the inactive lesions . CONCLUSIONS: The prevalence of eyelid involvement in paracoccidioidomycosis is low . Isolated active lesions are usually diagnosed as malignant tumors . Cicatricial changes are characterized by a high degree of fibrosis . If not treated, the mycosis can destroy the eyelid.

Microbes Infect, 2004 May, 6(6), 549 - 58
Binding of laminin to Paracoccidioides brasiliensis induces a less severe pulmonary paracoccidioidomycosis caused by virulent and low-virulence isolates; Andre DC et al.; The pathogenic fungus Paracoccidioides brasiliensis is the causative agent of paracoccidioidomycosis (PCM) . This pulmonary mycosis, acquired by inhalation of airborne propagules, may disseminate to several internal organs and tissues, leading to severe disease . Adhesion to host cell components is the first step involved in dissemination of pathogens . Previous studies showed that laminin, the most abundant glycoprotein of the basement membrane, binds to P . brasiliensis yeast cells, enhancing their pathogenicity in the hamster testicle model . As PCM is primarily a pulmonary infection, we studied the influence of previous treatment of yeast cells with laminin on the course of the intratracheal infection of resistant and susceptible mice using high-virulence (Pb18) and low-virulence (Pb265) P . brasiliensis isolates . Laminin treatment did not alter fungal loads, delayed-type hypersensitivity reactions, levels of pulmonary cytokines and production of specific antibodies in any group of Pb18-infected mice . However, early in the infection, a less intense inflammatory reaction was detected in the lungs of the laminin-treated groups . In addition, laminin treatment of Pb265 resulted in a less severe infection as revealed by the lower fungal loads recovered from lungs . Antibody and cytokine levels, however, did not change after laminin treatment . Altogether, our results demonstrate that laminin binding to yeast cells diminishes P . brasiliensis pathogenicity . The lower inflammatory response observed with the virulent isolate and the decreased pulmonary fungal burden with the low-virulence isolate indicate an inhibitory effect of laminin treatment on P . brasiliensis infectivity and interaction with pulmonary host cells or extracellular matrix proteins.

Mol Genet Genomics, 2004 Jul, 271(6), 667 - 77 Epub 2004 May 08.
Identification of genes preferentially expressed in the pathogenic yeast phase of Paracoccidioides brasiliensis, using suppression subtraction hybridization and differential macroarray analysis; Marques ER et al.; Paracoccidioides brasiliensis, a thermodimorphic fungus, is the causative agent of paracoccidioidomycosis (PCM), the most prevalent systemic mycosis in Latin America . Pathogenicity appears to be intimately related to the dimorphic transition from the hyphal to the yeast form, which is induced by a shift from environmental temperature to the temperature of the mammalian host . Little information is available on the P . brasiliensis genes that are necessary during the pathogenic phase . We have therefore undertaken Suppression Subtraction Hybridization (SSH) and macroarray analyses with the aim of identifying genes that are preferentially expressed in the yeast phase . Genes identified by both procedures as being more highly expressed in the yeast phase are involved in basic metabolism, signal transduction, growth and morphogenesis, and sulfur metabolism . In order to test whether the observed changes in gene expression reflect the differences between the growth conditions used to obtain the two morphological forms rather than differences intrinsic to the cell types, we performed real-time RT-PCR experiments using RNAs derived from both yeast cells and mycelia that had been cultured at 37 degrees C and 26 degrees C in either complete medium (YPD or Sabouraud) or minimal medium . Twenty genes, including AGS1 (alpha-1,3-glucan synthase) and TSA1 (thiol-specific antioxidant), were shown to be more highly expressed in the yeast cells than in the hyphae . Although their levels of expression could be different in rich and minimal media, there was a general tendency for these genes to be more highly expressed in the yeast cells.

FEBS Lett, 2004 May 7, 565(1-3), 48 - 52
Paracoccus pantotrophus NapC can reductively activate cytochrome cd1 nitrite reductase; Zajicek RS et al.; The oxidized "as isolated" form of Paracoccus pantotrophus cytochrome cd1 nitrite reductase has a bis-histidinyl coordinated c heme and a histidine/tyrosine coordinated d1 heme . This form of the enzyme has previously been shown to be kinetically incompetent . Upon reduction, the coordination of both hemes changes and the enzyme is kinetically activated . Here, we show that P . pantotrophus NapC, a tetraheme c-type cytochrome belonging to a large family of such proteins, is capable of reducing, and hence activating, "as isolated" cytochrome cd1 . NapC is the first protein from P . pantotrophus identified as being capable of this activation step and, given the periplasmic co-location and co-expression of the two proteins, is a strong candidate to be a physiological activation partner.

Appl Microbiol Biotechnol, 2004 Jul, 65(1), 124 - 31 Epub 2004 May 07.
Isolation and characterization of novel bacteria degrading polycyclic aromatic hydrocarbons from polluted Greek soils; Zhang H et al.; Three bacterial strains, designated as Wphe1, Sphe1, and Ophe1, were isolated from Greek soils contaminated with polycyclic aromatic hydrocarbon (PAH)-containing waste from the wood processing, steel, and oil refinery industries . Wphe1, Sphe1, and Ophe1 were characterized and identified as species of Pseudomonas, Microbacterium, and Paracoccus, respectively, based on Gram staining, biochemical tests, phospholipid analysis, FAME analysis, G+C content and 16S rRNA gene sequence analysis . The results of gas chromatography showed that strain Wphe1 degraded naphthalene, phenanthrene, and m-cresol over a wide temperature range; strain Sphe1 was a degrader of phenanthrene and n-alkanes; most interestingly, strain Ophe1 degraded anthracene, phenanthrene, fluorene, fluoranthene, chrysene, and pyrene, as well as cresol compounds and n-alkanes as sole carbon source . This is the first report of a representative of the genus Paracoccus capable of degrading PAHs with such versatility . These three strains may be useful for bioremediation applications.

Med Mycol, 2004 Apr, 42(2), 119 - 21
Differential expression of actin modulated by temperature in mycelial and yeast cells of Paracoccidioides brasiliensis; Goldani LZ et al.; We used yeast and mycelial forms of Paracoccidioides brasiliensis to evaluate the effect of heat shock stress on actin expression . P . brasiliensis yeasts harvested during the exponential growth phase showed more expression of the actin mRNA when incubated at 40 degrees C than when incubated at 37 degrees C, the usual temperature at which these yeasts grow . In contrast, expression of actin mRNA was lower in yeasts incubated at 25 degrees C than in yeasts incubated at 37 degrees C . Mycelium harvested at 25 degrees C, an approximation of its normal growth temperature, and then exposed to 37 degrees C and 40 degrees C showed progressively higher expression of actin mRNA . Mycelial and yeast forms showed a similar pattern of response to exposure to supra-optimal temperatures: both showed the same increase in expression of actin . This suggests that actin may play a role not only in cellular differentiation but also in this species' rapid adaptive response to heat stress, a mechanism necessary to deal with a potentially hostile environment.

FEMS Microbiol Lett, 2004 May 1, 234(1), 93 - 7
Detection of gp43 of Paracoccidioides brasiliensis by the loop-mediated isothermal amplification (LAMP) method; Endo S et al.; 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.

Eur J Epidemiol, 2004, 19(2), 189 - 93
Epidemiological survey of histoplasmine and paracoccidioidine skin reactivity in an agricultural area in Bolívar state, Venezuela; Cermeno JR et al.; Paracoccidiodomycosis and histoplasmosis are endemic diseases in the south of Venezuela, representing a public health problem . Prevalence of Paracoccidiodes brasiliensis and Histoplasma capsulatum infections were estimated in Monte Ralo, a rural community area of Bolivar state using paracoccidiodine and histoplasmine skin tests . Paracoccidiodine was intradermically injected to 173 persons and readings were made at 24 and 48 h afterwards to 167 persons (97.85%) . Reaction was positive in 8.5% (n = 14) at 24 h post-injection and 13.2% (n = 20) at 48 h . Farmers showed the higher percentage of positivity at 24 and 48 h . One hundred-seventy five persons were intradermically injected with histoplasmine but 167 and 157 of them attended for reading of the dermal reaction at 24 and 48 h post-injection respectively . Tests were positive in 25.7% (n = 43) at 24 h and 42.7% (n = 67) at 48 h . Further studies are needed in surrounding places to delimit the endemic area of these mycosis in the Bolivar state . However, epidemiological vigilance of PCM and H should be considered by local health authorities.

Curr Microbiol, 2004 Apr, 48(4), 291 - 4
Genetic organization of the basic replicon of plasmid pMTH4 of a facultatively methylotrophic bacterium Paracoccus methylutens DM12; Szymanik M et al.; Two functional regions within the basic replicon of plasmid pMTH4 of Paracoccus methylutens DM12 have been distinguished that are responsible for the replication of the plasmid (REP) and its stabilization (STA) . In the REP region, a gene encoding the putative replication initiation protein RepA has been identified, with the highest similarity to the replication protein of plasmid pALC1 (Paracoccus alcaliphilus) . The potential origin of replication (oriV), consisting of five long repeated sequences (iterons) as well as putative DnaA and IHF boxes, has been localized in the promoter region of the gene repA . The STA region was found to ensure stability for heterogeneous plasmid pABW3 that is unstable itself in paracocci . The mini-STA region (850 bp) contains two short open reading frames, one of which shows similarity to the RelB protein of Escherichia coli . Our investigations suggest that the stabilizing system of pMTH4 is based on the toxin and antidote principle.

Rev Inst Med Trop Sao Paulo, 2004 Jan-Feb, 46(1), 47 - 50 Epub 2004 Mar 29.
Disseminated paracoccidioidomycosis with peripleuritis in an AIDS patient; Corti M et al.; Paracoccidioidomycosis is one of the most frequent systemic and endemic mycoses of Latin America caused by a dimorphic fungus . In AIDS patients, paracoccidioidomycosis appears as a severe and disseminated disease with a wide spectrum of clinical findings . The CD4 counts are usually less than 200 cell/mu L . We present a case of disseminated paracoccidioidomycosis with peripleuritis and subcutaneous abscesses on the chest wall as initial manifestation of AIDS . In endemic countries, paracoccidioidomycosis should be included as an opportunistic infection in AIDS.

Rev Inst Med Trop Sao Paulo, 2004 Jan-Feb, 46(1), 37 - 9 Epub 2004 Mar 29.
Fatal disseminated paracoccidioidomycosis in a two-year-old child; Pereira RM et al.; A two year-old female child was admitted at the Pediatric Intensive Care Unit in a septic shock associated with a lymphoproliferative syndrome, with history of fever, adynamia and weight loss during the last two months . On admission, the main clinical and laboratory manifestations were: pallor, jaundice, disseminated enlarged lymph nodes, hepatosplenomegaly, crusted warts on face, anemia, eosinophilia, thrombocytopenia, increased direct and indirect bilirubin, alkaline phosphatase, and gammaglutamyl transpeptidase . A parenteral administration of fluids, dobutamine and mechanical ventilation was started, without improvement of the clinical conditions . A direct examination of exsudate collected from cervical lymph node revealed numerous oval-to-around cells with multiple budding, like a "pilot wheel" cell, suggesting Paracoccidioides brasiliensis . Even though treatment with intravenous sulfamethoxazole-trimethoprine was soon started, the child died 36 hours after hospital admission . Disseminated paracoccidioidomycosis was confirmed in the autopsy . This is the youngest case of paracoccidioidomycosis in children reported in the literature.

Gen Dent, 2004 Jan-Feb, 52(1), 48 - 50
Oral paracoccidioidomycosis or squamous cell carcinoma?
Kaminagakura E, Graner E, de Almeida OP, Di Hipolito Junior O, Lopes MA.
Paracoccidioidomycosis is a deep, systemic, and progressive mycosis caused by Paracoccidioides brasiliensis . Oral lesions normally are multiples with a mulberry-like appearance . This article reviews an unusual case involving a chronic, solitary, and ulcerated lesion whose clinical aspects were similar to squamous cell carcinoma . Viewed microscopically, the lesion showed pseudoepitheliomatous hyperplasia and non-necrotizing granulomas . The patient was treated with systemic ketoconazole . Over the next 11 years, follow-up examinations were performed but no recurrence was observed.

Fungal Genet Biol, 2004 May, 41(5), 510 - 20
Pbhyd1 and Pbhyd2: two mycelium-specific hydrophobin genes from the dimorphic fungus Paracoccidioides brasiliensis; Albuquerque P et al.; Paracoccidioides brasiliensis, the etiologic agent of paracoccidioidomycosis, is a dimorphic fungus which is found as mycelia (M) at 26 degrees C and as yeasts (Y) at 37 degrees C, or after the invasion of host tissues . Although the dimorphic transition in P . brasiliensis and other dimorphic fungi is an essential step in the establishment of infection, the molecular events regulating this process are yet poorly understood . Since the differential gene expression is a well-known mechanism which plays a central role in the dimorphic transition as well as in other biological process, in this work we describe the identification and characterization of two differentially expressed P . brasiliensis hydrophobin cDNAs (Pbhyd1 and Pbhyd2) . Hydrophobins are small hydrophobic proteins related to a variety of important functions in fungal biology, including cell growth, development, infection, and virulence . These two hydrophobin genes are present as single copy in P . brasiliensis genome and Northern blot analysis revealed that both mRNAs are mycelium-specific and highly accumulated during the first 24 h of M to Y transition.

Rev Soc Bras Med Trop, 2003 Nov-Dec, 36(6), 747 - 9
{Paracoccidioidomycosis and gastrectomy}; da Silva PZ et al.; In a review of the clinical records of 757 cases of paracoccidioidomycosis 11 (1.5%) of the patients had been previously submitted to gastrectomy . It is suggested that after gastrectomy individuals present a higher risk for the development of such infections, similar to that which occurs in tuberculosis.

Infect Immun, 2004 Apr, 72(4), 2369 - 78
Absence of interleukin-4 determines less severe pulmonary paracoccidioidomycosis associated with impaired Th2 response; Pina A et al.; Host resistance to paracoccidiodomycosis, the main deep mycosis in Latin America, is mainly due to cellular immunity and gamma interferon (IFN-gamma) production . To assess the role of interleukin-4 (IL-4), a Th2-inducing cytokine, pulmonary paracoccidioidomycosis was studied in IL-4-deficient (IL-4(-/-)) and wild-type (WT) C57BL/6 mice at the innate and acquired phases of immune response . Forty-eight hours after infection, equivalent numbers of viable Paracoccidioides brasiliensis yeast cells were recovered from the lungs of IL-4(-/-) and WT mice intratracheally infected with one million fungal cells . Alveolar macrophages from infected IL-4(-/-) mice controlled in vitro fungal growth more efficiently than macrophages from WT mice and secreted higher levels of nitric oxide . Compared with WT mice, IL-4(-/-) animals presented increased levels of pulmonary IFN-gamma and augmented polymorphonuclear leukocyte influx to the lungs . Decreased pulmonary fungal loads were characterized in deficient mice at week 2 postinfection, concomitant with diminished presence of IL-10 . At week 8, lower numbers of yeasts were recovered from lungs and liver of IL-4(-/-) mice associated with increased production of IFN-gamma but impaired synthesis of IL-5 and IL-10 . However, a clear shift to a Th1 pattern was not characterized, since IL-4(-/-) mice did not alter delayed-type hypersensitivity anergy or IL-2 levels . In addition, IL-4 deficiency resulted in significantly reduced levels of pulmonary IL-12, granulocyte-macrophage colony-stimulating factor, IL-3, monocyte chemotactic protein 1, and specific antibody isotypes . In IL-4(-/-) mice, well-organized granulomas restraining fungal cells replaced the more extensive lesions containing high numbers of fungi and inflammatory leukocytes developed by IL-4-sufficient mice . These results clearly showed that genetically determined deficiency of IL-4 can exert a protective role in pulmonary paracoccidioidomycosis.

Mycopathologia, 2004 Jan, 157(1), 53 - 7
Asymptomatic presentation of chronic pulmonary paracoccidioidomycosis: case report and review; dos Santos JW et al.; Asymptomatic presentations of chronic pulmonary paracoccidiodomycosis have been reported since 1959, usually published in case series of paracoccidiodomycosis . Incidental radiographic findings on chest roentgenogram led to diagnostic evaluation in these cases . An unusual case in a female patient is described, and 24 previously reported cases are reviewed.

Med Mycol, 2004 Feb, 42(1), 51 - 7
Isolation of the CHS4 gene of Paracoccidioides brasiliensis and its accommodation in a new class of chitin synthases; Nino-Vega GA et al.; The nucleotide sequence of a chitin synthase gene (PbrCHS4) of the dimorphic fungal human pathogen Paracoccidioides brasiliensis has been determined . A homology search with the deduced amino acid sequence of PbrChs4 (1744 aa) reveals the presence of two distinct domains, an N-terminal domain showing up to 30% homology to myosin motor-like domains and a C-terminal domain with up to 68% homology to chitin synthases, as has been reported for some class V chitin synthases . However, unlike class V chitin synthases with myosin motor-like domains, PbrChs4 does not present characteristic signatures of myosin motor-like domains . Also, although the Chs domain presents the closest homology to other fungal class V enzymes, it is low enough to consider PbrChs4 as belonging to a new class, which we propose as class VII.

Med Mycol, 2004 Feb, 42(1), 43 - 9
Viability of yeast form cells of Paracoccidioides brasiliensis after sonication; Dias MF et al.; To perform in-vitro studies with Paracoccidioides brasiliensis yeast cells it is necessary to avoid the presence of clumps of cells while maintaining their integrity . Because of the multiple budding type of growth, the bud cells are always attached to the mother cell and the yeast cells keep growing, resulting in the formation of large clumps . In order to obtain free cells, the cultures are usually sonicated . The present study shows that sonication induces lesions in a significant number of cells, as evaluated by labelling of the cells with acridine orange and Janus green vital dyes . In some cases labelling was initially observed in only one cell of the clump; however, the other cells also became labelled after a few minutes . These observations were confirmed by scanning and transmission electron microscopy of treated cells . Colony forming units (c.f.u.) on BHI plates also confirmed the decrease in cell viability following sonication.

Biomedica, 2003 Dec, 23(4), 424 - 36
Molecular cloning and characterization of two hsp 70 homologous genes from the dimorphic fungus Paracoccidioides brasiliensis; Florez AM et al.; Paracoccidioides brasiliensis, a dimorphic fungus, is the etiologic agent of Paracoccidioidomycosis (PCM), one of the most important systemic mycosis in Latin America . Two genes (2.2 and 1DB5) were cloned, characterized and sequenced; they showed homology with members of hsp70 gene family . By using several probe fragments derived from these genes, levels of expression for each gene were determined by Northern blot during transition to the yeast phase . The highest level of hsp70 transcript occurred between 30 min to 6 hours after temperature shift, with significant reduction after 36-48 hours . However, after 72 hours, the level of the transcription increased until yeast phase was reached . As a response to temperature increase, hsp 70 genes are expressed during the transition phase and possibly play a role in the differentiation process.

Yeast, 2004 Feb, 21(3), 211 - 8
Cloning and expression analysis of the ornithine decarboxylase gene (PbrODC) of the pathogenic fungus Paracoccidioides brasiliensis; Nino-Vega GA et al.; We describe the isolation and sequencing of PbrODC, the gene encoding ornithine decarboxylase (ODC) in Paracoccidioides brasiliensis . The gene contains a single open reading frame made of 1413 bp with a single intron (72 bp), and encodes a 447 amino acid polypeptide with a predicted molecular weight of 50.0 kDa, an isoelectric point of 4.9 and a high similarity to other fungal ornithine decarboxylases . Functionality of the gene was demonstrated by transformation into a Saccharomyces cerevisiae odc null mutant . A phylogenetic tree generated with several fungal ODCs provided additional evidence to favour a taxonomic position for P . brasiliensis as an ascomycetous fungus, belonging to the order Onygenales . Expression of the PbrODC gene was determined by Northern analyses during growth of the mycelial and yeast forms, and through the temperature-regulated dimorphic transition between these two extreme phases . Expression of PbrODC remained constant at all stages of the fungal growth, and did not correlate with a previously observed increase in the activity of ornithine decarboxylase at the onset of the budding process in both yeast growth and mycelium-to-yeast transition . Accordingly, post-transcriptional regulation for the product of PbrODC is suggested .

Yeast, 2004 Jan 30, 21(2), 173 - 82
Monofunctional catalase P of Paracoccidioides brasiliensis: identification, characterization, molecular cloning and expression analysis; Moreira SF et al.; Within the context of studies on genes from Paracoccidioides brasiliensis (Pb) potentially associated with fungus-host interaction, we isolated a 61 kDa protein, pI 6.2, that was reactive with sera of patients with paracoccidioidomycosis . This protein was identified as a peroxisomal catalase . A complete cDNA encoding this catalase was isolated from a Pb cDNA library and was designated PbcatP . The cDNA contained a 1509 bp ORF containing 502 amino acids, whose molecular mass was 57 kDa, with a pI of 6.5 . The translated protein PbCATP revealed canonical motifs of monofunctional typical small subunit catalases and the peroxisome-PTS-1-targeting signal . The deduced and the native PbCATP demonstrated amino acid sequence homology to known monofunctional catalases and was most closely related to catalases from other fungi . The protein and mRNA were diminished in the mycelial saprobic phase compared to the yeast phase of infection . Protein synthesis and mRNA levels increased during the transition from mycelium to yeast . In addition, the catalase protein was induced when cells were exposed to hydrogen peroxide . The identification and characterization of the PbCATP and cloning and characterization of the cDNA are essential steps for investigating the role of catalase as a defence of P . brasiliensis against oxygen-dependent killing mechanisms . These results suggest that this protein exerts an influence in the virulence of P . brasiliensis .

J Leukoc Biol, 2004 Apr, 75(4), 649 - 56 Epub 2004 Jan 14.
PTX3 function as an opsonin for the dectin-1-dependent internalization of zymosan by macrophages; Diniz SN et al.; Pentraxin 3 (PTX3) is a tumor necrosis factor and interleukin-1beta-stimulated gene that encodes a long PTX with proinflammatory activity . Here, we show that peritoneal macrophages derived from PTX3 transgenic (Tg) mice express higher levels of PTX3 mRNA than macrophages from wild-type (WT) mice, at basal level as well as upon stimulation with zymosan (Zy) . Macrophages from Tg mice also showed improved opsonin-independent phagocytosis of Zy particles and the yeast form of the fungus Paracoccidioides brasiliensis . In the case of P . brasiliensis, an enhanced microbicidal activity accompanied by higher production of nitric oxide was also observed in macrophages from Tg mice . Using fluorescein-activated cell sorter analysis and reverse transcriptase-polymerase chain reaction, we demonstrated that basal level of Toll-like receptor-6 and Zy-induced dectin-1 expression was slightly but consistently higher in macrophages from Tg mice than in macrophages from WT mice . Recombinant (r)PTX3 protein binds to Zy particles as well as to yeast cells of P . brasiliensis and addition of rPTX3, to a culture of WT-derived macrophages containing Zy leads to an increase in the phagocytic index, which parallels that of Tg-derived macrophages, demonstrating the opsonin-like activity of PTX3 . It is important that blockade of dectin-1 receptor inhibited the phagocytosis of Zy particles by WT and PTX3 Tg macrophages, pointing out the relevant role of dectin-1 as the main receptor involved in Zy uptake . Our results provide evidence for a role of PTX3 as an important component of the innate-immune response and as part of the host mechanisms that control fungal recognition and phagocytosis.

Med Mycol, 2003 Dec, 41(6), 539 - 42
Simplified method for producing Paracoccidioides brasiliensis exoantigens for use in immunodiffusion tests; Camargo ZP et al.; A simplified method to produce Paracoccidioides brasiliensis exoantigens for immunodiffusion testing is proposed . It uses technology accessible for small laboratories with few resources in Latin America, where paracoccidioidomycosis is endemic . This procedure may replace the more complex procedure, originally proposed by Camargo et al . in 1988, that is currently commonly used . It is based on the production of exoantigen by P . brasiliensis isolate B339, a good secretor of the characteristic 43000-Da glycoprotein gp43.

Scand J Immunol, 2004 Jan, 59(1), 58 - 65
Synthesis and immunological activity of a branched peptide carrying the T-cell epitope of gp43, the major exocellular antigen of Paracoccidioides brasiliensis; Taborda CP et al.; The 43 kDa glycoprotein (gp43) of Paracoccidioides brasiliensis is the major diagnostic antigen of paracoccidioidomycosis (PCM), a prevalent fungal infection in South America . A 15-mer sequence from gp43, denominated P10, induced T-CD4+ T helper 1 cellular immune responses in mice of three different haplotypes and protected against intratracheal challenge by a virulent isolate of P . brasiliensis . In an attempt to improve delivery of P10, a promiscuous antigen also presented by human leucocyte antigen-DR alleles, aiming at immunotherapy, we synthesized a multiple antigen peptide with the protective T-cell epitope expressed in a tetravalent 13-mer analog of P10 (M10) . M10 induced specific lymph node cell proliferation in mice preimmunized with peptides in complete Freund's adjuvant (CFA) . In addition, M10 immunization without CFA significantly protected intratracheally infected mice . We conclude that M10 is a candidate for an anti-PCM vaccine . In this report we describe: (1) the synthesis of M10; (2) the induction of M10-elicited T-cell response and (3) in vivo protection of mice immunized with M10 and challenged by a virulent strain of P . brasiliensis.

J Infect, 2004 Feb, 48(2), 193 - 8
Central nervous system paracoccidioidomycosis: clinical features and laboratorial findings; de Almeida SM et al.; OBJECTIVE: To study prospectively the clinical features and laboratorial characteristics of 24 patients with central nervous system (CNS) involvement with paracoccidioidomycosis (PCM) . PCM is an infectious disease caused by the dimorphic fungus Paracoccidioides brasiliensis, endemic in subtropical areas of Central and South America . METHODS: From 173 cases of PCM, 24 (13.9%) had CNS involvement (NPCM) and were studied prospectively from 1993 to 1997 . In all the patients, the diagnosis of systemic PCM was made by the demonstration of the P . brasiliensis organisms or positive serology, DID (double immunodiffusion) . In seven cases the diagnosis was made by means of a CNS biopsy . CNS clinical manifestations, neuroimaging (CT or MRI) and CSF cytochemical characteristics were reported . RESULTS: The mean age was 44 years (range 25-72 years); 23 patients were male, only one was female . Neurological symptoms began before systemic symptoms in 21%; simultaneously in 33%, and after systemic symptoms in 46% . Epilepsy was the more frequent neurological presentation (44%) . Twenty-three cases had parenchymatous involvement and in two of these cases there was an association with meningitis and one case had spinal cord involvement . Lesions were more frequent in the brain hemispheres (69%), in 65% there were multiple granuloma characterized by hypodense images with annular or nodular enhancing . All cases were treated with sulphamethoxazole-trimethoprin . Four patients died, while 20 patients showed a good therapeutic response . CONCLUSION: NPCM should always be considered in the differential diagnosis of expanding lesions of the CNS and meningoencephalitis . Being alert to this diagnosis depends on knowledge of epidemiology . There was good response to sulphamethoxazole-trimethoprin treatment.

Rev Port Pneumol, 2003 Mar-Apr, 9(2), 173 - 8
{Paracoccidiodomycosis, disseminated chronic form--case report}; Pereira JC et al.; Case report of paracoccidiodomycosis Chronic Form, adult type, with genital lesions and residual form presented as dysphagia and dysphonia at the same patient . Diagnosis accomplished by direct examination of clinical specimens, in this case cutaneous lesions . After specific treatment there was regression of lesions and symptoms had disappeared.

Eur Respir J, 2003 Dec, 22(6), 895 - 9
Evaluation of bronchoalveolar cells in pulmonary paracoccidioidomycosis; Fornazim MC et al.; To investigate the local immune response, the cellular infiltrate and cytokine levels were analysed in bronchoalveolar lavage (BAL) from patients with pulmonary paracoccidioidomycosis . The group consisted of 19 patients aged 34-65 yrs . The diagnosis was confirmed by demonstration of the fungus in the sputum or BAL fluid and by serological tests . Cytospin preparations showed an increased number of lymphocytes and neutrophils in BAL . A higher number of CD8+ lymphocytes were observed in BAL compared with peripheral blood . Alveolar macrophages (AM) expressed approximately three-fold more major histocompatibility class II, intercellular adhesion molecule-1 and B7-2 molecules on their surfaces than their circulating counterparts, indicating that they had differentiated into activated macrophages inside the lungs . Cultured AM produced higher levels of interleukin (IL)-6, tumour necrosis factor (TNF)-alpha and macrophage inflammatory protein (MIP)-1alpha than peripheral blood monocytes . BAL fluid contained low but detectable amounts of IL-6, TNF-alpha and MIP-1alpha, and specific antibodies to Paracoccidioides brasiliensis, mainly of the immunoglobulin G2 isotype . As macrophage inflammatory protein-1alpha was shown to selectively attract CD8+ T-cells and this population was elevated in bronchoalveolar lavage, the data suggest that, besides macrophages, CD8+ T-cells may have an important role in the pathogenesis of pulmonary paracoccidioidomycosis.

J Biol Chem, 2004 Mar 12, 279(11), 10293 - 303 Epub 2003 Dec 12.
Soluble CuA domain of cyanobacterial cytochrome c oxidase; Paumann M et al.; The genomes of several cyanobacteria show the existence of gene clusters encoding subunits I, II, and III of aa(3)-type cytochrome c oxidase . The enzyme occurs on both plasma and thylakoid membranes of these oxygenic phototrophic prokaryotes . Here we report the expression and purification of a truncated subunit II copper A (Cu(A)) domain (i.e . the electron entry and donor binding site) of cytochrome c oxidase from the cyanobacterium Synechocystis PCC 6803 in high yield . The water-soluble purple redox-active bimetallic center displays a relatively low standard reduction potential of 216 mV . Its absorption spectrum at pH 7 is similar to that of other soluble fragments from aa(3)-type oxidases, but the insensitivity of both absorbance and circular dichroism spectra to pH suggests that it is less exposed to the aqueous milieu compared with other Cu(A) domains . Oxidation of horse heart cytochrome c by the bimetallic center follows monophasic kinetics . At pH 7 and low ionic strength the bimolecular rate constant is (2.1 +/- 0.3) x 10(4) m-1 s(-1), and the rates decrease upon the increase of ionic strength . Sequence alignment and modeling of cyanobacterial Cu(A) domains show several peculiarities such as: (i) a large insertion located between the second transmembrane region and the putative hydrophobic cytochrome c docking site, (ii) the lack of acidic residues shown to be important in the interaction between cytochrome c and Paracoccus Cu(A) domain, and (iii) an extended C terminus similar to Escherichia coli ubiquinol oxidase.

Microbes Infect, 2003 Dec, 5(15), 1373 - 9
Study of bronchoalveolar lavage fluid in paracoccidioidomycosis: cytopathology and alveolar macrophage function in response to gamma interferon; comparison with blood monocytes; Calvi SA et al.; Patients with paracoccidioidomycosis (PCM) present marked involvement of the lungs during the course of the mycosis . The purpose of this work was to obtain bronchoalveolar lavage (BAL) fluid from these patients to study the cytopathology, TNF levels and the oxidative and fungicidal response of alveolar macrophages (AMs) to in vitro incubation with recombinant IFN-gamma . To compare the lung and blood compartments, these determinations were also made in plasma and blood monocytes (BMs) obtained from the same patients . The cytopathology of BAL fluid revealed a predominance of macrophages, but with the presence of neutrophil exudation, and rare lymphocytes and epithelioid and giant cells . Comparison of the oxidative status and fungicidal activity of AMs and circulating BMs demonstrated that both cell types are highly activated for these two functions when compared to control cells . However, TNF levels were higher in BAL fluid than in plasma . The possible mechanisms involved in the hyperresponsiveness of cells from PCM patients are discussed.

J Biol Chem, 2004 Mar 5, 279(10), 9532 - 8 Epub 2003 Dec 09.
A superfamily of voltage-gated sodium channels in bacteria; Koishi R et al.; NaChBac, a six-alpha-helical transmembrane-spanning protein cloned from Bacillus halodurans, is the first functionally characterized bacterial voltage-gated Na(+)-selective channel . As a highly expressing ion channel protein, NaChBac is an ideal candidate for high resolution structural determination and structure-function studies . The biological role of NaChBac, however, is still unknown . In this report, another 11 structurally related bacterial proteins are described . Two of these functionally expressed as voltage-dependent Na(+) channels (Na(V)PZ from Paracoccus zeaxanthinifaciens and Na(V)SP from Silicibacter pomeroyi) . Na(V)PZ and Na(V)SP share approximately 40% amino acid sequence identity with NaChBac . When expressed in mammalian cell lines, both Na(V)PZ and Na(V)SP were Na(+)-selective and voltage-dependent . However, their kinetics and voltage dependence differ significantly . These single six-alpha-helical transmembrane-spanning subunits constitute a widely distributed superfamily (Na(V)Bac) of channels in bacteria, implying a fundamental prokaryotic function . The degree of sequence homology (22-54%) is optimal for future comparisons of Na(V)Bac structure and function of similarity and dissimilarity among Na(V)Bac proteins . Thus, the Na(V)Bac superfamily is fertile ground for crystallographic, electrophysiological, and microbiological studies.

Microbiology, 2003 Dec, 149(Pt 12), 3587 - 93
A functional plasmid-borne rrn operon in soil isolates belonging to the genus Paracoccus; Battermann A et al.; Plasmid analysis of isolates from a small Paracoccus population revealed that all 15 representatives carried at least one endogenous plasmid of 23 or 15 kb in size, in addition to further plasmids of different sizes . It was shown by restriction analysis and hybridization that the 23 and 15 kb plasmids from the different isolates were identical or very similar to each other . By partial sequencing of pOL18/23, one of the 23 kb plasmids, a complete rrn operon with the structural genes for 16S, 23S and 5S rRNA, two genes for tRNA(Ile) and tRNA(Ala) within the spacer between the 16S and 23S rRNA genes, and a final tRNA(fMet) at the end of the operon were discovered . Expression of a green fluorescent protein gene (gfp) after insertion of a DNA fragment from the region upstream of the rRNA genes into a promoter-probe vector demonstrated that the rrn promoter region is functional . The rrn operon encoded by plasmid pOL18/23 is the first complete rrn operon sequenced from a strain of the genus Paracoccus, and only the second example of an rrn operon on a small plasmid.

J Clin Microbiol, 2003 Dec, 41(12), 5735 - 7
Comparison of the sequences of the internal transcribed spacer regions and PbGP43 genes of Paracoccidioides brasiliensis from patients and armadillos (Dasypus novemcinctus); Hebeler-Barbosa F et al.; Paracoccidioides brasiliensis isolates from 10 nine-banded armadillos (Dasypus novemcinctus) were comparable with 19 clinical isolates by sequence analysis of the PbGP43 gene and ribosomal internal transcribed spacer 1 (ITS1) and ITS2 and by random amplified polymorphic DNA . In this original ITS study, eight isolates differed by one or three sites among five total substitution sites.

Med Mycol, 2003 Oct, 41(5), 427 - 36
Immunity and hypersensitivity to gp43 antigen in susceptible and resistant mice infected with Paracoccidioides brasiliensis; Godoy LC et al.; Pathogenic mechanisms underlying paracoccidioidomycosis are still poorly understood . A well-established murine model of resistance (mouse lineage A/Sn) and susceptibility (lineage B10.A) to P . brasiliensis pulmonary infection was here employed to compare immune response to gp43, the major antigenic component of the fungus . Mice were infected and their cellular and humoral immunity to gp43 were investigated for up to 16 weeks . In both mouse strains, challenge with gp43 indistinguishably evoked a typical immediate-hypersensitivity response, followed by a 24-h late-phase reaction consistent with the same type of immunological activation . IL-4 was detected in cultures of gp43-stimulated lymph node cells only in susceptible animals 2 weeks post-infection, while IL-5 was found throughout the study in both mouse strains . IL-10 appeared in the supernatants of stimulated cells from resistant and susceptible animals in increasing amounts as infection advanced . Conversely, interferon (IFN)-gamma was produced under gp43 stimulation only by cells from A/Sn animals . The humoral response was characterized by low levels of anti-gp43 . Titration of IgG isotypes, however, revealed a predominance of IgG1 . IgG2a levels were highest in resistant animals, whereas IgG2b levels were highest in susceptible mice . In conclusion, immunity induced by gp43 exhibits common features in A/Sn and B10.A phenotypes, such as immediate hypersensitivity, late phase reaction and high levels of IL-10, but some differences between the strains are also seen.

Med Mycol, 2003 Oct, 41(5), 391 - 9
Production of pro-inflammatory cytokines during the early stages of experimental Paracoccidioides brasiliensis infection; Gonzalez A et al.; Pro-inflammatory cytokines play an important role in both recruitment and activation of leukocytes migrating into tissues in response to invading pathogens . In this study the production of pro-inflammatory cytokines, determined by ELISA assays, and the recruitment of leukocytes into the lungs of BALB/c mice infected with Paracoccidioides brasiliensis conidia were evaluated during the early stages of infection . The results showed that infected mice had a significant increase in leukocytes in the lung during the first 4 days with a peak at day 2 post-challenge; infiltrates were composed mainly of polymorphonuclear neutrophils (PMN) . Pro-inflammatory cytokines such as tumour necrosis factor alpha (TNF-alpha), interleukin (IL) 6, IL-1beta and macrophage inflammatory protein (MIP) 2 were produced at elevated levels during the first 4 days post-challenge, but only in pulmonary samples and not in sera . Additionally, during the early stages of infection, overall weight loss was recorded in infected mice . These results suggest that pro-inflammatory cytokines could be responsible for the recruitment of leukocytes into the lung during the early stages of P . brasiliensis infection . In addition, both pro-inflammatory cytokine production and leukocyte recruitment may participate in the control of infection by influencing the organization of the immune response in the host exposed to P . brasiliensis conidia.

Biochem Biophys Res Commun, 2003 Dec 26, 312(4), 1011 - 8
Sulfur oxidation in Paracoccus pantotrophus: interaction of the sulfur-binding protein SoxYZ with the dimanganese SoxB protein; Quentmeier A et al.; The central protein of the sulfur-oxidizing enzyme system of Paracoccus pantotrophus, SoxYZ, formed complexes with subunits associated and covalently bound . In denaturing SDS-polyacrylamide gel electrophoresis (PAGE) SoxY migrated at 12 and SoxZ at 16kDa . SDS-PAGE of homogeneous SoxYZ without reductant separated dimeric complexes of 25, 29, and 32kDa identified by the N-terminal amino acid sequences as SoxY-Y, SoxY-Z, and SoxZ-Z, and subunit cleavage by reduction suggested their linkage via protein disulfide bonds . SoxYZ was reversibly redox active between -0.25 and 0.2V, as monitored by a combined electrochemical and FTIR spectroscopic approach . The dimanganese SoxB protein (58.611Da) converted the covalently linked heterodimer SoxY-Z to SoxYZ with associated subunits which in turn aggregated to the heterotetramer Sox(YZ)(2) . This reaction depended on time and the SoxB concentration, and demonstrated the interaction of these two Sox proteins.

J Biol Chem, 2004 Feb 20, 279(8), 6252 - 60 Epub 2003 Nov 26.
Cytochrome c551 from Starkeya novella: characterization, spectroscopic properties, and phylogeny of a diheme protein of the SoxAX family; Kappler U et al.; Cytochromes from the SoxAX family have a major role in thiosulfate oxidation via the thiosulfate-oxidizing multi-enzyme system (TOMES) . Previously characterized SoxAX proteins from Rhodovulum sulfidophilum and Paracoccus pantotrophus contain three heme c groups, two of which are located on the SoxA subunit . In contrast, the SoxAX protein purified from Starkeya novella was found to contain only two heme groups . Mass spectrometry showed that a disulfide bond replaced the second heme group found in the diheme SoxA subunits . Apparent molecular masses of 27,229 +/- 10.3 Da and 20,258.6 +/- 1 Da were determined for SoxA and SoxX with an overall mass of 49.7 kDa, indicating a heterodimeric structure . Optical redox potentiometry found that the two heme cofactors are reduced at similar potentials (versus NHE) that are as follows: +133 mV (pH 6.0); +104 mV (pH 7.0); +49 (pH 7.9) and +10 mV (pH 8.7) . EPR spectroscopy revealed that both ferric heme groups are in the low spin state, and the spectra were consistent with one heme having a His/Cys axial ligation and the other having a His/Met axial ligation . The His/Cys ligated heme is present in different conformational states and gives rise to three distinct signals . Amino acid sequencing was used to unambiguously assign the protein to the encoding genes, soxAX, which are part of a complete sox gene cluster found in S . novella . Phylogenetic analysis of soxA- and soxX-related gene sequences indicates a parallel development of SoxA and SoxX, with the diheme and monoheme SoxA sequences located on clearly separated branches of a phylogenetic tree.

Microbes Infect, 2003 Nov, 5(13), 1205 - 11
Paracoccidioides brasiliensis exoantigens: recognition by IgG from patients with different clinical forms of paracoccidioidomycosis; Panunto-Castelo A et al.; Serum antibodies against antigens of Paracoccidioides brasiliensis have been one of the major diagnostic indicators of paracoccidioidomycosis (PCM) . In the present study, released antigen preparations (exoAg) obtained from P . brasiliensis isolates were characterized in terms of their protein components electrophoretically detectable and recognizable by sera (IgG) of patients . Among five different isolates (DGO, C-9, BAT, Pb-18 and B-339) the electrophoretic profiles of exoAg varied greatly . A total of 28 different components were detected, 11 of them shared by all isolates . The most representative preparation was BAT-exoAg, which presented the largest number of protein bands (23) and the highest frequency of reacting bands (19) with sera from patients with active PCM (n = 40) . Six bands reacted with more than 20% of sera . Independently of clinical forms, the sera recognized the 43-kDa (97% of tested sera), 160-kDa (78% of tested sera) and 70-kDa (60% of tested sera) antigens more frequently . Sera from patients with severe forms of acute (n = 14) or chronic (n = 10) PCM recognized a greater number of antigens, with a higher frequency, than those from moderate forms . The most pronounced reduction in reactivity was provided by sera of patients that became asymptomatic at the beginning of treatment . Remnant reactivity with BAT-exoAg was detected after clinical recovery, especially with those of 43, 70 and 160 kDa . The latter presented a stable recognition frequency (60%) during the entire follow-up, allowing us to suppose that the IgG reactivity against the 160-kDa antigen constitutes a possible persistent marker of P . brasiliensis infection.

Mycoses, 2003, 46(9-10), 412 - 7
Oral manifestations of paracoccidioidomycosis . Report of 21 cases from Argentina; Godoy H et al.; The present study describes 21 Argentinian patients living in the province of Corrientes, who had developed oral manifestations due to Paracoccidioides brasiliensis infection . Of these, 20 patients were men and one a woman . Patients were of an average age of 39 years (range 25 to 72 years) . Approximately, 76.2% of the patients were farmers . Gingival lesions were observed in 76% . Also, the tongue (71%) and the lips (62%) were frequently affected . Cytological smears and histopathology showed the characteristic fungal cells with the characteristic granulomatous inflammatory reaction consisting of lymphocytes, epithelioid cells and giant cells of the Langhans type . All patients except one had detectable pulmonary involvement . Therapy consists of long-term administration of itraconazole . Oral manifestations of paracoccidioidomycosis are characteristic in their clinical presentation . Early diagnosis and adequate therapy may prevent extensive tissue destructions . Long-term follow-up is mandatory.

Mycoses, 2003, 46(9-10), 407 - 11
Imported paracoccidioidomycosis in Spain; Ginarte M et al.; The report deals with three patients, who developed paracoccidioidomycosis in Europe after having been in South America 50, 25 and 38 years before, respectively . The conditions for the long period of quiescent infection and for the late manifestations are discussed.

Mycoses, 2003, 46(9-10), 397 - 401
Decreased monocytic phagosomal acidification among chronic paracoccidioidomycosis patients; Guereno MT et al.; To determine the ability of monocyte phagosomal acidification in chronic paracoccidioidomycosis, 13 patients were recruited at different times during follow-up and compared with 18 normal controls . Eight patients were studied at diagnosis, six of them also during treatment and five additional patients after ending treatment . Phagosomal acidification of monocytes, triggered by challenge with opsonized zymosan, was evaluated with acridine orange and expressed as percentage of orange-stained intracellular particles, as mean +/- SE . In comparison with controls, acidification was severely impaired before treatment (33 +/- 11% vs . 67 +/- 6%) and reached values similar to controls during treatment (73 +/- 6%, n = 6) . In addition, phagosomal acidification of the patients studied after treatment (63 +/- 4%) had no difference when compared with controls . This study demonstrates that phagosomal acidification is perturbed among chronic paracoccidioidomycosis patients and reverses with antifungal treatment.

Inorg Chem, 2003 Nov 3, 42(22), 7249 - 57
Kinetic stability of the peroxidase activity of unfolded cytochrome c: heme degradation and catalyst inactivation by hydrogen peroxide; Diederix RE et al.; Unfolding converts Paracoccus versutus cytochrome c-550 into a potent peroxidase (Diederix, R . E . M.; Ubbink, M.; Canters, G . W . ChemBioChem 2002, 3, 110-112) . The catalytic activity is accompanied by peroxide-driven inactivation that is prevented, in part, by reducing substrate . Here, the kinetics of inactivation are described, and evidence is presented for the occurrence of a labile intermediate on the catalytic peroxidase pathway of unfolded cytochrome c-550 . This intermediate represents a branching point, whereby the protein proceeds along either the productive pathway or self-inactivates . Reducing substrate suppresses inactivation by decreasing the steady-state concentration of the labile intermediate . Inactivation is accompanied by heme degradation . Its chemical reactivity, UV-vis, and EPR properties identify the first intermediate as hydroxyheme-cytochrome c-550, i.e . with heme hydroxylated at one of the heme meso positions . The occurrence of this species argues for the peroxo-iron species in the peroxidase mechanism as the labile intermediate leading to inactivated cytochrome c-550.

Infect Immun, 2003 Nov, 71(11), 6534 - 42
Characterization of gp70 and anti-gp70 monoclonal antibodies in Paracoccidioides brasiliensis pathogenesis; de Mattos Grosso D et al.; Paracoccidioidomycosis (PCM) is a systemic granulomatous mycosis whose agent is Paracoccidioides brasiliensis . In the culture supernatant, the fungus expresses glycoproteins of from 13 to 148 kDa . A cell surface glycoprotein of 43 kDa is the major antigenic component of P . brasiliensis . Another expressed glycoprotein, gp70, is recognized by 96% of sera from PCM patients and is able to induce lymphoproliferation . Since, little is known about this glycoprotein, we produced monoclonal antibodies (MAbs) against gp70 to isolate the molecule from total fungus extracts and to investigate its possible role in the pathogenesis of PCM . Using these MAbs, it was observed by confocal microscopy that gp70 is located mainly in the intracellular compartment of the fungus, although it was also detected in the culture supernatant . Based on observations showing that gp43 has a down-regulatory effect on mouse peritoneal macrophages, we tested the effects of gp70 on their phagocytic ability . Purified gp70 was able to inhibit the activity of macrophages through the mannose receptors and also through the Fc receptors; the latter effect was not observed with gp43 . gp70 inhibits NO and H(2)O(2) liberation by peritoneal macrophages in vitro, as does gp43 . Results obtained with gp43 led us to hypothesize that gp70 could act as an escape mechanism for fungal establishment in primary infections . To corroborate this hypothesis, we analyzed the effect of passive immunization of mice during infection with P . brasiliensis using anti-gp70 MAbs . This treatment almost completely abolished granuloma formation in the lungs, suggesting that the protein facilitates fungal establishment and progression of lesions in primary infection.

Crit Rev Oral Biol Med, 2003, 14(5), 377 - 83
Paracoccidioidomycosis of the mouth: an emerging deep mycosis; Almeida OP et al.; Oral fungal infections (mycoses) have come into particular prominence since the advent of infection with Human Immunodeficiency Virus (HIV), and recognition of the Acquired Immune Deficiency Syndrome (AIDS), as well as the phenomenal increase in world travel with increased exposure to infections endemic in the tropics . Paracoccidioidomycosis is a rare mycosis worldwide but common in Brazil and some other areas in Latin America . It can be life-threatening and can manifest with a spectrum of clinical presentations, including frequent oral lesions . This paper reviews the more recent information on Paracoccidioidomycosis, emphasizing those areas most relevant in dental science.

Clin Infect Dis, 2003 Oct 1, 37(7), 898 - 904 Epub 2003 Sep 08.
Residual pulmonary abnormalities in adult patients with chronic paracoccidioidomycosis: prolonged follow-up after itraconazole therapy; Tobon AM et al.; Itraconazole effectively controls active paracoccidioidomycosis but appears not to hinder lung fibrosis . Clinical records and chest radiographs from 47 itraconazole-treated patients with prolonged posttherapy follow-up (mean follow-up period, 5.6 years) were analyzed; the radiographs were interpreted following pneumoconiosis standards that consider the lungs as 6 fields and grade damage according to the number of fields involved . Infiltrative lesions were observed at diagnosis in 93.6% of the patients . Fibrosis was observed in 31.8% of the patients at diagnosis and had not cleared at the end of the observation period in any of these patients . Fibrosis also developed de novo in 11 patients (25%), so that by the end of the follow-up period it was seen in 53.2% of patients overall . Fibrosis correlated with severity of infiltrates at diagnosis: fibrosis was present in 83% of patients with very severe infiltration and in 12.5% of patients with minor infiltration . Among patients with severe infiltration, fibrosis was present in 30%; this increased (to 75%) when bullae were concomitantly present at diagnosis . Prompt initiation of treatment is necessary to avoid the development of fibrosis.

Clin Diagn Lab Immunol, 2003 Sep, 10(5), 802 - 7
Negative immunodiffusion test results obtained with sera of paracoccidioidomycosis patients may be related to low-avidity immunoglobulin G2 antibodies directed against carbohydrate epitopes; Neves AR et al.; Immunodiffusion (ID) is the serologic test most frequently used for the diagnosis and posttherapy follow-up of patients with paracoccidioidomycosis (PCM) . The ID test is highly specific (100%), but its sensitivity is relatively low (90%), leading to false-negative results . The aim of this study was to determine the profiles of antibodies in sera from patients with proven PCM and with negative results in the ID test (IDneg) versus positive results in the ID test (IDpos) . We analyzed 46 sera from patients with active PCM for total immunoglobulin G (IgG) and IgG subclass responses to Paracoccidioides brasiliensis gp43 antigen (treated or not treated with sodium metaperiodate) by enzyme-linked immunosorbent assay and immunoblotting . Immunoblotting showed that both IDneg and IDpos sera recognized predominantly the gp43 fraction of the P . brasiliensis antigen used in the ID test . IDneg sera contain low-avidity antibodies, low levels of specific IgG (total) and IgG1, and high levels of IgG2 compared with IDpos sera . The antibodies present in IDneg sera were predominantly directed against carbohydrate epitopes, since treatment with sodium metaperiodate resulted in a significant decrease in antibody reactivity . These data suggest that the lack of reactivity of sera from PCM patients in the ID test may be related to the production of low-avidity IgG2 antibodies directed against carbohydrate epitopes.

Med Mycol, 2003 Apr, 41(2), 131 - 6
Synergistic antifungal effect of fluconazole and human polymorphonuclear leukocytes on Paracoccidioides brasiliensis: effect of interferon-gamma and granulocyte-macrophage colony-stimulating factor; Kurita N et al.; To better understand the in vivo efficacy of fluconazole (FCZ), we investigated the possible synergy of fungistatic FCZ with human polymorphonuclear leukocytes (PMN) against Paracoccidioides brasiliensis (Pb) . The effect of interferon-gamma (IFN-gamma) and granulocyte-macrophage colony-stimulating factor (GM-CSF) in this system was also studied . For this purpose, FCZ, PMN, PMN + FCZ, PMN + IFN-gamma, PMN + IFN-gamma + FCZ, PMN + GM-CSF and PMN + GM-CSF + FCZ were co-cultured with Pb and the cfu of Pb was measured . The antifungal effect of FCZ on yeast cells of Pb was concentration-dependent . At 0.1 microg ml(-1), FCZ had no effect on the growth of Pb . At 0.2 microg ml(-1) FCZ showed a growth-inhibitory effect on three isolates of Pb in a long-term (120 h) assay, and at 0.6 microg ml(-1) or higher FCZ was fungicidal . Fungistatic concentration of FCZ (0.4 microg ml(-1)) acted synergistically with fungistatic PMN for killing isolate Bt-4 during the first 24 h of co-culture . Moreover, IFN-gamma and GM-CSF substantially enhanced the synergistic antifungal effect of PMN and FCZ . These findings provide a better understanding of why FCZ is more efficacious in in vivo models of paracoccidioidomycosis than is predicted by in vitro susceptibility tests.

Med Mycol, 2003 Apr, 41(2), 89 - 96
Virulence profiles of ten Paracoccidioides brasiliensis isolates obtained from armadillos (Dasypus novemcinctus); Hebeler-Barbosa F et al.; Paracoccidioides brasiliensis is the etiologic agent of paracoccidioidomycosis (PCM), the most important systemic mycosis in Latin America . The armadillo, Dasypus novemcinctus, has been confirmed as the primary natural reservoir of this fungus . Its geographic distribution is similar to that of human PCM . In this study, virulence profiles of 10 P . brasiliensis isolates from different armadillos and of two clinical isolates were tested in an experimental hamster model . Pathogenicity was evaluated by counting cfu and performing histopathological analysis in the testis, liver, spleen and lung . Circulating specific antibodies were measured using enzyme-linked immunosorbent assay (ELISA) . All isolates from armadillos were virulent in the model, with dissemination to many organs . The clinical isolates, which had long been stored in cultured collections, were less virulent . The isolates were classified into four virulence categories according to number of cfu per gram of tissue: very high, high, intermediate and low . This study confirms that armadillos harbor pathogenic genotypes of P . brasiliensis, probably the same ones that infect humans.

Med Mycol, 2003 Apr, 41(2), 83 - 7
Thymus invasion and atrophy induced by Paracoccidioides brasiliensis in BALB/c mice; Brito VN et al.; Literature has shown that immunosuppression observed in systemic mycosis can be related to damage in primary lymphoid organs . We have studied the immunopathological alterations induced experimentally by Paracoccidioides brasiliensis in these organs . In this work, thymic alterations induced in BALB/c mice during acute and chronic stages of infection are described . It was observed that P . brasiliensis is able to invade the thymic microenvironment, inducing severe atrophy characterized by degeneration of the cortical area, organ weight decrease, loss of corticomedullary delimitation and increase in histiocyte number . Occurrence of polymorphonuclear infiltration in the subcapsular area was also observed . Our results demonstrate that P . brasiliensis induces profound thymic atrophy and raises the question of whether this could be a fungal strategy to achieve successful establishment in the host over the long term.

Med Mycol, 2003 Jun, 41(3), 265 - 8
Experimental paracoccidioidomycosis in dogs; Ono MA et al.; The aim of this study was to evaluate the susceptibility of dogs to develop paracoccidioidomycosis by experimental infection . Puppies were inoculated with Paracoccidioides brasiliensis by an intravenous route and two out of four died 1 week postinoculation, showing, at histopathological analysis, granulomas in the lungs, spleen and liver . P . brasiliensis was isolated from these organs . The animals that survived the infection showed a strong reaction when skin was tested with gp43, a specific antigen of P . brasiliensis . These animals were killed at 1 and 5 months after infection, and no lesions, macroscopic or microscopic, were observed in the lungs, spleen or liver; furthermore no P . brasiliensis culture was obtained from these organs . These results suggest that dogs can develop paracoccidioidomycosis and reinforces the importance of this animal as a sensitive indicator of P . brasiliensis in the environment.

Med Mycol, 2003 Jun, 41(3), 259 - 63
Paracoccidioidomycosis associated with human immunodeficiency virus infection . Report of 10 cases; Silva-Vergara ML et al.; We describe here the epidemiological and clinical characteristics of 10 HIV-infected patients with paracoccidioidomycosis . All patients were adult males from small towns in Brazil and had a previous history of work or residence in a rural area . The two infections were diagnosed concomitantly in six of the ten patients, and for six of the patients, the mycosis was the first clinical manifestation of HIV infection . Risk factors for HIV infection were injection drug use in some patients and multiple sexual partners in others . Six patients died and autopsy revealed severe disseminated paracoccidioidomycosis in three . Exuberant and severe clinical pictures suggest an alteration in the natural history of this mycosis as a result of HIV immunosuppression . The frequency of paracoccidioidomycosis in the HIV-infected population is not known to differ from that reported for this mycosis in non-HIV patients.

Med Mycol, 2003 Jun, 41(3), 217 - 23
High frequency of Paracoccidioides brasiliensis infection in armadillos (Dasypus novemcinctus): an ecological study; Bagagli E et al.; The fungus Paracoccidioides brasiliensis has been isolated from nine-banded armadillos (Dasypus novemcinctus) in different regions where paracoccidiodomycosis (PCM) is endemic . The link between PCM and these animals has provided the first valuable clue in the effort to elucidate the ecological niche of P . brasiliensis . The present study was aimed at correlating P . brasiliensis infection in armadillos with local ecological features and, if possible, the presence of the fungus in the soil in the Botucatu hyperendemic area of PCM . In this region the mean temperature ranges from 14.8 to 25.8 degrees C and the annual average precipitation is 1520 mm . The sites where 10 infected animals (positive group) were collected were studied and compared with the sites where five uninfected animals were found . The occurrence of the fungus in soil samples collected from the positive armadillos' burrows and foraging sites was investigated by the indirect method of animal inoculation . Environmental data from the sites of animal capture, such as temperature, rainfall, altitude, vegetation, soil composition, presence of water and proximity of urban areas, were recorded . All 37 soil samples collected from the sites had negative fungal cultures . Positive animals were found much more frequently in sites with disturbed vegetation, such as riparian forests and artificial Eucalyptus or Pinus forests, in altitudes below 800 m, near water sources . The soil type of the sites of positive animals was mainly sandy, with medium to low concentrations of organic matter . The pH was mainly acidic at all the sites, although the concentrations of aluminum cations (H+Al) were lower at the sites where positive animals were found . Positive armadillos were also captured in sites very close to urban areas . Our data and previous studies indicate that P . brasiliensis occurs preferentially in humid and shady disturbed forests in a strong association with armadillos.

Mycoses, 2003 Sep, 46(8), 342 - 7
Disseminated paracoccidioidomycosis: correlation between clinical and in vitro resistance to ketoconazole and trimethoprim sulphamethoxazole; Hahn RC et al.; The present study relates to a case of subacute multifocal paracoccidioidomycosis where the upper intestinal tract is involved . The involvement of the upper digestive tract is uncommon . The recommended therapeutic treatment plans and the difficulty in the treatment of paracoccidioidomycosis are discussed in association with susceptibility tests to antifungal drugs in vitro . This is the first report available in the literature showing, in parallel, clinical and in vitro resistance to ketoconazole and trimethoprim sulphamethoxazole, studied during the course of the disease.

Scand J Immunol, 2003 Sep, 58(3), 290 - 7
Regulation of T helper cell differentiation in vivo by GP43 from Paracoccidioides brasiliensis provided by different antigen-presenting cells; Ferreira KS et al.; Paracoccidioidomycosis, endemic in Latin America, is a progressive systemic mycosis caused by Paracoccidioides brasiliensis . The infection can evolve into different clinical forms that are associated with various degrees of suppressed cell-mediated immunity . Assuming that the effector immune response is a consequence of the preferential activation of either Th1 or Th2 subsets, in the present work we evaluated whether the nature of antigen-presenting cells (APCs) can influence the Th1/Th2 balance in vivo . It was observed that the injection of mature dendritic cells (DCs), macrophages and B cells primed the mice and induced a proliferation of T cells in vitro . It was seen that DCs from resistant mice stimulated predominantly interleukin-2 (IL-2) and interferon-gamma (IFN-gamma), whereas macrophages activated IL-10, IL-4 and IFN-gamma-secreting T cells and B cells IL-4 and IL-10 only . Results presented here clearly demonstrate that DC drives the development of cells secreting Th1-derived cytokines, whereas B cells induce the differentiation of a Th2 phenotype in vivo.

Rev Soc Bras Med Trop, 2003 Jul-Aug, 36(4), 455 - 9 Epub 2003 Aug 13.
{Paracoccidioidomycosis: a clinical and epidemiological study of 422 cases observed in Mato Grosso do Sul}; Paniago AM et al.; Clinical and epidemiological features of 422 cases of paracoccidioidomycosis attended at University Hospital of Universidade Federal de Mato Grosso do Sul (Campo Grande, Mato Grosso do Sul, Brazil) from January 1980 to August 1999, were analysed . The mean age was 43.4 years old and the male: female ratio was 10:1 . Nearly half (45.5%) of the patients were agricultural workers at the moment of diagnosis . In the acute/subacute form (juvenile type) the phagocytic-monocytic system was very much impaired and mainly marked by lymphadenopathy (95.4%), hepatomegaly (40%), splenomegaly (23.1%) . The chronic form (adult type) presents more lesions in oropharynx (66.4%), dysphonia (31.4%) and cough (50.7%) . Mycological diagnosis was obtained by direct microscopy of wet mounts in 185/365 (50.7%) patients and by histopathological examination of biopsies in 294/302 (97.3%) patients . The treatment of choice was Sulfamethoxazole/ trimethoprim (Co-trimoxazole), used in 90.3% patients . Sequelae occurred in 30.3% and death in 7.6% of the cases.

Antimicrob Agents Chemother, 2003 Sep, 47(9), 2966 - 70
S-Adenosyl-L-methionine inhibitors delta(24)-sterol methyltransferase and delta(24(28))-sterol methylreductase as possible agents against Paracoccidioides brasiliensis; Visbal G et al.; We studied the antiproliferative effects of three azasterol analogs {piperidyl-2-yl-5alpha-pregnan-3beta,20(R)-diol (AZA-1), 22-piperidin-2-yl-pregnan-22(S),3beta-diol (AZA-2), and 22-piperidin-3-yl-pregnan-22(S),3beta-diol (AZA-3)} and their effects on the lipid composition of the pathogenic yeastlike phase of the dimorphic fungus Paracoccidioides brasiliensis . Inhibition was 100% for AZA-1 at 5 microM, 62% for AZA-2 at 10 microM, and 100% for AZA-3 at 0.5 microM . The analogs inhibited different stages of the sterol biosynthesis pathway.

Biochim Biophys Acta, 2003 Aug 22, 1622(3), 151 - 60
Dietary supplementation with docosahexaenoic acid, but not with eicosapentaenoic acid, reduces host resistance to fungal infection in mice; Oarada M et al.; The effect of dietary docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) on host resistance to Paracoccidioides brasiliensis infection was investigated . Mice fed palm oil supplemented with DHA showed reduced antifungal activity in the spleen and liver, as compared with mice fed palm oil or soybean oil without supplementation with DHA . Mice fed DHA-supplemented soybean oil also showed reduced antifungal activity in the liver, but the extent of reduction was less profound . This reduction in antifungal activity was not observed with EPA-supplemented palm or EPA-supplemented soybean oil . These results suggest that two factors, DHA and palm oil in combination, are involved in reducing the host resistance . DHA-enriched palm oil was also responsible for an increase in DHA concentration and a marked decrease in arachidonic acid content in the spleen and liver . However, this group did not show elevated spleen and liver phospholipid hydroperoxide levels compared with the other groups, excluding the possibility that the reduction in antifungal activity observed with DHA-enriched palm oil is due to acceleration of in vivo lipid peroxidation . Greater infection-induced increases in spleen and serum interferon-gamma concentrations were observed in mice fed DHA-enriched palm oil compared with the other groups.

Genet Mol Res, 2003 Mar 31, 2(1), 169 - 77
Mining microorganism EST databases in the quest for new proteins; Faria-Campos AC et al.; Microorganisms with large genomes are commonly the subjects of single-round partial sequencing of cDNA, generating expressed sequence tags (ESTs) . Usually there is a great distance between gene discovery by EST projects and submission of amino acid sequences to public databases . We analyzed the relationship between available ESTs and protein sequences and used the sequences available in the secondary database, clusters of orthologous groups (COG), to investigate ESTs from eight microorganisms of medical and/or economic relevance, selecting for candidate ESTs that may be further pursued for protein characterization . The organisms chosen were Paracoccidioides brasiliensis, Dictyostelium discoideum, Fusarium graminearum, Plasmodium yoelii, Magnaporthe grisea, Emericella nidulans, Chlamydomonas reinhardtii and Eimeria tenella, which have more than 10,000 ESTs available in dbEST . A total of 77,114 protein sequences from COG were used, corresponding to 3,201 distinct genes . At least 212 of these were capable of identifying candidate ESTs for further studies (E . tenella) . This number was extended to over 700 candidate ESTs (C . reinhardtii, F . graminearum) . Remarkably, even the organism that presents the highest number of ESTs corresponding to known proteins, P . yoelii, showed a considerable number of candidate ESTs for protein characterization (477) . For some organisms, such as P . brasiliensis, M . grisea and F . graminearum, bioinformatics has allowed for automatic annotation of up to about 20% of the ESTs that did not correspond to proteins already characterized in the organism . In conclusion, 4093 ESTs from these eight organisms that are homologous to COG genes were selected as candidates for protein characterization.

Rev Soc Bras Med Trop, 2003 May-Jun, 36(3), 427 - 30 Epub 2003 Jul 31.
{Evaluation of cytopathologic exam for diagnosis of oral chronic paracoccidioidomycosis}; de Araujo MS et al.; With the aim of evaluating exfoliative cytology for the diagnosis of paracoccidioidomycosis oral lesions, eight patients that presented the disease were studied . The presence of fungi was demonstrated in all these cases . It was concluded that the oral exfoliative cytology exam can be effectively used in the diagnosis of paracoccidioidomycosis and contribute to the therapeutic control of oral forms of this mycosis.

Crit Rev Oral Biol Med, 2003, 14(4), 268 - 74
Paracoccidioidomycosis of the mouth: an emerging deep mycosis; Almeida OP et al.; Oral fungal infections (mycoses) have come into particular prominence since the advent of infection with Human Immunodeficiency Virus (HIV), and recognition of the Acquired Immune Deficiency Syndrome (AIDS), as well as the phenomenal increase in world travel with increased exposure to infections endemic in the tropics . Paracoccidioidomycosis is a rare mycosis worldwide but common in Brazil and some other areas in Latin America . It can be life-threatening and can manifest with a spectrum of clinical presentations, including frequent oral lesions . This paper reviews the more recent information on Paracoccidioidomycosis, emphasizing those areas most relevant in dental science.

Fungal Genet Biol, 2003 Aug, 39(3), 204 - 10
Functional and genetic characterization of calmodulin from the dimorphic and pathogenic fungus Paracoccidioides brasiliensis; de Carvalho MJ et al.; 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.

Braz J Med Biol Res, 2003 Aug, 36(8), 1073 - 8 Epub 2003 Jul 23.
Immunohistochemical demonstration of TGF-beta and decorin in paracoccidioidal granulomas; Nishikaku AS et al.; Different patterns of granulomas have been observed in 6- to 8-week-old mice after ip inoculation with 5 x 10(6) yeast cells of Paracoccidioides brasiliensis . Transforming growth factor-beta (TGF-beta) is a cytokine that has been shown to participate in fibrosis and granuloma formation; its activities seem to be modulated by the small proteoglycan decorin . In the present study, TGF-beta and decorin expression in epiploon granulomas was assessed by immunohistochemistry in susceptible (B10.A) and resistant (A/J) mice after 15, 30, 120 and 150 days of P . brasiliensis ip infection . The epiploon was collected, fixed in Methacarn solution and embedded in paraffin, and 5-microm thick sections were used for immunohistochemical analysis employing the streptavidin-biotin-peroxidase technique . The former mouse strain developed fatal disease with many disseminated lesions increasing in size and number during the infection and the latter developed mild disease with the presence of encapsulated granulomas . In the epiploon, TGF-beta was present on macrophages, giant cells, lymphocytes and fibroblasts, and absent on neutrophils . It was also detected in areas of fibrosis and necrosis, as well as disperse in amorphous extracellular matrix, mostly in resistant mice . Decorin was present circumscribing macrophages and giant cells containing fungi, but absent on these cells . In both mouse strains, decorin was found at the periphery of the lesions, and markedly in milky spot granulomas . In resistant mice, positivity was found around fibrotic and necrotic areas of encapsulated and residual lesions containing lysed fungi . Decorin was found associated with thick fibers around encapsulated lesions . In susceptible mice, the size and number of lesions increased with the progression of the disease and were correlated with the weaker expression of decorin . We suggest an association of decorin with the fibrogenic process observed in paracoccidioidal granulomas.

Am J Pathol, 2003 Aug, 163(2), 583 - 90
Chemokine production and leukocyte recruitment to the lungs of Paracoccidioides brasiliensis-infected mice is modulated by interferon-gamma; Souto JT et al.; Chemokines and chemokine receptors play a role in cell recruitment during granulomatous inflammatory reactions . Here, we evaluated the expression of chemokines and chemokine receptors and their regulation by IFN-gamma in the course of Paracoccidioides brasiliensis (Pb) infection in mice . We found an association between KC and MIP-1alpha (CCL3) production and neutrophil infiltration in the lungs of Pb-infected mice during the early acute phase of infection . High levels of RANTES/CCL5, MCP-1/CCL2, IP-10/CXCL10, and Mig/CXCL9 simultaneously with mononuclear cell infiltration in the lungs was found . In the absence of IFN-gamma (GKO mice) we observed increased production of KC and MIP-1alpha and chronic neutrophilia . Moreover, we found a change in the chemokine receptor profiles expressed by wild-type (WT) versus GKO animals . Increased expression of CXCR3 and CCR5, and low levels of CCR3 and CCR4 were observed in the lungs of Pb-infected WT mice, whereas the opposite effect was observed in the lungs of GKO mice . Consistent with these results, infected cells from WT mice preferentially migrated in response to IP-10 (CXCR3 ligand), while those from GKO mice migrated in response to eotaxin/CCL11 (CCR3 ligand) . These results suggest that IFN-gamma modulates the expression of chemokines and chemokine receptors as well as the kind of cells that infiltrate the lungs of Pb-infected mice.

Protein Eng, 2003 Jun, 16(6), 435 - 41
Role of tryptophan 121 in the soluble CuA domain of cytochrome c oxidase: structure and electron transfer studies; Song AX et al.; To investigate the contribution of tryptophan-121 (Trp121) residue to the structure and function of soluble CuA domain of cytochrome c oxidase, three mutant proteins, Trp121Tyr, Trp121Leu and Trp121-deleted mutant of the soluble domain of Paracoccus versutus cytochrome c oxidase, were constructed and expressed in Escherichia coli BL21 (DE3) . Optical spectral studies showed that both the coordination structure of the CuA center and the secondary structure of the protein were changed significantly in the Leu substitution and deletion mutants of Trp121 . Their electron transfer activity with cytochrome c was inhibited severely, as shown in stopped-flow kinetic studies . However, the CuA center can be reconstructed in the Trp121Tyr mutant although its stability decreases compared with the wild-type protein . This mutant keeps the same secondary structure as the wild-type protein, but can only transfer electrons with cytochrome c at a rate of one-seventh-fold . Based on the information on the structure, we also investigated and analyzed the possible factors that affect electron transfer . It appears that the aromatic ring, the size of the side chain and the hydrogen bonding ability of the Trp121 are crucial to the structure and function of the soluble CuA domain.

Yeast, 2003 Jul 30, 20(10), 865 - 80
The pfr1 gene from the human pathogenic fungus Paracoccidioides brasiliensis encodes a half-ABC transporter that is transcribed in response to treatment with fluconazole; Gray CH et al.; We have isolated a gene that encodes a half-ABC-transporter, designated Pfr1, from the dimorphic human pathogenic fungus Paracoccidioides brasiliensis, which has high identity with members of the ABC-superfamily involved in multidrug resistance . The pfr1 gene is predicted to encode a 827 amino acid protein that, in common with mammalian Mdr1, has a TM-NBD topology . The transcription of the pfr1 gene is induced by the triazole drug fluconazole but not by amphotericin B, suggesting a role in transport-mediated azole resistance . However, Pfr1 has greatest identity to the mitochondrial ABC transporters Mdl1 and Mdl2 from Saccharomyces cerevisiae and mammalian ABC-me, with identities of 47.2%, 40.6% and 39.5%, respectively, over the length of these proteins . Furthermore, the N-terminus of Pfr1 is rich in positively charged residues, a feature of mitochondrial targeting sequences . Considering these features, it seems likely that Pfr1 is a mitochondrial protein . Previous studies have revealed that the acquisition of azole resistance in S . cerevisiae is linked to mitochondrial loss and, conversely, that mitochondrial dysfunction can lead to the upregulation of PDR transporters mediated by the transcription factor Pdr3 . Our studies suggest that a mitochondrial ABC transporter is induced as part of the cellular response to drug treatment . The promoter region of pfr1 contains a PDRE-like consensus sequence to which Pdr3 binds, which may be the element responsible for the upregulation of Pfr1 in response to fluconazole . The nucleotide binding domain of Pfr1 was expressed and purified from Escherichia coli and shown to retain ATPase activity, consistent with Pfr1 functioning as a homodimeric transport ATPase .

Exp Parasitol, 2002 Nov-Dec, 102(3-4), 170 - 7
Involvement of prostaglandins in the immunosuppression occurring during experimental infection by Paracoccidioides brasiliensis; Michelin MA et al.; We investigated whether PGE2 mediates the immunosuppression observed during Paracoccidioides brasilensis infection . Con-A-stimulated splenocytes, isolated from mice on days 15 and 60 of infection, release high amounts of PGE2, this release was inhibited by the treatment of animals with indomethacin, sodium salicylate or meloxicam . The treatment of the animals with salicylate or meloxicam, but not indomethacin, enhanced the release of IL-2 by splenocytes from animals on day 15, but not on day 60 of infection . Furthermore, we demonstrated that the productions of TNF-alpha, IFN-gamma, IL-4, and IL-10 by Con-A-stimulated splenocytes from mice at 15 days of infection were inhibited by treatment with salicylate or meloxicam . Indomethacin inhibited only TNF-alpha and IFN-gamma production . The three treatments caused reduction of granuloma areas in the liver and lungs of infected mice . In conclusion, results suggest that the PGE2 released by COX-2 mediates the immunosuppression early on (day 15), but not during the later phase (60 days) of P . brasiliensis infection by a mechanism dependent upon IL-4 and IL-10.

J Clin Microbiol, 2003 Jul, 41(7), 3419 - 22
Evaluation of fungal burden in experimental paracoccidioidomycosis by using the fluorescent dye Blankophor; Nishikaku AS et al.; The fungal load in organs and blood of susceptible and resistant mice infected with Paracoccidioides brasiliensis was quantitated by using the optical brightener Blankophor and compared with CFU counts . Fluorescent staining of fungal cells proved to be a quick and easy procedure, suitable for evaluation of paracoccidioidomycotic infection.

J Clin Microbiol, 2003 Jul, 41(7), 2849 - 54
Randomly amplified polymorphic DNA as a valuable tool for epidemiological studies of Paracoccidioides brasiliensis; Hahn RC et al.; Randomly amplified polymorphic DNA (RAPD) has been successfully used to detect genetic variations among isolates of Paracoccidioides brasiliensis . However, the usefulness of this technique for assessing important parasitic properties is still unconfirmed . In the present work we further investigated the applicability of RAPD in revealing important intrinsic and extrinsic features of this fungus associated with geographical origin, time of isolation, source of clinical specimen, clinical forms of human disease and also in vitro and in vivo susceptibility to antimicrobial and antifungal drugs . The RAPD patterns allowed us to distinguish all of the analyzed strains, which included 26 clinical isolates, 2 animal isolates, and 1 environmental isolate of P . brasiliensis obtained from different geographic regions, confirming the strong discriminating power of this technique . A phenetic tree, build from the RAPD data, showed that although the two nonclinical Brazilian strains were set together the majority of the clinical Brazilian strains were randomly distributed through different sub-branches of a major cluster without any correlation to any of the parameters analyzed . A second major cluster, however, has grouped isolates from Mato Grosso and Roraima (Brazil) that not only were susceptible in vitro to trimethoprim-sulfamethoxazole but also produced a good in vivo response . These results open new vistas for epidemiological and clinical studies of P . brasiliensis.

Med Microbiol Immunol (Berl), 2004 Feb, 193(1), 53 - 9 Epub 2003 Jun 28.
Paracoccidioidomycosis: reduction in fungal load and abrogation of delayed-type hypersensitivity anergy in susceptible inbred mice submitted to therapy with trimethoprim-sulfamethoxazole; Scavone R et al.; Isogenic mouse strains have previously been characterized as susceptible or resistant to Paracoccidioides brasiliensis infection; the former presented anergy in delayed-type hypersensitivity reactions (DTH) and progressive disease with high numbers of colony-forming units (CFU), while the later presented preserved DTH responses and control of the infectious process . Here, we studied whether susceptible mice infected with P . brasiliensis and treated with the antifungal drug trimethoprim-sulfamethoxazole (SXT) had their behavior pattern altered to the one observed in infected resistant mice . Therapy with either 30 or 150 mg SXT day(-1 )kg(-1), instituted 24 h after infection, elicited more adequate DTH responses than those of non-treated mice, and also diminished the number of viable fungi in the spleen and lungs, but not in epiploo and liver, indicating a partial control of the infectious process . This phenomenon was confirmed by histopathological analyses, in which the spleen was found to be the organ in which differences between the treated and non-treated groups were most remarkable . In control non-treated mice, the spleen parenchyma showed multiple granulomatous foci presenting giant cells, plasmocytes and many yeasts of P . brasiliensis with well-preserved morphology and abundant budding, whereas SXT-treated mice, independently of the dosage used, had no granulomas within the parenchyma and only few capsular lesions, mainly composed of pseudoxantomatous macrophages . Treatment with 150 mg day(-1 )kg(-1) (the dose considered to evoke best responses in CFU assays when therapy was instituted 24 h after infection), initiated at different times after infection, did not led to sustained DTH reactions, but provided an effective control of the disease when therapy began until the 15(th) day post infection, as showed by CFU assays . We conclude that reversal from the susceptible to the resistant pattern in experimental paracoccidioidomycosis can occur, but only when therapy with an adequate SXT dosage is instituted at a very initial phase of the infection . These protocols may constitute a model for further investigations concerning responses during antifungal therapy.

Cytokine, 2003 Mar 7, 21(5), 234 - 41
Immunocytochemical localization of cytokines and inducible nitric oxide synthase (iNOS) in oral mucosa and lymph nodes of patients with paracoccidioidomycosis; Neworal EP et al.; Paracoccidioidomycosis (PCM) is a deep mycosis caused by Paracoccidioides brasiliensis, with high incidence in Brazil . In order to examine the immune response in lesional tissue from patients with PCM, we analyzed cytokines as well as the phenotype of the cell infiltrate . Paraffin-embedded tissue from the oral mucosa of eight patients with the localized adult form (AF) of PCM and from the lymph nodes of 10 patients with the juvenile form (JF) of PCM was analyzed by immunohistochemistry to detect tumor necrosis factor-alpha (TNF-alpha), inducible nitric oxide synthase (iNOS), transforming growth factor-beta (TGF-beta) and interleukin-10 (IL-10) . Most of the inflammatory cells in the lymph nodes were CD68+ (macrophages, epithelioid and giant cells), while a mixed infiltrate with macrophages, plasma cells and neutrophils was detected in the oral mucosa . TNF-alpha as well as iNOS expression was similar in lymph nodes and oral mucosa, whereas TGF-beta and IL-10 were observed in a larger number of macrophages, epithelioid and giant cells in the lymph nodes, where numerous yeast cells were visualized . The higher expression of anti-inflammatory cytokines (IL-10 and TGF-beta) in lesions of patients with the JF of PCM (lymph nodes) may represent a mechanism by which the fungus evades the host immune response, contributing to a more severe and disseminated form of the disease.

Mol Microbiol, 2003 Jul, 49(1), 69 - 79
Electron transport to periplasmic nitrate reductase (NapA) of Wolinella succinogenes is independent of a NapC protein; Simon J et al.; The rumen bacterium Wolinella succinogenes grows by respiratory nitrate ammonification with formate as electron donor . Whereas the enzymology and coupling mechanism of nitrite respiration is well known, nitrate reduction to nitrite has not yet been examined . We report here that intact cells and cell fractions catalyse nitrate and chlorate reduction by reduced viologen dyes with high specific activities . A gene cluster encoding components of a putative periplasmic nitrate reductase system (napA, G, H, B, F, L, D) was sequenced . The napA gene was inactivated by inserting a kanamycin resistance gene cassette . The resulting mutant did not grow by nitrate respiration and did not reduce nitrate during growth by fumarate respiration, in contrast to the wild type . An antigen was detected in wild-type cells using an antiserum raised against the periplasmic nitrate reductase (NapA) from Paracoccus pantotrophus . This antigen was absent in the W . succinogenes napA mutant . It is concluded that the periplasmic nitrate reductase NapA is the only respiratory nitrate reductase in W . succinogenes, although a second nitrate-reducing enzyme is apparently induced in the napA mutant . The nap cluster of W . succinogenes lacks a napC gene whose product is thought to function in quinol oxidation and electron transfer to NapA in other bacteria . The W . succinogenes genome encodes two members of the NapC/NirT family, NrfH and FccC . Characterization of corresponding deletion mutants indicates that neither of these two proteins is required for nitrate respiration . A mutant lacking the genes encoding respiratory nitrite reductase (nrfHA) had wild-type properties with respect to nitrate respiration . A model of the electron transport chain of nitrate respiration is proposed in which one or more of the napF, G, H and L gene products mediate electron transport from menaquinol to the periplasmic NapAB complex . Inspection of the W . succinogenes genome sequence suggests that ammonia formation from nitrate is catalysed exclusively by periplasmic respiratory enzymes.

Mycoses, 2003 Jun, 46(5-6), 229 - 32
Paracoccidioidomycosis and larynx carcinoma; Maymo Arganaraz M et al.; Report of a case of paracoccidioidomycosis associated with a carcinoma: both located in the larynx in a patient whose therapeutic response to antifungal treatment produced a recovery of physical conditions . This case shows the importance of taking into account the diagnosis of paracoccidioidomycosis in all patients with problems in the larynx, especially those who inhabit or inhabited endemic areas of Paracoccidioides brasiliensis.

Can J Microbiol, 2003 Mar, 49(3), 197 - 206
Nitrate reductase from the magnetotactic bacterium Magnetospirillum magnetotacticum MS-1: purification and sequence analyses; Taoka A et al.; We purified the nitrate reductase from the soluble fraction of Magnetospirillum magnetotacticum MS-1 . The enzyme was composed of 86- and 17-kDa subunits and contained molybdenum, non-heme iron, and heme c . These properties are very similar to those of the periplasmic nitrate reductase found in Paracoccus pantotrophus . The M . magnetotacticum nap locus was clustered in seven open reading frames, napFDAGHBC . The phylogenetic analyses of NapA, NapB, and NapC suggested a close relationship between M . magnetotacticum nap genes and Escherichia coli nap genes, which is not consistent with the 16S rDNA data . This is the first finding that the alpha subclass of Proteobacteria possesses a napFDAGHBC-type nap gene cluster . The nap gene cluster had putative fumarate and nitrate reduction regulatory protein (Fnr) and NarL protein binding sites . Furthermore, we investigated the effect of molybdate deficiency in medium on the total iron content of the magnetosome fraction and discussed the physiological function of nitrate reductase in relation to the magnetite synthesis in M . magnetotacticum.

Biosci Biotechnol Biochem, 2003 Apr, 67(4), 720 - 8
Purification and characterization of formate dehydrogenase from Ancylobacter aquaticus strain KNK607M, and cloning of the gene; Nanba H et al.; Ancylobacter aquaticus strain KNK607M, which had high NAD-dependent formate dehydrogenase (FDH) activity, was newly isolated . The enzyme, purified to homogeneity, was a dimer composed of identical subunits with a molecular mass of 44 kDa . The specific activity was 9.5 u/mg, and the enzyme was optimum at pH 6.3 and 50 degrees C, most stable at pH 7.0, and stable at 50 degrees C or lower . The apparent Km values for formate and NAD+ were 2.4 and 0.057 mM, respectively . The enzyme was specific to formate and was inhibited by SH reagents and heavy metal ions . The cloned gene of FDH contained one open reading frame (ORF) of 1206 base pairs, predicted to encode a polypeptide of 401 amino acids, with a calculated molecular weight of 43,895; this gene was highly expressed in E . coli cells . The FDH had high identity to other FDHs, i.e., those of Pseudomonas, Mycobacterium, Moraxella, and Paracoccus, which were 91.3%, 90.8%, 84.2%, and 82.3%, respectively.

Microbiology, 2003 Jun, 149(Pt 6), 1533 - 40
Characterization of the expression and activity of the periplasmic nitrate reductase of Paracoccus pantotrophus in chemostat cultures; Ellington MJ et al.; The periplasmic nitrate reductase (Nap) from Paracoccus pantotrophus has a role in cellular redox balancing . Previously, transcription from the nap promoter in P . pantotrophus was shown to be responsive to the oxidation state of the carbon substrate . During batch culture, expression was higher during growth on reduced substrates such as butyrate compared to more oxidized substrates such as succinate . In the present study the effect of growth rate on nap expression in succinate-, acetate- and butyrate-limited chemostat cultures was investigated . In all three cases transcription from the nap promoter and Nap enzyme activity showed a strong correlation . At the fastest growth rates tested for the three substrates nap expression and Nap activity were highest when growth occurred on the most reduced substrate (butyrate > acetate > succinate) . However, in all three cases a bell-shaped pattern of expression was observed as a function of growth rate, with the highest levels of nap expression and Nap activity being observed at intermediate growth rates . This effect was most pronounced on succinate, where an approximately fivefold variation was observed, and at intermediate dilution rates nap expression and Nap activity were comparable on all three carbon substrates . Analysis of mRNA prepared from the succinate-grown cultures revealed that different transcription initiation start sites for the nap operon were utilized as the growth rate changed . This study establishes a new regulatory feature of nap expression in P . pantotrophus that occurs at the level of transcription in response to growth rate in carbon-limited cultures.

Nippon Ishinkin Gakkai Zasshi, 2003, 44(2), 133 - 8
The role of chlamydospores of Paracoccidioides brasiliensis; Miyaji M et al.; The role of chlamydospores in the conversion process from a mycelial-to-yeast form using the slide culture method was studied . Three clinical isolates and two other isolates from armadillo, belonging to the fungal species Paracoccidioides brasiliensis, were cultured on Sabouraud dextrose agar (SDA), potato dextrose agar (PDA) and brain heart infusion dextrose agar (BHIDA) . Initially, the mycelial forms of each isolate were grown at 25 degrees C for 7, 14, 30 or 60 days on slide cultures and then the temperature was shifted to 35 degrees C . Interestingly, the slide cultures of all the isolates at 25 degrees C formed chlamydospores on either SDA or BHIDA, whereas, on PDA medium, aleurioconidia were formed . If the slide cultures on BHIDA were incubated at 35 degrees C for 7 to 14 days, multiple budding forms could be observed . This phenomenon was not evident in the slide cultures of SDA or PDA . The results of this morphological study indicate that in P . brasiliensis, chlamydospores may play an important role in the conversion process from a mycelial-to-yeast form.

J Med Microbiol, 2003 Jun, 52(Pt 6), 479 - 81
Biosynthesis of chondroitinase and hyaluronidase by different strains of Paracoccidioides brasiliensis; de Assis CM et al.; The biosynthesis of chondroitinase and hyaluronidase by different isolates of Paracoccidioides brasiliensis was investigated in 20 strains isolated from patients (17 strains), a penguin (Pygocelis adeliae, one strain), an armadillo (Dasypus novemcinctus, one strain) and the environment (dog food, one strain) . All the P . brasiliensis isolates studied had the ability to produce chondroitinase and hyaluronidase, although differences in colony morphology and enzyme production were detected among them . These results suggest that further investigations should be carried out in the clinical field in order to clarify the potential role of P . brasiliensis enzyme production in the pathogenesis of paracoccidioidomycosis.

Fungal Genet Biol, 2003 Jun, 39(1), 60 - 9
Chromosomal polymorphism, syntenic relationships, and ploidy in the pathogenic fungus Paracoccidioides brasiliensis; Feitosa Ldos S et al.; Pulsed field gel electrophoresis (PFGE) and DNA hybridization were used to establish and compare the electrophoretic karyotypes of 12 clinical and environmental Paracoccidioides brasiliensis isolates from different geographic areas . Gene mapping allowed the identification of synteny groups and the use of isolated whole chromosomal bands to probe chromoblots indicated the existence of repetitive sequences, contributing to a better understanding of the structure and organization of the fungus genome . This represents the first comparative mapping study among different isolates . The results are indicative of the existence of genetic differences among natural isolates . DNA content of DAPI-stained nuclei of each isolate was estimated by confocal microscopy . Comparison of the genome sizes estimated by PFGE with those calculated by microfluorometry indicated the possible existence of haploid and diploid (or aneuploid) isolates of the fungus.

Microbes Infect, 2003 Apr, 5(5), 413 - 8
Production of pro- and anti-inflammatory cytokines by monocytes from patients with paracoccidioidomycosis; Peracoli MT et al.; Monocytes and macrophages can produce a large repertoire of cytokines and participate in the pathogenesis of granulomatous diseases . We investigated the production of pro- and anti-inflammatory cytokines by monocytes from patients with active paracoccidioidomycosis . Peripheral blood monocytes from 37 patients and 29 healthy controls were cultivated with or without 10 microg/ml of lipopolysaccharide (LPS) for 18 h at 37 degrees C, and the cytokine levels were determined in the culture supernatants by enzyme immunoassay . The results showed that the endogenous levels of tumor necrosis factor alpha (TNF-alpha), interleukin-1beta (IL-1beta), IL-6, IL-8, IL-10 and transforming growth factor beta detected in the supernatant of patient monocytes cultivated without stimulus were significantly higher than those produced by healthy controls . These data demonstrated that monocytes from patients with active paracoccidioidomycosis produce high levels of cytokines with both inflammatory and anti-inflammatory activities . However, patient monocytes produced significantly lower TNF-alpha and IL-6 levels in response to LPS when compared to normal subjects, suggesting an impairment in their capacity to produce these cytokines after LPS stimulation . Concentrations of IL-1beta, IL-8 and IL-10 in cultures stimulated with LPS were higher in patients than in controls . These results suggest that an imbalance in the production of pro- and anti-inflammatory cytokines might be associated with the pathogenesis of paracoccidioidomycosis.

J Infect Dis, 2003 May 1, 187(9), 1496 - 505 Epub 2003 Apr 15.
Fas-Fas ligand (CD95-CD95L) and cytotoxic T lymphocyte antigen-4 engagement mediate T cell unresponsiveness in patients with paracoccidioidomycosis; Campanelli AP et al.; The mechanism that leads to the remarkable T cell unresponsiveness to antigens in paracoccidioidomycosis is unknown . We investigated the involvement of cytokines, of Fas-Fas ligand (Fas-FasL)-induced apoptosis, and of cytotoxic T lymphocyte antigen 4 (CTLA-4) engagement, in the mediation of this phenomenon . T cell unresponsiveness was not associated with imbalanced cytokine production or with absence of CD28 expression . Only patient T cells expressed higher levels of CTLA-4, Annexin V(+), and FasL . The addition of anti-FasL decreased the levels of apoptosis, suggesting an activation-induced cell death triggered through the Fas-FasL pathway . Blockage of CTLA-4 and FasL resulted in increased production of interferon-gamma . Moreover, concomitant inhibition of FasL and of CTLA-4, but not of transforming growth factor-beta, resulted in significant T cell proliferation in patients, in response to phytohemagglutinin . Together, these data show that apoptosis mediated by Fas-FasL and engagement of CTLA-4 are involved in modulation of the immune response in patients infected with Paracoccidioides brasiliensis.

Rev Iberoam Micol, 2002 Mar, 19(1), 49 - 51
Characterization of Paracoccidioides brasiliensis atypical isolates by random amplified polymorphic DNA analysis; Hahn RC et al.; Two atypical Paracoccidioides brasiliensis strains (yeast form at room temperature) have been isolated from chronically infected patients living in Brazil . Different random primers were used to characterize these isolates and compare them to typical strains . The RAPD patterns allowed the differentiation of all the selected isolates . Their genetic distance ranged from 5% to 80% of non-shared bands depending on the strains and the primer used . The RAPD data were used to build a Wagner phenogram, which showed two major branched with more than 56% of genetic distance separating them . No significant difference was observed between the atypical isolates and the others suggesting that specific genes are involved in the dimorphism phenomenon.

Rev Soc Bras Med Trop, 2003 Jan-Feb, 36(1), 11 - 6 Epub 2003 Apr 22.
{Prevalence of paracoccidioidomycosis infection determined by sorologic test in donors' blood in the Northwest of Paraná, Brazil}; Maluf ML et al.; In this paper the classical ELISA test was used, which is a highly sensitive method, with the objective of estimating the anti-Paracoccidioides brasiliensis antibodies level in healthy people, residing in supposedly endemic regions for PCM, Northwest Paran State, Brazil . The sera was analyzed from 680 blood donors' who were approved by the classical serologic screening by blood banks . Four cities were studied (Campo Mour o, Maring , Cianorte and Umuarama) with a sample of 170 donors each . The antigen used was Ag7days, diluted serum 1/400, anti human IgG-conjugate marked with peroxidase and revealed with O-Phenylenediamine . It was possible to detect 181 (27%) sera with antibody level above the cut off threshold . The percentage varied between the cities . These results allied with the favorable climatic and geographical conditions and also previous reports of PCM indigenous to the region, indicate that the Northwest of Paran is an endemic region for PCM.

Int J Syst Evol Microbiol, 2003 Mar, 53(Pt 2), 443 - 7
Paracoccus seriniphilus sp . nov., an L-serine-dehydratase-producing coccus isolated from the marine bryozoan Bugula plumosa; Pukall R et al.; A novel marine Gram-negative, non-motile, non-spore-forming, aerobic bacterium, associated with the bryozoan Bugula plumosa, was isolated in a screening programme for strains containing enzymes able to convert the amino acid L-serine . Strain MBT-A4T produced L-serine dehydratase and was able to grow on L-serine as the sole carbon and nitrogen source . The nearest phylogenetic neighbour was Paracoccus marcusii, as determined by 16S rDNA sequence analysis (97.8% similarity) . The DNA-DNA reassociation value obtained for Paracoccus marcusii DSM11574T and MBT-A4T was 32.6% . The major ubiquinone was 0-10 . Based on genotypic, chemotaxonomic and physiological characteristics, a new species of the genus Paracoccus is proposed, Paracoccus seriniphilus sp . nov., the type strain being strain MBT-A4T (=DSM 14827T =CIP 107400T).

Eur J Nucl Med Mol Imaging, 2003 Jun, 30(6), 888 - 94 Epub 2003 Apr 23.
The role of gallium-67 scan in defining the extent of disease in an endemic deep mycosis, paracoccidioidomycosis: a predominantly multifocal disease; Yamaga LY et al.; The tools currently used to evaluate the extent of paracoccidioidomycosis (PCM) may be of limited value in detecting subclinical lesions . The aim of this study was to verify the role of gallium-67 whole-body scan in evaluating the extent of disease of 65 patients with active PCM . The (67)Ga scan findings were compared with the results of clinical evaluation, chest radiography and/or high-resolution computed tomography (CT), abdominal ultrasound (US) or CT, laryngoscopy, CT or magnetic resonance imaging (MRI) of the head, and technetium-99m methylene diphosphonate bone scan, obtained before treatment . Clinically unsuspected lesions were detected by imaging procedures in 21 patients (32%), mainly in the lungs (n=11), adrenals (n=6), and superficial (n=3) and deep lymph nodes (n=14) . (67)Ga scan detected 100% of the cases with subclinical involvement in the lungs . Scintigraphy was superior to chest radiography in demonstrating lung disease (94% vs 81%) . The lymphatic lesions were demonstrated by (67)Ga scan in all the clinically suspected cases and in nearly all unsuspected cases, and also revealed more extensive involvement than was clinically suspected in many of them . There was good agreement between (67)Ga scan and the other imaging procedures for the initial detection of thoracic and abdominal lymph nodes and bone involvement . (67)Ga imaging detected most cases of laryngopharyngeal disease with active inflammatory lesions found at indirect laryngoscopy . On the other hand, (67)Ga scan failed to demonstrate most of the adrenal and CNS lesions detected by abdominal US/CT and head CT/MRI . In conclusion, (67)Ga imaging is a useful tool for evaluating the location and extent of suspected and unsuspected lesions in PCM . It could serve as a screening method before the use of other diagnostic procedures, particularly in the detection of lung, superficial and deep lymph node and bone involvement.

Cad Saude Publica, 2003 Jan-Feb, 19(1), 245 - 53 Epub 2003 Apr 01.
{Association between smoking and paracoccidioidomycosis: a case-control study in the State of Espírito Santo, Brazil}; dos Santos WA et al.; Paracoccidioidomycosis, the main systemic mycosis in Brazil, requires long-term, high-cost treatment and leaves serious sequelae in the lungs, the organ most frequently affected and further subject to aggressive external risk factors like smoking . The influence of tobacco and alcohol consumption on chronic paracoccidioidomycosis was investigated using a case-control study . Data on occupation, place of residence, and living habits were obtained from 70 cases and 180 controls residing in the same geographic areas . The risk of becoming ill was 14 times greater among smokers and 3.6 times greater among individuals with an alcohol intake of more than 50 g/day . Logistic regression showed as significant variables: tobacco consumption for more than 20 years (OR = 10.1), smoking manufactured (not hand-rolled) cigarettes (OR = 4.8), and alcohol intake > 50 g/day (OR = 2.9) . Cases who smoked 20 or more cigarettes/day became ill on average eight years before others (p = 0.002) . Alcohol intake > 50 g/day had no statistically significant impact on age at onset of illness (p = 0.78) . The study concludes that smoking stands as an important risk factor for the development of chronic paracoccidioidomycosis . As for alcoholism, there is evidence that it acts as a co-factor, together with smoking.

J Clin Microbiol, 2003 Apr, 41(4), 1536 - 42
Combined use of Paracoccidioides brasiliensis recombinant 27-kilodalton and purified 87-kilodalton antigens in an enzyme-linked immunosorbent assay for serodiagnosis of paracoccidioidomycosis; Diez S et al.; The diagnosis of paracoccidioidomycosis (PCM) has relied on the identification of the host's humoral response by using a variety of immunological methods, such as complement fixation and immunodiffusion . Although these approaches are useful, historically their sensitivity and specificity have often been compromised by the use of complex mixtures of undefined antigens . The use of combinations of purified, well-characterized antigens appears preferable and may yield optimum results . Accordingly an indirect enzyme-linked immunosorbent assay (ELISA) using combinations of the previously described 27-kDa recombinant antigen and the 87-kDa heat shock protein were used for diagnosis and follow-up of patients with PCM . A total of 37 patients classified according to their clinical presentations (7 with the acute or subacute form of the disease, 22 with the chronic form of the disease, and 8 with the chronic unifocal form) were studied . Eighteen of these patients were also evaluated at every follow-up appointment . Forty serum samples from patients with other diseases and 50 serum samples from healthy individuals were also studied . Detection of anti-27-kDa and anti-87-kDa antibodies in sera of patients with PCM by ELISA using a combination of the two purified proteins showed a sensitivity of 92% with a specificity of 88% in comparison with normal human sera and 90% in comparison with the heterologous sera . These results demonstrated a significant increase in sensitivity and specificity compared to results when the antigens were used separately . Thus, the use of combinations of well-defined antigens appears to offer clear advantages over the use of single antigens when diagnosing PCM.

Med Microbiol Immunol (Berl), 2003 Nov, 192(4), 225 - 9 Epub 2003 Apr 08.
Programmed cell death in thymus during experimental paracoccidioidomycosis; Souto PC et al.; Many works have shown that immunosuppressive effects induced by systemic mycosis can be related to primary lymphoid organ damage . Previous studies in our laboratory showed that Paracoccidioides brasiliensis was able to invade the thymus, inducing a severe atrophy with significant reduction of cortical area along with a loss of cortico-medullary boundary . The objective of the present study was to investigate whether thymic atrophy is caused by programmed cell death (PCD) and to examine the ultrastructural characteristics of the thymus in experimentally infected BALB/c mice . The results revealed an eightfold increase in the apoptotic index occurring by day 5 post infection, i.e., during early stages of the infection, shown by immunohistochemistry . In addition, typical cell alterations of autophagic PCD were observed by transmission electron microscopy . Taken together, these results reinforce the idea that thymic alterations may be involved in the immunosuppressive phenomenon frequently associated with paracoccidioidomycotic infection.

Am J Trop Med Hyg, 2003 Mar, 68(3), 301 - 3
Case report: severe juvenile type paracoccidioidomycosis with hepatitis C; Pellegrino A et al.; We present the case of a 34-year-old man with previous history of hepatitis C and severe juvenile type of paracoccidioidomycosis characterized by involvement of the reticuloendothelial system, eosinophilia, lung compromise (pleural effusion), retinal hemorrhage, and blood culture positive to Paracoccidioides brasiliensis . The immune evaluation showed four precipitation lines to P . brasiliensis by the double immunodiffusion test . Treatment with amphotericin B and itraconazol resulted in resolution of the disease.

J Infect Chemother, 2003 Mar, 9(1), 16 - 20
The trend of imported mycoses in Japan; Kamei K et al.; Pathogenic fungi that are non-native in Japan are highly virulent and present a significant health hazard to persons in the environment into which they are introduced . Little is known, however, about the precise trend of infection by imported mycoses in Japan . To clarify this issue, all available cases were collected through a search of MEDLINE and Japana Centra Revuo Medicine and analyzed . Also included in this analysis were cases not reported in the literature for which the authors provided identification of the fungi, diagnosis, or treatment . The analysis revealed that, for three diseases, the number of imported mycoses cases in Japan is much higher than previously reported: 31 cases of coccidioidomycosis, 34 cases of histoplasmosis, and 17 cases of paracoccidioidomycosis . Additionally, one case of penicilliosis marneffei was found . The most rapid increase in the incidence of these mycoses occurred from 1991 to 1995 . Analysis of the patients' profiles provided the following information: (1) coccidioidomycosis infection in Japan is increasing very rapidly, (2) Japan might be an endemic area of histoplasmosis infection, (3) histoplasmosis is a potentially fatal disease; and (4) reliable serodiagnostic methods have been used only infrequently . Because of the increase of international travel and immigration, the incidence of imported mycoses in Japan is expected to continue rising, and mycoses that have never been reported in Japan, such as blastomycosis, might also be encountered in the near future . To cope with this newly emerging health problem to residents of Japan, the Japanese medical system must train its members to identify and treat mycoses.

Glycobiology, 2003 Nov, 13(11), 743 - 7 Epub 2003 Apr 02.
Structural differences between the alkali-extracted water-soluble cell wall polysaccharides from mycelial and yeast phases of the pathogenic dimorphic fungus Paracoccidioides brasiliensis; Ahrazem O et al.; Paracoccidioides brasiliensis is a pathogenic dimorphic fungus causing paracoccidioidomycosis, the most widespread systemic mycosis in Latin America . We have studied the structure of the alkali-extracted water-soluble cell wall polysaccharides (F1SS) from both mycelial and yeast phases of this fungus by using chemical analysis and NMR spectroscopic techniques . The F1SS polysaccharide from the mycelial phase consists of a trisaccharidic repeating unit of -->6)-{alpha-Galf -(1-->6)-alpha-Manp-(1-->2)}-alpha-Manp-(1--> . The F1SS polysaccharide of the yeast phase maintains 10% of the structure of the mycelium phase, but the main structure contain a disaccharide repeating unit of -->6)-{-alpha-Manp-(1-->2)}-alpha-Manp-(1-->, alternating with a trisaccharide repeating block of -->6)-{beta-Galf -(1-->6)-alpha-Manp-(1-->2)}-alpha-Manp-(1-->.

Antimicrob Agents Chemother, 2003 Apr, 47(4), 1376 - 81
In vitro antifungal activity of Micafungin (FK463) against dimorphic fungi: comparison of yeast-like and mycelial forms; Nakai T et al.; The characteristics of in vitro micafungin (FK463) antifungal activity against six species of dimorphic fungi were investigated in accordance with the NCCLS M27-A microdilution methods . MICs of micafungin, amphotericin B, itraconazole, and fluconazole for Histoplasma capsulatum var . capsulatum, Blastomyces dermatitidis, Paracoccidioides brasiliensis, Penicillium marneffei, and Sporothrix schenckii were determined both for the yeast-like form and mycelial form . Coccidioides immitis was tested only in its mycelial form . We have clearly demonstrated that the in vitro activity of micafungin depends considerably on the growth form of dimorphic fungi . Micafungin exhibited potent activity against the mycelial forms of H . capsulatum, B . dermatitidis, and C . immitis (MIC range, 0.0078 to 0.0625 micro g/ml), while it was very weakly active against their yeast-like forms (MIC range, 32 to >64 micro g/ml) . Micafungin was also more active against the mycelial forms than the yeast-like forms of Paracoccidioides brasiliensis, Penicillium marneffei, and S . schenckii . The MICs of amphotericin B were 2 to 5 dilutions lower for the mycelial forms than for the yeast-like forms of B . dermatitidis and Paracoccidioides brasiliensis . There was no apparent difference in the activity of itraconazole between the two forms . The MICs of fluconazole for the yeast-like forms were generally lower than those for the mycelial forms, and considerably so for B . dermatitidis . These results suggest that the growth form employed in antifungal susceptibility testing of dimorphic fungi can considerably influence the interpretation of results . At present, it cannot be judged whether micafungin has clinical usefulness for dimorphic fungus infections, since for most fungi it remains uncertain which growth form correlates better with therapeutic outcome . However, the results of this study warrant further investigations of micafungin as a therapeutic agent for infections caused by dimorphic fungi.

Am J Dermatopathol, 2003 Apr, 25(2), 107 - 12
Dendritic cells and pattern of cytokines in paracoccidioidomycosis skin lesions; Pagliari C et al.; We demonstrated and quantified by immunohistochemistry epidermal Langerhans cells, CD34+ dermal dendrocytes (DDs), and cells expressing TNFalpha, interferon-gamma (IFNgamma), IL-5, and IL-10 in skin lesions of paracoccidioidomycosis (PCM) . Sixty-one biopsies were classified in three groups according to the pattern of tissue response: Group 1, well-organized granuloma; Group 2, poorly organized granuloma; and Group 3, both kinds of granuloma . Langerhans cells had short and irregular dendrites in all groups and were decreased in number in Groups 1 and 2 . CD34+ DDs did not differ in number from the control group . Group 1 was characterized by many cells expressing IFNgamma . Groups 2 and 3 exhibited large numbers of cells expressing IL-5 and IL-10 . The data obtained suggest that well-organized granulomas reflect a better cellular immune response, and the large number of cells expressing IL-5 and IL-10 in Group 2 indicate an ineffective response in PCM skin lesions . Both kinds of granuloma in the same cutaneous lesion probably represent an intermediate response between the anergic and hyperergic poles . Group 3 also showed higher numbers of cells expressing TNFalpha when compared with the control group . Some cells expressing TNFalpha were dendritic and localized around the granuloma similar to the factor XIIIa+ DD localization that we previously described.

Microbes Infect, 2003 Feb, 5(2), 107 - 13
Effect of cytokines on the in vitro fungicidal activity of monocytes from paracoccidioidomycosis patients; Calvi SA et al.; Peripheral blood monocytes obtained from paracoccidioidomycosis patients and healthy individuals were preactivated with recombinant gamma interferon (IFN-gamma) in different concentrations (250, 500 and 1000 U/ml) and evaluated for fungicidal activity against Paracoccidiodes brasiliensis strain 18 (Pb 18, high-virulence strain) and strain 265 (Pb 265, low-virulence strain) by plating of cocultures and counting of colony-forming units, after 10 d . Monocytes from healthy individuals failed to present fungicidal activity against P . brasiliensis even after IFN-gamma activation at the three concentrations . However, patient monocytes activated with IFN-gamma (1000 U/ml) showed a significant fungicidal activity when compared to that obtained with non-activated or activated cells with other IFN-gamma concentrations (250 and 500 U/ml) . Moreover, patient monocytes presented higher fungicidal activity than the control, even before the activation process . These results may be explained by the activation state of patients' cells as a function of the in vivo contact with the fungus, which was confirmed by their higher capacity to release H(2)O(2) in vitro . Unlike the results obtained with Pb 18, patient and control cells presented a significant fungicidal activity against Pb 265, after priming with IFN- gamma . These results are explained by the higher levels of TNF-alpha in supernatants of cultures challenged with Pb 265 . Moreover, higher levels of the cytokine were obtained in patient cell supernatants . Taken together, our results suggest that for effective killing of P . brasiliensis by monocytes, an initial activation signal induced by IFN-gamma is necessary to stimulate the cells to produce TNF-alpha . This cytokine may be involved, through an autocrine pathway, in the final phase activation process . The effectiveness of this process seems to depend on the virulence of the fungal strain and the activation state of the challenged cells.

Nippon Ishinkin Gakkai Zasshi, 2003, 44(1), 17 - 23
Direct invasion of bones by highly pathogenic fungi in an in vitro model and its ecological significance; Sharmin S et al.; Animal bones after being devitalized at death are strongly resistant to wear and tear and remain in the soil or environment much longer than other organic components from dead animals . Yet over the course of time they seem to disappear and thus our ecological surroundings are not cluttered with bone remnants . Mechanical factors creating compression or friction and chemical factors like pH of the soil and surroundings must together have provided concerted degrading effects . Microorganisms in the soil also help in this process by utilizing the organic components of devitalized bones . Certain highly pathogenic fungi that have been collected from soil from time to time and many other environmental fungi may take part in the degrading of the bone remnants . In this study, several strains from the highly pathogenic dimorphic fungi Coccidioides immitis, Blastomyces dermatitidis, Histoplasma spp., Paracoccidioides brasiliensis and also some strains of dematiaceous fungi (Exophiala spp . and Foncecaea pedrosoi) were inoculated to dissected and devitalized murine long bones that had been placed on solidified water agar plates to see if they would survive, grow and invade the bones . After being kept for 12 weeks at 25 degrees C all the parts of the histological sections of these bones showed invasion by most of the strains used in this study, although the cortical component of the bony architecture seemed to be comparatively resistant to invasion . Their ability to grow and sporulate in the aforementioned nutrient-limiting condition hinted at a possible role of these fungi in the degradation of devitalized bones.

Eukaryot Cell, 2003 Feb, 2(1), 34 - 48
Expressed sequence tag analysis of the human pathogen Paracoccidioides brasiliensis yeast phase: identification of putative homologues of Candida albicans virulence and pathogenicity genes; Goldman GH et al.; Paracoccidioides brasiliensis, a thermodimorphic fungus, is the causative agent of the prevalent systemic mycosis in Latin America, paracoccidioidomycosis . We present here a survey of expressed genes in the yeast pathogenic phase of P . brasiliensis . We obtained 13,490 expressed sequence tags from both 5' and 3' ends . Clustering analysis yielded the partial sequences of 4,692 expressed genes that were functionally classified by similarity to known genes . We have identified several Candida albicans virulence and pathogenicity homologues in P . brasiliensis . Furthermore, we have analyzed the expression of some of these genes during the dimorphic yeast-mycelium-yeast transition by real-time quantitative reverse transcription-PCR . Clustering analysis of the mycelium-yeast transition revealed three groups: (i) RBT, hydrophobin, and isocitrate lyase; (ii) malate dehydrogenase, contigs Pb1067 and Pb1145, GPI, and alternative oxidase; and (iii) ubiquitin, delta-9-desaturase, HSP70, HSP82, and HSP104 . The first two groups displayed high mRNA expression in the mycelial phase, whereas the third group showed higher mRNA expression in the yeast phase . Our results suggest the possible conservation of pathogenicity and virulence mechanisms among fungi, expand considerably gene identification in P . brasiliensis, and provide a broader basis for further progress in understanding its biological peculiarities.

Arq Neuropsiquiatr, 2002 Dec, 60(4), 1015 - 8 Epub 2003 Jan 15.
{Paracoccidioidomycosis meningitis: case report}; Lorenzoni PJ et al.; The involvement of the central nervous system in paracoccidioidomycosis and its meningeal form of clinical presentation have rarely been described . This is a case report of a 23 years old man who developed paracoccidioidomycosis meningitis achieved by fungus' presence in direct research and culture of cerebrospinal fluid . The meningeal form of paracoccidioidomycosis, its laboratorial and imaging diagnostic are discussed, and the importance of the routine investigation of the fungus is emphasized.

Yeast, 2003 Feb, 20(3), 263 - 71
Transcriptome characterization of the dimorphic and pathogenic fungus Paracoccidioides brasiliensis by EST analysis; Felipe MS et al.; Paracoccidioides brasiliensis is a pathogenic fungus that undergoes a temperature-dependent cell morphology change from mycelium (22 degrees C) to yeast (36 degrees C) . It is assumed that this morphological transition correlates with the infection of the human host . Our goal was to identify genes expressed in the mycelium (M) and yeast (Y) forms by EST sequencing in order to generate a partial map of the fungus transcriptome . Individual EST sequences were clustered by the CAP3 program and annotated using Blastx similarity analysis and InterPro Scan . Three different databases, GenBank nr, COG (clusters of orthologous groups) and GO (gene ontology) were used for annotation . A total of 3,938 (Y = 1,654 and M = 2,274) ESTs were sequenced and clustered into 597 contigs and 1,563 singlets, making up a total of 2,160 genes, which possibly represent one-quarter of the complete gene repertoire in P . brasiliensis . From this total, 1,040 were successfully annotated and 894 could be classified in 18 functional COG categories as follows: cellular metabolism (44%); information storage and processing (25%); cellular processes-cell division, posttranslational modifications, among others (19%); and genes of unknown functions (12%) . Computer analysis enabled us to identify some genes potentially involved in the dimorphic transition and drug resistance . Furthermore, computer subtraction analysis revealed several genes possibly expressed in stage-specific forms of P . brasiliensis . Further analysis of these genes may provide new insights into the pathology and differentiation of P . brasiliensis .

J Biol Chem, 2003 Apr 4, 278(14), 11773 - 81 Epub 2003 Jan 28.
Structure and kinetic properties of Paracoccus pantotrophus cytochrome cd1 nitrite reductase with the d1 heme active site ligand tyrosine 25 replaced by serine; Gordon EH et al.; The 1.4-A crystal structure of the oxidized state of a Y25S variant of cytochrome cd(1) nitrite reductase from Paracoccus pantotrophus is described . It shows that loss of Tyr(25), a ligand via its hydroxy group to the iron of the d(1) heme in the oxidized (as prepared) wild-type enzyme, does not result in a switch at the c heme of the unusual bishistidinyl coordination to the histidine/methionine coordination seen in other conformations of the enzyme . The Ser(25) side chain is seen in two positions in the d(1) heme pocket with relative occupancies of approximately 7:3, but in neither case is the hydroxy group bound to the iron atom; instead, a sulfate ion from the crystallization solution is bound between the Ser(25) side chain and the heme iron . Unlike the wild-type enzyme, the Y25S mutant is active as a reductase toward nitrite, oxygen, and hydroxylamine without a reductive activation step . It is concluded that Tyr(25) is not essential for catalysis of reduction of any substrate, but that the requirement for activation by reduction of the wild-type enzyme is related to a requirement to drive the dissociation of this residue from the active site . The Y25S protein retains the d(1) heme less well than the wild-type protein, suggesting that the tyrosine residue has a role in stabilizing the binding of this cofactor.

J Comput Assist Tomogr, 2003 Jan-Feb, 27(1), 12 - 7
Central nervous system paracoccidioidomycosis: imaging findings in 17 cases; Gasparetto EL et al.; PURPOSE: To quantify the imaging findings of 17 patients with central nervous system (CNS) paracoccidioidomycosis . METHOD: Retrospective analysis of the clinical data, films, and laboratory findings of 17 patients with CNS paracoccidioidomycosis . The clinical data were obtained by medical records review . Plain chest films, cranial computed tomography scans (CT), and serum and liquor analysis were evaluated . RESULTS: All the patients were male, with a mean age of 46.5 years . Headache and hemiparesis were the most frequent symptoms . The head CT scans revealed two or more granulomas in 53% of the cases, frequently confined to the parietal lobes (35%) and cerebellar hemispheres (35%) . The lesions were hypodense (53%) and irregular (76%) and showed ring-like contrast enhancement (94%) . Perilesional edema was seen in 82% of the patients, and hydrocephalus was seen in 41% . CONCLUSION: Although CNS paracoccidioidomycosis might represent a suspicious lesion pattern on CT scan examination, correlation of the CT scan findings, clinical data, plain chest films, and laboratory results is necessary to define the diagnosis of this uncommon entity.

Diagn Microbiol Infect Dis, 2002 Dec, 44(4), 383 - 6
Molecular identification of Paracoccidioides brasiliensis by 5' nuclease assay; Semighini CP et al.; A PCR assay based on the 5' nuclease assay using a fluorescent probe derived from the sequence of the gene coding for the 43,000-Da (gp43) antigen was developed to detect Paracoccidioides brasiliensis . The assay could detect at least 10 copies of this DNA sequence, providing efficient accuracy to be useful for diagnosis of paracoccidioidomycosis.

J Infect Dis, 2003 Jan 1, 187(1), 124 - 7 Epub 2002 Dec 13.
Adrenocortical hormones and interleukin patterns in paracoccidioidomycosis; Leal AM et al.; The functional status of adrenocortical hormones and their relationship to the pattern of inflammatory cytokines in paracoccidioidomycosis were investigated in a prospective study . Patients were evaluated before treatment and 1 and 6 months after receiving antifungal therapy . Interleukin (IL)-1beta, IL-6, and tumor necrosis factor-alpha plasma levels, C-reactive protein (CRP) concentrations, and erythrocyte sedimentation rate (ESR) were significantly higher in untreated patients than in control subjects . After 6 months of treatment, levels of the 3 cytokines, CRP concentrations, and the ESR decreased significantly . Both baseline and stimulated adrenocorticotropic hormone and cortisol plasma levels were not different between patients and control subjects . In contrast, adrenal androgen dehydroepiandrosterone sulfate (DHEA-S) plasma levels were significantly lower in patients than in sex- and age-matched control subjects . There was a significant inverse correlation between DHEA-S and IL-6 plasma levels . This finding may be of pathogenetic significance in this disease and in other inflammatory states.

Clin Immunol, 2002 Nov, 105(2), 215 - 22
The role of apoptosis in the antigen-specific T cell hyporesponsiveness of paracoccidioidomycosis patients; Cacere CR et al.; Paracoccidioidomycosis is a deep endemic mycosis associated with an antigen-specific immunodeficiency . To examine the role of apoptosis in this immunodeficiency, peripheral blood mononuclear cells (PBMC) of patients with paracoccidioidomycosis and controls were stimulated with the main antigen of Paracoccidioides brasiliensis (gp43) and an unrelated fungal antigen (from Candida albicans, CMA) and analyzed for annexin V and propidium iodide staining by flow cytometry . Control PBMC proliferated well with both antigens . Patients' PBMC proliferated only with CMA, but presented higher levels of apoptosis with gp43 and CMA than in their own unstimulated cultures . Moreover, gp43-triggered apoptosis in control PBMC was lower than in those of the patients . Thus, patient but not control gp43-stimulated T cells apparently remained anergized and subsequently underwent apoptosis . While CMA-induced apoptosis is likely triggered by activation-induced cell death, this is apparently not the case in gp43-induced apoptosis because of the lack of cell cycling and IL-2 in the gp43-stimulated cultures . However, higher IL-10 levels were found in gp43-stimulated patient PBMC cultures . Addition of a neutralizing anti-IL-10 antibody to the cultures resulted in increased apoptosis levels only in gp43-stimulated patient PBMC cultures . Our results suggest that apoptosis plays a role in the patients' antigen-specific hyporesponsiveness and that IL-10 may have an antiapoptotic role.

Am J Clin Pathol, 2002 Dec, 118(6), 864 - 8
Anti-gp43 antibodies in the cerebrospinal fluid of patients with central nervous system involvement by paracoccidioidomycosis; de Almeida SM et al.; Paracoccidioidomycosis (PCM) is a chronic granulomatous infectious disease, endemic in subtropical areas of Central and South America . The diagnosis of the central nervous system (CNS) involvement with PCM (neuroparacoccidioidomycosis {NPCM}) frequently is difficult . A definitive diagnosis usually is made by visualization or isolation of Paracoccidioides brasiliensis from CNS biopsy or necropsy material . In the present study, we determined the presence of anti-gp43 antibodies in the cerebrospinalfluid (CSF) of patients with CNS involvement in PCM by enzyme-linked immunosorbent assay (ELISA) in 9 cases of NPCM and 15 control cases . ELISA anti-gp43 was compared with double immunodiffusion (DID) . ELISA anti-gp43 was positive in 8 (89%) of 9 CSF samples from patients with NPCM and negative in all CSF samples of the control group . DID was negative in all CSF samples from patients with NPCM and control samples . ELISA anti-gp43 in CSF samples is better than DID for the diagnosis of NPCM . It is a sensitive and specific diagnostic method and has high predictive values . To our knowledge, this is thefirst time ELISA anti-gp43 was applied to CSF.

Cell Immunol, 2002 Jul-Aug, 218(1-2), 87 - 94
GP43 from Paracoccidioides brasiliensis inhibits macrophage functions . An evasion mechanism of the fungus; Flavia Popi AF et al.; Macrophages constitute one of the primary cellular mechanisms that impairs parasite invasion of host tissues . The phagocytic and microbicidal properties of these cells can be modulated by specific membrane receptors involved in cell-microorganism interactions . Gp43, the main antigen secreted by Paracoccidiodes brasiliensis (Pb), the causative agent of Paracoccidioidomycosis, is a high mannose glycoprotein . The role played by gp43 in the pathogenesis of the disease is not completely known . Here, we describe the influence of this molecule on the interaction between peritoneal murine macrophages and Pb . Phagocytosis of Pb, live or heat-killed, by adherent peritoneal cells from both, B10.A (susceptible) and A/Sn (resistant) mice, was evaluated . Addition of different concentrations of gp43 to the culture medium inhibited, in a dose-dependent pattern, phagocytosis of live or heat-killed Pb by peritoneal macrophages from both B10.A and A/Sn mice . Gp43 also inhibits phagocytosis of zymosan particles but did not interfere with the uptake of opsonized sheep red blood cells . It was also shown that both gp43 and heat-killed Pb have an inhibitory effect on the release of NO by zymosan stimulated macrophages . Finally, we demonstrated that gp43 inhibits the fungicidal ability of macrophages from both lineages . Based on these data, it is suggested that gp43 can be considered one of the evasion mechanisms for the installation of primary infection in susceptible hosts.

Med Mycol, 2002 Oct, 40(5), 493 - 9
Inhibition of Paracoccidioides brasiliensis by pesticides: is this a partial explanation for the difficulty in isolating this fungus from the soil?
Ono MA, Itano EN, Mizuno LT, Mizuno EH, Camargo ZP.
The aim of this study was to evaluate the effect of ten pesticides on Paracoccidioides brasiliensis, and thereby to investigate the possible association between these data and the difficulty in isolating P . brasiliensis from agricultural soil . Six fungicides (Alto 100, Benlate, Captan, Dithane, Plantacol, Rovral), two herbicides (Pivot, Roundup) and two insecticides (Azodrin and Curacron) were evaluated . Five P . brasiliensis isolates from the environment (labelled group 'N,' for 'nature') and five isolates from patients (group 'P'), were grown on Sabouraud's dextrose agar, at 35 degrees C, with ten different concentrations of each pesticide . The dose of pesticide that causes 50% growth inhibition (ED50) was calculated for each isolate . All pesticides assayed inhibited P . brasiliensis in a dose-dependent manner, and great variability among ED50 values was observed in isolates from both groups . No statistically significant difference was observed between averages of ED50 from groups N and P, except with Alto 100 . The inhibitory effect of pesticides on P . brasiliensis suggests that they can interfere with attempts to isolate P . brasiliensis from soil, where tonnes of pesticides are applied over large areas planted with various crops.

Biochemistry, 2002 Dec 3, 41(48), 14293 - 8
Thermodynamics of the acid transition in blue copper proteins; Battistuzzi G et al.; The thermodynamic parameters of the conformational transition occurring at low pH (acid transition, AT) in blue copper proteins, involving protonation and detachment from the Cu(I) ion of one histidine ligand, have been determined electrochemically for spinach and cucumber plastocyanins, Rhus vernicifera stellacyanin, cucumber basic protein (CBP), and Paracoccus versutus amicyanin . These data were obtained from direct protein electrochemistry experiments carried out at varying pH and temperature . For all species but CBP, the overall conformational change turns out to be exothermic . The entropy change is remarkably species-dependent . In particular, we found that (i) the balance of bond breaking/formation favors the acid transition in plastocyanins, which show remarkably negative DeltaH degrees '(AT) values, and (ii) the transition enthalpy turns out to be much less negative (or even positive) for the two phytocyanins (stellacyanin and CBP): for these species, the transition turns out to be observable thanks to the favorable (positive) entropy change . Thus, it is apparent that the thermodynamic "driving force" for this transition is enthalpic for the plastocyanins and entropic for the phytocyanins . Amicyanin is an intermediate case in which both enthalpic and entropic terms favor the transition . Under the assumption that the transition entropy originates from solvent reorganization effects, which are known to involve compensative enthalpy and entropy changes, the free energy change of the transition would also correspond to the enthalpy change due to bond breaking/formation in the first coordination sphere of the metal and in its immediate environment . Indeed, this term turns out to be very similar for the proteins investigated, in line with the conservation of the Cu(I)-His bond strengths in these species, except for amicyanin, for which the greater exothermicity of the transition can be ascribed to peculiar features of the active site.

Oral Surg Oral Med Oral Pathol Oral Radiol Endod, 2002 Nov, 94(5), 609 - 13
Paracoccidioidomycosis: report of 2 cases mimicking squamous cell carcinoma; Meneses-Garcia A et al.; Paracoccidioidomycosis is an endemic fungal infection in Latin America . This mucocutaneous disease often involves the oral mucosa and may clinically resemble other infectious and neoplastic processes . Paracoccidioidomycosis that clinically suggested squamous cell carcinoma was diagnosed in 2 patients with a history of heavy alcohol and tobacco use . Antifungal therapy with ketoconazole and itraconazole resulted in resolution of the oral lesions . Interestingly, 1 patient had a pulmonary lesion that persisted after antifungal therapy, and biopsy proved this to be a squamous cell carcinoma of the lung.

Mycoses, 2002 Nov, 45(9-10), 351 - 7
Histopathologic and histochemical characterization of calcified structures in hamsters inoculated with Paracoccidioides brasiliensis; Essayag SM et al.; In pathogenicity studies of 31 Paracoccidioides brasiliensis isolates preserved using Castellani's method we intraperitoneally inoculated 104 young adult hamsters and found laminated concentric structures and calcified appearance that resembled Schaumann bodies, in 43 of them, especially in animals with apparently good condition . We characterized these structures histologically and histochemically using different stains (PAS, Grocott, haematoxylin-eosin, Von Kossa) . The Von Kossa staining revealed calcium in these structures . Similar structures have been described in patients with sarcoidosis and also in hamsters inoculated with P . brasiliensis . We found no correlation between the presence of these calcifications and serum calcium levels.

Clin Diagn Lab Immunol, 2002 Nov, 9(6), 1200 - 4
Expression in bacteria of the gene encoding the gp43 antigen of paracoccidioides brasiliensis: immunological reactivity of the recombinant fusion proteins; Diniz SN et al.; gp43 is the major diagnostic antigen of Paracoccidioides brasiliensis, the agent of paracoccidioidomycosis (PCM) in humans . In the present study, cDNA of the gp43 gene (PbGP43) was obtained by reverse transcriptase PCR, inserted into a pGEX vector in frame with the glutathione S-transferase (GST) gene, and expressed in Escherichia coli as inclusion bodies . Immunoblotting showed that all sera from patients with chronic pulmonary and acute lymphatic forms of PCM reacted with the recombinant fusion protein of the mature gp43 (381 amino acids) . Reactivity with fusion proteins containing subfragments of the N-terminal, internal, or C-terminal regions occurred eventually, and the C-terminal region was the most antigenic . Lack of reactivity with the subfragments may be due to the conformational nature of the gp43 epitopes . Sera from patients with aspergillosis, candidiasis, and histoplasmosis did not react with the gp43-GST fusion protein . Our results suggest that recombinant gp43 corresponding to the processed antigen can be a useful tool in the diagnosis of PCM.

Biomedica, 2002 Sep, 22(3), 280 - 6
{Adrenal gland insufficiency secondary to paracoccidioidomycosis}; Onate JM et al.; Paracoccidioidomycosis is regularly associated with adrenal insufficiency in 10-15% of symptomatic cases, and in some instances, diagnosis of the mycosis precedes the adrenal manifestation . To establish the frequency of this association, records were reviewed of 207 cases diagnosed with mycosis at the Mycology Service of the Corporacion para Investigaciones Biologicas . Six cases (2.9%) were found to have adrenal insufficiency . Patients were all males with a mean age of 67.2 years (range 48-75) and most worked in agriculture . The duration of the symptoms of adrenal damage was 4.1 months (range 2-6) . All patients experienced weight loss and malaise; all had abnormal lung X-rays . Major clinical improvement was recorded after initiation of the specific treatments consisting of itraconazole, prednisolone and fluorcortisone . Diminished antibody titers against Paracoccidioides brasiliensis were also recorded after treatment . Prompt treatment re-established adrenal function and effected recovery of normal gland morphology . Consequently, early detection of hypoadrenalism in patients living in the endemic areas is necessary to avoid further adrenal damage and permits a shorter hormonal treatment period in patients afflicted by the mycosis.

Gene, 2002 Sep 4, 297(1-2), 129 - 39
Genetics of isoprenoid biosynthesis in Paracoccus zeaxanthinifaciens; Humbelin M et al.; The genes coding for all the enzymes involved in the conversion of acetyl-CoA to farnesyl diphosphate (FPP) in the zeaxanthin-producing bacterium Paracoccus zeaxanthinifaciens were cloned and characterized . Two genes encoding enzymes catalysing the condensation of two acetyl-CoA molecules to acetoacetyl-CoA were found . The six enzymes involved in the conversion of acetyl-CoA and acetoacetyl-CoA to isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) are grouped in an operon, designated the mevalonate operon . The gene encoding the enzyme catalysing two consecutive condensations, IPP and DMAPP to geranyl diphosphate (GPP) and IPP and GPP to FPP, is not clustered with any other gene encoding an enzyme of the isoprenoid pathway . Genes encoding enzymes involved in the biosynthesis of poly-hydroxyalkanoate and non-carotenoid isoprenoids found in P . zeaxanthinifaciens are also presented.

Plasmid, 2002 Sep, 48(2), 130 - 41
Comparative characterization of repABC-type replicons of Paracoccus pantotrophus composite plasmids; Bartosik D et al.; The repABC replicons have an unusual structure, since they carry genes coding for partitioning (repA, repB) and replication (repC) proteins, which are organized in an operon . So far, the presence of these compact bi-functional modules has been reported only in the megaplasmids of the Rhizobiaceae and within the plasmid pTAV1 (107kb) of Paracoccus versutus . We studied the distribution of repABC-type replicons within bacteria belonging to the genus Paracoccus . We found that repABC replicons occur only in the group of pTAV1-like plasmids: pKLW1, pHG16-a, pWKS2, and pPAN1, harbored by different strains of Paracoccus pantotrophus . A partial sequencing approach followed by phylogenetic analysis revealed that these replicons constitute a distinct evolutionary branch of repABC replicons . Incompatibility studies showed that they represent two incompatibility groups designated IncABC1 (pTAV1, pKLW1, and pHG16-a) and IncABC2 (pPAN1) . Sequence comparison using available databases allowed the identification, within plasmid pRS241d of Rhodobacter sphaeroides 2.4.1, of an additional sequence highly homologous to the paracoccal repABC replicons, which has been included in comparative analyses.

Yeast, 2002 Oct, 19(14), 1221 - 31
The kex2 gene from the dimorphic and human pathogenic fungus Paracoccidioides brasiliensis; Venancio EJ et al.; Kexin-like protein is a component of the subtilase family of proteinases involved in the processing of proproteins to their active forms . Kexin-like proteins are also synthesized as a propeptide and this is involved in (auto)inhibition, correct folding and subcellular sorting of proteins . The kexin-like protein was described as the product of the kex2 gene for Aspergillus niger, Candida albicans, Saccharomyces cerevisiae, Yarrowia lipolytica and other fungi . Disruption of the kex2 gene in C . albicans and Y . lipolytica affects hyphae production and induces morphological cell defects, strongly suggesting a possible role of kexin-like proteins in dimorphism of human pathogenic fungi . In this work, we report the nucleotide sequence of the kex2 gene cloned from the dimorphic and human pathogenic fungus Paracoccidioides brasiliensis (Pbkex2) . An open reading frame (ORF) of 2622 bp was identified in the complete sequence, interrupted by only one intron of 93 bp . The 5' non-coding region contains consensus sequences such as canonical TATA, CAAT boxes and putative motifs for transcriptional factors binding sites, such as HSE-like regulating genes involved in thermo-dependent processes; Xbp1, reported as a transcriptional factor that may control genes involved in cell morphology; and StuAp, which may regulate spore differentiation and pseudohyphal growth in fungi . In the 3' non-coding region were observed the canonical motifs necessary for correct mRNA processing and polyadenylation . The deduced protein sequence consists of 842 amino acid residues, showing identity to kexin-like proteinases from A . niger (55%), Emericella nidulans (53%) and C . albicans (48%) . Comparative sequence analysis of P . brasiliensis kexin-like protein reveals the presence of homologous regions related to a signal peptide, a propeptide, a subtilisin-like catalytic domain, a P domain, a S/T rich region and a transmembrane domain . A putative Golgi retrieval signal (YEFEMI) has also been found in the cytoplasmic tail . The complete nucleotide sequence of Pbkex2 and its flanking regions have been submitted to GenBank database under Accession No . AF486805 .

Cad Saude Publica, 2002 Sep-Oct, 18(5), 1441 - 54
Paracoccidioidomycosis mortality in Brazil (1980-1995); Coutinho ZF et al.; This study analyzes 3,181 deaths from paracoccidioidomycosis in Brazil, based on 16 years of sequential data (from 1980 to 1995) . During this period paracoccidioidomycosis showed considerable magnitude and low visibility, representing the eighth most common cause of death from predominantly chronic or recurrent types of infectious and parasitic diseases . It also had the highest mortality rate among the systemic mycoses . The mean annual mortality rate was 1.45 per million inhabitants, indicating a downward long-term trend (reduction of 31.28%), while spatial distribution among the different regions and States of Brazil was non-homogenous . The South (with the highest regional rate) and the Southeast showed a downward trend, while the Central West had the second highest rate in the country . At least one-fifth of Brazilian municipalities (or 22.71% of the country's total area) reported deaths from paracoccidioidomycosis . Overall nationwide mortality per area was 3.73/10,000km2 . The disease was endemic in non-metropolitan areas . The majority of deaths occurred in males (84.75%), and there was a sex ratio of 562 men/100 women . The 30-59-year and over-60-year age groups were the most affected . The study showed that the mortality rate justifies classifying this disease as a major health problem in Brazil.

Med Mycol, 2002 Aug, 40(4), 411 - 7
Randomized trial with itraconazole, ketoconazole and sulfadiazine in paracoccidioidomycosis; Shikanai-Yasuda MA et al.; Forty-two patients with active paracoccidioidomycosis were randomized to receive itraconazole (50-100 mg d(-1)), ketoconazole (200-400 mg d(-1)) or sulfadiazine (100-150 mg kg d(-1) up to 6 g d(-1)) for 4-6 months, followed by slow release sulfa until negativity of serological tests . All 14 patients in itraconazole and sulfadiazine groups and 13 in the ketoconazole group showed an adequate clinical response to the chemotherapy . One patient in the latter group showed treatment failure according to clinical and mycological criteria . The test of the hypothesis that the drugs reduced antibody levels up to ten months of treatment showed a p value equal to 0.0001 for itraconazole, 0.017 for ketoconazole and 0.0012 for sulfadiazine; this reduction was similar for the three groups . In this first randomized study for the treatment of paracoccidioidomycosis we could not show superiority of any one regimen over the others in the clinical and serological responses of patients with the moderately severe form of the disease.

Med Mycol, 2002 Aug, 40(4), 407 - 10
Correlation of factor XIIIa+ dermal dendrocytes with paracoccidioidomycosis skin lesions; Pagliari C et al.; We demonstrated and quantified by immunohistochemistry the factor XIIIa+ dermal dendrocytes (FXIIIa+ DD) in paracoccidioidomycosis skin lesions . Sixty-one biopsies were classified according to the tissue response in well-organized granulomas (group 1), poorly organized granulomas (group 2) and samples showing both kinds of granuloma (group 3) . Ten biopsies from normal skin were used as controls . In order to verify the internalization of Paracoccidioides brasiliensis antigens by FXIIIa+ DD, we performed a double immunostaining technique . FXIIIa+ DD were hypertrophied with prominent dendrites and their number in the test groups was higher than in the control group, especially in the dermal papillae . P . brasiliensis yeasts were seen within the cytoplasm of FXIIIa+ DD in 40% of the immunostained biopsies . We could correlate these findings with the probable role of FXIIIa+ DD as antigen-presenting cells in the pathogenesis of skin lesions in paracoccidioidomycosis.

Med Mycol, 2002 Aug, 40(4), 387 - 91
Paracoccidioides brasiliensis-reactive antibodies in Brazilian blood donors; Botteon FA et al.; In a survey for primary paracoccidioidomycosis (PCM) infection (and not the clinical disease), two groups of blood donors were analyzed . One study group was drawn from donors living in a rural area where PCM is endemic, and the other group from urban residents of a large city, Sao Paulo . Anti-Paracoccidioides brasiliensis (Pb) specific antibodies (IgG) in sera were analyzed by ELISA, using crude Pb exoantigens (exoAg) and purified specific Pb 43 kDa glycoprotein (gp43) . The results showed that 21% of 700 rural samples and 0.9% of 350 urban samples were positive for exoAg and gp43 . To avoid cross-reactions, the sera were adsorbed first with Histoplasma capsulatum antigens and secondly with Leishmania amazonensis antigens . In the first adsorption with H . capsulatum, reactivity to gp43 fell to 12.8% in the rural group and to 0% in the urban group . In the succeeding adsorption with L . amazonensis, this reactivity fell to 12.3% in the rural group . There was a statistically greater proportion of persons with gp43-reactive antibodies in rural group than in the urban group, indicating that rural residents had frequently become exposed to Pb and contracted primary, subclinical PCM . The present report is the first epidemiological study using ELISA to detect antibodies against gp43 in blood donors.

Med Mycol, 2002 Aug, 40(4), 377 - 82
Role of Paracoccidioides brasiliensis cell wall fraction containing beta-glucan in tumor necrosis factor-alpha production by human monocytes: correlation with fungicidal activity; Anjos AR et al.; The polysaccharide fraction of Paracoccidioides brasiliensis mycelial cell wall (F1 fraction), the active component of which is composed of beta-glucan, was investigated in regard to the activation of human monocytes for fungal killing . The cells were primed with interferon-gamma (IFN-gamma) or F1 (100 and 200 microg ml(-1)) or F1 (100 and 200 microg ml(-1)) plus IFN-gamma for 24 h and then evaluated for H2O2 release . In other experiments, the cells were pretreated with the same stimuli, challenged with a virulent strain of P . brasiliensis and evaluated for fungicidal activity and levels of tumor necrosis factor (TNF-alpha) in the supernatants . F1 increased the levels of H2O2 in a similar manner to IFN-gamma . However, a synergistic effect between these two activators was not detected . On the contrary, a significant fungicidal activity was only obtained after priming with IFN-gamma plus F1 . This higher activity was associated with high levels of TNF-alpha in the supernatants of the cocultures . Overall, P . brasiliensis F1 fraction induced human monocytes to release relatively high levels of TNF-alpha, which, in combination with IFN-gamma, is responsible for the activation of human monocytes for effective killing of P . brasiliensis.

Rev Gastroenterol Peru, 1998 Sep, 18(3), 285 - 291
{COLONIC PARACOCCIDIOIMYCOSIS: A REPORT ON ONE CASE}; Verona R et al.; We present a 36-year-old patient with colonic paracoccidiodomycosis . The epidemiological, clinical, laboratory, radiological, pathological features and treatment of this unusual disease were reviewed.

Am J Ophthalmol, 2002 Sep, 134(3), 456 - 9
Ocular and central nervous system paracoccidioidomycosis in a pregnant woman with acquired immunodeficiency syndrome; Finamor LP et al.; PURPOSE: To describe an atypical case of central nervous system and ocular paracoccidioidomycoses simulating ocular toxoplasmosis in a pregnant woman with acquired immunodeficiency syndrome (AIDS) . DESIGN: Interventional case report . METHODS: Case report . RESULTS: A 25-year-old pregnant woman with AIDS, presented with a severe ocular inflammation in the right eye involving the choroid, retina, and the optic disk, which rapidly progressed to retinal detachment, iris neovascularization, and neovascular glaucoma . The left eye was normal . Magnetic resonance imaging (MRI) showed a focal hypodense contrast-enhanced ring lesion in the brain . Serum antibody titers were negative for Toxoplasma gondii, but the polymerase chain reaction was positive for the parasite in the vitreous sample . The patient responded partially to specific treatment for toxoplasmosis, and there was a small reduction in size of the brain lesion . She progressed to a blind painful eye, which was enucleated . Paracoccidioides brasiliensis was found in the histopathological studies of the eye and oropharynx . With the diagnosis of disseminated ocular paracoccidioidomycoses, the patient was treated with trimethoprim-sulfamethoxazole with a satisfactory outcome and reduction in size of the brain lesion . CONCLUSION: Although ocular infection with ocular paracoccidioidomycoses is rare, this diagnosis should be considered when investigating ocular inflammation in a patient with AIDS.

Eur J Radiol, 2002 Sep, 43(3), 204 - 18
Percutaneous drainage of abdominal abcess; Men S et al.; The mortality in undrained abdominal abscesses is high with a mortality rate ranging between 45 and 100% . The outcome in abdominal abscesses, however, has improved due to advances in image guided percutaneous interventional techniques . The main indications for the catheter drainage include treatment or palliation of sepsis associated with an infected fluid collection, and alleviation of the symptoms that may be caused by fluid collections by virtue of their size, like pancreatic pseudocele or lymphocele . The single liver abscesses may be drained with ultrasound guidance only, whereas the multiple abscesses usually require computed tomography (CT) guidance and placement of multiple catheters . The pancreatic abscesses are generally drained routinely and urgently . Non-infected pancreatic pseudocysts may be simply observed unless they are symptomatic or cause problems such as pain or obstruction of the biliary or the gastrointestinal tract . Percutaneous routes that have been described to drain pelvic abscesses include transrectal or transvaginal approach with sonographic guidance, a transgluteal, paracoccygeal-infragluteal, or perineal approach through the greater sciatic foramen with CT guidance . Both the renal and the perirenal abscesses are amenable to percutaneous drainage . Percutaneous drainage provides an effective and safe alternative to more invasive surgical drainage in most patients with psoas abscesses as well.

Microbes Infect, 2002 Aug, 4(10), 1027 - 34
Characterization of a gene which encodes a mannosyltransferase homolog of Paracoccidioides brasiliensis; Costa AA et al.; We screened an expression library of the yeast form of Paracoccidioides brasiliensis with a pool of human sera that was pre-adsorbed with mycelium, from patients with paracoccidioidomycosis (PCM) . A sequence (PbYmnt) was obtained and characterized . A genomic clone was obtained by PCR of P . brasiliensis total DNA . The sequence contained a single open reading frame (ORF) encoding a protein of 357 amino acid residues, with a molecular mass of 39.78 kDa . The deduced amino acid sequence exhibited identity to mannosyl- and glycosyltransferases from several sources . A DXD motif was present in the translated gene and this sequence is characteristic of the glycosyltransferases . Hydropathy analysis revealed a single transmembrane region near the amino terminus of the molecule that suggested a type II membrane protein . The PbYmnt was expressed preferentially in the yeast parasitic phase . The accession number of the nucleotide sequence of PbYmnt and its flanking regions is AF374353 . A recombinant protein was generated in Escherichia coli . Our data suggest that PbYmnt encodes one member of a glycosyltransferase family of proteins and that our strategy was useful in the isolation of differentially expressed genes.

Curr Genet, 2002 Jul, 41(4), 261 - 7 Epub 2002 Jun 27.
chsZ, a gene for a novel class of chitin synthase from Aspergillus oryzae; Chigira Y et al.; We cloned and characterized a novel Aspergillus oryzae chitin synthase gene, chsZ, encoding a polypeptide containing a new myosin motor-like domain in its N-terminal half . Alignment analysis revealed that ChsZ was less homologous to known class V enzymes, except for its probable chitin synthase conserved region in the C-terminal half . We also found a chsY gene and found that ChsY showed higher similarity to the class V enzymes than did ChsZ . Phylogenetic analysis clearly demonstrated that the A . oryzae ChsZ, together with Chs4 of Paracoccidioides brasiliensis and Chs6 of Ustilago maydis, formed a new subclass distinct from A . oryzae ChsY and known class V chitin synthases, including A . nidulans CsmA (ChsD) and A . fumigatus ChsE . In conclusion, we propose a new class, class VI chitin synthases, represented by A . oryzae ChsZ, P . brasiliensis Chs4 and U . maydis Chs6 . Expression analysis suggested that the regulation of chsZ expression is distinct from that of chsY expression.

J Bacteriol, 2002 Sep, 184(17), 4767 - 74
Hierarchy of carbon source selection in Paracoccus pantotrophus: strict correlation between reduction state of the carbon substrate and aerobic expression of the nap operon; Ellington MJ et al.; Paracoccus pantotrophus can express a periplasmic nitrate reductase (Nap) during aerobic growth . A proposed role for this enzyme is the dissipation of excess redox energy during oxidative metabolism of reduced carbon substrates . To investigate the regulation of nap expression, a transcriptional fusion between the nap promoter region of P . pantotrophus and the lacZ gene was constructed . When this fusion was used, analyses showed that transcription from the nap promoter increases as the average reduction state of the carbon atoms increases . Thus, beta-galactosidase activities increase as the carbon source changes in the order succinate-acetate-butyrate . This result was obtained regardless of which of the three carbon sources was used for culture of the inoculum . If two carbon sources were presented together, the beta-galactosidase activity was always the same as it was when the least-reduced carbon source was added alone . This suggests that the regulation is dependent upon metabolism of the more-reduced carbon sources rather than just their presence in the medium . Analysis of culture medium by (1)H nuclear magnetic resonance showed that for aerobic growth P . pantotrophus strictly selected its carbon source in the order succinate-acetate-butyrate . This was reflected by diauxic growth kinetics on medium containing mixed carbon substrates . The regulatory mechanism underpinning such a selection is unknown but is likely to be related to the mechanism which controls the transcription of the nap operon.

J Clin Microbiol, 2002 Aug, 40(8), 2828 - 31
In vitro comparison of activities of terbinafine and itraconazole against Paracoccidioides brasiliensis; Hahn RC et al.; In vitro, terbinafine is highly active against a broad spectrum of pathogenic fungi . We evaluated the activities of terbinafine and itraconazole against 31 isolates of Paracoccidioides brasiliensis . The tests were conducted by using a broth macrodilution procedure . MICs, in micrograms per milliliter, were as follows: terbinafine, 0.015 to 1.0 (geometric mean, 0.1188); itraconazole, 0.007 to 0.5 (geometric mean, 0.03165) . The usual therapy for paracoccidioidomycosis is sulfonamides, amphotericin B, and azole derivatives (ketoconazole, itraconazole, and fluconazole) . In comparison to amphotericin B, azole derivatives allow shorter treatment courses, can be administered orally, and are equally effective . Itraconazole has as high efficacy as ketoconazole, but with superior tolerance . It is the current drug of choice for treatment of paracoccidioidomycosis . The data obtained in this study indicate that terbinafine is active against P . brasiliensis in vitro and suggest that this allylamine can be considered a new option as drug therapy for paracoccidioidomycosis.

Biochim Biophys Acta, 2002 Jul 29, 1598(1-2), 65 - 73
The cytochrome complex SoxXA of Paracoccus pantotrophus is produced in Escherichia coli and functional in the reconstituted sulfur-oxidizing enzyme system; Rother D et al.; The heterodimeric c-type cytochrome complex SoxXA of Paracoccus pantotrophus was produced in Escherichia coli . The soxX and soxA genes, separated by two genes in the sox gene cluster of P . pantotrophus, were fused with ribosome binding sites optimal for E . coli and combined to give soxXA in pRD133.27 . The cytochrome complex SoxXA was produced in E . coli M15 containing pRD133.27, pREP4 encoding the Lac repressor and plasmid pEC86, carrying essential cytochrome c maturation genes . SoxX and SoxA were formed in a ratio of about 2.5:1 . SoxA appeared to be unstable when not complexed with SoxX . The cytochrome complex SoxXA, purified to homogeneity from periplasmic extracts of E . coli M15 (pRD133.27, pREP4, pEC86), exhibited identical biochemical and biophysical properties as compared to SoxXA of P . pantotrophus . Moreover, this cytochrome complex was shown to be equally catalytically active with respect to rates and reactivity with different sulfur substrates in the reconstituted sulfur-oxidizing enzyme system using homogeneous Sox-proteins of P . pantotrophus . Homogeneous SoxX was catalytically inactive.

Med Mycol, 2002 Jun, 40(3), 225 - 42
Paracoccidioides brasiliensis and paracoccidioidomycosis: molecular approaches to morphogenesis, diagnosis, epidemiology, taxonomy and genetics; San-Blas G et al.; Paracoccidioides brasiliensis is an amenable model to study the molecular and biochemical events that lead to morphological transition in fungi, because temperature seems to be the only factor regulating this process . It is the causative agent of paracoccidioidomycosis, a systemic mycosis that affects humans and that is geographically confined to Latin America, where it constitutes one of the most prevalent deep mycoses . With the help of molecular tools, events leading to the morphological transition have been traced to genes that control cell wall glucan and chitin syntheses, and other metabolic processes such as production of heat shock proteins and ornithine decarboxylase activity . Molecular diagnosis and epidemiology of paracoccidioidomycosis are also the focus of intensive research, with several primers being proposed as specific probes for clinical and field uses . Although P . brasiliensis is refractory to cytogenetic analysis, electrophoretic methods have allowed an approximation of its genomic organization and ploidy . Finally, the recognition of P . brasiliensis as an anamorph in the phylum Ascomycota, order Onygenales, family Onygenaceae, has been accomplished by means of molecular tools . This phylogenetic placement has revised the taxonomic position of this fungus, which was traditionally included within now-abandoned higher anamorph taxa, the phylum Deuteromycota and the class Hyphomycetes.

Br J Nutr, 2002 Aug, 88(2), 151 - 8
Effect of timing of food deprivation on host resistance to fungal infection in mice; Oarada M et al.; Mice were deprived of food for a period of 72 h at varying times relative to the time of infection with Paracoccidioides brasiliensis . Host resistance was diminished profoundly when the period of food deprivation was from 48 h before to 24 h after infection (group B) . When food deprivation was initiated immediately after infection (group C), host resistance was reduced less profoundly . When food deprivation was initiated at 24 and 48 h post-infection, reductions in host resistance were only moderate or not observed respectively . These results suggest that the earlier in the course of infection starvation occurs, the more profoundly host resistance is impaired . When food deprivation was initiated 72 h before infection, finishing at the time of infection (group A), the reduction in host resistance was considerably less profound compared with group B mice, suggesting that refeeding initiated immediately after infection is responsible for rapid restoration of the antifungal resistance in starved mice . Infection-induced responses of corticosterone and interferon-gamma were changed according to the timing of food deprivation . Group A mice, similar to non-fasted controls, showed an infection-induced increase in serum corticosterone concentration, while groups B and C did not . Group C mice showed a substantially greater infection-induced increase in serum interferon-gamma compared with the other fasted and non-fasted control groups.

Cytokine, 2002 May 7, 18(3), 149 - 57
IL-12 and neutralization of endogenous IL-10 revert the in vitro antigen-specific cellular immunosuppression of paracoccidioidomycosis patients; Romano CC et al.; Treatment of patients with paracoccidioidomycosis is still a challenge . Patients present defective lymphoproliferation and IFN-gamma responses to the main Paracoccidioides brasiliensis antigen (gp43), which correlates with disease severity . Here, we demonstrated that the patients show also a defective synthesis of interleukin (IL)-12 . Therefore, we attempted to revert this immune disfunction by adding IL-12 and neutralizing anti-IL-10 antibody to gp-43-stimulated peripheral blood mononuclear cell cultures . Both treatments increased IFN-gamma secretion to levels observed with healthy sensitized individuals, but affected proliferation only modestly . When combined, the treatments further increased IFN-gamma synthesis and cell proliferation . The addition of suboptimal concentrations of IL-2 also further increased the IL-12-mediated secretion of IFN-gamma . Interestingly, the immune modulation was mostly antigen-specific, since the responses to Candida albicans' antigen were not affected . These results suggest that appropriate immune intervention with cytokines and/or anti-cytokines may help in the treatment of PCM.

Yeast, 2002 Aug, 19(11), 963 - 72
Characterization of a chaperone ClpB homologue of Paracoccidioides brasiliensis; Jesuino RS et al.; We report the cloning and sequence analysis of a genomic clone encoding a Paracoccidioides brasiliensis ClpB chaperone homologue (PbClpB) . The clpb gene was identified in a lambda Dash II library . Sequencing of Pbclpb revealed a long open reading frame capable of encoding a 792 amino acid, 87.9 kDa protein, pI of 5.34 . The predicted polypeptide contains several consensus motifs of the ClpB proteins . Canonical sequences such as two putative nucleotide-binding sites, chaperonins ClpA/B signatures and highly conserved casein kinase phosphorylation domains are present . ClpB is 69% to 49% identical to members of the ClpB family from several organisms from prokaryotes to eukaryotes . The transcript of PbclpB was detected as a mRNA species of 3.0 kb, preferentially expressed in the yeast parasitic phase of the fungus . A 89 kDa protein was also detected in yeast cells of P . brasiliensis .

FEMS Immunol Med Microbiol, 2002 Jul 12, 33(3), 151 - 7
Evaluation of DNA polymorphisms amplified by arbitrary primers (RAPD) as genetically associated elements to differentiate virulent and non-virulent Paracoccidioides brasiliensis isolates; Motta TR et al.; Randomly amplified polymorphic DNA (RAPD) analysis of 35 Paracoccidioides brasiliensis isolates was carried out to evaluate the correlation of RAPD profiles with the virulence degree or the type of the clinical manifestations of human paracoccidioidomycosis . The dendrogram presented two main groups sharing 64% genetic similarity . Group A included two isolates from patients with chronic paracoccidioidomycosis; group B comprised the following isolates showing 65% similarity: two non-virulent, six attenuated, five virulent, eight from patients with chronic paracoccidioidomycosis and two from patients with acute paracoccidioidomycosis . The virulent Pb18 isolate and six attenuated or non-virulent samples derived from it were genetically indistinguishable (100% of similarity) . Thus, in our study, RAPD patterns could not discriminate among 35 P . brasiliensis isolates according to their differences either in the degree of virulence or in the type of the clinical manifestation of this fungal infection.

Mycoses, 2002 Jun, 45(5-6), 174 - 9
Paracoccidioides brasiliensis: virulence and an attempt to induce the dimorphic process with fetal calf serum; De Moraes Borba C et al.; The virulence of Paracoccidioides brasiliensis strains and the induction of the dimorphic process of the fungus were investigated . The strains were kept under mineral oil for different periods of time, except for one that was successively subcultured on agar slants . Four strains with transitional morphology at room temperature were avirulent to experimental animals . Two strains with typical morphology at 36 degrees C, one of them preserved under mineral oil for 16 years and the other maintained by successive subcultures for 10 years, were virulent to mice . These strains caused macro- and microscopic lesions in various organs, with enlargement of the spleen and heart . An attempt to induce the dimorphic process with fetal calf serum (FCS) in P.brasiliensis strains with transitional morphology failed . FCS was probably unable to stimulate the synthesis of Y cell wall alpha-1,3-glucan . The results demonstrate that keeping strains under mineral oil for a long period of time may have altered the enzymatic activities of the proteinases and other virulence factors that participate in the transition process and in pathogenesis.

Biochem J, 2002 Sep 15, 366(Pt 3), 883 - 8
A novel, kinetically stable, catalytically active, all-ferric, nitrite-bound complex of Paracoccus pantotrophus cytochrome cd1; Allen JW et al.; The oxidized form of Paracoccus pantotrophus cytochrome cd(1) nitrite reductase, as isolated, has bis-histidinyl co-ordination of the c haem and His/Tyr co-ordination of the d(1) haem . On reduction, the haem co-ordinations change to His/Met and His/vacant respectively . If the latter form of the enzyme is reoxidized, a conformer is generated in which the ferric c haem is His/Met co-ordinated; this can revert to the 'as isolated' state of the enzyme over approx . 20 min at room temperature . However, addition of nitrite to the enzyme after a cycle of reduction and reoxidation produces a kinetically stable, all-ferric complex with nitrite bound to the d(1) haem and His/Met co-ordination of the c haem . This complex is catalytically active with the physiological electron donor protein pseudoazurin . The effective dissociation constant for nitrite is 2 mM . Evidence is presented that d(1) haem is optimized to bind nitrite, as opposed to other anions that are commonly good ligands to ferric haem . The all-ferric nitrite bound state of the enzyme could not be generated stoichiometrically by mixing nitrite with the 'as isolated' conformer of cytochrome cd(1) without redox cycling.

Med Mycol, 2002 Apr, 40(2), 213 - 6
A case of imported paracoccidioidomycosis in a German legionnaire; Horre R et al.; We report on a case of the chronic form of paracoccidioidomycosis with swelling and ulcerations of the mouth in a German legionnaire who also suffered from a chronic bronchitis . The patient had worked for many years in Brazil, an area endemic for the disease . Infection due to Paracoccidioides brasiliensis was diagnosed in Germany, more than 10 years after the patient's return . Diagnosis was established by the presence of yeast cells with multipolar budding in the tissue of the oral lesion . Furthermore, the fungus was grown in a liquid Leishmania culture medium . Identification of the fungus was based on morphology and genetic sequencing . Furthermore, IgG antibodies against a 43-kDa antigen of P . brasiliensis were detected in a western blot . After itraconazole therapy (400 mg day(-1)) for 4 weeks, the lesions had disappeared almost completely, but the therapy was continued for further 5 months to avoid relapse of the infection.

Med Mycol, 2002 Apr, 40(2), 169 - 78
Experimental paracoccidioides brasiliensis infection in mice: influence of the hormonal status of the host on tissue responses; Aristizabal BH et al.; We have previously proposed that 17beta-estradiol may be responsible in part for the decreased frequency of clinical paracoccidioidomycosis in females via a blocking of the initial morphological transformation necessary to initiate infection . Here we examined the course of infection in male and female mice in relation to their hormonal status . After pulmonary infection with conidia, normal males showed progressive infection, whereas normal females restricted proliferation and progressive disease . In contrast, castrated animals exhibited lesser capacity to restrict disease progression . Castrated male mice reconstituted with 17beta-estradiol initially restricted proliferation, but showed disease progression later in infection, whereas castrated female mice reconstituted with testosterone were unable to restrict disease . Quantitative histological analyses demonstrated that only normal male and castrated reconstituted mice developed granulomas, which decreased in number and size with time correlating with increasing numbers of CFU in the lungs . Greater numbers of chronic inflammatory foci did not correlate with higher CFU . These results further support a role for 17beta-estradiol during early innate resistance of females to paracoccidioidomycosis.

Med Mycol, 2002 Apr, 40(2), 153 - 9
Enhanced production of specific IgG4, IgE, IgA and TGF-beta in sera from patients with the juvenile form of paracoccidioidomycosis; Mamoni RL et al.; Paracoccidioidomycosis (PCM) occurs in two distinct forms, the acute or juvenile form (JF), and the chronic or adult form (AF) . To clarify the basis of this dichotomy, specific IgG subclasses, IgA and IgE anti-gp43 were measured by enzyme-linked immunosorbent assay, in patients with different forms of PCM . Serum levels of tumor necrosis factor-alpha, interleukin (IL)-6, IL-8, macrophage inflammatory protein (MIP)-1alpha and transforming growth factor (TGF)-beta were also quantified . We show here that JF patients have significantly higher titers of IgE antibodies against gp43, an immunodominant antigen specific for Paracoccidioides brasiliensis, than do patients with the unifocal adult form (UF-AF, isolated lesions) . Patients with the multifocal adult form (MF-AF, lesions in more than one organ) also produced elevated levels of anti-P . brasiliensis IgE . Furthermore, specific IgE levels were correlated with IgG4, IgA and eosinophilia . Patients with JF showed eosinophilia and increased levels of TGF-beta, a switching factor for IgA . These results indicate a T helper (Th)-2 pattern of cytokine expression in both the JF and the MF-AF of PCM . On the other hand, patients with UF-AF had a significantly lower production of specific IgE, IgG4 and IgA than was seen in the other patient groups.

Immunol Lett, 2002 Aug 1, 83(1), 61 - 6
Characterization of B-1b cells as antigen presenting cells in the immune response to gp43 from Paracoccidioides brasiliensis in vitro; Vigna AF et al.; Antigen presentation is an essential stage in the development of immune response to a specific antigen . This response can lead to the production of antibodies and/or effector T lymphocyte activation . Macrophages, dendritic cells and B-lymphocytes, among others, act as antigen presenting cells . B-lymphocytes capture antigenic particles through a surface receptor of IgM nature . The interaction IgM-antigen leads to endocytosis of the complex and antigen processing which culminates in presentation of the antigen on the cell surface associated with a class II MHC molecule . At least three B cell subsets, B-1a (Ly-1B), B-1b and B-2, are present in the mouse periphery . B-1a and B1-b cells represent a small population in the adult spleen and are abundant in the peritonial and pleural cavities . It has been demonstrated in our laboratory that B-1b cells spontaneously proliferated in stationary cultures of adherent peritonial cells . Further, that these cells migrate to a non-specific inflammatory focus . Based on these findings, we investigated whether these cells are antigen presenting cells in vitro using as antigenic stimulus gp43 from Paracoccidioides brasiliensis . Results showed that B1-b cells express constitutively high levels of class II MHC and costimulatory molecules inducing an efficient proliferation of gp43 sensitized T lymphocytes.

Med Mycol, 2001 Oct, 39(5), 457 - 62
Expression of alpha tubulin during the dimorphic transition of Paracoccidioides brasiliensis; Silva WP et al.; In this study we analyzed the expression of (alpha-tubulin during the dimorphic transition of the human-pathogenic fungus Paracoccidioides brasiliensis . The alpha-tubulin from P . brasiliensis was recognized by a commercially available anti-tubulin antibody and was developmentally regulated during the dimorphic form transition . We detected at least two alpha-tubulin isoforms in the mycelial state and only one isoform in the yeast forms . This finding suggests specific roles for the alpha-tubulin isoforms in P . brasiliensis's yeast and mycelial forms.

Med Mycol, 2001 Oct, 39(5), 445 - 55
Molecular cloning, characterization and expression of the heat shock protein 60 gene from the human pathogenic fungus Paracoccidioides brasiliensis; Izacc SM et al.; A gene encoding the heat shock protein (HSP) 60 from Paracoccidioides brasiliensis (Pb) was cloned and characterized . The hsp60 gene is composed of three exons divided by two introns . Structural analysis of the promoter detected canonical sequences characteristic of regulatory regions from eukaryotic genes . The deduced amino acid sequence of the Pb hsp60 gene and the respective cloned cDNA consists of 592 residues highly homologous to other fungal HSP60 proteins . The hsp60 gene is present as a single copy in the genome, as shown by Southern blot analysis . The HSP60 protein was isolated from Pb yeast cellular extracts . N-terminal amino acid sequencing of HSP60 confirmed that the cloned hsp60 gene correlated to the predicted protein in Pb . HSP60 expression appeared to be regulated during form transition in Pb, as different levels of expression were detected in in vitro labeling of cells and northern blot analysis . The complete coding region of Pb hsp60 was fused with plasmid pGEX-4T-3 and expressed in Escherichia coli as a glutathione S-transferase-tagged recombinant protein . The protein reacted with a mouse monoclonal antibody raised to a human recombinant HSP60 . Western immunoblot experiments demonstrated that the recombinant protein and the native HSP60 were recognized by sera from humans with paracoccidioidomycosis (PCM).

Med Mycol, 2001 Oct, 39(5), 423 - 8
Levels of specific antigen (gp43), specific antibodies, and antigen-antibody complexes in saliva and serum of paracoccidioidomycosis patients; Miura CS et al.; The present study analyses human immunoglobulin G (IgG) antibodies directed against the Paracoccidioides brasiliensis exoantigen, gp43, as well as the presence of gp43-IgG immune complexes (ICs) in 31 samples of saliva and serum from 19 patients with paracoccidioidomycosis (PCM) and 12 normal donors . Additional analysis of secretory IgA (sIgA) was performed on the same saliva samples . Consistent with previous findings, a significant increased specific IgG level was observed in PCM patients' saliva and serum (P < 0.05) . The analysis of serum gp43 and gp43-IgG IC demonstrated a higher level in patients with PCM (P < 0.05); however, this difference was not statistically significant with regard to gp43 and gp43-IgG in saliva when compared to the healthy donors . A high level of sIgA in saliva of PCM patients compared to that of normal donors was also observed (P < 0.05) . Patients exhibiting low levels of serum IgG but with high titres of IC were observed, thus strengthening the idea of the necessity to use more than one marker for diagnosis and treatment monitoring of PCM . This is the first report of sIgA in PCM patients' saliva and may be indicative of a protective role in neutralizing antigens on mucosal surfaces.

Biochem J, 2002 Jun 15, 364(Pt 3), 807 - 15
Multiple forms of the catalytic centre, CuZ, in the enzyme nitrous oxide reductase from Paracoccus pantotrophus; Rasmussen T et al.; Nitrous oxide reductase catalyses the reduction of nitrous oxide to dinitrogen at a unique tetranuclear copper site, called Cu(Z), which has a central inorganic sulphide ligand . Limited incubation with oxygen during the preparation of nitrous oxide reductase from Paracoccus pantotrophus results in changed redox properties of the catalytic centre by comparison with anaerobic preparations . While the anaerobically purified enzyme has a catalytic centre which performs a single electron step at a midpoint potential of E(m)=+60 mV versus the standard hydrogen electrode (n=1), the altered centre shows no redox change under similar experimental conditions . Spectroscopic properties of this 'redox fixed' centre are similar to spectra of the reduced 'redox active' form of CuZ, although the positions and intensities of a number of transitions are changed in the optical spectrum . These observations are interpreted in terms of two forms of the catalytic centre, called CuZ and CuZ* . The structural relationship between these forms is unclear . EPR and magnetic circular dichroism spectra suggest that the basic Cu4S structure is common to both . Curiously, steady-state activity of the aerobic enzyme preparation is slightly increased despite the fact the catalytic centre does not undergo detectable redox changes.

Biochem J, 2002 Jun 15, 364(Pt 3), 659 - 67
Expression of human electron transfer flavoprotein-ubiquinone oxidoreductase from a baculovirus vector: kinetic and spectral characterization of the human protein; Simkovic M et al.; Electron transfer flavoprotein-ubiquinone oxidoreductase (ETF-QO) is an iron-sulphur flavoprotein and a component of an electron-transfer system that links 10 different mitochondrial flavoprotein dehydrogenases to the mitochondrial bc1 complex via electron transfer flavoprotein (ETF) and ubiquinone . ETF-QO is an integral membrane protein, and the primary sequences of human and porcine ETF-QO were deduced from the sequences of the cloned cDNAs . We have expressed human ETF-QO in Sf9 insect cells using a baculovirus vector . The cDNA encoding the entire protein, including the mitochondrial targeting sequence, was present in the vector . We isolated a membrane-bound form of the enzyme that has a molecular mass identical with that of the mature porcine protein as determined by SDS/PAGE and has an N-terminal sequence that is identical with that predicted for the mature holoenzyme . These data suggest that the heterologously expressed ETF-QO is targeted to mitochondria and processed to the mature, catalytically active form . The detergent-solubilized protein was purified by ion-exchange and hydroxyapatite chromatography . Absorption and EPR spectroscopy and redox titrations are consistent with the presence of flavin and iron-sulphur centres that are very similar to those in the equivalent porcine and bovine proteins . Additionally, the redox potentials of the two prosthetic groups appear similar to those of the other eukaryotic ETF-QO proteins . The steady-state kinetic constants of human ETF-QO were determined with ubiquinone homologues, a ubiquinone analogue, and with human wild-type ETF and a Paracoccus-human chimaeric ETF as varied substrates . The results demonstrate that this expression system provides sufficient amounts of human ETF-QO to enable crystallization and mechanistic investigations of the iron-sulphur flavoprotein.

Mol Microbiol, 2002 Jun, 44(5), 1153 - 66
The vbs genes that direct synthesis of the siderophore vicibactin in Rhizobium leguminosarum: their expression in other genera requires ECF sigma factor RpoI; Carter RA et al.; A cluster of eight genes, vbsGSO, vbsADL, vbsC and vbsP, are involved in the synthesis of vicibactin, a cyclic, trihydroxamate siderophore made by the symbiotic bacterium Rhizobium leguminosarum . None of these vbs genes was required for symbiotic N2 fixation on peas or Vicia . Transcription of vbsC, vbsGSO and vbsADL (but not vbsP) was enhanced by growth in low levels of Fe . Transcription of vbsGSO and vbsADL, but not vbsP or vbsC, required the closely linked gene rpoI, which encodes an ECF sigma factor of RNA polymerase . Transfer of the cloned vbs genes, plus rpoI, to Rhodobacter, Paracoccus and Sinorhizobium conferred the ability to make vicibactin on these other genera . We present a biochemical genetic model of vicibactin synthesis, which accommodates the phenotypes of different vbs mutants and the homologies of the vbs gene products . In this model, VbsS, which is similar to many non-ribosomal peptide synthetase multienzymes, has a central role . It is proposed that VbsS activates L-N5-hydroxyornithine via covalent attachment as an acyl thioester to a peptidyl carrier protein domain . Subsequent VbsA-catalysed acylation of the hydroxyornithine, followed by VbsL-mediated epimerization and acetylation catalysed by VbsC, yields the vicibactin subunit, which is then trimerized and cyclized by the thioesterase domain of VbsS to give the completed siderophore.

Clin Immunol, 2002 May, 103(2), 185 - 95
Interleukin-12 protects mice against disseminated infection caused by Paracoccidioides brasiliensis but enhances pulmonary inflammation; Arruda C et al.; Paracoccidioides brasiliensis is a facultative, intracellular pathogen causing the most important deep mycosis in Latin America . As the production of IFN-gamma and induction of cell-mediated immunity to P . brasiliensis is of critical importance in host defense, the immunotherapeutic effect of exogenous IL-12 administration was studied in a murine model of susceptibility to pulmonary infection . rIL-12 treatment led to a less disseminated disease, as confirmed by decreased fungal loads in liver and spleen . Administration of rIL-12 did not affect fungal growth in the lungs, although it did induce an augmented pulmonary mononuclear cell inflammation . IL-12 treatment induced an early (week 1) increase in pulmonary IFN-gamma, but decreased cytokine and specific antibody (IgG1 and IgG3) production at week 8 after infection . These results show that IL-12 administration induces a less severe infection, but the high inflammatory response detected in the lungs precludes its possible use as a new therapeutic tool for severe paracoccidioidomycosis . (c) 2002 Elsevier Science (USA).

Bioelectrochemistry, 2002 May 15, 56(1-2), 17 - 8
Enzyme-catalysed nitrate reduction-themes and variations as revealed by protein film voltammetry; Butt JN et al.; Protein film voltammetry has been used to define the catalytic performance of two nitrate reductases: the respiratory nitrate reductase, NarGH, from Paracoccus pantotrophus and the assimilatory nitrate reductase, NarB, from Synechococcus sp . PCC 7942 . NarGH and NarB present distinct "fingerprints" of catalytic activity when viewed in this way . Potentials that provide insufficient driving force for significant rates of nitrate reduction by NarB result in appreciable rates of nitrate reduction by NarGH . However, both enzymes display complex modulations in their rate of substrate reduction when viewed across the electrochemical potential domain.

Biosci Biotechnol Biochem, 2002 Feb, 66(2), 271 - 6
Cloning, nucleotide sequencing, and expression in Escherichia coli of the gene for formate dehydrogenase of Paracoccus sp . 12-A, a formate-assimilating bacterium; Shinoda T et al.; The gene for the NAD-dependent formate dehydrogenase (FDH) of Paracoccus sp . 12-A, a formate-assimilating bacterium, was cloned through screening of the genomic library with activity staining . The FDH gene included an open reading frame of 1,200 base pairs, and encoded a protein of 43,757 Da, which had high amino acid sequence identity with known FDHs, in particular, with bacterial enzymes such as those of Moraxella sp . (86.5%) and Pseudomonas sp . 101 (83.5%) . The gene was highly expressed in Escherichia coli cells using an expression plasmid with the pUC ori and tac promoter . The recombinant enzyme was somewhat inactive in the stage of the cell-free extract, but its activity markedly increased with purification, in particular, with the step of heat-treatment at 50 degrees C . The purified enzyme showed essentially the same properties as the enzyme from the original Paracoccus cells.

J Immunol, 2002 May 1, 168(9), 4593 - 600
Dual role for nitric oxide in paracoccidioidomycosis: essential for resistance, but overproduction associated with susceptibility; Nascimento FR et al.; Using a murine model of susceptibility and resistance to paracoccidioidomycosis, we have previously demonstrated that immunosuppression occurs in susceptible (B10.A), but not in resistant (A/Sn), mouse strains . Accumulating evidence shows that NO is involved in the induction of T cell immunosuppression during infection as well as in the killing of Paracoccidioides brasiliensis . In the present work, we focused on NO and other macrophage products that could be associated with resistance or susceptibility to paracoccidioidomycosis . A striking difference was related to NO and TNF production . Macrophages from B10.A mice produced high and persistent NO levels, while in A/Sn animals, TNF production predominated . In in vitro cultures, P . brasiliensis-infected macrophages from A/Sn mice also produced large amounts of TNF, while B10.A macrophages only produced NO . TNF production by B10.A macrophages appeared to be suppressed by NO, because the addition of aminoguanidine sulfate, an inducible NO synthase (NOS2) inhibitor, resulted in TNF production . These results suggested that enhanced TNF or NO production is associated with resistance and susceptibility, respectively . However, regardless of the mouse strain, NOS2-deficient or aminoguanidine sulfate-treated mice presented extensive tissue lesions with increased fungal load in lungs and liver compared with their controls . We conclude that NOS2-derived NO is essential for resistance to paracoccidioidomycosis, but overproduction is associated with susceptibility.

Biochem J, 2002 May 1, 363(Pt 3), 817 - 23
Mo(V) co-ordination in the periplasmic nitrate reductase from Paracoccus pantotrophus probed by electron nuclear double resonance (ENDOR) spectroscopy; Butler CS et al.; The first electron nuclear double resonance (ENDOR) study of a member of the Mo-bis-molybdopterin guanine dinucleotide family of molybdoenzymes is presented, using the periplasmic nitrate reductase from Paracoccus pantotrophus . Rapid freeze-quenched time-resolved EPR revealed that during turnover the intensity of a Mo(V) EPR signal known as High-g {resting} increases . This signal is split by two interacting protons that are not solvent-exchangeable . X-band proton-ENDOR analysis resolved broad symmetrical resonance features that arose from four classes of protons weakly coupled to the Mo(V) . Signals from two of these were lost upon exchange into deuterated buffer, suggesting that they may originate from OH(-) or H(2)O groups . One of these signals was also lost when the enzyme was redox-cycled in the presence of azide . Since these protons are very weakly coupled OH/H(2)O groups, they are not likely to be ligated directly to the Mo(V) . This suggests that protonation of a Mo(VI)zO group does not occur on reduction to Mo(V), but most probably accompanies reduction of Mo(V) to Mo(IV) . A resonance feature from a more strongly coupled proton, that was not lost following exchange into deuterated buffer, could also be resolved at 22-24 MHz . The anisotropy of this feature, determined from ENDOR spectra collected at a range of field positions, indicated a Mo-proton distance of approx . 3.2 A, consistent with this being one of the beta-methylene protons of a Mo-Cys ligand.

FEMS Microbiol Lett, 2002 Feb 19, 208(1), 111 - 6
Nitrite reduction in Paracoccus sp . is affected by a novel plasmid pYR1; Barak Y et al.; Two relatively low-copy plasmids of 9 and 16 kb were found to comprise the extrachromosomal DNA of a Paracoccus strain . Reduction of nitrate by plasmid-cured cells resulted in a significant intermediate nitrite accumulation as compared to wild-type cells . By examining nitrate reduction by transformants containing one of the two plasmids, it was found that nitrite accumulation was influenced by the 9.0-kb plasmid, designated as pYR1 . Subcloning analysis showed that a 1.8-kb fragment of this plasmid affected nitrite accumulation . Sequence analysis of this fragment revealed the presence of five open reading frames . One of the six deduced proteins showed a strong homology to ABC transporters.

Int J Syst Evol Microbiol, 2002 Mar, 52(Pt 2), 679 - 82
Emended description of Paracoccus kondratievae; Doronina NV et al.; An aerobic, facultatively chemolithotrophic and methylotrophic strain, GB, was isolated from a maize rhizosphere . On the basis of comparative analysis of its phenotypic and genotypic properties, it is proposed that strain GB(T) (= VKM B-2222T = NCIMB 13773T) be assigned to the genus Paracoccus as Paracoccus kondratievae sp . nov.

Rev Inst Med Trop Sao Paulo, 2002 Jan-Feb, 44(1), 49 - 52
Parinaud'S oculoglandular syndrome associated with paracoccidioidomycosis; Costa PS et al.; The authors report one case of Parinaud's oculoglandular syndrome associated with Paracoccidioides brasiliensis infection . No other medical report of this condition was found in the medical literature available at Index Medicus and Medline . The eye involvement has been rather uncommon in paracoccidioidomycosis and this report emphasizes the possibility of this kind of presentation making it also necessary to include paracoccidioidomycosis among the several known causes of Parinaud's oculoglandular syndrome.

Microbes Infect, 2002 Feb, 4(2), 139 - 44
Cytokines and lymphocyte proliferation in juvenile and adult forms of paracoccidioidomycosis: comparison with infected and non-infected controls; Oliveira SJ et al.; Cellular immune response to Paracoccidiodes brasiliensis antigens (PbAg) was evaluated in patients with the juvenile (JF) and adult (AF) forms of paracoccidioidomycosis as well as in a group of infected individuals living in the endemic area but without any clinical manifestation of the disease . The immune profile of this group of paracoccidioidomycosis-infected individuals was characterized by: 1) a positive skin test to P . brasiliensis antigen; 2) absence of specific antibodies; 3) a vigorous lymphoproliferative response to PbAg; and 4) a typical Th1 pattern of cytokines, with production of IFN-gamma and basal levels of IL-4, IL-5 and IL-10 . At the opposite end of the spectrum were the JF patients whose proliferative response to PbAg was significantly impaired and whose cytokine pattern was characteristically Th2, i.e . lower IFN-gamma secretion and significantly higher levels of IL-4, IL-5 and IL-10 . These profiles are compatible with forms of higher and lower resistance, respectively . Intermediate immune responses were observed in AF patients, whose specific lymphoproliferative response was lower than in the paracoccidioidomycosis-infected group but higher than in the JF patients . The secretion of IFN-gamma and IL-10 did not differ from the JF group, although IL-4 and IL-5 levels were significantly lower . Since AF patients are able to control fungal dissemination for decades, they can be considered more resistant than JF patients, who manifest the disease soon after infection.

Nippon Ishinkin Gakkai Zasshi, 2002, 43(1), 29 - 35
Detection of the gp43 gene and (1-3)-beta-D-glucan of Paracoccidioides brasiliensis in the blood of experimentally infected mice; Nakagawa Itano E et al.; Paracoccidioidomycosis (PCM) is a deep mycosis caused by the thermo-dependent dimorphic fungus Paracoccidioides brasiliensis and is prevalent in Latin American countries . Diagnosis of PCM is sometimes difficult outside the endemic countries, thus a rapid and conclusive method for diagnosis of PCM has been anticipated . We compared the sensitivities of a nested PCR method for detecting the gp43 gene and a commercial kit for detecting (1-3)-beta-D-glucan in the blood of experimentally infected mice . Blood samples were collected from mice at 0 (soon after inoculation), 6, 12, 24, 48, and 72 hours and 5, 7, 10, 14, 17, 21, 24, 28 and 56 days after the intravenous inoculation of 10(6) yeast cells of P . brasiliensis, and were separated into clots and plasma . The (1-3)-beta-D-glucan detection kit in the plasma showed positive reactions in some samples within 7 days and 28 and 56 days after infections . In contrast, the PCR method was more sensitive than the (1-3)-beta-D-glucan detection kit throughout the observation period . The clot samples yielded more sensitive PCR-results than did the plasma samples . Although 24 hours is required for the PCR detection, it was confirmed to provide an accurate diagnosis of PCM.

Med Mycol, 2002 Feb, 40(1), 45 - 51
Identification of differentially expressed transcripts in the human pathogenic fungus Paracoccidioides brasiliensis by differential display; Venancio EJ et al.; Paracoccidioides brasiliensis is a dimorphic human pathogenic fungus that is the causal agent of paracoccidioidomycosis, a systemic disease that predominantly affects rural communities in South and Central America . Dimorphism is a common characteristic of systemic human pathogenic fungi . Here we describe the use of differential display (DD) to isolate and identify differentially expressed genes of P . brasiliensis, in the two cell types, yeast (Y) and mycelium (M), as well as at different time intervals during temperature-induced M to Y transition . Using two oligo-deoxythymidine-anchored primers combined with 10 arbitrary ones, we were able to detect the presence of at least 20 differentially transcribed cDNA fragments . Some of these fragments were further analysed by reverse-northern blot and northern blot in order to confirm their differential expression . The M32, M51 and M73 cDNA fragments were specific for the mycelial form of P . brasiliensis . Furthermore, we found two cDNA fragments (M-Y1 and M-Y2) that were upregulated during M-Y transition . This method was efficient and useful in the detection of differentially expressed genes in P . brasiliensis.

J Biol Inorg Chem, 2002 Jan, 7(1-2), 94 - 100 Epub 2001 Aug 09.
The pH-dependent redox inactivation of amicyanin from Paracoccus versutus as studied by rapid protein-film voltammetry; Jeuken LC et al.; The redox properties of the blue copper protein amicyanin have been studied with slow and fast scan protein-film cyclic voltammetry . At slow scan rates, which reveal the thermodynamics of the redox reactions, the reduction potential of amicyanin depends on pH in a sigmoidal manner, and the data can be analysed in terms of electron transfer being coupled to a single protonatable group with pKa(red)=6.3 and pKa(ox) < or = 3.2 at 22 degrees C . Voltammetry at higher scan rates reveals the kinetics and shows that the low-pH reduced form of amicyanin is not oxidised directly; instead, oxidation occurs only after conversion to the high-pH form . Simulations show that this conversion, which gates the electron transfer, occurs with a rate constant >750 s-1 at 25 degrees C . In order to decrease the rate of the coupled reaction, the experiments were performed at 0 degrees C, at which the rate constant for this conversion was determined to be 35 +/- 20 s-1 . Together with evidence from NMR, the results lead to a mechanism involving protonation and dissociation of the copper coordinating histidine-96 in the reduced form.

J Ethnopharmacol, 2002 Mar, 79(3), 331 - 4
Effects of propolis from Brazil and Bulgaria on fungicidal activity of macrophages against Paracoccidioides brasiliensis; Murad JM et al.; Paracoccidioidomycosis is the most important systemic mycosis in Latin America . Its etiological agent, Paracoccidoides brasiliensis, affects individuals living in endemic areas through inhalation of airborne conidia or mycelial fragments . The disease may affect different organs and systems, with multiple clinical features, with cell-mediated immunity playing a significant role in host defence . Peritoneal macrophages from BALB/c mice were stimulated with Brazilian or Bulgarian propolis and subsequently challenged with P . brasiliensis . Data suggest an increase in the fungicidal activity of macrophages by propolis stimulation, independently from its geographic origin.

J Bacteriol, 2002 Mar, 184(5), 1314 - 23
Periplasmic nitrate reductase (NapABC enzyme) supports anaerobic respiration by Escherichia coli K-12; Stewart V et al.; Periplasmic nitrate reductase (NapABC enzyme) has been characterized from a variety of proteobacteria, especially Paracoccus pantotrophus . Whole-genome sequencing of Escherichia coli revealed the structural genes napFDAGHBC, which encode NapABC enzyme and associated electron transfer components . E . coli also expresses two membrane-bound proton-translocating nitrate reductases, encoded by the narGHJI and narZYWV operons . We measured reduced viologen-dependent nitrate reductase activity in a series of strains with combinations of nar and nap null alleles . The napF operon-encoded nitrate reductase activity was not sensitive to azide, as shown previously for the P . pantotrophus NapA enzyme . A strain carrying null alleles of narG and narZ grew exponentially on glycerol with nitrate as the respiratory oxidant (anaerobic respiration), whereas a strain also carrying a null allele of napA did not . By contrast, the presence of napA+ had no influence on the more rapid growth of narG+ strains . These results indicate that periplasmic nitrate reductase, like fumarate reductase, can function in anaerobic respiration but does not constitute a site for generating proton motive force . The time course of phi(napF-lacZ) expression during growth in batch culture displayed a complex pattern in response to the dynamic nitrate/nitrite ratio . Our results are consistent with the observation that phi(napF-lacZ) is expressed preferentially at relatively low nitrate concentrations in continuous cultures (H . Wang, C.-P . Tseng, and R . P . Gunsalus, J . Bacteriol . 181:5303-5308, 1999) . This finding and other considerations support the hypothesis that NapABC enzyme may function in E . coli when low nitrate concentrations limit the bioenergetic efficiency of nitrate respiration via NarGHI enzyme.

Trends Microbiol, 2002 Feb, 10(2), 80 - 7
The pathobiology of Paracoccidioides brasiliensis; Borges-Walmsley MI et al.; Paracoccidioides brasiliensis causes one of the most prevalent systemic mycoses in Latin America--paracoccidioidomycosis . It is a dimorphic fungus that undergoes a complex transformation in vivo, with mycelia in the environment producing conidia, which probably act as infectious propagules upon inhalation into the lungs, where they transform to the pathogenic yeast form . This transition is readily induced in vitro by temperature changes, resulting in modulation of the composition of the cell wall . Notably, the polymer linkages change from beta-glucan to alpha-glucan, possibly to avoid beta-glucan triggering the inflammatory response . Mammalian oestrogens inhibit this transition, giving rise to a higher incidence of disease in males . Furthermore, the susceptibility of individuals to paracoccidioidomycosis has a genetic basis, which results in a depressed cellular immune response in susceptible patients; resistance is conferred by cytokine-stimulated granuloma formation and nitric oxide production . The latency period and persistence of the disease and the apparent lack of efficacy of humoral immunity are consistent with P . brasiliensis existing as a facultative intracellular pathogen.

J Clin Microbiol, 2002 Feb, 40(2), 359 - 65
Paracoccidioides brasiliensis 87-kilodalton antigen, a heat shock protein useful in diagnosis: characterization, purification, and detection in biopsy material via immunohistochemistry; Diez S et al.; The 87-kDa antigen derived from the fungal pathogen Paracoccidioides brasiliensis can be detected in the sera of infected patients, and its levels have been shown to correlate well with response to treatment and with clinical cure . Despite its potential importance, the antigen has been poorly characterized . The 87-kDa antigen was purified to homogeneity via preparative gel electrophoresis; N-terminal amino acid sequencing revealed substantial homology with heat shock proteins (hsps) from a variety of organisms . A monoclonal antibody (MAb) raised against a Histoplasma capsulatum 80-kDa hsp showed cross-reactivity to the purified 87-kDa antigen via Western blotting, and the 87-kDa-specific MAb P1B demonstrated that the antigen was expressed at higher levels in yeast than in mycelia by the same technique . Enzyme-linked immunosorbent assay and immunofluorescence reactivity using P1B confirmed increased expression of the 87-kDa antigen during the temperature-induced transformation of mycelia to yeast . Yeast-to-mycelium transformation was accompanied by a fall in expression, although the 87-kDa antigen was clearly constitutively expressed in both phases . Immunochemical staining of tissues from patients with MAb P1B who were infected with P . brasiliensis confirmed in vivo expression of the 87-kDa antigen by yeasts, and identification of this antigen via this method appears to be a useful adjunct to other methods used to diagnose paracoccidioidomycosis.

Hum Immunol, 2002 Feb, 63(2), 149 - 54
Patients with active infection with Paracoccidioides brasiliensis present a Th2 immune response characterized by high Interleukin-4 and Interleukin-5 production; Marques Mello L et al.; Paracoccidiodomycosis (PCM) is caused by the dimorphic fungus Paracoccidioides brasiliensis . In humans, the disease presents a broad spectrum of clinical manifestations, ranging from localized mucocutaneous lesions to a widespread manifestation with involving the mononuclear phagocyte system . In attempt to better understand the regulation of immune response during the infection, this study analyzed the production of regulatory and inflammatory cytokines in 25 infected patients and 19 health controls . Regulatory and inflammatory cytokines were analyzed in supernatants of peripheral blood mononuclear cells (PBMC) stimulated with mitogens or soluble P . brasiliensis antigens . A pattern of Th2 immune response was observed in patients, mainly attributed to a higher production of IL-4 and IL-5 than to a lower production of IFN-gamma . Patients with disseminated infection presented undetectable levels of IFN-gamma after antigen stimulation and high levels of IL-1beta, which were probably associated with the inflammatory reaction observed in multifocal infection.

Rev Soc Bras Med Trop, 2001 Nov-Dec, 34(6), 583 - 6
Anal paracoccidioidomycosis: an unusual presentation of disseminated disease; Costa Vieira RA et al.; We report a patient with an unusual anal ulceration . The biopsy of an anal lesion and subsequent studies revealed a disseminated form of paracoccidioidomycosis, observed in the lungs, small and large bowel . The anorectal disease frequently represents a secondary site of disease, and the patient must be better evaluated.

Mycopathologia, 2001, 152(3), 125 - 33
Expression of complement receptor type 1 (CR1) on erythrocytes of paracoccidiodomycosis patients; Teixeira JE et al.; Complement receptor type 1 (CR1) is a membrane glycoprotein that acts as a receptor for the C3b, iC3b and C4b fragments of complement . In primates, one function of erythrocytes is to promote safe clearance of immunocomplexes (IC) from the circulation through CR1 . Theoretically, in diseases characterized by high levels of circulating IC, an erythrocyte CR1 (CR1/E) deficiency may favor IC deposition in tissues or facilitate inappropriate activation of leukocytes in the circulation . Depression of the cell immune response occurs in paracoccidioidomycosis (PCM), especially in the more severe cases, and is frequently associated with high serum IC levels . In the present study we quantified the number of CR1/E in patients with the acute and chronic forms of PCM before and after treatment and correlated it with serum IC levels and CD4+ and CD8+ T cell concentration in the peripheral blood of these patients . Patients with PCM, particularly those with active disease and who had received treatment for shorter periods of time, had low numbers of CR1/E . In addition, an increase in serum IC concentration and a reduction in the CD4+/CD8+ T cell ratio were observed . After treatment there was a significant increase in mean CR1/E number and a reduction in serum IC levels . In patients with the chronic form of the disease the CD4+/CD8+ T cell ratio tended to increase after treatment and was associated with increased CR1/E levels . These results suggest that the reduction in CR1/E observed in patients is a phenomenon acquired with the disease and that CR1 could play a role in the pathogenesis of PCM.

FEBS Lett, 2002 Jan 16, 510(3), 185 - 8
Replacement of the methionine axial ligand in cytochrome c(550) by a lysine: effects on the haem electronic structure; Louro RO et al.; The prosthetic group of low-spin haem proteins is an iron porphyrin with two axial ligands, typically histidine, methionine or lysine . Determining the geometry of the axial ligands is an important step in structural characterisation, particularly in the paramagnetic oxidised forms . This work extends earlier studies of the hyperfine nuclear magnetic resonance (NMR) shifts of haem substituents in bis-His and His-Met cytochromes to His-Lys co-ordination in the M100K mutant of Paracoccus versutus cytochrome c(550) . The electronic structure of the His-Lys haem is shown to be similar to that produced by His-cyanide co-ordination, such that NMR can be used to determine the geometry of the His ligand.

Med Mycol, 2001 Dec, 39(6), 475 - 8
IgE antibody response to the main antigenic component of Paracoccidioides brasiliensis in patients with paracoccidioidomycosis; Biselli PJ et al.; Paracoccidioidomycosis (PCM) has two main clinical presentations, a chronic form (CF) and an acute, more severe form (AF) . The AF is associated with a more marked dysfunction of the patient's immune response, and a distinct anti-Paracoccidioides brasiliensis immunoglobulin (Ig)A and IgG antibody subclass expression, compared with that seen in the CF . In this study we investigated the presence of IgE antibodies against the main P . brasiliensis antigen (a 43-kDa molecule) in the serum of PCM patients using an enzyme-linked immunosorbent assay . We found that 100% of the AF patients (n = 16) produced IgE antibodies, mostly at high levels, whereas only 9 (27%) out of 33 CF patients produced this isotype . Interestingly, these nine patients presented higher serological titers on the counter-immunoelectrophoresis assay than did those who did not produce IgE; a finding that suggests that they had a relatively more severe disease . As IgE is a characteristic feature of the AF patients, and switching to a positive IgE response is dependent on interleukin-4, our results support the notion that the relatively more severe impairment of cellular immunity in the AF is probably related to a Th-2 pattern of immune response.

J Biol Chem, 2002 Feb 1, 277(5), 3093 - 100 Epub 2001 Nov 14.
Cytochrome cd1, reductive activation and kinetic analysis of a multifunctional respiratory enzyme; Richter CD et al.; Paracoccus pantotrophus cytochrome cd(1) is an enzyme of bacterial respiration, capable of using nitrite in vivo and also hydroxylamine and oxygen in vitro as electron acceptors . We present a comprehensive analysis of the steady state kinetic properties of the enzyme with each electron acceptor and three electron donors, pseudoazurin and cytochrome c(550), both physiological, and the non-physiological horse heart cytochrome c . At pH 5.8, optimal for nitrite reduction, the enzyme has a turnover number up to 121 s(-1) per d(1) heme, significantly higher than previously observed for any cytochrome cd(1) . Pre-activation of the enzyme via reduction is necessary to establish full catalytic competence with any of the electron donor proteins . There is no significant kinetic distinction between the alternative physiological electron donors in any respect, providing support for the concept of pseudospecificity, in which proteins with substantially different tertiary structures can transfer electrons to the same acceptor . A low level hydroxylamine disproportionase activity that may be an intrinsic property of cytochromes c is also reported . Important implications for the enzymology of P . pantotrophus cytochrome cd(1) are discussed and proposals are made about the mechanism of reduction of nitrite, based on new observations placed in the context of recent rapid reaction studies.

J Trop Pediatr, 2001 Oct, 47(5), 311 - 5
Osteomyelitis caused by Paracoccidioides brasiliensis in a child from the metropolitan area of Rio de Janeiro; Nogueira SA et al.; The authors describe a case of paracoccidioidomycosis in a 7-year-old girl from the city of Rio de Janeiro who initially presented to her physician with a lesion in her calcaneous which was misdiagnosed and treated as bacterial osteomyelitis . Later, cutaneous manifestations, lymph node enlargement, and hepatosplenomegaly developed and biopsy of the skin and cervical lymph nodes showed the fungus which was also present in the sputum . It is emphasized that Paracoccidioides brasiliensis can be the cause of bone lesions in endemic areas of Latin America and that response to treatment with amphotercin B is good.

Oral Dis, 2001 Jul, 7(4), 217 - 20
Exfoliative cytology: a helpful tool for the diagnosis of paracoccidioidomycosis; Cardoso SV et al.; OBJECTIVE: To describe the main cytological findings associated with smears collected from oral lesions of paracoccidioidomycosis and to appraise the use of cytology as a diagnostic tool for the disease . STUDY DESIGN: Cytological smears and biopsies were collected from 40 lesions with a clinical suspicion of paracoccidioidomycosis . Evaluation of the sensitivity, specificity, positive and negative predictive values, accuracy and the positive likeness ratio of the oral smear when compared with the histological diagnosis, was performed . The latter is considered the 'gold standard' for comparison . RESULTS: The main morphological findings were the rounded-shaped, birefringent and multiple-budded fungi, Langhans' giant cells and epithelioid cells . The following associative measures were found: sensitivity, 67.9%; specificity, 91.7%; positive predictive value, 95.0%; negative predictive value, 55.0%; accuracy, 75.0%; and positive likeness ratio, 8.14 . CONCLUSION: The cytological findings of paracoccidioidomycosis are characteristic and cytology is accurate in the diagnosis of the disease . Positive patients should be treated . Negative patients should be submitted to biopsy to confirm or to dismiss the diagnosis of this mycosis.

Mem Inst Oswaldo Cruz, 2001 Aug, 96(6), 771 - 2
The marsupial Didelphis albiventris is an improbable host of Paracoccidioides brasiliensis in an endemic area of paracoccidioidomycosis in Minas Gerais, Brazil; Silva-Vergara ML et al.; To determine whether Didelphis albiventris is naturally infected with Paracoccidioides brasiliensis, 20 specimens of this mammal were studied by both direct cultivation of their viscera (spleen, liver and lungs) and by inoculation of Swiss mice by the intraperitoneal route with a suspension of fragments of these viscera . No fungal growth or structures similar to this fungus were detected . Probably D . albiventris is not frequently infected with P . brasiliensis.

Am J Trop Med Hyg, 2001 Sep, 65(3), 237 - 41
Capture enzyme-linked immunosorbent assay to detect specific immunoglobulin E in sera of patients with paracoccidioidomycosis; Mamoni RL et al.; Paracoccidioidomycosis (PCM) is the most frequent systemic mycosis in South America . The disease is characterized by a polyclonal activation of B cells, resulting in hyperimmunoglobulinemia . The production of immunoglobulin (Ig) E in deep mycosis has been related to the severity of the disease . However, the detection of specific IgE in sera of patients is difficult because of the competition with the IgG . We compared a capture and an indirect enzyme-linked immunosorbent assay (ELISA) technique to detect Paracoccidioides brasiliensis IgE . We found that the capture ELISA presented higher performance and lower background values than the indirect assay, resulting in a significant quantitative discrimination between sera from patients with the 2 major clinical forms of PCM . Patients with the juvenile form presented significantly higher levels of P . brasiliensis IgE, as compared with patients with the adult form . The capture ELISA was used in the follow-up of patients receiving treatment, showing that the levels of specific IgE decreased as the patient's clinical conditions improved.

Biochemistry, 2001 Sep 25, 40(38), 11294 - 307
Catalytic protein film voltammetry from a respiratory nitrate reductase provides evidence for complex electrochemical modulation of enzyme activity; Anderson LJ et al.; The first step in the respiratory reduction of nitrate to dinitrogen in Paracoccus pantotrophus is catalyzed by the quinol-nitrate oxidoreductase NarGHI . This membrane-anchored protein directs electrons from quinol oxidation at the membrane anchor, NarI, to the site of nitrate reduction in the membrane extrinsic {Fe-S} cluster and Mo-bis-MGD containing dimer, NarGH . Liberated from the membrane, NarGH retains its nitrate reductase activity and forms films on graphite and gold electrodes within which direct and facile exchange of electrons between the electrode and the enzyme occurs . Protein film voltammetry has been used to define the catalytic behavior of NarGH in the potential domain and a complex pattern of reversible, nitrate concentration dependent modulation of activity has been resolved . At low nitrate concentrations the local maximum observed in the catalytic current-potential profile reveals how NarGH can catalyze nitrate reduction via two pathways having distinct specificity constants, k(obs)(cat)/K(obs)(M) . Catalysis is directed to occur via one of the pathways by an electrochemical event within NarGH . On increasing the nitrate concentration, the local maximum in the catalytic current becomes less distinct, and the catalytic waveform adopts an increasingly sigmoidal form . A pattern of voltammetry similar to that observed during nitrate reduction is observed during reduction of the stereochemically distinct substrate chlorate . Centers whose change of oxidation state may define the novel catalytic voltammetry of NarGH have been identified by EPR-monitored potentiometric titrations and mechanisms by which the electrochemistry of Mo-bis-MGD or {Fe-S} clusters can account for the observed behavior are discussed.

Med Mycol, 2001 Aug, 39(4), 373 - 7
Interpretation and clinical correlation of serological tests in paracoccidioidomycosis; Do Valle AC et al.; In order to correlate the findings of two serological tests, double immunodiffusion (IDD) and immunoblotting (IB), with the clinical diagnosis and follow-up of paracoccidioidomycosis (PCM), 325 serum samples from PCM patients were tested at the beginning of specific therapy and after its completion . Group I included 245 PCM patients at the onset of symptoms without treatment . In 221 cases (90.2%) the IDD showed positive reactions and in 24 (9.8%) the results were negative . Of the 24 IDD negative samples, 23 were investigated by IB and were positive . Group II included 80 PCM patients under follow-up after treatment . There were four cases of relapse in which the IDD and IB tests were positive (100%) . Among the 76 cases with inactive mycotic infection, the IDD was negative in 71.2% and positive in 28.8%; the IB was positive in all cases (100%) . The control group (Group III) included 27 samples from patients with other mycoses, tuberculosis and from healthy individuals . All showed negative IDD tests but positive reactions with IB, which could be abolished by serum dilutions without altering the PCM reactivity . Therefore, the utilization of the IB, an immunoenzymatic method for the diagnosis of PCM, raised the sensitivity to 100%.

Med Mycol, 2001 Aug, 39(4), 359 - 68
Regulation of serine-type exoproteinases by endogenous inhibitors present in exoantigens of the mycelial form of Paracoccidioides brasiliensis; Zambrano EA et al.; We have partially characterized some biochemical properties of exoproteinases secreted into culture medium by the mycelial form of Paracoccidioides brasiliensis, a dimorphic fungus that causes human disease in Latin America . Proteinase activity was analyzed in solid- and liquid-phase systems using zymography and Azocoll, respectively . Minimal or no gelatinase activity was observed by zymography in the crude filtrates among proteins with a relative mobility greater than 200 kDa . When the crude filtrate was fractionated by isoelectric focusing or ion exchange chromatography, we observed striking activation of gelatinases, both those of high apparent molecular mass and alkaline isoelectric points (pI), as well as those of lower molecular mass and acidic pI . The apparent high molecular mass gelatinases, pI 10, showed optimal activity at pH 7.0 . They were totally inhibited by phenylmethylsulfonylfluoride and partially inhibited by incubation with previously neutralized fractions of pI 5.4 and 6.1 . The latter inhibition could be reversed by exposure to 10% isopropanol . These results provide evidence of regulatory mechanisms controlling proteinase activity in secreted proteins . The principal mechanism appears to be the formation of reversible complexes with endogenous inhibitors.

Scand J Immunol, 2001 Oct, 54(4), 348 - 56
Immunostimulatory DNA from Paracoccidioides brasiliensis acts as T-helper 1 promoter in susceptible mice; Souza MC et al.; Th1 immune responses afford protection against some pathogens like the fungus P . brasiliensis (P.b.), etiological agent of Paracoccidioidomycosis (PCM) . It is well known that nonmethylated CpG sequences from bacterial DNA have immunomodulatory properties and can be used as a Th1-promoting adjuvant . By analyzing the available gene sequences of P.b . we observed a high number of unmethylated CpG dinucleotides . In a murine model of the PCM infection, the isogenic mouse strain known to be susceptible presents a predominant Th2 pattern . In order to access the possibility of the genomic DNA to act as a Th1-promoting adjuvant, in vitro assays were made and indicated a significant increase in phagocytosis when the macrophages were stimulated with DNA from P.b . and in vivo assays of a decreased production of antibodies antigp43, the main antigen of the PCM system . The analysis of the antibody isotypes and the cytokine production suggested a Th1 modulation in the susceptible animals . Thus, when mice were infected with fungus plus synthetic oligodeoxynucleotide (ODN), made from the available sequence of gp43, a decrease in the fungus dissemination was observed . Results herein described suggest that genomic DNA from P.b . could have a immunostimulatory function as a Th-1-promoting adjuvant in susceptible mice.

Mycopathologia, 2001, 151(2), 57 - 62
The oral route in the pathogenesis of paracoccidioidomycosis: an experimental study in BALB/c mice infected with P . brasiliensis conidia; Camilo Roldan J et al.; Due to the high frequency of oral mucosal lesions observed in paracoccidioidomycosis patients, it was advocated that the infection was acquired by the traumatic implantation of the etiologic agent Paracoccidioides brasiliensis . Although at present this theory is considered invalid, it has not yet been excluded in experimental studies . In order to determine if intra-oral inoculation could explain the pathogenesis of paracoccidioidomycosis, 64 BALB/c mice were inoculated intra-orally with 850.000 viable P . brasiliensis conidia into the mandibular body . Animals were sacrificed at various time intervals up to 20 weeks and cultures were made from gingiva, lungs, spleen, and liver . Additionally, histopathological studies of the mandibular body were also performed . P . brasiliensis was isolated from all gingival tissues during the interval 24-72 h, indicating that the infection was active . During the 5-10 week period, the infection appeared to have been controlled at the inoculation site as cultures showed a significant reduction in colony forming units (CFU); however, at the 15-20 week period such control was lost and the fungus was recovered once more . Dissemination to other body sites was rare; thus, the lungs were involved in just one animal (2%), the liver in two (3%) and the spleen in seven (11%) . The infection became established as proven by positive organ cultures, but the dissemination pattern did not correspond to the one observed in humans . Based on these findings, the intra-oral traumatic route does not appear to mimic the natural history of paracoccidioidomycosis.

Arch Microbiol, 1986, 145, 202 - 8
The purification and properties of a cd-cytochrome nitrite reductase from Paracoccus halodenitrificans; Mancinelli RL et al.; Paracoccus halodenitrificans, grown anaerobically in the presence of nitrite, contained membrane and cytoplasmic nitrite reductases . When assayed in the presence of phenazine methosulfate and ascorbate, the membrane-bound enzyme produced nitrous oxide whereas the cytoplasmic enzyme produced nitric oxide . When both enzymes were assayed in the presence of methyl viologen and dithionite, the cytoplasmic enzyme produced ammonia . Following solubilization, the membrane-bound enzyme behaved like the cytoplasmic enzyme, producing nitric oxide in the presence of phenazine methosulfate and ascorbate, and ammonia when assayed in the presence of methyl viologen and dithionite . The cytoplasmic and membrane-bound enzymes were purified to essentially the same specific activity . Only a single nitrite-reductase activity was detected on electrophoretic gels and the electrophoretic behavior of both enzymes suggested they were identical . The spectral properties of both enzymes suggested they were cd-type cytochromes . These data suggest that the products of nitrite reduction by the cd-cytochrome nitrite reductase are determined by the location of the enzyme and the redox potential of the electron donor.

FEMS Microbiol Lett, 1996 Jun 15, 140(1), 55 - 60
Is the Paracoccus halodenitrificans ATPase a chimeric enzyme?
Hochstein LI.
Membranes from Paracoccus halodenitrificans contain an ATPase that is most active in the absence of NaCl . The most unusual characteristic of the enzyme is its pattern of sensitivity to various inhibitors . Azide and rhodamine 6G, inhibitors of F1F0-ATPases, inhibit ATP hydrolysis as do bafilomycin A1, concanamycin A (folimycin), N-ethylmaleimide, and p-chloromercuriphenylsulfonate which are inhibitors of vacuolar ATPases . This indiscriminate sensitivity suggests that this ATPase may be a hybrid and that caution should be exercised when using inhibition as a diagnostic for distinguishing between F1F0-ATPases and vacuolar ATPases.

Can J Microbiol, 1984 Jun, 30(6), 837 - 40
The growth of Paracoccus halodenitrificans in a defined medium; Hochstein LI et al.; A synthetic medium, consisting of inorganic salts and any of a number of carbon sources, supported the aerobic growth of Paracoccus halodenitrificans when supplemented with thiamine . The same medium plus an appropriate nitrogenous oxide supported anaerobic growth when additionally supplemented with methionine . The observation that vitamin B12 or betaine replaced methionine suggested that P . halodenitrificans had a defect in the cobalamin-dependent pathway for methionine biosynthesis, as well as the inability to synthesize betaine when growing anaerobically.

Can J Microbiol, 1984 May, 30(5), 572 - 7
Electron transport in Paracoccus halodenitrificans and the role of ubiquinone; Hochstein LI et al.; The membrane-bound NADH oxidase of Paracoccus halodenitrificans was inhibited by dicoumarol, 2-n-heptyl-4-hydroxy-quinoline-N-oxide (HQNO), and exposure to ultraviolet light (at 366 nm) . When the membranes were extracted with n-pentane, NADH oxidase activity was lost . Partial restoration was achieved by adding the ubiquinone fraction extracted from the membranes . Succinate oxidation was not inhibited by dicoumarol or HQNO, but was affected by ultraviolet irradiation or n-pentane extraction . However, the addition of the ubiquinone fraction to the membranes extracted with n-pentane did not restore enzyme activity . These observations suggested that NADH and succinate were not oxidized through a common ubiquinone pool.

Clin Diagn Lab Immunol, 2001 Sep, 8(5), 1036 - 8
High serum interleukin-10 and tumor necrosis factor alpha levels in chronic paracoccidioidomycosis; Fornari MC et al.; In patients with chronic paracoccidioidomycosis (n = 10), levels of tumor necrosis factor alpha, interleukin-10, and interleukin-2 in serum, measured by enzyme-linked immunosorbent assay (in picograms per milliliter, as mean +/- standard error of the mean), were higher than in normal controls (n = 8): 186 +/- 40 versus 40 +/- 7 (P < 0.05), 203 +/- 95 versus 20 +/- 8 (P = 0.001), and 96.3 +/- 78.57 versus 1.19 +/- 1.19 (P = 0.045), respectively . Gamma interferon and interleukin-4 levels were similar in patients and controls.

FEMS Microbiol Lett, 2001 Aug 21, 202(2), 215 - 20
The shxVW locus is essential for oxidation of inorganic sulfur and molecular hydrogen by Paracoccus pantotrophus GB17: a novel function for lithotrophy; Bardischewsky F et al.; The shxVW genes of Paracoccus pantotrophus were identified to be essential for lithotrophic oxidation of sulfur and hydrogen . shxV predicts a membrane protein which is 42% identical to CcdA of P . pantotrophus essential for cytochrome c biogenesis . shxW predicts a periplasmic thioredoxin . Disruption of shxV by an Omega-kanamycin interposon disabled the resulting mutant GB(Omega)V to grow with thiosulfate or molecular hydrogen and to express ShxW while cytochrome c formation was not affected . Mixotrophic growth with succinate and thiosulfate of strain GB(Omega)V revealed 2% of the thiosulfate-dependent oxygen uptake rate as compared to the wild-type while antigens of proteins essential for sulfur oxidation were present in both strains . Mixotrophic growth of strain GB(Omega)V with succinate and molecular hydrogen revealed neither hydrogenase activity nor antigens . Complementation analysis with plasmid pBHP6 carrying the shxVW genes revealed the wild-type phenotype of strain GB(Omega)V(pBHP6).

FEBS Lett, 2001 Aug 17, 503(2-3), 168 - 72
The cysteine residue of the SoxY protein as the active site of protein-bound sulfur oxidation of Paracoccus pantotrophus GB17; Quentmeier A et al.; Four proteins of Paracoccus pantotrophus are required for hydrogen sulfide-, sulfur-, thiosulfate- and sulfite-dependent horse heart cytochrome c reduction . The lack of free intermediates suggested a protein-bound sulfur oxidation mechanism . The SoxY protein has a novel motif containing a cysteine residue . Electrospray ionization and matrix-assisted laser desorption ionization mass spectrometry of the SoxYZ protein revealed one mass for SoxZ and different masses for SoxY, indicating native SoxY (10977 Da) and SoxY with additional masses of +32, +80, +112 and +144 Da, suggesting addition of sulfur, sulfite, thiosulfate and thioperoxomonosulfate . Reduction of SoxY removed the additional masses, indicating a thioether or thioester bond . N-Ethylmaleimide inhibited thiosulfate-oxidation and the kinetics suggested a turn-over-dependent mode of action . These data were evidence that the sulfur atom to be oxidized was covalently linked to the thiol moiety of the cysteine residue of SoxY and the active site of sulfur oxidation.

Infect Immun, 2001 Sep, 69(9), 5760 - 7
Detection of melanin-like pigments in the dimorphic fungal pathogen Paracoccidioides brasiliensis in vitro and during infection; Gomez BL et al.; Melanins are implicated in the pathogenesis of several human diseases, including some microbial infections . In this study, we analyzed whether the conidia and the yeasts of the thermally dimorphic fungal pathogen Paracoccidioides brasiliensis produce melanin or melanin-like compounds in vitro and during infection . Growth of P . brasiliensis mycelia on water agar alone produced pigmented conidia, and growth of yeasts in minimal medium with L-3,4-dihydroxyphenylalanine (L-DOPA) produced pigmented cells . Digestion of the pigmented conidia and yeasts with proteolytic enzymes, denaturant, and hot concentrated acid yielded dark particles that were the same size and shape as their propagules . Immunofluorescence analysis demonstrated reactivity of a melanin-binding monoclonal antibody (MAb) with the pigmented conidia, yeasts, and particles . Electron spin resonance spectroscopy identified the yeast-derived particles produced in vitro when P . brasiliensis was grown in L-DOPA medium as a melanin-like compound . Nonreducing polyacrylamide gel electrophoresis of cytoplasmic yeast extract revealed a protein that catalyzed melanin synthesis from L-DOPA . The melanin binding MAb reacted with yeast cells in tissue from mice infected with P . brasiliensis . Finally digestion of infected tissue liberated particles reactive to the melanin binding MAb that had the typical morphology of P . brasiliensis yeasts . These data strongly suggest that P . brasiliensis propagules, both conidia and yeast cells, can produce melanin or melanin-like compounds in vitro and in vivo . Based on what is known about the function of melanin in the virulence of other fungi, this pigment may play a role in the pathogenesis of paracoccidioidomycosis.

Eur J Biochem, 2001 Aug, 268(15), 4207 - 16
The peroxidase activity of cytochrome c-550 from Paracoccus versutus; Diederix RE et al.; Next to their natural electron transport capacities, c-type cytochromes possess low peroxidase and cytochrome P-450 activities in the presence of hydrogen peroxide . These catalytic properties, in combination with their structural robustness and covalently bound cofactor make cytochromes c potentially useful peroxidase mimics . This study reports on the peroxidase activity of cytochrome c-550 from Paracoccus versutus and the loss of this activity in presence of H2O2 . The rate-determining step in the peroxidase reaction of cytochrome c-550 is the formation of a reactive intermediate, following binding of peroxide to the haem iron . The reaction rate is very low compared to horseradish peroxidase (approximately one millionth), because of the poor accessibility of the haem iron for H2O2, and the lack of a base catalyst such as the distal His of the peroxidases . This is corroborated by the linear dependence of the reaction rate on the peroxide concentration up to at least 1 M H2O2 . Steady-state conversion of a reducing substrate, guaiacol, is preceded by an activation phase, which is ascribed to the build-up of amino-acid radicals on the protein . The inactivation kinetics in the absence of reducing substrate are mono-exponential and shown to be concurrent with haem degradation up to 25 mM H2O2 (pH 8.0) . At still higher peroxide concentrations, inactivation kinetics are biphasic, as a result of a remarkable protective effect of H2O2, involving the formation of superoxide and ferrocytochrome c-550.

Hum Immunol, 2001 Aug, 62(8), 799 - 808
In vitro granuloma formation, NO production and cytokines profile from human mononuclear cells induced by fractionated antigens of Paracoccidioides brasiliensis; Diniz SN et al.; Paracoccidioides brasiliensis causes a chronic granulomatous mycosis prevalent in South America, and cell-mediated immunity represents the main mode of protection against this fungal infection . We investigated in vitro the response of peripheral blood mononuclear cells (PBMC) from paracoccidioidomycosis (PCM) patients presenting different clinical forms to antigenic fractions from P . brasiliensis yeast cell lysate (PbAg) . These fractions designated F0 to FV were obtained using anion-exchange chromatography on a FPLC system . Our studies showed variation in the cellular responses induced by different antigenic fractions . The fraction F0 caused significant decrease in cellular proliferation, granuloma formation, accompanied by significant elevation in the production of IL-10 . The fractions FII and FIII increased in vitro granuloma formation associated with high production of TNF-alpha . Besides that, FII and FIII evoked decrease in NO production but not F0 that induced very high levels, among patients with PCM from acute form . The findings suggest that P . brasiliensis antigenic components participate in the modulation or activation of PBMC response in PCM, and IL-10 and NO could be important in the regulation of in vitro granuloma formation.

Biochemistry, 2001 Jul 24, 40(29), 8542 - 7
Intramolecular electron transfer from c heme to d1 heme in bacterial cytochrome cd1 nitrite reductase occurs over the same distances at very different rates depending on the source of the enzyme; Kobayashi K et al.; Intramolecular electron transfer over 12 A from heme c to heme d(1) was investigated in cytochrome cd(1) nitrite reductase from Pseudomonas aeruginosa, following reduction of the c heme by pulse radiolysis . The rate constant for the transfer is relatively slow, k = 3 s(-1) . The present observations contrast with a corresponding rate of electron transfer, 1.4 x 10(3) s(-1), measured for cytochrome cd(1) from Paracoccus pantotrophus, though the relative positions of the two heme groups are the same in both enzymes . The rate of intramolecular electron transfer within the enzyme from P . aeruginosa was accelerated 10(4)-fold (1.4 x 10(4) s(-1)) by the binding of cyanide to the d(1) heme . A coordination change at the d(1) heme upon its reduction is suggested to be a major factor in determining the slow rate of electron transfer in the P . aeruginosa enzyme in the absence of cyanide.

Int J Dermatol, 2001 Apr, 40(4), 281 - 3
Hard palate perforation: an unusual finding in paracoccidioidomycosis; Castro LG et al.; A 36-year-old black man presented to his dermatologist in May 1996 complaining of mucosal lesions in the mouth, as well as perforation of the hard palate . The lesions had started approximately 7 months before and had worsened gradually . Other complaints included odynophagia, dysphagia, mild dyspnea, and dry cough . The patient was in good general health, but reported a 3 kg weight loss over the previous semester . The hard and soft palate presented erythematous ulcers with a finely granulated base and irregular, but clearly defined margins . A perforation (diameter, 0.5 cm) of the hard palate was seen in the center of the ulcerated region (Fig . 1) . Direct examination of 10% KOH cleared specimens showed typical double-walled, multiple budding yeast structures . Paracoccidioidomycosis (PCM) serologic reactions tested positive for double immunodiffusion (DI), complement fixation (CF) 1 : 256 and counterimmunoelectrophoresis (CIE) 1 : 128 . Hematoxylin and eosin-stained sections of oral lesions showed an ulcer covered by a fibrous leukocytic crust, with a lymphoplasmacytic infiltrate, as well as multinuclear giant cells containing round bodies with a double membrane . Gomori-Grocott staining showed budding and blastoconidia suggestive of PCM . Lung computed tomography (CT) exhibited findings consistent with pulmonary PCM . Diagnosis of the chronic multifocal form of PCM with oral and pulmonary manifestations was established . Drug therapy was initiated with ketoconazole (KCZ) 200 mg twice daily, which led to clinical cure in approximately 2 months . Serum antibody values rose 30 days after institution of therapy (CIE 1 : 256; CF 1 : 512), peaking at day 60 (CIE 1 : 1024; CF 1 : 1024) . Three months later the daily dose was reduced to 200 mg and titers declined slowly . The diameter of the perforation remained unchanged (Fig . 2) . The hard palate perforation was corrected with a palatoplasty 27 months after initiation of drug therapy (Fig . 3) . KCZ was discontinued when serologic cure was achieved after 34 months of treatment (DI weakly positive; CIE 1 : 8; CF not measurable) . The patient was discharged 46 months after the first visit.

Yeast, 2001 Jul, 18(10), 981 - 8
Cloning and characterization of a LON gene homologue from the human pathogen Paracoccidioides brasiliensis; Barros TF et al.; A LON gene homologue from the human pathogen Paracoccidioides brasiliensis (PbLON) has been cloned, sequenced and characterized . It encodes a putative ATP-dependent proteinase Lon, which in Saccharomyces cerevisisae (PIM1) is a heat-inducible protein involved in the degradation of abnormal or short-lived proteins in the mitochondria . The PbLON ORF is within a 3369 bp fragment interrupted by two introns located in the 3'segment . The 5' and 3' regions flanking the ORF contain sequences which resemble known transcription elements . Several transcription binding factor motifs have also been found, including sites for heat shock/stress response and nitrogen control . The deduced protein consists of 1063 residues containing a mitochondrial import signal at the N-terminus and conserved ATP-binding (GPPGVGKT) and serine catalytic (KDGPSAG) sites . It shares high identity with Lon homologues from S . cerevisiae (73%), Homo sapiens (62%) and Escherichia coli (56%) . In P . brasiliensis, an MDJ1 putative gene has also been partially sequenced adjacent to PbLON, possibly sharing divergently orientated promoter elements . This chromosomal organization is interesting, since Mdj1p is a heat shock chaperone essential for substrate degradation by PIM1 in yeast .

Med Mycol, 2001 Jun, 39(3), 277 - 82
Canine paracoccidioidomycosis: a seroepidemiologic study; Ono MA et al.; Sera from 305 dogs were analyzed by enzyme-linked immunosorbent assay (ELISA) to determine presence of the antibody anti-gp43, which reacts to a specific antigen of Paracoccidioides brasiliensis . The dogs were divided into three groups according to their origin: urban dogs (animals with little or no contact with rural areas); suburban dogs (from the urban outskirts); and rural dogs . There was a significant difference between groups (P < 0.05) . Rural dogs reacted positively in 89.5% of cases, followed by suburban (48.8%) and urban dogs (14.8%) . There were no differences between male and female dogs . In an attempt to verify the feasibility of skin testing with gp43 to determine sensitization against P . brasiliensis in dogs, suburban (n = 61) and rural (n = 21) dogs were tested, showing positivity of 13.1 and 38.1%, respectively . Six dogs that had higher ELISA titers and also showed strong reactions in skin testing were killed in an attempt to isolate P . brasiliensis . The fungus was not detected by culture or histopathological analysis in these dogs, suggesting that dogs have a natural resistance or that they encounter an inoculum level that is insufficient to cause disease . These results indicate that ELISA and skin testing can be useful in the epidemiological study of paracoccidioidomycosis in dogs and that encounter with the fungus in nature is a frequent event.

Med Mycol, 2001 Jun, 39(3), 233 - 41
The habitat of Paracoccidioides brasiliensis: how far from solving the riddle?
Restrepo A, McEwen JG, Castaneda E.
When trying to understand the pathophysiology of any infectious agent, one key piece of information is the determination of its habitat . In the case of Paracoccidioides brasiliensis, the precise location of the fungus' environmental niche remains undefined despite the efforts of various research groups . This review summarizes recent studies on the ecology of P . brasiliensis and certain facets of paracoccidioidomycosis . Studies on the juvenile form of paracoccidioidomycosis in children less than 13 years of age, the characterization of the ecological factors in the 'reservarea' where the infection is acquired and the presence of P . brasiliensis in the nine-banded armadillo (Dasypus novemcinctus), are all helping to pinpoint the microniche of this pathogen . The application of molecular biology techniques based on the amplification of nucleic acids will also hopefully help in establishing the precise habitat of P . brasiliensis.

J Bacteriol, 2001 Aug, 183(15), 4499 - 508
Novel genes of the sox gene cluster, mutagenesis of the flavoprotein SoxF, and evidence for a general sulfur-oxidizing system in Paracoccus pantotrophus GB17; Rother D et al.; The novel genes soxFGH were identified, completing the sox gene cluster of Paracoccus pantotrophus coding for enzymes involved in lithotrophic sulfur oxidation . The periplasmic SoxF, SoxG, and SoxH proteins were induced by thiosulfate and purified to homogeneity from the soluble fraction . soxF coded for a protein of 420 amino acids with a signal peptide containing a twin-arginine motif . SoxF was 37% identical to the flavoprotein FccB of flavocytochrome c sulfide dehydrogenase of Allochromatium vinosum . The mature SoxF (42,832 Da) contained 0.74 mol of flavin adenine dinucleotide per mol . soxG coded for a novel protein of 303 amino acids with a signal peptide containing a twin-arginine motif . The mature SoxG (29,657 Da) contained two zinc binding motifs and 0.90 atom of zinc per subunit of the homodimer . soxH coded for a periplasmic protein of 317 amino acids with a double-arginine signal peptide . The mature SoxH (32,317 Da) contained two metal binding motifs and 0.29 atom of zinc and 0.20 atom of copper per subunit of the homodimer . SoxXA, SoxYZ, SoxB, and SoxCD (C . G . Friedrich, A . Quentmeier, F . Bardischewsky, D . Rother, R . Kraft, S . Kostka, and H . Prinz, J . Bacteriol . 182:4476-4487, 2000) reconstitute a system able to perform thiosulfate-, sulfite-, sulfur-, and hydrogen sulfide-dependent cytochrome c reduction, and this system is the first described for oxidizing different inorganic sulfur compounds . SoxF slightly inhibited the rate of hydrogen sulfide oxidation but not the rate of sulfite or thiosulfate oxidation . From use of a homogenote mutant with an in-frame deletion in soxF and complementation analysis, it was evident that the soxFGH gene products were not required for lithotrophic growth with thiosulfate.

FEBS Lett, 2001 Jun 29, 500(1-2), 71 - 4
Assignment of haem ligands and detection of electronic absorption bands of molybdenum in the di-haem periplasmic nitrate reductase of Paracoccus pantotrophus; Butler CS et al.; The periplasmic nitrate reductase (NAP) from Paracoccus pantotrophus is a soluble two-subunit enzyme (NapAB) that binds two c-type haems, a {4Fe-4S} cluster and a bis-molybdopterin guanine dinucleotide cofactor that catalyses the reduction of nitrate to nitrite . In the present work the NapAB complex has been studied by magneto-optical spectroscopy to probe co-ordination of both the NapB haems and the NapA active site Mo . The absorption spectrum of the NapAB complex is dominated by features from the NapB c-type cytochromes . Using a combination of electron paramagnetic resonance spectroscopy and magnetic circular dichroism it was demonstrated that both haems are low-spin with bis-histidine axial ligation . In addition, a window between 600 and 800 nm was identified in which weak absorption features that may arise from Mo could be detected . The low-temperature MCD spectrum shows oppositely signed bands in this region (peak 648 nm, trough 714 nm) which have been assigned to S-to-Mo(V) charge transfer transitions.

Microbes Infect, 2001 Jun, 3(7), 535 - 42
Two-dimensional electrophoresis and characterization of antigens from Paracoccidioides brasiliensis; da Fonseca CA et al.; Paracoccidioides brasiliensis is a fungal pathogen of humans . To identify antigens from P . brasiliensis we fractionated a crude preparation of proteins from the fungus and detected the IgG reactive proteins by immunoblot assays of yeast cellular extracts with sera of patients with paracoccidioidomycosis (PCM) . We identified and characterized six new antigens by amino acid sequencing and homology search analyses with other proteins deposited in a database . The newly characterized antigens were highly homologous to catalase, fructose-1,6-biphosphate aldolase (aldolase), glyceraldehyde-3-phosphate dehydrogenase, malate dehydrogenase and triosephosphate isomerase from several sources . The characterized antigens presented preferential synthesis in yeast cells, the host fungus phase.

Int J Syst Evol Microbiol, 2001 May, 51(Pt 3), 1051 - 8
Albibacter methylovorans gen . nov., sp . nov., a novel aerobic, facultatively autotrophic and methylotrophic bacterium that utilizes dichloromethane; Doronina NV et al.; A novel genus, Albibacter, with one species, Albibacter methylovorans sp . nov., is proposed for a facultatively chemolithotrophic and methylotrophic bacterium (strain DM10T) with the ribulose bisphosphate (RuBP) pathway of C1 assimilation . The bacterium is a Gram-negative, aerobic, asporogenous, nonmotile, colourless rod that multiplies by binary fission . The organism utilizes dichloromethane, methanol, methylamine, formate and CO2/H2, as well as a variety of polycarbon compounds, as carbon and energy sources . It is neutrophilic and mesophilic . The major cellular fatty acids are straight-chain unsaturated C18:1, saturated C16:0 and cyclopropane C19:0 acids . The main ubiquinone is Q-10 . The dominant phospholipids are phosphatidyl ethanolamine, phosphatidyl glycerol, phosphatidyl choline and cardiolipin . The DNA G+C content is 66.7 mol% . Strain DM10T has a very low degree of DNA-DNA hybridization (4-7%) with the type species of the genera Paracoccus, Xanthobacter, Blastobacter, Angulomicrobium, Ancylobacter and Ralstonia of RuBP pathway methylobacteria . Another approach, involving comparative 16S rDNA analysis, has shown that the novel isolate represents a separate branch within the alpha-2 subgroup of the Proteobacteria . The type species of the new genus is Albibacter methylovorans sp . nov.; the type strain is DM10T (= VKM B-2236T = DSM 13819T).

Mycoses, 2001, 44(1-2), 9 - 12
Cerebriform colonies of Paracoccidioides brasiliensis isolated from nine-banded armadillos (Dasypus novemcinctus) at room temperature; Tanaka R et al.; Twelve isolates of Paracoccidioides brasiliensis generated cerebriform colonies at room temperature on potato glucose agar slants (PDA) . These isolates contained abundant chlamydospores and yeast-like cells and are a subset of the 65 isolates obtained from nine-banded armadillos (Dasypus novemcinctus) . They grew as a yeast form with typical multiple buddings at 37 degrees C on brain heart infusion agar supplemented with 1% glucose . After replating on PDA and culturing at room temperature for 2 months, the mutants appeared as cottonous colonies, which indicated that the morphological characteristics were unstable.

Biochemistry, 2001 Jun 5, 40(22), 6570 - 9
Calcium-dependent conformation of a heme and fingerprint peptide of the diheme cytochrome c peroxidase from Paracoccus pantotrophus; Pauleta SR et al.; The structural changes in the heme macrocycle and substituents caused by binding of Ca(2+) to the diheme cytochrome c peroxidase from Paracoccus pantotrophus were clarified by resonance Raman spectroscopy of the inactive fully oxidized form of the enzyme . The changes in the macrocycle vibrational modes are consistent with a Ca(2+)-dependent increase in the out-of-plane distortion of the low-potential heme, the proposed peroxidatic heme . Most of the increase in out-of-plane distortion occurs when the high-affinity site I is occupied, but a small further increase in distortion occurs when site II is also occupied by Ca(2+) or Mg(2+) . This increase in the heme distortion explains the red shift in the Soret absorption band that occurs upon Ca(2+) binding . Changes also occur in the low-frequency substituent modes of the heme, indicating that a structural change in the covalently attached fingerprint pentapeptide of the LP heme occurs upon Ca(2+) binding to site I . These structural changes may lead to loss of the sixth ligand at the peroxidatic heme in the semireduced form of the enzyme and activation.

Acta Cytol, 2001 May-Jun, 45(3), 360 - 4
Oral exfoliative cytology in the diagnosis of paracoccidioidomycosis; de Araujo MS et al.; OBJECTIVE: To assess the efficacy of conventional oral exfoliative cytology as a diagnostic tool in paracoccidioidomycosis . STUDY DESIGN: Cytologic smears and incisional biopsies were obtained from 10 patients with a clinical suspicion and oral manifestations of paracoccidioidomycosis . Cytologic smears and sections of the incisional biopsy underwent methenamine silver staining for fungi according to the Gomori-Grocott method . The dry glass slides were examined at 400 or 1,000 x magnification, and the presence and shape of yeasts of Paracoccidioides brasiliensis were investigated . RESULTS: Yeasts of the fungus P brasiliensis were clearly identified in cytologic smears and sections from incisional biopsies in all cases analyzed (100.0%) . CONCLUSION: Cytology of oral samples proved an effective diagnostic method for the detection of paracoccidioidomycosis in humans.

J Biol Chem, 2001 Aug 3, 276(31), 29450 - 5 Epub 2001 May 23.
The Structure of an alternative form of Paracoccus pantotrophus cytochrome cd(1) nitrite reductase; Sjogren T et al.; Cytochrome cd(1) nitrite reductase is a bifunctional enzyme, which can catalyze the 1-electron reduction of nitrite to nitric oxide and the 4-electron reduction of dioxygen to water . Here we describe the structure of reduced nitrite reductase, crystallized under anaerobic conditions . The structure reveals substantial domain rearrangements with the c domain rotated by 60 degrees and shifted by approximately 20 A compared with previously known structures from crystals grown under oxidizing conditions . This alternative conformation gives rise to different electron transfer routes between the c and d(1) domains and points to the involvement of elements of very large structural changes in the function in this enzyme . In the present structure, the c heme has a His-69/Met-106 ligation, and this ligation does not change upon oxidation in the crystal . The d(1) heme is penta-coordinated, and the d(1) iron is displaced from the heme plane by 0.5 A toward the proximal ligand, His-200 . After oxidation, the iron moves into the d(1) heme plane . A surprising finding is that although reduced nitrite reductase can be readily oxidized by dioxygen in the new crystal, it cannot turnover with its other substrate, nitrite . The results suggest that the rearrangement of the domains affects catalysis and substrate selectivity.

Oral Dis, 2001 Jan, 7(1), 56 - 60
Oral paracoccidioidomycosis: a retrospective study of 62 Brazilian patients; Bicalho RN et al.; OBJECTIVE: The purpose of this study was to evaluate retrospectively the profile of patients with oral paracoccidioidomycosis referred to two Dental Schools in Belo Horizonte (MG, Brazil) between 1955 and 1998 . Despite the importance of the oral manifestations of this disease, few papers in the English literature have provided epidemiological data . SUBJECTS AND METHODS: The medical records of 62 patients presenting oral paracoccidioidomycosis were reviewed in detail . Patient age, gender, race, occupation, site of lesion and type of clinical manifestation of the disease were tabulated . RESULTS: There was a predominance of male patients (97%), with a male:female ratio of 30:1 . The mean age was 40 years . Most of the patients were farm workers (53%) . Some patients presented multiple oral lesions (19 cases, 30%) . The fungal lesions were found principally in the alveolar process and gingiva, but were also seen on the palate, lip and buccal mucosa . All patients had chronic proliferative mulberry-like ulcerated oral lesions and the diagnosis was confirmed histologically . The clinical records did not contain notes about pulmonary involvement by the lesions . CONCLUSION: The purpose of this study was to describe the epidemiological profile of a specific population with the diagnosis of oral paracoccidioidomycosis . The major goal is to establish a scientific basis for initiating educational programs for prevention and early diagnosis of oral paracoccidioidomycosis.

Epidemiol Infect, 2001 Apr, 126(2), 309 - 15
Paracoccidioidomycosis in Colombia: an ecological study; Calle D et al.; The natural habitat of Paracoccidioides brasiliensis, agent of paracoccidioidomycosis (PCM), remains unknown . This study is aimed at establishing associations between the ecological variables present in all Colombian municipalities and the incidence of PCM . Records of 940 patients were studied and several ecological variables analysed, as well as their association to amount of patients per total rural population in each municipality, determined through a multivariate analysis . All 940 patients came from 216 municipalities (20-3%), out of which, 93 were birthplace and place of long-term residence for 121 patients . The Incidence Rate Ratio (IRR) was determined for these 93 municipalities . The following variables fitted the model: altitude from 1,000 to 1,499 metres above sea level (IRR = 6.37), rainfall from 2000 to 2999 mm (IRR = 2.15), presence of humid forests (Holdridge) (IRR = 1.79) and coffee (IRR = 1.95), tobacco (IRR = 3.59) crops . These results indicate that these municipalities constitute reservareas for P . brasiliensis (Borelli).

Mikrobiologiia, 2001 Jan-Feb, 70(1), 92 - 7
{Phylogenetic analysis of aerobic methylotrophic bacteria, using dichloromethane}; Turova TP et al.; The phylogenetic relationships of 12 aerobic dichloromethane-degrading bacteria that implement different C1-assimilation pathways was determined based on 16S ribosomal RNA sequences and DNA-DNA hybridization data . The restricted facultative methylotroph "Methylophilus leisingerii" DM11 with the ribulose monophosphate pathway was found to belong to the genus Methylophilus cluster of the beta subdivision of the phylogenetic kingdom Proteobacteria . The facultative methylotroph Methylorhabdus multivorans DM13 was assigned to a separate branch of the alpha-2 group of Proteobacteria . Paracoccus methylutens DM12, which utilizes C1-compounds via the Calvin cycle was found to belong to the alpha-3 group of the Proteobacteria (more precisely, to the genus Paracoccus cluster) . Thus, phylogenetic analysis confirmed the taxonomic status of these recently characterized bacteria . According to the degree of DNA homology, several novel strains of methylotrophic bacteria were divided into three genotypic groups within the alpha-2 group of the Proteobacteria . Genotypic group 1, comprising strains DM1, DM3, and DM5 through DM9, and genotypic group 3, comprising strain DM10, were phylogenetically close to the methylotrophic bacteria of the genus Methylopila, whereas genotypic group 2 (strain DM4) was close to bacteria of the genus Methylobacterium . The genotypic groups obviously represent distinct taxa of methylotrophic bacteria, whose status should be confirmed by phenotypic analysis.

Immunology, 2001 Apr, 102(4), 480 - 5
Modulatory effect of prostaglandins on human monocyte activation for killing of high- and low-virulence strains of Paracoccidioides brasiliensis; Soares AM et al.; The effect of indomethacin (Indo), a cyclo-oxygenase inhibitor, on the monocyte-mediated killing of a low- (Pb265) and a high- (Pb18) virulence strain of Paracoccidioides brasiliensis was examined . The Pb18 strain was not killed by either non-activated or interferon-gamma (IFN-gamma) -activated human monocytes but these cells did show fungicidal activity if pretreated with Indo . In contrast with IFN-gamma, tumour necrosis factor-alpha (TNF-alpha) was very effective at stimulating the fungicidal activity of monocytes . While the low-virulence strain, Pb265, could not be killed by monocytes, cells preincubated with IFN-gamma demonstrated fungicidal activity . The killing of this strain was also induced by pretreatment of monocytes with Indo . The results suggest a negative role for prostaglandins, which are synthesized via the cyclo-oxygenase pathway, in the regulation of monocyte-mediated killing of virulent and avirulent strains of P . brasiliensis and that TNF-alpha generation during the fungus-monocyte interaction is more important in the killing of Pb265 than Pb18.

Mikrobiologiia, 2000 Sep-Oct, 69(5), 706 - 11
{A novel plant-associated thermotolerant alkalophilic methylotroph of the genus Paracoccus}; Doronina NV et al.; Strain GB isolated from the maize rhizosphere is a gram-negative, aerobic, non-spore-forming, nonpigmented, nonmotile, chemolithotrophic, facultatively methylotrophic bacterium . Cells are cocci or short rods . The strain does not require vitamins . Optimum growth in a medium with methanol occurs at 38-42 degrees C at pH 8.0-9.2 . The doubling time is 12 h . In addition to methanol, the bacterium can grow on methylamine, dimethylformamide, acetone, thiosulfate + NaHCO3, and in an atmosphere of H2 + CO2 + O2 . Methanol and methylamine are oxidized by the respective dehydrogenases to CO2 via formaldehyde and formate, respectively . The CO2 produced is assimilated via the ribulose bisphosphate pathway . Fatty acids are dominated by cyclopropanoic (58-61%), palmitic (24-26%), and octadecanoic (8-9%) acids . The main phospholipids are phosphatidylglycerol, phosphatidylethanolamine, and phosphatidylcholine . The major ubiquinone is Q10 . The bacterial genome contains genes controlling the synthesis and secretion of cytokinins . The culture liquid exhibits cytokinin activity . The G + C content of DNA is 62.5 mol %, as determined from the DNA thermal denaturation temperature (Tm) . Strain GB shows a moderate degree of DNA-DNA homology (< 40%) with the type representatives of the genus Paracoccus . Based on the data obtained, the bacterium was classified as a new species of this genus, named P . kondratievae.

FEMS Microbiol Lett, 2001 Apr 13, 197(2), 171 - 8
Phylogeny and distribution of the soxB gene among thiosulfate-oxidizing bacteria; Petri R et al.; A PCR protocol for the detection of sulfur-oxidizing bacteria based on soxB genes that are essential for thiosulfate oxidation by sulfur-oxidizing bacteria of various phylogenetic groups which use the 'Paracoccus sulfur oxidation' pathway was developed . Five degenerate primers were used to specifically amplify fragments of soxB genes from different sulfur-oxidizing bacteria previously shown to oxidize thiosulfate . The PCR yielded a soxB fragment of approximately 1000 bp from most of the bacteria . Amino acid and nucleotide sequences of soxB from reference strains as well as from new isolates and environmental DNA from a hydrothermal vent habitat in the North Fiji Basin were compared and used to infer relationships of soxB between sulfur-oxidizing bacteria belonging to various 16S rDNA-based phylogenetic groups . Major phylogenetic lines derived from 16S rDNA were confirmed by soxB phylogeny . Thiosulfate-oxidizing green sulfur bacteria formed a coherent group by their soxB sequences . Likewise, clearly separated branches demonstrated the distant relationship of representatives of alpha-, beta-, and gamma-Proteobacteria including representative species of the former genus Thiobacillus (now Halothiobacillus - gamma-Proteobacteria, Thiobacillus - beta-Proteobacteria and Starkeya - alpha-Proteobacteria) . This general picture emerged although apparent evidence for lateral transfer of the soxB gene is indicated and comparison of soxB phylogeny and 16S rDNA phylogeny points to the significance of this gene transfer in hydrothermal vent bacterial communities of the North Fiji Basin.

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