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.