Antigenotoxic and antimutagenic effects of glutamine supplementation on mice treated with cisplatin.

We evaluated the effects of glutamine on clastogenic and genotoxic damage prevention caused by the administration of cisplatin. Forty Swiss mice were divided into 8 experimental groups: G1 and G2, which were control groups; G3, G4, and G5, which were administered [2 doses of glutamine (orally)] separated by a 24-h period (150, 300, and 600 mg/kg, respectively), and a dose of phosphate-buffered saline by intraperitoneal injection; G6, G7, and G8, which were treated in the same manner as the previous groups, but received cisplatin rather than phosphate-buffered saline. The antimutagenicity groups showed damage reduction percentages of 79.05, 80.00, and 94.27% at the time point T1, 53.18, 67.05, and 64.74 at time point T2 for the 150, 300, and 600 mg/kg doses of glutamine, respectively. Antigenotoxic activity was evident for all 3 doses with damage reduction percentages of 115.05, 119.06, and 114.38 for the doses of glutamine of 150, 300, and 600 mg/ kg, respectively. These results suggest that further studies are needed to confirm the clastogenic activity of glutamine. However, our results may lead to rational strategies for supplementation of this antioxidant as an adjuvant in cancer treatment or for preventing genomic lesions.

Chronic paracoccidioidomycosis of the intestine as single organ involvement points to an alternative pathogenesis of the mycosis.

Current knowledge on the natural history of paracoccidioidomycosis states that the chronic form of the disease results from reactivation of quiescent foci established years or decades before during the primary lung infection. Once reactivated, the fungi can disseminate to virtually any organ or system. We present herein two chronic paracoccidioidomycosis patients with a single organ involvement that points to an alternative pathogenesis of the mycosis. These patients suggest that the chronic form may also arise from reactivation of foci not confined to the lungs, due to the early dissemination of yeast cells during the primary infection.

Retrospective seroepidemiological analysis of patients with suspicion of paracoccidioidomycosis in São Paulo State, Brazil

The geographic distribution of paracoccidioidomycosis (PCM) in the Brazilian state of São Paulo was evaluated in a retrospective study using secondary data from serological analyses, carried out by double immunodiffusion assay of patients with PCM suspicion, from January 1999 to May 2010. Sixty percent of 10,176 patients, from 239 cities, were serologically reactive to P. brasiliensis. The cities that showed the most serological reactivity among patients were São João da Boa Vista (85%), Piracicaba (75%), Sorocaba (73%), Campinas (72%) and São Paulo (62%). São Paulo state has an area of 248,209.4 km²; the climate is tropical and sub-tropical with annual temperatures between 18 and 24ºC, high rainfall (900 to 1800 mm/year), rainy summers and mild winters. It also features large areas composed of acidic soils, and is one of the greatest contributors to Brazilian agricultural production and, separately, the largest producer of orange juice and, the ninth greatest producer of soy and sugar cane and the fourth largest coffee producer. We suggest that the climatic characteristics associated with soil type and development of primary activities can contribute to the endemic potential of PCM in São Paulo state.

Reactivity of antibodies from patients with acute and chronic paracoccidioidomycosis to a high molecular mass antigen from Paracoccidioides brasiliensis.

Yeast forms of Paracoccidioides brasiliensis produce polydispersed high molecular mass (h-MM) antigens. We investigated the antibodies to an h-MM antigen from P. brasiliensis by immunoblotting and ELISA in sera from paracoccidioidomycosis (PCM) patients. IgG from the sera of chronic PCM patients was able to recognize the h-MM antigen at a higher frequency in the cell-free antigen (CFA) (8/13) than in the somatic antigen (SA) (2/13), as assessed by immunoblotting. The CFA was fractionated by Sephadex G-200 chromatography, and fraction 17 (F17) with the h-MM antigen of approximately 366 kDa was used in ELISA to analyze specific levels of IgG and IgE. Patients with the chronic form showed significantly higher levels of IgG (P<0.05) but not IgE (P>0.05) to F17 by ELISA, compared to patients with the acute form or to healthy donors. In conclusion, CFA is better than SA as a source of the P. brasiliensis h-MM antigen. This study reveals a new characteristic to differentiate between the acute and chronic forms of PCM, by demonstrating a higher level of seric IgG to h-MM antigen in chronic compared to acute PCM patients.

High-level soluble production and characterization of porcine ribonuclease inhibitor.

Ribonucleases can be cytotoxic if they retain their ribonucleolytic activity in the cytosol. The cytosolic ribonucleolytic activity of ribonuclease A (RNase A) and other pancreatic-type ribonucleases is limited by the presence of excess ribonuclease inhibitor (RI). RI is a 50-kDa cytosolic scavenger of pancreatic-type ribonucleases that competitively inhibits their ribonucleolytic activity. RI had been overproduced as inclusion bodies, but its folding in vitro is inefficient. Here, porcine RI (pRI) was overproduced in Escherichia coli using the trp promoter and minimal medium. This expression system maintains pRI in the soluble fraction of the cytosol. pRI was purified by affinity chromatography using immobilized RNase A and by anion-exchange chromatography. The resulting yield of 15 mg of purified RI per liter of culture represents a 60-fold increase relative to previously reported recombinant DNA systems. Differential scanning calorimetry was used to study the thermal denaturation of pRI, RNase A, and the pRI-RNase A complex. The conformational stability of the complex is greater than that of the individual components.

Ribonuclease 4, an evolutionarily highly conserved member of the superfamily.

The structural and enzymatic properties of RNase 4 are reviewed. This RNase shows a much higher interspecies similarity (approximately 90%) than the other members of the RNase A superfamily. The enzyme is ubiquitous, with the highest amounts present in liver and lung. Its unique uridine specificity results from alterations in and around the pyrimidine-binding site. In particular, the shortened C-terminus and the side chains of Phe-42, Asp-80 and Arg-101 appear to be involved. RNase 4 binds tightly to the intracellular RNase inhibitor, with a Kd of 4 x 10(-15) M.

A single amino acid substitution changes ribonuclease 4 from a uridine-specific to a cytidine-specific enzyme.

The structural features underlying the strong uridine specificity of ribonuclease 4 (RNase 4) are largely unknown. It has been hypothesized that the negatively charged alpha-carboxylate is close to the pyrimidine binding pocket, due to a unique C-terminal deletion. This would suppress the cleavage of cytidine-containing substrates [Zhou, H.-M., and Strydom, D. J. (1993) Eur. J. Biochem. 217, 401-410]. Replacement of the alpha-carboxylate by an alpha-carboxamide in a fragment complementation system decreased both (kcat/Km)CpA and (kcat/Km)UpA , thus refuting the hypothesis. However, model building showed that the deletion allowed the side chain of Arg-101 to reach the pyrimidine binding pocket. From the 386-fold reduction in (kcat/Km)UpA in RNase 4;R101N, it is concluded that this residue functions in uridine binding, analogous to Ser-123 in RNase A. In addition, it may have an effect on Asp-80. The 2-fold increase in (kcat/Km)CpA in the mutant R101N and the close proximity of the side chains of Arg-101 and Asp-80 suggested that the latter could be involved in suppressing CpA catalysis. The substrate specificity of RNase 4;D80A was completely reversed: (kcat/Km)UpA decreased 159-fold, whereas (kcat/Km)CpA increased 233-fold. The effect on CpA was unexpected, because the corresponding residue in bovine pancreatic RNase A (Asp-83) hardly affects cytidine-containing substrates. Furthermore, the residue is conserved in nearly all sequences of mammalian RNase 1. Thus, an evolutionary highly conserved residue does not necessarily function in the same way in homologous enzymes. A model, which proposes that the structure of RNase 4 has been optimized to permanently fix the position of Asp-80 and impede its movement away from the pyrimidine binding pocket, is presented.

Recognition signal for C-mannosylation of Trp-7 in RNase 2 consists of sequence Trp-x-x-Trp.

C2-alpha-Mannosyltryptophan was discovered in human RNase 2, an enzyme that occurs in eosinophils and is involved in host defense. It represents a novel way of attaching carbohydrate to a protein in addition to the well-known N- and O-glycosylations. The reaction is specific, as in RNase 2 Trp-7, but never Trp-10, which is modified. In this article, we address which structural features provide the specificity of the reaction. Expression of chimeras of RNase 2 and nonglycosylated RNase 4 and deletion mutants in HEK293 cells identified residues 1-13 to be sufficient for C-mannosylation. Site-directed mutagenesis revealed the sequence Trp-x-x-Trp, in which the first Trp becomes mannosylated, as the specificity determinant. The Trp residue at position +3 can be replaced by Phe, which reduces the efficiency of the reaction threefold. Interpretation of the data in the context of the three-dimensional structure of RNase 2 strongly suggests that the primary, rather than the tertiary, structure forms the determinant. The sequence motif occurs in 336 mammalian proteins currently present in protein databases. Two of these proteins were analyzed protein chemically, which showed partial C-glycosylation of recombinant human interleukin 12. The frequent occurrence of the protein recognition motif suggests that C-glycosides could be part of the structure of more proteins than assumed so far.

C-Mannosylation of human RNase 2 is an intracellular process performed by a variety of cultured cells.

C2-alpha-Mannosyltryptophan was discovered in RNase 2 from human urine, representing a novel way of attaching carbohydrate to a protein. Here, we have addressed two questions related to the biosynthesis of this modification: (i) is C-mannosylation part of the normal intracellular biosynthetic route, and (ii) how general is it, i.e. which organisms perform this kind of glycosylation? To answer the first question, RNase 2, which is identical to the eosinophil-derived neurotoxin, was isolated from intracellular stores of cultured human HL-60 cells. The enzyme was C-mannosylated at Trp-7, showing that the modification occurs intracellularly, before secretion of the protein. The second question was investigated by immunological and chemical analysis of RNase 2 purified from the supernatant of transiently transformed cells from different organisms. This revealed that C-mannosylation occurs in cells from man, green monkey, pig, mouse, and hamster. The observation that pig kidney cells contain the machinery for C-mannosylation of Trp-7 of human RNase 2 but that the homologous RNase from porcine kidney is not a substrate, since it does not contain a tryptophan at position 7, strongly suggests that C-mannosylated proteins other than RNase 2 exist. Recombinant RNase 2 isolated from insect cells, plant protoplasts, and Escherichia coli was not C-mannosylated. These results not only form the basis for further studies on the biochemical aspects of C-mannosylation but also have implications for the choice of cells for production of recombinant glycoproteins.

Laminin-binding epitope on gp43 from Paracoccidioides brasiliensis is recognized by a monoclonal antibody raised against Staphylococcus aureus laminin receptor.

Adhesion is regarded as an important step in the pathogenesis of several microorganisms. Thus, the ability to recognize extracellular matrix proteins, such as laminin or fibronectin, has been correlated with invasiveness. Studying the already characterized laminin-binding protein of Paracoccidioides brasiliensis, the 43 kDa glycoprotein (gp43), we evaluated whether MAb 1.H12, raised against the laminin-binding protein from Staphylococcus aureus, cross-reacts with that fungal protein. By immunoblot analysis we show that MAb 1.H12 recognizes gp43. This interaction is able to inhibit the laminin-mediated adhesion to epithelial cells as well as the P. brasiliensis infection in vivo. Moreover, through immunoenzymatic assays, we show that MAb 1.H12 recognizes gp43 in solid phase and that this interaction is partially inhibited by the addition of anti-gp43 MAbs. These results show that MAb 1.H12 recognizes the gp43, suggesting the presence of an epitope similar to those found in the other laminin-binding proteins from phylogenetically very distant cells. These findings reinforce the possibility of evolutionary conservation of such epitopes.

Monoclonal antibodies against the 43,000 Da glycoprotein from Paracoccidioides brasiliensis modulate laminin-mediated fungal adhesion to epithelial cells and pathogenesis.

The surface glycoprotein gp43, a highly immunogenic component of Paracoccidioides brasiliensis, is used in the serodiagnosis of paracoccidioidomycosis (PCM) and has recently been shown to specifically bind the extracellular matrix protein laminin. Binding to laminin induces the increased adhesion of the fungus to epithelial cells; a hamster testicle infection model has shown that the gp43-dependent binding of fungal cells to laminin enhances their pathogenicity in vivo. We report on the production and characterization of 12 monoclonal antibodies against the gp43 that recognize peptide sequences in the molecule detecting at least three different epitopes as well as different isoforms of this antigen. MAbs interfered in the fungal pathogenicity in vivo either by inhibiting or enhancing granuloma formation and tissue destruction. Results suggest that P. brasiliensis propagules may start infection in man by strongly adhering to human lung cells. Thus, laminin-mediated fungal adhesion to human lung carcinoma (A549) cells was much more intense than to Madin-Darby canine kidney cells (MDCK), indicating differences in binding affinity. Subsequent growth of fungi bound to the lung cells could induce the granulomatous inflammatory reaction characteristic of PCM. Both steps are greatly stimulated by laminin binding in infective cells expressing gp43.

Structural determinants of the uridine-preferring specificity of RNase PL3.

RNase PL3 is a structurally highly conserved, pyrimidine-specific RNase, which strongly prefers to cleave at the 3'-side of uridine. Here, question of which residues are involved in determining substrate specificity is addressed. The difference in the rate of cleavage of UpA and CpA was found to result from a 375-fold larger kcat for the former substrate, whereas the values of Km were essentially the same. The pyrimidine specificity of this class of RNases is thought to result from hydrogen bonds between the base and a threonine residue in the B1 subsite. Mutation of this residue (Thr-44) in RNase PL3 resulted in strongly reduced activity with UpA and poly(U). However, the activity with CpA and poly(C) had increased. Comparison with the effect of the same mutation in RNase A [delCardayre, S. B., & Raines, R. T. (1994) Biochemistry 33, 6031-6037] and angiogenin [Curran et al. (1993) Biochemistry 32, 2307-2313] showed that the function of this threonine in substrate recognition is different in three RNase subfamilies. Previous studies have shown that the 36-42 region contains one or more residues that are involved in substrate recognition [Vicentini et al. (1994) Protein Sci. 3, 459-466]. Site-directed mutagenesis of amino acids in this region identified Phe-42 as the only single residue that affected the cytidine/uridine specificity ratio. The mutation F42V resulted in a 10-fold increase in kcat and a 1.9-fold decrease in Km for CpA. The properties of the double mutant F42V/T44A suggested that a suboptimal binding of cytidine is caused by Phe-42, partially through an effect on Thr-44.

Intravenous propafenone in paroxysmal atrial fibrillation: a randomized, placebo-controlled, double-blind, multicenter clinical trial. Paroxysmal Atrial Fibrillation Italian Trial (PAFIT)-2 Investigators.

BACKGROUND: Pharmacological conversion of paroxysmal atrial fibrillation is frequently necessary. The aim of this study was to compare intravenous propafenone, a class Ic antiarrhythmic agent, with placebo in paroxysmal atrial fibrillation (AF) of recent onset (< 72 h). PATIENTS AND METHODS: We randomly allocated 75 patients, aged 18 to 70 years, with paroxysmal AF to receive intravenous propafenone (2 mg/kg in 15 min followed by 1 mg/kg in 2 h) or the matching placebo. Patients were followed for 3 h. Exclusion criteria were the presence of one of the following: clinical heart failure, recent acute myocardial infarction, hypotension, atrioventricular block, Wolff-Parkinson-White syndrome, or current treatment with antiarrhythmic agents or digitalis. RESULTS: No sign of heart disease was found in 74.7% of the patients. Echocardiographically determined left atrium diameter was similar in the two groups. Conversion to sinus rhythm occurred in 24 of 41 patients allocated to propafenone and in 10 of 34 patients allocated to placebo (odds ratio 3.2, 95% confidence intervals 1.3-7.9; p < 0.01). Mean conversion time was 34 +/- 29 and 71 +/- 55 min, respectively, for propafenone and placebo. Mean heart rate in nonconverters decreased from 146 to 109 beats/min in patients treated with propafenone while it remained virtually unchanged in those treated with placebo. Only minor side effects were noted. CONCLUSIONS: Intravenous propafenone is an effective therapeutic option for restoring sinus rhythm in patients with paroxysmal AF of recent onset.

Monoclonal antibody capture enzyme immunoassay for detection of Paracoccidioides brasiliensis antibodies in paracoccidioidomycosis.

Four murine monoclonal antibodies (MAbs 17C, 21A, 21F, and 32B) raised against the 43-kDa glycoprotein of Paracoccidioides brasiliensis were tested in a capture enzyme immunoassay (EIA) for the detection of specific human anti-gp43 immunoglobulin G in patients with paracoccidioidomycosis (PCM). All MAbs reacted similarly in the assay. These MAbs, which detected anti-gp43 at levels of as low as 500 pg/ml, were demonstrated to specifically recognize at least two different epitopes in gp43 binding assays. Specific antibodies in the sera of patients with active PCM were detected at dilutions of as high as 1:819,200, and the reactivities of patient sera, as measured by optical densities, were found to be significantly higher than those of control sera. The comparison between classical ELISA and our capture enzyme immunoassay showed that both sensitivity and specificity were greatly improved by the latter. These MAbs represent the first specific reagents to P. brasiliensis described for use in serological tests for PCM.

Characterization of glycoprotein gp43, the major laminin-binding protein of Paracoccidioides brasiliensis.

We have demonstrated that laminin mediates the adhesion of P. brasiliensis to monolayers of epithelial cells through specific binding to the surface glycoprotein gp43. This binding seems to be related to the fungal pathogenesis. We now report the confirmation of these findings by scanning electron microscopy and show that some isolates that do not secrete gp43 do express the protein as seen by studying whole cell extracts. These results confirm the ability of these strains to produce paracoccidioidomycosis but should not be used for serological purposes since the absence of gp43 in exoantigens may lead to false negative results.

Characterization of glycoprotein gp43, the major laminin-binding protein of Paracoccidioides brasiliensis

We have demonstrated that laminin mediates the adhesion of P. brasiliensis to monolayers of epithelial cells through specific binding to the surface glycoprotein gp43. This binding seems to be related to the fungal pathogenesis. We now report the confirmation of these findings by scanning electron microscopy and show that some isolates that do not secrete gp43 do express the protein as seen studying whole cell extracts. This results confirm the ability of these strains to produce paracoccidioidomycosis but should not be used for serological purposes since the absence of gp43 in exoantigens may lead to false negative results

Binding of Paracoccidioides brasiliensis to laminin through surface glycoprotein gp43 leads to enhancement of fungal pathogenesis.

Extracellular matrix protein laminin binds specifically to yeast forms of Paracoccidioides brasiliensis and enhances adhesion of the fungus to the surface of epithelial Madin-Darby canine kidney cells in vitro. Immunoblotting of fungal extracts showed that the gp43 glycoprotein is responsible for adhesion. This was confirmed by binding assays using purified gp43, with a Kd of 3.7 nM. The coating of P. brasiliensis yeast forms with laminin before injection into hamster testicles enhanced the fungus virulence, resulting in a faster and more severe granulomatous disease. These results indicate that interaction of fungi with extracellular matrix elements may constitute a basis for the evolution of fungal infection toward regional spreading and dissemination.

Residues 36-42 of liver RNase PL3 contribute to its uridine-preferring substrate specificity. Cloning of the cDNA and site-directed mutagenesis studies.

Within the superfamily of homologous mammalian ribonucleases (RNases) 4 distinct families can be recognized. Previously, representative members of three of these have been cloned and studied in detail. Here we report on the cloning of a cDNA encoding a member of the fourth family, RNase PL3 from porcine liver. The deduced amino acid sequence showed the presence of a signal peptide, confirming the notion that RNase PL3 is a secreted RNase. Expression of the cDNA in Escherichia coli yielded 1.5 mg of purified protein/liter of culture. The recombinant enzyme was indistinguishable from the enzyme isolated from porcine liver based on the following criteria: amino acid analysis, N-terminal amino acid sequence, molecular weight, specific activity toward yeast RNA, and kinetic parameters for the hydrolysis of uridylyl(3',5')adenosine and cytidylyl(3',5')adenosine. Interestingly, the kinetic data showed that RNase PL3 has a very low activity toward yeast RNA, i.e., 2.5% compared to pancreatic RNase A. Moreover, using the dinucleotide substrates and homopolymers it was found that RNase PL3, in contrast to most members of the RNase superfamily, strongly prefers uridine over cytidine on the 5' side of the scissile bond. Replacement, by site-directed mutagenesis, of residues 36-42 of RNase PL3 by the corresponding ones from bovine pancreatic RNase A resulted in a large preferential increase in the catalytic efficiency for cytidine-containing substrates. This suggests that this region of the molecule contains some of the elements that determine substrate specificity.