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.

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.

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.

Rapid construction of large synthetic genes: total chemical synthesis of two different versions of the bovine prochymosin gene.

We have tested several different synthesis designs and assembly methodologies to develop an improved gene synthesis strategy which enables significantly longer nucleotide sequences to be easily constructed. This strategy, based in part upon our ability to synthesize high-quality extended-length oligodeoxynucleotides (over 100-mer in length), together with the use of chemical 5'-phosphorylation, and simplified low-melting-temperature agarose gel purification methods, combines ease, speed and high overall efficiency. We show that it is now feasible to synthesize routinely even long genes (at least 1-2 kb). To demonstrate this capability we have chemically synthesized and assembled two different versions of the gene encoding the bovine enzyme prochymosin (prorennin). One gene is essentially the natural bovine prochymosin gene sequence. In the second gene the codons have been optimized with regard to the codon bias of highly expressed yeast genes. Each synthetic gene was in excess of 1100 bp, yet they were assembled from only 13 or 14 pairs of complementary oligodeoxynucleotides (oligos), the average lengths of which were 87 and 82 bp, respectively. The 'mutation' rate was low enough to assess that more than 75% of all such oligo pairs (160-170 total nt) were error-free.

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.

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.

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.

[Leiomyosarcoma of the duodenum (description of a case and review of the literature)]. / Leiomiosarcoma del duodeno (descrizione di un caso e revisione della letteratura).

The Authors report on a case of duodenal leiomyosarcoma of small dimensions in a paravaterian site and presenting a low degree of histological malignity. They summarize the main anatomoclinical features of the neoplasm and conclude by indicating less aggressive surgical therapy as adequate in such cases, accompanied, however, by a protracted post-operative follow-up.

[Ductal adenoma of the breast: correlations between clinico-radiologic and histo-pathologic aspects (considerations on a case)]. / L'adenoma duttale della mammella: correlazioni tra aspetti clinico-radiologici ed isto-patologici (considerazioni su di un caso).

The authors report on a case of ductal adenoma of the breast, pointing out that routine clinico-instrumental diagnostic signs, though suggesting a picture of malignancy, may actually relate to this rare benign form of tumor. In the presence of cases of this type, we therefore advise a cautions approach to surgery, in that oncological radicality is subordinate to confirmation in the form of a traditional histological preparation, as freezer examinations are unable to provide wholly reliable responses.

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.

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.

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.

[Physical work capacity of the patient with myocardial infarction submitted to intensive rehabilitative training (author's transl)]. / Capacita' fisica funzionale dell'infartuato sottoposto a training riabilitativo intensivo.

The Authors have examined two homogeneous groups of patients with myocardial infarction; one underwent (61 patient) to a rehabilitating training, the other underwent (42 patient) to a domiciliary physical program. Patients have been considered by means of bicycle ergometer test at 2, 4, 12 and 24 months from myocardial infarction. They have been taken into consideration clinical and Ecg factors and also, as an index of physical work capacity was considered the complete work developed, the VO2 max/Kg per min., the product B.P. x H. R. and the pulse O2. The average value of parameters have been statistically compared by means of the t of Student, analysis of variation x2, and the test of Kolmogorov-Smirnov; moreover the correlation with linear coefficient has been studied. Afterwards the many-varied discriminating analysis and the test F di Snedelor have been carried out just for the index of functionality. From the statistical analysis of the data considered it results an increase of the functional capacity and its maintenance long after in the group that underwent to the rehabilitating training. However, two years after, this increase of capacity did not result greater than that obtained, in the same period of time, by the group of patients who underwent to a domiciliary physical rehabilitating program. These considerations point out the necessity of keeping physical exercise indefinitely or at least of repeating it periodically. In that event they suggest the right time to programme further cycles of training.

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.

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.

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.