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Objectives: This study compared three methods of Streptococcus mutans and Lactobacillus spp. detection in the oral cavity: saliva swab (SS)-sample of stimulated saliva collected with swab; whole saliva (WS)-sample of 2 ml of stimulated saliva; and the dental plaque method (DP)-plaque sample of all dental surfaces., Methods: Thirty children were included in this study. In the first 15 children, the SS and WS methods were carried out before the dental plaque collection, and in the following 15, the sequence was inverted to evaluate possible interference of the methods sequence. The samples were diluted and inoculated in SB20 and Rogosa agar, respectively for S.mutans and Lactobacillus spp., at 37 degrees C for 48 h., Results: The results (cfu/mL) of S. mutans were analysed by the statistical Friedman's test. The levels of Lactobacillus spp. were analysed by descriptive statistics due to the high proportion of zero counts in the culture. In the first sequence of methods, the number of S. mutans counted for the SS method was inferior to DP and WS (P<0.05), and the results for the WS and DP methods were similar. The detection of Lactobacillus spp. was observed just by the WS (100%) and SS (14.3%) methods. However, in the second experimental set the number of S. mutans detected by the DP method was similar to those of the SS and WS, however, the WS method showed higher values than SS (P<0.05). A greater number of Lactobacillus spp. was detected by the WS method (100%), followed by SS (55.5%) and DP (33.3%)., Conclusions: The dental plaque collection and the sample of stimulated whole saliva presented similar results in the S. mutans count. The most suitable method to detect the Lactobacillus spp. level in the oral cavity is the stimulated whole saliva method.

The last step of the production of four phthalimide-derived acids, designed to act as antiasthma drugs, was performed by enzymatic hydrolysis of the respective methyl or ethyl esters. The esters 4-ethyl-[2-(1,3-dioxo-1,3-dihydro- 2-isoindoylyl)]-phenoxyacetic methyl ester (PHT-MET), 4-ethyl-[2- (1,3-dioxo-1,3-dihydro-2-isoindoylyl)]-phenoxyacetic ethyl ester, 4-(1, 3-dioxo-1,3-dihydro-2-isoindoylyl)-phenoxyacetic ethyl ester, and 2-(1,3-dioxo-1, 3-dihydro-2-isoindoylyl)-phenoxyacetic ethyl ester were hydrolyzed by immobilized lipase. The enzymatic reaction could be used only to produce the desired 4-substituted compounds. The best result that was found to hydrolysis of PHT-MET, and, therefore, that ester was selected for optimization experiments in a three-phase system. Reactions were performed with solid biocatalyst (Lipozyme RM IM), organic solvent phase (ethyl acetate), and aqueous phase (saturated Na2CO3 solution). To optimize the reaction conditions, an experimental design optimization procedure was used. The variables studied were the amount of enzyme, the temperature, and the volume of the aqueous solution. Time course experiments were then performed for different initial enzyme concentrations (0.5, 0.9, and 1.4 UH/mL of solvent). The optimized reaction conditions found were 20 mg of Lipozyme (0.9 UH/mL solvent) and 5.0 mL of Na2CO3(sat) at 40 degrees C for 6 h.

As part of an ongoing effort to develop new thalidomide analogues as antiinflammatory lead-candidates, this paper describes the synthesis and antiinflammatory activity of novel N-phenyl-phthalimide functionalized derivatives (4a-d, 5a,b, 6a,b). The target compounds were assayed in an acute lung inflammatory model and all compounds were able to inhibit TNF-alpha production and subsequent neutrophil recruitment in the LPS-acute lung inflammatory model.

Protein-nucleic acid interactions are responsible for the regulation of key biological events such as genomic transcription and recombination and viral replication. However, the recognition mechanisms involved in these processes are not completely understood. Here, we investigate the dominant forces involved in protein-protein and protein-DNA interactions for the 80-amino-acid C-terminal domain of the E2 protein (E2c) from human papillomavirus (HPV-16). The E2c protein is a homodimer that specifically binds to double-stranded DNA containing the consensus sequence ACCG-N(4)-CGGT, where N is any nucleotide. DNA binding affinity is reduced by lowering water chemical potential, accompanied by an increase in cooperativity. Wyman linkage relations between affinity and water chemical potential indicate that 11 additional water molecules are bound in the formation of the complex between E2c and DNA. Salt dissociation isotherms showed that 10 counterions are released upon association, even at low water activity, indicating that this latter variable does not change the electrostatic component of the interaction. Further analysis demonstrates a strong dependence of cooperativity of binding on the protein concentration. Altogether, these results reveal a novel binding pathway in which the consolidated complex may achieve its final form via a monomer-DNA intermediate, which favors the binding of a second monomer. This molecular mechanism reveals the contributions of multiple conformers in a tight virus genome modulation that seems to be important in the cell infection scenario.

The role of tumor suppressor protein p53 in cell cycle control depends on its flexible and partially unstructured conformation, which makes it crucial to understand its folding landscape. Here we report an intermediate structure of the core domain of the tumor suppressor protein p53 (p53C) during equilibrium and kinetic folding/unfolding transitions induced by guanidinium chloride. This partially folded structure was undetectable when investigated by intrinsic fluorescence. Indeed, the fluorescence data showed a simple two-state transition. On the other hand, analysis of far ultraviolet circular dichroism in 1.0 M guanidinium chloride demonstrated a high content of secondary structure, and the use of an extrinsic fluorescent probe, 4,4'-dianilino-1,1' binaphthyl-5,5'-disulfonic acid, indicated an increase in exposure of the hydrophobic core at 1 M guanidinium chloride. This partially folded conformation of p53C was plagued by aggregation, as suggested by one-dimensional NMR and demonstrated by light-scattering and gel-filtration chromatography. Dissociation by high pressure of these aggregates reveals the reversibility of the process and that the aggregates have water-excluded cavities. Kinetic measurements show that the intermediate formed in a parallel reaction between unfolded and folded structures and that it is under fine energetic control. They are not only crucial to the folding pathway of p53C but may explain as well the vulnerability of p53C to undergo departure of the native to an inactive state, which makes the cell susceptible to malignant transformation., (Copyright 2004 Biophysical Society)

A set of tetraaza macrocycles containing pyridine and methylcarboxylate (ac(3)py14) or methylphosphonate (MeP(2)py14 and P(3)py14) pendant arms were prepared and their stability constants with La(3+), Sm(3+), Gd(3+) and Ho(3+) determined by potentiometry at 25 degrees C and 0.10 M ionic strength in NMe(4)NO(3). The metal:ligand ratio for (153)Sm and (166)Ho and for ac(3)py14, MeP(2)py14 and P(3)py14, as well as the pH of the reaction mixtures, were optimized to achieve a chelation efficiency higher than 98%. These radiocomplexes are hydrophilic and have a significant plasmatic protein binding. In vitro stability was studied in physiological solutions and in human serum. All complexes are stable in saline and PBS, but 20% of radiochemical impurities were detected after 24 h of incubation in serum. Biodistribution studies in mice indicated a slow rate of clearance from blood and muscle, a high and rapid liver uptake and a very slow rate of total radioactivity excretion. Some bone uptake was observed for complexes with MeP(2)py14 and P(3)py14, which was enhanced with time and the number of methylphosphonate groups. This biological profile supports the in vitro instability found in serum and is consistent with the thermodynamic stability constants found for these complexes.

The effects of high hydrostatic pressure (HHP) and urea on conformational transitions of human alpha-thrombin structure were studied by fluorescence spectroscopy and by measuring the catalytic activity of the enzyme. Treatment of thrombin with urea produced a progressive red shift in the center of mass of the intrinsic fluorescence emission spectrum, with a maximum displacement of 650 cm(-1). HHP (270 MPa) shifted the centre of mass by only 370 cm(-1). HHP combined with a subdenaturing urea concentration (1.5 m) displaced the centre of mass by approximately 750 cm(-1). The binding of the fluorescent probe bis(8-anilinonaphthalene-1-sulfonate) to thrombin was increased by 1.8-, 4.0-, and 2.7-fold after treatment with high urea concentration, HHP or HHP combined with urea, respectively, thus suggesting that all treatments convert the enzyme to partially folded intermediates with exposed hydrophobic regions. On the other hand, treatment of thrombin with urea (but not HHP) combined with dithiothreitol progressively displaced the fluorescent probe, thus suggesting that this condition converts the enzyme to a completely unfolded state. Urea and HHP also led to different conformations when changes in the thrombin catalytic site environment were assessed using the fluorescence emission of fluorescein-d-Phe-Pro-Arg-cloromethylketone-alpha-thrombin: addition of urea up to 2 m gradually decreased the fluorescence emission of the probe to 65% of the initial intensity, whereas HHP caused a progressive increase in fluorescence. Hydrolysis of the synthetic substrate S-2238 was enhanced (35%) in 2 m urea and gradually abolished at higher concentrations, while HHP (270 MPa) inhibited the enzyme's catalytic activity by 45% and abolished it when 1.5 m urea was also present. Altogether, analysis of urea and HHP effects on thrombin structure and activity indicates the formation of dissimilar intermediate states during denaturation by these agents.

The main hypothesis for prion diseases proposes that the cellular protein (PrP(C)) can be altered into a misfolded, beta-sheet-rich isoform (PrP(Sc)), which undergoes aggregation and triggers the onset of transmissible spongiform encephalopathies. Here, we compare the stability against pressure and the thermomechanical properties of the alpha-helical and beta-sheet conformations of recombinant murine prion protein, designated as alpha-rPrP and beta-rPrP, respectively. High temperature induces aggregates and a large gain in intermolecular antiparallel beta-sheet (beta-rPrP), a conformation that shares structural similarity with PrP(Sc). alpha-rPrP is highly stable, and only pressures above 5 kilobars (1 kilobar = 100 MegaPascals) cause reversible denaturation, a process that leads to a random and turnrich conformation with concomitant loss of alpha-helix, as measured by Fourier transform infrared spectroscopy. In contrast, aggregates of beta-rPrP are very sensitive to pressure, undergoing transition into a dissociated species that differs from the denatured form derived from alpha-rPrP. The higher susceptibility to pressure of beta-rPrP can be explained by its less hydrated structure. Pressure perturbation calorimetry supports the view that the accessible surface area of alpha-rPrP is much higher than that of beta-rPrP, which explains the lower degree of hydration of beta-rPrP. Our findings shed new light on the mechanism of prion conversion and show how water plays a prominent role. Our results allow us to propose a volume and free energy diagram of the different species involved in the conversion and aggregation. The existence of different folded conformations as well as different denatured states of PrP may explain the elusive character of its conversion into a pathogenic form.

Two N-methylphosphonic acid derivatives of a 14-membered tetraazamacrocycle containing pyridine have been synthesized, H(4)L(1) and H(6)L(2). The protonation constants of these compounds and the stability constants of complexes of both ligands with Ni(2+), Cu(2+) and Zn(2+) were determined by potentiometric methods at 298 K and ionic strength 0.10 mol dm(-3) in NMe(4)NO(3). The high overall basicity of both compounds is ascribed to the presence of the phosphonate arms. (1)H and (31)P NMR spectroscopic titrations were performed to elucidate the sequence of protonation, which were complemented by conformational analysis studies. The complexes of these ligands have stability constants of the order of or higher than those formed with ligands having the same macrocyclic backbone but acetate arms. At pH = 7 the highest pM values were found for solutions containing the compound with three acetate groups, followed immediately by those of H(6)L(2), however, as expected, the increasing pH favours the complexes of ligands containing phosphonate groups. The single-crystal structure of Na(2)[Cu(HL(1))]NO(3)x8H(2)O has shown that the coordination geometry around the copper atom is a distorted square pyramid. Three nitrogen atoms of the macrocyclic backbone and one oxygen atom from one methylphosphonate arm define the basal plane, and the apical coordination is accomplished via the nitrogen atom trans to the pyridine ring of the macrocycle. To achieve this geometric arrangement, the macrocycle adopts a folded conformation. This structure seems consistent with Uv-vis-NIR spectroscopy for the Ni(2+) and the Cu(2+) complexes and with the EPR for the latter.

The prion protein (PrP) is the major agent implicated in the diseases known as transmissible spongiform encephalopathies. The onset of transmissible spongiform encephalopathy is related to a change in conformation of the PrP(C), which loses most of its alpha-helical content, becoming a beta-sheet-rich protein, known as PrP(Sc). Here we have used two Syrian hamster prion domains (PrP 109-141 and PrP 109-149) and the murine recombinant PrP (rPrP 23-231) to investigate the effects of anilino-naphtalene compounds on prion oligomerization and aggregation. Aggregation in the presence of bis-ANS (4,4'-dianilino-1,1'-binaphthyl-5,5'-sulfonate), ANS (1-anilinonaphthalene-8-sulfonate), and AmNS (1-amino-5-naphtalenesulfonate) was monitored. Bis-ANS was the most effective inhibitor of prion peptide aggregation. Bis-ANS binds strongly to rPrP 23-231 leading to a substantial increase in beta-sheet content and to limited oligomerization. More strikingly, the binding of bis-ANS to full-length rPrP is diminished by the addition of nanomolar concentrations of oligonucleotides, demonstrating that they compete for the same binding site. Thus, bis-ANS displays properties similar to those of nucleic acids, causing oligomerization and conversion to beta-sheet (Cordeiro, Y., Machado, F., Juliano, L., Juliano, M. A., Brentani, R. R., Foguel, D., and Silva, J. L. (2001) J. Biol. Chem. 276, 49400-49409). This dual effect of bis-ANS on prion protein makes this compound highly important to sequester crucial conformations of the protein, which may be useful to the understanding of the disease and to serve as a lead for the development of new therapeutic strategies.

Purpose: The aim of this investigation was to quantitatively evaluate C-cells in colloid goiters, analyzing 36 thyroids that were obtained through thyroidectomy from 24 patients with goiter and 12 normal glands from adult patients without thyroid disease, which were used as the control group., Material and Methods: On average, 6 different thyroid areas were sampled and labeled by immunohistochemistry with a monoclonal anticalcitonin antibody, utilizing the avidin-biotin-peroxidase complex. C-cells were counted in fields measuring 1 square centimeter, and the mean number of cells per field was then calculated. Data were statistically analyzed using the Mann-Whitney test., Results: In the colloid goiter group, the number of C-cells ranged from 0 to 23 per field, while in normal controls they ranged from 20 to 148 per field., Conclusions: These results demonstrate a significant decrease of C-cell number in the colloid goiter group compared with control group, indicating that the hyperplastic process is restricted to follicular cells, to the detriment of C-cells, which probably cease to receive trophic stimuli.

The toxic effect of the hydroalcoholic crude extract (HACE) from the fruits of the Solanum grandiflorum was evaluated following subchronic treatment (p.o.) in Wistar rats for 65 days using the dose of 2 mg/kg. The LD(50) in mice was 50 mg/kg (p.o.). At the end of treatment, the consumption of water remained within the normal patterns. On the other hand, the consumption of food had 40% reduction in the period between the 18th and the 40th day after the beginning of the study. The diuresis showed a wide variation in volume which started at the 8th day of treatment until the end of the study. This finding was correlated with macroscopic observations of the kidneys which presented nephromegaly (70%) when compared to the control group. Moreover, for the histopathological study, it was observed degeneration and glomerular necrosis (100%), tubular lesions (80%), and hypercellular lesions (100%) in the treated group. The ponderal development of the treated group was increased compared to the control group. The weight of some of the organs, including kidneys, lungs, and pancreas, was also increased in the treated group (P<0.05, Student "t" test). These data suggest a nephrotoxicity following a subchronic treatment of the hydroalcoholic crude extract from Solanum grandiflorum.

The purpose of this study was to compare the effects of 5 weeks of physioball core stability and balance exercises with conventional floor exercises in women. The experimental group (n = 15) performed curl-ups and back extensions on the physioball while the control group (n = 15) performed the same exercises on the floor. Baseline and post-training tests included electromyography (EMG) recordings of the rectus abdominus and erector spinae muscles; abdominal, back, and knee strength measurements with the Cybex Norm System; and 2 unilateral stance balance tests. The physioball group was found to have significantly greater mean change in EMG flexion and extension activity (p = 0.04 and p = 0.01, respectively) and greater balance scores (p < 0.01) than the floor exercise group. No significant changes (p > 0.05) were observed for heart rate or Cybex strength measurements. Early adaptations in a short-term core exercise program using the physioball resulted in greater gains in torso balance and EMG neuronal activity in previously untrained women when compared to performing exercises on the floor.

Transgenic Medicago truncatula plants were produced harboring chimeric gene constructs of the glutamine synthetase (GS) cDNA clones (MtGS1a or MtGS1b) fused in sense or antisense orientation to the nodule-specific leghemoglobin promoter Mtlb1. A series of transgenic plants were obtained showing a 2- to 4-fold alteration in nodule GS activity when compared with control plants. Western and northern analyses revealed that the increased or decreased levels of GS activity correlate with the amount of cytosolic GS polypeptides and transcripts present in the nodule extracts. An analysis of the isoenzyme composition showed that the increased or decreased levels of GS activity were attributable to major changes in the homo-octameric isoenzyme GS1a. Nodules of plants transformed with antisense GS constructs showed an increase in the levels of both asparagine synthetase (AS) polypeptides and transcripts when compared with untransformed control plants, whereas the sense GS transformants showed decreased AS transcript levels but polypeptide levels similar to control plants. The polypeptide abundance of other nitrogen metabolic enzymes NADH-glutamic acid synthase and aspartic acid amino-transferase as well as those of major carbon metabolic enzymes phosphoenolpyruvate carboxylase, carbonic anhydrase, and sucrose synthase were not affected by the GS-gene manipulations. Increased levels of AS polypeptides and transcripts were also transiently observed in nodules by inhibiting GS activity with phosphinothricin. Taken together, the results presented here suggest that GS activity negatively regulates the level of AS in root nodules of M. truncatula. The potential role of AS in assimilating ammonium when GS becomes limiting is discussed.

The effects of LASSBio596, a phosphodiesterase type-4 and -5 inhibitor, were tested in Escherichia coli lipopolysaccharide (LPS)-induced acute lung injury. Twenty-four BALB/c mice were randomly divided into four groups. In the control group, saline (0.05 mL) was injected intratracheally (i.t.). The LPS group received LPS (10 microg i.t., 0.05 mL). In the LASSBio596 groups, LASSBio596 (10 mg x kg(-1), 0.2 mL) was injected intraperitoneally 1 h before or 6 h after LPS administration. After 24 h, in vivo (lung resistive and viscoelastic pressures, and static and dynamic elastances) and in vitro (tissue resistance, elastance and hysteresivity) pulmonary mechanics, lung morphometry and collagenous fibre content were computed. Neutrophils and tumour necrosis factor (TNF)-alpha levels were evaluated in the bronchoalveolar lavage fluid. LASSBio596 prevented the changes in lung mechanics, and inhibited neutrophilic recruitment, TNF-alpha release, bronchoconstriction, alveolar collapse and the increment of collagen fibre content induced by LPS, independently of the moment of injection. In conclusion, LASSBio596 modulated the lung inflammatory process and had the potential to block fibroproliferation. Thus, agents that inhibit phosphodiesterase 4 and 5 simultaneously may be a useful adjunct therapy for acute lung injury.

Troponin C (TnC) is the Ca(2+)-binding subunit of the troponin complex of vertebrate skeletal muscle. It consists of two structurally homologous domains, N and C, connected by an exposed alpha-helix. The C-domain has two high-affinity sites for Ca(2+) that also bind Mg(2+), whereas the N-domain has two low-affinity sites for Ca(2+). Previous studies using isolated N- and C-domains showed that the C-domain apo form was less stable than the N-domain. Here we analyzed the stability of isolated N-domain (F29W/N-domain) against urea and pressure denaturation in the absence and in the presence of glycerol using fluorescence spectroscopy. Increasing the glycerol concentration promoted an increase in the stability of the protein to urea (0-8 M) in the absence of Ca(2+). Furthermore, the ability to expose hydrophobic surfaces normally promoted by Ca(2+) binding or low temperature under pressure was partially lost in the presence of 20% (v/v) glycerol. Glycerol also led to a decrease in the Ca(2+) affinity of the N-domain in solution. From the ln K(obs) versus ln a(H)2(O), we obtained the number of water molecules (63.5 +/- 8.7) involved in the transition N <=>N:Ca(2) that corresponds to an increase in the exposed surface area of 571.5 +/- 78.3 A(2). In skinned fibers, the affinity for Ca(2+) was also reduced by glycerol, although the effect was much less pronounced than in solution. Our results demonstrate quantitatively that the stability of this protein and its affinity for Ca(2+) are critically dependent on protein hydration.

In this paper, we report the cloning and characterization of the plastid-located glutamine synthetase (GS) of Medicago truncatula Gaertn (MtGS2). A cDNA was isolated encoding a GS2 precursor polypeptide of 428 amino acids composing an N-terminal transit peptide of 49 amino acids. Expression analysis, by Westerns and by northern hybridization, revealed that MtGS2 is expressed in both photosynthetic and non-photosynthetic organs. Both transcripts and proteins of MtGS2 were detected in substantial amounts in root nodules, suggesting that the enzyme might be performing some important role in this organ. Surprisingly, about 40% of the plastid GS in nodules occurred in the non-processed precursor form (preGS2). This precursor was not detected in any other organ studied and moreover was not observed in non-fixing nodules. Cellular fractionation of nodule extracts revealed that preGS2 is associated with the plastids and that it is catalytically inactive. Immunogold electron microscopy revealed a frequent coincidence of GS with the plastid envelope. Taken together, these results suggest a nodule-specific accumulation of the GS2 precursor at the surface of the plastids in nitrogen-fixing nodules. These results may reflect a regulation of GS2 activity in relation to nitrogen fixation at the level of protein import into nodule plastids.

Purpose: To study quantitatively C cells in the thyroids of non-isogenic rats to determine the possible effects of pinealectomy on the number of these cells, and consequently on the synthesis and secretion of calcitonin., Methods: Twenty male rats of an outbred strain (200-300 g) were used in the present study. One group of 10 animals was pinealectomized 50 days prior to sacrifice. Thyroid tissue was stained for calcitonin (Dako Corporation) at a 1:1500 dilution. The number of C cells observed was expressed as number of cells/cm . Data were analyzed statistically by Mann-Whitney test., Results: The number of C cells in pinealectomized and normal animals ranged from 489 to 2084 per cm and 227 to 1584 per cm , respectively, a difference that was statistically significant (P <0.05)., Conclusions: These results showed consistent differences in the number of C cells after pinealectomy when compared to controls. We believe that pinealectomy increases the number of C cells in the rat thyroid.

This paper describes the synthesis and anti-inflammatory activity of new N-phenyl-phthalimide sulfonamides (3a-e) and the isosters N-phenyl-phthalimide amides (4a-e), designed as hybrids of thalidomide (1) and aryl sulfonamide phosphodiesterase inhibitor (2). In these series, compound 3e (LASSBio 468), having a sulfonyl-thiomorpholine moiety, showed potent inhibitory activity on LPS-induced neutrophil recruitment with ED(50)=2.5mg kg(-1), which was correlated with its inhibitory effect on TNF-alpha level.

A series of phthalimide acid derivatives was synthesized and evaluated as leukotriene D(4) receptor antagonists. The tetrazolephthalimide LASSBio 552 (7) was shown to be able to inhibit the contractile activity induced by 100 nM of LTD(4) in guinea-pig tracheal strips with an IC(50) = 31.2 microM. In addition, LASSBio 552 (7) has been showed to present a better efficacy than zafirlukast (1) used as standard.

Protein-nucleic acid interactions are crucial for a variety of fundamental biological processes such as replication, transcription, restriction, translation and virus assembly. The molecular basis of protein-DNA and protein-RNA recognition is deeply related to the thermodynamics of the systems. We review here how protein-nucleic acid interactions can be approached in the same way as protein-protein interactions involved in protein folding and protein assembly, using hydrostatic pressure as the primary tool and employing several spectroscopic techniques, especially fluorescence, circular dichroism and high-resolution nuclear magnetic resonance. High pressure has the unique property of stabilizing partially folded states or molten-globule states of a protein. The competition between correct folding and misfolding, which in many proteins leads to formation of insoluble aggregates is an important problem in the biotechnology industry and in human diseases such as amyloidosis, Alzheimer's, prion and tumor diseases. The pressure studies reveal that a gradient of partially folded (molten globule) conformations is present between the unfolded and fully folded structure of several bacteria, plant and mammalian viruses. Using pressure, we have detected the presence of a ribonucleoprotein intermediate, where the coat protein is partially unfolded but bound to RNA. These intermediates are potential targets for antiviral compounds. Pressure studies on viruses have direct biotechnological applications. The ability of pressure to inactivate viruses has been evaluated with a view toward the applications of vaccine development and virus sterilization. Recent studies demonstrate that pressure causes virus inactivation while preserving the immunogenic properties. There is substantial evidence that a high-pressure cycle traps a virus in the 'fusion intermediate state', not infectious but highly immunogenic.

Background: Rabeprazole, a substituted benzimidazole, represents a new generation of proton pump inhibitors that has recently been approved by the FDA and European Union for treatment of acid-related diseases., Objectives: To assess the efficacy and tolerability of rabeprazole 20 mg in actual conditions of use in everyday clinical practice on subjects with diagnosis of erosive gastroesophageal reflux disease and/or gastric and/or duodenal ulcer., Patients/methods: A total of 171 outpatients (55% men, 45% women) with a mean age of 42.5 years were enrolled in this trial. The majority of subjects (81.0%) were Caucasians. Patients with endoscopically confirmed erosive/ulcerative gastroesophageal reflux disease (Savary-Miller classification), duodenal ulcer and/or benign gastric ulcer were eligible to receive rabeprazole 20 mg once daily for 4 to 8 weeks, depending on the diagnosis and at investigators' discretion. Patients were requested to record their symptoms in a diary card in a daily basis., Results: One hundred and sixty two patients completed the study in accordance with the protocol. Reflux esophagitis was diagnosed in 78 (48.1%) patients, duodenal ulcer in 39 (24.1%) and gastric ulcer in 24 (14.8%). Eleven (6.8%) patients presented reflux esophagitis associated with duodenal ulcer and 7 (4.3%) associated with gastric ulcer. Finally, 3 (1.9%) presented both gastric and duodenal ulcer. Fifty-three percentage of patients were free of symptoms on the first day of treatment and 89.5% after a week. The healing rate was 84.4% for patients with reflux esophagitis, 90.6% for duodenal ulcer and 90.9% for gastric ulcer. The adverse effects were minimal and transitory., Conclusions: Rabeprazole is highly effective and well tolerated in acute healing of reflux esophagitis and peptic ulcers. In addition, it provides fast symptoms relief.

To investigate the influence of the pineal gland through melatonin secretion on the physiological and morphological parameters of pancreatic islets, we studied the plasma biochemistry and morphological and morphometric characteristics of the endocrine pancreas of male Wistar rats. The animals were distributed into five groups of ten rats each: NC - normal control group; NS -- sham-operated group; Px (25) -- pinealectomised group, studied 15--25 days after surgery; Px (70) -- pinealectomised group, studied 60-70 days after surgery; ALX - alloxan monohydrate-treated group. Data are analyzed statistically by ANOVA and by the Kruskal-Wallis test. Although there was no significant difference in plasma glucose or insulin levels between the Px (25), Px (70) and NC groups, Px (25) animals showed a tendency to increased glucose and reduced insulin levels. The ALX group showed a clear elevation of plasma glucose and a reduction of plasma insulin compared to the other groups. Morphometric analysis showed a larger pancreatic islet area and a lower pancreatic islet density in the pancreas of Px (70) animals and an increase in degenerative pathological processes in the pancreatic islets of the Px (25) and ALX groups. The present results suggest that melatonin, in addition to acting on tissue sensitivity to insulin (as reported in other studies), affects the secretory action of beta cells, as demonstrated by the morphological and morphometric changes observed in pinealectomised animals.

The plasma membrane (Ca(2+) + Mg(2+))ATPase hydrolyzes pseudo-substrates such as p-nitrophenylphosphate. Except when calmodulin is present, Ca(2+) ions inhibit the p-nitrophenylphosphatase activity. In this report it is shown that, in the presence of glycerol, Ca(2+) strongly stimulates phosphatase activity in a dose-dependent manner. The glycerol- and Ca(2+)-induced increase in activity is correlated with modifications in the spectral center of mass (average emission wavenumber) of the intrinsic fluorescence of the enzyme. It is concluded that the synergistic effect of glycerol and Ca(2+) is related to opposite long-term hydration effects on the substrate binding domain and the Ca(2+) binding domain.

The recognition of palindromic specific DNA sequences by the human papillomavirus (HPV) E2 proteins is responsible for regulation of virus transcription. The dimeric E2 DNA-binding domain of HPV-16 (E2c) dissociates into a partially folded state under high hydrostatic pressure. We show here that pressure-induced monomers of E2c are highly structured, as evidenced by NMR hydrogen-deuterium exchange measurements. On binding to both specific and nonspecific DNA, E2c becomes stable against pressure. Competitive binding studies using fluorescence polarization of fluorescein-labeled DNA demonstrate the reversibility of the specific binding. To assess the thermodynamic parameters for the linkage between protein dissociation and DNA binding, urea denaturation curves were obtained at different pressures in the presence of specific and nonspecific DNA sequences. The change in free energy on denaturation fell linearly with increase in pressure for both protein-DNA complexes, and the measured volume change was similar to that obtained for E2c alone. The data show that the free energy of dissociation increases when E2c binds to a nonspecific DNA sequence but increases even more when the protein binds to the specific DNA sequence. Thus, specific complexes are tighter but do not entail variation in the volume change. The thermodynamic data indicate that DNA-bound E2c dissociates into monomers bound to DNA. The existence of monomeric units of E2c bound to DNA may have implications for the formation of DNA loops, as an additional target for viral and host factors binding to the loosely associated dimer of the N-terminal module of the E2 protein.

The C-terminal DNA binding domain of the E2 protein is involved in transcriptional regulation and DNA replication in papillomaviruses. At low ionic strength, the domain has a tendency to form aggregates, a process readily reversible by the addition of salt. While fluorescence anisotropy measurements show a 1:1 stoichiometry at pH 5.5, we observed that a second HPV-16 E2 C-terminal dimer can bind per DNA site at pH 7.0. This was confirmed by displacement of bis-ANS binding, tryptophan fluorescence, native electrophoresis, and circular dichroism. The two binding events are nonequivalent, with a high-affinity binding involving one E2C dimer per DNA molecule with a K(D) of 0.18 +/- 0.02 nM and a lower affinity binding mode of 2.0 +/- 0.2 nM. The bovine (BPV-1) E2 C-terminal domain binds to an HPV-16 E2 site with 350-fold lower affinity than the human cognate domain and binds 7-fold less tightly even to a bovine-derived DNA site. The ability to discriminate between cognate and noncognate sequences is 50-fold higher for the human domain, and the latter is 180-fold better than the bovine at discriminating specific from nonspecific DNA. A substantial conformational change in bound DNA is observed by near-UV circular dichroism. The bovine domain imposes a different DNA conformation than that caused by the human counterpart, which could be explained by a more pronounced bent. Structure-function differences and biochemical properties of the complexes depend on the protein domain rather than on the DNA, in line with crystallographic evidence. Despite the strong sequence homology and overall folding topology, the differences observed may explain the distinctive transcriptional regulation in bovine and human viruses.

The dimeric beta-barrel is a characteristic topology initially found in the transcriptional regulatory domain of the E2 DNA binding domain from papillomaviruses. We have previously described the kinetic folding mechanism of the human HPV-16 domain, and, as part of these studies, we present a structural characterization of the urea-denatured state of the protein. We have obtained a set of chemical shift assignments for the C-terminal domain in urea using heteronuclear NMR methods and found regions with persistent residual structure. Based on chemical shift deviations from random coil values, 3'J(NHN alpha) coupling constants, heteronuclear single quantum coherence peak intensities, and nuclear Overhauser effect data, we have determined clusters of residual structure in regions corresponding to the DNA binding helix and the second beta-strand in the folded conformation. Most of the structures found are of nonnative nature, including turn-like conformations. Urea denaturation at equilibrium displayed a loss in protein concentration dependence, in absolute parallel to a similar deviation observed in the folding rate constant from kinetic experiments. These results strongly suggest an alternative folding pathway in which a dimeric intermediate is formed and the rate-limiting step becomes first order at high protein concentrations. The structural elements found in the denatured state would collide to yield productive interactions, establishing an intermolecular folding nucleus at high protein concentrations. We discuss our results in terms of the folding mechanism of this particular topology in an attempt to contribute to a better understanding of the folding of dimers in general and intertwined dimeric proteins such as transcription factors in particular.

Previously we demonstrated that Kalanchoe pinnata (KP) leaf extracts inhibited in vitro lymphocyte proliferation and showed in vivo immunosuppressive activity. Here we attempt to identify the immunosuppressive substances present in KP guided by the lymphoproliferative assays. From the ethanolic extract was purified a fraction (KP12SA) twenty-fold more potent to block murine lymphocyte proliferation than the crude extract. Chemical analysis by 1H- and 13C-NMR, IR and GC-MS of KP12SA (methylated sample) showed 89.3% of palmitic acid (C16), 10.7% of stearic acid (C18) and traces of arachidic (C20) and behenic acids (C22). This study provides evidence that fatty acids present in Kalanchoe pinnata may be responsible, at least in part, for its immunosuppressive effect in vivo.

Anew series of antinociceptive compounds belonging to the N-acylarylhydrazone (NAH) class were synthesized from natural safrole (7). The most analgesic derivative represented by 10f, [(4'-N,N-dimethylaminobenzylidene-3-(3', 4'-methylenedioxyphenyl)propionylhydrazine], was more potent than dipyrone and indomethacin, used as standards. The NAH compounds described herein were structurally planned by molecular hybridization and classical bioisosterism strategies on previously reported analgesic NAH in order to identify the pharmacophoric contribution of the N-acylarylhydrazone moiety and investigate the structure-activity relationship (SAR) in these series.

In the scope of a research program aiming at the synthesis and pharmacological evaluation of novel possible antiplatelet prototype compounds, exploring bioisosterism principles for molecular design, we describe in this paper the synthesis of new aryl-sulfonamides derivatives, structurally similar to known thromboxane A2 receptor antagonists. The synthetic route used to access the new compounds described herein starts from safrole, an abundant Brazilian natural product, which occurs in Sassafras oil (Ocotea pretiosa). The results from preliminary evaluation of these novel aryl-sulfonamide compounds by the platelet aggregation inhibitory test, using rabbit PRP, induced by ADP, collagen, arachidonic acid, and U46619, identified the N-[2-(4-carboxymethoxyphenyl)ethyl]-6-methyl-3,4-methylenedioxyphe nyl- sulfonamido derivative as the most active among them, presenting in IC50 value for the U-46619-induced platelet aggregation in rabbit platelet-rich plasma: 329 microM.

We are investigating the folding of the 81-residue recombinant dimeric DNA binding domain of the E2 protein from human papillomavirus and how it is coupled to the binding of its DNA ligand. Modifications in buffer composition, such as ionic strength and phosphate, cause an approximately 5.0 kcal mol-1 stabilization of the domain to urea unfolding, based on very similar conformational changes as measured by far UV circular dichroism. Binding of DNA produces an even greater stabilization, magnitude similar to that caused by the nonspecific polymer ligand heparin, which shifts the urea midpoint 2.5-fold. The DNA-bound complex displays substantial changes similar to those caused by ionic strength and phosphate in terms of overall secondary structure. Bis-8-anilino-1-naphthalenesulfonate provides a very sensitive conformational probe, which shows alterations in the domain caused by the above mentioned compounds. In general terms, binding of DNA involves an overall conformational readjustment in the protein but maintains the beta-barrel scaffold intact. This conformational plasticity seems to be of importance in the regulatory functions of this type of DNA-binding protein. The extremely long half-life of the E2-DNA complex, together with its very high stability, suggests that, in the absence of other factors that may affect its stability in vivo, the possibility of dissociation once formed is restricted.

This study's objective was to search for Cryptosporidium sp. in diarrheic feces from children aged zero to 12 years and cared for at medical units within Universidade Federal de Uberlândia or at a private practice in Uberlândia, State of Minas Gerais, Brazil, from September 1992 to August 1993. Three fecal samples preserved in 10% formalin, were collected from 94 children. Oocyst concentration was performed through Ritchie's (modified) method and staining of fecal smears for each sample (total of 1128 slides) was done by the "Safranin/Methylene Blue" and the "Kinyoun (modified)" techniques. The Hoffmann, Pons & Janer method was also employed to look for other enteroparasites. From 94 children, 4.26% excreted fecal Cryptosporidium oocysts. The infection seemed to vary according to age: 5.08% of patients aged zero to two years old; 33.33% of those aging eight to ten years (P > 0.05). Cryptosporidium appeared in November, December and March, during the rainy season. 20.21% of the children harbored at least one enteroparasite different from Cryptosporidium, mainly Giardia intestinalis (12.77%). From Cryptosporidium infected patients, two had only this kind, another harbored Giardia intestinalis; the last one hosted Strongyloides stercoralis.

The authors performed a prospective study of 86 patients with septic arthritis of the knee between 1990 e 1994. The age, sex, predisposing factors, time between onset of symptoms and treatment, etiologic organism and their route of entrance in the joint, previous use of antibiotic the data from of treatment, stay at the hospital and complications were evaluated and compared with the literature.

A case of hemolytic disease in a newborn infant caused by anti-Dia is described. The parents are Japanese. The antibody was an IgG of subclasses IgG1 and IgG3, did not bind complement, and gave a 27 percent phagocytosis rate when used to sensitize red blood cells for a macrophage binding assay. The antibody was not detected during antenatal serologic testing. This case suggests the need for reagent red blood cell panels selected to match antibody specificities likely to be encountered in a particular population.

A rare case of septal gallbladder with a long spiral cystic duct crossing the common hepatic duct and joining it at the papilla of Vater is described. These anomalies wer observed by oral cholecystography after a fatty meal.

The serum of 134 patients infected with Schistosoma mansoni, including both acute and chronic forms of the disease, were tested for the presence of cold lymphocytotoxins. These were found in 4.5% of acute forms of the disease, in 88.4% of chronic infections and in only 9.8% of the uninfected control group (blood donors). An analysis of the results suggests a probable role for cold lymphocytotoxins in the immune response of the different phases of human schistosomiasis.

One case of Budd-Chiari syndrome is described in a woman taking for 10 months oral contraceptive. The diagnosis was established by liver biopsy and inferior cavography. A review of the pertinent literature was made and the causal relationship between Budd-Chiari syndrome and oral contraceptive was considered.