Caries Diagnosis in Dental Practices: Results From Dentists in a Brazilian Community.

The aim of this study was to assess practices related to diagnosis of dental caries among dentists (n=217) from Araraquara, São Paulo State, Brazil. Data on sociodemographic information and practitioner characteristics were collected using a pretested questionnaire, and data on practices related to caries diagnosis were gathered by using a translated and culturally adapted questionnaire from the US National Dental Practice-Based Research Network. Descriptive statistics and regression analyses were used for data analysis. Respondents reported using in most of their patients radiographs (Rx) to diagnose proximal caries (59%), explorer (Ex) for the diagnosis of occlusal caries (64%) and on the margins of existing restorations (79%), as well as air jet (AJ) with drying (92%). Magnification (M) (25%), fiber optic transillumination (FOTI; 14%), and laser fluorescence (LF) (3%) were used in the minority of patients. Regression analysis revealed that the following dentists' characteristics were significantly associated (p<0.05) with the use of diagnostic methods on a greater percentage of their patients: advanced degree (Rx, FOTI), higher percentage of patients with individualized caries prevention (Rx, FOTI, M), more years since dental school graduation (Ex, M), and work in an exclusively private practice model (LF). In conclusion, most Brazilian dentists from Araraquara reported they most commonly use visual, tactile, and radiographic imaging for the diagnosis of dental caries. Some dentists' characteristics, such as time from dental school graduation and having a postgraduation course, were associated with the use of certain diagnostic methods.

Dificultades para la colaboración en investigación entre cardiólogos y médicos de urgencias / Difficulties for research collaboration between cardiologists and emergency service doctors

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Comentario al artículo original. Unidades de soporte vital básico y avanzado en España: análisis de la situación actual / S. Ballesteros / Coment on the original article. Basic and advanced life support units in Spain: analysis and current situations

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Diseño y redacción de una trayectoria clínica. Herniorrafia inguino-crural y umbilical en el adulto mediante cirugía mayor ambulatoria / No disponible

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Characterization of BphF, a Rieske-type ferredoxin with a low reduction potential.

BphF is a small, soluble, Rieske-type ferredoxin involved in the microbial degradation of biphenyl. The rapid, anaerobic purification of a heterologously expressed, his-tagged BphF yielded 15 mg of highly homogeneous recombinant protein, rcBphF, per liter of cell culture. The reduction potential of rcBphF, determined using a highly oriented pyrolytic graphite (HOPG) electrode, was -157+/- 2 mV vs the standard hydrogen electrode (SHE) (20 mM MOPS, 80 mM KCl, and 1 mM dithiothreitol, pH 7.0, 22 degrees C). The electron paramagnetic resonance spectrum of the reduced rcBphF is typical of a Rieske cluster while the close similarity of the circular dichroic (CD) spectra of rcBphF and BedB, a homologous protein from the benzene dioxygenase system, indicates that the environment of the cluster is highly conserved in these two proteins. The reduction potential and CD spectra of rcBphF were relatively independent of pH between 5 and 10, indicating that the pK(a)s of the cluster's histidinyl ligands are not within this range. Gel filtration studies demonstrated that rcBphF readily oligomerizes in solution. Crystals of rcBphF were obtained using sodium formate or poly(ethylene glycol) (PEG) as the major precipitant. Analysis of the intermolecular contacts in the crystal revealed a head-to-tail interaction that occludes the cluster, but is very unlikely to be found in solution. Oligomerization of rcBphF in solution was reversed by the addition of dithiothreitol and is unrelated to the noncovalent crystallographic interactions. Moreover, the oligomerization state of rcBphF did not influence the latter's reduction potential. These results indicate that the 450 mV spread in reduction potential of Rieske clusters of dioxygenase-associated ferredoxins and mitochondrial bc(1) complexes is not due to significant differences in their solvent exposure.

Characterization of an alkaline transition intermediate stabilized in the Phe82Trp variant of yeast iso-1-cytochrome c.

In general, mutation of the phylogenetically conserved residue Phe82 in yeast iso-1-cytochrome c destabilizes the native conformation of the protein by facilitating the ligand exchange reactions that are associated with the alkaline conformational transitions of the ferricytochrome. Of the Phe82 variants surveyed thus far, Phe82Trp is unique in that it adopts a thermodynamically stable, high-spin conformation at mildly alkaline pH. This species exhibits spectroscopic features that can only be detected transiently in other ferricytochromes c within the first 100 ms immediately after a pH-jump from neutrality to pH >10. Spectroscopic characterization of this high-spin reaction intermediate suggests that in addition to an obligatory pentacoordinate heme iron, a group within the heme pocket coordinates the heme iron but is then replaced either by Met80, to revert to the native conformation, or by Lys73 or Lys79, to yield one of the conventional alkaline conformers. Evidence is presented to suggest that this group is either a hydroxide ion or Tyr67 rather than a loosely bound Met80.

Investigation of the role of a surface patch in the self-association of Chromatium vinosum high potential iron-sulfur protein.

The role of a flattened, relatively hydrophobic surface patch in the self-association of Chromatium vinosum HiPIP was assessed by substituting phenylalanine 48 with lysine. The reduction potential of the F48K variant was 26 mV higher than that of the wild-type (WT) recombinant (rc) HiPIP, consistent with the introduction of a positive charge close to the cluster. Nuclear magnetic resonance spectroscopy (NMR) revealed that the electronic structure of the oxidized cluster in these two proteins is very similar at 295 K. In contrast, the electron transfer self-exchange rate constant of F48K was at least 15-fold lower than that of the WT rcHiPIP, indicating that the introduction of a positive charge at position 48 diminishes self-association of the HiPIP in solution. Moreover, the substitution at position 48 abolished the fine structure in the g(z) region of the electron paramagnetic resonance (EPR) spectrum of oxidized C. vinosum rcHiPIP recorded in the presence of 1 M sodium chloride. These results support the hypothesis that the flattened, relatively hydrophobic patch mediates interaction between two molecules of HiPIP and that freezing-induced dimerization of the HiPIP mediated by this patch is responsible for the unusual fine structure observed in the EPR spectrum of the oxidized C. vinosum HiPIP.

[Simplified periodontal record for pregnant women]. / Registro periodontal simplificado em gestantes.

PURPOSE: The assessment using the PSR (Periodontal Screening and Recording) of the prevalence and severity of and the basic treatment needs for periodontal disease in a group of pregnant women who attended the Preventive Dentistry Clinic at the School of Dentistry of Araraquara--UNESP. METHODS: Forty-one pregnant women of 16 to 37 years of age, were examined. The PSR index was evaluated with a suitable periodontal probe (Trinity-model 621-WHO) with index codes scores of from 0 to 4, capable of indicating the presence of the following conditions: periodontal health, bleeding on probing, calculus, shallow and deep pockets. These codes were attributed to each sextant and could be marked with an asterisk (*) to indicate the presence of gingival recession, furcation lesions, mobility or any other mucogingival alterations. RESULT: It is shown that 100% of the pregnant women had some kind of gingival alteration, represented mainly by PSR code 2 (56.1%) and "*" (19.5%). The women in the youngest age groups, 15-19 and 20-24 years, had code 2 as their highest score with no sextant excluded. In the 25-29 age group, the PSR code 2 still prevailed (54.5%) although codes 3 and 4 were already appearing. The code "*" and the occurrence of excluded sextants tended to increase in the oldest age group (30-37). In general, the affected sextants showed codes 1 and 2 more frequently, corresponding to 41.6% and 39.8% respectively, which represented a mean of 2.49 and 2.39 sextants affected in each pregnant woman. Regarding the treatment needs, 90.2% of the women needed some treatment beyond the preventive measures begun, including scaling and root planning and/or corrections of defective restorative margins (61%), and more complex treatment (29.2%). CONCLUSION: The meeting of the treatment needs during pregnancy must include special efforts to increase motivation and promote oral health, minimizing the possibility of vertical transmission of pathogenic microrganisms to the child, and thus contributing to the primary prevention of the main oral diseases.

The structural and functional role of lysine residues in the binding domain of cytochrome c in the electron transfer to cytochrome c oxidase.

The interactions of yeast iso-1 cytochrome c with bovine cytochrome c oxidase were studied using cytochrome c variants in which lysines of the binding domain were substituted by alanines. Resonance Raman spectra of the fully oxidized complexes of both proteins reveal structural changes of both the heme c and the hemes a and a3. The structural changes in cytochrome c are the same as those observed upon binding to phospholipid vesicles where the bound protein exists in two conformers, B1 and B2. Whereas the structure of B1 is the same as that of the unbound cytochrome c, the formation of B2 is associated with substantial alterations of the heme pocket. In cytochrome c oxidase, the structural changes in both hemes refer to more subtle perturbations of the immediate protein environment and may be a result of a conformational equilibrium involving two states. These changes are qualitatively different to those observed for cytochrome c oxidase upon poly-l-lysine binding. The resonance Raman spectra of the various cytochrome c/cytochrome c oxidase complexes were analyzed quantitatively. The spectroscopic studies were paralleled by steady-state kinetic measurements of the same protein combinations. The results of the spectra analysis and the kinetic studies were used to determine the stability of the complexes and the conformational equilibria B2/B1 for all cytochrome c variants. The complex stability decreases in the order: wild-type WT > J72K > K79A > K73A > K87A > J72A > K86A > K73A/K79A (where J is the natural trimethyl lysine). This order is not exhibited by the conformational equilibria. The electrostatic control of state B2 formation does not depend on individual intermolecular salt bridges, but on the charge distribution in a specific region of the front surface of cytochrome c that is defined by the lysyl residues at positions 72, 73 and 79. On the other hand, the conformational changes in cytochrome c oxidase were found to be independent of the identity of the bound cytochrome c variant. The maximum rate constants determined from steady-state kinetic measurements could be related to the conformational equilibria of the bound cytochrome c using a simple model that assumes that the conformational transitions are faster than product formation. Within this model, the data analysis leads to the conclusion that the interprotein electron transfer rate constant is around two times higher in state B2 than in B1. These results can be interpreted in terms of an increase of the driving force in state B2 as a result of the large negative shift of the reduction potential.

Class I heme peroxidases: characterization of soybean ascorbate peroxidase.

An efficient expression system [D. A. Dalton et al. Arch. Biochem. Biophys. 328, 1-8, 1996) for soybean nodule ascorbate peroxidase (APX) has, for the first time, been used to generate enzyme in large enough quantities for detailed biophysical analysis. The recombinant APX has been characterized by electronic absorption, EPR, NMR and circular dichroism spectroscopies, and by electrochemistry. Electronic, EPR, and NMR spectra are consistent with a high-spin ferric resting state for the enzyme at 298 K. Low-temperature EPR (7 K) and electronic absorption (77 K) experiments indicate formation of a low-spin heme derivative at these temperatures. The midpoint reduction potential for the Fe(III)/Fe(II) redox couple, determined by spectroelectrochemistry, is -159 +/- 2 mV vs SHE (pH 7.0, 25.0 degrees C, mu = 0.10 M). Circular dichroism spectra of pea and soybean APXs are very similar, indicating common structural features for the two enzymes. The melting temperature of soybean APX, as monitored by circular dichroism spectroscopy, is 49 degrees C. These results represent the first detailed spectroscopic and electrochemical analysis of soybean ascorbate peroxidase and are discussed in the broader context of other class I peroxidases.

Spectroscopic and functional studies of a novel quadruple myoglobin variant with increased peroxidase activity.

A quadruple variant of horse heart myoglobin (Thr39Ile/Lys45Asp/Phe46Leu/Ile107Phe) that exhibits significantly (approximately 25-fold) greater peroxidase activity than the wild-type protein has been studied to determine its midpoint reduction potential (24(2) mV vs. SHE; pH 6.0, mu = 0.1 M, 25 degrees C) and to characterize the kinetics of its reaction with hydrogen peroxide. In addition, Fourier transform infrared (FTIR) spectra of the carbonyl and azide adducts of the protein have been obtained to gain initial insight into the effects of these substitutions on the ligand binding properties of the reduced and oxidized variant. All of the results obtained in this work are consistent with a variant heme binding pocket with increased hydrophilic character.

N epsilon,N epsilon-dimethyl-lysine cytochrome c as an NMR probe for lysine involvement in protein-protein complex formation.

The reductively dimethylated derivatives of horse and yeast iso-1-ferricytochromes c have been prepared and characterized for use as NMR probes of the complexes formed by cytochrome c with bovine liver cytochrome b5 and yeast cytochrome c peroxidase. The electrostatic properties and structures of the derivatized cytochromes are not significantly perturbed by the modifications; neither are the electrostatics of protein-protein complex formation or rates of interprotein electron transfer. Two-dimensional 1H-13C NMR spectroscopy of the complexes formed by the derivatized cytochromes with cytochrome b5 and cytochrome c peroxidase has been used to investigate the number and identity of lysine residues of cytochrome c that are involved in interprotein interactions of cytochrome c. The NMR data are incompatible with simple static models proposed previously for the complexes formed by these proteins, but are consistent with the presence of multiple, interconverting complexes of comparable stability, consistent with studies employing Brownian dynamics to model the complexes. The NMR characteristics of the Nepsilon,Nepsilon-dimethyl-lysine groups, their chemical shift dispersion, oxidation state and temperature dependences and the possibility of chemical exchange phenomena are discussed with relevance to the utility of Nepsilon, Nepsilon-dimethyl-lysine's being a generally useful derivative for characterizing protein-protein complexes.

Bacterial expression of a mitochondrial cytochrome c. Trimethylation of lys72 in yeast iso-1-cytochrome c and the alkaline conformational transition.

Saccharomyces cerevisiae iso-1-cytochrome c has been expressed in Escherichia coli by coexpression of the genes encoding the cytochrome (CYC1) and yeast cytochrome c heme lyase (CYC3). Construction of this expression system involved cloning the two genes in parallel into the vector pUC18 to give the plasmid pBPCYC1(wt)/3. Transcription was directed by two promoters, Lac and Trc, that were located upstream from CYC1. Both proteins were expressed in the cytoplasm of E. coli cells harboring the plasmid. Semianaerobic cultures grown in a fermentor produced 15 mg of recombinant iso-1-cytochrome c per liter of culture. Attempts to increase production by addition of IPTG suppressed the number of copies of the CYC1 gene within the population. Wild-type iso-1-cytochrome c expressed with pBPCYC1(wt)/3 in E. coli was compared to the same protein expressed in yeast. At neutral pH, the two proteins exhibit indistinguishable spectroscopic and physical (Tm, Em') characteristics. However, electrospray mass spectrometry revealed that the lysyl residue at position 72 is not trimethylated by E. coli as it is by S. cerevisiae. Interestingly, the pKa of the alkaline transition of the protein expressed in E. coli is approximately 0.6 pKa unit lower than that observed for the cytochrome expressed in yeast (8.5-8.7). 1H NMR spectroscopy of the bacterially expressed cytochrome collected at high pH revealed the presence of a third alkaline conformer that is not observed in the corresponding spectrum of the cytochrome expressed in yeast. These observations suggest that Lys72 can serve as an axial ligand to the heme iron of alkaline iso-1-ferricytochrome c if it is not modified posttranscriptionally to trimethyllysine.

Bacterial expression and spectroscopic characterization of soybean leghaemoglobin a.

A gene encoding leghaemoglobin a from soybean has been constructed and the soluble recombinant protein expressed in E. coli. The integrity of the recombinant protein has been assessed by a range of spectroscopic techniques. Electrospray mass spectrometry of the protein indicates that the molecular mass of the protein corresponds to the predicted amino acid sequence. Circular dichroism spectra of the ferric derivative and UV-visible spectra of various ferric and ferrous derivatives (pH 6.99, mu = 0.10 M, 25.0 degrees C) are consistent with published data for the wild-type protein. For the ferric derivative, UV-visible (298 and 77 K) and EPR (10 K) spectra indicate the existence of a thermal equilibrium between high- and low-spin forms. Titration of the protein (0.10 M NaCl, mu = 0.10 M, 25.0 degrees C) between pHs 6.68 and 10.35 indicate formation (pKa = 8.3+/-0.03) of a 6-coordinate, hydroxide-bound form of the protein at high pH. All of the above data are consistent with the behaviour of the wild-type protein.

Acid-induced denaturation of myoglobin studied by time-resolved electrospray ionization mass spectrometry.

The acid-induced denaturation of holo-myoglobin (hMb) following a pH-jump from 6.5 to 3.2 has been studied by electrospray ionization (ESI) mass spectrometry in combination with a continuous flow mixing technique (time-resolved ESI MS). Different protein conformations are detected by the different charge state distributions that they generate during ESI. The changes in intensity of the peaks in the mass spectrum as a function of time can be described by two exponential lifetimes of 0.38 +/- 0.06 s and 6.1 +/- 0.5 s, respectively. The acid-induced denaturation of hMb was also studied in stopped-flow experiments by monitoring changes in the Soret absorption. The lifetimes measured by this method are in good agreement with those obtained by time-resolved ESI MS. The shorter lifetime is associated with the formation of a transient intermediate which shows the mass of the intact heme-protein complex but leads to the formation of much higher charge states during ESI than native hMb at pH 6.5. This form of hMb has an absorption spectrum similar to that of the native protein, indicating a relatively unperturbed chromophore environment inside the heme binding pocket. The intermediate can thus be characterized as an unfolded form of hMb with essentially intact heme-protein interactions. The longer of the two lifetimes is associated with the formation of a product which has a blue-shifted absorption spectrum with a much lower maximum absorption coefficient than observed for native hMb. In the ESI mass spectrum, this product appears as the apoprotein with high charge states which indicates the disruption of the native heme-protein interactions and a considerable degree of unfolding compared to native apo-myoglobin. The mechanism of acid-induced denaturation of hMb, therefore, appears to follow the sequence (heme-protein)native --> (heme-protein)unfolded --> heme + (protein)unfolded.