Inflammatory profile of apical periodontitis associated with liver fibrosis in rats: histological and immunohistochemical analysis.

AIM: To investigate the effects of liver fibrosis (LF) on the pro-inflammatory mediators and periapical bone resorption of apical periodontitis (AP) in rats. METHODOLOGY: Forty male Wistar rats were distributed into four groups: C - control, AP - rats with AP, LF - rats with LF, AP + LF - rats with AP and LF. LF was induced by carbon tetrachloride administration for 8 weeks and surgical bile duct ligation for 4 weeks; AP was induced in the teeth of rats by dental pulp exposure to the oral environment for 30 days. Jaws and livers were removed after euthanasia. Haematoxylin and Eosin (H&E) and Picrosirius Red (PSR) staining were used to confirm fibrosis in the livers. The jaws were analysed using H&E staining, immunohistochemical assays of interleukin (IL)-1ß, IL-6 and tumour necrosis factor-alpha (TNF-α). Student's t-test and Mann-Whitney's U-test were used for statistical analysis (P < 0.05). RESULTS: Inflammatory infiltrate was moderate in the AP group and severe in the AP + LF group (P < 0.05). Periapical bone resorption was significantly larger in the AP + LF group compared with the AP group (P < 0.05). IL-1ß, IL-6 and TNF-α levels were significantly higher in AP + LF group when compared to the AP group (P < 0.05). CONCLUSION: More intense inflammatory infiltrate, greater amounts of pro-inflammatory cytokines and increased periapical bone resorption were observed in the presence of liver fibrosis in rats with exposed pulps.

Palatogingival groove and root canal instrumentation.

AIM: To evaluate the morphology and impact of root canal preparation in maxillary incisors with palatogingival grooves (PGG). METHODOLOGY: Twenty extracted human maxillary incisors with PGG were subjected to macroscopic analysis and scanning electron microscopy analysis (SEM). The following characteristics of the PGG were evaluated: depth, point of origin in the cingulum, extension and position on the lingual surface. Furthermore, the presence of calculus, communications between the root canal system and the PGG, and root resorptions were investigated. The root canals were subsequently instrumented with K-files of three consecutive sizes. The teeth were sectioned, and the axial plane of each tooth section was imaged using SEM before and after instrumentation. The distance between the root canal walls and the PGG was calculated according to the location. Additionally, the distance between canal walls and cementum was measured at three different sites, to verify if instrumentation influenced dentine removal on a specific wall in teeth with PGG. Statistical analysis was performed using the Mann-Whitney or Student's t-test (P < 0.05). RESULTS: Macroscopic analysis revealed that a deep groove was most frequently observed (75%), followed by a depression/shallow groove (25%) (P < 0.05). PGG typically originated in the distal margin ridge of the cingulum (65%) (P < 0.05), extending only to the middle (45%) or up to the apical (50%) third of the root (P < 0.05). Additionally, PGGs were typically located on the distal aspect of the lingual surface (70%) (P < 0.05). Calculus was concentrated on the surface of the crown and cementum-enamel junction (P < 0.05). Communication between the root canal and PGG was present in 35% of teeth, and root resorptions were noted in 50% of teeth. The distance between the external root surface and the pulp cavity was significantly narrower after instrumentation (P < 0.05); however, root canal preparation did not influence dentine removal on the specific wall associated with the groove (P > 0.05). CONCLUSIONS: Palatogingival grooves were characteristically deep and originated from the distal margin of the cingulum. Although it has been associated with a thinner root wall, root canal preparation did not influence the thickness of the specific wall in the maxillary incisors with PGG.

3D map distribution of metallic nanoparticles in whole cells using MeV ion microscopy.

In this work, a new tool was developed, the MORIA program that readily translates Rutherford backscattering spectrometry (RBS) output data into visual information, creating a display of the distribution of elements in a true three-dimensional (3D) environment. The program methodology is illustrated with the analysis of yeast Saccharomyces cerevisiae cells, exposed to copper oxide nanoparticles (CuO-NP) and HeLa cells in the presence of gold nanoparticles (Au-NP), using different beam species, energies and nuclear microscopy systems. Results demonstrate that for both cell types, the NP internalization can be clearly perceived. The 3D models of the distribution of CuO-NP in S. cerevisiae cells indicate the nonuniform distribution of NP in the cellular environment and a relevant confinement of CuO-NP to the cell wall. This suggests the impenetrability of certain cellular organelles or compartments for NP. By contrast, using a high-resolution ion beam system, discretized agglomerates of Au-NP were visualized inside the HeLa cell. This is consistent with the mechanism of entry of these NPs in the cellular space by endocytosis enclosed in endosomal vesicles. This approach shows RBS to be a powerful imaging technique assigning to nuclear microscopy unparalleled potential to assess nanoparticle distribution inside the cellular volume.

Selection of reference genes for expression analyses of red-fleshed sweet orange (Citrus sinensis).

Red-fleshed oranges (Citrus sinensis) contain high levels of carotenoids and lycopene. The growing consumer demand for products with health benefits has increased interest in these types of Citrus cultivars as a potential source of nutraceuticals. However, little is known about the physiology of these cultivars under Brazilian conditions. Transcriptome and gene expression analyses are important tools in the breeding and management of red-fleshed sweet orange cultivars. Reverse transcription quantitative polymerase chain reaction is a method of quantifying gene expression, but various standardizations are required to obtain precise, accurate, and specific results. Among the standardizations required, the choice of suitable stable reference genes is fundamental. The objective of this study was to evaluate the stability of 11 candidate genes using various tissue and organ samples from healthy plants or leaves from citrus greening disease (Huanglongbing)-symptomatic plants of a Brazilian red-fleshed cultivar ('Sanguínea de Mombuca'), in order to select the most suitable reference gene for investigating gene expression under these conditions. geNorm and NormFinder identified genes that encoded translation initiation factor 3, ribosomal protein L35, and translation initiation factor 5A as the most stable genes under the biological conditions tested, and genes coding actin (ACT) and the subunit of the PSI reaction center subunit III were the least stable. Phosphatase, malate dehydrogenase, and ACT were the most stable genes in the leaf samples of infected plants.

Effects of sodium hypochlorite associated with EDTA and etidronate on apical root transportation.

AIM: To evaluate the influence of sodium hypochlorite associated with EDTA and etidronate on apical root transportation. METHODOLOGY: Forty-five roots of human mandibular molars with curvatures of 15-25° were embedded in acrylic resin to allow standardized angulation of the initial and final radiographs. The pre-instrumentation radiographs of the mesiobuccal canal of each root were taken using a radiograph digital sensor with a size 15 K-file in the canal. The canals were prepared with the ProTaper Universal system (Dentsply Maillefer, Ballaigues, Switzerland), using one of the following irrigation regimens during the instrumentation (n = 15): G1 - irrigation with 20 mL of saline solution (control); G2 - alternating irrigation with 2.5% hypochlorite solution (NaOCl) (15 mL); and 17% ethylenediaminetetraacetic acid (EDTA) (5 mL). During instrumentation, the canal was filled with NaOCl and then between each exchange of instrument filled with EDTA for 1 min, and G3 - irrigation with 20 mL of 5% NaOCl and 18% etidronate solution (HEBP) mixed in equal parts. The postinstrumentation radiographs were made with a F3 instrument in the canal. The images were magnified and superposed with Adobe Photoshop software (Adobe Systems, Mountain View, CA, USA). Apical transportation was determined with AutoCAD 2012 software (Autodesk Inc., San Rafael, CA, USA) by measuring the distance in millimetres between the tips of the instruments. The results were subjected to the nonparametric statistical Kruskal-Wallis test (α < 0.05). RESULTS: The median transportation and interquartile range values were 0.00 ± 0.05 for G1, 0.08 ± 0.23 for G2 and 0.13 ± 0.14 for G3. Comparison between groups showed that apical transportation in G3 was significantly greater than in G1 (P < 0.05). CONCLUSION: The use of NaOCl associated with etidronate increased apical transportation in the canals of extracted teeth.

Phenotypic and genetic parameters compared during repeated measures of longissimus muscle area and subcutaneous fat thickness in Nelore cattle.

Real-time ultrasound is currently used for in vivo carcass evaluation of beef cattle. We estimated heritability and repeatability coefficients for ultrasound-measured carcass traits in Nelore cattle. We measured longissimus muscle area, backfat thickness, and rump fat thickness in male and female animals ranging in age from 10 to 26 months. The variance components were estimated by single-trait analysis using the derivative-free restricted maximum likelihood method, under an animal model. The complete data file included 3077 records of 1172 animals born between 2003 and 2008. Two analyses were performed using a repeatability model: a) all records of longissimus muscle area, backfat thickness, and rump fat thickness of animals aged 10 to 26 months, and b) considering two age periods (11 to 17 and 18 to 24 months). The heritability estimates ranged from 0.33 to 0.46 for longissimus muscle area, from 0.20 to 0.26 for backfat, and from 0.26 to 0.29 for rump fat, demonstrating that direct selection for these traits can provide genetic gain. The repeatability estimates were moderate, ranging from 0.42 to 0.73. The highest repeatability estimates were obtained for longissimus muscle area in both the first (0.70) and second (0.73) analyses. The repeatability estimates suggest that ultrasound measures are precise; higher precision was obtained for longissimus muscle area than for subcutaneous fat thickness. The latter could be obtained during periods of greater feed availability to increase precision.

Establishing references for gene expression analyses by RT-qPCR in Theobroma cacao tissues.

Lack of continuous progress in Theobroma cacao (Malvaceae) breeding, especially associated with seed quality traits, requires more efficient selection methods based on genomic information. Reverse transcript quantitative PCR (RT-qPCR) has become the method of choice for gene expression analysis, but relative expression analysis requires various reference genes, which must be stable across various biological conditions. We sought suitable reference genes for various tissues of cacao, especially developing seeds. Ten potential reference genes were analyzed for stability at various stages of embryo development, leaves, stems, roots, flowers, and pod epicarp; seven of them were also evaluated in shoot tips treated either with hormones (salicylate; ethefon; methyl-jasmonate) or after inoculation with the fungus Moniliophthora perniciosa (Marasmiaceae sensu lato). For developing embryos, the three most stable genes were actin (ACT), polyubiquitin (PUB), and ribosomal protein L35 (Rpl35). In the analyses of various tissues, the most stable genes were malate dehydrogenase (MDH), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and acyl-carrier protein B (ACP B). GAPDH, MDH and tubulin (TUB) were the most appropriate for normalization when shoot apexes were treated with hormones, while ACT, TUB and Rpl35 were the most appropriate after inoculation with M. perniciosa. We conclude that for each plant system and biological or ontogenetical condition, there is a need to define suitable reference genes. This is the first report to define reference genes for expression studies in cacao.

Microsatellite polymorphisms in cassava landraces from the Cerrado biome, Mato Grosso do sul, Brazil.

Using nine microsatellite loci, we investigated genetic structure and diversity in 83 Brazilian cassava accessions, including several landraces, in the Cerrado biome in Mato Grosso do Sul, Brazil. All nine loci were polymorphic, averaging 6.00 alleles per locus. Treating each of seven municipalities as a cassava group or population, they averaged 3.5 alleles per locus, with 97% polymorphic loci, high values for observed heterozygosity (0.32) and gene diversity (0.56). Total genetic variability was high (0.668), and most of this genetic variability was concentrated within municipalities (0.577). Cluster and structure analyses divided accessions into two major clusters or populations (K = 2). Also, a significant genetic versus geographic correlation was found (r = 0.4567; P < 0.0260). Migratory routes in the Cerrado are considered main contributors to the region's high cassava diversity and spatial genetic structure, amplifying interactions between traditional farmers and the evolutionary dynamics of this crop.

Predictors of nutritional and inflammation risk in hemodialysis patients.

BACKGROUND: Malnutrition and chronic inflammation are prevalent complications in hemodialysis (HD) patients. Different nutritional assessment tools are used to identify patients at risk. A composite and comprehensive malnutrition inflammation score (MIS) has been correlated with morbidity and mortality, and appears to be a robust and quantitative tool. OBJECTIVES: Determine malnutrition risk profile in a sample of portuguese HD patients; determine the association of clinical and laboratory factors with MIS, and the impact of each parameter on MIS. METHODS AND RESULTS: We performed, between September 15th of 2015 and January 31st of 2016, a cross sectional analysis of 2975 patients, representing 25% of portuguese HD patients. 59% were men (66.7 ± 14.8 years); 31% diabetic; 79% and 21% performed, respectively, high-flux HD and HDF. A MIS >5 was considered to indicate higher risk and was present in 1489 patients (50%). Amongst all parameters, comorbilities/dialysis vintage, transferrin, functional capacity, changes in body weight and decreased fat stores showed the higher impact, while albumin had one of the lowest impact on the nutritional risk. MULTIVARIABLE ANALYSIS: Higher age (>75 years, OR 1.71, p < 0.001), diabetes (OR 1.25, p = 0.026), lower P levels (OR 1.57,p = 0.001), higher Ca levels (OR 1.51, p < 0.001), higher ERI (OR 1.05, p < 0.001), higher Kt/V (OR 2.14, p < 0.001) and higher CRP (OR 1.01, p < 0.001) were independently associated with a higher risk of MIS>5; higher nPNA (OR 0.29, p < 0.001) and higher Pcreat (OR 0.88, p < 0.001) were associated with a risk reduction of MIS>5 (95% CI). CONCLUSIONS: Routine clinical and analytic parameters were found to be associated with MIS range that might indicate higher risk, and may represent a simple alert sign for the need of further assessments.

Stratum corneum is an effective barrier to TiO2 and ZnO nanoparticle percutaneous absorption.

BACKGROUND: There is increasing concern over the local and systemic side effects of TiO(2) and ZnO coated nanoparticles widely used in sun blockers. OBJECTIVE: To determine the localization and possible skin penetration of TiO(2) and ZnO nanoparticles, dispersed in 3 sunscreen formulations, under realistic in vivo conditions in normal and altered skin. METHODS: Nuclear microscopy techniques provided spatially resolved quantitative analysis of Ti and Zn nanoparticle distributions in transversal cryosections of skin obtained by biopsy with no further treatment. A test hydrophobic formulation containing coated 20-nm TiO(2) nanoparticles and 2 commercial sunscreen formulations containing TiO(2) alone or in combination with ZnO were tried, taking into account realistic use conditions by consumers and compared with the recommended standard condition for the sun protection factor test. The protocols consisted of an open test. RESULTS: Following a 2-hour exposure period of normal human skin to TiO(2)- and ZnO-containing sunscreens, detectable amounts of these physical blockers were only present at the skin surface and in the uppermost stratum corneum regions. Layers deeper than the stratum corneum were devoid of TiO(2) or exogenous ZnO, even after 48 h of exposure to the sunscreen, under occlusion. Deposition of TiO(2) and ZnO nanoparticles in the openings of the pilosebaceous follicles was also observed, suggesting a preferential fixation area. Penetration of nanoparticles into viable skin tissue could not be detected. CONCLUSIONS: TiO(2) or ZnO nanoparticles are absent or their levels are too low to be tested under the stratum corneum in human viable epidermal layers. Therefore, significant penetration towards the underlying keratinocytes is unlikely.

Cytogenetic study in sand spiders (Sicariidae) from the brazilian Caatinga: sex chromosome system diversity in closely related species / Estudio citogenético en arañas de arena (Sicariidae) de la Caatinga brasileña: diversidad del sistema de cromosomas sexuales en especies estrechamente relacionadas

ABSTRACT In this study, we investigated the chromosomes of three species of Sicarius spiders from the Brazilian Caatinga, using classical and molecular cytogenetic techniques. Based on the phylogenetic approach, we also discussed about the variation of diploid number, types of sex chromosome system and changes in the localization of ribosomal genes of Scytodoidea. Sicarius are Synspermiata spiders that together with the genera Loxosceles and Hexophthalma constitute the family Sicariidae. In this group, the available cytogenetic data showed a low diploid number range (2n♂=18 to 2n♂=23) and the presence of only multiple sex chromosome systems (X1X2Y and X1X20). Mitotic metaphase cells exhibited 2n♂=16+X1X2Y for Sicarius cariri and S. ornatus, and 2n♂=18+XY for S. tropicus. In these species, silver impregnation revealed nucleolar organizer region (Ag-NOR) on the terminal region of pair 1. In S. ornatus and S. tropicus, the results obtained with fluorescent in situ hybridization (FISH) using 18S rDNA probe were similar to Ag-NOR, however in S. cariri, the ribosomal sites were localized in the terminal region of the X1 sex chromosome. In this work, we presented the first description of a simple sex chromosome system for Sicariidae, helping to understand how the XY sex chromosome system evolved from the X1X2Y system. Additionally, FISH data incongruous with Ag-NOR indicate that the cytogenetic studies in Sicariidae allow investigating the relation between the karyotype evolution and the distribution and the activity of rDNA genes.

RESUMEN En este estudio, investigamos los cromosomas de tres especies de arañas Sicarius de la Caatinga brasileña, utilizando técnicas de citogenética clásica y molecular. Usando un enfoque filogenético, también discutimos la variación del número diploide, los tipos de sistema cromosómico sexual y los cambios en la localización de los genes ribosómicos en Scytodoidea. Los Sicarius son arañas Synspermiata que, junto con los géneros Loxosceles y Hexophthalma, constituyen a la familia Sicariidae. En este grupo, los datos citogenéticos disponibles mostraron un rango de número diploide bajo (2n♂=18 a 2n♂=23) y únicamente la presencia de sistemas de cromosomas sexuales múltiples (X1X2Y y X1X20). Las células mitóticas en metafase mostraron 2n♂=16+X1X2Y para Sicarius cariri y S. ornatus, y 2n♂=18+XY para S. tropicus. En estas especies, la impregnación de plata reveló la región organizadora nucleolar (Ag-NOR) en la región terminal del par 1. En S. ornatus y S. tropicus, los resultados obtenidos con la hibridación in situ fluorescente (FISH) utilizando la sonda de ADNr 18S fueron similares a los de Ag-NOR, sin embargo, en S. cariri los sitios ribosomales se localizaron en la región terminal del cromosoma sexual X1. En este trabajo, presentamos la primera descripción de un sistema cromosómico sexual simple para Sicariidae, ayudando a entender cómo el sistema cromosómico sexual XY evolucionó a partir del sistema X1X2Y. Además, los datos de FISH incongruentes con Ag-NOR indican que los estudios citogenéticos en Sicariidae permiten investigar la relación entre la evolución del cariotipo y la distribución y la actividad de los genes de ADNr.

An assessment of time-dependent effects of lead exposure in algerian mice (Mus spretus) using different methodological approaches.

Time-dependent effects of lead (Pb) toxicity were studied in Algerian mice (Mus spretus) treated with Pb acetate via drinking water (1 g Pb acetate/L) for different periods of exposure (15, 45, and 90 d). End points included the determination of hepatic Pb concentration and the assessment of some morphophysiological, biochemical and cytogenetical parameters. A control group receiving distilled water was also monitored for comparative purposes. Hepatic Pb accumulation increased with the time of exposure and was significantly higher in treated mice when compared to controls. In association with significant body mass loss in Pb-exposed mice, for 15 and 45 d, a significant increase in the relative spleen mass was observed after 45 d of intoxication. Pb-exposed mice also showed significant decreases in red blood cells, hematocrit, and mean corpuscular hemoglobin. On the contrary, changes in plasma transferases (aspartate aminotransferase and alanine aminotransferase) and hepatic superoxide dismutase activities did not reach statistical significance. A significant increase in the frequency of micronucleated polychromatic bone marrow erythrocytes was also found in the 90-d-exposed mice, compared to nontreated mice and the other exposed groups. Exposure to Pb acetate resulted also in a slight time-dependent decrease of the polychromatic-normochromatic ratio. These results support the concept that a long-term chronic exposure to Pb induced alterations upon some morphophysiological and genetic parameters in Algerian mice.

Hypertensive disorders during pregnancy and health outcomes in the offspring: a systematic review.

UNLABELLED: The hypertensive disorders of pregnancy complicate up to 10% of pregnancies worldwide and are a leading cause of maternal, foetal, and neonatal morbidity and mortality. The aim of this study was to present an overview of recent studies addressing offspring's medium and long-term health outcomes after intrauterine exposure to maternal hypertension. A search on PubMed/MEDLINE and Bireme databases was conducted to identify observational studies that reported any offspring outcome measured after the 6th month of life. The search was limited to studies published after May 2008. Forty-five articles were included and categorized into four groups of outcomes: cardiovascular, immune, metabolic and behavioural/neurological effects. According to our findings, hypertensive disorders of pregnancy had an overall negative impact on offspring's cardiovascular, immune and neurological health, although not all parameters analysed in each group had consistent results among studies. The most prominent and reliable associations were verified between gestational hypertension and higher offspring's blood pressure and between preeclampsia and offspring's lower cognitive functioning. In the metabolic outcomes, body composition had conflicting results among papers, while all studies that examined blood biomarkers showed no evidence that preeclampsia or gestational hypertension could be associated with an alteration of this metabolic outcomes. Most included studies were highly heterogeneous regarding the measure of outcomes and covariables used for adjustments. Future studies should consider using the same protocols and cut-off points already published so that results can be better compared and summarized. This review was registered in PROSPERO. REGISTRATION NUMBER: CRD42015020838.

Direct evidence for the cooperative unfolding of cytochrome c in lipid membranes from H-(2)H exchange kinetics.

The interaction of cytochrome c (cyt c) with anionic lipid membranes is known to disrupt the tightly packed native structure of the protein. This process leads to a lipid-inserted denatured state, which retains a native-like alpha-helical structure but lacks any specific tertiary interactions. The structural and dynamic properties of cyt c bound to vesicles containing an anionic phospholipid (DOPS) were investigated by amide H-(2)H exchange using two-dimensional NMR spectroscopy and electrospray ionisation mass spectrometry. The H-(2)H exchange kinetics of the core amide protons in cyt c, which in the native protein undergo exchange via an uncorrelated EX2 mechanism, exchange in the lipid vesicles via a highly concerted global transition that exposes these protected amide groups to solvent. The lack of pH dependence and the observation of distinct populations of deuterated and protonated species by mass spectrometry confirms that exchange occurs via an EX1 mechanism with a common rate of 1(+/-0.5) h(-1), which reflects the rate of transition from the lipid-inserted state, H(l), to an unprotected conformation, D(i), associated with the lipid interface.

Elemental characterization of tissues of Octopus vulgaris along the Portuguese coast.

The concentrations of V, Mn, Fe, Ni, Cu, Zn, As, Se, Mo, and Pb were measured in digestive gland (DG), branchial hearts (BH), gill (G), and muscle (M) of Octopus vulgaris Cuvier, 1797 caught in three fishing areas of the Portuguese coast, Viana do Castelo, Cascais, and Santa Luzia, for 2 years. The elemental concentrations measured for the different tissues were in accordance with values reported in the literature. The digestive gland presented high concentration levels of Fe, Cu, and Zn, while the branchial hearts showed elevated levels of V, Ni, Mo, as well as Fe and Cu. Significant variations in As, V, Cu, Mo, and Pb tissue concentrations were observed for animals originated from different sampling sites. Pb and As determined in the digestive gland and branchial hearts of animals from Cascais and Santa Luzia, can reflect local environmental characteristics. The variability observed in the elemental concentrations may be useful to further assess the species susceptibility to environmental conditions.

Unfolding and refolding of cytochrome c driven by the interaction with lipid micelles.

Binding of native cyt c to L-PG micelles leads to a partially unfolded conformation of cyt c. This micelle-bound state has no stable tertiary structure, but remains as alpha-helical as native cyt c in solution. In contrast, binding of the acid-unfolded cyt c to L-PG micelles induces folding of the polypeptide, resulting in a similar helical state to that originated from the binding of native cyt c to L-PG micelles. Far-ultraviolet (UV) circular dichroism (CD) spectra showed that this common micelle-associated helical state (HL) has a native-like alpha-helix content, but is highly expanded without a tightly packed hydrophobic core, as revealed by tryptophan fluorescence, near-UV, and Soret CD spectroscopy. The kinetics of the interaction of native and acid-unfolded cyt c was investigated by stopped-flow tryptophan fluorescence. Formation of H(L) from the native state requires the disruption of the tightly packed hydrophobic core in the native protein. This micelle-induced unfolding of cyt c occurs at a rate approximately 0.1 s(-1), which is remarkably faster in the lipid environment compared with the expected rate of unfolding in solution. Refolding of acid-unfolded cyt c with L-PG micelles involves an early highly helical collapsed state formed during the burst phase (<3 ms), and the observed main kinetic event reports on the opening of this early compact intermediate prior to insertion into the lipid micelle.

Folding of apocytochrome c induced by the interaction with negatively charged lipid micelles proceeds via a collapsed intermediate state.

Unfolded apocytochrome c acquires an alpha-helical conformation upon interaction with lipid. Folding kinetic results below and above the lipid's CMC, together with energy transfer measurements of lipid bound states, and salt-induced compact states in solution, show that the folding transition of apocytochrome c from the unfolded state in solution to a lipid-inserted helical conformation proceeds via a collapsed intermediate state (I(C)). This initial compact state is driven by a hydrophobic collapse of the polypeptide chain in the absence of the heme group and may represent a heme-free analogue of an early compact intermediate detected on the folding pathway of cytochrome c in solution. Insertion into the lipid phase occurs via an unfolding step of I(C) through a more extended state associated with the membrane surface (I(S)). While I(C) appears to be as compact as salt-induced compact states in solution with substantial alpha-helix content, the final lipid-inserted state (Hmic) is as compact as the unfolded state in solution at pH 5 and has an alpha-helix content which resembles that of native cytochrome c.

The interaction of horse heart cytochrome c with phospholipid bilayers. Structural and dynamic effects.

The interaction of cytochrome c with phospholipid bilayers is reviewed. Special emphasis is given to the structural and dynamic perturbations induced, either in the membrane lipid component or protein itself, by the lipid-protein interaction. The lipid-induced perturbations in the structure of cytochrome c involve: i) conformational changes in and around the heme crevice, converting the heme iron to a high-spin state: and ii) a destabilisation/loosening of the overall tertiary and secondary structure. This highly mobile, partially unfolded intermediate of cytochrome c has a remarkable resemblance to partially folded membrane-bound intermediates of the precursor protein. The functional implications of lipid-protein intermediates for (apo) cytochrome c in (protein-translocation) electron-transfer are discussed.

Action of polygodial, a sesquiterpene isolated from Drymis winteri, in the guinea-pig ileum and trachea 'in vitro'.

This study describes the action of the sesquiterpene polygodial, the major constituent isolated from the bark of Drymis winteri in the guinea pig ileum and trachea in vitro. Polygodial (5 to 128 microM), added for 20 min, did not affect the resting tone of the preparations, but caused graded inhibition, associated in some cases with rightward displacement of the acetylcholine, histamine (1 nM to 10 microM), bradykinin (0.1 nM to 1 microM) and KCl (1 to 100 mM)-contraction response curves. When assessed in the guinea-pig trachea, polygodial (5 to 342 microM) caused significant inhibition of bradykinin (10 pM to 1 microM), 9,11-dideoxy-9alpha,11alpha-methano-epoxy prostaglandin F2alpha (0.1 to 1000 nM) and KCl (1 to 100 mM)-induced contractions, although the action against bradykinin was not concentration-dependent. Polygodial (5 to 80 microM) caused a small but significant shift to the right of substance P and also the selective agonist of tachykinin NK2 receptor [beta-Ala8]neurokinin A-(4-10)-induced contractions in guinea pig trachea. This action of polygodial seems to be quite selective towards tachykinin NK2 receptors since up to 432 microM, polygodial had no effect against contraction caused by tachykinin NK1 receptor agonist, substance P methyl ester. When tested in the guinea-pig trachea from animals which had been actively sensitised to ovalbumin, polygodial (30 to 40 microM) caused time and concentration-dependent inhibition of ovalbumin-mediated contraction. In addition, polygodial (85 to 342 microM) inhibited contraction induced by compound 48/80 (1 to 1000 microg/ml), in the guinea-pig trachea from non-sensitised animals. These findings and those from our previous study are consistent with the notion that the main sesquiterpene polygodial isolated from the bark of D. winteri is responsible for most, if not all, of the relevant pharmacological action reported previously for the extract of this plant. Thus, polygodial could be of potential value in the development of a new drug for the treatment of asthma, allergy and other inflammatory processes.

Antinociceptive properties of the hydroalcoholic extract and preliminary study of a xanthone isolated from Polygala cyparissias (Polygalaceae).

Polygala cyparissias (Polygalaceae) grows abundantly on Brazil's Atlantic coast, belonging to the typical underbrush vegetation of dunes and have been used in folk medicine for treatment of several diseases, such as disturbances of bowel and kidney. The hydroalcoholic extract of P. cyparissias (HE, 3 to 60 mg kg(-1), i.p. or 25 to 200 mg kg(-1), p.o.) produced significant and graded inhibition of acetic acid-induced abdominal constrictions, with mean ID50 values of 6 and 72 mg kg(-1), respectively. The HE (at this same range of doses) also produced dose-related inhibition of both the early and the late phase of formalin-induced licking. The calculated mean ID50 values for the early phase were: >60 and >200 mg kg(-1), while for the late phase they were 11 and 101 mg kg(-1), respectively, by i.p. and p.o. routes. The HE also caused dose-related inhibition of formalin-induced edema formation (P<0.01). The HE (3 to 60 mg kg(-1), i.p. or 50 to 200 mg kg(-1), p.o.) produced significant and dose-related inhibition of the neurogenic nociception caused by topical injection of capsaicin, with mean ID50 values of 12 and 71 mg kg(-1), respectively. Given orally, the HE (50 to 200 mg kg(-1)) prevented in a dose-dependent manner, bradykinin (3 nmol/paw) and substance P (10 nmol/paw)-induced hyperalgesia in the rat paw, with mean ED50 values of 122 and 121 mg kg(-1), respectively, but was ineffective in the hot-plate model of nociception. The antinociception caused by the HE, in contrast to that of morphine (5 mg kg(-1), s.c.), was not reversed by naloxone (5 mg kg(-1), i.p.) when assessed in the acetic acid writhing test. The HE, at antinociceptive doses, did not affect motor coordination of animals when assessed in the rota-rod model. The xanthone isolated from P. cyparissias, identified as 1,7-dihydroxy-2,3-dimethoxy xanthone (0.3 to 30 mg kg(-1), i.p.), produced dose-related inhibition of acetic acid-induced abdominal constriction, with mean ID50 value of 1.5 mg kg(-1). These data show that the active principle(s) present in the HE of P. cyparissias, elicited pronounced antinociception when assessed by i.p. or p.o. routes, against both inflammatory and neurogenic nociception, and was able to prevent bradykinin and substance P-induced hyperalgesia. Its precise mechanism of action still remains unclear.