Inflammation markers in healthy and periodontitis patients: a preliminary data screening

Advances in diagnostic research are moving towards methods whereby the periodontal risk can be identified and quantified by objective measures using biomarkers. Patients with periodontitis may have elevated circulating levels of specific inflammatory markers that can be correlated to the severity of the disease. The purpose of this study was to evaluate whether differences in the serum levels of inflammatory biomarkers are differentially expressed in healthy and periodontitis patients. Twenty-five patients (8 healthy patients and 17 chronic periodontitis patients) were enrolled in the study. A 15 mL blood sample was used for identification of the inflammatory markers, with a human inflammatory flow cytometry multiplex assay. Among 24 assessed cytokines, only 3 (RANTES, MIG and Eotaxin) were statistically different between groups (p<0.05). In conclusion, some of the selected markers of inflammation are differentially expressed in healthy and periodontitis patients. Cytokine profile analysis may be further explored to distinguish the periodontitis patients from the ones free of disease and also to be used as a measure of risk. The present data, however, are limited and larger sample size studies are required to validate the findings of the specific biomarkers.

Crystallization, data collection and data processing of maltose-binding protein (MalE) from the phytopathogen Xanthomonas axonopodis pv. citri

Maltose-binding protein is the periplasmic component of the ABC transporter responsible for the uptake of maltose/maltodextrins. The Xanthomonas axonopodis pv. citri maltose-binding protein MalE has been crystallized at 293 Kusing the hanging-drop vapour-diffusion method. The crystal belonged to the primitive hexagonal space group P6(1)22, with unit-cell parameters a = 123.59, b = 123.59, c = 304.20 angstrom, and contained two molecules in the asymetric unit. It diffracted to 2.24 angstrom resolution.

Screening of Filamentous Fungi to Identify Biocatalysts for Lupeol Biotransformation

The goal of the study was to evaluate the ability of filamentous fungi to biotransform the pentacyclic triterpene lupeol. The microbial transformations were carried out in shake flasks in different media. Experiments were also run with control flasks. Samples of each culture were taken every 24 hours, extracted with ethyl acetate, and analyzed by GC-MS. The biotransformation of lupeol by Aspergillus ochraceus and Mucor rouxii afforded two compounds in each culture, which were detected in the cultures developed for more than seven days only in the Koch's K1 medium. The obtained data demonstrated that A. ochraceus is a good biocatalyst to introduce double bonds in the lupeol structure, whereas M. rouxii exhibits ability to biocatalyze oxygen insertions in that pentacyclic triterpene. Mass spectrometry was demonstrated to be an efficient analytical method to select promising biocatalysts for the compound investigated in this study. The biotransformation processes were influenced by the culture medium and incubation period. The obtained results open the perspective of using A. ochraceus and M. rouxii in pentacyclic triterpene biotransformations.

A rational protocol for the successful crystallization of l-amino-acid oxidase from Bothrops atrox

Despite the valuable contributions of robotics and high-throughput approaches to protein crystallization, the role of an experienced crystallographer in the evaluation and rationalization of a crystallization process is still crucial to obtaining crystals suitable for X-ray diffraction measurements. In this work, the difficult task of crystallizing the flavoenzyme l-amino-acid oxidase purified from Bothrops atrox snake venom was overcome by the development of a protocol that first required the identification of a non-amorphous precipitate as a promising crystallization condition followed by the implementation of a methodology that combined crystallization in the presence of oil and seeding techniques. Crystals were obtained and a complete data set was collected to 2.3 A resolution. The crystals belonged to space group P2(1), with unit-cell parameters a = 73.64, b = 123.92, c = 105.08 A, beta = 96.03 degrees. There were four protein subunits in the asymmetric unit, which gave a Matthews coefficient V (M) of 2.12 A3 Da-1, corresponding to 42% solvent content. The structure has been solved by molecular-replacement techniques.

Expression, crystallization and preliminary crystallographic analysis of PilZ(XAC1133) from Xanthomonas axonopodis pv. citri

Proteins containing PilZ domains are widespread in Gram-negative bacteria and have recently been shown to be involved in the control of biofilm formation, adherence, aggregation, virulence-factor production and motility. Furthermore, some PilZ domains have recently been shown to bind the second messenger bis(3'-> 5') cyclic diGMP. Here, the cloning, expression, purification and crystallization of PilZ(XAC1133), a protein consisting of a single PilZ domain from Xanthomonas axonopodis pv. citri, is reported. The closest PilZ(XAC1133) homologues in Pseudomonas aeruginosa and Neisseria meningitidis control type IV pilus function. Recombinant PilZ(XAC1133) containing selenomethionine was crystallized in space group P6(1). The unit-cell parameters were a = 62.125, b = 62.125, c = 83.543 angstrom. These crystals diffracted to 1.85 angstrom resolution and a MAD data set was collected at a synchrotron source. The calculated Matthews coefficient suggested the presence of two PilZ(XAC1133) molecules in the asymmetric unit.

Crystallization and preliminary structural analysis of the giant haemoglobin from Glossoscolex paulistus at 3.2 angstrom

Glossoscolex paulistus is a free-living earthworm encountered in south-east Brazil. Its oxygen transport requirements are undertaken by a giant extracellular haemoglobin, or erythrocruorin (HbGp), which has an approximate molecular mass of 3.6 MDa and, by analogy with its homologue from Lumbricus terrestris (HbLt), is believed to be composed of a total of 180 polypeptide chains. In the present work the full 3.6 MDa particle in its cyanomet state was purified and crystallized using sodium citrate or PEG8000 as precipitant. The crystals contain one-quarter of the full particle in the asymmetric unit of the I222 cell and have parameters of a = 270.8 angstrom, b = 320.3 angstrom and c = 332.4 angstrom. Diffraction data were collected to 3.15 angstrom using synchrotron radiation on beamline X29A at the Brookhaven National Laboratory and represent the highest resolution data described to date for similar erythrocruorins. The structure was solved by molecular replacement using a search model corresponding to one-twelfth of its homologue from HbLt. This revealed that HbGp belongs to the type I class of erythrocruorins and provided an interpretable initial electron density map in which many features including the haem groups and disulfide bonds could be identified.

Purification, crystallization and preliminary crystallographic analysis of the catalytic domain of the extracellular cellulase CBHI from Trichoderma harzianum

The filamentous fungus Trichoderma harzianum has a considerable cellulolytic activity that is mediated by a complex of enzymes which are essential for the hydrolysis of microcrystalline cellulose. These enzymes were produced by the induction of T. harzianum with microcrystalline cellulose (Avicel) under submerged fermentation in a bioreactor. The catalytic core domain (CCD) of cellobiohydrolase I (CBHI) was purified from the extracellular extracts and submitted to robotic crystallization. Diffraction-quality CBHI CCD crystals were grown and an X-ray diffraction data set was collected under cryogenic conditions using a synchrotron-radiation source.

Protein preparation, crystallization and preliminary X-ray analysis of Trypanosoma cruzi nucleoside diphosphate kinase 1

The flagellated protozoan parasite Trypanosoma cruzi is the aetiological agent of Chagas disease. Nucleoside diphosphate kinases (NDPKs) are enzymes that are involved in energy management and nucleoside balance in the cell. T. cruzi TcNDPK1, a canonical isoform, was overexpressed in Escherichia coli as an N-terminally poly-His-tagged fusion protein and crystallized. Crystals grew after 72 h in 0.2 M MgCl(2), 20% PEG 3350. Data were collected to 3.5 angstrom resolution using synchrotron X-ray radiation at the National Synchrotron Light Laboratory (Campinas, Brazil). The crystals belonged to the trigonal space group P3, with unit-cell parameters a = b = 127.84, c = 275.49 angstrom. Structure determination is under way and will provide relevant information that may lead to the first step in rational drug design for the treatment of Chagas disease.

Crystallization and preliminary X-ray diffraction analysis of human IL-22 bound to its soluble decoy receptor IL-22BP

Interleukin-22 (IL-22) is a pleiotropic cytokine that is involved in inflammatory responses. Human IL-22 was incubated with its soluble decoy receptor IL-22BP (IL-22 binding protein) and the IL-22 -IL-22BP complex was crystallized in hanging drops using the vapour-diffusion method. Suitable crystals were obtained from polyethylene glycol solutions and diffraction data were collected to 2.75 angstrom resolution. The crystal belonged to the tetragonal space group P41, with unit-cell parameters a = b = 67.9, c = 172.5 angstrom, and contained two IL-22-IL- 22BP complexes per asymmetric unit.

Crystallization and preliminary X-ray diffraction analysis of recombinant chlorocatechol 1,2-dioxygenase from Pseudomonas putida

Chlorocatechol 1,2-dioxygenase from the Gram-negative bacterium Pseudomonas putida (Pp 1,2-CCD) is considered to be an important biotechnological tool owing to its ability to process a broad spectrum of organic pollutants. In the current work, the crystallization, crystallographic characterization and phasing of the recombinant Pp 1,2-CCD enzyme are described. Reddish-brown crystals were obtained in the presence of polyethylene glycol and magnesium acetate by utilizing the vapour-diffusion technique in sitting drops. Crystal dehydration was the key step in obtaining data sets, which were collected on the D03B-MX2 beamline at the CNPEM/MCT - LNLS using a MAR CCD detector. Pp 1,2-CCD crystals belonged to space group P6(1)22 and the crystallographic structure of Pp 1,2-CCD has been solved by the MR-SAD technique using Fe atoms as scattering centres and the coordinates of 3-chlorocatechol 1,2-dioxygenase from Rhodococcus opacus (PDB entry 2boy) as the search model. The initial model, which contains three molecules in the asymmetric unit, has been refined to 3.4 A resolution.

Clarification of a wheat straw-derived medium with ion-exchange resins for xylitol crystallization

BACKGROUND: Xylitol bioproduction from lignocellulosic residues comprises hydrolysis of the hemicellulose, detoxification of the hydrolysate, bioconversion of the xylose, and recovery of xylitol from the fermented hydrolysate. There are relatively few reports on xylitol recovery from fermented media. In the present study, ion-exchange resins were used to clarify a fermented wheat straw hemicellulosic hydrolysate, which was then vacuum-concentrated and submitted to cooling in the presence of ethanol for xylitol crystallization. RESULTS: Sequential adsorption into two anion-exchange resins (A-860S and A-500PS) promoted considerable reductions in the content of soluble by-products (up to 97.5%) and in medium coloration (99.5%). Vacuum concentration led to a dark-colored viscous solution that inhibited xylitol crystallization. This inhibition could be overcome by mixing the concentrated medium with a commercial xylitol solution. Such a strategy led to xylitol crystals with up to 95.9% purity. The crystallization yield (43.5%) was close to that observed when using commercial xylitol solution (51.4%). CONCLUSION: The experimental data demonstrate the feasibility of using ion-exchange resins followed by cooling in the presence of ethanol as a strategy to promote the fast recovery and purification of xylitol from hemicellulose-derived fermentation media. (c) 2008 Society of Chemical Industry.

Phase Equilibrium and Optimization Tools: Application for Enhanced Structured Lipids for Foods

Solid-liquid phase equilibrium modeling of triacylglycerol mixtures is essential for lipids design. Considering the alpha polymorphism and liquid phase as ideal, the Margules 2-suffix excess Gibbs energy model with predictive binary parameter correlations describes the non ideal beta and beta` solid polymorphs. Solving by direct optimization of the Gibbs free energy enables one to predict from a bulk mixture composition the phases composition at a given temperature and thus the SFC curve, the melting profile and the Differential Scanning Calorimetry (DSC) curve that are related to end-user lipid properties. Phase diagram, SFC and DSC curve experimental data are qualitatively and quantitatively well predicted for the binary mixture 1,3-dipalmitoyl-2-oleoyl-sn-glycerol (POP) and 1,2,3-tripalmitoyl-sn-glycerol (PPP), the ternary mixture 1,3-dimyristoyl-2-palmitoyl-sn-glycerol (MPM), 1,2-distearoyl-3-oleoyl-sn-glycerol (SSO) and 1,2,3-trioleoyl-sn-glycerol (OOO), for palm oil and cocoa butter. Then, addition to palm oil of Medium-Long-Medium type structured lipids is evaluated, using caprylic acid as medium chain and long chain fatty acids (EPA-eicosapentaenoic acid, DHA-docosahexaenoic acid, gamma-linolenic-octadecatrienoic acid and AA-arachidonic acid), as sn-2 substitutes. EPA, DHA and AA increase the melting range on both the fusion and crystallization side. gamma-linolenic shifts the melting range upwards. This predictive tool is useful for the pre-screening of lipids matching desired properties set a priori.

Rapid Screening and Identification of Polar Constituents from Brazilian Arnica Lychnophora sp by LC-UV/DAD-ESI-MS and LC-UV/DAD-ESI-MS/MS Analysis

This paper describes an analytical method for the rapid screening and identification of the phenolic constituents present in the polar extracts of different Lychnophora spp. using LC-UV/DAD-ESI-MS and LC-UV/DAD-ESI-MS/MS. Compounds were identified based on UV, retention time, MS experiments and MS/MS of precursor ion or standard. On-line phytochemical investigation of Lychnophora spp. allowed for the identification of flavonoids, chlorogenic acid derivatives and lactones. Some of the observed compounds were for the first time identified in Lychnophora species in a fast analytical procedure. The data obtained here may be helpful to the investigation of polar constituents from other Lychnophora species.

Use of virtual screening, flexible docking, and molecular interaction fields to design novel HMG-CoA reductase inhibitors for the treatment of hypercholesterolemia

Dietary changes associated with drug therapy can reduce high serum cholesterol levels and dramatically decrease the risk of coronary artery disease, stroke, and overall mortality. Statins are hypolipemic drugs that are effective in the reduction of cholesterol serum levels, attenuating cholesterol synthesis in liver by competitive inhibition regarding the substrate or molecular target HMG-CoA reductase. We have herewith used computer-aided molecular design tools, i.e., flexible docking, virtual screening in large data bases, molecular interaction fields to propose novel potential HMG-CoA reductase inhibitors that are promising for the treatment of hypercholesterolemia.

The actual incidence of congenital adrenal hyperplasia in Brazil may not be as high as inferred - An estimate based on a public neonatal screening program in the state of Goias

The incidence of 21-hydroxylase deficiency (CYP21 D) congenital adrenal hyperplasia (CAH) in Brazil is purportedly one of the highest in the world (1:7,533). However, this information is not based on official data. The aim of this study was to determine the incidence of CYP21 D CAH in the state of Goias, Brazil, based on the 2005 results of government-funded mandatory screening. Of the live births during this period, 92.95% were screened by heel-prick capillary 17 alpha-hydroxyprogesterone (17-OHP). Of these, 82,343 were normal, 28 were at high risk for CAH and 232 at low risk for CAH. Eight cases, all from the high risk group, were confirmed. Eight asymptomatic children at 6-18 months of age still have high 17-OHP levels and await diagnostic definition. Based on the number of confirmed CYP21 D CAH cases among the 82,603 screened, the estimated annual incidence of the disease was 1:10,325, lower than the previously reported rate in Brazil.