Mapping of suramin binding sites on the group IIA human secreted phospholipase A(2)

Suramin is a polysulphonated napthylurea used as an antiprotozoal/anthelminitic drug, which also inhibits a broad range of enzymes. Suramin binding to recombinant human secreted group IIA phospholipase A(2) (hsPLA(2)GIIA) was investigated by molecular dynamics simulations (MD) and isothermal titration calorimetry (ITC). MD indicated two possible bound suramin conformations mediated by hydrophobic and electrostatic interactions with amino-acids in three regions of the protein. namely the active-site and residues located in the N- and C-termini, respectively. All three binding sites are located on the phospholipid membrane recognition surface, suggesting that suramin may inhibit the enzyme, and indeed a 90% reduction in hydrolytic activity was observed in the presence of 100 nM suramin. These results correlated with ITC data, which demonstrated 2.7 suramin binding sites on the hsPLA(2)GIIA, and indicates that suramin represents a novel class of phosphohpase A(2) inhibitor. (C) 2009 Elsevier Inc. All rights reserved.

CD4+T cells from HIV-1-infected patients recognize wild-type and mutant human immunodeficiency virus-1 protease epitopes

P>Human immunodeficiency virus (HIV)-1 protease is a known target of CD8+ T cell responses, but it is the only HIV-1 protein in which no fully characterized HIV-1 protease CD4 epitopes have been identified to date. We investigated the recognition of HIV-1 protease by CD4+ T cells from 75 HIV-1-infected, protease inhibitor (PI)-treated patients, using the 5,6-carboxyfluorescein diacetate succinimidyl ester-based proliferation assay. In order to identify putative promiscuous CD4+ T cell epitopes, we used the TEPITOPE algorithm to scan the sequence of the HXB2 HIV-1 protease. Protease regions 4-23, 45-64 and 73-95 were identified; 32 sequence variants of the mentioned regions, encoding frequent PI-induced mutations and polymorphisms, were also tested. On average, each peptide bound to five of 15 tested common human leucocyte antigen D-related (HLA-DR) molecules. More than 80% of the patients displayed CD4+ as well as CD8+ T cell recognition of at least one of the protease peptides. All 35 peptides were recognized. The response was not associated with particular HLA-DR or -DQ alleles. Our results thus indicate that protease is a frequent target of CD4+ along with CD8+ proliferative T cell responses by the majority of HIV-1-infected patients under PI therapy. The frequent finding of matching CD4+ and CD8+ T cell responses to the same peptides may indicate that CD4+ T cells provide cognate T cell help for the maintenance of long-living protease-specific functional CD8+ T cells.

A catecholamine transporter from the human parasite Schistosoma mansoni with low affinity for psychostimulants

The trematode Schistosoma mansoni is the primary cause of schistosomiasis, a devastating neglected tropical disease that affects 200 million individuals. Identifying novel therapeutic targets for the treatment of schistosomiasis is therefore of great public interest. The catecholamines norepinephrine (NE) and dopamine (DA) are essential for the survival of the parasite as they cause muscular relaxation and a lengthening in the parasite and thereby control movement. Here we characterize a novel dopamine/norepinephrine transporter (SmDAT) gene transcript, from S. mansoni. The SmDAT is expressed in the adult form and in the sporocyst form (infected snails) of the parasite, and also in the egg and miracidium stage. It is absent in the cercariae stage but curiously a transcript missing the exon encoding transmembrane domain 8 was identified in this stage. Heterologous expression of the cDNA in mammalian cells resulted in saturable, dopamine transport activity with an apparent affinity for dopamine comparable to that of the human dopamine transporter. Efflux experiments reveal notably higher substrate selectivity compared with its mammalian counterparts as amphetamine is a much less potent efflux elicitor against SmDAT compared to the human DAT. Pharmacological characterization of the SmDAT revealed that most human DAT inhibitors including psychostimulants such as cocaine were significantly less potent in inhibiting SmDAT. Like DATs from other simpler organisms the pharmacology for SmDAT was more similar to the human norepinephrine transporter. We were not able to identify other dopamine transporting carriers within the completed parasite genome and we hypothesize that the SmDAT is the only catecholamine transporter in the parasite and could be responsible for not only clearing DA but also NE. (C) 2011 Elsevier B.V. All rights reserved.

Cannabidiol inhibitory effect on marble-burying behaviour: involvement of CB1 receptors

Cannabidiol (CBD) is a major nonpsychotomimetic component of Cannabis sativa that has been shown to have an anxiolytic effect in human and animal models. Earlier studies suggest that these effects involve facilitation of serotonin, a neurotransmitter that has also been related to obsessive-compulsive disorder. On the basis of this evidence, this study investigated the effects of CBD in C57BL/6J mice submitted to the marble-burying test (MBT), an animal model proposed to reflect compulsive behaviour. CBD (15, 30 and 60 mg/kg) induced a significant decrease in the number of buried marbles compared with controls (34, 41 and 48%, respectively). A similar, although larger, decrease was also found after the serotonin selective reuptake inhibitor paroxetine (10 mg/kg, 77% decrease) and the benzodiazepine diazepam (2.5 mg/kg, 84% decrease). The effect of CBD (30 mg/kg) was still significant after 7 days of daily repeated administration, whereas the effect of diazepam disappeared. Pretreatment with WAY100635 (3 mg/kg), a 5HT1A receptor antagonist, prevented the effects of paroxetine but failed to alter those of CBD. These latter effects, however, were prevented by pretreatment with the CB1 receptor antagonist AM251 (1 mg/kg). These results indicated that CBD and paroxetine decrease the number of buried marbles in the MBT through distinct pharmacological mechanisms. They also suggest a potential role of drugs acting on the cannabinoid system in modulating compulsive behaviour. Behavioural Pharmacology 21: 353-358 (C) 2010 Wolters Kluwer Health | Lippincott Williams & Wilkins.

Effects of flunixin meglumine, recombinant bovine somatotropin and/or human chorionic gonadotropin on pregnancy rates in Nelore cows

The objective was to compare pharmacological strategies aiming to inhibit prostaglandin F2 alpha (PGF(2 alpha)) synthesis (flunixin meglumine; FM), stimulate growth of the conceptus (recombinant bovine somatotropin; bST) and progesterone (P(4)) synthesis (human chorionic gonadotropin; hCG), as well as their combinations, regarding their ability to improve pregnancy rates in beef cattle. Lactating Nelore cows (N = 975), 35 to 70 days postpartum, were synchronized and inseminated by timed artificial insemination (TAT) on Day 0. On Day 7, cattle were allocated into eight groups and received one of the following treatments: saline (S) on Days 7 and 16 (Group Control); S on Day 7 and FM on Day 16 (Group FM); bST on Day 7 and S on Day 16 (Group bST); bST on Day 7 and FM on Day 16 (Group bST + FM); hCG on Day 7 and S on Day 16 (Group hCG); hCG on Day 7 and FM on Day 16 (Group hCG + FM); bST and hCG on Day 7 and S on Day 16 (Group bST + hCG), or bST and hCG on Day 7 and FM on Day 16 (Group bST + hCG + FM). The aforementioned treatments were administered at the following doses: 2.2 mg/kg FM (Banamine (R); Intervet Schering-Plough, Cotia, SP, Brazil), 500 mg bST (Boostin (R); Intervet Schering-Plough), and 2500 IU hCG (Chorulon (R); Intervet Schering-Plough). Pregnancy diagnosis was performed 40 days after TAI by transrectal ultrasonography. Pregnancy rates were not significantly different among treatments. However, there was a main effect of hCG treatment to increase pregnancy rates (63.0 vs. 55.4%; P = 0.001). Concentrations of P(4) did not differ significantly among groups on Day 7 or on Day 16. However, consistent with the higher pregnancy rates, hCG increased P(4) concentrations on Day 16 (10.6 vs. 9.6 ng/mL, respectively; P = 0.05). We concluded that hCG treatment 7 days after TAI improved pregnancy rates of lactating Nelore cows, possibly via a mechanism leading to induction of higher P(4) concentrations, or by reducing the luteolytic stimulus during maternal recognition of pregnancy. (C) 2011 Elsevier Inc. All rights reserved.

Human alveolar bone cell proliferation, expression of osteoblastic phenotype, and matrix mineralization on porous titanium produced by powder metallurgy

Objective: This study aimed at investigating the influence of the porous titanium (Ti) structure on the osteogenic cell behaviour. Materials and methods: Porous Ti discs were fabricated by the powder metallurgy process with the pore size typically between 50 and 400 mm and a porosity of 60%. Osteogenic cells obtained from human alveolar bone were cultured until subconfluence and subcultured on dense Ti (control) and porous Ti for periods of up to 17 days. Results: Cultures grown on porous Ti exhibited increased cell proliferation and total protein content, and lower levels of alkaline phosphatase (ALP) activity than on dense Ti. In general, gene expression of osteoblastic markers-runt-related transcription factor 2, collagen type I, alkaline phosphatase, bone morphogenetic protein-7, and osteocalcin was lower at day 7 and higher at day 17 in cultures grown on porous Ti compared with dense Ti, a finding consistent with the enhanced growth rate for such cultures. The amount of mineralized matrix was greater on porous Ti compared with the dense one. Conclusion: These results indicate that the porous Ti is an appropriate substrate for osteogenic cell adhesion, proliferation, and production of a mineralized matrix. Because of the three-dimensional environment it provides, porous Ti should be considered an advantageous substrate for promoting desirable implant surface-bone interactions.

Mycoplasmal lipid-associated membrane proteins and Mycoplasma arthritidis mitogen recognition by serum antibodies from patients with rheumatoid arthritis

Mycoplasmal lipid-associated membrane proteins (LAMPs) and Mycoplasma arthritidis mitogen (MAM superantigen) are potent stimulators of the immune system. The objective of this work was to detect antibodies to MAM and LAMPs of Mycoplasma hominis and M. fermentans in the sera of patients affected by rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE) to identify mycoplasmal products that can be involved in the etiopathogenesis of these autoimmune diseases. Serum samples from female RA and SLE patients and controls, recombinant MAM, and LAMPs of M. hominis PG21 and M. fermentans PG18 were used in Western blot assays. A similar frequency of sera from patients and controls reactive to MAM was detected. A larger number of M. hominis and M. fermentans LAMPs were recognized by sera from RA patients than controls, but no differences were detected between sera from SLE patients and controls. Among the LAMPs recognized by IgG antibodies from RA patients, proteins of molecular masses in a range of < 49 and a parts per thousand yen20 KDa (M. hominis) and < 102 and a parts per thousand yen58 KDa (M. fermentans) were the most reactive. These preliminary results demonstrate the strong reactivity of antibodies of RA patients with some M. hominis and M. fermentans LAMPs. These LAMPs could be investigated as mycoplasmal antigens that can take part in the induction or amplification of human autoimmune responses.

Expression and purification of human respiratory syncytial virus recombinant fusion protein

The Human Respiratory Syncytial Virus (HRSV) fusion protein (F) was expressed in Escherichia call BL21A using the pET28a vector at 37 degrees C. The protein was purified from the soluble fraction using affinity resin. The structural quality of the recombinant fusion protein and the estimation of its secondary structure were obtained by circular dichroism. Structural models of the fusion protein presented 46% of the helices in agreement with the spectra by circular dichroism analysis. There are only few studies that succeeded in expressing the HRSV fusion protein in bacteria. This is a report on human fusion protein expression in E. call and structure analysis, representing a step forward in the development of fusion protein F inhibitors and the production of antibodies. (c) 2008 Elsevier Inc. All rights reserved.

Effect of a calcium-channel blocker (verapamil) on the morphology, cytoskeleton and collagenase activity of human skin fibroblasts

The effects of verapamil modulating collagen biosynthesis have prompted us to study the role of this drug in cultured fibroblasts. In this article, we describe the effects of verapamil on fibroblast behaviour, with special emphasis to phenotypic modifications, reorganisation of actin filaments and secretion of MMP1. Human dermal fibroblasts treated with 50-mu M verapamil changed their normal spindle-shaped morphology to stellate. Treated cells showed discrete reorganisation of actin filaments, as revealed by fluorescein isothiocyanate (FITC)-phalloidin staining and confocal microscopy. We hypothesised that these effects would be associated to lower levels of cytosolic Ca(2+). Indeed, short time loading with calcium green confirmed that verapamil-treated fibroblasts exhibited lower intracellular calcium levels compared to controls. We also observed that verapamil increases the secretion of MMP1 in cultured fibroblasts, as demonstrated by zymography, specific substrate assays and immunoblot. The morphological alterations induced by verapamil are neither cytotoxic nor associated with other dramatic cytoskeleton alterations. Thus we may conclude that this drug enhances collagenase secretion and does not disrupt the major tracks necessary to deliver these enzymes in the extracellular space. The present results suggested that verapamil could be used at physiological levels to enhance collagen I breakdown, and maybe considered a potential candidate for intralesional therapy of wound healing and fibrocontractive diseases. (C) 2010 Elsevier Ltd and ISBI. All rights reserved.

Dynamic Changes in the Mental Rotation Network Revealed by Pattern Recognition Analysis of fMRI Data

We investigated the temporal dynamics and changes in connectivity in the mental rotation network through the application of spatio-temporal support vector machines (SVMs). The spatio-temporal SVM [Mourao-Miranda, J., Friston, K. J., et al. (2007). Dynamic discrimination analysis: A spatial-temporal SVM. Neuroimage, 36, 88-99] is a pattern recognition approach that is suitable for investigating dynamic changes in the brain network during a complex mental task. It does not require a model describing each component of the task and the precise shape of the BOLD impulse response. By defining a time window including a cognitive event, one can use spatio-temporal fMRI observations from two cognitive states to train the SVM. During the training, the SVM finds the discriminating pattern between the two states and produces a discriminating weight vector encompassing both voxels and time (i.e., spatio-temporal maps). We showed that by applying spatio-temporal SVM to an event-related mental rotation experiment, it is possible to discriminate between different degrees of angular disparity (0 degrees vs. 20 degrees, 0 degrees vs. 60 degrees, and 0 degrees vs. 100 degrees), and the discrimination accuracy is correlated with the difference in angular disparity between the conditions. For the comparison with highest accuracy (08 vs. 1008), we evaluated how the most discriminating areas (visual regions, parietal regions, supplementary, and premotor areas) change their behavior over time. The frontal premotor regions became highly discriminating earlier than the superior parietal cortex. There seems to be a parcellation of the parietal regions with an earlier discrimination of the inferior parietal lobe in the mental rotation in relation to the superior parietal. The SVM also identified a network of regions that had a decrease in BOLD responses during the 100 degrees condition in relation to the 0 degrees condition (posterior cingulate, frontal, and superior temporal gyrus). This network was also highly discriminating between the two conditions. In addition, we investigated changes in functional connectivity between the most discriminating areas identified by the spatio-temporal SVM. We observed an increase in functional connectivity between almost all areas activated during the 100 degrees condition (bilateral inferior and superior parietal lobe, bilateral premotor area, and SMA) but not between the areas that showed a decrease in BOLD response during the 100 degrees condition.

Structure, Dynamics, and RNA Interaction Analysis of the Human SBDS Protein

Shwachman-Bodian-Diamond syndrome is an autosomal recessive genetic syndrome with pleiotropic phenotypes, including pancreatic deficiencies, bone marrow dysfunctions with increased risk of myelodysplasia or leukemia, and skeletal abnormalities. This syndrome has been associated with mutations in the SBDS gene, which encodes a conserved protein showing orthologs in Archaea and eukaryotes. The Shwachman-Bodian-Diamond syndrome pleiotropic phenotypes may be an indication of different cell type requirements for a fully functional SBDS protein. RNA-binding activity has been predicted for archaeal and yeast SBDS orthologs, with the latter also being implicated in ribosome biogenesis. However, full-length SBDS orthologs function in a species-specific manner, indicating that the knowledge obtained from model systems may be of limited use in understanding major unresolved issues regarding SBDS function, namely, the effect of mutations in human SBDS on its biochemical function and the specificity of RNA interaction. We determined the solution structure and backbone dynamics of the human SBDS protein and describe its RNA binding site using NMR spectroscopy. Similarly to the crystal structures of Archaea, the overall structure of human SBDS comprises three well-folded domains. However, significant conformational exchange was observed in NMR dynamics experiments for the flexible linker between the N-terminal domain and the central domain, and these experiments also reflect the relative motions of the domains. RNA titrations monitored by heteronuclear correlation experiments and chemical shift mapping analysis identified a classic RNA binding site at the N-terminal FYSH (fungal, Yhr087wp, Shwachman) domain that concentrates most of the mutations described for the human SBDS. (C) 2010 Elsevier Ltd. All rights reserved.

Human endogenous RNAs: Implications for the immunomodulation of Toll-like receptor 3

Toll-like receptors (TLRs), a family of mammalian receptors, are able to recognize nucleic acids. TLR3 recognizes double-stranded (ds)RNA, a product of the replication of certain viruses. Polyinosinic-polycytidylic acid, referred to as poly(I:C), an analog of viral dsRNA, interacts with TLR3 thereby eliciting immunoinflammatory responses characteristic of viral infection or down-regulating the expression of chemokine receptor CXCR4. It is known that dsRNA also directly activates interferon (IFN)-induced enzymes, such as the RNA-dependent protein kinase (PKR). In the present study, the mRNA expression of TLR3, CXCR4, IFN gamma and PKR was investigated in a culture of peripheral blood mononuclear cells (PBMCs) stimulated with poly(I:C) and endogenous RNA from human PBMCs. No cytotoxic effect on the cells or on the proliferation of CD3(+), CD4(+) and CD8(+) cells was observed. TLR3 expression in the PBMCs in the presence of poly(I:C) was up-regulated 9.5-fold, and TLR3 expression in the PBMCs treated with endogenous RNA was down-regulated 1.8-fold (p=0.002). The same trend was observed for IFN gamma where in the presence of poly(I:C) an 8.7-fold increase was noted and in the presence of endogenous RNA a 3.1-fold decrease was observed. In the culture activated with poly(1:C), mRNA expression of CXCR4 increased 8.0-fold and expression of PKR increased 33.0-fold. Expression of these genes decreased in the culture treated with endogenous RNA when compared to the culture without stimulus. Thus, high expression of mRNA for TLR3, IFN gamma, CXCR4 and PKR was observed in the presence of poly(I:C) and low expression was observed in the cells cultured with endogenous RNA. In conclusion, TLR3 may play major physiological roles that are not in the context of viral infection. It is possible that RNA released from cells could contain enough double-stranded structures to regulate cell activation. The involvement of endogenous RNA in endogenous gene expression and its implications in the regulation thereof, are still being studied, and will have significant implications in the future.

Stoichiometry and thermodynamics of the interaction between the C-terminus of human 90 kDa heat shock protein Hsp90 and the mitochondrial translocase of outer membrane Tom70

A large majority of the 1000-1500 proteins in the mitochondria are encoded by the nuclear genome, and therefore, they are translated in the cytosol in the form and contain signals to enable the import of proteins into the organelle. The TOM complex is the major translocase of the outer membrane responsible for preprotein translocation. It consists of a general import pore complex and two membrane import receptors, Tom20 and Tom70. Tom70 contains a characteristic TPR domain, which is a docking site for the Hsp70 and Hsp90 chaperones. These chaperones are involved in protecting cytosolic preproteins from aggregation and then in delivering them to the TOM complex. Although highly significant, many aspects of the interaction between Tom70 and Hsp90 are still uncertain. Thus, we used biophysical tools to study the interaction between the C-terminal domain of Hsp90 (C-Hsp90), which contains the EEVD motif that binds to TPR domains, and the cytosolic fragment of Tom70. The results indicate a stoichiometry of binding of one monomer of Tom70 per dimer of C-Hsp90 with a K(D) of 360 30 nM, and the stoichiometry and thermodynamic parameters obtained suggested that Tom70 presents a different mechanism of interaction with Hsp90 when compared with other TPR proteins investigated. (C) 2011 Elsevier Inc. All rights reserved.

Quality evaluation of DNA obtained from stored human saliva and its applicability to identification in Forensic Dentistry

PURPOSE: This study evaluated the quality of DNA obtained from stored human saliva and its applicability to human identification. METHODS: The saliva samples of 20 subjects, collected in the form of saliva in natura and from mouth swabs and stored at -20ºC, were analyzed. After 7 days, the DNA was extracted from the 40 saliva samples and subjected to PCR and electrophoresis. After 180 days, the technique was repeated with the 20 swab samples. RESULTS: The first-stage results indicated that DNA was successfully extracted in 97.5% of reactions, 95% of saliva in natura and 100% of swab saliva samples, with no statistically significant difference between the forms of saliva. In the second phase, the result was positive for all 20 analyzed samples (100%). Subsequently, in order to analyze the quality of the DNA obtained from human saliva, the SIX3-2 gene was tested on the 20 mouth swab samples, and the PCR products were digested using the MbO1 restriction enzyme to evaluate polymorphisms in the ADRA-2 gene, with positive results for most samples. CONCLUSION: It was concluded that the quantity and quality of DNA from saliva and the techniques employed are adequate for forensic analysis of DNA.

Pulpotomies with portland cement in human primary molars

Two clinical cases in which Portland cement (PC) was applied as a medicament after pulpotomy of mandibular primary molars in children are presented. Pulpotomy using PC was carried out in two mandibular first molars and one mandibular second molar, which were further followed-up. At the 3, 6 and 12-month follow-up appointments, clinical and radiographic examinations of the pulpotomized teeth and their periradicular area revealed that the treatments were successful in maintaining the teeth asymptomatic and preserving pulpal vitality. Additionally, the formation of a dentin bridge immediately below the PC could be observed in the three molars treated. PC may be considered as an effective alternative for primary molar pulpotomies, at least in a short-term period. Randomized clinical trials with human teeth are required in order to determine the suitability of PC before unlimited clinical use can be recommended.