Proteome analysis of schizophrenia patients Wernicke's area reveals an energy metabolism dysregulation

Background: Schizophrenia is likely to be a consequence of DNA alterations that, together with environmental factors, will lead to protein expression differences and the ultimate establishment of the illness. The superior temporal gyrus is implicated in schizophrenia and executes functions such as the processing of speech, language skills and sound processing. Methods: We performed an individual comparative proteome analysis using two-dimensional gel electrophoresis of 9 schizophrenia and 6 healthy control patients' left posterior superior temporal gyrus (Wernicke's area - BA22p) identifying by mass spectrometry several protein expression alterations that could be related to the disease. Results: Our analysis revealed 11 downregulated and 14 upregulated proteins, most of them related to energy metabolism. Whereas many of the identified proteins have been previously implicated in schizophrenia, such as fructose-bisphosphate aldolase C, creatine kinase and neuron-specific enolase, new putative disease markers were also identified such as dihydrolipoyl dehydrogenase, tropomyosin 3, breast cancer metastasis-suppressor 1, heterogeneous nuclear ribonucleoproteins C1/C2 and phosphate carrier protein, mitochondrial precursor. Besides, the differential expression of peroxiredoxin 6 (PRDX6) and glial fibrillary acidic protein (GFAP) were confirmed by western blot in schizophrenia prefrontal cortex. Conclusion: Our data supports a dysregulation of energy metabolism in schizophrenia as well as suggests new markers that may contribute to a better understanding of this complex disease.

Identification of Novel Immunoregulatory Molecules in Human Thymic Regulatory CD4(+)CD25(+) T Cells by Phage Display

Thymic CD4(+)CD25(+) cells play an important role in immune regulation and are continuously developed in the thymus as an independent lineage. How these cells are generated, what are their multiple pathways of suppressive activity and which are their specific markers are questions that remain unanswered. To identify molecules involved in the function and development of human CD4(+)CD25(+) T regulatory cells we targeted thymic CD4(+)CD25(+) cells by peptide phage display. A phage library containing random peptides was screened ex vivo for binding to human thymic CD4(+)CD25(+) T cells. After four rounds of selection on CD4(+)CD25(+) enriched populations of thymocytes, we sequenced several phage displayed peptides and selected one with identity to the Vitamin D Receptor (VDR). We confirmed the binding of the VDR phage to active Vitamin D in vitro, as well as the higher expression of VDR in CD4(+)CD25(+) cells. We suggest that differential expression of VDR on natural Tregs may be related to the relevance of Vitamin D in function and ontogeny of these cells.

No-match ORESTES explored as tumor markers

Sequencing technologies and new bioinformatics tools have led to the complete sequencing of various genomes. However, information regarding the human transcriptome and its annotation is yet to be completed. The Human Cancer Genome Project, using ORESTES (open reading frame EST sequences) methodology, contributed to this objective by generating data from about 1.2 million expressed sequence tags. Approximately 30 of these sequences did not align to ESTs in the public databases and were considered no-match ORESTES. On the basis that a set of these ESTs could represent new transcripts, we constructed a cDNA microarray. This platform was used to hybridize against 12 different normal or tumor tissues. We identified 3421 transcribed regions not associated with annotated transcripts, representing 83.3 of the platform. The total number of differentially expressed sequences was 1007. Also, 28 of analyzed sequences could represent noncoding RNAs. Our data reinforces the knowledge of the human genome being pervasively transcribed, and point out molecular marker candidates for different cancers. To reinforce our data, we confirmed, by real-time PCR, the differential expression of three out of eight potentially tumor markers in prostate tissues. Lists of 1007 differentially expressed sequences, and the 291 potentially noncoding tumor markers were provided.

Direct effect of Plasmodium vivax recombinant vaccine candidates AMA-1 and MSP-1(19) on the innate immune response

The recombinant apical membrane antigen 1 (AMA-1) and 19-kDa fragment of merozoite surface protein (MSP-1(19)) are the lead candidates for inclusion in a vaccine against blood stages of malaria due to encouraging protective studies in humans and animals. Despite the importance of an efficacious malaria vaccine, vaccine-related research has focused on identifying antigens that result in protective immunity rather than determining the nature of anti-malarial immune effector mechanisms. Moreover, emphasis has been placed on adaptive rather than innate immune responses. In this study, we investigated the effect of Plasmodium vivax vaccine candidates Pv-AMA-1 and Pv-MSP-1(19) on the immune response of malaria-naive donors. Maturation of dendritic cells is altered by Pv-AMA-1 but not Pv-MSP-1(19), as observed by differential expression of cell surface markers. In addition, Pv-AMA-1 induced an increased production of MIP-1 alpha/CCL3 and decreased production of TARC/CCL17 levels in both dendritic cells (DCs) and peripheral blood mononuclear cells (PBMCs). Finally, a significant pro-inflammatory response was elicited by Pv-AMA-1-stimulated PBMCs. These results suggest that the recombinant vaccine candidate Pv-AMA-1 may play a direct role on innate immune response and might be involved in parasite destruction. (C) 2007 Elsevier Ltd. All rights reserved.

Time course proteomic profiling of human myocardial infarction plasma samples: An approach to new biomarker discovery

Background: The aim of this study was to identify novel candidate biomarker proteins differentially expressed in the plasma of patients with early stage acute myocardial infarction (AMI) using SELDI-TOF-MS as a high throughput screening technology. Methods: Ten individuals with recent acute ischemic-type chest pain (< 12 h duration) and ST-segment elevation AMI (1STEMI) and after a second AMI (2STEMI) were selected. Blood samples were drawn at six times after STEMI diagnosis. The first stage (T(0)) was in Emergency Unit before receiving any medication, the second was just after primary angioplasty (T(2)), and the next four stages occurred at 12 h intervals after T(0). Individuals (n = 7) with similar risk factors for cardiovascular disease and normal ergometric test were selected as a control group (CG). Plasma proteomic profiling analysis was performed using the top-down (i.e. intact proteins) SELDI-TOF-MS, after processing in a Multiple Affinity Removal Spin Cartridge System (Agilent). Results: Compared with the CG, the 1STEMI group exhibited 510 differentially expressed protein peaks in the first 48 h after the AMI (p < 0.05). The 2STEMI group, had similar to 85% fewer differently expressed protein peaks than those without previous history of AMI (76, p < 0.05). Among the 16 differentially-regulated protein peaks common to both STEMI cohorts (compared with the CG at T(0)), 6 peaks were persistently down-regulated at more than one time-stage, and also were inversed correlated with serum protein markers (cTnI, CK and CKMB) during 48 h-period after IAM. Conclusions: Proteomic analysis by SELDI-TOF-MS technology combined with bioinformatics tools demonstrated differential expression during a 48 h time course suggests a potential role of some of these proteins as biomarkers for the very early stages of AMI, as well as for monitoring early cardiac ischemic recovery. (C) 2011 Elsevier B.V. All rights reserved.

A Sex-Specific Association between a 15q25 Variant and Upper Aerodigestive Tract Cancers

Background: Sequence variants located at 15q25 have been associated with lung cancer and propensity to smoke. We recently reported an association between rs16969968 and risk of upper aerodigestive tract (UADT) cancers (oral cavity, oropharynx, hypopharynx, larynx, and esophagus) in women (OR = 1.24, P = 0.003) with little effect in men (OR = 1.04, P = 0.35). Methods: In a coordinated genotyping study within the International Head and Neck Cancer Epidemiology (INHANCE) consortium, we have sought to replicate these findings in an additional 4,604 cases and 6,239 controls from 10 independent UADT cancer case-control studies. Results: rs16969968 was again associated with UADT cancers in women (OR = 1.21, 95% CI = 1.08-1.36, P = 0.001) and a similar lack of observed effect in men [OR = 1.02, 95% CI = 0.95-1.09, P = 0.66; P-heterogeneity (P(het)) = 0.01]. In a pooled analysis of the original and current studies, totaling 8,572 UADT cancer cases and 11,558 controls, the association was observed among females (OR = 1.22, 95% CI = 1.12-1.34, P = 7 x 10(-6)) but not males (OR = 1.02, 95% CI = 0.97-1.08, P = 0.35; P(het) = 6 x 10(-4)). There was little evidence for a sex difference in the association between this variant and cigarettes smoked per day, with male and female rs16969968 variant carriers smoking approximately the same amount more in the 11,991 ever smokers in the pooled analysis of the 14 studies (P(het) = 0.86). Conclusions: This study has confirmed a sex difference in the association between the 15q25 variant rs16969968 and UADT cancers. Impact: Further research is warranted to elucidate the mechanisms underlying these observations. Cancer Epidemiol Biomarkers Prev; 20(4); 658-64. (C) 2011 AACR.

New transposons to generate GFP protein fusions in Candida albicans

Transposon elements are important tools for gene function analysis, for example they can be used to easily create genome-wide collections of insertion mutants. Transposons may also carry sequences coding for an epitope or fluorescent marker useful for protein expression and localization analysis. We have developed three new Tn5-based transposons that incorporate a GFP (green fluorescent protein) coding sequence to generate fusion proteins in the important fungal pathogen Candida albicans. Each transposon also contains the URA3 and Kan(R) genes for yeast and bacterial selection, respectively. After in vitro transposition, the insertional allele is transferred to the chromosomal locus by homologous recombination. Transposons Tn5-CaGFP and Tn5-CaGFP-URA3:FLIP can generate C-terminal truncated GFP fusions. A URA3 flipper recycling cassette was incorporated into the transposon Th5-CaGFP-UFRA3:FLIP. After the induction of Flip recombinase to excise the marker, the heterozygous strain is transformed again in order to obtain a GFP-tagged homozygous strains. In the Tn5-CaGFP-FL transposon the markers are flanked by a rare-cutting enzyme. After in vitro transposition into a plasmid-borne target gene, the markers are eliminated by restriction digestion and religation, resulting in a construct coding for full-length GFP-fusion proteins. This transposon can generate plasmid libraries of GFP insertions in proteins where N- or C-terminal tagging may alter localization. We tested our transposon system by mutagenizing the essential septin CDC3 gene. The results indicate that the Cdc3 C-terminal extension is important for correct septin filament assembly. The transposons described here provide a new system to obtain global gene expression and protein localization data in C. albicans. (c) 2008 Elsevier B.V. All rights reserved.

The genetic structure of 3 ` untranslated region of the HLA-G gene: polymorphisms and haplotypes

The HLA-G gene is predominantly expressed at the maternal-fetal interface. It has been associated with maternal-fetal tolerance and in the inhibition of cytotoxic T lymphocyte and natural killer cytolytic functions. At least two variations in the 3` untranslated region (UTR) of HLA-G locus are associated with HLA-G expression levels, the 14-bp deletion/insertion polymorphism and the +3142 single-nucleotide polymorphism (SNP). However, this region has not been completely characterized yet. The variability of the 3`UTR of HLA-G gene and its haplotype structure were characterized in 155 individuals from Brazil, as well as HLA-G alleles associated with each of the 3`UTR haplotype. The following eight variation sites were detected: the 14-bp polymorphism and SNPs at the positions +3003T/C, +3010C/G, +3027A/C, +3035C/T, +3142G/C, +3187A/G and +3196C/G. Similarly, 11 different 3`UTR haplotypes were identified and several HLA-G alleles presented only one 3`UTR haplotype. In addition, a high linkage disequilibrium among the variation sites was detected, especially among the 14-bp insertion and the alleles +3142G and +3187A, all previously associated with low mRNA availability, demonstrating that their effects are not independent. The detailed analyses of 3`UTR of the HLA-G locus may shed some light into mechanisms underlying the regulation of HLA-G expression. Genes and Immunity (2010) 11, 134-141; doi: 10.1038/gene.2009.74; published online 1 October 2009

Chemokines and chemokine receptors coordinate the inflammatory immune response in human cutaneous leishmaniasis

Cutaneous leishmaniasis (CL) includes different clinical manifestations displaying diverse intensities of dermal Inflammatory infiltrate Diffuse CL (DCL) cases are hyporesponsive and lesions show very few lymphocytes and a predominance of macrophages In contrast localized CL (LCL) cases are responsive to leishmanial antigen and lesions exhibit granulocytes and mononuclear cell infiltration in the early phases changing to a pattern with numerous lymphocytes and macrophages later in the lesion Therefore different chemokines may affect the predominance of cell infiltration in distinct clinical manifestations In lesions from LCL patients we examined by flow cytometry the presence of different chemokines and their receptors in T cells and we verified a higher expression of CXCR3 in the early stages of LCL (less than 30 days of infection) and a higher expression of CCR4 in the late stages of disease (more than 60 days of infection) We also observed a higher frequency of T cells producing IL-10 in the late stage of LCL Using immunohistochemistry we observed a higher expression of CCL7 CCL17 in lesions from late LCL as well as CCR4 suggesting a preferential recruitment of regulatory T cells in the late LCL Comparing lesions from LCL and DCL patients we observed a higher frequency of CCL7 in DCL lesions These results point out the Importance of the chemokines defining the different types of cells recruited to the site of the infection which could be related to the outcome of infection as well as the clinical form observed (C) 2010 American Society for Histocompatibility and Immunogenetics Published by Elsevier Inc All rights reserved

Proteomic analysis of kidney in rats chronically exposed to fluoride

Two-dimensional gel electrophoresis (2-DE) was used to better understand alterations in renal metabolism induced by fluoride (F). Three groups of weanling male Wistar rats were treated with drinking water containing 0 (control), 5, or 50 ppm F for 60 days (n=6/group). Kidneys were collected for proteomic and histological (HE) analysis. After protein isolation, renal proteome profiles were examined using 2-DE and Colloidal Coomassie Blue staining. Protein spots with a 2-fold significant difference as detected by quantitative intensity analysis (image Master Platinum software) and t-test (p < 0.05) were excised and analyzed by MALDI-TOF MS (matrix assisted laser desorption ionization-time-of-flight mass spectrometry). The histological analysis revealed no damage in kidneys induced by F, except for a vascular congestion in the 50 ppm F group. Between control vs 50 ppm F, and control vs 5 ppm F groups, 12 and 6 differentially expressed proteins were detected, respectively. Six proteins, mainly related with metabolism, detoxification and housekeeping, were successfully identified. At the high F group, pyruvate carboxylase, a protein involved in the formation of oxaloacetate was found to be downregulated, while enoyl coenzyme A hydratase, involved in fatty acids oxidation, was found to be upregulated. Thus, proteomic analysis can provide new insights into the alterations in renal metabolism after F exposure, even in low doses. (C) 2009 Elsevier Ireland Ltd. All rights reserved.

Rat forming incisor requires a rigorous ECM remodeling modulated by MMP/RECK balance

Reversion-inducing-cysteine-rich protein with Kazal motifs (RECK) is a single membrane-anchored MMP-regulator and regulates matrix metalloproteinases (MMP) 2, 9 and 14. In turn, MMPs are endopeptidases that play a pivotal role in remodeling ECM. In this work, we decided to evaluate expression pattern of RECK in growing rat incisor during, specifically focusing out amelogenesis process. Based on different kinds of ameloblasts, our results showed that RECK expression was conducted by secretory and post-secretory ameloblasts. At the secretory phase, RECK was localized in the infra-nuclear region of the ameloblast, outer epithelium, near blood vessels, and in the stellate reticulum. From the transition to the maturation phases, RECK was strongly expressed by non-epithelial immuno-competent cells (macrophages and/or dendritic-like cells) in the papillary layer. From the transition to the maturation stage, RECK expression was increased. RECK mRNA was amplified by RT-PCR from whole enamel organ. Here, we verified the presence of RECK mRNA during all stages of amelogenesis. These events were governed by ameloblasts and by non-epithelial cells residents in the enamel organ. Concluding, we found differential expression of MMPs-2, -9 and RECK in the different phases of amelogenesis, suggesting that the tissue remodeling is rigorously controlled during dental mineralization.

High Matrix Metalloproteinase Activity Is a Hallmark of Periapical Granulomas

Introduction: The inability to distinguish periapical cysts from granulomas before performing root canal treatment leads to uncertainty in treatment outcomes because cysts have lower healing rates. Searching for differential expression of molecules within cysts or granulomas could provide information with regard to the identity of the lesion or suggest mechanistic differences that may form the basis for future therapeutic intervention. Thus, we investigated whether granulomas and cysts exhibit differential expression of extracellular matrix (ECM) molecules. Methods: Human periapical granulomas, periapical cysts, and healthy periodontal ligament tissues were used to investigate the differential expression of ECM molecules by microarray analysis. Because matrix metalloproteinases (MMP) showed the highest differential expression in the microarray analysis, MMPs were further examined by in situ zymography and immunohistochemistry. Data were analyzed by using one-way analysis of variance followed by the Tu-key test. Results: We observed that cysts and granulomas differentially expressed several ECM molecules, especially those from the MMP family. Compared with cysts, granulomas exhibited higher MMP enzymatic activity in areas stained for MMP-9. These areas were composed of polymorphonuclear cells (PMNs) in contrast to cysts. Similarly, MMP-13 was expressed by a greater number of cells in granulomas compared with cysts. Conclusion: Our findings indicate that high enzymatic MIMP activity in PMNs together with MMP-9 and MMP-13 stained cells could be a molecular signature of granulomas unlike periapical cysts. (J Endod 2009;35:1234-1242)

CONSTRUCTING QUANTUM OBSERVABLES AND SELF-ADJOINT EXTENSIONS OF SYMMETRIC OPERATORS. III. SELF-ADJOINT BOUNDARY CONDITIONS

This paper completes the review of the theory of self-adjoint extensions of symmetric operators for physicists as a basis for constructing quantum-mechanical observables. It contains a comparative presentation of the well-known methods and a newly proposed method for constructing ordinary self-adjoint differential operators associated with self-adjoint differential expressions in terms of self-adjoint boundary conditions. The new method has the advantage that it does not require explicitly evaluating deficient subspaces and deficiency indices (these latter are determined in passing) and that boundary conditions are of explicit character irrespective of the singularity of a differential expression. General assertions and constructions are illustrated by examples of well-known quantum-mechanical operators like momentum and Hamiltonian.

Self-adjoint extensions and spectral analysis in the Calogero problem

In this paper, we present a mathematically rigorous quantum-mechanical treatment of a one-dimensional motion of a particle in the Calogero potential alpha x(-2). Although the problem is quite old and well studied, we believe that our consideration based on a uniform approach to constructing a correct quantum-mechanical description for systems with singular potentials and/or boundaries, proposed in our previous works, adds some new points to its solution. To demonstrate that a consideration of the Calogero problem requires mathematical accuracy, we discuss some `paradoxes` inherent in the `naive` quantum-mechanical treatment. Using a self-adjoint extension method, we construct and study all possible self-adjoint operators (self-adjoint Hamiltonians) associated with a formal differential expression for the Calogero Hamiltonian. In particular, we discuss a spontaneous scale-symmetry breaking associated with self-adjoint extensions. A complete spectral analysis of all self-adjoint Hamiltonians is presented.

Differential gene expression profiling in mucus glands of honey bee (Apis mellifera) drones during sexual maturation

The mating sign that each drone leaves when mating with a queen essentially consists of mucus gland proteins. We employed a Representational Difference Analysis (RDA) methodology to identify genes that are differentially expressed in mucus glands during sexual maturation of drones. The RDA library for mucus glands of newly emerged drones was more complex than that of 8 day-old drones, with matches to 20 predicted genes. Another 26 reads matched to the Apis genome but not to any predicted gene. Since these ESTs were located within ORFs they may represent novel honey bee genes, possibly fast evolving mucus gland proteins. In the RDA library for mucus glands of 8 day-old drones, most reads corresponded to a capsid protein of deformed wing virus, indicating high viral loads in these glands. The expression of two genes encoding venom allergens, acid phosphatase-1 and hyaluronidase, in drone mucus glands argues for their homology with the female venom glands, both associated with the reproductive system.