Mutation and genomic amplification of the PIK3CA proto-oncogene in pituitary adenomas

The tumorigenesis of pituitary adenomas is poorly understood. Mutations of the PIK3CA proto-oncogene, which encodes the p110-α catalytic subunit of PI3K, have been reported in various types of human cancers regarding the role of the gene in cell proliferation and survival through activation of the PI3K/Akt signaling pathway. Only one Chinese study described somatic mutations and amplification of the PIK3CA gene in a large series of pituitary adenomas. The aim of the present study was to determine genetic alterations of PIK3CA in a second series that consisted of 33 pituitary adenomas of different subtypes diagnosed by immunohistochemistry: 6 adrenocorticotropic hormone-secreting microadenomas, 5 growth hormone-secreting macroadenomas, 7 prolactin-secreting macroadenomas, and 15 nonfunctioning macroadenomas. Direct sequencing of exons 9 and 20 assessed by qPCR was employed to investigate the presence of mutations and genomic amplification defined as a copy number ≥4. Previously identified PIK3CA mutations (exon 20) were detected in four cases (12.1%). Interestingly, the Chinese study reported mutations only in invasive tumors, while we found a PIK3CA mutation in one noninvasive corticotroph microadenoma. PIK3CA amplification was observed in 21.2% (7/33) of the cases. This study demonstrates the presence of somatic mutations and amplifications of the PIK3CA gene in a second series of pituitary adenomas, corroborating the previously described involvement of the PI3K/Akt signaling pathway in the tumorigenic process of this gland.

RET haplotype, not linked to the C620R activating mutation, associated with Hirschsprung disease in a novel MEN2 family

Hirschsprung disease is a congenital form of aganglionic megacolon that results from cristopathy. Hirschsprung disease usually occurs as a sporadic disease, although it may be associated with several inherited conditions, such as multiple endocrine neoplasia type 2. The rearranged during transfection (RET) proto-oncogene is the major susceptibility gene for Hirschsprung disease, and germline mutations in RET have been reported in up to 50% of the inherited forms of Hirschsprung disease and in 15-20% of sporadic cases of Hirschsprung disease. The prevalence of Hirschsprung disease in multiple endocrine neoplasia type 2 cases was recently determined to be 7.5% and the cooccurrence of Hirschsprung disease and multiple endocrine neoplasia type 2 has been reported in at least 22 families so far. It was initially thought that Hirschsprung disease could be due to disturbances in apoptosis or due to a tendency of the mutated RET receptor to be retained in the Golgi apparatus. Presently, there is strong evidence favoring the hypothesis that specific inactivating haplotypes play a key role in the fetal development of congenital megacolon/Hirschsprung disease. In the present study, we report the genetic findings in a novel family with multiple endocrine neoplasia type 2: a specific RET haplotype was documented in patients with Hirschsprung disease associated with medullary thyroid carcinoma, but it was absent in patients with only medullary thyroid carcinoma. Despite the limited number of cases, the present data favor the hypothesis that specific haplotypes not linked to RET germline mutations are the genetic causes of Hirschsprung disease.

Assessing the gain of biological data integration in gene networks inference

Background: A current challenge in gene annotation is to define the gene function in the context of the network of relationships instead of using single genes. The inference of gene networks (GNs) has emerged as an approach to better understand the biology of the system and to study how several components of this network interact with each other and keep their functions stable. However, in general there is no sufficient data to accurately recover the GNs from their expression levels leading to the curse of dimensionality, in which the number of variables is higher than samples. One way to mitigate this problem is to integrate biological data instead of using only the expression profiles in the inference process. Nowadays, the use of several biological information in inference methods had a significant increase in order to better recover the connections between genes and reduce the false positives. What makes this strategy so interesting is the possibility of confirming the known connections through the included biological data, and the possibility of discovering new relationships between genes when observed the expression data. Although several works in data integration have increased the performance of the network inference methods, the real contribution of adding each type of biological information in the obtained improvement is not clear. Methods: We propose a methodology to include biological information into an inference algorithm in order to assess its prediction gain by using biological information and expression profile together. We also evaluated and compared the gain of adding four types of biological information: (a) protein-protein interaction, (b) Rosetta stone fusion proteins, (c) KEGG and (d) KEGG+GO. Results and conclusions: This work presents a first comparison of the gain in the use of prior biological information in the inference of GNs by considering the eukaryote (P. falciparum) organism. Our results indicates that information based on direct interaction can produce a higher improvement in the gain than data about a less specific relationship as GO or KEGG. Also, as expected, the results show that the use of biological information is a very important approach for the improvement of the inference. We also compared the gain in the inference of the global network and only the hubs. The results indicates that the use of biological information can improve the identification of the most connected proteins.

Combined snake venomics and venom gland transcriptomic analysis of Bothropoides pauloensis

Unraveling the repertoire of venom toxins of Bothropoides pauloensis was assessed by snake venomics and venom gland transcriptomic surveys. Both approaches yielded converging overall figures, pointing to metalloproteinases (similar to 37%), PLA(2)s (26-32%), and vasoactive (bradykinin-potentiating) peptides (12-17%) as the major toxin classes. The high occurrence of SVMPs, PLA(2) molecules, vasoactive peptides, along with serine proteinases, explains the local and systemic effects observed in envenomations by B. pauloensis. Minor (<3%) C-type lectin, serine proteinase, L-amino acid oxidase, nerve growth factor, and CRISP molecules were also identified in the transcriptome and the proteome. Low abundance (0.3%) EST singletons coding for vascular endothelial growth factor (svVEGF), ohanin, hyaluronidase, and 5' nucleotidase were found only in the venom gland cDNA library. At the molecular level, the transcriptomic and proteomic datasets display low compositional concordance. In particular, although there is good agreement between transcriptome and proteome in the identity of BPPs, PLA(2) molecules and L-amino acid oxidase, both datasets strongly depart in their C-type lectin and SVMP complements. These data support the view that venom composition is influenced by transcriptional and translational mechanisms and emphasize the value of combining proteomic and transcriptomic approaches to acquire a more complete understanding of the toxinological profile and natural history of the snake venom. (C) 2012 Elsevier B.V. All rights reserved.

Biochemical, physicochemical and molecular characterization of a genuine 2-Cys-peroxiredoxin purified from cowpea [Vigna unguiculata (L.) Walpers] leaves

Background: Peroxiredoxins have diverse functions in cellular defense-signaling pathways. 2-Cys-peroxiredoxins (2-Cys-Prx) reduce H2O2 and alkyl-hydroperoxide. This study describes the purification and characterization of a genuine 2-Cys-Prx from Vigna unguiculata (Vu-2-Cys-Prx). Methods: Vu-2-Cys-Prx was purified from leaves by ammonium sulfate fractionation, chitin affinity and ion exchange chromatography. Results: Vu-2-Cys-Prx reduces H2O2 using NADPH and DTT. Vu-2-Cys-Prx is a 44 kDa (SDS-PAGE)/46 kDa (exclusion chromatography) protein that appears as a 22 kDa molecule under reducing conditions, indicating that it is a homodimer linked intermolecularly by disulfide bonds and has a pI range of 4.56-4.72; its NH2-terminal sequence was similar to 2-Cys-Prx from Phaseolus vulgaris (96%) and Populus tricocarpa (96%). Analysis by ESI-Q-TOF MS/MS showed a molecular mass/pI of 28.622 kDa/5.18. Vu-2-Cys-Prx has 8% alpha-helix, 39% beta-sheet, 22% of turns and 31% of unordered forms. Vu-2-Cys-Prx was heat stable, has optimal activity at pH 7.0, and prevented plasmid DNA degradation. Atomic force microscopy shows that Vu-2-Cys-Prx oligomerized in decamers which might be associated with its molecular chaperone activity that prevented denaturation of insulin and citrate synthase. Its cDNA analysis showed that the redox-active Cys(52) residue and the amino acids Pro(45), Thr(49) and Arg(128) are conserved as in other 2-Cys-Prx. General significance: The biochemical and molecular features of Vu-2-Cys-Prx are similar to other members of 2-Cys-Prx family. To date, only one publication reported on the purification of native 2-Cys-Prx from leaves and the subsequent analysis by N-terminal Edman sequencing, which is crucial for construction of stromal recombinant 2-Cys-Prx proteins. (C) 2012 Elsevier B.V. All rights reserved.

Serological detection of Plasmodium vivax malaria using recombinant proteins corresponding to the 19-kDa C-terminal region of the merozoite surface protein-1

Abstract Background Serological tests to detect antibodies specific to Plasmodium vivax could be a valuable tool for epidemiological studies, for screening blood donors in areas where the malaria is not endemic and for diagnosis of infected individuals. Because P. vivax cannot be easily obtained in vitro, ELISA assays using total or semi-purified antigens are rarely used. Based on this limitation, we tested whether recombinant proteins representing the 19 kDa C-terminal region of the merozoite surface protein-1 of P. vivax (MSP119) could be useful for serological detection of malaria infection. Methods Three purified recombinant proteins produced in Escherichia coli (GST-MSP119, His6-MSP119 and His6-MSP119-PADRE) and one in Pichia pastoris (yMSP119-PADRE) were compared for their ability to bind to IgG antibodies of individuals with patent P. vivax infection. The method was tested with 200 serum samples collected from individuals living in the north of Brazil in areas endemic for malaria, 53 serum samples from individuals exposed to Plasmodium falciparum infection and 177 serum samples from individuals never exposed to malaria. Results Overall, the sensitivity of the ELISA assessed with sera from naturally infected individuals was 95%. The proportion of serum samples that reacted with recombinant proteins GST-MSP119, His6-MSP119, His6-MSP119-PADRE and yMSP119-PADRE was 90%, 93.5%, 93.5% and 93.5%, respectively. The specificity values of the ELISA determined with sera from healthy individuals and from individuals with other infectious diseases were 98.3% (GST-MSP119), 97.7% (His6-MSP119 and His6-MSP119-PADRE) or 100% (yMSP119-PADRE). Conclusions Our study demonstrated that for the Brazilian population, an ELISA using a recombinant protein of the MSP119 can be used as the basis for the development of a valuable serological assay for the detection of P. vivax malaria.

Characterization of yolk sac proteins of Bos indicus cattle embryos

The yolk sac is an embryonic membrane that is essential for the embryo's initial survival in many mammals. It also plays an important role in the production of proteins necessary for development. We studied proteins of the yolk sac in bovine embryos at up to 40 days of gestation. We examined the yolk sac of 17 bovine embryos at different gestational periods, measuring a-fetoprotein, alpha-1-antitrypsin, and transferrin. This experiment was carried out by Western blot technique, associated with electrophoresis on a 6% sodium dodecyl sulfate polyacrylamide gel. Mouse monoclonal antibody anti-human-alpha-fetoprotein, mouse antibody anti-human-transferrin and rabbit polyclonal anti-human-alpha-1-antitrypsin were used as primary antibodies, and conjugated peroxidase as a secondary antibody. We detected the three proteins in some of the yolk sac samples; however, the bands in some specimens (samples) were weak, maybe a result of poor antigen-antibody reaction, since the antibodies used in this study were not specific to bovine proteins. The fact that weak bands appeared might be due to a weak cross-reaction.

Associations among genotype, clinical phenotype, and intracellular localization of trafficking proteins in ARC syndrome

Arthrogryposisrenal dysfunctioncholestasis (ARC) syndrome is a rare autosomal recessive multisystem disorder caused by mutations in vacuolar protein sorting 33 homologue B (VPS33B) and VPS33B interacting protein, apicalbasolateral polarity regulator (VIPAR). Cardinal features of ARC include congenital joint contractures, renal tubular dysfunction, cholestasis, severe failure to thrive, ichthyosis, and a defect in platelet alpha-granule biogenesis. Most patients with ARC do not survive past the first year of life. We report two patients presenting with a mild ARC phenotype, now 5.5 and 3.5 years old. Both patients were compound heterozygotes with the novel VPS33B donor splice-site mutation c.1225+5G>C in common. Immunoblotting and complementary DNA analysis suggest expression of a shorter VPS33B transcript, and cell-based assays show that c.1225+5G>C VPS33B mutant retains some ability to interact with VIPAR (and thus partial wild-type function). This study provides the first evidence of genotypephenotype correlation in ARC and suggests that VPS33B c.1225+5G>C mutation predicts a mild ARC phenotype. We have established an interactive online database for ARC (https://grenada.lumc.nl/LOVD2/ARC) comprising all known variants in VPS33B and VIPAR. Also included in the database are 15 novel pathogenic variants in VPS33B and five in VIPAR. Hum Mutat 33:16561664, 2012. (c) 2012 Wiley Periodicals, Inc.

Natural intracellular peptides can modulate the interactions of mouse brain proteins and thimet oligopeptidase with 14-3-3e and calmodulin

Protein interactions are crucial for most cellular process. Thus, rationally designed peptides that act as competitive assembly inhibitors of protein interactions by mimicking specific, determined structural elements have been extensively used in clinical and basic research. Recently, mammalian cells have been shown to contain a large number of intracellular peptides of unknown function. Here, we investigate the role of several of these natural intracellular peptides as putative modulators of protein interactions that are related to Ca2+-calmodulin (CaM) and 14-3-3 epsilon, which are proteins that are related to the spatial organization of signal transduction within cells. At concentrations of 1-50 mu M, most of the peptides that are investigated in this study modulate the interactions of CaM and 14-3-3 epsilon with proteins from the mouse brain cytoplasm or recombinant thimet oligopeptidase (EP24.15) in vitro, as measured by surface plasmon resonance. One of these peptides (VFDVELL; VFD-7) increases the cytosolic Ca2+ concentration in a dose-dependent manner but only if introduced into HEK293 cells, which suggests a wide biological function of this peptide. Therefore, it is exciting to suggest that natural intracellular peptides are novel modulators of protein interactions and have biological functions within cells.

Plasma levels of complement proteins from the alternative pathway in patients with age-related macular degeneration are independent of Complement Factor H Tyr(402)His polymorphism

Purpose: To investigate the influence of the Factor H (CFH) Tyr(402)His polymorphism on the plasma levels of the alternative pathway proteins CFH, C3, Factor B (FB), Factor D (FD), and Factor I (FI) and the inflammatory marker C-reactive protein (CRP) in 119 patients with age-related macular degeneration (AMD) and 152 unrelated control individuals. Methods: Patients with AMD and the control group were separated according to CFH polymorphism, age, and gender. Plasma complement proteins and CRP concentrations were determined with enzyme-linked immunosorbent assay, immunodiffusion, or nephelometry. Results: Significant differences in the concentrations of FD and FI were observed between the patients with AMD and the control individuals. We observed significantly reduced FD plasma levels in patients with AMD. We also identified a significant decrease in CFH plasma levels in female patients with AMD in relation to female controls. Plasma FI levels were significantly increased in patients with AMD compared to the control group. Regarding gender, a significant increase in FI plasma levels was observed in male patients. Finally, we found no significant correlation between the CFH Tyr(402)His polymorphism and the CFH, C3, FB, FD, FI, and CRP plasma levels. Conclusions: Patients with AMD present altered levels of FD and FI in a manner independent of this CFH polymorphism, and gender apparently contributes to the plasma levels of these two proteins in patients with AMD and control individuals.

Analysis of three Xanthomonas axonopodis pv. citri effector proteins in pathogenicity and their interactions with host plant proteins

Xanthomonas axonopodis pv. citri, the bacterium responsible for citrus canker, uses effector proteins secreted by a type III protein secretion system to colonize its hosts. Among the putative effector proteins identified for this bacterium, we focused on the analysis of the roles of AvrXacE1, AvrXacE2 and Xac3090 in pathogenicity and their interactions with host plant proteins. Bacterial deletion mutants in avrXacE1, avrXacE2 and xac3090 were constructed and evaluated in pathogenicity assays. The avrXacE1 and avrXacE2 mutants presented lesions with larger necrotic areas relative to the wild-type strain when infiltrated in citrus leaves. Yeast two-hybrid studies were used to identify several plant proteins likely to interact with AvrXacE1, AvrXacE2 and Xac3090. We also assessed the localization of these effector proteins fused to green fluorescent protein in the plant cell, and observed that they co-localized to the subcellular spaces in which the plant proteins with which they interacted were predicted to be confined. Our results suggest that, although AvrXacE1 localizes to the plant cell nucleus, where it interacts with transcription factors and DNA-binding proteins, AvrXacE2 appears to be involved in lesion-stimulating disease 1-mediated cell death, and Xac3090 is directed to the chloroplast where its function remains to be clarified.

Venomics Profiling of Thamnodynastes strigatus Unveils Matrix Metalloproteinases and Other Novel Proteins Recruited to the Toxin Arsenal of Rear-Fanged Snakes

Rear-fanged and aglyphous snakes are usually considered not dangerous to humans because of their limited capacity of injecting venom. Therefore, only a few studies have been dedicated to characterizing the venom of the largest parcel of snake fauna. Here, we investigated the venom proteome of the rear-fanged snake Thamnodynastes strigatus, in combination with a transcriptomic evaluation of the venom gland. About 60% of all transcripts code for putative venom components. A striking finding is that the most abundant type of transcript (similar to 47%) and also the major protein type in the venom correspond to a new kind of matrix metalloproteinase (MMP) that is unrelated to the classical snake venom metalloproteinases found in all snake families. These enzymes were recently suggested as possible venom components, and we show here that they are proteolytically active and probably recruited to venom from a MMP-9 ancestor. Other unusual proteins were suggested to be venom components: a protein related to lactadherin and an EGF repeat-containing transcript. Despite these unusual molecules, seven toxin classes commonly found in typical venomous snakes are also present in the venom. These results support the evidence that the arsenals of these snakes are very diverse and harbor new types of biologically important molecules.

Biological evaluation of the bone healing process after application of two potentially osteogenic proteins: an animal experimental model

doi: 10.1111/j.1741-2358.2011.00526.x Biological evaluation of the bone healing process after application of two potentially osteogenic proteins: an animal experimental model Objective: The aim of this work was to analyse qualitatively and quantitatively the newly formed bone after insertion of rhBMP-2 and protein extracted from Hevea brasiliensis (P-1), associated or not with a carrier in critical bone defects created in Wistar rat calvarial bone, using histological and histomorphometrical analyses. Materials and methods: Eighty-four male Wistar rats were used, divided into two groups, according to the period of time until the sacrifice (2 and 6 weeks). Each one of these groups was subdivided into six groups with seven animals each, according to the treatments: (1) 5 mu g of pure rhBMP-2, (2) 5 mu g of rhBMP-2/monoolein gel, (3) pure monoolein gel, (4) 5 mu g of pure P-1, (5) 5 mu g of P-1/monoolein gel and (6) critical bone defect controls. The animals were euthanised and the calvarial bone tissue removed for histological and histomorphometrical analyses. Result and conclusion: The results showed an improvement in the bone healing process using the rhBMP-2 protein, associated or not with a material carrier in relation to the other groups, and this process demonstrated to be time dependent.

Septin C-Terminal Domain Interactions: Implications for Filament Stability and Assembly

Septins form a conserved family of filament forming GTP binding proteins found in a wide range of eukaryotic cells. They share a common structural architecture consisting of an N-terminal domain, a central GTP binding domain and a C-terminal domain, which is often predicted to adopt a coiled-coil conformation, at least in part. The crystal structure of the human SEPT2/SEPT6/SEPT7 heterocomplex has revealed the importance of the GTP binding domain in filament formation, but surprisingly no electron density was observed for the C-terminal domains and their function remains obscure. The dearth of structural information concerning the C-terminal region has motivated the present study in which the putative C-terminal domains of human SEPT2, SEPT6 and SEPT7 were expressed in E. coli and purified to homogeneity. The thermal stability and secondary structure content of the domains were studied by circular dichroism spectroscopy, and homo- and hetero-interactions were investigated by size exclusion chromatography, chemical cross-linking, analytical ultracentrifugation and surface plasmon resonance. Our results show that SEPT6-C and SEPT7-C are able to form both homo- and heterodimers with a high alpha-helical content in solution. The heterodimer is elongated and considerably more stable than the homodimers, with a K (D) of 15.8 nM. On the other hand, the homodimer SEPT2-C has a much lower affinity, with a K (D) of 4 mu M, and a moderate alpha-helical content. Our findings present the first direct experimental evidence toward better understanding the biophysical properties and coiled-coil pairings of such domains and their potential role in filament assembly and stability.