Redox Activation of Nox1 (NADPH Oxidase 1) Involves an Intermolecular Disulfide Bond Between Protein Disulfide Isomerase and p47phox in Vascular Smooth Muscle Cells.

Objective- PDI (protein disulfide isomerase A1) was reported to support Nox1 (NADPH oxidase) activation mediated by growth factors in vascular smooth muscle cells. Our aim was to investigate the molecular mechanism by which PDI activates Nox1 and the functional implications of PDI in Nox1 activation in vascular disease. Approach and Results- Using recombinant proteins, we identified a redox interaction between PDI and the cytosolic subunit p47phox in vitro. Mass spectrometry of crosslinked peptides confirmed redox-dependent disulfide bonds between cysteines of p47phox and PDI and an intramolecular bond between Cys 196 and 378 in p47phox. PDI catalytic Cys 400 and p47phox Cys 196 were essential for the activation of Nox1 by PDI in vascular smooth muscle cells. Transfection of PDI resulted in the rapid oxidation of a redox-sensitive protein linked to p47phox, whereas PDI mutant did not promote this effect. Mutation of p47phox Cys 196, or the redox active cysteines of PDI, prevented Nox1 complex assembly and vascular smooth muscle cell migration. Proximity ligation assay confirmed the interaction of PDI and p47phox in murine carotid arteries after wire injury. Moreover, in human atheroma plaques, a positive correlation between the expression of PDI and p47phox occurred only in PDI family members with the a' redox active site. Conclusions- PDI redox cysteines facilitate Nox1 complex assembly, thus identifying a new mechanism through which PDI regulates Nox activity in vascular disease.

Sequesterpene Lactones Isolated from a Brazilian Cerrado Plant (Eremanthus spp.) as Anti-Proliferative Compounds, Characterized by Functional and Proteomic Analysis, are Candidates for New Therapeutics in Glioblastoma.

Gliomas are responsible for more than 60% of all primary brain tumors. Glioblastoma multiforme (GBM), a grade IV tumor (WHO), is one of the most frequent and malignant gliomas. Despite two decades of advances in the discovery of new markers for GBM, the chemotherapy of choice falls to temozolomide after surgery and radiotherapy, which are not enough to increase the survival of patients to more than 15 months. It is urgent to discover new anti-glioma compounds. Many compounds derived from natural products have been used in the development of anti-tumor drugs. In this work, we have screened six low molecular weight sesquiterpene lactones, isolated from Eremanthus spp., and studied their function as anti-proliferative agents against GBM strains. We demonstrated that two of them, goyazensolide and lychnofolide, were effective in reducing cell viability, preventing the formation of anchorage-dependent colony and were able to pass through a mimetic blood-brain barrier making them candidates for glioma therapy, being more potent than temozolomide, according to in vitro assays for the cell lines tested. Proteomic analysis revealed a number of altered proteins involved in glycolytic metabolism and cellular catabolism.

Peptides with antioxidant properties identified from casein, whey, and egg albumin hydrolysates generated by two novel fungal proteases.

There are many diseases linked to oxidative stress, including cancer. Importantly, endogenous antioxidants are insufficient to protect against this process. Peptides derived from food proteins produced by hydrolysis have been investigated as exogenous antioxidants. The present study aimed to identify novel peptides with antioxidant potential produced from egg and milk proteins hydrolysis with two new fungal proteases isolated from Eupenicillium javanicum and Myceliophthora thermophila. The degree of hydrolysis at several time points was calculated and correlated to DPPH scavenging and metal chelating assays, all hydrolysates presented antioxidant activity. Casein hydrolyzed by the M. thermophila protease showed the best antioxidant activity. The identified sequences showed that the proportions of amino acids that influence antioxidant activity support the antioxidant assay. Our data reveal the conditions necessary for the successful generation of antioxidant peptides using two novel fungal proteases. This opens a potential new avenue for the design and manufacture of antioxidant molecules.

A soluble recombinant form of human leucocyte antigen-G 6 (srHLA-G6).

Human Leucocyte Antigen-G (HLA-G) is a non classical major histocompatibility complex (MHC) molecule that through RNA splicing can encode seven isoforms which are membrane bound (-G1, -G2, -G3 and -G4) and soluble (-G5, -G6 and -G7). HLA-G is described as important immune suppressor endogenous molecule to favor maternal-fetal tolerance, transplant survival and tumor immune scape. HLA-G shows low protein variability and a unique structural complexity that is related with the expression of different isoforms followed by biochemical processes, such as, proteolytic cleavage, molecular interactions, and protein ubiquitination. Studies with HLA-G have shown difficult to assess the role of the individual isoforms. Thus, the aim of this work was to obtain a HLA-G6 recombinant form. The results indicated the production of high homogeneous preparations of soluble recombinant HLA-G6 (srHLA-G6) with molecular mass 23,603.76 Da, determined by MALD-TOF/TOF. In addition, native and denatured srHLA-G6 were detected by ELISA, using commercial monoclonal antibodies. Finally, we developed a suitable methodology to express srHLA-G6 that could contribute in structural and functional studies involving specific isoforms.

Medium pH in submerged cultivation modulates differences in the intracellular protein profile of Fusarium oxysporum.

Fusarium oxysporum is a filamentous fungus that damages a wide range of plants and thus causes severe crop losses. In fungal pathogens, the genes and proteins involved in virulence are known to be controlled by environmental pH. Here, we report the influence of culture-medium pH (5, 6, 7, and 8) on the production of degradative enzymes involved in the pathogenesis of F. oxysporum URM 7401 and on the 2D-electrophoresis profile of intracellular proteins in this fungus. F. oxysporum URM 7401 was grown in acidic, neutral, and alkaline culture media in a submerged bioprocess. After 96 hr, the crude extract was processed to enzyme activity assays, while the intracellular proteins were obtained from mycelium and analyzed using 2D electrophoresis and mass spectrometry. We note that the diversity of secreted enzymes was changed quantitatively in different culture-medium pH. Also, the highest accumulated biomass and the intracellular protein profile of F. oxysporum URM 7401 indicate an increase in metabolism in neutral-alkaline conditions. The differential profiles of secreted enzymes and intracellular proteins under the evaluated conditions indicate that the global protein content in F. oxysporum URM 7401 is modulated by extracellular pH.

Stathmin involvement in the maternal embryonic leucine zipper kinase pathway in glioblastoma.

BACKGROUND: Maternal Embryonic Leucine Zipper Kinase (MELK) is a serine/threonine kinase involved in cell cycle, differentiation, proliferation, and apoptosis. These multiple features are consistent with it being a potential anticancer target. Nevertheless, the MELK pathway in tumorigenesis is not yet completely understood. This study aims to identify proteins associated with MELK pathway in astrocytomas. To this end, proteomic data of the human glioma cell line U87MG transfected with siRNA for MELK were compared with non-target transfected control cells and compared with oligonucleotide microarray data. RESULTS: In both assays, we identified stathmin/oncoprotein 18 (STMN1), involved in cell cycle. STMN1 gene expression was further assessed in a series of 154 astrocytomas and 22 non-neoplastic brain samples by qRT-PCR. STMN1 expression was significantly increased in malignant diffusely infiltrative astrocytomas compared with pilocytic astrocytoma (p < 0.0001). A strong correlation between MELK and STMN1 expressions was observed (r = 0.741, p < 0.0001) in glioblastoma (GBM) samples. However, no difference on survival times was found when compared GBM cases with upregulated and downregulated STMN1 (Breslow = 0.092, median survival time: 11 and 13 months, respectively). Functional assays knocking down MELK by siRNA in GBM cell line showed that gene and protein expression of both MELK and stathmin were diminished. On the other hand, when the same analysis was performed for STMN1, only stathmin gene and protein was silenced. CONCLUSIONS: The results presented herein point stahtmin as a downstream target in the MELK pathway that plays a role in malignant progression of astrocytomas.

The effects of photodynamic treatment with new methylene blue N on the Candida albicans proteome.

Candida albicans is a human pathogenic fungus mainly affecting immunocompromised patients. Resistance to the commonly used fungicides can lead to poor treatment of mucosal infections which, in turn, can result in life-threatening systemic candidiasis. In this scenario, antimicrobial photodynamic treatment (PDT) has emerged as an effective alternative to treat superficial and localized fungal infections. Microbial death in PDT is a consequence of the oxidation of many cellular biomolecules, including proteins. Here, we report a combination of two-dimensional electrophoresis and tandem mass spectrometry to study the protein damage resulting from treating C. albicans with PDT with new methylene blue N and red light. Two-dimensional gels of treated cells showed an increase in acidic spots in a fluence-dependent manner. Amino acid analysis revealed a decrease in the histidine content after PDT, which is one plausible explanation for the observed acidic shift. However, some protein spots remained unchanged. Protein identification by mass spectrometry revealed that both modified and unmodified proteins could be localized to the cytoplasm, ruling out subcellular location as the only explanation for damage selectivity. Therefore, we hypothesize that protein modification by PDT is a consequence of both photosensitizer binding affinity and the degree of exposure of the photooxidizable residues on the protein surface.

PROTEINS OF THE INTEGUMENTARY SYSTEM OF THE HONEYBEE, Apis mellifera.

The integument of insects and other arthropods is composed of an inner basal lamina coated by the epidermis, which secretes the bulk of the outer integument layer, the cuticle. The genome sequencing of several insect species has allowed predicting classes of proteins integrating the cuticle. However, only a small proportion of them, as well as other proteins in the integumentary system, have been validated. Using two-dimensional gel electrophoresis coupled with mass spectrometry, we identified 45 different proteins in a total of 112 selected gel spots derived from thoracic integument samples of developing honeybee workers, including 14 cuticular proteins (AmelCPR 3, AmelCPR 12, AmelCPR 16, AmelCPR 27, apidermin 2, apidermin 3, endocuticle structural glycoprotein SgAbd-8-like, LOC100577363, LOC408365, LOC413679, LOC725454, LOC100576916, LOC725838, and peritrophin 3-C analogous). Gene ontology functional analysis revealed that the higher proportions of the identified proteins have molecular functions related to catalytic and structural molecule activities, are involved in metabolic biological processes, and pertain to the protein class of structural or cytoskeletal proteins and hydrolases. It is noteworthy that 26.7% of the identified proteins, including five cuticular proteins, were revealed as protein species resulting from allelic isoforms or derived from posttranslational modifications. Also, 66.7% of the identified cuticular proteins were expressed in more than one developmental phase, thus indicating that they are part of the larval, pupal, and adult cuticle. Our data provide experimental support for predicted honeybee gene products and new information on proteins expressed in the developing integument.

A brain proteome profile in rats exposed to methylmercury or thimerosal (ethylmercury).

Exposure to organomercurials has been associated with harmful effects on the central nervous system (CNS). However, the mechanisms underlying organomercurial-mediated neurotoxic effects need to be elucidated. Exposure to toxic elements may promote cellular modifications such as alterations in protein synthesis in an attempt to protect tissues and organs from damage. In this context, the use of a "proteomic profile" is an important tool to identify potential early biomarkers or targets indicative of neurotoxicity. The aim of this study was to investigate potential modifications in rat cerebral cell proteome following exposure to methylmercury (MeHg) or ethylmercury (EtHg). For MeHg exposure, animals were administered by gavage daily 140 µg/kg/d of Hg (as MeHg) for 60 d and sacrificed 24 h after the last treatment. For EtHg exposure, 800 µg/kg/d of Hg (as EtHg) was given intramuscularly (im) in a single dose and rats were sacrificed after 4 h. Control groups received saline either by gavage or im. After extraction of proteins from whole brain samples and separation by two-dimensional electrophoresis (2-DE), 26 differentially expressed proteins were identified from exposed animals by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF/TOF). Both MeHg and EtHg exposure induced an overexpression of calbindin, a protein that acts as a neuroprotective agent by (1) adjusting the concentration of Ca(2+) within cells and preventing neurodegenerative diseases and (2) decreasing expression of glutamine synthetase, a crucial protein involved in regulation of glutamate concentration in synaptic cleft. In contrast, expression of superoxide dismutase (SOD), a protein involved in antioxidant defense, was elevated in brain of MeHg-exposed animals. Taken together, our data provide new valuable information on the possible molecular mechanisms associated with MeHg- and EtHg-mediated toxicity in cerebral tissue. These observed protein alterations may be considered as biomarkers candidates for biological monitoring of organomercurial poisoning.

Secretome Analysis of Metarhizium anisopliae Under Submerged Conditions Using Bombyx mori Chrysalis to Induce Expression of Virulence-Related Proteins.

The entomopathogenic fungus Metarhizium anisopliae is used to control insect pests. This species is specialized for the secretion of an enzymatic complex consisting of proteases, lipases, and chitinases related to pathogenicity and virulence. In this context, the secretomes of strains IBCB 167 and IBCB 384 of M. anisopliae var. anisopliae, grown under submerged fermentation in the presence of chrysalis as an inducer, were analyzed. Analysis of two-dimensional gels showed qualitative and quantitative differences between secreted proteins in both isolates. Around 102 protein spots were analyzed, and 76 % of the corresponding proteins identified by mass spectrometry were grouped into different classes (hydrolases, oxidases, reductases, isomerases, kinases, WSC domains, and hypothetical proteins). Thirty-three per cent of all the proteins analyzed were found to be common in both strains. Several virulence-related proteins were identified as proteases and mannosidases. Endo-N-acetyl-ß-D-glucosaminidase expression was observed to be 10.14-fold higher for strain IBCB 384 than for strain IBCB 167, which may be an important contributor to the high virulence of IBCB 384 in Diatraea ssaccharalis. These results are important for elucidation of the host-pathogen relationship and the differences in virulence observed between the two strains.

Quantitative proteomic analysis shows differentially expressed HSPB1 in glioblastoma as a discriminating short from long survival factor and NOVA1 as a differentiation factor between low-grade astrocytoma and oligodendroglioma.

BACKGROUND: Gliomas account for more than 60 % of all primary central nervous system neoplasms. Low-grade gliomas display a tendency to progress to more malignant phenotypes and the most frequent and malignant gliomas are glioblastomas (GBM). Another type of glioma, oligodendroglioma originates from oligodendrocytes and glial precursor cells and represents 2-5 % of gliomas. The discrimination between these two types of glioma is actually controversial, thus, a molecular distinction is necessary for better diagnosis. METHODS: iTRAQ-based quantitative proteomic analysis was performed on non-neoplastic brain tissue, on astrocytoma grade II, glioblastoma with short and long survival and oligodendrogliomas. RESULTS: We found that expression of nucleophosmin (NPM1), glucose regulated protein 78 kDa (GRP78), nucleolin (NCL) and heat shock protein 90 kDa (HSP90B1) were increased, Raf kinase inhibitor protein (RKIP/PEBP1) was decreased in glioblastoma and they were associated with a network related to tumor progression. Expression level of heat shock protein 27 (HSPB1/HSP27) discriminated glioblastoma presenting short (6 ± 4 months, n = 4) and long survival (43 ± 15 months, n = 4) (p = 0.00045). Expression level of RNA binding protein nova 1 (NOVA1) differentiated low-grade oligodendroglioma and astrocytoma grade II (p = 0.0082). Validation were done by Western blot, qRT-PCR and immunohistochemistry in a larger casuistry. CONCLUSION: Taken together, our quantitative proteomic analysis detected the molecular triad, NPM1, GRP78 and RKIP participating together with NCL and HSP27/HSPB1 in a network related to tumor progression. Additionally, two new important targets were uncovered: NOVA1 useful for diagnostic refinement differentiating astrocytoma from oligodendroglioma, and HSPB1/HSP27, as a predictive factor of poor prognosis for GBM.

Gender-specific effects of intrauterine growth restriction on the adipose tissue of adult rats: a proteomic approach.

BACKGROUND: Intrauterine growth restriction (IUGR) may program metabolic alterations affecting physiological functions and lead to diseases in later life. The adipose tissue is an important organ influencing energy homeostasis. The present study was aimed at exploring the consequences of IUGR on the retroperitoneal adipose tissue of adult male and female rats, using a proteomic approach. METHODS AND RESULTS: Pregnant Wistar rats were fed with balanced chow, either ad libitum (control group) or restricted to 50 % of control intake (restricted group) during the whole gestation. The offspring were weaned to ad libitum chow and studied at 4 months of age. Retroperitoneal fat was analyzed by two-dimensional gel electrophoresis followed by mass spectrometry. Both male and female restricted groups had low body weight at birth and at weaning but normal body weight at adulthood. The restricted males had normal fat pads weight and serum glucose levels, with a trend to hyperinsulinemia. The restricted females had increased fat pads weight with normal glucose and insulin levels. The restricted males showed up-regulated levels of proteasome subunit α type 3, branched-chain-amino-acid aminotransferase, elongation 1- alpha 1, fatty acid synthase levels, cytosolic malate dehydrogenase and ATP synthase subunit alpha. These alterations point to increased proteolysis and lipogenesis rates and favoring of ATP generation. The restricted females showed down-regulated levels of L-lactate dehydrogenase perilipin-1, mitochondrial branched-chain alpha-keto acid dehydrogenase E1, and transketolase. These findings suggest impairment of glycemic control, stimulation of lipolysis and inhibition of proteolysis, pentose phosphate pathway and lipogenesis rates. In both genders, several proteins involved in oxidative stress and inflammation were affected, in a pattern compatible with impairment of these responses. CONCLUSIONS: The proteomic analysis of adipose tissue showed that, although IUGR affected pathways of substrate and energy metabolism in both males and females, important gender differences were evident. While IUGR males displayed alterations pointing to a predisposition to later development of obesity, the alterations observed in IUGR females pointed to a metabolic status of established obesity, in agreement with their increased fat pads mass.

Acerola (Malpighia emarginata DC.) juice intake protects against alterations to proteins involved in inflammatory and lipolysis pathways in the adipose tissue of obese mice fed a cafeteria diet.

BACKGROUND: Obesity has been studied as a metabolic and an inflammatory disease and is characterized by increases in the production of pro-inflammatory adipokines in the adipose tissue.To elucidate the effects of natural dietary components on the inflammatory and metabolic consequences of obesity, we examined the effects of unripe, ripe and industrial acerola juice (Malpighia emarginata DC.) on the relevant inflammatory and lipolysis proteins in the adipose tissue of mice with cafeteria diet-induced obesity. MATERIALS/METHODS: Two groups of male Swiss mice were fed on a standard diet (STA) or a cafeteria diet (CAF) for 13 weeks. Afterwards, the CAF-fed animals were divided into five subgroups, each of which received a different supplement for one further month (water, unripe acerola juice, ripe acerola juice, industrial acerola juice, or vitamin C) by gavage. Enzyme-linked immunosorbent assays, Western blotting, a colorimetric method and histology were utilized to assess the observed data. RESULTS: The CAF water (control obese) group showed a significant increase in their adiposity indices and triacylglycerol levels, in addition to a reduced IL-10/TNF-α ratio in the adipose tissue, compared with the control lean group. In contrast, acerola juice and Vitamin C intake ameliorated the weight gain, reducing the TAG levels and increasing the IL-10/TNF-α ratio in adipose tissue. In addition, acerola juice intake led to reductions both in the level of phosphorylated JNK and to increases in the phosphorylation of IκBα and HSLser660 in adipose tissue. CONCLUSIONS: Taken together, these results suggest that acerola juice reduces low-grade inflammation and ameliorates obesity-associated defects in the lipolytic processes.

Peptide structure modifications: effect of radical species generated by controlled gamma ray irradiation approach.

The present work aimed at evaluating the radiolysis effect upon a set of peptides, most of them involved in physiological functions. To generate reactive radical species, a Co(60) source (up to 15 kGy) was used for controlled gamma irradiation of some peptide solutions including derivatives attaching the stable free radical Toac (2,2,6,6-tetramethypiperidine-1-oxyl-4-amino-4-carboxylic acid). Regardless of the peptide sequence, a nonlinear and progressive degradation of a total of nine peptides was detected. The results were interpreted in the light of the half-life dose (D(1/2)) parameter which represents the dose necessary for 50% peptide structure degradation. The vasoactive angiotensin II (AngII)'s analogue Ang-(1-7) showed greater stability towards gamma ray radiation than bradykinin (BK), Toac(0)-BK, Pro(4)-BK (D(1/2) around 4 and 2 kGy, respectively) which decreased to about 0.5-1.0 kGy in the case of acetyl-α-melanocyte-stimulating hormone (Ac-α-MSH) and substance P (SP). In terms of peptide structural modifications, the data acquired from different analytical methods suggested a Phe to Tyr (or its ortho and/or meta isomers) transformation as a consequence of the hydroxyl moiety insertion. Noteworthy, this effect seemed to be position-dependent as only Phe located at or near the C-terminal portion seemed to display this transformation. In contrast, Met is comparatively more easily oxidized, thus allowing to conclude that gamma irradiation may induce a complex position and/or sequence-dependent effect on peptides. As previously applied for BK, some irradiated peptides were submitted to their by-products purification, indeed a complementary target of the present approach for development of uncommon analogues for further structure-function investigation.

Quantitative proteomic analysis and functional studies reveal that nucleophosmin is involved in cell death in glioblastoma cell line transfected with siRNA.

Previously, we reported that nucleophosmin (NPM) was increased in glioblastoma multiforme (GBM). NPM is a phosphoprotein related to apoptosis, ribosome biogenesis, mitosis, and DNA repair, but details about its function remain unclear. We treated U87MG and A172 cells with small interference RNA (siRNA) and obtained a reduction of 80% in NPM1 expression. Knockdown at the protein level was evident after the 4th day and was maintained until the 7th day of transfection that was investigated by quantitative proteomic analysis using isobaric tags. The comparison of proteomic analysis of NPM1-siRNA against controls allowed the identification of 14 proteins, two proteins showed increase and 12 presented a reduction of expression levels. Gene ontology assigned most of the hypoexpressed proteins to apoptosis regulation, including GRP78. NPM1 silencing did not impair cell proliferation until the 7th day after transfection, but sensitized U87MG cells to temozolomide (TMZ), culminating with an increase in cell death and provoking at a later period a reduction of colony formation. In a large data set of GBM patients, both GRP78 and NPM1 genes were upregulated and presented a tendency to shorter overall survival time. In conclusion, NPM proved to participate in the apoptotic process, sensitizing TMZ-treated U87MG and A172 cells to cell death, and in association with upregulation of GRP78 may be helpful as a predictive factor of poor prognosis in GBM patients.

Both adiponectin and interleukin-10 inhibit LPS-induced activation of the NF-κB pathway in 3T3-L1 adipocytes.

Adiponectin and interleukin 10 (IL-10) are adipokines that are predominantly secreted by differentiated adipocytes and are involved in energy homeostasis, insulin sensitivity, and the anti-inflammatory response. These two adipokines are reduced in obese subjects, which favors increased activation of nuclear factor kappa B (NF-κB) and leads to elevation of pro-inflammatory adipokines. However, the effects of adiponectin and IL-10 on NF-κB DNA binding activity (NF-κBp50 and NF-κBp65) and proteins involved with the toll-like receptor (TLR-2 and TLR-4) pathway, such as MYD88 and TRAF6 expression, in lipopolysaccharide-treated 3T3-L1 adipocytes are unknown. Stimulation of lipopolysaccharide-treated 3T3-L1 adipocytes for 24h elevated IL-6 levels; activated the NF-κB pathway cascade; increased protein expression of IL-6R, TLR-4, MYD88, and TRAF6; and increased the nuclear activity of NF-κB (p50 and p65) DNA binding. Adiponectin and IL-10 inhibited the elevation of IL-6 levels and activated NF-κB (p50 and p65) DNA binding. Taken together, the present results provide evidence that adiponectin and IL-10 have an important role in the anti-inflammatory response in adipocytes. In addition, inhibition of NF-κB signaling pathways may be an excellent strategy for the treatment of inflammation in obese individuals.

Changes in the expression of proteins associated with aerobic glycolysis and cell migration are involved in tumorigenic ability of two glioma cell lines.

BACKGROUND: The most frequent and malignant brain cancer is glioblastoma multiforme (GBM). In gliomas, tumor progression and poor prognosis are associated with the tumorigenic ability of the cells. U87MG cells (wild-type p53) are known to be tumorigenic in nude mice, but T98G cells (mutant p53) are not tumorigenic. We investigated the proteomic profiling of these two cell lines in order to gain new insights into the mechanisms that may be involved in tumorigenesis. RESULTS: We found 24 differentially expressed proteins between T98G and U87MG cells. Gene Ontology supports the notion that over-representation of differentially expressed proteins is involved in glycolysis, cell migration and stress oxidative response. Among those associated with the glycolysis pathway, TPIS and LDHB are up-regulated in U87MG cells. Measurement of glucose consumption and lactate production suggests that glycolysis is more effective in U87MG cells. On the other hand, G6PD expression was 3-fold higher in T98G cells and this may indicate a shift to the pentose-phosphate pathway. Moreover, GRP78 expression was also three-fold higher in T98G than in U87MG cells. Under thapsigargin treatment both cell lines showed increased GRP78 expression and the effect of this agent was inversely correlated to cell migration. Quantitative RT-PCR and immunohistochemistry of GRP78 in patient samples indicated a higher level of expression of GRP78 in grade IV tumors compared to grade I and non-neoplastic tissues, respectively. CONCLUSIONS: Taken together, these results suggest an important role of proteins involved in key functions such as glycolysis and cell migration that may explain the difference in tumorigenic ability between these two glioma cell lines and that may be extrapolated to the differential aggressiveness of glioma tumors.

Insights into structure and function of CdcVEGFs, the vascular endothelial growth factor from Crotalus durissus collilineatus snake venom.

Vascular endothelial growth factors (VEGFs) are crucial molecules involved in the modulation of angiogenesis. Snake venom-derived VEGFs (svVEGFs) are known to contribute significantly to the envenoming due to their capacity of increasing vascular permeability. In our work, we isolated and analyzed the biochemical and functional properties of the VEGF from Crotalus durissus collilineatus venom (CdcVEGF). The venom was fractionated by reversed phase chromatography on FPLC system (Fast Protein Liquid Chromatography) and the eluted fractions were submitted to an ELISA assay using an anti-VEGF-F antibody, for identification of svVEGF. Positive fractions for svVEGF were submitted to SDS-PAGE and to an anion exchange chromatography to isolate the molecule. The subfractions were analyzed by ELISA and SDS-PAGE and six of them presented svVEGFs, named CdcVEGF1 (Q23-3), CdcVEGF2 (Q24-3), CdcVEGF3 (Q24-4), CdcVEGF4 (Q25-3), CdcVEGF5 (Q25-4), and CdcVEGF6 (Q25-5). Their structural characterization was accomplished by mass spectrometry analysis using MALDI-TOF to determine their molecular masses and UPLC-ESI-QTOF to determine their amino acid sequence. Interestingly, all isolated CdcVEGFs induced angiogenesis on HUVEC cells through tube formation on Matrigel when compared to culture medium (negative control). Moreover, CdcVEGF2 and CdcVEGF3 also induced a significant increase in tube formation when compared to the positive control (basic fibroblast growth factor - bFGF). Additionally, crotalid antivenom produced by the Instituto Butantan was able to recognize CdcVEGFs, demonstrating to be immunogenic. This study demonstrates that snake venom cocktail can reveal novel and important molecules, which are potential molecular tools to study diverse biological processes, such as angiogenesis.

Isolation, functional, and partial biochemical characterization of galatrox, an acidic lectin from Bothrops atrox snake venom.

Snake venom lectins have been studied in regard to their chemical structure and biological functions. However, little is known about lectins isolated from Bothrops atrox snake venom. We report here the isolation and partial functional and biochemical characterization of an acidic glycan-binding protein called galatrox from this venom. This lectin was purified by affinity chromatography using a lactosyl-sepharose column, and its homogeneity and molecular mass were evaluated by high-performance liquid chromatography, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry. The purified galatrox was homogeneous and characterized as an acidic protein (pI 5.2) with a monomeric and dimeric molecular mass of 16.2 and 32.5 kDa, respectively. Alignment of N-terminal and internal amino acid sequences of galatrox indicated that this protein exhibits high homology to other C-type snake venom lectins. Galatrox showed optimal hemagglutinating activity at a concentration of 100 µg/ml and this effect was drastically inhibited by lactose, ethylenediaminetetraacetic acid, and heating, which confirmed galatrox's lectin activity. While galatrox failed to induce the same level of paw edema or mast cell degranulation as B. atrox crude venom, galatrox did alter cellular viability, which suggested that galatrox might contribute to venom toxicity by directly inducing cell death.

Proteomic analysis of low- to high-grade astrocytomas reveals an alteration of the expression level of raf kinase inhibitor protein and nucleophosmin.

Proteomic approaches have been useful for the identification of aberrantly expressed proteins in complex diseases such as cancer. These proteins are not only potential disease biomarkers, but also targets for therapy. The aim of this study was to identify differentially expressed proteins in diffuse astrocytoma grade II, anaplastic astrocytoma grade III and glioblastoma multiforme grade IV in human tumor samples and in non-neoplastic brain tissue as control using 2-DE and MS. Tumor and control brain tissue dissection was guided by histological hematoxylin/eosin tissue sections to provide more than 90% of tumor cells and astrocytes. Six proteins were detected as up-regulated in higher grade astrocytomas and the most important finding was nucleophosmin (NPM) (p<0.05), whereas four proteins were down-regulated, among them raf kinase inhibitor protein (RKIP) (p<0.05). We report here for the first time the alteration of NPM and RKIP expression in brain cancer. Our focus on these proteins was due to the fact that they are involved in the PI3K/AKT/mTOR and RAS/RAF/MAPK pathways, known for their contribution to the development and progression of gliomas. The proteomic data for NPM and RKIP were confirmed by Western blot, quantitative real-time PCR and immunohistochemistry. Due to the participation of NPM and RKIP in uncontrolled proliferation and evasion of apoptosis, these proteins are likely targets for drug development.