Argininosuccinate Synthetase Is a Functional Target for a Snake Venom Anti-hypertensive Peptide ROLE IN ARGININE AND NITRIC OXIDE PRODUCTION

Bj-BPP-10c is a bioactive proline-rich decapeptide, part of the C-type natriuretic peptide precursor, expressed in the brain and in the venom gland of Bothrops jararaca. We recently showed that Bj-BPP-10c displays a strong, sustained anti-hypertensive effect in spontaneous hypertensive rats (SHR), without causing any effect in normotensive rats, by a pharmacological effect independent of angiotensin-converting enzyme inhibition. Therefore, we hypothesized that another mechanism should be involved in the peptide activity. Here we used affinity chromatography to search for kidney cytosolic proteins with affinity for Bj-BPP-10c and demonstrate that argininosuccinate synthetase (AsS) is the major protein binding to the peptide. More importantly, this interaction activates the catalytic activity of AsS in a dose-dependent manner. AsS is recognized as an important player of the citrulline-NO cycle that represents a potential limiting step in NO synthesis. Accordingly, the functional interaction of Bj-BPP-10c and AsS was evidenced by the following effects promoted by the peptide: (i) increase of NO metabolite production in human umbilical vein endothelial cell culture and of arginine in human embryonic kidney cells and (ii) increase of arginine plasma concentration in SHR. Moreover, alpha-methyl-DL-aspartic acid, a specific AsS inhibitor, significantly reduced the anti-hypertensive activity of Bj-BPP-10c in SHR. Taken together, these results suggest that AsS plays a role in the anti-hypertensive action of Bj-BPP-10c. Therefore, we propose the activation of AsS as a new mechanism for the anti-hypertensive effect of Bj-BPP-10c in SHR and AsS as a novel target for the therapy of hypertension-related diseases.

HPV16 Tumor Associated Macrophages Suppress Antitumor T Cell Responses

Purpose: High-risk human papillomavirus (HPV) is the main etiologic factor for cervical cancer. The severity of HPV-associated cervical lesions has been correlated to the number of infiltrating macrophages. The objective of this work is to characterize the role of tumor-associated macrophages (TAM) on the immune cellular response against the tumor. Experimental Design: We used the HPV16 E6- and E7-expressing TC-1 mouse tumor model to study the effect of TAM on T-cell function in vitro, and depleted TAM, using clodronate-containing liposomes, to characterize its role in vivo. Results: TAM, characterized by the positive expression of CD45, F4/80, and CD11b, formed the major population of infiltrating tumor cells. TAM displayed high basal Arginase I activity, producing interleukin-10 (IL-10); they were resistant to iNOSll activity induction, therefore reversion to M1 phenotype, when stimulated in vitro with lipopolysaccharide/IFN gamma, indicating an M2 phentoype. In cultures of isolated TAM, TAM induced regulatory phenotype, characterized by IL-10 and Foxp3 expression, and inhibited proliferation of CD8 lymphocytes. In vivo, depletion of TAM inhibited tumor growth and stimulated the infiltration of tumors by HPV16 E7(49-57)-specific CD8 lymphocytes, whereas depletion of Gr1(+) tumor-associated cells had no effect. Conclusions: M2-like macrophages infiltrate HPV16-associated tumors causing suppression of antitumor T-cell response, thus facilitating tumor growth. Depletion or phenotype alteration of this population should be considered in immunotherapy strategies.

Glypican-3 regulates migration, adhesion and actin cytoskeleton organization in mammary tumor cells through Wnt signaling modulation

Glypican-3 (GPC3) is a proteoglycan involved in migration, proliferation and cell survival modulation in several tissues. There are many reports demonstrating a downregulation of GPC3 expression in some human tumors, including mesothelioma, ovarian and breast cancer. Previously, we determined that GPC3 reexpression in the murine mammary adenocarcinoma LM3 cells induced an impairment of their in vivo invasive and metastatic capacities together with a higher susceptibility to in vitro apoptosis. Currently, the signaling mechanism of GPC3 is not clear. First, it was speculated that GPC3 regulates the insulin-like growth factor (IGF) signaling system. This hypothesis, however, has been strongly challenged. Recently, several reports indicated that at least in some cell types GPC3 serves as a selective regulator of Wnt signaling. Here we provide new data demonstrating that GPC3 regulates Wnt pathway in the metastatic adenocarcinoma mammary LM3 cell line. We found that GPC3 is able to inhibit canonical Wnt signals involved in cell proliferation and survival, as well as it is able to activate non canonical pathway, which directs cell morphology and migration. This is the first report indicating that breast tumor cell malignant properties can be reverted, at least in part, by GPC3 modulation of Wnt signaling. Our results are consistent with the potential role of GPC3 as a metastasis suppressor.

Identification of protein-coding and intronic noncoding RNAs down-regulated in clear cell renal carcinoma

The clear cell subtype of renal cell carcinoma (RCC) is the most lethal and prevalent cancer of the urinary system. To investigate the molecular changes associated with malignant transformation in clear cell RCC, the gene expression profiles of matched samples of tumor and adjacent non-neoplastic tissue were obtained from six patients. A custom-built cDNA microarray platform was used, comprising 2292 probes that map to exons of genes and 822 probes for noncoding RNAs mapping to intronic regions. Intronic transcription was detected in all normal and neoplastic renal tissues. A subset of 55 transcripts was significantly down-regulated in clear cell RCC relative to the matched nontumor tissue as determined by a combination of two statistical tests and leave-one-out patient cross-validation. Among the down-regulated transcripts, 49 mapped to untranslated or coding exons and 6 were intronic relative to known exons of protein-coding genes. Lower levels of expression of SIN3B, TRIP3, SYNJ2BP and NDE1 (P<0.02), and of intronic transcripts derived from SND1 and ACTN4 loci (P<0.05), were confirmed in clear cell RCC by Real-time RT-PCR. A subset of 25 transcripts was deregulated in additional six nonclear cell RCC samples, pointing to common transcriptional alterations in RCC irrespective of the histological subtype or differentiation state of the tumor. Our results indicate a novel set of tumor suppressor gene candidates, including noncoding intronic RNAs, which may play a significant role in malignant transformations of normal renal cells. (C) 2008 Wiley-Liss, Inc.

A two-component system, an anti-sigma factor and two paralogous ECF sigma factors are involved in the control of general stress response in Caulobacter crescentus

The extracytoplasmic function sigma factor sigma(T) is the master regulator of general stress response in Caulobacter crescentus and controls the expression of its paralogue sigma(U). In this work we showed that PhyR and NepR act, respectively, as positive and negative regulators of sigma(T) expression and function. Biochemical data also demonstrated that NepR directly binds sigma(T) and the phosphorylated form of PhyR. We also described the essential role of the histidine kinase gene CC3474, here denominated phyK, for expression of sigma(T)-dependent genes and for resistance to stress conditions. Additionally, in vivo evidence of PhyK-dependent phosphorylation of PhyR is presented. This study also identified a conserved cysteine residue (C95) located in the periplasmic portion of PhyK that is crucial for the function of the protein. Furthermore, we showed that PhyK, PhyR and sigma(T) regulate the same set of genes and that sigma(T) apparently directly controls most of its regulon. In contrast, sigma(U) seems to have a very modest contribution to the expression of a subset of sigma(T)-dependent genes. In conclusion, this report describes the molecular mechanism involved in the control of general stress response in C. crescentus.

Tempol ameliorates murine viral encephalomyelitis by preserving the blood-brain barrier, reducing viral load, and lessening inflammation

Multiple sclerosis (MS) is a progressive inflammatory and/or demyelinating disease of the human central nervous system (CNS). Most of the knowledge about the pathogenesis of MS has been derived from murine models, such as experimental autoimmune encephalomyelitis and vital encephalomyelitis. Here, we infected female C57BL/6 mice with a neurotropic strain of the mouse hepatitis virus (MHV-59A) to evaluate whether treatment with the multifunctional antioxidant tempol (4-hydroxy-2,2,6,6-tetramethyl-1-piperidinyloxy) affects the ensuing encephalomyelitis. In untreated animals, neurological symptoms developed quickly: 90% of infected mice died 10 days after virus inoculation and the few survivors presented neurological deficits. Treatment with tempol (24 mg/kg, ip, two doses on the first day and daily doses for 7 days plus 2 mM tempol in the drinking water ad libitum) profoundly altered the disease outcome: neurological symptoms were attenuated, mouse survival increased up to 70%, and half of the survivors behaved as normal mice. Not Surprisingly, tempol substantially preserved the integrity of the CNS, including the blood-brain barrier. Furthermore, treatment with tempol decreased CNS vital titers, macrophage and T lymphocyte infiltration, and levels of markers of inflammation, such as expression of inducible nitric oxide synthase, transcription of tumor necrosis factor-alpha and interferon-gamma, and protein nitration. The results indicate that tempol ameliorates murine viral encephalomyelitis by altering the redox status of the infectious environment that contributes to an attenuated CNS inflammatory response. overall, our study supports the development of therapeutic strategies based on nitroxides to manage neuroinflammatory diseases, including MS. (C) 2009 Elsevier Inc. All rights reserved.

Arginine vasopressin controls p27(KiP1) protein expression by PKC activation and irreversibly inhibits the proliferation of K-Ras-dependent mouse Y1 adrenocortical malignant cells

The neurohypophyseal hormone arginine vasopressin (AVP) is a classic mitogen in many cells. In K-Ras-dependent mouse Y1 adrenocortical malignant cells, AVP elicits antagonistic responses such as the activation of the PKC and the ERK1/2 mitogenic pathways to down-regulate cyclin D1 gene expression, which induces senescence-associated beta-galactosidase (SA-beta Gal) and leads to cell cycle arrest. Here, we report that in the metabolic background of Y1 cells, PKC activation either by AVP or by PMA inhibits the PI3K/Akt pathway and stabilises the p27(Kip1) protein even in the presence of the mitogen fibroblast growth factor 2 (FGF2). These results suggest that p27(Kip1) is a critical signalling node in the mechanisms underlying the survival of the Y1 cells. In Y1 cells that transiently express wild-type p27(Kip1), AVP caused a severe reduction in cell survival, as shown by clonogenic assays. However, AVP promoted the survival of Y1 cells transiently expressing mutant p27-S10A or mutant p27-T187A, which cannot be phosphorylated at Ser10 and Thr187, respectively. In addition, PKC activation by PMA mimics the toxic effect caused by AVP in Y1 cells, and inhibition of PKC completely abolishes the effects caused by both PMA and AVP in clonogenic assays. The vulnerability of Y1 cells during PKC activation is a phenotype conditioned upon K-ras oncogene amplification because K-Ras down-regulation with an inducible form of the dominant-negative mutant H-RasN17 has resulted in Y1 cells that are resistant to AVP`s deleterious effects. These data show that the survival destabilisation of K-Ras-dependent Y1 malignant cells by AVP requires large quantities of the p27(Kip1) protein as well as phosphorylation of the p27(Kip1) protein at both Ser10 and Thr187. (C) 2011 Elsevier B.V. All rights reserved.

The isatin-Schiff base copper(II) complex Cu(isaepy)(2) acts as delocalized lipophilic cation, yields widespread mitochondrial oxidative damage and induces AMP-activated protein kinase-dependent apoptosis

We previously demonstrated that Bis[(2-oxindol-3-ylimino)-2-(2-aminoethyl) pyridine-N, N`] copper(II) [Cu(isaepy)(2)] was an efficient inducer of the apoptotic mitochondrial pathway. Here, we deeply dissect the mechanisms underlying the ability of Cu(isaepy)(2) to cause mitochondriotoxicity. In particular, we demonstrate that Cu(isaepy)(2) increases NADH-dependent oxygen consumption of isolated mitochondria and that this phenomenon is associated with oxy-radical production and insensitive to adenosine diphosphate. These data indicate that Cu(isaepy)(2) behaves as an uncoupler and this property is also confirmed in cell systems. Particularly, SH-SY5Y cells show: (i) an early loss of mitochondrial transmembrane potential; (ii) a decrease in the expression levels of respiratory complex components and (iii) a significant adenosine triphosphate (ATP) decrement. The causative energetic impairment mediated by Cu(isaepy)(2) in apoptosis is confirmed by experiments carried out with rho(0) cells, or by glucose supplementation, where cell death is significantly inhibited. Moreover, gastric and cervix carcinoma AGS and HeLa cells, which rely most of their ATP production on oxidative phosphorylation, show a marked sensitivity toward Cu(isaepy)(2). Adenosine monophosphate-activated protein kinase (AMPK), which is activated by events increasing the adenosine monophosphate: ATP ratio, is deeply involved in the apoptotic process because the overexpression of its dominant/negative form completely abolishes cell death. Upon glucose supplementation, AMPK is not activated, confirming its role as fuel-sensing enzyme that positively responds to Cu(isaepy)(2)-mediated energetic impairment by committing cells to apoptosis. Overall, data obtained indicate that Cu(isaepy)(2) behaves as delocalized lipophilic cation and induces mitochondrial-sited reactive oxygen species production. This event results in mitochondrial dysfunction and ATP decrease, which in turn triggers AMPK-dependent apoptosis.

Volsurf Descriptors to Analyse Anti-HCV and Cytotoxic Activities of Sesquiterpene Lactones from Asteraceae Family

Various significant anti-HCV and cytotoxic sesquiterpene lactones (SLs) have been characterized. In this work, the chemometric tool Principal Component Analysis (PCA) was applied to two sets of SLs and the variance of the biological activity was explored. The first principal component accounts for as much of the variability in the data as possible, and each succeeding component accounts for as much of the remaining variability as possible. The calculations were performed using VolSurf program. For anti-HCV activity, PC1 (First Principal Component) explained 30.3% and PC2 (Second Principal Component) explained 26.5% of matrix total variance, while for cytotoxic activity, PC1 explained 30.9% and PC2 explained 15.6% of the total variance. The formalism employed generated good exploratory and predictive results and we identified some structural features, for both sets, important to the suitable biological activity and pharmacokinetic profile.

Nasal immunization of mice with Lactobacillus casei expressing the Pneumococcal Surface Protein A: induction of antibodies, complement deposition and partial protection against Streptococcus pneumoniae challenge

Strategies for the development of new vaccines against Streptococcus pneumoniae infections try to overcome problems such as serotype coverage and high costs, present in currently available vaccines. Formulations based on protein candidates that can induce protection in animal models have been pointed as good alternatives. Among them, the Pneumococcal Surface Protein A (PspA) plays an important role during systemic infection at least in part through the inhibition of complement deposition on the pneumococcal surface, a mechanism of evasion from the immune system. Antigen delivery systems based on live recombinant lactic acid bacteria (LAB) represents a promising strategy for mucosal vaccination, since they are generally regarded as safe bacteria able to elicit both systemic and mucosal immune responses. In this work, the N-terminal region of clade I PspA was constitutively expressed in Lactobacillus casei and the recombinant bacteria was tested as a mucosal vaccine in mice. Nasal immunization with L. casei-PspA 1 induced anti-PspA antibodies that were able to bind to pneumococcal strains carrying both clade 1 and clade 2 PspAs and to induce complement deposition on the surface of the bacteria. In addition, an increase in survival of immunized mice after a systemic challenge with a virulent pneumococcal strain was observed. (C) 2008 Elsevier Masson SAS. All rights reserved.

Expression, Purification, Bioactivity, and Partial Characterization of a Recombinant Human Bone Morphogenetic Protein-7 Produced in Human 293T Cells

Bone morphogenetic protein-7 (BMP-7) is a secreted multifunctional growth factor of the TGF-beta superfamily, which is predominantly known for its osteoinductive properties and emerging potential for treatment of kidney diseases. The mature 34-38 kDa disulfide-linked homodimer protein plays a key role in the differentiation of mesenchymal cells into bone and cartilage. In this study, the full-length sequence of hBMP-7 was amplified and, then, cloned, expressed, and purified from the conditioned medium of 293T cells stably transfected with a lentiviral vector. The mature protein dimer form was properly secreted and recognized by anti-BMP-7 antibodies, and the protein was shown to be glycosilated by treatment with exoglycosidase, followed by western blotting. Moreover, the activity of the purified protein was demonstrated both in vitro, by alkaline phosphatase activity in C2C12 cells, and in vivo by induction of ectopic bone formation in Balb/c Nude mice after 21 days, respectively. This recombinant protein platform may be very useful for expression of different human cytokines and other proteins for medical applications.

Expression of human papillomavirus type 16 E7 oncoprotein alters keratinocytes expression profile in response to tumor necrosis factor-alpha

Acute expression of E7 oncogene from human papillomavirus (HPV) 16 or HPV18 is sufficient to overcome tumor necrosis factor (TNF)-alpha cytostatic effect on primary human keratinocytes. In the present study, we investigated the molecular basis of E7-induced TNF resistance through a comparative analysis of the effect of this cytokine on the proliferation and global gene expression of normal and E7-expressing keratinocytes. Using E7 functional mutants, we show that E7-induced TNF resistance correlates with its ability to mediate pRb degradation and cell transformation. On the other hand, this effect does not depend on E7 sequences required to override DNA damage-induced cell cycle arrest or extend keratinocyte life span. Furthermore, we identified a group of 66 genes whose expression pattern differs between normal and E7-expressing cells upon cytokine treatment. These genes are mainly involved in cell cycle regulation suggesting that their altered expression may contribute to sustained cell proliferation even in the presence of a cytostatic stimulus. Differential expression of TCN1 (transcobalamin I), IFI44 (Interferon-induced protein 44), HMGB2 (high-mobility group box 2) and FUS [Fusion (involved in t(12; 16) in malignant liposarcoma)] among other genes were further confirmed by western-blot and/or real-time polymerase chain reaction. Moreover, FUS upregulation was detected in HPV-positive cervical high-grade squamous intraepithelial lesions when compared with normal cervical tissue. Further evaluation of the role of such genes in TNF resistance and HPVassociated disease development is warranted.

Overexpression of Nrp/b (nuclear restrict protein in brain) suppresses the malignant phenotype in the C6/ST1 glioma cell line

Upon searching for glucocorticoid-regulated cDNA sequences associated with the transformed to normal phenotypic reversion of C6/ST1 rat glioma cells, we identified Nrp/b (nuclear restrict protein in brain) as a novel rat gene. Here we report on the identification and functional characterization of the complete sequence encoding the rat NRP/B protein. The cloned cDNA presented a 1767 nucleotides open-reading frame encoding a 589 aminoacids residues sequence containing a BTB/POZ (broad complex Tramtrack bric-a-brac/Pox virus and zinc finger) domain in its N-terminal region and kelch motifs in its C-terminal region. Sequence analysis indicates that the rat Nrp/b displays a high level of identity with the equivalent gene orthologs from other organisms. Among rat tissues, Nrp/b expression is more pronounced in brain tissue. We show that overexpression of the Nrp/b cDNA in C6/ST1 cells suppresses anchorage independence in vitro and tumorigenicity in vivo, altering their malignant nature towards a more benign phenotype. Therefore, Nrp/b may be postulated as a novel tumor suppressorgene, with possible relevance for glioblastoma therapy. (C) 2009 Elsevier Ltd. All rights reserved.

Human regulatory protein Ki-1/57 has characteristics of an intrinsically unstructured protein

The human protein Ki-1/57 was first identified through the cross reactivity of the anti-CD30 monoclonal antibody Ki-1; in Hodgkin lymphoma cells. The expression of Ki-1/57 in diverse cancer cells and its phosphorylation in peripheral blood leukocytes after mitogenic activation suggested its possible role in cell signaling. Ki-1/57 interacts with several other regulatory proteins involved in cellular signaling, transcriptional regulation and RNA metabolism, suggesting it may have pleiotropic functions. In a previous spectroscopic analysis, we observed a low content of secondary structure for Ki-1/57 constructs. Here, Circular dichroism experiments, in vitro RNA binding analysis, and limited proteolysis assays of recombinant Ki-1/57(122-413) and proteolysis assays of endogenous full length protein from human HEK293 cells suggested that Ki-1/57 has characteristics of an intrinsically unstructured protein. Small-angle X-ray scattering (SAXS) experiments were performed with the C-terminal fragment Ki-1/57(122-413). These results indicated an elongated shape and a partially unstructured conformation of the molecule in solution, confirming the characteristics of an intrinsically unstructured protein. Experimental curves together with ab initio modeling approaches revealed an extended and flexible molecule in solution. An elongated shape was also observed by analytical gel filtration. Furthermore, sedimentation velocity analysis suggested that Ki-1/57 is a highly asymmetric protein. These findings may explain the functional plasticity of Ki-1/57, as suggested by the wide array of proteins with which it is capable of interacting in yeast two-hybrid interaction assays.