The Function and Three-Dimensional Structure of a Thromboxane A(2)/Cysteinyl Leukotriene-Binding Protein from the Saliva of a Mosquito Vector of the Malaria Parasite

The highly expressed D7 protein family of mosquito saliva has previously been shown to act as an anti-inflammatory mediator by binding host biogenic amines and cysteinyl leukotrienes (CysLTs). In this study we demonstrate that AnSt-D7L1, a two-domain member of this group from Anopheles stephensi, retains the CysLT binding function seen in the homolog AeD7 from Aedes aegypti but has lost the ability to bind biogenic amines. Unlike any previously characterized members of the D7 family, AnSt-D7L1 has acquired the important function of binding thromboxane A(2) (TXA(2)) and its analogs with high affinity. When administered to tissue preparations, AnSt-D7L1 abrogated Leukotriene C(4) (LTC(4))-induced contraction of guinea pig ileum and contraction of rat aorta by the TXA(2) analog U46619. The protein also inhibited platelet aggregation induced by both collagen and U46619 when administered to stirred platelets. The crystal structure of AnSt-D7L1 contains two OBP-like domains and has a structure similar to AeD(7). In AnSt-D7L1, the binding pocket of the C-terminal domain has been rearranged relative to AeD7, making the protein unable to bind biogenic amines. Structures of the ligand complexes show that CysLTs and TXA(2) analogs both bind in the same hydrophobic pocket of the N-terminal domain. The TXA(2) analog U46619 is stabilized by hydrogen bonding interactions of the omega-5 hydroxyl group with the phenolic hydroxyl group of Tyr 52. LTC(4) and occupies a very similar position to LTE(4) in the previously determined structure of its complex with AeD7. As yet, it is not known what, if any, new function has been acquired by the rearranged C-terminal domain. This article presents, to our knowledge, the first structural characterization of a protein from mosquito saliva that inhibits collagen mediated platelet activation.

Crystallization, data collection and data processing of maltose-binding protein (MalE) from the phytopathogen Xanthomonas axonopodis pv. citri

Maltose-binding protein is the periplasmic component of the ABC transporter responsible for the uptake of maltose/maltodextrins. The Xanthomonas axonopodis pv. citri maltose-binding protein MalE has been crystallized at 293 Kusing the hanging-drop vapour-diffusion method. The crystal belonged to the primitive hexagonal space group P6(1)22, with unit-cell parameters a = 123.59, b = 123.59, c = 304.20 angstrom, and contained two molecules in the asymetric unit. It diffracted to 2.24 angstrom resolution.

Genetic variability and natural selection at the ligand domain of the Duffy binding protein in brazilian Plasmodium vivax populations

Background: Plasmodium vivax malaria is a major public health challenge in Latin America, Asia and Oceania, with 130-435 million clinical cases per year worldwide. Invasion of host blood cells by P. vivax mainly depends on a type I membrane protein called Duffy binding protein (PvDBP). The erythrocyte-binding motif of PvDBP is a 170 amino-acid stretch located in its cysteine-rich region II (PvDBP(II)), which is the most variable segment of the protein. Methods: To test whether diversifying natural selection has shaped the nucleotide diversity of PvDBP(II) in Brazilian populations, this region was sequenced in 122 isolates from six different geographic areas. A Bayesian method was applied to test for the action of natural selection under a population genetic model that incorporates recombination. The analysis was integrated with a structural model of PvDBP(II), and T-and B-cell epitopes were localized on the 3-D structure. Results: The results suggest that: (i) recombination plays an important role in determining the haplotype structure of PvDBP(II), and (ii) PvDBP(II) appears to contain neutrally evolving codons as well as codons evolving under natural selection. Diversifying selection preferentially acts on sites identified as epitopes, particularly on amino acid residues 417, 419, and 424, which show strong linkage disequilibrium. Conclusions: This study shows that some polymorphisms of PvDBP(II) are present near the erythrocyte-binding domain and might serve to elude antibodies that inhibit cell invasion. Therefore, these polymorphisms should be taken into account when designing vaccines aimed at eliciting antibodies to inhibit erythrocyte invasion.

Sustained release carriers used to delivery bone morphogenetic proteins in the bone healing process

Bone morphogenetic proteins (BMPs) are multi-functional growth factors belonging to the transforming growth factor beta superfamily, especially BMP-2, induce bone formation in vivo, and clinical application in repair of bone fractures and defects is expected. However, appropriate systems to delivery BMPs for practical use need to be developed with the objective to heal cartilage and bone-related diseases in medical, dental and veterinary practice. Thus, the aim of this article was to present an overview of the principals carriers used to delivery BMPs and alternative delivery systems for these proteins.

Functional characterization of the putative Aspergillus nidulans DNA damage binding protein homologue DdbA

Nucleotide excision repair (NER) eliminates helix-distorting DNA base lesions. Seven XP-deficient genetic complementation groups (XPA to XPG) have already been identified in mammals, and their corresponding genes have been cloned. Hereditary defects in NER are associated with several diseases, including xeroderma pigmentosum (XP). UV-DDB (XPE) is formed by two associated subunits, DDB1 and DDB2. UV-DDB was identified biochemically as a protein factor that exhibits very strong and specific binding to ultraviolet (UV)-treated DNA. As a preliminary step to characterize the components of the NER in the filamentous fungus Aspergillus nidulans, here we identified a putative DDB1 homologue, DdbA. Deletion and expression analysis indicated that A. nidulans ddbA gene is involved in the DNA damage response, more specifically in the UV light response and 4-nitroquinoline oxide (4-NQO) sensitivity. Furthermore, the Delta ddbA strain cannot self-cross and expression analysis showed that ddbA can be induced by oxidative stress and is developmentally regulated in both asexual and sexual processes. The Delta ddbA mutation can genetically interact with uvsB(ATR), atmA(ATM), nkuA(KU70), H2AX-S129A (a replacement of the conserved serine in the C-terminal of H2AX with alanine), and cshB (a mutation in CSB Cockayne`s syndrome protein involved in the transcription-coupled repair subpathway of NER) mutations. Finally, to determine the DdbA cellular localization, we constructed a GFP:DdbA strain. In the presence and absence of DNA damage, DdbA was mostly detected in the nuclei, indicating that DdbA localizes to nuclei and its cellular localization is not affected by the cellular response to DNA damage induced by 4-NQO and UV light.

Fibroblast Growth Factor 23 in Hemodialysis Patients: Effects of Phosphate Binder, Calcitriol and Calcium Concentration in the Dialysate

Background: Fibroblast growth factor 23 (FGF23) concentrations increase early in chronic kidney disease (CKD), and the influence of current CKD-mineral and bone disorder (MBD) therapies on serum FGF23 levels is still under investigation. Methods: In this post-hoc analysis of a randomized clinical trial, phosphate binders and calcitriol were washed out of 72 hemodialysis patients who were then submitted to bone biopsy, coronary tomography and biochemical measures, including FGF23. They were randomized to receive sevelamer or calcium acetate for 1 year and the prescription of calcitriol and the calcium concentration in the dialysate were adjusted according to serum calcium, phosphate and PTH and bone biopsy diagnosis. Results: At baseline, bone biopsy showed that 58.3% had low-turnover bone disease, whereas 38.9% had high-turnover bone disease, with no significant differences between them with regard to FGF23. Median baseline FGF23 serum levels were elevated and correlated positively with serum phosphate. After 1 year, serum FGF23 decreased significantly. Repeated measures ANOVA analysis showed that the use of a 3.5-mEq/l calcium concentration in the dialysate, as well as the administration of calcitriol and a calcium-based phosphate binder were associated with higher final serum FGF23 levels. Conclusions: Taken together, our results confirm that the current CKD-MBD therapies have an effect on serum levels of FGF23. Since FGF23 is emerging as a potential treatment target, our findings should be taken into account in the decision on how to manage CKD-MBD therapy. Copyright (C) 2010 S. Karger AG, Basel

Insulin-like growth factor-1 and 17 beta-estradiol down-regulate prostate apoptosis response-4 expression in MCF-7 breast cancer cells

The PKC apoptosis WTI regulator gene, also named prostate apoptosis response-4 (PAR-4), encodes a pro-apoptotic protein that sensitizes cells to numerous apoptotic stimuli. Insulin-like growth factor-1 (IGF-1) and 17 beta-estradiol (E2), two important factors for breast cancer development and progression, have been shown to down-regulate PAR-4 expression and inhibit apoptosis induced by PAR-4 in neuronal cells. In this study, we sought to investigate the mechanisms of regulation of PAR-4 gene expression in MCF-7 cells treated with E2 or IGF-1. E2 (10 nM) and IGF-1 (12.5 nM) each down-regulated PAR-4 expression in MCF-7 cells after 24 h of treatment. The effect of E2 was dependent on ER activation, as demonstrated by an increase in PAR-4 expression when cells were pretreated for 1 h with 1 mu M ICI-182,780 (ICI) before receiving E2 plus ICI. The effect of IGF-1 was abolished by pre-treatment for 1 h with 30 mu M LY294002 (a specific PI3-K inhibitor), and significantly inhibited by 30 mu M SB202190 (a specific p38MAPK inhibitor). We also demonstrated that E2 acts synergistically with IGF-1, resulting in greater down-regulation of PAR-4 mRNA expression compared with E2 or IGF-1 alone. Our results show for the first time that E2 and IGF-1 inhibit PAR-4 gene expression in MCF-7 cells, suggesting that this down-regulation may provide a selective advantage for breast cancer cell survival.

Enolase from Paracoccidioides brasiliensis: isolation and identification as a fibronectin-binding protein

Paracoccidioides brasiliensis yeast cells can enter mammalian cells and may manipulate the host cell environment to favour their own growth and survival. Moreover, fibronectin and several other host extracellular matrix proteins are recognized by various components of the yeast cell extracts. The present study was designed to isolate and characterize a fibronectin-binding protein from P. brasiliensis. We also compared P. brasiliensis strain 18, tested before (Pb18a) and after (Pb18b) animal passage, in relation to its adhesion and invasion processes. Extracts from both samples, when cultured on blood agar solid medium, showed higher levels of protein expression than when the same samples were cultured on Fava-Netto solid medium, as demonstrated by two-dimensional electrophoresis and SDS-PAGE. Also, both Pb18a and Pb18b exhibited stronger adhesion to A549 epithelial cells when cultured on blood agar medium than when cultured on Fava-Netto medium. Ligand affinity binding assays revealed a protein of 54 kDa and pl 5.6 in P. brasiliensis cell-free extracts with the properties of a fibronectin-binding adhesin, which was characterized by tryptic digestion and mass spectroscopy as a homologue of enolase from P. brasiliensis. Antibody raised against this 54 kDa protein abolished 80 % of P. brasiliensis adhesion to A549 epithelial cells. Our results demonstrate that P. brasiliensis produces a fibronectin-binding adhesin, irrespective of the culture medium, and that this activity can be inhibited by a specific antibody and is involved in the adhesion of the fungus to pulmonary epithelial cells.

Involvement of Astroglial Fibroblast Growth Factor-2 and Microglia in the Nigral 6-OHDA Parkinsonism and a Possible Role of Glucocorticoid Hormone on the Glial Mediated Local Trophism and Wound Repair

We have observed in previous studies that 6-hydroxydopamine (6-OHDA)-induced lesions in the nigrostriatal dopamine (DA) system promote increases of the astroglial basic fibroblast growth factor (FGF-2, bFGF) synthesis in the ascending DA pathways, event that could be modified by adrenosteroid hormones. Here, we first evaluated the changes of microglial reactivity in relation to the FGF-2-mediated trophic responses in the lesioned nigrostriatal DA system. 6-OHDA was injected into the left side of the rat substantia nigra. The OX42 immunohistochemistry combined with stereology showed the time course of the microglial activation. The OX42 immunoreactivity (IR) was already increased in the pars compacta of the substantia nigra (SNc) and ventral tegmental area (VTA) 2 h after the 6-OHDA injection, peaked on day 7, and remained increased on the 14th day time-interval. In the neostriatum, OX42 immunoreactive (ir) microglial profiles increased at 24 h, peaked at 72 h, was still increased at 7 days but not 14 days after the 6-OHDA injection. Two-colour immunofluorescence analysis of the tyrosine hydroxylase (TH) and OX42 IRs revealed the presence of small patches of TH IR within the activated microglia. A decreased FGF-2 IR was seen in the cytoplasm of DA neurons of the SNc and VTA as soon as 2 h after 6-OHDA injection. The majority of the DA FGF-2 ir cells of these regions had disappeared 72 h after neurotoxin. The astroglial FGF-2 IR increased in the SNc and VTA, which peaked on day 7. Two-colour immunofluorescence and immunoperoxidase analyses of the FGF-2 and OX42 IRs revealed no FGF-2 IR within the reactive or resting microglia. Second, we have evaluated in a series of biochemical experiments whether adrenocortical manipulation can interfere with the nigral lesion and the state of local astroglial reaction, looking at the TH and GFAP levels respectively. Rats were adrenalectomized (ADX) and received a nigral 6-OHDA stereotaxical injection 2 days later and sacrificed up to 3 weeks after the DA lesion. Western blot analysis showed time-dependent decrease and elevation of TH and GFAP levels, respectively, in the lesioned versus contralateral midbrain sides, events potentiated by ADX and worsened by corticosterone replacement. ADX decreased the levels of FGF-2 protein (23 kDa isoform) in the lesioned side of the ventral midbrain compared contralaterally. The results indicate that reactive astroglia, but not reactive microglia, showed an increased FGF-2 IR in the process of DA cell degeneration induced by 6-OHDA. However, interactions between these glial cells may be relevant to the mechanisms which trigger the increased astroglial FGF-2 synthesis and thus may be related to the trophic state of DA neurons and the repair processes following DA lesion. The findings also gave further evidence that adrenocortical hormones may regulate astroglial-mediated trophic mechanisms and wound repair events in the lesioned DA system that may be relevant to the progression of Parkinson`s disease.

Effects of bone marrow-derived mononuclear cells on airway and lung parenchyma remodeling in a murine model of chronic allergic inflammation

We hypothesized that bone marrow-derived mononuclear cells (BMDMC) would attenuate the remodeling process in a chronic allergic inflammation model. C57BL/6 mice were assigned to two groups. In OVA, mice were sensitized and repeatedly challenged with ovalbumin. Control mice (C) received saline under the same protocol. C and OVA were further randomized to receive BMDMC (2 x 10(6)) or saline intravenously 24 h before the first challenge. BMDMC therapy reduced eosinophil infiltration, smooth muscle-specific actin expression, subepithelial fibrosis, and myocyte hypertrophy and hyperplasia, thus causing a decrease in airway hyperresponsiveness and lung mechanical parameters. BMDMC from green fluorescent protein (GFP)-transgenic mice transplanted into GFP-negative mice yielded lower engraftment in OVA. BMDMC increased insulin-like growth factor expression, but reduced interleukin-5, transforming growth factor-beta, platelet-derived growth factor, and vascular endothelial growth factor mRNA expression. In conclusion, in the present chronic allergic inflammation model, BMDMC therapy was an effective pre-treatment protocol that potentiated airway epithelial cell repair and prevented inflammatory and remodeling processes. (C) 2010 Elsevier B.V. All rights reserved.

High-Resolution Genomic Mapping Reveals Consistent Amplification of the Fibroblast Growth Factor Receptor Substrate 2 Gene in Well-Differentiated and Dedifferentiated Liposarcoma

Well-differentiated liposarcoma (WDLS) is one of the most common malignant mesenchymal tumors and dedifferentiated liposarcoma (DDLS) is a malignant tumor consisting of both WDLS and a transformed nonlipogenic sarcomatous component. Cytogenetically, WDLS is characterized by the presence of ring or giant rod chromosomes containing several amplified genes, including MDM2, TSPAN31 CDK4, and others mainly derived from chromosome bands 12q13-15. However, the 12q13-15 amplicon is large and discontinuous. The focus of this study was to identify novel critical genes that are consistently amplified in primary (nonrecurrent) WDLS and with potential relevance for future targeted therapy. Using a high-resolution (5.0 kb) ""single nucleotide polymorphism""/copy number variation microarray to screen the whole genome in a series of primary WDLS, two consistently amplified areas were found on chromosome 12: one region containing the MDM2 and CPM genes, and another region containing the FRS2 gene. Based on these findings, we further validated FRS2 amplification in both WDLS and DDLS. Fluorescence in situ hybridization confirmed FRS2 amplification in all WDLS and DDLS tested (n = 57). Real time PCR showed FRS2 mRNA transcriptional upregulation in WDLS (n = 19) and DDLS (n = 13) but not in lipoma (n = 5) and normal fat (n = 9). Immunoblotting revealed high expression levels of phospho-FRS2 at 1436 and slightly overexpression of total FRS2 protein in liposarcoma but not in normal fat or preadipocytes. Considering the critical role of FRS2 in mediating fibroblast growth factor receptor signaling, our findings indicate that FRS2 signaling should be further investigated as a potential therapeutic target for liposarcoma. (C) 2011 Wiley-Liss, Inc.

Exercise training inhibits inflammatory cytokines and more than prevents myocardial dysfunction in rats with sustained beta-adrenergic hyperactivity

Myocardial hypertrophy and dysfunction occur in response to excessive catecholaminergic drive. Adverse cardiac remodelling is associated with activation of proinflammatory cytokines in the myocardium. To test the hypothesis that exercise training can prevent myocardial dysfunction and production of proinflammatory cytokines induced by beta-adrenergic hyperactivity, male Wistar rats were assigned to one of the following four groups: sedentary non-treated (Con); sedentary isoprenaline treated (Iso); exercised non-treated (Ex); and exercised plus isoprenaline (Iso+Ex). Echocardiography, haemodynamic measurements and isolated papillary muscle were used for functional evaluations. Real-time RT-PCR and Western blot were used to quantify tumour necrosis factor alpha, interleukin-6, interleukin-10 and transforming growth factor beta(1) (TGF-beta(1)) in the tissue. NF-kappa B expression in the nucleus was evaluated by immunohistochemical staining. The Iso rats showed a concentric hypertrophy of the left ventricle (LV). These animals exhibited marked increases in LV end-diastolic pressure and impaired myocardial performance in vitro, with a reduction in the developed tension and maximal rate of tension increase and decrease, as well as worsened recruitment of the Frank-Starling mechanism. Both gene and protein levels of tumour necrosis factor alpha and interleukin-6, as well as TGF-beta(1) mRNA, were increased. In addition, the NF-kappa B expression in the Iso group was significantly raised. In the Iso+Ex group, the exercise training had the following effects: (1) it prevented LV hypertrophy; (ii) it improved myocardial contractility; (3) it avoided the increase of proinflammatory cytokines and improved interleukin-10 levels; and (4) it attenuated the increase of TGF-beta(1) mRNA. Thus, exercise training in a model of beta-adrenergic hyperactivity can avoid the adverse remodelling of the LV and inhibit inflammatory cytokines. Moreover, the cardioprotection is related to beneficial effects on myocardial performance.

NOVEL 65 kDa RNA-BINDING PROTEIN IN SQUID PRESYNAPTIC TERMINALS

A polyclonal antibody (C4), raised against the head domain of chicken myosin Va, reacted strongly towards a 65 kDa polypeptide (p65) on Western blots of extracts from squid optic lobes but did not recognize the heavy chain of squid myosin V. This peptide was not recognized by other myosin Va antibodies, nor by an antibody specific for squid myosin V. In an attempt to identify it, p65 was purified from optic lobes of Loligo plei by cationic exchange and reverse phase chromatography. Several peptide sequences were obtained by mass spectroscopy from p65 cut from sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) gels. BLAST analysis and partial matching with expressed sequence tags (ESTs) from a Loligo pealei data bank indicated that p65 contains consensus signatures for the heterogeneous nuclear ribonucleoprotein (hnRNP) A/B family of RNA-binding proteins. Centrifugation of post mitochondrial extracts from optic lobes on sucrose gradients after treatment with RNase gave biochemical evidence that p65 associates with cytoplasmic RNP complexes in an RNA-dependent manner. Immunohistochemistry and immunofluorescence studies using the C4 antibody showed partial co-labeling with an antibody against squid synaptotagmin in bands within the outer plexiform layer of the optic lobes and at the presynaptic zone of the stellate ganglion. Also, punctate labeling by the C4 antibody was observed within isolated optic lobe synaptosomes. The data indicate that p65 is a novel RNA-binding protein located to the presynaptic terminal within squid neurons and may have a role in synaptic localization of RNA and its translation or processing. (C) 2010 IBRO. Published by Elsevier Ltd. All rights reserved.

Factors involved in the T helper type 1 and type 2 cell commitment and osteoclast regulation in inflammatory apical diseases

Periapical chronic lesion formation involves activation of the immune response and alveolar bone resorption around the tooth apex. However, the overall roles of T helper type 1 (Th1), Th2, and T-regulatory cell (Treg) responses and osteoclast regulatory factors in periapical cysts and granulomas have not been fully determined. This study aimed to investigate whether different forms of apical periodontitis, namely cysts and granulomas, show different balances of Th1, Th2 regulators, Treg markers, and factors involved in osteoclast chemotaxis and activation. Gene expression of these factors was assessed using quantitative real-time polymerase chain reaction, in samples obtained from healthy gingiva (n = 8), periapical granulomas (n = 20), and cysts (n = 10). Periapical cysts exhibited a greater expression of GATA-3, while a greater expression of T-bet, Foxp3, and interleukin-10 (IL-10) was seen in granulomas. The expression of interferon-gamma, IL-4, and transforming growth factor-beta was similar in both lesions. Regarding osteoclastic factors, while the expression of SDF-1 alpha/CXCL12 and CCR1 was higher in cysts, the expression of RANKL was significantly higher in granulomas. Both lesions exhibited similar expression of CXCR4, CK beta 8/CCL23, and osteoprotegerin, which were significantly higher than in control. Our results showed a predominance of osteoclast activity in granulomas that was correlated with the Th1 response. The concomitant expression of Treg cell markers suggests a possible suppression of the Th1 response in granulomas. On the other hand, in cysts the Th2 activity is augmented. The mechanisms of periradicular lesion development are still not fully understood but the imbalance of immune and osteoclastic cell activity in cysts and granulomas seems to be critically regulated by Treg cells.

Expression of transcription factors NF-kappa B and AP-1 in nasal polyposis

Background The treatment and prognosis of nasal polyposis (NP) may be influenced by transcription factors, but their expression is poorly understood. Objective To determine the expression of transcription factors [(nuclear factor-kappa B) NF-kappa B and (activator protein) AP-1], cytokines [IL-1 beta, TNF-alpha and (granulocytes and macrophage colony-stimulating factor) GM-CSF], growth factor (b-FGF), chemokine (eotaxin-2) and adhesion molecule (ICAM-1) in NP in comparison with nasal mucosa controls. Methods Cross-sectional study. Twenty biopsies of nasal polyps were compared with eight middle turbinate biopsies. p65, c-Fos, IL-1 beta, TNF-alpha, ICAM-1, b-FGF, eotaxin-2 and GM-CSF were analysed through RQ-PCR, and p65 and c-Fos were also analysed through Western blotting. Results NF-kappa B expression was increased in patients with NP when compared with control mucosa (P < 0.05), whereas AP-1 expression did not differ significantly between groups. Expressions of IL-1 beta, eotaxin-2 and b-FGF were also increased in patients with NP compared with controls (P < 0.05). Conclusions The transcription factor NF-kappa B is more expressed in NP than in control mucosa. This is important in NP because NF-kappa B can induce the transcription of cytokines, chemokines and adhesion molecules, which play an important role in the inflammatory process. Moreover, transcription factors influence the response to corticosteroids, which are the basis of NP treatment. Transcription factor AP-1 does not seem to have a significant role in the pathological process.