Stimulation of mouse hematopoietic stem cells by angiogenin and DNA preparations.

Immature hematopoietic progenitors are a constant source for renewal of hemocyte populations and the basic component of the tissue and cell repair apparatus. A unique property of these cells of internalizing extracellular double-stranded DNA has been previously shown. The leukostimulatory effect demonstrated in our pioneering studies was considered to be due to the feature of this cell. In the present research, we have analyzed the effects of DNA genome reconstructor preparation (DNAgr), DNAmix, and human recombinant angiogenin on both hematopoietic stem cells and multipotent progenitors. Treatment with bone marrow cells of experimental mice with these preparations stimulates colony formation by hematopoietic stem cells and proliferation of multipotent descendants. The main lineage responsible for this is the granulocyte-macrophage hematopoietic lineage. Using fluorescent microscopy as well as FACS assay, co-localization of primitive c-Kit- and Sca-1-positive progenitors and the TAMRA-labeled double-stranded DNA has been shown. Human recombinant angiogenin was used as a reference agent. Cells with specific markers were quantified in intact bone marrow and colonies grown in the presence of inducers. Quantitative analysis revealed that a total of 14,000 fragment copies of 500 bp, which is 0.2% of the haploid genome, can be delivered into early progenitors. Extracellular double-stranded DNA fragments stimulated the colony formation in early hematopoietic progenitors from the bone marrow, which assumed their effect on cells in G0. The observed number of Sca1+/c-Kit+ cells in colonies testifies to the possibility of both symmetrical and asymmetrical division of the initial hematopoietic stem cell and its progeny.

Nuanced role for dendritic cell intrinsic IRE1 RNase in the regulation of antitumor adaptive immunity.

In cancer, activation of the IRE1/XBP1s axis of the unfolded protein response (UPR) promotes immunosuppression and tumor growth, by acting in cancer cells and tumor infiltrating immune cells. However, the role of IRE1/XBP1s in dendritic cells (DCs) in tumors, particularly in conventional type 1 DCs (cDC1s) which are cellular targets in immunotherapy, has not been fully elucidated. Here, we studied the role of IRE1/XBP1s in subcutaneous B16/B78 melanoma and MC38 tumors by generating loss-of-function models of IRE1 and/or XBP1s in DCs or in cDC1s. Data show that concomitant deletion of the RNase domain of IRE1 and XBP1s in DCs and cDC1s does not influence the kinetics of B16/B78 and MC38 tumor growth or the effector profile of tumor infiltrating T cells. A modest effect is observed in mice bearing single deletion of XBP1s in DCs, which showed slight acceleration of melanoma tumor growth and dysfunctional T cell responses, however, this effect was not recapitulated in animals lacking XBP1 only in cDC1s. Thus, evidence presented here argues against a general pro-tumorigenic role of the IRE1/XBP1s pathway in tumor associated DC subsets.

Construction of polymer monolithic columns in polypropylene ink-pen tubes for separation of proteins by cation-exchange chromatography.

We describe the synthesis of polymer monoliths inside polypropylene tubes from ink pens. These tubes are cheap, chemically stable, and resistant to pressure. UV-initiated grafting with 5 wt% benzophenone in methanol for 20 min activated the internal surface, thus enabling the covalent binding of ethylene glycol dimethacrylate, also via photografting. The pendant vinyl groups attached a poly(glycidyl methacrylate-co-ethylene glycol dimethacrylate) monolith prepared via photopolymerization. These tubes measured 100-110 mm long, with 2 mm of internal diameter. The parent monoliths were functionalized with Na2 SO3 or iminodiacetate to produce strong and weak cation exchangers, respectively. The columns exhibited permeabilities varying from 2.7 to 3.3 × 10-13  m2 , which enabled the separation of proteins at 500 µL/min and back pressures <2.8 MPa. Neither structure collapse nor monolith detachment occurred at flow rates as high as 2.0 mL/min, which produced back pressures between 6.9 and 9.0 MPa. The retention times of ovalbumin, ribonuclease A, cytochrome C, and lysozyme in salt gradient at pH 7.0 followed the order of increasing isoelectric points, confirming the cation exchange mechanism. Separation and determination of lysozyme in egg white proved the applicability of the columns to the analysis of complex samples.

Fast construction of polymer monolithic columns inside fluorinated ethylene propylene (FEP) tubes for separation of proteins by reversed-phase liquid chromatography.

This paper describes the preparation of polymer monolithic columns in the confines of fluorinated ethylene propylene (FEP) tubes. These tubes are cheap, chemically stable, and widely used in flow analysis laboratories. UV-initiated grafting with 5 wt% benzophenone in methanol for 1 h activated the internal surface walls, thus enabling the further covalent binding of ethylene glycol dimethacrylate (EDMA) from a 15 wt% solution in methanol, also via photografting. Both steps used 254 nm radiation under a potency of 120 mJ cm2. ATR-FTIR measurements revealed the presence of carbonyl, alkyl and vinyl groups in the functionalized FEP. The density of vinyl groups was high enough to firmly attach a poly(lauryl methacrylate-co-ethylene glycol dimethacrylate) monolith in 120 × 1.57 mm i.d. tubes, prepared via photopolymerization. The total preparation lasts less than 2-h. The columns were permeable, (1.58 ± 0.06) × 10-13 m2, providing reproducible chromatographic parameters of retention times, retention factor, selectivity, and resolution. The monoliths were stable at flow rates of 500 µL min-1, collapsing only at flow rates >700 µL min-1, a condition that increased the backpressure over 1000 psi (experiments at the room temperature). The separation of proteins by reversed-phase liquid chromatography demonstrated the efficiency of the columns. Determination of egg white proteins (ovalbumin and lysozyme) and myoglobin in spiked urine proved the applicability to the analysis of real samples.

Nailfold capillary changes in adult new-onset dermatomyositis: a prospective cross-sectional study.

OBJECTIVES: It is to prospectively analyze nailfold capillaroscopy (NC) findings in new-onset dermatomyositis (DM) and to correlate NC findings with serum angiogenic cytokines and DM clinical and laboratory features. MATERIALS AND METHODS: Twenty-three patients with DM who experienced < 12 months of symptoms were included in the study. To assess serum cytokine levels, 23 age-, sex-, and ethnicity-matched healthy volunteers were used. NC characteristics and DM activity parameters were analyzed. RESULTS: Significantly higher serum angiogenin (ANG) and vascular endothelial growth factor-1 (VEGF1) levels were observed in DM patients than in controls. Capillary density and avascular areas correlated positively and negatively, respectively, with serum levels of ANG. Moreover, the capillary density correlated inversely with the number of enlarged and giant capillaries and avascular areas. The number of enlarged capillaries correlated positively with patient and physician visual analogue scales (VAS), the presence of a facial rash, giant capillaries, and microhemorrhages. Giant capillaries had a positive correlation with physician and cutaneous VAS, enlarged capillaries, avascular areas, microhemorrhages and bushy capillaries, and a negative correlation with capillary density. Microhemorrhages correlated positively with the "V-neck" sign and physician VAS. VEGF1 showed no relationship with the NC parameters with DM-related clinical and laboratory features. Additionally, 15 out of 23 patients were assessed prospectively after 3.21 years. All patients had a major clinical response with significant improvement in all NC parameters, except for enlarged and bushy capillaries. CONCLUSIONS: The NC may be a useful tool to assess disease activity in recent-onset DM, and it can also reinforce the role of ANG in the angiogenesis of this myopathy.

Relevance of serum angiogenic cytokines in adult patients with dermatomyositis.

BACKGROUND: Until now, there are few studies evaluating serum levels of angiogenic cytokines in dermatomyositis (DM). Therefore, the aims of the present study were: (a) to analyze systematically and simultaneously serum levels of angiogenin (ANG), angiopoietin (ANGPT)-1, vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF)-1 and - 2, platelet derived growth factor (PDGF)-AA and -BB in DM; (b) to correlate the serum level of these cytokines with the DM clinical and laboratory features. METHODS: This is a cross sectional study, in which 48 patients with DM aged 18 to 45 years were gender-, age- and ethnicity-matched with 48 healthy individuals (control group). The serum levels of cytokines analyses were performed by multiplex immunoassay. The parameters of DM activity were based on the scores established by the International Myositis Assessment & Clinical Studies Group. RESULTS: The mean ages, gender frequencies and ethnicities were comparable between the patients with DM and the control group. A significantly higher serum FGF-1 and FGF-2 levels (P < 0.001 and P < 0.001, respectively), lower VEGF and PDGF-AA levels (P = 0.009 and P = 0.022), and comparable ANG, ANGPT-1 and PDGF-BB levels were observed in DM patients compared to controls. There was a tendency for cytokines (with the exceptions of VEGF and PDGF-BB) to correlate positively with the DM activity parameters, whereas FGF-2 correlated inversely. Moreover, FGF-1 strongly correlated with DM cutaneous manifestations. CONCLUSIONS: The present data provide the relevance of different serum angiogenic cytokines in patients with DM. Additional studies will be needed to validate the data obtained in this work.

Calcitriol promotes proangiogenic molecules in keratinocytes in a diabetic foot ulcer model.

Foot ulceration is one of the most common and complex sequelae of diabetes mellitus, generally posing a therapeutic challenge due to poor healing responses and high rates of complications, including peripheral vascular disease, ischemia and infections. Calcitriol, the most active vitamin D metabolite, induces antimicrobial peptides production in keratinocytes from diabetic foot ulcers (DFU); however, little is known about its effects on angiogenic factors in this pathology. Herein we aimed at studying whether calcitriol induces angiogenic molecules in keratinocytes under normoxic and hypoxic conditions, and if these molecules are able to improve cell migration in vitro. Evaluation of DFU samples by immunohistochemistry showed increased VEGF and decreased angiogenin and HIF-1α expression compared to controls, suggesting an altered pattern of angiogenic factors in DFU. Interestingly, incubation of keratinocytes with calcitriol significantly upregulated VEGFA, HIF-1α and angiogenin gene expression, while the resulting cell culture media stimulated both endothelial cells and keratinocytes migration in an in vitro wound closure assay under a normoxic environment (p<0.05). Moreover, the culture media of calcitriol-treated keratinocytes stimulated cell migration in a similar extent as exogenous VEGF or EGF in endothelial and keratinocytes cells. These results suggest that the altered profile of angiogenic molecules in DFU might be improved by local or systemic treatment with calcitriol under normoxic conditions, which could probably be achieved with hyperbaric oxygen therapy. Given that calcitriol not only augments proangiogenic factors but also induces antimicrobial peptides expression, this hormone should be further investigated in clinical trials of DFU.

Evaluation of nicotinamide as an anti-inflammatory and anti-angiogenic agent in uveal melanoma cell lines.

PURPOSE:: To investigate the effect of nicotinamide on the secretion of pro-an giogenic and pro-inflammatory cytokines in uveal melanoma cell lines. METHODS:: Two human uveal melanoma cell lines (92.1 and OCM-1) were treated with nicotinamide (10 mmol/L) or control media for 48 hours in culture. The su perna tant from each culture was used in sandwich enzyme-linked immuno sorbent assay-based angiogenesis and inflammation arrays to evaluate the effects of exogenously administered nicotinamide on the secretion of a total of 20 pro-an gio genic and pro-inflammatory proteins. RESULTS:: Seven pro-angiogenic cytokines were detected under control conditions for both uveal melanoma cell lines. Treatment with nicotinamide resulted in a significant decrease in secretion of the following pro-angiogenic cytokines: angiogenin, angiopoietin-2, epidermal growth factor, and vascular epithelial growth factor-A in the 92.1 cells; basic fibroblast growth factor in the OCM-1 cells; and placenta growth factor in both cell lines. Among the pro-inflammatory proteins, monocyte chemotactic protein-1 and interleukin-8 were expressed in both untreated cell lines and both were significantly reduced when treated with nicotinamide. CONCLUSIONS:: Results from this in vitro model suggest that nicotinamide may have anti-inflammatory and anti-angiogenic properties, which may open the possibility of using it as a chemopreventive agent for uveal melanoma; however, further studies including animal models are warranted.

Benchmarking a Fast Proton Titration Scheme in Implicit Solvent for Biomolecular Simulations.

pH is a key parameter for technological and biological processes, intimately related to biomolecular charge. As such, it controls biomolecular conformation and intermolecular interactions, for example, protein/RNA stability and folding, enzyme activity, regulation through conformational switches, protein-polyelectrolyte association, and protein-RNA interactions. pH also plays an important role in technological systems in food, brewing, pharma, bioseparations, and biomaterials in general. Predicting the structure of large proteins and complexes remains a great challenge experimentally, industrially, and theoretically, despite the variety of numerical schemes available ranging from Poisson-Boltzmann approaches to explicit solvent based methods. In this work we benchmark a fast proton titration scheme against experiment and several theoretical methods on the following set of representative proteins: [HP36, BBL, HEWL (triclinic and orthorhombic), RNase, SNASE (V66K/WT, V66K/PHS, V66K/Δ+PHS, L38D/Δ+PHS, L38E/Δ+PHS, L38K/Δ+PHS), ALAC, and OMTKY3]; routinely used in similar tests due to the diversity of their structural features. Our scheme is rooted in the classical Tanford-Kirkwood model of impenetrable spheres, where salt is treated at the Debye-Hückel level. Treating salt implicitly dramatically reduces the computation time, thereby circumventing sampling difficulties faced by other numerical schemes. In comparison with experimental measurements, our calculated pKa values have the average, maximum absolute, and root-mean-square deviations of 0.4-0.9, 1.0-5.2, and 0.5-1.2 pH units, respectively. These values are within the ranges commonly observed in theoretical models. They are also in the large majority of the cases studied here more accurate than the NULL model. For BBL, ALAC, and OMTKY3, the predicted pKa are closer to experimental results than other analyzed theoretical data. Despite the intrinsic approximations of the fast titration scheme, its robustness and ability to properly describe the main system physics is confirmed.

Evaluation of nicotinamide as an anti-inflammatory and anti-angiogenic agent in uveal melanoma cell lines / Avaliação da nicotinamida como agente anti-inflamatório e anti-angiogênico em linhas celulares de melanoma uveal

ABSTRACT Purpose: To investigate the effect of nicotinamide on the secretion of pro-an giogenic and pro-inflammatory cytokines in uveal melanoma cell lines. Methods: Two human uveal melanoma cell lines (92.1 and OCM-1) were treated with nicotinamide (10 mmol/L) or control media for 48 hours in culture. The su perna tant from each culture was used in sandwich enzyme-linked immuno sorbent assay-based angiogenesis and inflammation arrays to evaluate the effects of exogenously administered nicotinamide on the secretion of a total of 20 pro-an gio genic and pro-inflammatory proteins. Results: Seven pro-angiogenic cytokines were detected under control conditions for both uveal melanoma cell lines. Treatment with nicotinamide resulted in a significant decrease in secretion of the following pro-angiogenic cytokines: angiogenin, angiopoietin-2, epidermal growth factor, and vascular epithelial growth factor-A in the 92.1 cells; basic fibroblast growth factor in the OCM-1 cells; and placenta growth factor in both cell lines. Among the pro-inflammatory proteins, monocyte chemotactic protein-1 and interleukin-8 were expressed in both untreated cell lines and both were significantly reduced when treated with nicotinamide. Conclusions: Results from this in vitro model suggest that nicotinamide may have anti-inflammatory and anti-angiogenic properties, which may open the possibility of using it as a chemopreventive agent for uveal melanoma; however, further studies including animal models are warranted.

RESUMO Objetivo: Acredita-se que a nicotinamida (NIC) seja capaz de diminuir a angiogênese induzida pelo fator de crescimento endotelial vascular (VEGF). Investigar os efeitos da nicotinamida sobre a secreção de citocinas pró-angiogênicas e pró-inflamatórias em linhagens de células de melanoma uveal humano (UM). Métodos: Duas linhagens de células humanas de UM (92,1 e OCM-1) foram tratadas com NIC (10 mmol/L) ou apenas com meio de cultura por 48 horas. O sobrenadante das culturas obtido após a administração de nicotinamida foi comparado com o sobrenadante das culturas controle quanto à expressão de 20 fatores pró-angiogênicos e pró-inflamatórios, pela técnica de enzyme-linked immunosorbent assay (ELISA). Resultados: Sete citocinas pró-angiogênicas foram detectadas nas condições de controle em ambas as linhagens de células de UM. O tratamento com nicotinamida promoveu uma redução significativa da secreção das seguintes citocinas angiogênicas: Angiogenina, ANG2, EGF e VEGF-A em células 92.1; bFGF em células OCM-1; PIGF em ambas as linhagens celulares. Quanto às proteínas pró-inflamatórias, a expressão de MCP-1 e IL-8 foi significativamente reduzida com a administração de nicotinamida em relação às culturas de células que não receberam o tratamento. Conclusões: Nicotinamida apresenta propriedades anti-inflamatórias e anti-angiogênicas em modelo experimental in vitro. Tais efeitos sugerem a possibilidade de utilizar esta substância na quimioprevenção do UM. Entretanto, estudos com modelos experimentais in vivo são necessários para melhor avaliar o benefício do tratamento do UM com nicotinamida.

Inkjet Printing of Proteins: an Experimental Approach.

Peptides and proteins represent a promissory group of molecules used by the pharmaceutical industry for drug therapy with great potential for development. However, the administration of these molecules presents a series of difficulties, making necessary the exploration of new alternatives like the buccal route of administration to improve drug therapy compliance. Although drop-on demand printers have been explored for small molecule drugs with promising results, the development of delivery systems for peptides and proteins through inkjet printing has seen little development. Therefore, the aim of this study was to assess the feasibility of using a thermal inkjet printing system for dispensing lysozyme and ribonuclease-A as model proteins. To address the absorption limitations of a potential buccal use, a permeation enhancer (sodium deoxycholate) was also studied in formulations. We found that a conventional printer successfully printed both proteins, exhibiting very high printing efficiency. Furthermore, the protein structure was not affected and minor effects were observed in the enzymatic activity after the printing process. In conclusion, we provide evidence for the usage of an inexpensive, easy to use, reliable, and reproducible thermal inkjet printing system to dispense proteins solutions for potential buccal application. Our research significantly contributes to present an alternative for manufacturing biologics delivery systems, with emphasis in buccal applications. Next steps of developments will be aimed at the use of new materials for printing, controlled release, and protection strategies for proteins and peptides.

The HEX 110 Hexamerin Is a Cytoplasmic and Nucleolar Protein in the Ovaries of Apis mellifera.

Hexamerins are insect storage proteins abundantly secreted by the larval fat body into the haemolymph. The canonical role of hexamerins consists of serving as an amino acid reserve for development toward the adult stage. However, in Apis mellifera, immunofluorescence assays coupled to confocal laser-scanning microscopy, and high-throughput sequencing, have recently shown the presence of hexamerins in other organs than the fat body. These findings have led us to study these proteins with the expectation of uncovering additional functions in insect development. We show here that a honeybee hexamerin, HEX 110, localizes in the cytoplasm and nucleus of ovarian cells. In the nucleus of somatic and germline cells, HEX 110 colocalized with a nucleolar protein, fibrillarin, suggesting a structural or even regulatory function in the nucleolus. RNase A provoked the loss of HEX 110 signals in the ovarioles, indicating that the subcellular localization depends on RNA. This was reinforced by incubating ovaries with pyronin Y, a RNA-specific dye. Together, the colocalization with fibrillarin and pyronin Y, and the sensitivity to RNase, highlight unprecedented roles for HEX110 in the nucleolus, the nuclear structure harbouring the gene cluster involved in ribosomal RNA production. However, the similar patterns of HEX 110 foci distribution in the active and inactive ovaries of queens and workers preclude its association with the functional status of these organs.

Dielectrophoretic behavior of PEGylated RNase A inside a microchannel with diamond-shaped insulating posts.

Ribonuclease A (RNase A) has proven potential as a therapeutic agent, especially in its PEGylated form. Grafting of PEG molecules to this protein yields mono-PEGylated (mono-PEG) and di-PEGylated (di-PEG) RNase A conjugates, and the unreacted protein. Mono-PEG RNase A is of great interest. The use of electrokinetic forces in microdevices represents a novel alternative to chromatographic methods to separate this specie. This work describes the dielectrophoretic behavior of the main protein products of the RNase A PEGylation inside a microchannel with insulators under direct current electric fields. This approach represents the first step in route to design micro-bioprocesses to separate PEGylated RNase A from unreacted native protein. The three proteins exhibited different dielectrophoretic behaviors. All of them experienced a marked streaming pattern at 3000 V consistent with positive dielectrophoresis. Native protein was not captured at any of the conditions tested, while mono-PEG RNase A and di-PEG RNase A were captured presumably due to positive dielectrophoresis at 4000 and 2500 V, respectively. Concentration of mono-PEG RNase A with a maximal enrichment efficiency of ≈9.6 times the feed concentration was achieved in few seconds. These findings open the possibility of designing novel devices for rapid separation, concentration, and recovery of PEGylated RNase A in a one-step operation.

Separation of proteins by cation-exchange sequential injection chromatography using a polymeric monolithic column.

Since sequential injection chromatography (SIC) emerged in 2003, it has been used for separation of small molecules in diverse samples, but separations of high molar mass compounds such as proteins have not yet been described. In the present work a poly(glycidyl methacrylate-co-ethylene dimethacrylate) (GMA-co-EDMA) monolithic column was prepared by free radical polymerization inside a 2.1-mm-i.d. activated fused silica-lined stainless steel tubing and modified with iminodiacetic acid (IDA). The column was prepared from a mixture of 24% GMA, 16% EDMA, 20% cyclohexanol, and 40% 1-dodecanol (all% as w/w) containing 1% of azobisisobutyronitrile (AIBN) (in relation to monomers). Polymerization was done at 60 °C for 24 h. The polymer was modified with 1.0 M IDA (in 2 M Na2CO3, pH 10.5) at 80 °C for 16 h. Separation of myoglobin, ribonuclease A, cytochrome C, and lysozyme was achieved at pH 7.0 (20 mM KH2PO4/K2HPO4) using a salt gradient (NaCl). Myoglobin was not retained, and the other proteins were separated by a gradient of NaCl created inside the holding coil (4 m of 0.8-mm-i.d. PTFE tubing) by sequential aspiration of 750 and 700 µL of 0.2 and 0.1 M NaCl, respectively. As the flow was reversed toward the column (5 µL s(-1)) the interdispersion of these solutions created a reproducible gradient which separated the proteins in 10 min, with the following order of retention: ribonuclease A < cytochrome C < lysozyme. The elution order was consistent with a cation-exchange mechanism as the retention increased with the isoelectric points.

Cytotoxic Activities of Eosinophil Cationic Protein and Eosinophil-derived Neurotoxin: In Silico Analysis.

BACKGROUND: Eosinophil cationic protein (ECP) and eosinophil derived-neurotoxin (EDN) are homologous ribonuclease (RNAse) A family proteins. The objective of the present study was to in silico characterize ECP and EDN with respect to their cytotoxic activities. MATERIALS AND METHODS: Structural, physicochemical, and conserved domain characterizations were carried-out using open-source software, such as InterProScan, NetOGlyc, NetPhos and Discovery Studio 3.1. RESULTS: The proteins did not have atypical conserved domains. EDN had a greater number of glutamine amino acid residues, whereas ECP had a predominance of arginine. ECP had four possible N-glycosylation, three O-glycosylation and four phosphorylation sites. EDN had five putative N-glycosylation, three phosphorylation and no O-glycosylation sites. CONCLUSION: The greater cationicity of ECP may be related to its higher cytotoxicity and to the fact that the varying post-translational modification profiles can generate functional differences from structural alteration. In vivo and in vitro studies need to be performed in order to confirm these predictions.

Preeclampsia in high risk women is characterized by risk group-specific abnormalities in serum biomarkers.

OBJECTIVE: To determine if early pregnancy serum biomarkers in high-risk women who develop preeclampsia vary according to risk factor. STUDY DESIGN: We performed a secondary analysis of the Maternal-Fetal Medicine Units Network randomized controlled trial of low-dose aspirin for the prevention of preeclampsia in high-risk women. Serum biomarker levels at enrollment (before initiation of aspirin or placebo) were compared between women who did and did not develop preeclampsia, both for the group as a whole and within each of 4 high-risk groups (insulin-dependent diabetes, hypertension, multiple gestation, and previous preeclampsia) using a regression model adjusting for gestational age at collection and prepregnancy body mass index. RESULTS: 1258 women were included (233 with insulin-dependent diabetes, 387 with chronic hypertension, 315 with a multiple gestation, 323 with previous preeclampsia). Multiple early pregnancy serum biomarkers differed between women who did and did not develop preeclampsia. Each high-risk group had a unique and largely nonoverlapping pattern of biomarker abnormality. Differences between those who did and did not develop preeclampsia were noted in vascular cell adhesion molecule in the diabetes group; human chorionic gonadotropin, soluble tumor necrosis factor receptor-2, tumor necrosis factor-alpha, selectin and angiogenin in the chronic hypertension group; interleukin-6, placental growth factor, soluble fms-like tyrosine kinase plus endoglin to placental growth factor ratio in the multiple gestation group; and angiogenin in the previous preeclampsia group. CONCLUSION: Patterns of serum biomarkers vary by high-risk group. These data support the hypothesis that multiple pathogenic pathways lead to the disease recognized clinically as preeclampsia.

Urea's effect on the ribonuclease A catalytic efficiency: a kinetic, 1H NMR and molecular orbital study.

Understanding of protein-urea interactions is one of the greatest challenges to modern structural protein chemistry. Based in enzyme kinetics experiments and (1)H NMR spectroscopic analysis we proposed that urea, at low concentrations, directly interacts with the protonated histidines of the active center of RNase A, following a simple model of competitive inhibition. These results were supported by theoretical analysis based on the frontier molecular orbital theory and suggest that urea might establish a favorable interaction with the cationic amino acids. Our experimental evidence and theoretical analysis indicate that the initials steps of the molecular mechanism of Urea-RNase A interaction passes through the establishment of a three center four electron adduct. Also, our results would explain the observed disruption of the (1)H NMR signals corresponding to H12 and H119 (involved in catalysis) of the RNase A studied in the presence of urea. Our interaction model of urea-amino acids (cationic) can be extended to explain the inactivation of other enzymes with cationic amino acids at the active site.

Hydrophobic interaction chromatography for purification of monoPEGylated RNase A.

The chromatographic methods used for the purification of PEGylated proteins are mainly Size Exclusion (SEC) and Ion Exchange Chromatography (IEX). Although the PEGylation affects the protein hydrophobicity, Hydrophobic Interaction Chromatography (HIC) has not been extensively applied for the separation of these proteins. Purification of monoPEGylated Ribonuclease A (RNase A) using HIC is studied in this work. The products of the PEGylation reaction of RNase A with 20 kDa methoxy-poly(ethylene glycol) were separated using three resins with different degrees of hydrophobicity: Butyl, Octyl and Phenyl sepharose. The effects of resin type, concentration and salt type (ammonium sulphate or sodium chloride), and gradient length on the separation performance were evaluated. Yield and purity were calculated using the plate model. Under all conditions assayed the native protein was completely separated from PEGylated species. The best conditions for the purification of monoPEGylated RNase A were: Butyl sepharose, 1 M ammonium sulphate and 35 column volumes (CVs); this resulted in a yield as high as 85% with a purity of 97%. The purity of monoPEGylated RNase A is comparable to that obtained when the separation is performed using SEC, but the yield increases from 65% with SEC to ~85% with HIC. This process represents a viable alternative for the separation of PEGylated proteins.

Characterization of Trypanosoma cruzi telomerase.

High telomerase activity is always associated with actively dividing cells, however the detection of this activity in dividing Leishmania and Trypanosoma cruzi cells has always been disappointingly low. Recently, we have found that Leishmania major telomerase activity can be activated by heat, which combined with dilutions of the nuclear extracts produced an increase in activity comparable to cancer cells. Here we examined whether T. cruzi telomerase shares the same physicochemical properties of primer specificity and overall features of the L. major. Our studies revealed that no telomerase inhibitory factors were present in the nuclear lysates of T. cruzi however the enzyme was activated by heat and was very resilient to heat denaturation. We also showed the extension primer specificity, susceptibility to RNase-A and RNase-H digestion, and the effect of telomerase inhibitors.

Entamoeba histolytica: biochemical characterization of a protein disulfide isomerase.

Protein disulfide isomerase (PDI) enzymes are eukaryotic oxidoreductases that catalyze oxidation, reduction and isomerization of disulfide bonds in polypeptide substrates. Here, we report the biochemical characterization of a PDI enzyme from the protozoan parasite Entamoeba histolytica (EhPDI). Our results show that EhPDI behaves mainly as an oxidase/isomerase and can be inhibited by bacitracin, a known PDI inhibitor; moreover, it exhibits chaperone-like activity. Albeit its physiological role in the life style of the parasite (including virulence and survival) remains to be studied, EhPDI could represent a potential drug target for anti-amebic therapy.