Diagnostic value of serum activin A and follistatin levels in women with peritoneal, ovarian and deep infiltrating endometriosis

Activin A is a growth factor, produced by the endometrium, whose actions are modulated by the binding protein follistatin. Both proteins are detectable in the peripheral serum and their concentrations may be increased in women with endometriosis. The present study was designed to evaluate whether serum levels of activin A and follistatin are altered, and therefore have a potential diagnostic value, in women with peritoneal, ovarian and deep infiltrating endometriosis. We performed a multicenter controlled study evaluating simultaneously serum activin A and follistatin concentrations in women with and without endometriosis. Women with endometriosis (n 139) were subdivided into three groups: peritoneal endometriosis (n 28); ovarian endometrioma (n 61) and deep infiltrating endometriosis (n 50). The control group (n 75) consisted of healthy women with regular menstrual cycles. Blood samples were collected from a peripheral vein and assayed for activin A and follistatin using commercially available enzyme immunoassay kits. The ovarian endometrioma group had serum activin A levels significantly higher than healthy controls (0.22 0.01 ng/ml versus 0.17 0.01 ng/ml, P 0.01). None of the endometriosis groups had serum follistatin levels which were significantly altered compared with healthy controls; however, levels found in the endometrioma group (2.34 0.32 ng/ml) were higher than that in the deep endometriosis group (1.50 0.17 ng/ml, P 0.05). The area under the receiver operating characteristic curve of activin A was 0.700 (95 confidence interval: 0.6050.794), while that of follistatin was 0.620 (95 confidence interval: 0.5100.730) for the diagnosis of ovarian endometrioma. The combination of both markers into a duo marker index did not improve significantly their diagnostic accuracy. The present study demonstrated that serum activin A and follistatin are not significantly altered in peritoneal or deep infiltrating endometriosis and have limited diagnostic accuracy in the diagnosis of ovarian endometrioma.

Homeobox gene expression profile indicates HOXA5 as a candidate prognostic marker in oral squamous cell carcinoma

The search for molecular markers to improve diagnosis, individualize treatment and predict behavior of tumors has been the focus of several studies. This study aimed to analyze homeobox gene expression profile in oral squamous cell carcinoma (OSCC) as well as to investigate whether some of these genes are relevant molecular markers of prognosis and/or tumor aggressiveness. Homeobox gene expression levels were assessed by microarrays and qRT-PCR in OSCC tissues and adjacent non-cancerous matched tissues (margin), as well as in OSCC cell lines. Analysis of microarray data revealed the expression of 147 homeobox genes, including one set of six at least 2-fold up-regulated, and another set of 34 at least 2-fold down-regulated homeobox genes in OSCC. After qRT-PCR assays, the three most up-regulated homeobox genes (HOXA5, HOXD10 and HOXD11) revealed higher and statistically significant expression levels in OSCC samples when compared to margins. Patients presenting lower expression of HOXA5 had poorer prognosis compared to those with higher expression (P=0.03). Additionally, the status of HOXA5, HOXD10 and HOXD11 expression levels in OSCC cell lines also showed a significant up-regulation when compared to normal oral keratinocytes. Results confirm the presence of three significantly upregulated (>4-fold) homeobox genes (HOXA5, HOXD10 and HOXD11) in OSCC that may play a significant role in the pathogenesis of these tumors. Moreover, since lower levels of HOXA5 predict poor prognosis, this gene may be a novel candidate for development of therapeutic strategies in OSCC.

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

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

Transcription profiling of Prss16 (Tssp) can be used to find additional peptidase genes that are candidates for self-peptide generation in the thymus

Positive selection (PS) in the thymus involves the presentation of self-peptides that are bound to MHC class II on the surface of cortical thymus epithelial cells (cTECs). Prss16 gene corresponds to one important element regulating the PS of CD4(+) T lymphocytes, which encodes Thymus-specific serine protease (Tssp), a cTEC serine-type peptidase involved in the proteolytic generation of self-peptides. Nevertheless, additional peptidase genes participating in the generation of self-peptides need to be found. Because of its role in the mechanism of PS and its expression in cTECs, the Prss16 gene might be used as a transcriptional marker to identify new genes that share the same expression profile and that encode peptidases in the thymus. To test this hypothesis, we compared the differential thymic expression of 4,500 mRNAs of wild-type (WT) C57BL/6 mice with their respective Prss16-knockout (KO) mutants by using microarrays. From these, 223 genes were differentially expressed, of which 115 had known molecular/biological functions. Four endopeptidase genes (Casp1, Casp2, Psmb3 and Tpp2) share the same expression profile as the Prss16 gene; i.e., induced in WT and repressed in KO while one endopeptidase gene, Capns1, features opposite expression profile. The Tpp2 gene is highlighted because it encodes a serine-type endopeptidase functionally similar to the Tssp enzyme. Profiling of the KO mice featured down-regulation of Prss16, as expected, along with the genes mentioned above. Considering that the Prss16-KO mice featured impaired PS, the shared regulation of the four endopeptidase genes suggested their participation in the mechanism of self-peptide generation and PS.

Immbolization of uricase enzyme in Langmuir and Langmuir-Blodgett films of fatty acids: Possible use as a uric acid sensor

Preserving the enzyme structure in solid films is key for producing various bioelectronic devices, including biosensors, which has normally been performed with nanostructured films that allow for control of molecular architectures. In this paper, we investigate the adsorption of uricase onto Langmuir monolayers of stearic acid (SA), and their transfer to solid supports as Langmuir Blodgett (LB) films. Structuring of the enzyme in beta-sheets was preserved in the form of 1-layer LB film, which was corroborated with a higher catalytic activity than for other uricase-containing LB film architectures where the beta-sheets structuring was not preserved. The optimized architecture was also used to detect uric acid within a range covering typical concentrations in the human blood. The approach presented here not only allows for an optimized catalytic activity toward uric acid but also permits one to explain why some film architectures exhibit a superior performance. (C) 2011 Elsevier Inc. All rights reserved.

Improving the performance of a glucose biosensor using an ionic liquid for enzyme immobilization. On the chemical interaction between the biomolecule, the ionic liquid and the cross-linking agent

The effect of the room temperature ionic liquid (1-butyl-2,3-dimethylimidazolium tetrafluoroborate ([BMMI][BF4])) on the immobilization of glucose oxidase (GOx) was studied. The electrochemical performance of biosensors prepared following different protocols indicated a beneficial effect of the ionic liquid on the analytical parameters. The chemical interaction between GOx, [BMMI][BF4] and glutaraldehyde was investigated using UV-visible spectroscopy (UV-vis) and circular dichroism (CD). Structural changes of the biomolecule were observed to depend on the method used for the immobilization. (C) 2011 Elsevier Ltd. All rights reserved.

Lupus anticoagulant: a marker for stroke and venous thrombosis in primary Sjogren's syndrome

Antiphospholipid antibodies (aPL) and antiphospholipid syndrome (APS) have been described in primary Sjogren's syndrome (pSS) with controversial findings regarding aPL prevalence and their association with thrombotic events. We evaluated 100 consecutive pSS patients (American-European criteria) and 89 age-gender-ethnicity-matched healthy controls for IgG/IgM anticardiolipin (aCL), IgG/IgM anti-beta2-glycoprotein-I (a beta 2GPI), and lupus anticoagulant (LA) (positivity according to APS Sydney's criteria). Clinical analysis followed standardized interview and physical examination assessing thrombotic and nonthrombotic APS manifestations and thrombosis risk factors. aPLs were detected in 16 % patients and 5.6 % controls (p = 0.035). LA was the most common aPL in patients (9 %), followed by a beta 2GPI (5 %) and aCL (4 %). Thrombotic events occurred in five patients [stroke in two, myocardial infarction in one and deep-vein thrombosis (DVT) in four], but in none of controls (p = 0.061). Mean age at time of stroke was 35 years. Three patients with thrombotic events (including the two with stroke) had APS (Sydney's criteria) and were positive exclusively for LA. Comparison of patients with (n = 16) and without (n = 84) aPL revealed similar mean age, female predominance, and ethnicity (p > =0.387). Frequencies of livedo reticularis (25 vs. 4.8 %, p = 0.021), stroke (12.5 vs. 0 %, p = 0.024), and DVT (18.8 vs. 1.2 %, p = 0.013) were significantly higher in APL + patients. Conversely, frequencies of hypertension, dyslipidemia, diabetes, obesity, smoking, sedentarism, and hormonal contraception were similar in patients with or without aPL (p a parts per thousand yenaEuro parts per thousand 0.253). Our study identified LA as an important marker for APS in pSS, particularly for stroke in young patients, warranting routine evaluation of these antibodies and rigorous intervention in modifiable risk factors.

A marker-and-cell approach to free surface 2-D multiphase flows

This work describes a methodology to simulate free surface incompressible multiphase flows. This novel methodology allows the simulation of multiphase flows with an arbitrary number of phases, each of them having different densities and viscosities. Surface and interfacial tension effects are also included. The numerical technique is based on the GENSMAC front-tracking method. The velocity field is computed using a finite-difference discretization of a modification of the NavierStokes equations. These equations together with the continuity equation are solved for the two-dimensional multiphase flows, with different densities and viscosities in the different phases. The governing equations are solved on a regular Eulerian grid, and a Lagrangian mesh is employed to track free surfaces and interfaces. The method is validated by comparing numerical with analytic results for a number of simple problems; it was also employed to simulate complex problems for which no analytic solutions are available. The method presented in this paper has been shown to be robust and computationally efficient. Copyright (c) 2012 John Wiley & Sons, Ltd.

Relationship between global structural parameters and Enzyme Commission hierarchy: Implications for function prediction

In protein databases there is a substantial number of proteins structurally determined but without function annotation. Understanding the relationship between function and structure can be useful to predict function on a large scale. We have analyzed the similarities in global physicochemical parameters for a set of enzymes which were classified according to the four Enzyme Commission (EC) hierarchical levels. Using relevance theory we introduced a distance between proteins in the space of physicochemical characteristics. This was done by minimizing a cost function of the metric tensor built to reflect the EC classification system. Using an unsupervised clustering method on a set of 1025 enzymes, we obtained no relevant clustering formation compatible with EC classification. The distance distributions between enzymes from the same EC group and from different EC groups were compared by histograms. Such analysis was also performed using sequence alignment similarity as a distance. Our results suggest that global structure parameters are not sufficient to segregate enzymes according to EC hierarchy. This indicates that features essential for function are rather local than global. Consequently, methods for predicting function based on global attributes should not obtain high accuracy in main EC classes prediction without relying on similarities between enzymes from training and validation datasets. Furthermore, these results are consistent with a substantial number of studies suggesting that function evolves fundamentally by recruitment, i.e., a same protein motif or fold can be used to perform different enzymatic functions and a few specific amino acids (AAs) are actually responsible for enzyme activity. These essential amino acids should belong to active sites and an effective method for predicting function should be able to recognize them. (C) 2012 Elsevier Ltd. All rights reserved.

Enzyme activity in the small intestine of goat kids during the period of passive immunity acquisition

Enzyme activity of protein and carbohydrate degradation in small intestinal mucosa was investigated in goat kids fed with lyophilized bovine and goat colostrum. At 0,7 and 14 h of life 15 male newborns received 5% of body weight of lyophilized bovine colostrum and 14 goat colostrum, both with 55 mg/mL of IgG. Duodenum, jejunum and ileum samples were collected at 18,36 and 96 h of life. Three animals were sampled at birth, without colostrum intake. Activity of aminopeptidase N and A, dipeptidil peptidase IV, lactase, maltase and sucrase was determined as one international unit per gram of tissue. Intracellular enzymatic activity of acid phosphatase was observed by histochemistry in tissue section. Only the activity of aminopeptidase A in the ileum was affected by treatment, with a greater value for LBC than for GC (P < 0.05). The aminopeptidase N activity was the highest at 36 h in the duodenum (P < 0.05) and lowest at 96 h in the jejunum (P < 0.05). Dipeptidil peptidase IV activity was highest at 36 h in the duodenum (P < 0.05), lowest at 96 h in the jejunum (P < 0.05) and higher at 36 h than at 96 h in the ileum (P < 0.05). Aminopeptidase A activity in the ileum was highest at 36 h (P < 0.05), followed by 18 and 96 h of life (P < 0.05). Lactase activity in the duodenum increased from 18 to 36 h and from 36 to 96 h in the jejunum (P < 0.05). Maltase activity increased only in the duodenum from 18 to 96 h (P < 0.05). Sucrase activity in the jejunum decreased from 18 to 36 h and from 36 to 96 h in the ileum (P < 0.05). At birth, activity of most enzymes was similar to that at later times (P < 0.05). Histochemistry analyses showed a higher frequency of lysosomes with acid phosphatase activity in the duodenum, especially at 36 h of life. In the jejunum, the presence of lysosomes with acid phosphatase activity was the highest at 96 h, followed by 36 and 18 h of life. In the ileum, all samples showed low presence of lysosomes with acid phosphatase activity. These results indicate that lyophilized bovine colostrum, as a heterologous source of antibodies or nutrients, is a possible alternative management tool for goats. The present work also suggests that in the first 4 days of life, enzyme activity in the intestinal epithelium of goats is still not fully stimulated, which is an important characteristic for these animals that depend on macromolecule absorption to acquire passive protection after birth. (C) 2012 Elsevier B.V. All rights reserved.

Enzyme replacement prevents enamel defects in hypophosphatasia mice

Hypophosphatasia (HPP) is the inborn error of metabolism characterized by deficiency of alkaline phosphatase activity, leading to rickets or osteomalacia and to dental defects. HPP occurs from loss-of-function mutations within the gene that encodes the tissue-nonspecific isozyme of alkaline phosphatase (TNAP). TNAP knockout (Alpl-/-, aka Akp2-/-) mice closely phenocopy infantile HPP, including the rickets, vitamin B6-responsive seizures, improper dentin mineralization, and lack of acellular cementum. Here, we report that lack of TNAP in Alpl-/- mice also causes severe enamel defects, which are preventable by enzyme replacement with mineral-targeted TNAP (ENB-0040). Immunohistochemistry was used to map the spatiotemporal expression of TNAP in the tissues of the developing enamel organ of healthy mouse molars and incisors. We found strong, stage-specific expression of TNAP in ameloblasts. In the Alpl-/- mice, histological, mu CT, and scanning electron microscopy analysis showed reduced mineralization and disrupted organization of the rods and inter-rod structures in enamel of both the molars and incisors. All of these abnormalities were prevented in mice receiving from birth daily subcutaneous injections of mineral-targeting, human TNAP at 8.2?mg/kg/day for up to 44 days. These data reveal an important role for TNAP in enamel mineralization and demonstrate the efficacy of mineral-targeted TNAP to prevent enamel defects in HPP. (C) 2012 American Society for Bone and Mineral Research.

Characterization of Ectonucleotidases in Human Medulloblastoma Cell Lines: ecto-5 ' NT/CD73 in Metastasis as Potential Prognostic Factor

Medulloblastoma (MB) is the most common malignant brain tumor in children and occurs mainly in the cerebellum. Important intracellular signaling molecules, such those present in the Sonic Hedgehog and Wnt pathways, are involved in its development and can also be employed to determine tumor grade and prognosis. Ectonucleotidases, particularly ecto-5'NT/CD73, are important enzymes in the malignant process of different tumor types regulating extracellular ATP and adenosine levels. Here, we investigated the activity of ectonucleotidases in three malignant human cell lines: Daoy and ONS76, being representative of primary MB, and the D283 cell line, derived from a metastatic MB. All cell lines secreted ATP into the extracellular medium while hydrolyze poorly this nucleotide, which is in agreement with the low expression and activity of pyrophosphate/phosphodiesterase, NTPDases and alkaline phosphatase. The analysis of AMP hydrolysis showed that Daoy and ONS76 completely hydrolyzed AMP, with parallel adenosine production (Daoy) and inosine accumulation (ONS76). On the other hand, D283 cell line did not hydrolyze AMP. Moreover, primary MB tumor cells, Daoy and ONS76 express the ecto-5'NT/CD73 while D283 representative of a metastatic tumor, revealed poor expression of this enzyme, while the ecto-adenosine deaminase showed higher expression in D283 compared to Daoy and ONS76 cells. Nuclear beta-catenin has been suggested as a marker for MB prognosis. Further it can promotes expression of ecto-5'NT/CD73 and suppression of adenosine deaminase. It was observed that Daoy and ONS76 showed greater nuclear beta-catenin immunoreactivity than D283, which presented mainly cytoplasmic immunoreactivity. In summary, the absence of ecto-5'NT/CD73 in the D283 cell line, a metastatic MB phenotype, suggests that high expression levels of this ectonucleotidase could be correlated with a poor prognosis in patients with MB.

Glutathione Peroxidase 1 Pro198Leu Polymorphism in Brazilian Alzheimer's Disease Patients: Relations to the Enzyme Activity and to Selenium Status

Background/Aims: Oxidative stress plays a central role in Alzheimer's disease (AD). Pro198Leu cytosolic glutathione peroxidase (GPx1) polymorphism seems to be associated with a lower activity of this enzyme, but there are no studies with AD patients. Thus, the aim was to determine the frequency of the GPx1 Pro198Leu polymorphism in AD patients and to verify its relation to glutathione peroxidase (GPx) activity and selenium (Se) status. Methods:The study was carried out in a group of AD elderly (n = 28) compared to a control group (n = 29). Blood Se concentrations were measured through hydride generation atomic absorption spectroscopy. GPx activity was determined using a commercial kit, and the polymorphism using amplified DNA sequencing. Results:The distribution of genotypes was not different between groups. The variant allele frequency was 0.179 (AD group) and 0.207 (control group). Although no differences regarding GPx activity were found between individuals with different genotypes, lower blood Se levels were found in Pro/Pro AD patients compared to Pro/Pro control subjects, which was not found in the Pro/Leu groups. Moreover, the association between the erythrocyte Se concentration and GPx activity was affected by the Pro198Leu genotype. Conclusions: Results indicate that this polymorphism had apparently affected Se status in AD patients and that more studies in this field are necessary. Copyright (c) 2012 S. Karger AG, Basel

Enzyme Microheterogeneous Hydration and Stabilization in Supercritical Carbon Dioxide

Supercritical carbon dioxide is a promising green-chemistry solvent for many enzyme-catalyzed chemical reactions, yet the striking stability of some enzymes in such unconventional environments is not well understood. Here, we investigate the stabilization of the Candida antarctica Lipase B (CALB) in supercritical carbon dioxide-water biphasic systems using molecular dynamics simulations. The preservation of the enzyme structure and optimal activity depend on the presence of small amounts of water in the supercritical dispersing medium. When the protein is at least partially hydrated, water molecules bind to specific sites on the enzyme surface and prevent carbon dioxide from penetrating its catalytic core. Strikingly, water and supercritical carbon dioxide cover the protein surface quite heterogeneously. In the first solvation layer, the hydrophilic residues at the surface of the protein are able to pin down patches of water, whereas carbon dioxide solvates preferentially hydrophobic surface residues. In the outer solvation shells, water molecules tend to cluster predominantly on top of the larger water patches of the first solvation layer instead of spreading evenly around the remainder of the protein surface. For CALB, this exposes the substrate-binding region of the enzyme to carbon dioxide, possibly facilitating diffusion of nonpolar substrates into the catalytic funnel. Therefore, by means of microheterogeneous solvation, enhanced accessibility of hydrophobic substrates to the active site can be achieved, while preserving the functional structure of the enzyme. Our results provide a molecular picture on the nature of the stability of proteins in nonaqueous media.

Inflammatory milieu as an early marker of kidney injury in offspring rats from diabetic mothers

The present study investigated the early presence of inflammatory response in renal tissue of young offspring from diabetic mothers. The effect of L-arginine (L-arg) supplementation was also investigated. The offspring was divided into four groups: group CO (controls); group DO (diabetic offspring); group CA (CO receiving 2% L-arg solution) and group DA (DO receiving the 2% L-arg solution). Glycemia, arterial pressure and renal function were evaluated; gene and protein expression of pro-inflammatory cytokines were also measured. Blood pressure levels were significantly increased in 2 and 6 month-old DO rats, whereas L-arg administration caused a significant decrease in the DA group, at both ages. DO rats showed a significantly blunted glycemic response to exogenous insulin. In 2 month-old DO animals, renal protein expression of pro-inflammatory molecules was significantly increased. At six months of age, we also observed an increase in gene expression of pro-inflammatory molecules, whereas L-arg supplementation prevented this increase at both ages. Our data suggest that activation of inflammatory pathways is present early in the kidney of DO rats, and that L-arg can attenuate the expression of these markers of tissue inflammation. Our results also reinforce the concept that intrauterine environmental factors are a fundamental determinant in the development of metabolic and vascular diseases later in life. (C) 2012 Elsevier B.V. All rights reserved.