Importance of Protein-tyrosine Phosphatase-α Catalytic Domains for Interactions with SHP-2 and Interleukin-1-induced Matrix Metalloproteinase-3 Expression

Interleukin-1 (IL-1) induces extracellular matrix degradation as a result of increased expression of matrix metalloproteinases (MMPs). We examined adhesion-restricted signaling pathways that enable IL-1-induced MMP release in human gingival and murine fibroblasts. Of the seven MMPs and three tissue inhibitors of MMPs screened, IL-1 enhanced release only of MMP3 when cells formed focal adhesions. Inhibition of protein-tyrosine phosphatases (PTPs), which are enriched in focal adhesions, blocked IL-1-induced MMP3 release. Accordingly, in contrast to wild-type cells, fibroblasts null for PTPα did not exhibit IL-1-induced MMP3 release. IL-1 treatment enhanced the recruitment of SHP-2 and PTPα to focal adhesions and the association of PTPα with SHP-2. Pulldown assays confirmed a direct interaction between PTPα and SHP-2, which was dependent on the intact, membrane-proximal phosphatase domain of PTPα. Interactions between SHP-2 and PTPα, recruitment of SHP-2 to focal adhesions, IL-1-induced ERK activation, and MMP3 expression were all blocked by point mutations in the phosphatase domains of PTPα. These data indicate that IL-1-induced signaling through focal adhesions leading to MMP3 release and interactions between SHP-2 and PTPα are dependent on the integrity of the catalytic domains of PTPα.     

Expression of Serum Amyloid A in Human Ovarian Epithelial Tumors: Implication for a Role in Ovarian Tumorigenesis

Serum amyloid A (SAA) is an acute phase protein which is expressed primarily in the liver as a part of the systemic response to various injuries and inflammatory stimuli; its expression in ovarian tumors has not been described. Here, we investigated the expression of SAA in human benign and malignant ovarian epithelial tumors. Non-radioactive in situ hybridization applied on ovarian paraffin tissue sections revealed mostly negative SAA mRNA expression in normal surface epithelium. Expression was increased gradually as epithelial cells progressed through benign and borderline adenomas to primary and metastatic adenocarcinomas. Similar expression pattern of the SAA protein was observed by immunohistochemical staining. RT-PCR analysis confirmed the overexpression of the SAA1 and SAA4 genes in ovarian carcinomas compared with normal ovarian tissues. In addition, strong expression of SAA mRNA and protein was found in the ovarian carcinoma cell line OVCAR-3. Finally, patients with ovarian carcinoma had high SAA serum levels, which strongly correlated with high levels of CA-125 and C-reactive protein. Enhanced expression of SAA in ovarian carcinomas may play a role in ovarian tumorigenesis and may have therapeutic application. (J Histochem Cytochem 58:1015–1023, 2010)     

Heparanase 2 Interacts with Heparan Sulfate with High Affinity and Inhibits Heparanase Activity

Heparanase activity is highly implicated in cell dissemination associated with tumor metastasis, angiogenesis, and inflammation. Heparanase expression is induced in many hematological and solid tumors, associated with poor prognosis. Heparanase homolog, termed heparanase 2 (Hpa2), was cloned based on sequence homology. Detailed characterization of Hpa2 at the biochemical, cellular, and clinical levels has not been so far reported, and its role in normal physiology and pathological disorders is obscure. We provide evidence that unlike heparanase, Hpa2 is not subjected to proteolytic processing and exhibits no enzymatic activity typical of heparanase. Notably, the full-length Hpa2c protein inhibits heparanase enzymatic activity, likely due to its high affinity to heparin and heparan sulfate and its ability to associate physically with heparanase. Hpa2 expression was markedly elevated in head and neck carcinoma patients, correlating with prolonged time to disease recurrence (follow-up to failure; p = 0.006) and inversely correlating with tumor cell dissemination to regional lymph nodes (N-stage; p = 0.03). Hpa2 appears to restrain tumor metastasis, likely by attenuating heparanase enzymatic activity, conferring a favorable outcome of head and neck cancer patients.     

Agmatine stimulates hepatic fatty acid oxidation: a possible mechanism for up-regulation of ureagenesis

We demonstrated previously in a liver perfusion system that agmatine increases oxygen consumption as well as the synthesis of N-acetylglutamate and urea by an undefined mechanism. In this study our aim was to identify the mechanism(s) by which agmatine up-regulates ureagenesis. We hypothesized that increased oxygen consumption and N-acetylglutamate and urea synthesis are coupled to agmatine-induced stimulation of mitochondrial fatty acid oxidation. We used 13C-labeled fatty acid as a tracer in either a liver perfusion system or isolated mitochondria to monitor fatty acid oxidation and the incorporation of 13C-labeled acetyl-CoA into ketone bodies, tricarboxylic acid cycle intermediates, amino acids, and N-acetylglutamate. With [U-13C16] palmitate in the perfusate, agmatine significantly increased the output of 13C-labeled beta-hydroxybutyrate, acetoacetate, and CO2, indicating stimulated fatty acid oxidation. The stimulation of [U-13C16]palmitate oxidation was accompanied by greater production of urea and a higher 13C enrichment in glutamate, N-acetylglutamate, and aspartate. These observations suggest that agmatine leads to increased incorporation and flux of 13C-labeled acetyl-CoA in the tricarboxylic acid cycle and to increased utilization of 13C-labeled acetyl-CoA for synthesis of N-acetylglutamate. Experiments with isolated mitochondria and 13C-labeled octanoic acid also demonstrated that agmatine increased synthesis of 13C-labeled beta-hydroxybutyrate, acetoacetate, and N-acetylglutamate. The current data document that agmatine stimulates mitochondrial beta-oxidation and suggest a coupling between the stimulation of hepatic beta-oxidation and up-regulation of ureagenesis. This action of agmatine may be mediated via a second messenger such as cAMP, and the effects on ureagenesis and fatty acid oxidation may occur simultaneously and/or independently.     

Intermediate filament protein profiles of human testicular non-seminomatous germ cell tumors: correlation of cytokeratin synthesis to cell differentiation

The patterns of cytoskeletal differentiation were studied in 20 testicular non-seminomatous germ cell tumors by immunohistochemistry, using diverse monoclonal antibodies specific for different intermediate filament (IF) proteins and for desmoplakin. Immunofluorescence and immunoperoxidase methods on both formalin-fixed and frozen tissues were applied, in some cases together with a gel electrophoretic analysis of IF proteins. The tumors examined included embryonal carcinoma (EC), endodermal sinus tumor (EST), choriocarcinoma and teratoma. Nine of the tumors were composed of only one histological type, the others showed mixed components. Cytokeratins 8 and 18 were identified in all these neoplasms, but their immunostaining was weak in ECs. Cytokeratin 19 was absent or very scarce in ECs, but strongly expressed in ESTs, choriocarcinomas and teratomas, thus allowing the identification of small EST and choriocarcinoma elements in ECs even when they were morphologically not obvious. Occasionally, some cells in ECs and ESTs also stained for cytokeratins 4 and/or 17, indicating potential for epithelial stratification. The majority of the germ cell tumors showed varied amounts of vimentin, often in co-existence with cytokeratins. Neurofilaments were demonstrated in scattered tumor cells in a single case of EST. In the teratomas studied, each type of tissue component present showed the expected IF protein. However, in many germ cell tumors some stromal cells and blood vessels contained, in addition to vimentin and desmin, also cytokeratins 8 and 18. This heterogeneity of the cytoskeletal profile of germ cell tumors is indicative of the varied differentiation potential inherent in these neoplasms.(ABSTRACT TRUNCATED AT 250 WORDS)     

Evidence of the participation of remote residues in the catalytic activity of Co-type nitrile hydratase from Pseudomonas putida

Active sites may be regarded as layers of residues, whereby the residues that interact directly with substrate also interact with residues in a second shell and these in turn interact with residues in a third shell. These residues in the second and third layers may have distinct roles in maintaining the essential chemical properties of the first-shell catalytic residues, particularly their spatial arrangement relative to the substrate binding pocket, and their electrostatic and dynamic properties. The extent to which these remote residues participate in catalysis and precisely how they affect first-shell residues remains unexplored. To improve our understanding of the roles of second- and third-shell residues in catalysis, we used THEMATICS to identify residues in the second and third shells of the Co-type nitrile hydratase from Pseudomonas putida (ppNHase) that may be important for catalysis. Five of these predicted residues, and three additional, conserved residues that were not predicted, have been conservatively mutated, and their effects have been studied both kinetically and structurally. The eight residues have no direct contact with the active site metal ion or bound substrate. These results demonstrate that three of the predicted second-shell residues (α-Asp164, β-Glu56, and β-His147) and one predicted third-shell residue (β-His71) have significant effects on the catalytic efficiency of the enzyme. One of the predicted residues (α-Glu168) and the three residues not predicted (α-Arg170, α-Tyr171, and β-Tyr215) do not have any significant effects on the catalytic efficiency of the enzyme.     

Detection of cross-infection associated to a Brazilian PCR-ribotype of <Emphasis Type="Italic">Clostridium difficile</Emphasis> in a university hospital in Rio de Janeiro,Brazil

Clostridium difficile is an important nosocomial enteric pathogen and is the etiological agent of pseudomembranous colites. Recently, the rates of C. difficile infection (CDI) have increased worldwide, but in Brazil few data about this situation and the incidence of clonal types of C. difficile exist. This study aimed to isolate and characterize C. difficile strains from samples obtained of a university hospital (HUCFF) in Rio de Janeiro city, Brazil. CDI was identified by ELISA in 27.1% of HUCFF-in-patients enrolled in the study, and the bacterium was recovered from eight of these fecal samples. All strains, except one, presented tcdA and tcdB genes and presented neither the cdtA and cdtB genes nor any significant deletions in the tcdC gene. All strains were sensitive to metronidazole, vancomycin and moxifloxacin, and resistant to clindamycin, ciprofloxacin and levofloxacin. PCR-ribotyping and PFGE revealed four different clonal types among the isolates. The Brazilian PCR-ribotype 133 accounted for 50% of strains isolated, and PCR-ribotype 233 strains were obtained from 25% of the in-patients. The prevalence and resurgence of the Brazilian PCR-ribotype 133 among the hospitalized patients of HUCFF was established, and cross-infection of different patients associated to the same PCR-ribotypes was detected. Our results emphasize the importance of the diagnosis and control of CDI in order to prevent the emergence of specific clones that can lead to C. difficile-associated outbreaks in Brazilian hospitals.     

Extensive metabolic cross-talk in melon fruit revealed by spatial and developmental combinatorial metabolomics

? Variations in tissue development and spatial composition have a major impact on the nutritional and organoleptic qualities of ripe fleshy fruit, including melon (Cucumis melo). To gain a deeper insight into the mechanisms involved in these changes, we identified key metabolites for rational food quality design. ? The metabolome, volatiles and mineral elements were profiled employing an unprecedented range of complementary analytical technologies. Fruits were followed at a number of time points during the final ripening process and tissues were collected across the fruit flesh from rind to seed cavity. Approximately 2000 metabolite signatures and 15 mineral elements were determined in an assessment of temporal and spatial melon fruit development. ? This study design enabled the identification of: coregulated hubs (including aspartic acid, 2-isopropylmalic acid, β-carotene, phytoene and dihydropseudoionone) in metabolic association networks; global patterns of coordinated compositional changes; and links of primary and secondary metabolism to key mineral and volatile fruit complements. ? The results reveal the extent of metabolic interactions relevant to ripe fruit quality and thus have enabled the identification of essential candidate metabolites for the high-throughput screening of melon breeding populations for targeted breeding programmes aimed at nutrition and flavour improvement.     

Predicting diabetic nephropathy using a multifactorial genetic model

Aims

The tendency to develop diabetic nephropathy is, in part, genetically determined, however this genetic risk is largely undefined. In this proof-of-concept study, we tested the hypothesis that combined analysis of multiple genetic variants can improve prediction.

Methods

Based on previous reports, we selected 27 SNPs in 15 genes from metabolic pathways involved in the pathogenesis of diabetic nephropathy and genotyped them in 1274 Ashkenazi or Sephardic Jewish patients with Type 1 or Type 2 diabetes of >10 years duration. A logistic regression model was built using a backward selection algorithm and SNPs nominally associated with nephropathy in our population. The model was validated by using random “training” (75%) and “test” (25%) subgroups of the original population and by applying the model to an independent dataset of 848 Ashkenazi patients.

Results

The logistic model based on 5 SNPs in 5 genes (HSPG2, NOS3, ADIPOR2, AGER, and CCL5) and 5 conventional variables (age, sex, ethnicity, diabetes type and duration), and allowing for all possible two-way interactions, predicted nephropathy in our initial population (C-statistic = 0.672) better than a model based on conventional variables only (C = 0.569). In the independent replication dataset, although the C-statistic of the genetic model decreased (0.576), it remained highly associated with diabetic nephropathy (χ² = 17.79, p<0.0001). In the replication dataset, the model based on conventional variables only was not associated with nephropathy (χ² = 3.2673, p = 0.07).

Conclusion

In this proof-of-concept study, we developed and validated a genetic model in the Ashkenazi/Sephardic population predicting nephropathy more effectively than a similarly constructed non-genetic model. Further testing is required to determine if this modeling approach, using an optimally selected panel of genetic markers, can provide clinically useful prediction and if generic models can be developed for use across multiple ethnic groups or if population-specific models are required.     

Reading Biochips by Raman and Surface-Enhanced Raman Spectroscopies

Biochips are a rapidly increasing research field, driven by the versatility of sensing devices and the importance of their applications. The regular approaches for creating biochips and for reading them suffer from some limitations, motivating development of miniature biochips and label-free formats. To push forward these challenges, we have chosen to combine the methods of printing of droplets of synthetic receptors by pipettes or nanofountain pens with detection by Raman spectroscopy or its surface-assisted plasmon variant, namely, surface-enhanced Raman spectroscopy (SERS). The selected receptors included molecularly imprinted polymers (MIPs), produced by polymerization of functional and cross-linking monomers around a molecular template, the β-blocking drug propranolol. The measured Raman and SERS spectra of the MIP constituents enabled identification of the template presence and consequently chemical imaging of individual and multiple dots in an array. This concept, combining nanolithography techniques with SERS paves the road toward miniaturized arrayed MIP sensors with label-free, specific and quantitative molecular recognition.