The Eph Tyrosine Kinase Receptors EphB2 and EphA2 Are Novel Proteolytic Substrates of Tissue Factor/Coagulation Factor VIIa

Tissue factor (TF) binds the serine protease factor VIIa (FVIIa) to form a proteolytically active complex that can trigger coagulation or activate cell signaling. Here we addressed the involvement of tyrosine kinase receptors (RTKs) in TF/FVIIa signaling by antibody array analysis and subsequently found that EphB2 and EphA2 of the Eph RTK family were cleaved in their ectodomains by TF/FVIIa. We used N-terminal Edman sequencing and LC-MS/MS analysis to characterize the cleaved Eph isoforms and identified a key arginine residue at the cleavage site, in agreement with the tryptic serine protease activity of FVIIa. Protease-activated receptor 2 (PAR2) signaling and downstream coagulation activity was non-essential in this context, in further support of a direct cleavage by TF/FVIIa. EphB2 was cleaved by FVIIa concentrations in the subnanomolar range in a number of TF expressing cell types, indicating that the active cellular pool of TF was involved. FVIIa caused potentiation of cell repulsion by the EphB2 ligand ephrin-B1, demonstrating a novel proteolytical event to control Eph-mediated cell segregation. These results define Eph RTKs as novel proteolytical targets of TF/FVIIa and provide new insights into how TF/FVIIa regulates cellular functions independently of PAR2.     

Changes in Intake of Fruits and Vegetables and Weight Change in United States Men and Women Followed for Up to 24 Years: Analysis from Three Prospective Cohort Studies

BackgroundCurrent dietary guidelines recommend eating a variety of fruits and vegetables. However, based on nutrient composition, some particular fruits and vegetables may be more or less beneficial for maintaining or achieving a healthy weight. We hypothesized that greater consumption of fruits and vegetables with a higher fiber content or lower glycemic load would be more strongly associated with a healthy weight.ConclusionsIncreased consumption of fruits and non-starchy vegetables is inversely associated with weight change, with important differences by type suggesting that other characteristics of these foods influence the magnitude of their association with weight change.     

The effects of different training modalities on monocarboxylate transporters MCT1 and MCT4, hypoxia inducible factor-1α (HIF-1α), and PGC-1α gene expression in rat skeletal muscles

Molecular Biology Reports - The research literature suggests that different training modalities cause various patterns in training-induced genes expression. This study aimed to investigate the...     

A novel function for a redox-related LEA protein (SAG21/AtLEA5) in root development and biotic stress responses

SAG21/AtLEA5 belongs to the late embryogenesis-associated (LEA) protein family. Although it has been implicated in growth and redox responses, its precise roles remain obscure. To address this problem, we characterized root and shoot development and response to biotic stress in SAG21/AtLEA5 over-expressor (OEX) and antisense (AS) lines. AS lines exhibited earlier flowering and senescence and reduced shoot biomass. Primary root length was reduced in AS lines, as was the number of laterals relative to the primary root. Root hair number was unchanged but root hair length was proportional to SAG21/AtLEA5 expression level, with longer root hairs in OEX lines and shorter root hairs in AS, relative to wild type. Growth of the fungal nectroph, Botrytis cinerea and of a virulent bacterial pathogen (Pseudomonas syringae pv. tomato) was affected by SAG21/AtLEA5 expression; however, growth of an avirulent P.syringae strain was unaffected. A SAG21/AtLEA5-YFP fusion was localized to mitochondria, raising the intriguing possibility that SAG21 interacts with proteins involved in mitochondrial ROS signalling, which in turn, impacts on root development and pathogen responses.     

Efficiency of adult mouse spermatogonial stem cell colony formation under several culture conditions

The aim of this study was to compare the in vitro effects of glial cell line-derived neurotrophic factor, stem cell factor, granulocyte macrophage-colony stimulating factor, and co-culture with Sertoli cells on the efficiency of adult mouse spermatogonial stem cells colony formation. For these purpose, both Sertoli and spermatogonial cells were isolated from adult mouse testes. The identity of the cells was confirmed through analysis of alkaline phosphatase activity, immunocytochemistry against OCT-4, c-kit, and vimentin, and also by transplantation of these cells in the recipient testes. The isolated spermatogonial cells were treated either with various concentrations of the above mentioned factors or co-cultured with Sertoli cells for 3 wk. The spermatogonial cells of the resulting colonies were transplanted via rete testis into the mouse testes, which were irradiated with 14 Gy. The results indicated that glial cell line-derived neurotrophic factor is the most appropriate factor for in vitro colonization of adult mice spermatogonial cells compared with other cytokines and growth factors. A short-term co-culture with Sertoli cells showed a significant increase in the number and diameter of the colonies compared with the treated growth factors and the control group. We have also demonstrated that mouse spermatogonial stem cells in the colonies after co-culturing with Sertoli cells could induce spermatogenesis in the recipient testes after transplantation.     

Fungal glucoamylases

Fungi are employed to produce industrially important glucoamylases. Most glucoamylases are glycosylated. Glycosylation enhances the enzyme stability. Glucoamylases contain both starch binding and catalytic binding domains, the former being responsible for activity on raw (insoluble) starch. Proteases may act on this domain causing the enzyme to lose its activity on insoluble starch. Optimal activity is observed at pH 4.5 to 6.5 and 50 to 70 degrees C. Glucoamylases contain up to 7 sub-sites with highly varying affinity. They can be produced by different methods including submerged, solid state and semi-solid state fermentation processes.     

Isolation of Bacteria Able to Metabolize High Concentrations of Formaldehyde

Summary Nineteen bacterial strains able to degrade and metabolize formaldehyde as a sole carbon source were isolated from soil and wastewater of a formaldehyde production factory. The samples were cultured in complex and mineral salts media containing 370 mg formaldehyde/l. The bacterial strains were identified to be Pseudomonas pseudoalcaligenes, P. aeruginosa, P. testosteroni, P. putida, and Methylobacterium extorquens. After adaptation of these microorganisms to high concentrations of formaldehyde; two isolated strains of M. extorquens (strains ESS and PSS) and four strains of P. pseudoalcaligenes (strains LSW, SSW, NSW and OSS) degraded 1850 mg formaldehyde/l, where as P. pseudoalcaligenes strain OSS completely consumed 3700 mg of formaldehyde/l after 24 h and degraded 70% of 5920 mg of formaldehyde/l after 72 h.     

Coordination and binding geometry of methyl-coenzyme M in the red1m state of methyl-coenzyme M reductase

Methane formation in methanogenic Archaea is catalyzed by methyl-coenzyme M reductase (MCR) and takes place via the reduction of methyl-coenzyme M (CH₃-S-CoM) with coenzyme B (HS-CoB) to methane and the heterodisulfide CoM-S–S-CoB. MCR harbors the nickel porphyrinoid coenzyme F₄₃₀ as a prosthetic group, which has to be in the Ni(I) oxidation state for the enzyme to be active. To date no intermediates in the catalytic cycle of MCR_red1 (red for reduced Ni) have been identified. Here, we report a detailed characterization of MCR_red1m (“m” for methyl-coenzyme M), which is the complex of MCR_red1a (“a” for absence of substrate) with CH₃-S-CoM. Using continuous-wave and pulse electron paramagnetic resonance spectroscopy in combination with selective isotope labeling (¹³C and ²H) of CH₃-S-CoM, it is shown that CH₃-S-CoM binds in the active site of MCR such that its thioether sulfur is weakly coordinated to the Ni(I) of F₄₃₀. The complex is stable until the addition of the second substrate, HS-CoB. Results from EPR spectroscopy, along with quantum mechanical calculations, are used to characterize the electronic and geometric structure of this complex, which can be regarded as the first intermediate in the catalytic mechanism. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.