Soluble and Catalytically Active Endothelin Converting Enzyme-1 is Present in Cerebrospinal Fluid of Subarachnoid Hemorrhage Patients

Endothelin converting Enzyme-1 (ECE-1) is essential for the production of Endothelin-1 (ET-1), which is associated with vasospasm following subarachnoid hemorrhage (SAH). We have previously demonstrated the presence of a catalytically active soluble form of ECE-1 in the media of endothelial cells. We aimed to determine if this form of ECE-1 exists in vivo, in cerebrospinal fluid (CSF) of SAH patients. We examined CSF taken from SAH subjects for the presence of soluble ECE-1 using a bradykinin based quenched fluorescent substrate assay. We obtained further confirmation by characterizing the CSF mediated cleavage products of BigET-1 and BigET_18–34 (6 μg/ml) using mass spectrometry. The specificity of cleavage was confirmed using the ECE-1 inhibitor {"type":"entrez-protein","attrs":{"text":"CGS35066","term_id":"877962710","term_text":"CGS35066"}}CGS35066 5nmol/L. SAH CSF samples had mean ECE-1 activity of 0.127 ± 0.037 μmols of substrate cleaved/μl of CSF/24 h. The C-terminal peptides generated upon the cleavage of BigET-1 and BigET_18–34 were detected 48 h after incubation of these substrates with CSF. Cleavage of these substrates was inhibited by {"type":"entrez-protein","attrs":{"text":"CGS35066","term_id":"877962710","term_text":"CGS35066"}}CGS35066. Results of Western blots also produced strong evidence for the presence of truncated soluble ECE-1 in CSF. These results strongly suggest the presence of a truncated but catalytically active form of ECE-1 in the CSF of SAH subjects. Further studies are necessary to determine the biological significance of soluble ECE-1 in CSF of SAH subjects, including an association with vasospasm after SAH.Endothelin-1 (ET-1)¹ is the most potent vasoconstrictor in the central nervous system. Elevated levels of ET-1 in cerebrospinal fluid (CSF) have been implicated in the pathogenesis of cerebral vasospasm following subarachnoid hemorrhage (SAH) (1). However, it is not known whether the production of ET-1 within the CSF space contributes to the pathogenesis of vasospasm. ET-1 is produced upon the cleavage of its precursor BigET-1 by the highly specific metalloprotease Endothelin Converting Enzyme-1 (ECE-1). The rate-limiting step of ET-1 production is the expression and localization of ECE-1, whose catalytic activity is confined to the extracellular C-terminal domain. Previously, we have demonstrated that the catalytically active C terminus can be shed from the cell surface (2). This results in the release of a truncated but catalytically active form of ECE-1 into the extracellular milieu.Although the presence of a soluble form of ECE-1 has been demonstrated in vitro, it is yet to be shown in any human biological fluid. In this study, we have used a combination of mass spectrometry, Western blotting as well as quenched fluorescent substrate (QFS) based enzyme assays to demonstrate for the first time the presence of catalytically active, soluble form of ECE-1 in CSF of SAH subjects.     

Solution NMR structure of the 48-kDa IIAMannose-HPr complex of the Escherichia coli mannose phosphotransferase system

The solution structure of the 48-kDa IIA(Man)-HPr complex of the mannose branch of the Escherichia coli phosphotransferase system has been solved by NMR using conjoined rigid body/torsion angle-simulated annealing on the basis of intermolecular nuclear Overhauser enhancement data and residual dipolar couplings. IIA(Man) is dimeric and has two symmetrically related binding sites per dimer for HPr. A convex surface on HPr, formed primarily by helices 1 and 2, interacts with a deep groove at the interface of the two subunits of IIA(Man). The interaction surface on IIA(Man) is predominantly helical, comprising helix 3 from the subunit that bears the active site His-10 and helices 1, 4, and 5 from the other subunit. The total buried accessible surface area at the protein-protein interface is 1450 A(2). The binding sites on the two proteins are complementary in terms of shape and distribution of hydrophobic, hydrophilic, and charged residues. The active site histidines, His-10 of IIA(Man) and His-15 (italics indicate HPr residues) of HPr, are in close proximity. An associative transition state involving a pentacoordinate phosphoryl group with trigonal bipyramidal geometry bonded to the N-epsilon2 atom of His-10 and the N-delta1 atom of His-15 can be readily formed with negligible displacement in the backbone coordinates of the residues immediately adjacent to the active site histidines. Comparing the structures of complexes of HPr with three other structurally unrelated phosphotransferase system proteins, enzymes I, IIA(glucose), and IIA(mannitol), reveals a number of common features that provide a molecular basis for understanding how HPr specifically recognizes a wide range of diverse proteins.     

On/Off-regulation of phospholipase C-γ1-mediated signal transduction

Alterations in the production and regulation of lipid second messengers can give rise to key molecular lesions that trigger tumorigenesis and cancer progression. Especially, the hydrolysis of membrane phospholipid, phosphatidylinositol 4,5-bisphosphate (PIP2), is mediated by a specific phospholipase C (PLC), which plays important roles in the regulation of various cell functions. PLC generates two intracellular messengers, diacylglycerol and inositol 1,4,5-trisphosphate, which mediate the activation of protein kinase C (PKC) and intracellular Ca²⁺ release, respectively. Among the PLC isozymes, PLC-γ1 contains two src homology (SH) 2 domains and one SH3 domain between the X and Y catalytic domains. The SH2 domains of PLC-γ1 have been implicated in the association between PLC-γ1 and activated receptor tyrosine kinases, and the SH3 domain of PLC-γ1 has been reported to be responsible for the mitogenic effect of PLC-γ1, suggesting that PLC-γ1 exerts other actions that are independent of its lipase activity and appears to be involved in the SH domains. However, the physiological role of SH domains in the regulation of PLC-γ1 is still unclear. We have recently characterized the regulation mechanism of PLC-γ1 through protein-protein interactions. We have elucidated that PLC-γ1 can serve as a guanine nucleotide exchange factor (GEF) for dynamin-1 through direct interaction via its SH3 domain. These results indicate that GEF function of PLC-γ1 for dynamin-1 may link with PLC-γ1's mitogenic actions. The SH3 domain of PLC-γ1 can mediate Sos and PIKE activation by acting as GEF, suggesting that PLC-γ1 plays an essential role on cellular proliferation through protein–protein interaction independent of its enzymatic activity. On the other hand, we have found that Cbl directly interacts with the SH3 domain of PLC-γ1 and inhibits its tyrosine phosphorylation and enzymatic activity, suggesting that PLC-γ1 can be off-regulated by protein–protein interaction. In addition, we demonstrate that Grb2 interacts with tyrosine phosphorylated PLC-γ1 and acts as an inhibitor on PLC-γ1-mediated signaling. These results suggest that Grb2, one of the key regulators of Ras/Raf/MAPK signaling pathway, may participate in the regulation of PLC-γ1. Lastly, PLC-γ1 forms a ternary complex with Jak2 and PTP-1B and negatively regulates GH-induced Jak2 phosphorylation. Taken together, our data strengthen the hypothesis that the interaction between PLC-γ1 and effector proteins plays a key role in on- or off-regulating PLC-γ1-mediated cellular proliferation independent its enzymatic activity. These results can provide novel insights to understand how PLC-γ1 is regulated and involved in cellular growth and proliferation.     

Application of a combined approach involving classical random mutagenesis and metabolic engineering to enhance FK506 production in Streptomyces sp. RM7011

FK506 production by a mutant strain (Streptomyces sp. RM7011) induced by N-methyl-N′-nitro-N-nitrosoguanidine and ultraviolet mutagenesis was improved by 11.63-fold (94.24 mg/l) compared to that of the wild-type strain. Among three different metabolic pathways involved in the biosynthesis of methylmalonyl-CoA, only expression of propionyl-CoA carboxylase (PCC) pathway led to a 1.75-fold and 2.5-fold increase in FK506 production and the methylmalonyl-CoA pool, respectively, compared to those of the RM7011 strain. Lipase activity of the high FK506 producer mutant increased in direct proportion to the increase in FK506 yield, from low detection level up to 43.1 U/ml (12.6-fold). The level of specific FK506 production and lipase activity was improved by enhancing the supply of lipase inducers. This improvement was approximately 1.88-fold (71.5 mg/g) with the supplementation of 5 mM Tween 80, which is the probable effective stimulator in lipase production, to the R2YE medium. When 5 mM vinyl propionate was added as a precursor for PCC pathway to R2YE medium, the specific production of FK506 increased approximately 1.9-fold (71.61 mg/g) compared to that under the non-supplemented condition. Moreover, in the presence of 5 mM Tween 80, the specific FK506 production was approximately 2.2-fold (157.44 mg/g) higher than that when only vinyl propionate was added to the R2YE medium. In particular, PCC expression in Streptomyces sp. RM7011 (RM7011/pSJ1003) together with vinyl propionate feeding resulted in an increase in the FK506 titer to as much as 1.6-fold (251.9 mg/g) compared with that in RM7011/pSE34 in R2YE medium with 5 mM Tween 80 supplementation, indicating that the vinyl propionate is more catabolized to propionate by stimulated lipase activity on Tween 80, that propionyl-CoA yielded from propionate generates methylmalonyl-CoA, and that the PCC pathway plays a key role in increasing the methylmalonyl-CoA pool for FK506 biosynthesis in RM7011 strain. Overall, these results show that a combined approach involving classical random mutation and metabolic engineering can be applied to supply the limiting factor for FK506 biosynthesis, and vinyl propionate could be successfully used as a precursor of important methylmalonyl-CoA building blocks.     

Comparison of the synthetic biodegradable polymers, polylactide (PLA), and polylactic-co-glycolic acid (PLGA) as scaffolds for artificial cartilage

Chondrocytes are easily de-differentiated when cultured in monolayer, and tissue-engineered cartilage can be generated by seeding chondrocytes onto three-dimensional porous synthetic biodegradable polymers. In this study, we investigated the biochemical and molecular aspects of chondrocytes in a monolayer-culture system and selected the optimal subculture passages based on their de-differentiation. We also compared two commonly used synthetic biodegradable polymers, polylactide (PLA), and polylactic-co-glycolic acid (PLGA), for their suitability as scaffolds for artificial cartilage. De-differentiated chondrocytes were observed after two passages. These results suggested that the first cell passage was optimal for seeding as only a few chondrocytes secreted extracellular matrix components to form homogeneously compact cartilage. Substantially increased glycosaminoglycan and total collagen levels revealed that PLGA scaffolds were a better option for inducing cartilage tissue formation compared to the PLA scaffolds. Histological and immunohistochemical results showed that chondrocytes seeded into PLGA retained their morphological phenotype to a greater extent than those seeded into PLA.     

Crystal structure of filamentous aggregates of human DJ-1 formed in an inorganic phosphate-dependent manner

Mutations in the DJ-1 gene have been implicated in the autosomal recessive early onset parkinsonism. DJ-1 is a soluble dimeric protein with critical roles in response to oxidative stress and in neuronal maintenance. However, several lines of evidence suggest the existence of a nonfunctional aggregated form of DJ-1 in the brain of patients with some neurodegenerative diseases. Here, we show that inorganic phosphate, an important anion that exhibits elevated levels in patients with Parkinson disease, transforms DJ-1 into filamentous aggregates. According to the 2.4-A crystal structure, DJ-1 dimers are linearly stacked through P(i)-mediated interactions to form protofilaments, which are then bundled into a filamentous assembly.     

CD166 promotes the cancer stem-like properties of primary epithelial ovarian cancer cells

Cancer stem cells (CSCs) or tumor-initiating cells are thought to play critical roles in tumorigenesis, metastasis, drug resistance, and tumor recurrence. For the diagnosis and targeted therapy of CSCs, the molecular identity of biomarkers or therapeutic targets for CSCs needs to be clarified. In this study, we identified CD166 as a novel marker expressed in the sphere-forming CSC population of A2780 epithelial ovarian cancer cells and primary ovarian cancer cells. The CD166⁺ cells isolated from A2780 cells and primary ovarian cancer cells highly expressed CSC markers, including ALDH1a1, OCT4, and SOX2, and ABC transporters, which are implicated in the drug resistance of CSCs. The CD166⁺ cells exhibited enhanced CSC-like properties, such as increased sphere-forming ability, cell migration and adhesion abilities, resistance to conventional anti-cancer drugs, and high tumorigenic potential in a xenograft mouse model. Knockdown of CD166 expression in the sphere-forming ovarian CSCs abrogated their CSC-like properties. Moreover, silencing of CD166 expression in the sphere-forming CSCs suppressed the phosphorylation of focal adhesion kinase, paxillin, and SRC. These results suggest that CD166 plays a key role in the regulation of CSC-like properties and focal adhesion kinase signaling in ovarian cancer.     

Effect of rpoS Mutation on the Stress Response and Expression of Virulence Factors in Pseudomonas aeruginosa

The sigma factor RpoS (sigmaS) has been described as a general stress response regulator that controls the expression of genes which confer increased resistance to various stresses in some gram-negative bacteria. To elucidate the role of RpoS in Pseudomonas aeruginosa physiology and pathogenesis, we constructed rpoS mutants in several strains of P. aeruginosa, including PAO1. The PAO1 rpoS mutant was subjected to various environmental stresses, and we compared the resistance phenotype of the mutant to that of the parent. The PAO1 rpoS mutant was slightly more sensitive to carbon starvation than the wild-type strain, but this phenotype was obvious only when the cells were grown in a medium supplemented with glucose as the sole carbon source. In addition, the PAO1 rpoS mutant was hypersensitive to heat shock at 50 degrees C, increased osmolarity, and prolonged exposure to high concentrations of H2O2. In accordance with the hypersensitivity to H2O2, catalase production was 60% lower in the rpoS mutant than in the parent strain. We also assessed the role of RpoS in the production of several exoproducts known to be important for virulence of P. aeruginosa. The rpoS mutant produced 50% less exotoxin A, but it produced only slightly smaller amounts of elastase and LasA protease than the parent strain. The levels of phospholipase C and casein-degrading proteases were unaffected by a mutation in rpoS in PAO1. The rpoS mutation resulted in the increased production of the phenazine antibiotic pyocyanin and the siderophore pyoverdine. This increased pyocyanin production may be responsible for the enhanced virulence of the PAO1 rpoS mutant that was observed in a rat chronic-lung-infection model. In addition, the rpoS mutant displayed an altered twitching-motility phenotype, suggesting that the colonization factors, type IV fimbriae, were affected. Finally, in an alginate-overproducing cystic fibrosis (CF) isolate, FRD1, the rpoS101::aacCI mutation almost completely abolished the production of alginate when the bacterium was grown in a liquid medium. On a solid medium, the FRD1 rpoS mutant produced approximately 70% less alginate than did the wild-type strain. Thus, our data indicate that although some of the functions of RpoS in P. aeruginosa physiology are similar to RpoS functions in other gram-negative bacteria, it also has some functions unique to this bacterium.     

Photoperiodic influence on the body mass of bumblebee, Bombus terrestris and its copulation duration

Abstract:  The body mass of Bombus terrestris individuals is an important trait for their behavioural performance and colony organization. In this study, colonies were reared under four different photoperiodic regimes, viz. 0 : 24, 8 : 16, 16 : 8 and 24 : 0 h light : darkness (L : D) at 28°C and 50% relative humidity. The changes in body mass were observed at the stages of larvae, pupae and on the day of adult eclosion. Both the wet and dry mass of sexuals gradually decreased with increasing day length. The relationship between body mass and copulation duration revealed that copulation duration was negatively correlated with male body mass, but positively with queen body mass. Higher number of matings by males resulted in significantly higher duration of copulation.     

Cbl competitively inhibits epidermal growth factor-induced activation of phospholipase C-gamma1

Phospholipase C-gamma1 (PLC-gamma1) plays pivotal roles in cellular growth and proliferation through its two Src homology (SH) 2 domains and its single SH3 domain, which interact with signaling molecules in response to various growth factors and hormones. However, the role of the SH domains in the growth factor-induced regulation of PLC-gamma1 is unclear. By peptide-mass fingerprinting analysis we have identified Cbl as a binding protein for the SH3 domain of PLC-gamma1 from rat pheochromatocyte PC12 cells. Association of Cbl with PLC-gamma1 was induced by epidermal growth factor (EGF) but not by nerve growth factor (NGF). Upon EGF stimulation, both Cbl and PLC-gamma1 were recruited to the activated EGF receptor through their SH2 domains. Mutation of the SH2 domains of either Cbl or PLC-gamma1 abrogated the EGF-induced interaction of PLC-gamma1 with Cbl, indicating that SH2-mediated translocation is essential for the association of PLC-gamma1 and Cbl. Overexpression of Cbl attenuated EGF-induced tyrosine phosphorylation and the subsequent activation of PLC-gamma1 by interfering competitively with the interaction between PLC-gamma1 and EGFR. Taken together, these results provide the first indications that Cbl may be a negative regulator of intracellular signaling following EGF-induced PLC-gamma1 activation.