Identification of a novel proline-rich peptide-binding domain in prolyl 4-hydroxylase

Prolyl 4-hydroxylase (EC 1.14.11.2) catalyzes the hydroxylation of -X-Pro-Gly- sequences and plays a central role in the synthesis of all collagens. The [alpha(I)]2beta2 type I enzyme is effectively inhibited by poly(L-proline), whereas the [alpha(II)]2beta2 type II enzyme is not. We report here that the poly(L-proline) and (Pro-Pro-Gly)10 peptide substrate-binding domain of prolyl 4-hydroxylase is distinct from the catalytic domain and consists of approximately 100 amino acids. Peptides of 10-19 kDa beginning around residue 140 in the 517 residue alpha(I) subunit remained bound to poly(L-proline) agarose after limited proteolysis of the human type I enzyme tetramer. A recombinant polypeptide corresponding to the alpha(I) subunit residues 138-244 and expressed in Escherichia coli was soluble, became effectively bound to poly(L-proline) agarose and could be eluted with (Pro-Pro-Gly)10. This polypeptide is distinct from the SH3 and WW domains, and from profilin, and thus represents a new type of proline-rich peptide-binding module. Studies with enzyme tetramers containing mutated alpha subunits demonstrated that the presence of a glutamate and a glutamine in the alpha(II) subunit in the positions corresponding to Ile182 and Tyr233 in the alpha(I) subunit explains most of the lack of poly(L-proline) binding of the type II prolyl 4-hydroxylase. Keywords: collagen/dioxygenases/peptide-binding domain/ proline-rich/prolyl hydroxylase     

Cloning and characterization of the polyhydroxybutyrate depolymerase gene of Pseudomonas stutzeri and analysis of the function of substrate-binding domains

BACKGROUND: Overexpression of the 70 kDa heat shock protein (Hsp70) protects myocytes and neural cells from hypoxic injury. In contrast, Hsp70 induction in the kidney after ischemic or thermal preconditioning does not correlate well with protection from hypoxic injury. Herein, we directly tested if Hsp70 overexpression protects LLC-PK1 porcine tubular epithelial cells from hypoxic or thermal injury. METHODS: LLC-PK1 cells were either cotransfected with an Hsp70 and a luciferase expression vector or singly transfected with the luciferase expression vector. Loss of intracellular luciferase activity was used to assess injury after exposure to hypoxia or hyperthermia and after recovery under normal growth conditions. RESULTS: Overexpression of Hsp70 decreased loss of and improved restoration of intracellular luciferase activity in LLC-PK1 cells exposed to hyperthermia. In contrast, Hsp70 overexpression did not decrease the loss of or improve restoration of luciferase activity in cells exposed to hypoxia. CONCLUSIONS: These results suggest that Hsp70 overexpression is sufficient to protect LLC-PK1 proximal tubular cells from hyperthermia but is not sufficient for protection from hypoxia.     

简易实验性细菌性心内膜炎模型的制作方法1

目的 用α-甲型溶血链球菌建立兔细菌性心内膜炎模型。方法 自麻醉兔颈总动脉插一导管至左心室, 24 h 后随机分为空白对照组和感染组。对照组耳缘静脉注射1 ml 生理盐水, 感染组注射10⁸CFU甲型溶血链球菌, 7 d 后抽血进行血液培养, 处死后观察主动脉瓣赘生物和左心室腔内壁性赘生物的情况。结果 感染组的每个动物在主动脉瓣和左心室中底部均有大小不等的赘生物生长, 血液中均有细菌。对照组则没有。结论 用这种方法制作的兔实验性细菌性心内膜炎成功率高且简单易行。     

Using nitrogen isotope fractionation to assess abiotic reduction of nitroaromatic compounds

Compound-specific isotope analysis (CSIA) is an increasingly important tool for the qualitative and quantitative assessment of transformations of organic compounds in contaminated environments. To date, the use of CSIA has been mainly restricted to the elements C and H, although Nconstitutes a very important reactive centerfor many priority contaminants. To evaluate the potential use of N isotope effects in the fate assessment of organic contaminants, we investigated the N isotope enrichment during the abiotic reduction of 4 substituted nitroaromatic compounds (NACs), using two abiotic model reductants, namely Fe(ll) sorbed to goethite (alpha-FeOOH) and juglone (8-hydroxy-1,4-naphthoquinone) in the presence of H2S. Substantial and virtually identical isotope enrichment factors, EpsilonN, of about -30%, indicative of the breaking of one N-O bond, were found for all NACs, regardless of the reductant involved and the substitution of the NAC. These results indicate that the EpsilonN-values determined in our study could be representative for the reduction of aromatic NO2-groups and thus be used to assess the abiotic transformation of NACs qualitatively and quantitatively in complex anoxic environments.     

δ¹⁵N enrichment suggests possible source for halogenated 1'-methyl-1,2'-bipyrroles (MBPs)

Polyhalogenated 1'-methyl-1,2'-bipyrroles are natural products that biomagnify into upper trophic levels of marine food webs. Here we demonstrate that they are unusually enriched in (15)N (δ(15)N from +19.3‰ to +28.1‰) relative to other biosynthetic organic compounds measured to date and the mammals from which the compounds were isolated. We argue the (15)N enrichment likely stems from enriched precursors and/or fractionation during biosynthesis and is not from MBP degradation. We also consider possible sources of MBPs in light of these results.     

Use of Glandless Cottonseed and Sunflower Seed Products in Batter/Breading for Fried Beef Patties

This study indicates that defatted glandless cottonseed flour (GCF) 01 sunflower seed protein isolate (SI) incorporated in the batter/breading system can impart their antioxidant effect to the meat of fried beef patties. Coarsely ground lean beef patties were coated consecutively with dry ingredient (wheat flour for control and 1:1 mixture of wheat flour-GCF or wheat flour-SI for experimental samples), batter made of 1 part of dry ingredient and 2 parts of water, and finally dry ingredient. Ground beef and dry ingredients each contained 1% salt. Use of GCF or SI in the coating mixes greatly reduced the thiobarbituric acid values of the meat when the coated patties were fried and stored at 4°C for 5 days.     

Inhibitory effects of natural plants of Jeju Island on elastase and MMP-1 expression

In order to search for new active cosmetic ingredients of natural origin, we screened about 60 plants collected from Jeju Island, which is located in the southernmost part of the Republic of Korea. We investigated their free radical scavenging activity, elastase inhibition activity, and reduction of MMP-1 mRNA expression for the development of anti-aging ingredients as raw materials for use in cosmetics. In the free radical scavenging capacity assay, 12 extracts, including Typha orientalis (seed) and Torreya nucifera (leaf), showed significant free radical scavenging activity (up to SC50 < 30 μg/ml). Among these extracts, Nymphaea tetragona (rhizome) extract showed the highest free radical scavenging activity (SC50 = 4.7 μg/ml). In the anti-elastase inhibition assay, seven extracts, including T. orientalis (seed) and Persicaria hydropiper (whole plant), showed high inhibitory activity (>50% at 100 μg/ml). Among these extracts, P. hydropiper (whole plant) extract showed the highest elastase inhibition activity (IC50 = 46.7 μg/ml). In the MMP-1 expression assay using RT-PCR, T. orientalis (seed), Pyrrosia hastata (root), and Capsicum annum (whole plant) showed slightly lower inhibition activity than EGCG, which was used as a control. Furthermore, four extracts, including P. hydropiper (whole plant), Filipendula glaberrima (root), N. tetragona (root), and Camellia japonica (leaf), completely inhibited the expression of MMP-1 in human fibroblast cells. The results showed that four of the 60 plant extracts may hold potential for use as natural active ingredients for anti-aging cosmetics.     

Flow Properties of Solutions of Oat β-Glucans

The flow properties of aqueous solutions of oat β-glucans were studied using a Bohlin rheometer (concentric cylinder geometry) over the shear rate range 0.734–1500 1/sec at temperatures between 15 and 75 °C. The β-glucan powder concentrations employed were 0.20–1.56% (w/w). The power law model was satisfactory for describing the flow behavior of β-glucans over the shear rate range 18.6–232 1/sec. The magnitudes of activation energy were in the range 2.41–4.61 (kcal/ mol). Sucrose decreased the flow behavior index and increased the consistency index of β-glucans and decreased the activation energy at lower β-glucan concentrations, these effects being most pronounced at a sucrose concentration of 25%. The D-value for solutions of β-glucans (0.65%) at 100 °C was 145 hr.     

Graphene bimetallic-like cantilevers: probing graphene/substrate interactions

The remarkable mechanical properties of graphene, the thinnest, lightest, and strongest material in existence, are desirable in applications ranging from composite materials to sensors and actuators. Here, we demonstrate that these mechanical properties are strongly affected by the interaction with the substrate onto which graphene is deposited. By measuring the temperature-dependent deflection of graphene/substrate "bimetallic" cantilevers we determine strain, thermal expansion coefficient, and the adhesion force acting on graphene films attached to a substrate. Graphene deposited on silicon nitride (SiN(x)) is under much larger strain, ε(g) ～ 1.5 × 10(-2), compared to graphene on gold (Au), ε(g) < 10(-3). The thermal expansion coefficient α(g) of graphene attached to SiN(x) is found to be negative, in the range from (- 5... - 1) × 10(-6)K(-1) and smaller in magnitude than α(g) of suspended graphene. We also estimate the interfacial shear strength of the graphene/SiN(x) interface to be ～1 GPa at room temperature.