USP10 exacerbates neointima formation by stabilizing Skp2 protein in vascular smooth muscle cells

The underlying mechanism of neointima formation remains unclear. Ubiquitin-specific peptidase 10 (USP10) is a deubiquitinase that plays a major role in cancer development and progression. However, the function of USP10 in arterial restenosis is unknown. Herein, USP10 expression was detected in mouse arteries and increased after carotid ligation. The inhibition of USP10 exhibited thinner neointima in the model of mouse carotid ligation. In vitro data showed that USP10 deficiency reduced proliferation and migration of rat thoracic aorta smooth muscle cells (A7r5) and human aortic smooth muscle cells (HASMCs). Mechanically, USP10 can bind to Skp2 and stabilize its protein level by removing polyubiquitin on Skp2 in the cytoplasm. The overexpression of Skp2 abrogated cell cycle arrest induced by USP10 inhibition. Overall, the current study demonstrated that USP10 is involved in vascular remodeling by directly promoting VSMC proliferation and migration via stabilization of Skp2 protein expression.     

Isolation and characterization of a porin-like protein of 45 kilodaltons from Bacteroides fragilis

Resistance to the combination of amoxicillin and clavulanic acid in some Bacteroides fragilis strains may be associated with a lack of porin proteins. Comparison of outer membrane protein profiles from one resistant strain ( B. fragilis CFPL 358) and two susceptible strains of B . fragilis (ATCC 25285 and CFPL 92125) showed that a few proteins were missing in the resistant strain, especially a 45-kDa protein. To determine whether this protein was a porin-like protein, we attempted to isolate it from the two susceptible strains by using gel filtration (Sephacryl S-200, Superose 6) and ion exchange chromatographies (DEAE Trisacryl, DEAE Sepharose Fast Flow). Elution from DEAE resins was poor compared to the 60–67-kDa region, which suggested that the 45-kDa protein exhibited stronger cationic forms. The use of sodium dodecyl sulfate during elution improved the recovery of the 45-kDa protein, showing that detergent modified its conformation and its ionic bounds with the chromatographic matrices but it was not sufficient for good purification. Superose 6 gel filtration also failed to separate this protein from the 60–67-kDa region. The only method resulting in the positive recovery of a purified 45-kDa band from both susceptible B. fragilis strains was electroelution from SDS-PAGE. The swelling assay showed that the 45-kDa protein was a porin-like protein. From this study, we concluded that the 45-kDa protein from B. fragilis was a porin-like protein which might be involved in the antibiotic resistance of a strain in which this protein was missing.     

Characterization of DNA binding activities of over-expressedKpnI restriction endonuclease and modification methylase

The genes encoding theKpnI restriction endonuclease and methyltransferase fromKlebsiella pneumoniae have been cloned and expressed inEscherchia coli using a two plasmid strategy. The gene forKpnI methylase with its promoter was cloned and expressed in pACYC184. Even though the methylase clone is in a low copy number plasmid pACMK, high level expression of methylase is achieved. A hyper-expressing clone ofKpnI endonuclease, pETRK was engineered by cloning the R gene into the T7 expression system. This strategy resulted in over-expression ofKpnI endonuclease to about 15–30% of cellular protein. Both the enzymes were purified using a single Chromatographic step to apparent homogeneity. The yield of purified endonuclease and methylase from one liter of culture was approximately 30 and 6 mg respectively. Electrophoretic mobility shift assays show that both the enzymes are capable of binding to specific recognition sequence in the absence of any cofactors. The complexes ofKpnI methyl transferase and endonuclease with their cognate site exhibit distinctive behaviour with respect to ionic requirement.     

Nutritional and hormonal effects on storage proteins in the silkworm,Bombyx mori L.

Starvation of 48 h old fifth instar larvae depressed storage protein titres initially for 48 h but retained the levels comparable to control thereafter, possibly due to nutrients obtained during the 48 h feeding after fourth ecdysis. After an initial decline ligated larvae accumulated maximum storage proteins in haemolymph. This is because of inhibitory juvenile hormone titre at the basal level besides the appropriate release of 20-hydroxyecdysone from the ectopic source(s). Injection of methoprene (10 Μg/larva) repressed accumulation of storage proteins while 20-hydroxyecdysone (10 Μg/larva) increased the same. P-soyatose injection to starved and ligated larvae accelerated storage protein accumulation in haemolymph, signalling nutrient indispensability for initiation of storage protein synthesis at the appropriate time of last instar development inBombyx mori.     

INCREASED PHOSPHORYLATION OF DARPP-32 BY D_1 AGONISTIC ACTION OF l-STEPHOLIDINE IN THE 6-OHDA-LESIONED RAT STRIATUM

为了进一步阐明ＳＰＤ对大鼠纹状体突触后Ｄ１受体的激动作用特性,本文应用反磷酸化在体内测定及放射配体结合方法,分别观察ＳＰＤ对６ＯＨＤＡ损毁大鼠纹状体ＤＡＲＰＰ３２体内磷酸化作用及突触后Ｄ１受体密度的影响。结果表明：皮下给予ＳＰＤ（２０,４０ｍｇ／ｋｇ,２１ｄ）,损毁侧纹状体ＤＡＲＰＰ３２体外［３２Ｐ］的掺入量较健侧下降５０％（Ｐ＜００１）。换言之,损毁侧纹状体内ＤＡＲＰＰ３２的磷酸化程度增加了。然而,ＳＰＤ使损毁导致Ｄ１受体上调的作用减弱（Ｂｍａｘ从３８５０±２６１ｆｍｏｌ／ｍｇ降至３１９７±２０１ｆｍｏｌ／ｍｇ水平）。因此,ＳＰＤ激动Ｄ１受体,使６ＯＨＤＡ损毁大鼠纹状体内ＤＡＲＰＰ３２磷酸化作用加强,而受体密度减少。这是ＳＰＤ调节脑内Ｄ１受体信号转导功能的重要机制。     

CeLINC,a fluorescence-based protein–protein interaction assay in Caenorhabditis elegans

Interactions among proteins are fundamental for life and determining whether two particular proteins physically interact can be essential for fully understanding a protein’s function. We present Caenorhabditis elegans light-induced coclustering (CeLINC), an optical binary protein–protein interaction assay to determine whether two proteins interact in vivo. Based on CRY2/CIB1 light-dependent oligomerization, CeLINC can rapidly and unambiguously identify protein–protein interactions between pairs of fluorescently tagged proteins. A fluorescently tagged bait protein is captured using a nanobody directed against the fluorescent protein (GFP or mCherry) and brought into artificial clusters within the cell. Colocalization of a fluorescently tagged prey protein in the cluster indicates a protein interaction. We tested the system with an array of positive and negative reference protein pairs. Assay performance was extremely robust with no false positives detected in the negative reference pairs. We then used the system to test for interactions among apical and basolateral polarity regulators. We confirmed interactions seen between PAR-6, PKC-3, and PAR-3, but observed no physical interactions among the basolateral Scribble module proteins LET-413, DLG-1, and LGL-1. We have generated a plasmid toolkit that allows use of custom promoters or CRY2 variants to promote flexibility of the system. The CeLINC assay is a powerful and rapid technique that can be widely applied in C. elegans due to the universal plasmids that can be used with existing fluorescently tagged strains without need for additional cloning or genetic modification of the genome.     

Preparation of fluorescence quenched libraries containing interchain disulphide bonds for studies of protein disulphide isomerases

Protein disulphide isomerase is an enzyme that catalyses disulphide redox reactions in proteins. In this paper, fluorogenic and interchain disulphide bond containing peptide libraries and suitable substrates, useful in the study of protein disulphide isomerase, are described. In order to establish the chemistry required for the generation of a split-synthesis library, two substrates containing an interchain disulphide bond, a fluoroescent probe and a quencher were synthesized. The library consists of a Cys residue flanked by randomized amino acid residues at both sides and the fluoroescent Abz group at the amino terminal. All the 20 natural amino acids except Cys were employed. The library was linked to PEGA‒beads via methionine so that the peptides could be selectively removed from the resin by cleavage with CNBr. A disulphide bridge was formed between the bead‒linked library and a peptide containing the quenching chromophore (Tyr(NO₂)) and Cys(pNpys) activated for reaction with a second thiol. The formation and cleavage of the interchain disulphide bonds in the library were monitored under a fluoroescence microscope. Substrates to investigate the properties of protein disulphide isomerase in solution were also synthesized. © 1998 European Peptide Society and John Wiley & Sons, Ltd.     

Species-dependent enantioselective pharmacokinetics of PNU-103017, a Pyrone HIV protease inhibitor

PNU-103017, 4-Cyano-N-(3-(cyclopropyl(5,6,7,8,9,10-hexahydro-4-hydroxy-2-oxo-2H-cycloocta(b) pyran-3-yl)methyl)phenyl)-benzenesulfonamide, is a selective HIV aspartyl protease inhibitor under evaluation as a potential oral treatment of Acquired Immunodeficiency Diseases. PNU-103017 is a racemic mixture of two enantiomers, designated PNU-103264 (R-) and PNU-103265 (S-). Stereoselective pharmacokinetics of the two enantiomers of PNU-103017 were observed in the dog, rat, and human after single and multiple dose administration of the racemate and were apparently species-dependent. Mean enantiomeric ratios of plasma concentrations (R-/S-) at each time point were greater than 1 in the dog, ranging from 1.22 to 3.06, but less than 1 in the rat and in the human, ranging from 0.44 to 0.80 and 0.23 to 0.73, respectively. A trend towards increased or decreased (farther from 1:1, R-/S-) enantiomeric ratio of plasma concentrations with time after each administration was also observed. The enantiomeric ratio remained unchanged after multiple dose administration in the rat, dog, and human although enzyme induction and increased plasma clearance were observed for both enantiomers. Chirality 10:210–216, 1998. © 1998 Wiley-Liss, Inc.     

Semiquantitative bioluminescent assay of glutathione

A novel technique has been developed for semiquantitative detection of glutathione (GSH) in small volumes of liquid samples. GSH is detected via enzymatic linkage to the NADP/NADPH + H⁺ redox system through glutathione reductase. Accumulated NADPH is measured via the bioluminescent FMN oxidoreductase bacterial luciferase reaction. A linear correlation is obtained between bioluminescence intensity of the luciferase reaction and the GSH content of the liquid sample. Possible applications of this procedure are discussed. © 1998 John Wiley & Sons, Ltd.