Determination of the intracellular protein thiol distribution of hepatocytes using monobromobimane derivatisation of intact cells and isolated subcellular fractions

The derivatisation of intact rat hepatocytes with monobromobimane resulted in rapid labelling of accessible protein thiols in several subcellular fractions. The derivatisation procedure did not cause acute cytotoxicity, nor did it alter the buoyant densities of the fractions or their gross protein compositions. Quantitation of the fluorescence irreversibly associated with the fractions demonstrated considerable intracellular heterogeneity in this pool of thiols. Values were highest in cytosol (ca. 90 nmol/mg protein), intermediate in microsomes (ca. 65 nmol/mg protein) and mitochondria (ca. 45 nmol/mg protein) and lowest in a crude fraction containing both nuclei and plasma membrane (ca. 35 nmol/mg protein). Similar values were obtained from microsomes and cytosol derivatised after fractionation but there were significant increases of ca. 100% in corresponding values from isolated mitochondria and the nuclear/plasma membrane fraction. These results are discussed in terms of the dynamic fluxes in monobromobimane protein thiols during fractionation and the applicability of this noninvasive method to studies of the mechanism(s) of toxicity of reactive xenobiotics and the role(s) of protein thiols in normal cellular function.     

Identification of a stromelysin cleavage site within the interglobular domain of human aggrecan. Evidence for proteolysis at this site in vivo in human articular cartilage.

Products generated by the digestion of human aggrecan with recombinant human stromelysin have been purified and analyzed by N-terminal sequencing and C-terminal peptide isolation. N-terminal analysis of chondroitin sulfate-bearing fragments revealed a clearly identifiable sequence initiating at residue Phe342 of human aggrecan, providing evidence for a cleavage site at the Asn341-Phe342 bond located within the interglobular domain. This cleavage site, which separates the G1 domain from the remainder of the molecule, was confirmed by isolation from the liberated G1 domain of a C-terminal tryptic peptide with the sequence YDAICYTGEDFVDIPEN (in which the C-terminal residue is Asn341). This peptide was also isolated from tryptic digests of hyaluronan-binding proteins (A1D4 samples) prepared by CsCl gradient centrifugation of extracts of mature human articular cartilages. Since these A1D4 samples contain G1 domain which accumulates as a result of aggrecan catabolism in vivo, these results clearly indicate that stromelysin cleaves the Asn341-Phe342 bond of human aggrecan in situ.     

Env gene products of AKR dual-tropic viruses: examination of peptide maps and cell surface expression.

The env gene products of nine AKR dual-tropic murine leukemia viruses were compared by peptide mapping and were assayed for expression on the cell surface of infected fibroblasts. Seven virus isolates expressed the env gene polyprotein on the cell surface. The env gene products of six of the seven viruses had identical peptide maps. The analysis of structure and expression of env gene products carried out in this study characterizes a subset of dual-tropic murine leukemia viruses shown by others to be thymotropic.     

Heteroaryl urea inhibitors of fatty acid amide hydrolase: Structure–mutagenicity relationships for arylamine metabolites

The structure–activity relationships for a series of heteroaryl urea inhibitors of fatty acid amide hydrolase (FAAH) are described. Members of this class of inhibitors have been shown to inactivate FAAH by covalent modification of an active site serine with subsequent release of an aromatic amine from the urea electrophile. Systematic Ames II testing guided the optimization of urea substituents by defining the structure–mutagenicity relationships for the released aromatic amine metabolites. Potent FAAH inhibitors were identified having heteroaryl amine leaving groups that were non-mutagenic in the Ames II assay.     

SECRETION OF LIPASES IN THE DIGESTIVE TRACT OF THE CRICKET Gryllus bimaculatus

Little is known concerning the sites and the ratios of the lipase secretions in insects, therefore we undertook an examination of the lipase secretion of fed and unfed adult female Gryllus bimaculatus. The ratio of triacylglyceride lipase, diacylglyceride lipase, and phosphatidylcholine lipase secreted by fed females in the caecum and ventriculus is 1:1.4:0.4. These activities decrease in the caecum by 30–40% in unfed females. The total lipase activity (TLA) in the caecum is about 10 times that in the ventriculus. Minimal lipase secretion occurs before and during the final moult, and remains at this level in unfed crickets, indicating a basal secretion rate. In 2‐day‐old fed females, about 10% of the TLA in the entire gut is found in the crop, about 70% in the caecum, 20% in the ventriculus, and 3% in the ileum. Lipases in the ventriculus are recycled back to the caecum and little is lost in the feces. Oleic acid stimulated in vitro lipase secretion, but lipids did not. Feeding stimulated lipase secretion, starvation reduced lipase secretion, but this does not prove a direct prandal regulation of secretion, because feeding also induced a size and volume increase of the caecum.     

Single and dual task gait training in people with Parkinson's Disease: A protocol for a randomised controlled trial

Background  

Difficulty performing more than one task at a time (dual tasking) is a common and disabling problem experienced by people with Parkinson disease (PD). If asked to perform another task when walking, people with PD often take shorter steps or walk more slowly. Currently there is uncertainty about whether clinicians should teach people with PD to avoid dual tasking or whether they should encourage them to practice dual tasking with the hope that practice will lead to enhanced performance. This study will address this issue by comparing single to dual task gait training.     

Adamts5, the gene encoding a proteoglycan-degrading metalloprotease,is expressed by specific cell lineages during mouse embryonic development and in adult tissues

The secreted metalloprotease ADAMTS5 is implicated in destruction of the cartilage proteoglycan aggrecan in arthritis, but its physiological functions are unknown. Its expression profile during embryogenesis and in adult tissues is therefore of considerable interest. β-Galactosidase (β-gal) histochemistry, enabled by a LacZ cassette inserted in the Adamts5 locus, and validated by in situ hybridization with an Adamts5 cRNA probe and ADAMTS5 immunohistochemistry, was used to profile Adamts5 expression during mouse embryogenesis and in adult mouse tissues. Embryonic expression was scarce prior to 11.5 days of gestation (E11.5) and noted only in the floor plate of the developing brain at E9.5. After E11.5 there was continued expression in brain, especially in the choroid plexus, peripheral nerves, dorsal root ganglia, cranial nerve ganglia, spinal and cranial nerves, and neural plexuses of the gut. In addition to nerves, developing limbs have Adamts5 expression in skeletal muscle (from E13.5), tendons (from E16.5), and inter-digital mesenchyme of the developing autopod (E13.5–15.5). In adult tissues, there is constitutive Adamts5 expression in arterial smooth muscle cells, mesothelium lining the peritoneal, pericardial and pleural cavities, smooth muscle cells in bronchi and pancreatic ducts, glomerular mesangial cells in the kidney, dorsal root ganglia, and in Schwann cells of the peripheral and autonomic nervous system. Expression of Adamts5 during neuromuscular development and in smooth muscle cells coincides with the broadly distributed proteoglycan versican, an ADAMTS5 substrate. These observations suggest the major contexts in which developmental and physiological roles could be sought for this protease.     

The cell cycle-regulated protein human GTSE-1 controls DNA damage-induced apoptosis by affecting p53 function

GTSE-1 (G2 and S phase-expressed-1) protein is specifically expressed during S and G2 phases of the cell cycle. It is mainly localized to the microtubules and when overexpressed delays the G2 to M transition. Here we report that human GTSE-1 (hGTSE-1) protein can negatively regulate p53 transactivation function, protein levels, and p53-dependent apoptosis. We identified a physical interaction between the C-terminal regulatory domain of p53 and the C-terminal region of hGTSE-1 that is necessary and sufficient to down-regulate p53 activity. Furthermore, we provide evidence that hGTSE-1 is able to control p53 function in a cell cycle-dependent fashion. hGTSE-1 knock-down by small interfering RNA resulted in a S/G2-specific increase of p53 levels as well as cell sensitization to DNA damage-induced apoptosis during these phases of the cell cycle. Altogether, this work suggests a physiological role of hGTSE-1 in apoptosis control after DNA damage during S and G2 phases through regulation of p53 function.