Cellular thiols as a determinant of responsiveness to menadione in cardiomyocytes

The role of intracellular thiols in menadione-mediated toxicity was studied in neonatal rat cardiomyocytes. The sensitivity of cardiomyocytes to menadione was greater than that of skeletal muscle cells and 3T3 fibroblasts. Before cell degeneration, menadione induced marked depletion of intracellular thiols and an increase of oxidized glutathione. The sensitivity of these cells to menadione correlated with the level of depletion of intracellular thiols. After incubation of cardiomyocytes with menadione, glutathione reductase activity was inhibited and lipid peroxidation was increased. Both dicumarol (an inhibitor of DT-diaphorase) and diethyldithiocarbamate (an inhibitor of superoxide dismutase) enhanced the capacity of menadione to induce cellular damage and to cause depletion of intracellular glutathione. Decreasing intracellular glutathione by pretreatment of cells with N-ethylmaleimide or buthionine sulphoximine also increased menadione-induced cell degeneration. Preincubation with cysteine or dithiothreitol suppressed the capacity of menadione to damage the cells. Menadione-induced lipid peroxidation was also suppressed by the same treatment. These results show that the oxidative stress induced by menadione in cardiomyocytes results in the depletion of glutathione and protein thiols. Both DT-diaphorase and superoxide dismutase can protect cells from the toxicity of menadione. Cellular thiols are determinants of the responsiveness to menadione.     

Isolation and characterization of dendritic cells from mouse heart and kidney

Dendritic cells (DC) are thought to be distributed throughout lymphoid and most nonlymphoid tissues. Single cell suspensions were prepared from mouse hearts and kidneys. Subsets of MHC class II-positive (Ia+) leukocytes from both sources expressed markers such as CDw32 Fc receptors, F4/80, and complement receptor type 3 (CD11b/CD18). The capacity of these cells to initiate primary in vitro immune responses was assessed using oxidative mitogenesis and allogeneic mixed leukocyte responses. After fractionation by density centrifugation, cell sorting, immunomagnetic bead separation, or cell panning, the stimulatory activity of kidney cell suspensions was found to reside in the low density, Ia+ leukocyte fractions after overnight culture (day 1). In contrast, freshly isolated (day 0) cells had considerably less or no activity in these assays. However, depletion of Ia+ or CD45+ cells on day 0 followed by overnight culture removed the stimulatory activity on day 1. Therefore, day 0 kidney cells contain Ia+ leukocytes that can acquire or up-regulate their stimulatory activity during overnight culture. Similar observations were made for cells isolated from hearts, except that a population of uncharacterized nonleukocytes with stimulatory activity was detected on day 0 but not day 1. The phagocytic capacity of the leukocytes was then examined. Subsets of Ia+ cells phagocytosed zymosan, as shown by two-color flow cytometry and other immunofluorescence studies, and the zymosan-positive cells from kidney were able to initiate primary responses. Overall, these data demonstrate the existence of DC in kidneys and hearts, and suggest that in situ these cells resemble immature rather than mature DC.     

A COMPREHENSIVE BIOMECHANICAL MODEL USING STRENGTH, STABILITY, AND COF CONSTRAINTS TO PREDICT HAND FORCE EXERTION CAPABILITY UNDER SAGITTALLY SYMMETRIC STATIC CONDITIONS

A new mathematical biomechanics] model has been developed to comprehensively estimate feasible hand force exertion capability under sagittally symmetric static conditions. In the model, a set of 15 linear constraint equations has been developed in three constraint classes: coefficient of friction, stability, and strength. This set of constraints defines a feasible solution space for combinations of horizontal and vertical forces exerted by the hands. Inputs to the model include posture, anthropometry, strength capability, coefficient of static friction, and gender. Examples of lifting and pushing demonstrate some features and advantages of the model. The most promising aspect of this model appears to be its ability to comprehensively combine multiple factors that can affect hand force exertion capability.     

Capacitative Ca2+ entry and the regulation of smooth muscle tone

In many non-excitable cells, activation of phospholipase C-linked receptors results in a biphasic increase in the cytosolic Ca2+ concentration; an initial transient increase, owing to the release of Ca2+ from the endoplasmic/sarcoplasmic reticulum (ER/SR), is followed by a much smaller but sustained elevation, which often involves capacitative Ca2+ entry, where depletion of Ca2+ within the ER signals the opening of store-operated Ca2+ channels in the plasma membrane. However, in excitable cells such as smooth muscle, the role of capacitative Ca2+ entry is less clear and the main Ca2+ entry mechanisms responsible for sustained cellular activation have been considered to be either voltage-operated or receptor-operated Ca2+ channels. Although store-regulated Ca2+ entry was known to occur following agonist activation of smooth muscle, it was believed to be important only for the re-filling of the depleted SR and not as a source of activator Ca2+ for the contractile mechanisms. Here, Alan Gibson, Ian McFadzean, Pat Wallace and Christopher Wayman review recent evidence that capacitative Ca2+ entry might indeed be important for the regulation of smooth muscle tone, and that it might provide an important for pharmacological intervention.     

Crystal structure of human uroporphyrinogen decarboxylase

Uroporphyrinogen decarboxylase (URO-D) catalyzes the fifth step in the heme biosynthetic pathway, converting uroporphyrinogen to coproporphyrinogen by decarboxylating the four acetate side chains of the substrate. This activity is essential in all organisms, and subnormal activity of URO-D leads to the most common form of porphyria in humans, porphyria cutanea tarda (PCT). We have determined the crystal structure of recombinant human URO-D at 1.60 A resolution. The 40.8 kDa protein is comprised of a single domain containing a (beta/alpha)8-barrel with a deep active site cleft formed by loops at the C-terminal ends of the barrel strands. Many conserved residues cluster at this cleft, including the invariant side chains of Arg37, Arg41 and His339, which probably function in substrate binding, and Asp86, Tyr164 and Ser219, which may function in either binding or catalysis. URO-D is a dimer in solution (Kd = 0.1 microM), and this dimer also appears to be formed in the crystal. Assembly of the dimer juxtaposes the active site clefts of the monomers, suggesting a functionally important interaction between the catalytic centers.     

Intracellular maturation of the mouse metalloprotease disintegrin MDC15

Metalloprotease disintegrins are a family of membrane-anchored glycoproteins that play a role in fertilization, myoblast fusion, neuronal development, and cleavage of the membrane-anchored cytokine tumor necrosis factor-alpha. Here, we report the cloning and cDNA sequencing of the mouse metalloprotease disintegrin MDC15 and an analysis of its processing in the secretory pathway. A notable difference between mMDC15 and its putative human orthologue (hMDC15, metargidin) is the presence of the peptide sequence TDDC instead of the RGDC found in the disintegrin domain of hMDC15. In a Western blot analysis the majority of mMDC15 was found to lack the pro-domain in all mouse tissues examined. Pulse-chase experiments in transiently transfected COS-7 cells suggest that mMDC15 is processed by a pro-protein convertase in a late Golgi compartment, since (i) addition of brefeldin A or monensin blocks pro-domain removal, (ii) all detectable processed mMDC15 is endoglycosidase H -resistant, and (iii) a recombinant soluble form of the trans-Golgi network pro-protein convertase furin can mimic mMDC15 processing in vitro. Cell-surface trypsinization revealed that more than half of mature mMDC15 is intracellular. Immunolocalization provided evidence for a strong perinuclear accumulation in a region resembling the trans-Golgi network and/or endosomal compartments. This study provides the first characterization of the intracellular processing of a metalloprotease disintegrin, and highlights the potential role of pro-protein convertases in removal of the inhibitory pro-domain. These results further suggest possible intracellular functions for mMDC15, such as in protein maturation, in addition to a potential role in cell-surface proteolysis or cell adhesion.     

Antioxidant depletion during aortic aneurysm repair

Ischaemia-reperfusion injury generates oxygen-derived free radicals leading to local and distant damage. A simple method of following oxidative activity is to measure the consumption of endogenous scavenging antioxidants; an enhanced chemiluminescent assay was used to study this phenomenon in 21 patients undergoing surgery for abdominal aortic aneurysm (AAA). Samples of peripheral venous blood were taken before induction of anaesthesia and then from a central venous line and the inferior mesenteric vein before, during, and after clamping of the aorta. Further specimens were taken from the central line at 2, 6 and 24 h after operation. Antioxidant concentration in the peripheral, central and inferior mesenteric blood were similar, indicating that anaesthesia and surgical dissection had no effect. Levels decreased significantly in central and inferior mesenteric blood during and after clamping, but returned to normal by 24 h. These results confirm ischaemia-reperfusion phenomena in AAA repair.     

Differentiation-specific enhancer activity in transduced keratinocytes: a model for epidermal gene therapy

Active sequences from the laminin alpha1 and alpha2 chain carboxyl-terminal globular domains (G domain) have been identified by screening overlapping synthetic peptides in a number of biological assays (Nomizu et al. [1995] J. Biol. Chem. 270:20583-20590; Nomizu et al. [1996] FEBS Lett. 396:37-42). We have tested the activity of these peptides in submandibular gland explants of embryonic day 13 mice to determine the functional sites involved in organ development. The laminin alpha1 chain peptide, RKRLQVQLSIRT (residues 2719-2730 and designated AG-73), significantly inhibited epithelial branching morphogenesis. In contrast, other cell adhesive laminin alpha1 chain peptides including the AASIKVAVSADR and NRWHSIYITRFG failed to inhibit the branching. MG-73, a homologue of AG-73 from the laminin alpha2 chain, did not inhibit the branching. The alpha2 chain peptide had no effect, which may be due to the low levels of this laminin chain in day 13 mice. Laminin alpha2 chain-specific monoclonal antibodies strongly reacted with the basement membranes of developed acini but only weakly stained embryonic day 13 submandibular epithelium. The expression of E-cadherin and alpha6 integrin, as detected by immunofluorescence, were unchanged in both AG-73 and control scramble peptide-treated epithelial cells of the explants. In contrast, immunostaining of nidogen/entactin showed that explants treated with AG-73 for 3 days had a discontinuous basement membrane. Explants treated for 3 days with control peptide showed a normal basement membrane. These results suggest that the region containing the AG-73 sequence of the laminin alpha1 chain is crucial for development of submandibular gland at early embryonic stages. The discontinuous basement membrane in AG-73-treated explants may indicate an important role for this region in basement membrane assembly.     

Molecular cloning and characterization of an invertebrate homologue of a neuropeptide Y receptor

Neuropeptide Y is an abundant and physiologically important peptide in vertebrates having effects on food intake, sexual behaviour, blood pressure and circadian rhythms. Neuropeptide Y homologues have been found in invertebrates, where they are very likely to play similar, important roles. Although five neuropeptide Y-receptor subtypes have been identified in mammals, none has been reported from invertebrates. Here we describe the cloning of a neuropeptide Y-receptor from the brain of the snail Lymnaea stagnalis. The identity of the receptor was deduced by expressing the neuropeptide Y-receptor-encoding cDNA in Chinese Hamster Ovary cells, which were subsequently challenged with size-fractionated Lymnaea brain extracts. An active peptide, selected on the basis of its ability to induce changes in cAMP levels, was purified to homogeneity, analysed by mass spectrometry and amino acid sequence determination, and turned out to be a Lymnaea homologue of neuropeptide Y.