Micromechanical coupling between cell surface receptors and RGD peptides

Contact between an adherent cell and the extracellular matrix (ECM) promotes the recruitment of structural and signaling molecules to the cytoplasmic domain of integrins, which mediate cell adhesion, cell migration, and cell growth. It is unclear whether the intracellular recruitment of these cytoplasmic molecules enhances the affinity between the ECM and the extracellular domain of the cell surface receptors (integrins). Using soft microneedles coated with Arg-Gly-Asp (RGD) peptides, a sequence commonly shared by ECM proteins, we apply a localized ramp shear stress to the surface of a HeLa cell and measure the cell stiffness and the collective (or apparent) unbinding lifetime of its surface receptors to RGD. These measurements demonstrate that both cell stiffness and the collective cell surface receptor-RGD unbinding lifetime increase with the duration of the pre-shear cell-microneedle contact and with the rate of shear applied to the cell membrane. These parameters are also crucially dependent on the integrity of the actin filament network. Our results are consistent with a model of positive feedback signaling where RGD-mediated initial recruitment of cytoskeletal proteins to the cytoplasmic domain of integrins directly enhances the interaction between the extracellular domain of integrins and the RGD sequence of ECM molecules.     

Synthesis of palmitoyl-thioester T-cell epitopes of myelin proteolipid protein (PLP). Comparison of two thiol protecting groups (StBu and Mmt) for on-resin acylation.

In order to test the effect of thiopalmitoylation on the encephalitogenic properties of two proteolipid protein (PLP) T-cell epitopes, we have studied the on-resin S-palmitoylation of peptides, synthesized using the Fmoc/tBu strategy. The use of two Cys protecting groups was investigated: the tert-butylsulfenyl (StBu) and the methoxytrityl (Mmt). Our studies show that the ease of deprotection of the thiol protected with StBu was sequence dependent. The deprotection of Cys(StBu) was difficult in the case of the two peptides PLP(104-117) and PLP(139-151). Neither of the two Cys(StBu) (Cys108 and Cys140, respectively) could be deprotected with tributylphosphine. Beta-mercaptoethanol was only efficient for the deprotection of Cys(StBu)140 at 85 degrees C and at 135 degrees C for Cys108. The two palmitoylated peptides could be obtained in good yield starting from Cys protected with Mmt. Our conclusion is that the Mmt group is the more versatile protecting group of the thiol for use in the on-resin synthesis of thiopalmitoylated peptides.     

Methionine transsulfuration is increased during sepsis in rats

Methionine transsulfuration in plasma and liver, and plasma methionine and cysteine kinetics were investigated in vivo during the acute phase of sepsis in rats. Rats were infected with an intravenous injection of live Escherichia coli, and control pair-fed rats were injected with saline. Two days after injection, the rats were infused for 6 h with [(35)S]methionine and [(15)N]cysteine. Transsulfuration was measured from the transfer rate of (35)S from methionine to cysteine. Liver cystathionase activity was also measured. Infection significantly increased (P < 0.05) the contribution of transsulfuration to cysteine flux in both plasma and liver (by 80%) and the contribution of transsulfuration to plasma methionine flux (by 133%). Transsulfuration measured in plasma was significantly (P < 0.05) higher in infected rats than in pair-fed rats (0.68 and 0.25 micromol. h(-1). 100 g(-1), respectively). However, liver cystathionase specific activity was decreased by 17% by infection (P < 0.05). Infection increased methionine flux (16%, P < 0.05) less than cysteine flux (38%, P < 0.05). Therefore, the plasma cysteine flux was higher than that predicted from estimates of protein turnover based on methionine data, probably because of enhanced glutathione turnover. Taken together, these results suggest an increased cysteine requirement in infection.     

The cytotoxicity of chrysotile asbestos fibers to pulmonary alveolar macrophages I. Effects of inhibitors of ADP-ribosyl transferase

Pulmonary alveoler macrophages exposedto very short chrysotile asbestos fibers present a typical cytotoxic response: extracellular releases of lactate dehydrogenase and -galactosidase, and a decrease in cellular ATP content. The objective of this study was to determine if nicotinamide and 3-aminobenzamide, two inhibitors of the ADP-ribosyl transferase, could modify the in vitro toxicity of chrysotilee fibers. After 30 min of pre-exposure with each of the two inihibitors, pulmonary alveolar macrophage monolayers were concominantly exposed for 18 hours to 50g of fibers. It was observed that, in a dose-effect relationship (5 to 30 mM), nicotinamide was very effective in reducing the extracellular liberation of the marker enzymes. At 30 mM, the enzyme releases in the medium had returned to control values; the restoration of cell viability was confirmed by ATP levels. Up to 5 mM 3-aminobenzamide did not provide any protection against chrysotile cytotoxicity. Nicotinic acid, a structural analogue of nicotinamide, but not an inhibitor of the ADP-ribosyl transferase, also showed no protective effect. Nicotinamide and 3-aminobenzamide increased the intracellular NAD⁺ pools, respectively by 350% and 250%. However, with or without additives, the chrysotile fibers caused a constant and significant decrease in NAD⁺ levels (40–55 pmoles). These results suggest that the inhibition of the nuclear ADP-ribosyl transferase is not the major mechanism by which nicotinamide protects pulmonary alveolar macrophages against the chrysotile asbestos fibers.Abbreviations 3-AB 3-aminobenzamide - ADPRT ADP-ribosyl transferase - -GAL -galactosidase - DTT dithiothreitol - FBS fetal bovine serum - FMN flavin mononucleotide - HEPES N-2-hydroxyethyl piperazine-N-2-ethanesulfonic acid - LDH lactate dehydrogenase - NAD⁺ nicotinamide adenine dinucleotide (oxidized form) - NADH nicotimide adenine dinucleotide (reduced forms) - NADPH nicotimide adenine dinucleotide phosphate (reduced form) - NAM nicotinamide - NIC nicotinic acid - ORS oxygen radical species - PAM pulmonary alveolar macrophages - S.E. standard error of the mean - TAPS tris (hydroxymethyl) methylamino-propane sulfonic acid - TRIS tris (hydroxymethyl) aminomethane - VSF very short chrysotile fibers     

5-heteroatom substituted pyrazoles as canine COX-2 inhibitors. Part 1: Structure-activity relationship studies of 5-alkylamino pyrazoles and discovery of a potent, selective, and orally active analog

Structure-activity relationship (SAR) studies of the novel 2-[3-di and trifluoromethyl-5-alkylamino pyrazo-1-yl]-5-methanesulfonyl (SO(2)Me)/sulfamoyl (SO(2)NH(2))-pyridine derivatives for canine COX enzymes are described. The studies led to the identification of 2e as lead with potent in vitro activity, selectivity, and in vivo activity in dogs and cats.     

Metabolic response to feeding in Tupinambis merianae: circadian rhythm and a possible respiratory constraint

The diurnal tegu lizard Tupinambis merianae exhibits a marked circadian variation in metabolism that is characterized by the significant increase in metabolism during part of the day. These increases in metabolic rate, found in the fasting animal, are absent during the first 2 d after meal ingestion but reappear subsequently, and the daily increase in metabolic rate is added to the increase in metabolic rate caused by digestion. During the first 2 d after feeding, priority is given to digestion, while on the third and following days, the metabolic demands are clearly added to each other. This response seems to be a regulated response of the animal, which becomes less active after food ingestion, rather than an inability of the respiratory system to support simultaneous demands at the beginning of digestion. The body cavity of Tupinambis is divided into two compartments by a posthepatic septum (PHS). Animals that had their PHS surgically removed showed no significant alteration in the postprandial metabolic response compared to tegus with intact PHS. The maximal metabolic increment during digestion, the relative cost of meal digestion, and the duration of the process were virtually unaffected by the removal of the PHS.