Synthesis and evaluation of amphiphilic RGD derivatives: uses for solvent casting in polymers and tissue engineering applications.

Derivatives containing arginine-glycine-aspartic acid (RGD) inhibit fibrinogen binding to activated platelets and promote endothelial and smooth muscle cell attachment. An amphiphilic derivative of RGD that can be dissolved in an organic solvent has potential in the development of non-thrombogenic biomaterials. Such a derivative, LA-GRGD, was synthesised by coupling glycine-arginine-glycine-aspartic acid (GRGD) with lauric acid (LA). Its solubility and antithrombotic, cytotoxic and cell-binding effects were then evaluated in comparison with heparin (which is used clinically) and a fibronectin-engineered protein polymer (FEPP). Thromboelastography (TEG) was used to measure blood clotting time using fresh whole blood from healthy volunteers. Tissue factor (TF) activity was measured using plasma with a standard prothrombin time assay (PT). Cytotoxicity was assessed on human umbilical cord endothelial cells (HUVECs) using an Alamar blue assay. Solubility of the conjugate was assessed in a co-solvent. These techniques were used to study LA-GRGD, using heparin and FEPP as controls. The amphiphilic property of LA-GRGD was dependent on the feed mole ratio of GRGD to LA. LA-GRGD was soluble in acetone:water and water. LA-GRGD inhibited TF by >90% and prolonged TEG-r by 8.2+/-3.3 min (200 microg ml(-1)). Heparin inhibited TF by >90%, but prolonged TEG-r by 97.4+/-1.6 min (1 U ml(-1)); FEPP inhibited TF by >90% (100 microg ml(-1)) and prolonged TEG-r by 73.7+/-8.4 min (10 microg ml(-1)). Heparin had no cytotoxic effect on EC metabolism and viability at the concentrations studied (0.1-100 U ml(-1)). No significant cytotoxic effect was produced by LA-GRGD or FEPP at concentrations ranging from 0.1 microg ml(-1) to 50 microg ml(-1), but, at higher concentrations (100 microg ml(-1) and 200 microg ml(-1)), a detrimental effect was observed. Cell binding studies showed that LA-GRGD bound 29% of ECs compared with FEPP (60%) and heparin (22%). This new approach for synthesising amphiphilic RGD and its analogues has potential as a drug delivery system for the manufacture of new polymer formulations for use in bypass grafts and other tissue-engineered devices.

Studies of the pore-forming domain of a voltage-gated potassium channel protein.

Recent mutagenesis studies have identified a stretch of amino acid residues which form the ion-selective pore of the voltage-gated potassium channel. It has been suggested that this sequence of amino acids forms a beta-barrel structure making up the structure of the ion-selective pore [Hartman, H.A., Kirsch, G.E., Drewe, J.A., Taglialatela, M., Joho, R.H. and Brown, A.M. (1991) Science, 251, 942-944; Yellen, G., Jurman, M.E., Abramson, T. and MacKinnon, R. (1991) Science, 251, 939-942; Yool, A.J. and Schwarz, T.L. (1991) Nature, 349, 700-704]. We have synthesized a polypeptide corresponding to this amino acid sequence (residues 431-449 of the ShA potassium channel from Drosophila). A tetrameric version of this sequence was also synthesized by linking together four of these peptides onto a branching lysine core. Fourier transform infrared (FT-IR) and circular dichroism (CD) spectroscopy have been used to investigate the structure of these peptides after their reconstitution into lyso phosphatidylcholine micelles and lipid bilayers composed of dimyristoyl phosphatidylcholine and dimyristoyl phosphatidylglycerol. The spectroscopic studies show that these peptides are predominantly alpha-helical in these lipid environments. When incorporated into planar lipid bilayers both peptides induce ion channel activity. Molecular modelling studies based upon the propensity of these peptides to form an alpha-helical secondary structure in a hydrophobic environment are described. These results are discussed in the light of recent mutagenesis and binding studies of the Drosophila Shaker potassium ion channel protein.

The effect of chronic ethanol consumption on iron absorption in rats.

Alcoholics often have an increased amount of iron in the liver which may contribute to the development of alcoholic liver disease, although the mechanism is unknown. It has been shown that chronic ethanol intake decreases the enterocyte turnover and enhances galactose absorption. Whether it affects iron absorption is still controversial. The aim of this study was to investigate the effect of chronic ethanol ingestion on whole body iron absorption in rats. Twenty-eight adult male Sprague-Dawley rats were pair-fed a liquid diet containing either ethanol as 36% of total calories or an isocaloric diet where fat was substituted for ethanol. On the 28th day, four-hour fasted rats were given an oral dose of 59Fe (0.5 microCi) and were immediately counted by a whole body counter. 59Fe levels were then monitored over the following nine days. Although ethanol- and control-fed rats had a similar hepatic iron content (59.5 +/- 5.8 vs 60.2 +/- 7.4 micrograms/100 mg dry liver weight) (mean +/- S.E.M.), the 59Fe total body content was greater in the ethanol group (75% +/- 3%) compared with the control group (45% +/- 4%). These results show that chronic ethanol ingestion increased iron absorption in rats. A reduction of enterocyte turnover may play a role in determining this effect.

Haem-binding proteins of the rat liver cytosol.

Gel filtration of soluble supernatant fraction obtained from livers of rats 10 min after an injection of the haem precursor 5-amino[3H]laevulinic acid shows the presence of a major radioactive fraction which upon gel filtration is similar in elution volume to ligandin. 20 min after administration of the precursor four previously minor components also come into prominence. This pattern is a characteristic of in vivo binding since a different elution pattern is obtained if soluble supernatant fraction from rat liver is labelled in vitro by incubation either with [3H]haem-labelled mitochondria, [3H]haem-labelled microsomes or with [3H]haemin. These results are discussed with particular reference to ligandin.