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.

Axial distribution and characterization of basolateral P2Y receptors along the rat renal tubule.

BACKGROUND: Several groups have identified P2Y receptors in the basolateral membrane of the rat nephron. These studies have not covered all segments of the nephron and have relied solely on the relative potency of receptor agonists for classification. METHODS: We measured purine and pyrimidine-induced changes in intracellular free calcium concentration ([Ca(2+)](i)) in anatomically defined segments of the rat nephron. To complement these functional studies, we have used reverse transcription-polymerase chain reaction methodology to identify specific P2Y receptor transcripts in these segments. RESULTS: Adenosine 5'-triphosphate (ATP) mobilized [Ca(2+)](i) in all nephron segments, except for the thick ascending limb of Henle, which was poorly responsive. Adenosine (100 micromol/L) was without effect, confirming that the effect of ATP was mediated by P2 receptors. In the proximal convoluted tubule (PCT) and outer medullary collecting duct (OMCD), there was evidence for two receptor subtypes with characteristics of P2Y(1)- and either P2Y(2)- or P2Y(4)-like receptors. A novel finding in the thin limbs was the presence of a receptor with properties of both P2Y(2) and P2Y(4) receptor subtypes. To aid classification, we identified P2Y receptor mRNA in rat nephron segments. In the PCT and OMCD and thin ascending limb of Henle, we found expression of P2Y(1), P2Y(2), and P2Y(4) receptors. In the descending limb of Henle, P2Y(1) and P2Y(2) mRNA was found, but P2Y(4) was not expressed. CONCLUSION: These data suggest that extracellular ATP can influence tubular cell function in all segments of the rat nephron, through P2Y receptors via multiple (and coexpressed) P2Y receptor subtypes.

Interactions of the flavonoid naringenin in the gastrointestinal tract and the influence of glycosylation.

We have studied interactions in the gastrointestinal tract of flavonoids and the influence of glycosylation on their subsequent metabolism by examining the urinary recoveries of the flavonoid naringenin-7-glucoside and its aglycone, in the conscious rat model, after oral and intravenous administration. Absorption studies were also conducted using an in vitro isolated rat jejunum. The results show that ca. 10% of the administered dose of naringenin was recovered after oral dosing, the majority as naringenin glucuronide, whereas, after intravenous administration, the recovery of the glucuronide was ca. 20%. In contrast, after oral dosing of naringenin-7-glucoside, its hydrolysis product naringenin (0.5%) and naringenin glucuronide (12.7%) were detected. After intravenous dosing the majority of that identified in the urine was as the native glucoside. These findings suggest that, via the oral route, the glycoside group is cleaved by an intestinal enzyme prior to glucuronidation within the epithelium. This is substantiated by the urinary elimination of the native glucoside and the lack of detection of glucuronide after intravenous administration. Transport studies with isolated intestine showed that neither unchanged naringenin nor the 7-glucoside was absorbed in significant quantities across the gut wall. The major metabolite detected in both cases was naringenin glucuronide, thus supporting the notion that glucuronidation as well as hydrolysis can occur at the intestinal epithelium.

A precursor of the nitric oxide donor SIN-1 modulates the stress protein heme oxygenase-1 in rat liver.

In this study the effect of increased nitric oxide (NO) production on the expression of rat liver heme oxygenase-1, an inducible stress protein responsible for the catalysis of heme to biliverdin and carbon monoxide, was investigated. Rats were injected intraperitoneally with molsidomine (SIN-10), a long acting drug that is enzymatically converted in the liver to yield the active NO-releasing agent 3-morpholinosydnonimine (SIN-1). Administration of SIN-10 resulted in a significant time- and dose-dependent increase in plasma levels of nitrite/nitrate, an index of NO release. A time course of heme oxygenase-1 mRNA levels in liver showed a gradual increase in the expression of the gene encoding for this protein, which was maximal at 4 hours and returned to normal levels by 6 hours after SIN-10 treatment. Heme oxygenase activity also increased by 50% at 4 hours and was maximal 12 hours after SIN-10 administration (63% increase over baseline). These results indicate a possible role for locally generated NO in the modulation of hepatic stress response in vivo suggesting that NO mediates cell adaptation to stress by activation of endogenous defensive mechanisms.

Complex I, iron, and ferritin in Parkinson's disease substantia nigra.

Elevated iron levels, enhanced oxidative damage, and complex I deficiency have been identified in the substantia nigra of Parkinson's disease patients. To understand the interrelationship of these abnormalities, we analyzed iron levels, ferritin levels, and complex I activity in the substantia nigra of patients with Parkinson's disease. Total iron levels were increased significantly, ferritin levels were unchanged, and complex I activities were decreased significantly in the substantia nigra samples. The failure of ferritin levels to increase with elevated iron concentrations suggests that the amount of reactive iron may increase in the substantia nigra of Parkinson's disease patients. There was no correlation between the iron levels and complex I activity or the iron-ferritin ratio and complex I activity in the substantia nigra samples.

Measles virus induction of human endothelial cell tissue factor procoagulant activity in vitro.

Measles virus infection of microvascular endothelium in vivo and ensuing endothelial cell activation may be important in the pathogenesis of subsequent inflammation in target organs. This study investigated the capacity of measles virus to induce procoagulant activity, in vitro, in endothelial cells isolated from human umbilical cord veins. Endothelial cells were infected with a clinical isolate of measles virus propagated in Vero cells. Cells were also incubated with bacterial lipopolysaccharide (10 micrograms/ml), herpes simplex virus type 1, cytomegalovirus or culture medium alone as positive and negative controls, respectively. Endothelial cell procoagulant activity was measured in a one-stage clotting assay. Measles virus stimulated both a time and dose-dependent endothelial cell procoagulant response by the induction of tissue factor synthesis, confirmed by both immunocytochemistry and its dependence on factor VII for activity. This activity was reduced by u.v.-irradiation of the virus. Infected cells were analysed by double immunofluorescent staining for both tissue factor and measles virus N-protein, and examined using confocal scanning laser microscopy. Cells expressing tissue factor were also positive for the measles virus N-protein. Low levels of interleukin-1 were detected in some viral inocula derived from measles virus-infected Vero cells, however neutralising antibody to interleukin-1 failed to inhibit the endothelial cell procoagulant response to measles virus, whereas it significantly reduced procoagulant activity induced in endothelial cells by recombinant interleukin-1. The capacity of measles virus to induce endothelial tissue factor in vitro, may be relevant to the thrombotic vasculopathy associated with measles virus infection in vivo.

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.