Developing a self-healing supramolecular nucleoside hydrogel.

Low molecular weight gelator hydrogels provide a viable alternative to traditional polymer based drug delivery platforms, owing to their tunable stability and in most cases inherent biocompatibility. Here we report the first self-healing nucleoside hydrogel using N4-octanoyl-2'-deoxycytidine (0.5% w/v) for drug delivery. The hydrogel's cross-linked nanofibrillar structure, was characterised using oscillatory rheology and confirmed using SEM and TEM imaging. The potential of this gel for drug delivery was explored in vitro using fluorescently labelled tracers. Cell viability assays were conducted using pancreatic cell lines which tolerated the gels well; whilst no adverse effects on the viability or proliferation of cells were observed for fibroblast cell lines.

Gelation properties of self-assembling N-acyl modified cytidine derivatives.

In this study we report the synthesis of new cytidine derived gelators possessing acyl chains of different lengths. These low molecular weight gelators were shown to form self-supporting gels at 0.5% (w/v) in binary systems of aqueous miscible polar organic solvent and water. The representative gels were studied using rheology and their fibrillar structure confirmed by TEM imaging and FTIR. We further demonstrated the use of these gels as potential drug delivery platforms by monitoring release characteristics of both high and low molecular weight fluorescently labelled tracers.

Agonist-occupied A3 adenosine receptors exist within heterogeneous complexes in membrane microdomains of individual living cells.

G protein-coupled receptors are known to be organized within different membrane compartments or microdomains of individual cells. Here, we have used a fluorescent A3 adenosine receptor (A3-AR) agonist, ABEA-X-BY630, and the technique of fluorescence correlation spectroscopy (FCS) to investigate the diffusional characteristics of functional agonist-occupied A3-AR complexes in single living cells. In Chinese hamster ovary cells expressing the human A3-AR, the fluorescent A3-AR agonist was able to inhibit forskolin-stimulated [3H]cAMP production (pEC50=8.57), and this was antagonized by the A3-selective antagonist MRS1220 (pK(B)=9.32). The fluorescent ligand also stimulated phosphoinositide hydrolysis (pEC50=7.34). Ligand binding to the A3-AR on the membranes of single cells and subsequent increases in single cell [Ca2+]i were monitored simultaneously in real time using confocal microscopy. FCS measurements in small-membrane microdomains (approximately 0.2 microm2) revealed two agonist-occupied A3-AR components with differing diffusion characteristics (diffusion coefficients=2.65x10(-8) and 1.19x10(-9) cm2/s, respectively). The binding of ligand to these two components was reduced from 5.1 and 14.9 to 2.6 and 3.3 receptors/microm2, respectively, by MRS1220 (100 nM). These data provide direct evidence for at least two populations of agonist-occupied A3-receptor complexes, showing different motilities within the membrane of single living cells.

Quantitative analysis of the formation and diffusion of A1-adenosine receptor-antagonist complexes in single living cells.

The A1-adenosine receptor (A1-AR) is a G protein-coupled receptor that mediates many of the physiological effects of adenosine in the brain, heart, kidney, and adipocytes. Currently, ligand interactions with the A1-AR can be quantified on large cell populations only by using radioligand binding. To increase the resolution of these measurements, we have designed and characterized a previously undescribed fluorescent antagonist for the A1-AR, XAC-BY630, based on xanthine amine congener (XAC). This compound has been used to quantify ligand-receptor binding at a single cell level using fluorescence correlation spectroscopy (FCS). XAC-BY630 was a competitive antagonist of A1-AR-mediated inhibition of cAMP accumulation [log10 of the affinity constant (pKb) = 6.7)] and stimulation of inositol phosphate accumulation (pKb = 6.5). Specific binding of XAC-BY630 to cell surface A1-AR could also be visualized in living Chinese hamster ovary (CHO)-A1 cells by using confocal microscopy. FCS analysis of XAC-BY630 binding to the membrane of CHO-A1 cells revealed three components with diffusion times (tauD) of 62 micros (tauD1, free ligand), 17 ms (tauD2, A1-AR-ligand), and 320 ms (tauD3). Confirmation that tauD2 resulted from diffusion of ligand-receptor complexes came from the similar diffusion time observed for the fluorescent A1-AR-Topaz fusion protein (15 ms). Quantification of tauD2 showed that the number of receptor-ligand complexes increased with increasing free ligand concentration and was decreased by the selective A1-AR antagonist, 8-cyclopentyl-1,3-dipropylxanthine. The combination of FCS with XAC-BY630 will be a powerful tool for the characterization of ligand-A1-AR interactions in single living cells in health and disease.

Surface engineering of living myoblasts via selective periodate oxidation.

Cell surface molecules are vital for normal cell activity. To study the functions of these molecules or manipulate cell behavior, the ability to decorate cell surfaces with bioactive molecules of our choosing is a potentially powerful technique. Here, we describe the molecular engineering of living L6 myoblast monolayers via selective periodate oxidation of sialic acid residues and the application of this surface modification in the artificial aggregation of cells. The aldehyde groups generated by this reaction were used to selectively ligate a model molecule, biotin hydrazide, to the cell surfaces. Flow cytometry analysis after staining with fluorescently conjugated avidin revealed a concentration-dependent increase in fluorescence compared to untreated cells with a maximal shift of 345.1 +/- 27.4-fold and an EC(50) of 17.4 +/- 1.1 microM. This mild oxidation reaction did not affect cell number, viability, or morphology. We then compared this chemical technique with the metabolic incorporation of reactive cell surface ketone groups using N-levulinoylmannosamine (ManLev). In this cell line, only a 22.3-fold fluorescence shift was observed compared to untreated cells when myoblasts were incubated with a high concentration of ManLev for 48 hours. Periodate oxidation was then used to modify myoblast surfaces to induce cell aggregation. Crosslinking biotinylated myoblasts, which do not spontaneously aggregate in culture, with avidin resulted in the rapid formation of millimeter-sized, multicellular structures. These data indicate that sodium periodate treatment is an effective, noncytotoxic method for the in vitro molecular engineering of living cell surfaces with the potential for cell biology and tissue engineering applications.

Tenderfoot Preemie vs a manual lancet: a clinical evaluation.

PURPOSE: To compare the amount of time required to collect a blood specimen, the number of heel punctures required, and the rate of hematology re-collections required when using a Monolet lancet vs a Tenderfoot Preemie device. DESIGN: Randomized, two-group, quasi-experimental. SAMPLE: Neonates with a birth weight >800 gm were eligible to participate in the study. Twenty subjects were randomized to the Monolet lancet (control) group and 20 to the Tenderfoot Preemie (experimental) group. A total of 157 blood specimens was collected, 89 of which were for hematology testing. RESULTS: For this sample population of preterm infants, less collection time was required, fewer heel punctures were necessary, and a lower re-collection rate occurred with use of the Tenderfoot Preemie than with use of the Monolet lancet.

Isolation and characterization of a previously unrecognized myosin heavy chain gene present in the Syrian hamster.

A full length (25,000 base-pair) myosin heavy chain gene completely contained within a single cosmid clone was isolated from a Syrian hamster cosmid genomic library. Sequence comparison of the 3' untranslated region indicated the presence of a 75% homology with the rat embryonic myosin heavy chain gene. Extensive 5' flanking region regulatory element conservation was also found when the sequence was compared to the rat myosin heavy chain gene. S1 nuclease digestion analysis, however, indicated that the Syrian hamster myosin heavy chain gene exhibited expression in adult Syrian hamster ventricular tissue, as well as the adult vastus medialis, a fast twitch skeletal muscle. Expression also appears to be enhanced in myopathic relative to control hearts. This myosin heavy chain gene is neither the alpha nor beta cardiac myosin heavy chain gene, but is a unique, previously unrecognized, myosin heavy chain gene present in both myocardial and skeletal muscle tissues.

Clot lysis for thrombosed central venous catheters in pediatric patients.

Central venous catheters (CVC) in pediatric patients provide a reliable method for administration of total parenteral nutrition and chemotherapy. Catheter thrombotic occlusion is a major complication and, until recently, the only therapeutic option was removal and surgical replacement of the catheter. Two fibrinolytic agents, streptokinase and urokinase, have been used successfully in adults to dissolve the clots. Few side effects have been reported when these agents were administered for this purpose. The Physician's Desk Reference advises against the use of such agents in the pediatric population. However, several reports of successful use of these agents in pediatric patients have been reported. They have also been infused systemically to relieve both arterial and venous thrombi. We prospectively evaluated the safety and efficacy of thrombolytic drugs in infants and children with CVCs who were receiving parenteral nutrition and/or hemodialysis. Abbokinase was used on 14 occasions to unclot silastic catheters in 10 pediatric patients. All catheters restored to patency were cleared within 50 minutes with an average clearance time of 19.3 minutes. Only one catheter could not be salvaged. Protime levels were obtained whenever possible before and after administration of the abbokinase. No significant elevations were noted after abbokinase administration. No allergic reactions or other complications occurred. Abbokinase was found to clear clotted central lines in a shorter time frame than has previously been reported in this patient population.