Activation of thromboxane receptors and the induction of vasomotion in the hamster cheek pouch microcirculation

1. The present study was designed to investigate a possible role of thromboxane A2 (TXA2) on arteriolar vasomotion (spontaneous rhythmic variations of the vessel diameter). Therefore the microcirculatory effects of the thromboxane-receptor (TP-receptor) agonist, U 46619, as well as the effects of the TP-receptor antagonists S 17733 and Bay U3405 were evaluated in the hamster cheek pouch microcirculation. For comparison some effects of angiotensin II were also investigated. 2. For microcirculatory measurements, the cheek pouch preparation was placed under an intravital microscope coupled to a closed circuit TV system. The TV monitor display was used to obtain arteriolar internal diameter measurements by means of an image shearing device. 3. Superfusion (0.1 nM to 1 microM) or bolus application (1 pmol to 10 nmol) of U 46619 concentration- or dose-dependently decreased the arteriolar diameter and induced vasomotion in arterioles with a mean initial diameter of 24+/-2 microm. Both the vasoconstriction and the vasomotion induced by U 46619 were inhibited by the TP-receptor antagonists S 17733 (100 mg kg(-1), i.v.) and Bay U3405 (10 mg kg(-1), i.v.). 4. Bolus applications of angiotensin II (0.1 pmol to 1 nmol) induced transient vasoconstriction followed by vasodilatation in the cheek pouch arterioles. The dilatation but not the constriction, was sensitive to treatment with the NO-synthase inhibitor N(omega)-nitro-L-arginine (L-NOARG; 100 microM). Angiotensin II did not induce vasomotion in control conditions or in the presence of L-NOARG. 5. Bolus application of phenylephrine (10 pmol) induced vasoconstriction but no vasomotion in previously quiescent hamster cheek pouch arterioles. 6. These results indicate that activation of TP-receptors causes vasomotion in the hamster cheek pouch arterioles. These spontaneous rhythmic variations in arteriolar diameter are not observed with equipotent doses of angiotensin II and phenylephrine. Thus, the vasoconstriction by itself cannot explain the occurrence of vasomotion observed with the TP-receptor agonist.     

Adhesion properties of mutants of Staphylococcus aureus defective in fibronectin-binding proteins and studies on the expression of fnb genes

Staphylococcus aureus 8325-4 has the potential to express two distinct cell wall-associated fibronectin-binding proteins called FnBPA and FnBPB. In order to test if both proteins are expressed in S. aureus and if both are required for promoting bacterial adhesion to fibronectin-coated surfaces, insertion mutations were isolated in each gene. A DNA fragment encoding tetracycline resistance was inserted into fnbA and a fragment encoding erythromycin resistance was inserted into fnbB. A double fnbAfnbB mutant was also constructed. The fnbA and fnbB single mutants showed no significant reduction in their adhesion to polymethylmethacrylate coverslips that had been coated in vitro with fibronectin. However, the double mutant was completely defective in adhesion. Monospecific antibodies directed against the non-conserved N-terminal regions of both proteins confirmed the lack of expression of FnBPs in the mutant strains. Wild-type fnbA and fnbB genes cloned seperately on a multicopy plasmid were each able to restore fully the adhesion-defective phenotype of the 8325-4 fnbAfnbB mutant. This demonstrates that both fnb genes are expressed in S. aureus and that both contribute to the ability of strain 8325-4 to adhere to fibronectin-coated surfaces. The double mutant was also defective in adhesion to coverslips that had been removed from tissue cages implanted subcutaneously in guinea-pigs, which suggests that fibronectin is important in promoting attachment of S. aureus to biomaterial in vivo.     

Exacerbation of atopic dermatitis after bacillus Calmette-Guérin vaccination

In two children with atopic dermatitis, routine vaccination with bacillus Calmette-Guérin (BCG) was followed by severe exacerbation of skin disease. If the sequence is cause and effect, a possible mechanism is stimulation of a Th2 lymphocyte cytokine profile by the vaccine, with migration of activated lymphocytes to inflamed skin. In children with active atopic dermatitis, BCG vaccination is best deferred until remission.     

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.     

Characterization of glucose transport activity reconstituted from heart and other tissues

We examined several aspects of glucose transport reconstituted in liposomes, with emphasis on transporters of rat heart (mostly GLUT4) compared to those of human erythrocytes (GLUT1), and on effects of agents that modulate transport in intact cells. Several types of samples gave higher reconstituted activity using liposomes of egg lipids rather than soybean lipids. Diacylglycerol, proposed to activate transporters directly as part of the mechanism of insulin action, increased the intrinsic activity of heart transporters by only 25%, but increased the size of the reconstituted liposomes by 90%. The dipeptide Cbz-Gly-Phe-NH2 inhibited GLUT4 with a Ki of 0.2 mM, compared to 2.5 mM for GLUT1, which explains its preferential inhibition of insulin-stimulated glucose transport in adipocytes. Verapamil, which inhibits insulin- and hypoxia-stimulated glucose transport in muscle, had no effect on reconstituted transporters. Heart transporters had a higher Km for glucose uptake (13.4) than did GLUT1 (1.6 mM), in agreement with a recent study of GLUT1 and GLUT4 expressed in yeast and reconstituted in liposomes. Transporters reconstituted from heart and adipocytes were 40-70% inactivated by external trypsin, suggesting the presence of trypsin-sensitive sites on the cytoplasmic domain of GLUT4. NaCl and KCl both reduced reconstituted transport activity, but KCl had a much smaller effect on the size of the liposomes.     

In vivo modulation of iododeoxyuridine metabolism and incorporation into cellular DNA by 5'-amino-5'-deoxythymidine in normal mouse tissues and two human colon cancer xenografts

This in vivo study examines the ability of 5'-amino-5'-deoxythymidine (5'-AdThd) to modulate 5-iododeoxyuridine (IdUrd) cellular metabolism in two human colon cancer xenografts (HT 29 and HCT-116), two actively proliferating normal mouse tissues (bone marrow and intestine), and a quiescent normal mouse tissue (liver). 5'-AdThd is a thymidine analogue that at low concentrations (<30 micrometer) can increase thymidine kinase activity, which is the rate-limiting enzyme for activation of IdUrd. We reported recently that the in vitro incubation of HT 29 and HCT-116 cells in 5'-AdThd + IdUrd resulted in an enhancement of 5-iodo-2'-dUTP pools, IdUrd DNA incorporation, and subsequent radiosensitization compared with incubation with IdUrd alone (Clin. Cancer Res., 1: 407-416, 1995). These in vitro effects were more significant in the radioresistant cell line HT 29. Using a 6-day continuous infusion of IdUrd (50 or 100 mg/kg/day) and/or 5'-AdThd (200 mg/kg/day), no increase in systemic toxicity (percentage of body weight loss) was observed in athymic nude mice with 5'-AdThd alone or when combined with IdUrd. There was significant dose-dependent, systemic toxicity with IdUrd, which was reversible within 3 days of completing the lower-dose IdUrd infusion. However, a comparison of plasma levels during the 6-day continuous infusion of IdUrd +/- 5'-AdThd showed a significant interaction of IdUrd and 5'-AdThd, resulting in higher plasma levels by day 6 of both compounds and the principal metabolites, iodouracil and deoxyuridine, which is consistent with nonlinear saturating effects on dihydrouracil dehydrogenase. Coadministration of IdUrd and 5'-AdThd resulted in an increase in the percentage of IdUrd DNA incorporation in the two proliferating normal tissues, which was significant only with the lower IdUrd dose. No effect on IdUrd DNA incorporation was found in normal liver at either IdUrd dose +/- 5'-AdThd. Similar to our in vitro data, the continuous infusion of IdUrd and 5'-AdThd showed a significant effect by increasing the percentage of IdUrd DNA incorporation in HT-29 xenografts at both IdUrd doses, whereas coadministration of 5'-AdThd had no such effect in HCT-116 xenografts.     

Intrapreneurial nursing: the Comprehensive Lower Extremity Assessment Form

The Comprehensive Lower Extremity Assessment Form was developed in response to the need for a screening tool in a nurse-managed foot care clinic. It differs from other such tools because it includes clinical measures that identify the potential for foot pathology. The Comprehensive Lower Extremity Assessment Form also serves as an assessment teaching guide in a foot care course and is included as part of a home-study program. The authors demonstrate how the Comprehensive Lower Extremity Assessment Form has generated revenue as part of an intrapreneurial outgrowth of their foot clinic and provides a comprehensive approach to lower extremity assessment. The form can be tailored to meet the needs of the advanced practice nurse, the clinical setting, or patient population.