Delivery of superoxide dismutase to pulmonary epithelium via pH-sensitive liposomes.

Respiratory insufficiency, when treated with oxygen supplementation, or exposure to diverse pulmonary toxins can cause lung damage as a result of increased oxygen radical production. Enzymes such as superoxide dismutase (SOD) may attenuate this pathological process, but the intracellular delivery and antioxidant action of SOD is impeded by its inability to cross cellular membranes. One approach for facilitating intracellular delivery of macromolecules is to entrap SOD into liposomes. The delivery of SOD to lung cells was accomplished using pH-sensitive liposomes, made with 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) and 1-oleoyl-2-oleoyl-sn-glycero-3-succinate (DOSG), added to cultured fetal rat lung distal epithelial (FRLE) cells. FRLE cells, obtained from fetuses at day 19 gestation, expressed a high-affinity receptor for surfactant protein A (SP-A) with an apparent dissociation constant (Kd) = 3.6 +/- 0.2 micrograms/ml (5.5 x 10(-9) M) and a capacity of 130 +/- 3 ng/10(6) cells (125,000 +/- 3,000 binding sites/cell). This receptor was utilized for targeting liposomes to cells, after incorporating SP-A during liposome membrane formation. Liposomes were uniformly small (180 +/- 77 nm; mean +/- SD) and stable at 4 degrees C for 1 wk, entrapping 10 +/- 4% of initially added SOD. After incubation of pH-sensitive liposomes containing entrapped SOD with cultured FRLE cells, cell-associated SOD activity was increased 5.1-fold from 7.8 +/- 2.5 to 40.1 +/- 3.3 U SOD/mg cell protein. Incorporation of SP-A into liposomes increased by 6.2-fold the delivery of liposomal SOD to cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Potential of Loperamide Oxide in the Reduction of Ileostomy and Colostomy Output.

The effects of a single oral dose of loperamide oxide on stoma output were evaluated in an open trial that included 22 patients - 4 with a colostomy and 18 with an ileostomy, in whom daily stoma output was usually more than 500g. Antidiarrhoeal therapy was stopped from days 2 to 7, and from days 5 to 8 a standardised high-fibre diet was given. Stoma effluent was collected for 24 hours on day 7. On day 8, patients took one dose of loperamide oxide 6mg. In 20 of the 22 patients, stoma output was reduced by 13 to 75% after administration of loperamide oxide. The mean output was reduced by 45% (p = 0.0001). There were no adverse experiences associated with administration of loperamide oxide. The majority of drug recovered in stoma effluent was loperamide, suggesting extensive conversion of loperamide oxide to loperamide. These preliminary findings suggest that a single 6mg dose of loperamide oxide is effective in reducing stoma output in patients with an ileostomy or colostomy.

Effects of argon beam coagulation on split thickness skin graft survival in the swine model.

Argon beam coagulation (ABC) uses argon gas to evacuate blood in an operative field and simultaneously to transport a coagulating electric current to bleeding sites. This allows a 'no touch' method of coagulation which is faster, more precise, and less destructive than conventional electrocautery. The technique has been used to reduce blood loss in liver surgery, trauma surgery and neck dissections. In this study, the effect of ABC treatment of the recipient graft beds on the survival of split thickness skin grafts was assessed in pigs, testing the hypothesis that ABC treatment of the recipient bed would have no adverse effect on skin graft survival. Nine 20 kg mixed breed pigs had split thickness skin grafts raised on each side of the paraspinous area. The graft beds on one side were prepared using ABC and the contralateral side served as controls. The percentage of graft survival on the ABC treated sides was compared to control graft survival on the other side at 12 days postsurgery. Our results confirm the hypothesis that treatment of the recipient bed with ABC does not adversely affect the survival of split thickness skin grafts.

Superoxide and peroxynitrite in atherosclerosis.

The role of reactive oxygen species in the vascular pathology associated with atherosclerosis was examined by testing the hypothesis that impaired vascular reactivity results from the reaction of nitric oxide (.NO) with superoxide (O2-), yielding the oxidant peroxynitrite (ONOO-). Contractility studies were performed on femoral arteries from rabbits fed a cholesterol-supplemented diet. Cholesterol feeding shifted the EC50 for acetylcholine (ACh)-induced relaxation and impaired the maximal response to ACh. We used pH-sensitive liposomes to deliver CuZn superoxide dismutase (SOD; superoxide:superoxide oxidoreductase, EC 1.15.1.1) to critical sites of .NO reaction with O2-. Intravenously injected liposomes (3000 units of SOD per ml) augmented ACh-induced relaxation in the cholesterol-fed group to a greater extent than in controls. Quantitative immunocytochemistry demonstrated enhanced distribution of SOD in both endothelial and vascular smooth muscle cells as well as in the extracellular matrix. SOD activity in vessel homogenates of liposome-treated rabbits was also increased. Incubation of beta very low density lipoprotein with ONOO- resulted in the rapid formation of conjugated dienes and thiobarbituric acid-reactive substances. Our results suggest that the reaction of O2- with .NO is involved in the development of atherosclerotic disease by yielding a potent mediator of lipoprotein oxidation, as well as by limiting .NO stimulation of vascular smooth muscle guanylate cyclase activity.

Improved RNA isolation from cells in tissue culture using a commercial nucleic acid extractor.

The Applied Biosystems 340A Nucleic Acid Extractor automates isolation of either DNA or RNA from tissue or cells in culture. We have found that several modifications to the manufacturer's recommended protocol greatly improve the quality of RNA that can be routinely isolated from cells in culture. These modifications include lysis of monolayer cells directly on plates, centrifuging samples after homogenization to remove precipitable RNase contaminants and purging the instrument's reagent lines with 0.1% diethyl pyrocarbonate. These simple modifications enhance both RNA quality and reproducibility of yield.

Salmonella neck abscess.

Salmonella species represent uncommon causative agents in focal infections of the head and neck. The case of a 24-year-old diabetic man with a neck abscess caused by Salmonella enteritidis is presented, and the relevant literature is reviewed. Salmonella should be included in the differential diagnosis of head and neck abscesses.

Comparison of propranolol-binding plasma proteins in sheep with those in humans, dogs and rats.

Alpha 1-acid glycoprotein (AGP), a plasma protein responsible for the binding of a variety of basic lipophilic drugs including propranolol, is different from other plasma proteins in being nonprecipitable after treatment with 1.2M perchloric acid (PCA). To assess the contribution of AGP to drug disposition in sheep and three other species (rats, dogs, and humans), the binding of [3H]propranolol was measured before and after PCA precipitation. PCA precipitation reduced propranolol binding 14-fold in sheep, compared to 2- to 3-fold in the other species. This implied either that sheep AGP binds less propranolol than other species, or that the AGP in sheep is more precipitable. It was not due to inherently poor propranolol binding, as whole sheep plasma bound a higher fraction than the other species. When samples of PCA-precipitated sheep plasma were analyzed using polyacrylamide gel electrophoresis, the concentration of AGP was 10-20% that of the other species. Phenobarbital induction was used as a tool to examine the changes in the plasma protein profile. Phenobarbital induced propranolol binding and AGP along with two other proteins in sheep. One of these proteins migrated similarly to AGP deglycosylated by peptide-N-glycosidase F. It is postulated that the greater precipitability of propranolol binding in sheep is due to a less glycosylated form of AGP which is not important in other species.

Silver accumulation in Pseudomonas stutzeri AG259.

Silver toxicity to Pseudomonas stutzeri AG259 was strongly dependent on the NaCl concentration in the medium, which reduced the availability of Ag+ by precipitation as AgCl. Accumulation of Ag by growing cultures was low being less than or equal to 7.5 nmol (mg dry mass)-1 over all treatments examined. The presence of NaCl in the growth medium did not markedly affect the amounts of Ag accumulated by the cells but influenced toxicity as manifest by a lag period which was greatest at low NaCl concentrations (less than or equal to 0.1% mass/vol.). In NaCl-free medium, P. stutzeri did not grow in the presence of 0.5 mM AgNO3 in contrast to Ag-free controls. The majority of Ag accumulation by resting cells of P. stutzeri occurred within 1 min of incubation and there was little difference in uptake capacities between cells previously grown in the absence or presence of AgNO3. Lowest amounts of Ag uptake by resting cells occurred when suspended in 1 mM Mes pH 6.5, containing 1% (mass/vol.) NaCl. Prior exposure of P. stutzeri to Cu(NO3)2 resulted in a marked reduction in Ag uptake when suspended in 1 mM Mes pH 6.5, containing 0.5 mM AgNO3.

Temperature dependence of glucose transport in erythrocytes from normal and alloxan-diabetic rats.

Alloxan diabetes increased 3-O-methylglucose transport rates in rat red blood cells (RBC) at temperatures below 30 degrees C and decreased them above 30 degrees C. Preincubation of RBC from control rats with 20 mM glucose, 3-O-methylglucose, 2-deoxyglucose or xylose greatly elevated transport at 14 degrees C by increasing Vmax. The effect was slight at 40 degrees C. Preincubation with glucose or deoxyglucose alone caused a 50% depression of transport rates at 40 degrees C as a result of a rise in the Km, which is similar to findings in cells from alloxan-diabetic rats. Measurement of intracellular glucose metabolites suggested inhibition of glycolysis in cells from diabetic rats and a positive correlation between the level of intracellular hexose monophosphates and transport inhibition. Membrane fatty-acid and cholesterol composition and membrane lipid-ordering as monitored by electron paramagnetic resonance were not altered by alloxan diabetes. It is concluded that intracellular sugar and sugar metabolism alter the temperature dependence of glucose transport kinetics. Glucose metabolism can feed back to inhibit transport by increasing the transport Km at physiological temperatures only.

Lipoprotein composition as a component in the lipoprotein clearance defect in experimental diabetes.

The hypertriglyceridemia associated with streptozotocin-induced diabetes in rats is largely reflected in the plasma lipoproteins of density less than 1.006 g/ml. Analysis of the plasma apolipoproteins of these rats indicated marked alterations in both the total levels and in the lipoprotein distribution of the major apolipoproteins. In whole plasma, diabetes was associated with significant increases in apolipoprotein (apo)-AIV, apo-AI, and apo-B (mainly in the intestinally derived apo-B240) and a marked decrease in apo-E. In the d less than 1.006 g/ml lipoprotein fraction (very-low-density lipoproteins (VLDL], there were significant increases in apo-B240, apo-AI, and apo-AIV and decreased levels of apo-E and the C apolipoproteins. The decrease in apo-C was primarily due to lower levels of apo-CII, and the ratio of the lipoprotein lipase inhibitor, apo-CIII, to the lipoprotein lipase activator, apo CII, was significantly increased over that in controls. The comparative clearance of triglycerides of VLDL particles from control and diabetic rat plasma was tested in recirculating heart perfusion in vitro. During 45-min perfusions of hearts from control donor rats, lipolysis of triglycerides of VLDL from diabetic rats was only 63-64% of that using plasma VLDL from control rats. Perfusion of hearts from diabetic rats with VLDL from control rats gave lipolysis values of only 53% of that obtained with normal hearts. Where both the VLDL and hearts were obtained from diabetic rats, lipolysis was 23% of that observed when both the lipoprotein and the organ were from control rats. The data suggest that in addition to depressed lipoprotein lipase activity in the tissue from diabetic rats, there are also major compositional changes in circulating lipoproteins which may contribute to defective triglyceride clearance from the circulation.