Application of alginate gel as a vehicle for liposomes. II. Erosion of alginate gel beads and the release of loaded liposomes

Short chain fatty acids (SCFAs) stimulate electroneutral sodium absorption by activation of apical Na/H exchange in colonocytes. It is often assumed that activation of Na/H exchange is via an intracellular acidification caused by SCFA uptake. These lecture notes review shortcomings in this model of SCFA-stimulated sodium absorption, revealed by recent reports in the literature. This is supplemented by information generated in our laboratory using both a tissue culture model of colonocytes (HT29-C1 cells) and a native tissue preparation (mouse distal colonic mucosa). In both preparations, evidence suggests that physiologic SCFA gradients may generate pH heterogeneity in aqueous microdomains near the plasma membrane of colonocytes. Finally, direct observation of such extracellular microdomains with confocal microscopy is used to support a new model, in which pH microdomains play an important role in regulating both SCFA fluxes and sodium absorption.     

Incorporation of hydrophobic aminopeptidase from hog kidney into egg lecithin liposomes: number and orientation of aminopeptidase molecules in the lecithin vesicles

The effects of xanthomegnin and O-methylxanthomegnin on the oxidative phosphorylation of rat liver mitochondria were compared. The n-octanol/water partition coefficient of xanthomegnin was markedly enhanced by O-methylation, but O-methylation of xanthomegnin reduced the uncoupling effect on the respiratory system of mitochondria. Analogous results were obtained in the uncoupling action of 5-hydroxy-1, 4-naphthoquinone (juglone) and 5-methoxy-1, 4-naphthoquinone (O-methyljuglone) on the oxidative phosphorylation of rat liver mitochondria. These data indicate that the phenolic hydroxyl groups of xanthomegnin might contribute to its uncoupling action on the oxidative phosphorylation of mitochondria. Bovine serum albumin (BSA) improved the efficiency of oxidative phosphorylation of mitochondria which were uncoupled by xanthomegnin. Spectroscopic observations revealed that xanthomegnin interacted with BSA by means of hydrophobic and ionic forces but O-methylxanthomegnin showed only hydrophobic interaction. Analogous interactions between mitochondria and xanthomegnin or O-methylxanthomegnin were observed. These results indicate that the uncoupling action of xanthomegnin on the respiratory system in mitochondria might involve ionic interaction of xanthomegnin with cationic residues in the hydrophobic region of mitochondrial membrane proteins.     

Structure of gel phase saturated lecithin bilayers: temperature and chain length dependence

Systematic low-angle and wide-angle x-ray scattering studies have been performed on fully hydrated unoriented multilamamellar vesicles of saturated lecithins with even chain lengths N = 16, 18, 20, 22, and 24 as a function of temperature T in the normal gel (L beta') phase. For all N, the area per chain Ac increases linearly with T with an average slope dAc/dT = 0.027 A2/degree C, and the lamellar D-spacings also increase linearly with an average slope dD/dT = 0.040 A/degree C. At the same T, longer chain length lecithins have more densely packed chains, i.e., smaller Ac's, than shorter chain lengths. The chain packing of longer chain lengths is found to be more distorted from hexagonal packing than that of smaller N, and the distortion epsilon of all N approaches the same value at the respective transition temperatures. The thermal volume expansion of these lipids is accounted for by the expansion in the hydrocarbon chain region. Electron density profiles are constructed using four orders of low-angle lamellar peaks. These show that most of the increase in D with increasing T is due to thickening of the bilayers that is consistent with a decrease in tilt angle theta and with little change in water spacing with either T or N. Because of the opposing effects of temperature on area per chain Ac and tilt angle 0, the area expansivity alpha A is quite small. A qualitative theoretical model based on competing head and chain interactions accounts for our results.     

Surface charge control of electropermeabilization and glycophorin electroinsertion with 1,2-diacyl-sn-glycero-3-phosphocholine (lecithin) liposomes

beta-Carotene has been studied widely as a potential cancer-preventing agent. Recent studies found that subjects who took beta-carotene supplements orally had increases in their serum concentrations of alpha-carotene and lycopene that were large (> 150% increase) and significantly greater than such increases in subjects who received placebo and that similar supplementation was associated with a decrease of approximately 37% in plasma lutein concentrations. A biologic interaction between beta-carotene and other carotenoids was suggested. We measured concentrations of retinol, alpha-tocopherol, and five carotenoids in serum specimens from a random sample of subjects enrolled in a clinical trial of the use of antioxidant vitamins in preventing colonic adenomas. We used serum specimens obtained at enrollment and after the subjects took placebo (n = 54) or 25 mg beta-carotene/d (n = 54) orally for 4 y. In a multivariate analysis, baseline serum concentrations of the analytes, sex, body mass index, diet, smoking status, and age were associated with variable changes in some analytes over the 4-y period but supplementation with beta-carotene was related only to a mean increase in serum beta-carotene itself of 151%. We excluded with 95% confidence an increase in lycopene > 4.9%, an increase in alpha-carotene > 17.6%, and a decrease in lutein > 14.7% in subjects given beta-carotene. These results confirm previous findings that supplementation with beta-carotene given orally does not alter serum concentrations of retinol or alpha-tocopherol. The findings also indicate that beta-carotene supplementation, which results in a moderate increase in serum beta-carotene concentration, does not significantly change serum concentrations of other carotenoids.     

Sensory irritation, pulmonary irritation and structure-activity relationships of alcohols

Sensory irritation due to inhalation of n-pentanol, n-heptanol, sec-butanol and tert-pentanol was determined from the reflexively induced decrease in respiratory rate in CF-1 mice. The concentration-effect relations followed Michaelis-Menten equations, complying with receptor mediated processes. The relations were transformed into nearly rectilinear relationships in log concentration-effect plots, and the extrapolated threshold concentrations (RD-0) from the lines were 120, 28, 640 and 1210 ppm, respectively, obtained from the first 2 min of the exposure period. These values were comparable to those found in Swiss-Webster mice and to those obtained by electrophysiological experiments in Sprague-Dawley rats. The hydrophobic properties of the receptor biophase were found to approach that of the internal part of the bilayer membrane. Estimates on threshold limit values (TLV) were obtained and were found in reasonable agreement with the established values. The nose has a scrubbing effect, which reduces the concentration in the lungs in normal mice. n-Pentanol, sec-butanol and tert-pentanol decreased tidal volume in normal mice, explained either by an activation of receptors in the upper airways or by a sensitization of the stretch receptors. Two types of pulmonary responses were seen in tracheal-cannulated mice, which could be explained by an effect on stretch receptors and another type of lung receptors.     

Human transfer factor prepared by dialysis, ultrafiltration and gel chromatography: biological activity in local transfer of skin sensitivity

Human transfer factor (TF) was prepared by a variety of methods including dialysis using cellophane tubing, ultrafiltration through a membrane of known pore size. Sephadex G25 chromatography or combinations of some of these methods. In general the various preparations when injected locally into human skin gave greater delayed-type responses than antigen (PPD or Candida) alone. The combination of either vacuum dialysis, or ultrafiltration, with G-25 chromatography gave as good or better TF activity when compared with unchromatographed materials. Since ultrafiltration and concentration is rapid procedure and eliminates the need for freeze-drying, in contrast to vacuum dialysis against water, these results indicate that ultrafiltration and G-25 chromatography provide a convenient method for preparing large batches of relatively pure TF from leucocyte extracts.     

Silicone gel sheeting for stabilization of skin grafts

BACKGROUND: Success of skin grafts depends on sufficient immobilization and early intervention for hematoma, seroma, or infection. OBJECTIVE: To stabilize and cover skin grafts with a tie-over technique using translucent silicone gel sheet. METHODS: Twenty-seven skin defects were resurfaced with skin grafts. A sterile silicone gel sheet was placed over the skin graft. Gel was fixed to the wound edges with skin staplers. RESULTS: All grafts healed without any complication. CONCLUSION: Using silicone gel sheeting on 27 skin grafts, we found that it is an effective method for stabilization and allows direct visualization of the graft in order to inspect hematoma-like complications.     

Relationship between the amount of propranolol permeating through the stratum corneum of guinea pig skin after application of propranolol adhesive patches and skin irritation

In the present study we evaluated the relationship between the cumulative amount of propranolol permeating through the stratum corneum and the formation of erythema, a skin irritation reaction, after transdermal application of adhesive patches containing propranolol to the skin of guinea pigs. The intensity of erythema was expressed in terms of a* values measured with a chromameter. The a* values increased in guinea pigs after application of the adhesive patches containing 0.4 mg/cm2 of propranolol to the skin. Since the adhesive patches showed good adhesion to the skin (propranolol content is less than the saturated concentration in the adhesive base) and the cumulative amount of propranolol permeating through the stratum corneum is small, the development of erythema was considered to be mainly due to physical factors such as peeling. Even in adhesive patches containing 0.8 mg/cm2 or 1.2 mg/cm2 of propranolol, a* values increased, although adhesion to the skin is low because of crystallization of propranolol in the adhesive base. On the other hand, in these two adhesive patches, the cumulative amount of propranolol permeating through the stratum corneum increased up to 24 h after application. These findings suggest that the skin irritation reaction is due to propranolol mainly absorbed transdermally, because there is a high correlation between the cumulative amount of propranolol permeating through the stratum corneum and the a* values (r = 0.928).     

Reduction of skin flap necrosis by transdermal application of buflomedil bound to liposomes

The influence of the vasoactive drug buflomedil hydrochloride bound to liposomes (2 mg/ml) was investigated in an arterial pattern skin flap model using the ear of hairless mice. For flap creation, the ear is cut at four-fifths of its base, which leaves the anterior artery as the only feeding vessel of the flap. Liposomes were locally applied daily for 30 minutes up to 5 days after flap creation. Microvascular perfusion in the proximal, central, and distal parts of the flap was measured by laser Doppler flowmetry. The border between perfused and nonperfused tissue was visualized by intravital fluorescence microscopy using fluorescein isothiocyanate (FITC)-labeled dextran (Mr 150,000) for contrast enhancement of microvessels. The area of nonperfused tissue was assessed by digital planimetry. Five days after flap creation the nonperfused area amounted to 23.8 +/- 3.1 percent of total flap surface in treated ears compared with 46.1 +/- 5.6 percent in untreated ears (p < 0.05) of the contralateral side. Additional preoperative treatment for 5 days did not further reduce the area of nonperfused tissue (treated ears, 23.0 +/- 1.3 percent; control ears, 44.6 +/- 5.1 percent). Microvascular perfusion was higher in the postoperatively treated ears in all parts of the flap from day 1 after flap creation until termination of the experiment. Five days after flap creation, perfusion as measured by laser Doppler flowmetry was reduced to 46.0 +/- 10.8 percent in the distal part in control ears compared with 91.9 +/- 8.3 percent (p < 0.05) in treated animals. Additional preoperative treatment for 5 days did not result in further improvement. It is concluded that local application of the vasoactive drug buflomedil docked to liposomes could be of therapeutic use in the treatment of ischemic tissue, including skin flaps.     

Analysis of collagen in burned skin with SDS-polyacrylamide gel electrophoresis

Type III collagen was determined by SDS-polyacrylamide gel electrophoresis in normal rabbit skin, skin with deep II degrees burn 72h after injury, skin one month after burn, and healed burn 1.5 month after injury. Results showed that there was a reduction of type III collagen in the process of healing of burn wounds.     

Evaluation of skin irritation and sensitization of two diol solutions used as experimental dentin primers in humans and guinea pigs

The purpose of the present study was to evaluate the safety of ethylene glyco (EG) and 1,6-hexanediol (HD) solutions as experimental dentin primers when subjected to the guinea pig maximization test (GPMT), primary irritation test, cumulative skin irritation test and human patch test. No primary and cumulative skin irritation resulting from the use of 62.5% EG or 45% HD solutions was observed. In the case of GPMT, the animals sensitized with 2-hydroxyethyl methacrylate (2-HEMA) responded to 100% HD. 62.5% EG and 45% HD as dentin primers were safer than 2-HEMA such as a methacrylic primer.     

Transmission of two novel mutations in a pedigree with familial lecithin:cholesterol acyltransferase deficiency: structure-function relationships and studies in a compound heterozygous proband

Two novel mutations were identified in a compound heterozygous male with lecithin:cholesterol acyltransferase (LCAT) deficiency. Exon sequence determination of the LCAT gene of the proband revealed two novel heterozygous mutations in exons one (C110T) and six (C991T) that predict non-conservative amino acid substitutions (Thr13Met and Pro307Ser, respectively). To assess the distinct functional impact of the separate mutant alleles, studies were conducted in the proband's 3-generation pedigree. The compound heterozygous proband had negligible HDL and severely reduced apolipoprotein A-I, LCAT mass, LCAT activity, and cholesterol esterification rate (CER). The proband's mother and two sisters were heterozygous for the Pro307Ser mutation and had low HDL, markedly reduced LCAT activity and CER, and the propensity for significant reductions in LCAT protein mass. The proband's father and two daughters were heterozygous for the Thr13Met mutation and also displayed low HDL, reduced LCAT activity and CER, and more modest decrements in LCAT mass. Mean LCAT specific activity was severely impaired in the compound heterozygous proband and was reduced by 50% in individuals heterozygous for either mutation, compared to wild type family members. It is also shown that the two mutations impair both catalytic activity and expression of the circulating protein.     

Human lymphocyte cDNA ordered library analyzed by 2D gel electrophoresis. 1. Pooling strategy and matching of gel patterns

beta-Citryl-L-glutamate-hydrolysing enzyme (beta-CGHE) was purified from rat testis particulate fraction 13,000-fold, at a yield of 7%. The enzyme was purified by ammonium sulfate fractionation, hydroxyapatite, chelating Sepharose, beta-CG-Sepharose affinity chromatography and Sephacryl S-300 gel filtration. The purified enzyme usually migrated as two periodic acid Schiff's-stained bands on native polyacrylamide gel-electrophoresis (PAGE) with molecular weights of 350 and 420 kDa. Both bands hydrolyzed beta-citryl-L-glutamate (beta-CG) to citrate and glutamate. The 420 kDa band was changed by digestion with N-glycosidase F, into a 350 kDa band on native PAGE. The purified enzyme was composed of 90, 100, 115 and 130 kDa subunits on SDS-PAGE under non-reduced conditions. The purified enzyme was pharmacologically similar to the beta-CGHE activity partially purified from rat testis. This enzyme required manganese ions for full activity and it was strongly inhibited by nucleotides such as ATP or GTP and phosphate ions. beta-CGHE was also potently inhibited by an excitatory amino acid agonist, L-quisqualate, but not by another agonists, N-methyl-D-aspartate and kinate. It had high substrate specificity for beta-CG. The antibodies against the purified enzyme reacted mainly to the 115 kDa band on the SDS-PAGE and precipitated the enzyme activity from the crude and purified enzyme solution.