A spectrophotometric method for the determination of hydroperoxides in liposomes

A modification of the Asakawa-Matsushita iodometric assay method for the determination of the content of lipid hydroperoxides was developed which permits the simultaneous processing of many samples of high lipid content. The method has the advantages of simplicity as well as good reproducibility, so it is not necessary to process standards with each determination. Our technique exceeds the sensitivity attained with other spectrophotometric determinations reported in the literature. The method requires the total elimination of water from the samples, and this was accomplished using an azeotropic mixture of ethanol:water of 96:4. The results obtained with liposomes indicate that the method is applicable to biological material limited to small volume samples, ranging 5-50 microliters. We want to emphasize that this method permits the study of the peroxidation process as function of time.     

Enzymatic method for the simultaneous determination of hyaluronan and chondroitin sulfates using high-performance liquid chromatography

Biological samples have a high dielectric constant that can shorten RF wavelengths by a factor of 8 relative to the vacuum. At high field strengths, finite wavelength effects within larger samples are the dominant cause of RF field nonuniformity. A coil design is presented that can reduce and even eliminate this inhomogeneity; 4-T images in phantoms and in the head of a normal volunteer are presented, which demonstrate improved homogeneity relative to a standard coil. This coil design should aid in realizing the potential advantages of imaging large samples at high field strengths.     

Liposome-cell interactions in vitro: effect of liposome surface charge on the binding and endocytosis of conventional and sterically stabilized liposomes

The cellular uptake of liposomes is generally believed to be mediated by adsorption of liposomes onto the cell surface and subsequent endocytosis. This report examines the effect of liposome surface charge on liposomal binding and endocytosis in two different cell lines: a human ovarian carcinoma cell line (HeLa) and a murine derived mononuclear macrophage cell line (J774). The large unilamellar liposomes were composed of 1, 2-dioleolyl-sn-glycero-3-phosphatidylcholine with and without the addition of either a positively charged lipid, 1, 2-dioleoyl-3-dimethylammonium propanediol (DODAP), or a negatively charged lipid, 1,2-dioleolyl-sn-glycero-3-phosphatidylserine. In some experiments 5 mol % of the anionic PEG2000-PE or a neutral PEG lipid of the same molecular weight was added. HeLa cells were found to endocytose positively charged liposomes to a greater extent than either neutral or negatively charged liposomes. This preference was not lipid-specific since inclusion of a cationic cyanine dye, DiIC18(3), to impart positive charge in place of DODAP resulted in a similar extent of endocytosis. In contrast the extent of liposome interaction with J774 cells was greater for both cationic and anionic liposomes than for neutral liposomes. The greater uptake of positively charged liposomes by HeLa cells was also observed with sterically stabilized liposomes (PEG liposomes). Although the overall amount of endocytosis for all the PEG liposomes examined was attenuated relative to conventional liposomes, the extent of endocytosis was greatest for positively charged PEG liposomes, whereas negatively charged PEG2000-PE liposomes were hardly endocytosed by the HeLa cells. Incorporation of a neutral PEG lipid into liposomes permits the independent variation of liposome steric and electrostatic effects in a manner that may allow interactions with cells of the reticuloendothelial system to be minimized, yet permit strong interactions between liposomes and proliferating cells.     

The influence of the route of administration and liposome composition on the potential of liposomes to protect tissue against local toxicity of two antitumor drugs

Pulling tasks require the torso to act as a rigid link in order to facilitate the force transmission between the ground and the hands. In this study, we tested the hypothesis that a lifting belt increases the rigidity of the torso, thereby increasing pulling strength or reducing trunk muscle forces, or both, as pulling tasks are performed. Twelve volunteers performed maximal and submaximal isometric pulling exertions; the latter were performed on nonslippery and slippery surfaces. Electromyographic data from 8 trunk muscles, trunk kinematic data, and ground reaction forces were sampled during each exertion. Results indicated that the lifting belt had no effect during maximal exertions on the maximal pull forces generated or the muscle recruitment levels, irrespective of the pulling posture. The lifting belt did not affect the EMG data obtained during the submaximal (40% of maximum) exertions, even when participants pulled on a slippery surface. However, the slippery surface increased the coactivation within the trunk musculature, perhaps stiffening the torso in the event of a slip. The absence of a statistical interaction effect between the lifting belt and the footing condition (slipperiness) indicates that the belt did not alter the coactivation pattern and hence was not relied upon by the participants as a protective mechanism. The data presented here will assist those who must make decisions regarding lifting-belt use and those who train individuals in manual materials handling techniques.     

A method of determining the specific volume of liposomes with Turnbull blue]

OBJECTIVES: To present the results obtained in patients with stress urinary incontinence treated with periurethral collagen injection. METHODS: 26 female patients with stress urinary incontinence were treated with bovine collagen injection; mean volume 10.8 cc. The results achieved by this therapeutic modality are described herein. RESULTS: Control evaluations performed during a period of one year showed highly satisfactory results had been achieved initially and the success rate gradually increased over the 12 months follow-up. Overall the final results showed a success rate of 34.6%, 38.4% showed frank improvement and 26.9% had a failed procedure. There were no significant differences in the results for both types of stress urinary incontinence. The results correlated with the severity of incontinence; the success rate was higher in the patients with low grade incontinence. CONCLUSIONS: Periurethral collagen injection is indicated in patients with type I and type III stress urinary incontinence who cannot benefit from surgery. Patients with type II stress urinary incontinence, however, do not benefit from this therapeutic modality.     

Microencapsulation of human insulin DEAE-dextran complex and the complex in liposomes by the emulsion non-solvent addition method

The blood levels of ethanol, acetaldehyde, acetate, methanol, acetone, lactate, pyruvate, and glucose were measured in 23 male alcohol-dependent patients on days 2 to 6 after hospitalization and in 22 healthy male blood donors. Correlations between the biochemical parameters and 17 symptoms of the alcohol-withdrawal syndrome (AWS) were calculated. Abnormally high levels of ethanol, methanol, acetate, and acetone as well as hypoglycaemia were found on day 2, but lactacidaemia and pyruvataemia were pronounced throughout the observation period. AWS severity correlated positively with the acetone content on day 2 and with the acetate content on days 2 to 6. Negative correlations were found between ethanol levels and craving for alcohol, methanol levels and craving for alcohol, and between psychopathologic disorders and the total AWS severity score. The results suggest that concentrations of blood ethanol, methanol, acetate, and acetone exceeding their normal endogenous levels can be used only as indicators of recent heavy drinking. Linear discriminant analysis using the levels of the nine parameters studied enabled the correct classification of 91% to 96% of alcoholic patients during 1 week of abstinence and 100% of control subjects. The most informative parameters in the discrimination between alcoholics and controls were lactate, pyruvate, the lactate/pyruvate ratio, acetate, and acetone.     

Intracellular hyperthermia for cancer using magnetite cationic liposomes: an in vivo study

OBJECTIVE: Only two previous studies have assessed the effects of long-term GH replacement therapy on bone mineral density (BMD) in patients with adult onset GH deficiency. To date no study has looked at the long-term impact on BMD after a short course (6-12 months) of GH replacement. In two groups of patients with adult onset GH deficiency we have studied BMD either (a) after 3 years of continuous GH replacement or (b) 2 years after completion of a short course of GH. DESIGN: An open GH therapeutic study in which patients were recruited from a previous double-blind placebo-controlled study. The BMD status of all patients was unknown to the physician and patient at the time of recruitment. PATIENTS: Group A (n = 7, three females) all received GH replacement continuously for 3 years. Group B (n = 8, five females) included six patients who received GH replacement for 6 months and two who received GH replacement for 12 months with BMD being measured at 6-monthly intervals. METHODS: Single photon absorptiometry (SPA) and later single X-ray absorptiometry (SXA) were used to measure forearm cortical BMD. Dual-energy X-ray absorptiometry (DXA) was used to measure lumbar spine, trochanteric, femoral neck and Ward's area BMD. RESULTS: In group A lumbar spine and trochanter BMD had increased significantly from baseline by 3.7% (DXA: median change = 0.045 g/cm2; P = 0.028) and 4.0% (DXA: median change = 0.031 g/cm2; P = 0.046), respectively. There were non-significant decreases in femoral neck (1.9%) (DXA: median change = -0.02 g/cm2; P = 0.39), Ward's area (6.5%) (DXA: median change = -0.06 g/cm2; P = 0.09) and forearm (2.6%) (SPA/SXA: median change = -0.013 g/cm2; P = 0.18). In group B, compared with baseline, only trochanter BMD changed significantly, increasing by 5.9% (DXA: median change = 0.0485 g/cm2; P = 0.049). Lumbar spine (DXA: median change = -0.001 g/cm2) Ward's area (DXA: median change = 0.0135 g/cm2), femoral neck (DXA: median change = -0.005 g/cm2) and forearm cortical (SPA/SXA; median change = -0.01 g/cm2) BMD did not change significantly (P = 0.67, P = 0.57, P = 0.86 and P = 0.31, respectively). Median percentage changes compared with baseline were -0.1%, 1.8%, -0.5% and -2.1%, respectively. From the time of completion of GH therapy however, BMD increased significantly at lumbar spine, (median change = 0.023 g/cm2), Ward's area (median change = 0.03 g/cm2) and trochanter (median change = 0.056 g/cm2) (P = 0.036, P = 0.049 and P = 0.012, respectively) but not at the femoral neck (median change = 0.017 g/cm2; P = 0.31) or forearm (median change = 0 g/cm2; P = 0.75). CONCLUSION: Long-term GH replacement therapy for three years appears to have beneficial effects on bone in patients with adult onset GH deficiency particularly at the lumbar spine and trochanter; the effects on femoral neck and forearm cortical BMD, however, are less impressive. A short course (6-12 months) of GH replacement therapy results in an increase in trochanter BMD several years later, and after an initial decline in BMD whilst on GH replacement, lumbar spine and Ward's area BMD return towards their baseline values. These results emphasize that not all types of bone and skeletal sites respond to GH therapy identically. Furthermore a short course of GH replacement over 6-12 months may result in significant changes in BMD several years later.     

Development of a gas chromatography-mass spectrometry method for the analysis of aminoglycoside antibiotics using experimental design for the optimisation of the derivatisation reactions

Although blepharoplasty has long been the traditional method of improving the aesthetic appearance of the eyelid area, it yields little improvement in several of the major signs of periorbital aging including (1) wrinkling of the infrabrow and lower lid skin, (2) crow's foot wrinkles, (3) malar bags and wrinkles, (4) crepey changes in the periorbital skin texture, (5) pigment spots and other actinic damage, (6) elongation of the apparent vertical height of the lower lid, and (7) loss of the gentle, indistinct transition between the lower lid and cheek skin. All of these indications can be improved by the combination of laser blepharoplasty and laser resurfacing of the periorbital region. In addition to resurfacing, the author prefers to use the laser for the incisional aspects of the blepharoplasty, as it is quick, causes less sensory nerve stimulation, is less likely to damage the inferior oblique muscle, and has essentially no bleeding--and therefore, less ecchymosis--postoperatively. This technique is described and illustrated photographically. Its outcomes include a high-quality result that maintains itself well over time, improvement in the signs of periorbital aging not treated by traditional blepharoplasty, a low rate of complication, and high patient satisfaction.     

A column centrifugation method for the reconstitution in liposomes of the mitochondrial F0F1 ATP synthase/ATPase

A method for reconstitution of membrane proteins into unilamellar liposomes is described. The model enzyme was the F0F1 ATP synthase from mitochondria when in complex or free from its inhibitor protein. The enzymes were first solubilized with either of two detergents, i.e., n-dodecyl-beta-D maltoside or lauryldimethylamine oxide. After solubilization, the enzymes were passed through a column of Sepharose-AH using an ADP/sodium cholate selective elution buffer. The enzymes recovered from the column were subsequently passed through a centrifuge column of Sephadex G-50 fine. The eluate contained liposomes in which the F0F1 complex (with and without inhibitor protein) had been reconstituted. The reconstituted enzymes were capable of hydrolyzing ATP with formation of electrochemical H+ gradients. They also catalyzed the ATP-Pi exchange reactions. Thus the F0F1 complex which is formed by 18 subunits can be rapidly reconstituted into liposomes in a fully functional state. Moreover the data show that the interactions between the enzyme and its inhibitor protein are not perturbed in the reconstitution procedure.     

Expression of the human dystrophin gene in mdx mouse muscle fibers after transfection using liposomes and synthetic oligopeptides

The number of dysrophin-positive fibers appearing in the femoral quadriceps muscle of mdx mice after injection of the full-length human dystrophin cDNA within the pHSADy plasmid was examined by means of immunohystochemical techniques. Transfection was carried out using lipofectamine (LFA), or synthetic oligopeptide complexes that provided the condensation of plasmid DNA (K8) and its release from endosomes gopeptide complexes that provided the condensation of plasmid DNA (K8) and its release from endosomes (JTS1). The LFA + pHSADy at a dose of 10 micrograms DNA did not affect the number of dystrophin-positive fibers at the site of injection (0.6-0.8%), whereas it caused a statistically significant increase in the number of these fibers in the same muscle of the contralateral leg (up to 2.3%). Injection of the SO + pHSADy complex resulted in the occurrence of dystrophin-positive muscle fibers characterized by a heterogeneous content and the distribution of dystrophin. The greatest number of dystrophin-positive fibers (about 16%) was observed under a ratio of pHSADy to K8 of 1:3 or 1:4. The observed maximal number of dystrophin-positive fibers after a single injection of SO + pHSADy was 3.8%, and it was 17.7% after three injections. These values were statistically significantly higher compared to intact mice (0.6%), the injection of pure plasmid (2.2%), or the intramuscular injection of sucrose (from 0.7 to 1.3%). A relatively high level of transfection (about 5%) was observed after an intracardiac injection of a large dose of the pHSADy (70 micrograms DNA). The perspectives of the targeted delivery of the dystrophin gene into muscles under conditions of parenteral administration are discussed.     

ESR study of the liposome membrane physical parameters in the heating-cooling cycles

Changes of dynamic and structural parameters of egg yolk lecithin (EYL) liposome membranes in the heating-cooling cycles have been studied using the ESR spin probe method. The investigations were conducted in the range of temperatures from -18 degrees C to +60 degrees C. It has been found that in the range of temperatures -15 degrees C to +45 degrees C in both the heating and the cooling run the spectroscopic parameters changed practically along the same curve (reversible changes). However, after exceeding this range of temperatures one of the parameters (partition coefficient of the spin probe 2,2,6,6--tetramethylpiperidine -1-oxyl; TEMPO) changed along a closed curve, showing the phenomenon of thermal hysteresis. In the heating process the TEMPO content in liposome membranes was smaller than this in the cooling process. We assume that during the heating, the lipid molecules of the outer liposome layers dissolve in the aqueous medium. In the cooling process they can aggregate and form new liposomes, what in turn increases the surface of liposome membranes, accessible for the TEMPO probe (active surface).     

Enzymatic reactions of methionine sulfoximine. Conversion to the corresponding alpha-imino and alpha-keto acids and to alpha-ketobutyrate and methane sulfinimide

L-Methionine sulfoximine is a substrate of L-amino acid oxidase (Crotalus adamanteus), glutamine transaminase, and gamma-cystathionase. In the reaction catalyzed by L-amino acid oxidase, methionine sulfoximine is converted to aplph-imino-gamma-methylsulfoximinylbutyrate, which undergoes rapid gamma elimination yielding methane sulfinimide and 2-imino-3-butenoic acid. Methane sulfinimide is converted to methane sulfonamide, methane sulfinic acid, and methane sulfonic acid; 2-imino-3-butenoic acid is hydrolyzed to vinylglyoxylate, which polymerizes to an insoluble product. When the reaction is carried out in the presence of semicarbazide, the imine formed initially is quantitatively trapped as alpha-keto-gamma-methylsulfoximinylbutyrate semicarbazone, from which the free alpha-keto acid may be obtained. When the reaction is carried out in the presence of a mercaptan (RSH), a gamma exchange reaction occurs leading to formation of a new alpha-keto acid substituted in the gamma position by an SR-group; thus, alpha-keto-gamma-(beta-hydroxyethiol)butyric acid (S-(hydroxyethyl)-2-keto-4-mercaptobutyric acid) was obtained when L-methionine sulfoximine was oxidized in the presence of 2-mercaptoethanol, and enzymatic transamination of this alpha-keto acid with L-glutamine gave the new amino acid, L-omega-hydroxyethionine. The reaction of L-methionine sulfoximine with gamma-cystathionase yields 1 mol each of alpha-ketobutyrate and methane sulfinimide; the latter is hydrolyzed almost exclusively to methane sulfinic acid. Transamination of L-methionine sulfoximine yields the corresponding alpha-keto acid (alpha-keto-gamma-methylsulfoximinylbutyrate), which is stable. Some of these reactions may occur in vivo, and thus contribute to the toxicity of L-methionine sulfoximine.     

Two-dimensional blood flow determinations in allergic reactions using laser Doppler scanning

The new technique of laser Doppler scanning (LDS) provides a 2-dimensional pattern of cutaneous microcirculation, which offers a visual image and can quantify the intensity and expansion of perfusion. With the help of this technique, we examined the microcirculatory pattern of Type IV reactions to recall antigens, which were applied using a test stamp (Multitest Merieux). The measurements were performed before application of the test stamp as well as 10 min, 24, 48 and 72 h afterwards. The inflammatory hyperemia was evaluated using LDS and unidimensional laser Doppler fluxmetry. The diameter of the inflammatory infiltrate was quantified by means of palpation, the thickness by means of high-resolution 20 MHz sonography. The clinically visible erythema was measured planimetrically. An unspecific hyperemia resulting from the trauma of the stamp revealed no evident infiltrate under sonography 10 min after the test application. Depending of the individual reaction, the mean flux and the expansion of the hyperemia were at their peak after 48 h. The flux values were at a maximum in the center of the inflammatory reaction and dropped continuously toward the periphery. The area of the hyperemia seen in the LDS image was significantly larger than the expansion of the erythema measured planimetrically, but there was a significant correlation. The perfusion correlated significantly with the infiltration diameter (24 h, 48 h, 72 h) and the infiltration thickness 48 h after testing. All in all, it was possible to measure directly and without touching the skin and to quantify a subclinical pattern of skin perfusion as a response to and inflammatory reaction on a 2-dimensional display.     

Protective effect of liposome encapsulation on paclitaxel developmental toxicity in the rat

Paclitaxel is an anticancer drug that has demonstrated severe embryotoxicity in chicks. This embryotoxicity is reduced by liposome encapsulation of the drug. The current study was designed to evaluate the potential of liposome encapsulation for reducing paclitaxel embryotoxicity in rats. Wistar rats were treated with paclitaxel on day 8 of pregnancy (plug = day 0) at doses of 0.67, 2.0, or 10.0 mg/kg intravenously. The same doses of paclitaxel encapsulated in liposomes were administered intravenously to other groups of animals. Control animals were given blank liposomes. Free paclitaxel produced maternal and embryotoxicity at 10.0 mg/kg with three of seven dams dying and resorption of all embryos in surviving dams. Liposome encapsulation at 10.0 mg/kg was not associated with maternal death and there were live fetuses on evaluation at term, although litter size was reduced and malformations occurred in surviving fetuses. At 2.0 mg/kg free paclitaxel, fetal weight was decreased and resorptions increased. Liposome encapsulation at 2.0 mg/kg produced litter results similar to those obtained in control animals given empty liposomes. Malformations were prominent at 2.0 mg/kg free paclitaxel and at 10.0 mg/kg paclitaxel in liposomes and included exencephaly/anencephaly, ventral wall defects, facial clefts, anophthalmia, diaphragmatic hernia, and defects of the kidney, cardiovascular system, and tail. Liposome encapsulation appeared to shift the developmental response to paclitaxel such that 10 mg/kg encapsulated drug produced effects similar to 2.0 mg/kg free drug. These results may have implications for drug delivery of therapeutic agents used during human pregnancy.     

Therapy of a xenografted human colonic carcinoma using cisplatin or doxorubicin encapsulated in long-circulating pegylated stealth liposomes

The present paper describes AMmtDB, a database collecting the multi-aligned sequences of vertebrate mitochondrial genes coding for proteins and tRNAs, as well as the multiple alignment of the mammalian mtDNA main regulatory region (D-loop) sequences. The genes coding for proteins are multi-aligned based on the translated sequences and both the nucleotide and amino acid multi-alignments are provided. As far as the genes coding for tRNAs are concerned, the multi-alignments based on the primary and the secondary structures are both provided; for the mammalian D-loop multi-alignments we report the conserved regions of the entire D-loop (CSB1, CSB2, CSB3, the central region, ETAS1 and ETAS2) as defined by Sbisà et al. [ Gene (1997), 205, 125-140). A flatfile format for AMmtDB has been designed allowing its implementation in SRS (http://bio-www.ba.cnr.it:8000/BioWWW/#AMMTDB ). Data selected through SRS can be managed using GeneDoc or other programs for the management of multi-aligned data depending on the user's operative system. The multiple alignments have been produced with CLUSTALV and PILEUP programs and then carefully optimized manually.     

Enhancement of computed tomography liver contrast using iomeprol-containing liposomes and detection of small liver tumors in rats

RATIONALE AND OBJECTIVES: We evaluated iomeprol-containing liposomes (Lipiom), a new contrast medium for computed tomography (CT) liver scanning, in an animal model of chemically induced hepatocellular carcinomas and other liver tumors in rats. METHODS: Liver tumors were induced by administration of carcinogens to rats, either 0.55% (w/w) 1'-hydroxysafrole in the diet or induction by 3'-methyl-4-diethylaminoazobenzene followed by promotion with carbon tetrachloride. CT scanning was performed 1-3 hr after intravenous injection of iomeprol-containing liposomes. RESULTS: After injection of iomeprol-containing liposomes at a dose of 70 mg of liposome-entrapped iodine per kilogram of body weight, the normal liver parenchyma showed a contrast enhancement, in Hounsfield units, of more than 60% over the control value before bolus. Liver tumors with no or few Kupffer cells were not enhanced and appeared as dark areas within the normal parenchyma. Tumors and pretumoral lesions devoid of Kupffer cells, as small as 3 mm in diameter, could be distinguished using this non-invasive method. CONCLUSION: CT liver scanning after injection of iomeprol-containing liposomes appears to be promising method for detecting liver tumors and focal liver lesions.