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PubMed2000

Damage to liposomal lipids: protection by antioxidants and cholesterol-mediated dehydration.

Liposomes composed of egg phosphatidylcholine (EPC) (13.4%, of the acyl chains being polyunsaturated fatty acids (PUFA)) and EPC/cholesterol (10:1 mol/mol) were studied for factors that affect liposomal lipid oxidative damage and hydrolysis upon long-term (16 months) storage. Factors studied include: (1) levels of lipid/water interface hydration, related to the presence of cholesterol in the lipid bilayer; (2) the membrane-associated antioxidant vitamin E; (3) the water-soluble antioxidant Tempol; and (4) exposure to light. Liposomal dispersions were stored at room temperature, either exposed to or protected from daylight, for a period of 16 months. Chemical and physical changes were monitored at several time points to assess oxidative and hydrolytic degradation of liposomal lipids. The conclusions of the study are: (1) PUFA are the most sensitive component of the liposome bilayer to oxidative degradation damage during long-term storage; (2) EPC liposomes are more sensitive to degradation during storage than EPC cholesterol liposomes, the presence of cholesterol in the lipid bilayer having a protective effect, probably due to its effect in decreasing the lipid-bilayer hydration; (3) oxidative degradation is the major process during long-term storage, having an earlier onset than the hydrolytic degradation: and (4) Tempol provided significantly better protection than vitamin E to EPC liposomal PUFA against oxidative damage during long-term storage. The relevance of cholesterol's presence, as a 'drying agent', in membranes containing PUFA to resistance of biological membranes to oxidative damage is discussed.

2-Naphthylamine↗
PubMed2004

Phospholipid phase transitions in homogeneous nanometer scale bilayer discs.

Nanoscale protein supported phospholipid bilayer discs, or Nanodiscs, were produced for the purpose of studying the phase transition behavior of the incorporated lipids. Nanodiscs and vesicles were prepared with two phospholipids, dipalmitoyl phosphatidylcholine and dimyristoyl phosphatidylcholine, and the phase transition of each was analyzed using laurdan fluorescence and differential scanning calorimetry. Laurdan is a fluorescent probe sensitive to the increase of hydration in the lipid bilayer that accompanies the gel to liquid crystalline phase transition. The emission intensity profile can be used to derive the generalized polarization, a measure of the relative amount of each phase present. Differential scanning calorimetry was used to further quantitate the phase transition of the phospholipids. Both methods revealed broader transitions for the lipids in Nanodiscs compared to those in vesicles. Also, the transition midpoint was shifted 3-4 degrees C higher for both lipids when incorporated into Nanodiscs. These findings are explained by a loss of cooperativity in the lipids of Nanodiscs which is attributable to the small size of the Nanodiscs as well as the interaction of boundary lipids with the protein encircling the discs. The broad transition of the Nanodisc lipid bilayer better mimics the phase behavior of cellular membranes than vesicles, making Nanodiscs a 'native-like' lipid environment in which to study membrane associated proteins.

1,2-Dipalmitoylphosphatidylcholine↗
PubMed2001

Laurdan fluorescence: a simple method to evaluate sperm plasma membrane alterations.

OBJECTIVE: To determine, by a simple fluorescence method, sperm plasma membrane alterations related with changes of lipid bilayer that, together with routine semen analysis, could help to elucidate the causes of the unexplained male infertility problems. DESIGN: Pilot study. SETTING: Andrology laboratory and biochemistry institute, medical school. PATIENT(S): Men whose semen was studied for infertility problems. INTERVENTIONS(S): No therapeutic intervention was performed on patients. MAIN OUTCOME MEASURE(S): Presence of spermatozoa plasma membrane alterations evidenced by evaluation of Laurdan fluorescence Generalized Polarization (GP) and reported as a function of increasing cell concentration, spermatozoa total motility, linear speed, and vitality. RESULT(S): Reporting GP values as a function of increasing sperm cell concentration, it is evident that the samples are distributed in two distinct areas: at >32 x 10(6) cells per milliliter, mean GP value was 0.303 +/- 0.015, whereas for lower sperm cell concentrations, the mean GP was 0.365 +/- 0.026 (P<.001). These data indicate that the spermatozoa plasma membranes are characterized by liquid-crystalline phases with different ordering degree and polarity and that about 50% of samples with normal semen characteristics (> or =20 x 10(6) cells per milliliter) show high GP values. CONCLUSION(S): Laurdan fluorescence can be used as a simple method to evaluate spermatozoa plasma membrane alterations, particularly in a group of infertile men presenting normal semen parameters. In these samples, Laurdan could be used as a simple tool for infertility assessment. In fact, it is known that compositional and physicochemical alterations of bilayer features can be important for the fertilizing ability of spermatozoa because they are necessary for a proper physiological membrane activity.

2-Naphthylamine↗
PubMed2000

Effect of organochlorine insecticides on nicotinic acetylcholine receptor-rich membranes.

The so-called generalized polarization (GP) of the fluorescent probe Laurdan and the steady-state fluorescence anisotropy of the probe diphenylhexatriene (DPH) and its phenylpropionic derivative (PA-DPH) were used to study the effects of several organochlorine insecticides of the chlorophenylethane, chlorinated cyclohexane and chlorinated cyclodiene families on the Torpedo nicotinic acetylcholine receptor (AChR)-rich native membrane. All insecticides, with the exception of Lindane, augmented Laurdan GP both in the native membrane and in model lipid systems. Most organochlorine compounds produced a concentration-dependent decrease of DPH and PA-DPH anisotropy in the AChR-rich membrane. These compounds exhibited a dual behavior vis-à-vis the native AChR-rich membrane, exerting disordering effects at the bilayer core while ordering and/or excluding water molecules from the lipid-protein interface region, as sensed by DPH anisotropy and Laurdan GP, respectively. Furthermore, all insecticides decreased the efficiency of fluorescence resonance energy transfer between the intrinsic protein and Laurdan, albeit to different extents. On the basis of all these observations, the existence of potential target sites for insecticides in the protein-lipid interface region is postulated.

2-Naphthylamine↗
PubMed1997

In K562 and HL60 cells membrane ageing during cell growth is associated with changes in cholesterol concentration.

In a cell culture model of aging we have previously shown that there is an age-related decrease in the lipid dynamics of the proerythropoetic K562 cell membranes, as determined by the generalized polarization (GP) of the phase-sensitive lipid probe 2-dimethylamino-6-lauroylnaphthalene (Laurdan) (T. Parasassi, M. Di Stefano, G. Ravagnan, O. Sapora and E. Gratton. Exp. Cell Res., 202 (1992) 432-439). In the present study we also extended our observations to the lymphoblastoid HL60 cell line. In both K562 and HL60 cells during the four days after the last cell culture medium renewal the GP Laurdan value increased in a linear fashion indicating a time-dependent decrease in lipid dynamics. The initial membrane physical properties were almost completely restored upon renewal of the cell culture medium. We measured lipid composition, including individual and total phospholipids, free and esterified cholesterol at the first ('young') and at the fourth ('aged') day after culture medium renewal. We found that the decreased membrane lipid 'fluidity' at the fourth day of cell growth was associated with a 40% increase in cholesterol concentration in both cell lines. This increase in cholesterol concentration was reversible 24 h following the culture medium change. We conclude that in K562 and HL60 cells the 'age-related' decrease in membrane lipid dynamics is mediated by an 'age-related' increase in cell cholesterol content.

2-Naphthylamine↗
PubMed2002

Inhibition of bacterial foodborne pathogens by the lactoperoxidase system in combination with monolaurin.

The lactoperoxidase system (LPS) and monolaurin (ML) are potential natural antimicrobial agents for use in foods. The LPS is considered to have greatest activity against Gram-negative bacteria while ML is usually considered to have greatest activity against Gram-positive bacteria. An LPS-ML combination system (utilizing lactoperoxidase (LPX) in the range 5-200 mg kg(-1) and ML in the range 50-1,000 ppm) inhibited growth of Escherichia coli O157:H7 and Staphylococcus aureus. Growth of S. aureus was inhibited more strongly in broth than in milk, in milk than in ground beef A similar pattern was observed for E. coli O157:H7, though enhanced inhibition by LPS-ML systems over that obtained in comparable LPS only systems was not observed in ground beef The inhibitory action of the LPS in combination with other lipids was also examined, with progressively weaker inhibition observed in combinations including palmitoleic acid, monopalmitolein, lauric acid, caprylic acid, and sodium lauryl sulphate.

Animals↗
PubMed2002

Evaluation of antilisterial action of cilantro oil on vacuum packed ham.

Cilantro oil is an essential oil preparation extracted from the plant Coriandrum sativium. A series of experiments were conducted to evaluate the ability of cilantro oil to control the growth of Listeria monocytogenes on vacuum-packed ham. The in vitro minimal inhibitory concentration for five strains of L. monocytogenes was found to vary from 0.074% to 0.018% depending on strain. Cilantro oil treatments were then tested on ham disks inoculated with a cocktail of the five L. monocytogenes strains. The treatments studied were 0.1%, 0.5%, and 6% cilantro oil diluted in sterile canola oil or incorporated into a gelatin gel in which lecithin was used to enhance incorporation of the cilantro oil. Gelatin gel treatments were also conducted with 1.4% monolaurin with or without 6% cilantro oil to determine if an interaction between the antimicrobials could increase inhibition of L. monocytogenes. Treated ham was then vacuum-packed and stored at 10 degrees C for up to 4 weeks. The only cilantro oil treatment which inhibited growth of L. monocytogenes on the ham samples was 6% cilantro oil gel. Samples receiving this treatment had populations of L. monocytogenes 1.3 log CFU/ml lower than controls at week 1 of storage, though there was no difference between treatments from week 2 onward. It appears that immobilization of the antimicrobial in a gel enhanced the effect of treatments. Cilantro oil does not appear to be a suitable agent for the control of L. monocytogenes on ham. The possible reasons for reduced effectiveness of cilantro oil against L. monocytogenes on ham are discussed.

Animals↗
PubMed2003

Development of a method to quantify in vitro the synergistic activity of "natural" antimicrobials.

Despite numerous papers being published on the use of hurdle technology to control food-borne pathogens or spoilage organisms, there is no commonly accepted methodology to quantify the level of synergistic activity. This paper describes a method to quantify in vitro the synergistic activity of antibacterial agents against bacteria. Initially, a microtiter plate growth assay was used to determine the inhibitory concentrations of four "natural" antimicrobials (nisin, lauricidin, totarol, and the lactoperoxidase system (LPS)) against a panel of eight bacteria. Using the same microtiter system, the impact of various combinations of antimicrobials was assessed. The degree of synergy was based on the analysis of three criteria: (1) increase in lag phase, (2) reduction in culture density after 24 h, (3) and residual viability at 24 h. Only the lactoperoxidase system was active against all the Gram-positive and Gram-negative bacteria tested. Nisin, lauricidin, and totarol were only effective against the Gram-positive bacteria. The method successfully identified three combinations (nisin-lauricidin, LPS-nisin, and LPS-lauricidin) previously reported to have synergistic activity and highlighted the synergistic activity of two novel combinations (nisin-totarol and LPS-totarol). The development of a quick and reliable method to identify and quantify synergistic activity is a useful screening tool to establish preservative techniques that could have potential antimicrobial synergy in food-based systems.

Abietanes↗
PubMed2004

Enhancement of nisin, lysozyme, and monolaurin antimicrobial activities by ethylenediaminetetraacetic acid and lactoferrin.

A microtiter plate assay was employed to systematically assess the interaction between ethylenediaminetetraacetic acid (EDTA) or lactoferrin and nisin, lysozyme, or monolaurin against strains of Listeria monocytogenes, Escherichia coli, Salmonella enteritidis, and Pseudomonas fluorescens. Low levels of EDTA acted synergistically with nisin and lysozyme against L. monocytogenes but EDTA and monolaurin interacted additively against this microorganism. EDTA synergistically enhanced the activity of nisin, monolaurin, and lysozyme in tryptic soy broth (TSB) against two enterohemorrhagic E. coli strains. In addition, various combinations of nisin, lysozyme, and monolaurin with EDTA were bactericidal to some gram-negative bacteria whereas none of the antimicrobials alone were bactericidal. Lactoferrin alone (2000 microg ml(-1)) did not inhibit any of the bacterial strains, but did enhance nisin activity against both L. monocytogenes strains. Lactoferrin in combination with monolaurin inhibited growth of E. coli O157:H7 but not E. coli O104:H21. While lactoferrin combined with nisin or monolaurin did not completely inhibit growth of the gram-negative bacteria, there was some growth inhibition. All combinations of EDTA or lactoferrin with antimicrobials were less effective in 2% fat UHT milk than in TSB. S. enteritidis and P. fluorescens strains were consistently more resistant to antimicrobial combinations. Resistance may be due to differences in the outer membrane and/or LPS structure.

Animals↗
PubMed1998

Interaction of monolaurin, eugenol and sodium citrate on growth of common meat spoilage and pathogenic organisms.

Interactions of monolaurin, eugenol (phenolic compound) and sodium citrate (chelator) on the growth of six organisms including common meat spoilage (Lactobacillus curvatus, Lactobacillus sake, Leuconostoc mesenteroides, Brochothrix thermosphacta) and pathogenic (Escherichia coli O157:H7 and Listeria monocytogenes) organisms were investigated. The combinations of 100 to 250 ppm monolaurin with 500 and 1000 ppm eugenol, and 0.2 and 0.4% sodium citrate were more effective than each component separately. More than one combination prevented detectable growth of each organism. Lactic acid bacteria (LAB) and E. coli O157:H7 were most resistant and L. monocytogenes and B. thermosphacta most sensitive to control by the chosen combinations. The presence of sodium citrate was necessary to yield potent inhibition of Lb. curvatus and Lb. sake growth by the monolaurin and eugenol combinations.

Bacteria↗
PubMed2000

P450 in biotechnology: zinc driven omega-hydroxylation of p-nitrophenoxydodecanoic acid using P450 BM-3 F87A as a catalyst.

Cytochrome P450 enzymes require the delivery of two electrons to the heme protein for their enzymatic function. NADPH or NADH are usually used as reduction equivalents. In the absence of a substrate, NADPH may inactivate P450 enzymes. Furthermore, it is expensive, making it unsuitable for the preparative synthesis of fine chemicals. Approaches for replacing NADPH with an electrochemically generated reduction by using platinum-electrodes and different mediators are known. In the present study, NADPH was substituted by the mediator cobalt(III)sepulchrate and zinc dust that serves as an electron source. The mutated fatty acid hydroxylase P450 BM-3 F87A from Bacillus megaterium was chosen as a catalyst, since it shows a three-fold higher sensitivity and a nearly five-fold higher activity for p-nitrophenoxydodecanoic acid (12-pNCA) than the wild-type enzyme. The formation of p-nitrophenolate can easily be monitored using a photometer at 410 nm. The turnover rate of the zinc/cobalt(III)sepulchrate system reaches 20% of the NADPH activity. Compared to the electrochemical approaches the activity is at least 77% higher (turnover 125 eq min-1). The presented alternative cofactor system can be used instead of NADPH or expensive electrochemical devices (platinum electrodes) for fine chemical synthesis.

Bacillus megaterium↗
PubMed2003

[Cannabis arteritis: four new cases].

INTRODUCTION: The responsibility of cannabis in juvenile thromboangeitis has been suggested for few years. We describe four new cases. EXEGESIS: Young men presented with distal arteriopathy of the lower limbs in 3 cases, and of the left upper limb in the remaining patient. Symptoms occurred progressively, distal pulses had disappeared, and distal necrosis was constant. Three patients suffered from Raynaud phenomenon, none of them presented with venous thrombosis. Radiologic evaluation revealed distal abnormalities in all cases, and proximal arterial thrombosis in one case. The four patients were cannabis smokers for at least four years. With cannabis interruption and symptomatic treatment, lesions improved for three patients. For one of them, recurrence of arteriopathy occurred when he resumed to smoke cannabis. For the fourth one who never stopped cannabis, an amputation was necessary. CONCLUSION: Search for cannabis use is important because interruption may improve prognosis.

Adult↗
PubMed2002

[Follow-up of monoclonal gammopathy of undetermined significance in a population of 51 patients older than 70 years ].

UNLABELLED: Monoclonal gammopathy of undetermined significance (MGUS) are very frequent in the elderly. Its incidence is about 3% after 70 years. We have performed a retrospective study of patients aged of more than 70 years who were examined until 1995 for a MGUS. METHODS: The study included 51 patients: 24 men and 27 women. Information about evolution was obtained by medical file or by asking medical practitioner. We know the evolution of all patients until January 1st 2000. The median follow-up is 5.8 years (70 months). RESULTS: The MGUS remained stable for 34 patients (67%) with a median follow-up of 83 months (12 to 180 months). Nine patients (17%) developed malignant transformation of MGUS (6 multiple myeloma, 2 Waldenström macroglobulinemia, 1 malignant lymphoma). Eight (15%) developed a cancer. Twelve died without evidence of multiple myeloma or related disorder. The actuarial probability of malignant transformation at 5 years was 12%. CONCLUSION: The risk of malignant transformation of MGUS doesn't decrease in the elderly. A regular and prolonged follow-up is necessary.

Aged↗
PubMed2002

[Nonsurgical treatment of chronic subdural hematoma with steroids: two case reports].

INTRODUCTION: Hemorrhages beneath the dura define a subdural hematoma. EXEGESIS: The course of chronic subdural hematoma may be asymptomatic for many months before diagnosis. Recovery is observed in 80% of patients after surgery. We report two cases of chronic subdural hematoma successfully treated with steroids. CONCLUSION: Steroids can be an alternative treatment of subdural hematoma, specially in elderly or alcoholic patients.

Anti-Inflammatory Agents↗