Corneal epithelial wound healing promoted by verbascoside-based liposomal eyedrops.

Different liposomal formulations were prepared to identify those capable of forming eyedrops for corneal diseases. Liposomes with neutral or slightly positive surface charge interact very well with the cornea. Then these formulations were loaded with verbascoside to heal a burn of corneal epithelium induced by alkali. The cornea surface affected involved in wound was monitored as a function of time. Experimental results were modeled by balance equation between the rate of healing, due to the flow of phenylpropanoid, and growth of the wound. The results indicate a latency time of only three hours and furthermore the corneal epithelium heals in 48 hours. Thus, the topical administration of verbascoside appears to reduce the action time of cells, as verified by histochemical and immunofluorescence assays.

Ocular tissues and fluids oxidative stress in hares fed on verbascoside supplement.

The influence of a prolonged diet supplemented with the powerful antioxidant verbascoside on the oxidative state of 20 healthy hares eye fluids and tissues has been studied. Verbascoside was dosed at 2, 3, 4 mg/die and the impact on the oxidative state of ocular tissues and fluids was tested by TBARS (thio barbituric acid reactive substances) and TEAC (trolox equivalent antioxidant capacity) assays. The percentage of change in antioxidant activity increased largely in retina and lenses at a daily verbascoside dose of 3 mg, whereas for optic nerve and vitreous humor the higher antioxidant capacity was measured at 4 mg/die verbascoside dose. The present findings demonstrate that verbascoside supplementation is able to protect ocular tissue and fluids from naturally occurring oxidation and that its protective effect depends on the daily dose, being maximum up to 3 mg/die.

Evidence for the role of hydrophobic forces on the interactions of nucleotide-monophosphates with cationic liposomes.

In this work, the interaction of nucleotide-monophosphates (NMPs) with unilamellar liposomes made of 1,2-Dioleoyl-3-Trimethylammonium-Propane (DOTAP) and 1,2-Dioleoyl-sn-Glycero-3-Phosphoethanolamine (DOPE) was investigated. Here, we demonstrate how adsorption is affected by the type of nucleotide-monophosphate. Dynamic light scattering (DLS) results revealed, for each NMP, that a distinguishable concentration exists at which a significant growth of the aggregates occurs. Adenosine 5'-monophosphate (AMP) and guanosine 5'-monophosphate (GMP) have shown a higher propensity to induce liposome aggregation process and in particular GMP appears to be the most effective. From ζ-potential experiments we found that liposomes loaded with purine based nucleotides (AMP and GMP) are able to decrease the ζ-potential values to a greater extent in comparison with the pyrimidine based nucleotides thimydine 5'-monophosphate (TMP) and uridine 5'-monophosphate (UMP). Moreover, a careful analysis of nucleotide-liposome interactions revealed that nucleotides have different capacity to induce the formation of nucleotide-liposome complexes, and purine based nucleotides have higher affinities with lipid membranes. On the whole, the data emphasize that the mechanisms driving the interactions between liposomes and NMPs are also influenced by the existence of hydrophobic forces.

Oligonucleotides and polynucleotides condensation onto liposome surface: effects of the base and of the nucleotide length.

The association behavior of different nucleic acids with cationic liposomes has been monitored, in order to find out how the polymer length, the type of base and the charge density affect the lipoplex formation. In particular the associative features displayed by the homopolymer 20-mer of adenine, Oligo (dA), of timine, Oligo (dT), and of guanine, Oligo (dG), were compared to understand the role of the base. The effects of the nucleic acid length and of the charge density were evaluated taking account of the association of the polyadenylic acid and of the DNA onto the liposomes. The results show that the homopolymer Oligo (dG) is able to interact with the cationic liposomes to the same extent as DNA, in spite of the fact that Oligo (dG) is a short polymer made of 20 residues and DNA is a longer and dual strand polymer having a higher charge density.

Effects of verbascoside-based diet on blood and plasma constituents of rabbits.

OBJECTIVE: Oxidative stress brought on by free radicals can lead to an increased risk of some chronic pathologies. Antioxidants can scavenge free radicals by turning them into nonradical and nontoxic metabolites. The purpose of this study is to investigate the effect of a phenylpropanoid glycosides-based prolonged diet on blood constituents in animals. METHODS: Tests were carried out on healthy New Zealand white rabbits and the following parameters were evaluated at baseline and after 90 days' follow-up: plasma triglycerides, total cholesterol, low-density lipoprotein cholesterol, high-density lipoprotein cholesterol, aspartate transaminase, alanine aminotransferase, bilirubin, the reactive oxygen metabolites, thiobarbituric acid-reactive substances, vitamin A, and vitamin E. The same parameters were analyzed in an age- and sex-matched animal control group. RESULTS: We first defined the concept of average rate and then used it to calculate, by experimental data fitting, the formation or destruction rate of some blood or plasma constituents as a function of the daily dose. The results indicate that the effects can be categorized into 2 classes. The first includes the effects that produce monotonously continuous changes with daily dose, and the second includes those that exhibit a saturating trend. CONCLUSIONS: The experimental results suggest that high doses of verbascoside can potentially cause adverse effects through prooxidative effects. Risk is increased by the use of pharmacological doses of polyphenols in prevention, treatment, and as dietary supplements.

Effect of membrane composition on lipid oxidation in liposomes.

To study the effect of membrane composition on the oxidation of liposomes, different systems were prepared by adding one component at time to phosphatidylcholine (Epikuron 200). In particular, the effect of cholesterol and its ester, cholesterol stearate, on membrane structure and oxidation was studied. A first screening of the structure and net charge of the different preparation was made by means of z-potential and size measurements. Then the liposomes were oxidized by using a hydrophilic radical initiator, the (2,2-azobis(2-amidinopropane) hydrochloride, AAPH, which thermally decomposes to give a constant radical flux in water. The oxidation of liposomes, monitored by following the absorbance of the primary products of oxidation at 234 nm, was shown to be dependent on the composition of the liposomal bilayer and so on its biophysical properties. In addition, size and z-potential measurements gathered in the time course of the peroxidation reaction, revealed that the oxidation induced a modification of the superficial characteristics of the membrane bilayer so as to change its charge at the shear plane (z-potential). This behaviour was shared by all liposomal preparations independent of the composition. The change in sizes of the different liposomal preparation, instead, followed different trends, being more stable both in control samples and in oxidized ones when cholesterol was present. From the analysis of the results, it can be concluded that cholesterol affects the oxidation induced by hydrophilic radical initiator of model membranes by changing the biophysical properties of the phospholipid bilayer. The rigidity induced by cholesterol at temperatures above the T(m) makes the membrane more resistant to radical attack from an external aqueous phase and this in turn delays the start of the reaction. The decrease of z-potential of the liposomal particles induced by the oxidation process can be an important clue to understand the mechanisms involved in the etiology of important diseases.

Lipid oxidation in water-in-olive oil emulsions initiated by a lipophilic radical source.

The lipophilic 2,2'-azobis(2,4-dimethylvaleronitrile) (AMVN) was used to study thoroughly the oxidation reaction in a model water-in-olive oil emulsion system. This radical species decomposes thermally generating a constant flux of radicals in the oil phase. The dissociation constant k(d) in olive oil at 40 degrees C for AMVN was calculated as 2.5 x 10(-4) min(-1) and the rate of initiation of the oxidation reaction, R(i) was calculated by using vitamin E as antioxidant. The olive oil oxidation in emulsion was monitored by measuring the hydroperoxide concentration by a sensitive fluorimetric method. The DPPP (diphenyl-1-pyrenylphosphine) was used as a probe because it reacts stoichiometrically with hydroperoxides to yield a fluorescent product, the diphenyl-1-pyrenylphosphine oxide (DPPP-O). Oxidation data together with emulsion droplet size data showed that in the presence of radical initiator and a large interface, the oxidation reaction is accelerated in W/Olive oil emulsion with respect to whole oil. The mediation of the surface area of water droplets is surely involved in this process because the addition of saturated solutions of ascorbic acid (AA) dispersed in the oil brings about the strong reduction of the oxidation rate even in the presence of the highest AMVN quantity.

Biocompatible water-in-oil emulsion as a model to study ascorbic acid effect on lipid oxidation.

A biocompatible water-in-oil (W/O) emulsion has been used as a model to study the effect of ascorbic acid (AA) on the oxidation of the oil (glycerol trioleate, GTO) continuous phase. The model system consisted of 3 wt % water dispersed in GTO containing 0.5 wt % sodium oleate (NaO)/oleic acid (OA) mixture (NaO/OA = 20/80 mol/mol %) as a stabilizer. To study the ascorbic acid effect on GTO light-promoted oxidation, we added aqueous solutions of ascorbic acid to GTO in place of distilled water. Results obtained as peroxide values show that ascorbic acid activity depends on its concentration and it is affected by the characteristics of the W/O interface. In the presence of ascorbyl palmitate (AP) or sorbitan trioleate (Span 85) in the continuous phase, ascorbic acid activity increases in the first few hours of oxidation. The effect of ascorbic acid has been related to emulsion structure by calculating characteristic parameters of the droplet size distributions by means of optical microscopy.

Biocompatible lipid-based liquid crystals and emulsions.

Due to its potential relevance as a fully biocompatible formulation useful in cosmetic, food, and pharmaceutical applications, the glycerol trioleate/sodium oleate/water ternary system was investigated via optical microscopy and NMR methods. The ternary diagram is dominated by monophasic and biphasic regions where a lamellar phase coexists with different isotropic phases. A broad emulsion region, characterized by small oil droplets dispersed within the lamellar phase, extends from the center toward the water corner of the diagram. Information on the inner structure of these emulsion-like samples is supplied by modeling water and oil NMR self-diffusion data. Sizing of oil droplets was provided at different storage times. A highly polydisperse log-normal distribution was observed. The presence of the liquid crystalline phase is called into play for the negligible differences found in the droplets size distribution upon samples aging. Indeed, samples within this region stored at 25 degrees C did not show phase separation after several months from their preparation.

The FU gene and its possible protein isoforms.

BACKGROUND: FU is the human homologue of the Drosophila gene fused whose product fused is a positive regulator of the transcription factor Cubitus interruptus (Ci). Thus, FU may act as a regulator of the human counterparts of Ci, the GLI transcription factors. Since Ci and GLI are targets of Hedgehog signaling in development and morphogenesis, it is expected that FU plays an important role in Sonic, Desert and/or Indian Hedgehog induced cellular signaling. RESULTS: The FU gene was identified on chromosome 2q35 at 217.56 Mb and its exon-intron organization determined. The human developmental disorder Syndactyly type 1 (SD1) maps to this region on chromosome 2 and the FU coding region was sequenced using genomic DNA from an affected individual in a linked family. While no FU mutations were found, three single nucleotide polymorphisms were identified. The expression pattern of FU was thoroughly investigated and all examined tissues express FU. It is also clear that different tissues express transcripts of different sizes and some tissues express more than one transcript. By means of nested PCR of specific regions in RT/PCR generated cDNA, it was possible to verify two alternative splicing events. This also suggests the existence of at least two additional protein isoforms besides the FU protein that has previously been described. This long FU and a much shorter isoform were compared for the ability to regulate GLI1 and GLI2. None of the FU isoforms showed any effects on GLI1 induced transcription but the long form can enhance GLI2 activity. Apparently FU did not have any effect on SUFU induced inhibition of GLI. CONCLUSIONS: The FU gene and its genomic structure was identified. FU is a candidate gene for SD1, but we have not identified a pathogenic mutation in the FU coding region in a family with SD1. The sequence information and expression analyses show that transcripts of different sizes are expressed and subjected to alternative splicing. Thus, mRNAs may contain different 5'UTRs and encode different protein isoforms. Furthermore, FU is able to enhance the activity of GLI2 but not of GLI1, implicating FU in some aspects of Hedgehog signaling.

Tissue expression and translational control of rat kynurenine aminotransferase/glutamine transaminase K mRNAs.

Kynurenic acid (KA) is an endogenous glutamate receptor antagonist at the level of the different ionotropic glutamate receptors. One of the enzymes responsible for the production of KA, kynurenine aminotransferase I (KATI), also catalyses the reversible transamination of glutamine to oxoglutaramic acid (GTK, EC 2.6.1.15). The enzyme exists in a cytosolic and in a mitochondrial form because of the presence of two different KATI mRNAs coding for a protein respectively with and without leader sequence targeting the protein into mitochondria. We have cloned from a phage library of rat kidney cDNA four new KATI cDNAs containing different 5' untranslated regions (UTRs). One of the transcripts (+14KATI cDNA) contains an alternative site of initiation of translation. The tissue distribution of the different transcripts was studied by RT-PCR. The study demonstrated that several KATI mRNAs are constitutively expressed in ubiquitous manner, while +14KATI mRNA is present only in kidney. The translational efficiency of the different transcripts was studied in vitro and enzymatic activities were measured in transiently transfected Cos-1 cells. Each KATI mRNA exhibits a different in vitro translational efficiency, which corresponds to different levels of KAT enzymatic activity in transfected cells. Both findings correlate with the predicted accessibility of the ribosomal binding sites of the different mRNAs. The structure of the rat KATI/GTK gene was also studied. The expression of several KATI mRNAs with different 5'UTRs represents an interesting example of transcriptional/translational control on the expression of pyridoxal phosphate (PLP)-dependent aminotransferases.