Combined effects of VX-770 and VX-809 on several functional abnormalities of F508del-CFTR channels.

BACKGROUND: The most common cystic fibrosis-associated mutation, the deletion of phenylalanine 508 (F508del), results in channels with poor membrane expression and impaired function. VX-770, a clinically approved drug for treatment of CF patients carrying the G551D mutation, and VX-809, a corrector shown in vitro to increase membrane expression of mutant channels, are currently undergoing clinical trials, but functional data at the molecular level is still lacking. METHODS: The effect of VX-770 and VX-809 on the multiple functional defects of F508del-CFTR was assessed via excised inside-out patch-clamp experiments. RESULTS: VX-770 completely restores the low opening-rate of F508del-CFTR, with smaller open-time increase, in temperature-corrected and VX-809-treated channels. The shorter locked-open time of hydrolysis-deficient F508del-CFTR is also prolonged by VX-770. VX-809 does not improve channel function by itself as previously reported. CONCLUSIONS: The results from these studies can be interpreted as an equilibrium shift toward the open-channel conformation of F508del-CFTR channels.

Periodontitis is associated with altered plasma fatty acids and cardiovascular risk markers.

BACKGROUND AND AIMS: In periodontitis it has been found that some perturbation exists in lipid biomarkers, such as increased serum total cholesterol and low-density lipoprotein cholesterol. Nevertheless, the relationship between fatty acids and periodontitis has been demonstrated only in a few studies and remains controversial. The aim of this investigation was to explore the effects of periodontitis on a cluster of traditional and novel cardiovascular risk factors such as plasma-lipids profile, types of plasma fatty acids, adhesion molecules and systemic inflammatory markers. METHODS AND RESULTS: At a university dental school, 56 patients all over 35 years old were enrolled and invited to participate in the study. Total plasma fatty acids, saturated, n-6 polyunsaturated and monounsaturated fatty acids, peroxidability index, soluble VCAM, TNF-alpha, cholesterol, triacylglycerols, and VLDL-c were significantly higher in the periodontitis group compared to the non-periodontitis group. CONCLUSIONS: This close association found between plasma triacylglycerols, LDL-c, saturated fatty acids, polyunsaturated fatty acids, total amount of fatty acids and coenzyme Q(10) with some periodontal data such as periodontal probing depth, recession of the gingival margin and clinical attachment level (Pearson correlation between 0.3 and 0.6), leads to the conclusion that there is an inter-relationship between periodontitis, plasma fatty acids profile and the increase in metabolic risk factors for cardiovascular diseases.

A stochastic formulation of the gompertzian growth model for in vitro bactericidal kinetics: parameter estimation and extinction probability.

Time-kill curves have frequently been employed to study the antimicrobial effects of antibiotics. The relevance of pharmacodynamic modeling to these investigations has been emphasized in many studies of bactericidal kinetics. Stochastic models are needed that take into account the randomness of the mechanisms of both bacterial growth and bacteria-drug interactions. However, most of the models currently used to describe antibiotic activity against microorganisms are deterministic. In this paper we examine a stochastic differential equation representing a stochastic version of a pharmacodynamic model of bacterial growth undergoing random fluctuations, and derive its solution, mean value and covariance structure. An explicit likelihood function is obtained both when the process is observed continuously over a period of time and when data is sampled at time points, as is the custom in these experimental conditions. Some asymptotic properties of the maximum likelihood estimators for the model parameters are discussed. The model is applied to analyze in vitro time-kill data and to estimate model parameters; the probability of the bacterial population size dropping below some critical threshold is also evaluated. Finally, the relationship between bacterial extinction probability and the pharmacodynamic parameters estimated is discussed.

In vitro antioxidant activities of antioxidant-enriched toothpastes.

Several forms of periodontal diseases (PD) are often associated with modified phagocytosing leukocytes and contemporary free radical production. Host antioxidant defenses could benefit from toothpastes used as adjuncts to counteract plaque-associated bacteria. The aim of the present study was to determine possible antioxidant activity (AA) of 12 differently antioxidant-enriched toothpastes, regardless of their efficacy as antimicrobial agents. Toothpastes were enriched alternatively with sodium ascorbyl phosphate, alpha-tocopherol acetate, pycnogenol, allantoin and methyl salycilate or a mixture of these. AA was tested in a cell-free system with a ABTS-decolorization assay improved by means of a flow injection analysis device. Comet assay, using NCTC 2544 keratinocytes, was performed to test if it was possible to identify any protection against in vitro DNA fragmentation provoked by a challenge with H(2)O(2) in cultures pre-incubated with toothpaste extracts. Only toothpastes containing sodium ascorbyl phosphate displayed clear AA with I(50) values ranging between 50 and 80 mg of toothpaste/ml water. COMET analysis of cells challenged with H(2)O(2) in presence of toothpaste extracts revealed a limited protection exerted by sodium ascorbyl phosphate. The results described herein indicate that toothpastes containing sodium ascorbyl phosphate possess AA. All the data were obtained in systems in vitro and the demonstration of in vivo AA is desirable. These findings could be useful in the treatment and maintenance of some forms of PD and should be considered when arranging new toothpaste formulations.

Coenzyme Q, Vitamin E and Apo-E alleles in Alzheimer Disease.

Neurodegenerative Diseases represent the most common cause of Dementia, about 5-10% of the population aged above 65 years and about 30% above 80 years. A study about Apo-E alleles, Coenzyme Q and Vitamins E as biological indicators was performed in plasma samples of patients aged from 30 to 85 years, affected by Neurodegenerative Diseases. The results were compared with control subjects of approximately the same ages as the reference group. A frequency of 21.7% of epsilon4 allele in control group was estimated, against 15.8% observed in patients. The frequency of epsilon2 and epsilon3 alleles was 13.0% and 65.2% in the control group against 10.5% and 73.7% in patients. No significant differences were observed between the frequency of epsilon3/epsilon3 genotype and epsilon3/epsilon4 genotype in the control group compared to patients' group. The frequencies observed in epsilon2/epsilon3 genotype groups were 8.7% vs 15.8% and of e2/e4 genotype 17.4% vs 5.3%. The epsilon2/epsilon2 and epsilon4/epsilon4 genotypes were not identified in any groups. Plasma CoQ10 concentrations were similar in patient and control groups and no differences were found even taking into account the distribution of male and female subjects in the two groups. Also, vitamin E did not provide evidence of any differences between groups and the analysis among sexes revealed that again vitamin E concentrations were similar in between subgroups.

Coenzymes Q9 and Q10, vitamin E and peroxidation in rat synaptic and non-synaptic occipital cerebral cortex mitochondria during ageing.

Great attention has been devoted both to ageing phenomena at the mitochondrial level and to the antioxidant status of membrane structures. These kinds of investigations are difficult to perform in the brain because of its heterogeneity. It is known that synaptic heavy mitochondria (HM) may represent an aged mitochondrial population characterized by a partial impairment of their typical mitochondrial function. We arranged a novel system requiring no extraction procedure, very limited handling of the samples and their direct injection into the HPLC apparatus, to carry out, for the first time, a systematic and concomitant determination of vitamin E, Coenzyme Q9 (CoQ9) and Coenzyme Q10 (CoQ10) contents in rat brain mitochondria. The trends found for CoQ9 and CoQ10 levels in synaptic and non-synaptic occipital cerebral cortex mitochondria during rat ageing are consistent with previous data. Hydroperoxides (HP) differed with age and it was confirmed that in the HM fraction the summation of contributions results in an oxidatively jeopardized subpopulation. We found that vitamin E seems to increase with age, at least in non-synaptic free (FM) and synaptic light (LM) mitochondria, while it was inclined to remain substantially constant in HM.

Elevated hydroperoxide levels and antioxidant patterns in Papillon-Lefèvre syndrome.

BACKGROUND: Since it has been found that reactive oxygen species seem to be involved in the pathogenesis of both periodontitis and hyperkeratotic syndromes, we studied a group of patients belonging to 3 generations of a family with different degrees of severity of Papillon-Lefèvre syndrome (PLS) to ascertain whether altered concentrations of the most important hydrophobic and hydrophilic plasma antioxidants as well as products of oxidative damage are present in PLS. METHODS: Coenzyme Q (CoQ), vitamin E, glutathione (GSH), and uric acid were evaluated by high-performance liquid chromatography (HPLC) (supplied with electrochemical detector) techniques and hydroperoxides by a spectrophotometric method. RESULTS: GSH and uric acid were in the range of reference values; CoQ was very low in both the child of the third generation and his mother, and these 2 subjects had the highest hydroperoxide levels. The child also had extremely low values of vitamin E. In general, all family members showed abnormally high hydroperoxide levels, with the exception of those members who are phenotypically healthy. CONCLUSIONS: Since the subjects with the lowest hydroperoxide contents are phenotypically healthy, whereas the affected individuals presented lower antioxidant levels and very high hydroperoxide concentrations, it has been suggested that a specific antioxidant therapy could be a promising approach in treating some PLS subjects. Moreover, unexpected manifestations of heterozygosity in the child of the third generation were also detected.

Parameter estimation in a Gompertzian stochastic model for tumor growth.

The problem of estimating parameters in the drift coefficient when a diffusion process is observed continuously requires some specific assumptions. In this paper, we consider a stochastic version of the Gompertzian model that describes in vivo tumor growth and its sensitivity to treatment with antiangiogenic drugs. An explicit likelihood function is obtained, and we discuss some properties of the maximum likelihood estimator for the intrinsic growth rate of the stochastic Gompertzian model. Furthermore, we show some simulation results on the behavior of the corresponding discrete estimator. Finally, an application is given to illustrate the estimate of the model parameters using real data.

Total oxidant scavenging capacity (TOSC) of microsomal and cytosolic fractions from Antarctic, Arctic and Mediterranean scallops: differentiation between three potent oxidants.

The enhanced formation of reactive oxygen species (ROS) is a common pathway of toxicity induced by stressful environmental conditions. In polar environments, characterization of antioxidant defences in key sentinel species may be of particular value as early detection biomarkers of unforeseen effects of human activities which are progressively increasing in these remote areas.The complexities associated with predicting the consequences at the 'organism level' of variations of specific antioxidant defences have been recently overcome by the ability to quantify an index of specific biological resistance to various kinds of ROS.The total oxyradical scavenging capacity (TOSC) assay has been used in three species of scallops for quantifying their ability to neutralize peroxyl (ROO(&z.rad;)) and hydroxyl (&z.rad;OH) radicals and peroxynitrite (HOONO). Adamussium colbecki and Chlamys islandicus represent key organisms for monitoring Antarctic and Arctic regions while Pecten jacobaeus was chosen for a comparison with a related temperate species. TOSC values for ROO&z.rad; were significantly higher in A. colbecki indicating this species as the most efficient scavenger of ROO&z.rad;. Mediterranean scallops had the lowest TOSC for ROO(&z.rad;). A. colbecki also exhibited the highest scavenging capacity for &z.rad;OH with values more than 2-fold greater than for C. islandicus and P. jacobaeus. TOSC for HOONO was lower for all scallops as compared to those for ROO&z.rad; or &z.rad;OH. TOSC for microsomes was not significantly different among the species for any ROS studied, and the percentage contribution to the specific TOSC for the various oxidants of microsomes of all scallops accounted for 1-3% of the total TOSC of the post-mitochondrial fraction. The specific TOSC of scallop microsomes for &z.rad;OH was approximately ten times lower than that for ROO&z.rad; or HOONO.The higher basal capability of the Antarctic scallop to neutralize different reactive oxygen species is discussed in terms of a possible adaptation to this extreme environment and TOSC is validated as a quantifiable measure of susceptibility to oxidative stress in marine organisms.

Antiangiogenic, antitumoural and antimetastatic effects of two distamycin A derivatives with anti-HIV-1 Tat activity in a Kaposi's sarcoma-like murine model.

The antiangiogenic, antitumoural and antimetastatic effects of two novel sulphonic derivatives of distamycin A, PNU145156E and PNU153429, were studied in a Kaposi's sarcoma-like tumour model obtained by injecting nude mice with cells releasing extracellular HIV-Tat protein, derived from a tumour which developed in a BK virus/tat transgenic mouse. Both PNU145156E and PNU153429 were administered intraperitoneally every fourth day for three weeks at doses of 100 or 50 mg/kg of body weight respectively, starting one day after injecting the tumour cells. Both drugs delayed tumour growth in nude mice, preventing neovascularization induced by the Tat protein. PNU153429 also significantly reduced the number and size of spontaneous tumour metastases. Both effects on tumour growth and metastases were augmented by treating simultaneously nude mice with 7.5 mg/kg of body weight of minocycline given per os daily for four weeks starting four days after injecting the tumour cells. Neither acute nor chronic toxic side-effects were observed during the life span of treated nude mice. Due to their antiangiogenic and anti-Tat effects, these drugs are promising for the treatment of Kaposi's sarcoma in AIDS patients.

Determination of meropenem in serum by high-performance liquid chromatography with column switching.

A rapid, accurate and sensitive liquid chromatographic assay with on-line solid-phase extraction for determination of meropenem in serum is described. Sample was directly injected onto the extraction column for sample clean-up and extraction. Thereafter, using an on-line column-switching system the drug was quantitatively transferred and separated on a C18 analytical column. Ultraviolet absorption at 298 nm was used for detection. The assay was linear from 1 to 100 micrograms/ml. Recovery was 98.5%. Based on a 20-microliters sample volume (serum- water, 1:1, v/v), detection limit was 0.1 microgram/ml. An application of the method to study the pharmacokinetics of meropenem is given.

Oxidation of LDL and their subfractions: kinetic aspects and CoQ10 content.

Coenzyme Q10 in its reduced form, ubiquinol-10, although present in LDL at concentrations considerably lower than that of alpha-tocopherol, exerts a potent antioxidant action in this class of lipoproteins. Previous studies indicated that the content of CoQ10 is the lowest in the densest subfraction of LDL, i.e. LDL3, which is commonly regarded as the most peroxidizable and atherogenic one. These levels were associated with the highest levels of hydroperoxides detectable in the three subclasses. Enrichment of LDL with CoQ10, by means of exogenous supplementation, resulted in a significant increase of CoQ10 in LDL, mainly in LDL3, and in a lower extent of peroxidizability. Spontaneous oxidation of ubiquinol was monitored in plasma and in LDL of unsupplemented and of supplemented subjects and the time-course of oxidation was found considerably slower in CoQ10-enriched LDL. The lagphase of conjugated dienes formation upon induced oxidation was significantly correlated with the absolute content of ubiquinol-10. Distribution of CoQ10 among different classes of plasma lipoproteins was also studied: about 60% of plasma CoQ10 was found associated with LDL.

The protective role of ubiquinol-10 against formation of lipid hydroperoxides in human seminal fluid.

Defective sperm function in infertile men has been associated with increased lipid peroxidation and impaired function of antioxidant defenses in spermatozoa. Evidence strongly suggests that CoQ10, a lipid-soluble component of the respiratory chain acts, in its reduced form (ubiquinol), as a potent antioxidant in various biological systems, such as lipoproteins and membranes. In this study we assayed CoQ10 content in both the reduced and oxidized form (ubiquinol/ubiquinone), and hydroperoxide levels in seminal plasma and seminal fluid from 32 subjects with a history of infertility. Our results showed a significant correlation between ubiquinol content and sperm count (r = 0.62; P < 0.05) in seminal plasma. An inverse correlation between ubiquinol content and hydroperoxide levels both in seminal plasma and in seminal fluid (r = -0.56; P = 0.01) was found. Using multiple regression analysis we also found a strong correlation among sperm count, motility and ubiquinol-10 content (P < 0.01) in seminal fluid. An inverse correlation between ubiquinol/ubiquinone ratio and percentage of abnormal morphology was also observed in total fluid. These results suggest that ubiquinol-10 inhibits hydroperoxide formation in seminal fluid and in seminal plasma. Since peroxidation in sperm cells is an important factor affecting male infertility, ubiquinol could assume a diagnostic and/or a therapeutic role in these patients.

Coenzyme Q homologs and vitamin E in synaptic and non-synaptic occipital cerebral cortex mitochondria in the ageing rat.

The coenzyme Q8 (CoQ8) and alpha-tocopherol contents of different mitochondrial fractions were investigated from occipital cerebral cortices of different ages. The highest CoQ8 and vitamin E concentrations were found in non-synaptic free mitochondria (FM) fractions. In several cases heavy mitochondria (HM) fractions displayed the lowest values. Occipital cerebral cortex mitochondria contained higher CoQ9 and lower CoQ10 amounts than those typical for other brain regions.

On-line solid-phase extraction of ceftazidime in serum and determination by high-performance liquid chromatography.

A column-switching high-performance liquid chromatographic assay is described for the determination of ceftazidime (a third-generation cephalosporin) in human serum. The method does not require prior sample pretreatment. Serum is directly injected in a first chromatographic column for sample clean-up and extraction. Thereafter, using an on-line column-switching system, the drug is quantitatively transferred and separated on a second, analytical column followed by determination using ultraviolet absorption at 258 nm. The technique allows direct, rapid, precise, and simple determination of ceftazidime in serum over the range of 1-250 micrograms/ml using 12.5 microliters of serum. This method was applied to study the pharmacokinetics of the drug in patients undergoing vascular surgery.

A Bayesian approach to drug disposition evaluation: application to teicoplanin.

We suggest a Bayesian method in order to evaluate the disposition kinetics of the drug plasma concentrations after intravenous (i.v.) and/or after extravascular administration of the drug in the same subject pooled with information obtained taking into account previous pharmacokinetics of the drug. This approach was applied to the investigation of teicoplanin pharmacokinetics after 200 mg i.v. administration to 10 healthy subjects.