Identification and Functional Analysis of APOB Variants in a Cohort of Hypercholesterolemic Patients.

Mutations in APOB are the second most frequent cause of familial hypercholesterolemia (FH). APOB is highly polymorphic, and many variants are benign or of uncertain significance, so functional analysis is necessary to ascertain their pathogenicity. Our aim was to identify and characterize APOB variants in patients with hypercholesterolemia. Index patients (n = 825) with clinically suspected FH were analyzed using next-generation sequencing. In total, 40% of the patients presented a variant in LDLR, APOB, PCSK9 or LDLRAP1, with 12% of the variants in APOB. These variants showed frequencies in the general population lower than 0.5% and were classified as damaging and/or probably damaging by 3 or more predictors of pathogenicity. The variants c.10030A>G;p.(Lys3344Glu) and c.11401T>A;p.(Ser3801Thr) were characterized. The p.(Lys3344Glu) variant co-segregated with high low-density lipoprotein (LDL)-cholesterol in 2 families studied. LDL isolated from apoB p.(Lys3344Glu) heterozygous patients showed reduced ability to compete with fluorescently-labelled LDL for cellular binding and uptake compared with control LDL and was markedly deficient in supporting U937 cell proliferation. LDL that was carrying apoB p.(Ser3801Thr) was not defective in competing with control LDL for cellular binding and uptake. We conclude that the apoB p.(Lys3344Glu) variant is defective in the interaction with the LDL receptor and is causative of FH, whereas the apoB p.(Ser3801Thr) variant is benign.

Assessment of the ptxD gene as a growth and selective marker in Trichoderma atroviride using Pccg6, a novel constitutive promoter.

BACKGROUND: Trichoderma species are among the most effective cell factories to produce recombinant proteins, whose productivity relies on the molecular toolkit and promoters available for the expression of the target protein. Although inducible promoter systems have been developed for producing recombinant proteins in Trichoderma, constitutive promoters are often a desirable alternative. Constitutive promoters are simple to use, do not require external stimuli or chemical inducers to be activated, and lead to purer enzyme preparations. Moreover, most of the promoters for homologous and heterologous expression reported in Trichoderma have been commonly evaluated by directly assessing production of industrial enzymes, requiring optimization of laborious protocols. RESULTS: Here we report the identification of Pccg6, a novel Trichoderma atroviride constitutive promoter, that has similar transcriptional strength as that of the commonly used pki1 promoter. Pccg6 displayed conserved arrangements of transcription factor binding sites between promoter sequences of Trichoderma ccg6 orthologues genes, potentially involved in their regulatory properties. The predicted ccg6-encoded protein potentially belongs to the SPE1/SPI1 protein family and shares high identity with CCG6 orthologue sequences from other fungal species including Trichoderma reesei, Trichoderma virens, Trichoderma asperellum, and to a lesser extent to that of Neurospora crassa. We also report the use of the Pccg6 promoter to drive the expression of PTXD, a phosphite oxidoreductase of bacterial origin, which allowed T. atroviride to utilize phosphite as a sole source of phosphorus. We propose ptxD as a growth reporter gene that allows real-time comparison of the functionality of different promoters by monitoring growth of Trichoderma transgenic lines and enzymatic activity of PTXD. Finally, we show that constitutive expression of ptxD provided T. atroviride a competitive advantage to outgrow bacterial contaminants when supplied with phosphite as a sole source of phosphorus. CONCLUSIONS: A new constitutive promoter, ccg6, for expression of homologous and heterologous proteins has been identified and tested in T. atroviride to express PTXD, which resulted in an effective and visible phenotype to evaluate transcriptional activity of sequence promoters. Use of PTXD as a growth marker holds great potential for assessing activity of other promoters and for biotechnological applications as a contamination control system.

Functional Characterization of TvCyt2, a Member of the p450 Monooxygenases From Trichoderma virens Relevant During the Association With Plants and Mycoparasitism.

Secondary metabolites are crucial for the establishment of interactions between plants and microbes, as in the case of Trichoderma-plant interactions. In the biosynthetic pathway of secondary metabolites, specific enzymes participate in the formation of hydroxyl and epoxy groups, belonging to the p450 monooxygenases family. Here, we show that the product of the gene TvCyt2 from Trichoderma virens encodes a new protein homologous to the cytochrome p450, which is down-regulated at the beginning of Trichoderma-Arabidopsis interaction. To investigate its role in the interactions established by Trichoderma spp., we analyzed the metabolic profile obtained from the overexpressing (OETvCyt2) and null mutant (Δtvcyt2) strains, observing that the OETvCyt2 strains produce a higher concentration of some metabolites than the wild-type (WT) strain. Δtvcyt2 strains showed a decreased antagonistic activity against Rhizoctonia solani in antibiosis assays. Arabidopsis plants cocultivated with the OETvCyt2 strains showed stronger induction of systemic acquired resistance than plants cocultivated with the WT strain, as well as increases in biomass and fitness. Our data suggest that the product of the TvCyt2 gene is involved in secondary metabolite biosynthesis, which can increase antagonistic activity with phytopathogenic fungi and the capacity to promote plant growth.

The real role of prediagnostic high-density lipoprotein cholesterol and the cancer risk: a concise review.

BACKGROUND: In several observational and clinical studies, the association between serum cholesterol levels and cancer is still unsettled although serum total cholesterol has been associated with increased mortality from cancer. Moreover, the importance of abnormal levels of serum lipid components as the main features of dyslipidemia and the risk of individual cancers is unclear. The prevalence of dyslipidemia is increasing worldwide but, the precise aetiology of the link between risk of cancer and the behaviour of lipid profile, prior diagnosis, has yet to be determinated. Low levels of high-density lipoprotein cholesterol (HDL) at baseline of many of the studies analyzed has to be taken into account, and continued low levels of HDL without explanation should be considered by clinicians. AIMS: The main aim of this review was to undertake the assessment of the most recent studies implying the lipid profile and cancer risk, and focused on low HDL levels at baseline and follow up, and also analyzing this behaviour on the different cancer types. MATERIAL AND METHODS: A literature search was performed to identify publications. The most recent prospective and case-control studies with multivariate Cox models were analyzed and also were considered some recent meta-analyses. RESULTS AND CONCLUSIONS: The findings exposed in this review suggest that the association with low HDL levels at baseline of different studies of cancer risk is shared among many types of cancer, and it is mainly linked to obesity and inflammation, suggesting a common pathway.

Performance evaluation of biochemical and ultrasound markers for the screening of chromosomal numerical abnormalities in the first trimester of pregnancy.

BACKGROUND: To estimate the effectiveness of the first-trimester combined screening test in our population, departing from the results of diagnostic sensitivity and false positive rate (FPR), and checking some important parameters in prenatal screening. METHODS: The test was evaluated on 14250 pregnant women. The following variables were studied: the number of invasive techniques and the reasons for using such techniques, newborns with chromosomal abnormalities, total number of births, variation of biochemical markers throughout the gestational weeks, and MoM (multiple of the median) for biochemical and ultrasound markers. RESULTS: An important coverage and a decreased number of invasive techniques were obtained. For our population of pregnant women, the best gestational week to determine free beta-hCG and PAPP-A would be week 11 in which the best discrimination was found between affected and non affected fetuses for the three trisomies researched. We propose the cut-off 1/350, because it is the best one to increase sensitivity without exceeding the 5% FPR. CONCLUSIONS: Combined screening should be offered to pregnant woman, preferentially at week 11. Although different cut-offs for this prenatal test have been recommended by scientific societies, biochemical laboratories should set their own cut-off for getting the best sensitivity and FPR results. There should be a good level of collaboration between the laboratory and each participating ultrasound unit in order to ensure an optimal use of the first-trimester combined screening test.

Kidney stones in a Mediterranean population from the south of Spain.

BACKGROUND: Kidney stones have become increasingly prevalent in the developed countries over the past 100 years. The incidence of urolithiasis in a population depends on the geographical area, racial distribution, socio-economic status and dietary habits. During the past decades, these factors have changed affecting the incidence and also the chemical composition of calculi; nowadays in our region, the most common stones composition is calcium oxalate. The identification of the calculi composition enables superior treatment, lower (decreased) cost and a better quality of life for the patients. METHODS: We analyzed the composition and the evolution of all of the cases concerning calculi received at Biochemical Clinical Analysis Laboratory from 2007 to 2010, using Interferometry with Fourier transformation (FTIR). The relationship between composition, gender and age was studied for an aleatory group in 2010 (n=657, 431 men and 226 women). RESULTS: The stone composition obtained was mixtures 24.7% and only one component 75.3%. Calcium oxalate monohydrate (COM) 41.5%, calcium oxalate dihydrate (COD) 7.6%, anhydrous uric acid (AUA) 12.4%, uric acid dehydrate (UAD) 6.7%, urates 1.4%, carbonate-apatite (CA) 2.9%, and others 2.8%. The male to female ratio was 1.9 and the largest number of stones was found in patients between the ages of 40 and 49, for both men and women. CONCLUSIONS: The most common composition (relative percentage) was COM, mixtures and AUA. Presence of calculi is more common in men than in women with the exception of carbonate apatite stones. Stones follow a Gaussian distribution throughout the lifetime of a patient, with particular incidence in those between 40 to 49 years old.

A Global Analysis of Photoreceptor-Mediated Transcriptional Changes Reveals the Intricate Relationship Between Central Metabolism and DNA Repair in the Filamentous Fungus Trichoderma atroviride.

Light provides critical information for the behavior and development of basically all organisms. Filamentous fungi sense blue light, mainly, through a unique transcription factor complex that activates its targets in a light-dependent manner. In Trichoderma atroviride, the BLR-1 and BLR-2 proteins constitute this complex, which triggers the light-dependent formation of asexual reproduction structures (conidia). We generated an ENVOY photoreceptor mutant and performed RNA-seq analyses in the mutants of this gene and in those of the BLR-1, CRY-1 and CRY-DASH photoreceptors in response to a pulse of low intensity blue light. Like in other filamentous fungi BLR-1 appears to play a central role in the regulation of blue-light responses. Phenotypic characterization of the Δenv-1 mutant showed that ENVOY functions as a growth and conidiation checkpoint, preventing exacerbated light responses. Similarly, we observed that CRY-1 and CRY-DASH contribute to the typical light-induced conidiation response. In the Δenv-1 mutant, we observed, at the transcriptomic level, a general induction of DNA metabolic processes and strong repression of central metabolism. An analysis of the expression level of DNA repair genes showed that they increase their expression in the absence of env-1. Consistently, photoreactivation experiments showed that Δenv-1 had increased DNA repair capacity. Our results indicate that light perception in T. atroviride is far more complex than originally thought.

Oxidized LDL and its correlation with lipid profile and oxidative stress biomarkers in young healthy Spanish subjects.

The biological effects of oxidized LDL (oxLDL) may contribute to initiation and progression of the atherosclerotic process, and the association between cardiovascular disease and oxidation of LDL has been largely demonstrated. The objectives of this study were to establish the reference values of oxidative stress biomarkers in a young healthy Spanish population to determine the concentration of oxLDL and its relationship with lipid profile and with these biomarkers. oxLDL, F(2)-isoprostanes, protein carbonyls (PC), and 8-hydroxy-2'-deoxyguanosine (8-OHdG) were determinate by ELISA in 72 healthy subjects. Antioxidant enzymes, superoxide dismutase (SOD), glutathione peroxidase (GPx), and glutathione reductase (GR) were carried out on a Hitachi 912 analyzer; lipid profile were assayed using automated systems (Cobas 711, Roche Diagnostics). All statistics were analyzed by using SPSS for Windows 15.0. SPSS Inc, Chicago, IL, USA. (Normal mean reference values): oxLDL (63.23 +/- 16.23 U/L), (Male/Female 68.06 +/- 17.69/58.39 +/- 13.6 U/L), F(2)-isoprostanes (2.26 +/- 0.9 microg/g creatinine), PC (0.34 +/- 0.15 nmol/mg), 8-OHdG (23.27 +/- 10.58 ng/ml), SOD (931.97 +/- 271.09 U/g Hb), GR (46.56 +/- 11.68 U/L), GPx (27.58 +/- 6.89 U/gHb (Male/Female 25.91 +/- 5.03/29.2 +/- 8.07 U/L)). OxLDL (63.23 U/L) was significantly (p < 0.05) positively correlated with BMI (22.53 Kg/m(2)), total cholesterol (175.79 mg/dl), triglycerides (87.58 mg/dl), LDL cholesterol (96.25 mg/dl), and uric acid (4.78 mg/dl), while negatively correlated with HDL-cholesterol (62.25 mg/dl). We have found different correlation between oxLDL and isoprostanes by gender with the rest of parameters. Normal reference values have been found significantly different for oxLDL and GPx by gender. Oxidized LDL is correlated with lipid profile but not with the oxidative stress biomarkers. Urinary isoprostanes are positively correlated with triglycerides and negatively with GR and GPx.

Urinary oxylipin signature as biomarkers to monitor the allograft function during the first six months post-renal transplantation.

Oxylipins such as isoprostanes (IsoPs), prostaglandins (PGs) and thromboxanes (TXs) are lipid mediators derived from the oxidation of polyunsaturated fatty acids, which regulate the magnitude of oxidative stress and inflammation processes and play an important role in pathophysiological processes in the kidney. A total of 36 oxylipins were analyzed by UHPLC-QqQ-MS/MS in the urine of 41 renal recipients from cadaveric donors of the Nephrology Unit of the University Hospital Virgen de la Arrixaca during the first six months after renal transplantation, in order to investigate several candidate oxylipins as more accurate and predictive biomarkers in renal transplantation than classical biological variables. A decrease in nine PGs, mostly from the AA-D pathway (p < 0.05) and one IsoP: 15-keto-15-F2t-IsoP (p < 0.001) was observed. Moreover, two PGs (2,3-dinor-11ß-PGF2α and 17-trans-PGF3α) increased between five days and six months after renal transplantation (p < 0.05). In addition, when kidney function improved, a positive correlation between oxylipin levels and the excretion of urine proteins was observed. These results suggest that oxylipins could be useful markers for monitoring renal function in the post-renal transplantation period. These findings could be of utility not only for the development of strategies for long-term preservation of graft function, but also for innovative and alternative therapies -using oxylipins as predictive markers-to avoid organ rejection.

[LDL concentration and particle size after treatment with rosiglitazone in patients with diabetes mellitus type 2]. / Concentración y tamaño de las partículas de LDL tras el tratamiento con rosiglitazona en pacientes con diabetes mellitus tipo 2.

BACKGROUND AND OBJECTIVE: The effects of rosiglitazone on the lipid profile are controversial, with related increases in the concentration of total and LDL cholesterol. Our objective is to evaluate the evolution of the concentration and size of LDL particles in a group of patients with type 2 diabetes mellitus taking rosiglitazone. PATIENTS AND METHODS: We studied 30 patients under treatment with oral antidiabetics to whom rosiglitazone was added to their initial therapy. The following tests were determined before and after 6 months of treatment: glucose, total cholesterol, HDL, LDL, triglycerides, C reactive protein, lipoprotein (a) and glycosylated haemoglobin. The average diameter of the particles LDL was also estimated, as well as the probability of cardiovascular events up to ten years, according to the Framingham and SCORE model. RESULTS: Statistically significant reductions of glucose, HbA(1C) and CRP levels, and an increase of total cholesterol, cholesterol LDL and triglycerides concentrations were observed, with statistical significance for total cholesterol. A significant increase in the estimation of cardiovascular risk up to ten years was found. No modifications either in the concentrations of HDL-c and Lp (a) or in the average size of LDL particles were detected. CONCLUSIONS: After treatment with rosiglitazone, there is an increase of total cholesterol concentrations without variation in the mean size of LDL particles. Nevertheless, the reduction of CRP, and thus of inflammation is clear, with prevention of the progression of atherosclerosis.

Cancer-associated differences in acetylcholinesterase activity in bronchial aspirates from patients with lung cancer.

In non-neuronal contexts, ACh (acetylcholine) is thought to be involved in the regulation of vital cell functions, such as proliferation, differentiation, apoptosis and cell-cell interaction. In airways, most cells express the non-neuronal cholinergic system, each containing a specific set of components required for synthesis, signal transduction and ACh hydrolysis. The aim of the present study was determine the expression of cholinergic system components in bronchial aspirates from control subjects and patients with lung cancer. We conducted an analysis of cholinergic components in the stored soluble and cellular fraction of bronchial aspirates from non-cancerous patients and patients diagnosed with lung cancer. The results show that the fluid secreted by human lung cells contains enough AChE (acetylcholinesterase) activity to control ACh levels. Thus these findings demonstrate that: (i) AChE activity is significantly lower in aspirates from squamous cell carcinomas; (ii) the molecular distribution of AChE in both bronchial cells and fluids consisted of amphiphilic monomers and dimers; and (iii) choline acetyltransferase, nicotinic receptors and cholinesterases are expressed in cultured human lung cells, as demonstrated by RT-PCR (reverse transcriptase-PCR). It appears that the non-neuronal cholinergic system is involved in lung physiology and lung cancer. The physiological consequences of the presence of non-neuronal ACh will depend on the particular cholinergic signalling network in each cell type. Clarifying the pathophysiological actions of ACh remains an essential task and warrants further investigation.

Melatonin and hydroxytyrosol protect against oxidative stress related to the central nervous system after the ingestion of three types of wine by healthy volunteers.

Adrenic acid (AdA) and docosahexaenoic acid (DHA) peroxidation produces F2-dihomo-IsoPs and neuroprostanes, which have been related to oxidative damage in the central nervous system. Besides polyphenols, melatonin (MEL) and hydroxytyrosol (OHTyr) could be partly responsible for the antioxidant benefits of red wine (excluding colon derivatives). In order to elucidate whether these compounds are responsible for the protective antioxidant effects of red wine, a double-blind, crossover, placebo-controlled in vivo study - involving the intake of red wines and their native musts by healthy volunteers - was performed. The urinary metabolites decreased after the administration of red wines, to a greater extent than after the intake of their corresponding musts or ethanol. Melatonin is the most effective compound that protects adrenic acid from oxidative attack, judged by the reduction in the formation of F2-dihomo-isoprostanes. Similarly, hydroxytyrosol, being the most effective bioactive compound in reducing the formation of F3-neuroprostanes n-6 DPA and F4-neuroprostanes, protected docosahexaenoic and eicosapentaenoic acids from oxidative attack.

Snapshot situation of oxidative degradation of the nervous system, kidney, and adrenal glands biomarkers-neuroprostane and dihomo-isoprostanes-urinary biomarkers from infancy to elderly adults.

We analyzed biomarkers of lipid peroxidation of the nervous system -F2-dihomo-isoprostanes, F3-neuroprostanes, and F4-neuroprostanes- in urine samples from 158 healthy volunteers ranging from 4 to 88 years old with the aim of analyzing possible associations between their excretion values and age (years). Ten biomarkers were screened in the urine samples by UHPLC-QqQ-MS/MS. Four F2-dihomo-isoprostanes (ent-7-(R)-7-F2t-dihomo-isoprostane, ent-7-epi-7-F2t-dihomo-isoprostane, 17-F2t-dihomo-isoprostane, 17-epi-17-F2t-dihomo-isoprostane), and one DPA-neuroprostane (4-F3t-neuroprostane) were detected in the samples. On the one hand, we found a significant, positive correlation (Rho: 0.197, P=0.015) between the age increase and the amount of total F2-dihomo-IsoPs. On the other hand, the values were significantly higher in the childhood group (4-12 years old), when compared to the adolescence group (13-17 years old) and the young adult group (18-35 years old). Surprisingly, no significant differences were found between the middle-aged adults (36-64 years old) and the elderly adults (65-88 years old). We display a snapshot situation of excretory values of oxidative stress biomarkers of the nervous system, using healthy volunteers representative of the different stages of human growth and development. The values reported in this study could be used as a basal or starting point in clinical interventions related to aging processes and/or pathologies associated with the nervous system.

Effect of the dietary intake of melatonin- and hydroxytyrosol-rich wines by healthy female volunteers on the systemic lipidomic-related oxylipins.

Oxylipins are lipid mediators involved in the physiopathology of all organs. Moreover, isoprostanes have been established as general and reliable in vivo oxidative stress biomarkers. Red wine has proved to exert several benefits through the maintenance of the oxidative balance of the organism. Antiradical scavenging capacity has been mainly attributed to polyphenols. However, melatonin and hydroxytyrosol should be taken into account as potent antiradical agents. The present research aimed to clarify the situation of enzymatic and oxidative injury and eicosanoid urinary excretion related to the intake of three kinds of red wines and their primary musts. Judging by the reduction in the excretion of isoprostanes, red wine consumption exhibited the highest antioxidant protection against oxidative stress, attributed to its OHTyr content (p < 0.05), and to a lesser extent to its MEL content. Similarly, the intake of red wine leads to the cardioprotective effect due to the reduction in the urinary excretion of the pro-inflammatory prostaglandin 2,3-dinor-11-ß-PGF2α, besides the increase in the vasodilator prostaglandin PGE1, mediated by the melatonin (p < 0.05) and hydroxytyrosol (p < 0.05) contents. In conclusion, red wine (especially non-aged wine) exerts a higher in vivo antioxidant capacity than must or alcohol.

Potential applications of lipid peroxidation products - F4-neuroprostanes, F3-neuroprostanesn-6 DPA, F2-dihomo-isoprostanes and F2-isoprostanes - in the evaluation of the allograft function in renal transplantation.

F4-neuroprostanes, F3-neuroprostanesn-6 DPA, and F2-dihomo-isoprostanes, metabolites of non-enzymatic lipid peroxidation of polyunsaturated fatty acids [docosahexaenoic acid, n-6 docosapentanoic acid, and adrenic acid respectively], have become important biomarkers for oxidative stress in several diseases like epilepsy and alzheimer. These biomarkers and the 15-F2t-isoprostane (also known as 8-iso-PGF2α), a F2-isoprostane isomer measured as reference oxidative marker at systemic level, were analyzed by UHPLC-QqQ-MS/MS in the urine of 60 renal recipients from cadaveric donors of the Nephrology Unit of the University Hospital Virgen de la Arrixaca, at six different times during the first six months after renal transplantation, and were compared with a control group of 60 healthy subjects from the same hospital. A total of 11 metabolites were analyzed and different patterns were observed. A tendency to decrease was observed in three metabolites (4-epi-4-F3t- NeuroPn-6 DPA, ent-7(RS)-7-F2t-dihomo-IsoP, and ent-7(S)-7-F2t-dihomo-IsoP) and in our reference oxidative marker (15-F2t-IsoP) when kidney function improved and the excretion of urine proteins decreased. These results suggest that these three biomarkers of oxidative stress could be useful to assess renal function in the postransplant phase. Unfortunately, little is known about this kind of biomarker in this cohort of patients, so further investigation would be required in the clinical field to clarify the relationship between oxidative stress and the graft function, as well as the usefulness of these biomarkers as rejection markers.

Molecular characterization and clinical interpretation of BRCA1/BRCA2 variants in families from Murcia (south-eastern Spain) with hereditary breast and ovarian cancer: clinical-pathological features in BRCA carriers and non-carriers.

This is the first study performed in Murcia (south-eastern Spain) in which 592 families with hereditary breast and ovarian cancer were identified thanks to Genetic Counselling Units from this area over 6 years. Diagnostic performance was 18.1% and 194 different genetic variants were obtained. Variants with uncertain significance accounted for only 5.6% of the total number of reports, so our population has been well characterised. In BRCA1 gene, two novel variants were found (c.1859delT and c.3205C > T) and the most frequently detected mutations were c.68_69delAG, c.212 + 1G > A, c.5123C > A, c.211A > G and c.1918C > T, which together represented 56.67% of total pathogenic mutations. In BRCA2 gene, four recurrent variants were described (deletion of entire exon 2, c.9117G > A, c.3264dupT and c.3455T > G) representing 43.5% of the mutations in this gene. Mutation c.68_69delAG and deletion of entire exon 2 in BRCA1 and BRCA2 genes respectively were the most prevalent variants in our population. Regarding the genotype-phenotype relation, mutation c.212 + 1G > A appeared in an important percentage of breast and ovarian cancer cases, c.5123C > A in bilateral breast cancer and c.9117G > A in bilateral breast cancer and ovarian cancer. With respect to clinical-pathological characteristic, BRCA1/BRCA2 mutation carriers showed earlier onset age of breast tumour and higher risk of developing contra lateral breast cancer than non-informative cases. Moreover, association between either molecular subtype triple negative breast cancer or ovarian cancer and BRCA1 carriers was obtained.

Real time PCR assay with fluorescent hybridization probes for genotyping intronic polymorphism in presenilin-1 gene.

BACKGROUND: Identification of all susceptibility loci for Alzheimer's disease has been a major goal in resolving the pathogenesis of this disease. METHODS: A PCR assay with fluorescently labeled oligonucleotide hybrinization probes with subsequent fluorescent probe melting point analysis was developed. RESULTS: Allelic discrimination of intronic polymorphism of presenilin-1 gene and the restriction fragment length polymorphism method yielded identical results, proving its usefulness for genotyping PS1 gene. CONCLUSIONS: This method provides excellent robustness, speed, and accuracy, and is well suited for determination of the polymorphism in both small and large numbers of samples. This assay could help to overcome the controversy regarding the association between the PS1 s165932 intronic polymorphism and Alzheimer's disease.

Novel mutations in the GPIHBP1 gene identified in 2 patients with recurrent acute pancreatitis.

BACKGROUND: Glycosylphosphatidylinositol-anchored high-density lipoprotein-binding protein 1 (GPIHBP1) has been demonstrated to be essential for the in vivo function of lipoprotein lipase (LPL), the major triglyceride (TG)-hydrolyzing enzyme involved in the intravascular lipolysis of TG-rich lipoproteins. Recently, loss-of-function mutations of GPIHBP1 have been reported as the cause of type I hyperlipoproteinemia in several patients. METHODS: Two unrelated patients were referred to our Lipid Units because of a severe hypertriglyceridemia and recurrent pancreatitis. We measured LPL activity in postheparin plasma and serum ApoCII and sequenced LPL, APOC2, and GPIHBP1. RESULTS: The 2 patients exhibited very low LPL activity not associated with mutations in LPL gene or with ApoCII deficiency. The sequence of GPIHBP1 revealed 2 novel point mutations. One patient (proband 1) was found to be homozygous for a C>A transversion in exon 3 resulting in the conversion of threonine to lysine at position 80 (p.Thr80Lys). The other patient (proband 2) was found to be homozygous for a G>T transversion in the third base of the ATG translation initiation codon in exon 1, resulting in the conversion of methionine to isoleucine (p.Met1Ile). CONCLUSION: In conclusion, we have identified 2 novel GPIHBP1 missense mutations in 2 unrelated patients as the cause of their severe hypertriglyceridemia.

Melatonin and hydroxytyrosol-rich wines influence the generation of DNA oxidation catabolites linked to mutagenesis after the ingestion of three types of wine by healthy volunteers.

The Mediterranean Diet (MD) has been proved to exert benefits with respect to the maintenance of the redox balance, and wine is a representative component. Bioactive compounds such as polyphenols, melatonin and hydroxytyrosol act as radical scavengers and regulate the oxidation status of organisms. Oxidative damage to DNA yields a large range of end products. The repair of oxidized DNA entails the removal of the useless bases and/or nucleotides as well as the release of circulating nucleotides and nucleosides. The current research aims to elucidate, for the first time, the DNA protection against oxidative stress provided by three types of red wine - relating it to the intake of bioactive compounds - after the intake of a serving of red wine/must by 18 healthy female volunteers during a short term double-blind, crossover and placebo-controlled study. The novelty of our work is to describe the importance of melatonin and hydroxytyrosol and its metabolites (from gut microflora) in comparison with polyphenols in a red wine matrix (excluding colon derivatives). The results show that the intake of red wine and must secondarily reduces oxidative stress and carcinogenesis due to their content of homovanillic acid, as measured by decreases in the plasmatic concentration of 8-hydroxy-2'deoxyguanosine, 8-hydroxyguanine, and 8-nitroguanosine. Moreover, the intake of wine appears to exert vasodilatory effects, mediated by the action of nitric oxide and increased plasma guanosine-3'-5'-cyclic monophosphate plasmatic levels, owing to the intake of wines higher in melatonin and homovanillic acid. Therefore, the results obtained in the present study revealed that polyphenols, despite being the major compounds in the red wine matrix, are not the most effective compounds protecting DNA from oxidative attack.