Novel TMPRSS3 variants in Pakistani families with autosomal recessive non-syndromic hearing impairment.

Mutations in the TMPRSS3 gene are known to cause autosomal recessive non-syndromic hearing impairment (ARNSHI). After undergoing a genome scan, 10 consanguineous Pakistani families with ARNSHI were found to have significant or suggestive evidence of linkage to the TMPRSS3 region. In order to elucidate if the TMPRSS3 gene is responsible for ARNSHI in these families, the gene was sequenced using DNA samples from these families. Six TMPRSS3 variants were found to cosegregate in 10 families. None of these variants were detected in 500 control chromosomes. Four novel variants, three of which are missense [c.310G>A (p.Glu104Lys), c.767C>T (p.Ala256Val) and c.1273T>C (p.Cys425Arg)] and one nonsense [c.310G>T (p.Glu104Stop)], were identified. The pathogenicity of novel missense variants was investigated through bioinformatics analyses. Additionally, the previously reported deletion c.208delC (p.His70ThrfsX19) was identified in one family and the known mutation c.1219T>C (p.Cys407Arg) was found in five families, which makes c.1219T>C (p.Cys407Arg) as the most common TMPRSS3 mutation within the Pakistani population. Identification of these novel variants lends support to the importance of elements within the low-density lipoprotein receptor A (LDLRA) and serine protease domains in structural stability, ligand binding and proteolytic activity for proper TMPRSS3 function within the inner ear.

Plitidepsin to treat multiple myeloma.

While remaining relatively rare, multiple myeloma (MM) accounts for approximately 10% of all hematological malignancies, being an insidious disease with an overall 5-year survival rate of 52%. In addition to other associated complications, myeloma bone disease further aggravates MM patients, the majority of whom suffer from lytic lesions, leading to pain, fractures, mobility issues and neurological deficits. Patients not responding or becoming resistant to prior therapies have now a novel therapeutic tool with an unprecedent mode of action, differing from those currently in use. The anticancer effects of the marine-derived antitumor agent plitidepsin primarily rely on the interaction with elongation factor 1-α 2 (eEF1A2), known to be overexpressed in breast cancer and MM cells, targeting the noncanonical role of the protein and leading to a proapoptotic response. Following the drug's approval from Australian regulatory authorities, eligible patients will have access to a new first-in-class drug to treat MM, expanding the current anti-MM portfolio. Plitidepsin (Aplidin; PharmaMar) was approved in combination with the corticosteroid agent dexamethasone, to treat MM patients who failed or became resistant to other therapies, covering the third- and fourth-line treatment setting.

Further knowledge on barley (Hordeum vulgare L.) leaves O-glycosyl-C-glycosyl flavones by liquid chromatography-UV diode-array detection-electrospray ionisation mass spectrometry.

Thirty-seven flavonoids and a hydroxycynnamic acid have been characterized in barley leaves (Hordeum vulgare L.) by liquid chromatography-UV diode-array coupled to ion trap mass spectrometry with electrospray ionisation interface (negative mode). Their structures have been determined by the study of the ion mass fragmentation which characterizes C-glycosyl flavones and O-glycosyl-C-glycosyl flavones, and differentiates di-O-glycosyl flavones from O-diglycosyl-flavones. The majority of them are described for the first time in barley. Saponarin (isovitexin-7-O-glucoside), lutonarin (isoorientin-7-O-glucoside) and isoscoparin-7-O-glucoside derivatives constitute the major part of the detected compounds. Some acylated derivatives are also described, namely, 7-O-[6-acyl]-glucoside and -7-O-[6-acyl]-glc-4'-glucoside of isovitexin, isoorientin and isoscoparin.

Expression of fibroblast growth factor receptors during development and regression of the bovine corpus luteum.

There is evidence that fibroblast growth factors (FGFs) are involved in the regulation of growth and regression of the corpus luteum (CL). However, the expression pattern of most FGF receptors (FGFRs) during CL lifespan is still unknown. The objective of the present study was to determine the pattern of expression of 'B' and 'C' splice variants of FGFRs in the bovine CL. Bovine CL were collected from an abattoir and classed as corpora hemorrhagica (Stage I), developing (Stage II), developed (Stage III) or regressed (Stage IV) CL. Expression of FGFR mRNA was measured by semiquantitative reverse transcription-polymerase chain reaction and FGFR protein was localised by immunohistochemistry. Expression of mRNA encoding the 'B' and 'C' spliced forms of FGFR1 and FGFR2 was readily detectable in the bovine CL and was accompanied by protein localisation. FGFR1C and FGFR2C mRNA expression did not vary throughout CL lifespan, whereas FGFR1B was upregulated in the developed (Stage III) CL. FGFR3B, FGFR3C and FGFR4 expression was inconsistent in the bovine CL. The present data indicate that FGFR1 and FGFR2 splice variants are the main receptors for FGF action in the bovine CL.

Assessing the antioxidative properties and chemical composition of Linaria vulgaris infusion.

The ability of Linaria vulgaris (Scrophulariaceae) infusion to act as a scavenger of 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical, reactive oxygen species (superoxide radical, hydroxyl radical, hypochlorous acid (HOCl)) and nitric oxide was investigated. The obtained data indicate that the infusion has a good scavenging activity against superoxide radical and is a very potent nitric oxide and DPPH scavenger. In hydroxyl radical assay a pro-oxidant capacity was noticed, especially for concentrations higher than 31.25 microg mL(-1). No effect was found against HOCl. A phytochemical study of this extract was also performed. The HPLC/UV analysis allowed the identification and quantification of eight organic acids (oxalic, aconitic, citric, ketoglutaric, ascorbic, malic, shikimic and fumaric acids). The phenolic composition of the lyophilised infusion was also determined by HPLC/DAD and four compounds were quantified, but, despite its high content, only linarin was managed to be identified.

Evaluation of the neuroprotective and antidiabetic potential of phenol-rich extracts from virgin olive oils by in vitro assays.

In this work, phenol-rich extracts from 'Cornicabra' and 'Picual' virgin-olive oils (EVOOs) were examined, for the first time, to establish their capacity to inhibit key enzymes involved in Alzheimer's disease (AD) (acetylcholinesterase (AChE), butyrylcholinesterase (BuChE) and 5-lipoxygenase (LOX)), major depressive disorder (MDD) and Parkinson's disease (PD) (monoamine oxidases: hMAO-A and hMAO-B respectively), and diabetes mellitus (DM) (α-glucosidase and α-amylase). 'Cornicabra' displayed the best inhibitory activity against all enzymes, when compared to 'Picual': BuChE (IC50 = 156 ±â€¯4 and 308 ±â€¯33 mg mL-1), LOX (IC50 = 26 ±â€¯0.5 and 37 ±â€¯3 mg mL-1), hMAO-A (IC50 = 20 ±â€¯2 and 37 ±â€¯0.2 mg mL-1), hMAO-B (IC50 = 131 ±â€¯7 and 215 ±â€¯13 mg mL-1) and α-glucosidase (IC50 = 154 ±â€¯17 and 251 ±â€¯31 mg mL-1), respectively. The behaviour observed can be associated with the higher content of secoiridoids, lignans and phenolic acids in 'Cornicabra' EVOO.

Characterization of C-glycosyl flavones O-glycosylated by liquid chromatography-tandem mass spectrometry.

Fifty three O-glycosyl-C-glycosyl flavones with O-glycosylation on phenolic hydroxyl or on the C-glycosyl residue or combination of both forms have been studied by liquid chromatography-UV diode array detection-electrospray ionisation mass spectrometry ion trap in the negative mode. The study of the relative abundance of the main ions from the MS preferential fragmentation on -MS2 and/or -MS3 events allows the differentiation of the position of the O-glycosylation, either on phenolic hydroxyl or on the sugar moiety of C-glycosylation. In addition, it is possible to discriminate between O-glycosylation at 2'' and at 6'' positions. The occurrence of an abundant ion Y(0)(-) ([(M-H)-132/-146/-162](-), mono-O-pentosyl/rhamnosyl/hexosyl-C-glycosyl derivatives) after -MS2 fragmentation characterizes the O-glycosylation on phenolic hydroxyls. The preferential fragmentation leading to a relevant Z(1)(-) ([Y(1)-18](-)) fragment is characteristic of 2''-O-glycosyl-C-glycosyl derivatives. The 6''-O-glycosyl-C-glycosyl derivatives are characterized by (0,2)X(0)(-), which is generated by a global loss of the sugar moiety from the O-glycosylation at 6'' and the glycosidic fraction that involves the carbons 6''-3'' of the C-glycosyl residue ([(M-H)-162-120](-), in the case of 6''-O-hexosyl-C-hexosyl derivatives). Regarding the combined O-glycosylated compounds (both on phenolic hydroxyl and on sugar moiety at C-glycosylation), the main fragmentation on -MS2 events produces a Y(0)(-) characterizing the O-glycosylation on the phenolic hydroxyl, and the -MS3[(M-H)-->Y(0)](-) fragmentation of the O-glycosylation on the C-glycosyl residue.

HPLC-DAD-ESI/MS(n) profiling of phenolic compounds from Lathyrus cicera L. seeds.

Lathyrus cicera L. seeds are of interest for food and feed purposes. Despite the recognized antioxidant activity of the seeds, arising from the phenolic fraction, their phenolic compounds have not been studied in depth yet. Therefore, to determine the phenolics profile of these seeds, a target analysis was performed using high-performance liquid chromatography coupled to photodiode-array detection and electrospray ionization/ion trap mass spectrometry (HPLC-DAD-ESI/MS(n)). Thirty-seven glycosylated flavonoids were identified for the first time in the seeds of this species and, according to their MS fragmentation, clustered in flavonol-3-O-di-/tri-glycosides-7-O-rhamnosides and other flavonol-glycosides, and flavonol-3-O-(cinnamoyl)glycoside-7-O-rhamnosides, flavonol-3-O-(dihydrophaseoyl, cinnamoyl)glycoside-7-O-rhamnosides and flavonol-3-O-(malonyl)glycoside-7-O-rhamnosides. Glycosides of kaempferol were the main flavonoids found (10 non-acylated and 21 acylated), followed by those of quercetin (3) and those of isorhamnetin, apigenin and luteolin (1). The most abundant flavonols were identified as kaempferol-3-O-(2-hexosyl)hexoside-7-O-rhamnosides. The methodology used allowed to increase the knowledge on a relevant phytochemical class of seeds from L. cicera.

Functional reconstitution of Arabidopsis thaliana plant uncoupling mitochondrial protein (AtPUMP1) expressed in Escherichia coli.

The Arabidopsis thaliana uncoupling protein (UCP) gene was expressed in Escherichia coli and isolated protein reconstituted into liposomes. Linoleic acid-induced H+ fluxes were sensitive to purine nucleotide inhibition with an apparent K(i) (in mM) of 0.8 (GDP), 0.85 (ATP), 0.98 (GTP), and 1.41 (ADP); the inhibition was pH-dependent. Kinetics of AtPUMP1-mediated H+ fluxes were determined for lauric, myristic, palmitic, oleic, linoleic, and linolenic acids. Properties of recombinant AtPUMP1 indicate that it represents a plant counterpart of animal UCP2 or UCP3. This work brings the functional and genetic approaches together for the first time, providing strong support that AtPUMP1 is truly an UCP.

Inhibition of membrane lipid peroxidation by a radical scavenging mechanism: a novel function for hydroxyl-containing ionophores.

In the present study we show that K+/H+ hydroxyl-containing ionophores lasalocid-A (LAS) and nigericin (NIG) in the nanomolar concentration range, inhibit Fe2+-citrate and 2,2'-azobis(2-amidinopropane) dihydrochloride (ABAP)-induced lipid peroxidation in intact rat liver mitochondria and in egg phosphatidylcholine (PC) liposomes containing negatively charged lipids--dicetyl phosphate (DCP) or cardiolipin (CL)--and KCl as the osmotic support. In addition, monensin (MON), a hydroxyl-containing ionophore with higher affinity for Na+ than for K+, promotes a similar effect when NaCl is the osmotic support. The protective effect of the ionophores is not observed when the osmolyte is sucrose. Lipid peroxidation was evidenced by mitochondrial swelling, antimycin A-insensitive O2 consumption, formation of thiobarbituric acid-reactive substances (TBARS), conjugated dienes, and electron paramagnetic resonance (EPR) spectra of an incorporated lipid spin probe. A time-dependent decay of spin label EPR signal is observed as a consequence of lipid peroxidation induced by both inductor systems in liposomes. Nitroxide destruction is inhibited by butylated hydroxytoluene, a known antioxidant, and by the hydroxyl-containing ionophores. In contrast, valinomycin (VAL), which does not possess alcoholic groups, does not display this protective effect. Effective order parameters (Seff), determined from the spectra of an incorporated spin label are larger in the presence of salt and display a small increase upon addition of the ionophores, as a result of the increase of counter ion concentration at the negatively charged bilayer surface. This condition leads to increased formation of the ion-ionophore complex, the membrane binding (uncharged) species. The membrane-incorporated complex is the active species in the lipid peroxidation inhibiting process. Studies in aqueous solution (in the absence of membranes) showed that NIG and LAS, but not VAL, decrease the Fe2+-citrate-induced production of radicals derived from piperazine-based buffers, demonstrating their property as radical scavengers. Both Fe2+-citrate and ABAP promote a much more pronounced decrease of LAS fluorescence in PC/CL liposomes than in dimyristoyl phosphatidylcholine (DMPC, saturated phospholipid)-DCP liposomes, indicating that the ionophore also scavenges lipid peroxyl radicals. A slow decrease of fluorescence is observed in the latter system, for all lipid compositions in sucrose medium, and in the absence of membranes, indicating that the primary radicals stemming from both inductors also attack the ionophore. Altogether, the data lead to the conclusion that the membrane-incorporated cation complexes of NIG, LAS and MON inhibit lipid peroxidation by blocking initiation and propagation reactions in the lipid phase via a free radical scavenging mechanism, very likely due to the presence of alcoholic hydroxyl groups in all three molecules and to the attack of the aromatic moiety of LAS.

Antioxidant activity of Centaurium erythraea infusion evidenced by its superoxide radical scavenging and xanthine oxidase inhibitory activity.

Centaurium erythraea Rafin. (Gentianaceae) has long been used in traditional medicine. This plant contains considerable amounts of polyphenolic compounds, namely, xanthones and phenolic acids as the main constituents. Because phenolic groups exhibit activity as radical scavengers and/or metal chelators, this study evaluated the superoxide radical scavenging properties of a lyophilized infusion obtained from C. erythraea flowering tops. Superoxide radical scavenging activity was assayed using enzymatic (xanthine/xanthine oxidase) and nonenzymatic (NADH/phenazine methosulfate) superoxide generating systems. This study provided evidence that C. erythraea exhibits interesting antioxidant properties, expressed either by the capacity to scavenge superoxide radical or to noncompetitively inhibit xanthine oxidase. The main phenolic compounds present in this extract were several esters of hydroxycinnamic acids, namely, p-coumaric, ferulic, and sinapic acids.

Flavonoids and phenolic acids of sage: influence of some agricultural factors.

A reversed-phase high-performance liquid chromatography/diode-array detector procedure is proposed for the determination of six phenolic compounds (caffeic acid, luteolin 7-O-glucoside, rosmarinic acid, apigenin, hispidulin, and cirsimaritin) in sage. The chromatographic separation was achieved using a reversed-phase Spherisorb ODS2 (5-microm particle size, 25.0 x 0.46 cm) column. Of the several extractive solvents assayed only ethyl ether, ethyl acetate, and acetone were able to extract all the compounds mentioned. Best resolution was obtained using a gradient of water/phosphoric acid (999:1) and acetonitrile. Ten samples cultivated in two experimental fields (1997-1999) were analyzed and the individual compounds quantified. Four commercially available samples were also analyzed and the results are discussed.

Analysis of vervain flavonoids by HPLC/Diode array detector method. Its application to quality control.

A reversed-phase HPLC procedure is proposed for the determination of seven flavonoids (luteolin, nepetin, hispidulin, jaceosidin, cirsimaritin, cirsilineol, and eupatorin) in vervain samples. A simple extractive technique was developed, involving only extraction with ethyl ether, which allowed the elimination of phenolic acids, yielding a sufficiently purified flavonoid fraction. All of the analyzed vervain samples showed a common flavonoid pattern, in which hispidulin and jaceosidin were the major compounds and nepetin was the minor compound.

Analysis of phenolic compounds in the evaluation of commercial quince jam authenticity.

The phenolic compounds present in 17 samples of Portuguese commercial and three homemade quince jams were analyzed by reversed-phase HPLC/DAD, to determine their authenticity. Two different extraction methods were needed for the complete definition of quince jams profiles, one of them including an Amberlite XAD-2 cleaning step. These analyses showed that all the samples presented a similar profile composed of at least eight identified phenolic compounds, several unidentified characteristic procyanidin polymers, and sodium benzoate as preservative of quince jams. Several samples also contained arbutin, suggesting that these quince jam samples were fraudulently adulterated with pear puree.

The use of flavonoids in central nervous system disorders.

Neurodegenerative, neurological and psychiatric diseases are a group of pathologies with huge social and economic impacts. Since brain disorders continue to be pathological conditions for which corrective surgery cannot be widely used, treatments are based on drugs that only alleviate the symptoms. Despite all efforts in finding more efficient therapeutic agents, the requirement for neuroprotective drugs able to cross the blood-brain barrier (BBB) has been a hard challenge to overcome. Flavonoids are known by their antioxidant activities thus preventing oxidative stress, which is believe to be one of the causes of disorders affecting the central nervous system. Moreover, they can also modulate both enzymes and receptors activities, being regarded as multi-target botanical therapeutics or drugs. This review will give emphasis to the benefits of flavonoids found in the diet in the treatment of Alzheimer's disease, Parkinson's disease, epilepsy, depression, and schizophrenia. The antioxidant effect of several flavonoids, as well as their effects not related with antioxidant activity, in the above mentioned diseases will be reviewed. Aspects concerning structure-activity relationships, but also the bioavailability of these compounds in the brain will be referred.

Alzheimer's disease and antioxidant therapy: how long how far?

Alzheimer's disease (AD) has become a health problem to societies worldwide affecting millions of people. AD normally ensues in middle and late life but its specific cause remains unknown. Besides amyloid-ß deposition and hyperphosphorylated tau protein, increased production of reactive species (RS) has also been described to be a hallmark in early steps of this disorder. Antioxidant therapy has received considerable attention over the last years as a promising approach to delay or slow the neurodegeneration progression in AD either by boosting the pool of endogenous antioxidants (e.g.vitamins, coenzyme Q10 or melatonin) or by the intake of dietary antioxidants, such as phenolic compounds of flavonoid or non-flavonoid type. However, the majority of antioxidants studied so far have limited success in clinical trials, a fact that could be related to their poor distribution and with the inherent difficulties to cross the blood brain barrier and attain the target sites. Despite the evidence that different classes of antioxidants are neuroprotectants in vitro, the clinical data is not consistent. Alzheimer's disease and antioxidant therapy is still an open question: the research is far from the end but the success may not be so time-consuming if the data obtained so far are gathered and rationally analyzed either by checking new targets or by the obtention of new and effective compounds, for instance by the rational modification of the previous ones.

Avaliação anatômica das artérias radiais e ulnares em adultos submetidos à intervenção coronariana percutânea / Anatomical evaluation of radial and ulnar arteries in adults undergoing percutaneous coronary intervention

INTRODUÇÃO: A artéria radial tornou-se, recentemente, uma alternativa de acesso vascular muito popular nos procedimentos percutâneos coronarianos. Contudo, apesar do uso de cateteres com diâmetro maior ser fator de risco para oclusão da artéria radial, a mensuração do tamanho da mesma não é realizada rotineiramente. MÉTODOS: Com uso da ultrassonagrafia, realizou-se, prospectivamente, a mensuração do diâmetro das artérias radiais e ulnares de 317 pacientes submetidos à angiocoronariografia e/ou intervenção coronariana percutânea em serviços de referência em cardiologia no sul, sudeste e centro oeste do País. Além disso foi verificado as características demográficas e clínicas desta população. Os dados foram tabulados e analisados no programa Statistical Package for the Social Sciences (SPSS). Version 18.0, conforme frequência, média e desvio padrão. RESULTADOS: O diâmetro interno médio das artérias radial e ulnar direitas foi de 2,836±0,7mm e 2,155±0,58mm, respectivamente. Os resultados das artérias contralaterais foram similares (radial 2,53±0,63mm, ulnar 2,15±0,55mm). 20,5% dos pacientes apresentaram diâmetro da artéria radial direita inferiores à 2,3mm e 38,5% menor que 2,5mm, equivalente aos diâmetros dos introdutores Terumo® GSS 5Fr e 6Fr, respectivamente. A circunferência média do punho direito foi de 17,273±1,39cm, também semelhante ao punho esquerdo (17,33±1,39cm). A média de idade foi de 60±10,89 anos. O peso médio encontrou-se na faixa de 76,34±15 kg e a altura 164,1±8,9cm. Houve predomínio do sexo masculino (61%), a comorbidade mais prevalente foi a hipertensão (73%) e 70% dos pacientes apresentaram-se como síndrome coronariana aguda. CONCLUSÃO: Em aproximadamente 40% dos pacientes da amostra foi verificado um diâmetro inferior à 2,5mm da artéria radial direita, sendo este um valor menor ao do introdutor utilizado para sua abordagem nos casos de intervenção coronariana percutânea. (AU)

Water extracts of Brassica oleracea var. costata potentiate paraquat toxicity to rat hepatocytes in vitro.

Tronchuda cabbage extracts have been proven to have antioxidant potential against various oxidative species in cell free systems, though its antioxidant potential in cellular models remained to be demonstrated. In the present study, we used primary cultures of rat hepatocytes for the cellular assay system and paraquat PQ exposure as a pro-oxidant model agent, to test whether tronchuda cabbage hydrolysed water extracts provide protective or aggravating effects towards PQ-induced oxidative stress and cell death. For this purpose cellular parameters related to oxidative stress were measured, namely the generation of superoxide anion, glutathione oxidation, lipid peroxidation, intracellular ATP levels, activation of nuclear factor-kappaB (NF-kappaB), activity of antioxidant enzymes, and cell death. The obtained results demonstrated that the studied hydrolysed water extracts of tronchuda cabbage, especially rich in kaempferol (84%) and other polyphenols, namely hydroxycinnamic acids and traces of quercetin, can potentiate the toxicity of PQ in primary cultures of rat hepatocytes. These results highlight that prospective antioxidant effects of plant extracts, observed in vitro, using non-cellular systems, are not always confirmed in cellular models, in which the concentrations required to scavenge pro-oxidant species may be highly detrimental to the cells.

Principal components of phenolics to characterize red Vinho Verde grapes: anthocyanins or non-coloured compounds?

Phenolic profile of 10 different varieties of red "Vinho Verde" grapes (Azal Tinto, Borraçal, Brancelho, Doçal, Espadeiro, Padeiro de Basto, Pedral, Rabo de ovelha, Verdelho and Vinhão), from Minho (Portugal) were studied. Nine Flavonols, four phenolic acids, three flavan-3-ols, one stilben and eight anthocyanins were determined. Malvidin-3-O-glucoside was the most abundant anthocyanin while the main non-coloured compound was much more heterogeneous: catechin, epicatechin, myricetin-3-O-glucoside, quercetin-3-O-glucoside or syringetin-3-O-glucoside. Anthocyanin contents ranged from 42 to 97%. Principal component analysis (PCA) was applied to analyse the date and study the relations between the samples and their phenolic profiles. Anthocyanin profile proved to be a good marker to characterize the varieties even considering different origin and harvest. "Vinhão" grapes showed anthocyanins levels until twenty four times higher than the rest of the samples, with 97% of these compounds.