Long-chain polyunsaturated fatty acids profile in plasma phospholipids of hyperphenylalaninemic children on unrestricted diet

IntroductionThe aim of the present study was to examine whether hyperphenylalaninemic children on unrestricted diet (MHP) may exhibit a different LCPUFA profile from PKU or healthy children in plasma phospholipids.Patients and methodsForty-five MHP children (age 9–14 years) were age and sex matched with 45 PKU and 45 healthy children. Fatty acids were determined and expressed as % of total fatty acids.ResultsMHP children showed docosahexaenoic acid (DHA) levels higher than PKU children (mean difference, 0.2%; 95% confidence interval, 0.02%–0.38%), although difference was not significant after correction for multiple comparisons, and lower levels than healthy children (?0.8%; ?1.01% to ?0.59%). Concentration of n-3 PUFA was higher in MHP than PKU children (0.6%; 0.4% to 0.8%),ConclusionsThe results suggest that low DHA levels in plasma phospholipids not only are evident in PKU but also may occur in MHP children, who are on unrestricted diet, as compared to healthy children.     

A hybrid process for chiral separation of compound‐forming systems

The resolution of chiral compound‐forming systems using hybrid processes was discussed recently. The concept is of large relevance as these systems form the majority of chiral substances. In this study, a novel hybrid process is presented, which combines pertraction and subsequent preferential crystallization and is applicable for the resolution of such systems. A supported liquid membrane applied in a pertraction process provides enantiomeric enrichment. This membrane contains a solution of a chiral compound acting as a selective carrier for one of the enantiomers. Screening of a large number of liquid membranes and potential carriers using the conductor‐like screening model for realistic solvation method led to the identification of several promising carriers, which were tested experimentally in several pertraction runs aiming to yield enriched (+)‐(S)‐mandelic acid (MA) solutions from racemic feed solutions. The most promising system consisted of tetrahydronaphthalene as liquid membrane and hydroquinine‐4‐methyl‐2‐quinolylether (HMQ) as chiral carrier achieving enantiomeric excesses of 15% in average. The successful production of (+)‐(S)‐MA with a purity above 96% from enriched solutions by subsequent preferential crystallization proved the applicability of the hybrid process. Chirality, 2011. © 2010 Wiley‐Liss, Inc.     

Basic science track. Entry and release of canine coronavirus from polarized epithelial cells

Virus entry into and release from epithelial cells are polarized as a result of the distribution of the viral receptors. In order to establish the polarity of entry and release of CCoV from epithelial cells, the interactions of the virus with A72 and CrFK cells grown on permeable supports was evaluated, and the amount of infective virus in the apical and in the basolateral media was determined and compared. Infection of A72 cells after different times post seeding demonstrated that CCoV grow after infection from both apical and basolateral sides. In CrFK cells, CCoV was observed in both compartments only in the later phase of the infection. To establish the reciprocal binding of CCoV on plasma membrane, A72 cells on a permeable support were preincubated with a mAb specific for CCoV. Infection from the apical side was blocked by mAb applied to that side; in contrast, such treatment on the basolateral side had no effect on the infectious process. Similarly, the low levels of CCoV observed after basolateral exposure to virus was abolished following mAb treatment of that side. The identification of CCoV into the basolateral medium could play an important role in the viral pathogenesis.     

Fluorescence in situ hybridization mapping of six loci containing genes involved in the dioxin metabolism of domestic bovids

Six loci containing genes involved in the dioxin metabolism (ARNT, AHR, CYP1A1, CYP1A2, CYP1B1 and AHRR) were assigned, for the first time, to cattle (Bos taurus, 2n = 60, BTA), river buffalo (Bubalus bubalis, 2n = 50, BBU), sheep (Ovis aries, 2n = 54, OAR) and goat (Capra hircus, 2n = 60, CHI) chromosomes by comparative FISH-mapping and R-banding using bovine BAC-clones. The following chromosome locations were found: ARNT to BTA3q21, BBU6q21, OAR1p21 and CHI3q21, AHR to BTA4q15, BBU8q15, OAR4q15 and CHI4q15; CYP1A1 and CYP1A2 to BTA21q17, BBU20q17, OAR18q17 and CHI21q17; CYP1B1 to BTA11q16, BBU12q22, OAR3p16 and CHI11q16, AHRR to BTA20q24, BBU19q24, OAR16q24 and CHI20q24. All loci were mapped at the same homoeologous chromosomes and chromosome bands of the four bovid species. Comparisons with corresponding human locations were also reported.     

GJB2 Gene Mutations in Syndromic Skin Diseases with Sensorineural Hearing Loss

The GJB2 gene is located on chromosome 13q12 and it encodes the connexin 26, a transmembrane protein involved in cell-cell attachment of almost all tissues. GJB2 mutations cause autosomal recessive (DFNB1) and sometimes dominant (DFNA3) non-syndromic sensorineural hearing loss. Moreover, it has been demonstrated that connexins are involved in regulation of growth and differentiation of epidermis and, in fact, GJB2 mutations have also been identified in syndromic disorders with hearing loss associated with various skin disease phenotypes. GJB2 mutations associated with skin disease are, in general, transmitted with a dominant inheritance pattern. Nonsyndromic deafness is caused prevalently by a loss-of-function, while literature evidences suggest for syndromic deafness a mechanism based on gain-of-function. The spectrum of skin manifestations associated with some mutations seems to have a very high phenotypic variability. Why some mutations can lead to widely varying cutaneous manifestations is poorly understood and in particular, the reason why the skin disease-deafness phenotypes differ from each other thus remains unclear. This review provides an overview of recent findings concerning pathogenesis of syndromic deafness imputable to GJB2 mutations with an emphasis on relevant clinical genotype-phenotype correlations. After describing connexin 26 fundamental characteristics, the most relevant and recent information about its known mutations involved in the syndromic forms causing hearing loss and skin problems are summarized. The possible effects of the mutations on channel expression and function are discussed.     

Copper exposure effects on yeast mitochondrial proteome

Mitochondria play an important role on the entire cellular copper homeostatic mechanisms. Alteration of cellular copper levels may thus influence mitochondrial proteome and its investigation represents an important contribution to the general understanding of copper-related cellular effects. In these study we have performed an organelle targeted proteomic investigation focusing our attention on the effect of non-lethal 1mM copper concentration on Saccharomyces cerevisiae mitochondrial proteome. Functional copper effects on yeast mitochondrial proteome were evaluated by using both 2D electrophoresis (2-DE) and liquid chromatography coupled with tandem mass spectrometry. Proteomic data have been then analyzed by different unsupervised meta-analysis approaches that highlight the impairment of mitochondrial functions and the activation of oxidative stress response. Interestingly, our data have shown that stress response generated by 1mM copper treatment determines the activation of S. cerevisiae survival pathway. To investigate these findings we have treated yeast cells responsiveness to copper with hydrogen peroxide and observed a protective role of this metal. These results are suggestive of a copper role in the protection from oxidative stress possibly due to the activation of mechanisms involved in cellular survival and growth.     

The effect of solvent extraction on antioxidant properties of apricot fruit

Two solvent extraction procedures were used to investigate the extraction efficiency in terms of total antioxidant capacity and total phenols in apricot fruit. Samples were either sequentially extracted with aqueous ethanol (ethanol/water 80% v/v) and tetrahydrofuran or directly extracted with tetrahydrofuran. Each extract was analyzed for total antioxidant capacity by the Trolox Equivalent Antioxidant Capacity (TEAC) assay and total phenols by the Folin-Ciocalteu assay. The results showed that using sequential solvent extraction, the majority (85%) of the total antioxidant capacity and total phenols was due to hydrophilic compounds. In tetrahydrofuran direct extractions, the total antioxidant capacity and total phenols were higher than values obtained with aqueous ethanol and the sum of results obtained from sequential extracts for either total antioxidant capacity or total phenols was similar to the tetrahydrofuran-extract antioxidant values. A linear correlation between total antioxidant capacity and total phenols was found and was independent of the solvent extraction method. In conclusion, the choice of solvent is related to the antioxidant potential of fruit and depends on the food hydrophilic/lipophilic composition.     

Designing nanoconjugates to effectively target pancreatic cancer cells in vitro and in vivo

Background

Pancreatic cancer is the fourth leading cause of cancer related deaths in America. Monoclonal antibodies are a viable treatment option for inhibiting cancer growth. Tumor specific drug delivery could be achieved utilizing these monoclonal antibodies as targeting agents. This type of designer therapeutic is evolving and with the use of gold nanoparticles it is a promising approach to selectively deliver chemotherapeutics to malignant cells.Gold nanoparticles (GNPs) are showing extreme promise in current medicinal research. GNPs have been shown to non-invasively kill tumor cells by hyperthermia using radiofrequency. They have also been implemented as early detection agents due to their unique X-ray contrast properties; success was revealed with clear delineation of blood capillaries in a preclinical model by CT (computer tomography). The fundamental parameters for intelligent design of nanoconjugates are on the forefront. The goal of this study is to define the necessary design parameters to successfully target pancreatic cancer cells.

Methodology/Principal Findings

The nanoconjugates described in this study were characterized with various physico-chemical techniques. We demonstrate that the number of cetuximab molecules (targeting agent) on a GNP, the hydrodynamic size of the nanoconjugates, available reactive surface area and the ability of the nanoconjugates to sequester EGFR (epidermal growth factor receptor), all play critical roles in effectively targeting tumor cells in vitro and in vivo in an orthotopic model of pancreatic cancer.

Conclusion

Our results suggest the specific targeting of tumor cells depends on a number of crucial components 1) targeting agent to nanoparticle ratio 2) availability of reactive surface area on the nanoparticle 3) ability of the nanoconjugate to bind the target and 4) hydrodynamic diameter of the nanoconjugate. We believe this study will help define the design parameters for formulating better strategies for specifically targeting tumors with nanoparticle conjugates.     

Biotransformation of CL-20 by a dehydrogenase enzyme from Clostridium sp. EDB2

In a previous study, a marine isolate Clostridium sp. EDB2 degraded 2,4,6,8,10,12-hexanitro-2,4,6,8,10,12-hexaazaisowurtzitane (CL-20) under anaerobic conditions (Bhushan B, Halasz A, Thiboutot S, Ampleman G, Hawari J (2004c) Chemotaxis-mediated biodegradation of cyclic nitramine explosives RDX, HMX, and CL-20 by Clostridium sp. EDB2. Biochem Biophys Res Commun 316:816–821); however, the enzyme responsible for CL-20 degradation was not known. In the present study, we isolated and purified an enzyme, from strain EDB2, responsible for CL-20 degradation. The enzyme was membrane-associated and NADH-dependent and had a molecular weight of 56 kDa (with SDS-PAGE). N-terminal amino acid sequence of enzyme revealed that it belonged to dehydrogenase class of enzymes. The purified enzyme degraded CL-20 at a rate of 18.5 nmol/h mg protein under anaerobic conditions. Carbon and nitrogen mass balance of the products were 100 and 64%, respectively. In LC–MS–MS studies, we detected three different initial metabolites from CL-20, i.e., mono-nitroso derivative, denitrohydrogenated product, and double-denitrated isomers with molecular weight of 422, 393, and 346 Da, corresponding to presumed empirical formulas of C₆H₆N₁₂O₁₁, C₆H₇N₁₁O₁₀, and C₆H₆N₁₀O₈, respectively. Identity of all the three metabolites were confirmed by using ring-labeled [¹⁵N]CL-20 and the nitro-group-labeled [¹⁵NO₂]CL-20. Taken together, the above data suggested that the enzyme degraded CL-20 via three different routes: Route A, via two single electron transfers necessary to release two nitro-groups from CL-20 to produce two double-denitrated isomers; Route B, via a hydride transfer necessary to produce a denitrohydrogenated product; and Route C, via transfer of two redox equivalents to CL-20 necessary to produce a mono-nitroso derivative of CL-20. This is the first biochemical study which showed that CL-20 degradation can be initiated via more than one pathway.     

Oxidative stress and phytochelatin characterisation in bread wheat exposed to cadmium excess.

In this work, we first investigated if the bread wheat (Triticum aestivum L.) cv. Albimonte can be defined as "shoot cadmium excluder"--by comparing the cadmium (Cd) content in leaves and roots and by calculating the shoot-to-root Cd concentration ratio. Furthermore, we evaluated if the exposure to Cd excess could generate oxidative stress in leaves and roots of this cv., in terms of hydrogen peroxide (H(2)O(2)) accumulation, NAD(P)H oxidation rate, and variations in reduced glutathione (GSH) content and peroxidase (POD, EC 1.11.1.7) activity. Finally, we surveyed possible quali- quantitative differences in thiol-peptide compound pattern between roots and leaves, in order to verify whether phytochelatins (PCs) and related thiol-peptides could contribute in limiting the Cd-induced oxidative stress. Unambiguous characterisation of PCs and related forms present in the root samples was obtained by electrospray ionisation mass spectrometry (ESI-MS) and ESI-tandem MS (ESI-MS/MS). Our results indicate that in leaves the stress generated by the low accumulation of Cd (due to a moderate translocation in planta) seems to be counteracted by the antioxidant response and by the PC biosynthesis. On the contrary, in roots, in spite of the elevated presence of PCs and related thiol-peptide-compounds, the excess of Cd causes a decline in the antioxidant protection of the organ, with the consequent generation of considerable amounts of H(2)O(2), a direct agent of oxidative stress.