Effects of age and caloric restriction on glutathione redox state in mice

The main purpose of this study was to determine whether the aging process in the mouse is associated with a pro-oxidizing shift in the redox state of glutathione and whether restriction of caloric intake, which results in the extension of life span, retards such a shift. Amounts of reduced and oxidized forms of glutathione (GSH and GSSG, respectively) and protein-glutathione mixed disulfides (protein-SSG) were measured in homogenates and mitochondria of liver, kidney, heart, brain, eye, and testis of 4, 10, 22, and 26 month old ad libitum-fed (AL) mice and 22 month old mice fed a diet containing 40% fewer calories than the AL group from the age of 4 months. The concentrations of GSH, GSSG, and protein-SSG vary greatly (approximately 10-, 30-, and 9-fold, respectively) from one tissue to another. During aging, the ratios of GSH:GSSG in mitochondria and tissue homogenates decreased, primarily due to elevations in GSSG content, while the protein-SSG content increased significantly. Glutathione redox potential in mitochondria became less negative, i.e., more pro-oxidizing, as the animal aged. Caloric restriction (CR) lowered the GSSG and protein-SSG content. Results suggest that the aging process in the mouse is associated with a gradual pro-oxidizing shift in the glutathione redox state and that CR attenuates this shift.     

Effect of phytosterols and phytostanols on the solubilization of cholesterol by dietary mixed micelles: an in vitro study

The effect of a plant sterol, beta-sitosterol (SI), and a plant stanol, sitostanol (SS), on the solubilization of cholesterol (CH) by model dietary mixed micelles was examined under in vitro conditions with the use of gas chromatography, isothermal titration calorimetry, NMR spectroscopy and cryogenic transmission electron microscopy techniques. Free SI and SS were shown to reduce the concentration of CH in dietary mixed micelles via a dynamic competition mechanism. CH, SI and SS affect the microstructure of lipid vesicles and influence the process of amphiphilic self-assembly of nutrients in the gut with the formation of dietary mixed micelles in a similar manner. Therefore, substitution of CH by phytosterols and phytostanols in the diet does not lead to the notable changes in the mechanism of dietary mixed micelle formation and does not affect the process of the intestinal transport of nutrients and drugs via the micellar diffusion mechanism. Our experimental findings demonstrate that the introduction of plant sterols and plant stanols into the diet is clearly beneficial for the reduction of the intestinal uptake of cholesterol. Due to the limited capacity of dietary mixed micelles to embody hydrophobic sterol/stanol molecules, the micellar concentration of cholesterol is reduced and hence, its transport towards the intestinal brush border membrane decreases.     

Factors Affecting the Tailing of Blunt End DNA with Fluorescent Pyrimidine dNTPs

The transferase activity of non-proofreading DNA polymerases is a well-known phenomenon that has been utilized in cloning and sequencing applications. The non-templated addition of modified nucleotides at DNA blunt ends is a potentially useful feature of DNA polymerases that can be used for selective transformation of DNA 3′ ends. In this paper, we characterized the tailing reaction at perfectly matched and mismatched duplex ends with Cy3- and Cy5-modified pyrimidine nucleotides. It was shown that the best DNA tailing substrate does not have a perfect Watson–Crick base pair at the end. Mismatched duplexes with a 3′ dC were the most efficient in the Taq DNA polymerase-catalysed tailing reaction with a Cy5-modified dUTP. We further demonstrated that the arrangement of the dye residue relative to the nucleobase notably affects the outcome of the tailing reaction. A comparative study of labelled deoxycytidine and deoxyuridine nucleotides showed higher efficiency for dUTP derivatives. The non-templated addition of modified nucleotides by Taq polymerase at a duplex blunt end was generally complicated by the pyrophosphorolysis and 5′ exonuclease activity of the enzyme.     

A set of fluorescent protein‐based markers expressed from constitutive and arbuscular mycorrhiza‐inducible promoters to label organelles,membranes and cytoskeletal elements in Medicago truncatula

Medicago truncatula is widely used for analyses of arbuscular mycorrhizal (AM) symbiosis and nodulation. To complement the genetic and genomic resources that exist for this species, we generated fluorescent protein fusions that label the nucleus, endoplasmic reticulum, Golgi apparatus, trans‐Golgi network, plasma membrane, apoplast, late endosome/multivesicular bodies (MVB), transitory late endosome/ tonoplast, tonoplast, plastids, mitochondria, peroxisomes, autophagosomes, plasmodesmata, actin, microtubules, periarbuscular membrane (PAM) and periarbuscular apoplastic space (PAS) and expressed them from the constitutive AtUBQ10 promoter and the AM symbiosis‐specific MtBCP1 promoter. All marker constructs showed the expected expression patterns and sub‐cellular locations in M. truncatula root cells. As a demonstration of their utility, we used several markers to investigate AM symbiosis where root cells undergo major cellular alterations to accommodate their fungal endosymbiont. We demonstrate that changes in the position and size of the nuclei occur prior to hyphal entry into the cortical cells and do not require DELLA signaling. Changes in the cytoskeleton, tonoplast and plastids also occur in the colonized cells and in contrast to previous studies, we show that stromulated plastids are abundant in cells with developing and mature arbuscules, while lens‐shaped plastids occur in cells with degenerating arbuscules. Arbuscule development and secretion of the PAM creates a periarbuscular apoplastic compartment which has been assumed to be continuous with apoplast of the cell. However, fluorescent markers secreted to the periarbuscular apoplast challenge this assumption. This marker resource will facilitate cell biology studies of AM symbiosis, as well as other aspects of legume biology.     

Multivalency: the hallmark of antibodies used for optimization of tumor targeting by design

High-precision tumor targeting with conventional therapeutics is based on the concept of the ideal drug as a "magic bullet"; this became possible after techniques were developed for production of monoclonal antibodies (mAbs). Innovative DNA technologies have revolutionized this area and enhanced clinical efficiency of mAbs. The experience of applying small-size recombinant antibodies (monovalent binding fragments and their derivatives) to cancer targeting showed that even high-affinity monovalent interactions provide fast blood clearance but only modest retention time on the target antigen. Conversion of recombinant antibodies into multivalent format increases their functional affinity, decreases dissociation rates for cell-surface and optimizes biodistribution. In addition, it allows the creation of bispecific antibody molecules that can target two different antigens simultaneously and do not exist in nature. Different multimerization strategies used now in antibody engineering make it possible to optimize biodistribution and tumor targeting of recombinant antibody constructs for cancer diagnostics and therapy.     

New species of oribatid mites of the genera Lepidozetes and Scutozetes (Acari,Oribatida, Tegoribatidae) from?Nepal

Two new species of oribatid mites, Lepidozetes acutirostrum sp. n. and Scutozetes clavatosensillus sp. n., are described from Nepal. The genera Lepidozetes and Scutozetes are recorded for the first time for the Oriental region. The identification keys to the known species of these genera are provided.     

Inhibition of biofouling by marine microorganisms and their metabolites

Development of microbial biofilms and the recruitment of propagules on the surfaces of man-made structures in the marine environment cause serious problems for the navies and for marine industries around the world. Current antifouling technology is based on the application of toxic substances that can be harmful to the natural environment. For this reason and the global ban of tributyl tin (TBT), there is a need for the development of "environmentally-friendly" antifoulants. Marine microbes are promising potential sources of non-toxic or less-toxic antifouling compounds as they can produce substances that inhibit not only the attachment and/or growth of microorganisms but also the settlement of invertebrate larvae and macroalgal spores. However, so far only few antilarval settlement compounds have been isolated and identified from bacteria. In this review knowledge about antifouling compounds produced by marine bacteria and diatoms are summarised and evaluated and future research directions are highlighted.     

DNA barcoding and minibarcoding as a powerful tool for feather mite studies

Feather mites (Astigmata: Analgoidea and Pterolichoidea) are among the most abundant and commonly occurring bird ectosymbionts. Basic questions on the ecology and evolution of feather mites remain unanswered because feather mite species identification is often only possible for adult males, and it is laborious even for specialized taxonomists, thus precluding large‐scale identifications. Here, we tested DNA barcoding as a useful molecular tool to identify feather mites from passerine birds. Three hundred and sixty‐one specimens of 72 species of feather mites from 68 species of European passerine birds from Russia and Spain were barcoded. The accuracy of barcoding and minibarcoding was tested. Moreover, threshold choice (a controversial issue in barcoding studies) was also explored in a new way, by calculating through simulations the effect of sampling effort (in species number and species composition) on threshold calculations. We found one 200‐bp minibarcode region that showed the same accuracy as the full‐length barcode (602 bp) and was surrounded by conserved regions potentially useful for group‐specific degenerate primers. Species identification accuracy was perfect (100%) but decreased when singletons or species of the Proctophyllodes pinnatus group were included. In fact, barcoding confirmed previous taxonomic issues within the P. pinnatus group. Following an integrative taxonomy approach, we compared our barcode study with previous taxonomic knowledge on feather mites, discovering three new putative cryptic species and validating three previous morphologically different (but still undescribed) new species.