Dimer–monomer equilibrium of human thymidylate synthase monitored by fluorescence resonance energy transfer

An ad hoc bioconjugation/fluorescence resonance energy transfer (FRET) assay has been designed to spectroscopically monitor the quaternary state of human thymidylate synthase dimeric protein. The approach enables the chemoselective engineering of allosteric residues while preserving the native protein functions through reversible masking of residues within the catalytic site, and is therefore suitable for activity/oligomerization dual assay screenings. It is applied to tag the two subunits of human thymidylate synthase at cysteines 43 and 43′ with an excitation energy donor/acceptor pair. The dimer–monomer equilibrium of the enzyme is then characterized through steady‐state fluorescence determination of the intersubunit resonance energy transfer efficiency.     

A Modular Mind? A Test Using Individual Data from Seven Primate Species

It has long been debated whether the mind consists of specialized and independently evolving modules, or whether and to what extent a general factor accounts for the variance in performance across different cognitive domains. In this study, we used a hierarchical Bayesian model to re-analyse individual level data collected on seven primate species (chimpanzees, bonobos, orangutans, gorillas, spider monkeys, brown capuchin monkeys and long-tailed macaques) across 17 tasks within four domains (inhibition, memory, transposition and support). Our modelling approach evidenced the existence of both a domain-specific factor and a species factor, each accounting for the same amount (17%) of the observed variance. In contrast, inter-individual differences played a minimal role. These results support the hypothesis that the mind of primates is (at least partially) modular, with domain-specific cognitive skills undergoing different evolutionary pressures in different species in response to specific ecological and social demands.     

Feeding postures of suspension-feeding larval black flies: the conflicting demands of drag and food acquisition

Summary We tested whether larval black flies actively control the positioning of their feeding appendages (labral fans), and if so, whether their posture represents a balance between the conflicting demands of drag and feeding. We compared the postures of live larvae with the postures of larvae killed by heat-shock in three different flow regimes in a laboratory experiment; we assumed that the postures of heat-killed larvae approximated a passive response to drag. The average height of the labral fans above the bed declined significantly in faster flows, and was significantly greater in live than dead larvae. There was also a significant interaction effect, since the difference between the fan heights of live and dead larvae was greater in slower flows. Two mechanisms may contribute to this result. Larvae in slower flows have to increase their fan heights more than larvae living in faster flows to achieve comparable increases in velocity and thus particle flux. In addition, muscular strength may limit the feeding postures larvae can assume. The fan heights of live larvae also varied depending on the concentration of food particles: larvae exposed to low food concentrations held their fans higher above the bed than did larvae exposed to high food concentrations in the same flow regime. This change in posture is due neither to an uneven particle concentration in the boundary layer nor to added drag from particles trapped in the labral fans. Collectively, our results indicate that these suspension feeders actively control their feeding posture, and suggest that these varying postures represent a dynamic balance between the conflicting needs of minimizing drag and maximizing feeding.     

Cytometry and flow cytometry of 4',6-diamidino-2-phenylindole (DAPI)-stained suspensions of nuclei released from fresh and fixed tissues of plants

Cytometry and flow cytometry were used to study characteristics of fluorescence of the DNA-DAPI complex in nuclei released from different fresh and formaldehyde-fixed pea ( Pisum sativum L. cv. Lincoln) tissues. The two methods of isolation are compared and discussed as well as their possible use for quantitative analysis of DNA in plant tissues. With fixed tissues it is possible to obtain a number of nuclei sufficient for the flow cytometric analysis, even using small amounts of plant tissue.     

Myocardial hypertrophy in rats exposed to simulated high altitude

Myocardial hypertrophy in Sprague-Dawley adult rats exposed to hypobaric hypoxia (0.40 atmosphere of air/18 h daily for 7 days) in a hypobaric chamber was investigated. Changes in the myocardial mass were evaluated on the basis of the dry heart weight and expressed as mg/100 g of total body weight (mean +/- SEM). Data are presented indicating that: chronic hypobaric hypoxia causes a significant degree of myocardial hypertrophy in rats; hypertrophic process involves both ventricles (the right more than the left); removal of the hypoxic stimulus leads to the disappearance of hypertrophy when evaluated as an increase in dry heart weight; hypoxia affects the synthesis of a significant amount of connective tissue in the left ventricle, which is not exposed to pressure load. The r?le of neurohumoral factors (i.e., adrenergic stimulation and catecholamines) in the development of the ventricular hypertrophy is suggested.     

Biosynthesisin vitro of neolactotetraosylceramide by a galactosyltransferase from mouse T-lymphoma: purification and kinetic studies; synthesis of neolacto and polylactosamine core

The galactosyltransferase, GalT-4, which catalyses the biosynthesisin vitro of neolactotetraosylceramide, nLcOse4Cer (Gal1-4GleNAc1-3Gal1-4Glc-Cer) from lactotriaosylceramide, LcOse3Cer (GlcNAc1-3Gal1-4Glc-Cer), and UDP-galactose has been purified 107 500-fold from a mineral oil induced mouse T-lyphoma P-1798, using affinity columns. The purified enzyme is partially stabilized in the presence of phospholipid liposomes. Two closely migrating protein bands of apparent molecular weights 56 kDa and 63 kDa were observed after sodium dodecyl sulfate polyacrylamide gel electrophoresis of highly purified mouse GalT-4. These two protein bands, when subjected to limited proteolysis, resulted in three peptides with identical mobilities indicating amino acid sequence identity between the proteins. Both protein bands from P-1798 gave a positive immunostain when tested with polyclonal antibody against bovine lactose synthetase (UDP-Gal:Glc 4-galactosyltransferase) following Western blot analysis on nitrocellulose paper. The enzyme has a pH optimum between 6.5 and 7.0 and like all other galactosyltransferases, GalT-4 has absolute requirements for divalent cation (Mn²⁺). TheK _m values for the substrate LcOse3Cer and donor UDP-galactose are 110 and 250 µm, respectively. Substrate competition studies with LcOse3Cer and either asialo-agalacto-1-acid glycoprotein orN-acetylglucosamine revealed that these reactions might be catalysed by the same protein. The only other glycolipid which showed acceptor activity toward the purified GalT-4 was iLcOse5Cer (GlcNAc1-1-3Gal1-4Lc3), the precursor for polylactosamine antigens. However, competition studies with these two active substrates using the most purified enzyme fraction, revealed that these two reactions might be catalysed by two different proteins since the experimental values were closer to the theoretical values calculated for two enzymes. Interestingly however, it seems that the GalT-4 from P-1798 has an absolute requirement for anN-acetylglucosamine residue in the substrate since the lyso-derivative (GlcNH₂1-3Gal1-4Glc-sphingosine) of the acceptor glycolipid LcOse3Cer is completely inactive as substrate while theK _m andV _max of the reacetylated substrate (GlcNac1-3Gal1-4Glc-acetylsphingosine) was comparable with LcOse3Cer. Autoradiography of the radioactive product formed by purified P-1798 GalT-4 confirmed the presence of nLcOse4Cer, as the product cochromatographed with authentic glycolipid. The monoclonal antibody IB-2, specific for nLcOse4Cer, also produced a positive immunostained band on TLC as well as giving a positive ELISA when tested with radioactive product obtained using a highly purified enzyme from mouse P-1798 T-lymphoma.Abbreviations EDTA ethylenediamine tetraacetate - ME -mercaptoethanol - PEG polyethylene glycol - PBS phosphate buffered saline - Suc sucrose - Mn²⁺ manganese - Gal galactose - GlcNAc N-acetylglucosamine - UDP-Gal Uridine diphosphate galactose - Ab antibody - SDS sodium dodecyl sulphate - PAGE polyacrylamide gel electrophoresis - ECB embryonic chicken brain - Cer ceramide - nLc4 or NlcOse4Cer Gal1-4GleNac1-3Gal1-4Glc-Cer, neoLactotetraosylceramide - Lc3 or LcOse3Cer GlcNac1-3Gal1-4Glc-Cer, lactotriaosylceramide - iLc5 iLcOse5Cer, GlcNAc1-3nLcOse4Cer - nLc6 nLcOse6Cer, Gal1-4iLcOse5Cer - SA^–Gal^–₁AGP asialo-agalacto1-acid glycoprotein - TLC thin layer chromatography     

Photomorphogenic effects on in vitro rooting of Prunus roostock GF 655-2

The morphogenic effect of different light wavelengths on in vitro rooting of Prunus insititia GF655-2 in relation to the presence of napthaleneacetic acid (NAA) in the culture medium was investigated. Results of experiments in which plantlets were rooted in NAA enriched medium showed that the presence of auxin induced rooting even in the dark after an initial lag period. Illumination of the cultures with Red light was as effective in promoting rooting as treatment with 0.5 M NAA; Red was more active in stimulating rooting in the short term than was NAA. The pattern of root formation resulting from the addition of NAA appeared to dominate development under White, Blue and Far Red treatments. Although it was possible to correlate the rooting response to the phytochrome photoequilibrium induced by the light treatments used, there arises a possible interference of specific Blue absorbing photoreceptors.Abbreviations B Blue - FR Far Red - HIR High Irradiance Response - P_fr active (far-red absorbing) form of phytochrome - P_tot total phytochrome - R Red - W White - NAA -naphtaleneacetic acid - BA benzyladenine - IAA indole 3-acetic acid     

Oxyprenylated secondary metabolites: a survey of their innovative extraction methodologies

Phytochemistry Reviews - Oxyprenylated secondary metabolites of plant, fungal, and microbial origin have emerged as biologically active natural compounds with a great potential for the next future....     

PYK2 is overexpressed in chronic lymphocytic leukaemia: A potential new therapeutic target

Chronic Lymphocytic Leukaemia (CLL) is the most common adult B-cell leukaemia and despite improvement in patients' outcome, following the use of targeted therapies, it remains incurable. CLL supportive microenvironment plays a key role in both CLL progression and drug resistance through signals that can be sensed by the main components of the focal adhesion complex, such as FAK and PYK2 kinases. Dysregulations of both kinases have been observed in several metastatic cancers, but their role in haematological malignancies is still poorly defined. We characterized FAK and PYK2 expression and observed that PYK2 expression is higher in leukaemic B cells and its overexpression significantly correlates with their malignant transformation. When targeting both FAK and PYK2 with the specific inhibitor defactinib, we observed a dose–response effect on CLL cells viability and survival. In vivo treatment of a CLL mouse model showed a decrease of the leukaemic clone in all the lymphoid organs along with a significant reduction of macrophages and of the spleen weight and size. Our results first define a possible prognostic value for PYK2 in CLL, and show that both FAK and PYK2 might become putative targets for both CLL and its microenvironment (e.g. macrophages), thus paving the way to an innovative therapeutic strategy.