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     

Transfer of a marine DNA virus fromEctocarpus toFeldmannia (Ectocarpales,Phaeophyceae): aberrant symptoms and restitution of the host

Summary The marine brown algaEctocarpus siliculosus is invaded by a polyhedric virus, whose genome consists of circular, double-stranded DNA. In laboratory experiments this virus can infect a different host species,Feldmannia simplex. InfectedFeldmannia plants show severe somatic malformations. However, no functional virus particles are formed. SuchFeldmannia plants recover to resume a normal, symptom-free appearance. This result raises the possibility of intergeneric gene transfer in the natural habitat.     

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.     

Early detection of cell metabolism with a silicon microsensor

A silicon microsensor (ISFET — Ion Sensitive Field Effect Transistor) has been used to detect the metabolism of a cell population cultured on a coverslip and positioned close to the sensor surface. The system output is analyzed as a function of cell density.     

Locomotor Activity in the Freshwater Crab Potamon fluviatile: The Analysis of Temporal Patterns by Radio-telemetry

Locomotor activity in a field population of the freshwater crab, Potamon fluviatile, was studied during the breeding season by means of radio-telemetry and by direct counts of active animals along a transect of the stream. The basic pattern of crabs' locomotor activity can be described as a sequence of short distance movements around the shelters (foraging movements), followed by rarer long distance displacements (wandering movements). Whilst direct counting showed that the crabs exhibit a broadly nocturnal rhythm in foraging movements, no daily periodicity in wandering activity was revealed by telemetry. There is sexual difference in these latter excursions: females move farther along the stream and into the surrounding terrestrial habitat than do males. This behaviour is in contrast to observations made during the non-breeding season and is probably related to the stage of female reproduction.     

An elongation factor Tu (EF-Tu) resistant to the EF-Tu inhibitor GE2270 in the producing organism Planobispora rosea

SecA protein, the ATPase promoting translocation of proteins across the Escherichia coli inner membrane, contains two ATP-binding domains that differ greatly in their affinity for bound nucleotide. In order to define more precisely the location of the high-affinity nucleotide-binding site, oligonucleotide-directed mutagenesis was used to introduce cysteine residues into the SecA sequence, and a cysteine-specific cleavage reagent was employed to generate defined peptides of SecA protein after photocross-linking with [α-³²P]-ATP. This analysis revealed that the nucleotide was cross-linked between amino acid residues 75 and 97 of SecA protein. The biochemical function of the high affinity ATP-binding domain was explored by subcellular fractionation studies which demonstrated that SecA proteins defective in this region were found almost exclusively in their integral membrane form, while SecA proteins with defects in the low-affinity ATP-domain showed a normal distribution of cytosolic, peripheral and integral membrane forms. Interestingly, the SecA51(Ts) protein that has a Leu to Pro substitution at amino acid residue 43 bound ATP with high affinity, but its fractionation pattern and translocation ATPase activity were similar to those of proteins with defects in the high-affinity ATP-binding site. These results delimit more precisely the high-affinity ATP-binding domain of SecA, indicate the importance of the early amino-terminal region of SecA protein in the functioning of this domain, and demonstrate the role of this domain in regulating penetration of SecA protein into the inner membrane. Our results lead to a simple model for the regulation of a cycle of SecA insertion into, and de-insertion from, the inner membrane by the activity of the high-affinity ATP-binding domain.     

Intracellular Ionic Variations in the Apoptotic Death of L Cells by Inhibitors of Cell Cycle Progression

Treatment with VP-16 (1-50 μM) or excess thymidine (5 mM) caused a block of L cells at different steps in their progression through the replicative cycle. The arrest was followed by an asynchronous process of cell death that conformed to criteria for apoptosis. Careful monitoring of this process in the whole cell population by flow cytometry showed a virtual absence of necrosis, an increase in side light scattering, followed by the occurrence of a population with subdiploid DNA fluorescence as well as reduced forward and side light seattering. The development of apoptosis required sufficient time and adequate ion gradients in the cells. By the combined use of flow cytometry and fluorescence microscopy data were obtained suggesting that (i) intracellular free Ca²⁺ and pH and/or their drug-induced alterations had to be adequately controlled for the apoptotic process to evolve; (ii) mitochondria were compromised earlier than the plasma membrane or lysosomes; and (iii) K⁺ extrusion possibly played a role in the final loss of cell volume. Interfering with the control of ion gradients and/or their changes in drug-treated cells resulted in cell death by necrosis.     

Leaf characteristics and optical properties of different woody species

 Light partition has been examined and evaluated on five woody species (Olea europaea, Ficus carica, Pittosporum tobira, Hedera helix maculata, Persica vulgaris) in relation to their leaf morpho-histological characteristics, water and chlorophyll contents. Leaf parameters and optical properties (reflectance, transmittance, absorbance) in PAR, FR and NIR wavebands (400–1100 nm) were preliminarily submitted to a canonical correlation analysis where lamina thickness and water content showed a leading role in determining all the optical properties, while chlorophyll, influential in the PAR region, was remarkably effective only in an extreme pigment situation when green and albino patches of ivy leaves were compared. Transmittance appeared inversely related to lamina thickness in accordance with the Lambert Beer law. Significant correlations were found also between mesophyll water content and both transmittance (positive) and reflectance (negative). Olive leaves showed peculiar optical patterns because of the dense and continuous trichome layer on their abaxial surface. Received: 3 January 1997 / Accepted: 5 May 1997