Analysis of an activatable promoter: sequences in the simian virus 40 late promoter required for T-antigen-mediated trans activation.

The late promoter of simian virus 40 (SV40) is activated in trans by the viral early gene product, T antigen. We inserted the wild-type late-promoter region, and deletion mutants of it, into chloramphenicol acetyltransferase transient expression vectors to identify promoter sequences which are active in the presence of T antigen. We defined two promoter activities. One activity was mediated by a promoter element within simian virus 40 nucleotides 200 to 270. The activity of this element was detectable only in the presence of an intact, functioning origin of replication and accounted for 25 to 35% of the wild-type late-promoter activity in the presence of T antigen. The other activity was mediated by an element located within a 33-base-pair sequence (simian virus nucleotides 168 to 200) which spans the junction of the 72-base-pair repeats. This element functioned in the absence of both the origin of replication and the T-antigen-binding sites and appeared to be responsible for trans-activated gene expression. When inserted into an essentially promoterless plasmid, the 33-base-pair element functioned in an orientation-dependent manner. Under wild-type conditions in the presence of T antigen, the activity of this element accounted for 65 to 75% of the late-promoter activity. The roles of the 33-base-pair element and T antigen in trans-activation are discussed.     

Un biofacies à Trilobites Homalonotidae dans I'Ordovicien de la marge nord-gondwanienne: implications paléobiologiques et paléogéographiques

During the Llandeilo, the sandy lithological units which develop locally in the Armorican Massif, Spain and Morocco, are all characterized by similar trilobite associations in which endobenthic homalonotids. well adapted to shallow water and sandy substrate, are predominant. During the Llandeilo, on the northern Gondwanan margin, the wide geographical distribution of most of the trilobites, controlled by environmental factors, shows that the existence of a 'Proto-Tethys' ocean is unlikely. □ North Gondwana, Trilobita, Homalonotidae, Ordovician, Llandeilo. palaeoenvironment.     

Characterisation of the first enzymes committed to lysine biosynthesis in Arabidopsis thaliana

In plants, the lysine biosynthetic pathway is an attractive target for both the development of herbicides and increasing the nutritional value of crops given that lysine is a limiting amino acid in cereals. Dihydrodipicolinate synthase (DHDPS) and dihydrodipicolinate reductase (DHDPR) catalyse the first two committed steps of lysine biosynthesis. Here, we carry out for the first time a comprehensive characterisation of the structure and activity of both DHDPS and DHDPR from Arabidopsis thaliana. The A. thaliana DHDPS enzyme (At-DHDPS2) has similar activity to the bacterial form of the enzyme, but is more strongly allosterically inhibited by (S)-lysine. Structural studies of At-DHDPS2 show (S)-lysine bound at a cleft between two monomers, highlighting the allosteric site; however, unlike previous studies, binding is not accompanied by conformational changes, suggesting that binding may cause changes in protein dynamics rather than large conformation changes. DHDPR from A. thaliana (At-DHDPR2) has similar specificity for both NADH and NADPH during catalysis, and has tighter binding of substrate than has previously been reported. While all known bacterial DHDPR enzymes have a tetrameric structure, analytical ultracentrifugation, and scattering data unequivocally show that At-DHDPR2 exists as a dimer in solution. The exact arrangement of the dimeric protein is as yet unknown, but ab initio modelling of x-ray scattering data is consistent with an elongated structure in solution, which does not correspond to any of the possible dimeric pairings observed in the X-ray crystal structure of DHDPR from other organisms. This increased knowledge of the structure and function of plant lysine biosynthetic enzymes will aid future work aimed at improving primary production.     

Amyloid beta-peptide effects on synaptosomes from apolipoprotein E-deficient mice

Apolipoprotein E (apoE) is present in the brain and may contribute to neurophysiologic or neuropathologic events, depending on environmental and genetic influences. Recent studies indicate a role for apoE in synaptic plasticity and maintenance of synaptic membrane symmetry, suggesting that apoE may be involved in regulating synaptic homeostasis. In the present study, cerebrocortical synaptosomes were prepared from transgenic mice lacking apoE (apoE KO) to analyze the possible contribution of apoE toward maintaining homeostasis in synaptosomes. Synaptosomal preparations from apoE KO and wild-type mice exhibited similar basal levels of reactive oxygen species, mitochondrial function, and caspase activity; however, following application of amyloid beta-peptide [Abeta(1-40)], apoE KO synaptosomes displayed increased levels of oxidative stress, mitochondrial dysfunction, and caspase activation compared with synaptosomes from wild-type mice. Synaptosomal membranes from apoE KO mice were more fluid than wild-type synaptosomes and contained higher levels of thiobarbituric acid-reactive substances, consistent with elevated levels of lipid peroxidation occurring in the synapses of apoE KO mice. Together, these data are consistent with a role for apoE in maintaining homeostasis by attenuating oxidative stress, caspase activation, and mitochondrial homeostasis in synapses.     

Biofilm stratification during simultaneous nitrification and denitrification (SND) at a biocathode

The aeration of the cathode compartment of bioelectrochemical systems (BESs) was recently shown to promote simultaneous nitrification and denitrification (SND). This study investigates the cathodic metabolism under different operating conditions as well as the structural organization of the cathodic biofilm during SND. Results show that a maximal nitrogen removal efficiency of 86.9 ± 0.5%, and a removal rate of 3.39 ± 0.08 mg N L⁻¹ h⁻¹ could be achieved at a dissolved oxygen (DO) level of 5.73 ± 0.03 mg L⁻¹ in the catholyte. The DO levels used in this study are higher than the thresholds previously reported as detrimental for denitrification. Analysis of the cathodic half-cell potential during batch tests suggested the existence of an oxygen gradient within the biofilm while performing SND. FISH analysis corroborated this finding revealing that the structure of the biofilm included an outer layer occupied by putative nitrifying organisms, and an inner layer where putative denitrifying organisms were most dominant. To our best knowledge this is the first time that nitrifying and denitrifying microorganisms are simultaneously observed in a cathodic biofilm.     

Stepwise mass extinctions and impact events: Late Eocene to early Oligocene

Species ranges and relative abundances of dominant planktonic foraminifers of eight late Eocene to early Oligocene deep-sea sections are discussed to determine the nature and magnitude of extinctions and to investigate a possible cause-effect relationship between impact events and mass extinctions.Late Eocene extinctions are neither catastrophic nor mass extinctions, but occur stepwise over a period of about 1–2 million years. Four stepwise extinctions are identified at the middle/late Eocene boundary, the upperGlobigerapsis semiinvoluta zone, theG. semiinvoluta/Globorotalia cerroazulensis zone boundary and at the Eocene/Oligocene boundary. Each stepwise extinction event represents a time of accelerated faunal turnover characterized by generally less than 15% species extinct and in itself is not a significant extinction event. Relative species abundance changes at each stepwise extinction event, however, indicate a turnover involving > 60% of the population implying major environmental changes.There microtektite horizons are present in late Eocene sediments; one in the upperG. semiinvoluta zone (38.2 Ma) and two closely spaced layers only a few thousand years apart in the lower part of theGloborotalia cerroazulensis zone (37.2 Ma). Each of the three impact events appears to have had some effect on microplankton communities. However, the overriding factor that led to the stepwise mass extinctions may have been the result of multiple causes as there is no evidence of impacts associated with the step preceding, or the step following the deposition of the presently known microtektite horizons.     

Immunological evidence of a common structure between Paramecium surface antigens and Trypanosoma variant surface glycoproteins

The surface antigens (SAgs) of Paramecium and the variant surface antigens (VSGs) of Trypanosoma can be purified in two distinct molecular forms: a soluble form (solubilized in dilute ethanolic solution in the case of Paramecium, or in water for Trypanosoma) and a membranal form, amphiphile (solubilized in SDS). In trypanosomes, the enzymatic conversion of the membrane form into the soluble form is accompanied by the unmasking of a particular immunological determinant, called cross-reacting determinant (CRD), which is located in the COOH-terminal phospho-ethanolamine glycopeptide. We demonstrate immunological homologies between Paramecium SAgs and Trypanosoma VSGs. A determinant corresponding to the CRD of VSGs is borne by the ethanol-soluble form of the SAgs and by two cross-reacting light chains also present in ethanolic cellular extracts (together with the soluble form), and not by the membranal form of SAgs. Furthermore, we show that the membranal form of Paramecium SAgs can be converted into soluble form and that this enzymatic conversion also yields cross-reacting light chains. We also demonstrate that the membranal form is the physiological form in paramecia stably expressing a given SAg.     

Functional characterization of an insulin-responsive glucose transporter (GLUT4) from fish adipose tissue

Glucose transport across the plasma membrane is mediated by a family of glucose transporter proteins (GLUTs), several of which have been identified in mammalian, avian, and, more recently, in fish species. Here, we report on the cloning of a salmon GLUT from adipose tissue with a high sequence homology to mammalian GLUT4 that has been named okGLUT4. Kinetic analysis of glucose transport following expression in Xenopus laevis oocytes demonstrated a 7.6 +/- 1.4 mM K(m) for 2-deoxyglucose (2-DG) transport measured under zero-trans conditions and 14.4 +/- 1.5 mM by equilibrium exchange of 3-O-methylglucose. Transport of 2-DG by okGLUT4-injected oocytes was stereospecific and was competed by D-glucose, D-mannose, and, to a lesser extent, D-galactose and D-fructose. In addition, 2-DG uptake was inhibited by cytochalasin B and ethylidene glucose. Moreover, insulin stimulated glucose uptake in Xenopus oocytes expressing okGLUT4 and in isolated trout adipocytes, which contain the native form of okGLUT4. Despite differences in protein motifs important for insulin-stimulated translocation of mammalian GLUT4, okGLUT4 was able to translocate to the plasma membrane from intracellular localization sites in response to insulin when expressed in 3T3-L1 adipocytes. These data demonstrate that okGLUT4 is a structural and functional fish homolog of mammalian GLUT4 but with a lower affinity for glucose, which could in part explain the lower ability of fish to clear a glucose load.     

Proteome analysis of human colon cancer by two-dimensional difference gel electrophoresis and mass spectrometry

Two-dimensional difference gel electrophoresis (2-D DIGE) coupled with mass spectrometry (MS) was used to investigate tumor-specific changes in the proteome of human colorectal cancers and adjacent normal mucosa. For each of six patients with different stages of colon cancer, Cy5-labeled proteins isolated from tumor tissue were combined with Cy3-labeled proteins isolated from neighboring normal mucosa and separated on the same 2-D gel along with a Cy2-labeled mixture of all 12 normal/tumor samples as an internal standard. Over 1500 protein spot-features were analyzed in each paired normal/tumor comparison, and using DIGE technology with the mixed-sample internal standard, statistically significant quantitative comparisons of each protein abundance change could be made across multiple samples simultaneously without interference due to gel-to-gel variation. Matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) and tandem (TOF/TOF) MS provided sensitive and accurate mass spectral data for database interrogation, resulting in the identification of 52 unique proteins (including redundancies due to proteolysis and post-translationally modified isoforms) that were changing in abundance across the cohort. Without the benefit of the Cy2-labeled 12 sample mixture internal standard, 42 of these proteins would have been overlooked due to the large degree of variation inherent between normal and tumor samples.