Molecular characterization of the mouse agouti locus.

The agouti (a) locus acts within the microenvironment of the hair follicle to regulate coat color pigmentation in the mouse. We have characterized a gene encoding a novel 131 amino acid protein that we propose is the one gene associated with the agouti locus. This gene is normally expressed in a manner consistent with a locus function, and, more importantly, its structure and expression are affected by a number of representative alleles in the agouti dominance hierarchy. In addition, we found that the pleiotropic effects associated with the lethal yellow (Ay) mutation, which include pronounced obesity, diabetes, and the development of neoplasms, are accompanied by deregulated overexpression of the agouti gene in numerous tissues of the adult animal.     

High-performance liquid chromatographic method for the determination of di(2-ethylhexyl) phthalate in total parenteral nutrition and in plasma

A simple, rapid and sensitive reversed-phase high-performance liquid chromatographic method with UV detection was developed for the quantification of di(2-ethylhexyl) phthalate (DEHP) in parenteral nutrition admixtures containing fat emulsion and in plasma samples of children daily treated by total parenteral nutrition. The analyte and the internal standard, di-n-heptyl phthalate, were extracted twice using hexane and the organic layer separated and dried under nitrogen. The residues were reconstituted with acetonitrile and 20 μl was injected into a Waters Spherisorb C₁₈ column, the UV detector was set at 202 nm. The mobile phase was acetonitrile–aqueous buffer (triethylamine 0.08% adjusted to pH 2.8 with 1 M phosphoric acid) mixture (88:12, v/v) and it was pumped at 1 ml/min. Average recoveries were 97% or greater. This method was successfully used to investigate the amounts of DEHP which can leach from bags and tubing into fat emulsion and which could contaminate children under long-term parenteral nutrition. On the other hand, the circulating DEHP concentrations were estimated in four children under regular long-term parenteral nutrition.     

Pathways of carbon cycling in marine surface waters: the fate of small-sized phytoplankton in the Northeast Water Polynya

The fate of small-sized phytoplankton (<5 µm) and pathwaysof carbon cycling in surface waters, i.e. recycling within orexport out of the mixed layer, were investigated in the NortheastWater (NEW) Polynya (77–81°N) from 23 May to 22 July1993. The sampling covered a wide range of ice, hydrographicand nutrient conditions. Chlorophyll a concentrations, phytoplanktonproduction rates and zooplankton abundances were determinedin the field, and potential rates of grazing by protozoa, copepodsand appendicularians were calculated from abundances, usingassumptions from the literature. To our knowledge, this is thefirst published attempt to assess concurrently the grazing ofthese three plankton groups in the Arctic. The production rateof small-sized phytoplankton was significantly higher in ice-freecompared with ice-covered areas, but the biomasses of small-sizedphytoplankton and zooplankton were not. Potential recycling,downward export and horizontal advection of phytoplankton werecalculated by resolving carbon budgets for the mixed layer.A large fraction of the small-sized phytoplankton produced insidethe polynya was advected horizontally to the ice-covered partof the NEW, where these algae were necessary to sustain theheterotrophic community. We conclude that the fate of small-sizedphytoplankton production was mostly recycling (>70%). Downwardexport would have occurred infrequently, as a result of intensegrazing by appendicularians. Size-differential pathways of carboncycling in planktonic food webs are discussed.     

Over-production, purification and properties of the uridine-diphosphate-N-acetylmuramoyl-L-alanyl-D-glutamate: meso-2,6-diaminopimelate ligase from Escherichia coli

The UDP-N-acetylmuramoyl-L-alanyl-D-glutamate:meso-2,6-diaminopimelate ligase was over-produced and purified from two plasmid-harbouring strains of Escherichia coli. The first strain, E. coli JM83(pHE5), gave a 15-fold over-production relative to parental strain. The enzyme could be partially purified (8.8-fold) by ion-exchange chromatography. With the second strain, E. coli JM83(pMLD25), a very strong over-production was obtained, since the enzyme represented about 20% of the cytoplasmic proteins. Purification yielded 77% protein homogeneity. However, the enzymatic activity, which was very unstable, was lost during the purification procedure. Several properties of the enzyme were studied. The enzyme gave maximal activity around pH 8. The isoelectric point was 5.2. The activity was increased by potassium phosphate. Reverse and exchange reactions could be catalysed. The N-terminal sequence of the protein was determined and correlated with the nucleotide sequence of the murE gene. The actual initiation codon was assigned.     

Early Deficits in Glycolysis Are Specific to Striatal Neurons from a Rat Model of Huntington Disease

In Huntington disease (HD), there is increasing evidence for a link between mutant huntingtin expression, mitochondrial dysfunction, energetic deficits and neurodegeneration but the precise nature, causes and order of these events remain to be determined. In this work, our objective was to evaluate mitochondrial respiratory function in intact, non-permeabilized, neurons derived from a transgenic rat model for HD compared to their wild type littermates by measuring oxygen consumption rates and extracellular acidification rates. Although HD striatal neurons had similar respiratory capacity as those from their wild-type littermates when they were incubated in rich medium containing a supra-physiological glucose concentration (25 mM), pyruvate and amino acids, respiratory defects emerged when cells were incubated in media containing only a physiological cerebral level of glucose (2.5 mM). According to the concept that glucose is not the sole substrate used by the brain for neuronal energy production, we provide evidence that primary neurons can use lactate as well as pyruvate to fuel the mitochondrial respiratory chain. In contrast to glucose, we found no major deficits in HD striatal neurons’ capacity to use pyruvate as a respiratory substrate compared to wild type littermates. Additionally, we used extracellular acidification rates to confirm a reduction in anaerobic glycolysis in the same cells. Interestingly, the metabolic disturbances observed in striatal neurons were not seen in primary cortical neurons, a brain region affected in later stages of HD. In conclusion, our results argue for a dysfunction in glycolysis, which might precede any defects in the respiratory chain itself, and these are early events in the onset of disease.     

Multiparasitism of stink bug eggs: competitive interactions between Ooencyrtus pityocampae and Trissolcus agriope

Females of Trissolcus agriope (Platygastridae) avoid host (Brachynema signatum) eggs parasitized by conspecifics or Ooencyrtus pityocampae (Encyrtidae), but females of the latter species will superparasitize and multiparasitize, although they mostly attack unparasitized eggs. Females of T. agriope were more efficient in the laboratory and parasitized more hosts in 24 and 48 h. In multiparasitized hosts, O. pityocampae was a superior larval competitor and could complete also development as a facultative hyperparasitoid. When females of both species foraged together, T. agriope parasitized significantly more than O. pityocampae, about 50 % in both cases, although O. pityocampae almost doubled its parasitism rate as the exposure period was lengthened from 24 to 48 h. When O. pityocampae followed T. agriope in sequential foraging bouts, the former species successfully parasitized more hosts than the latter. The advisability of co-releases of both species to improve biological control of first generation B. signatum in Iranian pistachio orchards is discussed.     

Protection of Recombinant Mammalian Antibodies from Development-Dependent Proteolysis in Leaves of Nicotiana benthamiana

The expression of clinically useful proteins in plants has been bolstered by the development of high-yielding systems for transient protein expression using agroinfiltration. There is a need now to know more about how host plant development and metabolism influence the quantity and quality of recombinant proteins. Endogenous proteolysis is a key determinant of the stability and yield of recombinant proteins in plants. Here we characterised cysteine (C1A) and aspartate (A1) protease profiles in leaves of the widely used expression host Nicotiana benthamiana, in relation with the production of a murine IgG, C5-1, targeted to the cell secretory pathway. Agroinfiltration significantly altered the distribution of C1A and A1 proteases along the leaf age gradient, with a correlation between leaf age and the level of proteolysis in whole-cell and apoplast protein extracts. The co-expression of tomato cystatin SlCYS8, an inhibitor of C1A proteases, alongside C5-1 increased antibody yield by nearly 40% after the usual 6-days incubation period, up to ∼3 mg per plant. No positive effect of SlCYS8 was observed in oldest leaves, in line with an increased level of C1A protease activity and a very low expression rate of the inhibitor. By contrast, C5-1 yield was greater by an additional 40% following 8- to 10-days incubations in younger leaves, where high SlCYS8 expression was maintained. These findings confirm that the co-expression of recombinant protease inhibitors is a promising strategy for increasing recombinant protein yields in plants, but that further opportunity exists to improve this approach by addressing the influence of leaf age and proteases of other classes.     

Autophagy inhibition radiosensitizes in vitro,yet reduces radioresponses in vivo due to deficient immunogenic signalling

Clinical oncology heavily relies on the use of radiotherapy, which often leads to merely transient responses that are followed by local or distant relapse. The molecular mechanisms explaining radioresistance are largely elusive. Here, we identified a dual role of autophagy in the response of cancer cells to ionizing radiation. On one hand, we observed that the depletion of essential autophagy-relevant gene products, such as ATG5 and Beclin 1, increased the sensitivity of human or mouse cancer cell lines to irradiation, both in vitro (where autophagy inhibition increased radiation-induced cell death and decreased clonogenic survival) and in vivo, after transplantation of the cell lines into immunodeficient mice (where autophagy inhibition potentiated the tumour growth-inhibitory effect of radiotherapy). On the other hand, when tumour proficient or deficient for autophagy were implanted in immunocompetent mice, it turned out that defective autophagy reduced the efficacy of radiotherapy. Indeed, radiotherapy elicited an anti-cancer immune response that was dependent on autophagy-induced ATP release from stressed or dying tumour cells and was characterized by dense lymphocyte infiltration of the tumour bed. Intratumoural injection of an ecto-ATPase inhibitor restored the immune infiltration of autophagy-deficient tumours post radiotherapy and improved the growth-inhibitory effect of ionizing irradiation. Altogether, our results reveal that beyond its cytoprotective function, autophagy confers immunogenic properties to tumours, hence amplifying the efficacy of radiotherapy in an immunocompetent context. This has far-reaching implications for the development of pharmacological radiosensitizers.     

Contingent vs. noncontingent EMG feedback and hand temperature in relation to anxiety and locus of control

This study was designed to measure the effects of contingent and noncontingent EMG feedback on hand temperature, anxiety, and locus of control. Two groups of six subjects each were selected on the basis of high test-anxiety scores. The groups participated in a reverse design study in which Group 1 received five sessions of contingent EMG ffedback followed by five sessions of noncontingent feedback. Group 2 received noncontingent feedback followed by contingent feedback. Results indicate a significant order of treatment effect. Subjects who received contingent feedback first produced lower EMG readings, lower test-anxiety scores, and higher hand temperatures during noncontingent feedback sessions. Receiving noncontingent feedback first may actually have interfered with utilizing contingent feedback.