The activation state of nitrate reductase is not always correlated with total nitrate reductase activity in leaves

The relation between nitrate reductase (NR; EC 1.6.6.1) activity, activation state and NR protein in leaves of barley (Hordeum vulgare L.) seedlings was investigated. Maximum NR activity (NRA_max) and NR protein content (Western blotting) were modified by growing plants hydroponically at low (0.3 mM) or high (10 mM) nitrate supply. In addition, plants were kept under short-day (8 h light/16 h dark) or long-day (16 h light/8 h dark) conditions in order to manipulate the concentration of nitrate stored in the leaves during the dark phase, and the concentrations of sugars and amino acids accumulated during the light phase, which are potential signalling compounds. Plants were also grown under phosphate deficiency in order to modify their glucose-6-phosphate content. In high-nitrate/long-day conditions, NRA_max and NR protein were almost constant during the whole light period. Low-nitrate/long-day plants had only about 30% of the NRA_max and NR protein of high-nitrate plants. In low-nitrate/long-day plants, NRA_max and NR protein decreased strongly during the second half of the light phase. The decrease was preceded by a strong decrease in the leaf nitrate content. Short daylength generally led to higher nitrate concentrations in leaves. Under short-day/low-nitrate conditions, NRA_max was slightly higher than under long-day conditions and remained almost constant during the day. This correlated with maintenance of higher nitrate concentrations during the short light period. The NR activation state in the light was very similar in high-nitrate and low-nitrate plants, but dark inactivation was twice as high in the high-nitrate plants. Thus, the low NRA_max in low-nitrate/long-day plants was slightly compensated by a higher activation state of NR. Such a partial compensation of a low NR_max by a higher dark activation state was not observed with phosphate-depleted plants. Total leaf concentrations of sugars, of glutamine and glutamate and of glucose-6-phosphate did not correlate with the NR activation state nor with NRA_max. Received: 24 March 1999 / Accepted: 31 May 1999     

Modulation of social interactions by immune stimulation in honey bee, <Emphasis Type="Italic">Apis mellifera</Emphasis>, workers

Background  

Immune response pathways have been relatively well-conserved across animal species, with similar systems in both mammals and invertebrates. Interestingly, honey bees have substantially reduced numbers of genes associated with immune function compared with solitary insect species. However, social species such as honey bees provide an excellent environment for pathogen or parasite transmission with controlled environmental conditions in the hive, high population densities, and frequent interactions. This suggests that honey bees may have developed complementary mechanisms, such as behavioral modifications, to deal with disease.     

Association between pygmy right whales (Caperea marginata) and areas of high marine productivity off Australia and New Zealand

Abstract

Opportunistic sightings and strandings of Caperea marginata (n=196) from the vicinity of Australia and New Zealand (1884 to early 2007) were used to relate geographic and temporal patterns to oceanographic and broad-scale climatic variability. Records were not uniformly distributed along the coast and more (69%) were from Australia than New Zealand. Seven coastal whale ‘hotspots’ were identified which accounted for 61% of records with locality data. Half of the hotspot records were from southeast (37) and northwest (20) Tasmania—others each had 9–15 events. Upwelling and/or high zooplankton abundance has been documented near all whale hotspots. Records of C. marginata occurred in all months, with 75% in spring and summer. Inter-annual variability showed broad agreement between increased whale records (usually in spring/summer) and strongly positive ‘Niño 3.4’ during 1980–1995 but not thereafter. Coastal upwelling and productivity increase during climatic phenomena such as El Niño and are likely to be quickly beneficial to plankton-feeding whales such as C. marginata.     

A focus on fixation

Three fixation issues related to immunostaining are discussed here: 1) Generally, a tissue block is fixed, then embedded and sectioned (pre-fixation). The type of fixative applied, crosslinking or coagulating, has an impact on selecting an epitope retrieval method. Individual antigens have a fixation–retrieval characteristic. 2) A long fixation time, especially with crosslinking fixatives, may compromise the result of immunostaining. This negative effect varies among different antigens and can be partially restored by applying a more sensitive/efficient detection system such as tyramide amplification. 3) Sections cut from a fresh frozen tissue block usually are acetone fixed (post-fixation). This was accepted as the “gold standard” for a long time. Post-fixation, however, may have serious consequences for preservation of small peptides leaking from the cut open cells, whereas this is not the case with pre-fixed intact cells. Consequently, the concept of an acetone post-fixed cryostat tissue section as “gold standard” no longer exists and a more appropriate use of the terms immunohistochemistry and immunocytochemistry therefore seems justified. For many antibodies, it is not known whether a formalin fixed, paraffin embedded tissue specimen is appropriate. Suggestions are made for creating a positive control cell block for testing such antibodies.     

Characterization of an endogenous substrate of the insulin receptor in cultured cells

Using antiphosphotyrosine antibodies, we have characterized the tyrosine phosphorylation of an endogenous substrate of the insulin receptor in Fao hepatoma cells and in Chinese hamster ovary cells transfected with a eukaryotic expression vector containing the human insulin receptor cDNA. In Fao cells, besides the beta-subunit of the insulin receptor, a protein with a molecular mass between 170 and 210 kDa designated pp185, undergoes tyrosine phosphorylation immediately after insulin stimulation reaching a maximum level within 30 s. After 4 h of continuous insulin stimulation, the labeling of pp185 decreased to less than half of its original intensity, whereas the insulin receptor was unchanged. After 24 h of insulin stimulation, the phosphotyrosine-containing insulin receptor decreased by 75% owing to down-regulation, whereas the pp185 was completely undetectable. By several biochemical and physiological criteria, the pp185 is distinct from the insulin receptor. The pp185 and the beta-subunit of the insulin receptor were strongly labeled with [32P]orthophosphate, but in contrast to the insulin receptor, the pp185 was not labeled by cross-linking with 125I-insulin or surface 125I iodination. Unlike the insulin receptor, the pp185 was extracted from Fao cells without detergent, and tryptic phosphopeptide mapping of the pp185 and the insulin receptor yielded distinct patterns. Thus, the pp185 is not located at the external face of the plasma membrane and does not bind insulin. Treatment of Fao cells with the phorbol ester, phorbol 12-myristate 13-acetate, stimulated the phosphorylation of two proteins with molecular weights of 170 and 210 kDa which were immunoprecipitated with the anti-phosphotyrosine antibody. Subsequent insulin stimulation increased the phosphorylation of the 210 kDa protein, but the pp185 was not detected. Increasing the concentration of the human insulin receptor in the Chinese hamster ovary cells by transfection with a plasmid containing the human insulin receptor cDNA caused a higher level of tyrosine phosphorylation of the beta-subunit and the pp185. These data support the notion that the insulin signal may be transmitted to a cellular substrate (pp185) which may initiate insulin action at intracellular sites.     

Spontaneous lipid vesicle fusion with electropermeabilized cells

Fusion is obtained between electropermeabilized mammalian cells and intact large unilamellar lipid vesicles. This is monitored by a fluorescence assay. Prepulse contact is obtained by Ca2+ when negatively charged lipids are present in the liposomes. The mixing of the liposome content in the cell cytoplasm is observed under conditions preserving cell viability. Electric conditions are such that free liposomes are not affected by the external field. Therefore destabilization of only one of the two membranes of the partners is sufficient for fusion. The comparison between the efficiency of dye delivery for different liposome preparations (multilamellar vesicles, large unilamellar vesicles, small unilamellar vesicles) is indicative that more metastable liposomes are more fusable with electropulsated cells. This observation is discussed within the framework of the recent hypothesis that occurrence of a contact induced electrostatic destabilization of the plasma membrane is a key step in the exocytosis process.     

The ancestor of the <Emphasis Type="Italic">Paulinella</Emphasis> chromatophore obtained a carboxysomal operon by horizontal gene transfer from a <Emphasis Type="Italic">Nitrococcus</Emphasis>-like γ-proteobacterium

Background  

Paulinella chromatophora is a freshwater filose amoeba with photosynthetic endosymbionts (chromatophores) of cyanobacterial origin that are closely related to free-living Prochlorococcus and Synechococcus species (PS-clade). Members of the PS-clade of cyanobacteria contain a proteobacterial form 1A RubisCO (ribulose-1,5-bisphosphate carboxylase/oxygenase) that was acquired by horizontal gene transfer (HGT) of a carboxysomal operon. In rDNA-phylogenies, the Paulinella chromatophore diverged basal to the PS-clade, raising the question whether the HGT occurred before or after the split of the chromatophore ancestor.     

A study on the minimum number of loci required for genetic evaluation using a finite locus model

For a finite locus model, Markov chain Monte Carlo (MCMC) methods can be used to estimate the conditional mean of genotypic values given phenotypes, which is also known as the best predictor (BP). When computationally feasible, this type of genetic prediction provides an elegant solution to the problem of genetic evaluation under non-additive inheritance, especially for crossbred data. Successful application of MCMC methods for genetic evaluation using finite locus models depends, among other factors, on the number of loci assumed in the model. The effect of the assumed number of loci on evaluations obtained by BP was investigated using data simulated with about 100 loci. For several small pedigrees, genetic evaluations obtained by best linear prediction (BLP) were compared to genetic evaluations obtained by BP. For BLP evaluation, used here as the standard of comparison, only the first and second moments of the joint distribution of the genotypic and phenotypic values must be known. These moments were calculated from the gene frequencies and genotypic effects used in the simulation model. BP evaluation requires the complete distribution to be known. For each model used for BP evaluation, the gene frequencies and genotypic effects, which completely specify the required distribution, were derived such that the genotypic mean, the additive variance, and the dominance variance were the same as in the simulation model. For lowly heritable traits, evaluations obtained by BP under models with up to three loci closely matched the evaluations obtained by BLP for both purebred and crossbred data. For highly heritable traits, models with up to six loci were needed to match the evaluations obtained by BLP.