A gibberellin-regulated gene from wheat with sequence homology to cathepsin B of mammalian cells

A previous report described several cDNAs corresponding to mRNAs which accumulated in wheat aleurone layers treated with gibberellic acid (GA) (Baulcombe and Buffard, 1983). The protein sequence deduced from one of these clones (2529) has extensive similarity to the thiol protease, cathepsin B from mammalian cells. Southern analysis of wheat DNA has shown that the 2529 mRNA is encoded by a small family of genes carried on the group 4 chromosome. The nucleotide sequence of a member of the gene family expressed at a low level in aleurone layers and the use of a primer extension assay to identify a clone of a member of the gene family producing an abundant mRNA are reported. The 2529 mRNA accumulates in the scutellum and the aleurone layer of germinating grains where its expression is regulated by GA. In the scutellum the expression was restricted to the parenchyma, suggesting that the 2529 product may have a role other than for mobilization of the endosperm.     

Nitrate permease from Azotobacter chroococcum

Active transport systems in bacteria can be divided into two groups: those that are osmotic shock-resistant with one single membrane protein, and those that are shock-sensitive and have a membrane-bound protein complex plus a soluble periplasmic protein. Whether the bacterial assimilatory nitrate transport falls into the one or the other of these two groups has not been studied before. We report that nitrate uptake by the strictly aerobic, N₂-fixing heterotrophic bacterium Azotobacter chroococcum is sensitive to osmotic shock. The polypeptide composition of cytoplasmic membranes changes in response to the nitrogen source available to the cells. Incorporation of [³⁵S]-methionine into proteins as well as use of the A. chroococcum TRI mutant, which is defective in nitrate transport, and the A. choococcum MCD1 strain, a mutant unable to use nitrate as a nitrogen source, suggest that nitrate transport into A. chroococcum cells is mediated by a multicomponent system tightly bound to the cytoplasmic membrane.     

Dexamethasone Rescues Neurovascular Unit Integrity from Cell Damage Caused by Systemic Administration of Shiga Toxin 2 and Lipopolysaccharide in Mice Motor Cortex

Shiga toxin 2 (Stx2)-producing Escherichia coli (STEC) causes hemorrhagic colitis and hemolytic uremic syndrome (HUS) that can lead to fatal encephalopathies. Neurological abnormalities may occur before or after the onset of systemic pathological symptoms and motor disorders are frequently observed in affected patients and in studies with animal models. As Stx2 succeeds in crossing the blood-brain barrier (BBB) and invading the brain parenchyma, it is highly probable that the observed neurological alterations are based on the possibility that the toxin may trigger the impairment of the neurovascular unit and/or cell damage in the parenchyma. Also, lipopolysaccharide (LPS) produced and secreted by enterohemorrhagic Escherichia coli (EHEC) may aggravate the deleterious effects of Stx2 in the brain. Therefore, this study aimed to determine (i) whether Stx2 affects the neurovascular unit and parenchymal cells, (ii) whether the contribution of LPS aggravates these effects, and (iii) whether an inflammatory event underlies the pathophysiological mechanisms that lead to the observed injury. The administration of a sub-lethal dose of Stx2 was employed to study in detail the motor cortex obtained from a translational murine model of encephalopathy. In the present paper we report that Stx2 damaged microvasculature, caused astrocyte reaction and neuronal degeneration, and that this was aggravated by LPS. Dexamethasone, an anti-inflammatory, reversed the pathologic effects and proved to be an important drug in the treatment of acute encephalopathies.     

The nucellus degenerates by a process of programmed cell death during the early stages of wheat grain development

The nucellus, which is the maternal tissue of the wheat grain, degenerates during the early stages of development. We have investigated whether or not this degenerative process may be considered as programmed cell death (PCD). The analysis of DNA of tissues dissected from developing wheat (Triticum aestivum L. cv Chinese Spring) grains at 5-20 days post anthesis (dpa) showed the presence of DNA laddering, which is indicative of internucleosomal fragmentation of nuclear DNA, in maternal tissues but not in the endosperm. The TUNEL assay showed in-situ internucleosomal fragmentation of DNA in nuclei of parenchymal and epidermal cells of the nucellus, as well as in the pericarp, during the early stages of grain development (5 dpa). Furthermore, internucleosomal fragmentation of nuclear DNA was observed in nucellar projection cells in the middle stages of grain development (13-18 dpa), thus showing a process of PCD in these maternal tissues. Electron-transmission microscopy analysis allowed the morphology of PCD to be characterized in this plant tissue. Initially, fragmentation of the cytoplasm was observed, the nuclear envelope appeared dilated and to be forming vacuoles, and the content of heterochromatin increased. A progressive degradation of the cytosolic contents and organelles was observed, and the plasma membrane was disrupted. However, the Golgi apparatus remained intact and apparently functional even in the final stages of cell death.     

Characterization of the expression of a wheat cystatin gene during caryopsis development

A cDNA coding for phytocystatin, a protease inhibitor, was isolated from wheat embryos by differential display RT-PCR and the corresponding full-length cDNA (named WC5 for wheat cystatin gene 5) subsequently obtained by RACE. The deduced primary sequence of the protein suggests the presence of a 28 amino acid N-terminal signal sequence and a 100 amino acid mature protein containing the three consensus motifs known to interact with the active site of cysteine peptidases. Northern and western analysis revealed a spatio-temporal pattern of the cystatin gene expression during caryopse development. In the embryo, WC5 was only expressed during early embryogenesis whereas, in seed covering layers, WC5 expression was restricted to the maturation stage of grain development. In addition, immunolocalization experiments showed that cystatin accumulated in the aleurone layer of the maturating seed and in the parenchymal tissues of the embryo scutellum. A recombinant form of the wheat cystatin was shown to be able to inhibit peptidase activities present in whole seed protein extracts. In addition, immunological techniques allowed us to identify two putative target peptidases. The possible roles of the cystatin protein are discussed in relation with tissular localization and putative peptidase targets during seed maturation.     

Cloning, biochemical characterisation, tissue localisation and possible post-translational regulatory mechanism of the cytosolic phosphoglucose isomerase from developing sunflower seeds

Lipid biosynthesis in developing sunflower (Helianthus annuus L.) seeds requires reducing power. One of the main sources of cellular NADPH is the oxidative pentose phosphate pathway (OPPP), generated from the oxidation of glucose-6-phosphate. This glycolytic intermediate, which can be imported to the plastid and enter in the OPPP, is the substrate and product of cytosolic phosphoglucose isomerase (cPGI, EC 5.3.1.9). In this report, we describe the cloning of a full-length cDNA encoding cPGI from developing sunflower seeds. The sequence was predicted to code for a protein of 566 residues characterised by the presence of two sugar isomerase domains. This cDNA was heterologously expressed in Escherichia coli as a His-tagged protein. The recombinant protein was purified using immobilised metal ion affinity chromatography and biochemically characterised. The enzyme had a specific activity of 1,436 μmol min⁻¹ mg⁻¹ and 1,011 μmol min⁻¹ mg⁻¹ protein when the reaction was initiated with glucose-6-phosphate and fructose-6-phosphate, respectively. Activity was not affected by erythrose-4-phosphate, but was inhibited by 6-P gluconate and glyceraldehyde-3-phosphate. A polyclonal immune serum was raised against the purified enzyme, allowing the study of protein levels during the period of active lipid synthesis in seeds. These results were compared with PGI activity profiles and mRNA expression levels obtained from Q-PCR studies. Our results point to the existence of a possible post-translational regulatory mechanism during seed development. Immunolocalisation of the protein in seed tissues further indicated that cPGI is highly expressed in the procambial ring.     

Modelling the niche for a marine vertebrate: a case study incorporating behavioural plasticity,proximate threats and climate change

The integration of satellite telemetry, remotely sensed environmental data, and habitat/environmental modelling has provided for a growing understanding of spatial and temporal ecology of species of conservation concern. The Republic of Cape Verde comprises the only substantial rookery for the loggerhead turtle Caretta caretta in the eastern Atlantic. A size related dichotomy in adult foraging patterns has previously been revealed for adult sea turtles from this population with a proportion of adults foraging neritically, whilst the majority forage oceanically. Here we describe observed habitat use and employ ecological niche modelling to identify suitable foraging habitats for animals utilising these two distinct behavioural strategies. We also investigate how these predicted habitat niches may alter under the influence of climate change induced oceanic temperature rises. We further contextualise our niche models with fisheries catch data and knowledge of fisheries ‘hotspots’ to infer threat from fisheries interaction to this population, for animals employing both strategies. Our analysis revealed repeated use of coincident oceanic habitat, over multiple seasons, by all smaller loggerhead turtles, whilst larger neritic foraging turtles occupied continental shelf waters. Modelled habitat niches were spatially distinct, and under the influence of predicted sea surface temperature rises, there was further spatial divergence of suitable habitats. Analysis of fisheries catch data highlighted that the observed and modelled habitats for oceanic and neritic loggerhead turtles could extensively interact with intensive fisheries activity within oceanic and continental shelf waters of northwest Africa. We suggest that the development and enforcement of sustainable management strategies, specifically multi‐national fisheries policy, may begin to address some of these issues; however, these must be flexible and adaptive to accommodate potential range shift for this species.