Ca2+ regulation of thyroid NADPH-dependent H2O2 generation

A thyroid particulate fraction contains an NADPH-dependent H2O2-generating enzyme which requires Ca2+ for activity. A Chaps solubilized extract of the thyroid particulate fraction partially purified by DEAE chromatography did not show a dependence on Ca2+ for activity. Preincubation of the particulate fraction with Ca2+ yielded a preparation insensitive to Ca2+. The non-particulate fraction obtained after incubation of the particles in the presence of Ca2+ was able to inhibit, in the presence of EGTA, the Ca2+-desensitized particulate fraction and the enzyme isolated on DEAE. It is concluded that the reversible Ca2+ activation of the NADPH-dependent H2O2 generation was modulated in porcine thyroid tissue by (a) calcium-releasable inhibitor protein(s).     

Generation of an Fsp1 (fibroblast‐specific protein 1)‐Flpo transgenic mouse strain

Recombination systems represent a major breakthrough in the field of genetic model engineering. The Flp recombinases (Flp, Flpe, and Flpo) bind and cleave DNA Frt sites. We created a transgenic mouse strain ([Fsp1‐Flpo]) expressing the Flpo recombinase in fibroblasts. This strain was obtained by random insertion inside mouse zygotes after pronuclear injection. Flpo expression was placed under the control of the promoter of Fsp1 (fibroblast‐specific protein 1) gene, whose expression starts after gastrulation at Day 8.5 in cells of mesenchymal origin. We verified the correct expression and function of the Flpo enzyme by several ex vivo and in vivo approaches. The [Fsp1‐Flpo] strain represents a genuine tool to further target the recombination of transgenes with Frt sites specifically in cells of mesenchymal origin or with a fibroblastic phenotype.     

NewLeaf Plus® Russet Burbank potatoes: replicase-mediated resistance to potato leafroll virus

Potato leafroll poleovirus and the Colorado potato beetle (Leptinotarsa decemlineata (Say)) are major pests of potato in the USA. The US Department of Agriculture estimates that over 50% of annual insecticide use on potato is applied to control the Colorado potato beetle and aphids that transmit potato leafroll virus (PLRV). To address this issue, Russet Burbank potatoes have been genetically modified for a high level of resistance to infection and the resulting disease symptoms caused by PLRV and to feeding damage caused by the Colorado potato beetle. This resistance was achieved by the expression of the unmodified full-length replicase gene of PLRV and the cry3A insect control protein gene from Bacillus thuringiensis var. tenebrionis. Plant expression constructs containing various modifications of the PLRV replicase gene were produced during the development of this product. The genes in these constructs were a full-length unmodified replicase (open reading frame 2a/2b), an antisense orientation of the full-length cDNA, an open reading frame 1 translation of the full-length gene, and a gene truncation containing the 3 sense coding portion of the replicase gene. Growth chamber experiments demonstrated that transformation of plants with the full-length and 3 sense coding constructs substantially protected these potato plants from infection and disease symptoms caused by PLRV. The Russet Burbank potato expressing the full-length PLV replicase gene and the cry3A gene is a new potato product from NatureMark called NewLeaf Plus®.     

Resistance to wheat streak mosaic virus in transgenic wheat engineered with the viral coat protein gene

Wheat (Triticum aestivum) plants were stably transformed with the coat protein (CP) gene of wheat streak mosaic virus (WSMV) by the biolistic method. Eleven independently transformed plant lines were obtained and five were analyzed for gene expression and resistance to WSMV. One line showed high resistance to inoculations of two WSMV strains. This line had milder symptoms and lower virus titer than control plants after inoculation. After infection, new growth did not show symptoms. The observed resistance was similar to the recovery type resistance described previously using WSMV NIb transgene and in other systems. This line looked morphologically normal but had an unusually high transgene copy number (approximately 90 copies per 2C homozygous genome). Northern hybridization analysis indicated a high level of degraded CP mRNA expression. However, no coat protein expression was detected.     

Targeting of the dual oxidase 2 N-terminal region to the plasma membrane

Dual oxidase 2 (Duox2) is a cell surface glycoprotein that probably provides thyroperoxidase with the H2O2 required to catalyze thyroid hormone synthesis. No functional H2O2-generating system has yet been obtained after transfecting Duox2 into non-thyroid cell lines, because it is retained in the endoplasmic reticulum (ER). We investigated the level of maturation of various Duox2 truncated proteins in an attempt to identify the region of Duox2 responsible for its remaining in the ER. Duox2-Q686X mutant, corresponding to the N-terminal ectodomain including the first putative transmembrane domain, was expressed in different cell lines. Carbohydrate content analysis revealed that complex type-specific Golgi apparatus (GA) oligosaccharides were present on pig Duox2-Q686X, whereas human truncated Duox2 carried only high mannose-type sugar chains characteristic of the ER. Further characterization using surface biotinylation and flow cytometry assays indicated that pig Duox2-Q686X was present at the plasma membrane, whereas human Duox2-Q686X remained inside the cell. The replacement of the last 90 residues of the human Duox2-Q686X with the pig equivalent region allowed the chimerical peptide to reach the Golgi apparatus. Pig mutants containing the complete first intracellular loop with or without the second transmembrane domain accumulated in the ER. These findings show that 1) the human Duox2-Q686X region encompassing residues 596-685 prevents mutant exportation from the ER and 2) there is a pig Duox2 retention domain in the first intracellular loop. In addition, missense mutations of four cysteines (Cys-351, -370, -568, or -582) completely inhibited the emergence of pig Duox2-Q686X from the ER compartment, indicating their importance in Duox2 maturation.     

Man, vegetation and climate during the Holocene in the territory of Sagalassos, Western Taurus Mountains, SW Turkey

Past vegetation change and the influence of climate change and anthropogenic pressure during the Holocene is constructed from a series of palynological records sampled from three locations within the territory of the antique site of Sagalassos. The disappearance of the original deciduous oak woodlands and increases in anthropogenic indicator species around 5300 and 4300 b.c. correspond with an increase in settlements in the region. A period of drought following the deforestation may have hampered the recovery of deciduous oak. The timing of the onset of the Bey?ehir Occupation Phase (BO-Phase) in the territory differs between locations, estimates ranging from ca. 1000–800 b.c. to the start of the Hellenistic period (334 b.c.). The most intense period of arboriculture coincides with the Roman and late-Roman periods. Increases in human pressure on the landscape as reflected in the pollen record correspond with an increased rate of sedimentation and fire activity. The timing of the end of the BO-Phase again differs between locations. Estimates range from the 4th century a.d. to the mid 7th century a.d., when a region-wide shift to dry environmental conditions is observed. Numerical analyses show that post BO-Phase vegetation change is largely driven by climate and displays a succession of dry and wet periods that coincided with well-defined European climate shifts, including the Medieval Climate Anomaly and the Little Ice Age. Current agricultural activities in the region are of a very recent (20th century) origin.     

Madonna dell'Arma (San Remo, Italie) : expression locale de la végétation ligure au cours du Paléolithique moyen

Pollen analysis of Madonna dell’Arma cave inside part (Liguria, Italy) allowed us to reconstitute, at local scale, the Ligurian coastal vegetation between the end of the last interglacial period and the beginning of the pleniglacial. This transition period shows an important representation of arboreal cover and a persistence of thermophilous elements that allowed us to considerate the western Liguria as a periglacial refuge. During the end of the last interglacial, xerophytic, halophytic herbs and shrubs taken over from a Mediterranean pre-forest unit in the first slopes constituted the coastal zone. The nearness of Argentina torrent and the Armea influenced, in the coast, the existence of marshy zones colonized by hygrophilous trees. In the Ligurian lowlands spread out sclerophyllous and mesophilous forests according to altitude. The beginning of the pleniglacial is indicated in the sequence by increases of Pinus, Cupressaceae and Artemisia in the context of a decline in the thermophilous components. This picture of vegetation has become integrated into the multidisciplinary studies of the site and contributed to the elaboration of palaeoclimatic hypothesis similar as those revealed by the fauna.     

Santa Lucia superiore (Toirano, Ligurie) : reconstitution locale de la végétation ligure durant le Pléniglaciaire ancien

Pollen analysis of Santa Lucia superiore cave (Italy) reports at local scale the lowland Ligurian vegetation during the Lower Pleniglacial (75 to 57 Kyr BP). The pollen profile shows two AP extensions during a steppe-landscape episode in Liguria. The dry and cold phases caused the establishment of a steppe-landscape (NAP 92%) with Artemisia, Ephedra, Poaceae and Chenopodiaceae, similar to those mentioned in the Latium during the Pleniglacial. The increase of moisture generated a first arboreal extension (Pinus, Betula, Corylus, Ulmus), which engendered the formation of an open-forest landscape (AP 43%). The second arboreal extension (AP 55%) was due to an increase of moisture and higher temperatures, which allowed the development of Mediterranean trees and shrubs (Quercus ilex, Olea, Phillyrea). This warming up occurred probably simultaneously with a secondary transgression during the glacial sea-level change. The upper part of the profile shows an open vegetation, which indicates the return of a steppe-landscape. Pollen data, replaced in the multidisciplinary studies of the site, are well correlated with the fauna and sediment data.     

Vulnerability of Mediterranean Ecosystems to Long-Term Changes along the Coast of Israel

Although human activity is considered to be a major driving force affecting the distribution and dynamics of Mediterranean ecosystems, the full consequences of projected climate variability and relative sea-level changes on fragile coastal ecosystems for the next century are still unknown. It is unclear how these waterfront ecosystems can be sustained, as well as the services they provide, when relative sea-level rise and global warming are expected to exert even greater pressures in the near future (drought, habitat degradation and accelerated shoreline retreat). Haifa Bay, northern Israel, has recorded a landward sea invasion, with a maximum sea penetration 4,000 years ago, during an important period of urban development and climate instability. Here, we examine the cumulative pressure of climate shifts and relative sea-level changes in order to investigate the patterns and mechanisms behind forest replacement by an open-steppe. We provide a first comprehensive and integrative study for the southern Levant that shows that (i) human impact, through urbanization, has been the main driver behind ecological erosion in the past 4,000 years; (ii) climate pressures have reinforced this impact; and (iii) local coastal changes have played a decisive role in eroding ecosystem resilience. These three parameters, which have closely interacted during the last 4,000 years in Haifa Bay, clearly indicate that for an efficient management of the coastal habitats, anthropogenic pressures linked to urban development must be reduced in order to mitigate the predicted effects of Global Change.     

NADPH-dependent H2O2 generation catalyzed by thyroid plasma membranes. Studies with electron scavengers

Hog thyroid plasma membrane preparations containing a Ca2+-regulated NADPH-dependent H2O2-generating system were studied. The Ca2+-dependent reductase activities of ferricytochrome c, 2,6-dichloroindophenol, nitroblue tetrazolium, and potassium ferricyanide were tested and the effect of these scavengers on H2O2 formation, NADPH oxidation and O2 consumption were measured, with the following results. 1. Thyroid plasma membrane Ca2+-independent cytochrome c reduction was not catalyzed by the NADPH-dependent H2O2-generating system. This activity was superoxide-dismutase-insensitive. 2.Of the three other electron scavengers tested, only K3Fe(CN)6 was clearly, but partially reduced in a Ca2+-dependent manner. 3. Though the NADPH-dependent reduction of nitroblue tetrazolium was very low and superoxide-dismutase-insensitive, nitroblue tetrazolium inhibited O2 consumption, H2O2 formation and NADPH oxidation, indicating that nitroblue tetrazolium inhibits the H2O2-generating system. We conclude that the thyroid plasma membrane H2O2-generating system does not or liberate O2- and that Ca2+ controls the first step (NADPH oxidation) of the H2O2-generating system.