Mechanism(s) of action of heavy metals to investigate the regulation of plastidic glucose-6-phosphate dehydrogenase

The regulation of recombinant plastidic glucose-6P dehydrogenase from Populus trichocarpa (PtP2-G6PDH - EC 1.1.1.49) was investigated by exposing wild type and mutagenized isoforms to heavy metals. Nickel and Cadmium caused a marked decrease in PtP2-G6PDH WT activity, suggesting their poisoning effect on plant enzymes; Lead (Pb++) was substantially ineffective. Copper (Cu++) and Zinc (Zn++) exposition resulted in strongest decrease in enzyme activity, thus suggesting a physiological competition with Magnesium, a well-known activator of G6PDH activity. Kinetic analyses confirmed a competitive inhibition by Copper, and a mixed inhibition by (Cd++). Mutagenized enzymes were differently affected by HMs: the reduction of disulfide (C175-C183) exposed the NADP+ binding sites to metals; C145 participates to NADP+ cofactor binding; C194 and C242 are proposed to play a role in the regulation of NADP+/NADPH binding. Copper (and possibly Zinc) is able to occupy competitively Magnesium (Mg++) sites and/or bind to NADP+, resulting in a reduced access of NADP+ sites on the enzyme. Hence, heavy metals could be used to describe specific roles of cysteine residues present in the primary protein sequence; these results are discussed to define the biochemical mechanism(s) of inhibition of plant plastidic G6PDH

The blue lizard spandrel and the island syndrome

<p>Abstract</p> <p>Background</p> <p>Many small vertebrates on islands grow larger, mature later, lay smaller clutches/litters, and are less sexually dimorphic and aggressive than their mainland relatives. This set of observations is referred to as the 'Island Syndrome'. The syndrome is linked to high population density on islands. We predicted that when population density is low and/or fluctuating insular vertebrates may evolve correlated trait shifts running opposite to the Island Syndrome, which we collectively refer to as the 'reversed island syndrome' (RIS) hypothesis. On the proximate level, we hypothesized that RIS is caused by increased activity levels in melanocortin receptors. Melanocortins are postranslational products of the proopiomelanocortin gene, which controls pleiotropically pigmentation, aggressiveness, sexual activity, and food intake in vertebrates.</p> <p>Results</p> <p>We tested the RIS hypothesis performing a number of behavioral, genetic, and ontogenetic tests on a blue colored insular variant of the Italian Wall lizard <it>Podarcis sicula</it>, living on a small island off the Southern Italian coast. The population density of this blue-colored variant was generally low and highly fluctuating from one year to the next.</p> <p>In keeping with our predictions, insular lizards were more aggressive and sexually dimorphic than their mainland relatives. Insular males had wide, peramorphic heads. The growth rate of insular females was slower than growth rates of mainland individuals of both sexes, and of insular males. Consequently, size and shape dimorphism are higher on the Island. As predicted, melanocortin receptors were much more active in individuals of the insular population. Insular lizards have a higher food intake rate than mainland individuals, which is consistent with the increased activity of melanocortin receptors. This may be adaptive in an unpredictable environment such as Licosa Island. Insular lizards of both sexes spent less time basking than their mainland relatives. We suspect this is a by-product (spandrel) of the positive selection for increased activity of melanocortins receptors.</p> <p>Conclusions</p> <p>We contend that when population density is either low or fluctuating annually as a result of environmental unpredictability, it may be advantageous to individuals to behave more aggressively, to raise their rate of food intake, and allocate more energy into reproduction.</p

Transgenic chloroplasts are efficient sites for high-yield production of the vaccinia virus envelope protein A27L in plant cells.

Orthopoxviruses (OPVs) have recently received increasing attention because of their potential use in bioterrorism and the occurrence of zoonotic OPV outbreaks, highlighting the need for the development of safe and cost-effective vaccines against smallpox and related viruses. In this respect, the production of subunit protein-based vaccines in transgenic plants is an attractive approach. For this purpose, the A27L immunogenic protein of vaccinia virus was expressed in tobacco using stable transformation of the nuclear or plastid genome. The vaccinia virus protein was expressed in the stroma of transplastomic plants in soluble form and accumulated to about 18% of total soluble protein (equivalent to approximately 1.7 mg/g fresh weight). This level of A27L accumulation was 500-fold higher than that in nuclear transformed plants, and did not decline during leaf development. Transplastomic plants showed a partial reduction in growth and were chlorotic, but reached maturity and set fertile seeds. Analysis by immunofluorescence microscopy indicated altered chlorophyll distribution. Chloroplast-synthesized A27L formed oligomers, suggesting correct folding and quaternary structure, and was recognized by serum from a patient recently infected by a zoonotic OPV. Taken together, these results demonstrate that chloroplasts are an attractive production vehicle for the expression of OPV subunit vaccines

Abscisic acid effects on activity and expression of barley (Hordeum vulgare) plastidial glucose-6-phosphate dehydrogenase

Total glucose-6-phosphate dehydrogenase (G6PDH) activity, protein abundance, and transcript levels of G6PDH isoforms were measured in response to exogenous abscisic acid (ABA) supply to barley (Hordeum vulgare cv Nure) hydroponic culture. Total G6PDH activity increased by 50% in roots treated for 12 h with exogenous 0.1 mM ABA. In roots, a considerable increase (35%) in plastidial P2-G6PDH transcript levels was observed during the first 3 h of ABA treatment. Similar protein variations were observed in immunoblotting analyses. In leaves, a 2-fold increase in total G6PDH activity was observed after ABA treatment, probably related to an increase in the mRNA level (increased by 50%) and amount of protein (increased by 85%) of P2-G6PDH. Together these results suggest that the plastidial P2-isoform plays an important role in ABA-treated barley plants

Cloning, expression and characterization of P2-glucose-6P dehydrogenase from barley (Hordeum vulgare) and poplar (Populus trichocarpa. Effects of Abscissic acid (ABA) on activity, occurrence and expression of different isoforms of glucose-6P dehydrogenase in barley

Glucose 6-phosphate dehydrogenase (G6PDH, EC 1.1.1.49) is the main regulatory enzyme of the oxidative pentose phosphate pathway (OPPP), an important provider of NADPH. The effect of exogenous ABA on the activity, expression level and protein amount of different G6PDH isoforms has been studied in barley plants. G6PDH activity increased by 50% in ABA treated roots for 12h; this increase resembles a similar (+35%) effect observed in P2-G6PDH transcript levels .The transcript Cy-G6PDH levels did not change appreciably upon ABA supply. Similar variations were observed in protein levels in immunoblotting analyses. In leaves, ABA did not affect the cytosolic protein levels, while a sudden decrease was observed for chloroplastic P1-G6PDH protein; therefore the two-fold increase in G6PDH activity observed after ABA treatment is likely related to an increase in mRNA level (+ 50%) and protein amount (+ 85%) of leaf P2-G6PDH. Altogether, these results suggest a specific role for the plastidial P2-isoform in ABA treated barley plants, encouraging a better characterization of this plastidic isoform. Therefore, genes of the plastidic isoform of G6PDH (P2-G6PDH) from two different organisms, barley (Hordeum vulgare) and poplar (Populus trichocarpa) have been cloned in two different expression vectors, pET3d and pET15b (His-Tag), and overexpressed in E.coli. The purified enzyme was checked by mass spectrometry analysis, and for the reactivity against specific P2—G6PDH antibodies; the main kinetic parameters, and differential sensitivity to reduction by DTT were determined.The recombinant P2-G6PDHs exhibits molecular weights of 55.5 kDa and 61kDa for barley and poplar, respectively. The main kinetic parameters measured for both HvP2-G6DPH and PtP2G6PDH are in agreement (e.g. high KiNADPH) with the values known for the most of other P2-type G6PDHs. The recombinant barley protein is moderately sensitive to reductants (DTT); moreover the Populus trichocarpa enzyme presents a redox potential (-280 mV) favourable for control by either thioredoxins m or f