Glutamate 59 is critical for transport function of the amino acid cotransporter KAAT1

KAAT1 is a neutral amino acid transporter activated by K⁺ or by Na⁺ (9). The protein shows significant homology with members of the Na⁺/Cl^–-dependent neurotransmitter transporter super family. E59G KAAT1, expressed in Xenopus oocytes, exhibited a reduced leucine uptake [20–30% of wild-type (WT)], and kinetic analysis indicated that the loss of activity was due to reduction of V_max and apparent affinity for substrates. Electrophysiological analysis revealed that E59G KAAT1 has presteady-state and uncoupled currents larger than WT but no leucine-induced currents. Site-directed mutagenesis analysis showed the requirement of a negative charge in position 59 of KAAT1. The analysis of permeant and impermeant methanethiosulfonate reagent effects confirmed the intracellular localization of glutamate 59. Because the 2-aminoethyl methanethiosulfonate hydrobromid inhibition was not prevented by the presence of Na⁺ or leucine, we concluded that E59 is not directly involved in the binding of substrates. N-ethylmaleimide inhibition was qualitatively and quantitatively different in the two transporters, WT and E59G KAAT1, having the same cysteine residues. This indicates an altered accessibility of native cysteine residues due to a modified spatial organization of E59G KAAT1. The arginine modifier phenylglyoxal effect supports this hypothesis: not only cysteine but also arginine residues become more accessible to the modifying reagents in the mutant E59G. In conclusion, the results presented indicate that glutamate 59 plays a critical role in the three-dimensional organization of KAAT1. amino acid transport; structure/function; amino acid modifiers; Manduca sexta     

Adenoviral-mediated overexpression of catalase inhibits endothelial cell proliferation

Although hydrogen peroxide (H(2)O(2)) induces proliferation of vascular smooth muscle cells, its role in endothelial cell proliferation is unclear. Our aim was to study the role of hydrogen peroxide in endothelial cell proliferation by overexpressing catalase. Human aortic endothelial cells were transduced with adenoviral vectors encoding beta-galactosidase (Adbetagal) or catalase (AdCat) or were exposed to diluent alone (control). Transgene expression was demonstrated by beta-galactosidase staining, Western analysis, and significantly increased enzyme activity in AdCat-transduced cells. Overexpression of catalase decreased DNA synthesis in AdCat compared with control and Adbetagal-transduced cells (536.8 +/- 31 vs. 1,875.1 +/- 132.9 vs. 1,347.5 +/- 93.7 dpm/well, respectively; P < 0.05 vs. control and Adbetagal). Six days after transduction with AdCat (multiplicity of infection = 50), cell numbers were significantly reduced (AdCat: 38 +/- 1.8% of cell counts in control, P < 0.05; and 45 +/- 2% of cell count in Adbetagal, P < 0.05). Incubation with aminotriazole 10 mmol/l, an inhibitor of catalase, prevented this effect. The number of apoptotic cells was increased one- and threefold 2 and 4 days, respectively, after transduction with AdCat. Exogenous administration of low concentrations of H(2)O(2) (50 microM) significantly increased cell proliferation, whereas it was inhibited by higher concentrations. These results suggest that H(2)O(2) is an important modulator of endothelial cell proliferation.     

A framework for the identification of hotspots of climate change risk for mammals

As rates of global warming increase rapidly, identifying species at risk of decline due to climate impacts and the factors affecting this risk have become key challenges in ecology and conservation biology. Here, we present a framework for assessing three components of climate‐related risk for species: vulnerability, exposure and hazard. We used the relationship between the observed response of species to climate change and a set of intrinsic traits (e.g. weaning age) and extrinsic factors (e.g. precipitation seasonality within a species geographic range) to predict, respectively, the vulnerability and exposure of all data‐sufficient terrestrial non‐volant mammals (3,953 species). Combining this information with hazard (the magnitude of projected climate change within a species geographic range), we identified global hotspots of species at risk from climate change that includes the western Amazon basin, south‐western Kenya, north‐eastern Tanzania, north‐eastern South Africa, Yunnan province in China, and mountain chains in Papua‐New Guinea. Our framework identifies priority areas for monitoring climate change effects on species and directing climate mitigation actions for biodiversity.     

Life in a fragment: Evolution of foraging strategies of translocated collared brown lemurs,Eulemur collaris,over an 18-year period

While the drivers of primate persistence in forest fragments have been often considered at the population level, the strategies to persist in these habitats have been little investigated at the individual or group level. Considering the rapid variation of fragment characteristics over time, longitudinal data on primates living in fragmented habitats are necessary to understand the key elements for their persistence. Since translocated animals have to cope with unfamiliar areas and face unknown fluctuations in food abundance, they offer the opportunity to study the factors contributing to successful migration between fragments. Here, we illustrated the evolution of the foraging strategies of translocated collared brown lemurs (Eulemur collaris) over an 18-year period in the Mandena Conservation Zone, south-east Madagascar. Our aim was to explore the ability of these frugivorous lemurs to adjust to recently colonized fragmented forests. Although the lemurs remained mainly frugivorous throughout the study period, over the years we identified a reduction in the consumption of leaves and exotic/pioneer plant species. These adjustments were expected in frugivorous primates living in a degraded area, but we hypothesize that they may also reflect the initial need to cope with an unfamiliar environment after the translocation. Since fragmentation is often associated with the loss of large trees and native vegetation, we suggest that the availability of exotic and/or pioneer plant species can provide an easy-to-access, nonseasonal food resource and be a key factor for persistence during the initial stage of the recolonization.     

A novel alpha-D-galactosynthase from Thermotoga maritima converts beta-D-galactopyranosyl azide to alpha-galacto-oligosaccharides

The large-scale production of oligosaccharides is a daunting task, hampering the study of the role of glycans in vivo and the testing of the efficacy of novel glycan-based drugs. Glycosynthases, mutated glycosidases that synthesize oligosaccharides in high yields, are becoming important chemo-enzymatic tools for the production of oligosaccharides. However, while β-glycosynthase can be produced with a rather well-established technology, examples of α-glycosynthases are thus far limited only to enzymes from glycoside hydrolase 29 (GH29), GH31 and GH95 families. α-L-Fucosynthases from GH29 use convenient glycosyl azide derivatives as a strategic alternative to glycosyl fluoride donors. However, the general applicability of this method to other α-glycosynthases is not trivial and remains to be confirmed. Here, β-D-galactopyranosyl azide was converted to α-galacto-oligosaccharides with good yields and high regioselectivity, catalyzed by a novel α-galactosynthase based on the GH36 α-galactosidase from the hyperthermophilic bacterium Thermotoga maritima. These results open a new avenue to the practical synthesis of biologically interesting α-galacto-oligosaccharides and demonstrate more widespread use of β-glycosyl-azide as donors, confirming their utility to expand the repertoire of glycosynthases.     

Helminth communities of loggerhead turtles (Caretta caretta) from Central and Western Mediterranean Sea: The importance of host's ontogeny

We investigated the factors providing structure to the helminth communities of 182 loggerhead sea turtles, Caretta caretta, collected in 6 localities from Central and Western Mediterranean. Fifteen helminth taxa (10 digeneans, 4 nematodes and 1 acanthocephalan) were identified, of which 12 were specialist to marine turtles; very low numbers of immature individuals of 3 species typical from fish or cetaceans were also found. These observations confirm the hypothesis that phylogenetic factors restrict community composition to helminth species specific to marine turtles. There were significant community dissimilarities between turtles from different localities, the overall pattern being compatible with the hypothesis that parasite communities reflect the ontogenetic shift that juvenile loggerheads undergo from oceanic to neritic habitats. The smallest turtles at the putative oceanic, pelagic-feeding stage harboured only the 2 digenean species that were regionally the most frequent, i.e. Enodiotrema megachondrus and Calycodes anthos; the largest turtles at the putative neritic, bottom-feeding stage harboured 11 helminth taxa, including 3 nematode species that were rare or absent in turtles that fed partially on pelagic prey. Mean species richness per host was low (range: 1.60–1.89) and did not differ between localities. Variance ratio tests indicated independent colonization of each helminth species. Both features are expected in ectothermic and vagrant hosts living in the marine environment.     

A PCR-based diagnostic tool for distinguishing grape skin color mutants

Berry skin color mutants are phenotypically different from their original cultivars, but they show identical molecular profile if analysed by using microsatellite markers. This work gives an easy, inexpensive and quick diagnostic tool to discriminate these somatic variants. We distinguished some grape (Vitis vinifera L.) skin color mutants from white to red or pink and from black to grey, pink or white and we investigated their molecular bases by single-strand conformational polymorphism (SSCP), single base primer extension and coding sequence analysis of anthocyanin biosynthetic enzyme genes and by polymerase chain reaction (PCR) analysis of VvmybA1 regulatory gene. Analyses of structural genes did not reveal polymorphisms between wild type and mutant cultivars but only among different varieties, whereas the study of VvmybA1 regulatory gene has given important outcomes for color mutants characterisation. The discrimination between white wild type and its derived colored mutant and between black wild type and white mutant has been obtained through a simple test of amplification for presence/absence. The discrimination between black wild type and less colored mutant has occurred through a quantitative result on agarose gel confirmed by real-time PCR analysis: the amount of functional allele in less colored somatic variants genome was about one-fourth of the correspondent quantity in original black cultivars genome.     

Glucokinase gene mutations: structural and genotype-phenotype analyses in MODY children from South Italy

Background

Maturity onset diabetes of the young type 2 (or GCK MODY) is a genetic form of diabetes mellitus provoked by mutations in the glucokinase gene (GCK).

Methodology/Principal Findings

We screened the GCK gene by direct sequencing in 30 patients from South Italy with suspected MODY. The mutation-induced structural alterations in the protein were analyzed by molecular modeling. The patients'' biochemical, clinical and anamnestic data were obtained. Mutations were detected in 16/30 patients (53%); 9 of the 12 mutations identified were novel (p.Glu70Asp, p.Phe123Leu, p.Asp132Asn, p.His137Asp, p.Gly162Asp, p.Thr168Ala, p.Arg392Ser, p.Glu290X, p.Gln106_Met107delinsLeu) and are in regions involved in structural rearrangements required for catalysis. The prevalence of mutation sites was higher in the small domain (7/12: ∼59%) than in the large (4/12: 33%) domain or in the connection (1/12: 8%) region of the protein. Mild diabetic phenotypes were detected in almost all patients [mean (SD) OGTT = 7.8 mMol/L (1.8)] and mean triglyceride levels were lower in mutated than in unmutated GCK patients (p = 0.04).

Conclusions

The prevalence of GCK MODY is high in southern Italy, and the GCK small domain is a hot spot for MODY mutations. Both the severity of the GCK mutation and the genetic background seem to play a relevant role in the GCK MODY phenotype. Indeed, a partial genotype-phenotype correlation was identified in related patients (3 pairs of siblings) but not in two unrelated children bearing the same mutation. Thus, the molecular approach allows the physician to confirm the diagnosis and to predict severity of the mutation.