Human serine racemase: moleular cloning, genomic organization and functional analysis

High levels of D-serine are found in mammalian brain, where it is an endogenous agonist of the strichinine-insensitive site of N-methyl D-aspartate type of glutamate receptors. D-serine is enriched in protoplasmic astrocytes that occur in gray matter areas of the brain and was shown to be synthesized from L-serine. We now report cloning and expression of human serine racemase, an enzyme that catalyses the synthesis of D-serine from L-serine. The enzyme displays a high homology to the murine serine racemase. It contains a pyridoxal 5'-phosphate attachment sequence similar to bacterial biosynthetic threonine dehydratase. Northern-blot analysis show high levels of human serine racemase in areas known to contain large amounts of endogenous D-serine, such as hippocampus and corpus callosum. Robust synthesis of D-serine was detected in cells transfected with human serine racemase, demonstrating the conservation of D-amino acid metabolism in humans. Serine racemase may be a therapeutic target in psychiatric diseases as supplementation of D-serine greatly improves schizophrenia symptoms. We identify the human serine racemase genomic structure and show that the gene encompasses seven exons and localizes to chromosome 17q13.3. Identification of the intron-exon boundaries of the human serine racemase gene will be useful to search for mutations in neuropsychiatric disorders.     

Selective Cytotoxicity of 1,3,4-Thiadiazolium Mesoionic Derivatives on Hepatocarcinoma Cells (HepG2)

In this work, we evaluated the cytotoxicity of mesoionic 4-phenyl-5-(2-Y, 4-X or 4-X-cinnamoyl)-1,3,4-thiadiazolium-2-phenylamine chloride derivatives (MI-J: X=OH, Y=H; MI-D: X=NO₂, Y=H; MI-4F: X=F, Y=H; MI-2,4diF: X=Y=F) on human hepatocellular carcinoma (HepG2), and non-tumor cells (rat hepatocytes) for comparison. MI-J, M-4F and MI-2,4diF reduced HepG2 viability by ~ 50% at 25 μM after 24-h treatment, whereas MI-D required a 50 μM concentration, as shown by 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyltetrazolium bromide assays. The cytotoxicity was confirmed with lactate dehydrogenase assay, of which activity was increased by 55, 24 and 16% for MI-J, MI-4F and MI-2,4diF respectively (at 25 μM after 24 h). To identify the death pathway related to cytotoxicity, the HepG2 cells treated by mesoionic compounds were labeled with both annexin V and PI, and analyzed by flow cytometry. All compounds increased the number of doubly-stained cells at 25 μM after 24 h: by 76% for MI-J, 25% for MI-4F and MI-2,4diF, and 11% for MI-D. It was also verified that increased DNA fragmentation occurred upon MI-J, MI-4F and MI-2,4diF treatments (by 12%, 9% and 8%, respectively, at 25 μM after 24 h). These compounds were only weakly, or not at all, transported by the main multidrug transporters, P-glycoprotein, ABCG2 and MRP1, and were able to slightly inhibit their drug-transport activity. It may be concluded that 1,3,4-thiadiazolium compounds, especially the hydroxy derivative MI-J, constitute promising candidates for future investigations on in-vivo treatment of hepatocellular carcinoma.     

Dung Beetle and Terrestrial Mammal Diversity in Forests, Indigenous Agroforestry Systems and Plantain Monocultures in Talamanca, Costa Rica

In order to explore the importance of indigenous agroforestry systems for biodiversity conservation, we compared the abundance, species richness and diversity of dung beetles and terrestrial mammals across a gradient of different land use types from agricultural monocultures (plantains) to agroforestry systems (cocoa and banana) and forests in the BriBri and Cabécar indigenous reserves in Talamanca, Costa Rica. A total of 132,460 dung beetles of 52 species and 913 tracks of 27 terrestrial mammal species were registered. Dung beetle species richness and diversity were greatest in the forests, intermediate in the agroforestry systems and lowest in the plantain monocultures, while dung beetle abundance was greatest in the plantain monocultures. The number of mammal tracks per plot was significantly higher in forests than in plantain monocultures, whereas mammal species richness was higher in forests than in either cocoa agroforestry systems or plantain monocultures. Species composition of both terrestrial mammals and dung beetles also varied across the different land use types. Our study indicates that indigenous cocoa and banana agroforestry systems maintain an intermediate level of biodiversity (which is less than that of the original forest but significantly greater than that of plantain monocultures) and provide suitable habitat for a number of forest-dependent species. Although the agroforestry systems appear to serve as favorable habitats for many terrestrial mammal species, their potential positive contribution to mammal conservation is being offset by heavy hunting pressure in the reserves. As in other agricultural landscapes, the conservation of biodiversity in Talamanca will depend not only on maintaining the existing forest patches and reducing the conversion of traditional agroforestry systems to monocultures, but also on reducing hunting pressure.     

Active Surveillance of Hansen's Disease (Leprosy): Importance for Case Finding among Extra-domiciliary Contacts

Hansen''s disease (leprosy) remains an important health problem in Brazil, where 34,894 new cases were diagnosed in 2010, corresponding to 15.3% of the world''s new cases detected in that year. The purpose of this study was to use home visits as a tool for surveillance of Hansen''s disease in a hyperendemic area in Brazil. A total of 258 residences were visited with 719 individuals examined. Of these, 82 individuals had had a previous history of Hansen''s disease, 209 were their household contacts and 428 lived in neighboring residences. Fifteen new Hansen''s disease cases were confirmed, yielding a detection rate of 2.0% of people examined. There was no difference in the detection rate between household and neighbor contacts (p = 0.615). The two groups had the same background in relation to education (p = 0.510), household income (p = 0.582), and the number of people living in the residence (p = 0.188). Spatial analysis showed clustering of newly diagnosed cases and association with residential coordinates of previously diagnosed multibacillary cases. Active case finding is an important tool for Hansen''s disease control in hyperendemic areas, enabling earlier diagnosis, treatment, decrease in disability from Hansen''s disease and potentially less spread of Mycobacterium leprae.     

Identification of a Bifunctional Maize C- and O-Glucosyltransferase

Flavonoids accumulate in plant vacuoles usually as O-glycosylated derivatives, but several species can also synthesize flavonoid C-glycosides. Recently, we demonstrated that a flavanone 2-hydroxylase (ZmF2H1, CYP93G5) converts flavanones to the corresponding 2-hydroxy derivatives, which are expected to serve as substrates for C-glycosylation. Here, we isolated a cDNA encoding a UDP-dependent glycosyltransferase (UGT708A6), and its activity was characterized by in vitro and in vivo bioconversion assays. In vitro assays using 2-hydroxyflavanones as substrates and in vivo activity assays in yeast co-expressing ZmF2H1 and UGT708A6 show the formation of the flavones C-glycosides. UGT708A6 can also O-glycosylate flavanones in bioconversion assays in Escherichia coli as well as by in vitro assays with the purified recombinant protein. Thus, UGT708A6 is a bifunctional glycosyltransferase that can produce both C- and O-glycosidated flavonoids, a property not previously described for any other glycosyltransferase.     

SP3-Enabled Rapid and High Coverage Chemoproteomic Identification of Cell-State–Dependent Redox-Sensitive Cysteines

Proteinaceous cysteine residues act as privileged sensors of oxidative stress. As reactive oxygen and nitrogen species have been implicated in numerous pathophysiological processes, deciphering which cysteines are sensitive to oxidative modification and the specific nature of these modifications is essential to understanding protein and cellular function in health and disease. While established mass spectrometry-based proteomic platforms have improved our understanding of the redox proteome, the widespread adoption of these methods is often hindered by complex sample preparation workflows, prohibitive cost of isotopic labeling reagents, and requirements for custom data analysis workflows. Here, we present the SP3-Rox redox proteomics method that combines tailored low cost isotopically labeled capture reagents with SP3 sample cleanup to achieve high throughput and high coverage proteome-wide identification of redox-sensitive cysteines. By implementing a customized workflow in the free FragPipe computational pipeline, we achieve accurate MS1-based quantitation, including for peptides containing multiple cysteine residues. Application of the SP3-Rox method to cellular proteomes identified cysteines sensitive to the oxidative stressor GSNO and cysteine oxidation state changes that occur during T cell activation.     

Zooplankton and ichthyoplankton communities in a temperate estuary: spatial and temporal patterns

Zooplankton and ichthyoplankton assemblages were studied fromJanuary 2003 to June 2004 in a temperate shallow estuary (Mondegoestuary, Portugal). Monthly sampling was performed at five stationsat high and low tides, with subsurface tows with 335 and 500µm mesh Bongo nets. Analysis of variance (ANOVA) showeda significant effect (P < 0.05) of the mesh size of the neton the abundance of main zooplankton groups. On average, theabundance of the 500 µm taxocenosis was 67 and 102 timeslower than the 335 µm taxocenosis at high and low tidesrespectively, especially in the south arm. More than 80 specieswere identified in the zooplankton samples. The upper reachesof northern arm are dominated by freshwater crustacean mesozooplanktonlike Acanthocyclops robustus and Diaptomus spp. and the cladoceransDaphnia, Ceriodaphnia and Bosmina, often being codominant. Inthe southern arm, the resident estuarine copepod Acartia tonsawas dominant, eventually being the most abundant taxon. Marinereaches of estuary are usually dominated by the marine calanoidcopepods Acartia clausi and Temora longicornis and the siphonophoresMuggiaea atlantica. Concerning the ichthyoplankton, this wasdominated by the larvae of estuarine resident species, mainlyPomatoschistus sp., and eggs of Engraulis encrasicolus. Theabundance of Pomatoschistus sp. larvae was positively correlatedwith water temperature. Statistical analysis (canonical correspondenceanalysis) used to determine the spatiotemporal structure ofthe zooplankton assemblages and its correlation with environmentalvariables showed that salinity and temperature were the mainfactors influencing the distribution of zooplankton. The resultsobtained also showed that abundance was strongly influencedby the hydrological circulation pattern and direct or indirecthuman impacts that occur in each arm of the estuary. This article was presented at Plankton Symposium III, held atFiguera da Foz, portugal, between 17 and 20 March 2005, underthe auspices of the University of Coimbra and the Universityof Aveiro, and coordinated by Mário Jorge Pereira andUlisses M. Azeiteiro.