Involvement of thiaminase II encoded by the THI20 gene in thiamin salvage of Saccharomyces cerevisiae

The physiological significance of thiaminase II, which catalyzes the hydrolysis of thiamin, has remained elusive for several decades. The C-terminal domains of THI20 family proteins (THI20/21/22) and the whole region of PET18 gene product of Saccharomyces cerevisiae are homologous to bacterial thiaminase II. On the other hand, the N-terminal domains of THI20 and THI21 encode 2-methyl-4-amino-5-hydroxymethylpyrimidine kinase and 2-methyl-4-amino-5-hydroxymethylpyrimidine phosphate kinase involved in the thiamin synthetic pathway. In this study, it was first indicated that the C-terminal domains of the THI20 family and PET18 are not required for de novo thiamin synthesis in S. cerevisiae, using a quadruple deletion strain expressing the N-terminal domain of THI20. Biochemical analysis using cell-free extracts and recombinant proteins demonstrated that yeast thiaminase II activity is exclusively encoded by THI20. It appeared that Thi20p has an affinity for the pyrimidine moiety of thiamin, and HMP produced by the thiaminase II activity is immediately phosphorylated. Thi20p was found to participate in the formation of thiamin from two synthetic antagonists, pyrithiamin and oxythiamin, by hydrolyzing both antagonists and phosphorylating HMP to give HMP pyrophosphate. Furthermore, 2-methyl-4-amino-5-aminomethylpyrimidine, a presumed naturally occurring thiamin precursor, was effectively converted to HMP by incubation with Thi20p. It is proposed that the thiaminase II activity of Thi20p is involved in the thiamin salvage pathway by catalyzing the hydrolysis of HMP precursors in S. cerevisiae.     

Size Distribution and Molecular Associations of Plasma Fibronectin and Fibronectin Crosslinked by Transglutaminase 2

Fibronectin (FN) is a ubiquitously expressed cell adhesion protein capable of assembling into large, extended fibrillar networks as part of an extracellular matrix (ECM) that regulates cell behavior. FN is a substrate for certain members of the transglutaminase family of protein-crosslinking enzymes-enzymes which can modify the ability of FN to support cell adhesion. In this study, we have analyzed the thermo-chemical stability of plasma FN in its noncrosslinked form, and after crosslinking by transglutaminase 2 (TG2), using dynamic light scattering. We report that FN is found in a generally globular (8.7 nm hydrodynamic radius), dimerized form in aqueous solutions, but unfolds into a linear arrangement at high ionic (1 M NaCl) and chaotropic (5 M urea) environments. FN conformation remained stable after multiple heating and cooling cycles ranging from 4 to 60 degrees C. Crosslinking of FN with TG2 formed large, multimeric complexes having high chemical stability in aqueous, high ionic and chaotropic environments, demonstrating that this covalent modification stabilizes FN. Given recent data that substrate (e.g. ECM) rigidity profoundly affects cell differentiation and behavior, we further studied how TG2 crosslinking affects the molecular rigidity of FN by obtaining atomic force microscopy nanoindentation measurements from untreated and crosslinked FN samples embedded in acrylamide gels. We demonstrate that TG2-mediated crosslinking of FN significantly increases Young's modulus (of elasticity), an observation of increased rigidity having important implications with respect to the biological role of ECM protein-crosslinking in cell signaling and guiding cell differentiation.     

Expression of Sox and fibrillar collagen genes in lamprey larval chondrogenesis with implications for the evolution of vertebrate cartilage

Lampreys possess unique types of cartilage in which elastin-like proteins are the dominant matrix component, whereas gnathostome cartilage is mainly composed of fibrillar collagen. Despite the differences in protein composition, the Sox-col2a1 genetic cascade was suggested to be conserved between lamprey pharyngeal cartilage and gnathostome cartilage. We examined whether the cascade is conserved in another type of lamprey cartilage, the trabecular cartilage. We found that SoxD and SoxE are expressed in both trabecular and pharyngeal cartilages. However, trabecular cartilage shows no clade A fibrillar collagen gene expression, including genes expressed in pharyngeal cartilage of this animal. On the basis of these observations, we propose that lampreys possess an ancestral type of cartilage that is similar to amphioxus gill cartilage, and in this respect, gnathostome cartilage can be regarded as derived for the loss of elastin-like protein as a cartilage component and recruitment of fibrillar collagen, which is included as a minor component in the ancestral cartilage, as the main component.     

Reintroduction of a Rare Plant (Gladiolus imbricatus) Population to a River Floodplain—How Important is Meadow Management?

In seminatural grasslands, the success of reintroduction of locally extinct rare plant species may depend on the ambient management regime. We aimed to study to what extent the success of the restoration of a rare species (Gladiolus imbricatus) depends on management conditions. A management experiment with traditional cutting by scythe and hay removal, mowing (machine cutting and hay removal), mulching (machine cutting without hay removal), spring burning, and unmanaged control, combined with reintroduction of seeds of Gladiolus, was conducted in an Estonian flooded meadow in which the species had become extinct. Seeds were reintroduced in 2003 in all management treatments and populations monitored until 2006. Mulching, mowing, and traditional management resulted in the greatest establishment, whereas the subsequent mortality was not influenced greatly by management regime. The population started to increase in mulching treatment in the third season due to vegetative growth. The results indicate that the establishment of G. imbricatus is primarily seed limited under current conditions, whereas favorable management significantly enhances establishment in a river floodplain meadow. Successful restoration depends on seed addition and proper grassland management—mowing to a height of approximately 15 cm and mulching.     

L-Amino acid oxidase from Naja naja oxiana venom

A new l-amino acid oxidase (LAAO) was isolated from the Central Asian cobra Naja naja oxiana venom by size exclusion, ion exchange and hydrophobic chromatography. The N-terminal sequence and the internal peptide sequences share high similarity with other snake venom l-amino acid oxidases, especially with those isolated from elapid venoms. The enzyme is stable at low temperatures (− 20 °C, − 70 °C) and loses its activity by heating at 70 °C. Specific substrates for the isolated protein are l-phenylalanine, l-tryptophan, l-methionine and l-leucine. The enzyme has antibacterial activity inhibiting the growth of Gram-positive (Bacillus subtilis) and Gram-negative (Escherichia coli) bacteria. N. naja oxiana LAAO dose-dependently inhibited ADP- or collagen-induced platelet aggregation with IC₅₀ of 0.094 μM and 0.036 μM, respectively. The antibacterial and anti-aggregating activity was abolished by catalase.     

Systematic bioinformatic analysis of expression levels of 17,330 human genes across 9,783 samples from 175 types of healthy and pathological tissues

Our knowledge on tissue- and disease-specific functions of human genes is rather limited and highly context-specific. Here, we have developed a method for the comparison of mRNA expression levels of most human genes across 9,783 Affymetrix gene expression array experiments representing 43 normal human tissue types, 68 cancer types, and 64 other diseases. This database of gene expression patterns in normal human tissues and pathological conditions covers 113 million datapoints and is available from the GeneSapiens website.     

Integration of gene dosage and gene expression in non-small cell lung cancer, identification of HSP90 as potential target

Background

Lung cancer causes approximately 1.2 million deaths per year worldwide, and non-small cell lung cancer (NSCLC) represents 85% of all lung cancers. Understanding the molecular events in non-small cell lung cancer (NSCLC) is essential to improve early diagnosis and treatment for this disease.

Methodology and Principal Findings

In an attempt to identify novel NSCLC related genes, we performed a genome-wide screening of chromosomal copy number changes affecting gene expression using microarray based comparative genomic hybridization and gene expression arrays on 32 radically resected tumor samples from stage I and II NSCLC patients. An integrative analysis tool was applied to determine whether chromosomal copy number affects gene expression. We identified a deletion on 14q32.2-33 as a common alteration in NSCLC (44%), which significantly influenced gene expression for HSP90, residing on 14q32. This deletion was correlated with better overall survival (P = 0.008), survival was also longer in patients whose tumors had low expression levels of HSP90. We extended the analysis to three independent validation sets of NSCLC patients, and confirmed low HSP90 expression to be related with longer overall survival (P = 0.003, P = 0.07 and P = 0.04). Furthermore, in vitro treatment with an HSP90 inhibitor had potent antiproliferative activity in NSCLC cell lines.

Conclusions

We suggest that targeting HSP90 will have clinical impact for NSCLC patients.     

Variation in species richness within and between calcareous (alvar) grassland stands: the role of core and satellite species

Anoda cristata is a widelydistributed annual weed in Mexico, which grows as agrestal or naturally indisturbed and undisturbed vegetation, respectively. Plants of this species aretolerated in orchards and corn-bean fields by Mexican ethnic groups ofCentral Mexico. Leaves of the plants are used as a food source, andoccasionallyseeds are sown in orchards. Because Mexicans have used Anodacristata for a long time, it is possible that ecological andmorphological characteristics of managed populations differ from those of wildpopulations. In this study, we analyzed phenotypic responses of two populationsof A. cristata (wild and agrestal) growing in twohabitats (forest and cultivated field) comparing survivorship, andlife history traits. Natural progenies from wild and agrestal populations ofA. cristata were transplanted into a cultivated field andin the understory of a pine forest in a reciprocal transplant experiment.Results showed that the habitat of transplant and the origin of populations hadsignificant effect on all plant characters measured (growth, phenology, andbiomass allocation), but the habitat x origin interaction wasnon-significant. In general, plants from the agrestal population grewfaster, reproduced earlier, and allocated more biomass to reproduction, thanplants from the wild population. Similarly, significant effects of habitat andorigin on plant survivorship were detected, but the origin of populationsexplained the largest proportion of variance in plant survival. Most traitswerephenotypically plastic but there were no differences in the magnitude ordirection of the response between populations. In contrast both populationsshowed differentiation for most character mean values. Populationdifferentiation is possibly the result of genetic differences driven byprocesses other than incipient domestication.     

Improved cider fermentation performance and quality with newly generated <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> × <Emphasis Type="Italic">Saccharomyces eubayanus</Emphasis> hybrids

Yeast cryotolerance may be advantageous for cider making, where low temperatures are usually employed. Here, we crossed the cryotolerant S. eubayanus with a S. cerevisiae wine strain and assessed the suitability of the hybrids for low-temperature cider fermentation. All strains fermented the juice to 5% ABV, but at different rates; hybrid strains outperformed S. cerevisiae, which was sensitive to low temperatures. The best hybrid fermented similarly to S. eubayanus. S. eubayanus produced sulphurous off flavours which masked a high concentration of fruity ester notes. This phenotype was absent in the hybrid strains, resulting in distinctly fruitier ciders. Aroma was assessed by an independent consumer panel, which rated the hybrid ciders as identical to the wine strain cider. Both were significantly more pleasant than the S. eubayanus cider. Interspecific hybridization can apparently be used effectively to improve low-temperature fermentation performance without compromising product quality.