Evidence for essential catalytic determinants for human erythrocyte pyrimidine 5′-nucleotidase

Human erythrocyte pyrimidine 5′-nucleotidase, PN-I, catalyzes the dephosphorylation of pyrimidine nucleoside monophosphates. The enzyme also possesses phosphotransferase activity, transferring phosphate groups between pyrimidine nucleoside monophosphates and various pyrimidine nucleosides. Deficiency of the enzyme activity is associated with a hemolytic anemia. PN-I cDNA has been expressed in Escherichia coli, yielding a fully active recombinant enzyme, which was purified to homogeneity and extensively characterized. Multiple sequence alignment of PN-I and homologues proteins revealed the existence of conserved regions, whose importance in catalysis was examined by performing experiments designed to intercept covalent intermediates as strongly suggested by our previous kinetic studies. Furthermore, a functional analysis of the enzyme was carried out through site-directed mutagenesis designed on the basis of the sequence of the identified conserved regions as well as mutations observed in PN-I-deficient patients.Received 25 March 2005; received after revision 3 May 2005; accepted 13 May 2005     

Baker's yeast cytidine deaminase: Substrate and inhibitor specificity,and a hypothesis on metabolic and regulatory significance

Riassunio La citidina deaminasi, parzialmente purificata da lievito di pane, è capace di deaminare sia la citidina che la deossicitidina. I valori delleK _m per ambedue i 2 substrati sono 25×10^–5 M e 9.1×10^–5 M rispettivamente. Inoltre l'enzima è inibito da numerosi nucleosidi monofosfati, difosfati e trifosfati. È molto significativa il tipo di inibizione allosterica esercitata dal dTTP. Si riporta una ipotesi sul ruolo metabolico della citidina deaminasi.     

Presence of a specific uridine 5'-monophosphate pyrophosphorylase in baker's yeast.

Uridine 5'-monophosphate pyrophosphorylase was found to be present in baker's yeast. The enzyme preparation, purified about 30-fold, shows a strict specificity toward uracil and requires Mg++ for its activity.     

Tas1r3, encoding a new candidate taste receptor, is allelic to the sweet responsiveness locus Sac

The ability to taste the sweetness of carbohydrate-rich foodstuffs has a critical role in the nutritional status of humans. Although several components of bitter transduction pathways have been identified, the receptors and other sweet transduction elements remain unknown. The Sac locus in mouse, mapped to the distal end of chromosome 4 (refs. 7-9), is the major determinant of differences between sweet-sensitive and -insensitive strains of mice in their responsiveness to saccharin, sucrose and other sweeteners. To identify the human Sac locus, we searched for candidate genes within a region of approximately one million base pairs of the sequenced human genome syntenous to the region of Sac in mouse. From this search, we identified a likely candidate: T1R3, a previously unknown G protein-coupled receptor (GPCR) and the only GPCR in this region. Mouse Tas1r3 (encoding T1r3) maps to within 20,000 bp of the marker closest to Sac (ref. 9) and, like human TAS1R3, is expressed selectively in taste receptor cells. By comparing the sequence of Tas1r3 from several independently derived strains of mice, we identified a specific polymorphism that assorts between taster and non-taster strains. According to models of its structure, T1r3 from non-tasters is predicted to have an extra amino-terminal glycosylation site that, if used, would interfere with dimerization.     

Burrow plugging by prairie dogs in response to Siberian polecats

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Food spectrum analysis of the Asian toad,Duttaphrynus melanostictus (Schneider, 1799) (Anura: Bufonidae), from Timor Island,Wallacea

The Asian toad, Duttaphrynus melanostictus (Schneider, 1799), is widespread throughout tropical Asia and very abundant where it occurs. It was relatively recently introduced to Timor, the second largest island in the biogeographic region called Wallacea. Timor Island shows an exceptionally high level of endemism in a wide range of faunal groups and there are concerns that D. melanostictus may have a negative impact on this diversity, including vertebrates, through direct predation. To evaluate the impact the diet of D. melanostictus might have on the local fauna, gut contents of 83 preserved toad specimens from five habitat types in Timor-Leste, a country occupying the eastern half of Timor Island, were examined. We identified 5581 prey items, comprising the following animal groups: annelids; snails and slugs; spiders and harvestmen; woodlice; millipedes and centipedes; grasshoppers, crickets and earwigs; termites; thrips and true bugs; beetles; ants; hymenopterans other than ants; true flies; butterflies; unidentified insects; and insect larvae. Small eusocial insects (ants and termites) constituted the major part of the diet (61.6% and 23.4%, respectively). No vertebrate prey was recorded. Prey item composition did not differ between habitats. The wide prey spectrum well indicates that D. melanostictus is a generalist invertebrate feeder, as other studies, from regions where this species occurs naturally, have already shown. Although the Asian toad seems to not generally prey on vertebrates, vertebrate species that are morphologically similar to invertebrates in their overall appearance may be consumed. Hence, a negative effect on some taxa (e.g. blindsnakes) may be possible. We also present some limited data on intestinal parasites occurring in D. melanostictus.     

Sequencing of neuroblastoma identifies chromothripsis and defects in neuritogenesis genes

Neuroblastoma is a childhood tumour of the peripheral sympathetic nervous system. The pathogenesis has for a long time been quite enigmatic, as only very few gene defects were identified in this often lethal tumour. Frequently detected gene alterations are limited to MYCN amplification (20%) and ALK activations (7%). Here we present a whole-genome sequence analysis of 87 neuroblastoma of all stages. Few recurrent amino-acid-changing mutations were found. In contrast, analysis of structural defects identified a local shredding of chromosomes, known as chromothripsis, in 18% of high-stage neuroblastoma. These tumours are associated with a poor outcome. Structural alterations recurrently affected ODZ3, PTPRD and CSMD1, which are involved in neuronal growth cone stabilization. In addition, ATRX, TIAM1 and a series of regulators of the Rac/Rho pathway were mutated, further implicating defects in neuritogenesis in neuroblastoma. Most tumours with defects in these genes were aggressive high-stage neuroblastomas, but did not carry MYCN amplifications. The genomic landscape of neuroblastoma therefore reveals two novel molecular defects, chromothripsis and neuritogenesis gene alterations, which frequently occur in high-risk tumours.     

Mutations in the profilin 1 gene cause familial amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a late-onset neurodegenerative disorder resulting from motor neuron death. Approximately 10% of cases are familial (FALS), typically with a dominant inheritance mode. Despite numerous advances in recent years, nearly 50% of FALS cases have unknown genetic aetiology. Here we show that mutations within the profilin 1 (PFN1) gene can cause FALS. PFN1 is crucial for the conversion of monomeric (G)-actin to filamentous (F)-actin. Exome sequencing of two large ALS families showed different mutations within the PFN1 gene. Further sequence analysis identified 4 mutations in 7 out of 274 FALS cases. Cells expressing PFN1 mutants contain ubiquitinated, insoluble aggregates that in many cases contain the ALS-associated protein TDP-43. PFN1 mutants also display decreased bound actin levels and can inhibit axon outgrowth. Furthermore, primary motor neurons expressing mutant PFN1 display smaller growth cones with a reduced F/G-actin ratio. These observations further document that cytoskeletal pathway alterations contribute to ALS pathogenesis.