The NADH diphosphatase encoded by the Saccharomyces cerevisiae NPY1 nudix hydrolase gene is located in peroxisomes

The NPY1 nudix hydrolase gene of Saccharomyces cerevisiae has been cloned and shown to encode a diphosphatase (pyrophosphatase) with NADH as the preferred substrate, giving NMNH and AMP as products. NADPH, diadenosine diphosphate, NAD+, NADP+, and ADP-ribose were also utilized efficiently. Km values for NADH, NAD+, and ADP-ribose were 0.17, 0.5, and 1.3 mM and kcat values 1.5, 0.6, and 0.6 s(-1), respectively. NPY1 has a potential C-terminal tripeptide PTS1 peroxisomal targeting signal (SHL). By fusing NPY1 to the C-terminus of yeast-enhanced green fluorescent protein, the enzyme was found to be targeted to peroxisomes. Colocalization with peroxisomal thiolase was also shown by indirect immunofluorescence. Related sequences in other organisms also have potential PTS1 signals, suggesting an important peroxisomal function for this protein. This function may be the regulation of nicotinamide coenzyme concentrations independently of those in other compartments or the elimination of oxidized nucleotide derivatives from the peroxisomal environment.     

Cloning, expression, and characterization of a human inosine triphosphate pyrophosphatase encoded by the itpa gene

ITP and dITP exist in all cells. dITP is potentially mutagenic, and the levels of these nucleotides are controlled by inosine triphosphate pyrophosphatase (EC ). Here we report the cloning, expression, and characterization of a 21.5-kDa human inosine triphosphate pyrophosphatase (hITPase), an enzyme whose activity has been reported in many animal tissues and studied in populations but whose protein sequence has not been determined before. At the optimal pH of 10.0, recombinant hITPase hydrolyzed ITP, dITP, and xanthosine 5'-triphosphate to their respective monophosphates whereas activity with other nucleoside triphosphates was low. K(m) values for ITP, dITP, and xanthosine 5'-triphosphate were 0.51, 0.31, and 0.57 mm, respectively, and k(cat) values were 580, 360, and 640 s(-1), respectively. A divalent cation was absolutely required for activity. The gene encoding the hITPase cDNA sequence was localized by radiation hybrid mapping to chromosome 20p in the interval D20S113-D20S97, the same interval in which the ITPA inosine triphosphatase gene was previously localized. A BLAST search revealed the existence of many similar sequences in organisms ranging from bacteria to mammals. The function of this ubiquitous protein family is proposed to be the elimination of minor potentially mutagenic or clastogenic purine nucleoside triphosphates from the cell.     

ABC: software for interactive browsing of genomic multiple sequence alignment data

Background  

Alignment and comparison of related genome sequences is a powerful method to identify regions likely to contain functional elements. Such analyses are data intensive, requiring the inclusion of genomic multiple sequence alignments, sequence annotations, and scores describing regional attributes of columns in the alignment. Visualization and browsing of results can be difficult, and there are currently limited software options for performing this task.     

Procathepsin and acid phosphatase are stored in Musca domestica yolk spheres

Yolk spheres present in mature invertebrate oocytes are composed of yolk proteins and proteolytic enzymes. In the fly Musca domestica, yolk proteins are degraded during embryogenesis by a cathepsin-like proteinase that is stored as a zymogen. An acid phosphatase is also active in the yolk spheres during Musca embryogenesis. In this paper we show that procathepsin and acid phosphatase are initially stored by a different pathway from the one followed by yolk protein precursors. Both enzymes are taken up by the oocytes and transitorily stored into small vesicles (lysosomes) surrounding the early yolk spheres. Fusion of both structures, the early yolk spheres and lysosomes, creates the mature yolk spheres.     

The IalA invasion gene of Bartonella bacilliformis encodes a (de)nucleoside polyphosphate hydrolase of the MutT motif family and has homologs in other invasive bacteria

The product of the ialA invasion gene of Bartonella bacilliformis has been expressed as a thioredoxin fusion protein. It is a (di)nucleoside polyphosphate hydrolase of the MutT motif protein family with strong sequence similarity to plant diadenosine tetraphosphate hydrolases. It hydrolyses nucleoside and dinucleoside polyphosphates with four or more phosphate groups, always producing an NTP as one product. Diadenosine tetraphosphate (Ap4A) is the preferred substrate with a Km of 10 microM and a kcat of 3.0 s-1. It is inhibited by Ca2+ and F- (Ki = 30 microM). Hydrolysis of Ap4A in H218O yielded [18O]AMP as the only labelled product. In terms of sequence, reaction mechanism and properties, IalA is very similar to eukaryotic Ap4A hydrolases and unlike previously described bacterial Ap4A hydrolases. Homologs are present in the genomes of other invasive pathogens. They may function to reduce stress-induced dinucleotide levels during invasion and so enhance pathogen survival.     

Nudix hydrolases that degrade dinucleoside and diphosphoinositol polyphosphates also have 5-phosphoribosyl 1-pyrophosphate (PRPP) pyrophosphatase activity that generates the glycolytic activator ribose 1,5-bisphosphate

A total of 17 Nudix hydrolases were tested for their ability to hydrolyze 5-phosphoribosyl 1-pyrophosphate (PRPP). All 11 enzymes that were active toward dinucleoside polyphosphates with 4 or more phosphate groups as substrates were also able to hydrolyze PRPP, whereas the 6 that could not and that have coenzyme A, NDP-sugars, or pyridine nucleotides as preferred substrates did not degrade PRPP. The products of hydrolysis were ribose 1,5-bisphosphate and P(i). Active PRPP pyrophosphatases included the diphosphoinositol polyphosphate phosphohydrolase (DIPP) subfamily of Nudix hydrolases, which also degrade the non-nucleotide diphosphoinositol polyphosphates. K(m) and k(cat) values for PRPP hydrolysis for the Deinococcus radiodurans DR2356 (di)nucleoside polyphosphate hydrolase, the human diadenosine tetraphosphate hydrolase, and human DIPP-1 (diadenosine hexaphosphate and diphosphoinositol polyphosphate hydrolase) were 1 mm and 1.5 s(-1), 0.13 mm and 0.057 s(-1), and 0.38 mm and 1.0 s(-1), respectively. Active site mutants of the Caenorhabditis elegans diadenosine tetraphosphate hydrolase had no activity, confirming that the same active site is responsible for nucleotide and PRPP hydrolysis. Comparison of the specificity constants for nucleotide, diphosphoinositol polyphosphate, and PRPP hydrolysis suggests that PRPP is a significant substrate for the D. radiodurans DR2356 enzyme and for the DIPP subfamily. In the latter case, generation of the glycolytic activator ribose 1,5-bisphosphate may be a new function for these enzymes.     

Differentiation of naive CD4<Superscript>+</Superscript> T cells towards T helper 2 cells is not impaired in rheumatoid arthritis patients

An impaired differentiation of naive CD4+ T cells towards Th2 cells may contribute to the chronic tissue-destructive T-cell activity in rheumatoid arthritis (RA). The differentiation of naive CD4+ T cells into memory Th2 cells by IL-7 in comparison with that by IL-4 was studied in RA patients and in healthy controls. Naive CD4+ T cells from peripheral blood were differentiated by CD3/CD28 costimulation in the absence of or in the presence of IL-7 and/or IL-4. The production of IFN-gamma and IL-4 was measured by ELISA and by single-cell FACS analysis to indicate Th1 and Th2 cell activity. CD3/CD28 costimulation and IL-7 were early inducers of IL-4 production, but primarily stimulated IFN-gamma production. In contrast, in short-term cultures exogenously added IL-4 did not prime for IL-4 production but suppressed IL-7-induced IFN-gamma production. Upon long-term stimulation of naive CD4+ T cells, IFN-gamma production was differentially regulated by IL-7 and IL-4, but IL-4 production was increased by both IL-7 and IL-4. IL-7 and IL-4 additively induced polarization towards a Th2 phenotype. This susceptibility of naive CD4+ T cells to become Th2 cells upon culture with IL-7 and IL-4 was increased in RA patients compared with that in healthy controls. These findings demonstrate that, in RA patients, differentiation of naive CD4+ T cells towards a Th2 phenotype by CD3/CD28 costimulation, IL-7 and IL-4 is not impaired. The perpetuation of arthritogenic T-cell activity in RA therefore seems not to be the result of intrinsic defects of naive CD4+ T cells to develop towards suppressive memory Th2 cells.     

Spinal motor axons and neural crest cells use different molecular guides for segmental migration through the rostral half-somite

The peripheral nervous system in vertebrates is composed of repeating metameric units of spinal nerves. During development, factors differentially expressed in a rostrocaudal pattern in the somites confine the movement of spinal motor axons and neural crest cells to the rostral half of the somitic sclerotome. The expression patterns of transmembrane ephrin-B ligands and interacting EphB receptors suggest that these proteins are likely candidates for coordinating the segmentation of spinal motor axons and neural crest cells. In vitro, ephrin-B1 has indeed been shown to repel axons extending from the rodent neural tube (Wang & Anderson, 1997). In avians, blocking interactions between EphB3 expressed by neural crest cells and ephrin-B1 localized to the caudal half of the somite in vivo resulted in loss of the rostrocaudal patterning of trunk neural crest migration (Krull et al., 1997). The role of ephrin-B1 in patterning spinal motor axon outgrowth in avian embryos was investigated. Ephrin-B1 protein was found to be expressed in the caudal half-sclerotome and in the dermomyotome at the appropriate time to interact with the EphB2 receptor expressed on spinal motor axons. Treatment of avian embryo explants with soluble ephrin-B1, however, did not perturb the segmental outgrowth of spinal motor axons through the rostral half-somite. In contrast, under the same treatment conditions with soluble ephrin-B1, neural crest cells migrated aberrantly through both rostral and caudal somite halves. These results indicate that the interaction between ephrin-B1 and EphB2 is not required for patterning spinal motor axon segmentation. Even though spinal motor axons traverse the same somitic pathway as neural crest cells, different molecular guidance mechanisms appear to influence their movement.