Purine nucleoside phosphorylase (PNP), the enzyme schematically next to adenosine deaminase in the purine salvage pathway, has been demonstrated cytochemically in peripheral blood lymphocytes of healthy subjects and chronic lymphocytic leukemia (CLL) patients. The enzyme activity is confined to the cytosol. In healthy subjects the majority of lymphocytes are strongly reactive for PNP, whereas the rest are devoid of cytochemically demonstrable activity. The percentage of PNP-positive cells largely corresponds to the number of E rosette-forming cells and is inversely proportional to the number of Ig-bearing cells. In six of seven CLL patients studied only a minor percentage of the lymphocytes showed strong PNP activity, whereas the large majority (88%--98%) possessed trace activity. Such patients have a high number of Ig-bearing cells and a low number of E rosette-forming cells. A different pattern of markers was found in the lymphocytes of the seventh CLL patient: 66% were strongly reactive for PNP, an important number formed E rosettes, and a minor percentage were Ig bearing. These data indicate that PNP can be useful as a "nonmembrane" marker in the differentiation of the B and T cell origin in CLL and deserves to be studied in other lymphoproliferative disorders. (+info)
(2/935) Expression of pyrimidine nucleoside phosphorylase mRNA plays an important role in the prognosis of patients with oesophageal cancer.
To clarify the significance of the expression of pyrimidine nucleoside phosphorylase (PyNPase) mRNA as a predictive factor for the prognosis of patients with oesophageal carcinoma, the PyNPase mRNA in the tumours and normal tissues from 55 resected cases of oesophageal carcinoma was examined by a reverse transcription polymerase chain reaction (RT-PCR). As a result, a positive correlation was observed between the tumour/normal (T/N) ratio of the expression of PyNPase mRNA by RT-PCR and that of the enzyme activity of PyNPase based on the findings of an enzyme linked immunosolvent assay (r = 0.594, P = 0.009). The T/N ratio of the expression of PyNPase mRNA was significantly higher in the cases with lymph vessel invasion (P = 0.013), lymph node metastasis (P = 0.0016), and an advanced stage of the disease (P = 0.021) than those without these factors. The patients with a higher T/N ratio of PyNPase mRNA showed significantly worse prognosis than those with a lower T/N ratio (P = 0.023 with log-rank tests). A multivariate analysis for the cumulative survival rates revealed that a high T/N ratio of the expression of PyNPase mRNA was independently related to a poor prognosis. These findings suggested that the determination of PyNPase mRNA by RT-PCR thus appears to be a new useful parameter for identifying both a poor prognosis and a highly malignant potential of oesophageal carcinoma. (+info)
(3/935) Structural motif of phosphate-binding site common to various protein superfamilies: all-against-all structural comparison of protein-mononucleotide complexes.
In order to search for a common structural motif in the phosphate-binding sites of protein-mononucleotide complexes, we investigated the structural variety of phosphate-binding schemes by an all-against-all comparison of 491 binding sites found in the Protein Data Bank. We found four frequently occurring structural motifs composed of protein atoms interacting with phosphate groups, each of which appears in different protein superfamilies with different folds. The most frequently occurring motif, which we call the structural P-loop, is shared by 13 superfamilies and is characterized by a four-residue fragment, GXXX, interacting with a phosphate group through the backbone atoms. Various sequence motifs, including Walker's A motif or the P-loop, turn out to be a structural P-loop found in a few specific superfamilies. The other three motifs are found in pairs of superfamilies: protein kinase and glutathione synthetase ATPase domain like, actin-like ATPase domain and nucleotidyltransferase, and FMN-linked oxidoreductase and PRTase. (+info)
(4/935) Identification and characterization of a DeoR-specific operator sequence essential for induction of dra-nupC-pdp operon expression in Bacillus subtilis.
The deoR gene located just upstream the dra-nupC-pdp operon of Bacillus subtilis encodes the DeoR repressor protein that negatively regulates the expression of the operon at the level of transcription. The control region upstream of the operon was mapped by the use of transcriptional lacZ fusions. It was shown that all of the cis-acting elements, which were necessary for full DeoR regulation of the operon, were included in a 141-bp sequence just upstream of dra. The increased copy number of this control region resulted in titration of the DeoR molecules of the cell. By using mutagenic PCR and site-directed mutagenesis techniques, a palindromic sequence located from position -60 to position -43 relative to the transcription start point was identified as a part of the operator site for the binding of DeoR. Furthermore, it was shown that a direct repeat of five nucleotides, which was identical to the 3' half of the palindrome and was located between the -10 and -35 regions of the dra promoter, might function as a half binding site involved in cooperative binding of DeoR to the regulatory region. Binding of DeoR protein to the operator DNA was confirmed by a gel electrophoresis mobility shift assay. Moreover, deoxyribose-5-phosphate was shown to be a likely candidate for the true inducer of the dra-nupC-pdp expression. (+info)
(5/935) Up-regulated pyrimidine nucleoside phosphorylase in breast carcinoma correlates with lymph node metastasis.
BACKGROUND: The clinical significance of pyrimidine nucleoside phosphorylase (PyNPase) activity in breast carcinomas has never been determined. MATERIALS AND METHODS: In 41 cases of breast carcinoma, the enzyme activity of PyNPase was determined by the high performance liquid chromatography (HPLC) assay and its value was analyzed with clinicopathologic variables. The expression level of mRNA was examined by the semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) assay and compared with the enzyme activity. RESULTS: The higher activity of PyNPase was significantly correlated not only with the presence of vascular permeation (P = 0.02) but of lymph node metastasis (P = 0.02). The mRNA expression correlated well with the enzyme activity (r = 0.74, P < 0.01). A multivariate analysis disclosed the PyNPase factor to be associated with lymph node metastasis. In addition, 17 (41%) showed positive staining only in the tumor stromal cells and 18 (44%) cases showed positive staining in both the tumor stromal cells and the carcinoma cells by immunohistochemical study. CONCLUSIONS: These findings suggest that PyNPase activity is a new marker predicting the malignant potential of breast carcinomas, especially with respect to lymph node metastasis, and that the RT-PCR assay is a more useful method than direct evaluation of PyNPase activity. (+info)
(6/935) Uridine diphosphate xylosyltransferase activity in cartilage from manganese-deficient chicks.
The glycosaminoglycan content of cartilage is decreased in manganese deficiency in the chick (perosis). The activity of xylosyltransferase, the first enzyme in the biosynthetic pathway of sulphated glycosaminoglycans, was studied in the epiphysial cartilage of 4-week-old chicks which had been maintained since hatching on a manganese-deficient diet. Enzymic activity was measured by the incorporation of [14C]xylose from UDP-[14C]xylose into trichloroacetic acid precipitates. Optimal conditions for the xylosyltransferase assay were established and shown to be the same for both control and manganese-deficient cartilage. Assay of the enzyme by using an exogenous xylose acceptor showed no difference in xylosyltransferase activity between control and manganese-deficient tissue. Further, the extent of xylose incorporation was greater in manganese-deficient than in control cartilage preparations, suggesting an increase in xylose-acceptor sites on the endogenous acceptor protein in the deficient cartilage. 35S turnover in the manganese-deficient cartilage was also increased. The data suggest that the decreased glycosaminoglycan content in manganese-deficient cartilage is due to decreased xylosylation of the acceptor protein plus increased degradation of glycosaminoglycan. (+info)
(7/935) Developmental changes in purine phosphoribosyltransferases in human and rat tissues.
1. The hypoxanthine/guanine and adenine phosphoribosyltransferase activities in a wide variety of human tissues were studied during their growth and development from foetal life onward. A wide range of activities develop after birth, with especially high values in the central nervous system and testes. 2. Postnatal development of hypoxanthine/guanine phosphoribosyltransferase was also defined in the rat. Although there were increases in the central nervous system and testes, there was also a rise in activity in the liver, which was less marked in man. 3. A sensitive radiochemical assay method, using dTTP to inhibit 5'-nucleotidase activity, suitable for tissue extracts, was developed. 4. No definite evidence of the existence of tissue-specific isoenzymes of hypoxanthine/guanine or adenine phosphoribosyltransferase was found. Hypoxanthine/guanine phosphoribosyltransferase in testes, however, had a significantly different thermal-denaturation rate constant. 5. The findings are discussed in an attempt to relate activity of hypoxanthine/guanine phosphoribosyltransferase to biological function. Growth as well as some developmental changes appear to be related to increase in the activity of this enzyme. (+info)
(8/935) Isolation and characterization of the Saccharomyces cerevisiae XPT1 gene encoding xanthine phosphoribosyl transferase.
A new Saccharomyces cerevisiae gene, XPT1, was isolated as a multicopy suppressor of a hypoxanthine phosphoribosyl transferase (HPRT) defect. Disruption of XPT1 affects xanthine utilization in vivo and results in a severe reduction of xanthine phosphoribosyl transferase (XPRT) activity while HPRT is unaffected. We conclude that XPT1 encodes XPRT in yeast. (+info)