The complete genomic sequence of Mycoplasma penetrans, an intracellular bacterial pathogen in humans.

The complete genomic sequence of an intracellular bacterial pathogen, Mycoplasma penetrans HF-2 strain, was determined. The HF-2 genome consists of a 1 358 633 bp single circular chromosome containing 1038 predicted coding sequences (CDSs), one set of rRNA genes and 30 tRNA genes. Among the 1038 CDSs, 264 predicted proteins are common to the Mycoplasmataceae sequenced thus far and 463 are M.penetrans specific. The genome contains the two-component system but lacks the essential cellular gene, uridine kinase. The relatively large genome of M.penetrans HF-2 among mycoplasma species may be accounted for by both its rich core proteome and the presence of a number of paralog families corresponding to 25.4% of all CDSs. The largest paralog family is the p35 family, which encodes surface lipoproteins including the major antigen, P35. A total of 44 genes for p35 and p35 homologs were identified and 30 of them form one large cluster in the chromosome. The genetic tree of p35 paralogs suggests the occurrence of dynamic chromosomal rearrangement in paralog formation during evolution. Thus, M.penetrans HF-2 may have acquired diverse repertoires of antigenic variation-related genes to allow its persistent infection in humans.

Deoxycytidine kinase, thymidine kinase and cytidine deaminase and the formation of Ara-CTP in leukemic cells in different phases of the cell cycle.

In this study we investigated the Ara-CTP-forming capacity of leukemic cells in different phases of the cell cycle. Cells from two leukemic cell lines and leukemic bone marrow cells from patients and rats (BNML model) with acute myelocytic leukemia were separated according to cell cycle phase by means of an albumin density gradient in a specially designed sedimentation chamber. We found that the activity of CdR kinase and Cyt deaminase is much less influenced by cell-cycle phase progression than TdR kinase activity. For the leukemic cell lines HL-60 and BNML-CL/O CdR kinase activity is even independent of cell-cycle phase. In addition, Ara-CTP formation is not restricted to cells in S-phase. Cell cycle phase-independent Ara-CTP formation creates a situation in which cells which are not in S-phase during exposure to Ara-C might undergo the cytotoxic effects of Ara-C as soon as they enter S-phase.

Enzyme pathology of human mesotheliomas.

In samples of 16 surgically resected mesotheliomas arising from the pleura of the human lung, 6 enzymes from different metabolic pathways, DNA, and mitotic frequency were quantified. The mesotheliomas, irrespective of cell type or grade, showed lower gamma-glutamyl transpeptidase (GGT) concentration than 36 of the 38 pulmonary adenocarcinomas. The mean concentration of this enzyme in the 15 mesotheliomas was an eighth of that in the 56 carcinomas, whereas their DNA content was similar. The quantitative correlation of thymidine kinase (TK), uridine kinase (UK), and phosphoserine phosphatase to mitotic frequency was highly significant for mesotheliomas, as well as for carcinomas. As estimated from their TK [and its recently established quantitative correlation to volume doubling time (DT)], the DT of the 16 mesotheliomas ranged from 50 to over 700 days, with a somewhat longer median than the median for pulmonary carcinomas. Subject survival, though shortest for the 2 sarcomatous mesothelioma cases, varied over an overlapping range for mesotheliomas with epithelial or mixed cell type. The biopsy samples' TK and UK concentrations, however, showed a significant inverse correlation with months of survival after diagnosis. Survival time after the first appearance of symptoms decreased linearly (on log scales) with TK concentration (P less than .001) over the 14 cases. The results of this first quantitative study of a spectrum of biochemical constituents of mesotheliomas identify GGT as an enzyme whose measurement guards against mistaking mesotheliomas and adenocarcinomas for one another and show that the TK concentrations of these mesothelioma samples bear a highly significant, inverse correlation to the postdiagnosis survival time of the individual subjects.

Mechanisms of potentiation of antitumor activity of 5-fluorouracil by ribothymidine in a mouse tumor system.

In the presence of ribothymidine, the uptake of 5-fluorouracil (5-FU) was enhanced in Ehrlich ascites carcinoma cells in vitro, and was efficiently converted to FU-nucleotides. In these cells, almost all ribothymidine was found to be converted to thymine by, presumably, uridine phosphorylase. The enhanced uptake of 5-FU and its efficient conversion to FU-nucleotides were also observed in Ehrlich ascites carcinoma of ribothymidine-coadministered mice. The radioactivity of 5-FU-14C in the carcinoma cells, which was detected mainly in the acid-soluble fraction immediately after the administration and then shifted to the ribonucleic acid fraction, was approximately 2-fold higher in the ribothymidine-coadministered group than in the control group. On the other hand, ribothymidine-14C was also taken up by the carcinoma cells and an appreciable portion of its radioactivity appeared in the thymine fraction. Thymine produced from ribothymidine-14C was also detected in the extracellular ascitic fluid. The conversion of ribothymidine to thymine was assumed to be catalyzed by uridine phosphorylase, suggesting that ribose 1-phosphate was coproduced and served as a ribose donor for 5-FU. These results suggest that ribothymidine may act as a ribose donor for 5-FU to form FU-nucleotides via 5-fluorouridine in carcinoma cells, resulting in the potentiation of 5-FU activity.

Enhancement of pyrimidine nucleoside uptake into K562 and YAC-1 cells by cadeguomycin.

Cadeguomycin markedly stimulated the uptake of thymidine, deoxycytidine and uridine into the acid-insoluble fraction of K562 human leukemic cells, but did not significantly affect adenosine incorporation. The enhancement of pyrimidine nucleoside uptake was 6 approximately 17 fold over the control. Aspartate incorporation into nucleic acid was not significantly blocked by the antibiotic, suggesting that the stimulation of pyrimidine nucleoside incorporation is not due to the inhibition of de novo pyrimidine nucleotide synthesis. Net DNA and RNA syntheses, observed by [32P]phosphate uptake, were not significantly affected by cadeguomycin. The enzymatic activity of thymidine, deoxycytidine and uridine kinases was higher in cadeguomycin-treated cells than in untreated cells, suggesting that the enhancement of pyrimidine nucleoside uptake occurs in the phosphorylation process. The stimulatory activity of cadeguomycin of thymidine uptake was reversed by guanosine and deoxyguanosine, but not by adenosine and deoxyadenosine, suggesting that intracellular metabolism and/or action of cadeguomycin is related to that of guanosine and deoxyguanosine. The stimulation of pyrimidine nucleoside incorporation by cadeguomycin was also found with YAC-1 cells, but not with the other cell lines. The enhancement effect of the antibiotic seems to be not directly related to its cytotoxicity.

Biochemical measure of the volume doubling time of human pulmonary neoplasms.

The volume doubling time (DT) of human lung neoplasms, determined from sequential, presurgery roentgenograms, was compared with biochemical and histologic observations on biopsy samples of the same tumors obtained during surgery. The DTs of the 16 neoplasms ranged from 24 days in an oat cell carcinoma to over 500 in a pulmonary carcinoid tumor, and showed a statistically significant, inverse correlation to the TK (thymidine kinase) and the UK (uridine kinase) concentration of the biopsy samples per g wet weight or mg DNA. Log DT bore a linear relationship to log TK (r = 0.75, P = 0.0008) and to log UK (r = 0.69, P = 0.0067), and an even better fit to the straight line was found when plotting the log of the standardized average of the two enzymes against log DT. The results demonstrate the feasibility of a biochemical method for determining the unknown DT of the many tumors that are not amenable (on account of shape, location, or lack of prior chest x-rays) to the direct, radiologic determination of this useful, dynamic parameter of clinical malignancy.

Effects of N4-methyl and N4,N4-dimethyl derivatives of 5-azacytidine on liver RNA synthesis and gastric secretion in rats.

Of different N-alkyl substituted derivatives of 5-azacytidine tested for their action on gastric secretion in rats with ligated pylorus the most effective beside 5-azacytidine were N4-methyl and N4,N4-dimethyl derivatives. The drugs blocked gastric secretion, gastric acidity, the extent of hemorrhage and the number and size of gastric defects. Simultaneously a higher synthesis of RNA in the liver of drug-treated animals was observed.

Decreased host toxicity in vivo during chronic treatment with 5-flourouracil.

Chronic weekly administration of FUra to CD8F1 female mice bearing spontaneous mammary tumors produced body weight loss during the first 2 weeks of treatment, which became less severe during subsequent weeks of therapy. To our knowledge, the development of such a decrease in FUra toxicity in vivo during chronic treatment with the drug has not been described previously, and a study of this phenomenon was therefore undertaken in tumor-free CD8F1 female mice. Weekly administration of FUra at 85 mg/kg resulted in toxicity expressed in body weight loss and in depressed peripheral WBC levels; however, the magnitude of these toxic effects decreased significantly by the 5th week of treatment. Pretreatment of normal mice with FUra for 7 weeks resulted in a dose-related shift in the LD50 of FUra administered as a subsequent challenge. Compared with an LD50 of 240 mg/kg for FUra in normal mice, the LD50 in mice pretreated with FUra at 50 or 85 mg/kg per week was found to be significantly elevated to 370 and 460 mg/kg, respectively. Pretreatment with FUra at 85 mg/kg for 7 weeks did not alter the activity of the enzymes responsible for the activation of FUra, namely uridine kinase or orotate phosphoribosyltransferase, in the intestinal epithelium or bone marrow, but it did decrease the 24-h urinary excretion of intact [3H]FUra by almost 40% (P less than 0.01). In addition, the FUra pretreatment schedule resulted in a 31% (P = 0.14) increase in the activity of dihydrouracil dehydrogenase in the liver. These results suggest that increased degradation of FUra can be induced by chronic treatment with the drug. Finally, knowledge of the development of increased drug catabolism was used to increase the therapeutic effectiveness of FUra by its incorporation into an increasing-dose regimen. Mice bearing 24-h transplants of the murine breast tumor were treated with a constant dose of FUra for 12 weeks or with a dose that was increased, after 7 weeks, to a dose normally causing a high degree of drug-related mortality. The group receiving the incremented FUra dose had a significantly slower tumor growth rate without an increase in drug-related toxicity. These results are discussed in light of their obvious clinical implications.

Metabolic effects of acarbose administration in normal and diabetic rats.

The effect of acarbose on cardiac and hepatic metabolism was investigated in normal and diabetic rats. Groups of rats were fed one of the three following diets for 7 days: (1) ground Purina chow, (2) ground Purina chow fortified with raw corn starch and sucrose, and (3) the above high carbohydrate diet, with added acarbose (40 mg/100 g food). At the end of the dietary period the rats were decapitated, and a sample of liver tissue was removed and frozen in liquid nitrogen. The heart was extirpated for subsequent perfusion by the Langendorff technique. Increases in liver and heart glycogen produced by the high carbohydrate diet in the normal rats were prevented completely when acarbose was incorporated into the food. In diabetic animals, liver glycogen was uniformly lower than normal, irrespective of the diet or the presence of acarbose. With animals fed the control diet, cardiac glycogen was higher in diabetic than in normal rats. The high carbohydrate diet caused a lowering of heart glycogen in diabetic rats and this reduction in glycogen content was reversed by including acarbose in the diet. Effects of isoproterenol on myocardial phosphorylase a activity were determined in hearts from normal and diabetic rats given one of the three diets. The high carbohydrate diet decreased the enzymatic response to the catecholamine in hearts from both normal and diabetic animals, and this phenomenon was prevented by the presence of acarbose in the diet. In diabetic rats fed any of the three diets, the activation of cardiac phosphorylase by isoproterenol was greatly accentuated. Measurements of heart uridine kinase showed that the activity of this enzyme was lower than normal in hearts from diabetic rats given either the control or the high carbohydrate diet. The presence of acarbose in the latter diet resulted in a significant decrease in cardiac uridine kinase activity in hearts from normal rats. The results of this study demonstrate the effectiveness of acarbose in modulating tissue metabolism in normal and diabetic animals.

Uridine kinase molecular species and uridine uptake in some variants of rat hepatomas.

In a family of clonal lines derived from the Reuber H 35 rat hepatoma, four electrophoretically distinct molecular forms of uridine kinase (UK I, II, III, and IV) have been characterized. They are the same as those found in foetal rat liver. Different UK profiles occur in these cell lines, and no strict correlation could be established between the state of differentiation of the cells and the form of UK expressed. A clone of somatic hybrid cells between line p4 (form 1 only) and Fu5-5 (forms II, III, and IV) that does not express form I indicates that p4 cells may lack a factor controlling the polymerization of form I. This variety of clonal cell lines was used to study the uptake and phosphorylation of labeled uridine. The results suggest a relationship between the UK form present and the rate uridine phosphorylation by the intact cells.

High uridine phosphorylase activity in human melanoma tumor.

Uridine (Urd) phosphorylase and Urd kinase activities were examined in 4 human tumor types including melanoma and human and mouse melanoma cell lines. Urd phosphorylase activity in melanoma tumor specimens was higher than in specimens of colon, ovarian, and breast tumors. Urd kinase activity levels were similar in the 4 tumor types. In 3 human melanoma cell lines examined, Urd phosphorylase activity was markedly greater than in mouse B16 melanoma cells, while Urd kinase activity did not differ appreciably in the human and mouse cell lines. Urd phosphorylase activity in crude extract preparations from different melanoma cell lines showed similar substrate affinity and sensitivity to 1-(2'-deoxy-beta-D-glucopyranosyl)-thymine, a specific inhibitor. The high Urd phosphorylase activity found in melanoma tumor tissue may be exploited in the treatment of malignant melanoma with antipyrimidine agents. Cultured human melanoma cells retain this biochemical characteristic and may serve as appropriate in vitro models for the human tumor in studies concerning pyrimidine metabolism.

Multiple forms and inhibitors of uridine-cytidine kinase in neoplastic cells.

1. Two forms (isozymes) of uridine (urd)-cytidine (cyd) kinase are present in the 30-50% ammonium sulfate fraction of the cytosols of L1210 ascites leukemia cells and a human malignant lymphoma. 2. These findings confirm those which described multiple forms of urd-cyd kinase in tumors with rapid growth rate. 3. Studied of inhibitors (nucleoside analogs) of urd-cyd kinase derived from L1210 and 6410 leukemia cells resulted in the finding of four possible inhibitors of this enzyme.

Determination of pyrimidine phosphoribosyltransferase and uridine kinase activities by an assay with DEAE-paper discs.

Rapid (6 min washing step) and reliable methods for determining uridine kinase and pyrimidine phosphoribosyltransferase activities were devised. These procedures, consisting of a spotting technique on DEAE-discs followed by washing and elution, permitted the consistent recovery of about 90% of the nucleoside 5'-phosphate esters formed from radioactive precursors, either uridine or 5-fluorouracil, respectively. Of these precursors, less than 2% were retained on the discs. Direct counting of the discs (without elution), filtration by either gravity or suction, and the use of so-called activation of discs have not proved advantageous.

Electrophoretically distinct forms of uridine kinase in the rat. Tissue distribution and age-dependence.

By the use of polyacrylamide-gel electrophoresis, uridine kinase from foetal rat liver was separated into four types designated I, II, III and IV in decreasing order of mobility towards the anode. The most anodic (type I) was found only in rapidly growing tissues, such as foetal liver and brain, postnatal spleen and tumour cells. In adult tissues, types II, III and IV were found in the kidney, and types III and IV in the spleen and the liver, whereas type IV was the sole form of uridine kinase present in the brain.

Incorporation characteristics of uracil, uridine, and orotic acid into ribonucleic acid of neoplastic cells.

Incorporation of uracil and uridine into ribonucleic acid (RNA) was compared among the ascitic and solid forms of Ehrlich mouse tumor, Morris hepatoma, Rhodamine sarcoma, gastric cancer and ulcer from human patients, and several normal rat tissues. Of these cells tested, the cells of Ehrlich ascites and solid tumors, human gastric cancer and ulcer, and certain tissues of a normal rat showed a considerably high activity. Furthermore, Ehrlich ascites tumor cells indicating a high incorporation activity was also high in activities of both phosphorylase and kinase for uridine, while Rhodamine sarcoma as a representative having a low incorporation activity was considerably low in these two enzymic activities. RNA synthesis from uridine phosphates by Rhodamine sarcoma was maintained to a fairly high extent contrary to its low activities of the phosphorylase and the kinase. Consequently, the low utilization of uracil and uridine by certain tumors was suggested to be due to the extremely low activities of both enzymes.

Pyrimidine metabolism during restorative brain growth after neonatal undernutrition in the rat.

Three litters of 20 Wistar rat pups each were maintained until age 6 days at which time only the 4 lightest and 4 heaviest pups from each litter were left with the mother until age 13 days. Three control litters of eight pups each were also maintained for 13 days. At that time, the undernourished light pups showed body weight, cerebellar weight, and cerebellar DNA, respectively, of 79.2%, 86.6%, and 90.4% compared with a "combined control" group consisting of control pups plus undernourished heavy pups which were statistically indistinguishable with regard to these three measurements. After the week of "catch-up" or restorative body and brain growth, activities of enzymes from metabolic pathways leading to pyrimidine and nucleic acid biosynthesis were measured in cerebella from all three groups (control, undernourished heavy, and undernourished light). The salvage pathway enzyme thymidine kinase (TK) and the inter-conversion pathway enzyme thymidylate synthetase (TS) in the undernourished light group showed significant elevations of 32% and 11%, respectively, above activity in the combined control group. The salvage pathway enzyme uridine kinase (UK) and the de novo pathway enzyme aspartate transcarbamylase were not significantly different in cerebella from these two groups. The significant elevation in TK and TS in undernourished pups suggest that these enzymes are critical for restorative brain growth. The significant elevation of TS indicates that the inter-conversion pathway converting available uridylate, a ribonucleotide, to thymidylate, a deoxyribonucleotide, is activated in order to augment DNA biosynthesis.

Thymidine phosphotransferase and nucleotide phosphohydrolase of the fern Asplenium nidus. General properties and inhibition by adenosine 3':5'-cyclic monophosphate.

1. Extracts of several plant species contained nucleoside-AMP phosphotransferase activity. The ratio of activity with thymidine to that with uridine as nucleoside substrate was essentially constant, both between species and throughout plant development. Evidence is presented that the total thymidine-AMP phosphotransferase activity of the leaves of Asplenium nidus (bird's-nest fern) and of Helianthus tuberosus (Jerusalem artichoke) increases during maturation. 2. Thymidine-AMP phosphotransferase was purified 22-fold from a very rich source of this activity, extracts of A. nidus. 3. A broad specificity towards both nucleoside and nucleoside 5'-monophosphate substrates is displayed by this preparation, and the evidence suggests that all could be due to a single enzyme. 4. Nucleosides that act as substrates will also inhibit phosphotransfer to other nucleosides, with Ki values close to the corresponding Km values found when utilized as substrates. 5. Ca2+-activated ATP phosphohydrolase was separated from the phosphotransferase by differential complexing to Blue Dextran in the presence of urea, whereas an AMP phosphohydrolase activity was closely associated with thymidine-AMP phosphotransferase through all separation techniques used. 6. Metal ions did not activate either of the latter two activities, and 1,10-phenanthroline was found to inhibit the phosphotransferase. 7. Km values for AMP for the respective activities were 0.11 mM (thymidine phosphotransferase) and 0.20 mM (AMP phosphohydrolase) and for thymidine (phosphotransferase only) 0.88 mM. 8. 3':5'-Cyclic AMP was found to inhibit both phosphotransferase and AMP phosphohydrolase activities, with Ki values of 0.056 mM and 0.15 mM respectively. It is suggested that this inhibitor would be of value in revealing the existence of thymidine kinase in plant extracts with high thymidine phosphotransferase activity.