Molecular cloning of a unique CMP-sialic acid synthetase that effectively utilizes both deaminoneuraminic acid (KDN) and N-acetylneuraminic acid (Neu5Ac) as substrates.

2-keto-3-deoxy-D-glycero-D-galacto-nononic acid (KDN) is a sialic acid (Sia) that is ubiquitously expressed in vertebrates during normal development and tumorigenesis. Its expression is thought to be regulated by multiple biosynthetic steps catalyzed by several enzymes, including CMP-Sia synthetase. Using crude enzyme preparations, it was shown that mammalian CMP-Sia synthetases had very low activity to synthesize CMP-KDN from KDN and CTP, and the corresponding enzyme from rainbow trout testis had high activity to synthesize both CMP-KDN and CMP-N-acetylneuraminic acid (Neu5Ac) (Terada et al. [1993] J. Biol. Chem., 268, 2640-2648). To demonstrate if the unique substrate specificity found in the crude trout enzyme is conveyed by a single enzyme, cDNA cloning of trout CMP-Sia synthetase was carried out by PCR-based strategy. The trout enzyme was shown to consist of 432 amino acids with two potential nuclear localization signals, and the cDNA sequence displayed 53.8% identity to that of the murine enzyme. Based on the Vmax/Km values, the recombinant trout enzyme had high activity toward both KDN and Neu5Ac (1.1 versus 0.68 min(-1)). In contrast, the recombinant murine enzyme had 15 times lower activity toward KDN than Neu5Ac (0.23 versus 3.5 min(-1)). Northern blot analysis suggested that several sizes of the mRNA are expressed in testis, ovary, and liver in a tissue-specific manner. These results indicate that at least one cloned enzyme has the ability to utilize both KDN and Neu5Ac as substrates efficiently and is useful for the production of CMP-KDN.

Can earnings decline cause a retirement flight of physicians? Financial compensation and the decision to stay in practice.

The authors analyze the historical correlation between annual change in the population of inactive physicians and annual change in the real net income earned by the average physician per hour of patient care. For a sample of nine census divisions across 8 years (1986-1989 and 1994-1997), two regression models conclude with 99% confidence that a fall in net income increases the outflow of physicians from active practice. Regression coefficients estimate that a $1.00 fall in hourly net income increases the population of inactive physicians by 1.46 percent after a 2-year period. Based on 1999 population data, the authors project that an earnings decline of $10.00 per patient care hour motivates 11,000 physicians to retire early. With projections of between 50,000 and 150,000 excess practitioners in the U.S. health care system, the analysis suggests that deterioration in financial compensation can erase part but not all of a physician surplus through early retirement.

Reduction of acetals with samarium diiodide in acetonitrile in the presence of Lewis acids.

Transformation of acetals into ethers by partial reduction using a samarium diiodide-Lewis acids-acetonitrile system is described. The reaction with aromatic acetals occurred in good yields in the presence of aluminum chloride (2 eq) whereas the corresponding aliphatic, vinylic, and alkynyl derivatives did not afford ethers under the same conditions. Beta-elimination to give an enol ether becomes predominant when aliphatic acetals that possess a hydrogen at the 2-position are treated with iodotrimethylsilane in the presence of SmI2 or SmI3.

Increased oxidative DNA damage in mammary tumor cells by continuous epidermal growth factor stimulation.

BACKGROUND: Growth factors can enhance the malignant potential of tumor cells. To examine the relationship between growth factors and tumor progression, we previously established a weakly malignant cell line, ER-1. We found that a 24-hour exposure of ER-1 cells to epidermal growth factor (EGF) induced malignant properties (tumor progression) that were reversible but that, after a 1-month exposure, these changes were irreversible. In this study, we investigated the irreversible changes induced in ER-1 cells by a 1-month exposure to EGF and the possible involvement of oxidative stress. METHODS: ER-1 cells were treated with EGF (100 ng/mL) for 1 month in the presence or absence of an antioxidant, N-acetylcysteine or selenium, and compared with untreated control ER-1 cells. We assessed tumor progression by measuring intracellular peroxide levels, 8-hydroxydeoxyguanosine (a marker for oxidative DNA damage) levels, in vitro invasiveness, and in vivo tumorigenicity and metastatic ability. All statistical tests are two-sided. RESULTS: After ER-1 cells were treated for 1 month with EGF, levels of intracellular peroxide and 8-hydroxyguanosine in the DNA of treated cells were higher than those in the DNA of control cells, and treated ER-1 cells were more tumorigenic and metastatic in vivo and more invasive in vitro than untreated control cells (all P<.001). Levels of 8-hydroxyguanosine in DNA increased as the length of the EGF treatment increased (P<.001). However, when N-acetylcysteine or selenium was added with EGF for 1 month, levels of intracellular peroxide and 8-hydroxyguanosine in DNA were comparable to those in control cells (r =.795). Both tumorigenicity (P =.008) and metastatic ability (P<.001) decreased after addition of N-acetylcysteine or selenium. CONCLUSION: The irreversible changes caused by continuous EGF stimulation of ER-1 cells result from increased oxidative damage in the DNA, which generates tumor cells with more malignant characteristics.

Molecular cloning and expression of the mouse N-acetylneuraminic acid 9-phosphate synthase which does not have deaminoneuraminic acid (KDN) 9-phosphate synthase activity.

A cDNA of the mouse homologue of Escherichia coli N-acetylneuraminic acid (Neu5Ac) synthase (neuB gene product) was cloned by the PCR-based method. The mouse homologue consists of 359 amino acids, and the cDNA sequence displays 33% identity to that of the E. coli Neu5Ac synthase. The recombinant mouse homologue which is transiently expressed in HeLa cells does not exhibit the Neu5Ac synthase activity, which catalyzes condensation of phosphoenolpyruvate (PEP) and N-acetylmannosamine (ManNAc) to synthesize Neu5Ac, but the Neu5Ac 9-phosphate (Neu5Ac-9-P) synthase activity, which catalyzes condensation of PEP and ManNAc 6-phosphate (ManNAc-6-P) to synthesize Neu5Ac-9-P. Thus, the mouse homologue of E. coli Neu5Ac synthase is the Neu5Ac-9-P synthase. The Neu5Ac-9-P synthase is a cytosolic enzyme and ubiquitously distributed in mouse various tissues. Notably, the Neu5Ac-9-P synthase can not catalyze the synthesis of deaminoneuraminic acid (KDN) or KDN-9-P from PEP and Man or ManNAc-6-P, thus suggesting that the enzyme is not involved in the synthesis of KDN. This is consistent with the previous observation that only a very low activity to synthesize KDN is found in mouse B16 cells [Angata, T., et al. (1999) Biochem. Biophys. Res. Commun. 261, 326-331].

Transcriptional slippage of p53 gene enhanced by cellular damage in rat liver: monitoring the slippage by a yeast functional assay.

The Long-Evans Cinnamon (LEC) rat is a mutant strain characterized by abnormal copper metabolism and a high incidence of hepatitis and hepatoma. Using a yeast-based assay which scores mutants in p53 gene transcripts as red colonies, we detected frequent mutations in the liver of LEC rats. The majority (50-60%) of these were frameshift mutations caused by the insertion of an extra adenine (A) in the regions containing six consecutive adenines. The rate of A insertion was calculated to be 6.9-9.0% of the total p53 cDNA. Insertions of an extra adenine were found almost exclusively in the mRNA (cDNA), especially in the (A)(6) tract located at the most 5'-side (exon 4) among the three (A)(6) tracts (exons 4, 7, and 8), but rarely in the corresponding sites of genomic DNA. Wild-type p53 cDNA was transcribed in vitro into mRNA with the use of SP6 RNA polymerase and tested by the yeast functional assay. Subsequent sequencing detected A insertions at an overall rate of 1.6% in exons 7 and 8 but none in exon 4. This indicates that the A insertion in the exon 4 (A)(6) tract was an in vivo phenomenon rather than an artifact in reverse transcription or polymerase chain reaction. The percentage of red colonies increased sharply to about 20% of the liver samples in the acute hepatitis stage, and returned to control level of those in the chronic hepatitis stage, and increased again slightly to those in the neoplastic stage. The percentage of red colonies correlated with the serum GOT level (r=0.96, p<0.001) but not with the contents of copper and 8-hydroxydeoxyguanosine in the liver of LEC rats. Ethanol treatment of hepatic cell lines also increased the rate of transcriptional slippage at the (A)(6) tract. These findings indicate that cellular damage is responsible for the increase in the rate of mutation at the transcriptional level, and suggest that cellular damage degrades transcriptional fidelity, thereby further impairing cellular functions.

Adenovirus type 5 E1A immortalizes primary rat cells expressing wild-type p53.

Adenovirus (Ad) E1A induces apoptosis in cells expressing wild-type p53, and stable transformation by Ad E1A requires the co-introduction of an anti-apoptotic gene such as Ad E1B 19K. Thus, cells immortalized by Ad E1A alone might have lost functional p53. In order to analyze the p53 in rat cells expressing Ad E1A, we established rat cell lines by transfecting primary rat embryo fibroblast (REF) and baby rat kidney (BRK) cells with cloned Ad5 E1A. By using a yeast functional assay, we analyzed p53 in six primary REF and three BRK cell lines immortalized by Ad5 E1A as well as five spontaneously immortalized rat cell lines (REF52, NRK, WFB, Rat-1 and 3Y1). The yeast functional assay revealed that all of the spontaneously and Ad5 ElA-immortalized rat cell lines except for 3Y1 expressed wild-type p53. All of the Ad5 E1A-immortalized rat cell lines contained p53 detectable by immunoprecipitation. Recombinant adenovirus expressing rat p53 cloned from a REF cell line immortalized by Ad5 E1A, as well as that expressing murine wild-type p53, induced apoptosis in p53-null cells in collaboration with E1A. Thus, it is suggested that the mutation of p53 appears to be not frequent in the spontaneous immortalization of primary rat cells, and that the functional loss of wild-type p53 is not a prerequisite of E1A-mediated immortalization.

A functional and quantitative mutational analysis of p53 mutations in yeast indicates strand biases and different roles of mutations in DMBA- and BBN-induced tumors in rats.

In order to analyze the mutational events and to understand the biological significance of the p53 gene in chemical carcinogenesis, we applied a new yeast-based p53 functional assay to ovarian tumors induced by 7, 12-dimethylbenz[a]anthracene (DMBA), as well as to transitional cell carcinomas of the urinary bladder induced by N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN) in rats. The assay demonstrated that 15 of 19 DMBA induced tumors harbored clonal p53 mutations, which is consistent with the expectations of the "clonal expansion" hypothesis. The majority of the mutations were purine (AG) to pyrimidine (CT) transversions (12/19) on the non-transcribed (sense) strand (NTS), which is likely to be due to depurination created by DMBA adduct formation on the NTS. In contrast, we found no pyrimidine to purine [corrected] transversion on the NTS. After cessation of BBN treatment, BBN-induced multifocal lesions in the bladder contained heterogeneous p53 mutations at an early stage. In the later stage, however, clonal p53 mutations were identified in 4 out of 7 bladders analyzed, conforming with the concept of "field cancerization". The observed base substitutions were G-->A (1/6) or C -->T transitions (2/6), and mutations at T (3/6) on the NTS in clonal mutations, together with non-clonal mutations, showing a preference of C-->T to G-->A (17 vs. 0). Thus, preferential repair was found in the transcribed strand of the p53 gene, whether modified by DMBA or by BBN carcinogens. Very similar mutation patterns were observed between clonal and non-clonal mutations in the DMBA- and BBN-induced tumors, indicating that the rat yeast p53 functional assay can be a potential tool for the characterization of in vivo mutation patterns of p53, when modified by chemical carcinogens.

Biosynthesis of KDN (2-keto-3-deoxy-D-glycero-D-galacto-nononic acid). Identification and characterization of a KDN-9-phosphate synthetase activity from trout testis.

Although the deaminoneuraminic acid or KDN glycotope (2-keto-3-deoxy-D-glycero-D-galacto-nononic acid) is expressed in glycoconjugates that range in evolutionary diversity from bacteria to man, there is little information as to how this novel sugar is synthesized. Accordingly, biosynthetic studies were initiated in trout testis, an organ rich in KDN, to determine how this sialic acid is formed. These studies have shown that the pathway consists of the following three sequential reactions: 1) Man + ATP --> Man-6-P + ADP; 2) Man-6-P + PEP --> KDN-9-P + P(i); 3) KDN-9-P --> KDN + P(i). Reaction 1, catalyzed by a hexokinase, is the 6-O-phosphorylation of mannose to form D-mannose 6-phosphate (Man-6-P). Reaction 2, catalyzed by KDN-9-phosphate (KDN-9-P) synthetase, condenses Man-6-P and phosphoenolpyruvate (PEP) to form KDN-9-P. Reaction 3, catalyzed by a phosphatase, is the dephosphorylation of KDN-9-P to yield free KDN. It is not known if a kinase specific for Man (Reaction 1) and a phosphatase specific for KDN-9-P (Reaction 3) may exist in tissues actively synthesizing KDN. In this study, the KDN-9-P synthetase, an enzyme that has not been previously described, was identified as at least one key enzyme that is specific for the KDN biosynthetic pathway. This enzyme was purified 50-fold from rainbow trout testis and characterized. The molecular weight of the enzyme was estimated to be about 80,000, and activity was maximum at neutral pH in the presence of Mn(2+). N-Acetylneuraminic acid 9-phosphate (Neu5Ac-9-P) synthetase, which catalyzes the condensation of N-acetyl-D-mannosamine 6-phosphate and phosphoenol-pyruvate to produce Neu5Ac-9-P, was co-purified with the KDN-9-P synthetase. Substrate competition experiments revealed, however, that syntheses of KDN-9-P and Neu5Ac-9-P were catalyzed by two separate synthetase activities. The significance of these studies takes on added importance with the recent discovery that the level of free KDN is elevated in human fetal cord but not matched adult red blood cells and in ovarian cancer cells (Inoue, S., Lin, S-L., Chang, T., Wu, S-H., Yao, C-W., Chu, T-Y., Troy, F. A., II, and Inoue, Y. (1998) J. Biol. Chem. 273, 27199-27204). This unexpected finding emphasizes the need to understand more fully the role that free KDN and KDN-glycoconjugates may play in normal hematopoiesis and malignancy.

Elevated expression of free deaminoneuraminic acid in mammalian cells cultured in mannose-rich media.

Deaminoneuraminic acid (KDN, 2-keto-3-deoxy-D-glycero-D-galacto-nononic acid) is a member of the family of sialic acids in which an acylamino group at the C-5 position of N-acylneuraminic acid (Neu5Acyl) is replaced by a hydroxyl group. It has recently been shown that KDN is synthesized de novo from its precursor, mannose (Man), in trout testis (Angata, T., Nakata, D., Matsuda, T., Kitajima, K., and Troy, F. A. (1999) J. Biol. Chem. 274, in press). In this study, we examined the effect of extracellular free Man on biosynthesis of KDN in mouse melanoma B16 and African green monkey kidney COS-7 cell lines. The following new findings are reported. First, the levels of free and bound forms of KDN increased when the cells were cultured in the presence of 20 mM Man. The level of intracellular free KDN in COS-7 and B16 cells increased 47- and 66-fold respectively, compared with the levels in control cells. Second, the elevated expression of free KDN was proportional to the intracellular concentration of free Man. Third, KDN 9-phosphate (KDN-9-P) synthase, which condenses Man 6-phosphate and phosphoenolpyruvate (PEP), forming KDN-9-P, was detected in cell lysates from both cell lines. Fourth, the de novo synthesis of KDN in both cell lines in the Man-rich media was unaffected by the addition of N-acetylmannosamine (ManNAc), the hexosamine precursor for synthesis of N-acetylneuraminic acid (Neu5Ac). These results show that KDN is synthesized using free Man as its hexose precursor in these mammalian cells. Thus, the KDN biosynthetic pathway utilizes enzymes distinct, at least in part, from those involved in Neu5Ac biosynthesis. This is the first report showing that in vivo synthesis of KDN can be manipulated by growing cells in the presence of Man. This now provides a useful method to study the metabolism and function of the KDN glycotope.

Inhibitory effects of malotilate on invasion and metastasis of rat mammary carcinoma cells by modifying the functions of vascular endothelial cells.

Malotilate (diisopropyl,1,3-dithiol-2-ylidenemalonate, MT) is clinically used as a hepatoprotective agent. Because we noticed that MT induced the differentiation of cultured vascular endothelial cells, we have examined its effects on lung metastasis of the highly metastatic rat mammary carcinoma c-SST-2. MT was orally administered to syngeneic SHR rats from 7 days before or after s.c. inoculation of c-SST-2 cells to the end of the experiments. In the MT-treated rats, pulmonary metastasis was markedly suppressed compared with the non-treated rats. In the rats treated with MT for 19 days after i.v. inoculation of c-SST-2 cells, lung metastasis was also significantly suppressed. An in vitro invasion assay using a rat lung endothelial (RLE) cell monolayer revealed that pretreatment of the RLE cells with MT, but not c-SST-2 cells, significantly reduced the invasion of the RLE monolayer by c-SST-2 cells. An in vitro vascular permeability assay demonstrated that MT prevented the increase in permeability of the RLE monolayer by serum starvation. On the other hand, in vivo and in vitro growth, gelatinase production and adhesion to the RLE cell monolayer of c-SST-2 cells were not affected by MT treatment. These findings suggest that MT suppressed tumour metastasis by intensifying the cell-to-cell contact of endothelial cells, thus preventing tumour cells from invading vascular endothelium.

Characterization of the progressive sublines derived from a weakly malignant cloned cell line, ER-1, co-inoculated subcutaneously with a foreign body.

We previously established an experimental model of tumor progression using a weakly malignant rat mammary carcinoma cell line, ER-1. Using this model, we demonstrated that ER-1 cells converted into highly tumorigenic and metastatic cells, ERpP, by s.c. co-inoculation with plastic plates. We here compared in vitro biological properties associated with malignancy of ER-1 cells with those of ERpP cells which were highly malignant when inoculated into syngeneic rats. In vitro growth rate of ERpP cells was higher than that of ER-1 cells under a low nutrient condition. Invasion capacity of ERpP cells to rat lung endothelial cell monolayer or reconstituted basement membrane, Matrigel, was higher than that of ER-1 cells. Migration of ERpP cells toward fibronectin or laminin was also significantly higher than that of ER-1 cells. There was no difference in gelatinolytic or plasminogen activator activity detected in conditioned media between ER-1 and ERpP cells. Furthermore, we found that ER-1 cells communicated better among themselves and with normal fibroblasts through gap junctions compared to ERpP cells. These results suggest that growth advantage in a poor nutrient condition, enhancement of cell motility, and loss or decrease of junctional communication may be associated with tumor progression of ER-1 cells.

Reversible and irreversible tumor progression of a weakly malignant rat mammary carcinoma cell line by in vitro exposure to epidermal growth factor.

We examined the effects of epidermal growth factor (EGF) on tumor progression of a weakly malignant rat mammary carcinoma cell line, ER-1. In vitro treatment with EGF enhanced tumorigenicity, metastatic capacity and in vitro invasive capacity of ER-1 cells. The increased malignancy of ER-1 cells was reversible, when the cells were pretreated with EGF for 24 h, whereas it was irreversible when pretreated with EGF for 1 month. EGF treatment elevated the intracellular peroxide level in ER-1 cells. When ER-1 cells were treated with EGF in the presence of N-acetylcysteine, a chemical antioxidant, the reversible or irreversible EGF-induced progression was inhibited. These results suggest that the reversible or irreversible tumor progression in ER-1 cells occur in accordance with the duration of exposure to EGF, and that reactive oxygen species may be involved in the progression.

Characterization and Solubilization of Kaurenoic Acid Hydroxylase from Gibberella fujikuroi.

A key step in gibberellin biosynthesis is the conversion of ent-kaurenoic acid to ent-7[alpha]-hydroxykaurenoic acid, mediated by the enzyme kaurenoic acid hydroxylase. A cell-free system obtained from Gibberella fujikuroi (Saw.) Wr. was used to characterize kaurenoic acid hydroxylase activity. Microsomal preparations from disrupted fungal cells, in the presence of O2 and NADPH, converted [17-14C]ent-kaurenoic acid to oxidation products that were separated by high-performance liquid chromatography and identified as ent-7[alpha]-hydroxykaurenoic acid and gibberellin A14 by combined gas chromatography-mass spectrometry. Flavin adenine dinucleotide and the chloride salts of several monovalent cations stimulated the conversion of ent-kaurenoic acid to these products, whereas CO and a number of known inhibitors of cytochrome P-450-dependent reactions, including paclobutrazol, tetcyclacis, BAS 111.W, flurprimidol, triarimol, metyrapone, and 1-phenylimida-zole, significantly reduced kaurenoic acid hydroxylase activity. Kaurenoic acid hydroxylase was solubilized from fungal microsomes by treatment with 1 M KCl. The properties of the enzyme noted above suggest that kaurenoic acid hydroxylase from G. fujikuroi is a cytochrome P-450-dependent monooxygenase.

Defective function of a microsomal UDP-glucuronyltransferase in Gunn rats.

The kinetic parameters of the p-nitrophenol-metabolizing form of UDP-glucuronyltransferase [-UDPglucuronosyltransferase; UDPglucuronate beta-glucuronosyltransferase (acceptor-unspecific), EC 2.4.1.17] have been compared in liver microsomes from the Gunn strain of rat and from normal; Wistar rats. The abnormally low rate of glucuronidation of p-nitrophenol in the Gunn rats, as compared with Wistar rats, is due to decreased affinity of UDP-glucuronyltransferase for UDP-glucuronic acid. Activities at Vmax and the Michaelis constant for p-nitrophenol, KPNP, of UDP-glucuronyltransferase are the same for enzyme from either strain of rat. Studies of the kinetic parameters of the reverse reaction catalyzed by UDP-glucuronyltransferase indicate that the enzyme from Gunn rats also has decreased affinity for UDP. Calculated values of deltaG degrees for the binding of the UDP portion of UDP-glucuronic acid suggest that the defect of UDP-glucuronyltransferase of Gunn rats appears limited to abnormal interactions between the enzyme and the UDP portion of UDP-glucuronic acid. Studies of the extent of UDP-induced inhibition of the forward reaction support this idea. Diethylnitrosamine, added to microsomes in vitro, enhances the affinity of UDP-glucuronyltransferase for the UDP portion of UDP-glucuronic acid. Despite the defective conformation of the UDP-glucuronic acid binding site of UDP-glucuronyltransferase from Gunn rats this enzyme is activated in the normal way by UDP-N-acetylglucosamine, which is a K-type effector with regard to UDP-glucuronic acid.