Retinoid metabolism in spontaneously transformed mouse fibroblasts (Balb/c 3T12-3 cells): enzymatic conversion of retinol to anhydroretinol.

Spontaneously transformed mouse fibroblasts (Balb/c 3T12-3 cells) displayed an increased adhesion when cultured in the presence of 10(-6) M all-trans retinol and acquired morphological characteristics of the normal phenotype. Thus it was of interest to investigate the metabolism of [15-(14)C]retinol in this system. Within 24 hours of culture, approximately 4.25% of the [(14)C]retinol was taken up by the cells. The hydrocarbon [(14)C]anhydroretinol was a major metabolic product and was identified by gas-liquid chromatography and by its typical ultraviolet absorption spectrum with maxima at 386, 364, and 346 nm. At 24 and 40 hours anhydroretinol represented 27% and 55%, respectively, of the total nonpolar metabolites or approximately 16% and 30% of the total radioactive products. Formalin-fixed fibroblasts or cultured intestinal mucosal cells did not convert retinol into anhydroretinol. A more polar product with a UV absorption maximum at 310 nm was also found. The time course of the synthesis of this product by 3T12 cells suggested a precursor-product relationship with anhydroretinol. A microsomal preparation from 3T12 cells was also active in synthesizing [(14)C]anhydroretinol and [(14)C]metabolite-310 from [(14)C]retinol. Moreover incubation of metabolite-310 with the 3T12 microsomes yielded anhydroretinol (40% conversion in 30 minutes), suggesting that metabolite-310 is an intermediate in the synthesis of anhydroretinol by these cells. Anhydroretinol appears to be an end product of the metabolism of retinol in 3T12-3 cells, as suggested by the finding that over 90% of [(14)C]anhydroretinol incubated for 30 hours with 3T12-3 cells was recovered unaltered, without the formation of detectable retroretinol, retinol, or retinoic acid.-Bhat, P. V., L. M. De Luca, S. Adamo, I. Akalovsky, C. S. Silverman-Jones, and G. L. Peck. Retinoid metabolism in spontaneously transformed mouse fibroblasts (Balb/c 3T12-3 cells): enzymatic conversion of retinol to anhydroretinol.     

Cytotoxicity, genotoxicity and transforming activity of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) in rat tracheal epithelial cells.

The cytotoxicity, genotoxicity and transforming activity of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) were studied by the assays of colony-forming efficiency (CFE), micronucleus formation (MN), and cell transformation in rat tracheal epithelial (RTE) cells both in vitro and in vivo. Liver S9, primary hepatocytes and RTE cells from normal and Aroclor-1254 induced rats were compared for bioactivation of NNK using Salmonella mutagenesis as the endpoint. Results from the in vitro experiments indicated that low concentrations of NNK (0.01-25 micrograms/ml) caused from 15% to greater than 100% increases in CFE of RTE cells. At high concentrations (100-200 micrograms/ml), NNK was significantly toxic to RTE cells. NNK treatment in vitro (50-200 micrograms/ml) increased MN frequency as much as 3-fold above background and significantly increased the transformation frequency (TF) in 4/5 (50 micrograms/ml) and 6/8 (100 micrograms/ml) experiments. The in vivo exposure of rats to NNK (150-450 mg/kg, given i.p.) resulted in a 60-85% reduction in CFE and a 3-5-fold increase in MN formation in RTE cells. In vivo treatment with cumulative doses of 150 and 300 mg/kg of NNK produced significant increases in TF of tracheal cells from 3/3 and 2/3 rats, respectively. Without activation, NNK was not mutagenic in Salmonella TA1535. The bioactivation of NNK to a mutagenic metabolite was achieved by incubation of NNK with liver S9 fraction from Aroclor-1254 induced rats or primary hepatocytes from both untreated and Aroclor-1254 pretreated rats. RTE cells did not produce sufficient quantities of mutagenic NNK metabolites to be detected by the Salmonella assay.     

Poly(ADP-ribose) polymerase activity in mouse cells which exhibit temperature-sensitive DNA synthesis

The poly(ADP-ribose) polymerase activity of wild-type mouse L cells and of Balb/C-3T3 mouse fibroblasts remained relatively unchanged (at approx. 400 nmol substrate utilized/mg DNA per h) in actively-growing cells incubated at 34 degrees C or at 38.5 degrees C for at least 72 h. A similar result was obtained with the following temperature-sensitive cells grown at the permissive temperature (34 degrees C): ts A1S9 mouse L cells, ts C1 mouse L cells and Balb/C-3T3 ts mouse fibroblasts. The poly(ADP-ribose) polymerase activity of the temperature-sensitive cells was little affected during incubation for 20-24 h at the non-permissive temperature of 38.5 degrees C under which conditions temperature-inactivation of DNA replication was complete. Thereafter, this enzyme activity was found to increase some 2-fold, at a time when normal semi-conservative DNA synthesis was totally suppressed and replaced by repair replication (Sheinin, R. and Guttman, S. (1977) Biochim. Biophys. Acta 479, 105-118; Sheinin, R., Dardick, I. and Doane, F.W. (1980) Exp. Cell. Res., in the press).     

The (6-4) photoproduct of thymine-thymine induces targeted substitution mutations in mammalian cells.

Two major ultraviolet-induced photolesions of TpT, a (6-4) photoproduct [T(6-4)T] and a cis-syn cyclobutane TT dimer (T=T), were incorporated into a predetermined site of one of the leading and lagging template strands of a double-stranded vector, and the modified DNAs were transfected into simian COS-7 cells. The DNAs replicated in the cells were recovered and were transfected again into Escherichia coli. The DNA replication efficiencies of plasmids containing T(6-4)T and T=T in the template strand for lagging strand synthesis were 93 and 79%, respectively, as compared with the unmodified DNA. Similar inhibitory effects were observed in the cases of the photoproducts in the template strand for leading strand synthesis (71 and 58%, respectively). These results indicated that T(6-4)T blocked DNA replication more weakly than T=T during leading and lagging strand syntheses in mammalian cells. The mutation frequencies of T(6-4)T were 2.3 and 4.7% in the leading and lagging template strands, respectively. The T=T lesion was less mutagenic and induced mutations with 0.2-0.7% frequencies. The T(6-4)T lesion primarily elicited 3'-T-->C substitutions, and T=T induced various types of mutations. These results indicate that T(6-4)T is more mutagenic than T=T during leading and lagging strand syntheses in simian cells. Moreover, this is the first evidence that shows T(6-4)T mainly elicits targeted substitutions at its 3'-T site in mammalian cells.     

Studies of CD4- CD8- alpha beta bone marrow T cells with suppressor activity.

The predominant T cell subset in the bone marrow of specific pathogen-free C57BL/Ka and BALB/c mice expressed the alpha beta+ TCR CD4- CD8- surface phenotype. Purified C57BL/Ka alpha beta+ TCR CD4- CD8- marrow cells obtained by cell sorting suppressed the MLR of C57BL/Ka responder and BALB/c stimulator spleen cells. Although the percentage of typical T cells in the spleen was markedly reduced in adult nude mice or normal neonatal mice as compared to the normal adult, the percentage of alpha beta+ TCR CD4- CD8- cells in the spleen and marrow was not. The percentage of "self-reactive" V beta 5+ T cells in the BALB/c spleen was markedly reduced as compared to that in the C57BL/Ka spleen. However, the percentages in the bone marrow were similar. The results indicate that the predominant subset of marrow T cells in these pathogen-free mice differ with regard to surface marker phenotype, function, dependence on the adult thymus, and deletion of certain self-reactive V beta receptors as compared to typical spleen T cells. The marrow T cells appear to develop directly from marrow precursors without rearranged beta chain genes during a 48 hour in vitro culture.     

Mutations induced by 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) in the lacZ and cII genes of Muta Mouse

4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) found in chewing tobacco, snuff, cigarettes, and cigars is a tobacco-specific nitrosamine and classified as a possible human carcinogen (Class 2B) by the International Agency for Research on Cancer (IARC). NNK given intraperitoneally was seen to induce lung and liver adenomas. To evaluate the genotoxicity of NNK in vivo, NNK was intraperitoneally administered to Muta Mouse at two concentrations (125 and 250 mg/kg, once a week for 4 weeks) followed by the measurement of mutant frequencies in the lacZ and cII genes from lung and liver in the same mice. Characterization of the types of the mutation was determined by sequencing the cII genes from mutant plaques. The mutant frequencies in both target genes from both organs dose-dependently increased up to 10 times compared to those of the control group. For the types of mutations, the ratio of the G:C to A:T mutation in the total number of mutants was less than the ratio of A:T to T:A and A:T to C:G transversion, contrary to a previous report. The A:T to T:A transversion was the most highly induced mutation both in the lung and liver cII genes. The increasing rate of mutant frequencies in lung and liver over the vehicle control was 55 and 56 times, respectively, while the increasing rate of G:C to A:T transition was only 1.9 and 2.8 times, respectively. These observations show that NNK predominantly induces DNA adducts leading to A:T to T:A and/or A:T to C:G mutations in the transgene.     

Role of Ser-65 in the activity of alpha-galactosidase A: characterization of a point mutation (S65T) detected in a patient with Fabry disease

Fabry disease is a genetic disorder caused by deficient activity of alpha-galactosidase A (alpha-Gal A). Recent gene analysis of a Fabry patient revealed a point mutation (S65T) resulting in a significant decrease of enzyme activity (Chen, C.-H., et al. (1998) Hum. Mutat. 11, 328-330). In order to evaluate the role of Ser-65 in the alpha-Gal A activity and the molecular mechanism of its deficient enzyme activity in mammalian cells, we prepared gene products of S65T, S65A, and E66D mutations of alpha-Gal A by using an expression system with baculovirus/insect cells and characterized the kinetic and physical properties of those purified enzymes. The Km values of mutant enzymes were 3.5 (S65T), 3.4 (S65A), and 2.3 mM (E66D), using 4-methylumbelliferyl alpha-D-galactoside as a substrate, and the Vmax values were 2.7 x 10(6) (S65T), 3.4 x 10(6) (S65A), and 2.5 x 10(6) units/mg (E66D), respectively, which were similar to those of the normal enzyme (Km, 2.3 mM; Vmax, 2.3 x 10(6) units/mg). The in vitro stability of mutant enzymes at neutral pH was significantly reduced (S65T, 4% of normal; S65A, 29%; E66D, 54%). The intracellular alpha-Gal A activities of S65T, S65A, and E66D in COS1 cells transfected with corresponding plasmid DNAs were markedly lower than the normal enzyme activity (9, 26, and 68% of normal, respectively). However, intracellular enzyme activities were enhanced to 34% (S65T), 44% (S65A), and 80% (E66D) of normal, respectively, by cultivation of the cells with 20 microM 1-deoxygalactonojirimycin (a potent inhibitor of alpha-Gal A) for 24 h. These results suggest that Ser-65 is responsible for the stability of alpha-Gal A but not for the enzyme function.     

Anti-tumor activity of new orally bioavailable 2-amino-5-(thiophen-2-yl)benzamide-series histone deacetylase inhibitors, possessing an aqueous soluble functional group as a surface recognition domain

New orally bioavailable 5-(thiophen-2-yl)-substituted 2-aminobenzamide-series histone deacetylase inhibitors were synthesized. These compounds possess a morpholine or piperadine-derived moiety as an aqueous soluble functional group. Among them, 8b, having a 4-ethyl-2,3-dioxopiperazine-1-carboxamide group as a surface recognition domain, showed promising inhibitory activities against HCT116 cell growth and HDAC1/2. Notably, unlike MS-275, this compound did not induce apoptosis in the cell cycle tests. We therefore conducted antitumor tests of 8b and MS-275 against HCT116 cell xenografts in nude mice. Compound 8b reduced the volume of tumor mass to T/C: 60% and 47% at 45 and 80mg/kg over 16days, respectively. These values were comparable to the rate (T/C: 51% at 45mg/kg) for MS-275. Furthermore, 8b, at neither 45 nor 80mg/kg, induced the weight loss which was observed in the mice given MS-275 at 45mg/kg.     

Comparison of the skeletal effects induced by daily administration of PTHrP (1-36) and PTHrP (107-139) to ovariectomized mice

We here compared the changes induced by subcutaneous injection of PTHrP (1-36) or PTHrP (107-139) (80 μg/kg/day, 5 days/week for 4 or 8 weeks) in bone histology and bone remodeling factors, and in bone marrow cells (BMCs) ex vivo, in ovariectomized (OVX) mice. We also examined the osteogenic effects of these peptides in mouse mesenchymal C3H10T1/2 cells under oxidative stress condition in vitro, which recapitulates the effects of OVX. We confirmed that PTHrP (1-36) exerts bone anabolic actions, as assessed by bone histology and osteoblast differentiation markers in the long bones and plasma from OVX mice. PTHrP (107-139) was also efficient in stimulating several bone formation parameters, and it dramatically decreased bone resorption markers. Moreover, both PTHrP peptides modulate DKK-1 and Sost/sclerostin in osteoblast-like UMR-106 cells highly expressing these Wnt pathway inhibitors, related to their osteogenic action in this in vivo scenario. Administration of either PTHrP peptide improved osteogenic differentiation in BMCs from OVX mice ex vivo and in mouse mesenchymal C3H10T1/2 cells under oxidative stress condition in vitro. These data demonstrate that PTHrP (1-36) and PTHrP (107-139) can exert similar osteogenic effects in the appendicular skeleton of OVX mice. Our results suggest that these effects might occur in part by modulating the Wnt pathway. These findings lend credence to the notion that the osteogenic action of PTHrP (107-139) is likely a consequence of its anti-resorptive and anabolic features, and further support the usefulness of PTHrP (1-36) as a bone anabolic peptide in the setting of estrogen-depletion.     

The -429 T/C and -374 T/A gene polymorphisms of the receptor of advanced glycation end products gene (RAGE) are not risk factors for coronary artery disease in Slovene population with type 2 diabetes

Receptor for advanced glycation end products (RAGE) plays a role in atherosclerosis in diabetics. There are two functional polymorphisms in the promoter of the RAGE gene (-429T/C and -374T/A). The aim of this study was to look for a relationship between the -429T/C and the -374T/A gene polymorphisms of the RAGE gene and the development of coronary artery disease (CAD) in the Slovene population with type 2 diabetes of duration longer than 10 years. One hundred and sixty-eight subjects with diabetes and CAD were compared to 241 diabetic subjects without CAD. The -429T/C and the -374T/A RAGE genotype distributions in patients with CAD (-429T/C: CC: 3%, TC: 31%, TT: 66.0%; -374T/A:AA: 7.7%, TA: 48.2%, TT: 44.1%) were not significantly different from those in patients without CAD (-429 T/C: CC: 1.7%, TC: 26.1%, TT: 72.2%; -374T/A: AA: 11.2%, TA: 43.2%, TT: 45.6%). Our study failed to demonstrate an association between either the -429T/C or the -374T/A gene polymorphism of the RAGE gene and CAD in the Slovene population with type 2 diabetes of duration longer than 10 years.     

Enhanced antitumor activity [correction for activitiy] of trans(+/-)-1,2-diaminocyclo- hexaneglutamatoplatinum(II) formulated with stealth liposome

The antitumor platinum(II) compound, [Pt(dach)(Glu)] (dach=trans(+/-)-1,2-diaminocyclohexane, Glu=glutamate) was formulated with a stealth liposome to improve its biological activity. Liposomes were composed of PC/PEG2000-PE/CH (PC=1,2-diacyl-glycero-3-phosphocholine; PEG2000-PE=poly(ethylene glycol)2000-1,2-diacyl-glycero-3-phosphoethanolamine; CH=cholesterol) involving different acyl moieties of phospholipids such as DO (dioleoyl), DM (dimyristoyl) or DS (distearoyl) group. Among the different acyl groups in the stealth liposomes, the DM formulation was optimal for the preparation of the liposomal [Pt(dach)(Glu)] at the mole ratio of DMPC/PEG2000-DMPE/CH=50/5/45 and at the weight ratio of drug/lipid=1/20, which is represented as L-[Pt(dach)(Glu)]. In vitro cytotoxicity was examined in sensitive A2780 and ME180 and their cisplatin-resistant A2780/PDD and ME180/PDD cancer cells. L-[Pt(dach)(Glu)] was 2 approximately 3 times more cytotoxic than the free complex [Pt(dach)(Glu)] and cisplatin in sensitive cells, and 4 approximately 8 times more cytotoxic in resistant cells. Thus, the resistance index of L-[Pt(dach)(Glu)] was 1.3 approximately 2 while those of the free complex and cisplatin were 5 approximately 6, which indicates that L-[Pt(dach)(Glu)] overcome the cisplatin resistance in both resistant cells. In vivo antitumor activity was assayed against the L1210/S leukemia. The optimal activities (% T/C) of the free complex and L-[Pt(dach)(Glu)] were >459/20 and >442/200 mg/kg, respectively. Considering the amount of the platinum complex in L-[Pt(dach)(Glu)], the liposomal [Pt(dach)(Glu)] displayed 2-fold higher drug potency than the free complex. The biodistribution experiment using LE52 tumor-bearing mouse showed excellent lung targeting property of L-[Pt(dach)(Glu)].     

Resistance performances of transgenic bt rice lines T(2A)-1 and T1c-19 against Cnaphalocrocis medinalis (Lepidoptera: Pyralidae)

The transgenic Bacillus thuringiensis (Bt) rice, Oryza sativa L., lines T(2A)-1 and T1c-19 expressing Cry2A* and Cry1C* from 'Minhui 63' (MH63) were evaluated for resistance to newly hatched and third-instar larvae of Cnaphalocrocis medinalis (Lepidoptera: Pyralidae), by using detached leaf laboratory bioassays. Both T(2A)-1 and T1c-19 rice showed high C. medinalis resistance; however, the lethal time (LT)50 of larvae fed with T(2A)-1 rice was significantly longer than that of larvae fed with T1c-19 rice, implying T1c-19 rice was more toxic to C. medinalis larvae. Larval mortality after 4 d on nitrogen-free MH63 was 25.5% compared with 2.4% mortality on high nitrogen fertilizer (250 kg N/ha) plants. Larval mortality on high nitrogen T(2A)-1 plants declined by 20% compared with nitrogen-free plants. However, resistance in T1c-19 plants was unaffected by nitrogen fertilizer. C. medinalis moths preferred MH63 at both the seedling and grain milk stages for oviposition but not the T1c-19 and T(2A)-1 Bt rice lines.     

T(Iat)- and B(Iab)-cell alloantigens determined by theH-2 linkedI region in mice

The specificity of an antiserum directed againstI region associated (Ia) antigens is described. The serum was raised in (DBA/1×B10.D2)F₁ mice against lymphocytes of AQR mice, differing from the responder for theI region only. The serum reacts with Ia antigens expressed on B cells (Iab) as well as with Ia antigens expressed on T cells (Iat). Absorption studies indicate that B cells possess at least two Ia antigens, and one of these is shared by T cells. However, this shared antigen is not present on the surface of lymphocytes of thymectomized mice. Analysis of the strain distribution of Iab and Iat antigens revealed that the Iab antigens are present on lymphocytes of mice carrying theIA ^k subregion and that the Iat antigens are present on lymphocytes of mice carryingI region genes of theH-2 ^k haplotype located between theIA andIB subregions. This conclusion is based on the analysis of the antiserum's reactivity with T and B cells of the strains B10.A(2R), B10.A(4R) and B10.HTT: the serum reacts with B and T cells of B10.A(2R) but only with B cells of B10.A(4R) mice and only weakly with T cells of B10.HTT mice.Abbreviations ALG antimouse lymphocyte globulin from rabbits - B cells bone marrow derived lymphocytes - B10 C57BL/10Sn mice - D1D2F₁ (DBA/1×B10.D2)F₁ hybrid mice - GVHR graft-vs-host reaction - Ia I region associated antigen - Iab on B cells - Iat on T cells - MLR mixed lymphocyte reaction - T cells thymus-derived lymphocytes - Thy-1 thymus antigen 1, formerly called theta - Tx-Lyc lymphocytes of thymectomized, ALG treated, lethally irradiated and anti-Thy-1 treated bone marrow reconstituted mice - 2R B10.A(2R)/SgSn mice - 4R B10.A(4R) mice     

Interleukin-2- and mitogen-activated NK-like killer cells from highly purified human peripheral blood T cell (CD3+ N901-) cultures

In this study, highly purified (HP) CD3-positive N901-negative T lymphocytes could be induced to become natural killer (NK)-like in culture in the presence of recombinant interleukin-2 (rIL-2) and phytohemagglutinin (PHA). Thus, purified CD3+ N901- T cells from fresh human peripheral blood were obtained by negative selection using an indirect panning technique. To ensure that T lymphocyte fractions were completely devoid of any detectable NK cells, two additional purification procedures were employed: incubation of post-pan T cells with the NK-cytotoxic lysomotropic agent L-leucinemethylester, and complement-mediated lysis using the NK cell specific NKH1a monoclonal antibody. Purity of CD3+ N901- cells could be confirmed by surface marker analysis, whereby two NK-associated antigens, N901 and H-25, were undetectable, while 94 +/- 1% of cells expressed the CD3 (Leu-4) antigen. On functional analysis, fresh HP CD3+ N901- cells exhibited no cytotoxic activity against the standard NK target K562. When HP NK-depleted T lymphocytes were cultured for 7 days in the presence of rIL-2 (100 U/ml), neither surface antigen expression nor cytotoxic activity against K562 changed significantly. However, significant cytotoxicity against K562 [18 +/- 5% specific lysis at 25:1 effector:target (E/T) ratio] could be induced when HP CD3+ N901- cells were grown for 7 days in the presence of rIL-2 and PHA (0.5% v/v). Concomitantly, antigens N901 and H-25 were found to be coexpressed on a minor proportion (22 +/- 16 and 22 +/- 6%, respectively) of CD3+ (88 +/- 2% on day 7) cells. Four-week long-term culture of HP NK-depleted T cells in the presence of rIL-2 and PHA yielded a continuous increase in cytotoxicity against K562 cells (0 up to 46% specific lysis at 25:1 E/T ratio). Of particular interest was the emergence of cytotoxicity against the NK-resistant Daudi cell target (15 +/- 8% specific lysis at 25:1 E/T ratio on day 21). Expression of antigens N901 and H-25 as well as CD3 remained essentially unchanged in long-term culture. In sorting experiments, the H-25+ cell fraction was significantly enriched for cytotoxicity against K562, when compared to both H-25- and unseparated cell fractions. In summary, our results suggest that a proportion of HP CD3+ N901- T lymphocytes may give rise to cells that exhibit NK-like functional and phenotypic properties.     

Synthesis of (S)- and (R)-3-hydroxy acids using cells or purified (S)-3-hydroxycarboxylate oxidoreductase from Clostridium tyrobutyricum and the NADP(H) regeneration system of Clostridium thermoaceticum

A purified and partially characterized novel NADP⁺-dependent oxidoreductase from Clostridium tyrobutyricum DSM 1460 was applied for the preparative reduction of several 3-oxo acids to (S)-3-hydroxy acids. (R)-3-Hydroxybutyrate was prepared by the same enzyme selectively dehydrogenating the S enantiomer of (R,S)-3-hydroxybutyrate. The enantiomeric purity of the (S)- and (R)-3-hydroxy acids was at least 98% enantiomeric excess (e.e). NADPH for reductions and NADP⁺ for dehydrogenations were regenerated by applying artificial mediator accepting pyridine nucleotide oxidoreductases in the form of a crude extract of C. thermoaceticum cells. For NADP⁺ regeneration also the system 2-oxoglutarate/glutamate dehydrogenase was used for comparison. Instead of the purified (S)-3-hydroxycarboxylate oxidoreductase, resting cells of C. tyrobutyricum were also applied for reductions and dehydrogenations with substrate concentrations of 200–400 mM leading to products with e.e. values above 96%.Dedicated to Prof. H.G. Floss on the occasion of his 60th birthday     

New orally bioavailable 2-aminobenzamide-type histone deacetylase inhibitor possessing a (2-hydroxyethyl)(4-(thiophen-2-yl)benzyl)amino group

New 2-aminobenzamide-type histone deacetylase (HDAC) inhibitors were synthesized. They feature a sulfur-containing bicyclic arylmethyl moiety—a surface recognition domain introduced to increase in cellular uptake—and a substituted tert-amino group which affects physicochemical properties such as aqueous solubility. Compound 22 with a (2-hydroxyethyl)(4-(thiophen-2-yl)benzyl)amino group reduced the volume of human colon cancer HCT116 xenografts in nude mice to T/C 67% by oral administration at 45 mg/kg, which was comparable to the rate (T/C 62%) for a positive control, MS-275. Western blot analyses as well as cell cycle and TUNEL assays by flow cytometry suggested that the two compounds inhibited the growth of cancer cells via similar mechanisms.     

Regiospecific hydroxylation of 3-(methylaminomethyl) pyridine to 5-(methylaminomethyl) -2 (1H) -pyridinone byArthrobacter ureafaciens

Microbial production of a 6-hydroxy-3-pyridylmethyl compound from 3-pyridylmethyl compound was investigated. The hydroxylation of 3-(methylaminomethyl)pyridine to 5-(methylaminomethyl)-2(1H)-pyridinone, tautomer of 2-hydroxy-5(methylaminomethyl)pyridine, by resting cells ofArthrobacter ureafaciens JCM3873 was found to proceed regio- and chemo-selectively with an almost quantitative yield. The addition of molybdate ion and nicotine as an inducer to the culture medium was required for the preparation of cells containing high hydroxylation activity. The optimal temperature and pH for the hydroxylation by using resting cells were 35°C and around 7, respectively. This hydroxylation enzyme does undergo inhibition by the substrate. The inhibitory effect could be eliminated by stepwise feeding of the substrate. Under adequate conditions, 23 mg/ml of 5-(methylaminomethyl)-2(1H)-pyridinone was produced with a molar yield of nearly 100% from 3-(methylaminomethyl)pyridine.     

Somatic hypermutation and class switch recombination in Msh6(-/-)Ung(-/-) double-knockout mice

Somatic hypermutation (SHM) and class switch recombination (CSR) are initiated by activation-induced cytosine deaminase (AID). The uracil, and potentially neighboring bases, are processed by error-prone base excision repair and mismatch repair. Deficiencies in Ung, Msh2, or Msh6 affect SHM and CSR. To determine whether Msh2/Msh6 complexes which recognize single-base mismatches and loops were the only mismatch-recognition complexes required for SHM and CSR, we analyzed these processes in Msh6(-/-)Ung(-/-) mice. SHM and CSR were affected in the same degree and fashion as in Msh2(-/-)Ung(-/-) mice; mutations were mostly C,G transitions and CSR was greatly reduced, making Msh2/Msh3 contributions unlikely. Inactivating Ung alone reduced mutations from A and T, suggesting that, depending on the DNA sequence, varying proportions of A,T mutations arise by error-prone long-patch base excision repair. Further, in Msh6(-/-)Ung(-/-) mice the 5' end and the 3' region of Ig genes was spared from mutations as in wild-type mice, confirming that AID does not act in these regions. Finally, because in the absence of both Ung and Msh6, transition mutations from C and G likely are "footprints" of AID, the data show that the activity of AID is restricted drastically in vivo compared with AID in cell-free assays.     

Aluminium(III), chromium(III) and iron(III) complexes with 5-iodouracil and 5-iodouracil-histidine and their antitumour activity

Complexes of the type [Al(HL)(OH)Cl(2)], [M(HL)(OH)(2)Cl] and [M'(HL)(L')(OH)Cl], where HL = 5-iodouracil; HL' = histidine; M = Cr(III), Fe(III) and M' = Al(III), Cr(III), Fe(III), were synthesized and characterized. The complexes are polymeric showing high decomposition points and are insoluble in water and common organic solvents. The mu(eff) values, electronic spectral bands and ESR spectra suggest a polymeric 6-coordinate spin-free octahedral stereochemistry for the Cr(III) and Fe(III) complexes. 5-Iodouracil acts as a monodentate ligand coordinating to the metal ion through the O atom of C((4)) = O while histidine through the O atom of -COO(- ) and the N atom of -NH(2) group. In vivo antitumour effect of 5-iodouracil and its complexes was examined on C(3)H /He mice against P815 murine mastocytoma. As evident from their T/C values, Cr(III) and Fe(III) complexes display significant and higher antitumour activity compared to the 5-iodouracil ligand. The in vitro results of the complexes on the same cells indicate that Cr(III) and Fe(III) complexes show higher inhibition on (3)H-thymidine and (3)H-uridine incorporation in DNA and RNA replication, respectively, at a dose of 5 microg/mL.