潮霉素抗性3T3细胞的建立和鉴定

目的　建立具有潮霉素 (hygromycin)抗性的 3T3细胞系 ,用于转染目的基因 (pTRE Ins human)的ES阳性细胞克隆筛选的饲养层。方法　通过脂质体转染的方法 ,将含有潮霉素B磷酸转移酶基因的质粒pHyg导入 3T3细胞中 ,利用潮霉素的药物选择特性 ,对转染细胞进行压力筛选 ,并对其进行PCR鉴定。结果　经 5 0 0 μg ml的潮霉素压力筛选后 ,获得了抗性细胞克隆。抗性 3T3细胞的形态和生长速度与正常 3T3细胞没有差异 ,特异性核苷酸引物检测抗性细胞基因组DNA ,可以扩增出对应的核苷酸片段。结论　成功地培育了潮霉素抗性的 3T3细胞 ,为进行目的基因 (pTRE Ins human)转染ES细胞的阳性细胞克隆筛选奠定了基础。     

The separation of functionally distinct forms of the third component of human complement (C3).

Complement component C3 prepared by the method of Tack & Prahl [(1976) Biochemistry 15, 4513-4521] was found to contain the following trace contaminants: C3b, haemolytically inactive C3 with intact alpha- and beta-chains (C3u) and degraded C3 (apparent mol.wt. 140000) with an intact beta-chain but with a fragmented alpha-chain. The proportion of C3u in the C3 is increased on standing and by freezing and thawing. These contaminants could be separated from each other and from native C3 by chromatography on sulphated Sepharose. They have been characterized by their susceptibility to C3b inactivator in the presence of beta 1H, their ability to be cleaved by C3 convertase and their ability to form alternative-pathway C3 convertase in solution. Incubation of C3b or C3u with beta 1H and C3b inactivator resulted in cleavage of the C3 species; the alpha'-chain of C3b was cleaved to fragments of apparent mol.wts. 67000 and 43000, the alpha-chain of C3u was cleaved to fragments of apparent mol.wt. 75000 and 43000. Native C3 and degraded C3 were unaffected by incubation with beta 1H and C3b inactivator. C3u, unlike C3, was not cleaved to C3b by the classical- or alternative-pathway C3 convertase in solution. When C3b or C3 was incubated with factors B and D, forming C3 convertase, the initial rate of factor-B cleavage was several order of magnitude lower in the presence of C3 than in the presence of C3b. The slow rate observed for C3 could be decreased by preincubation with beta 1H and C3b inactivator or by rechromatography of the C3. The degraded C3 did not support factor-B cleavage by factor D.     

Characterization of 14-3-3sigma Dimerization Determinants: Requirement of Homodimerization for Inhibition of Cell Proliferation

The seven highly conserved 14-3-3 proteins expressed in mammalian cells form a complex pattern of homo- and hetero-dimers, which is poorly characterized. Among the 14-3-3 proteins 14-3-3sigma is unique as it has tumor suppressive properties. Expression of 14-3-3sigma is induced by DNA damage in a p53-dependent manner and mediates a cell cycle arrest. Here we show that the 14-3-3sigma protein exclusively forms homodimers when it is ectopically expressed at high levels, whereas ectopic 14-3-3zeta formed heterodimers with the 5 other 14-3-3 isoforms. The x-ray structure of 14-3-3sigma?revealed 5 residues (Ser⁵, Glu²⁰, Phe²⁵, Q⁵⁵, Glu⁸⁰) as candidate determinants of dimerization specificity. Here we converted these amino-acids to residues present in 14-3-3zeta at the analogous positions. Thereby, Ser⁵, Glu²⁰ and Glu⁸⁰ were identified as key residues responsible for the selective homodimerization of 14-3-3sigma. Conversion of all 5 candidate residues was sufficient to switch the dimerization pattern of 14-3-3sigma to a pattern which is very similar to that of 14-3-3zeta. In contrast to wildtype 14-3-3sigma this 14-3-3sigma variant and 14-3-3zeta were unable to mediate inhibition of cell proliferation. Therefore, homodimerization by 14-3-3sigma is required for its unique functions among the 7 mammalian 14-3-3 proteins. As inactivation of 14-3-3sigma sensitizes to DNA-damaging drugs, substances designed to interfere with 14-3-3sigma dimerization may be used to inactivate 14-3-3sigma function for cancer therapeutic purposes.     

Residues of 14-3-3 zeta required for activation of exoenzyme S of Pseudomonas aeruginosa.

Exoenzyme S (ExoS) is a mono-ADP-ribosyltransferase secreted by the opportunistic pathogen Pseudomonas aeruginosa. ExoS requires a eukaryotic factor, the 14-3-3 protein, for enzymatic activity. Here, two aspects of the activation of the ADP-ribosyltransferase activity of ExoS by 14-3-3 proteins are examined. Initial studies showed that several isoforms of 14-3-3, including beta, zeta, eta, sigma, and tau, activated ExoS with similar efficiency. This implicates a conserved structure in 14-3-3 that contributes to the interaction between 14-3-3 and ExoS. One candidate structure is the conserved amphipathic groove that mediates the 14-3-3/Raf-1 interaction. The next series of experiments examined the role of individual amino acids of the amphipathic groove of 14-3-3 zeta in ExoS activation and showed that ExoS activation required the basic residues lining the amphipathic groove of 14-3-3 zeta without extensive involvement of the hydrophobic residues. Strikingly, mutations of Val-176 of 14-3-3 zeta that disrupted its interaction with Raf-1 did not affect the binding and activation of ExoS by 14-3-3. Thus, ExoS selectively employs residues in the Raf-binding groove for its association with 14-3-3 proteins.     

Metabolic engineering of Alcaligenes eutrophus through the transformation of cloned phbCAB genes for the investigation of the regulatory mechanism of polyhydroxyalkanoate biosynthesis

The regulatory mechanisms of the biosynthesis of in vivo poly-beta-hydroxybutyrate [PHB] and poly(3-hydroxybutyrate-3-hydroxyvalerate) [P(3HB-3HV)] of Alcaligenes eutrophus were investigated by using various transformants with enzyme activities that were modified through the transformation of cloned phbCAB genes. The biosynthesis rates of PHB and P(3HB-3HV) were controlled by beta-ketothiolase and acetoacetyl-CoA reductase, and especially by beta-ketothiolase condensing acetyl-CoA or propionyl-CoA. The contents of PHB and P(3HB-3HV) were controlled by PHB synthase, polymerizing 3-hydroxybutyrate to PHB or 3-hydroxybutyrate and 3-hydroxyvalerate to P(3HB-3HV). The molar fraction of 3-hydroxyvalerate in P(3HB-3HV) was also closely connected with PHB synthase. This may be due to the accelerated polymerization between 3-HB from glycolysis pathway and 3-HV converted from propionate supplied as precursor. Enforced beta-ketothiolase and acetoacetyl-CoA reductase to PHB synthase tended to enlarge the size of the PHB and P(3HB-3HV) granules, however, higher activity ratio of PHB synthase to beta-ketothiolase and acetoacetyl-CoA reductase than parent strain tended to induce the number of granules.     

Molecular determinants of proteolytic disassembly of the reovirus outer capsid

Following attachment and internalization, mammalian reoviruses undergo intracellular proteolytic disassembly followed by viral penetration into the cytoplasm. The initiating event in reovirus disassembly is the cathepsin-mediated proteolytic degradation of viral outer capsid protein σ3. A single tyrosine-to-histidine mutation at amino acid 354 (Y354H) of strain type 3 Dearing (T3D) σ3 enhances reovirus disassembly and confers resistance to protease inhibitors such as E64. The σ3 amino acid sequence of strain type 3 Abney (T3A) differs from that of T3D at eight positions including Y354H. However, T3A displays disassembly kinetics and protease sensitivity comparable with T3D. We hypothesize that one or more additional σ3 polymorphisms suppress the Y354H phenotype and restore T3D disassembly characteristics. To test this hypothesis, we engineered a panel of reovirus variants with T3A σ3 polymorphisms introduced individually into T3D-σ3Y354H. We evaluated E64 resistance and in vitro cathepsin L susceptibility of these viruses and found that one containing a glycine-to-glutamate substitution at position 198 (G198E) displayed disassembly kinetics and E64 sensitivity similar to those properties of T3A and T3D. Additionally, viruses containing changes at positions 233 and 347 (S233L and I347T) developed de novo compensatory mutations at position 198, strengthening the conclusion that residue 198 is a key determinant of σ3 proteolytic susceptibility. Variants with Y354H in σ3 lost infectivity more rapidly than T3A or T3D following heat treatment, an effect abrogated by G198E. These results identify a regulatory network of residues that control σ3 cleavage and capsid stability, thus providing insight into the regulation of nonenveloped virus disassembly.     

Regulation of expression of the 3β-hydroxysteroid dehydrogenases of human placenta and fetal adrenal

The appropriate expression of 3β-hydroxysteroid dehydrogenase/Δ^5→4-isomerase (3β-HSD) is vital for mammalian reproduction, fetal growth and life maintenance. Several isoforms of 3β-HSD, the products of separate genes, have been identified in various species including man. Current investigations are targeted toward defining the processes that regulate the levels of specific isoforms in various steroidogenic tissues of man. High levels of expression of 3β-HSD were observed in placental tissues. It has been generally considered that the multinucleated syncytiotrophoblastic cells are the principal sites of 3β-HSD expression and, moreover, that 3β-HSD expression is intimately associated with cyclic AMP-promoted formation of syncytia. Herein we report the presence of 3β-HSD immunoreactive and mRNA species in uninucleate cytotrophoblasts in the chorion laeve, similar to that in syncytia but not cytotrophoblast placenta. In vitro, 3β-HSD levels in chorion laeve cytotrophoblasts were not increased with time nor after treatment with adenylate cyclase activators, whereas villous cytotrophoblasts spontaneously demonstrated progressive, increased 3β-HSD expression. Moreover, 3β-HSD synthesis appeared to precede morphologic syncytial formation. Thus high steroidogenic enzyme expression in placenta is not necessarily closely linked to formation of syncytia. Both Western immunoblot and enzymic activity analyses also indicated that the 3β-HSD expressed in these cytotrophoblastic populations was the 3β-HSD type I gene product (M_r, 45K) and not 3β-HSD type II (M_r, 44K) expressed in fetal testis. In cultures of fetal zone and definitive zone cell of human fetal adrenal, 3β-HSD expression was not detected until ACTH was added. ACTH, likely acting in a cyclic AMP-dependent process, induced 3β-HSD type II activity and mRNA expression. The higher level of 3β-HSD mRNA in definitive zone compared with fetal zone cells was associated with parallel increases in cortisol secretion relative to dehydroepiandrosterone sulfate formation.     

Development of potent inhibitors of the coxsackievirus 3C protease

Coxsackievirus B3 (CVB3) 3C protease (3CP) plays essential roles in the viral replication cycle, and therefore, provides an attractive therapeutic target for treatment of human diseases caused by CVB3 infection. CVB3 3CP and human rhinovirus (HRV) 3CP have a high degree of amino acid sequence similarity. Comparative modeling of these two 3CPs revealed one prominent distinction; an Asn residue delineating the S2' pocket in HRV 3CP is replaced by a Tyr residue in CVB3 3CP. AG7088, a potent inhibitor of HRV 3CP, was modified by substitution of the ethyl group at the P2' position with various hydrophobic aromatic rings that are predicted to interact preferentially with the Tyr residue in the S2' pocket of CVB3 3CP. The resulting derivatives showed dramatically increased inhibitory activities against CVB3 3CP. In addition, one of the derivatives effectively inhibited the CVB3 proliferation in vitro.     

Synthesis and biological activity of 3 beta-fluorovitamin D3: comparison of the biological activity of 3 beta-fluorovitamin D3 and 3-deoxyvitamin D3

The alteration in the biologic activity of the vitamin D3 molecule resulting from the replacement of a hydrogen atom with a fluorine atom is a subject of fundamental interest. To investigate this problem we synthesized 3 beta-fluorovitamin D3 6 and its hydrogen analog, 3-deoxyvitamin D3 7, and tested the biologic activity of each by in vitro and in vivo methods. Contrary to previous reports which showed that 3 beta-fluorovitamin D3 was as active as vitamin D3 in vivo, we found that the fluoro-analog was less active than vitamin D3. With regard to stimulation of intestinal calcium transport and bone calcium mobilization in the D-deficient hypocalcemic rat, 3 beta-fluorovitamin D3 showed significantly greater biologic activity than its hydrogen analog, 3-deoxyvitamin D3. In the organ-cultured, embryonic chick duodenum, 3 beta-fluorovitamin D3 was approx 1/1000th as active as the native hormone, 1,25-dihydroxyvitamin D3, while 3-deoxyvitamin D3 was inactive even at microM concentrations, in the induction of the vitamin D-dependent, calcium-binding protein. With regard to in vitro activity in displacing radiolabeled 25-hydroxyvitamin D3 from vitamin D binding protein and radiolabelled 1,25-dihydroxyvitamin D3 from a chick intestinal cytosol receptor, 3 beta-fluorovitamin D3 and 3 beta-deoxyvitamin D3 both showed very poor binding efficiencies when compared with vitamin D3. Our results show that the substitution of a fluorine atom for a hydrogen atom at the C-3 position of the vitamin D3 molecule results in a fluorovitamin 6 with significantly more biological activity than its hydrogen analog, 3-deoxyvitamin D3 7.     

Identification of two types of naturally-occurring intertypic recombinants of Epstein-Barr virus

Two Epstein-Barr virus (EBV) types, type 1 and type 2, maintain the same allelic specificity at four genomic loci encoding the EBNA2, -3A, -3B, and -3C proteins. We have previously described 16 EBV-transformed B-lymphoblastoid cell lines derived from Korean cancer patients, and the EBNA2 types of the EBV isolates therein. In this study, the allelic types of the EBNA2, -3A, -3B, and -3C genes of these EBV isolates were determined. We report the identification of two distinct types of naturally occurring intertypic recombinants, one with genotype EBNA2 type 1/EBN3A, -3B, -3C type 2 and the other with genotype EBNA2, -3A type 1/EBNA3B, -3C type 2. The existence of these intertypic recombinants indicates that various intertypic EBV strains may be circulating in the human population, in addition to typical EBV-1 and EBV-2 strains.     

Biosynthesis of cholestanol: 5-alpha-cholestan-3-one reductase of rat liver

The 3-beta-hydroxysteroid dehydrogenase of rat liver which catalyzes the conversion of 5alpha-cholestan-3-one to 5alpha-cholestan-3beta-ol is localized mainly in the microsomal fraction. The enzyme required NADPH as hydrogen donor and differed from the known 3-beta-hydroxysteroid dehydrogenases of the C(19) series in being inactive in the presence of NADH. The microsomal preparations did not reduce the 3-keto groups of cholest-4-en-3-one, cholest-5-en-3-one, or 5beta-cholestan-3-one to the corresponding 3beta-hydroxy compounds. The conversion of 5alpha-cholestan-3-one to 5alpha-cholestan-3beta-ol was only slightly inhibited by the reaction product or by other monohydroxy steroids, but a strong inhibitory effect was noted with cholest-5-en-3-one, 5alpha-cholestane-3beta, 7alpha-diol and 5alpha-cholestan-7-on-3beta-ol. The microsomes, but not high speed supernatant solution, catalyzed the reverse of the cholestanone reductase reaction, namely the conversion of 5alpha-cholestan-3beta-ol to 5alpha-cholestan-3-one in the presence of oxygen and an NADP-generating system. The action of the microsomal preparations upon 5alpha-cholestan-3-one produced 5alpha-cholestan-3alpha-ol in addition to the 3beta-epimer. The 3-alpha-hydroxysteroid dehydrogenase involved functioned with either NADH or NADPH as hydrogen donor. The ratio of 5alpha-cholestan-3beta-ol to 5alpha-cholestan-3alpha-ol formed from 5alpha-cholestan-3-one was approximately 10:1 and was independent of the sex of the animal from which the microsomes were prepared.     

The contribution of thymine-thymine interactions to the stability of folded dimeric quadruplexes.

The loop of four thymines in the sodium form of the dimeric folded quadruplex [d(G3T4G3)]2 assumes a well-defined structure in which hydrogen bonding between the thymine bases appears to contribute to the stability and final conformation of the quadruplex. We have investigated the importance of the loop interactions by systematically replacing each thymine in the loop with a cytosine. The quadruplexes formed by d(G3CT3G3), d(G3TCT2G3), d(G3T2CTG3) and d(G3T3CG3) in the presence of 150 mM Na+ were studied by gel mobility, circular dichroism and 1H NMR spectroscopy. The major species formed by d(G3CT3G3), d(G3TCT2G3) and d(G3T3CG3) at 1 mM strand concentration at neutral pH is a dimeric folded quadruplex. d(G3T2CTG3) has anomalous behaviour and associates into a greater percentage of linear four-stranded quadruplex than the other three oligonucleotides at neutral pH and at the same concentration. The linear four-stranded quadruplex has a greater tendency to oligomerize to larger ill-defined structures, as demonstrated by broad 1H NMR resonances. At pH 4, when the cytosine is protonated, there is a greater tendency for each of the oligonucleotides to form some four-stranded linear quadruplex, except for d(G3T2CTG3), which has the reverse tendency. The experimental results are discussed in terms of hydrogen bonding within the thymine loop.     

Metabolism of β-ionone. Isolation, characterization and identification of the metabolites in the urine of rabbits

1. Rabbits dosed orally with beta-ionone excreted in the urine unchanged beta-ionone, 3-oxo-beta-ionone, 3-oxo-beta-ionol, dihydro-3-oxo-beta-ionol and 3-hydroxy-beta-ionol. 2. Excretion products were isolated as 2,4-dinitrophenylhydrazone derivatives (beta-ionone, 3-oxo-beta-ionone, 3-oxo-beta-ionol and dihydro-3-oxo-beta-ionol) and as p-nitrobenzoate derivatives (3-oxo-beta-ionol, dihydro-3-oxo-beta-ionol and 3-hydroxy-beta-ionol), which were characterized and identified by comparison with the synthetic authentic compounds. 3. The glucuronides of 3-oxo-beta-ionol and dihydro-3-oxo-beta-ionol were also detected in the urine. The latter compound was isolated as free glucuronide, sodium salt and 2,4-dinitrophenylhydrazone.     

Mutagenicity of the phenolic microsomal metabolites of 3-nitrofluoranthene and 1-nitropyrene in strains of Salmonella typhimurium

The environmental pollutant 3-nitrofluoranthene is metabolized in vitro and in vivo to several products including the phenolic metabolites 3-nitrofluoranthen-6-ol (3NF-6-ol), 3-nitrofluoranthen-8-ol (3NF-8-ol), and 3-nitrofluoranthen-9-ol (3NF-9-ol). Similarly, 1-nitropyrene is metabolized to the phenolic metabolites 1-nitropyren-3-ol (1NP-3-ol), 1-nitropyren-6-ol (1NP-6-ol), and 1-nitropyren-8-ol (1NP-8-ol). The mutagenicity of these compounds was investigated using strains of Salmonella typhimurium deficient in either certain nitroreductase or the aryl hydroxylamine O-esterificase. In TA98, 3-nitrofluoranthene and 3NF-8-ol were equally mutagenic at approximately 10³ revertants/nmole while 3NF-6-ol and 3NF-9-ol were 10-fold less mutagenic. 1-Nitropyrene and 1NP-3-ol likewise were equally mutagenic at approximately 700 revertants/nmole and 1NP-6-ol and 1NP-8-ol were 100-fold less mutagenic. The mutagenicity of 1-nitropyrene was dependent on the ‘classical nitroreductase’ which is absent in TA98NR, and that of 3-nitrofluoranthene, 3NF-8-ol, and 1NP-3-ol was less dependent on this nitroreductase. Using TA98/1,8DNP₆, it was determined that the mutagenicity of 3-nitrofluoranthene, 3NF-8-ol, and 1NP-3-ol but not 1-nitropyrene was dependent on the presence of the O-esterificase. 3-Nitrofluoranthene and 3NF-8-ol were mutagenic in TA100, while 3NF-6-ol and 3NF-9-ol were considerably less mutagenic. 3-Nitrofluoranthene was not mutagenic in TA100NR nor in TA100-Tn5-1,8-DNP1012. None of the phenolic metabolites of 3-nitrofluoranthene were mutagenic in TA100-Tn5-1,8DNP1012 indicating a strong dependence for mutagenicity of the O-esterificase of the 1,8-dinitropyrene nitroreductase which is absent in this strain. These results are discussed in view of possible mechanisms for the differences in the mutagenicity of the phenolic metabolites of these two nitrated arenes.     

PI3Kgamma is the dominant isoform involved in migratory responses of human T lymphocytes: effects of ex vivo maintenance and limitations of non-viral delivery of siRNA

Use of mice in which individual PI3K isoforms have been deleted or mutated by gene targeting, has determined that PI3Kγ provides a key migratory signal for T lymphocyte migration. Since PI3Kγ can be a dispensable signal for directional migration of human T cells, we have adopted a pharmacological and siRNA strategy to assess the contribution of individual PI3K isoforms to chemokine-stimulated migration of human T cells. The broad spectrum PI3K isoform inhibitor Ly294002 inhibits CXCL12-stimulated migration of freshly isolated T lymphocytes. Use of second generation inhibitors that can discriminate between individual PI3K isoforms, revealed that PI3Kγ was the major contributor to CXCL12-induced migration and PI3K/Akt signaling (as assessed by S6 phosphorylation). Non-viral delivery of siRNA targeting class I (PI3Kγ), class II (PI3KC2 and PI3KC2β) and class III PI3Ks, followed by 3 days ex vivo culture, reduces the levels of isoform mRNA, but is insufficient to impact on cell migration responses. However, ex vivo maintenance of T cells alone, independently of siRNA treatment, resulted in the migratory response of T cells toward CXCL12 becoming insensitive to Ly294002. Remarkably, random migration remains sensitive to Ly294002. This study therefore, highlights that the migratory response of freshly isolated human T cells is dependent on PI3K signals that are provided predominantly by PI3Kγ. However, the role of PI3K in cell migration is context-dependent and diminishes during ex vivo maintenance.     

Biotransformation of bufadienolides by cell suspension cultures of Saussurea involucrata

The biotransformation of three bioactive bufadienolides, namely, bufotalin (1), telocinobufagin (2), and gamabufotalin (3) by cell suspension cultures of Saussurea involucrata yielded 11 products. Bufotalin yielded 3-epi-bufotalin (1a), 3-epi-desacetylbufotalin (1b), 3-epi-bufotalin 3-O-β-D-glucoside (1c), 1β-hydroxybufotalin (1d), and 5β-hydroxybufotalin (1e); telocinobufagin yielded 3-dehydroscillarenin (2a), 3-dehydrobufalin (2b), and 3-epi-telocinobufagin (2c); and gamabufotalin yielded 3-epi-gamabufotalin (3a), 3-dehydrogamabufotalin (3b), and 3-dehydro-Δ1-gamabufotalin (3c), respectively. Among these 11 products, 1a, 1b, 1c, 1d, 3a and 3c are previously unreported. The structures of these metabolites were elucidated based on NMR spectroscopic analyses and mass spectrometry. Most metabolites showed significant cytotoxic activities against human hepatoma (HepG2) and breast cancer (MCF-7) cell lines. In addition, the time course for the biotransformation of 3 was investigated.     

Loss of density-dependent regulation of multiplication of BALB-3T3 cells chemically transformed in vitro

Balb/3T3 cells show density-dependent regulation of multiplication with the final cell density depending on serum concentration in the media. Chemically transformed Balb/3T3 cells (Balb/3T3-D) pile up on each other, multiply to a high cell density, but have decreased DNA synthesis at very high cell densities. Balb/3T3-D cells require less serum for multiplication compared with original Balb/3T3 cells. A rat serum fraction and a bovine β-globulin fraction stimulate the multiplication of Balb/3T3 cells but only slightly stimulate Balb/3T3-D cells indicating different serum factors stimulate growth of these two cell types. The multiplication properties of Balb/3T3-D cells are very similar to those of SV-40 transformed 3T3 cells, however, these properties were brought about by a single treatment by a chemical carcinogen, without an exogenous virus. The transformation altered the contact of cells to one another, indicating a permanent chemical change in the membrane structure.     

Identification of novel alternative splicing variants of interferon regulatory factor 3

Interferon regulatory factor 3 (IRF-3) plays a crucial role in host defense against viral and microbial infection as well as in cell growth regulation. IRF-3a is the only structurally and functionally characterized IRF-3 splicing variant and has been established to antagonize IRF-3 activity. Here, five novel splicing variants of IRF-3, referred to as IRF-3b, -3c, -3d, -3e, and -3f, were identified and shown to be generated by deletion of exons 2, 3, or 6 or some combination thereof. RT-PCR examination revealed that these novel splicing variants were more frequently expressed in human liver, esophagus, and cervical tumor tissues than in their normal counterparts. Additionally, electrophoretic mobility shift assay and subcellular localization showed only IRF-3 and IRF-3e were capable of binding the PRDI/III element of interferon-beta (IFNβ) promoter in vitro and underwent cytoplasm-to-nucleus translocation following Poly(I:C) stimulation. Coimmunoprecipitation assay revealed that only IRF-3c (3f) of novel splicing variants associated with IRF-3 in vivo. Further luciferase assay showed IRF-3c (3f) and IRF-3e failed to transactivate PRDI/III-containing promoter but appeared to inhibit transactivation potential of IRF-3 to varying degrees. Taken together, our findings suggest novel splicing variants may function as negative modulators of IRF-3 and may be correlated with pathogenesis of human tumors.     

The formation of dihydrodiols by chemical or enzymic oxidation of 3-methylcholanthrene.

The chemical oxidation of 3-methylcholanthrene in an ascorbic acid-ferrous sulphate-EDTA reaction mixture gave all five possible dihydrodiols. The structures and stereochemistry of the dihydrodiols were shown by UV, mass and NMR spectral studies and by chemical examination to be cis-2a,3-dihydroxy-3-methylcholanthrene, trans-4,5-dihydro-4,5-dihydroxy-3-methylcholanthrene, trans-7,8-dihydro-7,8-dihydroxy-3-methylcholanthrene, trans-9,10-dihydro-9,10-dihydroxy-3-methylcholanthrene, cis-11,12-dihydro-11,12-dihydroxy-3-methylcholanthrene and trans-11,12-dihydro-11,12-dihydroxy-3-methylcholanthrene. An examination by HPLC of the dihydrodiols formed in the metabolism of 3-methylcholanthrene by rat-liver microsomal preparations showed the presence of trans-4,5-dihydro-4,5-dihydoxy-3-methylcholanthrene, trans-7,8-dihydro-7,8-dihydroxy-3-methylcholanthrene, trans-9,10-dihydro-9,10-dihydroxy-3-methylcholanthrene and trans-11,12-dihydro-11,12-dihydroxy-3-methylcholanthrene, identified by comparison of their UV and chromatographic characteristics with those of authentic standards. Tentative identification of cis- and trans-1,2-dihydroxy-3-methylcholanthrene, cis-2a,3-dihydroxy-3-methylcholanthrene and cis-11,12-dihydro-11,12-dihydroxy-3-methylcholanthrene as metabolites were made from their mobilities using HPLC. A quantitative comparison of the dihydrodiols formed from 3H-labelled 3-methylcholanthrene by microsomal preparations from the livers of normal and 3-methylcholanthrene-treated rats was carried out. trans-9,10-Dihydro-9,10-dihydroxy-3-methylcholanthrene and cis- and trans-1,2-dihydroxy-3-methylcholanthrene were formed when 3-methylcholanthrene was incubated with mouse skin in organ culture.