Soft-hydrothermal processing of red cedar bedding reduces its induction of cytochrome P450 in mouse liver.

Red cedar-derived bedding materials cause changes in cytochrome P450-dependent microsomal enzyme systems in laboratory animals. We examined the effect of essential oil of red cedar (EORC), as well as the effect of bedding from which it had been removed, on the hepatic expression cytochrome P450s in mice. EORC was obtained from liquid extracts of red cedar bedding by a soft-hydrothermal process and was administered orally to mice. Between days 1 and 2 after administration, hepatic P450s were significantly induced as follows: CYP3As, 7.1x; CYP1As, 1.6x; CYP2E1, 1.5x; CYP2Cs, 1.6x. A housing study of mice indicated that red cedar bedding increased the levels of these P450s in mouse liver, whereas mice housed in cedar bedding from which EORC had been removed (ST-cedar bedding) showed significantly lower levels of P450s, especially CYP3As, CYP1As and CYP2E1. Soft-hydrothermal processing partially removed many components of EORC. In particular, several volatile sesquiterpenes, naphthalene-derived aromatics and 4,4-dimethyl-13alpha-androst-5-ene were decreased in the ST-cedar bedding, suggesting that these may be responsible for P450 induction. This study demonstrated that the removal of these volatile compounds by soft-hydrothermal processing can decrease the hepatic P450-inducing effect of red cedar bedding.

A further investigation and reappraisal of the thio effect in the cleavage reaction catalyzed by a hammerhead ribozyme.

We synthesized three types of 11mer substrate, namely the natural substrate S11O and the thio-substituted substrates S11 S pS and S11 R pS, in which the respective pro-S p and pro-R p oxygen atoms were replaced by sulfur, and subjected them to detailed kinetic analysis in the cleavage reaction catalyzed by a hammerhead ribozyme. In agreement with previous findings, in the presence of Mg(2+)or Ca(2+)ions the rate of ribozyme-catalyzed cleavage of S11 S pS was as high as that of S11O, whereas the corresponding rate for S11 R pS was nearly four orders of magnitude lower than that for either S11O or S11 S pS. However, the rate of the ribozyme-catalyzed reaction with each of the three substrates was enhanced by Cd(2+)ions. Such results have generally been taken as evidence that supports the direct interaction of the sulfur atom at the R p position of the cleavage site with the added Cd(2+)ion. However, our present analysis demonstrates that (i) the added Cd(2+)ion binds at the P9 site; (ii) the bound Cd(2+)ion at the P9 site replaces two Mg(2+)or two Ca(2+)ions, an observation that suggests a different mode of interaction with the added Cd(2+)ion; and, most importantly and in contrast to the conclusion reached by other investigators, (iii) the Cd(2+)ion does not interact with the sulfur atom at the R p position of the scissile phosphate either in the ground state or in the transition state.

Recent advances in the elucidation of the mechanisms of action of ribozymes.

The cleavage of RNA can be accelerated by a number of factors. These factors include an acidic group (Lewis acid) or a basic group that aids in the deprotonation of the attacking nucleophile, in effect enhancing the nucleophilicity of the nucleophile; an acidic group that can neutralize and stabilize the leaving group; and any environment that can stabilize the pentavalent species that is either a transition state or a short-lived intermediate. The catalytic properties of ribozymes are due to factors that are derived from the complicated and specific structure of the ribozyme-substrate complex. It was postulated initially that nature had adopted a rather narrowly defined mechanism for the cleavage of RNA. However, recent findings have clearly demonstrated the diversity of the mechanisms of ribozyme-catalyzed reactions. Such mechanisms include the metal-independent cleavage that occurs in reactions catalyzed by hairpin ribozymes and the general double-metal-ion mechanism of catalysis in reactions catalyzed by the Tetrahymena group I ribozyme. Furthermore, the architecture of the complex between the substrate and the hepatitis delta virus ribozyme allows perturbation of the pK(a) of ring nitrogens of cytosine and adenine. The resultant perturbed ring nitrogens appear to be directly involved in acid/base catalysis. Moreover, while high concentrations of monovalent metal ions or polyamines can facilitate cleavage by hammerhead ribozymes, divalent metal ions are the most effective acid/base catalysts under physiological conditions.

A growth-promoting factor for human myeloid leukemia cells from horse serum identified as horse serum transferrin.

A growth-promoting factor for human myeloid cells was purified to apparent homogeneity from horse serum by a combination of gel filtration, blue Sepharose affinity chromatography, Mono Q anion-exchange chromatography, Mono P chromatofocusing and sodium dodecyl sulfate polyacrylamide gel electrophoresis. The growth promoter was an iron-bound, single glycopolypeptide chain with a molecular weight of 84,000, an isoelectric point of 5.4 and an amino terminal sequence of Glu-Gln-Thr-Val-Arg-Trp-Cys-Thr-Val-Ser-Asn-His-Glu-Val-Ser-Lys-. According to the results of the amino acid sequence, iron binding ability and physicochemical properties, we identified the growth-promoting factor as horse serum transferrin. It was highly active in promoting the proliferation of a human monocytic leukemia cell line, THP-1, as well as of two other human myeloid cell lines, HL-60 and K-562. It had the same activity in proliferating THP-1 cells as 5% fetal calf serum-supplemented medium. Horse serum transferrin could be substituted for human or bovine serum transferrin.

Estrogen activation of the nuclear orphan receptor CAR (constitutive active receptor) in induction of the mouse Cyp2b10 gene.

The nuclear orphan receptor CAR (constitutively active receptor or constitutive androstane receptor) can be activated in response to xenochemical exposure, such as activation by phenobarbital of a response element called NR1 found in the CYP2B gene. Here various steroids were screened for potential endogenous chemicals that may activate CAR, using the NR1 enhancer and Cyp2b10 induction in transfected HepG2 cell and/or in mouse primary hepatocytes as the experimental criteria. 17beta-Estradiol and estrone activated NR1, whereas estriol, estetrol, estradiol sulfate, and the synthetic estrogen diethylstilbestrol did not. On the other hand, progesterone and androgens repressed NR1 activity in HepG2 cells, and the repressed NR1 activity was fully restored by estradiol. Moreover, estrogen treatment elicited nuclear accumulation of CAR in the mouse livers, as well as primary hepatocytes, and induced the endogenous Cyp2b10 gene. Ovariectomy did not affect either the basal or induced level of CAR in the nucleus of the female livers, while castration slightly increased the basal and greatly increased the induced levels in the liver nucleus of male mice. Thus, endogenous estrogen appears not to regulate CAR in female mice, whereas endogenous androgen may be the repressive factor in male mice. Estrogen at pharmacological levels is an effective activator of CAR in both female and male mice, suggesting a biological and/or toxicological role of this receptor in estrogen metabolism. In addition to mouse CAR, estrogens activated rat CAR, whereas human CAR did not respond well to the estrogens under the experimental conditions.

Significant change in the structure of a ribozyme upon introduction of a phosphorothioate linkage at P9: NMR reveals a conformational fluctuation in the core region of a hammerhead ribozyme.

A modified hammerhead ribozyme (R32S) with a phosphorothioate linkage between G(8) and A(9), a site that is considered to play a crucial role in catalysis, was examined by high-resolution 1H and (31)P nuclear magnetic resonance (NMR) spectroscopy. Signals due to imino protons that corresponded to stems were observed, but the anticipated signals due to imino protons adjacent to the phosphorothioate linkage were not detected and the (31)P signal due to the phosphorothioate linkage was also absent irrespective of the presence or absence of the substrate. (31)P NMR is known to reflect backbone mobility, and thus the absence of signals indicated that the introduction of sulfur at P9 had increased the mobility of the backbone near the phosphorothioate linkage. The addition of metal ions did not regenerate the signals that had disappeared, a result that implied that the structure of the core region of the hammerhead ribozyme had fluctuated even in the presence of metal ions. Furthermore, kinetic analysis suggested that most of the R32S-substrate complexes generated in the absence of Mg(2+) ions were still in an inactive form and that Mg(2+) ions induced a further conformational change that converted such complexes to an activated state. Finally, according to available NMR studies, signals due to the imino protons of the central core region that includes the P9 metal binding site were broadened or not observed, suggesting that this catalytically important region might be intrinsically flexible. Our present analysis revealed a significant change in the structure of the ribozyme upon the introduction of the single phosphorothioate linkage at P9 that is in general considered to be a conservative modification.

An ELISA to determine the biodistribution of human monoclonal antibody in tumor-xenografted SCID mice.

An ELISA technique was developed to assay the distribution of native human monoclonal antibodies (mAbs) in tumor-xenografted SCID mice. This was used in an investigation of its potential as an alternative to the conventional radioisotopic technique for mAb biodistribution assays which would be simpler to implement and might yield results in closer accord with actual mAb activity because it is based on the use and detection of the native mAbs rather than their radioisotope-coupled immunoconjugates. SCID mice bearing xenografted tumors of the human lung adenocarcinoma cell line A549 received injections via the tail vein of four human mAbs that had been obtained from human-mouse heterohybridomas and were known to be reactive with A549. The biodistribution of the mAbs was assayed by the ELISA technique seven days after the mAb injection. The assay yielded tumor/serum ratios for the four reactive mAbs which were in the range of three to six and tumor mAb levels which were in the range of 0.28 to 0.92 %ID/g (percent of injected mAb dose per gram of tumor). The tumor mAb levels were thus lower than the levels commonly found by radioisotopic assay, and further investigation is desirable to determine the cause of the difference. The results indicate that ELISA can provide a simple, practical means of investigating the biodistribution of human mAbs in mice bearing xenografted carcinomas. The application of this procedure would obviate the need for the complex facilities and procedures associated with radioisotopic labelling and assay.

Long-term production of human monoclonal antibodies by human-mouse heterohybridomas.

Production of monoclonal antibodies (mAbs) by fused somatic cells was first developed by Köhler and Milstein two decades ago, but its utilization for the production of human mAbs, particularly those bearing kappa chains, has been difficult because heterohybridomas formed with mouse myeloma cells are unstable and tend to lose certain of their human chromosomes. We have stabilized two such heterohybridomas over one year period and induced the production of kappa-bearing and lambda-bearing human mAb, respectively. Increased productivity was achieved by adding the Na+K(+)-ATPase inhibitor, ouabain and a cell mitosis inhibitor, cytochalasin B, to the cell culture media.

2-Methoxy-4-nitroaniline is a selective inducer of cytochrome P450IA2 (CYP1A2) in rat liver.

Male F344 rats were treated with a chemical (aniline, nitrobenzene, 2-methoxy-p-phenylenediamine, 2-methoxy-4-nitroaniline or 2-methoxy-4-nitroazobenzene) produced by the azo-reduction and/or N-oxidation of 2-methoxy-4-amino-azo-benzene, a selective inducer of cytochrome P450IA2 (CYP1A2), and their effects on the induction of CYP1A enzymes in the liver were examined in terms of the protein and mRNA amounts. 2-Methoxy-4-nitroaniline and 2-methoxy-4-nitroazo-benzene, but not other compounds tested, induced CYP1A enzymes, especially CYP1A2, as assessed by Western blotting and Northern blotting. It is noteworthy that 2-methoxy-4-nitroaniline was more selective than 2-methoxy-4-aminoazobenzene for induction of CYP1A2, and it has the smallest molecular size among the known CYP1A2 inducers.

DNA adduct formation of hepatocarcinogenic aromatic amines in rat liver: effect of cytochrome P450 inducers.

F344 rats were treated with an i.p. injection of 2-amino-6- methyldipyrido[1,2-a:3',2'-d]imidazole (Glu P-1) or 3-methoxy-4-aminoazobenzene (3-MeO-AAB) and examined for the formation of the DNA adduct in the liver. To examine the effect of pretreatment with a cytochrome P450 (CYP) inducer on the formation of DNA adduct, these rats were pretreated with 3-methylcholanthrene (MC; CYP1A1/1A2 inducer) or phenobarbital (PB; CYP2B inducer). Administration of Glu P-1 and 3-MeO-AAB gave 2 and 5 adducts, respectively, as determined by 32P-postlabeling assay. By Glu P-1 administration, pretreatment of rats with MC, but not with PB, increased the total amount of DNA adducts including 3 new adducts as minor products. In contrast, pretreatment of rats with PB increased the total amount of DNA adducts derived by 3-MeO-AAB. The increase of aromatic amine DNA adducts by pretreatment with a CYP inducer was proportional to the activity of induced CYP isozyme(s) responsible for the mutagenic activation of each aromatic amine.

Sulfotransferase catalyzing sulfation of heterocyclic amines.

Cytosolic sulfation of arylamines to form sulfamates is found to be mediated by sulfotransferases of three gene families (SULT1 to 3). Among them, a SULT3 form (ST3A1) showed a high selectivity for N-sulfation of N-substituted aryl and alicyclic compounds. SULT1 (phenol) and SULT2 (hydroxysteroid) sulfotransferases showed N-sulfating activities of carcinogenic heterocyclic amines. For N-hydroxyarylamine O-sulfation, SULT1 forms showed high activity. In rats, ST1C1 mediated the metabolic activation of N-hydroxyarylamines. However, the related form (ST1C2) in humans showed the negligible activity. Instead, ST1A3 showed high metabolic activating abilities among human sulfotransferases.

Human monoclonal antibody 28K29 highly reactive with lung adenocarcinomas of all grades of differentiation and with large cell carcinomas.

Human monoclonal antibody (mAb) 28K29 was previously established by fusing regional lymphocytes from a lung adenocarcinoma patient with human-mouse heteromyeloma. This mAb was shown to have an antigen localized in the membrane and cytoplasm of lung cancer cells. This mAb was investigated for reaction with tissue sections from formalin-fixed paraffin-embedded blocks from 100 patients with lung cancer. The mAb 28K29 reacted with 83% (5/6) of the well-differentiated, 79% (22/28) of the moderately-differentiated, and 67% (4/6) of the poorly-differentiated lung adenocarcinoma sections. The mAb also reacted with 35% (14/40) of the squamous cell carcinoma sections, 70% (7/10) of the large cell carcinoma sections, and 20% (2/10) of the small cell carcinoma sections. In Western blot analyses, a broad band at a molecular weight of approximately 600 kDa was detected in extracts from the lung cancer tissues positive for immunohistostaining with the mAb 28K29. The results of the study suggest that the expression of the 28K29 antigen is independent of the histological differentiation grade in lung adenocarcinoma and that this antigen might be a useful marker for detection of both large cell carcinoma and adenocarcinoma of the lung as well as for investigation of the putative transition of large cell carcinoma to adenocarcinoma proposed by Yesner.

Molecular cloning and expression of an amine sulfotransferase cDNA: a new gene family of cytosolic sulfotransferases in mammals.

A cDNA of amine sulfotransferase-RB1 (AST-RB1), which efficiently catalyzes 4-phenyl-1,2,3,6-tetrahydropyridine (PTHP) sulfation, has been isolated by immunoscreening of a rabbit liver cDNA library. The cDNA consisted of 1,117 base pairs and encoded a protein of 301 amino acids with a molecular weight of 35,876. The deduced amino acid sequence matched at six positions those of peptide fragments obtained from purified AST-RB1 protein. The sequence had less than 38% identity at the amino acid level with cytosolic sulfotransferases in mammals, although high degrees of similarity were observed with regions conserved throughout mammalian sulfotransferases. These results indicate that AST-RB1, arbitrarily named sulfotransferase 3A1 (ST3A1), constitutes a new and third gene family of cytosolic sulfotransferases in mammals. ST3A1 expressed in Escherichia coli as a fused protein catalyzed sulfation of amines such as PTHP, aniline, 4-chloroaniline, 2-naphthylamine, and desipramine, but barely O-sulfation of typical aryl and hydroxysteroid sulfotransferase substrates. These data unequivocally demonstrate the existence of a cytosolic sulfotransferase showing a high selectivity for amine substrates, and indicate that multiple forms of sulfotransferase mediate sulfation of xenobiotics in mammalian livers.

Molecular cloning, expression, and enzymatic characterization of rabbit hydroxysteroid sulfotransferase AST-RB2.

Cytosolic sulfotransferases, which consist of at least three gene families, play a major role in activation and detoxification of both endogenous and exogenous chemicals. We recently purified a rabbit sulfotransferase, AST-RB2, showing high activities to both hydroxysteroids and amines. To characterize this enzyme, a rabbit cDNA library was screened using anti-AST-RB2 antibodies. The isolated cDNA was judged to encode AST-RB2 (ST2A8) based on the amino acid sequences of peptide fragments obtained from purified AST-RB2. The cDNA showed high similarity to other mammalian hydroxysteroid sulfotransferases (ST2) at the amino acid level (58-68%), but low similarity to aryl sulfotransferases (ST1) (less than 37%). The protein expressed in Escherichia coli catalyzed sulfation of typical ST2 substrates. Therefore, ST2A8 was judged to belong to the ST2 family from both its primary structure and substrate specificity. The ST2A8 protein expressed in E. coli clearly differed from rat ST2A1 and ST2A2 on its localization (cytosol/insoluble fraction ratio). ST2A8 had no activity to lithocholate, but showed the highest catalysis on dehydroepiandrosterone and testosterone among the four forms (ST2A1, ST2A2, ST2A3, and ST2A8), indicating a clear difference between ST2A forms in substrate specificity to endogenous chemicals.

Identification of ST2A1 as a rat brain neurosteroid sulfotransferase mRNA.

A hydroxysteroid sulfotransferase (ST2A1) was identified as a form mediating neurosteroid sulfation in rat brain. The sole expression among known rat ST2A forms was indicated by brain RT-PCR. All nucleotide sequences of seven ST2A cDNA clones isolated from brain matched completely with that of hepatic ST2A1. The recombinant ST2A1 protein mediated neurosteroid sulfation. These data strongly suggest a functional role of ST2A1 as a neurosteroid sulfotransferase in rat brain.

Theoretical and numerical analysis of the behavior of ions injected into a quadrupole ion trap mass spectrometer

A numerical simulation method has been developed for the analysis of trapping ions injected into an ion trap mass spectrometer. This method was applied to clarify the effects of the following parameters on trapping efficiencies: (1) initial phase of the radio frequency (RF) drive voltage, (2) ion injection energy, and (3) RF peak voltage while injecting ions. The following conclusions were obtained by theoretical and simulation approaches. 1. The second and third dominant oscillations contribute significantly to the trapping mechanism of the injected ions, even for low q values. 2. A formula relating the operating parameters, which gives the maximum trapping efficiency, is derived. 3. Based on the above-mentioned formula, an advanced injection method is proposed, in which the RF peak voltage is decreased while injecting ions. The ability of this method to solve the problem of unequal sensitivity among different ion species is indicated by numerical simulation. Copyright 2000 John Wiley & Sons, Ltd.

Chemical and enzymatic probing of effector-mediated changes in the conformation of a maxizyme.

The protein encoded by chimeric BCR-ABL mRNA causes chronic myelogenous leukemia (CML). We showed previously that a novel allosterically controllable ribozyme, of the type known as a maxizyme, can cleave this mRNA, with high specificity and high-level activity in vivo. We designed the maxizyme in such a way that it was able to form an active core with which to capture the catalytically indispensable Mg2+ ions only in the presence of the BCR-ABL mRNA junction. In order to probe the putative conformational changes, we used a weakly alkaline solution (pH 9.2) in the presence of 25 mM Mg2+ ions to hydrolyze differentially phosphodiester bonds that were located in different environments. Phosphodiester bonds in single-stranded regions were clearly more susceptible to attack by alkali than those within a double-stranded helix. As indicated by earlier data obtained in vivo, our results demonstrated that the active conformation was achieved only in the presence of the junction within the chimeric BCR-ABL mRNA. Moreover, we demonstrated that the use of mild alkaline solutions to probe RNA structures is very informative.

Arylamine activating sulfotransferase in liver.

Carcinogenic N-hydroxy-arylamines and -arylamides undergo metabolic activation by several enzymes in mammals to cause the DNA damage. Cytosolic sulfotransferases in rat and human livers, which belong to the ST1 (SULT1) family, have been studied to assess their properties to mediate the metabolic activation. A human orthologue of rat ST1C1 from, which catalyzes sulfation of N-hydroxy-2-acetylaminofluorene, was screened in a EMBL genomic library with ST1C1 cDNA [Nagata, K., S. Ozawa, M. Miyata, M. Shimada, D.-W. Gong, Y. Yamazoe and R. Kato (1993) J. Biol. Chem., 268, 24720-24725]. Sequencing of the hybridized clones indicate that at least 3'-terminal region of human ST1C1 orthologue contains sequence highly homologus to rat ST1C1 at both nucleotide and deduced amino acid levels. The experiments using anti-rat ST1C1 antibody and nucleotide probes for human ST1C1 showed no detectable band in Western blots and an mRNA-detecting method with polymerase chain reaction, respectively, in human liver samples.

Efficient production of IgG human monoclonal antibodies by lymphocytes stimulated by lipopolysaccharide, pokeweed mitogen, and interleukin 4.

Extensive screening of the mitogens lipopolysaccharide (LPS), pokeweed mitogen (PWM), and Staphylococcus aureus Cowan I (SAC I), alone and in combination and with and without interleukin (IL) was performed for in vitro activation of regional lymph node lymphocytes from lung cancer patients for the production of human IgG, IgM, and IgA. As assessed by electrofusion of the lymphocytes following their exposure to these agents with mouse myeloma cells and incubation of the fused hybridoma, a remarkable stimulatory effect was shown by LPS and particularly by LPS plus IL-4, which was substantially greater than that of either SAC I or PWM with or without various IL. Optimization studies indicated that the addition of PWM to LPS and IL-4 in the culture medium further stimulated the human antibody (Ab) production, and that the optimal formulation for stimulation of human IgG production was a culture medium containing 20 micrograms/ml of LPS, 1/500 of PWM, and 100 u/ml of IL-4.

Panclicins, novel pancreatic lipase inhibitors. I. Taxonomy, fermentation, isolation and biological activity.

Panclicins A, B, C, D, and E are novel pancreatic lipase inhibitors isolated from Streptomyces sp. NR 0619. Structurally, panclicins A, B, C, D, and E are analogues of tetrahydrolipstatin (THL), which contains a beta-lactone and a N-formyl leucine ester, and the IC50s of panclicins A, B, C, D, and E for porcine pancreatic lipase are 2.9, 2.6, 0.62, 0.66, and 0.89 microM, respectively. The potency of the inhibitory activity of each compound is attributed to the amino acid moiety of each structure. The panclicins are either glycine-type compounds such as panclicins C, D, E, which are two to threefold more potent than THL, or they are alanine-type compounds such as panclicins A and B, which are less potent than the glycine compounds. The inhibitory profiles of the panclicins for other lipases such as post-heparin plasma lipases and bacterial lipases are similar to those for pancreatic lipase. Panclicins A, B, C, D, and E, in a manner similar to THL, irreversibly inhibit pancreatic lipase. However, the compounds don't irreversibly inhibit the enzyme as strongly as THL does.