Mutagenic activation of aflatoxin B1 by several forms of purified cytochrome P-450

Metabolic activation by several forms of purified cytochrome P-450 of aflatoxin B1 to a product(s) mutagenic to Salmonella typhimurium TA100 was examined. Of the 5 forms of cytochrome P-450 purified from liver microsomes of untreated and PCB-treated male rats, a constitutive form purified from untreated male rats, P-450-male, and a high-spin form of cytochrome P-450, P-448-H, from PCB-treated rats were highly active.     

Effects of phenobarbital, 3-methylcholanthrene and polychlorinated biphenyls on sex-specific forms of cytochrome P-450 in liver microsomes of rats

The effects of treatment with phenobarbital, 3-methylcholanthrene or polychlorinated biphenyls (PCB) on the amounts of sex-specific forms of cytochrome P-450, namely P-450-male and P-450-female, in male and female rats were studied. Although treatment with phenobarbital, 3-methylcholanthrene or PCB markedly increased the total amount of hepatic cytochrome P-450, P-450-male and P-450-female were rather decreased or not significantly changed. Thus, the percentages of P-450-male and P-450-female in the total cytochrome P-450 were decreased in liver microsomes from the treated rats. The increases in specific cytochrome P-450, such as P-448-H, P-448-L, and P-450I-c accounted for the increase in the total amount of cytochrome P-450 in the treated rats. The treatment with phenobarbital or PCB increased the activities of testosterone 16 alpha-hydroxylase, benzo(a)pyrene hydroxylase and aminopyrine N-demethylase more markedly in female rats than in male rats. Similarly, the treatment with 3-methylcholanthrene increased benzo(a)pyrene hydroxylase more markedly in female rats. Therefore, the sex-differences in testosterone 16 alpha-hydroxylase, benzo(a)pyrene hydroxylase, and aminopyrine N-demethylase activities became smaller after the drug treatment. These results indicate that sex-specific P-450-male and P-450-female were unaffected, or even depressed by the agents in some cases.     

Isolation and characterization of the complete complementary and genomic DNA sequences of human serum amyloid P component

Complementary and genomic DNA clones corresponding to the human serum amyloid P component (SAP) mRNA have been isolated and analyzed. The nucleotide sequences of the cDNA and the corresponding regions of the genomic SAP DNA reported here were identical, and revealed that after coding for a signal peptide of 19 amino acids and the first two amino acids of the mature SAP protein, there is one small intron of 115-base pairs (bp), followed by a nucleotide sequence coding for the remaining 202 amino acid residues. The SAP gene has an ATATAAA sequence 29-bp upstream from the cap site, but there is no CAAT box-like sequence. A possible polyadenylation signal sequence, ATTAAA, was found to be located 28-bp upstream from the polyadenylation site. A comparison of the genomic SAP DNA sequence with that of human C-reactive protein (CRP) revealed a striking overall homology which was not uniform: several highly conserved regions were bounded by non-homologous regions. This comparison provides further support for the hypothesis that SAP and CRP are products of a gene duplication event.     

Synthetic analogs of the active site of cytochrome P-450cam characterization of thiolpeptide fragment-hemin complexes by optical and ESR spectrometries

Thiol-containing penta (Leu-Ala-Cys-Ser-Leu) and nona (Leu-Ala-Cys-Ser-Leu-Ile-Glu-Ser-Leu) peptides corresponding to residues 132-136 and 132-140, respectively, of apo-P450 from Psuedomonas putida were synthesized to examine heme-binding by the enzymes in the oxidized (ferric) form. The peptide-hemin complexes prepared in solution were characterized by their optical and ESR spectra. In these complexes without nitrogenous ligands, no ferric complexes in the low-spin state were observed, suggesting that simultaneous axial coordination of Cys-134 (thiolate) and Ser-139 (hydroxyl) of apo-P450cam to the heme is unlikely to occur. In the presence of nitrogenous ligands, such as py, Im and MeIm, the resulting complexes were good models of apo-P450cam-nitrogenous ligand adducts in the low-spin ferric state retaining a thiolate-Fe(III)-nitrogen axial coordination mode, as judged by their spectral pattern as well as by crystal field analysis of ESR g-values.     

A temperature-sensitive Chinese hamster ovary cell mutant pleiotropically defective in protein export

We have developed a new selection procedure for mammalian cell mutants defective in protein export by the use of diphtheria toxin, and devised a new screening method for defective protein secretion using nitrocellulose membranes. By the combination of these procedures, we have isolated a temperature-sensitive mutant clone of Chinese hamster ovary cells which shows a pleiotropic defect in protein export. This mutant, designated DS28-6, is temperature-sensitive for growth. Secretion of a series of proteins is markedly inhibited at the non-permissive temperature. These proteins seem to be normally synthesized and accumulated within the cell at the non-permissive temperature and secreted upon shift down to the permissive temperature. When this mutant is infected with vesicular stomatitis virus, oligosaccharide processing of G-protein is arrested at an endoglycosidase-H-sensitive stage at the non-permissive temperature. The lesion of this mutant appears to be in the endoplasmic reticulum or the cis Golgi or both.     

Postnatal development of the anulospiral endings of Ia fibers in muscle spindles of mice

The formation of the anulospiral ending of Ia fibers in muscle spindles was investigated in the masseter muscle of developing mice. Before 15 days after birth, the complete anulospiral ending was not observed in almost all of the muscle spindles examined. With the growth of mice, the Ia fiber began to construct the spiral ending, and by the 40th postnatal day after weaning, almost all of the Ia fibers of the muscle spindles had complete coiled endings, though the formation still continued in some spindles. The continuous formation of anulospiral endings for a long period after weaning indicates that muscle spindle morphogenesis may be affected by muscle tension in the masseter muscle due to the movement activated after weaning.     

Promotion of Rooting in Azukia Cuttings by Possible Glycoproteins Extracted from Lentinus edodes Culture

Macromolecules purified from Lentinus edodes mycelia cultureenhanced adventitious root formation in Azukia epicotyl cuttings.Partial purification by sequential column chromatographies gavematerial composed of 71% polysaccharides and 29% proteins. Thesugar moiety consisted of mainly xylose, glucose and arabinose,the sum of their contents being more than 76% of the total carbohydrates.The protein moiety consisted of mainly glycine and serine, whichaccounted for more than 40% of the total amino acid residues. (Received June 9, 1984; Accepted November 12, 1984)     

Phorbol ester increases the level of interleukin 2 mRNA in mitogen-stimulated human lymphocytes

TPA alone did not induce the production of IL 2 in human tonsillar lymphocytes but enhanced the PHA-induced IL 2 production by seven-fold. That the effect of TPA was due to an increase in IL 2 mRNA was demonstrated by examining the amount of IL 2 mRNA translatable in Xenopus laevis oocytes, and by Northern blotting analysis using IL 2 cDNA as a probe. In these ways, it was shown that TPA alone did not induce any significant IL 2 mRNA synthesis, but when added together with PHA it increased the level of IL 2 mRNA by at least 10-fold, as compared with that induced by PHA alone.     

The Tropic Effect of Intrarectal Deoxycholate On Rat Colorectum Is Unaffected By Oral Metronidazole

Abstract. Intrarectal administration of sodium deoxycholate (SDC) enhances experimental colorectal carcinogenesis, an effect that is partly vitiated by oral metronidazole. the effect of topical SDC with or without concurrent metronidazole on colorectal cell proliferation was explored in male Sprague-Dawley rats ( n = 30) allocated to five groups. Two groups received thrice weekly intrarectal instillations of 1 ml N saline or 1 ml 0.12 m SDC. A third group received SDC plus metronidazole 22.5 mg/kg/day in the drinking water. Controls had no instillations or metronidazole alone. At time of killing (10 weeks), crypt cell production rate (CCPR) was determined by the stathmokinetic technique for four large-bowel segments. Saline had no significant effect on colorectal CCPR but SDC produced increases throughout, varying from 53% in the proximal colon to 222% in the rectum ( P < 0.01). Metronidazole did not reduce this effect, although given alone it reduced colonic CCPR by 40 to 50%. the direct tropic effect of bile acids could largely explain their cocarcinogenic properties. Since metronidazole does not prevent this increase in cell proliferation, its mildly protective role against cancer may reflect the presence of fewer anaerobes capable of degrading bile acids to carcinogenic metabolites.     

Isolation and characterization of a human interleukin 2 gene

An interleukin 2 (IL-2) gene was isolated from a Charon 4A human gene library. Electron microscopic examination of 15 heteroduplexes formed between the genomic DNAs and the IL-2 cDNAs demonstrated that the size of the IL-2 gene is about 5.1 +/- 0.5 kb and that there are at least two introns in this gene. Nucleotide sequence of the 5' flanking region of the IL-2 gene showed a homology with that of the corresponding region of the human immune interferon gene.