Sulfated glycoproteins synthesized by the corneal epithelium of chick embryo having the same molecular weights as cytokeratin polypeptides

The previous study from this laboratory demonstrated that the corneal epithelium of 19-d-old chick embryo synthesizes two classes of sulfated glycoconjugates consisting of sulfated glycoproteins and proteoglycans (Yonekura, H., Oguri, K., Nakazawa, K., Shimizu, S., Nakanishi, Y., & Okayama, M. (1982) J. Biol. Chem. 257, 11166-11175). The present study demonstrated that when the sulfated glycoproteins labeled metabolically with [35S]sulfate and [3H]glucosamine were analyzed by SDS-PAGE, the 70,000 component (accounting for approximately 30% of the 35S and 35% of the 3H of the total sulfated glycoprotein) co-migrated with five major proteins with apparent molecular weights (Mrs) of 70,000, 66,000, 58,000, 51,000, and 48,000, which together accounted for about 57% of the total tissue protein. All five proteins cross-reacted with an antibody against human sole keratin, indicating that they are cytokeratin polypeptides of the corneal epithelium. Amino acid analysis demonstrated that they had high contents of glycine, serine, glutamic acid, leucine, and aspartic acid. Two-dimensional tryptic peptide maps indicated that they were all different. Analysis of radiolabeled materials released by alkaline borohydride treatment of the sulfated glycoproteins which were synthesized in the presence and absence of tunicamycin and co-purified with the five cytokeratin polypeptides, revealed that they contained both N- and O-glycosidically linked sulfated oligosaccharides. All the results obtained in the present study indicate that the five sulfated glycoproteins are similar, if not identical, to the cytokeratin polypeptides. This is consistent with the result in the accompanying paper that these sulfated glycoproteins are localized intracellularly.     

The amino acid sequence of ribonuclease N1, a guanine-specific ribonuclease from the fungus Neurospora crassa

The complete amino acid sequence of ribonuclease N1 (RNase N1), a guanine-specific ribonuclease from a fungus, Neurospora crassa, was determined by conventional protein sequencing, using peptide fragments obtained by tryptic digestion of cyanogen bromide-treated RNase N1 and by Staphylococcus aureus V8 protease digestion of heat-denatured RNase N1. The results showed that the protein is composed of a single polypeptide chain of 104 amino acid residues cross-linked by two disulfide bonds and has a molecular weight of 11,174: (sequence; see text) (Disulfide bonds: C2-C10, C6-C103) The amino acid sequence was homologous with those of RNase T1 (65% identity) and related microbial RNases.     

Structure of the human neutrophil elastase gene

The gene for human neutrophil elastase (NE), a powerful serine protease carried by blood neutrophils and capable of destroying most connective tissue proteins, was cloned from a genomic DNA library of a normal individual. The NE gene consists of 5 exons and 4 introns included in a single copy 4-kilobase segment of chromosome 11 at q14. The coding exons of the NE gene predict a primary translation product of 267 residues including a 29-residue N-terminal precursor peptide and a 20-residue C-terminal precursor peptide. Analysis of the N-terminal peptide sequence suggests it contains a 27-residue "pre" signal peptide followed by a "proN" dipeptide, similar to that of other blood cell lysosomal proteases. The sequences for the mature 218-residue NE protein are included in exons II-V. The 5'-flanking region of the gene includes typical TATA, CAAT, and GC sequences within 61 base pairs (bp) of the cap site. The sequence 1.5 kilobases 5' to exon I contains several interesting repetitive sequences including six tandem repeats of unique 52- or 53-bp sequences. The 5'-flanking region also contains a 19-bp segment with 90% homology to a segment of the 5'-flanking region of the human myeloperoxidase (MPO) gene, a gene also expressed in bone marrow precursor cells and a protein stored in the same neutrophil granules as NE. In addition, like the MPO gene, the NE 5'-flanking region has several regions with greater than or equal to 75% homology to sequences 5' to c-myc, but there is no overlap between the NE-c-myc and MPO-c-myc homologous sequences.     

Effect of metal ions on the adaptive response induced by N-methyl-N-nitrosourea in Escherichia coli

Induction of the adaptive response was quantified by analysis of beta-galactosidase released after the treatment of Escherichia coli CHS26/pYM3 (ada'-lacZ') with N-methyl-N-nitrosourea (MNU). Of the 15 metal ions examined, only Cd++ and Hg++ inhibited induction of the adaptive response with neither severe suppression of cell growth nor inhibition of the induction of the SOS response by MNU. Mutagenicity of MNU was potentiated by the presence of these metal ions in an E. coli strain. These results suggest that the inhibition mechanism involves a specific interaction of Cd++ or Hg++ with O6-methyl-guanine-DNA methyltransferase.     

Acute reversible cataract due to nitrocompounds in Japanese quail (Coturnix coturnix japonica)

The present study was designed to examine the usefulness of the Japanese quail as an experimental model of cataractogenic activity. Chemicals, 2, 6-dibromo-4-nitro-phenol (2, 6-D), 2, 4-dinitroanisole (2, 4-DA), and 2, 4-dinitrophenol (2, 4-D; for the positive control), were administered singly through an oral route to 2-week old male Japanese quails to investigate the reversibility of cataracts. A single administration of 2, 4-D (36 and 43 mg/kg) produced reversible cataract in 14 of 16 animals (87.5%). This cataract was seen 1 or 2 hours after treatment and continued for 1 to 12 hours. Treatment with 2, 6-D (20 and 25 mg/kg) and 2, 4-DA (120 and 150 mg/kg) caused cataracts in 7 of 11 (63.6%) and 8 of 8 surviving animals (100%), respectively. Cataracts produced by 2, 6-D and 2, 4-DA, which were observed from 1 and 2 to 4 hours after the treatment, continued for 6 to 15 and 1 to 13 hours, respectively. Mortalities in the 25 mg/kg group of 2, 6-D, 120 mg/kg and 150 mg/kg group of 2, 4-DA were found in 2 of 5 animals, 1 of 5 animals and 5 of 9 animals, respectively. These results indicate that the Japanese quail is useful as an animal model to evaluate toxicity to the eye and cataractogenesis.     

Oxidation of lipids. XV. Role of hydrophilic diarylamines as antioxidants in the oxidations of lipids and biological tissues

The abilities of two kinds of water-soluble diarylamines, disodium 4-chloro-2,2'-iminodibenzoate (CCA) and disodium 4-chloro-3',6'-dimethyl-2,2'-iminodibenzoate (CCM), to protect lipids, membranes and biological tissues from oxidative damages have been studied. The experimental systems studied include the oxidations of methyl linoleate micelles and soybean phosphatidylcholine (Pc) liposomal membranes in aqueous dispersions, oxidative hemolysis of rabbit erythrocytes, and the in vivo oxidative damages of biological tissues all induced by free radicals generated from an azo radical initiator. The two diarylamines functioned as moderate chain-breaking antioxidants and retarded the above oxidations.     

Characterization of a glutathione conjugate of the 1,4-benzosemiquinone-free radical formed in rat hepatocytes

Rat hepatocytes treated with 1,4-benzoquinone formed 1,4-benzosemiquinone and 2-S-glutathionyl-1,4-benzosemiquinone radicals as detected by ESR spectroscopy. The 2-S-glutathionyl-1,4-benzosemiquinone radical was first obtained from the reaction of 1,4-benzoquinone with glutathione. Glutathione both reduced benzoquinone to form benzosemiquinone and conjugated benzoquinone to form 2-S-glutathionyl-1,4-benzosemiquinone radical. The ratio of these two radicals depended upon the ratio of 1,4-benzoquinone to glutathione. At near equimolar ratios, the 2-S-glutathionyl-1,4-benzosemiquinone radical was predominantly formed. This radical was characterized by computer simulation of the experimental spectra and identified by comparison of its hyperfine coupling constants with those of chemical analogues. The 2-S-glutathionyl-1,4-benzosemiquinone radicals formed inside hepatocytes, and then crossed the plasma membrane into the media.     

Analyses of constituents of feces and the effect of a vitamin B12 fortified diet on coprophagy in the mouse

In order to investigate coprophagy from the viewpoint of nutrition, fecal constituents were analyzed in freeze-dried samples. Feces were collected from 7:00 to 11:00 and from 19:00 to 23:00. Inorganic elements and crude fibers per unit weight were 3-4 times more concentrated in feces than in basal diet, whereas, crude proteins, crude fats and nitrogen-free extract showed various degrees of reduction. There were no differences in these tendencies with sampling time. As for some B vitamins, feces collected from 7:00 to 11:00 contained 22-92% more vitamins than feces collected from 19:00 to 23:00. In comparison with the dietary concentration, vitamin B12 was increased by 124-197 times (520-730 micrograms/100 g) in feces collected between 7:00 and 11:00. Folic acid in feces collected between 7:00 and 11:00 was 10 times greater than that in the diet. On the basis of the findings on vitamins, the effect of a vitamin B12 fortified diet (1,350 micrograms/100 g) on coprophagy was examined. Mean frequency of coprophagy per animal per day was 9.6 when animals were fed on the basal diet, whereas the frequency was immediately and significantly (p less than 0.05 approximately p less than 0.01) reduced to 4.7 after the diet had been replaced by the fortified one. However, coprophagy was not completely inhibited by vitamin B12 fortification. This indicates that some nutrient(s) in feces other than vitamin B12 might be of use to the host, and that otherwise, coprophagy might be a basically habitual form of behavior. Furthermore, under the fortified diet, the frequency of coprophagy increased gradually.(ABSTRACT TRUNCATED AT 250 WORDS)     

Carbohydrate structures of acetylcholine receptor from Torpedo californica and distribution of oligosaccharides among the subunits

The structure of carbohydrates in acetylcholine receptor (AChR) from Torpedo californica is reported. Oligosaccharides released quantitatively from the whole molecule by N-oligosaccharide glycopeptidase digestion were fractionated by thin-layer chromatography and further purified by high-performance liquid chromatography. We show that more than 70% of the total oligosaccharide chains in Torpedo AChR are of the high-mannose type with the structures (Man)8(GlcNAc)2 and (Man)9(GlcNAc)2. The structure of these oligosaccharides were determined by proton nuclear magnetic resonance spectroscopy. These two types of oligosaccharides were shown to be distributed different proportions in all subunits of Torpedo AChR. We also show that several kinds of complex-type oligosaccharides comprising the rest of the carbohydrate in the protein exist mainly in the gamma and delta subunits. The structure of the carbohydrate moiety that is distributed on the four subunits of AChR was also examined by susceptibility to endo-beta-N-acetylglucosaminidase and sialidase and by binding affinity to lectins, e.g. concanavalin A, leucoagglutinating phytohemagglutinin, and wheat germ agglutinin.     

DNA fragments of 300 base pairs released from metaphase chromosomes by digestion with deoxyribonuclease I

DNase I digestion of metaphase chromosomes, that have been extensively digested with Hae III, further released chromosomal DNA and proteins; 3.3% and 10.8% of the chromosomal DNA and proteins, respectively, remained insoluble. However, digestion of chromosomes first with DNase I followed by Hae III caused most of the proteins to remain in the insoluble fraction. DNase I released DNA fragments of 300 base pairs long which were not released by Hae III digestion. These DNA fragments may be protected by protein components from further fragmentation by DNase I.