Partial purification and characterization of a factor which stimulates an increase in ornithine decarboxylase activity in the liver from tumor cell-free ascites fluid

A factor responsible for stimulating an increase in ornithine decarboxylase activity in the liver of mice was found in tumor cell-free ascites fluid of mice 3 days after inoculation of tumor cells. The factor was purified about 70-fold in 25% yield from tumor cell-free ascites fluid. As little as 1 μg of protein of purified fraction, injected intraperitoneally into normal mice, significantly increased the activity of ornithine decarboxylase in the liver. The most active preparation of the factor formed two major protein bands on analytical polyacrylamide gel electrophoresis and both these bands stained with periodic acid-Schiff's reagent. The factor was a heat-labile, alkaline-stable, acidic protein with a molecular weight of more than 300 000. It was inactivated by treatment with 10 mM dithiothreitol, 5M urea, pronase or mixed glycosidase, but was stable on treatment with DNAase, RNAase or neuraminidase.     

Lack of association and linkage between HLA and familial polyposis coli

Summary We investigated possible association of and linkage between HLA and familial polyposis coli (FPC). In 182 individuals from 66 pedigrees of FPC and 108 individuals from a normal population, HLA-A,-B, and-C antigens were determined. When the frequencies of HLA antigens in 66 unrelated patients and in normal controls were compared, no association of FPC with HLA was observed. For the linkage analysis, HLA haplotypes of 17 affected sib pairs were investigated by the affected sib pair method. The number of pairs which shared two, one, and no haplotypes identical by descent was not significantly different from the number expected with random occurrence (P>0.95). Finally, seven families were analyzed using Morton's sequential test. A maximum lod score of-0.056 at a recombination fraction of 0.4, and a lod of-3.089 at a recombination fraction of 0.05 were obtained. Therefore, there is neither an association of nor linkage between FPC and HLA.     

Assignment of the human myeloperoxidase gene (MPO) to bands q21.3----q23 of chromosome 17

Using a human myeloperoxidase cDNA, we have mapped the human myeloperoxidase gene to chromosome 17 at q21.3----q23 by in situ hybridization to metaphase chromosomes from human lymphocyte preparations.     

Characteristics of settling matter and its role in nutrient cycles in a deep oligotrophic lake

The settling flux of seston (dry weight, DW), chlorophyll a (Chl a), particulate organic carbon (POC), particulate organic nitrogen (PON), and particulate phosphorus (PP) was measured monthly in 1981–1983 at 10 different depths in Lake Chuzenji, Japan; an oligotrophic lake with a maximum depth of 163 m. The Ti concentration in entrapped matter was used to separate the sedimentation flux into allochthonous and autochthonous components. Inflow loads of dissolved nutrients (DN: 4.5, DP: 0.48 g m^-2a^-1) were almost sufficient to supply the autochthonous fluxes at 30 m (PON: 2.9, PP: 0.51 g m^-2a^-1 ), and this flux of POC (26.6 g m^-2a ^-1) was about one-third of primary production (84 g C M^-2a^-1). Sedimentation of particulate matter was the main path of losing nutrients from lake water, explaining more than 80% removal of inflow loads (TN, TP). Decomposition rates during sedimentation which were calculated from the vertical difference in the autochthonous flux agreed very closely with the results obtained by laboratory experiments of a 100-day incubation (content ratios from field observations were: POC 0.67, PON 0.65, PP 0.85; and from laboratory experiments they were: POC 0.68, PON 0.70, PP 0.94). These decomposition rates and those near the sediment interface were used to explain dissolved oxygen depletion and nitrate increase in the hypolimnion during stratification. The average sinking velocities were 1.82m d^-1 for seston and 1.16 m d^-1 for Chl a at 30m, they were influenced by Chl a content of seston.     

Mast cell degranulation and its inhibition by an anti-allergic agent tranilast. An electron microscopic study

Degranulation of IgE-sensitized rat mast cells by antigen was studied quantitatively in vitro and in vivo by electron microscopy. The inhibition of this degranulation by an anti-allergic drug, N-(3,4-dimethoxycinnamoyl)anthranilic acid (Tranilast), was also examined both in vitro and in vivo. In the in vitro study using peritoneal mast cells, alteration of the granules, cavity formation by fusion of the perigranular membrane and granule discharge due to fusion of the cavity membrane with the cell membrane were observed and were accompanied by histamine release. Scanning electron microscopy disclosed the extrusion of smooth, round bodies from pores formed on the cell surface. In the in vivo study of passive cutaneous anaphylaxis (PCA), the characteristic features of mast cell degranulation were obvious 5 min after the injection of antigen; leakage of dye increased progressively from 5 to 30 min but was not found at 6 h. From quantitative analysis of the substructure of mast cells, it was demonstrated that degranulation of IgE-sensitized mast cell induced by antigen was achieved by sequential exocytosis both in vitro and in vivo. Tranilast inhibited these changes to a remarkable extent and it was concluded that the inhibition of mast cell degranulation by this drug might play an important role in anti-allergic treatment.     

Preparation ofN-acetylneuraminic acid from delipidated egg yolk

Egg yolk, a large proportion of the egg, was studied for the preparation ofN-acetylneuraminic acid (Neu5Ac). The delipidated hen egg yolk (DEY; 500 kg containing 0.2% w/w, Neu5Ac) was hydrolysed with HCl (pH 1.4) at 80 °C and neutralized with NaOH (pH 6.0). The mixture was filtered and electrodialysed until the conductivity was 240 µS cm^–1. The filtrate was applied on a column of Dowex HCR-W2 (20–50 mesh), followed by a column of Dowex 1-X8 (200–400 mesh). The latter column was washed with water, and then eluted with a linear gradient of HCO₂H (0–2m). The eluates containing Neu5Ac were concentrated using a reverse osmosis membrane and, finally, rotary evaporated at 40 °C. The residue was then lyophilized to yield 500 g Neu5Ac. The purity of Neu5Ac was >98% (TBA method). HPLC, NMR spectroscopy and TLC chromatography of the product obtained from the DEY showed that Neu5Ac was the sole derivative present in egg yolk. The DEY, a byproduct from egg processing plants, was found to be an excellent source for the large-scale preparation of Neu5Ac.Abbreviations Neu5Ac N-acetylneuraminic acid - Neu5Gc N-glycolylneuraminic acid - DEY delipidated egg yolk - HPLC high performance liquid chromatography - TLC thin layer chromatography - NMR nuclear magnetic resonance - IR infrared spectroscopy Presented at the 11th International Symposium on Glycoconjugates, Toronto, Canada.     

New mutations in cloned Escherichia coli umuDC genes: novel phenotypes of strains carrying a umuC125 plasmid

The umuDC locus of Escherichia coli is required for most mutagenesis by UV and many chemicals. Mutations in E. coli umuDC genes cloned on pBR322-derived plasmids wer e isolated by two methods. First, spontaneously-arising mutant umuDC plasmids that failed to confe cold-sensitive growth on a lexA51(Def) strain were isolated by selection. Second, mutant umuDC plasmids that affected apparent mutant yield after UV-irradiation in a strain carrying umuD⁺C⁺ in the chromosome were isolated by screening hydroxylamine-mutagenized umuD⁺C⁺ plasmids. pBR322-derived umuD⁺C⁺ plasmids inhibited the induction of the SOS response of lexA⁺ strains as measured by expression of din::Mu dl(lac) Ap) fusionsbut most mutant plasmids did not. Mutant plasmids defective in complementation of chromosomal umuD44, umuC36, or both were found among those selected for failure to confer cold-sensitivity, whereas those identified by the screening procedure yielded mostly mutant plasmids with more complex phenotypes. We studied in greater detail a plasmid pLM109, carrying the umuC125 mutation. This plasmid increased the sensitivity of lexA⁺ strainsto killing by UV-irradiation but was able to complement the deficiencies of umuC mutants in UV mutagenesis. pLM109 failed to confer cold-sensitive growth on lexA(Def) strains but inhibited SOS induction in lexA⁺ strains. The effect of pLM109 on the UV sensitivity of lexA(Def)strains was similar to that of the parental umuD⁺C⁺ plasmid. The mutation responsible for the phenotypes of pLM109 was localized to a 615-bp fragment. DNA sequencing revealed that the umuC125 mutation was a G:C → A:T transition that changed codon 39 of umuC from GCC → GTC thus changing Ala39 to Val39. The implications of the umuC125 mutation for umuDC-dependent effects on UV-mutagenesis and cell survival after UV damage are discussed.     

Type II restriction endonucleases cleave single-stranded DNAs in general.

Restriction endonucleases (13 out of 18 species used for the test) were certified to cleave single-stranded(ss)DNA. Such enzymes as AvaII, HaeII, DdeI, AluI, Sau3AI, AccII,TthHB8I and HapII were newly reported to cleave ssDNA. A model to account for the cleavage of ssDNA by restriction enzymes was proposed with supportive data. The essential part of the model was that restriction enzymes preferentially cleave transiently formed secondary structures (called canonical structures) in ssDNA composed of two recognition sequences with two fold rotational symmetry. This means that a restriction enzyme can cleave ssDNAs in general so far as the DNAs have the sequences of restriction sites for the enzyme, and that the rate of cleavage depends on the stabilities of canonical structures.     

Serologic dissection of HLA-D specificities by the use of monoclonal antibodies

To study the gene products of the HLA complex, we produced two monoclonal antibodies, termed HU-18 and HU-23. They were active in complement-dependent cytotoxicity and detected B-cell alloantigens encoded by a locus (or loci) linked to HLA. When three types of HLA-DR4 homozygous B-cell lines with different HLA-D specificities were tested for reactivity with HU-18 and HU-23, they displayed distinct reaction patterns depending on the HLA-D specificities they possessed: EBV-Wa (HLA-DYT homozygous), negative for both HU-18 and HU-23; KT2 and KOB (HLA-DKT2 homozygous), positive only for HU-18; and ER (HLA-Dw4 homozygous), positive for both. These differential reaction patterns were further confirmed by testing against a panel of 17 HLA-DR4-positive peripheral blood lymphocytes with known HLA-D specificities. Thus, these monoclonal antibodies allow us to identify HLA-DYT, HLA-DKT2, and HLA-Dw4 solely by serologic methods. This is the first clearcut serologic identification of these three HLA-DR4-associated HLA-D specificities, which have been indistinguishable by conventional serology and identified only by cellular techniques. It is hoped that immunochemical investigations using HU-18 and HU-23 will advance our understanding of the HLA-D region on a molecular level.