DNA ligase I mRNA and enzyme levels in human hematopoietic cells under dimethyl sulfoxide-induced growth-arrest and differentiation.

About half the activity level of DNA ligase I in cycling human lymphoblastoid cells (Raji and Akata) remained in the cells arrested at G1 by a 4-day treatment with 1.5% dimethyl sulfoxide (DMSO), and one-third the enzyme activity in actively growing promyelocytic leukemia cells HL-60 was detected in the terminally differentiated cells after DMSO-treatment. In contrast, DNA ligase I mRNA was negligible in the G1-arrested Raji and differentiated HL-60 cells. The steady-state mRNA level was increased 9 h after release from DMSO in the G1-arrested Raji cells and reached a maximum at 18 h. These results indicate that gene expression of human DNA ligase I, but not activity level of the enzyme, is closely correlated with activity of cell proliferation.     

Onset of the constrictive effect of indomethacin on the ductus arteriosus in fetal rats.

The time of onset of the constrictive effect of indomethacin on the ductus arteriosus (DA) in fetal rats was assessed by measurement of the caliber of the DA after maternal treatment with indomethacin on days 19-21 of gestation. The day following overnight mating was regarded as day 0 of gestation. Observation was performed by direct exposure of the DA by hand shaving of intact frozen fetuses. On days 20 and 21, the DA was significantly constricted 3 h after maternal treatment with 1 mg/kg of indomethacin. When the DA was examined at 19 1/2 and 19 2/3 days of gestation (3 h after indomethacin exposure), it was significantly constricted at 19 2/3 days but not at 19 1/2 days. Higher doses of indomethacin (10 and 100 mg/kg) induced a significant constriction of the DA at day 19 1/2, but not at the beginning of the same day (1.00 a.m.). These results suggest that the onset of the susceptibility of the DA to the constrictive effect of indomethacin occurs in the first half of day 19 of gestation.     

Active c-erbB-2 induces short-term growth of FDC-P2 cells after IL-3 depletion.

A retroviral expression vector carrying the c-erbB-2 gene was introduced into the FDC-P2 myeloid cell line, which is absolutely dependent on interleukin-3 (IL-3) for proliferation and survival. Since the c-erbB-2 protein appears to be the receptor of an as yet unidentified growth factor, epidermal growth factor receptor (EGFR) was used as a control of a ligand-dependent receptor. FDC-P2 cells expressing normal c-erbB-2 were unable to grow without IL-3 stimulation. The c-erbB-2 protein in these cells was under-phosphorylated on tyrosine residues in vivo. On the contrary, the active c-erbB-2 protein, in which Val-659 was replaced by Glu in the transmembrane domain, and EGF-stimulated EGFR showed significant levels of tyrosine phosphorylation in vivo. These active proteins could promote short-term growth of FDC-P2 cells without IL-3 stimulation, though not indefinitely. These findings suggested that immortalization of this factor-dependent cell line requires an additional oncogenic promoting process(es).     

An endonuclease activity in human polymorphonuclear neutrophils that removes 8-hydroxyguanine residues from DNA+

An endonuclease that specifically removes 8-hydroxyguanine (oh8Gua) from DNA has been isolated from Escherichia coli. As the amount of oh8Gua produced in DNA of X-ray-irradiated mice is known to decrease with time after irradiation, an attempt was made to find a similar activity in human polymorphonuclear neutrophils (PMNs) using a synthetic dsDNA containing oh8Gua as a substrate. The PMN enzyme was isolated free of other DNases, and found to cleave the substrate DNA simultaneously at 2 sites, the phosphodiester bonds 5' and 3' to oh8Gua, producing free hydroxyl and phosphate groups, respectively. The enzyme showed almost no activity on DNAs containing other kinds of modified base tested or mismatched DNA. Thus human cells also contain an endonuclease that specifically removes oh8Gua residues from DNA.     

Analysis of non-heme iron in arachidonate 12-lipoxygenase of porcine leukocytes

Arachidonate 12-lipoxygenase of porcine leukocytes, which was purified to homogeneity by immunoaffinity chromatography, was analyzed for iron content by atomic absorption spectrophotometry. The enzyme contained 0.70 +/- 0.09 g atom of iron per mol of enzyme (mean +/- S.D., n = 4). Inorganic iron, which was added to the enzyme solution as an internal standard, was recovered in almost 100% yield. Among various iron chelators tested, only 2,2'-dipyridyl at 1 mM inactivated the enzyme by 87%, but the enzyme was not reactivated by the addition of excess ferrous or ferric iron.     

Free radical damage to proteins: the influence of the relative localization of radical generation, antioxidants, and target proteins

Free radicals were generated at known rates in the aqueous phase (by means of 2,2'-azobis (2-amidinopropane) dihydrochloride [AAPH]) and in a membranous (lipid) phase (by means of 2,2'-azobis (2,4-dimethylvaleronitrile [AMVN]). A soluble protein (bovine serum albumin: BSA), and membranes of lysed mitochondria containing radioactively labeled monoamine oxidase (MAO), were exposed to the resultant radical fluxes. Antioxidants were added to the system, either in the aqueous phase (Trolox) or in a liposomal membrane phase (alpha-tocopherol). Protein damage was assessed as tryptophan oxidation and conformational changes in tryptophan fluorescence of the soluble protein, BSA, and as fragmentation of both BSA and monoamine oxidase. Radicals generated in the aqueous phase, by AAPH, were effective in damaging BSA and MAO. Radicals generated within the liposome membrane phase (by AMVN) were less effective against BSA than those deriving from AAPH. Liposomal AMVN radicals could damage MAO, present in a separate membranous phase, though again, less effectively than could AAPH-derived radicals. BSA could be protected by Trolox, the aqueous soluble antioxidant, but hardly by tocopherol itself. Damage to MAO was limited by Trolox, and also by the hydrophobic antioxidant, tocopherol. Damaging reactions due to radicals generated in a membrane phase were significantly accelerated when the membrane was peroxidizable (soybean phosphatidylcholine) rather than nonperoxidizable (saturated dimyristoyl phosphatidylcholine). Thus lipid radicals also played some role in protein damage in these systems. BSA was attacked similarly in the presence or absence of liposomes by AAPH. Correspondingly, BSA could inhibit the peroxidation of liposomes induced by AAPH and less efficiently that induced by AMVN.(ABSTRACT TRUNCATED AT 250 WORDS)     

Isolation and functional expression of pituitary peptidylglycine alpha-amidating enzyme mRNA. A variant lacking the transmembrane domain

We demonstrate that, in rat pituitary, peptidylglycine alpha-amidating enzyme was encoded by at least 5 distinct mRNAs. Southern blot and ribonuclease protection analyses revealed that the mRNAs arose through alternative splicing. A variant lacking the transmembrane domain-coding sequence was a major mRNA species for the enzyme in the pituitary. When the cDNAs were expressed in COS-7 cells, the variant was the most efficient in producing a secretory form (37 kDA) of the enzyme.     

A novel TIMP-insensitive type IV collagen-degrading metalloproteinase from murine metastatic sarcoma cells

A novel type IV collagen-degrading metalloproteinase was purified from the conditioned media of a murine metastatic sarcoma cell line. The molecular weight of the purified enzyme was determined to be 100 kDa by SDS-PAGE, while 700 kDa by gel filtration suggesting that the enzyme has a multimer structure. This enzyme degrades type IV collagen, but neither type I collagen nor casein. The failure of trypsin treatment to enhance the enzyme activity suggested that the purified enzyme did not require activation. Although the enzyme seems to be classified as a matrix metalloproteinase, it was inhibited by neither tissue inhibitor of metalloproteinases (TIMP) nor TIMP-2 and thus represents a novel type IV collagen-degrading metalloproteinase.     

Prevalence of spotted fever group rickettsia antibody in Apodemus speciosus captured in an endemic focus in Miyazaki Prefecture, Japan

Fourteen of Apodemus speciosus (large Japanese field mouse) were captured near the place where one of the patients with spotted fever group rickettsiosis had been infected, in Takaoka town, Miyazaki Prefecture. In the town, four human cases were reported. All of the mice had antibodies against Rickettsia japonica and R. montana. The incidence of the antibody was significantly higher in Apodemus mice in the area than in those from nonendemic area.     

Sugar-nucleotide donor specificity of histo-blood group A and B transferases is based on amino acid substitutions

Four amino acid substitutions (aa 176, 235, 266, and 268) have been found between the coding regions of cDNAs for histo-blood group A and B transferases (Yamamoto F., Clausen, H., White, T., Marken, J., and Hakomori, S. (1990) Nature 345, 229-233). Here we establish the basis of differential affinity of these glycosyltransferases to nucleotide-sugar (UDP-GalNAc or UDP-Gal). On the basis of gene reconstruction experiments and studies of expression in DNA transfected HeLa cells, the third as well as the fourth aa substitutions (leucine and glycine in A and methionine and alanine in B), which were calculated to modify flexibility of the protein, were found to be crucial in determining nucleotide-sugar specificity. The second substitution (glycine in A and serine in B) also affects the specificity. We have also created new enzymes which catalyze the transfer of both GalNAc and Gal, and may provide an explanation of the rare cis-AB phenotype.