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.     

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.     

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.     

Activation of human Hageman factor by Pseudomonas aeruginosa elastase in the presence or absence of negatively charged substance in vitro.

Human Hageman factor, a plasma proteinase zymogen, was activated in vitro under a near physiological condition (pH 7.8, ionic strength I = 0.14, 37 degrees C) by Pseudomonas aeruginosa elastase, which is a zinc-dependent tissue destructive neutral proteinase. This activation was completely inhibited by a specific inhibitor of the elastase, HONHCOCH(CH2C6H5)CO-Ala-Gly-NH2, at a concentration as low as 10 microM. In this activation Hagemen factor was cleaved, in a limited fashion, liberating two fragments with apparent molecular masses of 40 and 30 kDa, respectively. The appearance of the latter seemed to correspond chronologically to the generation of activated Hageman factor. Kinetic parameters of the enzymatic activation were kcat = 5.8 x 10(-3) s-1, Km = 4.3 x 10(-7) M and kcat/Km = 1.4 x 10(4) M-1 x s-1. This Km value is close to the plasma concentration of Hageman factor. Another zinc-dependent proteinase, P. aeruginosa alkaline proteinase, showed a negligible Hageman factor activation. In the presence of a negatively charged soluble substance, dextran sulfate (0.3-3 micrograms/ml), the activation rate by the elastase increased several fold, with the kinetic parameters of kcat = 13.9 x 10(-3) s-1, Km = 1.6 x 10(-7) M and kcat/Km = 8.5 x 10(4) M-1 x s-1. These results suggested a participation of the Hageman factor-dependent system in the inflammatory response to pseudomonal infections, due to the initiation of the system by the bacterial elastase.     

Gas chromatographic method for the determination of urinary acetylpolyamines

A gas chromatographic method was developed for the determination of monoacetylputrescine, monoacetylcadaverine, N¹-acetylspermidine and N⁵-acetylspermidine in human urine. The amines were isolated from urine by silica gel column chromatography. 1, 10-Diaminodecane was used as internal standard. The amines were reacted with ethyl chloroformate in aqueous medium to four ethyloxycarbonyl derivatives prior to application to gas chromatography using a flame ionization detector. Separation and determination of the derivatives were carried out on a Uniport HP column (1.0 m) impregnated with 0.5% SP-1000 under temperature-programmed conditions. The monoacetylpolyamines could be measured accurately at the nanomole level. The method was used for the determination of the monoacetylpolyamines in urine of healthy volunteers. The values obtained were in the range of the published data.     

Biological effects of anti-idiotypic antibodies on lymphocyte function: I. Analysis of the effects on B lymphocytes of combining site and framework-directed anti-T-15 idiotypic antibodies

Anti-idiotypic antibodies against TEPC-15 myeloma protein (BALB/c origin) were raised in allogeneic animals by immunization of A/J mice with the myeloma protein. The antibody activities were fractionated into two specificities by TEPC-15 immunoadsorbent affinity columns by elution with free hapten (phosphorylcholine, PC), followed by elution with acidic buffer (glycine- HCl, pH 2.3). Idiotype binding analysis indicated that the fraction eluted with hapten could be inhibited in its binding to TEPC-15 by free hapten (i.e., binding site-directed anti-idiotypic antibody), whereas the acid-eluted fraction could not (i.e., framework-directed anti-idiotypic antibody). When analyzed for their biological activities on PC-specific B lymphocytes producing T-15 idiotype-bearing antibodies, both anti-idiotypic antibody fractions had similar suppressive effects on the in vitro production of antiphosphorylcholine antibody in culture.