A highly sensitive chromogenic microtiter plate assay for plasminogen activators which quantitatively discriminates between the urokinase and tissue-type activators

A simple and highly sensitive chromogenic microtiter plate assay for plasminogen activators is described. The assay is based on plasmin cleavage of the synthetic tripeptide plasmin substrate H-D-norleucyl-hexahydrotyrosyl-lysine p-nitroaniline, which yields the yellow chromophore p-nitroanilide. Production of the latter compound is then quantitated spectrophotometrically at 405 nm on an ELISA plate reader. Linearity of the assay can be achieved over at least four orders of magnitude in a single experiment (0.01-100 milliPloug units) with appropriate incubation times. Capitalizing on tissue-type plasminogen activator's dependence on fibrin for enzymatic activity, the selective use of soluble fibrin products allows discrimination between urokinase and tissue-type activator. The utility of this aspect of the assay for the analysis of complex samples containing both types of plasminogen activators is demonstrated.     

STUDIES OF NECROTIC LESIONS IN CORN STALKS

Littlefield , Larry J., and Roy D. Wilcoxson . (U. Minnesota, St. Paul.) Studies of necrotic lesions in corn stalks . Amer. Jour. Bot. 49(10): 1072–1078. Illus. 1962.—In 3-day-old necrotic lesions in corn stalks caused by Fusarium graminearum, ground parenchyma cells were discolored and small amounts of a dark substance were present in the cells. The walls of phloem cells were also slightly discolored and a small amount of dark substance was present in the xylem cells. In older lesions the discoloration of parenchyma and phloem cells was more intense; many of the cells contained occluding substances; many phloem protoplasts collapsed, and xylem cells were partially to completely occluded. The occluding substance filling the cells appeared to be translocated from the lesion into the vessel elements extending beyond the lesion so that the bundles appeared as long, dark streaks in the stalk. The occluding substance in xylem, but not in phloem or parenchyma, stained with ruthenium red, a result indicating presence of pectin. Pectinase, however, did not remove the occluding substance. The pectinase dissolved the parenchyma cells in healthy tissues but not in the necrotic lesions. Necrosis in naturally infected plants began as small lesions, but the parenchyma cells quickly dissolved leaving the vascular bundles free of ground parenchyma. No occlusions were found in the central vascular system; a few xylem cells in the peripheral vascular system were occluded with the same substance observed in artificially inoculated plants. Phloem was entirely destroyed by the pathogen. The necrosis prevented upward movement of dye solution in the stalk, but did not measurably affect transpiration, probably because the lesions were not large. Yield was reduced in plants when lesions involved more than 50% of the tissue in inoculated internodes. Smaller lesions had no effect on yield.     

Different mechanisms contribute to simultaneous inhibition of urokinase and tissue-type plasminogen activators by glucocorticoids in human ovarian carcinoma cells

Previous studies from our laboratory have demonstrated that OVCA 433 human ovarian carcinoma cells are glucocorticoid responsive by several criteria and contain high affinity, saturable, steroid-specific glucocorticoid receptors. These cells secrete both mammalian plasminogen activators (PAs), urokinase (uPA) and tissue-type PA (tPA). Treatment of OVCA 433 cells with 1 x 10(-7) M dexamethasone (Dex) for 4 days led to 77% and 83% reductions in the extracellular activities of uPA and tPA, respectively, released into serum-free conditioned medium during a 1-h period. Dex treatment led to a 71% decrease in the rate of extracellular uPA antigen accumulation, as determined by enzyme-linked immunosorbent assay, as well as a 73% reduction in steady state uPA mRNA levels. In contrast, Dex treatment led to only a 42% decrease in the rate of extracellular tPA antigen accumulation and a 48% decrease in tPA mRNA levels; such decreases were insufficient to account for the 83% reduction in tPA activity. Thus, while Dex-induced decreases in uPA antigen and mRNA levels accounted for all but 6% of the decrease in uPA activity, a large discrepancy existed between the magnitudes of decreased tPA activity and decreased tPA antigen and mRNA levels. OVCA 433 cells produce both PAI-1 and PAI-2, two specific PA inhibitors. Treatment of cells with 1 x 10(-7) M Dex for 4 days led to a 3.3-fold increase in the rate of extracellular PAI-1 accumulation, with little or no effect on PAI-2 accumulation.(ABSTRACT TRUNCATED AT 250 WORDS)     

The synthesis of three 4-substitutedbenzo[b]thiophene-2-carboxamidines as potent and selective inhibitors of urokinase

Efficient syntheses of structurally novel 4-substituted benzo[b]thiophene-2-carboxamidmes 1–3, which selectively inhibit urokinase-type plasminogen activator (uPA) with IC₅₀ values of 70–320 nM, are described. The key intermediate, methyl 4-iodobenzo[b]thiophene-2-carboxylate (7), is prepared from 3-fluoroiodobenzene in two steps in 70% overall yield via fluorine-directed metalation/formylation and subsequent thiophene annulation. Amidination of ester 7 gives the 320 nM inhibitor 1. Palladium-catalyzed arylacetylene and vinyl stannane couplings with ester 7 or 4-iodobenzo[b]thiophene-2-carbonitrile (16, derived from 7), respectively, followed by amidination leads to the more potent inhibitors 2 (IC₅₀ = 133 nM) and 3 (IC₅₀ = 70 nM). These compounds represent an important new class of synthetic uPA inhibitors, with carboxamidine 3 being the most potent selective inhibitor currently described in the literature.     

Hypoxanthine transport in normal and hypoxanthine guanine phosphoribosyltransferase (HGPRT) deficient diploid human lymphoblasts

We have examined the possible relation between hypoxanthine guanine phosphoribosyltransferase (EC 2.4.2.7., HGPRT) activity and hypoxanthine transport in the normal human lymphoblast line MGL8 and two HGPRT- mutant lines derived from it. The mutant line MGL8A29 (L8A29) had considerable amounts of material cross-reacting immunologically to HGPRT, while mutant MGL8A18 (L8A18) had none. In the normal cells, hypoxanthine is taken up by both a saturable and non-saturable process. Kinetic studies show that the velocity of transport is much lower than HGPRT activity, while both have similar values of K_m. In the two mutant lines, we failed to demonstrate saturable transport, and the rates of hypoxanthine uptake by these cells were directly proportional to its concentration in the medium. Active HGPRT molecules appear to be related to the saturable transport process.     

Sister-chromatid exchanges in human lymphocytes exposed during Go to four classes of DNA-damaging chemicals.

Human lymphocytes were treated prior to mitogenic stimulation with varying concentrations of 6 cytostatic drugs representing 4 classes of DNA-damaging chemicals. Afterwards the cells were washed to remove residual chemical and cultured in the presence of bromodeoxyuridine for analysis of sister-chromatid exchanges (SCEs). A dose-related increase in SCEs was observed in cells exposed during Go to the alkylating chemicals mitomycin C, chlorambucil, and thiotepa, while significant increases in SCEs were not noted in cultures exposed to methotrexate, cytarabine, or bleomycin. These findings suggest that not all classes of clatogenic chemicals which induce SCEs in proliferative cells substituted with BUdR are capable of inducing long-lived lesions in the DNA of Go lymphocytes that can lead to SCE formation.     

Hydroxylation and deglycosylation of 2'-deoxyguanosine in the presence of magnesium and nickel

Nickel (Ni), a carcinogenic and genotoxic metal, has been shown to enhance deglycosylation and hydroxylation of 2'-deoxyguanosine (dG) that has been caused by ascorbic acid and H2O2. There is evidence that Mg is a competitive antagonist of the toxicological effects of Ni. A factorial design was used to examine the interactive influence of Mg and Ni on the deglycosylation and hydroxylation of dG under a range of pH conditions in which ascorbate (Ascb) and H2O2 were added. Formation of guanine (Gu) (deglycosylation) and 8-hydroxy-2'-deoxyguanosine (8-OH-dG) (hydroxylation) appeared in large amounts in samples in which both H2O2 and Ascb were present. The largest amounts of Gu appeared where both Ni or magnesium (Mg) were present. When Mg alone was present, the amounts of Gu was intermediate between these two. Slightly less 8-OH-dG was formed where only Mg was present. The reaction mixtures were more sensitive to the pH than to the respective presence or absence of metals. At slightly acid or neutral pH (6.2-7.0) large amounts of both Gu and 8-OH-dG were formed. Gu formation decreased dramatically between pH 7.0 and 7.2. There was no 8-OH-dG formed at pH 7.8 and only small amounts at pH 7.6. The formation of 8-OH-dG was generally less where Mg was present. When Ni was absent, 8-OH-dG formation was greater in the pH 6.8 mixtures. The formation of Gu and 8-OH-dG from 2'-deoxyguanosine are directly a function of pH. Slight changes in pH greatly effected the formation of these biomarkers of oxidatively damaged DNA. Additional research is needed to determine if this is a cause or effect, i.e. does pH enhance toxicity conditions, thus permitting formation of 8-OH-dG, or does pH permit the reaction to proceed.     

Partial trisomies in two spontaneously arising long-lived human keratinocyte lines

Summary During experiments concerning the introduction of oncogenes into normal human keratinocytes, we observed long-lived colonies arising spontaneously at the same low frequency in control cultures as in those transfected with Ha-rasEJ or activated c-myc or both. Two of these were karyotyped early in their life span and showed additional chromosomal material on the short arm of chromosome 9 in one case and of chromosome 18 in the other, whereas the parental cells had a normal karyotype. This indicates the presence of a partial trisomy in each line, although the origin of the extra chromosomal material is not known. A similarly long-lived human keratinocyte line containing an isochromosome of the long arm of chromosome 8 has been described elsewhere. Together these results suggest that the spontaneous occurrence of long-lived lines is more common in human keratinocytes than in fibroblasts and that a triple dose of one or more genes may be the initial event in this process. This work was supported by grant CA16754 from the National Cancer Institute, Bethesda, MD.