Normal human keratinocytes in culture exhibit a nitric oxide synthase (NOS) activity ranging from 50 to 150 pmol/min/mg of protein. The enzyme is cytosolic and requires the presence of calcium, nicotinamide adenine dinucleotide phosphate, reduced form (NADPH), and flavin adenine dinucleotide. Calmodulin antagonists (trifluoperazine and calmidazolium) inhibit the enzyme activity. We show that NG-nitro-L-arginine inhibits NOS more potently than NG-monomethyl-L-arginine and that L-canavanine is a weak inhibitor. NOS was partially purified using a 2',5'-ADP Sepharose affinity column eluted with NADPH. A partially purified fraction was analyzed by sodium dodecyl sulfate-polyacrylamide gel electro-phoresis and Western blotting. A protein with an apparent molecular weight of 152 kDa cross-reacted with monoclonal antibodies raised against the neuronal constitutive isoform of NOS. The enzyme had a Vmax of 7.3 nmol/min/mg of protein and a Km for L-arginine of 22.3 microM. These results indicate that normal human keratinocytes contain a constitutive nitric oxide synthase related to NOS I.

Hydrogen peroxide (H2O2), was able to nick the replicative form of the phage fd, without the addition of a reducing agent or of a metal. This DNA single-strand breakage decreased with an increase of the ionic strength, suggesting that H2O2 reacted with traces of metal bound to DNA. When cupric of ferric ions were added, the rate of DNA single-strand breakage by H2O2 greatly increased and it was 20-30 times faster with cupric than with ferric ions. The addition of EDTA at an equimolar ratio or in excess of metal prevented partially DNA single-strand cleavage by H2O2 in the presence of ferric ions and completely when cupric ions were used. Superoxide dismutase prevented DNA single-strand breakage by H2O2 and ferric ions. On the contrary, with cupric ions and H2O2, the addition of superoxide dismutase increased the rate of DNA single-strand breakage. That superoxide dismutase was acting catalytically was shown by the loss of its effects after heat inactivation of the enzyme. The results of the present study show that besides its involvement in the Fenton reaction, H2O2 is able to reduce the metal bound to DNA, generating the superoxide anion radical or/and its protonated form, the perhydroxyl radical involved in DNA nicking. On the other hand, the ability of cuprous ions unlike ferrous ions to dismutate the superoxide radical may explain some differences observed between iron and copper in the DNA single-strand breakage by H2O2.

No abstract

An indirect immunohistochemical technique was developed using a rabbit anti-abscissic acid (ABA) serum and the soluble peroxidase-antiperoxidase (PAP) complex for the localization of endogenous ABA in the aerial parts of Chenopodium. Terminal bud, axillary bud bearing nodes, and adult leaves were prefixed by a soluble carbodiimide to obtain the coupling of ABA on cellular proteins and postfixed by a conventional mixture of aldehydes. They were then embedded in paraffin or in plastic. Numerous controls were carried out on sections and on a model system to test the validity of the technique. Based on the staining patterns observed along the plant, an apico-basal gradient of ABA was revealed. In the older buds, ABA was mainly concentrated in the quiescent meristematic cells of the apex. Phloem cells of the main axis and chloroplasts of the leaves were specifically labeled. No reaction product was visualized in the parenchyma cells or in the cambial zone. Water stress, which is known to increase ABA content, induced an increase of immunoreactivity within the same compartments. This physiological test validates the stain.

Reaction to schistosomal antigen was studied in children born to mothers infected with Schistosoma mansoni. Values of immediate (15 min) skin reactions were more elevated and values of delayed (24 h) skin reactions were significantly higher in children born to infected mothers than in children born to uninfected mothers. The macrophage migration inhibition test, done on cord blood cells, was positive to schistosomal antigen in 40% of children born to infected mothers and negative in all children born to uninfected mothers. These results suggest prenatal sensitization to schistosomal antigen in children born to mothers infected with S. mansoni.