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.

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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.

The role of histidine on DNA breakage induced by hydrogen peroxide (H2O2) and ferric ions or by H2O2 and cupric ions was studied on purified DNA. L-histidine slightly reduced DNA breakage by H2O2 and Fe3+ but greatly inhibited DNA breakage by H2O2 and Cu2+. However, only when histidine was present, the addition of EDTA to H2O2 and Fe3+ exhibited a bimodal dose response curve depending on the chelator metal ratio. The enhancing effect of histidine on the rate of DNA degradation by H2O2 was maximal at a chelator metal ratio between 0.2 and 0.5, and was specific for iron. When D-histidine replaced L-histidine, the same pattern of EDTA dose response curve was observed. Superoxide dismutase greatly inhibited the rate of DNA degradation induced by H2O2, Fe3+, EDTA and L-histidine involving the superoxide radical. These studies suggest that the enhancing effect of histidine on the rate of DNA degradation by H2O2 and Fe3+ is mediated by an oxidant which could be a ferrous-dioxygen-ferric chelate complex or a chelate-ferryl ion.

Synopsis We have compared the in vitro cellular toxicity and the in vivo ocular irritation potency of 16 surfactants (7 non ionics, 3 anionics, 2 amphoterics, 4 cationics) ranking from very weakly irritant to strongly irritant. In vitro, the cellular toxicity was estimated on Chinese hamster lung fibroblasts (V79) using a cell mortality test and a cell growth inhibition test. For each surfactant, a lethal concentration 50% without foetal calf serum (LC.50-0) and with 10% foetal calf serum (LC50-I0), as well as the concentration required to reduce 50% of the growth (CI.50) were determined. In vivo, each surfactant was applied directly to the cornea of six albino rabbits. The maximal ocular irritation score (I0 max) and the ocular irritation score obtained seven days later (I0 J7) were collected. Comparison of in vitro results with those obtained in vivo showed good correlations, particularly when I0 max and the difference (LC.50-10 - LC50-0) were considered (r= 0.845, P<0.001). These results suggest that the use of cell culture tests as pre-screening systems to appreciate eye irritation potency of surfactants could be a reliable alternative method in order to reduce the use of the Draize rabbit eye test. They can provide a better knowledge of the irritative process induced by surfactants (cellular toxicity and protein interaction potency).

The effects of three macrolide antibiotics were studied on rat polymorphonuclear leukocyte chemotaxis. Rats were given 25 mg/kg twice a day of either erythromycin, josamycin or spiramycin by gastric intubation for 5 days. In all cases, chemotaxis was found to be impaired by 10–20% only. As macrolides are known to reach high intracellular concentrations within polymorphonuclear leukocytes, our results suggest that these antibiotics are unlikely to exert a deleterious influence on the chemotactic response of treated patients.