Influence of the embryonal-suspensor mass (ESM) sampling on development and proliferation of maritime pine somatic embryos.

Two maturation media with high and low concentration of gellan gum were used to evaluate the maturation performances of four maritime pine ESM (embryonal-suspensor mass) lines. The maturation performance is influenced by sampling modalities; the outer part of the ESM yielded more cotyledonary embryos than the inner part or the whole colony. ESM lines showing several stage 1 embryos at the periphery (spiky) were more productive than those for which stage 1 embryos were rarely visible (smooth). This latter group develop preferably stage 3 embryos on the maturation medium containing high concentration of gellan gum. Biomass production is higher on a medium containing low concentration of gellan gum. However, sampling modalities did not affect the biomass production, and no relation was found between the biomass production and the maturation performance of each line. Stage 3 embryos developed on the medium with low concentration of gellan gum (0.45%, w/v) were shorter than those developed on medium with 0.9% of gellan gum. These short embryos were not able to germinate whereas about 48% of germination was reached with the longest embryos. The ability to develop primary root is dependent on the genotype while epicotyl elongation was observed among all lines.

A possible role of the Na(+)/K(+)-ATPase for O(2) production from H(2)O(2).

We are attempting to supply a new insight on interaction between Na(+)/K(+)-ATPase and H(2)O(2). We demonstrate that in vitro the Na(+)/K(+)-ATPase, a non heme-protein, is able to disproportionate H(2)O(2) catalatically into dioxygen and water, as well as C(40) catalase. By polarography, we quantify O(2) production and by Raman spectroscopy H(2)O(2) consumption. A comparative analysis of kinetics parameters relative to O(2) production shows that for Na(+)/K(+)-ATPase the affinity of the catalytic site able to transform H(2)O(2) into O(2) is twice weaker than that for C(40) catalase. It also shows that the molar activity for O(2) production is 300-fold weaker for ATPase than for catalase. Inhibitors, pH and GSH studies highlight the differences between the heme- and nonheme-proteins. Indeed, for C(40), NaN(3) is strongly inhibiting, but much less for ATPase. The pH range for the catalatic activity of ATPase is wide (6.5 to 8.5), while it is not for C(40) catalase (optimum at pH 8). The Na(+)/K(+)-ATPase catalatic activity is reduced in presence of glutathione, while it is not the case with C(40) catalase.

Copper-adenine catalyst for O(2) production from H(2)O(2).

In solutions of CuCl2 and adenine copper can be bound to adenine. Two Cu(adenine)(2) complexes [Cu(C(5)H(5)N(5))(2)]2+/Cu(C(5)H(4)N(5))(2)] are in equilibrium with free adenine. Copper-adenine complexes present a catalytic activity (e.g., H(2)O(2) disproportionation into O(2) and water) but depending on complex concentration H(2)O(2) also strongly oxidizes the adenine within the complexes. Raman spectroscopy quantifies copper-adenine complex formation and H(2)O(2) consumption; polarography quantifies O(2) production. As for C(40) catalase, optimal catalytic capacities depend on physiological conditions, such as pH and temperature. The comparative analysis of kinetic parameters shows that the affinity for H(2)O(2) of Cu(adenine)(2) is 37-fold lower than that of C(40) catalase and that the molar activity for O(2) production is 200-fold weaker for Cu(adenine)(2) than for the enzyme. In the 10(-6)-10(-3) M range, the strong decrease of activity with raising complex concentration is explained by aggregation or stacking, which protects Cu(adenine)(2) entities from H(2)O(2) oxidation, but also decreases O(2) production.

Metabolic studies on eel (Anguilla anguilla L.) hepatocytes in primary culture: effect of 17 beta-estradiol and growth hormone.

Previous studies demonstrated that native and recombinant growth hormone from mammalian and fish species potentiate the estrogenic induction of vitellogenin synthesis by cultured eel hepatocytes. In the present study, the metabolic competence (respiratory activity and estradiol catabolism) of cultured hepatocytes and their functional capacity to synthesize a specific protein, vitellogenin, in the presence of estradiol and/or bovine growth hormone was investigated. In addition, we examined the possible role of insulin-like growth factors as mediators of growth hormone. Hepatocytes retain a high level of metabolic activity under the primary culture conditions applied. Estradiol has a half life of several hours in the hepatocyte culture, and is metabolized into conjugated forms. Estradiol and/or growth hormone had no effects on respiratory activity of the cultured hepatocytes. Moreover, the estradiol catabolic parameters were not affected by growth hormone. Finally, human and trout recombinant insulin-like growth factors do not potentiate vitellogenin synthesis induced by estradiol.

Retroviral-mediated correction of DNA repair defect in xeroderma pigmentosum cells is associated with recovery of catalase activity.

Xeroderma pigmentosum (XP) is a rare inherited disease associated with photosensitivity, a very high susceptibility to develop neoplasm on sun-exposed skin and neurological abnormalities for some patients. We previously reported that diploid cell lines established from XP skin biopsies present an abnormal low level of catalase activity, which is involved in the defense against oxygen free radicals. This biochemical dysfunction, probably involved in the skin cancer formation, has been difficult to be directly related to the nucleotide excision repair (NER) defect in XP. In this paper we report that the retroviral-mediated transduction of XP diploid cells by the XPC and XPD/ERCC2 cDNAs fully and stably corrects the NER defect in terms of survival and unscheduled DNA synthesis (UDS) after ultraviolet (UV) irradiation. The catalase activity in transduced cells was recovered up to normal levels only in cells transduced with repair genes correcting the repair defect. These results imply that: (i) the reduced catalase activity in XP, which might result from cellular depletion of its NADPH cofactor, is directly related to impaired DNA repair, and (ii) this depletion might be one of the multiple cellular consequences of XP inborn defect.

Partial molecular analysis of the psbA gene in Euglena gracilis mutants exhibiting resistance to DCMU and atrazine.

Mutations conferring herbicide resistance have been detected in two new strains (ZR250 and ZR480) of Euglena gracilis Z by partial gene cloning and sequencing. These mutants were originally derived from Z cells grown in medium containing progressively increasing concentrations of DCMU. Each of these strains have been characterized by measuring their growth kinetics, O2 evolution, and resistance to DCMU and atrazine. Partial sequences of the psbA gene of these strains were compared to those published for strains Z and ZR25. The ZR250 and ZR480 strains were found to be double mutants. Besides the expected mutation S265A, they showed an additional point mutation at codon 219 (equivalent to codon 218 of other organisms). This mutation results in leucine being substituted by phenylalanine. For each of the ZR strains, two growth conditions (with or without DCMU in the medium) have been compared. The presence of the second mutation (at codon 219) leads to notable increase (20-fold) in resistance to DCMU, whereas the resistance to atrazine is only 2-fold. The presence of DCMU, as the selective agent, was responsible for an enhanced herbicide resistance, irrespective of the concentrations used. Substantial modifications in the rate of cell growth and O2 yields were observed when the maximal concentration (480 microM) of DCMU was used. These modifications were reversible on withdrawal of the DCMU. Thus the reversible adaptative modifications also adds to the mutational effect observed.

Decrease in catalase activity and loss of the 11p chromosome arm in the course of SV40 transformation of human fibroblasts.

The activity of catalase, a key enzyme in cell detoxication of oxygen derivatives, was studied in SV40 transformed human fibroblasts. A cytogenetic study was performed in parallel to establish a quantification of 11p arm on which the corresponding gene is mapped. mRNA amounts were determined by Northern blotting. At early passages, catalase activity strongly decreased whereas the corresponding mRNA was present. No deletions of 11p arms were detected. At later passages, catalase activity remained low. 11p arm deletions were frequent, and the amount of mRNA was decreased. In these late passages, the good correlation between the number of 11p arms and catalase activity suggested a gene dosage effect. It is assumed that the decrease of catalase activity provides a selective advantage for the transformed cells. This decrease is related to a post-transcriptional change of regulation at early passages and to the loss of the corresponding gene at later passages.

Striking differences in cellular catalase activity between two DNA repair-deficient diseases: xeroderma pigmentosum and trichothiodystrophy.

Xeroderma pigmentosum (XP) and trichothiodystrophy (TTD) are two recessively transmitted human diseases characterized by DNA repair deficiency. While XP is associated with a very high incidence of cancer on skin exposed to sunlight, TTD is not a cancer-prone disease. Therefore, unrepaired UV-induced DNA lesions do not appear to be enough to give rise to tumors. In order to understand the differences between these two syndromes, we measured catalase activity in cellular extracts, UV irradiated or not, and quantified H2O2 production following in vitro UV irradiation. We confirmed on 21 different XP diploid fibroblast lines that catalase activity was decreased on average by a factor of five as compared to controls, while XP heterozygote lines exhibited intermediary responses. All seven TTD lines we tested were deficient in UV-induced lesion repair and exhibited a high level of catalase activity. However, molecular analysis of catalase transcription showed no difference between normal, XP and TTD cell lines. This was confirmed by Western blots where the amount of catalase subunits was identical in all cell lines studied. Finally, UV irradiation induces five and three times more H2O2 production in XP lines compared with TTD or controls respectively. These striking differences between TTD and XP indicate that UV light, directly or indirectly, together with defective oxidative metabolism may increase the initiation and/or the progression steps in the XP environment compared to TTD. This may partly explain the different tumoral phenotype observed between the two diseases.

Use of a microbial model for the determination of drug effects on cell metabolism and energetics: study of citrulline-malate.

Euglena gracilis can be used as a microbial model to study the effect of drugs on lactate metabolism and gluconeogenetic synthesis. The cell growth and metabolism have been characterized in a 33 mM lactate medium, non-supplemented or supplemented by dl-malate or by l-citrulline alone or by the compound formed by the stoichiometric combination of the two components: the citrulline-malate (Stimol). The malate of the complex accelerated the ammonium disappearance, while the citrulline facilitated the lactate consumption. A synergistic action of the complex, by comparison with the additive effects of the individual components, on most of the parameters studied was detected. A remarkable resistance to anoxia, and a quicker recovery under aeration of the cells supplemented with CM, were evident: after carbonation for 2 min the total nucleotides in the medium were increased by 44 per cent with an unchanged energy charge; and after a prolonged (20 min) anoxia followed by an aeration, the capacities of the cells to synthesize ATP in the presence of excesses of both ADP and phosphate were two-fold higher in Stimol treated cells than in control.

Catalase-associated abnormalities and H2O2 increase in pre-neoplastic and neoplastic lesions of the human lower female genital tract and their near adjacent epithelia.

We report that an internal and non-UV-dependent type of neoplasia, the human cervical intraepithelial neoplasia (SIL), is also deficient in catalase activity, like the UV-induced tumors in the autosomal recessive human epithelial disease, xeroderma pigmentosum (XP). Whether or not the lesions are papillomavirus (HPV) positive in the different categories of preneoplastic and neoplastic extracts, the following parameters are affected: i), catalase activity level; ii), kinetic profile of catalase activity; iii), H2O2 increase. Mathematical treatment of these parameters (CONSTEL-Program), unambiguously distinguishes between normal and pathological cases. Such analyses make it possible to grade the pathological samples into 4 classes, depending on their deviance from normality. These classes may be correlated with the gradual steps in the process of malignant transformation defined by histological and clinical diagnosis. We found conformity between catalase activity and histological analyses in 66 biopsies, out of a total of 100 biopsies (35 patients). Moreover, 23 patients presenting decreased catalase activities in 31 biopsies showed disease progression after 3 to 6 months contrary to surgery histological data. We show that ATP synthesis in the presence of catalase and H2O2 (further aspect of catalase function), may occur in neoplastic extracts at much lower concentrations of H2O2 than in normal extracts. Thus, the catalase abnormality seems to be a good tool to study pre-neoplastic to neoplastic evolution of lesions and their adjacent tissues of the lower female genital tract; furthermore, i) it provides an earlier, more powerful means of detecting micro-SIL in progression to squamous cell carcinoma, than combined clinical and histological examinations; ii) model for investigating drugs such as in situ H2O2 scavengers or agents increasing glutathione peroxidase activity (GSH).

Deficiency in catalase activity correlates with the appearance of tumor phenotype in human keratinocytes.

Biopsies isolated from various human stratified epitheliomas and cultures of the associated fibroblasts exhibit a breakdown in catalase activity (70 to 95%). We report here that primary human cultures of keratinocytes and various immortalized keratinocyte cell lines differ from one other in their catalase activity. Moreover, deficient catalase activity appears to be related to the intensity of tumor phenotype expression. Since catalase activity in cell lines transformed in vitro is not as weak as activity observed in cell lines isolated from tumors, catalase deficiency may follow the successive steps of tumor progression.

Catalatic capacities in heat-shocked Euglena cells.

1. Various heat treatments were applied to the wild strain Z. Klebs. of Euglena gracilis. 2. Samples of cells were taken at day 1 of the culture at 26 degrees C in a 33 mM lactate medium, when the catalatic capacities of the catalase were highest. 3. They were either submitted to heat treatments (36 and 38 degrees C), or heat-shocks (40, 42 degrees C) or non-permissive heat stress (45 degrees C) for 15 min, 1 and 2 hr. 4. After a 2-hr 45 degrees C treatment the cells were unable to recover normal physiological functions. 5. Heat treatments between 36 and 38 degrees C decreased the catalatic capacities of cells, while heat-shocks at 40 and 42 degrees C strongly reinforced these capacities of hydrogen peroxide dismutation. 6. Having been heat-shocked at 42 degrees C for 2 hr, the cells became different from control cells: (a) after several months of culture, they displayed catalatic capacities increased by 65%; (b) they were able from now on to survive a 2 hr heat shock at 45 degrees C.

Catalase activity in human spermatozoa and seminal plasma.

Catalase activity was determined in human semen by measuring the oxygen burst with a Clark electrode, after H2O2 addition. Significant catalase activities (mean +/- SD) were found in migrated, motile spermatozoa (44 +/- 17 nmoles O2/min/10(8) cells) and in seminal plasma of normozoospermic men (129 +/- 59 nmoles O2/min/ml). It has been demonstrated that seminal catalase originated from prostate; however, its activity was not correlated with the usual prostatic markers (such as citric acid and zinc). Our data suggest a multiglandular function secreted by this organ. The catalase activities measured in seminal samples from asthenozoospermic, infertile men were found lower than those from normozoospermic subjects. The understanding of the relative contribution of the different enzyme systems against O2 toxicity (superoxide dismutase, catalase, glutathione peroxidase) seem to be a priority area of research to understand disturbances of sperm function.

Effect of gossypol on the morphology, motility and metabolism of a flagellated protist, Dunaliella bioculata.

Gossypol, a natural polyphenolic compound extracted from cotton seed, is known for its contraceptive properties on human beings and several other mammals. Gossypol induces disorders of spermatogenesis and inhibition of spermatozoal motility. To determine the molecular mechanism inhibiting the motility of cells, a flagellar protist (Dunaliella bioculata) has been chosen as a cellular model because of the heterogeneity of spermatozoa populations. Inhibition of cell motility by gossypol, measured by Laser Doppler Velocimetry, was observed with these cells at the same doses as for spermatozoa (30-50 mumoles/l). Mitochondria were found to be swollen in gossypol-treated algae and, in parallel, respiratory exchanges were decreased and photosynthesis was inhibited, although the chloroplast did not seem to be morphologically altered. A diminution of the essential ATP production was also found at the same doses. Therefore, the inhibition of flagellar motility could be explained by such a decrease. The rapid and parallel inhibition of respiration and photosynthesis at non-lethal concentrations leads us to the hypothesis of a common target involving both ubiquinone and plastoquinone, although this remains to be proved.

Induction by antimycin A of cyanide-resistant respiration in heterotrophic Euglena gracilis: Effects of growth, respiration and protein biosynthesis.

The addition of antimycin A during the logarithmic phase of growth of heterotrophic Euglena gracilis cultures (in lactate or glucose medium) was immediately followed by decreased respiration and a cessation of grwoth. Induced cyanideresistent respiration appeared 5 h after the addition of the inhibitor then the cells started to grow again and could be cultured in the presence of antimycin A. Thus the cells exhibited a cyanide-and antimycin-resistant respiration which was, in addition, sensitive to salicylhydroxamic acid and propylgallate. Antimycin-adapted Euglena and control cells were compared for their biomass production and protein synthesis. The difference in growth yield between control and antimycin-adapted cells was not as high as would be expected if only the first phosphorylation site of the normal respiratory chain was active in the presence of antimycin A. Furthermore, the ability to incorporate labelled valine into proteins, under resting-cell conditions, was not changed. Strong correlations were established between the effects of respiratory effectors on O2 consumption and valine incorporation. These results suggest that sufficient energy for protein synthesis and growth is provided by the operation of the cyanide-resistant respiratory pathway in antimycin-adapted Euglena.

Deficiency in the catalase activity of xeroderma pigmentosum cell and simian virus 40-transformed human cell extracts.

It has been previously shown that skin biopsies isolated from various xeroderma pigmentosum (XP) patients present a permanent decline in catalase activity from the onset of the disease to the tumor formation. We report here that cultured XP cell strains are also markedly deficient in the catalase activity with about only 25% of the activity measured in normal human cells. No direct correlation between catalatic activity and excision repair ability has been found, since a XP variant line is as deficient as an XP-C strain. The exact cause of the catalase deficiency is still unknown but could be due to the synthesis of a modified enzyme or to an abnormal regulation leading to a limited enzyme synthesis. Furthermore, simian virus 40 transformation of normal and radiosensitive cells (XP, ataxia telangiectasia) provokes a decrease in catalase activity of about 80% compared to the control derivatives. Mathematical analysis performed on our data shows a clearcut distinction between XP and normal cells while some of the XP heterozygote cells exhibit an intermediate behavior. Although most of the XP syndrome could be explained by the impairment in the excision repair ability, the decrease in catalase activity leading to a probable increase in intracellular H2O2 concentration and/or to a higher sensitivity to any oxygen-activated species could represent an additive effect in inducing the carcinogenic process.

The possible role of the superoxide ion in the induction of heat-shock and specific proteins in aerobic Drosophila cells during return to normoxia after a period of anaerobiosis.

In vitro cultured Drosophila melanogaster cells were shown to be aerobic and several kinetic parameters of their respiration were measured. This allowed us to define experimental conditions for a transient period of anaerobiosis followed by a reexposure to normal oxygenation. This treatment, applied without any change of temperature, induced not only the heat-shock proteins, but also a new specific peptide of 27 000 daltons and a twofold increase of the maximal rate of O2 uptake. This evokes a common molecular mechanism activated either by heat or by O2, which could involve the increase of the products of oxygen reduction such as the superoxide ion.

[Xeroderma pigmentosum: development of photoformation of hydrogen peroxide and catalytic activity in skin cells; application to an early diagnosis and to a tentative treatment]. / Xeroderma pigmentosum: évolution de la photoformation d'eau oxygénée et de l'activité catalatique dans les cellules de la peau; application à un diagnostic précoce et à une tentative de traitement.

In Xeroderma pigmentosum (XP) a genetic-recessive ultraviolette-hypersensitive disease we found an accumulation of photoproduced H2O2 in skin cells accompanied by a breakdown of the catalatic activity. Both disorders intensified as the disease progressed. Concomitantly with these processes, one electrochemical peak of urine decreases. By the time epitheliomas appeared, this peak was no longer detectable. Several applications to the skin of a cream containing catalase seemed to prevent the formation and the evolution of neoplastic tumors during the treatment.

Paramylon synthesis by Euglena gracilis photoheterotrophically grown under low O2 pressure : Description of a mitochloroplast complex.

Special culture conditions for Euglena gracilis Z and ZR are described. They induce interactions between the chloroplast and mitochondrial metabolisms leading to paramylon synthesis. When grown in continuous light under pure nitrogen and in the presence of lactate as the sole carbon source, sugar synthesis occurs during the first 24 h of culture with the participation of both mitochondria (using lactate) and of chloroplasts (fixing CO2 from lactate decarboxylation). The activities of ribulose bisphosphate carboxylase, phosphoenolpyruvate carboxylase, and phosphoenolpyruvate carboxykinase are very high and mitochondria and chloroplasts develop then a common network of vesicles in which paramylon grains can be seen. Electron micrographs demonstrate membrane continuity between the two types of organelles. Occasionally the mitochondrial matrix and the chloroplast stroma are separated by only a unit membrane.

Evolution of enzymes involved in carbon metabolism (phosphoenolpyruvate and ribulose-bisphosphate carboxylases, phosphoenolpyruvate carboxykinase) during the light-induced greening of Euglena gracilis strains Z and ZR.

Phosphoenolpyruvate carboxykinase activity decreases when Euglena gracilis Z and ZR undergo light-induced chloroplast development in batch "resting" medium lacking utilizable organic carbon and CO2. This enzyme is present in heterotrophically grown cells (Briand et al. 1981) and assures gluconeogenesis. It was consistently more active in strain ZR. Decreased carboxykinase activities were accompanied by parallel increases in the activities of ribulose bisphosphate carboxylase and phosphoenolpyruvate carboxylase. The rates of O2 evolution in light were much lower than those of CO2 fixed simultaneously. The incorporation of (14)CO2 into early C-4 dicarboxylic acids was higher in green cells than in etiolated cells, and it was even higher in green cells assayed in light in the presence of (DCMU). A hypothesis has been proposed, according to which there is a possible cooperation of phosphoenolpyruvate carboxylase in photosynthetic CO2 fixation, especially under conditions of limiting CO2.High temperatures (34° C) depress carboxylation enzyme activities to a greater extent than that of the carboxykinase without a great effect on cellular chlorophyll content. In the presence of 25 µm DCMU, however, chlorophyll accumulation is reduced without any detectable changes in enzyme activities in the Z strain. The ZR strain displayed its characteristic resistance to DCMU.