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Influences of dietary restriction and age on liver enzyme activities and lipid peroxidation in mice.

Dietary restriction extends maximum life span in rodents by unknown mechanisms. We compared livers from 12- and 24-mo-old mice fed control (C, approximately 95 kcal/wk) or restricted (R, approximately 55 kcal/wk) amounts of diet since 3 wk of age. We hypothesized that dietary restriction might alter the activity levels of enzymes with possible relevance to aging processes. The enzymes included several xenobiotic metabolizers, radical scavengers (catalase, superoxide dismutase, glutathione peroxidase), superoxide sources (xanthine oxidase, peroxisomal beta-oxidation of palmitoyl-CoA) and glucose-6-phosphatase. Lipid peroxidation (LP) was also measured. Comparing 12- and 24-mo-old mice, the strongest diet or age effect was an increased catalase activity for group R (42% higher at 12 mo, 64% at 24 mo). LP was clearly lower in group R at 12 mo (a 30% decrease) and somewhat lower (13%) at 24 mo than in group C. Similarly, in 12-mo-old C and R mice injected with either the P-450 inducer beta-naphthoflavone (beta-NF in corn oil) or with corn oil alone. R mice showed higher catalase activity (40-44%) and lower LP (43-46%) in both beta-NF-injected and vehicle-injected groups. These data suggest that if free radical damage is involved in aging, it may be a particular kind of damage, that is, that in part prevented by a selective increase in catalase activity.

Aging↗

Synovial fluid polymorphonuclear leucocytes from patients with rheumatoid arthritis have reduced MPO and NADPH-oxidase activity.

Synovial fluid (SF) polymorphonuclear leucocytes (PMN) from patients with rheumatoid arthritis (RA) were compared to RA and normal circulating blood PMN. RA SF PMN were as viable as blood PMN and remained viable for up to 24 h in culture. Measurement of myeloperoxidase indicated that RA SF PMN had degranulated and secreted their myeloperoxidase prior to isolation, 26.5 +/- 11.7% being found extracellularly compared to less than 2.9% in RA and normal blood PMN. RA SF PMN alone showed a decrease in basal NADPH-oxidase activity as well as an increase in responsiveness to stimulation by chemotactic peptide during culture. Stimulation of PMN with phorbol-12-myrisitate-13-acetate evoked equivalent responses in each population before and after culture. These results demonstrate a major difference in resting and receptor-mediated activation of superoxide release by RA SF PMN. Together, these results have important implications in identifying the role of the activated PMN in RA.

Arthritis, Rheumatoid↗

Photoinactivation of Catalase Occurs under Both High- and Low-Temperature Stress Conditions and Accompanies Photoinhibition of Photosystem II.

Severe photoinactivation of catalase (EC 1.11.1.6) and a decline of variable fluorescence (F(v)), indicating photoinhibition of photosynthesis, were observed as rapid and specific symptoms in leaves exposed to a high heat-shock temperature of 40 degrees C as well as in leaves exposed to low chilling temperatures in white light of only moderately high photosynthetic photon flux density of 520 muE m(-2) s(-1). Other parameters, such as peroxidase (EC 1.11.1.7), glycolate oxidase (EC 1.1.3.1), glutathione reductase (EC 1.6.4.2), or the chlorophyll content, were hardly affected under these conditions. At a compatible temperature of 22 degrees C, the applied light intensity did not induce severe photoinactivations. In darkness, exposures to high or low temperatures did not affect catalase levels. Also, decline of F(v) in light was not related to temperature sensitivity in darkness. The effective low-temperature ranges inducing photoinactivation of catalase differed significantly for chilling-tolerant and chilling-sensitive plants. In leaves of rye (Secale cereale L.) and pea (Pisum sativum L.), photoinactivation occurred only below 15 degrees C, whereas inactivation occurred at 15 degrees C in cucumber (Cucumis sativus L.) and maize (Zea mays L.). The behavior of F(v) was similar, but the difference between chilling-sensitive and chilling-tolerant plants was less striking. Whereas the catalase polypeptide, although photoinactivated, was not cleaved at 0 to 4 degrees C, the D1 protein of photosystem II was greatly degraded during the low-temperature treatment of rye leaves in light. Rye leaves did not exhibit symptoms of any major general photodamage, even when they were totally depleted of catalase after photoinactivation at 0 to 4 degrees C, and catalase recovered rapidly at normal temperature. In cucumber leaves, the decline of catalase after exposures to bright light at 0 to 4 degrees C was accompanied by bleaching of chlorophyll, and the recovery observed at 25 degrees C was slow and required several days. Similar to the D1 protein of photosystem II, catalase differs greatly from other proteins by its inactivation and high turnover in light. Inasmuch as catalase and D1 protein levels depend on continuous repair synthesis, preferential and rapid declines are generally to be expected in light whenever translation is suppressed by stress actions, such as heat or chilling, and recovery will reflect the repair capacity of the plants.

Journal Article↗

Occurrence of some enzymes in starchy endosperm and hormonal regulation of isoperoxidase in aleurone of wheat.

Peroxidase, indoleacetic acid-oxidase, alkaline, and acid phosphatases were detected in dry starchy endosperm (minus aleurone) of wheat grain. The isoperoxidase pattern differed in different parts of the dry grain. Several new isoperoxidases were found in embryos after soaking. The intensity of isoperoxidases in aleurones was enhanced in the presence of embryo or 2 muM GA(3) after 24 hours of soaking, but decreased after 72 hours. Indoleacetic acid and kinetin had no effect on isoperoxidase of aleurone. Actinomycin D and cycloheximide had no effect on isoperoxidases of aleurones from embryonectomized or naturally occurring embryoless grains. However, these two inhibitors increased the intensity of isoperoxidases in aleurones of intact embryonated grains after soaking.

Journal Article↗

Polarity of Production of Polyphenols and Development of Various Enzyme Activities in Cut-injured Sweet Potato Root Tissue.

Investigation of polyphenol production in cut-injured sweet potato (Ipomoea batatas Lam. cv. Kokei 14) roots by histochemical and quantitative methods showed that polyphenols were produced in striking amounts in the proximal side of the tissue pieces (2 cm thick), but only in small amounts in cells of the distal side. In response to cut injury, formation of the enzymes related to polyphenol biosynthesis, phenylalanine ammonia-lyase and trans-cinnamic acid 4-hydroxylase, was also pronounced in the proximal side of the tissue pieces and slight in the distal side. The similar polarity was observed in the development of activities of various enzymes, such as NADPH-cytochrome c oxidoreductase, acid invertase, peroxidase, o-diphenol oxidase, and cytochrome c-O(2) oxidoreductase. Acropetal development of polyphenol contents and of various enzyme activities may be related to the acropetal movement of indoleacetic acid (IAA) in roots of various plants. Treatment of the distal surface of tissue pieces with IAA or 2,4-dichlorophenoxyacetic acid caused polyphenol production but treatment with gibberellic acid, abscisic acid, kinetin, or ethylene had little effect. The results suggest that IAA may play a role in the metabolic response to cut injury.

Journal Article↗

HEX, PAX-8 and TTF-1 gene expression in human thyroid tissues: a comparative analysis with other genes involved in iodide metabolism.

OBJECTIVE: Benign and malignant thyroid tumours are characterized by alterations of the expression level of thyroid-specific genes involved in the iodide metabolism. Imbalance in the levels of transcription factors has been recognized as a critical molecular event in the development of neoplasm. The delineation of eventual correlations existing between the expression of transcription factors and of putative target genes in physiological and pathological conditions could be relevant to better understand tumorigenesis. PATIENTS AND METHODS: We examined the expression levels of transcription factors involved in thyroid development [thyroid transcription factor 1 (TTF-1), paired box gene 8 (PAX-8) and haematopoietically expressed homeobox (HEX)] in 101 thyroid tissues, including 14 normal thyroid tissues, 13 hyperfunctioning tissues, 27 benign adenomas and 47 follicular or papillary carcinomas. Then, we compared their expression levels with those of thyroid-specific genes involved in iodide metabolism. RESULTS: In benign tumours, PAX-8 and TTF-1 gene expression levels were not significantly different from the expression levels in normal tissues. However, a significant decrease was found in carcinomas. Interestingly, HEX gene expression was significantly decreased in both hyper- and hypofunctioning benign tissues and also in carcinomas. Expression levels of Pendred syndrome (PDS), natrium iodine symporter (NIS), thyroglobulin (Tg), thyroid peroxidase (TPO) and dual oxidase 1 or 2 (DUOX2) genes were significantly correlated with the expression of PAX-8 and with that of HEX. Expression level of TTF-1 was weakly correlated only with the expression levels of PDS and DUOX2. CONCLUSION: Our findings suggest that alterations in the transcription factors PAX-8, TTF-1 and HEX gene expression, by acting individually or together, have a role in both thyroidal tumorigenesis and in the dedifferentiation process.

Case-Control Studies↗

Myeloperoxidase.

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Coloring Agents↗

Metabolism of nomifensine to a dihydroisoquinolinium ion metabolite by human myeloperoxidase, hemoglobin, monoamine oxidase A, and cytochrome P450 enzymes.

Nomifensine is an antidepressant agent that was removed from use because of a high incidence of hemolytic anemia. It contains an N-methyl-8-aminotetrahydroisoquinoline ring which has the potential to be oxidized to quaternary dihydroisoquinolinium and isoquinolinium ions, albeit such a transformation had not been previously observed. In this report, we demonstrate the conversion of nomifensine to a dihydroisoquinolinium ion metabolite by several human enzymes. Human liver microsomes supplemented with NADPH generated the dihydroisoquinolinium ion metabolite along with other hydroxylated metabolites, whereas when supplemented with t-butyl peroxide, only the dihydroisoquinolinium ion metabolite was observed. Monoamine oxidase A, but not monoamine oxidase B, catalyzed this reaction, as well as human hemoglobin supplemented with H2O2. Human myeloperoxidase catalyzed this reaction in the presence of H2O2, and activation of the reaction was observed when incubations were conducted in the presence of acetaminophen at concentrations relevant to those measured in humans. The reaction was also observed in human whole blood. The equilibrium between the dihydroisoquinolinium ion and carbinolamine was shown to have a pK of about 11.7. The dihydroisoquinolinium ion was shown to react with cyanide and borohydride, but not glutathione. These findings suggest that the electrophilic nomifensine dihydroisoquinolinium metabolite, which can be generated by several enzymes, could be behind toxic responses to nomifensine such as hemolytic anemia and hepatotoxicity.

Cytochrome P-450 Enzyme System↗

Enzyme immunoconjugates utilizing glucose oxidase and myeloperoxidase are cytotoxic to Candida tropicalis.

A dual-enzyme immunoconjugate system was evaluated for its cytotoxic effect on Candida tropicalis. Glucose oxidase, which generates hydrogen peroxide in the presence of glucose and oxygen and myeloperoxidase, which catalyzes the oxidation of halides in the presence of hydrogen peroxide, were each conjugated to a C. tropicalis-specific monoclonal antibody. Neither the glucose oxidase nor the myeloperoxidase conjugates exhibited any significant cytotoxic effect by themselves. A combination of glucose oxidase conjugate (3.2 ng/ml) and myeloperoxidase conjugate (12.8 ng/ml) in the presence of 5 mg of glucose per ml, 150 mM chloride, and 50 microM iodide was cytotoxic to C. tropicalis, killing 99.9% of the treated sample. Flow cytometry was used to characterize the binding of the conjugates to yeast cells and demonstrated that the binding of both conjugates to the yeast cell surface is required for cytotoxicity. In addition, the concentrations of conjugates required for a cytotoxic effect were below the concentrations required to saturate all of the yeast cell surface antibody-binding sites.

Antibodies, Monoclonal↗

Redundant contribution of myeloperoxidase-dependent systems to neutrophil-mediated killing of Escherichia coli.

Neutrophil microbicidal activity is a consequence of overlapping antimicrobial systems that vary in prominence according to the conditions of the neutrophil-microbe interaction, the nature of the microbe, and its metabolic state. In this study, normal, myeloperoxidase-deficient, and respiratory burst-deficient (chronic granulomatous disease [CGD]) neutrophils killed Escherichia coli with equivalent, high efficiencies. Killing by CGD and myeloperoxidase-deficient neutrophils was not augmented by supplements, such as exogenous H2O2 and myeloperoxidase, directed at ameliorating their metabolic defects, suggesting that nonoxidative microbicidal systems were sufficient for a full microbicidal effect. Neutrophils with an intact myeloperoxidase antimicrobial system (normal or appropriately supplemented deficient cells) were capable of rapidly suppressing E. coli DNA synthesis, while unsupplemented CGD or myeloperoxidase-deficient cells were far less effective, indicating that the myeloperoxidase system was active in normal neutrophils. The degree of DNA synthesis inhibition by myeloperoxidase-sufficient neutrophils could account, in a cell-free system, for most of the observed microbicidal activity. While the myeloperoxidase system was active and probably bactericidal, it was not rate limiting for microbicidal activity and appears to have been redundant with other microbicidal systems in the cell. Rapid and extensive inhibition of bacterial DNA synthesis appears to be an indicator of myeloperoxidase activity in neutrophils.

DNA, Bacterial↗

'Pseudohypouricosuria' in alcaptonuria: homogentisic acid interference in the measurement of urinary uric acid with the uricase-peroxidase reaction.

Urinary excretion of uric acid was found to be extremely low in a 58-year-old female patient with alcaptonuria. This was due to interference with the uricase-peroxidase method used, because analysis using high-performance liquid chromatography (HPLC) showed a normal urinary concentration of uric acid. In vitro experiments demonstrated that a high concentration of homogentisic acid in the patient's urine inhibited the peroxidase reaction, possibly due to inhibition of the colour development of 3-methyl-N-ethyl-N-(beta-hydroxyethyl)aniline (MEHA) and 4-aminoantipyrine, via the peroxidase reaction. A homogentisic acid concentration equivalent to that in plasma did not affect the uricase-peroxidase reaction. This result suggests that any assay based on a peroxidase method is affected by a high urinary concentration of homogentisic acid in patients with alcaptonuria.

Alkaptonuria↗

Neutrophil bactericidal dysfunction towards oxidant radical-sensitive microorganisms during experimental iron deficiency.

We developed a clear-cut nutritional iron deficiency anemia without concomittant malnutrition in rats given a low iron diet, and we restored normal iron and hemoglobin levels in these same animals with iron dextran injections. The neutrophil function studies performed during and after a period of iron deficiency showed the following: Phagocytosis of Staphylococcus aureus 502A, Streptococcus pneumoniae, and Salmonella typhimurium was not altered by iron deficiency or by the administration of iron; phagocytosis of Candida albicans was moderately abnormal during iron deficiency, and became normal with the restoration of iron sufficiency. Microbicidal activity towards Staphylococcus aureus 502A and Candida albicans, two catalase-positive microorganisms, was markedly decreased (to 50% of control values) and returned to normal when iron sufficiency was restored. Killing of a catalase-negative organism, Streptococcus pneumoniae was normal in iron-deficient rats. This pattern of differential bactericidal activities suggested an abnormality of the oxidant radical-generating machinery in neutrophils of iron-deficient animals. Indeed, iron deficiency caused a marked decrease of neutrophil nitroblue tetrazolium dye reduction, which disappeared after iron administration. Neutrophil myeloperoxidase activity was slightly decreased in iron deficient rats and returned to normal after iron administration. Microbicidal activity towards a gram-negative, catalase-positive organism, Salmonella typhimurium, was equal in iron deficient and iron sufficient animals. Our combined results suggest that a definite microbicidal defect is the consequence of nutritional iron deficiency, apart from any protein-calorie malnutrition. This defect affects the disposal in PMNs of two catalase-positive microorganisms (which require intracellular production of oxidant radicals for their destruction) but not of a catalase-negative bacterial species.(ABSTRACT TRUNCATED AT 250 WORDS)

Anemia, Hypochromic↗

Phorbol myristate acetate induces neutrophil death through activation of p38 mitogen-activated protein kinase that requires endogenous reactive oxygen species other than HOCl.

Stimulation of normal mouse neutrophils with phorbol 12-myristate 13-acetate resulted in an acceleration of chromatin condensation and phosphatidylserine externalization that was not associated with caspase-3 activation. Caspase-independent death was completely inhibited by GF109203X and SB202190, specific inhibitors for protein kinase C and p38 mitogen-activated protein kinase respectively. Activation of p38 mitogen-activated protein kinase was completely suppressed by GF109203X, indicating that this enzyme is regulated by protein kinase C. On the other hand, cell death was abolished in NADPH oxidase-deficient neutrophils lacking superoxide production. Of note, p38 mitogen-activated protein kinase was activated by phorbol 12-myristate 13-acetate in normal and myeloperoxidase-deficient neutrophils lacking production of HOCl, whereas no activation was observed in NADPH oxidase-deficient neutrophils. These results strongly suggest that activation of p38 mitogen-activated protein kinase is regulated by endogenously generated superoxide or its metabolites other than HOCl, a critical regulator of inducer-stimulated death of neutrophils.

Animals↗