Increased superoxide radicals generation from alveolar macrophages in immature guinea-pigs

To study the effect of maturation on abilities of superoxide radicals (O-2) generation in the airways, we compared stimuli-induced O-2 generation by alveolar macrophages in immature (aged 10+/-2 days) and adult (aged 90+/-2 days) guinea-pigs. The production of O-2 was assayed by chemiluminescence method, using a Cypridina luciferin analog as a highly sensitive and specific probe for O-2. Whereas no significant difference in cell components of bronchoalveolar lavage fluid was observed between immature and adult animals, O-2 generation induced by stimulation of alveolar macrophages was greater in immature than in adult animals, with significant differences observed after platelet-activating factor (100 nM) or phorbol myristate acetate (0.5 micro g/ml). The results suggest that alveolar macrophages from immature animals are far more potent O-2 generators than the same cells of adult animals.     

Effect of caffeine in Fanconi anemia

Summary In Fanconi anemia (FA) cells the duration of the G₂ phase of the cell cycle prolonged. Such a slowing of the G₂ phase can be induced in normal cells by irradiation with rays during S phase, which also further increases the duration of G₂ in FA cells. The addition of caffeine during the last 7h of culture shortens the G₂ phase in both nonirradiated and irradiated FA cells. In nonirradiated normal cells it may have no effect or may increase G₂ phase duration, but in irradiated normal reduces the slowing of G₂ induced by the radiation. This suggests that FA cells recognize and repair preexisting DNA lesions during G₂ phase and that caffeine inhibits this process. The principal anomaly in FA may be a deficient repair during S phase, as manifest in the prolonged postreplication repair period during G₂ phase required to repair the larger number of lesions passing through S phase.     

Fanconi anemia proteins in telomere maintenance

Mammalian chromosome ends are protected by nucleoprotein structures called telomeres. Telomeres ensure genome stability by preventing chromosome termini from being recognized as DNA damage. Telomere length homeostasis is inevitable for telomere maintenance because critical shortening or over-lengthening of telomeres may lead to DNA damage response or delay in DNA replication, and hence genome instability. Due to their repetitive DNA sequence, unique architecture, bound shelterin proteins, and high propensity to form alternate/secondary DNA structures, telomeres are like common fragile sites and pose an inherent challenge to the progression of DNA replication, repair, and recombination apparatus. It is conceivable that longer the telomeres are, greater is the severity of such challenges. Recent studies have linked excessively long telomeres with increased tumorigenesis. Here we discuss telomere abnormalities in a rare recessive chromosomal instability disorder called Fanconi Anemia and the role of the Fanconi Anemia pathway in telomere biology. Reports suggest that Fanconi Anemia proteins play a role in maintaining long telomeres, including processing telomeric joint molecule intermediates. We speculate that ablation of the Fanconi Anemia pathway would lead to inadequate aberrant structural barrier resolution at excessively long telomeres, thereby causing replicative burden on the cell.     

Antioxidant status of Fanconi anemia fibroblasts

Summary Several observations in the recent literature have indicated that Fanconi anemia (FA) cells may be primarily deficient in the detoxification of activated oxygen species. To evaluate the antioxidant status of FA fibroblasts, we measured Mn-containing superoxide dismutase (Mn-SOD), CuZn-containing superoxide dismutase (CuZn-SOD), catalase, and glutathione peroxidase activities, as well as cellular glutathione contents and total nonenzymatic antioxidant potential in Fa and control fibroblasts at multiple time point during a single passage. All parameters exhibited a characteristic pattern of changes during a period of 19 days following trypsinization. Unlike FA erythrocytes, which are known to be deficient in CuZn-SOD, FA fibroblasts exhibited normal CuZn-SOD activities. Also, the nonenzymatic antioxidant potential as well as glutathione levels were similar in FA and control fibroblasts. However, Mn-SOD, catalase, and glutathione peroxidase activities were consistently higher in FA fibroblasts. We hypothesize that the elevation of these enzyme activities might reflect a cellular prooxidant state in FA resulting from an increased formation of endogenous oxidizing molecular species that trigger enhanced synthesis of certain enzymatic antioxidant defenses.     

Alteration of a nuclease in Fanconi anemia.

Fanconi anemia is a cancer-prone disease characterized by progressive loss of blood cells, skeletal defects and stunted growth. Studies of a nuclease acting on double-stranded DNA have revealed an enzyme alteration in cells derived from Fanconi patients. A particulate fraction isolated from cultured human lymphoblasts and fibroblasts was solubilized with detergent and subjected to isoelectric focusing. Nuclease activity observed in four normal cell lines bands in a pH gradient with a pI of 6.3. Four cell lines belonging to complementation group A exhibit an increase in the pI of that nuclease to 6.8. These observations provide a new diagnostic for this disorder. Analysis of this enzyme in tetraploid cultures derived from fusion of normal and Fanconi cells suggest that the normal phenotype is dominant. That observation supports the hypothesis that the Fanconi A gene is required for modification of the nuclease pI. Definition of the molecular basis of this enzyme alteration should provide insight into the primary genetic lesion in this disorder.     

The cell cycle of lymphocytes in Fanconi anemia

Summary BrdU-incorporation techniques were used to study the cell cycle in 18 cases of Fanconi's anemia (FA).By comparison with controls, a significant slowing of the cell cycle of lymphocytes in vitro was observed in all FA patients, and possibly in FA heterozygotes, although to a lesser degree. It is probable that the demonstration of the slowing is dependent on the culture conditions. No slowing was observed in other patients affected by at least one of the symptoms of FA. The slow cell cycle of FA cells is mostly due to a very long G₂-phase. A relationship between slow cell cycle and chromatid anomalies exists, the slower cells being significantly more frequently carriers of radial figures than the faster cells, in the same patient.     

Increased rate of sodium-proton metabolism in erythrocytes of patients with occlusive thrombosis]

The authors studied the kinetics of the yield of protons from erythrocytes with preliminary acidified cytoplasma in the presence of the inhibitor of anionic transport. It was observed that in patients with occlusive venous thromboses the yield of H+ increased 4 times in comparison to the control group.     

Specific cellular defects in patients with Fanconi anemia

Measurements of plating efficiency, accumulation of metaphases and generation times have shown that fibroblast from patients with Fanconi anemia (FA) have decreased probability of completing a further division after successful mitosis. Thus FA cells show decreased growth rates and increased generation times. We have also measured the survival of FA fibroblasts and lymphoblasts after treatment with a variety of mutagens. All FA cells show an increased sensitivity to drugs such as MMC and psoralen plus long wave length UV which cause DNA interstrand crosslinks. FA strains show varying degrees of sensitivity to these drugs and the extent of this sensitivity seems to be characteristic of each patient. FA cells are equal to controls in their sensitivity to other alkylating agents such as ethyl methane sulfonate, N-methyl-N1-nitro-N-nitrosoguanidine and actinomycin D. Both the decreased growth and increased drug sensitivity may result from defect in DNA replication or repair.     

Cation-induced superoxide generation in tobacco cell suspension culture is dependent on ion valence

There have been many reports suggesting the involvement of reactive oxygen species (ROS), including superoxide anion (O₂^.–), in salt stress. Herein, direct evidence that treatments of cell suspension culture of tobacco (Nicotiana tabacum L.; cell line, BY‐2) with various salts of trivalent, divalent and monovalent metals stimulate the immediate production of O₂^.– is reported. Among the salts tested, LaCl₃ and GdCl₃ induced the greatest responses in O₂^.– production, whereas CaCl₂ and MgCl₂ showed only moderate effects; salts of monovalent metals such as KCl and NaCl induced much lower responses, indicating that there is a strong relationship between the valence of metals and the level of O₂^.– production. As the valence of the added metals increased from monovalent to divalent and trivalent, the concentrations required for maximal responses were lowered. Although O₂^.– production by NaCl and KCl required high concentrations associated with hyperosmolarity, the O₂^.– generation induced by NaCl and KCl was significantly greater than that induced simply by hyperosmolarity. Since an NADPH oxidase inhibitor, diphenyleneiodonium chloride, showed a strong inhibitory effect on the trivalent and divalent cation‐induced generation of O₂^.–, it is likely that cation treatments activate the O₂^.–‐generating activity of NADPH oxidase.     

Effect of calcium ion on fatty acid-induced generation of superoxide in guinea pig neutrophils

When phospholipases of plasma membranes are activated by certain stimuli, unsaturated fatty acids are liberated. Because unsaturated fatty acids enhance the transmembrane movement of calcium ions, the fatty acids released may modulate intracellular calcium homeostasis in various cells, including neutrophils. To determine the physiological function of these unsaturated fatty acids, we studied the effects of various fatty acids on superoxide generation and on changes in intracellular calcium contents of guinea pig neutrophils. Some unsaturated fatty acids, arachidonate and linoleate, stimulated the rate of superoxide generation concomitant with the increase in the amount of intracellular calcium. In contrast, the saturated fatty acid, myristate, stimulated the generation of superoxide without affecting the content of intracellular calcium. The stimulating actions of arachidonate and myristate were increased dramatically by the presence of a low concentration (1 microM) of extracellular calcium ion. The rate of superoxide generation in fatty acid-treated neutrophils was inhibited by chlorpromazine, an inhibitor of such calcium-binding proteins as C-kinase. These and other observations suggest that liberated unsaturated fatty acids increase the amount of intracellular calcium and enhance C-kinase activity also that the increased activity of the enzyme is involved in the chain of events leading to the stimulation of superoxide generation in fatty acid-treated neutrophils.     

Increased heart rate variability in mice overexpressing the Cu/Zn superoxide dismutase

Cu/Zn superoxide dismutase (SOD1) is implicated in various pathological conditions including Down's syndrome, neurodegenerative diseases, and afflictions of the autonomic nervous system (ANS). To assess the SOD1 contribution to ANS dysfunction, especially its influence on cardiac regulation, we studied the heart rate variability (HRV) and cardiac arrhythmias in conscious 12-month-old male and female transgenic mice for the human SOD1 gene (TghSOD1). TghSOD1 mice presented heart rate reduction as compared with control FVB/N individuals. All HRV parameters reflecting parasympathetic activity were increased in TghSOD1. Pharmacological studies confirmed that the parasympathetic tone was exacerbated and the sympathetic pathway was functional in TghSOD1 mice. A high frequency of atrioventricular block and premature ventricular contractions was observed in TghSOD1. By biochemical assays we found that SOD1 activities were multiplied by 9 and 4 respectively in the heart and brainstem of transgenic mice. A twofold decrease in cholinesterase activity was observed in the heart but not in the brainstem. We demonstrate that SOD1 overexpression induces an ANS dysfunction by an exacerbated vagal tone that may be related to impaired cardiac activity of the cholinesterases and may explain the high occurrence of arrhythmias.     

Hydrogen peroxide-independent generation of superoxide catalyzed by soybean peroxidase in response to ferrous ion

It is well documented that extracellular alkalization occurs in plants under the challenges by pathogenic microbes. This may eventually induce the pH-dependent extracellular peroxidase-mediated oxidative burst at the site of microbial challenges. By employing the purified proteins of horseradish peroxidase as a model, we have recently proposed a likely role for free Fe²⁺ in reduction of ferric enzyme of plant peroxidases into ferrous intermediate and oxygen-bound form of enzyme known as Compound III which may eventually releases superoxide anion radical (O₂^•−), especially under alkaline condition, possibly contributing to the plant defense mechanism. In the present study, we employed the purified protein of soybean peroxidase (SBP) as an additional model, and examined the changes in the redox status of enzyme accompanying the generation of O₂^•− in response to Fe²⁺ under alkaline condition.     

Differentiation of Fanconi anemia and aplastic anemia using mitomycin C test in Tunisia

Fanconi anemia (FA) is a recessive chromosomal instability syndrome that is clinically characterized by multiple symptoms. Chromosome breakage hypersensitivity to alkylating agents is the gold standard test for FA diagnosis. In this study, we provide a detailed laboratory protocol for accurate assessment of FA diagnosis based on mitomycin C (MMC) test. Induced chromosomal breakage study was successful in 171 out of 205 aplastic anemia (AA) patients. According to the sensitivity of MMC at 50 ng/ml, 38 patients (22.22%) were diagnosed as affected and 132 patients (77.17%) as unaffected. Somatic mosaicism was suspected in an 11-year-old patient with a FA phenotype. Twenty-six siblings of FA patients were also evaluated and five of them (19.23%) were diagnosed as FA. From this study, a standard protocol for diagnosis of FA was developed. It is routinely used as a diagnostic test of FA in Tunisia.     

Differentiation of Nijmegen breakage syndrome from Fanconi anemia

Nijmegen breakage syndrome (NBS) is a rare auto-somal recessive condition with chromosomal instability. Clinical and biological overlap between Fanconi anemia and ataxia telangiectasia has been reported. We report two cases of NBS born to consanguineous parents. Case one had NBS and Falconi anemia clinical features but relatively little chromosome breakage. The second case had mild NBS features, while cytogenetic evaluation with mitomycin C induction showed chromosome damage. Chromosomal analysis of bone marrow cells revealed tetraploidy, which indicates progression towards leukemia. On the basis of clinical and cytogenetic evaluation, these two cases were confirmed as NBS. However, detailed molecular studies are essential for accurate diagnosis and management of this disease.