Genetic complementation analysis of ataxia telangiectasia and Nijmegen breakage syndrome: a survey of 50 patients

Cultured cells from patients with ataxia telangiectasia (AT) or Nijmegen breakage syndrome (NBS) are hypersensitive to ionizing radiation. After radiation exposure, the rate of DNA replication is inhibited to a lesser extent than in normal cells, whereas the frequency of chromosomal aberrations is enhanced. Both of these features have been used in genetic complementation studies on a limited series of patients. Here we report the results of extended complementation studies on fibroblast strains from 50 patients from widely different origins, using the radioresistant DNA replication characteristic as a marker. Six different genetic complementation groups were identified. Four of these, called AB, C, D, and E (of which AB is the largest), represent patients with clinical signs of AT. Patients having NBS fall into two groups, V1 and V2. An individual with clinical symptoms of both AT and NBS was found in group V2, indicating that the two disorders are closely related. In AT, any group-specific patterns with respect to clinical characteristics or ethnic origin were not apparent. In addition to the radiosensitive ATs, a separate category of patients exists, characterized by a relatively mild clinical course and weak radiosensitivity. It is concluded that a defect in one of at least six different genes may underlie inherited radiosensitivity in humans. To facilitate research on defined defects, a complete list of genetically characterized fibroblast strains is presented.     

Ataxia-telangiectasia: Linkage analysis in highly inbred Arab and Druze families and differentiation from an ataxia-microcephaly-cataract syndrome

Summary Ataxia-telangiectasia (A-T) is a progressive autosomal recessive disease featuring neurodegeneration, immunodeficiency, chromosomal instability, radiation sensitivity and a highly increased proneness to cancer. A-T is ethnically widespread and genetically heterogeneous, as indicated by the existence of four complementation groups in this disease. Several A-T-like genetic diseases share various clinical and cellular characteristics with A-T. By using linkage analysis to study North American and Turkish A-O families, the ATA (A-T, complementation group A) gene has been mapped to chromosome 11q23. A number of Israeli Arab A-T patients coming from large, highly inbred families were assigned to group A In one of these families, an additional autosomal recessive disease was identified, characterized by ataxia, hypotonia, microcephaly and bilateral congenital cataracts. In two patients with this syndrome, normal levels of serum immunoglobulins and alpha-fetoprotein, chromosomal stability in peripheral blood lymphocytes and skin fibroblasts, and normal cellular response to treatments with X-rays and the radiomimetic drug neocarzinostatin indicated that this disease does not share, with A-T, any additional features other than ataxia. These tests also showed that another patient in this family, who is also mentally retarded, is affected with both disorders. This conclusion was further supported by linkage analysis with 11q23 markers. Lod scores between A-O and these markers, cumulated over three large Arab families, were significant and confirmed the localization of the ATA gene to aq23. However, another Druze family unassigned to a specific complementation group, showed several recombinants between A-T and the same markers, leaving the localization of the A-T gene in this family open.     

Competition between laboratory populations of green leafhoppers,Nephotettix spp. (Homoptera: Cicadellidae)

Intra- and interspecific competition between laboratory populations of four green leafhoppers, Nephotettix spp. was studied in the laboratory under three different temperature regimes of 24°C, 27°C and 30°C. For the single-species population of the three tropical species, the equilibrium density increased as the temperature increased. On the other hand, for the temperature species N. cincticeps, the highest equilibrium density was at the intermediate temperature and the lowest at high temperature. Interspecific interactions between two tropical (N. virescens vs. N. nigropictus), a tropical and a temperature (N. virescens vs. N. cincticeps) and a rice-feeding and a grass-inhabiting (N. virescens vs. N. malayanus) Nephotettix species were also studied in the laboratory at the three temperature regimes. Temperature differentially affected the outcome of competition between two Nephotettix species. Between N. virescens and N. nigropictus, the latter was more successful over the former at low and intermediate temperatures, while the former was more successful at high temperature. Between N. virescens and N. cincticeps, the temperate species inhibited the growth of the tropical species at low temperature while the tropical species inhibited the growth of the temperate species at high temperature. At intermediate temperature, the population of N. virescens persisted at a slightly higher density over the population of N. cincticeps. Between the rice-feeding N. virescens and the grass-inhabiting N. malayanus, regardless of temperature the population density of the latter was greatly reduced and later became extinct while the population of the former continued its growth. These consequences of competition between two Nephotettix species conformed fairly well to those predicted by theLotka-Volterra model using demographic parameters specified for each species.     

Structure and function of the mitochondrial bc1 complex. A mutational analysis of the yeast Rieske iron-sulfur protein

Respiratory-defective mutants of Saccharomyces cerevisiae assigned to a single complementation group (G12) have been determined to have lesions in the iron-sulfur protein (Rieske protein) of ubiquinol: cytochrome c reductase. Mutants capable of expressing the protein were chosen for further studies. The genes from 13 independent isolates were cloned and their mutations sequenced. Twelve mutations were ascertained to cause single amino acid substitutions in the carboxyl-terminal regions of the protein between residues 127 and 173. This region is proposed to be part of the catalytic domain with the ligands responsible for co-ordinating the two irons of the 2Fe-2S cluster. Based on the catalytic properties of the ubiquinol: cytochrome c reductase complex and the electron paramagnetic resonance (e.p.r.) signals of the iron-sulfur protein, the mutants describe two different phenotypes. A subset of mutants have no detectable iron-sulfur cluster and are completely deficient in ubiquinol: cytochrome c reductase activity. These strains identify mutations in residues considered to be essential for binding of the iron or for maintaining a proper tertiary structure of the catalytic domain. A second group of mutants have reduced levels of enzymatic activity and exhibit e.p.r. spectra characteristic of the Rieske iron-sulfur cluster. The mutations in the latter strains have been ascribed to residues that influence the redox properties of the cluster by distorting the iron-binding pocket. A secondary and tertiary structure model is presented of the carboxyl-terminal 65 residues constituting the catalytic domain of the iron-sulfur protein. It is postulated that the two irons of the cluster are co-ordinated by three cysteine and a single histidine residue located in a loop structure. The catalytic domain also contains two short alpha-helices and three beta-strands that form a partial beta-barrel. Most of the hydrophilic amino acids are present in turns that map to one pole of the domain. When viewed in the context of the model, mutations that abolish the iron-sulfur cluster are mostly in residues defining the boundaries of the alpha-helices and beta-strands. The notable exception is a cysteine residue that has been assigned to the loop with the iron ligands. This cysteine residue is proposed to co-ordinate one iron of the cluster. Mutations that reduce ubiquinol: cytochrome c reductase activity and alter the redox potential of the cluster occur in residues located in the loop that contains the ligands of the cluster.     

Tissue specificity of chromosomal rearrangements in ataxia-telangiectasia

Summary Cytogenetic studies of lymphocytes and fibroblasts from individuals with ataxia-telangiectasia (AT) demonstrate spontaneous chromosomal breakage. In the AT lymphocytes, this damage results in a high frequency of balanced rearrangements involving chromosome bands 7p14, 7q35, 14q12, and 14q32. The T-cell receptor , , and chain gene complexes and the immunoglobulin heavy chain gene complex, all of which may be functional in lymphocytes, have been localized to these bands. To assess the relationship between genes at these breakpoints and the entirety of the AT phenotype, we undertook a detailed cytogenetic analysis of fibroblasts and lymphocytes from seven AT homozygotes. Our findings indicate that the rearrangements present in the lymphocytes are not commonly observed in the fibroblasts, despite the increased instability of chromosomes from these cells relative to lymphocytes. Furthermore, the changes in the fibroblasts are neither consistent within nor between patients, suggesting that chromosome rearrangement occurs more randomly in this tissue. Therefore, differential site-specific damage in separate tissues may generate the distinct features of the disease in those tissues and may account for the pleiotrophic effects of the AT gene.     

A high frequency of distinct ATM gene mutations in ataxia-telangiectasia.

The clinical features of the autosomal recessive disorder ataxia-telangiectasia (AT) include a progressive cerebellar ataxia, hypersensitivity to ionizing radiation, and an increased susceptibility to malignancies. Epidemiological studies have suggested that AT heterozygotes may also be at increased risk for malignancy, possibly as a consequence of radiation exposure. A gene mutated in AT patients (ATM) has recently been isolated, making mutation screening in both patients and the general population possible. Because of the relatively large size of the ATM gene, the design of screening programs will depend on the types and distribution of mutations in the general population. In this report, we describe 30 mutations identified in a panel of unrelated AT patients and controls. Twenty-five of the 30 were distinct, and most patients were compound heterozygotes. The most frequently detected mutation was found in three different families and had previously been reported in five others. This corresponds to a frequency of 8% of all reported ATM mutations. Twenty-two of the alterations observed would be predicted to lead to protein truncation at sites scattered throughout the molecule. Two fibroblast cell lines, which displayed normal responses to ionizing radiation, also proved to be heterozygous for truncation mutations of ATM. These observations suggest that the carrier frequency of ATM mutations may be sufficiently high to make population screening practical. However, such screening may need to be done prospectively, that is, by searching for new mutations rather than by screening for just those already identified in AT families.     

Changes in non-synaptosomal and synaptosomal mitochondrial membrane-linked enzymatic activities after transient cerebral ischemia

Non-synaptosomal and synaptosomal mitochondrial membrane-linked enzymatic activities, NADH-cytochrome c reductase rotenone insensitive (marker of the outer membrane) and cytochrome oxidase (marker of the inner membrane), were measured in rat brain hippocampus and striatum immediately after and 1, 4, and 7 days following the induction of complete transient ischemia (15 min) by the four vessel occlusion method. Furthermore citrate synthetase activity was measured with and without Triton X-100 in order to qualitatively evaluate the membrane permeability. Nonsynaptosomal mitochondrial membranes showed reduction of both activities only in the late reperfusion phase: NADH-CCRRi decreased in striatal mitochondria after 4–7 days and only after 7 days in the hippocampus. COX activity decreased only in striatal mitochondria 7 days after ischemia. Non-synaptosomal mitochondrial membrane permeability did not show changes. Synaptosomal mitochondria showed a decrease of NADH-CCRRi only at 7 days of reperfusion both in hippocampus and striatum, while COX activity decreased only during ischemia and returned to normal levels in the following days in the two areas considered. In summary, free mitochondria showed insensitiveness to ischemia but they risulted damaged in the late reperfusion phase, while mitochondria from the synaptic terminal showed ischemic damage, partially restored during reperfusion. The striatal mitochondria showed a major susceptibility to ischemia/repefusion damage, showing changes earlier than the hippocampal ones.     

Branched chain amino acids chronic treatment and muscular exercise performance in athletes: a study through plasma acetyl-carnitine levels

Summary In relation to energy request during physical exercise, muscular tissue Branched Chain Amino Acids (BCAA) are metabolized particularly when the oxidation rises. But in the whole-human body, it is difficult to estimate, in a quantitative sense, the role played by BCAA in sustaining exercise. During a BCAA treatment, made on a group of athletes kept under observation, it was observed, through Conconi's test, that this treatment influenced physical performance. Aim of present work is to investigate if BCAA chronic treatment effect on physiological trial is confirmed on blood circulating biochemical energy parameters and in particular on acetyl-carnitine, since acetyl-linked compounds may be an important biochemical factor.Fourteen athletic well trained male subjects, were randomly divided into two subgroups; a first group was submitted to a chronic treatment (n = 7) of BCAA (oral intake was 0.2 g/Kg die) and a second group, as controls (n = 7), assumed oral placebo. Conconi's test demonstrated a significant difference (p < 0.005) in the exercise performance of the two sub-groups, comparing the measurements of ratios of deflection velocity (V _d ), before and after the treatment. Therefore we studied the athletes performing a muscular exercise test (40 Km/h, cycle race, for 90 min) after one month of treatment. During this treatment period the subjects followed a well standardized diet. Samples of blood were drawn before, at the end and during the recovery (60 min) to study if traditional biochemical parameters varied and confirmed the observed differences in Conconi's test. The measurements of concentrations of FFA, KB, free carnitine, acetyl-carnitine and BCAA were performed. Plasma BCAA levels did not demonstrate variations either before or after the exercise performance, or between the two groups. The biochemical factors, substrates and hormones, KB, FFA, lactate, insulin and growth hormone plasma levels did not demonstrate significant differences from the patterns present in literature. Plasma free and acetyl-carnitine followed the well known variations, but only acetyl-carnitine levels demonstrated, at the end increase in acetyl-carnitine levels could be related to a minor fatigue situation and to a larger energy supply availability perhaps present in BCAA treated athletes (Sahlin et al., 1990; May et al., 1989). Both mentioned hypothesis seem in concordance with a smaller acetyl-CoA substrate accumulation, or better for present study, is even more successful with athletes who give a better physical performance. In fact Conconi's test in the two sub-groups of athletes seems to suggest that BCAA treated athletes were able to give a better performance, furthermore out of curiosity we point out that the athletes treated with BCAA won more races than the untreated.We would also like to add in conclusion that although confirming the difficulties of studies in the whole-body, our work gives an interesting clue about the possibility to use acetyl-carnitine plasma levels to understand the biochemical importance of the BCAA as substrate able to influence physical performance, but further research is needed.The phenomenon presence might be showed better perhaps by studying untrained groups during prolonged exercise and with physical performance at exhaustion. If treatment were able to help the physical performance and to shift the fatigue, then confirmation might be a less raised plasma acetyl-carnitine level. In effect blood ammonium levels in present study did not demonstrate any variation in and between sub-groups; this latter observation could be caused by the quantity of work load, and training state of the athletes (Ji et al., 1987; Kirkendall, 1990). Moreover, as observed by Hageloch et al. (1990), the ammonia increases less during prolonged endurance exercise, and in fact the athletes of present study were all middle distance racing cyclists, and the physical performance was a prolonged endurance exercise.