Lentivirus-meditated frataxin gene delivery reverses genome instability in Friedreich ataxia patient and mouse model fibroblasts.

Friedreich ataxia (FRDA) is a progressive neurodegenerative disease caused by deficiency of frataxin protein, with the primary sites of pathology being the large sensory neurons of the dorsal root ganglia and the cerebellum. FRDA is also often accompanied by severe cardiomyopathy and diabetes mellitus. Frataxin is important in mitochondrial iron-sulfur cluster (ISC) biogenesis and low-frataxin expression is due to a GAA repeat expansion in intron 1 of the FXN gene. FRDA cells are genomically unstable, with increased levels of reactive oxygen species and sensitivity to oxidative stress. Here we report the identification of elevated levels of DNA double strand breaks (DSBs) in FRDA patient and YG8sR FRDA mouse model fibroblasts compared to normal fibroblasts. Using lentivirus FXN gene delivery to FRDA patient and YG8sR cells, we obtained long-term overexpression of FXN mRNA and frataxin protein levels with reduced DSB levels towards normal. Furthermore, γ-irradiation of FRDA patient and YG8sR cells revealed impaired DSB repair that was recovered on FXN gene transfer. This suggests that frataxin may be involved in DSB repair, either directly by an unknown mechanism, or indirectly via ISC biogenesis for DNA repair enzymes, which may be essential for the prevention of neurodegeneration.

The Friedreich's ataxia gene encodes a novel phosphatidylinositol-4- phosphate 5-kinase.

The STM7 gene on chromosome 9 was recently 'excluded' as a candidate for Friedreich's ataxia following the identification of an expanded intronic GAA triplet repeat in the adjacent gene, X25, in patients with the disease. Using RT-PCR, northern and sequence analyses, we now demonstrate that X25 comprises part of the STM7 gene, contributing to at least four splice variants, and report the identification of new coding sequences. Functional analysis of the STM7 recombinant protein corresponding to the reported 2.7-kilobase transcript has demonstrated PtdlnsP 5-kinase activity, supporting the idea that the disease is caused by a defect in the phosphoinositide pathway, possibly affecting vesicular trafficking or synaptic transmission.

Mapping the gene causing X-linked recessive nephrolithiasis to Xp11.22 by linkage studies.

X-linked recessive nephrolithiasis is associated with kidney stones and renal tubular dysfunction in childhood progressing to renal failure in adulthood. The primary defect causing this renal tubular disorder is unknown and determining the chromosomal location of the mutant gene would represent an important step toward defining the biochemical basis. We have performed linkage studies in 102 members (10 affected males, 47 unaffected males, 15 obligate heterozygote females, and 30 unaffected females) from five generations of one family. As genetic markers we used 10 cloned human X chromosome fragments identifying restriction fragment length polymorphisms and seven pairs of oligonucleotide primers identifying microsatellite polymorphisms. Linkage with the locus DXS255 was established with a peak LOD score = 5.91 at 3.6% recombination, thereby localizing the X-linked recessive nephrolithiasis gene to the pericentromeric region of the short arm of the X chromosome (Xp11.22). Multilocus analysis indicated that the mutant gene was distal to DXS255 but proximal to the Duchenne muscular dystrophy locus on Xp. Thus, the gene that causes X-linked recessive nephrolithiasis maps to the pericentromeric region of the short arm of the X chromosome (Xp11.22), and further characterization of this gene will help to elucidate the factors controlling renal tubular function and mineral homeostasis.

Association of somatotrophinomas with loss of alleles on chromosome 11 and with gsp mutations.

The molecular pathology of somatotrophinomas has been investigated by a combined search for dominant mutations of the gene encoding the Gs alpha protein and for recessive mutations involving chromosome 11q13, which contains the gene causing multiple endocrine neoplasia type 1 (MEN1). Somatotrophinomas and peripheral leukocytes were obtained from thirteen patients with acromegaly; one patient also suffered from MEN1. Five DNA probes identifying restriction fragment length polymorphisms from 11q revealed allele loss in pituitary tumors from five (four non-MEN1 and one MEN1) patients. Deletion mapping revealed that the region of allele loss common to the somatotrophinomas involved 11q13. An analysis for similar allelic deletions at 12 other loci from chromosomes 1-5, 7-9, 12-14, and 17 did not reveal generalized allele loss in the somatotrophinomas. These results, which represent the first report of chromosome 11 allele loss occurring in non-MEN1 somatotrophinomas, indicate that a recessive oncogene on 11q13 is specifically involved in the monoclonal development of somatotrophinomas. In addition Gs alpha mutations were detected in two non-MEN1 somatotrophinomas, one of which also revealed allele loss of chromosome 11. Thus, our results reveal that the development of somatotrophinomas is associated with alterations in both dominant and recessive oncogenes and further characterization of these genetic abnormalities will help to elucidate the multistep etiology and progression of somatotrophinomas.

'Mitochondrial energy imbalance and lipid peroxidation cause cell death in Friedreich's ataxia'.

Friedreich's ataxia (FRDA) is an inherited neurodegenerative disease. The mutation consists of a GAA repeat expansion within the FXN gene, which downregulates frataxin, leading to abnormal mitochondrial iron accumulation, which may in turn cause changes in mitochondrial function. Although, many studies of FRDA patients and mouse models have been conducted in the past two decades, the role of frataxin in mitochondrial pathophysiology remains elusive. Are the mitochondrial abnormalities only a side effect of the increased accumulation of reactive iron, generating oxidative stress? Or does the progressive lack of iron-sulphur clusters (ISCs), induced by reduced frataxin, cause an inhibition of the electron transport chain complexes (CI, II and III) leading to reactive oxygen species escaping from oxidative phosphorylation reactions? To answer these crucial questions, we have characterised the mitochondrial pathophysiology of a group of disease-relevant and readily accessible neurons, cerebellar granule cells, from a validated FRDA mouse model. By using live cell imaging and biochemical techniques we were able to demonstrate that mitochondria are deregulated in neurons from the YG8R FRDA mouse model, causing a decrease in mitochondrial membrane potential (▵Ψm) due to an inhibition of Complex I, which is partially compensated by an overactivation of Complex II. This complex activity imbalance leads to ROS generation in both mitochondrial matrix and cytosol, which results in glutathione depletion and increased lipid peroxidation. Preventing this increase in lipid peroxidation, in neurons, protects against in cell death. This work describes the pathophysiological properties of the mitochondria in neurons from a FRDA mouse model and shows that lipid peroxidation could be an important target for novel therapeutic strategies in FRDA, which still lacks a cure.

Defining a metabolic phenotype in the brain of a transgenic mouse model of spinocerebellar ataxia 3.

Many of the spinocerebellar ataxias (SCAs) are caused by expansions of CAG trinucleotide repeats encoding abnormal stretches of polyglutamine. SCA3 or Machado-Joseph disease (MJD) is the commonest dominant inherited ataxia disease, with pathological phenotypes apparent with a CAG triplet repeat length of 61-84. In this study a mouse model of SCA3 has been examined which was produced using a human yeast artificial chromosome containing the MJD gene with a CAG triplet expansion of 84 repeats. These mice have previously been shown to possess a mild progressive cerebellar deficit. NMR-based metabolomics/metabonomics in conjunction with multivariate pattern recognition identified a number of metabolic perturbations in SCA3 mice. These changes included a consistent increase in glutamine concentration in tissue extracts of the cerebellum and cerebrum and spectra obtained from intact tissue using magic angle spinning (1)H-NMR spectroscopy. Furthermore, these profiles demonstrated metabolic abnormalities were present in the cerebrum, a region not previously implicated in SCA3. As well as an increase in glutamine both brain regions demonstrated decreases in GABA, choline, phosphocholine and lactate (representing the summation of lactate in vivo, and postmortem glycolysis of glucose and glycogen). The metabolic changes are discussed in terms of the formation of neuronal intranuclear inclusions associated with SCA3. This study suggests high-resolution (1)H-NMR spectroscopy coupled with pattern recognition may provide a rapid method for assessing the phenotype of animal models of human disease.

Polymerase chain reaction analysis of transcriptional patterns of expression of class I HLA genes.

In order to study transcriptional patterns of expression of individual class I HLA genes we have constructed a series of cDNA libraries from human cell lines including normal lymphoblastoid cell lines MANN and HOM2, two colorectal carcinoma cell lines, WiDr and SW480, and a fetal lung fibroblast cell line, MRC-5. Between 0.5 and 1 x 10(6) independent clones were screened in each library using a class I HLA-specific DNA probe and the frequency of class I HLA cDNA clones was found to vary between 0.23% (WiDr) and 0.76% (HOM2). Polymerase chain reaction (PCR)-based analyses of possible alternative splicing events showed that each of 161 class I HLA cDNA clones which had insert sizes exceeding 0.6 kb exhibited normal splicing patterns for exons 5 and 6. Similar PCR-based analyses in clones with appropriately large inserts revealed no exceptions to the normal splicing patterns for each of exons 2, 3, 4, and 7. Sixty of the class I HLA cDNA clones selected from the WiDr, MRC-5, and MANN cDNA libraries were assigned to individual loci following identification of locus-specific DNA sequences by PCR sequencing across exon 5. The sequences obtained from the 60 clones were each interpreted to correspond to one of the classical loci, HLA-A, HLA-B, and HLA-C. While representatives of the HLA-A locus predominated in the MANN library, HLA-B-specific clones were the most abundant in the WiDr and MRC-5 libraries.

Characterization of an expressible nonclassical class I HLA gene.

Screening of a human cosmid library representing genomic DNA from an individual homozygous for the HLA-DR2 B7 A2 haplotype yielded 109 class I HLA-specific clones. One cosmid clone, Ice 6.23, had a full-length nonclassical class I gene within a 5.4-kb HindIII fragment. The Ice 6.23-5.4H gene was cloned into the unique NotI site of an expression vector pSV2.Not, a derivative of pSV2neo, which was constructed to contain a second SV40 early region promoter adjacent to an introduced NotI site. The resulting construct was transfected into the P815-B2M cell line, a derivative of the mouse mastocytoma P815 (HTR) line which expressed human beta2-microglobulin following stable transfection with a cloned human beta2-microglobulin gene. Following transfection the Ice 6.23-5.4 H gene was found to be expressed at both the mRNA and cell surface product levels. DNA sequencing of this gene suggests that it is allelic to the HLA-6.0 gene clone (HLA-G) of Geraghty et al. (Proceedings of the National Academy of Sciences USA, 84:9145, 1987); thereby revealing a HindIII restriction fragment length polymorphism at the HLA-G locus. An extraordinarily high degree of sequence similarity (99.92%) between these two genes, which derive from unrelated HLA haplotypes, suggests strong conservative selection pressure at the HLA-G locus. A flanking single copy sequence probe 4 kb distant from the Ice 6.23-5.4H gene was used to generate long-range restriction mapping at the HLA-G locus.

Evaluation of manual-based cognitive-behavioral therapy for bulimia nervosa in a service setting.

In the present study manual-based cognitive-behavioral therapy for bulimia nervosa was evaluated on an unselected sample of an out-patient service facility. A total of 73 female patients who asked for treatment received the primary diagnosis of bulimia nervosa. Of these, 67 took up treatment. Treatment was completed by 66 patients. Outcome variables were the number of binge episodes along with questionnaire scores for restraint eating, emotional eating, body dissatisfaction and depressiveness. At the end of treatment and 1 year after the end of treatment significant improvements were found in all outcome variables. Effect sizes for outcome variables were within the range of those of controlled research. Therefore, the present study delivered empirical evidence that manual-based cognitive-behavioral therapy is an effective treatment for bulimia nervosa not only within the restricted area of research.

Why do infertile males use psychological couple counselling?

The purpose of the present study was to identify characteristics of male patients that could be relevant for the uptake of psychological couple counselling for infertility. Therefore, 94 male patients who participated in psychological couple counselling were compared to 134 unselected infertility patients who attended an andrological clinic. Counselling users showed higher scores for depression and anxiety as well as a higher number of impaired sperm parameters. Multivariate analysis revealed that beyond the level of depression the number of impaired sperm parameters delivered additional information about the probability of a patient using counselling. For interpretation of these results the former research was broadly reviewed. It is suggested that an increased level of distress, the feeling of being responsible for infertility and few marital difficulties are relevant for the usage of couple counselling by male infertility patients. Practical consequences are discussed.

Bulimia nervosa: mood changes do have an impact on body width estimation.

OBJECTIVES: This study was designed to investigate the impact of mood changes on body width estimation in women with bulimia nervosa. DESIGN: A pre-post controlled experimental design was chosen. METHOD: Mood changes were induced in 40 women with bulimia nervosa, 20 women with panic disorder and 40 women with no diagnosis of a psychological disorder. A combination of autobiographical memory method and music induction method was used to induce positive and negative mood, respectively. Before and after mood induction a video distorting technique was used for body width estimation. RESULTS: Induction of negative mood increased and induction of positive mood decreased the body width estimations of women with bulimia. Patients with panic disorder and 'healthy' controls did not show these changes after mood induction. CONCLUSION: The findings suggest that change in mood state rather than the more habitual mood quality are relevant for bulimic women's body perception.

Platelets store laminins 411/421 and 511/521 in compartments distinct from α- or dense granules and secrete these proteins via microvesicles.

BACKGROUND: Blood platelets secrete upon activation of laminins 411/421 and 511/521, large adhesive proteins mainly found in the basement membranes of blood vessels and other tissues. At present, the subcellular localization and secretion mechanisms of platelet laminins are largely unknown. OBJECTIVES: Our aim was to compare the subcellular localization of laminins 411/421 and 511/521 and specific granule markers in platelets. We also elucidated the role of microvesicles and exosomes in laminin release in platelet activation. METHODS: We studied laminin and granule marker protein localization in platelets by using immunofluorescence confocal microscopy and immunoelectron microscopy. Microvesicles and exosomes were separated from material released from platelets on activation by thrombin. The expression of laminins in microvesicles and exosomes was studied by using SDS-PAGE and Western blotting as well as by flow cytometric analysis. The exosomes were immunoprecipitated with magnetic microbeads coated with anti-CD63 antibodies. RESULTS AND CONCLUSIONS: We demonstrate that laminins 411/421 and 511/521 are present in compartments of platelets that do not express α-granule, dense granule, or lysosome marker proteins. Moreover, laminins secreted by activated platelets are mostly found in microvesicles shed from the plasma membrane, while their presence in simultaneously released exosomes is minimum.

The impact of treatment experiences on the course of infertility distress in male patients.

BACKGROUND: Previous research on infertile males has delivered equivocal findings on the course of infertility distress in males. The present longitudinal study examines whether there are differentials associated with specific treatment experiences (i.e. duration of treatment, the diagnosis received, and treatment failure of assisted reproductive technologies). METHODS: The sample consisted of 118 patients who twice visited an andrology clinic on their own initiative for fertility work-ups. Baseline and follow-up examinations were > or = 6 months apart. Prior to each fertility work-up, patients completed a questionnaire assessing distress due to infertility. RESULTS: No uniform course of distress could be detected. A significant interaction between treatment experiences indicated that distress rises significantly only in those patients who were in treatment > or = 17 months and experienced treatment failure between the first and the second psychological evaluation. For the diagnosis of male infertility, however, neither a direct nor an indirect impact was identified. CONCLUSION: The present study indicates that the interaction of specific treatment experiences is associated with changes in distress of infertile males.

Personality, coping and sperm count.

Previous research has suggested an association between personality traits and coping, as well as between coping and sperm concentration. In the present study, both domains of research were combined, leading to the formulation of specific hypotheses. A total of 54 healthy volunteers were given questionnaires twice to assess personality traits and coping behaviour. Participants also produced up to three semen specimens. As hypothesized, active coping was correlated negatively with neuroticism (r = -0.59) and positively with conscientiousness (r = 0.56), whereas sperm concentration was correlated negatively with both active coping (r = -0.28) and conscientiousness (r = -0.37). The relationship between conscientiousness and sperm concentration did not appear to be mediated by active coping. Although the correlations were small, evidence is mounting that psychological aspects and male sperm parameters are not independent. The present findings, however, should not lead to the conclusion that conscientiousness and active forms of coping are characteristics of infertile patients.

Periodic variability in cetacean strandings: links to large-scale climate events.

Cetacean strandings elicit much community and scientific interest, but few quantitative analyses have successfully identified environmental correlates to these phenomena. Data spanning 1920-2002, involving a total of 639 stranding events and 39 taxa groups from southeast Australia, were found to demonstrate a clear 11-13- year periodicity in the number of events through time. These data positively correlated with the regional persistence of both zonal (westerly) and meridional (southerly) winds, reflecting general long-term and large-scale shifts in sea-level pressure gradients. Periods of persistent zonal and meridional winds result in colder and presumably nutrient-rich waters being driven closer to southern Australia, resulting in increased biological activity in the water column during the spring months. These observations suggest that large-scale climatic events provide a powerful distal influence on the propensity for whales to strand in this region. These patterns provide a powerful quantitative framework for testing hypotheses regarding environmental links to strandings and provide managers with a potential predictive tool to prepare for years of peak stranding activity.

Individual prognosis for changes in sperm quality on the basis of perceived stress.

BACKGROUND: In this study we examined whether stress has a negative influence on sperm quality. To investigate this issue we developed a scale assessing perceived stress resulting from infertility. METHODS AND RESULTS: The Infertility Distress Scale was constructed based on the data of 158 infertility patients contacting an andrological clinic for the first time. The Infertility Distress Scale consists of items assessing self-reported stress, different appraisals of infertility and cognitive involvement in infertility. The scale was shown to have good psychometric properties. Changes in sperm quality were predicted by this scale for 69 patients. Changes in sperm concentration and sperm motility were predicted correctly 75.4 and 65.6% of the time, respectively. While these prediction accuracies were significantly better than chance prediction, the Infertility Distress Scale had no predictive value for changes in morphology. CONCLUSION: Results indicate that distress caused by infertility is a risk factor for a decrease in sperm quality.

Coping with infertility: distress and changes in sperm quality.

Infertility represents a serious stressor for some patients as well as a risk factor for a decrease in sperm quality. The purpose of the present study was to identify coping strategies that went along with both better emotional and physical adjustment to infertility. The sample consisted of 63 patients who contacted an andrological clinic more than one time. Prior to clinical examination, patients filled out a questionnaire referring to the way in which they coped with their wives' previous menstruation. Participants also completed a scale assessing perceived distress due to infertility. Change in sperm concentration since baseline semen analysis and the level of distress were used to evaluate patient's adjustment. The better-adjusted patients showed less prominent overall coping efforts, and a higher proportion of distancing coping strategies. An improvement in sperm quality also was associated with a low cognitive involvement in infertility. Situational uncontrollability of infertility could be a moderator of the effectiveness of coping employed by the better-adjusted patients. In addition, the coping behaviour related to better adjustment could be due to a dispositional stress resistance factor. For clinical implementation of the findings, the attitudes of a patient and the expectations of his wife have to be taken into consideration.

Is infertility a risk factor for impaired male fertility?

BACKGROUND: Previous research has suggested an interaction between distress and male fertility. The present longitudinal study sought to deliver evidence for a negative impact of distress due to infertility on sperm concentration. METHODS: The sample consisted of 120 patients who twice visited an andrological clinic on their own initiative for fertility work-ups. Baseline and follow-up examinations were at least 6 months apart. Prior to each fertility work-up, patients completed a questionnaire assessing distress due to infertility. RESULTS: Path analyses revealed that the level of infertility distress at follow-up has a negative impact on the change in sperm quality from baseline to follow-up assessment. Distress scores were highly stable. As a consequence, the level of distress at baseline assessment provided only little additional information for the changes in sperm concentration. Further analysis suggested that the fertility status had no impact on infertility distress. CONCLUSION: The present study delivers the strongest evidence to date that distress due to infertility is a significant risk factor for a decrease in sperm quality.