Unravelling the etiology of sporadic late-onset cerebellar ataxia in a cohort of 205 patients: a prospective study.

BACKGROUND: Despite recent progress in the field of genetics, sporadic late-onset (> 40 years) cerebellar ataxia (SLOCA) etiology remains frequently elusive, while the optimal diagnostic workup still needs to be determined. We aimed to comprehensively describe the causes of SLOCA and to discuss the relevance of the investigations. METHODS: We included 205 consecutive patients with SLOCA seen in our referral center. Patients were prospectively investigated using exhaustive clinical assessment, biochemical, genetic, electrophysiological, and imaging explorations. RESULTS: We established a diagnosis in 135 (66%) patients and reported 26 different causes for SLOCA, the most frequent being multiple system atrophy cerebellar type (MSA-C) (41%). Fifty-one patients (25%) had various causes of SLOCA including immune-mediated diseases such as multiple sclerosis or anti-GAD antibody-mediated ataxia; and other causes, such as alcoholic cerebellar degeneration, superficial siderosis, or Creutzfeldt-Jakob disease. We also identified 11 genetic causes in 20 patients, including SPG7 (n = 4), RFC1-associated CANVAS (n = 3), SLC20A2 (n = 3), very-late-onset Friedreich's ataxia (n = 2), FXTAS (n = 2), SCA3 (n = 1), SCA17 (n = 1), DRPLA (n = 1), MYORG (n = 1), MELAS (n = 1), and a mitochondriopathy (n = 1) that were less severe than MSA-C (p < 0.001). Remaining patients (34%) had idiopathic late-onset cerebellar ataxia which was less severe than MSA-C (p < 0.01). CONCLUSION: Our prospective study provides an exhaustive picture of the etiology of SLOCA and clues regarding yield of investigations and diagnostic workup. Based on our observations, we established a diagnostic algorithm for SLOCA.

Evidence of positive selection towards Zebuine haplotypes in the BoLA region of Brangus cattle.

The Brangus breed was developed to combine the superior characteristics of both of its founder breeds, Angus and Brahman. It combines the high adaptability to tropical and subtropical environments, disease resistance, and overall hardiness of Zebu cattle with the reproductive potential and carcass quality of Angus. It is known that the major histocompatibility complex (MHC, also known as bovine leucocyte antigen: BoLA), located on chromosome 23, encodes several genes involved in the adaptive immune response and may be responsible for adaptation to harsh environments. The objective of this work was to evaluate whether the local breed ancestry percentages in the BoLA locus of a Brangus population diverged from the estimated genome-wide proportions and to identify signatures of positive selection in this genomic region. For this, 167 animals (100 Brangus, 45 Angus and 22 Brahman) were genotyped using a high-density single nucleotide polymorphism array. The local ancestry analysis showed that more than half of the haplotypes (55.0%) shared a Brahman origin. This value was significantly different from the global genome-wide proportion estimated by cluster analysis (34.7% Brahman), and the proportion expected by pedigree (37.5% Brahman). The analysis of selection signatures by genetic differentiation (F st ) and extended haplotype homozygosity-based methods (iHS and Rsb) revealed 10 and seven candidate regions, respectively. The analysis of the genes located within these candidate regions showed mainly genes involved in immune response-related pathway, while other genes and pathways were also observed (cell surface signalling pathways, membrane proteins and ion-binding proteins). Our results suggest that the BoLA region of Brangus cattle may have been enriched with Brahman haplotypes as a consequence of selection processes to promote adaptation to subtropical environments.

The F-box protein beta-TrCP associates with phosphorylated beta-catenin and regulates its activity in the cell.

Defects in beta-catenin regulation contribute to the neoplastic transformation of mammalian cells. Dysregulation of beta-catenin can result from missense mutations that affect critical sites of phosphorylation by glycogen synthase kinase 3beta (GSK3beta). Given that phosphorylation can regulate targeted degradation of beta-catenin by the proteasome, beta-catenin might interact with an E3 ubiquitin ligase complex containing an F-box protein, as is the case for certain cell cycle regulators. Accordingly, disruption of the Drosophila F-box protein Slimb upregulates the beta-catenin homolog Armadillo. We reasoned that the human homologs of Slimb - beta-TrCP and its isoform beta-TrCP2 (KIAA0696) - might interact with beta-catenin. We found that the binding of beta-TrCP to beta-catenin was direct and dependent upon the WD40 repeat sequences in beta-TrCP and on phosphorylation of the GSK3beta sites in beta-catenin. Endogenous beta-catenin and beta-TrCP could be coimmunoprecipitated from mammalian cells. Overexpression of wild-type beta-TrCP in mammalian cells promoted the downregulation of beta-catenin, whereas overexpression of a dominant-negative deletion mutant upregulated beta-catenin protein levels and activated signaling dependent on the transcription factor Tcf. In contrast, beta-TrCP2 did not associate with beta-catenin. We conclude that beta-TrCP is a component of an E3 ubiquitin ligase that is responsible for the targeted degradation of phosphorylated beta-catenin.

Prevention of bleomycin-induced pulmonary fibrosis after adenovirus-mediated transfer of the bacterial bleomycin resistance gene.

A serious limitation in the use of the DNA-cleaving, antitumoral-antibiotic, bleomycin during chemotherapy is pulmonary toxicity. Lung injury induced by bleomycin is characterized by an increased deposition of interstitial extracellular matrix proteins in the alveolar wall that compromises respiratory function. Several drugs have been tested in animal models to prevent the pulmonary toxicity of bleomycin, but have not led to a useful clinical treatment because of their adverse effects on other tissues. We have shown that transgenic mice expressing Streptoalloteichus hindustanus (Sh) ble bleomycin resistance protein in pulmonary epithelial cells in the lungs are protected against bleomycin-induced toxicity in lungs. In the present study, we used intranasal administration by adenovirus-mediated gene transfer of the bleomycin resistance Sh ble gene to mouse lung for prevention of bleomycin-induced pulmonary fibrosis. We constructed recombinant adenoviruses Ad.CMVble and Ad.RSVble harboring the bleomycin resistance Sh ble gene under the control of the cytomegalovirus early promoter and the Rous sarcoma virus early promoter, respectively. Transgene expression was detected in epithelia of conducting airways and alveolar septa by immunostaining with a rabbit polyclonal antibody directed against the bleomycin resistance protein and persisted for the duration of drug treatment; i.e., up to 17 d. No toxic effect was seen in adenovirus-treated mice. Pretreatment of mice with Ad.CMVble or Ad.RSVble completely prevented collagen deposition 42-133 d after bleomycin treatment, as measured by lung OH-proline content. Histologic studies indicated that there was little or no lung injury in the adenovirus/bleomycin-treated mice compared with the bleomycin-treated mice. These observations may lead to new approaches for the prevention of bleomycin-induced pulmonary fibrosis.

ATF4 degradation relies on a phosphorylation-dependent interaction with the SCF(betaTrCP) ubiquitin ligase.

The ubiquitin-proteasome pathway regulates gene expression through protein degradation. Here we show that the F-box protein betaTrCP, the receptor component of the SCF E3 ubiquitin ligase responsible for IkappaBalpha and beta-catenin degradation, is colocalized in the nucleus with ATF4, a member of the ATF-CREB bZIP family of transcription factors, and controls its stability. Association between the two proteins depends on ATF4 phosphorylation and on ATF4 serine residue 219 present in the context of DSGXXXS, which is similar but not identical to the motif found in other substrates of betaTrCP. ATF4 ubiquitination in HeLa cells is enhanced in the presence of betaTrCP. The F-box-deleted betaTrCP protein behaves as a negative transdominant mutant that inhibits ATF4 ubiquitination and degradation and, subsequently, enhances its activity in cyclic AMP-mediated transcription. ATF4 represents a novel substrate for the SCF(betaTrCP) complex, which is the first mammalian E3 ubiquitin ligase identified so far for the control of the degradation of a bZIP transcription factor.

Lp-PLA2 activity and PLA2G7 A379V genotype in patients with diabetes mellitus.

Lipoprotein associated phospholipase A2 (Lp-PLA2) modulates low-density lipoprotein (LDL) oxidation by hydrolysing oxidised phospholipids present on particle surfaces. We investigated whether Lp-PLA2 activity and PLA2G7 A379V genotype were related to mediators of atherosclerosis in a diabetic study. Plasma Lp-PLA2 activity (taken in men only) and A379V genotype were investigated with regards to metabolic syndrome (MS), UKPDS risk score, and oxidised LDL (oxLDL/LDL), in a cohort of Caucasian men and women (n=783, age 62.5+/-13.7 years). After adjustment for type of diabetes, CHD status, and statin use, those individuals with features defining the MS (WHO guidelines) had higher Lp-PLA2 activity (35.6+/-11.9 nmol/min/ml) compared to those without (33.0+/-10.8 nmol/min/ml) (p=0.02). Quartiles of UKPDS coronary heart disease (CHD) risk score were also positively associated with Lp-PLA2 activity (p=0.006, p=0.004 linear trend). Those men in the highest quartile of oxLDL/LDL level had the lowest Lp-PLA2 activity (31.3+/-10.5 nmol/min/ml) when compared to the middle two (32.3+/-9.8 and 35.9+/-10.9 nmol/min/ml, respectively) and lowest quartile (35.6 +/-12.5 nmol/min/ml; p=0.03, p=0.004 linear trend). There was no significant association between A379V genotype and Lp-PLA2 enzyme activity (p=0.34) or oxLDL/LDL (p=0.32). Lp-PLA2 activity is an independent predictor of CHD risk and MS in a sample of subjects with diabetes mellitus. The association of Lp-PLA2 activity with oxLDL/LDL suggests that Lp-PLA2 may be a modulating factor in the process of atherosclerosis.

Bleomycin resistance conferred by a drug-binding protein.

The protein coded by a bleomycin-resistance gene (ble) cloned from producing actinomycetes was purified from a culture of a recombinant E. coli strain and its action on bleomycin was determined by in vitro assays. The protein binds reversibly in a one to one ratio to bleomycin which can no longer cleave DNA. The bleomycin resistance of cells harboring a ble gene could be accounted for by a sequestering effect of the bleomycin-binding protein.

Cell signalling associated with fibrinolytic ligand binding to human colorectal carcinoma cells.

Addition of purified plasmin or plasminogen (0.1 microM) to serum-free culture media elevated cellular D-myo-inositol 1,4,5-trisphosphate (InsP3) levels in human colorectal carcinoma cells within 1 h to double those of control cells. This was accompanied by decreases in cellular phosphatidylinositol bisphosphate by 40% in cells exposed to fibrinolytic ligands for up to 1 h. The effect was not due to opening of Ca2+ channels of the type blocked by 5 microM nifedipine, and 100 microM EGTA, a Ca2+ chelator, did not suppress plasmin's ability to elevate InsP3. Binding assays at 4 degrees C with 125I-labelled plasmin indicated maximum binding within 1 h suggesting that the effects of plasmin may be associated with its cell-binding function. These cells could convert exogenous plasminogen to plasmin with endogenous activation and this was accompanied by a decrease in radioactive phosphatidylinositol well below control levels (13% of control). Our results contribute to evidence for the association of plasmin-binding sites with a signalling system. A cell signalling system indirectly or directly associated with plasmin binding, would permit carcinoma cells to coordinate extracellular fibrinolysis with cell migration and motility through second messengers.

Efficient secretion of human lysozyme fused to the Sh ble phleomycin resistance protein by the fungus Tolypocladium geodes.

Tolypocladium geodes strain NC50 was transformed by different integrating vectors bearing both a synthetic gene encoding human lysozyme (HLz) and the Sh ble phleomycin resistance marker, either in separate expression cassettes or in transcriptional or translational fusion configurations. Clones derived from all vectors were able to secrete HLz. The highest productivities in shake flasks (up to 150 mg l-1 in 5 days) were obtained when HLz was fused at the C-terminal end of the Sh ble protein. The fusion protein is efficiently secreted and release of active lysozyme occurs by extracellular proteolytic cleavage in the junction peptide.

Proteolytic events in the processing of secreted proteins in fungi.

Secreted heterologous proteins have been found to be produced much less efficiently by fungi than secreted homologous ones. This could be due, at least in part, to proteolytic cleavage by site-specific endoproteases of the secretory pathway, similar to the yeast KEX2 protease and the mammalian dibasic endoproteinases found in secretory pathways. Mature secreted fungal proteins may be protected from such cleavage due to the absence of cleavable sites in exposed regions. A comparison of the dipeptide distributions of 33 secreted and 34 cytoplasmic proteins from fungal producers of extracellular enzymes indicated a significant bias for some doublets, including the basic dipeptides Lys-Arg, Arg-Arg and Arg-Lys which have also been demonstrated to be KEX2 substrates. Other combinations were also found to be rare in secreted proteins, which could indicate either a broader specificity of the considered endopeptidase, or the presence either in the secretory organelles or among the secreted proteins of additional proteases with different specificities. Experimental evidence that the Lys-Arg site is processed in Tolypocladium geodes was provided by cloning a synthetic prosequence upstream of a phleomycin resistance (Sh ble) gene and analyzing the N-terminus of the corresponding protein purified from the culture supernatant. This system also provides a tool for further studies of specific proteases of fungi.

Relevance of urinalysis monitoring of methadone maintenance patients: a clinical-biological agreement on 41 patients.

Abstract This study evaluated the concordance of concomitant urinalysis and clinical assessments of drug abusers included in a methadone maintenance programme. The agreement between a clinical subjective score and an objective biological score, both measuring the evolution of illicit substance consumption over 12 months, was analysed. The clinical score, established by physicians and applied during patient interviews, was determined at entry into the programme and re-evaluated after 6 and 12 months. Forty-one patients were evaluated. The urinalysis score was based on regular screening of urine samples with the EMIT method. Agreement between the two scores was determined by using the kappa coefficient for each substance (opiates, benzodiazepines and cocaine) for each time-point. The calculated kappa coefficients showed poor agreement between the two scores, but could indicate the complementarity of these clinical and biological appraisals. Indeed, the urinalysis objectively detected change in drug use before the clinician. Thus, urinalysis monitoring should be considered as an additional and complementary biological procedure for patient follow-up by physicians.

Sutton's disease (periadenitis mucosa necrotica recurrens).

Sutton's disease is characterized by giant necrotizing ulcers around minor salivary glands and is of unknown cause. We report a case, review the medical literature, and discuss the treatment of this affliction.

Puccinia sorghi Virulent on Sweet Corn with the Rp1-D Gene in Southern France.

The Rp1-D gene, which conveys a chlorotic-fleck resistant reaction to Puccinia sorghi, effectively controlled common rust on sweet corn in North America for nearly 15 years. Biotypes of P. sorghi virulent on plants with the Rp1-D gene were widespread in North America for the first time in 1999 and again in 2000 (1,2). Many Rp-resistant sweet corn hybrids that are developed and grown in North America also are grown in Europe, including France where virulence against the Rp1-D gene has not been reported previously. In September 2000, uredinia of common rust were observed on and collected from sweet corn hybrids with the Rp1-D gene in commercial fields and hybrid trials in the Landes and Pyrénées Atlantiques departments of the Aquitaine region of southwestern France. Severity of rust generally was below 5% on these plants except for a few hybrids for which severity was about 20 to 30%. Common rust was not observed on hybrids with the Rp-G gene. Urediniospores were increased as a bulk population on the susceptible sweet corn hybrid Sterling in a greenhouse. Plants with each of 10 single Rp genes (Rp1-A, Rp1-C, Rp1-D, Rp1-E, Rp1-F, Rp1-I, Rp1-K, Rp1-L, Rp1-N, and Rp-G) or each of six compound rust resistance genes (Rp1-D5, Rp1-JC, Rp1-JFC, Rp-GDJ, Rp-GFJ, and Rp-G5JC) were assayed for reactions to this population of P. sorghi. Two to six different sources of seed of each single Rp gene and two different sources of seed of each compound rust resistance gene were replicates with a single pot of 6 to 18 plants grown from a specific seed source. Plants were inoculated three times on successive days by placing 2 or 3 ml of a urediniospore suspension in the whorl of two- to four-leaved seedlings. Reactions were rated 10 days after the last inoculation. Plants without symptoms or with chlorotic-fleck resistant reactions were inoculated again and rated 10 days later. Uredinia did not form on plants with compound rust resistance genes. Plants with the genes Rp1-E, Rp1-I, Rp1-K, and Rp-G also were resistant although a few, very small uredinia (i.e., type-1 uredinia) were observed on a few plants. Plants with the genes Rp1-A, Rp1-C, Rp1-D, Rp1-F, Rp1-L, and Rp1-N were fully susceptible. This pattern of virulence is the same as that observed during the past two years in North American populations of P. sorghi virulent against Rp1-D. Rp-resistance currently available in most sweet corn hybrids will not be effective in France if these virulent biotypes become prevalent. References: (1) J. K. Pataky et al. Plant Dis. 85:165, 2001. (2) M. C. Pate et al. Plant Dis. 84:1154, 2000.

[Determination of erythrocyte protoporphyrin by a spectrofluorometry method]. / Détermination de la protoporphyrine érythrocytaire par méthode spectrofluorimétrique.

Erythrocyte protoporphyrin (EP) measurement is useful for the diagnosis of protoporphyrin, iron deficiency or lead poisoning. The authors described a manual procedure for the spectrofluorimetric determination of EP. Erythrocytes were used, instead of whole blood. The following steps were performed: treatment with celite suspension, extraction using ethylacetate/acetic acid, estimation of the fluorescence of protoporphyrin IX extracted in HCl. Analytical results are following: a low detection limit (0.045 mumol/l), a linear response up to 2 mumol/l, within run and day-to-day precision respectively better than 3.5 p. cent and 5.8 p. cent. The procedure with predilution led to no significant difference with the spectrophotometric reference method (student t-test, t: 0.85) up to 42 mumol/l. Concentrations of EP according to age were studied for children (0.0470-1.000 mumol/l), middle-aged subjects (0.390-0.880) and elderly subjects (0.340-1.040). The proposed method is accurate, rapid and available for routine measurements.

[Withdrawal syndrome in 2 drug addicts after intravenous injection of buprenorphine?]. / Syndrome de manque chez deux toxicomanes après injection intraveineuse de buprénorphine?

Adequate dosage of sublingual buprenorphine is now recommended for substitution treatment of severe opioid dependance. We report two cases of acute discomfort, probably linked to withdrawal syndrome, after an IV injection of high doses of buprenorphine in opiate dependant patients. Data on the pharmacokinetics and neurobiochemical aspects of buprenorphine are compared with those of other opiates. A major issue of this work is a guideline for inducing substitution treatment with this "unique" partial agonist/antagonist of endorphinic receptors.

[Churg-Strauss syndrome. Retrospective study of 20 cases]. / Le syndrome de Churg et Strauss. Etude rétrospective de vingt observations.

Churg-Strauss syndrome (CSS) is a disorder characterised by hypereosinophilia and systemic vasculitis complicating a preexisting asthma. Twenty cases have been studied. Mean duration of asthma before CSS was 8 years, the peripheral-blood eosinophilia, always > 1,700/microL, went above 5,000/microL in 17 cases. The clinical manifestations were the following: 20 impairements of general state with fever, 13 peripheral neuropathies, 15 cutaneous injuries, 10 pericardial or myocardial attacks, 10 digestive impairements, 9 muscular and articular diseases, 7 renal diseases--all of them linked with the vasculitis--and 9 upper respiratory tract involvements. Pleuropulmonary or cardiac anomalies have been discovered at the chest X-ray in 14 cases. The diagnosis has been histologicaly confirmed in 15 cases. No clinical or biological or evolutive distinction was noticed between the 15 positive biopsy patients and the 5 negative ones. During 8.4 (+/- 7.9 years) the evolution was caracterised by relapses which have always been announced by increasing eosinophilia. Eighteen patients have been successfully treated with corticoids alone or associated with cyclophosphamid in 9. Survival at 5 years was 85%. Five deaths occured because of CSS (2 because of an unadapted treatment). We have to focus the need for an emergency treatment of CSS. The diagnosis can be done by clinical investigation only, before any anatomopathologic results.

Foreign meat identification by DNA breed assignment for the Chinese market.

Methods for individual identification are usually employed for traceability, whereas breed identification is useful to detect commercial frauds. In this study, Chinese Yellow Cattle (CYC) samples plus data from six Bos taurus breeds, two Bos indicus breeds, and one composite breed were used to develop an allocation test based on 22 microsatellites. The test allowed discriminating all foreign breeds from the CYC, although some CYC individuals were wrongly allocated as Limousin or Holstein, probably due to the recent introduction of these breeds into China. In addition, CYC evidenced a previously reported Zebu cline (south-north) and a possible structure within the B. taurus component that should be confirmed. An independent test performed with meat samples of unknown breed origin from Argentina allocated 92% of them to either Angus, Hereford, or their crossbreed, but none was identified as CYC. We conclude that the test is a suitable tool to certify meat of foreign breed origin and to detect adulterations of CYC beef labeled as imported meat.

Probiotics in animal nutrition and health.

The use of probiotics for farm animals has increased considerably over the last 15 years. Probiotics are defined as live microorganisms which can confer a health benefit for the host when administered in appropriate and regular quantities. Once ingested, the probiotic microorganisms can modulate the balance and activities of the gastrointestinal microbiota, whose role is fundamental to gut homeostasis. It has been demonstrated that numerous factors, such as dietary and management constraints, can strongly affect the structure and activities of the gut microbial communities, leading to impaired health and performance in livestock animals. In this review, the most important benefits of yeast and bacterial probiotics upon the gastrointestinal microbial ecosystem in ruminants and monogastric animals (equines, pigs, poultry, fish) reported in the recent scientific literature are described, as well as their implications in terms of animal nutrition and health. Additional knowledge on the possible mechanisms of action is also provided.