Identification of a mutation in the CHAT gene of Old Danish Pointing Dogs affected with congenital myasthenic syndrome

The presence of a recessive inherited muscle disease in Old Danish Pointing Dogs has been well known for years. Comparisons of this disease with myasthenic diseases of other dog breeds and humans have pointed toward a defect in the synthesis of the neurotransmitter acetylcholine possibly due to decreased activity of the enzyme choline acetyltransferase. We sequenced exons 5-18 of the gene encoding choline acetyltransferase (CHAT) in 2 affected and 2 unaffected dogs and identified a G to A missense mutation in exon 6. The mutation causes a valine to methionine substitution and segregates in agreement with the inheritance of the disease. The mutation was not detected in 50 dogs representing 25 other dog breeds. A DNA test has been developed and is now available to the breeders of Old Danish Pointing Dogs.     

Detoxification of cassava by Aspergillus niger B-1

 The effects of fermentation of cassava by Aspergillus niger B-1 β-glucosidase on its cyanide and protein content, and the optimal conditions for this enzyme’s activity, were examined. This fermentation process reduced the cyanide content of cassava by 95% to 2 mg/kg, and increased its total protein content by 50%, thereby improving its nutritional value. A significant decrease in cyanogenic glycosides was detected after 3 days of fermentation. The optimal pH for A. nigerβ-glucosidase activity on the cyanogenic glycoside linamarin was determined to be 3, the optimal temperature 55 °C, and its K _m 0.3 mM. The findings presented here will facilitate the development of an improved method for detoxification of cassava and for enhancement of its nutritional value. Received: 17 August 1995/Received revision: 27 October 1995/Accepted: 30 October 1995     

Cognition,Emotion and Behavior in Children with Tourette’s Syndrome and Children with ADHD-Combined Subtype—A Two-Year Follow-Up Study

Objective

This two-year follow-up study investigates the course of and association among measures of cognitive control, focused attention, decision-making and symptom severity (anxiety, depression and behavior) in children and adolescents with Tourette’s Syndrome (TS) or Attention-Deficit/Hyperactivity Disorder-Combined subtype (ADHD-C).

Method

19 children with TS, 33 with ADHD-C, and 50 typically developing children (TDC) were examined with a battery of psychometric measures and rating forms at baseline and two-years later.

Results

All three groups improved likewise in measures of cognitive control over time, whereas only the TDC improved in focused attention. The group of children with TS with comorbidities performed more similar to the children with ADHD-C in cognitive control at T1 and T2, whereas the children with TS without comorbidities performed more similar to the TDC in cognitive control at T1 and T2. In the decision-making task, the children with TS (with or without comorbidities) preferred a safer strategy in selecting advantageous choices than the children with ADHD-C and the TDC at T2. Children with TS and children with ADHD-C showed higher symptoms of anxiety and depression and more problems with emotional control compared with TDC at both time points. Finally, children with ADHD-C self-reported more depression symptoms than those with TS at both assessments. For the TS group, safer decision-making was related to better emotional control, and this relationship was stronger for the TS subgroup without comorbidities.

Conclusion

This study emphasizes the importance of addressing symptoms of anxiety and depression in children with TS or ADHD-C, identifying the effect of comorbidities in children with TS, and that children with TS or ADHD-C likely differ in their sensitivity to reinforcement contingencies.     

Extracellular compartments in tendon morphogenesis: collagen fibril, bundle, and macroaggregate formation

The formation of collagen fibrils, fibril bundles, and tissue-specific collagen macroaggregates by chick embryo tendon fibroblasts was studied using conventional and high voltage electron microscopy. During chick tendon morphogenesis, there are at least three extracellular compartments responsible for three levels of matrix organization: collagen fibrils, bundles, and collagen macroaggregates. Our observations indicate that the initial extracellular events in collagen fibrillogenesis occur within narrow cytoplasmic recesses, presumably under close cellular regulation. Collagen fibrils are formed within these deep, narrow recesses, which are continuous with the extracellular space. Where these narrow recesses fuse with the cell surface, it becomes highly convoluted with folds and processes that envelope forming fibril bundles. The bundles laterally associate and coalesce, forming aggregates within a third cell-defined extracellular compartment. Our interpretation is that this third compartment forms as cell processes retract and cytoplasm is withdrawn between bundles. These studies define a hierarchical organization within the tendon, extending from fibril assembly to fascicle formation. Correlation of different levels of extracellular compartmentalization with tissue architecture provides insight into the cellular controls involved in collagen fibril and higher order assembly and a better understanding of how collagen fibrils are collected into structural groups, positioned, and woven into functional tissue-specific collagen macroaggregates.     

Nitrogen availability in radiata pine plantations on former pasture sites in southern New South Wales

Studies of nitrogen availability were carried out in radiata pine (Pinus radiata D. Don) plantations on former pasture sites in N.S.W. in conjunction with studies of the effects of previous land use on tree form. Sites were selected on previously improved pastures (cleared with introduced legumes) and unimproved pastures (partially cleared without legumes) to form age sequences of stands which had been established for periods of up to fifteen years. Mineral-N pools in soils and forest floor samples were determined monthly for thirteen months and nitrification potentials were determined from periodic laboratory incubations.Nitrate and ammonium pools in 2-, 4-, 6-, 9- and 15-year-old radiata pine stands fluctuated seasonally, peaking in summer and autumn. Maximum total mineral-N concentrations of 20 to 40 g g^–1 soil occurred in the youngest, ex-improved pastures with nitrate-N concentrations of up to 25 g g^–1. In the 15-year-old stands, nitrate-N was only detected during autumn, at less than 5 g g^–1 soil. Net N-mineralization and nitrification potentials were consistently higher in the ex-improved pasture soils compared with the ex-unimproved pastures. N availability decreased with increasing stand age in the ex-improved pasture soils, but the pattern was less clear for the unimproved pasture sites. Suppression of clover by pines and the accumulation of nitrogen in the standing biomass are thought to be the major factors controlling the decline of available N during stand development.     

Identification of the Tyrosine-Protein Phosphatase Non-Receptor Type 2 as a Rheumatoid Arthritis Susceptibility Locus in Europeans

Objectives

Genome-wide association studies have facilitated the identification of over 30 susceptibility loci for rheumatoid arthritis (RA). However, evidence for a number of potential susceptibility genes have not so far reached genome-wide significance in studies of Caucasian RA.

Methods

A cohort of 4286 RA patients from across Europe and 5642 population matched controls were genotyped for 25 SNPs, then combined in a meta-analysis with previously published data.

Results

Significant evidence of association was detected for nine SNPs within the European samples. When meta-analysed with previously published data, 21 SNPs were associated with RA susceptibility. Although SNPs in the PTPN2 gene were previously reported to be associated with RA in both Japanese and European populations, we show genome-wide evidence for a different SNP within this gene associated with RA susceptibility in an independent European population (rs7234029, P = 4.4×10⁻⁹).

Conclusions

This study provides further genome-wide evidence for the association of the PTPN2 locus (encoding the T cell protein tyrosine phosphastase) with Caucasian RA susceptibility. This finding adds to the growing evidence for PTPN2 being a pan-autoimmune susceptibility gene.     

Alignment-Independent Comparisons of Human Gastrointestinal Tract Microbial Communities in a Multidimensional 16S rRNA Gene Evolutionary Space

We present a novel approach for comparing 16S rRNA gene clone libraries that is independent of both DNA sequence alignment and definition of bacterial phylogroups. These steps are the major bottlenecks in current microbial comparative analyses. We used direct comparisons of taxon density distributions in an absolute evolutionary coordinate space. The coordinate space was generated by using alignment-independent bilinear multivariate modeling. Statistical analyses for clone library comparisons were based on multivariate analysis of variance, partial least-squares regression, and permutations. Clone libraries from both adult and infant gastrointestinal tract microbial communities were used as biological models. We reanalyzed a library consisting of 11,831 clones covering complete colons from three healthy adults in addition to a smaller 390-clone library from infant feces. We show that it is possible to extract detailed information about microbial community structures using our alignment-independent method. Our density distribution analysis is also very efficient with respect to computer operation time, meeting the future requirements of large-scale screenings to understand the diversity and dynamics of microbial communities.     

Expression of wild-type and mutant medium-chain acyl-CoA dehydrogenase (MCAD) cDNA in eucaryotic cells

An effective EBV-based expression system for eucaryotic cells has been developed and used for the study of the mitochondrial enzyme medium-chain acyl-CoA dehydrogenase (MCAD). 1325 bp of PCR-generated cDNA, containing the entire coding region, was placed between the SV40 early promotor and polyadenylation signals in the EBV-based vector. Both wild-type MCAD cDNA and cDNA containing the prevalent disease-causing mutation A to G at position 985 of the MCAD cDNA were tested. In transfected COS-7 cells, the steady state amount of mutant MCAD protein was consistently lower than the amount of wild-type human enzyme. The enzyme activity in extracts from cells harbouring the wild-type MCAD cDNA was dramatically higher than in the controls (harbouring the vector without the MCAD gene) while only a slightly higher activity was measured with the mutant MCAD. The mutant MCAD present behaves like wild-type MCAD with respect to solubility, subcellular location, mature protein size and tetrameric structure. In immunoblot comparisons, the MCAD protein was present in normal fibroblasts, but essentially undetectable in patient fibroblasts homozygous for the prevalent mutation. We suggest that the MCAD protein carrying this mutation has an impaired ability to form correct tetramers, leading to instability and subsequent degradation of the enzyme. This finding is discussed in relation to the results from expression of human MCAD in Escherichia coli, where preliminary results show that production of mutant MCAD leads to the formation of aggregates.     

Necrotic enteritis challenge models with broiler chickens raised on litter: evaluation of preconditions, Clostridium perfringens strains and outcome variables

The effect of Clostridium perfringens challenge, number of challenge days, and pre-challenge antibiotic treatment on the induction of necrotic enteritis in broiler chickens raised on litter was studied, and the relationship between bacterial counts and frequency of gut lesions was evaluated. Specific intestinal lesions in randomly selected birds were present despite a lack of disease-specific mortality. Challenge, number of challenge days and frequency of lesions were associated with median counts of C. perfringens. The effect of pre-challenge C. perfringens counts and antibiotics cannot be evaluated unless procedures for the control of pre-challenge infection and methods for the differentiation between wild-type and challenge strains are established.