Morphological and morphometric studies of the dysmyelinating mutant,the Long Evans shaker rat

The Long Evans shaker (les) rat is a recently identified CNS myelin mutant with an autosomal recessive mode of inheritance. Although scattered myelin sheaths are present in some areas of the CNS, most notably the ventral spinal cord in the young neonatal rat, this myelin is gradually lost, and 8-12 weeks little myelin is present throughout the CNS. Despite this severe myelin deficiency, some mutants may live beyond one year of age. Rare, thin myelin sheaths that are present early in development lack myelin basic protein (MBP) and on ultrastructural examination are poorly compacted and lack a major dense line. Many oligodendrocytes develop an accumulation of vesicles and membranous bodies, but no abnormal cell death is observed. In the optic nerve, cell kinetic studies show an increase in proliferation at early time points in les, while total glial cell counts are also increased in les from 2 months of age. In situ hybridization studies demonstrate that the numbers of mature oligodendrocytes are similar to controls early in life and increase with time compared to controls. There is both a progressive astrocyte hypertrophy and microgliosis. While les has a mutation in the myelin basic protein (mbp) gene, it is dissimilar in both genotype and phenotype to the previously described mbp mouse mutants, shiverer (shi) and shiverermld. Unlike shi and its allele, where myelin increases with time and oligodendrocytes become ultrastructurally normal, les oligodendrocytes are permanently disabled, continue to demonstrate cytoplasmic abnormalities, and fail to produce myelin beyond the first weeks of life.     

Complete Chloroplast Genome of the Wollemi Pine (Wollemia nobilis): Structure and Evolution

The Wollemi pine (Wollemia nobilis) is a rare Southern conifer with striking morphological similarity to fossil pines. A small population of W. nobilis was discovered in 1994 in a remote canyon system in the Wollemi National Park (near Sydney, Australia). This population contains fewer than 100 individuals and is critically endangered. Previous genetic studies of the Wollemi pine have investigated its evolutionary relationship with other pines in the family Araucariaceae, and have suggested that the Wollemi pine genome contains little or no variation. However, these studies were performed prior to the widespread use of genome sequencing, and their conclusions were based on a limited fraction of the Wollemi pine genome. In this study, we address this problem by determining the entire sequence of the W. nobilis chloroplast genome. A detailed analysis of the structure of the genome is presented, and the evolution of the genome is inferred by comparison with the chloroplast sequences of other members of the Araucariaceae and the related family Podocarpaceae. Pairwise alignments of whole genome sequences, and the presence of unique pseudogenes, gene duplications and insertions in W. nobilis and Araucariaceae, indicate that the W. nobilis chloroplast genome is most similar to that of its sister taxon Agathis. However, the W. nobilis genome contains an unusually high number of repetitive sequences, and these could be used in future studies to investigate and conserve any remnant genetic diversity in the Wollemi pine.     

Influence of plasma osmolality on baroreflex control of sympathetic activity

The purpose of this study was to determine if plasma osmolality alters baroreflex control of sympathetic activity when controlling for a change in intravascular volume; we hypothesized that baroreflex control of sympathetic activity would be greater during a hyperosmotic stimulus compared with an isoosmotic stimulus when intravascular volume expansion was matched. Seven healthy subjects (25 +/- 2 yr) completed two intravenous infusions: a hypertonic saline infusion (HSI; 3% NaCl) and, on a separate occasion, an isotonic saline infusion (ISO; 0.9% NaCl), both at a rate of 0.15 ml x kg(-1) x min(-1). To isolate the effect of osmolality, comparisons between HSI and ISO conditions were retrospectively matched based on hematocrit; therefore, baroreflex control of sympathetic outflow was determined at 20 min of a HSI and 40 min of an ISO. Muscle sympathetic outflow (MSNA) was directly measured using the technique of peroneal microneurography; osmolality and blood pressure (Finometer) were assessed. The baroreflex control of sympathetic outflow was estimated by calculating the slope of the relationship between MSNA and diastolic blood pressure during controlled breathing. Plasma osmolality was greater during the HSI compared with the ISO (HSI: 292 +/- 0.9 mosmol/kg and ISO: 289 +/- 0.8 mosmol/kg, P < 0.05). Hematocrits were matched (HSI: 39.1 +/- 1% and ISO: 39.1 +/- 1%, P > 0.40); thus, we were successful in isolating osmolality. The baroreflex control of sympathetic outflow was greater during the HSI compared with the ISO (HSI: -8.3 +/- 1.2 arbitrary units x beat(-1) x mmHg(-1) vs. ISO: -4.0 +/- 0.8 arbitrary units x beat(-1) x mmHg(-1), P = 0.01). In conclusion, when controlling for intravascular volume, increased plasma osmolality enhances baroreflex control of sympathetic activity in humans.     

Noradrenaline modulates transmission at a central synapse by a presynaptic mechanism

The lateral division of the central amygdala (CeAL) is the target of ascending fibers from the pain-responsive and stress-responsive nuclei in the brainstem. We show that single fiber inputs from the nociceptive pontine parabrachial nucleus onto CeAL neurons form suprathreshold glutamatergic synapses with multiple release sites. Noradrenaline, acting at presynaptic alpha2 receptors, potently inhibits this synapse. This inhibition results from a decrease in the number of active release sites with no change in release probability. Introduction of a presynaptic scavenger of Gbetagamma subunits blocked the effects of noradrenaline, and botulinum toxin A reduced its effects, showing a direct action of betagamma subunits on the release machinery. These data illustrate a mechanism of presynaptic modulation where the output of a large multiple-release-site synapse is potently regulated by endogenously released noradrenaline and suggests that the CeA may be a target for the central nociceptive actions of noradrenaline.     

Assessment of algorithms for high throughput detection of genomic copy number variation in oligonucleotide microarray data

Background

Genomic deletions and duplications are important in the pathogenesis of diseases, such as cancer and mental retardation, and have recently been shown to occur frequently in unaffected individuals as polymorphisms. Affymetrix GeneChip whole genome sampling analysis (WGSA) combined with 100 K single nucleotide polymorphism (SNP) genotyping arrays is one of several microarray-based approaches that are now being used to detect such structural genomic changes. The popularity of this technology and its associated open source data format have resulted in the development of an increasing number of software packages for the analysis of copy number changes using these SNP arrays.

Results

We evaluated four publicly available software packages for high throughput copy number analysis using synthetic and empirical 100 K SNP array data sets, the latter obtained from 107 mental retardation (MR) patients and their unaffected parents and siblings. We evaluated the software with regards to overall suitability for high-throughput 100 K SNP array data analysis, as well as effectiveness of normalization, scaling with various reference sets and feature extraction, as well as true and false positive rates of genomic copy number variant (CNV) detection.

Conclusion

We observed considerable variation among the numbers and types of candidate CNVs detected by different analysis approaches, and found that multiple programs were needed to find all real aberrations in our test set. The frequency of false positive deletions was substantial, but could be greatly reduced by using the SNP genotype information to confirm loss of heterozygosity.     

SKN1 and KRE6 define a pair of functional homologs encoding putative membrane proteins involved in beta-glucan synthesis.

KRE6 encodes a predicted type II membrane protein which, when disrupted, results in a slowly growing, killer toxin-resistant mutant possessing half the normal level of a structurally wild-type cell wall (1-->6)-beta-glucan (T. Roemer and H. Bussey, Proc. Natl. Acad. Sci. USA 88:11295-11299, 1991). The mutant phenotype and structure of the KRE6 gene product, Kre6p, suggest that it may be a beta-glucan synthase component, implying that (1-->6)-beta-glucan synthesis in Saccharomyces cerevisiae is functionally redundant. To examine this possibility, we screened a multicopy genomic library for suppression of both the slow-growth and killer resistance phenotypes of a kre6 mutant and identified SKN1, which encodes a protein sharing 66% overall identity to Kre6p. SKN1 suppresses kre6 null alleles in a dose-dependent manner, though disruption of the SKN1 locus has no effect on killer sensitivity, growth, or (1-->6)-beta-glucan levels. skn1 kre6 double disruptants, however, showed a dramatic reduction in both (1-->6)-beta-glucan levels and growth rate compared with either single disruptant. Moreover, the residual (1-->6)-beta-glucan polymer in skn1 kre6 double mutants is smaller in size and altered in structure. Since single disruptions of these genes lead to structurally wild-type (1-->6)-beta-glucan polymers, Kre6p and Skn1p appear to function independently, possibly in parallel, in (1-->6)-beta-glucan biosynthesis.