1H and15N resonance assignments and secondary structure of the human thioredoxin C62A,C69A,C73A mutant

Summary The complete assignment of¹H and¹⁵N backbone resonances and near-complete¹H side-chain resonance assignments have been obtained for the reduced form of a mutant of human thioredoxin (105 residues) in which the three non-active site cysteines have been substituted by alanines: C62A, C69A, C73A. The assignments were made primarily on the basis of three-dimensional.¹⁵N-separated nuclear Overhauser and Hartmann-Hahn spectroscopy, in conjunction with two-dimensional homonuclear and heteronuclear correlation experiments. Based on comparisons of short-range and interstrand nuclear Overhauser effects, patterns of amide exchange, and chemical-shift differences, the structure appears essentially unchanged from that of the previously determined solution structure of the native protein [Forman-Kay. J.D. et al. (1991)Biochemistry, 30, 2685–2698). An assay for thioredoxin shows that the C62A, C69A, C73A mutant retains activity. The assignment of the spectrum for this mutant of human thioredoxin constitutes the basis for future studies aimed at comparing the details of the active-site conformation in the reduced and oxidized forms of the protein.     

A pivotal heme-transfer reaction intermediate in cytochrome c biogenesis

c-Type cytochromes are widespread proteins, fundamental for respiration or photosynthesis in most cells. They contain heme covalently bound to protein in a highly conserved, highly stereospecific post-translational modification. In many bacteria, mitochondria, and archaea this heme attachment is catalyzed by the cytochrome c maturation (Ccm) proteins. Here we identify and characterize a covalent, ternary complex between the heme chaperone CcmE, heme, and cytochrome c. Formation of the complex from holo-CcmE occurs in vivo and in vitro and involves the specific heme-binding residues of both CcmE and apocytochrome c. The enhancement and attenuation of the amounts of this complex correlates completely with known consequences of mutations in genes for other Ccm proteins. We propose the complex is a trapped catalytic intermediate in the cytochrome c biogenesis process, at the point of heme transfer from CcmE to the cytochrome, the key step in the maturation pathway.     

A novel transmembrane MSP-containing protein that plays a role in right ventricle development

We have identified and characterized a gene, Mospd3 on mouse chromosome 5 using gene trapping in ES cells. MOSPD3 is part of a family of proteins, including MOSPD1, which is defined by the presence of a major sperm protein (MSP) domain and two transmembrane domains. Interestingly Mospd3 is mammalian specific and highly conserved between mouse and man. Insertion of the gene trap vector at the Mospd3 locus is mutagenic and breeding to homozygosity results in a characteristic right ventricle defect and neonatal lethality in 50% of mice. The phenotypic defect is dependent on the genetic background, indicating the presence of genetic modifier loci. We speculate that the further characterization of Mospd3 will shed light on the complex genetic interactions involved in cardiac development and disease.     

Comparative genetic mapping reveals synteny and collinearity between the American cranberry and diploid blueberry genomes

Cranberry and blueberry are closely related and recently domesticated fruit crops in the genus Vaccinium. Both have a presumed American origin and likely evolved from a common ancestor; however, details of their adaptive radiation and the extent of their genomic divergence remains little understood. To better understand their evolutionary and genomic relationships, a set of 323 cross-transferable simple sequence repeat (SSR) markers were identified, added to existing marker datasets, and used to construct linkage maps for cranberry (582 SSRs) and an interspecific diploid blueberry population (V. darrowii x V. corymbosum) x V. corymbosum (409 markers, densest blueberry SSR map currently available). The maps allowed for the first comparative genetic mapping study in Vaccinium, and revealed a surprisingly high degree of macro-synteny and collinearity between the cranberry and blueberry genomes. Approximately 93% of the blueberry linkage map was collinear with cranberry, while the remaining 7% (66.3 cM) was spread across 15 non-collinear regions detected in eight of the 12 linkage groups. These observations suggest that large-scale genome differentiation between the cranberry and blueberry genomes has not occurred during their evolution, and that sequence information will be highly transferable between the species in future genetic research and breeding. Finally, the set of 323 cross-transferable SSRs and linkage maps they were used to construct can serve as a shared resource for the Vaccinium research community, enabling additional comparative mapping studies, the identification and transfer of quantitative trait loci and candidate genes between species, and future exploration of evolutionary relationships in Vaccinium.     

A Microfluidic Platform for Precision Small-volume Sample Processing and Its Use to Size Separate Biological Particles with an Acoustic Microdevice

A major advantage of microfluidic devices is the ability to manipulate small sample volumes, thus reducing reagent waste and preserving precious sample. However, to achieve robust sample manipulation it is necessary to address device integration with the macroscale environment. To realize repeatable, sensitive particle separation with microfluidic devices, this protocol presents a complete automated and integrated microfluidic platform that enables precise processing of 0.15–1.5 ml samples using microfluidic devices. Important aspects of this system include modular device layout and robust fixtures resulting in reliable and flexible world to chip connections, and fully-automated fluid handling which accomplishes closed-loop sample collection, system cleaning and priming steps to ensure repeatable operation. Different microfluidic devices can be used interchangeably with this architecture. Here we incorporate an acoustofluidic device, detail its characterization, performance optimization, and demonstrate its use for size-separation of biological samples. By using real-time feedback during separation experiments, sample collection is optimized to conserve and concentrate sample. Although requiring the integration of multiple pieces of equipment, advantages of this architecture include the ability to process unknown samples with no additional system optimization, ease of device replacement, and precise, robust sample processing.     

Selenium accumulation in durum wheat and spring canola as a function of amending soils with selenite, selenate and or sulphate

Aims

A comparison was performed between plant species to determine if extractable, rather than total soil Se, is more effective at predicting plant Se accumulation over a full growing season.

Methods

Durum wheat (Triticum turgidum L.) and spring canola (Brassica napus L.) were sown in potted soil amended with 0, 0.1, 1.0, or 5.0 mg kg^?1 Se as SeO₄ ^2? or SeO₃ ^2?. In addition, SeO₄ ^2?-amended soils were amended with 0 or 50 mg kg^?1 S as SO₄ ^2?. Soils were analyzed for extractable and total concentration of Se ([Se]). Twice during the growing season plants were harvested and tissue [Se] was determined.

Results

Plants exposed to SeO₃ ^2? accumulated the least Se. Fitted predictive models for whole plant accumulation based on extractable soil [Se] were similar to models based on total [Se] in soil (R²?=?0.73 or 0.74, respectively) and selenium speciation and soil [S] were important soil parameters to consider. As well, soil S amendments limited Se toxicity.

Conclusions

Soil quality guidelines (SQGs) based on extractable Se should be considered for risk assessment, particularly when Se speciation is unknown. Predictive models to estimate plant Se uptake should include soil S, a modifier of Se accumulation.     

Brain Cytochrome Oxidase in Alzheimer''s Disease

A recent demonstration of markedly reduced (-50%) activity of cytochrome oxidase (CO; complex 4), the terminal enzyme of the mitochondrial enzyme transport chain, in platelets of patients with Alzheimer's disease (AD) suggested the possibility of a systemic and etiologically fundamental CO defect in AD. To determine whether a CO deficiency occurs in AD brain, we measured the activity of CO in homogenates of autopsied brain regions of 19 patients with AD and 30 controls matched with respect to age, postmortem time, sex, and, as indices of agonal status, brain pH and lactic acid concentration. Mean CO activity in AD brain was reduced in frontal (-26%: p less than 0.01), temporal (-17%; p less than 0.05), and parietal (-16%; not significant, p = 0.055) cortices. In occipital cortex and putamen, mean CO levels were normal, whereas in hippocampus, CO activity, on average, was nonsignificantly elevated (20%). The reduction of CO activity, which is tightly coupled to neuronal metabolic activity, could be explained by hypofunction of neurons, neuronal or mitochondrial loss, or possibly by a more primary, but region-specific, defect in the enzyme itself. The absence of a CO activity reduction in all of the examined brain areas does not support the notion of a generalized brain CO abnormality. Although the functional significance of a 16-26% cerebral cortical CO deficit in human brain is not known, a deficiency of this key energy-metabolizing enzyme could reduce energy stores and thereby contribute to the brain dysfunction and neurodegenerative processes in AD.