Structure of the Pseudorabies Virus Capsid: Comparison with Herpes Simplex Virus Type 1 and Differential Binding of Essential Minor Proteins

The structure of pseudorabies virus (PRV) capsids isolated from the nucleus of infected cells and from PRV virions was determined by cryo-electron microscopy (cryo-EM) and compared to herpes simplex virus type 1 (HSV-1) capsids. PRV capsid structures closely resemble those of HSV-1, including distribution of the capsid vertex specific component (CVSC) of HSV-1, which is a heterodimer of the pUL17 and pUL25 proteins. Occupancy of CVSC on all PRV capsids is near 100%, compared to ~ 50% reported for HSV-1 C-capsids and 25% or less that we measure for HSV-1 A- and B-capsids. A PRV mutant lacking pUL25 does not produce C-capsids and lacks visible CVSC density in the cryo-EM-based reconstruction. A reconstruction of PRV capsids in which green fluorescent protein was fused within the N-terminus of pUL25 confirmed previous studies with a similar HSV-1 capsid mutant localizing pUL25 to the CVSC density region that is distal to the penton. However, comparison of the CVSC density in a 9-Å-resolution PRV C-capsid map with the available crystal structure of HSV-1 pUL25 failed to find a satisfactory fit, suggesting either a different fold for PRV pUL25 or a capsid-bound conformation for pUL25 that does not match the X-ray model determined from protein crystallized in solution. The PRV capsid imaged within virions closely resembles C-capsids with the addition of weak but significant density shrouding the pentons that we attribute to tegument proteins. Our results demonstrate significant structure conservation between the PRV and HSV capsids.     

Aggregated α-synuclein and complex I deficiency: exploration of their relationship in differentiated neurons

α-Synuclein becomes misfolded and aggregated upon damage by various factors, for example, by reactive oxygen species. These aggregated forms have been proposed to have differential toxicities and their interaction with mitochondria may cause dysfunction within this organelle that contributes to the pathogenesis of Parkinson''s disease (PD). In particular, the association of α-synuclein with mitochondria occurs through interaction with mitochondrial complex I and importantly defects of this protein have been linked to the pathogenesis of PD. Therefore, we investigated the relationship between aggregated α-synuclein and mitochondrial dysfunction, and the consequences of this interaction on cell survival. To do this, we studied the effects of α-synuclein on cybrid cell lines harbouring mutations in either mitochondrial complex I or IV. We found that aggregated α-synuclein inhibited mitochondrial complex I in control and complex IV-deficient cells. However, when aggregated α-synuclein was applied to complex I-deficient cells, there was no additional inhibition of mitochondrial function or increase in cell death. This would suggest that as complex I-deficient cells have already adapted to their mitochondrial defect, the subsequent toxic effects of α-synuclein are reduced.The pathological hallmark of Parkinson''s disease (PD) is the presence of α-synuclein aggregates, particularly within the substantia nigra (SN). These aggregations take the form of intracellular Lewy bodies, and also neuritic aggregations. However, both the effect of these inclusions on neuronal survival and the toxicity of different forms of α-synuclein are still debated. To aggregate α-synuclein must undergo a conformational change, however, the mechanism behind this change and subsequent aggregation in PD remains to be determined.Mutations within the α-synuclein gene (SNCA (MIM 163890)) were the first to be associated with autosomal dominant PD, while more recently genome-wide association studies have suggested that single-nucleotide polymorphisms in this gene are important for sporadic PD. A widely expressed protein α-synuclein is important for synaptic vesicle recycling and the modulation of dopamine transmission within SN neurons.^{1, 2, 3, 4, 5, 6, 7, 8} It interacts with curved cellular membranes including those of mitochondria suggesting a possible mode of its toxicity,^{9, 10, 11} and can be imported into mitochondria in an energy-dependent manner.⁹ The accumulation of α-synuclein within mitochondria leads to complex I impairment, decreased mitochondrial membrane potential (ΔΨ_m) and increased reactive oxygen species (ROS) production. The occurrence of these changes is also dependent on calcium homoeostasis.^{9, 12, 13}Mitochondrial dysfunction has also been heavily implicated in the pathogenesis of PD. Early studies showed a decrease in mitochondrial complex I in the SN of PD patients and studies involving the inhibition of this complex replicate many of the features of this disease. In addition, SN neurons show high levels of mitochondrial DNA deletions in old age,^{14, 15} which lead to respiratory deficiency, and the environment of the SN is believed to be particularly oxidative due to a number of processes, including the metabolism of dopamine. More recently a number of genes known to cause autosomal recessive forms of PD have been shown to encode proteins with functions associated with mitochondrial turnover (Parkin/Pink1 (MIM 602544, MIM 608309)) or oxidative stress (DJ-1 (MIM 602533)). However, the link between these two processes and the loss of dopaminergic neurons in PD remains to be elucidated.Several hypotheses have been suggested for what might cause α-synuclein to undergo the conformational change into more aggregate prone forms, from oxidative stress to gene mutations. Furthermore, the accumulation of mitochondrial DNA (mtDNA) mutations and dysfunctional mitochondria with advancing age are likely to have an effect on oxidative stress levels within the SN, which might contribute further to the misfolding and accumulation of this protein. Numerous studies have used rotenone and other toxins to induce mitochondrial dysfunction and monitor the accumulation of α-synuclein, despite the wealth of information that these studies provide they often do not reflect the subtleties of the slow accumulation of mitochondrial dysfunction within ageing SN neurons.Therefore, we investigated the relationship between mitochondria and aggregated α-synuclein, focussing on how these forms affect neurons with and without mitochondrial dysfunction. We wanted to understand how aggregated α-synuclein impacted on the survival of cells with mitochondrial dysfunction, to enable a deeper understanding of the effect of these two processes on neuronal survival. To investigate this we used cells with mutations in and partial inhibition of complexes I and IV.     

Microbial community analysis of rectal methanogens and sulfate reducing bacteria in two non-human primate species

Background  Methanogenesis by methanogenic Archaea and sulfate reduction by sulfate reducing bacteria (SRB) are the major hydrogenotrophic pathways in the human colon. Methanogenic status of mammals is suggested to be under evolutionary rather than dietary control. However, information is lacking regarding the dynamics of hydrogenotrophic microbial communities among different primate species.
Methods  Rectal swabs were collected from 10 sooty mangabeys ( Cercocebus atys ) and 10 baboons ( Papio hamadryas ). The diversity and abundance of methanogens and SRB were examined using PCR-denaturing gradient gel electrophoresis (DGGE) and real-time quantitative PCR (qPCR).
Results  The DGGE results revealed that intestinal Archaea and SRB communities differ between mangabeys and baboons. Phylogenetic analyses of Archaea DGGE bands revealed two distinct clusters with one representing a putative novel order of methanogenic Archaea. The qPCR detected a similar abundance of methanogens and SRB.
Conclusions  Intestinal Archaea and SRB coexist in these primates, and the community patterns are host species-specific.     

Uremia causes premature ageing of the T cell compartment in end-stage renal disease patients

ABSTRACT: BACKGROUND: End-stage renal disease (ESRD) patients treated with renal replacement therapy (RRT) have premature immunologically aged T cells which may underlie uremia-associated immune dysfunction. The aim of this study was to investigate whether uremia was able to induce premature ageing of the T cell compartment. For this purpose, we examined the degree of premature immunological T cell ageing by examining the T cell differentiation status, thymic output via T cell receptor excision circle (TREC) content and proliferative history via relative telomere length in ESRD patients not on RRT. RESULTS: Compared to healthy controls, these patients already had a lower TREC content and an increased T cell differentiation accompanied by shorter telomeres. RRT was able to enhance CD8+ T cell differentiation and to reduce CD8+ T cell telomere length in young dialysis patients. An increased differentiation status of memory CD4+ T cells was also noted in young dialysis patients. CONCLUSION: Based on these results we can conclude that uremia already causes premature immunological ageing of the T cell system and RRT further increases immunological ageing of the CD8+ T cell compartment in particular in young ESRD patients.     

Gene regulatory divergence among species estimated by altered developmental patterns in interspecific hybrids

Disturbances in the schedules of gene expression in developing interspecific fish hybrids have been used to draw inferences about the extent of gene regulatory divergence between species and about the degree to which this gene regulatory divergence is correlated with structural gene divergence, as estimated by genetic distance. Sperm from each of 10 different species representing six genera within the family Centrarchidae was used to fertilize eggs of the Florida largemouth bass (Micropterus salmoides floridanus). The genetic distances (D; Nei 1978) between the parental species used to form the hybrids ranged from 0.133 to 0.974. The developmental success and temporal patterns of gene expression of each of the hybrids were compared with those of the Florida largemouth bass. As the genetic distance between the paternal species and the Florida largemouth bass increased, there was a general decline in developmental success in the hybrid embryos as demonstrated by the observed reductions in the percentage of hatching and by progressively earlier and more extensive morphological abnormalities. Concomitantly, progressively more marked alterations in developmental schedules of expression of 15 enzyme loci occurred in the hybrids as the genetic distance between parental species increased. However, observed deviations from this trend for a few species may represent an uncoupling of the rates and modes of evolution of structural genes from those for genes regulating developmental processes.      

The Effect of Magnesium and Calcium Ions on Adenosine Triphosphatases from Wheat and Oat Roots at Different pH

Microsomal fractions from wheat (Triticum vulgare) and oat (Avena sativa) roots were used to study Mg²⁺ and Ca²⁺ activated adenosine triphosphatases, their dependence of pH, and how Mg²⁺ and Ca²⁺ compete or add in stimulation and inhibition. Wheat gives a high proportion of Ca²⁺ stimulated ATPase. Less effect is obtained with Mg²⁺. The characteristics of oar ATPase are the reverse. The ATPase from the wheat roots depends on the mineral nutrition. A kinetïc analysis shows one site, where Mg²⁺ and Ca²⁺ at low concentrations (or complexes between the di-valents and ATP) cooperate in the activation of the ATPase. The action of this site is more dearly expressed at pH 6.0 than at 6.8, and more clearly in the preparations from low salt roots than in those from high salt conditions. In another site, which is particularly evident in preparations from high salt roots tested at pH 6.8, high concentrations of Mg²⁺ inhibit the ATPase; this inhibition is competitively relieved by Ca²⁺. The specific activity of the ATPase from high salt roots of wheat is higher than that from low salt roots, although the amount of protein of the fraction studied remains the same, when calculated per g fresh weight of the roots.     

Poor physical fitness is independently associated with mild cognitive impairment in elderly Koreans

The purpose of this study was to investigate the association between physical fitness and mild cognitive impairment (MCI) in elderly Koreans. This was a cross-sectional study that involved 134 men and 299 women aged 65 to 88 years. Six senior fitness tests were used as independent variables: 30 s chair stand for lower body strength, arm curl for upper body strength, chair-sit-and-reach for lower body flexibility, back scratch for upper body flexibility, 8-ft up-and-go for agility/dynamic balance, and 2-min walk for aerobic endurance. Global cognitive function was assessed using the Korean version of the Mini-Mental State Examination (MMSE). Potential covariates such as age, education levels, blood lipids, and insulin resistance (IR) markers were also assessed. Compared to individuals without MMSE-based MCI, individuals with MMSE-based MCI had poor physical fitness based on the senior fitness test (SFT). There were significant positive trends observed for education level (p=0.001) and MMSE score (p<0.001) across incremental levels of physical fitness in this study population. Individuals with moderate (OR=0.341, p=0.006) and high (OR=0.271, p=0.007) physical fitness based on a composite score of the SFT measures were less likely to have MMSE-based MCI than individuals with low physical fitness (referent, OR=1). The strength of the association between moderate (OR=0.377, p=0.038) or high (OR=0.282, p=0.050) physical fitness and MMSE-based MCI was somewhat attenuated but remained statistically significant even after adjustment for the measured compounding factors. We found that poor physical fitness was independently associated with MMSE-based MCI in elderly Koreans.