The MS Risk Allele of CD40 Is Associated with Reduced Cell-Membrane Bound Expression in Antigen Presenting Cells: Implications for Gene Function

Human genetic and animal studies have implicated the costimulatory molecule CD40 in the development of multiple sclerosis (MS). We investigated the cell specific gene and protein expression variation controlled by the CD40 genetic variant(s) associated with MS, i.e. the T-allele at rs1883832. Previously we had shown that the risk allele is expressed at a lower level in whole blood, especially in people with MS. Here, we have defined the immune cell subsets responsible for genotype and disease effects on CD40 expression at the mRNA and protein level. In cell subsets in which CD40 is most highly expressed, B lymphocytes and dendritic cells, the MS-associated risk variant is associated with reduced CD40 cell-surface protein expression. In monocytes and dendritic cells, the risk allele additionally reduces the ratio of expression of full-length versus truncated CD40 mRNA, the latter encoding secreted CD40. We additionally show that MS patients, regardless of genotype, express significantly lower levels of CD40 cell-surface protein compared to unaffected controls in B lymphocytes. Thus, both genotype-dependent and independent down-regulation of cell-surface CD40 is a feature of MS. Lower expression of a co-stimulator of T cell activation, CD40, is therefore associated with increased MS risk despite the same CD40 variant being associated with reduced risk of other inflammatory autoimmune diseases. Our results highlight the complexity and likely individuality of autoimmune pathogenesis, and could be consistent with antiviral and/or immunoregulatory functions of CD40 playing an important role in protection from MS.     

Reactions of lignin peroxidase compounds I and II with veratryl alcohol. Transient-state kinetic characterization

Stopped-flow techniques were utilized to investigate the kinetics of the reaction of lignin peroxidase compounds I and II (LiPI and LiPII) with veratryl alcohol (VA). All rate data were collected from single turnover experiments under pseudo first-order conditions. The reaction of LiPI with VA strictly obeys second-order kinetics over the pH range 2.72-5.25 as demonstrated by linear plots of the pseudo first-order rate constants versus concentrations of VA. The second-order rate constants are strongly dependent on pH and range from 2.62 x 10(6) M-1 s-1 (pH 2.72) to 1.45 x 10(4) M-1 s-1 (pH 5.25). The reaction of LiPII and VA exhibits saturation behavior when the observed pseudo first-order rate constants are plotted against VA concentrations. The saturation phenomenon is quantitatively explained by the formation of a 1:1 LiPII-substrate complex. Results of kinetic and rapid scan spectral analyses exclude the formation of a LiPII-VA cation radical complex. The first-order dissociation rate constant and the equilibrium dissociation constant for the LiPII reaction are also pH dependent. Binding of VA to LiPII is controlled by a heme-linked ionizable group of pKa approximately 4.2. The pH profiles of the second-order rate constants for the LiPI reaction and of the first-order dissociation constants for the LiPII reaction both demonstrate two pKa values at approximately 3.0 and approximately 4.2. Protonated oxidized enzyme intermediates are most active, suggesting that only electron transfer, not proton uptake from the reducing substrate, occurs at the enzyme active site. These results are consistent with the one-electron oxidation of VA to an aryl cation radical by LiPI and LiPII.     

Development of a somatic cell hybrid mapping panel and molecular probes for human chromosome 3

A somatic cell hybrid mapping panel and molecular probes have been developed for human chromosome 3. This panel defines 11 regions for the short and long arms of the chromosome. Four hundred thirty-two probes have been mapped using these hybrids. One hundred thirty-one of these probes were derived from EcoRI and HindIII flow-sorted libraries. The remaining 301 probes were isolated from NotI boundary and random (partial MboI) libraries constructed from a hybrid that provided a relative enrichment in 3p DNA sequences. For some regions of the chromosome, significant differences in the distribution of probes were noted. This was observed for both the unique sequence flow-sorted and NotI probes. These differences are in agreement with previous suggestions that Giemsa light bands are GC-rich, and therefore gene-rich (especially housekeeping genes), and that the Giemsa dark bands may contain DNA that is more highly condensed. The isolation of probes from different types of libraries, or by different screening strategies, appears to reduce deficiencies that might arise from the use of probes derived with a more limited approach. These hybrids and probes should facilitate the construction of physical and genetic linkage maps to identify various disease loci involving chromosome 3.     

Anion exchanger is present in both luminal and basolateral renal membranes

Binding of the anion-exchange inhibitor 3H2-labeled 4,4'-diisothiocyano-2,2'-stilbene disulfonic acid (DIDS) to highly purified luminal and basolateral beef kidney tubular membranes was characterized. Specific binding of [3H2]DIDS is present in both luminal and basolateral membranes. Scatchard analysis revealed a Kd for [3H2]DIDS of 5.5 microM and 19.3 microM and a maximal number of binding sites of 10.9 nmol and 31.7 nmol DIDS/mg protein in basolateral and luminal membranes, respectively. To assess the role of this putative anion exchanger on transport we measured 35SO4 uptake by luminal and basolateral membranes. In both luminal and basolateral membranes sulfate uptake was significantly greater in the presence of an outward-directed Cl gradient, OH gradient or HCO3 gradient than in the absence of these gradients. There was an early anion-dependent sulfate uptake of five to ten times the equilibrium uptake at 60 min. The sulfate taken in could be released by lysis of the vesicles indicating true uptake and not binding of sulfate. No significant difference in SO4 uptake was found in the presence and in the absence of valinomycin, indicating that the anion exchanger is electroneutral. The anion-dependent sulfate uptake was completely inhibited by either DIDS or furosemide in both luminal and basolateral membranes. Dixon analysis of HCO3-dependent SO4 uptake by luminal membranes in the presence of different concentrations of DIDS revealed a Ki for DIDS of 20 microM. The similar values of the Kd for [3H2]DIDS binding and the Ki for DIDS inhibition of SO4 uptake might suggest an association between DIDS binding and the inhibition of SO4 transport. In addition, an inward-directed Na gradient stimulated sulfate uptake in luminal but not in basolateral membranes. The Na-dependent sulfate uptake in luminal membranes was also inhibited by DIDS. We conclude that, in addition to the well-known Na-dependent sulfate uptake in luminal membranes, there exists an anion exchanger in both basolateral and luminal membranes capable of sulfate transport.     

Characterization and Molecular Genetic Complementation of Mutants Affecting Dimorphism in the FungusUstilago maydis

Ustilago maydis,the causal agent of corn smut disease, displays dimorphic growth in which it alternates between a unicellular, nonpathogenic yeast-like form and a dikaryotic, pathogenic filamentous form. Previously, a constitutively filamentous haploid mutant was obtained. Complementation of this mutant led to the isolation of the gene encoding adenylate cyclase,uac1.Secondary mutagenesis of auac1disruption strain allowed the isolation of a large number of suppressor mutants, termedubc,forUstilagobypass of cyclase, lacking the filamentous phenotype. Analysis of one of these suppressor mutants previously led to the identification of theubc1gene, encoding the regulatory subunit of cAMP-dependent protein kinase. In this report we describe the isolation of cosmids containing three newubcgenes, termedubc2, ubc3,andubc4.We also describe the morphology of theubc2, ubc3,andubc4mutants in auac1⁻background as well as in a background with a functionaluac1gene. In addition, we describe several mutant strains not complemented with any of the genes currently in hand and that are thus presumed to possess mutations in additionalubcgenes.     

USEtox human exposure and toxicity factors for comparative assessment of toxic emissions in life cycle analysis: sensitivity to key chemical properties

Purpose

The aim of this paper is to provide science-based consensus and guidance for health effects modelling in comparative assessments based on human exposure and toxicity. This aim is achieved by (a) describing the USEtox? exposure and toxicity models representing consensus and recommended modelling practice, (b) identifying key mechanisms influencing human exposure and toxicity effects of chemical emissions, (c) extending substance coverage.

Methods

The methods section of this paper contains a detailed documentation of both the human exposure and toxic effects models of USEtox?, to determine impacts on human health per kilogram substance emitted in different compartments. These are considered as scientific consensus and therefore recommended practice for comparative toxic impact assessment. The framework of the exposure model is described in details including the modelling of each exposure pathway considered (i.e. inhalation through air, ingestion through (a) drinking water, (b) agricultural produce, (c) meat and milk, and (d) fish). The calculation of human health effect factors for cancer and non-cancer effects via ingestion and inhalation exposure respectively is described. This section also includes discussions regarding parameterisation and estimation of input data needed, including route-to-route and acute-to-chronic extrapolations.

Results and discussion

For most chemicals in USEtox?, inhalation, above-ground agricultural produce, and fish are the important exposure pathways with key driving factors being the compartment and place of emission, partitioning, degradation, bioaccumulation and bioconcentration, and dietary habits of the population. For inhalation, the population density is the key factor driving the intake, thus the importance to differentiate emissions in urban areas, except for very persistent and mobile chemicals that are taken in by the global population independently from their place of emission. The analysis of carcinogenic potency (TD₅₀) when volatile chemicals are administrated to rats and mice by both inhalation and an oral route suggests that results by one route can reasonably be used to represent another route. However, we first identify and mark as interim chemicals for which observed tumours are directly related to a given exposure route (e.g. for nasal or lung, or gastrointestinal cancers) or for which absorbed fraction by inhalation and by oral route differ greatly.

Conclusions

A documentation of the human exposure and toxicity models of USEtox? is provided, and key factors driving the human health characterisation factor are identified. Approaches are proposed to derive human toxic effect factors and expand the number of chemicals in USEtox?, primarily by extrapolating from an oral route to exposure in air (and optionally acute-to-chronic). Some exposure pathways (e.g. indoor inhalation, pesticide residues, dermal exposure) will be included in a later stage. USEtox? is applicable in various comparative toxicity impact assessments and not limited to LCA.     

Vertebral Pneumaticity in the Ornithomimosaur Archaeornithomimus (Dinosauria: Theropoda) Revealed by Computed Tomography Imaging and Reappraisal of Axial Pneumaticity in Ornithomimosauria

Among extant vertebrates, pneumatization of postcranial bones is unique to birds, with few known exceptions in other groups. Through reduction in bone mass, this feature is thought to benefit flight capacity in modern birds, but its prevalence in non-avian dinosaurs of variable sizes has generated competing hypotheses on the initial adaptive significance of postcranial pneumaticity. To better understand the evolutionary history of postcranial pneumaticity, studies have surveyed its distribution among non-avian dinosaurs. Nevertheless, the degree of pneumaticity in the basal coelurosaurian group Ornithomimosauria remains poorly known, despite their potential to greatly enhance our understanding of the early evolution of pneumatic bones along the lineage leading to birds. Historically, the identification of postcranial pneumaticity in non-avian dinosaurs has been based on examination of external morphology, and few studies thus far have focused on the internal architecture of pneumatic structures inside the bones. Here, we describe the vertebral pneumaticity of the ornithomimosaur Archaeornithomimus with the aid of X-ray computed tomography (CT) imaging. Complementary examination of external and internal osteology reveals (1) highly pneumatized cervical vertebrae with an elaborate configuration of interconnected chambers within the neural arch and the centrum; (2) anterior dorsal vertebrae with pneumatic chambers inside the neural arch; (3) apneumatic sacral vertebrae; and (4) a subset of proximal caudal vertebrae with limited pneumatic invasion into the neural arch. Comparisons with other theropod dinosaurs suggest that ornithomimosaurs primitively exhibited a plesiomorphic theropod condition for axial pneumaticity that was extended among later taxa, such as Archaeornithomimus and large bodied Deinocheirus. This finding corroborates the notion that evolutionary increases in vertebral pneumaticity occurred in parallel among independent lineages of bird-line archosaurs. Beyond providing a comprehensive view of vertebral pneumaticity in a non-avian coelurosaur, this study demonstrates the utility and need of CT imaging for further clarifying the early evolutionary history of postcranial pneumaticity.     

Cortical and Subcortical Grey and White Matter Atrophy in Myotonic Dystrophies Type 1 and 2 Is Associated with Cognitive Impairment,Depression and Daytime Sleepiness

Objectives

Central nervous system involvement is one important clinical aspect of myotonic dystrophy type 1 and 2 (DM1 and DM2). We assessed CNS involvement DM1 and DM2 by 3T MRI and correlated clinical and neuocognitive symptoms with brain volumetry and voxel-based morphometry (VBM).

Methods

12 patients with juvenile or classical DM1 and 16 adult DM2 patients underwent 3T MRI, a thorough neurological and neuropsychological examination and scoring of depression and daytime sleepiness. Volumes of brain, ventricles, cerebellum, brainstem, cervical cord, lesion load and VBM results of the patient groups were compared to 33 matched healthy subjects.

Results

Clinical symptoms were depression (more pronounced in DM2), excessive daytime sleepiness (more pronounced in DM1), reduced attention and flexibility of thinking, and deficits of short-term memory and visuo-spatial abilities in both patient groups. Both groups showed ventricular enlargement and supratentorial GM and WM atrophy, with prevalence for more GM atrophy and involvement of the motor system in DM1 and more WM reduction and affection of limbic structures in DM2. White matter was reduced in DM1 in the splenium of the corpus callosum and in left-hemispheric WM adjacent to the pre- and post-central gyrus. In DM2, the bilateral cingulate gyrus and subgyral medio-frontal and primary somato-sensory WM was affected.Significant structural-functional correlations of morphological MRI findings (global volumetry and VBM) with clinical findings were found for reduced flexibility of thinking and atrophy of the left secondary visual cortex in DM1 and of distinct subcortical brain structures in DM2. In DM2, depression was associated with brainstem atrophy, Daytime sleepiness correlated with volume decrease in the middle cerebellar peduncles, pons/midbrain and the right medio-frontal cortex.

Conclusion

GM and WM atrophy was significant in DM1 and DM2. Specific functional-structural associations related morphological changes to cognitive impairment, depression and daytime sleepiness, partly indicating involvement of complex neuronal networks.