Development of an innervated model of human skin

Neuronal cell responses and interactions with the epithelial and fibroblastic cells of the skin are a key factor in the production in vivo of the irritation/inflammatory response. Currently, few in vitro models are available that contain dermal, epidermal and the relevant neuronal components. The primary objective of this study was to produce and maintain a 3-D in vitro model of human skin containing these elements. The relevant neuronal component was supplied by adding sensory neurons derived from the dorsal root ganglion (DRG). Since adult neuronal cells do not grow significantly in vivo or in vitro, and since it is very difficult to obtain such cells from humans, it was necessary to employ embryonic rat DRG cells. The ultimate purpose of this model is to improve prediction of the in vivo skin irritancy potential of chemicals and formulations, without the need to use animal models. In addition, this approach has also been applied to the in vitro human eye and bronchial 3-D models being developed in the FRAME Alternatives Laboratory.     

Discovery of a new class of glucosylceramide synthase inhibitors

A novel series of potent inhibitors of glucosylceramide synthase are described. The optimization of biochemical and cellular potency as well as ADME properties led to compound 23c. Broad tissue distribution was obtained following oral administration to mice. Thus 23c could be another useful tool compound for studying the effects of GCS inhibition in vitro and in vivo.     

The development of an innervated epithelial barrier model using a human corneal cell line and ND7/23 sensory neurons

The corneal epithelium is a highly innervated tissue and hence in vitro models that mimic the effects of chemicals or radiation (e.g. ultra violet) on this important barrier should include consideration of the potential role of innervation. A sensory neural cell line, ND7/23, was incorporated into a 2D and 3D model of a corneal epithelium, using a human corneal cell line, and effects on barrier integrity were neither adverse nor stimulatory. In the 3D model the nerve cell bodies were separated from the corneal epithelium, via a porous polycarbonate insert membrane. The ND7/23 cells were induced to form neurites and cease division when cultured in the keratinocyte medium employed for the corneal cells. In the absence of calcium, the epithelial barrier function was lost, shown by enhanced fluorescein leakage and relocation of ZO-1 and E-cadherin from the cell membrane. At 60 microM calcium, and above, the corneal cells formed tight junctions, with peripheral membrane location of ZO-1 and E-cadherin. The presence of the ND7/23 cells did not compromise or enhance the time taken to form these junctions, when monitored at 24-h intervals over 72 h. Both male- and female-derived human corneal cell lines showed a similar tight junction functional response to different medium calcium concentrations in the presence or absence of the ND7/23 cells. Once differentiated in keratinocyte medium, patch-clamped ND7/23 cells were capable of producing a whole-cell current when exposed to low pH (5.4), indicative of the presence of active pH-gated ion channels.     

Evidence for allosteric regulation of pH-sensitive System A (SNAT2) and System N (SNAT5) amino acid transporter activity involving a conserved histidine residue

System A and N amino acid transporters are key effectors of movement of amino acids across the plasma membrane of mammalian cells. These Na+-dependent transporters of the SLC38 gene family are highly sensitive to changes in pH within the physiological range, with transport markedly depressed at pH 7.0. We have investigated the possible role of histidine residues in the transporter proteins in determining this pH-sensitivity. The histidine-modifying agent DEPC (diethyl pyrocarbonate) markedly reduces the pH-sensitivity of SNAT2 and SNAT5 transporters (representative isoforms of System A and N respectively, overexpressed in Xenopus oocytes) in a concentration-dependent manner but does not completely inactivate transport activity. These effects of DEPC were reversed by hydroxylamine and partially blocked in the presence of excess amino acid substrate. DEPC treatment also blocked a reduction in apparent affinity for Na+ (K0.5Na+) of the SNAT2 transporter at low external pH. Mutation of the highly conserved C-terminal histidine residue to alanine in either SNAT2 (H504A) or SNAT5 (H471A) produced a transport phenotype exhibiting reduced, DEPC-resistant pH-sensitivity with no change in K0.5Na+ at low external pH. We suggest that the pH-sensitivity of these structurally related transporters results at least partly from a common allosteric mechanism influencing Na+ binding, which involves an H+-modifier site associated with C-terminal histidine residues.     

Novel IL10 gene family associations with systemic juvenile idiopathic arthritis

Juvenile idiopathic arthritis (JIA) is the most common cause of chronic childhood disability and encompasses a number of disease subgroups. In this study we have focused on systemic JIA (sJIA), which accounts for approximately 11% of UK JIA cases. This study reports the investigation of three members of the IL10 gene family as candidate susceptibility loci in children with sJIA. DNA from 473 unaffected controls and 172 patients with sJIA was genotyped for a single nucleotide polymorphism (SNP) in IL19 and IL20 and two SNPs in IL10. We examined evidence for association of the four SNPs by single marker and haplotype analysis. Significant differences in allele frequency were observed between cases and controls, for both IL10-1082 (p = 0.031) and IL20-468 (p = 0.028). Furthermore, examination of the haplotypes of IL10-1082 and IL20-468 revealed greater evidence for association (global p = 0.0006). This study demonstrates a significant increased prevalence of the low expressing IL10-1082 genotype in patients with sJIA. In addition, we show a separate association with an IL20 polymorphism, and the IL10-1082A/IL20-468T haplotype. The two marker 'A-T' haplotype confers an odds ratio of 2.24 for sJIA. This positive association suggests an important role for these cytokines in sJIA pathogenesis.     

Five novel locations of Neocentromeres in human: 18q22.1, Xq27.1∼27.2, Acro p13, Acro p12, and heterochromatin of unknown origin

Since the first report in 1993, an ectopic centromere, i.e. neocentromere formation, has been reported in more than 100 small supernumerary marker chromosomes (sSMC), in 7 instances of centromere repositioning, and in about a dozen cases with more complex chromosomal rearrangements. Here we report 2 new cases with centromere repositioning and 3 neocentric sSMC consisting exclusively of heterochromatic material. Yet, no centromere formation was reported for the regions 18q22.1 and Xq27.1～27.2 as it was observed in the 2 cases with centromere repositioning here; in both cases, cytogenetically an inversion was suggested. Two of the 3 neocentric sSMC were derived from a short arm of an acrocentric chromosome. The remainder neocentric sSMC case was previously reported and was stainable only by material derived from itself.     

Bacterial Diversity of the Broadbalk ‘Classical’ Winter Wheat Experiment in Relation to Long-Term Fertilizer Inputs

With more than 160 years of contrasting fertilizer regimes, the Broadbalk winter wheat experiment represents a unique experimental resource for studying the effects of long-term fertilizer application on microbial population diversity. Using DGGE and clone library analysis, we report here on eubacterial species diversity (16S rRNA gene) and diversity within two sets of gene products associated with microbial N acquisition: NifH (nitrogen fixation) and AmtB (ammonium transport). Comparisons were made within and between soils treated with mineral N fertilizer, farmyard manure or receiving no fertilizer. Analysis of 16S rRNA gene DGGE profiles showed no clear patterns to qualitatively distinguish bacterial community structure between the three different treatments (P > 0.05), with all samples containing a range of eubacterial taxa similar to those that are characteristic of soil bacteria reported elsewhere. Intra-plot heterogeneity was high and of a similar magnitude to that between treatments. This lack of qualitative between plot differences was echoed in the representative sequences of 16S rRNA, nifH, and amtB genes in the various samples. Taken together, both phylogenetic and functional gene analyses showed bacterial communities in the Broadbalk-trial soil were very stable and relatively non-responsive to long-term management of balanced fertilizer inputs.