Doublecortin is a microtubule-associated protein and is expressed widely by migrating neurons.

Doublecortin (DCX) is required for normal migration of neurons into the cerebral cortex, since mutations in the human gene cause a disruption of cortical neuronal migration. To date, little is known about the distribution of DCX protein or its function. Here, we demonstrate that DCX is expressed in migrating neurons throughout the central and peripheral nervous system during embryonic and postnatal development. DCX protein localization overlaps with microtubules in cultured primary cortical neurons, and this overlapping expression is disrupted by microtubule depolymerization. DCX coassembles with brain microtubules, and recombinant DCX stimulates the polymerization of purified tubulin. Finally, overexpression of DCX in heterologous cells leads to a dramatic microtubule phenotype that is resistant to depolymerization. Therefore, DCX likely directs neuronal migration by regulating the organization and stability of microtubules.     

Synthesis and evaluation of the NSCLC anti-cancer activity and physical properties of 4-aryl-N-phenylpyrimidin-2-amines

Reported herein are efforts to profile 4-aryl-N-phenylpyrimidin-2-amines in terms of their anti-cancer activity towards non small-cell lung carcinoma (NSCLC) cells. We have synthesized new 4-aryl-N-phenylpyrimidin-2-amines and assessed them in terms of their cytotoxicity (A549, NCI-H187, MCF7, Vero & KB) and physicochemical properties (logD_7.4 and solubility). 13f and 13c demonstrated potent anti-cancer activity in A549 cells (0.2 µM), compared to 0.4 μM for the NSCLC drug Doxorubicin. 13f also displayed low experimental logD_7.4 (2.9) and the best solubility (～40 μM). Compounds 13b and 13d showed the best balance of A549 anti-cancer activity and selectivity. 13g showed good activity and selectivity comparable with the anti-cancer drug Doxorubicin.     

Transgenic Mouse Line with Green-fluorescent Protein-labeled Centrin 2 allows Visualization of the Centrosome in Living Cells

The centrosome plays diverse roles throughout the cellular mitotic cycle and in post-mitotic cells. Analysis of centrosome position and dynamics in living murine cells has been limited due to a lack of adequate reporters and currently requires either cell fixation/immunostaining or transfection with centrosome reporters. Here we describe the generation and characterization of a transgenic mouse line that constitutively expresses green fluorescent protein-labeled Centrin-2 (GFP-CETN2). The phenotype of the mouse is indistinguishable from wild-type and it displays a single pair of fluorescent centrioles in cells of every organ and time point examined. This model will be helpful for visualizing the centrosome in multiple experimental conditions.     

Growth factors associated with gastric mucosal hypertrophy in autoimmune gastritis

A prominent pathological feature of murine autoimmune gastritis is a pronounced mucosal hypertrophy. Here, we examined factors that may be responsible for inducing this hypertrophy. Because gastrin is known to be both an inducer of gastric mucosal cell proliferation and is elevated in autoimmune gastritis, mice deficient in gastrin were thymectomised at day 3 and assessed for autoimmune gastritis. Gastrin-deficient mice showed all the characteristic features of murine autoimmune gastritis, including gastric unit hypertrophy due to hyperproliferation and accumulation of immature epithelial cells, decreases in the number of zymogenic and parietal cells, and autoantibodies to the gastric H+/K+-ATPase. Hence, gastrin is not required for either the establishment of chronic gastritis or development of the typical pathological features of this disease. We also examined mRNA levels of a number of gastric mucosal growth factors in RNA samples from mice with hypertrophic autoimmune gastritis. Members of the Reg family, RegIIIbeta and RegIIIgamma, were greatly elevated in mice with hypertrophic gastritis, whereas RegI and amphiregulin (an EGF receptor ligand) were more modestly and/or inconsistently induced. These data demonstrate that induction of gastric mitogenic factors, such as members of the Reg family, can be achieved in inflammatory situations by gastrin-independent pathways. Members of the Reg family, in particular RegIIIbeta and RegIIIgamma, are good candidates to be involved in inducing the mucosal hyperproliferation in autoimmune gastritis. These findings are likely to be of relevance to other gastric inflammatory conditions.     

Interleukin-10 and sudden infant death syndrome

Uncontrolled pro-inflammatory responses to infections or bacterial toxins have been suggested to play a role in triggering the physiological events leading to sudden infant death syndrome (SIDS). We tested the hypothesis that these uncontrolled responses might be due to interactions between the gene polymorphisms inducing low levels of IL-10 and exposure to cigarette smoke. In vitro, the IL-10 (G-1082A) polymorphism was associated with low IL-10 levels and the -1082G allele was associated with high levels. The first objective was to assess the distribution of this polymorphism among SIDS infants, parents of SIDS infants and controls, and two ethnic groups: Aboriginal Australians who have a high incidence of SIDS; and Bangladeshis who in Britain have a low incidence of SIDS compared with Europeans. The second objective was to assess effects of human recombinant IL-10 on interleukin-6 (IL-6) and tumour necrosis factor-alpha (TNF-alpha) responses of human leukocytes to staphylococcal toxins implicated in SIDS. The third objective was to assess IL-10 responses to endotoxin and toxic shock syndrome toxin (TSST) from leukocytes of smokers and non-smokers in relation to the IL-10 (G-1082A) polymorphism. There were major differences in the distributions of these polymorphisms between Europeans and Bangladeshis (p=0.00) and between Europeans and Aboriginal Australians (p=0.00); however, they were similar for the Bangladeshi and Aboriginal Australian subjects. There were no significant differences in the distribution of these polymorphisms among SIDS infants or parents of SIDS infants compared to control groups. IL-10 significantly reduced IL-6 and TNF-alpha responses to TSST and staphylococcal enterotoxins A and C. At 50 ng ml(-1), IL-10 significantly increased TNF-alpha but not IL-6 responses to TSST and enterotoxin A. Although IL-10 responses to endotoxin were lower from leukocytes of smokers who were homozygous for the G allele, the differences were not significant; however, significantly lower IL-10 responses were found for smokers who were homozygous for the A allele (p=0.01) and heterozygotes (p=0.04). The pooled data found smokers had significantly lower levels of IL-10 responses to TSST, but there were no significant differences for smokers compared with non-smokers for the three genotypes. The high incidence of SIDS and serious respiratory infections among Aboriginal Australian infants and the low incidence of these conditions among Bangladeshi infants might be explained in part by our findings of differences in IL-10 responses between smokers and non-smokers. The lowest levels of IL-10 responses were observed among smokers who were homozygous for the A allele which is most prevalent among the Aboriginal Australians (83%) and Bangladeshis (84%). The major difference between the risk factors for SIDS in these two groups is the level of exposure of infants to cigarette smoke associated with maternal smoking.     

Interleukin 1-beta responses to bacterial toxins and sudden infant death syndrome

We tested the hypothesis that significantly higher IL-1beta responses to toxic shock syndrome toxin (TSST) noted for parents of sudden infant death syndrome (SIDS) infants might be due in part to genetic factors such as the IL-1beta (C-511T) and IL-1RN (T+2018C) single nucleotide polymorphisms (SNP). The first objective was to assess the distribution of these polymorphisms among SIDS infants, parents of SIDS infants and controls, and two ethnic groups: Aboriginal Australians who have a high incidence of SIDS; and Bangladeshis who in Britain have a low incidence of SIDS compared with Europeans. The second objective was to assess IL-1beta responses to endotoxin and toxic shock syndrome toxin (TSST) from leukocytes of smokers and non-smokers in relation to these polymorphisms. There were major differences in the distributions of the IL-1beta (C-511T) SNP between Europeans and Bangladeshis (p=0.00) and between Europeans and Aboriginal Australians (p=0.00); however, they were similar for the Bangladeshi and Aboriginal Australian subjects. The allele frequency distribution of the IL-1RN (T+2018C) SNP for the Aboriginal Australians was statistically different from the European group (p=0.00), but it was not different from the Bangladeshi group (p=0.09). Compared with controls of European origin, there were no significant differences in the distribution of these polymorphisms among SIDS infants or parents of SIDS infants. For the IL-1beta (C-511T) SNP, the highest IL-1beta responses to endotoxin were obtained with leukocytes of non-smokers with the heterozygous CT genotype. Smokers had significantly lower levels of IL-1beta in response to endotoxin (p=0.01) and these differences were significant for donors with the wild type CC (p=0.00) and CT (p=0.03) genotypes. Similar patterns were observed for IL-1beta responses to TSST, but the differences were not significant. For the IL-1RN (T+2018C) SNP, the highest IL-1beta responses to endotoxin were obtained with leukocytes from non-smoker donors with the wildtype TT genotype and significantly lower responses were found with leukocytes from donors with the TC genotype (p=0.02). The responses of smokers were lower but the differences were significant only for donors with the TT genotype (p=0.00). Similar patterns were observed for IL-1beta responses to TSST, but the differences were not significant. IL-1beta responses to both endotoxin and TSST were increased for the small number of smokers with the TT genotype of the IL-1beta (C-511T) SNP. The TT genotype of the IL-1beta (C-511T) was found predominantly among Aboriginal Australian and Bangladeshi individuals but only a small proportion of Europeans. Smokers with the AA genotype of the IL-10 (G-1082A) SNP which is found predominantly among these two groups had significantly lower levels of IL-10 responses. If cigarette smoke enhances pro-inflammatory responses and reduces anti-inflammatory responses in individuals with these genotypes, this might partly explain the increased susceptibility of Aboriginal Australian infants to infections and SIDS.     

Promiscuous thymic expression of an autoantigen gene does not result in negative selection of pathogenic T cells

"Promiscuous" thymic expression of peripheral autoantigens can contribute to immunological tolerance in some cases. However, in this study we show that thymic mRNA expression alone cannot predict a contribution to thymic tolerance. Autoimmune gastritis is caused by CD4+ T cells directed to the alpha (H/Kalpha) and beta (H/Kbeta) subunits of the gastric membrane protein the H+/K+ ATPase. H/Kalpha mRNA is expressed in the thymus, but H/Kbeta expression is barely detectable. In this study, we demonstrate that thymic H/Kalpha in wild-type mice or mice that overexpressed H/Kalpha did not result in negative selection of pathogenic anti-H/Kalpha T cells. However, negative selection of anti-H/Kalpha T cells did occur if H/Kbeta was artificially overexpressed in the thymus. Given that H/Kalpha cannot be exported from the endoplasmic reticulum and is rapidly degraded in the absence of H/Kbeta, we conclude that H/Kalpha epitopes are unable to access MHC class II loading compartments in cells of the normal thymus. This work, taken together with our previous studies, highlights that thymic autoantigen expression does not necessarily result in the induction of tolerance.     

Characterization of Fungal Community Structure on a Weathered Pegmatitic Granite

This study exploited the contrasting major element chemistry of adjacent, physically separable crystals of framework and sheet silicates in a pegmatitic granite to investigate the mineralogical influences of fungal community structure on mineral surfaces. Large intact crystals of variably weathered muscovite, plagioclase, K-feldspar, and quartz were individually extracted, together with whole-rock granite. Environmental scanning electron microscopy (ESEM) revealed a diversity of fungal structures, with microcolonial fungi and fungal hyphae clearly visible on surfaces of all mineral types. Fungal automated ribosomal intergenic spacer analysis (FARISA) was used to generate a ribotype profile for each mineral sample and a randomization test revealed that ribotype profiles, or community fingerprints, differed between different mineral types. Canonical correspondence analysis (CCA) revealed that mineral chemistry affected individual fungal ribotypes, and strong relationships were found between certain ribotypes and particular chemical elements. This finding was further supported by analysis of variance (ANOVA) of the 16 most abundant ribotypes within the community. Significantly, individual ribotypes were largely restricted to single mineral types and ribotypes clustered strongly on the basis of mineral type. CCA also revealed that Al, Si, and Ca had a significant impact on fungal community structure within this system. These results show that fungal community structure was driven by the chemical composition of mineral substrates, indicating selective pressure by individual chemical elements on fungal populations in situ.     

A novel mammalian retromer component, Vps26B

The mammalian retromer protein complex, which consists of three proteins--Vps26, Vps29, and Vps35--in association with members of the sorting nexin family of proteins, has been implicated in the trafficking of receptors and their ligands within the endosomal/lysosomal system of mammalian cells. A bioinformatic analysis of the mouse genome identified an additional transcribed paralog of the Vps26 retromer protein, which we termed Vps26B. No paralogs were identified for Vps29 and Vps35. Phylogenetic studies indicate that the two paralogs of Vps26 become evident after the evolution of the chordates. We propose that the chordate Vps26-like gene published previously be renamed Vps26A to differentiate it from Vps26B. As for Vps26A, biochemical characterization of Vps26B established that this novel 336 amino acid residue protein is a peripheral membrane protein. Vps26B co-precipitated with Vps35 from transfected cells and the direct interaction between these two proteins was confirmed by yeast 2-hybrid analysis, thereby establishing Vps26B as a subunit of the retromer complex. Within HeLa cells, Vps26B was found in the cytoplasm with low levels at the plasma membrane, while Vps26A was predominantly associated with endosomal membranes. Within A549 cells, both Vps26A and Vps26B co-localized with actin-rich lamellipodia at the cell surface. These structures also co-localized with Vps35. Total internal reflection fluorescence microscopy confirmed the association of Vps26B with the plasma membrane in a stable HEK293 cell line expressing cyan fluorescent protein (CFP)-Vps26B. Based on these observations, we propose that the mammalian retromer complex is located at both endosomes and the plasma membrane in some cell types.