Macropodid herpesvirus 1 encodes genes for both thymidylate synthase and ICP34.5

Macropodid herpesvirus 1 (MaHV-1) is an unclassified alphaherpesvirus linked with the fatal infections of kangaroos and other marsupials. During the characterisation of the internal repeat region of MaHV-1, an open reading frame (ORF) encoding for thymidylate synthase (TS) gene was identified and completely sequenced. Southern blot analysis confirmed the presence of two copies of the TS gene in the MaHV-1 genome as expected. Computer analysis of the TS ORF showed it was 948 nucleotides in length. A putative polyadenylation signal was identified 17-22 bp inside the ORF implying a minimal or absent 3' untranslated region. The predicted polypeptide was 316 amino acid residues in length and contained the highly conserved motifs for folate binding and F-dUMP binding, typical of all TS enzymes. Interestingly, MaHV-1 TS polypeptide had highest similarity to the human TS polypeptide (81%) compared to the TS polypeptides of other herpesviruses (72-75%). Immediately upstream of the TS gene, a second ORF of 510 bp, encoding a polypeptide with 170 amino acid residues, was identified. The carboxyl domain of this MaHV-1 polypeptide shared 68% similarity to a 59 amino acid motif of human herpesvirus 1 ICP34.5, identifying it as the MaHV-1 ICP34.5 homologue. This is the first report of a herpesvirus that encodes for both TS and ICP34.5.

Exogenous Slit2 does not affect ureteric branching or nephron formation during kidney development

In an attempt to elucidate the role of Slit2 invertebrate kidney development, the effect of adding exogenous human Slit2 protein (hSlit2) to developing murine metanephric kidney explants was examined. To confirm the activity of the recombinant Slit2 protein, neurons from 8 day old chick sympathetic nerve chain dorsal root ganglia were cultured with hSlit2 protein, which induced significant neurite branching and outgrowth. Using kidney explants as a model system, metanephric development in the presence of hSlit2 protein was examined. Addition of hSlit2 up to a final concentration of 1 mug/ml had no detectable effect on the formation of nephrons or on branching morphogenesis of the ureteric tree after 2 or 4 days in culture, as assessed via immunofluorescence for the markers WT1 and calbindin 28K respectively. Similarly, maturation of the nephrogenic mesenchyme occurred in a phenotypically normal fashion. In situ analysis of the Slit receptors, Robot and Robot, the vasculogenic markers VEGFA and Flk-1, and the stromal cell marker BF2 displayed no difference in comparison to controls.

Movement through slits: cellular migration via the Slit family

First isolated in the fly and now characterised in vertebrates, the Slit proteins have emerged as pivotal components controlling the guidance of axonal growth cones and the directional migration of neuronal precursors. As well as extensive expression during development of the central nervous system (CNS), the Slit proteins exhibit a striking array of expression sites in non-neuronal tissues, including the urogenital system, limb primordia and developing eye. Zebrafish Slit has been shown to mediate mesodermal migration during gastrulation, while Drosophila slit guides the migration of mesodermal cells during myogenesis. This suggests that the actions of these secreted molecules are not simply confined to the sphere of CNS development, but rather act in a more general fashion during development and throughout the lifetime of an organism. This review focuses on the non-neuronal activities of Slit proteins, highlighting a common role for the Slit family in cellular migration.

The role of conserved CXXXRXG motif in the expression and function of the human norepinephrine transporter

Highly conserved motifs in the monoamine transporters, e.g. the human norepinephrine transporter (hNET) GXXXRXG motif which was the focus of the present study, are likely to be important structural features in determining function. This motif was investigated by mutating the glycines to glutamate (causing loss of function) and alanine, and the arginine to glycine. The effects of hG117A, hR121G and hG123A mutations on function were examined in COS-7 cells and compared to hNET. Substrate K-m values were decreased for hG117A and hG123A, and their K values for inhibition of [3 H]nisoxetine binding were decreased 3-4-fold and 4-6-fold, respectively. Transporter turnover was reduced to 65% of hNET for hG117A and hR121G and to 28% for hG123A, suggesting that substrate translocation is impaired. K values of nisoxetine and desipramine for inhibition of [H-3]norepinephrine uptake were increased by 5-fold for hG117A, with no change for cocaine. The K-i value of cocaine was increased by 3-fold for hG123A, with no change for nisoxetine and desipramine. However, there were no effects of the mutations on the K-d of [H-3]nisoxetine binding or K-i values of desipramine or cocaine for inhibition of [H-3]nisoxetine binding. Hence, glycine residues of the GXXXRXG motif are important determinants of NET expression and function, while the arginine residue does not have a major role. This study also showed that antidepressants and psychostimulants have different NET binding sites and provided the first evidence that different sites on the NET are involved in the binding of inhibitors and their competitive inhibition of substrate uptake. (C) 2002 Elsevier Science B.V. All rights reserved.

Colony-stimulating factor-1 suppresses responses to CpG DNA and expression of toll-like receptor 9 but enhances responses to lipopolysaccharide in murine macrophages

During bacterial infections, the balance between resolution of infection and development of sepsis is dependent upon the macrophage response to bacterial products. We show that priming of murine bone marrow-derived macrophages (BMMs) with CSF-1 differentially regulates the response to two such stimuli, LPS and immunostimulatory (CpG) DNA. CSF-1 pretreatment enhanced IL-6, IL-12, and TNF-alpha production in response to LPS but suppressed the same response to CpG DNA. CSF-1 also regulated cytokine gene expression in response to CpG DNA and LPS; CpG DNA-induced IL-12 p40, IL-12 p35, and TNF-alpha mRNAs were all suppressed by CSF-1 pretreatment. CSF-1 pretreatment enhanced LPS-induced IL-12 p40 mRNA but not TNF-alpha and IL-12 p35 mRNAs, suggesting that part of the priming effect is posttranscriptional. CSF-1 pretreatment also suppressed CpG DNA-induced nuclear translocation of NF-kappaB and phosphorylation of the mitogen-activated protein kinases p38 and extracellular signal-related kinases-1/2 in BMMs, indicating that early events in CpG DNA signaling were regulated by CSF-1. Expression of Toll-like receptor (TLR)9, which is necessary for responses to CpG DNA, was markedly suppressed by CSF-1 in both BMMs and thioglycolate-elicited peritoneal macrophages. CSF-1 also down-regulated expression of TLR1, TLR2, and TLR6, but not the LPS receptor, TLR4, or TLR5. Hence, CSF-1 may regulate host responses to pathogens through modulation of TLR expression. Furthermore, these results suggest that CSF-1 and CSF-1R antagonists may enhance the efficacy of CpG DNA in vivo.

Temporal and spatial expression of fibroblast growth factor receptor 4 isoforms in murine tissues

Fibroblast growth factor receptors (FGFRs) undergo highly regulated spatial and temporal changes of expression during development. This study describes the use of quantitative reverse transcriptase-polymerase chain reaction and immunochemistry to assess the changes in expression of FGFR4 as compared to its FGFR4-17a and -17b isoforms in mouse tissues, from early embryogenesis through to adulthood. Compared to FGFR4, the expression of the isoforms is more restricted at all developmental stages tested. The reverse transcriptase-polymerase chain reaction demonstrated that FGFR4 is expressed in more tissue types than either of its isoforms: it was found predominantly in lung, liver, brain, skeletal muscle and kidney, whereas the FGFR4-17a form was detected in lung and skeletal muscle, and the FGFR4-17b form only in lung, liver, skeletal muscle and kidney. Immunohistochemistry confirmed strong FGFR4-17b expression in the postnatal lung. When combined, the results suggest that FGFR4 variants play important roles particularly in lung and skeletal muscle development.

Complementary and layered expression of Ephs and Ephrins in developing mouse inner ear

The distributions of the Eph-class receptors EphA4 and EphB 1, and their ligands ephrin-A2, ephrin-B1, and ephrin-B2, were analysed by immunostaining in the mouse inner ear. Complementary patterns of EphA4 and its potential ligand ephrin-A2 were found, with ephrin-A2 in many of the structures lining the cochlear duct and within the cochlear nerve cells, and EphA4 in the deeper structures underlying the cochlear duct and in the cells lining the nerve pathway. EphB1 and its potential ligands ephrin-B1 and ephrin-B2 showed a segregated layered expression in the lateral wall of the cochlear duct (the external sulcus), which together with EphA4 expressed in the area, form a four-layered structure with an alternating pattern of receptors and ligands in the different layers. This arrangement gives the potential for different bidirectional Eph-mediated interactions between each of the layers. The results suggest that the Eph system in the cochlea may have a role in maintaining cell segregation during phases of cochlear development. (C) 2002 Wiley-Liss, Inc.

Heterogeneity in olfactory neurons in mouse revealed by differential expression of glycoconjugates

Cell surface glycoconjugates have been implicated in the growth and guidance of subpopulations of primary olfactory axons. While subpopulations of primary olfactory neurons have been identified by differential expression of carbohydrates in the rat there are few reports of similar subpopulations in the mouse. We have examined the spatiotemporal expression pattern of glycoconjugates recognized by the lectin from Wisteria floribunda (WFA) in the mouse olfactory system. In the developing olfactory neuroepithelium lining the nasal cavity, WFA stained a subpopulation of primary olfactory neurons and the fascicles of axons projecting to the target tissue, the olfactory bulb. Within the developing olfactory bulb, WFA stained the synaptic neuropil of the glomerular and external plexiform layers. In adults, strong expression of WFA ligands was observed in second-order olfactory neurons as well as in neurons in several higher order olfactory processing centres in the brain. Similar, although distinct, staining of neurons in the olfactory pathway was detected with Dolichos biflorus agglutinin. These results demonstrate that unique subpopulations of olfactory neurons are chemically coded by the expression of glycoconjugates. The conserved expression of these carbohydrates across species suggests they play an important role in the functional organization of this region of the nervous system.

Decreased expression of the Id3 gene at 1p36.1 in ovarian adenocarcinomas

The molecular events that drive the initiation and progression of ovarian adenocarcinoma are not well defined. We have investigated changes in gene expression in ovarian cancer cell lines compared to an immortalized human ovarian surface epithelial cell line (HOSE) using a cDNA array. We identified 17 genes that were under-expressed and 10 genes that were over-expressed in the cell lines compared to the HOSE cells. One of the genes under-expressed in the ovarian cancer cell lines, Id3, a transcriptional inactivator, was selected for further investigation. Id3 mRNA was expressed at reduced levels in 6 out of 9 ovarian cancer cell lines compared to the HOSE cells while at the protein level, all 7 ovarian cancer cell lines examined expressed the Id3 protein at greatly reduced levels. Expression of Id3 mRNA was also examined in primary ovarian tumours and was found in only 12/38 (32%) cases. A search was conducted far mutations of Id3 in primary ovarian cancers using single stranded conformation polymorphism (SSCP) analysis. Only one nucleotide substitution, present also in the corresponding constitutional DNA, was found in 94 ovarian tumours. Furthermore no association was found between LOH at 1p36 and lack of expression of Id3. These data suggest that Id3 is not the target of LOH at 1p36. (C) 2001 Cancer Research Campaign.

Growth hormone regulates osteogenic marker mRNA expression in human periodontal fibroblasts and alveolar bone-derived cells

Background: Growth hormone (GH) is a potent regulator of bone formation. The proposed mechanism of GH action is through the stimulation of osteogenic precursor Cell proliferation and, following clonal expansion of these cells. promotion of differentiation along the osteogenic lineage. Objectives: We tested this hypothesis by studying the effects of GH on primary cell populations of human periodontal ligament cells (PLC) and alveolar bone cells (ABC), which contain a spectrum of osteogenic precursors. Method: The cell populations were assessed for mineralization potential after long-term culture in media containing beta-glycerophosphate and ascorbic acid, by the demonstration of mineral deposition by Von Kossa staining. The proliferative response of the cells to GH was determined over a 48-h period using a crystal violet dye-binding assay. The profile of the cells in terms of osteogcnic marker expression was established using quantitative reverse transcriptase polymerase chain reaction (RT-PCR) for alkaline phosphatase (ALP), osteopontin. osteocalcin, bone sialoprotein (BSP), as well as the bone morphogenetic proteins BMP-2, BMP-4 and BMP-7. Results: As expected, a variety of responses were observed ranging from no mineralization in the PLC populations to dense mineralized deposition observed in one GH-treated ABC population. Over a 48-h period GH was found to be non-mitogenic for all cell populations. Quantitative reverse transcriptase polymerase chain reaction (RT-PCR) BSP mRNA expression correlated well with mineralizing potential of the cells. The change in the mRNA expression of the osteogenic markers was determined following GH treatment of the cells over a 48-h period. GH caused an increase in ALP in most cell populations, and also in BMP expression in some cell populations. However a decrease in BSP. osteocalcin and osteopontin expression in the more highly differentiated cell populations was observed in response to GH. Conclusion: The response of the cells indicates that while long-term treatment with GH may promote mineralization, short-term treatment does not promote proliferation of osteoblast precursors nor induce expression of late osteogenic markers.

Single-nucleotide polymorphism in the CD14 promoter and periodontal disease expression in a Japanese population

It has been reported that there is a relationship between a single-nucleotide polymorphism (SNP) in the promoter region of the CD 14 gene at position -159 (C-->T) and infectious diseases. The aim of the present study was to test the hypthesis that expression of this SNP correlates with periodontal disease in a Japanese population. The CD14 genotype was determined in 163 subjects with periodontitis and in 104 age- and gender-matched control subjects without periodontitis. The genotype distribution and allele frequency within the periodontitis patients were not significantly different from those of control subjects. There was, however, a significant difference in the genotype distribution between young patients (< 35 yrs) and older patients (greater than or equal to 35 yrs). These findings suggest that CD14-159C/T polymorphism is not related to the development of periodontitis in a Japanese population, but that, within the periodontitis subjects, expression of the SNP may be related to early disease activity.

Prognostic use of growth characteristics of early gastric cancer and expression patterns of apoptotic, cell proliferation, and cell adhesion proteins

Background and Objectives: Selection of suitable treatment for early gastric cancers, such as endoscopic mucosal resection or the major surgical option of resection of the cancer together with a radical lymph node dissection, may be assisted by comparing the growth characteristics of the cancer with selected molecular characteristics. The results could be used to predict those cases that have a higher risk of developing secondary metastases. Methods: A total of 1,196 Japanese patients with early gastric cancers (648 mucosal cancers and 548 submucosal) were included in the selection of two groups: a metastatic group made up 57 cancers with lymph node metastasis (9 mucosal, 48 submucosal), and a nonmetastatic group of 61 cases (6 mucosal, 55 submucosal) without lymph node metastasis. Growth characteristics of the cancers (superficially spreading, penetrating or invasive, lymph node metastasis) were compared with immunohistochemical expression of single-stranded DNA (ssDNA) protein (apoptosis indicator), bcl-2 and p53 (apoptosis-associated), Ki-67 (cell proliferation), and E-cadherin (cell adhesion) proteins. Results: The lesions in the nonmetastatic group had higher levels of apoptosis and lower expression of bcl-2 than in the metastatic group, indicating an inhibitory role for apoptosis in malignant progression. Apoptosis was also higher in the superficial compared with the invasive lesions of both groups. The lesions in the metastatic group had higher p53 expression than that of the nonmetastatic group, whereas apoptosis in the metastatic group was lower than in the nonmetastatic group. An unproved explanation for this finding may be that, although increased, p53 was mutated and ineffective in promoting apoptotic control of metastatic progression. E-cadherin was decreased in the invasive lesions of both groups, indicating a greater ability of these cells to lose adhesion, to invade the submucosa, and to metastasize. Cell proliferation was highest in the superficial lesions of both metastatic and nonmetastatic groups. Conclusions: Early gastric cancers with low levels of apoptosis, increased bcl-2, and high levels of p53 expression are more likely to invade and metastasize. (C) 2003 Wiley-Liss, Inc.

How important are brain banks for alcohol research?

This article contains the proceedings of a symposium at the 2002 RSA/ISBRA Meeting in San Francisco, organized and chaired by Clive Harper and co-chaired by Izuru Matsumoto. The presentations were (1) Introduction, by Clive Harper; (2) The quality of tissue-a critical issue, by Therese Garrick; (3) The first systematic brain tissue donor program in Japan, by Izuru Matsumoto; (4) Brain scans after death-really! by Adolf Pfefferbaum, Elfar Adalsteinsson, and Edith Sullivan; (5) Capture that (genial) expression, by Joanne Lewohl and Peter Dodd; and (6) Neurochemical/pharmacological studies: experimental design and limitations, by Roger Butterworth.

Gene Expression in Brain: A Window on Ethanol Dependence, Neuroadaptation, and Preference

This article represents the proceedings of a symposium at the 2002 joint RSA/ISBRA Conference in San Francisco, California. The organizer was Paula L. Hoffman and the co-chairs were Paula L. Hoffman and Michael Miles. The presentations were (1) Introduction and overview of the use of DNA microarrays, by Michael Miles; (2) DNA microarray analysis of gene expression in brains of P and NP rats, by Howard J. Edenberg; (3) Gene expression patterns in brain regions of AA and ANA rats, by Wolfgang Sommer; (4) Patterns of gene expression in brains of selected lines of mice that differ in ethanol tolerance, by Boris Tabakoff; (5) Gene expression profiling related to initial sensitivity and tolerance in gamma-protein kinase C mutants, by Jeanne Wehner; and (6) Gene expression patterns in human alcoholic brain: from microarrays to protein profiles, by Joanne Lewohl.

Model-based clustering in gene expression microarrays: An application to breast cancer data

In microarray studies, the application of clustering techniques is often used to derive meaningful insights into the data. In the past, hierarchical methods have been the primary clustering tool employed to perform this task. The hierarchical algorithms have been mainly applied heuristically to these cluster analysis problems. Further, a major limitation of these methods is their inability to determine the number of clusters. Thus there is a need for a model-based approach to these. clustering problems. To this end, McLachlan et al. [7] developed a mixture model-based algorithm (EMMIX-GENE) for the clustering of tissue samples. To further investigate the EMMIX-GENE procedure as a model-based -approach, we present a case study involving the application of EMMIX-GENE to the breast cancer data as studied recently in van 't Veer et al. [10]. Our analysis considers the problem of clustering the tissue samples on the basis of the genes which is a non-standard problem because the number of genes greatly exceed the number of tissue samples. We demonstrate how EMMIX-GENE can be useful in reducing the initial set of genes down to a more computationally manageable size. The results from this analysis also emphasise the difficulty associated with the task of separating two tissue groups on the basis of a particular subset of genes. These results also shed light on why supervised methods have such a high misallocation error rate for the breast cancer data.