To analyze the expression profile of tumor endothelial marker 7 (TEM7) in the spinal cord and dorsal root ganglion (DRG).

To identify regulatory molecules which play key roles in the development of obesity, we investigated the transcriptional profiles in 3T3-L1 cells at early stage of differentiation and analyzed the promoter sequences of differentially regulated genes. One hundred and sixty-one (161) genes were found to have significant changes in expression at the 2nd day following treatment with differentiation cocktail. Among them, 86 transcripts were up-regulated and 75 transcripts were down-regulated. The 161 transcripts were classified into 10 categories according to their functional roles; cytoskeleton, cell adhesion, immune, defense response, metabolism, protein modification, protein metabolism, regulation of transcription, signal transduction and transporter. To identify transcription factors likely involved in regulating these differentially expressed genes, we analyzed the promoter sequences of up- or -down regulated genes for the presence of transcription factor binding sites (TFBSs). Based on coincidence of regulatory sites, we have identified candidate transcription factors (TFs), which include those previously known to be involved in adipogenesis (CREB, OCT-1 and c-Myc). Among them, c-Myc was also identified by our microarray data. Our approach to take advantage of the resource of the human genome sequences and the results from our microarray experiments should be validated by further studies of promoter occupancy and TF perturbation.

Spodoptera frugiperda (J.E. Smith) (Lepidoptera: Noctuidae), the fall armyworm is the most economically important maize pest in the western hemisphere. This research focused on the genetic variability of the maize host strain because there is a lack of information in this area of S. frugiperda research. Amplified fragment length polymorphism (AFLP) was used to assess the genetic variability of S. frugiperda over a large geographic area. Twenty populations were collected from the maize, one population was collected from princess tree, one population was collected from lemon tree, and one population was collected from bermudagrass. The 23 populations were from Mexico, the continental United States, Puerto Rico, Brazil, and Argentina. The objective of this research was to evaluate whether the majority of genetic variability was within populations or between populations. The AFLP results showed that the majority of the genetic variability is within populations and not between populations, indicating minor gene flow and suggesting that S. frugiperda in the Western Hemisphere are an interbreeding population.

The carob moth, Ectomyelois ceratoniae (Zeller, 1839) (Lepidoptera: Pyralidae), is the most important pest of pomegranate in Iran. As it has been rarely recorded on other host plants, control methods have mostly been focused on its populations on pomegranate. In this study, shapes and sizes of wings were compared in populations on 4 host plants (pomegranate, fig, pistachio and walnut) using a landmark-based geometric morphometric method, and analysis of partial warp scores and centroid sizes. The results showed significantly smaller wing size in populations on pomegranate and a significant host plant-associated shape difference among populations as a consequence of allometric growth. This suggests that the wing size and shape differences among test populations may not have a genetic basis and could happen because of differences in the nutritional content of host plants. The results of the analysis suggest that the female carob moth lays her eggs on host plants that provide suitable conditions for hatching. The larger size of moths on hosts other than pomegranate showed that some host plants such as fig, pistachio and walnut can provide for increased stored nutritional reserves by larvae that may result in more successful over-wintering and higher fecundity in adults. This suggests that in spite of the more extensive activity of carob moth on pomegranate in Iran, populations on other host plants can have an important effect on expanding pest population sizes in following years which should be considered in control methods.

Kamiti river virus (KRV) is an insect-only Flavivirus that was isolated from field-collected Ae. macintoshi mosquitoes in 1999, and is closely related to cell fusing agent virus. Both of these viruses belong to the family Flaviviridae, which also contains other viruses of medical importance, such as yellow fever virus, West Nile virus and dengue. Because Ae. macintoshi is the only known natural host to KRV, the main objective of this study was to establish the possibility that other mosquito hosts of the virus exist, by determining its ability to infect Ae. aegypti mosquitoes under laboratory conditions. The study also sought to determine the rates of infection and, subsequently, vertical transmission as a possible means of its maintenance and propagation in nature, given that it neither grows in vertebrate cells or mice. The mosquitoes were infected by the virus either as larvae or adults. Virus assay was done by re-isolation in tissue culture and indirect immunofluoresce assay methods. KRV infected Ae. aegypti mosquitoes, with the observed rates as high as 74 to 96 %. The virus was also transmitted vertically in these mosquitoes. Vertical transmission rates of 3.90 % were observed for the 2nd and 3rd ovarian cycles combined. These results suggest that Ae. aegypti mosquitoes are likely to be infected with KRV in nature, and that vertical transmission is the natural means by which it is maintained and propagated in this host, and possibly others.

Vitellogenin is a yolk precursor protein in most oviparous females. In the advanced eusocial honeybee, Apis mellifera (Hymenoptera: Apidae), vitellogenin has recently attracted much interest as this protein, in addition to a classical function in oocyte development in the reproductive queen caste, has evolved functions in the facultatively sterile female worker caste not documented in other species. However, research on the spatial dynamics of vitellogenin in various tissues is not easily performed with available tools. Here we present an immunogold staining procedure that visualizes honeybee vitellogenin in resin embedded tissue. To establish the protocol, we used ovaries of worker bees from colonies with and without a queen. Under the first condition, vitellogenin is assumed not to be present in the workers' ovaries. Under the second condition, the ovaries of worker bees become vitellogenic, with abundant opportunities for detection of complex patterns of vitellogenin uptake and storage. By use of this experimental setup, the staining method is shown to be both sensitive and specific. To demonstrate the functional significance of the protocol, it was subsequently used to identify vitellogenin protein in the hypopharyngeal glands (brood food producing head glands) of nursing worker bees and in adjacent head fat body cells for the first time. Localization of vitellogenin in these tissues supports previously hypothesized roles of vitellogenin in social behavior. This protocol thus provides deeper insights into the functions of vitellogenin in the honeybee.

The present study evaluated the effect of various dosages of soybean isoflavone extract on body weight changes, glucose tolerance and liver function in streptozotocin-induced diabetic rats. One group of normal rats (normal control) was fed an AIN-76-based experimental diet and four groups of diabetic rats were fed the same diet supplemented with four different levels of soybean isoflavone extract for seven weeks. The daily dosages of pure isoflavone for four diabetic groups were set to be 0 mg (diabetic control), 0.5 mg (ISO-I), 3.0 mg (ISO-II) and 30.0 mg (ISO-III) per kilogram of body weight, respectively. The daily consumption of isoflavone at the level of 3.0mg per kilogram of body weight resulted in the suppression of body weight loss and increased the survival rate of diabetic animals one and half times compared to that of the diabetic control group. Blood glucose levels in a fasting state and after the oral administration of glucose were significantly lower in the ISO-II group during the oral glucose tolerance test. The ISO-II group showed a tendency to elongate the gastrointestinal transit time. The activity of serum aminotransferases, indicator of liver function, was not negatively affected by any intake level of isoflavone. The present study demonstrated that the soybean isoflavone extract may be beneficial to diabetic animals by improving their glucose tolerance and suppressing weight loss without incurring hepatotoxicity at the daily dosage of 3.0 mg per kg of body weight.

To elucidate the roles of enteric bacteria and immunological interactions among liver, spleen and intestine in the pathogenesis of liver injury during obstructive jaundice, we studied the effects of antibiotics and splenectomy on bile-duct-ligated C57BL mice. When animals were subjected to bile-duct-ligation (BDL), plasma levels of bilirubin, alanine aminotransferase and aspartate aminotransferase increased markedly. However, the increases in plasma transaminases were significantly lower in splenectomized or antibiotics-treated groups than in the control BDL group. Histological examination revealed that liver injury was also low in the two groups. BDL markedly increased plasma level of interferon-gamma (IFN-gamma) and the expression of inducible nitric oxide synthase (iNOS) in liver and spleen. These changes were suppressed either by splenectomy or administration of antibiotics. Kinetic analysis revealed that BDL-induced liver injury and the increase of interleukin-10 (IL-10) and INF-gamma were lower in iNOS(-/-) than in wild type animals. BDL markedly increased the expression of IgA in colonic mucosa. These observations suggest that enteric bacteria, nitric oxide and cytokines including IFN-gamma and IL-10 derived from spleen and intestines form a critical network that determines the extent of liver injury during obstructive jaundice.

The current work presents results of experiments on the calcium response evoked by the stimulation by extracellular nucleotides occurring in control, nonstarved glioma C6 cells and in cells after long-term (96 h) serum starvation. Three nucleotide receptors were studied: P2Y(1), P2Y(2) and P2Y(12). Two of them, P2Y(1) and P2Y(2), directly stimulate calcium response. The protein level of the P2Y(2) receptor did not change during the serum starvation, while P2Y(1) protein level fell dramatically. Observed changes in the calcium response generated by P2Y(1) are directly correlated with the receptor protein level as well as with the amount of calcium present in the intracellular calcium stores, partially depleted during starvation process. The third receptor, P2Y(12), did not directly evoke calcium response, however it is activated by the same ligand as P2Y(1). The experiments with AR-C69941MX, the P2Y(12)-specific antagonist, indicated that in control and serum-starved cells, calcium response evoked by P2Y(1) receptor is potentiated by the activity of P2Y(12)-dependent signaling pathways. This potentiation may be mediated by P2Y(12) inhibitory effect on the plasma membrane calcium pump. The calcium influx enhanced by the cooperation of P2Y(1) and P2Y(12) receptor activity directly depends on the capacitative calcium entrance mechanism.

Glide is a ligand docking program for predicting protein-ligand binding modes and ranking ligands via high-throughput virtual screening. Glide utilizes two different scoring functions, SP and XP GlideScore, to rank-order compounds. Three modes of sampling ligand conformational and positional degrees of freedom are available to determine the optimal ligand orientation relative to a rigid protein receptor geometry. This unit presents protocols for flexible ligand docking with Glide, optionally including ligand constraints or ligand molecular similarities.

The excitatory amino acid domoic acid is the causative agent of amnesic shellfish poisoning in humans. The in vitro effects of domoic acid on rat neonatal brain microglia were compared with E. coli lipopolysaccharide (LPS), a known activator of microglia mediator release over a 4 to 24 hour observation period. LPS [3 ng/mL] but not domoic acid [1 mM] stimulated a statistically significant increase in TNF-alpha mRNA and protein generation. Furthermore, both LPS and domoic acid did not significantly affect TGF-beta1 gene expression and protein release. Finally, an in vitro exposure of microglia to LPS resulted in statistically significant MMP-9 expression and release, thus extending and confirming our previous observations. However, in contrast, no statistically significant increase in MMP-9 expression and release was observed after domoic acid treatment. Taken together our observations do not support the hypothesis that a short term (4 to 24 hours) in vitro exposure to domoic acid, at a concentration toxic to neuronal cells, activates rat neonatal microglia and the concomitant release of the pro-inflammatory mediators tumor necrosis factor-alpha (TNF-alpha) and matrix metalloproteinases-9 (MMP-9), as well as the anti-inflammatory cytokine transforming growth factor beta1 (TGF-beta1).

The pyridoacridine alkaloids kuanoniamines A and C were isolated together with 24 alpha-methylcholestanol, p-hydroxybenzaldehyde, p-hydroxybenzoic acid, phenylacetic acid and 3-formylindole from the ethyl acetate extract of the marine sponge Oceanapia sagittaria (Sollas), collected from the Gulf of Thailand. Kuanoniamines A and C were evaluated for their effect on the growth of five human tumour and a non-tumour cell lines, as well as on the proliferation of human lymphocytes. Kuanoniamine A was found to be a potent growth inhibitor of all the tumour and a non-tumour cell lines while kuanoniamine C was less potent but showed high selectivity toward the estrogen dependent (ER+) breast cancer cell line. Kuanoniamine A has shown to be a more potent inhibitor of DNA synthesis than kuanoniamine C. Kuanoniamine A was also found to cause an extensive reduction of the MCF-7 cells in G2/M phase as well as an increase in the apoptotic cells.

The genus Pseudoalteromonas is a marine group of bacteria belonging to the class Gammaproteobacteria that has come to attention in the natural product and microbial ecology science fields in the last decade. Pigmented species of the genus have been shown to produce an array of low and high molecular weight compounds with antimicrobial, anti-fouling, algicidal and various pharmaceutically-relevant activities. Compounds formed include toxic proteins, polyanionic exopolymers, substituted phenolic and pyrolle-containing alkaloids, cyclic peptides and a range of bromine-substituted compounds. Ecologically, Pseudoalteromonas appears significant and to date has been shown to influence biofilm formation in various marine econiches; involved in predator-like interactions within the microbial loop; influence settlement, germination and metamorphosis of various invertebrate and algal species; and may also be adopted by marine flora and fauna as defensive agents. Studies have been so far limited to a relatively small subset of strains compared to the known diversity of the genus suggesting that many more discoveries of novel natural products as well as ecological connections these may have in the marine ecosystem remain to be made.

Eight sesquiterpene quinones: ilimaquinone (1), smenospongidine (3), smenospongiarine (5), smenospongine (7), and their corresponding 5-epimers 2, 4, 6, and 8, isolated from the Palauan marine sponge Hippospongia sp., were examined regarding their effects on TNF-α production in LPS-stimulated RAW 264.7 cells. 5-Epi-smenospongine (8) promoted the production of TNF-α to a level three times greater than the control at 10 micron, but compounds 1-7 did not show apparent activity. The results suggest that the cis-decaline ring and a primary amine in the benzoquinone ring are necessary for activity. This is the first study to report the modulation of TNF-α production by a sesquiterpene quinone.

Rheumatoid arthritis is the most common systematic autoimmune disease and its etiology is believed to have both strong genetic and environmental components. We demonstrate the utility of including genetic and clinical phenotypes as covariates within a linkage analysis framework to search for rheumatoid arthritis susceptibility loci. The raw genotypes of 1302 affected relative pairs were combined from four large family-based samples (North American Rheumatoid Arthritis Consortium, United Kingdom, European Consortium on Rheumatoid Arthritis Families, and Canada). The familiality of the clinical phenotypes was assessed. The affected relative pairs were subjected to autosomal multipoint affected relative-pair linkage analysis. Covariates were included in the linkage analysis to take account of heterogeneity within the sample. Evidence of familiality was observed with age at onset (p << 0.001) and rheumatoid factor (RF) IgM (p << 0.001), but not definite erosions (p = 0.21). Genome-wide significant evidence for linkage was observed on chromosome 6. Genome-wide suggestive evidence for linkage was observed on chromosomes 13 and 20 when conditioning on age at onset, chromosome 15 conditional on gender, and chromosome 19 conditional on RF IgM after allowing for multiple testing of covariates.

We applied nonparametric quantitative trait linkage analysis to two rheumatoid arthritis quantitative phenotypes, IgM rheumatoid factor (RF) and anti-cyclic citrullinated peptide autoantibody titer measurements, using 5700 genome-wide Illumina single-nucleotide polymorphism genotypes on 658 Caucasian North American Rheumatoid Arthritis Consortium families. Peak LOD scores for both quantitative traits were located in the human leukocyte antigen region 6p21 (15.8 and 13.8 for RF and anti-cyclic citrullinated peptide, respectively) followed by 11p12 (3.2 and 3.6). In addition, there were LOD scores of 3.2 on 2q32 for RF and 3.6 on 4q24 for anti-cyclic citrullinated peptide. The resulting linkage signals for both phenotypes are very similar to previous results for rheumatoid arthritis as a qualitative variable, with rheumatoid factor measurements being most closely aligned. Interestingly, anti-cyclic citrullinated peptide exhibits a stronger linkage peak on 2p14 than rheumatoid factor and rheumatoid arthritis, and stronger linkage on 4q24. Finally, we used ordered subset analyses to determine if sub-ranges of these two traits increased rheumatoid arthritis linkage signals; however, our analyses did not reveal significant effects of the quantitative traits on rheumatoid arthritis linkage signals in this population.

The Genetic Analysis Workshop 15 rheumatoid arthritis data included a set of 460 cases and 460 controls genotyped at 2300 closely spaced markers on a 10 megabase region of chromosome 18q. We conducted a multilocus analysis of these data using a localized haplotype clustering method that adapts to linkage disequilibrium structure and can be applied to large, densely genotyped data sets such as this one. We found a protective haplotype carried by 33 individuals that was significantly associated with rheumatoid arthritis in these data after adjusting for multiple testing. This haplotype was located less than 500 base pairs upstream of the CCBE1 gene. The association was not detected using single-marker tests, but could be found using a variety of multilocus tests.

There is a growing interest in studying natural variation in human gene expression. Studies mapping genetic determinants of expression profiles are often carried out considering the expression of one gene at a time, an approach that is computationally intensive and may be prone to high false-discovery rate because the number of genes under consideration often exceeds tens of thousands. We present an exploratory method for investigating such data and apply it to the data provided as Problem 1 of Genetic Analysis Workshop 15 (GAW15). In multivariate analysis, canonical correlation analysis is a common way to inspect the relationship between two sets of variables based on their correlation. It determines linear combinations of all variables from each data set such that the correlation between the two linear combinations is maximized. However, due to the large number of genes, linear combinations involving all single-nucleotide polymorphism (SNP) loci and gene expression phenotypes lack biological plausibility and interpretability. We introduce sparse canonical correlation analysis, which examines the relationships of many genetic loci and gene expression phenotypes by providing sparse linear combinations that include only a small subset of loci and gene expression phenotypes. These correlated sets of variables are sufficiently small for biological interpretability and further investigation. Applying this method to the GAW15 Problem 1 data, we identified groups of 41 loci and 150 gene expressions with the highest between-group correlation of 43%.

The nuclear factor-kappaB (NF-kappaB) family of transcription factors regulates the expression of a variety of genes involved in apoptosis and immune response. We examined relationships between genotypes at five NF-kappaB subunits (NFKB1, NFKB2, REL, RELA, and RELB) and variable expression levels of 15 NF-kappaB regulated proteins with heritability greater than 0.40: BCL2A1, BIRC2, CD40, CD44, CD80, CFLAR, CR2, FAS, ICAM1, IL15, IRF1, JUNB, MYC, SLC2A5, and VCAM1. SNP genotypes and expression phenotypes from pedigrees of Utah residents with ancestry from northern and western Europe were provided by Genetic Analysis Workshop 15 and supplemented with additional genotype data from the International HapMap Consortium. We conducted association, linkage, and family-based association analyses between each candidate gene and the 15 heritable expression phenotypes. We observed consistent results in association and linkage analyses of the NFKB1 region (encoding p50) and levels of FAS and IRF1 expression. FAS is a cell surface protein that also belongs to the TNF-receptor family; signals through FAS are able to induce apoptosis. IRF1 is a member of the interferon regulatory transcription factor family, which has been shown to regulate apoptosis and tumor-suppression. Analyses in the REL region (encoding c-Rel) revealed linkage and association with CD40 phenotype. CD40 proteins belong to the tumor necrosis factor (TNF)-receptor family, which mediates a broad variety of immune and inflammatory responses. We conclude that variation in the genes encoding p50 and c-Rel may play a role in NF-kappaB-related transcription of FAS, IRF1, and CD40.

Large-scale genome-wide association studies are increasingly common, due in large part to recent advances in genotyping technology. Despite a dramatic drop in genotyping costs, it is still too expensive to genotype thousands of individuals for hundreds of thousands single-nucleotide polymorphisms (SNPs) for large-scale whole-genome association studies for many researchers. A two-stage design has been a promising alternative: in the first stage, only a small fraction of samples are genotyped and tested using a dense set of SNPs, and only a small subset of markers that show moderate associations with the disease will be genotyped in the second stage. In this report, I developed an approach to select and prioritize SNPs for association studies with a two-stage or multi-stage design. In the first stage, the method not only evaluates associations of SNPs with the disease of interest, it also explicitly explores correlations among SNPs. I applied the approach on the simulated Genetic Analysis Workshop 15 Problem 3 data sets, which have modeled the complex genetic architecture of rheumatoid arthritis. Results show that the method can greatly reduce the number of SNPs required in later stage(s) without sacrificing mapping precision.