Characterization of binding proteins that recognize oligoglucoside elicitors of phytoalexin synthesis in soybean

We are studying the cellular signaling pathway leading to pterocarpan phytoalexin biosynthesis in soybean that is induced by a branched hepta-β-glucoside originally isolated from the mycelial walls of the phytopathogenic oomycete Phytophthora sojae. Our research has focused on the specific recognition of the hepta-β-glucoside elicitor by binding proteins in soybean cells. Elicitor-binding proteins with properties expected of physiological receptors for the hepta-β-glucoside elicitor have been identified in soybean root membranes. These elicitor-binding proteins co-migrate with a plasma membrane marker (vanadate-sensitive H⁺-ATPase) on linear sucrose density gradients. Binding of a radio-iodinated derivative of the hepta-β-glucoside elicitor by membrane-localized elicitor-binding proteins is specific, reversible, saturable, and of high affinity (K_d? 1 nM). After solubilization with the nonionic detergent, n-dodecylsucrose, the elicitor-binding proteins retain their high affinity (K_d= 1.8 nM) for the radiolabeled elicitor and their binding specificity for elicitor-active oligoglucosides. A direct correlation is observed between the ability of oligoglucosides to displace labeled elicitor from the elicitor-binding proteins and the elicitor activity of the oligosaccharides. Thus, the elicitor-binding proteins recognize the same structural elements of the hepta-β-glucoside elicitor that are essential for its phytoalexin-inducing activity, suggesting that the binding proteins are physiological receptors for the elicitor. Current research is directed toward the purification of the hepta-β-glucoside elicitor-binding proteins by using ligand affinity chromatography. Purification and characterization of the hepta-β-glucoside binding proteins are among the first steps toward elucidating how the hepta-β-glucoside elicitor triggers the signal transduction pathway that ultimately leads to the synthesis of phytoalexins in soybean.     

Altered expression of glycosaminoglycans in metastatic 13762NF rat mamma adenocarcinoma cells

A difference in the expression and metabolism of sulfated glycosaminoglycans between rat mammary tumor cells derived from a primary tumor and those from its metastatic lesions has been observed. Cells from the primary tumor possessed about equal quantities of chondroitin sulfate and heparan sulfate on their cell surfaces but released fourfold more chondroitin sulfate than heparan sulfate into their medium. In contrast, cells from distal metastatic lesions expressed approximately 5 times more heparan sulfate than chondroitin sulfate in both medium and cell surface fractions. This was observed to be the result of differential synthesis of the glycosaminoglycans and not of major structural alterations of the individual glycosaminoglycans. The degree of sulfation and size of heparan sulfate were similar for all cells examined. However, chondroitin sulfate, observed to be only chondroitin 4-sulfate, from the metastases-derived cells had a smaller average molecular weight on gel filtration chromatography and showed a decreased quantity of sulfated disaccharides upon degradation with chondroitin ABC lyase compared to the primary tumor derived cells. Major qualitative or quantitative alterations were not observed for hyaluronic acid among the various 13762NF cells. The metabolism of newly synthesized sulfated glycosaminoglycans was also different between cells from primary tumor and metastases. Cells from the primary tumor continued to accumulate glycosaminoglycans in their medium over a 72-h period, while the accumulation of sulfated glycosaminoglycans in the medium of metastases-derived cells showed a plateau after 18-24 h. A pulse-chase kinetics study demonstrated that both heparan sulfate and chondroitin sulfate were degraded by the metastases-derived cells, whereas the primary tumor derived cells degraded only heparan sulfate and degraded it at a slower rate.(ABSTRACT TRUNCATED AT 250 WORDS)     

Genus-Wide Comparative Genomics of Malassezia Delineates Its Phylogeny,Physiology, and Niche Adaptation on Human Skin

Malassezia is a unique lipophilic genus in class Malasseziomycetes in Ustilaginomycotina, (Basidiomycota, fungi) that otherwise consists almost exclusively of plant pathogens. Malassezia are typically isolated from warm-blooded animals, are dominant members of the human skin mycobiome and are associated with common skin disorders. To characterize the genetic basis of the unique phenotypes of Malassezia spp., we sequenced the genomes of all 14 accepted species and used comparative genomics against a broad panel of fungal genomes to comprehensively identify distinct features that define the Malassezia gene repertoire: gene gain and loss; selection signatures; and lineage-specific gene family expansions. Our analysis revealed key gene gain events (64) with a single gene conserved across all Malassezia but absent in all other sequenced Basidiomycota. These likely horizontally transferred genes provide intriguing gain-of-function events and prime candidates to explain the emergence of Malassezia. A larger set of genes (741) were lost, with enrichment for glycosyl hydrolases and carbohydrate metabolism, concordant with adaptation to skin’s carbohydrate-deficient environment. Gene family analysis revealed extensive turnover and underlined the importance of secretory lipases, phospholipases, aspartyl proteases, and other peptidases. Combining genomic analysis with a re-evaluation of culture characteristics, we establish the likely lipid-dependence of all Malassezia. Our phylogenetic analysis sheds new light on the relationship between Malassezia and other members of Ustilaginomycotina, as well as phylogenetic lineages within the genus. Overall, our study provides a unique genomic resource for understanding Malassezia niche-specificity and potential virulence, as well as their abundance and distribution in the environment and on human skin.     

In Vivo Bioluminescent Imaging of Irradiated Orthotopic Pancreatic Cancer Xenografts in Nonobese Diabetic-Severe Combined Immunodeficient Mice: A Novel Method for Targeting and Assaying Efficacy of Ionizing Radiation

Adenocarcinoma of the pancreas is a lethal malignancy, and better models to study tumor behavior in vivo are needed for the development ofmore effective therapeutics. Ionizing radiation is a treatment modality that is commonly used in the clinical setting, in particular, for locally confined disease; however, good model systems to study the effect of ionizing radiation in orthotopic tumors have not been established. In an attempt to create clinically relevant models for studying treatments directed against pancreatic cancer, we have defined a methodology to measure the effect of varying doses of radiation in established human pancreatic cancer orthotopic xenografts using two different pancreatic cancer cell lines (Panc-1 and BXPC3) infected with a lentiviral vector expressing CMV promoter-driven luciferase to allow bioluminescence imaging of live animals in real time. Quantifiable photon emission from luciferase signaling in vivo correlated well with actual tumor growth. Bioluminescence imaging of the established pancreatic xenografts was used to direct delivery of radiation to the orthotopic tumors and minimize off-target adverse effects. Growth delay was observed with schedules in the range of 7.5 Gy in five fractions to 10 Gy in four fractions, whereas doses 3 Gy or higher produced toxic adverse effects. In conclusion, we describe a model in which the effects of ionizing radiation, alone or in combination with other therapeutics, in orthotopic xenografts, can be studied.     

Multiple alleles and low variability of glycerol-3-phosphate dehydrogenase in Aedes albopictus (Diptera: Culicidae) from Peninsular Malaysia

1. A total of 8 samples from three natural populations and a laboratory strain of Aedes albopictus were analysed for glycerol-3-phosphate dehydrogenase phenotypes by means of horizontal starch-gel electrophoresis. 2. The electrophoretic phenotypes were governed by three codominant Gpd alleles. 3. There was low variability, with the heterozygosity in the variable samples ranging from 0.02 to 0.12. 4. The commonest allele in all the population samples was GpdB which encoded an electrophoretic band with intermediate mobility. 5. There was no temporal or spatial variation.     

Genome-Wide Profiling of Structural Genomic Variations in Korean HapMap Individuals

Background

Structural genomic variation study, along with microarray technology development has provided many genomic resources related with architecture of human genome, and led to the fact that human genome structure is a lot more complicated than previously thought.

Methodology/Principal Findings

In the case of International HapMap Project, Epstein-Barr various immortalized cell lines were preferably used over blood in order to get a larger number of genomic DNA. However, genomic aberration stemming from immortalization process, biased representation of the donor tissue, and culture process may influence the accuracy of SNP genotypes. In order to identify chromosome aberrations including loss of heterozygosity (LOH), large-scale and small-scale copy number variations, we used Illumina HumanHap500 BeadChip (555,352 markers) on Korean HapMap individuals (n = 90) to obtain Log R ratio and B allele frequency information, and then utilized the data with various programs including Illumina ChromoZone, cnvParition and PennCNV. As a result, we identified 28 LOHs (>3 mb) and 35 large-scale CNVs (>1 mb), with 4 samples having completely duplicated chromosome. In addition, after checking the sample quality (standard deviation of log R ratio <0.30), we selected 79 samples and used both signal intensity and B allele frequency simultaneously for identification of small-scale CNVs (<1 mb) to discover 4,989 small-scale CNVs. Identified CNVs in this study were successfully validated using visual examination of the genoplot images, overlapping analysis with previously reported CNVs in DGV, and quantitative PCR.

Conclusion/Significance

In this study, we describe the result of the identified chromosome aberrations in Korean HapMap individuals, and expect that these findings will provide more meaningful information on the human genome.     

Membrane palmitoylated proteins regulate trafficking and processing of nectins

Nectins are cell-cell adhesion molecules involved in the formation of various intercellular junctions and the establishment of apical-basal polarity at cell-cell adhesion sites. To have a better understanding of the roles of nectins in the formation of cell-cell junctions, we searched for new cytoplasmic binding partners for nectin. We report that nectin-1α associates with membrane palmitoylated protein 3 (MPP3), one of the human homologues of a Drosophila tumor suppressor gene, Disc large. Two major forms of MPP3 at 66 and 98 kDa were detected, in conjunction with nectin-1α, suggesting that an association between the two may occur in various cell types. Nectin-1α recruits MPP3 to cell-cell contact sites, mediated by a PDZ-binding motif at the carboxyl terminus of nectin-1α. Association with MPP3 increases cell surface expression of nectin-1α and enhances nectin-1α ectodomain shedding, indicating that MPP3 regulates trafficking and processing of nectin-1α. Further study showed that MPP3 interacts with nectin-3α, but not with nectin-2α, showing that the association of nectins with MPP3 is isoform-specific. MPP5, another MPP family member, interacts with nectins with varying affinity and facilitates surface expression of nectin-1α, nectin-2α, and nectin-3α. These data suggest that wide interactions between nectins and MPP family members may occur in various cell-cell junctions and that these associations may regulate trafficking and processing of nectins.