Diversity and evolution of Rp1 rust resistance genes in four maize lines.

KEY MESSAGE: This manuscript provides genome-level analysis of disease resistance genes in four maize lines, including studies of haplotype and resistance gene number as well as selection and recombination. The Rp1 locus of maize is a complex resistance gene (R-gene) cluster that confers race-specific resistance to Puccinia sorghi, the causal agent of common leaf rust. Rp1 NB-LRR disease resistance genes were isolated from two Rp1 haplotypes (HRp1-B and HRp1-M) and two maize inbred lines (B73 and H95). Sixty-one Rp1 genes were isolated from Rp1-B, Rp1-M, B73 and H95 with a PCR-based approach. The four maize lines carried from 12 to 19 Rp1 genes. From 4 to 9 of the identified Rp1 genes were transcribed in the four maize lines. The Rp1 gene nucleotide diversity was higher in HRp1-B and HRp1-M than in B73 and H95. Phylogenic analysis of 69 Rp1 genes revealed that the Rp1 genes maintained in HRp1-B, HRp1-M and H95 are evolving independently of each other, while Rp1 genes in B73 and HRp1-D appear more like each other than they do genes in the other lines. The results also revealed that the analysed Rp1 R-genes were under positive selection in HRp1-M and B73. Intragenic recombination was detected in Rp1 genes maintained in the four maize lines. This demonstrates that a genetic process that has the potential to generate new resistance genes with new specificities is active at the Rp1 locus in the four analysed maize lines and that the new resistance genes may act against newly arising pathogen races that become prevalent in the pathogen population.

Luteinizing hormone-induced up-regulation of ErbB-2 is insufficient stimulant of growth and invasion in ovarian cancer cells.

The effects of luteinizing hormone (LH), a gonadotropic hormone implicated in the development of ovarian cancer, are mediated by specific binding to its G protein-coupled receptor, the LH receptor (LHR). Activated LHR initiates second messenger responses, including cyclic AMP (cAMP) and inositol phosphate. Because cAMP increases expression of ErbB-2, a receptor tyrosine kinase whose overexpression in cancers correlates with poor survival, we hypothesized that LH may regulate ErbB-2 expression. Cell surface LHR expression in stable transformants of the ErbB-2-overexpressing ovarian cancer cell line SKOV3 was confirmed by PCR and whole-cell ligand binding studies. Second messenger accumulation in the LHR-expressing cells confirmed signaling through Gs and Gq. Western blots of total protein revealed that LHR introduction up-regulated ErbB-2 protein expression 2-fold and this was further up-regulated in a time- and dose-dependent manner in response to LH. Forskolin and 8Br-cAMP also up-regulated ErbB-2 in both LHR-expressing and mock-transfected cells, indicating that regulation of ErbB-2 is a cAMP-mediated event. Kinase inhibitor studies indicated the involvement of protein kinase A-mediated, protein kinase C-mediated, epidermal growth factor receptor-mediated, and ErbB-2-mediated mechanisms. The LH-induced up-regulation of ErbB-2 was insufficient to overcome the negative effects of LH on proliferation, invasion, and migration. A molecular signature for this nonaggressive phenotype was determined by Taqman array to include increased and decreased expression of genes encoding adhesion proteins and metalloproteinases, respectively. These data establish a role for LH and LHR in the regulation of ErbB-2 expression and suggest that, in some systems, ErbB-2 up-regulation alone is insufficient in producing a more aggressive phenotype.

Yoked complexes of human choriogonadotropin and the lutropin receptor: evidence that monomeric individual subunits are inactive.

Human choriogonadotropin (hCG) contains an alpha-subunit, common to other members of the glycoprotein hormone family, and a unique beta-subunit that determines hormone specificity. It is generally thought that heterodimer formation is obligatory for full hormonal activity, although other studies have indicated that individual subunits and homodimeric hCGbeta were capable of low affinity binding to the LH receptor (LHR) and subsequent activation. Previously, we constructed two yoked hormone (hCG)-LHR complexes, where the two hormone subunits and the heptahelical receptor were engineered to form single polypeptide chains, i.e. N-beta-alpha-LHR-C and N-alpha-beta-LHR-C. Expression of both complexes led to constitutive stimulation of cAMP production. In the present study, we investigated whether the human alpha-subunit and hCGbeta can act as functional agonists when covalently attached to or coexpressed with the LH receptor. Our initial results showed that hCGbeta, but not alpha, was able to activate LHR with an increase in intracellular cAMP in human embryonic kidney 293 cells but not in Chinese hamster ovary or COS-7 cells. Further examination of this apparent cell-specific agonist activity of hCGbeta revealed that low levels of endogenous alpha-subunit were expressed in human embryonic kidney 293 cells, thus enabling sufficient amounts of active heterodimer to form with the transfected hCGbeta to activate LHR. The studies in Chinese hamster ovary and COS-7 cells clearly demonstrate that, even under experimental conditions where hormone-receptor interactions are maximized, individual subunits of hCG can not act as functional agonists, at least in their monomeric form.

Utility of a column-free cell sorting system for separation of plasma cells in multiple myeloma FISH testing in clinical laboratories.

Targeted FISH analysis is an essential component of the management of plasma cell myeloma for identification of cytogenetic abnormalities. The purpose of this study was to evaluate the column-free method, RoboSep® (RS), for sorting CD138-expressing cells in bone marrow aspirates. Comparative analysis of column-based and RS methodologies was carried out on 54 paired bone marrow aspirate validation samples from patients undergoing work-up for plasma cell dyscrasia. Abnormalities detected by FISH analysis using an IGH@/CCND1 probe set were seen in 54% with RS, and 44% with column-based. We found a statistically significant difference between the yield of abnormalities detected in paired positive cases (p = 0.0001). An additional 183 consecutive post-validation samples sorted by RS showed recurrent genetic abnormalities in 85/120 (71%) of successfully sorted samples with ≥ 1% plasma cells but in none of 63 samples in which FISH analysis was completed on samples that could not be sorted due to insufficient plasma cells upon cell sorting. The column-free method successfully sorted PC, when present in ≥ 1% of cells, for detection of abnormalities by FISH. Furthermore, our data suggest that FISH analysis should not be performed on samples with an inadequate yield at the cell selection step.

Improving geocoding outcomes for the Nebraska Cancer Registry: learning from proven practices.

This report summarizes geocoding improvement experiments in the Nebraska Cancer Registry. An initial assessment of previous geocoding suggests that some proven geocoding procedures have not been followed, and overall results were unacceptable. This study concluded that when updating different address files from different time periods, it is sufficient to use the most recent street centerline database. The combination of match score of 80 and spelling sensitivity of 80 in ESRI's ArcGIS geocoder is sufficient for most geocoding purposes. Given the sizable number of unmatched addresses, the Google Maps geocoding service was used. A comparison of 1500 high-quality addresses that were matched by both Google Maps and ArcGIS geocoders shows that, in most cases, the location discrepancies between the two were acceptable. The median distance between each pair of 1500 coded locations was 36.6 meters, with an average of 92.8 meters. Distance discrepancies were larger in urban fringe areas and smaller toward urban centers. It was concluded that by strictly following proven procedures including address coding specification, Internet-based White Pages for reverse address finding, and Internet-based geocoding, a 90% or even a 95% match rate is achievable.