Endothelin-1 regulates proliferative responses, both alone and synergistically with PDGF, in rat tracheal smooth muscle cells.

The peptide, endothelin-1 (ET-1) regulates proliferative responses in numerous cell types. Recently, a dual ET receptor antagonist was shown to prevent the increase in airway smooth muscle cell (SMC) proliferation that accompanies airway smooth muscle remodeling in a rat model of experimental asthma. Thus, we used [(3)H]-thymidine incorporation assays and western immunoblotting to identify signaling pathways that regulate proliferative responses in cultured rat tracheal SMC. Our data indicate that ET-1 activation of the ET A receptor subtype induced [(3)H]-thymidine incorporation and activation of ERK 1/2 in primary rat tracheal SMC. ET-1-induced [(3)H]-thymidine incorporation and activation of ERK 1/2 were inhibited by pretreatment of SMC with pertussis toxin or down regulation of phorbol ester responsive isoforms of PKC. While ET- 1-induced ERK 1/2 activation was unaffected following inhibition of Rho kinase, ET-1-induced [(3)H]-thymidine incorporation was abrogated. ET-1 also potentiated [(3)H]-thymidine incorporation as well as cell proliferation of SMC stimulated with PDGF-BB and this response did not appear to be regulated by ERK1/ 2. These data demonstrate that ET-1 induces activation of multiple G proteins that regulate rat tracheal SMC proliferative responses, likely through signaling pathways downstream of ERK1/2 and Rho kinase.     

Synthesis and biological evaluation of 4-imidazolylflavans as nonsteroidal aromatase inhibitors

A series of 4-imidazolylflavans having a variety of substituents on the 2-phenyl ring was synthesized and investigated for their inhibitory effect against aromatase. Structure-activity relationships of these compounds were determined. An additional chlorine atom or a cyano group at the 4' position did not enhance aromatase inhibition as well as a 3'-hydroxyl group. The influence of an additional 4'-hydroxyl group depends on the substitution pattern of A ring. Among these molecules, 4'-hydroxy-4-imidazolyl-7-methoxyflavan is only 2.2-fold less active than the letrozole (as assessed by IC50 values). Letrozole is used as the first-line therapy for metastatic breast cancer.     

Mutagenic and membranal effect of a phytotoxic molecule isolated from olive leaves parasitized by the fungusCycloconium oleaginum Cast

A phytotoxic substance (C₂₃H₄₄O₃) which is named Substance A, was purified from olive leaves infected withCycloconium oleaginum Cast. The mutagenic effect of this substance was detected using TA 100 and TA 102 strains ofSalmonella in the Ames test usingBacillus subtilis strains M45 rec^–, H17 rec⁺ in the rec assay. Another substance manifesting the mutagenic effect was found in the extract from theCycloconium oleaginum culture. This substance was not detected in the extract from contaminated olive leaves. Substance A increased electrolytes leakage from tissue of olive leaves, thus manifesting its phytotoxicity.     

Joint analysis for heading date QTL in small interconnected barley populations

The purpose of the present work is to validate the effect of the main QTL determining heading date in a set of 281 doubled haploid lines of barley, derived from 17 small interconnected populations, whose parents are cultivars commonly used in the Spanish barley breeding program. We used 72 molecular markers distributed across the seven chromosomes, particularly in regions known to contain flowering time genes or QTL. A combined linkage map over the 17 populations was constructed. The lines were evaluated in four field trials: two autumn sowings and two winter sowings, and in two treatments at a greenhouse trial, under controlled conditions of photoperiod and temperature. We have found that it is possible to carry out QTL detection in a complex germplasm set, representative of the materials used in an active breeding programme. In most cases two alleles per QTL were detected, though polymorphism of flanking markers was notably higher. The results revealed that there is a set of QTL that accounts for an important percentage of the phenotypic variation, suitable for marker assisted selection. Also, the role of the regions carrying the photoperiod response genes Ppd-H1 and Ppd-H2, the vernalization response genes Vrn-H1 and Vrn-H2, and the earliness per se locus Eam6, of which allele-specific or closely linked markers were available, was confirmed. These results support the use of this kind of approach for the validation of QTL found in single cross population studies, or to survey allelic diversity in plant breeding sets of materials. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Genome analysis at different ploidy levels allows cloning of the powdery mildew resistance gene Pm3b from hexaploid wheat

In wheat, race-specific resistance to the fungal pathogen powdery mildew (Blumeria graminis f. sp. tritici) is controlled by the Pm genes. There are 10 alleles conferring resistance at the Pm3 locus (Pm3a to Pm3j) on chromosome 1AS of hexaploid bread wheat (Triticum aestivum L.). The genome of hexaploid wheat has a size of 1.6 x 1010 bp and contains more than 80% of repetitive sequences, making positional cloning difficult. Here, we demonstrate that the combined analysis of genomes from wheat species with different ploidy levels can be exploited for positional cloning in bread wheat. We have mapped the Pm3b gene in hexaploid wheat to a genetic interval of 0.97 centimorgan (cM). The diploid T. monococcum and the tetraploid T. turgidum ssp. durum provided models for the A genome of hexaploid wheat and allowed to establish a physical contig spanning the Pm3 locus. Although the haplotypes at the Pm3 locus differed markedly between the three species, a large resistance gene-like family specific to wheat group 1 chromosomes was consistently found at the Pm3 locus. A candidate gene for Pm3b was identified using partial sequence conservation between resistant line Chul and T. monococcum cv. DV92. A susceptible Pm3b mutant, carrying a single-base pair deletion in the coding region of the candidate gene was isolated. When tested in a single cell transformation assay, the Pm3b candidate gene conferred race-specific resistance to powdery mildew. These results demonstrate that the candidate gene, a member of the coiled-coil nucleotide binding site leucine-rich repeat (NBS-LRR) type of disease resistance genes, is the Pm3b gene.     

NHERF2/SIP-1 interacts with mouse SRY via a different mechanism than human SRY

In mammals, male sex determination is controlled by the SRY protein, which drives differentiation of the bipotential embryonic gonads into testes by activating the Sertoli cell differentiation program. The morphological effects of SRY are well documented; however, its molecular mechanism of action remains unknown. Moreover, SRY proteins display high sequence variability among mammalian species, which makes protein motifs difficult to delineate. We previously isolated SIP-1/NHERF2 as a human SRY-interacting protein. SIP-1/NHERF2, a PDZ protein, interacts with the C-terminal extremity of the human SRY protein. Here we showed that the interaction of SIP-1/NHERF2 and SRY via the SIP-1/NHERF2 PDZ1 domain is conserved in mice. However, the interaction occurs via a domain that is internal to the mouse SRY protein and involves a different recognition mechanism than human SRY. Furthermore, we show that mouse and human SRY induce nuclear accumulation of the SIP-1/NHERF2 protein in cultured cells. Finally, a transgenic mouse line expressing green fluorescent protein under the control of the mouse Sry promoter allowed us to show that SRY and SIP-1/NHERF2 are co-expressed in the nucleus of pre-Sertoli cells during testis determination. Taken together, our results suggested that the function of SIP-1/NHERF2 as an SRY cofactor during testis determination is conserved between human and mouse.