The CRKL gene encoding an adaptor protein is amplified, overexpressed, and a possible therapeutic target in gastric cancer

<p>Abstract</p> <p>Background</p> <p>Genomic DNA amplification is a genetic factor involved in cancer, and some oncogenes, such as <it>ERBB2</it>, are highly amplified in gastric cancer. We searched for the possible amplification of other genes in gastric cancer.</p> <p>Methods and Results</p> <p>A genome-wide single nucleotide polymorphism microarray analysis was performed using three cell lines of differentiated gastric cancers, and 22 genes (including <it>ERBB2</it>) in five highly amplified chromosome regions (with a copy number of more than 6) were identified. Particular attention was paid to the <it>CRKL</it> gene, the product of which is an adaptor protein containing Src homology 2 and 3 (SH2/SH3) domains. An extremely high <it>CRKL</it> copy number was confirmed in the MKN74 gastric cancer cell line using fluorescence <it>in situ</it> hybridization (FISH), and a high level of CRKL expression was also observed in the cells. The RNA-interference-mediated knockdown of CRKL in MKN74 disclosed the ability of CRKL to upregulate gastric cell proliferation. An immunohistochemical analysis revealed that CRKL protein was overexpressed in 24.4% (88/360) of the primary gastric cancers that were analyzed. The <it>CRKL</it> copy number was also examined in 360 primary gastric cancers using a FISH analysis, and <it>CRKL</it> amplification was found to be associated with CRKL overexpression. Finally, we showed that MKN74 cells with <it>CRKL</it> amplification were responsive to the dual Src/BCR-ABL kinase inhibitor BMS354825, likely via the inhibition of CRKL phosphorylation, and that the proliferation of MKN74 cells was suppressed by treatment with a CRKL-targeting peptide.</p> <p>Conclusion</p> <p>These results suggested that CRKL protein is overexpressed in a subset of gastric cancers and is associated with <it>CRKL</it> amplification in gastric cancer. Furthermore, our results suggested that CRKL protein has the ability to regulate gastric cell proliferation and has the potential to serve as a molecular therapy target for gastric cancer.</p

Identification of candidate flowering and sex genes in white Guinea yam (D. rotundata Poir.) by SuperSAGE transcriptome profiling

Open Access JournalDioecy (distinct male and female individuals) combined with scarce to non-flowering are common features of cultivated yam (Dioscorea spp.). However, the molecular mechanisms underlying flowering and sex determination in Dioscorea are unknown. We conducted SuperSAGE transcriptome profiling of male, female and monoecious individuals to identify flowering and sex-related genes in white Guinea yam (D. rotundata). SuperSAGE analysis generated a total of 20,236 unique tags, of which 13,901 were represented by a minimum of 10 tags. Of these, 88 tags were significantly differentially expressed in male, female and monoecious plants. Of the 88 differentially expressed SuperSAGE tags, 18 corresponded to genes previously implicated in flower development and sex determination in multiple plant species. We validated the SuperSAGE data with quantitative real-time PCR (qRT-PCR)-based analysis of the expression of four candidate genes. Our findings suggest that mechanisms of flowering and sex determination are likely conserved in Dioscorea. We further investigated the flowering patterns of 1938 D. rotundata accessions representing diverse geographical origins over two years, revealing that over 85% of the accessions are either male or non-flowering, and that less than 15% are female, while monoecious plants are rare. Intensity of flowering appeared to be a function of sex, with male plants flowering more abundantly than female ones. Candidate genes identified in this study can be targeted with the aim to induce regular flowering in poor to non-flowering cultivars. Findings of the study provide important inputs for further studies aiming to overcome the challenge of flowering in yams and to improve the efficiency of yam breeding

Genome analyses reveal the hybrid origin of the staple crop white Guinea yam (Dioscorea rotundata)

西アフリカの主食作物ギニアヤムの起源を解明 --ギニアヤムはサバンナと熱帯雨林に生育する野生種の雑種起源--. 京都大学プレスリリース. 2020-12-11.White Guinea yam (Dioscorea rotundata) is an important staple tuber crop in West Africa. However, its origin remains unclear. In this study, we resequenced 336 accessions of white Guinea yam and compared them with the sequences of wild Dioscorea species using an improved reference genome sequence of D. rotundata. In contrast to a previous study suggesting that D. rotundata originated from a subgroup of Dioscorea praehensilis, our results suggest a hybrid origin of white Guinea yam from crosses between the wild rainforest species D. praehensilis and the savannah-adapted species Dioscorea abyssinica. We identified a greater genomic contribution from D. abyssinica in the sex chromosome of Guinea yam and extensive introgression around the SWEETIE gene. Our findings point to a complex domestication scenario for Guinea yam and highlight the importance of wild species as gene donors for improving this crop through molecular breeding

Differing Patterns of P-Selectin Expression in Lung Injury

Using two models of acute lung inflammatory injury in rats (intrapulmonary deposition of immunoglobulin G immune complexes and systemic activation of complement after infusion of purified cobra venom factor), we have analyzed the requirements and patterns for upregulation of lung vascular P-selectin. In the immune complex model, upregulation of P-selectin was defined by Northern and Western blot analysis of lung homogenates, by immunostaining of lung tissue, and by vascular fixation of (125)I-labeled anti-P-selectin. P-selectin protein was detected by 1 hour (long before detection of mRNA) and expression was sustained for the next 7 hours, in striking contrast to the pattern of P-selectin expression in the cobra venom factor model, in which upregulation was very transient (within the 1st hour). In the immune complex model, injury and neutrophil accumulation were P-selectin dependent. Upregulation of P-selectin was dependent on an intact complement system, and the presence of blood neutrophils was susceptible to the antioxidant dimethyl sulfoxide and required C5a but not tumor necrosis factor α. In contrast, in the cobra venom factor model, upregulation of P-selectin, which is C5a dependent, was also dimethyl sulfoxide sensitive but neutrophil independent. Different mechanisms that may explain why upregulation of lung vascular P-selectin is either transient or sustained are discussed

Comprehensive predictions of target proteins based on protein-chemical interaction using virtual screening and experimental verifications

<p>Abstract</p> <p>Background</p> <p>Identification of the target proteins of bioactive compounds is critical for elucidating the mode of action; however, target identification has been difficult in general, mostly due to the low sensitivity of detection using affinity chromatography followed by CBB staining and MS/MS analysis.</p> <p>Results</p> <p>We applied our protocol of predicting target proteins combining <it>in silico</it> screening and experimental verification for incednine, which inhibits the anti-apoptotic function of Bcl-xL by an unknown mechanism. One hundred eighty-two target protein candidates were computationally predicted to bind to incednine by the statistical prediction method, and the predictions were verified by <it>in vitro</it> binding of incednine to seven proteins, whose expression can be confirmed in our cell system.</p> <p>As a result, 40% accuracy of the computational predictions was achieved successfully, and we newly found 3 incednine-binding proteins.</p> <p>Conclusions</p> <p>This study revealed that our proposed protocol of predicting target protein combining <it>in silico</it> screening and experimental verification is useful, and provides new insight into a strategy for identifying target proteins of small molecules.</p