Small RNA interference-mediated ADP-ribosylation factor 6 silencing inhibits proliferation, migration and invasion of human prostate cancer PC-3 cells / 南方医科大学学报

<p><b>OBJECTIVE</b>To investigate the effects of silencing ADP-ribosylation factor 6 (Arf6) on the proliferation, migration, and invasion of prostate cancer cell line PC-3 and the possible molecular mechanisms.</p><p><b>METHODS</b>Three Arf6-specific small interfering RNA (siRNA) were transfected into cultured prostate cancer cell line PC-3. Arf6 expression was examined by real-time PCR and Western blotting. MTT assay, wound healing assay, and Transwell migration and invasion assay were used to observe the effect of Arf6 silencing on the proliferation, migration, and invasion ability of PC-3 cells. The levels of phosphorylated extracellular signal-regulated kinase 1/2 (p-ERK1/2), ERK1/2, p-AKT, AKT and Rac1 were detected by Western blotting.</p><p><b>RESULTS</b>Transfection of siRNA-3 resulted in significantly decreased Arf6 mRNA and protein expression with inhibition rates of (91.88±3.13)% and (86.37±0.57)%, respectively. Arf6 silencing by siRNA-3 markedly suppressed the proliferation, migration and invasion of PC-3 cells and reduced the expression levels of p-ERK1/2 and Rac1.</p><p><b>CONCLUSION</b>Silencing of Arf6 efficiently inhibits the proliferation, migration, and invasion of PC-3 cells in vitro, and the underlying mechanisms may involve the down-regulation of p-ERK1/2 and Rac1.</p>

Isolation and analysis of differentially expressed genes from peanut in response to challenge with Ralstonia solanacearum

Background: Bacterial wilt caused by Ralstonia solanacearum is the most devastating disease in peanut. Planting resistant peanut cultivars is deemed as the sole economically viable means for effective control of the disease. To understand the molecular mechanism underlying resistance and facilitate breeding process, differences in gene expression between seeds of Rihua 1 (a Virginia type peanut variety resistant to bacterial wilt) inoculated with the bacterial pathogen suspension (10(9) cfu ml-1) and seeds of the same cultivar treated with water (control), were studied using the GenefishingTM technology. Results: A total of 25 differentially expressed genes were isolated. Expression of genes encoding cyclophilin and ADP-ribosylation factor, respectively, were further studied by real time RT-PCR, and full length cDNAs of both genes were obtained by rapid amplification of cDNA ends. Conclusions: The study provided candidate genes potentially useful for breeding peanut cultivars with both high yield and bacterial wilt resistance, although confirmation of their functions through transgenic studies is still needed.

Role of phospholipase D1 in glucose-induced insulin secretion in pancreatic beta cells

As glucose is known to induce insulin secretion in pancreatic beta cells, this study investigated the role of a phospholipase D (PLD)-related signaling pathway in insulin secretion caused by high glucose in the pancreatic beta-cell line MIN6N8. It was found that the PLD activity and PLD1 expression were both increased by high glucose (33.3 mM) treatment. The dominant negative PLD1 inhibited glucose-induced Beta2 expression, and glucose-induced insulin secretion was blocked by treatment with 1-butanol or PLD1-siRNA. These results suggest that high glucose increased insulin secretion through a PLD1-related pathway. High glucose induced the binding of Arf6 to PLD1. Pretreatment with brefeldin A (BFA), an Arf inhibitor, decreased the PLD activity as well as the insulin secretion. Furthermore, BFA blocked the glucose-induced mTOR and p70S6K activation, while mTOR inhibition with rapamycin attenuated the glucose induced Beta2 expression and insulin secretion. Thus, when taken together, PLD1 would appear to be an important regulator of glucose-induced insulin secretion through an Arf6/PLD1/mTOR/p70S6K/Beta2 pathway in MIN6N8 cells.

The importance of Arabidopsis seed mutants in the elucidation of the molecular basis of Endosperm Balance Number in tuber-bearing Solanum species

The Endosperm Balance Number (EBN) is an important concept for potato breeding and has evolutionary importance in tuber-bearing Solanum species. The EBN is part of the post-zygotic hybridization barriers in the group and represents a reproductive isolating mechanism. Few genes have been proposed to be involved in its genetic control; until now, however, neither specific genes nor its molecular basis have been well established. Histological observations of embryo and endosperm development in inter-EBN crosses in tuber-bearing Solanum revealed phenotypes similar to those recently described in Arabidopsis seed mutants. The common feature between them is that the endosperm nuclei become greatly enlarged and that embryos are arrested at the globular stage. The proteins encoded by the Arabidopsis TITAN genes are related to chromosome dynamics and cell division. Based on the sequence of titan mutants, genes in potato species related to cell cycle and microtubule assembly were isolated. In this article a perspective model is proposed to explore the utility of Arabidopsis mutants associated with cell cycle control as a tool to elucidate the molecular basis of EBN in potato. Further research focused on the expression pattern of these genes in intra- and inter-EBN crosses in potato species will be performed.

Expression analysis of mouse homologous proteins with human aldose reductase like-1 / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To detect expression of mouse ARL-1 homologous proteins in mouse tissues, and analyze homology, genetic distance and phylogenetic relationship between human aldose reductase like-1 (ARL-1) and mouse homologous proteins.</p><p><b>METHODS</b>Homology of mouse ARL-1 homologous proteins with human ARL-1 was analyzed by software Clustal X 1.8 using GenBank and Swiss-Prot database; genetic distance and phylogenetic relationship between mouse ARL-1 homologous proteins and human ARL-1 were analyzed by software Mega 2.0; mouse tissues were detected by Western blotting using polyclonal antibodies against ARL-1 protein from domestic rabbits.</p><p><b>RESULTS</b>The amino acid sequence of human ARL-1 was 83%, 82%, 81%, 79%, 70%, 51%, 50% and 45% identical to that of the Chinese hamster ovary reductase (CHO-Red), the mouse fibroblast growth factor-regulated protein (FR-1), rat aldose reductase-like protein (rARLP), the mouse vas deferens protein (MVDP), rat lens aldose reductase (LeAR), delta4-3-ketosteroid-5beta-reductase (5beta-Red), rat aldo-keto reductase protein c (RaK-c) and 3alpha-hydroxysteroid dehydrogenase (3alpha-HSD). Among all the mouse ARL-1 homologous proteins, the genetic distance between CHO-Red and human ARL was the shortest (18.0%, P = 0.023), next was FR-1 (19.1%, P=0.023) and rARLP (19.9%, P = 0.025). From the phylogenetic tree, the protein whose relationship with human ARL-1 was the closest with CHO-Red, next was mouse FR-1, rARLP, MVDP and LeAR. Homologous proteins were found in mouse tissues including vas deferens, testis, bladder and uterus by Western blotting using polyclonal antibodies against ARL-1 protein from domestic rabbits.</p><p><b>CONCLUSIONS</b>CHO-Red has the highest homology, the shortest genetic distance and the closest relationship with human ARL-1, next is FR-1, rARLP, MVDP. The major distribution of mouse ARL-1 homologous proteins is in vas deferens, testis, bladder and uterus, deducing they might be CHO-Red, FR-1, rARLP or MVDP</p>

Cytohesins and centaurins control subcellular trafficking of macromolecular signaling complexes: regulation by phosphoinositides and ADP-ribosylation factors

The ADP-ribosylation factor family of small GTP-binding proteins are implicated in the regulation of vesicular transport and control of cytoskeletal and cell adhesion events. The phosphoinositide 3-kinase, phosphoinositide 4-P 5-kinase and phospholipase D signaling pathways are major regulators of ARF signaling cascades. Two families of ARF regulatory molecules, the cytohesin ARF-Guanine nucleotide Exchange Factors and the centaurin GTPase-Activating Proteins provide key targets for the action of these lipids signals. A critical feature of the regulation of ARF signaling is coordinated recruitment of exchange factors, ARFs and GAPs to appropriate subcellular locations. These complexes drive repetitive cycles of ARF activation and membrane association that underlie the processes of cell movement as well as endosomal uptake and intracellular redistribution of signaling molecules. Cytohesins and centaurins bind specifically to a variety of other signaling proteins and these interactions may provide routes for regulated recruitment to the sites of ARF activation. Through their ability to control endosomal trafficking/recycling of these supramolecular signaling complexes ARF and phospholipid signaling pathways may have consequences that reach as far as the regulation of gene transcription and control of cell fate

Activation of Neutrophil Menbrane Phospholipase D by Soluble Proteins: Comparison of Cytosolic Neutrophil 50 kDa Factor , ADP-ribosylation Factor and a Novel Brain Factor / 대한면역학회지

GTPrS-dependent phospholipase D activity in human neutrophils was investigated using exogenous phospholipid as a substrate. Both cytosolic and membrane- associated phospholipase D activities were identified. The previously described 50 kDa cytosolic activating factor was resolved chromatographically from the cytosolic phospholipase D. Using exogenous phospholipid as substrate along with chromatographically resolved 50 kDa factor and recombinant ADP-ribosylation factor 1, plasma membrane was required for activity, indicating that the activity which was previously seen using endogenous phospholipid substrate was due to a phospholipase D located in the plasma membrane. In addition, ADP-ribosylation factor and the 50 kDa factor activated synergistically. Using neutrophil plasma membranes, a third regulator of neutrophil membrane phospholipase D was identified from bovine brain cytosol. This factor was resolved from ADP-ribosylation factor and Rho A by successive column chromatographies. The brain factor showed a synergistic effect with the 50 kDa neutrophil activator but an additive effect with recombinant ADP- ribosylation factor. Whether or not ADP-ribosylation factor or the brain factor were present, high activities were seen only when the 50 kDa factor was present, indicating that the 50 kDa cytosolic factor is a major activating factor for the neutrophil plasma membrane phospholipase D.