马铃薯人工种子的研究

本研究以虎头（Ｈ）、克四（Ｋ）和Ｆａｖｏｒｉｔａ（Ｆ）三个品种的马铃薯不定芽在液体培养条件下诱导生成的微型薯为试材,经筛选后获得的大小一致的微型薯经过适度的低温处理后,用４％的海藻酸钠和２％的氯化钙溶液,在附加一定的植物激素条件下,进行人。种皮包埋形成人工种子。在无菌条件下,三个品种的人工种子萌发率均达９０％以上。在土壤中,人工种子萌发率可达６０％以上,萌发的人工种子中８０％能够形成生长发育正常的完整植株。     

白喉毒素类免疫毒素研究进展

白喉毒素类免疫毒素是将缺失天然受体结合活性的白喉毒素片段或突变体与抗体或细胞因子偶联而得到的一类新型导向药物,它可特异性识别并结合靶细胞,通过发挥其ADP核糖基化活性而抑制细胞蛋白合成,引发细胞凋亡。由于白喉毒素类免疫毒素能高效、特异地杀伤特定靶细胞,而使其在肿瘤等疾病的药物开发中暂露头角。综述了基于白喉毒素的免疫毒素的研制现状与应用前景 。     

DNMT3A rs36012910 A>G polymorphism and gastric cancer susceptibility in a Chinese population

DNA-methyltransferase (DNMT)-3A plays a crucial role in embryonic development and aberrant DNA methylation in carcinogenesis. Polymorphisms of the DNMT3A gene may influence its enzymatic activity and its contribution to susceptibility to cancer. This study evaluated the association of DNMT3A rs36012910 A>G with susceptibility to gastric cancer (GC) in a Chinese population. Genomic DNA was extracted from samples taken from 340 patients with GC and 251 healthy control subjects. The genotype frequency of DNMT3A rs36012910 A>G in all subjects was detected by polymerase chain reaction–restriction fragment length polymorphism and confirmed by sequencing. Stratification analyses were used to study subgroups by age and gender and to evaluate the association of rs36012910 A>G polymorphism with genetic susceptibility to GC. All patients and control individuals were successfully genotyped for the DNMT3A rs36012910 A>G polymorphism. The frequency of DNMT3A rs36012910 allele G is 3.39?% in healthy individuals and 7.78?% in GC patients, respectively. The rs36012910 AG genotype was significantly more common in the GC group than in the controls, although the rs36012910 GG genotype was only one case in GC patients. Further stratification indicated that AG+GG genotypes were associated with susceptibility to GC in males older than 60, but this polymorphism has no significant association with GC susceptibility in females. Male individuals who carried AG+GG genotypes had a 2.362-fold increased risk of GC compared to those who carried the AA genotype. The rs36012910 allele G was associated with an increased risk of GC compared to the rs36012910 allele A. This is the first report to investigate the distribution and evaluate the association of a rare SNP in DNMT3A with genetic susceptibility to GC. DNMT3A rs36012910 A>G might become a potential biomarker for use in GC prediction, although further studies in larger groups and different populations are needed for confirmation.     

A small-molecule activator induces ULK1-modulating autophagy-associated cell death in triple negative breast cancer

ULK1 (unc-51 like autophagy activating kinase 1) is well known to be required to initiate the macroautophagy/autophagy process, and thus activation of ULK1-modulating autophagy/autophagy-associated cell death (ACD) may be a possible therapeutic strategy in triple negative breast cancer (TNBC). Here, our integrated The Cancer Genome Atlas (TCGA) data set, tissue microarray-based analyses and multiple biologic evaluations together demonstrate a new small-molecule activator of ULK1 for better understanding of how ULK1, the mammalian homolog of yeast Atg1, as a potential drug target can regulate ACD by the ULK complex (ULK1-ATG13-RB1CC1/FIP200-ATG101), as well as other possible ULK1 interactors, including ATF3, RAD21 and CASP3/caspase3 in TNBC. Moreover, such new inspiring findings may help us discover that this activator of ULK1 (LYN-1604) with its anti-tumor activity and ACD-modulating mechanisms can be further exploited as a small-molecule candidate drug for future TNBC therapy.     

Identification of novel caspase/autophagy‐related gene switch to cell fate decisions in breast cancers

Objectives

Caspases, a family of cysteine proteases with unique substrate specificities, contribute to apoptosis, whereas autophagy‐related genes (ATGs) regulate cytoprotective autophagy or autophagic cell death in cancer. Accumulating evidence has recently revealed underlying mechanisms of apoptosis and autophagy; however, their intricate relationships still remain to be clarified. Identification of caspase/ATG switches between apoptosis and autophagy may address this problem.

Materials and methods

Identification of caspase/ATG switches was carried out using a series of elegant systems biology & bioinformatics approaches, such as network construction, hub protein identification, microarray analyses, targeted microRNA prediction and molecular docking.

Results

We computationally constructed the global human network from several online databases and further modified it into the basic caspase/ATG network. On the basis of apoptotic or autophagic gene differential expressions, we identified three molecular switches [including androgen receptor, serine/threonine‐protein kinase PAK‐1 (PAK‐1) and mitogen‐activated protein kinase‐3 (MAPK‐3)] between certain caspases and ATGs in human breast carcinoma MCF‐7 cells. Subsequently, we identified microRNAs (miRNAs) able to target androgen receptor, PAK‐1 and MAPK‐3, respectively. Ultimately, we screened a range of small molecule compounds from DrugBank, able to target the three above‐mentioned molecular switches in breast cancer cells.

Conclusions

We have systematically identified novel caspase/ATG switches involved in miRNA regulation, and predicted targeted anti‐cancer drugs. These findings may uncover intricate relationships between apoptosis and autophagy and thus provide further new clues towards possible cancer drug discovery.

     

Regulators of G-protein signaling (RGS) 4, insertion into model membranes and inhibition of activity by phosphatidic acid

Regulators of G-protein signaling (RGS) proteins are critical for attenuating G protein-coupled signaling pathways. The membrane association of RGS4 has been reported to be crucial for its regulatory activity in reconstituted vesicles and physiological roles in vivo. In this study, we report that RGS4 initially binds onto the surface of anionic phospholipid vesicles and subsequently inserts into, but not through, the membrane bilayer. Phosphatidic acid, one of anionic phospholipids, could dramatically inhibit the ability of RGS4 to accelerate GTPase activity in vitro. Phosphatidic acid is an effective and potent inhibitor of RGS4 in a G alpha(i1)-[gamma-(32)P]GTP single turnover assay with an IC(50) approximately 4 microm and maximum inhibition of over 90%. Furthermore, phosphatidic acid was the only phospholipid tested that inhibited RGS4 activity in a receptor-mediated, steady-state GTP hydrolysis assay. When phosphatidic acid (10 mol %) was incorporated into m1 acetylcholine receptor-G alpha(q) vesicles, RGS4 GAP activity was markedly inhibited by more than 70% and the EC(50) of RGS4 was increased from 1.5 to 7 nm. Phosphatidic acid also induced a conformational change in the RGS domain of RGS4 measured by acrylamide-quenching experiments. Truncation of the N terminus of RGS4 (residues 1-57) resulted in the loss of both phosphatidic acid binding and lipid-mediated functional inhibition. A single point mutation in RGS4 (Lys(20) to Glu) permitted its binding to phosphatidic acid-containing vesicles but prevented lipid-induced conformational changes in the RGS domain and abolished the inhibition of its GAP activity. We speculate that the activation of phospholipase D or diacylglycerol kinase via G protein-mediated signaling cascades will increase the local concentration of phosphatidic acid, which in turn block RGS4 GAP activity in vivo. Thus, RGS4 may represent a novel effector of phosphatidic acid, and this phospholipid may function as a feedback regulator in G protein-mediated signaling pathways.     

The extracellular loops of Smoothened play a regulatory role in control of Hedgehog pathway activation

The Hedgehog (Hh) signaling pathway plays an instructional role during development, and is frequently activated in cancer. Ligand-induced pathway activation requires signaling by the transmembrane protein Smoothened (Smo), a member of the G-protein-coupled receptor (GPCR) superfamily. The extracellular (EC) loops of canonical GPCRs harbor cysteine residues that engage in disulfide bonds, affecting active and inactive signaling states through regulating receptor conformation, dimerization and/or ligand binding. Although a functional importance for cysteines localized to the N-terminal extracellular cysteine-rich domain has been described, a functional role for a set of conserved cysteines in the EC loops of Smo has not yet been established. In this study, we mutated each of the conserved EC cysteines, and tested for effects on Hh signal transduction. Cysteine mutagenesis reveals that previously uncharacterized functional roles exist for Smo EC1 and EC2. We provide in vitro and in vivo evidence that EC1 cysteine mutation induces significant Hh-independent Smo signaling, triggering a level of pathway activation similar to that of a maximal Hh response in Drosophila and mammalian systems. Furthermore, we show that a single amino acid change in EC2 attenuates Hh-induced Smo signaling, whereas deletion of the central region of EC2 renders Smo fully active, suggesting that the conformation of EC2 is crucial for regulated Smo activity. Taken together, these findings are consistent with loop cysteines engaging in disulfide bonds that facilitate a Smo conformation that is silent in the absence of Hh, but can transition to a fully active state in response to ligand.     

Distinct roles of IL-22 in human psoriasis and inflammatory bowel disease

IL-22, an IL-10 family cytokine, is produced by different leukocyte subsets, including T cells, NK cells and lymphoid tissue inducer (LTi) cells. IL-22 mediates the crosstalk between leukocytes and tissue epithelia because its receptor is preferentially expressed on various tissue epithelial cells. IL-22 is essential for host defense against infections of extracellular pathogens, such as bacteria and yeasts, by eliciting various innate defensive mechanisms from tissue epithelial cells and promoting wound-healing responses. In autoimmune diseases, however, diverse tissue microenvironments and underlying pathogenic mechanisms may result in opposing contributions of IL-22 in disease progression. For example, in psoriasis, IL-22 can synergize with other proinflammatory cytokines to induce many of the pathogenic phenotypes from keratinocytes and exacerbate disease progression. In contrast, IL-22 plays a beneficial role in IBD by enhancing barrier integrity and epithelial innate immunity of intestinal tract.