Association of cortactin and fascin-1 expression in gastric adenocarcinoma: correlation with clinicopathological parameters.

Cortactin and fascin-1 are important factors in tumor progression. We tested the hypothesis that cortactin and fascin-1 expression correlates with clinicopathological parameters of gastric adenocarcinoma. Immunohistochemical analysis of cortactin and fascin-1 was done using tissue microarrays of 100 surgical specimens, including 20 well-differentiated, 20 moderately differentiated, and 60 poorly differentiated gastric adenocarcinomas. Among the 20 well-differentiated gastric adenocarcinomas, 15 cases (75%) showed negative or weak staining (1+); 5 cases (25%) had moderate (2+) or strong (3+) cortactin expression. Among the 60 poorly differentiated gastric adenocarcinomas, more than three-quarters of the cases (76.7%) had moderate or strong cortactin expression; 14 cases (23.3%) had weak staining. Of 20 well-differentiated gastric adenocarcinoma cases, 14 (70%) showed negative or weak staining of fascin-1, whereas nearly one-third (30%) had moderate or strong expression. Among the 60 poorly differentiated gastric adenocarcinomas, 32 (53.3%) exhibited moderate or strong fascin-1 expression; fewer than half of the cases showed negative or weak staining. Higher intensity of cortactin and fascin-1 staining correlated directly with more-advanced cancer stages (TNM) and inversely with survival rates. Our findings suggest the possibility that pharmacological inhibitors of cortactin and fascin-1 activity may slow down tumor progression and prolong survival time in patients with gastric adenocarcinomas.     

Expression and purification of an FGF9 fusion protein in E. coli,and the effects of the FGF9 subfamily on human hepatocellular carcinoma cell proliferation and migration

Fibroblast growth factor (FGF) 9 has oncogenic activity and plays an important role in the development of ovarian, lung, prostate, and gastric cancers. In the present study, with the aim of reducing the cost of utilizing growth factors in cancer research, a simple and efficient method for the preparation of recombinant human (rh)FGF9 in Escherichia coli was established. The rhFGF9 fusion protein (6 × His-TEV-rhFGF9) and the native protein released by tobacco etch virus (TEV) protease were obtained using a Ni-NTA system, with > 95% purity. Both purified forms of rhFGF9, with and without fusion tags, significantly stimulated the proliferation of NIH3T3 cells. The FGF9 subfamily, including FGF9, FGF16, and FGF20, in addition to rhFGF16, rhFGF9, and rhFGF20, were shown to stimulate the proliferation and migration of HuH7 human hepatocellular carcinoma (HCC) cells. Mechanistic studies revealed that the stimulation of HuH7 cell proliferation and migration with rhFGF9 and rhFGF20 were associated with the activation of the extracellular signal-regulated kinase (ERK) and nuclear factor κB (NF-κB) pathways and matrix metalloproteinase-26 (MMP26). Inhibition of the ERK and NF-κB pathways blocked cell migration, and NF-κB was demonstrated to be regulated by ERK. Therefore, the present study demonstrates a simple method for the preparation of biologically active rhFGF9 protein. Furthermore, the results indicate that exogenous rhFGF9- and rhFGF20-activated ERK/NF-κB signal transduction pathways play important roles in the regulation of HCC cell proliferation and migration, and this discovery helps to find the potential for new solutions of the treatment of liver cancer.

     

Plk1 Self-Organization and Priming Phosphorylation of HsCYK-4 at the Spindle Midzone Regulate the Onset of Division in Human Cells

Animal cells initiate cytokinesis in parallel with anaphase onset, when an actomyosin ring assembles and constricts through localized activation of the small GTPase RhoA, giving rise to a cleavage furrow. Furrow formation relies on positional cues provided by anaphase spindle microtubules (MTs), but how such cues are generated remains unclear. Using chemical genetics to achieve both temporal and spatial control, we show that the self-organized delivery of Polo-like kinase 1 (Plk1) to the midzone and its local phosphorylation of a MT-bound substrate are critical for generating this furrow-inducing signal. When Plk1 was active but unable to target itself to this equatorial landmark, both cortical RhoA recruitment and furrow induction failed to occur, thus recapitulating the effects of anaphase-specific Plk1 inhibition. Using tandem mass spectrometry and phosphospecific antibodies, we found that Plk1 binds and directly phosphorylates the HsCYK-4 subunit of centralspindlin (also known as MgcRacGAP) at the midzone. At serine 157, this modification creates a major docking site for the tandem BRCT repeats of the Rho GTP exchange factor Ect2. Cells expressing only a nonphosphorylatable form of HsCYK-4 failed to localize Ect2 at the midzone and were severely impaired in cleavage furrow formation, implying that HsCYK-4 is Plk1's rate-limiting target upstream of RhoA. Conversely, tethering an inhibitor-resistant allele of Plk1 to HsCYK-4 allowed furrows to form despite global inhibition of all other Plk1 molecules in the cell. Our findings illuminate two key mechanisms governing the initiation of cytokinesis in human cells and illustrate the power of chemical genetics to probe such regulation both in time and space.     

麒麟紫葳的组织培养与快速繁殖

1植物名称麒麟紫葳(Radermachera sinica),又名幸福树、菜豆树. 2材料类别植株带芽茎段. 3培养条件芽诱导、分化培养基:(1)MS 6-BA0.5 mg·L-1(单位下同) IBA 0.2;生根培养基:(2)1/2MS IBA 0.5.上述培养基均添加3%蔗糖、0.6%琼脂,pH(5.8 0.1).培养温度为(28 2)℃,光照时间10 h·d-1,光照度为2 000 1x.     

琼胶酶研究进展

琼胶酶是一种多糖水解酶, 根据其降解琼脂糖的作用方式不同, 可以分为a- 琼胶酶(EC 3. 2.1. -) 和b- 琼胶酶(EC 3.2.1.81)。本文结合自己的研究, 从琼胶酶的生物学研究、酶的分类、晶体结构、催化机理以及酶的应用等几个方面综述了琼胶酶的研究进展。     

鳜早期发育阶段骨骼肌纤维的增生与肥大生长

骨骼肌是鱼体的主要组成部分,也是衡量鱼体生长发育的重要指标.为了解鳜(Siniperca chuatsi)早期发育阶段骨骼肌纤维的生长发育特征,通过制作孵化后1 ～41日龄个体骨骼肌背右侧第一肌节石蜡切片,利用图像分析软件统计该肌节中肌纤维的数目和面积,分析了鳜骨骼肌纤维的增生和肥大生长特征.结果表明,鳜早期发育阶段骨...     

Cdc42 induces activation loop phosphorylation and membrane targeting of mixed lineage kinase 3

Mixed lineage kinase 3 (MLK3) functions as a mitogen-activated protein kinase kinase kinase to activate multiple mitogen-activated protein kinase pathways. Our current studies demonstrate that lack of MLK3 blocks signaling of activated Cdc42 to c-Jun N-terminal kinase, giving strong support for the idea that Cdc42 is a physiological activator of MLK3. We show herein that Cdc42, in a prenylation-dependent manner, targets MLK3 from a perinuclear region to membranes, including the plasma membrane. Cdc42-induced membrane targeting of MLK3 is independent of MLK3 catalytic activity but depends upon an intact Cdc42/Rac-interactive binding motif, consistent with MLK3 membrane translocation being mediated through direct binding of Cdc42. Phosphorylation of the activation loop of MLK3 requires MLK3 catalytic activity and is induced by Cdc42 in a prenylation-independent manner, arguing that Cdc42 binding is sufficient for activation loop autophosphorylation of MLK3. However, membrane targeting is necessary for full activation of MLK3 and maximal signaling to JNK. We previously reported that MLK3 is autoinhibited through an interaction between its N-terminal SH3 domain and a proline-containing sequence found between the leucine zipper and the CRIB motif of MLK3. Thus we propose a model in which GTP-bound Cdc42/Rac binds MLK3 and disrupts SH3-mediated autoinhibition leading to dimerization and activation loop autophosphorylation. Targeting of this partially active MLK3 to membranes likely results in additional phosphorylation events that fully activate MLK3 and its ability to maximally signal through the JNK pathway.     

A chemical-genetic approach to studying neurotrophin signaling

Trk tyrosine kinases are receptors for members of the neurotrophin family and are crucial for growth and survival of specific populations of neurons. Yet, the functions of neurotrophin-Trk signaling in postnatal development as well as maintenance and plasticity of the adult nervous system are less clear. We report here the generation of mice harboring Trk knockin alleles that allow for pharmacological control of Trk kinase activity. Nanomolar concentrations of either 1NMPP1 or 1NaPP1, derivatives of the general kinase inhibitor PP1, inhibit NGF and BDNF signaling in TrkA(F592A) and TrkB(F616A) neurons, respectively, while no such Trk inhibition is observed in wild-type neurons. Moreover, oral administration of 1NMPP1 leads to specific inhibition of TrkA(F592A), TrkB(F616A), and TrkC(F167A) signaling in vivo. Thus, Trk knockin mice provide valuable tools for selective, rapid, and reversible inhibition of neurotrophin signaling in vitro and in vivo.     

Chemical xenobiotics and mitochondrial autoantigens in primary biliary cirrhosis: identification of antibodies against a common environmental, cosmetic, and food additive, 2-octynoic acid

Emerging evidence has suggested environmental factors as causative agents in the pathogenesis of primary biliary cirrhosis (PBC). We have hypothesized that in PBC the lipoyl domain of the immunodominant E2 component of pyruvate dehydrogenase (PDC-E2) is replaced by a chemical xenobiotic mimic, which is sufficient to break self-tolerance. To address this hypothesis, based upon our quantitative structure-activity relationship data, a total of 107 potential xenobiotic mimics were coupled to the lysine residue of the immunodominant 15 amino acid peptide of the PDC-E2 inner lipoyl domain and spotted on microarray slides. Sera from patients with PBC (n = 47), primary sclerosing cholangitis (n = 15), and healthy volunteers (n = 20) were assayed for Ig reactivity. PBC sera were subsequently absorbed with native lipoylated PDC-E2 peptide or a xenobiotically modified PDC-E2 peptide, and the remaining reactivity analyzed. Of the 107 xenobiotics, 33 had a significantly higher IgG reactivity against PBC sera compared with control sera. In addition, 9 of those 33 compounds were more reactive than the native lipoylated peptide. Following absorption, 8 of the 9 compounds demonstrated cross-reactivity with lipoic acid. One compound, 2-octynoic acid, was unique in both its quantitative structure-activity relationship analysis and reactivity. PBC patient sera demonstrated high Ig reactivity against 2-octynoic acid-PDC-E2 peptide. Not only does 2-octynoic acid have the potential to modify PDC-E2 in vivo but importantly it was/is widely used in the environment including perfumes, lipstick, and many common food flavorings.