全自动生化分析仪测定血清AST同工酶

应用天冬氨酸氨基转移酶抑制剂“AMANO-3”水解样品中的线粒体型天冬氨酸氨基转移酶同工酶（m-AST）,测定血清中剩余的胞浆型AST同工酶（c-AST）活性,进而与总AST活性相比较并计算出受抑制剂水解的m-AST同工酶活性．由于此方法采用蛋白水解反应破坏m-AST,因此测定方法可直接应用于生化自动分析仪．亦建立了一个适于日立7150分析仪的AST同工酶联合测定方法．其测定和计算出的m-AST同工酶批内CV为3.5％～7.9％;其结果(y)与AST同工酶电泳迁移率(x)相关．y=1.019x-0.489,r＝0.996（n＝30）．测定了113名健康人m-AST和c-AST,其m-AST参考值范围1.64～9.64U/L,x±s＝(5.641±2.013)U/L;c-AST参考值范围5.69～16.81U/L,x±s＝(5.641±2.013)U/L．     

PLS3 predicts poor prognosis in pancreatic cancer and promotes cancer cell proliferation via PI3K/AKT signaling

Plastin-3 plays a key role in cancer cell proliferation and invasion, but its prognostic value in pancreatic cancer (PACA) remains poorly defined. In this study, we show that PLS3 messenger RNA is overexpressed in PACA tissue compared with normal tissue. We accumulated 207 cases of PACA specimens to perform immunohistochemical analysis and demonstrated that PLS3 levels correlate with T-classification (p < .001) and pathology (p < .001). Furthermore, overall survival rates (p < .001) in tumors with high PLS3 expression were poor, as assessed through Kaplan–Meier survival analysis. PLS3 was found to be an independent prognostic factor for PACA through multivariate Cox regression analysis. Moreover, we found that PLS3 enhances the proliferation and invasion of tumor cells as assessed through Cell Counting Kit-8, wounding healing assays, and Transwell assays. The upregulation of PLS3 also led to enhanced phosphatidylinositol-3 kinase/protein kinase B signaling in PACA cells. These data suggest that PLS3 is a biomarker to estimate PACA progression and represents a molecular target for PACA therapy.     

Jittery, a Mutator distant relative with a paradoxical mobile behavior: excision without reinsertion

The unstable mutation bz-m039 arose in a maize (Zea mays) stock that originated from a plant infected with barley stripe mosaic virus. The instability of the mutation is caused by a 3.9-kb mobile element that has been named Jittery (Jit). Jit has terminal inverted repeats (TIRs) of 181 bp, causes a 9-bp direct duplication of the target site, and appears to excise autonomously. It is predicted to encode a single 709-amino acid protein, JITA, which is distantly related to the MURA transposase protein of the Mutator system but is more closely related to the MURA protein of Mutator-like elements (MULEs) from Arabidopsis thaliana and rice (Oryza sativa). Like MULEs, Jit resembles Mutator in the length of the element's TIRs, the size of the target site duplication, and in the makeup of its transposase but differs from the autonomous element Mutator-Don Robertson in that it encodes a single protein. Jit also differs from Mutator elements in the high frequency with which it excises to produce germinal revertants and in its copy number in the maize genome: Jit-like TIRs are present at low copy number in all maize lines and teosinte accessions examined, and JITA sequences occur in only a few maize inbreds. However, Jit cannot be considered a bona fide transposon in its present host line because it does not leave footprints upon excision and does not reinsert in the genome. These unusual mobile element properties are discussed in light of the structure and gene organization of Jit and related elements.     

Determining Antioxidant Activities of Lactobacilli Cell-Free Supernatants by Cellular Antioxidant Assay: A Comparison with Traditional Methods

Antioxidant activity of lactic acid bacteria is associated with multiple health-protective effects. Traditional indexes of chemical antioxidant activities poorly reflect the antioxidant effects of these bacteria in vivo. Cellular antioxidant activity (CAA) assay was used in this study to determine the antioxidant activity of cell-free supernatants (CFSs) of 10 Lactobacillus strains. The performance of the CAA assay was compared with that of four chemical antioxidant activity assays, namely, DPPH radical scavenging, hydroxyl radical scavenging (HRS), reducing power (RP), and inhibition of linoleic acid peroxidation (ILAP). Results of the CAA assay were associated with those of DPPH and ILAP assays, but not with those of RP and HRS assays. The inter- and intra-specific antioxidant activities of CFS were characterized by chemical and CAA assays. L. rhamnosus CCFM 1107 displayed a high antioxidative effect similar to positive control L. rhamnosus GG ATCC 53103 in all of the assays. The CAA assay is a potential method for the detection of antioxidant activities of lactobacilli CFSs.     

Endogenous synthesis of n-3 polyunsaturated fatty acids in Fat-1 mice is associated with increased mammary gland and liver syndecan-1

Long chain n-3 PUFA have been shown to have chemopreventive properties against breast cancer through various mechanisms. One pathway, studied in human breast cancer cell lines, involves upregulation of the proteoglycan, syndecan-1 (SDC-1) by n-3 PUFA-enriched LDL. Using Fat-1 mice that are able to convert n-6 to n-3 PUFA, we tested whether SDC-1 level in vivo is elevated in mammary glands due to endogenously synthesized rather than LDL-derived n-3 PUFA. Female Fat-1 and wild type (wt) mice were fed an n-6 PUFA- enriched diet for 7 weeks. Fatty acid analysis of plasma lipoproteins showed that total n-6 PUFA reflected dietary intake similarly in both genotypes (VLDL, 36.2±2.2 and 40.9±3.9; LDL, 49.0±3.3 and 48.1±2.0; HDL, 54.6±1.2 and 58.2±1.3, mean ± SEM percent of total fatty acids for Fat-1 and wt animals respectively). Lipoprotein percent n-3 PUFA was also similar between groups. However, phospholipids and triglycerides extracted from mammary and liver tissues demonstrated significantly higher n-3 PUFA and a corresponding decrease in the ratio n-6/n-3 PUFA in Fat-1 compared to wt mice. This was accompanied by higher SDC-1 in mammary glands and livers of Fat-1 mice, thus demonstrating that endogenously synthesized n-3 PUFA may upregulate SDC-1 in the presence of high dietary n-6 PUFA.     

Long non‐coding RNA AFAP1‐AS1/miR‐320a/RBPJ axis regulates laryngeal carcinoma cell stemness and chemoresistance

AFAP1‐AS1 is a long non‐coding RNA that is associated with tumorigenesis and poor prognosis in a variety of cancers. We have been suggested that AFAP1‐AS1 increases tumorigenesis in laryngeal carcinoma specifically by enhancing stemness and chemoresistance. We assessed AFAP1‐AS1 expression in human laryngeal specimens, paired adjacent normal tissues and human HEp‐2 cells. Indeed, we found not only that AFAP1‐AS1 was up‐regulated in laryngeal carcinoma specimens and cells, but also that stemness‐associated genes were overexpressed. Silencing of AFAP1‐AS1 promoted HEp‐2 cell chemoresistance under cisplatin treatment. Expression of AFAP1‐AS1 was increased in drug‐resistant Hep‐2 cells. We then probed the mechanism of AFAP1‐AS1 activity and determined that miR‐320a was a potential molecular target of AFAP1‐AS1. Luciferase reporter and qRT‐PCR assays of AFAP1‐AS1 and miR‐320a levels in human specimens and cell cultures indicated that AFAP1‐AS1 negatively regulates miR‐320a. To discover the molecular mechanism of miR‐320a, we again used the DIANA Tools algorithm to predict its genetic target, RBPJ. After cloning the 3′‐untranslated regions (3′‐UTR) of RBPJ into a luciferase reporter, we determined that miR‐320a did in fact reduce RBPJ mRNA and protein levels. Ultimately, we determined that AFAP1‐AS1 increases RBPJ expression by negatively regulating miR‐320a and RBPJ overexpression rescues stemness and chemoresistance inhibited by AFAP1‐AS1 silencing. Taken together, these results suggest that AFAP1‐AS1 can serve as a prognostic biomarker in laryngeal carcinoma and that miR‐320a has the potential to improve standard therapeutic approaches to the disease, especially for cases in which cancer cell stemness and drug resistance present significant barriers to effective treatment.     

The Roles of Multiple UNC-40 (DCC) Receptor-Mediated Signals in Determining Neuronal Asymmetry Induced by the UNC-6 (Netrin) Ligand

The polarization of post-mitotic neurons is poorly understood. Preexisting spatially asymmetric cues, distributed within the neuron or as extracellular gradients, could be required for neurons to polarize. Alternatively, neurons might have the intrinsic ability to polarize without any preestablished asymmetric cues. In Caenorhabditis elegans, the UNC-40 (DCC) receptor mediates responses to the extracellular UNC-6 (netrin) guidance cue. For the HSN neuron, an UNC-6 ventral-dorsal gradient asymmetrically localizes UNC-40 to the ventral HSN surface. There an axon forms, which is ventrally directed by UNC-6. In the absence of UNC-6, UNC-40 is equally distributed and the HSN axon travels anteriorly in response to other cues. However, we find that a single amino acid change in the UNC-40 ectodomain causes randomly oriented asymmetric UNC-40 localization and a wandering axon phenotype. With UNC-6, there is normal UNC-40 localization and axon migration. A single UNC-6 amino acid substitution enhances the mutant phenotypes, whereas UNC-6 second-site amino acid substitutions suppress the phenotypes. We propose that UNC-40 mediates multiple signals to polarize and orient asymmetry. One signal triggers the intrinsic ability of HSN to polarize and causes randomly oriented asymmetry. Concurrently, another signal biases the orientation of the asymmetry relative to the UNC-6 gradient. The UNC-40 ectodomain mutation activates the polarization signal, whereas different forms of the UNC-6 ligand produce UNC-40 conformational changes that allow or prohibit the orientation signal.A major challenge for developmental neuroscience has been to understand how axons are able to detect and follow molecular gradients of different extracellular guidance cues. Attractive guidance cues are proposed to stimulate cytoplasmic signaling pathways that promote actin polymerization (Huber et al. 2003). Thus the direction of axon outgrowth is directly linked to the extracellular gradient of the guidance cue; i.e., there is greater extension on the side of the neuron that is closest to the source of the cue. Netrins are bifunctional guidance cues that are attractive to some axons but repulsive to others. Studies have shown that the axon response to netrin is determined by the composition of netrin receptors on the cell surface and the internal state of the growth cone (Round and Stein 2007). The UNC-6 (netrin) guidance cue in Caenorhabditis elegans interacts with the UNC-40 (DCC) receptor to mediate attraction (Hedgecock et al. 1990; Ishii et al. 1992; Chan et al. 1996). The AVM and HSN neurons are useful for studying UNC-40-mediated responses to UNC-6. The cell bodies of these neurons are situated on the lateral body wall and send a single axon ventrally during larval development.In AVM and HSN, a signaling module comprising UNC-6, UNC-40, phosphoinositide 3-kinase (PI3K), Rac, and MIG-10 (lamellipodin) is thought to transmit the directional information provided by the graded distribution of extracellular guidance cues to the internal cellular machinery that promotes directed outgrowth (Adler et al. 2006; Chang et al. 2006; Quinn et al. 2006, 2008). MIG-10 appears to provide an important link because this family of proteins can interact with proteins that promote actin polymerization, and it is associated with asymmetric concentrations of f-actin and microtubules in turning growth cones (Krause et al. 2004; Quinn et al. 2008). MIG-10 is observed as asymmetrically localized to the ventral site of axon outgrowth in developing HSN neurons. This MIG-10 localization is sensitive to the source of UNC-6. Normally, the source of UNC-6 is ventral; in the absence of UNC-6, there is an equal distribution of MIG-10 along the cell surface, whereas ectopic UNC-6 expression from dorsal muscles causes dorsal MIG-10 localization (Adler et al. 2006). The UNC-40 receptor is also asymmetrically localized in HSN, and this localization is also dependent on UNC-6 (Adler et al. 2006). UNC-40 signaling activates Rac GTPase, and MIG-10 interacts specifically with the activated Rac (Quinn et al. 2008). Therefore, the asymmetric activation of Rac through UNC-40 recruits asymmetric MIG-10 localization.By activating or directing components to the surface nearest the UNC-6 source, the asymmetric distribution of UNC-6 could polarize the neuron. However, an alternative idea is suggested from studies of chemotaxing cells. This model predicts that chemoattractant signaling involves two different elements: one that activates the intrinsic ability of cells to generate asymmetry and another that biases the orientation of the asymmetry (Wedlich-Soldner and Li 2003). The polarization signal does not depend on the spatial information provided by the chemoattractant gradient, whereas the orientation signal does. The asymmetric localization of the UNC-40 and MIG-10 signaling complex is suggestive of the segregation of signaling components into separate “front” and “rear” regions during chemotactic cell migration (Weiner 2002; Mortimer et al. 2008). It is hypothesized that this segregation is accomplished through short-range positive feedback mechanisms that promote the local production or recruitment of signaling molecules. In addition, a long-range inhibition mechanism globally increases the degradation of these molecules. Together such mechanisms could strongly amplify the asymmetric distribution of molecules needed for directed movement. This model has been put forth to explain why chemotactic cells polarize and move in a random direction when encountering a uniform chemoattractant concentration. Although the chemoattractant receptors may be uniformly stimulated across the surface of the cells, randomly oriented asymmetry can be established through these mechanisms.If the AVM and HSN neurons behave similarly to chemotactic cells, then uniformly stimulating UNC-40 receptors might similarly cause nonspecific asymmetric UNC-40 localization and axon migrations in varying directions. However, this is difficult to test in vivo. Unlike exposing chemotactic cells to a uniform concentration of a chemotractant in vitro, there is no reliable way to ensure that a neuron in vivo is exposed to a uniform concentration of UNC-6. The pseudocoelomic cavity of C. elegans is fluid filled, and UNC-6 expression patterns are spatially and temporally complex (Wadsworth et al. 1996). How the distribution of UNC-6 is affected by interactions with the extracellular matrix and cell surfaces is unknown.Using a genetic approach, we have found an UNC-40 mutation that triggers randomly oriented neuronal asymmetry. On the basis of the models proposed for chemotactic cells, we suggest that there is an UNC-6/UNC-40-mediated signal that specifically induces the neuron''s intrinsic ability to polarize. The UNC-40 mutation activates this signal; however, a second signal, which normally would concurrently orient asymmetry relative to the UNC-6 gradient, is not activated. Single amino acid changes within the UNC-6 ligand can enhance or suppress the randomly oriented asymmetry phenotype caused by the UNC-40 mutation. This suggests that specific UNC-40 conformations uncouple the activation of the different signals.