Inhibition of replicon initiation by 12-O-tetradecanoylphorbol-13-acetate

To investigate the inhibition of DNA replication by tumor promoters, we incubated HeLa cells with 12-O-tetradecanoylphorbol-13-acetate (TPA; 10^?8 to 10^?5 g/ml) and quantified DNA synthesis on alkaline sucrose gradients. TPA was found to selectively inhibit replicon initiation without affecting DNA chain elongation in replicons that had already initiated. No inhibition of DNA synthesis was seen when cells were exposed to the nonpromoting derivative of TPA, 4-α-phorbol 12,13-didecanoate. Superoxide dismutase did not prevent the TPA-induced inhibition of initiation.     

Promoting increased mechanical properties of tissue engineered neocartilage via the application of hyperosmolarity and 4α-phorbol 12,13-didecanoate (4αPDD)

Osteoarthritis, a degenerative disease of the load-bearing joints, greatly reduces quality of life for millions of Americans and places a tremendous cost on the American healthcare system. Due to limitations of current treatments, tissue engineering of articular cartilage may provide a promising therapeutic option to treat cartilage defects. However, cartilage tissue engineering has yet to recapitulate the functional properties of native tissue. During normal joint loading, cartilage tissue experiences variations in osmolarity and subsequent changes in ionic concentrations. Motivated by these known variations in the cellular microenvironment, this study sought to improve the mechanical properties of neocartilage constructs via the application of hyperosmolarity and transient receptor potential vanilloid 4 (TRPV4) channel activator 4α-phorbol 12,13-didecanoate (4αPDD). It was shown that 4αPDD elicited significant increases in compressive properties. Importantly, when combined, 4αPDD positively interacted with hyperosmolarity to modulate its effects on tensile stiffness and collagen content. Thus, this study supports 4αPDD-activated channel TRPV4 as a purported mechanosensor and osmosensor that can facilitate the cell and tissue level responses to improve the mechanical properties of engineered cartilage. To our knowledge, this study is the first to systematically evaluate the roles of hyperosmolarity and 4αPDD on the functional (i.e., mechanical and biochemical) properties of self-assembled neotissue. Future work may combine 4αPDD-induced channel activation with other chemical and mechanical stimuli to create robust neocartilages suitable for treatment of articular cartilage defects.     

Microelectrophoretic study of radiation-induced DNA damages in individual mammalian cells

Mammalian cells were after irradiation suspended in melted agarose, and casted on microscope slides. The slides were after gelling at 0°C immersed in a neutral detergent solution which lysed the cells. A weak electric field (5 V/cm) was then applied over the gel for 5 minutes. The DNA in the gel was stained with the fluorescent dye acridine orange and gives a green emission in a microscope photometer. DNA had migrated towards the anode and this migration was more pronounced in irradiated than in control cells. The differences in migration pattern were quantitatively measured. The lower detection limit was below 0.5 Gy and a plateau in the dose-effect curve was reached at about 3 Gy. In repair experiments residual DNA damage could be observed after postirradiation incubation for 60 minutes.The advantages of the method is: no radioactive labelling and only a few number of cells is required.     

Interaction between phorbol esters and phospholipid in a monolayer model membrane

The possible molecular interaction between phorbol esters and a phospholipid was examined in monolayer films at an air-water interface. The surface pressure isotherms indicated that, at close-to-physiological pressure, there existed a repulsive interaction between the phospholipid and biologically active phorbol esters, such as 12-O-tetradecanoylphorbol-13-acetate (TPA) and phorbol 12,13-didecanoate (PDD). There was a close parallelism between the relative biological potency of different phorbol esters, as tumour promoters, and the magnitude of their repulsive interaction with the phospholipid. These findings raise the possibility that a biophysical interference of phorbol esters with the phospholipid domain of biological membranes may represent an important determinant of their biological actions.     

桂皮醛衍生物的合成及其对CVB_3的作用

目的：合成桂皮醛衍生物,观测其对心肌细胞的毒性及抗CVB3病毒的作用。方法：化学合成6种桂皮醛衍生物,其中3种进行了红外、质谱等结构表征;MTT法检测被CVB3病毒感染和用桂皮醛衍生物治疗后的心肌细胞活性。结果：α-溴代对氯肉桂醛、α-溴代对甲基肉桂醛、对氯肉桂醛3种桂皮醛衍生物对心肌细胞的半数毒性浓度（TC50）分别为2151.28μg·mL-1,1475.32μg·mL-1,22460.32μg·mL-1;对CVB3病毒的半数抑制浓度（IC50）分别为178.94μg·mL-1、173.35μg·mL-1、6045.25μg·mL-1;对CVB3病毒的治疗指数（TI）分别为12.02,8.51,3.71。结论：桂皮醛3种衍生物具有直接抑制CVB3病毒作用,但对病毒的细胞合成和吸附无明显作用。     

Hsp105 but not Hsp70 family proteins suppress the aggregation of heat-denatured protein in the presence of ADP

Hsp105alpha and Hsp105beta are mammalian members of the Hsp105/110 family, a diverged subgroup of the Hsp70 family. Here, we show that Hsp105alpha and Hsp105beta bind non-native protein through the beta-sheet domain and suppress the aggregation of heat-denatured protein in the presence of ADP rather than ATP. In contrast, Hsc70/Hsp40 suppressed the aggregation of heat-denatured protein in the presence of ATP rather than ADP. Furthermore, the overexpression of Hsp105alpha but not Hsp70 in COS-7 cells rescued the inactivation of luciferase caused by ATP depletion. Thus, Hsp105/110 family proteins are suggested to function as a substitute for Hsp70 family proteins to suppress the aggregation of denatured proteins in cells under severe stress, in which the cellular ATP level decreases markedly.     

Effects of α,β-methylene-adenosine-5′-diphosphate on blood platelet aggregation

The effects on platelet aggregation of α,β-methylene-adenosine-5′-diphosphate (Ado-PCP) have been investigated. Using human citrated platelet-rich plasma it has been shown that: (i) at concentrations of 10^?3 M or higher Ado-PCP is able to induce platelet aggregation; (ii) the rate of Ado-PCP-induced aggregation increases on raising the pH of platelet-rich plasma above the pK_a for the secondary phosphonyl dissociation of Ado-PCP; (iii) at concentrations from 1 · 10^?4 to 5 · 10^?4 M Ado-PCP does not cause platelet aggregation itself, but it inhibits ADP-induced aggregation. This inhibition is also observed in washed platelet suspensions. The data suggest that Ado-PCP acts at the same site on the platelet membrane as does ADP and that ADP to AMP transformation is not a prerequisite for the process of aggregation. The observed effect of pH on the rate of Ado-PCP induced aggregation suggests that the ionization state of a nucleotide terminal acid group is important in the process of aggregation.     

Lipid vesicles affect the aggregation of 4-hydroxy-2-nonenal-modified α-synuclein oligomers

Parkinson's disease (PD) and other synucleinopathies are characterized by accumulation of misfolded aggregates of α-synuclein (α-syn). The normal function of α-syn is still under investigation, but it has been generally linked to synaptic plasticity, neurotransmitter release and the maintenance of the synaptic pool. α-Syn localizes at synaptic terminals where it can bind to synaptic vesicles as well as to other cellular membranes. It has become clear that these interactions have an impact on both α-syn functional role and its propensity to aggregate. In this study, we investigated the aggregation process of α-syn covalently modified with 4-hydroxy-2-nonenal (HNE). HNE is a product of lipid peroxidation and has been implicated in the pathogenesis of different neurodegenerative diseases by modifying the kinetics of soluble toxic oligomers. Although HNE-modified α-syn has been reported to assemble into stable oligomers, we found that slightly acidic conditions promoted further protein aggregation. Lipid vesicles delayed the aggregation process in a concentration-dependent manner, an effect that was observed only when they were added at the beginning of the aggregation process. Co-aggregation of lipid vesicles with HNE-modified α-syn also induced cytotoxic effects on differentiated SHSY-5Y cells. Under conditions in which the aggregation process was delayed cell viability was reduced. By exploring the behavior and potential cytotoxic effects of HNE-α-syn under acidic conditions in relation to protein-lipid interactions our study gives a framework to examine a possible pathway leading from a physiological setting to the pathological outcome of PD.     

Modification of steroidogenesis in a mouse adrenal cell line (Y-1) transformed by simian adenovirus SA-7

Transformation of a steroidogenic mouse adrenal cell line (Y-1) by simian adenovirus SA7 produced a cell line with low apparent steroidogenic activity. The effect of ACTH and cholera toxin on cyclic AMP production was similar in both not transformed and virus-transformed cells and activity of cyclic AMP-dependent protein kinase was also similar in both cells. In transformed cells, cholesterol was metabolized to delta 5-3 beta-hydroxysteroids, mainly 20 alpha-dihydropregnenolone while in not transformed cells, the major metabolites were delta 4-3 ketosteroids (20 alpha-dihydro- and 11 beta-hydroxy-20 alpha-dihydroprogesterone). In both cell lines ACTH increased the metabolism of cholesterol. Further studies with labelled pregnenolone and progesterone revealed a loss of delta 5-3 beta-hydroxysteroid dehydrogenase/isomerase and 11 beta-hydroxylase activity in the transformed cells.     

Relationships between the expression of hepatocyte nuclear factors and factors essential for lipoprotein production in a human mesenchymal stem cell line,UE7T-13

To clarify the mechanisms regulating lipoprotein production by hepatocyte nuclear factors (HNFs), we generated four kinds of transfectants in human bone marrow mesenchymal stem cells: UE7T-13, stably expressing FOXA2 (also known as HNF3β), HNF4α, HNF1α or co-expressing HNF4α, and HNF1α (HNF4α/HNF1α). In HNF4α/HNF1α transfectants, cellular contents of triglycerides (TG) and cholesterol were markedly higher than in UE7T-13 cells and comparable to those in human hepatoma HepG2 cells. However, TG and cholesterol, which are secreted from cells as components of lipoproteins, were hardly detected in the medium for any of the transfectants. ApoB100 and MTP, which are essential for the formation and secretion of lipoproteins, were undetectable and detected at low levels, respectively, in HNF4α/HNF1α transfectants. We suggest that enforced co-expression of HNF4α and HNF1α is effective for cellular lipid accumulation, while additional factors are probably required for lipoprotein formation and secretion.     

The effect of the disruption of a gene encoding a PI4 kinase on the developmental defect exhibited by Dictyostelium rasC(-) cells

The disruption of the gene encoding the Dictyostelium Ras subfamily protein, RasC results in a strain that fails to aggregate with defects in both cAMP signal relay and chemotaxis. Restriction enzyme mediated integration disruption of a second gene in the rasC(-) strain resulted in cells that were capable of forming multicellular structures in plaques on bacterial lawns. The disrupted gene, designated pikD(1), encodes a member of the phosphatidyl-inositol-4-kinase beta subfamily. Although the rasC(-)/pikD(1) cells were capable of progressing through early development, when starved on a plastic surface under submerged conditions, they did not form aggregation streams or exhibit pulsatile motion. The rasC(-)/pikD(1) cells were extremely efficient in their ability to chemotax to cAMP in a spatial gradient, although the reduced phosphorylation of PKB in response to cAMP observed in rasC(-) cells, was unchanged. In addition, the activation of adenylyl cyclase, which was greatly reduced in the rasC(-) cells, was only minimally increased in the rasC(-)/pikD(1) strain. Thus, although the rasC(-)/pikD(-) cells were capable of associating to form multicellular structures, normal cell signaling was clearly not restored. The disruption of the pikD gene in a wild type background resulted in a strain that was delayed in aggregation and formed large aggregation streams, when starved on a plastic surface under submerged conditions. This strain also exhibited a slight defect in terminal development. In conclusion, disruption of the pikD gene in a rasC(-) strain resulted in cells that were capable of forming multicellular structures, but which did so in the absence of normal signaling and aggregation stream formation.     

Stabilising normal and mis-sense variant alpha-glucosidase

alpha-Glucosidase (EC 3.2.1.3) is a lysosomal enzyme that hydrolyses alpha-1,4- and alpha-1,6-linkages of glycogen to produce free glucose. A deficiency in alpha-glucosidase activity results in glycogen storage disorder type II (GSD II), also called Pompe disease. Here, d-glucose was shown to be a competitive inhibitor of alpha-glucosidase and when added to culture medium at 6.0 g/L increased the production of this protein by CHO-K1 expression cells and stabilised the enzyme activity. D-Glucose also prevented alpha-glucosidase aggregation/precipitation and increased protein yield in a modified purification scheme. In fibroblast cells, from adult-onset GSD II patients, D-glucose increased the residual level of alpha-glucosidase activity, suggesting that a structural analogue of d-glucose may be used for enzyme enhancement therapy.     

Synthesis and evaluation of esterified Hsp70 agonists in cellular models of protein aggregation and folding

Over-expression of the Hsp70 molecular chaperone prevents protein aggregation and ameliorates neurodegenerative disease phenotypes in model systems. We identified an Hsp70 activator, MAL1-271, that reduces α-synuclein aggregation in a Parkinson’s Disease model. We now report that MAL1-271 directly increases the ATPase activity of a eukaryotic Hsp70. Next, twelve MAL1-271 derivatives were synthesized and examined in a refined α-synuclein aggregation model as well as in an assay that monitors maturation of a disease-causing Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) mutant, which is also linked to Hsp70 function. Compared to the control, MAL1-271 significantly increased the number of cells lacking α-synuclein inclusions and increased the steady-state levels of the CFTR mutant. We also found that a nitrile-containing MAL1-271 analog exhibited similar effects in both assays. None of the derivatives exhibited cellular toxicity at concentrations up to 100?μm, nor were cellular stress response pathways induced. These data serve as a gateway for the continued development of a new class of Hsp70 agonists with efficacy in these and potentially other disease models.     

Chemical chaperone 4-phenylbutyric acid alleviates the aggregation of human familial pulmonary fibrosis-related mutant SP-A2 protein in part through effects on GRP78

G231V and F198S mutations in surfactant protein A2 (SP-A2) are associated with familial pulmonary fibrosis. These mutations cause defects in dimer/trimer assembly, trafficking, and secretion, as well as cause mutant protein aggregation. We investigated the effects and mechanisms of chemical chaperones on the cellular and biochemical properties of mutant SP-A2. Chemical chaperones, including 4-phenyl butyric acid (4-PBA), could enhance secretion and decrease intracellular aggregation of mutant SP-A2 in a dose-dependent manner. Interestingly, increased levels of aggregated mutant SP-A2, resulting from MG-132-mediated proteasome inhibition, could also be alleviated by 4-PBA. 4-PBA treatment reduced the degradation of mutant SP-A2 to chymotrypsin digestion in CHO-K1 cells and up-regulated GRP78 (BiP) expression. Overexpression of GRP78 in SP-A2 G231V- or F198S-expressing cells reduced, whereas shRNA-mediated knockdown of GRP78 enhanced aggregation of mutant SP-A2, suggesting that GRP78 regulates aggregation of mutant SP-A2. Together, these data indicate chemical chaperone 4-PBA and upregulation of GRP78 can alleviate aggregation to stabilize and facilitate secretion of mutant SP-A2. The up-regulation expression of GRP78 might partially contribute to the aggregate-alleviating effect of 4-PBA.     

(1S,3S,7R)-3-methyl-alpha-himachalene from the male sandfly Lutzomyia longipalpis (Diptera: Psychodidae) induces neurophysiological responses and attracts both males and females

Lutzomyia longipalpis adult males form leks on or near hosts and release (1S,3S,7R)-3-methyl-alpha-himachalene from their tergal glands to lure females to the same site for mating and feeding. Here we have examined whether the male-produced attractant could also serve as a male aggregation stimulus. High resolution chiral capillary gas chromatography analysis of male tergal gland extracts, synthetic (1S,3S,7R)-3-methyl-alpha-himachalene, and a synthetic mixture of all isomers of 3-methyl-alpha-himachalene, was coupled to electrophysiological recordings from ascoid sensillum receptor cells in antennae of male and female sandflies. Receptor cells of both sexes responded only to the main component of the male tergal gland extract that eluted at the same retention time as (1S,3S,7R)-3-methyl-alpha-himachalene. Furthermore, of the eight 3-methyl-alpha-himachalene isomers in the synthetic mixture only the fraction containing (1S,3S,7R)-3-methyl-alpha-himachalene, co-eluting with an isomer of (1S*,3S*,7S*)-3-methyl-alpha-himachalene, elicited an electrophysiological response from male and female ascoid sensillum receptor cells. Both males and females flew upwind in a wind tunnel towards a filter paper disk treated with either 4-6 male equivalents of the tergal gland extract, pure (1S,3S,7R)-3-methyl-alpha-himachalene or the synthetic mixture of eight isomers. This indicates that (1S,3S,7R)-3-methyl-alpha-himachalene derived from L. longipalpis males may have a dual function in causing male aggregation as well as serving as a sex pheromone for females.     

Homotypic leukocyte aggregation triggered by a monoclonal antibody specific for a novel epitope expressed by the integrin β1 subunit: Conversion of nonresponsive cells by transfecting human integrin α4 subunit cDNA

The monoclonal antibody 33B6 was found to be specific for the β1 integrin subunit. Treatment of leukocytes with this antibody induced a vigorous homotypic aggregation that had similar physiologic conditions as aggregation induced by a monoclonal antibody specific for the α4 subunit. Expression of a β1 subunit on the cell surface was not sufficient for mAb 33B6-mediated aggregation to occur, since cells of the K562 erythroleukemia line failed to respond even though they expressed the β1 subunit and the 33B6 epitope. However, after transfection with cDNA encoding the α4 subunit, K562 cells acquired the ability to aggregate in response to mAb 33B6 binding. By contrast, mAb 33B6 blocked cell binding to the endothelial surface protein vascular cell adhesion molecule-1 and the extracellular matrix protein fibronectin. These results suggest that the β1 epitope defined by mAb 33B6 may play a novel role in regulating leukocyte adhesive interactions.     

NLS-RARα蛋白在重组腺病毒Ad-NE感染的NB4中定位的验证

该实验主要验证重组腺病毒Ad．NE感染NB4细胞后,NLS-RARa蛋白的表达及其定位。用重组腺病毒Ad-NE感染NB4细胞,检测感染效率,分别用RT-PCR和Westernblot法在mRNA水平和蛋白水平验证转染成功：提取转染成功的NB4细胞的核蛋白,Westernblot法检测细胞核中NLS—RARα蛋白的表达;FITC—AnnexinV／DAPI双染色免疫荧光法检测转染成功的NB4细胞NLS-RARα的表达及定位;FITC—AnnexinV／PI双染色激光共聚焦法检测转染成功的NB4细胞中PNLS-RARα的表达及定位。结果显示,重组腺病毒Ad—NE和阴性对照腺病毒Ad-KZ对NB4细胞的感染效率可达70％～80％。RT-PCR和Westernblot结果显示,感染了重组腺病毒Ad—NLS-RAR的NB4细胞成功表龇基因和NE蛋白,且有NLS．RARa的蛋白表达。用细胞免疫荧光法、激光共聚焦法检测出已感染的NB4细胞中NLS—RARer蛋白的表达,并推测其主要定位于胞核。综上所述,该文成功用重组腺病毒Ad-NE感染NB4细胞,并用Westernblot法、免疫荧光法、激光共聚焦法验证了NLS-RARα蛋白的存在并推测其定位,为进一步研究急性早幼粒细胞白血病的早期诊断及复发监测提供了新的思路。     

Oxaliplatin enhances TRAIL-induced apoptosis in gastric cancer cells by CBL-regulated death receptor redistribution in lipid rafts

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a member of the tumor necrosis factor family that selectively induces apoptosis in cancer cells. However, gastric cancer cells are insensitive to TRAIL. In the present study, we show that oxaliplatin enhanced TRAIL-induced apoptosis of MGC803, BGC823, and SGC7901 cells. Oxaliplatin promoted death receptor 4 (DR4) and death receptor 5 (DR5) clustering into aggregated lipid rafts, while the cholesterol-sequestering agent nystatin partially prevented lipid raft aggregation, DR4 and DR5 clustering, and reduced apoptosis. Furthermore, the expression of the casitas B-lineage lymphoma (Cbl) family was downregulated by oxaliplatin. Transfection of c-Cbl or Cbl-b partially reversed oxaliplatin-induced lipid raft aggregation. These results indicated that oxaliplatin enhanced TRAIL-induced gastric cancer cell apoptosis at least partially through Cbl-regulated death receptor redistribution in lipid rafts.