Differential effects of vitamin E and three hydrophilic antioxidants on the actinomycin D-induced and colcemid-accelerated apoptosis in human leukemia CMK-7 cell line

The actinomycin D (AD)-induced apoptosis in human leukemia CMK-7 cell line is greatly accelerated by microtubule disruption with colcemid (CL). We studied the effect of antioxidants on this apoptosis in order to learn how the universal signal mediators, reactive oxygen species (ROS), are involved. Caspase-3 activation and DNA fragmentation were both suppressed by vitamin E (VE), t-butylhydroxyanisole, and luteolin. The ROS formation in the AD treatment was evidenced by flow cytometry, and further supported by suppression of caspase-3 activation by superoxide radical-forming enzyme inhibitors (TTFA, rotenone, and DPI). The inhibition of apoptosis by VE was completed during the initial 1-h treatment with AD, but it did not appear when VE was added with CL to washed cells after AD treatment. Luteolin, an iron chelator PDTC, and a water-soluble VE analogue, trolox, inhibited the apoptosis when added with CL after the AD treatment. Western blot analysis showed that the proteolytic cleavage of procaspase-9 and procaspase-3 were both inhibited when VE was added with AD or when luteolin was added with CL, and that the cytochrome c liberation was suppressed by both antioxidants. This result implies that the ROS are initially formed in lipophilic environments (e.g. mitochondrial membrane) and then they diffuse into an aqueous environment (i.e. cytoplasm) where they promote the apoptotic process in combination with the cytoskeletal disruption. Thus, the different antioxidants are effective to scavenge ROS for preventing the apoptosis in its different phases.     

中国水龟类的核型与银带带型研究

以血培养细胞为材料,研究了三种水龟的核型与ＮＯＲｓ,结果表明,染色体数均为２ｎ＝５２,核型模式９＋５＋１２,属龟科核型中的原始类型,但眼斑水龟ＮＦ＝７６,而四眼斑水龟和黄喉拟水龟的ＮＦ＝７８。从核型结构看,眼斑水龟与四眼斑水龟相似,而黄喉拟水龟则有较大差别,作者还发现黄喉拟水龟核型的一些重要特征,即其Ａ组Ｎｏ．３和Ｎｏ．６染色体有次缢痕,Ｎ．别,作者还发现黄喉拟水龟核型的一些重要特征,即Ａ组这些是     

Intracellular delivery of antibodies using TAT fusion protein A

Internalization of antibodies into mammalian cells is a useful method for analyzing and regulating cellular function. In this study, we developed a novel method for the delivery of antibodies into cells using the TAT-fused protein. This fusion protein consists of two functional domains, the protein transduction domain of HIV-1 TAT and the B domain of staphylococcal protein A (SpA), which has an ability to bind to the IgG. The TAT-SpA fusion protein was mixed with fluorescence-labeled rabbit IgG and added to cells. The internalization of antibody was analyzed using confocal microscopy and flow cytometry in living cells. As a result, fluorescence-labeled IgG with the TAT-SpA fusion protein was observed intracellularly. Flow cytometry results demonstrated time course and dose dependence relationships of antibody internalization. These results suggest that the TAT-SpA fusion protein can be a useful reagent for the delivery of antibody into cells.     

Relining of prosthesis with auto‐polymerizing hard denture reline resins: effect of post‐polymerization treatment on flexural strength

Background: It has been suggested that microwave irradiation and prosthesis immersion in hot water after its polymerization may improve mechanical and viscoelastic properties of acrylic resins. Purpose: This study was proposed to verify the influence of microwave post‐polymerization (PP) treatment over the flexural strength of thermo‐polymerizing acrylic resin specimens (QC‐20) relined or not with two different composition hard chairside auto‐polymerizing reliners [Kooliner (K) and New Truliner (NT)]. Materials and Methods: For this study, 50 specimens of 64 × 10 × 3.3 mm were polymerized and distributed into five groups. G1 (control) specimens without relining and PP; G2 specimens relined with K, without PP; G3 specimens relined with NT, without PP; G4 specimens relined with K, with PP (microwave irradiation with 650 W for 5 min); G5 specimens relined with NT, with PP. Tests were performed on a universal testing machine Instron 4411 with compression speed of 5 mm/min. Results: Specimens of K without PP did not show statistically different results (p < 0.05) when compared with control. However, when submitted to PP these specimens showed a significant increase in flexural strength. Specimens of NT showed the lowest flexural strength of all groups, with or without PP when compared with control and K groups. Conclusion: Microwave PP (650 W for 5 min) proved to be an effective method of improving the flexural strength of K relined prosthesis. However, it did not seem to affect NT specimens.     

Serum levels of trace elements in Egyptian patients with chronic hepatitis C under interferon therapy

The relationships between chronic liver diseases and trace heavy metal contents in blood are debatable and have not been understood clearly. The present study was designed to determine the interaction of IFN α-2b with some trace elements as (Cu, Fe, Mn and Zn) in patients with chronic hepatitis C, and study the effect of this interaction on the response to therapy. This study was performed on 42 patients having positive HCV-Ab & HCV-RNA by PCR and treated with interferon and ribavirin. Besides, 20 healthy subjects served as control. ALT and trace elements were determined before and after treatment with IFN-α 2b. The results showed that the levels of Zinc and Manganese in hepatitis C virus (HCV) infected patients’ were decreased compared to healthy group and this decrement became more after treatment. While the levels of Iron and Copper increased compared to healthy group and these increment became more after treatment. Serum ALT levels in the patients after treatment was statistically significant decrease (p < 0.05) when compared to its level before treatment.Conclusion: Our findings imply that the levels of trace elements (manganese, iron, copper, zinc, and Cu: Zn ratios) might serve as biochemical parameters with a predictive value for the responsiveness of patients to interferon/ribavirin therapy as there were changes in the levels of some trace elements (Zn and Mn) between responder and non-responder patients. So trace element abnormalities may reflect the condition of liver dysfunction.     

Tightly winding structure of sequential model peptide for repeated helical region in Samia cynthia ricini silk fibroin studied with solid-state NMR

There are many kinds of silks from silkworms and spiders with different structures and properties, and thus, silks are suitable to study the structure-property relationship of fibrous proteins. Silk fibroin from a wild silkworm, Samia cynthia ricini, mainly consists of the repeated similar sequences by about 100 times where there are alternative appearances of the polyalanine (Ala)(12-13) region and the Gly-rich region. In this paper, a sequential model peptide, GGAGGGYGGDGG(A)(12)GGAGDGYGAG, which is a typical sequence of the silk fibroin, was synthesized, and the atomic-level conformations of Gly residues at the N- and C-terminal ends of the polyalanine region were determined as well as that of the central Ala residue using (13)C 2D spin diffusion solid-state nuclear magnetic resonance (NMR) under off-magic angle spinning. In the model peptide with alpha-helical conformation, the torsion angle of the central Ala residue, the 19th Ala, was determined to be (phi, psi) = (-60 degrees, -50 degrees ), which was a typical alpha-helical structure, but the torsion angles of two Gly residues, the 12th and 25th Gly residues, which are located at the N- and C-terminal ends of the polyalanine region, were determined to be (phi, psi) = (-70 degrees, -30 degrees ) and (phi, psi) = (-70 degrees, -20 degrees ), respectively. Thus, it was observed that the turns at both ends of polyalanine with alpha-helix conformation in the model peptide are tightly wound.     

AtDREB1A基因在菊花中的异源表达提高了植株对干旱和盐渍胁迫的耐性

将携带有AtDREB1A基因, 并以35S或rd29A启动子驱动的植物表达载体转入地被菊花(Dendranthema grandiflorum)的粉色品种‘Fall color’. 与野生型相比, 35S:DREB1A转基因植株表现出严重的生长抑制, 而rd29A:DREB1A植株生长正常. RT-PCR检测表明, 在胁迫条件下, AtDREB1A基因在35S: DREB1A转基因植株中呈现组成型过量表达, 而在rd29A:DREB1A植株中则是受胁迫诱导型过量表达. 这两种启动子驱动的转基因植株对干旱和盐渍胁迫都表现出较强的耐性, 其中rd29A:DREB1A植株耐性显著强于35S:DREB1A植株. rd29A:DREB1A植株中的脯氨酸含量和SOD活性都强烈地被胁迫诱导升高, 且高于35S:DREB1A植株. 这些结果表明, 在地被菊花中表达AtDREB1A基因可以提高植株对干旱和盐渍胁迫的耐性, 同时这些耐性的升高可能与脯氨酸含量和SOD活性的上升有关.     

Clostridium perfringens Alpha-Toxin Induces Gm1a Clustering and Trka Phosphorylation in the Host Cell Membrane

Clostridium perfringens alpha-toxin elicits various immune responses such as the release of cytokines, chemokines, and superoxide via the GM1a/TrkA complex. Alpha-toxin possesses phospholipase C (PLC) hydrolytic activity that contributes to signal transduction in the pathogenesis of gas gangrene. Little is known about the relationship between lipid metabolism and TrkA activation by alpha-toxin. Using live-cell fluorescence microscopy, we monitored transbilayer movement of diacylglycerol (DAG) with the yellow fluorescent protein-tagged C1AB domain of protein kinase C-γ (EYFP-C1AB). DAG accumulated at the marginal region of the plasma membrane in alpha toxin-treated A549 cells, which also exhibited GM1a clustering and TrkA phosphorylation. Annexin V binding assays showed that alpha-toxin induced the exposure of phosphatidylserine on the outer leaflet of the plasma membrane. However, H148G, a variant toxin which binds cell membrane and has no enzymatic activity, did not induce DAG translocation, GM1a clustering, or TrkA phosphorylation. Alpha-toxin also specifically activated endogenous phospholipase Cγ-1 (PLCγ-1), a TrkA adaptor protein, via phosphorylation. U73122, an endogenous PLC inhibitor, and siRNA for PLCγ-1 inhibited the formation of DAG and release of IL-8. GM1a accumulation and TrkA phosphorylation in A549 cells treated with alpha-toxin were also inhibited by U73122. These results suggest that the flip-flop motion of hydrophobic lipids such as DAG leads to the accumulation of GM1a and TrkA. We conclude that the formation of DAG by alpha-toxin itself (first step) and activation of endogenous PLCγ-1 (second step) leads to alterations in membrane dynamics, followed by strong phosphorylation of TrkA.     

A two‐step extraction method to measure fecal steroid hormones in female cynomolgus monkeys (Macaca fascicularis)

We developed a two‐step extraction method for measuring fecal steroid concentrations. In the first step, distilled water was used to extract steroids from fecal samples. In the second step, a mixture of organic solvents (hexane and ether) was used to re‐extract water extracts that had been transferred to a glass tube. A portion of the upper layer of the organic solvents was transferred to separate assay‐tubes for measurement of estradiol (E2) or progesterone (P), and the organic solvents were evaporated in vacuo. After phosphate‐buffered saline was added to each tube, commercially supplied radioimmunoassay (RIA) kits were used to determine the steroids. We demonstrated the advantages and reliability of this method by using it to assay the steroid hormone concentrations in fecal samples and serum samples collected on the same day from female cynomolgus monkeys who showed normal menstrual cycles and from monkeys who had induced hyperfunction of ovarian steroidgenesis. Different fecal samples from each monkey were used to determine the recovery rate of each steroid in water extraction from the fecal samples and the reproductivity of hormone concentrations in the fecal samples. The results demonstrate that this two‐step method is simple and effective for measuring fecal steroids for monitoring the reproductive status of cynomolgus monkeys, without having to collect serum samples. Am. J. Primatol. 48:291–298, 1999. © 1999 Wiley‐Liss, Inc.