K物质对心肌细胞收缩幅度的影响及其机制探讨

目的 :探讨K物质 (SK)对心肌细胞收缩的影响及机制。方法 :原代培养幼鼠心肌细胞 ,利用计算机图像分析系统测定SK处理前后心肌细胞收缩频率和收缩幅度的变化 ,同时观察预先加入速激肽受体拮抗剂DSP、β受体阻断剂心得安、α受体阻断剂酚妥拉明对SK作用的影响。结果 :当加入SK (1 .78× 1 0 - 5mol/L)到培养细胞中时 ,心肌细胞收缩幅度增强 ,但收缩频率变化不显著 ;且在 1 0 - 8～ 1 0 - 5mol/L浓度范围内心肌细胞收缩幅度变化呈剂量 效应关系 ;预先加入DSP可抑制SK对心肌细胞的影响 ,而心得安、酚妥拉明对SK的作用无影响。结论 :SK使心肌细胞的收缩幅度增强 ,其作用是由速激肽受体介导的     

淋巴细胞功能相关抗原-1在冻融PMN介导的VEC损伤中的作用

目的：探讨组织冻结融化造成血管内皮细胞（VEC）损伤的机理。方法：采用密度梯度离心法分离大鼠外周血嗜中性粒细胞（PMN）,速率冷冻仪冷冻PMN后水浴复温建立冻融PMN模型。冻融后4、12和24h,测定PMN表面淋巴细胞功能相关抗原－1（LFA－1）的表达;将冻融PMN与正常VEC共同孵育后测定培养液中乳酸脱氢酶活性及冻融PMN与VEC的粘附。结果：冻融后24h内,冻融PMN表面LFA－1的表面呈时间依赖性增强。（2）冻融PMN与VEC相互作用后,PMN－VEC粘附明显增强、VEC受到明显损伤。（3）抗LFA－1单克隆抗体明显抑制冻融PMN与VEC粘附的增强、明显减轻VEC损伤。结论：冻融可诱发PMN表面LFA－1的表达,进而增强PMN－VEC粘附而导致VEC损伤。     

Delay of ZGA initiation occurred in 2-cell blocked mouse embryos

One-cell mouse embryos from KM strain and B6C3F1 strain were cultured in M16 medium, in which2-cell block generally occurs. Embryos of KM strain exhibited 2-cell block, whereas B6C3F1 embryos,which are regarded as a nonblocking strain, proceeded to the 4-cell stage in our culture condition. It is oftenassumed that the block of early development is due to the failure of zygotic gene activation (ZGA) in culturedembryos. In this study we examined protein synthesis patterns by two-dimensional gel electrophoresis of[35^S] methionine radiolabeled 2-cell embryos. Embryos from the blocking strain and the nonblocking strainwere compared in their development both in vitro and in vivo. The detection of TRC expression, a markerof ZGA, at 42 h post hCG in KM embryos developed in vitro suggested that ZGA was also initiated even inthe 2-cell arrested embryos. Nevertheless, a significant delay of ZGA was observed in KM strain as comparedwith normally developed B6C3F1 embryos. At the very beginning of major ZGA as early as 36 h post hCG,TRC has already been expressed in B6C3F1 embryos developed in vitro and KM embryos developed in vivo.But for 2-cell blocked KM embryos, TRC was still not detectable even at 38 h post hCG. These evidences suggest that 2-cell-blocked embryos do initiate ZGA, and that 2-cell block phenomenon is due not to the disability in initiating ZGA, but to a delay of ZGA.     

Signaling via histamine receptors in cat duodenal smooth muscle cells

Histamine produced concentration-dependent contractions in cat duodenal smooth muscle cells that were obtained by enzymatic digestion of smooth muscle with collagenase F. Pyrilamine, an H1 receptor antagonist, inhibited the contractile response while famotidine, an H2 receptor antagonist, augmented it. In cells with selectively preserved H1 receptors, produced by pretreatment with pyrilamine followed by inactivation of all unprotected receptors with N-ethylmaleimide, histamine-induced contraction was significantly augmented as compared with control cells. Pertussis toxin (PTX) had no effect on contraction, suggesting that the H1 receptor is coupled to a PTX-insensitive G protein. Gi2, Gi3, Go, Gs, and Gq subunits were present in cat duodenum, and histamine-induced contraction was inhibited by Gq antibody after cell permeabilization. Neomycin, a PLC inhibitor, inhibited the histamine-induced cell contraction, but not rhoCMB, a PLD inhibitor, or DEDA, a PLA2 inhibitor. Heparin, an IP3 receptor inhibitor, inhibited contraction whereas chelerythrine, a PKC inhibitor, had no effect. We conclude that histamine-induced contraction in cat duodenal smooth muscle cells is mediated by H1 receptors coupled to a PTX-insensitive Gq protein and results in activation of phosphatidylinositol-specific phospholipase C (PI-PLC).     

Alterations and reversibility in the chromatin,cytoskeleton and development of pig oocytes treated with roscovitine

Germinal vesicle (GV) breakdown in mammalian oocytes is regulated by the activation of maturation promoting factor (MPF). We investigated a specific cdc2 kinase inhibitor, roscovitine, to maintain pig oocytes in the GV stage. Cumulus-oocyte complexes (COCs) were aspirated from slaughterhouse ovaries and cultured for 44 hr in NCSU#23 medium containing different levels of roscovitine (0, 10, 20, 30, 40, 50 microM in Experiment 1 and 0, 40, 60, 80, 100, 120 microM in Experiment 2). The COCs were cultured for another 44 hr after removal of the chemical. Twenty oocytes in each group were fixed at 44 hr for immunocytochemical labeling of the cytoskeleton and the rest (approximately 20/group) were fixed at the end of 88 hr after culture. Results showed that the inhibition of the oocyte in the GV stage was not effective when 10-50 microM (Experiment 1) of roscovitine were used (19-34%). When oocytes were released from the inhibitor, similar proportions (70-83%) of oocytes were observed in the MII or advanced stages among treatments. However, when higher concentrations of roscovitine were used (Experiment 2), significantly greater inhibitory effect was observed at the levels of 80-120 microM with 83-91% oocytes being blocked in the GV stage when compared to the control (9%) and the 40-60 microM (27-43%) groups (P < 0.05). Although 15-21% of the oocytes showed abnormal MII morphology with aberrant meiotic spindles and/or formation of cytoplasmic microtubules, a substantial number of oocytes resumed meiosis and reached MII stage at 44 hr after removal of this chemical. In Experiment 3, different concentrations of roscovitine (0, 20, 40, and 80 microM) were tested to examine the length of intervals (0, 11, 22, 33, and 44 hr) for an effective inhibition. Results showed that the inhibitory effect was significantly more prominent at 22 hr than that at 33 and 44 hr after roscovitine treatment in all treatment groups (P < 0.05). This study demonstrated that roscovitine-treated oocytes resumed meiosis after removal of the inhibitor. This could provide flexibility for studying porcine oocyte development and embryo cloning and may have application in other species.     

Break of neonatal Th1 tolerance and exacerbation of experimental allergic encephalomyelitis by interference with B7 costimulation

Ig-PLP1 is an Ig chimera expressing proteolipid protein-1 (PLP1) peptide corresponding to aa residues 139-151 of PLP. Newborn mice given Ig-PLP1 in saline on the day of birth and challenged 7 wk later with PLP1 peptide in CFA develop an organ-specific neonatal immunity that confers resistance against experimental allergic encephalomyelitis. The T cell responses in these animals comprise Th2 cells in the lymph node and anergic Th1 lymphocytes in the spleen. Intriguingly, the anergic splenic T cells, although nonproliferative and unable to produce IFN-gamma or IL-4, secrete significant amounts of IL-2. In this work, studies were performed to determine whether costimulation through B7 molecules plays any role in the unusual form of splenic Th1 anergy. The results show that engagement of either B7.1 or B7.2 with anti-B7 Abs during induction of EAE in adult mice that were neonatally tolerized with Ig-PLP1 restores and exacerbates disease severity. At the cellular level, the anergic splenic T cells regain the ability to proliferate and produce IFN-gamma when stimulated with Ag in the presence of either anti-B7.1 or anti-B7.2 Ab. However, such restoration was abolished when both B7.1 and B7.2 molecules were engaged simultaneously, indicating that costimulation is necessary for reactivation. Surprisingly, both anti-B7.1 and anti-B7.2 Abs triggered splenic dendritic cells to produce IL-12, a key cytokine required for restoration of the anergic T cells. Thus, recovery from neonatally induced T cell anergy requires B7 molecules to serve double functions, namely, costimulation and induction of cytokine production by APCs.     

The heparan sulfate proteoglycan agrin modulates neurite outgrowth mediated by FGF-2

Although the role of agrin in the formation of the neuromuscular junction is well established, other functions for agrin have remained elusive. The present study was undertaken to assess the role of agrin in neurite outgrowth mediated by the heparin-binding growth factor basic fibroblast growth factor (FGF-2), which we have shown previously to bind to agrin with high affinity and that has been shown to mediate neurite outgrowth from a number of neuronal cell types. Using both an established neuronal cell line, PC12 cells, and primary chick retina neuronal cultures, we find that agrin potentiates the ability of FGF-2 to stimulate neurite outgrowth. In PC12 cells and retinal neurons agrin increases the efficacy of FGF-2 stimulation of neurite outgrowth mediated by the FGF receptor, as an inhibitor of the FGF receptor abolished neurite outgrowth in the presence of agrin and FGF-2. We also examined possible mechanisms by which agrin may modulate neurite outgrowth, analyzing ERK phosphorylation and c-fos phosphorylation. These studies indicate that agrin augments a transient early phosphorylation of ERK in the presence of FGF-2, and augments and sustains FGF-2 mediated increases in c-fos phosphorylation. These data are consistent with established mechanisms where heparan sulfate proteoglycans such as agrin may increase the affinity between FGF-2 and the FGF receptor. In summary, our studies suggest that neural agrin contributes to the establishment of axon pathways by modulating the function of neurite promoting molecules such as FGF-2.     

A synthetic E7 gene of human papillomavirus type 16 that yields enhanced expression of the protein in mammalian cells and is useful for DNA immunization studies

A synthetic E7 gene of human papillomavirus (HPV) type 16 was generated that consists entirely of preferred human codons. Expression analysis of the synthetic E7 gene in human and animal cells showed levels of E7 protein 20- to 100-fold higher than those obtained with wild-type E7. Enhanced expression of E7 protein resulted from highly efficient translation, as well as increased stability of the E7 mRNA due to its codon optimization. Higher levels of E7 protein in cells transfected with synthetic E7 correlated with significant loss of cell viability in various human cell lines. In contrast, lower E7 protein expression driven by the wild-type gene resulted in a slight induction of cell proliferation. Furthermore, mice inoculated with plasmids expressing the synthetic E7 gene produced significantly higher levels of E7 antibodies than littermates injected with wild-type E7, suggesting that synthetic E7 may be useful for DNA immunization studies and the development of genetic vaccines against HPV-16. In view of these results, we hypothesize that HPVs may have retained a pattern of G + C content and codon usage distinct from that of their host cells in response to selective pressure. Thus, the nonhuman codon bias may have been conserved by HPVs to prevent compromising viability of the host cells by excessive viral early protein expression, as well as to evade the immune system.     

Peptides induce ATP hydrolysis at both subunits of the transporter associated with antigen processing

The transporter associated with antigen processing (TAP) plays a key role in the adaptive immune response by pumping antigenic peptides into the endoplasmic reticulum for subsequent loading of major histocompatibility complex class I molecules. TAP is a heterodimer consisting of TAP1 and TAP2. Each subunit is composed of a transmembrane domain and a nucleotide-binding domain, which energizes the peptide transport. To analyze ATP hydrolysis of each subunit we developed a method of trapping 8-azido-nucleotides to TAP in the presence of phosphate transition state analogs followed by photocross-linking, immunoprecipitation, and high resolution SDS-PAGE. Strikingly, trapping of both TAP subunits by beryllium fluoride is peptide-specific. The peptide concentration required for half-maximal trapping is identical for TAP1 and TAP2 and directly correlates with the peptide binding affinity. Only a background level of trapping was observed for low affinity peptides or in the presence of the herpes simplex viral protein ICP47, which specifically blocks peptide binding to TAP. Importantly, the peptide-induced trapped state is reached after ATP hydrolysis and not in a backward reaction of ADP binding and trapping. In the trapped state, TAP can neither bind nor exchange nucleotides, whereas peptide binding is not affected. In summary, these data support the model that peptide binding induces a conformation that triggers ATP hydrolysis in both subunits of the TAP complex within the catalytic cycle.