Identification of a novel microtubule-destabilizing motif in CPAP that binds to tubulin heterodimers and inhibits microtubule assembly

We have previously identified a new centrosomal protein, centrosomal protein 4.1-associated protein (CPAP), which is associated with the gamma-tubulin complex. Here, we report that CPAP carries a novel microtubule-destabilizing motif that not only inhibits microtubule nucleation from the centrosome but also depolymerizes taxol-stabilized microtubules. Deletion mapping and functional analyses have defined a 112-residue CPAP that is necessary and sufficient for microtubule destabilization. This 112-residue CPAP directly recognizes the plus end of a microtubule and inhibits microtubule nucleation from the centrosome. Biochemical and functional analyses revealed that this 112-residue CPAP also binds to tubulin dimers, resulting in the destabilization of microtubules. Using the tetracycline-controlled system (tet-off), we observed that overexpression of this 112-residue CPAP inhibits cell proliferation and induces apoptosis after G2/M arrest. The possible mechanisms of how this 112-residue motif in CPAP that inhibits microtubule nucleation from the centrosome and disassembles preformed microtubules are discussed.     

Links between CD147 function, glycosylation, and caveolin-1

Cell surface CD147 shows remarkable variations in size (31-65 kDa) because of heterogeneous N-glycosylation, with the most highly glycosylated forms functioning to induce matrix metalloproteinase (MMP) production. Here we show that all three CD147 N-glycosylation sites make similar contributions to both high and low glycoforms (HG- and LG-CD147). l-Phytohemagglutinin lectin binding and swainsonine inhibition experiments indicated that HG-CD147 contains N-acetylglucosaminyltransferase V-catalyzed, beta1,6-branched, polylactosamine-type sugars, which account for its excess size. Therefore, CD147, which is itself elevated on invasive tumor cells, may make a major contribution to the abundance of beta1,6-branched polylactosamine sugars that appear on invasive tumor cells. It was shown previously that caveolin-1 associates with CD147, thus inhibiting CD147 self-aggregation and MMP induction; now we show that caveolin-1 associates with LG-CD147 and restricts the biosynthetic conversion of LG-CD147 to HG-CD147. In addition, HG-CD147 (but not LG-CD147) was preferentially captured as a multimer after treatment of cells with a homobifunctional cross-linking agent and was exclusively recognized by monoclonal antibody AAA6, a reagent that selectively recognizes self-associated CD147 and inhibits CD147-mediated MMP induction. In conclusion, we have 1) determined the biochemical basis for the unusual size variation in CD147, 2) established that CD147 is a major carrier of beta1,6-branched polylactosamine sugars on tumor cells, and 3) determined that caveolin-1 can inhibit the conversion of LG-CD147 to HG-CD147. Because it is HG-CD147 that self-aggregates and stimulates MMP induction, we now have a mechanism to explain how caveolin-1 inhibits these processes. These results help explain the previously established tumor suppressor functions of caveolin-1.     

Real-time monitoring of nucleic acid ligation in homogenous solutions using molecular beacons

Nucleic acids ligation is a vital process in the repair, replication and recombination of nucleic acids. Traditionally, it is assayed by denatured gel electrophoresis and autoradiography, which are not sensitive, and are complex and discontinuous. Here we report a new approach for ligation monitoring using molecular beacon DNA probes. The molecular beacon, designed in such a way that its sequence is complementary with the product of the ligation process, is used to monitor the nucleic acid ligation in a homogeneous solution and in real-time. Our method is fast and simple. We are able to study nucleic acids ligation kinetics conveniently and to determine the activity of DNA ligase accurately. We have studied different factors that influence DNA ligation catalyzed by T4 DNA ligase. The major advantages of our method are its ultrasensitivity, excellent specificity, convenience and real-time monitoring in homogeneous solution. This method will be widely useful for studying nucleic acids ligation process and other nucleic acid interactions.     

Cardioprotection involves activation of NF-kappa B via PKC-dependent tyrosine and serine phosphorylation of I kappa B-alpha

Previous studies indicated that activation of PKC and Src tyrosine kinases by ischemic preconditioning (PC) may participate in the activation of NF-kappa B. However, the molecular mechanisms underlying activation of NF-kappa B during ischemic PC remain unknown. In the hearts of conscious rabbits, it was found that ischemic PC (6 cycles of 4-min coronary occlusion and 4-min reperfusion) significantly induced both tyrosine (+226.9 +/- 42%) and serine (+137.0 +/- 36%) phosphorylation of the NF-kappa B inhibitory protein I kappa B-alpha, concomitant with increased activation of the I kappa B-alpha kinases IKK alpha (+255.0 +/- 46%) and IKK beta (+173.1 +/- 35%). Furthermore, both tyrosine and serine phosphorylation of I kappa B-alpha were blocked by pretreatment with either the nonreceptor tyrosine kinase inhibitor lavendustin-A (LD-A) or the PKC inhibitor chelerythrine (Che) (both given at doses previously shown to block ischemic PC). Interestingly, Che completely abolished PC-induced activation of IKK alpha/beta, whereas LD-A had no effect. In addition, I kappa B-alpha protein level did not change during ischemic PC. Together, these data indicate that ischemic PC-induced activation of NF-kappa B occurs through both tyrosine and serine phosphorylation of I kappa B-alpha and is regulated by nonreceptor tyrosine kinases and PKC.     

Caveolin-1 null mice develop cardiac hypertrophy with hyperactivation of p42/44 MAP kinase in cardiac fibroblasts

Recently, development ofa caveolin-1-deficient (Cav-1 null) mouse model has allowed thedetailed analysis of caveolin-1's function in the context of awhole animal. Interestingly, we now report that the hearts ofCav-1 null mice are markedly abnormal, despite the fact that caveolin-1is not expressed in cardiac myocytes. However, caveolin-1 is abundantlyexpressed in the nonmyocytic cells of the heart, i.e., cardiacfibroblasts and endothelia. Quantitative imaging studies of Cav-1 nullhearts demonstrate a significantly enlarged right ventricular cavityand a thickened left ventricular wall with decreased systolic function.Histological analysis reveals myocyte hypertrophy withinterstitial/perivascular fibrosis. Because caveolin-1 is thought toact as a negative regulator of the p42/44 MAP kinase cascade, weperformed Western blot analysis with phospho-specific antibodies thatonly recognize activated ERK1/2. As predicted, the p42/44 MAP kinasecascade is hyperactivated in Cav-1 null heart tissue (i.e.,interstitial fibrotic lesions) and isolated cardiac fibroblasts. Inaddition, endothelial and inducible nitric oxide synthase levels aredramatically upregulated. Thus loss of caveolin-1 expression drivesp42/44 MAP kinase activation and cardiac hypertrophy.

     

高肺血流对大鼠肺血管结构及尾加压素Ⅱ表达的影响

目的：探讨新型血管活性肽人类尾加压素Ⅱ(hUⅡ)在高肺血流量所致肺动脉高压大鼠肺动脉中的表达及其作用。方法：对大鼠行腹主动脉-下腔静脉分流术。11周后,以右心导管法测肺动脉压力,观测肺血管显微结构的变化,以免疫组织化学方法检测肺动脉hUⅡ的表达。结果：分流术后11周,大鼠肺动脉高压形成,肺小血管肌化程度明显增强,肺中、小型肌型动脉相对中膜厚度明显增加。分流组大鼠肺动脉内皮细胞和平滑肌细胞hUⅡ蛋白表达上调,并且与肺动脉压力和肺血管结构的改变呈正相关。结论：肺动脉内皮细胞和平滑肌细胞hUⅡ的上调可能参与了高肺血流量所致肺血管结构重建和肺动脉高压的形成。     

Behavioral modification in choice process of<Emphasis Type="Italic">Drosophila</Emphasis>

In visual operant conditioning ofDrosophila at the flight simulator, only motor output of flies—yaw torque—is recorded, which is involved in the conditioning process. The current study used a newly-designed data analysis method to study the torque distribution ofDrosophila. Modification of torque distribution represents the effects of operant conditioning on flies’ behavioral mode. Earlier works^[10] showed that, when facing contradictory visual cues, flies could make choices based upon the relative weightiness of different cues, and it was demonstrated that mushroom bodies might play an important role in such choice behavior. The new “torque-position map” method was used to explore the CS-US associative learning and choice behavior inDrosophila from the aspect of its behavioral mode. Finally, this work also discussed various possible neural bases involved in visual associative learning, choice processing and modification processing of the behavioral mode in the visual operant conditioning ofDrosophila.     

CD28/B7 regulation of anti-CD3-mediated immunosuppression in vivo

FcR-binding "classical" anti-CD3 mAb is a potent immunosuppressive drug that alters CD4(+) and CD8(+) T cell function in vivo via anergy induction and programmed cell death (PCD). Anti-CD3-mediated PCD was Fas independent but was mediated by the mitochondria-initiated apoptosis that was abrogated in Bcl-x(L)-transgenic T cells. The PCD was more pronounced in CD28-deficient mice consistent with defective Bcl-x(L) up-regulation. Residual T cells isolated from anti-CD3-treated wild-type, CD28(-/-), and Bcl-x(L)-transgenic mice were hyporesponsive. The hyporesponsiveness was more pronounced in CD28(-/-) and wild-type mice treated with anti-B7-2, suggesting that CD28 interaction with B7-2 regulates T cell responsiveness in anti-CD3-treated animals. Finally, anti-CD3 treatment led to indefinite cardiac allograft survival in wild-type but not Bcl-x(L) animals. Together these results implicate CD28/B7 signaling in the regulation of both anti-CD3-induced T cell depletion and hyporesponsiveness in vivo, but T cell depletion, not hyporesponsiveness, appears to be critical for anti-CD3 mAb-mediated long-term immune regulation.     

Crystal structure of tabtoxin resistance protein complexed with acetyl coenzyme A reveals the mechanism for beta-lactam acetylation

Tabtoxin resistance protein (TTR) is an enzyme that renders tabtoxin-producing pathogens, such as Pseudomonas syringae, tolerant to their own phytotoxins. Here, we report the crystal structure of TTR complexed with its natural cofactor, acetyl coenzyme A (AcCoA), to 1.55A resolution. The binary complex forms a characteristic "V" shape for substrate binding and contains the four motifs conserved in the GCN5-related N-acetyltransferase (GNAT) superfamily, which also includes the histone acetyltransferases (HATs). A single-step mechanism is proposed to explain the function of three conserved residues, Glu92, Asp130 and Tyr141, in catalyzing the acetyl group transfer to its substrate. We also report that TTR possesses HAT activity and suggest an evolutionary relationship between TTR and other GNAT members.