胃癌细胞高尔基体的蛋白质组学技术平台的建立

采用蔗糖密度梯度超速离心法分离纯化高尔基体,双向凝胶电泳（2-DE）分离高尔基体蛋白质,用ImageMaster 2D软件分析所得图谱,基质辅助激光解吸离子化飞行时间质谱(MALDI—TOF MS)鉴定蛋白质点等一系列亚细胞器蛋白质组学方法建立胃癌细胞内高尔基体的蛋白图谱。结果显示分离出的纯度较高的高尔基体建立了分辨率和重复性均较好的双向电泳图谱,运用质谱技术鉴定出12个蛋白质,包括蛋白合成相关蛋白、膜融合蛋白、调节蛋白、凋亡相关蛋白、运输蛋白、细胞增殖分化相关蛋白。通过亚细胞器分离纯化,双向电泳的蛋白分离及MALDI-TOF MS蛋白鉴定分析,首次成功建立了胃癌细胞SGC7901中高尔基体的蛋白质组学技术路线,为胃癌细胞内高尔基体功能的深入研究奠定了基础。     

Devil inside: does plant programmed cell death involve the endomembrane system?

Eukaryotic cells have to constantly cope with environmental cues and integrate developmental signals. Cell survival or death is the only possible outcome. In the field of animal biology, tremendous efforts have been put into the understanding of mechanisms underlying cell fate decision. Distinct organelles have been proven to sense a broad range of stimuli and, if necessary, engage cell death signalling pathway(s). Over the years, forward and reverse genetic screens have uncovered numerous regulators of programmed cell death (PCD) in plants. However, to date, molecular networks are far from being deciphered and, apart from the autophagic compartment, no organelles have been assigned a clear role in the regulation of cellular suicide. The endomembrane system (ES) seems, nevertheless, to harbour a significant number of cell death mediators. In this review, the involvement of this system in the control of plant PCD is discussed in‐depth, as well as compared and contrasted with what is known in animal and yeast systems.     

1-Cinnamoyl-3,11-dihydroxymeliacarpin delays glycoprotein transport restraining virus multiplication without cytotoxicity

The 1-cinnamoyl-3,11-dihydroxymeliacarpin (CDM), isolated from extracts of Melia azedarach L., displays antiviral and immunomodulating properties. CDM is the first reported tetranortriterpenoid responsible for the alkalinization of intracellular compartments affecting both, viral endocytic and exocytic pathways. Considering that viral glycoprotein synthesis is completely dependent upon cellular membrane trafficking, we questioned whether CDM might also interfere with the normal transport of cellular glycoproteins. This study demonstrates that CDM promoted a transient block in the transport of two cellular glycoproteins, the transferrin receptor (TfR) and TNF-α. Nevertheless, CDM did not affect the transferrin binding ability of TfR and did not impede the TNF-α secretion. On the other hand, CDM disturbed the intracellular localization of capsid, glycoprotein and tegument proteins simultaneously in the same HSV-1 infected cells. Besides, we show that concanamycin A and monensin provoke a permanent blockage of viral and cellular glycoproteins, in contrast to the delay observed after CDM treatment. Thus, the delay on glycoprotein transport caused by CDM would account for the strong inhibition on virus multiplication without interfering with the bioactivity of cellular glycoproteins.     

Craniodental mechanics and diet in Asian colobines: Morphological evidence of mature seed predation and sclerocarpy

Folivory has been accepted as the general dietary pattern for colobines. However, recent ecological studies have revealed that extensive seed eating is found in some colobine species. The ripeness of foraged seeds is also reported to differ between seed eaters. As seeds are generally stress‐limited and may pose greater mechanical demands, seed‐eating species are predicted to exhibit morphological features adaptive for seed predation. In addition, species that feeds on seeds from unripe fruits with hard pericarp is predicted to exhibit increased leverage for anterior dentition. To test these hypotheses, we compared the craniodental morphology of seed‐eating Asian colobines (Presbytis rubicunda and Trachypithecus phayrei) with those of species that rarely exploit seeds (Presbytis comata, Trachypithecus obscurus, and Semnopithecus vetulus). The results show that the seed‐eating colobines possess a masticatory system with enhanced leverage at postcanine bite points. The sclerocarpic forager P. rubicunda also exhibits markedly greater masticatory leverage at anterior dental bite points, while the mature‐seed‐eating T. phayrei shows no such advantage for canine and incisor use. These observations suggest that P. rubicunda is well adapted to husking the resistant pericarps of unripe fruits, using the anterior dentition and to gain access to the immature seeds, whereas such sclerocarpic feeding behavior may be less important for T. phayrei. Our findings indicate that the distinctive craniodental variations of colobines may be linked to mature and/or immature seed eating and suggest the significance of seed predation for the evolution of colobine monkeys. Am J Phys Anthropol, 2010. © 2010 Wiley‐Liss, Inc.     

LOCALIZATION OF CA2+-STIMULATED ATPASE IN THE COCCOLITH-PRODUCING COMPARTMENT OF CELLS OF PLEUROCHRYSIS SP. (PRYMNESIOPHYCEAE)1

Cell homogenates of Pleurochrysis sp. (CCMP299) were fractionated by means of sucrose gradients. Ca²⁺-stimulated ATPase (EC 3.6.1.3., ATP phosphohydrolase) was associated primarily with the plasma membrane, Golgi, and high density (1.21 g·cm^?3) membranous structures. Ca²⁺-stimulated ATPase was highly enriched in the latter. Based on treatments with Triton X-100 and NBD ceramide, we conclude that the high-density structures were membrane-delimited organelles. These vesicle-like organelles contained complex polysaccharides, a high concentration of calcium, and, upon microscopic examination, structures resembling coccoliths. These findings are consistent with observations on the known composition of coccoliths and the presumed mineralizing function of the sub-cellular coccolith-producing compartment. The high-density vesicles were linked to the Golgi by means of colchicine-sensitive materials, presumably microtubules. These data and prior ultrastructural observations by other investigators indicating vectorial assembly and secretion suggest that the subcellular movement of the newly formed coccoliths may be directed and/or powered by colchicine-sensitive cytoskeletal elements. We interpret the data to mean that the high-density vesicles represent the coccolith-producing compartment previously observed by others in electron micrographs.     

Differences in the electrostatic surfaces of the type III secretion needle proteins PrgI, BsaL, and MxiH

Gram-negative bacteria use a needle-like protein assembly, the type III secretion apparatus, to inject virulence factors into target cells to initiate human disease. The needle is formed by the polymerization of approximately 120 copies of a small acidic protein that is conserved among diverse pathogens. We previously reported the structure of the BsaL needle monomer from Burkholderia pseudomallei by nuclear magnetic resonance (NMR) spectroscopy and others have determined the crystal structure of the Shigella flexneri MxiH needle. Here, we report the NMR structure of the PrgI needle protein of Salmonella typhimurium, a human pathogen associated with food poisoning. PrgI, BsaL, and MxiH form similar two helix bundles, however, the electrostatic surfaces of PrgI differ radically from those of BsaL or MxiH. In BsaL and MxiH, a large negative area is on a face formed by the helix alpha1-alpha2 interface. In PrgI, the major negatively charged surface is not on the "face" but instead is on the "side" of the two-helix bundle, and only residues from helix alpha1 contribute to this negative region. Despite being highly acidic proteins, these molecules contain large basic regions, suggesting that electrostatic contacts are important in needle assembly. Our results also suggest that needle-packing interactions may be different among these bacteria and provide the structural basis for why PrgI and MxiH, despite 63% sequence identity, are not interchangeable in S. typhimurium and S. flexneri.     

Interspecific analysis of covariance structure in the masticatory apparatus of galagos

The primate masticatory apparatus (MA) is a functionally integrated set of features, each of which performs important functions in biting, ingestive, and chewing behaviors. A comparison of morphological covariance structure among species for these MA features will help us to further understand the evolutionary history of this region. In this exploratory analysis, the covariance structure of the MA is compared across seven galago species to investigate 1) whether there are differences in covariance structure in this region, and 2) if so, how has this covariation changed with respect to size, MA form, diet, and/or phylogeny? Ten measurements of the MA functionally related to bite force production and load resistance were obtained from 218 adults of seven galago species. Correlation matrices were generated for these 10 dimensions and compared among species via matrix correlations and Mantel tests. Subsequently, pairwise covariance disparity in the MA was estimated as a measure of difference in covariance structure between species. Covariance disparity estimates were correlated with pairwise distances related to differences in body size, MA size and shape, genetic distance (based on cytochrome‐b sequences) and percentage of dietary foods to determine whether one or more of these factors is linked to differences in covariance structure. Galagos differ in MA covariance structure. Body size appears to be a major factor correlated with differences in covariance structure among galagos. The largest galago species, Otolemur crassicaudatus, exhibits large differences in body mass and covariance structure relative to other galagos, and thus plays a primary role in creating this association. MA size and shape do not correlate with covariance structure when body mass is held constant. Diet also shows no association. Genetic distance is significantly negatively correlated with covariance disparity when body mass is held constant, but this correlation appears to be a function of the small body size and large genetic distance for Galagoides demidoff. These exploratory results indicate that changing body size may have been a key factor in the evolution of the galago MA. Am. J. Primatol. 69:46–58, 2007. © 2006 Wiley‐Liss, Inc.     

Subcellular localization determines the delicate balance between the anti- and pro-apoptotic activity of Livin

Livin is a member of the Inhibitor of Apoptosis Protein family which inhibits apoptosis induced by a variety of stimuli. We previously identified Livin and demonstrated that following apoptotic stimuli, Livin is cleaved by effector caspases to produce a truncated form with paradoxical pro-apoptotic activity. In the present study, we reveal that while full-length Livin shows diffuse cytoplasmic localization, truncated Livin (tLivin) is found in a peri-nuclear distribution with marked localization to the Golgi apparatus. Using mutation analysis, we identified two domains that are crucial for the pro-apoptotic activity of tLivin: the N-terminal region of tLivin which is exposed by cleavage, and the RING domain. We demonstrate that, of the N-terminal sequence, only the first N-terminal glycine residue dictates the peri-nuclear distribution of tLivin. However, while the perinuclear localization of tLivin is essential, it is not sufficient for tLivin to exert its pro-apoptotic function. Once tLivin is properly localized, an intact RING domain enables its pro-apoptotic function. Electronic Supplementary Material Supplementary material is available in the online version of this article at .     

Morphological Alteration of Golgi Apparatus and Subcellular Compartmentalization of TGF-β1 in Golgi Apparatus in Gerbils Following Transient Forebrain Ischemia

Golgi apparatus (GA) is a very important organelle involved in the metabolism of numerous proteins. TGF-β1 plays an important role in supporting neuronal survival after ischemic insults. Little is known, however, about the morphological alteration of GA and subcellular compartmentalization of TGF-β1 in brain after ischemia. Therefore, our present study was designed to check for GA morphological alterations and TGF-β1 subcellular localization. GA immunoreactivities were examined in the somatosensory cortex of gerbils after 10 min transient forebrain ischemia. Confocal Immunofluorographs of TGF-β1 and TGN38 were also taken. Results indicated that no fragmentation of GA was found in gerbils of norm, shams and 6, 24 and 72 h postocclusion, but some of the cortical cells showed fragmentation of GA in gerbils 7 days postocclusion. TGF-β1 was colocalized with TGN38, a marker molecule for the GA. We conclude that there was morphological alterations of GA and TGF-β1 was present in GA in the somatosensory cortex after 10 min ischemia.     

Identification and characterization of a novel alternative splice variant of mouse GMx33alpha/GPP34

We have cloned and characterized a novel splice variant of mouse GMx33alpha/Golgi-associated protein of 34 kDa (GPP34), hereby designated GMx33alphaV/GPP34V. This splice variant skips the second and third exons, and the resulting frame shift generates a stop codon in the fourth exon. GMx33alphaV/GPP34V is comprised of 81 amino acid residues derived from the N-terminal end of the full length protein and corresponds to approximately one-third of the full length GMx33alpha/GPP34 sequence with a calculated molecular mass of 8900. In contrast to GMx33alpha/GPP34 mRNA which is expressed at similar levels in various tissues, GMx33alphaV/GPP34V mRNA was differentially expressed when examined by RT-PCR. Compared to other tissues, skeletal muscle showed relatively strong expression of GMx33alphaV/GPP34V mRNA. This splice variant cDNA was also detected in a human cell line.