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1.
Striepen B Soldati D Garcia-Reguet N Dubremetz JF Roos DS 《Molecular and biochemical parasitology》2001,113(1):45-53
Rhoptry and microneme organelles of the protozoan parasite Toxoplasma gondii are closely associated with host cell adhesion/invasion and establishment of the intracellular parasitophorous vacuole. In order to study the targeting of proteins to these specialized secretory organelles, we have engineered green fluorescent protein (GFP) fusions to the rhoptry protein ROP1 and the microneme protein MIC3. Both chimeras are correctly targeted to the appropriate organelles, permitting deletion analysis to map protein subdomains critical for targeting. The propeptide and a central 146 amino acid region of ROP1 are sufficient to target GFP to the rhoptries. More extensive deletions result in a loss of rhoptry targeting; the GFP reporter is diverted into the parasitophorous vacuole via dense granules. Certain MIC3 deletion mutants were also secreted into the parasitophorous vacuole via dense granules, supporting the view that this route constitutes the default pathway in T. gondii, and that specific signals are required for sorting to rhoptries and micronemes. Deletions within the cysteine-rich central region of MIC3 cause this protein to be arrested at various locations within the secretory pathway, presumably due to improper folding. Although correctly targeted to the appropriate organelles in living parasites, ROP1-GFP and MIC3-GFP fusion proteins were not secreted during invasion. GFP fusion proteins were readily secreted from dense granules, however, suggesting that protein secretion from rhoptries and micronemes might involve more than a simple release of organellar contents. 相似文献
2.
The invasion of Toxoplasma gondii tachyzoites and bradyzoites was followed in bovine kidney cells via electron microscopy. The process of invasion differed
between bradyzoites and tachyzoites. In the early stages of entry there was evidence of localised formation of membrane projections
in the host cell adjacent to the parasite. Parasite reorientation and rhoptry release appeared to be necessary for invasion;
however, the tight junction could not be clearly discerned and there was no evidence of constriction or of any membrane shedding
from the parasite. The resulting parasitophorous vacuole was smaller than the tachyzoite vacuole and parasites were frequently
found to lie immediately under the host cell membrane. The vacuole was rapidly adapted by the release and formation of an
intra-phagosomal membrane network, while the parasitophorous vacuole formed a relationship with host-cell endoplasmic reticulum.
Received: 28 March 1998 / Accepted: 28 April 1998 相似文献
3.
Brydges SD Sherman GD Nockemann S Loyens A Däubener W Dubremetz JF Carruthers VB 《Molecular and biochemical parasitology》2000,111(1):51-66
During invasion of host cells, Toxoplasma gondii discharges the contents of small, apically located secretory organelles called micronemes. Micronemal proteins are known to be necessary for both parasite motility and invasion of host cells. To further define the contents of Toxoplasma micronemes, we used cell fractionation and secretion-modulating drugs to identify six novel, putative micronemal proteins. In this paper we describe preliminary characterization of one of these novel proteins, TgMIC5. Molecular cloning and DNA sequence analysis of the TgMIC5 cDNA and gene revealed that it encodes a previously identified immunodominant antigen called H4. TgMIC5 also possesses a consensus sequence unique to members of the parvulin family of peptidyl-prolyl cis-trans isomerases (PPIases). TgMIC5 is expressed as a preproprotein, which is proteolytically processed to a proprotein by signal peptidase before being further processed to a mature protein of 22 kDa. Using a combination of protein secretion experiments, immunofluorescence and immunoelectron microscopy, we demonstrated that TgMIC2 is stored in the micronemes of T. gondii tachyzoites before it is secreted into the surrounding medium. Based on its homology with parvulin-like PPIases, TgMIC5 may assist in the folding of other micronemal proteins that function in invasion of host cells by T. gondii tachyzoites. 相似文献
4.
Summary The ethanolic phosphotungstic acid (E-PTA) technique was used to locate basic proteins in trophozoites ofToxoplasma gondii, obtained from the peritoneal exudate of infected mice. The reaction was observed mainly on the conoid, rhoptries, and micronemes of both extracellular and intracellular parasites. The strongest reaction was observed on rhoptries. Nuclei and ribosomes from parasites and host cells also reacted. The role of basic proteins on rhoptries and micronemes is discussed with a view on host-cell penetration by the parasite. 相似文献
5.
Glycosaminoglycans such as heparin, heparan sulphate and dermatan sulphate, are distributed widely in the human body. Several glycosaminoglycans form part of the extracellular matrix and heparan sulphate is expressed on all eukaryotic surfaces. The identification of specific binding to different glycosaminoglycan molecules by bacteria (e.g., Helicobacter pylori, Bordetella pertussis and Chlamydia trachomatis), viruses (e.g., herpes simplex and dengue virus), and protozoa (e.g., Plasmodium and Leishmania), is therefore of great interest. Expression of glycosaminoglycan-binding proteins depends on growth and culture conditions in bacteria, and differs in various phases of parasite development. Glycosaminoglycan-binding microbial proteins may mediate adhesion of microbes to eukaryotic cells, which may be a primary mechanism in mucosal infections, and are also involved in secondary effects such as adhesion to cerebral endothelia in cerebral malaria or to synovial membranes in arthritis caused by Borrelia burgdorferi. It has been suggested that they may enhance intracellular survival in macrophages. Microbial binding of heparin may interfere with heparin-dependent growth factors. Whether or not glycosaminoglycan-binding proteins mediate invasion of epithelial cells is a matter of controversy. Heparin and other glycosaminoglycans may have potential uses as therapeutic agents in microbial infections and could form part of future vaccines against such infections. 相似文献
6.
Purification of Toxoplasma dense granule proteins reveals that they are in complexes throughout the secretory pathway 总被引:1,自引:0,他引:1
Braun L Travier L Kieffer S Musset K Garin J Mercier C Cesbron-Delauw MF 《Molecular and biochemical parasitology》2008,157(1):13-21
Dense granules are Apicomplexa specific secretory organelles. In Toxoplasma gondii, the dense granules proteins, named GRA proteins, are massively secreted into the parasitophorous vacuole (PV) shortly after invasion. Despite the presence of hydrophobic membrane segments, they are stored as both soluble and aggregated forms within the dense granules and are secreted as soluble forms into the vacuolar space where they further stably associate with PV membranes. In this study, we explored the unusual biochemical behavior of GRA proteins during their trafficking. Conventional chromatography indicated that the GRA proteins form high globular weight complexes within the parasite. To confirm these results, DeltaGRA knocked-out parasites were stably complemented with their respective HA-FLAG tagged GRA2 or GRA5. Purification of the tagged proteins by affinity chromatography showed that within the parasite and the PV soluble fraction, both the soluble GRA2-HA-FLAG and GRA5-HA-FLAG associate with several GRA proteins, the major ones being GRA3, GRA6 and GRA7. Following their insertion into the PV membranes, GRA2-HA-FLAG associated with GRA5 and GRA7 while GRA5-HA-FLAG associated with GRA7 only. Taken together, these data suggest that the GRA proteins form oligomeric complexes that may explain their solubility within the dense granules and the vacuolar matrix by sequestering their hydrophobic domains within the interior of the complex. Insertion into the PV membranes correlates with the decrease of the GRA partners number. 相似文献
7.
整合素/黏着斑激酶信号转导通路与结直肠癌的侵袭和转移 总被引:5,自引:0,他引:5
结直肠癌是一种常见的恶性肿瘤 ,近年来其发病率有逐年上升的趋势。随着肿瘤分子生物学的进展 ,对结直肠癌分子水平的认识逐步深入。最近 ,细胞内信号转导已成为普遍关注的生物学问题。它调节着多细胞生物的生长、发育、分裂及死亡等生物学行为 ,并且与结直肠癌等恶性肿瘤的发生发展密切相关。整合素 (integrin)是一类重要的细胞表面受体 ,它主要介导细胞与胞外基质的黏附 ,有的还可介导细胞间的黏附。除此之外 ,近年来的研究表明 ,整合素还可作为介导信号传递的膜分子 ,通过独特的途径转导信号 ,参与细胞多种生理功能和病理变化 ,如细胞分… 相似文献
8.
Neovascularization in mucinous ductal carcinoma in situ suggests an alternative pathway for invasion
Aims: Ductal carcinoma in situ (DCIS) associated with invasive mucinous carcinoma (IMC) has not been well characterized. The aim was to characterize mucinous DCIS (mDCIS) of the breast and to describe, to our knowledge for the first time, neovascularization in mucin. Methods and results: The pathology reports and slides were reviewed from 44 patients treated between 2003 and 2006 at The University of Texas M. D. Anderson Cancer Center, whose diagnosis fulfilled the criteria of IMC or DCIS with mucin production. The patients, all female, had a mean age of 62 years. DCIS was present in 93% of cases and the predominant histological types were solid, cribriform and micropapillary. The DCIS was grade 1 in 12 of 41 cases (29.3%), grade 2 in 25 of 41 cases (61%) and grade 3 in four of 41 cases (9.8%). Mucin was seen in the lumen of the ducts involved by DCIS in 88% of cases, mucin and vessels in 63.4% of cases and neither mucin nor vessels in 12.2%. The DCIS was vascular endothelial growth factor‐positive, platelet‐derived growth factor receptor‐beta‐positive and CDX‐2‐negative (100%). Occasional luminal cells within the DCIS were immunopositive for CD68. Conclusions: A significant number of mDCIS showed neovascularization in intraluminal mucin. When identified on core needle biopsy, the presence of vascularized mucin should not be used alone to discriminate between invasive and in situ carcinoma. A hypothesis proposed for the source of recruitment of vessels in the mucin is that mucin can promote neovascularization and that tumour cells invade not into the adjacent fibroconnective tissue, but rather into the mucinous, richly vascularized stroma that they have induced. Alternatively, it is possible that both cells and their secretory product invade together. To our knowledge, this is the first study to characterize neovascularization within the mucinous component of DCIS associated with and without IMC. 相似文献
9.
目的 观察牙龈卟啉单胞菌(P.gingivalis)W83和ATCC33277株侵入在单核细胞对内皮细胞黏附作用中的影响,及在内皮细胞细胞间黏附分子l(ICAM-1)转录和翻译中的作用. 方法 建立体外P.gingivalis侵入内皮细胞模型,孟加拉玫瑰红活细胞染色法测定P.gingivalis侵入前后单核细胞对内皮细胞黏附的变化;RT-PCR和mRNA比色定量法检测内皮细胞ICAM-1基因表达;West-ern blot检测ICAM-1蛋白水平的变化. 结果 P.gingivalis W83和ATCC33277株侵入可增加单核细胞对内皮细胞的黏附,抗ICAM-1抗体部分抑制P.gingivalis侵入介导的单核细胞对内皮细胞黏附增加;P.gingivalis侵入上调内皮细胞ICAM-l基因和蛋白的表达,W83诱导单核细胞对内皮细胞黏附增强及内皮细胞ICAM-1表达的能力强于ATCC33277. 结论 ICAM-1在P.gingivalis介导的单核细胞对内皮细胞黏附增强过程中起部分作用,P.gingivalis侵入内皮细胞诱导ICAM-1表达可能是其诱发动脉粥样硬化疾病的机制之一. 相似文献
10.
Summary The ability to reproduce active site sequences from integrin receptor ligands as synthetic peptides has revolutionized the field of cell adhesion. Most studies with these peptides use them in soluble form as inhibitors of integrin function, however, other studies necessitate immobilization of the peptides onto plastic or glass surfaces for analyzing the promotion of cell adhesion. Unfortunately, peptides rarely adsorb well and adsorbed peptides may have their active residues masked by the substrate. An alternative approach is therefore to analyze peptide activity after covalent conjugation to an inert carrier protein. Methods for the production and characterization of peptide conjugates are described. 相似文献
11.
Summary A method is described for culturing invasive cell lines on crosslinked gelatin beads and preparing them for immunocytochemical and morphological observation. Very invasive cells such as Rous sarcoma virus-transformed chicken embryo fibroblasts, and human melanoma LOX and RPMI7951 and breast carcinoma MDA-MB-231 cells will actively degrade this matrix, extending cellular protrusions, called invadopodia, into the sites of degradation. Normal chicken embryo fibroblasts and other non-invasive cell lines do not disrupt the surface of these beads and do not form invadopodia. Invadopodia extending into the bead can be visualized by electron microscopy. Cellular removal of fluorescent fibronectin that has been covalently coupled to the bead surface can be monitored using fluorescence microscopy of frozen-thin-sections. In double label experiments, immunocytochemistry is used to localize antigens in invadopodia at sites of membrane invasion. The materials for bead preparation are inexpensive, and this method has the advantage that many cell types will attach and spread readily on the beads, while only highly invasive cells will invade into the bead. 相似文献
12.
The Toxoplasma gondii dense granule protein GRA7 is phosphorylated upon invasion and forms an unexpected association with the rhoptry proteins ROP2 and ROP4
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The obligate intracellular parasite Toxoplasma gondii infects warm-blooded animals throughout the world and is an opportunistic pathogen of humans. As it invades a host cell, Toxoplasma forms a novel organelle, the parasitophorous vacuole, in which it resides during its intracellular development. The parasite modifies the parasitophorous vacuole and its host cell with numerous proteins delivered from rhoptries and dense granules, which are secretory organelles unique to the phylum Apicomplexa. For the majority of these proteins, little is known other than their localization. Here we show that the dense granule protein GRA7 is phosphorylated but only in the presence of host cells. Within 10 min of invasion, GRA7 is present in strand-like structures in the host cytosol that contain rhoptry proteins. GRA7 strands also contain GRA1 and GRA3. Independently of its phosphorylation state, GRA7 associates with the rhoptry proteins ROP2 and ROP4 in infected host cells. This is the first report of interactions between proteins secreted from rhoptries and dense granules. 相似文献
13.
14.
Burrows Tanya Dee; King Ashley; Smith S.K.; Loke Y.W. 《Molecular human reproduction》1995,1(7):341-352
At the time of implantation, the extracellular matrix proteinslaminin and fibronectin are abundant in the decidua and aredistributed pericellularly around each individual stromal cell.First trimester human trophoblast expresses both laminin andfibronectin receptors, specifically the 1ß1 5ß16ß1 and 6ß4 integrin heterodimers. In thisstudy we have demonstrated that in-vitro adhesion of first trimesterhuman trophoblast to purified extracellular matrix proteinsand to purified decidual stromal cell monolayers can be inhibitedby monoclonal antibodies directed against appropriate integrinsubunits and by synthetic peptides containing an arginine-glycine-asparticacid sequence. Monoclonal antibodies (mAbs) to the 5 and ß1integrin subunits and a synthetic peptide significantly inhibitedadhesion to fibronectin. Binding of trophoblast to laminin wasblocked with mAbs to the 6 and ß1 but not 1 and ß4integrinsubunits. Similarly, integrin-mediated adhesion to monolayersof decidual stromal cells could be blocked with mAbs to the5, 6, ß6 and ß4 integrin subunits. Integrin-mediatedsignal transduction in normal and malignant trophoblast wasinvestigated by Western blotting. A 115 kDa protein was themajor tyrosine phosphorylated protein detected in trophoblastafter binding to laminin or fibronectin. The profile of tyrosinephosphorylated proteins differed for malignant trophoblast. integrins/matrix/signal transduction/trophoblast 相似文献
15.
Hiller E Zavrel M Hauser N Sohn K Burger-Kentischer A Lemuth K Rupp S 《International journal of medical microbiology : IJMM》2011,301(5):384-389
Infectious diseases have long been regarded as losing their threat to mankind. However, in the recent decades infectious diseases have been regaining grounds and are back in the focus of research. This is also due to the fact that medical progress has enabled us to treat and cure a much higher fraction of severe diseases or trauma, resulting in a significant proportion of temporarily or constantly immune-suppressed patients. Infectious diseases result from the interplay between pathogenic microorganisms and the hosts they infect, especially their defense systems. Consequently, immune-suppressed patients are at high risk to succumb from opportunistic infections, like Candida infections. To study the balance between host and C. albicans with regard to the establishment of disease or asymptomatic, commensal colonisation, we developed host-pathogen interaction systems to study both the adaptation of C. albicans to different epithelia as well as to investigate the sensors of the innate immune system, the pattern recognition receptors. These host-pathogen interaction systems, as well as some of the results gained are described in this review. 相似文献
16.
Capsule impedes adhesion to and invasion of epithelial cells by Klebsiella pneumoniae 总被引:6,自引:0,他引:6
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Sahly H Podschun R Oelschlaeger TA Greiwe M Parolis H Hasty D Kekow J Ullmann U Ofek I Sela S 《Infection and immunity》2000,68(12):6744-6749
The adhesion of K21a, K26, K36, and K50 capsulated Klebsiella strains to ileocecal (HCT-8) and bladder (T24) epithelial cell lines was significantly lower than that of their corresponding spontaneous noncapsulated variants K21a/3, K26/1, K36/3, and K50/3, respectively. Internalization of the bacteria by both epithelial cell lines was also significantly reduced. Similarly, a capsule-switched derivative, K2(K36), that exhibited a morphologically larger K36 capsule and formed more capsular material invaded the ileocecal epithelial cell line poorly compared to the corresponding K2 parent strain. None of the capsulated strains exhibited significant mannose-sensitive type 1 fimbriae, whereas two of the noncapsulated variants K21a/3 and K50/3 exhibited potent mannose-sensitive hemagglutinating activity. Although hemagglutinating activity that could be attributed to mannose-resistant Klebsiella type 3 fimbriae was weak in all strains, in several cases the encapsulated parent strains exhibited lower titers than their corresponding noncapsulated variants. Although the level of adhesion to the ileocecal cells is not different from adhesion to bladder cells, bacterial internalization by bladder cells was significantly lower than internalization by ileocecal cells, suggesting that bladder cells lack components required for the internalization of Klebsiella. 相似文献
17.
This paper will review a series of studies using the Experience Sampling Method that suggest that altered sensitivity to stress is an endophenotype for psychosis. The Experience Sampling Method is a structured diary technique allowing the assessment of emotional reactivity to stressors occurring in normal daily life. Elevated emotional reactivity to stress was found in subjects vulnerable to psychosis, suggesting that affective responses to stressors in the flow of daily life are an indicator of genetic and/or environmental liability to psychosis. Indeed, the small stressors in daily life associated with affective responses also predict more intense moment-to-moment variation of subtle positive psychotic experiences. Increased emotional reactivity was found to be independent from cognitive impairments, and argued to constitute evidence of an affective pathway to psychosis that may underlie a more episodic, reactive, good-outcome type of psychosis. Evidence for this hypothesis was found in data suggesting that the experience of stressful life events and early trauma were associated with increased stress-sensitivity, and that women were more likely to display elevated stress-reactivity. These findings are discussed in the light of recent biological and psychological mechanisms. 相似文献
18.
Toxoplasma gondii is an obligate intracellular parasite of animal cells. Infection of humans is common and may result in devastating disease, especially in immunocompromised individuals. Despite previous reports that N-glycosylation of proteins may be a rare post-translational modification in this and related organisms, we demonstrate that it is actually quite prevalent in Toxoplasma. N-Glycosylation is completely inhibited by treatment of parasites with tunicamycin, but this does not appear to exert its major effect on the parasites until they have egressed from their host cells. Although the tunicamycin-treated parasites appear structurally normal at this time they are not motile and mostly incapable of invading new host cells. The few tunicamycin-treated parasites that do invade are severely affected in their ability to replicate and accumulate with a distended endoplasmic reticulum, deformed nuclei, and without recognizable late secretory organelles. We provide experimental evidence that indicate that Toxoplasma N-glycans differ structurally from those in other eukaryotes. 相似文献
19.
20.
Folkert J. Van Kemenade Karel C. Kuijpers Ren de Waal-Malefijt Ren A. W. Van Lier Frank Miedema 《European journal of immunology》1993,23(3):635-639
The effect of influenza (FLU) infection on heterotypic conjugate formation between antigen-presenting cells and T lymphocytes has been studied with FLU-specific T cell clones and FLU-infected B-lymphoblastoid cells (B-LCL). Conjugate formation between FLU-infected B-LCL (FLU+ B-LCL) and T cells was found to be consistently enhanced in comparison with peptide-sensitized or uninfected B-LCL. Treatment of B-LCL with exogenous neuraminidase (NA-NAse) similarly enhanced conjugate formation indicating that increased conjugate formation may be mediated by the viral neuraminidase. Monoclonal antibody blocking experiments revealed that the contribution by CD2/LFA-3 is increased relative to that of LFA-l/ICAM-1 in conjugates between FLU+ B-LCL or NANAse-treated B-LCL and T cell clones. In contrast, both pathways of adhesion contributed equally to conjugate formation between peptide-sensitized B-LCL or control B-LCL and T cell clones. Thus, FLU infection causes increased conjugate formation between antigen-presenting cells and T cells and skews towards CD2/LFA-3-dependent adhesion, independent of T cell receptor signalling. 相似文献