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81.
Association of cytoskeletal re-organization with capping of the complement decay-accelerating factor on T lymphocytes 总被引:1,自引:0,他引:1
G M Kammer E I Walter M E Medof 《Journal of immunology (Baltimore, Md. : 1950)》1988,141(9):2924-2928
Recent studies have identified cell-associated proteins that are membrane anchored by glycosyl-inositol-phospholipid structures but the biologic implications of this mode of membrane attachment are incompletely understood. Among proteins anchored in this way is the decay-accelerating factor (DAF), a complement (C) regulatory factor that functions on blood cell surfaces to prevent autologous C attack. As one approach to investigate the functional consequences of glycosyl-inositol-phospholipid-anchoring of DAF in T lymphocytes, the effects of crosslinking surface DAF molecules were compared to those of crosslinking conventionally by anchored cluster of differentiation (CD) proteins. Upon incubation with anti-DAF mAb and anti-murine IgG, DAF re-distributed to a pole of the cell with a t1/2 at 37 degrees C of 4.4 min as compared to t1/2 of 3.5 to 7 min for CD3, CD4, and CD8. Re-distribution of DAF occurred independently of CD2, CD3, CD4, or CD8. Anti-DAF immunoprecipitates of membrane extracts of cells chemically cross-linked with dithiobis(succinimidylpropionate) contained only monomeric DAF. Immunofluorescent staining demonstrated clustered actin, tubulin, and vimentin beneath the capped DAF protein. Pre-treatment of cells with colchicine or 8-azidoadenosine 3',5'-cyclic phosphate, but not lumicolchicine, resulted in reduction of the t1/2 for DAF to 1 to 2.6 min. Conversely, treatment of cells with cytochalasins B or D completely blocked DAF capping. The results indicate that, upon cross-linking, glycosyl-inositol-phospholipid-anchored DAF molecules undergo capping similar to conventionally anchored CD molecules and that DAF capping is associated with cytoskeletal reorganization. 相似文献
82.
Centrosomes undergo cell cycle-dependent changes in shape and separations, changes that govern the organization of the cytoskeleton. The cytoskeleton is largely organized by the centrosome; however, this investigation explores the importance of cytoskeletal elements in directing centrosome shape. Since the sea urchin egg during fertilization and mitosis displays dramatic and synchronous changes in centrosome shape, the effects of cytoskeletal inhibitors on centrosome compaction, expansion, and separation were explored by the use of anticentrosome immunofluorescence microscopy. Centrosome expansion and separation was studied during two phases: the transition after sperm incorporation, when the compact sperm centrosome enlarges and the sperm aster develops, and from prometaphase to telophase, when the compact spindle poles enlarge. Compaction was investigated when the dispersed centrosome at interphase condenses into the two spindle poles at prometaphase. Although centrosome expansion and separation typically occur concurrently, beta-mercaptoethanol results in centrosome separation independent of expansion. Microtubule inhibitors prevent centrosome expansion and separation, and expanded centrosomes collapse. Since pronuclear union is arrested by microtubule inhibitors, this treatment also affords the opportunity to explore the relative attractiveness of the male and female pronuclei for these centrosomal antigens. Both pronuclei acquire centrosomal material; though only the male centrosome is capable of organizing a functional bipolar mitotic apparatus at first division, the female centrosome nucleates a monaster. Microfilament inhibition (cytochalasin D) prevents centrosome separation but not expansion or compaction. These results demonstrate that as the centrosome shapes the cytoskeleton, the cytoskeleton alters centrosome shape. 相似文献
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86.
High plasticity of multispecific DNA methyltransferases in the region carrying DNA target recognizing enzyme modules. 下载免费PDF全文
Multispecific cytosine C5 DNA methyltransferases (MTases) methylate more than one specific DNA target. This is due to the presence of several target recognizing domains (TRDs) in these enzymes. Such TRDs form part of a variable centre in the MTase primary sequence, which separates conserved enzyme core sequences responsible for general steps in the methylation reaction. By deleting, rearranging and exchanging several TRDs of multispecific MTases, we demonstrate their modular character; they mediate target recognition independent of a particular TRD or core sequence context. We show also that multispecific MTases can accommodate inert material of non-MTase origin within their variable region without losing their activity. The remarkable plasticity with respect to the material that can be integrated into this region suggests that the enzyme core sequences preceding or following it form separable functional domains. In spite of the documented flexibility multispecific MTases could not be endowed with novel specificities by integration of putative TRDs of monospecific MTases, pointing to differences between multi- and monospecific MTases in the way their core and TRD sequences interact. 相似文献
87.
Immunogold labelling of the cytoplasmic estradiol receptor in resting porcine endometrium 总被引:2,自引:0,他引:2
Summary Serial sections of resting porcine endometrium were analyzed with the monoclonal antibody 13H2 using goat antimouse IgG/5 nm gold as secondary reagent or with either polyclonal antibodies from goat #402 or the rat monoclonal antibody H222, both in combination with protein G/12 nm gold. A modestly higher labelling of nuclei than of cytoplasm was seen only with the monoclonal antibody H222. Polyclonal #402 and monoclonal 13H2 showed fewer attachments over nuclear than over cytoplasmic areas. The highest densities of attachment and of predominantly cytoplasmic labelling were obtained with the monoclonal antibody 13H2. The results confirm the earlier assumption of a restricted accessiblity of estradiol receptor in the cytoplasm of resting cells for immunoreagents. 相似文献
88.
Zulma Gazit David W. Weiss Daniel Shouval Michal Yechezkeli Volker Schirrmacher Michael Notter Jürgen Walter Eli Kedar 《Cancer immunology, immunotherapy : CII》1992,35(2):135-144
Summary The antitumor effects of chemotherapy, recombinant human interleukin-2 (IL-2), recombinant human interferon A/D (IFN), allogeneic human lymphokine-activated killer (LAK) cells, and antitumor monoclonal antibody (mAb), administered alone and in various combinations, were tested in athymic nude mice carrying human tumor xenografts. Treatment began 6–18 days after i.v. or i.p. inoculation of colorectal carcinoma or melanoma cell lines, when macroscopic growths were evident. Chemotherapy consisted of two or three courses of 5-fluorouracil (5-FU) or dacarbazine. IL-2 and/or IFN were administered three to five times weekly for 1–3 weeks, usually starting 2–5 days after chemotherapy. Human LAK cells were infused once or twice weekly for 2 or 3 weeks concurrently with IL-2. In some experiments, murine anticolorectal carcinoma mAb (SF25) was administered. In both tumor systems, chemotherapy alone or immunotherapy alone (IL-2, IL-2 + LAK cells, IFN, IL-2 + IFN ± LAK cells) had little or no therapeutic effects. Additive effects were obtained by combining chemotherapy with IL-2 and LAK cells or with IL-2 and IFN. In the majority of the experiments, the most effective combination was chemotherapy + IL-2 + IFN + LAK cells. Treatment with mAb was beneficial in the colorectal carcinoma system when combined with 5-FU + IL-2 or 5-FU + IL-2 + IFN. Homing experiments with radiolabeled human and mouse LAK cells injected i.v. showed increased early accumulation in the liver and lungs, whereas freshly explanted mouse splenocytes localized mostly in the spleen and liver. The tissue distribution pattern of human LAK cells was similar in normal and tumor-bearing mice (with lung metastases). These findings suggest that combination of chemotherapy with cytokines and LAK cells can be partially effective for advanced solid human tumors even in the absence of the host's T-cell immune response. Preliminary experiments showed that tumor-specific, anti-melanoma T-cell clones were effective in local (s.c.) tumor growth inhibition (Winn assay) following coinjection with the autologous tumor cells. 相似文献
89.
Stefan Jansson Eran Pichersky Roberto Bassi Beverley R. Green Masahiko Ikeuchi Anastasios Melis David J. Simpson Michael Spangfort L. Andrew Staehelin J. Philip Thornber 《Plant Molecular Biology Reporter》1992,10(3):242-253
We propose a nomenclature for the genes encoding the chlorophylla/b-binding proteins of the light-harvesting complexes of photosystem I and II. The genes encoding LHC I and LHC II polypeptides
are namedLhca1 throughLhca4 andLhcb1 throughLhcb6, respectively. The proposal follows the general format recommended by the Commision on Plant Gene Nomenclature. We also present
a table for the conversion of old gene names to the new nomenclature. 相似文献
90.
Regarding biological products, increasing awareness of potential side effects have placed great importance not only at protein
purity regarding other proteins but on the removal of biologicals such as DNA and especially virus the importance of which
may not be known. Monoclonal antibodies (Mab) have come to be an important class of molecules obtained from hybridoma cells,
i.e., nonrecombinant cells in culture. It has been noted during the last years, that with rare exceptions hybridoma cell lines
contain retrovirus like particles. The infectious nature of the EM-visible particles has been tested for, however, in most
cases not been substantiated. In order to bring these valuable biological reagents, Mab's, to good use in man for imaging
or therapy, the remaining concern about a potential retroviral infection has to be reduced to an acceptable minimum. We describe
experimental approaches for the validation of chromatographic and ultrafiltration steps used in the production of monoclonal
antibodies to remove and inactivate murine retrovirus.
Present day biotechnological manufacturing processes have been devised incorporating a number of strategic preventive measures
that have found wide spread acceptance. They permit to answer the question: how can a potentially harmful infection by an
unknown virus be excluded.
Knowledge of the efficacy of purification steps to clear infectious model virus is fundamental to devise biotechnological
manufacturing processes yielding a purified antibody for use in man. 相似文献