Acta histochemica. Supplementband最新文献

Actin filaments in mammalian cells form a number of different architectures in conjunction with a number of different actin-binding proteins. In motile cells these complex architectural arrangements of actin filaments and associated proteins continuously adjust their 3-dimensional organisation to modify the shape and behaviour of cells in response to external information. Microinjection experiments with fluorescently-labelled actin monomers suggest that there is a continual exchange of monomers between the actin filaments and a soluble pool such that individual monomers exist for only a few minutes within polymers. These data suggest that remodelling of the actin filament architectures occurs by the continuous assembly of new filaments which is balanced by the disassembly of obsolete structures. The mechanisms driving and regulating the assembly and disassembly cycle are not yet clearly understood. The properties of the actin assembly ATPase in vitro suggest that the intrinsic exchange of monomers between polymers and the monomer pool is driven by the stoichiometric ATP hydrolysis which is uncoupled from monomer addition and leads to both treadmilling and to the potential for mechanisms analogous to the dynamic instability models proposed for microtubules. Because of the relatively rapid rate of ATP hydrolysis by monomers in the filament (k = 0.05-0.02/s), it is assumed that most of the F-actin in cells is in its ADP form. ADP-F-actin binds inorganic phosphate with a Kd close to that of cytoplasmic concentrations to form an ADP.Pi-F-actin form which has different kinetic, structural and behavioural properties to ADP-F-actin.(ABSTRACT TRUNCATED AT 250 WORDS)

Paired helical filaments (PHF) were isolated from the cerebral cortex of patients with Alzheimer's disease (AD) by a combination of SDS treatment and density gradient centrifugation according to the method of Ihara et al. (1983). The protein component of the preparation was extracted with formic acid and Balb/c mice were used for immunization. Hybridoma supernatants were screened by immunocytochemical staining, by an ELISA assay, and by immunoblotting of SDS-PAGE, the latter both using the PHF preparation as antigen. One hybridoma which showed a strong reactivity with PHF in both the ELISA assay and immunocytochemistry was then used to produce ascites fluid in Balb/c mice. Antibodies reacted immunocytochemically with neurofibrillary tangles and neurites involved in plaque formation in AD but did not show a cross-reaction to human control brain and rat brain. The results indicate that the antibody which has been raised reacts with an antigen component of PHF.

The ultrastructural changes in the cytoskeleton of the goldfish Mauthner cells (M-cells) at various functional states induced by intracerebral microinjections of biologically active substances were studied. Under the action of kainic acid, a structural analog of the excitatory neurotransmitter of glutamate, the density of the cytoplasmic matrix increased. Cytotoxin II from the cobra toxin, which blocks acetylcholine transmission, had an opposite effect upon the M-cell cytoskeleton. Simultaneously, in some areas of the neural cytoplasm strands of an electron-dense material of various shapes appeared. They had an unique structure which did not resemble any known cytoskeleton element. The molecular composition of the strands is unknown, but similar strands appeared after injections of phalloidin or cytochalasin B, both disturbing the microfilamental component of the cytoskeleton. Decoration with myosin subfragment-1 revealed actin in intact M-cells which was organized as crossing loose filaments and bundles of parallel fibers. The morphology of the fiber bundles resembles the helical part of the strands appearing after the treatment with phalloidin, cytochalasin B, or cytotoxin II. It is suggested that the cytoplasmic matrix of M-cells is a dynamic system which responds to the functional changes by thickening or loosening of its cytoskeletal elements or by formation of new structures.

1. The disruption of microfilaments by cytochalasin is followed by an inhibition of gel contraction and a strong decline of DNA-synthesizing cells. 2. Colcemid is without effect on the structure and function of microfilaments but produces a disorganization of intermediate filaments. This is also accompanied by a strong inhibition of DNA synthesis. 3. The application of calmodulin antagonists (TFP, CMZ, W7) at high doses results in disintegration of microfilaments as well as inhibition of gel contraction and DNA synthesis. 4. The results presented demonstrate the involvement of the cytoskeleton in growth regulation, but further experiments are needed to elucidate the interrelationship of the various elements.

Thrombin stimulation of human gel-filtered platelets in an unstirred system (without aggregation) results in actin polymerisation. Concomitantly with actin polymerisation the activation markers (CD 63--glycoprotein 53, a 53 kD lysosomal protein and CD 62--GMP 140, a 140 kD alpha granule protein) increase on the platelet membrane as well as the specific binding of monoclonal antibodies to thrombospondin (P 10). Consequently, both assays run in parallel and indicate sensitively platelet activation. Our data also indicate that exposure of subcellular structures following granule fusion is a very early event when platelets are challenged by thrombin and involve a reorganisation of cytoskeletal structures. Cytochalasin E inhibits completely the thrombin-induced actin polymerisation and the platelet aggregation but only partially the thrombin-induced exposure of CD 63. The activation markers CD 62 and P 10 were not influenced.

Normal and transformed epithelial cells are characterized by the expression of a distinct class of intermediate filaments (IFs), the cytokeratin (CK) filaments. Their constituents, the CKs, comprise a complex multigene family of related proteins which can be subdivided into two sequence types (I and II). In the various epithelial cell types (excluding trichocytes), 19 different CK polypeptides (CKs 1-19) have been distinguished until recently. An additional cytoskeletal polypeptide of Mr 46,000 has been detected, by gel electrophoresis and by immunocytochemistry, in certain types of epithelia including gastric foveolar epithelium, small and large intestinal epithelium, urothelium, and epidermal Merkel cells. On the basis of its biochemical properties and considerable sequence homologies with several type I CKs, this new cytoskeletal protein is suggested to be included in the catalogue of human CKs as CK 20. The various CK polypeptides, as detected by gel electrophoretic and/or immunocytochemical analysis, are expressed in different epithelia and carcinomas in various combinations in a differentiation-dependent manner. Moreover, co-expressions of CKs with other IF classes (vimentin, neurofilaments, glial filaments) are also characteristic of certain epithelial differentiation lineages. Both non-neoplastic epithelial alterations as well as malignant transformation may result in similar modifications of the IF expression profiles although there is a considerable tendency of conservativity. In several instances, a reduced degree of differentiation is paralleled by an increased complexity of the IF protein pattern. CK (and IF) typing can be successfully used in the tracing of developmental lineages as well as in the histological differential diagnosis of primary and metastatic carcinomas. Certain CK polypeptides (CKs 5, 6, 14, 16, 17) identify squamous cell carcinomas including poorly differentiated ones, while other CKs, including CK 20, are typical of primary and metastatic urothelium-derived carcinomas. Among simple-epithelial tumors, CK analysis is of potential diagnostic value in the distinction of mesotheliomas from adenocarcinomas, the discrimination of different carcinomas within the gastrointestinal tract, and the distinction of certain gastrointestinal carcinomas from other adenocarcinoma types including breast and lung carcinomas. Among the individual CKs, CKs 5, 7, 13, 14, 19, and 20 appear to be particularly useful for diagnostic purposes. An important task in the future will be the development of additional monospecific CK-antibodies, particularly such which work on routine paraffin sections.

The centromere of mitotic chromosomes organizes the kinetochore, a proteinaceous matrix that interfaces with spindle microtubules at one plane and with the centromeric DNA at the other. Antibodies present in the sera from patients with CREST scleroderma recognize several proteins localized to the centromere. We have studied the ability of the two main human centromere proteins CENP-A (18 kD) and CENP-B (80 kD) to bind tubulin, in order to correlate with one of the putative functional roles in spindle microtubule attachment. CENP-A was partially solubilized from nuclear extracts by high salt treatment and then purified by reverse phase HPLC. CENP-B was obtained by gel electrophoresis and electroelution from nuclear insoluble extracts. CENP-B binds to tubulin while no significant interaction was found for CENP-A. CENP-B binds to the C-terminal region of tubulin, a characteristic similar to that found for other better characterized microtubule-associated proteins.

The cytokeratins and lamins of rat liver nuclei, nuclear lamina, and cytoskeleton preparations as well as of rat liver cell lines were studied by immunoblotting using the monoclonal broad-range cytokeratin antibodies A 45-B/B3 and A 51-B/H4. A 45-B/B3 is bound to 64-, 58-, 55-, 52.5-, 49-, 45-, 43-, 39-, and 35-kDa proteins, which are already known as rat liver cytokeratins, excepted the 64- and 35-kDa bands. This antibody can therefore be considered to be a pan-cytokeratin marker for the rat. A 51-B/H4 detected an epitope occurring in both lamins (70, 67, and 60-63 kDa) and cytokeratins (49, 45, 43, and 39 kDa). In a few experiments, this monoclonal antibody also indicated a 55-kDa protein. Furthermore, in some cell lines a 80-kDa protein was detected which possibly may correspond to a higher molecular member of the lamin family. In subcellular preparations, the full set of cytokeratins was found to be associated with the nuclei and the nuclear lamina fraction, suggesting a tight connection of the intermediate filaments with the nuclear lamina. Cell lines derived from fetal or neonatal rat liver exhibited cytokeratin patterns which resembled the phenotype of immature or atypically differentiated parenchymal liver cells.

The major microfilamental protein of human blood platelets is actin. It has been estimated to constitute 20-50% of total platelet proteins. The organization of actin filaments within the cell is regulated by their association with other proteins. This review will focus on the composition, the cellular organization and on the functions of the microfilamental system in unstimulated and stimulated platelets.