HUT 14 is a cloned transformed cell line derived from normal diploid human KD fibroblasts. HUT 14 cells have an altered actin phenotype. In addition to the two nonmuscle actins beta and gamma, also present in the parent KD cells, they show the stable expression of a novel actin species (Ax-actin). Amino acid sequence analysis has been used to identify the three actins of HUT 14 cells. beta- and gamma-actins are identified as normal mammalian nonmuscle actins whereas Ax-actin is characterized as a beta-actin mutant revealing a single amino acid substitution at position 244. The results obtained are compatible with a simple mutational event involving a point mutation in one of the two beta-nonmuscle actin genes assumed to be present in proliferating human diploid fibroblasts. Certain emerging principles of nonmuscle actin gene expression in higher vertebrates are discussed.

Actins↗

PubMed1980

Calcium control of the intestinal microvillus cytoskeleton: its implications for the regulation of microfilament organizations.

The microvillus core-filament bundle from intestinal epithelial cells is a highly ordered structure containing actin and four major associated proteins. Two of these, villin and calmodulin, bind calcium ions (Kd approximately 10(-6) M) in the physiologically important range. Because ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid is present throughout the purification and the isolated cores contain levels of calcium substoichiometric to calmodulin, the protein is bound in the structure without calcium saturation. 10-[3-(4-Methyl-1-piperazinyl)propyl]-2-trifluoromethylphenothiazine, a calmodulin-specific drug, removes the protein from the cores without visibly affecting their ultrastructure. Calmodulin-depleted cores rebind exogenously supplied brain calmodulin. Although the core filaments are stable when the calcium level is less than 10(-7) M, they dissassemble when it is greater than 10(-6) M. This appears to be due to the calcium-sensitive allosteric transition of villin from an F-actin bundling protein to an F-actin severing protein. The actions of the two calcium-binding proteins, villin and calmodulin, are discussed in terms of the calcium sensitivity of the filament bundle. We suggest that villin may act as a calcium-sensitive factor regulating microfilament assembly and disassembly and that calmodulin serves as a buffer modulating the free calcium concentration. This hypothesis may explain some aspects of the physiological process of calcium uptake in the intestine and of the effects of calcium fluxes on the submembranous organization of microfilaments in other cells and tissues.

Actins↗

PubMed1980

Intermediate-sized filaments of the prekeratin type in myoepithelial cells.

Myoepithelial cells from mammary glands, the modified sweat glands of bovine muzzle, and salivary glands have been studied by electron microscopy and by immunofluorescence microscopy in frozen sections in an attempt to further characterize the type of intermediate-sized filaments present in these cells. Electron microscopy has shown that all myoepithelial cells contain extensive meshworks of intermediate-sized (7--11-nm) filaments, many of which are anchored at typical desmosomes or hemidesmosomes. The intermediate-sized filaments are also intimately associated with masses of contractile elements, identified as bundles of typical 5--6-nm microfilaments and with characteristically spaced dense bodies. This organization resembles that described for various smooth muscle cells. In immunofluorescence microscopy, using antibodies specific for the various classes of intermediate-sized filaments, the myoepithelial cells are strongly decorated by antibodies to prekeratin. They are not specifically stained by antibodies to vimentin, which stain mesenchymal cells, nor by antibodies to chick gizzard desmin, which decorate fibrils in smooth muscle Z bands and intercalated disks in skeletal and cardiac muscle of mammals. Myoepithelial cells are also strongly stained by antibodies to actin. The observations show (a) that the epithelial character, as indicated by the presence of intermediate-sized filaments of the prekeratin type, is maintained in the differentiated contractile myoepithelial cell, and (b) that desmin and desmin-containing filaments are not generally associated with musclelike cell specialization for contraction but are specific to myogenic differentiation. The data also suggest that in myoepithelial cells prekeratin filaments are arranged--and might function--in a manner similar to the desmin filaments in smooth muscle cells.

Actins↗

PubMed1980

Distribution of tubulin-containing structures in the egg of the sea urchin Strongylocentrotus purpuratus from fertilization through first cleavage.

Eggs of the sea urchin Strongylocentrotus purpuratus were examined by indirect immunofluorescence microscopy for tubulin-containing structures at intervals from fertilization through first cleavage. The staining revealed that the monaster is made up not only of the sperm aster but also of tubulin-staining fibers originating elsewhere in the egg. The monaster does not divide directly but is broken down first before the amphiaster or interphase asters begin to form. The interphase asters reach a peak of development at the streak stage and are in turn broken down before the formation of the mitotic apparatus. The breakdown of the monaster, interphase asters, as well as the asters of the mitotic apparatus proceeds from the cell center or aster centers to the periphery of the cell and is followed by growth of new asters, also proceeding outward from the aster centers. The pattern suggests a transient wavelike movement of some condition, or factor, which favors microtubule depolymerization.

Animals↗

PubMed1980

Fimbrin, a new microfilament-associated protein present in microvilli and other cell surface structures.

A 68,000 mol wt polypeptide has been identified as one of the few major proteins in the microfilament bundles of the microvilli present on intestinal epithelial cells. Antibodies against the purified protein have been used in indirect immunofluorescence microscopy on several cultured cells. The protein have been used in indirect immunofluorescence microscopy on several cultured cells. The protein is found particularly prominent in membrane ruffles, microspikes, and microvilli.

Actins↗

PubMed1980

Permanently proliferating rat vascular smooth muscle cell with maintained expression of smooth muscle characteristics, including actin of the vascular smooth muscle type.

Cells of an established clonal line (RVF-SMC) derived from rat vena cava are described by light and electron microscope methods and biochemical analysis of the major proteins. The cells are flat, and they moderately elongate and form monolayers. They are characterized by prominent cables of microfilaments bundles decoratable with antibodies to actin and alpha-actinin. These bundles contain numerous densely stained bodies and are often flanked by typical rows of surface caveolae and vesicles. The cells are rich in intermediate-sized filaments of the vimentin type but do not show detectable amounts of desmin and cytokeratin filaments. Isoelectric focusing and protein chemical studies have revealed actin heterogeneity. In addition to the two cytoplasmic actins, beta and gamma, common to proliferating cells, two smooth muscle-type actins (an acidic alpha-like and a gamma-like) are found. The major (alpha-type) vascular smooth muscle actin accounts for 28% of the total cellular actin. No skeletal muscle or cardiac muscle actin has been detected. The synthesis of large amounts of actin and vimentin and the presence of at least three actins, including alpha-like actin, have also been demonstrated by in vitro translation of isolated poly(A)+ mRNAs. This is, to our knowledge, the first case of expression of smooth muscle-type actin in a permanently growing cell. We conclude that permanent cell growth and proliferation is compatible with the maintained expression of several characteristic cell features of the differentiated vascular smooth muscle cell including the formation of smooth muscle-type actin.

Actins↗

PubMed1980

Purification of smooth-muscle desmin and a protein-chemical comparison of desmins from chicken gizzard and hog stomach.

A fast and convenient procedure for the purification of polymerization-competent smooth-muscle desmin is described. Desmin from chicken gizzard and hog stomach were compared by fingerprint techniques. The two proteins show extensive amino acid sequence homology, although some clear differences in the peptide patterns are indicated. Comparative amino acid sequence analysis of some of the peptides obtained in pure form directly proves this conclusion.

Amino Acid Sequence↗

PubMed1980

Immunocytochemical detection of actin on fixed and embedded tissues: its potential use in routine pathology.

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Actins↗

PubMed1980

Variations in the distribution and migration of centriole duplexes in mitotic PtK2 cells studied by immunofluorescence microscopy.

The localization and migration of centriole duplexes have been studied in PtK2 cells by indirect immunofluorescence microscopy using specific tubulin antibodies. The study demonstrated the usefulness of the immunofluorescence technique to quantitate studies of centriole migration and concomitant events such as cytoplasmic microtubule breakdown in large populations of cells. Centriole duplex locations in normal and Colcemid-treated interphase populations have been compared with duplex locations in prophase cells. A higher percentage of duplexes were found close to the nucleus in prophase than in interphase cells, but approximately 5% of the duplexes remained in the cytoplasm far removed from the nucleus in prophase and throughout the course of duplex separation. Duplex separation occurred along a wide variety of paths and duplexes did not have to be closely juxtaposed to the nuclear envelope for separation to occur. Some duplexes separated in the cytoplasm with no detectable nuclear attachment, with spindles forming far to the side of the condensing chromosomes. The timing of duplex separation did not always coincide either with chromosome condensation or with nuclear membrane breakdown, and in a small percentage of the cells separation occurred as late as prometaphase. These data suggest that normal spindle formation can occur despite the large variability in initial and final centriole duplex location, their migration patterns, and the timing of the different events. Breakdown of cytoplasmic microtubules began in prophase and progressed until prometaphase; the last cytoplasmic microtubules disappeared soon after the loss of the nuclear membrane.

Cell Line↗

PubMed1980

Antibodies against merokeratin from sheep wool decorate cytokeratin filaments in non-keratinizing epithelial cells.

Merokeratin is an easily soluble proteolytic derivative of mature alpha-keratin. Guinea pig antibodies have been raised to merokeratin prepared from sheep wool. These antibodies decorate in immunofluorescence microscopy arrays of bundles of intermediate sized filaments present in established epithelial cell lines growing in culture. Thus, the highly helical soluble proteolytic fragments of mature alpha-keratin contains antigenic determinants shared by the cytokeratins present in non-epidermal cells, and antibodies to these keratin fragments can be used for the demonstration of at least some cytokeratin-containing structures in other cells and tissues.

Animals↗

PubMed1980

Phalloidin associates with microfilaments after microinjection into tissue culture cells.

Phalloidin is a drug, which specifically binds to F-actin. Tissue culture cells were microinjected with phalloidin and the intracellular display of microfilament-associated proteins in such cells and in cytoskeletons prepared from them was followed by immunofluorescence microscopy using antibodies against actin, myosin, tropomyosin and alpha-actinin. When phalloidin concentrations of 0.2 mM were used, cells and cytoskeletons revealed in addition to the stress fibers aberrant microfilament arrangements ("islands") underneath the upper membrane. These islands contain in addition to actin all the microfilament-associated proteins. Cytockeletons were stabilized by phalloidin against the actin-depolymerization effect of 0.6 M Kl. This resistance of phalloidin-bound actin was used to localize the drug not only in the induced islands but also in the stress fibers. Phalloidin-injected cells showed the same response to cytochalasin B as normal cells and phalloidin did not interfere with ATP-induced contraction of glycerinated models. These results indicate that phalloid-in-stabilized filamentous actin can still give rise to contraction within the stress fiber system, and that F-actin bound to phalloidin may be translocated within the cell.

Actinin↗

PubMed1980

Detection of myoglobin in urine after myocardial infarction by luminescence method.

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Acute Disease↗

PubMed1980

[Prenatal and postnatal diagnosis of triploidy in man (author's transl)].

Prenatal amniocentesis, chromosome analysis, as well as clinical and pathologicoanatomic approaches are some of the ways by which to diagnose triploidy. The methods are described against the background of triploidy (karyotype 69 XXX) in a bipara, 24 years of age. Prenatal examination of hydramnion, particularly in cases with concomitant gestosis, should include amnion cell culturing for early detection of triploidy. Postnatal diagnosis, in response to typical malformation of the foetus and placenta, is possible, provided that thought is given to that aspect.

Adult↗