Electron microscopic immunolocalization of a karyoskeletal protein of molecular weight 145 000 in nucleoli and perinucleolar bodies of Xenopus laevis

Amplified nucleoli of Xenopus laevis oocytes contain a major karyoskeletal protein of Mr 145 000 insoluble in low- and high-salt buffer as well as in non-denaturing detergents. Electron microscopic localization on native and high-salt extracted nucleoli using specific murine antibodies against this polypeptide and gold-coupled antibodies for visualization reveals that the Mr 145 000 protein is located in coils of filaments of ca 4 nm diameter. In addition, this protein occurs in the medusoid filament bodies (MFBs) present in the nucleolar cortex and free in the nucleoplasm. In somatic cells of tissues and in A6 kidney epithelial cells grown in vitro the Mr 145 000 polypeptide or an immunologically related protein is also organized in coiled aggregates of filaments 4-12 nm in diameter present both in the periphery of nucleoli and free in the nucleoplasm. We discuss a possible role of this protein as a karyoskeletal support involved in the storage and transport of preribosomal particles.     

Formation of vinculin plaques precedes other cytoskeletal changes during retinoic acid-induced teratocarcinoma cell differentiation

Immunofluorescence and immunoblotting techniques were used to study the presence and distribution of vimentin and keratin type intermediate filaments, actin, and vinculin (130 kD protein) during retinoic acid (RA)-induced differentiation of F9 embryonal carcinoma (EC) cells. The undifferentiated F9 cells regularly expressed vimentin, usually concentrated close to the nucleus, but not keratin. Actin appeared as short intracellular filaments and as spikes at the edges of the colonies, together with some diffuse cytoplasmic staining. F9 cells also showed a weak, diffuse cytoplasmic vinculin-specific fluorescence in addition to occasional small focal vinculin patches at the edges of the cell colonies. RA treatment led into a series of changes in the cytoskeletal organization of F9 cells. These changes were initiated by the appearance of distinct vinculin plaques and followed by formation of actin stress fibers and by profound changes in the organization of vimentin in the flattening cells. RA treatment finally led to the appearance and co-expression of keratin fibrils in many of the vimentin-containing F9 cells. This sequence of changes suggests that the vinculin-containing adhesion plaques may be important in the mechanism of RA-induced differentiation of EC cells.     

Characterization of human embryonal carcinoma cell lines derived from testicular germ-cell tumors

Four human embryonal carcinoma (EC) cell lines (ITO, NEC 8, NEC 14, NEC 15) derived independently from testicular germ-cell tumors were established in vitro. In their xenografted tumor tissues, all of them exhibited histological characteristics consistent with EC. The cell-biological characterization of these human EC cell lines was investigated with reference to well-known murine EC cell lines. This included examination of their morphology, growth, tumorigenic potential, karyotype, cell-aggregate formation, HLA expression, large glycopeptides, AFP and HCG production, plasminogen-activator secretion, and LDH profiles. Three (ITO, NEC 14, NEC 15) of these human EC cell lines shared cell-biological characteristics consistent with typical EC, but one of them (NEC 8) differed from the others with respect to its rapid growth, high tumorigenic potential, formation of solid cell aggregates, and less differentiated, solid histological pattern. Thus, it is suggested that there are several developmentally different types of human EC cells. The relationship between the properties of these human EC cell lines and their differentiation potential is discussed.     

The synthesis and intracellular localization of adenovirus hexon protein studied by microinjection of mRNA into human cells

Polyadenylated RNA isolated 18 h after infection of HeLa cells with adenovirus type 2 was both translated in vitro and microinjected into the cytoplasm of human transformed amnion cells. The hexon polypeptide could be specifically immunoprecipitated from the products of cell-free translation with a rabbit-anti-hexon serum. The same serum when used in immunofluorescence assays of microinjected cells revealed hexon protein synthesized 6 h after microinjection. The intensity of the staining persisted up to 16 h after injection of messenger RNA (mRNA). Newly-synthesized hexon protein was characteristically located mainly in the nucleus. Essentially the same results were obtained when normal amnion cells were microinjected.     

Cytokeratin patterns of human oral epithelia: Differences in cytokeratin synthesis in gingival epithelium and the adjacent alveolar mucosa

Human oral mucosa includes various epithelia that are commonly classified as lining, masticatory, and specialized epithelia. Although adjacent tissues, the gingiva and alveolar mucosa represent two different types of epithelia: the gingiva is cornified and exhibits high rate ridges, whereas the mucosa does not normally cornify and exhibits a relatively smooth-contoured borderline between the epithelium and the underlying connective tissue. We examined the cytokeratin patterns of both epithelia using one- and two-dimensional gel electrophoresis. The gingiva expresses a great complexity of cytokeratins, including significant amounts of components nos. 1, 2, 5, 6, 10, 11, 13, 14, 16, and 17, as well as traces of cytokeratins nos. 4 and 15, i.e., a pattern similar to those of vaginal mucosa and epidermis containing proliferative keratinocytes. In contrast, the alveolar mucosa contains only two major cytokeratins, i.e., nos. 4 and 13, together with two minor amounts of cytokeratins nos. 5, 6, 14, and 17, thus resembling the patterns of certain other stratified, noncornified epithelia, such as the esophagus. Immunofluorescence microscopy using monoclonal antibodies to cytokeratins nos. 4 and 13 revealed the presence of these proteins in the suprabasal layers of alveolar mucosa, whereas in the gingiva, only certain small, suprabasal clusters of cells appeared to contain these cytokeratins. The cytoskeletal differences between gingival and alveolar mucosa are discussed in relation to the differences in their morphology and function, and with respect to pathological processes characteristic of these epithelia.     

Progress through G1 and S in relation to net protein accumulation in human NHIK 3025 cells

We have investigated whether human NHIK 3025 cells are dependent upon a net increase in cellular protein content in order to traverse G1 and S. The increase in DNA and protein content was studied by means of two-parameter flow cytometry using populations of cells synchronized by mitotic selection. By adding 1 μM cycloheximide to the medium protein synthesis was partially inhibited, resulting in negligible net accumulation of protein. The cells were able to enter S and progress through S under such conditions. The latter was the case whether the cells had been accumulating protein during G1 or not. The results further indicate that the larger cells enter S earlier and traverse S at a higher rate than the smaller cells. Our conclusion is that net accumulation of protein does not seem to be a prerequisite for traverse through G1 and S, i.e. DNA replication may be dissociated from the general growth of cell mass.     

Size changes of protamine 1 mRNA provide a molecular marker to monitor spermatogenesis in wild-type and mutant mice

We utilized a cDNA encoding the cysteine-rich, tyrosine-containing mouse protamine, mouse protamine 1 (MP1), to detect the presence of several classes of differentiating germ cells in testicular extracts from wild-type and male sterile mutant mice. This assay is based on the changes in the poly (A) length of MP1-mRNA during spermatogenesis. Testicular extracts of sexually mature CD-1 mice contain a heterogeneous population of protamine-1 mRNA ranging in length from 450 to 580 nucleotides. When the protamine-1 probe was hybridized to testicular RNA preparations from 16- to 20-day-old animals, no MP1-mRNA was detected. Twenty-four-day-old mice contain only the 580-nucleotide form of MP1-mRNA. This size class of protamine mRNA is also present in purified populations of round spermatids, whereas elongating spermatids and residual bodies contain mRNAs ranging from 450 to 580 nucleotides in length, which are identical in size to those present in the testes of sexually mature animals. When the protamine cDNA probe was used to examine the progression of spermiogenesis in three male sterile mouse mutants, blind sterile (bs), quaking (qk) and testicular feminization (Tfm), the results demonstrated that each mutant is pathologically distinct. Analysis of the bs mutant revealed a diminution in the amount of both size classes of MP1-mRNA, in agreement with the cytological reports of reduced numbers of haploid spermatogenic cells in these animals. The presence of both size classes of protamine mRNA in the qk mutant indicates that germ-cell differentiation has proceeded at least to the step-12 spermatid in these animals.(ABSTRACT TRUNCATED AT 250 WORDS)     

The release of cytosolic proteins from cells treated with concanavalin A during scraping from plastic culture dishes

When rat hepatoma cells (HTC and R117-21B), treated with concanavalin A (conA) at 37 °C, were scraped from plastic culture dishes with a silicone-rubber policeman, the cell membranes were broken and the cytoplasm was released. This phenomenon was also observed in cells treated with conA at 4 °C, even though it took a longer time to show the same effect. The effect of 10 μg/ml of conA on the release of the cellular proteins reached a plateau when the treatment was carried out at 37 °C. Ninety percent of this effect was abolished by 10 mM of α-methyl-d-mannoside. The effect was completely nullified by 100 mM. At 4 °C, however, even 100 mM of this sugar could not abolish this effect. The apparent decrease in the cellular proteins with conA after scraping was observed not only in the logarithmic phase, but also in the stationary phase of cell growth. The breakdown of plasma membranes with conA eventually caused decrease in tyrosine aminotransferase activity, even though the lectin induced the enzyme activity in cultured cells.     

Bismuth staining of a nucleolar protein

A major nucleolar protein in Chinese hamster ovary cells with a molecular weight (MW) of 100 kD has been found to stain selectively with the bismuth tartrate technique of Locke & Huie [19]. After glutaraldehyde fixation and bismuth staining of electrophoretic transfers of total nucleolar proteins separated by SDS-PAGE, a single band corresponding to the 100 kD protein is revealed. When the technique is applied to whole cells, small punctate regions of the nucleoli are strongly stained. At the ultrastructural level, bismuth selectively contrasts the fibrillar centers and the adjoining cords of the dense fibrillar component. The remainder of the dense fibrillar component is not stained. It is proposed that the high phosphorylation level of the 100 kD protein is responsible for its glutaraldehyde-insensitive bismuth staining. The concentration of this protein in certain localized regions of the nucleolus suggests that it plays a metabolic rather than a structural role.     

Heparan sulfate proteoglycans made by different basement-membrane-producing tumors have immunological and structural similarities

Using immunological assays, we determined the relationship between the heparan sulfate proteoglycans produced by two different murine basement-membrane-producing tumors, i.e., the mouse Engelbreth-Holm-Swarm (EHS) tumor and the L2 rat yolk-sac tumor. Antibodies prepared against the heparan sulfate proteoglycans obtained from these two sources immunoprecipitated the same precursor protein with a molecular mass of 400,000 daltons from 35S-methionine pulse-labeled cells of both tumors. Immunohistochemistry showed the heparan sulfate proteoglycan to be distributed in the extracellular matrix and also in the native basement membrane of surrounding normal murine tissues. Blocking and ELISA assays demonstrated that the antibodies recognized both antigens. Using techniques involving the chemical and enzymatic degradation of 35S-sulfate-labeled glycosaminoglycans, the mouse EHS tumor cells were found to produce mainly heparan sulfate (75%) along with smaller amounts of chondroitin sulfate (19%), whereas the L2 rat yolk-sac tumor produced mainly chondroitin sulfate (76%) with smaller amounts of heparan sulfate (21%). We conclude that these two murine basement-membrane-producing tumors elaborate an immunologically and structurally similar type of high-molecular-weight heparan sulfate proteoglycan which subsequently becomes incorporated into basement-membrane-like material.     

Specific alterations in the pattern of histone-3 synthesis during conversion of human leukemic cells to terminally differentiated cells in culture

The presence of nano- to micromolar concentrations of 12-0-tetradecanoyl-phorbol-13-acetate (TPA) in suspension cultures of human promyelocytic leukemia cells, HL-60, or human monocytic leukemia cells, THP-1, resulted in the appearance of macrophage-like cells attached to the substratum. The terminally TPA-differentiated cells continued to synthesize histones at a low rate even though DNA replication had ceased. The pattern of synthesis of histone variants in differentiated cells differed from that in undifferentiated cells and resembled that of quiescent or density-arrested cells. In undifferentiated cells, all three histone-H3 variants are synthesized, while in quiescent cells, only the H3.3 variant is synthesized. When TPA-differentiated macrophages were placed in normal medium, the pattern of histone synthesis was not altered, thus substantiating previous findings that the differentiation is irreversible. Further, TPA-differentiated macrophages and macrophages isolated from a normal human donor exhibited identical pattern of histone synthesis. Altogether, the results indicate that changes in the synthetic rates of histones during the TPA-induced maturation of human leukemic cells is not directly due to TPA or terminal cell differentiation per se but is due to the cessation of cell proliferation and DNA replication.     

Retinoid suppression of transglutaminase activity and envelope competence in cultured human epidermal carcinoma cells

Growth of SCC-13 squamous carcinoma cultures in the presence of retinoids considerably reduced the expression of two differentiation markers, the cellular capability to form cross-linked envelopes, and the enzyme transglutaminase required for cross-linking. A limited survey of retinoids showed that all-trans retinoic acid, 13-cis retinoic acid, and arotinoid Ro 13-6298 were highly effective in the absence of hydrocortisone and were only slightly antagonized by its presence in the medium. In contrast, retinyl acetate, retinol, and retinol bound to its plasma binding protein were quite active in the absence of hydrocortisone but were essentially inactive in its presence. Dexamethasone was also highly effective in antagonizing the suppressive action of retinyl acetate on envelope formation, while the corticosteroid antagonists cortexolone and progesterone were inactive. These results suggest that there are separate pathways, which are differentially regulated by hydrocortisone, for either the metabolism or action of retinol and retinoic acid in SCC-13 cells.     

Adhesion of human amnion epithelial cells to extracellular matrix : Evidence for multiple mechanisms

Human amnion epithelial cells attach and flatten slowly (approximately 65 min) onto plastic in the presence of serum but much more rapidly (20-30 min) onto subcellular matrix (SCM) deposited by the same cells. This matrix contains both fibronectin and laminin, but neither molecule on its own can reproduce its adhesive properties. Cells attach on surfaces containing fibronectin and laminin and extend filopodial and lamellipodial areas of cytoplasm without extensive flattening in the perinuclear region. Matrix deposited onto plastic by amnion epithelial cells has trypsin-sensitive and trypsin-resistant, papain-sensitive adhesion-promoting components. Cell spreading triggered by the latter but not the former can be inhibited by pretreating the adhering cells with heparin. Other GAGs are without effect. The results are discussed in terms of multiple interactions between epithelial cells and basal laminae.     

Stage-specific cohesion of cell ghosts and plasma-membrane fragments from Dictyostelium discoideum

During fruiting-body construction by Dictyostelium discoideum, the formation and subsequent maintenance of the multicellular assembly involve two stage-specific cohesive systems that are acquired sequentially and are distinguishable on serological and genetic grounds. We demonstrated that both systems, termed aggregation related (AR) and postaggregation related (PAR), can function in vitro. Ghosts prepared from cells of the wild-type and of a cohesion-defective mutant that were harvested during growth and at aggregation and postaggregative stages of fruiting-body construction exhibited the same cohesive properties as the cells from which they were derived. Membrane fragments prepared from the ghosts by mechanical disruption retained these cohesive properties.     

The isolation of an antimycin A-resistant human cell line

An antimycin A-resistant derivative of the human cell line, D98, has been obtained by selective mutagenesis with N-methyl-N'-nitro-N-nitrosoguanidine. The derivative, designed MA65, is capable of continuous growth in 15 microM antimycin and the resistant phenotype is stable in the absence of selection. MA65 is not cross-resistant to chloramphenicol or triethyl tin. Crude membrane preparations from MA65 after propagation in medium containing antimycin have normal succinate-cytochrome c oxidoreductase activity and the respiratory activity of whole cells continues in the presence of the drug. The mitochondrially synthesized proteins of D98 and MA65 are similar when compared on sodium dodecyl sulphate (SDS), or isoelectric focusing gels, but there is a reproducible difference in the extent of labelling of one band detected by isoelectric focusing. Genetic analysis is consistent with the existence of a cytoplasmically localized determinant conferring resistance.     

The influence of contact guidance on chemotaxis of human neutrophil leukocytes

Chemotaxis of human neutrophil leukocytes moving on or in aligned 3D fibrin gels is more efficient if the cells are moving along the axis of fibre alignment than if they have to cross the fibres. This was shown by using two assays, one in which the cells were responding to a distant (600 micrometers) gradient source diffusing from a filter paper impregnated with formyl-Met-Leu-Phe and incorporated into the gel, the other in which the cells were responding to nearby (20--30 micrometers) Candida albicans spores in serum. In the former assay, impairment of chemotaxis across the axis of fibre alignment was highly significant. In the latter, cells showed efficient chemotaxis to the spores, but took more irregular paths when crossing the aligned fibres than when running along them. Neutrophils show contact guidance in aligned collagen or fibrin gels (Wilkinson et al., Exp cell res 140 (1982) 55) [1], thus the cells were subjected simultaneously to two directional cues in these experiments, one the chemotactic gradient and the other a contact guidance field. These cues may reinforce or interfere with each other depending on their relative orientation. Since many tissues in vivo show alignment or more complex forms of patterning, tissue architecture is likely to be an important determinant of the efficiency of cellular mobilization in inflamed or infected sites.     

Selective inhibition of calcium-stimulated cation-induced pinocytosis by starvation and inhibitors of protein synthesis in Amoeba proteus

The capacity of Amoeba proteus to form pinocytotic channels after pretreatment with either puromycin, cycloheximide, emetine or a long period of starvation was studied. The effect on pinocytosis of the three inhibitors of protein synthesis was similar. They preferentially affected pinocytosis induced by Na+ with little effect on K+-induced pinocytosis. In Ca2+-deficient media, Na+-induced pinocytosis was inhibited, while the addition of Ca2+ restored channel formation. The degree of inhibition of Na+-induced pinocytosis was influenced by the concentration of Ca2+ in the inducing solution. Selective Ca2+-reversible inhibition of Na+-induced pinocytosis also occurred after starvation or treatment with a proteolytic enzyme, subtilisin. The membrane potential in starved or emetine-treated cells in culture medium was normal and their depolarising response to inducers was not diminished in solutions containing Na+. The resting input resistance of these cells was higher than in normal amoebae, but no significant difference in electrical parameters was observed after pinocytosis was induced. It is suggested that starvation, inhibition of protein synthesis, and enzyme digestion deplete the membrane of structures which are necessary for normal Ca2+ functions during induction of pinocytosis by Na+-like inducers.     

Decreased longevity of human diploid cells after incorporation of latex spheres within their cytoplasm

The effects of cytoplasmic incorporation of latex spheres (a cytoplasmic marker) on the growth potential of human diploid cells was examined. After incorporation of latex spheres within their cytoplasm, GM2290 (diploid, Lesch-Nyhan) cells showed a reduced replicative potential. A lower percentage of cells exposed to latex particles incorporated [³H]thymidme during any subsequent 24-h test period when compared with comparable, untreated cells. The overall life expectancy of the cultures treated with spheres was reduced approx. 25%. A similarly treated and examined transformed cell line (HeLa) showed no similar adverse effects after incorporation of latex spheres. The results suggest that latex spheres should be used with caution in experiments on in vitro cellular senescence.     

Ultrastructural organization of nucleoproteins during aging of cultured human embryonic fibroblasts

Analysis of nucleoproteins in resting human embryonic fibroblasts in vitro at different population doubling levels (PDL) using electron microscopy revealed the disappearance of non-nucleolar ribonucleoprotein structures at high PDL, the nucleoli became larger and the filamentous masses containing the nascent nucleolar RNA displayed a fibrillo-granular pattern which has never been described previously. In addition, conventional fixation revealed the disappearance of most of the stainable chromatin whose threads were unusually spaced and shortened specially at the nuclear surface after loosening. We interpret these changes in chromatin organization as the consequence of the alkali-sensitive sites that accumulate during senescence.     

A teratocarcinoma-derived endoderm stem cell line (1H5) that can differentiate into extra-embryonic endoderm cell types

We investigated the ability of the teratocarcinoma-derived, epithelial-type cell line 1H5 to differentiate into either of the two pathways to primary endoderm, and tested the hypothesis that 1H5 represents a state similar to primitive endoderm in the late 4th-day blastocyst. Like other endodermal cell types, 1H5 cells mixed with embryonal-carcinoma cells sort out into "embryoid bodies" or structures that resemble 4th-day mouse embryos. The epithelial line conforms morphologically and biochemically to the few known characteristics typical of primitive endoderm. The present study demonstrates that the formation in vitro of overt visceral endoderm is readily achieved. The spontaneous arrangement of the cells into a cystic form is followed by the appearance of several markers of visceral endoderm, most notably alphafetoprotein, which is detected when 1H5 cells are cultured either in the presence of retinoic acid or when the cells interact with embryonal-carcinoma cells in a specific spatial arrangement after sorting out. However, some less specific properties of visceral endoderm are not expressed. Although 1H5 differentiates histologically into parietal-like endoderm in the tumor form, parietal cells cannot yet be identified with certainty in vitro because of the paucity of parietal-specific markers. The 1H5 cell line could provide a useful system for studying the characteristics and mechanisms underlying visceral-endoderm differentiation in vitro, since it has the distinct advantage that homogeneous cultures are produced, in contrast to other teratocarcinoma cell lines such as F9 which differentiate into a mixture of cell types.