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.     

Phosphorylation and dephosphorylation of human promyelocytic leukemia cell (HL-60) proteins by tumor promoter

Human promyelocytic leukemia cell line (HL-60) has been shown to be induced to the terminal differentiation into macrophage-like cells by a tumor promoter, 12-O-tetradecanoyl-phorbol-13-acetate (TPA). The present studies describe the effects of TPA on the phosphorylation of HL-60 cell proteins. A rapid decrease in the phosphorylation of a 75 kD protein was observed within a few minutes after treatment with TPA. On the other hand, TPA treatment of HL-60 cells caused rapid increase in the phosphorylation of a 67 kD protein and other minor proteins. Phorbol and 4α-phorbol-12,13-dodecanoate, both of which are biologically inactive derivatives of TPA, failed to cause any changes in protein phosphorylation in HL-60 cells. These results suggest that changes in protein phosphorylation are involved in mechanisms of the differentiation in HL-60 cells induced by TPA. Cell fractionation experiments revealed that 67K protein was located in cytosol. Though 75K protein also seemed to be located in cytosol, the phosphate moiety of 75K protein was almost lost during cell fractionation, suggesting that the phosphorylation of 75K protein was specifically regulated in HL-60 cells. Dimethyl sulfoxide (DMSO), retinoic acid (RA) and 1,25-dihydroxy-vitamin D₃, all of which induce the differentiation in HL-60 cells, did not cause any changes in protein phosphorylation. These results suggest that the changes in protein phosphorylation are specific for TPA. The possible mechanisms of changes in protein phosphorylation by TPA were discussed.     

Further evidence that phosphoprotein C23 (110 kD/pI 5.1) is the nucleolar silver staining protein

Previously we demonstrated a similar distribution between nucleolar organizing region-(NOR)-specific silver staining and localization of nucleolar phosphoprotein C23 (MW 110 kD/pI 5.1) [1, 2]. We now report that under fixation conditions which allow for antibody binding and subsequent silver staining, monoclonal antibody against protein C23 blocks NOR silver staining as well as silver staining in interphase nucleoli. Monoclonal antibody against nucleolar phosphoprotein B23 (MW 37 kD/pI 5.1) did not block silver staining in either NORs or interphase nucleoli. These, along with earlier observations, provide evidence that nucleolar phosphoprotein C23 is the major silver staining protein of the nucleolus and that it is directly or indirectly associated with rDNA.     

Ia-positive T lymphocytes are the producer cells of interferon gamma

The production of γ-interferon (IFNγ) in human peripheral blood T lymphocytes was induced by stimulation with PHA. For identification of the producer cell of IFNγ, double fluorescence studies were undertaken and titers of interferon were determined in preparatively separated T-cell subpopulations reactive with one of the monoclonal antibodies OKT3, OKT4, OKT8, and OKIa1. Production of IFNγ was found in OKT3⁺, OKT4⁺, and OKT8⁺ cells. However, IFNγ production occurred only in T cells also reactive with the monoclonal antibody OKIa1. Addition of macrophages had no substantial effect on interferon titers in these subpopulations. It is suggested that the T cell subset producing IFNγ is characterized by its reactivity with the monoclonal antibodies OKT3, OKT4 or OKT8, and OKIa1.     

Cell surface proteins of rat myoblasts

During the course of cell fusion of a rat myoblast cell line, L6, the relative amount of a cell surface protein of molecular weight (MW) 200 000-250 000 (L200), detected on the two-dimensional gel electrophoretogram, increases to one of a few major cell surface proteins in the older culture in which myotubes are being formed. In the non-fusing variant (M3A) of L6, such a spot is not detectable. Instead, one protein of MW 90 000-100 000 (M100) is found in large quantities in M3A, but in a small amount in L6. These results suggest the possibility that these cell surface proteins, particularly L200, may play a key role in the development of muscle cells.     

Aging of chick embryo fibroblasts in vitro. V. Time course studies on polyploid nucleus accumulation

Age-dependent polyploidization of cultured chick embryo fibroblasts was quantitated using flow microfluorometry. The results confirm the previous observation that ploidy classes developing as a function of fibroblast population doubling are defined as 2ⁿC. Immediately after isolation from embryos, the proportion of 2C nuclei was 95.2–35.7%, decreasing with advancing in vitro age. The proportion of 4C nuclei was only 3.8% at the onset of culture, increasing to 34.5% in senescent cells. The proportion of nuclei 8C and greater increased during the last stage of culture, the highest ploidy class being 128C. On the basis of the polyploidization index, which indicates relative DNA content/cell, chick cells were shown to be considerably polyploidized when they stopped growing.     

The effect of monensin on cell aggregation of normal and dystrophic human skin fibroblasts

Measurements of aggregation kinetics using couette viscometry show that freshly trypsinized skin fibroblasts from patients with Duchenne muscular dystrophy have values of intercellular adhesiveness approx. 40% those of normal cells. If cells are allowed to recover from the effects of trypsinization (by incubation for 2 h at 37 degrees C in serum-containing medium) the intercellular adhesiveness of both cell types increases, and normal and Duchenne cells aggregate to the same extent. Exposure to the ionophore monensin during the recovery phase leads to suppression of recovery in both cell types, and this effect of the drug is greater in Duchenne fibroblasts. These results are discussed in relation to other data on the reported differential effects of trypsin and monensin on normal and Duchenne fibroblasts.     

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.     

Differential effects of pure human alpha and gamma interferons on fibroblast cell growth and the cell cycle

Pure human alpha and recombinant gamma interferons had differential effects on two strains of fetal lung fibroblasts in vitro. Alpha interferon had little effect on long-term cell growth, whereas gamma interferons, both glycosylated and non-glycosylated, were cytotoxic. However, when synchronized cells were studied, alpha interferon prolonged both G1 and S + G2 phases of the cell cycle, whereas gamma interferon only affected the G1 phase.     

Detection of melanogenic proteins in cultured chick-embryo melanocytes

The phorbol ester, 12-0-tetradecanoylphorbol-13-acetate (TPA), was used as a reversible inhibitor of melanogenesis. Chick-melanocyte cultures of the black genotype, E/E, were grown in conditioned medium plus TPA. After growth in TPA and after its removal, the cells were pulse labeled with 3H-leucine. The membrane fraction, which included all tyrosinase activity as well as both mature and immature melanosomes, was solubilized with Triton X-100. The proteins were separated using two-dimensional electrophoresis and visualized by fluorography. One defined melanogenic protein, tyrosinase, was isolated, and its location was determined in the two-dimensional protein pattern. The protein patterns for both the TPA-inhibited cells and the cells in which the TPA effects were reversed after removal were compared. In addition to tyrosinase, at least nine TPA-sensitive proteins were found. These were designated as being putative melanogenic proteins which, along with tyrosinase, may be responsible for melanin-granule synthesis.     

The effects of retinoic acid on [14C]acetate incorporation into lipids of normal and transformed hamster fibroblasts

This study examined the effects of retinoic acid (RA) on [14C]acetate incorporation and fatty acid composition of hamster embryo fibroblasts (HEF) and two cell lines derived from the same inbred strain but transformed by herpes simplex-2 virus (HSV) or polyoma virus (HFT). Cells were exposed to all trans RA, or dimethylsulfoxide (DMSO), the vehicle for RA, and the lipids labeled with [14C]acetate. Lipids were extracted from the cells, separated by paper chromatography, located by autoradiography, and acetate incorporation determined by liquid scintillation spectrometry. The distribution of fatty acids in total cell lipids was examined by gas chromatography. HEF cells incorporated more acetate into cholesterol than either transformed cell type. The HFT line incorporated more acetate into triglycerides and less into total phospholipids than either the HSV line or the HEF line. RA caused a significant decrease in incorporation of acetate into cholesterol and sphingomyelin in all three cell lines. HEF and HSV cells had decreased incorporation into phosphatidyl inositol-phosphatidyl serine and increased incorporation into triglycerides, changes not evident in the HFT cell. The control fatty acid profiles of the HEF and HSV cells were similar, while the HFT cells had a larger proportion of C16:0 and 18:1 fatty acids. Following treatment with RA all three cell types showed an increase in palmitic and a decrease in oleic acids. The three related cell types showed different [14C]acetate labeling patterns which did not respond uniformly to RA. On the other hand, exposure elicited some like responses in all cell types.     

[3H]thymidine incorporation in bovine lymphocytes treated with the tumor promoter, 12-O-tetradecanoyl phorobol-13-acetate

Treatment of bovine lymph node lymphocytes with the tumor promoter, 12-O-tetradecanoyl phorbol-13-acetate (TPA) leads to depressed [³H]thymidine incorporation in response to phytohemagglutinin (PHA). Radioautographic and morphological analyses showed that depression was at the level of blast-cell formation. Isotope-dilution experiments, and the use of [³H]deoxycytidine to label DNA indicated that the inhibition was not due to a block in thymidine transport in the treated cells. These experiments, as well as a bioassay designed to measure thymidine in the culture medium, showed that the apparent inhibition of [³H]thymidine incorporation and DNA synthesis was not the result of production of cold thymidine in the cultures. The results taken together support the idea that most TPA-treated cells are inhibited from responding to the mitogenic lectins. Those cells which do respond appear to form blast cells and synthesize DNA at the same rate as do untreated cells.     

Identification of plectin in different human cell types and immunolocalization at epithelial basal cell surface membranes

The occurrence of plectin in various human tissues and cell lines was investigated using immunofluorescence microscopy and antibody gel overlay/immunoblotting techniques. Plectin was identified in all tissues and cell lines tested, namely placenta, kidney, cornea, foreskin and eyelid skin, skin fibroblasts, monocytes, keratinocytes and HeLa cells. In frozen sections of cornea and skin, plectin was found to be enriched at epithelial basal cell surface membranes. Consequently, antibodies to plectin could serve as a tool in the classification of mechanobullous diseases.     

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.     

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.     

Antibodies to the most tightly bound proteins in eukaryotic DNA : Formation of immuno-complexes with ‘nuclear matrix’ components

Chromosomal DNA is associated with polypeptides covalently bound to internal DNA ends. Since these polypeptides can only be released from chromosomal DNA by enzymes or other agents hydrolysing phosphodiester bonds they were termed 'the most tightly bound' (MTB) polypeptides in DNA. Antibodies developed against the MTB polypeptides are shown to form immunocomplexes with major 'nuclear matrix' polypeptides as well as with polypeptides which are still associated with 'nuclear matrix' DNA isolated by means of SDS/proteinase K and phenol. Immuno-complex formation is revealed by immunoblotting and by indirect immunofluorescence. Thus, since MTB polypeptides, major 'nuclear matrix' polypeptides and 'nuclear matrix' DNA-associated polypeptides share common antigenic sites they can be considered to be identical or at least closely related. This suggests that a fraction of distinct 'nuclear matrix' polypeptides is either transiently or permanently linked to DNA by covalent bonds. Consistently, isolated eukaryotic 'bulk' DNA is inevitably associated with residual 'nuclear matrix' polypeptides.     

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.     

Biosynthesis of EGF receptor, transferrin receptor and colligin by cultured human keratinocytes and the effect of retinoic acid

The biosynthesis of EGF and transferrin receptor by human keratinocytes in culture has been followed using specific monoclonal antibodies. In addition, keratinocytes are shown to synthesise a Mr 47 000 protein that binds to gelatin-Sepharose. Peptide mapping confirms the identity of this protein with colligin, a newly described cell surface-associated glycoprotein that also binds to native collagens (Kurkinen et al., J biol chem 259 (1984) 5915) [9]. Vitamin A and its analogues have profound effects on the differentiation, morphology and motility of human keratinocytes in culture. We show here that retinoic acid (RA) has no effect on the growth rate of the cells or the synthesis of EGF receptor and colligin, but stimulates the synthesis of transferrin receptor.     

Protein kinases of the human placental microvillous membrane. Their potential role in intrasyncytial communication

Protein phosphorylation has been shown to alter various plasma membrane functions. To investigate the role of phosphorylation in human placental trophoblast, microvillous membrane vesicles were incubated with [gamma-32-P]ATP and the phosphorylation of endogenous and exogenous protein substrates was measured. The microvillous membrane was shown to possess both adenosine 3',5'-cyclic monophosphate (cAMP)-independent and cAMP-dependent kinases. Both endogenous proteins and exogenous proteins were phosphorylated and these processes were enhanced by the presence of Triton or the ionophore alamethicin. The phosphorylation of histone and of endogenous peptides of molecular weights (MW) 147 000, 97 000 and 53 000 was increased by the addition of cAMP. cAMP stimulation required the presence of Triton or alamethicin. The cAMP-dependent kinases are apparently located at the internal (cytoplasmic) surface of the membrane. This location would allow stimulation by cAMP produced by the basal (fetal-facing) plasma membrane. cAMP-stimulated protein phosphorylation may serve as a means of communication between the syncytial plasma membranes facing the fetal and maternal surfaces.     

Newborn human skin fibroblasts senesce in vitro without acquiring adult growth factor requirements

Nuclear-envelope nucleoside triphosphatase activity (NTPase), an enzymatic activity thought to participate in RNA transport, was localized in rat liver in situ after brief perfusion with 3% paraformaldehyde. Reaction product was distributed along the nucleoplasmic side of the nuclear envelope (NE) in heterochromatin, was only occasionally found at nuclear pores, and nuclear deposition was selectively blocked by inhibitors of NE NTPase activity. Our results suggest that NTPases, which are active in the NE and which participate in RNA transport, are not specifically associated with nuclear-pore complexes.