The in vitro DNA-binding properties of purified nuclear lamin proteins and vimentin.

The ability of purified nuclear lamin A, lamin B, lamin C, and vimentin from Ehrlich ascites tumor cells to bind nucleic acids was investigated in vitro via a quantitative filter binding assay. At low ionic strength, vimentin bound more nucleic acid than the nuclear lamins and showed a preference for G-containing nucleic acids. Nuclear lamins A and C were quite similar in their binding properties and bound G- and C-containing nucleic acids preferentially. The binding of poly(dT) by the lamins A and C was reduced in competition experiments by both poly(dG) and poly(dC), but not by poly(dA). Lamin B bound only oligo and poly(dG); no other nucleic acids tested were bound or could compete with the binding of oligo(dG). Vimentin, lamin A, and lamin C specifically bound a synthetic oligonucleotide human (vertebrate) telomere model. The Ka for vimentin (2.7 X 10(7) M-1) was approximately 10-fold higher than those for lamin A (2.8 X 10(6) M-1) and lamin C (2.9 X 10(6) M-1). Lamin B did not bind detectable amounts of the telomere model. Washing of lamin A- and lamin C-nucleic acid complexes, formed at low ionic strength, with solutions containing 150 mM KCl resulted in the elution of 30% of bound poly(dG)12-18 and 70% of bound synthetic oligonucleotide telomere model. These results, using purified individual proteins, are in good agreement with data from competition experiments with vimentin but are at odds with data obtained previously using a crude preparation of nuclear matrix proteins containing all three nuclear lamin proteins (Comings, D. E., and Wallack, A. S. (1978) J. Cell Sci. 34, 233-246). The nuclear lamins A and C and vimentin possess nucleic acid-binding properties that might permit their binding to specific base sequences and/or unique DNA structure, such as that observed for the binding of the telomere model. The significance of the higher affinity binding of nucleic acids by the cytoplasmic protein vimentin (compared with the nuclear lamins) remains to be elucidated.

Efecto de la desagregación molecular por métodos físicos (politrón) de la molécula de coralan sobre su actividad inmunomoduladora in vitro / Effects of molecular disintegration by physical methods (politron) of coralan molecule on its immunomodulator activity in vitro

Elcoralan es un polisácarido que ha demostrado inducir en los ratones una alta capacidad de rechazo al trasplante de células del tumor ascítico de Ehrlich. Este producto provoca además un aumento significativo en la proliferación de linfocitos humanos como respuesta a la fitohemaglutinina (PHA). Este producto es de dificil esterilización por lo que se han buscado métodos para desagregar la molécula (Politrón) sin que pierda su actividad biológica

Protein chemical analysis of purified murine lamin B identifies two distinct polypeptides B1 and B2.

Lamin B purified from murine EAT cells was characterized by partial protein sequences. Contrary to the current view that mammals express only a single lamin B polypeptide corresponding to a characterized murine cDNA clone, our analysis documents two distinct B lamins. One protein follows the established cDNA sequence while the other identifies a novel murine lamin B. Comparison with the two chicken lamin B sequences established by cDNA cloning identifies the first murine lamin B sequence as a B1 type and the second as a B2 type. We conclude that mammals express two distinct lamin B forms as established by others for chicken.

Changes of the surface proteolytic activity in synchronized Ehrlich ascites tumor cells grown in vivo.

Changes of the cell surface proteolytic activity during the cell cycle in vitro were reported. Using an easy assay, with casein as a substrate, the proteolytic activity on the surface of Ehrlich ascites tumor (EAT) cells grown in vivo was determined. The cleavage of casein incubated with EAT cells increased linearly for 20 min and permitted reproducible enzyme activity determinations. If the proliferation of exponentially growing EAT cells was partially synchronized by an intraperitoneal bleomycin injection, a significant increase of the surface enzymatic activity was observed in cells with an increased DNA content. This finding supports the results obtained with transformed cells in vitro, indicating that elevated proteolytic surface activity occurs in the late synthesis phase and prior to mitosis. However, the observed effect may also be due to changes of gene expression caused by bleomycin.

Calcium-binding protein from mouse Ehrlich ascites-tumour cells is homologous to human calcyclin.

A Ca2+-binding protein was purified from mouse Ehrlich ascites-tumour cells. The protein forms monomers and disulphide-linked dimers, which can be separated by reverse-phase h.p.l.c. A partial amino acid sequence analysis demonstrated that the protein has an EF-hand structure. A striking homology was found to rat and human calcyclin (a member of the S-100 protein family), which is possibly involved in cell-cycle regulation.

Tissue specific distribution of calcyclin--10.5 kDa Ca2+-binding protein.

Expression of calcyclin in different cell lines and mouse tissues was determined with polyclonal antibodies raised against calcyclin from Ehrlich ascites tumour (EAT) cells. The protein was detected in mouse skeletal and cardiac muscle, in lung, kidney and spleen, and was especially enriched in mouse smooth muscle as well as in rat fibroblasts. No positive immunological reaction was detected in mouse brain, liver and intestine and some tumourigenic cell lines. The level of calcyclin mRNA found in different cells and tissues corresponded well to the calcyclin level estimated by immunoblotting. The calcyclin-like protein was purified from mouse stomach and appeared to be very similar to the EAT protein.

[A fluorometric method of determining DNA in cells]. / Fluorometricheskii metod opredeleniia DNK v kletkakh.

A convenient express method is proposed for cell lysis and DNA solubilization in the presence of sarcosyl and sodium citrate. The DNA content was determined in cell lysates by registration of fluorescence enhancement with Hoechst No 33258 ("Serva", FRG). In 0.5 M NaCl solution the Hoechst-RNA fluorescence is negligible. An optimal molar ratio of DNA and Hoechst is in the range from 0.2 to 2.0. Beyond the range the accuracy of DNA quantification become poor. An optimal range of DNA quantification in cells is 200-2000 ng/ml.

Direct determination of bound sialic acids in sialoglycoproteins by acidic ninhydrin reaction.

A simple and rapid method for sialic acid determination in sialoglycoproteins by acidic ninhydrin reaction is described. The method is based on the reaction of sialic acids with an acidic ninhydrin reagent (K. Yao and T. Ubuka (1987) Acta Med. Okayama 41, 237-241). By heating a sample solution containing sialoglycoprotein with the reagent at 100 degrees C for 10 min, a stable color with an absorption maximum at 470 nm was produced. The standard curve was linear in the range of 20 micrograms to 3 mg of fetuin, a sialoglycoprotein, per 3.0 ml of the reaction mixture. The reaction is specific only for sialoglycoproteins among various proteins examined. The acidic ninhydrin method was applied to the determination of sialic acids in sialoglycoproteins in ascites fluids of Ehrlich ascites tumor-bearing mice.

Isolation and reconstitution of furosemide-binding proteins from Ehrlich ascites tumor cells.

Furosemide-binding proteins were isolated from cholate-solubilized membranes of Ehrlich ascites tumor cells by affinity chromatography, using furosemide as ligand. Solubilized proteins retarded by the affinity material were eluted by furosemide. In reducing and denaturing gels, the major proteins eluted by furosemide were 100 and 45 kDa. In nonreducing, non-denaturing gels, homodimers of both polypeptides were found, whereas no oligomeric proteins containing both polypeptides were seen. It is concluded that the furosemide gel binds two distinct dimeric proteins. The isolated proteins were reconstituted into phospholipid vesicles and the K+ transport activity of these vesicles was assayed by measurement of 86Rb+ uptake against a large opposing K+ gradient. The reconstituted system was found to contain a K+ transporting protein, which is sensitive to Ba2+ like the K+ channel previously demonstrated to be activated in intact cells after cell swelling.

[Angiogenesis activity of tissues and cells of different malignancies].

Cancer cells are able to produce tumor angiogenesis factors (TAF), by which the tumor growth could be maintained. Angiogenetic activity of 12 kinds of cells and tissues with distinct character from different sources were detected by chick embryo chorioallantoic membrane. The results indicated that all the cells and tissues tested showed angiogenetic activity but they differed from one another, some were strong and other weak. Of them, the breast cancer was the strongest. The ascitic tumor didn't show any angiogenetic activity, but it reappeared when the ascitic cells were transformed to solid tumors. The level of angiogenetic activity of tumors was closely related to their biological properties and form of existence.

Interactions of factors bound at two different sites in the 5'-upstream region of the adenovirus 12 E1A gene.

A nuclear extract of Ehrlich ascites tumor cell contains factors binding to two distinct sites in the 5'-upstream region of the adenovirus 12 E1A gene. The gel shift assay was performed for characterization of the binding factors with oligo DNA probes containing sequences corresponding to these sites. The specific binding of a factor to one probe was enhanced when the other oligo DNA was present in excess in the binding reaction. Thus possibly, protein-protein interactions between factors may mutually prevent their binding to target sequences.

Measurement of cell swelling in a hypotonic medium as a rapid and sensitive test of cell injury.

A development and application of a novel test of cell damage is described. The test is based on the ability of undamaged cells to swell in the hypotonic medium. The test is much more rapid and sensitive than the tests based on staining with trypan blue or fluorescein diacetate.

[DNA-bound lipids of the cells of Zajdela ascites hepatoma and of Ehrlich ascites cancer]. / DNK-sviazannye lipidy kletok astsitnoi gepatomy Zaidela i astsitnogo raka Erlikha.

DNA-bound neutral lipids (NL) and phospholipids (PL) were isolated and characterized from the Zajdel ascites hepatoma (ZAH) and Ehrlich ascites carcinoma (EAC) cells. The lipids are represented by light- and tightly bound components. It was shown, that the tumour DNA contained minor amount of NL (25, 17 micrograms and 16.87 micrograms per mg DNA, respectively) and of PL (4.54 micrograms and 5.36 micrograms per mg DNA, respectively, for ZAH and EAC). The composition of the tumour DNA-bound lipids was shown to differ from that of DNA-bound lipids of liver and thymus of intact rats by the next parameters: NL/PL ratio is much more than one; increased content of FC; equal values of the three basic ratios--CE/FC, NL/PL, cholesterol/PL, presence of mono- and triglycerides.

Characterization of Ca2+-binding proteins from Ehrlich ascites tumor cell cytoplasm and their binding to membranes.

A set of proteins in the 33-37 kDa range have been isolated from the cytoplasm of the Ehrlich ascites tumor cell. The proteins are characterized by their Ca2+-dependent binding to cell membranes. This property has been used for isolation of the proteins by Ca2+-dependent affinity binding to inside-out vesicles of the human red cell membrane. The proteins display Ca2+-binding properties as shown by gel-filtration studies. The Ca2+-dependent binding of the 33 and 34 kDa proteins to red cell membranes was studied after labelling of the proteins with tritium by reductive methylation. The average number of Ca2+ bound per protein molecule was 4.8 with a Kd of 3.4.10(-4) M Ca2+. The proteins are distinct from most other Ca2+-binding proteins of comparable molecular weights by not incorporating phosphate.

Response of different mammary epithelial cell lines to a mammary derived growth inhibitor (MDGI).

MDGI, a 14.5-KDa protein, is a chemically defined growth inhibitor, which we have purified from lactating bovine mammary gland and characterized biologically in a mouse Ehrlich ascites mammary tumour (EAT) short term suspension culture. It has now been tested for its inhibitory activity on proliferation of four malignant mammary epithelial cell lines of human and mouse origin and normal human mammary epithelial cells. In all experiments, cells were brought to quiescence by serum or growth factor deprivation. Using [3H]TdR pulse labelling the effect of MDGI was measured on the restimulation of proliferation after medium change. MaTu and T47 D, human malignant mammary epithelial cell lines, as well as the mouse malignant mammary epithelial cell line mMaCa 20177 could be inhibited, whereas the human malignant mammary epithelial cell line MCF7 showed a slight stimulation. MDGI showed no activity on the residual DNA synthesis of all cell lines after starvation. Normal human mammary epithelial cells (HMEC) of different passages could also be inhibited. Their responsiveness seemed to be dependent on the number of passages. Cells from high passages (10-14) showed a higher sensitivity, which is also about 10 times higher than that of the malignant cell lines. Furthermore, growth factors like insulin, epidermal growth factor (EGF) and fetal calf serum (FCS), known to be potent antagonists to the MDGI activity in the EAT and, in the case of insulin, also in the MaTu culture (shown in the present study), do not abolish the inhibitory activity of MDGI on HMEC cells. These results demonstrate that the inhibitory activity of MDGI is not exclusively restricted to EAT cells studied so far.

Simultaneous analysis of mitochondrial activity and DNA content in Ehrlich ascites tumor cells by dual parameter flow cytometry.

Ehrlich ascites tumor cells were permeabilized using low concentrations of digitonin, 8 micrograms/10(6) cells. Permeabilization was monitored by the assay of lactate dehydrogenase released into the incubation medium and of hexokinase partially bound to mitochondria. Integrity of the cellular organelles was unaffected as determined by assay of the mitochondrial enzyme glutamate dehydrogenase. Cells were stained with rhodamine 123 as a mitochondrial specific dye and propidium iodide/mithramycin as DNA specific dyes. The green fluorescence of bound rhodamine 123 versus red fluorescence of DNA in individual cells was analysed by dual parameter flow cytometry. Incubation of cells with inhibitors of mitochondrial energy metabolism, such as, potassium cyanide and carbonyl cyanide m-chlorophenylhydrazone abolished binding of rhodamine 123. Flow cytometric data allowed a correlation between cell position in the mitotic cycle with total mitochondrial activity. In addition, comparison of the characteristics of propidium iodide and ethidium bromide staining further elucidated the molecular basis of the staining with the positively-charged fluorescent dye rhodamine 123.

Isolation and characterization of a 58-kDa membrane- and microfilament-associated protein from ascites tumor cell microvilli.

A 58-kDa protein is found in microvilli and in actin-containing transmembrane complexes of 13762 ascites tumor cells with immobile surface receptors; it is absent from sublines with mobile receptors. 58-kDa protein has been proposed to stabilize microvilli and restrict receptor mobility by stabilizing membrane-microfilament interactions. Antibodies against 58-kDa protein were blot-purified from antisera of rabbits injected with crude transmembrane complex and were used to monitor purification of the protein. 58-kDa protein was extracted from EDTA/EGTA-stripped microvillar microfilament cores with 1 M NaCl. A single depolymerization-polymerization cycle of the microfilaments, followed by solubilization of 58-kDa protein in 1 M NaCl and chromatography on hydroxyapatite-Sephadex G-150, purified the protein to greater than 95% homogeneity. The native molecular weight and frictional coefficient indicated a monomeric, asymmetric structure. 58-kDa protein bound F-actin in pelleting assays and inhibited polymerization of pyrenyl-actin. It also bound phosphatidylserine, phosphatidylinositol, and phosphatidylcholine vesicles in pelleting studies. Immunoblot analyses of endogenously and exogenously proteolyzed microvilli and their membranes and microfilament cores showed specific membrane and microfilament binding fragments of 28-30 kDa. The microfilament- and phospholipid-binding properties of 58-kDa protein and the localization of its proteolysis products are consistent with its proposed role in stabilizing membrane-microfilament interactions in the ascites cell microvilli.

Mercuric chloride induces autoantibodies against U3 small nuclear ribonucleoprotein in susceptible mice.

Autoantibodies to nucleolar components are a common serological feature of patients suffering from scleroderma, a collagen vascular autoimmune disease. While animal models, which spontaneously develop abundant anti-nucleolar antibodies, have not yet been described, high titers of such antibodies may be induced by treating susceptible strains of mice with mercuric chloride. We have identified the nucleolar autoantigen against which the HgCl2-induced IgG autoantibodies from mice of strain B10.S are directed. It is a protein with an apparent molecular mass of 36 kDa and a pI value of approximately 8.6, which is associated with the nucleolar small nuclear RNA U3, and by these criteria must be identical with a polypeptide called fibrillarin. It is striking that scleroderma patients spontaneously produce autoantibodies against the same U3 ribonucleoprotein (RNP). The HgCl2-induced murine and the scleroderma-specific human anti-U3 RNP autoantibodies were indistinguishable in their reactivities toward fibrillarin. They further resemble each other insofar as both recognize epitopes on the 36-kDa protein, which have been highly conserved throughout evolution. Our results provide a basis to investigate at the molecular level whether similar immunoregulatory dysfunctions may lead to the preferential anti-U3 RNP autoantibody production in the animal model and in scleroderma patients.

Simple and effective purification of a Na+-dependent amino acid transport system from Ehrlich ascites cell plasma membrane.

A reconstitution assay was used to measure transport activity during purification of a Na+-dependent amino acid transporter from Ehrlich cell plasma membrane. Cholate/urea-solubilized membranes were fractionated on a Sepharose 6B column and transport activity was recovered in the column void volume. Centrifugation of the void volume fraction at 105,000 X g and reextraction of the pellet with 1% octyl glucoside led to recovery of an extract whose specific transport activity was nearly 30-fold higher than that of the original solubilized extract with a recovery of 38% of the original activity. The properties of amino acid uptake in the purified reconstituted transporter were identical to those in native plasma membrane vesicles. The major component present in the purified fraction had a molecular mass of 120-130 kDa. Strong evidence that this 120- to 130-kDa peptide contains a component of the amino acid transporter was obtained by immunoprecipitation of transport activity from solubilized membranes with an antibody against the 120- to 130-kDa peptide. This study tentatively identifies a component of the Na+-dependent amino acid transporter as a peptide with an apparent molecular mass of 120-130 kDa.

Nucleolin (C23), a physiological substrate for casein kinase II.

Nucleolin (C23), a 110 kDa phosphoprotein, which is mainly found in the nucleolus has been shown to be a physiological substrate for casein kinase II (CKII). Nucleolin was identified and characterized by immunodetection using an anti-nucleolin antibody. Phosphopeptide patterns from nucleolin phosphorylated by purified casein kinase II and of phosphorylated nucleolin which had been isolated from tumor cells grown in the presence of [32P]-o-phosphate, were identical. The partial tryptic digest revealed nine phosphopeptides. Nucleolin isolated from Krebs II mouse ascites cells was phosphorylated by purified casein kinase II with about two moles phosphate per one mole of nucleolin.