A comparative study of RNA fractions mediating delayed sensitivity to a chemically defined antigen in vitro.

Hot-cold phenol extracts of RNA prepared from guinea pigs sensitized to mono (p-azobenzene-arsonate)-N-chloroacetyl-l-tyrosine (ARSNAT) contains limited but distinct fractions able to transfer ARSNAT or KLH sensitivity to guinea pig peritoneal exudate cells in vitro. Each of these fractions have been compared by oligo(dT) affinity chromatography and sucrose density gradient analysis. One RNA fraction initially obtained from a sucrose density gradient (designated as B fraction) possessed two separate peaks and contained polyadenylic acid sequences as evidenced by its ability to bind to an oligo (dT) column. Another fraction (Fraction II) initially isolated by oligo (dT) affinity chromatography possessed two peaks after sucrose density gradient analysis, contained poly-A sequences, and had an S-value range approximating the B fraction. RNA fractions prepared from the liver or skeletal muscle of sensitized guinea pigs fails to transfer ARSNAT sensitivity and all fractions are completely inactivated by bovine pancreatic RNase. The results suggest that portions of density gradient prepared B fraction and Fraction II binding to oligo (dT) cellulose may represent the same and/or similar moieties of immunobiologically active RNA.     

Comparison of isoelectric focusing in Sephadex vs immobiline flatbeds for the recovery of follicle regulatory protein from porcine follicular fluid

We describe and compare the use of isoelectric focusing (IEF) in a granulated Sephadex matrix and in polyacrylamide immobilized pH gradients to separate an aromatase inhibitor (follicle regulatory protein: FRP) in preparative amounts from porcine follicular fluid (PFF). The starting material for IEF was derived from pFF after passage through agarose immobilized textile dye Orange A (0.5 KC1 eluent). Before IEF, some Orange A bound (OAB) material was further purified on a FPLC employing a Mono-Q anion exchange column. Previous use of chromatofocusing indicated that aromatase inhibitory activity is largely concentrated in OAB fractions with a pI in the ranges of pH approximately 4.5 and approximately 6.5. The current study revises these findings to provide a more precise measure of the isoelectric points in question to pH 4.73 +/- 0.05 and pH 6.41 +/- 0.06. The use of Sephadex was limited by gradient instability and the selection of pH ranges available. IEF using immobilized pH gradients had several advantages over Sephadex: 1) broader selection of gradients from 0.1 to 7.0 pH units; greater resolving power, and enhanced stability. The principal disadvantage of the immobiline system was the recovery of focused material from the gel matrix. The use of isoelectric focusing with immobilized pH gradients on a preparative scale to purify FRP from OAB resulted in a greater than 50% recovery with a substantial increase in specific activity (from ID50 approximately 300 micrograms/ml to 20 ng/ml).     

Preparative isoelectric focusing of human serum very low-density and low-density lipoproteins.

Ten percent glycerol prevented the usual precipitation of human serum very low-density lipoproteins (VLDL) and low-density lipoproteins (LDL) at their isoelectric points during their preparative isoelectric focusing (IEF), IEF separated VLDL and LDL into two major fractions. The observed optical density peaks are not artifacts caused by binding of Ampholines to VLDL or LDL since no radioactivity accumulated in the fractions containing VLDL or LDL during IEF in the presence of [¹⁴C]Ampholine, and gel filtration completely separated the lipoproteins from [¹⁴C]Ampholine. These results suggest that IEF may separate subspecies of VLDL and LDL under suitable experimental conditions.     

DNA methyltransferase levels in tumorigenic and nontumorigenic cells in culture

The levels of DNA methyltransferase in nuclei from 9 tumorigenic and 9 nontumorigenic cell lines were examined. In all but 2 cases, the extractable methyltransferase activity was 4-3000-fold higher in tumorigenic than in nontumorigenic cells. Tumorigenic and nontumorigenic cells from four species were grown in the presence of various concentrations (10(-8)-10(-6) M) of an inhibitor of the methylase enzyme, 5-aza-2'-deoxycytidine (5-aza-dCyd). The reduction of 5-methylcytosine content in newly replicated DNA in the presence of 5-aza-dCyd was used to determine the relative methylase activity in each cell line. In all 4 cases, tumorigenic cells required larger doses of drug to inhibit DNA methylation to the same extent as their nontumorigenic counterparts. The relative rates of incorporation of [3H]5-aza-dCyd were determined for each cell line, and tumorigenic cells were shown to incorporate equal or greater amounts of 5-aza-dCyd into DNA compared to nontumorigenic cells. These results showed that the differences in the inhibition of DNA methylation in response to 5-aza-dCyd were not due to differences in the ability of these cells to incorporate the drug. Thus, it was demonstrated by two independent methods that tumorigenic cells contained higher levels of methylating capacity than nontumorigenic cells. This overabundance of methyltransferase may alter DNA methylation patterns and affect phenotypic stability.     

Characterization and preparation of chymosin from calf rennet samples by means of isoelectric focusing

Chymosin has been identified in rennet samples from stomaches of living calfs by means of it's isoelectric points (IP), molecular weight (Mol. Wt.) and enzymatic activity. The values obtained by isoelectric focusing (IEF) and a two-dimensional electrophoretic technique, amount 4,5…4,7 (IP) and 36 300 (Mol. Wt.) respectively. Furthermore the fractionation of calf rennet by IEF has been performed in a preparative scale. From one gramme of calf rennet we obtained 53 mg of purified chymosin, which shall be used for preparing antisera against chymosin.     

Purification of acetyl-CoA: 17-O-deacetylvindoline 17-O-acetyltransferase from Catharanthus roseus leaves

The enzyme acetyl-CoA: 17-O-deacetylvindoline 17-O-acetyltransferase which terminates vindoline biosynthesis has been isolated from Catharanthus roseus leaves, further characterized and purified to homogeneity by three step column chromatography and subsequent preparative isoelectric focusing. Kinetic properties concerning the enzyme reaction are discussed. Five multiple forms of the acetyl-transferase could be observed, each consisting of two subunits. This enzyme is now the best characterized of the enzymes involved in vindoline biosynthesis.Abbreviations DTE dithiothreitol - EDTA ethylenediamine-tetraacetic acid - HEPES N-(2-hydroxyethyl)-piperazine-N-2-ethanesulfonic acid - IEF isoelectric focusing - KP_i potassium phosphate - M_r rel.molecular mass - PEG polyethylene glycol - SDS-PAGE sodium dodecylsulfate polyacrylamide gel electrophoresis - Tris 2-amino-2-(hydroxymethyl)-1,3-propandiol     

Correlation of the inability to sustain growth in defined serum-free medium with the suppression of tumorigenicity in Wilms' nephroblastoma.

We report the investigation of the growth properties of tumorigenic and reverted nontumorigenic Wilms' nephroblastoma cells when cultured in serum-free medium. Wilms' tumor, a pediatric nephroblastoma, has been associated with deletions encompassing the p13 band of chromosome 11 and an independent loss of heterozygosity at 11p15. Weissman et al. (Science 236:175-180, 1987) transferred a human der(11) chromosome into the G401.6TG.6 Wilms' tumor cell line via the microcell-mediated chromosome transfer technique. The resulting microcell hybrids were nontumorigenic when assayed in nude mice; however these cells retained all of the in vitro growth and morphological characteristics of the tumorigenic parental cells in 10% fetal calf serum (FCS). Segregation of the der(11) chromosome from the nontumorigenic microcell hybrid cells resulted in the reappearance of the tumorigenic phenotype in vivo. In vitro culture of these cell lines in serum-free medium supplemented with 0.1% bovine serum albumin (BSA) and 10 ng/ml Na2O3Se resulted in sustained growth of both the tumorigenic parent and the tumorigenic segregant while the nontumorigenic microcell hybrids were unable to divide. The separate addition of either 10 ng/ml of epidermal growth factor (EGF) or 5 micrograms/ml of insulin did not alter this effect. However, the addition of 5 micrograms/ml of transferrin stimulated the nontumorigenic microcell hybrid cells to grow at a rate comparable to the tumorigenic cells. In addition, conditioned serum-free medium from the tumorigenic parental or tumorigenic segregant cell lines was able to stimulate the growth of the nontumorigenic microcell hybrid cells, whereas the reciprocal experiment had no effect on the growth of the tumorigenic cells. These data suggest that the inability of the microcell hybrid cells to grow in serum-free conditions is correlated with their genetic nontumorigenic phenotype and that a specific growth factor, transferrin, can bypass or alter this negative growth regulatory pathway(s) in vitro.     

Demonstration of apolipoprotein CII in guinea pigs. Functional characteristics, cDNA sequence, and tissue expression

In contrast to plasma from other mammals, guinea pig plasma does not stimulate the activity of lipoprotein lipases in vitro. This had led previously to the conclusion that guinea pigs lack an analogue to apolipoprotein CII (apoCII). By adsorption of lipid-binding proteins to lipid droplets, thereby separating them from other plasma components, we could demonstrate apoCII-like activity in guinea pig plasma. On electrophoresis, the CII-like activity co-migrated with one isoform of guinea pig apolipoprotein CIII, identified by amino-terminal amino acid sequence determination (40 residues). By isoelectric focusing in a narrow pH gradient, the activating protein was separated sufficiently from the dominating apoCIII isoform to allow sequence determination of 8 residues from the amino terminus. Six of these were identical to corresponding residues in apoCII from dog and monkey. With the aid of a human apoCII cDNA probe we identified one cross-hybridizing mRNA species (approximately 600 nucleotides) on Northern blots of guinea pig liver. Three positive clones were isolated from a guinea pig liver cDNA library using the same cDNA probe. The nucleotide sequence showed extensive similarities to the previously known human, monkey, and canine sequences, but the signal peptide was 3 amino acid residues longer in the guinea pig protein, and there was a deletion of 4 residues in the putative lipid binding domain. Northern blot analyses indicated that guinea pig apoCII is mainly expressed in the liver with little or no contribution from the intestine.     

Purification and characterization of extracellular chitinase from Aeromonas schubertii

A locally isolated stain Aeromonas schubertii was cultured and induced by powdered chitin for the production of chitinases. Extracellular proteins were purified by ammonium sulfate precipitation, dialysis to remove salts, and then preparative isoelectric focusing (IEF) to yield several chitinases. The purified enzymes were analyzed by SDS–PAGE (sodium dodecyl sulfate–polyacrylamide gel electrophoresis) with and without glycol chitin and were found to be SDS-resistant. The chitinase present in the highest abundance was the one with an estimated molecular weight of 75 kDa. The Michaelis constant and turnover number were determined to be 0.29 mM and 1 s⁻¹, respectively, for this enzyme using colloidal chitin azure as the substrate. However, the ethanol treatment of this enzyme could significantly increase its chitinolytic activity. Other chitinases obtained in the same IEF fraction were determined to have molecular weights of ca. 30, 38, and 110 kDa. Since the proteins with highest chitinase activity were collected from IEF fraction tube with pH value of 4.8, those chitinase were believed to be acidic. An activity assay method using colloidal chitin azure as the substrate was recommended since it possessed a broader range of linearity in comparison with conventional reducing sugar equivalent method.     

Heterogeneity of the vacuolar pyrophosphatase protein fromChenopodium rubrum

Summary Activities of the tonoplast ATPase (V-ATPase EC 3.6.1.3) and PPase (V-PPase EC 3.6.1.1) provide the proton gradient driving the accumulation of various metabolites, organic and inorganic ions in the plant vacuole. We used anion exchange chromatography, liquid-phase isoelectric focusing (IEF), and continuous-elution native polyacrylamide gel electrophoresis (preparative PAGE) to enrich the V-PPase from solubilized tonoplast proteins from suspension cultured cells ofChenopodium rubrum L.The fractions were identified by their enzymatic activity, sensitivity towards the specific PPase inhibitor aminomethylenediphosphonate, apparent molecular weight, and immunological reactivity with an antibody raised against mung bean V-PPase. All these different methods used for the separation of solubilized tonoplast proteins revealed the existence of two physically separable V-PPase proteins exhibiting substrate specific enzymatic activity and 66 kDa apparent molecular weight after sodium dodecyl sulfate(SDS)-PAGE. The isoelectric points of the active V-PPase forms were 5.05 and 5.48 (V-ATPase 6.1). On the basis of the observation of high recoveries of enzymatic activity after different preparations we suggest that the V-PPase proteins separated may represent physiologically occurring forms of the enzyme which cannot be distinguished by SDS-PAGE and Western blot.     

Rapid apolipoprotein E phenotyping by immunofixation in agarose

Conventional determination of apolipoprotein E isomorphs comprises ultracentrifugation of 1-5 ml serum, delipidation of very low density lipoproteins (VLDL), and isoelectric focusing (IEF) in polyacrylamide gels. In order to reduce the sample volume and to avoid nonspecific protein bands, immunoblotting was proposed. Now we describe a methodological variant that uses 25 microliters serum, replaces ultracentrifugation by precipitation of apoE-containing lipoproteins with polyethylene glycol, and delipidation by dissolution in detergent. IEF is carried out in agarose. This allows specific immunofixation of apoE-containing bands with 10 microliters antiserum per sample. This method yields apoE patterns that are specific and well resolved. Also, it offers considerable savings of time and equipment involved.     

Identification and characterization of gp TFA-1, a guinea pig T cell surface antigen associated with T cell function

Monoclonal antibodies (Ab) were produced that specifically recognized guinea pig T cells. FACS analysis revealed that Ab 188 bound to the majority of peripheral T lymphocytes of strain 2 and strain 13 guinea pigs and to a minor population of thymocytes. It failed to react with the Ia-bearing guinea pig B cell leukemia line EN-L2C, with macrophages, bone marrow cells, erythrocytes, or thrombocytes. Treatment of T cells with Ab 188 and complement prevented T cell activation. Culturing primed T cells with antigen- or mitogen-pulsed syngeneic or with allogeneic macrophages in the continuous presence of Ab 188 produced a marked, dose-dependent inhibition of T cell proliferation. The antigen defined by Ab 188 was therefore designated guinea pig T lymphocyte function-associated antigen-1, gp TFA-1. The magnitude of inhibition by Ab 188 varied between 65 and 85% whereas three other antibodies to guinea pig T cells had no inhibitory effect on T cell proliferation. Time course experiments revealed that gp TFA-1 is critically involved in an early phase of T cell activation. Maximal inhibition was achieved only if the antibody was present from the beginning of the cell culture; the addition of antibody after 24 hr of culture no longer had an inhibitory effect. Ab 188 did not induce T cell mitogenesis. Two-dimensional analysis (one-dimensional, IEF; two-dimensional, SDS-PAGE) of immunoprecipitates obtained from NP40 lysates of [35S]methionine-labeled T cell blasts indicated that a molecule was specifically precipitated that consisted of two noncovalently associated polypeptide chains with apparent m.w. of 43,000 and 38,000. Both subunits displayed extensive charge heterogeneity focusing at an average isoelectric point of 5.0 and 6.5, respectively. The gp TFA-1 molecule exhibits striking similarities in its functional and structural properties to recently described clonotypically expressed T cell glycoproteins, which were shown to be involved in antigen recognition by T cells in the murine and human systems.     

Fractionation of murine macrophage inhibitory factor into two distinct species

Murine migration inhibitory factor (MIF) produced by concanavalin A-stimulated lymph node cells from C57BL/6 mice was fractionated by Sephadex G-100 gel filtration, density gradient electrophoresis, and isoelectrofocusing in a sucrose density gradient and assayed on in vitro-cultivated bone marrow macrophages from C57BL/6 mice. Two major MIF species, pH3-MIF with an isoelectric point of 3.0–4.3 and pH5-MIF with an isoelectric point of 4.6 to 5.2, were obtained. The similarity of murine MIF to guinea pig and human MIF is discussed.     

Humoral factors in very young NZB mice that enhance the maturation of normal B cell precursors. Partial purification and characterization

Soluble factors that enhance maturation of murine B lineage precursor cells in vitro were partially purified from the serum of very young NZB mice and characterized biochemically and biologically. Activity was initially detected by induction of colony-forming activity and surface immunoglobulin (sIg) on normal sIg- marrow cells as well as responsiveness of a pre-B cell line. Pooled sera from 4- to 5-wk-old NZB mice were initially fractionated on Sephacryl S-300 and Sephadex G-100 superfine columns. Fractions with activity (corresponding to m.w. of 15,000 to 45,000) were pooled and further separated. The activity was eluted as a single peak by hydrophobic (phenyl-Sepharose, with 0.8 M (NH4)2SO4) and lentil lectin affinity chromatography but resolved into three distinct peaks in preparative isoelectric focusing (IEF), with pI values of 3.5, 7.8, and 8.4. The latter two merged into a single peak with a pI value of 8.8 when the sample was further treated with neuraminidase before IEF. These three IEF fractions, each of which were enriched at least 1000-fold in specific activity relative to starting serum, were then characterized. Each was stable at pH 2 but sensitive to trypsin, 10 M urea, and heat treatment (56 degrees C for 1 hr). In nonreduced SDS-poly-acrylamide gel electrophoresis, their mobilities corresponded to m.w. of 17,000 for peak I (pI 3.5), 15,000 for peak II (pI 7.8), and 15,000 for peak III (pI 8.4). Interleukin 1, interleukin 2, interleukin 3, colony-stimulating factor for granulocyte and macrophage progenitors, antiviral, or B cell growth factor type I-like activities were not demonstrable. Peaks II and III, but not peak I, induced Ig secretion of anti-stimulated B cells. Peak I was also less effective than peaks II and III in induction of sIg on an established pre-B cell line. However, all fractions were equally effective in enhancing maturation of normal sIg- B lineage cells. Thus, serum from 4- to 5-wk-old NZB mice contains at least two distinct soluble factors that can enhance the maturation of sIg- B lineage cells in vitro. The biologic and biochemical characteristics of these factors appear to differ from those of previously well-defined cytokines.     

Nuclear control of tumorigenicity in cells reconstructed by PEG-induced fusion of cell fragments

The techniques of somatic cell hybridization have provided a valuable means of studying mechanisms of regulation of mammalian cell differentiation and transformation. Most previous studies have indicated that fusions between tumorigenic and nontumorigenic cells result in hybrid cells that are usually tumorigenic. In recent years it has been demonstrated that the phenotypic expression of tumorigenicity is at least partially due to the extensive chromosome loss that occurs in most interspecific and some intraspecific hybrid cells. In the present study we have utilized enucleation techniques that permit cells to be divided into nuclear (karyoplast) and cytoplasmic (cytoplast) cell fragments. Even though these nuclear and cytoplasmic fragments are metabolically stable for short periods of time, in our hands they ultimately degenerate. Viable cells can be reconstructed by PEG-induced fusion of karyoplasts to cytoplasts. Since reconstructed cells apparently do not segregate chromosomes, they may provide a clearer understanding of the interactions between the nucleus and the cytoplasm in the control of the expression of tumorigenicity. We have reconstructed cells using karyoplasts from the tumorigenic Y-1 cell line and cytoplasts from a nontumorigenic cell line, A-MT-BU-A1. In addition we have reconstructed cells containing Y-1 cytoplasts and A-MT-BU-A1 karyoplasts. The reconstructed cells porduced were assayed for tumorigenicity by their ability to grow in soft agar and in nude mice. The results of these experiments indicate that the reconstructed cells containing a tumorigenic nucleus and a nontumorigenic cytoplasm ultimately are tumorigenic and conversely the reconstructed cells containing a nontumorigenic nucleus and a tumorigenic cytoplasm are nontumorigenic. These experiments support the concept that with these cell lines the nucleus (karyoplast) is sufficient to control the phenotypic expression of tumorigenicity.     

Separation of bacterial cells by isoelectric focusing, a new method for analysis of complex microbial communities.

A simple isoelectric focusing (IEF) method for whole bacterial cells was developed. In a pH gradient of 2 to 10 and an electric field of 11.5 V cm-1, mixtures of cells from the three different bacterial strains Chlorobium limicola 6230, Pseudomonas stutzeri DSM 50227, and Micrococcus luteus DSM 20030 could be separated. A density gradient of Ficoll prevented convective currents in the system. The method was tested with a concentrated mixture of bacteria from a shallow eutrophic lake and yielded up to 10 different bands. Species composition in each IEF band was analyzed by PCR plus denaturing gradient gel electrophoresis (DGGE). Each IEF band exhibited a different species composition. After the separation of cells by IEF three times more 16S ribosomal DNA signals could be detected by DGGE than in the unfractionated natural bacterial community. It is concluded that the resolution of these molecular biological methods is significantly enhanced if cells are first separated by IEF. At the same time, the IEF fractions are enriched for certain species, which can be used in subsequent cultivation experiments.