sepharose and Carcinoma--Ehrlich-Tumor

In order to obtain better understanding of radiation-induced alterations in intracellular metabolism, a dynamic and non-invasive experimental model system is required. A serial study in cultured tumor cell line followed by verification in the in vivo samples may be of considerable value for non-invasive prediction and/or detection of tumor response to therapy. The present study was undertaken to evaluate the radiation response of perfused Ehrlich ascites tumors cells (EATC) immobilized in agarose gel matrix to that observed in mouse bearing EATC tumor, in order to identify biomarkers of radiation response.. Perfused EAT cells, entrapped in agarose gel threads were irradiated in the perfusion assembly outside the magnet with fast electrons (6 Gy, 1 Gy/min) using 30 MeV Betatron. Solid EATC tumors implanted subcutaneously onto right hand limb of Swiss-albino strain "A" mice, were focally irradiated using 60Co teletherapy (10 Gy, 0.4 Gy/min). Metabolites changes were monitored by 31P MR spectroscopic techniques.. A post-irradiation decrease in the levels of ATP and ADP along with an increase in inorganic phosphate and glycerophosphocholine levels was observed. The ratios of beta-phosphate of ATP to inorganic phosphate (beta-ATP/Pi), and phosphocholine to glycerophosphocholine (PC/GPC), declined during 1-5 hours following irradiation, in perfused EAT cells and in the solid tumors implanted in mice.. Perfused cells could be used as a simple model of tumor for prediction of clinical radiotherapeutic response. The present study demonstrates that radiation damage may be occurring both at the DNA protein as well as the membrane lipid levels. Therefore, the bioenergetics and phospholipid profiles of tumor cells could be used as complimentary, reliable and sensitive indirect indicators for devising predictive assays for assessment and monitoring of radiation response, which will also facilitate the individualization and optimization of tumor radiotherapy.

Topics: Adenosine Diphosphate; Adenosine Triphosphate; Animals; Biomarkers, Tumor; Carcinoma, Ehrlich Tumor; Magnetic Resonance Spectroscopy; Mice; Phosphorus Isotopes; Phosphorylcholine; Predictive Value of Tests; Sepharose; Tumor Cells, Cultured

By affinity chromatography with Sepharose coupled to 2'-deoxy-1-beta-D-ribofuranosyl-N4-dodecanoylcytosine, deoxycytidine kinase and cytidine deaminase were purified 1,950- and 2,240-fold, respectively, from Ehrlich carcinoma cells, and their enzyme activities for several deoxycytidine analogs were investigated.

Topics: Animals; Carcinoma, Ehrlich Tumor; Chromatography, Affinity; Chromatography, High Pressure Liquid; Cytidine Deaminase; Deoxycytidine Kinase; Kinetics; Mice; Sepharose; Tumor Cells, Cultured

Bumetanide-binding proteins were isolated from membranes of Ehrlich ascites tumor cells by affinity chromatography. An affinity column was constructed with the active moiety of bumetanide as a ligand using 4'-azidobumetanide, a photoactive analogue which inhibits Na/Cl cotransport in Ehrlich cells with high specificity. Covalent binding of the 4'-azidobumetanide with Sepharose was promoted by photolysis. Membranes isolated from Ehrlich cells were solubilized with n-octylglucoside. Solubilized proteins retarded by the affinity column were readily eluted by bumetanide. In reducing gels the major proteins eluted by bumetanide were approximately 76 kDa and 38-39 kDa. There were also two proteins of 32 to 35 kDa eluted in lesser amounts. No proteins retarded by the affinity column were eluted with extensive washing without bumetanide. Furthermore, bumetanide eluted no proteins from a "control" column lacking the specific ligand. Upon rechromatography with bumetanide in solution, bumetanide-eluted proteins were not retarded, but their purity was increased by the retardation of contaminating proteins. Bumetanide-binding protein purified in this manner were characterized further by electrophoresis in nonreducing, nondenaturing gels.

Topics: Animals; Binding Sites; Biological Transport; Bumetanide; Carcinoma, Ehrlich Tumor; Carrier Proteins; Chromatography, Affinity; Diuretics; Electrophoresis, Polyacrylamide Gel; Membrane Proteins; Protein Binding; Sepharose; Sodium-Potassium-Chloride Symporters; Solubility

CL-Sepharose 4B column chromatography has been used for the separation of four major classes of mammalian nucleic acids in a single chromatographic run. Gel filtration at 2.5 M NaCl separated DNAs (containing RNA hybrids) from tRNAs. The 18 S RNA (containing 3-5 wt% of small 5 S RNA and RNA degradative products) was eluted at 0.7 M NaCl, and 28 S RNA (containing hnRNAs) was eluted at 0.1 M NaCl. Poly(A)+ mRNAs were detected in both 18 and 28 S RNA fractions. The present procedure is suitable for both analytical and preparative work and may serve as an initial step for the further isolation of ultrapure nucleic acid preparations.

Topics: Animals; Carcinoma, Ehrlich Tumor; Chromatography, Agarose; Chromatography, Gel; DNA; DNA, Neoplasm; Kidney; Liver; Mice; Rats; RNA; RNA, Neoplasm; RNA, Ribosomal; RNA, Transfer; Sepharose; Spectrophotometry, Ultraviolet

Normal human serum has been shown to contain IgM antibodies that are lytic to Ehrlich ascites tumor cells in the presence of complement. We call this activity ascitolysin (AL). Using a 51Cr release assay, the AL titers of 164 normal sera, 211 sera from patients with non-malignant disease, 62 sera from 25 pregnant women and 170 sera from cancer patients were estimated. The patient sera were taken preoperatively, and were assigned to the cancer or non-cancer group after histological diagnosis. In normals, higher titers were found in sera of A and O blood groups but not B and AB. The difference was due to a fraction absorbed by B red blood cells. Sera from untreated cancer patients showed significantly reduced titers when compared to normal, benign tumor, chronic and other non-malignant disease groups. In pregnancy, titers were positively correlated with duration and remained elevated for at least 6 weeks post partum. About 30% of AL activity was absorbed by the free galactose groups of agarose. This fraction was significantly lower in untreated cancer patients. Anti-T antigen antibodies do not contribute to AL levels. Galactophilic blocking factors were also found; eight times more frequently in cancer sera than in normals. The non-absorbed fraction was also lower in cancer sera. The data suggest that AL antibodies may be directed against human oncofetal antigens.

Topics: ABO Blood-Group System; Adult; Age Factors; Animals; Antibodies; Binding, Competitive; Carcinoma, Ehrlich Tumor; Cells, Cultured; Complement System Proteins; Cytotoxins; Female; Galactose; Humans; Immunoglobulin M; Male; Middle Aged; Pregnancy; Sepharose; Sex Factors; Time Factors

Monoclonal antibodies to bovine alpha-crystallin have been produced using hybridoma technology. Five selected hybridoma clones were maintained as ascites tumours in mice and gram quantities of the antibodies were purified from the ascites fluids by affinity chromatography on alpha m-crystallin covalently bound to Sepharose CL-2B. Preliminary characterization studies suggest that the antibodies are pure and monospecific. The antibodies could be divided into two groups on the basis of their reactivities towards iodinated alpha-crystallin, their ability to bind to Protein A-Sepharose, their immunoglobulin subclass, their immunoelectrophoretic patterns and their abilities to react with chicken and opossum alpha-crystallin. The availability of these monoclonal antibodies will greatly facilitate studies on the surface topography of alpha-crystallin.

Topics: Amino Acids; Animals; Antibodies, Monoclonal; Antibody Specificity; Carcinoma, Ehrlich Tumor; Cattle; Chromatography, Affinity; Crystallins; Electrophoresis, Agar Gel; Female; Hybridomas; Immunodiffusion; Immunoglobulin G; Isoelectric Focusing; Mice; Mice, Inbred BALB C; Multiple Myeloma; Neoplasms, Experimental; Pregnancy; Rabbits; Rats; Sepharose; Staphylococcal Protein A

1. The inhibition of beta-naphthylamidase activity of free trypsin, trypsin-Sepharose and a trypsin-like neutral protease on the surface of tumour cells have been studied in independent systems and with mixtures of free trypsin plus surface-bound trypsin. 2. Kinetic data have demonstrated that high-molecular-weight (protein) inhibitors of free trypsin are less effective inhibitors of trypsin-Sepharose and fail to inhibit the cell surface neutral protease. 3. Inhibition of mixtures of free trypsin plus trypsin-Sepharose follows independent kinetic plots for each component. The free trypsin is reacted before any Sepharose-bound trypsin reacts with high-molecular-weight inhibitors. 4. Low-molecular-weight inhibitors of trypsin also inhibit bound trypsin equally well. 5. Papain-derived peptides from high-molecular-weight inhibitors of trypsin inhibit free trypsin, trypsin-Sepharose and the cell-surface neutral protease almost equally well. 6. Fluorescence microscopy has shown that a high-molecular-weight inhibitor of trypsin does not bind to the tumour cell-surface neutral protease, but it does bind to trypsin-Sepharose. 7. The cell-surface neutral protease has been shown to be capable of activation of latent beta-naphthylamidase activity in the presence of excess extracellular inhibitors of free trypsin. 8. The mechanism by which trypsin-Sepharose remains partially active in the presence of excess inhibitor necessary to inhibit an equivalent quantity of free trypsin has been discussed. 9. These studies indicate that a search for inhibitors which are selectively active against the cell-surface neutral protease and have no action on trypsin-like enzymes in free solution must take into account the modifying effects of the cell surface on neutral protease activity.

Topics: Aminopeptidases; Animals; Carcinoma, Ehrlich Tumor; Chemical Phenomena; Chemistry; Enzymes, Immobilized; Kinetics; Mice; Protein Binding; Sepharose; Spectrometry, Fluorescence; Surface Properties; Trypsin; Trypsin Inhibitors

Ehrlich ascites cells have been shown to possess a protease with beta-naphthylamidase activity located on the surface of these cells. This enzyme is protected from the inhibitory action of protein inhibitors of trypsin (EC 3.4.21.4) in free solution, but is inhibited by high concentrations of active site-directed inhibitors of trypsin. We believe the protection against inhibition is provided by the location of this protease on the cell surface. We employed a model system of trypsin coupled to Sepharose to demonstrate the protective action of an inert surface, resulting in a marked reduction in inhibition of trypsin-Sepharose, compared to trypsin in free solution, when exposed to both high and low molecular weight inhibitors. This cell protease has been shown to play a role in activation of the zymogen of collagenase exported by tumour cells. This role may have important implications for tumour cell invasion of the intercellular matrix.

Topics: Aminopeptidases; Animals; Binding Sites; Carcinoma, Ehrlich Tumor; Cell Membrane; Enzymes, Immobilized; Mice; Microbial Collagenase; Peptide Hydrolases; Polysaccharides; Sepharose; Trypsin; Trypsin Inhibitors