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R Heimer

Publications and source records attributed to R Heimer.

At least 55 records · Page 3Linked to original sources
PubMed1990

Reductions in gamma-globin mRNA levels restricted to the cytoplasm of 5-fluorouridine treated K-562 human erythroleukemia cells.

RNA synthesis in K-562 human erythroleukemia cells was markedly curtailed by exposure to the uridine analogue 5-fluorouridine (FUrd). The inhibition of ribosomal RNA synthesis was accompanied by rapid declines in the steady-state levels of several, but not all, mRNAs, including gamma-globin mRNA. In this report, we demonstrate that gamma-globin mRNA species were decreased by as much as 40% within 2 hr of exposure to micromolar concentrations of FUrd. The decline in gamma-globin mRNA occurred at a rate that outstripped the normal rate of degradation of this mRNA by a factor of 25. The decline in cytoplasmic mRNA was not mirrored in the nucleus; northern blotting revealed that pre-mRNA levels were not reduced. Nuclease protection analyses of precursors from FUrd treated and untreated control cells did not reveal any qualitative differences. Thus, the decrease was not accounted for by drug-induced inhibition of new gamma-globin mRNA synthesis or misincorporation but must have been due to an FUrd-induced increase in gamma-globin mRNA degradation. Drug-induced instability of RNA was not a generalized feature of FUrd exposure, since neither beta-actin mRNA nor cytoplasmic rRNA, whose stabilities in untreated cells are similar to that of gamma-globin mRNA, were affected. Furthermore, the instability of gamma-globin mRNA did not decrease globin protein levels, presumably because the stability of the protein was not altered. The mechanism by which specific increased degradation of gamma-globin mRNA occurred is unknown, but it may have been due to the activation of cytoplasmic endonuclease.

Actins↗
PubMed1989

Proteoglycan profiles obtained by electrophoresis and triple immunoblotting.

Using synovial fluid of individuals with osteoarthritis as a prototypic biologic fluid containing one part proteoglycan per 100 to 1000 parts of other protein, profiles of proteoglycan were produced without preliminary purification through agarose-acrylamide gel electrophoresis, transfer to nitrocellulose, and triple immunoblotting. Chondroitin sulfate, keratan sulfate, and a hyaluronic acid binding region appear to be present on individual synovial fluid proteoglycans in variable amounts, and consequently a triple immunoblot using monoclonal antibodies to these three epitopes has the potential for developing a proteoglycan profile. The profile is assembled by means of densitometric scans of autoradiograms obtained after use of 125I-labeled anti-mouse immunoglobulin. By contrast to the profile of a relatively homogeneous proteoglycan purified from articular cartilage extracts, the proteoglycans of synovial fluid appeared to be quite heterogeneous with the bulk of keratan sulfate epitopes migrating ahead of the bulk of the chondroitin sulfate epitopes. Most of the proteoglycans appeared to possess a hyaluronate binding region.

Animals↗
PubMed1989

RNA polymerase II transcripts as targets for 5-fluorouridine cytotoxicity: antagonism of 5-fluorouridine actions by alpha-amanitin.

The cytotoxicity of 5-fluorouridine (FUrd) results from actions directed at the synthesis of both DNA and RNA. The role of mRNA as a target for FUrd was investigated by selectively decreasing the incorporation of FUrd into RNA polymerase II transcripts of K-562 erythroleukemia cells, which was accomplished by the addition of alpha-amanitin to cultures of K-562 cells permeabilized with lysolecithin. In these cells alpha-amanitin at concentrations of 1-5 micrograms/ml inhibited the incorporation of [3H]-uridine into polyadenylated RNA by up to 45% and decreased the steady-state levels of two specific mRNAs but had no effect on poly A- RNA synthesis. alpha-Amanitin decreased the incorporation of FUrd into poly A+ RNA by up to 60%. The decrease in FUrd incorporation produced by alpha-amanitin was accompanied by an antagonism of the growth inhibitory effects of the fluorinated pyrimidine nucleoside by the mycotoxin, as measured by both growth in suspension culture and colony formation in 0.12% agar. Antagonism between these agents increased as the concentration of alpha-amanitin was elevated; furthermore, it was sequence-dependent, occurring only when alpha-amanitin preceded FUrd. These findings provide evidence that the actions of FUrd directed against mRNA are antagonized when FUrd incorporation into mRNA transcripts is decreased and that the effects of FUrd on mRNA produce cytotoxic consequences.

Adenosine↗
PubMed1987

Detecting proteoglycans immobilized on positively charged nylon.

Proteoglycans (PG) immobilized on positively charged Nylon 66 are detected readily by staining with Alcian blue. With the exception of hyaluronic acid, free glycosaminoglycans appear unreactive when treated similarly. Immobilization was performed by dot blotting or by electrophoretic transblotting from various gel supports. When transblotted to positively charged Nylon 66 from large-pore agarose-acrylamide gels, levels of 10-50 ng of PG could be detected by Alcian blue staining. This procedure appeared to be nearly 10(2) times more sensitive than staining of gels with toluidine blue. The transblot and staining procedure also appears to be effective with PG separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and was applied to a preparation enriched in basement membrane components.

Alcian Blue↗
PubMed1987

Detection by 125I-cationized cytochrome c of proteoglycans and glycosaminoglycans immobilized on unmodified and on positively charged nylon 66.

We have examined the detection by a 125I-labeled basic protein, cationized cytochrome c, of selected proteoglycans (PGs) and standard preparations of glycosaminoglycans (GAGs) immobilized on Nylon 66 and also on positively charged Nylon 66. Immobilization on Nylon 66 appears to allow a relative freedom of interaction between PGs or GAGs and 125I-cationized cytochrome c, but a more restricted reaction was observed when PGs and GAGs were immobilized to positively charged Nylon 66. On this support PGs with large numbers of GAG side chains reacted well with 125I-cationized cytochrome c, but GAGs were minimally reactive. By taking advantage of some of the properties of large-pore agarose-acrylamide gels, rapid partial characterization of some PGs can be accomplished in the 10-ng range, and therefore at a sensitivity equal to PGs with internal biosynthetic labels.

Animals↗
PubMed1986

Levels of thymidylate synthetase during normal culture growth of L1210 cells.

The binding of 5-fluoro-2'-deoxyuridylate generated from 5-fluoro-2'-deoxyuridine in intact cells was used to measure changes in the level of thymidylate synthetase during the course of population growth of murine leukemia L 1210 cells. By the use of elutriation techniques and flow cytometric analysis, the amount and activity of thymidylate synthetase associated with the various phases of the cell cycle were determined for the L 1210 cells during unperturbed in vitro culture growth. Fluctuations of thymidylate synthetase levels were associated with the cell cycle; there was a positive correlation (P less than 0.001) between the percentage of the total cell population in S phase and the concentration of thymidylate synthetase, although there was an increase in the level of this enzyme in association with an increase in G2-M cells, this did not achieve statistical significance. A negative correlation between G1 cells and the concentration of thymidylate synthetase was also observed. The maximum amount of thymidylate synthetase was nearly 900 fmol/10(6) cells and occurred in cell populations during logarithmic growth when the percentage of the population in S phase and G2-M phase was greater than 50 and 20%, respectively. In late culture growth (plateau) when only 25% of the cell population was in S phase and nearly 75% of the population was in G1 phase, the level of enzyme was reduced to 200 fmol/10(6) cells.

Animals↗
PubMed1985

The specificity of antibodies to the F(ab')2 fragment of human IgG.

The specificity of IgG anti-F(ab')2 antibodies was examined by unfractionated sera of patients with rheumatoid arthritis and also with affinity-purified antibody preparations. Examination of the sera by an enzyme-linked immunosorbent assay, using pooled human F(ab')2 fragments absorbed to microtiter plates, revealed that IgG anti-F(ab')2 antibodies cross-react with human and rabbit IgG and rabbit F(ab')2. IgG anti-F(ab')2 antibodies were purified by affinity chromatography and, when tested by a fluid-phase inhibition enzyme-linked immunosorbent assay, were found to be of 2 types. One fraction, similar to pepsin agglutinator, reacted with human F(ab')2 fragment alone. The other fraction was cross-reactive with human IgG and yet failed to react with idiotopes on Fab or epitopes on Fc fragments. The IgG anti-F(ab')2 antibodies we purified had no reactivity toward a human immune complex prepared from tetanus toxoid and antitoxoid.

Antibodies, Anti-Idiotypic↗
PubMed1985

Lack of enhanced cytotoxicity of cultured L1210 cells using folinic acid in combination with sequential methotrexate and fluorouracil.

Previous studies from this laboratory have demonstrated that treatment of cultured cells with sequential methotrexate (MTX) and fluorouracil (FUra) leads to synergistic cell killing in several murine and human neoplasms in vitro. In this study leucovorin (folinic acid, LCV) was added to the MTX/FUra combination with the intention of generating elevated levels of methylenetetrahydrofolate to promote the formation of a stable fluorodeoxyuridylate-thymidylate synthetase ternary complex, thereby augmenting the cytotoxicity of the MTX-FUra sequence. The addition of 10 or 100 microM LCV concurrently with or after 10 microM FUra following MTX (1 microM) pretreatment did not augment the inhibition of L1210 cell growth or the clonigenicity compared with MTX prior to FUra without LCV. The effects of LCV scheduling on the sequential MTX and FUra-induced inhibition of thymidylate synthesis were measured by examining the rate of [6-3H] dUrd incorporation into the acid-precipitable cell fraction and by direct quantitation of the thymidylate synthetase ternary complex. Combination of 100 microM LCV with 10 microM FUra after 1 microM MTX resulted in significantly more ternary complex formation than did 1 microM MTX before 10 microM FUra alone. The inhibitory effects of FUra on thymidylate synthetase in the presence of MTX, however, could not be augmented by LCV as determined by [6-3H] incorporation into acid-precipitable material, nor did the addition of LCV result in increased cytotoxicity. Factors other than the inhibition of DNA synthesis may be critical to the cytotoxicity of sequential MTX and FUra in L1210 cells.

Animals↗
PubMed1985

Detection of glycosaminoglycans at the one-nanogram level by 125I-cytochrome c.

The basic protein cytochrome c forms stable ionic complexes with all known glycosaminoglycans. When labeled with 125I, cytochrome c is capable of detecting exceptionally small quantities of glycosaminoglycans. Subsequent to electrophoresis on cellulose acetate strips using pyridine formate buffer at pH 3, followed by ethanol fixation, and treatment with 125I-cytochrome c, all the known glycosaminoglycans are detected at minimum levels of 1 ng/0.25-microliter application. The method can be used for quantification of glycosaminoglycans in other electrophoretic buffer systems also.

Animals↗
PubMed1985

Analysis of immune complexes by two-dimensional gel electrophoresis.

High resolution two-dimensional (2D) gel electrophoresis is useful for analysis of constituents of immune complexes (IC) in serum, provided that the samples for the analysis are prepared by a standardized and effective purification protocol. The details of the protocol, which involve gel permeation chromatography and adsorption with protein A-Sepharose, were worked out with a model system of radiolabeled antigen bound to antibody. With this protocol one can attain an over 50% recovery of the antigen, in a protein preparation purified over 1000-fold with respect to starting amounts in an initial 0.5 ml serum. With silver staining of the 2D gel, the model antigen was detectable at levels of 100 ng initial input. The analysis of eight normal sera and eight sera of patients with systemic lupus erythematosus (SLE) showed no clearly demonstrable differences, suggesting that the latter sera did not contain homogeneous antigens exceeding 100 ng within IC. In addition to IgG, albumin was seen in all preparations, probably due to complexing with immunoglobulin. Trace amounts of other constituents of the samples appeared in some gels, and the presence of C3 related material was detectable only by Western blotting.

Antigen-Antibody Complex↗
PubMed1984

Specificity of IgM antibodies to pooled human F(ab')2 fragments.

An isotope specific immunoassay which minimizes interference by endogenous rheumatoid factors was used to determine the specificity of IgM anti-F(ab')2 antibodies in human serum. We underscore the heterogeneity of these antibodies. While one subset of IgM anti-F(ab')2 antibodies reacts only with intact F(ab')2, another recognizes determinants present following reduction and alkylation of F(ab')2 and separation of Fd' fragments from light chains. IgM anti-F(ab')2 antibodies in sera from rheumatoid arthritis patients do not react significantly with intact pooled IgG and, therefore, probably are not anti-idiotypic antibodies. Some sera, but not all, contain elevated levels of antibodies that are crossreactive with rabbit F(ab')2. Such crossreactive antibodies may interfere with assays which utilize F(ab')2 fragments of rabbit antibodies specific for antigens of clinical relevance.

Antibody Specificity↗
PubMed1983

Modulation of fluoropyrimidine metabolism in L1210 cells by L-alanosine.

L-Alanosine, an analogue of aspartic acid which inhibits the conversion of inosine monophosphate to adenosine monophosphate (AMP), was evaluated in L1210 cells as a modulator of 5-fluorouracil (FUra) and 5-fluorouridine (FUrd) metabolism. L-Alanosine resulted in increased intra-cellular levels of 5-phosphoribosyl-1-pyrophosphate (PRPP), enhanced FUra metabolism to ribonucleotide derivatives, and resulted in more FUra residues incorporated into RNA. Sequential addition of L-alanosine and FUra also resulted in synergistic cytotoxicity as determined by soft agar cloning. Adenine antagonized these biochemical and biological effects of L-alanosine. L-Alanosine also augmented the rate at which FUrd was metabolized and was also associated with a greater incorporation of the FUra residues into RNA. Cytotoxicity after sequential L-alanosine and FUrd was also synergistic. The mechanism by which L-alanosine altered the metabolism of FUrd, however, was different from the way in which it enhanced FUra metabolism in that aspartic acid and not adenine was able to reverse the effects of L-alanosine on FUrd metabolism and cytotoxicity. L-Alanosine appeared to augment the RNA-directed activity of FUra and FUrd in that there was no correlation between the enhanced metabolism and cytotoxicity of these two fluoropyrimidines and either levels of fluorodeoxyuridylate (FdUMP) which inhibits thymidylate (TMP) synthetase or inhibition of the ability of cells to incorporate deoxyuridylate into acid-precipitable material.

Adenine↗
PubMed1982

IgM and IgG anti-F(ab')2 antibodies in rheumatoid arthritis and systemic lupus erythematosus.

Radioimmunoassays for anti-F(ab')2 antibodies, which feature the use of goat anti-human Fc antibody for correcting potentiation of IgM anti-F(ab')2 antibody titers by endogenous IgM anti-Fc antibodies (rheumatoid factors), are described. Individuals with classic rheumatoid arthritis had significantly more IgM anti-F(ab')2 antibody (P less than 0.001) and IgG anti-F(ab')2 antibody (P = 0.05) than did individuals with systemic lupus erythematosus or normal volunteers. There is some similarity in patterns of isotype distribution of anti-F(ab')2 antibodies and rheumatoid factors.

Antibodies, Anti-Idiotypic↗
PubMed1981

The influence of methotrexate pretreatment on 5-fluorouracil metabolism in L1210 cells.

Pretreatment of L1210 cells with methotrexate in concentrations which produced free intracellular methotrexate and near maximal inhibition of dihydrofolate reductase resulted in an enhancement of intracellular 5-fluorouracil (FUra) accumulation. This enhancement of FUra accumulation was maximum (5-fold increase) after a 6-h exposure to 100 microM methotrexate. The nucleotide derivatives of FUra, including a 5-fluoro-2'-deoxyuridylate, and 5-fluorouridine-5'-triphosphate were also increased nearly 5-fold following methotrexate treatment. In cells pretreated with methotrexate, there was an increase in intracellular 5-phosphoribosyl-1-pyrophosphate pools which ranged from 2 to 8 times control values following concentrations of methotrexate between 0.1 microM and 10 microM. Both the increase in 5-phosphoribosyl-1-pyrophosphate and FUra accumulation could be prevented by the addition of Leucovorin (N5-formyltetrahydrofolate) at concentrations which rescued cells from the inhibitory effects of methotrexate. Pretreatment with 6-methylmercaptopurine riboside, which inhibits amidophosphoribosyltransferase, the first committed step in de novo purine synthesis, also resulted in a similar elevation in 5-phosphoribosyl-1-pyrophosphate pools and enhancement of FUra accumulation. If the 5-phosphoribosyl-1-pyrophosphate pools were reduced following methotrexate pretreatment by the addition to the cultures of hypoxanthine, which utilizes 5-phosphoribosyl-1-pyrophosphate for the conversion to IMP, the intracellular accumulation of FUra was not enhanced. Also, if the inhibitor of 5-phosphoribosyl-1-pyrophosphate synthetase, 7-deazaadenosine, was given to cultures with methotrexate, there was no increase in 5-phosphoribosyl-1-pyrophosphate pools, nor enhancement of FUra accumulation. In addition, when 5-fluoro-2'-deoxyuridine was added with the methotrexate to cell cultures, there was no increase in 5-phosphoribosyl-1-pyrophosphate pools, nor enhancement of intracellular FUra accumulation. These results indicate that the ability of methotrexate to enhance FUra accumulation was probably the consequence of the antipurine effect of methotrexate which resulted in a reduction of the complex feedback inhibition on 5-phosphoribosyl-1-pyrophosphate synthesis and utilization. The resultant increased 5-phosphoribosyl-1-pyrophosphate pools were then capable of being utilized for the conversion of FUra to 5-fluorouridylate, the possible rate-limiting step in FUra intracellular metabolism and the major determinant of the rate of intracellular FUra accumulation. When methotrexate preceded FUra, there was synergistic cell killing as determined by soft agar cloning. The exact mechanism of this sequential synergistic antitumor activity may be the result of the enhanced incorporation of FUra into RNA, since the increased 5-fluoro-2'-deoxyuridylate which is formed is unlikely to increase substantially the inhibition of dTMP synthesis induced by methotrexate pretreatment.

Animals↗
PubMed1981

The detection of antigens in immune complexes.

A procedure is described for the purification of soluble immune complexes (IC) from biologica fluids and for the detection of antibody-bound antigen. A model IC, prepared with various amounts of human serum albumin (HSA) and constant amounts of anti-HSA, gave a 44% recovery after gel filtration on Sephacryl S-300, affinity chromatography on protein A--Sepharose, and concentration of eluates there from before analysis by sodium dodecyl sulphate--polyacrylamide gel electrophoresis. It was calculated that 20 ng of antibody-bound HSA should be detectable when polyacrylamide gels are subjected to a direct radioimmunoassay involving the use of rabbit anti-HSA, 125I-protein A, and autoradiography, thus defining the sensitivity of the procedure for detecting IC-bound antigen in a model system. The procedure has direct relevance to the examination of IC of unknown composition present in sera of individuals with various diseases.

Animals↗