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Biomedical subjects

B F Erlanger

Publications and source records attributed to B F Erlanger.

At least 19 recordsLinked to original sources
PubMed2001

Overexpression of the A1 adenosine receptor in adipose tissue protects mice from obesity-related insulin resistance.

In-vitro studies have implicated the A(1) adenosine receptor (A(1)AR) of adipocytes in inhibition of lipolysis, stimulation of lipogenesis and enhancement of the action of insulin on glucose metabolism. To determine whether any of these activities were physiologically relevant in an intact animal, A(1)AR was overexpressed in adipose tissue of transgenic mice. Lower plasma free fatty acid (FFA) levels were observed in the transgenic mice relative to the litter-matched controls, supporting a significant physiological role for adipocyte A(1)AR in the control of lipolysis. However, no differences were observed in body weights or body composition. On a high fat diet, both the transgenic mice and the litter matched controls, male and female, became equally obese. Unlike the control mice, however, the transgenic mice did not develop insulin resistance, as demonstrated by serum glucose and insulin levels and glucose and insulin tolerance tests. These findings demonstrate that adipocyte A(1)AR plays an important physiological role in the control of insulin sensitivity in an intact animal and therefore should be considered to be a potential therapeutic target for the treatment of obesity-related insulin resistance and type 2 diabetes.

Adipose Tissue↗
PubMed2000

X-ray crystal structure of an anti-Buckminsterfullerene antibody fab fragment: biomolecular recognition of C(60).

We have prepared a monoclonal Buckminsterfullerene specific antibody and report the sequences of its light and heavy chains. We also show, by x-ray crystallographic analysis of the Fab fragment and by model building, that the fullerene binding site is formed by the interface of the antibody light and heavy chains. Shape-complementary clustering of hydrophobic amino acids, several of which participate in putative stacking interactions with fullerene, form the binding site. Moreover, an induced fit mechanism appears to participate in the fullerene binding process. Affinity of the antibody-fullerene complex is 22 nM as measured by competitive binding. These findings should be applicable not only to the use of antibodies to assay and direct potential fullerene-based drug design but could also lead to new methodologies for the production of fullerene derivatives and nanotubes as well.

Amino Acid Sequence↗
PubMed1999

A1 adenosine receptor of human and mouse adipose tissues: cloning, expression, and characterization.

The aberrant functioning of the A1 adenosine receptor of adipose tissue has been implicated as a factor in obesity. To begin to address questions concerning this relationship, the possibility of a unique A1 adenosine receptor in adipose tissue must be investigated. Therefore, cDNAs encoding the A1 adenosine receptors of adipose tissues of a mouse and an obese human were isolated, sequenced, and expressed in eukaryotic cells. We found their sequences to be 90% identical and each identical to published sequences of the receptors in brain preparations of the two species. The two cDNAs were transiently expressed in 293T cells, a human kidney cell line. Despite the 90% identity in their sequences, the ligand binding properties of the human and mouse cDNAs expressed in the 293T cell line differed markedly. With respect to amino acid differences in the extracellular loops, four occur in the second extracellular loop, which has been implicated in binding by other studies. The ligand binding characteristics of the recombinant receptors matched those of native receptors from human and mouse adipose tissue. The human A1 receptor cDNA was also expressed in ob17 preadipocyte cells to investigate reported influences of cellular environment on binding characteristics. We compared ligand binding of the expressed receptor in the two cell lines (ob17 and 293T). We also compared ligand binding of native receptors from mouse brain and adipose tissue preparations. In both studies, cellular environment had no affect on binding characteristics. This conclusive evidence resolves earlier conflicting reports in the literature.

Adipose Tissue↗
PubMed1999

Initiation of a process of differentiation by stable transfection of ob17 preadipocytes with the cDNA of human A1 adenosine receptor.

A process of differentiation was observed when ob17 preadipocyte cells were stably transfected with a vector containing the cDNA of the human A1 adenosine receptor of adipose tissue. Growth of the cell lines continued but was slowed relative to untransfected cells and cells transfected with vector alone, never attaining confluence. During this process, cells were observed to differentiate morphologically and to accumulate lipid droplets in their cytoplasm, droplets that stained with Oil red-O. During that same period of time, cells transfected with vector alone multiplied rapidly, attained confluence, and showed no signs of differentiation. We conclude that expression of the A1 adenosine receptor initiated a differentiation process that resembled aspects of the normal differentiation of preadipocytes. If so, a role for the A1 receptor in normal preadipocyte differentiation should be considered, perhaps after cell-to-cell contact occurs at confluence.

Adipocytes↗
PubMed1998

Antigenicity of fullerenes: antibodies specific for fullerenes and their characteristics.

The recent interest in using Buckminsterfullerene (fullerene) derivatives in biological systems raises the possibility of their assay by immunological procedures. This, in turn, leads to the question of the ability of these unprecedented polygonal structures, made up solely of carbon atoms, to induce the production of specific antibodies. Immunization of mice with a C60 fullerene derivative conjugated to bovine thyroglobulin yielded a population of fullerene-specific antibodies of the IgG isotype, showing that the immune repertoire was diverse enough to recognize and process fullerenes as protein conjugates. The population of antibodies included a subpopulation that crossreacted with a C70 fullerene as determined by immune precipitation and ELISA procedures. These assays were made possible by the synthesis of water-soluble fullerene derivatives, including bovine and rabbit serum albumin conjugates and derivatives of trilysine and pentalysine, all of which were characterized as to the extent of substitution and their UV-Vis spectra. Possible interactions of fullerenes with the combining sites of IgG are discussed based on the physical chemistry of fullerenes and previously described protein-fullerene interactions. They remain to be confirmed by the isolation of mAbs for x-ray crystallographic studies.

Animals↗
PubMed1997

Antibodies to oxidative DNA damage: characterization of antibodies to 8-oxopurines.

The 8-oxo-7,8-dihydropurines (8-oxopurines) are important cellular premutagenic lesions produced in DNA by free radicals. Specific antibodies were prepared to detect these lesions. For antigens, 8-oxo-7,8-dihydroadenosine (8-oxoAdo) and 8-oxo-7,8-dihydroguanosine (8-oxoGuo) were synthesized from the bromonucleosides, and the immunogens were produced by conjugating these to either bovine serum albumin or rabbit serum albumin by the periodate method. Polyclonal antibodies specific for the haptens were elicited from rabbits immunized with the BSA conjugates. The antibodies to 8-oxoAdo (anti-8-oxoAdo) and 8-oxoGuo (anti-8-oxoGuo) precipitated the homologous antigens in an Ouchterlony gel diffusion assay and no cross-reactivity was observed toward the normal nucleosides or to the heterologous 8-oxopurine. Specificity was also examined by hapten inhibition of antibody reactivity with the homologous conjugates using ELISA. For anti-8-oxoAdo, the IC50 for 8-oxoAdo was 8 mumol/L and 8-bromoadenosine, guanosine, and inosine did not inhibit, even at concentrations of 1.25 mmol/L. Similarly, the IC50 for anti-8-oxoGuo for 8-oxoGuo was 0.1 mumol/L. 8-Methoxyguanosine also inhibited the reaction but was about 500-fold less effective than the eliciting hapten. Other nucleosides tested did not inhibit at concentrations up to 100 mumol/L. Both antibodies could easily detect the corresponding damage in x-irradiated f1 DNA at a dose of 7.5 Gy and both antibodies recognized the corresponding lesion in duplex DNA; however, with anti-8-oxoGuo the signal was reduced about 50% compared to single-stranded DNA. In order to determine the exact amount of each lesion produced in irradiated DNA, and to standardize the ELISA signal, both products were measured after alkaline phosphatase digestion of x-irradiated calf thymus DNA using high-pressure liquid chromatography (HPLC) coupled to an electrochemical detector. Anti-8-oxoGuo could detect ten 8-oxoG residues and anti-8-oxoAdo could detect two 8-oxoA residues per 10,000 nucleotides. Thus, these antibodies should be useful for the detection and measurement of 8-oxopurines in cellular DNA.

Adenosine↗
PubMed1996

A strategy for immunohistochemical signal enhancement by end-product amplification.

We report a novel strategy, called end-product (EP) amplification, capable of enhancing the sensitivity of immunohistochemical procedures by about an order of magnitude or more. The strategy employs an antibody (anti-EP) to the product generated by the action of horseradish peroxidase on 3,3'-diaminobenzidine (DAB), and can be extended to the products of other enzymes as well, e.g., alkaline phosphatase. Amplification is the consequence of the ability of anti-EP to detect the multiplicity of product moelcules resulting from the turnover of substrate by a single enzyme molecule. The subsequent detection of anti-EP was by biotinylated goat anti-rabbit antibody, followed by avidin-peroxidase and DAB or by avidin-alkaline phosphatase and Vector Red. Further amplification can be accomplished by repeated cycles of the protocol. Anti-EP was produced by immunization with a bovine serum albumin (BSA) conjugate of a soluble polymer of DAB, prepared by a carefully controlled reaction of DAB with horseradish peroxidase and hydrogen peroxide. Coupling to BSA (and to RSA) was accomplished with glutaraldehyde. The titer of anti-EP was established by ELISA. Formalin-fixed, paraffin-embedded sections of five cases of Hodgkin's disease and five tonsils with follicular hyperplasia were immunolabeled for the following lymphoid markers: CD3, CD20, CD30, CD45RA, and CD68. EP amplification with anti-EP was also applied to cases of CMV pneumonia and cerebral toxoplasmosis to determine whether this procedure could improve detection of the infectious agents. Immunolabeling of the primary antibody was performed by the avidin-biotin-peroxidase technique with DAB as the reaction substrate. The specificity of EP amplification was tested by demonstrating binding of anti-EP with Vector Red with the generation of a fluorescence end-point. There was complete congruence in the distribution of the DAB signal and the red immunofluorescence representing EP amplification. The intensity of the DAB signal was increased as much as 16-fold by EP amplification, making possible a reduction in the amount of the primary antibody by as much as 85-90%. Sensitivity also increased with respect to weakly expressed antigens and low concentrations of infectious agents.

3,3'-Diaminobenzidine↗
PubMed1995

Cyclophilin binding to the human immunodeficiency virus type 1 Gag polyprotein is mimicked by an anti-cyclosporine antibody.

Human immunodeficiency virus type 1 is unique among retroviruses in that infectivity requires specific incorporation into virions of the cellular protein cyclophilin A through interactions with the Gag polyprotein. Here we show that monoclonal antibody B11 1.4, which recognizes a cyclophilin-binding epitope on cyclosporine, detects denatured or native human immunodeficiency virus type 1 capsid. B11 1.4 does not recognize the capsids of other retroviruses, and binding is inhibited by cyclosporine or by cyclophilin A.

Amino Acid Isomerases↗
PubMed1994

Idiotypic mimicry and the assembly of a supramolecular structure: an anti-idiotypic antibody that mimics taxol in its tubulin-microtubule interactions.

Taxol, originally extracted from the bark of the western yew, Taxus brevifolia, is reportedly the first of a new class of anti-cancer agents. It acts by promoting and irreversibly stabilizing microtubule assembly, thus interfering with the dynamic processes required for cell viability and multiplication. With the aim of using immunological techniques to study the mechanism of action of taxol, a monoclonal anti-idiotypic antibody that mimics taxol was prepared, using an auto-anti-idiotypic strategy. It and its Fab fragment inhibited the binding of [3H]taxol to microtubules. Moreover, like taxol, both promoted the assembly of tubulin into microtubules. These findings provide an example of an anti-idiotypic antibody capable of assembling an organized supramolecular structure from soluble cellular components. In addition, it further establishes the ability of anti-idiotypic antibodies to be functional mimics of ligand molecules bearing no structural similarity to immunoglobulins. The variable regions of the antibody have been sequenced. With the exception of the complementarity-determining region 3, the sequence of the heavy chain variable region is strikingly similar to that of an anti-idiotypic antibody raised to anti-insulin. The finding that a polypeptide can mimic taxol raises the possibility that taxol acts as a peptidomimetic compound that interferes with the function of an endogenous polypeptide.

Amino Acid Sequence↗
PubMed1993

Human mononuclear phagocytes express adenosine A1 receptors. A novel mechanism for differential regulation of Fc gamma receptor function.

Using monoclonal anti-adenosine A1 receptor antibodies that bind the A1 receptor ligand binding site, we demonstrate that A1 receptors are expressed on cultured monocytes and rheumatoid synovial fluid mononuclear phagocytes. This finding is associated with the acquisition of reactivity with selective adenosine A1 receptor agonists and is temporally coordinated with the induction of adenosine A2 receptors on cultured monocytes. In a rapid, concentration-dependent fashion, these two distinct adenosine receptors modulate Fc gamma receptor-mediated phagocytosis, a response critical to the pathogenesis of immune complex diseases. Occupancy of A1 receptors by N6-cyclopentyladenosine (an A1-specific adenosine analogue) or mAb AA1 (an anti-A1 mAb) results in a potent stimulation that is blocked by adenosine receptor antagonists. This A1 receptor-induced enhancement of Fc gamma receptor-mediated phagocytosis is a consequence of preferential augmentation of Fc gamma RI function, suggesting distinct mechanisms for receptor-effector coupling of Fc gamma receptor families. In contrast, ligation of A2 receptors by A2-specific agonists decreases Fc gamma receptor-mediated phagocytosis in cultured monocytes. The opposing effects of adenosine A1 and A2 receptors allow for a concentration-dependent feed-back loop that responds more rapidly than effects elicited by other endogenous modulators. Low concentrations of adenosine are proinflammatory providing enhanced Fc gamma receptor function via A1 receptors, whereas higher concentrations that can occur with tissue damage are anti-inflammatory providing inhibition via A2 receptors. This rapid and potent modulation of Fc gamma receptor-mediated function suggests that adenosine is an important local regulator of the inflammatory response.

Adenosine↗
PubMed1993

Characterization of polyclonal and monoclonal anti-taxol antibodies and measurement of taxol in serum.

Anti-taxol antibodies were generated in the rabbit using a taxol-bovine serum albumin conjugate prepared from 2'-succinyltaxol using a mixed anhydride procedure. Immunization with 2'-succinyltaxol-bovine serum albumin gave rise to polyclonal anti-taxol antibodies. By a radioimmunoassay using [3H]taxol, a standard curve gave a 50% inhibitory concentration of 1.0 nM. Taxol levels in human serum could be measured, with the lower limit of detection and measurement being 0.1 nM or 0.085 ng/ml. Two mouse monoclonal anti-taxol antibodies were isolated by immunizing BALB/c mice with the same antigen. One was an immunoglobulin G1 (69E4A8E) and the other was immunoglobulin M (29B7B3C). The specificity of these antibodies was determined by a competitive enzyme-linked immunosorbent assay with taxol and 10 different related derivatives and analogues. 29B7B3C had higher binding affinities for biologically active derivatives and markedly lower affinities for inactive derivatives; i.e., the specificity was consistent with the results of tubulin disassembly and cytotoxicity studies using the same taxol derivatives, making it suitable for screening for taxol or taxol-like compounds in extracts of natural products. 69E4A8E recognized the benzamidocarbamyl group at the C-3' position of taxol and had a lower affinity for other active compounds with different substitutions. Taxol levels in human serum could be detected and measured by 69E4A8E using a competitive enzyme-linked immunosorbent assay. The lower limit of measurement was about 50 nM or approximately 42 ng/ml. Similar measurements could be made by radioimmunoassay.

Animals↗
PubMed1992

A monoclonal anti-idiotypic 'internal image' antibody that recognizes the A1 adenosine receptor potentiates the alpha 1-adrenergic activation of phospholipase C in primary cultures of mouse striatal astrocytes.

To determine which subtype of adenosine receptor mediates the potentiating effect of 2-chloroadenosine on the noradrenaline-induced inositol-phosphate formation, we used the monoclonal anti-idiotypic antibody AA1 that acts as an 'internal image' of adenosine and specifically recognizes the A1 adenosine receptor. In cultured mouse striatal astrocytes, AA1 increased the noradrenaline-evoked inositol phosphate (IP) accumulation, thus demonstrating a biological activity of an anti-idiotypic antibody. This effect was inhibited by PACPX, a selective A1 antagonist. Inhibitors of phospholipase A2 activity prevented the potentiation. These results establish the involvement of A1 adenosine receptors in the modulation of phospholipase C activity.

2-Chloroadenosine↗
PubMed1992

Evidence for a functional receptor for cyclosporin A on the surface of lymphocytes.

Cyclosporin A (CsA) is an immunosuppressive agent that inhibits the synthesis of lymphokines by T lymphocytes at the level of transcription. A cytoplasmic protein, cyclophilin, is the most thoroughly studied CsA-binding protein, but its ubiquitous presence in cells of all types raises questions about its role in immunosuppression. In an attempt to ascertain the presence of a cell surface receptor, we synthesized two polyvalent macromolecular CsA derivatives, CsA-BBa-ovalbumin and CsA-BBa-aminodextran (CBD), from the product of the photochemical reaction of CsA and 4-benzoylbenzoic acid (CsA-BBa). (i) They inhibited the peptidylprolyl cis-trans isomerase activity of cyclophilin and the synthesis of interleukin 2 by phorbol ester-activated EL-4 cells. (ii) CBD also inhibited interleukin 2 secretion by Con A-activated T-cell-enriched mouse splenocytes. 4-Benzoylbenzoic acid (BBa)-aminodextran and aminodextran were inactive. (iii) Direct binding and competition studies with [3H]CsA indicated that CBD does not enter EL-4 cells (i.e., it acted at the surface). (iv) CBD caused agglutination of EL-4 cells, murine B and T lymphocytes, human thymocytes, and two T-cell hybridomas. Agglutination was inhibited by a monoclonal antibody to CsA and by CsA and CsA-BBa, but not by BBa. No agglutination was seen with BBa-aminodextran or aminodextran. HeLa cells, Vero (monkey kidney) cells, a mouse plasmacytoma, COS cells, and a poorly differentiated B-cell lymphoma were not agglutinated. (v) EL-4 cells failed to be agglutinated after treatment with trypsin or chymotrypsin. Specific agglutination was again possible after incubation for 5 h at 37 degrees C in the absence of enzyme. (vi) CBD covalently linked to crosslinked agarose beads inhibited interleukin 2 production by phorbol ester-stimulated EL-4 cells. No activity was seen if cell-to-bead contact was prevented by a 0.02-microns microporous filter that did not interfere with the passage of CBD. Our findings support the presence of a functional receptor on the surface of selected cells of the immune system.

Animals↗
PubMed1992

Peptide sequences from the hypervariable regions of two monoclonal anti-idiotypic antibodies against the thyrotropin (TSH) receptor are similar to TSH and inhibit TSH-increased cAMP production in FRTL-5 thyroid cells.

Monoclonal antibodies, D2 and 4G11, selected by the autoantiidiotypic approach following injection of thyrotropin (TSH) into mice, mimic TSH in binding to receptors on thyroid membranes. Based on TSH receptor transfection studies, D2 and 4G11 show unequivocal specificity for the TSH receptor. To see if the complementary determining regions (CDRs) of these antibodies share any primary sequence similarities to regions of TSH critical for receptor binding, we deduced the primary structure of the variable regions of D2 and 4G11 by sequencing the immunoglobulin mRNA. We found that CDR1 of 4G11K and CDR2 of D2 mu show sequence similarity to regions of TSH alpha and TSH beta that had been previously implicated in the interaction of the hormone with its receptor. We tested the inhibitory effects of synthetic peptides from D2 mu-CDR2 and 4G11K-CDR1 on the binding of the corresponding antibodies to rat thyroid FRTL-5 cells and found an EC50 of 0.1 and 1 microM, respectively. TSH-derived peptides with similarity to D2 mu-CDR2 and 4G11K-CDR1 showed a significant but lesser effect on the binding of 4G11 or D2 to thyroid cells. Additionally, we tested the effects of the CDR peptides and TSH-derived peptides on TSH-stimulated cAMP production in FRTL-5 cells and found that D2 mu-CDR2 and 4G11K-CDR1 inhibited this activity, D2 mu-CDR2 most strongly (EC50 10 microM). Thus, linear sequences from the CDRs of these autoantiidiotypic antibodies with similarity to sequences from both subunits of TSH appear to interact with the TSH receptor. These data support previous studies indicating the complexity of the interaction between TSH and its receptor and advance earlier findings that such immunologic approaches are useful in dissecting receptor-ligand interactions.

Animals↗
PubMed1992

Novel monoclonal antibodies to cyclosporine A: characterization and epitope mapping with cyclosporine analogs and cyclophilin.

Monoclonal antibodies to cyclosporine A (Cs), a potent immunosuppressant, were generated in BALB/c mice using a novel antigen prepared by linking Cs to a protein carrier via a photoactive cross-linking reagent, 4-benzoylbenzoic acid (BBa). Twenty-two monoclonal anti-Cs antibodies were generated, using Cs-BBa-bovine serum albumin (Cs-BBa-BSA) as the immunogen. They were characterized with respect to affinity by Scatchard analysis of a radioimmunoassay (RIA), and with respect to specificity by an ELISA in which a series of singly substituted Cs derivatives were examined as inhibitors. McAb affinities ranged from 5 x 10(-8) M to 2 x 10(-10) M. Based on ELISA inhibition data with Cs analogs, and on the binding to two Cs-BSA conjugates in which opposite sides of the Cs molecule are exposed, the antibodies fell into five epitope recognition groups. Binding to Cs was also studied by ELISA in competition with cyclophilin (CyP), a Cs-binding protein whose epitope specificity has been well characterized. Competition by CyP was found to correlate with antibody specificity, not with affinity, i.e. CyP competed best with antibodies having specificities most similar to that of CyP. Epitope mapping can, therefore, be accomplished in a system in which two different species of binding proteins compete for the same antigen. This type of characterization may be useful in identifying antibodies whose combining sites mimic those of a receptor.

Amino Acid Isomerases↗
PubMed1992

Do we know the site of action of cyclosporin?

The site and mode of action of cyclosporin A (CsA) have been subjects of study ever since CsA was discovered and demonstrated to be a selective suppressor of allograft rejection. In this article, Bernard Erlanger traces progress to date and presents evidence that the site of action is not cytoplasmic cyclophilin but a lymphocyte cell-surface receptor that might be related in structure to cyclophilin.

Amino Acid Isomerases↗