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Biomedical subjects

R Taub

Publications and source records attributed to R Taub.

At least 91 records · Page 5Linked to original sources
PubMed1989

A monoclonal antibody against the platelet fibrinogen receptor contains a sequence that mimics a receptor recognition domain in fibrinogen.

The binding of fibrinogen to its platelet receptor, the glycoprotein IIb-IIIa complex, is mediated, in part, by an Arg-Gly-Asp (RGD) sequence within the fibrinogen A alpha chain. PAC1 is an IgM-kappa murine monoclonal antibody that binds to the platelet fibrinogen receptor, and its binding is inhibited by both fibrinogen and RGD-containing peptides. To identify the regions of PAC1 that interact with the fibrinogen receptor, we determined the mRNA sequences of PAC1 immunoglobulin heavy and light chain variable regions. Five out of the six complementarity-determining regions (CDRs) of PAC1 had entirely germline sequences with no regions of similarity to fibrinogen. However, CDR3 of the PAC1 heavy chain (H-CDR3) was very large and unique due to the insertion of a novel D region segment. H-CDR3 contained a sequence, Arg-Tyr-Asp (RYD), that, if present in the proper conformation, might behave like the RGD sequence in fibrinogen. A 21-residue synthetic peptide encompassing the H-CDR3 region inhibited fibrinogen-dependent platelet aggregation as well as the binding of PAC1 (Ki = 10 microM) and fibrinogen (Ki = 5 microM) to activated platelets. The RYD region of H-CDR3 appeared to be central to its function, because substitution of the tyrosine with glycine increased the inhibitory potency of the peptide by 10-fold, while replacing the tyrosine with D-alanine or inverting the RYD sequence sharply reduced the inhibitory potency. Thus, the linear sequence, RYD, within H-CDR3 of PAC1 appears to mimic the RGD receptor recognition sequence in fibrinogen. This type of immunologic approach could be useful in studying the structural basis of other receptor-ligand interactions.

Amino Acid Sequence↗
PubMed1989

Insulin, insulin-like growth factor I and platelet-derived growth factor interact additively in the induction of the protooncogene c-myc and cellular proliferation in cultured bovine aortic smooth muscle cells.

Vascular smooth muscle cell (SMC) growth is under the influence of various growth factors. We demonstrate that platelet-derived growth factor (PDGF) stimulates DNA synthesis of cultured bovine aortic SMCs by 2.5- to 3.5-fold. PDGF also exhibits additivity with insulin and insulin-like growth factor I (IGF-I) for DNA synthesis and cellular proliferation. Insulin (2 x 10(-6) M), IGF-I (1 x 10(-8) M), and PDGF (1 x 10(-9) M) cause a 60-80% increase in cell numbers over basal, but PDGF with insulin or IGF causes a 40-150% increase over basal. No additivity between insulin and IGF-I is evident. PDGF also induces commitment to DNA synthesis earlier than insulin or IGF-I. After exposure to PDGF for 4 h, SMCs incorporate 3H-thymidine to 60% of maximum (with PDGF alone) levels (achieved after exposure of 12 h or longer). Insulin and IGF-I exposure for 4 h, on the other hand, achieves 3H-thymidine incorporation that is only a 20-30% of maximum (with insulin or IGF-I alone). Insulin, IGF-I, and PDGF increase mRNA levels of the protooncogene c-myc. This induction begins within 30 min of exposure to these growth factors which causes a 4- to 6-fold increase in c-myc mRNA levels. Additivity is also observed between PDGF with insulin or IGF-I, but not between insulin or IGF-I, in c-myc induction. C-myc mRNA levels remain elevated as long as the hormones are present, although there's a tendency for the mRNA levels to fall off with insulin and IGF-I.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1989

Characterization of induction of protooncogene c-myc and cellular growth in human vascular smooth muscle cells by insulin and IGF-I.

Insulin and insulin-like growth factor I (IGF-I) are structurally related polypeptides that stimulate DNA synthesis and cellular proliferation, probably through a common pathway. Human arterial smooth muscle cells in culture demonstrated the presence of high-affinity receptors for both these hormones. Insulin and IGF-I both exhibited cross-reactivity to each other's receptors but with an affinity that is 100-fold less than for the homologous receptor. To examine more closely the receptor responsible for producing the growth effects, we used the polyclonal antibody against the insulin receptor, B2, and a monoclonal antibody to the IGF-I receptor, alpha IR3. We studied the growth effects of insulin and IGF-I as measured by stimulation of c-myc, DNA synthesis, and cellular proliferation in the presence and absence of these antibodies. F(ab') fragments of the anti-insulin-receptor antibody at a concentration of 10 micrograms/ml were capable of displacing greater than 90% of the bound insulin, thus establishing an effective insulin-receptor blockade. Under such blockade, insulin and IGF-I were both capable of doubling the amount of DNA synthesis and cell number in cultured human arterial smooth muscle cells. However, in the presence of a 1:2500 dilution of the monoclonal antibody alpha IR3, which caused a 90% displacement of IGF-I bound to its receptor, both the insulin and IGF-I effects on stimulating DNA synthesis or cellular proliferation were inhibited by greater than 90%. These findings demonstrate that the IGF-I receptor is the common pathway for the growth effects of both insulin and IGF-I.(ABSTRACT TRUNCATED AT 250 WORDS)

Cell Division↗
PubMed1989

Insulin-receptor gene and its expression in patients with insulin resistance.

We studied the structure of the insulin-receptor gene in normal individuals and in four unrelated patients with leprechaunism (Minn-1, Ark-1, Ark-2, Can-1) and four unrelated patients with the type A syndrome of insulin resistance, both disorders associated with genetic alterations in affinity, binding capacity, and kinase activity of the insulin receptor. Genomic cloning and Southern blot analysis indicate that the normal human insulin-receptor gene is greater than or equal to 150 kilobases long and consists of a minimum of 17 exons, 6 in the genomic region of the alpha-subunit and 11 in the region of the beta-subunit. Three of the patients, one with leprechaunism and two with type A syndrome, have decreases in insulin-receptor mRNA but on genomic blot analysis have no obvious abnormalities in the insulin-receptor gene. No distinctive pattern of restriction-fragment-length polymorphisms or evidence for major insertion or deletion mutations of the insulin-receptor gene was found in any of the patients. These data indicate that the insulin-receptor gene is greater than 35 times larger than coding regions and has a complex structure. Although leprechaunism and type A syndrome are most likely due to defects in the structure and expression of the insulin-receptor gene, they are likely to be associated with specific point mutations rather than major changes in gene structure.

Cloning, Molecular↗
PubMed1988

Application of two-dimensional high-performance liquid chromatography in metabolism studies of ivermectin.

Two-dimensional high-performance liquid chromatography (HPLC) of alternate reversed-phase and normal-phase columns was used in the purification, quantification and identification of submicrogram quantities of drug residue and metabolites in tissues from animals dosed with ivermectin. In reverse isotope dilution assay of the parent drug, two-dimensional HPLC assured constant specific activity of the drug isolates. For identification of metabolites from liver tissue, HPLC in two dimensions not only facilitated the purification, but also provided information (capacity factor k') in both systems for the elimination of possible metabolite structures in the course of compound identification. These approaches exemplified by the ivermectin studies should be generally applicable in analyses of complex biological samples in which quantity is frequently a limiting factor.

Animals↗
PubMed1988

Altered expression and function of the insulin receptor in a family with lipoatrophic diabetes.

To determine the role of genetic defects in the insulin receptor in the insulin resistance of lipoatrophic diabetes mellitus, we studied insulin binding, insulin receptor autophosphorylation, and insulin receptor mRNA levels and performed Southern blot analysis of genomic DNA in four siblings, all of whom have some degree of insulin resistance and three of whom have lipoatrophy. The insulin receptor concentration in Epstein-Barr virus-transformed lymphocytes was about 30% of normal in all three lipoatrophic siblings (LA1, LA2, and LA3) and was 55% of normal in the nonlipoatrophic sibling (LAS). Insulin receptor mRNA concentrations in the lymphocytes paralleled insulin binding and ranged from 15-67% of the mean normal level. Insulin binding to fibroblasts was also reduced about 50% in the lipoatrophic siblings. In addition, insulin binding to fibroblasts of LAS and LA2 exhibited a rightward shift of the competition curve, suggesting reduced receptor affinity [ED50, 35 and 50 ng/mL (5845 and 8350 pmol/L); normal, 1-3 ng/mL (167-501 pmol/L)]. Receptor autophosphorylation determined using Triton X-100 extracts of the fibroblasts was decreased in LA1 and LA3, but normal in LA2 and LAS. Using restriction enzyme digests of genomic DNA and probes spanning the entire cDNA of the insulin receptor, no gross alterations in receptor gene structure were detected in any members of this family. In 2 of the lipoatrophic siblings (LA1 and LA3) and in the sibling with insulin resistance but no lipoatrophy (LAS), a unique variant BamHI site was detected using a probe to the alpha-subunit region. This site was not found in 200 normal or diabetic insulin receptor alleles. By use of probes 5' and 3' to the alpha-subunit probe and by genomic cloning analysis, this variant BamHI site was localized to an intron in the insulin receptor gene downstream of exon 3 which encodes amino acids 191-296 of the alpha-subunit of the receptor. These data indicate the complex nature of familial lipoatrophic diabetes mellitus, with alterations in insulin receptor expression and/or function in both clinically affected and non-affected siblings. Both the reduced insulin binding and reduced levels of insulin receptor mRNA in the lipoatrophic siblings suggest that an insulin receptor gene defect contributes to this syndrome. Several members of this family also carry a unique variant insulin receptor gene, which, however, could not be linked to a specific alteration in receptor expression or the presence of lipoatrophy.(ABSTRACT TRUNCATED AT 250 WORDS)

DNA Restriction Enzymes↗
PubMed1987

Insulin as a growth factor in rat hepatoma cells. Stimulation of proto-oncogene expression.

In a subline of Reuber H35 rat hepatoma cells that becomes quiescent under serum-deprived conditions, insulin acts as a growth factor. When added to serum-deprived H35 cells, physiologic concentrations of insulin stimulate DNA synthesis, demonstrating that insulin alone is capable of inducing a transition from G0/G1 into S phase. This response, which is induced by nanomolar concentrations of insulin, is mediated directly through the insulin receptor. Here we show that coincident with this growth response, insulin or serum induces dramatic increases in the steady-state levels of c-fos and c-myc mRNAs in serum-deprived H35 cells in a time course similar to that observed in the regenerating liver. Other growth factors, including epidermal growth factor, appear not to affect these cells either in terms of DNA synthesis or c-myc mRNA induction. The phorbol ester phorbol 12-myristate 13-acetate (PMA) also induces c-myc and c-fos mRNAs without inducing DNA synthesis. However, the mechanism of this induction appears to be different from the insulin-induced induction since pretreatment of cells with PMA blocks only the PMA-mediated, not the insulin-mediated, induction of c-myc and c-fos.

Actins↗
PubMed1986

A translocated human c-myc oncogene is altered in a conserved coding sequence.

We have cloned and characterized a c-myc (now designated MYC) oncogene that had been translocated into the mu switch region of the immunoglobulin heavy chain locus in a Burkitt lymphoma cell line. The breakpoint of the translocation occurs within the first intron of the c-myc gene, thereby separating the untranslocated first exon from the two coding exons. Transcription from the translocated gene arises from a cryptic promoter within the first intron, which produces a 438-nucleotide untranslated 5' region. The amino acid sequence of the protein encoded by the c-myc gene has been substantially altered. In particular, a compensating set of frameshift mutations alters a string of 24 amino acids in a region of the protein tightly conserved in human, mouse, and chicken c-myc genes as well as in the human N-myc and L-myc oncogenes. Despite this, the mutated gene retains a reduced transforming ability in a rat embryo fibroblast focus-formation assay.

Amino Acid Sequence↗
PubMed1984

Antibodies to human c-myc oncogene product: evidence of an evolutionarily conserved protein induced during cell proliferation.

Antisera to a synthetic c-myc peptide and to c-myc antigens synthesized from various portions of the human gene expressed in Escherichia coli were used in order to characterize the protein product of the human c-myc oncogene. Although the deduced molecular weight of the human c-myc protein is 49,000, these antisera precipitate a protein from human cells that migrates in sodium dodecyl sulfate-polyacrylamide gel as if its molecular weight were 65,000. In addition, the mouse c-myc protein, whether synthesized in cells or in a cell-free system directed by pure, synthetic messenger RNA, has analogous properties and is immunoprecipitated by the antiserum to the human c-myc protein. Similar proteins are immunoprecipitated from monkey, rat, hamster, and frog cells, suggesting evolutionary conservation of antigenic structure of the c-myc protein among vertebrates. In addition, and in a manner consistent with the behavior of its messenger RNA, the immunoprecipitable c-myc protein is sharply induced by the action of mitogens on resting human T cells.

Amino Acid Sequence↗
PubMed1984

Mapping of the human Blym-1 transforming gene activated in Burkitt lymphomas to chromosome 1.

Blym-1, a transforming gene detected by transfection of NIH 3T3 cells with DNA from Burkitt lymphomas, was mapped to the short arm of chromosome 1 (1p32) by chromosomal in situ hybridization. The Blym-1 gene was not physically linked to the cellular myc oncogene or to any of the immunoglobulin gene loci implicated in the characteristic chromosomal translocations in Burkitt lymphoma.

Base Sequence↗
PubMed1984

The effect of translocations on the cellular myc gene in Burkitt lymphomas.

Chromosomal translocations are found to be a characteristic feature of Burkitt lymphomas. Similar translocations are found in mouse plasmacytomas and both diseases involve interchanges between one of the immunoglobulin loci and DNA in the vicinity of the myc gene. The structure of the myc gene has been elucidated from studies on translocated versions of the gene. Activation of the myc gene may play a role in transformation by promoting growth of the cells bearing the rearranged chromosomes.

Animals↗
PubMed1984

Activation and somatic mutation of the translocated c-myc gene in burkitt lymphoma cells.

In contrast to other human tumors in which the c-myc gene and its transcript are greatly amplified, careful analysis of t(8;14) Burkitt cell lines indicates that the c-myc transcript is marginally, and in some cases not at all, increased by comparison to control lymphoblastoid cell lines. Instead, there is a more subtle alteration in the expression of the translocated c-myc gene characterized by a shift in promoter utilization and an apparent insensitivity to the regulation that inactivates the normal c-myc allele within these same cells. In some Burkitt cell lines, such deregulation might be because of the loss of a putative control region through removal of the large dual promoter/leader segment of the c-myc gene. In other cell lines, however, this deregulation may be explained by somatic mutations that occur within the putative control region even though it is located many hundreds of bases from the translocation breakpoint.

Alleles↗
PubMed1984

A novel alteration in the structure of an activated c-myc gene in a variant t(2;8) Burkitt lymphoma.

We have characterized a variant Burkitt lymphoma in which translocation joins the immunoglobulin kappa locus on chromosome 2 to the c-myc gene on chromosome 8. This Burkitt lymphoma is especially interesting because, in contrast to the more common lymphomas that carry 8;14 translocations, it carries a translocation that involves a light chain locus and occurs 3' to and at least 20 kb downstream of the c-myc gene. Furthermore, the c-myc gene from the translocated chromosome is abnormally expressed in that there is a characteristic shift in c-myc promoter utilization and an increase in c-myc transcript. These disturbances could be explained by novel structural alterations that occur in the c-myc gene and include a duplication of a 2.5 kb segment of DNA containing the two c-myc promoters and their untranslated leader exons. Interestingly, these alterations arise at a considerable distance from the translocation breakpoint.

Base Sequence↗
PubMed1984

Localization of the beta-globin gene by chromosomal in situ hybridization.

A 3.7-kilobase (kb) genomic clone of the human beta-globin gene, including 1.5-kb upstream and approximately 0.5-kb downstream, was utilized in chromosomal in situ hybridization for precise mapping of the beta-globin locus on peripheral blood lymphocyte-derived metaphases from a normal male, and for further evaluation of a clonal t(7;11) (q22;p15) translocation on bone marrow-derived metaphases from a 46-year-old male with erythroleukemia. Analyses of 205 midmetaphases from a normal male hybridized with the tritium-labeled beta-globin probe and stained with quinacrine mustard dihydrochloride revealed approximately 12% of spreads to have silver-grain deposition over the p15 band of chromosome 11. Of the 365 silver grains observed to be located on or beside chromosomes, 25 (approximately 7%) grains were localized in band p15. Karyotype analysis of a bone marrow specimen from the patient with erythroleukemia revealed hypodiploidy with various unidentified marker chromosomes as well as a presumably balanced translocation between 7q and 11p . Chromosomal in situ hybridization showed localization of silver grains at the junction between chromosomes 7 and 11 as well as to the normal chromosome 11, indicating that the beta-globin locus had not been translocated in the chromosomal rearrangement. This case demonstrates the value of chromosomal in situ hybridization in the definition of chromosome rearrangements and provides further evidence for the localization of the beta-globin gene to 11p15 .

Chromosome Banding↗
PubMed1983

Translocations among antibody genes in human cancer.

The characteristic chromosomal translocations that occur in certain human malignancies offer opportunities to understand how two gene systems can affect one another when they are accidentally juxtaposed. In the case of Burkitt lymphoma, such a translocation joins the cellular oncogene, c-myc, to a region encoding one of the immunoglobulin genes. In at least one example, the coding sequence of the rearranged c-myc gene is identical to that of the normal gene, implying that the gene must be quantitatively, rather than qualitatively, altered in its expression if it is to play a role in transformation. One might expect to find the rearranged c-myc gene in a configuration that would allow it to take advantage of one of the known immunoglobulin promoters or enhancer elements. However, the rearranged c-myc gene is often placed so that it can utilize neither of these structures. Since the level of c-myc messenger RNA is often elevated in Burkitt cells, the translocation may lead to a deregulation of the c-myc gene. Further, since the normal allele in a Burkitt cell is often transcriptionally silent in the presence of a rearranged allele, a model for c-myc regulation is suggested that involves a trans-acting negative control element that might use as its target a highly conserved portion of the c-myc gene encoding two discrete transcriptional promoters.

Base Sequence↗
PubMed1983

The human c-myc oncogene: structural consequences of translocation into the IgH locus in Burkitt lymphoma.

We have determined the sequence of the normal human c-myc gene and compared it to portions of a c-myc gene that has been translocated into the immunoglobulin heavy chain locus in a Burkitt lymphoma cell. The normal c-myc gene is encoded in three discrete exons divided by two large intervening sequences. Its mRNA is transcribed from two active promoters located about 150 nucleotides from one another. Each promoter initiates transcription of a long (approximately 550 bp) untranslatable leader sequence encoding the entire first exon. This exon and additional 5' flanking sequences are tightly conserved between mouse and man. In the Burkitt cell BL22, the rearranged c-myc gene retains both promoters and is unchanged in its amino acid coding domains. Translocation of this gene joins it to the immunoglobulin heavy chain switch region at a point approximately 1000 bp 5' to the dual c-myc promoters. These genes are joined in opposite transcriptional orientation. The structure of the translocated gene and the nature of its linkage to the immunoglobulin locus and the presence of two c-myc promoters and consequently two long leader sequences raise novel possibilities for the activation of an oncogene.

Amino Acid Sequence↗