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Biomedical subjects

B R Franza

Publications and source records attributed to B R Franza.

At least 55 records · Page 3Linked to original sources
PubMed1988

The Fos complex and Fos-related antigens recognize sequence elements that contain AP-1 binding sites.

The Fos protein complex and several Fos-related antigens bind directly or indirectly to a common sequence element that is similar to the consensus binding site for HeLa cell activator protein 1 (AP-1). This element is present in a negative regulatory sequence in the differentiation-sensitive adipocyte gene, aP2; in a transcriptional enhancer for the Gibbon ape leukemia virus; and in a region of the human immunodeficiency virus (HIV) long terminal repeat partially characterized as a negative regulatory element. The protein level and binding activity of Fos and Fos-related antigens increase rapidly after calcium ionophore treatment of a CD4+ human lymphoblast cell line, H9. These data suggest that several proteins may associate with the AP-1 binding site. Moreover, temporally regulated control of the level of each protein could represent a mechanism for modulation of these putative mediators of gene expression.

Base Sequence↗
PubMed1988

Molecular studies of HHV-6.

Methods for the purification of enveloped HHV-6 virions and the viral DNA are presented. The viral genome is estimated to be 170,000 base pairs in size and does not appear to contain inversions due to absence of submolar bands by restriction enzyme analyses. The genomes of two independent HHV-6 isolates, HHV-6GS and HHV-6Z29, showed restriction enzyme site pleomorphism. Large scale purification of enveloped HHV-6 was achieved by continuous flow centrifugation utilizing sucrose gradients, DNAse 1 treatment and banding on 10-30% Dextran T10 gradients. The viral proteins were visualized on high resolution two dimensional polyacrylamide gels and the proteins recognized by serum antibody from patient GS were detected by HR2D Western blot analysis and radioimmunoprecipitation assay. The major antigenic proteins were 200, 120, 80, 72, 30 and 19 kDa.

Blotting, Western↗
PubMed1988

Fos and Jun bind cooperatively to the AP-1 site: reconstitution in vitro.

The protein products of the fos (Fos) and jun (Jun) proto-oncogenes have been shown to associate with a DNA element known as the transcription factor activator protein-1 (AP-1) binding site. Jun (previously known as the Fos-binding protein p39) and Fos form a protein complex in the nucleus. To investigate the nature of the association of Fos and Jun with the AP-1 site, and to determine the role of protein complex formation in DNA-binding, we have reconstituted the protein-protein and protein-DNA interactions in vitro using Fos and Jun synthesized in reticulocyte lysates. The Fos-Jun complex formed extremely rapidly in vitro and possessed similar, though not identical, chromatographic and sedimentation properties to the complex isolated from cell extracts. Jun exhibited a low level of AP-1 binding activity; however, this was evident only at high concentrations of DNA. Fos did not bind to the AP-1 site on its own; however, it acted cooperatively with Jun to give enhanced DNA-binding activity. The increased affinity of the Fos-Jun complex for DNA resulted from a stabilization of the protein-DNA complex. These data demonstrate a cooperative interaction between the protein products of two proto-oncogenes with a DNA element involved in transcriptional regulation.

Allosteric Regulation↗
PubMed1987

A 100-kD HeLa cell octamer binding protein (OBP100) interacts differently with two separate octamer-related sequences within the SV40 enhancer.

Numerous eukaryotic upstream promoter and enhancer regions contain a functional octamer sequence ATGCAAAT. We have examined the interactions between an octamer binding protein isolated from HeLa cells and the SV40 and immunoglobulin heavy-chain (IgH) gene enhancers. A partially purified octamer binding activity forms a single complex with the IgH enhancer octamer in a gel retardation assay, but two complexes with a SV40 enhancer fragment containing a single 72-bp element. By using point mutants and both dimethyl sulfate and diethyl pyrocarbonate modification interference assays, we show that the SV40 complexes result from binding of a factor to the octamer-related sequence ATGCAAAG (Octa1) and to an adjacent previously unidentified octamer-related sequence ATGCATCT (Octa2). The base-specific interactions with Octa1 and Octa2 differ; chemical modifications over a 10-bp sequence TATGCAAAGC affect Octa1 binding whereas Octa2 binding is affected by modifications spanning a 13-bp sequence ATGCATCTCAATT in which the octamer-like sequence is not centered. The octamer binding activity has been purified extensively by a DNA affinity precipitation procedure and SDS-polyacrylamide gel electrophoresis. The purified protein, OBP100, has an apparent molecular weight of 100 kD and binds both SV40 Octa1 and Octa2, as well as the IgH enhancer. The distinct interactions of OBP100 with the differently sized Octa1 and Octa2 binding sites suggest remarkably flexible sequence recognition between OBP100 and its binding sites.

Base Sequence↗
PubMed1987

Expression of normal and mutant ras proteins in human acute leukemia.

The expression of normal and mutant ras genes in human acute leukemias was assessed by the direct analysis of p21ras polypeptides, using immunoprecipitation with monoclonal antibodies. High-resolution two-dimensional gel electrophoresis permits the identification of a wide array of activated ras alleles encoding proteins with single amino acid substitutions at any of several positions. The products of three ras genes, H-ras, N-ras, and K-ras, were detected in each of 33 specimens of fresh leukemic cells. The normal K-ras and N-ras polypeptides were substantially more abundant than H-ras p21 in all samples. In over three-fourths of the cases the total amount of p21ras exceeded that seen in control hematopoietic cell lines. The level of ras expression did not correlate simply with clinical parameters, although the two samples with the most abundant p21ras were obtained from patients with relapsed T-cell acute lymphocytic leukemia (ALL). Abnormal p21ras, consistent with oncogenic activation, was found in eight patients. Six of 11 samples from acute myelocytic leukemia (AML) patients displayed a mutant N-ras p21, while only one of 20 ALL specimens had abnormal N-ras, and one had a mutant H-ras. In every case the mutant protein comprised a minority of total p21ras. In two T-cell ALL cell lines both normal and activated N-ras gene products were expressed at equal levels. By contrast, in five fresh AML samples the abnormal N-ras protein was several-fold less abundant than the normal N-ras p21. This finding implies that only a proportion of leukemic cells in an individual patient may carry the mutant ras oncogene.

Acute Disease↗
PubMed1987

Analysis of Fos protein complexes and Fos-related antigens by high-resolution two-dimensional gel electrophoresis.

Protein complexes containing the c-fos protein (Fos) and (Fos)-related antigens were isolated from serum-stimulated fibroblasts and from nerve growth factor plus benzodiazepine-treated pheochromocytoma (PC12) cells, and investigated by high-resolution two-dimensional gel electrophoresis. The results show that Fos is complexed with a basic 39-kDa protein (p39) in fibroblasts, and primarily with an acidic 40-kDa protein (p40) in PC12 cells. Whole cells lysates from both cell types contain p40, suggesting that the interaction of Fos and other cellular proteins is dependent on the differentiated state of the cell. In addition to p39 and p40, a heterogeneous population of polypeptides of approximately 48 kDa are present in Fos complexes isolated from non-denatured extracts of either cell type. These proteins may represent a minor class of Fos-binding proteins. Analysis of extracts prepared under denaturing conditions using antisera raised against a Fos peptide (amino acids 127-152) reveals a series of Fos-related antigens. These antigens are induced, some with a slower kinetics than Fos, in fibroblasts and PC12 cells. Thus, c-fos may represent a marker for a family of genes, some of which are antigenically related, that are part of an early cellular transcriptional response to diverse extracellular stimuli.

Animals↗
PubMed1986

In vitro establishment is not a sufficient prerequisite for transformation by activated ras oncogenes.

Activated ras genes transform REF52 cells only at low frequencies and adenovirus early region 1A collaborates with ras oncogenes to convert REF52 cells to a tumorigenic phenotype. While failure to transform did not result from an absence of ras gene expression, E1A appeared to enhance expression of transfected ras genes by approximately tenfold. However, enhanced ras expression alone does not account for collaboration by E1A since overexpression of T24 Ha-ras p21 induced morphological crisis and cell growth arrest rather than stable transformation. These results indicate that E1A contributes complementing biochemical activities that enable ras genes to transform REF52 and suggest that the role of E1A in primary cell transformation may extend beyond facilitating in vitro establishment.

Adenoviridae↗
PubMed1986

Association of adenovirus early-region 1A proteins with cellular polypeptides.

Extracts from adenovirus-transformed human 293 cells were immunoprecipitated with monoclonal antibodies specific for the early-region 1A (E1A) proteins. In addition to the E1A polypeptides, these antibodies precipitated a series of proteins with relative molecular weights of 28,000, 40,000, 50,000, 60,000, 80,000, 90,000, 110,000, 130,000, and 300,000. The two most abundant of these polypeptides are the 110,000-molecular-weight protein (110K protein) and 300K protein. Three experimental approaches have suggested that the 110K and 300K polypeptides are precipitated because they form stable complexes with the E1A proteins. The 110K and 300K polypeptides do not share epitopes with the E1A proteins, they copurify with a subset of the E1A proteins, and they bind to the E1A proteins following mixing in vitro. The 110K and 300K polypeptides are not adenoviral proteins, but are encoded by cellular DNA. Both the 12S and the 13S E1A proteins bind to the 110K and 300K species, and these complexes are found in adenovirus-transformed and -infected cells.

Adenovirus Early Proteins↗
PubMed1985

Monoclonal antibodies specific for adenovirus early region 1A proteins: extensive heterogeneity in early region 1A products.

Hybridomas secreting monoclonal antibodies specific for the adenovirus early region 1A (E1A) proteins were prepared from BALB/c mice immunized with a bacterial trpE-E1A fusion protein. This protein is encoded by a hybrid gene that joins a portion of the Escherichia coli trpE gene and a cDNA copy of the E1A 13S mRNA (Spindler et al., J. Virol. 49:132-141, 1984). Eighty-three hybridomas that secrete antibodies which recognize the immunogen were isolated and single cell cloned. Twenty-nine of these antibodies are specific for the E1A portion of the fusion protein. Only 12 of the monoclonal antibodies can efficiently immunoprecipitate E1A polypeptides from detergent lysates of infected cells. E1A polypeptides were analyzed on one-dimensional, sodium dodecyl sulfate-polyacrylamide gels and two-dimensional, isoelectric focusing polyacrylamide gels. The E1A proteins that are specifically immunoprecipitated by the monoclonal antibodies are heterogeneous in size and charge and can be resolved into approximately 60 polypeptide species. This heterogeneity is due not only to synthesis from multiple E1A mRNAs, but also at least in part to post-translational modification. Several of the monoclonal antibodies divide the E1A polypeptides into immunological subclasses based on the ability of the antibodies to bind to the antigen. In particular, two of the monoclonal antibodies bind to the polypeptides synthesized from the 13S E1A mRNA, but not to other E1A proteins.

Adenoviridae↗
PubMed1984

The use of prothrombin activating snake venoms to measure human prethrombin 2: absence of prethrombin 2 in serum.

The activation of the prothrombin intermediate, Prethrombin 2, has been studied in order to establish test systems that would enable identification of Prethrombin 2 in serum and Factor IX concentrates. While activation of Prethrombin 2 by Taipan Snake Venom (TSV) was slow and incomplete, inclusion of approximately molar amounts of prothrombin fragments F1 or F1.2 markedly enhanced the amount of thrombin formed by TSV. This effect could also be obtained by the inclusion of serum. Neither normal serum nor Factor V deficient serum contain any identifiable Prethrombin 2. On the other hand substantial amounts of Prethrombin 2 are present in Factor IX concentrates used for the treatment of Christmas Disease (Hemophilia B).

Chromatography, DEAE-Cellulose↗
PubMed1980

Mutagenic activity of nitrosourea antitumor agents.

The relative mutagenic activities of chloroethyl-nitrosourea and methylnitrosourea antitumor agents in active clinical use were determined with the use of the Ames Salmonella typhimurium assay. The results indicated that the drugs induced base substitutions. 2-Deoxy-2-[[(methylnitrosoamino)carbonyl]amino]-D-glucopyranose (also called streptozotocin), a glucose-containing methylnitrosourea, was the most mutagenic of all compounds tested and showed at least a 250-fold increase in activity when compared to that of its chloroethyl analog, 2-[[[(2-chloroethyl) nitrosoamino]carbonyl]-amino]-2-deoxy-D-glucose (also called chlorozotocin). All nitrosoureas, with the exception of N'-[(4-amino-2-methyl-5-pyrimidinyl)methyl]-N-(2-chloroethyl)-N-nitrosourea monohydrochloride, a pyrimidine chloroethyl analog, demonstrated an increase in mutagenicity after incubation with induced Sprague-Dawley rat liver microsomes. No correlation between in vitro chemical alkylating activity and mutagenic potential was observed. Mutagenic activity was not observed to be of predictive value for antitumor activity in the L1210 leukemia model system.

Alkylation↗
PubMed1977

Generation of the combined prothrombin activation peptide (F1-2) during the clotting of blood and plasma.

We have investigated the pathway of prothrombin activation in blood and plasma. By means of a rapid purification procedure involving chromatography on DEAE-cellulose and hydroxyapatite, we demonstrated that the major prothrombin fragment in serum is that representing the amino-terminal half of prothrombin (i.e. F1-2). The F1-2 isolated was characterized by its size, amino acid and antigenic compositions, amino-terminal residue, and the peptides (designated F1 and F2, respectively) it yielded upon hydrolysis by thrombin. Measurements by the isotope dilution technique showed that F1-2 could account for the fate of at least 90% of the prothrombin originally present in plasma. By contrast, the serum concentration of the fragment representing the amino-terminal third of prothrombin (viz. F1) was less than 10% that of F1-2. These results demonstrated that the major route of prothrombin conversion in blood or plasma involves the removal of the combined activation fragment (F1-2) as a single peptide.

Amino Acid Sequence↗
PubMed1976

The pH dependence of quantitative ristocetin-induced platelet aggregation: theoretical and practical implications-a new device for maintenance of platelet-rich plasma pH.

Quantitative ristocetin-induced platelet aggregation of normal platelet-rich plasma (PRP) decreased with time after PRP preparation. An increase in p H of the PRP with time proved to be responsible for this finding. Diffusion of CO2from the plasma is the prime determinant of the change in pH. Since a complex combination of factors influences CO2 diffusion (surface area-to-volume relationship, capping, mixing, etc.) The change in pH is variable with time. Thus, quantitative ristocetin aggregation should be pH controlled. A simple device for maintaining PRP pH constant by control of the ambient pCO2 was designed and found effective in keeping both pH and quantitative ristocetin aggregation constant over a prolonged period of time. It can be adapted for use in platelet aggregation studies employing other reagents. The pH dependence of ristocetin-induced platelet aggregation is consistent with other data supporting an elctrostatic interaction between the platelet, von Willebrand factor, and ristocetin. We favor a model wherein ristocetin neutralizes some of the platelet's negative change and permits the von Willebrand factor to bridge sites on separate platelets to induce agglutination.

Blood Coagulation Tests↗