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Biomedical subjects

B Lüscher

Publications and source records attributed to B Lüscher.

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

Myc oncoproteins are phosphorylated by casein kinase II.

Casein kinase II (CK-II) is a ubiquitous protein kinase, localized to both nucleus and cytoplasm, with strong specificity for serine residues positioned within clusters of acidic amino acids. We have found that a number of nuclear oncoproteins share a CK-II phosphorylation sequence motif, including Myc, Myb, Fos, E1a and SV40 T antigen. In this paper we show that cellular myc-encoded proteins, derived from avian and human cells, can serve as substrates for phosphorylation by purified CK-II in vitro and that this phosphorylation is reversible. One- and two-dimensional mapping experiments demonstrate that the major phosphopeptides from in vivo phosphorylated Myc correspond to the phosphopeptides produced from Myc phosphorylated in vitro by CK-II. In addition, synthetic peptides with sequences corresponding to putative CK-II phosphorylation sites in Myc are subject to multiple, highly efficient phosphorylations by CK-II, and can act as competitive inhibitors of CK-II phosphorylation of Myc in vitro. We have used such peptides to map the phosphorylated regions in Myc and have located major CK-II phosphorylations within the central highly acidic domain and within a region proximal to the C terminus. Our results, along with previous studies on myc deletion mutants, show that Myc is phosphorylated by CK-II, or a kinase with similar specificity, in regions of functional importance. Since CK-II can be rapidly activated after mitogen treatment we postulate that CK-II mediated phosphorylation of Myc plays a role in signal transduction to the nucleus.

Amino Acid Sequence↗
PubMed1989

Regulation of transcription factor AP-2 by the morphogen retinoic acid and by second messengers.

The expression of the transcription factor AP-2 recently has been shown to be enhanced during retinoic acid (RA)-induced differentiation of NT2 cells, a human teratocarcinoma cell line. Here we show that this induction of AP-2 mRNA is at the level of transcription and is transient, reaching a peak 48-72 hr after the addition of RA and declining thereafter, even in the continuous presence of RA. Increased levels of AP-2 mRNA are reflected in a similar elevation of AP-2 protein and accompanied by an increase in the AP-2-binding site-dependent transcriptional activity of a reporter gene. AP-2 also has been proposed to confer TPA and cAMP inducibility on promoters/enhancers containing AP-2-binding sites. We investigated the effect of these agents on the expression of AP-2 protein and mRNA. Our experiments demonstrate that expression of the AP-2 gene in HeLa cells is not elevated significantly by TPA or by a calcium ionophore and is not enhanced at all by agents that increase intracellular cAMP concentration. In fact, AP-2 mRNA is repressed by both TPA and the calcium ionophore A23187 through a delayed response. These data suggest that the AP-2-binding site-mediated cAMP and TPA responses are not regulated at the level of AP-2 expression but, rather, achieved either by post-translational changes in AP-2 or in conjunction with another protein.

1-Methyl-3-isobutylxanthine↗
PubMed1989

A second c-myb protein is translated from an alternatively spliced mRNA expressed from normal and 5'-disrupted myb loci.

The major protein encoded by the c-myb oncogene in many species has been identified as an unstable, nuclear DNA-binding protein with an apparent molecular mass of 75 to 80 kilodaltons (p75c-myb). Recently, an alternatively spliced form of c-myb-encoded mRNA has been identified in murine cells containing either normal or rearranged c-myb genes. This mRNA includes a new exon, termed E6A, formed through use of cryptic splice sites located in the large intron between c-myb exons vE6 and vE7. E6A is predicted to contribute an internal 121-residue in-frame insertion into a region C terminal of the DNA-binding domain the c-myb-encoded protein. Here we report the identification of an 85-kilodalton (p85c-myb-E6A) protein as the translation product of the alternatively spliced E6A c-myb mRNA. This protein as well as p75c-myb were precipitated with anti-Myb antibodies raised against the conserved DNA-binding region of c-Myb. Proteolytic mapping studies showed that the two proteins are highly related but not identical. However, only the p85 protein reacted with an antiserum prepared against the E6A region expressed in bacteria, demonstrating that p85 but not p75 contains E6A sequences. In addition, the mobilities of both p85 and p75 were increased in myeloid tumor cell lines containing proviral integrations upstream of the 5' coding exons of v-myb, indicating that both proteins are truncated forms of c-Myb expressed from the same disrupted allele. p75c-myb and p85c-myb-E6A were indistinguishable with respect to nuclear localization and protein half-life. Furthermore, both forms of Myb were synthesized continuously throughout the cell cycle in 70Z ore-B cells. The contribution of the E6A domain to c-myb function remains to be elucidated.

Amino Acid Sequence↗
PubMed1989

The E7 protein of human papillomavirus type 16 is phosphorylated by casein kinase II.

The E7 protein of human papillomavirus type 16 (HPV16) transforms cultured cells and cooperates with the ras or fos oncogenes in the transformation of primary cells. In this study we have investigated the phosphorylation of E7. When we immunoprecipitated E7 from CaSki cells with a rabbit polyclonal antiserum to a bacterial fusion protein (trpE-E7), we found that E7 was phosphorylated at serine residues contained in five characteristic thermolysin peptides. Immunoprecipitated E7, and fusion proteins harboring the E7 protein from various HPV types, could all be specifically phosphorylated in vitro by the ubiquitous, growth factor-activated casein kinase II (CKII). Comparative peptide mapping showed that the sites of in vivo and in vitro phosphorylation are the same. CKII was shown previously to specifically phosphorylate serine or threonine residues within a cluster of acidic amino acids. The E7 protein contains such a sequence between amino acids 30 and 37. When a synthetic peptide corresponding to this region of E7 was phosphorylated by CKII in vitro, its thermolysin digestion products were the same as those in the phosphorylated E7 protein. We conclude that E7 is phosphorylated in vivo only at serines within the predicted CKII site and that CKII, or a CKII-like enzyme, participates in the reaction. Both the E1A and SV40 large T proteins contain similar CKII consensus sites proximal to the regions required for their associations with the retinoblastoma gene product (p105Rb). Thus it is conceivable that CKII phosphorylation can modulate the interaction between the transforming proteins and the retinoblastoma gene product.

Amino Acid Sequence↗
PubMed1988

Cloning and expression of AP-2, a cell-type-specific transcription factor that activates inducible enhancer elements.

Human AP-2 is a sequence-specific DNA-binding protein that interacts with inducible viral and cellular enhancer elements to stimulate transcription of selected genes. Here, we report the isolation and characterization of a human cDNA clone containing the entire protein-coding region of AP-2. The deduced primary amino acid sequence of AP-2 does not contain a domain resembling any previously identified DNA binding motif. However, an interesting feature of the AP-2 protein is a clustered arrangement of proline and glutamine residues that have been found recently within the activation domains of other transcription factors. Expression of the AP-2 clone in bacteria yields a protein that binds to DNA and activates transcription in vitro in a comparable manner to native human AP-2. Transfection of cDNA clones into Drosophila cells indicates that the AP-2 gene product can also activate gene expression in vivo in a DNA template-dependent manner. Expression of endogenous AP-2 is repressed in a hepatoma cell line and stimulated following retinoic-acid-induced differentiation of a human teratocarcinoma cell line. This indicates that AP-2 may be a transcription factor involved in the control of developmentally regulated gene expression.

Amino Acid Sequence↗
PubMed1988

c-myc and c-myb protein degradation: effect of metabolic inhibitors and heat shock.

The proteins encoded by both viral and cellular forms of the c-myc oncogene have been previously demonstrated to have exceptionally short in vivo half-lives. In this paper we report a comparative study on the parameters affecting turnover of nuclear oncoproteins c-myc, c-myb, and the rapidly metabolized cytoplasmic enzyme ornithine decarboxylase. The degradation of all three proteins required metabolic energy, did not result in production of cleavage intermediates, and did not involve lysosomes or ubiquitin. A five- to eightfold increase in the half-life of c-myc proteins, and a twofold increase in the half-life of c-myb proteins was detected after heat-shock treatment at 46 degrees C. In contrast, heat shock had no effect on the turnover of ornithine decarboxylase. Heat shock also had the effect of increasing the rate of c-myc protein synthesis twofold, whereas c-myb protein synthesis was decreased nearly fourfold. The increased stability and synthesis of c-myc proteins led to an overall increase in the total level of c-myc proteins in response to heat-shock treatment. Furthermore, treatments which reduced c-myc and c-myb protein turnover, such as heat shock and exposure to inhibitors of metabolic energy production, resulted in reduced detergent solubility of both proteins. The recovery from heat shock, as measured by increased turnover and solubility, was energy dependent and considerably more rapid in thermotolerant cells.

Animals↗
PubMed1987

RNA 3' processing regulates histone mRNA levels in a mammalian cell cycle mutant. A processing factor becomes limiting in G1-arrested cells.

Post-transcriptional regulation of histone gene expression in a mouse mastocytoma cell cycle mutant (21-Tb) depends largely on conserved DNA sequences that are essential for RNA 3' processing. We have analyzed whether this regulation occurs at the level of RNA 3' processing. We show, by RNase mapping, that nuclear H4 mRNA precursors, which are hardly detectable in total RNA from exponentially dividing cells, accumulate in G1-arrested cells, i.e. when mature mRNAs are drastically reduced. Furthermore, we show that a heat-labile component of the processing apparatus, recently identified in HeLa cell nuclear extracts, is limiting in extracts from G1-arrested 21-Tb cells. In contrast, this activity is in excess in extracts from exponentially dividing cells, whereas both extracts contain similar amounts of snRNPs of the Sm serotype. These fluctuations in the heat-labile activity may generally contribute to proliferation or cell cycle dependent histone gene regulation.

Animals↗
PubMed1986

A signal regulating mouse histone H4 mRNA levels in a mammalian cell cycle mutant and sequences controlling RNA 3' processing are both contained within the same 80-bp fragment.

Fragments from the 3' end of a mouse histone H4 gene, when introduced into transcription units controlled by the SV40 early promoter, yield correctly processed RNA with histone-specific 3' ends, both in monkey and mouse cell lines. The processed RNA is regulated in parallel with endogenous H4 mRNAs in 21-Tb cells, a temperature-sensitive mouse mastocytoma cell cycle mutant that is specifically blocked in G1 phase at the non-permissive temperature. Mutational analyses of the H4 gene fragment indicate that the minimal sequences for this regulation and for RNA 3' processing are both contained within the same 80 bp. This fragment contains two histone-specific, highly conserved sequence elements that are located at the 3' end of histone mRNA and in the adjacent spacer region, respectively. Our data suggest that the observed cell cycle regulation is achieved either at RNA 3' processing or at some later step involving the conserved 3'-terminal sequence element of mature histone mRNA.

Animals↗
PubMed1986

Proteins encoded by the c-myc oncogene: analysis of c-myc protein degradation.

We have examined one of the most striking characteristics of the c-myc oncogene protein product: its extremely short half-life relative to all but a few normal cellular proteins. Our studies indicate that the rapid degradation of c-myc proteins is not confined to human and avian cells, where it was first observed, but is found in cells derived from as evolutionarily widely separated species as murine and amphibian as well as in both normal and transformed cells. In addition the rate of degradation appears to be essentially the same throughout the G1, S, and G2 phases of the cell cycle as determined by "pulse-chase" analysis of cell cycle subpopulations separated by centrifugal elutriation. Kinetic analysis of c-myc protein turnover utilizing immunoprecipitation from labelled cells or immunoblotting of total nuclei indicate that there is not likely to be a significantly large pool of the protein which is protected from degradation and that no major degradation products can be detected. Evidence that the nucleus plays a role in degradation comes from preliminary experiments which indicate that in enucleated cells the c-myc proteins are stable. We also discuss evidence suggesting that myc proteins are stable in mitotic cells. Finally, using a series of chemical agents we have found conditions which inhibit in vivo degradation. These experiments indicate the involvement of both ATP and metal ions in degradation of c-myc proteins.

Animals↗
PubMed1985

Faithful cell-cycle regulation of a recombinant mouse histone H4 gene is controlled by sequences in the 3'-terminal part of the gene.

We have analyzed the expression of endogenous histone H4 genes and of a newly introduced H4 gene in 21-Tb cells, a mouse mastocytoma cell-cycle mutant. Endogenous H4 mRNAs were less abundant by a factor of 120-180 in G1-arrested than in exponentially multiplying cells. However, H4 transcription rates were only decreased by a factor of 3 under these conditions, as determined by in vitro elongation of nascent transcripts. This indicates that post-transcriptional control of histone mRNA levels is important, in accord with published data. We introduced a mouse H4 gene, modified by a 12-base-pair (bp) insertion in its coding sequence, into 21-Tb cells by DNA-mediated gene transfer. The levels of transcripts from this gene were regulated in parallel with those of the endogenous genes. Moreover, fusion of the simian virus 40 (SV40) early promoter to a 463-bp fragment containing the 3'-terminal half of the mouse H4 gene, including 230 bp of spacer sequences, led to the regulated expression of SV40/H4 fusion RNA. However, a small proportion of SV40-initiated transcripts were not processed to histone-specific 3' ends, but extended farther through the downstream Escherichia coli galactokinase gene to a SV40 polyadenylylation site. In contrast to the short SV40/H4 RNA, the levels of these longer transcripts were not reduced in G1-arrested cells. These results show that sequences in the 3'-terminal part of the H4 gene can regulate gene expression in the cell cycle, presumably at the post-transcriptional level, as long as they are not positioned much more distant from the terminus than normal.

Animals↗
PubMed1985

Biosynthesis and maturation of the Lyt-2/3 molecular complex in mouse thymocytes.

The biosynthesis and maturation of the three subunits alpha (Mr = 37,000), beta (Mr = 32,000), and gamma (Mr = 27,000) of the mouse Lyt-2/3 antigenic complex have been studied by using two monoclonal antibodies directed against a monomorphic determinant of the Lyt-2 antigen. Short time-pulse labeling of thymocytes reveals three different high mannose intermediates that give rise upon endo-beta-N-acetyl-glucosaminidase H digestion to three distinct precursor polypeptides of Mr = 22,000 (alpha P), Mr = 18,000 (beta P), and Mr = 19,500 (gamma P). Pulse-chase analysis indicates rapid posttranslational processing, because mature forms already appear after 10 min of chase. The half-life of the endo-H-sensitive early forms are in the range of 20 to 30 min. Both the alpha and beta subunits are suggested to contain three N-asparagine-linked oligosaccharides, one of which is of the high mannose type. In contrast, the gamma-chain contains only one such glycan unit of the complex type. Moreover, the results presented show that all three chains undergo additional posttranslational modifications. Finally, the data suggest that the cytoplasmic domains of these chains are of different size.

Animals↗
PubMed1985

Cell surface glycoproteins involved in the stimulation of interleukin 1-dependent interleukin 2 production by a subline of EL4 thymoma cells. II. Structure, biosynthesis, and maturation.

In the present study, we examined the biosynthesis and the maturation of two distinct membrane glycoproteins detected by two monoclonal antibodies (RL388 and RL119), which were selected on the basis of their ability to stimulate the production of interleukin 2 by a subline of the murine EL4 thymoma. RL388 detected a disulfide-linked heterodimer complex (Mr = 130,000) composed of a glycosylated heavy (Mr = 86,000) and a nonglycosylated light (Mr = 39,000) subunit. The unglycosylated precursor of the heavy chain was a polypeptide of Mr = 57,500, which was converted upon maturation into a Mr = 73,000 core-glycosylated intermediate, and then into the Mr = 86,000 surface-expressed molecule. Partial endo-H digestion of the core-glycosylated form suggested the presence of four N-linked glycan units. The antibody reacted with a protein determinant expressed on the mature form as well as the unglycosylated precursor of the heavy chain. Moreover, both subunits assembled rapidly during biosynthesis, and the glycosylation of the heavy chain was not required for this association. Taken together, these data suggest that the antigen detected by RL388 may be the murine homologue of the human 4F2 antigen. The antigen identified by RL119 was a surface glycoprotein of Mr = 55,000 with three to five N-linked glycan units. The unglycosylated precursor polypeptide was of Mr = 29,000. The fully core-glycosylated form of Mr = 41,000, which was detected after inhibition of glucosidase I with 1-deoxynojirimycin, was converted into a Mr = 39,000 intermediate, and upon further trimming, into a Mr = 36,000 endo-H-sensitive form. The latter could be detected for chase periods of over several hours, thus suggesting a low rate of intracellular processing. The wide cellular distribution of the molecules identified by RL388 and RL119 and their preferential expression on the surface of growing cells suggests that they may be associated with cell activation events.

Animals↗
PubMed1978

Pulpal and periodontal effects of electrosurgery involving cervical metallic restorations.

The pulpal and periodontal effects of electrosurgery involving teeth restored with Class V cervical amalgams and nonrestored teeth were evaluated in three cynomolgus monkeys. Electrosurgical current was delivered for 1 second with a fully rectified unit at an output intensity consistent with normal clinical usage. Experimental conditions included electrosurgery involving restored teeth, electrosurgery involving unrestored enamel, restored teeth not subjected to electrosurgery, and teeth which were neither restored nor subjected to electrosurgery. No pulpal or periodontal tissue changes were observed in the latter three groups of teeth. Electrosurgery involving cervical restorations consistently resulted in coagulation necrosis of the pulp and extensive resorption of cementum, dentin, and interradicular bone in the furcation area of multirooted teeth. The results suggest that inadvertent contact with cervical restorations during electrosurgical procedures may endanger both the pulp and the periodontal attachment apparatus.

Animals↗
PubMed1978

Microleakage and marginal adaptation of composite resin restorations.

It has been shown that the use of a sealant offers no advantage with respect to the retentive strength of a composite resin restoration. However, for the improvement of marginal seal and adaptation, the present findings indicate that enamel etching and the application of a sealant, in conjunction with use of a cavity geometry which reduces shrinkage strain, are absolute necessities.

Acid Etching, Dental↗
PubMed1978

Marginal adaptation and microleakage of thixotropic and injection-capsule composites, an in vitro study.

The marginal integrity of MOD adhesive restorations of a thixotropic test composite (ZKP-A-5) and of two injection-capsule composites (Concise -capsule composite and Compocap-S) was examined. Dy penetration tests measured microleakage and scanning electron microscope evaluated marginal adaptation. The test restorations showed no improved micromorphological marginal adaptation when compared to those of a standard paste-paste composite (Concise). Compocap-S demonstrated better prevention of microleakage than did the other test preparations. The marginal quality of these thixotropic and injection-capsule composite restorations was improved from 34% to 52% with utilization of a sealer application (ZKP-A-2), (Concise-Enamel Bond). The application of a sealer prior to insertion of thixotropic and injection-capsule composites is, therefore, definitely indicated when these materials are used. The resultant restorations obtained with these test materials plus sealer correspond qualitatively to those of standard composites Concise or Adaptic [6,7]. The newly developed, complete composite systems (Nimetic, GC's Epolite 100/Epobond) demonstrated in comparison to Concise and Adaptic [6,7] inferior results.

Adhesiveness↗
PubMed1977

[Follow-up studies on 15-month-old adhesive fillings].

Clinically, 450 Adhesive Restorations have been reexamined 15 months after insertion. Several of these restorations were also evaluated by SEM. The principles of cavity preparation for adhesive fillings deviate from those postulated by G.V. Black. They aim at a micromethanical retention of the restorative material to the tooth structure. A considerable amount of sound tooth structure can be saved. The combination of cavity design with enamel conditioning and the use of a low viscosity sealant prior to the insertion of the composite (Caulk Nuva System) resulted in restorations showing no microleakage and a perfect marginal adaptation. Their retentive strength seems to be sufficient as well as the durability of their excellent marginal properties. Pulpal responses to the application of etching agents were avoided by covering the dentin with an acid resistant base. Splinting of teeth using a strictly adhesive method without cavity preparations failed in more than 50%. The high rate of composite discoloration (43%) was probably due to factors inherent in the restorative material itself.

Adhesiveness↗