Biomedical subjects

P Menzel

Publications and source records attributed to P Menzel.

At least 19 recordsLinked to original sources

PubMed1994

Development and characterization of recombinant adenoviruses encoding human p53 for gene therapy of cancer.

We have constructed recombinant human adenoviruses that express wild-type human p53 under the control of either the Ad 2 major late promoter (MLP) or the human cytomegalovirus (CMV) immediate early gene promoter. Each construct replaces the Ad 5 E1a and E1b coding sequences necessary for viral replication with the p53 cDNA and MLP or CMV promoter. These p53/Ad recombinants are able to express p53 protein in a dose-dependent manner in infected human cancer cells. Tumor suppressor activity of the expressed p53 protein was assayed by several methods. [3H]Thymidine incorporation assays showed that the recombinant adenoviruses were capable of inhibiting DNA synthesis in a p53-specific, dose-dependent fashion. Ex vivo treatment of Saos-2 tumor cells, followed by injection of the treated cells into nude mice, led to complete tumor suppression using the MLP/p53 recombinant. Following a single injection of CMV/p53 recombinant adenovirus into the peritumoral space surrounding an in vivo established tumor derived from a human small cell lung carcinoma cell line (NIH-H69), we were able to detect p53 mRNA in the tumors at 2 and 7 days post-injection. Continued treatment of established H69 tumors with MLP/p53 recombinant led to reduced tumor growth and increased survival time compared to control treated animals. These results indicate that recombinant adenoviruses expressing wild-type p53 may be useful vectors for gene therapy of human cancer.

Adenoviruses, Human↗

PubMed1991

Characterization of motifs which are critical for activity of the cyclic AMP-responsive transcription factor CREB.

Cyclic AMP mediates the hormonal stimulation of a number of eukaryotic genes by directing the protein kinase A (PK-A)-dependent phosphorylation of transcription factor CREB. We have previously determined that although phosphorylation at Ser-133 is critical for induction, this site does not appear to participate directly in transactivation. To test the hypothesis that CREB ultimately activates transcription through domains that are distinct from the PK-A site, we constructed a series of CREB mutants and evaluated them by transient assays in F9 teratocarcinoma cells. Remarkably, a glutamine-rich region near the N terminus appeared to be important for PK-A-mediated induction of CREB since removal of this domain caused a marked reduction in CREB activity. A second region consisting of a short acidic motif (DLSSD) C terminal to the PK-A site also appeared to synergize with the phosphorylation motif to permit transcriptional activation. Biochemical experiments with purified recombinant CREB protein further demonstrate that the transactivation domain is more sensitive to trypsin digestion than are the DNA-binding and dimerization domains, suggesting that the activator region may be structured to permit interactions with other proteins in the RNA polymerase II complex.

Amino Acid Sequence↗

PubMed1990

Characterization of a bipartite activator domain in transcription factor CREB.

In this paper, we characterize a trans-activating region in CREB, termed alpha, that interacts cooperatively with the kinase A phosphorylation motif to stimulate transcription. The alpha region appears to be encoded by an alternate exon that is deleted in a CREB-related cDNA named delta CREB. Both proteins are expressed in eukaryotic cells, although the activity of CREB is 10-fold higher than that of delta CREB. Circular dichroism data on a synthetic "alpha peptide" combined with results from in vitro mutagenesis experiments support the hypothesis that the alpha region contains an amphipathic alpha helix whose structure is critical to CREB activity. We propose that phosphorylation by kinase A may stimulate CREB activity in part by modulating the structure of alpha and thus may stimulate its ability to interact with other proteins in the polymerase II complex.

Adrenal Gland Neoplasms↗

PubMed1989

A cluster of phosphorylation sites on the cyclic AMP-regulated nuclear factor CREB predicted by its sequence.

Cyclic AMP regulates the expression of a number of genes through a conserved promoter element, the CRE1. Moreover, transcriptional induction by cAMP requires the activation of cAMP-dependent protein kinase (protein kinase A). We have previously characterized the cAMP response element binding protein (CREB) in PC12 cells and brain tissue as a nuclear factor, of relative molecular mass 43,000, whose transcriptional efficacy is regulated by protein kinase A phosphorylation. CREB stimulates transcription on binding to the CRE as a dimer. Experiments suggesting that the dimerization and transcriptional efficacy of CREB are each stimulated by phosphorylation at distinct sites prompted us to suggest that CREB is regulated by multiple kinases in vivo. We now report the isolation of a cDNA clone for rat CREB using amino-acid sequence information from purified CREB protein. Sequence analysis of this CREB cDNA predicts a cluster of protein kinase A, protein kinase C and casein kinase II consensus recognition sites near the N terminus of the protein. The proximity of these potential phosphorylation sites to one another indicates that they may interact either positively or negatively to regulate CREB bioactivity.

Activating Transcription Factor 2↗

PubMed1988

Two guanosine binding sites exist in group I self-splicing IVS RNAs.

A shortened form of the self-splicing rRNA intervening sequence (IVS) of Tetrahymena thermophila can catalyze a transesterification reaction, termed G-exchange, between a monomeric guanosine derivative such as GTP and the substrate GpN (where N is A, C, G or U). The reaction is specific to the two guanosines involved, providing evidence that two guanosine binding sites exist in this group I IVS RNA. One binding site accommodates a guanosine which initiates self-splicing and the other recognizes the guanosine preceding the 3' splice site. Previously, only one guanosine binding site was thought to be involved in the mechanism of self-splicing. Based on the two functionally distinguishable guanosine binding sites, a new model is proposed to explain how the two independent transesterification reactions required for self-splicing might proceed in a concerted manner.

Animals↗

PubMed1982