Search PubMed⌕ Search

Biomedical subjects

F X Farrell

Publications and source records attributed to F X Farrell.

11 recordsLinked to original sources
PubMed1996

Small peptides as potent mimetics of the protein hormone erythropoietin.

Random phage display peptide libraries and affinity selective methods were used to isolate small peptides that bind to and activate the receptor for the cytokine erythropoietin (EPO). In a panel of in vitro biological assays, the peptides act as full agonists and they can also stimulate erythropoiesis in mice. These agonists are represented by a 14- amino acid disulfide-bonded, cyclic peptide with the minimum consensus sequence YXCXXGPXTWXCXP, where X represents positions allowing occupation by several amino acids. The amino acid sequences of these peptides are not found in the primary sequence of EPO. The signaling pathways activated by these peptides appear to be identical to those induced by the natural ligand. This discovery may form the basis for the design of small molecule mimetics of EPO.

Amino Acid Sequence↗
PubMed1993

Microinjection of Rap2B protein or RNA induces rearrangement of pigment granules in Xenopus oocytes.

Rap2B, a member of the ras superfamily of low-molecular-mass GTP-binding proteins, induced a characteristic rearrangement of the pigment granules in Xenopus oocytes following its microinjection, resulting in numerous unpigmented spots on the animal hemisphere. This phenomenon, termed 'mottling', was also induced by microinjection of in vitro-transcribed Rap2B RNA or of purified recombinant Rap2A. Following the microinjection of Rap2B, more than 90% of the oocytes showed signs of mottling within 10 h. The time course of mottling paralleled the association of the recombinant Rap2B with an oocyte membrane fraction. Like other members of the ras superfamily, Rap2B possesses a C-terminal CAAX motif that serves as a signal for post-translational processing. Mutation of the cysteine residue in the CAAX motif to serine prevents the association of Rap2B with oocyte membranes, and also prevents mottling. This result suggests that post-translational processing of Rap2B is required for the observed effect. Mottling was blocked by boiling Rap2B prior to its microinjection or by co-injection of the cytoskeletal reagent phalloidin.

Animals↗
PubMed1993

Prenyl group identification of rap2 proteins: a ras superfamily member other than ras that is farnesylated.

Rap proteins comprise a subset of the large family of ras-related proteins. They contain the C-terminal tetrapeptide sequence motif Cys-Ali-Ali-Xaa (Ali is an aliphatic amino acid and X is any amino acid), which has been found to be the site of membrane attachment via isoprenylation for ras, nuclear lamins and the gamma subunits of the heterotrimeric G-proteins. To investigate the isoprenylation of rap2a and rap2b, human cDNAs coding for these proteins were expressed in COS cells incubated in the presence of [3H]mevalonolactone. Both proteins incorporated a product of [3H]mevalonolactone, as judged by Western blot analysis. To identify the specific isoprenoid attached to each protein, the cDNAs were transcribed in vitro and the rap2 specific RNA was translated in a rabbit reticulocyte lysate system in the presence of [3H]mevalonolactone. The translation products were treated with methyl iodide and the released isoprenoid groups were analysed by h.p.l.c. Rap2b, which terminates in Cys-Val-Ile-Leu, is geranylgeranylated as predicted while rap2a, which terminates in Cys-Asn-Ile-Gln, incorporated farnesyl. A mutant construct generated by site-directed mutagenesis of rap2a cDNA yielding a protein terminating in leucine instead of glutamine incorporated geranylgeranyl, lending further support to the notion that isoprenoid specificity is governed by the terminal amino acid. In addition, when the CAAX motif cysteine at position 180 of rap2a was replaced by a serine residue no isoprenoid incorporation was observed. Thus rap2a and rap2b, despite showing 90% sequence identity, incorporate different isoprenoid groups. Thus glutamine is a signal for farnesylation, and rap2a is the first non-ras member of the ras superfamily that is farnesylated.

Amino Acid Sequence↗
PubMed1992

Partial purification of a GTPase-activating protein for rap2b from bovine brain membranes.

Rap2b is a ras-related GTP-binding protein isolated from a human platelet cDNA library. It shares 90% similarity to the previously described rap2a and is closely related to rap1a (Krev-1, smgp21), which has been shown to possess reversion of transformation activity in Kirsten ras transformed 3T3 cells. In this study we have partially purified a protein from bovine brain membranes which stimulates the GTPase activity of rap2b. This rap2b GTPase-activating protein (GAP) activity is not immunoreactive with antibodies specific for rap1 GAP or ras GAP, yet displays limited GTPase stimulatory activity toward rap1. This result differs from the previously described rap1 GAP which is highly specific for rap1. When the rap2 GAP activity is analyzed by coomassie staining, an enrichment of a approximately 55 kDa protein is observed providing further evidence of a distinct rap2 GAP.

Animals↗
PubMed1990

Regulation of vimentin gene expression in the ocular lens.

Vimentin expression in the lens is striking due to the reported mesenchymal preference of vimentin and the epithelial origin of the lens. The amount of chicken vimentin mRNA levels determined by Northern blot analysis increased 3-fold from 7 to 14 days of embryonic lens development and then decreased 10-fold at 16 days of development, suggesting that post-transcriptional processes may contribute to the level of cytoplasmic vimentin mRNA during lens development. To analyze the mechanisms governing vimentin gene expression in the lens at the level of transcription, a series of chicken vimentin 5'-flanking region deletions were fused to the bacterial CAT gene and transfected into fibroblasts and lens cultures derived from three species. The -160 to +1 sequence conferred equal promoter activity in cultured chicken lens epithelial cells and fibroblasts. The -321 to -160 sequences increased promoter activity in all cultures, but more strongly in fibroblasts than in lens cells. Sequence elements in the region -608 to -321 repressed promoter activity in lens cells and fibroblasts. Promoter activity was partially restored in fibroblasts but not in lens cells by -767 to -608 sequences. Vimentin gene expression in the lens thus appears to be controlled by multiple positive- and negative-acting elements in its 5'-flanking sequence.

Animals↗
PubMed1990

RAP2B: a RAS-related GTP-binding protein from platelets.

A platelet cDNA expression library was screened with the monoclonal antibody M90, which recognizes a specific epitope on RAS-encoded p21 proteins (amino acids 107-130). DNA sequence analysis of one clone revealed that it encoded a partial amino acid sequence of a protein closely related to RAP2, which we have named RAP2B. A repeated screening of the platelet cDNA library with an internal Ava I fragment of the RAP2B cDNA allowed the isolation of a full-length cDNA for the RAP2B sequence. RAP2B is 90% identical to RAP2 at the amino acid level with the most variability at the carboxyl terminus of the protein. Oligonucleotides were synthesized to complete the amino acid sequence of the RAP2B protein and the entire sequence was expressed in Escherichia coli. Analysis of crude soluble extracts indicated that RAP2B was a Mr 22,000 protein that specifically bound GTP on blots. Moreover, incubation of similar extracts with the catalytic subunit of cAMP-dependent protein kinase did not cause phosphorylation of RAP2B, as had been observed for the closely homologous proteins, RAP1A and RAP1B. These results suggest that RAP2B, like the other RAP proteins, is a low molecular weight GTP-binding protein in human platelets.

Amino Acid Sequence↗
PubMed1990

A negative element involved in vimentin gene expression.

Vimentin is one member of the intermediate filament multigene family which exhibits both tissue- and developmental stage-specific expression. In vivo, vimentin is expressed in cells of mesenchymal origin. Previously, we identified both enhancer and promoter elements in the chicken vimentin gene which regulate gene expression in a positive manner. In this report, we have identified a 40-base-pair region at -568 base pairs between the proximal and distal enhancer elements which represses transcriptional activity. This silencer region can also repress the heterologous herpes simplex virus thymidine kinase promoter, which is comparable to the vimentin promoter. In addition, the element is able to function in a position- and orientation-independent manner, and the amount of repression is increased by multiple copies. Here we show by gel retardation assays and DNase I footprinting that this region binds a protein in nuclear extracts from HeLa cells. Southwestern (DNA-protein) blot analysis indicates this protein is approximately 95 kilodaltons in size. Moreover, protein distribution and activity mimic the expression pattern of vimentin during myogenesis, i.e., protein binding increases as vimentin gene expression decreases. The silencer region shares strong sequence similarity with 5'-flanking sequences found in both the human and hamster vimentin genes and with other characterized silencer elements, including the human immunodeficiency virus long terminal repeat, rat growth hormone, chicken lysozyme, and rat insulin genes. Thus, a negative element appears to bind a 95-kilodalton protein involved in regulating the tissue-specific expression of the chicken vimentin gene.

Animals↗
PubMed1989

Down-regulation of vimentin gene expression during myogenesis is controlled by a 5'-flanking sequence.

During myogenesis, the intermediate filament proteins vimentin and desmin are differentially expressed. While desmin levels increase dramatically, vimentin mRNA levels decrease substantially. Here, we show that transfected whole- and mini-vimentin-coding genes (Vim) are expressed in fibroblasts (mouse L cells) and down-regulated during muscle cell differentiation in culture. Functional assays with 5'-end Vim::cat constructs demonstrate that this repression is controlled by a 5'-element (nt -321 to -160). This region is distinct from Vim promoter elements (nt -160 to +71) which do not contribute to vimentin's down-regulation during myogenesis.

Animals↗
PubMed1988

Multiple elements are required for expression of an intermediate filament gene.

The expression of vimentin is unique within the intermediate filament multigene family. It is the only member which deviates from its usual tissue-specific expression pattern and whose 5'-flanking region contains multiple GC boxes, the binding site for Sp1. The activity of vimentin 5'-end:CAT fusions has been compared in cells where vimentin is highly expressed (mouse L cells) or not expressed at all (MH1C1). In addition, CAT activity has been examined by microinjection into Xenopus oocytes. Both in vivo expression and in vitro binding studies implicate Sp1 as a general regulatory factor in vimentin gene expression. Increased expression of 5'-end:CAT fusions in mouse L cells suggests that a fibroblast-specific enhancer element resides in the region -321 to -160. Low transcriptional activity in MH1C1 cells may be due to either the lack of this positive transcription factor(s) or the presence of a repressor element. Here, we demonstrate that the unique and complex pattern of vimentin gene expression is controlled by multiple cis-acting elements.

Animals↗