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Biomedical subjects

J A Fischer

Publications and source records attributed to J A Fischer.

At least 19 recordsLinked to original sources
PubMed2005

Enhanced vascular responses to adrenomedullin in mice overexpressing receptor-activity-modifying protein 2.

Adrenomedullin (AM) levels are elevated in cardiovascular disease, but little is known of the role of specific receptor components. AM acts via the calcitonin receptor-like receptor (CLR) interacting with a receptor-activity-modifying protein (RAMP). The AM1 receptor is composed of CLR and RAMP2, and the calcitonin gene-related peptide (CGRP) receptor of CLR and RAMP1, as determined by molecular and cell-based analysis. This study examines the relevance of RAMP2 in vivo. Transgenic (TG) mice that overexpress RAMP2 in smooth muscle were generated. The role of RAMP2 in the regulation of blood pressure and in vascular function was investigated. Basal blood pressure, acute angiotensin II-raised blood pressure, and cardiovascular properties were similar in wild-type (WT) and TG mice. However, the hypotensive effect of IV AM, unlike CGRP, was enhanced in TG mice (P<0.05), whereas a negative inotropic action was excluded by left-ventricular pressure-volume analysis. In aorta relaxation studies, TG vessels responded in a more sensitive manner to AM (EC50, 8.0+/-1.5 nmol/L) than WT (EC50, 17.9+/-3.6 nmol/L). These responses were attenuated by the AM receptor antagonist, AM(22-52), such that residual responses were identical in all mice. Remaining relaxations were further inhibited by CGRP receptor antagonists, although neither affected AM responses when given alone. Mesenteric and cutaneous resistance vessels were also more sensitive to AM in TG than WT mice. Thus RAMP2 plays a key role in the sensitivity and potency of AM-induced hypotensive responses via the AM1 receptor, providing evidence that this receptor is a selective target for novel therapeutic approaches.

Adrenomedullin↗
PubMed2002

Functional relevance of G-protein-coupled-receptor-associated proteins, exemplified by receptor-activity-modifying proteins (RAMPs).

The calcitonin (CT) receptor (CTR) and the CTR-like receptor (CRLR) are close relatives within the type II family of G-protein-coupled receptors, demonstrating sequence identity of 50%. Unlike the interaction between CT and CTR, receptors for the related hormones and neuropeptides amylin, CT-gene-related peptide (CGRP) and adrenomedullin (AM) require one of three accessory receptor-activity-modifying proteins (RAMPs) for ligand recognition. An amylin/CGRP receptor is revealed when CTR is co-expressed with RAMP1. When complexed with RAMP3, CTR interacts with amylin alone. CRLR, initially classed as an orphan receptor, is a CGRP receptor when co-expressed with RAMP1. The same receptor is specific for AM in the presence of RAMP2. Together with human RAMP3, CRLR defines an AM receptor, and with mouse RAMP3 it is a low-affinity CGRP/AM receptor. CTR-RAMP1, antagonized preferentially by salmon CT-(8-32) and not by CGRP-(8-37), and CRLR-RAMP1, antagonized by CGRP-(8-37), are two CGRP receptor isotypes. Thus amylin and CGRP interact specifically with heterodimeric complexes between CTR and RAMP1 or RAMP3, and CGRP and AM interact with complexes between CRLR and RAMP1, RAMP2 or RAMP3.

Amino Acid Sequence↗
PubMed2002

Circulating procalcitonin and cleavage products in septicaemia compared with medullary thyroid carcinoma.

OBJECTIVE: Raised plasma levels of procalcitonin (proCT) represent an early marker for septicaemia. They are related to disease severity and inversely to outcome and response to treatment. ProCT is presumably synthesised in tIssues other than the thyroid C-cells which are the source of calcitonin (CT) in normal physiology. This study compares proCT and its cleavage products in the serum of patients with septicaemia with those in medullary thyroid carcinoma (MTC). METHODS: Immunoreactive proCT and its cleavage products were extracted from the serum of patients with septicaemia or MTC using octadecylsilyl silica columns and characterised by reversed phase HPLC and Western blot analysis. ProCT, CT(1-32) and the flanking peptides PAS-57 and PDN-21 were recognised with antibodies specific for the individual peptides. RESULTS: ProCT and a 10 kDa polypeptide were recognised with antibodies to PAS-57, CT(1-32) and PDN-21. An 8 kDa proCT fragment was detected with antibodies to CT and PDN-21. However, intact CT(1-32), PAS-57 and PDN-21, found in the serum of MTC patients, were undetectable. The results indicate partial cleavage of proCT in septicaemia different from that in MTC patients. CONCLUSIONS: ProCT and 10 and 8 kDa proCT fragments were recognised in the circulation of septic patients. They were different from the known proCT-processing products PAS-57, CT(1-32) and PDN-21 identified in the serum of normal subjects and of MTC patients. Distinct cleavage of proCT may contribute to the symptoms of septicaemia.

Aged↗
PubMed2002

Receptors for calcitonin gene-related peptide, adrenomedullin, and amylin: the contributions of novel receptor-activity-modifying proteins.

The discovery of receptor-activity-modifying proteins (RAMP) revealed a new principle for the function of G protein-coupled receptors. The initially orphan calcitonin receptor-like receptor (CRLR) was identified as a CGRP receptor when coexpressed with RAMP1. The same receptor is specific for adrenomedullin (ADM) in the presence of RAMP2. Calcitonin receptors (CTR) with 60% homology to the CRLR predominantly recognize calcitonin in the absence of RAMP. An amylin/CGRP receptor was recognized when a calcitonin receptor (CTR) was coexpressed with RAMP1. In the presence of RAMP3, the CTR only interacts with amylin. Noncovalent association of the RAMP with the CRLR or the CTR reveals heterodimeric RAMP/receptor complexes at the cell surface. Thus, two Class II G protein-coupled receptors, the CRLR and CTR, associate with three RAMP to form high affinity receptors for CGRP, ADM, or amylin. Here, the molecular composition and the functional properties of these receptors is reviewed.

Animals↗
PubMed2001

Mutations of the asparagine117 residue of a receptor activity-modifying protein 1-dependent human calcitonin gene-related peptide receptor result in selective loss of function.

The initially orphan human calcitonin (CT) receptor-like receptor (hCRLR) interacts with novel accessory receptor activity-modifying protein 1 (RAMP1) to reveal a functional CT gene-related peptide (CGRP) receptor. In mammalian cells, RAMP1 is required for mature N-glycosylation of the hCRLR predicted to occur at Asn(60), Asn(112), and/or Asn(117) in the amino-terminal extracellular domain. Here we have shown that the substitution of Asn(117) with Ala, Gln, Thr, or Pro abolished CGRP-evoked cAMP formation which was left unchanged when the Asn(117) was replaced with Asp. Moreover, the hCRLR and the Asn(117) mutants exhibited comparable N-glycosylation and cell surface expression, and the association with RAMP1 was only slightly impaired. In contrast, the hCRLR Asn(60,112) to Thr double mutant exhibited defective RAMP1-dependent N-glycosylation, and impaired cell surface expression and CGRP receptor function. Unlike Asn(60) and Asn(112), Asn(117) is normally not N-glycosylated, but essential for CGRP binding to the hCRLR-RAMP1 complex.

Amino Acid Substitution↗
PubMed2001

Methylation is involved in the Ustilago maydis mating response.

Methionine auxotrophs of Ustilago maydis were deficient in mating; unlike wild-type cells, they neither induced nor produced normal mating filaments in the presence of compatible cells. The deficiency was most severe when cells were located some distance apart, but when in direct contact with compatible cells methionine auxotrophs mated and infected plants fairly normally. The mating deficiency was genetically linked to the methionine auxotrophy, segregating with it through in planta crosses. Wild-type cells exposed to the methyltransferase inhibitors ethionine and homocysteine thiolactone were similarly impaired in mating. Exogenous methionine, S-adenosylmethionine (SAM), synthetic mating pheromone, or cAMP all compensated for the mating impairment of the auxotrophs to some extent. Although SAM-dependent methylation could influence activities of various molecules in diverse pathways, these observations indicate that the most likely cause of the mating deficiency in met(-) cells is failure to methylate a component of the U. maydis pheromone signal transduction pathway.

Cyclic AMP↗
PubMed2001

Adrenomedullin and related peptides: receptors and accessory proteins.

Adrenomedullin (AM), alpha- and beta-calcitonin gene-related peptide (CGRP), amylin and calcitonin (CT) are structurally and functionally related peptides. The structure of a receptor for CT (CTR) was elucidated in 1991 through molecular cloning, but the structures of the receptors for the other three peptides had yet to be elucidated. The discovery of receptor-activity-modifying proteins (RAMP) 1 and -2 and their co-expression with an orphan receptor, calcitonin receptor-like receptor (CRLR) has led to the elucidation of functional CGRP and AM receptors, respectively. RAMP1 and -3 which are co-expressed with CTR revealed two amylin receptor isotypes. Molecular interactions between CRLR and RAMPs are involved in their transport to the cell surface. Heterodimeric complexes between CRLR or CTR and RAMPs are required for ligand recognition.

Adrenomedullin↗
PubMed2000

Glycosylation of the calcitonin receptor-like receptor at Asn(60) or Asn(112) is important for cell surface expression.

The human calcitonin (CT) receptor-like receptor (hCRLR) of the B family of G protein-coupled receptors is N-glycosylated and associates with receptor-activity-modifying proteins for functional interaction with CT gene-related peptide (CGRP) or adrenomedullin (ADM), respectively. Three putative N-glycosylation sites Asn(60), Asn(112) and Asn(117) are present in the amino-terminal extracellular domain of the hCRLR. Tunicamycin dose-dependently inhibited the glycosylation of a myc-tagged hCRLR and in parallel specific [(125)I]CGRP and -ADM binding. Similarly, the double mutant myc-hCRLR(N60,112T) exhibited minimal N-glycosidase F sensitive glycosylation, presumably at the third Asn(117), and the cell surface expression and specific radioligand binding were impaired. Substitution of the Asn(117) by Thr abolished CGRP and ADM binding in the face of intact N-glycosylation and cell surface expression.

Adrenomedullin↗
PubMed2000

Receptor-activity-modifying protein 1 forms heterodimers with two G-protein-coupled receptors to define ligand recognition.

Receptor-activity-modifying proteins (RAMPs) with single transmembrane domains define the function of two G-protein-coupled receptors of the B family. Cell-surface complexes of human RAMP1 (hRAMP1) and human calcitonin (CT) receptor isotype 2 (hCTR2) or rat CT-receptor-like receptor (rCRLR) have now been identified through protein cross-linking, co-immunoprecipitation and confocal microscopy. They are two distinct CT-gene-related peptide (CGRP) receptors coupled to cAMP production and pharmacologically distinguished by the CT and CGRP antagonists salmon CT(8-32) and human or rat CGRP(8-37). Thus direct molecular interactions of hRAMP1 with hCTR2 or rCRLR are required for CGRP recognition. hCTR2, moreover, adopts non-traditional functions through its association with hRAMP1.

Animals↗
PubMed2000

Mouse receptor-activity-modifying proteins 1, -2 and -3: amino acid sequence, expression and function.

The calcitonin receptor-like receptor (CRLR) requires novel receptor-activity-modifying proteins (RAMPs) for its function as an adrenomedullin (ADM) or a calcitonin (CT) gene-related peptide (CGRP) receptor. Here, mouse cDNA clones representing expressed sequence tags (ESTs) in the GenEMBL database have been identified. They encode for proteins with 70, 68 and 84% amino acid sequence identity with respect to human RAMP1, -2 and -3. On Northern blot analysis of polyA(+) RNA mouse RAMP1 (mRAMP1) encoding mRNA with an apparent size of 0.8 kb was predominantly observed in embryonic and adult brain and lung and in adult skeletal muscle. Mouse RAMP2 encoding 0.8 and 1.2 kb mRNA were recognized in all tissues analyzed with the highest levels in embryonic brain, lung and gut and in adult heart, lung, skeletal muscle and brain. A single 1.2 kb mRAMP3 encoding transcript was mainly expressed in embryonic and adult brain. In COS-7 cells co-expressing rat CRLR (rCRLR) and mRAMP1, [125I]halphaCGRP binding was inhibited by ralphaCGRP(8-37), ralphaCGRP and rbetaCGRP with IC(50) of 1.4+/-0.5, 4.5+/-0.6 and 7+/-0.3 nM, respectively. CyclicAMP accumulation was maximally stimulated tenfold by rbetaCGRP and ralphaCGRP with EC(50) of 0. 65+/-0.67 and 0.86+/-0.6 nM. In the same cells co-expressing rCRLR and mRAMP2, binding of [125I]rADM was displaced by rADM and rADM(20-50) with IC(50) of 1.9+/-0.5 and 3.4+/-1.4 nM, respectively, and a maximal sevenfold stimulation of cAMP accumulation was observed with rADM with an EC(50) of 0.82+/-0.85 nM. In the cells co-expressing rCRLR and mRAMP3, [125I]halphaCGRP binding was inhibited by ralphaCGRP(8-37), rbetaCGRP, ralphaCGRP, rADM and rADM(20-50) with IC(50) between 4 and 22 nM. cAMP accumulation was stimulated by rADM with an EC(50) of 5.1+/-2.7 nM that was 12-fold and 11-fold lower than that of ralphaCGRP and rbetaCGRP. In conclusion, mouse RAMP1, -2 and -3 exhibit high amino acid sequence homology to the corresponding human RAMPs. Co-expression of rCRLR with mRAMP1, -2 or -3 in COS-7 cells revealed distinct CGRP-, ADM- or ADM/CGRP receptors.

Adrenomedullin↗
PubMed2000

Mammalian calcitonin receptor-like receptor/receptor activity modifying protein complexes define calcitonin gene-related peptide and adrenomedullin receptors in Drosophila Schneider 2 cells.

Differential glycosylation of human and rat (r) calcitonin (CT) receptor-like receptors (CRLR) as a result of interactions with accessory receptor activity-modifying proteins (RAMP)1 or -2 was considered to define CT gene-related peptide (CGRP) or adrenomedullin (ADM) receptors in mammalian cells. Here, Drosophila Schneider (S2) cells stably co-expressed rCRLR and RAMP1 or -2 as functional CGRP or ADM receptors. Different from mammalian cells, rCRLR expressed in S2 cells are uniformly glycosylated proteins independent of RAMP1 or RAMP2. Bis(sulfosuccinimidyl)suberate cross-linking revealed receptor components with the size of rCRLR, increased by the molecular weights of the corresponding RAMPs and [(125)I]CGRP or [(125)I]ADM. In conclusion, [(125)I]CGRP/rCRLR/RAMP1 and [(125)I]ADM/rCRLR/RAMP2 complexes have been recognized in Drosophila S2 cells.

Adrenomedullin↗
PubMed2000

On the conservation of function of the Drosophila fat facets deubiquitinating enzyme and Fam, its mouse homolog.

Fat facets is a Drosophila deubiquitinating enzyme required for eye development and early embryogenesis. Genetic evidence suggests that Fat facets deubiquitinates and thereby prevents the proteasomal degradation of specific substrates. The Drosophila Liquid facets protein is implicated as the critical substrate of Fat facets in the eye. A mouse homolog of Fat facets, called Fam, has been identified. The results of biochemical experiments implicate two different proteins, Af-6 and beta-catenin, as substrates for Fam. Here, the functional relationship between Fat facets and Fam is explored. We show that Fam can substitute for Fat facets in all of its essential functions in Drosophila. In addition, we tested the hypothesis that Canoe and Armadillo, the Drosophila homologs of Af-6 and beta-catenin, respectively, are important substrates for Fat facets in the Drosophila eye. We found no genetic evidence to support a role for either Canoe or Armadillo in the essential Fat facets pathways in Drosophila eye development. The significance of these results is discussed in light of the biochemical experiments that suggest that Af-6 and beta-catenin are substrates of Fam.

Animals↗
PubMed2000

Molecular motors and developmental asymmetry.

The generation of distinct cell fates can require movement of specific molecules or organelles to particular locations within the cell. These subcellular movements are often the jobs of motor proteins. Seemingly disparate developmental processes--determination of right and left in vertebrates, setting up the axes of polarity in insect embryos, mating-type switching in yeast, and coordinated organelle movements in Drosophila--converge in their dependence on motor proteins. The extent of possible regulatory complexity is only beginning to emerge.

Animals↗
PubMed2000

Genetic analysis of the Drosophila DNAprim gene. The function of the 60-kd primase subunit of DNA polymerase opposes the fat facets signaling pathway in the developing eye.

The Drosophila DNAprim gene encodes the large subunit (60 kD) of DNA primase, the part of DNA polymerase alpha that synthesizes RNA primers during DNA replication. The precise function of the 60-kD subunit is unknown. In a mutagenesis screen for suppressors of the fat facets (faf) mutant eye phenotype, we identified mutations in DNAprim. The faf gene encodes a deubiquitinating enzyme required specifically for patterning the compound eye. The DNA sequences of four DNAprim alleles were determined and these define essential protein domains. We show that while flies lacking DNAprim activity are lethal, flies with reduced DNAprim activity display morphological defects in their eyes, and unlike faf mutants, cell cycle abnormalities in larval eye discs. Mechanisms by which DNA primase levels might influence the faf-dependent cell communication pathway are discussed.

Alleles↗
PubMed2000

In vivo Structure/Function analysis of the Drosophila fat facets deubiquitinating enzyme gene.

The Drosophila Fat facets protein is a deubiquitinating enzyme required for patterning the developing compound eye. Ubiquitin, a 76-amino-acid polypeptide, serves as a tag to direct proteins to the proteasome, a protein degradation complex. Deubiquitinating enzymes are a large group of proteins that cleave ubiquitin-protein bonds. Fat facets belongs to a class of deubiquitinating enzymes called Ubps that share a conserved catalytic domain. Fat facets is unique among them in its large size and also because Fat facets is thought to deubiquitinate a specific substrate thereby preventing its proteolysis. Here we asked which portions of the Fat facets protein are essential for its function. P-element constructs that express partial Fat facets proteins were tested for function. In addition, the DNA sequences of 12 mutant fat facets alleles were determined. Finally, regions of amino acid sequence similarity in 18 Drosophila Ubps revealed by the Genome Project were identified. The results indicate functions for specific conserved amino acids in the catalytic region of Fat facets and also indicate that regions of the protein both N- and C-terminal to the catalytic region are required for Fat facets function.

Alleles↗
PubMed2000

How to promote pulmonary health with kinetic therapy.

Kinetic Therapy can improve outcomes for immobilized patients, but the treatment can be costly. Using a protocol designed by critical care nurses, nurses in your units can provide cost-effective Kinetic Therapy.

Algorithms↗
PubMed2000

The function of the Drosophila fat facets deubiquitinating enzyme in limiting photoreceptor cell number is intimately associated with endocytosis.

Fat facets is a deubiquitinating enzyme required in a cell communication pathway that limits to eight the number of photoreceptor cells in each facet of the Drososphila compound eye. Genetic data support a model whereby Faf removes ubiquitin, a polypeptide tag for protein degradation, from a specific ubiquitinated protein thus preventing its degradation. Here, mutations in the liquid facets gene were identified as dominant enhancers of the fat facets mutant eye phenotype. The liquid facets locus encodes epsin, a vertebrate protein associated with the clathrin endocytosis complex. The results of genetic experiments reveal that fat facets and liquid facets facilitate endocytosis and function in common cells to generate an inhibitory signal that prevents ectopic photoreceptor determination. Moreover, it is demonstrated that the fat facets mutant phenotype is extraordinarily sensitive to the level of liquid facets expression. We propose that Liquid facets is a candidate for the critical substrate of Fat facets in the eye.

Adaptor Proteins, Vesicular Transport↗