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Biomedical subjects

J M McCoy

Publications and source records attributed to J M McCoy.

At least 19 recordsLinked to original sources
PubMed2001

In situ hybridization screen in zebrafish for the selection of genes encoding secreted proteins.

An in situ hybridization expression screen using a signal sequence trap system has been conducted in zebrafish to isolate cDNAs that encode secreted proteins. Random clones (secreted expressed sequence tags; sESTs) were sequenced from zebrafish embryonic (18-24 hr postfertilization) and adult kidney libraries. From the two RNA sources, 627 random sEST cDNAs were identified as being homologous or identical to known genes and 166 clones encode currently unidentified genes. The sESTs represent a broad range of enzymes and other regulatory molecules. Whole-mount in situ hybridization analysis was carried out by using antisense probes generated from 244 selected sESTs, and a range of expression patterns was obtained. Genetic mapping undertaken with sEST sequences demonstrated that assignment of map position was attainable by using 5' primers. The signal sequence trap system used in this work has yielded a range of cDNAs that encode secreted proteins and, together with analysis of patterns of expression and genetic mapping, has the potential to facilitate analysis of signaling pathways central to development and physiology.

Animals↗
PubMed2001

Downregulation of placental syncytin expression and abnormal protein localization in pre-eclampsia.

Development of placentation and successful pregnancy depend on co-ordinated interactions between the maternal decidua and myometrium, and the invasive properties of the fetal trophoblast. Syncytin, a protein encoded by the envelope gene of a recently identified human endogenous defective retrovirus, HERV-W, is highly expressed in placental tissue. Previously, we have shown that the major site of syncytin expression is the placental syncytiotrophoblast, a fused multinuclear syncytium originating from cytotrophoblast cells. Here we present the first evidence that in pre-eclampsia, syncytin gene expression levels are dramatically reduced. Additionally, immunohistochemical examination of normal placentae and placentae from women with pre-eclampsia reveals that the syncytin protein in placental tissue from women with pre-eclampsia is localized improperly to the apical syncytiotrophoblast microvillous membrane as opposed to its normal location on the basal syncytiotrophoblast cytoplasmic membrane. Our previous results suggest that syncytin may mediate placental cytotrophoblast fusion in vivo and may play an important role in human placental morphogenesis. The present study suggests that altered expression of the syncytin gene, and altered cellular location of its protein product, may contribute to the aetiology of pre-eclampsia.

Female↗
PubMed2001

Investigation of the 'switch-epitope' concept with random peptide libraries displayed as thioredoxin loop fusions.

The 'FLITRX' random peptide library, consisting of dodecamer loop peptides displayed on a thioredoxin-flagellin scaffold on Escherichia coli, was used to select peptide sequences with affinity for a monoclonal antibody. These peptides were further screened for pH- and metal-sensitive antibody binding. Several zinc-sensitive peptides were identified, termed 'switch epitopes'. A soluble, monomeric thioredoxin loop ('Trxloop') insertion analog of a FLITRX switch epitope was constructed and its antibody binding properties were characterized by Western blots. Zinc-dependent antibody recognition was maintained in the Trxloop protein although the apparent antibody affinity was lower. This Trxloop protein bound to an immobilized metal affinity chromatography matrix, similar to a 'histidine-patch' thioredoxin variant, and was reversibly precipitated by 1 mM Zn(2+) or Cu(2+) ions. Residues important for zinc and antibody binding were determined by site-directed mutagenesis. The Trxloop antibody affinity was increased by saturation mutagenesis. Biotinylated Trxloop ('Biotrxloop') variants of the original and improved affinity Trxloop proteins were constructed and characterized by surface plasmon resonance measurements. Increased antibody affinity was partially due to a slower antibody desorption rate, although the relative adsorption rates were dependent on the amount of immobilized Biotrxloop protein, indicating an influence of avidity on the apparent affinity.

Amino Acid Sequence↗
PubMed2000

Syncytin is a captive retroviral envelope protein involved in human placental morphogenesis.

Many mammalian viruses have acquired genes from their hosts during their evolution. The rationale for these acquisitions is usually quite clear: the captured genes are subverted to provide a selective advantage to the virus. Here we describe the opposite situation, where a viral gene has been sequestered to serve an important function in the physiology of a mammalian host. This gene, encoding a protein that we have called syncytin, is the envelope gene of a recently identified human endogenous defective retrovirus, HERV-W. We find that the major sites of syncytin expression are placental syncytiotrophoblasts, multinucleated cells that originate from fetal trophoblasts. We show that expression of recombinant syncytin in a wide variety of cell types induces the formation of giant syncytia, and that fusion of a human trophoblastic cell line expressing endogenous syncytin can be inhibited by an anti-syncytin antiserum. Our data indicate that syncytin may mediate placental cytotrophoblast fusion in vivo, and thus may be important in human placental morphogenesis.

Amino Acid Sequence↗
PubMed1999

derrière: a TGF-beta family member required for posterior development in Xenopus.

TGF-beta signaling plays a key role in induction of the Xenopus mesoderm and endoderm. Using a yeast-based selection scheme, we isolated derrière, a novel TGF-beta family member that is closely related to Vg1 and that is required for normal mesodermal patterning, particularly in posterior regions of the embryo. Unlike Vg1, derrière is expressed zygotically, with RNA localized to the future endoderm and mesoderm by late blastula, and to the posterior mesoderm by mid-gastrula. The derrière expression pattern appears to be identical to the zygotic expression domain of VegT (Xombi, Brat, Antipodean), and can be activated by VegT as well as fibroblast growth factor (FGF). In turn, derrière activates expression of itself, VegT and eFGF, suggesting that a regulatory loop exists between these genes. derrière is a potent mesoderm and endoderm inducer, acting in a dose-dependent fashion. When misexpressed ventrally, derrière induces a secondary axis lacking a head, an effect that is due to dorsalization of the ventral marginal zone. When misexpressed dorsally, derrière suppresses head formation. derrière can also posteriorize neurectoderm, but appears to do so indirectly. Together, these data suggest that derrière expression is compatible only with posterior fates. In order to assess the in vivo function of derrière, we constructed a dominant interfering Derrière protein (Cm-Derrière), which preferentially blocks Derrière activity relative to that of other TGFbeta family members. Cm-derrière expression in embryos leads to posterior truncation, including defects in blastopore lip formation, gastrulation and neural tube closure. Normal expression of anterior and hindbrain markers is observed; however, paraxial mesodermal gene expression is ablated. This phenotype can be rescued by wild-type derrière and by VegT. Our findings indicate that derrière plays a crucial role in mesodermal patterning and development of posterior regions in Xenopus.

Amino Acid Sequence↗
PubMed1999

Recognize, reward, retain.

Staff recognition plans can be tools for retention or points of contention. The author shows the do's and don'ts for implementing a successful program on your unit.

Humans↗
PubMed1998

A plasmid expression system for quantitative in vivo biotinylation of thioredoxin fusion proteins in Escherichia coli.

The high affinity binding interaction of biotin to avidin or streptavidin has been used widely in biochemistry and molecular biology, often in sensitive protein detection or protein capture applications. However, in vitro chemical techniques for protein biotinylation are not always successful, with some common problems being a lack of reaction specificity, inactivation of amino acid residues critical for protein function and low levels of biotin incorporation. This report describes an improved expression system for the highly specific and quantitative in vivo biotinylation of fusion proteins. A short 'biotinylation peptide', described previously by Schatz, is linked to the N-terminus of Escherichia coli thioredoxin (TrxA) to form a new protein, called BIOTRX. The 'biotinylation peptide' serves as an in vivo substrate mimic for E. coli biotin holoenzyme synthetase (BirA), an enzyme which usually performs highly selective biotinylation of E.coli biotin carboxyl carrier protein (BCCP). A plasmid expression vector carrying the BIOTRX and birA genes arranged as a bacterial operon can be used to obtain high level production of soluble BIOTRX and BirA proteins and, under appropriate culture conditions, BIOTRX protein produced by this system is completely biotinylated. Fusions of BIOTRX to other proteins or peptides, whether these polypeptides are linked to the C-terminus or inserted into the BIOTRX active site loop, are also quantitatively biotinylated. Both types of BIOTRX fusion can be captured efficiently on avidin/streptavidin media for purification purposes or to facilitate interaction assays. We illustrate the utility of the system by measurements of antibody and soluble receptor protein binding to BIOTRX fusions immobilized on streptavidin-conjugated BIAcore chips.

Acetyl-CoA Carboxylase↗
PubMed1997

A genetic selection for isolating cDNAs encoding secreted proteins.

We describe a simple, rapid technique for simultaneously isolating large numbers of cDNAs encoding secreted proteins. The technique makes use of a facile genetic selection performed in a strain of Saccharomyces cerevisiae deleted for its endogenous invertase gene. A cDNA cloning vector which carries a modified invertase gene lacking its leader sequence is used in conjunction with this strain. Heterologous secreted genes fused appropriately upstream of this defective invertase provide the necessary signals to restore secretion, allowing the yeast to grow on sugars such as sucrose or raffinose. This microbial growth selection facilitates scanning cDNA libraries containing millions of clones, enabling the wholesale identification of novel secreted proteins without the need for specific bioassays. The technique is similar to one previously described (Klein et al. (1996) Proc. Natl. Acad. Sci. USA 93, 7108-7113). We describe results using a cDNA library derived from activated human peripheral blood mononuclear cells (PBMC). Genes identified from this library encoded signal sequences of proteins of diverse structure, function, and cellular location such as cytokines, type 1 and type 2 transmembrane proteins, and proteins found in intracellular organelles. In addition, a number of novel secreted proteins were identified, including a chemokine and a novel G-protein-coupled receptor. Since signal sequences possess features conserved throughout evolution, the procedure can be used to isolate genes encoding secreted proteins from both eukaryotes and prokaryotes.

Amino Acid Sequence↗
PubMed1996

Histidine patch thioredoxins. Mutant forms of thioredoxin with metal chelating affinity that provide for convenient purifications of thioredoxin fusion proteins.

A cluster of surface amino acid residues on Escherichia coli thioredoxin were systematically mutated in order to provide the molecule with an ability to chelate metal ions. The combined effect of two histidine mutants, E30H and Q62H, gave thioredoxin the capacity to bind to nickel ions immobilized on iminodiacetic acid- and nitrilotriacetic acid-Sepharose resins. Even though these two histidines were more than 30 residues apart in thioredoxin's primary sequence, they were found to satisfy the geometric constraints for metal ion coordination as a result of the thioredoxin tertiary fold. A third histidine mutation, S1H, provided additional metal ion chelation affinity, but the native histidine at position 6 of thioredoxin was found not to participate in binding. All of the histidine mutants exhibited decreased thermal stability as compared with wild-type thioredoxin; however, the introduction of an additional mutation, D26A, increased their melting temperatures beyond that of wild-type thioredoxin. The metal chelating abilities of these histidine mutants of thioredoxin were successfully utilized for convenient purifications of human interleukin-8 and -11 expressed in E. coli as soluble thioredoxin fusion proteins.

Amino Acid Sequence↗
PubMed1996

Molecular cloning and characterization of murine interleukin-11.

Human interleukin-11 (IL-11) has been shown to have pleiotropic action on hematopoietic, hepatic, stromal, epithelial, neural, and osteoclast cells. In the present work, the murine IL-11 cDNA has been isolated from a fetal thymic cell line, and its structure and function compared with human IL-11. The murine protein was demonstrated to have identical actions on the proliferation of a murine plasmacytoma cell line, murine primitive bone marrow progenitor cells, and megakaryocyte precursors. The murine IL-11 protein was synthesized as a soluble thioredoxin-IL-11 fusion in Escherichia coli and the expression of murine IL-11 was examined by pulse-chase radiolabeling in COS cells. The chromosomal location of the murine IL-11 gene was assigned to the proximal arm of chromosome 7.

Amino Acid Sequence↗
PubMed1995

Alanine-scanning mutagenesis of human interleukin-11: identification of regions important for biological activity.

We have identified functionally important regions of human interleukin-11 (hIL-11) by means of alanine-scanning mutagenesis. A total of 61 mutated forms of hIL-11 were produced in E. coli as thioredoxin fusion proteins and tested in a murine T10 plasmacytoma proliferation assay. Mutations made at several positions proximal to the hIL-11 C-terminus caused substantial reduction in biological activity. In addition a number of other mutations in this region affected either protein folding or stability. Both effects displayed a characteristic periodicity with respect to the primary sequence which suggested that residues close to the C-terminus of hIL-11 adopt a helical conformation. Mutations made proximal to the N-terminus of hIL-11 also exhibited reduced bioactivity, although no effects on protein folding or stability were observed. The N-terminal mutations with reduced activity also mapped with a periodicity suggestive of a helical conformation. We previously have proposed a four-helix bundle topology for the hIL-11 structure based on physical studies, selective chemical modifications, positions of intron/exon boundaries, limit proteolysis experiments and site-directed mutagenesis. The alanine-scanning mutagenesis data we report here provide additional support for this model.

Alanine↗
PubMed1995

Structure-function relationships in human interleukin-11. Identification of regions involved in activity by chemical modification and site-directed mutagenesis.

Chemical modification approaches combined with site-directed and deletion mutagenesis have been used to identify functionally critical regions/residues of recombinant human IL-11 (rhIL-11). Incubation of rhIL-11 with iodoacetic acid results in specific alkylation of a single methionine residue, Met58, and a 25-fold reduction of in vitro biological activity on mouse plasmacytoma cells. A similar decrease in activity is observed when Met58 is substituted with Ala, Leu, Gln, Glu, or Lys by site-directed mutagenesis. Treatment of rhIL-11 with succinic anhydride leads to modification of the amino-terminal amino group and partial labeling of 2 lysines, Lys41 and Lys98, and to a 3-fold decrease in activity. The activity losses can be attributed to modification of the lysine residues, since the succinyl derivative of the amino terminus is fully active. In addition, carboxyl-terminal deletion mutagenesis studies have demonstrated that removal of the last 4 residues reduces rhIL-11 activity 25-fold, whereas removal of 8 or more amino acids results in an inactive molecule. Based on secondary structure predictions and the location of exon/intron boundaries in the IL-11 genomic structure, we propose a four-helix bundle topology as a structural model for rhIL-11. This model has been tested by limited proteolysis using three side chain-specific endoproteinases. A limited number of protease-sensitive cleavage sites are present in rhIL-11, and all but two are located in the postulated helix interconnecting loops or at helix termini. alpha-Helices, which in the proposed structure form a compact core of the molecule, are inaccessible to digestion under limiting conditions. According to the model, Met58, Lys41 and Lys98 are located on the surface of the molecule, in agreement with their preferential accessibility to chemical modifications. By analogy with human growth hormone, we postulate that Met58 and the carboxyl terminus of rhIL-11 are involved in the primary receptor binding site (site I), whereas Lys41 and Lys98 may be a part of binding site II.

Amino Acid Sequence↗
PubMed1995

Improving patient quality of life with feedback to physicians about functional status.

OBJECTIVE: To improve functional status among primary care patients. INTERVENTION: 1) Computer-generated feedback to physicians about the patient's functional status, the patient's self-reported "chief complaint," and problem-specific resource and management suggestions; and 2) two brief interactive educational sessions for physicians. DESIGN: Randomized controlled trial. SETTING: University primary care clinic. PARTICIPANTS: All 73 internal medicine house officers and 557 of their new primary care patients. MEASURES: 1) Change in patient functional status from enrollment until six months later, using the Functional Status Questionnaire (FSQ); 2) management plans and additional information about functional status abstracted from the medical record; and 3) physician attitude about whether internists should address functional status problems. RESULTS: Emotional well-being scores improved significantly for the patients of the experimental group physicians compared with those of the control group physicians (p < 0.03). Limitations in social activities indicated as "due to health" decreased among the elderly (> or = 70 years of age) individuals in the experimental group compared with the control group (p < 0.03). The experimental group physicians diagnosed more symptoms of stress or anxiety than did the control group physicians (p < 0.001) and took more actions recommended by the feedback form (p < 0.02). CONCLUSIONS: Computer-generated feedback of functional status screening results accompanied by resource and management suggestions can increase physician diagnoses of impaired emotional well-being, can influence physician management of functional status problems, and can assist physicians in improving emotional well-being and social functioning among their patients.

Activities of Daily Living↗
PubMed1995

Gene fusion expression systems in Escherichia coli.

In recent years, Escherichia coli gene fusion expression systems have circumvented many of the problems inherent in the use of this bacterium for the production of recombinant proteins. These systems also provide a powerful means for identifying peptides or proteins with desired binding specificities. Gene fusion technology continues to expand with the introduction of new fusion partners, purification and detection tags, cleavage reagents and ways to display peptides on the surface of bacteria.

Cloning, Molecular↗