John Dignam

John Dignam
University of Toledo · Department of Biochemistry and Cancer Biology

About

52
Publications
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Publications

Publications (52)
Article
Full-text available
Bacteriophage T4 gp59 helicase assembly protein (gp59) is required for loading of gp41 replicative helicase onto DNA protected by gp32 single-stranded DNA-binding protein. The gp59 protein recognizes branched DNA structures found at replication and recombination sites. Binding of gp32 protein (full-length and deletion constructs) to gp59 protein me...
Article
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Febrifugine, the bioactive constituent of one of the 50 fundamental herbs of traditional Chinese medicine, has been characterized for its therapeutic activity, though its molecular target has remained unknown. Febrifugine derivatives have been used to treat malaria, cancer, fibrosis and inflammatory disease. We recently demonstrated that halofugino...
Article
Alanyl-tRNA synthetase, a dimeric class 2 aminoacyl-tRNA synthetase, activates glycine and serine at significant rates. An editing activity hydrolyzes Gly-tRNA(ala) and Ser-tRNA(ala) to ensure fidelity of aminoacylation. Analytical ultracentrifugation demonstrates that the enzyme is predominately a dimer in solution. ATP binding to full length enzy...
Article
The Raf-MEK-ERK pathway regulates many fundamental biological processes, and its activity is finely tuned at multiple levels. The Raf kinase inhibitory protein (RKIP) is a widely expressed negative modulator of the Raf-MEK-ERK signaling pathway. We have previously shown that RKIP inhibits the phosphorylation of MEK by Raf-1 through interfering with...
Article
Full-text available
Human platelet 12-lipoxygenase (hp-12LOX, 662 residues+iron nonheme cofactor) and its major metabolite 12S-hydroxyeicosatetraenoic acid have been implicated in cardiovascular and renal diseases, many types of cancer and inflammatory responses. However, drug development is slow due to a lack of structural information. The major hurdle in obtaining a...
Article
Full-text available
Capillary leak accompanying systemic inflammatory response conditions is a significant clinical problem. In the present study, we describe and verify a method for studying capillary leak that is based on the injection of proteins that differ significantly in size and have spectrally distinguishable fluorophores. Control (n=11) and post-CLP (caecal...
Article
The interaction of daunomycin with sodium dodecyl sulfate and Triton X-100 micelles was investigated as a model for the hydrophobic contribution to the free energy of DNA intercalation reactions. Measurements of visible absorbance, fluorescence lifetime, steady-state fluorescence emission intensity, and fluorescence anisotropy indicate that the ant...
Article
Rep68 and Rep78 DNA helicases, encoded by adeno-associated virus 2 (AAV2), are required for replication of AAV viral DNA in infected cells. They bind to imperfect palindromic elements in the inverted terminal repeat structures at the 3'- and 5'-ends of virion DNA. The ATPase activity of Rep68 and Rep78 is stimulated up to 10-fold by DNA containing...
Article
Adeno-associated virus 2 Rep40 helicase is involved in packaging single-stranded genomic DNA into virions. ATPase activity was stimulated 5-10-fold by DNA, depending upon assay conditions. The concentration dependence of Rep40 ATPase activity in the absence and presence of DNA indicates that the monomer is inactive and that the active enzyme is at...
Article
Full-text available
Adeno-associated virus (AAV) is a human parvovirus that normally requires a helper virus such as adenovirus (Ad) for replication. The four AAV replication proteins (Rep78, Rep68, Rep52, and Rep40) are pleiotropic effectors of virus integration, replication, transcription, and virion assembly. These proteins exert effects on Ad gene expression and r...
Article
The defective parvovirus, adeno-associated virus (AAV), is under close scrutiny as a human gene therapy vector. AAV's non-pathogenic character, reliance on helper virus co-infection for replication and wide tissue tropism, make it an appealing vector system. The virus' simplicity and ability to generate high titer vector preparations have contribut...
Article
Full-text available
Adeno-associated virus (AAV) and other parvoviruses inhibit proliferation of nonpermissive cells. The mechanism of this inhibition is not thoroughly understood. To learn how AAV interacts with host cells, we investigated AAV's interaction with adenovirus (Ad), AAV's most efficient helper virus. Coinfection with Ad and AAV results in an AAV-mediated...
Article
Aminoacyl tRNA synthetases (ARS) catalyze the ligation of amino acids to cognate tRNAs. Chordate ARSs have evolved distinctive features absent from ancestral forms, including compartmentalization in a multisynthetase complex (MSC), noncatalytic peptide appendages, and ancillary functions unrelated to aminoacylation. Here, we show that glutamyl-prol...
Article
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Systemic inflammatory response conditions are associated with capillary leak and haemodynamic compromise. Fluid resuscitation to reverse the ensuing hypovolaemia is, however, complicated by the decreased endothelium reflection coefficient to albumin and other colloids. We developed PEG-Alb (albumin covalently linked to polyethylene glycol) as a pot...
Article
Full-text available
The human adeno-associated virus (AAV) has generated much enthusiasm as a transfer vector for human gene therapy. Although clinical gene therapy trials have been initiated using AAV vectors, much remains to be learned regarding the basic mechanisms of virus replication, gene expression, and virion assembly. AAV encodes four nonstructural, or replic...
Article
The interaction of adenine nucleotides with glycyl-tRNA synthetase was examined by several experimental approaches. ATP and nonsubstrate ATP analogues render glycyl-tRNA synthetase more resistant to digestion by a number of proteases (thrombin, Arg-C, and chymotrypsin) at concentrations that correlate with their Michaelis constants or inhibition co...
Article
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Unfolding of Bombyx mori glycyl-tRNA synthetase was examined by multiple spectroscopic techniques. Tryptophan fluorescence of wild type enzyme and an N-terminally truncated form (N55) increased at low concentrations of urea or guanidine-HCl followed by a reduction in intensity at intermediate denaturant concentrations; a transition at higher denatu...
Article
Aminoacyl-tRNA synthetases are the family of enzymes responsible for correctly linking tRNA molecules with amino acids so that the latter may be subsequently incorporated into growing polypeptide chains. A unique feature of aminoacyl-tRNA synthetases from higher eukaryotes is the existence of a high molecular mass multisynthetase complex. This appr...
Article
An oligodeoxynucleotide (ODN) that includes elements found in secondary structures at the 5′- and 3′- ends of adenoassociated virus 2 virion DNA was synthesized by ligation of three overlapping ODNs. The most stable secondary structure was calculated to be branched, with a 61 bp duplex stem, terminating in a three-way junction with 9 bp arms. The e...
Article
Full-text available
A high molecular mass complex of aminoacyl-tRNA synthetases is readily isolated from a variety of eukaryotes. Although its composition is well characterized, knowledge of its structure and organization is still quite limited. This study uses antibodies directed against prolyl-tRNA synthetase for immunoelectron microscopic localization of the bifunc...
Article
Myosin binding protein C (MyBP-C) is a major myofibril-associated protein in cardiac muscle which is subject to reversible phosphorylation. Cardiac MyBP-C is a substrate in vivo and in vitro for cAMP-dependent protein kinase (PKA) and calcium/phospholipid-dependent protein kinase (PKC). Chicken cardiac MyBP-C was phosphorylated by PKA to 3.0 mol ph...
Article
Transforming growth factor beta1 (TGF-beta1) is a potent growth differentiation and morphogenesis factor. The amino-terminal 248 amino acid pro region of TGF-beta1, the beta1-latency-associated peptide (beta1-LAP), is noncovalently associated with TGF-beta1 in an inactive complex. Previous studies suggested that deglycosylated beta1-LAP can not for...
Article
Full-text available
The formation of a non-covalent complex between mature transforming growth factor beta 1 (TGF-beta 1) and its pro region, the beta 1-latency-associated peptide (beta 1-LAP), is important in regulating the activity of this multipotent growth factor. We have overexpressed simian beta 1-LAP in Chinese hamster ovary (CHO) cells to produce a cell line w...
Article
Bombyx mori glycyl-tRNA synthetase (GRS) was expressed as the full length protein and as N-terminally and C-terminally truncated forms. The intact enzyme and forms with deletions of 12, 27, 46, and 55 N-terminal residues were expressed, purified, and characterized. All were active, having 15-25% of both pyrophosphate exchange activity and aminoacyl...
Article
Bombyx mori glycyl-tRNA synthetase (GRS) was expressed as the full length protein and as N-terminally and C-terminally truncated forms. The intact enzyme and forms with deletions of 12, 27, 46, and 55 N-terminal residues were expressed, purified, and characterized. All were active, having 1525% of both pyrophosphate exchange activity and aminoacyl-...
Article
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Article
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Treatment of rats with isoproterenol resulted in elevated levels of prolyl- and glutamyl-tRNA synthetase activities in the parotid and submandibular glands. This increase in enzyme activity was accompanied by an increase in the bi-functional glutamyl-prolyl-tRNA synthetase and of a low molecular weight form of prolyl-tRNA synthetase. Isoproterenol...
Article
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The primary structure of the gene encoding Bombyx mori glycyl-tRNA synthetase was determined by sequence analysis of one cDNA and two genomic clones. The sequence of the protein deduced from the nucleotide sequence was verified by sequence analysis of eight peptides. The M(r) 77,667 protein is encoded in a single open reading frame of 2061 nucleoti...
Data
On Feb 15, 1995 this sequence version replaced gi:156001.
Article
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Rat liver prolyl-tRNA synthetase was purified as a dimer of M(r) 60,000 subunits not associated with other aminoacyl-tRNA synthetases and as a form associated with glutamyl-tRNA synthetase. Proteolysis of the dimeric enzyme generated a less active form with M(r) 52,000 subunits and an inactive form with M(r) 40,000 subunits. A second species was is...
Article
Alanyl-tRNA synthetase from Escherichia coli, Bombyx mori and rat were examined with respect to the following functional and structural properties: the effect of substrates on sensitivity to proteolysis, secondary structure as determined by circular dichroism, amino acid composition and, in the case of the rat and insect enzymes, partial amino acid...
Article
Full-text available
cDNA clones encoding Bombyx mori alanyl-tRNA synthetase were isolated from a library in lambda gt11 using antibody, synthetic oligonucleotides, and a characterized cDNA as probes. Analysis of the sequence revealed significant homology between the B. mori and Escherichia coli alanyl-tRNA synthetases, particularly in their amino-terminal domains. Nor...
Article
Successful preparation of crude extracts from eukaryotic cells and tissues for enzyme purification requires close attention to conditions that may alter the activity or native structure of an enzyme because nonspecific inactivation can result in irreproducibility that may make interpretation of such studies difficult. This chapter describes some ge...
Article
We have examined the levels of glycyl-, alanyl-, and seryl-tRNA synthetases and the levels of their corresponding translatable mRNAs in the posterior and middle silkglands of the silkworm, Bombyx mori. Analysis of Western blots reveals that the change in the abundance of these enzymes during the fifth instar in crude extracts derived from posterior...
Article
Full-text available
Seryl-tRNA synthetase has been purified from the middle silk glands of Bombyx mori by successive chromatography on DEAE-Sephacel, hydroxylapatite, and Bio-Rex 70. The high abundance of seryl-tRNA synthetase in the middle silk glands may result from an adaptation of this organ for the production of the serine-rich protein, sericin. The enzyme is a d...
Article
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We report here the identification of a common immunological determinant in Escherichia coli and Bombyx mori (silkworm) alanine tRNA synthetases. The E. coli protein is a tetramer of identical Mr = 95,000 chains, and the silkworm enzyme is a monomer of Mr = 115,000. Antibodies against the silkworm enzyme react with E. coli Ala-tRNA synthetase. Analy...
Article
Full-text available
Glycyl- and alanyl-tRNA synthetases have been purified from an extract of Bombyx mori posterior silk glands by a procedure that allows for the simultaneous isolation of both enzymes. Glycyl-tRNA synthetase is a dimer of Mr = 160,000 consisting of similar or identical subunits, whereas alanyl-tRNA synthetase is a monomer of Mr = 110,000 to 115,000....
Article
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We have developed a procedure for preparing extracts from nuclei of human tissue culture cells that directs accurate transcription initiation in vitro from class II promoters. Conditions of extraction and assay have been optimized for maximum activity using the major late promoter of adenovirus 2. The extract also directs accurate transcription ini...
Article
Threonyl-tRNA synthetase was purified approximately 500-fold from a high-speed supernatant fraction of rat liver. The purified enzyme was estimated to be > 95% pure from acrylamide gel electrophoresis under denaturing and nondenaturing conditions. Based on a native molecular weight from sedimentation equilibrium of 154000 and a subunit molecular we...
Article
A protein was purified from rat liver which stimulated a number of liver aminoacyl-tRNA synthetases. This stimulatory factor was identical with the "tRNA activator" of Dickman & Boll [(1976) Biochemistry 15, 3925] in its mechanism of action and chemical properties, although it was considerably more purified. The two preparations stimulated syntheta...
Article
Transplantable rat liver tumors 5123 t.c., 7288 ct.c., 5123 t.c.(H) and the Novikoff hepatoma have active mixed function oxidase systems capable of metabolizing a variety of drug and polycyclic hydrocarbon substrates. The tumor drug metabolism systems are at best 20% as active as rat liver. The tumor drug metabolism activities are induced by pretre...
Article
Publisher Summary This chapter provides information on the purification and properties of NADPH-cytochrome P-450 reductase. NADPH—cytochrome P-450 reductase, a flavoprotein component of the endoplasmic reticulum of liver and other organs, catalyzes the transfer of electrons from NADPH to cytochrome P-450. Cytochrome P-450 is the terminal oxidase of...
Article
(NADPH)-cytochrome P-450 reductase was purified to apparent homogeneity by a procedure utilizing nicotinamide adenine dinucleotide phosphate (NADP)-Sepharose affinity column chromatography. The purified flavoprotein has a molecular weight of 79 700 and catalyzes cytochrome P-450 dependent drug metabolism, as well as reduction of exogenous electron...
Article
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NADPH-cytochrome P-450 reductase was purified from hepatic microsomes of phenobarbital and hydrocortisone-treated rats by detergent solubilization and column chromatography. This membrane protein contains 31 mol per cent hydrophobic amino acid residues, 6 half-cystine residues, and a single tryptophan residue as determined by amino acid analysis af...
Article
NADPH-cytochrome P-450 reductase with capacity to support cytochrome P-450-dependent drug metabolism and to reduce artificial electron acceptors has been purified to apparent homogeneity by solubilization with Renex 690 and chromatography on DEAE-Sephadex, Agarose and QAE-Sephadex. The purified protein migrates as a single band on native and SDS-po...

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