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D A Roth

Publications and source records attributed to D A Roth.

At least 55 records · Page 3Linked to original sources
PubMed1995

Mutagenesis of vitamin K-dependent carboxylase demonstrates a carboxyl terminus-mediated interaction with vitamin K hydroquinone.

The gamma-glutamyl carboxylase and vitamin K epoxidase activities of a series of mutants of bovine vitamin K-dependent carboxylase with progressively larger COOH-terminal deletions have been analyzed. The recombinant wild-type (residues 1-758) and mutant protein carboxylases, Cbx 711, Cbx 676, and Cbx 572, representing residues 1-711, 1-676, and 1-572, respectively, were expressed in baculovirus-infected Sf9 cells. Wild-type carboxylase had a Km for the substrate Phe-Leu-Glu-Glu-Leu (FLEEL) of 0.87 mM; the carboxylation of FLEEL was stimulated 2.5-fold by proPT18, the propeptide of prothrombin. Its Km for vitamin K hydroquinone was 23 microM and the specific epoxidase activity of the carboxylase was 938 pmol vitamin KO/30 min/pmol of carboxylase. Cbx 711, which was also stimulated by proPT18, had a Km for FLEEL, a Km for vitamin K hydroquinone, and a specific epoxidase activity that was comparable to the wild-type carboxylase. In contrast Cbx 572 lacked both carboxylase and epoxidase activities. Although Cbx 676 had a normal carboxylase active site in terms of the Km for FLEEL and its stimulation by proPT18, the Km for vitamin K hydroquinone was 540 microM, and the specific epoxidase activity was 97 pmol KO/30 min/pmol of Cbx 676. The catalytic efficiencies of Cbx 676 for glutamate carboxylation and vitamin K epoxidation were decreased 15- and 400-fold, respectively, from wild-type enzyme reflecting the requirement for formation of an activated vitamin K species for carboxylation to occur. These data indicate that the truncation of COOH-terminal segments of the carboxylase had no effect on FLEEL or propeptide recognition, but in the case of Cbx 676, selectively affected the interaction with vitamin K hydroquinone and the generation of epoxidase activity. These data suggest that a vitamin K epoxidase activity domain may reside near the COOH terminus while the carboxylase active site domain resides toward the NH2 terminus.

Amino Acid Sequence↗
PubMed1995

Localization of the factor IX propeptide binding site on recombinant vitamin K dependent carboxylase using benzoylphenylalanine photoaffinity peptide inactivators.

The propeptide binding/activation site on the vitamin K dependent carboxylase has been localized to a region of carboxylase between residues Arg +50 and Glu +225 by photoinactivation studies using [125I]tyrosyl-labeled benzoylphenylalanine (Bpa)-containing analogs of proFIX19, a peptide containing residues -18 to +1 of factor IX. Four proFIX19 analogs with Bpa substituents at -16, -13, -7, and -6 were synthesized. These peptides were specific photoinactivators of carboxylase and were used to label a His6-carboxylase construct produced in baculovirus-infected insect cells. Fragments of the labeled carboxylase produced by V8 protease digestion were analyzed by peptide-specific antibodies and by autoradiography. The propeptide recognition site was localized to the N-terminal one-third of the 94 kDa carboxylase. This is consistent with previous studies using a carboxylase substrate affinity label, N-(bromoacetyl)-FLEELY [Kuliopulos, A., Nelson, N.P., Yamada, M., Walsh, C.T., Furie, B., Furie, B.C., & Roth, D.A. (1994) J. Biol. Chem. 269, 21364-21370], indicating that the propeptide binding site and the FLEEL binding site are both located within the N-terminal one-third of the vitamin K dependent carboxylase.

Affinity Labels↗
PubMed1995

Adrenergic desensitization in left ventricle from streptozotocin diabetic swine.

Patients with diabetes mellitus that exhibit cardiac pump failure display compromised stroke volume, ejection fraction, and slower rates of rise and fall of left ventricular (LV) dP/dt in the absence of ischemic injury. We hypothesized that diabetic cardiomyopathy may involve decrements in adrenergic sensitivity, with specific molecular alterations in the beta-adrenergic receptor (beta AR)- G protein- adenylyl cyclase (AC) signal transduction system. We assessed the effects of 3 months of streptozotocin-induced diabetes (125 mg/kg i.v.; DIAB, n = 10) on myocardial signal transduction in mini-pigs. DIAB were hyperglycemic compared to controls (CON, n = 10; 20.92 +/- 2.64 v 5.24 +/- 0.35 mM glucose), and had lower fasting insulin levels (6.46 +/- 0.97 v 13.68 +/- 3.91 microU/ml). Transmural LV free wall homogenates from DIAB exhibited similar beta AR density as CON, but decreased cAMP production (pmol cAMP/mg prot.min) using these pharmacological stimulators: 10 microM Isoproterenol plus 100 microM GTP (74 +/- 5 v 97 +/- 11); 100 microM Gpp(NH)p (116 +/- 7 v 161 +/- 17); 10 mM fluoride ion (266 +/- 16 v 324 +/- 25). No differences between DIAB and CON were observed when stimulated by 100 microM forskolin (440 +/- 20 v 429 +/- 33), suggesting no alterations in the catalytic subunit of AC. In DIAB, quantitative immunoblotting indicated slightly depressed levels of Gs (552 +/- 44 v 630 +/- 59 pmol/g ww; NS), but a significant redistribution of alpha s from the sarcolemma to the cytosol (32.7 +/- 0.82% v 25.9 +/- 1.7%). Significantly elevated levels of cardiac Gi were seen in DIAB homogenates compared to CON ventricles (2326 +/- 145 v 1522 +/- 181 pmol/g ww), with no alpha i subunit redistribution. We conclude that despite maintained beta AR density, receptor-dependent and G protein-dependent stimulation of AC is depressed so that streptozotocin-induced diabetic LV is affected by increased cardiac Gi, redistribution of Gs alpha to the cytosol, and an increase in the Gi/Gs ratio. These results help explain depressed catecholamine responsiveness and cardiac performance exhibited by diabetic patients.

Adenylyl Cyclases↗
PubMed1995

Identification of a plant-encoded analog of PKR, the mammalian double-stranded RNA-dependent protein kinase.

Plant virus or viroid infection stimulates the phosphorylation of a plant-encoded protein of M(r) 68,000 to 70,000 (now termed pPKR) that is associated with double-stranded RNA-stimulated protein kinase activity. Using various biochemical and immunological comparisons, we have demonstrated that this plant protein is an analog of the mammalian PKR enzymes. pPKR is both cytosolic and ribosome associated, similar to mammalian PKR, and appears to be capable of phosphorylating exogenous histones. Monoclonal anti-serum to the human PKR as well as antiserum to a conserved double-stranded RNA-binding domain present on mammalian PKR demonstrated cross-reactivity with pPKR. Likewise, polyclonal antiserum to the pPKR detected the mouse and human PKR in western blot analysis. Northern blot analysis of a mammalian PKR cDNA detected a specific 2.5-kb transcript present in plant poly (A)+ RNA.

Animals↗
PubMed1995

Reduced beta-adrenergic receptor activation decreases G-protein expression and beta-adrenergic receptor kinase activity in porcine heart.

To determine whether beta-adrenergic receptor agonist activation influences guanosine 5'-triphosphate-binding protein (G-protein) expression and beta-adrenergic receptor kinase activity in the heart, we examined the effects of chronic beta 1-adrenergic receptor antagonist treatment (bisoprolol, 0.2 mg/kg per d i.v., 35 d) on components of the myocardial beta-adrenergic receptor-G-protein-adenylyl cyclase pathway in porcine myocardium. Three novel alterations in cardiac adrenergic signaling associated with chronic reduction in beta-adrenergic receptor agonist activation were found. First, there was coordinate downregulation of Gi alpha 2 and Gs alpha mRNA and protein expression in the left ventricle; reduced G-protein content was also found in the right atrium. Second, in the left ventricle, there was a twofold increase in beta-adrenergic receptor-dependent stimulation of adenylyl cyclase and a persistent high affinity state of the beta-adrenergic receptor. Finally, there was a reduction in left ventricular beta-adrenergic receptor kinase activity, suggesting a previously unrecognized association between the degree of adrenergic activation and myocardial beta-adrenergic receptor kinase expression. The heart appears to adapt in response to chronic beta-adrenergic receptor antagonist administration in a manner that would be expected to offset reduced agonist stimulation. The mechanisms for achieving this extend beyond beta-adrenergic receptor upregulation and include alterations in G-protein expression, beta-adrenergic receptor-Gs interaction, and myocardial beta-adrenergic receptor kinase activity.

Adaptation, Biological↗
PubMed1994

Localization of the affinity peptide-substrate inactivator site on recombinant vitamin K-dependent carboxylase.

A recombinant His6-tagged vitamin K-dependent gamma-glutamyl carboxylase has been produced in baculovirus-infected insect cells. The His6-carboxylase shares nearly identical kinetic properties with the wild-type enzyme from bovine liver microsomes. The His6-carboxylase was irreversibly inactivated by the N-bromoacetyl-FLEEL-125I-Y peptide substrate/affinity label under pseudo-first order conditions. This inactivation could be abolished by coincubation with a high affinity peptide substrate consistent with an active site-directed inactivation. The inactivated His6-carboxylase-Ac-FLEEL-125I-Y, purified under denaturing conditions by Ni-chelation chromatography followed by preparative polyacrylamide gel electrophoresis, was subjected to proteolytic digestions with either Glu-C or Lys-C endoproteinases. The resulting polypeptide fragments were probed with three regiospecific antibodies which recognized epitopes present at the extreme N terminus (residues -23 to -13), at the hydrophobic N-terminal region (residues 86-99), and at the hydrophilic C-terminal region (residues 661-673). The site of attachment to the 125I-affinity label is located within the first 218 amino acid residues of the 758-residue carboxylase. This is the first evidence for the involvement of either the putative membrane-anchoring hydrophobic region (residues 50-314) or possibly the N-terminal hydrophilic region (residues 1-50) in gamma-carboxylation of glutamate-peptide substrates.

Amino Acid Sequence↗
PubMed1994

Influence of aging and endurance training on lactate dehydrogenase in liver and skeletal muscle.

The purpose of this investigation was to determine the effects of aging and endurance training on lactate dehydrogenase (LDH) activity and isozyme pattern in liver and skeletal muscle. Male Fischer 344 rats (n = 30) of three different age groups (young, 4 months; middle-aged, 12 months and old, 22 months) were trained on a treadmill at 75% running capacity for 1 h/day, five times per week for 10 weeks. Age-matched sedentary controls (n = 36) were used for comparison. Total LDH enzyme activity was measured spectrophotometrically; LDH isozymes were separated by native 5.5% polyacrylamide gel electrophoresis and quantified densitometrically. With increasing age, hepatic LDH activity decreased 28%. Old sedentary animals displayed significantly less (22%) hepatic LDH 5 than young and middle-aged animals, and significantly more (40%) hepatic LDH 4 than middle-aged animals. Training resulted in a significant decrease (38%) in total hepatic LDH activity in young rats only. Young animals displayed a significant increase in hepatic LDH 3 (28%), whereas middle-aged animals exhibited a significant decrease in hepatic LDH 3 (40%) with training. No change in total hepatic LDH activity was exhibited in middle-aged or old rats with training. Neither aging or training had a significant effect on LDH activity or isozyme pattern in extensor digitorum longus (EDL). Similarly, LDH activity was maintained in soleus with age, and isozyme pattern was only negligibly affected. We conclude that with age there is a decline in hepatic LDH activity and a decrease in the LDH 5 isozyme. Endurance training induced significant decreases in hepatic LDH activity of young animals. However, these decreases were not a result of shifts in isozymal pattern. Further, LDH activity was maintained in EDL and soleus muscle with age. Finally, endurance training did not have a significant effect on LDH activity or isozymal pattern of EDL or soleus.

Adaptation, Physiological↗
PubMed1993

Expression of bovine vitamin K-dependent carboxylase activity in baculovirus-infected insect cells.

A vitamin K-dependent carboxylase has recently been purified from bovine liver microsomes and candidate cDNA clones have been isolated. Definitive identification of the carboxylase remains circumstantial since expression of candidate carboxylase cDNAs in mammalian cells is confounded by the presence of endogenous carboxylase activity. To overcome this problem, a recombinant strain of baculovirus (Autographa california nuclear polyhedrosis virus, AcMNPV) encoding a putative carboxylase (vbCbx/AcMNPV) was used to infect Sf9 insect cells, which we demonstrate have no endogenous carboxylase activity. Infection with vbCbx/AcMNPV conferred vitamin K-dependent carboxylase activity to Sf9 insect cells. Carboxylase activity was demonstrated to peak 2-3 days after infection with vbCbx/AcMNPV. Metabolic radiolabeling with L-[35S]methionine revealed that the 90-kDa recombinant protein is the major protein synthesized at the time of peak activity after infection. An anti-peptide antibody directed against residues 86-99 reacted with bovine liver carboxylase on Western blot analysis and immunoprecipitated recombinant carboxylase from infected Sf9 microsomal protein preparations. Since Sf9 insect cells lack endogenous vitamin K-dependent carboxylase activity, expression of carboxylase activity in Sf9 insect cells with recombinant baculovirus demonstrates that the protein encoded by this cDNA is a vitamin K-dependent gamma-glutamyl carboxylase.

Animals↗
PubMed1993

Tobacco mosaic virus infection of transgenic Nicotiana tabacum plants is inhibited by antisense constructs directed at the 5' region of viral RNA.

Antisense (AS) versions of two 51-nucleotide (nt) sequences near the 5' end of tobacco mosaic virus (TMV) RNA have been shown to inhibit in vitro translation of the adjacent gene that encodes both the 126- and 183-kDa proteins. These DNA fragments have been cloned into the binary vector, pMON530, such that either the nopaline synthase (Nos) promoter or cauliflower mosaic virus (CaMV) 35S RNA promoter is used to drive synthesis of the corresponding sense and AS RNAs. Transgenic Nicotiana tabacum cv. Xanthi nn plants containing these constructs were challenged with TMV. Plants expressing the AS orientation of a 51-nt TMV leader sequence, under the control of the CaMV 35S promoter, were found to be resistant to infection when inoculated with up to 100 times the concentration of TMV which produced severe infections in control plants. Systemic accumulation of TMV RNA and progeny virus was diminished 15 to 30-fold in these plants. Accumulation of the viral coat protein was diminished 6 to 7-fold implying a selective inhibition of TMV replication.

Base Sequence↗
PubMed1993

In vitro and in vivo functional characterization of bovine vitamin K-dependent gamma-carboxylase expressed in Chinese hamster ovary cells.

Coagulation factor IX is a serine protease for which high-level expression of biologically active protein in heterologous cells is limited due to inefficient proteolytic removal of the propeptide as well as vitamin K-dependent carboxylation of multiple amino-terminal glutamic acid residues. We have overexpressed the vitamin K-dependent gamma-carboxylase cDNA and monitored its ability to improve factor IX processing in Chinese hamster ovary (CHO) cells. From amino acid sequence analysis of bovine liver vitamin K-dependent gamma-carboxylase, degenerate oligonucleotides were used to isolate a 3.5-kbp bovine cDNA that encoded a 758-residue open reading frame. Expression of the cDNA in COS-1 and CHO cells yielded 17- and 16-fold increases in the in vitro gamma-carboxylase activity of microsomal preparations, respectively. Anti-serum raised against a predicted peptide sequence reacted with a 94-kDa polypeptide in the partially purified bovine liver preparation as well as in stably transfected CHO cells. The amount of antibody reactivity correlated with the increased ability to carboxylate a peptide substrate in vitro. These results strongly support the conclusion that the cDNA encodes the vitamin K-dependent gamma-carboxylase. Transient transfection of the gamma-carboxylase expression vector into factor IX-expressing CHO cells did not improve the specific procoagulant activity of secreted factor IX. In contrast, transfection of an expression vector encoding the propeptide processing enzyme PACE (paired basic amino acid cleaving enzyme) did improve the specific activity of secreted factor IX by 3-fold. These results demonstrate that the ability of CHO cells to modify glutamic acid residues to gamma-carboxyglutamic acid in secreted factor IX is not limited by the expression of the vitamin K-dependent gamma-carboxylase alone.

Amino Acid Sequence↗
PubMed1993

Training does not affect zero-trans lactate transport across mixed rat skeletal muscle sarcolemmal vesicles.

Hindlimb muscle sarcolemmal vesicles were purified from three age-matched groups of female Sprague-Dawley rats: sedentary control (CON; n = 10), sprint trained (ST; n = 8), and endurance trained (ET; n = 9). Membrane isolations from the three groups were not significantly different in protein yield or purification index. Blood lactate concentration was determined in resting CON rats and running ET and ST rats during the final week. Both the ST and ET groups were significantly higher in citrate synthase (vs. CON) in the soleus and mid-vastus lateralis. The time course of 1 mM L-(+)-lactate uptake in vesicles from the three groups showed no significant difference at any of the five time points tested under zero-trans conditions. Saturation kinetics were examined at nine lactate concentrations, and Lineweaver-Burk plots revealed no difference between groups in apparent Michaelis-Menten constant or maximal transport velocity. Vesicles from CON and ET rats were used to investigate cis inhibition of 0.1 mM L-(+)-lactate transport by four unlabeled monocarboxylates: L-(+)-lactate, D-(-)-lactate, pyruvate, and alpha-cyanohydroxycinnamate at 0.1, 1.0, and 10 mM. Under pH gradient-stimulated L-(+)-lactate transport conditions, cis inhibition was affected by neither D-(-)-lactate nor endurance training. We conclude that the lactate transporter has distinct cis-inhibitory specificity, is stereospecific, and is stimulated when confronted with parallel lactate and proton gradients but that spring and endurance training do not alter lactate transport rate or capacity under these conditions.

Acidosis, Lactic↗
PubMed1993

Downregulation of cardiac guanosine 5'-triphosphate-binding proteins in right atrium and left ventricle in pacing-induced congestive heart failure.

The extent to which congestive heart failure (CHF) is dependent upon increased levels of the cardiac inhibitory GTP-binding protein (Gi), and the impact of CHF on the cardiac stimulatory GTP-binding protein (Gs) and mechanisms by which Gs may change remain unexplored. We have addressed these unsettled issues using pacing-induced CHF in pigs to examine physiological, biochemical, and molecular features of the right atrium (RA) and left ventricle (LV). CHF was associated with an 85 +/- 20% decrease in LV segment shortening (P < 0.001) and a 3.5-fold increase (P = 0.006) in the ED50 for isoproterenol-stimulated heart rate responsiveness. Myocardial beta-adrenergic receptor number was decreased 54% in RA (P = 0.004) and 57% in LV (P < 0.001), and multiple measures of adenylyl cyclase activity were depressed 49 +/- 8% in RA (P < 0.005), and 44 +/- 9% in LV (P < 0.001). Quantitative immunoblotting established that Gi and Gs were decreased in RA (Gi: 59% reduction; P < 0.0001; Gs: 28% reduction; P < 0.007) and LV (Gi: 35% reduction; P < 0.008; Gs: 28% reduction; P < 0.01) after onset of CHF. Reduced levels of Gi and Gs were confirmed by ADP ribosylation studies, and diminished function of Gs was established in reconstitution studies. Steady state levels for Gs alpha mRNA were increased in RA and unchanged in LV, and significantly more GS alpha was found in the supernatant (presumably cytosolic) fraction in RA and LV membrane homogenates after CHF, suggesting that increased Gs degradation, rather than decreased Gs synthesis, is the mechanism by which Gs is downregulated. We conclude that cardiac Gi content poorly predicts adrenergic responsiveness or contractile function, that decreased Gs is caused by increased degradation rather than decreased synthesis, and that alterations in beta-adrenergic receptors, adenylyl cyclase, and GTP-binding proteins are uniform in RA and LV in this model of congestive heart failure.

Adenylyl Cyclases↗
PubMed1993

Regional myocardial downregulation of the inhibitory guanosine triphosphate-binding protein (Gi alpha 2) and beta-adrenergic receptors in a porcine model of chronic episodic myocardial ischemia.

Regional myocardial ischemia is associated with increased levels of adenosine and norepinephrine, factors that may alter activation of the beta-adrenergic receptor (beta AR)-G protein-adenylyl cyclase pathway in the heart. We have used the ameroid constrictor model to determine whether alterations in myocardial signal transduction through the beta AR-G protein-adenylyl cyclase pathway occur in the setting of chronic episodes of reversible ischemia. Pigs were instrumented with ameroid occluders placed around the left circumflex coronary artery. 5 wk later, after ameroid closure, flow and function were normal in the ischemic bed, but flow (P = 0.001) and function (P < 0.03) were abnormal when metabolic demands were increased. The ischemic bed showed a reduction in myocardial beta AR number (P < 0.005). Despite regional downregulation of myocardial beta AR number, adenylyl cyclase activity was similar in the ischemic and control beds. Quantitative immunoblotting showed that the cardiac inhibitory GTP-binding protein, Gi alpha 2, was decreased in the ischemic bed (P = 0.02). In contrast, the cardiac stimulatory GTP-binding protein, Gs alpha, was increased in endocardial sections from the ischemic bed (P = < 0.05). Decreased Gi alpha 2 content was associated with decreased inhibition of adenylyl cyclase. Reduced Gi alpha 2 content, in conjunction with increased Gs alpha content in the endocardium, may provide a means by which adrenergic activation is maintained in the setting of chronic episodic myocardial ischemia.

Adenylyl Cyclases↗
PubMed1992

A substantial proportion of cardiac Gs is not associated with the plasma membrane.

The precise interactions between the subunits of Gs (alpha s, beta, gamma) and the plasma membrane remain to be established. If alpha s is associated loosely with the inner membrane, is labile during activation, or is always present to some extent in the cytoplasm, then it should fractionate to the supernatant of a high-speed centrifugation. We identified abundant alpha s (52-66% of total cellular) in the supernatant fraction of right atrial and left ventricular membrane preparations of porcine heart as shown by two distinct measures of alpha s (immunoblotting and ADP ribosylation by cholera toxin). However, functional assays utilizing reconstitution of cardiac alpha s with cyc- S49 membranes revealed that the supernatant fraction contained approximately 16% of total cellular alpha s activity. The alpha s present in the supernatant fraction did not result from contamination by sarcolemmal membrane fragments. We conclude that traditional methods for quantifying alpha s which utilize only detergent extracts from high-speed pellets do not account for a sizable proportion of total cellular alpha s, but that the majority of this population of cardiac alpha s may not be functional, at least with respect to adenylyl cyclase activation.

Adenosine Diphosphate Ribose↗
PubMed1992

Computer-generated bone models in the planning of osteotomy of multidirectional distal radius malunions.

Computer-assisted design and manufacturing technology has been used to create solid models of five unusually complex, multidirectional malunions of distal radius fractures. Preoperative planning was dramatically enhanced by the ability to perform the surgical procedure on these models, with a model of the uninjured limb used for comparison. All five patients had significant malunions, with malrotation in the horizontal plane in five and an impacted articular fragment in two. A satisfactory outcome was achieved in each case.

Adult↗