Activation of cholera toxin by ADP-ribosylation factors.
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ADP-Ribosylation Factors↗
Biomedical subjects
J Moss
Publications and source records attributed to J Moss.
Activation of cholera toxin by ADP-ribosylation factors: 20-kDa guanine nucleotide-binding proteins.
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ADP-Ribosylation Factors↗
Volatile anesthetics decrease peristalsis in the guinea pig ureter.
BACKGROUND: The origin of renal dysfunction associated with anesthesia and surgery is complex and incompletely understood. The effects of the volatile anesthetic agents isoflurane, enflurane, and halothane on the renal pacemaker and ureteral peristalsis may play an important role. METHODS: Guinea pig ureter and pelvis were dissected and placed in a sample chamber that allowed immersion in a temperature-controlled bath with gas (O2 with or without volatile agent) bubbled through the chamber continuously. The baseline frequency and amplitude of peristaltic contractions were measured on a polygraph recorder. The preparations were then exposed to up to 4 vol% volatile agent incrementally to generate a cumulative dose-response curve, and the subsequent frequency of peristaltic contractions was determined. The concentration of the volatile agent in solution was measured by gas chromatography. RESULTS: There was a significant dose-related decrease in the frequency of ureteral contractions for all three agents. A statistical model relating percentage baseline frequency to millimolar concentrations of volatile anesthetics showed that halothane produced a more pronounced decrease in frequency than did isoflurane or enflurane. However, the decrease was directly related to the MAC multiples and did not differ for the three agents. CONCLUSIONS: Ureteral peristaltic contractions are decreased in a dose-dependent manner by enflurane, halothane and isoflurane.
Animals↗
AA glomerular amyloid. An ultrastructural immunogold study of the colocalization of heparan sulphate proteoglycan and P component with amyloid fibrils together with changes in distribution of type IV collagen and fibronectin.
An ultrastructural investigation was undertaken on paraformaldehyde-fixed Lowicryl resin-embedded human kidneys of three patients with AA amyloidosis to investigate the association of various basement membrane components with amyloid fibrils. An immunogold technique was used and antibodies to serum amyloid A, heparan sulphate proteoglycan, type IV collagen, P component, and fibronectin were applied to human normal and amyloid glomeruli. The amyloid was identified as AA, and P component was shown to be intimately associated with the fibrils. In addition, heparan sulphate proteoglycan was associated with amyloid in all subendothelial, subepithelial and intramembranous glomerular basement membrane deposits, and those throughout the mesangial matrix. This contrasted with the distribution of the proteoglycan in the normal glomerulus where it was found predominantly on the epithelial aspect of the basement membrane and only in the more peripheral regions of the mesangium. The accumulation of heparan sulphate proteoglycan with amyloid resulted in a marked increase in its amount in the glomeruli. The amyloid deposits contained little or no type IV collagen or fibronectin. These findings demonstrate a strong association of heparan sulphate proteoglycan with amyloid and suggest different roles for the various glomerular basement membrane components in amyloidogenesis.
Amyloidosis↗
Construction and characterization of recombinant Vibrio cholerae strains producing inactive cholera toxin analogs.
The catalytic A subunit of cholera toxin (CT-A) is capable of ADP-ribosylating the guanine nucleotide-binding protein, which regulates cell adenylyl cyclase, leading to the life-threatening diarrhea of cholera. Amino acids involved in the enzymatic activity of CT-A have previously been identified. By means of site-directed mutagenesis, an analog of the CT-A subunit gene was created with codon substitutions for both Arg-7 and Glu-112, each of which has been shown to produce subunits lacking ADP-ribosyltransferase activity. The mutated gene fragment was exchanged for the wild-type copy in the previously cloned ctxAB operon from El Tor biotype, Ogawa serotype Vibrio cholerae strain 3083, which produces CT-2. Further, the zonula occludens toxin gene, zot, was inactivated by an insertional mutation to create the new plasmid construct pCT-2*. Additionally, a DNA fragment encoding the B subunit of CT-1 (CT produced by classical biotype, Inaba serotype V. cholerae strain 569B) was exchanged for the homologous part in pCT-2*, resulting in the creation of pCT-1*. These plasmid constructs were introduced into the CT-negative V. cholerae mutant strain JBK70 (E1 Tor biotype, Inaba serotype); CT-A-B+ derivatives CVD101 and CVD103 of classical biotype Ogawa and Inaba serotype strains 395 and 569B, respectively; El Tor biotype Inaba and Ogawa serotype strains C6706 and C7258, respectively, recently isolated in Peru; and O139 (synonym Bengal) strain SG25-1 from the current epidemic in India. Recombinant toxins (CT-1* and CT-2*), partially purified from culture supernatants of transformed JBK70, were shown to be inactive on mouse Y1 adrenal tumor cells and in an in vitro ADP-ribosyltransferase assay. CT-1* and CT-2* reacted with polyclonal and monoclonal antibodies against both A and B subunits of CT. The toxin analogs reacted with antibodies against CT-A and CT-B on cellulose acetate strips and in a GM1 enzyme-linked immunosorbent assay; they reacted appropriately with B-subunit epitype-specific monoclonal antibodies in checkerboard immunoblots, and they formed precipitin bands with GM1-ganglioside in Ouchterlony tests. However, the reactions of the modified proteins with anti-A-subunit monoclonal antibodies were weaker than the reactions with wild-type holotoxins. V, cholerae strains carrying ctxA*, with either ctxB-1 or ctxB-2, and inactivated zot genes were created by homologous recombination. The recombinant strains and the purified toxin analogs were inactive in the infant rabbit animal model.(ABSTRACT TRUNCATED AT 400 WORDS)
Animals↗
Is thickening of the basal lamina in the saphenous vein a hallmark of smoking?
OBJECTIVE: To investigate whether smoking causes ultrastructural changes in the intima of the proximal saphenous vein. DESIGN: Proximal saphenous veins from heavy smokers and non-smokers were examined with scanning and transmission electron microscopy to determine changes in surface ultrastructure, in the intercellular junction, and in the thickness of the basal lamina. Immunogold labelling was used to identify specific components of the endothelial basal lamina. MATERIAL: Vein specimens were obtained from patients undergoing varicose vein surgery (12 patients) or distal bypass surgery (eight patients). MAIN RESULTS: The only ultrastructural change that discriminated between specimens was thickening of the endothelial basal lamina. All specimens with a thickened basal lamina were from heavy smokers. Immunogold labelling studies showed that the thickened basal lamina contained specific accumulations of fibronectin but not heparan sulphate proteoglycans, type IV collagen, or laminin. CONCLUSIONS: Two ultrastructural characteristics are associated with smoking: thickening of the endothelial basal lamina and a specific accumulation of fibronectin in the thickened basal lamina. Such abnormalities in the saphenous veins from smokers may contribute to the poorer performance of these veins as bypass conduits.
Adult↗
Congenital insensitivity to pain: a 20 year follow up.
The exact nosological status of "congenital insensitivity to pain" remains in doubt. Possible pathological correlates of this clinical syndrome include sensory neuropathy, central lesions at the level of the reticular formation or dorsal horn of the spinal cord, or a central indifference to, or asymbolia for, pain. The reassessment of two members of a kindred previously reported more than 20 years ago as having congenital insensitivity to pain indicated that they in fact had an inherited sensory and autonomic neuropathy. Prolonged follow up and morphometric analysis of sequential nerve biopsies may be necessary to definitively establish this diagnosis.
Adult↗
Nitric oxide accounts for dose-dependent estrogen-mediated coronary relaxation after acute estrogen withdrawal.
BACKGROUND: Estrogen replacement therapy reduces the risk of coronary heart disease in postmenopausal women, and estrogen treatment modulates endothelium-dependent vasodilation in ovariectomized, atherosclerotic monkeys. Estradiol-17 beta also induces relaxation in isolated rabbit coronary arteries as well as cerebral basilar arteries. The estrogen concentrations required to induce such relaxation are in the pharmacological range (10(-6) to 10(-5) mol/L). METHODS AND RESULTS: The present study was designed to test whether the sensitivity and specificity of the relaxing response of coronary vascular smooth muscle to exogenous estradiol-17 beta is dependent on the sex hormone status of the animal. In coronary artery rings contracted with PGF2 alpha (3 x 10(-5) mol/L), estradiol-17 beta caused significant relaxation at a physiological estrogen concentration (10(-9) mol/L), in coronary artery rings from oophorectomized, estrogen-treated and acutely estrogen-withdrawn rabbits only. Relaxation induced by estradiol-17 beta at lower concentrations (10(-9) to 10(-6) mol/L) in these rings was 20 +/- 6%, 42 +/- 8%, 54 +/- 9%, and 75 +/- 8%, respectively, compared with 4 +/- 2%, 12 +/- 5%, 16 +/- 7%, and 25 +/- 12% and 5 +/- 2%, 12 +/- 5%, 18 +/- 8%, and 23 +/- 10% in rings from estrogen-maintained and oophorectomized rabbits, respectively (P < .01). The relaxation in coronary artery rings from estrogen-treated and acutely estrogen-withdrawn rabbits was endothelium and nitric oxide dependent since it was abolished by endothelium removal and the nitric oxide synthase inhibitor N omega-nitro-L-arginine. CONCLUSIONS: This study demonstrates that estrogen-induced, endothelium-dependent relaxation of coronary arteries may, in some species, depend on the sex hormone status of the animal. These findings may help to better understand the effects of ovarian steroids in the coronary circulation of females.
Animals↗
Are hypotension and rash after atracurium really caused by histamine release?
A prospective, randomized, double-blind study was performed in 40 patients (ASA class I-III) treated with atracurium to ascertain whether histamine release caused hemodynamic or cutaneous changes. The treated group of 20 patients was premedicated with the H1 antagonist dimetindene (0.2 mg/kg) and the H2 antagonist ranitidine (1.25 mg/kg); the control group of 20 patients received saline. Six minutes after the induction of anesthesia with thiopental/fentanyl, patients received atracurium 0.5 mg/kg over 5 s. Plasma histamine levels were measured fluorometrically 5 min after administration of thiopental/fentanyl and 2 and 5 min after atracurium. Arterial blood pressure and heart rate were recorded every 2 min. Histamine levels (0.24 ng/mL) did not change significantly after thiopental/fentanyl. In the control group, 2 min after injection of atracurium, plasma histamine levels were 0.76 +/- 0.76 ng/mL, and in the antihistamine-treated group, 0.39 +/- 0.24 ng/mL (P < 0.05 control versus treated), suggesting that pretreatment with antihistamines may attenuate atracurium-induced histamine release. Systolic and diastolic blood pressure decreased significantly in both groups after thiopental (P < 0.05), but did not decrease further after the administration of atracurium. There were cutaneous manifestations in 7 of 20 patients in the control group and in none of the 20 patients treated with H1 and H2 antagonists (P < 0.0005). We conclude that atracurium caused modest histamine release in our patients but that the decrease in arterial blood pressure may have been due, in part, to thiopental. Cutaneous manifestations of histamine release did not correlate with hemodynamic events or with plasma histamine levels, but were prevented with antihistamine pretreatment.
Adolescent↗
Timothy Murrell. The general practitioner as human ecologist.
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Ecology↗
Hypertensive episode associated with phenelzine and tap beer--a reanalysis of the role of pressor amines in beer.
A case report of a hypertensive crisis resulting from the ingestion of tap beer in a patient on an irreversible monamine oxidase inhibitor (MAOI; phenelzine) stimulated the investigation of different kinds of beer for tyramine concentration. The objective was to determine the tyramine concentration in tap and bottled beers. A total of 98 beer samples (79 different brands of beer) were analyzed by high-performance liquid chromatography for tyramine. Of these 98 beers, 49 were bottled or canned beers and 49 were beers on tap. All of the bottled beers analyzed had safe tyramine concentrations (< or = 10 mg/liter; range, 0 to 3.16 mg/liter) and, thus, do not require restriction in patients receiving MAOIs. Therefore, the consumption of canned or bottled beer, including dealcoholized beer, in moderation (fewer than four bottles or cans; 1.5 liters within a 4-hour period) appears to be safe and does not require restriction in patients receiving MAOIs. Only 4 of 98 beer samples studied were found to have a dangerous (> 10 mg/liter) tyramine concentration, one of which was the index beer. The tyramine concentration in these four beers ranged from 26.34 to 112.91 mg/liter. All four of these beers were tap beers produced by bottom fermentation (lagers) and brewed by a secondary fermentation process. Although we did not find any visible bacterial growth in the tap beers with high tyramine content, this finding does not preclude the possibility that bacterial contamination, bacterial growth, production of tyramine, and eventually bacterial death occurred at some earlier time. Therefore, to err on the side of caution, it is recommended that patients on irreversible MAOIs avoid beers on tap.
Adult↗
Integrin alpha 7 as substrate for a glycosylphosphatidylinositol-anchored ADP-ribosyltransferase on the surface of skeletal muscle cells.
An arginine-specific mono-ADP-ribosyltransferase is expressed on the surface of differentiated mouse skeletal muscle cells and is anchored in the membrane via a glycosylphosphatidylinositol tail. Following incubation of intact cells with [adenylate-32P]NAD and analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), a 97-kDa [32P]ADP-ribosylated protein was observed under reducing conditions and a 140-kDa complex under nonreducing conditions. The ADP-ribosylated protein was purified on a laminin affinity column. Based on its N-terminal sequence (FNLDVM-GAIRKEGEPGSLFGF) and a partial internal sequence (GLMRSEELSFVAGAP), the modified protein was identified as integrin alpha 7. Following partial trypsin digestion, a 39-kDa/79-kDa radiolabeled fragment was produced (reduced/nonreduced SDS-PAGE), narrowing the ADP-ribosylation site to a 39-kDa segment in the extracellular domain of integrin alpha 7. Labeling under optimal conditions was at least 0.4 mol of ADP-ribose/mol of integrin alpha 7. Selective expression of both ADP-ribosyltransferase and integrin alpha 7 in cardiac and skeletal muscle, a similar developmental appearance, and the apparently specific ADP-ribosylation, are consistent with a regulatory association between these proteins. ADP-ribosylation may modulate integrin receptor signaling and could play a significant role in the regulation of muscle cell function by the extracellular matrix.
ADP Ribose Transferases↗
Cloning and site-directed mutagenesis of human ADP-ribosylarginine hydrolase.
Mono-ADP-ribosylation of arginine is a reversible modification of proteins with NAD:arginine ADP-ribosyltransferases and ADP-ribosylarginine hydrolases catalyzing the opposing reactions in the cycle. ADP-ribosylarginine hydrolases differ in their dithiothreitol (DTT) requirements. Rat and mouse hydrolases require DTT for maximal activity, but calf, guinea pig, and human hydrolases are DTT-independent. To define the molecular basis for these differences, brain ADP-ribosylarginine hydrolases were cloned. Deduced amino acid sequences of mouse and rat hydrolases were 94% identical with 5 conserved cysteines. The human hydrolase sequence was 83% identical to that of rat but contained only 4 cysteines with cysteine 108 in rat corresponding to serine 103 in human. To investigate the role of rat cysteine 108, human and rat wild-type hydrolases and mutants, in which serine 103 in human was replaced by cysteine (S103C) and cysteine 108 in rat was replaced by serine (C108S), were expressed in Escherichia coli. Affinity-purified anti-rat brain hydrolase antibodies reacted with recombinant wild-type rat hydrolase, but only weakly with the C108S mutant. They did not react with human wild-type or the S103C mutant. Human hydrolase and rat C108S were DTT-independent; human S103C was, however, DTT-dependent. These data clearly show that cysteine 108 in rat hydrolase plays a critical role in DTT dependence and may be important in immunoreactivity.
Amino Acid Sequence↗
Nitric oxide-independent, thiol-associated ADP-ribosylation inactivates aldehyde dehydrogenase.
Nitric oxide inhibits the activity of glyceraldehyde-3-phosphate dehydrogenase and stimulates NAD-dependent automodification of a cysteine (Dimmeler, S., Lottspeich, F., and Brüne, B. (1992) J. Biol. Chem. 267, 16771-16774). Another NAD-utilizing dehydrogenase that has a catalytic cysteine, aldehyde dehydrogenase (ALDH), was also inhibited by nitric oxide. Unlike glyceraldehyde-3-phosphate dehydrogenase, ALDH was modified in a nitric oxide-independent process by ADP-ribose, but not by NAD. Modification, which proceeded to > 2 mol ADP-ribose.mol ALDH-1, was associated with an exponential decrease in enzyme activity to less than 10% of control. Two types of evidence suggested modification of the ALDH-active site: 1) ADP-ribose inhibited ALDH competitively (Ki = 0.46 mM) with respect to NAD (Km = 0.11 mM) in brief incubations and 2) the presence of substrates protected ALDH from both modification and inhibition by ADP-ribose. The ALDH-ADP-ribose bond was sensitive to base and mercuric ion and stable to acid and neutral hydroxylamine, properties shared with the ADP-ribosylcysteine linkage synthesized enzymatically by pertussis toxin. These data demonstrate a novel means of inactivation of an NAD-dependent enzyme, namely the affinity-based modification of the enzyme NAD site by ADP-ribose, and suggest that nonenzymatic ADP-ribosylation may be responsible for modification of cysteine residues.
Adenosine Diphosphate Ribose↗
An RNA-binding protein gene (RBP1) of Saccharomyces cerevisiae encodes a putative glucose-repressible protein containing two RNA recognition motifs.
A gene, termed RNA-binding protein (RBP1), was cloned from Saccharomyces cerevisiae. RBP1 contains an open reading frame of 2016 nucleotides that encodes a 672-amino acid protein with a calculated M(r) of approximately 75,000. Southern blots of genomic DNA from wild-type and RBP1-disrupted strains were consistent with the presence of homologous genes. RNA blots revealed a major 2.7-kb RNA band and two minor bands of 1.5 and 1.1 kb. The sequence of the putative RBP1 protein contains two copies of an RNA recognition motif, two glutamine stretches, an asparagine-rich region, a methionine-rich region, and two long potential alpha-helixes. In addition, recombinant RBP1 fusion protein can bind to RNA and single-stranded DNA but not double-stranded DNA. RBP1 is a glucose-repressible gene. Disruption of RBP1 increased cell growth rate in the early log phase. Overexpression of RBP1 or reduction in its translation by expression of antisense RNA decreased or increased the cell growth rate, respectively. From these observations, we infer that RBP1 may be involved in growth regulation, possibly through its participation in RNA metabolism.
Amino Acid Sequence↗
Characterization of recombinant and endogenous ADP-ribosylation factors synthesized in Sf9 insect cells.
ADP-ribosylation factors (ARFs) are a family of highly conserved, 20-kDa guanine nucleotide-binding proteins that participate in protein trafficking and enhance cholera toxin-catalyzed ADP-ribosylation. ARF 2 from bovine retinal cDNA was expressed in Sf9 insect cells using recombinant baculovirus and compared to the major insect cell ARF (Sf9 ARF) and to recombinant ARF 2 expressed in Escherichia coli (E. coli rARF 2). The 150000g supernatant and particulate fractions of freeze-thawed, recombinant ARF 2 baculovirus-infected cells contained immunoreactive proteins of 20 and 21 kDa at significantly higher levels than were found in uninfected cells. Infected Sf9 cells incorporated [3H]myristate only into the 20-kDa protein. Sf9 cell recombinant ARF 2 (Sf9 rARF 2) and Sf9 ARF were separated by isoelectric focusing or ion-exchange chromatography and identified by microsequencing of HPLC-purified tryptic peptides. Sf9 ARF displayed considerable sequence identity to mammalian class I ARFs. Both Sf9 ARF and Sf9 rARF 2 stimulated in a GTP-dependent manner cholera toxin-catalyzed ADP-ribosylation. The Ka for GTP of Sf9 ARF was, however, significantly lower than that of Sf9 rARF 2 or E. coli rARF 2. Myristoylation did not significantly affect the ability of ARF 2 to enhance cholera toxin-catalyzed ADP-ribosylation or the Ka for GTP. Despite the sequence identities and the fact that both were synthesized in insect cells, the endogenous Sf9 ARF was functionally different from Sf9 rARF 2.
ADP-Ribosylation Factors↗
Stimulation by nitric oxide of an NAD linkage to glyceraldehyde-3-phosphate dehydrogenase.
Nitric oxide-stimulated modification of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) by [adenylate-32P]NAD has been interpreted in recent reports as ADP-ribosylation. Incubations of GAPDH with the NO-releasing agent sodium nitroprusside (SNP) and NAD resulted, however, in essentially equal incorporation of radiolabel from the adenine, phosphate, and nicotinamide moieties to the extent of approximately 0.02 mol of NAD.mol of GAPDH-1. Modification of GAPDH by free adenosine 5'-diphosphoribose (ADP-ribose) was only 10% of that by NAD. Exposure of GAPDH modified by NAD in the presence of SNP to HgCl2, which acts at thiol linkages, released two products. Both contained nicotinamide and adenylate but did not cochromatograph with NAD. GAPDH activity was inhibited by SNP in a dose-dependent manner in the presence of NAD. When inhibition was 80%, with 1 mM SNP and 1 mM dithiothreitol, covalent modification with NAD was < 2%. This result is consistent with the conclusion that inhibition of GAPDH activity by SNP in the presence of NAD is due primarily to active-site nitrosylation, as reported by other workers, and is not due to the minor modification with NAD. These results demonstrate that NO-stimulated modification of GAPDH with NAD is not ADP-ribosylation as previously reported but rather is covalent binding of NAD through a NO-dependent thiol intermediate, possibly providing an example of an unexpected, altered reactivity of a nitrosylated protein.
Glyceraldehyde-3-Phosphate Dehydrogenases↗
Molecular characterization of a conserved, guanine nucleotide-dependent ADP-ribosylation factor in Drosophila melanogaster.
ADP-Ribosylation factors (ARFs) are ubiquitous approximately 20-kDa guanine nucleotide-binding proteins that stimulate cholera toxin-catalyzed ADP-ribosylation in vitro. Because the functional role(s) of ARF in mammalian systems is (are) elusive, we looked for ARF in Drosophila melanogaster, and report the partial purification and molecular cloning of an ARF from Drosophila. We cloned the Drosophila ARF 1 gene without library screening by a combination of 5 polymerase chain reactions (PCRs), yielding a 546-base open reading frame encoding 182 amino acids, which are > 93% identical to those of mammalian class I ARFs. This ARF gene maps to 79F3-6 in the proximal region of the left arm of Drosophila chromosome 3. The Drosophila ARF1 gene structure, including placement of introns, is highly conserved relative to mammalian class 1 ARF genes. A single ARF mRNA species of 1.8 kb was abundant in all Drosophila body segments. Recombinant Drosophila ARF 1 synthesized in Escherichia coli had biochemical and immunochemical activities similar to those of mammalian ARF. The similarities of sequence and biochemical properties between Drosophila and mammalian ARFs contrast with their differences from Drosophila arl (ARF-like protein), which does not stimulate cholera toxin-catalyzed ADP-ribosylation, and is only approximately 52-56% identical in amino acid sequence to mammalian ARFs.
ADP-Ribosylation Factor 1↗