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1.
Infect Immun ; 85(6)2017 06.
Article in English | MEDLINE | ID: mdl-28396323

ABSTRACT

Mycoplasma gallisepticum, known primarily as a respiratory pathogen of domestic poultry, has emerged since 1994 as a significant pathogen of the house finch (Haemorhousmexicanus) causing severe conjunctivitis and mortality. House finch-associated M. gallisepticum (HFMG) spread rapidly and increased in virulence for the finch host in the eastern United States. In the current study, we assessed virulence in domestic poultry with two temporally distant, and yet geographically consistent, HFMG isolates which differ in virulence for house finches-Virginia 1994 (VA1994), the index isolate of the epidemic, and Virginia 2013 (VA2013), a recent isolate of increased house finch virulence. Here we report a significant difference between VA1994 and VA2013 in their levels of virulence for chickens; notably, this difference correlated inversely to the difference in their levels of virulence for house finches. VA1994, while moderately virulent in house finches, displayed significant virulence in the chicken respiratory tract. VA2013, while highly virulent in the house finch, was significantly attenuated in chickens relative to VA1994, displaying less-severe pathological lesions in, and reduced bacterial recovery from, the respiratory tract. Overall, these data indicate that a recent isolate of HFMG is greatly attenuated in the chicken host relative to the index isolate, notably demonstrating a virulence phenotype in chickens inversely related to that in the finch host.


Subject(s)
Chickens/microbiology , Finches/microbiology , Mycoplasma Infections/epidemiology , Mycoplasma gallisepticum/isolation & purification , Mycoplasma gallisepticum/pathogenicity , Animals , Female , Mycoplasma Infections/microbiology , Mycoplasma Infections/veterinary , Phenotype , Phylogeny , Virginia , Virulence
2.
J Evol Biol ; 27(6): 1271-8, 2014 Jun.
Article in English | MEDLINE | ID: mdl-24750277

ABSTRACT

In the mid-1990s, the common poultry pathogen Mycoplasma gallisepticum (MG) made a successful species jump to the eastern North American house finch Haemorhous mexicanus (HM). Subsequent strain diversification allows us to directly quantify, in an experimental setting, the transmission dynamics of three sequentially emergent geographic isolates of MG, which differ in the levels of pathogen load they induce. We find significant among-strain variation in rates of transmission as well as recovery. Pathogen strains also differ in their induction of host morbidity, measured as the severity of eye lesions due to infection. Relationships between pathogen traits are also investigated, with transmission and recovery rates being significantly negatively correlated, whereas transmission and virulence, measured as average eye lesion score over the course of infection, are positively correlated. By quantifying these disease-relevant parameters and their relationships, we provide the first analysis of the trade-offs that shape the evolution of this important emerging pathogen.


Subject(s)
Bird Diseases/transmission , Finches/microbiology , Mycoplasma gallisepticum/pathogenicity , Animals , Communicable Diseases, Emerging/microbiology , Communicable Diseases, Emerging/transmission , Communicable Diseases, Emerging/veterinary , Mycoplasma gallisepticum/isolation & purification
3.
J Evol Biol ; 23(8): 1680-8, 2010 Aug.
Article in English | MEDLINE | ID: mdl-20561136

ABSTRACT

Host genetic diversity can mediate pathogen resistance within and among populations. Here we test whether the lower prevalence of Mycoplasmal conjunctivitis in native North American house finch populations results from greater resistance to the causative agent, Mycoplasma gallisepticum (MG), than introduced, recently-bottlenecked populations that lack genetic diversity. In a common garden experiment, we challenged wild-caught western (native) and eastern (introduced) North American finches with a representative eastern or western MG isolate. Although introduced finches in our study had lower neutral genetic diversity than native finches, we found no support for a population-level genetic diversity effect on host resistance. Instead we detected strong support for isolate differences: the MG isolate circulating in western house finch populations produced lower virulence, but higher pathogen loads, in both native and introduced hosts. Our results indicate that contemporary differences in host genetic diversity likely do not explain the lower conjunctivitis prevalence in native house finches, but isolate-level differences in virulence may play an important role.


Subject(s)
Bird Diseases/microbiology , Finches/genetics , Host-Pathogen Interactions/genetics , Mycoplasma Infections/veterinary , Mycoplasma gallisepticum/pathogenicity , Animals , Bird Diseases/epidemiology , Finches/immunology , Genetic Variation , Immunocompetence/immunology , Microsatellite Repeats/genetics , Mycoplasma Infections/epidemiology , Mycoplasma Infections/microbiology , Mycoplasma gallisepticum/isolation & purification , Prevalence , Time Factors
4.
Mol Endocrinol ; 8(3): 315-24, 1994 Mar.
Article in English | MEDLINE | ID: mdl-8015549

ABSTRACT

We have studied insulin and antireceptor antibody binding to mutated human insulin receptors deleted of residues 485-599 in the alpha-subunit by site-directed mutagenesis. Both normal and mutated receptors were expressed in rat HTC hepatoma cells. Cells expressing either the normal receptor or the mutated receptor retained the ability to bind insulin. In contrast to the normal receptor, however, the mutated receptor failed to interact with antireceptor alpha-subunit antibodies. The inability of the mutated receptor to interact with various antireceptor antibodies was further documented by photoaffinity labeling studies. In intact HTC cells expressing mutated receptors, basal insulin receptor tyrosine autophosphorylation was 2-fold elevated when compared to cells expressing normal receptors. In these cells, however, the response of this function to insulin was blunted. When receptors were isolated from these cells and assayed for both autophosphorylation and phosphotransferase activities toward the synthetic substrate poly(Glu, Tyr), the response to insulin was also blunted. To study the ability of the mutated receptor to transmembrane signal, insulin stimulation of S6 kinase activity was measured. In cells with mutated receptors, in concert with the insulin receptor kinase data, basal S6 kinase activity was elevated, and the response to insulin was blunted. The data suggest, therefore, that residues 485-599 in the alpha-subunit of the insulin receptor are critical for antireceptor antibody binding, but not for insulin binding. Moreover, these data suggest that residues 485-599 contain a regulatory domain for insulin regulation of receptor beta-subunit functions.


Subject(s)
Antibodies, Monoclonal/immunology , Antibodies, Monoclonal/metabolism , Gene Deletion , Protein-Tyrosine Kinases/metabolism , Receptor, Insulin , Receptor, Insulin/genetics , Animals , Base Sequence , Blotting, Western , DNA, Neoplasm/analysis , DNA, Neoplasm/genetics , Enzyme Activation/genetics , Humans , Insulin/metabolism , Insulin/physiology , Iodine Radioisotopes , Liver Neoplasms, Experimental/chemistry , Liver Neoplasms, Experimental/pathology , Liver Neoplasms, Experimental/ultrastructure , Molecular Sequence Data , Mutation , Phosphorylation , Precipitin Tests , Rats , Receptor, Insulin/immunology , Receptor, Insulin/metabolism , Tumor Cells, Cultured
5.
Biochem Biophys Res Commun ; 179(2): 912-8, 1991 Sep 16.
Article in English | MEDLINE | ID: mdl-1898412

ABSTRACT

In order to study the role of tyrosine autophosphorylation in insulin receptor signalling, we investigated a mutant human insulin receptor whereby the three major tyrosine autophosphorylation sites at positions 1158, 1162, and 1163 in the receptor beta-subunit were mutated to phenylalanines. When these mutant receptors were expressed in HTC rat hepatoma cells, there was no enhanced beta-subunit autophosphorylation and tyrosine kinase activity. In these cells there was enhanced insulin stimulation of [3H]AIB uptake and [3H]thymidine incorporation when compared to wild type HTC cells. The present study suggests therefore that the presence of the major insulin autophosphorylation sites is not a requirement for insulin stimulation of amino acid transport and mitogenesis.


Subject(s)
Insulin/physiology , Receptor, Insulin/physiology , Tyrosine/genetics , Aminoisobutyric Acids/metabolism , Animals , Gene Expression , Mutagenesis, Site-Directed , Phenylalanine/genetics , Phosphorylation , Protein-Tyrosine Kinases/physiology , Rats , Receptor, Insulin/genetics , Signal Transduction , Thymidine/metabolism , Transfection , Tumor Cells, Cultured
6.
J Biol Chem ; 265(9): 4902-7, 1990 Mar 25.
Article in English | MEDLINE | ID: mdl-2156858

ABSTRACT

We examined the effect of insulin treatment on HTC cells transfected with large numbers of either normal insulin receptors (HTC-IR) or insulin receptors defective in tyrosine kinase (HTC-IR/M-1030). In both HTC-IR and HTC-IR/M-1030 cells, 20 h of insulin treatment (1 microM) at 37 degrees C resulted in a 65% decrease in the number of binding sites with a reciprocal 6-fold increase in affinity. In contrast, treatment with 10 nM insulin (20 h, 37 degrees C) also increased receptor affinity but had a smaller effect on the number of binding sites. 125I-Insulin binding to soluble receptors from HTC-IR and HTC-IR/M-1030 cells pretreated with insulin showed results similar to those obtained in intact cells. In both HTC-IR and HTC-IR/M-1030 cells, insulin enhanced insulin receptor degradation. In HTC-IR/M-1030 cells a 1-h incubation with insulin did not change receptor number and had only a small effect on receptor affinity; also there was no effect of insulin after a 20-h incubation at 15 degrees C. Inhibiting protein synthesis by pretreatment with cycloheximide (100 microM) did not block either the decrease in receptor number or the increase in receptor affinity. Both HTC-IR and HTC-IR/M-1030 cells exhibited a very slow rate of insulin and insulin receptor internalization and no differences were seen in this parameter when HTC-IR cells were compared to HTC-IR/M-1030 cells. These studies indicate, therefore, that in cells expressing kinase-defective insulin receptors, insulin down-regulates insulin receptor number via enhanced receptor degradation, and up-regulates receptor affinity. These effects were time- and temperature-dependent, but not dependent on new protein synthesis, and suggest that activation of tyrosine kinase may not be a prerequisite for certain mechanisms whereby insulin regulates its receptor.


Subject(s)
Down-Regulation , Protein-Tyrosine Kinases/genetics , Receptor, Insulin/genetics , Up-Regulation , Animals , Cell Line , Cycloheximide/pharmacology , Down-Regulation/drug effects , Humans , Insulin/metabolism , Kinetics , Liver Neoplasms, Experimental , Phosphorylation , Protein-Tyrosine Kinases/metabolism , Rats , Receptor, Insulin/drug effects , Receptor, Insulin/metabolism , Transfection , Tumor Cells, Cultured/metabolism , Up-Regulation/drug effects
7.
J Biol Chem ; 264(32): 18951-9, 1989 Nov 15.
Article in English | MEDLINE | ID: mdl-2553727

ABSTRACT

The effects of species-specific monoclonal antibodies to the human insulin receptor on ribosomal protein S6 phosphorylation were studied in rodent cell lines transfected with human insulin receptors. First, Swiss mouse 3T3 fibroblasts expressing normal human insulin receptors (3T3/HIR cells) were studied. Three monoclonal antibodies, MA-5, MA-20, and MA-51, activated S6 kinase in these cells but had no effects in untransfected 3T3 cells. Both insulin and MA-5, the most potent antibody, activated S6 kinase in a similar time- and dose-dependent manner. To measure S6 phosphorylation in vivo, 3T3/HIR cells were preincubated with [32P]Pi and treated with insulin and MA-5. Both agents increased S6 phosphorylation, and their tryptic phosphopeptide maps were similar. MA-5 and the other monoclonal antibodies, unlike insulin, failed to stimulate insulin receptor tyrosine kinase activity either in vitro or in vivo. Moreover, unlike insulin, they failed to increase the tyrosine phosphorylation of the endogenous cytoplasmic protein, pp 185. Next, HTC rat hepatoma cells, expressing a human insulin receptor mutant that had three key tyrosine autophosphorylation sites in the beta-subunit changed to phenylalanines (HTC-IR-F3 cells), were studied. In this cell line but not in untransfected HTC cells, monoclonal antibodies activated S6 kinase without stimulating either insulin receptor autophosphorylation or the tyrosine phosphorylation of pp 185. These data indicate, therefore, that monoclonal antibodies can activate S6 kinase and then increase S6 phosphorylation. Moreover, they suggest that activation of receptor tyrosine kinase and subsequent tyrosine phosphorylation of cellular proteins may not be crucial for activation of S6 kinase by the insulin receptor.


Subject(s)
Antibodies, Monoclonal , Insulin/pharmacology , Mutation , Protein Kinases/metabolism , Protein-Tyrosine Kinases/metabolism , Receptor, Insulin/metabolism , Transfection , Animals , Base Sequence , Blotting, Western , Cells, Cultured , Electrophoresis, Gel, Two-Dimensional , Enzyme Activation , Humans , Kinetics , Mice , Molecular Sequence Data , Oligodeoxyribonucleotides , Peptide Mapping , Phosphopeptides/isolation & purification , Phosphorylation , Protein Kinases/immunology , Receptor, Insulin/genetics , Ribosomal Protein S6 Kinases
8.
Mol Endocrinol ; 3(10): 1634-42, 1989 Oct.
Article in English | MEDLINE | ID: mdl-2558298

ABSTRACT

Glucocorticoids regulate the trafficking of mouse mammary tumor virus (MMTV) glycoproteins to the cell surface in the rat hepatoma cell line M1.54, but not in the immunoselected sorting variant CR4. To compare the localization of MMTV glycoproteins to another proteolytically processed glycoprotein, both wild type M1.54 cells and variant CR4 cells were transfected with a human insulin receptor (hIR) expression vector, pRSVhIR. The production of cell surface hIR was monitored in dexamethasone-treated and -untreated wild type M1.54 and variant CR4 cells by indirect immunofluorescence, direct plasma membrane immunoprecipitation, and by [125I] insulin binding. In both wild type and variant rat hepatoma cells, hIR were localized at the cell surface in the presence or in the absence of 1 microM dexamethasone. In contrast, the glucocorticoid-regulated trafficking of cell surface MMTV glycoproteins occurred only in wild type M1.54 cells. We conclude that the hIR, which undergoes posttranslational processing reactions similar to MMTV glycoproteins, does not require glucocorticoids to be transported to the plasma membrane and is representative of a subset of cell surface glycoproteins whose trafficking is constitutive in rat hepatoma cells. Thus, MMTV glycoproteins and hIR provide specific cell surface markers to characterize the glucocorticoid-regulated and constitutive sorting pathways.


Subject(s)
Glucocorticoids/physiology , Liver Neoplasms, Experimental/metabolism , Membrane Glycoproteins/genetics , Animals , Dexamethasone/pharmacology , Fluorescent Antibody Technique , Humans , Iodine Radioisotopes , Liver Neoplasms, Experimental/genetics , Mammary Tumor Virus, Mouse/genetics , Mice , Rats , Receptor, Insulin/genetics , Receptor, Insulin/metabolism , Sulfur Radioisotopes , Transfection , Tumor Cells, Cultured
9.
J Biol Chem ; 264(27): 15900-4, 1989 Sep 25.
Article in English | MEDLINE | ID: mdl-2550426

ABSTRACT

Our previous studies indicated that amino acid residues 240-250 in the cysteine-rich region of the human insulin receptor alpha-subunit constitute a site in which insulin binds (Yip, C. C., Hsu, H., Patel, R. G., Hawley, D. M., Maddux, B. A., and Goldfine, I. D. (1988) Biochem. Biophys. Res. Commun. 157, 321-329). We have now constructed a human insulin receptor mutant in which 3 residues in this sequence were altered (Thr-Cys-Pro-Pro-Pro-Tyr-Tyr-His-Phe-Gln-Asp to Thr-Cys-Pro-Arg-Arg-Tyr-Tyr-Asp-Phe-Gln-Asp) and have expressed this mutant in rat hepatoma (HTC) cells. When compared with cells transfected with normal insulin receptors, cells transfected with mutant receptors had an increase in insulin-binding affinity and a decrease in the dissociation of bound 125I-insulin. Studies using solubilized receptors also demonstrated that mutant receptors had a higher binding affinity than normal receptors. In contrast, cells transfected with either mutant or normal receptors bound monoclonal antibodies against the receptor alpha-subunit with equal affinity. When receptor tyrosine kinase activity and alpha-aminoisobutyric acid uptake were measured, cells transfected with mutant insulin receptors were more sensitive to insulin than cells transfected with normal receptors. These findings lend further support therefore to the hypothesis that amino acid sequence 240-250 of the human insulin receptor alpha-subunit constitutes one site that interacts with insulin, and they indicate that mutations in this site can influence insulin receptor binding and transmembrane signaling.


Subject(s)
Cysteine , Mutation , Receptor, Insulin/metabolism , Signal Transduction , Amino Acid Sequence , Animals , Base Sequence , Cell Line , Cell Membrane/physiology , Genetic Vectors , Humans , Kinetics , Liver Neoplasms, Experimental , Macromolecular Substances , Molecular Sequence Data , Oligonucleotide Probes , Protein-Tyrosine Kinases/metabolism , Rats , Receptor, Insulin/genetics , Transfection
10.
J Biol Chem ; 264(5): 2438-44, 1989 Feb 15.
Article in English | MEDLINE | ID: mdl-2536710

ABSTRACT

HTC rat hepatoma cells were transfected with human insulin receptor cDNA to a level of 40,000 receptors/cell. In these cells, as well as in nontransfected cells, insulin stimulated the uptake of alpha-aminoisobutyric acid. Two monoclonal antibodies directed against the human insulin receptor alpha subunit, like insulin, stimulated amino acid uptake in transfected HTC cells, but not in nontransfected HTC cells. The antibodies, in contrast to insulin, failed to stimulate insulin receptor tyrosine kinase activity, both in intact transfected cells and in cell free extracts prepared from them. These data suggest, therefore, that activation of insulin receptor tyrosine kinase may not be an obligatory step in all of the transmembrane signaling mechanisms of the insulin receptor.


Subject(s)
Antibodies, Monoclonal , Insulin/pharmacology , Protein-Tyrosine Kinases/metabolism , Receptor, Insulin/metabolism , Aminoisobutyric Acids/metabolism , Animals , Antigen-Antibody Reactions , Biological Transport/drug effects , Cell Line , Humans , Insulin/metabolism , Kinetics , Liver Neoplasms, Experimental , Phosphorylation , Rats , Receptor, Insulin/genetics , Receptor, Insulin/immunology , Transfection
11.
Biochem Biophys Res Commun ; 157(1): 321-9, 1988 Nov 30.
Article in English | MEDLINE | ID: mdl-3058126

ABSTRACT

Affinity-purified insulin receptor was photoaffinity labeled with a cleavable radioactive insulin photoprobe. Exhaustive digestion of the labeled alpha-subunit with endoproteinase Glu-C produced a major radioactive fragment of 23 kDa as a part of the putative insulin-binding domain. This fragment could contain either residues 205-316 or 518-633 of the alpha-subunit. Rat hepatoma cells and Chinese hamster ovary cells were transfected with cDNA encoding a human insulin receptor mutant with a deletion of the cysteine-rich region spanning amino acid residues 124-319. Insulin binding by these cells was not increased in spite of high numbers of the mutant insulin receptors being expressed. A panel of monoclonal antibodies which was specific for the receptor alpha-subunit and inhibited insulin binding immunoprecipitated the photolabeled 23-kDa receptor fragment but not the receptor mutant. A synthetic peptide containing residues 243-251 was specifically bound by agarose-insulin beads. We therefore suggest that the 23-kDa fragment contains residues 205-316, and that insulin binding occurs, in part, in the cysteine-rich region of the alpha-subunit.


Subject(s)
Insulin/metabolism , Receptor, Insulin/metabolism , Affinity Labels , Binding Sites , Cysteine , DNA Mutational Analysis , Molecular Structure , Molecular Weight , Oligopeptides/metabolism , Photochemistry , Precipitin Tests , Transfection
12.
Diabetes ; 36(6): 779-81, 1987 Jun.
Article in English | MEDLINE | ID: mdl-3569675

ABSTRACT

We found with IM-9 human cultured lymphocytes, that the glucocorticoid dexamethasone increased insulin-receptor mRNA levels. This increase correlated in a time- and dose-dependent manner with the increase in the biosynthesis of the insulin-receptor precursor. In addition, in AR42J cultured rat pancreatic acinar cells, dexamethasone increased insulin-receptor mRNA levels. These studies suggest, therefore, that an increase in mRNA levels is an early step in the regulation of the insulin receptor by glucocorticoids in several cell types.


Subject(s)
Dexamethasone/pharmacology , RNA, Messenger/analysis , Receptor, Insulin/genetics , Dose-Response Relationship, Drug , Nucleic Acid Hybridization
13.
J Histochem Cytochem ; 35(1): 75-82, 1987 Jan.
Article in English | MEDLINE | ID: mdl-3025290

ABSTRACT

Phosphodiesterase I (PDE I) is an exonuclease capable of hydrolyzing a variety of phosphate ester and pyrophosphate bonds. Cell fractionation and histochemical studies in animal tissues have localized PDE I in the plasma membrane of various epithelia. This suggests a role for the enzyme in active transport. Distribution of PDE I in human tissues has not previously been studied. We have produced a polyclonal antiserum to bovine intestinal PDE I and have demonstrated crossreactivity with the human intestinal enzyme. This polyclonal antiserum was used in PAP immunocytochemistry to localize immunoreactive PDE I in a variety of human tissues. Localization was prominent in the gastrointestinal tract, including the cytoplasm of gastric mucosa parietal cells, cytoplasm of surface epithelium and isolated crypt cells in small intestine, and the colonic epithelial cytoplasm and brush border. Parotid gland acinar cells and scattered ductal cells showed positive cytoplasmic staining. Acinar and scattered pancreatic islet cells contained immunoreactive PDE I, as did Kupffer cells of the liver sinusoids. Immunoreactive PDE I was found in all vascular endothelia. The epithelium of the urinary tract showed extensive immunoreactivity. This included the distal convoluted and collecting tubules of the kidney, and ureteral and bladder urothelium. In previous histochemical studies of animal tissues, no evidence of PDE I activity was noted in male or female reproductive tract. In this study, immunoreactive PDE I was localized to human Sertoli cells and to basal epithelium of the epididymis and prostate acini. Fallopian tube epithelium of female reproductive tract also demonstrated immunoreactive PDI I, as did several cell types in term placenta. Our immunocytochemical results with human tissues differ significantly from previous histochemical studies in animal tissues, principally in the genitourinary system. This may be due in part to the different detection systems employed as well as the higher sensitivity of the immunoperoxidase technique. This underscores the importance of adjunct techniques in tissue surveys. The widespread epithelial distribution of immunoreactive PDE I detected by this polyclonal antibody implies an integral role in cell function, probably in active transport.


Subject(s)
Phosphoric Diester Hydrolases/analysis , Histocytochemistry , Humans , Immunochemistry , Phosphodiesterase I , Reference Values
14.
Anal Biochem ; 151(2): 375-80, 1985 Dec.
Article in English | MEDLINE | ID: mdl-3006537

ABSTRACT

Several bovine spleen enzymes with acid pH optima, some of which hydrolyze bis(p-nitrophenyl)phosphate and therefore fit the definition of "phosphodiesterase IV," were partially separated by isoelectric focusing and ion-exchange techniques. The activities were characterized by zymogram analysis with the aid of p-nitrophenyl and 4-methylumbelliferyl phosphate and phosphonate substrates. A number of these enzymes meet the criteria for phosphodiesterase I or other phosphodiesterases. However, the predominant phosphodiesterase I hydrolyzes the bis(p-nitrophenyl)-and 4-methylumbelliferyl phosphates, p-nitrophenyl and 4-methylumbelliferyl phenylphosphonate, and ATP at the beta-gamma bond, but not p-nitrophenyl or 4-methylumbelliferyl 5'-thymidylate (the usual PDE I substrates). These properties, as well as the pH optimum, distinguish the activity from the previously described, alkaline pH optimum PDE I. A second phosphodiesterase hydrolyzes only the phenylphosphonates. Several other activities, less well described, are apparent on zymograms. None of the phosphodiesterases IV was also a phosphodiesterase II (no hydrolysis of 4-methylumbelliferyl 3'-thymidylate).


Subject(s)
3',5'-Cyclic-AMP Phosphodiesterases , Phosphoric Diester Hydrolases/metabolism , Spleen/enzymology , Adenosine Triphosphatases/metabolism , Animals , Cattle , Cyclic Nucleotide Phosphodiesterases, Type 4 , Hydrolysis , Nitrophenols , Phosphoric Diester Hydrolases/isolation & purification , Substrate Specificity
15.
Enzyme ; 32(2): 105-9, 1984.
Article in English | MEDLINE | ID: mdl-6094176

ABSTRACT

Six of the substrates currently used for the detection and quantitation of phosphodiesterase I (PDE I) were examined. Michaelis-Menten kinetic analysis of the hydrolysis of each of these by venom and bovine intestinal PDE I was performed to allow a comparison of the sensitivity of detection for each substrate. The results confirm that phenolic esters of phenylphosphonate are hydrolyzed 2- to 4-fold more rapidly than are the same esters of thymidine 5'-phosphate when both are present in saturating concentrations. The identification of the bovine intestinal enzyme as a PDE I is verified by its extensive similarity to the paradigmatical PDE I from rattlesnake venom. A new definition for PDE I activity is proposed which takes into account its ability to hydrolyze several types of substrates.


Subject(s)
Phosphoric Diester Hydrolases/metabolism , Animals , Cattle , Hydrolysis , Hymecromone/analogs & derivatives , Hymecromone/metabolism , Kinetics , Nitrophenols/metabolism , Phosphodiesterase I , Phosphoric Diester Hydrolases/analysis , Substrate Specificity
16.
Clin Chim Acta ; 130(1): 31-7, 1983 May 09.
Article in English | MEDLINE | ID: mdl-6303633

ABSTRACT

Human blood serum was found to contain two enzymes which hydrolyze various phosphate diester and phosphonate ester bonds. The enzymes were isolated by butanol extraction, ammonium sulfate precipitation, column chromatography and gel electrophoresis. Zymograms showed that one of these enzymes is serum alkaline phosphatase and the other is a 'true' phosphodiesterase I. Serums of 25 persons showed no polymorphisms for either activity. Alkaline phosphatase hydrolyzes phenolic thymidine 5'-nucleotide esters readily, but phenylphosphonate esters very poorly. Serum phosphodiesterase I prefers phenylphosphonate esters to nucleotide diesters, and has no detectable monoesterase activity.


Subject(s)
Phosphoric Diester Hydrolases/isolation & purification , Chromatography, DEAE-Cellulose , Fluorescent Dyes , Humans , Neuraminidase , Phosphodiesterase I , Phosphoric Diester Hydrolases/blood
17.
Clin Chim Acta ; 130(1): 39-45, 1983 May 09.
Article in English | MEDLINE | ID: mdl-6303634

ABSTRACT

Extracts of human intestinal mucosa were examined for their ability to hydrolyze various phosphodiester, phosphomonoester and phenylphosphonate ester linkages. Enzymes carrying out these reactions were partially purified by butanol extraction, ammonium sulfate precipitation and DEAE-cellulose chromatography, and examined for polymorphism on polyacrylamide gels. Two species of alkaline phosphatase and at least five species of PDE I were identified. Antibodies to purified bovine intestinal phosphatase and phosphodiesterase were found specific for the respective human enzymes.


Subject(s)
Intestinal Mucosa/enzymology , Phosphoric Diester Hydrolases/isolation & purification , Alkaline Phosphatase/isolation & purification , Chromatography, DEAE-Cellulose , Electrophoresis, Polyacrylamide Gel , Fluorescent Dyes , Humans , Phosphodiesterase I , Phosphoric Diester Hydrolases/genetics , Polymorphism, Genetic
18.
Anal Biochem ; 129(2): 522-8, 1983 Mar.
Article in English | MEDLINE | ID: mdl-6189427

ABSTRACT

The 4-methylumbelliferyl analog of p-nitrophenyl phenylphosphonate was prepared and its cleavage by different phosphohydrolases was studied. The new compound, 4-methylumbelliferyl phenylphosphonate, is hydrolyzed by purified phosphodiesterase I (EC 3.1.4.1) from snake venom or bovine intestinal mucosa. This fact has been exploited to develop a zymogram method which allows the detection of these phosphohydrolases on gels, even when the enzymes are present in small amounts in crude extracts. This method is superior to chromogenic methods evolving p-nitrophenol in that it is far more sensitive and is superior to the 4-methylumbelliferyl thymidine 5'-phosphate method in that the substrate is easier to prepare and can be isolated in large quantity.


Subject(s)
Fluorescent Dyes/chemical synthesis , Phosphoric Diester Hydrolases/analysis , Animals , Cattle , Electrophoresis, Polyacrylamide Gel , Hydrolysis , Hymecromone/analogs & derivatives , Intestinal Mucosa/enzymology , Microchemistry , Phosphodiesterase I , Staining and Labeling , Viper Venoms/analysis
20.
Nucleic Acids Res ; 5(12): 4949-56, 1978 Dec.
Article in English | MEDLINE | ID: mdl-106367

ABSTRACT

A mass spectral method for the quantitation of the percentages of deoxyadenosine, deoxyguanosine, deoxycytidine, and thymidine in intact DNAs has been devised. Standard curves for each nucleoside have been constructed which are based upon the observation that a direct correlation exists between the heights (% deflection) of diagnostic peaks from these nucleosides in a mass spectrum and the published percent composition of specific DNAs. Analyses of DNA from Clostridiumperfringens, Micrococcusluteus, Escherichiacoli, Bacillussubtilis, Pseudomonasfluorescens, Drosophilamelanogaster, salmon sperm, and bacteriophage lambda were used to determine standard curves. The validity of the method was demonstrated by comparison of the results from the mass spectral procedure with results from the chemical analyses of the DNAs from calf thymus and wheat germ. Analysis of ØX-174 DNA yielded values consistent with the published values obtained via sequence analysis and indicated that the method is applicable to both single and double-stranded DNAs. Results from T2 DNA, which contains no cytidine, exhibited artificially high values for adenosine, guanosine and thymidine with concomitant alteration in the A/T and G/C molar ratios. Such skewed results are useful in predicting the presence of modified nucleosides. The extreme sensitivity of the method has been exploited in the analysis of subnanogram quantities of restriction endonuclease fragments from DNA.


Subject(s)
DNA , Animals , Bacteria , DNA, Bacterial , Deoxyribonucleotides/analysis , Drosophila melanogaster , Mass Spectrometry/methods , Species Specificity
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