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M P Anderson

Publications and source records attributed to M P Anderson.

At least 19 recordsLinked to original sources
PubMed2004

AFLP analysis of Cynodon dactylon (L.) Pers. var. dactylon genetic variation.

Cynodon dactylon (L.) Pers. var. dactylon (common bermudagrass) is geographically widely distributed between about lat 45 degrees N and lat 45 degrees S, penetrating to about lat 53 degrees N in Europe. The extensive variation of morphological and adaptive characteristics of the taxon is substantially documented, but information is lacking on DNA molecular variation in geographically disparate forms. Accordingly, this study was conducted to assess molecular genetic variation and genetic relatedness among 28 C. dactylon var. dactylon accessions originating from 11 countries on 4 continents (Africa, Asia, Australia, and Europe). A fluorescence-labeled amplified fragment length polymorphism (AFLP) DNA profiling method was used to detect the genetic diversity and relatedness. On the basis of 443 polymorphic AFLP fragments from 8 primer combinations, the accessions were grouped into clusters and subclusters associating with their geographic origins. Genetic similarity coefficients (SC) for the 28 accessions ranged from 0.53 to 0.98. Accessions originating from Africa, Australia, Asia, and Europe formed major groupings as indicated by cluster and principal coordinate analysis. Accessions from Australia and Asia, though separately clustered, were relatively closely related and most distantly related to accessions of European origin. African accessions formed two distant clusters and had the greatest variation in genetic relatedness relative to accessions from other geographic regions. Sampling the full extent of genetic variation in C. dactylon var. dactylon would require extensive germplasm collection in the major geographic regions of its distributional range.

Cynodon↗
PubMed1999

Diversity among Cynodon accessions and taxa based on DNA amplification fingerprinting.

The genus Cynodon (Gramineae), comprised of 9 species, is geographically widely distributed and genetically diverse. Information on the amounts of molecular genetic variation among and within Cynodon taxa is needed to enhance understanding of phylogenetic relations and facilitate germplasm management and breeding improvement efforts. Genetic relatedness among 62 Cynodon accessions, representing eight species, was assessed using DNA amplification fingerprinting (DAF). Ten 8-mer oligonucleotides were used to amplify specific Cynodon genomic sequences. The DNA amplification products of individual accessions were scored for presence (1) or absence (0) of bands. Similarity matrices were developed and the accessions were grouped by cluster (UPGMA) and principal coordinate analysis. Analyses were conducted within ploidy level (2x = 18 and 4x = 36) and over ploidy levels. Each primer revealed polymorphic loci among accessions within species. Of 539 loci (bands) scored, 496 (92%) were polymorphic. Cynodon arcuatus was clearly separated from other species by numerous monomorphic bands. The strongest species similarities were between C. aethiopicus and C. arcuatus, C. transvaalensis and C. plectostachyus, and C. incompletus and C. nlemfuensis. Intraspecific variation was least for C. aethiopicus, C. arcuatus, and C. transvaalensis, and greatest for C. dactylon. Accessions of like taxonomic classification were generally clustered, except the cosmopolitan C. dactylon var. dactylon and C. dactylon var. afganicus. Within taxa, accessions differing in chromosome number clustered in all instances indicating the 2x and 4x forms to be closely related. Little, if any, relationship was found between relatedness as indicated by the DAF profiles and previous estimates of hybridization potential between the different taxa.

Base Sequence↗
PubMed1993

Dysfunction of CFTR bearing the delta F508 mutation.

The cystic fibrosis transmembrane conductance regulator (CFTR) is mutated in patients with cystic fibrosis (CF). The most common CF-associated mutation is deletion of phenylalanine at residue 508, CFTR delta F508. When expressed in heterologous cells, CFTR bearing the delta F508 mutation fails to progress through the normal biosynthetic pathway and fails to traffic to the plasma membrane. As a result, CFTR delta F508 is mislocalized and is not present in the apical membrane of primary cultures of airway epithelia. Consequently, the apical membrane of CF airway epithelia is Cl- -impermeable, a defect that probably contributes to the pathogenesis of the disease.

Cell Membrane↗
PubMed1993

Regulation of the cystic fibrosis transmembrane conductance regulator chloride channel by MgATP.

These results begin to indicate that nucleoside triphosphates directly regulate CFTR Cl- channels by interacting with the NBDs. Thus, they may begin to explain why some CF-associated mutations in the NBDs may block Cl- channel function in the epithelia of CF patients. These results also suggest that the intracellular ATP/ADP ratio may be more important than the absolute concentration of ATP in regulating CFTR. Thus, changes in the metabolic state of the cell that alter the ATP-ADP ratio may regulate CFTR Cl- channel activity in vivo. These observations suggest that CFTR might be regulated in the physiologic range of nucleotides. Such a mechanism of regulation could provide a mechanism for coupling the metabolic status of the cell and the activity of the Na-K ATPase with the rate of transepithelial Cl- secretion as regulated by apical membrane CFTR Cl- channels.

Adenosine Triphosphate↗
PubMed1992

Regulation by ATP and ADP of CFTR chloride channels that contain mutant nucleotide-binding domains.

Regulation of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel is unusual in that phosphorylated channels require cytosolic adenosine triphosphate (ATP) to open. The CFTR contains two regions predicted to be nucleotide-binding domains (NBDs); site-directed mutations in each NBD have now been shown to alter the relation between ATP concentration and channel activity, which indicates that ATP stimulates the channel by direct interaction with both NBDs. The two NBDs are not, however, functionally equivalent: adenosine diphosphate (ADP) competitively inhibited the channel by interacting with NBD2 but not by interacting with NBD1. Four cystic fibrosis-associated mutations in the NBDs reduced absolute chloride channel activity, and one mutation also decreased the potency with which ATP stimulates channel activity. Dysfunction of ATP-dependent stimulation through the NBDs may be the basis for defective CFTR chloride channel activity in some cystic fibrosis patients.

Adenosine Diphosphate↗
PubMed1992

Processing of mutant cystic fibrosis transmembrane conductance regulator is temperature-sensitive.

Cystic fibrosis transmembrane conductance regulator (CFTR) is a plasma membrane Cl- channel regulated by cyclic AMP-dependent phosphorylation and by intracellular ATP. Mutations in CFTR cause cystic fibrosis partly through loss of cAMP-regulated Cl- permeability from the plasma membrane of affected epithelia. The most common mutation in cystic fibrosis is deletion of phenylalanine at residue 508 (CFTR delta F508) (ref. 10). Studies on the biosynthesis and localization of CFTR delta F508 indicate that the mutant protein is not processed correctly and, as a result, is not delivered to the plasma membrane. These conclusions are consistent with earlier functional studies which failed to detect cAMP-stimulated Cl- channels in cells expressing CFTR delta F508 (refs 16, 17). Chloride channel activity was detected, however, when CFTR delta F508 was expressed in Xenopus oocytes, Vero cells and Sf9 insect cells. Because oocytes and Sf9 cells are typically maintained at lower temperatures than mammalian cells, and because processing of nascent proteins can be sensitive to temperature, we tested the effect of temperature on the processing of CFTR delta F508. Here we show that the processing of CFTR delta F508 reverts towards that of wild-type as the incubation temperature is reduced. When the processing defect is corrected, cAMP-regulated Cl- channels appear in the plasma membrane. These results reconcile previous contradictory observations and suggest that the mutant most commonly associated with cystic fibrosis is temperature-sensitive.

3T3 Cells↗
PubMed1992

Chloride channels in the apical membrane of normal and cystic fibrosis airway and intestinal epithelia.

Cl- channels located in the apical membrane of secretory epithelia play a key role in epithelial fluid and electrolyte transport. Dysfunction of one of these channels, cystic fibrosis transmembrane conductance regulator (CFTR), causes the genetic disease cystic fibrosis (CF). We review here the properties and regulation of the different types of Cl- channels that have been reported in airway and intestinal epithelia. We begin by describing the properties of the CFTR Cl- channel and then use those properties as a point of reference. We focused particularly on the evidence that localizes specific types of Cl- channel to the apical membrane. With that background, we assess the biological function of various Cl- channels in airway and intestinal epithelia.

Animals↗
PubMed1991

Nucleoside triphosphates are required to open the CFTR chloride channel.

The CFTR Cl- channel contains two predicted nucleotide-binding domains (NBD1 and NBD2); therefore, we examined the effect of ATP on channel activity. Once phosphorylated by cAMP-dependent protein kinase (PKA), channels required cytosolic ATP to open. Activation occurred by a PKA-independent mechanism. ATP gamma S substituted for ATP in PKA phosphorylation, but it did not open the channel. Several hydrolyzable nucleotides (ATP greater than GTP greater than ITP approximately UTP greater than CTP) reversibly activated phosphorylated channels, but nonhydrolyzable analogs and Mg(2+)-free ATP did not. Studies of CFTR mutants indicated that ATP controls channel activity independent of the R domain and suggested that hydrolysis of ATP by NBD1 may be sufficient for channel opening. The finding that nucleoside triphosphates regulate CFTR begins to explain why CF-associated mutations in the NBDs block Cl- channel function.

Adenosine Triphosphate↗
PubMed1991

Calcium and cAMP activate different chloride channels in the apical membrane of normal and cystic fibrosis epithelia.

The genetic disease cystic fibrosis (CF) causes decreased Cl- transport in several epithelia. cAMP-dependent regulation of apical membrane Cl- channels is defective in CF airway epithelia; as a result, CF epithelia fail to secrete Cl-. In contrast, Ca(2+)-stimulated Cl- secretion is intact in CF airway epithelia and thus has the potential to bypass the CF Cl- secretory defect. For a Cl- channel to govern Cl- secretion, it must be located in the apical membrane. To specifically investigate apical membrane Cl- channels, we studied cells grown on permeable filter supports and measured Cl- currents across the apical membrane. We found that Ca2+ and cAMP activate different Cl- channels in the apical membrane. (i) Ca(2+)-activated Cl- channels were present in the apical membrane of airway but not in intestinal epithelia. (ii) cAMP- but not Ca(2+)-activated Cl- channels were defective in CF airway epithelia. (iii) Ca(2+)- but not cAMP-activated Cl- channels were blocked by 4,4'-diisothiocyanato-2,2'-stilbenedisulfonate. (iv) Ca(2+)- and cAMP-activated apical channels had different anion permeabilities. (v) An increase in both second messengers produced an additive increase in Cl- current. These results also explain the puzzling observation that Ca(2+)-stimulated Cl- secretion is defective in CF intestine: the Ca(2+)-activated Cl- channels that could circumvent the Cl- secretory defect in CF airway are missing from the apical membrane of intestinal epithelia.

Calcium↗
PubMed1991

Demonstration that CFTR is a chloride channel by alteration of its anion selectivity.

Expression of the cystic fibrosis transmembrane conductance regulator (CFTR) generates adenosine 3',5'-monophosphate (cAMP)-regulated chloride channels, indicating that CFTR is either a chloride channel or a chloride channel regulator. To distinguish between these possibilities, basic amino acids in the putative transmembrane domains were mutated. The sequence of anion selectivity of cAMP-regulated channels in cells containing either endogenous or recombinant CFTR was bromide greater than chloride greater than iodide greater than fluoride. Mutation of the lysines at positions 95 or 335 to acidic amino acids converted the selectivity sequence to iodide greater than bromide greater than chloride greater than fluoride. These data indicate that CFTR is a cAMP-regulated chloride channel and that lysines 95 and 335 determine anion selectivity.

Amino Acid Sequence↗
PubMed1991

Effect of deleting the R domain on CFTR-generated chloride channels.

The cystic fibrosis transmembrane conductance regulator (CFTR), which forms adenosine 3',5'-monophosphate (cAMP)-regulated chloride channels, is defective in patients with cystic fibrosis. This protein contains two putative nucleotide binding domains (NBD1 and NBD2) and an R domain. CFTR in which the R domain was deleted (CFTR delta R) conducted chloride independently of the presence of cAMP. However, sites within CFTR other than those deleted also respond to cAMP, because the chloride current of CFTR delta R increased further in response to cAMP stimulation. In addition, deletion of the R domain suppressed the inactivating effect of a mutation in NBD2 (but not NBD1), a result which suggests that NBD2 interacts with the channel through the R domain.

Binding Sites↗
PubMed1991

Generation of cAMP-activated chloride currents by expression of CFTR.

Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) cause cystic fibrosis. In order to evaluate its function, CFTR was expressed in HeLa, Chinese hamster ovary (CHO), and NIH 3T3 fibroblast cells, and anion permeability was assessed with a fluorescence microscopic assay and the whole-cell patch-clamp technique. Adenosine 3',5'-monophosphate (cAMP) increased anion permeability and chloride currents in cells expressing CFTR, but not in cells expressing a mutant CFTR (delta F508) or in nontransfected cells. The simplest interpretation of these observations is that CFTR is itself a cAMP-activated chloride channel. The alternative interpretation, that CFTR directly or indirectly regulates chloride channels, requires that these cells have endogenous cryptic, chloride channels that are stimulated by cAMP only in the presence of CFTR.

Animals↗
PubMed1991

Atrazine Resistance in a Velvetleaf (Abutilon theophrasti) Biotype Due to Enhanced Glutathione S-Transferase Activity.

We previously reported that a velvetleaf (Abutilon theophrasti Medic) biotype found in Maryland was resistant to atrazine because of an enhanced capacity to detoxify the herbicide via glutathione conjugation (JW Gronwald, Andersen RN, Yee C [1989] Pestic Biochem Physiol 34: 149-163). The biochemical basis for the enhanced atrazine conjugation capacity in this biotype was examined. Glutathione levels and glutathione S-transferase activity were determined in extracts from the atrazine-resistant biotype and an atrazine-susceptible or "wild-type" velvetleaf biotype. In both biotypes, the highest concentration of glutathione (approximately 500 nanomoles per gram fresh weight) was found in leaf tissue. However, no significant differences were found in glutathione levels in roots, stems, or leaves of either biotype. In both biotypes, the highest concentration of glutathione S-transferase activity measured with 1-chloro-2,4-dinitrobenzene or atrazine as substrate was in leaf tissue. Glutathione S-transferase measured with 1-chloro-2,4-dinitrobenzene as substrate was 40 and 25% greater in leaf and stem tissue, respectively, of the susceptible biotype compared to the resistant biotype. In contrast, glutathione S-transferase activity measured with atrazine as substrate was 4.4- and 3.6-fold greater in leaf and stem tissue, respectively, of the resistant biotype. Kinetic analyses of glutathione S-transferase activity in leaf extracts from the resistant and susceptible biotypes were performed with the substrates glutathione, 1-chloro-2,4-dinitrobenzene, and atrazine. There was little or no change in apparent K(m) values for glutathione, atrazine, or 1-chloro-2,4-dinitrobenzene. However, the V(max) for glutathione and atrazine were approximately 3-fold higher in the resistant biotype than in the susceptible biotype. In contrast, the V(max) for 1-chloro-2,4-dinitrobenzene was 30% lower in the resistant biotype. Leaf glutathione S-transferase isozymes that exhibit activity with atrazine and 1-chloro-2,4-dinitrobenzene were separated by fast protein liquid (anion-exchange) chromatography. The susceptible biotype had three peaks exhibiting activity with atrazine and the resistant biotype had two. The two peaks of glutathione S-transferase activity with atrazine from the resistant biotype coeluted with two of the peaks from the susceptible biotype, but peak height was three- to fourfold greater in the resistant biotype. In both biotypes, two of the peaks that exhibit glutathione S-transferase activity with atrazine also exhibited activity with 1-chloro-2,4-dinitrobenzene, with the peak height being greater in the susceptible biotype. The results indicate that atrazine resistance in the velvetleaf biotype from Maryland is due to enhanced glutathione S-transferase activity for atrazine in leaf and stem tissue which results in an enhanced capacity to detoxify the herbicide via glutathione conjugation.

Journal Article↗
PubMed1991

Maturation and function of cystic fibrosis transmembrane conductance regulator variants bearing mutations in putative nucleotide-binding domains 1 and 2.

One feature of the mutations thus far found to be associated with the disease cystic fibrosis (CF) is that many of them are clustered within the first nucleotide-binding domain (NBD) of the CF transmembrane conductance regulator (CFTR). We sought to discover the molecular basis for this clustering by introducing into the two NBDs of CFTR mutations either mimicking amino acid changes associated with CF or altering residues within highly conserved motifs. Synthesis and maturation of the mutant CFTR were studied by transient expression in COS cells. The ability of the altered proteins to generate cyclic AMP-stimulated anion efflux was assessed by using 6-methoxy-N-(sulfopropyl) quinolinium (SPQ) fluorescence measurements in HeLa cells expressing mutated plasmids. The results show that (i) all CF-associated mutants, with one exception, lack functional activity as measured in the SPQ assay, (ii) mutations in NBD1 are more sensitive to the effects of the same amino acid change than are the corresponding mutations in NBD2, (iii) cells transfected with plasmids bearing CF-associated mutations commonly but not exclusively lack mature CFTR, (iv) NBD mutants lacking mature CFTR fail to activate Cl- channels, and (v) the glycosylation of CFTR, per se, is not required for CFTR function. We reason that the structure of NBD1 itself or of the surrounding domains renders it particularly sensitive to mutational changes. As a result, most NBD1 mutants, but only a few NBD2 mutants, fail to mature or lack functional activity. These findings are consistent with the observed uneven distribution of CFTR missense mutations between NBD1 and NBD2 of CF patients.

Amino Acid Sequence↗