PubMed Health⌕ Search

Biomedical subjects

D M Roberts

Publications and source records attributed to D M Roberts.

At least 55 records · Page 3Linked to original sources
PubMed1991

Restoration of the calcium binding activity of mutant calmodulins toward normal by the presence of a calmodulin binding structure.

The altered calcium binding activity of calmodulins (CaM) with point mutations can be restored toward that of wild type CaMs by the formation of a complex between CaM and a CaM binding sequence. Three different site-specific mutations resulted in selective effects on the apparent stoichiometry and affinity of CaM for calcium, with maintenance of the ability to activate myosin light chain kinase. The effects on calcium binding, however, were suppressed when the mutant CaMs were complexed with RS20, a peptide analog of a myosin light chain kinase CaM binding site. The mutations included: 1) a Glu----Ala mutation at two phylogenetically conserved calcium ligands in the second (E67A-CaM) and fourth (E140A-CaM) sites; and 2) a Ser----Phe mutation at residue 101 (S101F-CaM) which affects ion channel regulation. The mutant CaMs bind 4 calciums in the absence of magnesium, but two sites have approximately 60- to 300-fold weaker binding than wild-type CaM (SYNCAM CaM). E67A-CaM and E140A-CaM bound only two calciums and S101F-CaM bound 4 calciums in the presence of magnesium. E67A-CaM and E140A-CaM recovered the ability to bind 4 calcium ions in the presence of the RS20 CaM binding peptide. These results are consistent with models in which the calcium binding activity of CaM within a supramolecular complex is different from purified CaM and raise the possibility that the selective functional effects of in vivo mutations in the calcium binding sites of CaM might be partially due to the ability of some CaM binding proteins to select and utilize CaM conformations with calcium ligation structures different from the so-called canonical EF-hand.

Amino Acid Sequence↗
PubMed1991

Calcium-dependent phosphorylation of symbiosome membrane proteins from nitrogen-fixing soybean nodules : evidence for phosphorylation of nodulin-26.

By using a peptide (CK-15) based on the COOH-terminal sequence of nodulin-26, we have demonstrated the presence of a Ca(2+)-dependent protein kinase in soluble as well as particulate fractions of nitrogen-fixing soybean (Glycine max) root nodules. Substantial enzyme activity was found in symbiosome membranes. The soluble enzyme was purified 1570-fold. The enzyme was fractionated from endogenous calmodulin and yet was fully activated by Ca(2+) (K(0.5) = 0.4 micromolar) in the absence of exogenous calmodulin, phosphatidylserine and 1,2-dioleylglycerol, oleic acid, and platelet activating factor. CK-15 was used to generate a site-specific antibody to nodulin-26. The antibody reacted with a protein in the symbiosome membrane with an apparent molecular mass of 27,000 daltons, consistent with the molecular mass predicted for nodulin-26 from the deduced amino acid sequence. A symbiosome membrane protein with an identical electrophoretic mobility was phosphorylated in vitro in a Ca(2+)-dependent manner. Additionally, this symbiosome membrane protein was phosphorylated when nodules were incubated with (32)P-phosphate. Overall, the results show the existence of a Ca(2+)-dependent and calmodulin/lipid-independent enzyme in nitrogen-fixing soybean root nodules and suggest that nodulin-26 is a substrate for Ca(2+)-dependent phosphorylation.

Journal Article↗
PubMed1990

Isolation and characterization of the Fc receptor from the fetal yolk sac of the rat.

The yolk sac of the fetal rat and the proximal small intestine of the neonatal rat selectively transport maternal IgG. IgG-Fc receptors are thought to mediate transport across the epithelium of both tissues. We used a mouse mAb (MC-39) against the 45-54-kD component of the Fc receptor of the neonatal intestine to find an antigenically related protein that might function as an Fc receptor in fetal yolk sac. In immunoblots of yolk sac, MC-39 recognized a protein band with apparent molecular mass of 54-58 kD. MC-39 bound to the endoderm of yolk sac in immunofluorescence studies. In immunogold-labeling experiments MC-39 was associated mainly with small vesicles in the apical cytoplasm and in the region near the basolateral membrane of endodermal cells. The MC-39 cross-reactive protein and beta 2-microglobulin, a component of the intestinal Fc receptor, were copurified from detergent-solubilized yolk sac by an affinity purification that selected for proteins which, like the intestinal receptor, bound to IgG at pH 6.0 and eluted at pH 8.0. In summary, the data suggest that we have isolated the Fc receptor of the yolk sac and that this receptor is structurally and functionally related to the Fc receptor of the neonatal intestine. An unexpected finding is that, unlike the intestinal receptor which binds maternal IgG on the apical cell surface, the yolk sac receptor appears to bind IgG only within apical compartments which we suggest represent the endosomal complex.

Animals↗
PubMed1990

Analysis of the state of posttranslational calmodulin methylation in developing pea plants.

A specific calmodulin-N-methyltransferase was used in a radiometric assay to analyze the degree of methylation of lysine-115 in pea (Pisum sativum) plants. Calmodulin was isolated from dissected segments of developing roots of young etiolated and green pea plants and was tested for its ability to be methylated by incubation with the calmodulin methyltransferase in the presence of [(3)H]methyl-S-adenosylmethionine. By this approach, the presence of unmethylated calmodulins were demonstrated in pea tissues, and the levels of methylation varied depending on the developmental state of the tissue tested. Calmodulin methylation levels were lower in apical root segments of both etiolated and green plants, and in the young lateral roots compared with the mature, differentiated root tissues. The incorporation of methyl groups into these calmodulin samples appears to be specific for position 115 since site-directed mutants of calmodulin with substitutions at this position competitively inhibited methyl group incorporation. The present findings, combined with previous data showing differences in the ability of methylated and unmethylated calmodulins to activate pea NAD kinase (DM Roberts et al. [1986] J Biol Chem 261: 1491-1494) raise the possibility that posttranslational methylation of calmodulin could be another mechanism for regulating calmodulin activity.

Journal Article↗
PubMed1990

How plants respond to stimuli. The Ninth Annual Symposium on Current Topics in Plant Biochemistry and Physiology sponsored by the University of Missouri Interdisciplinary Program in Plant Biochemistry and Physiology, the US Department of Agriculture, and the National Science Foundation, Columbia, MO, USA, April 4-7, 1990.

With the rapid progress in the identification of the molecular components of calcium and protein kinase regulatory systems, it is presently an exciting time for those studying plant signal transduction. A clear message that was conveyed at the meeting was the need for an interdisciplinary approach to the problems currently facing researchers in this area. Thus, while the powerful methods of plant molecular biology allow for rapid advances in the identification of new molecules, this approach needs to be combined with careful biochemical and physiological analyses. This was exemplified by the large number of protein kinases described at the meeting but the surprisingly few reports of physiological function of phosphorylation. Similarly, while calcium has emerged as a major regulatory molecule in plants, little is known regarding the exact molecular mechanisms of calcium and calcium-modulatory protein action. These are clearly areas for future study that should reveal the elusive pathways that lie between stimuli and responses in the plant cell.

Calcium↗
PubMed1989

Fluorescence characterization of VU-9 calmodulin, an engineered calmodulin with one tryptophan in calcium binding domain III.

Absorption and fluorescence properties of VU-9 calmodulin, an engineered calmodulin in which a tryptophan residue has been introduced in position 99, have been investigated. Tryptophan 99 fluoresces with a maximum around 348 nm and is easily quenched by fluorescence quenchers such as acrylamide, indicating that the chromophore is in a polar environment and well exposed to the solvent, a location which has been reported previously for tyrosine 99 in mammalian calmodulin [Kilhoffer, M. C., Demaille, J. G., & Gérard, D. (1981) Biochemistry 20, 4407-4414]. The quantum yields of tryptophan 99 were found to be 0.19 in the absence of calcium and 0.15 in its presence. These values indicate that the chromophore is in a particular microenvironment where it is protected from the quenching mechanisms normally occurring in proteins. Steady-state fluorescence polarization measurements indicate that the protein exhibits segmental mobility both in the absence and in the presence of calcium. Binding of calcium decreases the mobility of the chromophore, a good indication for a rigidification of the protein structure. A quite rigid structure of at least the carboxy-terminal part of VU-9 calmodulin in the presence of Ca2+ is also suggested by Förster energy-transfer measurements.

Binding Sites↗
PubMed1989

Calmodulin-binding proteins are developmentally regulated in gametes and embryos of fucoid algae.

Calcium-binding proteins and calmodulin-binding proteins were identified in gametes and zygotes of the marine brown algae Fucus vesiculosus, Fucus distichus, and Pelvetia fastigiata using gel (SDS-PAGE) overlay techniques. A calcium current appears to be important during cell polarization in fucoid zygotes (K.R. Robinson and L.F. Jaffe, 1975, Science 187, 70-72; K.R. Robinson and R. Cone, 1980, Science 207, 77-78), but there are no biochemical data on calcium-binding proteins in these algae. By using a sensitive 45Ca2+ overlay method designed to detect high-affinity calcium-binding proteins, at least 9-11 polypeptides were detected in extracts of fucoid gametes and zygotes. All samples had calcium-binding proteins with apparent molecular weights of about 17 and 30 kDa. A 17-kDa calcium-binding protein was purified by calcium-dependent hydrophobic chromatography and was identified as calmodulin by immunological and enzyme activator criteria. A 125I-calmodulin overlay assay was used to identify potential targets of calmodulin action. Sperm contained one major calmodulin-binding protein of about 45 kDa. Eggs lacked major calmodulin-binding activity. A 72-kDa calmodulin-binding protein was prominent in zygotes from 1-65 hr postfertilization. Both calmodulin-binding proteins showed calcium-dependent binding activity. Overall, the data suggest that the appearance and distribution of certain calcium-binding and calmodulin-binding proteins are under developmental regulation, and may reflect the different roles of calcium during fertilization and early embryogenesis.

Autoradiography↗
PubMed1989

Systemic lupus erythematosus. How to manage this chronic, complicated disorder.

Systemic lupus erythematosus is an autoimmune disease of unknown cause characterized by exacerbations and remissions. Disease manifestations are diverse, but the most common symptoms are fatigue, fever, weight loss, skin rash, arthritis or arthralgias, and anemia. Clinical findings and measurement of antibody titers are the most common diagnostic criteria. Individualized therapy with corticosteroids, immunosuppressives, or antimalarial agents and careful monitoring of disease activity are essential.

Anti-Inflammatory Agents, Non-Steroidal↗
PubMed1989

Detection of a calcium-activated protein kinase in mougeotia by using synthetic Peptide substrates.

By using a synthetic peptide, KM-14, a protein kinase was detected and partially purified from Mougeotia sp. The peptide contains the sequence of the regulatory light chain of smooth muscle myosin that is phosphorylated by calcium-calmodulin-dependent myosin light chain kinase (MLCK). The Mougeotia kinase was stimulated 40-fold by calcium with half-maximal stimulation occurring at 1.5 micromolar. The enzyme was fractionated from calmodulin and was depleted of calmodulin based on enzyme activator analysis. The calmodulin-depleted enzyme was fully active and calcium dependent, and was not stimulated further by exogenous calmodulin nor by the calcium effectors phosphatidylserine and diacylglycerol. The enzyme phosphorylated intact chicken gizzard myosin light chain as well as the KM-14 substrate. KM-13, a peptide analog of KM-14 with a deletion of a glutamine at position 5, was a poor substrate with a V(max)/K(m) ratio 200-fold lower than KM-14. Thus, similarly to vertebrate MLCK, the Mougeotia enzyme is very sensitive to changes in sequence surrounding the phosphorylation site. Calcium-dependent KM-14 kinase activity also was detected in two other algae, Mesotaenium caldariorum and Spirogyra sp., as well as in pea seedlings. The data suggest that plant and algal tissues possess an enzyme with a substrate specificity similar to MLCK, but unlike MLCK, does not appear to require calmodulin for activity.

Journal Article↗
PubMed1988

Investigation of the mechanism of calcium binding to calmodulin. Use of an isofunctional mutant with a tryptophan introduced by site-directed mutagenesis.

A mutant calmodulin, in which phenylalanine 99 of calcium binding site III was changed to a tryptophan by using cassette-based, site-directed mutagenesis, has been used to analyze the mechanism of calcium binding. The combined study of direct calcium binding, modification of tryptophan fluorescence properties upon calcium binding, and terbium titration allows some discrimination among proposed mechanisms of cation binding to calmodulin. Calmodulin appears to have six cation binding sites, four of which are selective for calcium, that seem to be coupled. Under a given set of conditions, these calcium-selective sites are not identical. In addition to providing insight into the mechanisms of calcium modulation of calmodulin, these studies demonstrate the feasibility of using isofunctional, tryptophan-containing mutants of proteins to gain insight into protein-ligand interaction.

Amino Acid Sequence↗
PubMed1988

Prevalence of giardiasis in patients with cystic fibrosis.

A group of 107 patients with cystic fibrosis and a control group of 64 normal members of households of patients with cystic fibrosis were surveyed for Giardia lamblia cysts and trophozoites by counterimmunoelectrophoresis of fecal samples. The patient group had a significantly higher rate of infestation than the control group (28.0% vs 6.3%, P = 0.0006), and the disparity between the two groups increased with age (P = 0.005). Aside from cystic fibrosis, all risk factors examined were without influence, except for the presence of household members less than or equal to 5 years of age. We conclude that our patients with cystic fibrosis have a previously unrecognized increased prevalence of giardiasis compared with that in a control population.

Adolescent↗
PubMed1988

Low temperature storage and pupal survival in the Simulium damnosum complex.

Pupae of the Simulium damnosum complex were exposed for different time periods to temperatures of 0, 4, 8 and 12 degrees C before returning to ambient temperature (24-28 degrees C). As the period of exposure to the low temperatures increased, pupal survival decreased. A one day exposure even to 0 degrees C had little effect on survival, whereas for a 4 day exposure, the optimum survival occurred at 8 degrees C with a total of 46% emergence. Young pupae (pale cuticle) had greatest emergence after exposure to 12 degrees C, the old ones (dark sclerotised cuticle) after exposure to 8 degrees C. Exposure to the different cold temperatures did not significantly affect the longevity of the emerging adults. Low temperature storage of pupae is thus a viable method for long distance transportation of living specimens of blackflies.

Animals↗
PubMed1988

Sheehan's syndrome.

Sheehan's syndrome, or postpartum pituitary necrosis, is rarely seen today, but it may occur in women who have more difficult deliveries. The syndrome may be subclinical in some patients and may manifest only if these patients are stressed. Diagnosis is based on laboratory studies, including hormone levels and hormone stimulation tests. Replacement hormone therapy, with careful follow-up of laboratory and clinical results, is the treatment.

Diagnosis, Differential↗
PubMed1987

Site-specific mutagenesis of the alpha-helices of calmodulin. Effects of altering a charge cluster in the helix that links the two halves of calmodulin.

Alteration of residues 82-84 in the alpha-helix that links the two halves of calmodulin results in a differential effect on activator activity. Previous studies (Lukas, T. J., Burgess, W. H., Prendergast, F. G., Lau, W., and Watterson, D. M. (1986) Biochemistry 25, 1458-1464) indicated the importance of positive charge clusters in the calmodulin-binding protein, myosin light chain kinase. This suggested the possible importance of complementary negative charge clusters in calmodulin. By using an efficient cassette mutagenesis approach and a synthetic calmodulin gene (Roberts, D. M., Crea, R., Malecha, M., Alvarado-Urbina, G., Chiarello, R. H., and Watterson, D. M. (1985) Biochemistry 24, 5090-5098), this possibility was directly addressed by engineering a new calmodulin, VU-8 calmodulin, in which the glutamate cluster at residues 82-84 in the synthetic gene product (VU-1 calmodulin) was replaced by three lysines. VU-8 calmodulin activated phosphodiesterase to the same maximal extent as VU-1 calmodulin, although there was an alteration in the concentration of calmodulin required for half-maximal stimulation. In contrast, myosin light chain kinase was activated to only 30% of maximal activity and NAD kinase was not activated. These results provide insight into the functional role of the unusual central helix structure found in the calmodulin family of proteins and indicate that different, although possibly overlapping, chemical complementarities are employed in the interaction between calmodulin and its various physiological targets.

Amino Acid Sequence↗