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D M Roberts

Publications and source records attributed to D M Roberts.

At least 37 records · Page 2Linked to original sources
PubMed1997

Altered methylation substrate kinetics and calcium binding of a calmodulin with a Val136-->Thr substitution.

Calmodulin is trimethylated on Lys115 by a specific calmodulin methyltransferase. Previously, it was shown that the cam2 mutant (Ile136-->Thr) of Paramecium has a decreased level of methylated Lys115 [Lukas, T. J., Friedman, M. W., Kung, C. & Watterson, D. M. (1989) Proc. Natl Acad. Sci. USA 86, 7331-7335]. To investigate how this substitution affects calmodulin structure, function and recognition by the calmodulin methyltransferase, a calmodulin with a Thr136 substitution ([Thr136]calmodulin) was expressed in Escherichia coli in an unmethylated form for in vitro enzyme activator, calcium binding and methylation kinetic analyses. [Thr136]calmodulin was indistinguishable from wild-type calmodulin in saturating (1 mM) calcium in its ability to activate calmodulin-dependent enzymes and in its steady-state kinetic properties with isolated calmodulin methyltransferase. However, [Thr136]calmodulin did show two defects: a complete inability to be methylated in the absence of calcium; and defective calcium binding. As a result, an approximate 10-fold shift in the K0.5 values for calcium dependence of enzyme activation (shifted from 1.1 microM to 9.1 microM of Ca2+ for NAD kinase) and methylation (from 0.71 microM to 7.2 microM of Ca2+ in 0.15 M K+, 2 mM Mg2+) were observed. Non-denaturing electrophoresis and Tyr138 spectroscopic measurements suggest a difference in the conformation of the calcium-depleted structures of normal calmodulin and [Thr136]calmodulin. Overall, the results suggest that the mutation in this conserved position in the COOH-terminal hydrophobic core lowers calcium-binding affinity and alters the calcium-depleted structure leading to decreased methylation at physiological Ca2+ concentrations.

Animals↗
PubMed1996

Effect of weather conditions on the flight activity of Nigerian blackflies (Diptera:Simuliidae).

A vehicle-mounted net was used to make hourly catches of blackflies at 700-1000 m altitude in the River Assob valley, central Nigeria, on 20 days during the dry season. Pearson Correlation Matrix analysis of the collection data and meteorology showed that the main factors affecting flight activity of each of the four most abundant blackfly species were primarily light intensity and secondly wind velocity, whereas relative humidity was the least important factor. Flight activity showed a negative linear regression against wind, with some activity occurring in wind speeds up to 15 km/h. Distance-weighted least-squares (DWLS) regressions showed little correlation of temperature with activity for Simulium hargreavesi and S.adersi, but S.squamosum and S.vorax had small peaks at 28 degrees C and 31 degrees C, respectively. DWLS regression against light intensity showed an activity peak at 6000 lux, except in Simulium adersi.

Animals↗
PubMed1995

Phosphorylation of nodulin 26 on serine 262 affects its voltage-sensitive channel activity in planar lipid bilayers.

Nodulin 26 is an symbiosome membrane protein of soybean nodules that shows ion channel activity in planar lipid bilayers. Serine 262 of nodulin 26 is phosphorylated by calmodulin-like domain protein kinase. To study the effects of phosphorylation, nodulin 26 with Ser, Ala, or Asp at position 262 were expressed in Escherichia coli. The expressed protein possessed a histidine-rich leader sequence for purification by Ni2+ chelate fast protein liquid chromatography. Upon reconstitution into planar lipid bilayers, the recombinant proteins showed a large single channel conductance (3.1 nanosiemens (nS) in cis0.2M/trans1.0 M KCl and 1.6 nS in cis 0.2M/trans0.2 M KCl) and weak anion selectivity, similar to native soybean nodulin 26. Nodulin 26 with Ser- or Ala-262 occupied the maximal open conductance state greater than 97% of the time (3.1 nS in cis0.2M/trans1.0 M KCl) regardless of applied voltage. However, nodulin 26 with Asp-262 showed increased gating and preferential occupancy of lower subconductance states (1.8 and 0.6 nS in cis0.2M/trans1.0 M KCl) at high applied voltages (e.g. 70 mV). In situ phosphorylation of Ser-262 of nodulin 26 by calmodulin-like domain protein kinase also resulted in increased voltage-dependent gating and preferential occupancy of lower subconductance states. These results suggest that phosphorylation of serine 262 of nodulin 26 modulates channel activity by conferring voltage sensitivity.

Base Sequence↗
PubMed1995

Determination of codeine in human plasma by high-performance liquid chromatography with fluorescence detection.

A rapid, reliable and rugged assay for determining codeine in human plasma using reversed-phase high-performance liquid chromatography with fluorescence detection was developed. This analytical method utilized an ion-exchange/mixed-mode solid-phase extraction procedure. The chromatographic separation was achieved using a 150 x 4.6 mm I.D., 3-microns reversed-phase C8 (deactivated for basic analytes) column at ambient temperature. Fluorescence detection (excitation at 214 nm and emission above 345 nm) for codeine and nalorphine allowed for a detectable limit of 5 ng/ml. The results showed that the method was linear from 10 to 300 ng/ml. The method had good reproducibility, precision, accuracy and recoveries of 91 and 90% for codeine and nalorphine, respectively. This method has been applied to study the pharmacokinetics of codeine in normal human subjects.

Chromatography, High Pressure Liquid↗
PubMed1995

Phylogenetic relationships among karyorelictids and heterotrichs inferred from small subunit rRNA sequences: resolution at the base of the ciliate tree.

Ciliate protozoa are among the most diverse and complex cells that have been described. Ciliates are characterized by nuclear dimorphism, possessing a macronucleus and a micronucleus which share the same cytoplasm. An understanding of the evolution of ciliate diversity depends upon knowledge of their phylogeny. In this study we attempted to resolve some of the relationships at the base of the ciliate tree by determining the phylogenetic position of a sample of heterotrich and hypothesized primitive karyorelictid ciliates. Karyorelictids are considered primitive because they possess a "simple" form of nuclear dualism whereby the macronucleus does not divide once it has differentiated from a micronucleus. We micromanipulated cells of two heterotrichs, Spirostomum ambiguum and Gruberia sp., and two karyorelictids, Loxodes magnus and Tracheloraphis sp., and amplified their small subunit (SSU) rDNA using PCR. The primary structure of the SSU rDNA was determined for each species and used to infer their positions in the ciliate phylogenetic tree. The results indicate, with strong support, that the aerobic heterotrichs and the karyorelictids sampled constitute a monophyletic group. The most parsimonious interpretation of the form of nuclear dimorphism in karyorelictids is that it is derived from the general condition as found in its sister group the aerobic heterotrichs. The two anaerobic heterotrichs, Metopus contortus and Metopus palaeformis, comprise a distinct clade, so that the subclass Heterotrichia, as currently conceived, is not a monophyletic group. The complex mouth architecture which characterizes all heterotrichs must be reassessed in light of this finding.

Animals↗
PubMed1995

Chloride transport activation by plasma osmolarity during rapid adaptation to high salinity of Fundulus heteroclitus.

Transition from low salt water to sea water of the euryhaline fish, Fundulus heteroclitus, involves a rapid signal that induces salt secretion by the gill chloride cells. An increase of 65 mOsm in plasma osmolarity was found during the transition. The isolated, chloride-cell-rich opercular epithelium of sea-water-adapted Fundulus exposed to 50 mOsm mannitol on the basolateral side showed a 100% increase in chloride secretion, which was inhibited by bumetanide 10(-4) M and 10(-4) M DPC (N-Phenylanthranilic acid). No effect of these drugs was found on apical side exposure. A Na+/H+ exchanger, demonstrated by NH4Cl exposure, was inhibited by amiloride and its analogues and stimulated by IBMX, phorbol esters, and epithelial growth factor (EGF). Inhibition of the Na+/H+ exchanger blocks the chloride secretion increase due to basolateral hypertonicity. A Cl-/HCO3- exchanger was also found in the chloride cells, inhibited by 10(-4) M DIDS but not involved in the hyperosmotic response. Ca2+ concentration in the medium was critical for the stimulation of Cl- secretion to occur. Chloride cell volume shrinks in response to hypertonicity of the basolateral side in sea-water-adapted operculi; no effect was found on the apical side. Fresh-water-adapted fish chloride cells show increased water permeability of the apical side. It is concluded that the rapid signal for adaptation to higher salinities is an increased tonicity of the plasma that induces chloride cell shrinkage, increased chloride secretion with activation of the Na+K+2Cl- cotransporter, the Na+/H+ exchanger and opening of Cl- channels.

Adaptation, Physiological↗
PubMed1995

Expression of soybean nodulin 26 in transgenic tobacco. Targeting to the vacuolar membrane and effects on floral and seed development.

Nodulin 26 is an integral membrane protein of the symbiosome membrane of nitrogen-fixing soybean nodules. We expressed a nodulin 26 cDNA in transgenic tobacco (TN26 tobacco) under the control of the cauliflower mosaic virus 35S promoter to study subcellular targeting and the physiological effect(s) of its expression. Based on Northern and Western blots, the expression of nodulin 26 mRNA and protein in transgenic plants is high in apical shoot sections, flowers, and stems, low in mature leaves, and absent in roots. Western blot analysis revealed high levels of transgenic nodulin 26 protein in tonoplast membranes. In contrast, nodulin 26 protein was not found in isolated plasma membranes, the soluble fraction, nor in chloroplast and mitochondria-enriched membrane fractions. About 50-60% of the flowers and pods from TN26 tobacco plants abscised prematurely. Seed capsule size and seed fill per capsule from the remainder of surviving flowers were about 50% of that of control plants. Pollen viability was found to be normal, but flowers from TN26 tobacco plants showed shorter anther filaments compared with control plants. Normal seed production and capsule size was restored by manually crossing the stigmas from TN26 plants with isolated pollen from either transgenic or control plants. Thus, the aberrant filament growth could have resulted in the reproductive defects associated with the plants.

Base Sequence↗
PubMed1995

Use of the PCR and fluorescent probes to recover SSU rRNA gene sequences from single cells of the ciliate protozoon Spathidium.

A two-stage heminested PCR approach was developed to amplify small subunit (SSU) rDNA sequences, via two overlapping fragments, from single cells of microbial eucaryotes. The method was evaluated using the ciliate protozoon Spathidium when PCR products were obtained from nine of 10 cells tested. Southern blotting demonstrated that all fragments contained the same sequence in a region of SSU rDNA which is normally highly variable between species. A fluorescent oligonucleotide probe was used to demonstrate that this sequence also occurred in fixed cells of Spathidium. Fixatives containing mercuric salts preserved cell shape and allowed probe binding with little background autofluorescence. The Spathidium sequence is closely related to that from the haptorid Homalozoon vermiculare.

Animals↗
PubMed1994

Nodulin 26, a nodule-specific symbiosome membrane protein from soybean, is an ion channel.

Nodulin 26 is an integral symbiosome membrane protein of nitrogen-fixing soybean nodules. Nodulin 26 is a member of a family of structurally homologous membrane proteins with diverse transport functions. Thus, it has been proposed to be involved in symbiosome membrane transport. Despite this claim, there has not been any evidence that nodulin 26 has a transport activity. In this study, nodulin 26 was purified from soybean nodules by a non-denaturing protocol and was reconstituted into liposomes for channel studies in planar lipid bilayers. Nodulin 26 readily incorporated into bilayers, forming single channels with a maximum unitary conductance of 3.1 nanosiemens (nS) in a recording buffer of 20 mM 3-(N-morpholino)propanesulfonate-NaOH, pH 7.4, 1 M KCl. Nodulin 26 also exhibited multiple, discreet lower conductance states ranging from 0.5 to 2.5 nS. Nodulin 26 channels were voltage-sensitive. The maximal 3.1-nS state was preferentially occupied at lower applied voltages, whereas the lower conductance states were more frequently occupied at higher voltage potentials. Nodulin 26 channels transported both cations and anions, but showed a weak selectivity for anions. These results represent the first purification and functional characterization of the nodulin 26 channel and support a role for this protein in symbiosome membrane transport.

Ion Channels↗
PubMed1994

Arabian sandflies (Diptera: Psychodidae) prefer the hottest nights?

A vehicle-mounted net was used to collect hourly samples of sandflies on 15 nights during June in northern Oman. Every half hour, the temperature, relative humidity, wind velocity and light intensity were measured (there was no cloud or rainfall during this period). The sandflies caught were mainly Phlebotomus alexandri and Sergentomyia clydei. Their circadian activity increased rapidly after sunset (18.50 hours). The high level of activity was fairly constant during 9h of darkness until dawn, when it decreased rapidly. A few flies were still active at 07.00 hours, 1.5h after sunrise. A multiple regression showed that the main factor affecting sandfly activity was light intensity. When this factor was removed, by considering only the 135 catches collected during the 9h of darkness, the second most important factor was low relative humidity, followed by low wind velocity. Temperature was not a significant factor in the analysis, because of its strong negative correlation with humidity. However, when the effect of humidity was removed from the regression, high temperature became significant, but less important than wind. The regressions showed that, for flight activity, the optimum humidity was around 10%; the probable maximum wind velocity was 3.5 m s-1 and 11 degrees C was the probable minimum temperature. Thus, when the 4 nights with highest catches (200-260 flies/night) were compared with the 4 nights with lowest catches (50-120 flies/night), the best nights had a low humidity (10-25%) and low wind speed (< 0.3 m s-1) in combination with highest temperatures (31-43 degrees C).

Animals↗
PubMed1993

Isolation and kinetic characterization of the calmodulin methyltransferase from sheep brain.

The methyltransferase that catalyzes the trimethylation of lysine 115 in calmodulin has been purified from sheep brain. The enzyme is a monomer with an apparent molecular weight of 38,000 on the basis of gel filtration chromatography and SDS-polyacrylamide electrophoresis. In the presence of calcium the methyltransferase exhibited a Km of 100 nM for unmethylated calmodulin and a kcat of 0.0278 s-1. The enzyme was able to use calcium-depleted calmodulin as a substrate, albeit with less efficiency. The methylation of calcium-depleted calmodulin was inhibited by increases in ionic strength, whereas methylation of calcium-saturated calmodulin was not affected. This suggests a difference in the mode of interaction of calcium-saturated and calcium-depleted calmodulins with the enzyme. As with calmodulin's interactions with other calmodulin-dependent enzymes, the oxidation of the methionines of calmodulin by performic acid treatment decreases the ability of the methyltransferase to recognize and methylate calmodulin. A calmodulin-binding peptide based on the calmodulin-dependent protein kinase II sequence and the naphthalenesulfonamide W-7 inhibit the calmodulin methyltransferase-calmodulin interaction in a calcium-dependent manner. Removal of the NH2-terminal lobe (residues 1-77) does not affect the ability of the calmodulin methyltransferase to recognize and methylate lysine 115. Thus, the determinants for calmodulin methyltransferase binding reside solely in the COOH-terminal lobe of calmodulin. Further, structural features within this region, in particular, the hydrophobic cleft, that are manifested upon calcium binding may contribute to the interaction of calmodulin with the enzyme.

Amino Acid Sequence↗
PubMed1993

Protein kinases with calmodulin-like domains: novel targets of calcium signals in plants.

Recently, a novel calcium-dependent protein kinase has been identified that is structurally distinguished by the localization of a calcium-binding regulatory domain fused to a serine/threonine catalytic domain. The regulatory domain is homologous to calmodulin and contains four helix-loop-helix calcium-binding sites. As a result, the kinase is directly activated by calcium without a requirement for other effector molecules.

Amino Acid Sequence↗
PubMed1992

Determination of the site of phosphorylation of nodulin 26 by the calcium-dependent protein kinase from soybean nodules.

Nodulin 26 is a nodule-specific protein that is associated with the symbiosome membrane of soybean root nodules. Nodulin 26 is an endogenous substrate for a novel calcium-dependent protein kinase (CDPK) of soybean root nodules. By phosphopeptide mapping of endoproteinase Lys-C-digested nodulin 26 and automated and manual peptide sequence analyses, we have identified the site on nodulin 26 phosphorylated by CDPK. We have also established that the phosphorylation site of nodulin 26 is identical to the phosphorylation site of CK-15, a synthetic peptide with the carboxyl-terminal sequence of nodulin 26. The phosphorylation of nodulin 26 occurs at position Ser262, and the phosphorylation of CK-15 occurs at the analogous position, Ser,6 in vitro. Thus, the CK-15 sequence apparently contains sufficient structural features of the phosphorylation site of nodulin 26 to be recognized by CDPK. On the basis of peptide mapping analysis of nodulin 26 from nodules that are metabolically labeled with [32P]phosphate, it appears that the site of nodulin 26 that is phosphorylated in vitro is also labeled in vivo. The data indicate that the carboxyl terminus of nodulin 26 is phosphorylated by CDPK and provide initial sequence data for the phosphorylation site of an endogenous substrate for a plant CDPK.

Amino Acid Sequence↗
PubMed1992

Expression of a calmodulin methylation mutant affects the growth and development of transgenic tobacco plants.

Transgenic plants were constructed that express two foreign calmodulins (VU-1 and VU-3 calmodulins) derived from a cloned synthetic calmodulin gene. VU-1 calmodulin, similar to endogenous plant calmodulin, possesses a lysine residue at position 115 and undergoes posttranslational methylation. VU-3 calmodulin is a site-directed mutant of VU-1 calmodulin that is identical in sequence except for the substitution of an arginine at position 115 and thus is incapable of methylation. Both calmodulin genes, under the control of the cauliflower mosaic virus 35S promoter, were expressed in transgenic tobacco. Foreign calmodulin protein accumulated in plant tissues to levels equivalent to that of the endogenous calmodulin. All transformed lines of VU-1 plants were indistinguishable from untransformed controls with respect to growth and development. However, all transformed lines of VU-3 plants were characterized by decreased stem internode growth, reduced seed production, and reduced seed and pollen viability. The data suggest that these phenotypes are the result of the expression of the calmodulin mutant rather than the position of transferred DNA insertion or the overall alteration of calmodulin levels. Analyses of the activity of the purified transgenic calmodulins suggest that calmodulin-dependent NAD kinase is among the potential targets that may have altered regulation in VU-3 transgenic plants.

Amino Acid Sequence↗
PubMed1992

Modulation of calmodulin levels, calmodulin methylation, and calmodulin binding proteins during carrot cell growth and embryogenesis.

Carrot cell cultures were used to study the dynamics of calmodulin protein levels, calmodulin methylation, and calmodulin-binding proteins during plant growth and development. Comparisons of proliferating and nonproliferating wild carrot cells show that, while calmodulin protein levels does not vary significantly, substantial variation in post-translational methylation of calmodulin on lysine-115 is observed. Calmodulin methylation is low during the lag and early exponential stages, but increases substantially as exponential growth proceeds and becomes maximal in the postexponential phase. Unmethylated calmodulin quickly reappears within 12 h of reinoculation of cells into fresh media, suggesting that the process is regulated according to the cell growth state. Calmodulin and calmodulin-binding proteins were also analyzed during the formation and germination of domestic carrot embryos in culture. Neither calmodulin methylation nor calmodulin protein levels varied significantly during somatic embryogenesis. However, upon germination of embryos, the level of calmodulin protein doubled. By calmodulin overlay analysis, we have detected a major 54,000 M(r) calmodulin-binding protein that also increased during embryo germination. This protein was purified from carrot embryo extracts by calmodulin-Sepharose chromatography. Overall, the data suggest that calmodulin methylation is regulated depending upon the state of cell growth and that calmodulin and its target proteins are modulated during early plant development.

Calmodulin↗
PubMed1992

Ophthalmic aspects of orbital injury. A comprehensive diagnostic and management approach.

Improved prognosis for eye injuries sustained during facial trauma depends on early assessment and accurate diagnosis. The surgeon responsible for management of the orbital fracture(s) must perform an ocular assessment at the time of initial examination. Abnormalities found on the five-point assessment outlined herein should lead to urgent ophthalmic consultation. Fracture repair should not be undertaken until the results of consultation have been discussed with the ophthalmologist. Vision should be carefully monitored in the preoperative period because delayed optic neuropathy may occur. Similarly, vision should be followed regularly in the postoperative period after reduction of fractures of the orbital bones. Decreased postoperative vision should lead to immediate ocular assessment and early ophthalmologic consultation.

Clinical Protocols↗
PubMed1991

Protein phosphorylation stimulates the rate of malate uptake across the peribacteroid membrane of soybean nodules.

Incubation of intact isolated symbiosomes with [gamma-32P]ATP, followed by isolation of the peribacteroid membrane and polypeptide analysis, showed that a single major polypeptide at 26 kDa was labelled. Antibodies raised against nodulin 26 reacted with a similar sized polypeptide. Incubation of the symbiosomes with alkaline phosphatase removed the label from this polypeptide. Pre-incubation with ATP stimulated malate accumulation by isolated symbiosomes, but only slightly (10-30%). Pre-treatment of symbiosomes with alkaline phosphatase inhibited malate uptake substantially and this inhibition was completely relieved by addition of ATP. The ATP stimulation of malate uptake was not affected by ATPase inhibitors. It is suggested that the rate of malate uptake across the peribacteroid membrane is controlled by phosphorylation of nodulin 26.

Alkaline Phosphatase↗