PubMed Health⌕ Search

Biomedical subjects

R G Burns

Publications and source records attributed to R G Burns.

At least 37 records · Page 2Linked to original sources
PubMed1993

Reflectance and Mossbauer spectroscopy of ferrihydrite-montmorillonite assemblages as Mars soil analog materials.

Spectroscopic analyses show that Fe(3+)-doped smectites prepared in the laboratory exhibit important similarities to the soils on Mars. Ferrihydrite has been identified as the interlayer ferric component in Fe(3+)-doped smectites by a low quadrupole splitting and magnetic field strength of approximately 48 tesla in Mossbauer spectra measured at 4.2 K, as well as a crystal field transition at 0.92 micrometer. Ferrihydrite in these smectites explains features in the visible-near infrared region that resemble the energies and band strengths of features in reflectance spectra observed for several bright regions on Mars. Clay silicates have met resistance in the past as Mars soil analogs because terrestrial clay silicates exhibit prominent hydrous spectral features at 1.4, 1.9, and 2.2 micrometers; and these are observed weakly, if at all, in reflectance spectra of Mars. However, several mechanisms can weaken or compress these features, including desiccation under low-humidity conditions. The hydration properties of the interlayer cations also effect band strengths, such that a ferrihydrite-bearing smectite in the Martian environment would exhibit a 1.9 micrometers H2O absorption that is even weaker than the 2.2 micrometers structural OH absorption. Mixing experiments demonstrate that infrared spectral features of clays can be significantly suppressed and that the reflectance can be significantly darkened by mixing with only a few percent of a strongly absorbing opaque material. Therefore, the absolute reflectance of a soil on Mars may be disproportionately sensitive to a minor component. For this reason, the shape and position of spectral features and the chemical composition of potential analogs are of utmost importance in assessing the composition of the soil on Mars. Given the remarkable similarity between visible-infrared reflectance spectra of soils in bright regions on Mars and Fe(3+)-doped montmorillonites, coupled with recent observations of smectites in SNC meteorites and a weak 2.2 micrometers absorption in some Mars soils, ferrihydrite-bearing smectites warrant serious consideration as a Mars soil analog.

Aluminum Silicates↗
PubMed1993

The highly divergent alpha- and beta-tubulins from Dictyostelium discoideum are encoded by single genes.

As a step in the characterization of the microtubule system of Dictyostelium discoideum, we have isolated and sequenced full-length cDNA clones that encode the Dictyostelium alpha- and beta-tubulins, as well as the Dictyostelium alpha-tubulin gene. Southern blot analysis suggests that Dictyostelium is unusual in that its genome contains single alpha- and beta-tubulin genes, rather than the multi-gene family common in most eukaryotic organisms. The complete alpha-tubulin cDNA contains 1558 nucleotides, with an open reading frame, that encode a protein of 457 amino acids. The complete beta-tubulin cDNA contains 1572 nucleotides and encodes a protein of 456 amino acids. Analysis of the deduced protein sequences indicates that while there is a significant degree of sequence similarity between the Dictyostelium tubulins and other known tubulins, the Dictyostelium alpha-tubulin displays the greatest sequence divergence yet described. Single alpha- and beta-tubulin transcripts are detected by northern blot analysis during all stages of Dictyostelium development. The highest levels of message accumulate late in germinating spores and vegetative amoebae. Despite changes in alpha- and beta-tubulin mRNA levels, protein levels remain constant throughout development. We have expressed the carboxy-terminal two-thirds of the alpha- and beta-tubulins as trpE fusions in Escherichia coli and used this protein to produce polyclonal antisera specific for the Dictyostelium alpha- and beta-tubulins. These antisera recognize one alpha- and two beta-tubulin spots on western blots of 2-D gels and, by indirect immunofluorescence, both recognize the interphase and mitotic microtubule arrays in vegetative amoebae.

Amino Acid Sequence↗
PubMed1992

Degradation of aliphatic halogen-substituted pesticides by dehalogenase isolated from Pseudomonas alcaligenes. Identification and properties of the enzyme.

Some characteristics of a 2,2-dichloropropionate dehalogenase induced in a bacterial strain capable of degrading high concentrations of the herbicide dalapon were studied. Polyacrilamide gel electrophoresis of the crude cell free extracts identified only one type of dehalogenase. The single enzymatic protein showed activity against a variety of chlorinated aliphatic acids but differed in their activity levels. Thus activity in mumol substrate converted (mg protein)-1 min-1 was 2-monochloropropionate 0.65, 2,2-dichloropropionate 0.56, 2-monochloroacetate 1.70 and 2,2-dichloroacetate 1.00. In the crude extracts, the enzyme activity against 2,2-dichloropropionate was optimal at a broad pH range with a mid-point at pH 9.5 and apparent Km values were within the range 0.23-0.73 mM.

Biodegradation, Environmental↗
PubMed1992

Phosphatidylinositol inhibits microtubule assembly by binding to microtubule-associated protein 2 at a single, specific, high-affinity site.

The effects of various anionic phospholipids on the in vitro assembly of MAP2/tubulin microtubules has been examined. We show that the potency to inhibit is related to the polarity of the phospholipids and that this is consistent with a mode of action involving the sequencing of microtubule-associated proteins (MAPs) by nonspecific electrostatic interactions. The inhibitory potency of phosphatidylinositol (PI) is, however, considerably larger than predicted by this model. The effects of PI on MAP2/tubulin microtubule assembly have therefore been examined in greater detail by preparing phosphatidylcholine (PC) liposomes doped with increasing amounts of PI. We show that when the PI is sufficiently dispersed by dilution with PC, it inhibits microtubule assembly by binding to MAP2 with an apparent stoichiometry, after correction for the bilamellar nature of the liposomes, of 1:1 mol.mol-1 PI:MAP2. Furthermore, we show that the Kd of this interaction is in the submicromolar range.

Animals↗
PubMed1992

Analysis of the colchicine-binding site of beta-tubulin.

Comparison of the beta-tubulin sequences with the equilibrium colchicine Ka and the Ki for inhibition by podophyllotoxin suggests that residue beta:316 is directly involved in binding the common trimethoxyphenyl-(or A-) ring. By contrast, the analysis indicates that the local hydrophobicity affects the rate of one of the two conformational changes associated with colchicine binding but does not determine the affinity of the colchicine-binding site.

Amino Acid Sequence↗
PubMed1991

Identification of an inhibitor of microtubule assembly present in juvenile brain which displays a novel mechanism of action involving suppression of self-nucleation.

An inhibitor of microtubule assembly has been identified and partially purified from microtubule-depleted brain extracts from day-old chicks and 4-month-old calf. This inhibitor suppresses the self-nucleation of microtubules in vitro with minimal effect upon the final extent of assembly. It may have a developmental role in vivo as it is not detected in adult brain from either cattle or rabbit.

Ammonium Sulfate↗
PubMed1991

Assembly of chick brain MAP2-tubulin microtubule protein. Characterization of the protein and the MAP2-dependent addition of tubulin dimers.

The principle proteins present in twice-cycled chick brain microtubule protein were characterized. The protein consists of a stoichiometric mixture of MAP2 and tubulin, together with a number of minor components. Its composition remains unaltered after a third cycle of assembly in a buffer supplemented with 67 mM-NaCl, with the exception of the phosphorylation of MAP2 to a low level (congruent to 1 mol.mol-1). The inclusion of 67 mM-NaCl dissociates the MAP2-tubulin oligomers, and restricts the assembly to the MAP2-dependent addition and loss of tubulin dimers, such that the assembly kinetics approximate to a simple pseudo-first-order reaction. The assembled microtubules exhibit dynamic instability, with no evidence for end-to-end annealing.

Animals↗
PubMed1991

Kinetics of GTP hydrolysis during the assembly of chick brain MAP2-tubulin microtubule protein.

The kinetics of GTP hydrolysis during microtubule assembly have been examined using chick brain MAP2-tubulin microtubule protein in a NaCl-supplemented buffer. The elongating microtubules terminate in a 'GTP cap', since the kinetics of GTP hydrolysis are slower than those of subunit addition. GTP hydrolysis is (a) stoichiometric, (b) occurs as a vectorial wave as the initial rate of hydrolysis is proportional to the molar concentration of microtubule ends and not to the initial rate of subunit addition, and (c) either does not occur, or occurs only at a much lower rate, in the terminal subunits.

Animals↗
PubMed1991

Assembly of chick brain MAP2-tubulin microtubule protein. Analysis of tubulin subunit flux rates by immunofluorescence microscopy.

A filter-based immunofluorescence-microscopy method for obtaining microtubule lengths has been developed and evaluated. Kinetic constants and mean lengths obtained show close agreement with those obtained by complementary methods applied to chick brain MAP2-tubulin microtubule protein in NaCl-supplemented buffer. The filter-based method has been used to estimate tubulin subunit flux (Jon) resulting from isothermal dilution of microtubule populations to various free tubulin concentrations, (c). This experimental Jon(c) plot is significantly different from that predicted by a variety of theoretical models, but is consistent with a 'lateral cap' model of dynamic instability [Bayley, Schilstra & Martin (1990) J. Cell. Sci. 95, 33-48] adapted to accommodate the observed vectorial GTP hydrolysis.

Animals↗
PubMed1991

The dynamic instability of microtubules is not modulated by alpha-tubulin tyrosinylation.

The tyrosinylation of chick brain alpha-tubulin and the effects of the tyrosinylation status on the assembly and dynamic instability of chick brain MAP2:tubulin microtubule protein have been examined. Each of the eight major alpha-isotypes can be tyrosinylated in vitro, irrespective of whether a C-terminal tyrosine is genetically encoded. The extent of tyrosinylation is however limited to congruent to 0.3 mol.mol-1. The tyrosinylation status (0 vs. 0.3 mol.mol-1) has no effect on either the assembly kinetics of chick brain microtubule protein or on the rate of length redistribution following assembly and shearing. It is therefore unlikely that the tyrosinylation status directly affects the intrinsic stability of assembled microtubules since the rate of length redistribution is both a sensitive assay and a function of the kinetic parameters governing dynamic instability.

Animals↗
PubMed1991

Alpha-, beta-, and gamma-tubulins: sequence comparisons and structural constraints.

Comparison of congruent to 160 alpha-, beta-, and gamma-tubulins, and excluding the highly divergent C-terminal peptide, indicates that the three subclasses have similar tertiary structures. Conserved sequences within or between the subclasses have been identified, together with the locations of known epitopes, chemical modifications, and mutations. Evidence is also reviewed concerning the identity of the GTP-binding sites, about which residues are exposed in the assembled microtubule and at subunit:subunit interfaces. These characteristics constrain the possible tertiary structure of the tubulin subunit.

Amino Acid Sequence↗
PubMed1990

Analysis of beta-tubulin sequences reveals highly conserved, coordinated amino acid substitutions. Evidence that these 'hot spots' are directly involved in the conformational change required for dynamic instability.

Vertebrate beta-tubulins have been classified into six classes on the basis of their C-terminal sequences [(1987) J. Cell Biol. 105, 1707-1720]. In particular, the sequences starting at residue 430 differ between isotypes of the same animal but are conserved between species. We extend this analysis and show that there are three 'hot spots', at residues 35, 55-57 and 124 which exhibit intra-species heterogeneity but inter-species conservation. There is a remarkable correlation between the identity of these residues and the C-terminal sequences, and suggests that the vertebrate beta-tubulins fall into three broad types. This correlation extends to those non-vertebrate organisms which have the Type 1 C-terminal sequence. We propose that these three 'hot spots' and the C-terminal peptide interact in the tertiary structure. We have also noted that the C-terminal peptide almost always contains a single phenylalanine or tyrosine residue, and that there is a strong correlation between this residue and the amino acids at positions 217/218, in both the vertebrate and non-vertebrate sequences. We propose that the C-terminal aromatic amino acid interacts with residues 217/218 in the tertiary structure. Analysis of conditions which stabilise microtubules and/or lower the steady state critical concentration strongly suggests that these two sets of coordinated amino acid substitutions are directly involved in effecting the conformational change associated with GTP hydrolysis which results in dynamic instability. We propose that there is an interaction between the highly acidic sequence between residue 430 and the aromatic amino acid (termed peptide A) and conserved basic amino acids located close to the 'hot spots'. We suggest that this interaction is altered in response to the assembly-dependent GTP hydrolysis, with the consequential increase in the subunit dissociation rate constant.

Amino Acid Sequence↗
PubMed1990

Stoichiometry of estramustine phosphate binding to MAP2 measured by the disassembly of chick brain MAP2:tubulin microtubules.

The concentration of estramustine phosphate required to inhibit the assembly or to induce the disassembly of chick brain MAP2:tubulin microtubules is markedly dependent upon the microtubule protein concentration. Analysis of this relationship shows that estramustine phosphate and tubulin compete for common MAP2 sites, that MAP2 can bind 5-6 moles.mole-1 estramustine phosphate, and that the Kd of these sites is congruent to 20 microM estramustine phosphate. It is proposed that two molecules of estramustine phosphate interact with each of the three tubulin-binding sites of MAP2 and inhibit the MAP2:tubulin interaction by neutralising two highly conserved basic residues.

Amino Acid Sequence↗
PubMed1989

Localization and immunogenicity of tubulin in the filarial nematodes Brugia malayi and B. pahangi.

Tubulin was identified in the filarial nematodes Brugia malayi and B. pahangi by several approaches. Initially, a monoclonal antibody (6D8) was selected for its unusual binding to B. malayi microfilariae in indirect immunofluorescence assays: 6D8 showed granular, heterogeneously dispersed fluorescence on fixed parasites but did not bind to unfixed microfilariae. The microfilarial sheath did not bind 6D8, although it did bind fluoresceinated wheatgerm agglutinin. By Western blotting against microfilarial sonicate, 6D8 reacted with a 50,000-55,000 mol. wt protein, and also bound to purified chicken brain beta-tubulin. Additionally, this monoclonal antibody reacted with a recombinant fusion protein expressed by a clone (Bpa-7) originally isolated from an adult B. pahangi cDNA expression library by its reaction with chronic human filariasis serum. This clone encodes a small 40 amino acid C-terminal segment corresponding to residues 409-449 of beta-tubulin, and shows complete amino acid sequence homology with vertebrate beta-tubulin from 409 to 430 but 55% divergence (six amino acid substitutions, four insertions and one deletion) from human and chicken beta-tubulin over positions 431-449 at the C terminus. Antibody to both parasite and vertebrate (chicken) tubulin was found in filarial infection sera, with higher levels of autoreactive antibody apparent in amicrofilaraemic individuals. Immunogold electron microscopy was then used to localize beta-tubulin in B. malayi microfilariae and adult worms. Tubulin was shown not to be exposed on the microfilarial sheath or in the cuticle of either stage, but was found to be abundant in the somatic tissues. In microfilariae, 6D8 bound myofibril structures under the hypodermal layer, and also bound within cell nuclei. In the adult stage, tubulin was associated with muscle blocks, as well as the intestinal brush border and the embryonic uterine microfilariae.

Amino Acid Sequence↗
PubMed1987

Specificity and biological significance of microtubule-associated protein-DNA interactions in chick.

The interactions between chick brain microtubule associated proteins (MAPs) and chick DNA have been examined using DNA-cellulose chromatography, cross-blotting, and nitrocellulose filter-binding. Comparison of nitrocellulose filter-binding and cross-blotting results show that while MAPs and a minor, Mr 48,000, protein show significant binding at 50 mM NaCl, only the latter continues to bind a significant amount of DNA at 150 mM NaCl, suggesting an ionic basis for the MAP-DNA interactions. MAP-DNA interactions also show weak preference for AT-rich fractions, and are sensitive to S1 nuclease digestion. We suggest that the MAPs bind preferentially to single-stranded DNA. The binding may involve an interaction between the DNA phosphates and the highly cationic tubulin-binding domain of the MAPs. Repetitive fractions of the chick genome prepared both by hydroxyapatite chromatography and by S1 nuclease digestion show binding to a number of minor proteins present in preparations of microtubule proteins, as well as to the MAPs. We conclude that the MAPs probably do not bind specifically to repetitive DNA, in contrast to earlier reports using mouse DNA. MAP-DNA interactions are therefore unlikely to be involved in the attachment of microtubules to mitotic chromosomes.

Animals↗