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B H Robinson

Publications and source records attributed to B H Robinson.

At least 91 records · Page 5Linked to original sources
PubMed1995

Preparative-scale kinetic resolutions catalysed by microbial lipases immobilised in AOT-stabilised microemulsion-based organogels: cryoenzymology as a tool for improving enantioselectivity.

Gelatin-containing microemulsion based organogels have been used as an immobilisation matrix for lipases from a number of different sources. Kinetic resolutions of octan-3-ol, 1-octen-3-ol and 1-octyn-3-ol by esterification with decanoic acid have been performed using Chromobacterium viscosum (CV) lipase. CV lipase is highly enantioselective in favour of the (R)-(-) isomer of octan-3-ol, but the enantioselectivity is both reversed and decreased by the introduction of unsaturation at the 1-position. Marked improvements in enantioselectivity were achieved by carrying out the reaction at -15 degrees C, the enantiomeric excess of the ester product increasing from 47% (E = 3) to 73% (E = 8) in the case of 1-octen-3-ol, and from 17% (E = 1.4) to 38% (E = 2.5) in the case of 1-octyn-3-ol. The enantiomeric excess was approximately 85% (E approximately 15) for octan-3-ol, and there was no marked improvement in enantioselectivity even at -15 degrees C. Apparent activation energies for the esterification using decanoic acid of octan-3-ol, 1-octen-3-ol and 1-octyn-3-ol by CV lipase were 32 kJ mol-1, 31 kJ mol-1 and 41 kJ mol-1, respectively. This compares to an activation energy of 21 kJ mol-1 for the esterification of octan-1-ol with decanoic acid using CV lipase under the same conditions. Lipases from Pseudomonas (Fluka), Pseudomonas (Genzyme) and lipoprotein lipase ex Microbial (Genzyme) also selectively esterified the (R)-(-) isomer of racemic octan-3-ol, the two Pseudomonas preparations yielding product with an enantiomeric excess of 90%. Candida cylindracea lipase did not exhibit activity in gelatin-containing MBGs. Large-scale syntheses were performed in a 1 dm3 batch reactor in which 200 cm3 of pelleted MBG (containing 350 mg of CV lipase) was used repeatedly for the kinetic resolution of octan-3-ol.

Calorimetry↗
PubMed1995

A one-step polymerase chain reaction site-directed mutagenesis method for large gene-cassettes with high efficiency, yield, and fidelity.

A one-step three-stage efficient PCR method for site-directed mutagenesis has been developed. This method involves one PCR reaction with no need to add more reagents during this reaction or to do a second PCR reaction. This method utilizes a three-stage PCR cycling profile, a ddNTP-blocked restriction endonuclease fragment, and Pfu DNA polymerase. This method allows the amplification of at least 2 kb of final mutant product via an intermediate megaprimer as large as 1.3 kb with high fidelity, high efficiency, high yield, and high success rate. A 3.2-kb large final mutant product has also been obtained by using Taq and Taq Extender (Stratagene) instead of Pfu. This method is particularly useful when there are no unique restriction sites in the gene of interest to reduce the large sizes of the final mutant product and/or the megaprimer.

DNA Primers↗
PubMed1995

Macrocyclic lactone synthesis by lipases in water-in-oil microemulsions.

Five microbial lipases from Chromobacterium viscosum, Candida cylindracea, Pseudomonas (source Fluka), Pseudomonas (source Genzyme) and lipoprotein lipase ex Microbial (Genzyme) have been screened for lactonisation activity towards 16-hydroxyhexadecanoic acid (HHA) in a variety of different w/o microemulsion systems. With the exception of Candida cylindracea (CC), all the lipases exhibited lactonisation activity although they were inherently more active in microemulsion systems based on the anionic surfactant sodium bis(2-ethylhexyl)sulphosuccinate (AOT) than in those based on the cationic surfactant cetyltrimethylammonium bromide (CTAB). Lactone yields are typically 50-60% and are markedly better than those reported previously using microemulsions in combination with chemical catalysts. Lipase stability is superior in the CTAB microemulsion systems, while lipase stability in the low water content AOT microemulsion systems was still good with the exception of CC lipase, which is rapidly inactivated. Buffering the water pools of AOT microemulsions using diglycine buffer at pH 8.0 improved biocatalyst stability. The lactonisation activity of lipases in CTAB w/o microemulsion systems compares favourably with that obtained using the same preparations as a solid suspension in the corresponding water-saturated organic solvent. In addition, the unusual solubility properties of microemulsions allowed the use of considerably higher concentrations of substrate in the microemulsion systems as compared to water-saturated organic solvents such as n-heptane. Lactone yields obtained at equivalent concentrations in the corresponding organic solvents containing conventional condensation catalysts were consistently measured at approx. 10%.

4-Aminopyridine↗
PubMed1995

Monitoring DNA dynamics using spin-labels with different independent mobilities.

The electron paramagnetic resonance (EPR) spectra of spin-labeled DNA duplexes, both bound to DEAE-Sephadex and free in solution, have been analyzed. The nitroxide spin-labels are covalently linked to a deoxyuridine residue using either a monoacetylene or diacetylene tether. This difference in tether length produces a dramatic difference in the independent mobility of the nitroxide relative to the DNA. In the case of the monoacetylene tether, the motion of the nitroxide has previously been shown to be tightly coupled to that of the DNA duplex. With the diacetylene tether, there is considerable independent motion of the probe. The diacetylene tether is intended to minimize the possibility of the nitroxide producing a perturbation of the dynamics of DNA. It is demonstrated here that, when coupled via the diacetylene tether, the nitroxide undergoes a rapid uniaxial rotation about the tether. A detailed analysis of the EPR spectrum of duplex DNA in solution, spin-labeled using the diacetylene tether, demonstrates that the motion of the nitroxide can be modeled in terms of this independent uniaxial rotation together with motion of the DNA which is consistent with the global tumbling of the duplex. As was previously found using the monoacetylene tether, there is no evidence of rapid, large-amplitude motions of the base pair in the EPR spectrum of a nitroxide coupled to duplex DNA via the diacetylene tether. This result confirms the small amplitudes of internal motion, local and collective, previously observed in duplex DNA with the monoacetylene-tethered nitroxide.

Acetylene↗
PubMed1995

Self and non-self antigen in diabetic autoimmunity: molecules and mechanisms.

In this article, we have summarized current facts, models and views of the autoimmunity that leads to destruction of insulin-producing beta-cells and consequent Type 1 (insulin-dependent) diabetes mellitus. The presence of strong susceptibility and resistance gene loci distinguishes this condition from other autoimmune disorders, but environmental disease factors must conspire to produce disease. The mapping of most of the genetic risk (or disease resistance) to specific alleles in the major histocompatibility locus (MHC class II) has direct functional implications for our understanding of autoimmunity in diabetes and directly implies that presentation of a likely narrow set of peptides is critical to the development of diabetic autoimmunity. While many core scientific questions remain to be answered, current insight into the disease process is beginning to have direct clinical impact with concerted efforts towards disease prevention or intervention by immunological means. In this process, identification of the critical antigenic epitopes recognized by diabetes-associated T cells has achieved highest priority.

Amino Acid Sequence↗
PubMed1995

Site-specific dynamics in DNA: theory.

This chapter reviews recent progress in understanding duplex DNA dynamics. The weakly bending rod model of Schurr and coworkers is described and compared to a model-free formulation of DNA dynamics. Numerical trajectory methods for obtaining dynamic information are also discussed. The general principles of magnetic resonance relaxation are the reviewed, and the methods by which molecular motions are incorporated into the calculation of relaxation rates or the simulation of experimental NMR and EPR data are described. The impact that the time scale of the dynamics exerts on various computational methods is considered, and in particular, the implementation of (a) the stochastic Liouville equation, (b) the Redfield relaxation matrix, and (c) tensorial preaveraging is described. Expressions for direct and cross-relaxation processes are developed by expanding the density matrix in terms of an irreducible tensor basis set.

Computer Simulation↗
PubMed1995

Mutations in the X-linked E1 alpha subunit of pyruvate dehydrogenase: exon skipping, insertion of duplicate sequence, and missense mutations leading to the deficiency of the pyruvate dehydrogenase complex.

Human pyruvate dehydrogenase (PDH)-complex deficiency is an inborn error of metabolism that is extremely heterogeneous in its presentation and clinical course. In a study of 14 patients (7 females and 7 males), we have found a mutation in the coding region of the E1 alpha gene in all 14 patients. Two female patients had the same 7-bp deletion at nt 927; another female patient had a 3-bp deletion at nt 931. Another female patient was found to have a deletion of exon 6 in her cDNA. Two other female patients were found to have insertions, one of 13 bp at nt 981 and one of 46 bp at nucleotide 1078. Two male patients were found to have a 4-bp insertion at nucleotide 1163. The remaining six patients all had missense mutations. A male patient and a female patient both had an A1133G mutation. The other missense mutations were C214T, C615A, and C787G (two patients). Five of these mutations are novel mutations, five have been previously reported in other patients, and two were published observations in other patients in an E1 alpha-mutation summary. In the four cases where parent DNA was available, only one mother was found to be a carrier of the same mutation as her child.

Base Sequence↗
PubMed1994

cDNA cloning of human kidney pyruvate carboxylase.

cDNA clones covering a total of 4017 bp were isolated corresponding to the full length of the message for human pyruvate carboxylase. The coding sequence consisted of a stretch of 3534 bp flanked by a 5' untranslated sequence of 82 bp and a 3' untranslated region of 389 bp excluding the poly-A tail. The translated amino acid sequence of 1178 residues contained consensus sequences for the covalent attachment for biotin, the binding of ATP and the binding of the substrate, pyruvate.

Amino Acid Sequence↗
PubMed1994

Barth syndrome: clinical observations and genetic linkage studies.

Barth syndrome is an X-linked recessive condition characterized by skeletal myopathy, cardiomyopathy, proportionate short stature, and recurrent neutropenia, but with normal cognitive function. Some, but not all patients, exhibit carnitine deficiency and/or the presence of 3-methylglutaconic and ethylhydracylic acids in urine. Recently the mutation causing Barth syndrome was localised to the Xq28 region by linkage analysis. We report 6 cases of Barth syndrome from 4 families and highlight the fact that neuromuscular and cardiovascular symptoms and the severity of infections tend to improve with age, while short stature persists. Also previously unreported was myopathic facies and nasal quality to speech in our cases. The urinary organic acid abnormalities and plasma carnitine deficiency were inconsistent findings. We propose that they may be epiphenomena rather than indicators of the primary metabolic defect, and that the primary defect or defects in this disorder may lie in the mitochondrial electron transport chain.

Abnormalities, Multiple↗
PubMed1994

Variations in mitochondrial ultrastructure and dynamics observed by high resolution scanning electron microscopy (HRSEM).

Rat adrenal cortex was processed for high resolution scanning electron microscopy (HRSEM) to confirm tubular cristae, reported by transmission electron microscopy to be present in cortex mitochondria. Mitochondria in several other tissue and cell types were also observed and their ultrastructure confirmed by using three-dimensional, stereo, high resolution scanning electron microscopy. The mitochondria in rat and human hepatocytes as well as human skin fibroblasts grown in culture contained tubular cristae approximately 30 nanometers in diameter. The fibroblast mitochondria proved to be long, up to 46 micrometers and branching, as compared to those in liver which were spherical in shape. Cold adapted brown fat cells were packed with mitochondria, these containing plate or shelf-like cristae. Branched, rat striated muscle mitochondria were observed to curve around contractile protein filament bundles. The muscle mitochondrial cristae were found to be both tubular and plate-like, within the same mitochondrion. The ratio of tubular cristae to plate-like cristae varied considerably between muscle mitochondria. In order to use ultrastructural changes in mitochondria for differential diagnosis, and because 3D reconstruction of mitochondria based on transmission electron microscopy serial sections is severely limited in resolution, it is imperative to first develop a correct understanding of tissue specific, normal mitochondrial ultrastructure based on three-dimensional, HRSEM methods.

Adipose Tissue↗
PubMed1994

Molecular dynamics in liquids: spin-lattice relaxation of nitroxide spin labels.

Time domain pulsed saturation recovery and electron-electron double resonance spectroscopies were used to measure the spin-lattice relaxation rates of the electron and the nitrogen nucleus in nitroxide spin labels in liquids. The rotational correlation time range covered is from picoseconds to milliseconds. These rates are quantitatively explained by isotropic rotational Brownian dynamics, which modulate the interactions between the electron spin and the molecular angular momentum; the nitrogen and electron spins; and the solvent protons with both the electron and the nitrogen spins. This solves a 20-year-old problem that has limited scientific applications of nitroxides.

Cyclic N-Oxides↗
PubMed1994

MtDNA and nuclear mutations affecting oxidative phosphorylation: correlating severity of clinical defect with extent of bioenergetic compromise.

Rates of ATP synthesis were studied in cultured skin fibroblasts treated with digitonin. In fibroblasts from patients with complex I deficiency, complex IV and complex V deficiency rates of ATP synthesis were decreased below the levels found in controls. In mitochondria isolated from cultured lymphoblasts, ATP synthesis was also decreased by 35-50% in cases of Leigh's disease due to complex I, complex IV, or complex V deficiency. Calculating the effect of the mutations in the various complexes on the overall efficiency of oxidative phosphorylation, we show that the mtDNA 8993 mutation which affects the activity of the F1F0 ATPase (complex V) has the strongest effect.

Adenosine Triphosphatases↗
PubMed1994

ICA1 encoding p69, a protein linked to the development of type 1 diabetes, maps to human chromosome 7p22.

The development of type 1 (insulin dependent) diabetes mellitus (IDDM) requires a genetically susceptible host and exposure, early in life, to environmental trigger molecules that induce diabetic autoimmunity to insulin producing islet cells. We previously identified islet cell protein p69 as a candidate autoimmune target in IDDM. Here we describe a human genomic p69 fragment which allowed us to map the gene (ICA1) to chromosome 7p22.

Blotting, Southern↗
PubMed1994

The 8993 mtDNA mutation: heteroplasmy and clinical presentation in three families.

The point mutation at bp 8993 of human mtDNA in the ATPase 6 gene is associated with neurogenic weakness, ataxia and retinitis pigmentosa, and with subacute necrotizing encephalomyelopathy (Leigh disease) when present at high copy number. In this study we describe three new multiplex families with the ATPase 8993 mtDNA mutation and demonstrate a correlation between the percentage heteroplasmy of this mutation and the clinical phenotype. By combining this study with previous data we produce a graph of age of onset of symptoms versus percentage heteroplasmy of the mutation. Finally, we determine that ATP synthesis with NAD-linked substrates in cultured lymphoblast mitochondria from three patients with Leigh disease who had a high percentage heteroplasmy was on average 66% of the rate seen in control lymphoblast mitochondria. Similar rates are observed in lymphoblast mitochondria isolated from patients with Leigh disease due to complex I deficiency. This percentage appears to be independent of the rate of electron transport in mitochondria from patient cell lines with the mtDNA 8993 mutation.

Adenosine Triphosphatases↗
PubMed1994

Maternally inherited hypertrophic cardiomyopathy due to a novel T-to-C transition at nucleotide 9997 in the mitochondrial tRNA(glycine) gene.

We report a unique heteroplasmic T-to-C transition at nucleotide 9997 in the mitochondrial tRNA(glycine) gene in a multiplex family who manifested nonobstructive cardiomyopathy. The degree of mtDNA heteroplasmy generally correlated with the severity of the symptoms. This T-to-C transition disrupts hydrogen bonding in the region adjacent to the acceptor stem of the tRNA molecule. The thymine residue at position 9997 is highly conserved in mammals, as well as in various vertebrates and invertebrates. A PCR diagnostic test for the presence of the 9997 T-to-C transition revealed that the base change was always present in high proportion in affected family members, not present in unaffected family members, and never present in control subjects from various ethnic groups (25 groups sampled, 42 individuals), thus ruling out the possibility that this change represents a polymorphic variant in the general population. The degree of heteroplasmy in lymphoblast cultures also correlated with the level of enzyme activity present for cytochrome c oxidase (complex IV) and succinate cytochrome c oxidoreductase (complexes II and III). The absence of previously reported mtDNA mutations associated with hypertrophic cardiomyopathy was verified by both PCR diagnostic procedures and sequence analysis. All mitochondrial tRNA genes, as well as genes encoding ATPase subunits 6 and 8, were sequenced and found not to possess base changes consistent with the clinical profile. More detailed biochemical and molecular biological investigations are discussed.

Adenosine Triphosphatases↗
PubMed1993

Lacticacidemia.

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Adenosine Triphosphatases↗