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M E Spencer

Publications and source records attributed to M E Spencer.

At least 19 recordsLinked to original sources
PubMed1998

A study of dipole localization accuracy for MEG and EEG using a human skull phantom.

OBJECTIVE: To investigate the accuracy of forward and inverse techniques for EEG and MEG dipole localization. DESIGN AND METHODS: A human skull phantom was constructed with brain, skull and scalp layers and realistic relative conductivities. Thirty two independent current dipoles were distributed within the 'brain' region and EEG and MEG data collected separately for each dipole. The true dipole locations and orientations and the morphology of the brain, skull and scalp layers were extracted from X-ray CT data. The location of each dipole was estimated from the EEG and MEG data using the R-MUSIC inverse method and forward models based on spherical and realistic head geometries. Additional computer simulations were performed to investigate the factors affecting localization accuracy. RESULTS: Localization errors using the relatively simpler locally fitted sphere approach are only slightly greater than those using a BEM approach. The average localization error over the 32 dipoles was 7-8 mm for EEG and 3 mm for MEG. CONCLUSION: The superior performance of MEG over EEG appears to be because the latter is more sensitive to errors in the forward model arising from simplifying assumptions concerning the conductivity of the skull, scalp and brain.

Computer Simulation↗
PubMed1996

Expression of the major bean proteins from Theobroma cacao (cocoa) in the yeasts Hansenula polymorpha and Saccharomyces cerevisiae.

The production in two yeast expression systems of recombinant forms of the major proteins from the cocoa bean is described. Three major protein species are found in the cocoa bean: an albumin of molecular mass 21 kDa (p21) and two insoluble vicilin-like proteins of molecular mass 31 kDa and 47 kDa (p31 and p47, respectively). The p31 and p47 species are known to be derived from a common 67-kDa precursor (p67) by post-translational processing that includes the deletion of a hydrophilic domain located immediately after an N-terminal signal sequence. All three proteins appear to be targeted to membrane-bound storage organelles by N-terminal signal sequences. The p21 and p67 coding sequences were expressed in Hansenula polymorpha using the powerful methanol oxidase (MOX) promoter and in Saccharomyces cerevisiae using the promoter of the pyruvate kinase (PYK) gene. The expression constructs contained the native plant signal sequence, or various yeast signals. The p21 protein was successfully expressed and secreted from both yeasts. The insoluble p67 protein proved more difficult. Species of the correct molecular mass were recovered internally and small amounts of a p47 species were secreted using a yeast leader sequence. However, proteolytic cleavage, probably due to Kex2p-like processing, led to the appearance of other protein species.

Amino Acid Sequence↗
PubMed1993

Error bounds for EEG and MEG dipole source localization.

General formulas are presented for computing a lower bound on localization and moment error for electroencephalographic (EEG) or magnetoencephalographic (MEG) current source dipole models with arbitrary sensor array geometry. Specific EEG and MEG formulas are presented for multiple dipoles in a head model with 4 spherical shells. Localization error bounds are presented for both EEG and MEG for several different sensor configurations. Graphical error contours are presented for 127 sensors covering the upper hemisphere, for both 37 sensors and 127 sensors covering a smaller region, and for the standard 10-20 EEG sensor arrangement. Both 1- and 2-dipole cases were examined for all possible dipole orientations and locations within a head quadrant. The results show a strong dependence on absolute dipole location and orientation. The results also show that fusion of the EEG and MEG measurements into a combined model reduces the lower bound. A Monte Carlo simulation was performed to check the tightness of the bounds for a selected case. The simple head model, the low power noise and the few strong dipoles were all selected in this study as optimistic conditions to establish possibly fundamental resolution limits for any localization effort. Results, under these favorable assumptions, show comparable resolutions between the EEG and the MEG models, but accuracy for a single dipole, in either case, appears limited to several millimeters for a single time slice. The lower bounds increase markedly with just 2 dipoles. Observations are given to support the need for full spatiotemporal modeling to improve these lower bounds. All of the simulation results presented can easily be scaled to other instances of noise power and dipole intensity.

Brain↗
PubMed1991

Effects of medroxyprogesterone acetate on some parameters of calcium metabolism in patients with glucocorticoid-induced osteoporosis.

Long-acting medroxyprogesterone acetate (MPA) effect on some important parameters of calcium metabolism in patients with glucocorticoid-induced osteoporosis (GCO) was evaluated. Twelve steroid-dependent asthmatic male patients with GCO were administered 200 mg of MPA (Depo-Provera) intramuscularly, and had fasting serum samples obtained at baseline and at weekly intervals for 5 consecutive weeks. Baseline serum samples were also obtained from 12 control healthy male subjects matched for age. The following measurements were made from each serum sample: osteocalcin (OC), skeletal (SAP) and total alkaline phosphatase (TAP), calcitonin (C), insulin-like growth factor I (IGF1), 1,25-dihydroxyvitamin D and 25-hydroxyvitamin D. Significantly lower baseline serum levels of OC and C were found in the patients with GCO than in controls (P less than 0.001). Following MPA administration in GCO patients statistically significant and sustained increases in OC, SAP and C were noticed during the next 5 weeks. No significant differences in baseline levels for TAP, IGF1, 1,25(OH)2D and 25(OH)D between GCO patients and controls were found, and no significant changes following MPA administration in GCO patients were obtained for these parameters. In conclusion, when administered to patients with GCO, MPA seems to stimulate the osteoblastic activity as suggested by sustained increases in OC and SAP serum levels, and also enhances the C production by the C-cells of the thyroid.

Aged↗
PubMed1987

Regulation of citric acid cycle genes in facultative bacteria.

In a facultative anaerobe such as Escherichia coli the citric acid cycle is an inducible pathway rather than a constitutive pathway. In spite of intensive study at the enzymological and molecular biological levels many aspects of the cycle, including its regulation during the aerobic/anaerobic switch, are not fully understood.

Citric Acid Cycle↗
PubMed1986

Cloning and expression of the succinyl-CoA synthetase genes of Escherichia coli K12.

The genes encoding both subunits of the succinyl-CoA synthetase of Escherichia coli have been identified as distal genes of the suc operon, which also encodes the dehydrogenase (Elo; sucA) and succinyltransferase (E2o; sucB) components of the 2-oxoglutarate dehydrogenase complex. The newly defined genes express polypeptides of 41 kDa (sucC) and 31 kDa (sucD), corresponding to the beta and alpha subunits of succinyl-CoA synthetase, respectively. The genes are thus located at 16.8 min in the E. coli linkage map, together with the citrate synthase (gltA) and succinate dehydrogenase (sdh) genes, in a cluster of nine citric acid cycle genes: gltA-sdhCDAB-sucABCD. Four deletion strains lacking all of these citric acid cycle enzymes were characterized. The succinyl-CoA synthetase activities of strains harbouring plasmids containing the sucC and sucD genes were amplified some fourfold. Further enzymological studies indicated that expression of succinyl-CoA synthetase is coordinately regulated with 2-oxoglutarate dehydrogenase.

Cloning, Molecular↗
PubMed1985

Primary structure of the succinyl-CoA synthetase of Escherichia coli.

The primary structure of the succinyl-CoA synthetase of Escherichia coli has been deduced from the nucleotide sequence of a 2451-base-pair segment of DNA containing the corresponding sucC (beta subunit) and sucD (alpha subunit) genes. The genes are located at one end of a gene cluster that encodes several citric acid cycle enzymes: gltA-sdhCDAB-sucABCD; gltA, citrate synthase; sdh, succinate dehydrogenase; sucA and sucB, the dehydrogenase (E1) and succinyltransferase (E2) components of the 2-oxoglutarate dehydrogenase complex. The sucC and sucD genes are separated from the sucA and sucB genes by a 273-base-pair segment containing four palindromic units, but they appear to be expressed from a sucABCD read-through transcript that extends from the suc promoter to a potential rho-independent terminator at the distal end of sucD. The stop codon of the sucC gene overlaps the sucD initiation codon by a single nucleotide, indicating close translational coupling of the sucC and sucD genes. The sucC gene comprises 1161 base pairs (388 codons, excluding the stop codon), and it encodes a polypeptide of Mr 41 390 corresponding to the beta subunit of succinyl-CoA synthetase. The sucD gene comprises 864 base pairs (288 codons, excluding the start and stop codons), and it encodes a product of Mr 29 644, corresponding to the alpha subunit of succinyl-CoA synthetase. The alpha subunit contains a 12-residue amino acid sequence that is identical with the histidine peptide previously isolated from the phosphoenzyme. This sequence forms part of one of the two potential nucleotide binding sites detected in the alpha subunit.

Amino Acid Sequence↗
PubMed1985

Transcription analysis of the sucAB, aceEF and lpd genes of Escherichia coli.

Transcript mapping of the Escherichia coli sucAB, aceEF and lpd genes, encoding the five components of the pyruvate and 2-oxoglutarate dehydrogenase complexes, was carried out using single-stranded M13 probes. The sucA and aceE genes encode the specific dehydrogenase components (E1o, E1p), and the sucB and aceF genes encode the specific dihydrolipoamide acyltransferases (E2o, E2p). The common lipoamide dehydrogenase (E3) component is encoded by a single lpd gene adjacent to the aceEF genes. The sucAB, aceEF and lpd genes were all expressed on independent transcripts, and the promoters and terminators were identified. In addition, readthrough transcription from the sucAB genes to a downstream gene designated sucC, and from the aceEF genes to the adjacent lpd gene, was found. The relative levels of transcription of the suc, ace and lpd genes, and of the three different transcript types covering the ace-lpd region, were quantified using RNA from cells grown on different substrates. Most of the E3 components supplying the pyruvate dehydrogenase complex appear to be synthesised from approximately 6415-base aceEF-lpd readthrough transcripts, but additional approximately 4640-base aceEF transcripts terminating after the aceF gene provide a transcriptional basis for the observed stoichiometric excess of E1p and E2p relative to E3 in the assembled complex. Conversely most of the E3 components required for the 2-oxo-glutarate dehydrogenase complex appear to be synthesised from the independent 1670-base lpd transcripts.

Acyltransferases↗
PubMed1984

Nucleotide sequence of the sucA gene encoding the 2-oxoglutarate dehydrogenase of Escherichia coli K12.

The nucleotide sequence of a 3180-base-pair segment of DNA, containing the sucA gene encoding the 2-oxoglutarate dehydrogenase component (E1o) of the 2-oxoglutarate dehydrogenase complex of Escherichia coli, has been determined by the dideoxy chain-termination method. The sucA structural gene contains 2796 base pairs (932 codons, excluding the initiation codon AUG) and encodes a polypeptide having a glutamine residue at the amino terminus, a glutamate residue at the carboxy-terminus and a calculated Mr = 104905. The predicted amino acid composition is in good agreement with published information obtained by hydrolysis of the purified enzyme. There is a striking lack of sequence homology between the 2-oxoglutarate dehydrogenase (E1o) and the corresponding pyruvate dehydrogenase (E1p), which suggests that the two components are not closely related in evolutionary terms. The location and polarity of the sucA gene, relative to the restriction map of the corresponding segment of DNA, are consistent with it being the proximal gene of the suc operon, as defined in previous genetic and post-infection labelling studies, but it could also form part of a more complex regulatory unit. The sucA gene is preceded by a segment of DNA that contains many substantial regions of hyphenated dyad symmetry including an IS-like sequence of the type that is thought to function as an intercistronic regulatory element. This segment also contains three putative RNA polymerase binding sites and a good ribosome binding site.

Amino Acid Sequence↗
PubMed1984

Nucleotide sequence of the sucB gene encoding the dihydrolipoamide succinyltransferase of Escherichia coli K12 and homology with the corresponding acetyltransferase.

The nucleotide sequence of the sucB gene, which encodes the dihydrolipoamide succinyltransferase component (E2o) of the 2-oxoglutarate dehydrogenase complex of Escherichia coli K12, has been determined by the dideoxy chain-termination method. The results extend by 1440 base pairs the previously reported sequence of 3180 base pairs, containing the sucA gene. The sucB structural gene comprises 1209 base pairs (403 codons excluding the initiating AUG), and it is preceded by a 14-base-pair intercistronic region containing a good ribosomal binding site. The absence of a typical terminator sequence and the presence of an IS-like sequence downstream of sucB suggest that there may be further gene(s) in the suc operon. The IS-like sequence is homologous with other intercistronic sequences including that between the sdhB and sucA genes, the overall gene organisation being: sdhB-IS-sucAsucB-IS-. The patterns of codon usage indicate that sucB may be more strongly expressed than sucA, consistent with the disproportionate contents of their products in the oxoglutarate dehydrogenase complex. The predicted amino acid composition and Mr (43 607) of the succinyltransferase component agree with previous studies on the purified protein. Comparison with the corresponding acetyltransferase component of the pyruvate dehydrogenase complex (E2p, aceF gene product) indicates that each contains two analogous domains, an amino-terminal lipoyl domain linked to a carboxy-terminal catalytic and subunit binding domain. The lipoyl domain of the acetyltransferase (E2p) comprises three tandemly repeated approximately 100-residue lipoyl binding regions containing two short (approximately 19 residues) internal repeats, whereas the lipoyl domain of the succinyltransferase (E2o) contains just one approximately 100-residue lipoyl binding region, with approximately 27% homology to each of the three comparable regions in E2p, and no detectable internal repeats. The catalytic and subunit binding domains, each approximately 300 residues, have an overall homology of 34% and, consistent with their combination of analogous and specific functions, some regions are more homologous than others. Both sequences feature segments rich in proline and alanine. In E2p these occur at the carboxy-terminal ends of each of the three lipoyl binding regions, there being a particularly extended sequence at the end of the third repeat, whereas in E2o the main proline-alanine segment is found approximately 50 residues into the subunit binding domain.(ABSTRACT TRUNCATED AT 400 WORDS)

Acetyltransferases↗
PubMed1983

Nucleotide sequence of the promoter region of the citrate synthase gene (gltA) of Escherichia coli.

The gltA gene, specifying the citrate synthase (EC 4.1.3.7) of Escherichia coli, has been isolated and the nucleotide sequence of a 752 basepair segment containing the gltA promoter and encoding 96 aminoterminal residues of the protein has been defined using the dideoxy/M13 method. The results confirm the location and transcriptional polarity of the gltA gene and indicate that the gltA transcript may contain a long leader sequence of 302-306 nucleotides upstream from the coding region.

Base Sequence↗
PubMed1982

Molecular cloning of four tricarboxylic acid cyclic genes of Escherichia coli.

A fragment of DNA (3.1 kilobases [kb]) from a ColE1 Escherichia coli DNA hybrid plasmid containing the bacterial citrate synthase gene (gltA) was subcloned in both orientations into phage lambda vectors by in vitro recombination. The resulting phages were able to transduce gltA and, as prophages, complemented the lesion of a gltA mutant, showing that a functional gltA gene is contained in the 3.1-kb fragment. The segment of E. coli DNA cloned in these lambda gltA phages was extended in vivo by prophage integration and aberrant excision in the gltA region. Plaque-forming derivatives, carrying up to three additional tricarboxylic acid cycle genes, succinate dehydrogenase (sdh), 2-oxoglutarate dehydrogenase (sucA), and dihydrolipoamide succinyltransferase (sucB), were isolated and characterized by their transducing and complementing activities with corresponding mutants, and the order of the genes was confirmed as gltA-sdh-sucA-sucB. Physical maps of a variety of the transducing phages showed that the four tricarboxylic acid cycle genes are contained in a 12.8-kb segment of bacterial DNA. The four gene products, plus a possible succinate dehydrogenase small subunit, were identified in postinfection labeling studies, and the polarities of gene expression were defined as counterclockwise for gltA and clockwise for sdh, sucA, and sucB, relative to the E. coli linkage map.

Acyltransferases↗
PubMed1976

Location of the Aspartase Gene (aspA) on the linkage map of Escherichia coli K12.

The aspartase (L-asparatate ammonia-lyase, EC. 4.3.I.I) structural gene, aspA, was mapped by two-factor and three-factor transductional crosses using phage PI. Cotransduction frequencies between aspA and other markers were: ampA (69%); frdA (48 TO 67%) mel (35%); purA (17%); fdp (I-6%). The sequence of markers within the corresponding segment (91 to 95 min) of the Escherichia coli linkage map was shown to be mel-aspA-ampA-frdA-purA-fdp.

Ammonia-Lyases↗
PubMed1974

Proteins of the inner membrane of Escherichia coli: identification of succinate dehydrogenase by polyacrylamide gel electrophoresis with sdh amber mutants.

The inner or cytoplasmic membrane fraction of the cell envelope of Escherichia coli was isolated by isopycnic centrifugation on sucrose gradients. The membrane proteins were analyzed by electrophoresis in sodium dodecyl sulfate-polyacrylamide gels (8.5%), and up to 56 bands were resolved. Different preparations gave very similar patterns of proteins. Succinate dehydrogenase mutants (sdh) were isolated which could not grow on succinate minimal medium, although growth on fumarate was unimpaired. The protein patterns of inner membrane preparations from sdh amber mutants were compared with the wild type, and one major band was greatly reduced in the mutants. This component, which represented approximately 5% of the inner membrane protein, was restored by introducing an amber suppressor gene (supU), which also restored the Sdh(+) phenotype. The band corresponded to a protein with a molecular weight of 67,000 daltons, which is close to that for the large subunits of the succinate dehydrogenases of Rhodospirillum rubrum and beef heart mitochondria.

Bacterial Proteins↗
PubMed1974

Proteins of the inner membrane of Escherichia coli: changes in composition associated with anaerobic growth and fumarate reductase amber mutation.

The inner membrane fractions of Escherichia coli grown anaerobically and aerobically were isolated, and their proteins were compared by electrophoresis in polyacrylamide gels. To maximimize the differences between the preparations, the anaerobic cultures were grown on complex medium with added glucose, but glucose was omitted from the aerobic cultures to prevent catabolite repression. The pattern of bands in the two types of preparation differed considerably, and changes in approximately 20 components were observed. In particular, the band identified as succinate dehydrogenase in aerobic preparations was greatly reduced in anaerobic preparations. Mutants lacking fumarate reductase were isolated, and inner membrane preparations of an frd amber mutant were deficient in a major component of 75,000 daltons and possibly a minor one of 87,500 daltons. The former was also present in greater amounts in anaerobic preparations and could represent a fumarate reductase subunit.

Aerobiosis↗
PubMed1973

Isolation and properties of fumarate reductase mutants of Escherichia coli.

Escherichia coli produces two enzymes which interconvert succinate and fumarate: succinate dehydrogenase, which is adapted to an oxidative role in the tricarboxylic acid cycle, and fumarate reductase, which catalyzes the reductive reaction more effectively and allows fumarate to function as an electron acceptor in anaerobic growth. A glycerol plus fumarate medium was devised for the selection of mutants (frd) lacking a functional fumarate reductase by virtue of their inability to use fumarate as an anaerobic electron acceptor. Most of the mutants isolated contained less than 1% of the parental fumarate reduction activity. Measurements of the fumarate reduction and succinate oxidation activities of parental strains and frd mutants after aerobic and anaerobic growth indicated that succinate dehydrogenase was completely repressed under anaerobic conditions, the assayable succinate oxidation activity being due to fumarate reductase acting reversibly. Fumarate reductase was almost completely repressed under aerobic conditions, although glucose relieved this repression to some extent. The mutations, presumably in the structural gene (frd) for fumarate reductase, were located at approximately 82 min on the E. coli chromosome by conjugation and transduction with phage P1. frd is very close to the ampA locus, and the order of markers in this region was established as ampA-frd-purA.

Anaerobiosis↗