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Biomedical subjects

D A Mills

Publications and source records attributed to D A Mills.

At least 19 recordsLinked to original sources
PubMed2004

Topographical memory impairments after unilateral lesions of the anterior thalamus and contralateral inferotemporal cortex.

Monkeys with crossed unilateral excitotoxic lesions of the anterior thalamus and unilateral inferotemporal cortex ablation were severely impaired at learning two tasks which required the integration of information about the appearance of objects and their positions in space. The lesioned monkeys were also impaired at learning a spatial task and a task which required the integration of information about the appearance of objects and the background on which the objects were situated. Monkeys with only one of the unilateral lesions were not impaired and previous work has shown that monkeys with bilateral lesions of the anterior thalamus were not impaired on these tasks. These results indicate that the whole of the inferotemporal cortex-anterior thalamic circuit, which passes via the hippocampus, fornix, mamillary bodies and mamillothalamic tract, is essential for the topographical analysis of information about specific objects in different positions in space. Together with previous work, the results show that a unilateral lesion may affect cognition in the presence of other brain damage when an equivalent bilateral lesion alone does not. The tasks required the slow acquisition of information into long term memory and therefore assessed semantic knowledge although other research has shown impairment on topographical processing within working or episodic memory following lesions of the hippocampal-diencephalic circuit. It is argued that the hippocampal-diencephalic circuit does not have a role in a specific form of memory such as episodic memory but rather is involved in topographical analysis of the environment in perception and across all types of declarative memory.

Animals↗
PubMed2003

Evaluation of PCR primers for denaturing gradient gel electrophoresis analysis of fungal communities in compost.

AIMS: Three previously published fungal specific PCR primer sets, referred to as the NS, EF and NL primer sets, were evaluated for use in compost microbial community analysis by PCR and denaturing gradient gel electrophoresis (DGGE). METHODS AND RESULTS: Primers were first evaluated based on their tolerance to PCR inhibitors. Due to its sensitivity to inhibitors, the NS primer set was determined to require a 10-fold smaller volume addition of compost DNA to PCR than the EF and NL primer sets, based on a logistic regression model for a 75% PCR success rate. Further evaluation of the EF and NL primer sets involved testing the resolution of PCR products from pure fungal cultures on DGGE. The NL primer set, which targets the more variable 28S rDNA, resulted in multiple bands for each pure culture. Thus, the EF primer set was used to monitor the microbial community during compost colonization studies, where three fungi were inoculated onto autoclaved grape pomace and rice straw compost. CONCLUSIONS: Of the three primer sets evaluated, the EF primer set was determined to be the best for PCR-DGGE of compost fungal populations; however, concerns with the EF primer set included the lack of sequence divergence in the targeted region of 18S rDNA and PCR artifacts which interfered with detection of inoculated fungi in the colonization studies. SIGNIFICANCE AND IMPACT OF THE STUDY: There are many factors related to PCR primers that need to be assessed prior to applying PCR-DGGE to fungal communities in complex environments such as compost.

DNA Primers↗
PubMed2002

Complementary therapy involvement of physicians: implications for practice and learning.

OBJECTIVE: We studied physicians' current and desired clinical role functions within the complementary health paradigm and their perceptions of the necessary educational programs to support them. DESIGN AND SETTING: A questionnaire to determine clinical activities within different complementary therapies was developed and mailed-out to 837 eligible physicians in Hamilton, Ontario, Canada, using a modified Dillman approach. RESULTS: The overall response rate to the mailed survey was 49.8% (417/837), with response rates of 50.2% (115/229) for family physicians and 49.7% (302/608) for specialists. The amount of interactions around complementary therapies between physicians, their patients and complementary therapists appears to be low. At the same time, there is a growing interest among physicians about complementary therapies, particularly with respect to developing their knowledge about efficacy and enhancing their skills in assessment and counselling. CONCLUSIONS: The differential levels of acceptance of different therapies by physicians will influence integration of complementary therapies in mainstream medicine.

Chi-Square Distribution↗
PubMed2001

C-terminal truncation and histidine-tagging of cytochrome c oxidase subunit II reveals the native processing site, shows involvement of the C-terminus in cytochrome c binding, and improves the assay for proton pumping.

To enable metal affinity purification of cytochrome c oxidase reconstituted into phospholipid vesicles, a histidine-tag was engineered onto the C-terminal end of the Rhodobacter sphaeroides cytochrome c oxidase subunit II. Characterization of the natively processed wildtype oxidase and artificially processed forms (truncated with and without a his-tag) reveals Km values for cytochrome c that are 6-14-fold higher for the truncated and his-tagged forms than for the wildtype. This lowered ability to bind cytochrome c indicates a previously undetected role for the C-terminus in cytochrome c binding and is mimicked by reduced affinity for an FPLC anion exchange column. The elution profiles and kinetics indicate that the removal of 16 amino acids from the C-terminus, predicted from the known processing site of the Paracoccus denitrificans oxidase, does not produce the same enzyme as the native processing reaction. MALDI-TOF MS data show the true C-terminus of subunit II is at serine 290, three amino acids longer than expected. When the his-tagged form is reconstituted into lipid vesicles and further purified by metal affinity chromatography, significant improvement is observed in proton pumping analysis by the stopped-flow method. The improved kinetic results are attributed to a homogeneous, correctly oriented vesicle population with higher activity and less buffering from extraneous lipids.

Amino Acid Sequence↗
PubMed2001

Mutagenesis in the post genomics era: tools for generating insertional mutations in the lactic acid bacteria.

The increasing availability of whole genome sequences has increased the demand for effective tools to generate insertional mutations in the lactic acid bacteria (LAB). Several novel approaches, such as shuttle-, transposome- and intron-based mutagenesis methods, are possible additions to the existing repertoire of transposon- and recombination-based tools available for mutagenesis of LAB.

Bacteria↗
PubMed2001

Transposon mutagenesis of Xylella fastidiosa by electroporation of Tn5 synaptic complexes.

Pierce's disease, a lethal disease of grapevine, is caused by Xylella fastidiosa, a gram-negative, xylem-limited bacterium that is transmitted from plant to plant by xylem-feeding insects. Strains of X. fastidiosa also have been associated with diseases that cause tremendous losses in many other economically important plants, including citrus. Although the complete genome sequence of X. fastidiosa has recently been determined, the inability to transform or produce transposon mutants of X. fastidiosa has been a major impediment to understanding pathogen-, plant-, and insect-vector interactions. We evaluated the ability of four different suicide vectors carrying either Tn5 or Tn10 transposons as well as a preformed Tn5 transposase-transposon synaptic complex (transposome) to transpose X. fastidiosa. The four suicide vectors failed to produce any detectable transposition events. Electroporation of transposomes, however, yielded 6 x 10(3) and 4 x 10(3) Tn5 mutants per microg of DNA in two different grapevine strains of X. fastidiosa. Molecular analysis showed that the transposition insertions were single, independent, stable events. Sequence analysis of the Tn5 insertion sites indicated that the transpositions occur randomly in the X. fastidiosa genome. Transposome-mediated mutagenesis should facilitate the identification of X. fastidiosa genes that mediate plant pathogenicity and insect transmission.

Animals↗
PubMed2000

Redesign of the proton-pumping machinery of cytochrome c oxidase: proton pumping does not require Glu(I-286).

One of the putative proton-transfer pathways leading from solution toward the binuclear center in many cytochrome c oxidases is the D-pathway, so-called because it starts with a highly conserved aspartate [D(I-132)] residue. Another highly conserved amino acid residue in this pathway, glutamate(I-286), has been indicated to play a central role in the proton-pumping machinery of mitochondrial-type enzymes, a role that requires a movement of the side chain between two distinct positions. In the present work we have relocated the glutamate to the opposite side of the proton-transfer pathway by constructing the double mutant EA(I-286)/IE(I-112). This places the side chain in about the same position in space as in the original enzyme, but does not allow for the same type of movement. The results show that the introduction of the second-site mutation, IE(I-112), in the EA(I-286) mutant enzyme results in an increase of the enzyme activity by a factor of >10. In addition, the double mutant enzyme pumps approximately 0.4 proton per electron. This observation restricts the number of possible mechanisms for the operation of the redox-driven proton pump. The proton-pumping machinery evidently does require the presence of a protonatable/polar residue at a specific location in space, presumably to stabilize an intact water chain. However, this residue does not necessarily have to be at a strictly conserved location in the amino acid sequence. In addition, the results indicate that E(I-286) is not the "proton gate" of cytochrome c oxidase controlling the flow of pumped protons from one to the other side of the membrane.

Catalysis↗
PubMed2000

Characterization of a phosphate binding domain on the alpha-subunit of chloroplast ATP synthase using the photoaffinity phosphate analogue 4-azido-2-nitrophenyl phosphate.

The photoaffinity phosphate analogue 4-azido-2 nitrophenyl phosphate (ANPP) was shown previously (Pougeois, R., Lauquin, G. J.-M., and Vignais, P. V. (1983) Biochemistry 22, 1241-1245) to bind covalently and specifically to a single catalytic site on one of the three beta-subunits of the isolated chloroplast coupling factor 1 (CF(1)). Modification by ANPP strongly inhibited ATP hydrolysis activity. In this study, we examined labeling of membrane-bound CF(1) by ANPP by exposing thylakoid membranes to increasing concentrations of the reagent. ANPP exhibited saturable binding to two sites on CF(1), one on the beta-subunit and one on the alpha-subunit. Labeling by ANPP resulted in the complete inhibition of both ATP synthesis and ATP hydrolysis by the membrane-bound enzyme. Labeling of both sites by ANPP was reduced by more than 80% in the presence of P(i) (> or = 10 mM) and ATP (> or = 0.5 mM). ADP was less effective in competing with ANPP for binding, giving a maximum of approximately 35% inhibition at concentrations > or = 2 mM. ANPP-labeled tryptic peptides of the alpha-subunit were isolated and sequenced. The majority of the probe was contained in three peptides corresponding to residues Gln(173) to Arg(216), Gly(217) to Arg(253), and His(256) to Arg(272) of the alpha-subunit. In the mitochondrial F(1) (Abrahams, J. P., Leslie, A. G. W., Lutter, R., and Walker, J. E. (1994) Nature 370, 621-628), all three analogous peptides are located within the nucleotide binding pocket and within close proximity to the gamma-phosphate binding site. The data indicate, however, that the azidophenyl group of bound ANPP is oriented at approximately 180 degrees in the opposite direction to the adenine binding site with reference to the phosphate binding site on the alpha-subunit. The study has confirmed that ANPP is a bona fide phosphate analogue and suggests that it specifically targets the gamma-phosphate binding site within the nucleotide binding pockets on the alpha- and beta-subunits of CF(1). The study also indicates that in the resting state of the chloroplast F(1)-F(0) complex both the alpha- and beta-subunits are structurally asymmetric.

Azides↗
PubMed2000

Direct profiling of the yeast dynamics in wine fermentations.

We present a method to directly characterize the yeast diversity present in wine fermentations by employing denaturing gradient gel electrophoresis (DGGE) of polymerase chain reaction (PCR)-amplified 26S ribosomal RNA (rRNA) genes. PCR-DGGE of a portion of the 26S rRNA gene was shown to distinguish most yeast genera associated with the production of wine. With this method the microbial dynamics in several model wine fermentations were profiled. PCR-DGGE provided a qualitative assessment of the yeast diversity in these fermentations accurately identifying populations as low as 1000 cells ml(-1). PCR-DGGE represents an attractive alternative to traditional plating schemes for analysis of the microbial successions inherent in the fermentation of wine.

Ecosystem↗
PubMed2000

Where is 'outside' in cytochrome c oxidase and how and when do protons get there?

Cytochrome c oxidase moves both electrons and protons in its dual role as a terminal electron acceptor and a contributor to the proton motive force which drives the formation of ATP. Although the sequence of electron transfer events is well-defined, the correlated mechanism and routes by which protons are translocated across the membrane are not. A recent model [Michel, Proc. Natl. Acad. Sci. USA 95 (1998) 12819] offers a detailed molecular description of when and how protons are translocated through the protein to the outside, which contrasts with previous models in several respects. This article reviews the behavior of site-directed mutants of Rhodobacter sphaeroides cytochrome c oxidase in the context of these different models. Studies of the internally located lysine 362 on the K channel and aspartate 132 on the D channel, indicate that D132, but not K362, is connected to the exterior region. Analysis of the externally located arginine pair, 481 and 482, and the Mg/Mn ligands, histidine 411 and aspartate 412, which are part of the hydrogen-bonded network that includes the heme propionates, indicates that alterations in this region do not strongly compromise proton pumping, but do influence the pH dependence of overall activity and the control of activity by the pH gradient. The results are suggestive of a region of 'sequestered' protons: beyond a major energetic gate, but selectively responsive to the external environment.

Aspartic Acid↗
PubMed1998

Proton uptake and release in cytochrome c oxidase: separate pathways in time and space?

Analysis of mutant forms of cytochrome c oxidase in conjunction with knowledge from high resolution crystal structures is providing important clues as to the location and specificity of proton channels and the timing of proton movements with respect to electron transfer events. Mutant forms of Rhodobacter sphaeroides cytochrome c oxidase at the highly conserved aspartate 132, in the 'D-channel' and at lysine 362, in the 'K-channel', are compared with respect to the nature of their residual activity and their reactions with H2O2. The results argue for physical separation and specificity in these two proton input routes, due to their distinctive kinetics with peroxide and the apparent connection of the D-channel, but not the K-channel, to the proton exit pathway. The reversible nature and possible location of the exit pathway are discussed in the context of direct and indirect mechanisms of energy coupling.

Animals↗
PubMed1998

An origin of transfer (oriT) on the conjugative element pRS01 from Lactococcus lactis subsp. lactis ML3.

Previous analysis of the Tra1 region of the conjugative element pRS01 from Lactococcus lactis subsp. lactis ML3 suggested that an origin of transfer (oriT) was present. Deletion derivatives of this cloned Tra1 region were assayed for mobilization in the presence of the wild-type pRS01 element in trans. The pRS01 oriT was localized to a 446-nucleotide segment in the intergenic region between open reading frames ltrD and ltrE. Sequence analysis of this region revealed a cluster of direct and inverted repeat structures characteristic of oriT regions associated with other conjugative systems.

Amino Acid Sequence↗
PubMed1997

Homing of a group II intron from Lactococcus lactis subsp. lactis ML3.

Ll.ltrB is a functional group II intron located within a gene (ltrB) encoding a conjugative relaxase essential for transfer of the lactococcal element pRSO1. In this work, the Ll.ltrB intron was shown to be an independent mobile element capable of inserting into an intronless allele of the ltrB gene. Ll.ltrB was not observed to insert into a deletion derivative of the ltrB gene in which the intron splice site was removed. In contrast, a second vector containing a 271-nucleotide segment of ltrB spanning the Ll.ltrB splice site was shown to be a proficient recipient of intron insertion. Efficient homing was observed in the absence of a functional host homologous recombination system. This work demonstrates that the Ll.ltrB intron is a novel site-specific mobile element in lactococci and that group II intron self-transfer is a mechanism for intron dissemination among bacteria.

Alleles↗
PubMed1996

Site-directed mutagenesis of residues lining a putative proton transfer pathway in cytochrome c oxidase from Rhodobacter sphaeroides.

Several putative proton transfer pathways have been identified in the recent crystal structures of the cytochrome oxidases from Paracoccus denitrificans [Iwata et al. (1995) Nature 376, 660-669] and bovine [Tsukihara (1996) Science 272, 1138-1144]. A series of residues along one face of the amphiphilic transmembrane helix IV lie in one of these proton transfer pathways. The possible role of these residues in proton transfer was examined by site-directed mutagenesis. The three conserved residues of helix IV that have been implicated in the putative proton transfer pathway (Ser-201, Asn-207, and Thr-211) were individually changed to alanine. The mutants were purified, analyzed for steady-state turnover rate and proton pumping efficiency, and structurally probed with resonance Raman spectroscopy and FTIR difference spectroscopy. The mutation of Ser-201 to alanine decreased the enzyme turnover rate by half, and was therefore further characterized using EPR spectroscopy and rapid kinetic methods. The results demonstrate that none of these hydrophilic residues are essential for proton pumping or oxygen reduction activities, and suggest a model of redundant or flexible proton transfer pathways. Whereas previously reported mutants at the start of this putative channel (e.g., Asp-132-Asn) dramatically influence both enzyme turnover and coupling to proton pumping, the current work shows that this is not the case for all residues observed in this channel.

Carbon Monoxide↗
PubMed1996

Splicing of a group II intron involved in the conjugative transfer of pRS01 in lactococci.

Analysis of a region involved in the conjugative transfer of the lactococcal conjugative element pRS01 has revealed a bacteria] group II intron. Splicing of this lactococcal intron (designated Ll.ltrB) in vivo resulted in the ligation of two exon messages (ltrBE1 and ltrBE2) which encoded a putative conjugative relaxase essential for the transfer of pRS01. Like many group II introns, the Ll.ltrB intron possessed an open reading frame (ltrA) with homology to reverse transcriptases. Remarkably, sequence analysis of ltrA suggested a greater similarity to open reading frames encoded by eukaryotic mitochondrial group II introns than to those identified to date from other bacteria. Several insertional mutations within ltrA resulted in plasmids exhibiting a conjugative transfer-deficient phenotype. These results provide the first direct evidence for splicing of a prokaryotic group II intron in vivo and suggest that conjugative transfer is a mechanism for group II intron dissemination in bacteria.

Amino Acid Sequence↗
PubMed1995

ADP binding induces long-distance structural changes in the beta polypeptide of the chloroplast ATP synthase.

Binding of ADP to the beta polypeptide isolated from the catalytic F1 portion (CF1) of the chloroplast ATP synthase caused an increase of 10-20% in the steady state fluorescence intensity of fluorescent maleimides attached to the cysteine residue at position 63. Fluorescence lifetime distributions indicated that the beta polypeptide switched between two conformational states depending on the presence or absence of bound ADP. The fluorescence enhancement induced by ADP binding allowed a direct calculation of the dissociation constant for ADP of 0.7 microM. ATP did not cause a fluorescence enhancement but competed with ADP for binding to the same site. An apparent dissociation constant of 2 microM was obtained for ATP binding. Fluorescence resonance energy transfer experiments indicated that Cys63 is 42 A away from the nucleotide binding site on the beta polypeptide, confirming a previous measurement [(Colvert, K.K., Mills, D.A., Richter, M.L. (1992) Biochemistry 31, 3930-3935]. Frequency domain fluorescence anisotropy measurements indicated that the beta polypeptide has an irregular, elongated shape which is in good agreement with the conformation found in the crystal structure of the beef heart mitochondrial F1 enzyme [Abrahams, J.P., Leslie, A.G.W., Lutter, R., & Walker, J.E. (1994) Nature 370, 621-628]. The rotational correlation time did not change significantly upon ADP binding, indicating that ADP did not induce a large change in the overall shape of the beta polypeptide. The results show that the nucleotide binding domain and the N-terminal domain of the beta polypeptide communicate with each other over a significant distance via conformational changes.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Diphosphate↗
PubMed1995

Ototoxicity associated with intravenous bisphosphonate administration.

The development of ototoxicity in association with intravenous pamidronate treatment of Paget's disease is described. The patient developed hearing loss, vertigo, and tinnitus which were exacerbated by subsequent infusions. This potentially serious drug side-effect does not appear to have been reported previously in patients with Paget's disease.

Diphosphonates↗