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Biomedical subjects

D Mills

Publications and source records attributed to D Mills.

At least 37 records · Page 2Linked to original sources
PubMed2000

RHIZOSCAN: A semiautomatic image processing system for characterization of the morphology and secondary metabolite concentration in hairy root cultures.

We describe the application of the newly developed RHIZOSCAN software for accurate morphological analysis and determination of overall and local secondary metabolite concentrations in two clones of Beta vulgaris throughout the growth period. Local secondary metabolite concentrations may be determined at any point in the root, and pigment gradients in each lateral root can be followed during culture and saved to the computer. Throughout the entire analysis process, an image appears in a graphical result window on the screen, which enables visual evaluation of the numerical output at each stage of the analysis. Biosynthetic data on concentrations obtained by image analysis were validated by spectrophotometric analysis. The importance of determining appropriate scanning and analysis conditions (scanning resolution, background color, threshold value, segmentation plane in the hue-saturation-intensity color system and pruning length) for obtaining accurate morphological measurements is examined and the means of fixing these parameters is described. Our results show that, using RHIZOSCAN, detailed and accurate information on root architecture and secondary metabolite concentrations can be obtained in a short time.

Automation↗
PubMed2000

Mutations in the putative H-channel in the cytochrome c oxidase from Rhodobacter sphaeroides show that this channel is not important for proton conduction but reveal modulation of the properties of heme a.

As the final electron acceptor in the respiratory chain of eukaryotic and many prokaryotic organisms, cytochrome c oxidase catalyzes the reduction of oxygen to water, concomitantly generating a proton gradient. X-ray structures of two cytochrome c oxidases have been reported, and in each structure three possible pathways for proton translocation are indicated: the D-, K-, and H-channels. The putative H-channel is most clearly delineated in the bovine heart oxidase and has been proposed to be functionally important for the translocation of pumped protons in the mammalian oxidase [Yoshikawa et al. (1998) Science 280, 1723-1729]. In the present work, the functional importance of residues lining the putative H-channel in the oxidase from Rhodobacter sphaeroides are examined by site-directed mutagenesis. Mutants were generated in eight different sites and the enzymes have been purified and characterized. The results suggest that the H-channel is not functionally important in the prokaryotic oxidase, in agreement with the conclusion from previous work with the oxidase from Paracoccus denitrificans [Pfitzner et al. (1998) J. Biomembr. Bioenerg. 30, 89-93]. Each of the mutants in R. sphaeroides, with an exception at only one position, is enzymatically active and pumps protons in reconstituted proteoliposomes. This includes H456A, where in the P. denitrificans oxidase a leucine residue substituted for the corresponding residue resulted in inactive enzyme. The only mutations that result in completely inactive enzyme in the set examined in the R. sphaeroides oxidase are in R52, a residue that, along with Q471, appears to be hydrogen-bonded to the formyl group of heme a in the X-ray structures. To characterize the interactions between this residue and the heme group, resonance Raman spectra of the R52 mutants were obtained. The frequency of the heme a formyl stretching mode in the R52A mutant is characteristic of that seen in non-hydrogen-bonded model heme a complexes. Thus the data confirm the presence of hydrogen bonding between the heme a formyl group and the R52 side chain, as suggested from crystallographic data. In the R52K mutant, this hydrogen bonding is maintained by the lysine residue, and this mutant enzyme retains near wild-type activity. The heme a formyl frequency is also affected by mutation of Q471, confirming the X-ray models that show this residue also has hydrogen-bonding interactions with the formyl group. Unlike R52, however, Q471 does not appear to be critical for the enzyme function.

Alanine↗
PubMed2000

Telomere-associated RFLPs and electrophoretic karyotyping reveal lineage relationships among race-specific strains of Ustilago hordei.

The inheritance of telomere-associated restriction fragment length polymorphisms (tel-RFLP) and chromosome-length polymorphisms (CLPs) were criteria used for the identification of strains of Ustilago hordei that form a direct lineage. Teliospore collections of race 8 strains and strains reported to be derived from race 8 through inbreeding were used in these analyses. None of the race 8 strains examined in this study, representing three consecutive inbred generations, was polymorphic for any terminal BamHI and BglII chromosomal loci, nor did they have any apparent CLPs. Strains from a teliospore collection obtained in 1971 and designated 447, representing the first inbred generation of race 8 strains and a shift to increased virulence on cultivar Hannchen, had tel-RFLP arrays indistinguishable from the race 8 strains isolated in this study; and they had no obvious CLPs. Strains from the presumed second inbred generation, the 1279 teliospore line, which was pathogenic on six additional cultivars, had numerous CLPs and a tel-RFLP array that differed from race 8 strains at more than 50% of the terminal chromosomal BamHI and BglII restriction sites. The tel-RFLP array of each 1279 strain was identical and indistinguishable from the arrays of strains representing races 10 and 13, indicating that they share a direct lineage. A race 14 strain, also presumed to be derived from race 8 strains by inbreeding, had a unique tel-RFLP array and an electrophoretic karyotype that distinguished it from all other strains. The tel-RFLP arrays alone eliminate the 1279 and race 14 strains from being direct descendants from race 8 strains by inbreeding and suggest that this approach can identify a strain lineage among other inbred sexually reproducing fungi, or isolates that comprise different asexual clonal populations.

Blotting, Southern↗
PubMed2000

Profound hearing loss associated with hydrocodone/acetaminophen abuse.

OBJECTIVES: To describe profound hearing loss associated with hydrocodone overuse and the successful rehabilitation of these patients with cochlear implantation. STUDY DESIGN: Retrospective review. SETTING: A tertiary otologic referral center. PATIENTS: Twelve patients with rapidly progressive hearing loss and a concurrent history of hydrocodone overuse. INTERVENTIONS: Comprehensive medical histories, physical findings, audiometric tests, and, in those patients undergoing cochlear implantation, postimplantation performance data were reviewed. MAIN OUTCOME MEASURES: Clinical characteristics of hydrocodone-related hearing loss and open set word and sentence performance in those patients undergoing cochlear implantation. RESULTS: Hydrocodone overuse was associated with rapidly progressive sensorineural hearing loss in 12 patients. In four patients the initial presentation was unilateral, and two of the patients experienced vestibular symptoms. None of the 12 patients experienced improved thresholds after high-dose prednisone. Seven of the eight patients undergoing cochlear implantation have demonstrated early success with their devices. CONCLUSIONS: Hydrocodone is frequently prescribed in combination with acetaminophen for the relief of pain and has a side effects profile similar to other medications in its class. Although not described previously, overuse or abuse can be associated with a rapidly progressive sensorineural hearing loss. These patients can be successfully rehabilitated with cochlear implantation.

Acetaminophen↗
PubMed2000

Selective small molecule inhibitors of glycogen synthase kinase-3 modulate glycogen metabolism and gene transcription.

BACKGROUND: Glycogen synthase kinase-3 (GSK-3) is a serine/threonine protein kinase, the activity of which is inhibited by a variety of extracellular stimuli including insulin, growth factors, cell specification factors and cell adhesion. Consequently, inhibition of GSK-3 activity has been proposed to play a role in the regulation of numerous signalling pathways that elicit pleiotropic cellular responses. This report describes the identification and characterisation of potent and selective small molecule inhibitors of GSK-3. RESULTS: SB-216763 and SB-415286 are structurally distinct maleimides that inhibit GSK-3alpha in vitro, with K(i)s of 9 nM and 31 nM respectively, in an ATP competitive manner. These compounds inhibited GSK-3beta with similar potency. However, neither compound significantly inhibited any member of a panel of 24 other protein kinases. Furthermore, treatment of cells with either compound stimulated responses characteristic of extracellular stimuli that are known to inhibit GSK-3 activity. Thus, SB-216763 and SB-415286 stimulated glycogen synthesis in human liver cells and induced expression of a beta-catenin-LEF/TCF regulated reporter gene in HEK293 cells. In both cases, compound treatment was demonstrated to inhibit cellular GSK-3 activity as assessed by activation of glycogen synthase, which is a direct target of this kinase. CONCLUSIONS: SB-216763 and SB-415286 are novel, potent and selective cell permeable inhibitors of GSK-3. Therefore, these compounds represent valuable pharmacological tools with which the role of GSK-3 in cellular signalling can be further elucidated. Furthermore, development of similar compounds may be of use therapeutically in disease states associated with elevated GSK-3 activity such as non-insulin dependent diabetes mellitus and neurodegenerative disease.

Adenosine Triphosphate↗
PubMed1999

Mass determination of the unit cell of the innermost chorionic layer in Drosophilidae by scanning transmission electron microscopy.

The innermost chorionic layer (ICL) in eggshells of Drosophila melanogaster is a naturally occurring patchwork of thin three-dimensional crystalline plates located between the inner endochorion and the vitelline envelope. The mass-per-unit area of the ICL has been measured from scanning transmission electron microscope images of isolated unstained material and it was possible to distinguish up to four layers with the majority of the crystalline sheets being one to three layers thick. Taking into account the unit cell areas for the different crystals, we have estimated the mean ICL subunit sizes to be 36 kDa for Drosophila melanogaster, 35 kDa for Drosophila auraria, and 33 kDa for Drosophila teissieri. The results suggest that the three different Drosophilidae species have very similar average subunit masses.

Animals↗
PubMed1999

Bridging cognition, the brain and molecular genetics: evidence from Williams syndrome.

Williams syndrome (WMS) is a rare sporadic disorder that yields a distinctive profile of medical, cognitive, neurophysiological, neuroanatomical and genetic characteristics. The cognitive hallmark of WMS is a dissociation between language and face processing (relative strengths) and spatial cognition (profound impairment). Individuals with WMS also tend to be overly social, behavior that is opposite to that seen in autism. A genetic hallmark of WMS is a deletion on chromosome band 7q11.23. Williams syndrome is also associated with specific neuromorphological and neurophysiological profiles: proportional sparing of frontal, limbic and neocerebellar structures is seen using MRI; and abnormal functional organization of the neural systems that underlie both language and face processing is revealed through studies using event-related potentials. The non-uniformity in the cognitive, neuromorphological and neurophysiological domains of WMS make it a compelling model for elucidating the relationships between cognition, the brain and, ultimately, the genes.

Brain↗
PubMed1998

Retrohoming of a bacterial group II intron: mobility via complete reverse splicing, independent of homologous DNA recombination.

The mobile group II intron of Lactococcus lactis, Ll.LtrB, provides the opportunity to analyze the homing pathway in genetically tractable bacterial systems. Here, we show that Ll.LtrB mobility occurs by an RNA-based retrohoming mechanism in both Escherichia coli and L. lactis. Surprisingly, retrohoming occurs efficiently in the absence of RecA function, with a relaxed requirement for flanking exon homology and without coconversion of exon markers. These results lead to a model for bacterial retrohoming in which the intron integrates into recipient DNA by complete reverse splicing and serves as the template for cDNA synthesis. The retrohoming reaction is completed in unprecedented fashion by a DNA repair event that is independent of homologous recombination between the alleles. Thus, Ll.LtrB has many features of retrotransposons, with practical and evolutionary implications.

Bacterial Proteins↗
PubMed1998

Polysaccharide production by plant cells in suspension: experiments and mathematical modeling.

Symphytum officinale L cells were grown in Erlenmeyer flasks at four different temperatures: 15, 20, 25, and 30 degrees C. A mathematical model of the culture growth is presented. The intracellular and extracellular products are considered in separate equations. An interrelation between fresh weight, dry weight, and viability is considered in the balances. The model includes a description of the changes in time of wet and dry biomass, cell viability, substrate concentration and polysaccharide concentration, both intra- and extracellular. The model was tested by fitting the numerical results to the data obtained.

Biomass↗
PubMed1998

Control of pigmentation of Ustilago hordei: the effect of pH, thiamine, and involvement of the cAMP cascade.

The intensely pigmented teliospores of Ustilago hordei that are produced on susceptible barley cultivars contain nondiffusible deposits of a melanin-like pigment. Expression of pigmentation differed among haploid sporidial cultures and could be shown to be influenced by culture conditions. Pigmentation of strain 8.2a was black at acidic pH and repressed in medium adjusted to neutral or basic pH, and growth at elevated pH triggered a concomitant accumulation of a diffusible red pigment. Pigment formation by some strains was also determined to be under the control of thiamine and was completely inhibited when thiamine was present at levels above 0. 02 microM. The second messenger, cAMP, transiently repressed pigment formation, whereas the cAMP phosphodiesterase inhibitor isobutryl-3-methyl xanthine completely repressed pigment formation. Like cAMP, the expression of the Galpha subunit gene FIL1 from a multicopy vector resulted in transient inhibition of pigment formation. However, pigment formation was not observed in cells expressing the mutant allele FIL1(Q206R), which ostensibly renders the gene constitutively active. The means by which pigment formation is repressed suggested that numerous genes were involved. Upon examination of a wild-type strain transformed with random cosmid clones of a genomic library, it was estimated that approximately 30 cosmid members per genome equivalent caused repression of the melanin-like pigments, whereas approximately 6 cosmid members induced pigment formation.

1-Methyl-3-isobutylxanthine↗
PubMed1998

Insertion specificity and trans-activation of IS801.

The transposable element IS801, isolated from plasmid pMMC7105 of Pseudomonas syringae pv. phaseolicola, transposes in Escherichia coli to plasmid targets, expressing a relatively relaxed target specificity. The target sequences are tetramers with homology with the left terminus (GAAC) of the transposing unit, the alternative targets being GAAC, GGAC, CAAG, and CGAC. In the areas flanking IS801 in 13 different locations, no similarities other than the target tetramer were observed. The transposase is physically and functionally separable from the transposing unit since transposition of constructs carrying marker genes occurs with the transposase expressed in trans. The IS801 transposase shows amino acid sequence homology to the transposases of the E. coli elements IS91 and IS1294. These transposases contain conserved amino acid motifs found in the replicases of certain plasmids that replicate as rolling circles.

Base Sequence↗
PubMed1998

Auditory-nerve integrity after middle-fossa acoustic-tumor removal.

We sought to investigate the functional integrity of the auditory nerve in patients with postoperative hearing loss after middle cranial fossa acoustic-tumor removal in a case-series descriptive study. The study setting was a tertiary referral center, a private otologic practice. The study population comprised seven patients who underwent a middle-cranial-fossa approach for unilateral acoustic-tumor resection and sustained postoperative anacusis with an anatomically intact auditory nerve. Four were men and three women; they ranged in age from 30 to 60 years; all underwent surgery between 1990 and 1994 and agreed to return to the center to participate in the study during 1995. Diagnostic electrical promontory stimulation was used to determine the functional integrity of the auditory nerve. Our main outcome measures were the presence or absence of discrete tone perception, electrical threshold, maximum acceptable level and dynamic range, gap detection and temporal difference limen during electrical promontory stimulation. Three of the seven patients demonstrated positive responses to electrical promontory testing (e.g., discrete tone perception). All three were able to perform the gap-detection and temporal difference limen tests. None of the preoperative characteristics was related to performance on promontory stimulation testing. We conclude that the middle cranial fossa approach permits anatomic--and, in some cases, functional auditory-nerve preservation. These data suggest that auditory rehabilitation in some patients who sustain anacusis after the middle cranial fossa approach to acoustic tumors, as in neurofibromatosis 2, may be provided with cochlear implantation.

Adult↗
PubMed1998

The Greater Cincinnati/Northern Kentucky Stroke Study: preliminary first-ever and total incidence rates of stroke among blacks.

BACKGROUND AND PURPOSE: The Greater Cincinnati/Northern Kentucky Stroke Study was designed to be the first large, population-based metropolitan study of temporal trends in stroke incidence rates and outcome within a biracial population. METHODS: We are identifying all hospitalized and autopsied cases of stroke and transient ischemic attack (TIA) among the 1.3 million inhabitants of a five-county region of Greater Cincinnati/Northern Kentucky for the period 7/1/93-6/30/94. We have already prospectively monitored for out-of-hospital stroke and TIAs for this same time period at 128 screening sites, including a random sample of all primary care physicians and nursing homes in the region. We have already identified all hospitalized and autopsied cases of stroke and TIA among blacks for 1/1/93-6/30/93 and report preliminary incidence rates for this 6-month period. RESULTS: The overall incidence rate for all first-ever hospitalized or autopsied stroke (excluding TIAs) among blacks in the Greater Cincinnati region was 288 per 100000 (95% CI, 250 to 325, age- and sex-adjusted to 1990 US population). The overall incidence rate for first-ever and recurrent stroke (excluding TIAs) was 411 per 100000 (95% CI, 366 to 456). By comparison, the overall incidence rate of first-ever stroke among whites in Rochester, Minn, during the period 1985-1989 was 179 per 100000 (95% CI, 164 to 194, age- and-sex adjusted to 1990 US population). The incidence rates among blacks in Greater Cincinnati were substantially greater than the rates among whites in Rochester, Minn, for all age categories except ages 75 and older, for which the rates were similar. CONCLUSIONS: We conservatively estimate that 731100 first-ever or recurrent strokes occurred in the United States during 1996. Studies of first-ever as well as total stroke among biracial and representative populations are critical for understanding temporal trends in the incidence rate and the burden of stroke in the US population.

Adult↗
PubMed1997

Aspartate-407 in Rhodobacter sphaeroides cytochrome c oxidase is not required for proton pumping or manganese binding.

Several pathways for proton transport in cytochrome c oxidase have been proposed on the basis of mutational analysis and X-ray structure: at least one for moving "pumped" protons from the interior to exterior of the membrane and a separate route for transporting "substrate" protons from the interior to the binuclear metal center to combine with oxygen to make H2O. According to the crystal structures of cytochrome c oxidase, Asp407 (Rhodobacter sphaeroides numbering) is at the interface of subunit I and subunit II of the oxidase, in a negative patch proposed to be the proton exit site in a pumping pathway, as well as a possible ligand to Mg [Iwata et al. (1995) Nature 376, 660-669]. Three mutants at the Asp407 position of R. sphaeroides cytochrome oxidase, Asp407Ala, Asp407Asn, and Asp407Cys, have been purified and characterized. All showed electron transfer activity, and pH dependence of activity, similar to that of the wild type enzyme and no major structural changes, as evidenced by visible, EPR, and resonance Raman spectroscopy. When reconstituted into artificial vesicles, the purified mutants pumped protons with normal efficiency and responded to the membrane pH and electrical gradients in a manner similar to that of wild type. Furthermore, the EPR spectra and Mn quantitation analysis of mutants grown in high Mn indicated no significant alteration in the Mn/Mg site. These results suggest that Asp407 does not play a critical role in proton translocation or in Mn/Mg binding.

Aspartic Acid↗