Biomedical subjects

David M Miller

Publications and source records attributed to David M Miller.

12 recordsLinked to original sources

PubMed2006

Computational inference of the molecular logic for synaptic connectivity in C. elegans.

MOTIVATION: The nematode C. elegans is an ideal model organism in which to investigate the biomolecular mechanisms underlying the connectivity of neurons, because synaptic connections are described in a comprehensive wiring diagram and methods for defining gene expression profiles of individual neurons are now available. RESULTS: Here we present computational techniques linking these two types of information. A systems-based approach (EMBP: Entropy Minimization and Boolean Parsimony) identifies sets of synergistically interacting genes whose joint expression predicts neural connectivity. We introduce an information theoretic measure of the multivariate synergy, a fundamental concept in systems biology, connecting the members of these gene sets. We present and validate our preliminary results based on publicly available information, and demonstrate that their synergy is exceptionally high indicating joint involvement in pathways. Our strategy provides a robust methodology that will yield increasingly more accurate results as more neuron-specific gene expression data emerge. Ultimately, we expect our approach to provide important clues for universal mechanisms of neural interconnectivity.

Animals↗

PubMed2006

Kinetics of insulin-like growth factor II (IGF-II) interaction with domain 11 of the human IGF-II/mannose 6-phosphate receptor: function of CD and AB loop solvent-exposed residues.

Ligands of the IGF-II/mannose 6-phosphate receptor (IGF2R) include IGF-II and mannose 6-phosphate modified proteins. Disruption of the negative regulatory effects of IGF2R on IGF-II-induced growth can lead to embryonic lethality and cancer promotion. Of the 15 IGF2R extracellular domains, domains 1-3 and 11 are known to have a conserved beta-barrel structure similar to that of avidin and the cation-dependent mannose 6-phosphate receptor, yet only domain 11 binds IGF-II with high specificity and affinity. In order to define the functional basis of this critical biological interaction, we performed alanine mutagenesis of structurally determined solvent-exposed loop residues of the IGF-II-binding site of human domain 11, expressed these mutant forms in Pichia pastoris, and determined binding kinetics with human IGF-II using isothermal calorimetry and surface plasmon resonance with transition state thermodynamics. Two hydrophobic residues in the CD loop (F1567 and I1572) were essential for binding, with a further non-hydrophobic residue (T1570) that slows the dissociation rate. Aside from alanine mutations of AB loop residues that decrease affinity by modifying dissociation rates (e.g. Y1542), a novel mutation (E1544A) of the AB loop enhanced affinity by threefold compared to wild-type. Conversion from an acidic to a basic residue at this site (E1544K) results in a sixfold enhancement of affinity via modification principally of the association rate, with enhanced salt-dependence, decreased entropic barrier and retained specificity. These data suggest that a functional hydrophobic binding site core is formed by I1572 and F1567 located in the CD loop, which initially anchors IGF-II. Within the AB loop, residues normally act to either stabilise or function as negative regulators of the interaction. These findings have implications for the molecular architecture and evolution of the domain 11 IGF-II-binding site, and the potential interactions with other domains of IGF2R.

Amino Acid Sequence↗

PubMed2006

Predominance of char sorption over substrate concentration and soil pH in influencing biodegradation of benzonitrile.

Incomplete combustion of field crop residues results in the production of char, a material rich in charcoal-type substances. Consequently, char is an effective adsorbent of organic compounds and when incorporated into soil may adsorb soil-applied pesticides, thereby altering their susceptibility to biodegradation. We investigated the relative importance of char, soil pH and initial substrate concentration in biodegradation of pesticides in soils by measuring the biodegradation of benzonitrile in soil as a function of soil char content (0% and 1% by weight), initial benzonitrile concentration (0.1, 1.06, and 10.2 mg l(-1)) and soil pH (5.2, 6.9 and 8.5). Preliminary experiments revealed that wheat straw char had a much greater benzonitrile sorption capacity than did soil to which the char was added. The extent of benzonitrile degradation decreased as initial benzonitrile concentration increased in both buffer solution and soil slurry. In contrast, the degradation increased as initial benzonitrile concentration increased in char-amended slurry. In un-amended soil slurry, the benzonitrile degradation was lower at pH 5.2 than at pH 6.9 or 8.5, but in char-amended soil slurry the degradation was not affected by pH, again presumably due to adsorption of benzonitrile by the char. Adsorption by soil char appears to be more important than either initial substrate concentration or soil pH in controlling benzonitrile degradation in char-amended soil slurry. The presence of crop residue-derived chars may alter pesticide degradation patterns normally observed in soils and thus significantly affect their environmental fate.

Adsorption↗

PubMed2006

The motor circuit.

Caenorhabditis elegans motor neurons control a range of activities including locomotion, foraging, defecation, and gender-specific functions. In this chapter,we focus primarily on motor neurons that regulate body movement, with particular emphasis on those in the ventral nerve cord (VNC). We describe the basic architecture and development of the motor circuit, genes that specify motor neuron fates, and models of how the motor circuit controls locomotion. We identify surprising similarities between the structure and development of the nematode and vertebrate axial nerve cords and speculate about the potential roles of conserved families of transcription factors in the evolution of these motor circuits.

Animals↗

PubMed2005

Role of wheat-residue-derived char in the biodegradation of benzonitrile in soil: nutritional stimulation versus adsorptive inhibition.

Previous studies demonstrated that crop-residue-derived chars are effective adsorbents for pesticides and substantially reduce their biodegradation in soils. In contrast, the nutrients in the chars may stimulate cell growth and hence enhance the biodegradation. This work investigated the role of a wheat-residue-derived char in the nutritional stimulation and adsorptive inhibition of biodegradation of benzonitrile in a soil. The biodegradation was measured at 78 mg/L of benzonitrile, an initial concentration much higher than the half-saturation constant of the degrading organism (approximately 18 mg/L). The degradation was much faster in the extract of char-amended soil (CAS) than in those of soil and washed-char-amended soil (WCAS). The degradation was also faster in CAS slurry than in slurries of soil and WCAS until approximately 88% of benzonitrile degraded in char-containing slurries, where the aqueous-phase concentration was approximately 0.25 mg/L. Cell density was higher in extracts and slurries in the presence of the char nutrients than in the absence of them. Analysis of nutrient elements in extracts, along with measuring degradation in soil extract with nutrient supplements and subsequent stepwise multiple-regression, suggested that the char nutrients stimulated cell growth and degradation of benzonitrile, for which P was primarily responsible. Further degradation of benzonitrile beyond 88% in the presence of char was slow, suggestive of the adsorptive inhibition. The biodegradation of benzonitrile in CAS thus occurred through a fast-to-slow process, the former step being due primarily to the stimulation by soluble P of char and the latter phase to the adsorptive inhibition.

Adsorption↗

PubMed2005

acr-16 encodes an essential subunit of the levamisole-resistant nicotinic receptor at the Caenorhabditis elegans neuromuscular junction.

The Caenorhabditis elegans neuromuscular junction (NMJ) contains three pharmacologically distinct ionotropic receptors: gamma-aminobutyric acid receptors, levamisole-sensitive nicotinic receptors, and levamisole-insensitive nicotinic receptors. The subunit compositions of the gamma-aminobutyric acid- and levamisole-sensitive receptors have been elucidated, but the levamisole-insensitive acetylcholine receptor is uncharacterized. To determine which of the approximately 40 putative nicotinic receptor subunit genes in the C. elegans genome encodes the levamisole-resistant receptor, we utilized MAPCeL, a microarray profiling strategy. Of seven nicotinic receptor subunit transcripts found to be enriched in muscle, five encode the levamisole receptor subunits, leaving two candidates for the levamisole-insensitive receptor: acr-8 and acr-16. Electrophysiological analysis of the acr-16 deletion mutant showed that the levamisole-insensitive muscle acetylcholine current was eliminated, whereas deletion of acr-8 had no effect. These data suggest that ACR-16, like its closest vertebrate homolog, the nicotinic receptor alpha7-subunit, may form homomeric receptors in vivo. Genetic ablation of both the levamisole-sensitive receptor and acr-16 abolished all cholinergic synaptic currents at the NMJ and severely impaired C. elegans locomotion. Therefore, ACR-16-containing receptors account for all non-levamisole-sensitive nicotinic synaptic signaling at the C. elegans NMJ. The determination of subunit composition for all three C. elegans body wall muscle ionotropic receptors provides a critical foundation for future research at this tractable model synapse.

Action Potentials↗

PubMed2005

A gene expression fingerprint of C. elegans embryonic motor neurons.

BACKGROUND: Differential gene expression specifies the highly diverse cell types that constitute the nervous system. With its sequenced genome and simple, well-defined neuroanatomy, the nematode C. elegans is a useful model system in which to correlate gene expression with neuron identity. The UNC-4 transcription factor is expressed in thirteen embryonic motor neurons where it specifies axonal morphology and synaptic function. These cells can be marked with an unc-4::GFP reporter transgene. Here we describe a powerful strategy, Micro-Array Profiling of C. elegans cells (MAPCeL), and confirm that this approach provides a comprehensive gene expression profile of unc-4::GFP motor neurons in vivo. RESULTS: Fluorescence Activated Cell Sorting (FACS) was used to isolate unc-4::GFP neurons from primary cultures of C. elegans embryonic cells. Microarray experiments detected 6,217 unique transcripts of which approximately 1,000 are enriched in unc-4::GFP neurons relative to the average nematode embryonic cell. The reliability of these data was validated by the detection of known cell-specific transcripts and by expression in UNC-4 motor neurons of GFP reporters derived from the enriched data set. In addition to genes involved in neurotransmitter packaging and release, the microarray data include transcripts for receptors to a remarkably wide variety of signaling molecules. The added presence of a robust array of G-protein pathway components is indicative of complex and highly integrated mechanisms for modulating motor neuron activity. Over half of the enriched genes (537) have human homologs, a finding that could reflect substantial overlap with the gene expression repertoire of mammalian motor neurons. CONCLUSION: We have described a microarray-based method, MAPCeL, for profiling gene expression in specific C. elegans motor neurons and provide evidence that this approach can reveal candidate genes for key roles in the differentiation and function of these cells. These methods can now be applied to generate a gene expression map of the C. elegans nervous system.

Animals↗

PubMed2004

Immunogenicity of a highly attenuated MVA smallpox vaccine and protection against monkeypox.

The potential use of smallpox as a biological weapon has led to the production and stockpiling of smallpox vaccine and the immunization of some healthcare workers. Another public health goal is the licensing of a safer vaccine that could benefit the millions of people advised not to take the current one because they or their contacts have increased susceptibility to severe vaccine side effects. As vaccines can no longer be tested for their ability to prevent smallpox, licensing will necessarily include comparative immunogenicity and protection studies in non-human primates. Here we compare the highly attenuated modified vaccinia virus Ankara (MVA) with the licensed Dryvax vaccine in a monkey model. After two doses of MVA or one dose of MVA followed by Dryvax, antibody binding and neutralizing titres and T-cell responses were equivalent or higher than those induced by Dryvax alone. After challenge with monkeypox virus, unimmunized animals developed more than 500 pustular skin lesions and became gravely ill or died, whereas vaccinated animals were healthy and asymptomatic, except for a small number of transient skin lesions in animals immunized only with MVA.

Animals↗

PubMed2002

Neurotoxin-induced degeneration of dopamine neurons in Caenorhabditis elegans.

Parkinson's disease is a complex neurodegenerative disorder characterized by the death of brain dopamine neurons. In mammals, dopamine neuronal degeneration can be triggered through exposure to neurotoxins accumulated by the presynaptic dopamine transporter (DAT), including 6-hydroxydopamine (6-OHDA) and 1-methyl-4-phenylpyridinium. We have established a system for the pharmacological and genetic evaluation of neurotoxin-induced dopamine neuronal death in Caenorhabditis elegans. Brief (1 h) exposure of green fluorescent protein-tagged, living worms to 6-OHDA causes selective degeneration of dopamine neurons. We demonstrate that agents that interfere with DAT function protect against 6-OHDA toxicity. 6-OHDA-triggered neural degeneration does not require the CED-3/CED-4 cell death pathway, but is abolished by the genetic disruption of the C. elegans DAT.

Animals↗

PubMed2002

A primary culture system for functional analysis of C. elegans neurons and muscle cells.

C. elegans has provided important insights into neuromuscular system function and development. However, the animal's small size limits access to individual neurons and muscle cells for physiological, biochemical, and molecular study. We describe here primary culture methods that allow C. elegans embryonic cells to differentiate into neurons and muscle cells in vitro. Morphological, electrophysiological, and GFP reporter studies demonstrate that the differentiation and functional properties of cultured cells are similar to those observed in vivo. Enriched populations of cells expressing specific GFP reporters can be generated by fluorescence-activated cell sorting. Addition of double-stranded RNA to the culture medium induces dramatic knockdown of targeted gene expression. Primary nematode cell culture provides a new foundation for a wide variety of experimental opportunities heretofore unavailable in the field.

Animals↗

PubMed2002

The C. elegans even-skipped homologue, vab-7, specifies DB motoneurone identity and axon trajectory.

Locomotory activity is defined by the specification of motoneurone subtypes. In the nematode, C. elegans, DA and DB motoneurones innervate dorsal muscles and function to induce movement in the backwards or forwards direction, respectively. These two neurone classes express separate sets of genes and extend axons with oppositely directed trajectories; anterior (DA) versus posterior (DB). The DA-specific homeoprotein UNC-4 interacts with UNC-37/Groucho to repress the DB gene, acr-5 (nicotinic acetylcholine receptor subunit). We show that the C. elegans even-skipped-like homoedomain protein, VAB-7, coordinately regulates different aspects of the DB motoneurone fate, in part by repressing unc-4. Wild-type DB motoneurones express VAB-7, have posteriorly directed axons, express ACR-5 and lack expression of the homeodomain protein UNC-4. In a vab-7 mutant, ectopic UNC-4 represses acr-5 and induces an anteriorly directed DB axon trajectory. Thus, vab-7 indirectly promotes DB-specific gene expression and posteriorly directed axon outgrowth by preventing UNC-4 repression of DB differentiation. Ectopic expression of VAB-7 also induces DB traits in an unc-4-independent manner, suggesting that VAB-7 can act through a parallel pathway. This work supports a model in which a complementary pair of homeodomain transcription factors (VAB-7 and UNC-4) specifies differences between DA and DB neurones through inhibition of the alternative fates. The recent findings that Even-skipped transcriptional repressor activity specifies neurone identity and axon guidance in the mouse and Drosophila motoneurone circuit points to an ancient origin for homeoprotein-dependent mechanisms of neuronal differentiation in the metazoan nerve cord.

Animals↗

PubMedDate not supplied

Nutrient input and removal trends for agricultural soils in nine geographic regions in Arkansas.

Knowledge of the balance between nutrient inputs and removals is required for identifying regions that possess an excess or deficit of nutrients. This assessment describes the balance between the agricultural nutrient inputs and removals for nine geographical districts within Arkansas from 1997 to 2001. The total N, P, and K inputs were summed for each district and included inorganic fertilizer and collectable nutrients excreted as poultry, turkey, dairy, and hog manures. Nutrients removed by harvested crops were summed and subtracted from total nutrient inputs to calculate the net nutrient balance. The net balances for N, P, and K were distributed across the hectarage used for row crop, hay, pasture, or combinations of these land uses. Row-crop agriculture predominates in the eastern one-third and animal agriculture predominates in the western two-thirds of Arkansas. Nutrients derived from poultry litter accounted for >92% of the total transportable manure N, P, and K. The three districts in the eastern one-third of Arkansas contained 95% of the row-crop hectarage and had net N and P balances that were near zero or negative. The six districts in the western two-thirds of Arkansas accounted for 89 to 100% of the animal populations, had positive net balances for N and P, and excess P ranged from 1 to 9 kg P ha(-1) when distributed across row-crop, hay, and pasture hectarage. Transport of excess nutrients, primarily in poultry litter, outside of the districts in western Arkansas is needed to achieve a balance between soil inputs and removals of P and N.

Agriculture↗