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David J Miller

Publications and source records attributed to David J Miller.

At least 37 records · Page 2Linked to original sources
PubMed2005

Detecting protein-protein interaction in live yeast by flow cytometry.

BACKGROUND: The yeast Saccharomyces cerevisiae is the most commonly used organism for studying protein- protein interactions. In this report we demonstrate the use of flow cytometry in observing fluorescence resonance energy transfer (FRET) between cyan and yellow fluorescent fusion proteins (CFP and YFP, respectively) as a marker for protein interaction in live yeast cells. Probability binning is also employed to provide a statistical confirmation of our observations. METHODS: We coexpressed CFP and YFP fusions containing the N-terminal transmembrane domain (NTM) of Tom70p in yeast and analyzed FRET in live cells with a multilaser flow cytometer. The Tom70p NTM was previously shown to be sufficient for mitochondrial localization and protein-protein interaction (Millar and Shore, 1994, J Biol Chem 269:12229-12232). RESULTS: FRET was observed only in cells that expressed CFP and YFP fusions that each contained the wild-type NTM. The introduction of mutations previously shown to disrupt NTM interaction eliminated FRET. Probability binning confirmed that differences between the FRET channels of experimental and control samples were statistically and physiologically significant. CONCLUSION: Flow cytometric analysis of FRET in yeast is a powerful technique for studying protein-protein interactions. The use of flow cytometry allows FRET data to be gathered from a large number of individual cells, thus providing important advantages unavailable to other techniques. Its application to yeast presents a new method to a popular system widely used in proteomic studies.

Bacterial Proteins↗
PubMed2005

Reconstitution of the peridinin-chlorophyll a protein (PCP): evidence for functional flexibility in chlorophyll binding.

The coding regions for the N-domain, and full length peridinin-chlorophyll a apoprotein (full length PCP), were expressed in Escherichia coli. The apoproteins formed inclusion bodies from which the peptides could be released by hot buffer. Both the above constructs were reconstituted by addition of a total pigment extract from native PCP. After purification by ion exchange chromatography, the absorbance, fluorescence excitation and CD spectra resembled those of the native PCP. Energy transfer from peridinin to Chl a was restored and a specific fluorescence activity calculated which was approximately 86% of that of native PCP. Size exclusion analysis and CD spectra showed that the N-domain PCP dimerized on reconstitution. Chl a could be replaced by Chl b, 3-acetyl Chl a, Chl d and Bchl using the N-domain apo protein. The specific fluorescence activity was the same for constructs with Chl a, 3-acetyl Chl a, and Chl d but significantly reduced for those made with Chl b. Reconstitutions with mixtures of chlorophylls were also made with eg Chl b and Chl d and energy transfer from the higher energy Qy band to the lower was demonstrated.

Carotenoids↗
PubMed2005

Cnidarians and ancestral genetic complexity in the animal kingdom.

Eleven of the twelve recognized wingless (Wnt) subfamilies are represented in the sea anemone Nematostella vectensis, indicating that this developmentally important gene family was already fully diversified in the common ancestor of 'higher' animals. In deuterostomes, although duplications have occurred, no novel subfamilies of Wnts have evolved. By contrast, the protostomes Drosophila and Caenorhabditis have lost half of the ancestral Wnts. This pattern -- loss of genes from an ancestrally complex state -- might be more important in animal evolution than previously recognized.

Animals↗
PubMed2005

The cellular chaperone heat shock protein 90 facilitates Flock House virus RNA replication in Drosophila cells.

The assembly of viral RNA replication complexes on intracellular membranes represents a critical step in the life cycle of positive-strand RNA viruses. We investigated the role of the cellular chaperone heat shock protein 90 (Hsp90) in viral RNA replication complex assembly and function using Flock House virus (FHV), an alphanodavirus whose RNA-dependent RNA polymerase, protein A, is essential for viral RNA replication complex assembly on mitochondrial outer membranes. The Hsp90 chaperone complex transports cellular mitochondrial proteins to the outer mitochondrial membrane import receptors, and thus we hypothesized that Hsp90 may also facilitate FHV RNA replication complex assembly or function. Treatment of FHV-infected Drosophila S2 cells with the Hsp90-specific inhibitor geldanamycin or radicicol potently suppressed the production of infectious virions and the accumulation of protein A and genomic, subgenomic, and template viral RNA. In contrast, geldanamycin did not inhibit the activity of preformed FHV RNA replication complexes. Hsp90 inhibitors also suppressed viral RNA and protein A accumulation in S2 cells expressing an FHV RNA replicon. Furthermore, Hsp90 inhibition with either geldanamycin or RNAi-mediated chaperone downregulation suppressed protein A accumulation in the absence of viral RNA replication. These results identify Hsp90 as a host factor involved in FHV RNA replication and suggest that FHV uses established cellular chaperone pathways to assemble its RNA replication complexes on intracellular membranes.

Animals↗
PubMed2005

In vivo self-interaction of nodavirus RNA replicase protein a revealed by fluorescence resonance energy transfer.

Flock house virus (FHV) is the best-characterized member of the Nodaviridae, a family of small, positive-strand RNA viruses. Unlike most RNA viruses, FHV encodes only a single polypeptide, protein A, that is required for RNA replication. Protein A contains a C-proximal RNA-dependent RNA polymerase domain and localizes via an N-terminal transmembrane domain to the outer mitochondrial membrane, where FHV RNA replication takes place in association with invaginations referred to as spherules. We demonstrate here that protein A self-interacts in vivo by using flow cytometric analysis of fluorescence resonance energy transfer (FRET), spectrofluorometric analysis of bioluminescence resonance energy transfer, and coimmunoprecipitation. Several nonoverlapping protein A sequences were able to independently direct protein-protein interaction, including an N-terminal region previously shown to be sufficient for localization to the outer mitochondrial membrane (D. J. Miller and P. Ahlquist, J. Virol. 76:9856-9867, 2000). Mutations in protein A that diminished FRET also diminished FHV RNA replication, a finding consistent with an important role for protein A self-interaction in FHV RNA synthesis. Thus, the results imply that FHV protein A functions as a multimer rather than as a monomer at one or more steps in RNA replication.

Animals↗
PubMed2005

Mixture modeling with pairwise, instance-level class constraints.

The goal of semisupervised clustering/mixture modeling is to learn the underlying groups comprising a given data set when there is also some form of instance-level supervision available, usually in the form of labels or pairwise sample constraints. Most prior work with constraints assumes the number of classes is known, with each learned cluster assumed to be a class and, hence, subject to the given class constraints. When the number of classes is unknown or when the one-cluster-per-class assumption is not valid, the use of constraints may actually be deleterious to learning the ground-truth data groups. We address this by (1) allowing allocation of multiple mixture components to individual classes and (2) estimating both the number of components and the number of classes. We also address new class discovery, with components void of constraints treated as putative unknown classes. For both real-world and synthetic data, our method is shown to accurately estimate the number of classes and to give favorable comparison with the recent approach of Shental, Bar-Hillel, Hertz, and Weinshall (2003).

Algorithms↗
PubMed2004

Male mice that do not express group VIA phospholipase A2 produce spermatozoa with impaired motility and have greatly reduced fertility.

The Group VIA Phospholipase A(2) (iPLA(2)beta) is the first recognized cytosolic Ca(2+)-independent PLA(2) and has been proposed to participate in arachidonic acid (20:4) incorporation into glycerophosphocholine lipids, cell proliferation, exocytosis, apoptosis, and other processes. To study iPLA(2)beta functions, we disrupted its gene by homologous recombination to generate mice that do not express iPLA(2)beta. Heterozygous iPLA(2)beta(+/-) breeding pairs yield a Mendelian 1:2:1 ratio of iPLA(2)beta(+/+), iPLA(2)beta(+/-), and iPLA(2)beta(-/-) pups and a 1:1 male:female gender distribution of iPLA(2)beta(-/-) pups. Several tissues of wild-type mice express iPLA(2)beta mRNA, immunoreactive protein, and activity, and testes express the highest levels. Testes or other tissues of iPLA(2)beta(-/-) mice express no iPLA(2)beta mRNA or protein, but iPLA(2)beta(-/-) testes are not deficient in 20:4-containing glycerophosphocholine lipids, indicating that iPLA(2)beta does not play an obligatory role in formation of such lipids in that tissue. Spermatozoa from iPLA(2)beta(-/-) mice have reduced motility and impaired ability to fertilize mouse oocytes in vitro and in vivo, and inhibiting iPLA(2)beta with a bromoenol lactone suicide substrate reduces motility of wild-type spermatozoa in a time- and concentration-dependent manner. Mating iPLA(2)beta(-/-) male mice with iPLA(2)beta(+/+), iPLA(2)beta(+/-), or iPLA(2)beta(-/-) female mice yields only about 7% of the number of pups produced by mating pairs with an iPLA(2)beta(+/+) or iPLA(2)beta(+/-) male, but iPLA(2)beta(-/-) female mice have nearly normal fertility. These findings indicate that iPLA(2)beta plays an important functional role in spermatozoa, suggest a target for developing male contraceptive drugs, and complement reports that disruption of the Group IVA PLA(2) (cPLA(2)alpha) gene impairs female reproductive ability.

Alleles↗
PubMed2004

A proteomic approach to identify phosphoproteins encoded by cDNA libraries.

We report a method for large-scale rapid analysis of phosphoproteins in tissues or cells by combining immobilized metal affinity chromatography (IMAC) with phage display cDNA library screening. We expressed a testis cDNA library as fusion proteins on phage and, using IMAC, enriched for sequences encoding phosphoproteins. Selected clones were polymerase chain reaction amplified and sequenced. The majority of the clones sequenced (80%) encoded known proteins previously identified as phosphoproteins. Immunoblotting with phosphotyrosine antibodies confirmed that some of the selected sequences encoded tyrosine phosphorylated proteins when expressed on phage. An advantage of this method is the rapid identification of phosphoproteins encoded by a cDNA library, which can identify proteins that are potentially phosphorylated in vivo. When this method is combined with limited enzymatic digestion and tandem mass spectrometric techniques, the specific phosphorylation site in a protein can be identified. This technique can be used in proteomics studies to effectively detect phosphorylated proteins and avoid time-consuming and expensive peptide sequencing.

Chromatography, Affinity↗
PubMed2004

snail expression during embryonic development of the coral Acropora: blurring the diploblast/triploblast divide?

Although corals are nominally diploblastic, the early development of Acropora millepora involves a process that clearly resembles gastrulation in higher metazoans. This similarity at the morphological level led us to search for the Acropora equivalents of genes whose key roles in gastrulation are conserved across the higher Metazoa. We here report the characterisation of one such gene, snail, which in both Drosophila and the mouse is expressed in cells undergoing an epithelial-mesenchyme transition and/or morphogenetic movements. In addition to an N-terminal SNAG domain, the Acropora snail protein contains four zinc fingers with sequences diagnostic for members of the snail protein subfamily. In situ hybridisation reveals expression in epithelial tissue in the central portion of one side of the flattened pre-gastrulation embryo, which continues to express snail as it is engulfed by its opposite layer. Comparison to snail expression during gastrulation in bilaterians such as Drosophila reveals striking similarities and suggests mechanistic, and possibly evolutionary, links between the processes of mesoderm formation in bilaterians and endoderm formation in the Cnidaria.

Amino Acid Sequence↗
PubMed2004

Removal of the phosphate group in mechanism-based inhibitors of inositol monophosphatase leads to unusual inhibitory activity.

Inositol monophosphatase is widely held to be the therapeutic target for inhibition by lithium ion in the treatment of bipolar disorder. In a continued effort to improve the bioavailability of alternative inhibitors, we have designed and tested two new series of compounds; phosphonates and product-like mimics. Phosphonate substrate mimics were competitive inhibitors of reduced potency as compared to phosphate based inhibitors. Product mimics however, showed various inhibitory modes of action. The 6-butylamino derivative 6p was an uncompetitive inhibitor when acting alone (K(i)= 0.3 mM) but displayed non-competitive inhibition in the presence of inorganic phosphate. This compound represents a new lead in the search for a viable replacement for lithium ion therapy.

Animals↗
PubMed2004

Altered oscillatory work by ventricular myofilaments from a rabbit coronary artery ligation model of heart failure.

OBJECTIVES: Understanding the changed ability of cardiac myofilaments to produce pump work requires knowledge of kinetics of crossbridge function as well as more widely studied parameters such as Ca-sensitivity and isometric force development. We tested the hypothesis that altered crossbridge kinetics contribute to reduced myofilament work in early-stage heart failure (left ventricular dysfunction, LVD). METHODS: The sinusoidal oscillation technique can yield insights into crossbridge function. Dynamic stiffness, oscillatory work and power were assessed in chemically skinned, Ca-activated trabeculae from rabbit ventricles in early-stage failure, 8 weeks after infarction induced by coronary artery ligation (LIG). Results were compared with sham-operated controls (SH). LVD was assessed by echocardiography. RESULTS: Ca-activated force and myofilament Ca-sensitivity were not significantly altered at this early stage of LVD. In maximally Ca-activated preparations, the frequency of minimal dynamic stiffness (f(min)) was 23% lower in LIG. f(min) increases by >80% between pCa 5.8 and 4 in SH but not in LIG. Maximal phase lead and lag angles (between length and tension) were lower in LIG at frequencies near f(min), lowering oscillatory work and power. The Lissajous figures (oscillatory work loops) of imposed length vs. tension are often asymmetric near f(min). The degree of asymmetry was greater in LIG. CONCLUSIONS: Reduced capacity for mechanical power, consistent with depressed haemodynamic performance in LVD hearts, is only partially attributable to crossbridge slowing; changes in the phase relationship will also contribute. These changes are not readily attributable to known alterations in contractile protein isoforms. Some deductions are drawn about which steps in the crossbridge cycle are modified in this model of LVD. Altered cardiac myocyte Ca-transients, reported to be associated with LVD, will be translated into pump work by a contractile machinery that is functionally altered, even though isometric force and myofilament Ca-sensitivity might remain near-normal at this stage.

Actin Cytoskeleton↗
PubMed2004

CASK is in the mammalian sperm head and is processed during epididymal maturation.

Upon adhesion to the zona pellucida or egg extracellular matrix, sperm undergo regulated exocytosis of the acrosomal vesicle. CASK is an adaptor protein that has been implicated in coupling neuronal cell adhesion to regulated exocytosis. In neurons, this scaffolding molecule is associated with several types of transmembrane receptor complexes and connects cell adhesion molecules with ion channels, the actin cytoskeleton, and the cell's exocytotic machinery. We hypothesized CASK might also be an important link between zona pellucida binding and the sperm acrosome reaction. RT-PCR experiments indicated CASK is transcribed in mouse testis. The full size (120 kDa) CASK protein was present in testis from mouse and pig. Immunoblots of mature porcine and murine sperm revealed that the 120 kDa molecule was much less abundant than in testis but the antibody also recognized a group of smaller proteins migrating at 55-65 kDa. Immunofluorescence experiments indicated both the full length and smaller CASK immunoreactive products were found only in the acrosomal region of spermatids and mature sperm and not in other testicular cell types. CASK immunofluorescence was lost following the acrosome reaction. During epididymal maturation, the abundance of the full size CASK decreased and the CASK fragments increased. These results suggest that CASK may be proteolytically processed during epididymal maturation. Because sperm acquire the ability to bind the zona pellucida, acrosome react, and fertilize eggs during epididymal maturation, CASK processing may play a role in the acquisition of these functions.

Acrosome↗
PubMed2003

EST analysis of the cnidarian Acropora millepora reveals extensive gene loss and rapid sequence divergence in the model invertebrates.

A significant proportion of mammalian genes are not represented in the genomes of Drosophila, Caenorhabditis or Saccharomyces, and many of these are assumed to have been vertebrate innovations. To test this assumption, we conducted a preliminary EST project on the anthozoan cnidarian, Acropora millepora, a basal metazoan. More than 10% of the Acropora ESTs with strong metazoan matches to the databases had clear human homologs but were not represented in the Drosophila or Caenorhabditis genomes; this category includes a surprising diversity of transcription factors and metabolic proteins that were previously assumed to be restricted to vertebrates. Consistent with higher rates of divergence in the model invertebrates, three-way comparisons show that most Acropora ESTs match human sequences much more strongly than they do any Drosophila or Caenorhabditis sequence. Gene loss has thus been much more extensive in the model invertebrate lineages than previously assumed and, as a consequence, some genes formerly thought to be vertebrate inventions must have been present in the common metazoan ancestor. The complexity of the Acropora genome is paradoxical, given that this organism contains apparently few tissue types and the simplest extant nervous system consisting of a morphologically homogeneous nerve net.

Animals↗
PubMed2003

Subcritical (hot/liquid) water dechlorination of PCBs (Aroclor 1254) with metal additives and in waste paint.

No disposal option exists for "mixed wastes" such as paint scrapings that are co-contaminated with polychlorinated biphenyls (PCBs) and radioactive metals. Either removal or destruction of the PCBs is required prior to disposal. Comparison of subcritical water dechlorination (350 degrees C, 1 h) of Aroclor 1254 in paint scrapings (180 ppm) and of standard Aroclor 1254 showed significantly enhanced dechlorination in the presence of paint. While no significant degradation was observed for standard Aroclor (no paint), the dechlorination of PCBs in paint was 99, 99, and 80% for the hepta-, hexa-, and pentachlorinated congeners, respectively, indicating that metals in the paint enhanced the dechlorination reactions. Adding metals to the standard Aroclor (no paint) reactions enhanced PCB dechlorination in subcritical water in descending order of activity: Pb approximately = Cu > Al > Zn > Fe. In the presence of both zerovalent and divalent lead and zerovalent copper in subcritical water (350 degrees C, 1 h), 99% of the Aroclor 1254 mixture (tetra- to heptachlorinated biphenyls) was dechlorinated. High dechlorination (ca. 95%) was also achieved with zerovalent aluminum. In contrast to other metals, lead retained its degradation ability at a lower temperature of 250 degrees C after 18 h. The high degradation efficiency achieved using metal additives in water at reasonable temperatures and pressures demonstrates the potential for subcritical water dechlorination of PCBs in paint scrapings and, potentially, in other solid and liquid wastes.

Chlorodiphenyl (54% Chlorine)↗
PubMed2003

DNA-binding characteristics of cnidarian Pax-C and Pax-B proteins in vivo and in vitro: no simple relationship with the Pax-6 and Pax-2/5/8 classes.

Cnidarians are the simplest animals in which distinct eyes are present. We have previously suggested that cnidarian Pax-Cam might represent a precursor of the Pax-6 class. Here we show that when expressed in Drosophila imaginal discs, Pax-Cam chimeric proteins containing the C-terminal region of EY were capable of eye induction and driving expression of a reporter gene under the control of a known EY target (the sine oculis gene). Whilst these results are consistent with a Pax-6-like function for Pax-Cam, in band shift experiments we were unable to distinguish the DNA-binding behaviour of the Pax-Cam Paired domain from that of a second Acropora Pax protein, Pax-Bam. The ability of a Pax-Bam/EY chimera to also induce eye formation in leg imaginal discs, together with the in vitro data, cast doubt on previously assumed direct relationships between cnidarian Pax genes and the Pax-6 and Pax-2/5/8 classes of bilateral animals.

Animals↗