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David J Miller

Publications and source records attributed to David J Miller.

53 records · Page 3Linked to original sources
PubMed2003

In vitro and in vivo comparisons of constant resistance AC iontophoresis and DC iontophoresis.

A previous in vitro constant electrical resistance alternating current (AC) iontophoresis study with human epidermal membrane (HEM) and a model neutral permeant has shown less inter- and intra-sample variability in iontophoretic transport relative to conventional constant direct current (DC) iontophoresis. The objectives of the present study were to address the following questions. (1) Can the skin electrical resistance be maintained at a constant level by AC in humans in vivo? (2) Are the in vitro data with HEM representative of those in vivo? (3) Does constant skin resistance AC iontophoresis have less inter- and intra-sample variability than conventional constant current DC iontophoresis in vivo? (4) What are the electrical and the barrier properties of skin during iontophoresis in vivo? In the present study, in vitro HEM experiments were carried out with the constant resistance AC and the conventional constant current DC methods using mannitol and glucose as the neutral model permeants. In vivo human experiments were performed using glucose as the permeant with a constant skin resistance AC only protocol and two conventional constant current DC methods (continuous constant current DC and constant current DC with its polarity alternated every 10 min with a 3:7 on:off duty cycle). Constant current DC iontophoresis was conducted with commercial constant current DC devices, and constant resistance AC iontophoresis was carried out by reducing and maintaining the skin resistance at a constant target value with AC supplied from a function generator. This study shows that (1) skin electrical resistance can be maintained at a constant level during AC iontophoresis in vivo; (2) HEM in vitro and human skin in vivo demonstrate similar electrical and barrier properties, and these properties are consistent with our previous findings; (3) there is general qualitative and semi-quantitative agreement between the HEM data in vitro and human skin data in vivo; and (4) constant skin resistance AC iontophoresis generally provides less inter- and intra-subject variability than conventional constant current DC.

Diffusion Chambers, Culture↗
PubMed2003

Evidence for very tight sequestration of BTEX compounds in manufactured gas plant soils based on selective supercritical fluid extraction and soil/water partitioning.

Benzene, toluene, ethylbenzene, o-, m-, and p-xylenes (BTEX), and polycyclic aromatic hydrocarbons (PAHs) were extracted from eight manufactured gas plant (MGP) soils from sites that had been abandoned for several decades. Supercritical fluid extraction (SFE) with pure carbon dioxide demonstrated the presence of BTEX compounds that were highly sequestered in both coal gas and oil gas MGP soils and soots. Benzene was generally the slowest compound to extract from all samples and was even more difficult to extract than most two- to five-ring PAHs found on the same samples. Since the solubility of benzene in carbon dioxide is 2-5 orders of magnitude higher than the solubilities of PAHs, these results demonstrate that benzene was more tightly sequestered than toluene, ethylbenzene, xylenes, or the multi-ring PAHs. Additional evidence for very tight binding was based on the fact that BTEX concentrations determined using either SFE or with methylene chloride sonication were much higher than those obtained by the U.S. EPA purge-and-trap method, especially for benzene (whose concentration was underestimated by as much as 1000-fold by the EPA method). However, soil/water desorption showed little benzene mobility, and Kd values for benzene were 1-2 orders of magnitude higher than those calculated based on literature sorption K(OC) values. These results indicate that environmentally relevant concentrations of benzene may be better represented by mild extraction methods than by methods capable of extracting tightly bound benzene.

Benzene↗
PubMed2003

Pseudogenes contribute to the extreme diversity of nuclear ribosomal DNA in the hard coral Acropora.

One characteristic of Indo-Pacific Acropora spp. is extremely high diversity in rDNA sequences at both the species and individual levels. In order to test the hypothesis that pseudogenes may contribute to this diversity, three kinds of analyses were conducted. First, for A. millepora (the species containing the most diverse suite of rDNA types), RT-PCR was used to determine which 5.8S rDNA types are expressed. Second, as previous studies have indicated that interspecific hybridization has occurred in the genus Acropora and silencing of rDNA loci via nucleolar dominance has been shown in some cases to involve methylation, patterns of variation were examined at methylation-susceptible sites. Third, patterns of substitution at conserved sites (including those that are likely to contribute to secondary structure in rRNA) in the 5.8S rDNA were examined. These analyses consistently indicated that one rDNA sequence type present in a broad range of Indo-Pacific Acropora species is likely to consist predominantly of pseudogenes. Patterns of variation also suggest that species may differ with respect to which rDNA sequence types have been silenced and which are active. These pseudogenes are likely to have arisen as a consequence of the introduction of highly divergent rDNA types into single genomes by interspecific hybridization events, and we attribute the extreme rDNA diversity characteristic of many Acropora species to both the independent evolution of these silenced rDNA types and to the suppressive effects of high sequence diversity on homogenization processes acting on functional loci.

Animals↗
PubMed2003

Investigation of properties of human epidermal membrane under constant conductance alternating current iontophoresis.

Previous studies in our laboratory have shown that enhanced, constant permeant fluxes across human skin can be achieved by applying an alternating current (AC) to maintain skin electrical conductance at a constant level. Relative to conventional direct current (DC) iontophoresis, for which current is maintained at a constant level, this newly developed constant conductance alternating current (CCAC) method achieves constant fluxes with less inter- and intra-sample variability. The present study focused upon further investigating the permeability properties of human skin during CCAC iontophoresis at a variety of target resistance/conductance values. A three-stage experimental protocol was used with flux measurements determined on 3 consecutive days. Stage I was an AC only protocol (symmetrical AC square-wave signal), stage II was an AC plus DC protocol (AC square-wave with DC offset voltage), and stage III was a repeat of stage I. During this three-stage protocol, the skin electrical resistance was maintained at a constant target value by manually adjusting the applied AC voltage. Radiolabeled mannitol and urea were model permeants in all experiments. Their fluxes were determined and used to characterize the permeability properties of human skin. The results from the present study established that: (i) the CCAC protocol made it possible to reduce HEM electrical resistance to different target levels as low as 0.8 kOmega cm(2) and maintain the specific resistance level throughout the flux experiment, (ii) permeant fluxes are proportional to skin electrical conductance, (iii) under the studied CCAC passive conditions, membrane pore size tends to increase as skin resistance decreases, and (iv) as the membrane breaks down, its pore sizes become larger.

Administration, Cutaneous↗
PubMed2003

A 'fair go' for coral hybridization.

Hybridisation between coral species clearly occurs in vitro, but the evolutionary significance of this cross-fertility is still the subject of much debate. Compelling genetic and reproductive evidence support introgressive hybridization amongst Indo-Pacific members of the scleractinian genus Acropora. Although population genetic analyses indicate that interspecific hybridization events are relatively rare, they are likely be important on evolutionary time scales, creating the capacity for adaptive evolution by increasing genomic diversity and heterozygosity. However, in a recent paper based exclusively on the three endemic Caribbean Acropora species, Vollmer and Palumbi (2002) dispute the occurrence of reticulation in corals. Here we use data from both the Vollmer and Palumbi study and our earlier paper on the same species (van Oppen et al., 2000) to show that reticulation has occurred amongst the Caribbean Acropora species. Furthermore, conclusions based on the limited Caribbean Acropora fauna cannot simply be extrapolated to Indo-Pacific corals, and it is inappropriate to view some coral species as 'immortal mules'.

Animals↗
PubMed2003

Engineered retargeting of viral RNA replication complexes to an alternative intracellular membrane.

Positive-strand RNA virus replication complexes are universally associated with intracellular membranes, although different viruses use membranes derived from diverse and sometimes multiple organelles. We investigated whether unique intracellular membranes are required for viral RNA replication complex formation and function in yeast by retargeting protein A, the Flock House virus (FHV) RNA-dependent RNA polymerase. Protein A, the only viral protein required for FHV RNA replication, targets and anchors replication complexes to outer mitochondrial membranes in part via an N-proximal sequence that contains a transmembrane domain. We replaced the FHV protein A mitochondrial outer membrane-targeting sequence with the N-terminal endoplasmic reticulum (ER)-targeting sequence from the yeast NADP cytochrome P450 oxidoreductase or inverted C-terminal ER-targeting sequences from the hepatitis C virus NS5B polymerase or the yeast t-SNARE Ufe1p. Confocal immunofluorescence microscopy confirmed that protein A chimeras retargeted to the ER. FHV subgenomic and genomic RNA accumulation in yeast expressing ER-targeted protein A increased 2- to 13-fold over that in yeast expressing wild-type protein A, despite similar protein A levels. Density gradient flotation assays demonstrated that ER-targeted protein A remained membrane associated, and in vitro RNA-dependent RNA polymerase assays demonstrated an eightfold increase in the in vitro RNA synthesis activity of the ER-targeted FHV RNA replication complexes. Electron microscopy showed a change in the intracellular membrane alterations from a clustered mitochondrial distribution with wild-type protein A to the formation of perinuclear layers with ER-targeted protein A. We conclude that specific intracellular membranes are not required for FHV RNA replication complex formation and function.

Amino Acid Sequence↗
PubMed2002

Improvement on conventional constant current DC iontophoresis: a study using constant conductance AC iontophoresis.

The purpose of the present study was to compare conventional constant direct current (DC) transdermal iontophoresis with a new constant conductance alternating current (AC) iontophoresis method. The new method was developed with the intent of reducing flux drift during iontophoresis and minimizing skin-to-skin variability. The constant conductance AC iontophoresis studies involved three electrical components: (1) an initial applied potential used to decrease the human epidermal membrane (HEM) electrical resistance to a target level of either 1.5 or 3.0 k Omega cm(2), (2) an applied 50 Hz square-wave AC with a variable potential adjusted to maintain the HEM conductance at the target level during the transport study, and (3) a low voltage DC offset of 0 (passive), 0.25, or 0.40 V applied simultaneously with the AC to assist permeant transport. Current densities of 0.13 and 0.26 mA/cm(2) were chosen for the conventional constant current DC iontophoresis studies. Mannitol was used as the probe permeant for all studies. The constant current DC studies showed significant increases in mannitol flux with time during a given experiment and large skin-to-skin variability. Compared to the constant current DC experiments, the mannitol flux remained more constant during the constant conductance AC iontophoresis and skin-to-skin variability was significantly reduced. On a mechanistic level, changes in the transport properties during constant current DC iontophoresis indicate changes in the membrane parameters such as porosity, effective pore size, and/or pore surface charge density during the conventional method of iontophoresis. The results from the constant conductance AC iontophoresis transport studies imply that this method effectively maintains the membrane parameters that affect transport at a constant state this providing for a relatively constant permanent flux.

Biological Transport↗
PubMed2002

Localized expression of a dpp/BMP2/4 ortholog in a coral embryo.

As the closest outgroup to the Bilateria, the Phylum Cnidaria is likely to be critical to understanding the origins and evolution of body axes. Proteins of the decapentaplegic (DPP)/bone morphogenetic protein (BMP) 2/4 subfamily are central to the specification of the dorsoventral (D/V) axis in bilateral animals, albeit with an axis inversion between arthropods and chordates. We show that a dpp/BMP2/4 ortholog (bmp2/4-Am) is present in the reef-building scleractinian coral, Acropora millepora (Class Anthozoa) and that it is capable of causing phenotypic effects in Drosophila that mimic those of the endogenous dpp gene. We also show that, during coral embryonic development, bmp2/4-Am expression is localized in an ectodermal region adjacent to the blastopore. Thus, a representative of the DPP/BMP2/4 subfamily of ligands was present in the common ancestor of diploblastic and triploblastic animals where it was probably expressed in a localized fashion during development. A localized source of DPP/BMP2/4 may have already been used in axis formation in this ancestor, or it may have provided a means by which an axis could evolve in triploblastic animals.

Amino Acid Sequence↗
PubMed2002

The mitochondrial genome of Acropora tenuis (Cnidaria; Scleractinia) contains a large group I intron and a candidate control region.

The complete nucleotide sequence of the mitochondrial genome of the coral Acropora tenuis has been determined. The 18,338 bp A. tenuis mitochondrial genome contains the standard metazoan complement of 13 protein-coding and two rRNA genes, but only the same two tRNA genes (trnM and trnW) as are present in the mtDNA of the sea anemone, Metridium senile. The A. tenuis nad5 gene is interrupted by a large group I intron which contains ten protein-coding genes and rns; M. senile has an intron at the same position but this contains only two protein-coding genes. Despite the large distance (about 11.5 kb) between the 5?-exon and 3?-exon boundaries, the A. tenuis nad5 gene is functional, as we were able to RT-PCR across the predicted intron splice site using total RNA from A. tenuis. As in M. senile, all of the genes in the A. tenuis mt genome have the same orientation, but their organization is completely different in these two zoantharians: The only common gene boundaries are those at each end of the group I intron and between trnM and rnl. Finally, we provide evidence that the rns-cox3 intergenic region in A. tenuis may correspond to the mitochondrial control region of higher animals. This region contains repetitive elements, and has the potential to form secondary structures of the type characteristic of vertebrate D-loops. Comparisons between a wide range of Acropora species showed that a long hairpin predicted in rns-cox3 is phylogenetically conserved, and allowed the tentative identification of conserved sequence blocks.

Animals↗
PubMed2002

Spawning times, reproductive compatibilities and genetic structuring in the Acropora aspera group: evidence for natural hybridization and semi-permeable species boundaries in corals.

Species boundaries among five sympatric coral species of the Indo-Pacific Acropora aspera group were examined by a combination of in vitro breeding trials, comparisons of spawning times and DNA sequence analysis of ribosomal DNA internal transcribed spacer (rDNA ITS) and 5.8S regions. The breeding trials showed that reproductive compatibility exists between at least some colonies of all the species pairs tested, suggesting a large potential for natural hybridization and introgression. The Acropora ITS regions exhibited extremely high levels of variability (up to approximately 62% for ITS1, approximately 11% for 5.8S and approximately 43% for ITS2), but most of the variation was shared among four of the five species, A. millepora, A. papillare, A. pulchra and A. spathulata, consistent with extensive introgression. Phylogenetic analyses did not resolve these four species as distinct clusters across a wide biogeographic region stretching from the southern Great Barrier Reef to Papua New Guinea. However, most colonies of the fifth species, A. aspera, constituted a distinct clade in phylogenetic analyses. This is consistent with our observations of a semi-permeable temporal barrier involving differences in spawning times between this and the other four species. Although the majority of colonies of all five species generally spawned within 90 min of each other, in two out of four years, gametes were absent prior to mass spawning episodes from at least some A. aspera colonies. Hence, our data suggest that transient reproductive barriers may be the result of year-to-year variation in the date of spawning and that this difference in spawning time contributes to the genetic structure detected among Acropora species in this group. Occasional leakage through the reproductive barrier was confirmed by the observation of A. aspera xA. pulchra F1 hybrids, identified based on additivity of ITS sequences.

Animals↗
PubMed2002

Flock house virus RNA polymerase is a transmembrane protein with amino-terminal sequences sufficient for mitochondrial localization and membrane insertion.

Localization of RNA replication to intracellular membranes is a universal feature of positive-strand RNA viruses. Replication complexes of flock house virus (FHV), the best-studied alphanodavirus, are located on outer mitochondrial membranes in infected Drosophila melanogaster cells and are associated with the formation of membrane-bound spherules, similar to structures found for many other positive-strand RNA viruses. To further study FHV replication complex formation, we investigated the subcellular localization, membrane association, and membrane topology of protein A, the FHV RNA-dependent RNA polymerase, in the yeast Saccharomyces cerevisiae, a host able to support full FHV RNA replication and virion formation. Confocal immunofluorescence revealed that protein A localized to mitochondria in yeast, as in Drosophila cells, and that this mitochondrial localization was independent of viral RNA synthesis. Nycodenz gradient flotation and dissociation assays showed that protein A behaved as an integral membrane protein, a finding consistent with a predicted N-proximal transmembrane domain. Protease digestion and selective permeabilization after differential epitope tagging demonstrated that protein A was inserted into the outer mitochondrial membrane with the N terminus in the inner membrane space or matrix and that the C terminus was exposed to the cytoplasm. Flotation and immunofluorescence studies with deletion mutants indicated that the N-proximal region of protein A was important for both membrane association and mitochondrial localization. Gain-of-function studies with green fluorescent protein fusions demonstrated that the N-terminal 46 amino acids of protein A were sufficient for mitochondrial localization and membrane insertion. We conclude that protein A targets and anchors FHV RNA replication complexes to outer mitochondrial membranes, in part through an N-proximal mitochondrial localization signal and transmembrane domain.

Amino Acid Sequence↗
PubMed2002

Molecular basis of mammalian gamete binding.

Despite the importance of fertilization for controlling human reproduction, regulating animal production, and promoting preservation of endangered species, the molecular basis underlying gamete binding and fertilization has been perplexing. More progress has been made in the mouse than in other mammals and, recently, targeted deletion of specific genes in the mouse has yielded intriguing results. This review will emphasize research performed by our laboratory and others done primarily with mouse gametes but will include some interesting observations from other mammals. Studies of murine fertilization indicate that oligosaccharides on the egg coat glycoprotein ZP3 bind sperm. The precise oligosaccharides that bind sperm are the subject of considerable debate. ZP3 also induces exocytosis of the sperm acrosome, allowing sperm to penetrate through the egg coat (zona pellucida). A number of candidate ZP3 receptors have been proposed and studies of beta1,4galactosyltransferase-I (GalT-I) are reviewed here in the most detail. Sperm from mice with a targeted deletion of GalT-I still are able to bind the zona pellucida but are unable to acrosome react and penetrate through the zona. Therefore, the unique role of GalT-I appears to be in signal transduction. GalT-I forms a complex with heterotrimeric G proteins and activates signaling, leading to exocytosis in sperm and in heterologous cells expressing GalT-I. Other signaling steps triggered by GalT-I are under active investigation; this receptor forms a complex with a protein kinase anchoring protein. After exocytosis of the acrosome, sperm penetrate the zona pellucida and fuse with the oocyte plasma membrane using ADAM family members on sperm and integrins on oocytes. These proteins, along with the tetraspanins on oocytes, may form a complex web at gamete fusion. Targeted deletion of specific genes in this putative complex has provided important information about their redundancy. After the oocyte is fertilized, the binding site for GalT-I is lost from ZP3, preventing additional sperm from binding to the zona pellucida. New technical advances and creative ideas offer the opportunity to make important advances and to solve the conundrum of fertilization.

Acrosome Reaction↗
PubMed2002

Coral development: from classical embryology to molecular control.

The phylum Cnidaria is the closest outgroup to the triploblastic metazoans and as such offers unique insights into evolutionary questions at several levels. In the post-genomic era, a knowledge of the gene complement of representative cnidarians will be important for understanding the relationship between the expansion of gene families and the evolution of morphological complexity among more highly evolved metazoans. Studies of cnidarian development and its molecular control will provide information about the origins of the major bilaterian body axes, the origin of the third tissue layer, the mesoderm, and the evolution of nervous system patterning. We are studying the cnidarian Acropora millepora, a reef building scleractinian coral, and a member of the basal cnidarian class, the Anthozoa. We review ourwork on descriptive embryology and studies of selected transcription factor gene families, where our knowledge from Acropora is particularly advanced relative to other cnidarians. We also describe a recent preliminary whole genome initiative, a coral EST database.

Animals↗
PubMedDate not supplied

Onchocerca volvulus: expression and immunolocalization of a nematode cathepsin D-like lysosomal aspartic protease.

The N-terminal region of the cathepsin D-like aspartic protease from the human filarial parasite Onchocerca volvulus was expressed as His-tag fusion protein. Light and electron microscopic immunohistology using antibodies against the recombinant protein showed labeling of lysosomes in the hypodermis and epithelia of the intestine and the reproductive organs of Onchocerca. While developing oocytes were negative, mature oocytes and early morulae showed strong labeling. In older embryos and mature microfilariae, stained lysosomes were only found in a few cells. Cell death in degenerating microfilariae of patients untreated and treated with microfilaricidal drugs was associated with strong expression of aspartic protease. IgG1, IgG4, and IgE antibodies reactive with the recombinant protein were demonstrated in sera from onchocerciasis patients indicating exposure and recognition of the enzyme by the host's defence system. The aspartic protease of O. volvulus appears to function in intestinal digestion and tissue degradation of the filaria.

Animals↗
PubMedDate not supplied

A DM domain protein from a coral, Acropora millepora, homologous to proteins important for sex determination.

The identification and functional studies of DM domain-containing proteins Doublesex, MAB-3, and DMRT1 indicated that flies, nematodes, and humans share at least some of the molecular mechanisms of sex determination. We identified a gene, AmDM1, from the coral Acropora millepora that encodes a homologous DM domain-containing protein. Molecular analyses show that the AmDM1 primary transcript is processed to generate four different messenger RNAs. Alternative use of two polyadenylation sites produces transcripts that vary only in the 3' untranslated regions, whereas alternative splicing generates transcripts with and without the region coding for the DM domain. All the transcripts include a second motif, the DMA domain, which is found in a number of other proteins containing a DM domain. Hermaphroditic A. millepora differentiates sexual cells seasonally before the spring spawn, and Northern blot analysis shows that the AmDM1 transcripts are present at higher levels during sexual differentiation. The non-DM domain-containing messages are also present at significant levels in late embryos, but DM domain transcripts are extremely rare at this stage. These data suggest that the association of DM domain proteins and sexual determination or differentiation predates the separation of the Cnidaria from the rest of the Metazoa.

Amino Acid Sequence↗
PubMedDate not supplied

Examination of the binding ability of bovine spermatozoa to the zona pellucida as an indicator of fertility.

Despite the development of many new techniques, laboratory assays still do not predict male fertility accurately. To identify targets for laboratory assessment, we first need to determine which steps in fertilization are most often defective in subfertile males. We developed a competitive in vitro fertilization assay in which spermatozoa from 2 different males, stained with different lipophilic dyes, are incubated together with oocytes in a droplet. By exposing mixed spermatozoa to the same oocytes, this assay controls for many of the variables of in vitro fertilization and should allow identification of the most common faulty steps in fertilization. The relationship of zona-binding ability to fertility is controversial. Therefore, as a first step, we determined if zona pellucida-binding ability, measured by this competitive assay, was related to bovine spermatozoal fertility. Fertility data were collected from 2 groups of bulls by 2 means of evaluation, nonreturn to estrus rates postinsemination and competitive insemination. In the nonreturn to estrus study, semen samples from 15 bulls were effectively ranked by zona-binding ability, using pairwise competitive in vitro zona-binding assays (R(2) = 0.84). However, this ranking was not significantly correlated with nonreturn rates (r = -0.04). In the competitive insemination study, semen samples from 8 bulls were effectively ranked by pairwise comparison using the competitive zona-binding assay (R(2) = 0.67). Again, this ranking was not significantly correlated to the competitive insemination index calculated for these bulls (r = 0.29). In the third study, we tested 3 bulls to determine if in vivo zona binding, assessed by the number of accessory spermatozoa, was correlated with in vitro zona binding. The number of accessory spermatozoa on oocytes recovered from cows after mating was not correlated with in vitro competitive binding of the spermatozoa. In conclusion, in vitro competitive zona binding was not correlated with bovine fertility or binding of accessory spermatozoa to oocytes in vivo.

Animals↗