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J R Miller

Publications and source records attributed to J R Miller.

At least 73 records · Page 4Linked to original sources
PubMed1997

Changes in the pattern of adherens junction-associated beta-catenin accompany morphogenesis in the sea urchin embryo.

beta-Catenin was originally identified biochemically as a protein that binds E-cadherin in cultured cells and that interaction was later shown to be essential for cadherin function. Independently, armadillo, the beta-catenin homolog in Drosophila melanogaster, was identified as a segment polarity gene necessary for the transduction of wingless (Wnt) signals during embryonic and larval development. Recently, several investigations have also shown that beta-catenin plays a critical role in axial patterning of early Xenopus, zebrafish, and mouse embryos. In these systems, the localization of beta-catenin to the plasma membrane, cytosol, and nucleus is predictive of its role in cell adhesion and signaling. In order to examine the potential role of beta-catenin in regulating cell adhesion during embryogenesis, we cloned beta-catenin in the sea urchin Lytechinus variegatus and characterized its subcellular distribution in cells undergoing morphogenetic movements. Indicative of a role in the establishment and maintenance of cell adhesion, beta-catenin is associated with lateral cell-cell contacts and accumulates at adherens junctions from cleavage stages onward. At gastrulation, changes in junctional beta-catenin localization accompany several morphogenetic events. The epithelial-mesenchymal conversion that characterizes the ingression of both primary and secondary mesenchyme cells coincides with a rapid and dramatic loss of adherens junction-associated beta-catenin. In addition, epithelial cells in the archenteron display a significant decrease in adherens junction-associated beta-catenin levels as they undergo convergent-extension movements. These data are consistent with a role for beta-catenin in regulating cell adhesion and adherens junction function during gastrulation in the sea urchin embryo.

Amino Acid Sequence↗
PubMed1997

Characterization of the role of cadherin in regulating cell adhesion during sea urchin development.

During development, the modulation of cadherin adhesive function is proposed to control various morphogenetic events including epithelial-mesenchymal conversions and tubulogenesis, although the mechanisms responsible for regulating cadherin activity during these events remain unclear. In order to gain insights into the regulation of cadherin function during morphogenesis, we utilized the sea urchin embryo as a model system to study the regulation of cadherin localization during epithelial-mesenchymal conversion and convergent-extension movements. Polyclonal antibodies raised against the cytoplasmic domain of a cloned sea urchin cadherin recognize three major polypeptides of M(r) 320, 140, and 125 kDa and specifically stain adherens junctions, and to a lesser extent, lateral membrane domains in all epithelial tissues of the embryo. Analysis of embryos during gastrulation demonstrates that changes in cadherin localization are observed in cells undergoing an epithelial-mesenchymal conversion. Ingression of primary mesenchyme cells is accompanied by the rapid loss of junctional cadherin staining and the coincident accumulation of cadherin in intracellular organelles. These data are consistent with the idea that the deadhesion of mesenchymal cells from neighboring epithelial cells involves the regulated endocytosis of cell surface cadherin molecules. Conversely, neither cadherin abundance nor localization is altered in cells of the gut which undergo convergent-extension movements during the formation of the archenteron. This observation indicates that these movements do not require the loss of junctional cadherin molecules. Instead, the necessary balance between adhesion and motility may be achieved by regulating the expression of different subtypes of cadherin molecules or modifying interactions between cadherins and catenins, proteins that bind the cytoplasmic domain of cadherin and are necessary for cadherin adhesive function. To address cadherin function at the molecular level, we used a partial cDNA representing the conserved cytoplasmic domain to identify a novel cadherin molecule in the sea urchin Lytechinus variegatus. The deduced amino acid sequence of LvG-cadherin (for Goliath-cadherin) predicts that it is a transmembrane protein with an apparent relative molecular mass of 303 kDa. The cytoplasmic domain shows significant sequence identity to that of vertebrate classic cadherins. However, the extracellular domain is distinguished from its vertebrate counterparts by both an increased number of cadherin-specific repeats and the presence of four EGF-like repeats proximal to the transmembrane domain. Taken together, these data are consistent with the hypothesis that the sea urchin possesses several cadherins, including a novel member of the cadherin family, and that the dynamic regulation of cadherin localization plays a role in epithelial to mesenchymal conversions during gastrulation.

Amino Acid Sequence↗
PubMed1997

Analysis of the signaling activities of localization mutants of beta-catenin during axis specification in Xenopus.

In Xenopus embryos, beta-catenin has been shown to be both necessary and sufficient for the establishment of dorsal cell fates. This signaling activity is thought to depend on the binding of beta-catenin to members of the Lef/Tcf family of transcription factors and the regulation of gene expression by this complex. To test whether beta-catenin must accumulate in nuclei to establish dorsal cell fate, we constructed various localization mutants that restrict beta-catenin to either the plasma membrane, the cytosol, or the nucleus. When overexpressed in Xenopus embryos, the proteins localize as predicted, but surprisingly all forms induce an ectopic axis, indicative of inducing dorsal cell fates. Given this unexpected result, we focused on the membrane-tethered form of beta-catenin to resolve the apparent discrepancy between its membrane localization and the hypothesized role of nuclear beta-catenin in establishing dorsal cell fate. We demonstrate that overexpression of membrane-tethered beta-catenin elevates the level of free endogenous beta-catenin, which subsequently accumulates in nuclei. Consistent with the hypothesis that it is this pool of non-membrane-associated beta-catenin that signals in the presence of membrane-tethered beta-catenin, overexpression of cadherin, which binds free beta-catenin, blocks the axis-inducing activity of membrane- tethered beta-catenin. The mechanism by which ectopic membrane-tethered beta-catenin increases the level of endogenous beta-catenin likely involves competition for the adenomatous polyposis coli (APC) protein, which in other systems has been shown to play a role in degradation of beta-catenin. Consistent with this hypothesis, membrane-tethered beta-catenin coimmunoprecipitates with APC and relocalizes APC to the membrane in cells. Similar results are observed with ectopic plakoglobin, casting doubt on a normal role for plakoglobin in axis specification and indicating that ectopic proteins that interact with APC can artifactually elevate the level of endogenous beta-catenin, likely by interfering with its degradation. These results highlight the difficulty in interpreting the activity of an ectopic protein when it is assayed in a background containing the endogenous protein. We next investigated whether the ability of beta-catenin to interact with potential protein partners in the cell may normally be regulated by phosphorylation. Compared with nonphosphorylated beta-catenin, beta-catenin phosphorylated by glycogen synthase kinase-3 preferentially associates with microsomal fractions expressing the cytoplasmic region of N-cadherin. These results suggest that protein-protein interactions of beta-catenin can be influenced by its state of phosphorylation, in addition to prior evidence that this phosphorylation modulates the stability of beta-catenin.

Adenomatous Polyposis Coli Protein↗
PubMed1997

Zyme, a novel and potentially amyloidogenic enzyme cDNA isolated from Alzheimer's disease brain.

The deposition of the beta amyloid peptide in neuritic plaques and cerebral blood vessels is a hallmark of Alzheimer's disease (AD) pathology. The major component of the amyloid deposit is a 4.2-kDa polypeptide termed amyloid beta-protein of 39-43 residues, which is derived from processing of a larger amyloid precursor protein (APP). It is hypothesized that a chymotrypsin-like enzyme is involved in the processing of APP. We have discovered a new serine protease from the AD brain by polymerase chain reaction amplification of DNA sequences representing active site homologous regions of chymotrypsin-like enzymes. A cDNA clone was identified as one out of one million that encodes Zyme, a serine protease. Messenger RNA encoding Zyme can be detected in some mammalian species but not in mice, rats, or hamster. Zyme is expressed predominantly in brain, kidney, and salivary gland. Zyme mRNA cannot be detected in fetal brain but is seen in adult brain. The Zyme gene maps to chromosome 19q13.3, a region which shows genetic linkage with late onset familial Alzheimer's disease. When Zyme cDNA is co-expressed with the APP cDNA in 293 (human embryonic kidney) cells, amyloidogenic fragments are detected using C-terminal antibody to APP. These co-transfected cells release an abundance of truncated amyloid beta-protein peptide and shows a reduction of residues 17-42 of Abeta (P3) peptide. Zyme is immunolocalized to perivascular cells in monkey cortex and the AD brain. In addition, Zyme is localized to microglial cells in our AD brain sample. The amyloidogenic potential and localization in brain may indicate a role for this protease in amyloid precursor processing and AD.

Adult↗
PubMed1997

Purification and characterization of secreted human leptin produced in baculovirus-infected insect cells.

The product of the human ob (obesity) gene, leptin, appears to function in the maintenance of body weight in vivo. When injected into mice, this hormone reduces food consumption and causes weight loss. This work has been done with recombinant leptin (re-leptin) purified and renatured from inclusion bodies in Escherichia coli. We have expressed the human obesity gene encoding the predicted full-length leptin in Spodoptera frugiperda (Sf-9) cells by infection with the recombinant baculovirus system. Protein corresponding to re-leptin was secreted into the culture medium and purified in sufficient quantity for testing biological activity. The secreted re-protein was characterized and found to be unmodified except for correct cleavage of the signal peptide during export from the cells. The resulting molecule is expected to be properly folded and has been purified to a high level of homogeneity. The re-leptin secreted from Sf-9 cells should be an appropriate source of protein for study of the native structure.

Amino Acid Sequence↗
PubMed1997

Establishment of the dorso-ventral axis in Xenopus embryos is presaged by early asymmetries in beta-catenin that are modulated by the Wnt signaling pathway.

Eggs of Xenopus laevis undergo a postfertilization cortical rotation that specifies the position of the dorso-ventral axis and activates a transplantable dorsal-determining activity in dorsal blastomeres by the 32-cell stage. There have heretofore been no reported dorso-ventral asymmetries in endogenous signaling proteins that may be involved in this dorsal-determining activity during early cleavage stages. We focused on beta-catenin as a candidate for an asymmetrically localized dorsal-determining factor since it is both necessary and sufficient for dorsal axis formation. We report that beta-catenin displays greater cytoplasmic accumulation on the future dorsal side of the Xenopus embryo by the two-cell stage. This asymmetry persists and increases through early cleavage stages, with beta-catenin accumulating in dorsal but not ventral nuclei by the 16- to 32-cell stages. We then investigated which potential signaling factors and pathways are capable of modulating the steady-state levels of endogenous beta-catenin. Steady-state levels and nuclear accumulation of beta-catenin increased in response to ectopic Xenopus Wnt-8 (Xwnt-8) and to the inhibition of glycogen synthase kinase-3, whereas neither Xwnt-5A, BVg1, nor noggin increased beta-catenin levels before the mid-blastula stage. As greater levels and nuclear accumulation of beta-catenin on the future dorsal side of the embryo correlate with the induction of specific dorsal genes, our data suggest that early asymmetries in beta-catenin presage and may specify dorso-ventral differences in gene expression and cell fate. Our data further support the hypothesis that these dorso-ventral differences in beta-catenin arise in response to the postfertilization activation of a signaling pathway that involves Xenopus glycogen synthase kinase-3.

Animals↗
PubMed1996

A frizzled homolog functions in a vertebrate Wnt signaling pathway.

BACKGROUND: Wnts are secreted proteins implicated in cell-cell interactions during embryogenesis and tumorigenesis, but receptors involved in transducing Wnt signals have not yet been definitively identified. Members of a large family of putative transmembrane receptors homologous to the frizzled protein in Drosophila have been identified recently in both vertebrates and invertebrates, raising the question of whether they are involved in transducing signals for any known signaling factors. RESULTS: To test the potential involvement of frizzled homologs in Wnt signaling, we examined the effects of overexpressing rat frizzled-1 (Rfz-1) on the subcellular distribution of Wnts and of dishevelled, a cytoplasmic component of the Wnt signalling pathway. We demonstrate that ectopic expression of Rfz-1 recruits the dishevelled proten-as well as Xenopus Wnt-8 (Xwnt-8), but not the functionally distinct Xwnt-5A-to the plasma membrane. Moreover, Rfz-1 is sufficient to induce the expression of two Xwnt-8-responsive genes, siamois and Xnr-3, in Xenopus explants in a manner which is antagonized by glycogen synthase kinase-3, which also antagonizes Wnt signaling. When Rfz-1 and Xwnt-8 are expressed together in this assay, we observe greater induction of these genes, indicating that Rfz-1 can synergize with a Wnt. CONCLUSIONS: The results demonstrate that a vertebrate frizzled homolog is involved in Wnt signaling in a manner which discriminates between functionally distinct Wnts, which involves translocation of the dishevelled protein to the plasma membrane, and which works in a synergistic manner with Wnts to induce gene expression. These data support the likely function of frizzled homologs as Wnt receptors, or as components of a receptor complex.

Adaptor Proteins, Signal Transducing↗
PubMed1996

The axis-inducing activity, stability, and subcellular distribution of beta-catenin is regulated in Xenopus embryos by glycogen synthase kinase 3.

The serine/threonine kinase Xgsk-3 and the intracellular protein beta-catenin are necessary for the establishment of the dorsal-ventral axis in Xenopus. Although genetic evidence from Drosophila indicates that Xgsk-3 is upstream of beta-catenin, direct interactions between these proteins have not been demonstrated. We demonstrate that phosphorylation of beta-catenin in vivo requires an in vitro amino-terminal Xgsk-3 phosphorylation site, which is conserved in the Drosophila protein armadillo. beta-catenin mutants lacking this site are more active in inducing an ectopic axis in Xenopus embryos and are more stable than wild-type beta-catenin in the presence of Xgsk-3 activity, supporting the hypothesis that Xgsk-3 is a negative regulator of beta-catenin that acts through the amino-terminal site. Inhibition of endogenous Xgsk-3 function with a dominant-negative mutant leads to an increase in the steady-state levels of ectopic beta-catenin, indicating that Xgsk-3 functions to destabilize beta-catenin and thus decrease the amount of beta-catenin available for signaling. The levels of endogenous beta-catenin in the nucleus increases in the presence of the dominant-negative Xgsk-3 mutant, suggesting that a role of Xgsk-3 is to regulate the steady-state levels of beta-catenin within specific subcellular compartments. These studies provide a basis for understanding the interaction between Xgsk-3 and beta-catenin in the establishment of the dorsal-ventral axis in early Xenopus embryos.

Amino Acid Sequence↗
PubMed1996

Pig genome analysis: differential distribution of SINE and LINE sequences is less pronounced than in the human and mouse genomes.

The distribution of SINE and LINE sequences in the pig genome was examined by fluorescence in situ hybridization (FISH), interspersed repeat PCR, and restriction analysis of high molecular weight DNA. FISH revealed a largely uniform hybridization to the euchromatic chromosome regions with both interspersed repeats, although a bias toward the G-bands was observed for the LINE probe. Southern blots of inter-SINE and inter-LINE PCR products showed strong hybridization to LINE and SINE probes, respectively. High molecular weight DNA derived from a pig x hamster hybrid cell line was cut with a panel of G + C and A + T rich rare cutter restriction enzymes, then run on a pulsed field gel and Southern blotted. Sequential hybridization with SINE and LINE probes showed that SINE hybridization was to relatively low molecular weight fragments with the G + C rich enzymes, whereas the LINE probe gave hybridization to significantly larger fragments produced by these enzymes. DNA samples digested with A + T rich enzymes gave essentially similar patterns with SINE and LINE probes. We conclude that the pattern of differential distribution of SINEs and LINEs, which has been described in man and mouse, does exist in the pig but is much less pronounced.

Animals↗
PubMed1996

Construction and analysis of an hn-cDNA library derived from the p-arm of pig chromosome 12.

Our aim is to find unidentified genes on specific pig chromosomes or chromosome fragments. Our approach has involved the construction of a heterogeneous nuclear complementary (hn-c) DNA library of the p-arm of pig Chromosome (Chr) 12, the only pig chromosome present in the pig x hamster hybrid cell line 8990. Total RNA was extracted from the cells and first-strand synthesis of hn-cDNA carried out with random and oligo dT primers. Pig hn-cDNA was isolated by amplification of first-strand synthesized hn-cDNA with primers specific for Short Interspersed Repeat Elements (SINEs) via the polymerase chain reaction (PCR). Hn-cDNAs were size selected and cloned in E. coli XL-1 blue cells with PCR-Script as the vector. The library consisted of 6000 clones. Clone inserts were amplified by PCR with vector-specific primers, and randomly picked inserts greater than 600 bp were sequenced. Homology searches were carried out with the FASTA search program on the GenEmbl database. Thirty clones were sequenced, and of these three showed strong homologies to GenEmbl sequences: (1) to sheep, mouse, human, and rat mammary gland factor (MGF); (2) to MLN-50, a gene that is amplified in human familial breast cancer and is present on human Chr 17; the latter is homologous to pig chromosome 12; (3) to a family of unassigned overlapping human ESTs. Of the other sequenced clones, seven were over 80% homologous with pig SINE sequences; three were over 75% homologous to human LINE sequences; six displayed open reading frames over a mean distance equivalent to 50 amino acids, although these showed no significant similarities with sequences in the databases. Using this approach, we have been able to identify several new genes on the p-arm of pig Chr 12. This is the first report of gene isolation from a library derived from a pig chromosome fragment.

Animals↗
PubMed1996

A three-year clinical trial using a glass ionomer cement for the bonding of orthodontic brackets.

Recent clinical studies measuring orthodontic bracket failure, when using glass ionomer cement as an adhesive, have reported a wide range of percentages of bracket failure. The present study recorded bracket failure over a 3-year period, longer than had been previously measured. Seventeen participants were randomly assigned to one of two treatment groups, either using glass ionomer cement or composite resin for bonding. In each group, brackets were bonded to incisors, canines, and premolars. Bracket failure was measured over the duration of comprehensive orthodontic treatment for all participants. Brackets bonded with the glass ionomer cement were more likely to fail (log-rank test; P < or = 0.022). This difference was clinically significant. At the present time, the disadvantage of extra bracket failures appears to outweigh potential advantages when considering glass ionomer cement for the routine bonding of orthodontic brackets.

Adhesives↗
PubMed1996

The Harvard Nursing Research Institute's conference summary.

The Harvard Nursing Research Institute organized a national invitational conference on executive nursing leadership in major teaching hospitals and academic health centers. The conference brought together many of the nation's eminent nurse executives and other prominent individuals in healthcare to analyze the unique and complex challenges facing these organizations, expose participants to other perspectives, and articulate specific aims and strategies that might be taken to increase their effectiveness in leading the nursing profession's efforts to shape the ongoing transformation of the healthcare system.

Academic Medical Centers↗
PubMed1996

Superconducting receiver coils for sodium magnetic resonance imaging.

We present the results from sodium magnetic resonance imaging (MRI) experiments using high-temperature superconducting (HTS) receiver coils. Sodium imaging has been shown to have great potential for the assessment of cell integrity but suffers from a substantially lower signal-to-noise ratio (SNR) than that of a hydrogen imaging. The use of an HTS receiver coil was found to significantly increase the SNR relative to an equivalent copper receiver coil at room temperature. The SNR gains afforded by HTS coils can also be used to decrease the imaging time.

Copper↗