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Biomedical subjects

M E Clark

Publications and source records attributed to M E Clark.

At least 19 recordsLinked to original sources
PubMed2000

ADAMTS9, a novel member of the ADAM-TS/ metallospondin gene family.

ADAM-TS/metallospondin genes encode a new family of proteins with structural homology to the ADAM metalloprotease-disintegrin family. However, unlike other ADAMs, these proteins contain thrombospondin type 1 (TSP1) repeats at the carboxy-terminal end and are secreted proteins instead of being membrane bound. Members of the ADAM-TS family have been implicated in the cleavage of proteoglycans, the control of organ shape during development, and the inhibition of angiogenesis. We have cloned a new member of the ADAM-TS/metallospondin family designated here as ADAMTS9. This protein has a metalloprotease domain, a disintegrin-like domain, one internal TSP1 motif, and three carboxy-terminal TSP1-like submotifs. In contrast to other ADAM-TS family members, ADAMTS9 is expressed in all fetal tissues examined as well as some adult tissues. Using FISH and radiation hybrid analysis, we have localized ADAMTS9 to chromosome 3p14.2-p14.3, an area known to be lost in hereditary renal tumors.

ADAM Proteins↗
PubMed2000

The copper transport protein Atox1 promotes neuronal survival.

Atox1, a copper transport protein, was recently identified as a copper-dependent suppressor of oxidative damage in yeast lacking superoxide dismutase. We have previously reported that Atox1 in the rat brain is primarily expressed in neurons, with the highest levels in distinct neuronal subtypes that are characterized by their high levels of metal, like copper, iron, and zinc. In this report, we have transfected the Atox1 gene into several neuronal cell lines to increase the endogenous level of Atox1 expression and have demonstrated that, under conditions of serum starvation and oxidative injury, the transfected neurons are significantly protected against this stress. This level of protection is comparable with the level of protection seen with copper/zinc superoxide dismutase and the anti-apoptotic gene bcl-2 that had been similarly transfected. Furthermore, neuronal cell lines transfected with a mutant Atox1 gene, where the copper binding domain has been modified to prevent metal binding, do not afford protection against serum starvation resulting in apoptosis. Therefore, Atox1 is a component of the cellular pathways used for protection against oxidative stress.

Amino Acid Sequence↗
PubMed2000

Improved phase-contrast flow quantification by three-dimensional vessel localization.

In this paper, a method of three-dimensional (3D) vessel localization is presented to allow the identification of a vessel of interest, the selection of a vessel segment, and the determination of a slice orientation to improve the accuracy of phase-contrast magnetic resonance (PCMR) angiography. A marching-cube surface-rendering algorithm was used to reconstruct the 3D vasculature. Surface-rendering was obtained using an iso-surface value determined from a maximum intensity projection (MIP) image. This 3D vasculature was used to find a vessel of interest, select a vessel segment, and to determine the slice orientation perpendicular to the vessel axis. Volumetric flow rate (VFR) was obtained in a phantom model and in vivo using 3D localization with double oblique cine PCMR scanning. PCMR flow measurements in the phantom showed 5. 2% maximum error and a standard deviation of 9 mL/min during steady flow, 7.9% maximum error and a standard deviation of 13 mL/min during pulsatile flow compared with measurements using an ultrasonic transit-time flowmeter. PCMR VFR measurement error increased with misalignment at 10, 20, and 30 degrees oblique to the perpendicular slice in vitro and in vivo. The 3D localization technique allowed precise localization of the vessel of interest and optimal placement of the slice orientation for minimum error in flow measurements.

Blood Flow Velocity↗
PubMed2000

The Otx2 homeoprotein regulates expression from the gonadotropin-releasing hormone proximal promoter.

The GnRH gene is expressed exclusively in a highly restricted population of approximately 800 neurons in the mediobasal hypothalamus in the mouse. The Otx2 homeoprotein has been shown to colocalize with GnRH in embryonic mouse brain. We have identified a highly conserved bicoid-related Otx target sequence within the proximal promoter region of the GnRH gene from several species. This element from the rat GnRH promoter binds baculovirus-expressed Otx2 protein and Otx2 protein in nuclear extracts of a hypothalamic GnRH-expressing neuronal cell line, GT1-7. Transient transfection assays indicate that the GnRH promoter Otx/bicoid site is required for specific expression of the GnRH gene in GT1-7 cells and that it can confer specificity to a neutral Rous sarcoma virus (RSV) promoter in GT1-7 cells but not in NIH3T3 cells. Overexpression of mouse Otx2 in GT1-7 cells induces expression of a GnRH promoter plasmid, an effect that is dependent upon the Otx binding site. Thus, the GnRH proximal promoter is regulated by the Otx2 homeoprotein. Finally, we have now demonstrated the presence of Otx2 protein in the GnRH neurons of the adult mouse hypothalamus. These data suggest that Otx2 is important in the development of the GnRH neuron and/or in the maintenance of GnRH expression in the adult mouse hypothalamus.

3T3 Cells↗
PubMed1998

Solubility enhancement of phenol and phenol derivatives in perfluorooctyl bromide.

Perfluorinated solvents are gaining popularity as pulmonary ventilation fluids, but they suffer from poor solvent quality in concurrent drug delivery applications. The present study examines the use of a hydrophobic solubilizing agent capable of interacting with model drug solutes by hydrogen bonding with the purpose of enhancing solubility in perfluorooctyl bromide (PFOB). A series of solubilizing agents containing a ketone carbonyl to act as a hydrogen bond acceptor and a perfluoroalkyl chain to maintain the solubility of the putative complex in PFOB are investigated. The solubility of phenol in PFOB is enhanced to the greatest extent by 1-(4-perfluorobutyl phenyl)-1-hexanone (III) where the ketone carbonyl is protected from the electron withdrawing effects of the perfluorobutyl chain by a phenyl ring. Experiments with solubilizers lacking the ketone group suggest that pi-pi bond interactions of III with phenol do not significantly enhance solubility. For a series of phenol derivatives, a rank-order correlation exists between the magnitude of solubility enhancement by III, as reflected by the calculated association constants, and the Hammett sigma parameter of the phenols. Because the O-methyl-substituted phenols do not have the ability to hydrogen bond, their solubility is not enhanced by the presence of III. The results of the present study indicate that solubility of model drug hydrogen bond donating compounds can be enhanced in PFOB by the presence of fluorocarbon-soluble hydrogen bond acceptors.

Chromatography, High Pressure Liquid↗
PubMed1998

Mortality, size of the gonads, and ultrastructure of primordial germ cell in chick embryos treated with gamma-irradiation or injected with donor cells.

The effects of injection and/or gamma-irradiation prior to injection on mortality, size of the gonads, and ultrastructure of primordial germ cell (PGC) were examined after 5 d of incubation. The mortality of embryos injected with donor cells was significantly higher than that of control and irradiated embryos. All irradiated embryos were alive, although their development was delayed compared to those not exposed to irradiation. The size of the gonads of embryos injected with donor cells were similar to those of control embryos, however, the size of the gonads in irradiated embryos was significantly smaller than those of control embryos. The number of PGC in the gonads was significantly decreased by irradiation. There was no notable effect of irradiation or injection on the nuclei and cytoplasmic organelles in PGC.

Animals↗
PubMed1998

Use of music to decrease aggressive behaviors in people with dementia.

The purpose of this study was to examine the effects of recorded, preferred music in decreasing occurrences of aggressive behavior among individuals with Alzheimer's type dementia during bathing episodes. Eighteen older adults, age 55 to 95, with severe levels of cognitive impairment, participated in the study. They were randomly scheduled for observation during bath time under either a control (no music) condition or an experimental condition in which recorded selections of preferred music were played via audiotape recorder during the bathing episode. Following a 2-week (10 episode) observation period, conditions were reversed. A total of 20 observations were recorded for each individual. Results indicated that during the music condition, decreases occurred in 12 of 15 identified aggressive behaviors. Decreases were significant (p < 0.05) for the total number of observed behaviors and for hitting behaviors. During the music condition, caregivers frequently reported improved affect and a general increase in cooperation with the bathing task. The implications of these findings for improving the overall quality of care for severely cognitively impaired older adults are discussed.

Aged↗
PubMed1997

Production of chicken chimeras from injection of frozen-thawed blastodermal cells.

To execute a strategy for reconstituting genetic resources from cryopreserved blastodermal cells, experiments were conducted to optimize conditions for producing chimeric chickens from frozen-thawed blastodermal cells. Stage X blastodermal cells were collected from Barred Plymouth Rock embryos and dispersed. Cells were resuspended in 10% dimethyl sulfoxide in Dulbecco's modified Eagle's medium (DMEM) containing 20% fetal bovine serum, and distributed into plastic ampules. Cell suspensions were seeded to induce ice formation at -7 C, cooled from -7 to -35 C at 1 C/min and then ampules were plunged into liquid nitrogen. Thawing was done by plunging the ampules into warm water (37 C) for 3 min. After centrifugation, the supernatant was replaced with DMEM, and dead or broken cells were removed by density gradient centrifugation. Approximately 500 cells were injected into irradiated Stage X White Leghorn recipient embryos. Following incubation, several somatic chimeras were produced. The frequency of somatic chimerism when fresh (unfrozen) cells, or cells that were frozen and selected by density gradient centrifugation on Percoll or Nycoprep were injected into recipient embryos was 84, 79, and 85%, respectively. The percentage of donor-derived pigmentation in the down of these chimeric chickens was 79, 50, and 58%, respectively. Germline chimerism was determined by mating the chimeras that survived to sexual maturity to Barred Plymouth Rocks. Nine of 16 birds (56.2%) injected with fresh cells, 2 of 26 birds (7.7%) injected with cells that were frozen and selected by density gradient centrifugation on a Percoll gradient, and 3 of 26 birds (11.5%) injected with cells that were frozen and selected on a Nycoprep gradient showed germline transmission; the percentage of donor-derived progeny in these chimeras were 29.5, 5.2, and 6.8%, respectively. The Barred Plymouth Rock donor stock was "reconstituted" by inter se mating of germline male and female chimeras. These data demonstrate that the strategy described here for reconstituting genetic resources from cryopreserved blastodermal cells via chimeric intermediates can be performed successfully.

Animals↗
PubMed1997

Manipulation of blastodermal cells.

Blastodermal cells isolated from newly laid, unincubated eggs are virtually uncommitted cells that exhibit many of the properties of pluripotential stem cells. They can be transferred from donor to recipient embryos and contribute to both somatic tissues and the germline. Blastodermal cells that have been maintained in culture for 7 d express the epitopes ECMA-7 and SSEA-1, which are also expressed by mouse embryonic stem cells. After culture for up to at least 7 d, blastodermal cells retain the ability to differentiate into somatic tissues and the germline both in vivo and in vitro. Proliferation in the absence of differentiation of blastodermal cells is stimulated by the presence of Leukemia Inhibitory Factor (LIF) and other ligands that interact with the gp130 receptor, and differentiation is stimulated by exposure to retinoic acid. Blastodermal cells also possess high levels of telomerase activity, which is shared by immortalized cells and cells within the germline. Blastodermal cells can be transfected and will express foreign genes both in vivo and in vitro. Transfected cells can be isolated by fluorescence activated cell sorting and can be cryopreserved without losing their ability to contribute to either somatic tissues or the germline. These properties of blastodermal cells make them ideal vectors for introducing genetic modifications to the germline.

Alkaline Phosphatase↗
PubMed1996

Contributions to somatic and germline lineages of chicken blastodermal cells maintained in culture.

Chicken blastodermal cells were cultured for 48 hr as explanted intact embryos, as dispersed cells in a monolayer, or with a confluent layer of mouse fibroblasts. The cells were then dispersed and injected into stage X (E-G&K) recipient embryos that were exposed to 600 rads of irradiation from a 60Co source. Regardless of the conditions in which the cells were cultured, chimeras with contributions to both somatic tissues and the germline were observed. When blastodermal cells were co-cultured with mouse embryonic fibroblasts, significantly more somatic chimeras were observed and the proportion of feather follicles derived from donor cells was increased relative to that observed following the injection of cells derived from explanted embryos or monolayer cultures. Culture of blastodermal cells in any of the systems, however, yielded fewer chimeras that exhibited reduced contributions to somatic tissues in comparison to the frequency and extent of somatic chimerism observed following injection of freshly prepared cells. Contributions to the germline were observed at an equal frequency regardless of the conditions of culture, but were significantly reduced in comparison to the frequency and rate of germline transmission following injection of cells obtained directly from stage X (E-G&K) embryos. These data demonstrate that some cells retain the ability to contribute to germline and somatic tissues after 48 hr in culture and that the ability to contribute to the somatic and germline lineages is not retained equally.

Animals↗
PubMed1996

Technical basis for EPA's proposed regulation on the cleanup of sites contaminated with radioactivity.

The U.S. Environmental Protection Agency is proposing a regulation for the protection of the public from radioactive contamination at sites that are to be cleaned up and released for public use. The rule will apply to sites under the control of Federal agencies, and will impose limits on radiation doses to individuals living or working on a site following cleanup; it will thereby provide site owners and managers with uniform, consistent cleanup criteria for planning and carrying out remediation. This paper presents an overview of EPA's approach to assessing some of the beneficial and adverse effects associated with various possible values for the annual dose limit. In particular, it discusses the method developed to determine how the choice of cleanup criterion affects (1) the time-integrated potential numbers of non-fatal and fatal radiogenic cancers averted among future populations, (2) the occurrence of radiogenic cancers among remediation workers and the public caused by the cleanup process itself, and (3) the volumes of contaminated soil that may require remediation. The analytic methods described here were used to provide input data and assumptions for the Regulatory Impact Analysis (RIA) that supports the proposed regulation; the RIA also considered non-radiological benefits and costs (i.e., public health, economic, and ecological) of the standards.

Radiation Injuries↗
PubMed1996

Long-term in vitro culture and characterisation of avian embryonic stem cells with multiple morphogenetic potentialities.

Petitte, J.N., Clarck, M.E., Verrinder Gibbins, A. M. and R. J. Etches (1990; Development 108, 185-189) demonstrated that chicken early blastoderm contains cells able to contribute to both somatic and germinal tissue when injected into a recipient embryo. However, these cells were neither identified nor maintained in vitro. Here, we show that chicken early blastoderm contains cells characterised as putative avian embryonic stem (ES) cells that can be maintained in vitro for long-term culture. These cells exhibit features similar to those of murine ES cells such as typical morphology, strong reactivity toward specific antibodies, cytokine-dependent extended proliferation and high telomerase activity. These cells also present high capacities to differentiate in vitro into various cell types including cells from ectodermic, mesodermic and endodermic lineages. Production of chimeras after injection of the cultivated cells reinforced the view that our culture system maintains in vitro some avian putative ES cells.

Alkaline Phosphatase↗
PubMed1995

Sexual differentiation of chimeric chickens containing ZZ and ZW cells in the germline.

The developmental fate of male and female cells in the ovary and testis was evaluated by injecting blastodermal cells from Stage X (Eyal-Gliadi and Kochav, 1976: Dev Biol 49:321-337) chicken embryos into recipients at the same stage of development to form same-sex and mixed-sex chimeras. The sex of the donor was determined by in situ hybridization of blastodermal cells to a probe derived from repetitive sequences in the W chromosome. The sex of the recipient was assigned after determination of the chromosomal composition of erythrocytes from chimeras at 10, 20, 40, and 100 days of age. If the sex chromosome complement of all of the erythrocytes was the same as that of blastodermal cells from the donor, the sex of the recipient was assumed to be the same as that of the donor. Conversely, if the sex-chromosome complement of a portion of the erythrocytes of the chimera differed from that of the donor blastodermal cells, the sex of the recipient was assumed to differ from that of the donor. Injection of male blastodermal cells into female recipients produced both male and female chimeras in equal proportions whereas injection of female cells into male recipients produced only by male chimeras. One phenotypically male chimera developed with a left ovotestis and a right testis although sexual differentiation was usually resolved into an unambiguous sexual phenotype during development when ZZ and ZW cells were present in a chimera. Donor cells contributed to the germline of 25-33% of same-sex chimeras whereas 67% of male chimeras produced by injecting male donor cells into female recipients incorporated donor cells into the germline. When ZW cells were incorporated into chimeric males, W-chromosome-specific, DNA sequences were occasionally present in DNA extracted from semen. To examine the potential of W-bearing spermatozoa to fertilize ova, males producing ZW-derived offspring and semen in which W-chromosome-specific DNA was detected by Southern analysis were mated to sex-linked albino hens. Since sex-linked albino female progeny were not obtained from this mating, it was concluded that the W-bearing sperm cells were unable to fertilize ova. The production of Z-derived, but not W-derived, offspring from ZW spermatogonia indicates that female primordial germ cells can become spermatogonia in the testes. In the testes, ZW spermatogonia enter meiosis I and produce functional ZZ spermatocytes. The ZZ spermatocytes complete the second meiotic division, continue to differentiate during spermiogenesis, and leave the seminiferous tubules as functional spermatozoa. By contrast, the WW spermatocytes do not appear to complete spermiogenesis and, therefore, spermatozoa bearing the W-chromosome are not produced. When cells from male embryos were incorporated into a female chimera, ZZ "oogonia" were included within the ovarian follicles and the chromosome complement of genetically male oogonia was processed normally during meiosis. Following ovulation, the male-derived ova were fertilized and produced normal offspring. This is the first reported evidence that genetically male avian germ cells can differentiate into functional ova and that genetically female germ cells can differentiate into functional sperm.

Animals↗
PubMed1995

The POU homeodomain transcription factor Oct-1 is essential for activity of the gonadotropin-releasing hormone neuron-specific enhancer.

The mechanisms of specification of gene expression in a complex tissue such as the brain remain poorly understood. To provide a model system for the study of gene regulation in a specific subpopulation of differentiated neurons, we have derived cell lines from tumors created in transgenic mice by targeting simian virus 40 T antigen expression by using the regulatory regions of the gene for gonadotropin-releasing hormone (GnRH), a decapeptide released from specialized neurons in the hypothalamus. Transfections into the cultured GnRH-secreting hypothalamic neuronal cell line GT1 have identified a neuron-specific enhancer, 1.5 kb upstream of the GnRH gene, which binds multiple GT1 nuclear proteins. In particular, one AT-rich protein-binding region, AT-a, is critical for enhancer activity. In this study, we used electrophoretic mobility shift assays to detect a GT1 nuclear protein complex that binds the AT-a region. Close inspection of the AT-a bottom-strand sequence revealed homology to the octamer motif, a sequence known to bind members of the POU homeodomain transcription factor family. Although we demonstrate expression of a number of POU homeodomain genes in GT1 cells, a supershift assay with Oct-1 antibody demonstrates that Oct-1 is the protein binding the enhancer. Finally, specific mutations in the AT-a region that affected Oct-1 binding were correlated with decreased transcription. Thus, Oct-1 binds to the GnRH enhancer in vitro, and this binding is critical to the transcriptional activity of this neuron-specific enhancer in GT1 cells.

Animals↗
PubMed1993

Direct cleavage of human TATA-binding protein by poliovirus protease 3C in vivo and in vitro.

Host cell RNA polymerase II (Pol II)-mediated transcription is inhibited by poliovirus infection. This inhibition is correlated to a specific decrease in the activity of a chromatographic fraction which contains the transcription factor TFIID. To investigate the mechanism by which poliovirus infection results in a decrease of TFIID activity, we have analyzed a component of TFIID, the TATA-binding protein (TBP). Using Western immunoblot analysis, we show that TBP is cleaved in poliovirus-infected cells at the same time postinfection as when Pol II transcription is inhibited. Further, we show that one of the cleaved forms of TBP can be reproduced in vitro by incubating TBP with cloned, purified poliovirus encoded protease 3C. Protease 3C is a poliovirus-encoded protease that specifically cleaves glutamine-glycine bonds in the viral polyprotein. The cleavage of TBP by protease 3C occurs directly. Finally, incubation of an uninfected cell-derived TBP-containing fraction (TFIID) with protease 3C results in significant inhibition of Pol II-mediated transcription in vitro. These results demonstrate that a cellular transcription factor can be directly cleaved both in vitro and in vivo by a viral protease and suggest a role of the poliovirus proteinase 3C in host cell Pol II-mediated transcription shutoff.

3C Viral Proteases↗
PubMed1993

Germline chimeric chickens from dispersed donor blastodermal cells and compromised recipient embryos.

Stage-X blastoderms, within intact eggs from White Leghorn hens, were exposed to 500-700 rads of gamma radiation from a 60Co source prior to injection, into the subgerminal cavity, of approximately 100 or 200-400 dispersed cells from stage-X blastoderms isolated from eggs laid by Barred Plymouth Rock hens. Embryos developing past day 14 of incubation and hatched chicks were assessed for donor and recipient cell contribution to the melanocyte population through examination of black and yellow down pigmentation, respectively (Barred Plymouth Rocks have a recessive allele at the I locus while the White Leghorns have a dominant allele at the I locus). Of the 809 embryos injected with approximately 100 cells, 192 developed past day 14 and black pigmentation, indicating somatic chimerism, was observed on 118 of the 192 (58%) embryos and chicks. Of the 296 embryos injected with 200-400 donor cells, 86 developed past day 14 of incubation. Somatic chimerism was observed on 55 of the 86 (64%) embryos and chicks. To test for germline chimerism, birds surviving to maturity were mated to Barred Plymouth Rocks. Five somatically chimeric females were produced when approximately 100 cells were injected, and one was a germline chimera. Six somatic female chimeras were produced following the injection of 200-400 cells, three of which proved to be germline chimeras by the presence of Barred Rock chicks among their offspring. Two of the nine males produced by injecting approximately 100 cells were germline chimeras.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗