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D Locke

Publications and source records attributed to D Locke.

13 recordsLinked to original sources
PubMed2001

Expression of the platelet receptor GPVI confers signaling via the Fc receptor gamma -chain in response to the snake venom convulxin but not to collagen.

The mechanism of signal transduction underlying the activation of platelets by collagen has been actively investigated for over 30 years, but the receptors involved remain incompletely understood. Studies of human platelets, which are unresponsive to collagen, mouse knockout models, and platelet biochemical studies support the hypothesis that the recently cloned platelet surface protein GPVI functions as a signaling receptor for collagen. To directly test this hypothesis, we have expressed wild-type and mutant forms of GPVI in RBL-2H3 cells, which express the Fcepsilon receptor gamma-chain (Fc Rgamma), the putative signaling co-receptor for GPVI in platelets, but lack GPVI itself. Expression of GPVI in RBL-2H3 cells confers strong adhesive and signaling responses to convulxin (a snake venom protein that directly binds GPVI) and weak responsiveness to collagen-related peptide but no responsiveness to collagen. To elucidate the mechanism of GPVI intracellular signaling, mutations were introduced in the receptor's transmembrane domain and C-terminal tail. Unlike reported studies of other Fc Rgamma partners, these studies reveal that both the GPVI transmembrane arginine and intracellular C-tail are necessary for coupling to Fc Rgamma and for signal transduction. To our knowledge, these studies are the first to demonstrate a direct signaling role for GPVI and the first to directly test the role of GPVI as a collagen receptor. Our results suggest that GPVI may be necessary but not sufficient for collagen signaling and that a distinct ligand-binding collagen receptor such as the alpha(2)beta(1) integrin is likely to play a necessary role for collagen signaling as well as adhesion in platelets.

Amino Acid Sequence↗
PubMed2000

The RGD sequence in the cytomegalovirus DNA polymerase accessory protein can mediate cell adhesion.

The murine cytomegalovirus (MCMV) polymerase processivity factor ppM44 (also referred to as pp50) is an abundant phosphoprotein found in MCMV-infected cells. Sequence analysis of the MCMV M44 open reading frame revealed an "RGD" motif that is also present in the human cytomegalovirus (HCMV) UL44 open reading frame. In this report, histidine-tagged M44 protein produced in Escherichia coli or the vaccinia/T7 expression system was purified to near homogeneity by metal chelation affinity chromatography using His*Bind resins. We demonstrated that recombinant M44 protein could mediate cell adhesion via its conserved "RGD" motif, because a single amino acid change (RGD to RGE) abolished cell attachment. In addition, cell adhesion was abolished in the presence of EDTA. We next showed that recombinant HCMV UL44, but not human herpesvirus type 6 p41, which lacks the RGD motif, could mediate cell adhesion in a similar manner. We also provided evidence that ppM44 was present in the culture medium during virus infection. Thus these results suggested that in addition to its primary role as the polymerase processivity factor, MCMV ppM44 may serve as a substrate for integrin-binding via its conserved RGD motif, with the potential for a novel role in the MCMV replication cycle.

Amino Acid Sequence↗
PubMed2000

Developmental expression and assembly of connexins into homomeric and heteromeric gap junction hemichannels in the mouse mammary gland.

During the development of the mammary gland, duct-lining epithelial cells progress through a program of expansive proliferation, followed by a terminal differentiation that allows for the biosynthesis and secretion of milk during lactation. The role of gap junction proteins, connexins, in the development and function of this secretory epithelium was investigated. Connexins, Cx26 and Cx32, were differentially expressed throughout pregnancy and lactation in alveolar cells. Cx26 poly-(A)(+) RNA and protein levels increased from early pregnancy, whereas Cx32 was detectable only during lactation. At this time, immunolocalization of connexins by confocal microscopy and immunogold labeling of high-pressure frozen freeze-substituted tissue showed that both connexins colocalized to the same junctional plaque. Analysis of gap junction hemichannels (connexons) isolated from lactating mammary gland plasma membranes by a rate-density centrifugation procedure, followed by immunoprecipitation and by size-exclusion chromatography, showed that Cx26 and Cx32 were organized as homomeric and heteromeric connexons. Structural diversity in the assembly of gap junction hemichannels demonstrated between pregnant and lactating mammary gland may account for differences in ionic and molecular signaling that may physiologically influence the onset and/or maintenance of the secretory phenotype of alveolar epithelial cells.

Animals↗
PubMed1998

Gap junctions in normal and neoplastic mammary gland.

Progress in the characterization of gap junctions and their constituent connexin sub-units is leading to a greater understanding of the structure, function, and regulation of this cell-cell communication channel. Although much of the experimental evidence generated to date is correlative, recent work utilizing reverse genetic approaches to manipulate connexin gene function has provided direct evidence that intercellular communication via gap junctions plays key roles in development, cellular differentiation, and organogenesis. Pathogenic mutations in human connexin genes have now been identified. Furthermore, a considerable body of experimental evidence correlates a loss of junctional communication with progression to a malignant phenotype. Although the cell biology of the mammary gland has been extensively studied, the role(s) of gap junctions in the development, differentiation, and maintenance of this tissue are unknown. Gap junctions were first reported in the mammary gland following freeze-fracture and electron microscopic analyses. The development of anti-connexin antibodies and the cloning of individual connexin isoforms have enabled this work to be extended, but there are contradictory reports in the temporal expression patterns of these proteins within mammary epithelium. In addition, a recent report in this Journal has implied by immunocytochemistry that there is up-regulation of connexin protein in some human breast tumours, a novel observation which may be inconsistent with the proposed tumour suppressor role for gap junctions.

Animals↗
PubMed1997

Pig lymphocytes utilise mouse MAdCAM-1 to enter fetal gut xenografts in SCID mice.

Ileocecal junction (ICJ) and proximal intestine (PI) fragments from CD45(323-) allovariant fetal pigs were grafted subcutaneously into SCID mice. The xenografts were examined 8-12 weeks later using two-color immunohistology and the ICJ, but not PI, xenografts were found to contain three types of vessels. The first (the majority) was lined with mouse endothelium (mAb 9F1+), the second was lined with pig endothelium, and the third was chimeric. The ICJ vessels were specifically lined with mouse endothelium expressing MAdCAM-1, the mucosal addressin. Vessels lined with pig endothelium alone did not express the MAdCAM-1 epitopes. Radiolabeled allovariant pig peripheral blood lymphocytes (PBL) were introduced i.v. into the xenografted SCID mice, and entry into xenografts studied. Pig PBL were occasionally seen in MECA-367+ vessel walls after 4 h and within the ICJ but not PI xenografts after 24 h. This entry was specifically blocked by coinjection of the anti-MAdCAM-1 mAb MECA-367. The results demonstrate reendothelialization of xenografts by host endothelium that expresses its own addressin and is functional for xenogenic PBL.

Animals↗
PubMed1986

Distribution of immunoglobulin G subclasses in anti-A and anti-B sera.

Sera from 117 immunologically normal subjects, who had been selected for the presence of high titre ABO system antibody on routine screening, were further evaluated for the presence of IgG and its subclasses IgG1, IgG2, IgG3, and IgG4 using an indirect antihuman globulin technique. Subjects of all ABO groups had the capacity to produce IgG antibodies within each subclass, but those of group O produced the broadest spectrum of IgG subclasses and greatest strength of reactions.

ABO Blood-Group System↗
PubMed1982

Pharmacokinetics and tissue concentrations of tylosin in selected avian species.

Tissue and plasma concentrations and the biological half-life of tylosin in avian species of a variety of body sizes and metabolic rates were studied. The species chosen were eastern bobwhite quail (Colinus virginianus virginianus), pigeons (Columba livia), greater sandhill cranes (Grus canadensis tabida), and emus (Dromaius novaehollandiae). In the 1st phase of this study, tylosin was administered IM to quail, pigeons, and emus at a dosage rate of 25 mg/kg of body weight and to cranes at a dosage rate of 15 mg/kg. The average peak plasma concentrations of tylosin in quail, pigeons, cranes, and emus were 4.31, 5.63, 3.62, and 3.26 microgram/ml, respectively. These peak concentrations occurred at 0.5 to 1.5 hours after administration. The biological half-life of tylosin averaged 1.2 hours in quail, pigeons, and cranes, and was 4.7 hours in emus. In the 2nd phase of this study, tylosin concentrations in the tissues of quail, pigeons, and cranes were markedly higher than were plasma concentrations at corresponding sampling times. Six hours after antibiotic administration, tissue concentrations of tylosin in all species remained within the minimum inhibitory concentration for most pathogenic organisms. Dosage regimens of 25 mg of tylosin/kg 4 times daily for quail and pigeons, 15 mg/kg 3 times daily for cranes, and 25 mg/kg 3 times daily for emus would be needed to establish and maintain therapeutic tissue concentrations.

Animals↗
PubMed1981

Pharmacokinetics of cephalothin and cephalexin in selected avian species.

Plasma concentrations and the biological half-lives of cephalothin and cephalexin in avian species of a variety of body sizes and metabolic rates were studied. The species chosen were eastern bobwhite quail (Colinus v virginianus), pigeons (Columba livia), hybrid rosybill ducks (Netta sp), greater sandhill cranes (Grus canadensis tabida), and emus (Dromiceius novaehollandiae). In the 1st phase of the study, cephalothin sodium was given IM in a dose of 100 mg/kg of body weight. Plasma concentrations reached peak (av 18 micrograms/ml) at 0.5 hour and were measurable 2.5 to 5.5 hours after drug administration. The biological half-life of cephalothin was 16 to 54 minutes; the half-life varied directly with increased species body weight, with the exception of the ducks studied. In the 2nd phase, cephalexin monohydrate was given orally in doses of 25, 35, and 50 mg/kg of body weight. Plasma concentrations reached peak (av 20 micrograms/ml) at 0.5 to 1 hour and were measurable 2.5 to 5.5 hours after drug administration. The biological half-life of cephalexin was 36 to 126 minutes. In the 3rd phase, differences in plasma concentrations and the half-lives of cephalexin between fed quail and fasted quail were insignificant. Dosage regimens for cephalothin of 100 mg/kg 4 times a day and for cephalexin of 35 to 50 mg/kg 4 times a day would be expected to establish and maintain therapeutic plasma concentrations in large birds (pigeons, cranes, and emus). These same doses, administered every 2 to 3 hours, would be expected to establish and maintain therapeutic plasma concentrations in smaller birds (quail, ducks).

Animals↗
PubMed1981

Gentamicin tissue concentration in various avian species following recommended dosage therapy.

Plasma and tissue drug concentrations were compared in eastern bobwhite quail (Colinus virginianus virginianus) and pigeons (Columba livia) given gentamicin by IM administration at the dosage of 10 mg/kg, and in greater sandhill cranes (Grus canadensis tabida) and hybrid rosybill ducks (Netta sp) given the same antibiotic at a dosage of 5 mg/kg. Quail and cranes had significantly higher liver concentrations of gentamicin at 6 hours after injection than did pigeons and ducks. Cranes had significantly higher plasma concentrations than did ducks at 6 hours after injection. Compared with plasma values, gentamicin concentrations were significantly higher in the liver of cranes at 12 hours after injection, and in the kidneys at 18 hours.

Animals↗
PubMed1980

Alteration of lymphocyte function due to anesthesia: in vivo and in vitro suppression of mitogen-induced blastogenesis by sodium pentobarbital.

The mechanism of decreased lymphocyte responsiveness after major surgery is unclear. Because sodium pentobarbital, and intermediately long-acting barbiturate, will reproducibly induce anesthesia in experimental animals, we utilized a canine model to investigate its effect on lymphocyte proliferation induced by the mitogenic lectins erythroagglutinating phytohemagglutinin (E-PHA) and leukoagglutinating phytohemagglutinin (L-PHA). Although no effect was observed at 10 minutes or 1 hour after an anesthetic dose of sodium pentobarbital, after 1 and 3 hours of anesthesia, canine lymphocytes were significantly suppressed, as demonstrated by decreased responsiveness to E-PHA and L-PHA mitogen stimulation. After 3-hours the majority of animals had mitogenesis values of less than 50% of the preanesthetic control values. Recovery, as measured by a return to at least 70% of the preanesthetic mitogenesis value, was noted in the majority of animals at 24, 48, and 72 hours. In order to investigate the machanisms of the in vivo capability of sodium pentobarbital to induce immunosuppression of lymphocyte transformation, in vitro studies were carried out. Sodium pentobarbital was found to significantly inhibit mitogen-induced canine mononuclear cell blastogenesis at anesthetic (1.5 to 3.0 mg%) drug concentrations in vitro. Lymphocytes pretreated with barbiturate and washed prior to plating did not show this inhibiting effect. Our findings suggest that depression of the immune response reported in patients after operation could result from short-acting barbiturates administered during the induction phase of clinical anesthesia. Furthermore, the suppression may involve in vivo metabolism of pentobarbital, hormones or other in vivo factors, since washed lymphocytes from the in vivo but not the in vitro experiments demonstrated suppression. These results indicate that anesthesia may be an important factor in the immunosuppression reported after major surgery.

Animals↗
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