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Biomedical subjects

W L Dean

Publications and source records attributed to W L Dean.

At least 55 records · Page 3Linked to original sources
PubMed1990

U3 snRNPs and nucleolar development during oocyte maturation, fertilization and early embryogenesis in the mouse: U3 snRNA and snRNPs are not regulated coordinate with other snRNAs and snRNPs.

U3 small nuclear ribonucleic acids (snRNA) and U3 small nuclear ribonucleoprotein (snRNP), which are thought to be responsible for ribosomal RNA processing, are quantitated and localized during oocyte maturation, fertilization, and early embryogenesis in the mouse. On the basis of Northern blot and nuclease protection experiments, it is estimated that there are about 5 x 10(4) U3 snRNA molecules in an ovulated oocyte and in a two-cell embryo. This number then increases roughly 50-fold to 2.7 x 10(6) molecules per embryo by the blastocyst stage. At all stages of development U3 snRNP antigens colocalize with nucleoli, as defined by differential interference contrast microscopy and an antibody to a nucleolar epitope. The synthesis and distribution of U3 snRNA and U3 snRNP follow a pattern independent from other major U snRNPs and snRNAs.

Animals↗
PubMed1990

Relocalization of small ribonucleoprotein particles (snRNPs) during the first cell cycle of mouse embryo development is independent of RNA synthesis, DNA synthesis and cytokinesis.

The process of localization of small nuclear ribonucleoprotein particles (snRNPs) during the first cell cycle of mouse embryo development was investigated following treatment of fertilized eggs with cytochalasin D, aphidicolin and alpha-amanitin. The pattern of accumulation of snRNPs in nuclei of treated embryos as assessed by indirect immunofluorescence was unaffected by the inhibitors. The results demonstrate that the localization of snRNPs during the first cell cycle does not require ongoing cytokinesis, DNA replication or transcription of RNA polymerase II genes. These findings suggest that maternally derived snRNPs become localized to the nucleus of the fertilized ovum prior to the reinitiation of transcription from the zygote genome and are required for processing of messenger RNA precursors when genetic activity of the embryonic genome is activated at the early 2-cell stage.

Amanitins↗
PubMed1989

Role of intracellular calcium in priming of human peripheral blood monocytes by bacterial lipopolysaccharide.

To determine the role of intracellular calcium ([Ca2+]i) in the priming of monocytes (M phi) by bacterial lipopolysaccharide (LPS), the membrane expression of two functional proteins and phagocytosis and respiratory burst were examined by microfluorimetry. LPS induced a significant increase in HLA-DR and C3bi receptor (CR3) expression within 2 h of its addition to whole blood. The enhanced expression of both antigens by LPS was dose-dependent, with concentrations as low as 0.1 ng/ml producing a response. The involvement of [Ca2+]i was demonstrated by loading isolated M phi with the intracellular calcium chelator quin-2 or the inhibitor of intracellular calcium redistribution TMB-8 prior to addition of LPS. Both compounds inhibited the LPS-induced increase in HLA-DR and CR3 expression. No role for extracellular calcium, for calcium slow channel flux, or for the calcium-calmodulin complex in LPS priming was demonstrated when LPS was added in the presence of EGTA, trifluperazine (TFP), or verapamil. The addition of the calcium ionophores A23187 or ionomycin failed to increase expression of either antigen. Prior exposure to LPS primed M phi for enhanced phagocytosis and respiratory burst activity. These functions were inhibited by TMB-8, but not by TFP or verapamil. Addition of LPS to isolated M phi increased [Ca2+]i by 23% at 30 sec and 42% at 5 min, as measured by the calcium-sensitive, intracellular probe indo-1. These results suggest that intracellular Ca2+ mobilization is necessary, but not sufficient, for LPS-induced priming of human peripheral blood monocytes.

Aminoquinolines↗
PubMed1989

Structure, function and subcellular localization of a human platelet Ca2+-ATPase.

Human platelets contain a Ca2+-ATPase in internal membranes that is essential for Ca2+ homeostasis. This Ca2+ pump has enzymatic properties quite similar to the sarcoplasmic reticulum (SR) Ca2+ pumps. Antibodies against the SR Ca2+ pump crossreact with the human platelet protein. However, the platelet Ca2+-ATPase is approximately 10 kD larger than the SR pumps and exhibits a larger mRNA coding for the protein in a megakaryocyte tumor cell line. In addition, the platelet Ca2+-pump may be localized in specialized internal membrane structures that function in Ca2+ uptake and release. These results suggest that the platelet Ca2+-ATPase may represent a new class of internal membrane Ca2+-pumps.

Antibodies↗
PubMed1989

The small nuclear RNAs for pre-mRNA splicing are coordinately regulated during oocyte maturation and early embryogenesis in the mouse.

The abundance and localization of snRNAs and snRNPs involved in processing and splicing of pre-mRNA has been studied during early mouse embryogenesis. The amount of U1, U2, U4, U5 and U6 RNA remains relatively constant between the postovulatory oocyte and 2-cell stage but increases three- to ten-fold in quantity between the 2-cell and blastocyst stages. Localization was examined by in situ hybridization with U1, U2 and U6 riboprobes and immunofluorescence microscopy using a monoclonal antibody to snRNP antigens. The snRNAs and snRNPs are primarily localized to the germinal vesicle in the preovulatory oocyte but are released and diluted within the cytoplasm of the oocyte during germinal vesicle breakdown and meiotic maturation. They subsequently relocalize to both pronuclei following fertilization and the nuclei of the 2-cell embryo following the first cleavage division. Since the amount of snRNA is constant during the first cleavage, the small amount of pre-mRNA that is synthesized at the time of transcriptional activation in the 2-cell embryo may be spliced and processed by snRNPs of maternal origin.

Animals↗
PubMed1988

Immunocytochemical localization of the Ca2+-ATPase polypeptide in human platelets.

Specific polyclonal antibodies raised against purified human platelet Ca2+-ATPase were used with protein A-gold immunocytochemistry to localize this protein in human platelets. Immunolabeling specifically detected Ca2+-ATPase over the surface connected membrane system (SCS) in sections of paraformaldehyde-fixed, Lowicryl-embedded platelets. The maximum density of label, determined by quantitative morphometric techniques, was observed over electron-dense regions within the SCS which may represent specialized structures for uptake and release of Ca2+. Less intense immunolabeling was observed over cytosol and may represent localization over the dense tubular system (DTS) which was not readily visualized under the processing procedures employed.

Blood Platelets↗
PubMed1988

Structural and functional comparison of a 22 kDa protein from internal human platelet membranes with cardiac phospholamban.

We have shown that a platelet 22 kDa phosphoprotein is similar to the cardiac regulator phospholamban, in that both are phosphorylated by cAMP- and calmodulin-dependent protein kinases, and that both can be phosphorylated simultaneously by these two classes of protein kinases to yield an additive stimulation of the respective Ca2+ pumps (Adunyah, S.E. and Dean, W.L. (1987) Biochim. Biophys. Acta 930, 401-409). However, whereas phosphorylation of phospholamban increases the affinity of the cardiac Ca2+-ATPase for Ca2+, phosphorylation of the platelet 22 kDa protein increased the Vmax of the pump. In addition, antibodies raised against canine phospholamban did not crossreact with the human platelet 22 kDa protein. Finally, it was not possible to dissociate the platelet protein into lower-molecular-weight subunits by boiling in sodium dodecylsulfate which is characteristic of cardiac phospholamban. These results show that although phosphorylation of low-molecular-weight membrane-associated regulator proteins in cardiac muscle and platelets appears to stimulate the respective Ca2+ pumps, these proteins have different chemical and physical properties.

Blood Platelets↗
PubMed1988

Bronchial leukocyte proteinase inhibitor: hydrodynamic properties and interaction with alpha 2-macroglobulin-bound elastase.

Bronchial leukocyte proteinase inhibitor (BLPI) is an 11.7 kDa, acid-stable protein found in mucous secretions, which inhibits neutrophil elastase. The Stoke's radius of BLPI calculated from sedimentation equilibrium and sedimentation velocity centrifugation data was in good agreement with the value determined by gel filtration. These data indicate that BLPI exists in a compact globular conformation at both neutral and acidic pH. BLPI, due to its small compact size, can inhibit neutrophil elastase after the enzyme has been complexed with alpha 2-macroglobulin (A-2-M) but alpha 1-proteinase inhibitor failed to inactivate A-2-M-bound elastase. The apparent association rates of BLPI and Eglin C with A-2-M-bound elastase were found to be 6.3 X 10(2) M-1s-1 and 1.1 X 10(3) M-1s-1, respectively. These apparent association rates decreased 168-fold for BLPI and 909-fold for Eglin C, relative to the association rates of these inhibitors with free elastase. These changes probably result from a combination of effects, such as inhibitor accessibility to the enzyme and/or reaction rate, but regardless of the mechanism these data suggest that BLPI may function to control both free and A-2-M-bound elastase.

Bronchi↗
PubMed1988

Localization and expression of U1 RNA in early mouse embryo development.

We have studied the accumulation and localization of U1 RNA during mouse embryo development by in situ hybridization with a U1 RNA probe and immunofluorescence microscopy using a mouse monoclonal antibody to U1 snRNP. There is a substantial amount of U1 RNA present in the oocyte that is present in both the germinal vesicle and the cytoplasm although the concentration is higher in the nuclear compartment. Following the germinal vesicle breakdown that accompanies ovulation and meiotic maturation, the U1 RNA is uniformly distributed throughout the unfertilized oocyte. In the fertilized egg, the silver grain density from in situ hybridization is higher over pronuclei and this enrichment is maintained at the two-cell and later stages. Similar results were obtained for the distribution of the U1 snRNP as assayed by immunofluorescence microscopy: U1 RNA is predominantly localized in all nuclei except polar body nuclei. The U1 RNA in the oocyte and two-cell embryo is predominantly (greater than 85%) U1a RNA. By the eight-cell stage there is a two to three-fold increase in the amount of total U1 RNA and the proportion of U1b RNA has increased to about 40%. The amount of U1 RNA continues to increase through the blastocyst stage and the proportion of the U1b RNA increases to 60%.

Animals↗
PubMed1988

Calcium-binding and aggregation properties of parathyroid secretory protein-I (chromogranin A).

Secretory protein-I (SP-I, also referred to as chromogranin A) is a major acidic, sulfated glycoprotein of the parathyroid gland secretory granule. It is cosecreted with parathormone under calcium regulation. SP-I is similar if not identical to chromogranin A of the adrenal medulla chromaffin cell and has been found in most endocrine cells. Its function is unknown, but it may play a role in the packaging of secreted substances. We have examined the calcium-binding properties of purified bovine SP-I and find that it binds 14 or more Ca2+ at low affinity. Measurements of sedimentation equilibrium and light scattering reveal that in the absence of added Ca2+ SP-I is monomeric (molecular weight about 52,000), but as Ca2+ binding proceeds it aggregates. The adherence of the molecule to biological cell membranes is also enhanced by Ca2+. These properties are consonant with earlier speculation that SP-I serves as a 'condensing' agent within secretory granules.

Animals↗
PubMed1987

The effect of 2,3-diphosphoglycerate on the tetramer-dimer equilibrium of carbon monoxide hemoglobin in dilute solution. Correlation between sedimentation and kinetic behavior.

The effect of 2,3-diphospho-D-glycerate on the sedimentation coefficient of carbon monoxide hemoglobin was correlated with the fraction of rapidly reacting hemoglobin observed subsequent to flash photolysis at 23 degrees C at pH 7.30 in buffers of 0.1 M ionic strength. Concentrations of the organic phosphate up to about 5 mM resulted in an increase in S20,w, consistent with an increase in the fraction of tetrameric hemoglobin. A decrease in rapidly reacting hemoglobin parallelled the increase in the sedimentation coefficient. Between 5 and 20 mM 2,3-diphosphoglycerate, S20,w decreased, suggesting that dissociation to dimers was enhanced. An increase in rapidly reacting hemoglobin was also observed in this concentration range. Similar sedimentation results were obtained with oxyhemoglobin at pH 7.00 and carbon monoxide hemoglobin at pH 7.06. Assuming single binding sites on each species, the dissociation constants for 2,3-diphosphoglycerate binding to tetrameric and dimeric HbCO are 0.2-0.3 mM and 2-5 mM at pH 7.30. This biphasic effect of this physiologically important organic phosphate on the state of aggregation of R state hemoglobin has not been previously reported, but it is similar to that previously noted with inositol hexaphosphate, which enhanced tetramer formation at low concentrations, while at higher concentrations it promoted hemoglobin dissociation to dimers (White, S. L. (1976) J. Biol. Chem. 251, 4763-4769; Gray, R. D. (1980) J. Biol. Chem. 255, 1812-1818).

2,3-Diphosphoglycerate↗
PubMed1987

Regulation of human platelet membrane Ca2+ transport by cAMP- and calmodulin-dependent phosphorylation.

We have examined the effects of added cAMP-dependent protein kinase and endogenous calmodulin-dependent kinase on Ca2+ transport in purified internal membranes from human platelets. Both Ca2+ uptake and Ca2+-ATPase activity were maximally stimulated about 2-fold by addition of cAMP-dependent protein kinase. Cyclic AMP-dependent protein kinase inhibitor reduced both Ca2+ uptake and Ca2+-ATPase activities at concentrations which also inhibited cAMP-dependent protein phosphorylation. In addition, concerted stimulation of Ca2+-ATPase by exogenous calmodulin and added catalytic subunit of cAMP-dependent protein kinase was observed. A 22-kDa protein was phosphorylated by both cAMP-dependent and calmodulin-dependent kinases at the same rate as stimulation of the Ca2+-ATPase. Cyclic AMP-dependent phosphorylation of the 22-kDa polypeptide was inhibited by the protein kinase inhibitor and calmodulin-dependent phosphorylation was inhibited by chlorpromazine and EGTA. These results are consistent with the hypothesis that one mode of control of Ca2+ homeostasis in platelets may be similar to the phospholamban system in cardiac muscle.

Blood Platelets↗
PubMed1987

Potential mechanisms of cytosolic calcium modulation in interferon-gamma treated U937 cells.

Interferon-gamma (IFN-gamma) at a concentration of 50 U/ml increased internal Ca2+ in the monocyte-like cell line U937 by about 100% within 3 min of addition, as determined by indo-1 fluorescence. This IFN-gamma-induced increase was reduced to 30-40% of basal (Ca2+) by the addition of diltiazem (1 microM) or incubation in Ca2+-free buffer. Ai crude membrane preparation obtained by differential centrifugation of sonicated U937 cells possessed Ca2+-ATPase activity (10 nmol ATP hydrolyzed/min/mg protein at 30 C) and sequestered Ca2+ to a level of 8 nmol/mg protein in 30 min. Addition of inositol trisphophate (IP3) (10 microM) after accumulation of Ca2+ resulted in release of a portion of the sequestered Ca2+ within 30 s, which was then resequestered. Although mitochondrial contamination was indicated by partial inhibition of Ca2+ uptake by oligomycin A, this mitochondrial inhibitor had no effect on the IP3-induced Ca2+ release. These results suggest that the increase in U937 cell cytoplasmic Ca2+ induced by IFN-gamma results from both intracellular redistribution of Ca2+, probably via polyphosphoinositide metabolism, and the entry of extracellular Ca2+ through slow channels.

Biological Transport, Active↗
PubMed1987

Zwitterionic detergent mediated interaction of purified cytochrome P-450LM4 from 5,6-benzoflavone-treated rabbits with MADPH-cytochrome P-450 reductase.

Hydroxylation of acetanilide catalyzed by purified cytochrome P-450LM4 and NADPH-cytochrome P-450 reductase was reconstituted with the zwitterionic detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS). The optimum rate of production of 4-hydroxyacetanilide was observed between 3 and 7 mM CHAPS and was about half that with 0.05 mM dilauroylglyceryl-3-phosphocholine (di-12-GPC). At higher detergent concentrations, hydroxylase activity decreased until at 15-20 mM CHAPS the system was inactive. The effect of CHAPS on the state of aggregation of P-450LM4 and on interaction between the cytochrome and P-450 reductase alone and under turnover conditions was investigated by ultracentrifugation. At 4 mM CHAPS, P-450LM4 was hexameric to heptameric (Mr 369,000). Neither reductase nor reductase plus acetanilide and NADPH altered the state of P-450LM4 aggregation, suggesting that a stable 1:1 P-450/reductase complex did not form under turnover conditions. Replacing CHAPS with 0.05 mM di-12-GPC resulted in formation of heterogeneous P-450 oligomers (Mr greater than 480,000). At CHAPS concentrations where substrate hydroxylation did not occur (15 and 22 mM), P-450LM4 was shown by sedimentation equilibrium measurements to be dimeric and monomeric, respectively. P-450 reductase was shown to reduce monomeric P-450LM4 in the presence of NADPH. Thus, the dependence of hydroxylase activity on [CHAPS] may be related to the state of aggregation of the cytochrome. An apparent correlation between P-450 aggregation state and NADPH-supported hydroxylation was also observed with phenobarbital-inducible P-450LM2 in the presence of detergents [Dean, W.L., & Gray, R.D. (1982) J. Biol. Chem. 257, 14679-14685; Wagner, S.L., Dean, W.L., & Gray, R.D. (1984) J. Biol. Chem. 259, 2390-2395].

Animals↗
PubMed1987

Biochemical basis of HLA-DR and CR3 modulation on human peripheral blood monocytes by lipopolysaccharide.

The biochemical events leading to enhanced membrane expression of HLA-DR and CR3 by human peripheral blood monocytes (MO) following exposure to bacterial lipopolysaccharide (LPS) were examined. In a previous study we demonstrated that an increase in intracellular calcium was necessary, but not sufficient, for MO to increase membrane expression of both antigens within 1 hr of addition of LPS. The present study was initiated to examine the other biochemical requirements which lead to the MO response to LPS. Enhanced expression of both antigens following addition of LPS was dependent on microfilament function, but independent of microtubule function and of protein synthesis. Inhibition of formation of cyclooxygenase or lipoxygenase metabolites of arachidonic acid had no effect on HLA-DR or CR3 modulation by LPS. A role for phosphatidylinositol metabolism was suggested by the inhibition of the MO response to LPS by dibutyryl cAMP and theophylline and by the enhanced expression of both antigens following addition of phorbol diesters. However, H-7, a putative inhibitor of protein kinase C, did not alter the MO response to LPS or phorbol diesters. These results suggest that LPS enhances expression of HLA-DR and CR3 by inducing redistribution of these antigens from an intracellular pool. The data also support a role for the generation of hydrolysis products of phosphatidylinositol, leading to calcium redistribution and activation of protein kinase C or other kinases, in the MO response to LPS.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1987

New suggestions for the physiological role of secretory protein-I.

A major endocrine secretory protein of unknown function termed secretory protein-I or, alternatively, chromogranin A, is attracting increasing attention. Because of its abundance and apparently universal presence in endocrine and neuroendocrine cells, many suggestions for a physiological role have been advanced. Recent data provide the first firm bases for some proposed functions that include a regulator of a novel protease capable of converting prohormones to hormones, a hormone precursor, and through its calcium-binding and aggregating properties, a secretory granule condensing protein.

Animals↗