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Biomedical subjects

R M Schultz

Publications and source records attributed to R M Schultz.

At least 19 recordsLinked to original sources
PubMed1992

Fibroblasts transformed by different ras oncogenes show dissimilar patterns of protease gene expression and regulation.

NIH 3T3 cells transformed by different activated ras genes showed different patterns of protease gene expression, indicating the existence of least two pathways for NIH 3T3 transformation from mutated ras genes. In cells transformed by activated mammalian EJ-ras and chimeric EJ/vHa-ras, high constitutive levels of urokinase plasminogen activator (uPA) mRNA and/or phorbol-12-myristate-13-acetate (PMA) inducibility of the uPA mRNA was observed. However, PMA did not induce cathepsin L (CL) mRNA levels in these same cell lines. In contrast, NIH 3T3 cells transformed by homologous yeast RAS1Leu sequences showed low levels of uPA mRNA and a lack of PMA inducibility of uPA mRNA, but did show high constitutive levels of the mRNA for CL and/or PMA inducibility of CL mRNA expression. Based on their differences in PMA inducibility these two phenotypes are designated rasuPA+/CL- and rasCL+/uPA-, respectively. Run-on assays indicated the differences in the levels of CL and uPA mRNA with ras transformation and phorbol ester induction are due to changes in transcription rates. Based on the observation of the two ras-transformed phenotypes for protease expressions, we asked whether uPA and CL can substitute for each other in the promotion of experimental metastasis. Injection of in vitro antisense inhibited cells in nude mice showed an inhibition of lung colonization by anti-uPA only in the rasuPA+/CL- phenotype and by anti-CL only in the rasCL+/uPA- phenotype. The data thus show the existence of two distinct activated ras-transformed metastatic phenotypes induced in the same parental cell line and that uPA or CL protease expressions alternatively facilitate the metastasis of cells with one ras phenotype and not with the other.

3T3 Cells↗
PubMed1992

Zygotic gene activation in the mouse embryo: involvement of cyclic adenosine monophosphate-dependent protein kinase and appearance of an AP-1-like activity.

Protein phosphorylation catalyzed by the cyclic adenosine monophosphate (cAMP)-dependent protein kinase (PKA) is implicated in regulating zygotic gene activation in the two-cell mouse embryo (Poueymirou and Schultz; Dev Biol 133:588-599, 1989). We now provide evidence that H8, which is a PKA inhibitor, inhibits expression of an hsp70-driven beta-galactosidase reporter gene and that the concentration-dependence of this inhibition is similar to that for inhibiting expression of a stage-specific gene(s) that is a product of zygotic gene activation. We also demonstrate that neither cAMP nor serum can stimulate the expression, as detected by a histochemical assay, of a cAMP response element (CRE)- or serum response element (SRE)-driven beta-galactosidase reporter gene, respectively, in either germinal vesicle-intact oocytes or aphidicolin-arrested one-cell embryos that are chronologically at the tw-cell stage. In contrast, although 12-O-tetradecanoyl phorbol-13-acetate (TPA) does not stimulate expression of a TPA response element (TRE)-driven beta-galactosidase reporter gene in germinal vesicle-intact oocytes, it stimulates such expression in aphidicolin-arrested one-cell embryos. Moreover, TPA can stimulate the expression of either a CRE- or an SRE-driven beta-galactosidase reporter gene in such embryos. Results of these studies further implicate protein phosphorylation in regulating zygotic gene activation, along with its role in modulating enhancer function in the early mouse embryo.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1992

Changes in cAMP phosphodiesterase activity and cAMP concentration during mouse preimplantation development.

Cyclic nucleotide phosphodiesterase (PDE) activity and cAMP amounts were measured in mouse preimplantation embryos at the 1-cell, 2-cell, 8-cell/morula, and mid-blastocyst stages. PDE activity remained constant between the 1-cell and 2-cell stages. It decreased by the 8-cell stage and continued to decrease by the mid blastocyst stage to about 14% of the 1- and 2-cell values. By contrast, cAMP amounts remained essentially constant at 0.05 fmole/embryo (0.3 microM) from the 1-cell to the blastocyst stage and increased to 0.175 fmole in the fully expanded blastocyst that was close to hatching. Measurements of embryo volume indicated that intracellular volume remained essentially constant up to the blastocyst stage. The morphological changes in cell shape that accompany differentiation of the trophectoderm and that are coupled with blastocoel expansion decreased the intracellular volume. This decrease resulted in an increase in the cAMP concentration to about 0.4 microM by the mid-blastocyst stage. Previous studies indicate that either cAMP or TGF-alpha/EGF can stimulate the rate of blastocoel expansion. Although TGF-alpha/EGF can elevate cAMP levels in other cell types, TGF-alpha, at a concentration that maximally stimulates the rate of blastocoel expansion, did not elevate cAMP in blastocysts. Thus, it was unlikely that elevation of cAMP is the mechanism by which TGF-alpha stimulates the rate of blastocoel expansion.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1992

Colocalization of transforming growth factor-alpha and a functional epidermal growth factor receptor (EGFR) to the inner cell mass and preferential localization of the EGFR on the basolateral surface of the trophectoderm in the mouse blastocyst.

Results of previous studies suggested that responses of mouse blastocysts to TGF-alpha/EGF treatment are mediated by EGF receptors (EGFR) located on the apical surface of the trophectoderm (TE). We report here results of experiments using gold-labeled EGF that confirm the presence of these apically located EGFRs. In addition, immunoelectron microscopy (IEM) studies using anti-EGFR antibodies indicate that the receptor is preferentially distributed on the basolateral surface of the TE. Furthermore, the receptor is also present on the inner cell mass (ICM) and is likely to be functional, since treatment of isolated ICMs with TGF-alpha affects [35S]methionine uptake and incorporation into acid-insoluble material. IEM was also used to demonstrate that EGF, which is not synthesized by the mouse preimplantation embryo, is present in both the oviduct and the uterus. Maternally derived EGF is present in both ICM and TE cells in freshly isolated blastocysts, but is present in greatly reduced amounts following overnight culture of blastocysts in vitro. Last, IEM was also used to demonstrate that TGF-alpha is preferentially localized to the ICM and polar TE. The co-localization of TGF-alpha and functional EGFRs to the ICM and polar TE suggests potential autocrine, juxtacrine, and paracrine roles for TGF-alpha in blastocyst development.

Amino Acids↗
PubMed1992

Temporal pattern of synthesis of the mouse cortical granule protein, p75, during oocyte growth and maturation.

We previously demonstrated that a protein of M(r) 75,000 (p75) is localized to cortical granules (CGs) in mouse oocytes and eggs and is released upon activation or fertilization of eggs (K.E. Pierce, M. C. Siebert, G. S. Kopf, R. M. Schultz, and P. G. Calarco, 1990, Dev. Biol. 141, 381-392). To examine the temporal pattern of synthesis of p75 during the early stages of CG formation, growing oocytes, which were isolated from juvenile mice, were incubated for 4 hr in medium containing [35S]methionine, and radiolabeled proteins were immunoprecipitated using an antiserum that detects p75. Synthesis of p75 is detected at low levels in the smallest oocytes examined (less than 20 microns). Synthesis of p75 relative to total protein synthesis increases about 12-fold during oocyte growth from the 20-40 microns size and then remains constant throughout the remaining period of oocyte growth (40-70 microns). In the fully grown, germinal vesicle (GV)-intact oocyte (70-80 microns), immunoprecipitated p75 comprises approximately 1.5% of the total amount of radiolabeled protein. Three hours after the transfer of these oocytes to a medium that supports resumption of meiosis and GV breakdown in vitro, oocytes subjected to a 1-hr labeling pulse display a 35% decrease in the relative level of p75 synthesis. By 15 hr of maturation, p75 synthesis was reduced to 14% of that in the fully grown, GV-intact oocyte and this is similar to the level of p75 synthesis in ovulated eggs. The level of p75 synthesis following in vitro translation of total egg RNA is only 38% lower than that obtained from total oocyte RNA. In addition, synthesis of p75 is observed following in vitro translation of oocyte, but not egg, poly(A)+ RNA. These results are consistent with p75 synthesis during oocyte maturation being under translational control.

Animals↗
PubMed1992

Role of G proteins in mouse egg activation: stimulatory effects of acetylcholine on the ZP2 to ZP2f conversion and pronuclear formation in eggs expressing a functional m1 muscarinic receptor.

Sperm-mediated egg activation may be analogous to ligand-mediated signal transduction through G protein-coupled receptors. We investigated this possibility in the mouse egg by microinjecting mouse oocytes with an m1 muscarinic receptor mRNA. Following oocyte maturation in vitro, the metaphase II-arrested eggs were treated with acetylcholine and its effect was examined on zona pellucida modifications and pronuclear formation, which are end points of early and late egg activation, respectively. Treatment of these eggs with acetylcholine reveals that both the ZP2 to ZP2f conversion and pronuclear formation occur. Atropine and microinjected GDP beta S block the acetylcholine-induced ZP2 conversion, suggesting that the acetylcholine effects are mediated via a functional G protein-coupled m1 receptor. The acetylcholine-induced ZP2 conversion, however, is not inhibited by pertussis toxin under conditions in which greater than 90% of the endogenous Gi is inactivated by ADP ribosylation. The presence of a pertussis toxin-insensitive G protein, Gq, is detected by immunoblotting; this G protein could be a candidate to mediate the pertussis toxin-insensitive effects of acetylcholine. Results of these experiments are consistent with the hypothesis that receptor-mediated G protein activation may play a role in egg activation.

Acetylcholine↗
PubMed1992

Acquisition of a transcriptionally permissive state during the 1-cell stage of mouse embryogenesis.

Zygotic gene transcription initiates during the 2-cell stage of mouse embryogenesis. To learn more of the nature and timing of events leading up to transcriptional activation, we evaluated the ability of enucleated 1-cell-stage embryos to support transcription of the 2-cell-stage-specific gene(s) encoding the 70,000-Da transcription-requiring complex (TRC). Nuclei were transplanted from transcriptionally inhibited alpha-amanitin or N-[2-(methylamino)ethyl]-5-isoquinolinesulfonamide (H8)-treated 2-cell-stage embryos to either late or early enucleated 1-cell-stage recipients. Expression of the TRC gene(s) was much greater following transfer to late 1-cell than early 1-cell-stage recipients. In addition, treatment of early 1-cell-stage recipients with N6-monobutyryl cyclic AMP following transplantation of a nucleus from an H8-treated donor increased the rate of TRC synthesis to a value similar to that observed for late 1-cell-stage recipients. These results indicate that during the first cell cycle and prior to initiation of zygotic gene expression, the embryonic cytoplasm undergoes a transition from a transcriptionally nonpermissive to permissive state.

Amanitins↗
PubMed1992

Flow cytometric evaluation of the effects of leukotriene B4 receptor antagonists (LY255283 and SC-41930) on calcium mobilization and integrin expression of activated human neutrophils.

Leukotriene B4 (LTB4) is a naturally occurring eicosanoid mediator which chemoattracts and stimulates human neutrophils to an activated state. In an attempt to identify novel antiinflammatory drugs, synthetic LTB4 receptor antagonists have been developed in several laboratories. In this study, the effects of two such LTB4 receptor antagonists were examined for their influences on two elements of human neutrophil activation using flow cytometric techniques. Quantitative flow cytometric assays of human neutrophil intracellular calcium mobilization and up-regulation of integrin (CD11b/CD18) cell surface expression were developed and used to determine the potency and selectivity of compounds LY255283 and SC-41930 on these activities. Our results indicate that both compounds preferentially block these functions of LTB4-induced human neutrophil activation in a concentration dependent manner and fall in the 1-2 microM range of antagonist activity. Compound SC-41930 was approximately twice as potent as LY255283 in blocking the targeted agonist effects. Both compounds were approximately 100-fold less potent in blocking the same functions of interleukin-8-induced human neutrophil activation.

Anti-Inflammatory Agents, Non-Steroidal↗
PubMed1992

Requirements for extracellular metabolism of pulmonary surfactant: tentative identification of serine protease.

Pulmonary alveolar surfactant is secreted by the alveolar epithelium in the form of lamellar bodylike structures that evolve sequentially into tubular myelin and vesicular forms that can be separated by centrifugation. Using an in vitro procedure by which the extracellular metabolism of pulmonary surfactant can be mimicked, namely cyclic variation in surface area, we previously reported that serine protease activity, which we called "convertase," was required for the conversion of tubular myelin to the vesicular form. In the present studies we explored the biochemical requirements of this activity and sought the enzyme in alveolar products. Convertase activity has unusual requirements; in addition to being dependent on repetitive variations in surface area (cycling), it requires the presence of a high g fraction of lung secretions that is heat stable and not inhibitable by diisopropyl fluorophosphate (DFP) or alpha 1-antitrypsin, both typical serine protease inhibitors. The enzyme does not require calcium ions and has a pH optimum of 7.4. Convertase appears to be a component of surfactant itself because the ability of purified surfactant to convert to the vesicular form on cycling is impaired by pretreating it with DFP. A protein of Mr 75,000 that reacts with DFP and is heat sensitive was found in alveolar lavage, lamellar body preparations, and lung homogenate. It copurifies with lung surfactant in sucrose gradients. A similar DFP-reactive protein was observed in stable human neoplastic peripheral airway cell lines that express type II properties, suggesting that it may be a product of type II cells. We tentatively conclude that surfactant convertase is a 75,000 serine protease that is closely associated with surfactant phospholipid and that may be a product of alveolar type II cells.

Animals↗
PubMed1991

Activation of a two-cell stage-specific gene following transfer of heterologous nuclei into enucleated mouse embryos.

Zygotic gene activation occurs at the two-cell stage in the mouse embryo, resulting in the appearance of many new proteins, including a stage-specific family of related proteins of Mr 70,000. The mechanisms that regulate the stage-specific expression of these proteins were examined by transplanting nuclei from oocytes, four-cell-stage blastomeres, inner cell mass cells cultured embryonic stem cells, or differentiated endoderm-like PYS2 cells to enucleated one-cell embryos. Although none of these cell types synthesizes the 70 kDa complex, all were able to direct the synthesis of the 70 kDa complex following transplantation and overnight culture to the two-cell stage. These results suggest that the embryonic cytoplasm can exert a dominant, positive regulatory influence on a variety of heterologous nuclei that results in the transcription of a stage-specific gene. In addition, these results indicate that activation of the gene(s) coding for the 70 kDa complex is not dependent on prior programming during oogenesis and oocyte maturation, and that repression of the gene(s) coding for this complex after the two-cell stage does not involve irreversible gene inactivation.

Animals↗
PubMed1991

Stimulatory effect of okadaic acid, an inhibitor of protein phosphatases, on nuclear envelope breakdown and protein phosphorylation in mouse oocytes and one-cell embryos.

Treatment of one-cell mouse embryos with okadaic acid (OA), which is an inhibitor of protein phosphatases 1 and 2A, induces a concentration-dependent precocious nuclear envelope breakdown (NEBD) of the pronuclei; at 10 microM okadaic acid, NEBD starts to occur after 1 hr and the embryos become committed to NEBD after about 45 min. Correlated with NEBD is the conversion of a protein of Mr 32,000 (p32) to more highly phosphorylated forms. One-cell embryos cultured continuously in OA-containing medium do not cleave, whereas one-cell embryos incubated for 15-60 min prior to transfer to OA-free medium reveal a time-dependent inhibition in their ability to cleave. OA treatment of oocytes that are arrested from resuming spontaneous maturation by either a phosphodiesterase inhibitor or biologically active phorbol diester results in germinal vesicle breakdown and the maturation-associated changes in the pattern of protein phosphorylation, which include the apparent phosphorylation of p32. Results of these experiments implicate protein phosphatases in the G2 to M transition of the cell cycle in both meiotic and mitotic cells.

Animals↗
PubMed1991

Changes in temporal and spatial patterns of Gi protein expression in postimplantation mouse embryos.

We previously demonstrated the presence of GTP-binding proteins, G proteins, in the preimplantation mouse embryo (Jones and Schultz, 1990. Dev. Biol. 139, 250-262). These studies have been extended to the Day 6.5, 7.5, and 8.5 gestation embryo by employing PT-catalyzed ADP-ribosylation and immunoblotting techniques. We report here that the amount of embryonic alpha i increases from Day 6.5 to Day 7.5 of gestation, and remains at about the same level at Day 8.5. In contrast, the extent of PT-catalyzed ADP-ribosylation of Gi alpha protein(s) decreases between Days 6.5 and 7.5--this decrease is global and not restricted to a particular germ layer of the Day 7.5 embryo--and then dramatically increases by Day 8.5 of gestation. In the Day 8.5 gestation embryo, the extent of PT-catalyzed ADP-ribosylation of Gi alpha proteins increases along the anterior-posterior axis, whereas the amount of immunoreactive alpha i subunit decreases along this axis. By using a combination of PT-catalyzed ADP-ribosylation and immunoprecipitation with antisera specific for alpha i1, alpha i2, or alpha i3, we report that all three alpha i subtypes are present in the Day 8.5 gestation mouse embryo. Results of these experiments suggest that an activation of Gi proteins occurs between Days 6.5 and 7.5 of gestation in the postimplantation embryo, a time during which the embryo is gastrulating, and that a decreasing gradient of activation exists along the anterior to posterior axis in the Day 8.5 gestation embryo. Last, we report that oocytes, eggs, and preimplantation embryos possess all three subtypes of alpha i.

Amino Acid Sequence↗
PubMed1991

Regulation of hsp70 mRNA levels during oocyte maturation and zygotic gene activation in the mouse.

Protein phosphorylation catalyzed by the cAMP-dependent protein kinase is implicated in transcriptional activation of the embryonic genome in the two-cell mouse embryo, while heat shock protein (hsp70) has been identified as one of the first products of zygotic gene activation. Using reverse transcription-polymerase chain reaction we have analyzed relative changes in the amount of hsp70 mRNA during oocyte maturation and early embryogenesis. We report that the amount of hsp70 mRNA decreases after germinal vesicle breakdown, while inhibiting germinal vesicle breakdown inhibits this maturation-associated decrease. The amount of hsp70 mRNA increases between the one- and two-cell stages. This increase is inhibited by either alpha-amanitin or the cAMP-dependent protein kinase inhibitor H-8; the same concentration of H-7, which is a more potent inhibitor of protein kinase C, has little inhibitory effect on this increase in the relative amount of hsp70 mRNA. Last, addition of cycloheximide to one-cell embryos late in G2 inhibits neither cleavage to the two-cell stage nor the increase in the relative amount of hsp70 mRNA. These results strengthen the previous proposal that protein phosphorylation is involved in zygotic gene activation in the two-cell mouse embryo.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1991

Stage-specific expression of a family of proteins that are major products of zygotic gene activation in the mouse embryo.

Transcriptional activation of the embryonic genome occurs during the two-cell stage in the mouse embryo and is marked by the synthesis of a set of alpha-amanitin-sensitive proteins of Mr 73,000, 70,000, and 68,000. We have characterized these three proteins by two-dimensional gel electrophoresis of [35S]methionine radiolabeled two-cell embryos. Their isoelectric points range from 6.2 to 6.8 and their synthesis, which can constitute 5-10% of total protein synthesis, is restricted to the two-cell stage. These proteins are not heat shock proteins that have previously been reported as major products of transcriptional activation. Peptide mapping by limited proteolysis indicates that these three proteins are highly related to one another and the results of pulse-chase experiments indicate that they are likely to be degraded by the eight-cell stage. These proteins are nuclear-associated and insoluble in 2% Triton X-100/0.3 M KCl. Although these proteins share some features with somatic lamins--they exhibit solubility properties similar to somatic lamins--they do not cross-react with polyclonal antibodies to either lamins A/C or B, nor do they comigrate with somatic lamins on two-dimensional gels. Additional evidence that these proteins are not lamins is that although treatment of two-cell embryos with okadaic acid, which is an inhibitor of protein phosphatases 1 and 2A, results in precocious nuclear envelope breakdown, the proteins remain insoluble in 2% Triton X-100/0.3 M KCl.

Animals↗
PubMed1991

Effects of two leukotriene B4 (LTB4) receptor antagonists (LY255283 and SC-41930) on LTB4-induced human neutrophil adhesion and superoxide production.

Leukotriene B4 (LTB4) induces a number of functional changes in human neutrophils, including both superoxide release and CD11b/CD18 (Mo1)-mediated adherence to various substrates, such as keyhole limpet hemocyanin (KLH). These effects are both time- and concentration-dependent. Neutrophil adhesion was at least 10-fold more sensitive to the stimulatory action of LTB4 than superoxide production. Two LTB4 receptor antagonists, LY255283 (1-(5-ethyl-2-hydroxy-4-(6-methyl-6-(1H-tetrazol-5-yl)heptyloxy )- phenyl)ethanone) and the sodium salt of SC-41930 (7-[3-(4-acetyl-3-methoxy-2-propylphenoxy)-propoxy]-3,4-dihydro-8- propyl-2H- 1-benzopyran-2-carboxylic acid) were evaluated for effects on human neutrophil superoxide production and adhesion. Despite being more sensitive to LTB4-induced stimulation, neutrophil adhesion was at least 100-fold less sensitive to inhibition by LY255283 and SC-41930 than superoxide production. Both LTB4 receptor antagonists behaved similarly in these models. These compounds did not inhibit neutrophil responses induced by granulocyte/macrophage colony-stimulating factor (GM-CSF).

Adult↗