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Biomedical subjects

R M Schultz

Publications and source records attributed to R M Schultz.

At least 91 records · Page 5Linked to original sources
PubMed1995

Modulation of gene expression in the preimplantation mouse embryo by TGF-alpha and TGF-beta.

The effect of growth factors on regulating gene expression in the preimplantation mouse embryo was examined, since results of previous experiments revealed a stimulatory effect of exogenously-added growth factors on preimplantation development in vitro. Treatment of early cavitating blastocysts with either 250 pM TGF-alpha or TGF-beta results in changes in the pattern of total protein synthesis as assessed by high-resolution two-dimensional gel electrophoresis. In some cases, the synthesis of a particular polypeptide is either up- or downregulated by each growth factor, whereas in other instances the synthesis of a polypeptide is modulated by one but not the other growth factor. Use of the mRNA differential display method permitted the identification of genes whose expression is either up- or downregulated by these growth factors. Treatment of mouse blastocysts with either TGF-alpha or TGF-beta results in the increased expression of the b subunit of the F0ATPase. TGF-beta also stimulates the expression of the DNA polymerase alpha. TGF-alpha treatment results in the increase in expression of a gene homologous to the human HEPG2 cDNA, as well as in a decrease in expression of fibronectin.

Animals↗
PubMed1995

Preimplantation development of mouse embryos in KSOM: augmentation by amino acids and analysis of gene expression.

Simplex optimization has generated several media that have improved the development of mouse preimplantation embryos in vitro. One objective of this study was to compare the development of preimplantation mouse embryos in one of these computer-optimized media, KSOM, with embryos that developed in vivo, in terms of the relative abundances of specific mRNAs involved in metabolism, transcription, and cell proliferation. First, however, since studies have indicated an improvement of other simple embryo culture media by addition of amino acids, the effects of the addition of amino acids to KSOM (KSOM/AA) on preimplantation development were assessed. We find that addition of both essential and nonessential amino acids to KSOM augments development in vitro, as compared to development supported by KSOM without amino acids. This augmentation is observed starting at the blastocyst stage, and is associated with increased rate of development to the blastocyst stage, increased frequency of hatching, and increased number of cells in the blastocysts. Reverse-transcription PCR was then used to assess the relative abundance of mRNAs for actin, glyceraldehyde-3-phosphate dehydrogenase, Na+, K(+)-ATPase, Sp1, TATA box-binding protein TBP, IGF-I, IGF-II, IGF-I receptor, and IGF-II receptor in embryos that developed in vivo and in vitro using KSOM/AA. Eight out of 9 of these mRNAs were present in the 8-cell embryos and blastocysts raised in KSOM/AA in amounts that were indistinguishable from those in embryos that developed in vivo. It is concluded that KSOM/AA provides an environment in which preimplantation mouse embryos can undergo development that is quantitatively similar to that occurring in vivo.

Amino Acids↗
PubMed1995

Differential effect of activators of protein kinase C on cytoskeletal changes in mouse and hamster eggs.

Treatment of metaphase II-arrested hamster eggs with activators of protein kinase C has been reported to promote resumption of the cell cycle, second polar body emission, and pronucleus formation (G.I. Gallicano, S.M. Schwarz, R.W. McGaughey, and D.G. Capco, 1993, Dev. Biol. 156, 94-106). In contrast, we have not observed these responses in mouse eggs obtained from CF-1 mice treated with these activators. In this report, we evaluated if this difference was due to differences in the technique used for PKC stimulation in the two different laboratories or due to species differences. Metaphase II-arrested hamster or mouse eggs were treated with phorbol diesters for 5 min or with a membrane-permeable diacylglycerol for 1 hr. Treatment of hamster eggs resulted in (1) the formation of "second polar body-like structures" commencing 5 min after treatment and reaching a maximum by 20-40 min; (2) a remarkable increase in the staining of filamentous actin in the region of these polar body-like structures; and (3) the disassembly of spindle microtubules. A reduction in cdc2/cyclin B1 kinase activity, as assessed by a decrease in H1 kinase activity, as well as progression from metaphase to anaphase were not observed. Treatment of mouse eggs from either CF-1 or CD-1 mice with these activators of PKC did not result in the formation of these polar body-like structures, did not cause an increase in filamentous actin, and did not result in a reduction in histone H1 kinase activity. This treatment, however, did induce disassembly of the spindle microtubules and the formation of multiple "pronucleus-like structures" that were more discernible in eggs from CD-1 mice. We conclude that the "apparent" activation of hamster eggs by activators of PKC is due to the effect of these agents on the cytoskeleton, which gives rise to structures that appear similar to polar bodies, but without any evidence of cell cycle resumption. The different responses seen in mouse and hamster eggs are mainly due to differences in the sensitivity of the cytoskeleton to rearrangements induced by these agents.

Amino Acid Sequence↗
PubMed1995

Role of chromatin structure in zygotic gene activation in the mammalian embryo.

Changes in chromatin structure, rather than changes in the activity of the transcriptional apparatus, may underlie the timing and basis for zygotic gene activation in the mouse embryo. The transcriptional capacity of the male and female pronuclei differs and the first mitosis is associated with development of a transcriptionally repressive state such that efficient gene expression requires a functional enhancer. This repressive state is also relieved by increasing the level of histone H4 hyperacetylation. Zygotic gene activation is also associated with the transient enrichment of chromatin containing hyperacetylated histone H4 and RNA polymerase II at the nuclear periphery. Depletion of maternally-derived histones as the signal for zygotic gene activation is explored.

Animals↗
PubMed1995

Composition of the human zona pellucida and modifications following fertilization.

The composition of individual human zonae pellucidae and modifications to this extracellular coat both before and after fertilization were analysed using a rapid, sensitive, non-radioactive biotinylation- or lectin-based detection system; these assays use commercially available reagents and can be performed on fragments of individual zonae pellucidae. The zona pellucida from unfertilized eggs is composed of three glycoprotein species designated as huZP1, huZP2 and huZP3. Under non-reducing conditions, the molecular weights of these proteins are approximately 150 kDa, approximately 100 kDa, and approximately 55-65 kDa respectively. Following fertilization, huZP1 was not detected under either non-reducing or reducing conditions. In contrast, after fertilization huZP2 was detected under non-reducing conditions, but not under reducing conditions. The ability to detect pre- and post-fertilization changes in a single human zona pellucida is discussed in relation to its value in assessing deficiencies in clinical and laboratory protocols used for in-vitro fertilization.

Adult↗
PubMed1995

Potential role of mitogen-activated protein kinase in pronuclear envelope assembly and disassembly following fertilization of mouse eggs.

Changes in the activities of the p34cdc2/cyclin B complex and mitogen-activated protein (MAP) kinase were analyzed after insemination of mouse eggs in vitro. Whereas histone H1 kinase activity (p34cdc2/cyclin B) fell to negligible levels by 90 min postinsemination, a decrease to negligible levels of myelin basic protein kinase activity (i.e., MAP kinase) was not observed until about 7 h postinsemination. The decrease in MAP kinase activity appeared to be linked to the prior decline in p34cdc2/cyclin B kinase activity, since inhibiting the fertilization-induced destruction of cyclin B by treating eggs with the microtubule inhibitor nocodazole prevented the decrease in each of these protein kinases; an intact spindle is required for cyclin destruction. Moreover, experimental elevation of MAP kinase activity by okadaic acid treatment under conditions that maintain negligible levels of p34cdc2/cyclin B kinase activity suggested that MAP kinase could be involved in pronuclear envelope dynamics. Specifically, preventing the fertilization-induced decrease in MAP kinase activity was correlated with inhibiting pronucleus formation, and elevating MAP kinase activity subsequent to pronucleus formation resulted in precocious pronuclear envelope breakdown prior to entry into M phase.

Amino Acid Sequence↗
PubMed1995

Regulation of mouse egg activation: presence of ryanodine receptors and effects of microinjected ryanodine and cyclic ADP ribose on uninseminated and inseminated eggs.

Sperm-induced activation of mammalian eggs is associated with a transient increase in Ca2+ concentrations thought to be derived from inositol 1,4,5-trisphosphate-sensitive and -insensitive intracellular stores. Whereas the importance of inositol 1,4,5-trisphosphate-sensitive Ca2+ stores has been evaluated, the identity and role of inositol 1,4,5-trisphosphate-insensitive stores are poorly understood. To explore the role of the ryanodine-sensitive Ca2+ store, we first used reverse transcription-polymerase chain reaction to identify transcripts of the ryanodine receptor in eggs and determined that transcripts for the type 2 and 3 receptor were present. Immunoprecipitation of radioiodinated egg extracts with an antibody that recognizes both type 2 and 3 receptors detected specifically a band of Mr = 520,000. Immunolocalization of the receptor(s) using laser-scanning confocal microscopy revealed that the receptor(s) was uniformly distributed in the cortex of the germinal vesicle-intact oocyte, but became asymmetrically localized to the cortex in a region apposed to the meiotic spindle in the metaphase II-arrested egg; this asymmetrical localization developed by metaphase I. The role of the ryanodine receptor in mouse egg activation was examined by determining the effects of microinjected ryanodine or cyclic ADP ribose on endpoints of egg activation in either uninseminated or inseminated eggs. Ryanodine induced the conversion of the zona pellucida glycoprotein ZP2 to its postfertilization form ZP2f in a biphasic concentration-dependent manner; nanomolar concentrations stimulated this conversion, whereas micromolar concentrations had no stimulatory effect. Cyclic ADP ribose also promoted the ZP2 conversion, but with a hyperbolic concentration dependence. Neither of these compounds induced cell cycle resumption. Inhibiting the inositol 1,4,5-trisphosphate-sensitive Ca2+ store did not inhibit the ryanodine-induced ZP2 conversion and, reciprocally, inhibiting the ryanodine-sensitive Ca2+ store did not inhibit the inositol 1,4,5-trisphosphate-induced ZP2 conversion. Last, treatment of eggs under conditions that would block the release of Ca2+ from the ryanodine-sensitive store had no effect on any event of egg activation following fertilization. Results of these experiments suggest that although ryanodine receptors are present and functional, release of Ca2+ from this store is not essential for sperm-induced egg activation.

Adenosine Diphosphate Ribose↗
PubMed1995

Temporally restricted spatial localization of acetylated isoforms of histone H4 and RNA polymerase II in the 2-cell mouse embryo.

Using immunofluorescent labeling and laser-scanning confocal microscopy, we show that isoforms of histone H4 acetylated on lysine 5, 8 and/or 12 (H4.Ac5-12), as well as RNA polymerase II, become enriched at the nuclear periphery around the time of zygotic gene activation, i.e., the 2-cell stage, in the preimplantation mouse embryo. In contrast, DNA and H4 acetylated on lysine 16 are uniformly distributed throughout the cytoplasm. Culture of embryos with inhibitors of histone deacetylase trichostatin A and trapoxin results in an increase in the (1) amount of acetylated histone H4 detected by immunoblotting, (2) intensity and sharpness of the peripheral staining for H4.Ac5-12, and (3) relative rate of synthesis of proteins that are markers for zygotic gene activation. The enhanced staining for H4.Ac5-12 at the nuclear periphery seems to require DNA replication, but appears independent of cytokinesis or transcription, since its development is inhibited by aphidicolin but not by either cytochalasin D or alpha-amanitin. Lastly, the restricted localization of H4.Ac 5-12 is not observed in the 4-cell embryo or at later stages of preimplantation development. These results suggest that changes in chromatin structure underlie, at least in part, zygotic gene activation in the mouse.

Acetylation↗
PubMed1995

Mouse sperm-egg plasma membrane interactions: analysis of roles of egg integrins and the mouse sperm homologue of PH-30 (fertilin) beta.

The guinea pig sperm protein, PH-30 (also known as fertilin), is postulated to participate in the interaction between the sperm and egg plasma membranes. The beta subunit of guinea pig PH-30 (gpPH-30 beta) contains a domain with homology to disintegrins, snake venom proteins that bind to integrins via an integrin-binding domain containing the tripeptide RGD. This raises the question of whether an egg integrin serves as a receptor for PH-30. Although mouse eggs express integrin subunits, their role in mouse fertilization is unresolved. Therefore, we examined fertilization for two different hallmarks of integrin function, namely, dependence of ligand binding on divalent cations and the ability to inhibit ligand binding with RGD peptides. We demonstrate that sperm binding to zona pellucida-free eggs is supported by Ca2+, Mg2+, or Mn2+. Ca2+ was necessary and sufficient for sperm-egg fusion, with 2.5 mM Ca2+ being the most effective concentration. In addition, fertilization could be partially inhibited with various RGD peptides, which caused a decrease in sperm-egg fusion by 30-58%. This partial inhibition of fusion with RGD peptides prompted the cloning of the mouse homologue of gpPH-30 beta (hereafter referred to as mPH-30 beta) to determine if it possessed the tripeptide RGD or a different amino acid sequence in its disintegrin domain. mPH-30 beta, which is expressed during meiotic and post-meiotic phases of spermatogenesis, shares significant similarities to gpPH-30 beta throughout the length of the molecule, from the signal sequence to the cytoplasmic tail. The full-length deduced amino acid sequence of mPH-30 beta. The disintegrin domain of mPH-30 beta has the tripeptide QDE (instead of RGD) in its cell recognition region. Peptides containing this QDE sequence decrease the binding and fusion of sperm with zona pellucida-free eggs by approximately 70%, suggesting that the disintegrin domain of mPH-30 beta participates in the interaction between sperm and egg membranes.

ADAM Proteins↗
PubMed1995

Role of membrane-associated folate binding protein in the cytotoxicity of antifolates in KB, IGROV1, and L1210A cells.

Based on differential levels of membrane-associated folate binding protein (mFBP) expression, murine L1210 leukemia, human KB epidermoid carcinoma, and human IGROV1 ovarian carcinoma cells maintained under low (physiological) folate conditions (2 nM folinic acid) were used as model systems to investigate the potential role of mFBP in antifolate transport. In addition, L1210 parental cells were compared to a subline, L1210A, expressing high levels of mFBP and defective reduced folate carrier. Antifolates for which KB-derived mFBP has high affinity (5, 10-dideazatetrahydrofolic acid [DDATHF] and homo-DDATHF [0.24 and 0.78 respectively relative to folic acid]) and low affinity (methotrexate [0.002]) were chosen for this study. Protection against DDATHF/homo-DDATHF induced cytotoxicity was achieved preferentially by folic acid compared to folinic acid in IGROV1 and L1210A cells. In IGROV1 cells, cytotoxicity IC50s were increased 18- and 5.5-fold for DDATHF and homo-DDATHF respectively by 20 nM folic acid. Moreover, greater protection was observed in L1210A cells, where IC50s were increased 354- and 80-fold for these same compounds by 20 nM folic acid. Similar protection was not observed in KB cells, suggesting that KB mFBP was not functional in DDATHF transport. Although mFBP expression may be an important determinant in the cytotoxicity of antifolates for certain tumor cells, our data demonstrate a lack of correlation between levels of mFBP and function of mFBP for DDATHF transport in the models studied.

Animals↗
PubMed1995

Substrate specificity of mammalian folylpolyglutamate synthetase for 5,10-dideazatetrahydrofolate analogs.

The metabolism of 5,10-dideazatetrahydrofolate (DDATHF [lometrexol]) to polyglutamate derivatives by folylpoly-gamma-glutamate synthetase (FPGS) plays a central role in the activity of this compound as an antineoplastic agent. The availability of a series of DDATHF derivatives differing in structure throughout the molecule has allowed a study of the structural requirements for substrate activity with mouse liver and hog liver FPGS. Kinetics of the polyglutamation reaction in vitro have been related to the potency of these compounds as inhibitors of the growth of human CEM leukemic cells. The structure-activity relationships for enzyme from both sources were nearly identical. FPGS from both species showed a broad acceptance for structural changes in the pyridopyrimidine ring, in the phenyl group, and in the intermediate bridge region, with structural changes in these regions being reflected in changes in Km for FPGS but much more modest alterations in Vmax. The data suggested that the phenyl ring was not contributing to any pi-pi hydrophobic interactions. It appeared to function primarily in maintaining a favorable distance between the pyridopyrimidine ring and the glutamate side chain. The lowest Km values were found for DDATHF analogs in which there were small alterations at the 10 position, e.g., 5-deazatetrahydrofolate, 10-methyl-DDATHF, and 10-formyl-5-deazatetrahydrofolate; the first-order rate constants for these substrates were the highest in this series, an indication of the efficiency of polyglutamation at low substrate concentrations. After correction for the intrinsic inhibitory activity of the parent DDATHF analog as an inhibitor of the target enzyme, the first-order rate constants for FPGS were found to be predictive of the potency of tumor cell growth inhibition for most of the compounds in this structural series.

Acyltransferases↗
PubMed1994

Impact of dietary folic acid on reduced folates in mouse plasma and tissues. Relationship to dideazatetrahydrofolate sensitivity.

To investigate the role of dietary folic acid in dideazatetrahydrofolate (DDATHF) sensitivity, reduced folates were estimated in plasma and tissue of mice following dietary depletion and repletion. Previous studies showed that DDATHF, a new folate antagonist targeted against glycinamide ribonucleotide transformylase, produced unexpectedly severe toxicity in humans compared with mice. However, toxicity in the animal model also became pronounced upon the removal of folic acid from the diet. Further, modest dietary restoration of folic acid in the drinking water showed that toxicity could be alleviated while antitumor activity was maintained. To investigate the role of dietary folic acid levels on tissue folates in this system, all the natural reduced folates were evaluated by a ternary complex based assay in mice placed on folic acid deplete and replete diets. After 2 weeks on a folic acid deplete diet, total plasma folate had decreased by 85%, whereas red blood cell, liver, and intestinal folate fell by only 50%. Repletion of folic acid in the drinking water at a low level (0.0003%) caused partial restoration of reduced folates, while a higher repletion level (0.003%) resulted in restoration to control levels or above. Administration of folic acid and leucovorin by oral gavage to DDATHF-treated mice resulted in elevation of tissue folates in mice maintained on folic acid deplete and replete diets. Relatively high levels of folic acid were present in plasma following oral gavage of folic acid, while essentially no [S]5-formyltetrahydrofolate was observed after leucovorin. Reduced folate pools in a subcutaneously implanted mouse mammary adenocarcinoma responded more extensively to dietary folic acid depletion than folate pools in liver. Likewise, these pools were more sensitive to restoration by folic acid or leucovorin. This greater reduced folate response of tumor versus normal tissue, if confirmed in other systems, suggests a possible basis for selective antitumor activity.

Animals↗
PubMed1994

Analysis of gene expression in the preimplantation mouse embryo: use of mRNA differential display.

The analysis of differential gene expression during preimplantation embryogenesis has been hindered by the paucity of biological material. We report modifications of the recently described mRNA differential display method (Liang, P. & Pardee, A. B. (1992) Science 257, 967-971) to analyze differential gene expression during mouse preimplantation development. The method detects the appropriate changes in the temporal pattern of expression of an amplicon that by DNA sequence analysis is the cytokeratin endo A, a gene whose temporal pattern of expression has been previously determined by S1 nuclease digestion. In addition, this method identifies amplicons that likely represent genes (i) that encode maternal mRNAs, (ii) that are products of early and late zygotic gene activation, (iii) whose expression is greatest during the eight-cell stage (i.e., expressed in a stage-specific manner), and (iv) whose expression is greatest in the blastocyst. In addition to endo A, sequence analysis of these amplicons reveals that an amplicon that displays a temporal pattern of expression consistent with it being a maternal mRNA is the alpha subunit of the mitochondrial F1 ATP synthase.

Animals↗
PubMed1994

A novel class of monoglutamated antifolates exhibits tight-binding inhibition of human glycinamide ribonucleotide formyltransferase and potent activity against solid tumors.

Tight-binding inhibition of recombinant human monofunctional glycinamide ribonucleotide formyltransferase by Lometrexol (6R-5,10-dideazatetrahydrofolate) requires polyglutamation. LY254155 and LY222306 differ from 5,10-dideazatetrahydrofolate in the replacement of the 1',4'- phenylene moiety by a 2',5'-thiophene and a 2',5'-furan, respectively. Compared to Lometrexol, the thiophene and furan analogues had 25- and 75-fold greater inhibitory potencies against human monofunctional glycinamides ribonucleotide formyltransferase (Ki = 2.1 and 0.77 nM, respectively). The binding affinities of the thiophene and furan analogues for membrane folate-binding protein from human KB cells were 6- and 350-fold weaker than Lometrexol, respectively. Both the thiophene analogue and 5,10-dideazatetrahydrofolate inhibited the in vivo growth of murine 6C3HED lymphosarcoma, murine C3H mammary carcinoma, and human xenograft HXGC3, HC1, and VRC5 colon carcinomas by 95-100%. The thiophene analogue was efficacious against human xenograft PANC-1, a pancreatic carcinoma which was completely resistant to 5,10- dideazatetrahydrofolate. These novel antifolates represent the first monoglutamated tight-binding inhibitors of glycinamide ribonucleotide formyltransferase. By eliminating the need for polyglutamation, this class of antifolates may have clinical activity in the treatment of solid tumors expressing low levels of folylpolyglutamate synthetase or tumors resistant to antifolate therapy due to increased gamma-glutamyl hydrolase activity.

Acyltransferases↗
PubMed1994

Igf2r and Igf2 gene expression in androgenetic, gynogenetic, and parthenogenetic preimplantation mouse embryos: absence of regulation by genomic imprinting.

Genomic imprinting in mammals is believed to result from modifications to chromosomes during gametogenesis that inactivate the paternal or maternal allele. The genes encoding the insulin-like growth factor type 2 (Igf2) and its receptor (Igf2r) are reciprocally imprinted and expressed from the paternal and maternal genomes, respectively, in the fetal and adult mouse. We find that both genes are expressed in androgenetic, gynogenetic, and parthenogenetic preimplantation mouse embryos. These results indicate that inactivation of imprinted genes occurs postfertilization (most likely postimplantation) and that genomic imprinting and gene inactivation are separate processes. We propose that imprinting marks the chromosome so that regulatory factors expressed in cells at later times can recognize the imprint and selectively inactivate the maternal or paternal allele. For these genes, this finding invalidates models of genomic imprinting that require them to be inactive from the time of fertilization.

Alleles↗
PubMed1994

Temporal pattern of IGF-I expression during mouse preimplantation embryogenesis.

Although mouse preimplantation embryos express transcripts for the insulin-like growth factor-I receptor (IGF-IR), IGF-I transcripts were not detected by reverse transcription-PCR (RT-PCR) at any stage of preimplantation development in a previous study (Rappolee et al., Genes Dev 6:939-952, 1992). We report that IGF-I transcripts are detected in the preimplantation embryo by RT-PCR and describe the temporal pattern of expression of this transcript from the oocyte to blastocyst stages; the level declines from the oocyte to 8-cell embryo and then increases from the 8-cell to blastocyst stages. An explanation is offered to account for the differences in detecting the IGF-I transcript, and the results are discussed in the context of an autocrine/paracrine IGF-I circuit in the preimplantation mouse embryo.

Animals↗