Biomedical subjects

Richard M Schultz

Publications and source records attributed to Richard M Schultz.

At least 73 records · Page 4Linked to original sources

PubMed2003

A convergent approach to cryptophycin 52 analogues: synthesis and biological evaluation of a novel series of fragment a epoxides and chlorohydrins.

Cryptophycin 52 is a synthetic derivative of Cryptophycin 1, a potent antimicrotubule agent isolated from cyanobacteria. In an effort to increase the potency and water solubility of the molecule, a structure-activity relationship study (SAR) was initiated around the phenyl ring of fragment A. These Cryptophycin 52 analogues were accessed using a Wittig olefination reaction between various triphenylphosphonium salts and a key intermediate aldehyde prepared from Cryptophycin 53. Substitution on the phenyl ring of fragment A was well tolerated, and several of these analogues were equally or more potent than Cryptophycin 52 when evaluated in vitro in the CCRF-CEM leukemia cell line and in vivo against a murine pancreatic adenocarcinoma.

Adenocarcinoma↗

PubMed2003

Synthesis, structure-activity relationship, and biological studies of indolocarbazoles as potent cyclin D1-CDK4 inhibitors.

Novel substituted indolocarbazoles were synthesized, and their kinase inhibitory capability was evaluated in vitro. 6-Substituted indolocarbazoles 4 were found to be potent and selective D1/CDK4 inhibitors. 4d and 4h exhibited potent and ATP-competitive D1/CDK4 activities with IC50 values of 76 and 42 nM, respectively. Both compounds had high selectivity against the other kinases. These D1/CDK4 inhibitors inhibited tumor cell growth, arrested tumor cells at the G1 phase, and inhibited pRb phosphorylation.

Antineoplastic Agents↗

PubMed2003

Chromatin-mediated cortical granule redistribution is responsible for the formation of the cortical granule-free domain in mouse eggs.

A cortical granule-free domain (CGFD) overlies the metaphase chromatin in fully mature mouse eggs. Although a chromatin-induced localized release of cortical granules (CG) during maturation is thought to be a major contributing factor to its formation, there are indications that CG redistribution may also be involved in generating the CGFD. We performed experiments to determine the relative contributions of CG exocytosis and redistribution in generating the CGFD. We found that the CGFD-inducing activity was not specific to female germ cell chromatin and was heat stable but sensitive to DNase and protease treatment. Surprisingly, chelation of egg intracellular Ca(2+) levels did not prevent CGFD formation in response to microinjection of exogenous chromatin, suggesting that development of the CGFD was not a result of CG exocytosis. This finding was confirmed by the lack of CG exudate on the plasma membrane surface of the injected eggs and the absence of conversion of ZP2 to ZP2(f) during formation of the new CGFD. Moreover, clamping intracellular Ca(2+) did not prevent the formation of the CGFD during oocyte maturation, but did inhibit the maturation-associated release of CGs between metaphase I and II. Results of these experiments suggest that CG redistribution is the dominant factor in formation of the CGFD.

Animals↗

PubMed2003

Requirement of Cks2 for the first metaphase/anaphase transition of mammalian meiosis.

We generated mice lacking Cks2, one of two mammalian homologs of the yeast Cdk1-binding proteins, Suc1 and Cks1, and found them to be viable but sterile in both sexes. Sterility is due to failure of both male and female germ cells to progress past the first meiotic metaphase. The chromosomal events up through the end of prophase I are normal in both CKS2-/- males and females, suggesting that the phenotype is due directly to failure to enter anaphase and not a consequence of a checkpoint-mediated metaphase I arrest.

Anaphase↗

PubMed2003

Synthesis of quinolinyl/isoquinolinyl[a]pyrrolo [3,4-c] carbazoles as cyclin D1/CDK4 inhibitors.

A novel series of pyrrolo[3,4-c] carbazoles fused with a quinolinyl/isoquinolinyl moiety were synthesized and their D1/CDK4 inhibitory and antiproliferative activity were evaluated. Compound 8H, 14H-isoquinolinyl[6,5-a]-pyrrolo[3,4-c]carbazole-7,9-dione (1d) was found to be a highly potent D1/CDK4 inhibitor with an IC(50) of 69 nM. Compound 1d also inhibited tumor cell growth, arrested tumor cells in G1 phase and inhibited pRb phosphorylation.

Antineoplastic Agents↗

PubMed2003

Transgenic RNAi in mouse oocytes: a simple and fast approach to study gene function.

Double-strand RNA (dsRNA)-mediated posttranscriptional gene silencing, also known as RNA interference (RNAi), is a powerful tool to inhibit gene expression in several experimental model systems, including Arabidopsis, Caenorhabditis, and Drosophila. We previously described that the microinjection of Mos dsRNA into fully grown mouse oocytes results in the specific degradation of Mos mRNA in a time- and concentration-dependent manner. We report here a transgenic RNAi approach that is suitable to study gene function during mouse oocyte development and differentiation. The oocyte-specific Zp3 promoter was used to drive the expression of a long hairpin dsRNA ( approximately 500 bp) targeting Mos mRNA. Transgenic founder animals appeared healthy, but while males were fertile, females were not, in accordance with the known Mos null phenotype. The amount of Mos mRNA in the transgenic F(1) females was reduced by >90%, whereas there was no decrease in the nontargeted tissue plasminogen activator (Plat) mRNA. Moreover, the maturation-associated increase in mitogen-activated protein (MAP) kinase activity was not observed, and the metaphase II eggs underwent spontaneous parthenogenetic activation, thus recapitulating the Mos null phenotype. This approach provides a powerful method to study the functions of any oocyte-synthesized gene during oocyte development and early embryogenesis.

Animals↗

PubMed2003

Requirement for RNA-binding activity of MSY2 for cytoplasmic localization and retention in mouse oocytes.

MSY2, a mouse germ cell-specific Y-box protein, is implicated in the global regulation of the stability and/or translation of maternal mRNAs in the mouse oocyte. We report here that in the oocyte approximately 75% of MSY2 protein is associated with a Triton-insoluble preparation, whereas in either male germ cells or when exogenously expressed in transfected somatic cells almost all MSY2 is soluble. This retention in the oocyte, which is unlikely mediated either by microfilaments or by microtubules, markedly decreases beyond the two-cell stage of development. By microinjecting mutant MSY2-EGFP chimeric mRNAs into mouse oocytes and then assaying the expressed protein's localization by laser-scanning confocal microscopy, we find that an intact cold-shock domain (CSD), containing two RNA-binding motifs, is required to localize MSY2 to the oocyte cytoplasm. In addition, an additional basic/aromatic amino acid island (B/A), which can also interact with RNA, in the C-terminal tail domain is necessary to retain MSY2 following Triton permeabilization. Intact mRNA appeared required for this retention, since RNase A treatment of Triton-permeabilized oocytes or microinjection of RNase A into the oocyte released essentially all of the endogenous MSY2 protein. Furthermore, there is a positive correlation between the ability of the mutant MSY2-EGFP protein to remain associated with the Triton-insoluble preparations and its increased affinity for RNA, as determined by RNA electrophoretic mobility shift assays. These results suggest that binding of intact maternal mRNA by MSY2 is required for its cytoplasmic retention.

3T3 Cells↗

PubMed2003

Protamine 2 deficiency leads to sperm DNA damage and embryo death in mice.

Cytokinesis is incomplete in spermatogenic cells, and the descendants of each stem cell form a clonal syncytium. As a result, a heterozygous mutation in a gene expressed postmeiotically affects all of the haploid spermatids within a syncytium. Previously, we have found that disruption of one copy of the gene for either protamine 1 (PRM1) or protamine 2 (PRM2) in the mouse results in a reduction in the amount of the respective protein, abnormal processing of PRM2, and inability of male chimeras to transmit either the mutant or wild-type allele derived from the 129-genotype embryonic stem cells to the next generation. Although it is believed that protamines are essential for compaction of the sperm nucleus and to protect the DNA from damage, this has not been proven experimentally. To test the hypothesis that failure of chimeras to transmit the 129 genotype to offspring was due to alterations in the organization and integrity of sperm DNA, we used the single-cell DNA electrophoresis (comet) assay, ultrastructural analysis, and the intracytoplasmic sperm injection (ICSI) procedure. Comet assay demonstrated a direct correlation between the fraction of sperm with haploinsufficiency of PRM2 and the frequency of sperm with damaged DNA. Ultrastructural analysis revealed reduced compaction of the chromatin. ICSI with PRM2-deficient sperm resulted in activation of most metaphase II-arrested mouse eggs, but few were able to develop to the blastocyst stage. These findings suggest that development fails because of damage to paternal DNA and that PRM2 is crucial for maintaining the integrity of sperm chromatin.

Animals↗

PubMed2003

Maturation-associated increase in IP3 receptor type 1: role in conferring increased IP3 sensitivity and Ca2+ oscillatory behavior in mouse eggs.

Maturation of mouse oocytes is accompanied by an increase in sensitivity to inositol 1,4,5-trisphosphate (IP(3))-mediated release of intracellular calcium. To test the hypothesis that the maturation-associated 1.5- to 2.0-fold increase in the mass of the type 1 IP(3) receptor (IP(3)R-1) confers this increase in IP(3) sensitivity, we employed RNA interference to prevent this change in IP(3)R-1 protein level. Microinjection into germinal vesicle (GV)-intact oocytes of dsRNA corresponding to the IP(3)R-1 sequence resulted in a >90% reduction in the amount of maternal IP(3)R-1 mRNA and prevented the maturation-associated increase in the mass of the IP(3)R-1 protein. These injected oocytes matured to metaphase II, and there was no effect on the maturation-associated increases in p34(cdc2)/cyclin B kinase and MAP kinase activities or the global pattern of protein synthesis. IP(3)-induced cortical granule exocytosis was significantly decreased in these eggs when compared with controls previously injected with enhanced green fluorescent protein (EGFP) dsRNA. Following insemination, the IP(3)R-1 dsRNA-injected eggs displayed significantly fewer Ca(2+) transients than controls, and the duration of the first Ca(2+) transient was about half that of controls. These results support the hypothesis that the maturation-associated increase in the mass of IP(3)R-1 confers the increase in IP(3)-sensitivity that is observed following oocyte maturation and is necessary for the proper Ca(2+) oscillatory pattern following insemination.

Animals↗

PubMed2003

Acquisition of transcriptional competence in the 1-cell mouse embryo: requirement for recruitment of maternal mRNAs.

We previously suggested that the transcriptional machinery is rate-limiting for genome activation in the preimplantation mouse embryo (Aoki et al., 1997: Dev Biol 181:296-307), suggesting that genome activation requires the synthesis of some proteins following fertilization. To test this hypothesis, transcriptional activity was measured in 1-cell embryos in which protein synthesis was inhibited by cycloheximide from the time shortly after insemination. Transcription in both the male and female pronuclei, as assessed by BrUTP incorporation, was markedly inhibited when assayed 14 hr post-insemination. This result suggested that newly synthesized proteins derived from maternally recruited mRNA, and not maternally derived proteins that were post-translationally modified following fertilization/egg activation, were critical for the acquisition of transcriptional competence. To test this hypothesis, mobilization of maternally recruited mRNAs was inhibited by treating the embryos with 3'-deoxyadenosine (3'-dA), which prevents polyadenylation of mRNA. The results demonstrated that the transcriptional activity was markedly decreased in the embryos cultured with 2 mM 3'-dA, whereas 3'-deoxyguanosine had little inhibitory effect, and suggest that recruitment of maternal mRNAs is essential for acquisition of transcriptional competence.

Animals↗

PubMed2003

Potential of p38 MAP kinase inhibitors in the treatment of cancer.

The involvement of chronic inflammation in tumor development and progression is reviewed. Based on the natural history of certain diseases and epidemiology studies, a strong association has been established between particular chronic inflammatory conditions and eventual tumor appearance. Solid tumors require a stroma for their growth and recruit macrophages to synthesize essential growth and angiogenic factors that they do not have the capacity to produce. The microenvironment of the local host tissue appears to be an active participant in exchanging cytokines and enzymes with tumor cells that modify the local extracellular matrix, stimulate migration, and promote tumor angiogenesis, proliferation and survival. The role of p38 MAP kinase as a therapeutic target for treating cancer is discussed.

Angiogenesis Inhibitors↗

PubMed2003

Gene expression in mouse oocytes and preimplantation embryos: use of suppression subtractive hybridization to identify oocyte- and embryo-specific genes.

The paucity of biological material has inhibited identifying genes that are differentially expressed during mammalian oogenesis and preimplantation development. We report here the linear amplification of mRNA from small numbers of mouse oocytes and preimplantation embryos to generate amounts of sense RNA that are sufficient for suppression subtractive hybridization. The resulting oocyte-specific and 8-cell-specific cDNA libraries were partially characterized, and the known oocyte-specific ZP1, ZP2, GDF-9, BMP15, and H1(oo) genes were found in the oocyte-specific cDNA library but not in the 8-cell-specific library. Further characterization of the subtracted oocyte and 8-cell embryo cDNA libraries should furnish a trove of information regarding temporal changes in gene expression during oogenesis and preimplantation development in the mouse.

Animals↗

PubMed2003

RNAi: mammalian oocytes do it without RNA-dependent RNA polymerase.

Studies in mutant organisms deficient in RNA interference (RNAi) and related post-transcriptional gene silencing implicated a role for a single class of RNA-dependent RNA polymerases (RdRp). Nevertheless, sequence homologs to these RdRps have not been found in coelomate organisms such as Drosophila or mammals. This lack of homologous sequences does not exclude that an RdRp functions in RNAi in these organisms because an RdRp could be acquired by horizontal transfer from an RNA virus. In fact, such a sequence is found in mice (Aquarius) and we observe that it is expressed in mouse oocytes and early embryos, which exhibit RNAi. We report here that cordycepin, an inhibitor of RNA synthesis, does not prevent Mos double-strand RNA (dsRNA) to target endogenous Mos mRNA in mouse oocytes and that targeting a chimeric Mos-EGFP mRNA with dsRNA to EGFP does not reduce the endogenous Mos mRNA, but does target the chimeric mRNA. These results indicate that an RdRp is not involved in dsRNA-mediated mRNA degradation in mammalian oocytes, and possibly in mammals in general, and therefore that only homologous sequences to the dsRNA are targeted for degradation.

Animals↗

PubMed2002

p38 isoforms have opposite effects on AP-1-dependent transcription through regulation of c-Jun. The determinant roles of the isoforms in the p38 MAPK signal specificity.

p38 MAPK pathway signaling is known to participate in cell proliferation, apoptosis, and differentiation, in a manner dependent on the cellular context. The factors that determine the specific biological response in a given cell type, however, remain largely unknown. We report opposite effects of the p38 isoforms on regulation of AP-1-dependent activities by p38 activators MAPK kinase 6 (MKK6) and/or arsenite in human breast cancer cells. The p38beta isoform increases the activation of AP-1 transcriptional activities by MKK6 and/or arsenite, whereas p38gamma/p38delta inhibits or has no effect on the stimulation. The p38beta does so by increasing the levels of phosphorylated c-Jun, whereas the p38gamma and -delta isoforms may act by regulating the c-jun transcription. AP-1-dependent processes such as vitamin D receptor gene promoter activation and cellular proliferation were similarly activated by the p38beta or inhibited by the p38gamma and/or -delta isoforms. Whereas the human breast cancer cells express all four isoforms, mouse NIH 3T3 and EMT-6 cells express only some of the p38 family members, with p38beta higher in 3T3 cells but p38delta only detected in the EMT-6 line. Consistent with the positive and negative roles of p38beta and p38delta in AP-1 regulation, MKK6 stimulates AP-1-dependent transcription in NIH 3T3 but not EMT-6 cells. In support of a role of c-Jun regulation by p38 isoforms in determining AP-1 activity, the levels of endogenous c-Jun and its phosphorylated form on p38 activation are higher in NIH 3T3 cells. These results demonstrate the contrasting activities of the different p38 isoforms in transmitting the upstream signal to AP-1 and show that the expression profile of p38 isoforms determines whether the p38 signal pathway activates or inhibits AP-1-dependent processes.

3T3 Cells↗

PubMed2002

Egg-to-embryo transition is driven by differential responses to Ca(2+) oscillation number.

Ca(2+) oscillations and signaling represent a basic mechanism for controlling many cellular events. Activation of mouse eggs entrains a temporal series of Ca(2+)-dependent events that include cortical granule exocytosis, cell cycle resumption with concomitant decreases in MPF and MAP kinase activities, and recruitment of maternal mRNAs. The outcome is a switch in cellular differentiation, i.e., the conversion of the egg into the zygote. By activating mouse eggs with experimentally controlled and precisely defined Ca(2+) transients, we demonstrate that each of these events is initiated by a different number of Ca(2+) transients, while their completion requires a greater number of Ca(2+) transients than for their initiation. This combination of differential responses to the number of Ca(2+) transients provides strong evidence that a single Ca(2+) transient-driven signaling system can initiate and drive a cell into a new developmental pathway, as well as can account for the temporal sequence of cellular changes associated with early development.

Animals↗

PubMed2002

The science of ART.

The methods of gamete manipulation used in assisted reproductive technology (ART) are rapidly proliferating and in some instances outpacing the underlying science. In this review, we discuss two major advances in the ART laboratory-intracytoplasmic sperm injection and extended embryo culture before embryo transfer. We outline the rationale for these approaches, discuss results of experiments obtained from animal model systems and human preimplantation embryos that provide the scientific basis for these procedures, and point out potential concerns that have arisen from these studies.

Animals↗

PubMed2002

The p38 and JNK pathways cooperate to trans-activate vitamin D receptor via c-Jun/AP-1 and sensitize human breast cancer cells to vitamin D(3)-induced growth inhibition.

The signaling connection between mitogen-activated protein kinases(MAPKs) and nuclear steroid receptors is complex and remains mostly unexplored. Here we report that stress-activated protein kinases p38 and JNK trans-activate nuclear steroid vitamin D receptor (VDR) gene and increase vitamin D(3)-dependent growth inhibition in human breast cancer cells. Activation of p38 and JNK by an active MAPK kinase 6 stimulates VDR promoter activity independently of the ligand vitamin D(3) and estrogen receptor expression. Moreover, stimulation of the endogenous stress pathways by adenovirus-mediated delivery of recombinant MAPK kinase 6 also activates VDR and sensitizes MCF-7 cells to vitamin D(3)-dependent growth inhibition. Both the p38 and JNK MAPK pathways and the downstream transcription factor c-Jun/AP-1 are required for the VDR stimulation, as revealed by application of their dominant negatives, the specific p38 inhibitor SB203580, and site-directed mutagenesis of the AP-1 element in the VDR promoter. The essential role of the p38 and JNK stress pathways in up-regulation of VDR expression is further confirmed by using the chemical stimulator arsenite. These results establish a signaling connection between the stress MAPK pathways and steroid hormone receptor VDR expression and thereby offer new insights into regulation of cell growth by the MAPK pathways through regulation of vitamin D(3)/VDR activity.

Breast Neoplasms↗

PubMed2002

Cdc25b phosphatase is required for resumption of meiosis during oocyte maturation.

In a wide variety of animal species, oocyte maturation is arrested temporarily at prophase of meiosis I (ref. 1). Resumption of meiosis requires activation of cyclin-dependent kinase-1 (CDK1, p34cdc2), one component of maturation-promoting factor (MPF). The dual specificity phosphatases Cdc25a, Cdc25b and Cdc25c are activators of cyclin-dependent kinases; consequently, they are postulated to regulate cell-cycle progression in meiosis and mitosis as well as the DNA-damage response. We generated Cdc25b-deficient (Cdc25b-/-) mice and found that they are viable. As compared with wildtype cells, fibroblasts from Cdc25b-/- mice grew vigorously in culture and arrested normally in response to DNA damage. Female Cdc25b-/- mice were sterile, and Cdc25b-/- oocytes remained arrested at prophase with low MPF activity. Microinjection of wildtype Cdc25b mRNA into Cdc25b-/- oocytes caused activation of MPF and resumption of meiosis. Thus, Cdc25b-/- female mice are sterile because of permanent meiotic arrest resulting from the inability to activate MPF. Cdc25b is therefore essential for meiotic resumption in female mice. Mice lacking Cdc25b provide the first genetic model for studying the mechanisms regulating prophase arrest in vertebrates.

Animals↗