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  • 1
    ISSN: 1040-452X
    Keywords: Mouse ; Denuded oocytes ; Purines ; Germinal vesicle breakdown ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: The potential action of purines, such as hypoxanthine and adenosine, in meiotic arrest was examined using denuded mouse oocytes. The spontaneous meiotic maturation of denuded oocytes was significantly inhibited by hypoxanthine and/or adenosine in a dose-dependent manner. Germinal vesicle breakdown (GVBD) was inhibited even at a low concentration (1 nM) of hypoxanthine, when hypoxanthine was microinjected into the cytoplasm of denuded oocytes. This inhibitory action was potentiated by co-injection with allopurinol, a metabolic blocker of hypoxanthine that can block a metabolic pathway to uric acid. By contrast, a microinjection of adenosine was no longer effective in inhibiting GVBD. Inhibitory action of purines in meiotic maturation was correlated with sustaining intracellular cAMP levels. GVBD was resumed by econazole, one of the nitroimidazole derivatives which act as inhibitors of catalytic subunit of adenylate cyclase. This compound was effective in counteracting the effect of adenosine, but not the action of 3-isobutyl-1-methylxanthine (IBMX) on GVBD, indicating that adenosine is probably exerted at the level of oocyte plasmalemma. These data suggest that the inhibitory action of hypoxanthine and adenosine in oocyte meiotic maturation may be involved in the regulation of cAMP metabolism in a differential manner.
    Additional Material: 6 Ill.
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  • 2
    ISSN: 1040-452X
    Keywords: Thymidine kinase ; Preimplantation mouse embryos ; Embryonic gene expression ; Cell cycle ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Thymidine kinase (TK) activity was examined during the development of preimplantation mouse embryos. TK activity was increased approximately 20-fold from day 2 embryos (2-cell) to day 5 embryos (late blastocyst). TK activity did not change along with the progression into S-phase of the first and the second cell cycles but increased sharply at S-phase of the third cell cycle. Analysis of TK mRNA with a reverse transcription-polymerase chain reaction (RT-PCR) method showed that the level of TK mRNA was low in ovulated eggs and 1-cell embryos and was hardly detectable in day 2 embryo (2-cell), but sharply increased in day 3 embryos (mixture of 5- to 8-cell and morula). The functional role of 5′-flanking sequence of TK gene was also investigated in preimplantation embryos after microinjection with the DNA construct of 5′-flanking sequence of TK (2.4 kb) linked to bacterial lacZ gene (TK2.5lacZ) into the pronucleus of 1-cell and subsequently by histochemical staining with X-gal. β-Galactosidase activity was first detected in day 3 embryos (8-cell), and 30% of embryos were stained with X-gal in day 4 and day 5 embryos, respectively. These results show that an increase in TK activity occurred after 2-cell stage, and this increase was primarily due to the embryonic activation of TK gene expression. Also, it appears that the 5′-flanking sequence of TK may directly regulate the TK gene expression at the transcriptional level during preimplantation murine development. © 1994 Wiley-Liss, Inc.
    Additional Material: 6 Ill.
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  • 3
    Electronic Resource
    Electronic Resource
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 38 (1988), S. 213-227 
    ISSN: 0730-2312
    Keywords: recessive oncogene ; cancer genetics ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: The retinoblastoma susceptibility (RB) gene is unique among other cloned cancer genes because its causal role in a human cancer, retinoblastoma, was established by classical genetic methods before its isolation. Earlier hypotheses and experimental data suggested that inactivation of a gene in chromosome band 13q14 resulted in retinoblastoma formation. A gene in this region was identified as the RB gene on the basis of mutations found specifically in retinoblastoma tumors; however, its proposed biological activity in suppressing neoplasia has yet to be demonstrated. The RB gene product was identified as a nuclear phosphoprotein of 110 kD associated with DNA binding activity, suggesting that the RB protein may regulate other genes. Probes for the RB gene and gene product will be useful for genetic diagnosis of retinoblastoma susceptibility in affected families; for direct detection of mutant RB alleles; and, potentially, for genetic diagnosis of susceptibility to osteosarcoma and other tumors tentatively linked to RB-gene dysfunction. Continued study of the RB gene should yield further insight into mechanisms of oncogenesis, development, and gene regulation.
    Additional Material: 3 Ill.
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  • 4
    ISSN: 0730-2312
    Keywords: human hepatoma ; dedifferentiation/progression ; PDPK ; overexpression ; kinase FA/GSK-3α ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Computer analysis of protein phosphorylation sites sequence revealed that transcriptional factors and viral oncoproteins are prime targets for regulation of proline-directed protein phosphorylation, suggesting an association of the proline-directed protein kinase (PDPK) family with neoplastic transformation and tumorigenesis. In this report, an immunoprecipitate activity assay of protein kinase FA/glycogen synthase kinase-3α (kinase FA/GSK-3α) (a member of PDPK family) has been optimized for human hepatoma and used to demonstrate for the first time significantly increased (P 〈 0.01) activity in poorly differentiated SK-Hep-1 hepatoma (24.2 ± 2.8 units/mg) and moderately differentiated Mahlavu hepatoma (14.5 ± 2.2 units/mg) when compared to well differentiated Hep 3B hepatoma (8.0 ± 2.4 units/mg). Immunoblotting analysis revealed that increased activity of kinase FA/GSK-3α is due to overexpression of the protein. Elevated kinase FA/GSK-3α expression in human hepatoma biopsies relative to normal liver tissue was found to be even more profound. This kinase appeared to be ∼fivefold overexpressed in well differentiated hepatoma and ∼13-fold overexpressed in poorly differentiated hepatoma when compared to normal liver tissue. Taken together, the results provide initial evidence that overexpression of kinase FA/GSK-3α is involved in human hepatoma dedifferentiation/progression. Since kinase FA/GSK-3α is a PDPK, the results further support a potential role of this kinase in human liver tumorigenesis, especially in its dedifferentiation/progression. © 1996 Wiley-Liss, Inc.
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  • 5
    ISSN: 0730-2312
    Keywords: retinoic acid (RA) ; cervical intraepithelial neoplasia (CIN) ; HPV ; dysplasia ; colposcopic examination ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Retinoids, a family of molecules capable of profound impact on many biological functions, have antiproliferative, differentiative, and immunomodulatory properties. The present study assessed the effect of 13-cis- retinoic acid (13-CRA) treatment in 13 chronic cervicitis and 52 cervical intraepithelial neoplasia patients. We examined low- and high-risk human papilloma virus titer (using the hybrid capture method) and made a colposcopic and cervicographic examination before and after treatment with 13-CRA at 1 mg/kg for 4 to 12 weeks. Patients were between 27 and 64 years, the average age being 36.6 years. Histology revealed chronic cervicitis in 13 cases, mild dysplasia in 18 cases, moderate dysplasia in 18 cases, and severe dysplasia in 16 cases, totaling 65 cases. The expression rate of high-risk human papilloma virus (HPV 16, 18) was 9 of 13 cases (69%) in chronic cervicitis, 7 of 18 cases (38%) in mild dysplasia, 9 of 18 cases (50%) in moderate dysplasia, and 12 of 16 cases (75%) in severe dysplasia, with the overall expression rate being 37 of 65 cases (57%). Following 13-CRA treatment, decreases in high-risk titer were observed in 6 of 9 cases (66%) of chronic cervicitis, 4 of 11 cases (36%) of mild dysplasia, 7 of 9 cases (77%) of moderate dysplasia, and 8 of 12 cases (75%) of severe dysplasia. Overall, HPV titer decreased in 25 of 41 cases (61%). Minimal changes were found in colposcopic and cervicographic observations during the study. In summary, high-risk HPV titer decreased after treatment with 13-CRA in the majority of patients with cervical intraepithelial neoplasia. This study supports the potential of retinoids to interrupt multi-step carcinogenesis, possibly by down-regulation of gene products (E6, E7) produced by HPV infection. J. Cell. Biochem. Suppls. 28/29:133-139. © 1998 Wiley-Liss, Inc.
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  • 6
    ISSN: 0730-2312
    Keywords: mRNA export ; cell cycle ; gene transfection ; cultured mammalian cells ; hnRNP L ; nuclear transport ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: The pre-mRNA processing enhancer (PPE) element is an RNA sequence element derived from the intronless HSV-TK gene. Insertion of the element into the highly intron-dependent human β-globin gene leads to efficient expression in the absence of splicing. We have analyzed the effect of the PPE element on the expression of mouse thymidylate synthase (TS) minigenes. We have previously shown that the expression of intronless TS minigenes is moderately (up to 20-fold) stimulated by the inclusion of introns. Furthermore, S phase-specific expression of TS minigenes in growth-stimulated cells depends on the presence of a spliceable intron as well as the TS promoter. The goal of our study was to determine if the PPE element would overcome the dependence on introns for efficient expression and for S phase-specific expression of transfected TS minigenes. We found that insertion of the PPE element into an intronless TS minigene partially overcame intron dependence. However, the increase in expression was much less than that observed for the intronless β-globin gene. We also found that intronless TS or HSV-TK genes that contained the PPE element and that were driven by the TS promoter were expressed at a constant level in serum-stimulated cells. However, when an intron was included in these genes, they were expressed in an S phase-specific manner. Thus the PPE element was not able to overcome the dependence on introns for S phase-specific expression of TS minigenes. J. Cell. Biochem. 69:104-116, 1998. © 1998 Wiley-Liss, Inc.
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  • 7
    ISSN: 0003-276X
    Keywords: Rat ; DHEA ; Ovarian follicular cyst ; Ultrastructure ; Androgens ; Estrogens ; Prolactin ; Radioimmunoassay ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: Immature 27-day-old female Sprague-Dawley rats were administered daily subcutaneous injections of dehydroepiandrosterone (DHEA, 5 mg/100 g BW) to induce the formation of ovarian follicular cysts. Groups of rats were killed on days 0, 10, 15, 20, 25, and 30. Ovaries from each group of rats were processed for light and electron microscopy and for follicular or cystic fluid hormone analysis. Normal antral follicle fluid, PMSG-treated preovulatory follicular fluid, and cystic fluids were analyzed for progesterone (P), estrone (E1), estradiol (E2), testosterone (T), Δ4-androstenedione (Δ4-A), 5α-dihydrotestosterone (DHT), luteinizing hormone (LH), follicle stimulating hormone (FSH), and prolactin (PRL).DHEA induced anovulation, acyclicity, and the formation of follicular cysts. In certain antral follicles, there was a dramatic increase in the quantities of smooth endoplasmic reticulum (SER) in the granulosa cells and many mitochondria had tubular cristae. Further depletion of granulosa cell number was associated with intense blebbing of the cytoplasm into the follicle antrum. Formation of the ovarian follicular cyst was completed when the entire cyst was lined by a single layer of transformed granulosa cells in contact via adhering, gap, and tight junctions. These cells had little cytoplasm, mitochondria with lamellar cristae, vast basal and apical bands of microfilaments, and an extensive array of smooth-surfaced endocytotic invaginations on the basal plasma membrane. These endocytotic pits may subsequently form smooth-surfaced vesicles and thereby serve as one mechanism for moving fluid from the ovarian interstitium into the cyst. Theca interna cells were rarely observed in the peripheral regions of the cyst. Abundant smooth muscle cells were located beneath the basement membrane of the epithelial cells comprising the cyst wall. These acquired morphological and physiological features may ensure persistence of the ovarian cyst and thus potentiate a chronic pathological condition.In this study it was also shown that progesterone, estrone, and estradiol as well as androgen concentration increased in the follicle after PMSG treatment. With DHEA treatment, the follicular cystic fluid concentrations of these steroids progressively increased to extremely high levels concurrent with the development of the follicular cysts. With respect to pituitary protein hormones, there was no appreciable difference in FSH after PMSG treatment, while there was a decrease in LH and an increase in PRL.The information reported in this and a previous study (Lee et al., Anat. Rec., 231:185-192, 1991) suggests that the ovarian cystic condition that developed after DHEA treatment in rats is associated with elevated levels of circulating serum prolactin and androgens, and extremely high concentrations of steroids as well as FSH and LH in the follicular/cystic fluid.© Willey-Liss, Inc.
    Additional Material: 26 Ill.
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  • 8
    ISSN: 0730-2312
    Keywords: heat-shock proteins ; stress response ; vimentin ; intermediate filament ; withangulatin A ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Withangulatin A induced cell rounding up and the morphological alteration resulted from the reorganization of all of the major cytoskeletal components, i.e., vimentin, tubulin, and actin, as revealed by immunofluorescence techniques. When the withangulatin A-treated cells changed to a round-up morphology, vimentin intermediate filaments were found to be collapsed and clustered around the nucleus. The alteration was accompanied by characteristic changes of vimentin molecules, including augmentation of phosphorylation, retardation of electrophoretic mobility, and decrease in detergent extractability. The levels of vimentin phosphorylation were augmented by 2.5- and 1.8-fold in cells incubated with 50 μM withangulatin A for 1 and 3 h, respectively. The electrophoretic mobility of vimentin was partially retarded in cells treated with withangulatin A for 1 h at 10 μM and a completely upshift mobility was observed after 5 h treatment at 50 μM. In addition, vimentin molecules became less extractable by nonident P-40 after the cells were treated with withangulatin A and this effect was dose dependent. The decrease in solubility of vimentin was accompanied by the redistribution of HSP72 into the detergent nonextractable fraction and these two events were well correlated. Our results suggest that withangulatin A induced the modification of vimentin, which resulted in the alteration of cell morphology and redistribution of intracellular HSP72, an event that may play an important role in the induction of heat-shock response.
    Additional Material: 9 Ill.
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  • 9
    ISSN: 0003-276X
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: A newly established cell line of a granulocytosis/hypercalcemiainducing murine mammary carcinoma (CE mammary carcinoma) grown in serum-free culture medium secretes factors that stimulate proliferation of granulocytes and embryonal bone cells. These cultured cells retain the ability to produce granulocytosis and hypercalcemia when they are transplanted back into mice. In culture these cells form clusters of organized cells. Studies by scanning, transmission, and freeze-fracture electron microscope techniques reveal that these in vitro tumor cells retain the structural epithelial characteristics of mammary epithelia. They maintain cellular polarity, microvilli, and complete tight junctions. Both the in vivo and in vitro tumor cells produce viral particles with the ultrastructural features of murine mammary tumor viruses. In both in vivo and in vitro conditions, A-type particles are present intracellularly. B viral particles are present predominantly in the intercellular spaces. Since the structural characteristics of the cultured tumor cells are consistent with the features of mammary adenocarcinomas, this is a prime culture system for studying the tumor-derived soluble factors.
    Additional Material: 7 Ill.
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  • 10
    ISSN: 0003-276X
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: This investigation was designed to study ovarian and hormonal changes in the after treatment with dehydroeplandrosterone (DHEA). We identified a heterogeneous experimental group of animals with respect to ovarian histology: group I, corpora lutea (Cls) + cysts; group II, CLs + no cysts; group III, no CLs + cysts; group IV, no CLs + no cysts. Histological sections of these ovaries showed healthy and atretic follicles in different stages of cytomorphosis and degeneration. The aforementioned histological groups were also heterogeneous according to their hormonal profiles. Serum androgens, estrogens, and prolactin concentrations are significantly increased in DHEA-treated animals as compared with controls. There was no significant difference in follicle stimulating hormone between the with cysts and rats without cysts after DHEA treatment. After 20 days of DHEA treatment, rats with CLs have very high levels of luteinizing hormone. Luteinizing hormone and prolactin levels are significantly higher in rats with cysts than in rats without cysts after 10 days of DHEA treatment. As has been shwon in this inquiry, androgens and estradiol levels in rats with cysts after DHEA treatment are higher than those in rats without cysts after DHEA treatment. Therefore, this study suggests that the ovarian cystic condition developed after DHEA treatment in rats, is associated with higher levels of circulating androgens, estradiol, and prolactin.
    Additional Material: 11 Ill.
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