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  • 1
    ISSN: 0730-2312
    Keywords: osteocalcin ; transcriptional regulation ; homeodomain protein ; Msx ; bone-specific ; OC box ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Bone-specific expression of the osteocalcin gene is transcriptionally controlled. Deletion analysis of osteocalcin promoter sequences by transient transfection of osseous (ROS 17/2.8) and nonosseous (R2 fibroblast) cells revealed that the most proximal 108 nucleotides are sufficient to confer tissue-specific expression. By gel mobility shift assays with wild-type and mutated oligonucleotides and nuclear extracts from several different cell lines we identified a novel transcription factor complex which exhibits sequence-specific interactions with the primary transcriptional element, the OC box (nt -99 to -76). This OC box binding protein (OCBP) is present only in osteoblast-like cells. Methylation interference demonstrated association of the factor with OC box sequences overlapping the Msx homeodomain consensus binding site. By assaying several mutations of the OC box, both in gel shift and transient transfection studies using ROS 17/2.8, we show the following. First, binding of OCBP correlates with osteocalcin promoter activity in ROS 17/2.8 cells. Increased binding leads to a 2-3-fold increase in transcription, while decreased binding results in transcription 30-40% of control. Second, homeodomain protein binding suppresses transcription. However, Msx expression is critical for full development of the bone phenotype as determined by antisense studies. Last, we show that one of the mutations of the OC box permits expression of osteocalcin in non-osseous cell lines. In summary, we demonstrate association of at least two classes of tissue-restricted transcription factors with the OC box element, the OCBP and Msx proteins, supporting the concept that these sequences contribute to defining tissue specificity. © 1996 Wiley-Liss, Inc.
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  • 2
    ISSN: 0730-2312
    Keywords: epitope mapping ; monoclonal antibodies ; linear epitope ; immuno-dominant ; immuno-recessive ; ELISA ; competitive ELISA ; recombinant GST-PSP94 ; N-terminal and C-terminal peptides ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: PSP94 has shown potential to be a serum biomarker for evaluating prostate cancer. Studies of the epitope structure is crucial for this endeavour. In this article, we have used 15 different monoclonal antibodies (MAb) to analyse the epitope structure of PSP94 and to compare with the results obtained from our previous work using polyclonal antibody and recombinant PSP94. Firstly, we determined the relative activities of the 15 MAb population by direct and competitive ELISA. The two predominant MAbs (MAb PSP-6 and -19) in 15 MAbs were selected for further studies of the epitope structure. By comparing the binding activities of recombinant GST-PSP94 and natural PSP94 with MAbs, and by comparing their affinity with MAbs in an in vitro denaturing experiment, PSP94 was shown to have a similar, prevalently linear epitope structure as we demonstrated by polyclonal antibody. Using recombinant GST fusion protein with PSP94 and with each half of the N- and C-terminal 47 amino acids (GST-PSP-N47/C47) in E. coli cells, the different epitopes recognized by 15 monoclonal antibodies were delineated and the polar distribution of the epitope structure of PSP94 was characterized. Results of direct ELISA of recombinant N47 and C47 and their competitive binding against natural PSP94 (competitive ELISA) showed that the N- and C-termini represent the immuno-dominant and immuno-recessive area separately. A majority of the monoclonal antibodies (12/15) showed preferential binding of the N-terminal sequence of the PSP94 protein. Using GST-PSP-N47 as a standard protein, an epitope map of the 15 monoclonal antibodies was obtained. The results of this study will help to define the clinical utility of PSP94. J. Cell. Biochem. 65:186-197. © 1997 Wiley-Liss, Inc.
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  • 3
    Electronic Resource
    Electronic Resource
    New York, NY [u.a.] : Wiley-Blackwell
    Journal of Cellular Physiology 163 (1995), S. 312-320 
    ISSN: 0021-9541
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: We have investigated the promoter element(s) required by the cell cycle regulated FO108 human histone H4 gene for control of gene expression during adipocyte proliferation and differentiation. Stable 3T3L1 cell lines were established that express fusion genes in which the histone H4 promoter is joined to chloramphenicol acetyltransferase (cat) as a reporter gene. Expression of the H4CAT fusion genes was monitored in proliferating and confluent 3T3L1 preadipocytes and in differentiating 3T3L1 adipocytes. The results indicate that the H4 cell cycle element (CCE), which mediates S phase-specific stimulation of H4 gene transcription, is not required for transcriptional regulation during differentiation. Instead, a minimal H4 promoter (nucleotides -46 to -11) is sufficient to mediate the complex transcriptional response of H4 gene expression observed during the process of adipocyte differentiation of 3T3L1 cells. In addition, the data suggest that down-regulation of histone gene expression during cellular differentiation may be mediated by passive inactivation of the promoter due to loss of positive regulatory factor(s). © 1995 Wiley-Liss, Inc.
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  • 4
    Electronic Resource
    Electronic Resource
    New York, NY [u.a.] : Wiley-Blackwell
    Journal of Cellular Physiology 177 (1998), S. 402-410 
    ISSN: 0021-9541
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Potassium (K+) conductances are known to be involved in cell proliferation of a number of nonexcitable cell types. The nature of the mechanism by which K+ channel inhibition reduces cell proliferation has remained elusive despite intensive search. We investigated whether such a phenomenon could be demonstrated in excitable cells, using the GH3 pituitary cell line as a cell model. Our aims were: (1) to study the effect of K+ channel inhibition on the proliferation of GH3 cells; and (2) to investigate the putative intracellular signals involved in this inhibition. Tetraethylammonium chloride (TEA), a blocker of the calcium (Ca2+)-dependent K+ conductances of GH3, was found to reversibly inhibit cell proliferation, as measured by 3H-thymidine incorporation. Cell cycle block specifically occurred at the G1/S phase of the cell cycle. This inhibition of proliferation was observed for 1-4 mM TEA, which suppressed most of the Ca2+-activated K+ current and part of the inward rectifying K+ current, as shown by electrophysiological experiments. Increasing extracellular K+ concentrations with KCl also inhibited cell proliferation in a dose-dependent manner. Both TEA and KCl depolarized the cells and increased intracellular Ca2+ levels ([Ca2+]i), showing that, in this type of excitable cell, inhibition of cell proliferation can be associated with elevated Ca2+ levels. Ca2+ and membrane resting potential (MRP) were considered as possible messengers of this inhibition. Our results suggest that cell cycle arrest of GH3 cells by K+ channel block probably involves an additional pathway, distinct from those of Ca2+ and MRP. J. Cell. Physiol. 177:402-410, 1998. © 1998 Wiley-Liss, Inc.
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  • 5
    ISSN: 0197-8462
    Keywords: radiofrequency radiation ; heart rate ; blood pressure ; cardiovascular system ; Life and Medical Sciences ; Occupational Health and Environmental Toxicology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Physics
    Notes: Exposure to fast-rise-time ultra-wideband (UWB) electromagnetic pulses has been postulated to result in effects on biological tissue (including the cardiovascular system). In the current study, 10 anesthetized Sprague-Dawley rats were exposed to pulses produced by a Sandia UWB pulse generator (average values of exposures over three different pulse repetition rates: rise time, 174-218 ps; peak E field, 87-104 kV/m; pulse duration, 0.97-0.99 ns). Exposures to 50, 500 and 1000 pulses/s resulted in no significant changes in heart rate or mean arterial blood pressure measured every 30 s during 2 min of exposure and for 2 min after the exposure. The results suggest that acute UWB whole-body exposure under these conditions does not have an immediate detrimental effect on these cardiovascular system variables in anesthetized rats. Bioelectromagnetics 19:330-333, 1998. Published 1998 Wiley-Liss, Inc.
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  • 6
    Electronic Resource
    Electronic Resource
    Chichester [u.a.] : Wiley-Blackwell
    Developmental Genetics 16 (1995), S. 367-374 
    ISSN: 0192-253X
    Keywords: X-chromosome inactivation ; Gpd expression ; marsupial ; development ; opossum ; triplaid ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Metatherian (marsupial) mammals possess a non-random form of X-chromosome inactivation in which the paternally-derived X is always the one inactivated. To examine the progression of X-linked gene expression during metatherian development, we compared relative levels of the maternally and paternally encoded Gpd gene products in heterozygous female Virginia opossums (Didelphis virginiana) across a moior portion of the developmental period. Panels of tissues obtained from fetuses, newborns, and pouch young were examined via polyacrylamide gel electrophoresis of the G6PD protein. As in adults, G6PD phenotypes in these developmental stages were highly skewed in favor of the maternal allele product, but in some tissues there was a marked increase in paternal allele expression with advancing developmental age. However, even by 42 days of post-partum development, expression of the paternal Gpd allele had not attained the adult, tissue-specific activity pattern. Our findings indicate remarkable developmental changes in the activity of the paternal allele in several tissues/organs continuing well into mid pouch-life stages and beyond. Specifically we found that 1) a substantially repressed paternal Gpdgene is present in the cells of female stage 29 fetuses and later developmental stages, 2) the activity state of the paternal Gpd gene is not fixed during early embryonic development in this species, 3) maior changes in paternal Gpd expression occur in advanced developmental stages and comprise a maturation of the gene expression pattern during ontogeny, and 4) alterations of paternal Gpd allele activity during development occur in a tissue-specific manner. © 1995 Wiley-Liss, Inc.
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  • 7
    Electronic Resource
    Electronic Resource
    New York, NY [u.a.] : Wiley-Blackwell
    Bioelectromagnetics 16 (1995), S. 119-123 
    ISSN: 0197-8462
    Keywords: EMF ; melatonin ; puberty ; sheep ; transmission line ; Life and Medical Sciences ; Occupational Health and Environmental Toxicology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Physics
    Notes: In an earlier study, we found no effects of 60 Hz electric and magnetic fields (EMF) from a 500 kV transmission line on serum melatonin patterns or on puberty in ten female Suffolk lambs (Ovis aries). We conducted a larger replicate study of 15 lambs exposed to a mean electric field of 6.3 kV/m and a mean magnetic field of 3.77 μT and 15 controls exposed to EMF two orders of magnitude weaker than in the line area. The replicate produced essentially the same results as our previous study. © 1995 Wiley-Liss, Inc.
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  • 8
    ISSN: 0197-8462
    Keywords: RF radiation ; industrial solvents ; glycol ethers ; developmental toxicity ; synergism ; Life and Medical Sciences ; Occupational Health and Environmental Toxicology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Physics
    Notes: Radiofrequency (RF) radiation is used in a variety of workplaces. In addition to RF radiation, many workers are concurrently exposed to numerous chemicals; exposed workers include those involved with the microelectronics industry, plastic sealers, and electrosurgical units. The developmental toxicity of RF radiation is associated with the degree and duration of hyperthermia induced by the exposure. Previous animal research indicates that hyperthermia induced by an elevation in ambient temperature can potentiate the toxicity and teratogenicity of some chemical agents. We previously demonstrated that combined exposure to RF radiation (10 MHz) and the industrial solvent, 2-methoxyethanol (2ME), produces enhanced teratogenicity in rats. The purpose of the present research is to determine the effects of varying the degree and duration of hyperthermia induced by RF radiation (sufficient to maintain colonic temperatures at control [38.5], 39.0, 40.0, or 41.0 °C for up to 6 h) and 2ME (100 mg/kg) administered on gestation day 13 of rats. Focusing on characterizing the dose-response pattern of interactions, this research seeks to determine the lowest interactive effect level. Day 20 fetuses were examined for external and skeletal malformations. The results are consistent with previous observations. Significant interactions were observed between 2ME and RF radiation sufficient to maintain colonic temperatures at 41 °C for 1 h, but no consistent interactions were seen at lower temperatures even with longer durations. These data indicate that combined exposure effects should be considered when developing both RF radiation and chemical exposure guidelines and intervention strategies. Bioelectromagnetics 18:349-359, 1997. © 1997 Wiley-Liss, Inc.
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  • 9
    ISSN: 0192-253X
    Keywords: X-chromosome inactivation ; imprinting ; retinoblastoma ; transmission-ratio distortion ; methylation ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: We have begun a search for heritable variation in X-chromosome inactivation pattern in normal females to determine whether there is a genetic effect on the imprinting of X-chromosome inactivation in humans. We have performed a quantitative analysis of X-chromosome inactivation in lymphocytes from mothers in normal, three-generation families. Eight mothers and 12 grandmothers exhibited evidence of highly skewed patterns of X-chromosome inactivation. We observed that the male offspring of females with skewed X-inactivation patterns were three times more likely to inherit alleles at loci that were located on the inactive X chromosome (Xi) than the active X chromosome (Xa). The region of the X chromosome for which this phenomenon was observed extends from XP11 to -Xq22. We have also examined X-chromosome inactivation patterns in 21 unaffected mothers of male bilateral sporadic retinoblastoma patients. Six of these mothers had skewed patterns of X-chromosome inactivation. In contrast to the tendency for male offspring of skewed mothers from nondisease families to inherit alleles from the inactive X chromosome, five of the six affected males inherited the androgen receptor alleles from the active X chromosome of their mother. © 1995 Wiley-Liss, Inc.
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  • 10
    Electronic Resource
    Electronic Resource
    New York, NY [u.a.] : Wiley-Blackwell
    Molecular Reproduction and Development 40 (1995), S. 69-83 
    ISSN: 1040-452X
    Keywords: SGP-1 ; Hypophysectomy ; Castration ; Efferent ducts ; Lysosomes ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: The objective of this study was to define the factors regulating the endogenous production of sulfated glycoprotein-1 (SGP-1) in nonciliated cells of the efferent ducts. To this end we examined five different groups of animals undergoing the following experimental procedures: (1) hypophysectomized animals at 7, 14, and 28 days, (2) 7-day hypophysectomized rats receiving testosterone implants given at various time intervals thereafter, (3) castration at various time intervals up to 7 days, (4) 7-day castrated rats receiving testosterone implants at various time intervals thereafter, and (5) castrated rats given testosterone implants immediately after castration and sacrificed at different time intervals thereafter. Efferent ducts were fixed by perfusion with 4% paraformaldehyde and 0.5% glutaraldehyde in phosphate buffer for quantitative immunocytochemical analysis at the level of the electron microscope. For each experimental condition and their controls, the number of gold particles/μm2 within the endosomal and lysosomal compartments was calculated taking into account the changes in both the volume of the cell and organelles being quantified and expressed as labeling content. The results revealed that hypophysectomy (up to 4 weeks) caused a marked significant decrease in the SGP-1 labeling content of the endosomal and lysosomal compartments. The labeling content of the lysosomal compartment of efferent ducts from rats castrated for up to 1 week did not change significantly. However, there was a significant decrease in the labeling content of endosomes. This decrease is due to SGP-1, which is secreted by Sertoli cells, not being available for uptake in the efferent aucts. These results suggested that testosterone is not required for maintaining the high labeling content of SGP-1 within lysosomes of nonciliated cells, but that a pituitary factor appears to be needed. The administration of testosterone at different intervals to 7-day castrated animals resulted in a significant decrease of lysosomal SGP-1, suggesting that testosterone under these experimental conditions inhibits the production of a pituitary factor that maintains the high labeling content of SGP-1 within lysosomes of the nonciliated cells. Testosterone administered to 7-day hypophysectomized animals over a 24-hr period had no effect on the labeling content of SGP-1 within lysosomes. However, the administration of testosterone to animals immediately following castration showed no differences in the labeling content of SGP-1 within compared to controls. Together these results suggest that the labeling content of SGP-1 within lysosomes of nonciliated cells of the efferent ducts is not dependent on luminal or circulating androgens, nor is it dependent on a testicular factor entering the lumen of the ducts. It does appear, however, that SGP-1 synthesis and targeting to secondary lysosomes is dependent on a pituitary factor that may have a direct or an indirect effect on the nonciliated cells. © 1995 Wiley-Liss, Inc.
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