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  • 1
    ISSN: 1432-0827
    Keywords: Key words: Osteoclast — Monocyte — Macrophage — Differentiation — Bone Resorption.
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Physics
    Notes: Abstract. Mononuclear precursors of the human osteoclast have been identified in both bone marrow and the circulation in man, but osteoclast membership of the mononuclear phagocyte system (MPS) and its precise cellular ontogeny remain controversial. We isolated human hematopoietic marrow cells, blood monocytes, and peritoneal macrophages and incubated each of these cell populations with UMR106 osteoblast-like cells on glass coverslips and dentine slices in both the presence and absence of 1,25 dihydroxyvitamin D3 (1,25(OH)2D3), macrophage-colony stimulating factor (M-CSF), and dexamethasone. Cells isolated from peripheral blood and peritoneal dialysis fluid were positive only for monocyte/macrophage markers (CD11a, CD11b, CD14, and HLA-DR) and negative for osteoclast markers [tartrate-resistant acid phosphatase (TRAP), vitronectin reception (VNR), and calcitonin (CT) receptors and did not form resorption pits on dentine slices after 24 hours in culture. Similarly marrow cells did not form resorption pits on dentine slices after 24 hours in culture. However, after 14 days in co-culture with UMR106 cells, in the presence of 1,25(OH)2D3 and M-CSF, numerous TRAP, CT receptor, and VNR-positive multinucleated cells capable of extensive lacunar resorption were formed in co-cultures of all these preparations. The presence of 1,25 (OH)2D3, M-CSF, and UMR106 were absolute requirements for osteoclast differentiation. It is concluded that precursor cells capable of osteoclast differentiation are present in the marrow compartment, the monocyte fraction of peripheral blood, and in the macrophage compartment of extraskeletal tissues and that these cells are capable of differentiating into mature functional osteoclasts. These findings argue in favor of osteoclast membership of the human MPS.
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  • 2
    ISSN: 1432-0827
    Keywords: Key words: Osteoclast — Differentiation — Bone resorption — Prostaglandins.
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Physics
    Notes: Abstract. The effect of prostaglandins (PGs) on osteoclast differentiation, an important point of control for bone resorption, is poorly understood. After an initial differentiation phase that lasts at least 4 days, murine monocytes, cocultured with UMR106 osteoblastic cells (in the presence of 1,25-dihydroxyvitamin D3) give rise to tartrate-resistant acid phosphatase (TRAP) positive osteoclast-like cells that are capable of lacunar bone resorption. PGE2 strongly inhibits TRAP expression and bone resorption in these cocultures. To examine further the cellular mechanisms associated with this inhibitory effect, we added PGE2 to monocyte/UMR106 cocultures at specific times before, during, and after this initial 4-day differentiation period. To determine whether this PGE2 inhibition was dependent on the type of stromal cell supporting osteoclast differentiation, we also added PGE2 to cocultures of monocytes with ST2 preadipocytic cells. Inhibition of bone resorption was greatly reduced when the addition of PGE2 to monocyte/UMR106 cocultures was delayed until the fourth day of incubation; when delayed until the seventh day, inhibition did not occur. PGE2 inhibition of bone resorption was concentration-dependent and at 10−6 M was also mediated by PGE1 and PGF2α. In contrast to its effects on monocyte/UMR106 cocultures, PGE2 stimulated bone resorption in monocyte/ST2 cocultures. Both ST2 cells and UMR106 cells were shown to express functional receptors for PGE2. These results show that PGs strongly influence the differentiation of osteoclast precursors and that this effect is dependent not only on the type and dose of PG administered, but also on the nature of the bone-derived stromal cell supporting this process.
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  • 3
    ISSN: 1573-904X
    Keywords: trilostane ; ketotrilostane ; reversible metabolism ; pharmacokinetics ; metabolic interconversion
    Source: Springer Online Journal Archives 1860-2000
    Topics: Chemistry and Pharmacology
    Notes: Abstract The pharmacokinetics of trilostane and one of its metabolites ketotrilostane are described and characterized in the rat following the separate intravenous administration of trilostane and ketotrilostane. It was noted during these studies that the parent compound and its metabolite undergo metabolic interconversion–trilostane producing ketotrilostane and ketotrilostane generating trilostane. This result means that trilostane is conserved in the body by interconversion-being metabolized to ketotrilostane and then subsequently back to the “parent” drug, trilostane.
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  • 4
    ISSN: 1617-4623
    Keywords: Homologous recombination ; Protoplast transformation ; β-Glucuronidase ; Maize
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary The requirements for homologous recombination between plasmid DNA molecules have been studied using the PEG (polyethylene glycol)-mediated transformation system of maize (Zea mays L.) protoplasts coupled with the transient expression assay for β-glucuronidase (GUS). Two plasmids were introduced into maize protoplasts; one plasmid (pB×26) contained a genomic clone of the Adh1 maize gene; the other plasmid (piGUS) was a promoterless construction containing part of intron A of the Adhl gene fused to the gusA coding sequence. Thus, the two vectors shared an effective homologous region consisting of a 459 by (Hindlll—PvuII) fragment of the yAdh1 intron A sequence. An active gusA fusion gene would result upon homologous recombination between the plasmids within the intron A sequence, and indeed GUS activity was observed in extracts following co-transformation of maize protoplasts with the two plasmids. The presence of recombinant DNA molecules in protoplast DNA isolated 1 day after co-transformation was verified using polymerase chain reactions (PCR) and Southern blots. For efficient homologous recombination, both plasmids had to be linearized. The recombination reaction was induced by restriction of the plasmid molecules either inside the effective homologous region or at the borders of the intron sequence. However, the presence of even small, terminal, nonhomologous sequences at the 3′ end of the pB×26 fragment inhibited the recombination reaction. Also, both ends of the linearized piGUS DNA molecules were involved in the recombination reaction. The results revealed some features of homologous recombination reactions occurring in plant cells which cannot be accommodated by mechanisms postulated for similar reactions in animal system and in lower eukaryotes.
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  • 5
    ISSN: 1573-6865
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Summary The feasibility of using scanning electron microscopy (SEM) to identify the position of specific DNA sequences was examined using a Y chromosome ‘specific’ probe (pHY2.1). Tests were carried out on chromosome spreads hybridizedin situ with biotinylated pHY2.1. Chromosomal sites of hybridization of the probe were localized by an indirect immunohistochemical procedure which resulted in a gold product which could be amplified by silver precipitation. In the SEM, the specific location of the probe was easily identified due to the enhanced signal produced by the gold—silver complex. The probe was localized both on the long arm of the Y chromosome and within interphase nuclei. It was found that SEM was more sensitive than light microscopy since the probe could be identified without silver amplification. With refinements to the technique, SEM could provide a useful method for high resolution localizing of unique DNA sequences (i.e. single copy genes).
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  • 6
    ISSN: 1573-6865
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Summary It is generally assumed that nucleic acid association duringin situ hybridization reactions is similar to that of nucleic acid association in solution. This assumption has been investigated by detecting closely homologous human papillomavirus types 6 and 11 byin situ hybridization as a model for the evaluation of stringency conditions in clinical biopsies. By examining matched and mismatched, labelled and target sequences under various stringency conditions, empirical DNA-DNA stability curves and their derivative equations for tissue melting temperatures (Tmt) were derived. The corresponding values for Tmt are 10–20°C higher than their solution equivalents. These data, supported by polymerase chain reaction experiments, demonstrate that closely homologous viral DNAs cross linked in tissue by formaldehyde fixation do not interact with the corresponding labelled probes as predicted from solution kinetic equations. This not only has theoretical implications but is also relevant to the accuracy of clinical diagnostic testing.
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  • 7
    ISSN: 1573-6865
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Summary The parameter Tmt has been defined by non-isotopic in situ hybridization and describes the tissue melting temperature (Tmt) of human papillomavirus (HPV) DNA sequences. In this study, multiple in situ hybridization signals for HPV types 16, 31 and 33 in individual archival biopsies hybridized with genomic probes are shown by polymerase chain reactions to be due to cross-hybridization of probe sequences to a single tissue target. Tmt is independent of viral type but depends on the homology between probe and target when using nick-translated whole genomic probes. The difference between Tm and Tmt is not due to the presence of viral capsid protein. Multiple HPV signals in archival material should not therefore be interpreted as indicative of multiple HPV infection unless adequate stringency conditions have been employed or they are present in morphologically distinct areas of the biopsy. Furthermore, extrapolation of calculated DNA homologies to non-isotopic in situ hybridization analysis may not be appropriate. A hybridization signal does not imply probe and target identity: this has implications for HPV typing in clinical material.
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  • 8
    ISSN: 1573-6865
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Summary The experimentally derived parameter Tmt (tissue Tm) was defined previously to describe the end-point used for evaluation of the stringency of non-isotopic in situ hybridization and was found to differ from the theoretical melting temperature (Tm) for several HPV types. In this paper, the reasons for this discrepancy were investigated by performing a series of experiments with a variety of probes for both human genomic and integrated viral sequences in isolated and cultured normal and abnormal cells in addition to paraffin-embedded material. Tmt was shown to be dependent on several parameters of probe and target, and on the sensitivity of the detection system used but was not affected by aldehyde fixation or paraffin wax embedding under optimal conditions of nucleic acid unmasking. These data support the hypothesis that differences between Tmt and Tm may be due to the use of a different end-point for in situ hybridization analysis rather than biochemical alteration of DNA-DNA interactions in intact cells. Appropriate stringency conditions should therefore be determined by experiment rather than calculated theoretically for gene evaluation in cells and tissues.
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  • 9
    ISSN: 1476-5535
    Keywords: Phosphorylation ; Oleandomycin ; Macrolide 2′-phosphotransferase
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Summary An enzyme that catalyzes 2′-O-phosphorylation of oleandomycin and several other macrolide antibiotics has been purified approximately 47-fold from cell-free extracts ofStreptomyces coelicolor Müller, NRRL 3532 (UC™ 5240). The reaction product was verified as being oleandomycin-2′-O-phosphate by mass spectrometry. As a result of purification, the enzyme was separated from two lincosaminide inactivating enzyme activities also present in the cell-free extract.
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