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Biomedical subjects

V L Church

Publications and source records attributed to V L Church.

6 recordsLinked to original sources
PubMed2006

Differential regulation of GDF-5 and FGF-2/4 by immobilisation in ovo exposes distinct roles in joint formation.

Members of the fibroblast growth factor (FGF) family and growth and differentiation factor 5 (GDF-5) have been implicated in joint specification, but their roles in subsequent cavity formation are not defined. Cavity formation (cavitation) depends upon limb movement in embryonic chicks and factors involved in joint formation are often identified by their expression at the joint-line. We have sought support for the roles of FGF-2, FGF-4, and GDF-5 in cavitation by defining expression patterns, immunohistochemically, during joint formation and establishing whether these are modified by in ovo immobilisation. We found that FGF-2 exhibited low level nuclear expression in chondrocytes and fibrocartilage cells close to presumptive joints, but showed significantly higher expression levels in cells at, and directly bordering, the forming joint cavity. This high-level joint line FGF-2 expression was selectively diminished in immobilised limbs. In contrast, we show that FGF-4 does not exhibit differential joint-line expression and was unaffected by immobilisation. GDF-5 protein also failed to show joint-line selective labelling, and although immobilisation induced a cartilaginous fusion across presumptive joints, it did not affect cellular GDF-5 expression patterns. Examining changes in GDF-5 expression in response to a direct mechanical strain stimulus in primary embryonic chick articular surface (AS) cells in vitro discloses only small mechanically-induced reductions in GDF-5 expression, suggesting that GDF-5 does not exert a direct positive contribution to the mechano-dependent joint cavitation process. This notion was supported by retroviral overexpression of UDPGD, a characteristic factor involved in hyaluronan (HA) accumulation at presumptive joint lines, which was also found to produce small decreases in AS cell GDF-5 expression. These findings support a direct mechano-dependent role for FGF-2, but not FGF-4, in the cavitation process and indicate that GDF-5 is likely to influence chondrogenesis positively without contributing directly to joint cavity formation.

Animals↗
PubMed2000

Cloning and expression of the Wnt antagonists Sfrp-2 and Frzb during chick development.

The Wnt genes are known to play fundamental roles during patterning and development of a number of embryonic structures. Receptors for Wnts are members of the Frizzled family of proteins containing a cysteine-rich domain (CRD) that binds the Wnt protein. Recently several secreted frizzled-related proteins (Sfrps) that also contain a CRD have been identified and some of these can both bind and antagonise Wnt proteins. In this paper we report the expression patterns of the chick homologues of Frzb, a known Wnt antagonist, and Sfrp-2. Both genes are expressed in areas where Wnts are known to play a role in development, including the neural tube, myotome, cartilage, and sites of epithelial-mesenchymal interactions. Initially, Sfrp-2 and Frzb are expressed in overlapping areas in the neural plate and neural tube, whereas later, they have distinct patterns. In particular Sfrp-2 is associated with myogenesis while Frzb is associated with chondrogenesis, suggesting that they play different roles during development. Finally, we have used the early Xenopus embryo as an in vivo assay to show that Sfrp-2, like Frzb, is a Wnt antagonist. These results suggest that Sfrp-2 and Frzb may function in the developing embryo by modulating Wnt signalling.

Amino Acid Sequence↗
PubMed1990

DNA contents of replication without DNA density labeling.

A new method for determining the timing of DNA replication in specific regions of the mammalian genome without the use of DNA density labeling and DNA density centrifugation is described. The method is based on determination of average relative DNA copy numbers in specific genomic regions as cells progress through S phase, and "time of replication" for a specific region is described in terms of the cell's DNA content when the region is replicated. DNA is isolated from synchronized populations of G1 and S phase cells, it is slot-blotted at the same DNA concentration(s) for each population, and it is hybridized with 32P-labeled DNA probes that are specific to the regions of interest. Quantitation of the slot blot autoradiograms and flow cytometric analysis allows determination of (a) average relative DNA copy numbers for the regions of interest in synchronized cell populations, and (b) the average total DNA content in each population of synchronized cells. This information and the flow cytometry histograms are then used to calculate the cellular DNA content at which each region of interest is replicated. The results have a precision of less than or equal to +/- 10% of S phase for Chinese hamster (line CHO) rhodopsin, metallothionein II, the 5'-end of dihydrofolate reductase, the telomeric repeated sequence, pHuR-093 (also located near the centromeres in CHO chromosomes), and the c-Ki-ras family.

Cell Cycle↗
PubMed1986

Changes in H1 content, nucleosome repeat lengths and DNA elongation under conditions of hydroxyurea treatment that reportedly facilitate gene amplification.

Depletion of histone H1, changes in nucleosome repeat lengths, and extents of DNA elongation were investigated in synchronized Chinese hamster (line CHO) cells using the general conditions of hydroxyurea treatment that appear to increase the frequency of gene amplification, i.e., synchronized cultures of G1 cells were allowed to begin to enter S phase before treatment with hydroxyurea was effected to retard DNA synthesis (Mariani, B.D. and Schimke, R.T. (1984) J. Biol. Chem. 259, 1901-1910). During the time that synchronized G1 cells begin to enter S phase, there occur considerable synchrony decay and accumulation of new DNA that increase with time before treatment with hydroxyurea is initiated. During exposure to hydroxyurea, there occur depletion of histone H1 and shortened repeat lengths for the DNA synthesized in the presence of hydroxyurea. In contrast, DNA synthesized in S phase before exposure to hydroxyurea has essentially the same repeat lengths as bulk chromatin at both the time that hydroxyurea treatment is effected and after 6 h in its presence. Sedimentation measurements indicate that the early replicating DNA undergoes considerable elongation both before and during 6 h of exposure to 0.3 mM hydroxyurea. Thus, nearly all of the early replicating DNA is elongated to greater than average replicon size under those conditions of hydroxyurea treatment that appear to favor gene amplification. Because the extents of DNA synthesis and cell cycle progression vary as functions of drug concentration, treatment times, and unknown factors (from experiment to experiment), it would appear that the parameters must be carefully monitored in each experiment if biochemical results are to be related to the position of cells in the growth cycle.

Animals↗
PubMed1978

The stimulation of DNA synthesis by cytochalasin B in proliferative epidermal and dermal cells.

Cytochalasin B influences a variety of cellular events that are associated with the contractile microfilament system and the formation of binucleate cells. Along with the formation of binucleate cells, cytochalasin B also causes an acceleration of cells from G1 to S in the cell cycle. By pulsing the cytochalasin B for 30 minutes and allowing for a previously established lag time (17.5 hours) a stimulation of thymidine incorporation into DNA of proliferative epidermal and dermal cells was found in both control and stripped epidermis. Autoradiographic analysis confirmed that the stimulation was due to an increased number of basal cells accelerated from G1 to S phase. A minimal number of binucleate basal cells, 1 in 300, was observed, which suggests that the stimulated synthesis is independent of binucleate cell formation. The amount of stimulation is maximum with cytochalasin B concentration pulse between 5gamma and 30gamma/ml. The results suggest a possible link in coupling cell membrane and surface events with subsequent increased cell nuclei synthetic activity.

Animals↗