Biomedical subjects

D E Mosedale

Publications and source records attributed to D E Mosedale.

3 recordsLinked to original sources

PubMed1998

Transforming growth factor-beta dynamically regulates vascular smooth muscle differentiation in vivo.

Variations in the levels of smooth muscle-specific isoforms of contractile proteins have been reported to occur in many different vascular diseases. However, although much work has been done in vitro to investigate the regulation of smooth muscle cell differentiation, the molecular mechanisms which regulate the differentiation of vascular smooth muscle tissue in vivo are unknown. Using quantitative immunofluorescence, we show that in rat arteries levels of smooth muscle differentiation markers correlate with the levels of the cytokine TGF-beta. In young mice with one allele of the TGF-beta1 gene deleted, the levels of both TGF-beta1 and smooth muscle differentiation markers are reduced compared to wild-type controls. This regulation of smooth muscle differentiation by TGF-beta during post-natal development also occurs dynamically in the adult animal. Following various pharmacological or surgical interventions, including treatment of mice with tamoxifen and balloon injury of rat carotid arteries, there is a strong correlation between the changes in the levels of TGF-beta and changes in the levels of smooth muscle differentiation markers (r=0. 9, P<0.0001 for n=26 experiments). We conclude that TGF-beta dynamically regulates smooth muscle differentiation in rodent arteries in vivo.

Animals↗

PubMed1996

Optimization of immunofluorescence methods by quantitative image analysis.

There is a growing trend towards the objective quantification of immunohistochemical staining. However, quantification has not been used previously to optimize the original published immunohistochemical methods. We present a quantitative method for analyzing immunofluorescence staining employing the Applied Imaging MAGISCAN image analysis system, which has then been used to optimize major aspects of the standard immunofluorescent staining protocols. The optimization process resulted in a method that increased specific staining up to fivefold over typical published protocols, with no increase in nonspecific staining. The method is extremely reproducible. For slides stained by a single experimenter in one batch on one day, the coefficient of variation between replicate means is 1.2%. The image analysis protocol gave a linear response with increasing antigen concentration, as determined by using purified antigen dried onto slides. The revisions to the standard protocol presented here can also be applied to nonquantitative staining. It will help users of immunofluorescence to maximize their staining and may enable the detection of previously undetected antigens.

Actins↗

PubMed1995

Active and acid-activatable TGF-beta in human sera, platelets and plasma.

Assays which measure active and latent forms of transforming growth factor beta (TGF-beta) separately in human serum and plasma are required to investigate the biological role of TGF-beta in a variety of human diseases. We have developed an enzyme-linked immunosorbent assay (ELISA) using two polyclonal antibodies against TGF-beta which rapidly determines the amount of active plus acid-activatable, latent TGF-beta forms ((a+l)TGF-beta) present in human serum and plasma in the range 4 pmol/l to 2000 pmol/l. To measure active TGF-beta alone, we have developed a second ELISA using the extracellular domain of the TGF-beta type II receptor as the capture reagent which detects active TGF-beta in serum and plasma samples in the range 20 pmol/l to 4000 pmol/l. Both assays detect TGF-beta 1 and TGF-beta 3 with similar sensitivity, are > 10-fold less sensitive to TGF-beta 2 and are not affected by a range of other peptide growth factors. The mean (a+l)TGF-beta present in human serum was 330 pmol/l but the range was very large (< 4 pmol/l to 1400 pmol/l). The mean active TGF-beta present was 230 pmol/l (range < 20 pmol/l to 1400 pmol/l) and the proportion of the (a+l)TGF-beta present which was active [a/(a+l)] varied from < 10% to 100%. The concentration of (a+l)TGF-beta and the proportion of TGF-beta which was active were very similar in the serum and platelet-poor plasma prepared from the same whole blood sample. The clot formed during serum preparation retained all of the TGF-beta which was detected by the (a+l)TGF-beta ELISA in the corresponding platelet releasate, although the PDGF in platelets was released into the serum. In contrast, platelet-poor plasma contained no detectable PDGF demonstrating that the (a+l)TGF-beta assayed in the plasma was not due to platelet degranulation after bleeding. Serum active TGF-beta and (a+l)TGF-beta concentrations therefore provide a reliable estimate of these forms of TGF-beta present in plasma.

Adult↗