The effect of thyroid hormone-dependent dermal fibroblast proliferation: an investigation of connective tissue growth factor and proliferative cell nuclear antigen

Thesis (M.A.)--Boston UniversityThyroid hormone has significant impact on skin homeostasis and cutaneous wound healing. Previous research has demonstrated both in vitro proliferation of keratinocytes and fibroblasts and in vivo stimulation of epidermal and dermal layers in response to triiodothyronine (T3) administration. However, the physiological mechanism of action involving T3 signaling and the specific intermediate factors of T3-induced cell proliferation are poorly understood. Currently, there is no working model of T3-dependent dermal fibroblast proliferation. In order to gain a more complete understanding of thyroid hormone regulation in wound healing, two known proliferative growth factors, connective tissue growth factor (CTGF) and proliferative cell nuclear antigen (PCNA), were chosen as potential mediators of T3-stimulated fibroblast proliferation. In vitro dermal fibroblast cultures were dosed with one of three experimental T3 concentrations (10-9 M, 10-8 M and 10-7 M) and western blot analysis was conducted to determine whether CTGF and PCNA expression are regulated by T3 stimulation. The results indicated no significant change in CTGF or PCNA expression dependent on T3 concentration. The implications of the findings were addressed and suggestions for future research directions have been proposed. It is still unclear which growth factors are involved in T3-regulated fibroblast proliferation. Once these mediators are identified, it will be possible to construct a mechanism of action to integrate the findings and ultimately develop a complete understanding of cutaneous physiology

How protein - Protein interactions contribute to pyrenoid formation in Chlamydomonas

© The Author(s) 2019. The chloroplast pyrenoid, an important component of the CO2 concentrating mechanism of algae, is a structure composed primarily of Rubisco. In Chlamydomonas, Rubisco in the pyrenoid is held together by the linker protein EPYC1. Atkinson et al., (2019) determined the regions of the Rubisco small subunit and EPYC1 that are important for the protein-protein interaction, thus making progress towards reconstruction of a pyrenoid in higher plants. Why is a protein soluble in one organism while its homologue in another species becomes part of a liquidlike cell structure? That is the question being addressed by Atkinson et al., (2019) in this issue of the Journal of Experimental Botany. It is even more striking when the protein is ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco), the most abundant soluble enzyme in plants and algae. In terrestrial plants, Rubisco behaves as a soluble protein found throughout the chloroplast stroma of leaf mesophyll cells. However, in most algae, Rubisco is found in a structure within the chloroplast called the pyrenoid

Membrane lipid biosynthesis in Chlamydomonas reinhardtii: Ethanolaminephosphotransferase is capable of synthesizing both phosphatidylcholine and phosphatidylethanolamine

Phosphatidylethanolamine, but not phosphatidylcholine, is found in Chlamydomonas reinhardtii. A cDNA coding for diacylglycerol: CDP-ethanolamine ethanolaminephosphotransferase (EPT) was cloned from C. reinhardtii. The C. reinhardtii EPT appears phylogenetically more similar to mammalian aminoalcoholphosphotransferases than to those of yeast and the least close to those of plants. Similar membrane topography was found between the C. reinhardtii EPT and the aminoalcoholphosphotransferases from mammals, yeast, and plants. A yeast mutant deficient in both cholinephosphotransferase and ethanolaminephosphotransferase was complemented by the C. reinhardtii EPT gene. Enzymatic assays of C. reinhardtii EPT from the complemented yeast microsomes demonstrated that the C. reinhardtii EPT synthesized both PC and PE in the transformed yeast. The addition of either unlabeled CDP-ethanolamine or CDP-choline to reactions reduced incorporation of radiolabeled CDP-choline and radiolabeled CDP-ethanolamine into phosphatidylcholine and phosphatidylethanolamine. EPT activity from the transformed yeast or C. reinhardtii cells was inhibited nearly identically by unlabeled CDP-choline, CDP-ethanolamine, and CMP when [ 14C]CDP-choline was used as the primary substrate, but differentially by unlabeled CDP-choline and CDP-ethanolamine when [ 14C]CDP-ethanolamine was the primary substrate. The K m value of the enzyme for CDP-choline was smaller than that for CDP-ethanolamine. This provides evidence that C. reinhardtii EPT, similar to plant aminoalcoholphosphotransferase, is capable of catalyzing the final step of phosphatidylcholine biosynthesis, as well as that of phosphatidylethanolamine in the Kennedy pathway. © 2004 Elsevier Inc. All rights reserved

A new chloroplast protein is induced by growth on low CO\u3csub\u3e2\u3c/sub\u3e in Chlamydomonas reinhardtii

The biosynthesis of a 36 kilodalton polypeptide of Chlamydomonas reinhardtii was induced by photoautotrophic growth on low CO2. Fractionation studies using the cell-wall-deficient strain of C. reinhardtii, CC-400, showed that this polypeptide was different from the low CO2-induced periplasmic carbonic anhydrase. In addition, the 36 kilodalton polypeptide was found to be localized in intact chloroplasts isolated from low CO2-adapting cultures. This protein may, in part, account for the different inorganic carbon uptake characteristics observed in chloroplasts isolated from high and low CO2-grown C. reinhardtii cells

The Intracellular Localization of Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase in Chlamydomonas reinhardtii

The pyrenoid is a proteinaceous structure found in the chloroplast of most unicellular algae. Various studies indicate that ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) is present in the pyrenoid, although the fraction of Rubisco localized there remains controversial. Estimates of the amount of Rubisco in the pyrenoid of Chlamydomonas reinhardtii range from 5% to nearly 100%. Using immunolocalization, the amount of Rubisco localized to the pyrenoid or to the chloroplast stroma was estimated for C. reinhardtii cells grown under different conditions. It was observed that the amount of Rubisco in the pyrenoid varied with growth condition; about 40% was in the pyrenoid when the cells were grown under elevated CO2 and about 90% with ambient CO2. In addition, it is likely that pyrenoidal Rubisco is active in CO2 fixation because in vitro activity measurements showed that most of the Rubisco must be active to account for CO2-fixation rates observed in whole cells. These results are consistent with the idea that the pyrenoid is the site of CO2 fixation in C. reinhardtii and other unicellular algae containing CO2-concentrating mechanisms