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The LD(_50) of 10-day-old blowflies differed significantly in two different stocks, and were found to be 38.12 ± 0.07ºC for the Durham stock and 40.8 ± 0.18ºC for the Cambridge stock. A transitory increase in heat resistance occurred following the exposure of adult blowflies to a sublethal heat shock. This thermotolerance was apparent 1h after the application of heat shock, was maximal 2-3 h later and had disappeared after 6 h. Oxidative phosphorylation by flight muscle mitochondria from non-thermotolerant control flies was impaired by an LD(_50) dose in vivo. Respiration using glycerol-3- phosphate was more heat sensitive than that with pyruvate plus proline. State III respiration was markedly inhibited, acceptor control (RCI) was lost with (G 3P) as substrate and so ADP:0 ratios were not measurable; whereas with pyruvate + proline as substrates, although State III respiration was inhibited by 50% and acceptor control was significantly reduced, ADP:0 remained measurable. Uncoupling of oxidative phosphorylation was obvious only with pyruvate + proline where State IV was significantiy increased. The development of thermotolerance protected oxidative phosphorylation against heat damage. With G-3-P respiration State III was largely restored and acceptor control was not significantly different from controls, but ADP:0 remained lower. With pyruvate + proline as substrates State III respiration was inhibited, but State IV was also lower without evidence of uncoupling of oxidative phosphorylation. Acceptor control was restored to control levels but ADP:0 values were lower. The lower ADP:0 ratios indicate some impairment of mitochondrial function occurred. The effect of experimental temperature in vitro on respiratory performance of mitochondria from non-pretreated control and thermotolerant LD(_50) flies was also determined between 19 and 39ºC. State III respiration was markedly temperature- dependent in mitochondria from non-pretreated control flies with both substrates; it was maximal at 24-29ºC and fell progressively at higher measuring temperatures. In mitochondria from thermotolerant flies, State III respiration was less temperature dependent with both substrates, but this effect was more marked for G-3-P. The effect of experimental temperature on State IV respiration was similar in mitochondria from non- pretreated control and thermotolerant LD(_50) flies with the same substrate, but differed between the two substrates. With G 3P as substrate, respiration rate rose with temperature with a Q(_10) of approximately 1.5; however, with pyruvate + proline as substrate, the trend was for respiration rate to fall as experimental temperature rose. Differences in the temperature sensitivities of mitochondria from control and thermotolerant flies, in terms of acceptor control, were found. Using G-3-P, acceptor control was lost in mitochondria from control flies above 29ºC, but was still measurable at 34ºC in mitochondria from thermotolerant flies. With pyruvate + proline as substrate acceptor control was demonstrable in mitochondria from both non-pre-treated control and thermotolerant flies at all experimental temperatures. The thermal sensitivities of the respiratory complexes were studied using the inhibitors rotenone and antimycin A. In mitochondria from LD(_50) treated control flies respiration uncoupled with FCCP was not restored to State II levels. However, in LD(_50) treated mitochondria from thermotolerant flies respiration uncoupled with FCCP was not different from State III respiration. These data suggest that the reduction in State III respiration after heating is owing to an inhibition of oxidation rather than phosphorylation. Complex I, NADH coenzyme Q reductase, was shown to be the most temperature sensitive of the respiratory complexes.

This document only includes an excerpt of the corresponding thesis or dissertation. To request a digital scan of the full text, please contact the Ruth Lilly Medical Library's Interlibrary Loan Department (rlmlill@iu.edu).

Dietary phosphate is essential for normal fracture healing and bone growth. Previous studies have established that mice given a phosphate deficient diet after a fracture demonstrate delayed cartilage maturation and callus mineralization, as well as changes in gene expression consistent with oxidative phosphorylation dysfunction. This study was undertaken in order to examine the role of inorganic and organic phosphate availability on chondrocyte differentiation and mineralization, and to define the relationship between these processes and changes in chondrocyte metabolic function. ATDC5 murine chondroprogenitor cell line, which has been shown to undergo in vitro differentiation and extracellular matrix mineralization, was cultured under both differentiating and non-differentiating media conditions under conditions in 1mM -0.25mM sodium phosphate monobasic (inorganic phosphate) in the presence or absence of 4mM β-glycerol phosphate (organic phosphate). In the first series of studies, overall cell growth (total DNA and protein contents), mineralization (calcium accumulation), and cell-normalized oxidative metabolism (basal respiration, maximal respiration, ATP turnover, spare capacity, proton leak, and non-mitochondrial respiration rates) were measured over a 28 day time course in cultures grown in differentiating (ascorbic acid, insulin-transferrin-selenium, and β-glycerol phosphate) conditions in 1mM phosphate. These studies found that when the cells were induced to differentiate, there was a measurable increase in protein content while DNA content decreased by 30%, indicating a fraction of the cells underwent cell death. Differentiation was further associated with an overall two-fold increase in oxidative respiration. Next we assessed how differentiation, the promotion of matrix mineralization, and inorganic phosphate availability affected oxidative respiration. When differentiation was not induced with ascorbic acid and β-glycerol phosphate, there was no over growth in the cultures nor any change in total extracellular matrix mineralization or oxidative respiration. In the absence only of β-glycerol phosphate, differentiation proceeded but matrix mineralization did not occur. However, overall protein content and oxidative respiration were statistically two- and 1.5-fold higher, respectively, independent of the inorganic phosphate contents of the growth media. These results suggest that both differentiation and overall protein accumulation are strongly associated with increased oxidative metabolism while mineralization of the matrix decreased oxidative function. Only at the lowest phosphate levels were changes in basal oxidative function observed. These results are consistent with previous in vivo findings suggesting that diminished expression of mitochondrial associated genes in callus tissues from hypophosphatemic mice were associated with an overall decrease in chondrocyte differentiation.

Thesis (Ph. D.)--University of Wisconsin--Madison, 1956. / Typescript. Vita. eContent provider-neutral record in process. Description based on print version record. Includes bibliographies: leaves 69-73, 98.

This document only includes an excerpt of the corresponding thesis or dissertation. To request a digital scan of the full text, please contact the Ruth Lilly Medical Library's Interlibrary Loan Department (rlmlill@iu.edu).

<p> The present investigation was concerned with mitochondrial energy transfer reactions and their relationship to mitochondrial structural integrity. Experiments with azide demonstrated a close relationship between oxidative phosphorylation and large amplitude mitochondrial volume changes. Azide inhibited energy transfer and energy-linked mitochondrial swelling by competing with adenine nucleotide for a site on the terminal phosphorylating enzyme. As a permeant anion azide exerted secondary effects on mitochondrial structure and function.</p> <p> Experiments with mitochondria treated with phlorizin and phloretin emphasized the importance of Mg++ as a controlling factor in maintaining the integrity of mitochondrial energy transfer processes. The results indicated that these compounds interfered directly with oxidative phosphorylation, and that mitochondrial swelling was either a consequence of impaired energy transfer, or a separate phenomenon.</p> / Thesis / Doctor of Philosophy (PhD)

京都大学 / 新制・課程博士 / 博士(医学) / 甲第23415号 / 医博第4760号 / 新制||医||1052(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 武藤 学, 教授 浅野 雅秀, 教授 川口 義弥 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第20568号 / 医博第4253号 / 新制||医||1022(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 原田 浩, 教授 武藤 学, 教授 戸井 雅和 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

BACKGROUND/OBJECTIVE: Converging evidence in recent years suggests growth chondrocytes, involved in the integral process of endochondral bone formation and fracture healing, exhibit a dynamic bioenergetic profile despite residing in the nutrient poor cartilaginous environment. Specifically, chondrocytes show an increased dependence on mitochondrial derived oxidative phosphorylation during differentiating, collagen product, but to a differing extent when mineralizing. Therefore, quantitative analysis of mitochondrial dynamics during these varying processes serves to corroborate existing metabolic studies and further elucidate the role of oxidative metabolism during the endochondral process. METHODS: The murine chondroprogenitor cell line ATDC5 was used, and groups were cultured in differentiating, collagen promoted, and mineralizing conditions. Fluorescence confocal 3D image acquisition and bioimaging analysis was used to quantify changes in mitochondrial volume and branch length per mitochondria along with organization and colocalization changes of the actin cytoskeleton to mitochondria in the various conditions over 21 days. RESULTS: We showed that chondrocyte differentiation resulted in significantly increased mitochondrial volume and fusion when compared to non-differentiating groups, and in collagen promoted groups, mitochondrial volume was significantly higher. Additionally, we showed that the process of mineralization resulted in a significant decrease in mitochondrial volume and branch length per mitochondria by day 21 of the experiment. Finally, colocalization analyses of the actin cytoskeleton to mitochondria showed significantly increased overlap in non-differentiating cells when compared to differentiating conditions. CONCLUSIONS: These findings suggest that collagen production is likely an energetically taxing process and mineralization does not heavily rely on oxidative metabolism. Furthermore, the actin cytoskeleton likely plays a role in mitochondrial remodeling that coincides with mitochondrial fission and fusion; increased fission is associated with actin accumulation to mitochondria and fusion is associated with actin disassociation from the outer mitochondrial membrane.