The study of ligand binding specificities of the lipid binding proteins : recombinant human a-tocopherol transport protein (a-ttp), supernatant protein factor (spf) and S. cerevisiae Sec 14p for vitamin e (rrr-a-tocopherol) and other hydrophobic ligands.

One of the various functions of proteins in biological systems is the transport of small molecules, for this purpose proteins have naturally evolved special mechanisms to allow both ligand binding and its subsequent release to a target site; a process fundamental to many biological processes. Transport of Vitamin E (a-tocopherol), a lipid soluble antioxidant, to membranes helps in the protection of polyunsaturated fatty acids against peroxidative damage. In this research, the ligand binding characteristics of several members of the CRALTRIO family of lipid binding proteins was examined; the recombinant human a-Tocopherol Transfer Protein (a-TIP), Supernatant Protein Factor (SPF)ffocopherol Associated Protein (TAP), Cellular Retinaldehyde Binding Protein (CRALBP) and the phosphatidylinositol transfer protein from S. cerevisiae Sec 14p. Recombinant Sec 14p was expressed and purified from E. coli for comparison of tocopherol binding to the two other recombinant proteins postulated to traffic a-tocopherol. Competitive binding assays using [3H]-a-tocopherol and Lipidex-l000 resin allowed determination of the dissociation constants ~) of the CRAL-TRIO proteins for a-tocopherol and - 20 hydrophobic ligands for evaluation of the possible biological relevance of the binding interactions observed. The KIs (nM) for RRR-a-tocopherol are: a-TIP: 25.0, Sec 14p: 373, CRALBP: 528 and SPFffAP: 615. This indicates that all proteins recognize tocopherol but not with the same affinity. Sec 14p bound its native ligand PI with a KI of381 whereas SPFffAP bound PI (216) and y-tocopherol (268) similarly in contrast to the preferential binding ofRRR-a-tocopherol by a-TIP. Efforts to adequately represent biologically active SPFff AP involved investigation of tocopherol binding for several different recombinant proteins derived from different constructs and in the presence of different potential modulators (Ca+2, Mg+2, GTP and GDP); none of these conditions enhanced or inhibited a-tocopherol binding to SPF. This work suggests that only aTTP serves as the physiological mediator of a-tocopherol, yet structural homology between proteins allows common recognition of similar ligand features. In addition, several photo-affmity analogs of a-tocopherol were evaluated for their potential utility in further elucidation of a-TTP function or identification of novel tocopherol binding proteins.

Adaptations of skeletal muscle pyruvate dehydrogenase kinase in response to food-restriction in mitochondrial subpopulations

University, 2006 Dr. Sandra J. Peters Pyruvate dehydrogenase (PDH) catalyses the decarboxylation of pyruvate, to form acetyl-CoA. PDH activity is down-regulated by intrinsic PDH kinases (predominantly PDK2 and PDK4 isoforms), but the understanding of the PDK isoform distribution and adaptation to nutritional stresses has been restricted to mixed mitochondrial populations, and not delineated between subsarcolemmal (SS) and intermyofibrillar (IMF) subpopulations. SS and IMF mitochondria exhibit distinct morphological and biochemical properties; however the functional differences are not well understood. This study investigated the effect of fed (FED) versus 48 h total foodrestriction (FR) on rat red gastrocnemius muscle PDK2 and 4 isoform content in SS and IMF mitochondria. PDK4 content was ~3-5 fold higher in SS mitochondria compared to IMF (p=0.001), and increased with FR -3-4- fold in both subpopulations (p<0.001). PDK2 was -2.5-4 fold higher in SS mitochondria compared to IMF (p=0.001), but PDK2 was unaltered with FR. Citrate synthase activity (|imol/min/mg mitochondrial protein) was not different between either subpopulation. As well there were no significant differences between mitochondrial subpopulations in PDH complex components in both fed and FR states. These results demonstrate that there is a markedly higher content of both PDK isofonns in SS compared to IMF mitochondria. Although PDK2 does not increase in either subpopulation in response to FR, PDK4 increases to a similar extent in both SS and IMF after 48 h food-restriction.

Abrasion, transport and distribution of sediment in selected streams of Southern Ontario and Western New York /

The rate of decrease in mean sediment size and weight per square metre along a 54 km reach of the Credit River was found to depend on variations in the channel geometry. The distribution of a specific sediment size consist of: (1) a transport zone; (2) an accumulation zone; and (3) a depletion zone. These zones shift downstream in response to downcurrent decreases in stream competence. Along a .285 km man-made pond, within the Credit River study area, the sediment is also characterized by downstream shifting accumulation zones for each finer clast size. The discharge required to initiate movement of 8 cm and 6 cm blocks in Cazenovia Creek is closely approximated by Baker and Ritter's equation. Incipient motion of blocks in Twenty Mile Creek is best predicted by Yalin's relation which is more efficient in deeper flows. The transport distance of blocks in both streams depends on channel roughness and geometry. Natural abrasion and distribution of clasts may depend on the size of the surrounding sediment and variations in flow competence. The cumulative percent weight loss with distance of laboratory abraded dolostone is defined by a power function. The decrease in weight of dolostone follows a negative exponential. In the abrasion mill, chipping causes the high initial weight loss of dolostone; crushing and grinding produce most of the subsequent weight loss. Clast size was found to have little effect on the abrasion of dolostone within the diameter range considered. Increasing the speed of the mill increased the initial amount of weight loss but decreased the rate of abrasion. The abrasion mill was found to produce more weight loss than stream action. The maximum percent weight loss determined from laboratory and field abrasion data is approximately 40 percent of the weight loss observed along the Credit River. Selective sorting of sediment explains the remaining percentage, not accounted for by abrasion.

Amino acid transport : the special case of a H/L-glutamate cotransport system in Asparagus sprengeri mesophyll cells /

The addition of L-Glutamate (L-GLU) and L-Hethionine ~ulfoximine (L-HSO) to mechanically isolated. photosynthetically competent, Asparagus sprengeri mesophyll cells ~u~pended in 1mM CaS04 cau~ed an immediate transient alkalinization of the cell su~pension medium in both the light and dark. The alkalinization response was specific and stereospecific as none of the L-isomers of the other 19 protein amino acids tested or D-GLU gave this response. Uptake of 14C-L-GLU was stimulated by the light. The addition of non-radioactive L-GLU. or L-GLU analogs together with 14C-L-GLU showed that only L-GLU and L-HSO stimulated alkalinization whilst inhibiting the uptake of 14C-L-GLU. Both the L-GLU dependent alkalinization and the upt~ke of 14C-L-GLU were stimulated when the external pH was decreased from 6.5 to 5.5. Increasing external K+ concentrations inhibited the uptake of 14C-L-GLU. Fusicoccin (FC) stimulated uptake. The L-GLU dependent alkalinization re~ponse exhibited monophasic saturation kinetics while the uptake of 14C-L-GLU exhibited biphasic saturation kinetics. In addition to a saturable component. the uptake kinetics also showed a linear component of uptake. Addition of L-GLU and L-MSO caused internal acidification of the cell as measured by a change in the distribution of 14C-DMO. There was no change in K+ efflux when L-GLU was added. A H+ to L-GLUinflux stoichiometry of 3:1 wa~ mea~ured at an external I.-GLU concentration of O.5mM and increased with increasing external 13 L-QLU concentration. Metabolism of L-GLU was detected manometrlcally by observing an increase in COa evolution upon the addition of L-QLU and by detection of i*C02 evolution upon the addition of »*C-L-GLU. »*C02 evolution was higher in the dark than in the light. The data are consistent with the operation of a H+/L-QLO cotransport system. The data also show that attempts to quantify the stoichlometry of the process were complicated by the metabolism of L-GLU.

Significance of DNA base excision repair in the maintenance of mitochondrial function in Saccharomyces cerevisiae /

Mitochondria have an important role in cell metabolism, being the major site of ATP production via oxidative phosphorylation (OXPHOS). Accumulation of mtDNA mutations have been linked to the development of respiratory dysfunction, apoptosis, and aging. Base excision repair (BER) is the major and the only certain repair pathway existing in mitochondria that is in responsible for removing and repairing various base modifications as well as abasic sites (AP sites). In this research, Saccharomyces cerevisiae (S. cerevisiae) BER gene knockout strains, including 3 single DNA glycosylase gene knockout strains and Ap endonuclease (Apn 1 p) knockout strain were used to examine the importance of this DNA repair pathway to the maintenance of respiratory function. Here, I show that individual DNA glycosylases are nonessential in maintenance of normal function in yeast mitochondria, corroborating with previous research in mammalian experimental models. The yeast strain lacking Apn 1 p activity exhibits respiratory deficits, including inefficient and significantly low intracellular ATP level, which maybe due to partial uncoupling of OXPHOS. Growth of this yeast strain on respiratory medium is inhibited, but no evidence was found for increased ROS level in Apn 1 p mitochondria. This strain also shows an increased cell size, and this observation combined with an uncoupled OXPHOS may indicate a premature aging in the Apnlp knockout strain, but more evidence is needed to support this hypothesis. However, the BER is necessary for maintenance of mitochondrial function in respiring S.cerevisiae.

Comparison of two measures of PSI electron transport in winter rye

The number of P700 (the reaction centre of Photosystem I) converted to P700+, in winter rye, was determined by measuring the absorbance change at 820nm . It was found, with a single turnover flash, that thylakoids isolated from cold grown plants have a 50% greater number of P700 oxidized than thylakoids isolated from warm grown plants. Incubation of thylakoids in the dark at 35 C did not change the number of P700 oxidized. The conversion of P700 to P700+ with a single flash can be compared to a steady state rate of electron transport using a Clark electrode. The results for P700 oxidation using the absorbance change at 820 nm measure effects within the PSI complex whereas the results obtained from a Clark electrode measures steady state electron transport between the cytochrome blf complex and the PSI complex. In contrast to the results for P700 oxidation it was shown, using a Clark electrode, that both thylakoids from cold grown plants and thylakoids incubated at in the dark 35 C exhibited 50% higher rates of electron transport than thylakoids from warm grown plants. The correlation between the higher rate of steady state PSI electron transport observed in thylakoids isolated from cold grown winter rye and number of active PSI reaction centres localizes the site of the increase to the PSI reaction centre. In contrast the lack of correlation after incubation at 35 C indicates the increase in the rate of light saturated electron transport in thylakoids isolated from cold grown plants and thylakoids incubated in the dark at 35 C occur by different mechanisms.

Effects of large anphiphilic ligands upon the spectra and kinetics of cytochrome C oxidase

Cytoch ro me c oxidase (ferrocytochrome c : 02 oxidoreductase ; EC 1.9. 3.1) is the terminal enzyme in the mitochondrial electron transport chain, catalyzing the transfer of electrons from ferrocytochrome c to molecular oxygen. The effects of two large amphiphilic molecules .. valinomycin and dibucaine upon the spectra of the isolated enzyme and upon the activity of both isolated enzyme and enzyme in membrane systems are investigated by using spectrophotometric and oxygen electrode techniques. The results show that both valinomycin and dibucaine change the Soret region of the spectrum and cause a partial inhibition in a concentration range higher than that in which they act as ionophores. It is concluded that both valinomycin and dibucain~ binding induce a conformational change of the protein structure which modifies the spectrum of the a3 CUB centre and diminishes the rate of electron transfer between cytochrome a and the binuclear centre.

Glucosamine resistance in the yeast, Saccharomyces cerivisae [sic]

By using glucosamine resistant mutants of Saccharomyces ceriv~sa~ an attempt was made to discover the mechanisms which cause glucose repression and/or the Crabtree effect. The strains used are 4B2, GR6, lOP3r, GR8l and GRI08. 4B2 is a wild type yeast while the others are its mutants. To characterize the biochemical reactions which made these mutants resistant to glucosamine poisoning the following experiments were done~ 1. growth and respiration; 2. transport of sugars; 3. effect of inorganic phosphate (Pi): 4. Hexokinase; 5. In yivo phosphorylation. From the above experiments the following conclusions may be drawn: (i) GR6 and lOP3r have normal respiratory and fermentative pathways. These mutants are resistant to glucosamine poisoning due to a slow rate of sugar transport which is due to change in the cell membrane. (ii) GR8l has a normal respiratory pathway. The slow growth on fermentable carbon sourCEE indicates that in GR8l the lesion is in or associated with the glycolytic pathway. The lower rate of sugar transport may be due to a change in energy metabolism. The invivo phosphorylation rate indicates that in GR81 facilitated diffusion is the dominant transport mechanism. (iii) GR108 msa normal glycolytic pathway but the respiratory pathway is abnormal. The slow rate of sugar transport is due to a change in energy metabolism. The lower percentage of in vivo phosphorylation is probably due to a lowered availability of ATP because of the mitochondrial lesion. In all mutants resistance to glucosamine poisoning is due to a lower rate of utilization of ATP. which is caused by various mechanisms (see above), making less ADP available for phosphorylation via ATP synthase which utilizes inorganic phosphate. Because of the lower utilization of Pi, the concentration of intra-mitochondrial Pi does not go down thus protecting mutants from glucosamine poisoning.

Mitochondrial inheritance in ustilago violacea

The anther smut fungus U stilago violacea has been developed as an important model organIsm for genetic, morphological and physiological studies. Valuable information on the nuclear genetics on U stilago violacea has been obtained in the last 20-25 years. However, in this organism almost nothing is known about mitochondria which make up an important aspect of the fungal genetic system. One fundamental aspect, mitochondrial inheritance, was addressed by this investigation. Mitochondrial DNA (mtDNA) of U. violacea was purified and restriction fragments cloned. MtDNA restriction fragment length polymorphisms (RFLPs) were identified among different isolates and were used as genetic markers for studying mitochondrial inheritance in crosses between polymorphic isolates. Matings of the yeast-like haploid cells of opposite mating types resulted in dikaryons containing mitochondria from both parents. The dikaryons were induced to form hyphae and then allowed to revert to haploid growth, resulting 1ll a colony that is bisectored for the two nuclear types. Both nuclear-type progeny of each cross were examined for parental mitochondrial type: Either mitochondrial type was observed 1ll the progeny. Thus, mitochondrial inheritance is biparental in this organism. The recovery of both mitochondrial types in the progeny was non-random. In progeny with the nuclear genotype of the al mating type parent mitochondria from both parents were inherited equally well. However, 1ll progeny with the a2 mating type, mitochondria were inherited almost exclusively (94%) from the a2 parent.

Response rainbow trout (Salmon gairdneri) blood gas transport system to temperature, oxygen availability and photoperiod

Hematological status in rainbow trout, Salmo gairdneri, was examined in relation to eight combinations of three environmental fa ctors; temperature (5°, 20°C), oxygen availability «35%, >70% saturation) and photoperiod (16L:8D, 8L:16D) and evaluated by 3-factor analysis of variance. Hemog l obin and hematocrit , indicators of oxygenc arrying capacity increased significantly at the higher temperature, following exposure to hypoxia and in relation to reduced light period. Significant variations in mean corpuscular hemoglobin concentration were not detected. The effects of temperature and oxygen availability were more pronounced than that of photoperiod which was generally masked. Although oxygen availability and photoperiod did not interact with temperature, the interaction of the former fac tors was significant. Elec trophoresis revealed twelve hemoglobin isomorphs. Relative concentration changes were found in re lation to the factors c onsidered with temperature>hypoxia>photoperiod. Howeve r , in terms of absolute concentration, effects were hypoxia>temperature>photoperiod. Photoperiod effects were again masked by temperature and (or) hypoxia. Red cell +2 l eve ls of [CI ] and [Mg ], critical elements in the hemoglobin-oxygen affinity regulating system, were also significantly altered. Red cell CI +2 was influenced only by temperature ; Mg by temper ature and oxygen. No photoperiod influence on either ions was observed. Under nominal 'summer' conditions, these changes point to the likelihood of increases in oxygen-c arrying c apac ity coupled with low Hb-02 affinity adjustments which would be expected to increase oxygen delivery rates to their more rapidly metabolising tissues.

Thermoacclimatory variations in the activities of enzymes implicated in ion transport in the rainbow trout, salmo gairdneri

Two groups of rainbow trout were acclimated to 20 , 100 , and 18 o C. Plasma sodium, potassium, and chloride levels were determined for both. One group was employed in the estimation of branchial and renal (Na+-K+)-stimulated, (HC0 3-)-stimulated, and CMg++)-dependent ATPase activities, while the other was used in the measurement of carbonic anhydrase activity in the blood, gill and kidney. Assays were conducted using two incubation temperature schemes. One provided for incubation of all preparations at a common temperature of 2S oC, a value equivalent to the upper incipient lethal level for this species. In the other procedure the preparations were incubated at the appropriate acclimation temperature of the sampled fish. Trout were able to maintain plasma sodium and chloride levels essentially constant over the temperature range employed. The different incubation temperature protocols produced different levels of activity, and, in some cases, contrary trends with respect to acclimation temperature. This information was discussed in relation to previous work on gill and kidney. The standing-gradient flow hypothesis was discussed with reference to the structure of the chloride cell, known thermallyinduced changes in ion uptake, and the enzyme activities obtained in this study. Modifications of the model of gill lon uptake suggested by Maetz (1971) were proposed; high and low temperature models resulting. In short, ion transport at the gill at low temperatures appears to involve sodium and chloride 2 uptake by heteroionic exchange mechanisms working in association w.lth ca.rbonlc anhydrase. G.l ll ( Na + -K + ) -ATPase and erythrocyte carbonic anhydrase seem to provide the supplemental uptake required at higher temperatures. It appears that the kidney is prominent in ion transport at low temperatures while the gill is more important at high temperatures. 3 Linear regression analyses involving weight, plasma ion levels, and enzyme activities indicated several trends, the most significant being the interrelationship observed between plasma sodium and chloride. This, and other data obtained in the study was considered in light of the theory that a link exists between plasma sodium and chloride regulatory mechanisms.

Effects of large anphiphilic ligands upon the spectra and kinetics of cytochrome C oxidase

Cytoch ro me c oxidase (ferrocytochrome c : 02 oxidoreductase ; EC 1.9. 3.1) is the terminal enzyme in the mitochondrial electron transport chain, catalyzing the transfer of electrons from ferrocytochrome c to molecular oxygen. The effects of two large amphiphilic molecules - valinomycin and dibucaine upon the spectra of the isolated enzyme and upon the activity of both isolated enzyme and enzyme in membrane systems are investigated by using spectrophotometric and oxygen electrode techniques. The results show that both valinomycin and dibucaine change the Soret region of the speetrum and cause a partial inhibition in a concentration range higher than that in which they act as ionophores. It is concluded that both valinomycin and dibucaine binding induce a conformational change of the protein structure which modifies the spectrum of the a3 CUB centre and diminishes the rate of electron transfer between cytochrome a and the binuclear centre.

Magnetic and transport properties of (Ba1-xKx)Fe2As2

Single crystals of (Bal - xKx)Fe2As2 were prepared using the Sn flux method. Two heating methods were used to prepare the single crystals: the slow heating and rapid heating methods. It was found that the single crystals grown using the slow heating method were not superconducting due to a significant loss of potassium. When the rapid heating method was used, the single crystals were observed to be superconducting with the desired potassium concentration. The energy dispersive X-ray spectroscopy analysis indicated the presence of multiple phases in the single crystals. Using single crystal X-ray diffraction, the crystal structure of the single crystals was found to be 14/mmm tetragonal at room temperature. The magnetic measurements on the single crystals indicated the presence of multiple phases and magnetic impurities.

Cellular Mechanisms of Resveratrol's Interaction with Mitochondrial Reactive Oxygen Species Metabolism

Resveratrol, a polyphenol found naturally in red wines, has attracted great interest in both the scientific community and the general public for its reported ability to protect against many of the diseases facing Western society today. While the purported health effects of resveratrol are well characterized, details of the cellular mechanisms that give rise to these observations are unclear. Here, the mitochondrial antioxidant enzyme Mn superoxide dismutase (MnSOD) was identified as a proximal target of resveratrol in vitro and in vivo. MnSOD protein and activity levels increase significantly in cultured cells treated with resveratrol, and in the brain tissue of mice given resveratrol in a high fat diet. Preventing the increase in MnSOD levels eliminates two of resveratrol’s more interesting effects in the context of human health: inhibition of proliferative cell growth and cytoprotection. Thus, the induction of MnSOD is a critical step in the molecular mechanism of resveratrol. Mitochondrial morphology is a malleable property that is capable of impeding cell cycle progression and conferring resistance against stress induced cell death. Using confocal microscopy and a novel ‘cell free’ fusion assay it was determined that concurrent with changes in MnSOD protein levels, resveratrol treatment leads to a more fused mitochondrial reticulum. This observation may be important to resveratrol’s ability to slow proliferative cell growth and confer cytoprotection. Resveratrol's biological activities, including the ability to increase MnSOD levels, are strikingly similar to what is observed with estrogen treatment. Resveratrol fails to increase MnSOD levels, slow proliferative cell growth and confer cytoprotection in the presence of an estrogen receptor antagonist. Resveratrol's effects can be replicated with the specific estrogen receptor beta agonist diarylpropionitrile, and are absent in myoblasts lacking estrogen receptor beta. Four compounds that are structurally similar to resveratrol and seven phytoestrogens predicted to bind to estrogen receptor beta were screened for their effects on MnSOD, proliferative growth rates and stress resistance in cultured mammalian cells. Several of these compounds were able to mimic the effects of resveratrol on MnSOD levels, proliferative cell growth and stress resistance in vitro. Thus, I hypothesize that resveratrol interacts with estrogen receptor beta to induce the upregulation of MnSOD, which in turn affects cell cycle progression and stress resistance. These results have important implications for the understanding of RES’s biological activities and potential applications to human health.

Investigation into the relationships between molecular chaperones, mitochondrial antioxidant enzymes, and endothermic vertebrate longevity

The maximum lifespan (MLSP) of endothermic vertebrates can range from as little as a year to over two centuries, yet the underlying phenotype of aging is very similar amongst this group of organisms. One organelle that may be important in the phenotype of aging is the mitochondrion. When damaged, this organelle is thought to contribute to many of the neurodegenerative diseases of aging. For this thesis, mitochondria from brain tissues of 7 mammalian and 2 avian species were isolated to assess whether the antioxidant glutathione system and major molecular chaperone, HSP60, is correlated to species MLSP. Furthermore, HSP60, and the major endoplasmic reticulum chaperone, GRP78, were measured under basal conditions, and following the introduction of an oxidative stress (hydrogen peroxide) in cultured mammalian myoblasts from 10 different species. My results indicate that the enzymes involved in the glutathione defense system are not correlated to species MLSP in brain mitochondria; however HSP60 levels are indeed higher in the longer-lived species. HSP60 levels are also higher at the basal level in cultured mammalian myoblasts and after 1 hour of hydrogen peroxide exposure. GRP78 induction is not correlated to species MLSP at the basal level or following hydrogen peroxide exposure. Therefore, these results suggest that HSP60 is a correlate of longevity in endothermic vertebrate species, but neither the glutathione antioxidant defense system, nor GRP78, correlates to species longevity.