Potassium channel-interacting protein 1 (KChIP1), a Ca²⁺ sensor protein, regulates the function of A-type Kv4 potassium channels and increases their cell surface expression. Myristoylation at the N-terminus of KChIP1 has been suggested to facilitate membrane-binding, but was not sufficient for stable membrane assaociation. The aim of the present study is to investigate whether EF-hand motifs of KChIP1 are crucial for membranal targeting in addition to the N-terminal myristoyl group, and how the membrane association of KChIP1 is influenced by lipid compositions. According to hydropathy profile, EF-hands 3 and 4 of KChIP1 showed highly hydrophobicity. After deleting EF-hands 3 and 4, the altered microenvironment of Trp residue and decreased hydrophobicity were found in truncated KChIP1, but it still maintained £\-helix structure. Furthermore, truncated KChIP1 exhibited lower lipid-binding ability, affecting intracellular membrane localization and was almost diminished underlying increasing membrane permeability by digitonin in cells, suggesting that intact EF-hands 3 and 4 may be related to the anchorage of KChIP1 on cellular membrane. KChIP1, but not mutant, specifically bound with phosphatidylserine by lipid binding assay and the FTIR spectra showed the change of £\-helix structure by binding lipid large unilamellar vesicles was dependent on phosphatidylserine. Either phosphatidylserine or potassium channels enhanced KChIP1 to form tetramer for targeting to phospholipids by using chemical cross-linking assay. Taken together, our data highly suggest that intact of EF-hands 3 and 4 should structurally and functionally involve in fulfilling the physiological activity of KChIP1.

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Almost all mammalian cells contain energy-producing organelles called mitochondria. Phosphatidylethanolamine (PE) is a phospholipid which has been implicated to be important for mitochondrial function. The majority of mitochondrial PE is synthesized in mitochondria using the phosphatidylserine decarboxylase (PSD) pathway. To test the hypothesis that PE made from the PSD pathway is required for mitochondrial function, three Chinese Hamster Ovary Cell lines with different PSD-pathway defects were studied. These three cell lines referred to as PSB-2, R-41, and PSD knockdown cells all had ~35% reductions in mitochondrial PE levels compared to the parental cell line. As a result, the mitochondria from all three cell lines have abnormally high sedimentation densities and increased membrane potentials. However, the energy production, motility, and morphologies of each type of mutant mitochondria were each distinctly different from their parental cell line. / Experimental Medicine

Many factors influence male fertility, and conventional fertility evaluations are not able to reliably identify sub-fertile animals. The overall goal of this work was to explore the expression of phosphatidylserine (PS) on bovine sperm and investigate what factors may impact it, as previous research demonstrated that phosphatidylserine (PS) plays a role in murine fertilization. Despite conventionally being an apoptotic marker, it is present on viable and fertilization-competent murine sperm, however, less is known of the possible role of PS in bovine fertilization. In experiment 1, viable bovine sperm cells expressing PS were identified and PS levels in fresh and frozen semen were compared. Phosphatidylserine levels in frozen samples were significantly less than in fresh samples. We conclude that the cryopreservation process has an impact on PS expression in sperm by altering the proportion of sperm cells which are capable of fertilization. Experiment 2 examined PS levels in bulls with varying fertility levels based on sire conception rate (SCR). There was no difference in PS levels between high and low fertility bulls. There was a significant difference in PS levels of uncapacitated samples and those capacitated for one hour. These results warrant further investigation into the role of phosphatidylserine in bovine fertilization. / Master of Science / Improving the fertility of cattle is incredibly important to meet ever-growing consumer demands for animal protein. Researchers and producers can utilize a variety of reproductive technologies to improve their herds' reproductive efficiency. Phosphatidylserine (PS) is a glycerophospholipid which makes up a part of all cellular plasma membranes. Typically, it is used as a marker for cell death or apoptosis, however, some cells expose it on their surface temporarily while still viable, including sperm. Phosphatidylserine was found to be exposed on sperm from mice that were still viable and able to fertilize oocytes. Following that, the expression of PS in bovine sperm was investigated. Using bulls as a model, fresh semen was collected and analyzed for the level of PS expression then frozen and reanalyzed. We saw that there was a significant decrease in the level of PS expression in sperm that had been previously frozen, possibly due to damage to their membranes during the freezing process. Frozen semen from beef bulls with either high or low fertility was also analyzed. No difference was observed between bulls with varying levels of fertility. Addressing fertility issues in bulls is a complicated and multi-faceted issue which requires the use of many technologies and fertility markers. Further developing the knowledge of PS exposure in bulls and its relation to fertility and fertilization is worthwhile to attempt to improve the reproductive efficiency of cattle herds.

No description available.

Intrinsic association constants were determined for a series of aminopyridines on phosphatidylserine vesicles, through determination of electrophoretic mobility. The magnitudes of the constants were such that depolarization of the nerve terminal through binding to negative phospholipids seems unlikely to occur. The aminopyridines all had association constants between one half and one sixtieth the association constant of calcium. The aminopyridines probably block potassium channels in their enhancement of synaptic transmission. An interesting correlation was noted in that the ranking of the aminopyridines in terms of their affinity for these lipids was approximately the same as the ranking of their enhancement of synaptic transmission in a wide variety of biological preparations. A possible mechanism for this correlation is that adsorption of these drugs 2 to lipid membranes is the initial and rate-limiting step in their action.

Die durch Phosphatidylserin Decarboxylase (PSD) katalysierte Decarboxylierung von Phosphatidylserin (PS) zu Phosphatidylethanolamin (PE) ist für Mitochondrien in Hefe und Mäusen von essentieller Bedeutung. Im Rahmen der vorliegenden Dissertation wurde erstmals die Rolle dieses PE-Syntheseweges in Pflanzen untersucht. Die drei in Arabidopsis identifizierten PSD Gene atPSD1, atPSD2, atPSD3 codieren für Enzyme, die in Membranen der Mitochondrien (atPSD1), der Tonoplasten (atPSD2) und des Endoplasmatischen Retikulums (atPSD3) lokalisiert sind. Der Beitrag der einzelnen PSDs zur PE-Synthese wurde anhand von psd Null-Mutanten untersucht. Dabei stellte sich atPSD3 als das Enzym mit der höchsten Aktivität heraus. Alternativ zum PSD-Weg wird in Arabidopsis PE auch mittels Aminoalkohol-phosphotransferase synthetisiert. Der Verlust der gesamten PSD-Aktivität, wie es in der erzeugten psd Dreifachmutante der Fall ist, wirkt sich ausschließlich auf die Lipidzusammensetzung in der Mitochondrienmembran aus. Demzufolge wird extramitochondriales PE hauptsächlich über die Aminoalkoholphosphotransferase synthetisiert. Die veränderte Lipidzusammensetzung der Mitochondrienmembran hatte jedoch keinen Einfluss auf die Anzahl, Größe und Ultrastruktur der Mitochondrien sowie auf das ADP/ATP-Verhältnis und die Respiration. Neben der Bereitstellung von Reduktionsäquivalenten beeinflusst die Funktionalität der Mitochondrien auch die Bildung von Blüten- und Staubblättern. Diese Blütenorgane waren in der psd Dreifachmutante stark verändert, und der Blütenphänotyp ähnelte der APETALA3-Mutante. Dieses homöotische Gen ist für die Ausbildung von Blüten- und Staubblättern verantwortlich. Für die Erzeugung der Mutanten psd2-1 und psd3-1 wurde ein T-DNA Vektor verwendet, der den Promotor des APETALA3 Gens enthielt, welcher in den Mutanten psd2-1, psd3-1 sowie psd2-1psd3-1 und der psd1psd2-1psd3-1 Dreifachmutante eine vergleichbare Co-Suppression des APETALA3 Gens hervorruft. Der Blütenphänotyp trat jedoch nur in der psd Dreifachmutante auf, da nur in ihr die Kombination von geringen Funktionstörungen der Mitochondrien, hervorgerufen durch veränderte Lipidzusammensetzung, mit der Co-Suppression von APETALA3 auftritt. / Decarboxylation of phosphatidylserine (PS) to form phosphatidylethanolamine (PE) catalyzed by phosphatidylserine decarboxylase (PSD) is an essential reaction for mitochondria in yeast and mice. This dissertation describes the role of this biosynthesis pathway in plants for the first time. Three PSD genes were identified in Arabidopsis, atPSD1, atPSD2, atPSD3. The gene products localize to mitochondria (atPSD1), tonoplast (atPSD2) and endoplasmatic retikulum (atPSD3). Contribution to PE-synthesis of each PSD was analyzed using T-DNA insertion mutants. Thereby, atPSD3 was found to be the most active isoform. Alternatively, PE is also synthesized by the action of aminoalcohol phosphotransferase. Complete loss of PSD activity, like in the psd triple mutant, resulted in changes in lipid composition of mitochondria membranes exclusively. In conclusion the bulk of PE is synthesized by aminoalcohol phosphotransferase. Changed lipid composition of mitochondria did not result in changes of mitochondria number, structure, ADP/ATP ratio and respiration. Mitochondria functionality was formerly shown to effect formation of petals and stamens. These flower organs were drastically morphologically changed in psd triple mutants and showed strong similarities to APETALA3 mutants. APETALA3 is a homeotic gene responsible for specifying petals and stamens. Mutants psd2-1 and psd3-1 used for crossing psd double and triple mutants contained a T-DNA vector which include the promoter for APETALA3. This promoter caused co-suppression of the endogenous APETALA3 gene in all mutants isolated from the Arabidopsis Knockout Facility, whereas changed flower morphology occurred only in the triple mutant concluding a combined effect of co-suppression and a reduced functionality of mitochondria, caused by changed lipid composition.

京都大学 / 0048 / 新制・論文博士 / 博士(生命科学) / 乙第13281号 / 論生博第19号 / 新制||生||55(附属図書館) / 京都大学大学院生命科学研究科高次生命科学専攻 / (主査)教授 井垣 達吏, 教授 垣塚 彰, 教授 藤田 尚志 / 学位規則第4条第2項該当 / Doctor of Philosophy in Life Sciences / Kyoto University / DFAM

Lipid oxidation is one of the major challenges faced by the food industry as it contributes to the loss of nutritional quality and loss of flavor in food products. Studies have shown that naturally occurring phospholipids like phosphatidylserine (PS) and phosphatidylethanolamine (PE) can regenerate oxidized tocopherols and help delay the lipid oxidation in bulk oils and oil-in-water emulsions. Since consumers desire simpler and cleaner labels, without chemically synthesized antioxidants, this research is of great interest. The combination of PS and PE with tocopherols has already been studied. However, PS was a better antioxidant in combination with tocopherols in the oil-in-water emulsion system whereas PE was a better antioxidant in combination with tocopherols in bulk oils. But obtaining pure phospholipids is an expensive deal, therefore, this study uses the more economical alternative, high phosphatidylserine (PS) lecithin in combination with mixed tocopherols in soybean oil-in-water system. PS (30 µmol/kg emulsion) along with mixed tocopherols (3 µmol/kg emulsion) were dissolved in oil and emulsions stabilized by Tween20 were prepared. To determine the most effective concentration of mixed tocopherols, 0.5, 1.0, and 3 µmole of tocopherols/kg emulsion were used at pH of 3 and 7. Tocopherol with a concentration of 3 µmole/kg emulsion was found to be the most effective at pH 3. Tocopherols showed an extended lag phase at lower pH. The synergistic activities of authentic PS and high PS lecithin were compared in combination with tocopherol under similar conditions. They both had an almost similar lag phase. This combination was then tested for different pH of 3 and 7 and different salt concentrations of (0.5, 1, and 1.5 wt% of the emulsion) at pH 7 to determine the effects of external factors on the synergistic antioxidant combination. It was observed that the combination had extended antioxidant ability at lower pH of 3 whereas salt had no effect on the combination. The results showed that high PS lecithin forms a synergistic combination with mixed tocopherols to increase the lag phase in oil-in-water emulsions and can be used as a clean label antioxidant for oil-in-water emulsions.