京都大学 / 新制・課程博士 / 博士(理学) / 甲第23720号 / 理博第4810号 / 新制||理||1688(附属図書館) / 京都大学大学院理学研究科化学専攻 / (主査)教授 寺嶋 正秀, 教授 林 重彦, 教授 渡邊 一也 / 学位規則第4条第1項該当 / Doctor of Science / Kyoto University / DGAM

Two separate but related projects make up the work of this thesis. The growing amount of sequence data available in public databases provides an opportunity to compare species in new ways. It can be shown that there is a systematic change in amino acid composition in a dataset of sequences from 69 species possessing a range of optimal growth temperatures. By creating a phylogenetic tree of all available Archaea, pairs may be selected that contain a relatively closely related mesophile and (hyper)thermophile. In addition, pairs may be selected from Bacteria to include psychrophiles as well as other thermophiles. An evolutionary model is derived here that detects amino acid asymmetries in these species pairs beyond what might be expected to be caused by differences in GC content. This amino acid asymmetry can then be plausibly explained by temperature adaptation occurring in these species since they diverged from a common ancestor. In the second part, similarity searches using molecular sequences are drawn as networks, where open reading frames in one species may be linked to a corresponding sequence in another species if the similarity search score is above a given threshold. This process is similar to that used to identify orthologous sequences for use in evolutionary models. When drawn as a network of distinct clusters of similarity, patterns emerge that can be spurious or have some biological relevance. This work identifies the need to develop better methods of analyzing these network clusters. / Thesis / Master of Science (MSc)

PEA3, ERM, and ER81 comprise a subfamily of ETS transcription factors that upregulate genes correlated with an increased metastastic potential of tumors. In mouse embryo fibroblast (MEF) cells, PEA3 is required for transformation by activated Ras or Neu, but the means by which PEA3 mediates Ras-transformation is not clear. Osteopontin (OPN) expression is induced upon B-ras-transformation and purified PEA3 can bind the OPN promoter by gel-shift analysis. In this study, OPN expressed higher transcript levels in the wildtype MEF 4 cell line than the PEA3 null MEF 1 cell line and was further characterized as a potential PEA3 target gene by Northern blot analyses and transient transfection studies. Northern blot analyses of 4 wildtype MEF (4, 100, 101, 104), 5 FEA3 null MEF (1, 115, B5, B10, B12), and 5 MEF 1 retransformant cell lines that stably reexpress PEA3 showed a good correlation between OPN and ERM transcript levels in 9/11 cell lines although at least 2 PEA3 subfamily members were coexpressed in 8/11 cell lines that expressed high OPN transcript levels. This suggested that the PEA3 subfamily additively regulated OPN and that ERM protein was more abundant than PEA3 and ER81 protein levels in the MEF cell lines. The relative PEA3 subfamily protein levels remain to be clarified. Transient transfection assays in the HEK 293-1 C cell line indicated that the OPN promoter was responsive to PEA3 and that the promoter region between -258 to -88 was required for maximal OPN promoter activity. There are 16 candidate core ETS binding sites in the -777 /+79 OPN promoter which could be responsible for PEA3 subfamily transactivation. The OPN promoter was more active in the MEF 4 cell line than the MEF 1 cell line, corresponding to their relative number of expressed PEA3 subfamily members. Ectopic expression of dominant negative PEA3 suppress ed OPN promoter activity in the MEF 4 cell line. Furthermore, ectopic expression of PEA3, ERM, or ER81 increased OPN promoter activity in the MEF 1 or COS-1 cell line. Thus OPN is transcriptionally regulated by the PEA3 subfamily and represents a target gene that can mediate the progression of tumor cells. / Thesis / Master of Science (MSc)

The purpose of this study was to correlate the extent of myofibrillar disruption with muscle protein synthetic rate (MPS) following an isolated bout of concentric or eccentric elbow flexor resistance exercise. Six strength-trained males performed 8 unilateral sets of 8 repetitions at 80% concentric 1RM. The absolute amount of work performed by each arm was controlled by having both the concentrically-exercised (CON) and eccentrically-exercised (ECC) arms lift or lower the same weight through the same range and magnitude. Biopsies from biceps brachii of each arm, extracted ~21 h post -exercise, were analyzed electron microscopically to quantify myofibrillar disruption. The severity of disruption was classified as focal (FOC), moderate (MOD), or extreme (EXT). MPS of both arms was calculated from the increment in L- [1,2-¹³C₂] leucine abundance in biopsy samples relative to the mean plasma [1, 2-¹³C₂] -α-KIC enrichment at isotopic plateau using the primed-constant infusion technique over ~10 h so that the midpoint of the assessment period was ~24 h post-exercise. The severity of disruption was significantly (P < 0.001) greater in both the FOC (11.2%) and MOD (12.2%) compared to the EXT (1.6%) rating. Absolute disruption of fibers was significantly greater (P = 0.007) in the ECC (44.7%) as compared to baseline (BASE) samples (3.9%), obtained following 5 d where no arm training had occurred. In addition, ECC samples showed ~40% greater total disruption than CON samples (44.7% vs. 26.7%). Despite this, a positive correlation (r = 0.89) was found between individual values for MPS and the percentage of disrupted fibers in tissue from the ECC but not CON arm. These findings indicate that, in strength-trained males, residual myofibrillar disruption from a previous training session is essentially repaired within 5 d, but that resistance exercise induced muscle damage did not appear to provide the activating signal for elevating MPS between -21-29 h post-exercise. / Thesis / Master of Science (MSc)

Protein kinase C (PKC) is translocated to a phospholipid bilayer by calcium. Once at the membrane protein kinase C undergoes a conformational change which results in the removal of the pseudosubstrate domain from the active site. The enzyme then phosphorylates Ser/Thr residues on positively charged substrates. Certain substrates, however, can undergo cofactor independent phosphorylation by producing a conformational change in the enzyme in the absence of phospholipid and calcium. Studying the conformational change in PKC by physical techniques is difficult to perform with a phospholipid bilayer present. To study the conformational change in PKC in the absence of a membrane, the interactions between an Arginine-rich peptide (ARP), which underwent cofactor independent phosphorylation, and PKC was investigated. The Kₘ and kcₐₜ of the enzyme for ARP, in the absence of cofactors, was around 10 μM and 0.38 s⁻¹, respectively. The Kₘ did not significantly change upon the addition of phosphoipid and calcium. However, the kcₐₜ increased 2-3 fold in the presence of phospholipid and calcium. In the absence of phospholipid and calcium, ARP induced the exposure of hydrophobic site(s) on the enzyme. Additionally, ARP was able to promote the translocation of PKC to the membrane in the absence of calcium. PKC translocated to the membrane by ARP displayed the same susceptibility as the calcium membrane bound enzyme to limited proteolytic cleavage. Therefore, both ARP and calcium induce a similar membrane bound conformation in PKC. Additionally, the binding of ARP to PKC seems to occur through at least one high affinity site apart for the active site. These results demonstrate new insight into cofactor independent phosphorylation by PKC as well as illustrate a novel mechanism by which a substrate can promote the translocation of PKC in the absence of calcium. / Thesis / Master of Science (MSc)

Creatine-monohydrate (CrM) supplementation has been shown to increase body mass and fat-free mass (FFM), however, the mechanism by which CrM affects body composition has not been determined. We investigated the effects of short-term CrM supplementation on whole-body protein turnover in 27 recreationally active male and female volunteers. Subjects underwent measurements prior to and following 9-10 days of CrM (20 g/d x 5 d, 5 g/d x 4-5 d) (n =14), or placebo (PL) (n =13) supplementation. Protein turnover was assessed using L-[1-¹³C] leucine stable isotope tracer, urinary urea nitrogen (N) excretion, and N-balance (Nbal) techniques. Total body mass (TBM), leucine flux, leucine oxidation, non-oxidative leucine disposal (NOLD), 24-hr urinary urea N excretion, and Nbal were determined before and after treatment. Additionally, the effects of CrM supplementation on renal function and metabolite clearance were evaluated by measuring creatinine (CTN) excretion, plasma CTN concentration, and CTN clearance. There was no effect for CrM as compared to PL on TBM, leucine flux, leucine oxidation, or NOLD. However, leucine oxidation was lower for the CrM-treated males as compared to the PL-treated males following supplementation (P < 0.05). Leucine flux and NOLD were higher for the males vs. the females (P < 0.05). Neither urinary urea N excretion nor Nbal were affected by treatment. Plasma [CTN], CTN excretion, and CTN clearance were also unchanged for CrM vs. PL. These findings suggested that CrM supplementation may have an effect upon leucine oxidation in males, yet there were no effects seen in females, nor were other indices of leucine turnover altered by CrM supplementation. Furthermore, short-term CrM supplementation did not have any adverse effects on renal function. / Thesis / Master of Science (MSc)

Background & Rationale: During high intensity muscle contractions phosphocreatine is enzymatically degraded by creatine kinase (CK) to form creatine and the free energy which is released used to phosphorylate ADP to ATP. Creatine is then rephosphorylated during periods of relative ATP abundance (ie. rest) by CK back to phosphocreatine. Recognition of the importance of the phosphocreatine system to energy transduction has led many to believe that creatine monohydrate supplementation, which may lead to increases in phosptocreatine (PCr), may be beneficial during high intensity exercise. Several studies have demonstrated that creatine monohydrate supplementation for as few as three days can result in significant performance gains during exercise such as sprinting, or a weight lifting program. A common observation during these studies is a 1-2 kg increase in lean body mass (LBM). Although most researchers have speculated that this increase in LBM is due to water retention, some 𝘪𝘯 𝘷𝘪𝘵𝘳𝘰 work has demonstrated that creatine may stimulate protein synthesis. The purpose of this investigation was to examine whether a loading dose of creatine (20g/d x 7d) would have any affect on mixed muscle protein synthesis (MPS) in resting human skeletal muscle. Methods: A total of 22 young healthy subjects (n = 11 male, n = 11 female) were included in the study. On the day of measurement, subjects were provided with a meat free pre-packaged diet that was based upon individual diet records. Measurements of mixed muscle protein fractional synthetic rate (FSR) were completed over a 14 h resting period using a primed constant infusion of L[1-¹³C]leucine and muscle biopsies of 𝘲𝘶𝘢𝘥𝘳𝘪𝘤𝘦𝘱𝘴 𝘧𝘦𝘮𝘰𝘳𝘪𝘴 at isotopic plateau. Subjects were then randomly assigned to either a creatine (20g/d x 7d) or a placebo (isoenergetic glucose polymer) group. Following 7 days of supplementation, subjects reported to the lab under the same conditions as in the pre-trial, and resting mixed muscle protein FSR was again determined. Results: There were no significant between group differences in the baseline subject characteristics. No significant difference in FSR was observed with regards to condition (Pl: pre-0.63 ± 0.02 %/h; pst-0.71 ± 0.016 %/h; Cr: pre-0.56 ± 0.02 %/h; pst-0.58 ± 0.023 %/h) (creatine supplementation), time, or gender (Males: pre-0.06 ± 0.02 %/h; pst-0.068 ± 0.023 %/h; Females: pre-0.057 ± 0.02 %/h; pst-0.058 ± 0.015 %/h) Creatine supplementation resulted in a 13.1% increase in total creatine, however, no significant increases; in PCr or free Cr were observed. Similarly, no significant increases for fat free mass (FFM), or total mass were observed. Conclusion: It is concluded that creatine monohydrate supplementation for 7 d at 20g/d significantly increases muscle total creatine concentration, however, does not significantly affect muscle protein FSR in males or females. / Thesis / Master of Science (MSc)

Substantia nigra neurons produce dopamine in response to cholinergic stimuli that may involve receptor operated Ca²⁺ -entry that has been associated with the transient receptor potential (Trp) proteins. There were 6 Trp isoforms reported when I started this work. I set out to determine which isoforms of Trp mRNA were expressed in the substantia nigra using the whole brain for comparison. I initially used RT-PCR to determine the Trp mRNA expression. Subsequently, I used competitive RT-PCR for quantifying the major isoforms. Finally, I confirmed my results by Co-RT-PCR of the major isoforms. Trp3 and Trp6 were found to be the predominant forms expressed in the substantia nigra and whole brain, while the levels of Trps 1, 2, 4 and 5 were very low in both. Estimation of mRNA levels using competitive RT-PCR showed that the Trp6 mRNA levels in substantia nigra and the whole brain were similar while those for Trp3 were significantly lower in the substantia nigra than in the while brain. Thus substantia nigra differs from the whole brain in its Trp expression. Properties of Trps 3 and 6 are not fully known. Trp3 is regulated by IP₃-receptor activation but both Trp 3 and 6 can be activated by diacylglycerol. How this relates to the signal transduction events in substantia nigra remains to be determined. / Thesis / Master of Science (MS)

Sequences at the 5' end of the nascent RNA are known to be important as signals for encapsidation of the genome of vesicular stomatitis virus. In order to define the specific sequences involved in this process and to develop an in vitro encapsidation system, in vitro transcription from SP64-based plasmids was used to synthesize RNA molecules corresponding to various portions of the viral 5' plus strand sequence. Some of these RNAs were tested for their ability to bind the capsid N protein in vitro. N protein in this assay was provided either from VSV mRNA programmed reticulocyte lysates or from infected cell extracts or, in collaboration with Dr. Sue Moyer (Gainesville, Florida), purified from viral nucleocapsids. This thesis describes the construction of the SP64-based plasmids and the use of their RNA transcription products in the experiments described above. I also constructed a series of plasmids that could direct the synthesis of RNA molecules which have many of the features of VSV defective particle genomes. Two of the constructs generate a defective-like RNA carrying a reporter gene capable of expressing the bacterial lac Z protein. These RNAs have the potential, after in vitro encapsidation and transfection into mammalian cells, of producing readily detectable helper-dependent virions. / Thesis / Master of Science (MS)

Insufficient supply and pathogen safety concerns regarding plasma-derived therapeutic proteins, such as fibrinogen and immunoglobulins, have been the impetus for the development of genetic engineering techniques and separations methods for the economical and safe production of these proteins. This study is concerned with the isolation of these important therapeutics from complex media. Immunoaffinity chromatography has been an important method in the isolation of these products, typically being implemented as the final cleanup step yielding an extremely pure, homogenous final product. However, the use of immunoaffinity chromatography in large-scale purification processes have been precluded due to high capital costs and the inherent lability of immunosorbents. Peptide-based affinity sorbents are being developed in order to surmount the inherent limitations posed by monoclonal antibodies that are used as ligands in immunosorbents. The objective of this research is to quantitatively assess the impact of affinity ligand orientation, local density and transport phenomena on peptide-based affnity sorbent performance. The peptides under study herein can form high-affinity complexes with their protein targets, thus these ligands are one of the newest technologies arising from combinatorial chemistry with applications to the difficult problem of purifying high-molecular weight proteins from complex mixtures. Two types of structural motifs which are common to small peptide affinity ligands derived from combinatorial chemistry are studied here: a linear peptide which is comprised of the affinity recognition sequence in its entirety and a chain structure which displays multiple branches of the recognition sequence emanating from a central lysinic core structure. Two recognition sequences are studied here which bind plasma proteins. One peptide recognition sequence forms a high affinity complex with fibrinogen. Another peptide recognition sequence binds the Fc region of immunoglobulins. Immunglobulins are plasma proteins which range in molecular weight from 155 to 900-kDa and are valuable for therapeutic uses for imparting passive immunity. This study seeks to identify factors analogous to those manifested in immunosorbent performance that may also be important in the optimal design of peptide-based affinity sorbents. In general, previous research with the design of immunosorbents have found that immunosorbent performance, i.e., target-binding efficiency or activity, is substantially dependent upon several factors which include effects associated with ligand orientation, and local density as related to steric incumbrance of target binding sites, and transport phenomena as related to under utilization of intra matrix volume. In summary, this study asks the questions: (1) What factors regarding ligand orientation, local ligand density, and intraparticle transport phenomena, are important in the optimal design of peptide affinity sorbents?; and (2) Are these effects analogous to those manifested in immunosorbent performance? This study seeks to investigate the use of techniques used to mitigate the effects associated with these negative factors upon immunosorbent performance in order to elucidate the nature of these same effects upon peptide-based affinity sorbents. For example, oriented ligand immobilization can be facilitated through selective coupling chemistries and the premasking of ligand binding domains prior to immobilization. In addition, the manipulation of local ligand density using novel spatially controlled matrix activation and ligand immobilization methods can be assessed and implemented for the optimization of the performance and design of peptide-based affinity sorbents. This study has found that enhanced transport phenomena into the matrix interior volume can be achieved by using low solids content cellulose matrices having a low extent of crosslinking. This study demonstrates the effective use of these large-particle diameter, low-solids content cellulose hydrogel matrices in immunoaffinity, peptide-based affinity and ion exchange chromatography in the separation of high-molecular weight therapeutic proteins. / Ph. D. / This report presents an evaluation of the design of low-solids content, large-particle diameter beaded cellulose supports for column-mode protein purification. The study presented here optimizes the molecular accessibility of the cellulose support to high molecular weight proteins relative to the mechanical stability of the support at high operating linear velocities by the manipulation of bead particle diameter and solids content. A novel epoxidegradient activation method (epoxy-GAM) is developed for creating a gradient of support activation for support crosslinking and affinity ligand installation in the preparation of DEAE hydrogel matrices. These cellulose hydrogel supports were evaluated with regards to structure, dynamic and static binding capacity, pressure-flow stability, chemical stability and intraparticle transport phenomena. The utility of the low-solids content, large-particle diameter DEAE hydrogel matrices was demonstrated in a column-mode protein purification using albumin and fibrinogen mixtures.