Konsumtion av kosttillskott i samband med träning är något som ökat i Sverige de senaste åren. Det är främst produkter med högt proteininnehåll som konsumeras, även om behovet av extra protein är under diskussion. Men det kan finnas andra ämnen med positiv påverkan på träning. Exempelvis mjölkfettkulemembran (MFGM) som är en viktig del av bröstmjölk då det bland annat verkar bidra till spädbarnets kognitiva utveckling. Senare studier på möss har visat att MFGM tycks ha positiva effekter på träning. Syftet med denna litteraturstudie var därför att undersöka om MFGM även har positiva effekter på människor i samband med träning. Artiklar togs fram genom sökning i databasen PubMed. Artiklarna skulle undersökt effekten av MFGM på människors träning för att inkluderas, samt vara randomiserade, dubbelblindade och placebokontrollerade. Totalt inkluderades fem studier. Samtliga studier visade på positiva effekter av MFGM i samband med träning, dock skiljde sig resultaten mellan studierna beroende på vilken effekt MFGM hade. Om MFGM har någon effekt på styrka är fortfarande oklart då resultaten är oeniga, däremot kan det tänkas att rörlighet och neuromuskulär utveckling förbättras av ett tillskott av MFGM, men ytterligare studier behövs för att bekräfta detta. / Consumption of dietary supplements associated with exercise has increased in Sweden the last couple of years. It is mainly products with high protein that are consumed, although the need for additional protein is questionable. However, there may be other substances with positive effects on exercise. For example milk fat globule membrane (MFGM) which is an important part of breast milk that seems to contribute to infant's cognitive development. Studies on mice have shown that MFGM seems to have positive effects on exercise. The purpose of this literature study was therefore to investigate whether MFGM also has positive effects on humans in combination with exercise. Articles for this study was selected by searching the PubMed database. The articles were included if they were studying the effect of MFGM on exercise on humans, they also had to be randomized, double blinded and placebo-controlled. A total of five studies were included. All studies showed positive effects of MFGM combined with exercise, however, the results differed between studies in terms of what kind of effect. If MFGM has any effect on strength is still unclear since the results varies. Agility and neuromuscular development may be improved by a dietary supplement of MFGM, but further studies are needed to confirm this.

Recent experiments suggest that the human genome (all of our DNA) is organised as a so-called fractal globule. The fractal globule is a knot--free dense polymer that easily folds and unfolds any genomic locus, for example a group of nearby genes. Proteins often need to locate specific target sites on the DNA, for instance to activate a gene. To understand how proteins move through the DNA polymer, we simulate diffusion of particles through a fractal globule. The fractal globule was generated on a cubic lattice as spheres connected by cylinders. With the structure in place, we simulate particle diffusion and measure how their mean squared displacement ($\langle R^2(t)\rangle$) grows as function of time $t$ for different particle radii. This quantity allows us to better understand how the three dimensional structure of DNA affects the protein's motion. From our simulations we found that $\langle R^2(t)/t\rangle$ is a decaying function when the particle is sufficiently large. This means that the particles diffuse slower than if they were free. Assuming that $\langle R^2(t) \rangle \propto t^\alpha$ for long times, we calculated the growth exponent $\alpha$ as a function of particle radius $r_p$. When $r_p$ is small compared to the average distance between two polymer segments $d$, we find that $\alpha \approx 1$. This means the polymer network does not affect the particle's motion. However, in the opposite limit $r_p\sim d$ we find that $\alpha<1$ which means that the polymer strongly slows down the particle's motion. This behaviour is indicative of sub-diffusive dynamics and has potentially far reaching consequences for target finding processes and biochemical reactions in the cell.

5-Aminolevulinate synthase (ALAS) catalyzes the pyridoxal 5'-phosphate (PLP)-dependent condensation between glycine and succinyl-CoA to generate coenzyme A (CoA), CO2, and 5-aminolevulinate (ALA). The chemical mechanism of this reaction, which represents the first and regulated step of heme biosynthesis in mammals, involves the formation of a short-lived glycine quinonoid intermediate and an unstable 2-amino-3-ketoadipate intermediate. Using liquid chromatography coupled with tandem mass spectrometry to analyze the products from the reaction of murine erythroid ALAS (mALAS2) with O-methylglycine and succinyl-CoA, we directly identified the chemical nature of the inherently unstable 2-amino-3-ketoadipate intermediate, which predicates the glycine quinonoid species as its precursor. With stopped-flow absorption spectroscopy, we detected and confirmed the formation of the quinonoid intermediate upon reacting glycine with ALAS. Significantly, in the absence of the succinyl-CoA substrate, the external aldimine predominates over the glycine quinonoid intermediate. When instead of glycine, L-serine was reacted with ALAS, a lag phase was observed in the progress curve for the L-serine external aldimine formation, indicating a hysteretic behavior in ALAS. Hysteresis was not detected in the T148A-catalyzed L-serine external aldimine formation. These results with T148A, a mALAS2 variant, which, in contrast to the wild-type enzyme, is active with L-serine, suggest that the active site T148 modulates the strict amino acid substrate specificity of ALAS. The rate of ALA release is also controlled by a hysteretic kinetic mechanism (observed as a lag in the ALA external aldimine formation progress curve), consistent with conformational changes governing the dissociation of ALA from ALAS. In Rhodobacter capsulatus ALAS, apart from coordinating the positioning of succinyl-CoA, N85 has an important role in regulating the opening of an active site channel. Here, we have mutated the analogous asparagine of murine erythroid ALAS to a histidine (N150H) and assessed its effects on catalysis through steady-state and pre-steady-state kinetic studies. Quinonoid intermediate formation occurred with a significantly reduced rate for the N150H-catalyzed condensation of glycine with succinyl-CoA during a single turnover. When the same forward reaction was examined under multiple turnovers, the progress curve of the N150H reaction displayed a prolonged decay of the quinonoid intermediate into the steady-state, distinct from the steep decay in the wild-type ALAS reaction. This prolonged decay results from an accelerated transformation of the product, ALA, into the quinonoid intermediate during the reverse N150H-catalyzed reaction. In fact, while wild-type ALAS catalyzes the conversion of ALA into the quinonoid intermediate at a rate 6.3-fold lower than the formation of the same quinonoid intermediate from glycine and succinyl-CoA, the rate for the N150H-catalyzed reverse reaction is 1.7-fold higher than that of the forward reaction. We conclude that N150 is important in establishing a catalytic balance between the forward and reverse reactions, by favoring ALA synthesis over its non-productive transformation into the quinonoid intermediate. Mutations at this position could perturb the delicate heme biosynthetic equilibrium. Circular dichroism (CD) and fluorescence spectroscopies were used to examine the effects of pH (1.0-3.0 and 7.5-10.5) and temperature (20 and 37 °C) on the structural integrity of ALAS. The secondary structure, as deduced from far-UV CD, is mostly resilient to pH and temperature changes. Partial unfolding was observed at pH 2.0, but further decreasing pH resulted in acid-induced refolding of the secondary structure to nearly native levels. The tertiary structure rigidity, monitored by near-UV CD, is lost under acidic and specific alkaline conditions (pH 10.5 and pH 9.5/37 °C), where ALAS populates a molten globule state. As the enzyme becomes less structured with increased alkalinity, the chiral environment of the internal aldimine is also modified, with a shift from a 420 nm to 330 nm dichroic band. Under acidic conditions, the PLP cofactor dissociates from ALAS. Reaction with 8-anilino-1-naphtalenesulfonic acid corroborates increased exposure of hydrophobic clusters in the alkaline and acidic molten globules, although the reaction is more pronounced with the latter. Furthermore, quenching the intrinsic fluorescence of ALAS with acrylamide at pH 1.0 and 9.5 yielded subtly different dynamic quenching constants. The alkaline molten globule state of ALAS is catalytically active (pH 9.5/37 °C), although the kcat value is significantly decreased. Finally, the binding of 5-aminolevulinate restricts conformational fluctuations in the alkaline molten globule. Overall, our findings prove how the structural plasticity of ALAS contributes to reaching a functional enzyme.

La forte prévalence des hémoglobinopathies, notamment l'HbAS, l'HbC et l'α-thalassémie dans des zones d'endémie palustre est considérée comme la conséquence de la protection qu'elles procurent contre les formes létales du paludisme. De nombreux mécanismes ont été évoqués pour expliquer cette protection, ne sont pas cohérentes avec toutes les observations épidémio-cliniques disponibles. La première partie de ces travaux de thèse a abordé la réduction de la cytoadhérence des globules rouges (GR) parasités comme mécanisme potentiel commun aux trois hémoglobinopathies. Pour explorer plus en profondeur ce mécanisme, nous avons mené une étude comparative des phénotypes de cytoadhérence des isolats primaires de P. falciparum obtenus chez des patients HbAS et HbAC et HbAA. Cela avait pour but de déterminer si l'HbAS et l'HbAC jouaient un rôle dans la sélection et le maintien des souches virulentes de P. falciparum dans la nature. La deuxième partie de la thèse a concerné l'influence du trait drépanocytaire (HbAS) sur la déformabilité des GR non parasités d'une part et sur la rétention splénique des GR non parasités et parasités par les formes jeunes de P. falciparum d'autre part. Nous avons observé que l'adhésion des GR parasités aux cellules endothéliales micro-vasculaires humaines ainsi qu'aux monocytes était réduite avec les GR α-thalassémiques par rapport aux GR HbAA. Aucune différence statistiquement significative n’a en revanche été observée entre les profils de cytoadhérence des isolats primaires de P. falciparum issus de sujets HbAA, HbAS ou HbAC. L’étude de la déformabilité des GR a montré que les GR HbAS sont légèrement mais significativement moins déformables que les GR HbAA. En revanche, les GR HbAS parasités par les jeunes parasites de P. falciparum (anneaux) n’étaient pas plus retenus que leurs homologues HbAA dans la rate humaine isolé perfusée ex vivo, ou en microsphiltration, quelles que soient les conditions d’oxygénation. Nous n’avons observé aucune différence au niveau du taux de falciformation entre les GR parasités et non parasités que ce soit avec les GR HbAS ou avec les GR HbSS. En conclusion, nous proposons que la réduction de la cythoadhérence et la rétention splénique des GR contenant les formes matures de P. falciparum constituent un mécanisme commun à la protection des sujets HbAS, HbC et α-thalassémiques contre le paludisme. Ces deux phénomènes interconnectés peuvent rendre compte de l’ensemble des observations épidemio-cliniques disponibles sur la protection conférée par ces hémoglobinopathies. / The high prevalence of several inherited hemoglobin disorders, namely sickle cell trait (HbAS), HbAC and α-thalassemia, in malaria endemic areas is thought to be the consequence of their protective effects against malaria life-threatening manifestations. Numerous potential mechanisms have been proposed to explain this protective effect although many of them are not fully consistent with all available epidemiologic and clinical data. The first part of this thesis work explored the reduction of cytoadherence of infected RBC as a potential common mechanism for α-thalassemia-, HbAS- and HbAC-induced protection against malaria. To further explore this mechanism, and determine whether HbAS and HbAC select and maintain virulent P. falciparum parasite in nature, we compared the cytoadherence phenotype of P. falciparum isolates obtained from HbAS/HbAC and controls HbAA patients. The second part of the thesis work addressed the influence of HbAS on the deformability of uninfected RBC as well as the splenic retention of both uninfected RBCs and ring-infected RBCs. We observed a reduced adherence of α-thalassemic infected RBCs to human micro-vascular endothelial cells and monocytes compared to controls HbAA infected RBCs. The reduction was correlated to the number of non functional α- gene. Expression of PfEMP-1 on the surface of α- thalassemic infected RBCs was lower than on the surface of HbAA infected RBCs. There was no statistically significant difference between the cytoadherence of P. falciparum isolates obtained either from HbAS/HbAC or control HbAA malaria patients. The deformability of uninfected HbAS RBCs was slightly but significant lower than that of control uninfected HbAA RBCs. Retention rates of ring-infected HbAS and HbAA RBCs were similar either in human isolated spleen perfusion ex vivo and in microsphilters in vitro regardless of the oxygenation level. We did not observe any enhanced sickling of ring-infected RBCs compared to non infected RBCs, both in HbAS and HbSS samples. Based on these results along with available epidemiologic and previous experimental data, we propose a common malaria-protective mechanism of HbAS, HbAC and α-thalassemia whereby these hemoglobin disorders reduce the cytoadherence of mature P. falciparum-infected RBCs that stay in circulation where they are exposed to an enhanced splenic retention. These 2 mechanisms would act in conjunction to slower the rise of parasites loads in infected patients and protect them from sequestration-related complications of malaria.

No description available.

Colicin A is a water-soluble toxin that forms a voltage-gated channel in the cytoplasmic membrane of target bacteria. In the present thesis, we aimed at studying the closed channel state, the membrane insertion mechanism, the acidic pH induced molten globule state and the interaction of colicin A in living E. coli cells. For that, we used Electron Paramagnetic Resonance (EPR) spectroscopy in combination with site-directed spin labeling (SDSL) method to explore the structural details of colicin A. The EPR studies of the membrane-bound colicin A (reconstituted into proteoliposomes) suggest the transmembrane orientation of the hydrophobic hairpin in the closed channel state. The pH dependent membrane insertion studies indicate that the membrane binding efficiency is significantly enhanced at pH < 3. Moreover, in the presence of a membrane potential, the pH induced membrane-bound state is able to open channels in the liposomes. The membrane-bound conformation (induced by acidic pH) is similar to the conformation of reconstituted colicin A which support the umbrella model for the closed channel state of colicin A. The studies on pH dependent conformational changes suggest that colicin A forms a molten globule at pH 2. The molecular details of pH induced conformational changes were analyzed by molecular dynamic simulations. The results of the MD simulations agree with the EPR results. Conformational changes of colicin A upon interaction with living E. coli cells could also be followed. Comparison between colicin A in wild type (WT) cells and tolB knock-out mutants suggest that the observed conformational changes originate from colicin A which has been already translocated to the inner membrane.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第19365号 / 医博第4042号 / 新制||医||1011(附属図書館) / 32379 / 新制||医||1011 / 京都大学大学院医学研究科医学専攻 / (主査)教授 椛島 健治, 教授 佐藤 俊哉, 教授 山田 泰広 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

CLIC1 is an intracellular membrane protein that is unusual in that it can exist in both a soluble and an integral membrane form. The manner in which this protein inserts into membranes is unknown although it is proposed to undergo a change in structure whereby it initially experiences a degree of unfolding and then refolds into its new membrane-bound conformation. This study focuses on the characterisation of CLIC1 in terms of its secondary, tertiary and quaternary structure, the determination of its conformational stability at equilibrium and the establishment of its unfolding kinetics, all under conditions of varying pH, polarity, redox conditions, temperature and ionic strength. CLIC1 was found to be most stable at pH 7.0 / 20oC. The unfolding process is two-state and cooperative, producing a DG(H2O) of ~10 kcal/mol and a m-value of ~2 kcal/mol per molar urea. A decrease in pH to 5.5 or an increase in temperature to 37oC resulted in the stabilisation of an equilibrium intermediate species under mild denaturing conditions and a destabilisation of the native state. This was further evidenced by an increase in the rate of unfolding of CLIC1 from the native state to the denatured state under these conditions. A state with similar properties to the intermediate species was detected in the absence of urea at pH 5.5 / 37oC and under non-reducing conditions at both pH 7.0 / 20oC and pH 5.5 / 20oC. The intermediate species is more hydrophobic than either the native or denatured state; it is stabilised by salts, has a reduced secondary structure, increased flexibility and a buried Trp35 relative to the native state. The rate of formation of the intermediate species is a slow process which may involve an oligomerisation step. The results from this study provide an interpretation for the structure and mechanism of CLIC1 pore formation in vivo by comparing the effects of the environment on the structure and stability of the protein.

No description available.

Lignocellulosic biomass represents the largest potential volume and lowest cost for biofuel and biochemical production. Harnessing the full potential of the lignocellulosic biomass for low-carbon energy requires the knowledge of efficient breakdown and fractionation of its carbohydrates and lignin. Organic solvent pretreatment is recognized as an emerging way ahead because of its inherent advantages, such as the ability to fractionate lignocellulosic biomass into cellulose, lignin, and hemicellulose components with high purity, as well as easy solvent recovery and solvent reuse. Through all-atom MD simulation, we analyze the conformational transition of diverse lignin molecules in varying concentration of Methanol/water , DMSO/water mixtures and neat DMSO , neat methanol and water. From our work, it appears that in 40 mol% DMSO and 40 mol% methanol mixture (’theta solvent’) hardwood lignin(G/S=1.35) conforms random coil like structure, while 60 mol% DMSO and 60 mol% methanol solution (at 300 K) appears to be ’good solvent’ forhardwood lignin since it conforms extended chain like structure. While 80 mol% methanol is proven to be ’theta solvent’ and 80 mol% DMSO is proven to be ’good solvent’ for softwood lignin. We find that, major functional moieties of both lignin preferentially coordinated by methanol and DMSO molecules in increased organic solvents concentration which induces the conformational transition from crumbled globule to coil and prevent self-aggregation of lignin in binary mixtures. Chain dynamics of lignin explain the relaxation and subsequently elongated in addition of organic solvents into water.