Effects of solvent, water activity and temperature on lipase and hydroxynitrile lyase enantioselectivity

The influence of the reaction conditions on the enantioselectivity of reactions catalysed by lipases or hydroxynitrile lyases (HNLs) in organic solvents was investigated. The lipases catalysed kinetic resolution of chiral secondary alcohols or chiral carboxylic acids and the HNLs catalysed asymmetric addition of hydrogen cyanide to aldehydes.

The temperature effects on enantioselectivity were studied in detail. From measurements of the enantiomeric ratio (E) at different temperatures the activation parameters ΔΔH^# and ΔΔS^# were determined. In the lipase-catalysed reactions the enthalpic and entropic effects on E always counteracted, while in a few of the HNL-catalysed reactions, ΔΔH^# and ΔΔS^# had opposite signs and therefore the effects cooperated to give high E values (−RTlnE = ΔΔG^# = ΔΔH^# − TΔΔS^#). In all the HNL-catalysed reactions and most of the lipase-catalysed ones, the enantioselectivity increased with decreasing reaction temperature. However, in one of the lipase-catalysed reactions, the enantioselectivity decreased with decreasing temperature. The theoretical background of these observations was discussed.

In the HNL-catalysed reactions, the enantioselectivity increased with increasing water content up to water saturation, while in the lipase-catalysed reactions the opposite trend was found in one case and in the others no significant effect was observed. Solvent mixtures of diisopropylether and hexane were used to obtain solvents with different log P values. The log P value of the solvent did not influence the enantioselectivity in the HNL-catalysed reactions, while the enantioselectivity increased with increasing log P value in two of the lipase-catalysed reactions. The reaction temperature was shown to be a very useful way to influence enzyme selectivity and the effects obtained could be rationalised. The influence of the reaction medium (solvent and water activity) is much more difficult to rationalise and predict.     

Enzymatic conversions of polar lipids. Principles, problems and solutions

This text provides a brief overview of the principles of enzymatic lipid conversion and some recent advances in the enzymatic conversion of glycerophospholipids and galactolipids. Lipases and phospholipases are used to exchange fatty acids or the polar group in the lipids. The reactions can be carried out either as hydrolysis–esterification sequences or as one-step transferase reactions. The scope and limitations of the different methods are discussed.     

A theoretical study of myoglobin working as a nitric oxide scavenger

The mechanism for the reaction between nitric oxide (NO) and O₂ bound to the heme iron of myoglobin (Mb), including the following isomerization to nitrate, has been investigated using hybrid density functional theory (B3LYP). Myoglobin working as a NO scavenger could be of importance, since NO reversibly inhibits the terminal enzyme in the respiration chain, cytochrome c oxidase. The concentration of NO in the cell will thus affect the respiration and thereby the synthesis of ATP. The calculations show that the reaction between NO and the heme-bound O₂ gives a peroxynitrite intermediate whose O–O bond undergoes a homolytic cleavage, forming a NO₂ radical and myoglobin in the oxo-ferryl state. The NO₂ radical then recombines with the oxo-ferryl, forming heme-bound nitrate. Nine different models have been used in the present study to examine the effect on the reaction both by the presence and the protonation state of the distal His64, and by the surroundings of the proximal His93. The barriers going from the oxy-Mb and nitric oxide reactant to the peroxynitrite intermediate and further to the oxo-ferryl and NO₂ radical are around 10 and 7 kcal/mol, respectively. Forming the product, nitrate bound to the heme iron has a barrier of less than ~7 kcal/mol. The overall reaction going from a free nitric oxide and oxy-Mb to the heme bound nitrate is exergonic by more than 30 kcal/mol.     

A unique histone 3 lysine 14 chromatin signature underlies tissue-specific gene regulation

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An Integrated Understanding of the Rapid Metabolic Benefits of a Carbohydrate-Restricted Diet on Hepatic Steatosis in Humans

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Musical improvisation skill in a prospective partner is associated with mate value and preferences,consistent with sexual selection and parental investment theory: Implications for the origin of music

Music is a human universal, which suggests a biological adaptation. Several evolutionary explanations have been proposed, covering the entire spectrum of natural, sexual, and group selection. Here we consider the hypothesis that musical behaviour constitutes a reliable or even costly signal of fitness, and thus may have evolved as a human trait through sexual selection. We experimentally tested how musical performance quality (MPQ), in improvisations on the drums, saxophone, and violin, affects mate values and mate preferences perceived by a prospective partner. Swedish student participants (27 of each sex) saw a face of a person of the opposite sex and heard a piece of improvised music being played. The music occurred in three levels of MPQ and the faces in three levels of facial attractiveness (FA). For each parametric combination of MPG and FA, the participants rated four mate value scales (intelligence, health, social status, and parenting skill) and four mate preference scales (date, intercourse, and short- and long term relationship). Consistent with sexual selection theory, mate value ratings were generally increased by MPQ for raters of both sexes. Consistent with more specific hypotheses that follow from combining sexual selection and parental investment theory, women’s but not men’s preference for a long-term, but not short-term, relationship was significantly increased by MPQ, MPQ generally affected women’s ratings more than men’s, FA generally affected men’s ratings more than women’s, and women’s ratings of intelligence were even more influenced by MPQ than by FA.     

Structure and positioning comparison of two variants of penetratin in two different membrane mimicking systems by NMR.

The Antennapedia homeodomain protein of Drosophila has the ability to penetrate biological membranes and the third helix of this protein, residues 43-58, known as penetratin (RQIKIWFQNRRMKWKK-amide) has the same translocating properties as the entire protein. The variant, RQI KIFFQNRRMKFKK-amide, here called penetratin (W48F,W56F) does not have the same ability. We have determined a solution structure of penetratin and investigated the position of both peptides in negatively charged bicelles. A helical structure is seen for residues Lys46 through Met54. The secondary structure of the variant penetratin(W48F,W56F) in bicelles appears to be very similar. Paramagnetic spin-label studies and analysis of NOEs between penetratin and the phospholipids show that penetratin is located within the bicelle surface. Penetratin (W48F,W56F) is also located inside the phospholipid bicelle, however, with its N-terminus more deeply inserted than that of wild-type penetratin. The subtle differences in the way the two peptides interact with a membrane in an equilibrium situation could be important for their translocating ability. As a comparison we have also investigated the secondary structure of penetratin(W48F,W56F) in SDS micelles and the results show that the structure is very similar in SDS and bicelles. In contrast, penetratin(W48F,W56F) and penetratin appear to be located differently in SDS micelles. This clearly shows the importance of using realistic membrane mimetics for investigating peptide-membrane interactions.     

Peptide degradation is a critical determinant for cell-penetrating peptide uptake

Cell-penetrating peptide mediated uptake of labels appears to follow an equilibrium-like process. However, this assumption is only valid if the peptides are stabile. Hence, in this study we investigate intracellular and extracellular peptide degradation kinetics of two fluorescein labeled cell-penetrating peptides, namely MAP and penetratin, in Chinese hamster ovarian cells. The degradation and uptake kinetics were assessed by RP-HPLC equipped with a fluorescence detector. We show that MAP and penetratin are rapidly degraded both extracellularly and intracellularly giving rise to several degradation products. Kinetics indicates that intracellularly, the peptides exist in (at least) two distinct pools: one that is immediately degraded and one that is stabile. Moreover, the degradation could be decreased by treating the peptides with BSA and phenanthroline and the uptake was significantly reduced by cytochalasin B, chloroquine and energy depletion. The results indicate that the extracellular degradation determines the intracellular peptide concentration in this system and therefore the stability of cell-penetrating peptides needs to be evaluated.     

The structure and dynamics of ring chromosomes in human neoplastic and non-neoplastic cells

Acquired ring chromosomes have been found in most types of human neoplasia, with a frequency approaching 10% in malignant mesenchymal tumours. In this study, the composition and dynamics of ring chromosomes were analysed in eight cases of acute myelogenous leukaemia, 17 solid tumours, and five cases with constitutional rings. Chromosomal banding and fluorescence in situ hybridisation were performed to determine the content and the structural heterogeneity of the rings. Telomeric repeats were detected using peptide nucleic acid probes or primed in situ labelling, whereas centromeric activity was evaluated by detection of kinetochore proteins. Mitotic instability was assessed by the frequency of anaphase bridges. The results suggest that human ring chromosomes can be structurally and functionally divided into two categories. In the first of these, size variation is minimal and rearrangement at cell division is uncommon. The majority of such rings contain subtelomeric sequences. Constitutional ring chromosomes and most rings in leukaemias belong to this group, whereas only a few mesenchymal tumours exhibit rings of this type. The second category consists of rings with amplified sequences, primarily from chromosome 12, characteristically occurring in atypical lipomatous tumours and other subtypes of low or borderline malignant mesenchymal neoplasms. Variation in size and number is extensive, and breakage-fusion-bridge events occur at a high frequency. Abnormalities in pericentromeric sequences are common and, in some cases, kinetochores assemble in the absence of alphoid DNA. We conclude that it is not only the ring structure per se or the neoplastic nature of the host cell that determines ring instability, but probably also the functional role of the genes carried in the ring. Received: 24 November 1998 / Accepted: 18 January 1999