Protein kinase C α and ε phosphorylation of troponin and myosin binding protein C reduce Ca2+ sensitivity in human myocardium

Previous studies indicated that the increase in protein kinase C (PKC)-mediated myofilament protein phosphorylation observed in failing myocardium might be detrimental for contractile function. This study was designed to reveal and compare the effects of PKCα- and PKCε-mediated phosphorylation on myofilament function in human myocardium. Isometric force was measured at different [Ca2+] in single permeabilized cardiomyocytes from failing human left ventricular tissue. Activated PKCα and PKCε equally reduced Ca2+ sensitivity in failing cardiomyocytes (ΔpCa50 = 0.08 ± 0.01). Both PKC isoforms increased phosphorylation of troponin I- (cTnI) and myosin binding protein C (cMyBP-C) in failing cardiomyocytes. Subsequent incubation of failing cardiomyocytes with the catalytic subunit of protein kinase A (PKA) resulted in a further reduction in Ca2+ sensitivity, indicating that the effects of both PKC isoforms were not caused by cross-phosphorylation of PKA sites. Both isozymes showed no effects on maximal force and only PKCα resulted in a modest significant reduction in passive force. Effects of PKCα were only minor in donor cardiomyocytes, presumably because of already saturated cTnI and cMyBP-C phosphorylation levels. Donor tissue could therefore be used as a tool to reveal the functional effects of troponin T (cTnT) phosphorylation by PKCα. Massive dephosphorylation of cTnT with alkaline phosphatase increased Ca2+ sensitivity. Subsequently, PKCα treatment of donor cardiomyocytes reduced Ca2+ sensitivity (ΔpCa50 = 0.08 ± 0.02) and solely increased phosphorylation of cTnT, but did not affect maximal and passive force. PKCα- and PKCε-mediated phosphorylation of cMyBP-C and cTnI as well as cTnT decrease myofilament Ca2+ sensitivity and may thereby reduce contractility and enhance relaxation of human myocardium

Enzyme Promiscuity in Enolase Superfamily. Theoretical Study of o-Succinylbenzoate Synthase Using QM/MM Methods

The promiscuous activity of the enzyme o-succinylbenzoate synthase (OSBS) from the actinobacteria Amycolatopsis is investigated by means of QM/MM methods, using both density functional theory and semiempirical Hamiltonians. This enzyme catalyzes not only the dehydration of 2-succinyl-6R-hydroxy-2,4-cyclohexadiene-1R-carboxylate but also catalyzes racemization of different acylamino acids, with N-succinyl-R-phenylglycine being the best substrate. We investigated the molecular mechanisms for both reactions exploring the potential energy surface. Then, molecular dynamics simulations were performed to obtain the free energy profiles and the averaged interaction energies of enzymatic residues with the reacting system. Our results confirm the plausibility of the reaction mechanisms proposed in the literature, with a good agreement between theoretical and experimentally derived activation free energies. Our simulations unravel the role played by the different residues in each of the two possible reactions. The presence of flexible loops in the active site and the selection of structural modifications in the substrate seem to be key elements to promote the promiscuity of this enzyme.This work was supported by the Spanish Ministerio de Economia y Competitividad project CTQ2012-36253-C03-03 ́ and FEDER funds. K.S. thanks the Polish National Science Center (NCN) for Grant 2011/02/A/ST4/00246. The authors acknowledge computational facilities of the Servei d’Informatica ̀ de la Universitat de Valencia in the ̀ “Tirant” supercomputer, which is part of the Spanish Supercomputing Network

Transcendental-Phenomenological Proof and Descriptive Metaphysics

Following P.F. Strawson's reading of Kant, the majority of the literature on transcendental arguments seeks to divorce such arguments from their original Kantian context. This thesis is concerned with Mark Sacks's recent defence of transcendental arguments, which takes a different approach. A critique is given of Sacks's work and extensions and modifications of his approach are recommended. It is proposed that certain difficulties encountered by Kant's transcendentally-ideal approach can be overcome with Hegelian solutions

Wstępne obserwacje dotyczące zachowania się dwóch dzikich koniowatych: konia Przewalskiego (Equus ferus przewalskii ) i zebry Hartmanna (Equus zebra hartmannae) utrzymywanych w zmienionych pod wzgledem społecznym grupach w ZOO Warszawa

Preliminary observations of the behaviour in two wild equids : Przewalski horse (Equus ferus przewalskii) and Hartmann`s zebra (Equus zebra hartmannae) kept in socially changed groups at Warsaw Zoo Investigations were carried out at Warsaw Zoo to examine general behaviour in two wild equids after social changes. This changes were caused by management procedures. Three Hartmann`s zebra and four Przewalski horses were observed for 117 hours throughout the whole year. Three methods of sampling were used: ad libitum, scan and focal. Abnormal behaviour in these wild equids was not found. There were several significant differences in frequency of behaviour between Przewalski horse and Hartmann`s zebra . In both species coordination of behaviour also occured. Social interaction frequency was very low and gave little support for the speculation about social structures in these groups.Wstępne obserwacje dotyczące zachowania się dwóch dzikich koniowatych: konia Przewalskiego (Equus ferus przewalskii ) i zebry Hartmanna (Equus zebra hartmannae ) utrzymywanych w zmienionych pod wzgledem społecznym grupach w ZOO Warszawa. Badania przeprowadzono w ZOO Warszawa by zbadać ogólne zachowanie się dwóch dzikich koniowatych po zmianie społecznej. Zmiany te podyktowane były procedurami zarządzania zwierzętami w zoo. Trzy zebry Hartmanna i cztery konie Przewalskiego obserwowano przez 117 godzin w ciągu całego roku. Zastosowano trzy metody próbkowania: ad libitum, scan i focal. Nie stwierdzono występowania zachowania anormalnego. Odnotowano natomiast kilka wysoce istotnych różnic pomiędzy częstością występowania form zachowania pomiędzy zebrą Hartmanna a koniem Przewalskiego. U obydwóch gatunków zaobserwowano również koordynację behawioru w obrębie grupy. Częstość zachowań społecznych u obu gatunków była niska i nie dała podstaw do rozważań na temat struktury społecznej

Relationship between transferrin and globulin antigen polymorphism and sheep resistance to mastitis

The effect of transferrin genotypes and genes and globulin antigens on sheep resistance/susceptibility to mastitis was analysed. The udder health condition was diagnosed on the basis of the somatic cell count in 1 ml of milk and results of bacteriological tests. It was found that sheep with the transferrin genotype AC, AD and CC were characterised by a better health of the mammary gland than the remaining ewes. Among 9 transferrin alleles observed in the examined sheep, only the presence of transferrin I allele in the ewes genotype was connected with the somatic cell count below the mean value of this trait. The presence of transferrin A allele in the sheep's genotype was connected with a small infection of milk with mastitis pathogens. The serum globulin antigens analysed did not significantly affect the somatic cell count in the milk of the examined ewes. However, the antigens A2, NS1 (antigens of ß-globulin) and GB2 (antigen of class IgG immunoglobulins) as well as the globulin antigen A6 and GA1 antigen of class IgG immunoglobulins showed to have a significant effect on the level of sheep milk infection