No description available.

The elucidation of the molecular mechanism of the cell response to moderate heating is of importance for understanding the events that occur in the cell upon use of heating for therapeutic purpose or during illnesses that are associated with fever. The aim of this work was to investigate and to compare the effects of mild (fever) and severe hyperthermia on functional properties of oxidative phosphorylation system in normal tissue mitochondria. Modular kinetic analysis for the first time was applied to evaluate effects of hyperthermia on oxidative phosphorylation in rat heart and liver mitochondria. We demonstrated that changes in mitochondrial functions induced by mild hyperthermia (42 ºC) are reversible but more severe hyperthermia (45 ºC) causes partially irreversible uncoupling and inhibition of mitochondrial respiration in state 3, hyperthermia remarkably (3.6-2.1 fold) activates ROS generation in heart mitochondria and that maximal increase in rate of H2O2 production and lipid peroxidation is observed in the fever temperature range. We show that the response of liver mitochondria and hepatocytes to hyperthermia is to certain extent dependent on gender and temperature. Specific differences of male rat liver and heart mitochondrial components phase transitions have been revealed by DSC analysis. / Ląstelių atsako į nuosaikią hipertermiją molekulinio mechanizmo išaiškinimas yra labai svarbus norint suprasti procesus, kurie vyksta ląstelėse jas kaitinant gydymo tikslais ar organizmui karščiuojant. Šio darbo tikslas buvo nustatyti ir palyginti švelnios (karščiavimo) ir šiurkščios hipertermijos poveikį oksidacinės fosforilinimo sistemos funkcijoms normalių audinių mitochondrijose. Pirmą kartą panaudojome modulių kinetinę analizę hipertermijos poveikiui širdies ir kepenų mitochondrijų oksidacinio fosforilinimo sistemai tirti. Mes nustatėme, kad švelnios hipertermijos (42 ºC) poveikis širdies mitochondrijų funkcijoms yra grįžtamas, bet šiurkštesnė hipertermija (45 ºC) sukelia dalinai negrįžtamą kvėpavimo ir fosforilinimo atskyrimą bei mitochondrijų kvėpavimo greičio trečioje metabolinėje būsenoje slopinimą. Hipertermija didino ROS gamybos greitį ir lipidų peroksidaciją, kurie buvo didžiausi karščiavimo temperatūroje. Nustatėme, kad kepenų mitochondrijų ir hepatocitų atsakas į hipertermiją priklauso nuo žiurkės lyties ir temperatūros. Atlikome palyginamąjį širdies ir kepenų mitochondrijų sandų fazinių virsmų analizę diferencine skenuojamaja kalorimetrija ir nustatėme būdingus skirtumus.

Dimorphic Leishmania donovani parasites exist as promastigotes in the sandfly vector and differentiate into amastigotes once injected into the skin of human hosts during a blood meal. The mechanisms and signals that are involved in triggering differentiation are not well understood in Leishmania. We have investigated whether tyrosine phosphorylation is a possible signalling component. Differential levels of tyrosine-phosphorylated proteins were observed in extracts from in vitro promastigote and amastigote cultures, with an overall reduction in the latter stage. Following this observation, the inhibition of tyrosine phosphorylation was examined in promastigotes using Tyrphostin AG1433, a broad-spectrum tyrosine phosphorylation inhibitor. AG1433 treated in vitro promastigote cultures differentiate into amastigote-like morphology, have reduced tyrosine phosphorylation level, and express the amastigote-specific marker A2 proteins. Our studies demonstrate that signal transduction mechanisms involving tyrosine phosphorylation/dephosphorylation events are involved in controlling L. donovani promastigote differentiation into amastigote forms.

Šiame darbe siekta išsiaiškinti riebalų rūgščių vaidmenį reguliuojant oksidacinį fosforilinimą saponinu permeabilizuotose žiurkės širdies raumens skaidulose. Pagrindiniai darbo uždaviniai: 1).Įvertinti įvairios struktūros riebalų rūgščių vaidmenį reguliuojant oksidacinį fosforilinimą saponinu permeabilizuotose žiurkės širdies raumens skaidulose;2) Naudojant egzogeninę ADP-suvartojančią piruvato kinazės ir fosfoenolpiruvato sistemą įvertinti, ar oksiduojantis riebalų rūgštims kinta išorinės mitochondrijų membranos laidumas ADP-ui; 3) Tirti, ar riebalų rūgščių oksidacija veikia funkcinę sąveiką tarp kreatino kinazės ir ADP/ATP nešiklio; 4).Nustatyti riebalų rūgščių oksidacijos poveikį mitochondrijų in situ morfologijai bei įvertinti dekstrano T70 poveikį mitochondrijų in situ kvėpavimo parametrams ir morfologijai. Mitochondrijose in situ oksiduojantis įvairios struktūros riebalų rūgštims, vienoms ar mišinyje su piruvatu+malatu, oksidacinio fosforilinimo tariamoji KmADP sumažėja panašiu laipsniu lyginant su piruvato+malato oksidacija. Šis poveikis yra grįžtamas, t.y. riebalų rūgščių oksidacija nedaro įtakos po jos vykstančiai neriebalinės kilmės substratų oksidacijai. Oksiduojantis riebalų rūgštims išsaugoma funkcinė sąveika tarp kreatino kinazės ir ANT, nepaisant ženklaus tar. KmADP reikšmės sumažėjimo. Elektroninės mikroskopijos metodu įvertinome, kad riebalų rūgščių sąlygotas KmADP sumažėjimas gali būti susijęs su mitochondrijų struktūros pokyčiais, kuriuos sukelia riebalų... [to full text] / The aim of this study was to investigate the influence of fatty acid oxidation on the regulation of oxidative phosphorylation in permeabilized rat cardiac fibers. The objectives of the study:1). To evaluate the influence of different fatty acids in the regulation of oxidative phosphorylation in fibers; 2). To evaluate the changes in outer mitochondrial membrane permeability for ADP during fatty acid oxidation by the means of exogenous ADP consuming system consisting of pyruvate kinase and phosphoenolpyruvate;3); To investigate the effect of fatty acid oxidation on the functional coupling between mitochondrial creatine kinase and adenine nucleotide translocase; 4). To investigate the effects of fatty acid oxidation and dextran T70 on the morphology and respiration of mitochondria in saponin-permeabilized rat cardiac fibers. The apparent Km of oxidative phosphorylation for ADP in saponin-permeabilized rat cardiac fibers is decreased several fold during oxidation of fatty acids alone or in the mixture with pyruvate compared to oxidation of pyruvate+malate. This effect is reversible, and fatty acid oxidation does not influence the subsequent oxidation of non-fatty substrates. The functional coupling between creatine kinase and adenine nucleotide translocase is not influenced by fatty acid oxidation and the efficiency of creatine kinase system does not depend on the nature of respiratory substrates. Analysis of electron microscopy images of fibres indicates that morphological... [to full text]

Functional metabolism of storage carbohydrates is vital to plants and animals. The water-soluble glycogen in animal cells and the amylopectin which is the major component of water-insoluble starch granules residing in plant plastids are chemically similar as they consist of α-1,6 branched α-1,4 glucan chains. Synthesis and degradation of transitory starch and of glycogen are accomplished by a set of enzymatic activities that to some extend are also similar in plants and animals. Chain elongation, branching, and debranching are achieved by synthases, branching enzymes, and debranching enzymes, respectively. Similarly, both types of polyglucans contain low amounts of phosphate esters whose abundance varies depending on species and organs. Starch is selectively phosphorylated by at least two dikinases (GWD and PWD) at the glucosyl carbons C6 and C3 and dephosphorylated by the phosphatase SEX4 and SEX4-like enzymes. In Arabidopsis insufficiency in starch phosphorylation or dephosphorylation results in largely impaired starch turnover, starch accumulation, and often in retardation of growth. In humans the progressive neurodegenerative epilepsy, Lafora disease, is the result of a defective enzyme (laforin) that is functional equivalent to the starch phosphatase SEX4 and capable of glycogen dephosphorylation. Patients lacking laforin progressively accumulate unphysiologically structured insoluble glycogen-derived particles (Lafora bodies) in many tissues including brain. Previous results concerning the carbon position of glycogen phosphate are contradictory. Currently it is believed that glycogen is esterified exclusively at the carbon positions C2 and C3 and that the monophosphate esters, being incorporated via a side reaction of glycogen synthase (GS), lack any specific function but are rather an enzymatic error that needs to be corrected. In this study a versatile and highly sensitive enzymatic cycling assay was established that enables quantification of very small G6P amounts in the presence of high concentrations of non-target compounds as present in hydrolysates of polysaccharides, such as starch, glycogen, or cytosolic heteroglycans in plants. Following validation of the G6P determination by analyzing previously characterized starches G6P was quantified in hydrolysates of various glycogen samples and in plant heteroglycans. Interestingly, glucosyl C6 phosphate is present in all glycogen preparations examined, the abundance varying between glycogens of different sources. Additionally, it was shown that carbon C6 is severely hyperphosphorylated in glycogen of Lafora disease mouse model and that laforin is capable of removing C6 phosphate from glycogen. After enrichment of phosphoglucans from amylolytically degraded glycogen, several techniques of two-dimensional NMR were applied that independently proved the existence of 6-phosphoglucosyl residues in glycogen and confirmed the recently described phosphorylation sites C2 and C3. C6 phosphate is neither Lafora disease- nor species-, or organ-specific as it was demonstrated in liver glycogen from laforin-deficient mice and in that of wild type rabbit skeletal muscle. The distribution of 6-phosphoglucosyl residues was analyzed in glycogen molecules and has been found to be uneven. Gradual degradation experiments revealed that C6 phosphate is more abundant in central parts of the glycogen molecules and in molecules possessing longer glucan chains. Glycogen of Lafora disease mice consistently contains a higher proportion of longer chains while most short chains were reduced as compared to wild type. Together with results recently published (Nitschke et al., 2013) the findings of this work completely unhinge the hypothesis of GS-mediated phosphate incorporation as the respective reaction mechanism excludes phosphorylation of this glucosyl carbon, and as it is difficult to explain an uneven distribution of C6 phosphate by a stochastic event. Indeed the results rather point to a specific function of 6-phosphoglucosyl residues in the metabolism of polysaccharides as they are present in starch, glycogen, and, as described in this study, in heteroglycans of Arabidopsis. In the latter the function of phosphate remains unclear but this study provides evidence that in starch and glycogen it is related to branching. Moreover a role of C6 phosphate in the early stages of glycogen synthesis is suggested. By rejecting the current view on glycogen phosphate to be a stochastic biochemical error the results permit a wider view on putative roles of glycogen phosphate and on alternative biochemical ways of glycogen phosphorylation which for many reasons are likely to be mediated by distinct phosphorylating enzymes as it is realized in starch metabolism of plants. Better understanding of the enzymology underlying glycogen phosphorylation implies new possibilities of Lafora disease treatment. / Pflanzen und Tiere speichern Glukose in hochmolekularen Kohlenhydraten, um diese bei Bedarf unter anderem zur Gewinnung von Energie zu nutzen. Amylopectin, der größte Bestandteil des pflanzlichen Speicherkohlenhydrats Stärke, und das tierische Äquivalent Glykogen sind chemisch betrachtet ähnlich, denn sie bestehen aus verzweigten Ketten, deren Bausteine (Glukosylreste) auf identische Weise miteinander verbunden sind. Zudem kommen in beiden Kohlenhydraten kleine aber ähnliche Mengen von Phosphatgruppen vor, die offenbar eine tragende Rolle in Pflanzen und Tieren spielen. Ist in Pflanzen der Einbau oder die Entfernung von Phosphatgruppen in bzw. aus Stärke gestört, so ist oft der gesamte Stärkestoffwechsel beeinträchtigt. Dies zeigt sich unter anderem in der übermäßigen Akkumulation von Stärke und in Wachstumsverzögerungen der gesamten Pflanze. Beim Menschen und anderen Säugern beruht eine schwere Form der Epilepsie (Lafora disease) auf einer Störung des Glykogenstoffwechsels. Sie wird durch das erblich bedingte Fehlen eines Enzyms ausgelöst, das Phosphatgruppen aus dem Glykogen entfernt. Während die Enzyme, die für die Entfernung des Phosphats aus Stärke und Glykogen verantwortlich sind, hohe Ähnlichkeit aufweisen, ist momentan die Ansicht weit verbreitet, dass der Einbau von Phosphat in beide Speicherkohlenhydrate auf höchst unterschiedliche Weise erfolgt. In Pflanzen sind zwei Enzyme bekannt, die Phosphatgruppen an unterschiedlichen Stellen in Glukosylreste einbauen (Kohlenstoffatome 6 und 3). In Tieren soll eine seltene, unvermeidbare und zufällig auftretende Nebenreaktion eines Enzyms, das eigentlich die Ketten des Glykogens verlängert (Glykogen-Synthase), den Einbau von Phosphat bewirken, der somit als unwillkürlich gilt und weithin als „biochemischer Fehler“ (mit fatalen Konsequenzen bei ausbleibender Korrektur) betrachtet wird. In den Glukosylresten des Glykogens sollen ausschließlich die C-Atome 2 und 3 phosphoryliert sein. Die Ergebnisse dieser Arbeit zeigen mittels zweier unabhängiger Methoden, dass Glykogen auch am Glukosyl-Kohlenstoff 6 phosphoryliert ist, der Phosphatposition, die in der Stärke am häufigsten vorkommt. Die Tatsache, dass in dieser Arbeit Phosphat neben Stärke auch erstmals an Glukosylresten von anderen pflanzlichen Kohlenhydraten (wasserlösliche Heteroglykane) nachgewiesen werden konnte, lässt vermuten, dass Phosphorylierung ein generelles Phänomen bei Polysacchariden ist. Des Weiteren wiesen die Ergebnisse darauf hin, dass Phosphat im Glykogen, wie auch in der Stärke, einem bestimmten Zweck dient, der im Zusammenhang mit der Regulation von Kettenverzweigung steht, und dass kein zufälliges biochemisches Ereignis für den Einbau verantwortlich sein kann. Aufgrund der grundlegenden Ähnlichkeiten im Stärke- und Glykogenstoffwechsel, liegt es nahe, dass die Phosphorylierung von Glykogen, ähnlich der von Stärke, ebenfalls durch spezifische Enzyme bewirkt wird. Ein besseres Verständnis der Mechanismen, die der Glykogen-Phosphorylierung zugrunde liegen, kann neue Möglichkeiten der Behandlung von Lafora disease aufzeigen.

Protein phosphorylation is one of the major post-translational modifications to allow for signal transmission and fine tuning of metabolism on the cellular proteomic level. As such it is “one of the last instances” to modulate the activity of enzymes and hence to impact the cellular life irrespective of the basic conditions provided by the genome – and epigenome– controlled gene expression. The evolutionary increase in cellular complexity is reflected by highly sophisticated regulatory networks in multicellular eukaryotes based on the transfer of phosphate mostly onto the side chains of serine, threonine and tyrosine residues. Nature has chosen phosphate for inter- and intracellular communication, which is also an integral component of nucleic acids and can be regarded as the molecule of choice for life. Currently, life science is interested to unravel the network of reversible protein phosphorylation that is catalyzed by two antagonistic enzyme classes: the protein kinases and protein phosphatases. We are currently in the era of proteomics and enormously benefit from the progress of mass-spectrometry methods. This is documented by a huge number of “proteomic studies” that mostly provide a simple inventory of the existence of proteins – and/or their phosphorylated forms – under more or less defined conditions. So far, the physiological correlations could be established only in a few cases, e.g. by comparing two physiological conditions. Another strategy, which was addressed in this work, is the systematic screening of mutants defective in genes encoding either protein kinases or protein phosphatases. This approach benefits from the ease to predict these enzymes due to the presence of characteristic protein motifs. In combination with the major goal of this work – to shed light on the impact of protein phosphorylation in the mitochondrial (mt) compartment – the yeast Saccharomyces cerevisiae was chosen as a model system because of its respiro-fermentative metabolism, that allows for the maintenance of respiratory defective mutants. Indeed, this reverse genetic approach successfully revealed two kinases (Pkp1p, Pkp2p) and two phosphatases (Ppp1p, Ppp2p) as the key components regulating the pyruvate dehydrogenase complex by phosphorylation of serine 313 of its α- subunit Pda1p. In addition, evidence is provided that Pkp1p has an additional role in the assembly process of the PDH complex. Also, the effect of the deletion of the COQ8 gene (gene engaged in coenzyme Q synthesis; originally named ABC1) leading to respiratory deficiency, could be correlated with the phosphorylation of subunit Coq3p of the mitochondrial ubiquinone biosynthesis complex. Finally, in the case of the kinase Sat4p (protein involved in salt tolerance), overexpression of the enzyme was used as an alternative approach to unravel the molecular basis of the originally observed salt sensitivity of sat4 mutants. The data suggest that Sat4p has a direct or indirect role in the late steps of iron-sulfur (Fe/S) cluster assembly of the so-called “aconitase-type” enzymes in mitochondria, accompanied by a strongly reduced steady state concentration of the Fe/S-cluster protein aconitase. Interestingly, a secondary phenotype became apparent upon overexpression of Sat4p: the abundance of the lipoic acid containing mitochondrial proteome was markedly reduced. Most likely this phenotype is due to the fact that the synthesis and/or attachment of lipoic acid depend on a Fe/S-cluster bearing enzyme. In the course of the work it became clear that the regulatory (mt) protein phosphorylation network of yeast evolved to meet the criteria of a life adapted to the ecological niche on temporarily available sugar rich sources. Clearly, the transfer of the respective data to higher eukaryotes is limited. However, it shows that yeast is primarily an excellent model system for the principal molecular reactions shared with higher eukaryotes. / Phosphorylierungen von Aminosäuren ist eine der verbreitetsten post-translationalen Modifikationen für zelluläre Signalübertragungswege und zur Regulation des Metabolismus auf Proteom-Ebene. Mit der reversiblen Protein-Phosphorylierung eng verbunden ist die unabhängige Modulation der Aktivität von Enzymen ungeachtet der Genom- und Epigenom-basierten Genexpression. Die evolutionäre Zunahme der zellularen Komplexität äußert sich in zunehmend komplexeren Regulations-Netzwerken in mehrzelligen eukaryotischen Organismen basierend auf dem Transfer von Phosphatgruppen vorzugsweise auf die Aminosäuren Serin, Threonin und Tyrosin. Die Natur hat evolutionär als Baustein der inter- und intrazellulären Kommunikation Phosphat gewählt, welches auch ein integraler Bestandteil der Nukleinsäuren ist und somit als das „Molekül der Wahl“ für das Leben bezeichnet werden darf. Die Lebenswissenschaften sind gegenwärtig daran interessiert das Netzwerk der Proteinphosphorylierung aufzuklären, welches durch zwei antagonistisch wirkende Enzymklassen, die Proteinkinasen und Proteinphosphatasen charakterisiert ist. Dabei profitieren wir gegenwärtig von den Fortschritten der „Proteomics-Ära“ auf dem Gebiet der massenspektrometrischen Proteinidentifizierung. Ausdruck dessen ist eine Vielzahl von Proteom-Studien, die jedoch meist nur eine einfache Inventarisierung der unter mehr oder weniger gut definierten zellulären Bedingungen existierenden Proteine in ihrer Phosphat-modifizierten oder unphosphorylierten Form darstellen. Die beteiligten Enzyme werden dabei kaum berücksichtigt. Insbesondere gilt dies für extra-cytoplasmatische Ereignisse. Bisher gelang es nur in wenigen Fällen eine Korrelation der physiologischen Rolle dieser Proteinmodifikation, z.B. durch den Vergleich der Phospho-Proteome unter zwei unterschiedlichen physiologischen Bedingungen, herzustellen. Eine andere Strategie, die auch Gegenstand dieser Arbeit ist, sieht ein Screening von Mutanten vor, die durch Deletionen von Genen, die für Proteinkinasen bzw. –phosphatasen kodieren, gekennzeichnet sind. Dieser Ansatz profitiert von der Existenz und leichten bioinformatischen Vorhersagbarkeit charakteristischer Kinase- bzw. Phosphatase- Sequenzmotive. In Kombination mit dem Hauptziel der Arbeit – Licht ins Dunkel der Proteinphosphorylierung im mitochondrialen Kompartiment zu bringen – wurde die Hefe Saccharomyces cerevisiae als Modellsystem gewählt, insbesondere vor dem Hintergrund ihres fermentativen Metabolismus. Als Beleg der prinzipiellen Funktionalität des vorgeschlagenen Ansatzes konnten zwei Kinasen (Pkp1p, Pkp2p) und zwei Phosphatasen (Ppp1p, Ppp2p) als Schlüsselkomponenten der Regulation des Pyruvatdehydrogenase (PDH) Komplexes identifiziert und charakterisiert werden. Darüber hinaus konnte sowohl das Zielprotein der Phosphorylierung, Pda1p, die α-Untereinheit des Komplexes, als auch die modifizierte Aminosäure (Serin 313) experimentell bestätigt werden. Ferner konnte der Atmungsdefekt von Stämmen mit einer nicht-funktionellen Abc1p-Kinase mit dem Phosphorylierungszustand der Untereinheit Coq3p des Ubiquinon-Biosynthese Komplexes und dem Ausfall der Ubiquinonsynthese korreliert werden. Eine alternative Herangehensweise, die Überexpression einer Kinase, führte zur Identifizierung möglicher Zielproteine von Sat4p. Vergleichende Analysen des 2D-gelelektrophoretisch separierten mitochondrialen Genoms mit dem des Wildtyps legen die Vermutung nahe, dass Sat4p eine direkte oder indirekte Rolle bei der Regulation der „Aconitase-Typ“ Eisen-Schwefel (Fe/S) Proteine besitzt. Der darüber hinaus beobachtete Effekt einer Abnahme von Liponsäure-tragenden mitochondrialen Enzymen, ist wahrscheinlich sekundärer Natur und kann durch die Zugehörigkeit der Liponsäure-Synthase zur oben erwähnten Gruppe der „Aconitase-Typ“ -Fe/S-Proteine erklärt werden. Im Verlauf der Arbeit wurde deutlich, dass das regulatorische Netzwerk der Proteinphosphorylierung der Hefe eher den Kriterien einer evolutionären Adaptation an eine spezifische ökologische Nische – der temporären Verfügbarkeit zuckerreicher Substanzen – entsprechen. Das schränkt die Übertragbarkeit der gewonnen Einsichten in die Regulation des mitochondrialen Metabolismus auf höhere Eukaryonten ein. Es zeigt jedoch, dass Hefe in erster Linie ein exzellentes Modellsystem für die prinzipiellen molekulare Mechanismen ist, die sie mit den höheren Eukaryonten teilt.

Ląstelių atsako į nuosaikią hipertermiją molekulinio mechanizmo išaiškinimas yra labai svarbus norint suprasti procesus, kurie vyksta ląstelėse jas kaitinant gydymo tikslais ar organizmui karščiuojant. Šio darbo tikslas buvo nustatyti ir palyginti švelnios (karščiavimo) ir šiurkščios hipertermijos poveikį oksidacinės fosforilinimo sistemos funkcijoms normalių audinių mitochondrijose. Pirmą kartą panaudojome modulių kinetinę analizę hipertermijos poveikiui širdies ir kepenų mitochondrijų oksidacinio fosforilinimo sistemai tirti. Mes nustatėme, kad švelnios hipertermijos (42 ºC) poveikis širdies mitochondrijų funkcijoms yra grįžtamas, bet šiurkštesnė hipertermija (45 ºC) sukelia dalinai negrįžtamą kvėpavimo ir fosforilinimo atskyrimą bei mitochondrijų kvėpavimo greičio trečioje metabolinėje būsenoje slopinimą. Hipertermija didino ROS gamybos greitį ir lipidų peroksidaciją, kurie buvo didžiausi karščiavimo temperatūroje. Nustatėme, kad kepenų mitochondrijų ir hepatocitų atsakas į hipertermiją priklauso nuo žiurkės lyties ir temperatūros. Atlikome palyginamąjį širdies ir kepenų mitochondrijų sandų fazinių virsmų analizę diferencine skenuojamaja kalorimetrija ir nustatėme būdingus skirtumus. / The elucidation of the molecular mechanism of the cell response to moderate heating is of importance for understanding the events that occur in the cell upon use of heating for therapeutic purpose or during illnesses that are associated with fever. The aim of this work was to investigate and to compare the effects of mild (fever) and severe hyperthermia on functional properties of oxidative phosphorylation system in normal tissue mitochondria. Modular kinetic analysis for the first time was applied to evaluate effects of hyperthermia on oxidative phosphorylation in rat heart and liver mitochondria. We demonstrated that changes in mitochondrial functions induced by mild hyperthermia (42 ºC) are reversible but more severe hyperthermia (45 ºC) causes partially irreversible uncoupling and inhibition of mitochondrial respiration in state 3, hyperthermia remarkably (3.6-2.1 fold) activates ROS generation in heart mitochondria and that maximal increase in rate of H2O2 production and lipid peroxidation is observed in the fever temperature range. We show that the response of liver mitochondria and hepatocytes to hyperthermia is to certain extent dependent on gender and temperature. Specific differences of male rat liver and heart mitochondrial components phase transitions have been revealed by DSC analysis.

We hypothesized that alterations in cardiac myofilaments are associated with hereditary canine DCM. DCM myofilaments demonstrated a reduction in EC50 and a modest decrease in maximum activity compared to non-failing dog samples. Treatment of myofilaments with the calcium sensitizer, bepridil, showed a reduction in EC50. Desmin and tropomyosin phosphorylation was increased in DCM. Desmin protein levels were increased in DCM. Total troponin I phosphorylation was unchanged, but S23/S24 phosphorylation was reduced in DCM. Myofilament-associated PKC-δ and -ζ were elevated in DCM, PKC- ε was modestly reduced, and PKC-α showed no change. These data are the first investigation of cardiac myofilaments in naturally occurring canine DCM, and support the hypothesis that alterations in cardiac myofilaments are associated with DCM. / OVC Pet Trust (operating funds)

Storage starch is synthesized in sub-cellular organelles called amyloplasts in higher plants. The synthesis of the starch granule is a result of the coordinated activity of several groups of starch biosynthetic enzymes. There are four major groups of these enzymes, ADP-glucose pyrophosphorylase (AGPase), starch synthases (SS), starch branching enzymes (SBE), and starch debranching enzymes (SDE). Starch phosphorylase (SP) exists as both dimeric and tetrameric forms in plastids in developing cereal endosperm and catalyses the reversible transfer of glucosyl units from glucose-1-phosphate to the non-reducing end of α-1-4 linked glucan chains, although the precise role in the pathway remains unclear. The present study was conducted to investigate the role and regulation of SP and SSIV in starch biosynthesis in developing maize endosperm. The results of this study showed that the tetrameric form of SP accounts for the majority of measurable catalytic activity, with the dimeric form being barely active and the monomer catalytically inactive. A catalytically active recombinant maize SP was heterologously expressed and used as an affinity ligand with amyloplast lysates to test protein-protein interactions in vitro. Results showed that the different multimeric status of SP influenced interactions with other enzymes of starch synthesis. Tetrameric SP interacted with SBEI and SSIIa, whilst the dimeric form of the enzyme interacted with SBEI, SBEIIb. All of these interactions were enhanced when amyloplasts were pre-treated with ATP, and broken following treatment with alkaline phosphatase (APase), indicating these interactions are regulated by protein phosphorylation. In addition, the catalytic activity of SSIV was reduced following treatment with APase, indicating a role for protein phosphorylation in the regulation of SSIV activity. Protein-protein interaction experiments also suggested a weak interaction between SSIV and SP. Multimeric forms of SP regulated by protein-protein interactions and protein phosphorylation suggested a role for SP in starch biosynthesis in maize endosperm.

Several factors including an unidentified endogenous substance were found to stimulate microsomal diacylglycerol acyltransferase (DGAT, EC 2.3.1.20) from a microspore-derived cell suspension culture of oilseed rape (Brassica napus L. cv Jet Neuf). Mg2+ salts were found to stimulate microsomal DGAT 14 to 23-fold. ATP and CoA were also found to stimulate the enzyme 2.4 and 12 fold respectively, although the effects were decreased in the presence of high Mg2+ concentrations. While microsomal DGAT activity was only slightly increased by the concentration of exogenous diacylglycerol in the reaction mixture it was increased substantially by the addition of exogenous phosphatidate. Other phospholipids tested were not found to have this stimulatory effect. During attempts to investigate possible covalent modification of the enzyme, the soluble fraction obtained from cell suspension homogenate was found to contain a small metastable organic molecule(s) which stimulated DGAT activity. Stimulation of microsomal DGAT by this factor was concentration dependent but not dependent on preincubation time. / xiii, 95 leaves : ill. ; 28 cm.