Regulatory phosphorylation of cyclin-dependent kinase 2: insights from molecular dynamics simulations

The structures of fully active cyclin-dependent kinase-2 (CDK2) complexed with ATP and peptide substrate, CDK2 after the catalytic reaction, and CDK2 inhibited by phosphorylation at Thr14/Tyr15 were studied using molecular dynamics (MD) simulations. The structural details of the CDK2 catalytic site and CDK2 substrate binding box were described. Comparison of MD simulations of inhibited complexes of CDK2 was used to help understand the role of inhibitory phosphorylation at Thr14/Tyr15. Phosphorylation at Thr14/Tyr15 causes ATP misalignment for the phosphate-group transfer, changes in the Mg²⁺ coordination sphere, and changes in the H-bond network formed by CDK2 catalytic residues (Asp127, Lys129, Asn132). The inhibitory phosphorylation causes the G-loop to shift from the ATP binding site, which leads to opening of the CDK2 substrate binding box, thus probably weakening substrate binding. All these effects explain the decrease in kinase activity observed after inhibitory phosphorylation at Thr14/Tyr15 in the G-loop. Interaction of the peptide substrate, and the phosphorylated peptide product, with CDK2 was also studied and compared. These results broaden hypotheses drawn from our previous MD studies as to why a basic residue (Arg/Lys) is preferred at the P₊₂ substrate position. Figure View of the substrate binding site of the fully active cyclin-dependent kinase-2 (CDK2) (pT160-CDK2/cyclin A/ATP). The pThr160 activation site is located in the T-loop (yellow secondary structure). The G-loop, which partly forms the ATP binding site, is shown in blue. The Thr14 and Tyr15 inhibitory phosphorylation sites located in the G-loop are shown in licorice representation     

Structural and evolutionary aspects of two families of non-catalytic domains present in starch and glycogen binding proteins from microbes, plants and animals

Starch-binding domains (SBDs) comprise distinct protein modules that bind starch, glycogen or related carbohydrates and have been classified into different families of carbohydrate-binding modules (CBMs). The present review focuses on SBDs of CBM20 and CBM48 found in amylolytic enzymes from several glycoside hydrolase (GH) families GH13, GH14, GH15, GH31, GH57 and GH77, as well as in a number of regulatory enzymes, e.g., phosphoglucan, water dikinase-3, genethonin-1, laforin, starch-excess protein-4, the β-subunit of AMP-activated protein kinase and its homologues from sucrose non-fermenting-1 protein kinase SNF1 complex, and an adaptor-regulator related to the SNF1/AMPK family, AKINβγ. CBM20s and CBM48s of amylolytic enzymes occur predominantly in the microbial world, whereas the non-amylolytic proteins containing these modules are mostly of plant and animal origin. Comparison of amino acid sequences and tertiary structures of CBM20 and CBM48 reveals the close relatedness of these SBDs and, in some cases, glycogen-binding domains (GBDs). The families CBM20 and CBM48 share both an ancestral form and the mode of starch/glycogen binding at one or two binding sites. Phylogenetic analyses demonstrate that they exhibit independent behaviour, i.e. each family forms its own part in an evolutionary tree, with enzyme specificity (protein function) being well represented within each family. The distinction between CBM20 and CBM48 families is not sharp since there are representatives in both CBM families that possess an intermediate character. These are, for example, CBM20s from hypothetical GH57 amylopullulanase (probably lacking the starch-binding site 2) and CBM48s from the GH13 pullulanase subfamily (probably lacking the starch/glycogen-binding site 1). The knowledge gained concerning the occurrence of these SBDs and GBDs through the range of taxonomy will support future experimental research.     

Morphological and molecular diversity of the monocercomonadid genera Monocercomonas, Hexamastix, and Honigbergiella gen. nov

The family Monocercomonadidae (Parabasala, Trichomonadida) is characterized by the absence of a costa and in most species also of an undulating membrane; both of which are typical structures of trichomonadids. We have examined 25 isolates of Monocercomonadidae species by sequencing of the SSU rDNA and the ITS region and by light and transmission electron microscopy. The isolates formed three distinct phylogenetically unrelated clades: (1) Monocercomonas colubrorum, (2) Monocercomonas ruminantium together with a strain ATCC 50321 designated as Pseudotrichomonas keilini, and (3) Hexamastix. Twenty isolates of Monocercomonas colubrorum split into three clades with no host-specificity. The morphological differences among clades were insufficient to classify them as a separate species. Non-monophyly of the cattle commensal Monocercomonas ruminantium with the type species Monocercomonas colubrorum and absence of Pseudotrichomonas characters in the free-living strain ATCC 50321 led to their reclassification into a new genus (Honigbergiella gen. nov.). The close relationship of these strains indicates a recent switch between a free-living habit and endobiosis. Two strains of Hexamastix represented different species -Hexamastix kirbyi Honigberg 1955 and Hexamastix mitis sp. nov. Polyphyly of the Monocercomonadidae confirmed that the absence of a costa and an undulating membrane are not taxonomically significant characters and were probably secondarily lost in some or all clades. Our observations, however, indicated that other characters - infrakinetosomal body, comb-like structure, marginal lamella, and the type of axostyle - are fully consistent with the position of Monocercomonadidae species in the parabasalian tree and are, therefore, reasonable taxonomic characters.     

Relationships between membrane lipids and ion transport in red blood cells of Dahl rats

Distinct changes of membrane lipid content could contribute to the abnormalities of ion transport that take part in the development of salt hypertension in Dahl rats. The relationships between lipid content and particular ion transport systems were studied in red blood cells (RBC) of Dahl rats kept on low- and high-salt diets for 5 weeks since weaning. Dahl salt-sensitive (SS/Jr) rats on high-salt diet had increased blood pressure, levels of plasma triacylglycerols and total plasma cholesterol compared to salt-resistant (SR/Jr) rats. Furthermore, RBC of SS/Jr rats differed from SR/Jr ones by increased content of total membrane phospholipids, but membrane cholesterol was not changed significantly. SS/Jr rats had higher RBC intracellular Na+ (Na(i)+) content and enhanced bumetanide-sensitive Rb+ uptake. RBC membrane content of cholesterol and phospholipids correlated positively with RBC Na(i)+ content, with the activity of Na+-K+ pump and Na+-K+-2Cl- cotransport and also with Rb+ leak. The content of phosphatidylserines plus phosphatidylinositols was positively associated with RBC Na(i)+ content, with the activity of Na+-K+ pump and Na+-K+-2Cl- cotransport and with Rb+ leak. The content of sphingomyelins was positively related to Na+-K+-2Cl- cotransport activity and negatively to ouabain-sensitive Rb+-K+ exchange. We can conclude that observed relationships between ion transport and the membrane content of cholesterol and/or sphingomyelins, which are known to regulate membrane fluidity, might participate in the pathogenesis of salt hypertension in Dahl rats.     

The photosynthesis of individual algal cells during the cell cycle of Scenedesmus quadricauda studied by chlorophyll fluorescence kinetic microscopy

A microscope for imaging of chlorophyll fluorescence kinetics was equipped with a chamber that allows the growth of an immobilised population of algae and their study under well-defined conditions. Single cells of the chlorococcal alga Scenedesmus quadricauda were grown and recorded for periods of whole cell cycles (up to 48 h) displaying a normal course of cell development. Heterogeneity in fluorescence yield among individual coenobia in the population and among different cells in one coenobium were analysed. Differences were observed both in the shape of Kautsky transients and in the modulation of fluorescence parameter values during the progress of the cell cycle. The extent of heterogeneity in fluorescence parameters was cell cycle dependent – in some phases of the cycle, the population was almost homogeneous, while distinct heterogeneity was observed, in particular between the protoplast division and the release of the daughter coenobia. The heterogeneity was not random but reflected developmental processes.     

Die submontanen krautreichen Buchenwälder auf Silikatböden der westlichen Tschechoslowakei

Die submontanen krautreichen Buchenwälder auf Silikatböden in der westlichen Tschechoslowakei (Tschechische Sozialistische Republik) zerfallen in vier Assoziationen: 1.Tilio cordatae-Fagetum Mráz 1960 em.Moravec 1977, 2.Tilio platyphylli-Fagetum Klika 1939, 3.Melico-Fagetum Seibert 1954 und 4.Carici pilosae-Fagetum Oberdorfer 1957. DasTilio cordatae-Fagetum ist an niedrigere Bergländer Mittel-, Süd- und Westböhmens gebunden. DasTilio platyphylli-Fagetum ist eine lokal bedingte Assoziation des Westteils des Gebirges ?eské st?edoho?í. DasMelico-Fagetum kommt in Nord- bis Ostböhmen und Nord- und Mittelmähren vor. DasCarici pilosae-Fagetum ist auf die Karpaten beschränkt (ausnahmsweise greift es auf den Ostrand des Böhmischen Hochlandes über). Die Buchenwälder des Hügellandes bei Jevany (Mittelböhmen) und des Gebirges ?elezné hory gehören, trotz dem Vorkommen in submontanen Lagen, demDentario enneaphylli-Fagetum Oberdorfer ex W. etA. Matuszkiewicz 1960 an. Die Zusammensetzung der Assoziationen ist durch pflanzensoziologische Tabellen belegt und ihre Verbreitung auf einer Punktkarte erfasst.     

Rearing of germfree rabbits

The present work describes in detail experimental procedures and conditions required for rearing of germfree rabbits. Newborn germfree rabbits were obtained using the “wet” hysterectomy technique. The animals were successfully maintained under germfree conditions up to four months after delivery in flexible plastic isolators. Infant rabbits were first fed a sterile VA2-b diet and after weaning, a modifled diet L-478 according to Reddyet al. (1965). Both diets were shown to be suitable as the development of megacoecum was significantly reduced. The infant germfree rabbits were characterized by a good general clinical condition and body weights were comparable to those found in their conventional counterparts fed mother’s milk.     

Physical properties of microbial suspensions

A way of characterizing cell size distribution in suspensions of single-cell microorganisms is suggested, based on the first and second moments around origin. Suspension density may be predicted on the basis of the mixing law and the density of microbial dry matter, for suspensions ofSaccharomyces cerevisiae, Candida lipolytica, andAspergillus niger. Newtonian viscosities were correlated using regression analysis by η=Aexp(BC _m)exp(D/t); another correlation is presented for the calculation of surface tension in single-cell microbial suspensions. All the relations are valid in the range of concentrations up to 15% (w/v) and for temperatures between 15° and 35°C. The formulae presented may be used in other hydraulic studies.     

Joined suppression of rough phenotype and of red colour ofade2-1 mutants inSaccharomyces cerevisiae

A reciprocal suppression of rough phenotype was found in rough mutants of two prototrophic strains ofSaccharomyces cerevisiae on medium containing 1 % glucose. Suppression affects at least three independent rough loci and is strongly influenced by the genetic background. It is accompanied by suppression of red pigmentation inade2-1 mutants, and this too responds similarly to the genetic background. There is evidence for at least two independent loci that modify the action of the suppressor of both rough and red. The suppressor segregates regularly and is linked to one of the tested rough loci. It is not a super-suppressor and probably is not involved in the regulatory mechanism of purine biosynthesis either. An involvement of glucose metabolism in the suppression can be assumed from the lifting of the suppression of rough phenotype on lactate medium.