Thermus thermophilus glycoside hydrolase family 57 branching enzyme: crystal structure, mechanism of action, and products formed

Branching enzyme (EC 2.4.1.18; glycogen branching enzyme; GBE) catalyzes the formation of α1,6-branching points in glycogen. Until recently it was believed that all GBEs belong to glycoside hydrolase family 13 (GH13). Here we describe the cloning and expression of the Thermus thermophilus family GH57-type GBE and report its biochemical properties and crystal structure at 1.35-Å resolution. The enzyme has a central (β/α)₇-fold catalytic domain A with an inserted domain B between β2 and α5 and an α-helix-rich C-terminal domain, which is shown to be essential for substrate binding and catalysis. A maltotriose was modeled in the active site of the enzyme which suggests that there is insufficient space for simultaneously binding of donor and acceptor substrates, and that the donor substrate must be cleaved before acceptor substrate can bind. The biochemical assessment showed that the GH57 GBE possesses about 4% hydrolytic activity with amylose and in vitro forms a glucan product with a novel fine structure, demonstrating that the GH57 GBE is clearly different from the GH13 GBEs characterized to date.     

Prenatal diagnosis--principles of diagnostic procedures and genetic counseling

The frequency of inherited malformations as well as genetic disorders in newborns account for around 3-5%. These frequency is much higher in early stages of pregnancy, because serious malformations and genetic disorders usually lead to spontaneous abortion. Prenatal diagnosis allowed identification of malformations and/or some genetic syndromes in fetuses during the first trimester of pregnancy. Thereafter, taking into account the severity of the disorders the decision should be taken in regard of subsequent course of the pregnancy taking into account a possibilities of treatment, parent's acceptation of a handicapped child but also, in some cases the possibility of termination of the pregnancy. In prenatal testing, both screening and diagnostic procedures are included. Screening procedures such as first and second trimester biochemical and/or ultrasound screening, first trimester combined ultrasound/biochemical screening and integrated screening should be widely offered to pregnant women. However, interpretation of screening results requires awareness of both sensitivity and predictive value of these procedures. In prenatal diagnosis ultrasound/MRI searching as well as genetic procedures are offered to pregnant women. A variety of approaches for genetic prenatal analyses are now available, including preimplantation diagnosis, chorion villi sampling, amniocentesis, fetal blood sampling as well as promising experimental procedures (e.g. fetal cell and DNA isolation from maternal blood). An incredible progress in genetic methods opened new possibilities for valuable genetic diagnosis. Although karyotyping is widely accepted as golden standard, the discussion is ongoing throughout Europe concerning shifting to new genetic techniques which allow obtaining rapid results in prenatal diagnosis of aneuploidy (e.g. RAPID-FISH, MLPA, quantitative PCR).     

Physical properties of the lipid bilayer membrane made of calf lens lipids: EPR spin labeling studies

The physical properties of a membrane derived from the total lipids of a calf lens were investigated using EPR spin labeling and were compared with the properties of membranes made of an equimolar 1-palmitoyl-2-oleoylphosphatidylcholine/cholesterol (POPC/Chol) mixture and of pure POPC. Conventional EPR spectra and saturation-recovery curves show that spin labels detect a single homogenous environment in all three membranes. Profiles of the order parameter, hydrophobicity, and oxygen transport parameter are practically identical in lens lipid and POPC/Chol membranes, but differ drastically from profiles in pure POPC membranes. In both lens lipid and POPC/Chol membranes, the lipids are strongly immobilized at all depths, which is in contrast to the high fluidity of the POPC membrane. Hydrophobicity and oxygen transport parameter profiles in lens lipid and POPC/Chol membranes have a rectangular shape with an abrupt change between the C9 and C10 positions, which is approximately where the steroid ring structure of cholesterol reaches into the membrane. At this position, hydrophobicity increases from the level of methanol to the level of hexane, and the oxygen transport parameter increases by a factor of 2-3. These profiles in POPC membranes are bell-shaped. It is concluded that the high level of cholesterol in lens lipids makes the membrane stable, immobile, and impermeable to both polar and nonpolar molecules.     

An estimation of the effects of synthetic auxin and cytokinin and the time of their application on some morphological and physiological characteristics of Medicago x varia T. Martyn

The aim of the experiment was to determine the effects of synthetic auxin and cytokinin and the time of their application on some morphological and physiological characteristics of Medicago x varia T. Martyn grown under controlled conditions. The experiment was to check whether an application of exogenous hormones during vegetative and generative stages of the plant had an effect on above-ground mass development, on nitrate reductase activity and on plastid pigments content. Experiment factor was synthetic auxin and cytokinin and the date of their application. Auxin was applied in the form of a synthetic indole-3-butyric acid, while cytokinin was sprayed as synthetic 6-benzylaminopurine. The control plants were treated with distilled water. Depending on the experimental variant, spraying was applied at the sixth true leaf stage and at the first flower bud stage. The research showed that the response of the alfalfa plants to the application of cytokinin and auxin was not uniform. It seems that the most effective was the application of a mixture of them both but only during the vegetative stage.Additionally, cytokinin caused an increase in plastid pigments content in alfalfa leaves. On the other hand, a mixture of auxin and cytokinin triggered the highest nitrate reductase activity in alfalfa roots and raised the ratio of total chlorophyll content to carotenoids. Synthetic auxin caused the decrease of the levels of most parameters compared to the control.     

Comparison of sterol and fatty acid profiles of chytrids and their hosts reveals trophic upgrading of nutritionally inadequate phytoplankton by fungal parasites

Chytrids are ubiquitous fungal parasites in aquatic ecosystems, infecting representatives of all major phytoplankton groups. They repack carbon from inedible phytoplankton hosts into easily ingested chytrid propagules (zoospores), rendering this carbon accessible to zooplankton. Grazing on zoospores may circumvent bottlenecks in carbon transfer imposed by the dominance of inedible or poorly nutritious phytoplankton (mycoloop). We explored qualitative aspects of the mycoloop by analysing lipid profiles (fatty acids, sterols) of two chytrids infecting two major bloom-forming phytoplankton taxa of contrasting nutritional value: the diatom Asterionella formosa and the filamentous cyanobacterium Planktothrix agardhii. The polyunsaturated fatty acid composition of chytrids largely reflected that of their hosts, highlighting their role as conveyors of otherwise inaccessible essential lipids to higher trophic levels. We also showed that chytrids are capable of synthesizing sterols, thus providing a source of these essential nutrients for grazers even when sterols are absent in their phytoplankton hosts. Our findings reveal novel qualitative facets of the mycoloop, showing that parasitic chytrids, in addition to making carbon and essential lipids available from inedible sources, also upgrade their host's biochemical composition by producing sterols de novo, thereby enhancing carbon and energy fluxes in aquatic food webs.     

SCA14 mutation V138E leads to partly unfolded PKCγ associated with an exposed C‐terminus,altered kinetics,phosphorylation and enhanced insolubilization

The protein kinase C γ (PKCγ) undergoes multistep activation and participates in various cellular processes in Purkinje cells. Perturbations in its phosphorylation state, conformation or localization can disrupt kinase signalling, such as in spinocerebellar ataxia type 14 (SCA14) that is caused by missense mutations in PRKCG encoding for PKCγ. We previously showed that SCA14 mutations enhance PKCγ membrane translocation upon stimulation owing to an altered protein conformation. As the faster translocation did not result in an increased function, we examined how SCA14 mutations induce this altered conformation of PKCγ and what the consequences of this conformational change are on PKCγ life cycle. Here, we show that SCA14‐related PKCγ‐V138E exhibits an exposed C‐terminus as shown by fluorescence resonance energy transfer‐fluorescence lifetime imaging microscopy in living cells, indicative of its partial unfolding. This conformational change was associated with faster phorbol 12‐myristate 13‐acetate‐induced translocation and accumulation of fully phosphorylated PKCγ in the insoluble fraction, which could be rescued by coexpressing PDK1 kinase that normally triggers PKCγ autophosphorylation. We propose that the SCA14 mutation V138E causes unfolding of the C1B domain and exposure of the C‐terminus of the PKCγ‐V138E molecule, resulting in a decrease of functional kinase in the soluble fraction.

     

The Vanadium Iodoperoxidase from the Marine Flavobacteriaceae Species Zobellia galactanivorans Reveals Novel Molecular and Evolutionary Features of Halide Specificity in the Vanadium Haloperoxidase Enzyme Family

Vanadium haloperoxidases (VHPO) are key enzymes that oxidize halides and are involved in the biosynthesis of organo-halogens. Until now, only chloroperoxidases (VCPO) and bromoperoxidases (VBPO) have been characterized structurally, mainly from eukaryotic species. Three putative VHPO genes were predicted in the genome of the flavobacterium Zobellia galactanivorans, a marine bacterium associated with macroalgae. In a phylogenetic analysis, these putative bacterial VHPO were closely related to other VHPO from diverse bacterial phyla but clustered independently from eukaryotic algal VBPO and fungal VCPO. Two of these bacterial VHPO, heterogeneously produced in Escherichia coli, were found to be strictly specific for iodide oxidation. The crystal structure of one of these vanadium-dependent iodoperoxidases, Zg-VIPO1, was solved by multiwavelength anomalous diffraction at 1.8 Å, revealing a monomeric structure mainly folded into α-helices. This three-dimensional structure is relatively similar to those of VCPO of the fungus Curvularia inaequalis and of Streptomyces sp. and is superimposable onto the dimeric structure of algal VBPO. Surprisingly, the vanadate binding site of Zg-VIPO1 is strictly conserved with the fungal VCPO active site. Using site-directed mutagenesis, we showed that specific amino acids and the associated hydrogen bonding network around the vanadate center are essential for the catalytic properties and also the iodide specificity of Zg-VIPO1. Altogether, phylogeny and structure-function data support the finding that iodoperoxidase activities evolved independently in bacterial and algal lineages, and this sheds light on the evolution of the VHPO enzyme family.     

A novel insight into the structure of amphivasal secondary bundles on the example of Dracaena draco L. stem

Key message

Pattern of tracheids found along the bundles extends understanding of their cross - sectional anatomy and sheds a new light on the issue of radial transport in monocotyledons with secondary growth.

Abstract

Secondary growth of Dracaena draco L. stem is connected with the formation of amphivasal vascular bundles in which a centrally located phloem is surrounded by a ring of xylem cells (tracheids). However, as visible in a single transverse section, there is a tendency towards variation among the secondary bundles from such with a xylem ring to ones in which the tracheids do not completely surround the phloem, i.e., are separated by vascular parenchyma cells. We aimed to elucidate the cross-sectional anatomy of amphivasal secondary bundles using the method of serial sectioning (with sections 3 μm thick), which allowed us to follow very precisely the bundle structure along its length. The analysis revealed that the xylem arrangement in these bundles depends on the position of a section in the bundle path. Each amphivasal bundle is composed of sectors where tracheids form a ring, as well as of such where tracheids are separated by vascular parenchyma cells. We hypothesize that this structure of amphivasal vascular bundles facilitates radial transport of assimilates to the sink tissues. The result of the anatomical analysis is discussed in a physiological context.