Cell signalling by reactive lipid species: new concepts and molecular mechanisms

The process of lipid peroxidation is widespread in biology and is mediated through both enzymatic and non-enzymatic pathways. A significant proportion of the oxidized lipid products are electrophilic in nature, the RLS (reactive lipid species), and react with cellular nucleophiles such as the amino acids cysteine, lysine and histidine. Cell signalling by electrophiles appears to be limited to the modification of cysteine residues in proteins, whereas non-specific toxic effects involve modification of other nucleophiles. RLS have been found to participate in several physiological pathways including resolution of inflammation, cell death and induction of cellular antioxidants through the modification of specific signalling proteins. The covalent modification of proteins endows some unique features to this signalling mechanism which we have termed the 'covalent advantage'. For example, covalent modification of signalling proteins allows for the accumulation of a signal over time. The activation of cell signalling pathways by electrophiles is hierarchical and depends on a complex interaction of factors such as the intrinsic chemical reactivity of the electrophile, the intracellular domain to which it is exposed and steric factors. This introduces the concept of electrophilic signalling domains in which the production of the lipid electrophile is in close proximity to the thiol-containing signalling protein. In addition, we propose that the role of glutathione and associated enzymes is to insulate the signalling domain from uncontrolled electrophilic stress. The persistence of the signal is in turn regulated by the proteasomal pathway which may itself be subject to redox regulation by RLS. Cell death mediated by RLS is associated with bioenergetic dysfunction, and the damaged proteins are probably removed by the lysosome-autophagy pathway.     

Soluble, prolonged-acting insulin derivatives. III. Degree of protraction, crystallizability and chemical stability of insulins substituted in positions A21, B13, B23, B27 and B30

It was previously demonstrated that insulins to which positive charge has been added by substituting B13 glutamic acid with a glutamine residue, B27 threonine with an arginine or lysine residue, and by blocking the C-terminal carboxyl group of the B-chain by amidation, featured a prolonged absorption from the subcutis of rabbits and pigs after injection in solution at acidic pH. The phenomenon is ascribed to a low solubility combined with the readiness by which these analogs crystallize as the injectant is being neutralized in the tissue. However, acid solutions of insulin are chemically unstable as A21 asparagine both deamidates to aspartic acid and takes part in formation of covalent dimers via alpha-amino groups of other molecules. In order to circumvent the instability, substitutions were introduced in position A21, in addition to those in B13, B27 and B30, challenging the fact that A21 asparagine has been conserved in this position throughout the evolution. Biological potency was retained when glycine, serine, threonine, aspartic acid, histidine and arginine were introduced in this position, although to a varying degree. In the crystal structure of insulin a hydrogen bond bridges the alpha-nitrogen of A21 with the backbone carbonyl of B23 glycine. In order to investigate the importance of this hydrogen bond for biological activity a gene for the single-chain precursor B-chain(1-29)-Ala-Ala-Lys-A-chain(1-21) featuring an A21 proline was synthesized. However, this single-chain precursor failed to be properly produced by yeast, pointing to the formation of this hydrogen bond as an essential step in the folding process. The stability of the A21-substituted analogs in acid solutions (pH 3-4) with respect to deamidation and formation of dimers was approximately 5-10 times higher than that of human insulin in neutral solution. The rate of absorption of most insulins is decreased by increasing the Zn2+ concentration of the preparation. However, one analog with A21 glycine showed first-order absorption kinetics in pigs with a half-life of approximately 25 h, independent of the Zn2+ concentration. The day-to-day variation of the absorption of this analog was significantly lower than that of the conventional insulin suspensions, a property that might render such an insulin useful in the attempts to improve glucose control in diabetics by a more predictable delivery of basal insulin.     

Genetic diversity of oilseedBrassica napus germ plasm based on restriction fragment length polymorphisms

Oilseed rape (Brassica napus) is an important oilseed crop worldwide. Cultivars have been developed for many growing regions, however little is known about genetic diversity inB. napus germ plasm. The purpose of the research presented here was to study the genetic diversity and relationships ofB. napus accessions using restriction fragment length polymorphisms (RFLPs). Eighty threeB. napus accessions were screened using 43 genomic DNA clones which revealed 161 polymorphic fragments. Each accession was uniquely identified by the markers with the exception of the near-isogenic cvs Triton and Tower. The RFLP data were analyzed by cluster analysis of similarity coefficients and by principal component analysis. Overall, there were three major groups of cultivars. The first group included only spring accessions, the second mostly winter accessions and the third, rutabagas and oilseed rape accessions from China and Japan. These results indicate that withinB. napus, winter and spring cultivars represent genetically distinct groups. The grouping of accessions by cluster analysis was generally consistent with known pedigrees. This consistency included the grouping of lines derived both by backcrossing or self-pollination with their parents.     

Nucleotide sequence of a beta-1,3-glucanase isoenzyme IIA gene of Oerskovia xanthineolytica LL G109 (Cellulomonas cellulans) and initial characterization of the recombinant enzyme expressed in Bacillus subtilis.

The nucleotide sequence of the betaglIIA gene, encoding the extracellular beta-1,3-glucanase IIA (betaglIIA) of the yeast-lytic actinomycete Oerskovia xanthineolytica LL G109, was determined. Sequence comparison shows that the betaglIIA enzyme has over 80% identity to the betaglII isoenzyme, an endo-beta-1,3-glucanase having low yeast-lytic activity secreted by the same bacterium. The betaglIIA enzyme lacks a glucan- or mannan-binding domain, such as those observed in beta-1,3-glucanases and proteases having high yeast/fungus-lytic activity. It can be included in the glycosyl hydrolase family 16. Gene fusion expression in Bacillus subtilis DN1885 followed by preliminary characterization of the recombinant gene product indicates that betaglIIA has a pI of 3.8 to 4.0 and is active on both laminarin and curdlan, having an acid optimum pH activity (ca. 4.0).     

Der Feinbau des Spermatozoids von Sphaeroc arpos donnellii Aust. (Hepaticae)

Zusammenfassung Das reife, aus dem Antheridium entlassene Spermatozoid setzt sich aus den drei Abschnitten: Kopfteil, Kernabschnitt und dem daran anschließenden Plasmastück zusammen. Im Kopfabschnitt entspringen die beiden Geißeln. Jede Geißel zeigt den typischen Aufbau von neun randlichen Doppelfibrillen um zwei zentral gelegene Fibrillen. Zwischen den peripher und zentral liegenden Fibrillen, deren Durchmesser etwa 200 Å beträgt, bestehen Verbindungen, die zum Teil als etwa 70 Å breite, hohl erscheinende Fibrillen ausgebildet sind. Die Geißel wird von einer etwa 100 Å dicken Doppelmembran umgrenzt. Im Kopfabschnitt befindet sich ein membranhaltiger Körper, der als stark umgewandeltes Mitochondrium oder als Plastide anzusehen ist.Der Kernteil wird fast ganz von dem höchstens 0,4 breiten und etwa 13 langen Kern eingenommen. In der dichten Kernsubstanz sind etwa 25–40 Å breite Fibrillen erkennbar, die gelegentlich eng zu dickeren Bündeln zusammengepackt sein können. Eine typisch ausgebildete doppelte Kernmembran fehlt. Selten läßt sich eine nur 40–60 Å breite, stark kontrastierbare Linie feststellen, die möglicherweise als extrem reduzierte Abgrenzungsmembran des Kerns gedeutet werden kann. Zwischen dem Kern und der das ganze Spermatozoid umschlie\enden, doppelten, etwa 80–100 Å dicken Membran, die als etwas verbreitertes Plasmalemma anzusehen ist, kann sich ein schmaler Cytoplasmastreifen einschieben.Das Plasmastück umfaßt den großen Leukoplasten, Mitochondrien, Membranen des endoplasmatischen Reticulums, multivesikuläre Körper, kleinere Vesikel und gelegentlich Vacuolen. Im Innern des Leukoplasten befinden sich zahlreiche, bis zu 0,2 große Stärkekörner und nur selten einige Reste des Membransystems. Die Mitochondrien besitzen im Vergleich zu den entsprechenden Zellorganen der Spermatiden eine oft stark veränderte Struktur. Ferner treten im Plasmastück Körper auf, die von einer Doppelmembran umgeben sind und in ihrem Innern Doppelmembranen aufweisen. Sie gleichen in ihrem Bau völlig dem membranhaltigen Körper im Kopfabschnitt und dürften stark umgeformte Mitochondrien oder Plastiden darstellen.Unmittelbar unter der Spermatozoidmembran befindet sich eine für Pflanzenzellen ungewöhnliche Struktur, die als Fibrillenscheide bezeichnet wird, und die sich meistens vom Kernabschnitt bis in das Plasmastück der Zelle ausdehnt. Die 400–800 Å dicke Fibrillenscheide besteht aus bis zu 30 nebeneinander liegenden, hohl erscheinenden Fibrillen mit einem Durchmesser von etwa 180–220 Å. Die beiden endständigen Fibrillen jeder Scheide besitzen einen größeren Durchmesser von etwa 300 Å. Die Fibrillen werden von einer Doppelmembran gegen die Spermatozoidmembran und andere Teile der Zelle abgegrenzt.Spätestens beim Eindringen des Spermatozoids in den geöffneten Archegonhals wird das Plasmastück abgestreift. Das bis zur Eizelle vorgedrungene Spermatozoid besteht nur noch aus dem Kopfabschnitt und dem anschließenden Kernteil.

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The fine structure of the spermatozoid of Sphaeroc arpos donnellii Aust. (Hepaticae)

Summary The structure of the spermatozoid of the liverwort, Sphaerocarpos donnellii, was investigated under the electron microscope after fixation in potassium permanganate, osmium tetroxide or glutaraldehyde with postfixation in osmium tetroxide. Mature, newly emerged spermatozoids consist of three parts: Head end, nuclear piece and the attached cytoplasmic part. The two flagella originate in the head end. They show the typical structure of nine outer double fibers around two fibers in the middle. Connections exist between the central and the outer fibers. At least to some extent they are composed of thin, tubular fibers, about 70 Å in diameter. The head end contains a body which may be regarded as a modified mitochondrion or a plastid.Nearly the whole space of the nuclear piece is occupied by the nucleus, with a length of a about 13 and a thickness up to 0,4 . The dense nuclear content shows above all, approximately 25–40 Å thick fibers, which are often packed closely together. A typical double membrane as nuclear envelope is not recognizable. Rarely one observes a dark line, not thicker than 40–60 Å which may be interpreted as limiting membrane of the nucleus. A thin band of cytoplasmic material may be interposed between the nucleus and the double membrane, which has a thickness of about 80–100 Å and surrounds the whole spermatozoid.The cytoplasmic piece includes the big leucoplast, mitochondria, membranes of the endoplasmic reticulum, multivesicular bodies, small vesicles and occasionally vacuoles. The interior of the leucoplast is filled with numerous starch granules, up to 0,2 in diameter. Only rarely some remains of the thylacoid system appear. The mitochondria show a modified fine structure compared with the corresponding organelles in the spermatids. The cytoplasmic end comprises bodies which are limited by a double membrane and which contain double membranes. They are absolutely alike the membraneous body in the head piece and have to be regarded as modified mitochondria or plastids.Immediately below the membrane of the spermatozoid one recognizes a structure, named Fibrillenscheide=fibrous sheath, which in most cases expands from the nuclear piece into the cytoplasmic part. The 400–800 Å thick fibrous sheath consists of up to 30 fibers lying side by side, each with a diameter of about 180–220 Å. The fibers at both ends of the fibrous sheath possess a diameter of about 300 Å. A double membrane encloses all the fibers together and separates them from the limiting membrane and other components of the spermatozoid.The cytoplasmic end is lost, at the latest when the spermatozoid enters the open neck canal of the archegonium. The spermatozoid which has reached the egg cell is composed only of the head end and the nuclear piece.

     

Zur Frage des Aufbaus von Thylakoiden aus Vesikeln

Zusammenfassung In dem Oenotherenbastard Oe. (lamarckiana x hookeri) velans· ^h hookeri mit lamarckiana-Plastiden kommt es infolge einer Disharmonie zwischen Genom und Plastom in den Chloroplasten zu Störungen in der Differenzierung des Thylakoidsystems. Neben langen Thylakoiden und Thylakoidstapeln (Grana) können in ihnen noch Prolamellarkörper, Plastoglobuli und in großer Zahl rundliche Vesikel vorkommen. Die Vesikel können z.T. völlig voneinander isoliert und ungeordnet im Plastidenstroma liegen, z.T. sind mehrere von ihnen deutlich durch Fäden oder Kanäle verbunden.Häufig sind die Vesikel auf Einzelschnitten auch in Reihen angeordnet. Schnittserien zeigen, daß es sich dabei oft um ein unregelmäßiges, flächiges Netzwerk aus zusammenhängenden Vesikeln handelt. Zu solchen netzartigen Flächen können Thylakoidränder, aber anscheinend auch ganze Thylakoide umgestaltet sein. Dies wird auf eine Fixationslabilität von Thylakoiden oder von Thylakoidteilen, bedingt durch die Disharmonie zwischen Genom und Plastom, zurückgeführt.Das Auftreten von in Reihen oder Ebenen angeordneten Vesikeln zu bestimmten Zeiten auch der normalen Chloroplastendifferenzierung wurde schon verschiedentlich festgestellt, und es ist eine weitverbreitete Ansicht, daß dort die großflächigen Thylakoide durch Verschmelzung solcher Bläschen entstehen. Unsere Beobachtungen deuten aber darauf hin, daß auch diese bei der normalen Chloroplastendifferenzierung auftretenden Vesikelreihen aus großflächigen Thylakoiden hervorgehen könnten, die in bestimmten Entwicklungsstadien fixationslabil sind.

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On the formation of thylakoids from vesicles

Summary The Oenothera hybrid Oe. (lamarckiana x hookeri) velans· ^h hookeri with lamarckiana plastids is characterized by a disharmony between genom and plastom. This disharmony results in a disorder of the differentiation of the thylakoid system in the chloroplasts. Besides long single thylakoids and thylakoid stacks the chloroplasts may contain prolamellar bodies, plastoglobuli and many spherical vesicles. Some of these vesicles may be completely separated from one another and may show no recognizable arrangement in the plastid stroma whereas some are sometimes connected by thin fibrils or channels.In single sections the vesicles are frequently lined up in rows. Serial sections show that these rows of vesicles often belong to an irregular flat network of vesicles, all of which are connected with one another. The margins of the thylakoids, and apparently also whole thylakoids, may be transformed into such networks. Such a transformation can be attributed to an instability of the thylakoids as a whole or of parts of them during the fixation process. This instability may be caused or increased by the disharmony between genom and plastom.The occurrence of vesicles lined up in rows or planes in certain stages, even during the differentiation of the normal chloroplasts, was repeatedly observed, and it is a widespread opinion that there the expanded thylakoids are formed by the fusion of such vesicles. However, our observations indicate that during the normal differentiation of chloroplasts the thylakoids may also be temporarily unstable and may then be transformed into planes or rows of vesicles under the influence of the fixation agents.

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Herrn Prof. Dr. Leo Brauner zum 70. Geburtstag gewidmet.     

Über das Vorkommen kleiner netzartiger Strukturen in den Plastiden

Summary In the chlorotic leaves of the Oenothera hybrid Oe. (lamarckiana x hookeri) velans· ^h hookeri with lamarckiana plastids the differentiation of the chloroplasts is disordered in different ways because of a disharmony between genom and plastom. Some of the plastids possess numerous vesicles and plastoglobuli but only a few isolated grana instead of the normal thylakoid system. Furthermore, the plastids contain lattice-like structures consisting of fibrils with a thickness of approximately 5 to 11 nm. These networks are connected with thylakoids, vesicles or plastoglobuli. They are interpreted as fragments of prolamellar bodies. Sometimes prolamellar bodies are distinctly recognizable in the chloroplasts even though the thylakoid system is rather well differentiated.     

Yield improvement of heterologous peptides expressed in yps1-disrupted Saccharomyces cerevisiae strains

Heterologous protein expression levels in Saccharomyces cerevisiae fermentations are highly dependent on the susceptibility to endogenous yeast proteases. Small peptides, such as glucagon and glucagon-like-peptides (GLP-1 and GLP-2), featuring an open structure are particularly accessible for proteolytic degradation during fermentation. Therefore, homogeneous products cannot be obtained. The most sensitive residues are found at basic amino acid residues in the peptide sequence. These heterologous peptides are degraded mainly by the YPS1-encoded aspartic protease, yapsin1, when produced in the yeast. In this article, distinct degradation products were analyzed by HPLC and mass spectrometry, and high yield of the heterologous peptide production has been achieved by the disruption of the YPS1 gene (previously called YAP3). By this technique, high yield continuous fermentation of glucagon in S. cerevisiae is now possible.     

Calcium/calmodulin-regulated receptor-like kinase CRLK1 interacts with MEKK1 in plants

Recently we reported that CRLK1, a novel calcium/calmodulin-regulated receptor-like kinase plays an important role in regulating plant cold tolerance. Calcium/calmodulin binds to CRLK1 and upregulates its activity. Gene knockout and complementation studies revealed that CRLK1 is a positive regulator of plant response to chilling and freezing temperatures. Here we show that MEKK1, a member of MAP kinase kinase kinase family, interacts with CRLK1 both in vitro and in planta. The cold triggered MAP kinase activation in wild-type plants was abolished in crlk1 knockout mutants. Similarly, the cold induced expression levels of genes involved in MAP kinase signaling are also altered in crlk1 mutants. These results suggest that calcium/calmodulinregulated CRLK1 modulates cold acclimation through MAP kinase cascade in plants.Key words: calcium, calmodulin, cold stress, MAPK, Arabidopsis, protein phosphorylationCalcium, a universal second messenger in eukaryotic cells, mediates changes in external and internal signals leading to the physiological responses.^1–4 Calcium/calmodulin (Ca²⁺/CaM)-dependent protein kinases (CaMKs) are very important players in calcium/calmodulin mediated signaling in mammalian cells.⁵ In plants, Ca²⁺/CaM-dependent protein phosphorylation was observed more than 25 years ago.⁶ Several calmodulin-regulated protein kinases have been identified and characterized.^7,8 For example, plants have a unique chimeric Ca²⁺/CaM-dependent protein kinase (CCaMK), which exhibits Ca²⁺-dependent autophosphorylation and Ca²⁺/CaM-dependent substrate phosphorylation.⁹ CCaMK is required for bacterial and fungal symbioses in plants.^10–12 Recently, we characterized a novel plant-specific calcium/CaM-regulated receptor-like kinase, CRLK1.¹³ Ca²⁺/CaM binds to CRLK1 and stimulates its kinase activity. Functional studies with CRLK1 indicate that CRLK1 acts as a positive regulator in plant response to chilling and freezing temperatures. To further define the CRLK1-mediated signal pathway, we isolated CRLK1 interacting proteins by co-immunoprecipitation using an anti-CRLK1 antibody. Since cold increases the amount of CRLK1 protein, wildtype plants (WT) were treated at 4°C for 1 hr before co-immunoprecipitation. The resulting CRLK1 immunocomplex was separated by SDS-PAGE. We observed several bands of different sizes only in the wild-type but not in the crlk1 knockout mutant plants (Fig. 1A). Furthermore, the intensity of these bands increased upon cold treatment, suggesting that they are the putative partners or associated proteins of the CRLK1 immunocomplex.Open in a separate windowFigure 1CRLK1 Interacts with MEKK1. (A) One-dimension SDS-PAGE of anti-CRLK1 immunocomplexes from 3-week-old WT or crlk1 plants with or without cold treatment. One mg of total protein was used for immunoprecipitation. (B) A list of putative CRLK1-interacting proteins determined by MALDI-TOF-MS analysis. (C) CRLK1 interacts with MEKK1 as shown by GST pull-down assay. (D) BiFC analysis show that CR LK associates with MEKK1 in vivo. Upper row shows that CRLK and MEKK1 associate both on cell membrane and in endosomes. The middle and last rows are controls. Bar = 10 µm.To determine the identities of these proteins, mass spectrometric analysis was performed with the total immunocomplex.¹⁴ In addition to CRLK1, there were 12 other proteins which matched the Arabidopsis database. Several of them appeared in the pull-down complex from WT, but not from crlk1 mutants. These putative interacting proteins included MEKK1, another unknown protein kinase, a type 2C phosphatase and CaM (Fig. 1B). MEKK1 is one of the 60 putative MAPKKKs in the Arabidopsis genome, and sits on the top of mitogen-activated protein kinase (MAPK) cascade. The MAPK signaling consists of a cascade of three consecutively acting protein kinases, a MAP kinase kinase kinase (MAPKKK), a MAP kinase kinase (MAPKK) and a MAP kinase (MAPK). Plants possess multiple MAPKKKs, MAPKKs and MAPKs, which respond to different upstream signals and activate distinct downstream pathways.^15–17 The specific MAPK module responding to lower temperature has been determined in Arabidopsis.^18,19 MEKK1, a member of MAPKKKs, specifically interacts and phosphorylates MKK2 and regulates COR genes expression in response to cold stress.¹⁹ MEKK1 has been shown to play a role in mediating reactive oxygen species homeostasis.^20,21 Therefore we selected MEKK1 from the putative CRLK1 partners for further studies.     

Temporo-Spatial Dynamics of Event-Related EEG Beta Activity during the Initial Contingent Negative Variation

In the electroencephalogram (EEG), early anticipatory processes are accompanied by a slow negative potential, the initial contingent negative variation (iCNV), occurring between 500 and 1500 ms after cue onset over prefrontal cortical regions in tasks with cue-target intervals of about 3 s or longer. However, the temporal sequence of the distributed cortical activity contributing to iCNV generation remains unclear. During iCNV generation, selectively enhanced low-beta activity has been reported. Here we studied the temporal order of activation foci in cortical regions assumed to underlie iCNV generation using source reconstruction of low-beta (13–18 Hz) activity. During the iCNV, elicited by a cued simple reaction-time task, low-beta power peaked first (750 ms after cue onset) in anterior frontal and limbic regions and last (140 ms later) in posterior areas. This activity occurred 3300 ms before target onset and provides evidence for the temporally ordered involvement of both cognitive-control and motor-preparation processes already at early stages during the preparation for speeded action.