Engineering of cysteine and methionine biosynthesis in potato

Summary. Methionine and cysteine, two amino acids containing reduced sulfur, are not only an important substrate of protein biosynthesis but are also precursors of various other metabolites such as glutathione, phytochelatines, S-adenosylmethionine, ethylene, polyamines, biotin, and are involved as methyl group donor in numerous cellular processes. While methionine is an essential amino acid due to an inability of monogastric animals and human beings to synthesise this metabolite, animals are still able to convert methionine consumed with their diet into cysteine. Thus, a balanced diet containing both amino acids is necessary to provide a nutritionally favourable food or feed source. Because the concentrations of methionine and cysteine are often low in edible plant sources, e.g. potato, considerable efforts in plant breeding and research have been and are still performed to understand the physiological, biochemical, and molecular mechanisms that contribute to their synthesis, transport, and accumulation in plants. During the last decade molecular tools have enabled the isolation of most of the genes involved in cysteine and methionine biosynthesis, and the efficient plant transformation technology has allowed the creation of transgenic plants that are altered in the activity of individual genes. The physiological analysis of these transgenic plants has contributed considerably to our current understanding of how amino acids are synthesised. We focused our analysis on potato (Solanum tuberosum cv. Désirée) as this plant provides a clear separation of source and sink tissues and, for applied purposes, already constitutes a crop plant. From the data presented here and in previous work we conclude that threonine synthase and not cystathionine gamma-synthase as expected from studies of Arabidopsis constitutes the main regulatory control point of methionine synthesis in potato. This article aims to cover the current knowledge in the area of molecular genetics of sulfur-containing amino acid biosynthesis and will provide new data for methionine biosynthesis in solanaceous plants such as potato. Received December 19, 2001 Accepted January 7, 2002     

Elektronenmikroskopie der Chloroplasten-Feinstruktur physiologisch aktiver,vegetativer Zellen der AlgeBumilleriopsis

Zusammenfassung Die Feinstruktur der synchron gezogenen, mehrkernigen AlgeBumilleriopsis filiformis (Xanthophyceae) wurde nach Glutaraldehydfixierung elektronenmikroskopisch untersucht. Die Chloroplasten werden von Bändern aus 3–4 Thylakoiden in der Längsrichtung durchzogen. In den Chloroplasten befindet sich ein ribosomenfreierer, pyrenoidähnlicher Bereich, der die Chloroplastenmitte in Richtung Zellinnenraum vorwölbt. An der Grenze zu diesem Bereich treten oftmals Thylakoide von einem Band in ein benachbartes über. Hierbei erfolgt neben der Neuanordnung der Bänder auch die Ausbildung von Einzelthylakoiden. Dieser Übergangsbereich könnte der Neubildung von Bändern dienen. Das Auftreten von Thylakoid-Unterbrechungen ist augenscheinlich keine Erscheinung, die auf eine Chloroplastenteilung hindeutet, sondern vielmehr aus dem besonderen Verlauf der Bänder und einzelnen Thylakoide am Rande des ribosomenfreieren Bereichs (Thylakoidübergangsbereich) zu erklären.Asynchrone Zellen, die langsamer herangewachsen sind, zeigen erheblich mehr Thylakoide pro Band bzw. pro Fläche (entsprechend höherem Chlorophyllgehalt pro Zelle), eine wenig regelmäßige Anordnung der Bänder, keine pyrenoidähnlichen Bereiche, keine Zonen mit vermehrtem Übergang von Thylakoiden zwischen benachbarten Bändern, sowie eine geringere Anzahl von Mitochondrienanschnitten mit erheblich geringerer Tubuli-Anzahl.Auf die Struktur weiterer Zellorganelle in den physiologisch besonders aktiven Zellen der Synchronkultur wurde hingewiesen. Die Möglichkeit der Bildung einer 3konturigen Chloroplastenhülle aus einer zweifachen Doppelmembran wurde aufgezeigt. Strukturähnlichkeiten bzw. -unterschiede zuBumilleria undBotrydium wurden diskutiert.

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Electron microscopy of the chloroplast fine structure of the physiologically active, vegetative cells of Bumilleriopsis

Summary The fine structure of the synchronous, multinucleate algaBumilleriopsis filiformis (Xanthophyceae) has been studied in the electron microscope.It is typical that 3–4 thylakoids are grouped forming lamellae; these lamellae run for the whole length of the plastid in a very regular way.In the central region of the most chloroplasts the stroma has only few ribosomes. The thylakoids which enter this region are often leaving their bands and meet a neighbour-band. There is obviously a region with typical rearrangement of the thylakoids. In this area a synthesis of new thylakoids and also bands may be possible. The interruption of thylakoids and the occurence of smaller tubular thylakoids (Fig. 12) can be explained by the special course of the thylakoids through the region of rearrangement.Cells from asynchronous cultures, which are growing much slower, have more than 4 thylakoids in each band; they have a less regular arrangement of the bands. A typical rearrangement of the thylakoids resp. bands has not been found.

Die Arbeit wurde am Lehrstuhl für Physiologie und Biochemie der Pflanzen der Universität Konstanz durchgeführt.     

Differential Expression Levels of Integrin α6 Enable the Selective Identification and Isolation of Atrial and Ventricular Cardiomyocytes

Rationale

Central questions such as cardiomyocyte subtype emergence during cardiogenesis or the availability of cardiomyocyte subtypes for cell replacement therapy require selective identification and purification of atrial and ventricular cardiomyocytes. However, current methodologies do not allow for a transgene-free selective isolation of atrial or ventricular cardiomyocytes due to the lack of subtype specific cell surface markers.

Methods and Results

In order to develop cell surface marker-based isolation procedures for cardiomyocyte subtypes, we performed an antibody-based screening on embryonic mouse hearts. Our data indicate that atrial and ventricular cardiomyocytes are characterized by differential expression of integrin α6 (ITGA6) throughout development and in the adult heart. We discovered that the expression level of this surface marker correlates with the intracellular subtype-specific expression of MLC-2a and MLC-2v on the single cell level and thereby enables the discrimination of cardiomyocyte subtypes by flow cytometry. Based on the differential expression of ITGA6 in atria and ventricles during cardiogenesis, we developed purification protocols for atrial and ventricular cardiomyocytes from mouse hearts. Atrial and ventricular identities of sorted cells were confirmed by expression profiling and patch clamp analysis.

Conclusion

Here, we introduce a non-genetic, antibody-based approach to specifically isolate highly pure and viable atrial and ventricular cardiomyocytes from mouse hearts of various developmental stages. This will facilitate in-depth characterization of the individual cellular subsets and support translational research applications.     

Pollen walls of Araceae,with special reference to their fossilization potential

The pollen grains, the pistil, growth of the pollen tube and its pathway are described in Borago officinalis and Heliotropium europaeum. The exine is thick in both taxa but it is covered with dense gemmae in Borago. The intine seems to be thicker and contains more proteins in Borago than in Heliotropium. Starch is very abundant in the latter while it is lacking in the former. The style is hollow in Borago with a stylar canal running from the stigma to the ovary, while in Heliotropium it is broadly cone‐shaped with papillae located at the base of the cone (the “stigmatic ring") and not at the top of the style as usual. In Borago stigmatic papillae are unicellular, skittle‐shaped and have a thick pectocellulosic wall and an equally thick cuticular layer, while in Heliotropium the stigmatic papillae are unicellular, elongated cone‐shaped with a thin pecto‐cellulosic wall and have an apparently reduced cuticular layer. The stigmatic exudate is very abundant on the stigmas of Heliotropium even before anthesis while it is absent on those of Borago except when allo‐pollination occurs. Pollen tube growth has been followed from stigma to ovules in both taxa.     

Separation of fast from slow anabolism by site-specific PEGylation of insulin-like growth factor I (IGF-I)

Insulin-like growth factor I (IGF-I) has important anabolic and homeostatic functions in tissues like skeletal muscle, and a decline in circulating levels is linked with catabolic conditions. Whereas IGF-I therapies for musculoskeletal disorders have been postulated, dosing issues and disruptions of the homeostasis have so far precluded clinical application. We have developed a novel IGF-I variant by site-specific addition of polyethylene glycol (PEG) to lysine 68 (PEG-IGF-I). In vitro, this modification decreased the affinity for the IGF-I and insulin receptors, presumably through decreased association rates, and slowed down the association to IGF-I-binding proteins, selectively limiting fast but maintaining sustained anabolic activity. Desirable in vivo effects of PEG-IGF-I included increased half-life and recruitment of IGF-binding proteins, thereby reducing risk of hypoglycemia. PEG-IGF-I was equipotent to IGF-I in ameliorating contraction-induced muscle injury in vivo without affecting muscle metabolism as IGF-I did. The data provide an important step in understanding the differences of IGF-I and insulin receptor contribution to the in vivo activity of IGF-I. In addition, PEG-IGF-I presents an innovative concept for IGF-I therapy in diseases with indicated muscle dysfunction.     

From Inflammation to Wound Healing: Using a Simple Model to Understand the Functional Versatility of Murine Macrophages

Macrophages are fundamental cells of the innate immune system. Their activation is essential for such distinct immune functions as inflammation (pathogen-killing) and tissue repair (wound healing). An open question has been the functional stability of an individual macrophage cell: whether it can change its functional profile between different immune responses such as between the repair pathway and the inflammatory pathway. We studied this question theoretically by constructing a rate equation model for the key substrate, enzymes and products of the pathways; we then tested the model experimentally. Both our model and experiments show that individual macrophages can switch from the repair pathway to the inflammation pathway but that the reverse switch does not occur.     

Genome Announcement: The Draft Genome of the Carrot Cyst Nematode Heterodera Carotae

Heterodera carotae, the carrot cyst nematode, is a significant pest affecting carrot globally. Here we present the draft genome of H. carotae, which was generated from short read libraries from Illumina HiSeq technology, and the corresponding genome annotation.     

Hyperoxia modulates TGF-beta/BMP signaling in a mouse model of bronchopulmonary dysplasia

Prematurely born infants who require oxygen therapy often develop bronchopulmonary dysplasia (BPD), a debilitating disorder characterized by pronounced alveolar hypoplasia. Hyperoxic injury is believed to disrupt critical signaling pathways that direct lung development, causing BPD. We investigated the effects of normobaric hyperoxia on transforming growth factor (TGF)-beta and bone morphogenetic protein (BMP) signaling in neonatal C57BL/6J mice exposed to 21% or 85% O(2) between postnatal days P1 and P28. Growth and respiratory compliance were significantly impaired in pups exposed to 85% O(2), and these pups also exhibited a pronounced arrest of alveolarization, accompanied by dysregulated expression and localization of both receptor (ALK-1, ALK-3, ALK-6, and the TGF-beta type II receptor) and Smad (Smads 1, 3, and 4) proteins. TGF-beta signaling was potentiated, whereas BMP signaling was impaired both in the lungs of pups exposed to 85% O(2) as well as in MLE-12 mouse lung epithelial cells and NIH/3T3 and primary lung fibroblasts cultured in 85% O(2). After exposure to 85% O(2), primary alveolar type II cells were more susceptible to TGF-beta-induced apoptosis, whereas primary pulmonary artery smooth muscle cells were unaffected. Exposure of primary lung fibroblasts to 85% O(2) significantly enhanced the TGF-beta-stimulated production of the alpha(1) subunit of type I collagen (Ialpha(1)), tissue inhibitor of metalloproteinase-1, tropoelastin, and tenascin-C. These data demonstrated that hyperoxia significantly affects TGF-beta/BMP signaling in the lung, including processes central to septation and, hence, alveolarization. The amenability of these pathways to genetic and pharmacological manipulation may provide alternative avenues for the management of BPD.