Changes in the activities of ornithine transcarbarnylase and arginase, and concentrations of nitrogenous substances during germination and seedling development of Vitis vinifera L.

During germination and subsequent growth of seedlings of Vitis vinifera L. cv. Chenin blanc, marked changes occurred in the concentrations of the total nonprotein nitrogen fraction, amino nitrogen, and alnide nitrogen, and in the activities of ornithine transcarbamylase (OTC) and arginase. The level of total nonprotein nitrogen was lower in seeds than in seedlings, with maximum concentration reached at the 3rd stage of seedling growth (10 to 15 d after germination) and thereafter declining rapidly. Changes in the concentration of amino nitrogen and amide nitrogen fractions paralleled that of the total nonprotein nitrogen. The concentration of glutalnine exceeded that of asparagine at all stages of seedling development. The presence of OTC and arginase in seeds and seedlings, as well as the parallel changes between arginase activity and concentration of free arginine, suggested that the biosynthesis and degradation of this amino acid in grapevine tissues occurs through the KREBS-HENSELEIT pathway. The MICHAELIS constant for arginase, calculated from the LINEWEAVER-BURK plot, differed in seedlings at three different stages of seedling development. Veränderungen in der Aktivität der Ornithintranscarbamylase und der Arginase sowie der Konzentration der Stickstoffverbindungen während der Keimung und der Sämlingsentwicklung von Vitis vinifera L.Im Verlauf der Keimung und des anschließenden Wachstums der Sämlinge von Vitis vinifera L., cv . Chenin blanc, traten deutliche Veränderungen in der Konzentration des gesamten nicht-proteingebundenen Stickstoffs, des Amino- und des Amidstickstoffs sowie in der Aktivität der Ornithintranscarbamylase (OTC) und der Arginase auf. Die Samen besaßen einen niedrigeren Gehalt an gesamtem nicht-proteingebundenem Stickstoff als die Sämlinge, wobei die maximale Konzentration im 3. Stadium des Sämlingswachstums erreicht wurd~ (10-15 d nach der Keimung); danach nahm sie rasch ab. Die Verschiebungen in der Konzentration des Amino- und Amidstickstoffs verliefen parallel zu den Konzentrationsänderungen des gesamten nichtproteingebundenen Stickstoffs. In allen Stadien der Sämlingsentwicklung lag die Glutamin- über der Asparaginkonzentration. Das Vorkommen von OTC und Arginase in Samen und Sämlingen sowie die parallelen Veränderungen von Arginaseaktivität und Konzentration des freien Arginins la~sen vermuten, daß die Biosynthese und der Abbau dieser Aminosäure im Rebengewebe über den KREBS-HENSELEIT-Cyklus ablaufen. In den Sämlingsstadien 3, 4 und 5 wurden - nach LINEWEAVER-BURK - unterschiedliche MICHAELIS Konstanten der Arginase ermittelt

Ammonia assimilation in Vitis vinifera L.: III. Glutamate oxaloacetate transaminase from leaf and root tissue

Glutamate-oxaloacetate transaminase (GOT) activities in Vitis vinifera L. cv. Chenin blanc leaf and root tissues were associated only with the soluble fraction. Mean in vitro enzyme activity of both tissues was 4.3 ± 0.8 μmol oxaloacetate formed per g fresh tissue per hour. Km values of GOT from leaves and roots were (1.9 ± 0.4) · 10-4 M for α-ketoglutarate and (4.7 ± 0.5) · 10-3 M for L-aspartate. Optimum in vitro conditions for GOT activity were pH 7.4-7.8, amount of enzyme equivalent to 95-130 mg fresh tissue and incubation temperature 38-39 °C. Neither leaf nor root GOT responded to exogenous pyridoxal-5' phosphate.Die Ammonium-Assimilation bei Vitis vinifera L.:III. GlutamatcOxalacetat-Transaminase aus Blatt- und WurzelgewebeDie Aktivität der Glutamat-Oxalacetat-Transaminase (GOT) in Blatt- und Wurzelgewebe der Rebsorte Chenin blanc (Vitis vinifera L.) war auf die lösliche Fraktion beschränkt. In vitro betrug die mittlere Enzymaktivität in beiden Organen 4,3 ± 0,8 μmol Oxalacetat je g Frischgewicht und h. Die Km-Werte von GOT aus Blättern und Wurzeln betrugen (1,9±0,4) . 10-4 M für α-Ketoglutarat und (4,7±0,5) · 10-3 M für L-Aspartat. Die optimalen Bedingungen der GOT-Aktivität waren in vitro ein pH von 7,4-7,8, eine Enzymmenge, die dem Gehalt von 95-130 mg Frischmaterial entsprach, und eine Inkubationstemperatur von 38-39 °C. Weder Blatt- noch Wurzel-GOT reagierten auf zugesetztes Pyridoxal-5'-Phosphat

Ammonia assimilation in Vitis vinifera L.: I. Isolation and properties of leaf and root glutamate dehydrogenase

Glutamate dehydrogenase (GDH) activity in Vitis vinifera L. cv. Chenin blanc leaf and root tissues was associated with the particulate and the soluble fractions. In leaf extracts, about 66 % of the NADH-GDH activity was in the 10,000 g pellet and about 34 % was in the 23,500 gpellet fractions, whereas NADPH-GDH activity was associated mainly with the soluble fraction (23,500 g). In root extracts, about 53 % of the NADHGDH activity was in the soluble and 43 % in the 10,000 gpellet fractions, whereas about 47, 37, and 16 % of NADPH-GDH activity were in the soluble, 10,000 g and 23,500 gpellet fractions, respectively. GDH from the 10,000 gpellet of leaf and the soluble fraction of root extracts differed in their affinities to substrates. The Km values of leaf and root GDH were, respectively, 3.9 ± 1.1 and 0.7 ± 0.4 mM for a-ketoglutarate; 35.7 ± 7.1 and 61.3 ± 12.4 mM for NH4Cl; and 100.0 ± 7.4 and 36.2 ± 4.4 μM for NADH. GDH from leaf and root tissues showed Michaelis-Meuten kinetics with all. substrates except NH4Cl, which exhibited a sigmoid relationship in roots. Optimum in vitro reaction conditions were pH 7.90-8.10, incubation temperature of 38-40 °C, and amount of enzyme equivalent to 80-110 mg of fresh tissue. Enzyme from both leaves and roots was inhibited by EDTA and L-glutamate. Activation with Ca2+ was more pronounced in root GDH than in leaf GDH.Die Ammonium-Assimilation bei Vitis vinifera L.:1. Isolierung und Eigenschaften der Glutamatdehydrogenase aus Blättern und WurzelnBei der Rebsorte Chenin blanc (Vitis vinifera L.) wurde die Glutamatdehydrogenase- (GDH-)Aktivität der strukturierten und löslichen Fraktionen von Blattund Wurzelgeweben bestimmt. Die NADH-GDH-Aktivität von Blattextrakten verteilte sich zu ca. 66 % auf das nach Zentrifugation bei 10 000 g erhaltene Sediment und zu ca. 34 % auf das 23 500-g Sediment, während die NADPH-GDH-Aktivität hauptsächlich an die lösliche Fraktion (23 500 g) gebunden war. Bei Wurzelextrakten waren ca. 53 % der NADH-GDH-Aktivität in der löslichen Fraktion und 43 % im 10 000-g-Sediment enthalten, während jeweils 47, 37 und 16 % der NADPHGDH- Aktivität auf die lösliche Fraktion, das 10 000-g-Sediment bzw. das 23 500-g-Sediment entfielen. Die GDH aus dem 10 000-g-Sediment des Blattmaterials und aus der löslichen Fraktion der Wurzelextrakte unterschieden sich in ihrer Substrataffinität. Die Km-Werte der Blatt- und Wurzel GDH betrugen 3,9 ± 1,1 bzw. 0,7 ± 0,4 mM für a-Ketoglutarat, 35,7 ± 7,1 bzw. 61,3 ± 12,4 mM für NH4Cl sowie 100,0 ± 7,4 bzw. 36,2 ± 4,4 μM für NADH. Die GDH aus Blatt- und Wurzelgeweben folgte mit allen Substraten der Michaelis-Menten-Kinetik, ausgenommen mit NH4Cl; hier lag bei Wurzelextrakten eine sigmoide Beziehung vor. In vitro waren die optimalen Reaktionsbedingungen ein pH von 7,90-8,10, eine Inkubationstemperatur von 38-40 °C und eine Enzymmenge, die dem Gehalt von 80-110 mg Frischgewebe entsprach. Durch EDTA und L-Glutamat wurde das Enzym sowohl der Blätter als auch der Wurzeln inhibiert. Wurzel-GDH wurde durch Ca2+ stärker aktiviert als Blatt-GDH

Ammonia assimilation in Vitis vinifera L.: II. Leaf and root glutamine synthetase

Glutamine synthetase (GS) activity in Vitis vinifera L. cv. Chenin blanc leaf and root tissues was present in the supernatant and particulate fractions. The percentage distribution of GS activity in leaf and root extracts were respectively 39.5 and 49.2 % in the 10,000 gpellet, 38.6 and 41.1 % in the 2.3,500 gpellet, and 21.9 and 9.7 % in the 23,500 g supernatant fractions. Leaf GS activity was always greater than root enzyme activity. Kinetic studies revealed no significant differences between leaf and root GS from the 10,000 g pellet fraction. The Km values for L-glutamate, ATP and hydroxylamine were respectively 3.2 ± 0.7 mM, 0.8 ± 0.2 mM, and 0.8 ± 0.2 mM. Formation of y-glutamyl hydroxamate was linear for the first 35 min. Optimum in vitro reaction conditions were pH 7.70-8.10, incubation temperature 37 °C, and amount of enzyme equivalent to 75-105 mg of fresh tissue. L-arginine, L-ornithine and carbamyl phosphate at a concentration of 5 mM caused inhibition of 13.6 and 12 %, respectively.Die Ammonium-Assimilation bei Vitis vinifera L.:II. Die Glutaminsynthetase der Blätter und WurzelnBei der Rebsorte Chenin blanc (Vitis vinifera L.) lag nach differenzierter Zentrifugation sowohl in den löslichen wie in den strukturierten Fraktionen Glutaminsynthetase-(GS-)Aktivität vor. Die prozentuale Verteilung der GS-Aktivität auf Blatt- und Wurzelextrakte betrug jeweils 39,5 und 49,2 % im 10 000-g-Sediment, 38,6 und 41,1 % im 23 500-g-Sediment sowie 21,9 und 9,7 % im 23 500-g-Überstand. In den Blättern war die GS-Aktivität stets höher als in den Wurzeln. Untersuchungen der Enzymkinetik erbrachten bei dem 10 000-g-Sediment keine signifikanten Unterschiede zwischen der GS-Aktivität der Blätter und der Wurzeln. Die Km-Werte für L-Glutamat, ATP und Hydroxylamin betrugen 3,2 ± 0,7 mM, 0,8 ± 0,2 mM und 0,8 ± 0,2 mM. Die Bildung von y-Glutamylhydroxamat verlief in den ersten 35 min linear. In vitro waren die optimalen Reaktionsbedingungen ein pH von 7,70-8,10, eine Inkubationstemperatur von 37 °C und eine Enzymmenge, die dem Gehalt von 75-105 mg Frischgewebe entsprach. L-Arginin, L-Ornithin und Carbamylphosphat in Konzentrationen von 5 mM hemmten die Enzymwirkung ·um 13,6 bzw. 12%

AF-MSCs fate can be regulated by culture conditions

Human mesenchymal stem cells (hMSCs) represent a population of multipotent adherent cells able to differentiate into many lineages. In our previous studies, we isolated and expanded fetal MSCs from second-trimester amniotic fluid (AF) and characterized them based on their phenotype, pluripotency and proteomic profile. In the present study, we investigated the plasticity of these cells based on their differentiation, dedifferentiation and transdifferentiation potential in vitro. To this end, adipocyte-like cells (AL cells) derived from AF-MSCs can regain, under certain culture conditions, a more primitive phenotype through the process of dedifferentiation. Dedifferentiated AL cells derived from AF-MSCs (DAF-MSCs), gradually lost the expression of adipogenic markers and obtained similar morphology and differentiation potential to AF-MSCs, together with regaining the pluripotency marker expression. Moreover, a comparative proteomic analysis of AF-MSCs, AL cells and DAF-MSCs revealed 31 differentially expressed proteins among the three cell populations. Proteins, such as vimentin, galectin-1 and prohibitin that have a significant role in stem cell regulatory mechanisms, were expressed in higher levels in AF-MSCs and DAF-MSCs compared with AL cells. We next investigated whether AL cells could transdifferentiate into hepatocyte-like cells (HL cells) directly or through a dedifferentiation step. AL cells were cultured in hepatogenic medium and 4 days later they obtained a phenotype similar to AF-MSCs, and were termed as transdifferentiated AF-MSCs (TRAF-MSCs). This finding, together with the increase in pluripotency marker expression, indicated the adaption of a more primitive phenotype before transdifferentiation. Additionally, we observed that AF-, DAF- and TRAF-MSCs displayed similar clonogenic potential, secretome and proteome profile. Considering the easy access to this fetal cell source, the plasticity of AF-MSCs and their potential to dedifferentiate and transdifferentiate, AF may provide a valuable tool for cell therapy and tissue engineering applications

Resolving the Role of Plant Glutamate Dehydrogenase. I. in vivo Real Time Nuclear Magnetic Resonance Spectroscopy Experiments

In higher plants the glutamate dehydrogenase (GDH) enzyme catalyzes the reversible amination of 2-oxoglutarate to form glutamate, using ammonium as a substrate. For a better understanding of the physiological function of GDH either in ammonium assimilation or in the supply of 2-oxoglutarate, we used transgenic tobacco (Nicotiana tabacum L.) plants overexpressing the two genes encoding the enzyme. An in vivo real time 15N-nuclear magnetic resonance (NMR) spectroscopy approach allowed the demonstration that, when the two GDH genes were overexpressed individually or simultaneously, the transgenic plant leaves did not synthesize glutamate in the presence of ammonium when glutamine synthetase (GS) was inhibited. In contrast we confirmed that the primary function of GDH is to deaminate Glu. When the two GDH unlabeled substrates ammonium and Glu were provided simultaneously with either [15N]Glu or 15NH4+ respectively, we found that the ammonium released from the deamination of Glu was reassimilated by the enzyme GS, suggesting the occurrence of a futile cycle recycling both ammonium and Glu. Taken together, these results strongly suggest that the GDH enzyme, in conjunction with NADH-GOGAT, contributes to the control of leaf Glu homeostasis, an amino acid that plays a central signaling and metabolic role at the interface of the carbon and nitrogen assimilatory pathways. Thus, in vivo NMR spectroscopy appears to be an attractive technique to follow the flux of metabolites in both normal and genetically modified plants

Transcriptional Analysis of Fracture Healing and the Induction of Embryonic Stem Cell–Related Genes

Fractures are among the most common human traumas. Fracture healing represents a unique temporarily definable post-natal process in which to study the complex interactions of multiple molecular events that regulate endochondral skeletal tissue formation. Because of the regenerative nature of fracture healing, it is hypothesized that large numbers of post-natal stem cells are recruited and contribute to formation of the multiple cell lineages that contribute to this process. Bayesian modeling was used to generate the temporal profiles of the transcriptome during fracture healing. The temporal relationships between ontologies that are associated with various biologic, metabolic, and regulatory pathways were identified and related to developmental processes associated with skeletogenesis, vasculogenesis, and neurogenesis. The complement of all the expressed BMPs, Wnts, FGFs, and their receptors were related to the subsets of transcription factors that were concurrently expressed during fracture healing. We further defined during fracture healing the temporal patterns of expression for 174 of the 193 genes known to be associated with human genetic skeletal disorders. In order to identify the common regulatory features that might be present in stem cells that are recruited during fracture healing to other types of stem cells, we queried the transcriptome of fracture healing against that seen in embryonic stem cells (ESCs) and mesenchymal stem cells (MSCs). Approximately 300 known genes that are preferentially expressed in ESCs and ∼350 of the known genes that are preferentially expressed in MSCs showed induction during fracture healing. Nanog, one of the central epigenetic regulators associated with ESC stem cell maintenance, was shown to be associated in multiple forms or bone repair as well as MSC differentiation. In summary, these data present the first temporal analysis of the transcriptome of an endochondral bone formation process that takes place during fracture healing. They show that neurogenesis as well as vasculogenesis are predominant components of skeletal tissue formation and suggest common pathways are shared between post-natal stem cells and those seen in ESCs

Involvement of microRNAs in physiological and pathological processes in the lung

To date, at least 900 different microRNA (miRNA) genes have been discovered in the human genome. These short, single-stranded RNA molecules originate from larger precursor molecules that fold to produce hairpin structures, which are subsequently processed by ribonucleases Drosha/Pasha and Dicer to form mature miRNAs. MiRNAs play role in the posttranscriptional regulation of about one third of human genes, mainly via degradation of target mRNAs. Whereas the target mRNAs are often involved in the regulation of diverse physiological processes ranging from developmental timing to apoptosis, miRNAs have a strong potential to regulate fundamental biological processes also in the lung compartment. However, the knowledge of the role of miRNAs in physiological and pathological conditions in the lung is still limited. This review, therefore, summarizes current knowledge of the mechanism, function of miRNAs and their contribution to lung development and homeostasis. Besides the involvement of miRNAs in pulmonary physiological conditions, there is evidence that abnormal miRNA expression may lead to pathological processes and development of various pulmonary diseases. Next, the review describes current state-of-art on the miRNA expression profiles in smoking-related diseases including lung cancerogenesis, in immune system mediated pulmonary diseases and fibrotic processes in the lung. From the current research it is evident that miRNAs may play role in the posttranscriptional regulation of key genes in human pulmonary diseases. Further studies are, therefore, necessary to explore miRNA expression profiles and their association with target mRNAs in human pulmonary diseases