The isolation and purification of DNA from Vitis vinifera L. plants and in vitro cultures

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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

Methylated DNA changes associated with the initiation and maintenance of Vitis vinifera in vitro shoot and callus cultures: A possible mechanism for age-related changes

Tissue culture technologies are an important aspect of the genetic modification of grapevine (Vitis vinifera). The molecular basis of this phenomenon is not well understood, however, the extent of DNA methylation is recognised as a factor in the control of gene expression. This study explores the possibility that DNA methylation may have a role in grapevine culture responses. DNA methylation profiles were constructed for Vitis vinifera, cv. Sultanina, during plantlet micropropagation, callus induction and proliferation. Methylation of genomic DNA and ribosomal RNA genes (rDNA) was found in glasshouse-grown plants, micropropagated plantlets and callus cultures. An analysis of rDNA showed that glasshouse-grown plants had 74.6% of the recognition sequences for Hpa II methylated at the internal cytosine position CmCGG, whereas 7.7% of the recognition sequences appeared to be methylated at the external cytosine (mCmCGG). The rDNA profiles of micropropagated subcultures S0 (initial subcultures) and S4 (fourth subcultures), representing one year of in vitro growth, showed that the percentage of recognition sequences containing a methylated external cytosine increased from 7.7% in glasshouse-grown plants to 64.5% for S-0 and 72.5% for the S4 subculture. The implications of these findings for the in vitro manipulation of grapevine used in genetic modifications are discussed

Arginase from kiwifruit: properties and seasonal variation

The in vitro activity of arginase (EC 3.5.3.1) was investigated in youngest-mature leaves and roots (1-3 mm diameter) of kiwifruit vines (Actinidia deliciosa var. deliciosa) during an annual growth cycle, and enzyme from root material partially purified. No seasonal trend in the specific activity of arginase was observed in roots. Measurements in leaves, however, rose gradually during early growth and plateaued c. 17 weeks after budbreak. Changes in arginase activity were not correlated with changes in the concentration of arginine (substrate) or glutamine (likely end-product of arginine catabolism) in either tissue during the growth cycle. Purification was by (NH4)2SO4 precipitation and DEAE-cellulose chromatography. The kinetic properties of the enzyme, purified 60-fold over that in crude extracts, indicated a pH optimum of 8.8, and a Km (L-arginine) of 7.85 mM. Partially-purified enzyme was deactivated by dialysis against EDTA, and reactivated in the presence of Mn²⁺, Co²⁺, and Ni²⁺

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

Human Embryonic and Fetal Mesenchymal Stem Cells Differentiate toward Three Different Cardiac Lineages in Contrast to Their Adult Counterparts

Mesenchymal stem cells (MSCs) show unexplained differences in differentiation potential. In this study, differentiation of human (h) MSCs derived from embryonic, fetal and adult sources toward cardiomyocytes, endothelial and smooth muscle cells was investigated. Labeled hMSCs derived from embryonic stem cells (hESC-MSCs), fetal umbilical cord, bone marrow, amniotic membrane and adult bone marrow and adipose tissue were co-cultured with neonatal rat cardiomyocytes (nrCMCs) or cardiac fibroblasts (nrCFBs) for 10 days, and also cultured under angiogenic conditions. Cardiomyogenesis was assessed by human-specific immunocytological analysis, whole-cell current-clamp recordings, human-specific qRT-PCR and optical mapping. After co-culture with nrCMCs, significantly more hESC-MSCs than fetal hMSCs stained positive for α-actinin, whereas adult hMSCs stained negative. Furthermore, functional cardiomyogenic differentiation, based on action potential recordings, was shown to occur, but not in adult hMSCs. Of all sources, hESC-MSCs expressed most cardiac-specific genes. hESC-MSCs and fetal hMSCs contained significantly higher basal levels of connexin43 than adult hMSCs and co-culture with nrCMCs increased expression. After co-culture with nrCFBs, hESC-MSCs and fetal hMSCs did not express α-actinin and connexin43 expression was decreased. Conduction velocity (CV) in co-cultures of nrCMCs and hESC-MSCs was significantly higher than in co-cultures with fetal or adult hMSCs. In angiogenesis bioassays, only hESC-MSCs and fetal hMSCs were able to form capillary-like structures, which stained for smooth muscle and endothelial cell markers.Human embryonic and fetal MSCs differentiate toward three different cardiac lineages, in contrast to adult MSCs. Cardiomyogenesis is determined by stimuli from the cellular microenvironment, where connexin43 may play an important role