Developmental changes in carbohydrate metabolism during early tuberisation of potato

The potato tuber is a vegetative storage organ, which is initiated at the tip of an underground diagravitropic growing stem, called a stolon. The process of tuber formation comprises the induction, initiation and growth of a stolon, cessation of the longitudinal growth of the stolon, followed by the induction, initiation and growth of the tuber. Tuber initiation is characterized by swelling of the stolon tip. The formation of tubers is accompanied by a large accumulation of starch and of a specific set of storage proteins. How these major changes in carbohydrate metabolism interact and are coordinated with the developmental process of tuber formation is an important question from a fundamental point of view, not only for potato tubers, but also for the formation of starch-accumulating plant tissues in general. When a potato plant has started to form tubers, in principle all stolons present on the plant can be induced to initiate this developmental programme. However, this does not occur synchronously, creating problems for research as it is not possible to select externally those stolons, which are going to tuberize. Another problem is that the different developmental stages on one plant can influence each other. Therefore, for our research of tuberisation-related changes in carbohydrate metabolism, we have used a highly synchronous in vitro tuberisation system, providing plant material in well-defined developmental stages, which are not influenced by each other.Sucrose is the starting point of carbohydrate metabolism in growing stolons and tubers. It is synthesised in the leaves (source organs) and transported towards the underground parts via the phloem. When it arrives in a growing stolon it is most likely unloaded in an apoplastic way. In the apical region of the stolon a high invertase activity is present, which can be attributed to the cell wall-bound form (CWI). This apoplastic invertase isoform hydrolyses sucrose, resulting in the presence of glucose and fructose in the apoplast. The hydrolysis of sucrose steepens the sucrose gradient between phloem and the recipient cells, which is thought to play an important role in driving the unloading and short-distance transport of sucrose in apoplastically-supplied tissues. After the hexoses have entered the cells, they can be fed into carbohydrate metabolism by the activity of specific phosphorylating enzymes. During stolon growth, one of these enzymes, hexokinase (HK), shows a high correlation with CWI at the level of overall activity, suggesting a coordinated coarse control between these two enzymes.Furthermore, localisation of HK by specific enzyme activity staining showed, like CWI, a high activity in the apical region of the stolon. In this particular region of the stolon cell divisions occur. Therefore, the predominance of these two enzymes in this region in comparison with the rest of the stolon suggests a functional link with cell division.Cellular growth is sustained by a high flux of carbon through the respiratory(-related) pathways producing energy in the form of ATP, NAD(P)H, and carbon intermediates for important anabolic routes, such as the synthesis of DNA, RNA, proteins, lipids and cell wall-components. Measurements of activity of the main enzymes involved in glycolysis and the oxidative pentose phosphate pathway (OPPP) indicated that the extra demand for energy and carbon intermediates by dividing cells is accompanied with an elevated activity of these enzymes. Furthermore, from the patterns of activity of these enzymes it could be deduced that they are probably subject to a coordinated coarse control. Such a coordinated control mechanism may regulate the potential of respiratory(-related) pathways in response to the demand for their products. The high activities of CWI and HK in the mitotic-active region could ensure that sufficient carbon is directed into the respiratory(-related) pathways by enhancing the flow of carbon towards the dividing cells and by rapid conversion of sucrose into hexose-phosphates, the substrates for glycolysis and OPPP. In addition, these two enzymes may also be involved in the process of glucose-sensing, which is thought to mediate the transcriptional (and/or translational) regulation of genes involved in cell division. Interestingly, during stolon growth the enzyme alkaline pyrophosphatase (APPase) shows a similar pattern of overall activity as the main enzymes involved in glycolysis and OPPP.Moreover, in situ hybridisation with a RNA probe derived from a potato APPase revealed a clear presence of the corresponding transcript in all stolon tissues. This particular isoform has been proposed to be localised in the cytosol, which is in agreement with its temporal and spatial pattern of expression and with the pattern of overall APPase activity during stolon-to-tuber transition. In the dividing cells the biosynthesis of cell components produces a large amount of PPi, which is the substrate of this enzyme. Rapid conversion of PPi by a cytosolic APPase or another enzyme (e.g. tonoplast bound pyrophosphatase) is required to drive the biosynthetic reactions in the synthetic direction. The Pi released by this activity may serve as a metabolic pool of Pi for the conversion of ADP into ATP. In this way the observed apparent coordination between level of APPase activity and of respiratory(-related) pathways could be explained.When the cells have left the cell cycle and are no longer involved in cell division, their main cellular activity comprises enlargement, which continues until the cells are fully elongated and mature. In the region just below the apical part of the stolon, where cell divisions no longer occur, less invertase and HK staining is present. The reduction in the levels of activity of CWI and HK in this region, can also be deduced from their patterns of overall activity during stolon growth. The activity of a soluble vacuolar invertase, however, does not decrease but remains rather constant throughout the whole growing stolon. In these elongating parts of the stolon the combined activity of the soluble invertases will establish the sink strength, i.e. the capacity to draw sucrose from the phloem. Besides this function in maintaining a sucrose gradient between phloem and elongating cells and allowing sucrose to enter carbohydrate metabolism, the vacuolar invertase may also be involved in the process of cell elongation. Cell elongation is mechanistically driven by enlargement of the vacuole, which depends on the osmotic pressure of the vacuole. The hydrolysis of one osmotically-active molecule into two by the activity of this vacuolar isoform will enhance the osmotic pressure and the water influx into the vacuole. The elongation zone of the stolon probably has a lower requirement for energy and carbon intermediates in order to sustain the cellular growth than the mitotically active apical region, which is reflected by the decline in the overall activity of enzymes involved in glycolysis and OPPP during the period of consecutive stolon growth in our in vitro system.Tuber initiation is accompanied by a clear change in the patterns of several enzymes. In situ hybridisation revealed that the transcripts corresponding to the acid invertases progressively disappear from the parenchymatic tissues of the tuber. As a result, the overall activities of these two isoforms declined to very low levels, which was also indicated by the in situ activity staining. In contrast, the enzyme sucrose synthase (Susy) increases rapidly to high levels in the parenchymatic tissues of the initiated tuber as indicated by the in situ analysis at mRNA level and activity level and by overall activity measurements. This developmental change in the sucrose-degrading pathway is clearly associated with tuberisation and the onset of massive starch accumulation as it was absent in the two non-tuberising controls. Sucrose synthase catalyses in vivo a reversible, low-affinity reaction, which is subject to a high degree of metabolic regulation. Sucrose degradation by invertases is irreversible, not subject to metabolic regulation, and it occurs already at a low sucrose level. These differences between the two different sucrolytic pathways make the Susy-catalysed pathway probably more suitable than invertases for the complex regulation of sucrose degradation in coordination with tuber growth, symplastic sucrose unloading, and starch synthesis, as is explained in chapters 2 and 5. Furthermore, there are indications that a certain sucrose influx and/or sucrose levels are required for the upregulation of the expression of genes involved in sucrose-starch conversion. The effectiveness of this apparent sucrose-modulated mechanism depends largely on the type of sucrolytic route predominantly present in the cell. The down-regulation of CWI and the switch of an invertase-catalysed sucrose-degradation route into a Susy-mediated sucrolytic system allow import of carbon in the form of sucrose and allow high sucrose levels in the cytosol. Moreover, hexoses released by the activity of invertases appear to have an inducing effect on respiratory(-related) metabolism, which is thought to occur at the expense of the carbon flux toward starch synthesis.Concomitant with the developmental switch in the sucrose-degrading pathway an increase in the overall activity of fructokinase can be observed. In situ analysis at transcript level and activity level indicated that the spatial and temporal pattern of this enzyme show a close resemblance to that of Susy. The apparently closely coordinated coarse control indicates a functional linkage between these two enzymes. As already mentioned Susy catalyses a reversible reaction, which is severely inhibited by accumulation of fructose. Fructokinase may downregulate the fructose content, thereby allowing a large net flux of sucrose degradation through the Susy-catalysed reaction. The other product of the degrading reaction of Susy is UDPGlucose (UDPGlc). UDPGlc can be converted by the enzyme UGPase, which does not show a development-related increase in our in vitro system, but is present at a high activity throughout the whole period of culture. The coarse control of this enzyme, just like that of the enzyme PFP, seems subject to modulation by high sugar levels. Both enzymes catalyse a reversible reaction, use PPi as an energy-donor and are able to convert the substrate of the sucrose synthesising enzyme SPS. In combination with Susy (and SPS), the reversible character of these two enzymes allows a process of sucrose/hexose-P and hexose-P/triose-P cycling, which offers the cell a mechanism to adjust rapidly and automatically the net rate of sucrose degradation in response to the supply of sucrose and demand for carbon in the respiratory and biosynthetic pathways in the tuber parenchyma cells.Measurements of the overall level of different hexose-P's during stolon-to-tuber transition indicated that tuber initiation and successive growth is accompanied with a decline in the hexose-P pool, which is in contrast with an expected increase due to the tuberisation-initiated rise in sucrose supply and degradation rate. A plausible explanation for this unexpected observation is that the level of the hexose-P pool has to remain low during tuber growth, because an increase in this pool might lead to an enhanced rate of sucrose resynthesis by Susy and SPS. Such an increase in unidirectional sucrose synthesis will lead to a decrease in the net rate of sucrose degradation, thereby lowering the net flux of carbon towards starch synthesis. Therefore, a low hexose-P pool might be an important prerequisite for a high, but adjustable net rate of sucrose degradation in Susy-mediated sucrolytic systems. A possible role for the enzyme PFP in the maintenance of a low hexose-P content is discussed in chapter 5.During successive tuber growth the overall activities of enzymes involved in respiratory(-related) metabolism declined, indicating that tuber growth and starch accumulation does not require an increase in the potential of respiratory(-related) metabolism. Concomitant with this decline the overall activity of APPase decreased also. In situ hybridisation with the presumed corresponding RNA probe revealed a substantial decline in mRNA content in the whole growing tuber. The Susy-mediated pathway of sucrose degradation depends on the availability of PPi, which is required by the UDPGlc-converting reaction of UGPase. The decline in activity of the assumed cytosolic APPase may prevent that the sucrose-degrading reaction of Susy becomes restricted by the accumulation of UDPGlc.Three other enzymes show a clear and large increase in activity after the onset of tuberisation, which is not observed in the non-tuberising controls. Two of these enzymes are known to be involved in starch synthesis. The enzyme ADPglucose pyrophosphorylase (AGPase) synthesises the precursor for starch synthesis and is thought to contribute to a large extent to the total flux control of carbon towards starch synthesis. The other enzyme is starch phosphorylase (STP), which catalyses a reversible reaction and may, therefore, be involved in the synthesis of starch or in its degradation. The patterns of both enzyme-activities correlate highly with the accumulation of starch in our in-vitro tuberisation system, which is consistent with their known role in the (cycling) process of starch synthesis. Starch accumulation seems to be a process of simultaneous synthesis and degradation. Two possible routes of degradation may exist. The first one can be catalysed by STP, the second one involves the activity of amylases. The (final) endproduct of amylases, glucose, can only be redirected into starch metabolism by the activity of a hexokinase. This enzyme shows a considerable increase in its level of activity after tuberisation onset. Activity staining revealed that this enzyme is present in the main starch-accumulating tissues of the tuber. The product of its reaction, Glc6P, has to be converted into Glc1P, which serves as the substrate for AGPase and STP.The enzyme responsible for this reaction, phosphoglucomutase (PGM) also shows a considerable increase in overall activity after the initiation of tuberisation, although not that large as AGPase, STP and HK. Furthermore, the tuberisation-related increase in Glc1P content in comparison with the decline in the content of the other hexose-P's, suggests that the reversible reaction catalysed by the plastidial isoform of PGM lies in the direction of Glc1P synthesis.From this study on changes of carbohydrate metabolism during stolon-to-tuber transition of potato, it has become clear that the sucrolytic switch itself forms a central and important step in the differentiation process of starch-storing tubers. The direct implications and consequences of this metabolic switch for the regulation of carbon fluxes towards starch synthesis and other carbon-demanding process are discussed in chapter 2, 3, 4 and 5. In addition in chapter 5 an effort has been made to place this tuberisation-related switch in a broader context of metabolic induction of growth-related and storage-associated differentiation processes, including the role and importance of glucose and sucrose as different signal-inducing molecules, modulating the expression of genes involved in cell division and respiratory(-related) metabolism (glucose-responsive) versus differentiation into storage cells and sucrose-starch converting potential (sucrose-responsive). In chapter 5 is also discussed how these mechanisms might affect sink strength and thus the capacity to enhance the flow of photosynthate towards the cells of the two different types of growing sink organs and its importance for growth and/or storage.</p

Rad21-Cohesin Haploinsufficiency Impedes DNA Repair and Enhances Gastrointestinal Radiosensitivity in Mice

Approximately half of cancer-affected patients receive radiotherapy (RT). The doses delivered have been determined upon empirical experience based upon average radiation responses. Ideally higher curative radiation doses might be employed in patients with genuinely normal radiation responses and importantly radiation hypersensitive patients would be spared the consequences of excessive tissue damage if they were indentified before treatment. Rad21 is an integral subunit of the cohesin complex, which regulates chromosome segregation and DNA damage responses in eukaryotes. We show here, by targeted inactivation of this key cohesin component in mice, that Rad21 is a DNA-damage response gene that markedly affects animal and cell survival. Biallelic deletion of Rad21 results in early embryonic death. Rad21 heterozygous mutant cells are defective in homologous recombination (HR)-mediated gene targeting and sister chromatid exchanges. Rad21+/− animals exhibited sensitivity considerably greater than control littermates when challenged with whole body irradiation (WBI). Importantly, Rad21+/− animals are significantly more sensitive to WBI than Atm heterozygous mutant mice. Since supralethal WBI of mammals most typically leads to death via damage to the gastrointestinal tract (GIT) or the haematopoietic system, we determined the functional status of these organs in the irradiated animals. We found evidence for GIT hypersensitivity of the Rad21 mutants and impaired bone marrow stem cell clonogenic regeneration. These data indicate that Rad21 gene dosage is critical for the ionising radiation (IR) response. Rad21 mutant mice thus represent a new mammalian model for understanding the molecular basis of irradiation effects on normal tissues and have important implications in the understanding of acute radiation toxicity in normal tissues

Antioxidative protection of dietary bilberry, chokeberry and Lactobacillus plantarum HEAL19 in mice subjected to intestinal oxidative stress by ischemia-reperfusion

<p>Abstract</p> <p>Background</p> <p>Ischemia-reperfusion (I/R) in the intestines is an inflammatory condition which activates leukocytes and reactive oxygen species (ROS) and leads to lipid peroxidation and DNA damage. Bilberry and chokeberry fruits are rich sources of polyphenols which may act as antioxidants and prevent lipid peroxidation. Lactic acid bacteria (LAB) may improve microbial status in the intestines and increase the metabolic activity towards polyphenolic degradation. The aim of the study was to clarify antioxidative effects of bilberry and chokeberry fruits alone and with addition of a LAB-strain, <it>Lactobacillus plantarum </it>HEAL19, in an I/R-model in mice.</p> <p>Methods</p> <p>Male BALB/cJ mice were fed the experimental diets for 10 days. Diets consisted of standard chow supplemented with either bilberry (<it>Vaccinium myrtillus</it>) or chokeberry (<it>Aronia × prunifolia</it>) powder alone or in combination with the LAB-strain <it>Lactobacillus plantarum </it>HEAL19. I/R-injury was induced by holding superior mesenteric artery clamped for 30 minutes followed by reperfusion for 240 minutes. Thereafter, colonic and caecal tissues and contents were collected. Malondialdehyde (MDA) was used as indicator of lipid peroxidation and was measured by a calorimetric assay, lactobacilli were cultured on Rogosa agar plates and <it>Enterobacteriaceae </it>on VRBG agar plates, anthocyanins and phenolic acids were analysed by HPLC-DAD-ESI-MSn.</p> <p>Results</p> <p>MDA was significantly decreased in the colon of groups fed bilberry alone (p = 0.030) and in combination with <it>L. plantarum </it>HEAL19 (p = 0.021) compared to the IR-control but not in chokeberry-fed groups. Supplementation with bilberry or chokeberry alone reduced the total number of lactobacilli on the mucosa. Higher concentrations of anthocyanins were found in the colon than in the caecum content of mice. A more varied composition of different anthocyanins was also observed in the colon content compared to the caecum of bilberry-fed mice. Phenolic acids formed by microbial degradation of the dietary polyphenols in the gut could be detected. More phenolic metabolites were found in the intestines of bilberry-fed mice than in the chokeberry-fed ones.</p> <p>Conclusions</p> <p>Bilberry alone and in combination with <it>L. plantarum </it>HEAL19 exerts a better protection against lipid peroxidation than chokeberry. These dietary supplements may be used to prevent or suppress oxidative stress.</p

Molecular Characterization of the Pollen-Specific Genomic Clone Ntpg303 and in Situ Localization of Expression

Contains fulltext : 28834___.PDF (publisher's version ) (Open Access

Pharmacokinetics and Pharmacodynamics of Ruxolitinib: A Review

BACKGROUND AND OBJECTIVE: Ruxolitinib is a tyrosine kinase inhibitor targeting the Janus kinase (JAK) and signal transducer and activator of transcription (STAT) pathways. Ruxolitinib is used to treat myelofibrosis, polycythemia vera and steroid-refractory graft-versus-host disease in the setting of allogeneic stem-cell transplantation. This review describes the pharmacokinetics and pharmacodynamics of ruxolitinib. METHODS: Pubmed, EMBASE, Cochrane Library and web of Science were searched from the time of database inception to march 15, 2021 and was repeated on November 16, 2021. Articles not written in English, animal or in vitro studies, letters to the editor, case reports, where ruxolitinib was not used for hematological diseases or not available as full text were excluded. RESULTS: Ruxolitinib is well absorbed, has 95% bio-availability, and is bound to albumin for 97%. Ruxolitinib pharmacokinetics can be described with a two-compartment model and linear elimination. Volume of distribution differs between men and women, likely related to bodyweight differences. Metabolism is mainly hepatic via CYP3A4 and can be altered by CYP3A4 inducers and inhibitors. The major metabolites of ruxolitinib are pharmacologically active. The main route of elimination of ruxolitinib metabolites is renal. Liver and renal dysfunction affect some of the pharmacokinetic variables and require dose reductions. Model-informed precision dosing might be a way to further optimize and individualize ruxolitinib treatment, but is not yet advised for routine care due to lack of information on target concentrations. CONCLUSION: Further research is needed to explain the interindividual variability of the ruxolitinib pharmacokinetic variables and to optimize individual treatment

Partial characterization of the DNA repair protein complex, containing the ERCC1, ERCC4, ERCC11 and XPF correcting activities

The nucleotide excision repair (NER) protein ERCC1 is part of a functional complex, which harbors in addition the repair correcting activities of ERCC4, ERCC11 and human XPF. ERCC1 is not associated with a defect in any of the known human NER disorders: xeroderma pigmentosum, Cockayne's syndrome or trichothiodystrophy. Here we report the partial purification and characterization of the ERCC1 complex. Immunoprecipitation studies tentatively identified a subunit in the complex with an apparent MW of approximately 120 kDa. The complex has affinity for DNA, but no clear preference for ss, ds or UV-damaged DNA substrates. The size of the entire complex determined by non-denaturing gradient gels (approximately 280 kDa) is considerably larger than previously found using size separation on glycerol gradients (approximately 120 kDa). Stable associations of the ERCC1 complex with other known repair factors (XPA, XPC, XPG and TFIIH complex) could not be detected