First Steps Towards an Understanding of a Mode ofCarcinogenic Action for Vanadium Pentoxide

Inhalation of vanadium pentoxide clearly increases the incidence of alveolar/bronchiolar neoplasms in male and female B6C3F1 mice at all concentrations tested (1, 2 or 4 mg/m3), whereas responses in F344/N rats was, at most, ambiguous. While vanadium pentoxide is mutagenic in vitro and possibly in vivo in mice, this does not explain the species or site specificity of the neoplastic response. A nose-only inhalation study was conducted in female B6C3F1 mice (0, 0.25, 1 and 4 mg/m3, 6 h/day for 16 days) to explore histopathological, biochemical (α-tocopherol, glutathione and F2-isoprostane) and genetic (comet assays and 9 specific DNA-oxo-adducts) changes in the lungs. No treatment related histopathology was observed at 0.25 mg/m3. At 1 and 4 mg/m3, exposure-dependent increases were observed in lung weight, alveolar histiocytosis, sub-acute alveolitis and/or granulocytic infiltration and a generally time-dependent increased cell proliferation rate of histiocytes. Glutathione was slightly increased, whereas there were no consistent changes in α-tocopherol or 8-isoprostane F2α. There was no evidence for DNA strand breakage in lung or BAL cells, but there was an increase in 8-oxodGuo DNA lesions that could have been due to vanadium pentoxide induction of the lesions or inhibition of repair of spontaneous lesions. Thus, earlier reports of histopathological changes in the lungs after inhalation of vanadium pentoxide were confirmed, but no evidence has yet emerged for a genotoxic mode of action. Evidence is weak for oxidative stress playing any role in lung carcinogenesis at the lowest effective concentrations of vanadium pentoxide

Protein profiling and metabolite profiling of Arabidopsis thaliana in the context of systems biology

Die biologische Forschung der vergangenen Jahre konzentrierte sich im Wesentlichen auf die detaillierte qualitative Beschreibung ausgewählter molekularer Teilprozesse. Die technische und/oder medizinische Nutzung dieser Erkenntnisse setzt jedoch ein ganzheitliches Verständnis der Funktionszusammenhänge biologischer Systeme voraus. Da dieses bis heute bestenfalls im Ansatz gegeben ist, lag der Schwerpunkt der vorliegenden Arbeit in der Entwicklung und Validierung von Analysemethoden zur quantitativen und ganzheitlichen Beschreibung zellulärer Prozesse über einen interdisziplinären Ansatz mit Methoden und Konzepten der Statistik und Bioinformatik. Zur Methodenvalidierung wurden komplexe Extrakte pflanzlichen Blattgewebes mittels GC-TOF-MS und nano-HPLC-MS in einer Vielzahl technischer und biologischer Replikate analysiert, wobei insgesamt 80 polare Metabolite sowie 179 lösliche Proteine eindeutig identifiziert und relativ quantifiziert werden konnten. Des Weiteren wurden die mit dem diurnalen Rhythmus verbundenen Änderungen des Stoffwechsels von Arabidopsis thaliana sowie die Auswirkungen von Temperaturstress auf den pflanzlichen Metabolismus erstmals integrativ analysiert. Die Auswertung und Interpretation der gewonnenen Metabolit- und Proteindaten erfolgte mit Hilfe bioinformatorischer Ansätze wie der Hauptkomponentenanalyse, der unabhängigen Komponentenanalyse oder der Analyse von Korrelationsnetzwerken. Als Ergebnis dieser Untersuchungen konnten zahlreiche metabolische und proteomische Marker identifiziert werden, anhand welcher der genetische Hintergrund bzw. ein durch Temperaturstress veränderter Metabolismus angezeigt werden. Zudem erbrachte die Netzwerkanalyse einige gezielte Hinweise auf in verschiedenen pflanzlichen Spezies konservierte, oder infolge von Mutation oder veränderten Umweltbedingungen deflektierte regulatorische Mechanismen.Recent biological research essentially concentrated on detailed qualitative description of select molecular sub processes. However, the technical and/or medical application of these findings requires a holistic understanding of the functional coherence of biological systems. Since such work is still in its infancy, the present work focused on developing and validating analytical methods to quantitatively describe cellular processes by means of an interdisciplinary approach that uses methods and concepts from both, statistics and bioinformatics. To validate the developed methods, complex plant leaf extracts were analyzed using GC-TOF-MS and nano-HPLC-MS. This allowed for the unambiguously identification and quantification of 80 polar metabolites and 179 soluble proteins. For the first time changes in the metabolism of Arabidopsis thaliana resulting from its diurnal rhythm and from temperature stress were integratively analyzed. The analysis and interpretation of the metabolite and protein data obtained was performed using principal components analyses, independent components analysis, and the analysis of correlation networks. These analyses allowed for the identification of metabolic markers, which reveal the genetic background and/or changes in the metabolism caused by temperature stress. In addition, the network analysis points to several specific regulatory mechanisms conserved in various plant species or changed by mutation or altered environmental conditions

Integration of Metabolomic and Proteomic Phenotypes: Analysis of Data Covariance Dissects Starch and RFO Metabolism from Low and High Temperature Compensation Response in Arabidopsis Thaliana*S⃞

Statistical mining and integration of complex molecular data including metabolites, proteins, and transcripts is one of the critical goals of systems biology (Ideker, T., Galitski, T., and Hood, L. (2001) A new approach to decoding life: systems biology. Annu. Rev. Genomics Hum. Genet. 2, 343–372). A number of studies have demonstrated the parallel analysis of metabolites and large scale transcript expression. Protein analysis has been ignored in these studies, although a clear correlation between transcript and protein levels is shown only in rare cases, necessitating that actual protein levels have to be determined for protein function analysis. Here, we present an approach to investigate the combined covariance structure of metabolite and protein dynamics in a systemic response to abiotic temperature stress in Arabidopsis thaliana wild-type and a corresponding starch-deficient mutant (phosphoglucomutase-deficient). Independent component analysis revealed phenotype classification resolving genotype-dependent response effects to temperature treatment and genotype-independent general temperature compensation mechanisms. An observation is the stress-induced increase of raffinose-family-oligosaccharide levels in the absence of transitory starch storage/mobilization in temperature-treated phosphoglucomutase plants indicating that sucrose synthesis and storage in these mutant plants is sufficient to bypass the typical starch storage/mobilization pathways under abiotic stress. Eventually, sample pattern recognition and correlation network topology analysis allowed for the detection of specific metabolite-protein co-regulation and assignment of a circadian output regulated RNA-binding protein to these processes. The whole concept of high-dimensional profiling data integration from many replicates, subsequent multivariate statistics for dimensionality reduction, and covariance structure analysis is proposed to be a major strategy for revealing central responses of the biological system under study

d-GLYCERATE 3-KINASE, the Last Unknown Enzyme in the Photorespiratory Cycle in Arabidopsis, Belongs to a Novel Kinase Family

d-GLYCERATE 3-KINASE (GLYK; EC 2.7.1.31) catalyzes the concluding reaction of the photorespiratory C(2) cycle, an indispensable ancillary metabolic pathway to the photosynthetic C(3) cycle that enables land plants to grow in an oxygen-containing atmosphere. Except for GLYK, all other enzymes that contribute to the C(2) cycle are known by their primary structures, and the encoding genes have been identified. We have purified and partially sequenced this yet missing enzyme from Arabidopsis thaliana and identified it as a putative kinase-annotated single-copy gene At1g80380. The exclusive catalytic properties of the gene product were confirmed after heterologous expression in Escherichia coli. Arabidopsis T-DNA insertional knockout mutants show no GLYK activity and are not viable in normal air; however, they grow under elevated CO(2), providing direct evidence of the obligatory nature of the ultimate step of the C(2) cycle. The newly identified GLYK is both structurally and phylogenetically distinct from known glycerate kinases from bacteria and animals. Orthologous enzymes are present in other plants, fungi, and some cyanobacteria. The metabolic context of GLYK activity in fungi and cyanobacteria remains to be investigated

Deletion of Glycine Decarboxylase in Arabidopsis Is Lethal under Nonphotorespiratory Conditions1[W][OA]

The mitochondrial multienzyme glycine decarboxylase (GDC) catalyzes the tetrahydrofolate-dependent catabolism of glycine to 5,10-methylene-tetrahydrofolate and the side products NADH, CO2, and NH3. This reaction forms part of the photorespiratory cycle and contributes to one-carbon metabolism. While the important role of GDC for these two metabolic pathways is well established, the existence of bypassing reactions has also been suggested. Therefore, it is not clear to what extent GDC is obligatory for these processes. Here, we report on features of individual and combined T-DNA insertion mutants for one of the GDC subunits, P protein, which is encoded by two genes in Arabidopsis (Arabidopsis thaliana). The individual knockout of either of these two genes does not significantly alter metabolism and photosynthetic performance indicating functional redundancy. In contrast, the double mutant does not develop beyond the cotyledon stage in air enriched with 0.9% CO2. Rosette leaves do not appear and the seedlings do not survive for longer than about 3 to 4 weeks under these nonphotorespiratory conditions. This feature distinguishes the GDC-lacking double mutant from all other known photorespiratory mutants and provides evidence for the nonreplaceable function of GDC in vital metabolic processes other than photorespiration

A Cytosolic Pathway for the Conversion of Hydroxypyruvate to Glycerate during Photorespiration in Arabidopsis[W]

Deletion of any of the core enzymes of the photorespiratory cycle, one of the major pathways of plant primary metabolism, results in severe air-sensitivity of the respective mutants. The peroxisomal enzyme hydroxypyruvate reductase (HPR1) represents the only exception to this rule. This indicates the presence of extraperoxisomal reactions of photorespiratory hydroxypyruvate metabolism. We have identified a second hydroxypyruvate reductase, HPR2, and present genetic and biochemical evidence that the enzyme provides a cytosolic bypass to the photorespiratory core cycle in Arabidopsis thaliana. Deletion of HPR2 results in elevated levels of hydroxypyruvate and other metabolites in leaves. Photosynthetic gas exchange is slightly altered, especially under long-day conditions. Otherwise, the mutant closely resembles wild-type plants. The combined deletion of both HPR1 and HPR2, however, results in distinct air-sensitivity and a dramatic reduction in photosynthetic performance. These results suggest that photorespiratory metabolism is not confined to chloroplasts, peroxisomes, and mitochondria but also extends to the cytosol. The extent to which cytosolic reactions contribute to the operation of the photorespiratory cycle in varying natural environments is not yet known, but it might be dynamically regulated by the availability of NADH in the context of peroxisomal redox homeostasis

Metabolite Profiling in Arabidopsis thaliana with Moderately Impaired Photorespiration Reveals Novel Metabolic Links and Compensatory Mechanisms of Photorespiration

Photorespiration is an integral component of plant primary metabolism. Accordingly, it has been often observed that impairing the photorespiratory flux negatively impacts other cellular processes. In this study, the metabolic acclimation of the Arabidopsis thaliana wild type was compared with the hydroxypyruvate reductase 1 (HPR1; hpr1) mutant, displaying only a moderately reduced photorespiratory flux. Plants were analyzed during development and under varying photoperiods with a combination of non-targeted and targeted metabolome analysis, as well as C-13- and C-14-labeling approaches. The results showed that HPR1 deficiency is more critical for photorespiration during the vegetative compared to the regenerative growth phase. A shorter photoperiod seems to slowdown the photorespiratory metabolite conversion mostly at the glycerate kinase and glycine decarboxylase steps compared to long days. It is demonstrated that even a moderate impairment of photorespiration severely reduces the leaf-carbohydrate status and impacts on sulfur metabolism. Isotope labeling approaches revealed an increased CO2 release from hpr1 leaves, most likely occurring from enhanced non-enzymatic 3-hydroxypyruvate decarboxylation and a higher flux from serine towards ethanolamine through serine decarboxylase. Collectively, the study provides evidence that the moderate hpr1 mutant is an excellent tool to unravel the underlying mechanisms governing the regulation of metabolic linkages of photorespiration with plant primary metabolism

Integration of metabolomic and proteomic phenotypes: analysis of data covariance dissects starch and RFO metabolism from low and high temperature compensation response in Arabidopsis thaliana

Statistical mining and integration of complex molecular data including metabolites, proteins, and transcripts is one of the critical goals of systems biology (Ideker, T., Galitski, T., and Hood, L. (2001) A new approach to decoding life: systems biology. Annu. Rev. Genomics Hum. Genet. 2, 343372). A number of studies have demonstrated the parallel analysis of metabolites and large scale transcript expression. Protein analysis has been ignored in these studies, although a clear correlation between transcript and protein levels is shown only in rare cases, necessitating that actual protein levels have to be determined for protein function analysis. Here, we present an approach to investigate the combined covariance structure of metabolite and protein dynamics in a systemic response to abiotic temperature stress in Arabidopsis thaliana wild-type and a corresponding starch-deficient mutant (phosphoglucomutase-deficient). Independent component analysis revealed phenotype classification resolving genotype-dependent response effects to temperature treatment and genotype-independent general temperature compensation mechanisms. An observation is the stress-induced increase of raffinose-family-oligosaccharide levels in the absence of transitory starch storage/mobilization in temperature-treated phosphoglucomutase plants indicating that sucrose synthesis and storage in these mutant plants is sufficient to bypass the typical starch storage/mobilization pathways under abiotic stress. Eventually, sample pattern recognition and correlation network topology analysis allowed for the detection of specific metabolite-protein co-regulation and assignment of a circadian output regulated RNA-binding protein to these processes. The whole concept of high-dimensional profiling data integration from many replicates, subsequent multivariate statistics for dimensionality reduction, and covariance structure analysis is proposed to be a major strategy for revealing central responses of the biological system under study