Search

Aberrant expression of MYC transcription factor family members predicts poor clinical outcome in many human cancers. Oncogenic MYC profoundly alters metabolism and mediates an antioxidant response to maintain redox balance. Here we show that MYCN induces massive lipid peroxidation on depletion of cysteine, the rate-limiting amino acid for glutathione (GSH) biosynthesis, and sensitizes cells to ferroptosis, an oxidative, non-apoptotic and iron-dependent type of cell death. The high cysteine demand of MYCN-amplified childhood neuroblastoma is met by uptake and transsulfuration. When uptake is limited, cysteine usage for protein synthesis is maintained at the expense of GSH triggering ferroptosis and potentially contributing to spontaneous tumor regression in low-risk neuroblastomas. Pharmacological inhibition of both cystine uptake and transsulfuration combined with GPX4 inactivation resulted in tumor remission in an orthotopic MYCN-amplified neuroblastoma model. These findings provide a proof of concept of combining multiple ferroptosis targets as a promising therapeutic strategy for aggressive MYCN-amplified tumors.

Elevated blood lactate levels are frequently found in critically ill patients and thought to result from tissue hypoperfusion and cellular oxygen shortage. Considering the close relationship between immune cell function and intracellular metabolism, lactate is more than a glycolytic waste molecule but able to regulate the immune response. Our aim was to elucidate the temporal and mechanistic effect of extracellular lactate on monocytes. To this end, primary human monocytes and the human monocytic cell line MonoMac6 were stimulated with various toll-like-receptor agonists after priming with Na-L-lactate under constant pH conditions. As readout, cytokine production was measured, real-time assessment of intracellular energy pathways was performed, and intracellular metabolite concentrations were determined. Irrespective of the immunogenic stimulus, short-term Na-lactate-priming strongly reduced cytokine production capacity. Lactate and hexoses accumulated intracellularly and, together with a decreased glycolytic flux, indicate a lactate-triggered impairment of glycolysis. To counteract intracellular hyperglycemia, glucose is shunted into the branching polyol pathway, leading to sorbitol accumulation. In contrast, long-term priming with Na-L-lactate induced cellular adaption and abolished the suppressive effect. This lactate tolerance is characterized by a decreased cellular respiration due to a reduced complex-I activity. Our results indicate that exogenous lactate shapes monocyte function by altering the intracellular energy metabolism and acts as a metabolic checkpoint of monocyte activation.

Chronic hypoxia increases the resistance of pulmonary arteries by stimulating their contraction and augmenting their coverage by smooth muscle cells (SMCs). While these responses require adjustment of the vascular SMC transcriptome, regulatory elements are not well defined in this context. Here, we explored the functional role of the transcription factor nuclear factor of activated T-cells 5 (NFAT5/TonEBP) in the hypoxic lung. Regulatory functions of NFAT5 were investigated in cultured artery SMCs and lungs from control (Nfat5fl/fl) and SMC-specific Nfat5-deficient (Nfat5(SMC)−/−) mice. Exposure to hypoxia promoted the expression of genes associated with metabolism and mitochondrial oxidative phosphorylation (OXPHOS) in Nfat5(SMC)−/− versus Nfat5fl/fl lungs. In vitro, hypoxia-exposed Nfat5-deficient pulmonary artery SMCs elevated the level of OXPHOS-related transcripts, mitochondrial respiration, and production of reactive oxygen species (ROS). Right ventricular functions were impaired while pulmonary right ventricular systolic pressure (RVSP) was amplified in hypoxia-exposed Nfat5(SMC)−/− versus Nfat5fl/fl mice. Scavenging of mitochondrial ROS normalized the raise in RVSP. Our findings suggest a critical role for NFAT5 as a suppressor of OXPHOS-associated gene expression, mitochondrial respiration, and ROS production in pulmonary artery SMCs that is vital to limit ROS-dependent arterial resistance in a hypoxic environment.

Aberrant expression of MYC family members predicts poor clinical outcome in many human cancers. Oncogenic MYC profoundly alters metabolism and mediates an antioxidant response to maintain redox balance. Here we show that MYC induces massive lipid peroxidation upon depletion of cysteine, the rate-limiting amino acid for glutathione biosynthesis and sensitizes cells to ferroptosis, an oxidative, non-apoptotic and irondependent type of cell death. In MYCN-amplified childhood neuroblastoma, MYCN mediates resistance to ferroptosis by activating transsulfuration of methionine to cysteine. MYCN may contribute to spontaneous tumor regression in low-risk neuroblastomas by promoting ferroptosis in cells with epigenetically silenced cystathionine-beta-synthase, the rate-limiting enzyme for transsulfuration. We identified enzymes and antiporter proteins crucial to ferroptotic escape, providing multiple previously unknown sites that may be acted on therapeutically.

Background Hepatocellular carcinoma (HCC) is a major cause of cancer-related death. Paired related homeobox 1 (PRRX1) is a transcription factor that regulates cell growth and differentiation, but its importance in HCC is unclear. Methods We examined the expression pattern of PRRX1 in nine microarray datasets of human HCC tumour samples (n > 1100) and analyzed its function in HCC cell lines. In addition, we performed gene set enrichment, Kaplan-Meier overall survival analysis, metabolomics and functional assays. Results PRRX1 is frequently upregulated in human HCC. Pathway enrichment analysis predicted a direct correlation between PRRX1 and focal adhesion and epithelial-mesenchymal transition. High expression of PRRX1 and low ZEB1 or high ZEB2 significantly predicted better overall survival in HCC patients. In contrast, metabolic processes correlated inversely and transcriptional analyses revealed that glycolysis, TCA cycle and amino acid metabolism were affected. These findings were confirmed by metabolomics analysis. At the phenotypic level, PRRX1 knockdown accelerated proliferation and clonogenicity in HCC cell lines. Conclusions Our results suggest that PRRX1 controls metabolism, has a tumour suppressive role, and may function in cooperation with ZEB1/2. These findings have functional relevance in HCC, including in understanding transcriptional control of distinct cancer hallmarks.

Seeds preserve a far developed plant embryo in a quiescent state. Seed metabolism relies on stored resources and is reactivated to drive germination when the external conditions are favorable. Since the switchover from quiescence to reactivation provides a remarkable case of a cell physiological transition we investigated the earliest events in energy and redox metabolism of Arabidopsis seeds at imbibition. By developing fluorescent protein biosensing in intact seeds, we observed ATP accumulation and oxygen uptake within minutes, indicating rapid activation of mitochondrial respiration, which coincided with a sharp transition from an oxidizing to a more reducing thiol redox environment in the mitochondrial matrix. To identify individual operational protein thiol switches, we captured the fast release of metabolic quiescence in organello and devised quantitative iodoacetyl tandem mass tag (iodoTMT)-based thiol redox proteomics. The redox state across all Cys peptides was shifted toward reduction from 27.1% down to 13.0% oxidized thiol. A large number of Cys peptides (412) were redox switched, representing central pathways of mitochondrial energy metabolism, including the respiratory chain and each enzymatic step of the tricarboxylic acid (TCA) cycle. Active site Cys peptides of glutathione reductase 2, NADPH-thioredoxin reductase a/b, and thioredoxin-o1 showed the strongest responses. Germination of seeds lacking those redox proteins was associated with markedly enhanced respiration and deregulated TCA cycle dynamics suggesting decreased resource efficiency of energy metabolism. Germination in aged seeds was strongly impaired. We identify a global operation of thiol redox switches that is required for optimal usage of energy stores by the mitochondria to drive efficient germination.

Prostate cell metabolism exhibits distinct profiles pre- and post-malignancy. The malignant metabolic shift converts prostate cells from "citrate-producing" to "citrate-oxidizing" cells, thereby enhancing glucose metabolism, a phenotype that contrasts classical tumoral Warburg metabolism. An on-line biosensor chip system (BIONAS 2500) was used to monitor metabolic changes (glycolysis and respiration) in response to the putative anti-cancer nutraceutical 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], in different prostate cancer (PCa) cell lines (LNCaP, VCaP, DU145 and PC3). LNCaP cells exhibited profound metabolic responsiveness to the treatment and thus extensive analysis of metabolism-modulating effects of 1,25(OH)2D3 were performed, including mRNA expression analysis of key metabolic genes (e.g. GLUT1 and PDHK1), analysis of TCA cycle metabolites, glucose uptake/consumption measurements, ATP production, and mitochondrial biogenesis/activity. Altogether, data demonstrate a vivid disruption of glucose metabolism by 1,25(OH)2D3, illustrated by a decreased glucose uptake and an accumulation of citrate/isocitrate due to TCA cycle truncation. Depletion of glycolytic intermediates led to a consistent decrease in TXNIP expression in response to 1,25(OH)2D3, an effect that coincided with the activation of AMPK signaling and a reduction in c-MYC expression. Reduction in TXNIP levels in response to 1,25(OH)2D3 was rescued by an AMPK signaling inhibitor and mimicked by a MYC inhibitor highlighting the possible involvement of both pathways in mediating 1,25(OH)2D3's metabolic effects in PCa cells. Furthermore, pharmacological and genetic modulation of the androgen receptor showed similar and disparate effects on metabolic parameters compared to 1,25(OH)2D3 treatment, highlighting the AR-independent nature of 1,25(OH)2D3's metabolism-modulating effects.

The increased formation of methylglyoxal (MG) under hyperglycemia is associated with the development of microvascular complications in patients with diabetes mellitus; however, the effects of elevated MG levels in vivo are poorly understood. In zebrafish, a transient knockdown of glyoxalase 1, the main MG detoxifying system, led to the elevation of endogenous MG levels and blood vessel alterations. To evaluate effects of a permanent knockout of glyoxalase 1 in vivo, glo1(–/–) zebrafish mutants were generated using CRISPR/Cas9. In addition, a diet-induced–obesity zebrafish model was used to analyze glo1(–/–) zebrafish under high nutrient intake. Glo1(–/–) zebrafish survived until adulthood without growth deficit and showed increased tissue MG concentrations. Impaired glucose tolerance developed in adult glo1(–/–) zebrafish and was indicated by increased postprandial blood glucose levels and postprandial S6 kinase activation. Challenged by an overfeeding period, fasting blood glucose levels in glo1(–/–) zebrafish were increased which translated into retinal blood vessel alterations. Thus, the data have identified a defective MG detoxification as a metabolic prerequisite and glyoxalase 1 alterations as a genetic susceptibility to the development of type 2 diabetes mellitus under high nutrition intake.

Pyrophosphate (PPi), a byproduct of macromolecule biosynthesis is maintained at low levels by soluble inorganic pyrophosphatases (sPPase) found in all eukaryotes. In plants, H+-pumping pyrophosphatases (H+-PPase) convert the substantial energy present in PPi into an electrochemical gradient. We show here, that both cold- and heat stress sensitivity of fugu5 mutants lacking the major H+-PPase isoform AVP1 is caused by reduced SUMOylation. In addition, we show that increased PPi concentrations interfere with SUMOylation in yeast and we provide evidence that SUMO activating E1-enzymes are inhibited by micromolar concentrations of PPi in a non-competitive manner. Taken together, our results do not only provide a mechanistic explanation for the beneficial effects of AVP1 overexpression in plants but they also highlight PPi as an important integrator of metabolism and stress tolerance in eukaryotes.

The gene CNDP1 was associated with the development of diabetic nephropathy. Its enzyme carnosinase 1 (CN1) primarily hydrolyzes the histidine-containing dipeptide carnosine but other organ and metabolic functions are mainly unknown. In our study we generated CNDP1 knockout zebrafish, which showed strongly decreased CN1 activity and increased intracellular carnosine levels. Vasculature and kidneys of CNDP1-/- zebrafish were not affected, except for a transient glomerular alteration. Amino acid profiling showed a decrease of certain amino acids in CNDP1-/- zebrafish, suggesting a specific function for CN1 in the amino acid metabolisms. Indeed, we identified a CN1 activity for Ala-His and Ser-His. Under diabetic conditions increased carnosine levels in CNDP1-/- embryos could not protect from respective organ alterations. Although, weight gain through overfeeding was restrained by CNDP1 loss. Together, zebrafish exhibits CN1 functions, while CNDP1 knockout alters the amino acid metabolism, attenuates weight gain but cannot protect organs from diabetic complications.

The molecular structure of matter defines its properties and function. This is especially true for biological macromolecules such as proteins, which participate in virtually all biochemical processes. A three dimensional structural model of a protein is thus essential for the detailed understanding of its physiological function and the characterization of essential properties such as ligand binding and reaction mechanism. X-ray crystallography is a well-established technique that has been used for many years, but it is still by far the most widely used method for structure determination. A particular strength of this technique is the elucidation of atomic details of molecular interactions, thus providing an invaluable tool for a multitude of scientific projects ranging from the structural classification of macromolecules over the validation of enzymatic mechanisms or the understanding of host–pathogen interactions to structure-guided drug design. In the first part of this review, we describe essential methodological and practical aspects of X-ray crystallography. We provide some pointers that should allow researchers without a background in structural biology to assess the overall quality and reliability of a crystal structure. To highlight its potential, we then survey the impact X-ray crystallography has had on advancing an understanding of a class of enzymes that modify the bacterial cell wall. A substantial number of different bacterial amidase structures have been solved, mostly by X-ray crystallography. Comparison of these structures highlights conserved as well as divergent features. In combination with functional analyses, structural information on these enzymes has therefore proven to be a valuable template not only for understanding their mechanism of catalysis, but also for targeted interference with substrate binding.

The purpose of this article is to describe a technique for secondary reconstruction of traumatic orbital wall defects using titanium implants that act as three-dimensional (3D) puzzle pieces. We present three cases of large defect reconstruction using implants produced by Xilloc Medical B.V. (Maastricht, the Netherlands) with a 3D printer manufactured by LayerWise (3D Systems; Heverlee, Belgium), and designed using the biomedical engineering software programs ProPlan and 3-Matic (Materialise, Heverlee, Belgium). The smaller size of the implants allowed sequential implantation for the reconstruction of extensive two-wall defects via a limited transconjunctival incision. The precise fit of the implants with regard to the surrounding ledges and each other was confirmed by intraoperative 3D imaging (Mobile C-arm Systems B.V. Pulsera, Philips Medical Systems, Eindhoven, the Netherlands). The patients showed near-complete restoration of orbital volume and ocular motility. However, challenges remain, including traumatic fat atrophy and fibrosis.

Subcellular sugar partitioning in plants is strongly regulated in response to developmental cues and changes in external conditions. Besides transitory starch, the vacuolar sugars represent a highly dynamic pool of instantly accessible metabolites that serve as energy source and osmoprotectant. Here, we present the molecular identification and functional characterization of the vacuolar glucose (Glc) exporter Arabidopsis (Arabidopsis thaliana) Early Responsive to Dehydration-Like6 (AtERDL6). We demonstrate tonoplast localization of AtERDL6 in plants. In Arabidopsis, AtERDL6 expression is induced in response to factors that activate vacuolar Glc pools, like darkness, heat stress, and wounding. On the other hand, AtERDL6 transcript levels drop during conditions that trigger Glc accumulation in the vacuole, like cold stress and external sugar supply. Accordingly, sugar analyses revealed that Aterdl6 mutants have elevated vacuolar Glc levels and that Glc flux across the tonoplast is impaired under stress conditions. Interestingly, overexpressor lines indicated a very similar function for the ERDL6 ortholog Integral Membrane Protein from sugar beet (Beta vulgaris). Aterdl6 mutant plants display increased sensitivity against external Glc, and mutant seeds exhibit a 10% increase in seed weight due to enhanced levels of seed sugars, proteins, and lipids. Our findings underline the importance of vacuolar Glc export during the regulation of cellular Glc homeostasis and the composition of seed reserves.

The vacuolar membrane is involved in solute uptake into and release from the vacuole, which is the largest plant organelle. In addition to inorganic ions and metabolites, large quantities of protons and sugars are shuttled across this membrane. Current models suggest that the proton gradient across the membrane drives the accumulation and/or release of sugars. Recent studies have associated AtSUC4 with the vacuolar membrane. Some members of the SUC family are plasma membrane proton/sucrose symporters. In addition, the sugar transporters TMT1 and TMT2, which are localized to the vacuolar membrane, have been suggested to function in proton-driven glucose antiport. Here we used the patch-clamp technique to monitor carrier-mediated sucrose transport by AtSUC4 and AtTMTs in intact Arabidopsis thaliana mesophyll vacuoles. In the whole-vacuole configuration with wild-type material, cytosolic sucrose-induced proton currents were associated with a proton/sucrose antiport mechanism. To identify the related transporter on one hand, and to enable the recording of symporter-mediated currents on the other hand, we electrophysiologically characterized vacuolar proteins recognized by Arabidopsis mutants of partially impaired sugar compartmentation. To our surprise, the intrinsic sucrose/proton antiporter activity was greatly reduced when vacuoles were isolated from plants lacking the monosaccharide transporter AtTMT1/TMT2. Transient expression of AtSUC4 in this mutant background resulted in proton/sucrose symport activity. From these studies, we conclude that, in the natural environment within the Arabidopsis cell, AtSUC4 most likely catalyses proton-coupled sucrose export from the vacuole. However, TMT1/2 probably represents a proton-coupled antiporter capable of high-capacity loading of glucose and sucrose into the vacuole.

To understand carbon partitioning in roots and nodules of Datisca glomerata, activities of sucrose-degrading enzymes and sugar transporter expression patterns were analyzed in both organs, and plasmodesmal connections between nodule cortical cells were examined by transmission electron microscopy. The results indicate that in nodules, the contribution of symplastic transport processes is increased in comparison to roots, specifically in infected cells which develop many secondary plasmodesmata. Invertase activities are dramatically reduced in nodules as compared to roots, indicating that here the main enzyme responsible for the cleavage of sucrose is sucrose synthase. A high-affinity, low-specificity monosaccharide transporter whose expression is induced in infected cells prior to the onset of bacterial nitrogen fixation, and which has an unusually low pH optimum and may be involved in turgor control or hexose retrieval during infection thread growth. Electronic supplementary material The online version of this article (doi:10.1007/s00425-010-1285-8) contains supplementary material, which is available to authorized users.

AtSTP14, a new Arabidopsis sugar transporter, was identified and characterized on the molecular and physiological level. Reverse transcriptase-PCR analyses and reporter plants demonstrate high AtSTP14 expression levels in the seed endosperm and in cotyledons, as well as in green leaves. Thus, unlike previously characterized monosaccharide transporters, AtSTP14 is expressed in both source and sink tissues and represents the first monosaccharide transporter in the female gametophyte. Heterologous expression in yeast revealed that AtSTP14 does not transport glucose or fructose, but is the first plant transporter specific for galactose. Interestingly, AtSTP14 expression is regulated by factors which also induce cell wall degradation such as extended dark periods or changes in the sugar level, i.e. AtSTP14 is induced 3-fold by 24 h darkness and repressed 3-fold by 2% glucose and 2% sucrose. Two independent Atstp14 mutant lines were identified, but no effect on seed development or other differences during growth under normal conditions could be observed. A putative role for AtSTP14 in the recycling of cell wall-derived galactose during different developmental processes is discussed.

The availability of complete plant genomes has greatly influenced the identification and analysis of phylogenetically related gene clusters. In Arabidopsis, this has revealed the existence of a monosaccharide transporter(-like) gene family with 53 members, which play a role in long-distance sugar partitioning or sub-cellular sugar distribution and catalyze the transport of hexoses, but also polyols and in one case also pentoses and tetroses. An update on the currently available information on these Arabidopsis monosaccharide transporters, on their sub-cellular localization and physiological function will be given.

Metal mirrors are planned for optical diagnostic systems of ITER. However, erosion, deposition and particle implantation can change the performance of mirrors. Mirrors made from the single crystal (SC) materials are among the main candidates for use in ITER diagnostic systems operating under erosion-dominated conditions. Laboratory tests have confirmed good optical performance of SC mirrors under erosion, but the dedicated direct comparative test in tokamak environment was missing. Such a direct test was performed in TEXTOR. Single crystal molybdenum, tungsten and polycrystalline (PC) molybdenum mirrors were exposed under the same conditions in the SOL plasma of TEXTOR. Surface and optical properties of mirrors were characterized before and after exposure. Before exposure glow discharge cleaning in hydrogen restored the reflectivity of mirrors oxidized during storage on air. No significant changes in total reflectivity were observed for all mirrors after exposure. Drastic increase of diffuse reflectivity was measured for PC Mo mirror, no change for a SC one. Thus, specular reflectivity of single crystal is higher than of polycrystalline one. The most affected wavelength range is 250–1000 nm, no significant change of reflectivity was noticed in the range 1000–2000 nm. Negligible effect of the exposure on polarization characteristics was observed.

To orchestrate a complex life style in changing environments, the filamentous cyanobacterium Nostoc punctiforme facilitates communication between neighboring cells through septal junction complexes. This is achieved by nanopores that perforate the peptidoglycan (PGN) layer and traverse the cell septa. The N-acetylmuramoyl-l-alanine amidase AmiC2 (Npun_F1846; EC 3.5.1.28) in N. punctiforme generates arrays of such nanopores in the septal PGN, in contrast to homologous amidases that mediate daughter cell separation after cell division in unicellular bacteria. Nanopore formation is therefore a novel property of AmiC homologs. Immunofluorescence shows that native AmiC2 localizes to the maturing septum. The high-resolution crystal structure (1.12 A) of its catalytic domain (AmiC2-cat) differs significantly from known structures of cell splitting and PGN recycling amidases. A wide and shallow binding cavity allows easy access of the substrate to the active site, which harbors an essential zinc ion. AmiC2-cat exhibits strong hydrolytic activity in vitro. A single point mutation of a conserved glutamate near the zinc ion results in total loss of activity, whereas zinc removal leads to instability of AmiC2-cat. An inhibitory α-helix, as found in the Escherichia coli AmiCE. coli structure, is absent. Taken together, our data provide insight into the cell-biological, biochemical and structural properties of an unusual cell wall lytic enzyme that generates nanopores for cell–cell communication in multicellular cyanobacteria. The novel structural features of the catalytic domain and the unique biological function of AmiC2 hint at mechanisms of action and regulation that are distinct from other amidases. Database The AmiC2-cat structure has been deposited in the Protein Data Bank under accession number 5EMI.

The thermal stability of thiol-passivated gold colloids has been studied by means of X-ray photoelectron spectroscopy. Different colloids were chemically synthesized with thiol lengths from 3 to 8, and 16 carbon atoms. Depending on the synthesis parameters, the mean gold particle size varied from 1.6 to 4.9 nm for the particular colloid. Temperature-dependent measurements revealed a general tendency of shells with longer thiol chains (>5 carbon atoms) to be more stable in terms of desorption but to show a X-ray radiation-induced damage, which was absent with shorter thiols. Additionally, the self-assembling properties of the colloids were characterized by TEM, showing differences as a function of the chain lengths.

Pollen development, as well as pollen germination and pollen tube growth, requires a highly regulated supply of sugars. In this paper we describe the molecular, kinetic, and physiological characterization of AtSTP11, a new member of the H+/monosaccharide transporter family in Arabidopsis thaliana (L.) Heynh. Heterologous expression in yeast (Saccharomyces cerevisiae) showed that AtSTP11 is a high-affinity (Km = 25 microM), broad-spectrum, and uncoupler-sensitive monosaccharide transporter of the plasma membrane. In reverse transcription-polymerase chain reaction analyses we found that AtSTP11 expression is restricted to flowers. Furthermore, AtSTP11-promoter::GFP plants revealed that AtSTP11 expression is only found in pollen tubes. Using a specific antibody we could also detect the AtSTP11 protein exclusively in pollen tubes but not in other flower tissues or in pollen grains of any developmental stage. These results suggest that the newly identified AtSTP11 transporter plays a role in the supply of monosaccharides to growing pollen tubes.

Guard cell chloroplasts are unable to perform significant photosynthetic CO2 fixation via Rubisco. Therefore, guard cells depend on carbon supply from adjacent cells even during the light period. Due to their reversible turgor changes, this import cannot be mediated by plasmodesmata. Nevertheless, guard cells of several plants were shown to use extracellular sugars or to accumulate sucrose as an osmoticum that drives water influx to increase stomatal aperture. This paper describes the first localization of a guard cell-specific Arabidopsis sugar transporter involved in carbon acquisition of these symplastically isolated cells. Expression of the AtSTP1 H+-monosacharide symporter gene in guard cells was demonstrated by in situ hybridization and by immunolocalization with an AtSTP1-specific antiserum. Additional RNase protection analyses revealed a strong increase of AtSTP1 expression in the dark and a transient, diurnally regulated increase during the photoperiod around midday. This transient increase in AtSTP1 expression correlates in time with the described guard cell-specific accumulation of sucrose. Our data suggest a function of AtSTP1 in monosaccharide import into guard cells during the night and a possible role in osmoregulation during the day.

A genomic clone and the corresponding cDNA of a new Arabidopsis monosaccharide transporter AtSTP9 were isolated. Transport analysis of the expressed protein in yeast showed that AtSTP9 is an energy-dependent, uncoupler-sensitive, high-affinity monosaccharide transporter with a K m for glucose in the micromolar range. In contrast to all previously characterized monosaccharide transporters, AtSTP9 shows an unusual specificity for glucose. Reverse transcriptase-polymerase chain reaction analyses revealed that AtSTP9 is exclusively expressed in flowers, and a more detailed approach using AtSTP9 promoter/reporter plants clearly showed that AtSTP9 expression is restricted to the male gametophyte. AtSTP9 expression is not found in other floral organs or vegetative tissues. Further localization on the cellular level using a specific antibody revealed that in contrast to the early accumulation of AtSTP9 transcripts in young pollen, the AtSTP9 protein is only found weakly in mature pollen but is most prominent in germinating pollen tubes. This preloading of pollen with mRNAs has been described for genes that are essential for pollen germination and/or pollen tube growth. The pollen-specific expression found for AtSTP9 is also observed for other sugar transporters and indicates that pollen development and germination require a highly regulated supply of sugars.

This paper describes the molecular, kinetic, and physiological characterization of AtSTP6, a new member of the Arabidopsis H+/monosaccharide transporter family. The AtSTP6 gene (At3g05960) is interrupted by two introns and encodes a protein of 507 amino acids containing 12 putative transmembrane helices. Expression in yeast (Saccharomyces cerevisiae) shows that AtSTP6 is a high-affinity (K m = 20 μm), broad-spectrum, and uncoupler-sensitive monosaccharide transporter that is targeted to the plasma membrane and that can complement a growth deficiency resulting from the disruption of most yeast hexose transporter genes. Analyses ofAtSTP6-promoter::GUS plants and in situ hybridization experiments detected AtSTP6expression only during the late stages of pollen development. A transposon-tagged Arabidopsis mutant was isolated and homozygous plants were analyzed for potential effects of the Atstp6mutation on pollen viability, pollen germination, fertilization, and seed production. However, differences between wild-type and mutant plants could not be observed.

This case report illustrates a rare complex of symptoms leading to limited mouth opening in a young woman. The 28-year old suffered from a progressively limited mouth opening over several years that finally resulted in restricted alimentation and made dental treatment impossible. Clinical findings suggest a structural alteration, including a hyperplastic mandibular angle and marked hypertrophy of the masseter muscle. Further radiologic investigations reveal a thickened aponeurosis of the masticatory muscles and hyperplastic coronoid processes that are not interfering with the zygomatic bone. Primary therapeutic options for such conditions are mainly surgical, including reduction of the masseter muscles volume and aponeurorectomy as well as bony reductions, such as coronoidectomy and mandibular angle reduction. With this treatment, the outcome and prognosis are good. Long-term results depend on concomitant physical therapy. A uniform nomenclature for this condition is yet lacking and propositions such as “masticatory muscle tendon-aponeurosis hyperplasia” have been made. However, knowledge of this condition and its typical clinical signs can make the diagnosis and treatment straightforward, thus leading to an improved quality of life of affected patients.

The bifunctional major autolysin AtlA of Staphylococcus aureus cleaves the bacterium's peptidoglycan network (PGN) at two distinct sites during cell division. Deletion of the enzyme results in large cell clusters with disordered division patterns, indicating that AtlA could be a promising target for the development of new antibiotics. One of the two functions of AtlA is performed by the N-acetylmuramyl-l-alanine amidase AmiA, which cleaves the bond between the carbohydrate and the peptide moieties of PGN. To establish the structural requirements of PGN recognition and the enzymatic mechanism of cleavage, we solved the crystal structure of the catalytic domain of AmiA (AmiA-cat) in complex with a peptidoglycan-derived ligand at 1.55 A resolution. The peptide stem is clearly visible in the structure, forming extensive contacts with protein residues by docking into an elongated groove. Less well defined electron density and the analysis of surface features indicate likely positions of the carbohydrate backbone and the pentaglycine bridge. Substrate specificity analysis supports the importance of the pentaglycine bridge for fitting into the binding cleft of AmiA-cat. PGN of S. aureus with l-lysine tethered with d-alanine via a pentaglycine bridge is completely hydrolyzed, whereas PGN of Bacillus subtilis with meso-diaminopimelic acid directly tethered with d-alanine is not hydrolyzed. An active site mutant, H370A, of AmiA-cat was completely inactive, providing further support for the proposed catalytic mechanism of AmiA. The structure reported here is not only the first of any bacterial amidase in which both the PGN component and the water molecule that carries out the nucleophilic attack on the carbonyl carbon of the scissile bond are present; it is also the first peptidoglycan amidase complex structure of an important human pathogen.

Monosaccharide transport across the plant plasma membrane plays an important role both in lower and higher plants. Algae can switch between phototrophic and heterotrophic growth and utilize organic compounds, such as monosaccharides as additional or sole carbon sources. Higher plants represent complex mosaics of phototrophic and heterotrophic cells and tissues and depend on the activity of numerous transporters for the correct partitioning of assimilated carbon between their different organs. The cloning of monosaccharide transporter genes and cDNAs identified closely related integral membrane proteins with 12 transmembrane helices exhibiting significant homology to monosaccharide transporters from yeast, bacteria and mammals. Structural analyses performed with several members of this transporter superfamily identified protein domains or even specific amino acid residues putatively involved in substrate binding and specificity. Expression of plant monosaccharide transporter cDNAs in yeast cells and frog oocytes allowed the characterization of substrate specificities and kinetic parameters. Immunohistochemical studies, in situ hybridization analyses and studies performed with transgenic plants expressing reporter genes under the control of promoters from specific monosaccharide transporter genes allowed the localization of the transport proteins or revealed the sites of gene expression. Higher plants possess large families of monosaccharide transporter genes and each of the encoded proteins seems to have a specific function often confined to a limited number of cells and regulated both developmentally and by environmental stimuli.

Arabidopsis vacuoles harbor, besides sugar transporter of the TMT-type, an early response to dehydration like 6 (ERDL6) protein involved in glucose export into the cytosol. However, the mode of transport of ERDL6 and the plant's feedback to overexpression of its activity on essential properties such as, for example, seed germination or freezing tolerance, remain unexplored. Using patch-clamp studies on vacuoles expressing AtERDL6 we demonstrated directly that this carrier operates as a proton-driven glucose exporter. Overexpression of BvIMP, the closest sugar beet (Beta vulgaris) homolog to AtERDL6, in Arabidopsis leads surprisingly to impaired seed germination under both conditions, sugar application and low environmental temperatures, but not under standard conditions. Upon cold treatment, BvIMP overexpressor plants accumulated lower quantities of monosaccharides than the wild-type, a response in line with the reduced frost tolerance of the transgenic Arabidopsis plants, and the fact that cold temperatures inhibits BvIMP transcription in sugar beet leaves. With these findings we show that the tight control of vacuolar sugar import and export is a key requisite for cold tolerance and seed germination of plants.

Octopine synthase (ocs) elements are a group of promoter elements that have been exploited by plant pathogens to express genes in plants. ocs elements are components of the promoters of certain plant glutathione S-transferase genes and may function as oxidative stress response elements. Genes for ocs element binding factors (OBFs), which belong to a specific class of highly conserved, plant basic domain-leucine zipper transcription factors, have been isolated and include the Arabidopsis OBF4 and OBF5 genes. To characterize proteins that modulate the activity of the OBF proteins, we screened an Arabidopsis cDNA library with the labeled OBF4 protein and isolated OBP1 (for OBF binding protein). OBP1 contains a 51-amino acid domain that is highly conserved with two plant DNA binding proteins, which we refer to as the MOA domain. OBP1 is also a DNA binding protein and binds to the cauliflower mosaic virus 35S promoter at a site distinct from the ocs element in the 35S promoter. OBP1 specifically increased the binding of the OBF proteins to ocs element sequences, raising the possibility that interactions between these proteins are important for the activity of the 35S promoter.

The vacuolar membrane is involved in solute uptake into and release from the vacuole, which is the largest plant organelle. In addition to inorganic ions and metabolites, large quantities of protons and sugars are shuttled across this membrane. Current models suggest that the proton gradient across the membrane drives the accumulation and/or release of sugars. Recent studies have associated AtSUC4 with the vacuolar membrane. Some members of the SUC family are plasma membrane proton/sucrose symporters. In addition, the sugar transporters TMT1 and TMT2, which are localized to the vacuolar membrane, have been suggested to function in proton-driven glucose antiport. Here we used the patch-clamp technique to monitor carrier-mediated sucrose transport by AtSUC4 and AtTMTs in intact Arabidopsis thaliana mesophyll vacuoles. In the whole-vacuole configuration with wild-type material, cytosolic sucrose-induced proton currents were associated with a proton/sucrose antiport mechanism. To identify the related transporter on one hand, and to enable the recording of symporter-mediated currents on the other hand, we electrophysiologically characterized vacuolar proteins recognized by Arabidopsis mutants of partially impaired sugar compartmentation. To our surprise, the intrinsic sucrose/proton antiporter activity was greatly reduced when vacuoles were isolated from plants lacking the monosaccharide transporter AtTMT1/TMT2. Transient expression of AtSUC4 in this mutant background resulted in proton/sucrose symport activity. From these studies, we conclude that, in the natural environment within the Arabidopsis cell, AtSUC4 most likely catalyses proton-coupled sucrose export from the vacuole. However, TMT1/2 probably represents a proton-coupled antiporter capable of high-capacity loading of glucose and sucrose into the vacuole.

A novel IR method for measuring the kinetics of N(2)O photodecomposition has been devised and used to calibrate the flux of Lyman-alpha (10.2 eV) radiation from a H(2)/Ar microwave discharge lamp. The photodecomposition of N(2)O occurs with a weak pressure dependence due to the operation of a wall effect consuming some photogenerated active oxygen species. This effect is removed by working at high N(2)O pressures. The Lyman-alpha flux from the lamp is 1.28 +/- 0.36 x 10(15) photons cm(-2) s(-1).

Sugar compartmentation into vacuoles of higher plants is a very important physiological process, providing extra space for transient and long-term sugar storage and contributing to the osmoregulation of cell turgor and shape. Despite the long-standing knowledge of this subcellular sugar partitioning, the proteins responsible for these transport steps have remained unknown. We have identified a gene family in Arabidopsis consisting of three members homologous to known sugar transporters. One member of this family, Arabidopsis thaliana vacuolar glucose transporter 1 ( AtVGT1 ), was localized to the vacuolar membrane. Moreover, we provide evidence for transport activity of a tonoplast sugar transporter based on its functional expression in bakers' yeast and uptake studies in isolated yeast vacuoles. Analyses of Atvgt1 mutant lines indicate an important function of this vacuolar glucose transporter during developmental processes like seed germination and flowering.

The accumulation of sugars in grape berries requires the co-ordinate expression of sucrose transporters, invertases, and monosaccharide transporters. A monosaccharide transporter homologue (VvHT1, Vitis vinifera hexose transporter 1) has previously been isolated from grape berries at the veraison stage, and its expression was shown to be regulated by sugars and abscisic acid. The present work investigates the function and localization of VvHT1. Heterologous expression in yeast indicates that VvHT1 encodes a monosaccharide transporter with maximal activity at acidic pH (pH 4.5) and high affinity for glucose (K(m)=70 muM). Fructose, mannose, sorbitol, and mannitol are not transported by VvHT1. In situ hybridization shows that VvHT1 transcripts are primarily found in the phloem region of the conducting bundles. Immunofluorescence and immunogold labelling experiments localized VvHT1 in the plasma membrane of the sieve element/companion cell interface and of the flesh cells. The expression and functional properties of VvHT1 suggests that it retrieves the monosaccharides needed to provide the energy necessary for cell division and cell growth at an early stage of berry development.