Adsorption study of acetone on acid-doped ice surfaces between 203 and 233 K.

Adsorption studies of acetone on pure ice surfaces obtained by water freezing or deposition or on frozen ice surfaces doped either with HNO3 or H2SO4 have been performed using a coated wall flow tube coupled to a mass spectrometric detection. The experiments were conducted over the temperature range 203-233 K and freezing solutions containing either H2SO4 (0.2 N) or HNO3 (0.2-3 N). Adsorption of acetone on these ice surfaces was always found to be totally reversible whatever were the experimental conditions. The number of acetone molecules adsorbed per ice surface unit N was conventionally plotted as a function of acetone concentration in the gas phase. For the same conditions, the amount of acetone molecules adsorbed on pure ice obtained by deposition are about 3-4 times higher than those measured on frozen ice films, H2SO4-doped ice surfaces lead to results comparable to those obtained on pure ice. On the contrary, N increases largely with increasing concentrations of nitric acid in ice surfaces, up to about 300 times under our experimental conditions and for temperatures ranging between 213 and 233 K. Finally, the results are discussed and used to reestimate the partitioning of acetone between the ice and gas phases in clouds of the upper troposphere.

Medicago truncatula ENOD11: a novel RPRP-encoding early nodulin gene expressed during mycorrhization in arbuscule-containing cells.

Leguminous plants establish endosymbiotic associations with both rhizobia (nitrogen fixation) and arbuscular mycorrhizal fungi (phosphate uptake). These associations involve controlled entry of the soil microsymbiont into the root and the coordinated differentiation of the respective partners to generate the appropriate exchange interfaces. As part of a study to evaluate analogies at the molecular level between these two plant-microbe interactions, we focused on genes from Medicago truncatula encoding putative cell wall repetitive proline-rich proteins (RPRPs) expressed during the early stages of root nodulation. Here we report that a novel RPRP-encoding gene, MtENOD11, is transcribed during preinfection and infection stages of nodulation in root and nodule tissues. By means of reverse transcription-polymerase chain reaction and a promoter-reporter gene strategy, we demonstrate that this gene is also expressed during root colonization by endomycorrhizal fungi in inner cortical cells containing recently formed arbuscules. In contrast, no activation of MtENOD11 is observed during root colonization by a nonsymbiotic, biotrophic Rhizoctonia fungal species. Analysis of transgenic Medicago spp. plants expressing pMtENOD11-gusA also revealed that this gene is transcribed in a variety of nonsymbiotic specialized cell types in the root, shoot, and developing seed, either sharing high secretion/metabolite exchange activity or subject to regulated modifications in cell shape. The potential role of early nodulins with atypical RPRP structures such as ENOD11 and ENOD12 in symbiotic and nonsymbiotic cellular contexts is discussed.

A ray-traced texture mapping for enhanced virtuality in image-guided neurosurgery.

3D imaging systems and algorithms give virtual representations of the real world. New emergent hardware systems can combine virtual information and the real world. Virtual and real information must be also visually confronted in order to facilitate our comprehension of the data. We propose a solution which entails the superimposition of a real image of the anatomical areas visualised in a surgical operation with 3D digital data sets. Unlike other solutions which display virtual images in the real world, our method involves ray traced texture mapping which displays real images in a computed world.

Mechanisms of citrate oxidation by percoll-purified mitochondria from potato tuber.

The mechanisms and accurate control of citrate oxidation by Percoll-purified potato (Solanum tuberosum) tuber mitochondria were characterized in various metabolic conditions by recording time course evolution of the citric acid cycle related intermediates and O(2) consumption. Intact potato tuber mitochondria showed good rates of citrate oxidation, provided that nonlimiting amounts of NAD(+) and thiamine pyrophosphate were present in the matrix space. Addition of ATP increased initial oxidation rates, by activation of the energy-dependent net citrate uptake, and stimulated succinate and malate formation. When the intramitochondrial NADH to NAD(+) ratio was high, alpha-ketoglutarate only was excreted from the matrix space. After addition of ADP, aspartate, or oxaloacetate, which decreased the NADH to NAD(+) ratio, flux rates through the Krebs cycle dehydrogenases were strongly increased and alpha-ketoglutarate, succinate, and malate accumulated up to steady-state concentrations in the reaction medium. It was concluded that NADH to NAD(+) ratio could be the primary signal for coordination of fluxes through electron transport chain or malate dehydrogenase and NAD(+)-linked Krebs cycle dehydrogenases. In addition, these results clearly showed that the tricarboxylic acid cycle could serve as an important source of carbon skeletons for extra-mitochondrial synthetic processes, according to supply and demand of metabolites.

Enzymic capacities of purified cauliflower bud plastids for lipid synthesis and carbohydrate metabolism.

Isolated cauliflower (Brassica oleracea) bud plastids, purified by isopycnic centrifugation in density gradients of Percoll, were found to be highly intact, to be practically devoid of extraplastidial contaminations, and to retain all the enzymes involved in fatty acid, phosphatidic acid, and monogalactosyldiacylglycerol synthesis. Purified plastids possess all the enzymes needed to convert triose phosphate to starch and vice versa, and are capable of conversion of glycerate 3-phosphate to pyruvate for fatty acid synthesis. They are also capable of oxidation of hexose phosphate and conversion to triose phosphate via the oxidative pentosephosphate pathway. Cauliflower bud plastids prove to be, therefore, biochemically very flexible organelles.

Biochemical changes during sucrose deprivation in higher plant cells.

The mobilization of stored carbohydrates (sucrose and starch) during sucrose starvation was studied with sycamore (Acer pseudoplatanus) cells. When sucrose was omitted from the nutrient medium, vacuolar sucrose was first consumed. When a threshold of intracellular sucrose concentration was attained the cytoplasmic phosphorylated compounds decreased whereas cytoplasmic Pi increased symmetrically. Such a situation triggered starch breakdown. When almost all the intracellular sucrose pool had disappeared, the cell respiration rates (normal and uncoupled) declined progressively. The decrease in the rate of respiration triggered by sucrose starvation was attributable neither to the availability of substrate for mitochondrial respiration nor to a decrease in the maximal rate of O2 consumption by mitochondria expressed in terms of nanomole of O2 consumed per min/mg of mitochondrial protein. In fact, the uncoupled respiration rates decreased in parallel with the decrease in total intracellular cardiolipin or cytochrome aa3. These results demonstrate therefore that after a long period of sucrose starvation the progressive decrease in the uncoupled rate of O2 consumption by sycamore cells was attributable to a progressive diminution of the number of mitochondria/cell.

Functional heterogeneity of polyphosphoinositides in human erythrocytes.

After labelling of erythrocytes with [32P]P1 for 23 h, the specific radioactivities of the phosphomonoester groups of PtdIns4P and of PtdIns(4,5)P2 approached equilibrium values which were close to that of the gamma-phosphate of ATP (78-85%), showing that almost all of these phosphate groups were metabolically active. Phosphoinositidase C (PIC) activation, using Ca2+ and the ionophore A23187, of 32P-prelabelled erythrocytes was used to investigate a possible functional heterogeneity of the phosphoinositides. Hydrolysis of PtdIns(4,5)P2, measured from its radioactivity, decreased as function of the time of prelabelling up to a constant value equal to that measured from its content. In contrast, hydrolysis of PtdIns4P, determined both from radioactivity and from content, was always the same. These data suggest that newly labelled molecules of PtdIns(4,5)P2, initially accessible to PIC, then moved towards a PIC-resistant pool. This was further confirmed by measuring the fraction of labelled PtdIns(4,5)P2 molecules accessible to PIC after a prelabelling period of 5 min and different times of reincubation. Hydrolysis by PIC was also measured in erythrocytes in which the phosphoinositide content had been modified by activation (Mg2+-enriched cells) or inhibition (ATP-depleted cells) of the phosphoinositide kinases. The sizes of the PIC-resistant pools of polyphosphoinositides were not affected by these treatments, indicating that the kinases (and the phosphatases) act on the PIC-sensitive pools. This was also shown by the decrease in the production of Ins(1,4,5)P3 upon PIC activation in ATP-depleted erythrocytes. A model is presented in which the PIC-sensitive pools of polyphosphoinositides are those which are accessible to the kinases and the phosphatases and are rapidly turned over.

Role of Glutamate-oxaloacetate Transaminase and Malate Dehydrogenase in the Regeneration of NAD for Glycine Oxidation by Spinach leaf Mitochondria.

During glycine oxidation by spinach leaf mitochondria, oxygen consumption showed a strong and transient inhibition upon addition of oxaloacetate or aspartate plus alpha-ketoglutarate. During the course of the inhibition, aspartate and alpha-ketoglutarate were stoichiometrically transformed into malate and glutamate.It is concluded that oxaloacetate formed by transamination is reduced by the malate dehydrogenase, which allows the regeneration of NAD(+) for glycine oxidation and, thus, by-passes the respiratory chain. Efficiency of a malate-glutamate/aspartate-alpha-ketoglutarate shuttle upon illumination and under in vivo conditions is discussed.

Is the availability of substrate for the tricarboxylic acid cycle a limiting factor for uncoupled respiration in sycamore (Acer pseudoplatanus) cells?

Protoplasts obtained from sycamore (Acer pseudoplatanus) cell suspensions were found to be highly intact and to retain a high rate of O2 consumption. If the protoplasts were taken up and expelled through a fine nylon mesh, all the protoplasts were ruptured, leaving the fragile amyloplasts largely intact. Distribution of enzymes of glycolysis in plastids and soluble phase of sycamore protoplasts indicated that the absolute maximum activity for each glycolytic enzyme under optimum conditions exceeded the estimates of the maximal rate at which sycamore cells oxidize triose phosphate. Passage of protoplasts through the fine nylon mesh produced a 3-5-fold decrease in O2 consumption. However, addition of saturating amounts of respiratory substrates and ADP restored an O2 consumption equal to that observed with uncoupled intact protoplasts. Taken together, these results demonstrated that neither the overall capacity of the glycolytic enzymes in sycamore cells nor the availability of respiratory substrates for the mitochondria is ultimately responsible for determining the rate of uncoupled respiration in sycamore cells.

Synchronous expression of leghaemoglobin genes in Medicago truncatula during nitrogen-fixing root nodule development and response to exogenously supplied nitrate.

Two leghaemoglobin genes from the diploid, autogamous Medicago truncatula (Mtlb1 and Mtlb2) have been cloned and their nucleotide sequences determined. The deduced amino acid sequences encoded by these two genes differ significantly (18%), confirming that they belong to different sub-groups of Medicago leghaemoglobin genes. RNAse protection experiments have been used to show that both genes are transcriptionally active, and are expressed specifically in the nitrogen-fixing root nodule of M. truncatula. Whilst Mtlb1 mRNA is present at approximatively 3-fold higher steady-state levels than Mtlb2 mRNA, the transcription of both genes is triggered concomitantly during nodule development (5 days after inoculation with Rhizobium meliloti), and the ratio of the steady-state levels of the two mRNA species remains constant throughout nodule maturation. When the growth medium of nodulated M. truncatula is supplemented with 5 mM KNO3 over a period of 2-3 days there is a progressive drop in specific nitrogen fixation activity to only 20-25% of the original level. This is accompanied with a parallel and synchronous reduction in the quantities of mRNA corresponding to both Mtlb1 and Mtlb2. By contrast, the expression of the nodule parenchyma-specific gene ENOD2 is not significantly modified following nitrate treatment, clearly demonstrating differences in tissue-specific gene regulation in response to combined nitrogen.

Effect of NAD on Malate Oxidation in Intact Plant Mitochondria.

Potato tuber mitochondria oxidizing malate respond to NAD(+) addition with increased oxidation rates, whereas mung bean hypocotyl mitochondria do not. This is traced to a low endogenous content of NAD(+) in potato mitochondria, which prove to take up added NAD(+). This mechanism concentrates NAD(+) in the matrix space. Analyses for oxaloacetate and pyruvate (with pyruvate dehydrogenase blocked) are consistent with regulation of malate oxidation by the internal NAD(+)/NADH ratio.

The separation of [32P]inositol phosphates by ion-pair chromatography: optimization of the method and biological applications.

We have developed an ion-pair reverse-phase HPLC method to measure inositol phosphates in 32P-labeled cells. The different chromatographic parameters were analyzed to optimize the resolution of the 32P-labeled metabolites. Analysis of inositol phosphates in biological samples was improved by a single charcoal pretreatment which eliminated interfering nucleotides without removing inositol phosphates. The kinetics of production of inositol phosphates in calcium-activated erythrocytes, vasopressin-stimulated hepatocytes, and thrombin-activated platelets were analyzed. Original data on the activation of phosphoinositide phospholipase C were obtained in intact erythrocytes by direct measurement of inositol (1,4,5)P3. Data from agonist-stimulated hepatocytes and platelets were consistent with those from previous studies. In conclusion, this technique offers many advantages over the methodologies currently employed involving anion-exchange chromatography and [3H]inositol labeling: (i) 32P labeling is less expensive and more efficient than 3H labeling and can be used with all types of cells without permeabilization treatments and (ii) ion-pair HPLC gives good resolution of inositol phosphates from nucleotides with shorter retention times, and long reequilibration periods are not required.

Rhizobium meliloti elicits transient expression of the early nodulin gene ENOD12 in the differentiating root epidermis of transgenic alfalfa.

To study the molecular responses of the host legume during early stages of the symbiotic interaction with Rhizobium, we have cloned and characterized the infection-related early nodulin gene MtENOD12 from Medicago truncatula. In situ hybridization experiments have shown that, within the indeterminate Medicago nodule, transcription of the MtENOD12 gene begins in cell layers of meristematic origin that lie ahead of the infection zone, suggesting that these cells are undergoing preparation for bacterial infection. Histochemical analysis of transgenic alfalfa plants that express an MtENOD12 promoter-beta-glucuronidase gene fusion has confirmed this result and further revealed that MtENOD12 gene transcription occurs as early as 3 to 6 hr following inoculation with R. meliloti in a zone of differentiating root epidermal cells which lies close to the growing root tip. It is likely that this transient, nodulation (nod) gene-dependent activation of the ENOD12 gene also corresponds to the preparation of the plant for bacterial infection. We anticipate that this extremely precocious response to Rhizobium will provide a valuable molecular marker for studying early signal exchange between the two symbiotic organisms.

Rhizobium meliloti Nod factors elicit cell-specific transcription of the ENOD12 gene in transgenic alfalfa.

Extracellular lipo-oligosaccharides of Rhizobium, known as Nod factors, play a key role in the molecular signal exchange which leads to the specific nitrogen-fixing symbiotic association between the soil microbe and its host legume. The biological activity of Nod factors and their perception by the host plant during the earliest stages of the Rhizobium/legume interaction have been studied using transgenic alfalfa carrying a fusion between the promoter of the early nodulin gene MtENOD12 and the beta-glucuronidase (GUS) reporter gene. Histochemical staining has shown that GUS accumulates specifically in the differentiating root epidermis, prior to and during root hair emergence, within 2-3 h following the addition of purified Rhizobium meliloti Nod factors. This precocious transcriptional activation of the MtENOD12 gene, reminiscent of that observed after inoculation with intact Rhizobium, implies that the Nod factor signal can be perceived at a developmental stage preceding root hair formation. GUS activity can be detected following treatment with a wide range of R. meliloti Nod factor concentrations down to 10(-13) M, and furthermore, this rapid response to the bacterial elicitor appears to be non-systemic. Significantly, MtENOD12-GUS expression is not observed after inoculation with a R. meliloti nodH mutant which synthesizes exclusively non-sulphated Nod factors. Indeed purified Nod factors which lack the sulphate substituent are approximately 1000-fold less active than their sulphated counterparts. Thus, the triggering of ENOD12 transcription in the alfalfa root epidermis is a rapid molecular response which is subject to the same host-specificity determinant (Nod factor sulphation) that governs the interaction between alfalfa and its bacterial symbiont.

Four genes of Medicago truncatula controlling components of a nod factor transduction pathway.

Rhizobium nodulation (Nod) factors are lipo-chitooligosaccharides that act as symbiotic signals, eliciting several key developmental responses in the roots of legume hosts. Using nodulation-defective mutants of Medicago truncatula, we have started to dissect the genetic control of Nod factor transduction. Mutants in four genes (DMI1, DMI2, DMI3, and NSP) were pleiotropically affected in Nod factor responses, indicating that these genes are required for a Nod factor-activated signal transduction pathway that leads to symbiotic responses such as root hair deformations, expressions of nodulin genes, and cortical cell divisions. Mutant analysis also provides evidence that Nod factors have a dual effect on the growth of root hair: inhibition of endogenous (plant) tip growth, and elicitation of a novel tip growth dependent on (bacterial) Nod factors. dmi1, dmi2, and dmi3 mutants are also unable to establish a symbiotic association with endomycorrhizal fungi, indicating that there are at least three common steps to nodulation and endomycorrhization in M. truncatula and providing further evidence for a common signaling pathway between nodulation and mycorrhization.