Are sucrosyl-oligosaccharides synthesized in mesophyll protoplasts of mature leaves of Cucumis melo?

Biosynthesis of sucrosyl-oligosaccharides (raffinose, stachyose) was traced in source leaves of Cucumis melo after ¹⁴C-photoassimilation. The main carbon compound exported was ¹⁴C-labeled stachyose. No oligosaccharide synthesis was detected in young, importing leaves. Mesophyll protoplasts, isolated from mature leaves which had previously photosynthesized ¹⁴CO₂, did not contain ¹⁴C-oligosaccharides but contained [¹⁴C]-sucrose and ¹⁴C-hexoses. Isolated minor-vein-enriched fractions from the same leaves, however, showed nearly 30% of the ¹⁴C of the neutral fraction to be in oligosaccharides. Isolated, viable mesophyll protoplasts incubated with NaH¹⁴CO₃ also failed to incorporate radioactivity into oligosaccharides, although sucrose and galactinol synthesis was unimpaired. Galactinolsynthase activity in leaf extracts and in mesophyll protoplasts was 16.8 mol·h^-1·mg^-1 protein and 13.8 mol·h^-1·mg^-1 protein, respectively. Galactosyltransferase (EC 2.4.1.67), which synthesizes stachyose from raffinose and galactinol, had an activity of 50 nmol·h^-1·mg^-1 protein in leaf extracts and was also present in the minor-vein-enriched fraction, but could not be detected in mesophyll protoplast lysates. The results indicate that mesophyll cells may not be the site of stachyose synthesis although precursor compounds like sucrose and galactinol are synthesized there.Abbreviation HPLC high-performance liquid chromatography     

Plasmalemmal voltage-activated K + currents in protoplasts from tobacco BY-2 cells: possible regulation by actin microfilaments?

Summary.  Plasmalemmal ionic currents from enzymatically isolated protoplasts of suspension-cultured tobacco ‘Bright Yellow-2’ cells were investigated by whole-cell patch-clamp techniques. In all protoplasts, delayed rectifier outward K⁺ currents having sigmoidal activation kinetics, no inactivation, and very slow deactivation kinetics were activated by step depolarization. Tail current reversal potentials were close to equilibrium potential E_K when external [K⁺] was either 6 or 60 mM. Several channel blockers, including external Ba²⁺, niflumic acid, and 5-nitro-2-(3-phenylpropylamino)-benzoic acid, inhibited this outward K⁺ current. Among the monovalent cations tested (NH₄ ⁺, Rb⁺, Li⁺, Na⁺), only Rb⁺ had appreciable permeation (P_Rb/P_K ⁼ 0.7). In addition, in 60 mM K⁺ solutions, a hyperpolarization-activated, time-dependent, inwardly rectifying K⁺ current was observed in most protoplasts. This inward current activated very slowly, did not inactivate, and deactivated quickly upon repolarization. The tail current reversal potential was very close to E_K, and other monovalent cations (NH₄ ⁺, Rb⁺, Li⁺, Na⁺) were not permeant. The inward current was blocked by external Ba²⁺ and niflumic acid. External Cs⁺ reversibly blocked the inward current without affecting the outward current. The amplitude of the inward rectifier K⁺ current was generally small compared to the amplitude of the outward K⁺ current in the same cell, although this was highly variable. Similar amplitudes for both currents occurred in only 4% of the protoplasts in control conditions. Microfilament-depolymerizing drugs shifted this proportion to about 12%, suggesting that microfilaments participate in the regulation of K⁺ currents in tobacco ‘Bright Yellow-2’ cells. Received December 7, 2001; accepted April 15, 2002; published online July 4, 2002 RID="*" ID="*" Correspondence and reprints: Pharmacologie et Physicochimie, UMR CNRS 7034, Faculté de Pharmacie, Université Louis Pasteur, 74 route du Rhin, BP 24, 67401 Illkirch, France. Abbreviations: TBY-2 Tobacco ‘Bright Yellow-2’; DHCB dihydrocytochalasin B; I_Kin inward rectifier K⁺ current; I_Kout outward K⁺ current; MFs microfilaments; MTs microtubules; NPPB 5-nitro-2-(3-phenylpropylamino)-benzoic acid.     

Plasmalemmal voltage-activated K(+) currents in protoplasts from tobacco BY-2 cells: possible regulation by actin microfilaments?

Plasmalemmal ionic currents from enzymatically isolated protoplasts of suspension-cultured tobacco 'Bright Yellow-2' cells were investigated by whole-cell patch-clamp techniques. In all protoplasts, delayed rectifier outward K(+) currents having sigmoidal activation kinetics, no inactivation, and very slow deactivation kinetics were activated by step depolarization. Tail current reversal potentials were close to equilibrium potential E(K) when external [K(+)] was either 6 or 60 mM. Several channel blockers, including external Ba(2+), niflumic acid, and 5-nitro-2-(3-phenylpropylamino)-benzoic acid, inhibited this outward K(+) current. Among the monovalent cations tested (NH(4)(+), Rb(+), Li(+), Na(+)), only Rb(+) had appreciable permeation (P(Rb)/P(K) (=) 0.7). In addition, in 60 mM K(+) solutions, a hyperpolarization-activated, time-dependent, inwardly rectifying K(+) current was observed in most protoplasts. This inward current activated very slowly, did not inactivate, and deactivated quickly upon repolarization. The tail current reversal potential was very close to E(K), and other monovalent cations (NH(4)(+), Rb(+), Li(+), Na(+)) were not permeant. The inward current was blocked by external Ba(2+) and niflumic acid. External Cs(+) reversibly blocked the inward current without affecting the outward current. The amplitude of the inward rectifier K(+) current was generally small compared to the amplitude of the outward K(+) current in the same cell, although this was highly variable. Similar amplitudes for both currents occurred in only 4% of the protoplasts in control conditions. Microfilament-depolymerizing drugs shifted this proportion to about 12%, suggesting that microfilaments participate in the regulation of K(+) currents in tobacco 'Bright Yellow-2' cells.     

Perifosine sensitizes UVB-induced apoptosis in skin cells: New implication of skin cancer prevention?

We demonstrate here that a relative low dose of perifosine significantly enhanced UVB-induced apoptosis in skin cells (keratinocytes and fibroblasts), associated with a significant increase of reactive oxygen species (ROS) and ceramide production as well as multiple perturbations of diverse cell signaling pathways, shifting to a significant pro-apoptosis outcomes. Perifosine inhibited UVB-induced pro-survival Akt/mammalian target of rapamycin (mTOR) and ERK activation, while facilitating pro-apoptotic AMP-activated protein kinas (AMPK), c-Jun-NH(2)-kinase (JNK), and p53 activation; these signaling changes together promoted a striking increase in skin cell apoptosis and a significantly reduced amount of DNA damages. Our results suggest that perifosine may represent a novel skin cancer prevention strategy.     

Physiological characterisation of magnesium deficiency in sugar beet: acclimation to low magnesium differentially affects photosystems I and II

Magnesium deficiency in plants is a widespread problem, affecting productivity and quality in agriculture, yet at a physiological level it has been poorly studied in crop plants. Here, a physiological characterization of Mg deficiency in Beta vulgaris L., an important crop model, is presented. The impact of Mg deficiency on plant growth, mineral profile and photosynthetic activity was studied. The aerial biomass of plants decreased after 24 days of hydroponic culture in Mg-free nutrient solution, whereas the root biomass was unaffected. Analysis of mineral profiles revealed that Mg decreased more rapidly in roots than in shoots and that shoot Mg content could fall to 3 mg g^–1 DW without chlorosis development and with no effect on photosynthetic parameters. Sucrose accumulated in most recently expanded leaves before any loss in photosynthetic activity. During the development of Mg deficiency, the two photosystems showed sharply contrasting responses. Data were consistent with a down-regulation of PSII through a loss of antenna, and of PSI primarily through a loss of reaction centres. In each case, the net result was a decrease in the overall rate of linear electron transport, preventing an excess of reductant being produced during conditions under which sucrose export away from mature leaf was restricted.     

Do mitochondria in Dendrobium petal mesophyll cells form vacuole-like vesicles?

Using transmission electron microscopy, we investigated the ultrastructure of mitochondria in petal mesophyll cells of the orchid Dendrobium cv. Lucky Duan, from the time of floral opening to visible petal senescence. Cells close to the vascular bundle contained many mitochondria, some of which showed internal degeneration. This inner mitochondrial breakdown was accompanied by an eightfold increase in mitochondrial volume. Small electron-dense granules (approximately 0.04 μm in diameter) at the periphery of the mitochondrial matrix remained. These granules were used as an indicator of still later stages of mitochondrial development in these cells. The apparent final stage of mitochondrial degeneration was a single-membrane-bound vesicle, resembling a vacuole. No evidence was found for the idea that mitochondria became transferred (intact or degenerated) into a lytic vacuole. Taken together, the data suggest the hypotheses that (a) mitochondria in cells close to the vascular bundle in petals of open Dendrobium cv. Lucky Duan flowers undergo large-scale internal degeneration and that (b) such degenerating mitochondria form vacuole-like vesicles.     

Macrophage fusion: are somatic and cancer cells possible partners?

Macrophages are present in all tissues and can fuse with themselves to differentiate into multinucleate osteoclasts or giant cells that play a central role in osteoporosis and chronic inflammatory diseases, respectively. Yet, the mechanism by which they fuse remains uncharacterized. The macrophage fusion receptor (MFR) and its ligand CD47 might mediate homotypic fusion of macrophages and allow for their recognition as 'self' before fusion. Although a novel process and controversial idea, macrophages might exploit a similar mechanism for fusion with somatic cells or tumor cells, with resultant organ repair or metastasis, respectively. Hence, macrophages might be the 'double-edged swords' of tissues.     

Macrophage fusion: are somatic and cancer cells possible partners?

The fusion of cells is a fundamental biological event that plays a central role in a variety of developmental and homeostatic processes. Macrophages are present in all tissues and can initiate interaction and fusion. The putative macrophage-fusion machinery is still poorly understood, but some of its components have been identified. Macrophages recognize each other as < self > in order to fuse but some essential questions remain: do macrophages fuse with somatic cells to repair tissues and organs? Do macrophages fuse with tumor cells to trigger metastasis? Agnès Vignery discusses these novel and challenging ideas in this review.     

The presence and possible significance of cross-reactive antigens inRhizobium — legume associations

Soluble antigens of threeRhizobium species were compared by the agar-gel double-diffusion technique with those of eight legumes representing compatible and non-compatible hosts. Cross-reactive antigens were found between all the legume hosts and the three rhizobia. These common antigens among hosts and bacteria were not related to the specificity of compatibleRhizobium-legume associations. The cross-reactive antigens were absent between rhizobia and eight non-legume plants tested, but present between five out of eleven gramnegative phytopathogenic bacteria and legumes. The potential significance of cross-reactive antigens betweenRhizobium and legume hosts in nodule development is discussed.     

DNA fingerprinting in sugar beet (Beta vulgaris) — identification of double-haploid breeding lines

Summary The distribution and abundance of simple repetitive sequences complementary to the synthetic oligonucleotides (GACA)₄, (GATA)₄, (GTG)₅ and (CA)₈ in the genomes of several cultivars of Beta vulgaris and in the wild beet B. vulgaris ssp. maritima were investigated. Hybridization experiments revealed that all four motifs were present, though at different abundances, in the genomes of all of the investigated beet cultivars. Considerable intraspecific variation of the resulting DNA fingerprints was observed. The extent of polymorphism depends on the oligonucleotide probe. The most informative banding patterns were obtained with the (GATA)₄ probe hybridized to HinfI-, HaeIII-, or RsaI-restricted DNA, respectively. DNA fingerprinting with (GATA)₄ allowed a clear differentiation of double-haploid breeding lines (DH lines). We demonstrated that the application of oligonucleotide probes for DNA fingerprinting is a sensitive tool for genome diagnosis in cultivated beet.     

Branched respiratory chain in aerobically grown Staphylococcus aureus —oxidation of ethanol by cells and protoplasts

Addition of ethanol and some other primary alcohols, except methanol, to cells and protoplasts (but not membrane particles) considerably stimulated the rate of oxygen consumption. This additional respiration was strongly inhibited by 0.1 mM KCN. The cyanide inhibition curve of endogenous substrate oxidation was slightly biphasic while in the presence of ethanol it became clearly biphasic having K _i values of approx. 0.1 and 0.5 mM. Based on the steady-state cytochrome spectra in the presence of 0.1 mM KCN, we attributed the lower K _i to cytochrome a ₆₀₂. Proteolysis of protoplasts external membrane proteins did not change the rate of endogeneous substrate oxidation but prevented the inhibition of this respiration by low concentrations of KCN and stimulation of oxygen consumption by ethanol. The activity of NAD⁺-dependent ethanol dehydrogenase in the cytoplasm was found to be 520 nmol NADH-x min^–1 x mg^–1 protein. Proteolysis of external membrane proteins apparently inhibits the operation of the cytochrome a ₆₀₂-containing electron transport branch inducing the suppression of electron flow from NADH to oxygen.     

Resistance to metamitron in Chenopodium album from sugar beet. a tale of the (un)expected?

Metamitron is a key herbicide in modern low rate weed control programs in sugar beet. Fat hen (Chenopodium album, CHEAL) is a common, highly competitive, weed in sugar beet and one of the targets of metamitron. Recently, unsatisfactory control of fat hen has been reported in several sugar beet fields situated in various regions in Belgium. Weather conditions as well as the mere fact of using low rate systems have been blamed for these poor performances. To address the question "Is the recently recorded poor control of C. album due to decreased sensitivity to metamitron", greenhouse bioassays were carried out. A first experiment was conducted applying metamitron (0, 350, 700 and 1,400 g ai/ha) postemergence to three "suspected" C. album populations originating from sugar beet fields with unsatisfactory control by standard metamitron based treatment schemes ('Ligne', 'Outgaarden' and 'Boutersem I' respectively) and to one sensitive population originating from an untreated garden site ('Gent'). In a second experiment seven population, five "suspected" fat hen populations from sugar beet fields ('Boutersem I', 'Boutersem II', 'Postel', 'Vissenaken' and 'Kortessem' respectively), one sensitive reference population 'Herbiseed' and one atrazine-resistant reference population 'ME.85.01', were submitted to metamitron (0, 1, 2 and 4 mg ai/kg air-dry soil) and atrazine (1.5 mg ai/kg air-dry soil) preplant incorporated. All "suspected" C. album populations displayed a significantly lower sensitivity to metamitron compared to the sensitive populations ('Gent' and 'Herbiseed') that never had been exposed to this herbicide. As target site cross-resistance of atrazine-resistant C. album, selected by atrazine in maize, to metamitron has been known for a long time, cross-resistance of C. album populations in sugar beet grown on fields with "maize - atrazine" containing rotations might be expected to appear. The outcome of the experiment with atrazine preplant incorporated was the confirmation of resistance in all "suspected" populations ('Boutersem I', 'Boutersem II', 'Postel', 'Vissenaken' and 'Kortessem'). However, some "suspected" populations came from fields with no background of cropping with maize and use of atrazine. So, the question remains whether these triazine-resistant C. album had been imported, e.g. with slurry, or the rather unexpected possibility that metamitron itself did select for metamitron-resistant biotypes bearing cross-resistance to atrazine, had become reality.     

Subcellular localization and possible functions of acid β-glycerophosphatases and naphthol esterases in plant cells

Summary The subcellular localization of esterases and acid phosphatases in described for cells from roots of Vicia faba. The possible mode of transport of the esterases from a cytoplasmic site of synthesis to the cell wall is discussed.     

Identification and characterization of small cells in the adult pancreas: potential progenitor cells?

In this report we describe the identification of a novel cell type in human and canine pancreas using tissue culture techniques. These cells, representing less than 1% of total islet cells, are of a small size (7-10 microm) and highly quiescent. They display a fairly immature morphology, which is characterized by a weakly developed protein synthesis machinery, a few mitochondria and a small number of neuroendocrine granules. These cells, which we have termed "small cells," are usually organized into small clusters, which can be identified within the islets of predominantly small size. They can also be collected as separate structures from preparations of freshly isolated islets. Immunohistochemically, small cells are positive for PDX-1, synaptophysin, insulin, glucagon, somatostatin, pancreatic polypeptide, alpha-fetaprotein and Bcl-2 and negative for cytokeratin 19 and nestin. Insulin secretion studies demonstrated that these cells secrete insulin in a glucose-responsive fashion, although do not respond to secretagogues such as IBMX and arginine as do mature beta cells. Although this study does not provide evidence of the proliferative and differentiation potential of small cells, their immature morphology, along with a small size and quiescence, let us hypothesize that these cells may serve as progenitors contributing to the islet growth.     

Improved culture technique for strawberry (Fragaria × ananassa Duch.) protoplasts and the determination of DNA content in protoplast derived plants

A reproducible and efficient protoplast to plant-system has been developed for strawberry. By using young shoots growing on medium supplemented with cytokinin we were able to exclude the detrimental enzyme Pectolyase from our enzyme solution. The more gentle isolation procedure reported in this paper, together with the agarose-embedding system had a pronounced effect on protoplast viability and growth. Plant regeneration was enhanced by using thidiazuron instead of benzyladenine. Flow cytometric measurement of the DNA content in protoplast derived plants revealed that 27 out of 51 plants contained the amount of DNA corresponding to octoploid level. Fifteen plants exhibited chromosome doubling(16×), one was diplodecaploid(12×) and eight plants mixoploid.     

Backcrossing of nematode-resistant sugar beet: a second nematode resistance gene at the locus containing Hs1pro-1?

Beet cyst nematode-resistant sugar beet plants, containing the Hs1^pro-1 locus from Beta procumbens, show a female transmission frequency of the resistance of ca. 90%. Such plants often suffer from tumour formation on leaves and root systems, and from the occurrence of a so-called multi-top phenotype. With the aim of obtaining resistant sugar beet material lacking these negative traits, nematode-resistant plants with a reduced size of the chromosome segment of the wild beet that carries the Hs1^pro-1 gene were selected from backcrosses between the resistant stocks B883 or AN1-65-2 and susceptible sugar beet (Beta vulgaris). Analysis of such plants, referred to as Sat-minus plants, showed that the transmission frequency of the resistance to subsequent generations had dropped dramatically to ca. 0.5%. The multi-top phenotype was still present in the newly selected material, indicating that improvement of the resistant sugar beet material by further backcrossing will be hard to achieve. Two of the selected resistant offspring plants were analysed at the molecular level. With the aid of AFLP markers it was found that the size of the alien chromosome segment had decreased to 35% and 17% of the original size, respectively. Surprisingly, both plants had lost the Hs1^pro-1 nematode resistance gene that recently was isolated from the original introgression material. This shows that more than one gene conferring resistance must be present in the locus in B883 and AN1-65-2 carrying the resistance gene Hs1^pro-1.