Uptake of Lucifer Yellow CH into plant-cell protoplasts: a quantitative assessment of fluid-phase endocytosis

The highly fluorescent dye Lucifer Yellow CH (LYCH), now in common use in microinjection studies, has been shown to enter the vacuole of a range of plant-cell protoplasts from the external medium. Uptake was quantified by lysing the protoplasts following incubation and determining the amount of LYCH incorporated by spectrofluorimetry. Uptake was biphasic with respect to both time and substrate concentration, enhanced at low pH and inhibited by low temperature and metabolic inhibitors. The kinetics of uptake showed several similarities with those reported for the fluid-phase endocytosis of LYCH in animal cells and yeast cells. A calculated membrane permeability coefficient for LYCH, based on the observed rates of uptake, was too high to be consistent with simple diffusion of the undissociated form of the molecule and inconsistent with the membrane-impermeant properties of the dye. The data are discussed in the light of the possibility of fluid-phase endocytosis versus active transmembrane transport.Abbreviations CCCP carbonyl cyanide M-chlorophenyl hydrazone - LYCH Lucifer Yellow CH     

Vacuole motility and tubule-forming activity inPisolithus tinctorius hyphae are modified by environmental conditions

Summary Motile tubular vacuole systems have been visualised using DIC optics in living hyphae ofPisolithus tinctorius without loading of any fluorescent tracer. Adding new medium, with or without the tracer CFDA, alters the motility of this system and increases the number of tubules. This response has been shown in individual hyphal tip cells and quantified in populations of tip cells. Vacuoles with motile tubules are also demonstrated in more basal cells of the hyphae, within 600 m of the growing hyphal front. The vacuoles in these cells show more limited motility, but similarly respond to addition of new medium by increased motility and tubular activity. This demonstration that the vacuole system in more mature regions is both motile and interconnected as in the tips, and similarly responds to changes in external conditions, supports the hypothesis that the vacuole system may play a role in long-distance transport. Vacuoles in the most mature cells, more than 600 m behind the hyphal growth zone are not motile. They do not respond to these stimuli and remain spherical and isolated. There are many explanations for this and the present lack of response does not exclude the transport hypothesis. The findings further support the concept that tubular vacuole systems are equivalent to animal endosomal/lysosomal systems and have implications for their motility, especially their plasticity in response to external stimuli, such as fluorescent tracers.Abbreviations CFDA 6-carboxyfluorescein diacetate - DIC differential interference contrast - MMN modified Melin-Norkrans medium - SEM standard error of the mean     

Uptake of Lucifer Yellow CH into intact barley roots: Evidence for fluid-phase endocytosis

Intact barley (Hordeum vulgare L.) roots have been shown to take up the highly fluorescent dye Lucifer Yellow CH (LYCH) into their cell vacuoles. In the apical 1 cm of root tip, differentiating and dividing cells showed a prolific uptake of LYCH into their provacuoles. The LYCH was retained during fixation, apparently becoming bound to electron-dense material in the vacuoles. The dye freely entered the apoplast of roots in which the Casparian band was not developed, being taken up into the vacuoles of cells in both the cortex and stele. However, when LYCH was applied to a 1-cm zone approx. 6 cm behind the root tip the Casparian band on the radial walls of the endodermis completely prevented the dye from entering the cells of the stele, only the cell walls and vacuoles of the cortical cells taking up the dye. The inability of LYCH to cross the plasmalemma of the endodermal cells and enter the stele via the symplast substantiates previous claims that the dye is unable to cross the plasmalemma of plant cells. The results are discussed in the light of recent demonstrations that LYCH is a particularly effective marker for fluid-phase endocytosis in animal and yeast cells. A calculation of the energetic requirements for LYCH uptake into barley roots supports the contention that LYCH is taken up into the vacuoles of plant cells by fluid-phase endocytosis.Abbreviation LYCH Lucifer Yellow CH     

Some characteristics of anion transport at the tonoplast of oat roots,determined from the effects of anions on pyrophosphatedependent proton transport

The effects of anions on inorganicpyrophosphate-dependent H⁺-transport in isolated tonoplast vesicles from oat (Avena sativa L.) roots were determined. Both fluorescent and radioactive probes were used to measure formation of pH gradients and membrane potential in the vesicles. Pyrophosphate hydrolysis by the H⁺-translocating pyrophosphatase was unaffected by anions. Nonetheless, some anions (Cl^-, Br^- and NO₃-) stimulated H⁺-transport while others (malate, and iminodiacetate) did not. These differential effects were abolished when the membrane potential was clamped at zero mV using potassium and valinomycin. Stimulation of H⁺-transport by Cl^- showed saturation kinetics whereas that by NO₃- consisted of both a saturable component and a linear phase. For Cl^- and NO₃-, the saturable phase had a K _m of about 2 mol·m^-3. The anions that stimulated H⁺-transport also dissipated the membrane potential (.) generated by the pyrophosphatase. It is suggested that the stimulatory anions cross the tonoplast in response to the positive generated by the pyrophosphatase, causing dissipation of and stimulation of pH, as expected by the chemiosmotic hypothesis. The work is discussed in relation to recent studies of the effects of anions on ATP-dependent H⁺-transport at the tonoplast, and its relevance to anion accumulation in the vacuole in vivo is considered.Abbreviations and symools BTP 1,3-bis[tris(hydroxymethyl)-methylamino]-propane - EGTA ethylene glycol-bis(-aminoethyl ether)-N,N,N,N-tetraacetic acid - Hepes 4-(2-hydroxyethyl)-1-piperazine ethanesulphonic acid - IDA iminodiacetate - membrane potential - pH pH gradient - PPase inorganic pyrophosphatase - PPi morganic pyrophosphate     

Characterisation of chloride transport at the tonoplast of higher plants using a chloride-sensitive fluorescent probe

The characteristics of Cl^– transport in isolated tonoplast vesicles from red-beet (Beta vulgaris L.) storage tissue have been investigated using the Cl^–-sensitive fluorescent probe, 6-methoxy-1-(3-sulfonatopropyl)-quinolinium (SPQ). The imposition of (inside) positive diffusion potentials, generated with K⁺ and valinomycin, increased the initial rate of Cl^– transport, demonstrating that Cl^– could be electrically driven into the vesicles. Chloride influx was unaffected by SO ₄ ^2- , but was competitively blocked by NO ₃ ^– , indicating that both Cl^– and NO ₃ ^– may be transported by the same porter. In some preparations, increases in free-Ca²⁺ concentration from 10^–8 to 10^–5 mol·dm^–3 caused a significant decrease in Cl^– influx, which may indicate that cytosolic Ca²⁺ concentration has a role in controlling Cl^– fluxes at the tonoplast. However, this effect was only seen in about 50% of membrane preparations and some doubt remains over its physiological significance. A range of compounds known to block anion transport in other systems was tested, and some partially blocked Cl^– transport. However, many of these inhibitors interfered with SPQ fluorescence and so only irreversible effects could be tested. The results are discussed in the context of recent advances made using the patch-clamp technique on isolated vacuoles.Abbreviations and Symbols BTP 1,3-bis[tris(hydroxymethyl)-methylamino]propane - DTT dithiothreitol - EDTA ethylenediaminetetraacetic acid - membrane potential - pH pH gradient - SPQ 6-methoxy-1-(3-sulfonatopropyl)quinolinium - Tricine N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl] glycine     

Kinetic evidence for an anion binding pocket in the active site of nitronate monooxygenase

A series of monovalent, inorganic anions and aliphatic aldehydes were tested as inhibitors for Hansenula mrakii and Neurospora crassa nitronate monooxygenase, formerly known as 2-nitropropane dioxygenase, to investigate the structural features that contribute to the binding of the anionic nitronate substrates to the enzymes. A linear correlation between the volumes of the inorganic anions and their effectiveness as competitive inhibitors of the enzymes was observed in a plot of pK_is versus the ionic volume of the anion with slopes of 0.041 ± 0.001 mM/ų and 0.027 ± 0.001 mM/ų for the H. mrakii and N. crassa enzymes, respectively. Aliphatic aldehydes were weak competitive inhibitors of the enzymes, with inhibition constants that are independent of their alkyl chain lengths. The reductive half reactions of H. mrakii nitronate monooxygenase with primary nitronates containing two to four carbon atoms all showed apparent K_d values of 5 mM. These results are consistent with the presence of an anion binding pocket in the active site of nitronate monooxygenase that interacts with the nitro group of the substrate, and suggest a minimal contribution of the hydrocarbon chain of the nitronates to the binding of the ligands to the enzyme.     

Bicarbonate permeability and immunological evidence for an anion exchanger-like protein in the red blood cells of the sea lamprey,Petromyzon marinus

Physiological and immuno-blotting experiments were used to determine whether the red blood cell membrane of a primitive vertebrate, the sea lamprey Petromyzon marinus, contained a counterpart similar to the vertebrate anion exchange protein known as AE1 or band 3. Results of the physiological experiments which measured CO₂ production after adding H¹⁴CO ₃ ^- to the extracellular saline, indicated significant transmembrane bicarbonate movement in lamprey blood which unlike that in most vertebrates, was insensitive to inhibition by 4,4 diisothiocyanatostilbene-2,2 disulfonic acid. The present study also showed that lamprey red blood cells possess acetazolamide-sensitive carbonic anhydrase which is an important component of CO₂ production by vertebrate red blood cells. Polyclonal immunoglobulins against a 12 amino acid domain in the C-terminus of the mouse AE1 recognized a trout red blood cell membrane protein with a relative molecular mass of 97 kDa, but failed to immunoreact with any membrane proteins from the red blood cells of lamprey. Antibodies against trout AE1 immunoreacted with trout red blood cell membrane proteins of approximately 97 kDa, 200 kDa and >200 kDa. Interestingly, only a 200-kDa membrane protein from the red blood cells of the primitive lamprey immunoreacted with the trout anti-AE1 immunoglobulin proteins. Therefore, lamprey red blood cells appear to possess an AE1-like protein that may be physiologically different than that in most other vertebrates.     

The absence of cytoplasm ribosomes on the envelopes of chloroplasts and mitochondria in plants: Implications for the mechanism of transport of proteins into these organelles

Summary Chloroplasts and mitochondria ofChlamydomonas were examined by electron microscopy to determine if cytoplasmic ribosomes were associated with the envelopes of these organelles. Cells were treated with cycloheximide to prevent polypeptide chain completion, and resultant dissociation of envelope-ribosome associations. No extensive association of cytoplasm ribosomes with envelopes of chloroplasts, or mitochondria was detected in intact cells or in damaged cells in which cytoplasm was partly removed. Our results indicate that association of cytoplasm ribosomes with envelopes of chloroplasts or mitochondria is not an essential requirement for transport of polypeptides from cytoplasm to organelle.     

Kinetics and mechanism of iron release from the bacterial ferric binding protein<Emphasis Type="Italic"> n</Emphasis>Fbp: exogenous anion influence and comparison with mammalian transferrin

Ferric binding protein, Fbp, serves an essential biological function in shuttling naked (hydrated) Fe³⁺ across the periplasmic space of many Gram-negative bacteria. In this process, iron must be released at the cytoplasmic membrane to a permease. How iron is released from Fbp has yet to be resolved. Consequently, understanding the dynamics of iron release from Fbp is of both biological and chemical interest. Fbp requires an exogenous anion, e.g. phosphate when isolated from cell lysates, for tight iron sequestration. To address the role of exogenous anion identity and lability on Fe_aq ³⁺ dissociation from Fbp, the kinetics of PO₄ ^3– exchange in Fe³⁺ nFbp(PO₄) (nFbp=recombinant Fbp from Neisseria meningitidis) were investigated by dynamic ³¹P NMR and the kinetics of Fe³⁺ dissociation from Fe³⁺ nFbp(X) (X=PO₄ ^3–, citrate anion) were investigated by stopped-flow pH-jump measurements. We justify the use of non-physiological low-pH conditions because a high [H⁺] will drive the Fe_aq ³⁺ dissociation reaction to completion without using competing chelators, whose presence may complicate or influence the dissociation mechanism. For perspective, these studies of nFbp (which has been referred to as a bacterial transferrin) are compared to new and previously published kinetic and thermodynamic data for mammalian transferrin. Significantly, we address the lability of the Fe³⁺ coordination shell in nFbp, Fe³⁺ nFbp(X) (X=PO₄ ^3–, citrate), with respect to exogenous anion (X ^n–) exchange and dissociation, and ultimately complete dissociation of the protein to yield naked (hydrated) Fe_aq ³⁺. These findings are a first step in understanding the process of iron donation to the bacterial permease for transport across the cytoplasmic membrane.Electronic Supplementary Material Supplementary material is available in the online version of this article at . Abbreviations DTPP diethylenetriaminepenta(methylenephosphonic acid) - Fbp ferric binding protein - H₃cit citric acid - hFbp Fbp from Haemophilus influenzae - H₂ox oxalic acid - hTf human serum transferrin - 3,4-LICAMS N,N,N-tris(5-sulfo-2,3-dihydroxybenzoyl)-1,5,10-triazadecane - nFbp recombinant Fbp from Neisseria meningitidis - NTA nitrilotriacetic acid - TRENSOX tris[2-aminoethyl(8-hydroxyquinoline-5-sulfonato-7-carbonyl)]amine     

Degradation of some phenols and hydroxybenzoates by the imperfect ascomycetous yeastsCandida parapsilosis andArxula adeninivorans: evidence for an operative gentisate pathway

The imperfect ascomycetous yeastsCandida parapsilosis andArxula adeninivorans degraded 3-hydroxybenzoic acid via gentisate which was the cleavage substrate. 4-Hydroxybenzoic acid was metabolized via protocatechuate. No cleavage enzyme for the latter was detected. In stead of this NADH- and NADPH-dependent monooxygenases were present. In cells grown at the expense of hydroquinone and 4-hydroxygenzoic acid, enzymes of the hydroxyhydroquinone variant of the 3-oxoadipate pathway were demonstrated, which also took part in the degradation of 2,4-dihydroxybenzoic acid byC. parapsilosis.Abbreviations HHQ Hydroxyhydroquinone (1,2,4-trihydroxybenzene) - GSH reduced Glutathione     

Comparative analysis of the structure and chromosomal assignment ofCD1: an evidence for different evolutionary histories between classic CD1 and CD1D class genes

The non-MHC-encoded CD1 family has recently emerged as a novel antigen-presenting system that is distinct from MHC class I and class II molecules. In the present study, we determined the genomic structure of that rat CD1, and compared with those of other previously reported CD1 genes. Rat CD1 was extremely similar to mouse CD1 genes, especially to CD1D1. It is of interest that a tyrosine-based motif for endosomal localization, identified in the human CD1b cytoplasmic tail, was conserved in all CD1 molecules except for CD1a, that was encoded by a single short exon. Comparison of the overall exon-intron organization of CD1 genes revealed that the length of the introns was also characteristic to each of the two classes of CD1 genes; classic (CD1A, CD1B, CD1C and CD1E), and CD1D, which have been categorized by comparison of coding regions. These findings support a hypothesis that the two classes have different evolutionary histories. In contrast to the absence of the classic CD1 genes in rats and mice, the entire region of nonpolymorphic CD1D gene has been conserved through mammalian evolution. Furthermore, we determined chromosomal localization of rat CD1 gene using the fluorescence in situ hybridization method with several probes derived from genomic rat CD1 clones. Similar to human and mouse CD1, rat CD1 mapped outside the MHC loci despite the structural and functional resemblance to MHC. Conserved syntheny of chromosomal segments of RNO2 and MMU3 is implied.     

Green fluorescent protein and factorial approach: an effective partnership for screening the soluble expression of recombinant proteins in Escherichia coli

We report how the combined use of protein expression reporter green fluorescent protein (GFP), and of an incomplete factorial approach (“InFFact”) made of 12 combinations of different states of three expression variables (bacterial strains, culture media and expression temperatures) created a convenient tool for screening the soluble expression of recombinant proteins in Escherichia coli (E. coli).In the first part of this work, we used two recombinant proteins that could be easily detected by Western blotting in the soluble fraction of E. coli lysate in most of the 12 InFFact combinations. When these proteins were fused to GFP and used in the same experiment (“InFFact-GFP”), fluorescence signals proved as sensitive and reliable as those provided by Western blotting. A trend analysis based on Western blot signals or on fluorescence allowed finding expression conditions for successfully scaling up the production of both proteins. Thus, GFP allowed InFFact trend analysis to be performed without gel electrophoresis or Western blotting.In the second part, we compared the results obtained by InFFact and InFFact-GFP when two other recombinant proteins were used which, in contrast with the proteins used in the first part, were barely detectable by Western blotting. Surprisingly, InFFact-GFP but not InFFact was able to find expression conditions for successfully scaling up the production of both proteins, suggesting that GFP could increase the solubility of the fusion partner.In conclusion, GFP allowed InFFact to be performed without gel electrophoresis and with at least the same sensitivity and specificity as that of Western blotting.     

Proteins of the Drosophila melanogaster male reproductive system: Two-dimensional gel patterns of proteins synthesized in the XO,XY, and XYY testis and paragonial gland and evidence that the Y chromosome does not code for structural sperm proteins

Testes and paragonial glands of Drosophila melanogaster wild-type males were labeled in vitro using [³⁵S]methionine, and the proteins synthesized were analyzed by 2-dimensional gel electrophoresis. Testes and paragonial glands were also labeled in vivo by feeding male larvae ³⁵S-labeled yeast and then dissecting the adult males. Approximately 1200 proteins were resolved by autoradiography of the gels. The in vitro method was shown to be more sensitive and to allow faithful synthesis of all proteins produced in vivo. [³H]Proline was also used to label testes, and no significant differences from the ³⁵S pattern were noted. Testes and paragonial glands from XO and XYY males were labeled in vitro with [³⁵S]methionine, and the proteins synthesized were compared to those produced by wild-type males of identical autosomal background. No differences attributable to the Y chromosome could be detected in the testes or paragonial gland samples. Pure sperm were dissected manually from in vivo labeled males and the proteins analyzed. Ninety-two proteins were detected, which were all synthesized in comparable amounts by XO, XY, and XYY males, showing that the Y chromosome does not code for any of these structural sperm proteins. It is postulated that no Y chromosome products were detected because they are organizational or regulatory proteins present only in very small amounts in the adult testes. ³⁵S-labeled males were also mated to unlabeled females and the transferred proteins analyzed on two-dimensional PAGE. The contributions of the testis and paragonial gland to the ejaculate were determined.This work was supported by a grant from the Medical Research Council of Canada and by the U.S. National Institutes of Health (Grant No. GM-22753). J. I. B. is the recipient of a Medical Research Council of Canada studentship.     

Intra-species variation in transient accumulation of leaf anthocyanins in Cistus creticus during winter: evidence that anthocyanins may compensate for an inherent photosynthetic and photoprotective inferiority of the red-leaf phenotype

Leaf color in some individuals of Cistus creticus turns transiently to red during winter, while neighboring individuals occupying the same site remain green. We have examined whether anthocyanin accumulation can be associated with variations in photosynthetic and/or photoprotective characteristics between the two phenotypes, rendering the red phenotype more vulnerable to photoinhibition and, accordingly, needing additional protection in the form of anthocyanins. Towards this aim, maximum (pre-dawn) and effective (mid-day) PSII photochemical efficiencies, xanthophyll cycle pool sizes and leaf nitrogen contents were seasonably followed, encompassing both the green (spring, summer, autumn) and the red (winter) period of the year. Moreover, the distribution of the two phenotypes in exposed and shaded sites was assessed. The frequency of red individuals was considerably higher in fully exposed sites, pointing to a photoprotective function of leaf anthocyanins. Yet, the assumption was not corroborated by pre-dawn PSII yield measurements, since both phenotypes displayed similar high values throughout the year and a similar drop during winter. However, the red phenotype was characterized by lower light-saturated PSII yields, xanthophyll cycle pool sizes and leaf nitrogen, during both the green and the red period of the year. Based on this correlative evidence, we suggest that winter redness in C. creticus may compensate for an inherent photosynthetic and photoprotective inferiority, possibly through a light screen and/or an antioxidant function of leaf anthocyanins.     

Physical map and gene localization on sunflower (Helianthus annuus) chloroplast DNA: evidence for an inversion of a 23.5-kbp segment in the large single copy region

As a first step in the study of chloroplast genome variability in the genus Helianthus, a physical restriction map of sunflower (Helianthus annuus) chloroplast DNA (cpDNA) has been constructed using restriction endonucleases BamH I, Hind III, Pst I, Pvu II and Sac. I. Sunflower circular DNA contains an inverted repeat structure with the two copies (23 kbp each) separated by a large (86 kbp) and a small (20 kbp) single copy region. Its total length is therefore about 152 kbp. Sunflower cpDNA is essentially colinear with that of tobacco with the exception of an inversion of a 23.5-kbp segment in the large single copy region. Gene localization on the sunflower cpDNA and comparison of the gene map with that from tobacco chloroplasts have revealed that the endpoints of the inversion are located between the trnT and trnE genes on the one hand, and between the trnG and trnS genes on the other hand.Analysis of BamH I restriction fragment patterns of H. annuus, H. occidentalis ssp. plantagineus, H. grossesseratus, H. decapetalus, H. giganteus, H. maximiliani and H. tuberosus cpDNAs suggests that structural variations are present in the genus Helianthus.     

Acetate oxidation to CO2 via a citric acid cycle involving an ATP-citrate lyase: a mechanism for the synthesis of ATP via substrate level phosphorylation in Desulfobacter postgatei growing on acetate and sulfate

Desulfobacter postgatei is an acetate-oxidizing, sulfate-reducing bacterium that metabolizes acetate via the citric acid cycle. The organism has been reported to contain a si-citrate synthase (EC 4.1.3.7) which is activated by AMP and inorganic phosphate. It is show now, that the enzyme mediating citrate formation is an ATP-citrate lyase (EC 4.1.3.8) rather than a citrate synthase. Cell extracts (160,000xg supernatant) catalyzed the conversion of oxaloacetate (apparent K _m=0.2 mM), acetyl-CoA (app. K _m=0.1 mM), ADP (app. K _m=0.06 mM) and phosphate (app. K _m=0.7 mM) to citrate, CoA and ATP with a specific activity of 0.3 mol·min^-1·mg^-1 protein. Per mol citrate formed 1 mol of ATP was generated. Cleavage of citrate (app. K _m=0.05 mM; V _max=1.2 mol · min^-1 · mg^-1 protein) was dependent on ATP (app. K _m=0.4 mM) and CoA (app. K _m=0.05 mM) and yielded oxaloacetate, acetyl-CoA, ADP, and phosphate as products in a stoichiometry of citrate:CoA:oxaloacetate:ADP=1:1:1:1. The use of an ATP-citrate lyase in the citric acid cycle enables D. postgatei to couple the oxidation of acetate to 2 CO₂ with the net synthesis of ATP via substrate level phosphorylation.