Molecular Structure and Properties of Lectin from Tomato Fruit

A cDNA encoding tomato fruit lectin was cloned from an unripe cherry-tomato fruit cDNA library. The isolated lectin cDNA contained an open reading frame encoding 365 amino acids, including peptides that were sequenced. The deduced sequence consisted of three distinct domains: (i) an N-terminal short extensin-like domain; (ii) a Cys-rich carbohydrate binding domain composed of four almost identical chitin-binding domains; (iii) an internal extensin-like domain of 101 residues containing 15 SerPro₄ motifs inserted between the first and second chitin-binding domains. The molecular weight of the lectin was 65,633 and that of the deglycosylated lectin was 32,948, as determined by matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF MS). This correlated with the estimated molecular weight of the deduced sequence. Recombinant tomato lectin expressed in Pichia pastoris possessed chitin-binding but not hemagglutinating activity. These findings confirmed that the cDNA encoded tomato lectin.     

TheRelati磷饥饿时番茄幼苗根系质膜H~ - ATP酶活性的变化与磷吸收的关系(英文)

磷饥饿提高了番茄幼苗质膜H ATP酶活性并促进了番茄幼苗根部的H 分泌。动力学分析表明 ,磷饥饿使番茄幼苗根部质膜H ATP酶的Km 值明显降低 ,亦即提高了该酶对其底物的亲和力 ,但对该酶的Vmax影响不大。另外 ,磷饥饿并不改变ATP酶的最适 pH值 (最适 pH值为 6.5)。钒酸盐显著抑制番茄幼苗根部质膜ATP酶的活性以及H 分泌 ,也显著抑制番茄幼苗的Pi吸收。与对照相比 ,上述抑制作用在饥饿处理的植物中表现得更强。以上结果表明 ,磷饥饿时高亲和性Pi传递系统的诱导很可能包含质膜H ATP酶的参与。     

Hydroxylated jasmonates are commonly occurring metabolites of jasmonic acid and contribute to a partial switch-off in jasmonate signaling

In potato 12-hydroxyjasmonic acid (12-OH-JA) is a tuber-inducing compound. Here, it is demonstrated that 12-OH-JA, as well as its sulfated and glucosylated derivatives, are constituents of various organs of many plant species. All accumulate differentially and usually to much higher concentrations than jasmonic acid (JA). In wounded tomato leaves, 12-OH-JA and its sulfated, as well as glucosylated, derivative accumulate after JA, and their diminished accumulation in wounded leaves of the JA-deficient mutants spr2 and acx1 and also a JA-deficient 35S::AOCantisense line suggest their JA-dependent formation. To elucidate how signaling properties of JA/JAME (jasmonic acid methyl ester) are affected by hydroxylation and sulfation, germination and root growth were recorded in the presence of the different jasmonates, indicating that 12-OH-JA and 12-hydroxyjasmonic acid sulfate (12-HSO(4)-JA) were not bioactive. Expression analyses for 29 genes showed that expression of wound-inducible genes such as those coding for PROTEINASE INHIBITOR2, POLYPHENOL OXIDASE, THREONINE DEAMINASE or ARGINASE was induced by JAME and less induced or even down-regulated by 12-OH-JA and 12-HSO(4)-JA. Almost all genes coding for enzymes in JA biosynthesis were up-regulated by JAME but down-regulated by 12-OH-JA and 12-HSO(4)-JA. The data suggest that wound-induced metabolic conversion of JA/JAME into 12-OH-JA alters expression pattern of genes including a switch off in JA signaling for a subset of genes.     

Fructan synthesis is inhibited by phosphate in warm-grown, but not in cold-treated, excised barley leaves

The inhibition of fructan accumulation by phosphate was investigated in warm-grown and cold-treated barley (Hordeum vulgare) plants. Detached leaves were incubated in water or phosphate for 24 h under lighting or in darkness. Fructosyltransferase, sucrose phosphate synthase (SPS) and cytosolic fructose-1,6-bisphosphatase (FBPase) activities were subsequently analysed, as well as the content of carbohydrates, hexose-phosphates, phosphate, amino acids and protein. In warm-grown leaves, phosphate decreased fructan accumulation and total carbon in carbohydrates and did not affect protein content. Phosphate increased hexose-phosphates, phosphate and amino acids. Fructosyltransferase and FBPase activities were not affected by phosphate feeding, while SPS activity was inhibited by phosphate in incubations in both light and darkness. In cold-treated leaves, which before incubation had higher SPS activities than warm-grown leaves, phosphate had no inhibitory effect on fructan accumulation, carbohydrate content or total C in carbohydrates. The activities of SPS and FBPase were unaffected by phosphate. The results indicate that phosphate decreases fructan accumulation through an inhibition of SPS whenever this activity is not high before a rise in phosphate content.     

Isoflavonoid exudation from white lupin roots is influenced by phosphate supply, root type and cluster-root stage

The internal concentration of isoflavonoids in white lupin (Lupinus albus) cluster roots and the exudation of isoflavonoids by these roots were investigated with respect to the effects of phosphorus (P) supply, root type and cluster-root developmental stage.To identify and quantify the major isoflavonoids exuded by white lupin roots, we used high-pressure liquid chromatography (HPLC) coupled to electrospray ionization (ESI) in mass spectrometry (MS).The major exuded isoflavonoids were identified as genistein and hydroxygenistein and their corresponding mono- and diglucoside conjugates. Exudation of isoflavonoids during the incubation period used was higher in P-deficient than in P-sufficient plants and higher in cluster roots than in noncluster roots. The peak of exudation occurred in juvenile and immature cluster roots, while exudation decreased in mature cluster roots.Cluster-root exudation activity was characterized by a burst of isoflavonoids at the stage preceding the peak of organic acid exudation. The potential involvement of ATP-citrate lyase in controlling citrate and isoflavonoid exudation is discussed, as well as the possible impact of phenolics in repelling rhizosphere microbial citrate consumers.     

Effect of CPTA and cycocel on the biosynthesis of carotenoids by Phycomyces blakesleeanus mutants

CPTA and cycocel cause accumulation of lycopene and γ-carotene, simultaneously inhibiting the formation of β-carotene and β-zeacarotene in Phycomyces blakesleeanus mutant strain C115. Phytoene synthesis is enhanced. CPTA is more effective than cycocel. Kinetic studies show that with increasing concentrations of CPTA, lycopene and γ-carotene increase with the concomitant decrease in β-carotene, the total of these three carotenes being almost equal to β-carotene present in the control. When CPTA-treated mycelium is washed free of the chemical and resuspended in phosphate buffer solution containing 2·5% glucose (pH 5·6), β-carotene is formed at the expense of both γ-carotene and lycopene. β-Zeacarotene, which is not present in the mycelium, reappears upon resuspension. These results indicate that CPTA is inhibiting the enzymes causing cyclization both at neurosporene and lycopene levels. Studies on the effect of CPTA on the high lycopene mutant strain C9 reveal that with increasing concentrations of the compound, lycopene increases slightly and both β-carotene and γ-carotene decrease. Phytoene synthesis is stimulated up to a certain level of CPTA and then becomes steady. In the albino mutant strain C5, there is a slight increase in phytoene formation on the addition of CPTA to the medium. No other carotenoid is formed, suggesting that CPTA cannot remove the block caused by genetic mutation and exerts its influence in an already existing biosynthetic pathway.     

Comparison of the kinetic properties,inhibition and labelling of the phosphate translocators from maize and spinach mesophyll chloroplasts

The kinetic properties of the phosphate translocator from maize (Zea mays L.) mesophyll chloroplasts have been determined. We have used a double silicone-oil-layer centrifugation system in order to obtain true initial uptake rates in forward-reaction experiments. In addition, it was possible to perform back-exchange experiments and to study the effects of illumination and of preloading the chloroplasts with different substrates on transport. It is shown that the phosphate translocator from mesophyll chloroplasts of maize, a C₄ plant, transports inorganic phosphate and phosphorylated C₃ compounds in which the phosphate group is linked to the C₃ atom (e.g. 3-phosphoglycerate and triose phosphate). The affinities of the transported metabolites towards the translocator protein are about one order of magnitude higher than in mesophyll chloroplasts from the C₃ plant, spinach. In contrast to the phosphate translocator from C₃-mesophyll chloroplasts, that of C₄-mesophyll chloroplasts catalyzes in addition the transport of C₃ compounds where the phosphate group is attached to the C₂ atom (e.g. 2-phosphoglycerate, phosphoenolpyruvate). The phosphate translocator from both chloroplast types is strongly inhibited by pyridoxal-5-phosphate (PLP), 2,4,6-trinitrobenzenesulfonic acid and 4,4-diisothiocyanostilbene-2,2-disulfonic acid (DIDS). In the case of the spinach translocator protein these inhibitors were shown to react with the same amino-acid residue at the substrate binding site, and one molecule of either DIDS or PLP is obviously required per substrate binding site for the inactivation of the translocation process. In the functionally active dimeric translocator protein only one substrate-binding site appears to be accessible at a particular time, indicating that the site might be exposed to each side of the membrane in turn. Using [³H]-H₂DIDS for the labelling of maize mesophyll envelopes the radioactivity was found to be associated with two polypeptides of about 29 and 30 kDa. Since Western-blot analysis showed that only the 30 kDa polypeptide reacted with an antiserum directed against the spinach phosphate translocator protein it is suggested that this polypeptide presumably represents the phosphate translocator from maize mesophyll chloroplasts.Abbreviations DIDS 4,4-diisothiocyanostilbene-2,2-disulfonic acid - PEP phosphoenolpyruvate - 2-,3-PGA 2-,3-phosphoglycerate - PLP pyridoxal-5-phosphate - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis - TNBS 2,4,6-trinitrobenzenesulfonic acid - triose P triose phosphate This work was supported by the Deutsche Forschungsgemeinschaft     

The onset of sucrose accumulation in cold-stored potato tubers is caused by an increased rate of sucrose synthesis and coincides with low levels of hexose-phosphates, an activation of sucrose phosphate synthase and the appearance of a new form of amylase

These experiments investigate events involved in triggering sugar accumulation in the cold in tubers of Solanum tuberosum L. cv. Desirée. Sugar content, ¹⁴C-glucose metabolism, metabolite levels and activities of sucrose phosphate synthase (SPS) and starch-degrading enzymes were followed after transfer to 4°C. (i) Net sucrose accumulation began between 2 and 4 d. By 10 d, reducing sugars were also increasing. From 20 d onwards, sugar accumulation slowed. Sucrose fell, but reducing sugars continued to increase. (ii) To measure unidirectional sucrose synthesis, U-[¹⁴C]glucose was injected into tubers after various times at 4°C. The tubers were then incubated for 6 h. After 1 d at 4°C, both the absolute and the relative (expressed as a percentage of the metabolized label) rates of sucrose synthesis decreased compared to those at 20°C. Between 2 and 4 d at 4°C, labelling of sucrose increased 3-fold, to over 60% of the metabolized label. This high rate was maintained for up to 50 d in cold storage. When tissue slices were incubated with 2.5 mol m^?3 U-[¹⁴C]glucose, the rate of labelling of sucrose in slices from 6 d cold-stored material was higher than in slices from warm-stored material, irrespective of whether the incubation occurred at 4°C or at 20°C. (iii) Hexose-phosphates increased during the first day after transfer to 4°C. Their levels fell during the next 3 d, as sucrose synthesis increased. They then rose (until 20 d) and fell, in parallel with the rise and decline of sucrose levels. UDPglucose remained unaltered during the first 4 d, and then increased and decreased in parallel with sucrose. (iv) SPS activity assayed in optimal conditions and the total amount of SPS protein did not change. However, when assayed in the presence of phosphate and limiting substrate concentrations, activity rose 3–5-fold between 2 and 4 d. (v) Amylases and phosphorylases were investigated using zymograms to separate isoforms. Phosphorylases did not change. Between 2 and 4 d at 4°C, a new amylolytic activity appeared. (vi) Estimates of the specific activity of the phosphorylated intermediates and the absolute rate of sucrose synthesis (calculated from the ¹⁴C-labelling data and metabolite analysis) showed that changed kinetic properties of SPS and decreased levels of hexose-phosphate are accompanied by a 6–8-fold stimulation of sucrose synthesis. They also show that the final level of sugar is partly determined by a cycle of sugar synthesis and degradation. (vii) It is concluded that the onset of sugar accumulation in cold-stored tubers is initiated by a change in the kinetic properties of SPS and the appearance of a new amylolytic activity. It is discussed how other factors, including hexose-phosphate levels and subcellular compartmentalization, could also influence the final levels of sugars by altering the balance of sugar synthesis and remobilization.     

Leaching potential of heavy metals (Cd, Ni, Pb, Cu and Zn) from acidic sandy soil amended with dolomite phosphate rock (DPR) fertilizers

There is an increasing concern on heavy metal leaching from the soils amended with sewage sludge. A column study was conducted to examine the extent of leaching of five important heavy metals (Cd, Ni, Pb, Cu and Zn) from an acidic sandy soil amended with different dolomite phosphate rock (DPR) fertilizers (an application rate of 1% fertilizers) developed from DPR and N-Viro (consisting of biosolids and fly ash) at 0%, 10%, 20%, 30%, 40%, 50% and 100% DPR. Ten leaching events were carried out with each event done at an interval of 7 days and with total leaching volume of 1183mm, which is equivalent to the mean annual rainfall of this region during the period of 2001-2003. Leachate was collected after each leaching event and analyzed for heavy metals. The maximum leachate concentrations of Cd, Ni, Pb, Cu and Zn were all below drinking water quality guidance limits set by Florida Department of Environmental Protection and World Health Organization, suggesting that the application of DPR fertilizers may not pose a threat to water quality by leaching. Most of leachate concentrations of Cd, Ni and Pb were below their detection limits and there were no significant differences between the control and the treatments with different DPR fertilizers. By contrast, there were higher leachate concentrations of Cu and Zn (ranging from 0.7 to 37.1mug Cu/l and 5.1 to 205.6mug Zn/l for all treatments) due to their higher contents in both the soil and different DPR fertilizers compared with Cd, Ni and Pb. The leachate concentrations of Cu and Zn for each treatment decreased with increasing leaching events. The differences in leachate concentrations of Cu and Zn between the control and the treatments with different DPR fertilizers containing N-Viro were significant, especially in the first several leaching events and, moreover, they increased with increasing proportion of N-Viro in the DPR fertilizers. There were similar trends in total losses of Cu and Zn after ten leaching events. Greater differences in both leachate concentrations and total losses of Zn between the control and the treatments containing N-Viro were noted. Total losses of Zn for the treatments containing N-Viro were 3.0-5.1 times higher than those for the control compared with 1.4-2.2 times higher for total losses of Cu, suggesting that greater proportions of Zn losses came from the DPR fertilizers due to the greater mobility of Zn in the DPR fertilizers compared with Cu.     

Metastatic potential of B16-F10 melanoma cells is enhanced by extracellular S100A4 derived from RAW264.7 macrophages

S100A4, synthesized and secreted from both tumor and stroma cells, modulates an aggressive tumor phenotype in various cancers by intracellular and extracellular interactions which are not completely understood. Because of the high content of tumor-associated macrophages in melanoma, here, a syngeneic model (coculture of mouse B16-F10 melanoma cells (Mel) and RAW264.7 macrophages (M?); administration (i.v.) of Mel and M?/Mel in NMRI nu/nu mice) was used to investigate synthesis and secretion of (a) S100A4, (b) S100A4-mediated signaling and activation of NFκB, and (c) S100A4-mediated modulation of Mel invasiveness in vitro (transwell assay, transwell matrigel assay) and in vivo (metastatic lung colonization), respectively. In this model substantial S100A4 synthesis and secretion is demonstrated in M?. Macrophage-derived S100A4 promotes Mel invasiveness in a paracrine manner in vitro, which is further substantiated in control experiments using recombinant human S100A4 and Mel stably transfected with mouse S100A4. Moreover, the participation of S100A4-mediated signaling, e.g., via the receptor for advanced glycation endproducts (RAGE), resulting in activation of NFκB was demonstrated in all experimental settings. Finally, we demonstrated that interaction of macrophage-derived S100A4 with Mel results in increased metastatic lung colonization in vivo.     

Inorganic phosphate uptake in Trypanosoma cruzi is coupled to K cycling and to active Na extrusion

Background

Orthophosphate (P_i) is a central compound in the metabolism of all organisms, including parasites. There are no reports regarding the mechanisms of P_i acquisition by Trypanosoma cruzi.

Methods

³²P_i influx was measured in T. cruzi epimastigotes. The expression of P_i transporter genes and the coupling of the uptake to Na⁺, H⁺ and K⁺ fluxes were also investigated. The transport capacities of different evolutive forms were compared.

Results

Epimastigotes grew significantly more slowly in 2 mM than in 50 mM P_i. Influx of P_i into parasites grown under low P_i conditions took place in the absence and presence of Na⁺. We found that the parasites express TcPho84, a H⁺:P_i-symporter, and TcPho89, a Na⁺:P_i-symporter. Both P_i influx mechanisms showed Michaelis–Menten kinetics, with a one-order of magnitude higher affinity for the Na⁺-dependent system. Collapsing the membrane potential with carbonylcyanide-p-trifluoromethoxyphenylhydrazone strongly impaired the influx of P_i. Valinomycin (K⁺ ionophore) or SCH28028 (inhibitor of (H⁺ + K⁺)ATPase) significantly inhibited P_i uptake, indicating that an inwardly-directed H⁺ gradient energizes uphill P_i entry and that K⁺ recycling plays a key role in P_i influx. Furosemide, an inhibitor of the ouabain-insensitive Na⁺-ATPase, decreased only the Na⁺-dependent P_i uptake, indicating that this Na⁺ pump generates the Na⁺ gradient utilized by the symporter. Trypomastigote forms take up P_i inefficiently.

Conclusions

P_i starvation stimulates membrane potential-sensitive P_i uptake through different pathways coupled to Na⁺ or H⁺/K⁺ fluxes.

General significance

This study unravels the mechanisms of P_i acquisition by T. cruzi, a key process in epimastigote development and differentiation to trypomastigote forms.     

Mineral Release Dynamics of Tricalcium Phosphate and Waste Muscovite by Mineral-Solubilizing Rhizobacteria Isolated from Indo-Gangetic Plain of India

The aim of the present investigation was to evaluate the mineral release abilities of ten rhizobacterial strains isolated from rhizosphere of various crops growing in the Indo-Gangetic Plain (IGP) of India. Their abilities to solubilize inorganic phosphorus (P) and potassium (K) minerals from insoluble tricalcium phosphate (TCP) and waste muscovite (WM) revealed that rhizobacteria significantly solubilized different levels of inorganic P and fixed K, respectively. Some of the rhizobacterial stains have the ability to produce ammonia, indole-3-acetic acid (IAA), and hydrogen cyanide (HCN). The identification based on the 16S rDNA gene sequencing of selected mineral-solubilizing rhizobacteria (MSR) having greater potential to serve as bioagents were identified as Bacillus subtilis (BRHU01, BRHU03, and BHU20), Bacillus tequilensis (BRHU02), Bacillus licheniformis (BRHU04), Bacillus pumilus (BRHU05), Bacillus flexus (BHU02), Brevibacillus formosus (BHU16), Bacillus methylotrophicus (BHU29), and Bacillus amyloliquefaciens (BHU30). Interestingly, inorganic P and K solubilization by these strains belonging to genera Bacillus and Brevibacillus showed significant variations from 0.52 to 14.49 and from 1.62 to 8.60 µg mL^?1, respectively. However, generally, pH values of culture media decreased from near neutral (6.43) to acidic (3.83) with increasing incubation period, and this was inversely correlated with quantities of K solubilized by these rhizobacterial strains. Meanwhile, the electrical conductivity (EC) of broth culture increased from 0.09 to 0.23 dS m^?1 with increasing incubation period. Principal component analysis (PCA) of the MSR revealed three clusters, which exhibited high variance with respect to nutrient release. Taken together, these results suggest that Bacillus and Brevibacillus sp. identified in this study solubilized varying levels of inorganic P and fixed K from insoluble TCP and WM by acidolysis mechanisms.     

Similarity of the carbohydrate moieties of fibronectins derived from blood plasma and synthesised by cultured endothelial cells

Plasma and cellular fibronectins are reported to be very similar but not identical in chemical structure. We have compared bovine plasma fibronectin with fibronectin secreted by bovine aortal endothelial cells in culture. Techniques were chosen to highlight likely structural differences, particularly in the carbohydrate moieties. Both fibronectins were wholly reactive to monospecific antiserum and behaved identically in two-dimensional gel electrophoresis. The oligosaccharide chains were identical in proportion and degree of sialylation by anion-exchange HPLC. Fractionation of the glycopeptides on immobilised lectins and serotonin showed that both fibronectins contained (i)_predominantly biantennary oligosaccharides, (ii) exclusively N-acetylneuraminic acid residues in a non-clustered array and (iii) no L-fucose residues. The overriding structural similarities support the proposal that the endothelium is a site of synthesis of plasma fibronectin in vivo.     

When growing potato tubers are detached from their mother plant there is a rapid inhibition of starch synthesis,involving inhibition of ADP-glucose pyrophosphorylase

Labelling experiments in which high-specific-activity [U-¹⁴C]sucrose or [U-¹⁴C]hexoses were injected into potato (Solanum tuberosum L. cv. Desiree) tubers showed that within 1 d of detaching growing tubers from their mother plant, there is an inhibition of starch synthesis, a stimulation of the synthesis of other major cell components, and rapid resynthesis of sucrose. This is accompanied by a general increase in phosphorylated intermediates, an increase in UDP-glucose, and a dramatic decrease of ADP-glucose. No significant decline in the extracted activity of enzymes for sucrose degradation or synthesis, or starch synthesis is seen within 1 d, nor is there a significant decrease in sucrose, amino acids, or fresh weight. Over the next 7 d, soluble carbohydrates decline. This is accompanied by a decline in sucrose-synthase activity, hexose-phosphate levels, and the synthesis of structural cell components. It is argued that a previously unknown mechanism acting at ADP-glucose pyrophosphorylase allows sucrose-starch interconversions to be regulated independently of the use of sucrose for cell growth.