Ascorbic acid conjugates isolated from the phloem of Cucurbitaceae

Analysis of phloem exudates from the fruit of Cucurbitaceae revealed the presence of several compounds with UV-visible absorption spectra identical to that of l-ascorbic acid. In Cucurbita pepo L. (zucchini), the compounds could be isolated from phloem exudates collected from aerial parts of the plant but were not detected in whole tissue homogenates. The compounds isolated from the phloem exudates of C. pepo fruit were eluted from strong anion exchange resin in the same fraction as l-ascorbic acid and were oxidised by ascorbate oxidase (E.C. 1.10.3.3). The major compound purified from C. pepo fruit exudates demonstrated similar redox properties to l-ascorbic acid and synthetic 6-O-glucosyl-l-ascorbic acid (6-GlcAsA) but differed from those of 2-O-glucosyl-l-ascorbic acid (2-GlcAsA) isolated from the fruit of Lycium barbarum L. Parent and fragment ion masses of the compound were consistent with hexosyl-ascorbate in which the hexose moiety was attached to C5 or C6 of AsA. Acid hydrolysis of the major C. pepo compound resulted in the formation of l-ascorbic acid and glucose. The purified compound yielded a proton NMR spectrum that was almost identical to that of synthetic 6-GlcAsA. A series of l-ascorbic acid conjugates have, therefore, been identified in the phloem of Cucurbitaceae and the most abundant conjugate has been identified as 6-GlcAsA. The potential role of such conjugates in the long-distance transport of l-ascorbic acid is discussed.     

Evidence for graft transmission of structural phloem proteins or their precursors in heterografts of Cucurbitaceae

Interspecific and intergeneric grafts of Cucurbitaceae were used to study the mobility of structural P-proteins in the phloem. When Cucumis sativus L. scions were grafted onto Cucurbita rootstocks, at least nine additional proteins appeared on sodium dodecyl sulfate-polyacrylamide electrophoresis gels of scion exudate, 9–11 d after grafting. These proteins corresponded exactly to those of the respective Cucurbita sp. rootstock, including the filament-forming phloem protein PP1 and the phloem lectin PP2, as shown by the apparent molecular weights and peptide maps. According to probing at three sites, the additional proteins were evenly distributed within the scion. The appearance of additional proteins was correlated with the establishment of phloem bridges across the graft union. The developmental coincidence establishes that the structural proteins or their precursors are translocated in the phloem. This translocation was a universal phenomenon in Cucurbitaceae as shown by a comparative screening for additional proteins in eleven graft combinations, using Benincasa hispida (Thunb.) Cogn., Citrullus colocynthis (L.) Schrad., Cucumis melo L, C. sativus, Cucurbita ficifolia Bouché, Cucurbita maxima Duchesne ex Lam., and Trichosanthes cucumerina var. lobata Roxb. According to this screening, the direction of transmission of additional proteins depended upon the combination tested. While some graft partners failed to show exchange, some behaved as “donor” for additional proteins and still others could be both “donor” or “acceptor”. However, whether used as scion or stock, C. sativus was consistently identified as an acceptor. The occurrence of additional proteins in heterografts is discussed with regard to the transport mechanism of structural P-proteins in the phloem and its relationship to assimilate transport. Received: 18 February 1998 / Accepted: 12 May 1998     

Relatedness between Streptococcus pneumoniae and viridans streptococci: transfer of penicillin resistance determinants and immunological similarities of penicillin-binding proteins

The occurrence of highly variable penicillin-binding proteins (PBPs) in penicillin-resistant Streptococcus pneumoniae suggested that transfer of homologous genes from related species may be involved in resistance development. Antiserum and monoclonal antibodies raised against PBPs 1a and 2b from the susceptible S. pneumoniae R6 strain were used to identify related PBPs in 41 S. mitis, S. sanguis I and S. sanguis II strains mostly isolated in South Africa with MIC values ranging from less than 0.15 to 16 mg/ml. Furthermore, the possibility of genetic exchange was examined with 30 penicillin-resistant strains of this collection (MIC greater than 0.06 mg/ml) as donors using S. pneumoniae R6 as recipient in transformation experiments. The majority of S. mitis and S. sanguis II strains but none of the S. sanguis I strains could transform penicillin resistance genes into S. pneumoniae R6. All positive donor strains and all susceptible isolates of S. mitis and S. sanguis II strains contained PBPs which cross-reacted with the anti-PBP 1a and/or anti-PBP 2b antibodies. On the other hand, only five of the 14 S. sanguis I strains contained a PBP that reacted with one of the antibodies. This strongly suggested the presence of genes homologous to the pneumococcal PBP 1a and 2b genes in viridans streptococci, and documents that penicillin resistance determinants can be transformed from viridans streptococci into the pneumococcus.     

Intriguing similarities between two novel odorant-binding proteins of locusts

Two novel odorant-binding proteins (OBPs) of locust, LmigOBP2 and LmigOBP3 are very different from each other and from the previously reported LmigOBP1 in their amino acid sequences. Moreover, OBP3 contains three additional cysteines, a fact not previously recorded in standard length OBPs. However, these two proteins exhibit remarkably similar binding affinities to a set of organic compounds. Such behaviour is supported by three-dimensional models, showing very similar folding for LmigOBP2 and LmigOBP3, but clearly different for LmigOBP1. Also several amino acid residues lining the binding pockets of the three proteins appear conserved in LmigOBP2 and LmigOBP3, but not in LmigOBP1. Western blot experiments revealed the presence of LmigOBP2 in antennae, mouth parts and cerci, but could not detected LmigOBP3 in any of these tissues. In immunocytochemistry, antibodies against LmigOBP2 strongly stained the outer lymph of sensilla chaetica of the antennae, in contrast with LmigOBP1, previously reported in sensilla basiconica.     

Isolation and characterization of ribosome-inactivating proteins from Cucurbitaceae

Due to their RNA-N-glycosidase activity, ribosome-inactivating proteins (RIPs) are attractive candidates as antitumor and antiviral agents in biomedical and agricultural research. We have isolated and characterized two such proteins, foetidissimin II and texanin, from two Cucurbitaceae species. Foetidissimin II, obtained from the roots of Cucurbita foetidissima, was identified as a type-2 RIP, with a molecular weight of 61 kDa, as estimated by gel electrophoresis. It is composed of two chains, a 29-kDa chain A, and a 32-kDa chain B. Texanin, isolated from the fruits of Cucurbita texana, is a type-I RIP, with a single chain of molecular weight 29.7 kDa, as estimated by MALDI-TOF-MS. Both proteins exhibit RNA-N-glycosidase activity, with aniline playing a critical role in rRNA cleavage. The IC50 value of foetidissimin II, determined by cell-free protein-synthesis inhibition, was 0.251 muM. In an in vitro cytotoxicity assay, foetidissimin II exhibited IC50 values of ca. 70 nM to both adenocarcinoma and erythroleukemia cells. Texanin exhibited a weaker anticancer activity against erythroleukemia cells, with an IC50 value of 95 microM, but no activity against adenocarcinoma cells. The N-terminal sequences of both proteins were compared with those of reported RIPs.     

Structural differences between trypsin-inhibitors isolated from Cucurbitaceae family seeds: immunological, NMR and CD studies

By manifold immunizations of rabbits with virgin or modified trypsin inhibitor III from squash seeds and trypsin inhibitor II b from cucumber seeds, specific antibodies were produced. In double immunodiffusion the anti-squash inhibitor antibody also gave weak precipitate arcs with inhibitor I from squash, inhibitor II from summer squash and with inhibitor I from zucchini, but not with inhibitor II b from cucumber seeds. The genus of Cucurbita trypsin inhibitors, preincubated with the antibody, lost their antitrypsin activity. The antibody showed a significantly weaker effect on the activity of the inhibitor from cucumber sees. 1H-NMR and CD spectra also confirm structural differences between trypsin inhibitors from the genus of Cucurbita and the cucumber (genus of Cucumis) inhibitor.     

Finding weak similarities between proteins by sequence profile comparison

To improve the recognition of weak similarities between proteins a method of aligning two sequence profiles is proposed. It is shown that exploring the sequence space in the vicinity of the sequence with unknown properties significantly improves the performance of sequence alignment methods. Consistent with the previous observations the recognition sensitivity and alignment accuracy obtained by a profile–profile alignment method can be as much as 30% higher compared to the sequence–profile alignment method. It is demonstrated that the choice of score function and the diversity of the test profile are very important factors for achieving the maximum performance of the method, whereas the optimum range of these parameters depends on the level of similarity to be recognized.     

Chemical and immunological characterizations of equine infectious anemia virus gag-encoded proteins.

The viral core proteins (p15, p26, p11, and p9) of equine infectious anemia virus (EIAV) (Wyoming strain) were purified by reverse-phase high-pressure liquid chromatography. Each purified protein was analyzed for amino acid content, N-terminal amino acid sequence, C-terminal amino acid sequence, and phosphoamino acid content. The results of N- and C-terminal amino acid sequence analysis of each gag protein, taken together with the nucleotide sequence of the EIAV gag gene (R. M. Stephens, J. W. Casey, and N. R. Rice, Science 231:589-594, 1986), show that the order of the proteins in the precursor is p15-p26-*-p11-p9, where a pentapeptide also found in the virus is represented by the asterisk. The data are in complete agreement with the predicted structure of the gag polyprotein and show the peptide bonds cleaved during proteolytic processing. The N-terminus of p15 is blocked to Edman degradation. The p11 protein is identical to the nucleic acid-binding protein of EIAV previously isolated (C. W. Long, L. E. Henderson, and S. Oroszlan, Virology 104:491-496, 1980). High-titer rabbit antiserum was prepared against each purified protein. These antisera were used to detect the putative gag precursor (Pr55gag) and intermediate cleavage products designated Pr49 (p15-p26-*-p11), Pr40 (p15-p26), and Pr35 (p26-*-p11) in the virus and in virus-infected cells. High-titer antisera to EIAV p15 and p26 showed cross-reactivity with the homologous protein of human T-cell lymphotropic virus type III/lymphadenopathy-associated virus.     

Destination-selective long-distance movement of phloem proteins

The phloem macromolecular transport system plays a pivotal role in plant growth and development. However, little information is available regarding whether the long-distance trafficking of macromolecules is a controlled process or passive movement. Here, we demonstrate the destination-selective long-distance trafficking of phloem proteins. Direct introduction, into rice (Oryza sativa), of phloem proteins from pumpkin (Cucurbita maxima) was used to screen for the capacity of specific proteins to move long distance in rice sieve tubes. In our system, shoot-ward translocation appeared to be passively carried by bulk flow. By contrast, root-ward movement of the phloem RNA binding proteins 16-kD C. maxima phloem protein 1 (CmPP16-1) and CmPP16-2 was selectively controlled. When CmPP16 proteins were purified, the root-ward movement of CmPP16-1 became inefficient, suggesting the presence of pumpkin phloem factors that are responsible for determining protein destination. Gel-filtration chromatography and immunoprecipitation showed that CmPP16-1 formed a complex with other phloem sap proteins. These interacting proteins positively regulated the root-ward movement of CmPP16-1. The same proteins interacted with CmPP16-2 as well and did not positively regulate its root-ward movement. Our data demonstrate that, in addition to passive bulk flow transport, a destination-selective process is involved in long-distance movement control, and the selective movement is regulated by protein-protein interaction in the phloem sap.     

Structural and functional similarities between the nucleotide-binding domains of CFTR and GTP-binding proteins.

The opening and closing of the CFTR Cl- channel are regulated by ATP hydrolysis at its two nucleotide binding domains (NBDs). However, the mechanism and functional significance of ATP hydrolysis are unknown. Sequence similarity between the NBDs of CFTR and GTP-binding proteins suggested the NBDs might have a structure and perhaps a function like that of GTP-binding proteins. Based on this similarity, we predicted that the terminal residue of the LSGGQ motif in the NBDs of CFTR corresponds to a highly conserved glutamine residue in GTP-binding proteins that directly catalyzes the GTPase reaction. Mutations of this residue in NBD1 or NBD2, which were predicted to increase or decrease the rate of hydrolysis, altered the duration of channel closed and open times in a specific manner without altering ion conduction properties or ADP-dependent inhibition. These results suggest that the NBDs of CFTR, and consequently other ABC transporters, may have a structure and a function analogous to those of GTP-binding proteins. We conclude that the rates of ATP hydrolysis at NBD1 and at NBD2 determine the duration of the two states of the channel, closed and open, much as the rate of GTP hydrolysis by GTP-binding proteins determines the duration of their active state.     

Structural similarities between corresponding heat-shock proteins from different eucaryotic cells

Effects of heat treatments on chick embryo fibroblasts, Drosophila embryonic cells, and human lymphoblastoid cells have been compared. Cells from all three species synthesize large heat-shock proteins (hsps) with Mr = 70,000 and 84,000-85,000. Different small hsps with Mr between 22,000 and 27,000 are made at high rates in heat-treated chicken and Drosophila cells but could not be observed in human cells. The structural features of the large hsps from cells of the different organisms were compared by three methods of peptide mapping, namely the examination of tryptic digests by two-dimensional thin layer chromatography or by high pressure liquid chromatography and of incomplete V8 digests by polyacrylamide gel electrophoresis. The Mr = 84,000-85,000 polypeptides from all three organisms are closely related, the chicken and human polypeptides having many peptides in common. The relationship between the Mr = 70,000 polypeptides of the different organisms appears to be less close; possible explanations for this latter result are discussed. Rates of synthesis of total as well as poly(A)+ RNA are much lower in heat-treated than in untreated cells of all three organisms. Heat treatments induce dramatic changes in the shape of chick embryo fibroblasts as seen by microscopic examination. Human lymphoblastoid cells do not show changes in shape.     

Sequence and functional similarities between pro-apoptotic Bid and plant lipid transfer proteins

Pro-apoptotic proteins of the Bcl-2 family are known to act on mitochondria and facilitate the release of cytochrome c, but the biochemical mechanism of this action is unknown. Association with mitochondrial membranes is likely to be important in determining the capacity of releasing cytochrome c. The present work provides new evidence suggesting that some pro-apoptotic proteins like Bid have an intrinsic capacity of binding and exchanging membrane lipids. Detailed analysis indicates a significant sequence similarity between a subset of Bcl-2 family proteins including Bid and Nix and plant lipid transfer proteins. The similar structural signatures could be related to common interactions with membrane lipids. Indeed, isolated Bid shows a lipid transfer activity that is even higher than that of plant lipid transfer proteins. To investigate the possible relevance of these structure-function correlations to the apoptotic action of Bid, cell free assays were established with isolated mitochondria, recombinant Bid and a variety of exogenous lipids. Micromolar concentrations of lysolipids such as lysophosphatidylcholine were found to change the association of Bid with mitochondria and also stimulate the release of cytochrome c promoted by Bid. The changes in mitochondrial association and cytochrome c release were enhanced by the presence of liposomes of lipid composition similar to that of mitochondrial membranes. Thus, a mixture of liposomes, mitochondria and key lysolipids could reproduce the conditions enabling Bid to transfer lipids between donor and acceptor membranes, and also change its reversible association with mitochondria. Bid was also found to enhance the incorporation of a fluorescent lysolipid, but not of a related fatty acid, into mitochondria. On the basis of the results presented here, it is hypothesised that Bid action may depend upon its capacity of exchanging lipids and lysolipids with mitochondrial membranes. The hypothesis is discussed in relation to current models for the integrated action of pro-apoptotic proteins of the Bcl-2 family.     

Sequence and functional similarities between pro-apoptotic Bid and plant lipid transfer proteins

Pro-apoptotic proteins of the Bcl-2 family are known to act on mitochondria and facilitate the release of cytochrome c, but the biochemical mechanism of this action is unknown. Association with mitochondrial membranes is likely to be important in determining the capacity of releasing cytochrome c. The present work provides new evidence suggesting that some pro-apoptotic proteins like Bid have an intrinsic capacity of binding and exchanging membrane lipids. Detailed analysis indicates a significant sequence similarity between a subset of Bcl-2 family proteins including Bid and Nix and plant lipid transfer proteins. The similar structural signatures could be related to common interactions with membrane lipids. Indeed, isolated Bid shows a lipid transfer activity that is even higher than that of plant lipid transfer proteins. To investigate the possible relevance of these structure-function correlations to the apoptotic action of Bid, cell free assays were established with isolated mitochondria, recombinant Bid and a variety of exogenous lipids. Micromolar concentrations of lysolipids such as lysophosphatidylcholine were found to change the association of Bid with mitochondria and also stimulate the release of cytochrome c promoted by Bid. The changes in mitochondrial association and cytochrome c release were enhanced by the presence of liposomes of lipid composition similar to that of mitochondrial membranes. Thus, a mixture of liposomes, mitochondria and key lysolipids could reproduce the conditions enabling Bid to transfer lipids between donor and acceptor membranes, and also change its reversible association with mitochondria. Bid was also found to enhance the incorporation of a fluorescent lysolipid, but not of a related fatty acid, into mitochondria. On the basis of the results presented here, it is hypothesised that Bid action may depend upon its capacity of exchanging lipids and lysolipids with mitochondrial membranes. The hypothesis is discussed in relation to current models for the integrated action of pro-apoptotic proteins of the Bcl-2 family.     

Effects of the hydraulic coupling between xylem and phloem on diurnal phloem diameter variation

Measurements of diurnal diameter variations of the xylem and phloem are a promising tool for studying plant hydraulics and xylem-phloem interactions in field conditions. However, both the theoretical framework and the experimental verification needed to interpret phloem diameter data are incomplete. In this study, we analytically evaluate the effects of changing the radial conductance between the xylem and the phloem on phloem diameter variations and test the theory using simple manipulation experiments. Our results show that phloem diameter variations are mainly caused by changes in the radial flow rate of water between the xylem and the phloem. Reducing the hydraulic conductance between these tissues decreases the amplitude of phloem diameter variation and increases the time lag between xylem and phloem diameter variation in a predictable manner. Variation in the amplitude and timing of diameter variations that cannot be explained by changes in the hydraulic conductance, could be related to changes in the osmotic concentration in the phloem.     

Cell wall proteins of flax phloem fibers

A cell wall has been isolated from single-type cells, phloem fibers of flax (Linum usitatissimum L.) being at the stage of the active formation of a thick secondary cell wall. Weakly bound proteins of the cell wall of phloem fibers were extracted and separated, and their mass spectra were recorded. The identification of the proteins and their assignment to a particular cell compartment were performed using a variety of bioinformatics methods. In all, 93 proteins were identified of which many proteins were defined as predicted, putative, and hypothetical. Twenty one proteins were identified as cell-wall proteins. The absence of the marker proteins of primary cell walls such as xyloglucan endotransglycosylase and expansins indirectly confirms the predominance of the secondary cell wall in a sample for protein extraction.     

Phytomonas serpens: immunological similarities with the human trypanosomatid pathogens

The present review provides an overview of recent discoveries concerning the immunological similarities between Phytomonas serpens, a tomato parasite, and human trypanosomatid pathogens, with special emphasis on peptidases. Leishmania spp. and Trypanosoma cruzi express peptidases that are well-known virulence factors, named leishmanolysin and cruzipain. P. serpens synthesizes two distinct classes of proteolytic enzymes, metallo- and cysteine-type peptidases, that share common epitopes with leishmanolysin and cruzipain, respectively. The leishmanolysin-like and cruzipain-like molecules from P. serpens participate in several biological processes including cellular growth and adhesion to the salivary glands of Oncopeltus fasciatus, a phytophagous insect experimental model. Since previous reports demonstrated that immunization of mice with P. serpens induced a partial protective immune response against T. cruzi, this plant trypanosomatid may be a suitable candidate for vaccine studies. Moreover, comparative approaches in the Trypanosomatidae family may be useful to understand kinetoplastid biology, biochemistry and evolution.