Polygalacturonase-inhibiting protein accumulates in Phaseolus vulgaris L. in response to wounding, elicitors and fungal infection

Polygalacturonase-inhibiting protein (PGIP) is a cell wall-associated protein that specifically binds to and inhibits the activity of fungal endopolygalacturonases. The Phaseolus vulgaris gene encoding PGIP has been cloned and characterized. Using a fragment of the cloned pgip gene as a probe in Northern blot experiments, it is demonstrated that the pgip mRNA accumulates in suspension-cultured bean cells following addition of elicitor-active oligogalacturonides or fungal glucan to the medium. Rabbit polyclonal antibodies specific for PGIP were generated against a synthetic peptide designed from the N-terminal region of PGIP; the antigenicity of the peptide was enhanced by coupling to KLH. Using the antibodies and the cloned pgip gene fragment as probes in Western and Northern blot experiments, respectively, it is shown that the levels of PGIP and its mRNA are increased in P. vulgaris hypocotyls in response to wounding or treatment with salicylic acid. Using gold-labeled goat-anti-rabbit secondary antibodies in EM studies, it has also been demonstrated that, in bean hypocotyls infected with Colletotrichum lindemuthianum, the level of PGIP preferentially increases in those cells immediately surrounding the infection site. The data support the hypothesis that synthesis of PGIP constitutes an active defense mechanism of plants that is elicited by signal molecules known to induce plant defense genes.     

Association of a 76 kDa polypeptide with soluble starch synthase I activity in maize (cv B73) endosperm

Soluble starch synthase (SSS) I was purified 361-fold from hand-dissected endosperm tissue of inbred maize (Zea mays, cv. B73) to specific activities ranging between 5 and 9 µmol min⁻¹ mg⁻¹. A key to this purification protocol was the introduction of a size-exclusion chromatography step, a size-based fractionation which provided abundant levels of desalted SSS forms I and II. The native molecular masses calculated for SSS forms I and II were 75.5 kDa and 180 kDa, respectively. SSSI was then further purified by hydrophobic interaction chromatography on Phenyl-Superose and by FPLC on Mono Q. Analysis of column peaks by SDS—PAGE and scanning densitometry revealed that a 76 kDa polypeptide is strongly correlated with SSSI activity. Antibodies were then generated against a 76 kDa polypeptide extracted from starch granules. These antibodies, which were monospecific for the soluble 76 kDa polypeptide, neutralized greater than 90% of SSSI activity, and precipitated the 76 kDa protein. These results establish the 76 kDa protein as an SSSI in the B73 line of inbred maize. An immunologically similar 76 kDa protein also appears to be tightly associated with the starch granule.     

A (1→3,1→4)-β-glucan-specific monoclonal antibody and its use in the quantitation and immunocytochemical location of (1→3,1→4)-β-glucans

Monoclonal antibodies were raised against a (1→3,1→4)-β-glucan-bovine serum albumin (BSA) conjugate. One antibody (BG1) selected for further characterization, was specific for (1→3,1→4)-β-glucan, displaying no binding activity against a (1→3)-β-glucan-BSA conjugate and minimal binding against a cellopentaose-BSA conjugate. A range of oligosaccharides was prepared by enzymatic digestion of (1→3,1→4)-β-glucan, purified by size exclusion chromatography and characterized by ¹H-NMR and anion exchange chromatography. These (1→3,1→4)-β-oligoglucosides, together with (1→3)-β- and (1→4)-β-oligoglucosides were used to characterize the binding site of the monoclonal antibody (BG1) by competitive inhibition. The monoclonal antibody showed maximal binding to a heptasaccharide with the structure Glc(1→3) Glc(1→4) Glc(1→4) Glc(1→3) Glc(1→4) Glc(1→4) Glc and was determined to have an affinity constant of 3.8 × 10⁴ M⁻¹ for this oligoglucoside. The monoclonal antibody (BG1) has been used to develop a sensitive sandwich ELISA for the specific quantitation of (1→3,1→4)-β-glucans. The assay operates in the range 1–10 ng ml⁻¹ and shows no significant cross-reaction with tamarind xyloglucan, wheat endosperm arabinoxylan or carboxymethyl-pachyman ((1→3)-β-glucan). When used with a second-stage, rabbit anti-mouse gold conjugate and viewed under the electron microscope, the monoclonal antibody probe was found to bind strongly to the walls of the aleurone in thin sections of immature wheat (Triticum aestivum) cv. Millewa grains but not to the middle lamella region. A previously described specific anti-(1→3)-β-glucan antibody (Meikle et al., 1991) bound to discrete patches on the aleurone walls, believed to be plasmodesmata.     

Recruitment of helper T cells for induction of tumour rejection by cytolytic T lymphocytes

Immunotherapy of cancer could be possible in cases in which competent effector T cells can be induced. Such an approach depends on expression of tumour-specific antigens by the tumour cells and on the availability of sufficient costimulatory support for activation of cytotoxic T lymphocytes. Here, a strategy for helper T cell recruitment for induction of tumour-specific cytotoxic immune responses is presented. Allogenic MHC class II molecules were introduced into tumour cells by cell fusion. These hybrid cells, when injected into mice, induced rejection of an established tumour. The contribution of CD4-expressing helper T cells in the induction phase and of CD8-expressing T cells in the effector phase of the immune response was demonstrated. The approach described could be applicable to cases in which a suitable tumour antigen is present but not identified; it employs regulatory interactions that govern physiological immune responses and is designed to be minimally invasive.     

T cell recognition of melanoma antigens in association with HLA-A1 on allogeneic melanoma cells

Previous studies have shown that recognition of melanoma by cytotoxic T lymphocytes may be restricted by HLA-A1, A2 and other HLA antigens. The present study examined the cytotoxic specificity and major histocompatibility complex restriction of cloned cytotoxic T lymphocytes (CTL) isolated from a patient with the HLA phenotype A3,31 who had been immunized with a vaccine prepared from HLA-A1,3 melanoma cells. Cytotoxic assays against HLA-typed allogeneic melanoma cells indicated that cloned CTL from the patient were able to kill allogeneic melanoma cells expressing HLA-A1 but not other HLA-A1-positive cells. Studies on a representative clone indicated that proliferation and cytokine (tumour necrosis factor ) production in response to melanoma cells was also associated with HLA-A1 on melanoma cells. Response to the melanoma cells was associated with interleukin-4 (IL-4) rather than IL-2 production. The antigen recognized in the context of HLA-A1 on allogeneic melanoma cells was detected in cytotoxic assays on cells from 9 of 12 HLA-A1⁺ melanoma cell lines and did not appear to be the product of the MAGE-1 or-3 genes. These findings suggest that T cells can recognize melanoma antigens in the context of alloantigens and that allogeneic vaccines containing immunodominant alloantigens may generate CTL that are ineffective against autologous melanoma. The study does not, however, exclude the possibility that CTL with specificity to the latter may be activated by allogeneic vaccines, and further studies are needed to answer this question.     

Genes encoding homologues of three consecutive enzymes in the butyrate/butanol-producing pathway of Clostridium acetobutylicum are clustered on the Clostridium difficile chromosome

Abstract Screening of a Clostridium difficile ψEMBL3 gene library with antisera raised against C. difficile culture supernatant identified several clones expressing a 31-kDa protein. A 1.8-kb Hin dIII fragment subcloned from one of the clones was sufficient for expression of the 31-kDa polypeptide. Southern blot analysis showed a region homologous to this fragment to be present in all of 13 different C. difficile strains tested. Sequence analysis of the 1.8-kb fragment revealed three adjacent open reading frames. A database search showed that these three open reading frames appeared to encode homologues of three consecutive enzymes in the butanol/butyrate-producing pathway of Clostridium acetobutylicum (crotonase, β-hydroxybutyryl coenzyme A dehydrogenase and thiolase).     

Floral structure and evolution of primitive angiosperms: Recent advances

Concepts of primitive angiosperm flowers have changed in recent years due to new studies on relic archaic groups, new paleobotanical finds and the addition of molecular biological techniques to the study of angiosperm systematics and evolution.Magnoliidae are still the hot group, but emphasis is now on small primitive flowers with few organs and also on the great lability of organ number. Of the extant groups, a potential basal position of the paleoherbs has been discussed by some authors. Although some paleoherbs have a simple gynoecium with a single orthotropous ovule, anatropous ovules may still be seen as plesiomorphic in angiosperms. Anatropy is not necessarily a consequence of the advent of closed carpels. It may also exhibit biological advantages under other circumstances as is the case in podocarps among gymnosperms. Valvate anthers have now been found in most larger subgroups of theMagnoliidae (recently also in paleoherbs) and in some Cretaceous fossils. Nevertheless, as seen from its systematic distribution, valvate dehiscence is not necessarily plesiomorphic for the angiosperms, but may be a facultative by-product of the thick connectives and comparatively undifferentiated anther shape inMagnoliidae and lowerHamamelididae. A perianth is relatively simple in extantMagnoliidae or even wanting in some families. In groups with naked flowers the perianth may have been easily lost because integration in the floral architecture was less pronounced than in more advanced angiosperm groups. Problems with the comparison of paleoherb flowers with those ofGnetales are discussed. The rapid growth of information from paleobotany and molecular systematics requires an especially open attitude towards the evaluation of various hypotheses on early flower evolution in the coming years.     

Easy determination of ploidy level in Arabidopsis thaliana plants by means of pollen size measurement

Summary Cytogenetic examination of transgenic Arabidopsis thaliana (L.) Heynh. plants obtained by Agrobacterium-mediated gene transfer to cotyledon- and root-explants or by direct gene transfer into protoplasts revealed a high percentage of tetraploid or aneuploid transformants. Depending on the transformation procedure used, 13% (root explant transformation), 33% (cotyledon explant transformation), or 38% (direct gene transfer) of the transformants showed aberrant ploidy levels. A good correlation between the ploidy level of a plant and the size of its pollen grains was observed. This allows quick and simple testing of the ploidy level of transgenic Arabidopsis plants.Abbreviations AM Arabidopsis medium - ANOVA analysis of variance - DAPI 4,6-Diamidino-2-phenylindole - PEG polyethyleneglycol