The kinesin-like calmodulin binding protein is differentially involved in cell division.
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The kinesin-like calmodulin (CaM) binding protein (KCBP), a minus end-directed microtubule motor protein unique to plants, has been implicated in cell division. KCBP is negatively regulated by Ca(2)+ and CaM, and antibodies raised against the CaM binding region inhibit CaM binding to KCBP in vitro; therefore, these antibodies can be used to activate KCBP constitutively. Injection of these antibodies into Tradescantia virginiana stamen hair cells during late prophase induces breakdown of the nuclear envelope within 2 to 10 min and leads the cell into prometaphase. However, mitosis is arrested, and the cell does not progress into anaphase. Injection of antibodies later during cell division has no effect on anaphase transition but causes aberrant phragmoplast formation and delays the completion of cytokinesis by approximately 15 min. These effects are achieved without any apparent degradation of the microtubule cytoskeleton. We propose that during nuclear envelope breakdown and anaphase, activated KCBP promotes the formation of a converging bipolar spindle by sliding and bundling microtubules. During metaphase and telophase, we suggest that its activity is downregulated. (+info)
Evolution of C4 phosphoenolpyruvate carboxylase in Flaveria, a conserved serine residue in the carboxyl-terminal part of the enzyme is a major determinant for C4-specific characteristics.
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C4 phosphoenolpyruvate carboxylases have evolved from ancestral C3 isoforms during the evolution of angiosperms and gained distinct kinetic and regulatory properties compared with the C3 isozymes. To identify amino acid residues and/or domains responsible for these C4-specific properties the C4 phosphoenolpyruvate carboxylase of Flaveria trinervia (C4) was compared with its orthologue in the closely related C3 plant Flaveria pringlei. Reciprocal enzyme chimera were constructed and the kinetic constants, K(0.5) and k(cat), as well as the Hill coefficient, h, were determined for the substrate phosphoenolpyruvate both in the presence and absence of the activator glucose 6-phosphate. By this approach two regions were identified which determined most of the kinetic differences of the C4 and C3 ppcA phosphoenolpyruvate carboxylases with respect to the substrate PEP. In addition, the experiments suggest that the two regions do not act additively but interact with each other. The region between amino acids 296 and 437 is essential for activation by glucose 6-phosphate. The carboxyl-terminal segment between amino acids 645 and 966 contains a C4 conserved serine or a C3 invariant alanine at position 774 in the respective enzyme isoform. Site-directed mutagenesis shows that this position is a key determinant for the kinetic properties of the two isozymes. (+info)
Crystal structure of Urtica dioica agglutinin, a superantigen presented by MHC molecules of class I and class II.
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BACKGROUND: Urtica dioica agglutinin (UDA), a monomeric lectin extracted from stinging nettle rhizomes, is specific for saccharides containing N-acetylglucosamine (GlcNAc). The lectin behaves as a superantigen for murine T cells, inducing the exclusive proliferation of Vbeta8.3(+) lymphocytes. UDA is unique among known T cell superantigens because it can be presented by major histocompatibility complex (MHC) molecules of both class I and II. RESULTS: The crystal structure of UDA has been determined in the ligand-free state, and in complex with tri-acetylchitotriose and tetra-acetylchitotetraose at 1.66 A, 1.90 A and 1.40 A resolution, respectively. UDA comprises two hevein-like domains, each with a saccharide-binding site. A serine and three aromatic residues at each site form the principal contacts with the ligand. The N-terminal domain binding site can centre on any residue of a chito-oligosaccharide, whereas that of the C-terminal domain is specific for residues at the nonreducing terminus of the ligand. We have shown previously that oligomers of GlcNAc inhibit the superantigenic activity of UDA and that the lectin binds to glycans on the MHC molecule. We show that UDA also binds to glycans on the T cell receptor (TCR). CONCLUSIONS: The presence of two saccharide-binding sites observed in the structure of UDA suggests that its superantigenic properties arise from the simultaneous fixation of glycans on the TCR and MHC molecules of the T cell and antigen-presenting cell, respectively. The well defined spacing between the two binding sites of UDA is probably a key factor in determining the specificity for Vbeta8.3(+) lymphocytes. (+info)
Chemically prepared hevein domains: effect of C-terminal truncation and the mutagenesis of aromatic residues on the affinity for chitin.
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Chemically prepared hevein domains (HDs), N-terminal domain of an antifungal protein from Nicotiana tabacum (CBP20-N) and an antimicrobial peptide from Amaranthus caudatus (Ac-AMP2), were examined for their affinity for chitin, a beta-1,4-linked polymer of N-acetylglucosamine. An intact binding domain, CBP20-N, showed a higher affinity than a C-terminal truncated domain, Ac-AMP2. The formation of a pyroglutamate residue from N-terminal Gln of CBP20-N increased the affinity. The single replacement of any aromatic residue of Ac-AMP2 with Ala resulted in a significant reduction in affinity, suggesting the importance of the complete set of three aromatic residues in the ligand binding site. The mutations of Phe18 of Ac-AMP2 to the residues with larger aromatic rings, i.e. Trp, beta-(1-naphthyl)alanine or beta-(2-naphthyl)alanine, enhanced the affinity, whereas the mutation of Tyr20 to Trp reduced the affinity. The affinity of an HD for chitin might be improved by adjusting the size and substituent group of stacking aromatic rings. (+info)
Molecular and genetic analyses of the silky1 gene reveal conservation in floral organ specification between eudicots and monocots.
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The degree to which the eudicot-based ABC model of flower organ identity applies to the other major subclass of angrosperms, the monocots, has yet to be fully explored. We cloned silky1 (si1), a male sterile mutant of Zea mays that has homeotic conversions of stamens into carpels and lodicules into palea/lemma-like structures. Our studies indicate that si1 is a monocot B function MADS box gene. Moreover, the si1 zag1 double mutant produces a striking spikelet phenotype where normal glumes enclose reiterated palea/lemma-like organs. These studies indicate that B function gene activity is conserved among monocots as well as eudicots. In addition, they provide compelling developmental evidence for recognizing lodicules as modified petals and, possibly, palea and lemma as modified sepals. (+info)
Depressed pollination in habitat fragments causes low fruit set.
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In central New South Wales, Australia, flowers of Acacia brachybotrya and Eremophila glabra plants growing in linear vegetation remnants received less pollen than conspecifics in nearby reserves. Pollen supplementation increased fruit production by both species, indicating pollen limitation of fruit set. Together these observations explain why fruit production by these species was depressed in linear-strip populations relative to nearby reserves. This study confirms that habitat fragmentation can lead to decline in pollination and subsequent fruit set in wild plant populations. Disrupted pollination interactions of the kind documented in this study may offer a substantial challenge to the conservation of biodiversity in fragmented landscapes. (+info)
High-performance liquid chromatographic analysis of saponin compounds in Bupleurum falcatum.
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A mixture of saponin compounds (saikosaponin c, a, and d) in the 70% ethanol extract of a powdered sample of Bupleuri radix are analyzed by an Inertsil ODS-3 C(18) column at a flow rate of 1.0 mL/min and detection wavelength of 203 nm. Well resolved chromatograms of saikosaponin c, a, and d are obtained with a gradient elution of acetonitrile-water from 40:60 (v/v) to 50:50 (v/v). The total time required for a single analysis is approximately 20 min. Calibration curves for saikosaponin c, a, and d are linear up to 2.5 mg/mL. The coefficient of variability values for saikosaponins in the extract are below 4%, and the recoveries for saikosaponin c, a, and d are 95.2 +/- 1.1, 96.5 +/- 0.9, and 96.2 +/- 1.0%, respectively. The changes in saikosaponin contents for a two-year growth of Bupleurum falcatum are measured by the established high-performance liquid chromatography method. (+info)
ORCA3, a jasmonate-responsive transcriptional regulator of plant primary and secondary metabolism.
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Biosynthesis of many classes of secondary metabolites in plants is induced by the stress hormone jasmonate. The gene for ORCA3, a jasmonate-responsive APETALA2 (AP2)-domain transcription factor from Catharanthus roseus, was isolated by transferred DNA activation tagging. Orca3 overexpression resulted in enhanced expression of several metabolite biosynthetic genes and, consequently, in increased accumulation of terpenoid indole alkaloids. Regulation of metabolite biosynthetic genes by jasmonate-responsive AP2-domain transcription factors may link plant stress responses to changes in metabolism. (+info)