Scanning transmission X-ray, laser scanning, and transmission electron microscopy mapping of the exopolymeric matrix of microbial biofilms.
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Confocal laser scanning microscopy (CLSM), transmission electron microscopy (TEM), and soft X-ray scanning transmission X-ray microscopy (STXM) were used to map the distribution of macromolecular subcomponents (e.g., polysaccharides, proteins, lipids, and nucleic acids) of biofilm cells and matrix. The biofilms were developed from river water supplemented with methanol, and although they comprised a complex microbial community, the biofilms were dominated by heterotrophic bacteria. TEM provided the highest-resolution structural imaging, CLSM provided detailed compositional information when used in conjunction with molecular probes, and STXM provided compositional mapping of macromolecule distributions without the addition of probes. By examining exactly the same region of a sample with combinations of these techniques (STXM with CLSM and STXM with TEM), we demonstrate that this combination of multimicroscopy analysis can be used to create a detailed correlative map of biofilm structure and composition. We are using these correlative techniques to improve our understanding of the biochemical basis for biofilm organization and to assist studies intended to investigate and optimize biofilms for environmental remediation applications. (+info)
Polyhalogenated benzo- and naphthoquinones are potent inhibitors of plant and bacterial ureases.
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Polyhalogenated benzo- and naphthoquinones were found to be potent inhibitors of pure ureases from Bacillus pasteurii and Canavalia ensiformis. They also inhibited ureases in whole cells of Helicobacter pylori, Klebsiella oxytoca and Proteus mirabilis. Inhibition was non-competitive with K(i) values in the micromolar range or below. Inhibition was irreversible as shown by equilibrium dialysis. Inhibitory power decreased considerably when halogens were replaced by -OH, -CN, alkoxy or alkyl groups. (+info)
Unfolding studies on soybean agglutinin and concanavalin a tetramers: a comparative account.
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The unfolding pathway of two very similar tetrameric legume lectins soybean agglutinin (SBA) and Concanavalin A (ConA) were determined using GdnCl-induced denaturation. Both proteins displayed a reversible two-state unfolding mechanism. The analysis of isothermal denaturation data provided values for conformational stability of the two proteins. It was found that the DeltaG of unfolding of SBA was much higher than ConA at all the temperatures at which the experiments were done. ConA had a T(g) 18 degrees C less than SBA. The higher conformational stability of SBA in comparison to ConA is largely due to substantial differences in their degrees of subunit interactions. Ionic interactions at the interface of the two proteins especially at the noncanonical interface seem to play a significant role in the observed stability differences between these two proteins. Furthermore, SBA is a glycoprotein with a GlcNac2Man9 chain attached to Asn-75 of each subunit. The sugar chain in SBA lies at the noncanonical interface of the protein, and it is found to interact with the amino acid residues in the adjacent noncanonical interface. These interactions further stabilize SBA with respect to ConA, which is not glycosylated. (+info)
Crystallization and preliminary X-ray diffraction analysis of a lectin from Canavalia maritima seeds.
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A lectin from Canavalia maritima seeds (ConM) was purified and submitted to crystallization experiments. The best crystals were obtained using the vapour-diffusion method at a constant temperature of 293 K and grew in 7 d. A complete structural data set was collected to 2.1 A resolution using a synchrotron-radiation source. The ConM crystal belongs to the orthorhombic space group P2(1)2(1)2, with unit-cell parameters a = 67.15, b = 70.90, c = 97.37 A. A molecular-replacement search found a solution with a correlation coefficient of 69.2% and an R factor of 42.5%. Crystallographic refinement is under way. (+info)
Establishment of a heterologous system for the expression of Canavalia brasiliensis lectin: a model for the study of protein splicing.
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During its biosynthesis in developing Canavalia brasiliensis seeds, the lectin ConBr undergoes a form of protein splicing in which the order of the N- and C-domains of the protein is reversed. To investigate whether these events can occur in other eukaryotic organisms, an expression system based on Pichia pastoris cells was established. A DNA fragment encoding prepro-ConBr was cloned into the vector pPICZB, and the recombinant plasmid was transformed in P. pastoris strain GS115. Ten clones were screened for effective recombinant protein production. Based on Western blot analysis of the two clones with the highest level of protein expression: 1) diffuse high-molecular mass immunoreactive bands were produced as early as 24 h after induction; 2) a single-, high-molecular mass protein was secreted into the medium, and 3) a significant fraction of the recombinant polypeptides that cross-reacted with anti-ConBr antibodies comprised a band of approximately 34.5 kDa. Diffuse protein bands with high molecular masses are attributed to hyperglycosylation at the single potential N-glycosylation site located in the linker peptide of prepro-ConBr. In contrast, native ConBr is made up of three polypeptides, the intact alpha chain (aa 1-237) and the fragments beta (aa 1-118) and gamma (aa 119-237), which have apparent molecular masses of 30, 16 and 12 kDa, respectively. Apparently, the yeast P. pastoris is not able to carry out all the complex post-translational proteolytic processing necessary for the biosynthesis of ConBr. (+info)
Ureases display biological effects independent of enzymatic activity: is there a connection to diseases caused by urease-producing bacteria?
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Ureases are enzymes from plants, fungi and bacteria that catalyze the hydrolysis of urea to form ammonia and carbon dioxide. While fungal and plant ureases are homo-oligomers of 90-kDa subunits, bacterial ureases are multimers of two or three subunit complexes. We showed that some isoforms of jack bean urease, canatoxin and the classical urease, bind to glycoconjugates and induce platelet aggregation. Canatoxin also promotes release of histamine from mast cells, insulin from pancreatic cells and neurotransmitters from brain synaptosomes. In vivo it induces rat paw edema and neutrophil chemotaxis. These effects are independent of ureolytic activity and require activation of eicosanoid metabolism and calcium channels. Helicobacter pylori, a Gram-negative bacterium that colonizes the human stomach mucosa, causes gastric ulcers and cancer by a mechanism that is not understood. H. pylori produces factors that damage gastric epithelial cells, such as the vacuolating cytotoxin VacA, the cytotoxin-associated protein CagA, and a urease (up to 10% of bacterial protein) that neutralizes the acidic medium permitting its survival in the stomach. H. pylori whole cells or extracts of its water-soluble proteins promote inflammation, activate neutrophils and induce the release of cytokines. In this paper we review data from the literature suggesting that H. pylori urease displays many of the biological activities observed for jack bean ureases and show that bacterial ureases have a secretagogue effect modulated by eicosanoid metabolites through lipoxygenase pathways. These findings could be relevant to the elucidation of the role of urease in the pathogenesis of the gastrointestinal disease caused by H. pylori. (+info)
A comparative study of the expression of serine proteinases in quiescent seeds and in developing Canavalia ensiformis plants.
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An alkaline proteinase activity is present in quiescent seeds and up to the 24th day of development of Canavalia ensiformis DC (L.) plants. By a simple protocol consisting of cation exchange chromatography, followed by an anion exchange column, a serine proteinase (Q-SP) was purified to homogeneity from quiescent seeds. Q-SP consists of a 33 kDa chain with an optimum pH between 8.0 and 9.0. Arginine residues at P1 and P2 subsites favour binding to the substrate, as shown by the KM assay with N-alpha-benzoyl-DL-arginine-4-nitroanilide-hydrochloride and N-benzoylcarboxyl-L-arginyl-L-arginine-7-amido-4-methylcoumarin. The same protocol was used for partial purification of benzamidine-sensitive enzymes from the developing plant. On the 7th day, a new benzamidine-sensitive enzyme is synthesized in the seedling, seen as the second active peak appearing in anion exchange chromatography. A benzamidine-sensitive enzyme purified from cotyledons presented a similar gel filtration profile as Q-SP, although it was eluted at different salt concentrations in the anion exchange chromatography. None of the enzymes was inhibited by PMSF, APMSF, or SBTI, but they were inactivated by benzamidine, TLCK, and leupeptin. Q-SP did not cleave in vitro C. ensiformis urease, concanavalin A, or its main storage protein, canavalin. In conclusion, a ubiquitous benzamidine-sensitive proteolytic activity was found in C. ensiformis from quiescent seeds up to 24 d of growth, which apparently is not involved in the hydrolysis of storage proteins and might participate in an as yet unidentified limited proteolysis event. (+info)
Tailed forisomes of Canavalia gladiata: a new model to study Ca2+-driven protein contractility.
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BACKGROUND AND AIMS: Forisomes are Ca(2+)-dependent contractile protein bodies that form reversible plugs in sieve tubes of faboid legumes. Previous work employed Vicia faba forisomes, a not entirely unproblematic experimental system. The aim of this study was to seek to establish a superior model to study these intriguing actuators. METHODS: Existing isolation procedures were modified to study the exceptionally large, tailed forisomes of Canavalia gladiata by differential interference contrast microscopy in vitro. To analyse contraction/expansion kinetics quantitatively, a geometric model was devised which enabled the computation of time-courses of derived parameters such as forisome volume from simple parameters readily determined on micrographs. KEY RESULTS: Advantages of C. gladiata over previously utilized species include the enormous size of its forisomes (up to 55 microm long), the presence of tails which facilitate micromanipulation of individual forisomes, and the possibility of collecting material repeatedly from these fast-growing vines without sacrificing the plants. The main bodies of isolated Canavalia forisomes were box-shaped with square cross-sections and basically retained this shape in all stages of contraction. Ca(2+)-induced a 6-fold volume increase within about 10-15 s; the reverse reaction following Ca(2+)-depletion proceeded in a fraction of that time. CONCLUSIONS: The sword bean C. gladiata provides a superior experimental system which will prove indispensable in physiological, biophysical, ultrastructural and molecular studies on the unique ATP-independent contractility of forisomes. (+info)