Amylopectin molecular structure reflected in macromolecular organization of granular starch

For lintners with negligible amylose retrogradation, crystallinity related inversely to starch amylose content and, irrespective of starch source, incomplete removal of amorphous material was shown. The latter was more pronounced for B-type than for A-type starches. The two predominant lintner populations, with modal degrees of polymerization (DP) of 13-15 and 23-27, were best resolved for amylose-deficient and A-type starches. Results indicate a more specific hydrolysis of amorphous lamellae in such starches. Small-angle X-ray scattering showed a more intense 9-nm scattering peak for native amylose-deficient A-type starches than for their regular or B-type analogues. The experimental evidence indicates a lower contrasting density within the "crystalline" shells of the latter starches. A higher density in the amorphous lamellae, envisaged by the lamellar helical model, explains the relative acid resistance of linear amylopectin chains with DP > 20, observed in lintners of B-type starches. Because amylopectin chain length distributions were similar for regular and amylose-deficient starches of the same crystal type, we deduce that the more dense (and ordered) packing of double helices into lamellar structures in amylose-deficient starches is due to a different amylopectin branching pattern.     

Identification of the gene for beta-fructofuranosidase of Bifidobacterium lactis DSM10140(T) and characterization of the enzyme expressed in Escherichia coli

Bifidobacterium lactis is a moderately oxygen-tolerant, saccharolytic bacterium often used in combination with fructooligosaccharides (FOS) as a probiotic supplement in diverse dairy products. This is the first report describing the gene structure and enzymatic properties of a beta-fructofuranosidase [EC 3.2.1.26] from Bifidobacteria. BfrA was identified in Bifidobacterium lactis DSM 10140(T) and heterologously expressed in Escherichia coli. The G+C content was identical with the G+C content as determined for the total genomic DNA (61.9 mol %). The gene codes for a 532-aa residue polypeptide of 59.4 kDa. Surprisingly, the deduced aa sequence revealed only minor similarity to other fructofuranosidases (18% to E. coli cscA). The enzyme was purified to homogeneity after incorporation of a C-terminal 6 x HIS affinity tag. It hydrolased sucrose, 1-kestose, Raftilose, Actilight, inulin, and raffinose (100%, 91%, 84%, 80%, 37%, 4%). Fructose moieties were released in an exo-type fashion. Substrates with alpha-glycosidic linkages or residues other than fructose were not attacked. The kinetic parameters K(m) and V(max) for sucrose hydrolysis were 10.3 m M and 0.031 microM/min (pH 7.6; 37 degrees C). The activity was abolished by Zn(2+) (1 m M) and significantly inhibited by Fe(2+) and Ni(2+) (10 m M). The enzyme showed its maximal activity at 40 degrees C.     

Gene therapy: prospects for glycolipid storage diseases

Lysosomal storage diseases comprise a group of about 40 disorders, which in most cases are due to the deficiency of a lysosomal enzyme. Since lysosomal enzymes are involved in the degradation of various compounds, the diseases can be further subdivided according to which pathway is affected. Thus, enzyme deficiencies in the degradation pathway of glycosaminoglycans cause mucopolysaccharidosis, and deficiencies affecting glycopeptides cause glycoproteinosis. In glycolipid storage diseases enzymes are deficient that are involved in the degradation of sphingolipids. Mouse models are available for most of these diseases, and some of these mouse models have been used to study the applicability of in vivo gene therapy. We review the rationale for gene therapy in lysosomal disorders and present data, in particular, about trials in an animal model of metachromatic leukodystrophy. The data of these trials are compared with those obtained with animal models of other lysosomal diseases.     

Identification and characterization of the chaperone-subunit complex-binding domain from the type 1 pilus assembly platform FimD

The outer membrane protein FimD represents the assembly platform of adhesive type 1 pili from Escherichia coli. FimD forms ring-shaped oligomers of 91.4 kDa subunits that recognize complexes between the pilus chaperone FimC and individual pilus subunits in the periplasm and mediate subunit translocation through the outer membrane. Here, we have identified a periplasmic domain of FimD (FimD(N)) comprising the N-terminal 139 residues of FimD. Purified FimD(N) is a monomeric, soluble protein that specifically recognizes complexes between FimC and individual type 1 pilus subunits, but does not bind the isolated chaperone, or isolated subunits. In addition, FimD(N) retains the ability of FimD to recognize different chaperone-subunit complexes with different affinities, and has the highest affinity towards the FimC-FimH complex. Overexpression of FimD(N) in the periplasm of wild-type E.coli cells diminished incorporation of FimH at the tip of type 1 pili, while pilus assembly itself was not affected. The identification of FimD(N) and its ternary complexes with FimC and individual pilus subunits opens the avenue to structural characterization of critical type 1 pilus assembly intermediates.     

A novel type of deubiquitinating enzyme

A previous report from this laboratory described two novel proteins that have sequence similarity to A20, a negative regulator of NF-kappaB (Evans, P. C., Taylor, E. R., Coadwell, J., Heyninck, K., Beyaert, R., and Kilshaw, P. J. (2001) Biochem. J. 357, 617-623). One of these molecules, cellular zinc finger anti-NF-kappaB (Cezanne), a 100-kDa cytoplasmic protein, also suppressed NF-kappaB. Here we demonstrate that Cezanne is a novel deubiquitinating enzyme, distinct from the two known families of deubiquitinases, Types I and II. We show that Cezanne contains an N-terminal catalytic domain that belongs to the recently discovered ovarian tumor protein (OTU) superfamily, a group of proteins displaying structural similarity to cysteine proteases but having no previously described function. Recombinant Cezanne cleaved ubiquitin monomers from linear or branched synthetic ubiquitin chains and from ubiquitinated proteins. Mutation of a conserved cysteine residue in the catalytic site of the proteolytic domain caused Cezanne to co-precipitate polyubiquitinated cellular proteins. We also provide evidence for an additional ubiquitin binding site in the C-terminal part of the molecule. Our data provide the first demonstration of functional activity among OTU proteins.     

Two distinct Gb3/CD77 signaling pathways leading to apoptosis are triggered by anti-Gb3/CD77 mAb and verotoxin-1

Globotriasosylceramide (Gb3), a neutral glycosphingolipid, is the B-cell differentiation antigen CD77 and acts as the receptor for most Shiga toxins, including verotoxin-1 (VT-1). We have shown that both anti-Gb3/CD77 mAb and VT-1 induce apoptosis in Burkitt's lymphoma cells. We compared the apoptotic pathways induced by these two molecules by selecting cell lines sensitive to only one of these inducers or to both. In all these cell lines (including the apoptosis-resistant line), VT-1 was transported to the endoplasmic reticulum and inhibited protein synthesis similarly, suggesting that VT-1-induced apoptosis is dissociated from these processes. VT-1 triggered a caspase- and mitochondria-dependent pathway (rapid activation of caspases 8 and 3 associated with a loss of mitochondrial membrane potential (Deltapsim) and the release of cytochrome c from mitochondria). In contrast, the anti-Gb3/CD77 mAb-induced pathway was caspase-independent and only involved partial depolarization of mitochondria. Antioxidant compounds had only marginal effects on VT-1-induced apoptosis but strongly protected cells from anti-Gb3/CD77 mAb-induced apoptosis. VT-1- and anti-Gb3/CD77 mAb-treated cells displayed very different features on electron microscopy. These results clearly indicate that the binding of different ligands to Gb3/CD77 triggers completely different apoptotic pathways.     

The VirB4 family of proposed traffic nucleoside triphosphatases: common motifs in plasmid RP4 TrbE are essential for conjugation and phage adsorption

Proteins of the VirB4 family are encoded by conjugative plasmids and by type IV secretion systems, which specify macromolecule export machineries related to conjugation systems. The central feature of VirB4 proteins is a nucleotide binding site. In this study, we asked whether members of the VirB4 protein family have similarities in their primary structures and whether these proteins hydrolyze nucleotides. A multiple-sequence alignment of 19 members of the VirB4 protein family revealed striking overall similarities. We defined four common motifs and one conserved domain. One member of this protein family, TrbE of plasmid RP4, was genetically characterized by site-directed mutagenesis. Most mutations in trbE resulted in complete loss of its activities, which eliminated pilus production, propagation of plasmid-specific phages, and DNA transfer ability in Escherichia coli. Biochemical studies of a soluble derivative of RP4 TrbE and of the full-length homologous protein R388 TrwK revealed that the purified forms of these members of the VirB4 protein family do not hydrolyze ATP or GTP and behave as monomers in solution.     

Development and potential of starter lactobacilli resulting from exploration of the sourdough ecosystem

Lactic acid bacteria are widely used as starter organisms in food fermentations. The development of such cultures as organisms fulfilling all metabolic, technical and handling requirements is the result of a multidisciplinary approach, i.e. to analyse, follow and direct the microbial ecology in food fermentations by molecular biology tools, gene cloning, biochemical and physiological analyses, pilot trials and modelling of behaviour and metabolism. The possibilities and restrictions of such an approach is given for cereal fermentations, namely sourdoughs. In this environment highly adapted lactobacilli are predominant, sharing the environment with yeasts present in traditional preparations. The competitiveness of these lactobacilli and their contribution to flavour, machineability and prebiosis of doughs and bread relies on their maltose and amino acid metabolism, use of electron acceptors and EPS formation. Their reactions on environmental stresses can be used to embed these recalcitrant organisms into starter culture preparations. Beyond the cereal environment the described strategy can be generally applied to understand ecosystems in food fermentations and finally control them. This revised version was published online in August 2006 with corrections to the Cover Date.     

Archaeal histone tetramerization determines DNA affinity and the direction of DNA supercoiling

DNA binding and the topology of DNA have been determined in complexes formed by >20 archaeal histone variants and archaeal histone dimer fusions with residue replacements at sites responsible for histone fold dimer:dimer interactions. Almost all of these variants have decreased affinity for DNA. They have also lost the flexibility of the wild type archaeal histones to wrap DNA into a negative or positive supercoil depending on the salt environment; they wrap DNA into positive supercoils under all salt conditions. The histone folds of the archaeal histones, HMfA and HMfB, from Methanothermus fervidus are almost identical, but (HMfA)(2) and (HMfB)(2) homodimers assemble into tetramers with sequence-dependent differences in DNA affinity. By construction and mutagenesis of HMfA+HMfB and HMfB+HMfA histone dimer fusions, the structure formed at the histone dimer:dimer interface within an archaeal histone tetramer has been shown to determine this difference in DNA affinity. Therefore, by regulating the assembly of different archaeal histone dimers into tetramers that have different sequence affinities, the assembly of archaeal histone-DNA complexes could be localized and used to regulate gene expression.