Enzymatic fingerprinting reveals specific xyloglucan and pectin signatures in the cell wall purified with primary plasmodesmata.

Plasmodesmata (PD) pores connect neighbouring plant cells and enable direct transport across the cell wall. Understanding the molecular composition of these structures is essential to address their formation and later dynamic regulation. Here we provide a biochemical characterisation of the cell wall co-purified with primary PD of Arabidopsis thaliana cell cultures. To achieve this result we combined subcellular fractionation, polysaccharide analyses and enzymatic fingerprinting approaches. Relative to the rest of the cell wall, specific patterns were observed in the PD fraction. Most xyloglucans, although possibly not abundant as a group, were fucosylated. Homogalacturonans displayed short methylated stretches while rhamnogalacturonan I species were remarkably abundant. Full rhamnogalacturonan II forms, highly methyl-acetylated, were also present. We additionally showed that these domains, compared to the broad wall, are less affected by wall modifying activities during a time interval of days. Overall, the protocol and the data presented here open new opportunities for the study of wall polysaccharides associated with PD.

The scope and sequence of growth factor delivery for vascularized bone tissue regeneration.

Bone regeneration is a complex process, that in vivo, requires the highly coordinated presentation of biochemical cues to promote the various stages of angiogenesis and osteogenesis. Taking inspiration from the natural healing process, a wide variety of growth factors are currently being released within next generation tissue engineered scaffolds (in a variety of ways) in order to heal non-union fractures and bone defects. This review will focus on the delivery of multiple growth factors to the bone regeneration niche, specifically 1) dual growth factor delivery signaling and crosstalk, 2) the importance of growth factor timing and temporal separation, and 3) the engineering of delivery systems that allow for temporal control over presentation of soluble growth factors. Alternative methods for growth factor presentation, including the use of gene therapy and platelet-rich plasma scaffolds, are also discussed.

Plasmodesmata in Arabidopsis thaliana suspension cells.

A current challenge in plant biology is to identify the structural and functional components of plasmodesmata (PDs). The use of plant tissue as a source material for plasmodesmal characterisation has had limited success, so we have explored the frequency and features of PDs occurring in suspension cell cultures of Arabidopsis thaliana. This material has the advantages of homogeneity, quantity, and ease of disruption. Using light and electron microscopy and immunostaining for callose and calreticulin, we showed that suspension cells laid down abundant PDs in division walls, and that vestiges of these structures were retained as half PDs even when the cell-to-cell contacts were disrupted during culture growth. Although callose was a reliable marker for PD distribution, which was deposited in an organised collar around the neck of PDs, it was not abundant in unstressed cells. Calreticulin and the chemical stain 3,3'-dihexyloxacarbocyanine iodide also provided useful markers when monitoring PDs in cell wall preparations by light microscopy. Purified cell walls were shown to be virtually free of contamination from cytoplasmic components, except for the presence of small amounts of cortical endoplasmic reticulum attached to PDs. Hence, clean cell walls from A. thaliana suspension cells provide a valuable resource for a proteomic approach to the analysis of plasmodesmal components.

[Autoimmune liver disease: diagnosis and therapy]. / Autoimmune Lebererkrankungen: Diagnostik und Therapie.

Autoimmune Hepatitis (AIH), primary biliary cirrhosis (PBC), primary sclerosing cholangitis (PSC) and overlap syndromes of these three disease entities are regarded as autoimmune liver diseases. These conditions are important differential diagnoses of elevated liver function tests as about 10 % of liver transplantations in Europe and North America are for these indications. The diagnosis is often difficult but can be facilitated by sequential measurement of relevant autoantibodies, exclusion of other liver disease, ultrasound, ERCP and liver histology. In AIH immunosuppressive therapy has been shown to prevent or stop the development of cirrhosis and improve the prognosis of the patients decisively. In other autoimmune liver diseases this evidence is missing making individual therapeutic decisions necessary. Ursodesoxycholic acid (UDCA) seems to slow disease progression in particular in early stages of PBC.

Low temperature conversion of sludge and shavings from leather industry.

Abstract Brazil has one of the largest herds of cattle in the world, with more than 170 million heads. Over 400 farms have exported more than 2,875 ton (in 1997) of leather to Europe. The wet blue tanning process uses chemicals such as chromium compounds and produces liquid wastes that must be treated by physicochemical and biological systems. About 15,000 ton per month of dewatering sludge with 24% solids content is disposed of into landfills. During the process, pre-tanned skins (wet blue leather) are shaved to the desired thickness and the shavings, like sludge, are among the wastes that must have special attention. The organic content and chromium concentration are high. About 12% of the leather production from cattle hides are shavings, and its chromium concentration ranges from 3.5 to 5.5% of dry matter. The Environmentally friendly leather project, a co-operation between Brazilian and German tanneries, universities and technical schools, is looking for process optimisation, waste minimisation and adequate treatment for solid and liquid wastes from the leather industry. This work presents results of Low Temperature Conversion of chrome-containing sludge and shavings in a laboratory batch reactor, offering a solution for these hazardous wastes, recovering the energy content and transforming metals in insoluble sulphides.

Characterisation and some possible uses of Plukenetia conophora and Adenopus breviflorus seeds and seed oils.

Two non-conventional seeds, Plukenetia conophora (PKCP) and Adenopus breviflorus (ADB) were analysed for their proximate, fatty acids, sterols composition and physico-chemical characteristics. Crude protein was 25.65% for PKCP and 28.25% for ADB. ADB had lower moisture content (4.5%) than PKCP (8.0%) indicating that the former has better shelf life. Oil yields of the seeds were 49.58% for PKCP and 56.22% for ADB. The major sterols were stigmasterol and beta-sitosterol in PKCP and ADB respectively. PKCP oil had 98.8% unsaturated fatty acids with linolenic acid predominating (70.1%) while ADB had 85.1% unsaturated fatty acids with linoleic acid being most abundant (65.3%). The very high saponification and iodine values of PKCP oil suggest its utilisation in alkyd resin, shoe polish, liquid soap and shampoo production. There is the possibility of using ADB oil in these regards as well as for edible purposes.

Crystallization and preliminary X-ray analysis of W120K mutant of streptavidin.

Bacterial streptavidin and chicken avidin are homotetrameric proteins that share an exceptionally high affinity towards the vitamin biotin. The biotin-binding sites in both proteins contain a crucial tryptophan residue contributed from an adjacent subunit. This particular tryptophan (W110 in avidin and W120 in streptavidin) plays an important role in both biotin binding and in the quaternary stabilities of the proteins. An intriguing naturally occurring alteration of tryptophan to lysine was previously described in the C-terminal domain of sea-urchin fibropellins, which share a relatively high sequence similarity with avidin and streptavidin. Avidin (Avm-W110K) and streptavidin (Savm-W120K) mutations show substantially reduced affinities towards biotin as well as the dissociation of their tetrameric structure into stable avidin and streptavidin dimers. Savm-W120K was crystallized at 293 K using the hanging-drop vapour-diffusion method. The crystals diffract to 1.7 A resolution using synchrotron radiation and belong to the monoclinic space group P2(1), with unit-cell parameters a = 50.43, b = 100.41, c = 52.51 A, beta = 112.12 degrees. The asymmetric unit contains four molecules of Savm-W120K, with a corresponding V(M) of 2.3 A(3) Da(-1) and a solvent content of 46%.

Prediction of progressive liver fibrosis in hepatitis C infection by serum and tissue levels of transforming growth factor-beta.

Although many patients with chronic viral hepatitis C infection suffer from progressive liver disease, the rate of fibrosis progression is highly variable and some patients do not show any measurable progression. However, our ability to predict which patients progress is very limited. Since transforming growth factor-beta (TGF-beta) is a key mediator of liver fibrogenesis, we assessed the predictive role of TGF-beta for fibrogenesis in chronic hepatitis C. We studied 39 patients with chronic hepatitis C in whom two liver biopsies were taken at least 12 months apart, and who did not receive therapy during this period. TGF-beta was measured by bioassay and by ELISA in serum samples taken at the time of the first biopsies, and TGF-beta was determined semiquantitatively by immunostaining of liver biopsy sections. Fibrosis was scored blinded in the biopsy samples by two pathologists independently. There was a close correlation between TGF-beta serum levels and the rate of fibrosis progression. Patients with no progression of fibrosis had significantly lower (59 ng/mL +/- 22) TGF-beta serum levels than patients with progressive disease (115 ng/mL +/- 20), and a TGF-beta level below 75 ng/mL was predictive for stable disease. Immunohistology for TGF-beta in biopsy samples was also predictive for progressive liver disease with fibrosis progression found in those patients displaying staining of hepatocytes and sinusoidal cells. No such correlation was found with other markers such as procollagen III peptide, viral load or transaminase levels. These results further support the role of TGF-beta in liver fibrogenesis, and offer an opportunity to predict clinical disease progression, which may help in selecting patients who are in need of therapeutic interventions.

Subcellular distribution of glycanases and related components in Ruminococcus albus SY3 and their role in cell adhesion to cellulose.

AIMS: To compare the subcellular distribution of glycanase-related components between wild-type Ruminococcus albus SY3 and an adhesion-defective mutant, to identify their possible contribution to the adhesion process, and to determine their association with cellulosome-like complexes. METHODS AND RESULTS: Cell fractionation revealed that most of the cellulases and xylanases were associated with capsular and cell-wall fractions. SDS-PAGE and gel filtration indicated that most of the bacterial enzyme activity was not integrated into cellulosome-like complexes. The adhesion-defective mutant produced significantly less (5- to 10-fold) overall glycanase activity, and the 'true cellulase activity' appeared to be entirely confined to the cell membrane fractions. Antibodies specific for the cellulosomal scaffoldin of Clostridium thermocellum recognized a single 240 kDa band in R. albus SY3. CONCLUSIONS: The adhesion-defective mutant appeared to be blocked in exocellular transport of enzymes involved in true cellulase activity. A potential cellulosomal scaffoldin candidate was identified in R. albus SY3. SIGNIFICANCE AND IMPACT OF THE STUDY: Several glycanase-related proteins and more than one mechanism appear to be involved in the adhesion of R. albus SY3 to cellulose.

Influence of pressure upon coupling pressurized capillary electrochromatography with nuclear magnetic resonance spectroscopy.

In this work, the influence of supplementary pressure on the separation efficiency of pressurized capillary electrochromatography (pCEC) was examined. At low pressures of up to 30 bar, which is more than sufficient to prevent bubble formation, no significant loss in separation efficiency is observed. Even at 100 bar, the efficiency of pCEC is still significantly better than without application of an electric field. In addition, analysis times are drastically reduced compared to both capillary electrochromatography (CEC) and capillary HPLC. On the basis of these results, an improved interface for capillary NMR coupling is described and used for the separation and identification of a mixture of unsaturated fatty acid methyl esters. Under these conditions, the analysis time could be shortened by up to a factor of 10 when pCEC is coupled to NMR spectroscopy.

Chicken avidin exhibits pseudo-catalytic properties. Biochemical, structural, and electrostatic consequences.

Avidin and its bacterial analogue streptavidin exhibit similarly high affinities toward the vitamin biotin. The extremely high affinity of these two proteins has been utilized as a powerful tool in many biotechnological applications. Although avidin and streptavidin have similar tertiary and quaternary structures, they differ in many of their properties. Here we show that avidin enhances the alkaline hydrolysis of biotinyl p-nitrophenyl ester, whereas streptavidin protects this reaction even under extreme alkaline conditions (pH > 12). Unlike normal enzymatic catalysis, the hydrolysis reaction proceeds as a single cycle with no turnover because of the extremely high affinity of the protein for one of the reaction products (i.e. free biotin). The three-dimensional crystal structures of avidin (2 A) and streptavidin (2.4 A) complexed with the amide analogue, biotinyl p-nitroanilide, as a model for the p-nitrophenyl ester, revealed structural insights into the factors that enhance or protect the hydrolysis reaction. The data demonstrate that several molecular features of avidin are responsible for the enhanced hydrolysis of biotinyl p-nitrophenyl ester. These include the nature of a decisive flexible loop, the presence of an obtrusive arginine 114, and a newly formed critical interaction between lysine 111 and the nitro group of the substrate. The open conformation of the loop serves to expose the substrate to the solvent, and the arginine shifts the p-nitroanilide moiety toward the interacting lysine, which increases the electron withdrawing characteristics and consequent electrophilicity of the carbonyl group of the substrate. Streptavidin lacked such molecular properties, and analogous interactions with the substrate were consequently absent. The information derived from these structures may provide insight into the action of artificial protein catalysts and the evolution of catalytic sites in general.

On-line coupling of capillary gel electrophoresis with electrospray mass spectrometry for oligonucleotide analysis.

Homooligodeoxyribonucleotides differing one nucleotide in length from 12- to 15-mer and from 17- to 20-mer were separated by size with capillary gel electrophoresis (CGE) using an entangled polymer solution in coated capillaries. The resolved components were analyzed by on-line coupling of CGE with electrospray mass spectrometry (ES-MS), denoted as CGE/ES-MS, in the full-scan negative ion detection mode. Baseline separation was achieved for the 12-15-mer oligonucleotide mixtures. Both synthetic phosphodiester oligonucleotide mixtures as well as their phosphorothioate analogues, serving as model compounds for antisense oligonucleotides, could be analyzed by on-line CGE/ES-MS coupling. Terminally phosphorylated and nonphosphorylated synthetic failure sequences could be electrophoretically separated and mass spectrometically characterized as well. This methodology might be a useful tool for synthesis control of phosphodiester oligonucleotides as well as for analysis of phosphorothioate analogues as they are used in antisense drug development.

Recent advances in capillary electrophoresis/electrospray-mass spectrometry.

In this review, the progress in hyphenation of capillary electrophoresis (CE) with electrospray ionization-mass spectrometry (ESI-MS) since the article of Banks (Banks, J. F., Electrophoresis 1997, 18, 2255-2266) is reported. In all capillary-based electromigration techniques, such as capillary gel electrophoresis (CGE), capillary isotachophoresis (CITP), capillary isoelectric focussing (CIEF), micellar electrokinetic chromatography (MEKC), affinity capillary electrophoresis (ACE), as well as in the hybrid techniques capillary electrochromatography (CEC), and pressurized capillary electrochromatography (pCEC) progress has been made in experimental setups, and for many groups of analytes, such as peptides, proteins, nucleotides, saccharides, drugs and their metabolites, CE/ESI-MS has been successfully applied. Electromigration is further miniaturized. New preconcentration methods allow the investigation of compounds, which are not sensitively detected with ESI-MS. Coordination ion spray (CIS) MS is another method for sensitivity enhancement by on-line formation of charged coordination compounds.

Design and production of active cellulosome chimeras. Selective incorporation of dockerin-containing enzymes into defined functional complexes.

Defined chimeric cellulosomes were produced in which selected enzymes were incorporated in specific locations within a multicomponent complex. The molecular building blocks of this approach are based on complementary protein modules from the cellulosomes of two clostridia, Clostridium thermocellum and Clostridium cellulolyticum, wherein cellulolytic enzymes are incorporated into the complexes by means of high-affinity species-specific cohesin-dockerin interactions. To construct the desired complexes, a series of chimeric scaffoldins was prepared by recombinant means. The scaffoldin chimeras were designed to include two cohesin modules from the different species, optionally connected to a cellulose-binding domain. The two divergent cohesins exhibited distinct specificities such that each recognized selectively and bound strongly to its dockerin counterpart. Using this strategy, appropriate dockerin-containing enzymes could be assembled precisely and by design into a desired complex. Compared with the mixture of free cellulases, the resultant cellulosome chimeras exhibited enhanced synergistic action on crystalline cellulose.

Properties and applications of the (2-nitrofluoren-9-yl)methoxycarbonyl group.

This paper presents a new protecting group, the (2-nitrofluoren-9-yl)methoxycarbonyl group. Investigations on the properties of this new modification of the Fmoc-system, such as the solvent-dependent photochemical cleavage, and enhanced lability towards bases, are described, as well as UV-kinetic measurements of the cleavage reaction. In addition, the incorporation of the (2-nitrofluoren-9-yl)methoxycarbonyl group into two peptides, and a sequence-dependent photochemical cleavage reaction are reported.

Nonproteolytic cleavage of aspartyl proline bonds in the cellulosomal scaffoldin subunit from Clostridium thermocellum.

Previous work from our group [Morage (Morgenstern), E., Bayer, E. A., and Lamed, R. (1991), Appl. Biochem. Biotechnol. 30, 129-136] has demonstrated an anomalous electrophoretic mobility pattern for scaffoldin, the 210-kDa cellulosome-integrating subunit of Clostridium thermocellum. Subsequent evidence [Morag, E., Bayer, E. A., and Lamed, R. (1992), Appl. Biochem. Biotechnol. 33, 205-217] indicated that the effect could be attributed to a nonproteolytic fragmentation of the subunit into a defined series of lower-molecular-weight bands. In the present work, a recombinant segment of the scaffoldin subunit was employed to determine the site(s) of bond breakage. An Asp-Pro sequence within the cohesin domain was identified to be the sensitive peptide bond. This sequence appears quite frequently in the large cellulosomal proteins, and the labile bond may be related to an as yet undescribed physiological role in the hydrolysis of cellulose by cellulosomes.

Cellulosomal scaffoldin-like proteins from Ruminococcus flavefaciens.

Two tandem cellulosome-associated genes were identified in the cellulolytic rumen bacterium, Ruminococcus flavefaciens. The deduced gene products represent multimodular scaffoldin-related proteins (termed ScaA and ScaB), both of which include several copies of explicit cellulosome signature sequences. The scaB gene was completely sequenced, and its upstream neighbor scaA was partially sequenced. The sequenced portion of scaA contains repeating cohesin modules and a C-terminal dockerin domain. ScaB contains seven relatively divergent cohesin modules, two extremely long T-rich linkers, and a C-terminal domain of unknown function. Collectively, the cohesins of ScaA and ScaB are phylogenetically distinct from the previously described type I and type II cohesins, and we propose that they define a new group, which we designated here type III cohesins. Selected modules from both genes were overexpressed in Escherichia coli, and the recombinant proteins were used as probes in affinity-blotting experiments. The results strongly indicate that ScaA serves as a cellulosomal scaffoldin-like protein for several R. flavefaciens enzymes. The data are supported by the direct interaction of a recombinant ScaA cohesin with an expressed dockerin-containing enzyme construct from the same bacterium. The evidence also demonstrates that the ScaA dockerin binds to a specialized cohesin(s) on ScaB, suggesting that ScaB may act as an anchoring protein, linked either directly or indirectly to the bacterial cell surface. This study is the first direct demonstration in a cellulolytic rumen bacterium of a cellulosome system, mediated by distinctive cohesin-dockerin interactions.

Cohesin-dockerin interaction in cellulosome assembly: a single hydroxyl group of a dockerin domain distinguishes between nonrecognition and high affinity recognition.

The assembly of enzyme components into the cellulosome complex is dictated by the cohesin-dockerin interaction. In a recent article (Mechaly, A., Yaron, S., Lamed, R., Fierobe, H.-P., Belaich, A., Belaich, J.-P., Shoham, Y., and Bayer, E. A. (2000) Proteins 39, 170-177), we provided experimental evidence that four previously predicted dockerin residues play a decisive role in the specificity of this high affinity interaction, although additional residues were also implicated. In the present communication, we examine further the contributing factors for the recognition of a dockerin by a cohesin domain between the respective cellulosomal systems of Clostridium thermocellum and Clostridium cellulolyticum. In this context, the four confirmed residues were analyzed for their individual effect on selectivity. In addition, other dockerin residues were discerned that could conceivably contribute to the interaction, and the suspected residues were similarly modified by site-directed mutagenesis. The results indicate that mutation of a single residue from threonine to leucine at a given position of the C. thermocellum dockerin differentiates between its nonrecognition and high affinity recognition (K(a) approximately 10(9) m(-1)) by a cohesin from C. cellulolyticum. This suggests that the presence or absence of a single decisive hydroxyl group is critical to the observed biorecognition. This study further implicates additional residues as secondary determinants in the specificity of interaction, because interconversion of selected residues reduced intraspecies self-recognition by at least three orders of magnitude. Nevertheless, as the latter mutageneses served to reduce but not annul the cohesin-dockerin interaction within this species, it follows that other subtle alterations play a comparatively minor role in the recognition between these two modules.

Biotin induces tetramerization of a recombinant monomeric avidin. A model for protein-protein interactions.

Chicken avidin, a homotetramer that binds four molecules of biotin was converted to a monomeric form by successive mutations of interface residues to alanine. The major contribution to monomer formation was the mutation of two aspartic acid residues, which together account for ten hydrogen bonding interactions at the 1-4 interface. Mutation of these residues, together with the three hydrophobic residues at the 1-3 interface, led to stable monomer formation in the absence of biotin. Upon addition of biotin, the monomeric avidin reassociated to the tetramer, which exhibited properties similar to those of native avidin, with respect to biotin binding, thermostability, and protease resistance. To our knowledge, these unexpected results represent the first example of a small monovalent ligand that induces oligomerization of a monomeric protein. This study may suggest a biological role for low molecular weight ligands in inducing oligomerization and in maintaining the stability of multimeric protein assemblies.

Bcl-XL protects pancreatic adenocarcinoma cells against CD95- and TRAIL-receptor-mediated apoptosis.

In this study we sought to clarify the role of the proapoptotic potential of mitochondria in the death pathway emanating from the TRAIL (APO-2L) and CD95 receptors in pancreatic carcinoma cells. We focused on the role of the Bcl-2 family member Bcl-XL, using three pancreatic carcinoma cell lines as a model system, two of which have high (Panc-1, PancTuI) and one has low (Colo357) Bcl-XL expression. In these cell lines, the expression of Bcl-XL correlated with sensitivity to apoptosis induced by TRAIL or anti-CD95. Flow cytometric analysis revealed cell surface expression of TRAIL-R1 and TRAIL-R2 on PancTuI and Colo357, and TRAIL-R2 on Panc-1 cells. In Colo357 cells retrovirally transduced with Bcl-XL, caspase-8 activation in response to treatment with TRAIL or anti-CD95 antibody was not different from parental cells and EGFP-transfected controls, however, apoptosis was completely suppressed as measured by the mitochondrial transmembrane potential deltapsim, caspase-3 activity (PARP cleavage) and DNA-fragmentation. Inhibition of Bcl-XL function by overexpression of Bax or administration of antisense oligonucleotides against Bcl-XL mRNA resulted in sensitization of Panc-1 cells to TRAIL and PancTuI cells to anti-CD95 antibody-induced cell death. The results show that Bcl-XL can protect pancreatic cancer cells from CD95- and TRAIL-mediated apoptosis. Thus, in these epithelial tumour cells the mitochondrially mediated 'type II' pathway of apoptosis induction is not only operative regarding the CD95 system but also regarding the TRAIL system.