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PubMed2003

Development and maturation of Escherichia coli K-12 biofilms.

The development and maturation of E. coli biofilms in flow-chambers was investigated. We found that the presence of transfer constitutive IncF plasmids induced biofilm development forming structures resembling those reported for Pseudomonas aeruginosa. The development occurred in a step-wise process: (i). attachment of cells to the substratum, (ii). clonal growth and microcolony formation, and (iii). differentiation into expanding structures rising 70-100 microm into the water phase. The first two steps were the same in the plasmid-carrying and plasmid-free strains, whereas the third step only occurred in conjugation pilus proficient plasmid-carrying strains. The final shapes of the expanding structures in the mature biofilm seem to be determined by the pilus configuration, as various mutants affected in the processing and activity of the transfer pili displayed differently structured biofilms. We further provide evidence that flagella, type 1 fimbriae, curli and Ag43 are all dispensable for the observed biofilm maturation. In addition, our results indicate that cell-to-cell signalling mediated by autoinducer 2 (AI-2) is not required for differentiation of E. coli within a biofilm community. We suggest on the basis of these results that E. coli K-12 biofilm development and maturation is dependent on cell-cell adhesion factors, which may act as inducers of self-assembly processes that result in differently structured biofilms depending on the adhesive properties on the cell surface.

Bacterial Adhesion↗

PubMed1998

The use of 3-dimensional models in auricular reconstruction.

Reconstruction of the microtic auricle is a difficult process requiring considerable experience and dedication to detail. It is a multistage proposition requiring the talents of both the reconstructive surgeon and the otologic surgeon. Reconstruction of the external ear usually precedes the reconstruction of the middle ear. Often, a template is used by the reconstructive surgeon to aid in this complicated process. Traditionally, templates used by the reconstructive surgeon have been 2 dimensional (usually x-ray paper) and made from the opposite normal ear or another normal ear in cases of bilateral microtia. Use of a 2-dimensional model only provides a rough estimate of the cartilage framework needed. Considerable experience is therefore needed to get this cartilage framework "just right." We have developed a number of 3-dimensional synthetic templates to aid in the creation of an accurate cartilage framework implant. These templates serve as a more accurate guide in the complex cartilage carving and assembly process. The use of 3-dimensional templates has improved our technical reconstructive results in a small number of patients. We present these results and propose future application of these ideas.

Cartilage↗

PubMed1994

Fibrinogen assembly: insights from chicken hepatocytes.

In all vertebrate species studied, the complex, disulfide-linked structure of fibrinogen is essentially the same: a hexamer assembled from three different subunits (A alpha, B beta, gamma)2. This study utilized species differences in fibrinogen subunit monomer pools to address the question of how these surplus subunit pools may affect the assembly process. We used a chicken model system in which B beta and gamma-subunits are present in excess, in contrast to the A alpha and gamma-subunit surplus found in human model systems. Analysis was based on pulse-chase experiments with electrophoretic separation of intracellular forms and secreted fibrinogen on reducing and nonreducing gels. The chicken liver-derived cells employed for this purpose, primary hepatocytes and a hepatoma cell line with a fortuitous defect in fibrinogen synthesis, together offer advantages over human systems for resolving the complexes formed in the early stages of assembly. The results demonstrate that in chicken hepatocytes there is an initial binding of gamma to A alpha subunits rather than to B beta subunits, as occurs in human hepatoma cells. Nevertheless, the presence of similar intracellular fibrinogen-related forms in both chicken- and human-derived cells, in the context of their differing subunit monomer pools, suggests an assembly pathway common to both species, with the versatility to be regulated by limitation of A alpha or B beta subunit production.

Animals↗

PubMed2005

From bioremediation to biowarfare: on the impact and mechanism of type IV secretion systems.

Type IV secretion systems are employed by a wide variety of Gram-negative microorganisms for the translocation of macromolecules across the cell envelope. The translocated substrates (proteins, protein-DNA complexes and DNA) are as diverse as the organisms on the donor and recipient side of the translocation process. Over the course of evolution, these macromolecular transporters were adapted to many different purposes, but their basic mechanism was conserved. They impact human life in various ways, as there are driving forces of horizontal gene transfer, which spreads biodegradative capabilities of environmental bacteria as well as antibiotic resistance of pathogens in hospitals. Also, they translocate toxins and other effectors, which have an effect on host cell metabolism and are essential for the virulence of bacterial pathogens. We here present recent developments of research on the mechanism of type IV secretion focusing on the energetization of transport and assembly processes, formation of the translocation channel and of surface-exposed pili, which initiate host cell interactions.

Animals↗

PubMed2003

Layer-by-layer assembly of multilayer films composed of avidin and biotin-labeled antibody for immunosensing.

Protein multilayers composed of avidin and biotin-labeled antibody (bio-Ab) were prepared on gold surface by layer-by-layer assembly technology using the high specific binding constant (K(a): approximately 10(15) M(-1)) between avidin and biotin. The assembly process of the multilayer films was monitored by using real-time BIA technique based on surface plasmon resonance (SPR). The multilayer films were also characterized by electrochemical impedance spectroscopy (EIS) and reflection absorption Fourier transform infrared spectroscopy (FTIR). The results indicate that the growth of the multilayer is uniform. From response of SPR for each layer, the stoichiometry S for the interaction between avidin and bio-Ab is calculated to be 0.37 in the multilayer whereas 0.82 in the first layer. The protein mass concentration for each layer was also obtained. The schematic figure for the multilayer assembly was proposed according to the layer mass concentration and S value. The utility of the mutilayer films for immunosensing has been investigated via their subsequent interaction with hIgG. The binding ability of the multilayer increased for one to three layers of antibody, and then reach saturation after the fourth layer. These layer-by-layer constructed antibody multilayers enhance the binding ability than covalently immobilized monolayer antibody. This technology can be also used for construction of other thin films for immunosensing and biosensor.

Animals↗

PubMed2005

Comparative X-ray standing wave analysis of metal-phosphonate multilayer films of dodecane and porphyrin molecular square.

The nanoscale structures of multilayer metal-phosphonate thin films prepared via a layer-by-layer assembly process using Zr(4+) and 1,12-dodecanediylbis(phosphonic acid) (DDBPA) or porphyrin square bis(phosphonic acid) (PSBPA) were studied using specular X-ray reflectivity (XRR), X-ray fluorescence, and long-period X-ray standing wave (XSW) analysis. The films were prepared in 1, 2, 3, 4, 6, and 8 layer series on both Si(001) substrates for XRR and on 18.6 nm period Si/Mo layered-synthetic microstructure X-ray mirrors for XSW. After functionalizing the SiO(2) substrate surfaces with a monolayer film terminated with phosphonate groups, the organic multilayer films were assembled by alternating immersions in (a) aqueous solutions containing Zr(4+)or Hf(4+) (final metal layer only) cations and then (b) organic solvent solutions of PO(3)-R-PO(3)(4-), where R was DDBPA or PSBPA spacer molecule. The Hf(4+) cation served as the marker for the top surface of the films, whereas the Zr(4+) cation was present in all other layers. The PSBPA also contained Zn and Re atoms at its midline which served as heavy-atom markers for each layer. The long-period XSW generated by the 0th- (total external reflection) through 4th-order Bragg diffraction conditions made it possible to examine the Fourier transforms of the fluorescent atom distributions over a much larger q(z) range in reciprocal space which permitted simultaneous analysis of Hf, Zn/Re, and Zr atomic distributions.

Alkanes↗

PubMed2004

Pathway complexity of Alzheimer's beta-amyloid Abeta16-22 peptide assembly.

Recent studies suggest that both soluble oligomers and insoluble fibrils have toxic effects in cell cultures, raising the interest in determining the first steps of the assembly process. We have determined the aggregation mechanisms of Abeta(16-22) dimer using the activation-relaxation technique and an approximate free energy model. Consistent with the NMR solid-state analysis, the dimer is predicted to prefer an antiparallel beta sheet structure with the expected registry of intermolecular hydrogen bonds. The simulations, however, locate three other antiparallel minima with nonnative beta sheet registries and one parallel beta sheet structure, slightly destabilized with respect to the ground state. This result is significant because it can explain the observed dependency of beta sheet registry on pH conditions. We also find that assembly of Abeta(16-22) into dimers follows multiple routes, but alpha-helical intermediates are not obligatory. This indicates that destabilization of alpha-helical intermediates is unlikely to abolish oligomerization of Abeta peptides.

Amyloid beta-Peptides↗

PubMed2005

Controlling the morphology of cross beta-sheet assemblies by rational design.

Low molecular weight peptidomimetics with simple amphiphilic sequences can help to elucidate the structures of cross beta-sheet assemblies, such as amyloid fibrils. The peptidomimetics described herein comprise a dibenzofuran template, two peptide strands made up of alternating hydrophilic and hydrophobic residues, and carboxyl termini, each of which can be varied to probe the structural requirements for beta-sheet self-assembly processes. The dibenzofuran template positions the strands approximately 10 A apart, allowing corresponding hydrophobic side chains in the strands to pack into a collapsed U-shaped structure. This conformation is stabilized by hydrophobic interactions, not intramolecular hydrogen bonds. Intermolecular stacking of the collapsed peptidomimetics, enabled by intermolecular hydrogen bonding and hydrophobic interactions, affords 25-27 A wide protofilaments having a cross beta-sheet structure. Association of protofilaments, mediated by the dibenzofuran substructures and driven by the hydrophobic effect, affords 50-60 A wide filaments. These widths can be controlled by changing the length of the peptide strands. Further assembly of the filaments into fibrils or ribbons can be controlled by modification of the template, C-terminus, and buffer ion composition.

Amides↗

PubMedDate not supplied

A biochemical logic gate using an enzyme and its inhibitor. 1. The inhibitor as switching element.

Molecular-scale logic systems will allow for further miniaturization of information processing assemblies and contribute to a better understanding of brain function. Of much interest are the pertinent biological systems, some of the basic components of which are biomolecular switching elements and enzyme-based logic gates. In this series of accounts, results of investigations are presented on the implementation of an enzyme/inhibitor logic gate operating under the rules of Boolean algebra. In this report (part 1 of the series), consideration is given to the experimental conditions-particularly the irradiation mode-that affect the performance of proflavine as inhibitor of alpha-chymotrypsin. Also, assessments are made on the reversibility of the process involved and the long-term stability of the system. Moreover, using a theoretical conformational analysis of proflavine and its reduction products, detailed features were established regarding their three-dimensional structure, partial charge distribution, and hydrophobicity. Accordingly, an understanding was reached as to the factors affecting the interaction between these compounds and the enzyme. In part 2 of this series, the actual implementation of an AND logic gate will be presented. This gate involves proflavine and a chemically derivatized alpha-chymotrypsin, and its operation relies on the conclusions reached in this report regarding the optimal mode for controlling the inhibitory activity of proflavine.

Animals↗

PubMed2004

Specific assembly pathway of sarcoglycans is dependent on beta- and delta-sarcoglycan.

Mutations in sarcoglycans (SG) have been reported to cause autosomal-recessive limb-girdle muscular dystrophy (LGMD) and dilated cardiomyopathy. In skeletal and cardiac muscle, sarcoglycans exist as a complex of four transmembrane proteins (alpha-, beta-, gamma-, and delta-SG). In this study, the assembly of the sarcoglycan complex was examined in a heterologous expression system. Our results demonstrated that the assembly process occurs as a discrete stepwise process. We found that beta-SG appears to play an initiating role and its association with delta-SG is essential for the proper localization of the sarcoglycan complex to the cell membrane. The incorporation of alpha-SG into the sarcoglycan complex occurs at the final stage by interaction with gamma-SG. These findings were supported by chemical cross-linking of endogenous sarcoglycans in cultured myotubes. We have also provided evidence that glycosylation-defective mutations in beta-SG and a common mutation in gamma-SG (C283Y) disrupt sarcoglycan-complex formation. Our proposed model for the assembly and structure of sarcoglycans should generate important insight into their function in muscle as well as their role in muscular dystrophies and cardiomyopathies.

Animals↗

PubMed2006

Virus-like particles of the Ty3 retrotransposon assemble in association with P-body components.

Retroviruses and retrotransposons assemble intracellular immature core particles around a RNA genome, and nascent particles collect in association with membranes or as intracellular clusters. How and where genomic RNA are identified for retrovirus and retrotransposon assembly, and how translation and assembly processes are coordinated is poorly understood. To understand this process, the subcellular localization of Ty3 RNA and capsid proteins and virus-like particles was investigated. We demonstrate that mRNAs, proteins, and virus-like particles of the yeast Ty3 retrotransposon accumulate in association with cytoplasmic P-bodies, which are sites of mRNA translation repression, storage, and degradation. Deletions of genes encoding P-body proteins decreased Ty3 transposition and caused changes in the pattern of Ty3 foci, underscoring the biological significance of the association of Ty3 virus-like protein components and P-bodies. These results suggest the hypothesis that P-bodies may serve to segregate translation and assembly functions of the Ty3 genomic RNA to promote assembly of virus-like particles. Because Ty3 has features of a simple retrovirus and P-body functions are conserved between yeast and metazoan organisms, these findings may provide insights into host factors that facilitate retrovirus assembly.

Inclusion Bodies↗

PubMed1985

Defective assembly of the mitochondrial ribosomes in yeast cells grown in the presence of mitochondrial protein synthesis inhibitors.

The involvement of mitochondrial protein synthesis in the assembly of the mitochondrial ribosomes was investigated by studying the extent to which the assembly process can proceed in the presence of mitochondrial protein synthesis inhibitors erythromycin and chloramphenicol. Yeast cells grown in the presence of erythromycin (2 mg/ml) do not appear to contain any detectable amounts of the mitochondrial small (37 S) ribosomal subunit. Instead, a ribonucleoparticle with a sedimentation coefficient of 30 S was observed; this particle could be shown to be related to the mitochondrial small ribosomal subunit by two-dimensional gel electrophoretic analysis of its protein components. Since the var1 protein is the only mitochondrial translation product known to be associated with the mitochondrial ribosome, our results suggest that this protein is essential for the assembly of the mature small subunit, and that the var1 protein enters the pathway for the assembly of the small subunit at a late step. In at least one strain of yeast the accumulation of the 30-S particle appears to be very sensitive to catabolite repression. When yeast cells are grown in the presence of chloramphenicol instead of erythromycin, assembly of the small subunit appears to be only partially inhibited, and the presence of the 30-S particle could not be clearly demonstrated. This observation is consistent with the fact that in yeast, chloramphenicol inhibits mitochondrial protein synthesis by about 95% only and that the synthesis of the var1 protein appears to be the least sensitive to this inhibition.

Cell Fractionation↗

PubMed2000

The assembly pathway of outer membrane protein PhoE of Escherichia coli.

The assembly of the wild-type and several mutant forms of the trimeric outer membrane porin PhoE of Escherichia coli was investigated in vitro and in vivo. In in vivo pulse-chase experiments, approximately half of the wild-type PhoE molecules assembled within the 30-s pulse in the native conformation in the cell envelope. The other half of the molecules followed slower kinetics, and three intermediates in this multistep assembly process were detected: a soluble trypsin-sensitive monomer, a trypsin-sensitive monomeric form that was loosely associated with the cell envelope and a metastable trimer, which was integrated into the membranes and converted to the stable trimeric configuration within minutes. The metastable trimers disassembled during sample preparation for standard SDS/PAGE into folded monomers. In vitro, the isolated PhoE protein could efficiently be folded in the presence of N,N-dimethyldodecylamine-N-oxide (LDAO). A mutant PhoE protein, DeltaF330, which lacks the C-terminal phenylalanine residue, mainly followed the slower kinetic pathway observed in vivo, resulting in increased amounts of the various assembly intermediates. It appears that the DeltaF330 mutant protein is intrinsically able to fold, because it was able to fold in vitro with LDAO with similar efficiencies as the wild-type protein. Therefore, we propose that the conserved C-terminal Phe is (part of) a sorting signal, directing the protein efficiently to the outer membrane. Furthermore, we analysed a mutant protein with a hydrophilic residue introduced at the hydrophobic side of one of the membrane-spanning amphipathic beta strands. The assembly of this mutant protein was not affected in vivo or in vitro in the presence of LDAO. However, it was not able to form folded monomers in a previously established in vitro folding system, which requires the presence of lipopolysaccharides and Triton. Hence, a folded monomer might not be a true assembly intermediate of PhoE in vivo.

Biochemistry↗

PubMed1990

Deletions in epidermal keratins leading to alterations in filament organization in vivo and in intermediate filament assembly in vitro.

To investigate the sequences important for assembly of keratins into 10-nm filaments, we used a combined approach of (a) transfection of mutant keratin cDNAs into epithelial cells in vivo, and (b) in vitro assembly of mutant and wild-type keratins. Keratin K14 mutants missing the nonhelical carboxy- and amino-terminal domains not only integrated without perturbation into endogenous keratin filament networks in vivo, but they also formed 10-nm filaments with K5 in vitro. Surprisingly, keratin mutants missing the highly conserved L L E G E sequence, common to all intermediate filament proteins and found at the carboxy end of the alpha-helical rod domain, also assembled into filaments with only a somewhat reduced efficiency. Even a carboxy K14 mutant missing approximately 10% of the rod assembled into filaments, although in this case filaments aggregated significantly. Despite the ability of these mutants to form filaments in vitro, they often perturbed keratin filament organization in vivo. In contrast, small truncations in the amino-terminal end of the rod domain more severely disrupted the filament assembly process in vitro as well as in vivo, and in particular restricted elongation. For both carboxy and amino rod deletions, the more extensive the deletion, the more severe the phenotype. Surprisingly, while elongation could be almost quantitatively blocked with large mutations, tetramer formation and higher ordered lateral interactions still occurred. Collectively, our in vitro data (a) provide a molecular basis for the dominance of our mutants in vivo, (b) offer new insights as to why different mutants may generate different phenotypes in vivo, and (c) delineate the limit sequences necessary for K14 to both incorporate properly into a preexisting keratin filament network in vivo and assemble efficiently into 10-nm keratin filaments in vitro.

Amino Acid Sequence↗

PubMed2001

Hybrid organic/inorganic reverse osmosis (RO) membrane for bactericidal anti-fouling. 1. Preparation and characterization of TiO2 nanoparticle self-assembled aromatic polyamide thin-film-composite (TFC) membrane.

Hybrid organic/inorganic reverse osmosis (RO) membranes composed of aromatic polyamide thin films underneath titanium dioxide (TiO2) nanosized particles have been fabricated by a self-assembly process, aiming at breakthrough of biofouling problems. First, positively charged particles of the colloidal TiO2 were synthesized by a sol-gel process, and the diameter of the resulting particles in acidic aqueous solution was estimated to be approximately 2 nm by analyzing the UV-visible absorption characteristics with a quantum mechanical model developed by Brus. Transmission electron microscopy (TEM) further confirmed the formation of the quantum-sized TiO2 particles (approximately 10 nm or less). The TiO2 particles appeared to exist in the crystallographic form of anatase as observed with the X-ray diffraction (XRD) pattern in comparison with those of commercial 100% rutile and commercial 70:30% anatase-to-rutile mixture. The hybrid thin-film-composite (TFC) aromatic polyamide membranes were prepared by self-assembly of the TiO2 nanoparticles on the polymer chains with COOH groups along the surface. They showed improved RO performance in which the water flux even increased, though slightly. Field-emission scanning electron microscopy (FESEM) exhibited the TiO2 nanoparticles well adsorbed onto the surface. X-ray photoelectron spectroscopy (XPS) demonstrated quantitatively that a considerable amount of the adsorbed particles were tightly self-assembled at the expense of the initial loss of those that were loosely bound, and became stabilized even after exposure to the various washing and harsh RO operating conditions. The antibacterial fouling potential of the TiO2 hybrid membrane was examined and verified by measuring the viable numbers and determining the survival ratios of the Escherichia coli (E. coli) as a model bacterium, both with and without UV light illumination. The photocatalytic bactericidal efficiency was remarkably higher for the TiO2 hybrid membrane under UV illumination, compared to that of the same membrane in darkness, as well as those for the neat membranes under either light condition.

Biofilms↗

PubMed1989

Complementation of in vitro-assembled spliceosomes.

We describe the development and application of a system of in vitro-assembled splicing complexes that can be used for the identification of protein splicing factors which become associated with the spliceosome at the end of the assembly process ("late" splicing components). A splicing reaction performed in the presence of polyvinyl alcohol is interrupted after 15 to 20 minutes, before the appearance of splicing intermediates and products in significant amounts. Following low-speed centrifugation, a pellet is obtained containing splicing complexes that can be solubilized with 0.6 M-KCl. These complexes can be rapidly complemented for splicing in the presence of ATP and Mg2+ with protein factors that are present in HeLa cell nuclear extracts or in chromatographic extract fractions. Biochemical features of the complementation reactions, and conditions for reversible uncoupling of the two splicing steps, are described and discussed. These conditions are used to generate fully assembled spliceosomes in which splicing of the pre-mRNA can occur in the presence of ATP and Mg2+, but in the absence of nuclear extract ("autonomous splicing").

Adenosine Triphosphate↗

PubMed2002

Colloidal assemblies on patterned silane layers.

The site-selective assembly of colloidal polymer particles onto laterally patterned silane layers was studied as a model system for the object assembly process at mesoscale dimensions. The structured silane monolayers on silicon oxide substrates were fabricated by a combination of liquid- and gas-phase deposition of different trialkoxysilanes with a photolithographic patterning technique. By using this method various types of surface functionalizations such as regions with amino functions next to areas of the bare silica surface or positively charged regions of a quaternary ammonium silane surrounded by a hydrophobic octadecylsilane film could be obtained. Furthermore, a triethoxysilane with a photoprotected amino group was synthesized, which allowed direct photopatterning after monolayer preparation, leading to free NH(2) groups at the irradiated regions. The different silane monolayer patterns were used to study the surface assembly behavior of carboxylated methacrylate particles by optical and scanning electron microscopy. In dependence of the assembly conditions (different surface functionalizations, pH, and drying conditions), a selective preference of the particles for a specific surface type versus others was found. Site-specific colloid adsorption could be observed also on the photosensitive silane layers after local deprotection with light. From the photosensitive silane and positively charged ammonium silane, molecularly mixed monolayers were prepared, which allowed particle adsorption and photoactivation within the same monolayer as shown by fluorescence labeling.

Carboxylic Acids↗

PubMed2004

Chromophore selectivity in bacterial phytochromes: dissecting the process of chromophore attachment.

Bacterial phytochromes (Bphs) are ancestors of the well characterized plant photoreceptors. Whereas plant phytochromes perform their photoisomerization exclusively via a covalently bound bilin chromophore, Bphs are variable in their chromophore selection. This is demonstrated in the cyanobacterium Calothrix PCC7601 that expresses two Bphs, CphA and CphB. CphA binds phycocyanobilin (PCB) covalently, whereas CphB, lacking the covalently binding cysteine of the plant phytochromes, carries biliverdin IXalpha (BV) as the chromophore. Our experiments elucidate the different modes of chromophore-protein interaction in CphA and CphB and offer a rationale for their chromophore selectivity. The tight binding of BV by CphB prevents PCB from competing for the binding cavity. Even when the chromophore-binding cysteine has been inserted (CphB-mutant L266C), PCB replaces BV very slowly, indicating the tight, but not irreversible binding of BV. The mutant CphB L266C showed a redox-sensitivity with respect to its PCB binding mode: under reducing conditions, the chromoprotein assembly leads to spectra indicative for a covalent binding, whereas absence of dithiothreitol or its removal prior to assembly causes spectra indicative for noncovalent binding. Regarding the CphB-type Bphs lacking the covalently binding cysteine, our results support the involvement of the succeeding histidine residue in chromophore fixation via a Schiff base-like bond between the bilin A-ring carbonyl and the histidine imidazole group. The assembly process and the stability of the holo-proteins were strongly influenced by the concentration of added imidazole (mimicking the histidine side-chain), making the attachment of the chromophore via the histidine more likely than via another cysteine of the protein.

Amino Acid Substitution↗