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At least 631 records · Page 35Linked to original sources
PubMed2005

Molecular basis of antigenic polymorphism of EspA filaments: development of a peptide display technology.

Like many Gram-negative pathogens, enteropathogenic (EPEC) and enterohaemorrhagic Escherichia coli (EHEC) use a macromolecular type III secretion system (TTSS) to inject effector proteins into eukaryotic host cells. The membrane-associated needle complex (NC) of the TTSS, which shows broad similarity to the flagellar basal body, is conserved amongst bacterial pathogens. However, the extracellular part of the TTSS of EPEC and EHEC is unique, in that it has a hollow, approximately 12 nm in diameter, filamentous extension to the NC. EspA filaments are homo-polymers made of the translocator protein EspA. The three-dimensional structure of EspA filaments is comparable to that of flagella; the helical symmetry and packing of the subunits forming both filamentous structures are very similar. Like flagella, EspA filaments show antigenic polymorphism as EspA from different EPEC and EHEC clones show no immunological cross-reactivity. In this study, we determined the molecular basis of the antigenic polymorphism of EspA filaments and identified a surface-exposed hypervariable domain that contains the immunodominant EspA epitope. By exchanging the hypervariable domains of EspA(EPEC) and EspA(EHEC) we swapped the antigenic specificity of the EspA filaments. As for the flagellin D3 domain, which is known to tolerate insertions of natural and artificial amino acid sequences, we have inserted short peptides into the surface-exposed, hypervariable domain of EspA. We demonstrated that the inserted peptides are presented on the surface of the recombinant EspA filaments forming a new immunodominant epitope. Accordingly, EspA filaments have a potential to be developed into a novel epitope display system.

Actins↗
PubMed2003

The outs and ins of bacterial type IV secretion substrates.

Bacteria use type IV secretion systems (T4SS) to translocate macromolecular substrates destined for bacterial, plant or human target cells. The T4SS are medically important, contributing to virulence-gene spread, genome plasticity and the alteration of host cellular processes during infection. The T4SS are ancestrally related to bacterial conjugation machines, but present-day functions include (i) conjugal transfer of DNA by cell-to-cell contact, (ii) translocation of effector molecules to eukaryotic target cells, and (iii) DNA uptake from or release to the extracellular milieu. Rapid progress has been made toward identification of type IV secretion substrates and the requirements for substrate recognition.

Bacterial Proteins↗
PubMed1987

Reduction and catalytic properties of cytochrome P-450 LM2 in reconstituted system containing monomeric carriers.

The membrane microsomal monooxygenase system can be reconstituted in solution from NADPH-specific flavoprotein and cytochrome P-450 which exist in the monomeric state in the presence of Emulgen 913 at molar ratio of the proteins and detergent of 1:1:300. Oxidized and dithionite-reduced monomers of cytochrome P-450 were much less thermostable than its initial aggregates, while thermal stability of NADPH-specific flavoprotein did not depend on its aggregation state. Binding spectra of cytochrome P-450 monomers with benzphetamine were atypical and had an absorbance minimum at 422 nm only. The addition of benzphetamine and/or flavoprotein to cytochrome P-450 monomers did not cause the spin equilibrium shift and the low-spin form content was higher than 85% in all cases. Investigation of the dependence of the initial rates of NADPH-dependent cytochrome P-450 reduction and benzphetamine oxidation on the stoichiometry of the flavoprotein and cytochrome P-450 at their constant total concentration showed that the molar ratio of 1:1 was required for maximal activity. Thus this system works in full accordance with the mass action law.

Animals↗
PubMed2003

Recombinant Apoptin multimers kill tumor cells but are nontoxic and epitope-shielded in a normal-cell-specific fashion.

Apoptin, a protein derived from chicken anemia virus, induces apoptosis in human transformed or tumor cells but not in normal cells. When produced in bacteria as a recombinant fusion with maltose-binding protein (MBP-Apoptin), Apoptin forms a distinct, stable multimeric complex that is remarkably homogeneous and uniform. Here, using cytoplasmic microinjection, we showed that recombinant MBP-Apoptin multimers retained the characteristics of the ectopically expressed wild-type Apoptin; namely, the complexes translocated to the nucleus of tumor cells and induced apoptosis, whereas they remained in the cytoplasm of normal, primary cells and exerted no apparent toxic effect. In normal cells, MBP-Apoptin formed increasingly large, organelle-sized globular bodies with time postinjection and eventually lost the ability to be detected by immunofluorescence analysis. Costaining with an acidotrophic marker indicated that these globular structures did not correspond to lysosomes. Immunoprecipitation studies showed that MBP-Apoptin remained fully antibody-accessible regardless of buffer stringency when microinjected into tumor cells. In contrast, MBP-Apoptin in normal cells was only recoverable under stringent lysis conditions, whereas under milder conditions they became fully shielded with time on two epitopes spanning the entire protein. Further biochemical analysis showed that the long-term fate of Apoptin protein aggregates in normal cells was their eventual elimination. Our results provide the first example of a tumor-specific apoptosis-inducing aggregate that is essentially sequestered by factors or conditions present in the cytoplasm of healthy, nontransformed cells. This characteristic should reveal more about the cellular interactions of this viral protein as well as further enhance its safety as a potential tumor-specific therapeutic agent.

Antineoplastic Agents↗
PubMed2002

Genetic control of MHC class II expression.

The presentation of peptides to T cells by MHC class II molecules is of critical importance in specific recognition by the immune system. Expression of class II molecules is exquisitely controlled at the transcriptional level. A large set of proteins interact with the promoters of class II genes. The most important of these is CIITA, a master controller that orchestrates expression but does not bind directly to the promoter. The transcriptosome complex formed at class II promoters is a model for induction of gene expression.

Animals↗
PubMed2003

Nanotechnological approaches for the delivery of macromolecules.

In this overview, novel approaches are described for the controlled release and/or for the targeted delivery of macromolecules such as proteins and DNA. The building stones of these highly complex systems are (phospho)lipids and/or (biodegradable) polymers. They should be carefully chosen and preparation protocols should be rationally designed to maximize chances for success.

Animals↗
PubMed1978

Collagen biosynthesis by cultured Chinese hamster lung cells. Cell-free synthesis of procollagen alpha chains.

Cell-free extracts from the HTl clone of cultured Chinese hamster lung cells efficiently promote the incorporation of proline into newly synthesized material, 50% of which is digestible to small peptides by highly purified bacterial collagenase. The synthesis of the these products occurs under optimal protein synthesis conditions and is inhibited by puromycin. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the cell-free synthesized material reveals a major collagenase sensitive peak (20% of the total product) at mol wt 165 000 which is reflected by a collagenase sensitive material of similar size in the culture medium. Two additional collagenase digestible species (mol wt 95000 and 65000), each having a corresponding secreted product, are generated by the cell-free system. These results are consistent with the concept that procollagen is formed by the association of three individually translated pro alphachains. The data further constitute the report of a highly active homologous cell-free system capable of pro alpha chain biosynthesis derived from a cultured cell line that is a practical source for pro alphachain biosynthesis derived from a cultured cell line that is a practical source for proalpha chain mRNA as well as a unique system for elucidating regulatory mechanisms involved in collagen biosynthesis.

Cell Line↗
PubMed1990

Solute absorption from the airways of the isolated rat lung. III. Absorption of several peptidase-resistant, synthetic polypeptides: poly-(2-hydroxyethyl)-aspartamides.

A series of samples of the synthetic polypeptide poly-alpha, beta-[N(2-hydroxyethyl)-DL-aspartamide] (PHEA), containing covalently bound fluorophore, ethylcarbonyl-6-aminofluorescein, and exhibiting different molecular weight distributions with weight average molecular weights ranging from approximately 4 to 43 kD, was prepared and characterized. Aqueous solutions of the polymers were administered to the airways of isolated perfused rat lung preparations, and transfer to the perfusate was measured. Polymers administered directly to the perfusate were not degraded during the experiment. Polymer transfer rates were dependent upon starting molecular weight distribution, larger molecules being absorbed more slowly. In the case of a polymer with a median molecular weight of 7.2 kD, the absorbed species appeared to be smaller molecules than those which were originally administered. This was not the case for a 3.98-kD polymer; absorbed material had a gel permeation chromatography elution volume equivalent to that of the administered material. Absorption for the 3.98-kD polymer was found to be dose dependent. Approximately 70% absorption of a 0.2-mg dose occurred in 100 min. Much larger polymers (up to 11.65 kD) were also absorbed at finite rates. Results are discussed in the context of macromolecular delivery to the systemic circulation via the lung.

Absorption↗
PubMed1993

Crystal structure of a bacterial non-haem iron hydroxylase that catalyses the biological oxidation of methane.

The 2.2 A crystal structure of the 251K alpha 2 beta 2 gamma 2 dimeric hydroxylase protein of methane monooxygenase from Methylococcus capsulatus (Bath) reveals the geometry of the catalytic di-iron core. The two iron atoms are bridged by exogenous hydroxide and acetate ligands and further coordinated by four glutamate residues, two histidine residues and a water molecule. The dinuclear iron centre lies in a hydrophobic active-site cavity for binding methane. An extended canyon runs between alpha beta pairs, which have many long alpha-helices, for possible docking of the reductase and coupling proteins required for catalysis.

Amino Acid Sequence↗
PubMed2002

Selective apoptosis of tandemly duplicated FLT3-transformed leukemia cells by Hsp90 inhibitors.

An internal tandem duplication of the juxtamembrane (JM) domain of FLT3, a family of ligand-activated receptor tyrosine kinases, has been found in 20% of cases of acute myeloid leukemia (AML), and this mutation is correlated with leukocytosis and a poor prognosis. As a therapeutic approach, we previously reported that herbimycin A (HA) inhibited the growth of tandemly duplicated FLT3 (TDFLT3)-transformed cells (Leukemia 2000; 14: 374). Here, we have investigated the mechanism behind the cytotoxicity of HA, an ansamycin derivative which is now known to target Hsp90. The treatment with HA or another Hsp90 inhibitor, radicicol, induced selective apoptosis in TDFLT3-transformed 32D cells (TDFLT3/32D). The tyrosine-phosphorylation of TDFLT3 was inhibited by HA, whereas FLT3 ligand-induced phosphorylation of wild-type FLT3 (WtFLT3) was not. The downstream signal molecules MAPK, Akt and STAT5a were also dephosphorylated by HA in TDFLT3/32D. Immunoprecipitation analysis showed that TDFLT3 but not WtFLT3 formed a complex with Hsp90, and that the HA treatment dissociated TDFLT3 from the Hsp90 chaperone complex. These findings imply that targeting of Hsp90 will facilitate the development of anti-TDFLT3 therapy, and that Hsp90 is closely involved in the oncogenic activation of FLT3.

Animals↗
PubMed2005

Supramolecular gels: functions and uses.

In recent years there has been immense interest in studying gels derived from low molecular mass gelators (supramolecular, or simply molecular gels). The motivation for this is not only to understand the fundamental aggregate structures in the gels at different length scales, but also to explore their potential for futuristic technological applications. Gels have been made sensitive to external stimuli like light and chemical entities by incorporating a spectroscopically active or a receptor unit as part of the gelator molecule. This makes them suitable for applications such as sensing and actuating. The diversity of gel structural architectures has allowed them to be utilized as templates to prepare novel inorganic superstructures for possible applications in catalysis and separation. Gels derived from liquid crystals (anisotropy gels) that can act as dynamically functional materials have been prepared, for example, for (re-writable) information recording. Supramolecular gels can be important in controlled release applications, in oil recovery, for gelling cryogenic fuels etc. They can also serve as media for a range of applications. This tutorial review highlights some of the instructive work done by various groups to develop smart and functional gels, and covers a wide spectrum of scientific interest ranging from medicine to materials science.

Biocompatible Materials↗
PubMed2003

Preparation of small unilamellar vesicles (SUV) and biophysical characterization of their complexes with poly-l-lysine-condensed plasmid DNA.

Liposomes have numerous applications in the (bio)pharmaceutical industries as agents in the synthesis of new biomaterials for use in areas including gene delivery. There is currently a need to establish efficient scaleable methods for the manufacture of liposomes, and in the present paper we describe the operation of a new high-velocity jet homogenizer for downsizing of multilamellar large vesicles to produce small unilamellar vesicles (SUV). Measurements of size distribution of SUVs are presented and compared with mathematical simulations based on the solution of a population balance equation combined with computational-fluid-dynamics analysis of flow in the homogenizer. Anionic SUVs are produced by the new method and incubated with poly-L-lysine (PLL)-condensed plasmid DNA (pDNA) to generate complexes under different physico-chemical conditions. The colloidal properties of the resulting complexes, including their size and charge, are measured using a Zetasizer and the encapsulation efficiency is obtained experimentally using a Pico Green assay. The results show that between 85 and 95% of the PLL-pDNA condensed plasmids were encapsulated by the liposomes, the smaller liposomes being more effective in encapsulating the complexes.

Coated Materials, Biocompatible↗
PubMed2002

The neuropathogenic contributions of lysosomal dysfunction.

Multiple lines of evidence implicate lysosomes in a variety of pathogenic events that produce neurodegeneration. Genetic mutations that cause specific enzyme deficiencies account for more than 40 lysosomal storage disorders. These mostly pre-adult diseases are associated with abnormal brain development and mental retardation. Such disorders are characterized by intracellular deposition and protein aggregation, events also found in age-related neurodegenerative diseases including (i) Alzheimer's disease and related tauopathies (ii) Lewy body disorders and synucleinopathies such as Parkinson's disease, and (iii) Huntington's disease and other polyglutamine expansion disorders. Of particular interest for this review is evidence that alterations to the lysosomal system contribute to protein deposits associated with different types of age-related neurodegeneration. Lysosomes are in fact highly susceptible to free radical oxidative stress in the aging brain, leading to the gradual loss of their processing capacity over the lifespan of an individual. Several studies point to this lysosomal disturbance as being involved in amyloidogenic processing, formation of paired helical filaments, and the aggregation of alpha-synuclein and mutant huntingtin proteins. Most notably, experimentally induced lysosomal dysfunction, both in vitro and in vivo, recapitulates important pathological features of age-related diseases including the link between protein deposition and synaptic loss.

Amyloid↗
PubMed1998

Photomechanical transcutaneous delivery of macromolecules.

Transcutaneous drug delivery has been the subject of intensive research. In certain situations, rapid transcutaneous delivery is very desirable. A mechanical (stress) pulse generated by a single laser pulse was shown to transiently increase the permeability of the stratum corneum in vivo. The barrier function of the stratum corneum recovers within minutes. The increased permeability during these few minutes allows macromolecules to diffuse through the stratum corneum into the viable epidermis and dermis. Macromolecules (40 kDa dextran and 20 nm latex particles) were deposited into the skin using a photomechanical pulse generated by a single 23 ns laser pulse. This treatment can potentially be utilized in therapies that currently require occlusive dressings for hours or day(s).

Animals↗