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At least 217 records · Page 12Linked to original sources

PubMed2000

A synthetic oscillatory network of transcriptional regulators.

Networks of interacting biomolecules carry out many essential functions in living cells, but the 'design principles' underlying the functioning of such intracellular networks remain poorly understood, despite intensive efforts including quantitative analysis of relatively simple systems. Here we present a complementary approach to this problem: the design and construction of a synthetic network to implement a particular function. We used three transcriptional repressor systems that are not part of any natural biological clock to build an oscillating network, termed the repressilator, in Escherichia coli. The network periodically induces the synthesis of green fluorescent protein as a readout of its state in individual cells. The resulting oscillations, with typical periods of hours, are slower than the cell-division cycle, so the state of the oscillator has to be transmitted from generation to generation. This artificial clock displays noisy behaviour, possibly because of stochastic fluctuations of its components. Such 'rational network design may lead both to the engineering of new cellular behaviours and to an improved understanding of naturally occurring networks.

Bacterial Proteins↗

PubMed1992

Expression of cloned homologous fermentative genes in Clostridium acetobutylicum ATCC 824.

We have previously cloned the acetone-formation pathway gene, encoding acetoacetate decarboxylase (adc), and butyrate-formation pathway gene, encoding phosphotransbutyrylase (ptb), of Clostridium acetobutylicum ATCC 824 in Escherichia coli. Here we report their subcloning in Bacillus subtilis and transfer to strain ATCC 824 via electrotransformation, where the corresponding enzyme activities were expressed at elevated levels, using pFNK1, a new B. subtilis/C. acetobutylicum shuttle vector. Plasmid pFNK1 was used because shuttle vectors that function in E. coli were unable to electrotransform ATCC 824 unless they became deleted in the E. coli-plasmid regions. The difficulties with shuttle vectors that function in E. coli are probably due to the presence of a restriction endonuclease in ATCC 824. This endonuclease recognizes the sequence 5'-GCNGC-3', which is prevalent in E. coli plasmids but occurs infrequently in pFNK1 and C. acetobutylicum genes. Cloning of genes in C. acetobutylicum is critical for redirecting the cellular metabolism (metabolic engineering) as well as for genetic studies of this industrial organism.

Bacillus subtilis↗

PubMed2003

Colocalization of multiple DNA double-strand breaks at a single Rad52 repair centre.

DNA double-strand break repair (DSBR) is an essential process for preserving genomic integrity in all organisms. To investigate this process at the cellular level, we engineered a system of fluorescently marked DNA double-strand breaks (DSBs) in the yeast Saccharomyces cerevisiae to visualize in vivo DSBR in single cells. Using this system, we demonstrate for the first time that Rad52 DNA repair foci and DSBs colocalize. Time-lapse microscopy reveals that the relocalization of Rad52 protein into a focal assembly is a rapid and reversible process. In addition, analysis of DNA damage checkpoint-deficient cells provides direct evidence for coordination between DNA repair and subsequent release from checkpoint arrest. Finally, analyses of cells experiencing multiple DSBs demonstrate that Rad52 foci are centres of DNA repair capable of simultaneously recruiting more than one DSB.

Bacterial Proteins↗

PubMed2000

Tyrosine phosphorylation of the Helicobacter pylori CagA antigen after cag-driven host cell translocation.

Helicobacter pylori strains associated with severe tissue damage and inflammation possess a unique genetic locus, cag, containing 31 genes originating from a distant event of horizontal transfer and retained as a pathogenicity island. The cag system is an Helicobacter-specific type IV secretion engine involved in cellular responses like induction of pedestals, secretion of IL-8, and phosphorylation of proteic targets. It has previously been reported that cocultivation of epithelial cells with Helicobacter pylori triggers signal transduction and tyrosine phosphorylation of a 145-kDa putative host cell protein. Herein, we demonstrate that this protein is not derived from the host but rather is the bacterial immunodominant antigen CagA, a virulence factor commonly expressed in peptic ulcer disease and thought to be an orphan of a specific biological function. Thus, CagA is delivered into the epithelial cells by the cag type IV secretion system where it is phosphorylated on tyrosine residues by an as yet unidentified host cell kinase and wired to eukaryotic signal transduction pathways and cytoskeletal plasticity.

Antigens, Bacterial↗

PubMed2001

Down-modulation of type 1 interferon responses by receptor cross-competition for a shared Jak kinase.

In contrast to the large number of class I and II cytokine receptors, only four Janus kinase (Jak) proteins are expressed in mammalian cells, implying the shared use of these kinases by many different receptor complexes. Consequently, if receptor numbers exceed the amount of available Jak, cross-interference patterns can be expected. We have engineered two model cellular systems expressing two different exogenous Tyk2-interacting receptors. A receptor chimera was generated wherein the extracellular part of the interferon type 1 receptor (Ifnar1) component of the interferon-alpha/beta receptor is replaced by the equivalent domain of the erythropoietin receptor. Despite Tyk2 activation, erythropoietin treatment of cells expressing this erythropoietin receptor/Ifnar1 chimera did not evoke any detectable IFN-type response. However, a dose-dependent interference with signal transduction via the endogenous Ifnar complex was found for STAT1, STAT2, STAT3, Tyk2, and Jak1 activation, for gene induction, and for antiviral activity. In a similar approach, cells expressing the beta1 chain of the interleukin-12 receptor showed a reduced transcriptional response to IFN-alpha as well as reduced STAT and kinase activation. In both model systems, titration of the Tyk2 kinase away from the Ifnar1 receptor chain accounts for the observed cross-interference.

Animals↗

PubMed2001

Experimental culture conditions are critical for ex vivo expansion of hematopoietic cells.

The ex vivo expansion of hematopoietic stem cells (HSC) for clinical use is now recognized to be a feasible and very promising approach for hematotherapy. Expansion of specific HSC subsets is required for different clinical applications, for example, to increase the number of mature cells, to produce specific cells for adoptive therapy, or to increase the number of primitive stem cells available for engraftment. Although hematopoietic growth factors can play an important role in this setting, in this review we emphasize that other variables affect the outcome of stem and progenitor cell expansion. These variables include the serum supplement, the purity of CD34(+) cells, the initial cell concentration, and the duration of culture. It is also essential to define standard culture conditions for normal stem cells and to limit or prevent expansion of residual tumor cells. In clinical applications, determination of the hematopoietic value of the expanded population is mandatory. Thus, we have to demonstrate the expansion of primitive hematopoietic progenitor and stem cells, with maintenance of their hematopoietic potential as assessed by in vitro or in vivo assays. We draw attention to the challenges in the clinical application of ex vivo expansion. These include the establishment of well-defined experimental conditions and the determination of the hematopoietic value of the expanded grafts, whatever the graft source: bone marrow, mobilized peripheral blood, or cord blood. Future studies hopefully will optimize these procedures and allow not only expansion but engineering of defined cellular functions as HSCs grow under defined conditions.

Animals↗

PubMed1991

Recent progress in bone induction by osteogenin and bone morphogenetic proteins: challenges for biomechanical and tissue engineering.

Implantation of demineralized bone matrix results in local bone induction. Bone induction is a sequential biological chain reaction that consists of chemotaxis and proliferation of mesenchymal cells and differentiation of bone. Osteogenin, a bone morphogenetic protein has been purified and the amino acid sequence determined. Recently a family of bone morphogenetic proteins have been cloned and expressed by recombinant DNA technology. The availability of growth and morphogenetic factors will permit the rational design of new bone. The challenge for the biomechanical engineer is to attain mechanically optimal and functionally adaptive new bone for various skeletal prostheses. We are on the threshold for fabrication of new bone based on sound architectural design principles of tissue engineering based on cellular and molecular biology of growth and differentiation factors.

Animals↗

PubMed2001

Biosynthesis of complex polyketides in a metabolically engineered strain of E. coli.

The macrocyclic core of the antibiotic erythromycin, 6-deoxyerythronolide B (6dEB), is a complex natural product synthesized by the soil bacterium Saccharopolyspora erythraea through the action of a multifunctional polyketide synthase (PKS). The engineering potential of modular PKSs is hampered by the limited capabilities for molecular biological manipulation of organisms (principally actinomycetes) in which complex polyketides have thus far been produced. To address this problem, a derivative of Escherichia coli has been genetically engineered. The resulting cellular catalyst converts exogenous propionate into 6dEB with a specific productivity that compares well with a high-producing mutant of S. erythraea that has been incrementally enhanced over decades for the industrial production of erythromycin.

Acyl Coenzyme A↗

PubMed2002

In vivo analysis of retroviral enhancer mutations in hematopoietic cells: SP1/EGR1 and ETS/GATA motifs contribute to long terminal repeat specificity.

The objective of this work was to identify, in the context of chromosomally integrated DNA, the contribution of defined transcription factor binding motifs to the function of a complex retrovirus enhancer in hematopoietic cells in vivo. Repopulating murine hematopoietic cells were transduced with equal gene dosages of replication-incompetent retrovirus vectors encoding enhanced green fluorescent protein. Enhancer sequences were derived from mouse spleen focus-forming virus. Destruction of GC-rich sites representing overlapping targets for SP1 or EGR1 uniformly attenuated gene expression (approximately 25 to 70% of wild-type levels) in all hematopoietic lineages, as shown by multicolor flow cytometry of peripheral blood and bone marrow cells at various time points posttransplantation. In contrast, a point mutation within a dual ETS/GATA motif that abolished transactivation by ETS factors but not by GATA-1 slightly increased activity in erythroid cells and significantly attenuated enhancer function in T lymphocytes. This study shows that controlled gene transfer in transplantable hematopoietic cells allows a functional analysis of distinct cis elements within a complex retrovirus enhancer, as required for the characterization and engineering of various cellular and viral regulatory sequences in basic research and gene therapy.

Animals↗

PubMed2003

Body wall repair using small intestinal submucosa seeded with cells.

BACKGROUND/PURPOSE: Prosthetic repair of large ventral abdominal wall defects has been associated with high complication rates. This study was aimed at applying tissue engineering to body wall replacement. METHODS: Syngeneic Lewis rats underwent harvest of skeletal muscle specimens. Once expanded in vitro, skeletal muscle cells or fibroblasts were suspended in a collagen gel. All animals underwent creation of a 2.5- x 3-cm abdominal wall defect. The defect was repaired with the cell-seeded gel placed in between 2 pieces of small intestinal submucosa (SIS). The control group was repaired by SIS with acellular gel. Animals were killed at different time-points for histologic and mechanical examination. Statistical analysis was by analysis of variance (ANOVA). RESULTS: Abdominal wall hernia was present in 6 of 24 fibroblast-seeded constructs (25%), 5 of 21 skeletal muscle cell-seeded constructs (23.9%), and 16 of 21 acellular grafts (76.2%), respectively (P <.05). At harvest, cell-seeded constructs were thicker with better cellular infiltration, whereas acellular grafts were thin, low in cell density, and poor in mechanical resistance. CONCLUSIONS: Unlike acellular collagen matrices, engineered cellular constructs have better cell infiltration and mechanical performance. Tissue engineering may be a viable alternative for body-wall replacement.

Abdominal Wall↗

PubMed2001

Genetically encoded optical sensors of neuronal activity and cellular function.

Fluorescent proteins (FPs) have been engineered to produce an optical report in response to cellular signals. FP fluorescence can be made directly sensitive to the chemical environment, via specific mutations of or around the chromophore. Alternatively, FPs can be made indirectly sensitive to cellular signals by their fusion to 'detector' proteins that respond to specific cellular signals with structural rearrangements that act on the FP to alter fluorescence. These optical sensors of membrane voltage, neurotransmitter release, and intracellular messengers, including powerful new sensors of Ca(2+), cyclic nucleotides and nitric oxide, are likely to provide new insights into the workings of cellular signals and of information processing in neural circuits.

Animals↗

PubMed2006

RNA synthetic biology.

RNA molecules play important and diverse regulatory roles in the cell by virtue of their interaction with other nucleic acids, proteins and small molecules. Inspired by this natural versatility, researchers have engineered RNA molecules with new biological functions. In the last two years efforts in synthetic biology have produced novel, synthetic RNA components capable of regulating gene expression in vivo largely in bacteria and yeast, setting the stage for scalable and programmable cellular behavior. Immediate challenges for this emerging field include determining how computational and directed-evolution techniques can be implemented to increase the complexity of engineered RNA systems, as well as determining how such systems can be broadly extended to mammalian systems. Further challenges include designing RNA molecules to be sensors of intracellular and environmental stimuli, probes to explore the behavior of biological networks and components of engineered cellular control systems.

Computational Biology↗

PubMed2005

Smart materials as scaffolds for tissue engineering.

In this review, we focused our attention on the more important natural extracellular matrix (ECM) molecules (collagen and fibrin), employed as cellular scaffolds for tissue engineering and on a class of semi-synthetic materials made from the fusion of specific oligopeptide sequences, showing biological activities, with synthetic materials. In particular, these new "intelligent" scaffolds may contain oligopeptide cleaving sequences specific for matrix metalloproteinases (MMPs), integrin binding domains, growth factors, anti-thrombin sequences, plasmin degradation sites, and morphogenetic proteins. The aim was to confer to these new "intelligent" semi-synthetic biomaterials, the advantages offered by both the synthetic materials (processability, mechanical strength) and by the natural materials (specific cell recognition, cellular invasion, and the ability to supply differentiation/proliferation signals). Due to their characteristics, these semi-synthetic biomaterials represent a new and versatile class of biomimetic hybrid materials that hold clinical promise in serving as implants to promote wound healing and tissue regeneration.

Animals↗

PubMed1997

Engineering protein mechanics: inhibition of concerted motions of the cellular retinol binding protein by site-directed mutagenesis.

Recently we reported on the dynamic properties of the cellular retinol binding protein, a member of the fatty acid binding protein family. A few conserved glycines were identified as important for producing the conformational changes necessary for the uptake and release of retinol. Here, we describe a multidisciplinary analysis of a genetically engineered mutation of one of these glycines (Gly67), designed to inhibit an observed hinge bending motion. The correctly folded mutant protein is unable to bind retinol. Analysis of the molecular dynamics simulations of the mutant and wild type protein using the essential dynamics method shows that the mutation indeed inhibits the hinge bending motions which are important for retinol binding.

Binding Sites↗

PubMed1975

[Cellular responses to vibration during dental drilling with belt drive motor engine (in vitro) (author's transl)].

There have been many reports on the pulpal reaction caused by vibration during high speed dental drilling with air turbine hand-piece. It is, however, not clear pulpal responses to vibration of low speed drilling with 7 000 rpm of belt drive motor engine which produces a fundamental vibration of about 170 cps having most unpleasant sensation at vestibular apparatus. Here, cellular responses of L strain cells to vibration derived from 7 000 rpm and 15 000 rpm of belt drive motor engine were observed in vitro system. There is no difference between the both cellular responses to the vibration of 7 000 rpm and 15 000 rpm. Suspended free cells were easily influenced and degenerated by the vibration, and remarkably checked in their cell-multiplication. On the contrary, in the cells adhered to the substratum of glassware, no degeneration and no inhibitory effect were observed. And the cells showed reversible response of cytoplasmic shrinkage only.

Cell Adhesion↗

PubMed2003

Characterization of cellular carriers for use in injectable tissue-engineering composites.

Injectable composite tissue-engineering scaffolds are systems that incorporate individual cell carriers within a gel delivery matrix. This study assessed low-temperature casting as a possible method to produce synthetic cell-carrier beads. Porous poly-L-lactide beads were manufactured by low-temperature casting. Two porogens, either glucose or sodium chloride, were incorporated into the beads and subsequently leached. Beads were seeded with primary culture aortic smooth muscle rat cells and were evaluated over a 13-day period using a series of chemical, biochemical, and histological assays. Results indicate that low-temperature casting is a viable technique to produce injectable beads on the order of 1.5-2.0 mm. The manufactured beads supported smooth muscle cell attachment and proliferation; where the beads formed with sodium chloride allowed enhanced proliferation. Differences in physical qualities, namely buoyancy and topography, were dependent on porogen selection and may provide a mechanism for bead and composite customization.

Animals↗

PubMed2005

Microfabrication of poly (glycerol-sebacate) for contact guidance applications.

Controlling cell orientation and morphology through topographical patterning is a phenomenon that is applicable to a wide variety of medical applications such as implants and tissue engineering scaffolds. Previous work in this field, termed contact guidance, has demonstrated the application of this cellular response on a wide variety of material substrates such as silicon, quartz, glass, and poly(di-methyl siloxane) typically using ridge-groove geometries with sharp feature edges. One limitation of these studies in terms of biomedical applications is the choice of material. Therefore, demonstrating contact guidance and topography in a biodegradable material platform is a promising strategy for controlling cellular arrangements in tissue engineering scaffolds. This study investigates several strategies to advance contact guidance strategies and technology to more practical applications. Flexible biodegradable substrates with rounded features were fabricated by replica-molding poly(glycerol-sebacate) on sucrose-coated microfabricated silicon. Bovine aortic endothelial cells were cultured on substrates with microstructures between 2 and 5 microm in wavelength and with constant feature depth of 0.45 microm. Cells cultured on substrates with smaller pitches exhibited a substantially higher frequency of cell alignment and smaller circularity index. This work documents the first known use of using a flexible, biodegradable substrate with rounded features for use in contact guidance applications. The replica-molding technique described here is a general process that can be used to fabricate topographically patterned substrates with rounded features for many biomaterials. Furthermore, these results may lead to further elucidation of the mechanism of cell alignment and contact guidance on microfabricated substrates.

Animals↗

PubMed2002

Growth factors for sequential cellular de- and re-differentiation in tissue engineering.

A model system for the in vitro generation of cartilaginous constructs was used to study a tissue engineering paradigm whereby sequentially applied growth factors promoted chondrocytes to first de-differentiate into a proliferative state and then re-differentiate and undergo chondrogenesis. Early cultivation in medium with supplemental TGF-beta1/FGF-2 doubled cell fractions in 2-week constructs compared to unsupplemented controls. Subsequent culture with supplemental IGF-I yielded large 4-week constructs with high fractions of cartilaginous extracellular matrix (ECM) and high compressive moduli, whereas prolonged culture with supplemental FGF-2 yielded small 4-week constructs with low ECM fractions and moduli. Sequential supplementation with TGF-beta1/FGF-2 and then IGF-I yielded 4-week constructs with type-specific mRNA expression and protein levels that were high for type II and negligible for type I collagen, in contrast to other growth factor regimens studied. The data demonstrate that structural, functional, and molecular properties of engineered cartilage can be modulated by sequential application of growth factors.

Animals↗