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PubMed1992

[Vitamin D: biosynthesis, metabolism and mechanism of action at the cellular level].

The term vitamin D includes various chemical species. Vitamin D3 a true endogenous or alimentary prohormone is converted into its main metabolite, calcitriol, by successive hydroxylations in the liver in position 25 and in the kidney in position 1, the production of which is controlled by several factors including parathyroid hormone, blood calcium and phosphorus or insulin as well as by the metabolites of the hormone itself. It controls the synthesis of numerous peptides by acting on gene expression. Indeed, several structural proteins are involved including procollagen alpha 1l, core protein of proteoglycans, diverse regulatory peptides such as protooncogene c-myc and growth factors, "Tumor Necrosis Factor or TNF" and "Nerve Growth Factor or NGF" or hormones such as parathyroid hormone, and finally constitutive proteins of the mineralized tissues such as osteonectin, osteocalcin, osteopontin and calbindins. Therefore, it modulates very different cellular processes. It acts via a nuclear receptor the structure and function of which have been investigated by genetic engineering (cloning of genes encoding for the receptor and hormono-dependent peptides, transfection assays, directed mutagenesis). Actual studies investigate its role in the formation of the complex for transcription initiation near ADN sites, the "Vitamin D Responsive Element or VDRE", located upstream vitamin D-responsive genes and approximately RNA polymerase II. The receptor, which is present in many cell types at various concentrations, would determine spatial and temporal patterns of calcitriol action during development in conjunction with chromatin factors.

Cell Physiological Phenomena↗
PubMed2006

Vascular tissue engineering.

Reconstructive surgery using autologous vessels is the conventional approach for substitution of diseased vessels or for generation of bypass to improve blood supply downstream of stenosed vessels. In some circumstances the use of autologous material is not possible due to concomitant diseases or previous use, and artificial grafts must be used. Unfortunately, these grafts cannot substitute small-caliber arterial vessels because of thrombotic complications. The objective of tissue engineering at the vascular level is then to generate biological substitutes of arterial conduits with functional characteristics of native vessels, combining cellular components with biodegradable scaffolds. These research projects started in several laboratories, in the late 1990s, and have expanded in different directions using a number of experimental approaches. The objective of this review is to give an overview of the results so far obtained in this area of research, and to discuss the problems related to these investigations, at the experimental and clinical level. The article provides an overview of different biodegradable scaffolds used, experimental techniques for vessels maturation in vitro under mechanical stimulation, and of differentiated as well as precursors of vascular cells, which opens new opportunities for further development of this form of cell transplantation. Finally, the current available results in clinical research will be discussed.

Bioartificial Organs↗
PubMed2001

Antibody-based resistance to plant pathogens.

Plant diseases are a major threat to the world food supply, as up to 15% of production is lost to pathogens. In the past, disease control and the generation of resistant plant lines protected against viral, bacterial or fungal pathogens, was achieved using conventional breeding based on crossings, mutant screenings and backcrossing. Many approaches in this field have failed or the resistance obtained has been rapidly broken by the pathogens. Recent advances in molecular biotechnology have made it possible to obtain and to modify genes that are useful for generating disease resistant crops. Several strategies, including expression of pathogen-derived sequences or anti-pathogenic agents, have been developed to engineer improved pathogen resistance in transgenic plants. Antibody-based resistance is a novel strategy for generating transgenic plants resistant to pathogens. Decades ago it was shown that polyclonal and monoclonal antibodies can neutralize viruses, bacteria and selected fungi. This approach has been improved recently by the development of recombinant antibodies (rAbs). Crop resistance can be engineered by the expression of pathogen-specific antibodies, antibody fragments or antibody fusion proteins. The advantages of this approach are that rAbs can be engineered against almost any target molecule, and it has been demonstrated that expression of functional pathogen-specific rAbs in plants confers effective pathogen protection. The efficacy of antibody-based resistance was first shown for plant viruses and its application to other plant pathogens is becoming more established. However, successful use of antibodies to generate plant pathogen resistance relies on appropriate target selection, careful antibody design, efficient antibody expression, stability and targeting to appropriate cellular compartments.

Antibodies, Viral↗
PubMedDate not supplied

A novel rotating-shaft bioreactor for two-phase cultivation of tissue-engineered cartilage.

A novel rotating-shaft bioreactor (RSB) was developed for two-phase cultivation of tissue-engineered cartilage. The reactor consisted of a rotating shaft on which the chondrocyte/scaffold constructs (7.5 mm diameter x 3.5 mm thickness) were fixed and a reactor vessel half-filled with medium. The horizontal rotation of the shaft resulted in alternating exposure of the constructs to gas and liquid phases, thus leading to efficient oxygen and nutrient transfer, as well as periodically changing, mild shear stress exerting on the construct surfaces (0-0.32 dyn/cm2 at 10 rpm), as revealed by computer simulation. Strategic operation of the RSB (maintaining rotating speed at 10 rpm for 3 weeks and lowering the speed to 2 rpm in week 4) in combination with higher seeding density (6 x 10(6) chondrocytes/scaffold) and medium perfusion resulted in uniform cell distribution and increased glycosaminoglycan (3.1 mg/scaffold) and collagen (7.0 mg/scaffold) deposition. The 4-week constructs resembled native cartilages in terms of not only gross appearance and cell morphology but also distributions of glycosaminoglycan, total collagen, and type II collagen, confirming the maintenance of chondrocyte phenotype and formation of cartilage-like constructs in the RSB cultures. In summary, the novel RSB may be implicated for in vitro study of chondrogenesis and de novo cartilage development under periodic mechanical loading. With proper optimization of the culture conditions, a RSB may be employed for the production of cartilage-like constructs.

Animals↗
PubMed2004

Sintered hydroxyfluorapatites--IV: The effect of fluoride substitutions upon colonisation of hydroxyapatites by mouse embryonic stem cells.

Biodegradable scaffolds serve a central role for tissue engineering scaffolds and guiding tissue regeneration. Some of these scaffolds, including apatites, display a significant effect upon cell adhesion and cell proliferation. The incorporation of scaffold technology with the developing embryonic stem (ES) cell field and the capacity of ES cells for self-renewal and differentiation are believed to hold enormous potential for applications in biomedical research and regenerative medicine. The purpose of this work was to determine the effect of hydroxyapatite (HAP) and fluoride substitutions of HAP upon ES cell growth and colonisation. Sintered hydroxyfluorapatite discs were found to support cellular proliferation and colonisation, and the ES cells displayed a tendency for differentiation on the apatite surface as determined by reductions in colony Oct4 immunoreactivity. Fluoride-containing HAPs were found to provide equivalent support to gelatin in terms of cell numbers, yet superior support for cellular colonisation when compared to HAP. This study indicates that fluoride substitutions of HAP may represent a viable strategy for the development of certain engineered tissue replacements and tissue regeneration systems using ES cells.

Analysis of Variance↗
PubMed2006

Engineering of N-glycosylation of hepatitis C virus envelope protein E2 enhances T cell responses for DNA immunization.

N-linked oligosaccharides are known to be important in modulating immunogenicity of some viral proteins. However, the roles of N-glycans of hepatitis C virus (HCV) E2 envelope glycoprotein in specific cellular immune responses remain elusive. Previous studies showed that the epitopes aa481-500 and aa551-570 of E2 might be important for immunoreactivity and that the binding site of E2 for hCD81 is located at aa480-493 and aa544-551 within the E2 protein. Here, we made plasmids containing genes encoding either wild type or mutant E2 proteins in which N-glycosylation sites (N560NT and N576ST) close to these important regions were mutated separately or in combination. The immunogenicities of wild type E2 and three mutated E2 proteins were analyzed in BALB/c mice using DNA vaccination. The E2-M2 mutant (at N576ST) significantly enhanced (compared to control) E2-specific CTL activity (P<0.05), expression of IFN-gamma (P<0.05) and suppression of tumor growth (P<0.05). Also, high IgG2a/IgG1 ratios were elicited in a Th1-type response. These results indicate that engineering of the N-glycosylation site N576ST of HCV E2 protein enhances specific cellular immune responses, providing insights into the development of E2-based DNA vaccines with enhanced immunogenicity.

Animals↗
PubMed2000

Conditionally immortalized cell lines, engineered to produce and release GABA, modulate the development of behavioral seizures.

Transplantation of genetically engineered cells can provide sustained focal delivery of naturally occurring molecules, including neurotransmitters and growth factors. We have engineered immortalized mouse cortical neurons and glia to deliver GABA by driving GAD(65) expression. Engineered cell lines showed GAD(65) mRNA expression, enzymatic activity, and GABA release. In vitro, basal flux of GABA was approximately 20% of total cellular GABA. We transplanted these GABA-producing cells bilaterally into either the anterior or the posterior substantia nigra of 43 rats. The rats were subsequently kindled through an electrode placed in the entorhinal cortex. GABA-producing cells, but not beta-galactosidase-producing cells, affected kindling rates. The number of stimulations needed to reach the first stage-5 seizure and to achieve full kindling differed significantly between the anterior and posterior transplantation sites when GAD(65)-producing cells were transplanted but not when beta-galactosidase-producing cells were transplanted. Our data show that transplanted engineered cells can make and release GABA at physiologically meaningful concentrations.

Animals↗
PubMed1998

John von Neumann: the founding father of artificial life.

Aside from being known for his contributions to mathematics and physics, John von Neumann is considered one of the founding fathers of computer science and engineering. Not only did he do pioneering work on sequential computing systems, but he also carried out a major investigation of parallel architectures, leading to his work on cellular automata. His exceptional vision and daring, borrowing from biology the concept of genomic information even before the discovery of DNA's double helix, led him to propose the concept of self-reproducing automata.

Artificial Intelligence↗
PubMed2002

Metabolic flux analysis using mass spectrometry.

Detailed knowledge on carbon flux distributions is crucial for the understanding and targeted optimization of cellular systems. Analytical methods to identify the topology of metabolic networks and to quantify fluxes through its different pathways are therefore in the core of metabolic engineering. An elegant approach for metabolic flux analysis is provided by tracer experiments. In such studies tracer substrates with stable isotopes such as 13C are applied and the labeling pattern of metabolites is subsequently measured. Detailed flux distributions can be obtained by a combination of tracer experiments and stoichiometric balancing. In recent years, mass spectrometry (MS) has emerged as an interesting method for labeling measurements in metabolic flux analysis and provided valuable insights into the cellular metabolism. The present review provides an overview on current experimental and modeling tools for metabolic flux analysis by MS. The application of MS for flux analysis is illustrated by examples from the literature for various biological systems, including bacteria, fungi, tissue cultures and in vivo studies in humans.

Carbon Isotopes↗
PubMed2006

Collagen synthesis by mesenchymal stem cells and aortic valve interstitial cells in response to mechanical stretch.

OBJECTIVE: The synthesis of appropriate extracellular matrix by cells in tissue engineered heart valve constructs will be important for the maintenance of valve cusp integrity and function. We have examined and compared the capacity of mesenchymal stem cells to synthesise collagen in response to stretch in comparison with native aortic valve interstitial cells. METHODS: Cells were stretched on a Flexercell FX4000 apparatus and total collagen synthesis was measured by the incorporation of [3H]-proline. The effect of stretch on gene expression of different collagen types was assessed by RT-PCR. RESULTS: There was a significant (p<0.01) increase in [3H]-proline incorporation into stretched valve cells at 10%, 14% and 20% stretch. The response of mesenchymal stem cells at 14% stretch was similar to that seen in the valve cells. Incorporation of [3H]-proline into soluble proteins in the cell media was significantly higher (p<0.01) only at 14% and 20% stretch in valve interstitial cells. These effects were shared with mesenchymal stem cells at 14% stretch. RT-PCR experiments demonstrated that 14% stretch up-regulated levels of mRNA for COL3A1 gene (type III collagen) but did not increase the expression of COL1A1 gene (type I collagen) in valve interstitial cells. However, both collagen genes could be detected in non-stretched and stretched mesenchymal stem cells. There was no evidence that the mesenchymal stem cells had started to adopt an osteoblastic cell phenotype in response to stretch. CONCLUSIONS: Collagen synthesis by valve interstitial cells is dependent upon the degree and duration of stretch. This response can be mimicked closely by exposure of mesenchymal stem cells to the same stretching profile. These properties could have important implications for the choice of cells and programme of conditioning with which to tissue engineer heart valves.

Adolescent↗
PubMed1999

Interpretation of phenotype in genetically engineered mice.

BACKGROUND AND PURPOSE: In mice, genetic engineering involves two general approaches-addition of an exogenous gene, resulting in transgenic mice, and use of knockout mice, which have a targeted mutation of an endogenous gene. The advantages of these approaches is that questions can be asked about the function of a particular gene in a living mammalian organism, taking into account interactions among cells, tissues, and organs under normal, disease, injury, and stress situations. METHODS: Review of the literature concentrating principally on knockout mice and questions of unexpected phenotypes, lack of phenotype, redundancy, and effect of genetic background on phenotype will be discussed. CONCLUSION: There is little gene redundancy in mammals; knockout phenotypes exist even if none are immediately apparent; and investigating phenotypes in colonies of mixed genetic background may reveal not only more phenotypes, but also may lead to better understanding of the molecular or cellular mechanism underlying the phenotype and to discovery of modifier gene(s).

Animals↗
PubMed2005

Characterization of protein release from photocrosslinkable hyaluronic acid-polyethylene glycol hydrogel tissue engineering scaffolds.

The goal of this work was to utilize the naturally derived bioactive polymer hyaluronic acid (HA) to create a combination tissue engineering scaffold and protein delivery device. HA is a non-immunogenic, non-adhesive glycosaminoglycan that plays significant roles in several cellular processes, including angiogenesis and the regulation of inflammation. In previous work, we created photopolymerizable glycidyl methacrylate-hyaluronic acid (GMHA) hydrogels that had controlled degradation rates, were cytocompatible, and were able to be modified with peptide moieties. In the present studies, we characterized the release of a model protein, bovine serum albumin (BSA), from GMHA and GMHA-polyethylene glycol (PEG) hydrogels. Although BSA could be released rapidly (> 60% within 6 h) from 1% GMHA hydrogels, we found that increasing either the GMHA or the PEG concentrations could lengthen the duration of protein delivery. Preliminary size exclusion chromatography studies indicated that the released BSA was almost entirely in its native monomeric form. Lastly, protein release was extended to several weeks by suspending BSA-poly(lactic-co-glycolic acid) microspheres within the hydrogel bulk. These initial studies indicate that the naturally derived biopolymer HA can be employed to design novel photopolymerizable composites that are suitable for delivering stable proteins from scaffolding in tissue engineering applications.

Coated Materials, Biocompatible↗
PubMed2005

Plant-water relations and the fibre saturation point.

This review is about the behaviour of water in cell walls. The aim is to introduce to biologists the concept of the fibre saturation point (FSP), and the related research of material scientists and engineers on the thermodynamics and chemistry of water in timber and wood. In the review, we first summarise what the FSP is, why it is important and how the FSP is routinely used by engineers and material scientists to estimate the volume fractions of solid, liquid and gas phases in bulk timber. We then show that the FSP can be intuitively understood using equilibrium thermodynamics. That analysis shows that the FSP is based on the concept that a certain (and repeatable) amount of water is chemically bound to cellulose and other substances in wood. That water, sometimes called bound water, exists in a water-cell wall mixture. The noted physical chemist and wood scientist, A. J. Stamm, called this mixture a 'solid solution'. In timber, the 'solid solution' is considered a separate phase from adjacent water in either a pure liquid phase or a vapour phase. Following that, we examine the FSP and wood-water dynamics at the molecular and cellular level. Despite differences between timber and living trees, we conclude that the FSP-based framework long used by material scientists and engineers is likely to be useful to biologists.

Cell Wall↗
PubMed1990

Altered gene expression in plants due to trans interactions between homologous genes.

We have attempted to explain co-suppression in terms of a hypothesis whereby homologous sequences are able to interact somatically in trans, in a manner influenced by sequence context or location. We have speculated that there might be mechanistic similarities between co-suppression and some other trans interaction and epigenetic phenomena in plants, fungi and animals; and that it might be the sequence context in which a gene lies, or its location in the genome that influences the likelihood that it will participate in these phenomena. We suspect that elucidation of the mechanisms behind these phenomena will play a role in developing a better understanding of the relationship between nuclear architecture and gene expression. This, in turn, will be helpful in understanding the developmental regulatory mechanisms that exert control over somatic trans interactions in plants, and perhaps in understanding some aspects of the basis of cellular differentiation in development.

Acyltransferases↗
PubMed2002

Autologous and MHC class I-negative allogeneic tumor cells secreting IL-12 together cure disseminated A20 lymphoma.

Cytokine gene-modified tumor cells have increased immunogenicity and retain the antigenic repertoire of a particular neoplasia. However, practical concerns have led to an increased interest in allogeneic gene-transduced bystander cells as a broader source of cytokines for autologous tumor cell-based vaccines. Here, we show that allogeneic B78H1 major histocompatibility complex (MHC) class I-negative and -positive (H-2K(b)- and D(b)-transfected) cells induced cytotoxic T lymphocytes (CTLs) and protection in BALB/c mice at comparable levels in response to a challenge with C26 (H-2(d)) colon carcinoma cells sharing the tumor-associated antigen envelope glycoprotein 70 (env-gp70) with both cell lines. Class I-negative B78H1 cells transduced to express interleukin-12 (IL-12) and mixed with autologous A20 tumor cells led to eradication of preestablished A20 lymphoma in 50% or 100% of treated mice after 3 or 4 vaccinations, respectively, whereas A20 cells alone or mixed with nontransduced B78H1 cured none or 50% of mice after 3 or 4 vaccinations, respectively. Immunization with the IL-12-producing bystander cell line increased tumor-specific proliferation and type 1 cytokine production by CD4(+) T cells. By contrast, CD4 T-cell function appeared impaired after immunization with A20 cells alone or mixed with B78H1 cells. Indeed, only CD4(+) T cells from IL-12-treated mice could be restimulated with anti-OX40 monoclonal antibody (mAb) in place of a fourth cellular boost. Moreover, the IL-12-based tumor vaccine induced expansion of tumor-specific interferon-gamma (IFN-gamma)-producing CD8(+) T cells. These results are clinically relevant for the development of feasible IL-12 cancer vaccines based on engineered class I-negative bystander cells.

Animals↗
PubMed2000

Peptide-mediated cellular delivery.

Peptide-mediated molecular therapeutic delivery systems have recently emerged as an alternative means to effectively substitute or augment present gene therapy technologies, e.g., TAT, VP22, engineered peptides. These systems show great promise for the elimination of the main bottleneck to safe, efficient, targeted gene therapy delivery and are able to efficiently introduce DNA, antisense peptide nucleic acids, oligonucleotides, small molecules and proteins into cells both in vitro and in vivo. They are versatile and easily designed to incorporate a number of specific attributes required for efficient cargo delivery. A fundamentally new property of these moieties will allow the therapeutic intervention in the biochemistry of the target cell without the need to alter its genome.

Animals↗
PubMed2005

Observation of osteogenic differentiation cascade of living mesenchymal stem cells on transparent hydroxyapatite ceramics.

The use of bioceramics and cultured cells for tissue engineering is a novel approach, which is available in a wide variety of clinical situations. The approach requires apparent verification of the cellular functions occurring on the ceramic surface, and these functions could be monitored by microscopic observation of the cultured living cells on the ceramic material. However, such observation is difficult due to the opaque nature of ordinary ceramics. To overcome this drawback, we used transparent hydroxyapatite (tHA) ceramics as a culture substrate and a transgenic rat having an enhanced green fluorescent protein (EGFP)-expressing gene as the cell source. Marrow mesenchymal stem cells (MSC) were obtained from the rat and cultured on both tHA ceramics and a tissue culture polystyrene (TCPS) dish. One hour after the cell seeding, many MSC had attached and showed initial cell spreading. The attachment and spreading were more obvious 5h after the seeding. Following the culture in the osteogenic condition, the cells differentiated into osteoblasts, which fabricated bone matrix on the culture substrate. The phenomena were similarly observed on both the tHA ceramics and TCPS substrata. These results confirm the excellent properties of tHA ceramics, which support cell attachment, proliferation, and differentiation. Transparent materials make us know the biological usefulness of ceramics in tissue-engineering field.

Animals↗