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PubMed2006

Viral-based modelling and correction of neurodegenerative diseases by RNA interference.

Experimental recapitulation of recessive human genetic neurodegenerative disease in rodents can be classically addressed through genetic disruption of the related gene. Although very informative, this specific gene targeting is restricted to mice and precludes a species scale-up towards non-human primates. Concomitantly, this requirement to silence a specific gene in a broad range of animal models is important in the design of therapeutic approaches to dominantly inherited neurodegenerative diseases. The emergence of RNA interference (RNAi), a highly specific mechanism of post-translational gene silencing, has opened a plethora of biological application ranging from reverse genetic analysis to therapeutic schemes. Recombinant viral vectors, by promoting a long-lasting delivery of genetic instructions in a broad range of cellular types of different species origins, represent potential platforms mandating silencing of specific gene products through RNAi. This review aims at providing an overview of the different viral systems engineered so far for efficient in vitro and in vivo delivery of RNAi instructions. Additionally, the potential of RNAi for functional analysis and therapy for polyglutamine disorders or amyotrophic lateral sclerosis is discussed.

Animals↗

PubMed2003

Identification of novel protein-protein interactions using a versatile mammalian tandem affinity purification expression system.

Identification of protein-protein interactions is essential for elucidating the biochemical mechanism of signal transduction. Purification and identification of individual proteins in mammalian cells have been difficult, however, due to the sheer complexity of protein mixtures obtained from cellular extracts. Recently, a tandem affinity purification (TAP) method has been developed as a tool that allows rapid purification of native protein complexes expressed at their natural level in engineered yeast cells. To adapt this method to mammalian cells, we have created a TAP tag retroviral expression vector to allow stable expression of the TAP-tagged protein at close to physiological levels. To demonstrate the utility of this vector, we have fused a TAP tag, consisting of a protein A tag, a cleavage site for the tobacco etch virus (TEV) protease, and the FLAG epitope, to the N terminus of human SMAD3 and SMAD4. We have stably expressed these proteins in mammalian cells at desirable levels by retroviral gene transfer and purified native SMAD3 protein complexes from cell lysates. The combination of two different affinity tags greatly reduced the number of nonspecific proteins in the mixture. We have identified HSP70 as a specific interacting protein of SMAD3. We demonstrated that SMAD3, but not SMAD1, binds HSP70 in vivo, validating the TAP purification approach. This method is applicable to virtually any protein and provides an efficient way to purify unknown proteins to homogeneity from the complex mixtures found in mammalian cell lysates in preparation for identification by mass spectrometry.

Amino Acid Sequence↗

PubMed1990

Strategies for the analysis of oncogene overexpression. Studies of the neu oncogene in breast carcinoma.

The development of a consistent strategy for the analysis of oncogene expression at the cellular level is essential for understanding the roles of these genes in the development and progression of human neoplasia. Detection of the neu oncogene products in breast carcinoma was selected as a model for analysis of oncogene expression. Fifty-two primary human breast carcinomas were evaluated by quantitation of neu DNA amplification and mRNA expression and by localization of neu mRNA and protein (p 185) at the cellular level by in situ hybridization (ISH) and immunohistochemistry (IHC). The specificity and sensitivity of the molecular and immunologic probes for neu were established with the use of genetically engineered cell lines that overexpressed either neu or epidermal growth factor receptor (EGFR). Twenty-nine percent of breast carcinomas demonstrated neu DNA amplification and mRNA overexpression, and there was close correlation between the level of neu mRNA expression and detection of neu gene products by ISH and IHC. Thirty-two percent of carcinomas demonstrated neu mRNA overexpression by ISH. The immunohistochemical method using TA1 monoclonal antibody for p185 was exquisitely sensitive in acetone-fixed frozen sections and provided an excellent approach for judging overexpression as confirmed by the various molecular analyses. All areas of nonmalignant breast epithelium stained weakly, and a wide range of staining intensity was observed in malignant breast epithelium, with 31% of carcinomas judged to be p185 overexpressors. Heterogeneous expression of p185 was seen in some carcinomas. This study provides a strategic approach for the evaluation of oncogene expression in human tumors.

Antibodies, Monoclonal↗

PubMed2002

Flux through citrate synthase limits the growth of ethanologenic Escherichia coli KO11 during xylose fermentation.

Previous studies have shown that high levels of complex nutrients (Luria broth or 5% corn steep liquor) were necessary for rapid ethanol production by the ethanologenic strain Escherichia coli KO11. Although this strain is prototrophic, cell density and ethanol production remained low in mineral salts media (10% xylose) unless complex nutrients were added. The basis for this nutrient requirement was identified as a regulatory problem created by metabolic engineering of an ethanol pathway. Cells must partition pyruvate between competing needs for biosynthesis and regeneration of NAD(+). Expression of low-K(m) Zymomonas mobilis pdc (pyruvate decarboxylase) in KO11 reduced the flow of pyruvate carbon into native fermentation pathways as desired, but it also restricted the flow of carbon skeletons into the 2-ketoglutarate arm of the tricarboxylic acid pathway (biosynthesis). In mineral salts medium containing 1% corn steep liquor and 10% xylose, the detrimental effect of metabolic engineering was substantially reduced by addition of pyruvate. A similar benefit was also observed when acetaldehyde, 2-ketoglutarate, or glutamate was added. In E. coli, citrate synthase links the cellular abundance of NADH to the supply of 2-ketoglutarate for glutamate biosynthesis. This enzyme is allosterically regulated and inhibited by high NADH concentrations. In addition, citrate synthase catalyzes the first committed step in 2-ketoglutarate synthesis. Oxidation of NADH by added acetaldehyde (or pyruvate) would be expected to increase the activity of E. coli citrate synthase and direct more carbon into 2-ketoglutarate, and this may explain the stimulation of growth. This hypothesis was tested, in part, by cloning the Bacillus subtilis citZ gene encoding an NADH-insensitive citrate synthase. Expression of recombinant citZ in KO11 was accompanied by increases in cell growth and ethanol production, which substantially reduced the need for complex nutrients.

Citrate (si)-Synthase↗

PubMed2007

High content screening as a screening tool in drug discovery.

In most pharmaceutical and biotechnology companies there is a need to always improve the quality of lead candidates. This demand resulted in the use of cell-based screening as a method of choice in drug discovery. High content screening (HCS) is multiplexed, functional cell-based screening. HCS can be used in all aspects of drug discovery as an engine for driving lead discovery. The biological applications of HCS have been implemented in research in signaling, cell shape changes and toxicology. HCS has enabled an insight in the cellular effects of our clinical candidates in multiple cellular phenomena like dual reporter assay, subcellular target translocation and cellular morphology. Discovery of therapeutic protein and small molecule converge on diseases in therapeutic areas such neurological disorders and autoimmune diseases. HCS is used for assay development, primary, secondary screening and toxicology testing. In this chapter, the use of HCS assays in drug discovery is described and highlight the necessary step to set-up successfully these assays for screening.

Animals↗

PubMed2002

Archaeal tetraether lipids: unique structures and applications.

The extremely stable biomolecules manufactured by organisms from extreme environments are of great scientific and engineering interest in the development of robust and stable industrial biocatalysts. Identification of molecules that impart stability under extremes will also have a profound impact on our understanding of cellular survival. This review discusses isolation and characterization of archaeal tetraethers as well as target technologies for tetraether lipid application. The isolation and characterization of archaeal tetraether lipids has led to some interesting applications improving on ester lipid technologies. Potential applications include novel lubricants, gene-delivery systems, monolayer lipid matrices for sensor devices, and protein stabilization. Following this review, patent abstracts and additional literature pertaining to the isolation, characterization, and application of archaeal membrane lipids are listed.

Archaea↗

PubMed1989

Disease resistance and immune response genes in cattle: strategies for their detection and evidence of their existence.

The possibility of breeding or genetically engineering cattle for resistance to disease has tremendous potential for increasing the efficiency of milk and meat production. In cattle and other species, major genes that control humoral and cellular immune responses to a variety of antigens have been mapped to a chromosomal region known as the major histocompatibility complex. However, resistance or susceptibility to viral, bacterial, and parasitic diseases in noninbred species is often a complex phenotype, with age, stress, and physiologic status all being important factors in the outcome of infection. This paper reviews the function of major histocompatibility complex gene products and the relationship between polymorphism of these genes and infectious diseases. A discussion of strategies for detecting immune response genes and disease associations is presented, with particular reference to the problems and advantages of working with cattle. The present knowledge of the bovine major histocompatibility complex and its relationship to immune responsiveness and disease resistance are also reviewed, with special consideration given to enzootic bovine leukosis because of the significant relationship between alleles of the bovine lymphocyte antigen system and resistance or susceptibility to subclinical progression of bovine leukemia virus infection. Finally, potential applications of this research to genetic improvement and animal health are considered.

Animals↗

PubMed2004

The effects of age on monolayer culture of human keratinocytes for future use in skin engineering.

Skin is the largest organ in human system and plays a vital role as a barrier against environment and pathogens. Skin regeneration is important in tissue engineering especially in cases of chronic wounds. With the tissue engineering technology, these skins equivalent have been use clinically to repair burns and wounds. Consented redundant skin samples were obtained from patients aged 9 to 65 years old. Skin samples were digested with dispase, thus separating the epidermis and the dermis layer. The epidermis layer was trypsinized and cultured in DKSFM in 6-well plate at 37 degrees C and 5% CO2. Once confluent, the culture were trypsinized and the cells were pooled. Cells were counted using haemacytometer. Doubling time and viability were calculated and analysed. From the result, we conclude that doubling time and viability of in vitro keratinocytes cultured in DKSFM media is not age dependant.

Adolescent↗

PubMed2000

Myristoylation alters retinoic acid-induced down-regulation of MARCKS in immortalized hippocampal cells.

The myristoylated alanine-rich C kinase substrate (MARCKS) is a prominent PKC-substrate in the brain, which has been implicated in brain development, cytoskeletal remodeling, calcium/calmodulin signaling, and neuroplasticity. The sequence of the Macs gene codes for a protein that has three highly conserved domains including a 5' myristoylation region and a 25-amino-acid phosphorylation site domain (PSD), which are involved in anchoring MARCKS to the cellular membrane. In this study, we examined the role of the myristoylation signal in the regulation of MARCKS in transfected rat hippocampal cells (H19-7) following retinoic acid (RA) treatment. A mutant MARCKS lacking the myristoylation signal was engineered by substitution of alanine for glycine at position 2 of the Macs gene and was found to be exclusively expressed in the cytosol fraction of transfected cells. Exposure of the wild-type MARCKS-transfected cells to RA resulted in an apparent shift of MARCKS from the membrane to the cytosol, while the total protein of wild-type MARCKS was not significantly changed. In contrast, RA-exposed cells transfected with the mutant MARCKS revealed a dramatic reduction of expression of MARCKS protein in both cytosol and total protein fractions. These data suggest that the absence of the myristoyl moiety may not only alter the anchoring of the protein to the membrane but also play a novel role in modulating cellular levels of MARCKS protein in response to RA.

Animals↗

PubMed2000

Synthesis and characterization of polypyrrole-hyaluronic acid composite biomaterials for tissue engineering applications.

New tissue engineering technologies will rely on biomaterials that physically support tissue growth and stimulate specific cell functions. The goal of this study was to create a biomaterial that combines inherent biological properties which can specifically trigger desired cellular responses (e.g., angiogenesis) with electrical properties which have been shown to improve the regeneration of several tissues including bone and nerve. To this end, composites of the biologically active polysaccharide hyaluronic acid (HA) and the electrically conducting polymer polypyrrole (PP) were synthesized and characterized. Electrical conductivity of the composite biomaterial (PP/HA) was measured by a four-point probe technique, scanning electron microscopy was used to characterize surface topography, X-ray photoelectron spectroscopy and reflectance infrared spectroscopy were used to evaluate surface and bulk chemistry, and an assay with biotinylated hyaluronic acid binding protein was used to determine surface HA content. PP/HA materials were also evaluated for in vitro cell compatibility and tissue response in rats. Smooth, conductive, HA-containing PP films were produced; these films retained HA on their surfaces for several days in vitro and promoted vascularization in vivo. PP/HA composite biomaterials are promising candidates for tissue engineering and wound-healing applications that may benefit from both electrical stimulation and enhanced vascularization.

Animals↗

PubMed2005

Antibody engineering: facing new challenges in cancer therapy.

Antibody-based therapeutics are beginning to realize the promise enclosed in their early denomination as magic bullets. Initial disappointment has turned into clinical and commercial success, and engineered antibodies currently represent over 30% of biopharmaceuticals in clinical trials. Recent structural and functional data have allowed the design of a new generation of therapeutic antibodies, with strategies ranging from complement-mediated and antibody-dependant cellular cytotoxicity enhancement to improved cytotoxic payloads using toxins, drugs, radionucleids and viral delivery. This review considers the structure of different types of recombinant antibodies, their mechanism of action and how their efficacy has been increased using a broad array of approaches. We will also focus on the additional benefits offered by the use of gene therapy methods for the in vivo production of therapeutic antibodies.

Animals↗

PubMed2005

Resolving and classifying haematopoietic bone-marrow cell populations by multi-dimensional analysis of flow-cytometry data.

The study of normal or malignant haematopoiesis requires the analysis of heterogeneous cell populations using multiple morphological and molecular criteria. Flow cytometry has the capacity to acquire multi-parameter information of large haematopoietic cell populations, utilizing various combinations of >200 molecular markers (clusters of differentiation, CD). However, current flow cytometry analyses are based on serial gating of two-parametric scatter plots--a process that is inherently incapable to discriminate all subgroups of cells in the data. Here we studied the cellular diversity of normal bone marrows (BM) using multi-dimensional cluster analysis of six-parametric flow cytometry data (four CD, forward scatter and side scatter), focusing mainly on the myeloid lineage. Twenty-three subclasses of cells were resolved, many of them inseparable even when examined in all possible two-parametric scatter plots. The multi-dimensional analysis could distinguish the haematopoietic progenitors according to International Society of Haematotherapy and Graft Engineering criteria from other types of immature cells. Based on the defined clusters, we designed a classifier that assigns BM cells in samples to subclasses based on robust six-dimensional position and extended shape. The analysis presented here can manage successfully both the increasing numbers of haematopoietic cellular markers and sample heterogeneity. This should enhance the ability to study normal haematopoiesis, and to identify and monitor haematopoietic disorders.

Antigens, CD↗

PubMed2004

Cryoprotectant permeability parameters for cells used in a bioengineered human corneal equivalent and applications for cryopreservation.

A human corneal equivalent is being developed with applications in pharmaceutical testing and biomedical research, but the distribution of this engineered tissue, depends on successful cryopreservation. Cryopreservation of tissues depends on the presence of cryoprotectants, their addition and removal, and exposure to conditions during freezing and thawing, all of which depend on cellular membrane permeabilities to water and cryoprotectant. This study defines the permeability properties that define the rate of water and cryoprotectant movement across the plasma membrane of isolated human corneal endothelial, keratocyte, and epithelial cells. Cells were transferred from isotonic conditions (300 mosm/kg) to 0.5, 1, or 2 M dimethyl sulfoxide and propylene glycol solutions at constant temperature, and cell volumes monitored using an electronic particle counter. Histograms describing cell volume changes over time after cryoprotectant exposure allowed calculation of hydraulic conductivity (Lp), cryoprotectant permeability (Ps), and the reflection coefficient (sigma). Experimental values for Lp and Ps at 4, 13, 22, and 37 degrees C were used to determine the Arrhenius activation energy (Ea). Defining the permeability parameters and temperature dependencies allows simulation of responses of human corneal cells to addition and removal of cryoprotectants and to freezing conditions, allowing amount of supercooling, intracellular electrolyte concentration, and intracellular cryoprotectant concentration to be calculated. Simulations also show that the constituent cells in the bioengineered cornea respond differently to addition and removal of cryoprotectants and to freezing. This study has defined the requirements during cryopreservation for the corneal cells; future work will define the matrix requirements which will allow the development of a cryopreservation protocol.

Cell Membrane Permeability↗

PubMed2004

Effects of crosslinking degree of an acellular biological tissue on its tissue regeneration pattern.

It was reported that acellular biological tissues can provide a natural microenvironment for host cell migration and may be used as a scaffold for tissue regeneration. To reduce antigenicity, biological tissues have to be fixed with a crosslinking agent before implantation. As a tissue-engineering scaffold, it is speculated that the crosslinking degree of an acellular tissue may affect its tissue regeneration pattern. In the study, a cell extraction process was employed to remove the cellular components from bovine pericardia. The acellular tissues then were fixed with genipin at various known concentrations to obtain varying degrees of crosslinking. It was shown in the in vitro degradation study that after fixing with genipin, the resistance against enzymatic degradation of the acellular tissue increased significantly with increasing its crosslinking degree. In the in vivo subcutaneous study, it was found that cells (inflammatory cells, fibroblasts, endothelial cells, and red blood cells) were able to infiltrate into acellular tissues. Generally, the depth of cell infiltration into the acellular tissue decreased with increasing its crosslinking degree. Infiltration of inflammatory cells was accompanied by degradation of the acellular tissue. Due to early degradation, no tissue regeneration was observed within fresh (without crosslinking) and the 30%-degree-crosslinking acellular tissues. This is because the scaffolds provided by these two samples were already completely degraded before the infiltrated cells began to secrete their own extracellular matrix. In contrast, tissue regeneration (fibroblasts, neo-collagen fibrils, and neo-capillaries) was observed for the 60%- and 95%-degree-crosslinking acellular tissues by the histological examination, immunohistological staining, transmission electron microscopy, and denaturation temperature measurement. The 95%-degree-crosslinking acellular tissue was more resistant against enzymatic degradation than its 60%-degree-crosslinking counterpart. Consequently, tissue regeneration was limited in the outer layer of the 95%-degree-crosslinking acellular tissue throughout the entire course of the study (1-year postoperatively), while tissue regeneration was observed within the entire sample for the 60%-degree-crosslinking acellular tissue. In conclusion, the crosslinking degree determines the degradation rate of the acellular tissue and its tissue regeneration pattern.

Animals↗

PubMed2004

Cellular remodeling of depopulated bovine ureter used as an arteriovenous graft in the canine model.

BACKGROUND: One of the greatest challenges in hemodialysis access surgery is improving the durability of prosthetic grafts caused by structural deterioration. The depopulated bovine ureter SynerGraft (SG) (CryoLife, Inc) is a tissue-engineered vascular graft processed to remove the xenograft cells while maintaining an unfixed connective tissue matrix capable of autologous cell repopulation by the recipient. STUDY DESIGN: Nineteen 6-mm diameter bovine ureter SG conduits were implanted in 12 dogs as arteriovenous grafts between the carotid artery and jugular vein (n = 11) or between the femoral artery and vein (n = 8). Performance of these biologic conduits was compared with that of 15 IMPRA (Bard) ePTFE grafts implanted in 9 dogs, including 9 arteriovenous grafts between the carotid artery and jugular vein and 6 femoral artery to femoral vein grafts. After 14 days, the grafts were accessed once weekly. Histologic and immunohistochemical analyses were performed on grafts explanted between 10 to 60 weeks. RESULTS: The 6- and 12-month primary patency rates of the bovine SG were 72.6% and 58.6%, respectively, compared with 6- and 12-month primary patency for ePTFE conduits of 57.4% and 57.4%, respectively. None of the bovine SG grafts became infected, but synthetic conduits became infected within 54 days of implantation. At 10 weeks, bovine ureter SG conduit showed fibroblast cell migration and proliferation with incorporation into the surrounding subcutaneous tissue, and elongated cells expressing the contractile protein smooth muscle actin were also observed. After 24 weeks, procollagen synthesis was demonstrated in the fully colonized graft matrix. The ePTFE grafts had no evidence of cellular ingrowth and an absence of endothelium. CONCLUSIONS: The bovine SG was appropriately remodeled to its host environment through an organized process of recellularization and neovascularization. The absence of infection, similar patency rates, and cell repopulation of the matrix warrant further investigation.

Animals↗

PubMedDate not supplied

Bioresponsive phosphoester hydrogels for bone tissue engineering.

Bioresponsive and intelligent biomaterials are a vehicle for manipulating cell function to promote tissue development and/or tissue engineering. A photopolymerized hydrogel based on a phosphoester- poly(ethylene glycol) polymer (PhosPEG) was synthesized for application to marrow-derived mesenchymal stem cell (MSC) encapsulation and tissue engineering of bone. The phosphor-containing hydrogels were hydrolytically degradable and the rate of degradation increased in the presence of a bone-derived enzyme, alkaline phosphatase. Gene expression and protein analysis of encapsulated MSCs demonstrated that PhosPEG-PEG cogels containing an intermediate concentration of phosphorus promoted the gene expression of bone-specific markers including type I collagen, alkaline phosphatase, and osteonectin, without the addition of growth factors or other biological agents, compared with pure poly(ethylene glycol)-based gels. Secretion of alkaline phosphatase, osteocalcin, and osteonectin protein was also increased in the PhosPEG cogels. Mineralization of gels increased in the presence of phosphorus in both cellular and acellular constructs compared with PEG gels. In summary, phosphate-PEG-derived hydrogels increase gene expression of bone-specific markers, secretion of bone-related matrix, and mineralization and may have a potential impact on bone-engineering therapies.

Alkaline Phosphatase↗

PubMed2003

Mechanisms and prevention of plant tissue collapse during dehydration: a critical review.

The appearance and functional properties are primordial in the quality assessment of semifinished fruit and vegetable products. These properties are often associated with shrunken, shriveled, darkened materials of poor rehydration ability after been subjected to air-drying--the most used drying method in the food industry. Fruits and vegetables are cellular tissues containing gas-filled pores that tend to collapse when subjected to dehydration. Collapse is an overall term that has different meanings and scale-settings in the literature depending on whether the author is a plant physiologist, a food technologist, a chemical engineer, or a material scientist. Some clarifications are given in this particular but wide field. The purpose of this work was to make a state-of-the-art contribution to the structural and textural effects of different types of dehydration on edible plant products and give a basis for preventing this phenomenon. The plant tissue is described, and the primordial role of the cell wall in keeping the structural integrity is emphasized. Water and its functionality at macro and micro levels of the cellular tissue are reviewed as well as its transport during dehydration. The effects of both dehydration and rehydration are described in detail, and the term "textural collapse" is proposed as an alternative to structural collapse.

Desiccation↗

PubMed2005

Engineering a mimicry of bone marrow tissue ex vivo.

Hematopoietic stem cells reside in specific niches in the bone marrow and give rise to either more stem cells or maturing hematopoietic progeny depending on the signals provided in the bone marrow microenvironment. This microenvironment is comprised of cellular components as well as soluble constituents called cytokines. The use of cytokines alone for the ex vivo expansion of stem cells in flat, two-dimensional culture flasks, dishes or bags is inadequate and, given the three-dimensionality of the in vivo bone marrow microenvironment, inappropriate. Three-dimensional culture conditions can therefore provide an ex vivo mimicry of bone marrow, recapitulate the desired niche, and provide a suitable environment for stem cell expansion and differentiation. Choice of scaffold, manipulation and reproducibility of the scaffold properties and directed structuring of the niche, by choosing pore size and porosity may inform the resident stem cells of their fate in a directed fashion. The use of bioreactors for cultivation of hematopoietic cells will allow for culture control, optimization, standardization, scale-up, and a "hands-off" operation making the end-product dependable, predictable and free of contaminants, and therefore suitable for human use and therapeutic applications.

Biomimetics↗