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PubMed2005

Recent advances in engineering plant tolerance to abiotic stress: achievements and limitations.

Abiotic stresses, especially salinity and drought, are the primary causes of crop loss worldwide. Plant adaptation to environmental stresses is dependent upon the activation of cascades of molecular networks involved in stress perception, signal transduction, and the expression of specific stress-related genes and metabolites. Consequently, engineering genes that protect and maintain the function and structure of cellular components can enhance tolerance to stress. Our limited knowledge of stress-associated metabolism remains a major gap in our understanding; therefore, comprehensive profiling of stress-associated metabolites is most relevant to the successful molecular breeding of stress-tolerant crop plants. Unraveling additional stress-associated gene resources, from both crop plants and highly salt- and drought-tolerant model plants, will enable future molecular dissection of salt-tolerance mechanisms in important crop plants.

Adaptation, Physiological↗

PubMed2004

BMP-2 exerts differential effects on differentiation of rabbit bone marrow stromal cells grown in two-dimensional and three-dimensional systems and is required for in vitro bone formation in a PLGA scaffold.

Osteogenic differentiation of bone marrow stromal cells (BMSC) in a three-dimensional (3-D) scaffold has not been well studied. In this work, we studied expression of bone-related genes during differentiation of rabbit BMSCs in response to bone morphogenetic protein (BMP)-2 in both 2-D and 3-D culture systems. When BMSCs were cultured on films (2-D) of biodegradable poly(lactide-co-glycolide) (PLGA), increases in mRNA expression of type I collagen (Col I) and vascular endothelial growth factor (VEGF) became evident after 1 week. However, expression of both genes was only mildly stimulated by BMP-2. Expression of the osteopontin gene was highly stimulated by BMP-2 treatment. Expression of chordin, a BMP antagonist, increased significantly after 7 days. The increase was abrogated by BMP-2 treatment. BMP-2 was also able to stimulate mineralization of cultured BMSCs. After cells were switched to 3-D PLGA scaffolds for 24 h, expression of osteopontin and VEGF were markedly increased while expression of type I collagen and chordin remained unchanged. Expression of Col I did not increase with time in a 3-D culture as it did when cells were cultured on a 2-D film. We further explored the possibility of engineering bone tissue in vitro by seeding BMSCs into PLGA scaffolds. Cellular differentiation and bone formation in the scaffolds were analyzed histologically at 2 weeks and 2 months. Secretion of ECM by cells was evident at both 2-week and 2-month scaffolds, and was enhanced by rhBMP-2. Striking differences in 2-month scaffolds were observed between BMP-treated and untreated cells. A woven bone-like structure appeared in the scaffolds treated with BMP-2. The structure was verified to be bone-related by: (1) the presence of organized collagen fibrils; (2) the presence of mineral; and (3) morphological features of trabecular bone. Although collagen was abundant in the untreated 2-month scaffolds, it was disorganized. The untreated scaffolds also lacked mineral deposits, which were present in 2-D cultured cells even in the absence of BMP-2. Our results indicate that the requirement of osteo-inductive agents, such as BMP-2, is essential for bone tissue engineering.

Animals↗

PubMed2003

Principles of cell mechanics for cartilage tissue engineering.

The critical importance of mechanical signals to the health and maintenance of articular cartilage has been well demonstrated. Tissue engineers have taken a cue from normal cartilage physiology and incorporated the use of mechanical stimulation into their attempts to engineer functional cartilage. However, the specific types of mechanical stimulation that are most beneficial, and the mechanisms that allow a chondrocyte to perceive and respond to those forces, have yet to be elucidated. To develop a better understanding of these processes, it is necessary to examine the mechanical behavior of the single chondrocyte. This paper reviews salient topics related to chondrocyte biomechanics and mechanotransduction, and attempts to put this information into a context both appropriate and useful to cartilage tissue engineering. It also describes the directions this exciting field is taking, and lays out a vision for future studies that could have a significant impact on our understanding of cartilage health and disease.

Animals↗

PubMed2007

Shape-engineered fibroblasts: cell elasticity and actin cytoskeletal features characterized by fluorescence and atomic force microscopy.

The regulation of cell shape, which determines cell behaviors including adhesion, spreading, migration, and proliferation in an engineered artificial extracellular milieu, is an important task in tissue engineering and in development of functional biomaterials. To deepen the understandings of shape-dependent cell mechanics, the cell elasticity and structural features of the actin cytoskeleton (CSK) were characterized for shape-engineered fibroblasts; round and spindle-shaped cells cultured on photolithographically microprocessed surfaces, employing the cellular microindentation tests and fluorescence observation of actin CSK by the combination of atomic force microscopy (AFM) and fluorescence microscopy (FM). The relationships among cell elasticity, the structural features of actin CSK, and engineered cell shape were analyzed and compared with those of control cells that had been cultured on nonprocessed surfaces (termed naturally extended cells). Results showed that the spindle-shaped cells with sparse or no apical stress fibers (ASFs) exhibited similar stiffness to that of the naturally extended cells with dense ASFs. The elasticity of spindle-shaped cells was affected only slightly by the stress fiber (SF) density, which is in marked contrast to the significant correlation shown between cell elasticity and SF density in naturally extended cells. This result implies that the elasticity of regionally restricted adhesion-surface-induced shape-engineered cells, particularly of highly elongated cells, is affected predominantly by cell shape rather than by structural features of SFs.

3T3 Cells↗

PubMed1994

Symbiosis of biotechnology and biomaterials: applications in tissue engineering of bone and cartilage.

The three ingredients for the successful tissue engineering of bone and cartilage are regulatory signals, cells, and extracellular matrix. Recent advances in cellular and molecular biology of the growth and differentiation factors have set the stage for a symbiosis of biotechnology and biomaterials. Recent advances permit one to enunciate the rules of architecture for tissue engineering of bone and cartilage. The purification and cloning of bone morphogenetic proteins (BMPs) and growth factors such as platelet derived growth factors (PDGF), transforming growth factor-beta (TGF-beta), and insulin-like growth factors (IGF-I) will allow the design of an optimal combination of signals to initiate and promote development of skeletal stem cells into cartilage and bone. Successful and optimal bone and cartilage formation is a synergy of inductive and conductive strategies governed by biomechanics, optimal load bearing, and motion. BMPs function as inductive signals. Biomaterials (both natural and synthetic) mimic the extracellular matrix and play a role in conduction of bone and cartilage. Examples of biomaterials include hydroxyapatite, polyanhydrides, polyphosphoesters, polylactic acid, and polyglycolic acid. The prospects for novel biomaterials are immense, and they likely will be a fertile growth industry. Cooperative ventures between academia and industry and technology transfer from the federal government augur well for an exciting future for clinical applications.

Animals↗

PubMed2004

Inkjet printing for high-throughput cell patterning.

The adaptation of inkjet printing technology to the complex fields of tissue engineering and biomaterial development presents the potential to increase progress in these emerging technologies through the implementation of this high-throughput capability via automated processes to enable precise control and repeatability. In this paper, a method of applying high-throughput inkjet printing to control cellular attachment and proliferation by precise, automated deposition of collagen is presented. The results indicate that commercial inkjet printing technology can be used to create viable cellular patterns with a resolution of 350 microm through the deposition of biologically active proteins. This method demonstrates a combination of off-the-shelf inkjet printing and biomaterials and has potential to be adapted to tissue engineering and colony patterning applications. Adapting this method into the three-dimensional construction of cellular structures for eventual high-throughput tissue engineering using a bottom-up approach is possible.

Animals↗

PubMed2000

Control of hepatocyte function on collagen foams: sizing matrix pores toward selective induction of 2-D and 3-D cellular morphogenesis.

While microporous biopolymer matrices are being widely tested as cell culture substrates in hepatic tissue engineering, the microstructural basis for their control of cell differentiation is not well understood. In this paper, we studied the role of void size of collagen foams in directing the induction of liver-specific differentiated morphology and secretory activities of cultured rat hepatocytes. Hepatocytes cultured on collagen foams with subcellular sized pore diameters of 10 microm assumed a compact, cuboidal cell morphology, rapidly achieving monolayer coverage, and secreted albumin at the rate of 40 +/- 8 pg/cell/d. Increasing the pore size to 18 microm elicited a distinctly spread cellular phenotype, with discontinuous surface coverage, and albumin secretion rates declined precipitiously to 3.6 +/- 0.8 pg/cell/d. However, when collagen foams with an even higher average void size of 82 microm were used, hepatocytes exhibited high degree of spreading within an extensive three-dimensional cellular network, and exhibited high albumin secretory activity (26 +/- 0.6 pg/cell/d). The effect of void geometry on cellular ultrastructral polarity was further analyzed for the three void size configurations employed. The distribution of the cell-cell adhesion protein, E-cadherin, was primarily restricted to cell-cell contacts on the 10 microm foams, but was found to be depolarized to all membrane regions in cells cultured on the 18 and 82 microm foams. Vinculin-enriched focal adhesions were found to be peripherally clustered on cells cultured on 10 microm foams, but were found to redistribute to the entire ventral surface of cells cultured on the 18 and 82 microm foams. Overall, we demonstrate the significance of designing pore sizes of highly adhesive substrates like collagen toward selective cell morphogenesis in 2-D or 3-D. Subcellular and supercellular ranges of pore size promote hepatocellular differentiation by limiting 2-D cell spreading or effecting 3-D intercellular contacts, while intermediate range of pore sizes repress differentiation by promoting 2-D cell spreading.

Actins↗

PubMed2006

Engineering yeast transcription machinery for improved ethanol tolerance and production.

Global transcription machinery engineering (gTME) is an approach for reprogramming gene transcription to elicit cellular phenotypes important for technological applications. Here we show the application of gTME to Saccharomyces cerevisiae for improved glucose/ethanol tolerance, a key trait for many biofuels programs. Mutagenesis of the transcription factor Spt15p and selection led to dominant mutations that conferred increased tolerance and more efficient glucose conversion to ethanol. The desired phenotype results from the combined effect of three separate mutations in the SPT15 gene [serine substituted for phenylalanine (Phe(177)Ser) and, similarly, Tyr(195)His, and Lys(218)Arg]. Thus, gTME can provide a route to complex phenotypes that are not readily accessible by traditional methods.

Amino Acid Substitution↗

PubMed2006

Engineering plant-microbe symbiosis for rhizoremediation of heavy metals.

The use of plants for rehabilitation of heavy-metal-contaminated environments is an emerging area of interest because it provides an ecologically sound and safe method for restoration and remediation. Although a number of plant species are capable of hyperaccumulation of heavy metals, the technology is not applicable for remediating sites with multiple contaminants. A clever solution is to combine the advantages of microbe-plant symbiosis within the plant rhizosphere into an effective cleanup technology. We demonstrated that expression of a metal-binding peptide (EC20) in a rhizobacterium, Pseudomonas putida 06909, not only improved cadmium binding but also alleviated the cellular toxicity of cadmium. More importantly, inoculation of sunflower roots with the engineered rhizobacterium resulted in a marked decrease in cadmium phytotoxicity and a 40% increase in cadmium accumulation in the plant root. Owing to the significantly improved growth characteristics of both the rhizobacterium and plant, the use of EC20-expressing P. putida endowed with organic-degrading capabilities may be a promising strategy to remediate mixed organic-metal-contaminated sites.

Bacterial Proteins↗

PubMed2002

Control of calcium entry in human fibroblasts by frequency-dependent electrical stimulation.

Modulation of intracellular calcium ion concentration ((Ca2+)i) could be used to control cellular and molecular responses that are important in cell and tissue engineering. Electrical stimulation (ES) has been used to activate plasma membrane ion channels including Ca2+channels, and to induce changes in (Ca2+)i. Strong direct current (dc) ES depolarizes the membrane electrical potential (MEP) and, thereby, causes rapid increases in (Ca2+)i. Electrocoupling mechanisms that could control (Ca2+)i increases induced by modes of ES other than dc have not been elucidated, however. Here we report that 30 min of continuous exposure to a 1 or 10 Hz, 2 V/cm ES induces an (Ca2+)i increase by approximately 6-fold (baseline 25 nM) in human fibroblasts in culture. In contrast, a 100 Hz, 2 V/cm ES causes no significant (Ca2+)i increase. Either depletion of Ca2+from the extracellular medium or incubation of cells with verapamil inhibits the (Ca2+)i increase, indicating that Ca2+ influx through verapamil-sensitive Ca2+channels is required for the (Ca2+)i increase induced by oscillatory ES. More intense ES by a 1 Hz or a dc 10 V/cm electric field causes a rapid 20 to 25-fold (Ca2+)i increase. We hypothesize that selective, partial activation of Ca2+channels is likely to mediate Ca2+influx. These results suggest that optimal ES could be used to control Ca2+entry and, thereby, regulate cellular calcium homeostasis without adversely affecting cell viability.

Calcium↗

PubMed2026

Gene overexpression reduces inhibitory metabolites to enhance CHO cell growth and IgG1 production.

Controlling the generation of toxic by-products in mammalian bioprocess to maximize therapeutic protein production and glycosylation patterns is a challenge. Intracellular metabolism is often not well-regulated and known to secrete toxic intermediate by-products which hampers cellular performance and negatively impacts critical quality attributes (CQA) of cells. Previous studies have identified trigonelline (TRI), n-acetyl putrescine (NAP), aconitic acid (AA), and cytidine monophosphate (CMP) generated through CHO cell metabolism and verified their negative impacts on growth and antibody production. In this approach, a genetic engineering strategy was developed to control downstream accumulation of inhibitory metabolites. The study successfully identified four different metabolic genes in CHO cells, including Cat (nicotinate and nicotinamide metabolism) to control the generation of TRI, Got1 and Hoga1 (proline metabolism) to control the generation of NAP, Got1 (TCA cycle) to control the generation of AA, and Slc35a1 (n-glycan biosynthesis) to control the generation of CMP. Each target gene-of-interest (GOI) was cloned from CHO genomic library, inserted into linearized vector plasmid, and subsequently transfected into cells. CQA of the bioprocess realized 22-30% increase in peak cell density, 16-22% increase overall IVCD, with an improving growth rate during cellular expansion phase when comparing engineered cells against control cells. The study also conducted a follow-up quadruple transfection study where all four GOIs were co-transfected into cells at ¼ of the total DNA concentration per GOI. An increase in cellular performance was also realized, as increases in peak VCD (17% increase), cumulative IVCD (17% increase), and growth rate were achieved. Both studies also found higher IgG1 antibody synthesis when cell metabolism was better regulated, as the studies measured 4% to 40% titer increase across all engineered cells when compared against control cells. The study also measured higher levels of G1F and G2F glycans with decreased level of G0F across all transfected cells, further indicating improvement in bioprocess, as cells were able to produce a higher fraction of semi-complex and complex versus simple glycoforms. Further investigation revealed that Cat and Slc35a1 exhibited comparable expression levels in the MG condition to their single-gene conditions (within 1% and 10% difference, respectively), corresponding to modest titer improvements closest to the control. These findings suggest that when all four genes are co-expressed, Cat and Got1 may act as rate-limiting factors influencing both cellular phenotypes and titer production. In both studies, the concentrations of downstream metabolic inhibitors were measured to be significantly decreased when comparing engineered cells against control cells, further demonstrating that overexpression of genes to re-allocate metabolic fluxes away from synthesizing toxic by-products can significantly improve cellular growth and protein synthesis.

Animals↗

PubMed2003

Genetic neuroscience of mammalian learning and memory.

Our primary research interest is to understand the molecular and cellular mechanisms on neuronal circuitry underlying the acquisition, consolidation and retrieval of hippocampus-dependent memory in rodents. We study these problems by producing genetically engineered (i.e. spatially targeted and/or temporally restricted) mice and analysing these mice by multifaceted methods including molecular and cellular biology, in vitro and in vivo physiology and behavioural studies. We attempt to identify deficits at each of the multiple levels of complexity in specific brain areas or cell types and deduce those deficits that underlie specific learning or memory. We will review our recent studies on the acquisition, consolidation and recall of memories that have been conducted with mouse strains in which genetic manipulations were targeted to specific types of cells in the hippocampus or forebrain of young adult mice.

Animals↗

PubMed1997

Mutant adenovirus type 9 E4 ORF1 genes define three protein regions required for transformation of CREF cells.

Human adenovirus type 9 (Ad9) elicits exclusively estrogen-dependent mammary tumors in rats, and an essential oncogenic determinant for this virus is Ad9 E4 open reading frame 1 (9ORF1), which encodes a 125-residue cytoplasmic protein with cellular growth-transforming activity in vitro. In this study, we engineered 48 different mutant 9ORF1 genes in an attempt to identify regions of this viral protein essential for transformation of the established rat embryo fibroblast cell line CREF. In initial assays with CREF cells, 17 of the 48 mutant 9ORF1 genes proved to be severely defective for generating transformed foci but only 7 of these defective genes expressed detectable amounts of protein. To further examine the defects of the seven mutant proteins, we selected individual cell pools of stable CREF transformants for the wild-type and mutant 9ORF1 genes. Compared to cell pools expressing the wild-type 9ORF1 protein, most cell pools expressing mutant proteins displayed decreased growth in soft agar, and all generated significantly smaller tumors in syngeneic animals. The altered amino acid residues of the seven mutant 9ORF1 polypeptides clustered within three separate regions referred to as region I (residues 34 to 41), region II (residues 89 to 91), and C-terminal region III (residues 122 to 125). By using indirect immunofluorescence, we also assessed whether the mutant proteins localized properly to the cytoplasm of cells. The region I and region II mutants displayed approximately wild-type subcellular localizations, whereas most region III mutants aberrantly accumulated within the nucleus of cells. In summary, we have identified three 9ORF1 protein regions necessary for cellular transformation and have demonstrated that C-terminal region III sequences significantly influence the proper localization of the 9ORF1 polypeptide in cells.

Adenoviruses, Human↗

PubMedDate not supplied

Resorbable extracellular matrix grafts in urologic reconstruction.

PURPOSE: There is an increasingly large body of literature concerning tissue-engineering products that may be used in urology. Some of these are quite complex (such as multilayer patient-specific cell-seeded implants) yet the most simple and successful products to date are also the most uncomplicated: resorbable acellular extra-cellular matrices (ECMs) harvested from animals. ECMs have been used in a variety of difficult urologic reconstruction problems, and this review is intended to summarize this complex literature for the practicing urologist. METHODS: Medline search of related terms such as "SIS, small intestinal submucosa, ECM, extracellular matrix, acellular matrix and urologic reconstruction". Manuscripts missed in the initial search were taken from the bibliographies of the primary references. RESULTS: Full review of potential clinical uses of resorbable extra-cellular matrices in urologic reconstruction. CONCLUSIONS: Currently, the "state of the art" in tissue engineering solutions for urologic reconstruction means resorbable acellular xenograft matrices. They show promise when used as a pubovaginal sling or extra bolstering layers in ureteral or urethral repairs, although recent problems with inflammation following 8-ply pubovaginal sling use and failures after 1- and 4-ply SIS repair of Peyronie's disease underscore the need for research before wide adoption. Preliminary data is mixed concerning the potential for ECM urethral patch graft, and more data is needed before extended uses such as bladder augmentation and ureteral replacement are contemplated. The distant future of ECMs in urology likely will include cell-seeded grafts with the eventual hope of producing "off the shelf" replacement materials. Until that day arrives, ECMs only fulfill some of the requirements for the reconstructive urologist.

Animals↗

PubMed2002

Effective tumor immunotherapy: start the engine, release the brakes, step on the gas pedal,...and get ready to face autoimmunity.

Cellular immune responses can destroy cancer cells, achieving the cure of experimental malignancies. An expanding wealth of knowledge on the molecular basis of how to prime and amplify a T cell response has fueled a number of strategies successful at treating established tumors (rather than merely preventing tumor grafting). The most efficacious approaches operate at different stages, including: 1) priming the immune response using tumor antigen-expressing dendritic cells or tumor cells transfected with genes that render them immunogenic, 2) sustaining and amplifying immunity using agonistic monoclonal antibodies against costimulatory molecules or immune-potentiating cytokines, and 3) eliminating mechanisms that self-regulate the strength of the immune response, such as inhibitory receptors or regulatory T cells. A rational combination of such approaches holds great hope for cumulative and synergistic effects, but there is also evidence that they can open the flood-gates for unwanted inflammatory reactions. The next decade can be envisioned as the time when the first reproducibly efficacious combination regimes for cancer immunotherapy will become available and widely used in the clinic, as clinicians learn the best strategies and try to harness their potentially damaging effects.

Abatacept↗

PubMed1994

Cellular signaling by an agonist-activated receptor/Gs alpha fusion protein.

The consequences of agonist-dependent activation of guanine nucleotide-binding protein (G protein)-coupled receptors vary from cell to cell, depending on a complex network of regulations between components of the signaling cascade. Specific interactions between receptors, G proteins, and effectors are difficult to analyze in intact cells. Engineering of receptor-transducer fusion proteins might be an effective strategy to target cellular effectors more efficiently and specifically. As a model, we evaluated the ability of a fusion protein of beta 2-adrenergic receptor bound to the alpha subunit of adenylyl cyclase-stimulatory G protein (Gs alpha) to restore the defective activation of adenylyl cyclase in S49 cyc- cells that lack endogenous Gs alpha. The coupling between the two partners of the fusion protein was functional, and the agonist-dependent activation of the effector was more potent and more productive in transfected than in wild-type S49 cells. The covalent link between receptor and Gs alpha could thus convey an advantage over freely interacting components. Such receptor-G alpha fusion proteins may help to elucidate the complex interactions between members of signaling pathways and may also constitute a useful tool for studying the effects of single effector activation.

Adenylyl Cyclases↗

PubMedDate not supplied

Tissue engineering and the development of Apligraf, a human skin equivalent.

In recent years, skin grafting has evolved from the initial autograft and allograft preparations to biosynthetic and tissue-engineered living skin replacements. This review details the pioneering work of numerous investigators that led to the following precursors of tissue-engineered skin replacement: cultured autologous keratinocyte grafts, cultured allogeneic keratinocyte grafts, autologous/allogeneic composites, acellular collagen matrices, and cellular matrices. It also discusses the rationale for the development of the newer products and describes the technical advances leading to the development of Apligraf, a tissue-engineered human skin product.

Biomedical Engineering↗

PubMedDate not supplied

Tissue engineering and the development of Apligraf a human skin equivalent.

Biomedical Engineering↗