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At least 883 records · Page 49Linked to original sources
PubMed1998

Survival and toxicity of an allogeneic cytokine-secreting fibroblast vaccine in the central nervous system.

OBJECTIVE: In previous studies, we determined that C57BL/6 mice (H-2b) with intracerebral (i.c.) malignant glioma or melanoma treated with allogeneic fibroblasts genetically engineered to secrete interleukin (IL)-2 results in prolonged survival and a cellular antitumor response when the treatment is administered intratumorally (via i.c. injection). For this study, we evaluated the survival and toxicity of the cytokine-secreting cellular vaccine administered directly into the central nervous system in both syngeneic (C3H H-2k) and allogeneic (C57BL/6 H-2b) mice using bioassay and polymerase chain reaction techniques. METHODS: First, brain sections were prepared at varying time intervals (2-60 d) after i.c. injection of the IL-2-secreting fibroblasts (H-2k) into allogeneic and syngeneic mice, and the presence of the cells was detected by polymerase chain reaction for the neomycin gene, which was used as a selection marker for the gene transfer. As a second means of assessing survival of the IL-2-secreting cells, an in vitro bioassay technique was used. Brain sections were prepared at varying time intervals (2-60 d) after i.c. injection of the IL-2-secreting cells into allogeneic and syngeneic mice. Cells were disassociated and grown in media containing neomycin. RESULTS: A positive signal by polymerase chain reaction was no longer detectable 14 days after injection into allogeneic C57BL/6 (H-2b) mice. In contrast, under similar circumstances, the IL-2-secreting cellular vaccine could be detected for more than 55 days in histocompatible (C3H) syngeneic mice (H-2k). Allogeneic cells could be recovered from tissue culture for only 2 to 5 days after i.c. injection of the modified cells. In contrast, syngeneic cells were recovered for up to 28 days after i.c. injection. At no time did the mice exhibit signs of neurological deficit or adverse affects on their survival (>60 d). CONCLUSION: An allogeneic cytokine-secreting cellular vaccine has a limited lifespan in the central nervous system, surviving only 14 days or less, and has no apparent adverse effects. These results indicate the advantage of using a modified allogeneic cell line as the delivery vehicle for gene therapy in the treatment of an i.c. neoplasm.

Animals↗
PubMed2002

Development of magnetic particle techniques for long-term culture of bone cells with intermittent mechanical activation.

Magnetic particles were coated with RGD and adhered to primary human osteoblasts. During a 21-day culture, the osteoblasts plus adhered magnetic particles underwent a daily exposure to a time-varying magnetic field via a permanent NdFeB magnet, thus applying a direct mechanical stress to the cells (Bmax approximately 60 mT). After 21 days, preliminary results show that the cells plus magnetic particles were viable and had proliferated. A von-kossa stain showed mineralized bone matrix produced at 21 days in the experimental group whereas the control groups showed no mineralized matrix production. Real-time reverse transcription-polymerase chain reaction at 21 days showed an upregulation of osteopontin from the experimental group in comparison to the control group of cells with adhered particles and no magnet applied. These preliminary results indicate that adherence of RGD-coated 4.5 microm ferromagnetic particles to primary human osteoblasts does not initiate cell necrosis up to 21 days in vitro. Also, mechanical stimulation of human osteoblasts by magnetic particle technology appears to have an influence on osteoblastic activity.

Cell Culture Techniques↗
PubMed2006

Specificity of acyl-homoserine lactone synthases examined by mass spectrometry.

Many gram-negative bacteria produce a specific set of N-acyl-L-homoserine-lactone (AHL) signaling molecules for the purpose of quorum sensing, which is a means of regulating coordinated gene expression in a cell-density-dependent manner. AHLs are produced from acylated acyl-carrier protein (acyl-ACP) and S-adenosyl-L-methionine by the AHL synthase enzyme. The appearance of specific AHLs is due in large part to the intrinsic specificity of the enzyme for subsets of acyl-ACP substrates. Structural studies of the Pantoea stewartii enzyme EsaI and AHL-sensitive bioassays revealed that threonine 140 in the acyl chain binding pocket directs the enzyme toward production of 3-oxo-homoserine lactones. Mass spectrometry was used to examine the range of AHL molecular species produced by AHL synthases under a variety of conditions. An AHL selective normal-phase chromatographic purification with addition of a deuterated AHL internal standard was followed by reverse-phase liquid chromatography-tandem mass spectrometry in order to obtain estimates of the relative amounts of different AHLs from biological samples. The AHLs produced by wild-type and engineered EsaI and LasI AHL synthases show that intrinsic specificity and different cellular conditions influence the production of AHLs. The threonine at position 140 in EsaI is important for the preference for 3-oxo-acyl-ACPs, but the role of the equivalent threonine in LasI is less clear. In addition, LasI expressed in Escherichia coli produces a high proportion of unusual AHLs with acyl chains consisting of an odd number of carbons. Furthermore, these studies offer additional methods that will be useful for surveying and quantitating AHLs from different sources.

4-Butyrolactone↗
PubMed2005

Cell type-specific activation of metabolism reveals that beta-cell secretion suppresses glucagon release from alpha-cells in rat pancreatic islets.

Abnormal glucagon secretion is often associated with diabetes mellitus. However, the mechanisms by which nutrients modulate glucagon secretion remain poorly understood. Paracrine modulation by beta- or delta-cells is among the postulated mechanisms. Herein we present further evidence of the paracrine mechanism. First, to activate cellular metabolism and thus hormone secretion in response to specific secretagogues, we engineered insulinoma INS-1E cells using an adenovirus-mediated expression system. Expression of the Na+-dependent dicarboxylate transporter (NaDC)-1 resulted in 2.5- to 4.6-fold (P < 0.01) increases in insulin secretion in response to various tricarboxylic acid cycle intermediates. Similarly, expression of glycerol kinase (GlyK) increased insulin secretion 3.8- or 4.2-fold (P < 0.01) in response to glycerol or dihydroxyacetone, respectively. This cell engineering method was then modified, using the Cre-loxP switching system, to activate beta-cells and non-beta-cells separately in rat islets. NaDC-1 expression only in non-beta-cells, among which alpha-cells are predominant, caused an increase (by 1.8-fold, P < 0.05) in glucagon secretion in response to malate or succinate. However, the increase in glucagon release was prevented when NaDC-1 was expressed in whole islets, i.e., both beta-cells and non-beta-cells. Similarly, an increase in glucagon release with glycerol was observed when GlyK was expressed only in non-beta-cells but not when it was expressed in whole islets. Furthermore, dicarboxylates suppressed basal glucagon secretion by 30% (P < 0.05) when NaDC-1 was expressed only in beta-cells. These data demonstrate that glucagon secretion from rat alpha-cells depends on beta-cell activation and provide insights into the coordinated mechanisms underlying hormone secretion from pancreatic islets.

Animals↗
PubMed2005

An oncolytic HSV-1 mutant expressing ICP34.5 under control of a nestin promoter increases survival of animals even when symptomatic from a brain tumor.

Oncolytic herpes simplex virus-1 (HSV-1) mutants possessing mutations in the ICP34.5 and ICP6 genes have proven safe through clinical trials. However, ICP34.5-null viruses may grow poorly in cells due to their inability to prevent host-cell shut-off of protein synthesis caused by hyperphosphorylation of eukaryotic initiation factor 2alpha. To increase tumor selectivity, glioma-selective expression of ICP34.5 in the context of oncolysis may be useful. Malignant gliomas remain an incurable disease. One molecular marker of malignant gliomas is expression of the intermediate filament nestin. Expression of nestin mRNA was confirmed in 6 of 6 human glioma lines and in 3 of 4 primary glioma cells. Normal human astrocytes were negative. A novel glioma-selective HSV-1 mutant (rQNestin34.5) was thus engineered by expressing ICP34.5 under control of a synthetic nestin promoter. Replication, cellular propagation, and cytotoxicity of rQNestin34.5 were significantly enhanced in cultured and primary human glioma cell lines compared with control virus. However, replication, cellular propagation, and cytotoxicity of rQNestin34.5 in normal human astrocytes remained quantitatively similar to that of control virus. In glioma cell lines infected with rQNestin34.5, the level of phospho-eukaryotic initiation factor 2alpha was lower than that of cells infected by control rHsvQ1, confirming selective ICP34.5 expression in glioma cells. In vivo, rQNestin34.5 showed significantly more potent inhibition of tumor growth compared with control virus. Treatment in the brain tumor model was instituted on animal's display of neurologic symptoms, which usually led to rapid demise. rQNestin34.5 treatment doubled the life span of these animals. These results show that rQNestin34.5 could be a potent agent for the treatment of malignant glioma.

Animals↗
PubMed2003

Expression of the electrophysiological system during murine embryonic stem cell cardiac differentiation.

BACKGROUND: Stem cell based replacement therapy is envisioned as a method to repair failing hearts suffering from cardiomyocyte loss. To prevent potentially lethal arrhythmias, the donor cellular electrophysiological make-up should match with the acceptor tissue. To engineer the desired electrophysiological phenotype, the underlying molecular regulation of ion channels and gap-junction proteins should be clarified first. METHODS: We established the expression of seven main cardiac ion channel a-subunits and four b-subunits using semiquantitive RT-PCR and two major cardiac gap-junction proteins by immunohistochemistry during the differentiation process of murine ES cells into cardiomyocytes. RESULTS: Ion channel mRNA expression profiles display sequential upregulation. Connexin-40 and -43 expression is low in early cardiomyocytes, increased expression rates were found in subsequent differentiation phases. CONCLUSION: Cardiac differentiation of mouse embryonic stem cells is characterized by a sequential upregulation of the main cardiac ion channels and connexins.

Action Potentials↗
PubMed2004

At the crossroads of evolutionary computation and music: self-programming synthesizers, swarm orchestras and the origins of melody.

This paper introduces three approaches to using Evolutionary Computation (EC) in Music (namely, engineering, creative and musicological approaches) and discusses examples of representative systems that have been developed within the last decade, with emphasis on more recent and innovative works. We begin by reviewing engineering applications of EC in Music Technology such as Genetic Algorithms and Cellular Automata sound synthesis, followed by an introduction to applications where EC has been used to generate musical compositions. Next, we introduce ongoing research into EC models to study the origins of music and detail our own research work on modelling the evolution of melody.

Algorithms↗
PubMed2005

In vivo excision of a single targeted nucleotide from an mRNA by a trans excision-splicing ribozyme.

We have previously reported the development of a group I intron-derived ribozyme that can bind an exogenous RNA substrate and excise from that substrate an internal segment in vitro, which allows for sequence-specific modification of RNA molecules. In this report, the activity of this trans excision-splicing ribozyme in a cellular environment, specifically Escherichia coli, was investigated. The ribozyme was re-engineered to target for excision a single-base insertion in the transcript of a green fluorescent protein, and fluorescence was exploited as a reporter for trans excision-splicing. We show that the ribozyme is able to catalyze the trans excision-splicing reaction in vivo and can repair the mutant transcripts. On average, 12% correction is observed as measured by fluorescence and at least 0.6% correction as confirmed through sequence analysis. This represents the first report of a biomolecule (in this case a ribozyme) that can selectively excise a targeted nucleotide from within an mRNA transcript in vivo. This new class of biochemical tools makes possible a wide variety of new experimental strategies, perhaps including a new approach to molecular-based therapeutics.

Escherichia coli↗
PubMedDate not supplied

Systemic gene therapy for arthritis.

Based on the beneficial effects of antibodies to tumor necrosis factor (TNF)-alpha and inhibitory cytokines, gene delivery of cytokines and cytokine inhibitors has been tried and shown to be of therapeutic benefit in the collagen-induced arthritis model in DBA/1 mice. The clinical beneficial effects are accompanied by an effect at the level of immune functions. We have tested the cytokines transforming growth factor (TGF)-beta 1 and interferon (IFN)-beta, a small molecular weight dimeric p75 TNF receptor, as well as a p55 TNF receptor on an immunoglobulin (Ig) backbone. Inhibition of the complement system using a soluble form of complement receptor 1 also has been shown to block B- and T-cell functions. These molecules were expressed in vivo from syngeneic arthritogenic lymphocytes or immortalized fibroblasts. Because in human disease the antigen driving the autoimmune arthritic response is not defined, we have engaged in the targeting of T-cells to collagen type II, by engineering chimeric antibody-type recognition fused to cytoplasmic signaling domains of cellular receptors. From this spectrum of gene therapy approaches, effective in the mouse, it is hoped that some will be able to be transferred to humans.

Journal Article↗
PubMed2005

Cellular growth under hydrostatic pressure using bovine aortic EC-SMC co-cultured ePTFE vascular graft.

High blood pressure (hypertension) is implicated in the development of atherosclerosis. Blood vessels are constantly subjected to stretch due to blood pressure and changes in stretch usually instigate adaptive vascular remodeling, including abnormal growth and proliferation of vascular smooth muscle cells (VSMCs) as well as extracellular matrix (ECM). In this experiment, we used bovine aortic endothelial cells and smooth muscle cells (EC-SMC) co-cultured ePTFE vascular grafts subjected to normal atmospheric pressure (as a control), and 100 mmHg hydrostatic pressure for 7 d. The increase of cell layer thickness was observed. When measured, the cell layer thickness increased by 116.2%. The increase of collagen (Type IV) synthesis was also observed in the immunohistochemistry assay. When stained with toluidine blue, the cells showed metachromatic phenomenon.

Animals↗
PubMed2002

Evaluating the role of microbial stress response mechanisms in causing biological treatment system upset.

It is known that microbial stress mechanisms play a significant role in short-term microbial adaptation to environmental perturbations, and activation of these mechanisms enhance a cell's chance for surviving the perturbation with minimal damage. Although the target of these mechanisms is protective at the cellular level, the effect may be disruptive at the macroscopic level in engineered bioreactor systems. In this paper, it is proposed that these mechanisms are activated in response to wastewater influent perturbations and may be a significant cause of activated sludge treatment process upset. Selected microbial stress responses are reviewed and hypotheses indicating their potential role in treatment process upset are proposed. A research approach that was previously used to identify the mechanistic cause of deflocculation during perturbation by electrophilic chemicals is summarized, and a protocol for future experiments geared toward establishing source-cause-effect relationships for a range of wastewater upset conditions is put forth. Identifying source-cause-effect relationships will provide a basis for development of new monitoring technologies and operational strategies for systems under the influence of influent chemical perturbations.

Bacteria↗
PubMed2006

[Mechanical environment of rotating bioreactor and its effect on cell growth].

In this paper is presented an analysis of the mechanical effect of horizontal rotating bioreactor on cell culture. Getting the microgravity of the bioreactor and the shear stress on canine mesenchymal stem cells (cMSCs) with theoretic calculating model and differential equations, we have validated the density,growth rate and modality of cultured cell by scanning electron microscopy. The horizontal rotating bioreactor which we developed could create the mechanic environment of microgravity (K<8.38 X 10(-2))and low shear stress(r<1.62 dyn/cm2) in theory. The results of scanning electron microscopy indicated that the cells' growth-speed, quantity and modality in bioreactor were better than those of cells cultured in static 24-well plate. The mechanical environment of the rotating bioreactor is propitious for keeping better modality and more rapid proliferation of cMSCs. The rotating bioreactor is a novel approach and technique it is superior to static culture.

Animals↗
PubMed1997

Origin, dispersal, cultivation and variation of rice.

There are two cultivated and twenty-one wild species of genus Oryza. O. sativa, the Asian cultivated rice is grown all over the world. The African cultivated rice, O. glaberrima is grown on a small scale in West Africa. The genus Oryza probably originated about 130 million years ago in Gondwanaland and different species got distributed into different continents with the breakup of Gondwanaland. The cultivated species originated from a common ancestor with AA genome. Perennial and annual ancestors of O. sativa are O. rufipogon and O. nivara and those of O. glaberrima are O. longistaminata, O. breviligulata and O. glaberrima probably domesticated in Niger river delta. Varieties of O. sativa are classified into six groups on the basis of genetic affinity. Widely known indica rices correspond to group I and japonicas to group VI. The so called javanica rices also belong to group VI and are designated as tropical japonicas in contrast to temperate japonicas grown in temperate climate. Indica and japonica rices had a polyphyletic origin. Indicas were probably domesticated in the foothills of Himalayas in Eastern India and japonicas somewhere in South China. The indica rices dispersed throughout the tropics and subtropics from India. The japonica rices moved northward from South China and became the temperate ecotype. They also moved southward to Southeast Asia and from there to West Africa and Brazil and became tropical ecotype. Rice is now grown between 55 degrees N and 36 degrees S latitudes. It is grown under diverse growing conditions such as irrigated, rainfed lowland, rainfed upland and floodprone ecosystems. Human selection and adaptation to diverse environments has resulted in numerous cultivars. It is estimated that about 120,000 varieties of rice exist in the world. After the establishment of International Rice Research Institute in 1960, rice varietal improvement was intensified and high yielding varieties were developed. These varieties are now planted to 70% of world's riceland. Rice production doubled between 1966 and 1990 due to large scale adoption of these improved varieties. Rice production must increase by 60% by 2025 to feed the additional rice consumers. New tools of molecular and cellular biology such as anther culture, molecular marker aided selection and genetic engineering will play increasing role in rice improvement.

Africa, Western↗
PubMed1998

Three-dimensional extracellular matrix engineering in the nervous system.

Growing neurites are guided through their environment during development and regeneration via different cellular and extracellular matrix (ECM) molecular cues. To mimic cell-matrix interactions, a three-dimensional (3D) hydrogel-based ECM equivalent containing a covalently immobilized laminin oligopeptide sequence was designed to facilitate nerve regeneration. This study illustrates that the oligopeptide domain CDPGYIGSR covalently linked to an agarose gel as a bioartificial 3D substrate successfully supports neurite outgrowth from dorsal root ganglia (DRG) in vitro. The specificity of the neurite promoting activity was illustrated through the inhibition of neurite outgrowth from DRG in a CDPGYIGSR-derivatized gel in the presence of solubilized CDPGYIGSR peptide. Gels derivatized with CDPGYIGSK and CDPGRGSYI peptides stimulated a smaller increase of neurite outgrowth. In vivo experiments revealed the capability of a CDPGYIGSR-derivatized gel to enhance nerve regeneration in a transected rat dorsal root model compared to an underivatized gel, a CDPGRGSYI gel, and saline-filled nerve guidance channels. These data suggest the feasibility of a 3D hydrogel-based ECM equivalent capable of enhancing neurite outgrowth in vitro and in vivo.

Animals↗
PubMed2002

Regulatory barriers to tissue-engineered products.

Autologous tissue-engineering technology uses the body's own regenerative capacity to solve clinical problems. Human cellular- and tissue-based products include an array of medical products for repair, reproduction, replacement and other therapeutic purposes. However, the lack of European Union regulation for these products is putting the future of the technology at risk in Europe.

Costs and Cost Analysis↗
PubMed2026

Generation of Fibrin-Based Three-Dimensional Engineered Vascular Tissues from Human Aortic Smooth Muscle Cells for Proteomic Analysis.

Vascular smooth muscle cells (SMCs) reside within the medial layer of blood vessels, where they interact with an extracellular matrix (ECM) composed of collagen, elastin, and proteoglycans to maintain vascular structure and function. Aberrant ECM remodeling contributes to multiple vascular diseases; however, conventional two-dimensional culture systems do not adequately recapitulate the three-dimensional (3D) cellular and matrix environment required to study SMC-ECM interactions and matrix remodeling. This protocol describes the generation of engineered vascular tissues (EVTs) from primary human aortic SMCs cultured within fibrin-based 3D hydrogels. Following casting between flexible polydimethylsiloxane posts, EVTs undergo cellular alignment, contraction, and deposit de novo ECM, providing a physiologically relevant platform for studying vascular matrix biology. The protocol details tissue fabrication, culture, harvesting, and downstream analysis of newly deposited ECM by immunofluorescence staining. In addition, a workflow is presented for qualitative and quantitative characterization of EVT-derived proteins using Western blotting and mass spectrometry-based proteomics. Sequential protein extraction enables assessment of soluble and ECM-enriched protein fractions, facilitating in-depth evaluation of ECM composition. This platform provides a reproducible approach for investigating ECM production and remodeling by human SMCs in a 3D environment.

Humans↗
PubMed1999

Biomechanical factors in tissue engineered meniscal repair.

Damage to the meniscus after trauma or injury is associated with detrimental changes in joint function that can lead to pain, disability, and degenerative joint changes. Recently, tissue engineering strategies for meniscal repair have been suggested including using biocompatible grafts as a substrate for regeneration, and cellular supplementation to promote remodeling and healing. Little is known, however, about the contributions of these novel repair strategies to restoration of normal meniscal function. Biomechanical factors play a role in the design and synthesis of tissue engineered biomaterials and bioreactors, and also are important for evaluating the efficacy of these new strategies for restoring normal meniscal function. In this report, an overview is presented of biomechanical factors that are critical to meniscal function followed by a review of biomechanical considerations for the design and evaluation of tissue engineered strategies for meniscal repair. Recommendations for future study of biomechanical factors in tissue engineered meniscal repair also are provided.

Animals↗