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At least 631 records · Page 35Linked to original sources

PubMed2004

Impairment of Brucella growth in human macrophagic cells that produce nitric oxide.

In mice, nitric oxide (NO) production by inducible NO synthase (iNOS), is a component of the control of Brucella infection. In humans, the involvement of iNOS in infection is still a matter of debate. Based on in vitro experiments, it was recently postulated that in humans, Brucella infection tends to become chronic because NO cannot exert its deleterious effect. In fact, conditions allowing NO production by human macrophages in culture are poorly defined, rendering the in vitro study of NO function difficult. Using DFGiNOS U937 macrophagic cells engineered to produce NO and U937 cells activated by ligation of IgE receptors, we showed that the intracellular development of Brucella was impaired in human macrophages, which produced NO. Although Brucella-infected human macrophagic phagocytes did not release NO in commonly used models of infection, the machinery required to produce NO was expressed in these cells and could be triggered by cell membrane receptors present on the infected cells. Therefore, the lack of NO production in isolated human macrophages infected by Brucella under in vitro conditions did not exclude a possible involvement of NO in the control of human brucellosis.

Brucella↗

PubMedDate not supplied

An efficient enzymatic Baeyer-Villiger oxidation by engineered Escherichia coli cells under non-growing conditions.

Economical methods of supplying NADPH must be developed before biotransformations involving this cofactor can be considered for large-scale applications. We have studied the enzymatic Baeyer-Villiger oxidation of cyclohexanone as a model for this class of reactions and developed a simple approach that uses whole, non-growing Escherichia coli cells to provide high productivity (0.79 g epsilon-caprolactone/L/h = 18 micromol epsilon-caprolactone/min/g dcw) and an 88% yield. Glucose supplied the reducing equivalents for this process, and no exogenous cofactor was required. The volumetric productivity of non-growing cells was an order of magnitude greater than that achieved with growing cells of the same strain. Cells of an engineered E. coli strain that overexpresses Acinetobacter sp. cyclohexanone monooxygenase were grown under inducing conditions in rich medium until the entry to stationary phase; the subsequent cyclohexanone oxidation was carried out in minimal salts medium lacking a nitrogen source. After the biotransformation was complete, the lactone product was adsorbed to a solid support and recovered by washing with an organic solvent.

Caproates↗

PubMedDate not supplied

Improved degradation of organophosphorus nerve agents and p-nitrophenol by Pseudomonas putida JS444 with surface-expressed organophosphorus hydrolase.

Pseudomonas putida JS444, isolated from p-nitrophenol (PNP) contaminated waste sites, was genetically engineered to simultaneously degrade organophosphorus pesticides (OP) and PNP. A surface anchor system derived from the ice-nucleation protein (INP) from Pseudomonas syringae was used to target the organophosphorus hydrolase (OPH) onto the surface of Pseudomonas putida JS444, reducing the potential substrate uptake limitation. Engineered cells were capable of targeting OPH onto the cell surface as demonstrated by western blotting, cell fractionation, and immunofluorescence microscopy. The engineered P. putida JS444 degraded organophosphates as well as PNP rapidly without instability problems associated with the engineered Moraxella sp. The initial hydrolysis rate was 7.90, 3.54, and 1.53 micromol/h/mg dry weight for paraoxon, parathion, and methyl parathion, respectively. The excellent stability in combination with the rapid degradation rate for organophosphates and PNP make this engineered strain an ideal biocatalyst for complete mineralization of organophosphates.

Aryldialkylphosphatase↗

PubMed2005

Biosynthetic labeling of RNA with uracil phosphoribosyltransferase allows cell-specific microarray analysis of mRNA synthesis and decay.

Standard microarrays measure mRNA abundance, not mRNA synthesis, and therefore cannot identify the mechanisms that regulate gene expression. We have developed a method to overcome this limitation by using the salvage enzyme uracil phosphoribosyltransferase (UPRT) from the protozoan Toxoplasma gondii. T. gondii UPRT has been well characterized because of its application in monitoring parasite growth: mammals lack this enzyme activity and thus only the parasite incorporates (3)H-uracil into its nucleic acids. In this study we used RNA labeling by UPRT to determine the roles of mRNA synthesis and decay in the control of gene expression during T. gondii asexual development. We also used this approach to specifically label parasite RNA during a mouse infection and to incorporate thio-substituted uridines into the RNA of human cells engineered to express T. gondii UPRT, indicating that engineered UPRT expression will allow cell-specific analysis of gene expression in organisms other than T. gondii.

Animals↗

PubMed1997

SpoIIQ, a forespore-expressed gene required for engulfment in Bacillus subtilis.

A crucial step in converting an actively growing Bacillus subtilis cell into a dormant spore is the formation of a cell within a cell. This unusual structure is created by a phagocytosis-like process in which the larger mother cell progressively engulfs the adjacent smaller forespore. Only mutations blocking engulfment at an early stage and affecting genes expressed in the mother cell have been identified. Here we describe a new locus, spoIIQ, which is transcribed in the forespore and which encodes a membrane-bound protein required at a late stage of engulfment. Immunofluorescence microscopy analysis have shown that SpoIIQ is initially targeted to the septum at the boundary between the two cells and then spreads around the entire membrane of the forespore. Septum targeting requires only the first 52 residues of SpoIIQ as well as unidentified forespore-specific components. Electron-microscopy studies of cells engineered to activate the mother-cell program of gene expression independently of the forespore indicate that other as yet uncharacterized genes are involved in engulfment and that this morphological process is driven from both sides of the forespore envelope.

Amino Acid Sequence↗

PubMed2001

Tissue spreading on implantable substrates is a competitive outcome of cell-cell vs. cell-substratum adhesivity.

While the interactions of cells with polymeric substrata are widely studied, the influence of cell-cell cohesiveness on tissue spreading has not been rigorously investigated. Here we demonstrate that the rate of tissue spreading over a two-dimensional substratum reflects a competition or "tug-of-war" between cell-cell and cell-substratum adhesions. We have generated both a "library" of structurally related copolymeric substrata varying in their adhesivity to cells and a library of genetically engineered cell populations varying only in cohesiveness. Cell-substratum adhesivity was varied through the poly(ethylene glycol) content of a series of copolymeric substrata, whereas cell-cell cohesiveness was varied through the expression of the homophilic cohesion molecules N- and R-cadherin by otherwise noncohesive L929 cells. In the key experiment, multicellular aggregates containing about 600 cells were allowed to spread onto copolymeric surfaces. We compared the spreading behavior of aggregates having different levels of cell-cell cohesiveness in a series of copolymeric substrata having different levels of cell-substratum adhesivity. In these experiments, cell-cell cohesiveness was measured by tissue surface tensiometry, and cell-substratum adhesivity was assessed by a distractive method. Tissue spreading was assayed by confocal microscopy as the rate of cell emigration from similar-sized, fluorescence-labeled, multicellular aggregates deposited on each of the substrata. We demonstrate that either decreasing substratum adhesivity or increasing cell-cell cohesiveness dramatically slowed the spreading rate of cell aggregates.

Animals↗

PubMed2006

Nuclear reprogramming by cell fusion.

The use of cell fusion to study exchange of information at the molecular level between the nucleus and the cytoplasm of cells during regulation of gene expression was pioneered by Harris and Ringertz more than three decades ago. The ability to make heterokaryons with cells from different species or genetic strains is especially useful because genetic differences in gene products allow the origin of trans-acting regulatory factors to be determined. Heterokaryons between adult nucleated erythroid cells of one species and embryonic/larval nucleated erythroid cells of another species, for example, show cross-induction between the two types of nuclei, resulting in reprogramming of the adult nucleus to embryonic/larval globin gene expression and/or reprogramming of the embryonic/larval cell nucleus to adult globin expression. These experiments provided definitive evidence that developmental program switching is mediated by trans-acting factors. Other possible uses of this cell fusion protocol in stem cell biology and transplantation of genetically engineered cells for tissue regeneration are briefly discussed.

Animals↗

PubMed2003

Pharmacologically regulated production of targeted retrovirus from T cells for systemic antitumor gene therapy.

We aimed to use cell-based carriers to direct vector production to target sites for systemic therapy. We used T cells engineered to express a chimeric T cell receptor that can specifically recognize target cells expressing the tumor-associated carcinoembryonic antigen (CEA). These T cells were modified to produce a retrovirus under tight pharmacological control using the rapamycin-inducible transcriptional regulatory system. The retroviral vectors produced were transcriptionally targeted to CEA-expressing target cells. We found that vector production and transgene expression from these T cells in vitro was dependent on pharmacological induction and expression of CEA in target cells, respectively. Mice bearing metastatic tumors that received cell carriers delivering the HSVtk gene demonstrated a significant increase in survival, but only in response to pharmacological induction of vector production. Interestingly, the therapeutic effect required the presence of the tumor-specific chimeric receptor on T cells. Further studies demonstrated that systemic delivery of tumor-specific T cells to mice bearing metastatic tumors caused recruitment of nonspecific T cells to the tumor site. We hypothesize that this enhanced targeting to tumor sites is responsible for the efficiency of T cell-mediated retroviral gene transfer and that this principle can be used to enhance systemic therapies using immune-cell carriers.

Adenocarcinoma↗

PubMed2005

TRAIL decoy receptors mediate resistance of acute myeloid leukemia cells to TRAIL.

BACKGROUND AND OBJECTIVES: The tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is regarded as a potential anticancer agent. However, many cancer cells are resistant to apoptosis induction by TRAIL. The present study was designed to evaluate the sensitivity to TRAIL-induced apoptosis in acute myeloblastic leukemias (AML). DESIGN AND METHODS: TRAIL/TRAIL receptor (TRAIL-R) expression and sensitivity to TRAIL-mediated apoptosis were explored in 79 AML patients, including 17 patients with acute promyelocytic leukemia (APL). RESULTS: In non-APL AML we observed frequent expression of TRAIL decoy receptors (TRAIL-R3 and TRAIL-R4), while TRAIL-R1 and TRAIL-R2 expression was restricted to AML exhibiting monocytic features. Total leukemic blasts, as well as AML colony-forming units (AML-CFU), were invariably resistant to TRAIL-mediated apoptosis. APL express membrane-bound TRAIL on their surface and exhibit a pattern of TRAIL-R expression similar to that observed in the other types of AML. Before, during and after retinoic acid treatment APL cells are TRAIL-resistant. The induction of granulocytic maturation of APL cells by retinoic acid was associated with a marked decline of TRAIL expression. INTERPRETATION AND CONCLUSIONS: The analysis of experimental APL models (i.e., U937 cells engineered to express PML/RAR-Eo and NB4 cells) provided evidence that PML/RAR-Eo expression was associated with downmodulation of TRAIL-R1 and with resistance to TRAIL-mediated apoptosis. We suggest that AML blasts, including APL blasts, are resistant to TRAIL-mediated apoptosis, a phenomenon seemingly related to the expression of TRAIL decoy receptors on these cells. Finally, APL blasts express membrane-bound TRAIL that could confer an immunologic privilege to these cells.

Acute Disease↗

PubMedDate not supplied

Cell-based tissue engineering for the intervertebral disc: in vitro studies of human disc cell gene expression and matrix production within selected cell carriers.

BACKGROUND CONTEXT: Little is known about how disc cells attach, proliferate and form extracellular matrix (ECM) within carrier materials. Such information is needed to help formulate criteria for successful cell-carrier interactions in tissue engineering. PURPOSE: To compare proliferation, ECM production and gene expression in annulus cells cultured in a variety of cell carrier materials with potential application in tissue engineering of the disc. STUDY DESIGN: Human intervertebral disc cells from the annulus were used in a prospective study of proliferation, ECM production and gene expression within selected cell carriers. METHODS: Annulus cells from discs of 29 individuals were tested in collagen sponge, collagen gel, agarose, alginate or fibrin gel formulations. In situ hybridization assessed ECM gene expression of Types I and II collagen, aggrecan and chondroitin-6 sulfotransferase. Cell proliferation, cell shape, attachment and ECM production were evaluated. RESULTS: Collagen sponges provided the best microenvironment for disc cell ECM production and gene expression. Although collagen gels often could support good cell growth, such constructs did not result in either abundant ECM production or ECM gene expression, as shown by in situ hybridization. Growth and ECM production and gene expression in alginate, agarose and fibrin microenvironments were inferior. CONCLUSIONS: Tissue engineering techniques open new therapeutic possibilities for use of autologous disc cells, but fundamental questions on how these cells interact with cell carriers are unexplored. Results provide novel data on disc cell gene expression within diverse microenvironments. The collagen sponge proved to be a superior microenvironment.

Absorbable Implants↗

PubMed2006

Cell-free translation systems for protein engineering.

Cell-free translation systems have developed significantly over the last two decades and improvements in yield have resulted in their use for protein production in the laboratory. These systems have protein engineering applications, such as the production of proteins containing unnatural amino acids and development of proteins exhibiting novel functions. Recently, it has been suggested that cell-free translation systems might be used as the fundamental basis for cell-like systems. We review recent progress in the field of cell-free translation systems and describe their use as tools for protein production and engineering.

Animals↗

PubMed2002

Mathematical modeling of myoglobin facilitated transport of oxygen in devices containing myoglobin-expressing cells.

Low pO(2) is perhaps the most significant factor in artificial pancreas failure. In these environments, not only is the beta cell production of insulin reduced, but the cell death rate is also significantly higher. Mathematical models are developed to test the feasibility of facilitated oxygen transport in enhancing O(2) flux to genetically engineered cells in a bioartificial device such as a pancreas. For this device, it is proposed that beta cells be genetically engineered to express myoglobin throughout the cell. In addition, the significance of including myoglobin throughout the alginate matrix present to provide immuno-protection for the transplanted cells is considered. The mathematical analysis predicts that myoglobin facilitated oxygen transport has the potential of increasing the oxygen concentration at the centre of a cluster of cells (islet) with an effective radius of 100 microm by 50%. These theoretical models for myoglobin facilitated oxygen transport with homogeneous Michaelis-Menten consumption also indicate that including myoglobin in the alginate gel would beneficially improve the flux of oxygen to the transplanted cells.

Alginates↗

PubMed2003

Establishment and cultivation of cell line HB with high temperature resistance and fast growth in Porphyra yezoensis.

A new breeding technology with cell culture in Porphyra yezoensis was studied, to establish a system of fast breeding with cell engineering in Porphyra. By means of somatic cell isolation and multiple clone technique, 4 pure cell-lines (HA, HB, HC, HD) have been established in Porphyra yezoensis. With purely culturing the cell seedlings and conchocelis filaments of the cell lines, their growth rate and resistance to higher temperature were measured. Among cell line HA, HB, HC, HD, HB was the best for resisting higher temperature (at 19 degrees C, 21 degrees C, 23 degrees C, 25 degrees C). Also the growth rate of HB was faster than others. In 1998-2000, the HB was cultivated at sea field of Haifeng in Qidong County, Jiangsu Province. The yield of HB was higher than that of local cultivar. So the HB might be a good cell line for both resisting higher temperature and faster growth. It showed the breeding with cell culture was a fast breeding method.

Breeding↗

PubMed2003

Cellular engineering of HSV-tk transduced, expanded T lymphocytes for graft-versus-host disease management.

Engineering donor T lymphocytes with inducible 'suicide genes', such as herpes simplex virus thymidine kinase, has potential to improve safety and efficacy in allogeneic transplantation by facilitating management of graft-versus-host disease. Elective administration of a relatively nontoxic pro-drug would induce in vivo negative selection of engineered lymphocytes specifically, sparing other donor hematopoietic cells. The engineered cells must retain immunologic function, and undergo negative selection in response to clinically attainable plasma concentrations of pro-drug. The cell engineering process itself, typically involving activation, transduction, ex vivo expansion, and selection, must produce clinically useful numbers of genetically modified cells at high purity. We discuss development of a cellular engineering manufacturing process that yields transduced, expanded T lymphocytes meeting these requirements.

Adoptive Transfer↗

PubMed1992

Inhibition of tumor growth by histoincompatible cells expressing interleukin-2.

Murine tumor cells engineered to express IL-2 have been shown to be rejected by the syngeneic host, which is then protected against a subsequent tumorigenic challenge. To assess whether IL-2 has to be produced by the tumor cells themselves, or whether its local delivery would be sufficient to promote such beneficial effects, the syngeneic tumor cells were co-inoculated with allogeneic or xenogeneic cells secreting IL-2, selected after gene transfection. In several murine systems, it was observed that this is an efficient approach for controlling the growth of the syngeneic tumor. However, animals which rejected the tumor were not protected against a subsequent challenge. Several lines of evidence indicate that NK cells play a major role in tumor rejection induced by the IL-2 expressing histoincompatible vector cells. Thus, while local delivery of IL-2 in the vicinity of a tumor might not be sufficient to promote a systemic long-term specific antitumor immune response, it can control the growth of the primary syngeneic tumor. These experiments demonstrate the feasibility of using genetically engineered histoincompatible cells (which are rejected by the host's immune system) as a transient delivery system in vivo.

Animals↗

PubMed1990

Genetically engineered V79 Chinese hamster cells metabolically activate the cytostatic drugs cyclophosphamide and ifosfamide.

V79 cells, genetically engineered to express active cytochromes P450IIB1 and P450IA1, were used to study the cytotoxicity and mutagenicity of cyclophosphamide and ifosfamide. Cyclophosphamide, tested up to a concentration of 2 mM, was not cytotoxic in V79 nor in the P450IA1-expressing V79-derived cell line XEM2. Pronounced cytotoxicity was, however, observed in the P450IIB1-expressing V79-derived cell line SD1. Induction of gene mutations (acquisition of 6-thioguanine resistance) was observed in SD1 cells as well, but the effects were weak. Ifosfamide was inactive in V79 cells, but was cytotoxic in SD1 cells. Ifosfamide mustard, an active metabolite of ifosfamide, was equally cytotoxic and showed similar mutagenic effects in SD1 and parental V79 cells. The results indicate that cyclophosphamide and ifosfamide are metabolically activated by cytochrome P450IIB1. In contrast, cytochrome P450IA1 was not capable of activating cyclophosphamide. Thus, V79-derived cell lines defined for their expression of a specific form of cytochrome P-450 can be used as diagnostic tools to identify the cytochrome P-450 that is responsible for the metabolic activation of drugs.

Animals↗

PubMed2002

Permanent restoration of human skin treated with cultured epithelium grafting--wound healing by stem cell based tissue engineering--.

The technique of epidermal cell culture developed by Green and colleagues made a breakthrough in the treatment of massive wounds in vivo with grown cells in vitro. In the past two decades, progress of culture methods and clinical practice have been made and now it is possible to treat extensive skin defect with large amounts of cultured epithelium. Since 1985, we have been successfully used cultured epidermis as autografts for the permanent coverage of full-thickness burn wounds or excised burn scars, giant nevi, tattoos and so on. Furthermore, cultured epidermis has been available as allografts to promote the healing of chronic skin ulcers or deep dermal burn. In this paper we describe our clinical experience of cultured epithelium grafting for the treatment of wounds and predict new trial of wound management and regeneration based on tissue engineering concept.

Adult↗

PubMed1995

Extracellular signal protein triggering the proteolytic activation of a developmental transcription factor in B. subtilis.

We present biochemical evidence for an intercellular signal transduction pathway in B. subtilis. This pathway governs the conversion of the proprotein pro-sigma E to mature transcription factor sigma E. Proteolytic processing is mediated by the membrane protein SpollGA and is triggered by the inferred extracellular signal protein SpollR. A factor in conditioned medium from B. subtilis cells engineered to produce SpollR during growth triggered processing in protoplasts of B. subtilis cells that had been engineered to produce SpollGA and pro-sigma E. The factor was also detected in, and partially purified from, extracts of SpollR-producing cells of E. coli. We speculate that SpollGA is both a receptor and a protease and the SpollR interacts with SpollGA on the outside of the cytoplasmic membrane, activating the intracellular protease domain of SpollGA.

Amino Acid Sequence↗