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At least 703 records · Page 39Linked to original sources

PubMed2004

Three-dimensional electron microscopic imaging of membrane invaginations in Escherichia coli overproducing the chemotaxis receptor Tsr.

Electron tomography is a powerful method for determining the three-dimensional structures of large macromolecular assemblies, such as cells, organelles, and multiprotein complexes, when crystallographic averaging methods are not applicable. Here we used electron tomographic imaging to determine the molecular architecture of Escherichia coli cells engineered to overproduce the bacterial chemotaxis receptor Tsr. Tomograms constructed from fixed, cryosectioned cells revealed that overproduction of Tsr led to formation of an extended internal membrane network composed of stacks and extended tubular structures. We present an interpretation of the tomogram in terms of the packing arrangement of Tsr using constraints derived from previous X-ray and electron-crystallographic studies of receptor clusters. Our results imply that the interaction between the cytoplasmic ends of Tsr is likely to stabilize the presence of the membrane networks in cells overproducing Tsr. We propose that membrane invaginations that are potentially capable of supporting axial interactions between receptor clusters in apposing membranes could also be present in wild-type E. coli and that such receptor aggregates could play an important role in signal transduction during bacterial chemotaxis.

Bacterial Proteins↗

PubMed2002

[Tissue engineering--Quality and safety control of cultured human cells].

Tissue Engineering Research Center (TERC) at Ikeda city, Osaka has been established in April, 2001 and functions as Cell Processing Center (CPC) having biological clean rooms. At the center, many biomedical researchers as well as clinicians work to develop human cell-based medical devices. In the CPC, Medical Device Team in TERC deals with differentiated osteoblasts derived from cultured human bone marrow cells to create tissue-engineered implantable bone. We are focusing on patients treatments by direct application of the tissue engineered bone, therefore, we should manipulate the human cells under more restricted and controlled culture condition than ordinal cases. Here we introduce environment and technology of human bone marrow cell culture, and show quality control and safety evaluation of culture processes in TERC.

English Abstract↗

PubMed2006

Human sulphotransferases are involved in the activation of aristolochic acids and are expressed in renal target tissue.

Use of herbal preparations containing Aristolochia species has led to progressive nephropathy and urothelial cancer in humans. Analysis of DNA adducts formed in human target tissues and studies in animal models have pointed out a major role of the secondary plant metabolites, aristolochic acids, in these effects. Only a minority of the users of Aristolochia-containing products developed nephropathy and cancer, suggesting differences in individual susceptibility. Differences in metabolic activation and inactivation frequently affect the susceptibility towards chemicals. Others have shown that the activation of aristolochic acids to DNA-reactive and mutagenic metabolites requires reduction of their aryl nitro group. The biological activity of numerous nitro- and aminoarenes, after appropriate phase I metabolism, is strongly enhanced in the presence of acetyltransferases or sulphotransferases (SULTs). In the present study, we demonstrate that expression of human SULTs in bacterial and mammalian target cells reinforces the mutagenic activity of aristolochic acids. Using Salmonella typhimurium TA1538 as the recipient organism, we identified the expression of all 12 human SULT forms. SULT1A1 led to the strongest increase in the mutagenicity of aristolochic acids. Some activation was also observed with SULT1B1, but not with the remaining forms. The role of SULT1A1 in the activation of aristolochic acids was corroborated using S. typhimurium TA100- and Chinese hamster V79-derived target cells engineered for expression of human SULT1A1 when compared with control cells. Furthermore, pentachlorophenol, an inhibitor of SULT1A1, strongly reduced the mutagenic effect of aristolochic acids in V79-hCYP2E1-hSULT1A1 cells. Moreover, we demonstrate that SULT1A1 and SULT1B1 are expressed in human kidney using immunoblot analysis, but their levels are substantially lower than in liver. Finally, we discuss the possibility that reactive sulphuric acid conjugates produced in other tissues are transferred to kidney and ureter.

Aged↗

PubMed1994

Mirror image in vivo electroblotting technique, a new technique for visualizing virus particles electrophoretically transferred from infected leaves to nitrocellulose membranes.

A new technique was developed for visualizing virus particles electrophoretically transferred from infected leaves to nitrocellulose membranes. This technique was used to study the distribution of peanut mottle virus (PMV) particles in vivo. The immobilized transferable virus proteins from leaf tissue to nitrocellulose membranes were detected by an enzyme-linked immunobinding technique. The free binding groups on the nitrocellulose membrane were blocked with casein. The nitrocellulose membrane was then treated with PMV-specific antiserum. The virus-bound antibodies were located by protein A-peroxidase or protein A-alkaline phosphatase conjugate followed by the peroxidase substrate mixture, 4-chloro-1-naphthol and H2O2, in the first case or the alkaline phosphatase substrate mixture, 5-bromo-4-chloro-3-indolyl phosphate and Nitroblue tetrazolium in the second case. The locations of virus particles were detected by the corresponding reaction product. A mirror image was obtained on nitrocellulose membrane showing a pattern identical to that of necrotic lesions on leaf pieces but with a significantly larger size of local lesion copy, indicating the presence of virus particles in the apparently healthy tissue outside the necrotic lesion. The new technique is expected, because of its relative simplicity and sensitivity, to be useful for many other fields of biological research where the in vivo distribution of pathogens, antigenic molecules including drugs, or cells of specific antigenic molecules such as cancer or genetically engineered cells needs to be investigated.

Alkaline Phosphatase↗

PubMed2006

Manipulation of living cells by using PC-controlled micro-pattern projection system.

In order to meet the diversifying demand for the cell manipulation in the rapid progress of cell engineering, we developed a novel technique to capture the living cells on a culture substrate by irradiating light in a multiple manner. In clear contrast to the conventional cell patterning using the previously patterned substrate, the cell-retaining area can be defined even after cell seeding, and the captured cells can continue to grow freely beyond the defined area afterwards. After the light irradiation in arbitrary micropattern by using a newly developed apparatus and the process to remove non-captured cells including EDTA treatment, the highly contrasted cell patterns were formed with the precision of single cell size. Moreover, it was determined that the cell capturing arose just after light irradiation and diminished gradually in a time scale of 10h. It was confirmed that the cells maintained their viability well after the manipulation process including photo-induced cell capturing.

Animals↗

PubMed2004

Stromal cells expressing elevated VCAM-1 enhance survival of B lineage tumor cells.

In the current study we generated murine bone marrow stromal cells that constitutively express human VCAM-1. These stromal cell lines allow investigation of the contribution of VCAM-1 initiated signaling to tumor cell survival. Co-culture of ALL cell lines with stromal cells engineered to over express VCAM-1 enhanced survival of leukemic cells in a PI-3 kinase-dependent manner, compared to co-culture with parental stromal cells expressing only endogenous VCAM-1. These observations suggest that modulation of stromal cell VCAM-1 by specific chemotherapeutic drugs may have utility in decreasing residual disease. In addition, these novel lines provide an in vitro model in which other tumor types that interact with stromal cells in the bone marrow microenvironment may be evaluated to determine the contribution of VCAM-1 initiated signaling to modulation of treatment response.

Animals↗

PubMed2002

Human Dkk-1, a gene encoding a Wnt antagonist, responds to DNA damage and its overexpression sensitizes brain tumor cells to apoptosis following alkylation damage of DNA.

The human Dkk-1 (hDkk-1) gene, a transcriptional target of the p53 tumor suppressor, encodes a powerful inhibitor of the Wnt signaling pathway and regulates the spatial patterning/morphogenesis of the mammalian central nervous system. We investigated the p53-related functions of the hDkk-1 gene by studying its response to DNA damage and its modulation of apoptosis in human glioma cells. Various chemotherapeutic and other agents that induce DNA adducts and compromise its integrity (1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU), cisplatin, H(2)O(2) and UV rays) enhanced the expression of hDkk-1 significantly. The damage-induced increase in hDkk-1 mRNA levels occurred in many human tumor cell lines, irrespective of their p53 gene status. The human glioblastoma cell line, U87MG, which had undetectable hDkk-1 expression, was engineered to express moderate levels of the hDkk protein by stable transfection. The engineered cells did not show any morphological changes, but underwent marked apoptosis after ceramide treatment. Further, the DNA cross-linking drugs BCNU and cisplatin, but not the microtubule poison vincristine, induced significant cell death in U87MG/hDkk cells, and this was accompanied by altered Bcl-2/Bax expression and a reduction in the amount of telomere DNA as visualized by fluorescence in situ hybridization. These results show that hDkk-1 is a pro-apoptotic gene and suggest that it may play important roles in linking the oncogenic Wnt and p53 tumor suppressor pathways.

Alkylation↗

PubMed2005

Vascular endothelial growth factor selectively targets boronated dendrimers to tumor vasculature.

Tumor neovasculature is a potential but, until very recently, unexplored target for boron neutron capture therapy (BNCT) of cancer. In the present report, we describe the construction of a vascular endothelial growth factor (VEGF)-containing bioconjugate that potentially could be used to target up-regulated VEGF receptors (VEGFR), which are overexpressed on tumor neovasculature. A fifth-generation polyamidoamine dendrimer containing 128 reactive amino groups was reacted with 105 to 110 decaborate molecules to produce a macromolecule with 1,050 to 1,100 boron atoms per dendrimer. This was conjugated to thiol groups of VEGF at a 4:1 molar ratio using the heterobifunctional reagent sulfo-LC-SPDP. In addition, the boronated dendrimer was tagged with a near-IR Cy5 dye to allow for near-IR fluorescent imaging of the bioconjugate in vitro and in vivo. As would be predicted, the resulting VEGF-BD/Cy5 bioconjugate was not cytotoxic to HEK293 cells engineered to express 2.5 x 10(6) VEGFR-2 per cell. Furthermore, it showed binding and activation of VEGFR-2 comparable with that of native VEGF. Internalization of VEGF-BD/Cy5 by PAE cells expressing 2.5 x 10(5) VEGFR-2 per cell was inhibited by excess VEGF, indicating a VEGFR-2-mediated mechanism of uptake. Near-IR fluorescent imaging of 4T1 mouse breast carcinoma revealed selective accumulation of VEGF-BD/Cy5, but not BD/Cy5, particularly at the tumor periphery where angiogenesis was most active. Accumulation of VEGF-BD/Cy5 in 4T1 breast carcinoma was diminished in mice pretreated with a toxin-VEGF fusion protein that selectively killed VEGFR-2-overexpressing endothelial cells. Our data lay the groundwork for future studies using the VEGF-BD/Cy5 bioconjugate as a targeting agent for BNCT of tumor neovasculature.

Animals↗

PubMed2002

Current surgical treatment of Parkinson's disease.

There has been a renaissance in the surgical management of Parkinson's disease. This has been due to long-term effects of levodopa and a better understanding of the basal ganglia and its circuitry. Ablative surgery and neurostimulation are the only realistic surgical options at present. Although surgical treatments, such as ablation and stimulation are effective, they are not useful for stopping the progression or restoring the system. Neural transplantation helps restore the system by using a number of techniques. Targets mostly used are in the thalamus, globus pallidus and subthalamic nucleus. A number of factors must be considered including patient's age, disability and his wishes. Globus pallidus stimulation might be preferable for patients who suffer from dyskinesia as a major source of disability. Pallidotomy might be appropriate in cases where frequent stimulator adjustments are impractical. Subthalamic nucleus stimulation is more suitable for patients with significant off periods and in younger patients in whom it may be desirable to maintain intact circuitry. Fetal neural transplantation, stem cell transplantation, xenotransplantation, adrenal medullary transplantation and transplantation of genetically engineered cells are at various stages of development and research. Ethical issues surrounding these process are likely to arouse strong emotions and have to be carefully considered.

Cell Transplantation↗

PubMed2004

[Studies on the cell growth, differentiation and terpene lactone accumulation in Ginkgo biloba cell suspension cultures].

To provide supports for Ginkgo biloba cell engineering for production of Terpene lactones (Ginkgolides and bilobalide), the cell suspension were established from calli induced from zygote embryos and stems of 30-day-old seedlings respectively. The relationship between cell growth, differentiation and the terpene lactone accumulation in these suspension cultures were investigated. HPLC determination indicated that, the ginkgolide B was found in the embryo derived cell suspension cultures at 0.044% of cell dry weight, and this result was the first time reported in this study. The accumulation of terpene lactone in the suspension cultures derived from both the embryo and seedling stems are effected by the level of the cell differentiation. The ginkgolide B was only found in small cell aggregates in the size smaller than 2mm, and the highest level of ginkgolide B was accumulated in cell aggregates in the size smaller than 1mm; however, the cell aggregates in the size bigger than 3mm could only produced bilobalide and ginkgolide A. In the same size aggregates of the suspension cultures the terpene lactone accumulation is strongly effected by the source of the explant. When the size of cell aggregates was in less than 1mm, the concentration of bilobalide, ginkgolide A and B in the cell suspension cultures derived from the embryos was 2, 1.4 and 0.56-fold, respectively, higher than that of cell cultures derived from seedling stems.

Bilobalides↗

PubMedDate not supplied

Activation and phenotyping of LAK generated by exposure to product of cells transformed by interleukin 2 cDNA.

It has been previously found that local administration of X63-m-IL-2 cells transformed by interleukin 2 (IL-2) cDNA and constitutively producing large quantities of IL-2 mediated regressions of murine plasmacytomas and 3-methyl-cholanthrene-induced sarcomas transplanted in syngeneic mice. Here we show that killer cells generated by cultivation of spleen cells in supernatants from X63-m-IL-2 cultures (LAK) or by co-cultivation of murine splenocytes with X63-m-IL-2 cells were cytolytic for natural killer (NK)-sensitive as well as NK-resistant target cells, including the IL-2-producing X63-m-IL-2 cells. Spleen cells cultured in X63-m-IL-2 supernatants or co-cultivated with X63-m-IL-2 cells yielded predominantly Thy 1.2+, CD3+, LFA-1+ lymphocytes. The in vitro results suggest that the LAK cells generated due to the IL-2 production by genetically engineered cells probably help to terminate the treatment by killing the IL-2 producers.

Animals↗

PubMed1996

Hepatocyte growth factor and hepatocyte growth factor receptor in the lacrimal gland, tears, and cornea.

PURPOSE: The purpose of this study was to characterize the expression of hepatocyte growth factor (HGF) and HGF receptor proteins in lacrimal gland, tears, and cornea. METHODS: The reverse transcription-polymerase chain reaction method was used to detect HGF and HGF receptor messenger RNA in human lacrimal gland tissue. HGF and HGF (c-met) receptor monoclonal antibody specificity was demonstrated with fluorescent antibody sorting of cells engineered to express HGF or HGF receptor compared with control cell lines, enzyme-linked immunoadsorbent assay (ELISA), immunoprecipitation, and immunohistology with preabsorption. Immunohistochemistry was applied to study the distribution of HGF and HGF receptor expression in rabbit lacrimal gland tissue and in wounded and unwounded rabbit cornea. An ELISA was used to detect HGF in pooled samples of human tears and individual aliquots of tears collected from patients 1 day after anterior segment surgery. RESULTS: Amplification products of the expected size for HGF and HGF receptor mRNAs were detected in lacrimal tissue and were confirmed to be specific by hot blotting and nucleic acid sequencing. Hepatocyte growth factor protein was detected in interalveolar and interlobular connective tissue cells adjacent to glandular alveolar (acinar) cells and associated with the cells lining the interlobular ducts. Hepatocyte growth factor receptor protein was expressed in the glandular alveolar and interlobular ductal cells in the lacrimal gland and all three cell types of the cornea. It was detected in keratocyte and endothelial cells, and expression was increased in keratocytes after epithelial wounding. Hepatocyte growth factor was not present in corneal epithelial cells, but in the unwounded cornea a strong signal was associated with the epithelial cell surface. It was detected by ELISA in pooled normal tears at levels 186 to 290 pg/ml and in individual postoperative tear samples at 453 to 619 pg/ml. In some tear samples, HGF levels were below the sensitivity of the assay (97.5 pg/ml). CONCLUSIONS: The distribution of HGF receptor protein expression in the lacrimal gland suggests that HGF secreted by interalveolar connective tissue cells traverses the acinar cells and modulates functions in acinar and ductal epithelial cells. Hepatocyte growth factor likely collects within the interlobular ducts and becomes a component in normal tears. Thus, lacrimal gland HGF probably modulates corneal epithelial cell proliferation, motility, and differentiation. Its expression in keratocytes is upregulated after corneal epithelial wounding and probably contributes to the epithelial wound healing process.

Animals↗

PubMed1996

Targeting gamma interferon to tumor cells by a genetically engineered fusion protein secreted from myeloma cells.

The construction, synthesis and expression of a genetically engineered bifunctional antibody/cytokine fusion protein is described. To target IFN-tau to tumor cells, recombinant antibody techniques were used to construct a RM4/IFN-tau fusion protein containing the chimeric anti-tumor F(ab')2 (RM4) and the IFN-tau moiety. The recombinant cDNA of IFN-tau was linked to 3 prime end of the chimeric heavy-chain gene fragment (M4) containing the VH, the CH1 and the hinge region to form the fused heavy-chain gene fragment M4-IFN-tau. Transfection of the M4-IFN-tau gene fragment into a myeloma derived cell line VKCK which produced the chimeric light-chain of the same antibody, allowed the transfectant secreting the bifunctional fusion protein RM4/IFN-tau. The RM4/IFN-tau was purified by the affinity chromatography. Our data showed that the RM4/IFN-tau retained the TAG72 antigen-binding reactivity as well as the IFN-tau activity as measured in ELISA, FACS analysis of cell-surface TAG72 expression, immunohistochemical study, and up-regulation of cell-surface expression of CEA, HLA class I and class II antigens. Therefore, the bifunctional fusion protein RM4/IFN-tau may prove to be useful in targeting biological effects of the IFN-tau to tumor cells and in this way to stimulate the immune destruction of tumor cells.

Adenocarcinoma↗

PubMed2003

Discrete and complementary mechanisms of protection of beta-cells against cytokine-induced and oxidative damage achieved by bcl-2 overexpression and a cytokine selection strategy.

We have been investigating the potential utility of engineered cell lines as surrogates for primary islet cells in treatment of type 1 diabetes. To this end, two strategies that have emerged for procuring cell lines with resistance to immune-mediated damage are 1) selection of cytokine-resistant cell lines by growth of INS-1 insulinoma cells in iteratively increasing concentrations of interleukin (IL)-1beta + gamma-interferon (IFN-gamma), and 2) stable overexpression of the anti-apoptotic gene bcl-2 in INS-1 cells. Herein, we show that bcl-2-overexpressing cells are resistant to the cytotoxic effects of reactive oxygen and nitrogen species (ROS/RNS), but are only modestly protected against high concentrations of IL-1beta + INF-gamma, whereas the converse is true in cytokine selected cells. We also found that the combination of bcl-2 expression and cytokine selection confers a broader spectrum of resistance than either procedure alone, such that the resultant cells are highly resistant to cytokines and ROS/RNS, with no impairment in glucose-stimulated insulin secretion. INS-1-derived cells with combined bcl-2 expression and cytokine selection are also more resistant to damage induced by coculture with mitogen-activated peripheral blood mononuclear cells. Surprisingly, application of the cytokine selection procedure to bcl-2-overexpressing cells does not result in impairment of nuclear factor-kappaB translocation, iNOS expression, and NO production, as clearly occurs upon application of the selection procedure to cells without bcl-2 overexpression. Further investigation of the diverse pathways involved in the development of cytokine and ROS/RNS resistance may define simplified and specific strategies for preservation of beta-cell mass.

Animals↗

PubMed1993

Genetically engineered E. coli cells containing K. aerogenes gene, microencapsulated in artificial cells for urea and ammonia removal.

Microencapsulated genetically engineered E. coli cells can efficiently remove urea without any increase in the ammonia levels in the medium. A 100 mg. alginate encapsulated bacteria rapidly reduces urea in a 100 ml. solution. The original urea concentration 100.00 +/- 1.00 mg./dl. fell to 1.55 +/- 0.13 mg./dl. in 30 minutes. There was no increase in the ammonia in the reaction medium. Extrapolated results shows that urea depletion capacity of encapsulated bacteria is sufficient to remove urea during kidney failure. Using single pool model, 40 gm. of encapsulated genetically engineered E. coli can lower urea (100 mg./dl.) in 40 litres of the body water to 1.60 mg./dl. within 30 minutes. Also, 40.00 gm. bacteria can lower ammonia (758.00 microM/l), in 40 litres of body water, to 90.42 microM/l in 20 minutes. Further studies will be required for multi-compartmental models in the physiological conditions.

Ammonia↗

PubMed1992

Molecular neurosurgery for glial and neuronal disorders.

Molecular neurosurgery is the surgical use of molecules to destroy, sustain, or modulate or augment neurons, supporting cells or tumor cells within the nervous system. Molecules designed for surgical use may be engineered to be used in their molecular state or may be packaged as liposomes, viruses or genetically engineered cells. This article summarizes the current state of development of one aspect of molecular neurosurgery--the use of genetically engineered viruses to treat brain tumors. Both herpes virus and retrovirus strategies are discussed. Each virus has its own set of advantages and disadvantages that can be used in the design of an appropriate therapeutic strategy. Further application of this technology to intrinsic glial and neuronal disorders is discussed, and future perspectives are presented.

Brain Diseases↗

PubMed2006

The FunGenES consortium: functional genomics in engineered embryonic stem cells.

Although the complete sequence of a mammalian genome defines the information content of each cell, understanding the selective usage of this information during the development of specific cell types is limited. The fundamental questions that remain to be answered includes, which are the gene subsets that define the pluripotential self-renewing state of embryonic stem (ES) cells, partially and terminally differentiated developmental states, and how are transitions between these states regulated (lineage commitment)? The FunGenES consortium has been formed to address this challenge by mapping the gene subsets involved in pluripotent, lineage committed, and selected differentiated cell types using gene expression profiling and functional screens. They create an atlas of mammalian genome participating in early and late developmental processes. To fulfil the aim, mouse ES cells were used as an in vitro developmental model system that is very close to the human as they are pluripotent. They can be differentiated through the three major developmental pathways ecto-, meso-, and endoderm into many committed cell types and can be genetically engineered with relative ease. Knowledge of genetic pathways in mouse ES cell differentiation and development might be translated to human ES cells and the potential development of stem cell-based therapies.

Animals↗

PubMed1996

Immunologic and Ultrastructural Characterization of HIV Pseudovirions Containing Gag and Env Precursor Proteins Engineered in Insect Cells

Expression of human immunodeficiency virus (HIV) Gag precursor protein (Pr55) by recombinant baculoviruses in insect cells results in the assembly and budding of Pr55 as virus-like particles, or Gag pseudovirions. The ultrastructural morphology, size, and sucrose sedimentation rate of Gag pseudovirions are indistinguishable from immature lentivirus particles produced by HIV-infected human cells. Recombinant baculoviruses were engineered to express individually Pr55 and HIV Env glycoprotein precursor (gp160). These recombinant baculoviruses were used to co-infect insect cells to produce chimeric HIV Gag pseudovirions containing gp160 in experiments to develop methodologies for producing complex noninfectious particulate vaccines for HIV. Coexpression of HIV Pr55 and gp160 resulted in the apparent incorporation of gp160 into Gag pseudovirions as determined by immunoblotting with envelope-specific monoclonal antibodies. Furthermore, results from indirect immunogold electron microscopy using monoclonal antibodies to HIV gp120, a component of the Env glycoprotein precursor, suggested that HIV gp160 was specifically incorporated during the budding process into the outer surface of chimeric Gag pseudovirions. Parallel labeling experiments to localize gp120 and Pr55 epitopes on HIV-infected H9 lymphocytes provided results similar to those obtained with chimeric Gag pseudovirions producing recombinant baculovirus-infected insect cells. Parameters influencing immunoelectron microscopy results in cell-surface and postembedding labeling experiments are discussed.

Journal Article↗