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Results for “reverse engineering”

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At least 487 records · Page 27Linked to original sources

PubMed2006

Regulation of SR-BI-mediated selective lipid uptake in Chinese hamster ovary-derived cells by protein kinase signaling pathways.

Scavenger receptor, class B, type I (SR-BI) mediates binding and internalization of a variety of lipoprotein and nonlipoprotein ligands, including HDL. Studies in genetically engineered mice revealed that SR-BI plays an important role in HDL reverse cholesterol transport and protection against atherosclerosis. Understanding how SR-BI's function is regulated may reveal new approaches to therapeutic intervention in atherosclerosis and heart disease. We utilized a model cell system to explore pathways involved in SR-BI-mediated lipid uptake from and signaling in response to distinct lipoprotein ligands: the physiological ligand, HDL, and a model ligand, acetyl LDL (AcLDL). In Chinese hamster ovary-derived cells, murine SR-BI (mSR-BI) mediates lipid uptake via distinct pathways that are dependent on the lipoprotein ligand. Furthermore, HDL and AcLDL activate distinct signaling pathways. Finally, mSR-BI-mediated selective lipid uptake versus endocytic uptake are differentially regulated by protein kinase signaling pathways. The protein kinase C (PKC) activator PMA and the phosphatidyl inositol 3-kinase inhibitor wortmannin increase the degree of mSR-BI-mediated selective lipid uptake, whereas a PKC inhibitor has the opposite effect. These data demonstrate that SR-BI's selective lipid uptake activity can be acutely regulated by intracellular signaling cascades, some of which can originate from HDL binding to murine SR-BI itself.

Androstadienes↗

PubMed2002

Cutting edge: selection of B lymphocyte subsets is regulated by natural IgM.

Natural IgM has a wide range of actions in the immune system. Here we demonstrate that mice lacking serum IgM have an expansion in splenic marginal zone B cells with a proportionately smaller reduction in follicular B cells. The increase in the marginal zone-follicular B cell ratio (and an expansion in peritoneal B1a cells) is fully reversed by administration of polyclonal IgM, but not by two IgM monoclonals. Mice engineered to have a secreted oligoclonal IgM repertoire with an endogenous membrane IgM also exhibited a similar expansion of marginal zone B cells. We propose that natural IgM, by virtue of its polyreactivity, enhances Ag-driven signaling through the B cell receptor and promotes the formation of follicular B cells. These results demonstrate that natural IgM regulates the selection of B lymphocyte subsets.

Animals↗

PubMed2000

Properties and Synthetic Applications of Enzymes in Organic Solvents.

Biotransformations already represent an effective and sometimes preferable alternative to chemical synthesis for the production of fine chemicals and optically active compounds. To further widen the versatility of the biological approach, the so-called "nonaqueous enzymology", which now represents an important area of research and biotechnological development, has emerged in the last ten years or so. This new methodology is especially suitable for the modification of precursors of pharmaceutical compounds and fine chemicals, which, in most cases, are insoluble or poorly soluble in water. Even though the idea of carrying out an enzymatic process in organic solvent was initially considered with scepticism, biocatalysis in such media is now investigated and exploited in numerous academic and industrial laboratories. One of the reasons that makes enzymatic catalysis in nonaqueous media so appealing, is the important new properties that enzymes exhibit in organic solvents. For example, they are often more stable and can catalyze reactions that are impossible or difficult in water. Furthermore, enzyme selectivity can also differ from that in water and can change, or even reverse, from one solvent to another. This phenomenon, which can be called "medium engineering", can be exploited as a valid alternative to protein engineering. The first part of this review examines the thermodynamic, kinetic, spectroscopic, and physical approaches that have been adopted to investigate the factors that affect activity, stability, structure, and selectivity of enzymes in organic solvents. These combined studies have brought the understanding of enzyme catalysis in organic solvents to a level almost comparable to that reached for biocatalysis in aqueous media. The second part surveys a number of the synthetic applications of enzymes in organic media, which span from the preparation of milligrams of specifically labeled compounds to the modification of fats on multiton scale and from the preparation of complex key intermediates for the pharmaceutical industry to the synthesis of polymers.

Journal Article↗

PubMed2000

[Development of influenza vaccines against pandemics].

Influenza vaccine plays a crucial role as an effective measure for control of worldwide influenza pandemics. Currently licensed inactivated influenza vaccines can be used for the pandemic situation. Surveillance to isolate animal influenza viruses as vaccine candidate strains greatly contributes to the rapid production of vaccine. The reverse genetics approach can afford to attenuate a highly virulent virus by genetic engineering of any viral genes for the use of vaccine strain. The other approach to develop influenza vaccine should be encouraged to overcome the disadvantage of currently used inactivated vaccine.

Animals↗

PubMed2002

Cucumber: a model angiosperm for mitochondrial transformation?

Plants possess three major genomes, carried in the chloroplast, mitochondrion, and nucleus. The chloroplast genomes of higher plants tend to be of similar sizes and structure. In contrast both the nuclear and mitochondrial genomes show great size differences, even among closely related species. The largest plant mitochondrial genomes exist in the genus Cucumis at 1500 to 2300 kilobases, over 100 times the sizes of the yeast or human mitochondrial genomes. Biochemical and molecular analyses have established that the huge Cucumis mitochondrial genomes are due to extensive duplication of short repetitive DNA motifs. The organellar genomes of almost all organisms are maternally transmitted and few methods exist to manipulate these important genomes. Although chloroplast transformation has been achieved, no routine method exists to transform the mitochondrial genome of higher plants. A mitochondrial-transformation system for a higher plant would allow geneticists to use reverse genetics to study mitochondrial gene expression and to establish the efficacy of engineered mitochondrial genes for the genetic improvement of the mitochondrial genome. Cucumber possesses three unique attributes that make it a potential model system for mitochondrial transformation of a higher plant. Firstly, its mitochondria show paternal transmission. Secondly, microspores possess relatively few, huge mitochondria. Finally, there exists in cucumber unique mitochondrial mutations conditioning strongly mosaic (msc) phenotypes. The msc phenotypes appear after regeneration of plants from cell culture and sort with specific rearranged and deleted regions in the mitochondrial genome. These mitochondrial deletions may be a useful genetic tool to develop selectable markers for mitochondrial transformation of higher plants.

Cucumis sativus↗

PubMed2003

Gene expression profile of tissue engineered skin subjected to acute barrier disruption.

The main function of the skin is to protect the body from infection, dehydration, and other environmental insults by creating an impermeable barrier of cornified cell layers, the stratum corneum. In contrast to cells in culture, tissue-engineered skin equivalents contain well-developed basal, spinous, granular, and cornified cell layers providing an excellent model to study the tissue response to barrier disruption. After 7 d of culture at the air-liquid interface the barrier of the tissues was disrupted by short exposure to acetone and the global gene expression profile of the tissues was evaluated using DNA microarrays. We found that tissue-engineered skin responds to barrier disruption by a two-wave dynamic response. Early on, the cells upregulate signal transducing, stress, proliferation, and inflammation genes to protect the tissue and possibly to communicate the damage to the immune system and neighboring tissues. At later times, pro-inflammatory cytokines and some growth-related genes are significantly reduced but enzymes that participate in lipid synthesis increase, suggesting that the epidermal cells attempt to restore the lost barrier. Quantitative immunostaining for the proliferation antigen Ki67 revealed that barrier disruption by acetone increased proliferation by 4-fold in agreement with the microarray data and previous in vivo studies. Our work suggests that functional genomics may be used in tissue engineering to understand tissue development, wound regeneration, and response to environmental stimuli. A better understanding of engineered tissues at the molecular level may facilitate their application in the clinic and as biosensors for toxicologic testing.

Acetone↗

PubMed2006

Development of quantitative PCR methods to analyse neural progenitor cell culture state.

Stem cells have significant potential for tissue engineering and regeneration, and neural stem and progenitor cells have proven promising for neuroregeneration in numerous animal disease and injury models. However, improved approaches must be developed to culture, expand and control the cells. Therefore the development of enhanced methods to quantify cell differentiation would significantly aid both in the basic investigation of cell-fate control mechanisms and in the optimization and validation of cell culture and expansion conditions. Quantitative reverse transcription-PCR methods were developed to quantify cell differentiation state by monitoring the expression of several cell-lineage-specific markers. These methods provide more rapid and readily quantitative results when compared with immunostaining. These methods were also applied in a preliminary investigation of cell-culture conditions, and it was found that regular feeding of cells with fresh medium is necessary to maintain them in an undifferentiated and highly proliferative state. The present study may aid both basic efforts to study the control of neural stem and progenitor differentiation as well as endeavours to optimize cell culture and expansion conditions for biomedical applications.

Base Sequence↗

PubMed2005

Expressing engineered thymidylate kinase variants in human cells to improve AZT phosphorylation and human immunodeficiency virus inhibition.

The triphosphorylated form of the nucleoside analogue AZT (AZTTP) acts as a chain terminator during reverse transcription of the human immunodeficiency virus (HIV) genome. The bottleneck in the conversion of AZT to AZTTP is the phosphorylation of AZT monophosphate (AZTMP) by cellular thymidylate kinase. Human thymidylate kinase was engineered to exhibit highly improved activity for AZTMP to AZTDP conversion. It was demonstrated here that genetically modified human cells transiently expressing these enzyme variants show more than 10-fold higher intracellular concentrations of AZTDP and AZTTP. Stable clones expressing these enzymes appear to phosphorylate AZTMP less efficiently, but first experiments indicate they are still more potent in HIV inhibition than the parental cells. It was proposed that the concept of introducing into human cells a catalytically improved human enzyme, rather than an enzyme of viral, bacterial or yeast origin, may serve as a paradigm for ameliorating the metabolic activation of an established drug.

Genetic Engineering↗

PubMed1999

Quantitative RT-PCR to evaluate in vivo expression of multiple transgenes using a common intron.

An assay measuring RNA expression levels of a gene-encoded therapeutic must distinguish between endogenous mRNA and mRNA transcribed from the transgene. Specificity for the delivered transgene is especially critical when the treatment involves genes that are expressed in the target tissue. To facilitate uniform detection of transgene RNA without interference from endogenous mRNA, we have engineered expression vectors that include a 5' untranslated region (5' UTR) containing a synthetic intron (PGL3). The synthetic intron splice junction was the target sequence for a quantitative reverse transcription (RT)-PCR assay utilizing Taq-Man technology. In this study, we demonstrate that a quantitative RT-PCR assay designed to recognize an engineered intron splice site in the 5'UTR of expression constructs effectively measures the expression level of in vivo-delivered gene therapeutics.

5' Untranslated Regions↗

PubMed2006

Runx2 overexpression enhances osteoblastic differentiation and mineralization in adipose--derived stem cells in vitro and in vivo.

Like bone marrow stromal cells, adipose tissue-derived stem cells (ADSCs) possess multilineage potential, a capacity for self-renewal and long-term viability. To confirm whether ADSCs represent a promising source of cells for gene-enhanced bone tissue-engineering, the osteogenic potential of ADSCs under the control of certain osteoinductive genes has been evaluated. Runx2, a transcription factor at the downstream end of bone morphogenetic protein (BMP) signaling pathways, is essential for osteoblast differentiation and bone formation. In this study we used adenovirus vector to deliver Runx2 to ADSCs and then examined the enhancement of osteogenic activity. Overexpression of Runx2 inhibited adipogenesis, as demonstrated by suppression of LPL and PPARgamma expression at the mRNA level and reduced lipid droplet formation. Moreover, ADSCs transduced with Ad-Runx2 underwent rapid and marked osteoblast differentiation as determined by osteoblastic gene expression, alkaline phosphatase activity and mineral deposition. Additionally, histological examination revealed that implantation of Runx2 modified ADSCs could induce mineral deposition and bone-like tissue formation in vivo. These results confirmed, firstly, the ability of Runx2 to promote osteogenesis and cell differentiation and, secondly, the competence of ADSCs as target cells for bone tissue engineering. Our work demonstrates a potential new approach for bone repair using Runx2-modified ADSCs for bone tissue engineering.

Adenoviridae↗

PubMed2005

Mesenchymal progenitor cells derived from chorionic villi of human placenta for cartilage tissue engineering.

Human mesenchymal stem cells are currently being studied extensively because of their capability for self-renewal and differentiation to various connective tissues, which makes them attractive as cell sources for regenerative medicine. Herein we report the isolation of human placenta-derived mesenchymal cells (hPDMCs) that have the potential to differentiate into various lineages to explore the possibility of using these cells for regeneration of cartilage. We first evaluated the chondrogenesis of hPDMCs in vitro and then embedded the hPDMCs into an atelocollagen gel to make a cartilage-like tissue with chondrogenic induction media. For in vivo assay, preinduced hPDMCs embedded in collagen sponges were subcutaneously implanted into nude mice and also into nude rats with osteochondral defect. The results of these in vivo and in vitro studies suggested that hPDMCs can be one of the possible allogeneic cell sources for tissue engineering of cartilage.

Animals↗

PubMedDate not supplied

Fibrin-polyurethane composites for articular cartilage tissue engineering: a preliminary analysis.

In this study we investigated the use of a fibrin hydrogel to improve the potential of a polyurethane (PU) scaffold-based system for articular cartilage tissue engineering. PU-only ("no-fibrin") and PU-fibrin ("fibrin") composites were cultured for up to 28 days and analyzed for DNA content, glycosaminoglycan (GAG) content, type II collagen content, GAG release, and gene expression of aggrecan, collagen I, and collagen II. The use of fibrin allowed for higher viable cell-seeding efficiency (10% higher DNA content on day 2 in fibrin versus no-fibrin composites) and more even cell distribution on seeding, a more than 3-fold increase in the percentage of newly synthesized GAG retained in the constructs, and 2- to 6-fold higher levels of type II collagen and aggrecan gene expression through day 14. Addition of aprotinin to the medium inhibited fibrin degradation, most noticeably in the center of the constructs, but had little effect on biochemical composition or gene expression. Short-term mechanical compression (0-10% sinusoidal strain at 0.1 Hz for 1 h, applied twice daily for 3 days) doubled the rate of GAG release from the constructs, but had little effect on gene expression, regardless of the presence of fibrin. Although further work is needed to optimize this system, the addition of fibrin hydrogel to encapsulate cells in the stiff, macroporous PU scaffold is a step forward in our approach to articular cartilage tissue engineering.

Aggrecans↗

PubMed2026

Reversing-or Rewiring-Lineage Plasticity? Lessons from EZH2 Loss in Prostate Cancer.

Enhancer of zeste homolog 2 (EZH2) inhibitors have been proposed to counteract lineage plasticity (LP) in prostate cancer and thereby resensitize tumors to androgen receptor (AR) inhibition. In this issue of Cancer Research, Jacobi and colleagues provide new mechanistic insights into EZH2 biology across prostate cancer progression using a genetically engineered mouse model that recapitulates the transition toward a neuroendocrine (NE) phenotype. Unexpectedly, genetic deletion of Ezh2 did not reverse LP but instead promoted the diversification of transcription factor (TF) programs driving NE differentiation. In particular, the loss of EZH2 activated members of the KLF TF family, which contributed to this transcriptional diversification. Moreover, EZH2 deletion altered the chromatin-binding landscape of AR, redirecting it toward KLF-associated genomic sites. Collectively, these results refine our understanding of EZH2 function in prostate cancer: Rather than simply reversing LP, EZH2 loss rewires transcriptional networks and reshapes the AR cistrome. These findings are timely given the growing number of clinical trials testing EZH2 inhibitors in metastatic prostate cancer and highlight the need to define when and how to deploy EZH2 inhibition to exploit its effects on tumor lineage dynamics. See related article by Jacobi et al., p. 889.

Male↗

PubMedDate not supplied

Impact of electrical stimulation on three-dimensional myoblast cultures - a real-time RT-PCR study.

Several focal skeletal muscle diseases, including tumours and trauma lead to a limited loss of functional muscle tissue. There is still no suitable clinical approach for treating such defects. A promising approach could be the tissue engineering of skeletal muscle. However, a clinically reliable differentiation stimulus for three-dimensional (3-D) cultures is necessary for this process, and this condition has not yet been established. In order to quantify and analyze the differentiation potential of electrical cell stimulation, primary myoblasts were stimulated within a 3-D fibrin- matrix. Gene expression of MyoD, myogenin and AChR-epsilon were measured by real-time RT-PCR over a time period of eight days, showing immediate down-regulation of all marker genes. For tissue engineering approaches, cell multiplication is crucial for acquisition of sufficient tissue volumes for reconstruction. Therefore, all experiments were performed with high and low passaged myoblasts, demonstrating higher transcript rates of marker genes in lowpassage cells. Our findings strongly suggest a reconsideration of electrical stimulation in muscle tissue engineering.

Animals↗

PubMed2006

Increased safety with preserved antitumoral efficacy on hepatocellular carcinoma with dual-regulated oncolytic adenovirus.

PURPOSE: A dual-regulated adenovirus variant CNHK500, in which human telomerase reverse transcriptase promoter drove the adenovirus 5 (Ad5) E1a gene and hypoxia-response promoter controlled the E1b gene, was engineered. This virus has broad anticancer spectrum and higher specificity compared with mono-regulated adenovirus CNHK300. The objective of the current study is to show its antitumor selectivity and therapeutic potential. EXPERIMENTAL DESIGN: The antitumor specificity of human telomerase reverse transcriptase and hypoxia response promoters was evaluated in a panel of tumor and normal cells. Under the control of these promoters, the tumor-selective expression of E1a and E1b genes was evaluated. Further in vitro antitumor specificity and potency of this virus were characterized by viral replication and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Subsequently, hepatocellular carcinoma xenografts were established to evaluate CNHK500 antitumor efficacy in vivo by different routes of virus administration and different dosages. RESULTS: Human telomerase reverse transcriptase and hypoxia response promoters were activated in a tumor-selective manner or under hypoxia treatment in a broad panel of cells. Selective adenoviral early gene expression, efficient viral replication, and oncolysis were observed in all tested cancer cells with more attenuated replication capacity in normal cells. Significant regression of hepatocellular carcinoma xenografts and prolonged survival were observed by either i.t. or i.v. administration. CONCLUSIONS: CNHK500 greatly reduced side effects in normal cells via dual control of adenoviral essential genes while still preserving potent antitumor efficacy on broad-spectrum cancer cells in vitro and in vivo. It can be used as a powerful therapeutic agent not only for liver cancers but also for other solid tumors.

Adenoviridae↗

PubMed1998

Engineered erythrocytes: influence of P50 rightward shift and oxemia on oxygen transport to tissues.

The red blood cell (RBC) membrane may be reversibly opened using a lysis-resealing continuous flow method. The technology was adapted to the internalisation of an allosteric effector of haemoglobin, Inositol-Hexaphosphate (IHP). This molecule, occupying the allosteric site of 2,3 Bis-Phosphoglycerate with a very large affinity, induces a rightward shift of the oxyhaemoglobin dissociation curve (ODC). From ODC parameters in human volunteers, the potential effect of P50 (oxygen pressure at 50% haemoglobin saturation) on oxygen exchangeable fraction (OEF%), for various oxygen partial pressures (oxemia) was evaluated. For hyperoxic or normoxic arterial oxygen pressure (paO2), rightward shift greatly improved OEF%. In optimised conditions, engineered erythrocytes were potentially able to deliver two to three times more oxygen than normal cells. For patients with decreased paO2, as observed in chronic obstructive pulmonary deficiency (COPD), the reduction in arterial oxygen saturation (saO2%) reduces the benefit of the treatment for paO2 values between 60 and 80 mmHg. Below 60 mmHg, the saO2% reduction cannot be compensated by a corresponding reduction in svO2%, particularly for organs with physiologically low svO2%. In these organs, deleterious effects could be observed for a very large rightward shift of the ODC. Such engineered cells have unique properties for oxygen transport improvement and may be used for the treatment of patients suffering from diseases associated with hypoxia and ischemia.

Erythrocytes↗

PubMed2007

Human mesenchymal progenitor cell responses to a novel textured poly(L-lactide) scaffold for ligament tissue engineering.

Biocompatibility and cell seeding capability of a new cell scaffold made of textured polylactic acid (PLA) fibers was investigated as a new material for tissue engineering of anterior cruciate ligaments (ACL). Adhesion and proliferation of human mesenchymal progenitor cells (MPC) was investigated after 15 days by scanning electron microscopy and standard histology. Expression of collagen type I and III, fibronectin, tenascin C, decorin, smooth muscle actin, and the matrix metalloproteinases MMP-1 and MMP-2, as well as their tissue inhibitors TIMP-1 and TIMP-2 was analyzed using real-time PCR. Protein expression of collagen I and III, tenascin C, and proliferating nuclear antigen (PCNA) was determined by immunohistology. Apoptosis was analyzed by detection of p53 expression and TUNEL staining. MPC seeded the scaffold homogeneously and showed good cell growth and no increased rate of apoptosis. After 15 days, the matrix forming genes collagen type I, tenascin C, and decorin were upregulated, indicating the formation of a ligament-like matrix. MMP-1 and TIMP-1 were also significantly increased, suggesting initial matrix remodeling. It was concluded that the new porous PLA scaffold allowed homogeneous cell seeding, a fibroblastic phenotype and the production of a ligament-like matrix and, therefore, might be a suitable cell carrier for ACL tissue engineering.

Apoptosis↗

PubMed2006

Polyamines modulate nitric oxide production and COX-2 gene expression in response to mechanical loading in human adipose tissue-derived mesenchymal stem cells.

For bone tissue engineering, it is important that mesenchymal stem cells (MSCs) display a bone cell-like response to mechanical loading. We have shown earlier that this response includes increased nitric oxide (NO) production and cyclooxygenase-2 (COX-2) gene expression, both of which are intimately involved in mechanical adaptation of bone. COX-2 gene expression is likely regulated by polyamines, which are organic cations implicated in cell proliferation and differentiation. This has led to the hypothesis that polyamines may play a role in the response of adipose tissue-derived MSCs (AT-MSCs) to mechanical loading. The aim of this study was to investigate whether genes involved in polyamine metabolism are regulated by mechanical loading and to study whether polyamines modulate mechanical loading-induced NO production and COX-2 gene expression in human AT-MSCs. Human AT-MSCs displayed a bone cell-like response to mechanical loading applied by pulsating fluid flow (PFF), as demonstrated by increased NO production and increased gene expression of COX-2. Furthermore, PFF increased gene expression of spermidine/spermine N (1)-acetyltransferase, which is involved in polyamine catabolism, suggesting that mechanical loading modulates polyamine levels. Finally, the polyamine spermine was shown to inhibit both PFF-induced NO production and COX-2 gene expression, suggesting that polyamines modulate the response of human AT-MSCs to mechanical loading. In conclusion, this is the first study implicating polyamines in the response of human AT-MSCs to mechanical loading, creating opportunities for the use of polyamines in tissue engineering approaches targeting skeletal defects.

Acetyltransferases↗