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PubMed1999

Frontiers in transplantation of insulin-secreting tissue for diabetes mellitus.

Transplantation of insulin-secreting tissue represents a physiologic approach to reverse diabetes mellitus. Pancreas transplants yield a remarkable enhancement in quality of life and appear to modify the devastating neurovascular complications of diabetes. A more attractive approach is transplantation of insulin-secreting cells, a procedure of low invasiveness with the exciting prospect of modulating graft immunogenicity before transplantation, so as to minimize requirements for toxic immunosuppressive drugs. The Surgical-Medical Research Institute at the University of Alberta in Edmonton, and several others centres throughout the world, has demonstrated that islet cell transplants can reverse insulin dependence and induce remarkable glycemic stability for several years. However, widespread success has been denied because of insufficient donor tissue, early failures to reverse insulin dependence and the loss of graft function with time. Promising new research approaches to these problems are reviewed, including xenogeneic sources of cells, engineering islet cells with genes that induce expression of immunoprotective molecules, and neogenesis factors that may sustain populations of transplanted beta cells.

Alberta↗
PubMed2006

Techniques for biological characterization of tissue-engineered tendon and ligament.

Injuries to tendons and ligaments are prevalent and result in a significant decrease in quality of patient life. Tissue-engineering strategies hold promise as alternatives to current treatments for these injuries, which often fail to fully restore proper joint biomechanics and produce significant donor site morbidity. Commonly, tissue engineering involves the use of a three-dimensional scaffold seeded with cells that can be directed to form tendon/ligament tissue. When determining the success of such approaches, the viability and proliferation of the cells in the construct, as well as extracellular matrix production and structure should be taken into account. Histology and histochemistry, microscopy, colorimetric assays, and real-time reverse transcriptase-polymerase chain reaction (RT-PCR) are techniques that are employed to assess these biological characteristics. This review provides an overview of each of these methods, including specific examples of how they have been used in evaluation of tissue-engineered tendon and ligament tissue. Basic physical principles underlying each method and advantages and disadvantages of the various techniques are summarized.

Animals↗
PubMed2003

Recombinant Newcastle disease virus as a vaccine vector.

Veterinary vaccines remained conventional for more than fifty years. Recent advances in the recombinant genetic engineering techniques brought forward a leap in designing vaccines for veterinary use. A novel approach of delivering protective immunogens of many different pathogens in a single virus vector was made possible with the introduction of a "reverse genetics" system for nonsegmented negative-sense RNA viruses. Newcastle disease virus (NDV), a nonsegmented negative-sense virus, is one of the major viruses of economic importance in the poultry industry throughout the world. Despite the availability of live virus vaccines of good potency, the intrinsic ability of attenuated strains to revert in virulence makes control of this disease by vaccination difficult. Armed with the knowledge of virulence factors of this virus, it is now possible to produce genetically stable vaccines and to engineer mutations that enhance immunogenicity. The modular nature of the genome of this virus facilitates engineering additional genes from several different pathogens or tumor-specific antigens to design contemporary vaccines for animals and humans. This review will summarize the developments in using NDV as a vaccine vector and the potential of this approach in designing next generation vaccines for veterinary use.

Animals↗
PubMedDate not supplied

Molecular and cellular characterization during chondrogenic differentiation of adipose tissue-derived stromal cells in vitro and cartilage formation in vivo.

Human adipose tissue is a viable source of mesenchymal stem cells (MSCs) with wide differentiation potential for musculoskeletal tissue engineering research. The stem cell population, termed processed lipoaspirate (PLA) cells, can be isolated from human lipoaspirates and expanded in vitro easily. This study was to determine molecular and cellular characterization of PLA cells during chondrogenic differentiation in vitro and cartilage formation in vivo. When cultured in vitro with chondrogenic medium as monolayers in high density, they could be induced toward the chondrogenic lineages. To determine their ability of cartilage formation in vivo, the induced cells in alginate gel were implanted in nude mice subcutaneously for up to 20 weeks. Histological and immunohistochemical analysis of the induced cells and retrieved specimens from nude mice at various intervals showed obviously cartilaginous phenotype with positive staining of specific extracellular matrix (ECM). Correlatively, results of RT-PCR and Western Blot confirmed the expression of characteristic molecules during chondrogenic differentiation namely collagen type II, SOX9, cartilage oligomeric protein (COMP) and the cartilage-specific proteoglycan aggrecan. Meanwhile, there was low level synthesis of collagen type X and decreasing production of collagen type I during induction in vitro and formation of cartilaginous tissue in vivo. These cells induced to form engineered cartilage can maintain the stable phenotype and indicate no sign of hypertrophy in 20 weeks in vivo, however, when they cultured as monolayers, they showed prehypertrophic alteration in late stage about 10 weeks after induction. Therefore, it is suggested that human adipose tissue may represent a novel plentiful source of multipotential stem cells capable of undergoing chondrogenesis and forming engineered cartilage.

Adipose Tissue↗
PubMed2007

Evaluation of the biological responses of osteoblast-like UMR-106 cells to the engineered porous PHBV matrix.

Poly (3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) has been investigated for biomedical applications due to its many biologically favorable properties. However, to explore its application in bone tissue engineering, the poorly bioactive surface property of PHBV must be improved. To engineer PHBV to achieve a biologically active surface, in this study each porous PHBV matrix was prepared by solute leaching of salt/PHBV cast film and was treated with ozone followed by dip coating with type I collagen. The biological responses of osteoblast-like UMR-106 cells after being grown on the engineered PHBV matrix were evaluated. Confocal microscopy and the MTT assay were used to map and quantify the viable cell proliferation on the PHBV matrix, respectively. The cells were cultivated in osteogenic media containing beta-glycerophosphate and later stained with alizarin red to visualize mineralization of the matrix. RNA was extracted from the UMR-106 cells, and reverse transcriptase-polymerase chain reaction (RT-PCR) was applied to detect expression of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (a house keeping gene) and bone sialoprotein (BSP) (marker of the osteoblastic phenotype). The results showed that the UMR-106 cells after cultivation on the engineered PHBV matrix retained the osteoblastic phenotype characteristics, indicating that the porous PHBV matrix after ozone treatment and collagen dip coatings are a promising scaffold for bone tissue engineering applications.

Animals↗
PubMed2006

RRM1 modulated in vitro and in vivo efficacy of gemcitabine and platinum in non-small-cell lung cancer.

PURPOSE: RRM1 encodes the regulatory subunit of ribonucleotide reductase and is a molecular target of gemcitabine. Previous studies showed increased RRM1 expression on continuous exposure of cell lines to gemcitabine and suggested improved survival for patients with low as opposed to high tumoral RRM1 expression when treated with gemcitabine-containing chemotherapy. However, the principal hypothesis that intratumoral levels of gene expression are associated with disease response has not been addressed. PATIENTS AND METHODS: We constructed genetically modified lung cancer cell lines with increased and decreased RRM1 expression to investigate the in vitro 50% inhibitory concentration (IC50) for gemcitabine, cisplatin, and carboplatin. A prospective phase II clinical trial in patients with locally advanced non-small-cell lung cancer was conducted with pretreatment tumor collection for determination of RRM1 and ERCC1 expression by real-time reverse transcriptase polymerase chain reaction. The levels of gene expression were correlated with tumor response after two cycles of gemcitabine and carboplatin. RESULTS: In cell lines with a genetically engineered 15-fold RRM1 expression range, the gemcitabine IC50 had a 100-fold range, and the cisplatin and carboplatin IC50 had a two-fold range. They were highest in constructs with high RRM1 expression. In the prospective clinical trial, RRM1 expression was significantly (P = .002) and inversely correlated (r = -0.498) with disease response. ERCC1 expression showed a similar trend (P = .099). CONCLUSION: The results strongly suggest that tumoral RRM1 expression is a major predictor of disease response to gemcitabine/platinum chemotherapy. ERCC1 expression is predictive of response albeit to a lesser degree.

Antineoplastic Combined Chemotherapy Protocols↗
PubMed2001

The RNA/protein symmetry hypothesis: experimental support for reverse translation of primitive proteins.

Although the "RNA-world" theory, or the RNA-first theory is renowned for a promising theory of biogenesis, it is also possible that both RNAs and proteins have coevolved forming a stable metabolic complex from the very beginning. I investigated this possibility assuming that the genetic information flowed symmetrically in the era of the origin of life, i.e. the primitive translation machinery worked in both directions (from RNA to protein and from protein to RNA). According to this RNA/protein symmetry theory, the genetic information would have come from existing cellular proteins via reverse translation. This process would have been completed in a short period of time without searching an enormous RNA sequence space. Furthermore, reverse translation would have ensured biological continuity; proteins that were essential for cellular metabolism would have been utilized in the same way as before the protein sequence information would have been transferred into the RNA sequences. I also propose a possible mechanism for the process of reverse translation. The reverse translation would proceed in the 3' to 5' direction using a set of at least 20 reverse transfer RNAs (rtRNAs) that can recognize their specific amino acid residue and carry their corresponding codon. A source of genetic information would be a primary sequence of a protein molecule. Several basic steps of reverse translation were demonstrated using rtRNA(Arg).

3' Untranslated Regions↗
PubMed2006

Reference models for mitral valve tissue engineering based on valve cell phenotype and extracellular matrix analysis.

The advance of mitral valve repair techniques through tissue engineering is impeded by the lack of information regarding the cellular and extracellular components of the mitral valve. The present study aims to expand our understanding of the mitral valve structure by analysing the synthesis of extracellular matrix (ECM) proteins and the expression of nitric oxide synthase (NOS). Valvular endothelial cells (VECs) and valvular interstitial cells (VICs) were isolated from porcine mitral valves. Immunochemical staining of ECM components, including type I, II, III, IV and V collagen, laminin, fibronectin, elastin and chondroitin sulphate (CS), was performed on both mitral valve tissue and cell cultures. Reverse transcription polymerase chain reaction and immunochemistry were used to analyse NOS expression in native valve and in culture. Both VECs and VICs synthesised the basement membrane components, laminin and type IV collagen both in vivo and in vitro, amongst other fibrous ECM proteins. Synthesis of type I collagen and CS was absent in VEC cultures. Each cell type had a characteristic profile of NOS expression. VECs synthesised endothelial NOS both in vivo and in vitro, with a minority of VICs expressing neuronal NOS in vitro. The present study reports newly recognised aspects of the mitral valve structure and the in vitro behaviour of mitral valve cell populations based on ECM synthesis and NOS expression. The presented profiles can be used as base tools for the generation of data necessary for the selection of ideal cell sources and for the design of appropriate scaffolds for the development of effective tissue-engineered mitral valves.

Actins↗
PubMed2005

Immunophenotypic analysis of human articular chondrocytes: changes in surface markers associated with cell expansion in monolayer culture.

Cartilage tissue engineering relies on in vitro expansion of primary chondrocytes. Monolayer is the chosen culture model for chondrocyte expansion because in this system the proliferative capacity of chondrocytes is substantially higher compared to non-adherent systems. However, human articular chondrocytes (HACs) cultured as monolayers undergo changes in phenotype and gene expression known as "dedifferentiation." To gain a better understanding of the cellular mechanisms involved in the dedifferentiation process, our research focused on the characterization of the surface molecule phenotype of HACs in monolayer culture. Adult HACs were isolated by enzymatic digestion of cartilage samples obtained post-mortem. HACs cultured in monolayer for different time periods were analyzed by flow cytometry for the expression of cell surface markers with a panel of 52 antibodies. Our results show that HACs express surface molecules belonging to different categories: integrins and other adhesion molecules (CD49a, CD49b, CD49c, CD49e, CD49f, CD51/61, CD54, CD106, CD166, CD58, CD44), tetraspanins (CD9, CD63, CD81, CD82, CD151), receptors (CD105, CD119, CD130, CD140a, CD221, CD95, CD120a, CD71, CD14), ectoenzymes (CD10, CD26), and other surface molecules (CD90, CD99). Moreover, differential expression of certain markers in monolayer culture was identified. Up-regulation of markers on HACs regarded as distinctive for mesenchymal stem cells (CD10, CD90, CD105, CD166) during monolayer culture suggested that dedifferentiation leads to reversion to a primitive phenotype. This study contributes to the definition of HAC phenotype, and provides new potential markers to characterize chondrocyte differentiation stage in the context of tissue engineering applications.

Adult↗
PubMed1997

A yeast genetic system for selecting small molecule inhibitors of protein-protein interactions in nanodroplets.

Cellular processes are mediated by complex networks of molecular interactions. Dissection of their role most commonly is achieved by using genetic mutations that alter, for example, protein-protein interactions. Small molecules that accomplish the same result would provide a powerful complement to the genetic approach, but it generally is believed that such molecules are rare. There are several natural products, however, that illustrate the feasibility of this approach. Split-pool synthesis now provides a simple mechanical means to prepare vast numbers of complex, even natural product-like, molecules individually attached to cell-sized polymer beads. Here, we describe a genetic system compatible with split-pool synthesis that allows the detection of cell-permeable, small molecule inhibitors of protein-protein interactions in 100- to 200-nl cell culture droplets, prepared by a recently described technique that arrays large numbers of such droplets. These "nanodroplets" contain defined media, cells, and one or more beads containing approximately 100 pmol of a photoreleasable small molecule and a controlled number of cells. The engineered Saccharomyces cerevisiae cells used in this study express two interacting proteins after induction with galactose whose interaction results in cell death in the presence of 5-fluoroorotic acid (inducible reverse two-hybrid assay). Disruption of the interaction by a small molecule allows growth, and the small molecule can be introduced into the system hours before induction of the toxic interaction. We demonstrate that the interaction between the activin receptor R1 and the immunophilin protein FKBP12 can be disrupted by the small molecule FK506 at nanomolar concentrations in nanodroplets. This system should provide a general method for selecting cell-permeable ligands that can be used to study the relevance of protein-protein interactions in living cells or organisms.

Activin Receptors, Type I↗
PubMed2004

Creation and discovery of ligand-receptor pairs for transcriptional control with small molecules.

The nuclear receptor retinoid X receptor (RXR) is a ligand-activated transcription factor. To create receptors for a new ligand, a structure-based approach was used to generate a library of approximately 380,000 mutant RXR genes. To discover functional variants within the library, we used chemical complementation, a method of protein engineering that uses the power of genetic selection. Wild-type RXR has an EC50 of 500 nM for 9-cis retinoic acid (9cRA) and an EC50 of >10 microM for the synthetic retinoid-like compound LG335 in yeast. The library produced ligand-receptor pairs with LG335 that have a variety of EC50 values (40 nM to >2 microM) and activation levels (10-80% of wild-type RXR with 9cRA) in yeast. The variant I268V;A272V;I310L;F313M has an EC50 for LG335 of 40 nM and an EC50 for 9cRA of >10 microM in yeast. This variant has essentially the reverse ligand specificity of wild-type RXR and is transcriptionally active at a 10-fold-lower ligand concentration in yeast. This EC50 is 25-fold lower than the best receptor we have engineered through site-directed mutagenesis, Q275C;I310M;F313I. Furthermore, the variants' EC50 values and activation levels in yeast and mammalian cells correlate. This protein engineering method should be extendable to produce other functional ligand-receptor pairs, which can be selected and characterized from libraries within weeks. Coupling large library construction with chemical complementation could be used to engineer proteins that bind virtually any small molecule for conditional gene expression, applications in metabolic engineering, and biosensors and to engineer enzymes through genetic selection.

Amino Acid Substitution↗
PubMed2004

Identification and analysis of safener-inducible expressed sequence tags in Populus using a cDNA microarray.

Safeners are the chemicals used to protect plants from detrimental effects of herbicides, but their mode of action at the molecular level is not well understood. As an initial step towards understanding the molecular mechanism of safener action in trees, homologous genes in hybrid poplar (Populus nigra x Populus maximowiczii) that were induced by a safener were identified. We here describe the identification of differentially expressed genes in Populus that are induced by Concep-III, a herbicide safener. Expressed sequence tags (ESTs) enriched for transcriptionally induced genes were isolated by suppressive subtractive hybridization (SSH). The SSH library cDNA inserts were used to construct a cDNA microarray for high-throughput validation of the up-regulated expression of safener-induced genes. Single-pass and partial sequences of 1,344 safener-induced ESTs were assembled into 418 singletons and 328 clusters, but the putative functions of almost 53% of the ESTs are not known. Genes encoding proteins involved in all three different phases of safener action, viz., oxidation, conjugation, and sequestration, were found in the SSH library. Almost 75% of genes that showed greater than 2-fold expression upon safener treatment were redundant in the SSH library. The expression pattern for selected genes was validated by reverse transcription-polymerase chain reaction. A few safener-induced genes that were not previously reported to be induced by safeners, but which may have a role in herbicide metabolism, were identified. The newly identified genes could have potential for application in genetic engineering of plants for herbicide detoxification and tolerance.

Acetophenones↗
PubMed2005

In vitro growth and differentiation of osteoblast-like cells on hydroxyapatite ceramic granule calcified from red algae.

PURPOSE: The purpose of this study was to analyze the interaction between osteoblast-like cells isolated from mandibular bone and hydroxyapatite ceramic bone substitute obtained from calcified red algae to assess the growth and differentiation of adherent cells on this biomaterial. MATERIALS AND METHODS: The macroporous ceramic material C GRAFT/Algipore (The Clinician's Preference LLC, Golden, CO) is composed of 100% hydroxyapatite and possesses specific mechanical and physiochemical properties. Osteoblast-like cells were seeded on 200 mg of biomaterial and cultured for 6 and 21 days under osteogenic differentiation conditions. Specific alkaline phosphatase activity, DNA, and protein content of the proliferating cells were analyzed. The morphology of the cells in contact with the biomaterial was examined by scanning electron microscopy. The osteoblastic phenotype of the cells was confirmed by analysis of the expression of bone-specific genes (osteocalcin, osteopontin and collagen type I) by semi-quantitative reverse transcriptase polymerase chain reaction. RESULTS: The DNA and protein content increased over the culture period. Scanning electron microscopy showed cells spreading on the surface of the biomaterials, covering the macropores, and colonizing the depth of the particles. The analysis of the expression patterns of bone-related genes confirmed the osteoblastic phenotype of the cultured cells. CONCLUSION: The results of this study showed that hydroxyapatite ceramic bone substitute obtained from calcified red algae support the proliferation and differentiation of human osteoblast-like cells on its surface in vitro and might be suitable for use as scaffolds in tissue engineering strategies in vivo.

Bone Substitutes↗
PubMed2001

Differential effects of myeloid dendritic cells retrovirally transduced to express mammalian or viral interleukin-10 on cytotoxic T lymphocyte and natural killer cell functions and resistance to tumor growth.

BACKGROUND: Genetic engineering of dendritic cells (DC) to express immunosuppressive molecule(s) offers potential for therapy of allograft rejection and autoimmune disease. Viral (v) interleukin (IL)-10, encoded by the Epstein-Barr virus, is highly homologous to mammalian (m) IL-10, but lacks certain of its T-cell stimulatory activities. Our aim was to evaluate and compare the influence of vIL-10 and mIL-10 gene transfer on the T-cell and natural killer (NK) cell stimulatory activity of DC, and their impact on the growth of transplantable tumors. METHODS: Myeloid DC progenitors, propagated from the bone marrow of C57BL/6J (H2b) mice in granulocyte-macrophage colony-stimulating factor + IL-4, were transduced using retroviral supematant from the BOSC ecotropic packaging cell line. The function of the IL-b gene-modified DC was assessed by examining their ability to induce naive allogeneic T-cell proliferation and cytotoxic T lymphocyte (CTL) generation. MCA205 (H2b) sarcoma cells mixed with either vIL-10-, mIL-10-, or Zeo (control gene)-transduced DC were inoculated intradermally into C57BL/6J (syngeneic) or BALB/cJ (H2d) (allogeneic) recipients, which were monitored for tumor growth. The role of specific host effector cell populations in tumor resistance was determined by antibody depletion. RESULTS: Compared with control gene-modified DC, both vIL-10- and mIL-10-transduced DC, which secreted the transgene product, showed reduced surface expression of MHC class II and costimulatory molecules, and impaired ability to induce T-cell proliferation. vIL-10-transduced DC were also inhibited with respect to CTL induction but did not affect the generation of NK cells. By contrast, mIL-10-transduced DC augmented CTL generation and NK cell activity. In the tumor transplant model, vIL-10-transduced DC enhanced tumor growth both in syngeneic and allogeneic hosts, whereas mIL-10-transduced cells inhibited tumor development. Depletion of CD4+ or CD8+ T cells or NK cells in mice given mIL-10-transduced DC reversed this therapeutic effect. CONCLUSION: mIL-10 gene-modified myeloid DC promote CTL and NK cell-mediated responses and inhibit tumor growth. By contrast, vIL-10-engineered DC, which elicit diminished CTL responses and do not promote NK cell activity, seem to have therapeutic potential for inhibition of T cell-mediated immunity.

Animals↗
PubMed2005

Analysis of cell-seeded 3-dimensional bone constructs manufactured in vitro with hydroxyapatite granules obtained from red algae.

PURPOSE: Bone tissue engineering is a promising approach for the treatment of defective or lost bone in the maxillofacial region. Biocompatible and biodegradable scaffolds seeded with living cells are used to create functional tissue for load-bearing bone reconstruction. The aim of this study was to manufacture cell-seeded 3-dimensional bone constructs based on hydroxyapatite ceramic granule calcified from red algae and mesenchymal cambial-layer precursor cells. The ability of these cells to grow on hydroxyapatite ceramic was quantitatively investigated to evaluate 3-dimensional bone constructs for their potential use in bone tissue engineering. MATERIALS AND METHODS: Mesenchymal cambial-layer precursor cells were isolated from mandibular periosteum biopsy samples of 3 patients. To manufacture 72 bone constructs, these cells and hydroxyapatite ceramic granules (C GRAFT/Algipore; Clinician's Preference LLC, Golden, CO) were cultivated under osteogenic differentiation conditions in a rotating wall vessel system. After 6 and 21 days, histologic examination and scanning electron microscopy were performed. The absolute DNA content, protein synthesis, and alkaline phosphatase activity were also quantified. The osteoblastic phenotype of the constructs was confirmed by the expression of bone-specific genes (osteocalcin, osteonectin, osteopontin, and core binding factor alpha1) using semiquantitative reverse transcription-polymerase chain reaction and Western blot analysis. RESULTS: Cells within the constructs showed good viability, which was evidenced by an increase in DNA content over the culture period. The decrease in alkaline phosphatase-specific activity could be an indicator of the maturation of cells and the induction of mineralization. The osteoblastic phenotype of the constructs was demonstrated on protein and at the RNA level over the entire culture period. CONCLUSION: We observed a positive effect of hydroxyapatite ceramic granules on mesenchymal cambial-layer precursor cell behavior in cell-seeded 3-dimensional bone constructs, indicating the potential applicability of C GRAFT/Algipore composites in bone tissue engineering.

Blotting, Western↗
PubMed2005

End-labeled free-solution electrophoresis of DNA.

DNA is a free-draining polymer. This subtle but "unfortunate" property of highly charged polyelectrolytes makes it impossible to separate nucleic acids by free-flow electrophoresis. This is why one must typically use a sieving matrix, such as a gel or an entangled polymer solution, in order to obtain some electrophoretic size separation. An alternative approach consists of breaking the charge to friction balance of free-draining DNA molecules. This can be achieved by labeling the DNA with a large, uncharged molecule (essentially a hydrodynamic parachute, which we also call a drag-tag) prior to electrophoresis; the resulting methodology is called end-labeled free-solution electrophoresis (ELFSE). In this article, we review the development of ELFSE over the last decade. In particular, we examine the theoretical concepts used to predict the ultimate performance of ELFSE for single-stranded (ssDNA) sequencing, the experimental results showing that ELFSE can indeed overcome the free-draining issue raised above, and the technological advances that are needed to speed the development of competitive ELFSE-based sequencing and separation technologies. Finally, we also review the reverse process, called free-solution conjugate electrophoresis (FSCE), wherein uncharged polymers of different sizes can be analyzed using a short DNA molecule as an electrophoretic engine.

DNA↗
PubMed2006

Immortalization of bovine germ line stem cells by c-myc and hTERT.

The limited life span of bovine germ line stem cells in vitro is one of the obstacles to spermatogenesis analysis, genetic manipulation and generating transgenic animal. The aim of this study is to establish immortalized bovine germ line stem cells by c-myc or hTERT. We constructed pEMY and pETE expression vectors and transfected germ line cells from 5-month-old bovine. After G418 screening, four types of positive clones were obtained. The results showed that they expressed exogenous genes c-myc or hTERT at mRNA and protein level by RT-PCR and Western blotting detection. Presumable cell lines GM7, GT3, GMT5 all expressed germ-line-stem-cell-specific makers by immunocytochemical analysis, such as c-kit, Oct-4 and GFRalpha-1. The putative cell lines also had higher capacity of proliferation than freshly isolated bovine spermatogonial stem cells. So we can conclude that exogenous genes c-myc or hTERT have integrated into the genome of bovine germ cells and upregulated the expression of telomerase.

Animals↗
PubMed2004

Response of human cord blood cells to styrene exposure: evaluation of its effects on apoptosis and gene expression by genomic technology.

Styrene is one of the most important monomers produced worldwide, and it finds major use in the production of polystyrene, acrylonitrile-butadiene-styrene resins and unsaturated polystyrene resins. Epidemiological studies on styrene showed that the malignancies observed most frequently in humans after exposure are related to the lymphatic and haemopoietic system. IARC classified styrene a possible carcinogenic to humans (Group 2B). In this study, we evaluated the effect of styrene on gene expression profiles of human cord blood cells, as well as its activity on the apoptosis and bcl-2 related protein expression. Data demonstrated that, after 24 and 48 h of exposure, styrene (800 microM) induced an increase in the necrosis of mononuclear cord blood cells, whereas it did not cause any increase in the apoptotic process. Western blot analysis revealed a modified expression of Bax, BCl-2, c-Jun, c-Fos and Raf-1 proteins in the human cord blood cells after direct exposure to styrene, whereas p53 expression did not change. Furthermore, Macroarray analysis showed that styrene changed cord blood gene expression, inducing up-regulation of monocyte chemotactic protein 1 (MCP-1), and down-regulation of CC chemokine receptor type 1 (CCR-1) and SLP-76 tyrosine-phosphoprotein.

Apoptosis↗