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At least 1,423 records · Page 79Linked to original sources
PubMed2003

Replication of human immunodeficiency viruses engineered with heterologous Tat-transactivation response element interactions.

Human immunodeficiency viruses (HIVs) and the related bovine lentiviruses bovine immunodeficiency virus (BIV) and Jembrana disease virus (JDV) utilize the viral Tat protein to activate viral transcription. The arginine-rich RNA-binding domains of the Tat proteins bind to their cognate transactivation response element (TAR) RNA hairpins located at the 5' ends of the viral mRNAs, resulting in enhanced processivity of RNA polymerase II. It has previously been shown that HIV type 1 (HIV-1) Tat requires the cellular cyclin T1 protein for high-affinity RNA binding whereas BIV Tat and JDV Tat bind with high affinity on their own and adopt distinct beta-hairpin conformations when complexed to RNA. Here we have engineered the BIV and JDV Tat-TAR interactions into HIV-1 and show that the heterologous interactions support viral replication, correlating well with their RNA-binding affinities. Viruses engineered with a variant TAR able to bind all three Tat proteins replicate efficiently with any of the proteins. In one virus containing a noncognate Tat-TAR pair that neither interacts nor efficiently replicates (HIV-1 TAR and BIV Tat), viral revertants were isolated in which TAR had become mutated to generate a functional BIV Tat binding site. Our results support the view that incremental changes to TAR structure can provide routes for evolving new Tat-TAR complexes while maintaining active viral replication.

Amino Acid Sequence↗
PubMed2006

Chimeric DNA methyltransferases target DNA methylation to specific DNA sequences and repress expression of target genes.

Gene silencing by targeted DNA methylation has potential applications in basic research and therapy. To establish targeted methylation in human cell lines, the catalytic domains (CDs) of mouse Dnmt3a and Dnmt3b DNA methyltransferases (MTases) were fused to different DNA binding domains (DBD) of GAL4 and an engineered Cys2His2 zinc finger domain. We demonstrated that (i) Dense DNA methylation can be targeted to specific regions in gene promoters using chimeric DNA MTases. (ii) Site-specific methylation leads to repression of genes controlled by various cellular or viral promoters. (iii) Mutations affecting any of the DBD, MTase or target DNA sequences reduce targeted methylation and gene silencing. (iv) Targeted DNA methylation is effective in repressing Herpes Simplex Virus type 1 (HSV-1) infection in cell culture with the viral titer reduced by at least 18-fold in the presence of an MTase fused to an engineered zinc finger DBD, which binds a single site in the promoter of HSV-1 gene IE175k. In short, we show here that it is possible to direct DNA MTase activity to predetermined sites in DNA, achieve targeted gene silencing in mammalian cell lines and interfere with HSV-1 propagation.

Animals↗
PubMed1989

Overexpression of amyloid precursor protein A4 (beta-amyloid) immunoreactivity in genetically transformed cells: implications for a cellular model of Alzheimer amyloidosis.

Among the major obstacles to clarifying molecular mechanisms involved in amyloid metabolism in Alzheimer disease has been the unavailability of laboratory models for this uniquely human disorder. The present studies were aimed at establishing genetically engineered cell lines that overexpress amyloid immunoreactivity and that may be relevant to amyloid accumulation in the Alzheimer disease brain. We used cloned amyloid cDNA that contains a region encoding A4 (beta-polypeptide) amino acids along with recently developed tumor virus vectors derived from simian virus 40 to prepare transformed cells. After transient and permanent transfection, a variety of cell types overexpressed A4 immunoreactivity that was detected by highly specific monoclonal antibodies. We observed that the use of an amyloid subdomain containing the A4 region, but lacking the sequence of a Kunitz-type protease inhibitor found in amyloid precursor protein variants, was sufficient to obtain cells that overproduced an A4 epitope. The transformed cells were readily propagated in culture and may provide an experimental medium to elucidate aspects of the molecular pathogenesis of Alzheimer disease. The cellular models may also serve as tools for deriving potentially useful therapeutic agents.

Alzheimer Disease↗
PubMed2006

Human serum for tissue engineering of human nasal septal cartilage.

OBJECTIVE: To compare the chondrogenic and proliferative effects of pooled human serum (HS) and fetal bovine serum (FBS) on tissue-engineered human nasal septal chondrocytes. STUDY DESIGN AND SETTING: Human chondrocytes were expanded for one passage in monolayer in medium supplemented with 10% FBS, 2% HS, 10% HS, or 20% HS. Cells were then suspended in alginate beads for 3D culture for 2 weeks with 10% FBS, 2% HS, 10% HS, or 20% HS. RESULTS: Monolayer cell yields were greater with HS than FBS. In alginate, cellular proliferation, glycosaminoglycan production per cell, and type II collagen were significantly higher with 10% HS compared to 10% FBS controls. CONCLUSION: HS results in increased proliferation and production of cartilaginous extracellular matrix by tissue-engineered human nasal septal chondrocytes, compared to FBS controls. SIGNIFICANCE: Culture with human serum may facilitate creation of neocartilage constructs that more closely resemble native tissue.

Alginates↗
PubMed2004

Motif-grafted antibodies containing the replicative interface of cellular PrP are specific for PrPSc.

Prion diseases are closely associated with the conversion of the cellular prion protein (PrPC) to an abnormal conformer (PrPSc) [Prusiner, S. B. (1998) Proc. Natl. Acad. Sci. USA 95, 13363-13383]. Monoclonal antibodies that bind epitopes comprising residues 96-104 and 133-158 of PrPC potently inhibit this process, presumably by preventing heterodimeric association of PrPC and PrPSc, and suggest that these regions of PrPC may be critical components of the PrPC-PrPSc replicative interface. We reasoned that transplanting PrP sequence corresponding to these regions into a suitable carrier molecule, such as an antibody, could impart specific recognition of disease-associated forms of PrP. To test this hypothesis, polypeptides containing PrP sequence between residues 89-112 or 136-158 were used to replace the extended heavy chain complementarity-determining region 3 of an IgG antibody specific for the envelope glycoprotein of HIV-1. Herein the resulting engineered PrP-IgGs are shown to bind specifically to infective fractions of PrP in mouse, human, and hamster prion-infected tissues, but not to PrPC, other cellular components, or the HIV-1 envelope. PrPSc reactivity was abolished when the sequence of the PrP 89-112 and 136-158 grafts was mutated, scrambled, or N-terminally truncated. Our findings suggest that residues within the 89-112 and 136-158 segments of PrPC are key components of one face of the PrPC-PrPSc complex. PrPSc-specific antibodies produced by the approach described may find widespread application in the study of prion biology and replication and in the detection of infectious prions in human and animal materials.

Amino Acid Sequence↗
PubMed2005

Structural studies on the alphabeta T-cell receptor.

Alphabeta T-cell receptor (TcR) recognition of antigenic peptides bound to the major histocompatibility complex (pMHC), is integral to the cellular immune system. Crystallographic studies over the last decade have provided significant insight into this unique trimolecular recognition event. The TcR-pMHC structural information has been paralleled by biophysical studies that have further explored the emerging binding models in an attempt to answer fundamental immunological questions regarding MHC restriction, T-cell immunodominance and TcR cross-reactivity. However, despite the important data that has been generated regarding TcR-pMHC interactions, the scope of this information is still incomplete due to the limited range of TcRs that have been studied. These limitations are primarily due to difficulties in obtaining high yields of recombinant alphabeta TcR for crystallographic and biophysical analysis; here we will discuss some of the protein engineering strategies that have been employed to expand the pool of recombinant TcRs suitable for crystallographic studies and the subsequent studies that have utilized these proteins.

Biophysics↗
PubMed2006

Role of hepatocyte growth factor activator (HGF activator) in invasive growth of human glioblastoma cells in vivo.

Hepatocyte growth factor/scatter factor (HGF/SF) is a multifunctional growth factor that is involved in invasive growth of tumor cells via its receptor MET, a protein product of c-met proto-oncogene. HGF activator (HGFA) is a serine proteinase responsible for the activation of proform of HGF/SF (proHGF/SF). In our study, we examined the effects of engineered expression of HGFA on 2 human glioblastoma cell lines (YKG-1 and U251). Both cells expressed MET, while only YKG-1 expressed endogenous proHGF/SF. Enhanced MET phosphorylation and increased migratory activity were induced by the expression of HGFA in YKG-1 cells in vitro in the presence of thrombin, which is a known activator of proHGFA. In contrast, MET phosphorylation was consistently observed in U251 that lacked endogenous HGF/SF, suggesting ligand-independent activation of MET in this cell line. Consequently, the expression of HGFA in U251 did not enhance the MET phosphorylation and following cellular response even with the thrombin treatment. However, addition of exogenous proHGF/SF resulted in enhanced migratory activity of HGFA-expressing U251 cells in the presence of thrombin in vitro. The engineered HGFA expression resulted in significantly enhanced tumor growth with increased vascular density in vivo when YKG-1 cells were implanted in nude mouse brain. This effect was not observed in U251 lacking endogenous proHGF/SF. These results indicate the possible existence of multiple mechanisms of MET activation in glioblastomas and that the activation system of proHGF/SF is important in progression of glioblastomas that express endogenous proHGF/SF and require ligand-dependent MET activation.

Animals↗
PubMed2006

Regulated degradation is a mechanism for suppressing stochastic fluctuations in gene regulatory networks.

Cellular events that execute life programs are ordered and reproducible, despite the noise and randomness underlying their biochemical reactions. The identification of the processes that ensure this robust operation is essential not only to uncover the salient design principles in organisms, but also to forward-engineer reliable genetic networks for biotechnological and therapeutic purposes. The use of feedback for noise reduction has been suggested as a recurring motif in genetic networks. In this work, we show that regulated degradation of proteins implements a negative feedback loop that enhances robustness against stochastic fluctuations and cellular noise. The analysis is carried out in the context of the bacterial heat shock response where the tight control of the amount of heat shock proteins is achieved through an intricate architecture of feedback loops involving the sigma(32)-factor. The sigma(32) regulates the transcription of heat shock proteins under normal and stress conditions. An essential feature of the heat shock response is a feedback loop regulating the degradation of sigma(32). We investigate the noise-rejection properties of this loop, therefore illustrating our point in a biologically plausible example.

Computer Simulation↗
PubMed2006

Engineering of vascular grafts with genetically modified bone marrow mesenchymal stem cells on poly (propylene carbonate) graft.

Bone marrow mesenchymal stem cells (MSCs) have demonstrated their pluripotency to differentiate into different cell lineages and may be an alternative cell source for vascular tissue engineering. The objective of this study is to create small diameter vessels by seeding and culture of genetically modified MSCs onto a synthetic polymer scaffold produced by an electrospinning technique. A tubular scaffold (2 mm in diameter) with a microstructure of nonwoven fibers was produced by electrospinning of poly (propylene carbonate) (PPC). Rat MSCs obtained from bone marrow were expanded in culture and modified with vasculoprotective gene endothelial nitric oxide synthase (eNOS) or marker gene green fluorescent protein (GFP). These MSCs were seeded onto the electrospun fibrous grafts (internal diameter = 2 mm), and cultured in 5% CO(2) at 37 degrees C. The growth of MSCs in the scaffold was analyzed with scanning electron microscopy (SEM) and hematoxylin and eosin (H&E) staining. The gene transfer and transgenic gene expression were examined with fluorescence-activated cell sorting (FACS), immunochemical staining, reverse transcriptase-polymerase chain reaction (RT-PCR), and western blot. The production of nitric oxide (NO) by the engineered vessels was measured with an NO detection kit. Our data showed that the seeded cells integrated with the microfibers of the scaffold to form a three-dimensional cellular network, indicating a favorable interaction between this synthetic PPC scaffold with MSCs. High transduction efficiency was obtained with the use of concentrated retrovirus in the gene transfection of MSCs. The eNOS gene transcripts and protein were detected in the grafts seeded with eNOS-modified MSCs by RT-PCR and immunochemical staining. The amount of NO produced by grafts seeded with eNOS-modified MSCs was comparable to that produced by native blood vessels, and it was significantly higher than that in the grafts seeded with nonmodified MSCs. In summary, the vascular graft produced by culture of eNOS gene-modified MSCs onto the electrospun tubular scaffolds shows promising results in terms of function. The use of MSCs and therapeutic genes in tissue engineering of blood vessels could be helpful in improving vessel regeneration and patency.

Animals↗
PubMed2002

Cultivation of hematopoietic stem and progenitor cells: biochemical engineering aspects.

The ex vivo expansion of hematopoietic cells is one of the most challenging fields in cell culture. This is a rapidly growing area of tissue engineering with many potential applications in bone marrow transplantation, transfusion medicine or gene therapy. Over the last few years much progress has been made in understanding hematopoietic differentiation, discovery of cytokines, isolation and identification of cellular subtypes and in the development of a variety of bioreactor concepts. All this has led to a number of (preliminary) clinical trials that gave a hint of the benefits that can be obtained from the use of expanded hematopoietic cells in therapy. Moreover, as we understand the complexity and the regulation of hematopoiesis, it becomes obvious that highly sophisticated cultivation techniques and bioreactor concepts are needed: a new challenge for bioprocess engineering in cell culture.

Biocompatible Materials↗
PubMed2003

Phenotype modulation in vascular tissue engineering using biochemical and mechanical stimulation.

Biochemical stimulation was applied in combination with cyclic mechanical strain to engineered vascular constructs made of isolated smooth muscle cells in a three-dimensional (3D) collagen type 1 matrix. Platelet-derived growth factor (PDGF) and transforming growth factor beta (TGF-beta) were added exogenously to the medium used to culture the constructs. Mechanical stimulation was applied using a bioreactor system that imparted a 10% circumferential strain at a frequency of 1 Hz. The parameters studied were gel compaction, cell proliferation, and expression of the contractile protein smooth muscle alpha-actin (SMA). Mechanical stimulation caused a characteristic increase in gel compaction and cell proliferation, relative to statically cultured controls. Stimulation with PDGF increased cell proliferation and decreased SMA expression in 3D gels, but inhibited the effects of mechanical stimulation and produced a more open matrix structure. TGF-beta strongly inhibited cell proliferation and increased SMA expression, especially in the presence of mechanical strain, and resulted in a dense matrix. These results show that cell phenotype can be modulated in engineered blood vessels by applying selected combinations of biochemical and mechanical stimuli, and suggest that such control over cell function can be used to tailor the properties of engineered tissues.

Actins↗
PubMed2000

Tissue engineering of vascular grafts.

The challenge of tissue engineering blood vessels with the mechanical properties of native vessels, and with the anti-thrombotic properties required is immense. Recent advances, however, indicate that the goal of providing a tissue-engineered vascular graft that will remain patent in vivo for substantial periods of time, is achievable. For instance, collagen gels have been used to fabricate a tissue in vitro that is representative of a native vessel: an acellular collagen tubular structure, when implanted as a vascular graft, was able to function, and to become populated with host cells. A completely cellular approach culturing cells into tissue sheets and wrapping these around a mandel was able to form a layered tubular structure with impressive strength. Culture of cells onto a biodegradable scaffold within a dynamic bioreactor, generated a tissue-engineered vascular graft with substantial stiffness and, when lined with endothelial cells, was able to remain patent for up to 4 weeks in vivo. In our experiments, use of a non-degradable polyurethane scaffold and culture with smooth muscle cells generated a construct with mechanical properties similar to native vessels. This composite tissue engineered vascular graft with an endothelial layer formed using fluid shear stress to align the endothelial cells, was able to remain patent with an neointima for up to 4 weeks. These results show that tissue engineering of vascular grafts has true potential for application in the clinical situation.

Biocompatible Materials↗
PubMed2006

A century of progress in corticoneurogenesis: from silver impregnation to genetic engineering.

Within the past 125 years, we have witnessed great strides in understanding development and evolution of the cerebral cortex, arguably the structure that makes us human. Among the distinguishing features of cortical development are discoveries that its constituent neurons are not generated locally and that after assuming their proper areal, radial, and laminar position, they serve the individual throughout the lifespan. Although the basic cellular events and all major developmental phenomena have been discovered by the use of classical methods, advents of new, evermore sophisticated experimental methods that range from neuroimaging to molecular genetics enable elucidation of the molecular and cellular mechanisms underlying evolutionary elaboration of the cortex and opens the possibility for the prevention and treatment of congenital disorders of the highest cognitive functions in humans.

Animals↗
PubMed1986

Structure and function of p21 ras proteins.

Cancer is a malfunction of cellular growth control. The discovery of oncogenes, first in transforming retroviruses, and later in human and animal tumors, may have uncovered the key to understanding one of the most elusive subjects of basic cell biology, namely, the controlling mechanisms of cell growth. The ras gene family encodes a group of closely related 21,000 dalton (p21) proteins with special affinity for guanine nucleotides. Other cellular proteins with similar biochemical properties, collectively known as G-proteins, include the regulatory G proteins of adenylate cyclase, the alpha subunit of transducin of retina rod outer segments, the recently identified rho gene proteins, and perhaps also the elongation factors, EF-Tu and EF-G, of the protein synthesis system. These G-proteins have roles in cellular signal transduction; by analogy p21 may have a similar cellular function in mediating the flow of growth control signals. Recent progress in the cloning and sequencing of these genes, overproduction of gene products in E. coli, protein engineering, detailed biochemical characterization, and the molecular structure determined by high resolution X-ray crystallography, have helped to elucidate in great detail the structure and function of p21 ras proteins. p21 appears to have a small membrane binding domain at the C-terminus, which contains a palmitylation site at cysteine-186, four amino acid residues from the end. Separated by a variable "hinge" region, most of the rest of ras amino acid sequences are highly conserved in nature. Four regions of extensive sequence homology among G-proteins constitute the GTP/GDP binding domain. In the crystal structure of EF-Tu, four peptide loops connecting beta sheets and alpha helices form the pocket for binding GDP. Studies using site-directed mutagenesis and immnochemical probes, indicate that the basic structure of the GDP binding site is conserved between p21 and EF-Tu. Furthermore, these studies also conclude that GTP binding is crucial for p21 ras cellular function. Although the precise target molecules for p21 are still unknown, the finding of the on/off switch function for ras genes have provided a better understanding of the mechanism of proto-oncogene activation, and may also provide further impetus to explore means of cancer intervention by interfering with the switch function.

Amino Acid Sequence↗
PubMed2003

Overexpression of interleukin-12 enables dendritic cells to activate NK cells and confer systemic antitumor immunity.

Dendritic cells (DC) are initiators of T cell-mediated immunity. However, less is known about the relationship between DC and natural killer (NK) cells, and direct evidence of their interaction in vivo is scarce. Interleukin (IL)-12 is an activator of both DC and NK cells. We postulated that secretion of IL-12 by DC would enable them to activate NK cells. Bone marrow-derived DC propagated only in granulocyte macrophage colony-stimulating factor did not activate NK cells. In contrast, DC engineered to express IL-12 markedly stimulated NK cells as determined by coculture experiments in vitro, assays of NK cells isolated from treated animals, and survival experiments in a systemic tumor model. Activation depended on both DC-NK cellular interaction and secretion of IL-12. Adoptive transfer of DC expressing IL-12 to mice markedly increased NK cell interferon-gamma production and lytic activity in vivo. Treated mice were also protected against B16 melanoma hepatic metastases. The in vivo effects on NK cells were DC-specific. Administration of IL-12 protein alone or melanoma cells or fibroblasts engineered to secrete IL-12 were only weakly activating. Our findings demonstrate that IL-12 expression by DC enables them to activate NK cells and provide evidence for a substantial DC-NK relationship in vivo.

Animals↗
PubMed1998

Engineering smooth muscle tissue with a predefined structure.

Nonwoven meshes of polyglycolic acid (PGA) fibers are attractive synthetic extracellular matrices (ECMs) for tissue engineering and have been used to engineer many types of tissues. However, these synthetic ECMs lack structural stability and often cannot maintain their original structure during tissue development. This makes it difficult to design an engineered tissue with a predefined configuration and dimensions. In this study, we investigated the ability of PGA fiber-based matrices bonded at their fiber crosspoints with a secondary polymer, poly-L-lactic acid (PLLA), to resist cellular contractile forces and maintain their predefined structure during the process of smooth muscle (SM) tissue development in vitro. Physically bonded PGA matrices exhibited a 10- to 35-fold increase in the compressive modulus over unbonded PGA matrices, depending on the mass of PLLA utilized to bond the PGA matrices. In addition, the bonded PGA matrices degraded much more slowly than the unbonded matrices. The PLLA bonding of PGA matrices had no effect on the ability of cells to adhere to the matrices. After 7 weeks in culture, the bonded matrices maintained 101 +/- 4% of their initial volume and an approximate original shape while the unbonded matrices contracted to 5 +/- 1% of their initial volume with an extreme change in their shape. At this time the bonded PGA matrices had a high cellularity, with smooth muscle cells (SMCs) and ECM proteins produced by these cells (e.g., elastin) filling the pores between PGA fibers. This study demonstrated that physically bonded PGA fiber-based matrices allow the maintenance of the configuration and dimensions of the original matrices and the development of a new tissue in a predefined three-dimensional structure. This approach may be useful for engineering a variety of tissues of various structures and shapes, and our study demonstrates the importance of matching both the initial mechanical properties and the degradation rate of a matrix to the specific tissue one is engineering.

Animals↗
PubMed2003

Engineered CD20-specific primary human cytotoxic T lymphocytes for targeting B-cell malignancy.

BACKGROUND: Immunotherapy for B-cell lymphomas has evolved significantly with the advent of CD20-targeted Ab-based therapeutics. Strategies to invoke or augment cellular anti-lymphoma immune responses may also have considerable therapeutic potential and serve to further augment the clinical efficacy of MAbs. METHODS: We report here the aquisition by priming human cytotoxic T lymphocyte (CTL) effectors of re-directed CD20 specificity by their genetic modification to express a chimeric immunoreceptor consisting of an anti-CD20 single chain Ab extracellular domain molecularly fused to the T-cell receptor complex CD3-zeta cytoplasmic tail (scFvFczeta). Peripheral blood-derived human T-cells were transduced with naked DNA plasmid vector by electoporation then selected for G418 resistance. RESULTS: Following cloning in limiting dilution and ex vivo expansion to large numbers scFvFczeta+ TCRalpha/beta+ CD4- CD8+ CTL display re-directed HLA-unresricted CD20-specific lymphoma cell cytolysis proportional to the cell-surface density of the chimeric immunoreceptor. Engineered CTL clones are also activated through the chimeric immunoreceptor to produce Tc1 cytokines (IFN-gamma) upon co-culture with CD20+ lymphoma stimulator cells. Additionally, CD20-specific CTL proliferate in the presence of lymphoma stimulators and IL-2 (5 U/mL). DISCUSSION: These studies provide the rationale for exploring the clinical utility of adoptive therapy with CD20-specific CTL as a component of immunotherapeutic targeting of CD20+ malignancy.

Antigens, CD20↗
PubMed1997

Engineering unnatural nucleotide specificity for Rous sarcoma virus tyrosine kinase to uniquely label its direct substrates.

Protein phosphorylation plays a central role in controlling many diverse signal transduction pathways in all cells. Novel protein kinases are identified at a rapid rate using homology cloning methods and genetic screens or selections; however identification of the direct substrates of kinases has proven elusive to genetic methods because of the tremendous redundancy and overlapping of substrate specificities among protein kinases. We describe the development of a protein engineering-based method to identify the direct substrates of the prototypical protein tyrosine kinase v-Src, which controls fibroblast transformation by the Rous sarcoma virus. To differentiate the substrates of v-Src from all other kinase substrates, we mutated the ATP binding site of v-Src such that the engineered v-Src uniquely accepted an ATP analog. We show that the engineered v-Src kinase displayed catalytic efficiency with the ATP analog, N(6)-(cyclopentyl) ATP, which is similar to the wild-type kinase catalytic efficiency with ATP itself. However, the N(6)-(cyclopentyl) ATP analog was not accepted by the wild-type kinase. Furthermore, the engineered v-Src exhibited the same protein target specificity as wild-type v-Src despite the proximity of the reengineered nucleotide binding site to the phosphoacceptor binding site. The successful engineering of v-Src's active site to accept a unique nucleotide analog provides a unique handle by which the direct substrates of one kinase (v-Src) can be traced in the presence of any number of cellular kinases.

Amino Acid Sequence↗