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PubMed2004

Cytoskeletal-associated proteins in the migration of cortical neurons.

Neuronal migration is a hallmark of cerebral cortical development as neurons born deep within the brain migrate to the surface in a highly choreographed process. The cytoskeleton extends throughout the cell, mediating the dramatic morphological changes that accompany migration. On a cellular level, proper migration is accompanied by polarization of the cytoskeleton and cellular contents and by dynamic reorganization that generates the force for cell locomotion. Genetic analyses of human brain malformations, as well as genetically engineered mouse mutants, have highlighted a number of cytoskeletal-associated proteins underlying these functions, which are necessary for proper cortical development. While these proteins are involved in diverse molecular mechanisms, disruption during development results in the ectopic placement of neurons in the cortex. We review key cytoskeletal events and the critical cytoskeletal-associated proteins involved in cortical neuronal migration.

Animals↗
PubMed2004

Anti-prostate specific membrane antigen designer T cells for prostate cancer therapy.

BACKGROUND: Designer T cells are T lymphocytes engineered toward specific antibody-type membrane antigens through chimeric immunoglobulin-T-cell receptor (IgTCR) genes that have been used for adoptive cellular immunotherapy. We have extended this approach to prostate specific membrane antigen (PSMA) as a means to attack prostate cancer. METHODS: A chimeric anti-PSMA IgTCR gene was constructed based on an anti-PSMA monoclonal antibody, 3D8. Both T-cell lines and primary cultured human T lymphocytes were transduced with the chimeric anti-PSMA IgTCR construct and were analyzed for IgTCR expression, specific activation by PSMA, cytotoxicity against PSMA-expressing tumor cells in vitro, and retardation of tumor growth in an animal model. RESULTS: The IgTCR was incorporated into the TCR-CD3 complex and formed a functional chimeric complex. The IgTCR-modified T cells were specifically activated through the chimeric receptor with PSMA as measured by IL-2 production and increased CD25 expression and specifically lysed the PSMA-expressing prostate cancer cells in vitro as well as retarded tumor growth in an animal model. CONCLUSIONS: The anti-PSMA designer T cells exhibit an antibody-type specificity that can recognize PSMA expressing tumor cells in a MHC-independent fashion, resulting in T-cell activation, target cell lysis in vitro and inhibition of tumor growth in vivo.

Animals↗
PubMed1998

Cellular and cytoskeleton morphology and strength of adhesion of cells on self-assembled monolayers of organosilanes.

The objective of this study was to explore the potential use of self-assembled monolayers (SAMs) of alkylamine and arylalkyamine as well-defined, homogeneous, tailored in vitro model surfaces for exploring the effect of hydrodynamic flow on morphology and strength of adhesion of human umbilical vein endothelial cells. The cell surface area, shape, f-actin distribution, and adhesion strength of human umbilical vein endothelial cells cultured on self-assembled monolayers of organosilanes were found to be dependent on the chemical composition of the organosilane film and the magnitude of wall shear stress. The direct effects of the differences in chemistry between the two silanes, in modulating cellular response, are probably only secondary to the modulation of cellular functions mediated by differential protein adsorption and conformation on the two silanes. For short seeding times (30 min), prior to application of flow, both substrate chemistry and shear stress modulated the cellular morphology and cytoskeletal organization. For longer seeding times (24 h), prior to application of flow, the chemistry of the underlying surface was the dominant variable in modulating cellular morphology, while the hydrodynamic shear stress modulated the cytoskeleton organization. Cells on N-(2-aminoethyl)-3-aminopropyl trimethoxysilane (EDA) were pleomorphic, while cells on ((((aminoethyl)amino)methyl)phenylethyl)trimethoxysilane (PEDA) expressed a rounded morphology. Application of an incrementally loaded flow regime (0.07-1.25 ml/s) resulted in a time- and shear stress-dependent (10-180 dyn/cm2) detachment of cells, with the cells on EDA depicting higher resistance to wall shear stress. Cellular morphology correlated with the strength of adhesion; cells with rounded morphology on a hydrophobic silane, PEDA, were less tightly bound to the silane, while spread cells on a hydrophilic silane, EDA, were more tightly bound. The higher surface free energy of EDA is speculated to influence the increased cell spreading and strength of adhesion observed in these studies. The presence of the phenyl group in PEDA reduces the surface free energy and may account for the reduced spreading and lower strength of adhesion. The use of well-defined systems, such as monolayer organosilanes, with tunable surface physicochemical properties may serve to deconstruct the complex interaction of cells with extracellular matrix components: surface charge, surface hydrophobicity, and other short- and long-range forces can be individually controlled and correlated with cellular functions. The organosilane monolayers could serve as the building blocks for sequential addition of proteins or cell adhesive/cell repulsive cues to stepwise engineering and construction of more complex systems resembling ECM matrices.

Biocompatible Materials↗
PubMed1991

Cultured and immediately procured endothelial cells: current and future clinical applications.

Great progress has been made in the last several years in our ability to culture human endothelial cells. In addition, techniques to immediately procure and utilize these cells have also been developed. The purpose of this paper is to present an overview of the current and potential uses of these cells in both vascular and nonvascular conditions. It is likely that endothelial cells will be used in a variety of applications in the near future. Immediately procured and cultured cells will be used to resurface vascular prosthetic grafts. They may also be used on the surface of vessels following procedures such as balloon angioplasty or atherectomy. In addition, they may be placed upon the surface of implantable devices such as expandable stents. Through the mechanism of genetic engineering, these cells may be modified to produce proteins, which may modify thrombogenicity and perhaps decrease the rate of recurrent stenosis by influencing cellular hyperplasia. Genetically modified endothelial cells also have great potential in nonvascular disease. Their contact with circulating blood makes them an ideal cell for production of proteins to correct systemic conditions such as the insulin deficiency found in diabetes mellitus. The application of endothelial cell biology in both vascular and nonvascular science represents one of the most exciting fields of research active today.

Blood Vessel Prosthesis↗
PubMed2006

Current concepts in the biology of orthodontic tooth movement.

Adaptive biochemical response to applied orthodontic force is a highly sophisticated process. Many layers of networked reactions occur in and around periodontal ligament and alveolar bone cells that change mechanical force into molecular events (signal transduction) and orthodontic tooth movement (OTM). Osteoblasts and osteoclasts are sensitive environment-to-genome-to-environment communicators, capable of restoring system homeostasis disturbed by orthodontic mechanics. Five micro-environments are altered by orthodontic force: extracellular matrix, cell membrane, cytoskeleton, nuclear protein matrix, and genome. Gene activation (or suppression) is the point at which input becomes output, and further changes occur in all 5 environments. Hundreds of genes and thousands of proteins participate in OTM. Gene-directed protein synthesis, modification, and integration form the essence of all life processes, including OTM. Bone adaptation to orthodontic force depends on normal osteoblast and osteoclast genes that correctly express needed proteins at the right times and places. Cell membrane receptor-ligand docking is an important initiator of signal transduction and a discovery target for new bone-enhancing drugs. Despite progress in identification of regulatory molecules, the genetic mechanism of "orchestrated synthesis" between different cells, tissues, and systems remains largely unknown. Interpatient variation in mechanobiological response is most likely due to differences in periodontal ligament and bone cell populations, genomes, and protein expression patterns. Discovery of mutations in OTM-associated genes of orthodontic patients, including those regulating osteoclast bone-matrix acidification, chloride channel function, and osteoblast-derived mineral and protein matrices, will permit gene therapy to restore normal matrix and protein synthesis and function. Achieving selectivity in targeting abnormal genes, cells, and tissues is a major obstacle to safe and effective clinical application of gene engineering and stem-cell mediated tissue growth. Orthodontic treatment is likely to evolve into a combination of mechanics and molecular-genetic-cellular interventions: a change from shotgun to tightly focused communication with OTM cells.

Adaptation, Physiological↗
PubMed2004

Protein-protein interaction affinity plays a crucial role in controlling the Sho1p-mediated signal transduction pathway in yeast.

Protein-protein interactions are required for most cellular functions, yet little is known about the relationship between protein-protein interaction affinity and biological activity. To investigate this issue, we engineered a series of mutants that incrementally reduced the affinity of the yeast Sho1p SH3 domain for its in vivo target, the MAP kinase kinase Pbs2p. We demonstrate a strong linear correlation between the binding energy of these mutants and quantitative in vivo outputs from the HOG high-osmolarity response pathway controlled by Sho1p. In addition, we find that reduction in binding affinity for the correct target within this pathway causes a proportional increase in misactivation of the related mating pheromone response pathway and that strong binding affinity alone does not guarantee efficient biological activity. Our experiments also indicate that a second binding surface on the Sho1p SH3 domain is required for its proper in vivo function.

Amino Acid Sequence↗
PubMed2005

Transgenic mouse models for the prevention of breast cancer.

Breast cancer prevention research has made remarkable progress in the past decade. Much of this progress has come from clinical trials. However, in the future to test the many promising agents that are now available, pre-clinical models of breast cancer are needed. Such models are now available. Useful models include rat and mouse models, particularly, the genetically engineered mice (GEM). Many transgenic mouse models have been generated by manipulating growth factors and their receptors, cell cycle regulators, signal transduction pathways, cellular differentiation, oncogenes and tumor suppressor genes. The transgenes are induced to express in the mouse mammary glands under the control of various transgenic promoters, which have respective characteristics in expression pattern and other biological attributes. These models are providing invaluable insight on the molecular mechanisms of breast tumorigenesis. In this review, we discuss the relative relevance of the most commonly used transgenic mouse models for breast cancer prevention studies, and provide examples of how these transgenic models can be used to conduct cancer prevention research. Due to the multi-factor, multi-step nature of breast cancer, many factors should be incorporated into a valid prevention study. However, many barriers to progress must be overcome, including access to and availability of new cancer preventive drugs, and difficulties in conducting studies of combinations of preventive agents.

Animals↗
PubMed2003

Skeletal development: insights from targeting the mouse genome.

Manipulation of the mouse genome through mis-expressing, knocking out, and introducing mutations into genes of interest has provided important insights into the genetic pathways responsible for human skeletal development. These pathways contribute to the sequential phases of skeletal morphogenesis that include patterning, condensation, and overt organogenesis of the membranous and endochondral embryonic skeletons and to subsequent linear growth. Disturbances in these pathways account for many developmental syndromes and disorders of the human skeleton. Recurrent themes include establishment of interlocking regulatory circuits involving growth factors, receptors, signalling pathways, and transcription factors that control cellular programmes such as migration, adhesion, proliferation, differentiation, and apoptosis, and use of common molecules for different purposes. Technical advances suggest that genetic engineering in mice will continue to be highly instructive in the field of skeletal biology.

Animals↗
PubMed1999

Patterning proteins and cells using soft lithography.

This review describes the pattering of proteins and cells using a non-photolithographic microfabrication technology, which we call 'soft lithography' because it consists of a set of related techniques, each of which uses stamps or channels fabricated in an elastomeric ('soft') material for pattern transfer. The review covers three soft lithographic techniques: microcontact printing, patterning using microfluidic channels, and laminar flow patterning. These soft lithographic techniques are inexpensive, are procedurally simple, and can be used to pattern a variety of planar and non-planar substrates. Their successful application does not require stringent regulation of the laboratory environment, and they can be used to pattern surfaces with delicate ligands. They provide control over both the surface chemistry and the cellular environment. We discuss both the procedures for patterning based on these soft lithographic techniques, and their applications in biosensor technology, in tissue engineering, and for fundamental studies in cell biology.

Animals↗
PubMedDate not supplied

New role for the primate fovea: a retinal excavation determines photoreceptor deployment and shape.

In humans, an increasing density of foveal cone photoreceptors occurs slowly over several years after birth, and accounts for a region that subserves high visual acuity. Concurrently, inner retinal cells move centrifugally away from the foveal center. Such developmental rearrangements reflect complex cellular remodeling after the retinal neuronal cells have differentiated and have formed synapses. Explaining foveal morphogenesis is difficult, because differentiated neuronal cells seem incapable of moving actively. Presented here is a biomechanical explanation of how the above events occur. This hypothesis assumes that the cellular movements throughout the retinal layers occur passively as the eye grows and the retina is stretched. Retinal stretch was simulated using virtual engineering models that were analyzed with finite element analysis. A pit combined with retinal stretch causes the retinal layers to deform in a way that accounts for both the centrifugal and centripetal movement of various retinal cell types. Axially directed, tensile forces associated with stretching the retinal tissue surrounding the pit also accounts for the elongated morphology of foveal cone photoreceptors. These simulations suggest that a pit is required for both the centripetal displacement of cone cells toward the center of the fovea, and for the elongated foveal cone morphology. Since the primate fovea may have minimal impact on acuity, its primary role may be to initiate foveal morphogenesis in slowly developing eyes.

Animals↗
PubMed1999

Biomechanics of integrative cartilage repair.

Cartilage repair is required in a number of orthopaedic conditions and rheumatic diseases. From a macroscopic viewpoint, the complete repair of an articular cartilage defect requires integration of opposing cartilage surfaces or the integration of repair tissue with the surrounding host cartilage. However, integrative cartilage repair does not occur readily or predictably in vivo. Consideration of the 'integrative cartilage repair process', at least in the relatively early stages, as the formation of a adhesive suggests several biomechanical approaches for characterizing the properties of the repair tissue. Both strength of materials and fracture mechanics approaches for characterizing adhesives have recently been applied to the study of integrative cartilage repair. Experimental configurations, such as the single-lap adhesive test, have been adapted to determine the strength of the biological repair that occurs between sections of bovine cartilage during explant culture, as well as the strength of adhesive materials that are applied to opposing cartilage surfaces. A variety of fracture mechanics test procedures, such as the (modified) single edge notch, 'T' peel, dynamic shear, and trouser tear tests, have been used to assess Mode I, II, and III fracture toughness values of normal articular cartilage and, in some cases, cartilaginous tissue undergoing integrative repair. The relationships between adhesive biomechanical properties and underlying cellular and molecular processes during integrative cartilage repair remain to be elucidated. The determination of such relationships may allow the design of tissue engineering procedures to stimulate integrative cartilage repair.

Animals↗
PubMed2000

Genetic mosaic analysis based on Cre recombinase and navigated laser capture microdissection.

Defining molecular interactions that occur at the interface between "normal" and "abnormal" cell populations represents an important but often underexplored aspect of the pathogenesis of diseases with focal origins. Here, we illustrate an approach for conducting such analyses based on mosaic patterns of Cre recombinase expression in the adult mouse intestinal epithelium. Transgenic mice were generated that express Cre in the stem cell niche of crypts located in specified regions of their intestine. Some of these mice were engineered to allow for doxycycline-inducible Cre expression. Recombination in all pedigrees was mosaic: Cre-expressing crypts that supported recombination in all of their active multipotent stem cells were located adjacent to "control" crypts that did not express Cre at detectable levels. Cre-mediated recombination of a floxed LacZ reporter provided direct evidence that adult small-intestinal crypts contain more than one active multipotent stem cell, and that these cells can be retained in both small-intestinal and colonic crypts for at least 80 d. A method was developed to recover epithelial cells from crypts with or without recombination for subsequent gene expression profiling. Stained sections of intestine were used to create electronic image templates to guide laser capture microdissection (LCM) of adjacent frozen sections. This navigated form of LCM overcomes problems with mRNA degradation encountered when cells are marked directly by immunohistochemical methods. Combining Cre-engineered genetic mosaic mice with navigated-LCM will allow biology and pathobiology to be explored at the junction between normal and perturbed cellular cohorts.

Animals↗
PubMed1995

Molecular basis of common hereditary motor and sensory neuropathies in humans and in mouse models.

The Hereditary Motor and Sensory Neuropathies (HMSNs) are well known to be clinically, morphologically, and genetically heterogeneous. Yet, recent advances in the cellular and molecular biology of the peripheral nervous system coupled with remarkable progress in human and mouse genetics have provided a framework that has profoundly changed our understanding of the pathogenesis of these diseases. It now appears that most of the HMSNs are related to mutations affecting genes encoding Schwann cell proteins, specifically the Peripheral Myelin Protein PMP22, Myelin Protein Zero, and one of the gap junction proteins, connexin-32. Accordingly, these findings are discussed in the context of the clinical and pathologic features of the human HMSNs, but are interpreted in the context of basic research findings on the cellular and molecular biology of the peripheral nervous system derived from in vivo and in vitro studies in spontaneously-occurring and genetically engineered animal models for the HMSNs.

Animals↗
PubMedDate not supplied

Color-engineered rats and luminescent LacZ imaging: a new platform to visualize biological processes.

The rat represents an excellent mammalian model for broadening medical knowledge, and a wealth of information on its physiology has been obtained from its use as an experimental organism. Furthermore, its ample body size allows various surgical manipulations that cannot be performed on a mouse. Many rat models mimic human diseases and have therefore been used in a variety of biomedical studies, including physiology, pharmacology, and transplantation. In an effort to create specifically designed rats for new biomedical research and the field of regenerative medicine, we develop an engineered rat system on the basis of transgenic technology and succeed in establishing unique rats that possess genetically encoded color probes: green fluorescent protein (GFP), DsRed2 (red liver), Cre/LoxP (red to green), and LacZ (blue and luminescence). In this work, we highlight their characteristics and describe recent applications for tissue engineering and regeneration. Coupled with recent progress in modern imaging systems, these transgenic rats are providing powerful tools for the elucidation of many cellular processes in biomedical science, and may lead to innovative medical treatments.

Animals↗
PubMed2005

Engrafting periodontal fibroblasts with new 3-dimensional polylactide foams.

In clinical periodontics, "tissue engineering" techniques have been developed to guide the regenerative process following periodontal disease. We explored a new shaped biomaterial in order to promote cellular adhesion, proliferation and migration of periodontal cells. Granules of poly-D, L-lactide were foamed in specially designed moulds by a controlled gas-loading process. Explant cultures of periodontal tissue were seeded at different densities on the 3-dimensional scaffolds following analysis of cytotoxicity, cell proliferation and differentiation. The moulding procedure led to porous structures with predetermined tubes for cellular locomotion. Cells adhered to and populated the material's surface and inner cavities while retaining fibroblastic phenotype. An optimal ratio between cellular proliferation and mortality rate was achieved at a seeding density of > 10 6 cells/cm 3 scaffold. We designed modified polylactide scaffolds capable of acting as a stent and a cell carrier matrix. The foaming process is easy, cheap and suitable for commercial production.

Biocompatible Materials↗
PubMed2006

Discovery of new small molecules and targets towards angiogenesis via chemical genomics approach.

Chemical genetics/genomics is an inter- and multi-disciplinary research engine, which utilizes small molecules to explore the function of genes and accelerate the drug discovery. Bioactive small molecules that are permeable to cellular membrane and bind to its cognate target protein can exert the phenotype changes of the cells or organisms. Functional target proteins of these small molecules have been successfully identified by affinity, genetics and genomics based target identification. The specific molecular recognition of small molecule with target protein has facilitated to decipher the mode of actions of small molecules and developed better drug based on their structure activity relationship. Based on this idea, we have applied chemical genomics to angiogenesis, a new blood vessel formation, resulting in the identification of new small molecules as well as targets. In this review, our application of chemical genomics towards a cellular phenotype, angiogenesis, will be demonstrated.

Amino Acid Sequence↗
PubMed2002

Toward the engineering of minimal living cells.

The article focuses on the notion of a synthetic or semi-synthetic minimal cell, defined as a system that has the minimal and sufficient structural conditions for cellular life. It is emphasized that two complementary approaches are in principle possible, defined as "bottom-up" and "top-down" approaches. The first one aims at the construction of a minimal cell starting from scratch, and it is argued that a very serious bottle-neck to this pathway lies in the origination of specific macro-molecular sequences, as in nature those were constructed most likely by a particular contingent set of conditions. The top-down approaches utilize extant genes and enzymes, and the work in this case is based on the incorporation of the minimal and sufficient amount of such macromolecules into liposomes, as models for the shell of biological cells. The first phase of this ambitious project foresees the study of conditions under which complex molecular biology reactions takes place in the compartments of liposomes. Examples of these reactions are provided, for example, the production of RNA throughout Q-beta replicase in a self-reproducing vesicle system; or PC Reaction in phospholipid vesicles; or even the incorporation of ribosomes in liposomes, with the production of polypeptide chains. The use of giant vesicles is also illustrated. These systems, due to their large size, offer the advantage that by way of special micro-injection techniques, all sort of biochemical agents can be directly introduced in the compartment; and that the reaction can be followed by optical microscopy. In the final part of the article, the outlook of increasing the complexity of these liposome systems so as to arrive at first semi-synthetic cells is discussed.

Biopolymers↗
PubMed1995

Revising messages traveling along the cellular information superhighway.

Cells use RNA messages to carry instructions from the genome to ribosomes about the types of proteins that should be made. These messages are generally revised by splicing before translation. Engineering ribozymes that can use splicing to repair mutant transcripts may be a useful approach to gene therapy of several genetic diseases.

Animals↗