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At least 1,369 records · Page 76Linked to original sources
PubMed2006

GINsim: a software suite for the qualitative modelling, simulation and analysis of regulatory networks.

This paper presents GINsim, a Java software suite devoted to the qualitative modelling, analysis and simulation of genetic regulatory networks. Formally, our approach leans on discrete mathematical and graph-theoretical concepts. GINsim encompasses an intuitive graph editor, enabling the definition and the parameterisation of a regulatory graph, as well as a simulation engine to compute the corresponding qualitative dynamical behaviour. Our computational approach is illustrated by a preliminary model analysis of the inter-cellular regulatory network activating Notch at the dorsal-ventral boundary in the wing imaginal disc of Drosophila. We focus on the cross-regulations between five genes (within and between two cells), which implements the dorsal-ventral border in the developing imaginal disc. Our simulations qualitatively reproduce the wild-type developmental pathway, as well as the outcome of various types of experimental perturbations, such as loss-of-function mutations or ectopically induced gene expression.

Animals↗
PubMedDate not supplied

Development and analysis of a mathematical model for antibody-producing GS-NS0 cells under normal and hyperosmotic culture conditions.

The GS-NS0 cell line is industrially important and is currently used for the large-scale production of several therapeutic monoclonal antibodies. A novel hybrid model, consisting of both unstructured and structured elements, has been developed to describe cell growth and death, metabolism, and antibody production in the GS-NS0 system under normal culture conditions. A comparison between the hybrid model and a large-scale single-cell model (SCM) describing detailed metabolic processes verified the predictive ability of the hybrid model (when compared with experimental data) and highlighted the practical difficulties involved in utilizing complex models. Global sensitivity analysis (GSA) on the hybrid model identified the specific transcription and translation rates of heavy and light immunoglobulin chains as parameters with the largest impact on the antibody production process. This information, together with the addition of a 24-h lag phase, resulted in the successful extension of the hybrid model to represent GS-NS0 system behavior under hyperosmotic culture conditions.

Animals↗
PubMed2003

Protein semi-synthesis in living cells.

Incorporation of chemical probes into proteins is a powerful way to elucidate biological processes and to engineer novel function. Here we describe an approach that allows ligation of synthetic molecules to target proteins in an intracellular environment. A cellular protein is genetically tagged with one-half of a split intein. The complementary half is linked in vitro to the synthetic probe, and this fusion is delivered into cells using a transduction peptide. Association of the intein halves in the cytosol triggers protein trans-splicing, resulting in the ligation of the probe to the target protein through a peptide bond. This process is specific and applicable to cytosolic and integral membrane proteins. The technology should allow cellular proteins to be elaborated with a variety of abiotic probes.

Animals↗
PubMed2002

From mouse to man: understanding heart failure through genetically altered mouse models.

Human heart failure, a complex disease with heterogeneous etiologies, remains one of the most life-threatening diseases known. Identification of "candidate genes" and molecular and biochemical mediators of cardiac hypertrophy and failure has been vigorously pursued to dissect the pathogenesis and signaling pathways of this disease. With the availability of murine cardiac-specific promoters, transgenesis and gene targeting technologies have revolutionized the field of cardiac research. During the past decade, a large number of genetically engineered mouse models with altered cardiac function have been generated. The ability to engineer precise mutations in the heart, coupled with the technological sophistication to quantitate the effects of these mutations on cardiac function at cellular, organ and intact animal levels, has provided novel insights into the molecular mechanisms of heart failure and led to the recognition of a wide array of previously unknown molecular sensors, initiators, transducers, and effectors for the development of cardiac hypertrophy and its transition to heart failure.

Animals↗
PubMed1995

Immortalization of human primary B lymphocytes in vitro with DNA.

Epstein-Barr virus (EBV) is a human DNA tumor virus that efficiently immortalizes human primary B lymphocytes in vitro. Although viral genes that are expressed in latently infected B lymphocytes have been shown to function in cellular growth control, their detailed genetic analysis has been cumbersome for two reasons. The viral genome is too large to permit genetic engineering and human primary B lymphocytes, the only targets for infection by EBV in vitro, are both intractable in culture and recalcitrant to DNA transfection. To overcome these obstacles, we have assembled all the essential genes of EBV on a single recombinant vector molecule in Escherichia coli. We show here that this mini-EBV plasmid can yield immortalized B cells upon transfer of its naked DNA into human primary B lymphocytes. Established cell lines carry recombinant vector DNA and cannot support virus production. Because this DNA can be easily manipulated in E. coli, mutant mini-EBVs as well as foreign genes can now be introduced and studied successfully in recipient B lymphocytes from any human donors. These mini-EBVs therefore are potentially useful for human gene therapy.

B-Lymphocytes↗
PubMedDate not supplied

Geometric control of fibroblast growth on proton beam-micromachined scaffolds.

Circular three-dimensional (3D) micropatterns with grooves and ridges of various sizes on the circumference of the structure were micromachined in polymethylmethacrylate, using proton beam micromachining. Fibroblasts were seeded in the center smooth nonpatterned surface of the circle. The circumference grooves could retard the outward spreading of cells after they became confluent in the central smooth surface. The fibroblasts eventually migrated across the grooves and ridges several days later. Wider and deeper grooves were more effective in retarding fibroblast spreading. Our results indicate that groove structures in cellular dimensions can effectively retard fibroblasts invasion. Proton beam micromachining, which has the unique advantage of being the only technique capable of manufacturing direct-write precise 3D microstructure at cellular dimensions, has great potential in generating 3D microscaffolds for studying cell behavior in a 3D microenvironment, which is important for tissue engineering.

Animals↗
PubMed1999

Assessment of novel anti-p185HER-2 monoclonal antibodies for internalization-dependent therapies.

Novel therapies that require internalization of effector domains may be improved by assessing the efficacy of postbinding receptor-mediated endocytosis. To achieve targeted gene therapy of immunotoxin therapy, natural vector-host tropisms must be altered. Recent improvements in monoclonal antibody (MAb) engineering have expanded the potential range of host cells that can be targeted for therapeutic intervention. However, relatively little is known about cellular responses after binding of a vector construct. We have tested the utility of four novel MAbs recognizing the extracellular domain of p185HER-2, a membrane receptor protein, for use in internalization-dependent therapies. All four antibodies bound to p185HER-2 in a number of immunoassays. Two antibodies recognized accessible epitopes of p185HER-2 on viable cells. Radioimmunoassay demonstrated that antibody-membrane receptor complexes formed by two antibodies were internalized and trafficked through an endolysosomal degradative pathway. Two of the four antibodies evaluated were found to have favorable internalization characteristics suitable for incorporation in a targeting vector. This analytical approach could be applied to antibodies prior to and after fusion with various vectors or toxins to determine the potential utility of the antibodies for targeted therapy.

Animals↗
PubMed2005

Least absolute regression network analysis of the murine osteoblast differentiation network.

MOTIVATION: We propose a reverse engineering scheme to discover genetic regulation from genome-wide transcription data that monitors the dynamic transcriptional response after a change in cellular environment. The interaction network is estimated by solving a linear model using simultaneous shrinking of the least absolute weights and the prediction error. RESULTS: The proposed scheme has been applied to the murine C2C12 cell-line stimulated to undergo osteoblast differentiation. Results show that our method discovers genetic interactions that display significant enrichment of co-citation in literature. More detailed study showed that the inferred network exhibits properties and hypotheses that are consistent with current biological knowledge.

Animals↗
PubMed2005

A network of transcriptionally coordinated functional modules in Saccharomyces cerevisiae.

Recent computational and experimental work suggests that functional modules underlie much of cellular physiology and are a useful unit of cellular organization from the perspective of systems biology. Because interactions among modules can give rise to higher-level properties that are essential to cellular function, a complete knowledge of these interactions is necessary for future work in systems biology, including in silico modeling and metabolic engineering. Here we present a computational method for the systematic identification and analysis of functional modules whose activity is coordinated at the level of transcription. We applied this method, Search for Pairwise Interactions (SPIN), to obtain a global view of functional module connectivity in Saccharomyces cerevisiae and to provide insight into the biological mechanisms underlying this coordination. We also examined this global network at higher resolution to obtain detailed information about the interactions of particular module pairs. For instance, our results reveal possible transcriptional coordination of glycolysis and lipid metabolism by the transcription factor Gcr1p, and further suggest that glycolysis and phosphoinositide signaling may regulate each other reciprocally.

Gene Expression Profiling↗
PubMed2006

A three-dimensional model of differentiation of immortalized human bronchial epithelial cells.

A therapeutic approach being investigated for a variety of pathologies is tissue regeneration using a patient's own cells. Such studies have been hampered due to the difficulty in growing epithelial cells for prolonged periods in culture. Replicative senescence due to short telomeres and p16 induced by culture stress work together to inhibit cell growth. Forced expression of telomerase (hTERT) can prevent replicative senescence, and expression of the cell cycle protein cdk4 can sequester p16, thereby immortalizing epithelial cells in culture. In the present study, we used this method to immortalize human bronchial epithelial cells (HBECs) to determine whether immortalized HBECs retain the ability to differentiate normally. HBECs were plated atop contracted collagen gels containing lung fibroblasts. This three-dimensional (3D) tissue model was cultured initially submerged, then raised to the air/liquid interface for up to 28 days. Normal differentiation was assessed by the presence of ciliated cells, goblet (mucin-producing) cells, and basal epithelial cells. Scanning electron microscopic observations revealed both ciliated and non-ciliated cells in these 3D tissues. Histological examination revealed the presence of mucin-producing cells, and immunohistochemistry using antibodies against p63 and keratin 14 showed the presence of basal cells. These results demonstrate that immortalized HBECs retain the capacity to differentiate into each of three cell types: basal, mucin-producing, and columnar ciliated epithelial cells. Such cells will be useful cellular reagents for research in aging, cancer progression, as well as normal bronchial epithelial differentiation and will help progress the use of engineered cells to enhance tissue regeneration.

Biomarkers↗
PubMed2001

Complete in vitro assembly of the reovirus outer capsid produces highly infectious particles suitable for genetic studies of the receptor-binding protein.

Mammalian reoviruses, prototype members of the Reoviridae family of nonenveloped double-stranded RNA viruses, use at least three proteins--sigma1, mu1, and sigma3--to enter host cells. sigma1, a major determinant of cell tropism, mediates viral attachment to cellular receptors. Studies of sigma1 functions in reovirus entry have been restricted by the lack of methodologies to produce infectious virions containing engineered mutations in viral proteins. To mitigate this problem, we produced virion-like particles by "recoating" genome-containing core particles that lacked sigma1, mu1, and sigma3 with recombinant forms of these proteins in vitro. Image reconstructions from cryoelectron micrographs of the recoated particles revealed that they closely resembled native virions in three-dimensional structure, including features attributable to sigma1. The recoated particles bound to and infected cultured cells in a sigma1-dependent manner and were approximately 1 million times as infectious as cores and 0.5 times as infectious as native virions. Experiments with recoated particles containing recombinant sigma1 from either of two different reovirus strains confirmed that differences in cell attachment and infectivity previously observed between those strains are determined by the sigma1 protein. Additional experiments showed that recoated particles containing sigma1 proteins with engineered mutations can be used to analyze the effects of such mutations on the roles of particle-bound sigma1 in infection. The results demonstrate a powerful new system for molecular genetic dissections of sigma1 with respect to its structure, assembly into particles, and roles in entry.

Baculoviridae↗
PubMed2005

Characterization and gene expression profiling of a stable cell line expressing a cell cycle GFP sensor.

The use of stable cell lines expressing fusions with green fluorescent protein (GFP) has increased significantly in recent years. In this study we have used a range of complimentary analytical techniques to examine the characteristics of a cell line stably expressing a EGFP cell cycle sensor relative to parental U2OS cells. Analysis of cell cycle duration and cell cycle phase distribution by cell growth assays and flow cytometry revealed that the two cell lines had identical doubling times and cell cycle distributions. Measurement of EGFP fusion protein mRNA by quantitative RT-PCR indicated a EGFP sensor expression level equivalent to endogenous Cyclin B1 (7000 copies/cell in G2). Microarray analysis showed a 0.9% (>2 fold at p<0.001 across 20,000 genes) difference in global gene expression levels between parental and EGFP expressing U2OS cells, with no significant differences in expression of A, B, C, D, E, F, G, H, I, K, L, M or T type Cyclins between the two cell types. These results confirm that engineering a stable cell line for low expression of a EGFP cell cycle sensor is minimally perturbing to the cell cycle and cellular gene expression.

Cell Cycle↗
PubMed2001

[Cellular compatibility of three natural xenogeneic bone derived biomaterials].

OBJECTIVE: To evaluate the cellular compatibility of three natural xenogeneic bone derived biomaterials. METHODS: Three types of natural xenogeneic bone derived biomaterials were made with physical and chemical treatment, composite fully deproteinized bone(CFDB), partially deproteinized bone(PDPB) and partially decalcified bone(PDCB). Three types biomaterials were cocultured with human embryonic periosteal osteoblasts. The cell growth, attachment, cell cycle, alkaline phosphatase activity were detected to evaluate the cellular compatibility to biomaterials. RESULTS: Osteoblasts attached on all three biomaterials and grew well, the effect of three biomaterials on cell proliferation was PDCB > PDPB > CFDB. The cell cycle was not obviously affected by three biomaterials. The effect of three biomaterials on alkaline phosphatase activity of osteoblasts was PDCB > PDPB > CFDB. CONCLUSION: CFDB,PDPB,PDCB have good cellular compatibility without cytotoxic and tumorigenicity, CFDB is the best. The three biomaterials can be used as scaffold materials of bone tissue engineering.

Bone Substitutes↗
PubMed2001

A comprehensive computerized critical laboratory results alerting system for ambulatory and hospitalized patients.

Automatic alerting systems for key events in medical practice have been proven effective in improving the clinician's response to the event. Most of the systems described in current literature are hospital based, some in intensive care units. We designed a clinical alerting system that screens laboratory results of both hospitalized patients and most of Israel's Southern region ambulatory patients for critical results, using an integrative CDR and its interfaces to the hospital's laboratory information systems. New alerts can be defined and activated using a rule editor. Alerts are generated by screening all new laboratory results that are entered in to the CDR, by a decision engine. The alerts are dispatched to clinicians through a customizable preferred alerting device that can be either e-mail, fax or cellular phone SMS, and enables additional alerting devices as they become available. Clinicians may customize both the alerting method and the critical levels for alert generation, according to individual specific needs. We hope that this versatile and customizable alerting system will suite the needs of both the hospital and the community clinical staff. The system will be implemented and tested in the near future.

Ambulatory Care↗
PubMed1989

[Development of human monoclonal antibodies. Current status and future development].

The "state of the art" in the development of human monoclonal antibodies is reported. The classical cellular fusion method according to well established procedures, the Epstein-Barr virus mediated B-lymphocyte transformation and the modern genetic engineering method to obtain chimeric antibodies is reviewed in detail. Although they were praised in the last five years as the most efficient drugs against cancer and infectious diseases, no great success was clinically and experimentally reported in the past. The many experimental obstacles hindered the efficient development of these promising drugs. In the last two years, however, new ways were proposed to get this new therapeutic principle on a more efficient way in order to use them in the therapeutic treatment of patients with certain cancer and infectious diseases.

Allergy and Immunology↗
PubMed2006

Tissue-engineered temporary wound coverings. Important options for the clinician.

Finding an ideal temporary wound dressing is challenging. Although TransCyte, built on Biobrane, offers many of the characteristics of the ideal wound dressing, and may have added benefits from the delivery of extra-cellular matrix (ECM) and growth factors to the wound, the downside of high cost and less convenient storage and usage are considerable barriers to its broader adoption. The low cost and established clinical utility of Biobrane sets the bar high for new products.

Burns↗
PubMed2005

Ligand-receptor engineering and its application towards the complementation of genetic disease and target identification.

In some instances, small molecules can restore function to proteins that are impaired by genetic mutations. There are now many examples where non-specific molecules or specific ligands can act as chemical chaperones to fold proteins or stabilize folded proteins harboring genetic mutations. In contrast a few recent examples have shown that functionally impaired proteins that are stably folded can be "functionally rescued" by appropriate small molecules. Compounds that can rescue functionally impaired proteins may provide new strategies for the treatment of genetic diseases such as rickets and resistance to thyroid hormone (RTH). In addition mutant-complementing analogs and substrates that act exclusively on mutant proteins are providing important tools for the study of complex biological systems that are controlled by molecules that have multiple cellular targets.

Animals↗
PubMed2005

Bio-orthogonal affinity purification of direct kinase substrates.

Protein phosphorylation is a major mechanism of post-translational protein modification used to control cellular signaling. A challenge in phosphoproteomics is to identify the direct substrates of each protein kinase. Herein, we describe a chemical strategy for delivery of a bio-orthogonal affinity tag to the substrates of an individual protein kinase. The kinase of interest is engineered to transfer a phosphorothioate moiety to phosphoacceptor hydroxyl groups on direct substrates. In a second nonenzymatic step, the introduced phosphorothioate is alkylated with p-nitrobenzylmesylate (PNBM). Antibodies directed against the alkylated phosphorothioate epitope recognize these labeled substrates, but not alkylation products of other cellular nucleophiles. This strategy is demonstrated with Cdk1/cyclinB substrates using ELISA, western blotting, and immunoprecipitation in the context of whole cell lysates.

Adenosine Triphosphate↗