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At least 1,603 records · Page 89Linked to original sources
PubMed2001

Recombinant bacille Calmette-Guérin (BCG) expressing human interferon-alpha 2B demonstrates enhanced immunogenicity.

To increase its immunostimulatory properties, BCG was genetically engineered to secrete recombinant human interferon-alpha 2B (rhIFN-alpha) under control of the mycobacterial heat shock protein (hsp)60 promoter and the alpha antigen signal sequence. Expression of rhIFN-alpha was readily detectable by ELISA and on Western blotting. When compared with control BCG, rhIFN-alpha BCG was substantially more active in inducing the production of IFN-gamma and IFN-inducible protein 10 (IP-10) from human peripheral blood mononuclear cells, while IL-10 production was correspondingly decreased. These effects were reversible upon antibody neutralization of rhIFN-alpha. Among 10 patients tested, rhIFN-alpha BCG enhanced IFN-gamma production in all patients ranging from 1.4- to 23.7-fold with a general trend toward greatest enhancement among those with weakest baseline responses to control BCG. Correspondingly, rhIFN-alpha BCG decreased IL-10 production in all patients by 1.2-4.8-fold. The onset of IFN-gamma production induced by rhIFN-alpha BCG was also more rapid, occurring within 4 h after stimulation versus > 24 h with wild-type BCG. The observation that the maximum IFN-gamma induction depends on the simultaneous presence of both IFN-alpha and BCG highlights the advantages of rhIFN-alpha BCG. Taken together, these immunostimulatory properties of rhIFN-alpha BCG suggest that it may be a superior agent for immunotherapeutic protocols involving live BCG in humans.

BCG Vaccine↗
PubMed2002

A method for predicting the performance of packed columns operating with a reactive scrubbing liquid that control gaseous air pollutants.

A method for predicting the performance of packed columns that control gaseous air pollutants has been developed that exploits the advances in both computer software and hardware commonly used by practicing engineers. The solution of the simultaneous partial differential equations that describe the absorption process in packed columns that occurs in the presence of chemical reaction is obtained by converting the partial differential equations to systems of ordinary differential equations. These systems of ordinary differential equations are then solved using the method of lines along with a variable step, variable order numerical method. The method is applicable to systems in which there are multiple reactions within the liquid phase. The reactions can be of any order and can be reversible. The programming is simple and the machine running time is minimal. The method is illustrated here with an example.

Absorption↗
PubMed2006

Control of small inhibitory RNA levels and RNA interference by doxycycline induced activation of a minimal RNA polymerase III promoter.

RNA interference (RNAi) mediated by expression of short hairpin RNAs (shRNAs) is a powerful tool for efficiently suppressing target genes. The approach allows studies of the function of individual genes and may also be applied to human therapy. However, in many instances regulation of RNAi by administration of a small inducer molecule will be required. To date, the development of appropriate regulatory systems has been hampered by the few possibilities for modification within RNA polymerase III promoters capable of driving efficient expression of shRNAs. We have developed an inducible minimal RNA polymerase III promoter that is activated by a novel recombinant transactivator in the presence of doxycycline (Dox). The recombinant transactivator and the engineered promoter together form a system permitting regulation of RNAi by Dox-induced expression of shRNAs. Regulated RNAi was mediated by one single lentiviral vector, blocked the expression of green fluorescent protein (GFP) in a GFP-expressing HEK 293T derived cell line and suppressed endogenous p53 in wild-type HEK 293T, MCF-7 and A549 cells. RNA interference was induced in a dose- and time-dependent manner by administration of Dox, silenced the expression of both target genes by 90% and was in particular reversible after withdrawal of Dox.

Cell Line↗
PubMed1990

Platelet functions in recombinant hirudin-anticoagulated blood.

The influence of genetically engineered recombinant hirudin (r-hirudin) on platelet functions was studied. Depending on the concentration, r-hirudin inhibits the thrombin-induced aggregation and 14C-serotonin secretion to the same extent as native hirudin. Comparative studies in blood anticoagulated by r-hirudin, heparin or citrate show a significantly lower spontaneous platelet aggregation in r-hirudinized blood. The extent of the ADP-induced aggregation is nearly the same in r-hirudinized, heparinized or citrated plasma. In r-hirudinized plasma, however, aggregation is reversible. In contrast to heparinized or citrated plasma, adrenaline causes only a very slight aggregation in r-hirudinized plasma. ADP- or adrenaline-induced secretion of 14C-serotonin do not occur in r-hirudinized plasma. The collagen- as well as the PAF-induced aggregation and 14C-serotonin release in hirudinized plasma do not differ significantly from those in heparinized or citrated plasma. r-Hirudin is a suitable anticoagulant for studying platelet functions because it does not produce any alterations in platelet reactions and does not provoke any changes in the ionized calcium concentration in blood.

Anticoagulants↗
PubMedDate not supplied

Stimulation of growth and migration of vascular endothelial cells by vascular endothelial growth factor transduced smooth muscle cells in co-culture.

Vascular endothelial growth factor (VEGF) is a secreted mitogen with high specificity toward endothelial cells. Expression of VEGF by smooth muscle cells in vivo may be an important stimulus for the regrowth of the endothelium after damage caused by interventions such as angioplasty. The levels of VEGF secreted by cultured smooth muscle cells minimally stimulated growth of endothelial cells in co-culture. Full length cDNA for the 165 amino acid residue, bovine VEGF (VEGF165), was isolated from calf liver total RNA by reverse transcriptase polymerase chain reaction (RT-PCR) techniques, and used to generate plasmid constructs for transfection. Bovine aortic smooth muscle cells (BSMC), stably transfected with VEGF165 plasmid DNA, secreted mitogen into conditioned culture medium at levels that are physiologically relevant (2-4 ng/ml). Transformed BSMC stimulated growth of bovine aortic endothelial cells (BAEC) in co-culture, to a significantly greater extent than mock transfected BSMC. Migration of BAEC was also enhanced by the presence of VEGF transduced BSMC. These data suggest that smooth muscle cells, genetically engineered to produce VEGF, may provide biologic linings in cardiovascular prostheses that could promote the growth of endogenous endothelial cells.

Animals↗
PubMed2002

Thermosensitive sol-gel reversible hydrogels.

Aqueous polymer solutions that are transformed into gels by changes in environmental conditions, such as temperature and pH, thus resulting in in situ hydrogel formation, have recently attracted the attention of many investigators for scientific interest and for practical biomedical or pharmaceutical applications. When the hydrogel is formed under physiological conditions and maintains its integrity for a desired period of time, the process may provide various advantages over conventional hydrogels. Because of the simplicity of pharmaceutical formulation by solution mixing, biocompatibility with biological systems, and convenient administration, the pharmaceutical and biomedical uses of the water-based sol-gel transition include solubilization of low-molecular-weight hydrophobic drugs, controlled release, labile biomacromolecule delivery, such as proteins and genes, cell immobilization, and tissue engineering. When the formed gel is proven to be biocompatible and biodegradable, producing non-toxic degradation products, it will provide further benefits for in vivo applications where degradation is desired. It is timely to summarize the polymeric systems that undergo sol-gel transitions, particularly due to temperature, with emphasis on the underlying transition mechanisms and potential delivery aspects. This review stresses the polymeric systems of natural or modified natural polymers, N-isopropylacrylamide copolymers, poly(ethylene oxide)/poly(propylene oxide) block copolymers, and poly(ethylene glycol)/poly(D,L-lactide-co-glycolide) block copolymers.

Drug Delivery Systems↗
PubMed2005

Molecular breeding of white rot fungus Pleurotus ostreatus by homologous expression of its versatile peroxidase MnP2.

Using a DNA-mediated transformation technique, a molecular breeding approach to isolate Pleurotus ostreatus strains with enhanced productivity of its versatile peroxidase MnP2 was conducted. A recombinant mnp2 construct under the control of P. ostreatus sdi1 expression signals was introduced into the wild-type P. ostreatus strain by cotransformation with a carboxin-resistant marker plasmid. A total of 32 transformants containing the recombinant mnp2 sequence were isolated in a screening with specific amplification by PCR. Productivity of MnP2 in the recombinants was evaluated by the decolorization ability of Poly R-478 on agar plates in the absence of Mn2+. Recombinant P. ostreatus strains with elevated manganese peroxidase (MnP) productivity were successfully isolated. One of the recombinants, TM2-10, was demonstrated to secrete recombinant MnP2 predominantly on a synthetic medium containing 15 mM ammonium oxalate, which was confirmed by reverse transcription PCR (RT-PCR) and isozyme profile analysis using anion-exchange chromatography. The benzo[a]pyrene-removing activity by fungal treatment was also analyzed using the isolated recombinant strains.

Benzopyrenes↗
PubMed2005

MEPE is downregulated as dental pulp stem cells differentiate.

UNLABELLED: Previous studies on dental pulp cell culture have described heterogenous mixtures of cells that differentiate into odontoblasts and form mineralized dentin. OBJECTIVE: The aim of this study was to characterize the matrix extracellular phosphoglycoprotein (MEPE) expression by dental pulp stem cells (DPSC), related to cell differentiation. DESIGN: DPSC differentiation to form mineralized nodules was characterized by Alizarin red staining and micro-Raman spectroscopy. Osteogenesis SuperArray analysis was used to broadly screen for osteogenesis-related genes altered by DPSC differentiation. Relative levels of expression of MEPE and DSP were determined by semiquantitative RT-PCR and Western blot. RESULTS: Mineral analysis showed that as DPSC differentiated, they formed a carbonated hydroxyapatite mineral. Differentiation was initially marked by upregulation by Runx2, TGFbeta-related genes, EGFR and genes involved in collagen metabolism. ALP activity first increased, as DPSCs reached confluence but later decreased when cells further differentiated three weeks after confluence. MEPE was the only marker that was downregulated as DPSCs differentiated. CONCLUSION: DPSC differentiation can be characterized by downregulation of MEPE as other markers of DPSC differentiation, such as DSP, are upregulated. Expression of MEPE related to DSP and can be used to monitor DPSC as they are used for studies of odontoblast differentiation, tissue engineering or vital pulp therapy. The downregulation of MEPE as DPSC differentiate, suggests that MEPE is an inhibitor of mineralization.

Biomarkers↗
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↗
PubMed2005

Mimicking reverse protein splicing by three-segment tandem peptide ligation.

Here we report a bi-directional and interchangeable three-segment peptide ligation of N, M, and C-segments, mimicking the reverse process of protein splicing to form, in tandem, a tripartite NMC-peptide using a synthetic intein, a role served by the M-segment with an N-terminal Ser or Thr and a C-terminal thioester.

Amino Acid Sequence↗
PubMed2000

[Cloning of a full length cDNA of human thrombopoietin receptor c-Mpl and construction of engineered cells that stably express c-mpl].

A full length cDNA fragment encoding for human thrombopoietin receptor c-Mpl has been amplified by RT-PCR from the total RNA of human HEL cells. The complete sequence of the cloned cDNA was determined and is identical to that previously reported. Then the fragment was subcloned into the mammalian expression vector pcDNA3 and the resulting plasmid is designated as pcMPL. K562 cells, which do not express c-mpl, were transfected with pcMPL and pcDNA3, respectively. The transformants were selected with G418 and then tested by Northern and Southern blotting. A group of engineered cell lines stably expressing c-mpl have been obtained, which will facilitate further research on the signaling mediated by c-Mpl.

Cloning, Molecular↗
PubMed2002

Matrix-mediated cellular rejuvenation.

Biomaterial surface morphology and chemistry influence cell responses mediated via signaling cascades that regulate a wide range of metabolic processes. These responses may range from changes in surface adhesion and remodeling of the extracellular matrix to activation of cytokine, cytoskeletal and other biochemical pathways regulating or modulating cellular morphology and function. The present study has focused on collagen Type I, a key extracellular matrix protein, and its potential impact on the process of cellular aging. This study was undertaken for several reasons. First, several investigators reported that growth of cells on a collagen matrix markedly enhanced the resistance of cells to stresses. Second, a large body of accumulated data strongly indicated a relationship between the potential to respond to stresses and cellular aging with the former strongly influencing the rate of the latter. Finally, it has been recently demonstrated that in aged cells one of the key aging-related processes previously considered irreversible, attenuation of the expression of a major stress response protein, Hsp70, can be reversed. This fact together with a probable regulatory role of the stress response potential in cellular aging suggested a possibility that the cellular aging process as a whole can be altered. Indeed, in the present study, growth on a denatured collagen matrices reversed in aged cells not only the attenuation of Hsp70 expression but also other aging-related processes, such as beta-galactosidase expression, increase in protein oxidation and changes in cell morphology. Moreover, it appeared to reduce the rate of aging in young cells. Understanding the nature of collagen matrix-mediated cellular rejuvenation might suggest approaches for interfering with organismic aging. Some immediate applications include cell rejuvenation for purposes of cloning and reduction of the rate of aging during expansion of stem cells for purposes of tissue engineering.

Cell Line↗
PubMed2005

Molecular tailoring of farnesylation for plant drought tolerance and yield protection.

Protecting crop yield under drought stress is a major challenge for modern agriculture. One biotechnological target for improving plant drought tolerance is the genetic manipulation of the stress response to the hormone abscisic acid (ABA). Previous genetic studies have implicated the involvement of the beta-subunit of Arabidopsis farnesyltransferase (ERA1) in the regulation of ABA sensing and drought tolerance. Here we show that molecular manipulation of protein farnesylation in Arabidopsis, through downregulation of either the alpha- or beta-subunit of farnesyltransferase enhances the plant's response to ABA and drought tolerance. To test the effectiveness of tailoring farnesylation in a crop plant, transgenic Brassica napus carrying an ERA1 antisense construct driven by a drought-inducible rd29A promoter was examined. In comparison with the non-transgenic control, transgenic canola showed enhanced ABA sensitivity, as well as significant reduction in stomatal conductance and water transpiration under drought stress conditions. The antisense downregulation of canola farnesyltransferase for drought tolerance is a conditional and reversible process, which depends on the amount of available water in the soil. Furthermore, transgenic plants were more resistant to water deficit-induced seed abortion during flowering. Results from three consecutive years of field trial studies suggest that with adequate water, transgenic canola plants produced the same amount of seed as the parental control. However, under moderate drought stress conditions at flowering, the seed yields of transgenic canola were significantly higher than the control. Using protein farnesyltransferase as an effective target, these results represent a successful demonstration of engineered drought tolerance and yield protection in a crop plant under laboratory and field conditions.

Abscisic Acid↗
PubMed2004

Smart polymer-streptavidin conjugates.

The conjugation of stimuli-responsive, or "smart," polymers to streptavidin is described. The polymer is synthesized with a thiol-reactive end-group, which is used to end-graft the polymer to cysteine or lysine side-chains that are genetically engineered into controlled positions on the streptavidin surface. The conjugation positions are chosen on the basis of their location relative to the binding site, together with the criteria that they be solvent accessible and thus reactive. The polymer composition can be controlled to impart responsiveness to temperature, pH, and/or specific wavelengths of light. These signals are sent to the polymer, which serves as an antennae and actuator to gate biotin or biotinylated protein association with the streptavidin binding sites. The molecular switching and gating activity is directed by the reversible polymer transition between a hydrophilic, expanded coil and a more hydrophobic, collapsed state that is smaller in volume. The differences in the polymer steric properties serve to block or allow ligand access to the binding site. The control of polymer molecular weight is a particularly important design parameter for these molecular gates.

Acrylamides↗
PubMed2006

Increased expression of the 5-HT transporter confers a low-anxiety phenotype linked to decreased 5-HT transmission.

A commonly occurring polymorphic variant of the human 5-hydroxytryptamine (5-HT) transporter (5-HTT) gene that increases 5-HTT expression has been associated with reduced anxiety levels in human volunteer and patient populations. However, it is not known whether this linkage between genotype and anxiety relates to variation in 5-HTT expression and consequent changes in 5-HT transmission. Here we test this hypothesis by measuring the neurochemical and behavioral characteristics of a mouse genetically engineered to overexpress the 5-HTT. Transgenic mice overexpressing the human 5-HTT (h5-HTT) were produced from a 500 kb yeast artificial chromosome construct. These transgenic mice showed the presence of h5-HTT mRNA in the midbrain raphe nuclei, as well as a twofold to threefold increase in 5-HTT binding sites in the raphe nuclei and a range of forebrain regions. The transgenic mice had reduced regional brain whole-tissue levels of 5-HT and, in microdialysis experiments, decreased brain extracellular 5-HT, which reversed on administration of the 5-HTT inhibitor paroxetine. Compared with wild-type mice, the transgenic mice exhibited a low-anxiety phenotype in plus maze and hyponeophagia tests. Furthermore, in the plus maze test, the low-anxiety phenotype of the transgenic mice was reversed by acute administration of paroxetine, suggesting a direct link between the behavior, 5-HTT overexpression, and low extracellular 5-HT. In toto, these findings demonstrate that associations between increased 5-HTT expression and anxiety can be modeled in mice and may be specifically mediated by decreases in 5-HT transmission.

Animals↗
PubMed1989

Genetic engineering of EcoRI mutants with altered amino acid residues in the DNA binding site: physicochemical investigations give evidence for an altered monomer/dimer equilibrium for the Gln144Lys145 and Gln144Lys145Lys200 mutants.

We have genetically engineered the Arg200----Lys mutant, the Glu144Arg145----GlnLys double mutant, and the Glu144Arg145Arg200----GlnLysLys triple mutant of the EcoRI endonuclease in extension of previously published work on site-directed mutagenesis of the EcoRI endonuclease in which Glu144 had been exchanged for Gln and Arg145 for Lys [Wolfes et al. (1986) Nucleic Acids Res. 14, 9063]. All these mutants carry modifications in the DNA binding site. Mutant EcoRI proteins were purified to homogeneity and characterized by physicochemical techniques. All mutants have a very similar secondary structure composition. However, whereas the Lys200 mutant is not impaired in its capacity to form a dimer, the Gln144Lys145 and Gln144Lys145Lys200 mutants have a very much decreased propensity to form a dimer or tetramer depending on concentration as shown by gel filtration and analytical ultracentrifugation. This finding may explain the results of isoelectric focusing experiments which show that these two mutants have a considerably more basic pI than expected for a protein in which an acidic amino acid was replaced by a neutral one. Furthermore, while wild-type EcoRI and the Lys200 mutant are denatured in an irreversible manner upon heating to 60 degrees C, the thermal denaturation process as shown by circular dichroism spectroscopy is fully reversible with the Gln144Lys145 double mutant and the Gln144Lys145Lys200 triple mutant. All EcoRI endonuclease mutants described here have a residual enzymatic activity with wild-type specificity, since Escherichia coli cells overexpressing the mutant proteins can only survive in the presence of EcoRI methylase. The detailed analysis of the enzymatic activity and specificity of the purified mutant proteins is the subject of the accompanying paper [Alves et al. (1989) Biochemistry (following paper in this issue)].

Base Sequence↗
PubMed2003

N-linked glycan structures of mouse interferon-beta produced by Bombyx mori larvae.

The full-length mouse interferon-beta (mIFN-beta) cDNA, including the secretion signal peptide coding region under control of the polyhedrin promoter, was introduced into Bombyx mori nucleopolyhedrovirus (BmNPV). Recombinant mIFN-beta (rmIFN-beta) was accumulated in the haemolymph of infected silkworm larvae. Western blot analysis showed isoforms of rmIFN-beta, suggesting that rmIFN-beta is glycosylated. The glycan structures of purified rmIFN-beta were determined. The N-glycans were liberated by hydrazinolysis and the resulting oligosaccharides were labeled with 2-aminopyridine. The pyridylaminated (PA) glycans were purified by gel filtration, reversed-phase HPLC, and size-fractionation HPLC. The structures of the PA-sugar chains were identified by a combination of two-dimensional PA-sugar chain mapping, MS analysis, and exoglycosidase digestions.

Animals↗
PubMed2006

Primate disrupted-in-schizophrenia-1 (DISC1): high divergence of a gene for major mental illnesses in recent evolutionary history.

Here we analyze the species conservation of disrupted-in-schizophrenia-1 (DISC1) gene, a susceptibility gene for schizophrenia. We cloned cDNA of DISC1 and characterized DISC1 protein in monkey brains and compared their features with those in a variety of species, including humans, rodents and lower vertebrates. Sequences of human and monkey DISC1 are very similar for both nucleotides and amino acids, in sharp contrast to those of rodents; this is reminiscent of G72, another gene involved in major mental illnesses. Bioinformatic cross-species comparisons identified a portion of DISC1 sequences in chicken and Caenorhabditis elegans, but failed to find DISC1 in Drosophila. In contrast to sequence differences, the regional expression profile of DISC1 is well conserved between rodents and primates in that levels of DISC1 mRNA and protein are higher in the hippocampus and the cerebral cortex, and much lower in cerebellum in adult brains. The findings of this study may suggest overall patterns of evolution of genes for psychiatric disorders, and thus assist in production of genetically-engineered mice, and the interpretation of the underlying mechanisms of psychiatric conditions.

Amino Acid Sequence↗