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At least 1,171 records · Page 65Linked to original sources
PubMed2005

Receptor-mediated entry by equine infectious anemia virus utilizes a pH-dependent endocytic pathway.

Previous studies of human and nonhuman primate lentiviral entry mechanisms indicate a predominant use of pH-independent pathways, although more recent studies of human immunodeficiency virus type 1 entry appear to reveal the use of a low-pH-dependent entry pathway in certain target cells. To expand the characterization of the specificity of lentiviral entry mechanisms, we have in the current study examined the entry pathway of equine infectious anemia virus (EIAV) during infection of its natural target, equine macrophages, permissive equine fibroblastic cell lines, and an engineered mouse cell line expressing the recently defined equine lentivirus receptor-1. The specificity of EIAV entry into these various cells was determined by assaying the effects of specific drug treatments on the level of virus entry as measured by quantitative real-time PCR assay of early reverse transcripts or by measurements of virion production. The results of these studies demonstrated that EIAV entry into all cell types was substantially inhibited in a dose-dependent manner by treatment with the vacuolar H+-ATPase inhibitors concanamycin A and bafilomycin A1 or the lysosomotropic weak base ammonium chloride. In contrast, treatments with sucrose to block clathrin-mediated endocytosis or with chloroquine to block organelle acidification failed to inhibit EIAV entry into the same target cells. The observed inhibition of EIAV entry was shown not to be related to cytotoxicity. Taken together, these experiments reveal for the first time that EIAV receptor-mediated entry into target cells is via a low-pH-dependent endocytic pathway.

Ammonium Chloride↗
PubMed2002

Modular engineering of a Group I intron ribozyme.

All Group I intron ribozymes contain a conserved core region consisting of two helical domains, P4-P6 and P3-P7. Recent studies have demonstrated that the elements required for catalysis are concentrated in the P3-P7 domain. We carried out in vitro selection experiments by using three newly constructed libraries on a variant of the T4 td Group I ribozyme containing only a P3-P7 domain in its core. Selected variants with new peripheral elements at L7.1, L8 or L9 after nine cycles efficiently catalyzed the reversal reaction of the first step of self-splicing. The variants from this selection contained a short sequence complementary to the substrate RNA without exception. The most active variant, which was 3-fold more active than the parental wild-type ribozyme, was developed from the second selection by employing a clone from the first selection. The results show that the P3-P7 domain can stand as an independent catalytic module to which a variety of new domains for enhancing the activity of the ribozyme can be added.

Base Sequence↗
PubMed2005

Engineered single-chain dimeric streptavidins with an unexpected strong preference for biotin-4-fluorescein.

Streptavidin, a homotetrameric protein with extremely tight biotin binding (K(d) < or = 10(-14) M), has been widely used as an affinity reagent. Its utility would be increased by engineering single-chain mutants with a wide spectrum of affinities, more suitable for phage-display and chip technologies. By a circular permutation procedure, we converted streptavidin to a single-chain dimer (SCD) with two biotin-binding sites and introduced random mutations by error-prone PCR. Clones from a phagemid library, expressed as gene-3 fusion proteins on M13 bacteriophage, were panned with biotinylated beads, and SCD genes from affinity-enriched phage were subcloned to produce soluble proteins. Purification of products from the original gene and two mutants by FPLC and analysis by MALDI-TOF MS showed they exist in both dimeric (single-chain) and tetrameric (two-chain) forms, which were further characterized for their binding affinity to biotin-4-fluorescein (B4F) by fluorescence polarization and intensity measurements. K'(d) values for B4F ranged from approximately 10(-11) to 10(-10) M, although K(d) values for biotin ranged from 10(-6) to 10(-5) M. These results point to the possibility of combining an SCD streptavidin mutant with B4F derivatives to create a fluorescence-tagged affinity system with tight but still-reversible interaction that could be used sequentially with ordinary streptavidin-biotin for composite separation or analysis steps.

Affinity Labels↗
PubMed2001

Flexible genetic engineering using RecA protein.

With the explosion in genetic information and almost complete sequencing of the human genome, a shift in the experimental goals of molecular biologists is occurring. Instead of focusing on single genes, current attempts seek to divine the interactions of several genes and sequences. This requires increasingly complex genetic constructs and manipulations, often of very large DNA constructs, and these can be made with RecA protein-based techniques. When RecA protein combined with an oligonucleotide acts as a sequence-specific "masking tape" to block DNA from the action of DNA modifying enzymes, and can be used to direct the cleavage of DNA at single predetermined restriction endonuclease sites. This reaction is called RecA-Assisted Restriction Endonuclease (RARE) Cleavage. The reverse reaction, known as RecA-Assisted Ligation, can be used to join any two desired fragments. When one of those fragments is a vector, a desired fragment can be cloned directly without constructing a genomic library. The reagents and equipment needed are relatively inexpensive, and almost any desired genetic construct up to about 300 kb in size can be made in a straightforward manner.

Animals↗
PubMed2006

Osteogenic potential of human periosteum-derived progenitor cells in PLGA scaffold using allogeneic serum.

The use of periosteum-derived progenitor cells (PCs) combined with bioresorbable materials is an attractive approach for tissue engineering. The aim of this study was to characterize the osteogenic differentiation of PC in 3-dimensional (3D) poly-lactic-co-glycolic acid (PLGA) fleeces cultured in medium containing allogeneic human serum. PCs were isolated and expanded in monolayer culture. Expanded cells of passage 3 were seeded into PLGA constructs and cultured in osteogenic medium for a maximum period of 28 d. Morphological, histological and cell viability analyses of three-dimensionally cultured PCs were performed to elucidate osseous synthesis and deposition of a calcified matrix. Furthermore, the mRNA expression of type I collagen, osteocalcin and osteonectin was semi-quantitively evaluated by real-time reverse transcriptase-polymerase chain reaction (RT-PCR). The fibrin gel immobilization technique provided homogeneous PCs distribution in 3D PLGA constructs. Live-dead staining indicated a high viability rate of PCs inside the PLGA scaffolds. Secreted nodules of neo-bone tissue formation and the presence of matrix mineralization were confirmed by positive von Kossa staining. The osteogenic differentiation of PCs was further demonstrated by the detection of type I collagen, osteocalcin and osteonectin gene expression. The results of this study support the concept that this tissue engineering method presents a promising method for creation of new bone in vivo.

Biocompatible Materials↗
PubMed2000

The stress kit: a new method based on competitive reverse transcriptase-polymerase chain reaction to quantify the expression of human alphaB-crystallin, Hsp27, and Hsp60.

We describe a reverse transcriptase-polymerase chain reaction method for the semiquantitative detection of mRNAs encoding the human heat shock proteins alphaB-crystallin, Hsp27, and Hsp60. The method involves the coamplification of cellular mRNA-derived cDNA with a dilution series of a competitor fragment (internal standard), using 1 primer pair common to both templates. Internal standards were based on cellular-derived cDNA engineered to be slightly smaller to differentiate between the target and the standard on electrophoretic separation. Initial cDNA quantitations can be corrected for possible variations during cDNA synthesis by standardizing to the levels of beta-actin-encoding cDNA. We show that the coamplified templates accumulate in a parallel manner with the cellular-derived cDNA throughout both the exponential and the nonexponential phase of amplification. Furthermore, we illustrate the utility of this technique by quantifying increased expression of alphaB-crystallin, Hsp27, and Hsp60 mRNA in astroglioma cells on heat shock.

Astrocytoma↗
PubMed2000

In-vitro cyclic tensile loading of an immobilized and mobilized ligament autograft selectively inhibits mRNA levels for collagenase (MMP-1).

To test the hypothesis that loading conditions can be used to "engineer" ligament autograft behaviors, the effect of cyclic tension on the mRNA levels of matrix molecules and collagenase in in-vivo immobilized and mobilized 6-week rabbit medial collateral ligament (MCL) autografts was examined using an in-vitro system. Femur-[autograft MCL]-tibia complexes were subjected to a tensile stress of 4 MPa at 0.5 Hz for 1 min, followed by 14 min of rest. This 15-min testing cycle was repeated for 4 h. Semi-quantitative reverse transcrip-tase polymerase chain reaction (RT-PCR) was performed on RNA from mechanically treated MCL autografts, using rabbit-specific primer sets for types I and III collagen, biglycan, decorin, fibromodulin, lumican, versican, matrix metalloproteinase-1 (MMP-1, collagenase-1), MMP-13 (collagenase-3), and a housekeeping gene, glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Interestingly, 4 h of culture of normal control MCLs led to increased mRNA levels for MMP-1 (P < 0.05), but there were no significant changes in MMP-13 mRNA levels. Total RNA levels in that normal MCL tissue were, however, decreased after culture (P < 0.05). In-vitro tensile loading of in-vivo mobilized autografts resulted in a significant increase in total RNA (185% of in-vitro non-loaded autografts). On the other hand, in-vitro tensile loading of in-vivo immobilized autografts resulted in no significant changes in total RNA levels compared with levels in non-loaded control grafts. MMP-1 mRNA levels in both the in-vivo mobilized (47% of non-loaded autograft) and in-vivo immobilized (38% of non-loaded autograft) MCL autografts were significantly lower than those in non-loaded control tissue following in-vitro tensile loading, but there were no significant changes in the mRNA levels for the seven other matrix molecules assessed. These results show that it is possible to selectively inhibit MMP-1 mRNA levels in autograft ligaments by supplying mechanical stimuli in vitro. The results also demonstrate that in-vivo immobilization leads to a decrease in the effects of subsequent in-vitro mechanical loading in such autografts with respect to total RNA levels. Collectively, these results demonstrate that both in-vivo and in-vitro loading have implications in the engineering of an ideal ligament graft.

Animals↗
PubMed1990

Peptide building blocks from bacteriorhodopsin: isolation and physicochemical characterization of two individual transmembrane segments.

For protein engineering purposes, transmembrane segments of the structurally stable protein bacteriorhodopsin have been isolated and chemically characterized. Bacteriorhodopsin was cleaved by protease V8 from Staphylococcus aureus to two fragments, V-1 and V-2. The V-2 fragment was separated by gel filtration in organic solvents and purified by reversed-phase FPLC. The fragment has been identified as the C-terminal, partially truncated double-loop of bacteriorhodopsin, including amino acids Val-167-Glu-232/4. Cleavage of V-2 by cyanogen bromide at the single Met-209 yielded two subfragments, which were purified to homogeneity by FPLC procedures. The N-terminal subfragment psi, consisted of a single transmembrane segment (helix F) of bacteriorhodopsin (Val-167-Met(Hse)-209). The C-terminal amphipathic subfragment omega, (Val-210-Glu-232/4) was identified as part of the C-terminal seventh helix of bacteriorhodopsin. Secondary structures of V-2, psi, and omega were investigated in organic solvents and micellar solutions. Native helical structures were partially retained in the solvent systems mentioned.

Amino Acid Sequence↗
PubMed1995

RNA viruses as vectors for the expression of heterologous proteins.

RNA viruses comprise a wide variety of infectious agents, some of which are the cause of disease in humans, animals, and plants. Recombinant DNA technology is now making it feasible to modify these genomes and engineer them to express heterologous proteins. Several different schemes are being employed that depend on the genome organization of the virus and on the strategy of replication of the particular virus. Several different examples are illustrated and potential uses as well as possible problems are discussed. In the future reverse genetics may convert some of these viruses from agents of disease to agents of cure.

Animals↗
PubMed1992

Use of engineered proteins with internal tryptophan reporter groups and pertubation techniques to probe the mechanism of ligand-protein interactions: investigation of the mechanism of calcium binding to calmodulin.

Stopped-flow kinetic and fluorescence spectroscopic analyses, including solvent and temperature perturbations, of five isofunctional structural mutants of calmodulin indicate that calcium binding to calmodulin follows the order site III, site IV, site I, site II, with dissociation occurring in the reverse order. Each of the isofunctional structural mutants contains a single tryptophan residue, introduced by site-specific mutagenesis, as an internal spectroscopic reporter group that was used as a probe of local conformational change. Calcium binding was studied by using flow dialysis or by using fluorescence spectroscopy and monitoring the change in the single tryptophan residue in each calcium-binding site. Calcium removal was examined by using EDTA and monitoring tryptophan fluorescence or by using Quin 2 and monitoring the change in the chromophoric chelator. Computational analysis of the data suggests a rate-limiting step for dissociation between calcium removal from sites I/II and sites III/IV. Unexpected results with the site IV isofunctional mutant (Q135W-CaM) indicated cross-talk between the amino and carboxyl terminal halves of CaM during the calcium-binding mechanism. Studies with ethylene glycol provided empirical data that suggest the functional importance of the electrostatic potential of CaM, or the molarity of water, in the calcium-binding process. Altogether, the data allowed a kinetic extension of the sequential, cooperative model for calcium binding to calmodulin and provided values for additional parameters in the model of calcium binding to CaM, a prototypical member of the family of proteins required for calcium signal transduction in eukaryotic cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed2001

Proof of principle in a de novo designed protein maquette: an allosterically regulated, charge-activated conformational switch in a tetra-alpha-helix bundle.

New understanding of the engineering and allosteric regulation of natural protein conformational switches (such as those that couple chemical and ionic signals, mechanical force, and electro/chemical free energy for biochemical activation, catalysis, and motion) can be derived from simple de novo designed synthetic protein models (maquettes). We demonstrate proof of principle of both reversible switch action and allosteric regulation in a tetra-alpha-helical bundle protein composed of two identical di-helical subunits containing heme coordinated at a specific position close to the disulfide loop region. Individual bundles assume one of two switch states related by large-scale mechanical changes: a syn-topology (helices of the different subunits parallel) or anti-topology (helices antiparallel). Both the spectral properties of a coproporphyrin probe appended to the loop region and the distance-dependent redox interaction between the hemes identify the topologies. Beginning from a syn-topology, introduction of ferric heme in each subunit (either binding or redox change) shifts the topological balance by 25-50-fold (1.9-2.3 kcal/mol) to an anti-dominance. Charge repulsion between the two internal cationic ferric hemes drives the syn- to anti-switch, as demonstrated in two ways. When fixed in the syn-topology, the second ferric heme binding is 25-80-fold (1.9-2.6 kcal/mol) weaker than the first, and adjacent heme redox potentials are split by 80 mV (1.85 kcal/mol), values that energetically match the shift in topological balance. Allosteric and cooperative regulation of the switch by ionic strength exploits the shielded charge interactions between the two hemes and the exposed, cooperative interactions between the coproporphyrin carboxylates.

Allosteric Regulation↗
PubMed1999

Interaction of non-competitive blockers within the gamma-aminobutyric acid type A chloride channel using chemically reactive probes as chemical sensors for cysteine mutants.

Selected channel-lining cysteine mutants from the M2 segment of rat alpha1 gamma-aminobutyric acid (GABA) type A receptor subunit, at positions 257, 261, 264, and 272 were co-expressed with beta1 and gamma2 subunits in Xenopus oocytes. They generated functional receptors displaying conductance and response to both GABA and picrotoxinin similar to the wild type alpha1beta1gamma2 receptor. Three chemically reactive affinity probes derived from non-competitive blockers were synthesized to react with the engineered cysteines: 1) dithiane bis-sulfone derivative modified by an isothiocyanate function (probe A); 2) fiprole derivatives modified by an alpha-chloroketone (probe B) and alpha-bromoketone (probe C) moiety. These probes blocked the GABA-induced currents on all receptors. This blockade could be fully reversed by a washing procedure on the wild type, the alpha1T261Cbeta1gamma2 and alpha1L264Cbeta1gamma2 mutant receptors. In contrast, an irreversible effect was observed for all three probes on both alpha1V257Cbeta1gamma2 and alpha1S272Cbeta1gamma2 mutant receptors. This effect was probe concentration-dependent and could be abolished by picrotoxinin and/or t-butyl bicyclophosphorothionate. These data indicate a major interaction of non-competitive blockers at position 257 of the presumed M2 segment of rat alpha1 subunit but also suggest an interaction at the more extracellular position 272.

Animals↗
PubMed2004

A policy analysis of funding for ambitious interventional gerontology: the possibility of rejuvenation research at the National Institute on Aging.

Issues related to the development of ambitious interventional gerontology-rejuvenation research-at the National Institute on Aging (NIA) within the National Institutes of Health (NIH) are discussed. Creating a separate branch for rejuvenation research within the NIA is probably neither feasible nor desirable at this time. However, it may be both feasible and desirable to begin rejuvenation research by establishing a bioengineering laboratory offering technologies that complement the disease-orientated focus of other NIA intramural laboratories. Such a bioengineering lab could use modern engineering tools for cell and gene therapies to uncover mechanisms of aging and to attempt to repair age-associated pathogenic damage. It is argued that, even in the absence of a full understanding of the complex causes and manifestations of human aging, interventional bioengineering could create methods for reversing aging processes. Exploring technical interventions may both reverse aging processes and significantly advance current disease-specific research.

Aged↗
PubMed1999

Gap junctions regulate responses of tendon cells ex vivo to mechanical loading.

Avian digital flexor tendons were used with a device to apply load ex vivo to study the effects on deoxyribonucleic acid and collagen synthesis when cell to cell communication is blocked. Flexor digitorum profundus tendons from the middle toe of 52-day-old White Leghorn chickens were excised and used as nonloaded controls, or clamped in the jaws of a displacement controlled tissue loading device and mechanically loaded for 3 days at a nominal 0.65% elongation at 1 Hz for 8 hours per day with 16 hours rest. Tendon samples were radiolabeled during the last 16 hours with 3H-thymidine to monitor deoxyribonucleic acid synthesis or with 3H-proline to radiolabel newly synthesized collagen. Cyclic loading of whole avian flexor tendons stimulated deoxyribonucleic acid and collagen synthesis, which could be blocked with octanol, a reversible gap junction blocker. Cells from human digital flexor tendon were used to populate a rectangular, three-dimensional, porous, polyester foam that could be deformed cyclically in vitro. Together, these results support the hypothesis that tendon cells must communicate to sustain growth and matrix expression and that an engineered three-dimensional construct can be used to study responses to mechanical load in vitro.

Animals↗
PubMed1992

Phase I trial of an interleukin-2 (IL-2) fusion toxin (DAB486IL-2) in hematologic malignancies expressing the IL-2 receptor.

DAB486IL-2 is a recombinant fusion toxin in which the native receptor binding domain of diphtheria toxin has been replaced with human interleukin-2 (IL-2). It selectively binds and intoxicates only cells that bear the high-affinity receptor for IL-2. In the first clinical trial of a genetically engineered ligand fusion-toxin, we have treated 18 patients with chemotherapy-resistant IL-2 receptor expressing hematologic malignancies with escalating doses of DAB486IL-2. The maximal tolerated dose of a daily intravenous bolus of DAB486IL-2 was 0.1 mg/kg per day for 10 doses, established by asymptomatic, reversible elevations of hepatic transaminases without changes in other tests of liver function. Other mild reversible side effects noted were rash, nausea, elevated creatinine, chest tightness, and fever. Pharmacokinetic analysis showed a monophasic clearance of 5.8 +/- 0.7 minutes with peak levels of 3,549 +/- 1,041 mg/mL at the 0.1 mg/kg dose. Approximately 50% of patients developed an antibody response to diphtheria toxin or DAB486IL-2. The presence of such antibodies did not preclude patients from experiencing an antitumor response as four of the six patients with antitumor effect had detectable antibody titers. Although this was a phase I trial designed to define the safety of DAB486IL-2, remissions were observed in three patients lasting from 5 to over 18 months. The ability to achieve significant tumor reductions in this group of heavily treated patients is encouraging and suggests additional trials are warranted in hematologic malignancies.

Adult↗
PubMedDate not supplied

Adipose tissue model using three-dimensional cultivation of preadipocytes seeded onto fibrous polymer scaffolds.

A better understanding of the mechanism of adipose tissue differentiation is of paramount importance in the development of therapeutic strategies for the treatment and prevention of obesity and type 2 diabetes mellitus. Optimal results using tissue culture models can be expected only when the in vitro adipocyte resembles adipose tissue in vivo as closely as possible. In this study, we used tissue-engineering principles to develop a three-dimensional (3-D) culture system to mimic the geometry of adipose tissue in vivo. Mouse preadipocyte 3T3-L1 cells were seeded onto nonbiodegradable fibrous polyethylene terephthalate scaffolds and differentiated with a hormone cocktail consisting of insulin, dexamethasone, isobutylmethylxanthine, and fetal calf serum. Cell morphology, growth, differentiation, and function were studied by immunocytochemistry, reverse transcriptase-polymerase chain reaction (RT-PCR), Western blotting, enzyme-linked immunosorbent assay, and oil red O staining. Cells grown on 3-D fibrous scaffolds were differentiated in situ by hormone induction with high efficiency (approximately 90%) as shown by scanning electron microscopy. Immunocytochemistry, immunoblot analysis, and RT-PCR revealed that the 3-D constructs expressed adipocyte-specific genes, including peroxisome proliferator-activated receptor gamma, leptin, adipsin, aP2, adiponectin, GLUT4, and resistin. Adipocytes matured on 3-D constructs secreted leptin at levels even greater than that of fully differentiated adipocytes in 2-D conventional cell cultures. Finally, adipocyte-specific phenotypic function was demonstrated by accumulation of neutral lipids in larger fat droplets. In conclusion, preadipocytes grown on 3-D matrices acquire morphology and biological features of mature adipocytes. This new culture model should have significant utility for in vitro studies of adipocyte cell biology and development.

3T3-L1 Cells↗
PubMed2002

CD4 T-helper cells engineered to produce IL-10 prevent allergen-induced airway hyperreactivity and inflammation.

BACKGROUND: T(H)2 cells play a critical role in the pathogenesis of asthma, but the precise immunologic mechanisms that inhibit T(H)2 cell function in vivo are not well understood. OBJECTIVE: The purpose of our studies was to determine whether T cells producing IL-10 regulate the development of asthma. METHODS: We used gene therapy to generate ovalbumin-specific CD4 T-helper cells to express IL-10, and we examined their capacity to regulate allergen-induced airway hyperreactivity. RESULTS: We demonstrated that the CD4 T-helper cells engineered to express IL-10 abolished airway hyperreactivity and airway eosinophilia in BALB/c mice sensitized and challenged with ovalbumin and in SCID mice reconstituted with ovalbumin-specific T(H)2 effector cells. The inhibitory effect of the IL-10-secreting T-helper cells was accompanied by the presence of increased quantities of IL-10 in the bronchoalveolar lavage fluid, was antigen-specific, and was reversed by neutralization of IL-10. Moreover, neutralization of IL-10 by administration of anti-IL-10 mAb in mice sensitized and challenged with ovalbumin seriously exacerbated airway hyperreactivity and airway inflammation. CONCLUSION: Our results demonstrate that T cells secreting IL-10 in the respiratory mucosa can indeed regulate T(H)2-induced airway hyperreactivity and inflammation, and they strongly suggest that IL-10 plays an important inhibitory role in allergic asthma.

Allergens↗
PubMed1993

Studies on dicistronic polioviruses implicate viral proteinase 2Apro in RNA replication.

A dicistronic poliovirus W1-P1/E/P2,3-1 with the genotype [PV]5'NTR-P1-[EMCV]IRES-[PV]P2,3-3'NTR (Molla, Jang, Paul, Reuer, and Wimmer, 1992, Nature 356, 255) was used to investigate whether the viral proteinase 2Apro, whose primary function in proteolytic processing was erased through the insertion of an internal ribosomal entry site (IRES) element into the ORF of the polyprotein, had other function(s) in viral replication. Deletion of 2Apro from W1-P1/E/P2,3-1 rendered the corresponding transcripts unable to replicate whereas partial deletion of 2Apro or an exchange of Cys109 (an amino acid of the catalytic triad of the proteinase) to Ala reduced RNA replication. No cytopathic effects were observed after transfection with any of the three dicistronic constructs containing mutant 2A, and no virus was recovered after attempts to expand a possibly low yield of mutant virus. In contrast, insertion of the IRES of encephalomyocarditis virus (EMCV) into the ORF of the poliovirus polyprotein at the cleavage site between 2Apro and 2B yielded the novel dicistronic virus W1-P1,2A/E/2BC,P3-1 with the genotype [PV]5'NTR-P1-2A-[EMCV]IRES-[PV]2BC-P3-3'NTR, expressing a small plaque phenotype. These results indicate that neither the intact P2 polypeptide nor the cleavage fragment 2AB of P2 is required for viral proliferation. On the other hand, 2Apro appears to be an essential component in RNA replication as no viral RNA synthesis can be observed by reverse transcription/PCR in cells transfected with dicistronic RNA lacking this viral polypeptide.

Base Sequence↗