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At least 271 records · Page 15Linked to original sources
PubMed1999

Reovirus virion-like particles obtained by recoating infectious subvirion particles with baculovirus-expressed sigma3 protein: an approach for analyzing sigma3 functions during virus entry.

Structure-function studies with mammalian reoviruses have been limited by the lack of a reverse-genetic system for engineering mutations into the viral genome. To circumvent this limitation in a partial way for the major outer-capsid protein sigma3, we obtained in vitro assembly of large numbers of virion-like particles by binding baculovirus-expressed sigma3 protein to infectious subvirion particles (ISVPs) that lack sigma3. A level of sigma3 binding approaching 100% of that in native virions was routinely achieved. The sigma3 coat in these recoated ISVPs (rcISVPs) appeared very similar to that in virions by electron microscopy and three-dimensional image reconstruction. rcISVPs retained full infectivity in murine L cells, allowing their use to study sigma3 functions in virus entry. Upon infection, rcISVPs behaved identically to virions in showing an extended lag phase prior to exponential growth and in being inhibited from entering cells by either the weak base NH4Cl or the cysteine proteinase inhibitor E-64. rcISVPs also mimicked virions in being incapable of in vitro activation to mediate lysis of erythrocytes and transcription of the viral mRNAs. Last, rcISVPs behaved like virions in showing minor loss of infectivity at 52 degrees C. Since rcISVPs contain virion-like levels of sigma3 but contain outer-capsid protein mu1/mu1C mostly cleaved at the delta-phi junction as in ISVPs, the fact that rcISVPs behaved like virions (and not ISVPs) in all of the assays that we performed suggests that sigma3, and not the delta-phi cleavage of mu1/mu1C, determines the observed differences in behavior between virions and ISVPs. To demonstrate the applicability of rcISVPs for genetic studies of protein functions in reovirus entry (an approach that we call recoating genetics), we used chimeric sigma3 proteins to localize the primary determinants of a strain-dependent difference in sigma3 cleavage rate to a carboxy-terminal region of the ISVP-bound protein.

Animals↗
PubMed1995

Somatic gene transfer of NGF to the aged brain: behavioral and morphological amelioration.

Primary fibroblasts modified to secrete nerve growth factor (NGF) were implanted into the nucleus basalis magnocellularis (NBM) of aged memory impaired rats. The NGF-producing fibroblasts survived for 6 weeks following transplantation and continued expressing NGF mRNA through the duration of the experiment. A significant amelioration of the memory impairment and a significant increase in size and number of low-affinity NGF receptor (p75)-positive neurons in the basal forebrain were observed. Implantation of NGF-producing cells into normal young adult rats resulted in a transient but significant memory impairment and hypertrophy of low-affinity NGF receptor-positive neurons. These results show that naturally occurring age-related memory loss can be reversed by grafting cells engineered to secrete NGF directly to the NBM, and that either cholinergic hyper- or hypofunction may lead to cognitive impairments.

Aging↗
PubMed1997

Can Maryland become the first state to take heart disease out of first place where it has been since the turn of the century?

Heart disease has been the number one health problem in the United States since the turn of the century. It kills more Americans each year than all the American soldiers killed in all of this country's previous wars. Does it have to continue? Can this be reversed with re-engineering of information presently available? The hypothesis postulated here is that if a strategy can be successfully carried out in one state, then other states may be able to follow and pursue other strategies attempting to do the same. To accomplish this on a statewide level it is necessary to have individual hospitals proactively involved with their communities by having a game plan for penetration, identification, activation, and early management of patients with ischemic heart disease. Florida, under the leadership of Henry McIntosh, M.D., has attempted to put this into effect with a complicated strategy. Maryland attempts to logically put into place a chest pain strategy utilizing the principle of keeping it simple for widespread utilization.

Cardiovascular Diseases↗
PubMed2003

Engineered lymphocytes to treat dinitrobenzene sulphonic acid colitis in mice.

Current treatments of inflammatory bowel diseases are limited either by their lack of efficacy or their potential toxicity. In recent years, major advances have been obtained by the development of biological therapies. However, these types of treatment are systemic and can lead to serious adverse events. The new venue of local biological treatments would be most welcome. In this issue of the Journal, Castagliuolo et al. show that lymphocytes engineered to produce TGF-beta1 can reverse dinitrobenzene sulphonic acid-induced colitis in mice. These engineered lymphocytes selectively accumulate in the intestinal mucosa due to the homing properties of their alpha4beta7 integrins, a ligand for MAdCAM1. A local treatment restricted to the inflamed mucosa can thus be obtained. This opens a brand new area of research with the hope of restoring the immunoregulatory balance selectively in the inflamed tissues.

Animals↗
PubMed2004

Identification of the 5' sequences required for incorporation of an engineered ssRNA into the Reovirus genome.

Using a reovirus reverse genetics system, we have identified the 5' sequences required of an engineered s2 ssRNA for efficient incorporation into the dsRNA genome of Reovirus. Employing an engineered, functionally active reovirus S2/CAT gene retaining the first 198 5' terminal nucleotides and the last 284 3' terminal nucleotides of the wild-type S2 segment, we have determined the 5' sequence required by a ssRNA to be recognized, replicated to dsRNA, and stably incorporated into an infectious reovirus. The 5' sequence retains 96 nucleotides of the wild-type s2 ssRNA and a predicted sequence-structure element. Within these 96 nucleotides, we have identified three nucleotides A-U-U at positions 79-81 that are essential for the incorporation of in vitro-generated ssRNAs into new reovirus progeny viral particles. This study establishes a firm foundation for additional investigation into the assortment and encapsidation mechanism of all 10 ssRNAs into the dsRNA genome of reovirus.

5' Flanking Region↗
PubMed2005

The engineering of membrane-permeable peptides.

Reversible lipid attachment was investigated as a means to deliver small peptides into cells. Two labile straight chain alkyl motifs were developed: a cysteine dodecane disulfide (Cdd) building block and a tyrosine- or serine-myristate ester. Both moieties are cleaved on cell internalization and are compatible with Fmoc solid phase peptide synthesis. A series of fluorophore-labeled peptides that varied in lipophilic content, net charge, and charge distribution were synthesized. The peptides were screened for cellular uptake efficiency as monitored by fluorescence microscopy. Effective peptide transport is based on a distributed net positive charge introduced as lysine residues at the C and/or N terminus of the peptide and the presence of a hydrophobic domain exhibiting an estimated log P4.0. The incorporation of labile lipid motifs into peptides enhances lipophilic character of the peptides and contributes to cellular uptake with minimal alteration to the native sequence.

Amino Acid Sequence↗
PubMed1999

Reversal of diabetes in mice by implantation of human fibroblasts genetically engineered to release mature human insulin.

Autoimmune destruction of pancreatic beta cells in type I, insulin-dependent diabetes mellitus (IDDM) results in the loss of endogenous insulin secretion, which is incompletely replaced by exogenous insulin administration. The functional restoration provided by allogeneic beta-cell transplantation is limited by adverse effects of immunosuppression. To pursue an insulin replacement therapy based on autologous, engineered human non-beta cells, we generated a retroviral vector encoding a genetically modified human proinsulin, cleavable to insulin in non-beta cells, and a human nonfunctional cell surface marker. Here we report that this vector efficiently transduced primary human cells, inducing the synthesis of a modified proinsulin that was processed and released as mature insulin. This retrovirally derived insulin displayed in vitro biological activity, specifically binding to and phosphorylation of the insulin receptor, comparable to human insulin. In vivo, the transplantation of insulin-producing fibroblasts reverted hyperglycemia in a murine model of diabetes, whereas proinsulin-producing cells were ineffective. These results support the possibility of developing insulin production machinery in human non-beta cells for gene therapy of IDDM.

Animals↗
PubMed2006

Genetic engineering of a Ca(2+) dependent chemical switch into the linear biomotor kinesin.

Kinesin is a linear motor protein driven by energy released by ATP hydrolysis. In the present work, we genetically installed an M13 peptide sequence into Loop 12 of kinesin, which is one of the major microtubule binding regions of the protein. Because the M13 sequence has high affinity for Ca(2+)-calmodulin, the association of the engineered kinesin with microtubules showed a steep Ca(2+)-dependency in ATPase activity at Ca(2+) concentrations of pCa 6.5-8. The calmodulin-binding domain of plant kinesin-like calmodulin-binding protein is also known to confer Ca(2+)-calmodulin regulation to kinesins. Unlike this plant kinesin, however, our novel engineered kinesin achieves this regulation while maintaining the interaction between kinesin and microtubules. The engineered kinesin is switched on/off reversibly by an external signal (i.e., Ca(2+)-calmodulin) and, thus, can be used as a model system for a bio/nano-actuator.

Amino Acid Sequence↗
PubMed1992

Alteration of enzyme specificity and catalysis.

In the past year, site-directed mutagenesis and other forms of protein engineering have been used to reverse the substrate specificity of several pairs of enzymes, including disulphide oxidoreductases, proteases, sugar-processing enzymes, and nucleases, as well as the specificity of hormones and their receptors. Mutations have been found that affect rate-determining steps, allowing normally transient intermediates to accumulate. Other mutations endow enzymes with totally new chemical reactions, and even novel biological functions. A combination of molecular genetics and chemical modification has been used for protein engineering.

Animals↗
PubMed2000

A thermoacoustic-Stirling heat engine: detailed study

A new type of thermoacoustic engine based on traveling waves and ideally reversible heat transfer is described. Measurements and analysis of its performance are presented. This new engine outperforms previous thermoacoustic engines, which are based on standing waves and intrinsically irreversible heat transfer, by more than 50%. At its most efficient operating point, it delivers 710 W of acoustic power to its resonator with a thermal efficiency of 0.30, corresponding to 41% of the Carnot efficiency. At its most powerful operating point, it delivers 890 W to its resonator with a thermal efficiency of 0.22. The efficiency of this engine can be degraded by two types of acoustic streaming. These are suppressed by appropriate tapering of crucial surfaces in the engine and by using additional nonlinearity to induce an opposing time-averaged pressure difference. Data are presented which show the nearly complete elimination of the streaming convective heat loads. Analysis of these and other irreversibilities show which components of the engine require further research to achieve higher efficiency. Additionally, these data show that the dynamics and acoustic power flows are well understood, but the details of the streaming suppression and associated heat convection are only qualitatively understood.

Journal Article↗
PubMed2000

A novel method for the preparation of large cDNA fragments from dengue-3 RNA genome by long RT-PCR amplification.

For many years, dengue viruses were among the most difficult flaviviruses to isolate and to identify, but technical advances in the past 20 years have facilitated this process. Dengue viruses are usually recovered from specimens by the infection of mosquito-cell cultures. The virus may be passaged several times in cell cultures until a sufficient infectivity titer is attained. The viral nucleocapsid consists of capsid protein and an RNA genome. The dengue genome is a single stranded messenger (positive) sense RNA of approximately 11 kb in length. The isolation of dengue genomic RNA from various sources requires precautions to avoid RNases. RNases are released during cell disruption, and their activity must be inhibited as quickly as possible by using guanidinium thiocyanate in the presence of 2-mercaptoethanol. There has recently been a revolution in molecular biology with the development of the powerful reverse transcriptase (RT) and polymerase chain reaction (PCR) technology. Advanced studies on RT technique lead to much further improvement of the reverse transcriptase enzyme by genetic engineering. The Superscript II RNase H- RT (GIBCO BRL, USA) is genetically engineered DNA polymerase that synthesizes a complementary DNA strand from single-stranded RNA. DNA or an RNA-DNA hybrid. This enzyme is produced from a cloned M-MLV RT gene constructed by the introduction of point mutation in the RNase H active center. The selective mutations within the RNase H domain maintain full polymerase activity. This structural modification eliminates degradation of RNA molecules during the first strand cDNA synthesis. The combination of thermostable DNA polymerase with and without proofreading activity (3'-exonuclease activity), improved buffer conditions and thermal cycling profiles overcome the length limitation of PCR. On the basis of these findings, we have developed a long RT-PCR system for preparing large cDNA fragments of dengue 3 virus (H-87) by using the Superscript II RNase H- RT for reverse transcription and a mixture of Taq and Pwo DNA polymerases for PCR. Three large cDNA fragments covered the full genomic RNA from the 5'-end to the 3'-end of dengue-3 virus (H-87; 10,696 bps) could be successfully prepared as the lengths of 2.437 bps, 3,980 bps and 4,337 bps respectively. The ability of our developed long RT-PCR will bring speed and simplicity to genomic mapping and sequencing and facilitate studies in molecular genetics of dengue viruses.

Aedes↗
PubMed2005

Engineered proteins containing the cohesin and dockerin domains from Clostridium thermocellum provides a reversible, high affinity interaction for biotechnology applications.

The cohesin-dockerin interaction, which is responsible for the formation of the cellulosome complex of cellulolytic bacteria, is a calcium-dependent, high affinity interaction. In this study, the cohesin (Cip7) and dockerin (Doc) domains of Clostridium thermocellum were fused to the cellulose-binding domain (CBD) of C. cellulovorans and the antibody-binding domain, protein LG, respectively, to form CBD-Cip7 and LG-Doc. Immobilised CBD-Cip7 was able to bind LG-Doc and subsequently antibody as determined using surface plasmon resonance. Binding was reversed by the removal of Ca2+ with EDTA. The dockerin containing fusion protein was affinity purified using an immobilised cohesin domain. Elution of the LG-Doc from the cohesin column was with EDTA. This affinity chromatography was repeated using an LG-dockerin column for the purification of cohesin fusion protein. The fusion proteins created in this report have shown that the properties of the cohesin and dockerin domains can be transferred to other protein domains and that the interaction between the cohesin and dockerin is specific, Ca2+ -dependent and reversible. We have shown that the cohesin-dockerin interaction has several properties making it suitable for use in recombinant fusion protein production and purification.

Bacterial Proteins↗
PubMed2003

Evaluation of RNA messengers involved in lipid trafficking of human intestinal cells by reverse-transcription polymerase chain reaction with competimer technology and microchip electrophoresis.

We developed a semiquantitative reverse-transcription polymerase chain reaction (RT-PCR) procedure based on the combination of competimer technology with microchip electrophoresis. The approach was applied to total RNA extracts from the human colon carcinoma cell line CaCo-2 cultured for 22 days onto tissue plate inserts that allow the polarized cell growth. At time of experiment these cells were incubated for 2 h with lipid micelles containing either cholesterol or a mixture cholesterol/beta-sitosterol or serum-free Dulbecco's modified Eagle medium (DMEM) alone. Total RNA was extracted from cell pellets by silica membrane spin columns and reverse-transcribed by a genetically engineered M-MuLV enzyme and an oligo (dT)(15) primer. The cDNA underwent PCR amplification for the following genes: apolipoprotein A1 (ApoAI), apolipoprotein E (ApoE), ATP-binding cassette A1 (ABCA1), liver X receptor alpha (LXRalpha) and farnesoid X receptor (FXR). The beta-actin gene, used as an endogenous reference, was coamplified with mixtures of primer and competimer at a ratio leading to a reference band of intensity comparable to that of the gene under analysis. Amplification products were separated and quantitated by microchip electrophoresis in order to determine, for each gene, the optimum cycle numbers and primer to competimer ratios for the beta-actin therefore used for evaluating the relative variations of gene expressions in the different experiments. The incubation with cholesterol micelles stimulated both LXR and FXR expression that was accompanied by an increased expression of ABCA1 and ApoAI genes (1.4- and 1.5-fold, respectively) and halved the APOE expression. The effect on ABCA1 and ApoAI expression was even stronger (5.7- and 2.6-fold, respectively) with beta-sitosterol-containing micelles. Similar changes are in line with previous findings and suggest that the short incubation with beta-sitosterol-enriched micelles stimulates a specific mechanism that, via ABCA1 activation, can increase the cholesterol and phytosterol basolateral efflux.

Base Sequence↗
PubMed2005

In vivo transduction of HIV-1-derived lentiviral particles engineered for macrolide-adjustable transgene expression.

BACKGROUND: The molecular merger of latest-generation transduction technologies with advanced transgene control modalities may foster decisive advances in therapeutic reprogramming of somatic cell phenotypes. METHODS: We have engineered self-inactivating HIV-1-based lentiviral expression vectors for reversible macrolide-adjustable transgene expression. RESULTS: Lentiviral particles engineered for macrolide-responsive human vascular endothelial growth factor 121 (VEGF121) expression compared favourably with isogenic streptogramin- and tetracycline-responsive configurations and showed excellent growth-factor fine-tuning following transduction into a variety of mammalian cell lines and different human primary cells. Chicken embryos transduced for macrolide-controlled VEGF121 production exhibited dose-dependent neovascularization and exemplified lentivector-delivered transgene transcription fine-tuning in vivo. CONCLUSIONS: Macrolide-adjustable lentivectors enable robust and precise in vitro and in vivo transgene fine-tuning which may give future gene therapy trials a new impetus.

Animals↗
PubMed1994

[Genetic engineering of human insulin. IV. Development and optimization of an analysis system using reversed phase high pressure liquid chromatography].

The effectiveness of the RP HPLC application for the step-by-step analysis of the recombinant insulin production was studied. Properties of a number of commercial and experimental columns in different chromatographic conditions were considered. A three-dimension optimization of selectivity and resolution versus pH and ion strength was carried out. A mechanism of the resolution and selectivity control is suggested.

Chromatography, High Pressure Liquid↗
PubMed2004

FGF6 mediated expansion of a resident subset of cells with SP phenotype in the C2C12 myogenic line.

Fibroblast growth factor 6 (FGF6) is selectively expressed during muscle development and regeneration. We examined its effect on muscle precursor cells (mpc) by forcing stable FGF6 expression in C2C12 cells in vitro. FGF6 produced in genetically engineered mpc was active, inducing strong morphological changes, altering cell adhesion and compromising their ability to differentiate into myotubes. Expression of MyoD and myogenin, but not of Myf5, was abrogated in FGF6 engineered mpc. These effects were reversed by FGF inhibitors. Ectopic expression of MyoD also restored fiber formation indicating that FGF6 interferes with the myogenic differentiation pathway upstream of MyoD. We also report that in the presence of FGF6, the minor (0.5-2%) subpopulation of cells actively excluding Hoechst 33342 in a verapamil-dependent manner (SP phenotype) was increased to 15-20% and the expression of the mdr1a gene (but not mdr1b) was upregulated by 400-fold. Our data establish a previously undescribed link between FGF6--a muscle specific growth factor--and a multidrug resistance gene expressed in stem cells, and suggest a role for FGF6 in the maintenance of a reserve pool of progenitor cells in the skeletal muscle.

ATP Binding Cassette Transporter, Subfamily B↗