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PubMed2006

The 3' sequences required for incorporation of an engineered ssRNA into the Reovirus genome.

BACKGROUND: Understanding how an organism replicates and assembles a multi-segmented genome with fidelity previously measured at 100% presents a model system for exploring questions involving genome assortment and RNA/protein interactions in general. The virus family Reoviridae, containing nine genera and more than 200 members, are unique in that they possess a segmented double-stranded (ds) RNA genome. Using reovirus as a model member of this family, we have developed the only functional reverse genetics system for a member of this family with ten or more genome segments. Using this system, we have previously identified the flanking 5' sequences required by an engineered s2 ssRNA for efficient incorporation into the genome of reovirus. The minimum 5' sequence retains 96 nucleotides and contains 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. The work presented here builds on these findings and presents the results of an analysis of the required 3' flanking sequences of the s2 ssRNA. RESULTS: The minimum 3' sequence we localized retains 98 nucleotides of the wild type s2 ssRNA. These sequences do not interact with the 5' sequences and modifications of the 5' sequences does not result in a change in the sequences required at the 3' end of the engineered s2 ssRNA. Within the 3' sequence we discovered three regions that when mutated prevent the ssRNA from being replicated to dsRNA and subsequently incorporated into progeny virions. Using a series of substitutions we were able to obtain additional information about the sequences in these regions. We demonstrate that the individual nucleotides from, 98 to 84, 68 to 59, and 28 to 1, are required in addition to the total length of 98 nucleotides to direct an engineered reovirus ssRNA to be replicated to dsRNA and incorporated into a progeny virion. Extensive analysis using a number of RNA structure-predication software programs revealed three possible structures predicted to occur in all 10 reovirus ssRNAs but not predicted to contain conserved individual nucleotides that we could probe further by using individual nucleotide substitutions. The presence of a conserved structure would permit all ten ssRNAs to be identified and selected as a set, while unique nucleotides within the structure would direct the set to contain 10 unique members. CONCLUSION: This study completes the characterization and mapping of the 5' and 3' sequences required for an engineered reovirus s2 ssRNA to be incorporated into an infectious progeny virus and establishes a firm foundation for additional investigations into the assortment and encapsidation mechanism of all 10 ssRNAs into the dsRNA genome of reovirus. As researchers build on this work and apply this system to additional reovirus genes and additional dsRNA viruses, a complete model for genome assortment and replication for these viruses will emerge.

Animals↗
PubMed2006

A real-time RT-PCR assay for quantifying the fitness of tobacco etch virus in competition experiments.

Relative fitness determination has become a standard tool in experimental virus evolution studies. In this type of studies, the tested strain is mixed with a reference strain, which differs in an easy-to-score and genetically stable marker, and allowed to compete for a limited common pool of resources during a given number of generations. In this report, a TaqMan real-time PCR methodology is proposed for quantifying the relative fitness of tobacco etch potyvirus strains (TEV) in in planta mixed infections with a reference TEV strain. Two different forward primers along with a common reverse one are used into separated reactions mixes from the same RNA preparation. The reference strain, named TEV-PC1, was genetically engineered to carry a neutral marker in a highly conserved region of the RNA polymerase NIb gene. This marker allows tracking the frequency of both competitors during competition experiments by real-time quantitative PCR using specific primers. Both the reproducibility and sensitivity of the method have been explored. Reproducibility was assessed by running multiple competition experiments for the same genotype. Sensitivity was assessed by comparing the results of competition experiments against TEV-PC1 of 24 single-nucleotide substitutions mutants.

Gene Amplification↗
PubMed1996

Amplification of the full-length hepatitis A virus genome by long reverse transcription-PCR and transcription of infectious RNA directly from the amplicon.

The genetic study of RNA viruses is greatly facilitated by the availability of infectious cDNA clones. However, their construction has often been difficult. While exploring ways to simplify the construction of infectious clones, we have successfully modified and applied the newly described technique of "long PCR" to the synthesis of a full-length DNA amplicon from the RNA of a cytopathogenic mutant (HM 175/24a) of the hepatitis A virus (HAV). Primers were synthesized to match the two extremities of the HAV genome. The antisense primer, homologous to the 3' end, was used in both the reverse transcription (RT) and the PCR steps. With these primers we reproducibly obtained a full-length amplicon of approximately 7.5 kb. Further, since we engineered a T7 promoter in the sense primer, RNA could be transcribed directly from the amplicon with T7 RNA polymerase. Following transfection of cultured fetal rhesus kidney cells with the transcription mixture containing both the HAV cDNA and the transcribed RNA, replicating HAV was detected by immunofluorescence microscopy and, following passage to other cell cultures, by focus formation. The recovered virus displayed the cytopathic effect and large plaque phenotype typical of the original virus; this result highlights the fidelity of the modified long reverse transcription-PCR procedure and demonstrates the potential of this method for providing cDNAs of viral genomes and simplifying the construction of infectious clones.

Animals↗
PubMed2003

Structure-based design and engineering of a nontoxic recombinant pokeweed antiviral protein with potent anti-human immunodeficiency virus activity.

A molecular model of pokeweed antiviral protein (PAP)-RNA interactions was used to rationally engineer FLP-102((151)AA(152)) and FLP-105((191)AA(192)) as nontoxic PAPs with potent anti-human immunodeficiency virus (anti-HIV) activities. FLP-102 and FLP-105 have been produced in Escherichia coli and tested both in vitro and in vivo. These proteins depurinate HIV type 1 (HIV-1) RNA much better than rRNA and are more potent anti-HIV agents than native PAP or recombinant wild-type PAP. They are substantially less toxic than native PAP in BALB/c mice and exhibit potent in vivo activities against genotypically and phenotypically nucleoside reverse transcriptase inhibitor-resistant HIV-1 in a surrogate human peripheral blood lymphocyte (Hu-PBL) SCID mouse model of human AIDS. Rationally engineered nontoxic recombinant PAPs such as FLP-102 and FLP-105 may provide the basis for effective salvage therapies for patients harboring highly drug-resistant strains of HIV-1. The documented in vitro potencies of FLP-102 and FLP-105, their in vivo antiretroviral activities in the HIV-infected Hu-PBL SCID mouse model, and their favorable toxicity profiles in BALB/c mice warrant the further development of these promising new biotherapeutic agents.

Aniline Compounds↗
PubMed2004

The selective serotonin-2A receptor antagonist M100907 reverses behavioral deficits in dopamine transporter knockout mice.

A hyperdopaminergic state in humans has been hypothesized to contribute to the pathology of a number of psychiatric illnesses, including schizophrenia, bipolar disorder, and attention deficit hyperactivity disorder. Mice that display elevated synaptic levels of dopamine due to a genetically engineered deletion of the dopamine transporter (DAT) model behavioral deficits that simulate the above conditions. As novel treatment strategies for these disorders have focused on the serotonin (5-HT) 2A receptor, we determined the capacity of the highly selective 5-HT(2A) receptor antagonist M100907 to reverse behavioral deficits in DAT knockout (KO) mice. Prior to drug treatment, DAT KO mice exhibited increased levels of locomotor activity and highly linearized movement in a novel environment, as well as reduced prepulse inhibition (PPI) of acoustic startle, compared to wild-type littermates. Treatment with M100907 (0.3-1.0 mg/kg, but not 0.1 mg/kg) reversed locomotor deficits in DAT KO mice. Similarly, treatment with 1.0 mg/kg M100907 reversed the PPI deficits in DAT KO mice. These data indicate that selective 5-HT(2A) receptor antagonists, such as M100907, may represent a class of drugs that can be used to treat conditions in which a chronic, elevated dopaminergic tone is present and contributes to abnormal behavior and sensorimotor gating deficits.

Acoustic Stimulation↗
PubMed2002

A reverse genetics approach for recovery of recombinant influenza B viruses entirely from cDNA.

The recovery of recombinant influenza A virus entirely from cDNA was recently described (9, 19). We adapted the technique for engineering influenza B virus and generated a mutant bearing an amino acid change E116G in the viral neuraminidase which was resistant in vitro to the neuraminidase inhibitor zanamivir. The method also facilitates rapid isolation of single-gene reassortants suitable as vaccine seeds and will aid further investigations of unique features of influenza B virus.

Cloning, Molecular↗
PubMed1990

[Direct-action mutagens in exhausts of vehicles with diesel engines].

The genotoxic activity of exhausts from one-shaft gas-turbine GTE-5 engine (30 kW) and a standard D-54A diesel (40 kW) have been studied. Thus, the extracts of soot from GTE-5 and D-54A induced reversions in Salmonella typhimurium both with and without metabolic activation: furthermore, extracts of soot from GTE-5 demonstrated a higher mutagenic activity. The direct mutagenic effect of the exhausts depended neither on the presence of BP nor on the other polycyclic aromatic hydrocarbons (PAHs). Most probably, it was connected with the presence of nitro-PAHs. The need for studying the PAH content in vehicle engines' exhausts and for taking into account their effect in the control and standardization is established.

Benzopyrenes↗
PubMed1992

Vaccines for control of fertility and hormone dependent cancers.

Two vaccines, namely one inducing antibodies against hCG and the other against GnRH, are now in clinical trials. The hCG vaccine has entered Phase II clinical trials in three centres in India after successfully completing Phase I clinical studies in several centres in India and in four countries abroad. The vaccine was found to be devoid of side-effects; its effect was reversible. The available data on 179 cycles indicate that the vaccine prevents pregnancy at antibody titres above 50 ng/ml. A genetically engineered version of the vaccine has also been approved for trials in human lung cancer patients of the type which make hCG. hCG is observed to be a growth factor for such tumours. The GnRH vaccine is usable in both males and females as the deca-peptide is common to both sexes. Following suitable experimental and toxicology studies, the vaccine is currently in Phase I/Phase II clinical trials in patients of prostate carcinoma. Where antibody GnRH antibodies were induced, the LH, FSH and testosterone levels declined. This was accompanied by a reduction in prostate specific antigen. Clinical improvement was observed in many cases. The vaccine has also entered Phase I clinical studies in postpartum women, with the objective to extend the lactational amenorrhoea and extend inter-child interval.

Chorionic Gonadotropin↗
PubMed1999

Antibodies to human ZP3 induce reversible contraception in transgenic mice with 'humanized' zonae pellucidae.

The initial spermatozoon-egg interaction of mammalian fertilization is mediated by the zona pellucida, an extracellular matrix composed of three glycoproteins (ZP1, ZP2, ZP3). These proteins are sufficiently conserved between human and mouse to form chimeric zonae pellucidae, and genetically engineered mice in which the endogenous mouse ZP3 has been replaced by human ZP3 have 'humanized' zonae, but normal fertility. Administration of monoclonal antibodies to mouse ZP3 does not affect fertility in these animals, but administration of antibodies to human ZP3 results in long-term, reversible contraception. The antibodies coat the zonae pellucidae surrounding growing oocytes within the ovary and their presence in the zona matrix inhibits, but does not eliminate, sperm binding. The contraceptive effect is attributed to steric hindrance that decreases sperm binding and prevents penetration through the zona pellucida. The resumption of fertility is associated with the disappearance of antibodies from the zona matrix. No adverse effect on mating behaviour, ovarian histology or fetal development (if administered after fertilization) is detected in treated females. These results suggest that transgenic mice expressing human proteins will prove useful in assessing contraceptive efficacy of zona epitopes in the rational design of immunocontraception directed at the human zona pellucida.

Animals↗
PubMedDate not supplied

The potency of culture-expanded nasal septum chondrocytes for tissue engineering of cartilage.

Tissue engineering techniques to create extra autologous cartilage for reconstructive surgery receive more and more scientific and industrial attention. The objective of this experimental study was to assess the use of in vitro multiplied chondrocytes of the nasal septum for generation of cartilage grafts using tissue engineering techniques. Cells isolated from a biopsy of septal cartilage of rabbits and humans were expanded in culture to get a sufficient number of cells to engineer a cartilage graft. The drawback of the expansion procedure is that the cells lose their cartilaginous phenotype (dedifferentiation). We studied a method to reverse the dedifferentiation of expanded cells to stimulate them to produce cartilage matrix of good quality. Rabbit chondrocytes showed reversion of dedifferentiation (redifferentiation) when fetal calf serum was replaced by the growth factors IGF1 and TGFbeta2. This was expressed by increased glycosaminoglycan synthesis and increased numbers of collagen type II-producing cells. The redifferentiation capacity of septal cartilage cells of young rabbits was higher than that of adult rabbits. In human chondrocytes from the nasal septum redifferentiation could also be induced by replacement of serum with IGF1 and TGFbeta2. This method, however, was less efficient than in rabbits. Chondrocytes of older patients (>40 years old) were no longer sensitive to the growth factor treatment. In conclusion, our study demonstrates a method to regain cartilage phenotype in multiplied cells of nasal septum cartilage needed for tissue engineering of new cartilage. These results are promising for this technique to generate cartilage grafts for facial plastic surgery of the nasal septum.

Adult↗
PubMed2005

A novel mammalian expression system derived from components coordinating nicotine degradation in arthrobacter nicotinovorans pAO1.

We describe the design and detailed characterization of 6-hydroxy-nicotine (6HNic)-adjustable transgene expression (NICE) systems engineered for lentiviral transduction and in vivo modulation of angiogenic responses. Arthrobacter nicotinovorans pAO1 encodes a unique catabolic machinery on its plasmid pAO1, which enables this Gram-positive soil bacterium to use the tobacco alkaloid nicotine as the exclusive carbon source. The 6HNic-responsive repressor-operator (HdnoR-O(NIC)) interaction, controlling 6HNic oxidase production in A.nicotinovorans pAO1, was engineered for generic 6HNic-adjustable transgene expression in mammalian cells. HdnoR fused to different transactivation domains retained its O(NIC)-binding capacity in mammalian cells and reversibly adjusted transgene transcription from chimeric O(NIC)-containing promoters (P(NIC); O(NIC) fused to a minimal eukaryotic promoter [P(min)]) in a 6HNic-responsive manner. The combination of transactivators containing various transactivation domains with promoters differing in the number of operator modules as well as in their relative inter-O(NIC) and/or O(NIC)-P(min) spacing revealed steric constraints influencing overall NICE regulation performance in mammalian cells. Mice implanted with microencapsulated cells engineered for NICE-controlled expression of the human glycoprotein secreted placental alkaline phosphatase (SEAP) showed high SEAP serum levels in the absence of regulating 6HNic. 6HNic was unable to modulate SEAP expression, suggesting that this nicotine derivative exhibits control-incompatible pharmacokinetics in mice. However, chicken embryos transduced with HIV-1-derived self-inactivating lentiviral particles transgenic for NICE-adjustable expression of the human vascular endothelial growth factor 121 (VEGF121) showed graded 6HNic response following administration of different 6HNic concentrations. Owing to the clinically inert and highly water-soluble compound 6HNic, NICE-adjustable transgene control systems may become a welcome alternative to available drug-responsive homologs in basic research, therapeutic cell engineering and biopharmaceutical manufacturing.

Animals↗
PubMedDate not supplied

[Implantation of a mutant of Escherichia coli requiring diaminopimelic acid in the digestive tract of gnotobiotic mice].

A DAP- auxotroph mutant of Escherichia coli DP50 requiring DAP and thymidine for growth was used as the receptor strain in genetic engineering. It failed to be implanted in axenic mice. However, when an inoculum containing more than 10(7) bacteria/ml was used, the DAP+ reverse mutant devoid of requirement for DAP became implanted. When axenic mice were previously associated with Clostridium difficile containing DAP in the cell wall, the strain DAP- became implanted even when the inoculum was too small to permit implantation in axenic mice. Conversely, C. butyricum and C. perenne, whose cell walls also contain DAP, did not allow the establishment of a DAP- mutant. In animals associated with complex human flora without enterobacteria, neither of the 2 DAP- and DAP+ mutants became implanted.

Amino Acids, Diamino↗
PubMed2003

Expression of a bacterial aroA mutant, aroA-M1, encoding 5-enolpyruvylshikimate-3-phosphate synthase for the production of glyphosate-resistant tobacco plants.

Glyphosate is a non-selective broad-spectrum herbicide that inhibits 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), a key enzyme in the aromatic amino acid biosynthetic pathway in microorganisms and plants. We have previously reported a strategy for engineering glyphosate-resistant class I EPSPS based on staggered-PCR technology. Selected mutant enzymes exhibited high K(i)[glyphosate] and low K(m)[PEP] values compared to the parental enzymes from Escherichia coli ( EcaroA) and Salmonella typhimurium ( StaroA). One mutant, aroA-M1, was further engineered with a tobacco chloroplast leader sequence, and then placed in the binary vector pCAMBIA1300 for Agrobacterium-mediated gene transfer to tobacco ( Nicotiana tabacum cv. Xanthi). Transgenic plants with increased resistance to glyphosate were generated.

3-Phosphoshikimate 1-Carboxyvinyltransferase↗
PubMed2006

Use of recombinant calpain-2 siRNA adenovirus to assess calpain-2 modulation of lung endothelial cell migration and proliferation.

In this study, we developed an adenoviral vector harboring calpain-2 siRNA expression unit in which sense and anti-sense strands composing the siRNA duplex were connected by a loop and transcribed into a siRNA in porcine pulmonary artery endothelial cells (PAEC). We screened one efficient adenoviral vector Ad/si-m187 and found that Ad/si-m187 successfully exerted a gene knockdown effect on calpain-2 mRNA transcription and protein expression levels. The protein content of calpain-2 was reduced by 30%-80% in PAEC infected with Ad/si-m187 in comparison to a control adenoviral vector Ad/si-luc. The mRNA levels of calpain-2 were measured by real-time PCR and were decreased by 60%-100% and in a dose dependent manner. In correspondence to silencing calpain-2 gene expression, calpain-2 activity was decreased significantly. We further evaluated the role of calpain-2 in endothelial cell migration and proliferation. PAEC infected with Ad/si-m187 displayed impaired migration and cell proliferation in comparison to cells infected with control adenoviral vector (Ad/si-luc). These results indicate that adenoviral vector harboring calpain-2 siRNA expression unit is a valuable tool to study the biology of calpains and that calpain-2 plays an important role in lung endothelial cell migration and proliferation.

Adenoviridae↗
PubMed1993

Mechanism-based strategies for protein thermostabilization.

Strategies for stabilizing enzymes can be derived from a two-step model of irreversible inactivation that involves preliminary reversible unfolding, followed by an irreversible step. Reversible unfolding is best prevented by covalent immobilization, whereas methods such as covalent modification of amino acid residues or 'medium engineering' (by the addition of low-molecular-weight compounds) are effective against irreversible 'incorrect' refolding. Genetic modification of the protein sequence is the most effective approach for preventing chemical deterioration.

Enzyme Stability↗
PubMed2006

Osteogenic differentiation of adipose-derived stromal cells treated with GDF-5 cultured on a novel three-dimensional sintered microsphere matrix.

BACKGROUND CONTEXT: It is well known that under the proper conditions multipotential bone marrow stromal cells are capable of osteogenic differentiation. Recently studies have demonstrated that an analogous subpopulation of cells exist within adipose tissue. Although early studies characterizing these adipose-derived stromal (ADS) cells in culture exist, investigations exploring the characteristics and viability of these cells cultured on a three-dimensional sintered microsphere matrix are absent. PURPOSE: To characterize and investigate the viability of ADS cells cultured on bioengineered three-dimensional sintered microsphere matrices (SMM). STUDY DESIGN: Basic science, laboratory study. PATIENT SAMPLE: Sixty SMM total. Six underwent examination by scanning electron microscopy, 18 for cellular viability, 18 for biochemical assay, and 18 for evaluation by gene expression. OUTCOME MEASURES: The SMM were examined under scanning electron microscopy to evaluate for adherence, migration, and proliferation at 7, 14, and 28 days. Cellular viability was assessed using colorimetric assay for mitochondrial dehydrogenases activity in viable cells (MTS [3-(4,5-dimethylthiazol-2-yl)5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium] assay) at each corresponding time point. Osteoblastic differentiation was determined using biochemical assays for alkaline phosphatase activity and gene expression for alkaline phosphatase (ALP), osteocalcin (OC), and core binding factor alpha-1 (Cbfa1). METHODS: Multipotential ADS cells from adult Sprague Dawley rats were isolated and maintained in media. Sintered microsphere matrices of poly(lactide-co-glycolide) [85:15] were prepared using solvent evaporation technique followed by mechanical sieving and fabricated by heating in metal molds. ADS cells were then seeded on the SMM and cultured in media with growth and differentiation factor-5 (GDF-5). Treated samples and controls were evaluated at 7, 14, and 28 days. Statistical significance was set at p<.05. RESULTS: Multipotential ADS cells were capable of being isolated from adipose tissue. Scanning electron microscopy evaluation revealed cells adherent to the scaffold surface in a monolayer by 7 days. Cytoplasmic extensions were seen linking the cells on adjacent microspheres. Migration and proliferation resulting in extension of the cellular elements into the scaffold was apparent by 14 days. MTS confirmed cell viability within the scaffold throughout the 28-day study. Osteoblastic differentiation was confirmed using biochemical assays for alkaline phosphatase activity and gene expression for ALP, OC, and Cbfa1. CONCLUSIONS: This is the first study to investigate the fate of ADS seeded on a three-dimensional sintered microsphere matrix. The results of this study confirm that ADS cells, when treated with GDF-5, are not only capable of adhering to the bioengineered scaffold, but also remain viable and demonstrated the ability to migrate, proliferate, and subsequently undergo osteogenic differentiation under the conditions described. These early findings support the concept that ADS cells cultured on a SMM may serve as a viable alternative to more traditional methods of bone graft materials.

Adipose Tissue↗
PubMed2003

Expression of a novel regenerating gene product, Reg IV, by high density fermentation in Pichia pastoris: production, purification, and characterization.

Human regenerating (Reg) gene products are regionally expressed by gut-derived tissues, and are markedly up-regulated in cancer and in diseases characterized by mucosal injury. We recently identified Reg IV, a novel regenerating gene product that is uniquely expressed by the normal distal gastrointestinal mucosa. The function remains poorly understood due to the lack of significant purified Reg IV for biochemical and functional studies. Recombinant human Reg IV was efficiently expressed under the control of the AOX1 gene promoter in Pichia pastoris using the MutS strain KM71H. We describe the unique conditions that are required for efficient production of Reg IV protein in high density fermentation. Optimal protein expression was obtained by reduction of the fermentation temperature and addition of casamino acids as a supplemental nitrogen source and to minimize the activity of yeast produced proteases. Recombinant Reg IV protein was purified by tangential flow filtration and reverse phase chromatography. The purified protein was characterized by amino terminus sequence analysis and MALDI-TOFMS showing that the engineered protein had the expected sequence and molecular weight without secondary modification. Recombinant Reg IV was further characterized by specific monoclonal and polyclonal reagents that function for Western blot analysis and for immunolocalization studies.

Amino Acid Sequence↗