Cloning independent site-directed mutagenesis using total RNA as template.
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Amino Acid Substitution↗
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Results for “reverse engineering”
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[Incipient establishment of human bone marrow mesenchymal stem cells bank].
OBJECTIVE: To investigate the possibility of establishing the human bone marrow mesenchymal stem cells (hMSCs) bank as to provide an alternative source for the seed cells of tissue engineering. METHODS: The cell surface antigens of the purified, expanded hMSCs and the ones following cryopreservation were detected by flow cytometry, cultured in a special medium to induce the osteogenic and chondrocytic differentiation. Morphology was studied by light and electronic microscopes. The detection of alkaline phosphatase, collagen type I, osteocalcin, and collagen type II were also performed by immunochemistry and molecular biology. RESULTS: The phenotype and expansion possibility of hMSCs after cryopreservation were remained. It could expand for 10 generations. The doubling time was 40 h. CONCLUSION: The bank of hMSCs is incipiently established and can provide eligible seed cells for tissue engineering.
Alkaline Phosphatase↗
Relative replicative fitness of zidovudine-resistant human immunodeficiency virus type 1 isolates in vitro.
Replication of mixtures of two or more human immunodeficiency virus type 1 (HIV-1) variants would be expected to result in the eventual selection of the fittest virus due to Darwinian competition among the variants. The relative proportions of known HIV-1 variants (which may differ only by a single nucleotide from a standard "wild-type" virus, HIV-1HXB2) in mixed viral cultures were quantified by analysis of automated sequence signals of reverse transcriptase PCR products. With this method, the relative levels of replicative fitness of several zidovudine (3'-azidothymidine)-resistant HIV-1HXB2 variants were estimated under controlled in vitro conditions by measuring the rate of change in the proportions of viral variants as they replicated in cell cultures both in the presence and in the absence of drug selection pressure. These variants were engineered to contain commonly observed zidovudine resistance mutations in the HIV-1 reverse transcriptase (M41L, K70R, T215Y, and M41L+T215Y). In the absence of zidovudine, all variants tested displayed reduced replicative fitness compared to wild-type HIV-1HXB2. The order of relative fitness was wild type > K70R >> T215Y = M41L+T215Y > M41L. Mixed cultures in the presence of zidovudine showed a dose-dependent selection pressure against the wild-type virus which varied according to the resistance profile of each virus. The information gathered from this approach provides insight into competition among multiple HIV-1 variants, which likely occurs in vivo with drug selection pressure, and may be applicable in more complex mathematical models for predicting the emergence of HIV-1 variants after the initiation of antiretroviral therapy.
Adaptation, Biological↗
Gene expression of single articular chondrocytes.
Although previous studies in the field of tissue engineering have provided important information about articular cartilage, their conclusions are based on population averages and do not account for variations in cell subpopulations. To obtain a precise understanding of chondrocytes, we investigated the effects of cartilage zone and seeding duration on single chondrocyte gene expression to select an optimal zone for tissue engineering (Phase I), followed by an evaluation of growth factor exposure on the zone selected in Phase I (Phase II). In Phase I, superficial and middle/deep bovine articular chondrocytes were seeded in monolayers for 3 or 18 h. In Phase II, middle/deep chondrocytes (selected in Phase I) received 100 ng/ml insulin-like growth factor-I (IGF-I) for 3 h. Real-time reverse transcription/polymerase chain reaction was used to quantify the abundance of D-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and the relative abundances of aggrecan, collagens I and II, cartilage oligomeric matrix protein (COMP), matrix metalloproteinase-1 (MMP-1), and tissue inhibitor of metalloproteinase-1 (TIMP-1). GAPDH varied zonally, but neither time nor IGF-I had an effect on it, suggesting that GAPDH is a suitable housekeeping gene for comparisons within each zone, but not across zones. IGF-I increased the expression of aggrecan and collagen II in middle/deep chondrocytes seeded for 18 h. TIMP-1 expression increased with time in control cells, suggesting that chondrocytes enter a matrix protective state after seeding. IGF-I diminished this effect, suggesting that treatment with IGF-I refocuses chondrocytes on matrix production rather than on protection from metalloproteinases. Concomitant to increasing TIMP-1, MMP-1 was detectable by 18 h in superficial cells, providing further evidence of a trend toward matrix degradation with time. Collagen I was undetected in all cells, and no differences were observed for COMP, confirming that no dedifferentiation or osteoarthritic changes occurred. Taken together, these results establish a unique understanding of individual chondrocyte behavior.
Aggrecans↗
Intrabody construction and expression. I. The critical role of VL domain stability.
We have constructed a panel of hyperstable immunoglobulin VL domains by a rational approach of consensus sequence engineering and combining stabilizing point mutations. These prototype domains unfold fully reversibly, even when the conserved structural disulfide bridge is reduced. This has allowed us to probe the factors that limit the expression yield of soluble immunoglobulin domains in the reducing environment of the cytoplasm (intrabodies). The most important factor is thermodynamic stability, and there is an excellent quantitative correlation between stability and yield. Surprisingly, an unprocessed N-terminal methionine residue can severely compromise VL stability, but this problem can be overcome by changing the amino acid following the initiator methionine residue. Transcription from the strong T7 promoter does not increase the amount of soluble material over that obtained from the tetA promoter, but large amounts of inclusions bodies can be obtained. Elevated temperature shifts protein from a productive folding pathway to aggregation. The structural disulfide bridge does not form in the cytoplasm, but the two consensus cysteine residues can be quantitatively oxidized in vitro. In summary, stability engineering provides a plannable route to the high-yield cytoplasmic expression of functional intrabody domains.
Amino Acid Sequence↗
Tissue engineering of human cartilage in bioreactors using single and composite cell-seeded scaffolds.
Chondrocytes isolated from human fetal epiphyseal cartilage were seeded under mixed conditions into 15-mm-diameter polyglycolic acid (PGA) scaffolds and cultured in recirculation column bioreactors to generate cartilage constructs. After seeding, the cell distributions in thick (4.75 mm) and thin (2.15 mm) PGA disks were nonuniform, with higher cell densities accumulating near the top surfaces. Composite scaffolds were developed by suturing together two thin PGA disks after seeding to manipulate the initial cell distribution before bioreactor culture. The effect of medium flow direction in the bioreactors, including periodic reversal of medium flow, was also investigated. The quality of the tissue-engineered cartilage was assessed after 5 weeks of culture in terms of the tissue wet weight, glycosaminoglycan (GAG), total collagen and collagen type II contents, histological analysis of cell, GAG and collagen distributions, and immunohistochemical analysis of collagen types I and II. Significant enhancement in construct quality was achieved using composite scaffolds compared with single PGA disks. Operation of the bioreactors with periodic medium flow reversal instead of unidirectional flow yielded further improvements in tissue weight and GAG and collagen contents with the composite scaffolds. At harvest, the constructs contained GAG concentrations similar to those measured in ex vivo human adult articular cartilage; however, total collagen and collagen type II levels were substantially lower than those in adult tissue. This study demonstrates that the location of regions of high cell density in the scaffold coupled with application of dynamic bioreactor operating conditions has a significant influence on the quality of tissue-engineered cartilage.
Bioreactors↗
Glucocorticoid-inducible retrovector for regulated transgene expression in genetically engineered bone marrow stromal cells.
Transplantable bone marrow stromal cells can be utilized for cell therapy of mesenchymal disorders. They can also be genetically engineered to express synthetic transgenes and subsequently serve as a platform for systemic delivery of therapeutic proteins in vivo. Inducible production of therapeutic proteins would markedly enhance the usefulness of stromal cells for cell therapy applications. We determined whether synthetic corticosteroid hormones can be used to tightly control transgene expression via the glucocorticoid response pathway in primary bone marrow stromal cells. This regulatory mechanism does not require the presence of potentially immunogenic prokaryotic or chimeric "Trans-activators." Further, synthetic corticosteroids are pharmaceutical agents that can be readily used in vivo. We designed a self-inactivating retroviral vector in which expression of the green fluorescent protein (GFP) reporter is controlled by a minimal synthetic promoter composed of five tandem glucocorticoid response elements upstream of a TATAA box. Vesicular stomatitis virus G-pseudotyped retroparticles were synthesized and utilized to transduce cultured cell lines and primary rat bone marrow stromal cells. We have shown that primary rat bone marrow stromal cells could be efficiently engineered with our GRE-containing retrovector, basal reporter expression was low in the absence of exogenous synthetic corticosteroids, and GFP expression was dexamethasone inducible and reversible. To summarize, this strategy allows dexamethasone-induced, "on-demand" transgene expression from transplantable genetically engineered bone marrow stromal cells.
Animals↗
Engineering enzymes for stability.
There have been many recent developments in elaborating the approaches for stabilizing enzymes by stabilizing the folding state, destabilizing the unfolded state and altering the kinetics of unfolding. However, these represent a series of rules of thumb rather than the reliable principles that would be expected of 'engineering'. Stability is taken to include thermodynamic stability as measured by reversible denaturation, and kinetic stability as measured by the unfolding rate for enzymes that are subject to irreversible denaturation. As such, the factors that affect stability of the folded state versus the unfolded state, as well as factors affecting rates of folding and unfolding, all play a role in maintaining stability. Recent studies on the role of kinetics and the effect that site-specific substitutions have on transition-state free energies add to our understanding of the factors that determine whether or not a particular substitution will result in the measurable stabilization of a protein.
Enzyme Stability↗
Chronic inhibition of cyclic GMP phosphodiesterase 5A prevents and reverses cardiac hypertrophy.
Sustained cardiac pressure overload induces hypertrophy and pathological remodeling, frequently leading to heart failure. Genetically engineered hyperstimulation of guanosine 3',5'-cyclic monophosphate (cGMP) synthesis counters this response. Here, we show that blocking the intrinsic catabolism of cGMP with an oral phosphodiesterase-5A (PDE5A) inhibitor (sildenafil) suppresses chamber and myocyte hypertrophy, and improves in vivo heart function in mice exposed to chronic pressure overload induced by transverse aortic constriction. Sildenafil also reverses pre-established hypertrophy induced by pressure load while restoring chamber function to normal. cGMP catabolism by PDE5A increases in pressure-loaded hearts, leading to activation of cGMP-dependent protein kinase with inhibition of PDE5A. PDE5A inhibition deactivates multiple hypertrophy signaling pathways triggered by pressure load (the calcineurin/NFAT, phosphoinositide-3 kinase (PI3K)/Akt, and ERK1/2 signaling pathways). But it does not suppress hypertrophy induced by overexpression of calcineurin in vitro or Akt in vivo, suggesting upstream targeting of these pathways. PDE5A inhibition may provide a new treatment strategy for cardiac hypertrophy and remodeling.
3',5'-Cyclic-GMP Phosphodiesterases↗
Influenza vaccines generated by reverse genetics.
Influenza viruses cause annual epidemics and occasional pandemics of acute respiratory disease. Vaccination is the primary means to prevent and control the disease. However, influenza viruses undergo continual antigenic variation, which requires the annual reformulation of trivalent influenza vaccines, making influenza unique among pathogens for which vaccines have been developed. The segmented nature of the influenza virus genome allows for the traditional reassortment between two viruses in a coinfected cell. This technique has long been used to generate strains for the preparation of either inactivated or live attenuated influenza vaccines. Recent advancements in reverse genetics techniques now make it possible to generate influenza viruses entirely from cloned plasmid DNA by cotransfection of appropriate cells with 8 or 12 plasmids encoding the influenza virion sense RNA and/or mRNA. Once regulatory issues have been addressed, this technology will enable the routine and rapid generation of strains for either inactivated or live attenuated influenza vaccine. In addition, the technology offers the potential for new vaccine strategies based on the generation of genetically engineered donors attenuated through directed mutation of one or more internal genes. Reverse genetics techniques are also proving to be important for the development of pandemic influenza vaccines, because the technology provides a means to modify genes to remove virulence determinants found in highly pathogenic avian strains. The future of influenza prevention and control lies in the application of this powerful technology for the generation of safe and more effective influenza vaccines.
Animals↗
Three-dimensional nanohydroxyapatite/chitosan scaffolds as potential tissue engineered periodontal tissue.
The development of suitable three-dimensional scaffold for the maintenance of cellular viability and differentiation is critical for applications in periodontal tissue engineering. In this work, different ratios of porous nanohydroxyapatite/chitosan (HA/chitosan) scaffolds are prepared through a freeze-drying process. These scaffolds are evaluated in vitro by the analysis of microscopic structure, porosity, and cytocompatibility. The expression of type I collagen and alkaline phosphatase (ALP) activity are detected with real-time polymerase chain reaction (RT-PCR). Human periodontal ligament cells (HPLCs) transfected with enhanced green fluorescence protein (EGFP) are seeded onto the scaffolds, and then these scaffolds are implanted subcutaneously into athymic mice. The results indicated that the porosity and pore diameter of the HA/chitosan scaffolds are lower than those of pure chitosan scaffold. The HA/chitosan scaffold containing 1% HA exhibited better cytocompatibility than the pure chitosan scaffold. The expression of type I collagen and ALP are up-regulated in 1% HA/chitosan scaffold. After implanted in vivo, EGFP-transfected HPLCs not only proliferate but also recruit surrounding tissue to grow in the scaffold. The degradation of the scaffold significantly decreased in the presence of HA. This study demonstrated the potential of HA/ chitosan scaffold as a good substrate candidate in periodontal tissue engineering.
Alkaline Phosphatase↗
Preparation and characterization of a chimeric zebrafish-human neuroglobin engineered by module substitution.
Neuroglobin (Ngb) is a recently discovered vertebrate heme protein that can reversibly bind oxygen that is expressed in the brain. Zebrafish and human Ngb share about 50% amino acid sequence identity. These Ngb proteins consist of four compact protein structural unit "modules" referred to as M1-M4. In the present study, we investigated the effects of module substitution on the properties of Ngb. Specifically, we prepared and characterized a chimeric ZHZZ Ngb in which the heme-binding module M2 of zebrafish Ngb was replaced by the comparable human Ngb module. Our results showed that the chimeric ZHZZ was stable and formed almost the identical heme-environmental and alpha-helical structure as the human and zebrafish Ngb proteins, suggesting that the structure of Ngb has been evolutionarily conserved.
Amino Acid Sequence↗
[Plant lectin and its application in insect-resistant plant genetic engineering].
Plant lectins are proteins possessing at least one non-catalytic domain that binds reversibly to specific mono-or oligosaccharides. They distinguish themselves from other plant proteins by the ability of carbohydrate binding. Most plant lectins are directed to bind foreign polysaccharide. Plant lectin is believed to take part in the defense responses against invader. In this paper we presented a review on the classification, characters, functions, crystal structure and,functions related to the carbohydrate binding activity. The status and prospect of plant lectins utilization were also discussed.
English Abstract↗
RNase H activity is required for high-frequency repeat deletion during Moloney murine leukemia virus replication.
It has been postulated that retroviral recombination, like strong stop template switching, requires the RNase H activity of reverse transcriptase. To address this hypothesis, Moloney murine leukemia virus-based vectors, which were designed to test the recombination-related property of direct repeat deletion, were encapsidated in virions engineered to contain phenotypic mixtures of wild-type and RNase H catalytic site point mutant reverse transcriptase. Integrated provirus titers per milliliter were determined for these phenotypically mixed virions, and vector proviruses were screened to determine what percentage contained repeat deletions. The results revealed a steady decline in direct repeat deletion frequency that correlated with decreases in functional RNase H, with greater than fourfold decreases in repeat deletion frequency observed when 95% of virion reverse transcriptase was RNase H defective. Parallel experiments were performed to address effects of molar excesses of RNase H relative to functional DNA polymerase. These experiments demonstrated that increasing the stoichiometry of RNase H relative to the amount of functional DNA polymerase had minimal effects on direct repeat deletion frequency. DNA synthesis was error prone when directed principally by RNase H mutant reverse transcriptase, suggesting a role for RNase H catalytic integrity in the fidelity of intracellular reverse transcription.
3T3 Cells↗
Redesign of carnitine acetyltransferase specificity by protein engineering.
In eukaryotes, L-carnitine is involved in energy metabolism by facilitating beta-oxidation of fatty acids. Carnitine acetyltransferases (CrAT) catalyze the reversible conversion of acetyl-CoA and carnitine to acetylcarnitine and free CoA. To redesign the specificity of rat CrAT toward its substrates, we mutated Met564. The M564G mutated CrAT showed higher activity toward longer chain acyl-CoAs: activity toward myristoyl-CoA was 1250-fold higher than that of the wild-type CrAT, and lower activity toward its natural substrate, acetyl-CoA. Kinetic constants of the mutant CrAT showed modification in favor of longer acyl-CoAs as substrates. In the reverse case, mutation of the orthologous glycine (Gly553) to methionine in carnitine octanoyltransferase (COT) decreased activity toward its natural substrates, medium- and long-chain acyl-CoAs, and increased activity toward short-chain acyl-CoAs. Another CrAT mutant, M564A, was prepared and tested in the same way, with similar results. We conclude that Met564 blocks the entry of medium- and long-chain acyl-CoAs to the catalytic site of CrAT. Three-dimensional models of wild-type and mutated CrAT and COT support this hypothesis. We show for the first time that a single amino acid is able to determine the substrate specificity of CrAT and COT.
Acetyl Coenzyme A↗
Gene expression characteristic in human auricular cartilage tissue engineering.
This study was to assess collagen type II and collagen type I gene expression in tissue-engineered human auricular: cartilage formed via tissue engineering technique. Large-scale culture expansions were transformed into 3D in vitro construct and were implanted subcutaneously on the dorsal of athymic mice. After 8 weeks, explanted construct was processed in the same manner of native cartilage to facilitate cells for gene expression analysis. Isolated cells from in vivo construct demonstrated expression of type II collagen gene comparable to native cartilage. This study verified that tissue-engineered auricular cartilage expressed cartilage specific gene, collagen type II after in vivo maturation.
Actins↗
Adipose tissue-derived mesenchymal stem cell yield and growth characteristics are affected by the tissue-harvesting procedure.
BACKGROUND: Adipose tissue contains a stromal vascular fraction that can be easily isolated and provides a rich source of adipose tissue-derived mesenchymal stem cells (ASC). These ASC are a potential source of cells for tissue engineering. We studied whether the yield and growth characteristics of ASC were affected by the type of surgical procedure used for adipose tissue harvesting, i.e. resection, tumescent liposuction and ultrasound-assisted liposuction. METHODS: Frequencies of ASC in the stromal vascular fraction were assessed in limiting dilution assays. The phenotypical marker profile of ASC was determined, using flow cytometry, and growth kinetics were investigated in culture. ASC were cultured under chondrogenic and osteogenic conditions to confirm their differentiation potential. RESULTS: The number of viable cells in the stromal vascular fraction was affected by neither the type of surgical procedure nor the anatomical site of the body from where the adipose tissue was harvested. After all three surgical procedures, cultured ASC did express a CD34+ CD31- CD105+ CD166+ CD45- CD90+ ASC phenotype. However, ultrasound-assisted liposuction resulted in a lower frequency of proliferating ASC, as well as a longer population doubling time of ASC, compared with resection. ASC demonstrated chondrogenic and osteogenic differentiation potential. DISCUSSION: We conclude that yield and growth characteristics of ASC are affected by the type of surgical procedure used for adipose tissue harvesting. Resection and tumescent liposuction seem to be preferable above ultrasound-assisted liposuction for tissue-engineering purposes.
Adipocytes↗
Considerations on the use of ear chondrocytes as donor chondrocytes for cartilage tissue engineering.
Articular cartilage is often used for research on cartilage tissue engineering. However, ear cartilage is easier to harvest, with less donor-site morbidity. The aim of this study was to evaluate whether adult human ear chondrocytes were capable of producing cartilage after expansion in monolayer culture. Cell yield per gram of cartilage was twice as high for ear than for articular cartilage. Moreover, ear chondrocytes proliferated faster. Cell proliferation could be further stimulated by the use of serum-free medium with Fibroblast Growth Factor 2 (FGF2) in stead of medium with 10% serum. To evaluate chondrogenic capacity, multiplied chondrocytes were suspended in alginate and implanted subcutaneously in athymic mice. After 8 weeks the constructs demonstrated a proteoglycan-rich matrix that contained collagen type II. Constructs of ear chondrocytes showed a faint staining for elastin. Quantitative RT-PCR revealed that expression of collagen type II was 2-fold upregulated whereas expression of collagen type I was 2-fold down regulated in ear chondrocytes expanded in serum-free medium with FGF2 compared to serum-containing medium. Expression of alkaline phosphatase and collagen type X were low indicating the absence of terminal differentiation. We conclude that ear chondrocytes can be used as donor chondrocytes for cartilage tissue engineering. Furthermore, it may proof to be a promising alternative cell source to engineer cartilage for articular repair.
Adult↗