[Approximate calculation of the jet deviation in a memory element with positive reverse connections].
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Biomedical Engineering↗
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Results for “reverse engineering”
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Natural tooth intrusion and reversal in implant-assisted prosthesis: evidence of and a hypothesis for the occurrence.
Based on clinical observation, a hypothesis of the mechanism of intrusion of natural teeth in an implant-assisted prosthesis is suggested. Engineering principles are presented that establish an energy absorption model as it relates to the implant-assisted prosthesis. In addition, in the course of patient treatment it has been discovered that the intrusion of natural teeth can be reversed. Patient histories that demonstrate intrusion reversal are reviewed. The possible mechanisms for the intrusion/reversal phenomenon are presented and preventative recommendations are given.
Aged↗
Genetically engineered live attenuated influenza A virus vaccine candidates.
We have generated new influenza A virus live attenuated vaccine candidates by site-directed mutagenesis and reverse genetics. By mutating specific amino acids in the PB2 polymerase subunit, two temperature-sensitive (ts) attenuated viruses were obtained. Both candidates have 38 degrees C shutoff temperatures in MDCK cells, are attenuated in the respiratory tracts of mice and ferrets, and have very low reactogenicity in ferrets. Infection of mice or ferrets with either mutant conferred significant protection from challenge with the homologous wild-type virus. Three tests for genetic stability were used to assess the propensity for reversion to virulence: 14 days of replication in nude mice, growth at 37 degrees C in tissue culture, and serial passage in ferrets. One candidate, which contains mutations intended to reduce the ability of PB2 to bind to cap structures, was stable in all three assays, whereas the second candidate, which contains mutations found only in other ts strains of influenza virus, lost its ts phenotype in the last two assays. This approach has therefore enabled the creation of live attenuated influenza A virus vaccine candidates suitable for human testing.
Animals↗
Integrated production of human insulin and its C-peptide.
The potential for the development of an integrated process for production of human insulin and its C-peptide in Escherichia coli has been investigated. Human proinsulin was produced intracellularly in E. coli fused to two synthetic IgG-binding domains (ZZ) derived from staphylococcal protein A. High expression levels (3 g/l culture) of the gene product, which accumulated as inclusion bodies, was obtained. Solubilization of inclusion bodies by oxidative sulfitolysis and subsequent renaturation was performed directly after cell lysis and pellet wash. IgG affinity chromatography was used for efficient recovery of pure proinsulin fusion protein in a single step. Monomers of the proinsulin fusion protein constituted approximately 70%. A single step conversion of the fusion protein into insulin and C-peptide by trypsin and carboxypeptidase B treatment was achieved by engineering the junction between proinsulin and its affinity handle, ZZ. Characterization of the cleavage products by reversed phase chromatography (RPC) verified that human insulin and C-peptide were generated and that the ZZ affinity handle was resistant to cleavage. Human insulin and C-peptide were recovered with high yields by preparative reversed-phase high performance liquid chromatography (RP-HPLC). The potential use of the presented scheme for large-scale production of recombinant insulin and/or its C-peptide is discussed.
Biotechnology↗
Molecular cloning of the complementary DNA for an additional member of the family of aortic aneurysm antigenic proteins.
PURPOSE: We have purified and partially sequenced a protein from the adventitia of the human aorta (aortic aneurysm antigenic protein 40 kDa; AAAP-40) that has homologies to bovine aortic microfibril-associated glycoprotein (MAGP-36). It is immunoreactive with immunoglobulin G (IgGs) purified from the serum and aortic wall of patients with abdominal aortic aneurysms. AAAP-40 and MAGP-36 have fibrinogen-like and vitronectin-like motifs. Screening an expression library constructed from human aortic adventitial messenger RNA has resulted in the cloning of three complementary DNAs whose gene products are immunoreactive with immunoglobulin G from patients with abdominal aortic aneurysms. Two strongly resemble each other and have been described separately. The purpose of this article is to report the third clone. METHODS: Messenger RNA from a specimen of human aortic aneurysmal adventitia was reverse-transcribed for insertion into the phagemid Uni Zap XR (Stratagene). A strain of Escherichia coli, engineered for expression (XL 1-Blue MFR', Stratagene), was transfected, and rabbit antihuman vitronectin antibody as used to identify positive clones. Sequencing of the positive clones was performed by the Core Laboratories at Columbia University. RESULTS: The hypothetical protein of rAAAP-CL4 (clone 4) shares sequence motifs with known microfibril-associated glycoproteins (MAGPs). The recombinant protein (rAAAP-CL4) is immunoreactive with serum from patients (three of four abdominal aortic aneurysm sera). In addition, similarities have been detected with immunoglobulins of the kappa family and with a protein from cytomegalovirus that is a potential molecular mimic. CONCLUSIONS: There may several members of a novel family of human aortic autoantigenic proteins implicated in abdominal aortic aneurysm disease.
Amino Acid Sequence↗
Expression and characterization of chimeric rDNA proteins engineered for purification and enzymatic cleavage.
A strategy for the purification and cleavage of chimeric recombinant proteins based on a genetically engineered metal-binding peptide and a human renin cleavage site is described. Vectors were constructed to direct the synthesis of chimeric human immunodeficiency virus (HIV) reverse transcriptase (RT) or beta-galactosidase in Escherichia coli. As shown below, two control chimerics without the metal-binding peptide were also included: 1. Pro-Ile-His-Asp-His-Asp-His-Pro-Phe-His-Leu-Val-Ile-His-Ser-HIV RT 2. Pro-Ile-His-Asp-His-Asp-His-Pro-Phe-His-Leu-Leu-Tyr-Tyr-Ser-HIV RT 3. Pro-Ile-Pro-Phe-His-Leu-Val-Ile-His-Ser-HIV RT 4. Pro-Ile-Pro-Phe-His-Leu-Leu-Tyr-Tyr-Ser-HIV RT 5. Pro-Ile-His-Asp-His-Asp-His-Pro-Phe-His-Leu-beta-galactosidase Both N-terminal sequencing and an enzyme-linked immunosorbent assay utilizing antibodies to the metal-binding peptide were used to characterize the purified chimeric proteins. The relative RT activity of the chimeric protein was indistinguishable from the HIV-1 RT without the fusion sequence, indicating that the metal-binding and renin-cleavage sequences have no effect on the polymerase function of HIV-1 RT. The cleavage by recombinant human renin occurred at the expected site. A future paper will describe results on the use of genetically engineered alternating histidines in the purification of these chimerics by immobilized metal affinity chromatography.
Amino Acid Sequence↗
Inhibition of bovine leukaemia virus replication by the antisense RNA in cell line CC81.
A model system has been developed for quantitative evaluation of bovine leukaemia virus (BLV) replication in a permanent cell line CC81. Transfection of the BLV DNA into these cells evoked typical signs of retroviral infection: formation of syncytia, manifestation of reverse transcriptase activity and appearance of characteristic budding retroviral particles. To inhibit BLV replication, a recombinant plasmid pAGR with an antisense RNA gene targeted at the R-U5 region (147th-342th nt) of the viral genome has been engineered. Cotransfection of CC81 cells with infectious BLV DNA and pAGR led to effective inhibition of BLV replication by the antisense RNA, evidenced by a drop in the number of syncytia and reverse transcriptase activity. Maximal inhibition of BLV replication (95-97%) was observed at a weight ratio of input viral and plasmid DNAs equal to 1:10.
Animals↗
An improved method for the detection of hepatitis C virus RNA in plasma utilizing heminested primers and internal control RNA.
The majority of transfusion-associated, non-A, non-B hepatitis cases are caused by hepatitis C virus (HCV), a positive-stranded RNA virus. Although high titers of HCV in clinical specimens have been reported, in some cases extremely low titers of virus are not uncommon. Therefore, an extremely sensitive and reliable assay is required to determine viremia and replication of HCV accurately. We report here the systematic investigation of factors influencing the detection of HCV RNA by a reverse transcription-polymerase chain reaction (RT-PCR) assay utilizing "drop in-drop out" heminested primers derived from the conserved 5' non-coding region of the viral genome. A genetically engineered 5' noncoding region has been constructed and used as an internal control. Addition of the control RNA to each test not only allowed semiquantitation of positive reactions but also validated the performance of reverse transcription and PCR for every specimen. The optimized heminested PCR (HN-PCR) protocol is capable of amplifying one molecule of cloned HCV DNA or 10 molecules of in vitro-transcribed HCV RNA to levels detectable in ethidium bromide-stained agarose gels. We evaluated the improved method for the detection of HCV RNA on a human plasma sample containing the pedigreed strain H of HCV with a chimpanzee infectious dose of 10(6)/ml. Utilizing the internal control RNA, we calculated 2 x 10(7) virions in 1 ml of the original human plasma. The HN-PCR achieves the sensitivity and specificity of the double-nested PCR (DN-PCR) in a simplified format that avoids the false-positive results associated with DN-PCR.
Base Sequence↗
Aphid symbiotic virus engineered for in vivo expression of insecticidal effectors.
Microbial pesticides are eco-friendly alternatives to chemical pesticides. However, few viral pesticides have been developed. Insects harbor diverse symbiotic viruses, which have the potential to be engineered for translational applications in pest control. Here, we engineered Acyrthosiphon pisum virus (APV), a symbiotic virus of the pea aphid, to deliver anti-aphid effectors using reverse genetics technology. A cytomegalovirus (CMV) promoter-driven APV infectious clone was successfully rescued in pea aphids with the assistance of nanocarrier star polymer (SPc). Based on this infectious clone, the protein coding sequence of chymotrypsin inhibitor variant 8 (Chy8) and the double-stranded RNA sequence targeting the aphid clip-domain serine protease (SPLP) were separately assembled into the APV genome to generate APV-Chy8 and APV-dsSPLP infectious clones, respectively. The recombinant APV clones reduced aphid relative survival rates by 34% and 17% by microinjection, respectively. To enhance the transcriptional efficiency, the APV-Chy8 and APV-dsSPLP clones were transcribed in vitro using the T7 promoter. The in vitro-synthesized APV-Chy8 and APV-dsSPLP clones reduced aphid relative survival rates by 48% and 45% by microinjection, respectively. These results demonstrate that engineered APV can deliver cargos and reduce aphid survival under injection-based experimental conditions, highlighting the potential of symbiotic virus-based vectors for delivering insecticidal effectors.
Animals↗
Identification and isolation of human insulin A and B chains by high-performance liquid chromatography.
A method for the isolation, identification and quantification of human insulin A and B chains by high-performance liquid chromatography (HPLC) is described. These chains were isolated from a peptide mixture produced by E. coli with modified genes obtained by genetic engineering. The method is based on the use of hydrophilic reagents, forming ion pairs in a reversed-phase column. Because some undesirable effects resulting from the use of phosphoric acid were observed, especially with the B chain, a new HPLC method was developed for each of the two human insulin chains. The use of trifluoroacetic acid as a counter ion for the A chain and of formic acid for the B chain led to the rapid isolation and purification of each chain by HPLC. The advantage of this method is that it provides a highly pure product, which was identified by polyacrylamide gel electrophoresis and amino acid analysis.
Amino Acids↗
Small molecule binding to an artificially created cavity at the active site of cytochrome c peroxidase.
In the oxidized "ES" state of cytochrome c peroxidase, Trp-191 is reversibly oxidized to a stable cation free radical by the hypervalent heme. To explore the potential for engineering a binding site for heterocyclic compounds at this site, the mutant W191G was constructed. Two independent crystal structures of W191G at 2.1- and 2.3-A resolution show that W191G contains a well-defined, approximately 180-A3 cavity at the Trp-191 site. The cavity is occupied by five ordered water molecules which participate in an extensive hydrogen-bonding network with each other, with polar main-chain atoms, and with the carboxylate of Asp-235. After a number of heterocyclic compounds were screened, evidence was obtained that substituted imidazoles bind to the cavity of W191G. Titration of W191G with imidazole resulted in a perturbation of the Soret absorption band that was not observed for W191H, W191F, or the native enzyme. The dissociation constants for binding of benzimidazole, imidazole, 2-ethylimidazole, 1-methylimidazole, 2-methylimidazole, and 1,2-dimethylimidazole to W191G were respectively 2.58, 0.70, 0.36, 0.057, 0.047, and 0.027 mM at pH 6.0. The highest binding affinity was exhibited by 1,2-dimethylimidazole, indicating that steric interactions and the efficiency of filling the cavity are important determinants for specificity. The Kd for imidazole binding increased from 0.7 mM at pH 6 to 3.0 mM at pH 8 and could be fit to a single proton ionization curve with a pKa of 7.4, demonstrating the preferential binding by the imidazolium ion (pKa = 7.3). The binding of a number of substituted imidazoles to the cavity of W191G was verified by X-ray crystallographic analysis. The most clearly defined density was observed for W191G crystals soaked in 1 mM 1,2-dimethylimidazole and was consistent with an oriented occupation in which the unsubstituted nitrogen forms a hydrogen bond or ion pair interaction with Asp-235. Thus, enhanced binding of positively charged molecules may be the result of interactions with this carboxylate. An analogous interaction may stabilize the developing positive charge on the Trp-191 radical of the wild-type enzyme. While the oxidation of imidazoles by the ferryl intermediate of W191G was neither expected nor observed, this study has defined the structural determinants for small molecule binding to an artificially created cavity near a heme center which is capable of generating oxidized species at a potential of over 1 V, and these results will guide future attempts for novel substrate oxidation by CCP.
Amino Acid Sequence↗
Disulfide bonds and thermal stability in T4 lysozyme.
Disulfide bonds are thought to serve a stabilizing role in extracellular globular proteins, but little is known about the modes of stabilization or their mechanisms. Thermodynamic data presented here demonstrate that an engineered 3-97 disulfide bond previously shown to stabilize T4 lysozyme in vitro against irreversible thermal inactivation also stabilizes the molecule against reversible thermal unfolding. In this paper, we explore the relationship between the disulfide's thermodynamic contribution to protein folding and its role in providing resistance to irreversible thermal inactivation. In T4 lysozyme (C54V/C97S), a non-crosslinked mutant lacking the two cysteines found in the wild type, sensitivity toward irreversible thermal inactivation increases dramatically at temperatures above the melting temperature of the molecule. In addition, most of the lost activity can be restored by denaturation/renaturation with guanidine hydrochloride. In contrast, the crosslinked mutant T4 lysozyme (13C-97C/C54V) inactivates relatively slowly, even above its melting temperature, and the lost activity is not restored by denaturation/renaturation. These observations suggest that the predominant inactivation pathways for non-crosslinked T4 lysozymes are conformation related, while those for the crosslinked variant are insensitive to the conformational route and thus are susceptible only to slower processes of a chemical nature. We also show that multiple mutants, constructed to contain the 3-97 disulfide plus a temperature-sensitive lesion, are more stable than the wild type to irreversible inactivation even though they are less stable to reversible thermal unfolding. These findings together suggest that the 3-97 disulfide provides stability to irreversible inactivation primarily via a pathway that is independent of its thermodynamic contribution. The 3-97 disulfide may stabilize T4 lysozyme by restricting the unfolded state to a class of more compact structures with less exposed hydrophobic surface, compared to the unfolded states of non-crosslinked T4 lysozymes. The results have implications both for the use of the stabilizing potential of disulfide bonds in protein engineering and for their roles in protein function and evolution.
Disulfides↗
Contribution of a proline residue and a salt bridge to the stability of a type I reverse turn in chymotrypsin inhibitor-2.
The contributions of the components of a type I reverse turn to the stability of chymotrypsin inhibitor-2 (Lys43-Pro44-Gly45) have been determined by protein engineering methods. A double-mutant cycle was used to determine the interaction between Lys43 and Glu45 by replacing them with alanine. We also mutated Pro44, which gives the geometry of the turn, to alanine and analysed the stability of the resulting mutants compared with wild-type chymotrypsin inhibitor-2, using equilibrium denaturation induced by guanidinium chloride. There are decreases in stability (in kcal/mol) of 0.64 +/- 0.06 for Lys43-->Ala, 0.57 +/- 0.15 for Glu45-->Ala, 0.95 +/- 0.06 for Lys43-->Ala/Glu45-->Ala and 1.93 +/- 0.09 for Pro44-->Ala. The free energy of interaction between Lys43 and Glu45 is calculated to be only 0.25 +/- 0.09 kcal/mol. From the changes in denaturation midpoint, Tm measured by circular dichroism, we estimate the energy of interaction between Lys43 and Glu45 to be 0.36 +/- 0.07 kcal/mol whereas the contribution of Pro44 is approximately 2.0 kcal/mol. The contribution of the salt bridge to the stability of the protein is very small and the residue Pro44 plays the key role in stabilizing the turn.
Amino Acid Sequence↗
Introduction of foreign sequences into the genome of influenza A virus.
The ability to apply reverse genetics technologies to influenza virus now allows us to construct novel viruses containing heterologous sequences. We have engineered two neuraminidase (NA) genes of influenza A/WSN/33 virus containing additional sequences which were inserted downstream of the open reading frame of the NA. These NA genes, NA/EMC and NA/EMC-NS1, possess a 546 and a 917 nucleotide (nt) insertion respectively. Transfectant viruses were rescued following ribonucleoprotein (RNP) transfection of the engineered NA genes into influenza helper-virus-infected cells. The transfectant viruses maintained their artificially introduced sequences stably during three passages. The rescued virus containing the NA/EMC-NS1 gene produced one log fewer infectious virus particles in MDBK cells than did wild type A/WSN/33 virus. The growth characteristics in tissue culture of the virus containing the NA/EMC gene was indistinguishable from that of wild-type influenza virus. Based on these results we conclude that influenza A viruses can tolerate heterologous insertions of at least a kilobase in the NA gene.
Animals↗
Infectious transcripts of tick-borne encephalitis virus, generated in days by RT-PCR.
Construction of infectious clones of flaviviruses can be problematic owing to instability, toxicity, and recombination events occurring while cloning cDNA in the bacterial vectors. To overcome these difficulties we have devised a rapid and simple method for producing an infectious genetically engineered tick-borne encephalitis virus in less than 10 days using viral RNA from an unpurified virus suspension. The experimental protocol utilized the high fidelity reverse transcription-polymerase chain reaction to produce two long (5.7 and 5.2 kb) overlapping cDNA segments. To produce full-length cDNA the two overlapping segments were either ligated or fused by polymerase chain reaction. The cDNA was then transcribed and the derived full-length RNA was injected intracerebrally into young mice which reproduced the infectious virus within 8-20 days. To differentiate the engineered virus from parent virus, a Sunl restriction site was introduced by substituting nucleotides at positions 5688 and 5691 of the viral genome. This restriction site was present in the engineered virus recovered from infected mice. Antigenic and electrophoretic analysis of the proteins recovered from the engineered virus confirmed that it was indistinguishable from parent virus. In addition to its applicability as a rapid method of producing infectious engineered virus, this protocol offers the opportunity to introduce changes by site-directed mutagenesis without needing to clone the viral DNA. The method should be applicable to most viruses possessing an infectious RNA molecule and reduces the time required to produce a genetically engineered virus from years to days. When appropriate, the choice of mice for transfection of RNA has the advantage of being extremely simple, very sensitive, and producing high titers of stable virus.
Animals↗
A double mutant of sperm whale myoglobin mimics the structure and function of elephant myoglobin.
The functional, spectral, and structural properties of elephant myoglobin and the L29F/H64Q mutant of sperm whale myoglobin have been compared in detail by conventional kinetic techniques, infrared and resonance Raman spectroscopy, 1H NMR, and x-ray crystallography. There is a striking correspondence between the properties of the naturally occurring elephant protein and those of the sperm whale double mutant, both of which are quite distinct from those of native sperm whale myoglobin and the single H64Q mutant. These results and the recent crystal structure determination by Bisig et al. (Bisig, D. A., Di Iorio, E. E., Diederichs, K., Winterhalter, K. H., and Piontek, K. (1995) J. Biol. Chem. 270, 20754-20762) confirm that a Phe residue is present at position 29 (B10) in elephant myoglobin, and not a Leu residue as is reported in the published amino acid sequence. The single Gln64(E7) substitution lowers oxygen affinity approximately 5-fold and increases the rate of autooxidation 3-fold. These unfavorable effects are reversed by the Phe29(B10) replacement in both elephant myoglobin and the sperm whale double mutant. The latter, genetically engineered protein was originally constructed to be a blood substitute prototype with moderately low O2 affinity, large rate constants, and increased resistance to autooxidation. Thus, the same distal pocket combination that we designed rationally on the basis of proposed mechanisms for ligand binding and autooxidation is also found in nature.
Amino Acid Sequence↗
Chimeric influenza virus induces neutralizing antibodies and cytotoxic T cells against human immunodeficiency virus type 1.
Expression vectors based on DNA or plus-stranded RNA viruses are being developed as vaccine carriers directed against various pathogens. Less is known about the use of negative-stranded RNA viruses, whose genomes have been refractory to direct genetic manipulation. Using a recently described reverse genetics method, we investigated whether influenza virus is able to present antigenic structures from other infectious agents. We engineered a chimeric influenza virus which expresses a 12-amino-acid peptide derived from the V3 loop of gp120 of human immunodeficiency virus type 1 (HIV-1) MN. This peptide was inserted into the loop of antigenic site B of the influenza A/WSN/33 virus hemagglutinin (HA). The resulting chimeric virus was recognized by specific anti-V3 peptide antibodies and a human anti-gp120 monoclonal antibody in both hemagglutination inhibition and neutralization assays. Mice immunized with the chimeric influenza virus produced anti-HIV antibodies which were able to bind to synthetic V3 peptide, to precipitate gp120, and to neutralize MN virus in human T-cell culture system. In addition, the chimeric virus was also capable of inducing cytotoxic T cells which specifically recognize the HIV sequence. These results suggest that influenza virus can be used as an expression vector for inducing both B- and T-cell-mediated immunity against other infectious agents.
Amino Acid Sequence↗
Chemoenzymatic synthesis: application to the study of carbohydrate recognition.
Reported here are useful strategies recently developed for the large-scale synthesis of complex and polyfunctional molecules using native or engineered enzymes as catalysts. Several important issues in the field regarding the problems of substrate specificity, product inhibition, reaction reversibility, enzyme stability and catalytic efficiency are addressed in the representative synthesis of carbohydrates and carbohydrate mimetics designed for use to study carbohydrate-mediated cell adhesion.
Carbohydrates↗