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PubMed2003

Synthetic matrix metalloproteinase-sensitive hydrogels for the conduction of tissue regeneration: engineering cell-invasion characteristics.

Synthetic hydrogels have been molecularly engineered to mimic the invasive characteristics of native provisional extracellular matrices: a combination of integrin-binding sites and substrates for matrix metalloproteinases (MMP) was required to render the networks degradable and invasive by cells via cell-secreted MMPs. Degradation of gels was engineered starting from a characterization of the degradation kinetics (k(cat) and K(m)) of synthetic MMP substrates in the soluble form and after crosslinking into a 3D hydrogel network. Primary human fibroblasts were demonstrated to proteolytically invade these networks, a process that depended on MMP substrate activity, adhesion ligand concentration, and network crosslinking density. Gels used to deliver recombinant human bone morphogenetic protein-2 to the site of critical defects in rat cranium were completely infiltrated by cells and remodeled into bony tissue within 4 wk at a dose of 5 microg per defect. Bone regeneration was also shown to depend on the proteolytic sensitivity of the matrices. These hydrogels may be useful in tissue engineering and cell biology as alternatives for naturally occurring extracellular matrix-derived materials such as fibrin or collagen.

Animals↗
PubMed2000

An efficient recombination system for chromosome engineering in Escherichia coli.

A recombination system has been developed for efficient chromosome engineering in Escherichia coli by using electroporated linear DNA. A defective lambda prophage supplies functions that protect and recombine an electroporated linear DNA substrate in the bacterial cell. The use of recombination eliminates the requirement for standard cloning as all novel joints are engineered by chemical synthesis in vitro and the linear DNA is efficiently recombined into place in vivo. The technology and manipulations required are simple and straightforward. A temperature-dependent repressor tightly controls prophage expression, and, thus, recombination functions can be transiently supplied by shifting cultures to 42 degrees C for 15 min. The efficient prophage recombination system does not require host RecA function and depends primarily on Exo, Beta, and Gam functions expressed from the defective lambda prophage. The defective prophage can be moved to other strains and can be easily removed from any strain. Gene disruptions and modifications of both the bacterial chromosome and bacterial plasmids are possible. This system will be especially useful for the engineering of large bacterial plasmids such as those from bacterial artificial chromosome libraries.

Bacteriophage lambda↗
PubMed1995

Genetic engineering of vein grafts resistant to atherosclerosis.

Previously, researchers have speculated that genetic engineering can improve the long-term function of vascular grafts which are prone to atherosclerosis and occlusion. In this study, we demonstrated that an intraoperative gene therapy approach using antisense oligodeoxynucleotide blockage of medial smooth muscle cell proliferation can prevent the accelerated atherosclerosis that is responsible for autologous vein graft failure. Selective blockade of the expression of genes for two cell cycle regulatory proteins, proliferating cell nuclear antigen and cell division cycle 2 kinase, was achieved in the smooth muscle cells of rabbit jugular veins grafted into the carotid arteries. This alteration of gene expression successfully redirected vein graft biology away from neointimal hyperplasia and toward medial hypertrophy, yielding conduits that more closely resembled normal arteries. More importantly, these genetically engineered grafts proved resistant to diet-induced atherosclerosis. These findings establish the feasibility of developing genetically engineered bioprostheses that are resistant to failure and better suited to the long-term treatment of occlusive vascular disease.

Animals↗
PubMed1995

Scorpion toxins as natural scaffolds for protein engineering.

A compact, well-organized, and natural motif, stabilized by three disulfide bonds, is proposed as a basic scaffold for protein engineering. This motif contains 37 amino acids only and is formed by a short helix on one face and an antiparallel triple-stranded beta-sheet on the opposite face. It has been adopted by scorpions as a unique scaffold to express a wide variety of powerful toxic ligands with tuned specificity for different ion channels. We further tested the potential of this fold by engineering a metal binding site on it, taking the carbonic anhydrase site as a model. By chemical synthesis we introduced nine residues, including three histidines, as compared to the original amino acid sequence of the natural charybdotoxin and found that the new protein maintains the original fold, as revealed by CD and 1H NMR analysis. Cu2+ ions are bound with Kd = 4.2 x 10(-8) M and other metals are bound with affinities in an order mirroring that observed in carbonic anhydrase. The alpha/beta scorpion motif, small in size, easily amenable to chemical synthesis, highly stable, and tolerant for sequence mutations represents, therefore, an appropriate scaffold onto which polypeptide sequences may be introduced in a predetermined conformation, providing an additional means for design and engineering of small proteins.

Amino Acid Sequence↗
PubMed1995

Comparison of genetically engineered herpes simplex viruses for the treatment of brain tumors in a scid mouse model of human malignant glioma.

Genetically engineered viruses and viral genes inserted into retroviral vectors are increasingly being considered for experimental therapy of brain tumors. A primary target of these viruses and vectors is human gliomas, the most frequently occurring primary human brain tumor. To investigate the potential of genetically engineered herpes simplex viruses (HSVs) in the therapy of these tumors, we compared the attributes of two viruses, a recombinant from which the gamma 1(34.5) gene had been deleted (R3616) and a recombinant in which the gamma 1(34.5) gene had been interrupted by a stop codon (R4009). Previous studies have shown that these recombinants were completely devoid of the ability to multiply in the central nervous system of rodents. To pursue these studies, we developed a scid mouse glioma model. Tumor cell response (survival) for 10(3), 10(4), and 10(5) implanted MT539MG glioma cells was 38, 23, and 15 days, respectively. The results were as follows: (i) both R3616 and R4009 replicate and cause cytolysis in diverse glioma cell lines of murine and human origin in vitro, and (ii) Winn-type assays 10(5) MT539MG cells coinoculated with R3616 or R4009 as compared to saline significantly prolonged survival in a dose-dependent fashion. Mice that received only tumor cells or the wild-type parent strain of the recombinants, HSV-1(F), died within 15 days. Survival was greatest with R4009. These experiments define both a model for screening oncolytic viruses and a genetically engineered virus of significant potential use as an oncolytic agent.

Animals↗
PubMed1997

Characterization of a genetically engineered inactivation-resistant coagulation factor VIIIa.

Individuals with hemophilia A require frequent infusion of preparations of coagulation factor VIII. The activity of factor VIII (FVIII) as a cofactor for factor IXa in the coagulation cascade is limited by its instability after activation by thrombin. Activation of FVIII occurs through proteolytic cleavage and generates an unstable FVIII heterotrimer that is subject to rapid dissociation of its subunits. In addition, further proteolytic cleavage by thrombin, factor Xa, factor IXa, and activated protein C can lead to inactivation. We have engineered and characterized a FVIII protein, IR8, that has enhanced in vitro stability of FVIII activity due to resistance to subunit dissociation and proteolytic inactivation. FVIII was genetically engineered by deletion of residues 794-1689 so that the A2 domain is covalently attached to the light chain. Missense mutations at thrombin and activated protein C inactivation cleavage sites provided resistance to proteolysis, resulting in a single-chain protein that has maximal activity after a single cleavage after arginine-372. The specific activity of partially purified protein produced in transfected COS-1 monkey cells was 5-fold higher than wild-type (WT) FVIII. Whereas WT FVIII was inactivated by thrombin after 10 min in vitro, IR8 still retained 38% of peak activity after 4 hr. Whereas binding of IR8 to von Willebrand factor (vWF) was reduced 10-fold compared with WT FVIII, in the presence of an anti-light chain antibody, ESH8, binding of IR8 to vWF increased 5-fold. These results demonstrate that residues 1690-2332 of FVIII are sufficient to support high-affinity vWF binding. Whereas ESH8 inhibited WT factor VIII activity, IR8 retained its activity in the presence of ESH8. We propose that resistance to A2 subunit dissociation abrogates inhibition by the ESH8 antibody. The stable FVIIIa described here provides the opportunity to study the activated form of this critical coagulation factor and demonstrates that proteins can be improved by rationale design through genetic engineering technology.

Enzyme Activation↗
PubMed2001

Metabolic engineering of plant alkaloid biosynthesis.

Plant alkaloids, one of the largest groups of natural products, provide many pharmacologically active compounds. Several genes in the biosynthetic pathways for scopolamine, nicotine, and berberine have been cloned, making the metabolic engineering of these alkaloids possible. Expression of two branching-point enzymes was engineered: putrescine N-methyltransferase (PMT) in transgenic plants of Atropa belladonna and Nicotiana sylvestris and (S)-scoulerine 9-O-methyltransferase (SMT) in cultured cells of Coptis japonica and Eschscholzia californica. Overexpression of PMT increased the nicotine content in N. sylvestris, whereas suppression of endogenous PMT activity severely decreased the nicotine content and induced abnormal morphologies. Ectopic expression of SMT caused the accumulation of benzylisoquinoline alkaloids in E. californica. The prospects and limitations of engineering plant alkaloid metabolism are discussed.

Alkaloids↗
PubMed1996

The mechanism of velocity modulated allosteric regulation in D-3-phosphoglycerate dehydrogenase. Cross-linking adjacent regulatory domains with engineered disulfides mimics effector binding.

D-3-Phosphoglycerate dehydrogenase (PGDH) (EC 1.1.1.95) from Escherichia coli is an allosterically regulated enzyme of the Vmax type. It is a tetramer of identical subunits and each subunit is made up of three identifiable domains, the cofactor binding domain, the substrate binding domain, and the regulatory domain. Each subunit contacts two other subunits through adjacent cofactor binding domains and through adjacent regulatory domains. L-Serine, the physiological effector, inhibits catalytic activity by apparently tethering regulatory domains from adjacent subunits together through the formation of hydrogen bonds to each subunit. This investigation demonstrates that cross-linking adjacent regulatory domains with engineered disulfides produces catalytic inhibition in the absence of inhibitor in a manner similar to that produced by the inhibitor. The inhibition due to cross-linking can be completely reversed in a concentration dependent manner by dithiothreitol. The active mutant enzyme, containing the engineered cysteines in the reduced state, retains its ability to be inhibited by L-serine, although at a 100-fold higher concentration. Hill plots of the serine inhibition of mutant and native enzyme indicate that the number of interacting sites remains at 2 in the mutant enzyme. The reversible inhibition of enzyme activity that results from tethering adjacent regulatory domains with engineered disulfides suggests that these domains move in some manner relative to one another during the active to inhibited state transition. These observations support the model which predicts that catalytic activity is regulated by the movement of rigid domains about flexible hinges and that effector binding prevents this by locking the regulatory domains in a state that produces an open active site cleft.

Allosteric Regulation↗
PubMed1999

Conversion of an engineered potassium-binding site into a calcium-selective site in cytochrome c peroxidase.

We have previously shown that the K(+) site found in ascorbate peroxidase can be successfully engineered into the closely homologous peroxidase, cytochrome c peroxidase (CCP) (Bonagura, C. A. , Sundaramoorthy, M., Pappa, H. S., Patterson, W. R., and Poulos, T. L. (1996) Biochemistry 35, 6107-6115; Bonagura, C. A., Sundaramoorthy, M., Bhaskar, B., and Poulos, T. L. (1999) Biochemistry 38, 5538-5545). All other peroxidases bind Ca(2+) rather than K(+). Using the K(+)-binding CCP mutant (CCPK2) as a template protein, together with observations from structural modeling, mutants were designed that should bind Ca(2+) selectively. The crystal structure of the first generation mutant, CCPCA1, showed that a smaller cation, perhaps Na(+), is bound instead of Ca(2+). This is probably because the full eight-ligand coordination sphere did not form owing to a local disordering of one of the essential cation ligands. Based on these observations, a second mutant, CCPCA2, was designed. The crystal structure showed Ca(2+) binding in the CCPCA2 mutant and a well ordered cation-binding loop with the full complement of eight protein to cation ligands. Because cation binding to the engineered loop results in diminished CCP activity and destabilization of the essential Trp(191) radical as measured by EPR spectroscopy, these measurements can be used as sensitive methods for determining cation-binding selectivity. Both activity and EPR titration studies show that CCPCA2 binds Ca(2+) more effectively than K(+), demonstrating that an iterative protein engineering-based approach is important in switching protein cation selectivity.

Amino Acid Sequence↗
PubMed2001

Engineered biosynthesis of the peptide antibiotic bacitracin in the surrogate host Bacillus subtilis.

Nonribosomal peptides are processed on multifunctional enzymes called nonribosomal peptide synthetases (NRPSs), whose modular multidomain arrangement allowed the rational design of new peptide products. However, the lack of natural competence and efficient transformation methods for most of nonribosomal peptide producer strains prevented the in vivo manipulation of these biosynthetic gene clusters. In this study, we present methods for the construction of a genetically engineered Bacillus subtilis surrogate host for the integration and heterologous expression of foreign NRPS genes. In the B. subtilis surrogate host, we deleted the resident 26-kilobase srfA gene cluster encoding the surfactin synthetases and subsequently used the same chromosomal location for integration of the entire 49-kilobase bacitracin biosynthetic gene cluster from Bacillus licheniformis by a stepwise homologous recombination method. Synthesis of the branched cyclic peptide antibiotic bacitracin in the engineered B. subtilis strain was achieved at high level, indicating a functional production and proper posttranslational modification of the bacitracin synthetases BacABC, as well as the expression of the associated bacitracin self-resistance genes. This engineered and genetically amenable B. subtilis strain will facilitate the rational design of new bacitracin derivatives.

Anti-Bacterial Agents↗
PubMed2003

An engineered hyaluronan synthase: characterization for recombinant human hyaluronan synthase 2 Escherichia coli.

The Class I hyaluronan synthase (HAS) is a unique glycosyltransferase synthesizing hyaluronan (HA), a polysaccharide composed of GlcUA and GlcNAc, by using one catalytic domain that elongates two different monosaccharides. As for the synthetic mechanism, there are two alternative manners for the sugar elongation process. Some bacterial HASs add new sugars to the non-reducing end of the acceptor to grow polymers. On the other hand, some vertebrate enzymes seem to transfer sugars to the reducing end. Expression of vertebrate HASs as active and soluble proteins will accelerate further precise insight into mechanisms of sugar elongation reactions by natural HASs. Since large scale production of HA polymers and oligomers would become powerful tools both for basic studies and new biotechnology to create functional carbohydrates in medicinal purposes, advent of an efficient method for the expression of HASs in Escherichia coli is strongly expected. Here we communicate the first success of the production of recombinant human HAS2 proteins composed of only the catalytic region in E. coli as the active form. It was demonstrated that an engineered HAS2 expressed in E. coli exhibited significant activity to synthesize a mixture of HAS oligomers from 8-mer (HA8) to 16-mer (HA16). Engineered HAS2 prepared herein elongated sugars from exogenous tetrasaccharide to form polymers with a direction to the non-reducing end. According to the present results, large scale production of engineered recombinant HASs is to be performed using E. coli that will provide practical and economic advantages in manufacturing enzymes for use in the synthesis of various oligomeric HA molecules and their industrial applications.

Catalytic Domain↗
PubMed2003

An engineered CuA Amicyanin capable of intermolecular electron transfer reactions.

The type I copper center of amicyanin was replaced with a binuclear CuA center. To create this model CuA protein, a portion of the amino acid sequence that contains three of the ligands to the native type I copper center of Paracoccus denitrificans amicyanin was replaced with the corresponding portion of sequence that provides five ligands for the CuA center of cytochrome c oxidase from P. denitrificans. UV-visible and electron paramagnetic resonance spectroscopy confirm that the engineered protein as isolated possesses the mixed-valence Cu1.5Cu1.5 (purple) CuA center. Comparison of the spectroscopic properties of this CuA amicyanin with those of the CuA centers of other natural and engineered CuA proteins suggests that the spectroscopic features may be dictated more by the protein host than the sequence of the CuA loop. Novel reactions for a simple CuA model protein are also described. In contrast to other natural and engineered CuA proteins, the fully reduced CuA amicyanin may be reoxidized by molecular oxygen to the mixed-valence state. It is also shown that CuA amicyanin can serve as an electron donor and an electron acceptor for other redox proteins. The mixed-valence form accepts electrons from cytochromes c-551i and c-550 from P. denitrificans. The fully reduced form donates electrons to native and P94F amicyanin. The function as either an electron donor or acceptor is consistent with the measured redox potential of CuA amicyanin of +273 mV. These data indicate that this CuA amicyanin will be a particularly useful model protein for structure-function studies of reactivity and the electron transfer properties of the CuA redox center.

Amino Acid Sequence↗
PubMed2005

Engineering glutathione transferase to a novel glutathione peroxidase mimic with high catalytic efficiency. Incorporation of selenocysteine into a glutathione-binding scaffold using an auxotrophic expression system.

Glutathione peroxidase (GPx, EC 1.11.1.9) protects cells against oxidative damage by catalyzing the reduction of hydroperoxides with glutathione (GSH). Several attempts have been made to imitate its function for mechanical study and for its pharmacological development as an antioxidant. By replacing the active site serine 9 with a cysteine and then substituting it with selenocysteine in a cysteine auxotrophic system, catalytically essential residue selenocysteine was bioincorporated into GSH-specific binding scaffold, and thus, glutathione S-transferase (GST, EC 2.5.1.18) from Lucilia cuprina was converted into a selenium-containing enzyme, seleno-LuGST1-1, by genetic engineering. Taking advantage of the important structure similarities between seleno-LuGST1-1 and naturally occurring GPx in the specific GSH binding sites and the geometric conformation for the active selenocysteine in their common GSH binding domain-adopted thioredoxin fold, the as-generated selenoenzyme displayed a significantly high efficiency for catalyzing the reduction of hydrogen peroxide by glutathione, being comparable with those of natural GPxs. The catalytic behaviors of this engineered selenoenzyme were found to be similar to those of naturally occurring GPx. It exhibited pH and temperature-dependent catalytic activity and a typical ping-pong kinetic mechanism. Engineering GST into an efficient GPx-like biocatalyst provided new proof for the previous assumption that both GPx and GST were evolved from a common thioredoxin-like ancestor to accommodate different functions throughout evolution.

Catalysis↗
PubMed2005

Engineering of a recombinant vitamin K-dependent gamma-carboxylation system with enhanced gamma-carboxyglutamic acid forming capacity: evidence for a functional CXXC redox center in the system.

The vitamin K-dependent gamma-carboxylation system in the endoplasmic reticulum membrane responsible for gamma-carboxyglutamic acid modification of vitamin K-dependent proteins includes gamma-carboxylase and vitamin K 2,3-epoxide reductase (VKOR). An understanding of the mechanism by which this system works at the molecular level has been hampered by the difficulty of identifying VKOR involved in warfarin sensitive reduction of vitamin K 2,3-epoxide to reduced vitamin K(1)H(2), the gamma-carboxylase cofactor. Identification and cloning of VKORC1, a proposed subunit of a larger VKOR enzyme complex, have provided opportunities for new experimental approaches aimed at understanding the vitamin K-dependent gamma-carboxylation system. In this work we have engineered stably transfected baby hamster kidney cells containing gamma-carboxylase and VKORC1 cDNA constructs, respectively, and stably double transfected cells with the gamma-carboxylase and the VKORC1 cDNA constructs in a bicistronic vector. All engineered cells showed increased activities of the enzymes encoded by the cDNAs. However increased activity of the gamma-carboxylation system, where VKOR provides the reduced vitamin K(1)H(2) cofactor, was measured only in cells transfected with VKORC1 and the double transfected cells. The results show that VKOR is the rate-limiting step in the gamma-carboxylation system and demonstrate successful engineering of cells containing a recombinant vitamin K-dependent gamma-carboxylation system with enhanced capacity for gamma-carboxyglutamic acid modification. The proposed thioredoxin-like (132)CXXC(135) redox center in VKORC1 was tested by expressing the VKORC1 mutants Cys(132)/Ser and Cys(135)/Ser in BHK cells. Both of the expressed mutant proteins were inactive supporting the existence of a CXXC redox center in VKOR.

Animals↗
PubMed2006

Tissue engineered cartilage integration to live and devitalized cartilage: a study by reflectance mode confocal microscopy and standard histology.

This study investigated the in vivo formation of engineering cartilage within living or devitalized cartilage discs using reflectance mode confocal microscopy and conventional light microscopy. Pig articular chondrocytes were suspended in fibrin glue and placed between two cartilage discs. Four experimental groups were prepared: in groups 1 and 2, the cell-hydrogel composite was placed between two live or between two devitalized cartilage discs, respectively; in groups 3 and 4, acellular fibrin glue was placed between two live or between two devitalized cartilage discs, respectively. Samples were implanted in the back of nude mice and analyzed after 2, 5, and 8 weeks. Results showed that engineered cartilage seems to grow more homogenously when the cell-seeded gel was placed between devitalized cartilages than when it was placed between live cartilage matrices. Confocal microscopy provides valuable information on the integration of tissue-engineered cartilage with native tissue and could be useful for nondestructive imaging in vivo.

Animals↗
PubMed1997

Educating the rehabilitation engineer as a service provider.

This paper reviews a 12-month masters-level rehabilitation engineering training program to prepare engineers to be rehabilitation service providers. The extent of the rehabilitation engineering shortage is discussed. The curriculum is then provided with descriptions of 16 courses, the program's internship, and a comparison with other training programs. A listing of the intended customers of the program graduates is given along with a description of the advisory committee to help interface with these customers. Four educational goals consisting of disability and technology, major rehabilitation systems, applied skills, and life-long learning are discussed. The results of six years' experience with this program, including a survey of graduates, are presented.

Biomedical Engineering↗
PubMed1996

Subjective Annoyance Response to Diesel Engine Sound During Idling Conditions.

Subjective annoyance response to diesel engine sound during idling conditions was evaluated by 80 participants. Eight different sound spectra were presented to the participants at a constant level of 80 dB(A) in a paired comparison procedure. Stereo-recorded sound stimuli were played back through a pair of loudspeakers in an anechoic room. Four objective parameters of loudness, sharpness, impulsiveness, and roughness were found to be the determining factors that cause subjective annoyance. An annoyance prediction model for the test stimuli of an idling diesel engine was developed on the basis of these factors. The objective parameters and their interactions have a significant effect on the annoyance prediction model. The spectral distribution indicated by test participants to be pleasant can be used as a basis for appropriate modification of engine sound. A single microphone measurement in free field conditions can be used to estimate objective parameters for defining the cause of annoyance.

diesel engine↗
PubMedDate not supplied

Use of genetically engineered mice in drug discovery and development: wielding Occam's razor to prune the product portfolio.

Genetically engineered mice (GEMs) that either overexpress (transgenic) or lack (gene-targeted, or "knock-out") genes are used increasingly in industry to investigate molecular mechanisms of disease, to evaluate innovative therapeutic targets, and to screen agents for efficacy and/or toxicity. High throughput GEM construction in drug discovery and development (DDD) serves two main purposes: to test whether a given gene participates in a disease condition, or to determine the function(s) of a protein that is encoded by an expressed sequence tag (EST, an mRNA fragment for a previously uncharacterized protein). In some instances, phenotypes induced by such novel GEMs also may yield clues regarding potential target organs and toxic effects of potential therapeutic molecules. The battery of tests used in phenotypic analysis of GEMs varies between companies, but the goal is to define one or more easily measured endpoints that can be used to monitor the disease course--especially during in vivo treatment with novel drug candidates. In many DDD projects, overt phenotypes are subtle or absent even in GEMs in which high-level expression or total ablation of an engineered gene can be confirmed. This outcome presents a major quandary for biotechnology and pharmaceutical firms: given the significant expense and labor required to generate GEMs, what should be done with "negative" constructs? The 14th century philosophical principle known as Occam's razor-that the simplest explanation for a phenomenon is likely the truth-provides a reasonable basis for pruning potential therapeutic molecules and targets. In the context of DDD, Occam's razor may be construed to mean that correctly engineered GEMs lacking obvious functional or structural phenotypes have none because the affected gene is not uniquely essential to normal homeostasis or disease progression. Thus, a "negative" GEM construct suggests that the gene under investigation encodes a ligand or target molecule without significant therapeutic potential. This interpretation indicates that, at least in a market-driven industrial setting, such "negative" projects should be pruned aggressively so that resources may be redirected to more promising DDD ventures.

Animals↗