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Results for “reverse engineering”

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At least 145 records · Page 8Linked to original sources

PubMed1997

Short-term change of optic nerve head topography after trabeculectomy in adult glaucoma patients as measured by Heidelberg retina tomograph.

Using Heidelberg Retina Tomograph (HRT, Heidelberg Engineering, Heidelberg, Germany) we investigated whether reversal of glaucomatous optic disc cupping occurs in adult glaucoma patients following intraocular pressure (IOP) reduction by trabeculectomy, and whether topographic change in the optic disc differs according to region. Thirteen eyes of 13 glaucoma patients in whom IOP reduction was 25% or more at postoperative follow-up of 2 months were enrolled in the study. Cup/disc area ratio had decreased significantly, while rim area and rim volume showed significant increases (p < 0.05, Wilcoxon signed rank test). Among the optic disc sectors, rim area in the superior and inferior quadrants, especially in the superotemporal and inferotemporal octant, had increased significantly. The decrease in maximum cup death correlated significantly with the recent IOP reduction (r = 0.698, p = 0.016). Reversal of glaucomatous disc cupping after IOP reduction following trabeculectomy in adult patients as confirmed by HRT. Furthermore, the regions of selective reversal in the superior and inferior quadrants coincided with those parts of the optic nerve head in which early functional and structural glaucomatous damage has been known to occur.

Adult↗

PubMed1994

Purification, characterization and analysis of rolipram inhibition of a human type-IVA cyclic AMP-specific phosphodiesterase expressed in yeast.

Analyses were done on a human type-IV cyclic AMP (cAMP) phosphodiesterase (hPDE-IVA-h6.1) expressed in an engineered strain of Saccharomyces cerevisiae. This strain (YMS6) expressed soluble PDE activity, together with an insoluble activity which was not released by re-homogenization, treatment with high-ionic-strength solutions or with the detergent Triton X-100. Pellet and soluble PDE activities were typical of type-IV PDE. They were cAMP-specific, insensitive to the addition of either cGMP (1 microM) or Ca2+/calmodulin, and inhibited by rolipram. Thermostability studies showed both activities to decay as single exponentials, indicating the presence of homogeneous PDE protein species in each fraction. Pellet PDE activity was more thermostable than the soluble enzyme. Mg2+ and Mn2+ dose-dependently increased PDE activity and reversed the inactivating effect of EDTA.h6.1 was engineered to express a C-terminal five-histidine motif (h6.1his5). This allowed purification of the PDE to apparent homogeneity in a simple two-step process involving a rolipram affinity column and a Ni2(+)-chelate column. A single monomeric protein of subunit molecular mass approximately 73 kDa and native molecular mass approximately 74 kDa resulted after a approximately 53000-fold purification. This exhibited a Km for cAMP of 8 microM, a true Vmax. of 0.8 mumol of cAMP hydrolysed/min per mg of PDE protein, a kcat. of 3702 s-1, and a value of the specificity constant kcat/Km of 4.6 x 10(8) M-1.s-1, the last implying a diffusion controlled reaction. Rolipram (Ki 0.4 soluble; 0.7 microM pellet) and 3-isobutyl-1-methylxanthine (Ki 15 soluble; 19 microM pellet) served as simple competitive inhibitors for both soluble and pellet forms of h6.1, respectively.

1-Methyl-3-isobutylxanthine↗

PubMed1991

Metal affinity chromatography of recombinant HIV-1 reverse transcriptase containing a human renin cleavable metal binding domain.

A metal binding peptide, hexahistidine, preceding a renin cleavage sequence (Pro-Phe-His-Leu-Val-Ile-His-) was engineered on to the N-terminus of HIV-1 reverse transcriptase (RT). The chimeric protein was expressed in Escherichia coli and characterized after purification by DEAE chromatography and HPLC. Amino-terminal sequencing confirmed the presence of the first 15 amino acids of the chimeric protein. The chimeric exhibited RT activity like that of HIV-1 RT and was cleaved by human renin at the expected site. The potential of a hexa-histidine fusion in the purification of recombinant HIV-1 RT by immobilized metal affinity chromatography (IMAC) on the commonly used resin (IDA-Ni2+) was investigated. The chimeric gene product from a crude E. coli extract was strongly retarded on a immobilized nickel column, while most of the contaminating E. coli proteins were eliminated after elution with 20-35 mM imidazole. The bound chimeric protein was eluted with 300 mM imidazole and appeared predominantly as a single band on an SDS-polyacrylamide gel. The remarkable specificity of this affinity tail was further demonstrated by separating the chimeric protein from HIV-1 RT in a crude extract prepared by mixing extracts from cells expressing HIV-1 RT and the hexahistidine recombinant chimeric protein. The usefulness of a enzymatically cleavable metal binding peptide in the rapid purification and production of HIV-1 RT without proteolysis to a heterodimer is discussed.

Amino Acid Sequence↗

PubMed1993

Inhibition of HIV-1 replication by a high-copy-number vector expressing antisense RNA for reverse transcriptase.

We report the construction of a high-copy-number (hcn) expression vector for human cells. Amplification of this vector occurs due to the presence of an element derived from the murine DNA encoding ribosomal RNA (rDNA). HIV-1 replication in Jurkat T lymphocytes is nearly abolished when antisense RNA directed against the gene encoding reverse transcriptase is expressed from this hcn vector. The replication of the virus is only slightly reduced by the plasmid control version lacking the murine amplification-promoting element. This kind of hcn vector may represent an important improvement for the genetic engineering of eukaryotic cells and may also provide some ideas for the future gene therapy of some human diseases.

Culture Media, Conditioned↗

PubMed2000

Performance of discrete heat engines and heat pumps in finite time

The performance in finite time of a discrete heat engine with internal friction is analyzed. The working fluid of the engine is composed of an ensemble of noninteracting two level systems. External work is applied by changing the external field and thus the internal energy levels. The friction induces a minimal cycle time. The power output of the engine is optimized with respect to time allocation between the contact time with the hot and cold baths as well as the adiabats. The engine's performance is also optimized with respect to the external fields. By reversing the cycle of operation a heat pump is constructed. The performance of the engine as a heat pump is also optimized. By varying the time allocation between the adiabats and the contact time with the reservoir a universal behavior can be identified. The optimal performance of the engine when the cold bath is approaching absolute zero is studied. It is found that the optimal cooling rate converges linearly to zero when the temperature approaches absolute zero.

Journal Article↗

PubMed2001

Exhaust emissions from engines of the Detroit Diesel Corporation in transit buses: a decade of trends.

In the U.S.A., exhaust emissions from city buses fueled by diesel are not characterized well because current emission standards require engine tests rather than tests of whole vehicles. Two transportable chassis dynamometer laboratories developed and operated by West Virginia University (WVU) have been used extensively to gather realistic emission data from heavy-duty vehicles, including buses, tested in simulated driving conditions. A subset of these data has been utilized for a comprehensive introspection into the trends of regulated emissions from transit buses over the last 7 years, which has been prompted by continuously tightening restrictions on one hand, along with remarkable technological progress, on the other hand. Two widely used models of diesel engines manufactured by the Detroit Diesel Corporation (DDC) have been selected as a case-study for such an overview, based on full-scale, on-site testing of actual city buses, driven in accordance with the SAE J1376 standard of a Commercial Business District (CBD) cycle. The results provide solid, quantitative evidence that most regulated emissions from engines produced by DDC have declined over the years, especially with the transition from the 6V-92TA to the Series 50 models. This improvement is remarkable mainly for the emissions of particulate matter (PM), that are lower by over 70%, on average, for the Series 50 engines, though the emissions of nitrogen oxides (NOx) exhibit a reversed trend, showing a degradation of about 6%, on average, with the transition from 6V-92TA to the Series 50 engines. The expected trend of decreasing emission levels with the model year of the engine is clear and consistent for particulate matter (PM), hydrocarbons (HC), carbon monoxide (CO), and nitrogen oxides (NOx), starting with the 1990 models, although it is not conclusive for carbon dioxide (CO2) emissions.

Air Pollution↗

PubMed1996

Effects of exposure to 16.7 Hz magnetic fields on urinary 6-hydroxymelatonin sulfate excretion of Swiss railway workers.

The aim of our study was to examine the effects of 16.7 Hz electromagnetic-field exposure on pineal melatonin production in healthy humans. The study was based on comparing urinary 6-hydroxymelatonin sulfate (6-OHMS) levels of 108 male railway workers between leisure periods and days following the start of service on electrically powered engines (66 engineers) or working beneath transmission lines (42 railway employees such as train attendants and station managers; controls). A repeated measures design was used, i.e., each volunteer served as his own control. The exposure averaged 20 muTesla in the most exposed workers and around 1 muTesla in the least exposed. Apart from magnetic exposure the workers were subject to a shift work schedule with daily advances between 15 min and 1 hr. Melatonin was assessed by sampling urinary 6-OHMS both in the morning and the early evening. Evening 6-OHMS values appeared to be lowered by a factor of 0.81 (95%CI: 0.73-0.90) during work days compared to leisure days among engine drivers, but not in the controls. The lowering was not confined to certain types of shift work such as early, normal, or late shifts. During subsequent leisure periods evening values recovered significantly, mean ratio = 1.27 (95%CI: 1.03-1.56), i.e., the effects appeared to be reversible. In contrast, morning 6-OHMS samples of engineers and controls did not differ much between work and leisure days. There was, however, a tendency for a rebound of morning values in a leisure period following a work period both for engineers and controls. The observed pattern appears to be in line with predictions of the "phase response curve." No evidence for a dose-response relation was found. The results support the hypothesis that 16.7 Hz magnetic fields alter 6-OHMS excretion in humans exposed to magnetic fields. An alternative explanation that cannot be excluded in this study is that the difference between engineers and controls is due to differential exposure to day light at work.

Adult↗

PubMed1997

A new engine-driven canal preparation system with electronic canal measuring capability.

A new cordless engine-driven root canal preparation system has been developed that electronically monitors the location of the file tip and the torque applied to the file during all instrumentation procedures. The Root ZX is mounted inside the handpiece to measure the canal length. The engine is driven with a rechargeable battery, and it works more than 40 minutes without a recharge. Nickel-titanium files are used for this preparation system. The file is rotated at 240 to 280 rpms. When the file tip reaches the apical constriction, the revolution of the file can be automatically reversed (Auto-apical-reverse mechanism). If there is too much torque, the rotation is automatically reversed (Auto-torque-reverse mechanism). These automatic functions promise a safer engine preparation of the canal with nickel-titanium files.

Dental Alloys↗

PubMed1991

Expression of human recombinant cAMP phosphodiesterase isozyme IV reverses growth arrest phenotypes in phosphodiesterase-deficient yeast.

The low-Km cAMP-specific phosphodiesterases (PDEases) are of great pharmacological significance because of their involvement in regulating cAMP concentrations, which, in turn, are responsible for mediating the cellular response to extracellular signals such as hormones and neurotransmitters. We recently reported the isolation of a cDNA clone that encodes a human monocyte low-Km, rolipram-sensitive, cAMP PDEase (isozyme IV). We have engineered the inducible expression of this human PDEase in yeast. Cells of Saccharomyces cerevisiae contain two genes that encode cAMP PDEases. PDEase-deficient mutants are viable but exhibit specific growth arrest phenotypes associated with elevated intracellular cAMP content; these phenotypes include heat shock sensitivity and the inability to grow on acetate as a carbon source. We show that functional expression of our human cAMP PDEase in a genetically engineered PDEase-deficient strain of S. cerevisiae reverses these aberrant phenotypes. Furthermore, under conditions for growth arrest, rolipram is cytotoxic to PDEase-deficient mutants expressing the human cAMP PDEase, indicating that it is capable of inhibiting the human recombinant enzyme in vivo. This system can be used in the development of a yeast cell-based assay for isozyme-selective inhibitors of the human recombinant cAMP PDEase.

3',5'-Cyclic-AMP Phosphodiesterases↗

PubMed1991

Recombinant soluble human complement receptor type 1 inhibits inflammation in the reversed passive arthus reaction in rats.

The human CR1 was genetically engineered by site directed mutagenesis into a truncated form which was secreted from transfected Chinese hamster ovary cells. This soluble recombinant CR1 (sCR1) was purified from the supernatants of the Chinese hamster ovary cells cultured in a hollow fiber bioreactor. sCR1 inhibits the C3 and C5 convertases of the classical and the alternative pathways in vitro. The ability of sCR1 to inhibit the immune complex-mediated inflammation in vivo was tested in a rat reversed passive Arthus reaction model. Administration of sCR1 at the dermal sites reduced the Arthus vasculitis in a dose-dependent manner as judged by both gross and microscopic examination, as well as by immunohistologic localization of C3 and C5b-9 neoantigen deposits. These data suggest that sCR1 inhibits the Arthus reaction by interrupting the activation of the C cascade, hence limiting the detrimental immune complex-induced tissue damage in vivo.

Animals↗

PubMed2003

Crystal structure of a natural circularly permuted jellyroll protein: 1,3-1,4-beta-D-glucanase from Fibrobacter succinogenes.

The 1,3-1,4-beta-D-glucanase from Fibrobacter succinogenes (Fsbeta-glucanase) is classified as one of the family 16 glycosyl hydrolases. It hydrolyzes the glycosidic bond in the mixed-linked glucans containing beta-1,3- and beta-1,4-glycosidic linkages. We constructed a truncated form of recombinant Fsbeta-glucanase containing the catalytic domain from amino acid residues 1-258, which exhibited a higher thermal stability and enzymatic activity than the full-length enzyme. The crystal structure of the truncated Fsbeta-glucanase was solved at a resolution of 1.7A by the multiple wavelength anomalous dispersion (MAD) method using the anomalous signals from the seleno-methionine-labeled protein. The overall topology of the truncated Fsbeta-glucanase consists mainly of two eight-stranded anti-parallel beta-sheets arranged in a jellyroll beta-sandwich, similar to the fold of many glycosyl hydrolases and carbohydrate-binding modules. Sequence comparison with other bacterial glucanases showed that Fsbeta-glucanase is the only naturally occurring circularly permuted beta-glucanase with reversed sequences. Structural comparison shows that the engineered circular-permuted Bacillus enzymes are more similar to their parent enzymes with which they share approximately 70% sequence identity, than to the naturally occurring Fsbeta-glucanase of similar topology with 30% identity. This result suggests that protein structure relies more on sequence identity than topology. The high-resolution structure of Fsbeta-glucanase provides a structural rationale for the different activities obtained from a series of mutant glucanases and a basis for the development of engineered enzymes with increased activity and structural stability.

Amino Acid Sequence↗

PubMed1992

Chemical heterogeneity as a result of hydroxylamine cleavage of a fusion protein of human insulin-like growth factor I.

Recombinant DNA techniques were used to biosynthesize human insulin-like growth factor I (hIGF-I) as a fusion protein wherein the fusion polypeptide is an IgG-binding moiety derived from staphylococcal protein A. This fusion protein is produced in Escherichia coli and secreted into the fermentation broth. In order to release mature recombinant-derived hIGF-I (rhIGF-I), the fusion protein is treated with hydroxylamine, which cleaves a susceptible Asn-Gly bond that has been engineered into the fusion protein gene. Reversed-phase h.p.l.c. was used to estimate the purity of the rhIGF-I preparations, especially for the quantification of the methionine sulphoxide-containing variant. It was determined that hydroxylamine cleavage of the fusion protein produced, as a side reaction, hydroxamates of the asparagine and glutamine residues in rhIGF-I. Although isoelectric focusing was effective in detecting, and reversed-phase h.p.l.c. for producing enriched fractions of the hydroxamate variants, ion-exchange chromatography was a more definitive procedure, as it allowed quantification and facile removal of these variants. The identity of the variants as hydroxamates was established by Staphylococcus aureus V8 proteinase digestion, followed by m.s., as the modification was transparent to amino acid and N-terminal sequence analyses. The biological activity of rhIGF-I was established by its ability to incorporate [3H]thymidine into the DNA of BALB/c373 cells and by a radioreceptor assay utilizing human placental membranes. Both assays demonstrate that the native, recombinant and methionine sulphoxide and hydroxamate IGF-I variants are essentially equipotent.

3T3 Cells↗

PubMed2003

In vivo model for cross-species porcine endogenous retrovirus transmission using tissue engineered pulmonary arteries.

OBJECTIVE: Acellularised porcine scaffolds have been successfully used for cardiovascular tissue engineering. However, there is concern about the possibility of porcine endogenous retrovirus (PERV) transmission. In this study we developed an in vivo model for cross-species PERV transmission. METHODS: In vitro autologous repopulated porcine pulmonary arteries (n=6) were implanted in sheep in orthotopic position. Blood samples were collected regularly up to 6 months after implantation and tested for PERV by means of polymerase chain reaction and reverse transcriptase-polymerase chain reaction. Explanted tissue engineered pulmonary arteries were tested for PERV sequences. RESULTS: PERV DNA was detectable in acellularised porcine scaffolds. No PERV sequences were detectable 6 months after implantation of in vitro repopulated acellularised porcine pulmonary arteries and in all tested peripheral blood samples. CONCLUSIONS: Acellularised porcine matrix scaffolds can be used for cardiovascular tissue engineering of autologous grafts without risk of PERV transmission.

Animals↗

PubMed2002

Are beta-sheet breaker peptides dissolving the therapeutic problem of Alzheimer's disease?

Alzheimer's disease (AD) is a neurodegenerative disorder for which there is no cure or effective treatment. One of the major neuropathological signatures of AD is the deposition of amyloid plaques in the brain of affected people. Although the role of these structures in the pathogenesis of the disease is not fully understood, recent findings have provided evidence that amyloid may be a key player in the disease. Therefore, preventing and reversing cerebral amyloid deposition have become an attractive therapeutic strategy for AD. We have engineered synthetic beta-sheet breaker peptides to bind soluble amyloid peptide and prevent and reverse its conversion to the beta-sheet rich aggregated structure, precursor of the amyloid plaques. Results in vitro, in cell culture and in vivo suggest that beta-sheet breaker peptides might be candidates for an AD-therapy focused to reduce amyloid deposition.

Aged↗

PubMed2026

The future of TCR-Treg therapies is renewables.

Cell therapy has longstanding roots in haematopoietic stem cell transplantation and early immune cell transfers in infectious disease and transplantation, where patient- or donor-derived cells have achieved therapeutic benefit in selected contexts. The modern era has been driven largely by oncology, with engineered modalities such as tumour-infiltrating lymphocytes, CAR-T cells and TCR-engineered T cells delivering transformative responses but requiring complex, costly manufacturing. These platforms are now being adapted for autoimmune diseases to induce durable, antigen-specific immune tolerance, yet broad application is limited by safety concerns, process complexity and access. Non-engineered cell therapies for autoimmunity, including mesenchymal stem cells, polyclonal regulatory T cells and tolerogenic dendritic cells, have shown acceptable safety and proof-of-principle for immune re-education, but clinical responses have been modest and inconsistent, with limited scalability. Engineered approaches such as CAR-T cells can induce reversible B cell depletion in B cell-mediated rheumatic diseases but only addresses antibody-driven pathology and not T cell-mediated autoimmunity. TCR-engineered Tregs have emerged as a promising antigen-specific strategy, offering localized, antigen-linked suppression with bystander tolerance. Preclinical and early clinical data suggest superior potency, stability and disease control compared with polyclonal Tregs at similar or lower doses, but translation is constrained by the rarity and fragility of Tregs and by labour-intensive, CAR-T-like manufacturing. This review highlights emerging solutions for closed, automated and decentralised production, and discusses allogeneic approaches using gene-edited or banked Tregs with HLA engineering or matching. Together, these advances support the development of scalable, "off-the-shelf" TCR-Treg products with potential to provide safe, affordable tolerance-restoring therapies for autoimmune disease.

Humans↗

PubMed2003

New building blocks for the assembly of sequence selective molecular zippers.

Synthetic H-bonded molecular zippers contain no sequence information that can be used to engineer the selective binding interactions characteristic of biopolymers; reversing the sense of the amide bonds in the two binding partners generates a new orthogonal recognition motif and the mutually complementary binding partners form complexes an order of magnitude more stable than the corresponding mismatch complexes.

Amino Acid Motifs↗

PubMed1983

The iceperson cometh: cryonics, law and medicine.

The "New Biology" has already made profound impact on the law. Cryonics and genetic engineering represent technological triumphs. The natural, organic process of dying is being replaced by a humanly engineered technological process for living. The dying phase of life is prolonged until biological knowledge is available to reverse the dying phase and restore the living phase. Both cryonics and genetic engineering in their attempts to replace the organic process with the technological process disturb the delicate balance of the triad of life which each individual experiences--faith, health, and justice. Since law is a basic tool to achieve justice among human beings, how should it respond to the health argument of the cryonics' physician who views death as a disease which is curable? How should the law respond to the faith questions surrounding the cryonics patient? What am I? A block of ice. Who am I? A living, comatose patient or a dormant, static body with the possibility of a reverter. Why am I? A new human being now endowed with immortality through the triumph of life over death founded on man's current faith in the God called Technology. Cryonics through its unbalancing of the traditional triad of life poses formidable challenges to the major institutions of faith, health and justice. The practitioners in these institutions: clergy, physicians and lawyers must now reassess the rules of the game of life be they religious, medical or legal. This article offers insights to begin this reassessment.

Death↗

PubMed1987

Effects of engineering complementary charged residues into the hydrophobic subunit interface of tyrosyl-tRNA synthetase. Appendix: Kinetic analysis of dimeric enzymes that reversibly dissociate into inactive subunits.

Wild-type tyrosyl-tRNA synthetase (TyrTS) from Bacillus stearothermophilus is a symmetrical dimer. Four different heterodimeric enzymes have been produced by site-directed mutagenesis at the subunit interface so that the monomers are linked by a potential salt bridge in a hydrophobic environment. The two Phe-164 residues of wild-type TyrTS are on the axis of symmetry and interact in a hydrophobic region of the subunit interface. Mutation of Phe-164 to aspartate or glutamate in full-length TyrTS and to lysine or arginine in an active truncated enzyme (delta TyrTS) induces reversible dissociation of the enzyme into inactive monomers. Mixing mutants in equimolar amounts produces four different heterodimers: TyrTS(Asp-164)-delta TyrTS(Lys-164); TyrTS(Asp-164)-delta TyrTS(Arg-164); TyrTS(Glu-164)-delta TyrTS(Lys-164); TyrTS(Glu-164)-delta TyrTS(Arg-164). A general method is derived for analyzing the kinetics of dimeric enzymes that reversibly dissociate into inactive subunits. Application to mutants of TyrTS allows estimation of dissociation constants (Kd values) for the dimers. At pH 7.8, the heterodimers have Kd values of 6-14 microM, whereas for homodimers Kd = 120-4000 microM. These values decrease to about 30 microM for homodimers of TyrTS(Asp-164), TyrTS(Glu-164), and delta TyrTS(Lys-164) when the pH favors uncharged forms of the side chains at position 164. Each of the four salt bridges engineered into the hydrophobic subunit interface of TyrTS appears, therefore, to be weak. These engineered salt bridges may be compared with naturally occurring ones. In the latter, there are complementary interactions between the charges in the salt bridge with polar groups in the protein.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acyl-tRNA Synthetases↗