Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “reverse engineering”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 1,549 records · Page 86Linked to original sources
PubMed2001

CD40 ligation enhances IL-15 production by tubular epithelial cells.

Interleukin-15 (IL-15) is a potent T-cell growth factor and activator. Acute rejection of kidney allografts strongly correlated with elevated IL-15 mRNA in the graft. A role in the rejection process is also suggested for the interaction between CD40 ligand (CD154) expressed on membranes of activated T cells and its receptor (CD40). The effect of CD40 ligation on IL-15 production in human tubular epithelial cells (TEC) was investigated. TEC were co-cultured with a cell line genetically engineered to express CD154. CD154-expressing cells (CD40L cells) bind to TEC. Addition of the CD40L cells to the TEC culture resulted in elevated IL-15 levels. This enhanced production was not observed with control cells, when anti-CD154 antibody was added, and when direct contact between CD40L-cells and TEC was prevented with the use of a Trans-well system. CD40 activation resulted in a twofold increase of IL-15 mRNA transcripts as measured by reverse transcription-PCR and a concordant elevation in IL-15 protein production as measured by specific enzyme-linked immunosorbent assay. The intensity of activation by CD154 was linearly dependent on cell number, reaching plateau when the effector/target-ratio was 1:1. The increase of IL-15 levels was similar to that produced by stimulation with interferon-gamma (IFN-gamma). Combination of IFN-gamma and activation with CD154 resulted in an additive effect. To conclude, activated T cells may enhance IL-15 expression in two ways: by secreting IFN-gamma and by cell to cell contact using CD154. Each signal alone induces IL-15 in similar magnitudes, and both signals are additive. Because IL-15 is a major T-cell activator, this interaction may contribute to graft rejection.

CD40 Antigens↗
PubMed1994

Recombinant human hemoglobin inhibits both constitutive and cytokine-induced nitric oxide-mediated relaxation of rabbit isolated aortic rings.

A genetically engineered recombinant human hemoglobin (rHb1.1) was recently developed for use as a blood substitute (Nature 1992;356:258-60). Like other mammalian hemoglobin (Hb) molecules, it might bind and antagonize the actions of nitric oxide (NO). We used an isolated rabbit aortic ring preparation to examine the ability of rHb1.1 to inhibit acetylcholine (ACh)- and interleukin-1 beta (IL-1 beta)-induced reductions of vasoconstrictor responses to the alpha-adrenoceptor agonist phenylephrine (PE). rHb1.1 (0.04-4.4 microM) rapidly and reversibly inhibited, in a concentration-dependent manner, both ACh- and IL-1 beta-induced decreases in PE contractile responses. These inhibitory effects of rHb1.1 were non-competitive and were equipotent to those of purified, cell-free human Hb (p.hHb). These two forms of soluble Hb were at least 10 times more potent than Hb in erythrocytes (red blood cells: RBC-Hb). Both NG-nitro-L-arginine (10 microM) a NO synthase inhibitor, and LY-83583 (10 microM), a guanylyl cyclase inhibitor, mimicked the effects of rHb1.1. The inhibitory effects of rHb1.1 were not shared by either human serum albumin (HSA 44 microM), which combines with but does not deactivate NO, or cytochrome C (44 microM), a heme-containing protein that does not bind NO; neither were they reversed by L-arginine (L-ARG) (1 mM), the presumed NO precursor. These and other results suggest that the chemical antagonism of NO is likely to be the mechanism by which rHb1.1 and other Hbs inhibit ACh- and IL-1 beta-induced decreases in the response to PE in rabbit aortic rings.

Acetylcholine↗
PubMed2000

A phase I radioimmunotherapy trial evaluating 90yttrium-labeled anti-carcinoembryonic antigen (CEA) chimeric T84.66 in patients with metastatic CEA-producing malignancies.

Chimeric T84.66 (cT84.66) is a genetically engineered human/murine chimeric IgG, with high affinity and specificity to carcinoembryonic antigen (CEA). The purpose of this Phase I dose escalation therapy trial was to evaluate the toxicities, biodistribution, pharmacokinetics, tumor targeting, immunogenicity, and organ and tumor absorbed dose estimates of cT84.66 labeled with 90Y. Patients with metastatic CEA-producing malignancies were first administered 5 mCi 111In-labeled DTPA-cT84.66 (5 mg), followed by administration of the therapy dose of 90Y-labeled DTPA-cT84.66 1 week later. The therapy infusion was immediately followed by a 72-h administration of DTPA at 250 mg/m2/24 h. Dose levels of administered activity ranged from 5 to 22 mCi/m2 with three to six patients per level. Serial nuclear scans, blood samples, and 24-h urine collections were performed out to 5 days after infusion. Human antichimeric antibody response was assayed out to 6 months. Patients were administered up to 3 cycles of therapy every 6 weeks. Radiation absorbed doses to organs were estimated using a five compartment model and MIRDOSE3. Twenty-two patients received at least one cycle of therapy, with one individual receiving two cycles and two receiving three cycles of therapy. All were heavily pretreated and had progressive disease prior to entry in this trial. Reversible leukopenia and thrombocytopenia were the primary dose-limiting toxicities observed. Maximum tolerated dose was reached at 22 mCi/ m2. In general, patients with liver metastases demonstrated more rapid blood clearance of the antibody. Thirteen patients developed an immune response to the antibody. Average radiation doses to marrow, liver, and whole body were 2.6, 29, and 1.9 cGy/mCi 90Y, respectively. Dose estimates to tumor ranged from 66 to 1670 cGy (8.7 to 52.2 cGy/mCi 90Y) for each cycle of therapy delivered. Although no major responses were observed, three patients demonstrated stable disease of 12-28 weeks duration and two demonstrated a mixed response. In addition, a 41-100% reduction in tumor size was observed with five tumor lesions. 90Y-labeled cT84.66 was well tolerated, with reversible thrombocytopenia and leukopenia being dose limiting. Patients with extensive hepatic involvement by tumor demonstrated unfavorable biodistribution for therapy with rapid blood clearance and poor tumor targeting. Average tumor doses when compared with red marrow doses indicated a favorable therapeutic ratio. Stable disease and mixed responses were observed in this heavily pretreated population with progressive disease. This trial represents an important step toward further improving the therapeutic potential of this agent through refinements in the characteristics of the antibody and the treatment strategies used. Future trials will focus on the use of peripheral stem cell support to allow for higher administered activities and the use of combined modality strategies with radiation-enhancing chemotherapy drugs. Further efforts to reduce immunogenicity through humanization of the antibody are also planned. Finally, novel engineered, lower molecular weight, faster clearing constructs derived from cT84.66 continue to be evaluated in preclinical models as potential agents for radioimmunotherapy.

Animals↗
PubMed2005

Medical bioremediation: prospects for the application of microbial catabolic diversity to aging and several major age-related diseases.

Several major diseases of old age, including atherosclerosis, macular degeneration and neurodegenerative diseases are associated with the intracellular accumulation of substances that impair cellular function and viability. Moreover, the accumulation of lipofuscin, a substance that may have similarly deleterious effects, is one of the most universal markers of aging in postmitotic cells. Reversing this accumulation may thus be valuable, but has proven challenging, doubtless because substances resistant to cellular catabolism are inherently hard to degrade. We suggest a radically new approach: augmenting humans' natural catabolic machinery with microbial enzymes. Many recalcitrant organic molecules are naturally degraded in the soil. Since the soil in certain environments - graveyards, for example - is enriched in human remains but does not accumulate these substances, it presumably harbours microbes that degrade them. The enzymes responsible could be identified and engineered to metabolise these substances in vivo. Here, we survey a range of such substances, their putative roles in age-related diseases and the possible benefits of their removal. We discuss how microbes capable of degrading them can be isolated, characterised and their relevant enzymes engineered for this purpose and ways to avoid potential side-effects.

Aging↗
PubMed2005

Electrical stimulation of excitable tissue: design of efficacious and safe protocols.

The physical basis for electrical stimulation of excitable tissue, as used by electrophysiological researchers and clinicians in functional electrical stimulation, is presented with emphasis on the fundamental mechanisms of charge injection at the electrode/tissue interface. Faradaic and non-Faradaic charge transfer mechanisms are presented and contrasted. An electrical model of the electrode/tissue interface is given. The physical basis for the origin of electrode potentials is given. Various methods of controlling charge delivery during pulsing are presented. Electrochemical reversibility is discussed. Commonly used electrode materials and stimulation protocols are reviewed in terms of stimulation efficacy and safety. Principles of stimulation of excitable tissue are reviewed with emphasis on efficacy and safety. Mechanisms of damage to tissue and the electrode are reviewed.

Animals↗
PubMedDate not supplied

On the role of complement and Fc gamma-receptors in the Arthus reaction.

The contribution of either the complement system or the activation of Fc receptors for IgG (FcyRs) to the inflammatory response in immune complex (IC) disease is puzzling. A series of studies has been performed in mice with engineered deficiencies of either FcgammaRs, the complement components C3, C4 or the C5a receptor. In addition, different C5-deficient mice strains have been evaluated. Mice with gene targeted disruption of the gamma-subunit, which mediates surface expression and signal transduction of the high affinity Fc receptor type I for IgG (FcgammaRI), the low affinity receptor Fc receptor type III for IgG (FcgammaRIII) and the high affinity receptor type I for IgE (IgepsilonRI), showed an impaired inflammatory response in the reverse passive Arthus reaction in skin, peritoneum and lung. These data suggest, that the activation of FgammaRs is the initial event triggering the inflammatory cascade in IC disease. On the other hand, C5aR deficient mice are either protected from tissue injury induced by ICs, as in the lung, or the degree of the inflammatory response is markedly attenuated, as in peritoneum and skin. A detailed analysis of data obtained with the different knock-out strains revealed that both the activation of the complement system as well as the activation of different effector cells via FcgammaRs contribute to the inflammatory sequelae leading to tissue destruction in IC disease. The relative contributions of FcgammaRI or FcgammaRIII and the main effector cells through which these receptors mediate their effector functions are tissue dependent. The activation of the C5a receptor pathway appears to be the prominent contribution of the complement system.

Animals↗
PubMed2002

Blue myoglobin reconstituted with an iron porphycene shows extremely high oxygen affinity.

Myoglobin will be a good scaffold for engineering a function into proteins. To modulate the physiological function of myoglobin, almost all approaches have been demonstrated by site-directed mutagenesis, however, there are few studies which show a significant improvement in the function. In contrast, we focused on the replacement of heme in the protein with an artificial prosthetic group. Recently, we prepared a novel myoglobin reconstituted with an iron porphycene as a structural isomer of mesoheme. The bluish colored reconstituted myoglobin is relatively stable and the deoxymyoglobin reversibly binds ligands. Interestingly, the O2 affinity of the reconstituted myoglobin, 1.1 x 109 M-1, is a significant 1,400-fold higher than that of the native myoglobin. Furthermore, the unfavorable autoxidation kinetics show 7-fold decrease in rate for the reconstituted myoglobin relative to the native myoglobin, indicating the stable oxy-form against autoxidation. The net results come from the slow dissociation of the O2 ligand in the reconstituted myoglobin, koff = 0.11 s-1, because of the formation of strong hydrogen bond between His64 and negatively charged dioxygen. The present study indicates that the replacement of native heme with an artificially created prosthetic group will give us a unique function into a hemoprotein.

Heme↗
PubMed1993

Plants transformed with a cistron of a potato virus Y protease (NIa) are resistant to virus infection.

An oligonucleotide carrying signals for translation initiation in plants was engineered upstream to a cDNA clone containing nucleotides 5812-7260 of the potato virus Y (PVY) genome. This fragment contains all but the first 100 5' terminal bases of the cistron encoding one of the PVY proteases (NIa) as well as the first 251 bases of the next cistron (NIb). Nicotiana tabacum cv. SR1 plants were transformed with this fragment. The presence of the NIa sequences in transformed plants was determined by hybridization or PCR, and its expression was ascertained by reverse transcription coupled to PCR. Plants expressing NIa were self-pollinated, and the R1 kanamycin-resistant progeny were rechecked for NIa expression. Several of these plants were found to be resistant to PVY infection, inasmuch as they did not develop symptoms for at least 50 days (the duration of the experiments), and no viral accumulation could be detected in their leaves by ELISA. All of the descendents of resistant homozygous R2 plants were also resistant. Several of the plants transformed with the last three cistrons of PVY (bases 5812-9704; NIa-NIb-coat protein) were also resistant to PVY. None of the transformed plants exhibited resistance to tobacco mosaic virus. Exposure of the plants to 35 degrees C for 48 hr prior to inoculation lowered, but did not abolish, resistance.

Base Sequence↗
PubMed2000

Stopped-flow reaction kinetics of recombinant components of proton-translocating transhydrogenase with physiological nucleotides.

New information on the high resolution structure of the membrane proton pump, transhydrogenase, now provides a framework for understanding kinetic descriptions of the enzyme. Here, we have studied redox reactions catalyzed by mixtures of the recombinant NAD(H)-binding component (dI) of Rhodospirillum rubrum transhydrogenase, and the recombinant NADP(H)-binding component (dIII) of either the R. rubrum enzyme or the human enzyme. By recording changes in the fluorescence emission of native and engineered Trp residues, the rates of the redox reaction with physiological nucleotides have been measured under stopped-flow conditions, for the first time. Rate constants for the binding reaction between NAD(+)/NADH and the R. rubrum dI.dIII complex are much greater than those between nucleotide and isolated dI. For the redox step between the physiological nucleotides on the R. rubrum dI. dIII complex, the rate constant in the forward direction, k(f) approximately 2900 s(-1), and that for the reverse reaction, k(r) approximately 110 s(-1). Comparisons with reactions involving an analogue of NAD(H) indicate that the rate constants at this step are strongly affected by the redox driving force.

Dose-Response Relationship, Drug↗
PubMed2004

Receptor knock-out and knock-in strategies.

Accumulating examples have demonstrated that knock-out and knock-in mice of G-protein-coupled receptors (GPCRs) are useful in elucidating physiological functions of the receptor in vivo. GPCR knock-out and knock-in are achieved by either (1) manipulation of the endogenous locus of the receptor gene or (2) transgenic expression of the modified receptor. Historically speaking, the first generation knock-outs made the best use of homologous recombination in embryonic stem (ES) cells and their totipotency to introduce the desired mutation into the endogenous receptor locus. In the second-generation knock-outs using the Cre/loxP system, the disruption of the receptor gene is cell-type specific or region-specific but is irreversible in principle. In contrast, transgenic expression in the receptor knock-out mice of the wild-type receptor protein under a tissue- and stage-specific promoter (conditional "rescue" of the receptor knock-out) can be easily applied to create "reversible" or "inducible" knock-out of the receptor. This is called the third generation knock-out. In the following sections, we introduce examples of the materials and methods based on our in vivo analyses of the metabotropic glutamate receptor-subtype 1 (mGluR1).

Animals↗
PubMed1996

Bypassing the ribosome: peptide synthesis without translation.

Chemical peptide synthesis is well-established but has drawbacks, notably the need to protect side-chain functional groups during reactions. Proteolytic enzymes may be used 'in reverse' to catalyse peptide synthesis without side-chain protection and avoiding the danger of racemization. Enzymic syntheses may use either an equilibrium or a kinetic strategy. Equilibrium synthesis is simply the reversal of hydrolysis, using any type of protease. Kinetic synthesis involves time-dependent acyl transfer using activated substrates and a serine or cysteine protease. Enzymic synthesis often takes place in partially aqueous or non-aqueous reaction media; the medium can often desirably influence the protease's properties. It is possible to tailor a protease to improve its usefulness in peptide synthesis; one can alter the enzyme's properties by means such as chemical modification, PEG-coupling or protein engineering.

Binding Sites↗
PubMed2001

Sequence-specific detection of individual DNA strands using engineered nanopores.

We describe biosensor elements that are capable of identifying individual DNA strands with single-base resolution. Each biosensor element consists of an individual DNA oligonucleotide covalently attached within the lumen of the alpha-hemolysin (alphaHL) pore to form a "DNA-nanopore". The binding of single-stranded DNA (ssDNA) molecules to the tethered DNA strand causes changes in the ionic current flowing through a nanopore. On the basis of DNA duplex lifetimes, the DNA-nanopores are able to discriminate between individual DNA strands up to 30 nucleotides in length differing by a single base substitution. This was exemplified by the detection of a drug resistance-conferring mutation in the reverse transcriptase gene of HIV. In addition, the approach was used to sequence a complete codon in an individual DNA strand tethered to a nanopore.

Base Pair Mismatch↗
PubMedDate not supplied

High-throughput purification of single compounds and libraries.

The need for increasing productivity in medicinal chemistry and associated improvements in automated synthesis technologies for compound library production during the past few years have resulted in a major challenge for compound purification technology and its organization. To meet this challenge, we have recently set up three full-service chromatography units with the aid of in-house engineers, different HPLC suppliers, and several companies specializing in custom laboratory automation technologies. Our goal was to combine high-throughput purification with the high attention to detail which would be afforded by a dedicated purification service. The resulting final purification laboratory can purify up to 1000 compounds/week in amounts ranging from 5 to 300 mg, whereas the two service intermediate purification units take 100 samples per week from 0.3 to 100 g. The technologies consist of normal-phase and reversed-phase chromatography, robotic fraction pooling and reformatting, a bottling system, an automated external solvent supply and removal system, and a customized, high-capacity freeze-dryer. All work processes are linked by an electronic sample registration and tracking system.

Automation↗
PubMed2006

The long alpha-helix of SecA is important for the ATPase coupling of translocation.

SecA contains two ATPase folds (NBF1 and NBF2) and other interaction/regulatory domains, all of which are connected by a long helical scaffold domain (HSD) running along the molecule. Here we identified a functionally important and spatially adjacent pair of SecA residues, Arg-642 on HSD and Glu-400 on NBF1. A charge-reversing substitution at either position as well as disulfide tethering of these positions inactivated the translocation activity. Interestingly, however, the translocation-inactive SecA variants fully retained the ability to up-regulate the ATPase in response to a preprotein and the SecYEG translocon. The translocation defect was suppressible by second site alterations at the hinge-forming boundary of NBF2 and HSD. Based on these results, we propose that the motor function of SecA is realized by ligand-activated ATPase engine and its HSD-mediated conversion into the mechanical work of preprotein translocation.

Adenosine Triphosphatases↗
PubMed1991

Granulocyte colony-stimulating factor gene transfer suppresses tumorigenicity of a murine adenocarcinoma in vivo.

We have investigated the effect of granulocyte colony-stimulating factor (G-CSF) delivery at the site of tumor growth by transducing, via retroviral vector, the human (hu) G-CSF gene into the colon adenocarcinoma C-26 and assaying the ability of transduced cells to form tumors when injected into syngeneic mice. As a control, the same tumor cells were infected with retroviruses engineered to transduce an unrelated gene, the human nerve growth factor receptor, or carry the neomycin resistance gene only. Only cells transduced with the huG-CSF were unable to develop tumors, although huG-CSF was expressed and produced at low level as estimated by both RNA analysis and enzyme-linked immunosorbent assay, indicating that G-CSF can exert an antitumor effect at a physiological dose. Implication of G-CSF as mediator of tumor inhibition was proven by reversing the nontumorigenic phenotype of G-CSF-expressing cells with anti-huG-CSF monoclonal antibody injected at the tumor site. No tumors were formed by injecting C-26 infected cells into nu/nu mice, while neoplastic nodules appeared after injection into sublethally irradiated mice; such tumors, however, regressed when mice normalized their leukocyte counts after irradiation. Tumors were also formed after injection of a mixture of infected and uninfected C-26 cells, although critical delay in tumor formation occurred when infected cells were 10 times more represented in the mixture. Histological examination of tissues surrounding the site of injection showed infiltration of neutrophilic granulocytes, whose number correlated with that of G-CSF-expressing C-26 cells in the injected mixture. These results indicate that G-CSF may have a potent antitumoral activity when released, even at low doses, at the tumor site. The antitumoral effect is mediated by recruitment and targeting of neutrophilic granulocytes to G-CSF-releasing cells.

Adenocarcinoma↗
PubMed2006

Identification of 5' and 3' cis-acting elements of the porcine reproductive and respiratory syndrome virus: acquisition of novel 5' AU-rich sequences restored replication of a 5'-proximal 7-nucleotide deletion mutant.

We here demonstrate the successful engineering of the RNA genome of porcine reproductive and respiratory syndrome virus (PRRSV) by using an infectious cDNA as a bacterial artificial chromosome. Runoff transcription from this cDNA by SP6 polymerase resulted in capped synthetic RNAs bearing authentic 5' and 3' ends of the viral genome that had specific infectivities of >5 x 10(5) PFU/microg of RNA. The synthetic viruses recovered from the transfected cells were genotypically and phenotypically indistinguishable from the parental virus. Using our system, a series of genomic RNAs with nucleotide deletions in their 5' ends produced viruses with decreased or no infectivity. Various pseudorevertants were isolated, and acquisition of novel 5' sequences of various sizes, composed predominantly of A and U bases, restored their infectivities, providing a novel insight into functional elements of the 5' end of the PRRSV genome. In addition, our system was further engineered to generate a panel of self-replicating, self-limiting, luciferase-expressing PRRSV viral replicons bearing various deletions. Analysis of these replicons revealed the presence and location of a 3' cis-acting element in the genome that was required for replication. Moreover, we produced enhanced green fluorescent protein-expressing infectious viruses, which indicates that the PRRSV cDNA/viral replicon/recombinant virus can be developed as a vector for the expression of a variety of heterologous genes. Thus, our PRRSV reverse genetics system not only offers a means of directly investigating the molecular mechanisms of PRRSV replication and pathogenesis but also can be used to generate new heterologous gene expression vectors and genetically defined antiviral vaccines.

3' Flanking Region↗
PubMed2001

Bridging areas of injury in the spinal cord.

There is a devastating loss of function when substantial numbers of axons are interrupted by injury to the spinal cord. This loss may be eventually reversed by providing bridging prostheses that will enable axons to regrow across the injury site and enter the spinal cord beyond. This review addresses the bridging strategies that are being developed in a number of spinal cord lesion models: complete and partial transection and cavities arising from contusion. Bridges containing peripheral nerve, Schwann cells, olfactory ensheathing glia, fetal tissue, stem cells/neuronal precursor cells, and macrophages are being evaluated as is the administration of neurotrophic factors, administered by infusion or secreted by genetically engineered cells. Biomaterials may be an important factor in developing successful strategies. Due to the complexity of the sequelae following spinal cord injury, no one strategy will be effective. The compelling question today is: What combinations of the strategies discussed, or new ones, along with an initial neuroprotective treatment, will substantially improve outcome after spinal cord injury?

Animals↗
PubMed2002

[Molecular cloning of human IL-7cDNA and construction of eukaryotic vector expressing hIL-7]

OBJECTIVE: To construct a vector expressing eukaryotic human interluken-7(hIL-7). METHODS: hIL-7 DNA was identified and cloned (cDNA) from human spleen tissue using reverse transcription polymerase chain reaction (RT-PCR). We incorporated the cDNA into the pMD18-T plasmid. The pMD18-T plasmid was then inserted into a dual expression vector (prokaryotic and eukaryotic) pBK-CMV and called pBK-CMV-hIL-7. We used pBK-CMV-hIL-7 vector to infect E.coli DH5alpha. The expression of the recombinant hIL-7 protein (rhIL-7) by E.coli DH5alpha was analyzed using SDS-PAGE and western blot testing. RESULTS: The genetically engineered E.coli DH5alpha did express rhIL-7 confirmed by western blot. CONCLUSION: The successful construction of genetically engineered eukaryotic gene for hIL-7 was done, This will enable further research into therapeutic uses for hIL-7.

Journal Article↗