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At least 145 records · Page 8Linked to original sources
PubMed1995

The effect of fuel composition on the mutagenicity of diesel engine exhaust.

The effect of fuel composition on the mutagenicity of diesel engine emission was investigated. To this end, a fuel matrix comprising fuels with different contents of aromatic and naphthenic compounds was used. Extracts of the organic phase of raw exhausts obtained with different fuels were tested for mutagenicity in bacterial reversion assays. The results obtained demonstrate that the mutagenicity of diesel exhaust is largely dependent on the aromatic content of the fuel. In fact, mutagenicity was greatly reduced when the aromatic content of the fuel was lowered by hydrogen treatment. Conversely, mutagenicity was enhanced when the fuel was enriched with fractions of di- or triaromatic compounds. The addition of di- and trinaphthenic compounds only produced borderline mutagenicity. No clear relationship was observed between sulfur content of the fuel and mutagenicity of the exhaust. Assays in bacterial strains with different sensitivity to nitroaromatic compounds suggest a low contribution of the highly mutagenic dinitropyrenes to the responses observed, and a relatively greater contribution of 1-nitropyrene or other nitroaromatics processed by the same bacterial nitroreductase.

Mutagenicity Tests↗
PubMed1989

Tissue-type plasminogen activator variants with domain duplications and rearrangements.

The interactions between tPA domains that are important for catalysis are poorly understood. We have probed the function of interdomain interactions by generating tPA variants in which domains are duplicated or rearranged. The proteins were expressed in a transient mammalian expression system and tested in vitro for their ability to activate plasminogen, induce fibrinolysis and bind to a forming fibrin clot. Duplication of the heavy chain domains of tPA produced enzymatically active tPA variants, many of which demonstrated similar in vitro amidolytic and fibrinolytic activity and similar fibrin affinity to the parent molecule. Zymographic analysis of the domain duplication tPA variants showed one major active species for each variant. Selection of the residues duplicated and the interdomain spacing were found to be critical considerations in the design of tPA variants with duplicated domains. We also rearranged the domains of tPA such that kringle 1 replaced the second kringle domain and vice versa. An analysis of these variants indicates that the first kringle domain can confer fibrin affinity to a tPA variant and function in place of kringle 2. Therefore, in wild-type tPA, the functions of kringle 1 and kringle 2 must be dependent partially on their orientation within the heavy chain of the protein. The functional autonomy of the heavy and light chains of tPA is demonstrated by the activity of a tPA variant in which the order of the heavy and light chains was reversed.

Base Sequence↗
PubMed1994

Peptide mapping of subtilisins as a practical tool for locating protein sequence errors during extensive protein engineering projects.

Protein engineering based on rational design is an iterative process of sequential amino acid residue replacements. This requires a rapid and sensitive method for checking the protein sequence after each round of mutagenesis. As shown with subtilisin BL, acid treatment followed by urea denaturation renders the enzyme degradable by trypsin within 10 min. Separation of the peptides by reversed-phase HPLC produces a map that differentiates even the most conservative alteration on peptides as large as 48 amino acid residues. The method was used to uncover erroneous mutations; to determine the concentration of active protease relative to an internal standard of known specific activity; to measure the rate of oxidation of methionine-216 in the oxyanion hole of subtilisin BL; and to document that under these conditions no other methionine in the molecule is oxidized by hydrogen peroxide.

Amino Acid Sequence↗
PubMed1992

Behavior of large nucleic acids in reversed-phase high-performance liquid chromatography.

Large nucleic acids can be separated by reversed-phase high-performance liquid chromatography. Analysis shows that the retention time depends not only on the chain length but also on the base composition and the secondary structure of the molecule. A model is proposed to interpret their behavior. This model, called "multiple-point interaction theory" is based on the observation that macromolecules are flexible and very large compared to the hydrophobic phase (octadecylsilane) of the column. It explains the behavior of large nucleic acids in terms of an equilibrium of the macromolecule between the two phases through a multiple-point attachment to the chromatographic matrix, the parameters of the equilibrium being both the hydrophobicity of the base and the number of attachment points. This model fits the experimental data and can be applied to all types of flexible macromolecules, especially proteins and nucleic acids, when they are chromatographed on reversed-phase columns. The model is used to explain the separation of nucleic acids of importance in molecular biology.

Chromatography, High Pressure Liquid↗
PubMed1989

Isolation of recombinant hirudin by preparative high-performance liquid chromatography.

The purification of recombinant hirudin variant 2-Lys47 (rHV2-Lys47), produced by a genetically engineered yeast strain, is described. rHV2-Lys47 expressed and secreted into the culture medium was the starting material for the purification process of hirudin from the culture broth after cell harvesting by centrifugation. Initial purification of the product by preparative reversed-phase high-performance liquid chromatography (HPLC) using step-gradient elution, followed by precipitation of rHV2-Lys47 in the presence of acetone, removed most of the contaminants from the culture medium. The pure product was obtained by successive preparative anion-exchange and reversed-phase HPLC on silica based stationary phases. Characterization of the final product by analytical HPLC, isoelectric focusing gel electrophoresis, quantitative amino acid composition and sequence analysis did not reveal any contaminants. Liquid secondary ion mass spectrometry was used to confirm its primary structure. The isolated product was tested in an inhibition assay of human alpha-thrombin and proved to be fully active.

Chromatography, High Pressure Liquid↗
PubMed1983

Improved methods for structure probing in large RNAs: a rapid 'heterologous' sequencing approach is coupled to the direct mapping of nuclease accessible sites. Application to the 5' terminal domain of eukaryotic 28S rRNA.

We have developed a combined approach for probing native structures in large RNAs. In the first method, after digestion with a structure specific nuclease, accessible sites are mapped at sequence resolution along the entire RNA molecule which is used as a template for the reverse transcriptase elongation of a 5' end labelled selected primer (coding strand of a small restriction fragment of the cloned gene). This method circumvents any prior end-labelling of RNA, a technique with major limitations for large RNAs. In the second approach, a rapid "heterologous" sequencing can be easily applied to definite domains of an RNA molecule in a variety of species (or individuals), without additional DNA cloning nor end-labelling of RNA. By taking advantage of the presence of evolutionary conserved tracts within an RNA sequence, it allows a rapid analysis of RNA folding patterns in terms of phylogenetic comparisons : when located within such a conserved tract, selected restriction fragments from a cloned gene can be used as heterologous primers for sequencing the upstream divergent region in RNAs of other species by currently available technology, i.e. reverse transcriptase elongation in the presence of chain terminator dideoxynucleotides.

Animals↗
PubMed1995

Immunomodulation of transplant rejection using monoclonal antibodies and soluble receptors.

The main objective of our studies has been to optimize the effects of monoclonal antibodies (MAbs) and other immunosuppressive reagents to enhance organ graft survival. One such agent is OKT3, a MAb that is directed against the CD3 component of the human T-cell receptor (TCR) complex. Treatment of a rejection episode with OKT3 results in a rapid and efficient clearing of circulating T cells and reversal of most rejection episodes. Its wider use in transplantation and in the treatment of immune-mediated disease is limited by adverse reactions that follow the initial dose, the production of neutralizing Abs, and the transient nature of the immunosuppression. We have engineered CDR-grafted "humanized" anti-CD3 MAbs that lack Fc-receptor binding activity through mutagenesis of amino acids in the Fc portion of the MAb. This results in an immunosuppressive anti-CD3 MAb that is less antigenic and one that does not induce the first-dose side effects. In addition, we have pursued a goal of developing a therapy that will induce donor-specific tolerance while maintaining overall recipient immune competency. Because antigen-specific T-cell activation depends not only on TCR-ligand interaction, but also on additional costimulatory signals mediated by accessory molecules such as CD28, blocking the binding of CD28 on T cells to its ligand B7, during TCR engagement, might modulate transplantation responses. Using a soluble fusion protein of human CTLA4, CTLA4-Ig, that binds B7 with high affinity, inhibition of human pancreatic islet rejection that occurs, at least in part, by affecting T-cell recognition of human B7+ antigen-presenting cells has been demonstrated.(ABSTRACT TRUNCATED AT 250 WORDS)

Antibodies, Monoclonal↗
PubMed1995

Effects of beta 1-integrin antisense phosphorothioate-modified oligonucleotide on myoblast behaviour in vitro.

Myoblasts gene-engineered in vitro and then injected in vivo are safe, efficient options for gene therapy. While isolation of satellite cells is routinely achieved, their proliferation potential in vitro remains a limiting factor for cell transplantation under clinical conditions. We have studied the role of reversible inhibition of gene expression by antisense oligonucleotides on the proliferation of the myogenic cells. Addition of antisense oligonucleotides to myoblast cultures has been used to inhibit specifically the expression of the beta 1-integrin subunit gene. Here we show that the effects of multiple pulses of a phosphorothioate oligodeoxinucleotide antisense on the attachment to substrata and on the proliferation of myoblasts are dose-dependent. The addition of antisense to rat myoblasts caused rounding up of the cells and most of the cells became detached after several days in culture. A single pulse did not show any consistent effect, while in the presence of continuously administered antisense, the relative numbers of myoblasts in the treated muscle culture increased. We have no evidence of inhibition of myoblast fusion under these conditions. On the other hand, [3H]-TdR incorporation, total DNA and total number of cells decreased in antisense-treated cultures thus demonstrating an inhibitory effect of the phosphorothioate oligonucleotides on DNA synthesis. These side-effects could be overcome by substituting the phosphorothioate by unmodified oligonucleotides, so decreasing the half-life of the antisense, but also its toxicity. The overall results suggest a potential role of integrin antisense strategy in modulating the potential of myoblasts to proliferate.

Animals↗
PubMed2026

Current landscape of Cys-OxiPTMs in plants: from hormone signaling to phenotypic control and their potential in sustainable agriculture.

The integration of environmental and developmental cues into coherent physiological responses is fundamental to plant survival. Reactive oxygen, nitrogen, and sulfur species (ROS/RNS/RSS) are now recognized as essential signaling molecules, not merely cytotoxic byproducts. Their specificity is largely achieved through reversible, site-specific cysteine oxidative post-translational modifications (Cys-OxiPTMs), which constitute a dynamic and sophisticated "redox code". This review provides a systematic synthesis of the current landscape of Cys-OxiPTMs in plants, bridging chemistry, hormone biology, agronomy, detection, and engineering. The chemical and enzymatic basis of major Cys-OxiPTMs is detailed, along with a discussion of how their spatiotemporal interplay orchestrates signaling specificity. A critical examination is then presented on how these modifications decode and integrate plant hormone signaling networks to regulate key agronomic traits. Cutting-edge proteomic technologies that have revolutionized the identification of redox-sensitive cysteines are also evaluated. Finally, forward-looking strategies to "write" the redox code are explored. By moving the field from descriptive cataloging to predictive "redox breeding", this review establishes a foundational framework for manipulating Cys-OxiPTMs to develop climate-resilient, high-yielding crops for sustainable agriculture.

Agronomic traits↗
PubMed1992

Expression of murine interleukin 7 in a murine glioma cell line results in reduced tumorigenicity in vivo.

We have examined the immunoregulatory effect of local and continuous secretion of interleukin 7 (IL-7) from murine glioma cells (203-glioma) engineered by murine IL-7 gene transfection. Secretion of IL-7 from glioma cells did not result in morphology or growth rate changes but did reduce tumorigenicity in vivo in proportion to the amount of IL-7 produced. This reduction in tumorigenicity could be reversed in a dose-dependent fashion by injection of anti-IL-7 neutralizing monoclonal antibody at the tumor site. Mice immunized with IL-7-producing glioma cells showed a specific immune response to 203-glioma but not to two other syngeneic cell lines (B-16, a melanoma, and YM-12, a fibrosarcoma). IL-7-producing glioma cells were not rejected in mice depleted of CD8+ cells but were rejected in mice depleted of CD4+ or NK1.1+ cells. These results suggest that CD8+ T cells may play an important role in tumor rejection.

Animals↗
PubMed1990

A putative approach for gene therapy against human immunodeficiency virus (HIV).

It is proposed here that a form of intracellular immunity can be devised which would protect cells from virus infection and, in particular, could be used as a treatment for the human immunodeficiency virus (HIV) infected individual. Following in vitro immunization of naive human B lymphocytes with reverse-transcriptase (RT) or HIV transactivator protein (tat), messenger RNA (mRNA) would be isolated from these cells. Using the mRNA molecules as templates, copy DNA (cDNA) molecules encoding the RT or tat-specific immunoglobulins, are prepared and amplified by the polymerase chain reaction. After engineering of the antibody encoding cDNAs to provide appropriate intracellular addressing information, the cDNAs would be used to transfect stem cells of HIV infected individuals in vitro. The presence, in the cytoplasm and nucleus, of antibodies which had been selected to interfere with the reproduction of the virus, would protect these cells from infection. Autologous transplantation of such cells would confer resistance against HIV replication by these stem cells and their progeny in the treated individual. Such a strategy may also be useful against other retroviruses and could provide resistance against retrovirally triggered leukemia.

DNA↗
PubMed2026

Light-activated CRISPR/dCas9 nanomedicine for programmable control of renal fibrosis.

Renal fibrosis is the final common pathway of progressive chronic kidney disease and is maintained by spatially heterogeneous interactions among injured epithelial cells, activated fibroblasts, immune cells, extracellular matrix remodeling, metabolic stress, and persistent profibrotic transcriptional programs. Current therapies slow renal functional decline but do not directly control the regulatory circuits that stabilize maladaptive repair. Photoresponsive renal nanomedicine offers a potential strategy to add external control to anti-fibrotic intervention by combining kidney-directed delivery with light-gated release or activation of molecular payloads. This review examines the emerging interface between photoresponsive nanomaterials and CRISPR/dCas9-based gene regulation for renal fibrosis, with emphasis on upconversion nanoparticles, photoresponsive polymers, ROS- and pH-responsive matrices, optogenetic switches, and renal-compartment-directed carrier design. We argue that the most defensible therapeutic objective is not permanent genome editing or autonomous organ regeneration, but spatially confined, temporally limited, and reversible regulation of validated fibrotic or protective gene programs using CRISPRa, CRISPRi, or dCas9-based epigenome editors. The review therefore evaluates material requirements, optical-dosimetry constraints, payload architecture, renal biodistribution, target-selection logic, safety risks, and preclinical validation criteria. By defining the engineering and biological conditions required for controlled anti-fibrotic regulation, this framework positions photoresponsive renal nanomedicine as a translationally testable route toward localized modulation of fibrotic cell states rather than an overextended claim of kidney regeneration.

Anti-fibrotic gene regulation↗
PubMed1995

T-cell recognition of antigen. A process controlled by transient intermolecular interactions.

As recently as ten years ago, the nature of the T-cell receptor for antigen was a mystery, as was the precise role of histocompatibility molecules in antigen-presentation to T cells. Although T-cell receptors have now been cloned and crystal structures of MHC/peptide molecules exist, our understanding of the parameters that characterize this interaction and other interactions relevant to T-cell immunity are still unclear. The engineering of soluble forms of proteins that mediate T-cell recognition of antigen has allowed the first measurements of these parameters. Interestingly, many of these interactions are of a transient nature, with very rapid off-rates. These data suggest a model whereby highly reversible intermolecular interactions mediate the cell-cell association. The association of adhesion molecules is probably the first step in the stabilization of a conjugate, because they are more numerous than any antigen-specific interaction, followed later by TCR-MHC engagements. Diffusion within each lipid bilayer should allow the congregation of MHC/TCR interactions at the cell-cell interface, with peptide-specific TCR interactions outcompeting irrelevant interactions. Rapid off-rates for both the antigen-specific and nonspecific interactions may be necessary to maintain reversibility, yet allow a rapid approach to equilibrium and consequent signaling when a specific antigen is present or disengagement when it is not.

Animals↗
PubMed1992

Determination of the excited-state lifetimes of the tryptophan residues in barnase, via multifrequency phase fluorometry of tryptophan mutants.

A multifrequency phase fluorometric study is described for wild-type barnase and engineered mutant proteins in which tryptophan residues have been replaced by less fluorescent residues which do not interfere with the determination of the tryptophan emission spectra and lifetimes. The lifetimes of the three tryptophans in the wild-type protein have been resolved. Trp-35 has a single fluorescence lifetime, which varies in the different proteins between 4.3 and 4.8 ns and is pH-independent between pH 5.8 and 8.9. Trp-71 and Trp-94 behave as an energy-transfer couple with both forward and reverse energy transfer. The couple shows two fluorescence lifetimes: 2.42 (+/-0.2) and 0.74 (+/-0.1) ns at pH 8.9, and 0.89 (+/-0.05) and 0.65 (+/-0.05) ns at pH 5.8. In the mutant Trp-94----Phe the lifetime of Trp-71 is 4.73 (+/-0.008) ns at high pH and 4.70 (+/-0.004) ns at low pH. In the mutant Trp-71----Tyr, the lifetime of Trp-94 is 1.57 (+/-0.01) ns at high pH and 0.82 (+/-0.025) ns at low pH. From these lifetimes, one-way energy-transfer efficiencies can be calculated according to Porter [Porter, G.B. (1972) Theor. Chim. Acta 24, 265-270]. At pH 8.9, a 71% efficiency was found for forward transfer (from Trp-71 to Trp-94) and 36% for reverse transfer. At pH 5.8 the transfer efficiency was 86% for forward and 4% for reverse transfer (all +/-2%). These transfer efficiencies correspond fairly well with the ones calculated according to the theory of Förster [Förster, T. (1948) Ann. Phys. (Leipzig) 2, 55-75].(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed2025

New retron systems from environmental bacteria identify triggers of anti-phage defense and expand tools for genome editing.

Retrons are bacterial immune systems that protect a bacterial population against phages by killing infected hosts. Retrons typically comprise a reverse transcriptase (RT), a template noncoding RNA that is partially reverse transcribed into RT-DNA, and a toxic effector. The reverse transcriptase (RT), noncoding RNA, and RT-DNA complex sequester the toxic effector until triggered by phage infection, at which point the toxin is released to induce cell death. Due to their ability to produce single-stranded DNA in vivo, retrons have also been engineered to produce donor templates for genome editing in both prokaryotes and eukaryotes. However, the current repertoire of experimentally characterized retrons is limited, with most retrons sourced from clinical and laboratory strains of bacteria. To better understand retron biology and natural diversity, and to expand the current toolbox of retron-based genome editors, we developed a pipeline to isolate retrons and their bacterial hosts from a variety of environmental samples. Here, we identify seven new retron systems, each isolated from a different host bacterium. We characterize DNA production by these retrons and test their ability to defend against a panel of Escherichia coli phages. We find that two of these retrons are disrupted by other elements, in one case a group II intron and in another a separate defense system, yet both retrons still produce RT-DNA. For two other retrons, we further unravel their mechanism of defense by identifying the phage genes responsible for triggering abortive infection. Finally, we engineer these retrons for genome editing in E. coli, demonstrating their potential use in a biotechnological application.

Gene Editing↗
PubMed1986

Protein engineering of homodimeric tyrosyl-tRNA synthetase to produce active heterodimers.

Heterodimers of tyrosyl-tRNA synthetase from Bacillus stearothermophilus have been produced by mutagenesis at the subunit interface. Oppositely charged groups have been engineered into the subunits so that they can form a complementary pair. Wild-type tyrosyl-tRNA synthetase is a symmetrical dimer in which the side chains of the 2 Phe-164 residues interact at the subunit interface. Phe-164 was mutated to Asp in tyrosyl-tRNA synthetase and to Lys in a truncated enzyme (des-(321-419)tyrosyl-tRNA synthetase) which lacks the two tRNA-binding sites, but which can catalyze pyrophosphate exchange. The size difference allows subunit association to be studied by gel filtration chromatography. These changes induce reversible dissociation from active dimers into inactive monomers at pH values which favor ionization at position 164. A mixture of the two mutants near neutral pH is apparently fully active in pyrophosphate exchange and consists of a heterodimer of [Asp164]tyrosyl-tRNA synthetase and [Lys164]des-(321-419)tyrosyl-tRNA synthetase. Despite having only one binding site for tRNA, heterodimer has full aminoacylation activity at high concentrations of tyrosine. We have therefore produced a family of dimers that differ in stability near neutral pH. This novel approach using protein engineering allows specific dimerization of subunits of the same size that have different defined mutations, each subunit being tagged by the charge. Such hybrid proteins can be used to study subunit interaction.

Amino Acyl-tRNA Synthetases↗
PubMed1991

Engineering a bioluminescent indicator for cyclic AMP-dependent protein kinase.

cDNA coding for the luciferase in the firefly Photinus pyralis was amplified in vitro to generate cyclic AMP-dependent protein kinase phosphorylation sites. The DNA was transcribed and translated to generate light-emitting protein. A valine at position 217 was mutated to arginine to generate a site RRFS and the heptapeptide kemptide, the phosphorylation site of the porcine pyruvate kinase, was added at the N- or C-terminus of the luciferase. The proteins carrying phosphorylation sites were characterized for their specific activity, pI, effect of pH on the colour of the light emitted and effect of the catalytic subunit of protein kinase A in the presence of ATP. Only one of the recombinant proteins (RRFS) was significantly different from wild-type luciferase. The RRFS mutant had a lower specific activity, lower pH optimum, emitted greener light at low pH and when phosphorylated it decreased its activity by up to 80%. This latter effect was reversed by phosphatase. This recombinant protein is a good candidate to measure for the first time cyclic AMP-dependent phosphorylation in live cells.

Amino Acid Sequence↗
PubMed1989

Expression of an enzymatically active murine retroviral reverse transcriptase in human cells.

The region of the pol gene of the Moloney murine leukemia virus (M-MuLV) encoding the reverse transcriptase and RNase H activities was inserted in an eukaryotic expression vector and transiently expressed in human cultured cells. This results in the expression of high levels of reverse transcriptase activity. This enzyme, partially purified, also carries a RNase H activity, has the biochemical requirements of the viral enzyme and is recognized and inhibited by antibodies directed against a M-MuLV reverse transcriptase expressed in Escherichia coli.

Animals↗