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At least 127 records · Page 7Linked to original sources
PubMed1991

Transgenic mice that express the human multidrug-resistance gene in bone marrow enable a rapid identification of agents that reverse drug resistance.

The development of preclinical models for the rapid testing of agents that circumvent multidrug resistance in cancer is a high priority of research on drug resistance. A common form of multidrug resistance in human cancer results from expression of the MDR1 gene, which encodes a Mr 170,000 glycoprotein that functions as a plasma membrane energy-dependent multidrug efflux pump. We have engineered transgenic mice that express this multidrug transporter in their bone marrow and demonstrated that these animals are resistant to leukopenia by a panel of anticancer drugs including anthracyclines, vinca alkaloids, etoposide, taxol, and actinomycin D. Differential leukocyte counts indicate that both neutrophils and lymphocytes are protected. Drugs such as cisplatin, methotrexate, and 5-fluorouracil, which are not handled by the multidrug transporter, produce bone marrow suppression in both normal and transgenic mice. The resistance conferred by the MDR1 gene can be circumvented in a dose-dependent manner by simultaneous administration of agents previously shown to be inhibitors of the multidrug transporter in vitro, including verapamil isomers, quinidine, and quinine. Verapamil and quinine, both at levels suitable for human trials that produced only partial sensitization of the MDR1-transgenic mice, were fully sensitizing when used in combination. We conclude that MDR1-transgenic mice provide a rapid and reliable system to determine the bioactivity of agents that reverse multidrug resistance in animals.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1990

Effect of intravenous drug administration mode on drug distribution in a tumor slab: a finite Fourier transform analysis.

Cancer therapy using chemotherapeutic drugs frequently involves injection of the drug into the body through some intravenous mode of administration, viz, continuous (drip) infusion or single/multiple bolus injection(s). An understanding of the effect of the various modes of administration upon tumor penetration of drug is essential to rational design of drug therapy. This paper investigates drug penetration into a model tumor of slab geometry (between two capillaries) in which the overall transport rate of drug is limited by intra-tumor transport characterized by an effective diffusion coefficient. Employing the method of Finite Fourier Transforms (FFT), analytical solutions have been obtained for transient drug distribution in both the plasma and the tumor following three modes of administration, viz, continuous infusion, single bolus injection and equally-spaced equal-dose multiple bolus injections, of a given amount of drug. The qualitative trends exhibited by the plasma drug distribution profiles are consistent with reported experimental studies. Two concepts, viz, the dimensionless decay constant and the plasma/tumor drug concentration trajectories, are found to be particularly useful in the rational design of drug therapy. The dimensionless decay constant provides a measure of the rate of drug decay in the plasma relative to the rate of drug diffusion into the tumor and is thus characteristic of the tumor/drug system. The magnitude of this parameter dictates the choice of drug administration mode for minimizing drug decay in the plasma while simultaneously maximizing drug transport into the tumor. The concentration trajectories provide a measure of the plasma drug concentration relative to the tumor drug concentration at various times following injection. When the tumor drug concentration exceeds the plasma drug concentration, the drug will begin to diffuse out of the tumor. Knowledge of the time at which this diffusion reversal occurs is especially useful for optimum scheduling of subsequent bolus injections in a multiple bolus dosing regimen. There are no reported applications of the FFT method to solve repeated input functions in either the chemical engineering or pharmaceutical science literature. Thus, the application of FFT method to solve multiple bolus injections is a unique one. Use of this FFT based analysis as a predictor tool can limit the number of costly experiments which are being done now to achieve this purpose. Even though the model in its present form is simplified, the analysis thereof has nevertheless led to a better understanding of the various factors that must be taken into account for rational design of drug therapy.

Drug Administration Routes↗
PubMed1992

[Application of PCR and RT-PCR method to molecular biology study in nephrology].

Recent advance in molecular biology and genetic engineering has made a profound influence on basic and clinical medicine. Development of PCR (polymerase chain reaction) has transformed our trend of thought in molecular biology. Applications of PCR method has extended to nephrology study. RT (reverse transcription)-PCR method was elaborated to identify mRNA even in a small number of cells. It is also important and advantageous that PCR facilitates the utilization of a non-radioisotopic detection system. Newly developed chemiluminescent procedure yields as high sensitivity just as radioactive probes. Further applications are now being investigated including in situ PCR. PCR and RT-PCR methods are of great value for studying a molecular biological background of intricate kidney functions and diverse renal disorders.

Cloning, Molecular↗
PubMed1990

Unfolding-refolding of the domains in yeast phosphoglycerate kinase: comparison with the isolated engineered domains.

The role of domains as folding units was investigated with a two-domain protein, yeast phosphoglycerate kinase. Each of the domains was produced independently by site-directed mutagenesis. It has been previously demonstrated by several criteria that these domains are able to fold in vivo into a quasi-native structure [Minard et al. (1989a) Protein Eng. 3, 55-60; Fairbrother et al. (1989) Protein Eng. 3, 5-11]. In the present study, the reversibility of the unfolding-refolding process induced by guanidine hydrochloride was investigated for the intact protein and the isolated domains. The transitions were followed by circular dichroism for both domains and the intact protein and by the variations in enzyme activity for the intact protein. Tryptophan residues were used as intrinsic conformational probes of the C-domain. An extrinsic fluorescent probe, N-[[(iodoacetyl)amino]ethyl]-8-naphthylamine-1-sulfonic acid (IAEDANS), was bound to the unique cysteinyl residue Cys97 to observe the conformational events in the N-domain. The unfolding-refolding transitions of each domain in the intact protein and in the isolated domains prepared by site-directed mutagenesis were compared. It was shown that the two domains are able to refold in a fully reversible process. A hyperfluorescent intermediate was detected during the folding of both the isolated C-domain and the intact yeast phosphoglycerate kinase. The stability of each isolated domain was found to be similar, the free energy of unfolding being approximately half that of the intact molecule.

Fungal Proteins↗
PubMed1994

Reversible topology of a bifunctional transmembrane protein depends upon the charge balance around its transmembrane domain.

Hybrid genes were constructed to express bifunctional hybrid proteins in which staphyloccal nuclease A with or without an amino-terminal OmpA signal sequence was fused with TEM beta-lactamase (at the carboxyl terminal side) using the signal peptide of the major outer membrane lipoprotein of Escherichia coli as an internal linker. The hybrid proteins were found to be inserted in the membrane. Orientation of the hybrid protein with the OmpA signal peptide showed that the nuclease was translocated into the periplasm and the beta-lactamase remained in the cytoplasm. This indicates that the cleavable OmpA signal peptide served as a secretory signal for nuclease and the internal lipoprotein signal served as the transmembrane anchor. In the absence of the OmpA signal sequence the topology of the hybrid protein was reversed indicating that the internal lipoprotein signal peptide initially served as the signal peptide for the secretion of the carboxy terminal beta-lactamase domain across the membrane and subsequently as a membrane anchoring signal. The role of charged amino acids in the translocation and transmembrane orientation of membrane proteins was also analysed by introducing charged amino acids to either or both sides of the internal lipoprotein signal sequence in the bifunctional hybrid proteins in the absence of the amino-terminal signal sequence. Introduction of two lysine residues at the carboxy-terminal side of the internal signal sequence reversed the topology of the transmembrane protein by translocating the amino-terminal nuclease domain across the membrane, leaving the carboxyl terminal beta-lactamase domain in the cytoplasm. When three more lysine residues were added to the amino-terminal side of the internal signal sequence of the same construct the membrane topology flipped back to the original orientation. A similar reversion of the topology could be obtained by introducing negatively charged residues at the amino-terminal side of the internal signal sequence. Present results demonstrate for the first time that a bifunctional transmembrane protein can be engineered to assume either of the two opposite orientations and that charge balance around the transmembrane domain is a major factor in controlling the topology of a transmembrane protein.

Amino Acid Sequence↗
PubMed1988

Studies on the regulation of malignant phenotype and gene expression in human promyelocytic leukemia cell mutant (HL-60-AR).

The present study has investigated the regulation of malignant phenotype and gene expression in a human promyelocytic leukemia cell mutant (HL-60-AR) by means of cellular engineering technique of cybridization between the fusions of the mutant cells with enucleated mouse reticulocytes. Results indicate that the cybrid cells (HL-R) incorporated with reticulocyte cytoplasts become differentiated, and the malignancy is obviously suppressed or reversed to a certain degree when compared with those of parental tumor cells. They lose the growth ability to form colony soft agar medium, become non-tumorigenic under heterotransplantation to nude mice, and are accompanied by decrease in growth rate, cellular mitotic index and DNA synthesis. No gene transcripts or homologous sequence of mRNA corresponding to c-myc oncogene can be detected in cybrid cells by Northern blot technique with cloned c-myc gene probe, suggesting that the expression of originally active c-myc has been inhibited. On the other hand, analysis, by the same molecular hybridization technique with human globin gene probe and by PAGE method, of the expression of globin gene products in cybrid cells detected at the transcription (globin mRNA) and translation (hemoglobin) levels demonstrates consistently that originally inactive human globin gene has been activated to express in passages. These results suggest that some regulatory factors existing in reticulocyte cytoplasm can regulate gene expression and reverse malignant phenotype of leukemia tumor cells.

Animals↗
PubMed1992

Ligand-regulated internalization and recycling of human beta 2-adrenergic receptors between the plasma membrane and endosomes containing transferrin receptors.

Agonist-regulated redistribution of human beta 2-adrenergic receptors was examined in 293 cells. A specific antiserum recognizing the carboxyl-terminal hydrophilic domain of the receptor was developed, characterized, and used for immunocytochemical localization of receptors in fixed cells by conventional fluorescence and confocal fluorescence microscopy. The beta-adrenergic agonist isoproterenol induced redistribution of receptors from the surface of cells into small (less than 1 micron diameter) punctuate accumulations which were detected in cells within 2 min of agonist addition. The time course of receptor redistribution paralleled that of receptor sequestration measured by ligand binding, and receptor redistribution was reversible in the presence of the beta-adrenergic antagonist alprenolol. Optical sections imaged through cells by confocal microscopy localized receptor accumulations within the cytoplasm. To address the question of receptor internalization further, a mutant receptor possessing an engineered antigenic epitope in the amino-terminal hydrophilic domain was constructed, transfected into cells, and localized using both a monoclonal antibody recognizing the epitope tag (receptor ectodomain) and an antiserum recognizing the carboxyl terminus (receptor endodomain). In untreated cells most receptor antigen was detected at the cell surface, as assessed by accessibility to ectodomain antibodies in unpermeabilized specimens. In isoproterenol-treated cells, however, little receptor antigen was detected at the cell surface. Punctate receptor accumulations present in isoproterenol-treated cells were labeled by antibodies only following permeabilization of cells, as expected if these receptor accumulations were intracellular. Finally, internalized beta-adrenergic receptors colocalized with transferrin receptors, which are markers of endosomal membranes. These data provide several lines of evidence establishing that beta-adrenergic receptors undergo ligand-regulated internalization, they suggest that internalized receptors may be recycled back to the cell surface, and they provide the first direct indication that these processes involve the same endosomal membrane system passaged by constitutively recycling receptors.

Alprenolol↗
PubMed1993

Recombinant human hepatitis B virus reverse transcriptase is active in the absence of the nucleocapsid or the viral replication origin, DR1.

The double-stranded DNA genome of hepatitis B virus (HBV) is reverse transcribed from the viral pregenome RNA template by a virally encoded reverse transcriptase enzyme (RT) that possesses both priming and elongation activities. Prior efforts have failed to express an active form of HBV RT outside the nucleocapsid in animal cells or to release it from viral nucleocapsids, thus restricting the characterization of this important enzyme. Here, we have engineered epitope-tagged HBV RT proteins and expressed them in Xenopus oocytes via a synthetic RT mRNA which does not include the viral capsid protein or the known initiation site for viral DNA synthesis, DR1. We demonstrate the production of an immunoprecipitable 96-kDa HBV RT protein and show, using a simple in vitro RT assay, that oocyte lysates containing this protein possess an activity that (i) catalyzes an RNA-dependent deoxynucleotide triphosphate polymerization reaction by using an as-yet-unidentified RNA template and (ii) is sensitive to the RT inhibitors actinomycin D and phosphonoformate. Experiments with the chain terminator ddATP suggest that a significant amount of chain elongation occurs in our in vitro reaction. Electrophoretic analysis reveals a heterogeneous array of RT reaction products with sizes ranging from about 100 bases to far larger than that of the input RT mRNA. These products appear to contain covalently bound protein, consistent with the notion that the RT protein may have primed their synthesis. We conclude that HBV RT activity can be uncoupled from both the nucleocapsid and the replication origin, DR1. Our results raise the possibility that unless HBV employs novel mechanisms to regulate its constitutively active RT, cellular RNAs may be reverse transcribed during HBV infection, with potential implications for the development of HBV-related liver cancer. The use of the oocyte system should facilitate studies of HBV RT, including the development of HBV RT inhibitors for antiviral therapy.

Amino Acid Sequence↗
PubMed1985

Transition-state stabilization in the mechanism of tyrosyl-tRNA synthetase revealed by protein engineering.

The principal catalytic factor in the activation of tyrosine by the tyrosyl-tRNA synthetase is found to be improved binding of ATP in the transition state. The activation reaction involves the attack of the tyrosyl carboxylate on the alpha-phosphate group of ATP to generate a pentacoordinate transition state. Model building of this complex located a binding site for the gamma-phosphate group of ATP, consisting of hydrogen bonds with the side chains of Thr-40 and His-45. Removal of these groups by protein engineering shows that they contribute no binding energy with unreacted ATP but put all of their binding energy into stabilizing the [tyrosine-ATP] transition state [the mutant tyrosyl-tRNA synthetase (Thr-40----Ala-40; His-45----Gly-45) has the rate of formation of tyrosyl adenylate lowered by 3.2 X 10(5) but KS for ATP is lowered by only a factor of 5]. The side chains of these residues also provide a binding site for pyrophosphate in the reverse reaction. Thus, catalysis is accomplished by stabilization of the transition state by improved binding of a group on the substrate that is distant from the seat of reaction.

Adenosine Triphosphate↗
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↗