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At least 181 records · Page 10Linked to original sources
PubMed1992

Phase I trial of an interleukin-2 (IL-2) fusion toxin (DAB486IL-2) in hematologic malignancies expressing the IL-2 receptor.

DAB486IL-2 is a recombinant fusion toxin in which the native receptor binding domain of diphtheria toxin has been replaced with human interleukin-2 (IL-2). It selectively binds and intoxicates only cells that bear the high-affinity receptor for IL-2. In the first clinical trial of a genetically engineered ligand fusion-toxin, we have treated 18 patients with chemotherapy-resistant IL-2 receptor expressing hematologic malignancies with escalating doses of DAB486IL-2. The maximal tolerated dose of a daily intravenous bolus of DAB486IL-2 was 0.1 mg/kg per day for 10 doses, established by asymptomatic, reversible elevations of hepatic transaminases without changes in other tests of liver function. Other mild reversible side effects noted were rash, nausea, elevated creatinine, chest tightness, and fever. Pharmacokinetic analysis showed a monophasic clearance of 5.8 +/- 0.7 minutes with peak levels of 3,549 +/- 1,041 mg/mL at the 0.1 mg/kg dose. Approximately 50% of patients developed an antibody response to diphtheria toxin or DAB486IL-2. The presence of such antibodies did not preclude patients from experiencing an antitumor response as four of the six patients with antitumor effect had detectable antibody titers. Although this was a phase I trial designed to define the safety of DAB486IL-2, remissions were observed in three patients lasting from 5 to over 18 months. The ability to achieve significant tumor reductions in this group of heavily treated patients is encouraging and suggests additional trials are warranted in hematologic malignancies.

Adult↗
PubMed1993

Studies on dicistronic polioviruses implicate viral proteinase 2Apro in RNA replication.

A dicistronic poliovirus W1-P1/E/P2,3-1 with the genotype [PV]5'NTR-P1-[EMCV]IRES-[PV]P2,3-3'NTR (Molla, Jang, Paul, Reuer, and Wimmer, 1992, Nature 356, 255) was used to investigate whether the viral proteinase 2Apro, whose primary function in proteolytic processing was erased through the insertion of an internal ribosomal entry site (IRES) element into the ORF of the polyprotein, had other function(s) in viral replication. Deletion of 2Apro from W1-P1/E/P2,3-1 rendered the corresponding transcripts unable to replicate whereas partial deletion of 2Apro or an exchange of Cys109 (an amino acid of the catalytic triad of the proteinase) to Ala reduced RNA replication. No cytopathic effects were observed after transfection with any of the three dicistronic constructs containing mutant 2A, and no virus was recovered after attempts to expand a possibly low yield of mutant virus. In contrast, insertion of the IRES of encephalomyocarditis virus (EMCV) into the ORF of the poliovirus polyprotein at the cleavage site between 2Apro and 2B yielded the novel dicistronic virus W1-P1,2A/E/2BC,P3-1 with the genotype [PV]5'NTR-P1-2A-[EMCV]IRES-[PV]2BC-P3-3'NTR, expressing a small plaque phenotype. These results indicate that neither the intact P2 polypeptide nor the cleavage fragment 2AB of P2 is required for viral proliferation. On the other hand, 2Apro appears to be an essential component in RNA replication as no viral RNA synthesis can be observed by reverse transcription/PCR in cells transfected with dicistronic RNA lacking this viral polypeptide.

Base Sequence↗
PubMed1982

A multiple-dose phase I trial of recombinant leukocyte A interferon in cancer patients.

Eighty-one patients with a variety of refractory disseminated malignant neoplasms have been treated in the first multiple fixed-dose phase I trial of recombinant leukocyte A interferon (IFL-rA). Each patient received IFL-rA by intramuscular injection, three times weekly for 28 days. Dosages were escalated in different patients from 1 to 136 x 10(6) units per injection. The toxic reactions seen with IFL-rA resembled those of nonrecombinant leukocyte interferon and included fever, chills, fatigue, anorexia, myalgia, headache, occasional nausea and vomiting, and dose-dependent reversible leukopenia and hepatic transaminase elevations. The pharmacokinetics of IFL-rA were also comparable with nonrecombinant leukocyte interferon. Objective evidence of antitumor activity was seen in non-Hodgkin's lymphoma, chronic lymphocytic leukemia, Hodgkin's disease, breast cancer, and melanoma, indicating that IFL-rA, the first genetically engineered biological response modifier available for testing in cancer patients, is biologically active in vivo.

Anorexia↗
PubMed1991

Behavioural effects of genetically engineered cells releasing dopa and dopamine after intracerebral grafting in a rat model of Parkinson's disease.

The relative importance of synaptic versus paracrine dopamine transmission for the occurrence of functional effects following intrastriatal grafting is not fully established. In the present study we grafted cell lines, expressing the form I of human tyrosine hydroxylase after infection with a recombinant retrovirus and selection in tyrosine-free-medium, to the denervated striatum in order to analyse the extent to which extracellular dopamine levels can be restored and the effect of a diffuse release of dopamine on motor impairement in a rat model of Parkinson's disease. In petri dish, the modified fibroblast cells (NIH.3T3) release DOPA constitutively whereas the modified endocrine cells (RIN) store and release dopamine in a regulated way. Interestingly, in denervated striatum, grafts of modified fibroblast cells produce DOPA which was efficiently converted into dopamine by the host striatal tissue. In the grafted striatum, both fibroblast and endocrine cells restore subnormal levels of diffuse release of dopamine which is notably unaffected and stimulated, respectively, by high concentration of potassium, in connection with the in vitro properties of the grafted cells. The intrastriatal grafts of modified cells partially reversed the apomorphine-induced but not the amphetamine-induced motor asymmetry. We discuss the implications of these results in the context of Parkinson disease.

Animals↗
PubMed1994

Functional difference between the two oppositely oriented priming signals essential for the initiation of the broad host-range plasmid RSF1010 DNA replication.

The broad host-range plasmid RSF1010 contains two oppositely oriented priming signals, ssiA and ssiB, for DNA synthesis dependent on the origin of vegetative DNA replication (oriV). If either ssiA or ssiB was deleted or inverted, the RSF1010 miniplasmids containing engineered oriVs were maintained at low copy numbers, replicated abnormally as dimers, and accumulated specific single strands in the Escherichia coli strain supplying the three RSF1010-encoded RepA, RepB', and RepC proteins. Interestingly, an additional intracellular supply of the Sog primase (the sog gene product of plasmid CoIIb-P9) reversed the replication deficiency of these miniplasmids with respect to all three aspects described above. These were also true for the RSF1010 miniplasmids in which either ssiA or ssiB was replaced by the primosome assembly site (PAS) or by the G4-type ssi signal (G site). Furthermore, comparative analysis of the functional contribution of the two oppositely oriented ssi signals to the DNA replication of RSF1010 showed that, irrespective of their types, ssi signals conducting the initiation of DNA chain elongation away from the iterons were functionally more important than ones in the inverted orientation. We consider that this functional difference reflects the inherent properties of the initiation mechanism of RSF1010 DNA replication.

Bacterial Proteins↗
PubMed1987

Biological equivalence of natural bovine and recombinant human alpha-endothelial cell growth factors.

The cDNA encoding human alpha-endothelial cell growth factor (alpha-ECGF) has been engineered for high-level expression in Escherichia coli. Induction of bacterial cultures harboring the recombinant plasmid pMJ26 results in the appearance of a prominent 16-kDa polypeptide. This protein has been purified from bacterial lysates using a rapid, 2-step procedure employing heparin-Sepharose affinity based chromatography and reversed-phase high pressure liquid chromatography. Recombinant human alpha-ECGF was compared to bovine brain-derived alpha-ECGF in three biological assays: receptor binding on murine lung capillary endothelial cells (LE-II cells), stimulation of [3H]thymidine incorporation in LE-II cells, and stimulation of human umbilical vein endothelial cell proliferation. The results demonstrate that the recombinant human mitogen has the same biological potency as the bovine brain-derived material. Fluorescence spectroscopy was used to study the interaction between recombinant ECGF and heparin. Heparin-binding resulted in a 40% reduction in the intrinsic fluorescence of ECGF, consistent with a heparin-induced conformational change. The intrinsic fluorescence of ECGF also varied as a function of pH.

Amino Acids↗
PubMed1994

GMAP: a multi-purpose computer program to aid synthetic gene design, cassette mutagenesis and the introduction of potential restriction sites into DNA sequences.

A computer program called GMAP has been developed for i) mapping the potential restriction endonuclease (R.E.) sites that can be introduced in a nonambiguous DNA sequence; ii) predicting the mutations required to introduce unique R.E. sites in the nonambiguous DNA sequences; and iii) searching all R.E. sites in ambiguous DNA sequence obtained by reverse translation of a given amino acid sequence. This allows the design of synthetic genes as well as the modular redesign after introducing limited base pair mismatches in wild-type genes in order to adapt them for "cassette" mutagenesis. The GMAP program uses an algorithm based on set theory that reduces the degree of complexity from an exponential to linear function of sequence length. Therefore, the speed of searching for potential R.E. sites in reverse-translated gene sequences and the prediction of new R.E. sites in natural genes by mutations are rapid.

Algorithms↗
PubMed1989

Substantial increase of protein stability by multiple disulphide bonds.

Disulphide bonds can significantly stabilize the native structures of proteins. The effect is presumed to be due mainly to a decrease in the configurational chain entropy of the unfolded polypeptide. In phage T4 lysozyme, a disulphide-free enzyme, engineered disulphide mutants that crosslink residues 3-97, 9-164 and 21-142 are significantly more stable than the wild-type protein. To investigate the effect of multiple-disulphide bonds on protein stability, mutants were constructed in which two or three stabilizing disulphide bridges were combined in the same protein. Reversible thermal denaturation shows that the increase in melting temperature resulting from the individual disulphide bonds is approximately additive. The triple-disulphide variant unfolds at a temperature 23.4 degrees C higher than wild-type lysozyme. The results demonstrate that a combination of disulphide bonds, each of which contributes to stability, can achieve substantial overall improvement in the stability of a protein.

DNA Mutational Analysis↗
PubMed1989

Peristaltic transport of a particle-fluid suspension.

Peristaltic pumping by a sinusoidal traveling wave in the walls of a two-dimensional channel filled with a viscous incompressible fluid in which are distributed identical rigid spherical particles, is investigated theoretically. A perturbation solution is obtained which satisfies the momentum equations for the case in which amplitude ratio (wave amplitude/channel half width) is small. The results show that the fluid phase mean axial velocity decreases with increase in the particle concentration. The phenomenon of reflux (the mean flow reversal) is discussed. A reversal of velocity in the neighborhood of the centerline occurs when the pressure gradient is greater than that of the critical reflux condition. It is found that the critical reflux pressure is lower for the particle-fluid suspension than for the particle-free fluid. It is further observed that the mean flow reversal is strongly dependent on the particle concentration and the presence of particles in the fluid favors the reversal flow. A motivation of the present analysis has been the hope that such a theory of two-phase flow process is very useful in understanding the role of peristaltic muscular contraction in transporting bio-fluid behaving like a particle-fluid mixture. Also the theory is important to the engineering applications of pumping solid-fluid mixtures by peristalsis.

Gastrointestinal Motility↗
PubMed1987

The erythropoietin receptor of rat erythroid progenitor lens. Characterization and affinity cross-linkage.

Commercially available 125I-labeled erythropoietin, obtained by genetic engineering from a human gene, was used to characterize receptors for this hormone on the cell surface of rat erythroid progenitor cells. A low number of high affinity binding sites (487 +/- 32 sites/cell, Kd = 167 +/- 14 pm) were found. Nonerythroid cells and erythrocytes did not exhibit specific binding. The high affinity binding was reversible and displaced by unlabeled erythropoietin, but not by other hormones and growth factors. After incubation at 37 degrees C, nearly 35% of the specifically bound erythropoietin seemed to be internalized, as judged by resistance to acidic buffer treatment. Thus, binding showed characteristics of a hormone-receptor association. 125I-Erythropoietin-labeled cells were treated with the bifunctional reagent dissucinimidyl suberate. Analysis of the cellular extracts by polyacrylamide gel electrophoresis under denaturing and reducing conditions revealed that erythropoietin can be cross-linked to two molecules of 94 and 78 kDa, respectively. Both labeled bands disappeared when the cells were labeled in the presence of an excess of unlabeled erythropoietin. Under nonreducing conditions, a cross-linked band of 230-255 kDa was observed. The relationships between these bands are discussed.

Animals↗
PubMed1993

Structure of an engineered, metal-actuated switch in trypsin.

The X-ray crystal structure of the copper complex of the rat trypsin mutant Arg96 to His96 (trypsin R96H) has been determined in order to ascertain the nature of the engineered metal-binding site and to understand the structural basis for the metal-induced enzymatic inhibition. In the structure, the catalytically essential His57 residue is reoriented out of the active-site pocket and forms a chelating, metal-binding site with residue His96. The copper is bound to the N epsilon 2 atoms of both histidine residues with Cu-N epsilon 2 = 2.2 A and N epsilon 2-Cu-N epsilon 2 = 89 degrees. The metal is clearly bound to a third ligand leading to a distorted square planar geometry at Cu. The X-ray results do not unambiguously yield the identity of this third ligand, but chemical data suggest that it is a deprotonated, chelating Tris molecule which was used as a carrier to solubilize the copper in alkaline solution (pH 8.0). Upon reorientation of His57, a unique water molecule moves into the active site and engages in hydrogen-bonding with Asp102-O delta 2 and His57-N delta 1. Except for small movements of the peptide backbone near His96, the remainder of the trypsin molecule is isostructural with the native enzyme. These data support the notion that the effective inhibition of catalytic activity by metal ions observed in trypsin R96H is indeed caused by a specific and reversible reorganization of the active site in the enzyme.

Amino Acid Sequence↗
PubMed1994

Thermal stabilization of thymidylate synthase by engineering two disulfide bridges across the dimer interface.

Thermal inactivation of oligomeric enzymes is most often irreversible and is frequently accompanied by precipitation. We have engineered two symmetry related disulfide bridges (155-188' and 188-155') across the subunit interface of Lactobacillus casei thymidylate synthase, at sites chosen on the basis of an algorithm for the introduction of stereochemically unstrained bridges into proteins. In this communication, we demonstrate a remarkable enhancement in the thermal stability of the covalently cross-linked double disulfide containing dimeric enzyme. The mutant enzyme remains soluble and retains secondary structure even at 90 degrees C, in contrast to the wild-type enzyme which precipitates at 52 degrees C. Furthermore, the mutant enzyme has a temperature optimum of 55 degrees C and possesses appreciable enzymatic activity at 65 degrees C. Cooling restores complete activity, in the mutant protein, demonstrating reversible thermal unfolding. The results suggest that inter-subunit crosslinks can impart appreciable thermal stability in multimeric enzymes.

Amino Acid Sequence↗
PubMed1990

Genetic engineering of human lymphocytes for the production of monoclonal antibodies.

The production of human monoclonal antibodies is limited by a lack of good human fusion partners, the short-term stability of most human hybridomas, low fusion frequencies, and low secretion levels. An alternative method of immortalizing human immunoglobulin-secreting lymphocytes is presented here, which avoids many of the drawbacks associated with human hybridoma technology. Brief exposure of cells to a high intensity electric field results in the reversible permeabilization of cell membranes (electroporation). This technique allows the introduction of genes into virtually any cell type in a highly efficient manner. Specific genes or DNA sequences can thus be transferred into cells via the transient pores created in the membrane and, if the sequences are expressed, can yield cells with altered phenotypic properties (i.e., drug resistance). Electroporation of activated human lymphocytes in the presence of DNA sequences coding for immortalization functions and specifically targeted to B cells, would yield, via the stable integration of exogenous DNA into the cellular genome and the subsequent expression of the genes, immortalized, immunoglobulin-secreting lymphocytes, without the need for a fusion partner. Details of this technique, in addition to its advantages over the commonly used methods for generating hybridomas, are discussed.

Antibodies, Monoclonal↗
PubMed1988

Evidence for a polypeptide segment at the carboxyl terminus of recombinant human gamma interferon involved in expression of biological activity.

A panel of 18 murine monoclonal antibodies was raised in BALB/c mice to the full-length, 146 amino acid residue recombinant human gamma interferon (rHuIFN gamma-A). Two monoclonal antibodies, designated 47N3-6 and 30N47-1, were purified from ascites tumors and further characterized. Antibody 47N3-6 neutralized both the antiviral and antiproliferative activities of rHuIFN gamma-A. Both Western blotting and enzyme-linked immunosorbent assays indicated that antibody 47N3-6 could bind to rHuIFN gamma-A as well as to a genetically engineered truncated form lacking the first three amino-terminal residues (rHuIFN gamma-D) but did not recognize a genetically engineered variant terminating at residue 131 (rHuIFN gamma-B). This antibody also demonstrated binding to a 15 amino acid residue oligopeptide, designated F-1, corresponding to residues 132-146 at the carboxyl terminus of rHuIFN gamma-A. Chemical cleavage of peptide F-1 with cyanogen bromide produced two fragments that were separated by reversed-phase high-pressure liquid chromatography. Dot-blot analysis indicated that antibody 47N3-6 could bind to a fragment, KRKRSQHse, derived from residues 132-137 of rHuIFN gamma-A, but could bind only weakly to the cyanogen bromide fragment corresponding to residues 138-146. It was consistent with these results that antibody 47N3-6 demonstrated binding to a form lacking the five carboxyl-terminal amino acids (rHuIFN gamma-D') but did not bind to a synthetic polypeptide corresponding to residues 138-146. Peptide F-1 exhibited neither antiviral nor antiproliferative activity, and it did not antagonize the antiviral activity of rHuIFN gamma-A.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1989

Energetics of complementary side-chain packing in a protein hydrophobic core.

The energetics of complementary packing of nonpolar side chains in the hydrophobic core of a protein were analyzed by protein engineering experiments. We have made the mutations Ile----Val, Ile----Ala, and Leu----Ala in a region of the small bacterial ribonuclease barnase where the major alpha-helix packs onto the central beta-sheet. The destabilization resulting from the creation of cavities was determined by measuring the decrease in free energy of folding from reversible denaturation induced by urea, guanidinium chloride, or heat. The different methods give consistent and reproducible results. The loss in free energy of folding for the mutant proteins is 1.0-1.6 kcal/mol per methylene group removed. This exceeds by severalfold the values obtained from model experiments of the partitioning of relevant side chains between aqueous and nonpolar solvents. Much of this discrepancy arises because two surfaces are buried when a protein folds--both the amino acid side chain in question and the portions of the protein into which it packs. These experiments directly demonstrate that the interior packing of a protein is crucial in stabilizing its three-dimensional structure: the conversion of leucine or isoleucine to alanine in the hydrophobic core loses half the net free energy of folding of barnase with a concomitant decrease in yield of the expressed recombinant protein.

Amino Acids↗
PubMed1994

Functional expression and site-directed mutagenesis of a synthetic gene for alpha-bungarotoxin.

In order to explore the structure-function relationships of the curare mimetic alpha-neurotoxins we have constructed and cloned a synthetic gene for Bungarus multicinctus alpha-bungarotoxin which is expressed in Escherichia coli. The recombinant alpha-bungarotoxin is expressed as a fusion protein with alpha-bungarotoxin linked to the COOH-terminal end of the T7 Gene 9-encoded coat protein. After treatment of the fusion protein with Factor Xa protease, a recombinant alpha-bungarotoxin is released that co-migrates with authentic alpha-bungarotoxin upon reverse-phase high performance liquid chromatography and ion-exchange chromatography. Final yields of active recombinant alpha-bungarotoxin were about 0.4 mg/liter of starting bacterial culture. The recombinant alpha-bungarotoxin contains 10 additional residues linked to the NH2-terminal Ile of the alpha-bungarotoxin sequence due apparently to the inaccessibility of the engineered cleavage site to Factor Xa. Nevertheless, the recombinant alpha-bungarotoxin is capable of binding to the nicotinic acetylcholine receptor with an apparent affinity that is only decreased approximately 1.7-fold from that of authentic alpha-bungarotoxin. Alanine substitution of a residue, Asp30, highly conserved among alpha-neurotoxins and previously suggested to play a key role in receptor recognition, resulted in a recombinant alpha-bungarotoxin whose receptor binding activity is indistinguishable from authentic alpha-bungarotoxin.

Alanine↗
PubMed1993

Features of calmodulin that are important in the activation of the catalytic subunit of phosphorylase kinase.

Calmodulin (CaM) is an integral subunit, called delta, of the phosphorylase kinase hexadecamer, and the activity of the isolated catalytic gamma-subunit of the kinase is stimulated by CaM. We report here the first analysis of functionally important features of CaM for activation of the gamma-subunit. A set of genetically engineered CaMs, in which acidic residues in each of the four E-helices of the "EF-hands" were changed to basic lysine residues, was used to probe the relative importance of charge features in each domain of CaM. The maximal activation of the isolated gamma-subunit was diminished by all of the charge reversal mutations. The gamma-subunit was especially sensitive to reversals in the second and third E-helix of CaM (residues 45-47 and 82-84), the latter being present in the central helix. The results suggest the functional importance of electrostatics in the interactions between the delta-subunit (CaM) and the catalytic gamma-subunit of phosphorylase kinase, which is similar to results obtained with CaM-dependent myosin light chain kinase (MLCK) from chicken gizzard and CaM-dependent protein kinase II (CaMPK-II). However, novel features of the interaction between CaM and the gamma-subunit of phosphorylase kinase are the significant contribution of electrostatics throughout the CaM molecule, including residues in both halves and on more than one face of CaM, and the lack of a major effect of the CaM mutations on substrate kinetic parameters, unlike the effects observed with MLCK and CaMPK-II. These results are consistent with a model in which the delta-subunit (CaM) of phosphorylase kinase interacts with an extended region or multiple regions of the gamma-subunit and suggest that the mechanism of CaM activation of the gamma-subunit may have features that are distinct from those of MLCK and CaMPK-II.

Amino Acid Sequence↗