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At least 1,099 records · Page 61Linked to original sources
PubMed1989

A chimeric EGF-R-neu proto-oncogene allows EGF to regulate neu tyrosine kinase and cell transformation.

The neu oncogene, characterized by Weinberg and colleagues, is a transforming gene found in ethylnitrosourea-induced rat neuro/glioblastomas; its human proto-oncogene homologue has been termed erbB2 or HER2 because of its close homology with the epidermal growth factor receptor (EGF-R) gene (c-erbB1). Expression of the rat neu oncogene is sufficient for transformation of mouse NIH 3T3 fibroblasts in culture and for the development of mammary carcinomas in transgenic mice, but the neu proto-oncogene has not been associated with cell transformation. We constructed a vector for expression of a chimeric cDNA and hybrid protein consisting of the EGF-R extracellular, transmembrane and protein kinase C-substrate domains linked to the intracellular tyrosine kinase and carboxyl terminal domain of the rat neu cDNA. Upon transfection with the construct, NIH 3T3 cells gave rise to EGF-R antigen-positive cell clones with varying amounts of specific EGF binding. Immunofluorescence and immunoprecipitation using neu- and EGF-receptor specific antibodies demonstrated a correctly oriented and positioned chimeric EGF-R-neu protein of the expected apparent mol. wt on the surface of these cells. EGF or TGF alpha induced tyrosine phosphorylation of the chimeric receptor protein, stimulated DNA synthesis of EGF-R-neu expressing cells and led to a transformed cell morphology and growth in soft agar. In contrast, the neu proto-oncogene did not show kinase activity or transforming properties when expressed at similar levels in NIH 3T3 cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1990

The mannose 6-phosphate receptor cytoplasmic domain is not sufficient to alter the cellular distribution of a chimeric EGF receptor.

Unlike most receptors, 300 kd mannose 6-phosphate receptors (MPRs) are localized primarily in the trans-Golgi network (TGN) and endosomes, and they cycle constitutively between these compartments. Yet, when present at the cell surface, MPRs are internalized together with other cell surface receptors in clathrin-coated vesicles. We constructed a chimeric receptor, comprised of human EGF receptor extracellular and transmembrane domains joined to the bovine MPR cytoplasmic domain, to test whether the MPR cytoplasmic domain contained sufficient information to direct a cell surface receptor into both of these transport pathways. The expressed protein was stable, bound EGF with high affinity, and was efficiently endocytosed and recycled back to the cell surface, in the presence or absence of EGF. If the cytoplasmic domain alone is responsible for sorting native MPRs, chimeric receptors might have been expected to be located primarily in the TGN and in endosomes at steady state. Surprisingly, under conditions in which essentially all endogenous MPRs were intracellular, greater than 85% of the chimeric receptors were located at the cell surface. These experiments demonstrate that the MPR cytoplasmic domain is not sufficient to alter the distribution of the EGF receptor, and suggest a role for extracellular and transmembrane domains in MPR routing.

Animals↗
PubMed1990

Regulation by EGF is maintained in an overexpressed chimeric EGFR/neu receptor tyrosine kinase.

The effects of a ligand regulated neu tyrosine kinase were examined in NIH 3T3 cells. A chimeric construct encoding the human EGF receptor extracellular domain fused to the tyrosine kinase domain of the rat neu cDNA was expressed under the transcriptional control of the Moloney murine leukemia virus LTR promoter. This resulted in higher levels of expression of the chimeric receptor than were previously obtained from the SV40 virus early promoter in the same cells. The chimeric receptor showed strict ligand-dependent tyrosine kinase and signal transducing activities for the induction of growth-regulated biochemical activities and DNA synthesis in resting cells. The ligand-activated cells became morphologically transformed and grew in agar in the presence of EGF and TGF beta as efficiently as did the ligand-independent neu oncogene-transformed cells. Our results establish similarities between the signal pathways of the EGF receptor and the neu tyrosine kinase.

Animals↗
PubMed2004

Somatic gene targeting with RNA/DNA chimeric oligonucleotides: an analysis with a sensitive reporter mouse system.

BACKGROUND: Targeted gene correction provides a potentially powerful method for gene therapy. RNA/DNA chimeric oligonucleotides were reported to be able to correct a point mutation with a high efficiency in cultured rodent cells, in the body of mice and rats, and in plants. The efficiency of correction in the liver of rats was claimed to be as high as 20% after tail-vein injection. However, several laboratories have failed to reproduce the high efficiency. METHODS: In order to sensitively detect and measure sequence changes by the chimeric oligonucleotides, we used Muta Mouse, a transgenic mouse system for mutation detection in vivo. It carries, on its chromosome, multiple copies of the lambda phage genome with the lacZ(+) gene. Two chimeric oligonucleotides were designed to make a point mutation at the active site of the LacZ gene product. They were injected into the liver with HVJ liposomes, which were demonstrated to allow reliable gene delivery. One week later, DNA was extracted from the liver, and lambda::lacZ particles were recovered by in vitro packaging. The lacZ-negative phage was detected by selection with phenyl-beta-D-galactoside. RESULTS: The mutant frequency of the injected mice was at the same level as the control mouse (approximately 1/10000). Our further restriction analysis and sequencing did not detect the designed mutations. CONCLUSIONS: Gene correction frequency in mouse liver by these oligonucleotides was shown to be less than 1/20000 in our assay with the Muta Mouse system.

Animals↗
PubMed2007

Chimeric HIV-1 and HIV-2 lentiviral vectors with added safety insurance.

Lentiviruses are unique in their ability to infect both dividing and non-dividing cells. This makes the vectors derived from them particularly useful for gene transfer into non-dividing cells, including stem cells. Lentiviral vectors are becoming the vectors of choice for si/shRNA delivery. The utility of the lentiviral vectors will be enhanced if additional elements of safety are built into their design. One safety concern is the generation of replication competent virus by recombination. We reasoned that HIV-1 and HIV-2 hybrid or chimeric lentiviral vectors will have added safety insurance in this regard. This is based on the premise that HIV-1 and HIV-2 are dissimilar enough in sequence to curtail recombination, yet similar enough to complement functionally. For hybrid vectors, we found that both HIV-1 and HIV-2 transfer vector RNAs could be packaged to equivalent titer by the HIV-1 packaging machinery. However, HIV-2 packaging machinery was unable to package HIV-1 transfer vector as well as it did HIV-2 transfer vector. This non-reciprocacity suggested that the requirement for HIV-2 vectors was more stringent and that for HIV-1 vectors more promiscuous. When the HIV-1 transfer vector was packaged with the chimeric packaging construct where the leader-gag region of HIV-2 was replaced with that of HIV-1 packaging construct, the titer of the vector went up. This suggests that at least some of the determinants of specificity for vector assembly reside in the leader-gag region. Incorporation of central polypurine tract (cPPT) and woodchuck post-transcriptional enhance element (WPRE) into the HIV-2 vectors had only modest effect on vector titer. Thus, chimeric lentiviral vectors with added safety features can be designed without compromising transduction efficiency.

Cell Line↗
PubMed2004

Development of donor-specific chimerism and tolerance in composite tissue allografts under alphabeta-T-cell receptor monoclonal antibody and cyclosporine a treatment protocols.

Recently, we induced donor-specific tolerance to rat hindlimb allografts under a 35-day course of alphabeta-T-cell receptor monoclonal antibody (alphabeta-TCR mAb) and cyclosporine A (CsA). In this report, we investigated the role of shorter alphabeta-TCR/CsA protocols on tolerance induction. We performed 52 hindlimb transplantations, between Lewis-Brown-Norway (LBN, F1) donors and Lewis recipients to test the impact of 21-, 7-, and 5-day protocols of combined alphabeta-TCR/CsA treatment on tolerance induction. Donor-specific tolerance and immunocompetence were tested by mixed lymphocyte reaction (MLR) in vitro and by standard skin grafting in vivo. The efficacy of immunosuppressive protocol and donor-specific chimerism was assessed by flow cytometry. All transplants under 5, 7, and 21 days of combined alphabeta-TCR/CsA therapy survived over 350 days. Clinical tolerance and immunocompetence were confirmed by skin grafting in vivo and MLR in vitro. Flow cytometry revealed a high level of donor chimerism in the peripheral blood of long-term survivors. The extension of survival of limb allografts and allo-unresponsiveness were directly associated with the development of stable chimerism in the tolerant recipients.

Animals↗
PubMed2001

Effect of blastomere sex and fluorescent labelling on the development of bovine chimeric embryos reconstituted at the four-cell stage.

The development rate of bovine chimeric embryos reconstituted at the 4-cell stage is relatively low. If chimerism is to be used as an approach in producing transgenic livestock, it is important to investigate whether this rate is affected by the sex of the blastomeres being combined and if all blastomeres survive equally well. In Experiment 1, blastomeres from 4-cell stage embryos were inserted into surrogate zonae pellucidae either in pairs to reconstitute 4-cell chimeras, or as the original sets of four to make handled controls. The development of chimeras with one pair of blastomeres labelled with PKH26-GL was also investigated. The rate of development into blastocysts was similar in chimeras with unlabelled blastomeres (23%) and in those in which one pair of blastomeres was labelled (26%) and was lower (P < 0.001) than in the handled and IVF control groups (43 and 58%, respectively). Labelled cells were distributed approximately evenly between ICM and trophoblast. In Experiment 2, the effect of sex differences between pairs of blastomeres in chimeras was investigated; chimeras were reconstituted from pairs of blastomeres taken from 4-cell embryos in which the remaining pair was sexed by PCR. No significant differences according to the sex of constituent blastomeres were detectable (mixed sex, 27%; males, 24%; females, 21%; P > 0.05). These results suggest that, in addition to the negative effects of micromanipulation, factors other than the sex of the blastomeres are involved in the reduced rate of development of chimeric bovine embryos. They also confirm the usefulness of PKH26-GL labelling for tracking the progeny of cleaving bovine blastomeres at least to the blastocyst stage.

Animals↗
PubMed1988

XX/XY chimerism encountered during prenatal diagnosis.

46,XX/46,XY chimerism has previously been reported in patients with abnormal sexual development, and rarely in otherwise normal individuals. We report the first postnatally documented prenatal diagnosis of whole-body 46,XX/46,XY chimerism in humans, discovered by maternal age amniocentesis. The normal male phenotype in this child creates a dilemma in prenatal counselling, since genotypic male/female chimerism cannot be assumed to imply an abnormal sexual phenotype.

Adult↗
PubMed1996

Chimeric hemoglobins--hybrids of human and swine hemoglobin: assembly and stability of interspecies hybrids.

Transgenic swine expressing human HbA contained only one of two types of the anticipated interspecies hybrids, namely H alpha 2 P beta 2 (H = human, P = swine). In an attempt to establish whether the absence of the swine alpha and human beta (P alpha 2 H beta 2) hybrid in vivo is a reflection of the lack of complementarity between the interspecies chains to generate appropriate interfaces, we have undertaken the in vitro assembly of swine alpha and human beta chimeric tetramer. In contrast to the in vivo transgenic swine system, in vitro the hybrid of swine alpha human beta chain is assembled readily and the hybrid exhibits normal cooperative oxygen binding. Both the swine alpha human beta and the human alpha swine beta interspecies hybrids are stable around neutral pH and do not segregate into parent tetramers even when mixed together. On the other hand, nearly complete exchange of P alpha chain of P alpha 2 H beta 2 hybrid occurs in the presence of H alpha chain at pH 6.0 and room temperature, resulting in the formation of HbA. However, very little of such an exchange reaction takes place at pH 7.0. These results suggest that the thermodynamic stability of P alpha 2 H beta 2 hybrid is lower compared to that of HbA. In contrast, P beta chain of H alpha 2 P beta 2 hybrid is refractory to exchange with H beta chain at pH 7.0 as well as at pH 6.0, suggesting that the stability of H alpha 2 P beta 2 is higher compared to that of HbA (H alpha 2 H beta 2). The swine alpha human beta chimeric Hb undergoes subunit exchange reaction with human alpha-chain in the presence of 0.9 M MgCl2, at pH 7.0. This demonstrates the lower thermodynamic stability of the intradimeric interactions of the heterodimer even at neutral pH. A synergistic coupling of the intra- and interdimeric interactions of the swine alpha and human beta chain heterodimer is essential for the thermodynamic stability of the chimeric Hb under the physiological conditions. Accordingly, we speculate that the lower thermodynamic stability of P alpha H beta heterodimer (compared to the homodimers H alpha H beta and P alpha P beta) facilitates its segregation into the homodimers by subunit exchange reaction involving either H alpha or P beta. This molecular aspect by itself or possibly along with other cellular aspects of the swine system results in the absence of P alpha 2 H beta 2 hybrid in transgenic swine expressing HbA.

2,3-Diphosphoglycerate↗
PubMed1995

Functional organization of mammalian hexokinases: characterization of chimeric hexokinases constructed from the N- and C-terminal domains of the rat type I and type II isozymes.

Chimeric hexokinases consisting of either the N-terminal half of Type I hexokinase fused with the C-terminal half of the Type II isozyme (NICII) or the inverse pair (NIICI), along with the parental isozymes, were expressed in COS-1 cells. The thermal stability of the chimeras was intermediate between that of the highly labile Type II isozyme and the relatively stable Type I hexokinase. In their Kms for substrates, Glc and ATP, the chimeric enzymes were similar to the parental isozyme from which the C-terminal half was derived. Although the Type I and Type II isozymes were similar in their sensitivity to inhibition (competitive vs ATP) by the Glc-6-P analogs, 1,5-anhydroglucitol 6-phosphate (AnGlc-6-P), and Glc-1,6-bisphosphate, the chimeric enzymes differed markedly, with the NIICI chimera being much more sensitive and the NICII chimera much less sensitive than either parental form to these inhibitors. In contrast, the response of the chimeras to Pi, either as an antagonist of inhibition by AnGlc-6-P or, at higher concentrations, as an inhibitor, was correlated with the origin of the N-terminal domain. The results are consistent with the view that catalytic function is associated with the C-terminal domain of the Type I isozyme, with regulatory function--inhibition by Glc-6-P and its analogs and antagonism of this inhibition by Pi--being mediated by the N-terminal domain.

Adenosine Triphosphate↗
PubMed1994

Polystyrene reverse-phase ion-pair chromatography of chimeric ribozymes.

The use of a reverse-phase polystyrene resin for ion-pair HPLC purification of large amounts of synthetic chimeric DNA-RNA oligomers that is faster and more reliable than previously used techniques has been developed. The preparation of synthetic oligomers containing RNA requires the use of tetrabutylammonium fluoride in the final step, the cleavage of the tert-butyl-dimethyl silyl protecting group from the ribonucleotides. Cleavage is accompanied by the serendipitous formation of ion pairs between tetrabutylammonium cations and the oligomer phosphates. The formation of these ion pairs retards the elution of the oligomer during HPLC, which allows rapid removal of excess tetrabutylammonium fluoride and the concomitant purification of chimeric ribozymes. This technique is based on a correlation between the length of ion-paired oligomers and their retardation during HPLC. The advantages of reverse-phase ion-pair HPLC on polystyrene resin for the fast purification of oligoribonucleotides are discussed and illustrated through the examples of synthesized chimeric ribozymes.

Base Sequence↗
PubMed1997

Engineering of functional chimeric protein G-Vargula luciferase.

Luciferase of Vargula hilgendorfli is infinitely stable at room temperature in dried state, and its light-emitting reaction is very simple. These unique characteristics of Vargula luciferase have prompted us to engineer chimeric protein, the other moiety chosen for conjugation being streptococcal protein G. A single domain of protein G which binds to IgG of a wide range of species was fused at the N-terminal region of Vargula luciferase. Unexpectedly, we found that the chimeric protein expressed in mammalian COS-1 cells had no IgG-binding ability, probably due to some sort of interaction between the two moieties or some conformational preferences of the IgG-binding domain of protein G when fused to Vargula luciferase. Here we report how we regained the IgG binding of protein G, by the intervention of three alpha-helices of protein A between protein G and luciferase. To our knowledge, the new chimeric protein provides the first reported model of this kind.

Animals↗
PubMed1999

Chimeric G proteins allow a high-throughput signaling assay of Gi-coupled receptors.

G-protein-coupled receptors are a major target for potential therapeutics; yet, a large number of these receptors couple to the Gi pathway, generating signals that are difficult to detect. We have combined chimeric G proteins, automated sample handling, and simultaneous 96-well fluorometric imaging to develop a high-throughput assay system for Gi signaling. The chimeric G proteins alter receptor coupling so that signaling can occur through Gq and result in mobilization of intracellular calcium stores. An automated signaling assay device, the fluorometric imaging plate reader (FLIPR), can simultaneously measure this response in real time in 96-well microplates, allowing two people to process more than 10,000 points per day. We used the chimeric G protein/FLIPR system to characterize signaling by the Gi-coupled human opioid receptors. We show that the mu, delta, and kappa opioid receptors and the related nociceptin receptor, ORL1, each couple to Galphaqi5, Galphaqo5, and Galpha16 (Galphaqi5 and Galphaqo5 refer to Galphaq proteins containing the five carboxyl-terminal amino acids from Galphai and Galphao, respectively) and that different receptor/G protein combinations show different levels of maximal activation. We tested 31 opioid ligands for agonist activity at the opioid receptors (124 ligand-receptor combinations); all 31 activated at least one receptor type, and several activated multiple receptors with differing potencies. This high-throughput assay could be useful for dissecting the complex ligand-receptor relationships that are common in nature.

Amino Acid Sequence↗
PubMed1993

The chimeric receptor between interleukin-2 receptor beta chain and interleukin-4 receptor transduces interleukin-2 signal.

Transfectants expressing the chimeric receptor which has human interleukin-4 receptor (hIL-4R) in the extracellular domain and human interleukin-2 receptor beta chain (hIL-2R beta) in the cytoplasmic domain showed continuous growth in response to human interleukin-4 (hIL-4), whereas transfectants expressing hIL-4R responded only transiently to hIL-4. The pattern of tyrosine phosphorylation induced by hIL-4 in transfectants expressing the chimeric receptor was very similar to that in transfectants expressing hIL-2R beta, but different from that in transfectants expressing hIL-4R, indicating that the chimeric receptor transduces the interleukin-2 like growth signal by hIL-4.

Animals↗
PubMed1995

Characterization of chimeric heme-copper respiratory oxidases using subunits I of Escherichia coli cytochrome b o and Halobacterium salinarium cytochrome aa3.

We constructed chimeric enzymes with the Escherichia coli cytochrome bo and the Halobacterium salinarium cytochrome aa3 through recombinant DNA techniques and investigated their spectroscopic and biochemical properties. Although most of the chimeras could not retain hemes in the molecule, the chimeric enzyme containing helix VII of subunit I of the H. salinarium cytochrome aa3 showed the spectral properties similar to those of the native E. coli oxidase, suggesting that both the low-spin heme b and the high-spin heme o are associated with the chimeric subunit I. However, CuB was absent in the chimera. Helix VII of subunit I of the H. salinarium cytochrome aa3 is 70% similar to the counterpart of the E. coli cytochrome bo and further contains two invariant histidines which serve as the CuB ligands. These results indicate that helix VII must be arranged properly relative to helix VI which provides the third CuB ligand.

Amino Acid Sequence↗
PubMed1998

Cell-specific transfection of choriocarcinoma cells by using Sindbis virus hCG expressing chimeric vector.

The development of Sindbis virus vectors that can target specific cell types would provide an important gene therapy strategy. We explored the possibility of designing a Sindbis virus vector that can target human choriocarcinoma cells via ligand-receptor interaction. The Sindbis virus envelope gene was modified by insertion of the alpha- and beta-hCG genes. The chimeric helper RNA was then transfected into BHK cells along with a virus-based expression vector, allowing the production of virus particles containing hCG-envelope chimeras. The hCG-envelope chimeric virus vector has minimal infectivities against BHK cells and human cancer cells which do not contain LH/CG receptors on their surface. This vector can, however, infect and transfer a reporter gene to choriocarcinoma cells as well as other cells bearing LH/CG receptors. This chimeric Sindbis virus vector may provide a novel approach for gene therapy of gestational trophoblast disease and placental dysfunction.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1998

Ectopic expression of a chimeric colony-stimulating factor-1/TrkB-receptor promotes CSF-1-dependent survival of cultured sympathetic neurons.

The regulation of the density of innervation and the promotion of survival of neurons are the original effects depending on neurotrophins. Here we analyse such effects evoked by trkB tyrosine kinase in transfected PC12 cells and transfected sympathetic neurons. In order to exclude the previously described modulation of trk kinase activity by the extracellular activation of the low-affinity p75 neurotrophin receptor, we applied a chimeric receptor approach: The extracellular domain of colony-stimulating factor-1 (CSF-1) receptor was fused to the transmembrane and cytoplasmic domain of the trkB tyrosine kinase receptor, allowing its selective activation by the heterologous ligand. Protein expression and CSF-1-induced tyrosine phosphorylation of the chimeric receptor protein was demonstrated in transfected COS cells. After stable transfection into nerve growth factor (NGF)-responsive PC12 cells, CSF-1 mediated the K252a-sensitive induction of fiber outgrowth. Furthermore, we were able to show by heterologous expression of the chimeric receptor, that activation of trkB tyrosine kinase activity is sufficient to promote survival of neurotrophin deprived sympathetic neurons.

Adrenergic Fibers↗
PubMed1999

Chimeric virus-like particle formation of adeno-associated virus.

Adeno-associated virus (AAV) capsids are composed of three proteins, VP1, VP2, and VP3. These capsid proteins have a common amino acid sequence, being expressed from different initiation codons on the same open reading frame. Although VP1 is necessary for viral infection, it is not essential for capsid formation. The other capsid proteins, VP2 and VP3, are sufficient for capsid formation, but their functions are poorly understood. To investigate the role(s) of the capsid proteins in capsid formation, we used a baculovirus protein expression system to produce virus-like particles (VLPs). We found that varying the ratios of VP2 and VP3 did not affect VLP formation. Further, their physical properties were equivalent to those of empty wild-type particles. The function of VP3 was studied further by fusing a peptide tag, FLAG, to its N-terminus. This chimeric viral protein, in combination with VP2, could assemble into VLPs, indicating that the chimerism of VP3 did not affect VLP formation. Although the monomeric native form of the FLAG-VP3 chimera could react with anti-FLAG antibody, VLP containing the chimeric VP3 could not, suggesting that the N-terminal region of VP3 is located inside the VLP. These observations indicate that it may be possible to utilize AAV VLP as vectors of a broad range of drugs since fusion of the VP3 N-terminus with defined molecules could impose distinct physical properties onto the internal environment of the VLP.

Animals↗