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PubMed1991

Expression vectors for streptavidin-containing chimeric proteins.

We have constructed expression vectors for streptavidin-containing chimeric proteins. These vectors carry the DNA sequence corresponding to the core region of the streptavidin molecule, and have several unique cloning sites which facilitate construction of gene fusions of streptavidin with a target protein. A chimeric protein of streptavidin and the target protein should be expressible in Escherichia coli by using the T7 expression system. Because of the strong and specific biotin-binding affinity of the streptavidin moiety, such streptavidin-containing chimeric proteins should extensively expand the applications of the streptavidin-biotin system, and offer a variety of applications as new biological tools.

Amino Acid Sequence↗
PubMed1999

Preservation of Gi coupling of a chimeric EP3/I-type prostaglandin (IP) receptor.

For the EP3 subtype of prostaglandin E receptors, different C-terminal splice variants are known, which are coupled to distinct heterotrimeric GTP-binding proteins (G-proteins). To test the hypothesis that the C-terminal domain is essential for the G-protein-coupling specificity of the EP3 receptor, we exchanged the carboxyl-terminal tail of a porcine Gi-coupled EP3 receptor isoform for the corresponding C-terminal part of a Gs-coupled prostaglandin receptor. The porcine EP3 receptor was truncated at a lysine (K350) residue at the end of the seventh transmembrane region, representing the splicing site of the different EP3 receptor isoforms. The wild-type C-terminus (37 amino acids) was substituted by the C-terminal tail (89 amino acids) of the human I-type prostaglandin receptor (hIP-R). The G-protein coupling of the resulting chimeric receptor protein was studied in transfected Chinese hamster ovary (CHO) cells. Stimulation of the chimeric receptor protein with the EP3 receptor-specific agonist M&B 28.767 did not increase adenosine 3',5'-cyclic monophosphate (cAMP) formation but did reduce the forskolin-stimulated cAMP formation, indicating Gi coupling. Furthermore, the chimeric receptor did not show constitutive activity as demonstrated for the C-terminally truncated EP3 receptor. Thus, coupling specificity of the EP3 receptor is not exclusively mediated by the carboxyl-terminal tail, and constitutive activity of a C-terminally truncated EP3 receptor can be suppressed by the hIP-R C-terminus.

Amino Acid Sequence↗
PubMed1995

Coordinate frames for naming misoriented chimerics: a case study of visuo-spatial neglect.

A persistent line of inquiry for the students of visuo-spatial neglect has involved the perceptual frame of reference respect to which the neglected region of space is defined. On standard testing conditions viewer-centered and object-centered systems of coordinates are confounded. In order to disambiguate these two reference frames FB, a patient with severe left visual neglect consequent upon a right parieto-temporal haemorrhage, was asked to identify chimeric figures presented at different orientations. FB continued to recognize poorly the left side of chimeric figures even when the display was rotated 90 degrees clockwise or anticlockwise so that the 'left' of the chimeric fell on the patient's egocentric up or down, respectively. The result suggests that, at least under the present testing conditions, unilateral neglect is tied to the principal (top-bottom) axis of the object. Object-centered vs. (viewer-centered) representational accounts of this finding are discussed.

Aged↗
PubMed2002

Cleavage of a DNA-RNA-DNA/DNA chimeric substrate containing a single ribonucleotide at the DNA-RNA junction with prokaryotic RNases HII.

We have analyzed the cleavage specificities of various prokaryotic Type 2 ribonucleases H (RNases H) on chimeric DNA-RNA-DNA/DNA substrates containing one to four ribonucleotides. RNases HII from Bacillus subtilis and Thermococcus kodakaraensis cleaved all of these substrates to produce a DNA segment with a 5'-monoribonucleotide. Consequently, these enzymes cleaved even the chimeric substrate containing a single ribonucleotide at the DNA-RNA junction (5'-side of the single ribonucleotide). In contrast, Escherichia coli RNase HI and B. subtilis RNase HIII did not cleave the chimeric substrate containing a single ribonucleotide. These results suggest that bacterial and archaeal RNases HII are involved in excision of a single ribonucleotide misincorporated into DNA.

Bacillus subtilis↗
PubMed2004

cis-Effect of DnaJ on DnaK in ternary complexes with chimeric DnaK/DnaJ-binding peptides.

Chimeric peptides, comprising a DnaK-binding sequence of L-amino acid residues (motif k) and an exclusive DnaJ-binding sequence of D-amino acid residues (motif j) connected through a 22-residue linker, were examined as minisubstrates for the DnaK chaperone system. The DnaJ-stimulated ATPase activity of DnaK was three times higher in the presence of the chimeric peptides pjk or pkj than in the simultaneous presence of the corresponding single-motif peptides ala-p5 (k motif) plus D-p5 (j motif). Apparently, pjk and pkj mimic unfolded proteins by forming ternary (ATP x DnaK) x peptide x DnaJ complexes which favor cis-interaction of DnaJ with DnaK. Consistent with this interpretation, the specific stimulatory effect of the chimeric peptides was abolished by either single-motif peptide in excess.

2-Naphthylamine↗
PubMed1997

Cytotoxic activity of ribonucleolytic toxin restrictocin-based chimeric toxins targeted to epidermal growth factor receptor.

Targeted toxins represent a new approach to specific cytocidal therapy. The ribonucleolytic protein toxin restrictocin is a potent protein synthesis inhibitor produced by the fungus Aspergillus restrictus. In the present study we have constructed two restrictocin based chimeric toxins where human transforming growth factor alpha (TGF alpha) has been used as a ligand. TGF alpha is a single chain polypeptide, which binds to epidermal growth factor receptor (EGFR) and causes proliferation in a large number of cancers. The ligand has been separately fused either at the amino terminus or carboxyl terminus of restrictocin, giving rise to TGF alpha-restrictocin and restrictocin-TGF alpha respectively. The fusion proteins were overexpressed in Escherichia coli and purified from inclusion bodies by a denaturation-renaturation protocol. Both the chimeric toxins actively inhibited eukaryotic protein synthesis in a cell free in vitro translation assay system. These chimeric toxins selectively killed human epidermal growth factor receptor positive target cells in culture. Among the two proteins, restrictocin-TGF alpha was more active than TGF alpha-restrictocin on all the cell lines studied.

Allergens↗
PubMed1996

Induction of specific tolerance through mixed hematopoietic chimerism prevents chronic renal allograft rejection in a rat model.

BACKGROUND: Chronic rejection is the leading cause of late graft loss in kidney transplantation. We tested the ability of mixed hematopoietic chimerism to prevent chronic renal allograft rejection in an established rat model and described possible mechanisms responsible for this tolerance. METHODS: Mixed hematopoietic chimerism was established in lethally irradiated F-344 rats by reconstitution with Lewis bone marrow. Four groups (n = 5 each) received orthotopic kidney transplants: (1) allograft controls, (2) isograft controls, (3) experimental chimeras, and (4) specificity control. After 120 days kidney grafts were examined histologically, immunohistochemically, and for cytokine interferon-gamma, interleukin-2 (IL-2), IL-4, and IL-10 gene transcripts by means of reverse transcriptase polymerase chain reaction techniques. RESULTS: Allograft control group exhibited severe parenchymal fibrosis; isograft control and chimera groups failed to develop this lesion. Immunohistochemical analysis revealed increased CD8+ lymphocytes and ED-1+ monocyte-macrophages infiltrating the tubulointerstitium of control allografts. Interferon-gamma and IL-2 were absent in isografts. IL-4 was absent and IL-10 was positive in all grafts. Chimeras promptly rejected third-party allografts. CONCLUSIONS: Induction of specific tolerance through mixed hematopoietic chimerism prevents chronic renal allograft rejection. These results support the hypothesis of an immunologic basis of chronic rejection and advance previous observations that the induction of specific tolerance enables long-term solid organ transplantation without the use of immunosuppression.

Animals↗
PubMed1997

Rat renal allograft tolerance is associated with local TGF-beta and absence of IL-2r expression within chimeric immunocytic foci.

Tolerance was induced in Lewis (LEW) rat renal allograft recipients of Brown Norway kidneys by multiple pretransplant donor-blood transfusions and prior limited cyclosporine A. Rat renal allograft tolerance was associated with the induction of systemic donor T cells (10%), an early phase of nonspecific suppressor-cell generation, followed by maturation of systemic antigen-specific suppressor cells, and renal cellular infiltrates that develop long-term in situ in the kidney graft model. It was hypothesized that these infiltrates represent chimeric immunocytic foci that are locally regulated via a TGF-beta-dependent mechanism. Both immunohistochemical staining and digital image analysis for cellular and extracellular TGF-beta, IL-2 receptor (CD25), and the BN Class I-MHC marker (OX-27) were performed. Control rejecting (REJ) kidneys did not demonstrate any differences with respect to levels of infiltrating immunocyte area vs long-term surviving (TOL) kidneys (3.9% vs 4.5%, P = .303). Immunostaining with the BN Class I MHC marker (OX-27) demonstrated high levels of chimerism within immunocyte foci of the tolerant grafts (OX-27 BN+immunocytes 49.0% +/- 5.1%). In situ cellular IL-2 receptor (CD25) expression was demonstrated in REJ kidney infiltrates but not within TOL immunocytic infiltrating foci, when measured as percent of total lymphocytes (REJ = 5.0% vs TOL = 0.4%, P = .031). Conversely, TGF-beta expression was significantly higher in immunocytes of TOL kidneys when measured as the number of DAB chromogen-staining pixels per total immunocyte area (TOL = .076 vs REJ = .047, P = .003). In conclusion, these results suggested that stable mixed immune chimerism (SMIC) plays an important role in DST-CyA-induced tolerance in situ. SMIC-induced tolerance may involve a local TGF-beta-dependent mechanism that is associated with in situ TGF-beta (+) and IL-2r (-) immunocytes.

Animals↗
PubMed2000

60,XY/60,XX chimerism in the germ cell line of mature bulls born in heterosexual twinning.

According to present knowledge there is a germ cell chimerism (XY/XX) in young bulls born in heterosexual twinning due to exchange of primordial germ cells in embryonic life. These germ cells were believed to have been eliminated in the young bull. Two-color fluorescence in situ hybridization (FISH) identification of the sex chromosomes by biotinylated and digoxygenin labeled probes have been used. The material consisted of three bulls born in heterosexual twinning. The results obtained indicated that even mature bulls (more than two years old) demonstrate spermatogonial chimerism. Several authors state that the bulls with blood cell chimerism, originating from dizygous twinning, are characterized by decreased fertility. Changes of the sex ratio of offspring due to proliferation of the female cells have also been proposed. The present observations should give a renewed interest in checking the possibility of survival and differentiation of germ cells from the female partner in the germ cell lines.

Animals↗
PubMed2003

Chimeric flap in clinical use.

With the development of modern microsurgical reconstruction, there are several alternative methods for the complicated head and neck defect or injured extremity. The double-paddled flap, double flaps, and double flap with vascular flow-through linkage are good tools for extensive wide, composite, and three-dimensional defects. When faced with a difficult reconstruction, the expense, morbidity of the donor site, and operative time must be taken into consideration. Compared with other combined composite flaps and other methods, the chimeric flap has many advantages including: easy three-dimensional insetting, acceptable aesthetic appearance, reduced donor site morbidity, design with the least effort and operation time, and one pair of recipient vessel. The disadvantages of the chimeric flaps include: the variation of perforators, the requirement of a learning curve, and, sometimes, the need for a second venous drainage or shifting to double flaps. Overall, in selected cases, the chimeric flap is a good option for the reconstruction of extensive, composite, and three-dimensional defects.

Bone and Bones↗
PubMedDate not supplied

Chimerism in children with juvenile dermatomyositis.

Juvenile dermatomyositis is a disease with similarities to chronic graft-versus-host disease. To identify whether chimerism is present in juvenile dermatomyositis, we investigated the families of 15 children with the disorder. Chimerism was identified by PCR in 13 of the 15 affected children, compared with five of 35 siblings (p<0.0001). Maternal cells among peripheral-blood mononuclear cells were detected in 11 of the 15 boys, compared with five of 17 unaffected controls (p=0.02), and in muscle tissue of 12 of 15 compared with two of ten unaffected siblings (p=0.005). These results suggest that chimerism may be involved in juvenile dermatomyositis.

Case-Control Studies↗
PubMed1999

Mixed lymphohaemopoietic chimerism and graft-versus-lymphoma effects after non-myeloablative therapy and HLA-mismatched bone-marrow transplantation.

BACKGROUND: HLA-mismatched donor bone-marrow transplantation after standard myeloablative conditioning therapy for haematological malignant disorders has been limited by severe graft-versus-host disease (GVHD) and graft failure. We tested a new approach to find out whether lymphohaemopoietic graft-versus-host reactions could occur without excessive GVHD in mixed haemopoietic chimeras produced across HLA barriers with non-myeloablative conditioning. METHODS: Five patients with refractory non-Hodgkin lymphoma underwent bone-marrow transplantation from haploidentical related donors sharing at least one HLA A, B, or DR allele on the mismatched haplotype. Conditioning included cyclophosphamide and thymic irradiation before transplantation, and antithymocyte globulin before and after transplantation. The only other GVHD prophylaxis was cyclosporin. FINDINGS: Four of five patients were evaluable and showed engraftment. Mixed haemopoietic chimerism was established, with a predominance of donor lymphoid tissue and varying degrees of myeloid chimerism. Two patients were in GVHD-free states of complete and partial clinical remission at 460 and 103 days after bone-marrow transplantation. INTERPRETATION: Mixed chimerism can be induced in adult recipients of HLA-mismatched bone-marrow transplantation by a non-myeloablative conditioning regimen. The antilymphoma responses seen in two patients suggest that allogeneic bone-marrow transplantation without myeloablative conditioning might have potent immunotherapeutic benefits.

Adult↗
PubMed2001

Donor lymphocyte infusion followed by interferon-alpha plus low dose cyclosporine A for modulation of donor CD3 cells activity with monitoring of minimal residual disease and cellular chimerism in a patient with first hematologic relapse of chronic myelogenous leukemia after allogeneic bone marrow transplantation.

A 15-year-old girl with Ph-positive chronic myelogenous leukemia in first chronic phase received bone marrow from her human leukocyte antigen matched brother. Twenty three months after bone marrow transplantation hematological relapse occured which was treated with two infusions of donor lymphocytes (DLI) (0.5x10(8) CD3/kg b.w./infusion). To enforce the graft-versus-leukemia effect (GvL), the first DLI was followed by administration of interferon-alpha (INF-alpha) 6x10(6) U/day for 30 days, whereas, after the second infusion INF-alpha was given at the same dose until hematological remission was achieved (80 doses). Minimal residual disease (MRD) was detected by conventional cytogenetics (Ph chromosome), fluorescence in situ hybridization (FISH) cytogenetics (BCR/ABL translocation) and reverse transcriptase-polymerase chain reaction (RT-PCR) Ecotropic virus integration site-1 (EVI-1 gene expression), whereas cellular chimerism was monitored by assessment of microsatellite markers PCR and Y-chromosomal DNA content FISH. When hematological remission was achieved the pancytopenia was observed and the cytogenetic and molecular investigations revealed only partial remission and mixed chimerism, however, with predominance of donor origin hematopoiesis. To diminish the myelosupressive effect of donor CD3 cells without switching-off the GvL effect, a low dose of cyclosporine A was given. Further observation revealed significant improvement of hematopoiesis with parallel gradual decline of MRD and increase of donor hematopoiesis up to complete chimerism. Graft-versus-host disease was not observed at any stage of the treatment.

Adolescent↗
PubMed2002

Sequence-simplification and chimeric assembly: new models of peptide antigen modification.

Sequence-simplified variants of a 15-mer peptide antigen, identified by amino acid side chains in alternating positions were synthesized introducing glycine residues alternatively in the parent peptide sequence and used to induce antibodies in rabbit. They reacted to a significant extent with anti-parent peptide antibodies, and in addition, affinity purified antibodies against these halved forms recognized with similar affinity and specificity, the starting peptide in affinity chromatography, optical biosensor and enzyme linked immunosorbent assay (ELISA) experiments, while no cross-reactivity was detected between reduced antigens. These findings suggest that a peptide antigen can display two molecular surfaces of recognition, identified by side chains of residues in alternating positions. Each surface can even take part in antigen/antibody interaction independently, thus indicating the possibility to select and assembly sequence-simplified forms belonging to different epitopes, also deriving from different molecules, to generate new structures incorporating a two-fold antigen/antibody specificity. Two "chimeric" forms were then synthesized starting from the P15 and P13 complementary peptides, both able to bind interleukin 2. These structures, showing simultaneously trans-surfaces of recognition belonging to both parent forms, have been found to retain antigenic properties against antibodies of simplified P15 derivatives showing the same molecular surface of recognition. In addition, anti-chimeric antibodies recognized both P15 and P13 starting peptides, while no cross-antibody recognition was observed between chimeric antigens.

Amino Acid Sequence↗
PubMed1997

Humanized porcine VCAM-specific monoclonal antibodies with chimeric IgG2/G4 constant regions block human leukocyte binding to porcine endothelial cells.

Discordant xenografts surviving the initial hyperacute rejection phase may be subject to cellular rejection processes mediated by infiltrating leukocytes including T cells, NK cells and monocytes. The stable adhesion of these cell types to endothelial cells is due to the molecular interaction of the integrins VLA-4 and LFA-1 with their ligands vascular cell adhesion molecule (VCAM) and ICAM-1 present on the endothelial cells. Human VLA-4 binds to porcine VCAM, and blocking mAbs specific for porcine VCAM have been developed. We have localized the epitope of the anti-porcine VCAM blocking mAbs 2A2 and 3F4 to domains 1 and 2, respectively. Humanized antibodies (IgG4 isotype) were constructed from these anti-porcine VCAM antibodies and demonstrated to inhibit adhesion of Ramos, Jurkat and YT cells, as well as purified resting and activated human T cells, to porcine aortic endothelial cells (PAEC). These cell types express both LFA-1 as well as VLA-4, suggesting blockade of human VLA-4 interaction with porcine VCAM may alone be sufficient to significantly impair adhesion of human leukocytes to porcine endothelial cells. The chimeric anti-porcine VCAM (pVCAM) HuG4 antibodies promoted increased adhesion of Fc receptor (FcR) positive cells such as U937 monocytic cells to PAEC. In contrast, chimeric anti-porcine VCAM antibodies created using the CH1 and hinge region from human IgG2 and the CH2 and CH3 regions from human IgG4 (HuG2/G4 antibodies) inhibited binding of FcR positive cells to PAEC. These chimeric anti-pVCAM antibodies should allow delineation of the in vivo role of VLA-4/VCAM interaction in porcine-to-primate xenotransplants. Further, the design of the HuG2/G4 antibodies should render them efficacious in multiple settings requiring elimination of FcR binding.

Amino Acid Sequence↗
PubMed2003

Mixed chimeric hematopoietic stem cell transplant reverses the disease phenotype in canine leukocyte adhesion deficiency.

The genetic disease canine leukocyte adhesion deficiency (CLAD) is characterized by recurrent, severe bacterial infections, typically culminating in death by 6 months of age. CLAD is due to a mutation in the leukocyte integrin CD18 subunit, which prevents surface expression of the CD11/CD18 leukocyte integrin complex. We demonstrate that stable mixed donor:host hematopoietic chimerism, achieved by a non-myeloablative bone marrow transplant from a histocompatible littermate, reverses the disease phenotype in CLAD. Donor chimerism following the transplant was demonstrated both by flow cytometric detection of donor-derived CD18-positive leukocytes in the peripheral blood of the recipient, and by the demonstration of donor-derived DNA microsatellite repeats in the peripheral blood leukocytes of the recipient. These results indicate that mixed hematopoietic chimerism reverses the clinical phenotype in CLAD and represents a potential therapeutic approach for the human disease leukocyte adhesion deficiency.

Animals↗
PubMed2001

High-level expression of hepatitis C virus (HCV) structural proteins by a chimeric HCV/BVDV genome propagated as a BVDV pseudotype.

A chimeric cDNA genome was constructed in which the core, E1 and E2 genes of hepatitis C virus (HCV) replaced the core, E(rns), E1 and E2 genes of bovine viral diarrhea virus (BVDV). High levels of HCV structural proteins were expressed in a small number of human or bovine cells following transfection with chimeric RNA. However, in one cell line, bovine embryonic trachea cells [EBTr(A)], the number of cells expressing HCV proteins increased to greater than 70% following serial passage of culture medium. These cells were persistently infected with a non-cytopathogenic BVDV helper virus. In these cells, the chimeric genome was packaged into infectious particles that accumulated in the culture medium at a titer as high as 10(7)-10(9) genome equivalents per ml. The virus particles were pseudotypes, because they were neutralized by anti-BVDV but not by anti-HCV.

Animals↗
PubMed2003

A rapid and convenient variant of fusion-PCR to construct chimeric flaviviruses.

So far, full-length cDNAs of chimeric flaviviruses have been constructed by restriction-enzyme cleavage of the gene(s) to be exchanged or by fusion-PCR of two amplified PCR fragments. The construction of a chimeric flavivirus by a faster and more convenient variant of the standard fusion-PCR is reported. A Modoc/yellow fever chimeric virus was engineered in which the structural prM and E genes of yellow fever virus 17D were replaced by the homologous genes of Modoc virus. In two PCR steps, a fusion was made between the 3' end of the C gene of yellow fever virus and the 5' end of the prM gene of Modoc virus, and between the 3' end of the E gene of Modoc virus and the 5' end of the NS1 gene of yellow fever virus. For each of the two fusions between yellow fever and Modoc virus, a standard PCR was performed to amplify a short fragment with one overlapping end that could be used as one of the primers in the subsequent (fusion) PCR.

Animals↗