Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P30536 (PBS)
 9,886 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The objective of this research was to study efficiency of embryo development following transfer of blastomeres into the perivitelline space of oocytes. Single blastomeres from 8-, 16-, and 32-cell embryos were obtained following mucin coat and zona pellucida removal by combined treatments with pronase and acidic phosphate-buffered saline (PBS, pH = 2.5). Blastomeres were separated by pipetting with a fire-polished micropipette following incubation in Ca+(+)-free PBS for 15 min at 39 degrees C. This procedure resulted in over 97% blastomere separation. For ease of blastomere insertion, oocytes were placed in droplets of 0.5 M sucrose in PBS (SPBS) during micromanipulation. To functionally enucleate oocytes some were stained with Hoechst 33342 DNA stain and irradiated. A single 8- or 16-cell blastomere was aspirated into an injection pipette (35 microns or 25 microns at the tip, respectively) and inserted into the perivitelline space of an irradiated or non-irradiated oocyte, but not fused with the oocyte. This micromanipulation procedure did not affect development of individual blastomeres into blastocysts or trophectoderm vesicles when compared with cultured control single blastomeres (P greater than .05). When the inserted blastomere was induced to fuse with an intact non-irradiated oocyte under an electric field, 56-57% were fused and 39-45% of the fused and activated oocytes developed to morulae or blastocysts. When an inserted blastomere (from 8-32-cell embryos) was induced to fuse with a functionally enucleated oocyte treated by Hoechst 33342 staining, followed by washing and UV-light irradiation, 63-66% of them were fused, but only 15-22% developed to the morula or blastocyst stage. This research demonstrated that the use of hypertonic medium treated oocytes greatly improved the ease and success rate of blastomere subzona insertion, but the value of functionally enucleated oocytes as recipient cells for nuclear transfer requires further investigation.
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PMID:Potential of hypertonic medium treatment for embryo micromanipulation: II. Assessment of nuclear transplantation methodology, isolation, subzona insertion, and electrofusion of blastomeres to intact or functionally enucleated oocytes in rabbits. 224 75

A characteristic of activated T lymphocytes is the expression of high affinity IL-2R. We studied a new method of selective immunosuppression directed against activated T cells by using a chimeric recombinant protein (IL-2-PE40) composed of IL-2 fused to a modified Pseudomonas exotoxin lacking its cell recognition domain. As a model of T cell-mediated disease, we used experimental autoimmune uveoretinitis (EAU) produced in Lewis rats by active immunization with the retinal S-Ag. The treatment protocol consisted of i.p. injection of IL-2-PE40 at 0.25 micrograms/g every 12 h. Controls were PBS, PE40, or IL-2-PE40asp553 a mutant form of the molecule with reduced activity. Treatment with IL-2-PE40 resulted in a significant reduction of the incidence and severity of EAU over controls. The analysis of the effect of i.p. injection of IL-2-PE40 on the popliteal draining lymph nodes of immunized animals showed a marked reduction in the lymphocytes content. Transfer experiments demonstrated that IL-2-PE40 prevented the development of EAU effector T cells. Interestingly, although activated B cells were reported to express IL-2R, there was no significant reduction of antibody production against the immunizing Ag under IL-2-PE40 treatment, suggesting sparing of the B cells.
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PMID:Selective immunosuppression of activated T cells with the chimeric toxin IL-2-PE40. Inhibition of experimental autoimmune uveoretinitis. 251 Dec 43

Cells from adult Fasciola hepatica were fused with cells from a murine BALB/c myeloma Sp2 line. The hybrid cells were grown in HAT (hypoxanthine, aminopterin, and thymidine) medium, cloned and subcloned, and shown to express parasite antigen for 1 year after fusion. Expression of parasite antigen was demonstrated by the following: 2 histogram flow cytometric analyses, in which a population of hybrid cells in the population of 7 month cultured hybrid cells showed 57% more fluorescence when treated with an anti-F. hepatica serum followed by anti-rabbit immunoglobulin G coupled to fluorescein isothiocyanate as compared with the same hybrid cells washed and treated with normal rabbit serum; Sp2 myeloma cells treated with an anti-F. hepatica serum or normal rabbit serum followed by fluorescein-labeled anti-rabbit IgG had the same negative fluorescence; BALB/c mice immunized with PBS-washed cells from a subclone of these hybridomas developed anti-F. hepatica antibodies (shown by the Falcon assay screening test enzyme-linked immunosorbent assay); and antibodies recognized an F. hepatica antigenic polypeptide of 57,000 Mr in a Western immunoblot. These helminth:myeloma hybrids expressed murine host markers, further confirming the hybrid nature of this cell line. F. hepatica cells alone, like their Sp2 fusion partners, die in HAT supplemented medium by 9 days of culture. F. hepatica:Sp2 hybridomas have been grown continuously in HAT medium for greater than 1 year.
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PMID:Fasciola hepatica:Sp2/0 (helminth:myeloma) hybridoma expressing parasite antigen. 264 45

The ability of a tumor-specific T suppressor factor (TsF) isolated from a T cell hybridoma, A10, to act as an immunogen in DBA/2 mice was investigated. The TsF was affinity purified from ascites over an immunoadsorbent column containing a monoclonal antibody (B16G) that has specificity for the TsF molecule, or over columns containing membrane extracts of the P815 mastocytoma (the tumor for which A10 is specific). The specificity control was BW5147 (the fusion partner for A10) membrane extracts treated in the same way as A10. DBA/2 mice were immunized with the affinity-purified material or PBS and were subsequently challenged with either the P815 tumor or the L1210 DBA/2 thymoma. When mice were immunized with material affinity purified over B16G, eluted material from both A10 ascites and BW5147 membrane extracts enhanced resistance to both P815 and L1210 challenge, indicating that B16G was binding immunogenic material derived from both preparations, which exerted a tumor-protective effect. However, when a P815 affinity column was used, protective material was eluted only from A10 ascites, and this bestowed resistance to both P815 and L1210. When irradiated whole cells were used as immunogens, only A10 cells stimulated anti-tumor immunity, and this appeared to be directed specifically to the P815 tumor. The implications of these findings in terms of the potential for immune modulation with anti-suppressor therapy, and the specificity of the B16G monoclonal, are discussed. The demonstration of B16G binding material (TsF) in the membranes (but not the ascites) of the BW5147 line is also of significance to investigators using BW5147 fused suppressor hybridomas.
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PMID:Immunization of DBA/2 mice with a T cell hybridoma-derived TsF increases immune resistance to the syngeneic tumors P815 and L1210. 348 83

Fusion of blastomeres of 2-cell mouse embryos with an intact zona pellucida can be induced with electric pulses. Fusion was most frequent with the field strength of 1 kV/cm and direct current pulses of 100-250 microsec duration. An electrolyte solution (PBS) can be used instead of a non-electrolyte solution (0.3 M mannitol). The viability of blastomeres fused in these two types of solution is similar. Fused 2-cell blastomeres develop into tetraploid blastocysts but die after implantation. Embryos in which blastomeres failed to fuse despite the treatment (diploid controls) can develop till term. The technique can also be applied to 3- and 4-cell embryos and to zona-free oocytes and blastomeres.
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PMID:Electrofusion of mouse blastomeres. 388 49

We tested whether the in vivo infusion of recombinant, soluble CTLA4 fused with Ig heavy chains, as a surrogate ligand used to block CD28/CTLA4 T-cell costimulation, could prevent efficient T-cell activation and thereby reduce graft-versus-host disease (GVHD). Lethally irradiated B10.BR recipients of major histocompatibility complex disparate C57BL/6 donor grafts received intraperitoneal injections of human CTLA4-Ig (hCTLA4-Ig) or murine CTLA4-Ig (mCTLA4-Ig) in various doses and schedules beginning on day -1 or day 0 of bone marrow transplantation (BMT). In all five experiments, recipients of CTLA4-Ig had a significantly higher actuarial survival rate compared to mice injected with an irrelevant antibody control (L6) or saline alone. Survival rates in recipients of hL6 or PBS were 0% at 29 to 45 days post-BMT. In recipients of CTLA4-Ig, survival rates were as high as 63% mice surviving 3 months post-BMT. However, protection was somewhat variable and recipients of CTLA4-Ig were not GVHD-free by body weight, clinical appearance, and histopathologic examination. There were no significant differences in the survival rates in comparing injection dose, injection duration, or species of CTLA4-Ig (hCTLA4-Ig v mCTLA4-Ig). Splenic and peripheral blood flow cytometry studies of long-term hCTLA4-Ig-injected survivors showed a significant peripheral B-cell and CD4+ T-cell lymphopenia, consistent with GVHD. A kinetic study of splenic reconstitution was performed in mice that received hCTLA4-Ig and showed that mature splenic localized CD8+ T-cell repopulation was not significantly different in recipients of hCTLA4-Ig compared with hL6, despite the significant increase in actuarial survival rate in that experiment. These data suggest that the beneficial effect of hCTLA4-Ig on survival is not mediated by interfering with mature donor-derived T-cell repopulation post-BMT. Neither hCTLA4-Ig nor mCTLA4-Ig interfered with hematopoietic recovery post-BMT. We conclude that CTLA4-Ig (most likely in combination with other agents) may represent an important new modality for GVHD prevention.
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PMID:In vivo blockade of CD28/CTLA4: B7/BB1 interaction with CTLA4-Ig reduces lethal murine graft-versus-host disease across the major histocompatibility complex barrier in mice. 751 23

The Plasmodium falciparum clustered asparagine-rich protein (CARP) is a merozoite-associated antigen which contains approximately 30% asparagine. Analysis of the DNA sequences located 5' of the cloned 1.4-kb CARP gene in the P. falciparum genome suggests that this gene fragment may encode the complete CARP and that the gene product is a protein of M(r) 50,000. To analyze the immunogenicity of CARP, the gene was expressed as a fusion protein with staphylococcal protein A (SpA-CARP). Immunization of rabbits with SpA-CARP in Freund's complete adjuvant (FCA) resulted in a strong antibody response against CARP as measured in ELISA. This response was efficiently boosted and sustained over a long time while that induced by two immunizations with SpA-CARP in ISCOMs was weak and of shorter duration. In both instances, the antibody levels against CARP were further increased by a second booster injection consisting of either SpA-CARP or CARP fused to the serum albumin-binding region (BB) of streptococcal protein G (BB-CARP) in PBS, indicating that immunizations with SpA-CARP in FCA or ISCOMs had induced a CARP-specific immunological memory. Boosting with BB-CARP in PBS was more efficient than boosting with SpA-CARP in PBS. In all rabbits, the antibodies obtained after the booster with CARP in PBS were the most efficient inhibitors of merozoite invasion in vitro. The antisera reacted with the intracellular parasite in immunofluorescence and with a band of M(r) 50,000 in immunoblotting while several high-molecular-weight components as well as the one of M(r) 50,000 were immunoprecipitated. The specificity of the antibody responses varied between the different rabbits as indicated in ELISA, with short synthetic peptides representing different CARP sequences. Taken together, the results suggest that a previously cloned genomic DNA fragment may encode the complete P. falciparum blood-stage antigen CARP and that CARP is immunogenic in rabbits both when administered in FCA or ISCOMs.
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PMID:Plasmodium falciparum: the immune response in rabbits to the clustered asparagine-rich protein (CARP) after immunization in Freund's adjuvant or immunostimulating complexes (ISCOMs). 845 22

The present study was carried out to evaluate the effects of different activation protocols, enucleation methods, and culture media on the development of parthenogenetic and nuclear transfer (NT) rabbit embryos. Electroporation of 25 mM inositol 1,4, 5-trisphosphate (IP3) in calcium- and magnesium-free PBS immediately induced a single intracellular calcium transient in 6 out of 14 metaphase II-stage rabbit oocytes evaluated during a 10-min recording period. The percentage of oocytes treated with IP3 followed by 6-dimethylaminopurine (IP3 + DMAP) that cleaved (83.9%) and reached the blastocyst stage (50%) was significantly higher (p < 0.05) than those activated with multiple pulses (61.6% and 30.1%, respectively) or treated with ionomycin + DMAP (52.9% and 5.7%, respectively). Development of IP3 + DMAP-activated rabbit oocytes and in vivo-fertilized zygotes in different culture media was studied. Development of activated oocytes to the blastocyst stage in Earle's balanced salt solution (EBSS) supplemented with MEM nonessential amino acids, basal medium Eagle amino acids, 1 mM L-glutamine, 0.4 mM sodium pyruvate, and 10% fetal bovine serum (FBS) (EBSS-complete) (40.6%) was significantly higher (p < 0.05) than those that developed in either Dulbecco's Modified Eagle's medium (DMEM)/RPMI + 10% FBS (15.5%) or CR1aa + 10% FBS (4%) medium. In addition, 100% of in vivo-fertilized rabbit zygotes developed to the blastocyst stage in EBSS-complete. A third set of experiments was carried out to study the efficiency of blind versus stained (Hoechst 33342) enucleation of oocytes. Twenty-nine of 48 blind enucleated and IP3 + DMAP-activated oocytes cleaved (60.4%), and 15 (31.2%) subsequently reached the blastocyst stage, whereas 9 of 52 oocytes enucleated using epifluorescence (17.3%) cleaved, and none of these reached the blastocyst stage. When the above parameters that yielded the highest blastocysts were combined in an NT experiment using adult rabbit fibroblast nuclei, 72.2% (39 of 54) of the fused nuclear transplant embryos cleaved and 29.6% (16 of 54) reached the blastocyst stage.
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PMID:Development of nuclear transfer and parthenogenetic rabbit embryos activated with inositol 1,4,5-trisphosphate. 1008 54

A genetically engineered fusion toxin targeted to acute myeloid leukemic (AML) blasts was designed with the first 388 amino acid residues of diphtheria toxin with an H-M linker fused to human granulocyte-macrophage colony-stimulating factor. The cDNA was subcloned in the pRK bacterial expression plasmid and used to transform BL21 (DE3) Escherichia coli harboring pUBS500 plasmid. Transformants were grown in Superbroth and induced with IPTG. Inclusion bodies were isolated, washed, and denatured in guanidine hydrochloride with dithioerythritol. Recombinant protein was refolded by diluting 100-fold in cold buffer with arginine and oxidized glutathione. After dialysis, purified protein was obtained after anion-exchange, size exclusion on FPLC, and polymixin B affinity chromatography. The final material was filter sterilized, aseptically vialed, and stored at -80 degrees C. Fifty-four 3-liter bacterial culture preparations were made and pooled into 27 batches. The final product was characterized by Coomassie Plus protein assay, Coomassie-stained SDS-PAGE, limulus amebocyte lysate endotoxin assay, human AML HL60 cell cytotoxicity assay, HPLC TSK3000, N-terminal sequencing, E. coli DNA contamination, C57BL6 mouse toxicity, and immunohistochemistry. Yields were 23 mg/liter bacterial culture of denatured fusion toxin. After refolding and chromatography, final yields were 24 +/- 4% or 5 mg/liter. Vialed product was sterile and 1.7 +/- 0.4 mg/ml in PBS. Purity by SDS-PAGE was 99 +/- 1%. Aggregates by HPLC were <1%. Potency revealed a 24-h IC50 of 2.7 +/- 0.5 pM on HL60 cells. Endotoxin levels were 1 eu/mg. The N-terminal sequence was confirmed, and E. coli DNA was <113 pg/mg. The LD10 in mice was 110 microg/kg/day x5. There was no evidence of loss of solubility, proteolysis, aggregation, or loss of potency over 3 months at -80 and -20 degrees C. Further, the drug was stable at 4, 25, and 37 degrees C in human serum for 48 h. Drug reacted only with human monocytes, granulocytes, and myeloid precursors in frozen human tissue sections by immunohistochemistry. The synthesis of this protein drug should be useful for production for clinical phase I/II clinical trials and may be suitable for other diphtheria fusion toxins indicated for clinical development. This is the first report of the scaleup of a recombinant fusion toxin for clinical trials.
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PMID:High-level expression and purification of the recombinant diphtheria fusion toxin DTGM for PHASE I clinical trials. 1033 77

Transgene expression in intact animals now can be visualized by noninvasive techniques. However, the instruments and protocols developed so far have been formidable and expensive. We describe here a system for rapidly visualizing transgene expression in major organs of intact live mice that is simple, rapid, and eminently affordable. Green fluorescent protein (GFP) is expressed in the cells of brain, liver, pancreas, prostate, and bone, and its fluorescence is encoded in whole-body optical images. For low-magnification images, animals are illuminated atop a fluorescence light box and directly viewed with a thermoelectrically cooled color charge-coupled device camera. Higher-magnification images are made with the camera focused through an epi-fluorescence dissecting microscope. Both nude and normal mice were labeled by directly injecting 8 x 10(10) plaque-forming units/ml of adenoviral GFP in 20-100 microl PBS and 10% glycerol into either the brain, liver, pancreas, prostate, or bone marrow. Within 5-8 h after adenoviral GFP injection, the fluorescence of the expressed GFP in brain and liver became visible, and whole-body images were recorded at video rates. The GFP fluorescence continued to increase for at least 12 h and remained detectable in liver for up to 4 months. The system's rapidity of image acquisition makes it capable of real-time recording. It requires neither exogenous contrast agents, radioactive substrates, nor long processing times. The method requires only that the expressed gene or promoter be fused or operatively linked to GFP. A comparatively modest investment allows the study of the therapeutic and diagnostic potential of suitably tagged genes in relatively opaque organisms.
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PMID:Visualizing gene expression by whole-body fluorescence imaging. 1105 Feb 47


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