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)

Carcinoembryonic antigen (CEA) is expressed at greatly increased levels in nearly all human colorectal carcinomas. Anti-CEA antibodies have been proved to be useful for targeting several cancer types known to express CEA. A recombinant immunotoxin was constructed, in which the cell-binding domain of Pseudomonas exotoxin (PE) was replaced with the single-chain Fv (scFv) of anti-CEA monoclonal antibody for targeting to colorectal carcinomas. This single-chain immunotoxin was expressed in E. coli and purified under denaturing condition of 6M guanidine hydrochloride (GuHCl). It was found that the immunotoxin maintains a binding activity in denaturing condition of 6M GuHCl and the fused PE contributes to the stability of immunotoxin in such condition. Dialysis against PBS buffer after purification under 6M GuHCl keeps the binding activity of immunotoxin.
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PMID:Expression and purification of recombinant immunotoxin--a fusion protein stabilizes a single-chain Fv (scFv) in denaturing condition. 1250 88

Atomic force microscopy (AFM) and scanning electron microscopy (SEM) coupled with ellipsometry have been used to characterize the microscale and nanoscale structures of erodible multilayered films fabricated from degradable polyamine 1 and either sodium poly(styrene sulfonate) (SPS) or plasmid DNA. Striking differences were found in the topography, structures, and erosion profiles of these two materials upon incubation in PBS buffer at 37 degrees C. For films fabricated from SPS, AFM data are consistent with an erosion process that occurs uniformly without the generation of holes or pits over large, micrometer-scale areas. By contrast, films fabricated from plasmid DNA undergo structural rearrangements to present surface-bound particles ranging in size from 50 to 400 nm. Additional characterization of these particulate structures by SEM suggested that they are interpenetrated with or fused to underlying polyelectrolyte layers on the silicon surface, providing a potential mechanism to manipulate the adhesive forces with which these particles are bound to the surface. The erosion profile observed for polymer 1/SPS films suggests that it may be possible to design assemblies that release two film components with well-defined release kinetics. In the context of gene delivery, the presentation of condensed DNA as nanoparticles at these surfaces may be advantageous with respect to stimulating the internalization and processing of DNA by cells. A quantitative understanding of the factors influencing the fabrication, structure, and erosion profiles of these materials will be useful for the design of multilayered assemblies for specific applications in which controlled film erosion or the release of therapeutic materials is desired.
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PMID:Surface analysis of erodible multilayered polyelectrolyte films: nanometer-scale structure and erosion profiles. 1595 26

The present study was carried out to explore the feasibility of using buffalo fetal skin fibroblasts as donor nuclei and to find out the developmental competence of embryos following transfer of these nuclei to in vitro matured enucleated buffalo oocytes. Skin cells were isolated from 1 to 2-month-old fetuses obtained from slaughterhouse, by enzymatic digestion (0.5% w/v trypsin +0.05% w/v collagenase in Dulbecco's PBS) for 15-20 min. The cells were washed 4 times with Dulbecco's PBS and then once with RPMI-1640+10% FBS by centrifugation at 600 x g. The cells were then cultured in the same medium in a CO2 incubator (5% CO2 in air) at 38.5 degrees C for 2-3 days. Cumulus-oocyte complexes (COCs) collected from slaughterhouse buffalo ovaries were subjected to IVM in the IVM medium (TCM-199 + 5 microg/ml FSH-P + 10 microg/ml LH+10% FBS) for 20-22 h in a CO2 incubator (5% CO2 in air) at 38.5 degrees C. Oocytes were denuded with 0.1% trypsin followed by repeated pipetting and then enucleated by aspirating the first polar body with 10-15% of nearby cytoplasm with a micromanipulator. Two different types of donor cells (growing cells and those arrested with cytochalasin-B) were used for reconstruction of oocytes. The reconstructs were electro fused and incubated in the activation medium (TCM-199 + 8 microg/ml cytochalasin-B+10% FBS) for 4 h. These were then cultured in IVC medium (TCM-199+10% FBS) in a CO2 incubator (5% CO2 in air) at 38.5 degrees C for 48 h. The cleaved embryos were then co-cultured with buffalo oviduct cells in embryo development media (EDM). Out of 119 denuded matured oocytes which were enucleated and reconstructed with growing cells, 78 (65.5%) were electro fused, activated and cultured, out of which 4 (5.1%) reconstructs cleaved and developed to 2-cell stage, 3 (3.8%) reached to 4-cell stage and 3 (3.8%) reached to 8-cell stage. In the synchronized group, out of 62 denuded matured oocytes which were reconstructed with cytochalasin-B blocked cells, 40 (65%) were electrofused, activated and cultured, out of which 4 (10%) developed to 2-cell stage, 3 (7.50%) to 4-cell stage, 2 (5.0%) to early morula stage and 1 (2.50%) to blastocysts stage. These results suggest that buffalo fetal skin fibroblasts could be used as donor nuclei for the production of buffalo embryos after nuclear transfer to enucleated in vitro matured buffalo oocytes.
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PMID:Development of water buffalo (Bubalus bubalis) embryos from in vitro matured oocytes reconstructed with fetal skin fibroblast cells as donor nuclei. 1618 75

We evaluated the usefulness of fusion vaccine prepared from IL-2-gene-transduced splenic dendritic cells (DCs) and fibrosarcoma tumor cells (QRsP) in treating of lung metastasis. The IL-2 or LacZ gene was transferred into spleen-derived DCs using an adenoviral vector. Irradiated QRsP tumor cells were fused with IL-2 gene transduced DCs (fusion/IL-2) or LacZ gene transduced DCs (fusion/LacZ) by polyethyleneglycol. These fusion cells expressed major histocompatibility complex (MHC) class I and MHC class II, CD86, CD11c and CD8alpha. IFN-gamma and cytotoxic T lymphocyte (CTL) activity of splenic lymphocytes in mice vaccinated with fusion cells increased significantly as compared with those of DC or tumor cells vaccinated mice. CTL levels in fusion/IL-2-vaccinated mice were higher than that in fusion/LacZ-vaccinated mice. The number of lung metastasis in the fusion/IL-2 or fusion/LacZ-vaccineatd mice was significantly lower than that in mice vaccinated with DCs, tumor or PBS. The introduction of the IL-2 gene into fusion cells produced more potent therapeutic effects. Our results suggest that the fusion cells prepared from IL-2 gene transduced spleen derived DCs and tumor cells have the ability to induce therapeutic effect against lung metastasis.
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PMID:[Fusion vaccine therapy by IL-2-gene-transduced dendritic cells and tumor cells]. 1631 76

Proteins capable of selective and specific inhibition of cysteine protease have been identified as cystatins and are isolated from a variety of microbes and tissues of animals and plants. The physiological function of these proteins has been proposed to be the regulation of protein turnover and defense against pathogens as well as the balance of the host-parasite immune relationship. Genes encoding cystatins have been found in several species of ticks, but the function of cystatin in ticks is not understood. We cloned a gene encoding cystatin from tick H. longicornis and designated it as Hlcyst-2 (H. longicornis cystatin-2). Its full-length cDNA is 569 bp, and it encodes a putative 133 amino acid protein with an obvious signal peptide. Sequence analysis demonstrated that it has significant homology with the known cystatin. The recombinant protein was expressed in a GST-fused soluble form in Escherichia coli and purified by affinity chromatography. The inhibitory activity of the recombinant protein against papain, cathepsin L, and cathepsin B was identified by fluorogenic substrate analysis. Cystatin was mostly expressed in the tick midgut and hemocyte. Blood feeding induced significantly increased expression in the midgut. Real-time PCR confirmed that LPS-injected adult ticks expressed Hlcyst-2 1.6 more times than the PBS-injected control; Babesia gibsoni-infected larvae ticks expressed Hlcyst-2 1.8 more times than normal larvae ticks. The recombinant protein also showed a significant growth-inhibitory effect on Babesia bovis cultured in vitro. These results indicated this cystatin Hlcyst-2 is involved in tick innate immunity.
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PMID:A secreted cystatin from the tick Haemaphysalis longicornis and its distinct expression patterns in relation to innate immunity. 1683 18

A radiolabeled anti-HER2 Affibody molecule (Z(HER2:342)) targets HER2-expressing xenografts with high selectivity and gives good imaging contrast. However, the small size (approximately 7 kDa) results in rapid glomerular filtration and high renal accumulation of radiometals, thus excluding targeted therapy. Here, we report that reversible binding to albumin efficiently reduces the renal excretion and uptake, enabling radiometal-based nuclide therapy. The dimeric Affibody molecule (Z(HER2:342))(2) was fused with an albumin-binding domain (ABD) conjugated with the isothiocyanate derivative of CHX-A''-DTPA and labeled with the low-energy beta-emitter (177)Lu. The obtained conjugate [CHX-A''-DTPA-ABD-(Z(HER2:342))(2)] had a dissociation constant of 18 pmol/L to HER2 and 8.2 and 31 nmol/L for human and murine albumin, respectively. The radiolabeled conjugate displayed specific binding to HER2-expressing cells and good cellular retention in vitro. In vivo, fusion with ABD enabled a 25-fold reduction of renal uptake in comparison with the nonfused dimer molecule (Z(HER2:342))(2). Furthermore, the biodistribution showed high and specific uptake of the conjugate in HER2-expressing tumors. Treatment of SKOV-3 microxenografts (high HER2 expression) with 17 or 22 MBq (177)Lu-CHX-A''-DTPA-ABD-(Z(HER2:342))(2) completely prevented formation of tumors, in contrast to mice given PBS or 22 MBq of a radiolabeled non-HER2-binding Affibody molecule. In LS174T xenografts (low HER2 expression), this treatment resulted in a small but significant increase of the survival time. Thus, fusion with ABD improved the in vivo biodistribution, and the results highlight (177)Lu-CHX-A''-DTPA-ABD-(Z(HER2:342))(2) as a candidate for treatment of disseminated tumors with a high level of HER2 expression.
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PMID:Radionuclide therapy of HER2-positive microxenografts using a 177Lu-labeled HER2-specific Affibody molecule. 1736 99

We have recently expressed and characterized the product of the gene Rv2108 of Mycobacterium tuberculosis (MT), the p27 protein. Here, we investigated the immune responses against the p27 protein in the context of different pathogen associated molecular patterns (PAMPs). Different immunization protocols were used. BALB/c mice were immunized either with the p27 recombinant protein in Freund's adjuvant or in phosphate saline buffer (PBS), with a pcDNA3 plasmid containing the gene encoding the p27 protein, or with the Escherichia coli bacteria expressing the p27 protein genetically fused into the flagellin. We found that p27 expressed into the flagellin led to the strongest cellular responses, where we obtained the highest production of IFN-gamma and cell proliferation, an indication of specific Th1-like orientation of the immune response. We confirmed the role of flagellin in this response by using different immunization combinations.
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PMID:Flagellin as a good carrier and potent adjuvant for Th1 response: study of mice immune response to the p27 (Rv2108) Mycobacterium tuberculosis antigen. 1828 77

Cervical cancer is caused by infection by high-risk human papillomavirus (HPV), especially HPV16. Limitations in current treatments of cervical cancers call for the development of new and improved immunotherapies. This study aims at investigating the efficacy of a novel vaccine consisting of modified HPV 16E7 fused with human cytotoxic T-lymphocyte antigen 4 (CTLA4). The regions in HPV16 E7 gene associated with its transformation and CTL-enhanced response were modified; the resultant HPV16mE7 was fused with extracellular region of CTLA4 to generate HPVml6E7-eCTLA4 fusion protein. Binding of this fusion protein to B7 molecules expressed on antigen presenting-cells (APCs) was demonstrated. C57BL/6 (H-2b) mice immunized with low dose of the fusion protein (10 microg) produced higher titer antibody and stronger specific CTL response, and expressed higher levels of IFN-gamma and IL-12, compared with those immunized with HPVml6E7 only or admixture of HPVml6E7 and CTLA4, or PBS; and were protected from lethal dose tumor challenge. Tumor growth was retarded and survival prolonged in mouse models with the fusion protein treatment. Our results demonstrate that fusion of HPV16 E7 with eCTLA4 targeting APCs resulted in enhanced immunity, and that this fusion protein may be useful for improving the efficacy of immunotherapeutic treatments of cervical cancer and other HPV16 infection-associated tumors.
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PMID:Enhancement of immunotherapeutic effects of HPV16E7 on cervical cancer by fusion with CTLA4 extracellular region. 1910 4

Defensins are fundamental components of innate immune response. Current data favor that defensins play vital roles on both innate and adaptive immune responses. The aim of the present study was to investigate whether the chicken beta-defensin-1 (also named avian beta-defensin-1, AvBD1) has the potent adjuvant effects on DNA vaccine encoding IBDV VP2 gene, when genetically fused with VP2 gene. The recombinant vectors pcDNA3.1(+)-VP2 and pcDNA3.1(+)-AvBD1-VP2 were constructed as the DNA vaccines. Four groups of 14-day-old chickens were intramuscularly injected with PBS buffer, empty vector pcDNA3.1(+), recombinant pcDNA3.1(+)-VP2 and pcDNA3.1(+)-AvBD1-VP2. Results showed that VP2-specific antibody levels significantly increased following two recombinant DNA vaccine administrations (p<0.05), compared with the group of PBS and empty vector. The antibody level of group immunized with pcDNA3.1(+)-AvBD1-VP2 was significantly higher than that of group immunized with pcDNA3.1(+)-VP2 after second vaccination (p<0.05). The percentages of CD3+, CD4+ and CD8+ T-cell subtypes between groups of pcDNA3.1(+)-VP2 and pcDNA3.1(+)-AvBD1-VP2 obtained significantly different (p<0.05), the latter was higher, at 7 days post-booster. The protection from IBD challenged by immunized chickens with DNA vaccines encoding IBDV VP2 gene alone was lower than that by immunized IBDV VP2 gene together with AvBD1 gene. The results indicated that AvBD1 has an adjuvant effects on improvement the IBDV VP2-DNA vaccine effectiveness.
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PMID:The potent adjuvant effects of chicken beta-defensin-1 when genetically fused with infectious bursal disease virus VP2 gene. 2033 34

In the present study, we demonstrated site-specific immobilization and solid-phase refolding of single-chain Fv antibodies on hydrophilic polystyrene (phi-PS) plates that was mediated by novel polystyrene binding peptides (PS-tags: RIIIRRIRR), which were originally isolated and optimized in previous studies. Three PS-tag-fused scFvs, namely scFv-PS, scFv-(PS), and scFv-PSII, which were over-expressed in the insoluble fraction of Escherichia coli cells were denatured and site-specifically immobilized onto hydrophilic PS plates in the presence of 0.5-4 M urea and 0.1% Tween 20. The antigen-binding activity of the scFvs was efficiently recovered by washing the surface of the plate with PBS that contained 0.1% Tween 20 (PBST). The solid-phase refolding mediated by PS-tag was successfully applied to several scFvs such as mouse anti-CRP antibodies and an anti-RNase antibody, although further investigation of the versatility of scFv-PSII is needed. The maximal density of PS-tag-fused scFvs was increased more than 15-fold compared with a whole monoclonal antibody (mAb) immobilized on Maxisorp and, consequently, the sensitivity of PS-tag-fused scFvs for CRP in a sandwich ELISA was increased 25-fold. Thus, the novel, solid-phase, refolding method mediated by a PS-tag will be very useful for preparation of solid supports coated with recombinant antibody fragments, which can be used in immunoassays and immuno-separation.
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PMID:Novel solid-phase refolding method for preparation of scFv-immobilized polystyrene plates with high-antigen-binding activity. 2066 28


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