Protein A-Sepharose 4B Research Articles

The design of protein-imprinted polymers as antibody substitutes for investigating protein–protein interactions

Co-immunoprecipitation is a very effective method for studying protein–protein interactions. However, the preparation of antibodies in this method involves the injection of antigen into mammals, and requires the use of the expensive protein A-Sepharose 4B. Molecular imprinting polymer can compensate for these deficiencies. In this paper, a new strategy for studying protein interactions is reported; this method is based on the use of protein-imprinted polymers (PIPs). PIP is a proper substitute for antibody. We designed and synthesized assistant recognition polymer chains (ARPCs), which were limited length polymer chains with randomly distributed recognition and immobilizing sites. The template protein was selectively assembled with ARPCs. The assemblies were adsorbed by macroporous microspheres, and were immobilized by cross-linking polymerization. After removing the templates, the two kinds of synthesized PIPs were used to adsorb natural BiP or FKBP23 from ER extract; both showed high selectivity. Furthermore, we investigated the binding specificity of BiP to FKBP23, using synthesized PIPs. The results showed that FKBP23 could bind to BiP in ER in a process regulated by the concentration of Ca2+, which was consistent with the immunoprecipitation results. This strategy may provide a general solution for investigating protein interactions.

A Biotin–Streptavidin Amplified Enzyme-Linked Immunosorbent Assay with Improved Sensitivity for Rapid Detection of Ractopamine in muscular tissue

An effective biotin–streptavidin amplified enzyme-linked immunosorbent assay (BA-ELISA) was optimized and characterized for the rapid detection of Ractopamine (RAC) residue in muscular tissue. Purification of the RAC antiserum by protein A-Sepharose 4B followed with bovine serum albumin (BSA)-Sepharose 4B affinity chromatography enhanced the sensitivity and reduce the background adsorption. Blocking with 0.5% skimmed milk power and diluting streptavidin–HRP conjugates with 0.5% BSA/phosphate-buffered saline (PBS) effectively remove the nonspecific adsorption in biotin–streptavidin amplified ELISA system. The established method allowed RAC determination with an IC50 value of 0.3 ± 0.02 ng ml−1 and a limit of detection of 0.02 ± 0.003 ng ml−1, more sensitive than the other reported methods. The variation coefficients of intra-assay and inter-assay were all below 7%. RAC residue in pig muscular tissue could be quantified without matrix effects after a 5-fold extraction and 2-fold dilution with PBS. Recoveries of RAC in pig muscular tissue ranged from 75% to 82.75%. The results were also compared with those from HPLC and a good correlation was obtained (r 2 = 0.9822). The characters show that the established biotin–streptavidin amplified ELISA could be potentially useful in rapid detection of RAC in animal-derived foods.

Phospholipid Scramblase 1 Is Secreted by a Lipid Raft-dependent Pathway and Interacts with the Extracellular Matrix Protein 1 in the Dermal Epidermal Junction Zone of Human Skin

We examined the interaction of ECM1 (extracellular matrix protein 1) using yeast two-hybrid screening and identified the type II transmembrane protein, PLSCR1 (phospholipid scramblase 1), as a binding partner. This interaction was then confirmed by in vitro and in vivo co-immunoprecipitation experiments, and additional pull-down experiments with GST-tagged ECM1a fragments localized this interaction to occur within the tandem repeat region of ECM1a. Furthermore, immunohistochemical staining revealed a partial overlap of ECM1 and PLSCR1 in human skin at the basal epidermal cell layer. Moreover, in human skin equivalents, both proteins are expressed at the basal membrane in a dermal fibroblast-dependent manner. Next, immunogold electron microscopy of ultrathin human skin sections showed that ECM1 and PLSCR1 co-localize in the extracellular matrix, and using antibodies against ECM1 or PLSCR1 cross-linked to magnetic immunobeads, we were able to demonstrate PLSCR1-ECM1 interaction in human skin extracts. Furthermore, whereas ECM1 is secreted by the endoplasmic/Golgi-dependent pathway, PLSCR1 release from HaCaT keratinocytes occurs via a lipid raft-dependent mechanism, and is deposited in the extracellular matrix. In summary, we here demonstrate that PLSCR1 interacts with the tandem repeat region of ECM1a in the dermal epidermal junction zone of human skin and provide for the first time experimental evidence that PLSCR1 is secreted by an unconventional secretion pathway. These data suggest that PLSCR1 is a multifunctional protein that can function both inside and outside of the cell and together with ECM1 may play a regulatory role in human skin.

Complement Regulator Factor H Mediates a Two-step Uptake of Streptococcus pneumoniae by Human Cells

Streptococcus pneumoniae, a human pathogen, recruits complement regulator factor H to its bacterial cell surface. The bacterial PspC protein binds Factor H via short consensus repeats (SCR) 8-11 and SCR19-20. In this study, we define how bacterially bound Factor H promotes pneumococcal adherence to and uptake by epithelial cells or human polymorphonuclear leukocytes (PMNs) via a two-step process. First, pneumococcal adherence to epithelial cells was significantly reduced by heparin and dermatan sulfate. However, none of the glycosaminoglycans affected binding of Factor H to pneumococci. Adherence of pneumococci to human epithelial cells was inhibited by monoclonal antibodies recognizing SCR19-20 of Factor H suggesting that the C-terminal glycosaminoglycan-binding region of Factor H mediates the contact between pneumococci and human cells. Blocking of the integrin CR3 receptor, i.e. CD11b and CD18, of PMNs or CR3-expressing epithelial cells reduced significantly the interaction of pneumococci with both cell types. Similarly, an additional CR3 ligand, Pra1, derived from Candida albicans, blocked the interaction of pneumococci with PMNs. Strikingly, Pra1 inhibited also pneumococcal uptake by lung epithelial cells but not adherence. In addition, invasion of Factor H-coated pneumococci required the dynamics of host-cell actin microfilaments and was affected by inhibitors of protein-tyrosine kinases and phosphatidylinositol 3-kinase. In conclusion, pneumococcal entry into host cells via Factor H is based on a two-step mechanism. The first and initial contact of Factor H-coated pneumococci is mediated by glycosaminoglycans expressed on the surface of human cells, and the second step, pneumococcal uptake, is integrin-mediated and depends on host signaling molecules such as phosphatidylinositol 3-kinase.

The Legs at odd angles (Loa) Mutation in Cytoplasmic Dynein Ameliorates Mitochondrial Function in SOD1G93A Mouse Model for Motor Neuron Disease

Amyotrophic lateral sclerosis (ALS) is a debilitating and fatal late-onset neurodegenerative disease. Familial cases of ALS (FALS) constitute ∼10% of all ALS cases, and mutant superoxide dismutase 1 (SOD1) is found in 15–20% of FALS. SOD1 mutations confer a toxic gain of unknown function to the protein that specifically targets the motor neurons in the cortex and the spinal cord. We have previously shown that the autosomal dominant Legs at odd angles (Loa) mutation in cytoplasmic dynein heavy chain (Dync1h1) delays disease onset and extends the life span of transgenic mice harboring human mutant SOD1G93A. In this study we provide evidence that despite the lack of direct interactions between mutant SOD1 and either mutant or wild-type cytoplasmic dynein, the Loa mutation confers significant reductions in the amount of mutant SOD1 protein in the mitochondrial matrix. Moreover, we show that the Loa mutation ameliorates defects in mitochondrial respiration and membrane potential observed in SOD1G93A motor neuron mitochondria. These data suggest that the Loa mutation reduces the vulnerability of mitochondria to the toxic effects of mutant SOD1, leading to improved mitochondrial function in SOD1G93A motor neurons.

Lysyl Oxidase Binds Transforming Growth Factor-; and Regulates Its Signaling via Amine Oxidase Activity

Lysyl oxidase (LOX), an amine oxidase critical for the initiation of collagen and elastin cross-linking, has recently been shown to regulate cellular activities possibly by modulating the functions of growth factors. In this study, we investigated the interaction between LOX and transforming growth factor-beta1 (TGF-beta1), a potent growth factor abundant in bone, the effect of LOX on TGF-beta1 signaling, and its potential mechanism. The specific binding between mature LOX and mature TGF-beta1 was demonstrated by immunoprecipitation and glutathione S-transferase pulldown assay in vitro. Both proteins were colocalized in the extracellular matrix in an osteoblastic cell culture system, and the binding complex was identified in the mineral-associated fraction of bone matrix. Furthermore, LOX suppressed TGF-beta1-induced Smad3 phosphorylation likely through its amine oxidase activity. The data indicate that LOX binds to mature TGF-beta1 and enzymatically regulates its signaling in bone and thus may play an important role in bone maintenance and remodeling.

Lysophosphatidic Acid Protects Cancer Cells from Histone Deacetylase (HDAC) Inhibitor-induced Apoptosis through Activation of HDAC

Histone deacetylases (HDACs) catalyze the removal of acetyl groups from histones and contribute to transcriptional repression. In addition, the HDAC inhibitors induce apoptosis in cancer cells through alterations in histone acetylation and activation of the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) apoptotic pathway. Lysophosphatidic acid (LPA) is a growth factor that promotes survival of cancer cells through activation of G protein-coupled receptors. Here we show that HDAC inhibitors can induce apoptosis through activation of the TRAIL apoptotic pathway, and LPA prevented HDAC inhibitor-induced apoptosis and increased TRAIL receptor DR4 (death receptor 4) protein expression. This was associated with increased HDAC1 recruitment to the DR4 promoter following LPA treatment and a reduction in HDAC inhibitor-induced histone acetylation in the DR4 promoter. In addition, LPA induces HDAC enzyme activity in a dose- and time-dependent manner, and this is associated with HDAC1 activation and increased binding of HDAC1 to HDAC2. Reducing the expression of HDAC1 significantly lowered LPA-induced HDAC activity and increased histone acetylation. LPA induction of HDAC activity was blocked by the LPA receptor antagonist, Ki16425, or by inhibiting receptor activation with pertussis toxin. Reducing the expression of the LPA receptor LPA(1) also blocked LPA-induced HDAC activation. In addition, LPA reduced histone acetyltransferase enzymatic activity. Finally, LPA attenuated the ability of the HDAC inhibitor to reduce HDAC activity. Thus, LPA enhances survival of cancer cells by increasing HDAC activity and reducing histone acetylation.

Enzyme linked immunosorbent assay for PAT protein detection in genetically modified rape

AbstractWe have developed and applied an immunoassay method to detect genetically modified (GM) rape containing phosphinothricin acetyltransferase (PAT). The purified PAT was identified by Western blotting and enzymic activity analysis. The polyclonal antibody against purified PAT protein was obtained and purified by both a saturated ammonium sulphate method and protein A-Sepharose 4B. The sensitivity and cross-reactivity of the polyclonal antibody has been demonstrated in an enzyme-linked immunosorbent assay (ELISA). The result of the ELISA for antiserum sensitivity was about 2×10−5mg/ml and the cross-reactivity determined experimentally showed a high degree of specificity for the antiserum used, because values were all less than 0.1%. Detection of transgenic plants was evaluated using two transgenic rape lines (MS1/RF1 and MS8/RF3) which could be easily distinguished by ELISA.

Nephrocan, a Novel Member of the Small Leucine-rich Repeat Protein Family, Is an Inhibitor of Transforming Growth Factor-β Signaling

In a search of new, small leucine-rich repeat proteoglycan/protein (SLRP) family members, a novel gene, nephrocan (NPN), has been identified. The gene consists of three exons, and based on the deduced amino acid sequence, NPN has 17 leucine-rich repeat motifs and unique cysteine-rich clusters both in the N and C termini, indicating that this gene belongs to a new class of SLRP family. NPN mRNA was predominantly expressed in kidney in adult mice, and during mouse embryogenesis, the expression was markedly increased in 11-day-old embryos at a time when early kidney development takes place. In the adult mouse kidney, NPN protein was located in distal tubules and collecting ducts. When NPN was overexpressed in cell culture, the protein was detected in the cultured medium, and upon treatment with N-glycosidase F, the molecular mass was lowered by approximately 14 kDa, indicating that NPN is a secreted N-glycosylated protein. Furthermore, transforming growth factor-beta (TGF-beta)-responsive 3TP promoter luciferase activity was down-regulated, and TGF-beta-induced Smad3 phosphorylation was also inhibited by NPN, suggesting that NPN suppresses TGF-beta/Smad signaling. Taken together, NPN is a novel member of the SLRP family that may play important roles in kidney development and pathophysiology by functioning as an endogenous inhibitor of TGF-beta signaling.

ABCA7 expression is regulated by cellular cholesterol through the SREBP2 pathway and associated with phagocytosis

ABCA7 is highly homologous to ABCA1 and mediates cellular cholesterol and phospholipid release by apolipoproteins when transfected in vitro. However, expression of ABCA7 was downregulated by increased cellular cholesterol while ABCA1 was upregulated, and the results were consistent by forced expression or downregulation of sterol-responsive/regulatory element (SRE) binding proteins (SREBPs). We analyzed the promoter of the ABCA7 gene and identified the new exon encoding 96 bp (mouse) and 95 bp (human) of the 5' untranslated region and the transcription start site at 1,122 bp (mouse) and 1,260 bp (human) upstream of the initiation methionine codon. At 5' upstream of this exon is the ABCA7 proximal promoter containing multiple binding sites of transcription factors for hematopoiesis and SRE of 9 bp at 212 bp (mouse) and 179 bp (human) upstream of the new exon. The apolipoprotein A-I-mediated lipid release was not influenced by suppression of the endogenous ABCA7 with small interfering RNA in mouse fibroblasts or by its increase in ABCA1-deficient mouse cells. In contrast, phagocytic activity was altered in parallel to the ABCA7 expression in these cells. When phagocytosis was induced, the messages increased for SREBP2, ABCA7, and other SREBP2-regulated proteins. The ABCA1 message decreased in this condition. We conclude that the ABCA7 gene is regulated by sterol in the opposite direction to ABCA1 through SRE/SREBP2 and that expression of ABCA7 by this regulation is associated with phagocytic activity.

Discoidin Domain Receptor 2 Mediates Tumor Cell Cycle Arrest Induced by Fibrillar Collagen

During malignant invasion tumor cells establish contact with extracellular matrix proteins, including fibrillar collagen. In addition to providing a physical barrier against invasion, fibrillar collagen also restricts cell proliferation. It has been assumed that the growth regulatory activity of fibrillar collagen is the result of an indirect restrictive effect on cell spreading and cytoskeletal organization. Here we provide evidence for a direct inhibitory effect of fibrillar collagen on proliferation of human melanoma and fibrosarcoma cells that involves activation of the tyrosine kinase discoidin domain receptor 2 and is independent of effects on cell spreading. Cells plated in the presence of fibrillar collagen were growth arrested in the G0/G1 phase of the cell cycle. However treatment with the tyrosine kinase inhibitor genistein, down-regulation of discoidin domain receptor 2, or collagen deglycosylation that prevents discoidin domain receptor 2 activation allowed cells to enter the cell cycle in the presence of fibrillar collagen without a requirement for spreading and actin organization. Our data provide evidence for a novel direct mechanism by which cell contact with fibrillar collagen restricts proliferation.

Identification, Evolution, and Regulation of Expression of Guinea Pig Trappin with an Unusually Long Transglutaminase Substrate Domain

Trappins are found in human, bovine, hippopotamus, and members of the pig family, but not in rat and mouse. To clarify the evolution of the trappin genes and the functional significance of their products, we isolated the trappin gene in guinea pig, a species belonging to a rodent family distinct from rat and mouse. Guinea pig trappin was confirmed to encode the same domain structure as trappin, consisting of a signal sequence, an extra large transglutaminase substrate domain, and a whey acidic protein motif. Northern blot analysis and in situ hybridization histochemistry as well as immunohistochemistry demonstrated that guinea pig trappin is expressed solely in the secretory epithelium of the seminal vesicle and that its expression is androgen-dependent. We confirmed that guinea pig trappin is cross-linked by prostate transglutaminase and that the whey acidic protein motif derived from guinea pig trappin has an inhibitory activity against leukocyte elastase. Genome sequence analysis showed that guinea pig trappin belongs to the family of REST (rapidly evolving seminal vesicle transcribed) genes.

Type I Transforming Growth Factor β Receptor Binds to and Activates Phosphatidylinositol 3-Kinase

We have examined the interaction of transforming growth factor (TGF)beta receptors with phosphatidylinositol 3-(PI3) kinase in epithelial cells. In COS7 cells, treatment with TGFbeta increased PI3 kinase activity as measured by the ability of p85-associated immune complexes to phosphorylate inositides in vitro. Both type I and type II TGFbeta receptors (TbetaR) associated with p85, but the association of TbetaRII appeared to be constitutive. The interaction of TbetaRI with p85 was induced by treatment with TGFbeta. The receptor association with PI3 kinase was not direct as (35)S-labeled rabbit reticulocyte p85 did not couple with fusion proteins containing type I and type II receptors. A kinase-dead, dominant-negative mutant of TbetaRII blocked ligand-induced p85-TbetaRI association and PI3 kinase activity. In TbetaRI-null R1B cells, TGFbeta did not stimulate PI3 kinase activity. This stimulation was restored upon reconstitution of TbetaRI by transfection. In R1B and NMuMG epithelial cells, overexpression of a dominant active mutant form of TbetaRI markedly enhanced ligand-independent PI3 kinase activity, which was blocked by the addition of the TbetaRI kinase inhibitor LY580276, suggesting a causal link between TbetaRI function and PI3 kinase. Overexpressed Smad7 also prevented ligand-induced PI3 kinase activity. Taken together, these data suggest that 1) TGFbeta receptors can indirectly associate with p85, 2) both receptors are required for ligand-induced PI3 kinase activation, and 3) the activated TbetaRI serine-threonine kinase can potently induce PI3 kinase activity.

Increase of thyroid stimulating activity in Graves' immunoglobulin-G by high polyethylene glycol concentrations using porcine thyroid cell assay.

Cyclic adenosine monophosphate (cAMP) production during a 5-hour incubation using porcine thyroid cells (PTC) was stimulated significantly more by polyethylene glycol (PEG) 22.5% precipitated fractions (ppt frs) than by PEG 12.5% ppt frs from almost all Graves' sera. However, the thyrotropin (TSH) binding inhibition (TBI) activities of the PEG 12.5% and 22.5% ppt frs using porcine thyroid membranes were similar, and did not change in the 5-hour incubation. When the PEG 12.5% ppt fr from Graves' serum and the PEG 22.5% ppt fr from normal human serum (NHS) were coincubated, cAMP production was also stimulated as much as by the PEG 22.5% ppt fr from Graves' serum. When purified thyroid stimulating antibody (TSAb)-immunoglobulin G (IgG) and the PEG 22.5% ppt fr from NHS were coincubated, increased cAMP production was also observed, whereas bovine thyrotropin (bTSH) did not produce this effect. When purified TSAb-IgG and PEG solutions were coincubated, maximum increases in cAMP production (approximately 10-fold) with 5% PEG were found, whereas no increase was observed using bTSH. The stimulatory effect of high PEG concentrations on thyroid stimulating activity was observed by TSAb-IgG in salt-free or salt-containing medium (<0.15 mol/L NaCl concentration) but not by either TSAb-IgG conjugated to protein A-sepharose 4B or the inactivated TSAb-IgG by the treatment of 70 degrees C for 10 minutes. No stimulatory action by PEG was found with the thyroid stimulating substances such as GTPgammaS, forskolin, or pituitary adenylate-cyclase activating polypeptide (PACAP). The increased thyroid stimulating activity of Graves (IgG) at high PEG concentrations suggests the existence of some factors influencing the ability of TSAb to stimulate thyroid cells, although the exact mechanism remains to be clarified.

MONOCLONAL ANTIBODIES AGAINST RENAL TUMORS: THE POTENTIAL APPLICATION IN DISCRIMINATION OF AMBIGUOUS ADENOCARCINOMA OR TRANSITIONAL CELL CARCINOMA OF KIDNEY

Purpose We tested the discrimination ability of 2 monoclonal antibodies, mAB 90 and 2-2, in renal carcinomas, including renal cell carcinoma and transitional cell carcinoma of the kidney. Materials and Methods Two monoclonal antibodies raised in renal adenocarcinoma (mAB 90, IgG3 subclass and mAB 2-2, IgG1 subclass), have been generated in our laboratory by hybridoma technique. The tumor associated antigens recognized by these IgG subclass monoclonal antibodies are located in the cell membrane of tumor cells. Antibodies were purified from mice ascites through protein A-Sepharose 4B affinity column and then conjugated with fluorescein isothiocyanate fluorescence by dimethyl sulfoxide method. The binding activity of these antibodies was measured by direct immunofluorescence method and analyzed in a flow cytometer. Forty-five cases of renal cell carcinoma and 16 cases of transitional cell carcinoma of the renal pelvis were collected, and the recognition power of these 2 antibodies was tested. Frozen tumor tissues were prepared and allocated into single cell suspensions in phosphate buffered saline before adding diluted (1:10) conjugated antibodies. Irrelevant antibody and negative tumor cell lines without any reaction with these antibodies were used as background fluorescence control. Results Reactivities of mAB 90 and mAB 2-2 for renal cell carcinoma tissues were 80 and 91%, respectively. On the contrary the reactivities of mAB 90 and mAB 2-2 for transitional cell carcinoma of renal pelvis tissues were 0 and 69%, respectively. mAB 90 (+)/mAB 2-2 (-) expression pattern had 76% accurate diagnostic rate for renal cell carcinoma and mAB 90 (+)/mAB 2-2 (+) pattern had 69% accurate diagnostic rate for transitional cell carcinoma of the kidney. Conclusions when facing pathologically ambiguous kidney tumors, the binding specificity of mAB 90 and mAB 2-2 may be useful for discrimination between renal cell carcinoma tumors and transitional cell carcinoma of the renal pelvis.

Identification and Characterization of Galectin-9, a Novel β-Galactoside-binding Mammalian Lectin

A 36-kDa beta-galactoside mammalian lectin protein, designated as galectin-9, was isolated from mouse embryonic kidney by using a degenerate primer polymerase chain reaction and cloning strategy. Its deduced amino acid sequence had the characteristic conserved sequence motif of galectins. Endogenous galectin-9, extracted from liver and thymus, as well as recombinant galectin-9 exhibited specific binding activity for the lactosyl group. It had two distinct N- and C-terminal carbohydrate-binding domains connected by a link peptide, with no homology to any other protein. Galectin-9 had an alternate splicing isoform, exclusively expressed in the small intestine with a 31-amino acid insertion between the N-terminal domain and link peptide. Sequence homology analysis revealed that the C-terminal carbohydrate-binding domain of mouse galectin-9 had extensive similarity to that of monomeric rat galectin-5. The presence of galectin-5 in the mouse could not be demonstrated by polymerase chain reaction or by Northern or Southern blot genomic DNA analyses. Sequence comparison of rat galectin-5 and rat galectin-9 cDNA did not reveal identical nucleotide sequences in the overlapping C-terminal carbohydrate-binding domain, indicating that galectin-9 is not an alternative splicing isoform of galectin-5. However, galectin-9 had a sequence identical with that of its intestinal isoform in the overlapping regions in both species. Southern blot genomic DNA analyses, using the galectin-9 specific probe derived from the N-terminal carbohydrate-binding domain, indicated the presence of a novel gene encoding galectin-9 in both mice and rats. In contrast to galectin-5, which is mainly expressed in erythrocytes, galectin-9 was found to be widely distributed, i.e. in liver, small intestine, thymus > kidney, spleen, lung, cardiac and skeletal muscle > reticulocyte, brain. Collectively, these data indicate that galectin-9 is a new member of the galectin gene family and has a unique intestinal isoform.

The effect of microfluidization of protein-coated liposomes on protein distribution on the surface of generated small vesicles

Tetanus toxoid and immunoglobulin G (IgG), model proteins for vaccines and targeting ligands respectively, were covalently coupled to preformed dehydration-rehydration vesicles (DRV) to produce vesicles with surface-bound proteins (DRV-protein) or to preformed small unilamellar vesicles (SUV) which were used to generate DRV with bound protein [(SUV-protein)DRV]. Of the amount of protein employed for coupling (1 mg), 13.8–45.1% was recovered with the liposomes, depending on the type of preparations and the proteins used. Microfluidization of similar DRV-protein or (SUV-protein)DRV for up to 10 cycles led to the formation of smaller vesicles (98–136 nm diameter) which, however, had modestly reduced (estimated as 8.8–21.7%) bound proteins, again depending on the type of preparation and protein used. Treatment of DRV-protein and (SUV-protein) DRV with proteinase revealed that 32.9–45.6% of the total bound protein was exposed on the liposomal surface. With microfluidized liposomes, the proportion of surface-exposed protein increased to 63.1–76.2%. Incubation of intact and microfluidized DRV-IgG and (SUV-IgV) DRV with a protein A-Sepharose 4B CL gel confirmed the presence of IgG on the liposomal surface (47.1–68.4 and 80.5–82.1% of total bound protein respectively). These studies were supplemented with freeze-fracture electron microscopy of (SUV-toxoid)DRV which demostrated the presence of protein particles (up to 3; 12–14 nm diameter) on the surface of both intact and microfluidized individual liposomes.

Characterization of a murine-human chimeric antibody with specificity for the pre-S2 surface antigen of hepatitis B virus expressed in baculovirus-infected insect cells

We have produced and characterized a murine-human chimeric antibody with specificity for the pre-S2 surface antigen of hepatitis B virus (HBV) in baculovirus-infected insect cells. Recombinant baculovirus carrying the cDNA coding for the heavy or light chain of the chimeric antibody was constructed and co-infected into insect cells. The chimeric antibody (BV-S2) expressed in the cells was purified by an affinity chromatography on Protein A-Sepharose 4B column and characterized by N-terminal amino acid sequencing, affinity determination for pre-S2 peptide, endoglycosidase digestion and Clq binding assay, which were then compared with those of the chimeric antibody H69K that has the same amino acid sequence as BV-S2, but produced from transfected murine myeloma cells. The N-linked glycosylation of the BV-S2 antibody was also analyzed by culturing the baculovirus-infected cells in the presence of tunicamycin. The results showed that the BV-S2 was secreted following correct removal of the leader peptides, contained N-linked carbohydrate at the heavy chain, and had the same binding affinity and Clq binding ability as H69K, suggesting that the BV-S2 chimeric antibody is functional and thus may be useful in the prevention of HBV infection.

Tyrosine Phosphorylation of the c-cbl Proto-oncogene Protein Product and Association with Epidermal Growth Factor (EGF) Receptor upon EGF Stimulation

The murine retroviral oncogene v-cbl induces pre-B cell lymphomas and myelogenous leukemias. The protein product of the mammalian c-cbl proto-oncogene is a widely expressed cytoplasmic 120-kDa protein (p120cbl) whose normal cellular function has not been determined. Here we show that upon stimulation of human epidermal growth factor (EGF) receptor, p12ocbl becomes strongly tyrosine-phosphorylated and associates with activated EGF receptor in vivo. A GST fusion protein containing amino acids 1-486 of p120cbl, including a region highly conserved in nematodes, binds directly to the autophosphorylated carboxyl-terminal tail of the EGF receptor. Platelet-derived growth factor (PDGF), fibroblast growth factor (FGF), or nerve growth factor (NGF) stimulation also results in tyrosine phosphorylation of p120cbl. Recent genetic studies in Caenorhabditis elegans indicate that Sli-1, a p120cbl homologue, plays a negative regulatory role in control of the Ras signaling pathway initiated by the C. elegans EGF receptor homologue. Our results indicate that p120cbl is involved in an early step in the EGF signaling pathway that is conserved from nematodes to mammals.

Labeling conditions of antibody IgG2a (Mouse) with90Y and99mTc and in-vitro stability studies

Mouse serum was fractionated and isolated to pure mose sub-class immunoglobulin (IgG1, IgG2a, IgG2b) on protein A-sepharose 4B Microprecipition method was used to ascertain the purity of mouse IgG-sub class, labeling and coupling efficiecy with90Y and99mTc were determined by paper chromatography as a simple and rapid tool for analysis. The parameters investigated included the ratios of DTPA: IgG2a and DTPA: Sn, pH and protein concentrations. The optimal coupling efficiency for90Y was achieved at DTPA to IgG2a molar ratio of 1∶1 pH 8,4 and specific activity of 2 μCi/μg for IgG2a coupled with about 1 goup of DTPa per 1 molecule of IgG2a, while the optimal coupling efficiency for99mTc was achieved at a DTPA to IgG2a molar rato of 50:1, pH4 and specific activity of 1 μCi/μg for IgG2a. Affinity column chromatography with sephadex G50 and protein A sepharose 4B were used to determine the immunoreactive and non-immuoreactive mouse antibody IgG2a coupled with DTPA at different molar ratios and99mTc90Y atoms incorporated into each antibody. Anti-IgG2a affinity column and anti-human transferrin affinity column were used to determine in vitro stability of99mTc DPTa-IgG2a. Freeze-dried kit of IgG2a-Sn-DTPA to be labeled with99mTc was prepared under sterile condition Each vial contained 1.0 mg IgG2a-Sn-DTPA in about 100 μl.