Reactivity of an HIV gag gene polypeptide expressed in E. coli with sera from AIDS patients and monoclonal antibodies to gag

A segment of the gag gene of the human immunodeficiency virus (HIV) (HTLV-IIIB strain), the virus which causes acquired immunodeficiency syndrome (AIDS), has been cloned into the bacterial expression vector, pCQV2, and mapped to the right-hand portion of the gag gene containing the carboxyl-terminal portion of p24 and the amino-terminal portion of p15. Nucleic-acid sequencing of the insert-vector junctions further defined the 5'-terminal nucleotide of HIV sequence as nucleotide 997 and the 3'-terminal nucleotide as 1696. When used in an enzyme-linked immunosorbent assay (ELISA) with sera from HIV-infected patients, the cloned antigen reacted with a subset of sera which were positive on a standard ELISA using whole virus as antigen. Western-blot screening of these sera with whole virus indicated that all p24-positive sera were positive with the clone, suggesting that the carboxyl-terminal portion of p24 contains a highly antigenic epitope(s). A serum which was p24-negative p15-positive by Western blot analysis was also highly reactive, indicating that a p15 epitope is present in the cloned antigen. Epitope mapping with a series of monoclonal antibodies to gag resulted in positive ELISA with 2 of 3 anti-p24, 0 of 1 anti-p15, and 0 of 1 anti-p17 Western-blot-positive monoclonal antibodies, suggesting that one of the anti-p24 monoclonal antibodies reacts with epitopes amino-terminal to those coded from nucleotide 997, two anti-p24 monoclonals react with epitopes carboxyl-terminal to those coded from nucleotide 997, and the anti-p15 monoclonal reacts with epitopes carboxyl-terminal to those coded from nucleotide 1696.     

A cytoplasmic thyroid hormone binding protein: characterization using monoclonal antibodies

We have previously purified a cellular thyroid hormone binding protein (p58) from a human carcinoma cell line [Kitagawa, S., Obata, T., Hasumura, S., Pastan, I., & Cheng, S.-y. (1987) J. Biol. Chem. 262, 3903-3908]. In the present study, the binding characteristics, the molecular properties, and subcellular localization of p58 were further characterized. Binding of the purified p58 to thyroid hormones was examined. Analysis of binding data indicates that p58 binds to 3,3',5-triiodo-L-thyronine (T3) with a Kd of 24.3 +/- 0.3 nM and n = 0.71. p58 binds to L-thyroxine similarly as to T3. However, D-T3 and reverse-T3 bind to p58 with an affinity 4- and 20-fold less than that of T3, respectively. By use of the purified p58 as an immunogen, two hybridomas, J11 and J12, secreting monoclonal antibodies to p58 were isolated; both antibodies belong to the IgG1K subclass. J12 recognizes p58 from human, monkey, dog, hamster, and rat, but not mouse. J11 exhibits a similar species specificity except that it does not react with p58 from hamster. With these antibodies, p58 was found to be not posttranslationally modified by glycosylation, sulfation, or phosphorylation. It has a cellular degradation rate t1/2 congruent to 2.1 h. Immunocytochemical studies indicate that p58 is located in the nonmembranous cytoplasm (cytosol). These results are consistent with subcellular fractionation studies which show that greater than 95% of J11 and J12 reactivity and T3 binding activity can be found in the 110,000g supernatant.     

Down-modulation of an oncogene protein product and reversion of the transformed phenotype by monoclonal antibodies

Exposure of neu-oncogene-transformed NIH 3T3 cells to monoclonal antibodies reactive with the neu gene product, p185, results in the rapid and reversible loss of both cell-surface and total cellular p185. Although not directly cytotoxic, monoclonal anti-p185 antibody treatment causes neu-transformed NIH 3T3 cells to revert to a nontransformed phenotype, as determined by anchorage-independent growth. Isotype matched control antibodies of an unrelated specificity do not affect p185 levels or colony formation in soft agar by neu-transformed NIH 3T3 cells. Soft agar colony formation by NIH 3T3 cells transformed by ras oncogenes is not affected by anti-p185 antibody treatment. Anchorage-independent growth of cells from the ethylnitrosourea-induced rat neuroblastoma line in which neu was originally detected by DNA transfection is also inhibited in the presence of anti-p185 monoclonal antibodies. Collectively, these results suggest that p185 is required to maintain transformation induced by the neu oncogene.     

Distinct Roles for the p110α and hVPS34 Phosphatidylinositol 3′-Kinases in Vesicular Trafficking, Regulation of the Actin Cytoskeleton, and Mitogenesis

We have examined the roles of the p85/ p110α and hVPS34 phosphatidylinositol (PI) 3′-kinases in cellular signaling using inhibitory isoform-specific antibodies. We raised anti-hVPS34 and anti-p110α antibodies that specifically inhibit recombinant hVPS34 and p110α, respectively, in vitro. We used the antibodies to study cellular processes that are sensitive to low-dose wortmannin. The antibodies had distinct effects on the actin cytoskeleton; microinjection of anti-p110α antibodies blocked insulin-stimulated ruffling, whereas anti-hVPS34 antibodies had no effect. The antibodies also had different effects on vesicular trafficking. Microinjection of inhibitory anti-hVPS34 antibodies, but not anti-p110α antibodies, blocked the transit of internalized PDGF receptors to a perinuclear compartment, and disrupted the localization of the early endosomal protein EEA1. Microinjection of anti-p110α antibodies, and to a lesser extent anti-hVPS34 antibodies, reduced the rate of transferrin recycling in CHO cells. Surprisingly, both antibodies inhibited insulin-stimulated DNA synthesis by 80%. Injection of cells with antisense oligonucleotides derived from the hVPS34 sequence also blocked insulin-stimulated DNA synthesis, whereas scrambled oligonucleotides had no effect. Interestingly, the requirement for p110α and hVPS34 occurred at different times during the G1–S transition. Our data suggest that different PI 3′-kinases play distinct regulatory roles in the cell, and document an unexpected role for hVPS34 during insulin-stimulated mitogenesis.     

Modulation of two forms of tumor necrosis factor receptors and their cellular response by soluble receptors and their monoclonal antibodies.

Recently, two different receptors for human tumor necrosis factor (TNF) with molecular masses of 60 kDa (p60) and 80 kDa (p80) have been identified. In this report, we investigated the effect of the soluble forms of these receptors and monoclonal antibodies against them on ligand interaction, receptor down-regulation, and mediation of cellular response in U-937 cells. Our results indicate that p60 and p80 constitute 20-30 and 60-80% of the total TNF-binding sites on U-937 cells, respectively. However, by cross-linking, only the p80 form of the receptor could be detected. In contrast to unlabeled TNF, the anti-p60 and anti-p80 antibodies together only partially inhibited ligand binding, and this inhibition was not additive. Lack of additive inhibition of binding was found to be not due to stereo-chemical hindrance. TNF binding to cells can be completely displaced by soluble forms of either the p60 or p80 receptor. However, 100-fold more of the p80 than the p60 form of the soluble receptor is needed for equivalent displacement. Under optimum conditions, TNF and the anti-p80 and anti-p60 antibodies down-regulated 30, 80, and 20% of the TNF receptors, respectively. The anti-p60 and anti-p80 antibodies down-regulated not only their own receptors, but also reciprocal receptors, suggesting a cross-communication between the p60 and p80 forms of the TNF receptor. In spite of inhibiting as much as 80% of TNF binding, none of the receptor antibodies significantly inhibited the cytotoxic response to TNF in U-937 cells. Soluble forms of both receptors, however, completely abrogated the cellular response to TNF. Thus, overall, our results indicate that the antibodies against both receptors together inhibit the majority of the receptor-ligand interaction without any significant effect on the biological response to TNF.     

富含二硫键的膜蛋白p185胞外区在大肠杆菌中的可溶性表达和性质鉴定

Her2/c-erbB-2基因（其产物为膜蛋白p185）是表皮生长因子受体（EGFR）基因家族的一员,在约30％的乳腺癌中发现了其过量表达。为了鉴定抗p185单克隆抗体的抗原表位并进一步研究它们的相互作用,采用PCR的方法从含Her2/c_erbB_2基因的pBabe/erbB_2质粒中扩增了p185胞外区的富含二硫键的第一、二结构域和第四个结构域。产物克隆到pGEX/4T-1载体后,转化大肠杆菌Origami B（DE3）pLysS菌株,用低浓度IPTG进行低温过夜诱导后将菌体压力破碎,SDS-PAGE检测表达上清,得到了可溶性表达的融合有GST的目的蛋白。经ELISA、Western blot等方法鉴定,可溶性表达产物具有完全的抗体结合活性,且当用凝血酶把GST切掉后该活性仍然保留。P185胞外区融合蛋白的成功表达将为二硫键富含类蛋白的表达提供参考;并为将来具有肿瘤细胞生长抑制活性的抗p185单克隆抗体的抗原表位鉴定,以及为EGFR家族受体的结构和功能关系的研究打下基础。     

Immune response to individual maedi-visna virus gag antigens

The lesions caused by maedi-visna virus (MVV) are known to be immune mediated with a presumed contribution by the response to viral antigens. However, very little is known about the T-cell response to individual viral proteins. We have therefore expressed the three individual gag antigens of MVV strain EV1 (p16, p25, and p14) in a bacterial expression system and used the purified recombinant proteins to analyze the antibody and CD4+ T-cell response to MVV. Plasma samples were taken from sheep after 1 year of infection with MVV. The titers for antibodies in these samples were determined by indirect enzyme-linked immunosorbent assays and were as follows: anti-p25 antibody, 1:400 to >1:3,200; anti-p16 antibody, 1:400 to 1:3,200; and anti-p14 antibody, 1:<100 to 1:3,200. When the induction of antibodies was followed over time postinfection (p.i.), samples positive for anti-p25 were seen by day 24 p.i., followed by anti-p16 by day 45 p.i., and lastly anti-p14 by day 100 p.i. T-cell proliferative responses to all three gag antigens were detected in persistently infected sheep peripheral blood lymphocytes. The antigens were therefore used to raise T-cell lines from persistently infected sheep. These T-cell lines were shown to be specific for the recombinant gag antigens and for viral antigen expressed on infected macrophages. The proliferative response was restricted to major histocompatibility complex class II HLA-DR and so was due to CD4+ T lymphocytes. All three gag antigens may therefore play a role in immune-mediated lesion formation in MVV disease by presentation on infected macrophages in lesions.     

Dimers and complexes with p53 are the prevalent oligomeric forms of a transforming nonkaryophilic T antigen of simian virus 40.

The oligomers formed by a mutant nonkaryophilic large T antigen of simian virus 40, which lacks residues 110 through 152 of normal large T antigen and transforms only established cells (L. Fischer-Fantuzzi and C. Vesco, Proc. Natl. Acad. Sci. USA 82:1891-1895, 1985), were found to consist predominantly of dimers. Anti-p53 antibodies precipitated 14 to 16S complexes containing the mutant nonkaryophilic large T antigen and p53 from extracts of transformed cells, and anti-p53 indirect immunofluorescence stained these cells in the cytoplasm.     

Quantitative analysis of a nuclear antigen in interphase and mitotic cells

The quantification of an interchromatin-associated antigen, designated p 105, during cellular passage through mitosis is described. Indirect immunofluorescence microscopy and immunogold electron microscopy demonstrated a qualitative increase in p 105 within the mitotic cytoplasm. Multiparameter flow cytometric analysis was performed on fixed cells sequentially stained with anti-p 105 immunofluorescence and/or propidium iodide. This analysis demonstrated approximately a tenfold increase in intracellular p 105 content as a function of progression from the G2 to the M phase. This increase was corroborated by the quantitative immunoblot analysis of colchicine-treated cell cultures and of cells sorted on the basis of anti-p 105 immunofluorescence. The data reveal that the increased levels of anti-p 105 immunofluorescence in conjunction with flow cytometry may be used effectively to quantitate mitotic index and isolate mitotic cells. The function and modulation of p 105 throughout the cell cycle is discussed.     

Characterization of the antibodies to p62 nucleoporin in primary biliary cirrhosis using human recombinant antigen

Reactivity of sera from patients with primary biliary cirrhosis (PBC) with a 60 kDa component of nuclear pore complexes (NPCs), purified by affinity chromatography on wheat-germ agglutinin (WGA)-Sepharose, was previously detected. Recently, clinical significance of the anti-NPC antibodies in PBC became evident. In the light of recent reports, indicating the correlation of the anti-NPC antibodies with severity and progression of the disease, the characterization of the reactive antigens is becoming essential in the clinical management of patients with PBC. Since accurate autoantibody detection represents one of the fundamental requirements for a reliable testing, we have generated a human recombinant p62 protein and validated an immunoprecipitation assay for the detection of anti-p62. We also demonstrated that the generated human recombinant p62 nucleoporin was modified by N-acetylglucosamine residues. More than 50% of tested PBC sera precipitated (35)S-radioactively labeled p62 recombinant nucleoporin and 40% recognized this recombinant antigen by immunoblotting. We compared the reactivity of PBC sera with rat and human nucleoporin. The incidence of anti-p62 nucleoporin positive PBC sera increased by 15% when human recombinant antigen was used. The titer of autoantibodies in p62-positive PBC samples strongly varied. Preadsorption of the PBC sera with p62 recombinant protein completely abolished their reactivity with the antigen. In conclusion, this study unequivocally proves that autoantibodies reacting with the 60 kDa component of NPCs target p62 nucleoporin and, more importantly, provide a better antigen source for future evaluations of the clinical role of anti-p62 in PBC.     

A nuclear localization signal binding protein in the nucleolus

We used functional wild-type and mutant synthetic nuclear localization signal peptides of SV-40 T antigen cross-linked to human serum albumin (peptide conjugates) to assay their binding to proteins of rat liver nuclei on Western blots. Proteins of 140 and 55 kD (p140 and p55) were exclusively recognized by wild-type peptide conjugates. Free wild-type peptides competed for the wild-type peptide conjugate binding to p140 and p55 whereas free mutant peptides, which differed by a single amino acid from the wild type, competed less efficiently. The two proteins were extractable from nuclei by either low or high ionic strength buffers. We purified p140 and raised polyclonal antibodies in chicken against the protein excised from polyacrylamide gels. The anti-p140 antibodies were monospecific as judged by their reactivity with a single nuclear protein band of 140 kD on Western blots of subcellular fractions of whole cells. Indirect immunofluorescence microscopy on fixed and permeabilized Buffalo rat liver (BRL) cells with anti-p140 antibodies exhibited a distinct punctate nucleolar staining. Rhodamine- labeled wild-type peptide conjugates also bound to nucleoli in a similar pattern on fixed and permeabilized BRL cells. Based on biochemical characterization, p140 is a novel nucleolar protein. It is possible that p140 shuttles between the nucleolus and the cytoplasm and functions as a nuclear import carrier.     

Circulating anti-p53 antibodies in lung cancer and relationship to histology and smoking

Anti-p53 antibodies were examined in the plasma of 112 lung cancer patients by ELISA in order to study the distributions in lung cancer patients and the determinants of these antibodies in relation to lung cancer. Twenty (17.9 %) lung cancer patients were found to have anti-p53 antibodies. The distribution of the antibodies by histological type was 7/48 (14.6 %) adenocarcinoma, 8/32 (25.0 %) squamous cell carcinoma, 3/7 (42.9 %) small cell lung cancer, 0/4 large cell carcinoma, 0/8 adenosquamous cell carcinoma and 2/13 (15.4 %) other types. By ethnicity, 8/44 (18.2 %) Caucasians, 4/20 (20.0 %) Hispanics and 8/48 (16.7 %) African-Americans were positive for anti-p53 antibodies, with no significant differences among the groups (p=0.5137). The antibody positivity rates were higher in lung cancer patients 55 years or older (21.2 %) than in the patients under 55 years (7.4 %). The positive rates of the antibodies were 14.3 % in non-smokers, 16.7 % in ex-smokers and 19.1 % in current smokers, with heavy smokers (41 pack-years) having the highest positive rate (28.6 %), but none of these differences were statistically significant (p > 0.05). Seven controls who had anti-p53 antibodies were all ex-smokers or current smokers and some had occupational exposures. No anti-p53 antibodies were found in 41 non-smoking controls. These results suggest that the development of anti-p53 antibodies in pulmonary carcinogenesis and its association with smoking and other carcinogenic exposures deserve further study.     

Comparison of anti-p53 antibodies in immunoblotting

Some of the most important tools to study p53 protein are various anti-p53 antibodies and immunological methods based on antibody-antigen reactions. Critical comments on the specificity and sensitivity of anti-p53 antibodies have been published. Four monoclonal and two polyclonal anti-p53 antibodies, four of them from two different sources, were compared for their ability to detect in immunoblotting the benzo(a)pyrene-induced p53 from C57BL/6 mouse skin and MCF-7 human breast carcinoma cells. Multiple extra bands were seen with most antibodies. A theoretical comparison of the equivalent epitopes of p53 homologues with the known epitopes of p53 antibodies indicated that the extra bands seen with most antibodies are not due to cross-reactivity with these homologues. A careful adjustment of antibody dilutions is needed for each application utilizing commercial p53 antibodies, regardless of the recommendations of the supplier.     

Monoclonal antibody analysis of p53 expression in normal and transformed cells.

The cellular phosphoprotein p53 binds tightly and specifically to simian virus 40 T antigen and the 58,000-molecular-weight adenovirus E1b protein. Many human and murine tumor cell lines contain elevated levels of the p53 protein even in the absence of these associated viral proteins. Recently the cloned p53 gene, linked to strong viral promoters, has been shown to complement activated ras genes in transformation of primary rodent cell cultures. Overexpression of the p53 gene alone rescues some primary rodent cell cultures from senescence. We isolated three new monoclonal antibodies to the p53 protein, designated PAb242, PAb246, and PAb248, and mapped the epitopes they recognized on p53 in comparison with other previously isolated antibodies. At least five sterically separate epitopes were defined on murine p53. One of the antibodies, PAb246, recognizes an epitope on p53 that is unstable in the absence of bound simian virus 40 T antigen. This effect is demonstrable in vivo and in newly developed in vitro assays of T-p53 complex formation. Using the panel of anti-p53 antibodies and sensitive immunocytochemical methods, we found that p53 has a predominantly nuclear location in established but not transformed cells as well as in the vast majority of transformed cell lines. Several monoclonal antibodies to p53 showed cross-reactions with non-p53 components in immunocytochemical staining.     

Complex formation of human T-cell leukemia virus type I p40tax transactivator with cellular polypeptides.

We examined cellular components which associate with p40tax, the viral transactivation molecule of human T-cell leukemia virus type I. Such molecules were searched by immunoprecipitation with polyclonal and monoclonal antibodies specific for p40tax. Two cellular proteins with molecular masses of 95 kDa (p95) and 60 kDa (p60) were specifically coprecipitated with p40tax from extracts of all p40tax-producing cell lines but not from p40tax-negative cell lines. The p60 component was also shown to associate with p40tax in vitro, by using radiolabel-chase experiments. Rabbit antisera specific for p60 and p95 were prepared by immunization with electrophoretically purified molecules. While anti-p95 antiserum coprecipitated p40tax, no p40tax could be identified in immunoprecipitates by using a polyclonal anti-p60 antiserum. The partial amino acid sequence of p60 demonstrated that p60 is identical to the human 60-kDa heat shock protein (a member of the chaperonin family of proteins). Although the biological significance of the complex formation of p40tax with p95 and p60 has yet to be determined, it may be that the complex formation is one of the mechanisms by which the biological activity of p40tax can be regulated.     

Intracellular immunization. Cloning and intracellular expression of a monoclonal antibody to the p21ras protein

Following the demonstration that intracellular expression of antibodies ('intracellular immunization') may be utilized to engineer new traits in mammalian cells, we undertook experiments to perturb the function of p21ras proteins, by engineering the intracellular expression of the anti-p21ras antibody Y13-259. The variable regions of this antibody have been cloned and, after verifying their antigen binding activity, expressed in general purpose vectors for the intracellular expression of antibodies. The results confirmed that the cloned antibody has been efficiently expressed both in the secretory and the intracellular forms. Thus, intracellular immunization of mammalian cells against p21ras, or any other antigen for which a monoclonal antibody is available, can now be performed.     

Anti-P53 antibodies in Brazilian brain tumor patients

Gliomas of astrocytic origin are the most common primary brain tumors, accounting for over 40 to 50% of all central nervous system tumors. The TP53 tumor suppressor gene is the most frequently mutated gene found in human malignancies. A mutation of this gene can lead to an increased half-life of the resulting protein and loss of biological function. High levels of p53 have been detected in the serum of colon cancer patients, although p53 protein has not been detected in the serum of brain tumor patients. Besides circulating p53, several studies have detected antibodies against p53 in patients with lung and breast cancer, as well as those with other types of cancer. We studied p53 protein and anti-p53 antibodies in the plasma of Brazilian brain tumor patients. Plasma samples were drawn from 24 untreated brain tumor patients and from 15 healthy donors without clinical signs of cancer. Western blotting techniques were used to detect p53 protein and anti-p53 antibodies. We found anti-p53 antibodies in 5/24 brain tumor patients. Age appears to affect the immune response, as four of six tumor patients under 16 years old had detectable anti-p53 antibodies, while these were found in only 1 of 18 adults (over 16 years old). We found no p53 protein in any of the serum samples from the brain tumors. Possibly the presence of this protein is affected by tumor type or by the organs that are sampled.     

Analysis of recombinant DNA clones specific for the murine p53 cellular tumor antigen.

Three cDNA clones, corresponding to two non-overlapping regions of the mRNA coding for the mouse p53 cellular tumor antigen, were isolated and characterized. In hybridization-selection assays, these clones were capable of selectively binding p53 mRNA, as demonstrated by in vitro translation and immunoprecipitation with anti-p53 monoclonal antibodies. The p53 mRNA appeared to be the only messenger species specifically selected by these clones. The size of the p53 mRNA was found to be approximately 2 kb, and its levels to vary substantially among different types of transformed cells. Evidence was found for the existence of two distinct p53-specific genes in mouse genomic DNA. Two partially overlapping recombinant phage clones were obtained, both derived from the same p53-specific genomic DNA region. The orientation of the various cDNA clones relative to that of the p53 mRNA was established by S1 analysis and the relationship between the cDNA clones and the genomic ones was determined by comparative restriction enzyme mapping and nucleic acid hybridization.     

Localization of pepstatin's inhibitory action during Fc-mediated antibody internalization: possible implications for antibody-mediated viral transmission

Antibody internalization via Fc receptors is an important cellular mechanism, possibly facilitating the entry of antigenic peptides or viral particles into cells when specific antibodies are present at the periphery. Using an experimental model of trophoblast cells, we have shown that anti-p21(ras) monoclonal antibodies can use IFN-gamma-induced surface Fcgamma receptors to enter the cell. This entry of anti-p21(ras) antibodies ultimately inhibits IFN-gamma-mediated class II antigen induction. Since there may be obvious and inevitable harmful aspects of this mechanism, during which Fc-mediated viral particle or autoantigen transport may occur, we concentrated efforts on defining a potent inhibitor able to eliminate such uptake. The results presented here show that the protease inhibitor pepstatin A efficiently inhibits Fcgamma receptor induction by IFN-gamma and also blocks the endocytic pathway followed by an antibody when it enters the cell at the level of early endosomal compartments. We thus postulate that the use of pepstatin A, because of its inhibition of autoantigen presentation or viral transmission, including that of HIV, may find important applications in therapeutic protocols.