An immunoreactive peptide of the FSH involved in autoimmune infertility.

The purpose of this study was to identify autoantigens contained in human ovary extracts. Serum samples from 36 infertile women with anti-ovary antibodies as detected with an ELISA technique were tested in Western blot against human ovary extracts. A reactive protein with a molecular mass matching that of the FSH was detected in 34 cases. These serum samples also reacted strongly in Western blot and ELISA with purified FSH and, in immunofluorescence, with pituitary cells. Using the Pepscan approach, with overlapping peptides matching the amino acid sequence of the human FSH beta-chain, several immunoreactive regions were evidenced. The 78-93 amino acid sequence of the human FSH beta-chain appeared as one of the major epitopes. Synthetic peptides of this region were prepared and demonstrated to react with human serum samples from women with anti-ovary antibodies. These data demonstrate that FSH can be an autoantigen, recognized by autoantibodies associated with infertility.     

Immunoblot analysis of glutaminase peptides in intact and solubilized mitochondria isolated from various rat tissues.

Antibodies were prepared against isolated rat renal glutaminase and affinity-purified against the 65 kDa peptide contained in the purified rat brain glutaminase. The affinity-purified IgGs were then used to compare the glutaminase immunoreactive peptides contained in samples that had been subjected to SDS/polyacrylamide-gel electrophoresis and transferred to nitrocellulose. The purified brain glutaminase and isolated brain mitochondria contain 68 and 65 kDa peptides that exhibit nearly equivalent immunostaining. Partial proteolysis of the isolated 68 and 65 kDa peptides with Staphylococcus aureus V8 proteinase produced an identical pattern of immunoreactive proteolytic fragments. However, digestion of the two peptides with chymotrypsin resulted in similar, but slightly different, patterns. The pattern of immunostaining was unaltered even when the brain mitochondria were solubilized with Triton X-100 and stored for 2 days at 4 degrees C. A very similar pattern was observed when intact renal mitochondria were subjected to immunoblot analysis. However, when renal mitochondria were solubilized, the 68 kDa peptide was rapidly degraded to the 65 kDa form. At 4 degrees C this reaction occurs with apparent first-order kinetics and a t1/2 of 35 min. Degradation of the 65 kDa form of the renal glutaminase occurs with much slower kinetics, but is nearly complete after 24 h. Solubilization of mitochondria isolated from various zones of the kidney indicated that the responsible endogenous proteinase was localized primarily in the cortex. Mitochondria isolated from intestinal or renal papillary tissue contain four glutaminase immunoreactive peptides (Mr 68,000, 65,000, 61,000 and 58,000). The smallest of these peptides is identical in size with the single immunoreactive peptide observed in liver tissue.     

Mass spectrometric characterization of the human androgen receptor ligand-binding domain expressed in Escherichia coli

The ligand-binding domain (LBD) of the human androgen receptor (hAR LBD), encompassing amino acids (AAs) 647-919, was expressed in Escherichia coli with an N-terminal polyhistidine tag (His(10)-hAR LBD) from a pET-16b vector. The overexpressed protein was initially insoluble in inclusion bodies, and was subsequently solubilized in 8 M guanidine hydrochloride (GdnHCl). The solubilized His(10)-hAR LBD was purified to apparent homogeneity by metal ion affinity chromatography in the presence of 6 M GdnHCl. The isolated protein migrated as a single band in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with an apparent molecular mass of 33-34 kDa, as expected from the plasmid construct. Immunoblot analysis with C-terminal antibodies raised against a peptide corresponding to the last 19 AAs (AAs 901-919) of hAR revealed that the purified protein contained an immunoreactive epitope present within the AR and was of the appropriate size. Further characterization, using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI/TOF-MS), showed a single protein species of average mass 34 580 Da, confirming the size and purity of the purified His(10)-hAR LBD. Detailed tryptic peptide mapping analysis, using MALDI/TOF-MS, identified a total of eight peptides with a 30% coverage of the LBD, including the last tryptic peptide in the hAR sequence. These data confirm that the purified protein was the intact hAR LBD. AA sequencing of these tryptic peptides, using an HPLC-coupled electrospray ionization ion trap mass spectrometer (LC/ESI-ITMS and MS/MS), unambiguously confirmed that the peptides were from the hAR LBD. The purified His(10)-hAR LBD in 6 M GdnHCl could be renatured as determined by ligand-binding activity, with a similar equilibrium dissociation constant (K(d)) for [(3)H]-mibolerone and a similar steroid specificity to the AR isolated from rat ventral prostate.     

Antibodies Raised Against Synthetic Peptides React with Choline Acetyltransferase in Various Immunoassays and in Immunohistochemistry

Abstract: Antisera were raised in rabbits against five synthetic peptides. These peptides have been identified as potentially antigenic epitopes from the sequence of porcine choline acetyltransferase (ChAT) using primary and secondary structure analysis. All five antisera recognized immunoaffinity-purified antigen from porcine brain in an ELISA and on western blots. Four antisera recognized ChAT on dot blots, and another four antisera reacted with native and degraded enzyme in a sandwich ELISA using monoclonal antibodies as the capture antibody. One peptide antiserum was of similar avidity in this sandwich ELISA as a polyclonal antibody raised against immunoaffinity-purified ChAT. The same antiserum reacted with the enzyme from human placenta in an ELISA and on western and dot blots and recognized ChAT in rat, primate, and human neurons. Thus, a single peptide (amino acids 168- 189) provides the means for easy, reliable, and reproducible generation of antibodies against ChAT suitable for replacing conventional polyclonal and monoclonal antibodies.     

Tryptic digestion of the alpha subunit of human choriogonadotropin

In order to further characterize chemical, physicochemical, and immunochemical properties, as well as structure-function relationships, of the common alpha subunit of human glycoprotein hormones, a tryptic core was prepared from the alpha subunit of human choriogonadotropin. The core was purified in greater than 80% yield using gel permeation and anion-exchange chromatography, and, following reduction and S-carboxymethylation, the constituent peptides were purified by gel permeation and high performance liquid chromatography. The disulfide-bridged peptides comprising the alpha core were identified as residues 1-35 and residues 52-91 by amino acid composition and amino acid carboxyl sequence analyses of the reduced, S-carboxymethylated peptides. The alpha tryptic core contained both N-asparagine carbohydrate moieties, but was devoid of residues 36-51 and the carboxyl-terminal serine at position 92. The small peptides cleaved from residues 36-51, a known potential O-glycosylation region of the alpha subunit, were purified and identified. The tryptic core retained full immunopotency relative to the intact subunit in the binding to polyclonal and monoclonal antibodies directed against the alpha subunit. The region consisting of residues 36-51 is not part of the epitope recognized by these antibodies. With antisera generated to the reduced, S-carboxymethylated subunit, peptide 1-35, but not 52-91, was immunoreactive. This finding is consistent with the known dominant antigenicity of the amino-terminal region in the reduced, S-carboxymethylated molecule. The core exhibited no appreciable interaction with the complementary beta subunit, and, not surprisingly, was unable to compete with intact hormone binding in a radioreceptor assay using rat testicular homogenates. Circular dichroic spectroscopy was used to probe gross features of tertiary structure (240-300 nm) and secondary structure (190-240 nm). The tryptic core and each of the two constituent peptides exhibited spectra above 240 nm that resembled that of the reduced, S-carboxymethylated subunit more than that of the native material, thus suggesting a significant loss of tertiary structure in the core and isolated peptides. This finding is unexpected in consideration of the full retention of immunopotency by the alpha core although consistent with failure of the core to combine with intact complementary beta subunit. The intact subunit as well as the isolated constituent peptides exhibit little if any helicity in aqueous solution. Interestingly, the reduced, S-carboxymethylated chain and peptide 52-91 displayed helicity in 80% trifluoroethanol, a helicogenic solvent.     

Analysis of epitope structure of PSP94 (prostate secretory protein of 94 amino acids): (I) immuno-dominant and immuno-recessive area

PSP94 is a potential biomarker for evaluating patients with prostate carcinoma. We have systematically studied the epitope structure of PSP94 by using a polyclonal antibody against human PSP94. Results of peptide mapping and ELISA tests of dose response to rabbit antiserum against human PSP94 protein showed that only the N-terminal peptides (N30 and M23) are immunoreactive while all the synthetic peptides (C28, C10) located closer to the C-terminus are completely devoid of antigenic activity with the polyclonal antibody. These results were confirmed by analysis of reciprocal competitive binding of PSP94 polyclonal antibody by the N-terminal peptides (N30 and M23) v. either recombinant GST-PSP94 fusion protein, purified recombinant PSP94, or natural PSP94 protein. To further delineate the antigenic activity of the N- and C-termini, we have also expressed N- and C-terminal half of the whole PSP94 (each 47 peptides) using the E. coli GST expression system. The recombinant N47/C47 peptides were released by thrombin cleavage from the GST fusion protein and characterized by Western blotting experiments. Dose response of the recombinant GST-PSP-N47 and -C47 peptides to PSP94 polyclonal antibody showed differential binding activities. Competitive binding of these recombinant N47/C47 proteins against the GST-PSP94 protein demonstrates that the polyclonal antibody has a higher affinity for the N47 peptide than the C47 peptide. Based on the immunological studies of both synthetic peptides and recombinant PSP94- N/C terminal proteins, we propose an epitope structure of human PSP94 with an immno-dominant N-terminus and an immuno-recessive C-terminus. J. Cell. Biochem. 65:172–185. © 1997 Wiley-Liss, Inc.     

Presence of oxidized protein hydrolase in human cell lines, rat tissues, and human/rat plasma

Oxidized protein hydrolase (OPH), an 80 kDa serine protease whose activity is inhibited by diisopropyl fluorophosphate (DFP), has been isolated from human erythrocytes [Fujino, T. et al. (1998) J. Biochem. 124, 1077-1085]. The presence of OPH in various biological samples was examined by enzyme-linked immunosorbent assay (ELISA) and immunoblotting using an anti-OPH antibody raised against OPH purified from human erythrocytes, and by [(3)H]DFP-labeling and successive SDS-PAGE/fluorography. Solubilized samples of human cell lines including K-562 cells, THP-1 cells and Jurkat cells, and rat tissues including brain, heart, liver, kidney, and testis, inhibited the anti-OPH antibody binding to OPH in ELISA. Immunoblotting of lysates of K-562 cells, THP-1 cells and Jurkat cells showed four immunoreactive protein bands including an 80 kDa protein. Immunoprecipitation of the [(3)H]DFP-labeled K-562 cell lysate and successive SDS-PAGE/fluorography showed the presence of only the 80 kDa DFP-reactive protein with OPH antigenic activity. The level of the 80 kDa immunoreactive protein in K-562 cells rose as the cells differentiated toward erythrocytes. Immunoblotting of human and rat plasma showed two immunoreactive protein bands, including the 80 kDa protein, and SDS-PAGE/fluorography of [(3)H]DFP-labeled rat and human plasma showed the presence of only the 80 kDa DFP-reactive protein. The results indicate that OPH is present in a wide variety of biological samples.     

Radioimmunoassay and chemical properties of glucose-6-phosphate dehydrogenase and of a specific NADP(H)-binding protein (FX) from human erythrocytes

Two sensitive radioimmunoassays, based on a double-antibody technique, were developed which allow detection of nanogram amounts of glucose-6-phosphate dehydrogenase (EC 1.1.1.49) and of a so far unknown NADP(H)-binding protein present in human erythrocytes (designated FX).The two proteins isolated in homogeneous form from human erythrocytes were iodinated with ¹²⁵I by means of lactoperoxidase. Antisera to both purified proteins were raised in rabbits and sequentially adsorbed on human erythrocytes and on human serum before use. No cross-reaction between the two proteins was apparent.Hemolysates from normal as well as from glucose-6-phosphate dehydrogenase-deficient subjects were investigated for their content in both immunoreactive proteins using the two radioimmunoassay methods. This preliminary study showed significantly lowered levels of immunoreactive glucose-6-phosphate dehydrogenase in erythrocytes from subjects carrying the Mediterranean variant of this enzyme (characterized by severe deficiency of catalytic activity), compared with normal subjects. This figure was reversed as concerns the content of immunoreactive FX which was found to be twice as high in glucose-6-phosphate dehydrogenase Mediterranean erythrocytes as in normal ones.The two purified proteins were submitted to a comparative analysis of their chemical properties including NH₂-terminal residues, CNBr peptides and tryptic fingerprints. These studies revealed significant differences in the primary structures of the two proteins and therefore tend to exclude FX'x being a discrete product arising from degradation of native glucose-6-phosphate dehydrogenase. Moreover, amino axid analysis and tryptic fingerprints indicated that FX, as well as glucose-6-phosphatase dehydrogenase, is composed of very similar and possibly identical polypeptide chains.     

Proteomic identification, nucleotide sequence, heterologous expression and immunological reactivity of the triosephosphate isomerase of Paracoccidioides brasiliensis

An antigen of Paracoccidioides brasiliensis (Pb) was gel isolated and characterized. Endoproteinase Lys-C-digested peptides of the purified protein, which presented a molecular mass of 29 kDa and pI of 5.8, were subjected to sequence analysis of their amino acids. Searches at databases comparing the sequence of amino acids from the three peptides of the native protein revealed strong homology to triosephosphate isomerase (TPI: E.C. 5.3.1.1) from several sources. The complete cDNA and gene encoding PbTPI were obtained and both contained an open reading frame predicted to encode a 249-amino acid protein that presented all the peptides characterized in the native PbTPI. The Pbtpi gene contained six exons interrupted by five introns. Analysis performed with the deduced PbTPI suggested its usefulness in providing phylogenetic relatedness, as well as evidencing the correlation between the phylogeny provided by the deduced protein and intron positions in the cognate genes. The immunological reactivity of PbTPI was examined. The complete coding cDNA of PbTPI was overexpressed in an Escherichia coli host to produce high levels of recombinant fusion protein with glutathione S-transferase (GST) that had been purified by affinity chromatography. The purified recombinant TPI was recognized by sera of patients with confirmed paracoccidioidomycosis and not by sera of healthy individuals. Thus, recombinant PbTPI can be a valuable addition to the still small arsenal of P. brasiliensis immunoreactive proteins, which could be tested for incorporation into assays for serodiagnosis of the disease.     

Characterization of Mono- and Polyclonal Antibodies Against Highly Purified Choline Acetyltransferase: A Monoclonal Antibody Shows Reactivity in Human Brain

Choline acetyltransferase (ChAT) from porcine brain was purified by immunoaffinity chromatography, and the highly purified enzyme was subsequently used for immunization of mice and rabbits. After fusion of mouse spleen cells, 32 cultures producing monoclonal antibodies directed against ChAT were detected by an enzyme-linked immunosorbent assay (ELISA) with immunoaffinity-purified ChAT. Of these original 32, the most active 11 cultures were cloned and used for ascites production. The 11 clones generated monoclonal antibodies of the immunoglobulin (Ig) M class (three), the IgG1 subclass (seven), and the IgG2b subclass (one). The isoelectric points of the antibodies of the IgG class were different in each case. The monoclonal antibodies exhibited different binding characteristics in the above ELISA and on western blots. Two monoclonal antibodies demonstrated excellent immunohistological results with neurons of rat brain and spinal cord. One of them reacted well immunohistochemically with neurons of human brain and also recognized partially purified human placenta ChAT in the ELISA.     

Recombinant polypeptide from the endonuclease region of the acquired immune deficiency syndrome retrovirus polymerase (pol) gene detects serum antibodies in most infected individuals.

Sera from the majority of individuals that were positive in an enzyme-linked immunosorbent assay (ELISA) retrovirus (ARV), an isolate of the for antibodies to acquired immune deficiency syndrome (AIDS)-associated retrovirus (ARV), an isolate of the retrovirus identified as the etiologic agent of AIDS, were found to react with a 31,000-dalton protein (p31) in virus Western blot assays. To determine if this 31,000-dalton immunoreactive species originated from the putative endonuclease region of the polymerase (pol) gene of ARV, we cloned this portion of pol into bacterial expression vectors for direct expression and for expression as a fusion protein with human superoxide dismutase. Transformants from both constructions expressed immunoreactive protein detected in immunoblots with an AIDS patient's serum. Extracts from transformants expressing these sequences competed with the binding of antibodies from AIDS patients' sera to the 31,000-dalton protein in virus immunoblots, confirming that viral p31 originated from the endonuclease domain of the ARV polymerase gene. The superoxide dismutase-p31 fusion protein was purified, and an ELISA for detecting antibodies to p31 was developed. The majority (95%) of serum samples obtained from individuals seropositive in the virus ELISA were also positive in the p31 antibody ELISA.     

Isolation and Immunocytochemical Characterization of Three Tachykinin-Related Peptides from the Mosquito,Culex salinarius

Three myotropic peptides belonging to the Arg-amide insect tachykinin family were isolated from whole-body extracts of the mosquito, Culex salinarius. The peptides, APSGFMGMR-NH₂, APYGFTGMR-NH₂ and APSGFFGMR-NH₂ (designated culetachykinin I, II, and III) were isolated and purified on the basis of their ability to stimulate muscle contractions of isolated Leucophaea maderae hindgut. Biologically inactive methionine sulfoxides of two of the three peptides were isolated using an ELISA system based upon antiserum raised against APYGFTGMR-NH₂ and identified with mass spectrometry. Immunocytochemistry localized these peptides in cells in the brain, antennae, subesophageal, thoracic and abdominal ganglion, proventriculus and midgut. Nerve tracts containing these peptides were found in the median nerve of the brain, central body, nervi corpus cardiaci, cervical nerve, antennal lobe and on the surface of the midgut.     

Purification of phosphate-dependent glutaminase from isolated mitochondria of Ehrlich ascites-tumour cells.

Phosphate-dependent glutaminase was purified to homogeneity from isolated mitochondria of Ehrlich ascites-tumour cells. The enzyme had an Mr of 135,000 as judged by chromatography on Sephacryl S-300. SDS/polyacrylamide-gel electrophoresis displayed two protein bands, with Mr values of 64,000 and 56,000. Two major immunoreactive peptides of Mr values of 65,000 and 57,000 were found by immunoblot analysis using anti-(rat kidney glutaminase) antibodies. The concentration-dependences for both glutamine and phosphate were sigmoidal, with S0.5 values of 7.6 mM and 48 mM, and Hill coefficients of 1.5 and 1.6, respectively. The glutaminase pH optimum was 9. The activation energy of the enzymic reaction was 58 kJ/mol. The enzyme showed a high specificity towards glutamine. A possible explanation for the different kinetic behaviour found for purified enzyme and for isolated mitochondria [Kovacević (1974) Cancer Res. 34, 3403-3407] should be that a conformational change occurs when the enzyme is extracted from the mitochondrial inner membrane.     

Monoclonal antibodies to a nuclear protein (PCNA/cyclin) associated with DNA replication

Two hybridomas producing monoclonal antibodies to proliferating cell nuclear antigen. (PNCA)/cyclin were generated from spleen cells of BALB/c mice immunized with purified PCNA from rabbit thymus. The specificity of the monoclonal antibodies (19A2 and 19F4) was established by showing that they reacted in enzyme-linked immunosorbent assay (ELISA) with purified PCNA. Furthermore, they reacted in one-dimensional (ID) gel immunoblots with a 36 kD polypeptide which also reacted with human autoantibodies from lupus patients. Both monoclonals also recognized the nuclear polypeptide cyclin in two-dimensional (2D) gel immunoblots of HeLa cell proteins. Epitopes recognized by 19A2 and 19F4 were analysed by competitive inhibition test using a modified ELISA. The data suggested that the epitopes were closely related, but not identical. The data with human auto-antibodies were more difficult to interpret, although it suggested that some human anti-PCNA may share epitopes with 19A2 and 19F4, but in addition recognize different epitopes on the PCNA molecule.     

Complex formation between metarhodopsin II and GTP-binding protein in bovine photoreceptor membranes leads to a shift of the photoproduct equilibrium

Cap binding protein (CBP)-related polypeptides were identified in different cytoplasmic RNP particles of embryonic chick muscles using monoclonal antibody to purified CBP. A single immunoreactive peptide (M_r 78000) was present in preparations of both free mRNP particles and a novel 10 S translation inhibitory RNP particle. In contrast, proteins isolated from these particles showed two new low-M_r immunoreactive peptides (M_r 43000 and M_r 29000). No CBP related protein could be detected in polysomal mRNP, although an immunoreactive M_r 43000 CBP-related protein was present in polysomes. The relevance of the association of different CBP-related polypeptides with cytoplasmic RNP particles and polysomes are discussed.     

Retro-inverso peptide analogues of Trypanosoma cruzi B13 protein epitopes fail to be recognized by human sera and peripheral blood mononuclear cells

Retro inverso (RI) analogues of antigenic synthetic peptides, which are made of D-amino acids with a reversed sequence, may mimic the side chain conformation of natural all-L peptides. RI analogues were cross-reactively recognized by antibodies and CD4+ T cells reactive against natural all-L synthetic peptides or native proteins in animal models. Since peptides containing D-amino acids are highly resistant to proteolytic digestion, cross-reactive RI analogues may be ideal for in vivo administration to humans as synthetic peptide vaccines or immunomodulators. B13 is an immunodominant tandemly repetitive protein from Trypanosoma cruzi, a protozoan parasite that is the causative antigen of Chagas' disease. In order to test whether RI peptides can be recognized by human antibody and T cells, we synthesized two all-L peptides containing the immunodominant B (S12) and T (S15.7) cell epitopes of B13 protein from T. cruzi and their retro (R, made of all-L amino acids with reversed sequence), inverso (I, made of all-D amino acids) and RI analogues. Recognition of peptides S12, S12-R, S12-I and S12-RI by anti-B13 antibodies in sera from T. cruzi-infected patients was tested in competitive ELISA assay with recombinant B13 protein as the solid phase antigen. Peptides S15.7 and its topological analogues were tested at the 10-50 microM range in proliferation assays on peripheral blood mononuclear cells (PBMC) from S15.7-responder individuals. The median percentage inhibition of B13 ELISA for peptide S12 was 94%, while those of the RI analogue or the other topological analogues were below 12%. While peptide S15.7 was recognized by PBMC from all subjects tested, none recognized the RI analogue of the S15.7 T cell epitope. Our results indicate that cross-reactivity with natural epitopes is not an universal property of RI analogues. This may limit the general applicability of the use of cross-reactive RI analogues as human vaccines and immunotherapeutic agents.     

Advantage of Dimeric Peptide Antigens in Serodiagnosis of HIV-1 Infection

The reactivity of antibodies with dimeric and monomelic peptide antigens was compared by ELISA. A panel of highly purified synthetic peptides of HIV-1 representing defined regions, 598–609 and 524 533 (fusion domain) of gp41 and 306–320 of gpl20, were used as antigens in the ELISA. These peptides were selected and synthesized taking into account the level of sequence conservation of various strains and hydrophilicity. The analysis included sera from 52 HIV-1 infected individuals and 53 HIV-1 negative controls. Both peptides from gp41 were found to be particularly immunoreactive with sera from HIV-1 infected individuals. The frequency of reactivity to the selected peptide from gp120 (V₃ loop) in infected individuals was 82%. An interesting observation was that the dimeric peptide antigens had a detection rate more than 4-fold higher than the monomeric antigens. We found that lower levels of antibodies could be detected with dimeric antigens. The peptides reacted with few sera other than HIV-1 positive sera. These results implicate the potential dimeric peptide antigens to be utilized in the serodiagnosis of HIV-1 infection.     

Structural divergence of human ghrelin. Identification of multiple ghrelin-derived molecules produced by post-translational processing

Ghrelin, a novel 28-amino acid peptide with an n-octanoyl modification at Ser(3), was isolated from rat stomach and found to be an endogenous ligand for the growth-hormone secretagogue receptor (GHS-R). This octanoyl modification is essential for ghrelin-induced GH release. We report here the purification and identification of human ghrelin from the stomach, as well as structural analysis of the human ghrelin gene and quantitation of changes in plasma ghrelin concentration before and after gastrectomy. Human ghrelin was purified from the stomach by gel filtration and high performance liquid chromatography, using a ghrelin-specific radioimmunoassay and an intracellular calcium influx assay on a stable cell line expressing GHS-R to test the fractions. In the course of purification, we isolated human ghrelin of the expected size, as well as several other ghrelin-derived molecules. Classified into four groups by the type of acylation observed at Ser(3); these peptides were found to be non-acylated, octanoylated (C8:0), decanoylated (C10:0), and possibly decenoylated (C10:1). All peptides found were either 27 or 28 amino acids in length, the former lacking the C-terminal Arg(28), and are derived from the same ghrelin precursor through two alternative pathways. The major active form of human ghrelin is a 28-amino acid peptide octanoylated at Ser(3), as was found for rat ghrelin. Synthetic octanoylated and decanoylated ghrelins produce intracellular calcium increases in GHS-R-expressing cells and stimulate GH release in rats to a similar degree. Both ghrelin and the ghrelin-derived molecules were found to be present in plasma as well as stomach tissue. Plasma levels of immunoreactive ghrelin after total gastrectomy in three patients were reduced to approximately half of their pre-gastrectomy values, after which they gradually increased. This suggests that the stomach is the major source of circulating ghrelin and that other tissues compensate for the loss of ghrelin production after gastrectomy.     

p185胞外区及其亚区在大肠埃希菌中的表达及与单抗的结合特性分析

目的 旨在获得p185胞外区及其亚区基因在原核系统中表达的多肽,并分析其与单抗结合特性.方法 以pGEX-4T-1为载体,克隆p185胞外区及其亚区基因,并在原核系统中表达,经变性、复性后,以亲和层析法获得纯化的多肽.进一步通过ELISA方法分析了这些多肽与抗p185单抗A18的结合特性.结果 研究获得原核系统表达的p185胞外区及其亚区多肽,GST-E3和GST-E34与单抗A18具有特异结合活性.结论 原核系统表达的p185胞外区及其亚区多肽是可行的,初步确定为与单抗结合的亚区多肽,为进一步研究单抗识别的表位及其抑瘤机制奠定基础.