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1.
2.
Summary The distribution of chromogranin A and related peptides in rat tissues was investigated using sequence specific antisera. N- and C-terminal antisera and a presumptive C-terminal rat pancreastatin antiserum immunostained an extensive neuroendocrine cell population throughout the gastro-entero-pancreatic tract, anterior pituitary, thyroid and all adrenomedullary cells. However, mid- to C-terminal antisera immunostained a subpopulation of chromogranin A positive cells. Most notable of these was with the KELATE antiserum (R635.1) which immunostained discrete clusters of adrenomedullary cells and antiserum A87A which immunostained a subpopulation of cells in the anterior pituitary and throughout the gastrointestinal tract. The present study has demonstrated the widespread occurrence of chromogranin A and related peptides in rat neuroendocrine tissues and provides evidence of tissue and cell specific processing.  相似文献   

3.
The distribution of chromogranin A and related peptides in rat tissues was investigated using sequence specific antisera. N- and C-terminal antisera and a presumptive C-terminal rat pancreastatin antiserum immunostained an extensive neuroendocrine cell population throughout the gastro-entero-pancreatic tract, anterior pituitary, thyroid and all adrenomedullary cells. However, mid- to C-terminal antisera immunostained a subpopulation of chromogranin A positive cells. Most notable of these was with the KELTAE antiserum (R635.1) which immunostained discrete clusters of adrenomedullary cells and antiserum A87A which immunostained a subpopulation of cells in the anterior pituitary and throughout the gastrointestinal tract. The present study has demonstrated the widespread occurrence of chromogranin A and related peptides in rat neuroendocrine tissues and provides evidence of tissue and cell specific processing.  相似文献   

4.
Summary The presence of bombesin (gastrin-releasing peptide, GRP)-like immunoreactivity in mucosal endocrine cells of human fetal lung is well established. In this study we have investigated the localisation of pro-GRP mRNA and GRP gene products and compared the distribution and levels of extractable GRP-and C-terminal flanking peptide of human pro-GRP-like immunoreactivity in order to verify synthesis and to investigate their coexistence and molecular forms. Human fetal lungs (14 to 23 weeks gestation) were immunostained, and extracts were assayed using regionspecific antisera to pro-GRP. Additional antisera to chromogranin and protein gene product 9.5 (PGP 9.5) were used for immunostaining by the peroxidase anti-peroxidase technique and for double immunofluorescence staining using antisera raised in two species. Immunoreactivity for both bombesin (GRP) and flanking peptide was seen mainly in the same endocrine cells, but more cells were stained with antisera to flanking peptide than with antiserum to bombesin (GRP). In situ hybridisation showed that pro-GRP mRNA was present and thus synthesis of the peptides was taking place. Endocrine cells and nerve fibres were PGP 9.5-immunoreactive, and a subset of cells was immunoreactive for bombesin gene products. Radioimmunoassay and chromatography show that pro-GRP is present in both the uncleaved and cleaved forms, and, in agreement with immunocytochemistry results, that an excess of C-terminal peptide of pro-GRP is detectable. It is therefore concluded that GRP-like peptides and flanking peptide are co-local-ised in human pulmonary endocrine cells, but the latter is found in larger concentrations than free GRP. Thus GRP-like peptides may be secreted separately from the flanking peptide(s) of pro-GRP. Furthermore PGP 9.5 appears to be a useful marker for endocrine cells in the respiratory epithelium of human fetal lung.  相似文献   

5.
The occurrence of tachykinins in sensory neurons of the guinea-pig was studied by means of radioimmunoassay combined with ion-exchange and high-performance liquid chromatography as well as by immunohistochemistry. Antisera raised against kassinin (antiserum K12), neurokinin A (NKA) (antiserum NKA2) and substance P (SP) (antisera SP25 and SP2) were used. Antiserum K12 detected NKA, neuropeptide K (NPK) and a component eluting in the position of eledoisin (ELE) in extracts of the lung and ureter. Neurokinin B (NKB) was, however, not found. Neutral water extraction favored recovery of NKA and of the ELE-like component, while NPK was found only in acid extracts. The SP antisera detected two immunoreactive components of which the major form coeluted with synthetic SP. Capsaicin pretreatment depleted all these various forms of immunoreactivity in several peripheral organs including the ureter and lung. The immunoreactivity detected by antisera K12 or SP25 in radioimmunoassay had a similar regional distribution pattern in peripheral tissues. Immunohistochemical examination revealed that antiserum NKA2 stained the same spinal ganglion cells as the SP2 antiserum. The distribution of capsaicin-sensitive nerve fibers stained by these two antisera was also identical in peripheral organs such as the ureter, inferior mesenteric ganglion, heart and lung. It is concluded that multiple tachykinins, including SP, NKA, NPK and an ELE-like peptide, are present in capsaicin-sensitive sensory nerves in the guinea-pig. This finding can most likely be related to the origin of SP, NKA and NPK from the same precursor molecule, subsequent posttranslational tissue processing and axonal transport to terminal regions.  相似文献   

6.
Heterotrimeric G proteins are believed to play important roles as signal transducing components in various mammalian sperm functions. To assess the distribution of G proteins in bovine sperm tails, we purified membranes by hypoosmotic swelling of bovine spermatozoa followed by disruption of plasma membranes in a homogenizer and various centrifugation steps. Electron microscopy revealed highly purified membranes of bovine sperm tails. Subsequently, antisera against synthetic peptides were used to identify G proteins in immunoblots. An antiserum directed against the C-terminal decapeptide of Gi3 and detecting all known pertussis toxin-sensitive alpha-subunits, reacted specifically with a 40-kDa protein. In contrast, various other specific peptide antisera against alpha-subunits did not detect any G protein in enriched tail membranes. An antiserum recognizing the beta 2-subunit of G proteins and an antiserum reacting with both beta 1- and beta 2-subunits identified a 35-kDa protein in sperm tail membranes. In contrast, antisera against the 36-kDa beta 1-subunit did not detect any relevant proteins in the membrane fraction. Neither G protein alpha-subunits nor G protein beta-subunits were found in the cytosol. Our results suggest that G proteins in membranes of tails of bovine spermatozoa most likely belong to a novel subtype of G protein alpha-subunits, whereas the putative beta-subunit could be identified as a beta 2-subunit.  相似文献   

7.
The presence of bombesin (gastrin-releasing peptide, GRP)-like immunoreactivity in mucosal endocrine cells of human fetal lung is well established. In this study we have investigated the localisation of pro-GRP mRNA and GRP gene products and compared the distribution and levels of extractable GRP- and C-terminal flanking peptide of human pro-GRP-like immunoreactivity in order to verify synthesis and to investigate their coexistence and molecular forms. Human fetal lungs (14 to 23 weeks gestation) were immunostained, and extracts were assayed using region-specific antisera to pro-GRP. Additional antisera to chromogranin and protein gene product 9.5 (PGP 9.5) were used for immunostaining by the peroxidase anti-peroxidase technique and for double immunofluorescence staining using antisera raised in two species. Immunoreactivity for both bombesin (GRP) and flanking peptide was seen mainly in the same endocrine cells, but more cells were stained with antisera to flanking peptide than with antiserum to bombesin (GRP). In situ hybridisation showed that pro-GRP mRNA was present and thus synthesis of the peptides was taking place. Endocrine cells and nerve fibres were PGP 9.5-immunoreactive, and a subset of cells was immunoreactive for bombesin gene products. Radioimmunoassay and chromatography show that pro-GRP is present in both the uncleaved and cleaved forms, and, in agreement with immunocytochemistry results, that an excess of C-terminal peptide of pro-GRP is detectable. It is therefore concluded that GRP-like peptides and flanking peptide are co-localised in human pulmonary endocrine cells, but the latter is found in larger concentrations than free GRP. Thus GRP-like peptides may be secreted separately from the flanking peptide(s) of pro-GRP.(ABSTRACT TRUNCATED AT 250 WORDS)  相似文献   

8.
Phylogenetic Distribution of Peptides Related to Chromogranins A and B   总被引:3,自引:0,他引:3  
The presence of chromogranin-related peptides in a wide range of species was investigated by one and two-dimensional electrophoresis followed by immunoblotting. Antisera against bovine chromogranins A and B and the peptide WE-14 (chromogranin A316-329) were used. Chromogranins were identified by their heat stability, by their electrophoretic behavior, and by immunological cross-reaction with antisera. In all species investigated ranging from mammals to birds, amphibians, fish, and arthropods, chromogranin A- and B-like proteins could be demonstrated. For all species, there was an immunological cross-reaction with antisera against bovine chromogranins. The molecular sizes and isoelectric points of the chromogranins were similar in all species. The antiserum against WE-14 cross-reacted with pig, rat, and chicken chromogranins. It is concluded that the chromogranins A and B have a widespread phylogenetic distribution with a significant conservation of molecular size, isoelectric points, and immunological epitopes. This is consistent with the concept that these peptides have a specific function.  相似文献   

9.
Polyclonal and monoclonal antibodies were raised against pure human renin, but nothing was known about the regions against which they were directed. Using a three-dimensional model of mouse submandibular renin, we selected seven peptide sequences as belonging to potential epitopes. The main criteria for their choice were the location of the peptide sequences near the catalytic region and on the surface of the renin molecule and their hydrophilicity. After transposition of the regions to the 340-amino acid sequence of human renin, the seven peptides (corresponding to amino acids 50-60, 63-71, 81-90, 118-126, 162-169, 247-255, and 287-295) were synthesized, coupled to bovine serum albumin, and injected into rabbits. Five of these peptides elicited antibodies, and 50-68% binding of the corresponding iodinated peptide was obtained with a 1:25 dilution of antiserum. The antisera titers ranged from 1:5,000 to 1:100,000 when tested by enzyme-linked immunosorbent assay. The same antisera bound 15-65% of labeled pure human renin at a final dilution of 1:2.5, the highest percentage being obtained with peptide 81-90 antiserum. At a 1:5 dilution, the five antisera inhibited renin activity by 23-68% in human plasma with a high renin activity (40 ng of angiotensin I/h/ml). At a final dilution of 1:50, peptide 81-90 antiserum was still capable of producing 25% inhibition. Purified IgG (0.6 mg) from this antiserum inhibited pure human renin activity by up to about 40%, as measured by its reaction with pure synthetic human tetradecapeptide substrate. Antigenic peptides that mimic a part of the human renin sequence, especially peptide 81-90 representing the "flap" covering the cleft between the two renin lobes, constitute promising tools for the development of a synthetic antirenin vaccine.  相似文献   

10.
Immunochemical study on PHI/PHM with use of synthetic peptides   总被引:2,自引:0,他引:2  
We have synthesized PHI and PHM (human PHI) as well as their fragments, PHI (1-6), PHI (1-15), PHI (14-19), PHI (14-27), PHI (20-27), PHM (1-15) and PHM (13-27), by the solution or solid-phase method for peptide synthesis. Using the highly purified synthetic peptides as immunogens or haptenic immunogens, five kinds of PHI/PHM specific antisera were produced. The major antibody-recognition sites of the five antisera were located respectively in the PHI C-terminal (R8201), in the PHI N-terminal (R8403), in the PHM C-terminal (R8502), and in the PHM whole molecule (R8702 and R8703). Radioimmunoassays (RIAs) with antisera R8201, R8403 and R8502, respectively, showed a wide distribution of immunoreactive (IR) PHI/PHM in porcine and human gastrointestinal and brain tissues. The concentrations of IR-PHI in the porcine gastrointestinal tissues, however, differed between the R8201 and R8403 RIAs employed for measurement. By using these two different PHI RIAs, the IR-PHI in the porcine brain tissue extract was shown to be almost a single component coeluting with synthetic PHI in gel filtration. The IR-PHI in the extract of porcine lower intestine on the other hand, contained, besides a PHI-like component, unidentified component(s) eluting immediately after synthetic PHI in gel filtration; this crossreacted with the PHI C-terminal specific R8201 antiserum but not with the N-terminal specific R8403 antiserum, suggesting the presence of the C-terminal-related fragment(s) of PHI in the tissues.  相似文献   

11.
The vgf gene has been shown to be involved in several metabolic pathways. Because the pancreas is crucial to metabolism and food intake, we studied the VGF peptides in bovine, rat, and pig Langherans islets using antisera raised against specific sites along the primary sequence of the rat/mouse and human VGF protein precursor. Whereas almost all of the pancreatic endocrine cells expressed vgf mRNA, when using the VGF antisera a different staining pattern became apparent. VGF(556-565) and VGF(282-291) immunoreactivity were exclusively found in delta somatostatin-producing cells, whereas the human C-terminus antiserum selectively immunolabeled alpha glucagon and pancreatic polypeptide cells. The same cells were decorated with the VGF(443-588) antiserum, which also weakly labeled beta insulin-secreting cells. Finally, the VGF(298-306) peptide and the rat C terminus were found in virtually all pancreatic endocrine cells. Using bovine, swine, and rat pancreatic extracts, data from chromatography and ELISA assay showed the presence of a high molecular mass form compatible with the proVGF and lower molecular mass fractions corresponding to short VGF peptides. In conclusion, selective VGF distribution may suggest a multifaceted cell type-specific processing of proVGF, resulting in different peptides probably involved in neuroendocrine regulatory metabolic mechanisms.  相似文献   

12.
The majority of epitopes for TSH receptor (TSHR) stimulating autoantibodies are clustered around the Nterminal region of the TSH receptor. The characteristic feature of this region is the presence of four cysteine residues. It was proposed that cysteines in positions 29 and 41 in the receptor are connected by disulfide bonds and they are the target for receptor stimulating antibodies. The present study was aimed to check this possibility. The synthetic peptides: peptide corresponding to the part of TSHR containing the above 29-41 cysteine bond, the peptide similar to this peptide but without disulfide bond and the control peptide, containing sequence absent in the receptor were used for rabbit immunization. The thyroid status of all immunized rabbits was the same. Rabbits immunized with peptides related to TSHR generated antisera reactive with TSHR in immunoenzymatic assay. To check specificity of this reaction the influence of the peptides and the antisera on TSH binding to the receptor in competitive assay (TRAK) and their influence on adenylate cyclase activity were studied. It was found that neither synthetic peptides nor antiserum from any rabbit influenced TSH binding to the receptor in TRAK. In contrast low, but significant adenylate cyclase stimulating activity was noticed for antisera from two of six rabbit immunized by peptide containing the disulfide bond. We concluded that such a bond between cysteine residues 29 and 41 are present in TSHR in the site of stimulating antibodies epitope.  相似文献   

13.
1. Sperm-whale apomyoglobin was digested with chymotrypsin in a dialysis sac. The ultrafiltrate contained incompletely hydrolysed fragments which partially inhibited the precipitation of metmyoglobin and apomyoglobin by some antisera produced against metmyoglobin. The inhibitory activity was stable to heating at 100 degrees and depended on the peptide structure. 2. The fragments were fractionated according to molecular size and were purified by ion-exchange chromatography. Six pure peptides and two peptides which contained a minor impurity were isolated. Their amino acid compositions and N-terminal amino acid sequences were determined and their entire amino acid sequences deduced from the known amino acid sequence of sperm-whale myoglobin. 3. The peptides formed no detectable precipitates with the antisera. Five of the eight peptides partially inhibited the precipitation of apomyoglobin and/or metmyoglobin by one antiserum. Six of the peptides inhibited the precipitation of apomyoglobin by one or other of two antisera; at least two of these peptides inhibited both antisera. One peptide failed to inhibit the precipitation of either antigen by either antiserum. Two of the peptides possessed the same serological specificity. 4. The molar ratios of inhibitors to antigen for 50% of the maximum inhibition decreased as the molecular size of the inhibitor increased. With one antiserum and with apomyoglobin as the antigen, molar ratios 12 and 80 were obtained for peptides with molecular weights 2051 and 793 respectively. 5. The size and structure of an antigenic site is discussed in relation to the known steric configuration of myoglobin.  相似文献   

14.
Major antigenic determinants of F and ColB2 pili.   总被引:9,自引:7,他引:2       下载免费PDF全文
F-like conjugative pili are expressed by plasmids with closely related transfer systems. They are tubular filaments that are composed of repeating pilin subunits arranged in a helical array. Both F and ColB2 pilin have nearly identical protein sequences, and both contain an acetylated amino-terminal alanine residue. However, they differ by a few amino acid residues at their amino termini. Rabbit antisera raised against purified F and ColB2 pili are immunologically cross-reactive by only 25%, as measured by a competition enzyme-linked immunosorbent assay (ELISA). A tryptic peptide corresponding to the first 15 amino acid residues of ColB2 pilin was isolated and found to remove nearly 80% of ColB2 pilus-directed rabbit antibodies. The corresponding tryptic peptide from F pilin, which reacted with anti-F pilus antibodies to remove 80%, was less than 20% reactive with anti-ColB2 pilus antiserum. Cleavage of these peptides with cyanogen bromide (at a methionine residue approximately in the middle of the peptide) did not affect the antigenicity of these peptides. Synthetic N alpha-acetylated peptides corresponding to the first eight amino acids of F pilin (Ac-Ala-Gly-Ser-Ser-Gly-Gln-Asp-Leu-COOH) and the first six amino acids of ColB2 pilin (Ac-Ala-Gln-Gly-Gln-Asp-Leu-COOH) were prepared and tested by competition ELISA with homologous and heterologous anti-pilus antisera. The F peptide F(1-8) inhibited the interaction of F pili and anti-F pilus antiserum to 80%, while the ColB2 peptide ColB2(1-6) inhibited anti-ColB2 pilus antiserum reacting with ColB2 pili by greater than 60%. The two peptides F(1-8) and ColB2(1-6) were inactive by competition ELISAs with heterologous antisera. These results suggest that the major antigenic determinant of both F and ColB2 pili is at the amino terminus of the pilin subunit and that 80% of antibodies raised against these pili are specific for this region of the pilin molecule.  相似文献   

15.
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.  相似文献   

16.
Neurotensin-like immunoreactivity in the nervous system of hydra   总被引:3,自引:0,他引:3  
Summary Neurotensin-like immunoreactivity is found in nerve fibers present in all body regions of hydra. The nerve fibers are especially numerous in the ectoderm at the bases of the tentacles and in the ectoderm at a site just above the foot. Radioimmunoassays of acetic-acid extracts of hydra, using various region-specific antisera towards mammalian neurotensin, show the presence of multiple neurotensin-related peptides. The amounts of these peptides vary between 1 and 350 pmol per gram wet weight. Gel filtration on Sephadex G-25 reveals a fraction of neurotensin-like peptides that crossreacts equally well with an antiserum directed against sequence 1–8 and an antiserum directed against sequence 6–13 of neurotensin. This fraction elutes also at the position of neurotensin and might closely resemble the mammalian peptide. A fraction eluting with the void volume crossreacts preferentially with antisera directed against sequences 1–8 and 10–13 of neurotensin. Several components of apparent lower molecular weight than neurotensin crossreact preferentially with an antiserum against sequence 10–13. These last peptides represent the major portion of the neurotensin-like peptides in hydra.  相似文献   

17.
A number of peptides, fragments of the envelope protein E of the tick-borne encephalitis virus (Sofjin strain), were synthesized. Their binding to the polyclonal antiserum to protein E was studied. Rats were immunized with both the free peptides and their KLH-conjugates, and the resulting antisera were tested for their reactivity toward protein E and for their neutralizing activity toward the virus in cell culture. The only peptide corresponding to the 98-113 sequence of protein E was shown to be bound by the protein E antiserum in EIA. Two-fold immunization of rats with KLH-conjugates of the peptides corresponding to the 98-113, 130-143, and 394-403 sequences of protein E resulted in antipeptide antibodies capable of binding the native protein E, and the antibodies to the 98-113 and 394-403 peptides were capable of neutralizing the virus.  相似文献   

18.
Summary Results from a previous report demonstrate that more than one molecular form of neuropeptide Y-like peptide may be present in the islet organ of the anglerfish (Lophius americanus). Most of the neuropeptide Y-like immunoreactive material was anglerfish peptide YG, which is expressed in a subset of islet cells, whereas an additional neuropeptide Y-like peptide(s) was localized in islet nerves. To learn more about the neuropeptide Y-like peptides in islet nerves, we have employed immunohistochemical and biochemical methods to compare peptides found in anglerfish islets and brain. Using antisera that selectively react with either mammalian forms of neuropeptide Y or with anglerfish peptide YG, subsets of neurons were found in the brain that labelled with only one or the other of the antisera. In separate sections, other neurons that were labelled with either antiserum exhibited similar morphologies. Peptides from brains and islets were subjected to gel filtration and reverse-phase high performance liquid chromatography. Radioimmunoassays employing either the neuropeptide Y or peptide YG antisera were used to examine chromatographic eluates. Immunoreactive peptides having retention times of human neuropeptide Y and porcine neuropeptide Y were identified in extracts of both brain and islets. This indicates that peptides structurally similar to both of these peptides from the neuropeptide Y-pancreatic polypeptide family are expressed in neurons of anglerfish brain and nerve fibers of anglerfish islets. The predominant form of neuropeptide Y-like peptide in islets was anglerfish peptide YG. Neuropeptide Y-immunoreactive peptides from islet extracts that had chromatographic retention times identical to human neuropeptide Y and porcine neuropeptide Y were present in much smaller quantities. These results are consistent with the hypothesis that peptides having significant sequence homology with human neuropeptide Y and porcine neuropeptide Y are present in the nerve fibers that permeate the islet.  相似文献   

19.
Radioimmunoassays were developed for the tachykinins eledoisin (ELE) and kassinin (KAS) using antisera raised in rabbits. The antisera exhibited low (less than 0.1%) cross-reactivities to substance P (SP) and physalaemin (PHY), but crossreacted (with one exception, antiserum K7) to varying extents with neurokinin A (NKA) and neurokinin B (NKB). In the rat, the tissue distribution of the immunoreactive material detected by antiserum (E7) raised against ELE and by another antiserum (K1) raised against KAS both resembled that previously described for SP. Using the highly KAS-specific antiserum K7, no or only very low levels of immunoreactivity could be detected in extracts of various rat tissues. Gel permeation chromatography and ion-exchange chromatography of tissue extracts indicated that all antisera (except K7) detected the same population of immunoreactive molecules. One of the components was chromatographically indistinguishable from NKA. The tissue distribution of this component also resembled that of SP. Another immunoreactive component co-chromatographed with NKB at cation exchange chromatography. Acid tissue extracts, but not neutral tissue extracts, were found to contain immunoreactive components which appeared more basic than NKA and NKB. The total levels of immunoreactivity were higher in neutral than in acid tissue extracts. However, the ratio between the amounts of immunoreactivities in the two types of extracts varied considerably between tissues, indicating that tachykinin immunoreactive components may be present in different relative proportions in various tissues.  相似文献   

20.
The distribution of vasoactive intestinal peptide (VIP), bombesin and gastrin-cholecystokinin in the chicken was studied by radioimmunoassay of tissue extracts. VIP was present in high concentrations in colon (186 +/- 29 pmol/g), cloaca (116 +/- 27 pmol/g), jejunum (97 +/- 14 pmol/g) and pancreas (15 +/- 3 pmol/g) but not detected in lung, liver or thymus. The highest concentration of bombesin was in the proventriculus (92 +/- 13 pmol/g), negligible in remaining gut but found in brain. Gel chromatography indicated two forms of bombesin: one form eluting with bombesin-14 and the other with gastrin releasing peptide. Gastrin-like immunoreactivity was found in low levels in the gut and brain. The concentrations were higher with an antiserum which cross reacted with the carboxy terminus common to gastrin-17 and CCK compared to a gastrin specific antisera (P less than 0.01). This suggests that the carboxy terminal region has been conserved during evolution. Each distribution pattern of bombesin, VIP and gastrin CCK is different, and distinct from that found in mammals, suggesting specific roles for these peptides in birds.  相似文献   

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