Peaks Of Immunoreactive Material Research Articles

The distribution and partial characterization of FMRFamide-related peptides in the salivary glands of the locust, Locusta migratoria.

The distribution and partial characterization of FMRFamide-related peptides in the salivary glands of the locust, Locusta migratoria, were investigated by means of immunohistochemistry, radioimmunoassay and reversed-phase high performance liquid chromatography. Whole-mount preparations of glands stained positively against anti-FMRFamide antisera, and contained the equivalent of 837 +/- 80 fmol FMRFamide/gland pair, as determined by radioimmunoassay. FMRFamide-like immunoreactivity occurred in the processes of the transverse nerves of both the prothoracic and mesothoracic ganglia, but was not found in the salivary motoneurons 1 or 2 of the suboesophageal ganglion, both of which directly innervate the salivary glands via the salivary nerve 7b; nor was it found within the salivary nerve 7b itself, leading to the salivary glands. It was, however, found as a superficial nerve plexus on the surface of nerve 7 at the suboesophageal ganglion, but did not appear to extend to the salivary glands. The origin of this staining is unclear. High performance liquid chromatography of salivary gland tissue extracts, monitored by radioimmunoassay, revealed 4 peaks of immunoreactive material, 2 of which co-migrated with AFIRFamide and GQERNFLRFamide, previously isolated from the locust ventral nerve cord. These 2 synthetic peptides did not elevate basal levels of the second messengers cyclic AMP or cyclic GMP in the salivary glands.

Neurotensin in the Gut of the Amphibian Necturus maculosus and the Effects of Neurotensin on Stomach Motility

Neurotensin-like material was found throughout the gut of the amphibian Necturus maculosus, in both muscle layers and mucosa, and with the highest levels in the intestine. Ion-exchange high-performance liquid chromatography (HPLC) and reversed-phase HPLC revealed several peaks of immunoreactive material, with one closely related to neurotensin 1-6 occurring by both methods. Exogenous neurotensin was excitatory on longitudinal stomach muscle; this excitation was unaffected by tetrodotoxin, indicating a direct effect on the smooth muscle. An inhibitory effect of neurotensin on circular muscle preparations was abolished by tetrodotoxin, revealing an excitatory effect. Noradrenaline and adrenaline were in many cases inhibitory on the circular preparations, an effect that was blocked by the β-adrenergic anztagonist propranolol. Propranolol also blocked the inhibitory effect of neurotensin in approximately 50% of the tests, which suggests an involvement of an adrenergic link in the inhibitory effect of neurotensin on the circular muscle. The results are compatible with the view that a neurotensin-like peptide is involved in a simultaneous phase of shortening and widening of a segment of the stomach.

Metabolism of des(64,65)-human proinsulin in the rat. Evidence for the proteolytic processing to insulin.

The metabolism of des(64,65)-human proinsulin was examined in rats after subcutaneous administration. Profiles of circulating insulin-like immunoreactivity in rat plasma 25 min after subcutaneous administration were evaluated by anion exchange fast protein liquid chromatography and reversed-phase high-performance liquid chromatography. Both techniques indicated the presence of circulating immunoreactivity having retention characteristics of human insulin. This metabolite peak comprised 5-10% of circulating immunoreactivity; the remainder had retention characteristics of des(64,65)-human proinsulin. The peaks of immunoreactive material were isolated and their structure determined using reversed-phase high-performance liquid chromatography and electrospray ionization mass spectrometry. The major circulating component co-eluted with des(64,65)-human proinsulin and had an identical mass spectrum. Two circulating metabolites were identified. These metabolites co-eluted by reversed-phase high-performance liquid chromatography with human insulin and diarginyl(B31,32)-human insulin and had mass spectra identical to the standard compounds. The data indicate proteolytic processing of des(64,65)-human proinsulin involves an initial tryptic cleavage at the carboxy side of ArgB32, with the formation of human insulin by the subsequent action of a carboxypeptidase to remove the ArgB31-ArgB32 dipeptide from diarginyl(B31,32)-human insulin. The results suggest that some of the pharmacological activity of des(64,65)-human proinsulin may be mediated in part by circulating insulin-like metabolites.

Distribution of FMRFamide‐related peptides in the blood‐feeding bug Rhodnius prolixus

Immunohistochemistry was used to study the distribution of FMRFamide-like material in the central and peripheral nervous systems and visceral tissues of 5th instar Rhodnius prolixus. Over 200 immunoreactive cell bodies and their processes as well as extensive neuropile regions were distributed throughout the nervous system. Immunoreactive processes were seen over the cephalic aorta, corpus cardiacum/corpus allatum complex, and in neurohaemal sites on the abdominal nerves. In visceral tissues, immunoreactive processes were seen innervating the salivary glands, the foregut, and the hindgut. Immunoreactive cells were also found in the anterior midgut (i.e., the crop and the anterior intestine). A radioimmunoassay specific for "RFamide" carboxy-terminal peptides was used to quantify the amount and the distribution of FMRFamide-like material. Reversed-phase high performance liquid chromatography of nervous tissue extracts revealed several peaks of immunoreactive material. The results suggest the existence of a number of FMRFamide-related peptides in Rhodnius which may have roles in both central and peripheral transmission, may be released as neurohormones and may have endocrine functions in the gut.

γ-MSH-like immunoreactivity in porcine pituitary and adrenal medulla. An immunochemical and immunocytochemical study

A sensitive and specific radioimmunoassay for γ 2-melanotropin ( γ 2-MSH) has been developed that does not recognize α-, β-, γ 1- or γ 3-MSH. γ 2-MSH-like immunoreactivity could be demonstrated in the porcine pituitary and adrenal gland. The highest concentrations were detected in the neurointermediate lobe regardless of extraction procedure. The anterior lobe harboured lower concentrations and in adrenal gland extracts only small amounts were measured. Gel chromatography and high performance liquid chromatography of extracts of both pituitary and adrenal gland revealed several peaks of immunoreactive material, one of which eluted close to the position of synthetic γ 2-MSH. By immunocytochemistry γ 2-MSH immunoreactivity was localized to the adrenocorticotropin/α-MSH cells in the pituitary and to a subpopulation of the noradrenaline-storing cells in the adrenal medulla. Together, the immunocytochemical and immunochemical findings indicate the existence of γ 2-MSH-like material in the porcine pituitary and adrenal medulla.

Human pancreatic chymotrypsinogen A: a non-competitive enzyme immunoassay, and molecular forms in serum and amniotic fluid

A sandwich enzyme immunoassay has been developed for human pancreatic chymtrypsin(ogen) using polystyrene balls coated with specific IgG as the first antibody and peroxidase-labelled IgG as the second antibody. The detection limit was 0.5 μg/l. A good parallelism was observed with the curves obtained from standard chymotrypsinogen A and chymotrypsin(ogem) present in pancreatic juice: however, a slight discrepancy in parallelism with chymotrypsin(ogen) present in serum and amniotic fluid was noticed. Chymotrypsinogen concentration in pancreatic juice was evaluated to represent 9% of total proteins. Mean values of chymotrypsin(ogen) in human sera were 24.6±8.3 μg/l in adults and 20.9±8.8 μg/l in newborns. In amniotic fluid at the 18th week of pregnancy the values were scattered (5–70 μg/l). The molecular forms of immunoreactive chymotrypsin(ogen) in normal serum and amniotic fluid have been investigated by gel flitration of Sephadex G-100. Two peaks of immunoreactive chymotrypsin(ogen) were observed normal serum; the first peak elutes in a position consistent with a complex of chymotrypsin with serum ihibitor ( M r 76 000), and the second peak elutes with a molecular weight of approx. 25 000 corresponding to the elution position of free chymotyrpsin(ogen). In normal amniotic fluid three peaks of immunoreactive material were present; the first and second peaks elute same position as in serum, and the third peak with a molecular weight of about 14 500 may represent a degraded form of chymotrypsin.

Arginine vasopressin as an intragonadal hormone in Brattleboro rats: presence of a testicular vasopressin-like peptide and functional vasopressin receptors.

We have previously demonstrated the presence of an arginine vasopressin (AVP)-like peptide and AVP receptors in rat testis. We have also shown a direct inhibitory effect of AVP on androgen biosynthesis by cultured testicular cells. This study examined the presence of testicular AVP-like peptides and AVP receptors in homozygous (di/di) Brattleboro rats, a genetic mutant known to be deficient in hypothalamic, pituitary, and circulating AVP. The supernatant of homogenized 0.1 N acetic acid-extracted testis from adult homozygous Brattleboro rats was chromatographed on a Sephadex G-25 column. The elution profile of AVP immunoreactivity, as measured by a specific RIA, showed three distinct peaks. The first peak eluted close to the column void volume, a second peak eluted at the column total volume, while a third peak coeluted with synthetic AVP. The third peak of immunoreactive material (375 pg/g tissue) behaved similarly to authentic AVP on octadecylsilica adsorption chromatography, showed a competition curve parallel to that of AVP in the RIA, and comigrated with AVP on Sephadex G-25 and reverse phase TLC. The first, but not the second, immunoreactive peak contained enzyme activity that degraded labeled AVP in a time-dependent manner. Additional studies investigated the presence of AVP receptors in testes from Brattleboro rats. Saturable and specific [3H]AVP-binding sites were present in an enriched testicular interstitial cell preparation from these animals. Scatchard analysis indicated a Kd of 5.6 X 10(-10) M and a binding capacity of 9.7 fmol AVP bound/10(6) cells. These receptors were of the vasopressor (V1) subtype, as indicated by the potencies of selective AVP analogs for competition of [3H]AVP binding. The functionality of these receptors was shown by AVP inhibition of gonadotropin-induced androgen biosynthesis in cultured testicular cells derived from Brattleboro rats. Thus, testes from Brattleboro rats contain a high amount of an AVP-like peptide even though these animals lack hypothalamic, pituitary, and circulating AVP. Also, an AVP-degrading enzyme and AVP receptors with a Kd and binding capacity similar to those of Sprague-Dawley rats are present in the testes of Brattleboro rats. These findings add further support to the hypothesis that locally produced AVP acts as an intratesticular modulator of androgen biosynthesis.

Identification by specific radioimmunoassay of two novel peptides derived from the C-terminus of porcine preprogastrin

A radioimmunoassay has been developed using antibodies to a synthetic analogue of the C-terminal hexapeptide sequence of the porcine gastrin precursor. Boiling water extracts of porcine antral mucosa contained immunoreactive material that diluted in parallel with standard peptide. Concentrations of immunoreactivity were 5.5 ± 0.8 nmol · g −1 (mean ± S.E.M.) in antral mucosa and were closely similar to those of C-terminal heptadecapeptide gastrin immunoreactivity (5.0 ± 0.6 nmol · g −1). Approximately 30-fold lower concentrations were found in porcine duodenum. A similar distribution was found in ferret, but human, rat and chicken antrum did not contain significant quantities of immunoreactivity. Gel filtration of porcine antral extracts on Sephadex G-50 revealed a single peak of immunoreactivity eluting in a similar position to G17, but on anion-exchange chromatography two peaks of immunoreactive material were separated. These also differed in their retention time on reverse phase HPLC. Both peptides are probably derived by tryptic cleavage at the C-terminus of porcine preprogastrin. No evidence was found to suggest that there are significant quantities of unprocessed preprogastrin in hog antral mucosa. The precise chemical difference between the two immunoreactive peptides identified here remains to be established; together, however, they provide specific markers for progastrin synthesis.

Fetal and maternal plasma levels of immunoreactive somatostatin at delivery: evidence for its increase in the umbilical artery and its arterio-venous gradient in the feto-placental circulation.

The concentrations of immunoreactive somatostatin (IR-SRIF) in plasma samples taken from the umbilical artery (UA), umbilical vein (UV), and maternal antecubital vein (MV) were measured in 23 cases of normal delivery at term. High concentrations of IR-SRIF were detected in the plasma from the UA (mean +/- SD, 73.2 +/- 40.6 pg/ml), the value being about 2.5 times that in the UV (29.5 +/- 17.5 pg/ml; P less than 0.001). An arterio-venous gradient was observed in all 23 subjects. The plasma level of IR-SRIF in the MV (10.9 +/- 4.6 pg/ml) was significantly lower than those in the UA (P less than 0.001) and UV (P less than 0.001) and was almost the same as that in nonpregnant women (12.7 +/- 5.4 pg/ml). Chromatographic analysis of an extract of plasma from the UA gave only one peak of immunoreactive material, which was eluted in the same position as synthetic SRIF-14. A similar result was obtained in nonpregnant women. No correlation was found between the plasma level of IR-SRIF and that of GH, insulin, glucagon, or gastrin in the UA. The present findings suggest that the high concentration of IR-SRIF in the feto-placental circulation originates from the fetus and reflects a particular role of this peptide in the process of functional maturation of the neuroendocrine system in early human life.

Occurrence of higher molecular forms of LHRH in fractionated extracts from rat hypothalamus, cortex and placenta

Separation of higher molecular forms of LHRH-like immunoreactive material was attempted in homogenates and subcellular fractions of hypothalamus, cerebral cortex and placenta. Physiological activity was checked by means of antibodies directed against different sequences of LHRH and shown to recognize synthetic LHRH analogs extended on either C- or N-terminal portions of the molecule. After molecular-sieve filtration, 2 peaks of immunoreactive material corresponding apparently to sequences of LHRH extended on the C terminus were recovered. Peak I, with a molecular weight of about 26 000 dalton, was found exclusively in a cytoplasmic and axoplasmic supernatant (S 2), where it migrated alone with microsomes. Peak II(1800 dalton) was present both in S 2 and in a synaptosomal fraction (P 2) corresponding to nerve endings. Native LHRH was almost exclusively recovered from the synaptosomal fraction. Extracts from placenta or cerebral cortex contained little or no native LHRH; in contrast, a small amount of immunoreactive material corresponding to peak II was detected in the cerebral cortex, and fairly large amounts of both putative precursors were found in the placenta. Chromatography of tissues containing no LHRH, such as cerebellum or liver, yielded no immunoreactive material at either elution site, thus suggesting specific detection of LHRH-like material under our experimental conditions. The present data suggest that 2 higher molecular forms, one slightly heavier than the native peptide itself, and another corresponding to a much larger protein, could represent LHRH precursors and are present in the hypothalamus as well as the cerebral cortex and the placenta.

Sex differences in the content of beta-endorphin and enkephalin-like peptides in the pituitary of obese (ob/ob) mice.

The beta-endorphin content in pituitary extracts of male and female obese (ob/ob) and lean (+/?) mice was determined by radioimmunoassay. The amount of beta-endorphin-like material contained in the pituitary of 3-month-old ob/ob male mice is similar to that of lean male mice. In contrast, the pituitary glands of female ob/ob mice have a greater amount of beta-endorphin-like material than lean female mice. To determine with greater precision the molecular nature of the polypeptide that accounts for the increase in beta-endorphin immunoreactivity, the various molecular forms of beta-endorphin immunoreactivity were resolved by Biogel P-30 column chromatography. At least four peaks of immunoreactive material were detected. The first peak elutes in the void volume, and the second and the third peaks appear in the elution volumes of beta-lipotropin and beta-endorphin, respectively. That the material present in the void volume might be proopiocortin is supported by adrenocorticotropic hormone radioimmunoassay. The increased total beta-endorphin immunoreactivity in pituitary glands of ob/ob mice is accounted for mainly by beta-endorphin. The beta-endorphin content of various brain structures of ob/ob mice is similar to that of lean littermates.