Peak Of Immunoreactivity Research Articles

Deconvolution analysis of cardiac natriuretic peptides during acute volume overload.

Cardiac natriuretic peptides, especially amino terminal pro-Brain Natriuretic Peptide (NT-proBNP), are emerging as powerful circulating markers of cardiac function. However, the in vivo secretion and elimination (t1/2) of these peptides during acute volume overload have not been studied. We present the first report of the secretion and elimination of cardiac natriuretic peptides, based on deconvolution analysis of endogenous ovine plasma levels measured by specific radioimmunoassay. Four normal, conscious sheep underwent rapid right ventricular pacing (225 bpm) for 1 hour to stimulate acute cardiac natriuretic peptide release. Plasma samples and right atrial pressure measurements were taken at regular intervals 30 minutes before, during, and 4 hours after pacing. Baseline right atrial pressure significantly increased (P:=0.02) during the 1 hour of pacing in association with a prompt increase in plasma BNP (P:=0.03), atrial natriuretic peptide (P:=0.01), and NT-proBNP (P:=0.02). Deconvolution analysis showed that the t1/2 of NT-proBNP (69.6+/-10.8 minutes) was 15-fold longer than BNP (4.8+/-1. 0 minutes). Despite sustained increases in atrial pressure, cardiac secretion of natriuretic peptides (particularly atrial natriuretic peptide) fell during the pacing period, suggesting a finite source of peptide for secretion. Size-exclusion high-performance liquid chromatography revealed NT-proBNP to be a single immunoreactive peak, whereas BNP comprised at least 2 immunoreactive forms. These findings, especially the prompt secretion of BNP and the prolonged t1/2 of NT-proBNP, clarify the metabolism of BNP forms and help to explain the diagnostic value of NT-proBNP measurement as a sensitive marker of ventricular function.

Expression of Caspases and Their Substrates in the Rat Model of Focal Cerebral Ischemia

Experimental evidence suggests that the massive release of glutamate during experimental brain ischemia both directly and indirectly regulates downstream mechanisms of cell suicide. Cerebral ischemia was produced by distal, permanent occlusion of the middle cerebral artery (MCAO) in the rat. Sets of three animals and one sham-operated for each time-point were kept alive for 0–30 min, 1, 4, 12, 24, and 48 h, and 4 days. Additional animals were treated by local administration of a 10 μM (in 10 μl) cocktail of caspase inhibitors (YVAD-cmk, DEVD-fmk, IETD). Immunohistochemistry was performed on free-floating tissue sections with goat polyclonal antibodies to procaspase-1, -2, -3, -6, and -8. Some sections were processed for double-labeling procaspase immunohistochemistry and in situ end-labeling of nuclear DNA fragmentation (TUNEL method). Both immunohistochemistry and double-labeling procaspase immunohistochemistry and TUNEL method were carried out on formalin-fixed sections. For gel electrophoresis and Western blotting, we used antibodies to poly (ADP-ribose) polymerase (PARP), lamin B, and PKC-δ, as specific cleavage substrates of caspases. There was increased immunoreactivity ipsilaterally in the areas corresponding to the infarct and surrounding penumbra with the peak of immunoreactivity between 12 and 24 h for most of the procaspases. Procaspases were present early in the infarcted tissue neurones and their dendrites and axons. Additional procaspase expression occurred in astrocytes and microglial cells at different times following ischemia. Cells with positive in situ end-labeling of nuclear DNA fragmentation appeared in high number predominantly in the infarcted areas and at the edge of the infarction and colocalized with enhanced procaspase expression. These findings suggest increased procaspase expression in dying cells at the edge of the infarction. A major product of PARP degradation of about 89 kDa was found in the samples taken from the infarcted and penumbra areas. There was no difference in the intensity of the bands corresponding to lamin B or PKC-δ. Injection of procaspase inhibitors reduced the levels of major PARP products of 89 kDa and decreased the number of TUNEL-positive cells at 12 h post-MCAO. In conclusion, these results give support to further research on the use of caspase inhibitors as add-on therapeutic agents for the treatment of ischemia.

Proliferative activity of follicles and serum steroid concentration in Scotophilus heathi (vespertilionid bat) during periods of delayed ovulation

Follicular kinetics were studied using immunocytochemical localization of proliferating cell nuclear antigen (PCNA) and changes in circulating steroid concentrations to investigate the cause of prolonged survival of Graafian follicles and delayed ovulation in the vespertilionid bat Scotophilus heathi. PCNA immunoreactivity was limited to the granulosa cells and varied with stage of follicular development and reproductive phase. Two waves of follicular growth appear to be initiated in October and February. The first wave resulted in the appearance of some late antral follicles in November. These antral follicles grew slowly and differed from typical mammalian Graafian follicles, as the cells of their cumulus oophoricus were large and hypertrophied. The second wave of follicular development resulted in fresh recruitment and selection of dominant follicles in February. The steroid-hormone profile showed an unusually high concentration of circulating androstenedione from October to December. The estradiol concentration showed two peaks coinciding with two peaks of PCNA immunoreactivity. The results show that the unusually high androstenedione concentration might be responsible for the development of non-ovulatory Graafian follicles with hypertrophied granulosa cells, which survive for a prolonged period. The decline in androstenedione concentration allows the development of ovulatory follicles in S. heathi.

Immunohistochemical localization, biochemical characterization, and biological activity of neurotensin in the frog adrenal gland.

The primary structure of neurotensin has been recently determined for the frog Rana ridibunda (Endocrinology 139: 4140-4146, 1998). In the present study, we have investigated the distribution and biochemical characterization of neurotensin-like immunoreactivity in the frog adrenal gland, using an antiserum directed against the conserved C-terminal region of the peptide. Neurotensin-like immunoreactivity was detected in two populations of nerve fibers: numerous varicose fibers coursing between adrenal cells, and a few processes located in the walls of blood vessels irrigating the gland. Reversed-phase HPLC analysis of frog adrenal gland extracts revealed the existence of a major peak of neurotensin-like immunoreactivity that exhibited the same retention time as synthetic frog neurotensin. The possible involvement of neurotensin in the regulation of steroid secretion was studied in vitro using perifused frog adrenal slices. For concentrations ranging from 10(-10) to 10(-5) M, synthetic frog neurotensin increased corticosterone and aldosterone production in a dose-dependent manner (EC50 = 1.2 x 10(-9) M and 5.8 x 10(-10) M, respectively). Repeated administration of neurotensin induced a reproducible stimulation of steroid output without any tachyphylaxis. Prolonged administration (3 h) of frog neurotensin caused a transient increase in corticosterone and aldosterone secretion followed by a decline of corticosteroid secretion. Neurotensin also produced a significant stimulation of corticosteroid secretion from dispersed frog adrenal cells. This study demonstrates that neurotensin is located in nerve processes innervating the adrenal gland of amphibians. The results also show that synthetic frog neurotensin exerts a direct stimulatory effect on corticosteroid output. Taken together, these data support the view that neurotensin, released by nerve fibers, may act as a local regulator of corticosteroid secretion.

Juvenile hormone biosynthesis by corpora allata of larval tomato moth, Lacanobia oleracea, and regulation by Manduca sexta allatostatin and allatotropin

Juvenile hormone (JH) biosynthesis and the effects of synthetic Manduca sexta allatostatin (Mas-AS) and M. sexta allatotropin (Mas-AT) were investigated in isolated corpora allata (CA) of Vth stadium larvae of the tomato moth, Lacanobia oleracea. Reversed-phase high-performance liquid chromatography (RP-HPLC) of JH extracted from CA shows that larvae produce predominantly JH II and its corresponding acid. It appears that the acid homologue is a result of JH esterase activity in the CA (and other tissues) rather than the lack of JH acid methyltransferase. Mean rates of synthesis (100–200 fmol/pr/h) were inhibited ca. 70% by Mas-AS and stimulated in a dose-dependent manner up to three times by Mas-AT. However, Mas-AS had no significant effect on Mas-AT-stimulated rates of JH biosynthesis. Using RP-HPLC and an enzyme-linked immunosorbent assay (ELISA) to Mas-AT, a peak of Mas-AT-like immunoreactivity was detected in larval L. oleracea brain homogenates. Co-elution of this immunoreactive peak with synthetic Mas-AT suggests that this neuropeptide is also present in L. oleracea.

Ultraviolet B Exposure of Whole Leaves of Barley Affects Structure and Functional Organization of Photosystem II

This study examines the effects of ecologically important levels of ultraviolet B radiation on protein D1 turnover and stability and lateral redistribution of photosystem II. It is shown that ultraviolet B light supported only limited synthesis of protein D1, one of the most important components of photosystem II, whereas it promoted significant degradation of proteins D1 and D2. Furthermore, dephosphorylation of photosystem II subunits was specifically elicited upon exposure to ultraviolet B light. Structural modifications of photosystem II and changes in its lateral distribution between granum membranes and stroma-exposed lamellae were found to be different from those observed after photoinhibition by strong visible light. In particular, more complete dismantling of photosystem II cores was observed. Altogether, the data reported here suggest that ultraviolet B radiation alone fails to activate the photosystem II repair cycle, as hypothesized for visible light. This failure may contribute to the toxic effect of ultraviolet B radiation, which is increasing as a consequence of depletion of stratospheric ozone.

ELEVATED LEVELS OF SERUM SECRETONEURIN IN PATIENTS WITH THERAPY RESISTANT CARCINOMA OF THE PROSTATE

The majority of prostate cancers show some degree of neuroendocrine differentiation. It was previously demonstrated that chromogranin A, a constituent of large dense core vesicles of neuroendocrine cells, is frequently elevated in patients with metastatic prostate cancer. We evaluate the expression of secretoneurin, which is generated by proteolytic processing of secretogranin II (chromogranin C), in patients with prostate disease. Secretoneurin was measured in sera of 16 healthy men whose blood was drawn for prostate cancer screening (controls), and in 9 patients with prostatitis, 19 with benign prostate hyperplasia and 54 with prostate cancer detected by radioimmunoassay. Therapy resistant disease (clinical stage D3) was noted in 20 prostate cancer cases. Serum prostate specific antigen was measured in all patients and controls. In addition, chromogranin A, prostate acid phosphatase and interleukin-6 were determined in patients with D3 prostate cancer. Molecular properties of secretoneurin immunoreactivity were analyzed by gel filtration chromatography followed by radioimmunoassay. Mean secretoneurin was 58.9+/-8 fmol./ml. in patients with therapy resistant prostate cancer. Levels were significantly higher than those measured in sera from controls and patients with prostatitis, benign prostatic hyperplasia and pT2 or pT3 prostate cancer. There was a statistically significant correlation between secretoneurin and chromogranin A in patients with endocrine therapy failure (r = 0.543, p<0.05). There was no correlation between serum secretoneurin and prostate specific antigen, prostate acid phosphatase or interleukin-6. Gel filtration chromatography analysis of sera of 3 patients with D3 prostate cancer revealed a peak of secretoneurin immunoreactivity where intact secretoneurin elutes, thus showing that the processed peptide is circulating in the blood. Secretoneurin is elevated in sera of patients with endocrine therapy refractory prostate cancer. Our results support the concept that neuroendocrine differentiation is associated with prostate cancer progression.

Orexin-A in the human brain and tumor tissues of ganglioneuroblastoma and neuroblastoma

Regional distribution of orexin-A-like immunoreactivity in the human brain and pituitary, and the presence of orexin-A-like immunoreactivity in the tumor tissues of pheochromocytomas, ganglioneuroblastomas and neuroblastomas were studied by radioimmunoassay. Expression of orexin mRNA was studied by reverse transcriptase polymerase chain reaction (PCR) method. Orexin-A-like immunoreactivity was detected in every region of human brain, but not in the pituitary. The highest concentration of orexin-A-like immunoreactivity in the human brain was found in hypothalamus (17.8 ± 4.3 pmol/g wet weight, mean ± SEM, n = 7), followed by thalamus, medulla oblongata, and pons. Orexin-A-like immunoreactivity was detected in the tumor tissues of ganglioneuroblastoma and neuroblastoma, but not in the tumor tissues of pheochromocytoma. Reverse phase high performance liquid chromatographic analyses of the orexin-A-like immunoreactivity in the human brain extracts and neuroblastoma extracts showed a single immunoreactive peak, which was eluted in an identical position to synthetic human orexin-A. Orexin mRNA was expressed in the hypothalamus and in the tumor tissues of ganglioneuroblastoma and neuroblastoma. These findings suggest that orexin-A is produced in the hypothalamus and transported to various brain regions via axons. In addition, this study has shown for the first time the production of orexin-A by ganglioneuroblastomas and neuroblastomas.

Detection of N-terminal Pro-corticotrophin Releasing Hormone (CRH) and a ‘Novel’ CRH in Human Maternal Plasma and Placenta

Corticotrophin-releasing hormone (CRH) is released from the human placenta in high concentrations towards the end of the third trimester of pregnancy, but relative concentrations of other cleavage products derived from the CRH prohormone are unknown. We have measured CRH and N-terminal (1–100) and (1–127) proCRH peptides in maternal plasma in the second and third trimesters of pregnancy and in term placental extract using immunoradiometric assays (IRMAs) specific to different regions of the CRH precursor. Levels of N-terminal proCRH (amino acid residues 1–100) rose from 24±4pmol/l (mean±s.e.) in the second trimester to 378.8±65pmol/l (mean±s.e.) at term. Levels of intact proCRH and/or (1–127) proCRH remained relatively constant throughout the second and third trimesters, with a concentration of 29.3±3.8pmol/l (mean±s.e.). In the course of this work a novel form of CRH that cross-reacts within specific CRH immunoassays was observed. The use of two IRMAs developed for CRH (1–41) having different C-terminal epitope specificities provided evidence for two types of CRH coexisting in maternal plasma. Separation of term placental extract by HPLC and application of the two CRH IRMAs revealed two peaks of immunoreactivity one of which coeluted with synthetic CRH (1–41).

Processing of procorticotropin-releasing hormone (pro-CRH): molecular forms of CRH in normal and preeclamptic pregnancy.

This study examined the different molecular forms of CRH in normal and preeclampsia maternal plasma and protease-blocked placental extracts using antibodies to different regions of the CRH precursor, pro-CRH. In the absence of protease inhibitors, chromatographed normal placental extracts contained four peaks of immunoreactivity corresponding to unprocessed approximately 19-kDa pro-CRH, its approximately 8-kDa intermediate metabolite, pro-CRH125-194, its approximately 2.8-kDa midportion fragment, pro-CRH125-151, and 4.75-kDa CRH1-41. However, if protease inhibitors were included in the extraction medium, only pro-CRH and pro-CRH125-194 were found. Pro-CRH processing was more extensive in protease-blocked preeclampsia placentas than in those from normal pregnancy, with three peaks corresponding to pro-CRH, proCRH125-194, and mature CRH1-41 peptide found. Using quantitative competitive PCR, the messenger ribonucleic acid levels of CRH precursor in preeclampsia placentas were 1.7-fold higher than those in normal placentas (37.83 +/- 3.48 vs. 21.83 +/- 2.59 attomoles/microg total ribonucleic acid, respectively; P < 0.005). Preeclampsia placentas contained significantly more CRH1-41 cross-reactivity (4.72 +/- 1.22 pmol/g) than normal term placentas (1.52 +/- 0.39 pmol/g; P < 0.048) extracted in medium containing protease inhibitors. The content of pro-CRH(125+/-151)-reactive species in these extracts followed the same pattern, with more immunoreactivity detected in preeclampsia placentas (4.23 +/- 1.39 pmol/g) than in those from normal term pregnancies (1.44 +/- 0.32 pmol/g; P < 0.01). Sequential plasma samples from 10 women with normal pregnancy and 5 women with preeclampsia were assayed for pro-CRH(125-151)- and CRH(1-41)-immunoreactive species In normal pregnancy, maternal plasma CRH(1-41) immunoreactivity rose with increasing gestational age, reaching 460 +/- 48 pmol/L at term. In women with preeclampsia, CRH(1-41) levels at each gestational age point were higher than those at the equivalent stage of normal pregnancy. In contrast, the levels of pro-CRH(125-151)-immunoreactive species remained barely detectable throughout normal and preeclamptic pregnancy. Both pro-CRH and CRH(1-41), but not pro-CRH(125-151), were shown to bind to the plasma CRH-binding protein. Our findings highlight the importance of protection of placental tissue from degrading enzymes during extraction and show that most of the CRH in the human placenta exists as unprocessed pro-CRH, with very little in the form of CRH(1-41) except in preeclampsia. Our studies using maternal plasma indicate that CRH(1-41) is the only one of the pro-CRH fragments studied to be maintained in significant amounts in the maternal circulation and also the only fragment studied for which a specific plasma binding protein exists.

Binding of Ouabain and Human Ouabainlike Substance to Different Na+, K+-ATPase Isoforms

There is very little on the affinity of the human immunoreactive ouabainlike substance (OLS) to individual alpha-isoforms of Na+,K+-ATPase. The present study addresses this issue by comparing ouabain and OLS binding to dog kidney alpha1, rabbit kidney alpha1 and porcine cerebral cortex alpha3 Na+,K+-ATPase. OLS was initially isolated by solid phase extraction from human serum using C18 columns. The extract was further purified by reverse phase HPLC in an acetonitrile/water (containing 0.1% TFA) step-up gradient (16-80%). In this system, two distinct ouabain immunoreactive peaks were resolved. Peak I demonstrated a polarity identical with that of authentic ouabain. In contrast, peak II was relatively non-polar and eluted later in the run. The final step in the purification of OLS involved immuno-affinity chromatography of peak I using a specific sepharose immobilized mouse monoclonal anti-ouabain antiserum. Dose response curves (range 0-100 nmol/l) for ouabain with canine alpha1 and porcine alpha3 Na+,K+-ATPase showed similar inhibitory profiles (IC50=15 nmol/l), whilst rabbit alpha1 Na+,K+-ATPase was relatively insensitive to ouabain and purified peak I OLS. Two fold serial dilution of Peak I OLS, with subsequent analysis by canine and porcine Na+,K+-ATPase inhibition assays and RIA, demonstrated strong positive correlations between OLS determined by RIA and both canine (y=0.945x-2.532, r2=0.977) and porcine (y=0.428x-1.685; r2=0.993) Na+,K+-ATPase assays. The difference in the respective slopes suggests, however, that peak I OLS has a greater affinity for the canine derived enzyme compared to the porcine. In conclusion, these data suggest that like authentic ouabain, peak I OLS is a-isoform and species selective.

Release of angiotensin-(1-7) from the rat hindlimb: influence of angiotensin-converting enzyme inhibition.

The results of recent studies have demonstrated that angiotensin (Ang)-(1-7) contributes to the antihypertensive actions of either combined ACE/Ang II type 1 receptor blockade or ACE inhibition alone. The vasculature is a key site of action for either drug regimen, and evidence favors a local Ang system within these tissues. Because ACE may degrade Ang-(1-7), we determined whether ACE inhibition alters Ang-(1-7) release from the rat hindlimb perfused with Krebs-Ringer buffer containing Ficoll. Ang-(1-7) release averaged 36+/-13 fmol (period 1, 15-minute collection) and 44+/-11 fmol (period 2) in the control buffer. The addition of the ACE inhibitor lisinopril to the perfusion buffer augmented levels of Ang-(1-7) in periods 3 (144+/-39 fmol) and 4 (163+/-35 fmol; P<0.05 versus 1 or 2, n=8). HPLC and radioimmunoassay of effluent from control or lisinopril treatment demonstrated a single immunoreactive peak with a retention time identical to that of Ang-(1-7). The addition of the neprilysin inhibitor SCH 39370 reduced Ang-(1-7) release in the lisinopril buffer from 177+/-32 (period 1) and 173+/-39 (period 2) fmol to 112+/-24 (period 3) and 87+/-23 fmol (period 4; P<0.05 versus 1 or 2, n=6). Ang I metabolism in the collected perfusate revealed the formation of Ang-(1-7) that was sensitive only to thimet oligopeptidase inhibition; Ang II generation was not detected. The present study demonstrates the recovery of endogenous Ang-(1-7) from the perfused hindlimb. The release of Ang-(1-7) is significantly influenced by inhibition of ACE, which may reflect both increased substrate (Ang I) levels and reduced metabolism of the peptide. Neprilysin inhibition reduced but did not abolish Ang-(1-7) release, which suggests that other endopeptidases may contribute to the release of the peptide.

Isolation of mitogenically active C-terminal truncated pentapeptide of osteogenic growth peptide from human plasma and culture medium of murine osteoblastic cells.

The osteogenic growth peptide (OGP) is a 14-amino acid stromal cell mitogen that stimulates in vivo osteogenesis and hematopoiesis. In the blood circulation and cell culture conditioned medium immunoreactive OGP (irOGP), identified using antibodies raised against the OGP C-terminal region, presents free and bound forms. The bound form consists entirely of the full length peptide. The present study was designed to investigate the identity of free irOGP under nondenaturing conditions. Fresh human serum and culture medium conditioned with murine osteoblastic MC3T3 E1 cells were fractionated using ultrafiltration (3000 molecular weight cut-off). Hydrophobic chromatography of the ultrafiltrate, immunoscreening of chromatographic fractions with antibodies directed against the OGP C-terminal region and amino acid sequencing of immunoreactive peaks demonstrated the presence of two mitogens, the full length OGP and a C-terminal truncated form, OGP(10-14). The OGP(10-14) derived from both serum and conditioned medium, as well as the synthetic pentapeptide [sOGP(10-14)], shared the in vitro OGP proliferative activity. However, in a competitive binding assay, devised to assess the OGP-OGP binding protein (OGPBP) complex formation, sOGP(10-14) failed to compete out radiolabeled OGP from the complex. It is concluded that OGP(10-14) is a naturally occurring human and murine mitogen. In addition, the data suggests that the OGP(10-14) is generated from OGP by proteolytic cleavage upon dissociation of the OGP-OGPBP complexes.

Natriuretic peptides and the acclimation of aglomerular toadfish to hypo-osmotic media.

Since the aglomerular toadfish (Opsanus tau) experiences a natriuresis following transfer to 10% seawater, we examined the role of natriuretic peptides in the acclimation of toadfish to hypo-osmotic media. Gel filtration chromatography of acid extracts of toadfish heart and kidney identified a broad peak of atrial natriuretic peptide-like immunoreactivity in both tissues, with maximal immunoreactivity in fractions coeluting with human alpha-atrial natriuretic peptide. Using a homologous bioassay to measure changes in aortic ring tension, both vasorelaxing and, surprisingly, vasoconstricting bioactivities were identified in gel fractions of heart extract. No significant vasorelaxing activity was identified in kidney extract or fractions. Instead, a potent vasoconstricting activity was observed, with maximal activity in gel fractions with an estimated MW greater than 1000 Da. Levels of atrial natriuretic peptide immunoreactivity in plasma from the caudal vein were very low in seawater toadfish and were unchanged 12 h after transfer of toadfish to 10% seawater. We conclude, that natriuretic peptides are present in the heart and kidney of toadfish. However, atrial natriuretic peptide-like peptides of cardiac origin circulating to the kidney via the caudal vein do not appear responsible for the natriuresis that ensues upon the transfer of toadfish to 10% seawater. In the absence of glomeruli, this tubular natriuresis may be regulated by natriuretic peptides present in the kidney.

Determining Ovulation Frequency in Individually Penned Lactating Sows using a Faecal ‘Progestin’ Assay

ContentsTrials were performed to evaluate the validity of a modified faecal ‘progestin’ assay for monitoring ovarian activity (Trial 1) and to determine the occurrence of ovulation among individually housed lactating sows by repeated measurements of faecal ‘progestins’ (Trial 2). In trial 1 faecal and plasma samples were collected from six multiparous sows, from the day after weaning until oestrus occurred in the third oestrous cycle. A progesterone radioimmunoassay was applied to skimmed milk extracts of the faecal samples, and plasma progesterone concentrations were determined with a chemiluminescent assay. In addition, high performance liquid chromatography (HPLC) analysis was performed on faecal extracts and the immunoreactivity in the different fractions was determined. In Trial 2, faecal samples were collected from 88 multiparous sows at weekly intervals from the day of farrowing until 2 weeks after breeding. Samples were subjected to ‘progestin’ analysis. Trial 1 showed that the excretion patterns of faecal ‘progestins’ and plasma progesterone were significantly correlated (r = 0.79, p &lt; 0.05) during the oestrous cycles. Parallelism was obtained in the assay, but recovery was low (11.4%). The HPLC analysis revealed one major peak of immunoreactivity in a fraction, which corresponded with progesterone. This finding suggests that intact progesterone was a source for immunoreactivity in the assay. In Trial 2, a 10‐fold decrease in mean ‘progestin’ concentrations was noted from the day of farrowing to day 7 of lactation. The mean ‘progestin’ values remained low throughout lactation and started to increase 2 weeks after breeding. Large individual variations in luteal phase values were noted. Only 1.1% (one) of the sows ovulated during lactation.

Synthesis and Properties of an EGF-like Domain (residues 361-406) in the Extreme N-terminal Region of the Mouse EGF Precursor

Various proteins contain EGF-like domains that are not ligands for the EGF receptor. In the present study a cognate polypeptide for residues 361-406 of the mouse EGF precursor was synthesized by the solid-phase method. The product was renatured under oxidative conditions since it probably has an EGF-like array of three cystine disulfide bonds in its native state. HPLC analysis of the renaturation reaction revealed formation of a peak material with no apparent free-SH groups. Accordingly, the HPLC retention time of this product was readily increased by treatment(reduction of disulfides) with dithiothreitol. The renatured 46-mer (PEGF-1) did not displace 125 I-EGF bound to rat liver membranes and 125I-PEGF-l did not exhibit specific binding to membrane preparations from the mouse liver, mammary gland, or kidney, with or without Ca2+ in the binding medium. Although PEGF-1 contains a putative Ca2+ binding motif, specific binding of this cation by the polypeptide could not be demonstrated by electromobility shiff or incubation with 45Ca2+. Immunoassay of PEGF-1 and EGF in fractions obtained following gel filtration of mouse urine revealed multiple peaks of PEGF-1 immunoreactivity with the major peaks eluting at an Mr > 30 kDa. In contrast, virtually all the EGF immunoreactivity eluted at a volume similar to that of 125I-EGF. These data suggest that selective cleavage of the PEGF-1 domain from the precursor does not occur with the proclivity known for that of EGF. Instead, the PEGF-1 probably functions coordinately with other EGF-like domains while tethered to the precursor backbone. Finally, localization of PEGF-1 immunoreactivity occurred only in cell populations of the mouse previously demonstrated as sites for EGF/EGF precursor, which suggests that PEGF-1 is exclusively a domain of the EGF precursor.

Differential distribution of gonadotropin-releasing hormone variants in the brain of Hydrochaeris hydrochaeris (Mammalia, Rodentia).

1. In a previous paper we reported evidence for the presence of mGnRH- and sGnRH-like peptides in the preoptic-hypothalamic region of the capybara Hydrochaeris hydrochaeris (Montaner et al., 1998). In that study, the presence of a cGnRH-II like molecule in olfactory bulb extracts was suggested. 2. The capybara, the largest living rodent in the world, belongs to the order Hystricomorpha, which is considered to be one of the oldest groups of rodents. Some authors consider that this group is the ancestor of all remaining rodents. 3. In this study we have characterized GnRH molecular variants found in extracts from the olfactory bulbs and the mesencephalic region of capybara. These regions represent the two GnRH neuronal systems: the terminal nerve-septopreoptic and the midbrain systems. 4. An indirect method combining reverse-phase high-performance liquid chromatography (RP-HPLC) and radioimmunoassay (RIA) was used to characterize GnRH variants. The analysis of both extracts with two different RIA systems revealed three immunoreactive GnRH peaks, coeluting with mGnRH, cIIGnRH, and sGnRH synthetic standards. These results were additionally supported by serial dilution studies with specific antisera. 5. To our knowledge this the first report on the presence of three GnRH variants in the brain of an eutherian mammal. These results suggest that, similarly to other vertebrates, the expression of multiple GnRH variants may also be a common pattern in mammals.

Elevated plasma immunoreactive neuropeptide Y concentrations and its increased urinary excretion in patients with advanced diabetic nephropathy.

Neuropeptide Y (NPY) is a potent vasoconstrictor peptide that is abundant in the brain and the peripheral sympathetic nervous system. In the present study we investigated possible changes in plasma immunoreactive (IR)-NPY concentrations and urinary IR-NPY excretion in patients with non-insulin dependent diabetes mellitus (NIDDM) and the relationship to diabetic complications, such as nephropathy and neuropathy. IR-NPY in plasma and urine was measured by radioimmunoassay in 69 patients with NIDDM. Plasma IR-NPY concentrations in patients with advanced nephropathy (creatinine clearance <30 ml/min) (100.5 +/- 10.3 pmol/l, n=9, mean +/- SEM) were higher than in the control subjects (55.0 +/- 6.8 pmol/l, n=15) (P<0.02). Urinary excretion of IR-NPY and fractional excretion of NPY were also increased in the patients with advanced nephropathy. Sephadex G-50 column chromatography of the urine extracts obtained from healthy subjects, diabetic patients with renal failure and non-diabetic patients with renal failure showed an immunoreactive peak eluting in the NPY position. On the other hand, neither plasma nor urinary IR-NPY was high in patients with retinopathy, or in patients with peripheral neuropathy. The present study has, for the first time, shown high plasma IR-NPY concentrations and urinary IR-NPY excretion in NIDDM patients with advanced nephropathy.

Fecal Glucocorticoids Document Stress in Female Barbary Macaques (Macaca sylvanus)

Patterns of received aggression and the endocrine response were related to an increase in fecal glucocorticoid metabolites in an intact semifree-ranging group of Barbary macaque females in order to quantify the social stress incurred over a 20-week observation period. The novel result showed that immunoreactive cortisol and 11-oxoetiocholanolone found in the feces can indeed determine the endocrine response of the adrenal gland after a social stressor. After HPLC separation of pooled fecal samples, EIA analyses using three different assays (corticosterone, cortisol, and 11-oxoetiocholanolone) to quantify immunoreactive steroids showed that the corticosterone EIA had no distinctive immunoreactive peaks. Cortisol and 11-oxoetiocholanolone immunoassays showed respectively four and two immunoreactive substances. Time series analyses revealed a behaviorally initiated increase in concentrations of cortisol and 11-oxoetiocholanolone equivalents. Furthermore, both hormone curves exhibit comparable time functions. Either antibody is very suitable for determining glucocorticoid secretion after periods of stress.

Human thyrotropin-releasing hormone-associated peptide 3 (hTAP-3) in serum.

Human thyrotropin-releasing hormone (TRH)-associated peptide 3 (hTAP-3), one of the cryptic peptides resulting from the proteolytic processing of preproTRH to produce TRH, was measured in human plasma from normal, hyperthyroid, and hypothyroid subjects. The dilution curve of hTAP-3 immunoreactivity in the serum paralleled the standard curve of the radioimmunoassay. HPLC analysis revealed a single strong immunoreactive peak, which corresponded to the authentic peptide, hTAP-3. The half-life of hTAP-3 in serum was approximately 3.5 min, and the addition of aprotinin and EDTA completely prevented its degradation. In hyperthyroid patients, plasma concentrations of hTAP-3 were significantly higher than those in the control group and hypothyroid patients, but no correlation was found between its level and that of thyroid hormone. These findings indicate the existence of intact hTAP-3 in the human serum and increases in plasma hTAP-3 levels in hyperthyroid patients, suggesting that blood hTAP-3 may be derived from the peripheral organs rather than the hypothalamus.