The objective of this study was to isolate and purify prorelaxin or mature relaxin from the tammar wallaby corpus luteum (CL), determine their structure and bioactivity, and test the hypothesis that enzymatic cleavage of prorelaxin occurs in late gestation. Tammar relaxin peptides were extracted from pooled corpora lutea of late pregnant tammars using a combination of HPLC methods, and they were identified using Western blotting with a human (H2) relaxin antisera and matrix-assisted laser desorption ionization time of flight mass spectrometry. Although no prorelaxin was identified, multiple 6-kDa peptides were detected, which corresponded to the predicted mature tammar relaxin amino acid sequence, with an A chain of 24 amino acids, and different B chain lengths of 28, 29, 30, and 32 amino acids. Tammar relaxin bound with high affinity to rat cortical relaxin receptors and stimulated cAMP production in the human monocytic cell line, THP-1, which expresses the relaxin receptor. Analysis of individual CL indicated that equivalent amounts of mature relaxin peptides were present throughout gestation and also in unmated tammars at equivalent stages of the luteal phase in the nonpregnant cycle. Immunoreactive relaxin was localized specifically to the luteal cells of the CL and the intensity of immunostaining did not vary between gestational stages. These data show that the CL of both pregnant and unmated tammar wallabies produces mature relaxin and suggests that relaxin expression in this species is not influenced by the conceptus. Moreover, the presence of mature relaxin throughout gestation implies that prohormone cleavage is not limited to the later stages of pregnancy
Kidney failure is a major health problem worldwide. Patients with end-stage renal disease require intensive medical support by dialysis or kidney transplantation. Current methods for diagnosis of kidney disease are either invasive or insensitive, and renal function may decline by as much as 50% before it can be detected using current techniques. The goal of this study was, therefore, to identify biomarkers of kidney disease (associated with renal fibrosis) that can be used for the development of a non-invasive clinical test for early disease detection. We utilized two protein-profiling technologies (SELDI-TOF MS and 2-D) to screen the plasma and kidney proteome for aberrantly expressed proteins in an experimental mouse model of unilateral uretric obstruction, which mimics the pathology of human renal disease. Several differentially regulated proteins were detected at the plasma level of day-3-obstructed animals, which included serum amyloid A1, fibrinogen α, haptoglobin precursor protein, haptoglobin and major urinary proteins 11 and 8. Differentially expressed proteins detected at the tissue level included ras-like activator protein 2, haptoglobin precursor protein, malate dehydrogenase, α enolase and murine urinary protein (all p<0.05 versus controls). Immunohistochemistry was used to confirm the up-regulation of fibrinogen. Interestingly, these proteins are largely separated into four major classes: (i) acute-phase reactants (ii) cell-signaling molecules (iii) molecules involved in cell growth and metabolism and (iv) urinary proteins. These results provide new insights into the pathology of obstructive nephropathy and may facilitate the development of specific assay(s) to detect and monitor renal fibrosis.
Abstract: The primary structure of ovine Leydig cell insulin‐like peptide (Ley I‐L) was recently deduced from the corresponding cDNA sequence. It consists of two peptide chains and three disulphide bonds in an arrangement similar to both relaxin and insulin. As in relaxin B‐chain, an Arg‐X‐X‐X‐Arg sequence exists within the Ley I‐L B‐chain although it is located four residues towards the C‐terminus from the corresponding position within relaxin. This sequence of amino acids is known to be essential for relaxin biological activity and its presence in Ley I‐L suggested that the peptide might possess a relaxin‐like function. Ovine Ley I‐L was assembled by Fmoc‐solid‐phase synthesis of the separate chains followed by their combination in solution at high pH. The purity and identity of the chain‐combined peptide was confirmed by chemical characterization including mass spectrometry. At physiological concentrations, the peptide was shown not to possess relaxin‐like activity in the rat isolated atrial chronotropic and inotropic assay. This strongly suggests that Ley I‐L is not a relaxin in the sheep. In order to explore further a possible structural relationship between Ley I‐L and relaxin, we prepared a synthetic analogue of ovine Ley I‐L containing a single replacement of B‐chain residue 12, His, with Arg. This was found to possess significant relaxin‐like chronotropic and inotropic activity demonstrating that the tertiary structure of Ley I‐L is similar to that of relaxin and highlighting the key requirement for the five‐residue sequence, Arg‐X‐X‐X‐Arg, to be present in position B12–16 for characteristic relaxin activity.
Abstract: The Fmoc solid phase synthesis of Aβ(1–40), a strongly aggregating peptide found in Alzheimer’s disease brain, was performed using 2‐hydroxy‐4‐methoxybenzyl (Hmb) backbone amide protection. Hmb‐Gly residues were incorporated using N α ‐Fmoc‐Hmb‐Gly‐OH rather than N,O ‐ bis Fmoc‐Hmb‐Gly‐OPfp. Amino acid acylation of the sterically hindered Hmb‐amino acids was monitored using ‘semi‐on‐line’ MALDI‐TOF‐MS in a novel application of this technique which significantly simplified the successful incorporation of these residues. Standard coupling conditions in N,N‐ dimethylformamide (DMF) were used throughout the synthesis. Comparative structural studies of acetyl‐Hmb‐protected and native Aβ(1–40) were performed to investigate the structural basis of Hmb‐mediated disaggregation. The incorporation of backbone amide protection was observed by circular dichroism spectroscopy and gel electrophoresis to strongly affect the solution structure of Aβ(1–40). Despite the reported structure‐breaking activity of Hmb groups, penta(acetyl‐Hmb)Aβ(1–40) was found to adopt both α‐helix and intermolecular β‐sheet conformations. In 100% TFE a mixed α‐helix/random coil structure was formed by the protected peptide indicating reduced α‐helical propensity relative to Aβ(1–40). The protected peptide formed β‐sheet structures in aqueous buffer. Gel electrophoresis indicated that, unlike native Aβ(1–40), penta(acetyl‐Hmb)Aβ(1–40) did not form large aggregate species.
SUMMARY 1. The role of the kidneys in the maintenance of normal foetal plasma (FP) composition and hormone concentrations was examined in the present study. Five ovine foetuses were chronically cannulated and nephrectomized (nephx) at 100±1 days of gestation and maintained for 14 days. These were compared to five intact control foetuses. 2. Four hours after nephx, FP renin concentrations were significantly lower than in control foetuses. By 48 h, renin concentrations in nephx foetuses were below the level of detectability of the assay. Foetal plasma aldosterone concentrations declined in nephx foetuses, but were not significantly different to those in control foetuses ( P = 0.08). 3. During the second week, the nephx foetuses were significantly hypoxic, but FP erythropoietin concentrations were not increased. Adrenocorticotropic hormone (ACTH) and cortisol concentrations, when measured on day 14, were not different between the two groups. Adrenocorticotropic hormone levels were correlated with adrenal weight at post‐mortem. 4. Foetal plasma creatinine, magnesium and phosphate concentrations in nephx foetuses increased, eventually reaching values approximately twice that in controls. Foetal plasma chloride levels decreased continuously in nephx foetuses, eventually being 23 mmol/L lower than controls. Maternal plasma composition was unchanged. 5. Total foetal fluid (amniotic + allantoic) volumes were reduced when measured at post‐mortem on day 14 after nephx. The composition of both fluids was significantly altered in the nephx foetuses compared with controls. 6. Fetuses can survive in utero for 2 weeks after bilateral nephrectomy. However, there are multiple changes in plasma composition that may compromise foetal survival in the long term.
'/%3e%3c/defs%3e%3cpath fill='%23012169' d='M0 0h10080v5040H0z'/%3e%3cpath stroke='%23fff' stroke-width='.6' d='m0 0 6 3m0-3L0 3' clip-path='url(%23b)' transform='scale(840)'/%3e%3cpath stroke='%23e4002b' stroke-width='.4' d='m0 0 6 3m0-3L0 3' clip-path='url(%23c)' transform='scale(840)'/%3e%3cpath stroke='%23fff' stroke-width='840' d='M2520 0v2520M0 1260h5040'/%3e%3cpath stroke='%23e4002b' stroke-width='504' d='M2520 0v2520M0 1260h5040'/%3e%3cg fill='%23fff'%3e%3cuse xlink:href='%23d' x='2520' y='3780'/%3e%3cuse xlink:href='%23a' x='7560' y='4200'/%3e%3cuse xlink:href='%23a' x='6300' y='2205'/%3e%3cuse xlink:href='%23a' x='7560' y='840'/%3e%3cuse xlink:href='%23a' x='8680' y='1869'/%3e%3cuse xlink:href='%23e' x='8064' y='2730'/%3e%3c/g%3e%3c/svg%3e)
