Biomedical subjects
G W Tregear
Publications and source records attributed to G W Tregear.
Distribution of Fos immunoreactivity in the lamina terminalis and hypothalamus induced by centrally administered relaxin in conscious rats.
The effect of intracerebroventricular (ICV) injections of synthetic human or rat relaxin (25 or 250 ng) on the distribution of Fos detected immunohistochemically in the rat forebrain was investigated. Following ICV relaxin, many Fos-positive neurons were observed in the periphery of the subfornical organ, dorsal part of the organum vasculosum of the lamina terminalis, throughout the median preoptic nucleus, supraoptic nucleus and hypothalamic paraventricular nucleus. Such effects did not occur following ICV injection of artificial cerebrospinal fluid or the separated A and B chains of relaxin, nor following the intravenous injection of 250 ng of relaxin. Both vasopressin and oxytocin containing neurons identified immunohistochemically in the supraoptic and paraventricular nuclei exhibited Fos following ICV relaxin, and many neurons in the medial parvocellular part of the paraventricular nucleus contained Fos. The results indicate that centrally administered relaxin may increase neuronal activity in regions of the hypothalamus and lamina terminalis which are associated with cardiovascular and body fluid regulation and oxytocin secretion.
Expression of human relaxin genes: characterization of a novel alternatively-spliced human relaxin mRNA species.
Relaxin is a two-chain peptide hormone encoded by two non-allelic genes in humans and great apes, and by a single gene in all other species studied. We have characterized the expression of the human relaxin genes (H1 and H2) in placenta, decidua, prostate and ovary by reverse-transcription/polymerase chain reaction (RT/PCR). H2 relaxin mRNA was detected in the ovary, term placenta, decidua, and prostate gland. In contrast, H1 gene expression was detected only in the prostate gland. In addition to the relaxin PCR product of the predicted size (486 bp), a larger relaxin-specific product (587 bp) was detected in both H1 and H2 amplifications and in amplifications of chimpanzee relaxin from placenta and corpus luteum. Sequencing of human and chimpanzee PCR products, and human relaxin genomic clones, revealed that the larger product arises from an alternatively-spliced relaxin mRNA species incorporating an extra exon. This is the first evidence that the structure of the human and chimpanzee relaxin genes differ from that of other characterized relaxin genes, such as pig and rat. The novel peptide arising from this alternate message would be identical to prorelaxin in the B-chain and part of the C-peptide (extending to the position of the intron) but would differ from prorelaxin in the carboxy-terminal domain. Observation of a similar mRNA species in the chimpanzee suggests that this conserved relaxin-like peptide may have a significant biological role.
Purification and characterization of porcine prorelaxin.
Relaxin is a two-chain 6-kDa peptide hormone. It is a member of the insulin family of peptides and is produced mainly during pregnancy to prepare the reproductive tract for birth. In the pig, relaxin is produced mainly by ovarian luteal cells. It is processed via the regulated pathway from a larger (18 kDa) precursor, prorelaxin. Protocols have been described for the purification of mature relaxin from the ovaries of pregnant gilts. Multiple forms of relaxin have been detected during isolation due to exopeptidase trimming of the peptide chains. To date, such trimming events have prevented purification of the larger relaxin precursor. Described here is a method for the isolation of milligram amounts of homogeneous and bioactive prorelaxin from porcine ovaries.
Synthesis and characterization of human gene 1 relaxin peptides.
The peptide encoded for by one of the two relaxin genes found in the human genome, designated H1, has been synthesized by the Boc-polystyrene solid phase method. The two chains which constitute relaxin, A- and B-, were assembled separately and, after cleavage, deprotection and purification, combined in solution at high pH to form the one intra- and two intermolecular disulfide bonds. Comprehensive chemical characterization including ion spray mass spectrometry of the peptide confirmed both its correct identity and high purity. The synthetic H1 relaxin was analyzed by circular dichroism spectroscopy and shown to possess a greater alpha-helical conformation in water than the corresponding H2 relaxin. The peptide had powerful direct chronotropic and inotropic effects in the isolated rat heart assay as did an analogue of the peptide in which the C-terminus of the B-chain was extended by four residues.
Expression of the relaxin gene in rat tissues.
The peptide hormone relaxin is produced at high levels in the corpus luteum of the rat ovary during pregnancy. The biological effects of the hormone include remodelling of collagen in target tissues such as the cervix, and inhibition of uterine contractility. In addition, many paracrine actions for the hormone have been proposed, however sites of relaxin production outside the ovary have not been well characterized. We have investigated relaxin gene expression in a range of rat tissues using the techniques of reverse transcription/polymerase chain reaction (RT/PCR), RNase protection and immunohistochemistry. Relaxin mRNA was detected by RT/PCR in brain, uterus, prostate gland, pancreas and kidney, with other tissues giving weak signals. Relaxin gene expression in brain was detected by RNase protection, and relaxin-like immunoreactivity was observed in the arcuate nucleus of the hypothalamus of rat brain. This characterization of sites of relaxin gene expression provides further evidence for proposed paracrine actions of relaxin which may be important in non-pregnant and male rats in addition to the pregnant female.
Characterization of human relaxin gene regulation in the relaxin-expressing human prostate adenocarcinoma cell line LNCaP.FGC.
Relaxin is a peptide hormone which is produced in human reproductive tissues including the ovary and prostate gland. Little is known of the molecular events regulating relaxin gene transcription. We have studied this question using gene transfer of relaxin promoter/reporter gene constructs into a relaxin-expressing cell line. A number of human cells lines expressed relaxin as detected by reverse transcription-PCR. In one of these lines, the prostate adenocarcinoma cell line LNCaP.FGC, relaxin mRNA was also detected by Northern blot analysis. The DNA sequences of the proximal 5'-flanking regions (approximately 900 nucleotides) of the two human relaxin genes, H1 and H2, were determined. Deletion constructs containing portions of the 5'-flanking regions of H1 and H2 linked to the bacterial chloramphenicol acetyl transferase reporter gene were prepared. The expression of the reporter gene constructs was analysed in the LNCaP.FGC cell line and the results of these transient transfection assays have led to the identification of positive and negative regulatory regions within the 5'-flanking DNA. A difference in activity of the H1 and H2 gene promoters in this prostate cell line was observed, with the H2 promoter being more active. This situation may mimic that occurring in vivo since the relaxin secreted from the prostate gland into seminal fluid is the product of the H2 gene.
Primate relaxin: synthesis of gorilla and rhesus monkey relaxins.
The synthesis of the hormone relaxin from the species Gorilla gorilla (gorilla) and Macaca mulatta (rhesus monkey) has been achieved. Each of the two chains which constitute the peptide structures was assembled separately, the A-chains (24 amino acids) by the Boc-polystyrene solid-phase procedure and the B-chains (29 and 28 amino acids) by the Fmoc-polyamide (gorilla) and the Boc-polystyrene (rhesus monkey) solid-phase methods. After cleavage from the solid supports, the separate chains were purified to a high degree of homogeneity. Oxidative combination of the respective A- and B-chains in solution at high pH afforded the synthetic relaxins in low overall yield. Chemical and physiochemical characterization of the products confirmed both their purity and their conformational similarity to the human hormone. The synthetic gorilla and rhesus monkey relaxins were both found to possess potent chronotropic and inotropic activity in the isolated rat cardiac atrium assay.
Characterization of two relaxin genes in the chimpanzee.
Relaxin is a peptide hormone which has a variety of physiological effects on tissues of the reproductive tract as well as other organs such as the heart and brain. Whereas all non-primates so far examined have only a single relaxin gene, humans have two genes (H1 and H2, or gene 1 and gene 2). H2 relaxin is synthesized in the corpus luteum during pregnancy and is also found in the placenta and prostate, whereas expression of H1 has been very difficult to detect. We have begun a study of relaxin genes in the chimpanzee to assess whether this species may provide a suitable model in which to examine the roles of gene 1 relaxin. We find that the chimpanzee has two relaxin genes, one of which is very similar to H2. The second gene has an gene 1 type A chain but the B chain is of the gene 2 type, possibly due to a gene conversion event. The authentic chimpanzee gene 2 (Ch2) is expressed in the corpus luteum of pregnancy and in the placenta. Ch1 is not expressed in the placenta, but the mRNA can be detected by polymerase chain reaction in the corpus luteum.
A single-copy relaxin-like gene sequence is present in sheep.
Relaxin is a peptide hormone which consists of two polypeptide chains that are synthesized as a B-chain/C-peptide/A-chain precursor. We have used the polymerase chain reaction (PCR) to isolate and clone a relaxin-like cDNA from sheep placental RNA. This cDNA and two sheep genomic clones were characterised by nucleotide sequencing. A comparison of the sheep nucleotide sequence with exon II of pig relaxin revealed homology of 72%. The sheep sequence had numerous stop codons in the region corresponding to the C-peptide. Therefore, there is no open reading frame which would include the C-peptide and A-chain regions. Analysis of several animals indicates that the stop codons are not due to an allelic polymorphism and Southern blot analysis of genomic DNA reveals the presence of a single copy gene. The 5' RACE PCR protocol was used to obtain sequence information for the 5' relaxin-like RNA. This analysis reveals that unprocessed precursor RNA is the predominant RNA species in placenta. A small proportion of clones was isolated which contained novel 5' sequences. These sequences mostly appear to be generated from repetitive DNA elements upstream of exon II. No relaxin-like exon I sequence which encodes the B-chain was found after an extensive search of the 5' RACE PCR products. Therefore, this relaxin-like gene does not produce an RNA species in ovary, placenta or endometrial tissue which could give rise to a functional sheep relaxin hormone.
The mouse relaxin gene: nucleotide sequence and expression.
Relaxin is a polypeptide hormone that has a variety of physiological effects both on remodelling of collagen and on uterine contractility. These are most apparent during pregnancy. The sequences of relaxin cDNAs derived from ovaries of late-pregnant random-bred Swiss mice have been established. Multiple subclones obtained from three independent polymerase chain reaction experiments were found to encode relaxins which were identical except at position 11 in the A chain (Ile or Val). All mouse relaxin cDNAs expressed in the ovary during pregnancy had an extra tyrosine inserted prior to the final A chain cysteine residue, a result confirmed by direct sequencing of relaxin peptides. Whilst this tyrosine insertion must have local effects on the folding of the A chain, structure-activity studies will clarify whether it perturbs functional interaction with the relaxin receptor. We have shown that there is a single relaxin gene in the mouse genome, and that expression during pregnancy occurs in the ovary but is not detectable in the placenta, uterus or fetus.
Characterization of a major neutralization domain of Ross river virus using anti-viral and anti-peptide antibodies.
The E2 glycoprotein of the alphavirus Ross River virus (RRV) contains three defined neutralization epitopes (a, b1 and b2) with determinants located between amino acids 216 and 251 in the linear sequence (Vrati et al., 1988, Virology 162, 346-353). The antigenic structure of this region has been examined using hyperimmune mouse antiserum against RRV and antiserum against four synthetic peptides representing linear amino acid sequences in the neutralization region of E2. In plaque reduction neutralization tests using hyperimmune antiserum to RRV, an RRV mutant altered at all three neutralization epitopes was markedly more resistant than the parental virus; variants altered at single epitopes could not be distinguished in these tests. Sera from mice immunized with synthetic RRV E2 peptides conjugated to keyhole limpet haemocyanin reacted, in a direct ELISA, with the specific region of RRV represented by the peptide. The same sera did not neutralize or immunoprecipitate RRV in solution or bind to RRV in a capture ELISA. The RRV peptides did not prime mice to react to a subimmunogenic dose of RRV; they did not bind monoclonal or polyclonal antibodies to RRV. We conclude that a significant proportion of the neutralizing antibody response in mice is elicited by epitopes a, b1, and b2 of RRV E2 and that the sites to which neutralizing antibodies bind are formed by complex folding.
Electron microscopic evidence for the association of M2 protein with the influenza virion.
Immunogold electron microscopy revealed that site-specific antibodies elicited by a synthetic peptide representing the N-terminal sequence (residues 2-10) of influenza virus M2 protein were capable of binding to the surface of virions. Antibody binding was observed with two human influenza virus strains but not with an avian virus strain which has amino acid substitutions in the appropriate sequence of M2. These results provide direct evidence for the presence of M2 in the influenza virion.
The synthesis of polyamide-oligonucleotide conjugate molecules.
We have developed methods for the synthesis of peptide-oligodeoxyribonucleotide conjugate molecules in particular, and polyamide-oligonucleotide conjugates in general. Synthesis is carried out by a solid-phase procedure and involves the assembly of a polyamide on the solid support, conversion of the terminal amino group to a protected primary aliphatic hydroxy group by reaction with alpha, omega-hydroxycarboxylic acid derivatives, and finally oligonucleotide synthesis using phosphoramidite chemistry. The conjugate molecules can be used as DNA probes, with the polyamide component carrying one or more non-radioactive markers. These conjugates also have the potential to be used as anti-sense inhibitors of gene expression, with the peptide segment acting as a targeting moiety.
The preparation of polyamide-oligonucleotide probes containing multiple non-radioactive labels.
Oligonucleotide probes containing multiple non-radioactive labels have been prepared by utilising and extending the methods used to prepare polyamide-oligonucleotide conjugates. The probes were prepared by incorporating suitable amino acid residues, such as lysines, in the polyamide, which were then used as sites for the attachment of the non-radioactive labels. The procedures developed give control over the distance of the label from the oligonucleotide, and also the inter-label distance. The labels can be conveniently introduced while the substrate is still on the solid support. Even though fluorescent oligonucleotide probes prepared in this way carrying multiple carboxyfluorescein labels gave low levels of fluorescence due to quenching, the probes containing ten biotin labels gave a detection sensitivity of approximately 5 attomole (3 million molecules).
Relaxin in sera during the luteal phase of in-vitro fertilization cycles.
To identify the time when relaxin can first be detected in peripheral sera after in-vitro fertilization (IVF) and embryo transfer, blood samples were collected from 20 women up to 14 days after oocyte retrieval. Sixteen women did not become pregnant and in eight of them relaxin (but not beta-human chorionic gonadotrophin, beta-hCG) was measurable for the first time at days 6 to 12. Concentrations of other hormones measured were also different in these eight women compared with the remaining eight non-pregnant women; their serum concentrations of 17 alpha-OH progesterone, progesterone and oestradiol were higher but concentrations of luteinizing hormone and follicle-stimulating hormone were lower. Three women became pregnant; relaxin and beta-hCG were first detected on the same day (10 to 12). The remaining woman had increased beta-hCG levels but did not develop a clinical pregnancy. Measurement of serum relaxin during IVF cycles may allow assessment of corpora luteal function before its identification by levels of steroid hormones.
Relaxin in paired samples of serum and milk from women after term and preterm delivery.
In a study to determine if relaxin could be measured in milk and if so to correlate concentrations in milk and serum, paired samples of milk and serum were collected from 12 women 3 days after term delivery (term group), from 16 women 3 days after preterm delivery (preterm group), and from some of these patients 6 weeks after delivery (eight term and six preterm). Relaxin was measured by specific human relaxin radioimmunoassay. Relaxin from milk and sera behaved similarly in the relaxin radioimmunoassay and reverse-phase high-performance liquid chromatography. Concentrations of relaxin in sera and milk collected 3 days after delivery did not differ significantly within the term or preterm groups. Neither were there differences in relaxin levels in sera and milk between the term and preterm groups. At 6 weeks postpartum, relaxin was not measured in any sera but was measured in milk from six of eight patients in the term group and five of six patients in the preterm group. Relaxin concentrations in milk were higher in the preterm group. The presence of relaxin in milk at 6 weeks postpartum suggests a nonluteal site of synthesis.
Relaxin levels in antenatal patients following in vitro fertilization.
The aim of this study was to investigate the relationship between serum relaxin and pregnancy outcome in a group of patients pregnant after in vitro fertilization (IVF). Patients who delivered a single live infant at term after IVF had mean serum levels of relaxin more than double the mean levels in normal antenatal patients. The high relaxin levels were compatible with delivery at term. However, because of the proposed role of relaxin in the process of cervical ripening, the high serum levels may help explain the high rate of preterm labor observed among IVF patients.