A randomized, double-blind, single-dose study to assess bioequivalence of MB02 biosimilar after manufacturing iteration and reference bevacizumab.

To investigate and compare the pharmacokinetic (PK) profiles of MB02 products, before and after optimizing the manufacturing process, and reference bevacizumab to establish bioequivalence between them. In this randomized, double-blind, single dose, parallel study, 114 healthy male volunteers were randomized 1:1:1 to receive a 1 mg/kg intravenous dose of MB02-SP, MB02-DM, or US-bevacizumab. The follow-up period was 100 days. PK similarity between them was determined using the standard bioequivalence criteria (0.80-1.25) for the area under the serum concentration-time curve from time 0 extrapolated to infinity and the maximum observed serum concentration. Study results showed that the PK profiles of bevacizumab were similar. Statistical analysis demonstrated that for each pairwise comparison there were no differences. The 90% CIs for the ratios of geometric least squares means were fully contained within the predefined similarity acceptance limits and ranged from 0.899 to 1.12 for area under the curve and from 0.887 to 1.11 for maximum concentration. A total of 159 adverse events were reported by 76 subjects who received the study drug. The majority (90.6%) of the reported adverse events were grade 1 in severity, with 9.4% as grade 2 in severity. None were considered as grade 3, 4, or 5. Treatment-induced anti-drug antibodies incidence was 21.6%, 33.3%, and 23.7% for the treatment of MB02-SP, MB02-DM, and US-bevacizumab, respectively. No subjects showed treatment-induced neutralizing anti-drug antibodies. This study demonstrates the PK, safety, and immunogenicity similarity and bioequivalence of MB02-SP, MB02-DM, and the reference product bevacizumab.

The role of RXFP2 in mediating androgen-induced inguinoscrotal testis descent in LH receptor knockout mice.

LH receptor knockout (LhrKO) male mice exhibit a bilateral cryptorchidism resulting from a developmental defect in the gubernaculum during the inguinoscrotal phase of testis descent, which is corrected by testosterone replacement therapy (TRT). In vivo and in vitro experiments were conducted to investigate the roles of the androgen receptor (AR) and RXFP2 signals in regulation of gubernacular development in LhrKO animals. This study demonstrated that AR and RXFP2 proteins were expressed in the gubernaculum during the entire postnatal period. TRT normalized gubernacular RXFP2 protein levels inLhrKO mice. Organ and primary cell cultures of gubernacula showed that 5alpha-dihydrotestosterone (DHT) upregulated the expression of Rxfp2 which was abolished by the addition of an AR antagonist, flutamide. A single s.c. testosterone injection also led to a significant increase in Rxfp2 mRNA levels in a time-dependent fashion in LhrKO animals. DHT, natural and synthetic insulin-like peptide 3 (INSL3), or relaxin alone did not affect proliferation of gubernacular mesenchymal cells, while co-treatments of DHT with either INSL3 or relaxin resulted in an increase in cell proliferation, and they also enhanced the mesenchymal cell differentiation toward the myogenic pathway, which included a decrease in a mesenchymal cell marker, CD44 and the expression of troponin. These effects were attenuated by the addition of flutamide, siRNA-mediated Rxfp2 knockdown, or by an INSL3 antagonist. Co-administration of an INSL3 antagonist curtailed TRT-induced inguinoscrotal testis descent in LhrKO mice. Our findings indicate that the RXFP2 signaling pathway plays an important role in mediating androgen action to stimulate gubernaculum development during inguinoscrotal testis descent.

HIV/AIDS prevalence among South African health workers.

INTRODUCTION: Studies on HIV prevalence among health workers usually focus on occupational exposure to HIV. Little is known about HIV prevalence in this group. However, it is expected that HIV prevalence among health workers will reflect prevalence in their society. OBJECTIVE: To determine HIV prevalence among South African health workers. METHOD: A stratified cluster sample was drawn of 5% of health facilities in South Africa (N = 222) representative of the public and private health sectors in South Africa. The sample was designed to obtain a nationwide representative sample of medical professionals and non-professional health workers. A subsample comprising health workers in four provinces was tested for HIV status. The Orasure HIV-1 device in combination with the Vironostika HIV UNI-Form II plus O enzyme-linked immunosorbent assay (ELISA) kits were used to collect oral fluid specimens for HIV testing. RESULTS: Based on a sample of 721 health workers and a response rate of 82.5% (or 595 respondents), the study found that an estimated 15.7% (95% confidence interval (CI): 12.2-19.9%) of health workers employed in the public and private health facilities located in four South African provinces, were living with HIV/AIDS in 2002. Among younger health workers, the risk is much higher. This group (aged 18-35 years) had an estimated HIV prevalence of 20% (95% CI: 14.1-27.6%). Non-professionals had an HIV prevalence of 20.3%, while professionals had a prevalence of 13.7%. CONCLUSION: HIV prevalence among health workers in South Africa is high; this calls for the introduction of antiretroviral programmes targeting them. In addition, there is a need for the development of new policy regarding placement of infected health workers in tuberculosis (TB) wards, coupled with vigorous human resource planning to replace the health workers likely to die from AIDS. Infection control procedures also need to be reviewed.

Relaxin-like factor (RLF) serum concentrations and gubernaculum RLF receptor display in relation to pre- and neonatal development of rats.

Deletion of the relaxin-like factor (RLF) gene in mice causes retention of testicles and infertility. The development of a synthetic RLF has made it possible to investigate the events that connect the genomic event and the basic biological responses that cause gonadal positioning. Anti-RLF antibodies were raised against synthetic RLF, allowing determination of RLF concentrations during the critical period, testing for RLF receptors on the gubernaculum and exploration of the temporal relationship between receptor display and migration of the testes in developing rats. In male rat pups, serum RLF concentrations were high at day 2 before parturition (2.4 ng ml(-1)) and decreased sharply just before parturition. Thereafter, males and females had the same low serum concentrations until RLF concen-trations began to increase in males only, starting at day 10 after parturition and continuing until adult RLF concentrations (0.6 ng ml(-1)) were reached on day 39 after parturition. The testicles are descending into the scrotum during this phase of increasing RLF concentrations and are descended fully by day 19-21 after parturition, before adult hormone concentrations are established. The high prenatal serum RLF concentration coincides with high expression of RLF receptors in the gubernaculum tissue. Competitive binding of RLF per mg of membrane protein prepared from rat gubernacula at various developmental stages showed no increase in receptor density as sexual maturity was reached. Gubernaculum cells in primary culture showed an increased uptake of 5-bromo-2'-deoxyuridine in the presence of RLF compared with controls. These studies demonstrate that the synthetic RLF is biologically active and indicate that the cryptorchid phenotype INSL3(-/-) is a direct consequence of defective gubernaculum growth, caused by the absence of RLF during early phases of development.

Synthesis and conformational analysis of the insulin-like 4 gene product.

Insulin-like 4 (INSL-4) is a protein expressed in the early placenta. Its primary structure is insulin-like with reference to the distribution of cysteine residues and the single chain pro-form. Insulin-like 4 was generated by solid-phase peptide synthesis of the two chains followed by the sequential synthesis of the three disulfide bonds. Two disulfide isomers were produced, one with an insulin-like disulfide bonding pattern and the other with a reversed chain orientation. The CD spectra of the two disulfide isomers were indistinguishable without any features produced by periodic structures. In addition, the hydrodynamic properties of the two isomers were identical which implied a very open structure of the disulfide-bonded two-chain molecules. It appears that insulin-likeness cannot be defined solely on the basis of the primary structure of cDNA.

[Omphalocele and gastroschisis. Outcome--complications--follow-up--quality of life]. / Omphalocele und Gastroschisis. Ergebnisse--Komplikationen--Verlauf--Lebensqualität.

From 1970 to 1998, 35 children with omphalocele (OC) and 31 with gastroschisis (GS) were treated at the Department of Paediatric Surgery at Lübeck Medical University. Forty of 43 survivors were examined in 1990, the data of 30 patients were renewed in 1999 and 12 new cases added. Total follow-up was 1-28 years. Primary closure was possible in 25 OCs and 20 GSs. Eighteen children with OC and 8 with GS suffered from additional abnormalities, which were treated simultaneously. Twenty percent of the babies with OC died mostly because of severe congenital anomalies and 12.9% of GS because of infectious complications in combination with other diseases. There were no more deaths in the last decade. Accordingly, there was a reduction in consecutive operations. Improvements were due to better operative and perioperative treatment as well as abortions following improved ultrasound examinations. The results of the literature and our own experience show the benefit of primary closure. A two-stage approach with dura/amnion or a silo procedure prevents high intra-abdominal pressure, therefore, indirect measurements of intra-abdominal pressure can be used exceptionally. Umbilical preservation offers better cosmetic results. Long-term follow-up reveals normal growth and development of the children except for those with severe congenital anomalies. All the others are participating without problems in normal activities and education without reduction in their quality of life. Today an isolated OC or GS is not an indication for abortion. If prenatal OC or GS is diagnosed, paediatric surgeons should be involved in the consultations.

The relaxin receptor-binding site geometry suggests a novel gripping mode of interaction.

Relaxin has a unique, clearly identifiable, mixed function receptor-binding region comprising amino acid residues that evolve sequentially from the central portion of the B chain alpha-helix. Two arginine residues in positions B13 and B17 that project like forefinger and middle finger from the helix provide the electrostatic element opposed by the hydrophobic (thumb) element isoleucine (B20), offset from the arginines by about 40 degrees. The binding intensity of relaxin to its receptor decreases by 3 orders of magnitude if alanine is substituted for the newly discovered binding component isoleucine in position B20. The arginine residues cannot be replaced by other positive charges, nor can the guanidinium group be presented on a longer or shorter hydrocarbon chain. In contrast, the hydrophobic interaction is incremental in nature, and the contribution to the total binding energy is roughly proportional to the number of hydrocarbon units in the side chain. It appears that a hydrophobic surface exists on the receptor that offers optimal van der Waals' interaction with beta-branched hydrophobic amino acids. The binding energy increases roughly 10-fold with each methylene group whereby beta-branching is more effective per surface unit than chain elongation. Aromatic side chains appear to demarcate the extent of the binding region in so far as residues larger than phenylalanine decrease receptor binding. The exceptional clarity of binding site geometry in relaxin makes for an excellent opportunity to design peptido-mimetics.

The relaxin-like factor is a hormone.

The relaxin-like factor (RLF) circulates in the bloodstream of humans, interacts with a membrane protein with all the characteristics of ligand-receptor binding, and must therefore be considered a hormone by definition. The polyclonal antibody raised against synthetic human RLF showed no crossreactivity to other structurally related hormones, like insulin and relaxin. The sensitivity of this assay (ED50 at 100 pM) allowed the direct measurement of RLF concentrations in serum. The highest levels were detected in the serum of postpuberty males (190 pM), whereas in females and children, the RLF concentration was one order of magnitude lower.

Porcine relaxin, a 500 million-year-old hormone? the tunicate Ciona intestinalis has porcine relaxin.

The fossil record of tunicates reaches back to the upper Cambrian period. Ascidians have mobile, tadpole-like juvenile forms with a notochord, which inspired the classification of tunicates as Urochordata, i.e., predecessors of vertebrates. The genome of the tunicate Ciona intestinalis contains a relaxin coding region that is organized like a mammalian gene, i.e., signal peptide, B-chain domain, connecting peptide domain, followed by the A-chain domain with a stop codon after cysteine A-22. RNA-derived cDNA encodes a relaxin that is identical to the circulating form of the porcine hormone. In contrast to the porcine gene, the ascidian gene has no intron in the C-peptide domain, and in that respect is similar to the bombyxin gene of the silkworm. During the spawning period, only enough relaxin could be extracted and isolated from gonads of C. intestinalis for a partial sequence analysis. Remarkable as it may be, these findings suggest that relaxin is identical in pigs, whales, and the tunicate C. intestinalis.

Specific, high affinity relaxin-like factor receptors.

The relaxin-like factor (RLF) is a circulating hormone that binds to specific membrane-bound uterine receptors in the mouse. Mono-iodinated RLF tracers were produced and characterized specifically to study the properties of the RLF receptor. The tracers bound to the RLF receptor in uterine crude membrane preparations with high affinity (73 nM for (125)I-Tyr(A9) RLF and 90 nM for (125)I-Tyr(A26) RLF) as determined by Scatchard analysis. The specificity of binding was confirmed by chemical cross-linking experiments. Binding of (125)I-Tyr(A9) RLF to the putative receptor was inhibited in the presence of a 640-fold excess of unlabeled human RLF but not by the same excess of human relaxin. SDS-gel electrophoresis of the RLF-receptor complex revealed a molecular mass of >200 kDa, which remained unchanged upon reduction. The size and the lack of subunit structure of the receptor is similar to the features reported for the relaxin receptor. In this regard both, the RLF and the relaxin receptor are different from the insulin- and the insulin-like growth factor-type 1 receptors. This observation supports the relaxin-likeness of this new factor not only toward potential target tissues but also as regards receptor features.

[Wish and reality in installation of a clinic-wide system for image and documentation access]. / Wunsch und Wirklichkeit bei der Installation einer abteilungsübergreifenden Bild- und Befunddistribution.

PROBLEM: This report describes the problems that can occur in the representation of the radiological workplace in a digital environment. On one hand the radiologist can sometimes access good equipment in "stand-alone" surroundings (CT, laser printer, workstations,...); on the other hand, the existing insufficient communication between different components is only rarely qualified to support the radiological workflow. This unsatisfactory framework handicaps the required clinic-wide distribution of radiological information. METHODS: From the beginning we defined user groups requiring different radiological data closely associated with specific hard- and software: The radiological workstation in the department for reporting and image processing. The demonstration workstation in wards/outpatient departments for clinicians involved in treatment. Standard PCs with access to the digital medical document for clinicians involved in treatment. At all workstations the medical as well as the legal unity of digital radiological images and the corresponding report is ensured. RESULTS: Only the first two user groups have unrestricted access to the RIS database and to the PACS archive. We have decided that the RIS should be the master of the RIS/PACS-System. For an effective master/slave relationship between RIS and PACS archive and PACS workstations we suggest to mark images and/or series of images. CONCLUSION: The third user group depends on the information exported by the radiologist from PACS. After the report is written and signed by the radiologist, the digital report is transferred from the RIS to the HIS. The report is automatically attached to these images. Authorized personnel at the wards and outpatient are able to read the combination of validated report and exported radiological images as part of the digital medical record with an intranet browser on standard PCs.

Tryptophan B27 in the relaxin-like factor (RLF) is crucial for RLF receptor-binding.

The relaxin-like factor (RLF) is a circulating hormone that is synthesized in the gonads of mammals and released into the bloodstream. The distribution of its receptor and a trace of cross-reactivity to relaxin receptors implied that this relatively new factor is more relaxin- than insulin-like. The chemical synthesis of RLF analogues with specific modifications in positions B27 and B25, or the truncated form des(B27-31)RLF, clearly indicate that the intact indole ring in position B27 is crucial for high RLF receptor-binding. Receptor-binding was reduced by 2 orders of magnitude for Leu(B27)RLF (3%), Ala(B27)RLF (2.1%), and des(B27-31)RLF (0.4%), whereas slightly better binding was observed for His(B27)RLF (7.5%), Phe(B27)RLF (21%), D-Trp(B27) (26%), and the oxindole(B27)RLF (41%). On the basis of these observation it seems that an aromatic ring system in the beta- or gamma-position is required for binding. Structure prediction of the C-terminal region of the B chain indicated a possible type I or type III turn for residues C-G-G-P-R (B22-26) preceding the tryptophan. Exchanging Pro(B25) for D-Pro within the turn caused a severe structural rearrangement at the C terminus and a 96% drop in activity. It appears that the steric effect of L-Pro(B25) is important for the proper positioning of Trp(B27).

Antiporcine relaxin (antipRLX540) treatment decreases relaxin plasma concentration and disrupts delivery in late pregnant pigs.

Antibody against porcine relaxin (antipRLX540; 1:950,000) was produced in sheep and used to determine the effect on relaxin and progesterone secretion, and on parturition in late pregnant pigs. In group 1, Yorkshire gilts with normal estrous cycles were bred on the second observed estrus and fitted with an indwelling jugular cannula and an intraperitoneal cannula on day 100 of pregnancy. Gilts were infused at 6-h intervals with antipRLX540 (n = 10) or PBS (n = 10) beginning on day 103 until parturition. From days 103 to 120, daily blood samples (10 ml) were collected for RIA of relaxin, progesterone, and prolactin. In group 2, bred gilts were randomly assigned to antipRLX540 (n = 11), relaxin (n = 5), and PBS (n = 8) treatment on days 111, 113, and 115. Blood was collected twice daily from day 108 to 120, and every 20 min on days 111, 113, and 115 beginning 60 min before treatment and continuing 180 min. Parturition in gilts given antipRLX540 occurred on day 112.7 compared with day 114.0 in relaxin-treated gilts and day 114.3 in PBS controls (P < 0.05). Duration of delivery from first to last piglet was greatly delayed in antipRLX540 gilts (240 min) compared with PBS controls ([117 min] P < 0.005). Average number of stillborns was greater in antipRLX540- than in PBS-treated controls (2.4 vs. 1.0; P < 0.05). Relaxin concentration in peripheral plasma was lower in antipRLX540-treated gilts from day 105 to 110, but on day 113 the antipRLX540-treated group had a greater relaxin peak release compared with PBS-treated animals (P < 0.05). Plasma progesterone concentrations were similar in antipRLX540- and PBS-treated gilts throughout the period of the study. In group 2, by day 113, progesterone decreased in antipRLX540-treated gilts compared with relaxin- and PBS-treated gilts. Prolactin levels were similar in both antipRLX540- and PBS-treated gilts; however, from 1 to 3 days postpartum the antipRLX540 group had higher prolactin concentration (P < 0.05). The results indicate that antipRLX540 decreased circulating plasma concentrations of unbound or free relaxin during the last 10 days of pregnancy in Yorkshire gilts. AntipRLX540 markedly increased both the duration of delivery of piglets and the average number of stillbirths in this litter-bearing species compared with PBS-treated controls. This study provides strong evidence that increasing circulating concentrations of relaxin during late pregnancy is crucial for unimpaired parturition in the pig.

Dogfish shark (Squalus acanthias) testes contain a relaxin.

Relaxin is a 6-kd polypeptide that exerts important hormonal effects in many female mammals. Relaxin is produced by the ovary, placenta, or uterus in many mammalian species. The functions of relaxin in the male mammal are not yet firmly established, but there is some evidence suggesting an exocrine effect on sperm motility and fertilizability. In the male mammals that have been studied, relaxin is produced by the prostate gland (human) or seminal vesicles (boar). However, in the bird, the testis is the likely source of relaxin. Among the elasmobranchs, ovaries obtained from dogfish sharks have been shown to contain a polypeptide hormone that is structurally, biologically, and immunologically similar to mammalian relaxins, but the male reproductive tract of this species has not previously been investigated as a potential source of relaxin. Extracts of testes obtained from mature dogfish sharks have now been tested by a specific relaxin bioassay and by a homologous porcine radioimmunoassay for the presence of relaxin. Both crude and partially purified testicular extracts contained unmistakable guinea pig pubic symphysis-"relaxing" activity and relaxin-like immunoactivity. Following immunoaffinity purification, the shark testis polypeptide had an apparent specific activity of 88 microg porcine relaxin equivalents per milligram in the radioimmunoassay, which is similar to the immunoactivity of pure shark ovarian hormones. These data, therefore, strongly support the view that in dogfish sharks, the male as well as the female gonad produces relaxin. Furthermore, as the dogfish shark has existed as a species for about 200 million years, the data suggest that testicular relaxin appeared early in vertebrate evolution.

Benefits of growth hormone treatment on bone metabolism, bone density and bone strength in growth hormone deficiency and osteoporosis.

Bone mass is reduced in patients with GH deficiency (GHD) leading to an increased vertebral fracture rate and clinically significant osteoporosis. Patients with GHD of juvenile onset have reduced skeletal mineralization. When substituting GH in patients with GHD, bone turnover is increased and bone mineral density initially decreases during the first year due to the increase in remodelling space. From the experience in patients with acromegaly, cortical bone mass is increased and trabecular bone mass is normal in eugonadal or decreased in the hypogonadal patients. However, bone mineral content and bone area are increased leading to a higher biomechanical competence of bone as shown in rats. In patients with GHD of juvenile onset, mineralization and bone maturation are achieved during treatment with GH in adult life after having reached final body height leading to an increase in bone mass. The GH/ IGF-I system is dysregulated in patients with post-menopausal osteoporosis. This is shown by reduced systemic IGF and IGFBP-3-levels in osteoporosis suggesting a decrease of endogenous GH-secretion or a dysregulation of the GH receptor system which is beyond the normal ageing process of the GH/IGF system, the "somatopause". A premature somatopause may be responsible for the dysregulation in some patients with osteoporosis. However, 24-h GH profiles do not differ between patients suffering from osteoporosis or osteoarthritis. Treatment of osteoporosis with GH might be beneficial due to the increased bone metabolism and improved bone geometry which occurs with GH. The substantial increase of bone remodelling achieved with GH may be helpful during late post-menopause with decreased bone turnover and impaired osteoblastic function. Using GH to prevent physiological bone loss that occurs with age seems possible, but has to be discussed on an ethical and economic basis.

Identification of a glycosylated relaxin-like molecule from the male Atlantic stingray, Dasyatis sabina.

The alkaline gland fluid of the Atlantic stingray (Dasyatis sabina) contains a molecule that cross-reacts weakly to anti-porcine relaxin antibodies. This material was isolated and purified to homogeneity by reversed-phase high-performance liquid chromatography. In SDS gel electrophoresis, the molecule showed an apparent molecular mass of 13 kDa which upon reduction formed two polypeptide chains of 4 and 9 kDa, respectively. Sequence analyses revealed a 27-amino acid residue A chain and a 54-amino acid residue B chain which contained an N-glycosylation site in position B37. The distribution of the six cysteines and possibly the disulfide bonding is identical to that found in insulins and most relaxins. Although the stingray relaxin-like molecule contains the structurally relevant glycine residues within the A chain, in the midregion of the B chain it has only one of the two requisite binding site arginines, which explains the lack of relaxin bioactivity in standard mammalian assays. Stingray relaxin is the first member of the relaxin family identified in a nonhomeotherm male. Carbohydrate analysis of relaxin revealed an N-linked asialo, agalacto, bisected biantennary, and a core-fucosylated oligosaccharide in the position of Asn B37 which makes it the first reported glycosylated relaxin-like molecule.

Continuous infusion of relaxin on periparturient progesterone, oxytocin and relaxin plasma concentrations and time of parturition in beef heifers.

These studies were designed to determine whether continuous i.v. infusion of increasing dosages of porcine relaxin during late pregnancy in beef heifers would influence circulating blood concentrations of relaxin, progesterone and oxytocin, and time of onset of parturition. Beef heifers were bred by artificial insemination and, on Day 277, fitted with indwelling jugular cannulas for hormone infusion and blood sampling from Day 277 to Day 286. Intravenous infusion of purified porcine relaxin (pRLX, 3000 U mg-1) was started in heifers (n = 8) at increasing dosages (200 U h-1 on Days 277 and 278, 300 U h-1 on Days 279 and 280, 500 U h-1 on Day 281, 600 U h-1 on Day 282, and 700 U h-1 on Days 283-286). Phosphate-buffered saline (PBS, 10 ml h-1) was infused during these same times to control animals (n = 6). Relaxin treatment steadily increased the circulating plasma concentration of immunoreactive relaxin to more than 120 ng ml-1 compared with less than 0.5 ng ml-1 in PBS-treated controls. Relaxin infusion in increasing dosages over the treatment time was associated with a significant decrease (P < 0.01) in plasma progesterone concentration compared with the PBS controls. The rate of change in progesterone levels between pRLX and PBS groups differed (P < 0.05) at 300 U h-1, 600 U h-1 and 700 Uh-1 dosage intervals, respectively. Plasma levels of oxytocin at 4 h intervals remained similar (P > 0.05) during the pretreatment period and throughout continuous infusion of pRLX and PBS. Mean concentrations of oxytocin in PBS control heifers peaked at 0.95 pgml-1 during the corresponding infusion of 700 Uh-1 pRLX, which peaked at 0.77 pgml-1. Although continuous i.v. infusion of relaxin resulted in a decrease in circulating blood levels of progesterone, it did not significantly reduce the interval between the beginning of pRLX treatment and parturition compared with the PBS-infused control heifers. These results indicate that continuous i.v. infusion of high levels of porcine relaxin resulted in a decrease in progesterone secretion in late pregnant beef heifers.

The chemical synthesis of rat relaxin and the unexpectedly high potency of the synthetic hormone in the mouse.

Rat relaxin, as isolated from ovaries, has been described in the literature as a low potency hormone in the mouse symphysis pubis assay. Searching for an explanation, a helix-breaking glycine residue in the B chain seemed to be the most auspicious perturbation. Rat relaxin was chemically synthesized and analyzed by reverse-phase high performance liquid chromatography, amino acid composition, mass spectrometry and circular dichroic spectroscopy. Analogs of rat relaxin were synthesized either with aspartic acid in place of the helix-breaking glycine residue in the receptor-binding region of the B chain or with Asp-Leu-Val instead of Gly-Tyr-Val at positions B14-B16. In receptor-binding assays [B14D, B15L, B16V]relaxin was a better ligand than rat relaxin, whereas the [B14D]relaxin was less potent. In the mouse symphysis pubis assay, both analogs were less potent than unmodified rat relaxin, but the [B14D, B15L, B16V]relaxin was better than [B14D]relaxin. In contrast to previous reports on native rat relaxin, the chemically synthesized rat relaxin proved to be as active as human and porcine relaxin with respect to the standard mouse assay system. Glycine, which is considered to be a perturbator in an alpha helix, is not only tolerated in the B14 position but is required for full biological potency.

Chemical synthesis of a Zwitterhormon, insulaxin, and of a relaxin-like bombyxin derivative.

The structural motif of insulin and relaxin is frequently seen in molecules of divergent functions and origins. The insect developmental factor bombyxin, the relaxin-like factor from Leydig cells, and the insulin-like factor 4 (INSL4) all are made of two disulfide-linked chains and have one disulfide bond within the A-chain. The polyclonal antibody R6, which was raised against porcine relaxin, reacts with a wide variety of naturally occurring relaxins from primates, marine and terrestrial mammals, and elasmobranchs but does not recognize insulin. The antibody binds mainly to the arginines that occur in the N, N+4 positions in the B-chains of all relaxins which also constitute the receptor-binding site. The receptor-binding haptens were incorporated by total synthesis into human despentapeptide insulin and bombyxin II, a developmental factor from the silk moth Bombyx mori. In the process the insect factor became a perfect antigen for the anti-relaxin antibody, whereas the human insulin was transformed into a bona fide relaxin. The conversion was affected by changing four critical residues so that the insulin activity was retained to the extent of 10% of the original level. This, to the best of our knowledge, is the first designer protein to incorporate two unrelated biological functions in one primary sequence, and we are therefore proposing that, analogous to zwitterion, the generic name "Zwitterhormon" (German spelling) be used for this type of construct.

Use of synthetic canine relaxin to develop a rapid homologous radioimmunoassay.

Canine relaxin (cRlx) was synthesized by a combination of solid-phase methods and sequential site-directed disulfide bond formation. Proof that the intended molecule had been synthesized was obtained by analytical HPLC of the intact and reduced molecule, by amino acid and sequence analysis, and by receptor binding and in vivo mouse interpubic ligament bioassays. Antisera to synthetic cRlx were raised in six male rabbits; these cross-reacted with relaxins of other species, but not with insulin, LH, FSH, hCG, or prolactin (PRL). Three of the antisera neutralized relaxin-induced interpubic ligament formation in estrogen-primed mice in vivo. A new homologous cRlx RIA was developed through the use of rabbit antiserum 79888, synthetic cRlx for standards and 125l-labeled trace, and a goat anti-rabbit lgG-polyethylene glycol precipitant. The new RIA can be completed in 26 h and has a sensitivity of 0.195-0.39 ng cRlx/tube. Intra- and interassay coefficients of variation were 3% and 12.5%. During pregnancy in bitches, serum cRlx rose to about 10 micrograms/ml. Immunoactive cRlx was also detected in serum, colostrum, and milk of lactating bitches, but not in large volumes (100-300 microliters) of serum of pseudopregnant or estrous bitches. Immunoreactive cRlx was also found in seminal plasma, but not in serum, of male dogs. The new homologous cRlx RIA is simple, rapid, sensitive, and specific, and will be used in future studies of canine relaxin physiology.