The actin-based motility of the facultative intracellular pathogen Listeria monocytogenes

Drug resistance is a major obstacle to successful cancer chemotherapy. P-glycoprotein, which transports various antitumor agents outside the resistant tumor cells, plays a key role in multidrug resistance. We found that MRK-16, a monoclonal antibody against P-glycoprotein, and cyclosporine, synergistically enhanced the antitumor effects of vincristine and adriamycin in multidrug-resistant K562/ADM cells. On the other hand, the combined use of MRK-16 with verapamil or FK-506 did not show such synergistic effects. Drug accumulation studies revealed that MRK-16 remarkably increased the accumulation of cyclosporine, but not verapamil, in K562/ADM cells. This increased accumulation of cyclosporine by MRK-16 in K562/ADM cells directly resulted in the enhanced accumulation of vincristine and adriamycin in the cells. The synergistic effect of MRK-16 and cyclosporine was further confirmed by isobologram analysis in three different highly multidrug-resistant tumor cells. Moreover, while MRK-16 alone did not enhance the sensitivity of the KB-8-5 cells moderately resistant to vincristine, it increased two-fold the reversing effect of cyclosporine at 1 microM, an achievable blood concentration. Since MRK-16 alone showed therapeutic effects against multidrug-resistant tumors, the combined use of MRK-16, cyclosporine and antitumor agents would provide therapeutic benefits for the treatment of resistant tumors.     

The molecular mechanisms of actin-based intracellular motility by Listeria monocytogenes

A rapid, sensitive method was developed for the quantification of the R- and S-enantiomers of ketoprofen and their acyl glucuronide conjugates in the plasma and dialysate of hemodialysis-dependent anephric patients. Unconjugated R- and S-ketoprofen plasma concentrations were determined directly by liquid chromatography using a S,S-Whelk-O1 chiral stationary phase. R- and S-Ketoprofen glucuronide for use as standard were resolved using a C18 reversed-phase HPLC column with a mobile phase containing the ion-pair reagent tetrabutylammonium hydrogen sulfate. Plasma glucuronides, however, could not be directly quantified due to matrix interference. Therefore, the glucuronides were isolated using reversed-phase HPLC and quantified after alkaline hydrolysis using the S,S-Whelk-O1 chiral stationary phase column.     

Gelsolin, a protein that caps the barbed ends and severs actin filaments, enhances the actin-based motility of Listeria monocytogenes in host cells

The actin-based motility of Listeria monocytogenes requires the addition of actin monomers to the barbed or plus ends of actin filaments. Immunofluorescence micrographs have demonstrated that gelsolin, a protein that both caps barbed ends and severs actin filaments, is concentrated directly behind motile bacteria at the junction between the actin filament rocket tail and the bacterium. In contrast, CapG, a protein that strictly caps actin filaments, fails to localize near intracellular Listeria. To explore the effect of increasing concentrations of gelsolin on bacterial motility, NIH 3T3 fibroblasts stably transfected with gelsolin cDNA were infected with Listeria. The C5 cell line containing 2.25 times control levels of gelsolin supported significantly higher velocities of bacterial movement than did control fibroblasts (mean +/- standard error of the mean, 0.09 +/- 0.003 micro(m)/s [n = 176] versus 0.05 +/- 0.003 micro(m)/s [n = 65]). The rate of disassembly of the Listeria-induced actin filament rocket tail was found to be independent of gelsolin content. Therefore, if increases in gelsolin content result in increases in Listeria-induced rocket tail assembly rates, a positive correlation between gelsolin content and tail length would be expected. BODIPY-phalloidin staining of four different stably transfected NIH 3T3 fibroblast cell lines confirmed this expectation (r = 0.92). Rocket tails were significantly longer in cells with a high gelsolin content. Microinjection of gelsolin 1/2 (consisting of the amino-terminal half of native gelsolin) also increased bacterial velocity by more than 2.2 times. Microinjection of CapG had no effect on bacterial movement. Cultured skin fibroblasts derived from gelsolin-null mice were capable of supporting intracellular Listeria motility at velocities comparable to those supported by wild-type skin fibroblasts. These experiments demonstrated that the surface of Listeria contains a polymerization zone that can block the barbed-end-capping activity of both gelsolin and CapG. The ability of Listeria to uncap actin filaments combined with the severing activity of gelsolin can accelerate actin-based motility. However, gelsolin is not absolutely required for the actin-based intracellular movement of Listeria because its function can be replaced by other actin regulatory proteins in gelsolin-null cells, demonstrating the functional redundancy of the actin system.     

The bacterial actin nucleator protein ActA of Listeria monocytogenes contains multiple binding sites for host microfilament proteins

BACKGROUND: Several intracellular pathogens, including Listeria monocytogenes, use components of the host actin-based cytoskeleton for intracellular movement and for cell-to-cell spread. These bacterial systems provide relatively simple model systems with which to study actin-based motility. Genetic analysis of L. monocytogenes led to the identification of the 90 kD surface-bound ActA polypeptide as the sole bacterial factor required for the initiation of recruitment of host actin filaments. Numerous host actin-binding proteins have been localized within the actin-based cytoskeleton that surrounds Listeria once it is inside a mammalian cell, including alpha-actinin, fimbrin, filamin, villin, ezrin/radixin, profilin and the vasodilator-stimulated phosphoprotein, VASP. Only VASP is known to bind directly to ActA. We sought to determine which regions of the ActA molecule interact with VASP and other components of the host microfilament system. RESULTS: We used the previously developed mitochondrial targeting assay to determine regions of the ActA protein that are involved in the recruitment of the host actin-based cytoskeleton. By examining amino-terminally truncated ActA derivatives for their ability to recruit cytoskeletal proteins, an essential element for actin filament nucleation was identified between amino acids 128 and 151 of ActA. An ActA derivative from which the central proline-rich repeats were deleted retained its ability to recruit filamentous actin, albeit poorly, but was unable to bind VASP. CONCLUSIONS: Our studies reveal the initial interactions that take place between invading Listeria and host microfilament proteins. The listerial ActA polypeptide contains at least two essential sites that are required for efficient microfilament assembly: an amino-terminal 23 amino-acid region for actin filament nucleation, and VASP-binding proline-rich repeats. Hence, ActA represents a prototype actin filament nucleator. We suggest that host cell analogues of ActA exist and are important components of structures involved in cell motility.     

The Listeria monocytogenes iap gene as an indicator gene for the study of PrfA-dependent regulation

In this study the effects of hypoxanthine (HX) on meiotic maturation were compared using oocytes from mice possessing a hypoxanthine phosphoribosyltransferase null mutation (HPRT-) and from the corresponding HPRT-competent background strain (HPRT+). Oocyte-cumulus cell complexes and cumulus cell-enclosed oocytes (oocytes cultured while enclosed by cumulus cells) from HPRT+, but not HPRT-, mice took up HX and contained significant levels of HPRT activity. In addition, FSH increased, and HX suppressed, the de novo synthesis of purines in HPRT+ complexes, whereas de novo synthesis was elevated in HPRT complexes and was unaffected by FSH or HX. After 3 h of HX treatment, lower frequencies of germinal vesicle breakdown (GVB) were observed in cumulus cell-enclosed than in denuded HPRT+ oocytes; however, identical frequencies of maturation were observed in denuded and cumulus cell-enclosed HPRT oocytes. This demonstrates a direct inhibitory action of HX on the oocyte that does not depend on salvage, plus an additional action of the cumulus cells that requires HPRT activity. Nevertheless, cumulus cells from HPRT- mice are capable of exerting an additional inhibitory action of dibutyryl cAMP (dbcAMP) on the oocyte. A kinetics analysis of FSH action on HX-arrested cumulus cell-enclosed HPRT+ and HPRT- oocytes revealed, first, that the inhibitory effect of the cumulus cells is transient and, second, that HPRT activity is not required for FSH induction of GVB in HX-arrested oocytes. When dbcAMP- or HX-arrested oocytes were treated with FSH, GVB was blocked to the same extent in HPRT- oocytes with the purine de novo synthesis inhibitor, azaserine, but this drug was less effective in HX-treated HPRT+ oocytes. These results confirm the importance of the de novo pathway in hormone-induced maturation and also support a role for purine salvage as an alternative source of nucleotide in this process.     

The virulence gene cluster of Listeria monocytogenes is also present in Listeria ivanovii, an animal pathogen, and Listeria seeligeri, a nonpathogenic species

Most known Listeria monocytogenes virulence genes cluster within a 9.6-kb chromosomal region. This region is flanked on one end by two uncharacterized open reading frames (ORF A and ORF B) and ldh, an ORF presumably encoding the L. monocytogenes lactate dehydrogenase (J.-A. Vazquez-Boland, C. Kocks, S. Dramsi, H. Ohayon, C. Geoffroy, J. Mengaud, and P. Cossart, Infect. Immun. 60:219-230, 1992). We report here that the other end is flanked by prs, and ORF homologous to phosphoribosyl PPi synthetase genes. ORF B and prs were detected in all Listeria species and thus delimit the virulence region. This virulence gene cluster was detected exclusively in hemolytic Listeria species, Listeria ivanovii, an animal pathogen, and Listeria seeligeri, a nonpathogenic species.     

Efficient induction of cytotoxic CD8+ T cells against exogenous proteins: establishment and characterization of a T cell line specific for the membrane protein ActA of Listeria monocytogenes

The property of listeriolysin (LLO) to introduce soluble passenger proteins into the cytosol of antigen-presenting cells allows the induction of CD8+ cytotoxic T cells against such antigens. To overcome the potential problem of presentation of the immunodominant epitope LL091-99 by H-2Kd, a variant LLO92A was established in which Tyr 92 was replaced by Ala. Immunization of BALB/c mice with purified LLO92A failed to stimulate cytotoxic T cells specific for either the epitope LLO91-99 or for any other LLO-derived peptide. Injection of mixtures of purified LLO92A and soluble nucleoprotein (NP) of influenza virus into mice resulted in a strong cytotoxic T cell response exclusively directed against NP. The LLO92A variant was successfully used to generate, propagate and characterize a CD8 T cell line specific for the membrane-bound virulence factor ActA of Listeria monocytogenes. Interestingly, wildtype ActA bound to the surface of live L. monocytogenes was not presented by MHC class I molecules to the CD8+ T cell line.     

The gene cluster inlC2DE of Listeria monocytogenes contains additional new internalin genes and is important for virulence in mice

In this work we identified and characterized a gene cluster containing three internalin genes of Listeria monocytogenes EGD. These genes, termed inlG, inlH and inlE, encode proteins of 490, 548 and 499 amino acids, respectively, which belong to the family of large, cell wall-bound internalins. The inlGHE gene cluster is flanked by two listerial house-keeping genes encoding proteins homologous to the 6-phospho-beta-glucosidase and the succinyl-diaminopimelate desuccinylase of E. coli. A similar internalin gene cluster, inlC2DE, localised to the same position on the L. monocytogenes EGD chromosome was recently described in a different isolate (Dramsi S, Dehoux P, Lebrun M, Goossens PL, Cossart P (1997) Infect Immun 65: 1615-1625). Sequence comparison of the two inl gene clusters indicates that inlG is a new internalin gene, while inlH was generated by a site-specific recombination, leading to an in-frame deletion which removed the 3'-terminal end of inlC2 and the 5'-terminal part of inlD. The third gene of the inlGHE cluster, inlE, is almost identical to the previously reported inlE gene. Our data show that the inlGHE gene cluster is probably transcribed from a major PrfA-independent promoter located upstream of inlG. PCR analysis revealed the presence of the newly identified inl genes inlG and inlH in most L. monocytogenes isolates tested. A mutant which has lost inlG, inlH and inlE by an in-frame deletion exhibited, after oral infection of mice, a significant loss in virulence and shows drastically reduced numbers of viable bacteria in both liver and spleen when compared to the wild-type strain.     

Synthetic peptides derived from the Listeria monocytogenes p60 protein as antigens for the generation of polyclonal antibodies specific for secreted cell-free L. monocytogenes p60 proteins

All species of the genus Listeria secrete a major extracellular protein called p60. A comparison of the deduced amino acid sequences of all listerial p60 proteins previously indicated there were only a few regions which were unique to the pathogenic, food-borne species Listeria monocytogenes. Two of these p60 regions were chosen for the development of antibodies specific for the facultative intracellular species L. monocytogenes. Initially, these regions were characterized via epitope mapping, and this led to the development of two different synthetic peptides. Rabbits immunized with these synthetic peptides generated polyclonal antibodies that were then used in Western blot (immunoblot) analyses. Antiserum against peptide A (PepA) recognized the p60 protein in the supernatants collected from most L. monocytogenes serotypes except for several strains belonging to serotypes 4a and 4c. No p60-related protein was detected in the supernatants from other Listeria species with this anti-PepA antiserum. Antibodies raised against peptide D (PepD) reacted with p60 from all L. monocytogenes serotypes, including all 4a and 4c strains that were tested, and also showed no cross-reactivity with supernatant proteins from other Listeria species. Both antisera also detected p60 in supernatants of a large number of environmental isolates of L. monocytogenes. Besides Western blot analyses, these antisera to PepA and PepD reacted with secreted p60 in an enzyme-linked immunosorbent assay, indicating recognition of the native antigen in addition to the denatured form. These data suggest that synthetic peptides derived from the variable region of the L. monocytogenes p60 protein may be useful for the development of an immunological diagnostic assay.     

A homeotic transformation is generated in the rostral branchial region of the head by disruption of Hoxa-2, which acts as a selector gene

The Hoxa-2 gene was disrupted by homologous recombination. Homozygous mutant mice died at birth. Defects were found in the branchial region of the head, which corresponds to the Hoxa-2 rostral expression domain. While rhombomeric and neural crest cell (NCC) segmentation was not affected, mesenchymal NCC derivatives of the second arch were lacking, and second arch mesenchymal NCC identity was changed to first arch identity, resulting in homeotic transformation of second to first arch skeletal elements. These results reveal the existence of a skeletogenic ground pattern program common to at least the mesenchymal NCC that originated from rhombomeres 2 and 4. The appearance of an atavistic reptilian pterygoquadrate element in Hoxa-2 mutants suggests that this ground pattern is intermediate between reptiles and mammals. The ground pattern program appears to be modified in the mouse first arch by a Hox-independent process, whereas Hoxa-2 acts as a selector gene in the second arch.     

ClpE, a novel member of the HSP100 family, is involved in cell division and virulence of Listeria monocytogenes

We identified, in the facultative intracellular pathogen Listeria monocytogenes, a previously unknown Clp ATPase, unique among the HSP100 proteins because of the presence of a short N-terminal region with a potential zinc finger motif. This protein of 726 amino acids is highly homologous to ClpE of Bacillus subtilis, and is a member of a new subfamily of HSP100/Clp ATPases. The clpE gene is transcribed as a monocistronic mRNA from a typical consensus sigma A promoter. clpE is not stimulated by various stresses, but is upregulated in a clpC mutant. This is the first example of cross-regulation between Clp ATPases. By constructing a clpE mutant of L. monocytogenes, we found that ClpE is required for prolonged survival at 42 degrees C and is involved in the virulence of this pathogen. A double mutant deficient in both ClpE and ClpC was avirulent in a mouse model and completely eliminated in the liver. Electron microscopy studies did not show any morphological alterations in clpE or clpC mutants. In the clpE-clpC double mutant, however, cell division was affected, indicating that ClpE acts synergistically with ClpC in cell septation. These results show that the Clp chaperones play a crucial role in both cell division and virulence of L. monocytogenes.     

The N-terminal segment of antithrombin acts as a steric gate for the binding of heparin

The binding of heparin causes a conformational change in antithrombin to give an increased heparin binding affinity and activate the inhibition of thrombin and factor Xa. The areas of antithrombin involved in binding heparin and stabilizing the interaction in the high-affinity form have been partially resolved through the study of both recombinant and natural variants. The role of a section of the N-terminal segment of antithrombin, residues 22-46 (segment 22-46), in heparin binding was investigated using rapid kinetic analysis of the protein cleaved at residues 29-30 by limited proteolysis with thermolysin. The cleaved antithrombin had 5.5-fold lowered affinity for heparin pentasaccharide and 1.8-fold for full-length, high-affinity heparin. It was shown that, although the initial binding of heparin is slightly enhanced by the cleavage, it dissociates much faster from the cleaved form, giving rise to the overall decrease in heparin affinity. This implies that the segment constituting residues 22-46 in the N terminus of antithrombin hinders access to the binding site for heparin, hence the increased initial binding for the cleaved form, whereas, when heparin is bound, segment 22-46 is involved in the stabilization of the binding interaction, as indicated by the increased dissociation constant. When the heparin pentasaccharide is bound to antithrombin prior to incubation with thermolysin, it protects the N-terminal cleavage site, implying that segment 22-46 moves to interact with heparin in the conformational change and thus stabilizes the complex.     

The benefits of school screening for scoliosis in the central part of The Netherlands

The Netherlands has well-organized school health services, and children are assessed on a regular basis for scoliosis among other disturbances and pathologies. The purpose of this study was to assess the benefits of an annual screening programme for scoliosis in the Netherlands. Three cohorts of 10,000 children sampled at 10, 12 and 14 years of age, respectively, were followed for 3 years. Children with a positive bending sign were referred to a second screening stage, in which external asymmetry was quantified. Children diagnosed via the programme (group 1) were compared with those children who had been referred for treatment independently of the screening (group 2). The total number of children in these groups combined was then compared with the number that would have been expected on the basis of accepted prevalence figures for idiopathic scoliosis given in current literature. Over 30,000 children were screened. Although the programme established a total of 57 cases of definite scoliosis (0.18%), the 34 cases (0.11%) already known, mainly detected by previous school health checks, were more severe regarding the risk of progression and treatment. The annual screening programme did not detect a single case that needed surgery. These figures provide the basis on which to decide for or against adopting an annual screening programme for scoliosis; the decision is a socio-political one. Based on this study, we expect all scoliotic patients needing treatment should be detected in time if periodic health checks will be maintained biennially. On medical grounds, it is our view, that screening for scoliosis should not be performed in the Netherlands annually.     

A critical role for amino-terminal glutamine/asparagine repeats in the formation and propagation of a yeast prion

The yeast [PSI+] factor propagates by a prion-like mechanism involving self-replicating Sup35p amyloids. We identified multiple Sup35p mutants that either are poorly recruited into, or cause curing of, wildtype amyloids in vivo. In vitro, these mutants showed markedly decreased rates of amyloid formation, strongly supporting the protein-only prion hypothesis. Kinetic analysis suggests that the prion state replicates by accelerating slow conformational changes rather than by providing stable nuclei. Strikingly, our mutations map exclusively within a short glutamine/asparagine-rich region of Sup35p, and all but one occur at polar residues. Even after replacement of this region with polyglutamine, Sup35p retains its ability to form amyloids. These and other considerations suggest similarities between the prion-like propagation of [PSI+] and polyglutamine-mediated pathogenesis of several neurodegenerative diseases.     

The amino-terminal region of Tyk2 sustains the level of interferon alpha receptor 1, a component of the interferon alpha/beta receptor

Tyk2 belongs to the Janus kinase (JAK) family of receptor associated tyrosine kinases, characterized by a large N-terminal region, a kinase-like domain and a tyrosine kinase domain. It was previously shown that Tyk2 contributes to interferon-alpha (IFN-alpha) signaling not only catalytically, but also as an essential intracellular component of the receptor complex, being required for high affinity binding of IFN-alpha. For this function the tyrosine kinase domain was found to be dispensable. Here, it is shown that mutant cells lacking Tyk2 have significantly reduced IFN-alpha receptor 1 (IFNAR1) protein level, whereas the mRNA level is unaltered. Expression of the N-terminal region of Tyk2 in these cells reconstituted wild-type IFNAR1 level, but did not restore the binding activity of the receptor. Studies of mutant Tyk2 forms deleted at the N terminus indicated that the integrity of the N-terminal region is required to sustain IFNAR1. These studies also showed that the N-terminal region does not directly modulate the basal autophosphorylation activity of Tyk2, but it is required for efficient in vitro IFNAR1 phosphorylation and for rendering the enzyme activatable by IFN-alpha. Overall, these results indicate that distinct Tyk2 domains provide different functions to the receptor complex: the N-terminal region sustains IFNAR1 level, whereas the kinase-like domain provides a function toward high affinity ligand binding.     

Confocal microscopy as a tool for the investigation of the anterior part of the eye

In recent years, confocal microscopy has become a powerful tool for examining microscopic structures in the living eye. The decisive advantage of this technique is that it permits the investigation of optical sections of relatively thick (> 10 microns) specimens. Because confocal microscopy suppresses the out-of-focus blur, sharp three-dimensional images with excellent resolution can be obtained. Confocal microscopy is therefore able to provide more information than the classic methods--i.e., specular microscopy and slit-lamp biomicroscopy. This paper reviews recent applications of confocal microscopy in three fields of ophthalmology: the observation of the anatomy of the anterior parts of the eye, the investigation of these structures after local administration of drugs and, finally, the use of this technique for the diagnosis of infectious ocular diseases.     

The intermediate care unit as a cost-effective option for the treatment of medical patients in critical condition

Since the limited accessibility of general intensive care units creates a situation in which medical patients in critical condition continue to be cared for in the regular wards, we conducted a retrospective cohort study to assess the treatment outcomes in such patients referred to the medical intermediate care unit (MICU). At the Soroka Medical Center, a facility with 810 beds, of which 170 beds are in medical wards, including an 8-bed intensive cardiac care unit and a 5-bed general intensive care unit, 119 patients were referred to the MICU, directly from the emergency room or from medical wards, during the first half of 1994. Eighty percent of the patients were admitted to the MICU directly from the emergency room. The mean disease severity, as measured by the APACHE II score, was 12.9, and the mean intensity of care for these patients, as measured by the TISS scale, was 12.6. Twenty-one of the 119 patients died during hospitalization (17.6%). This mortality rate conformed to the mortality risk of 15.5%, which was calculated using prognostic formulae. The ratio of nursing staff to patient in the MICU was approximately 1:3, compared to 2:3 in the general intensive care unit and 1:12 in the wards. The mean cost of one day of hospitalization in the MICU was one-third that in the general intensive care unit and double the cost in a ward. Medical patients in critical condition can be treated in an MICU, with a savings in expenses and without impairing the patient's chances for survival.     

rOmpA is a critical protein for the adhesion of Rickettsia rickettsii to host cells

rOmpA and rOmpB are immunodominant, surface-exposed proteins of Rickettsia rickettsii. Prior evidence suggests that adhesion of R. rickettsii to the host cell is mediated by a rickettsial protein. Five monoclonal antibodies to rOmpA, five to rOmpB, and one to the rickettsial lipopolysaccharide (LPS) were tested for inhibition of rickettsial attachment. All the monoclonal antibodies to rOmpA inhibited adhesion of rickettsiae to the L-929 cells with some inhibition rates as high as 90%. In contrast, monoclonal antibodies to rOmpB and LPS did not block attachment. When Fab fragments of monoclonal antibodies against rOmpA and rOmpB were used, similar results were observed as for the intact monoclonals, non-adhesion and adhesion, respectively. Purified rOmpA showed a competitive inhibitive effect on the attachment of R. rickettsii to host cells. Trypsin completely digested rOmpA but not rOmpB from the surface of intact R. rickettsii, resulting in loss of the ability of the rickettsiae to attach to the host cell. rOmpA appears to play an important role in the initial adhesion of R. rickettsii to the host cell.     

Cholera toxin acts as a potent adjuvant for the induction of cytotoxic T-lymphocyte responses with non-replicating antigens

OBJECTIVE: To evaluate the relation between the development of the uteroplacental circulation as assessed by Doppler velocimetry and the maternal blood relaxin concentration. METHODS: Transvaginal color Doppler investigation of the uteroplacental circulation was performed in 42 healthy women at 6-15 weeks' gestation before termination of pregnancy for psychosocial reasons. The resistance index (RI), pulsatility index (PI), and maximum peak velocity were recorded at the level of the main uterine artery, and the presence of intervillous flow was noted. Relaxin, hCG, 17 beta-estradiol (E2), and progesterone levels were measured in maternal venous blood. RESULTS: Limited intervillous flow was noted from 10 weeks' gestation and continuous intervillous flow from 12 weeks. An inverse relation was observed between the circulating levels of both E2 and progesterone and uterine artery RI and PI, whereas the relaxin level correlated positively with uterine RI and PI. Estradiol and progesterone levels also correlated positively with uterine peak systolic velocity and intervillous blood flow. Multiple linear regression analysis indicated that both hormones contributed to the decrease in downstream resistance to uterine blood flow with advancing gestational age, as assessed by uterine RI. In addition, relaxin contributed to the uterine RI and PI and to the intervillous blood flow. CONCLUSION: These data suggest that relaxin, E2, and progesterone may influence the changes in uterine blood flow that occur in early pregnancy. The role played by E2 and progesterone in the development of the uteroplacental circulation may be modulated by relaxin, constituting a novel function for this ovarian peptide.