bluuebunny/crossref_metadata_2025 huggingface dataset:
Neighbourhood interactions and environmental factors influencing old‐pasture succession in the Central Pyrenees
Authors: Doležal Jiří , Št'astná Petra , Hara Toshihiko , Šrůtek Miroslav
Published: April 2009

Abstract.The shrubBuxus sempervirensand the treesAbies albaandFagus sylvaticahave recently recolonized old‐pastures in the Central Pyrenees. We mapped all live and dead individuals (> 1.3 m tall) in a large forest plot in Ordesa Valley to examine the importance of density‐dependent processes during recolonization. Biotic interactions were inferred from changes in horizontal structure and the influences of neighbours on tree survival.Buxusdifferentially influenced establishment and survival of tree species, thereby controlling future canopy composition and spatial structure. The rapidly invadingAbiesformed denser patches on elevated sites less occupied byBuxus, whereasFaguspreferentially established within shrubs.Abiesreached densities which led to intense intraspecific competition and high mortality rates among saplings. Self‐thinning inAbiesled to smaller numbers of regularly spaced survivors, and greater relative dominance ofFagus.Disregarding intraspecific effects and abiotic environment,Abiessurvival was significantly lower underBuxusshrubs, which suggests that the spatial location and abundance ofAbieswas constrained by the location ofBuxus. Fagussurvival was not related toBuxusdensity, but remained significantly lower in denserAbiespatches. The higher mortality ofFagusin denserAbiespatches, and the resulting spatial segregation of the species, reflects asymmetric interspecific competition. Inhibition fromBuxusshrubs and intraspecific competition prevent invadingAbiesfrom dominating and may therefore help in maintaining a mixedAbies‐Fagusstand.
Innovative procedure in surgical management of chronic lymphoedema
Authors: Mousavi Seyed Reza , Mehdikhah Zohreh , Kavyani Ali , Saberi Ali Reza
Published: April 2009

Background:  Lymphoedema is the result of impaired lymphatic drainage from the affected organ. This abnormality can be primary or secondary. Different non‐operative and operative approaches have been introduced to treat chronic lymphoedema. In the present study, we describe a new surgical technique and compare its results with other more commonplace methods.

Materials and Methods:  The study included 296 patients who had been diagnosed with chronic lower extremity lymphoedema and who had not responded to non‐operative management for at least 6 months. Data was collected over 15 years, between March 1987 and March 2002. Doppler ultrasonography of the deep venous system was routinely undertaken in all patients to confirm its patency. The 296 patients underwent surgery and their progress was followed for at least 1 year postoperatively.

Results:  All of the patients were operated on using our new technique, which is a modified form of the Homan techniques. The outcome was excellent, and 89.2% of patients were free of complication. The most common complication was wound seroma.

Conclusions:  Considering the difficulties associated with the treatment of chronic lymphoedema and the variety of surgical options, our method achieves excellent results, and could even become the standard operative procedure for treating intractable forms of disease.
Colonic perforation from computed tomographic colonography: A real complication from a virtual procedure
Authors: Ganesh Shanthan , Pathma‐nathan Nimalan , Loder Peter
Published: April 2009

Virtual colonoscopy or computed tomographic colonography was first described in 1994 as a non‐invasive test to visualize the colon. It requires bowel preparation and air or carbon dioxide insufflation through a rectal tube. We report a case of colonic perforation during virtual colonoscopy.
Compound depressed skull fracture caused by a flying model helicopter
Authors: Yung Yu‐Bun , Chan Kam‐Tong , Cheung Fung‐Ching
Published: April 2009

Compound depressed skull fractures are usually caused by high‐energy direct blows with sharp or blunt objects. A clear mechanism of injury is crucial in evaluating the degree of damage and formulating a management plan. We report an unusual cause of such a condition in this study. The patient, a 45‐year‐old male, suffered from head injury by a flying model helicopter. The flying of model helicopters is forbidden in the area where the incident occurred. He was confused on site and was found to be hemiplegic. Emergency wound debridement, craniectomy and external ventricular catheterization were done. Six months after the operation, the patient could walk with the assistance of a stick and cranioplasty has been planned for him. The potential dangers of flying model helicopters are well presented in this study. The authors strongly recommend implementation of safety measures to minimize the potential risk of the game or other similar games.
Factors for conversion in laparoscopic cholecystectomy for acute cholecystitis: Is timing important?
Authors: Li Wing‐Hong , Chu Colin Wai‐Ho , Cheung Moon‐Tong
Published: April 2009

Aim:  Laparoscopic cholecystectomy is regarded as the gold standard treatment for gallstones. Conversion to open cholecystectomy is still common, and preoperative factors to predict conversion are useful in clinical practice. The aim of this study was to evaluate preoperative factors that could predict conversion in acute cholecystitis.

Methods:  This is a retrospective review of 83 patients with a diagnosis of acute cholecystitis who had laparoscopic cholecystectomy carried out as an emergency operation. Clinical, biochemical, and operative factors were analyzed for association with conversion.

Results:  A total of 83 patients were recruited to this study. The overall conversion rate was 33.7% (28/83). A longer duration of symptoms before presentation (P = 0.005) and surgery that was carried out over 48 h after admission (P = 0.022) were associated with a higher conversion rate. Emergency operations that began between 20.00 hours and 08.00 hours were also associated with a higher rate of conversion (P = 0.003). Other factors that were associated with conversion included male sex (P = 0.004), low albumin level upon admission (P = 0.024), prolonged prothrombin time (P = 0.040), and a raised serum total bilirubin level (P = 0.024). ASA scores were found to be similar in both groups (P = 0.509). Multivariate analysis by logistic regression showed that the independent risk factors for conversion in emergency laparoscopic cholecystectomy were surgery >48 h after admission (P = 0.028), emergency operation started between 20.00 hours and 08.00 hours (P = 0.026), and longer duration of symptoms before presentation (P = 0.034).

Conclusions:  Laparoscopic cholecystectomy should be carried out within 48 h of the patient being admitted for acute cholecystitis. The operation should be carried out during the daytime.
The combination of polymorphisms within MCP‐1 and IL‐1β associated with ulcerative colitis
Authors: Li K.‐S. , Wang B.‐Y. , Liu S.‐Y. , Yao S.‐P. , Guo L. , Mao D.‐W.
Published: April 2009

Monocyte chemoattractant protein‐1 (MCP‐1) is a chemokine involved in monocyte recruitment to sites of inflammation. Raised level of MCP‐1 has been widely demonstrated in the intestinal mucosa of patients with ulcerative colitis (UC), suggesting an important role of MCP‐1 in the pathogenesis of UC. The –2518A/G polymorphism in the promoter region of MCP‐1 gene affecting its transcriptional activation has been reported recently. In order to assess the potential role of this polymorphism in UC, we examined its distribution in 162 unrelated UC patients and 203 healthy controls. In addition, considering the gene regulatory association between interleukin‐1β (IL‐1β) and MCP‐1, we further examined whether the gene polymorphisms between MCP‐1 and IL‐1β exert synergetic effects on risk of UC. Our results show that the distribution of MCP‐1 genotype or allele frequencies between UC patients and controls was not significantly different; however, the association between the polymorphism of MCP‐1 –2518 GG and the polymorphism of IL‐1β–511 T in UC patients is significant (OR 2.062, 95% CI 1.034–4.113, P = 0.038). This is the first report describing the association between MCP‐1 polymorphism and UC, and our data suggest that the MCP‐1 –2518 polymorphism itself does not represent an independent genetic risk factor for UC. In contrast, the combination polymorphisms between MCP‐1 and IL‐1β can increase UC risk significantly, which might help us understand the molecular mechanism underlying the development of UC.
Utility of MtCOI polymerase chain reaction‐restriction fragment length polymorphism in differentiating between Q and B whitefly Bemisia tabaci biotypes
Authors: Ma Wei‐Hua , Li Xian‐Chun , Dennehy Timothy J. , Lei Chao‐Liang , Wang Mo , Degain Benjamin A. , Nichols Robert L.
Published: April 2009

The invasive, insecticide‐resistant, Q whitefly biotype, has gradually spread to other countries including the US via human‐mediated movement of plant materials. We assessed the utility of the VspI‐based mtCOI (mitochondrion cytochrome oxidase I) polymerase chain reaction‐restriction fragment length polymorphism (PCR‐RFLP) technique as a rapid, cost‐effective, and reliable alternative for differentiating the Q from the dominant B biotype in Arizona. Using the standard mtCOI gene sequencing and mtCOI PCR‐RFLP techniques, we biotyped eight whitefly strains of five individuals each collected from poinsettia and cotton at different locations in Arizona. Complete concordance was observed between the two methods, with three strains being identified as the Q biotype and five samples as the B biotype. We also scanned the mtCOI gene sequences for VspI polymorphisms in the B and Q biotype whiteflies currently available in the GenBank database. This global screening revealed the existence of three and four VspI polymorphic types for the Q and B biotypes, respectively. Nevertheless, all three VspI polymorphic Q biotype whiteflies shared a common and unique VspI site that can be used to differentiate Q biotype from the four VspI polymorphic B biotype whiteflies identified. These results demonstrate that the VspI‐based mtCOI gene PCR‐RFLP provides a reliable diagnostic tool for differentiating the Q and B biotype whiteflies in the US and elsewhere.
Modes of action of fraxinellone against the tobacco budworm, Heliothis virescens
Authors: Liu Zhi Long , Ho Shuit Hung , Goh Swee Hock
Published: April 2009

Fraxinellone significantly reduced the relative growth rate, food consumption rate as well as the efficiency of conversion of ingested food into biomass of Heliothis virescens when incorporated into artificial diets at concentrations of 4.31 × 10−5 mol/L and above. After being fed with fraxinellone‐treated diets for 24 h, the larval midguts of H. virescens possess significantly lower activities of α‐amylase and non‐specific proteases and higher activities of cytochrome P450s. In vitro, the compound did not inhibit the activities of α‐amylase and non‐specific proteases extracted from the larval midguts. Clear evidence of post‐ingestive toxicity of fraxinellone to midgut cells was observed under an electron microscope. The modes of action of the compound against insects were discussed.
Chandra and Swift observations of the quasi-persistent neutron star transient EXO 0748—676 back to quiescence
Authors: Degenaar N. , Wijnands R. , Wolff M. T. , Ray P. S. , Wood K. S. , Homan J. , Lewin W. H. G. , Jonker P. G. , Cackett E. M. , Miller J. M. , Brown E. F.
Published: April 2009

The quasi-persistent neutron star X-ray transient and eclipsing binary EXO 0748−676 recently started the transition to quiescence following an accretion outburst that lasted more than 24 years. We report on two Chandra and 12 Swift observations performed within five months after the end of the outburst. The Chandra spectrum is composed of a soft, thermal component that fits to a neutron star atmosphere model with kT∞∼ 0.12keV, joined by a hard power-law tail that contributes ∼20 per cent of the total 0.5–10 keV unabsorbed flux. The combined Chandra/Swift data set reveals a relatively hot and luminous quiescent system with a temperature of kT∞∼ 0.11–0.13keV and a bolometric thermal luminosity of ∼8.1 × 1033–1.6 × 1034(d/7.4kpc)2ergs −1. We discuss our results in the context of cooling neutron star models.
Analytical Solution of Advective Mixing in a Conduit
Authors: Li Guangquan
Published: April 2009

The permeable conduit wall in a karst aquifer allows for water and solute to be exchanged between conduits and the limestone matrix. Contaminant sequestered in the limestone matrix is flushed into conduits following flood events. The contaminant released from the permeable wall will then mix with conduit water and will be transported downgradient in the conduit. A one‐dimensional advection‐dispersion equation is presented to describe this mixing‐transport incorporating water flow and solute flux through the conduit wall. An analytical solution ignoring conduit dispersion is derived using the method of characteristics. Scale analysis is performed to provide a general guideline to estimate when conduit dispersion can be neglected. The solution also can be used to compute the distribution of solute in the matrix before flushing.
The “Hot Wires” of the Relaxin‐Like Factor (Insl3)
Authors: Schwabe Christian , Büllesbach Erika E.
Published: April 2009

The discovery of the total separation of receptor‐binding and signal‐generating regions of the relaxin‐like factor (RLF) has provided an opportunity to investigate the mechanism of transmembrane signaling. The receptor‐binding residues of RLF are in the B chain of the two‐chain molecule and extend from the midregion of the central helix to the tryptophan in position B27. The signal initiation site resides in two residues before the N‐terminal cysteine in each chain. For optimal signaling the RLF requires five L‐α‐amino acids preceding cysteine A10, whereas the B chain requires only three. The nature of the side chains of these amino acids is not critical for the signaling function. Heuristic arguments lead us to suggest that the peptide bond is the signal‐generating feature of RLF and possibly of other peptide hormones.
Reversal of Cardiac Fibrosis and Related Dysfunction by Relaxin
Authors: Du Xiao‐Jun , Xu Qi , Lekgabe Edna , Gao Xiao‐Ming , Kiriazis Helen , Moore Xiao‐Lei , Dart Anthony M. , Tregear Geoffrey W. , Bathgate Ross A. D. , Samuel Chrishan S.
Published: April 2009

As a hallmark of heart disease, cardiac fibrosis contributes to the development of heart failure and arrhythmias and forms a key therapeutic target. There is a major unmet need for selective, potent, and safe antifibrotic drugs. Earlier studies revealed a cardiac fibrosis phenotype in relaxin‐1‐deficient mice. Recent studies in several rodent models of cardiac fibrosis have documented reversal of fibrosis by treatment with relaxin peptide or virally mediated relaxin gene delivery. In mice with surgically induced transmural myocardial infarction, relaxin therapy inhibited scar density. In these studies, however, functional benefits achieved by relaxin therapy were limited or less explored. Collectively, there is good experimental evidence that relaxin is able to reverse cardiac fibrosis due to distinct mechanisms. Future research needs to explore functional improvement following fibrosis reversal by relaxin and the usefulness of relaxin in antiarrhythmic or stem cell‐based therapy.
Prominent Role of Relaxin in Improving Postinfarction Heart Remodeling
Authors: Bani Daniele , Nistri Silvia , Formigli Lucia , Meacci Elisabetta , Francini Fabio , Zecchi‐Orlandini Sandra
Published: April 2009

Stem cell transplantation is a promising approach for treatment of the postinfarcted heart and prevention of deleterious cardiac remodeling and heart failure. We explored this issue by transplanting mouse C2C12 myoblasts, genetically engineered to express enhanced green fluorescent protein (eGFP) or eGFP and relaxin (eGFP/RLX), into swine with chronic myocardial infarction. One month later, C2C12 myoblasts selectively settled in the ischemic scar around blood vessels, showing an activated endothelium (ICAM‐1 and VCAM positive). Although unable to differentiate to a muscle phenotype, these cells induced extracellular matrix (ECM) remodeling by matrix metalloprotease secretion and increased microvessel density by vascular endothelial growth factor expression. C2C12/RLX myoblasts gave better results than C2C12/GFP. By echocardiography, C2C12‐engrafted swine, especially those that received C2C12/RLX, showed better heart contractility than the untreated controls. Hence, the advantage afforded by the grafted myoblasts on cardiac function is primarily dependent on their paracrine effects on ECM remodeling and vascularization.
Relaxin Reduces Fibrosis in Models of Progressive and Established Hepatic Fibrosis
Authors: Bennett Robert G. , Heimann Dean G. , Tuma Dean J.
Published: April 2009

The effect of relaxin administration before (prevention) or after (treatment) the establishment of hepatic fibrosis in a mouse model was examined. In the prevention study, relaxin reduced collagen and smooth muscle actin content and significantly reduced serum levels of the liver enzymes alanine aminotransferase and aspartate aminotransferase. In the treatment study, administration of relaxin for 1 week reduced collagen and smooth muscle actin but not liver enzyme levels. Relaxin administered for 2 weeks had no significant effect. In conclusion, the data suggest that relaxin treatment before fibrosis can reduce collagen and improve liver function but that there is little effect of short‐term relaxin treatment after fibrosis is established.
Mutations in INSL3 and RXFP2 Genes in Cryptorchid Boys
Authors: Ferlin Alberto , Zuccarello Daniela , Garolla Andrea , Selice Riccardo , Vinanzi Cinzia , Ganz Francesco , Zanon Gian Franco , Zuccarello Biagio , Foresta Carlo
Published: April 2009

Mutations in the INSL3 and RXFP2 genes have been associated with human cryptorchidism but with contrasting data. We analyzed the frequency of mutations in these genes in 600 newborns with cryptorchidism (396 unilateral and 204 bilateral) and 300 noncryptorchid subjects. We found five RXFP2 mutations in five bilateral cryptorchid boys, one INSL3 mutation in a unilateral cryptorchid boy, and one INSL3 mutation in a boy with unilateral cryptorchidism at birth and spontaneous descent during the first month of life. Overall, the frequency of INSL3 and RXFP2 mutations was therefore 7/600 at birth (1.2%) and 7/303 (2.3%) in persistent cryptorchid boys, with a higher prevalence of bilateral forms (5/120, 4.2%). No mutations were found in controls. This study confirmed the association between INSL3 and RXFP2 gene mutations and human cryptorchidism.
INSL3 Plays a Role in the Balance between Bone Formation and Resorption
Authors: Pepe Anastasia , Ferlin Alberto , Gianesello Lisa , Facciolli Arianna , Agoulnik Alexander I. , Foresta Carlo
Published: April 2009

We have recently demonstrated that the disruption of the INSL3 hormonal system, as observed in humans with mutations in the RXFP2 gene and in Rxfp2‐deficient mice, might affect the equilibrium of the osteoblast–osteoclast system, resulting in an imbalance between bone formation and bone resorption that may result in reduced bone mass. In the present study we have better characterized the in vitro effects of INSL3 on human osteoblasts. Stimulation of human primary osteoblasts with INSL3 at serial concentrations (1 pM, 1 nM, and 1 μM) induced a dose‐dependent increase in osteoblast proliferation and expression of specific osteoblast genes.
Role of Relaxin in Human Osteoclastogenesis
Authors: Facciolli Arianna , Ferlin Alberto , Gianesello Lisa , Pepe Anastasia , Foresta Carlo
Published: April 2009

Recently, we have demonstrated that INSL3 is important for bone metabolism, and in this study we analyzed the possible role of the cognate hormone relaxin on human osteoclasts. In fact, previous studies showed an effect of relaxin on peripheral blood mononuclear cells (PBMCs), the precursors of osteoclasts. Analysis of the expression of the relaxin receptor RXFP1 and RLN‐2 mRNA in primary cell cultures of human osteoclasts obtained from PBMCs showed by reverse transcriptase PCR and immunofluorescence only the presence of RXFP1 transcripts. Analysis of the in vitro effects of relaxin on osteoclastogenesis showed that relaxin is able alone to induce the differentiation of PBMCs into mature osteoclasts. This study shows, for the first time, that relaxin has an effect on bone metabolism, facilitating the differentiation of osteoclasts. A possible role for relaxin in osteolytic bone metastasis is also proposed.
A Randomized, Double‐Blind, Placebo‐Controlled Trial of Relaxin for Cervical Ripening in Post‐Delivery Date Pregnancies
Authors: Weiss Gerson , Teichman Sam , Stewart Dennis , Nader David , Wood Susan , Unemori Elaine
Published: April 2009

Recombinant human relaxin was used to attempt cervical ripening in post‐delivery date pregnancies. High doses of relaxin were safe but did not advance cervical ripening or induce labor.
Relaxin in Human Pregnancy
Authors: Goldsmith Laura T. , Weiss Gerson
Published: April 2009

Relaxin has beneficial effects upon the endometrium which are responsible for establishment of pregnancy. We have demonstrated that relaxin stimulates endometrial decidualization, the structural and biochemical changes in endometrial parenchymal cells, and the accompanying angiogenesis, modulation of matrix metalloproteinase activity, and increased concentrations in local immune cells which are required for implantation. Our recent data also demonstrate that either too much or too little relaxin can be detrimental. Elevated circulating maternal relaxin concentrations (hyperrelaxinemia) are associated with premature birth. This is likely due to the effects of relaxin at the level of the cervix, via upsetting the balance in the maintenance of cervical connective tissue architecture. In addition, the absence of circulating relaxin during pregnancy in women may have negative consequences upon glucose metabolism.
Evaluation of Systemic Relaxin Blood Profiles in Horses as a Means of Assessing Placental Function in High‐Risk Pregnancies and Responsiveness to Therapeutic Strategies
Authors: Ryan Peter L. , Christiansen David L. , Hopper Richard M. , Bagnell Carol A. , Vaala Wendy E. , Leblanc Michelle M.
Published: April 2009

Placental insufficiency is regarded as the primary factor contributing to late‐term abortion and perinatal death of foals. Often when problems associated with late‐term pregnancy in the horse are manifest the condition is well‐advanced and therapeutic intervention may not be effective in rescuing the pregnancy. If a compromised pregnancy due to placental insufficiency could be identified early, the pregnancy might be sustained through medical intervention. Because the placenta is the sole source of circulating relaxin in the mare, we hypothesized that systemic relaxin may serve as a biomarker of placental function and fetal well‐being and a predictor of pregnancy outcome at delivery. To test this hypothesis we monitored plasma relaxin in mares (light breeds) with normal and problematic pregnancies from clinical cases presented to the veterinary hospital and in pregnant mares experimentally inoculated withStreptococcus equi zooepidemicusto induce uterine infection. Upon establishment of placentitis, mares were assigned to different therapeutic strategies and responsiveness was monitored. Blood was collected during the third trimester of pregnancy, and relaxin content was determined using a homologous equine relaxin radioimmunoassay. The results reported here show a positive relationship between low circulating relaxin and poor pregnancy outcome in mares with compromised placental function. While relaxin may have value as a diagnostic assay for identifying mares with high‐risk pregnancies associated with placental dysfunction, the variable results obtained from mares undergoing drug treatment for experimentally induced placentitis make it difficult to determine the reliability of relaxin for evaluating therapeutic efficacy.
Relaxin Signaling in Uterine Fibroids
Authors: Li Zhen , Burzawa Jennifer K. , Troung Anne , Feng Shu , Agoulnik Irina U. , Tong Xiaowen , Anderson Matthew L. , Kovanci Ertug , Rajkovic Aleksandar , Agoulnik Alexander I.
Published: April 2009

Uterine fibroid is the most common tumor of female reproductive organs. The role of relaxin signaling in leiomyoma development was analyzed. We used 23 matched pairs of leiomyoma and normal myometrium samples to compare the expression of relaxin family peptide receptors RXFP1 and RXFP2, caveolin 1, desmin, and steroid receptors and their cofactors, NCOR1 and NCOR2. The expression of RXFP1, evaluated by quantitative reverse transcription‐PCR, was downregulated in fibroid tissues. Relaxin or insulin‐like peptide 3 treatment suppressed transforming growth factor β‐induced phosphorylation of SMAD2 in rat leiomyoma ELT‐3 cells in vitro, suggesting a possible involvement of relaxins in the etiology of leiomyoma.
Identification of Boar Testis as a Source and Target Tissue of Relaxin
Authors: Kohsaka Tetsuya , Kato Shinichi , Qin Si , Minagawa Itaru , Yogo Keiichiro , Kawarasaki Tatsuo , Sasada Hiroshi
Published: April 2009

The expression and cellular localization of relaxin and its own receptor, LGR7/RXFP1, were demonstrated in the boar testis, where relaxin was produced by the Leydig cells as 18‐kDa pro‐relaxin and LGR7/RXFP1 was detected in both Leydig cells and seminiferous epithelial cells, suggesting that a functional relaxin–LGR7/RXFP1 hormone–receptor network operates within the boar testis.
Relaxin Promotes Matrix Metalloproteinase‐2 and Decreases Wnt/β‐Catenin Expression in the Neonatal Porcine Heart
Authors: Ho Teh‐Yuan , Dilts Megan A. , Bartol Frank F. , Bagnell Carol A.
Published: April 2009

Relaxin (RLX), reported to play an important role in cardiovascular remodeling, is linked to activation of matrix metalloproteinases (MMPs). Interruption of the cardiac Wnt/β‐catenin signaling system is reported to have antihypertrophic effects. Thus, the objectives of this study were to determine the effects of RLX on the myocardial Wnt/β‐catenin signaling system and MMP expression in the postnatal day 2 (PND 2) porcine heart. Results showed that myocardial Wnt7a and Wnt4 gene expression at PND 2 decreased following treatment with RLX in vivo from birth, while there was no effect on Wnt5a expression. Immunoreactive myocardial β‐catenin protein was reduced in RLX‐treated animals. Zymographic analysis of medium from RLX‐treated heart explants showed an increase in pro‐MMP‐2 but not pro‐MMP‐9 activity. Western blot analysis indicated that RLX increased myocardial MMP‐2 protein compared to controls. Data suggest that the RLX‐induced decline in Wnt/β‐catenin expression at PND 2, together with increased MMP‐2 activity, may be important for neonatal porcine cardiac remodeling.
Relaxin Activates Multiple cAMP Signaling Pathway Profiles in Different Target Cells
Authors: Halls Michelle L. , Hewitson Tim D. , Moore Xiao‐Lei , Du Xiao‐Jun , Bathgate Ross A. D. , Summers Roger J.
Published: April 2009

Although RXFP1‐cAMP signaling in HEK293T cell systems is now relatively well‐defined, the signaling pathways activated by relaxin in its target cells and tissues are still unclear. This study aimed to examine the cAMP signaling of RXFP1 in cells that endogenously express the receptor. Seven cell types derived from various backgrounds were screened for receptor expression. Only in THP‐1 cells and rat cardiac fibroblasts was there activation of the Gαi3–Gβγ–phosphatidylinositol 3‐kinase–protein kinase Cζ pathway, leading to cAMP accumulation. In all other cells there was activation of a combination of the initial pathways to affect cAMP. T‐47D cells could activate only Gαs, whereas Colo 16 and rat renal fibroblasts from obstructed kidney could activate both Gαs and GαoB pathways. Thus, the signaling pathways activated by relaxin are highly dependent upon the cell type under investigation, and this may help to explain the varied physiological responses exerted by relaxin in its different target tissues.
Perinatal Zearalenone Exposure Affects RXFP1, RXFP2, and Morphoregulatory Gene Expression in the Neonatal Porcine Uterus
Authors: Chen Joseph C. , Wiley Anne A. , Kauffold Johannes , Wähner Martin , Bartol Frank F. , Bagnell Carol A.
Published: April 2009

The mycotoxin zearalenone (ZEA) is a selective estrogen receptor modulator that can contaminate cereal feeds and lead to reproductive disorders. To determine effects of perinatal ZEA exposure on uterine expression of genes associated with endometrial development in the neonatal gilt, pregnant sows were fed ZEA (1500 μg ZEA/kg of feed/day) or vehicle from 14 days before farrowing through postnatal day (PND) 20–21, when neonatal uterine tissues were collected. At birth, gilts were cross‐fostered to generate four ZEA exposure groups (n= 5–6/group): unexposed controls or exposures limited to prenatal, postnatal, or pre‐ and postnatal (continuous) periods. Results showed that at PND 20–21, uterine Wnt7a, Hoxa10, estrogen receptor α, and RXFP2 mRNA levels were decreased in neonates exposed continuously to ZEA (P < 0.05). Uterine RXFP1 transcripts were decreased in postnatally and continuously exposed groups (P < 0.05). Neonatal uterine Wnt4 mRNA levels were unchanged.
Development and Optimization of MicroRNA against Relaxin‐3
Authors: Callander Gabrielle E. , Thomas Walter G. , Bathgate Ross A. D.
Published: April 2009

Recent in vivo studies suggest a role for relaxin‐3 in feeding and stress. To further elucidate the function of relaxin‐3 in the central nervous system, we have employed a complementary approach, based on RNA interference, to modulate relaxin‐3 expression. We have designed, constructed, and characterized three microRNAs (miRNAs) targeting different regions of the relaxin‐3 transcript. These were tested to determine the amount of miRNA required to achieve the greatest knockdown and for their ability to reduce the expression of relaxin‐3 in transfected HEK293Tcells. All miRNA constructs significantly reduced relaxin‐3‐induced cAMP responses; however, miR499 was most effective. This should be a useful tool for in vitro studies and central targeting of relaxin‐3 in vivo using viral delivery systems.
Regulation of Rxfp2 (Lgr8) Expression in the Mouse Fetal Kidney by the Transcription Factor Pod1 (Tcf 21)
Authors: Familari Mary , Vu Duc , Parry Laura J.
Published: April 2009

Rxfp2 is present in putative mesangial cells at embryonic stage 18 (E18) in the rat kidney, suggesting that insulin‐like peptide 3 is involved in glomerulogenesis. One of the main regulatory factors in glomerulogenesis is the basic helix‐loop‐helix transcription factor Pod1. Therefore, we examined the expression of Rxfp2 relative to Pod1 in the fetal and neonatal mouse kidney and tested the hypothesis that Rxfp2 is a downstream target of Pod1. Rxfp2 gene expression in the neonatal kidney was highest at E14 and E16 but decreased significantly at E18. This was not correlated with increased Pod1 expression. However, there was a significant increase in Rxfp2 expression in the kidneys of Pod1−/− mice at E18. These data suggest that Pod1 may negatively regulate Rxfp2 expression in the developing glomerulus to promote cell proliferation.
Laser Microdissection of Neonatal Porcine Endometrium for Tissue‐Specific Evaluation of Relaxin Receptor (RXFP1) Expression in Response to Perinatal Zearalenone Exposure
Authors: Wiley Anne A. , Kauffold Johannes , Wähner Martin , Crean‐Harris Bethany , Miller Dori J. , Bagnell Carol A. , Bartol Frank F.
Published: April 2009

Porcine neonatal uterine relaxin receptor (RXFP1) expression is tissue compartment specific and estrogen sensitive. Here, procedures were established for laser microdissection, tissue capture, and quantification of the effects of perinatal exposure (14 days pre‐ to 21 days postnatal) to a selective estrogen receptor modulator of environmental origin, zearalenone (ZEA), on endometrial RXFP1 expression. Total RNA from captured endometrium was used to generate cDNA for quantitative reverse transcription‐PCR. Cycle threshold values indicated that ZEA reduced (P < 0.06) endometrial RXFP1 expression on postnatal days 20–21.
Role of Relaxin in Regulation of Fibrosis in the Lung
Authors: Tang Mimi L. K. , Samuel Chrishan S. , Royce Simon G.
Published: April 2009

Airway remodeling changes are a key pathological feature of asthma. They are associated with airway hyperresponsiveness and fixed airway obstruction. Aged, relaxin‐deficient mice display structural changes resembling airway remodeling in asthma, including thickening of the bronchial epithelium and extracellular matrix (ECM) deposition in the subepithelial region. Relaxin‐deficient mice also have exaggerated airway remodeling in models of allergic airway disease compared to wild‐type controls. Both in vitro and in vivo studies have suggested that airway fibrosis may drive other changes of airway remodeling, including epithelial changes. A suggested paradigm is that subepithelial fibrosis (deposition of ECM in the basement membrane region) may lead to increased epithelial cell proliferation and transforming growth factor‐β1 production, which in turn may lead to further fibrosis. Relaxin may therefore prevent or reverse activation of the epithelial and fibroblast remodeling in asthma. Control of epithelial remodeling and ECM production in the airway wall may play an important role in maintaining normal lung function. Understanding how relaxin protects against airway remodeling changes may have important implications in developing new therapies to treat airway remodeling in asthma.
Activation of Relaxin‐Related Receptors by Short, Linear Peptides Derived from a Collagen‐Containing Precursor
Authors: Shemesh Ronen , Hermesh Chen , Toporik Amir , Levine Zurit , Novik Amit , Wool Assaf , Kliger Yossef , Rosenberg Avi , Bathgate Ross A. D. , Cohen Yossi
Published: April 2009

In a screening effort based on algorithmic predictions for novel G‐protein‐coupled receptor (GPCR) peptide activators, we were able to identify and examine two novel peptides (P59 and P74) which are short, linear, and derived from a natural, previously unidentified precursor protein containing a collagen‐like repeat. Both peptides seemed to show an apparent cAMP‐related effect on CHO‐K1 cells transiently transfected with either LGR7 or LGR8, usually after treatment with cAMP‐generating forskolin, compared to the same cells treated with forskolin plus relaxin. This activation was not found for the relaxin‐3 receptor (GPR135). In a set of follow‐up experiments, both peptides were found to stimulate cAMP production, mostly upon initial stimulation of cAMP production by 5 μM forskolin in cells transfected with either LGR7 or LGR8. In a dye‐free cell impedance GPCR activation assay, we were able to show that these peptides were also able to activate a cellular response mediated by these receptors. Although untransfected CHO‐K1 cells showed some cellular activation by both relaxin and at least one of our newly discovered peptides, both LGR7‐ and LGR8‐transfected cells showed a stronger response, indicating stimulation of a cellular pathway through activation of these receptors. In conclusion, we were able to show that these newly discovered peptides, which have no similarity to any member of the relaxin–insulin‐like peptide family, are potential ligands for the relaxin‐related family of receptors and as such might serve as novel candidates for relaxin‐related therapeutic indications. Both peptides are linear and were found to be active after being chemically synthesized.
Roles of the Receptor, the Ligand, and the Cell in the Signal Transduction Pathways Utilized by the Relaxin Family Peptide Receptors 1–3
Authors: Summers Roger J. , Bathgate Ross A. D. , Wade John D. , Van Der Westhuizen Emma T. , Halls Michelle L.
Published: April 2009

The relaxin‐like peptides produce their effects by acting at four G‐protein‐coupled receptors (GPCRs) RXFP1 to 4. RXFP1 and 2 are characterized by large extracellular domains containing leucine‐rich repeats, whereas RXFP3 and 4 closely resemble small‐peptide‐liganded GPCRs. Studies with mutant RXFP1 receptors established that the final 10 amino acids of the C‐terminus and Arg752 in particular are obligatory for the second phase of cAMP signaling. Examination of the importance of cell type revealed different patterns of cAMP signaling related to the types of G‐proteins expressed in these cells. Studies of RXFP3 signaling using reporter genes revealed that both relaxin and relaxin‐3 activated the receptor but displayed different patterns of signaling. The studies suggest that the functional domains of the receptor, the cell type in which it is expressed, and the ligand used to activate the receptor all have important roles in determining the functional response observed.
Matrix Metalloproteinase Induction by Relaxin Causes Cartilage Matrix Degradation in Target Synovial Joints
Authors: Kapila Sunil , Wang Wei , Uston Karen
Published: April 2009

Our long‐term goal is to understand the mechanisms by which relaxin and estrogen potentially contribute to joint diseases, particularly those afflicting the fibrocartilaginous temporomandibular joint (TMJ). Previously, we showed that relaxin produces a dose‐dependent induction of tissue‐degrading enzymes of the matrix metalloproteinase (MMP) family, specifically MMP‐1 (collagenase‐1), MMP‐3 (stromelysin‐1), MMP‐9 (92‐kDa gelatinase), and MMP‐13 (collagenase‐3) in cell isolates and tissue explants from TMJ fibrocartilage. The induction of these MMPs is accompanied by loss of collagen and glycosaminoglycans (GAGs), which was blocked by a pan‐MMP inhibitor. We also found the targeted in vivo loss of collagen and GAGs in TMJ discs of ovariectomized rabbits treated with β‐estradiol, relaxin, or both hormones together. Progesterone attenuated the induction of MMPs and matrix loss by relaxin and estrogen. The modulation of matrix composition in TMJ fibrocartilage by these hormones was similar to that observed in the pubic symphysis and differed from that of the knee meniscus. The two target tissues showing the greatest modulation of MMPs and matrix loss, namely, the TMJ disc and pubic symphysis, had similar expression profiles of the estrogen receptors α and β, relaxin‐1 receptor (RXFP1, LGR7), and insulin‐like peptide 3 receptor (RXFP2, LGR8) and these profiles differed from those in cells from the knee meniscus. These findings suggest a novel model for targeted tissue turnover of cartilage of specific joints through hormone‐mediated induction of select MMPs.
Effects of Relaxin on Development of Mesangial Proliferative Nephritis
Authors: Ikegaya Naoki , Yoshida Takuya , Kohsaka Tetsuya , Suzuki Ayumi , Kobayashi Natsuki , Yamamoto Tatsuo , Fujigaki Yoshihide , Hishida Akira , Kumagai Hiromichi
Published: April 2009

The involvement of relaxin (RLX) in nephritis has not been fully elucidated. In this study, we examined the expression of the RLX receptor and effects of RLX administration in antithymocyte serum (ATS) nephritis. The nephritic glomeruli showed an increased immunostaining for LGR7 compared to normal glomeruli. The administration of RLX for 7 days to rats with ATS nephritis did not cause an alteration in blood pressure or body weight. On the sixth day, however, a significant reduction in urinary protein was recognized in rats treated with RLX. Histological studies revealed the amelioration of glomerular accumulation of Periodic acid–Schiff stain–positive matrix in rats treated with RLX. Significant reductions of phosphorylated SMAD2, mesangial cell proliferation, and α‐smooth muscle actin expression were observed in rats treated with RLX. These results suggest that RLX is a promising therapeutic tool for nephritic diseases.
Milk‐Borne Relaxin and the Lactocrine Hypothesis for Maternal Programming of Neonatal Tissues
Authors: Bagnell Carol A. , Steinetz Bernard G. , Bartol Frank F.
Published: April 2009

The fact that all newborn mammals drink milk extends the time frame of maternal influence on development into neonatal life. While the nutritional and immunological benefits of milk are clear, the role of milk as a conduit for bioactive factors with the potential to affect neonatal development is less well defined. Porcine and canine milk contain immunoreactive relaxin (RLX) that is transmitted into the circulation of nursing offspring. In the pig, a window of opportunity for transmission of milk‐borne RLX is open at birth and remains so for about the first 3 days of neonatal life. Recent studies have shown that pro RLX is the major form of RLX in milk and that milk‐borne porcine pro RLX is biologically active. Moreover, RLX receptor (RXFP1) expression is detectable in the porcine female reproductive tract and other somatic tissues at birth. The lactocrine hypothesis for maternal programming of neonatal development was proposed as a mechanism whereby RLX, a prototypical milk‐borne growth factor in the pig, is delivered to nursing offspring, where it can affect development of RXFP1‐positive target tissues. Data indicating that treatment of newborn gilts with RLX increased estrogen receptor‐α expression in the uterus and cervix by postnatal day 2 support a role for RLX in lactocrine programming of the female reproductive tract. Effects of RLX on Wnt/β‐catenin expression in neonatal porcine cardiac tissue support a role for RLX in developmental programming of nonreproductive target tissues as well. Ongoing studies will test the lactocrine hypothesis for maternal programming of development by RLX and related milk‐borne factors.
Estrogen and TCDD Influence RLN2 Gene Activity in Estrogen Receptor‐Positive Human Breast Cancer Cells
Authors: Kietz Silke , Feng Shu , Agoulnik Alexander , Hombach‐Klonisch Sabine
Published: April 2009

Expression of relaxin is increased in human breast cancer, and relaxin was shown to increasein vitroinvasiveness through increased production and secretion of matrix metalloproteinases in human breast cancer cells. The role of estrogen in the promotion of breast cancer is well‐known. The environmental toxin 2,3,7,8‐tetrachlorodibenzo‐p‐dioxin (TCDD) is a known carcinogen but has been shown to have antiestrogenic effects in human breast cancer cells. In this study, we have employed real‐time PCR and chromatin immunoprecipitation (ChIP) assays to investigate the influence of estrogen and TCDD on relaxin‐1 (RLN1) and relaxin‐2 (RLN2) gene expression in MCF‐7 and T47D human breast cancer cells. Estrogen increased RLN2 transcripts in T47D and MCF‐7 cells after just 4 h of exposure, whereas TCDD did not. RLN1 transcripts were only induced after 24 h of estrogen exposure. TCDD did have antiestrogenic activity and reduced the estrogen‐mediated increase in RLN2 and RLN1 mRNA. The estrogen‐mediated increase in RLN2 mRNA levels was not caused by changes in the mRNA stability. ChIP analysis revealed binding of estrogen receptor‐alpha (ERα) to promoter sequences of the RLN2 gene. Thus, we provide evidence that RLN2 gene activity is directly regulated by activated ERα in human breast cancer cells and we show that activation of the arylhydrocarbon receptor by TCDD inhibits this regulation by estrogen.
Structural Basis of Allosteric Ligand–Receptor Interactions in the Insulin/Relaxin Peptide Family
Authors: De Meyts Pierre , Gauguin Lisbeth , Svendsen Angela Manegold , Sarhan Mazen , Knudsen Louise , Nøhr Jane , Kiselyov Vladislav V.
Published: April 2009

The insulin/relaxin superfamily of peptide hormones comprises 10 members in humans. The three members of the insulin‐related subgroup bind to receptor tyrosine kinases (RTKs), while four of the seven members of the relaxin‐like subgroup are now known to bind to G‐protein‐coupled receptors (GPCRs), the so‐called relaxin family peptide receptors (RXFPs). Both systems have a long evolutionary history and play a critical role in fundamental biological processes, such as metabolism, growth, survival and longevity, and reproduction. The structural biology and ligand‐binding kinetics of the insulin and insulin‐like growth factor I receptors have been studied in great detail, culminating in the recent crystal structure of the insulin receptor extracellular domain. Some of the fundamental properties of these receptors, including constitutive dimerization and negative cooperativity, have recently been shown to extend to other RTKs and GPCRs, including RXFPs, confirming kinetic observations made over 30 years ago.
De Novo Design and Synthesis of Cyclic and Linear Peptides to Mimic the Binding Cassette of Human Relaxin
Authors: Hossain Mohammed Akhter , Bathgate Ross A. D. , Tregear Geoffery , Wade John D.
Published: April 2009

A synthetic cyclic regioselectively addressable functionalized template was used to prepare six analogs that presented the side chains of the key receptor‐binding residues of each of H2 and H3 relaxins and insulin‐like peptide 3. None showed any binding affinity for RXFP1, RXFP2, or RXFP3, indicating that the key residues were either incorrectly oriented or that additional residues are required for receptor binding.
Expression of LGR7 in the Primate Corpus Luteum Implicates the Corpus Luteum as a Relaxin Target Organ
Authors: Maseelall Priya B. , Seungdamrong Aimee , Weiss Gerson , Wojtczuk Andrea S. , Donnelly Robert , Stouffer Richard L. , Goldsmith Laura T.
Published: April 2009

In women, the corpus luteum is the source of circulating relaxin. No previous studies have addressed whether the corpus luteum is also a relaxin target organ. We determined relaxin receptor LGR7 mRNA expression in human term pregnancy corpora lutea and nonhuman primate corpora lutea obtained during the menstrual cycle. Real‐time reverse transcription‐PCR demonstrated the expression of LGR7 mRNA in both human and rhesus monkey corpora lutea. Rhesus monkey corpora lutea were obtained from naturally cycling animals following documented luteinizing hormone (LH) surges at early, mid‐, mid‐late, and late luteal phases. Luteal expression of LGR7 mRNA did not show temporal variation. Since the primate corpus luteum is LH dependent, we assessed LGR7 mRNA expression in corpora lutea from rhesus monkeys treated with a gonadotropin‐releasing hormone (GnRH) antagonist, which significantly suppressed pituitary LH levels. GnRH antagonist treatment, which also inhibits both progesterone and relaxin production, resulted in a fivefold increase in luteal LGR7 mRNA expression. These data suggest that luteal LGR7 mRNA expression may be regulated by relaxin and/or LH and that the primate corpus luteum is a target organ for relaxin.
Modeling the Primary Hormone‐Binding Site of RXFP1 and RXFP2
Authors: Scott Daniel J. , Tregear Geoffrey W. , Bathgate Ross A. D.
Published: April 2009

The primary binding sites of the relaxin and insulin‐like peptide 3 (INSL3) receptors, RXFP1 and RXFP2, are found within the leucine‐rich repeats (LRRs) of the ectodomains. Specific B‐chain residues in the peptides interact with residues in the inner β‐sheets of the LRRs of the receptors. Relaxin binds to RXFP2 with high affinity, although INSL3 has a very poor affinity for RXFP1. In this paper we present evidence that relaxin binds to the LRRs of RXFP2 in a manner similar to INSL3 binding to its receptor. Additionally, we introduce a model of this binding interaction and compare it to an alternate model for relaxin–RXFP1 binding.