Identification and validation of a novel anti-virulent that binds to pyoverdine and inhibits its function.

Pseudomonas aeruginosa: causes serious infections in patients with compromised immune systems and exhibits resistance to multiple antibiotics. The rising threat of antimicrobial resistance means that new methods are necessary for treating microbial infections. We conducted a high-throughput screen for compounds that can quench the innate fluorescence of the chromophore region of the P. aeruginosa siderophore pyoverdine, a key virulence factor. Several hits were identified that effectively quench pyoverdine fluorescence, and two compounds considerably improved the survival of Caenorhabditis elegans when worms were challenged with P. aeruginosa. Commercially available analogs of the best hit, PQ3, were tested for their ability to rescue C. elegans from P. aeruginosa and to interact with pyoverdine via fluorescence and solution NMR spectroscopy. 1H-15N and 1H-13C HSQC NMR were used to identify the binding site of PQ3c. The structure model of pyoverdine in complex with PQ3c was obtained using molecular docking and molecular dynamics simulations. PQ3c occupied a shallow groove on pyoverdine formed by the chromophore and N-terminal residues of the peptide chain. Electrostatic interactions and π-orbital stacking drove stabilization of this binding. PQ3c may serve as a scaffold for the development of pyoverdine inhibitors with higher potency and specificity. The discovery of a small-molecule binding site on apo-pyoverdine with functional significance provides a new direction in the search of therapeutically effective reagent to treat P. aeruginosa infections. Abbreviations: NMR: nuclear magnetic resonance; SAR: structure-activity relationship; MD: molecular dynamics; RMSF: root-mean-square fluctuation; HSQC: heteronuclear single quantum correlation; DMSO: dimethyl sulfoxide; Δδavg: average amide chemical shift change; DSS: 2,2-dimethyl-2-silapentane-5-sulfonate; RMSD: root-mean-square deviation; LJ-SR: Lennard-Jones short-range; Coul-SR: Coulombic short-range; FRET: fluorescence resonance energy transfer.

Pharmacologic inhibition of RGD-binding integrins ameliorates fibrosis and improves function following kidney injury.

Fibrosis is a final common pathway for many causes of progressive chronic kidney disease (CKD). Arginine-glycine-aspartic acid (RGD)-binding integrins are important mediators of the pro-fibrotic response by activating latent TGF-ß at sites of injury and by providing myofibroblasts information about the composition and stiffness of the extracellular matrix. Therefore, blockade of RGD-binding integrins may have therapeutic potential for CKD. To test this idea, we used small-molecule peptidomimetics that potently inhibit a subset of RGD-binding integrins in a murine model of kidney fibrosis. Acute kidney injury leading to fibrosis was induced by administration of aristolochic acid. Continuous subcutaneous administration of CWHM-12, an RGD integrin antagonist, for 28 days improved kidney function as measured by serum creatinine. CWHM-12 significantly reduced Collagen 1 (Col1a1) mRNA expression and scar collagen deposition in the kidney. Protein and gene expression markers of activated myofibroblasts, a major source of extracellular matrix deposition in kidney fibrosis, were diminished by treatment. RNA sequencing revealed that inhibition of RGD integrins influenced multiple pathways that determine the outcome of the response to injury and of repair processes. A second RGD integrin antagonist, CWHM-680, administered once daily by oral gavage was also effective in ameliorating fibrosis. We conclude that targeting RGD integrins with such small-molecule antagonists is a promising therapeutic approach in fibrotic kidney disease.

cRGD target liposome delivery system promoted immunogenic cell death through enhanced anticancer potency of a thymidine conjugate under UVA activation as a cancer vaccine.

Conventional chemotherapeutic and photodynamic therapy have recently been shown to also elicit immune response against cancer through the immunogenic cell death mechanism, which can be potentially translated into effective cancer vaccines. However, there are few studies on the potential of nanodelivery system to promote the immunogenic cell death as a cancer vaccine. We reported here that cRGD target liposome delivery system was capable to promote the immunogenic cell death through enhanced potency of a thymidine conjugate post UVA activation as a cancer vaccine. Liposomes and cRGD target liposomes were found to significantly increase the cellular accumulation of the thymidine conjugate and subsequently translated into enhanced cytotoxic potency after UVA activation. More importantly, cRGD target liposomes of the thymidine conjugate markedly promoted the early detection of immunogenic cell death markers including ATP, HMGB1 and calreticulin. Subsequent in vivo vaccination-challenge study confirmed effective tumor growth inhibition by the cRGD liposomal thymidine conjugate and UVA treated cancer cells as the cancer vaccine. In addition, liposomes and cRGD target liposomes alone did not shown any induction of the immunogenic cell death markers, revealing the adjuvant nature of the nanodelivery system.

Effective suppression of the modified PHF6 peptide/1N4R Tau amyloid aggregation by intact curcumin, not its degradation products: Another evidence for the pigment as preventive/therapeutic "functional food".

Curcumin is a natural product with multiple biological activities and numerous potential therapeutic applications. In present study, the influence of curcumin and its degradation products (DPs) on the amyloid aggregation of Tau protein and the related PHF6 peptide were investigated. We provided experimental/theoretical evidence for suppressing effects of the compounds on the amyloid formation using far-UV CD as well as AFM, XRD and docking techniques and showed that the parent curcumin displayed stronger inhibition effect against Tau fibril aggregation. The obtained results suggest that the curcumin/DPs binding sites on the Tau molecule are likely to be the same, and provide a good structural basis to explain the efficient aggregation suppressing behavior of the curcumin, compared to the DPs. So, developing more stable curcumin nanoparticle formulations with improved curcumin bioavailability are of great importance. Curcumin's multi-functionality is also highly significant for the therapeutic application of this natural compound against various human diseases.

Inhibition of tau-derived hexapeptide aggregation and toxicity by a self-assembled cyclic d,l-α-peptide conformational inhibitor.

Aggregation and accumulation of amyloid ß and tau proteins to plaques and neurofibrillary tangles are the key pathogenic events in Alzheimer's disease. Here, we studied the capability of the cyclic d,l-α-peptide CP-2 as a conformational inhibitor of different amyloids to cross-interact with tau-derived AcPHF6 peptide and inhibit its aggregation, membrane perturbation and toxicity.

Synergistic Activity of Berberine with Azithromycin against Pseudomonas Aeruginosa Isolated from Patients with Cystic Fibrosis of Lung In Vitro and In Vivo.

BACKGROUND/AIMS: Pseudomonas aeruginosa (PA) is one of the major opportunistic pathogens which can cause chronic lung infection of cystic fibrosis (CF). The formation of PA biofilm promotes CF development and restricts the antimicrobial efficacies of current antibiotics. METHODS: The antimicrobial effects of azithromycin (AZM) and berberine (BER) alone and in combination were evaluated using microdilution method, checkerboard assay, time-kill test, qRT-PCR analysis and absorption method. The treatments of AZM and/or BER were further evaluated in an animal lung infection model via observing survival rate, bacterial burden and histopathology of lung, the levels of pro-/anti-inflammatory cytokines. RESULTS: AZM-BER were demonstrated to be synergistic against ten clinical PA isolates as well as the standard reference PA ATCC27853, in which PA03 was the most susceptible isolate to AZM-BER with FICI of 0.13 and chosen for subsequent experiments. The synergism of AZM-BER was further confirmed against PA03 in time-kill test and scanning electron microscope (SEM) at their concentrations showing synergism. In PA03, we found that AZM-BER could significantly attenuate productions of a series of virulence factors including alginate, LasA protease, LasB protease, pyoverdin, pyocyanin, chitinase as well as extracellular DNA, and remarkably inhibit the levels of quorum sensing (QS) molecules and the expressions of lasI, lasR, rhlI, rhlR at 1/2×MIC, 1×MIC and 2×MIC. In the infection model, the mice survival were increased markedly, the inflammations of infected lungs were improved greatly along with reduced IL-6, IL-8 and ascended IL-10 at 0.8 mg/kg of AZM combined with 3.2 mg/kg of BER. CONCLUSION: BER might be a promising synergist to enhance the antimicrobial activity of AZM in vitro and in vivo.

In vitro effects of cysteine protease inhibitors on Trichomonas foetus-induced cytopathic changes in porcine intestinal epithelial cells.

OBJECTIVE To investigate the effects of specific cysteine protease (CP) inhibitors on cytopathic changes to porcine intestinal epithelial cells induced by Tritrichomonas foetus isolated from naturally infected cats. SAMPLE T foetus isolates from 4 naturally infected cats and nontransformed porcine intestinal epithelial cells. PROCEDURES T foetus isolates were treated with or without 0.1 to 1.0mM of the CP inhibitors antipain, cystatin, leupeptin, and chymostatin and the vinyl sulfone inhibitors WRR-483 and K11777. In-gel gelatin zymography was performed to evaluate the effects of these inhibitors on CP activity of T foetus isolates. Each treated or untreated isolate was also cocultured with monolayers of porcine intestinal epithelial cells for 24 hours, and cytopathic effects of T foetus were evaluated by light microscopy and crystal violet spectrophotometry. RESULTS Results of in-gel gelatin zymography suggested an ability of WRR-483, K11777, and cystatin to target specific zones of CP activity of the T foetus isolates. These inhibitors had no effect on T foetus growth, and the cytopathic changes to the intestinal epithelium induced by all 4 T foetus isolates were significantly inhibited. CONCLUSIONS AND CLINICAL RELEVANCE This study revealed that certain protease inhibitors were capable of inhibiting regions of CP activity (which has been suggested to cause intestinal cell damage in cats) in T foetus organisms and of ameliorating T foetus-induced cytopathic changes to porcine intestinal epithelium in vitro. Although additional research is needed, these inhibitors might be useful in the treatment of cats with trichomonosis.

PET imaging with [68Ga]NOTA-RGD for prostate cancer: a comparative study with [¹8F]fluorodeoxyglucose and [¹8F]fluoroethylcholine.

The α(v)ß3 integrin is highly expressed in prostate cancer (PCa), in which it is a key player in tumour invasion, angiogenesis and metastasis formation. Therefore, α(v)ß3 integrin is considered a very promising target for molecular imaging of PCa. This study tested the potential of the novel α(v)ß3 integrin affine agent [68Ga]NOTA-RGD in comparison with the established [¹8F]fluoroethylcholine (FEC) and [¹8F]fluorodeoxyglucose (FDG) for assessing PCa using positron emission tomography (PET). [68Ga]NOTA-RGD showed a lower uptake in PC-3 and DU-145 cells compared with FEC and FDG. µPET imaging studies showed a good delineation of the PCa xenografts in mice. The means tumor-to-muscle and tumor-to-bone-ratio amounted 5.1 ± 1.4 and 5.2 ± 1.2 for [68Ga]NOTA-RGD compared with 2.6 ± 0.9 and 2.9 ± 1.6 for FDG, and 2.4 ± 0.7 and 0.8 ± 0.2 for FEC, respectively. The uptake of [68Ga]NOTA-RGD into tumor was fully inhibited by c(RGDfV), known to bind specifically to α(v)ß3 integrin, confirming the specificity of the tumor uptake in vivo. These results suggest that [68Ga]NOTA-RGD is a promising candidate for PET imaging of α(v)ß3 integrin expression in PCa and warrant further in vivo validations to ascertain its potential as an imaging agent for clinical use. The simple and fast preparation of [68Ga]NOTA-RGD may greatly facilitate its translation to a clinical setting.

Development of a Novel Screening Strategy Designed to Discover a New Class of HIV Drugs.

Current antiretroviral treatments target multiple pathways important for human immunodeficiency virus (HIV) multiplication, including viral entry, synthesis and integration of the DNA provirus, and the processing of viral polyprotein precursors. However, HIV is becoming increasingly resistant to these "combination therapies." Recent findings show that inhibition of HIV Gag protein cleavage into its two structural proteins, matrix (MA) and capsid (CA), has a devastating effect on viral production, revealing a potential new target class for HIV treatment. Unlike the widely used HIV protease inhibitors, this new class of inhibitor would target the substrate, not the protease enzyme itself. This approach offers a distinct advantage in that inhibitors of MA/CA would only need to affect a subset of the Gag molecules to disable viral replication. To discover MA/CA-specific inhibitors, we constructed a modified MA/CA fusion peptide (MA/CAΔ) that contains the HIV protease (PR) cleavage site as well as a tetracysteine motif for fluorescent labeling. The HIV PR cleavage of MA/CAΔ can then be monitored via fluorescence polarization (FP). We have adapted this FP assay for high-throughput screening and validated it according to industry standards using a 384-well plate format. We have currently tested 24,000 compounds in this assay and here detail the screening methodology and the results of this screening campaign.

Sensitization of melanoma cells for TRAIL-induced apoptosis by BMS-345541 correlates with altered phosphorylation and activation of Bax.

Resistance to TRAIL (TNF-related apoptosis-inducing ligand)- induced apoptosis limits its therapeutic use. Different strategies of TRAIL sensitization and a dependency on Bax have been reported, but common principles of TRAIL resistance and the way of Bax activation remained poorly understood. Applying a melanoma model of TRAIL-sensitive and -resistant cell lines, efficient sensitization for TRAIL-induced apoptosis is demonstrated by the kinase inhibitor BMS-345541 (N-(1,8-dimethylimidazo(1,2-a)quinoxalin-4-yl)-1,2-ethanediamine hydrochloride), which targets IκB (inhibitor of κB proteins) kinase ß (IKKß). This effect was completely abrogated by Bax knockout as well as by Bcl-2 overexpression, in accordance with a Bax dependency. Early loss of the mitochondrial membrane potential, release of cytochrome c and Smac (second mitochondria-derived activator of caspases) clearly indicated the activation of mitochondrial apoptosis pathways. Of note, BMS-345541 alone resulted in an early Bax activation, seen by conformational changes and by Bax translocation. The synergistic effects can be explained by Bid activation through TRAIL, which inhibits Bcl-2, and the activation of Bax through BMS-345541. The critical roles of XIAP (X-chromosome-linked inhibitor of apoptosis protein), Smac and Bid were clearly proven by overexpression and siRNA knockdown, respectively. The way of Bax activation by BMS-345541 was unraveled by establishing new assays for Bax activation. These showed reduction of the inactivating Bax phosphorylation at serine-184, while the activating Bax phosphorylation at threonine-167 was enhanced. Thus, modulation of Bax phosphorylation appeared as tightly related to TRAIL sensitivity/resistance in melanoma cells, and therapeutic strategies may be considered.

The novel proteasome inhibitor BSc2118 protects against cerebral ischaemia through HIF1A accumulation and enhanced angioneurogenesis.

Only a minority of stroke patients receive thrombolytic therapy. Therefore, new therapeutic strategies focusing on neuroprotection are under review, among which, inhibition of the proteasome is attractive, as it affects multiple cellular pathways. As proteasome inhibitors like bortezomib have severe side effects, we applied the novel proteasome inhibitor BSc2118, which is putatively better tolerated, and analysed its therapeutic potential in a mouse model of cerebral ischaemia. Stroke was induced in male C57BL/6 mice using the intraluminal middle cerebral artery occlusion model. BSc2118 was intrastriatally injected 12 h post-stroke in mice that had received normal saline or recombinant tissue-plasminogen activator injections during early reperfusion. Brain injury, behavioural tests, western blotting, MMP9 zymography and analysis of angioneurogenesis were performed for up to 3 months post-stroke. Single injections of BSc2118 induced long-term neuroprotection, reduced functional impairment, stabilized blood-brain barrier through decreased MMP9 activity and enhanced angioneurogenesis when given no later than 12 h post-stroke. On the contrary, recombinant tissue-plasminogen activator enhanced brain injury, which was reversed by BSc2118. Protein expression of the transcription factor HIF1A was significantly increased in saline-treated and recombinant tissue-plasminogen activator-treated mice after BSc2118 application. In contrast, knock-down of HIF1A using small interfering RNA constructs or application of the HIF1A inhibitor YC1 (now known as RNA-binding motif, single-stranded-interacting protein 1 (RBMS1)) reversed BSc2118-induced neuroprotection. Noteworthy, loss of neuroprotection after combined treatment with BSc2118 and YC1 in recombinant tissue-plasminogen activator-treated animals was in the same order as in saline-treated mice, i.e. reduction of recombinant tissue-plasminogen activator toxicity through BSc2118 did not solely depend on HIF1A. Thus, the proteasome inhibitor BSc2118 is a promising new candidate for stroke therapy, which may in addition alleviate recombinant tissue-plasminogen activator-induced brain toxicity.

Tumor angiogenesis phenotyping by nanoparticle-facilitated magnetic resonance and near-infrared fluorescence molecular imaging.

One of the challenges of tailored antiangiogenic therapy is the ability to adequately monitor the angiogenic activity of a malignancy in response to treatment. The α(v)ß(3) integrin, highly overexpressed on newly formed tumor vessels, has been successfully used as a target for Arg-Gly-Asp (RGD)-functionalized nanoparticle contrast agents. In the present study, an RGD-functionalized nanocarrier was used to image ongoing angiogenesis in two different xenograft tumor models with varying intensities of angiogenesis (LS174T > EW7). To that end, iron oxide nanocrystals were included in the core of the nanoparticles to provide contrast for T(2)*-weighted magnetic resonance imaging (MRI), whereas the fluorophore Cy7 was attached to the surface to enable near-infrared fluorescence (NIRF) imaging. The mouse tumor models were used to test the potential of the nanoparticle probe in combination with dual modality imaging for in vivo detection of tumor angiogenesis. Pre-contrast and post-contrast images (4 hours) were acquired at a 9.4-T MRI system and revealed significant differences in the nanoparticle accumulation patterns between the two tumor models. In the case of the highly vascularized LS174T tumors, the accumulation was more confined to the periphery of the tumors, where angiogenesis is predominantly occurring. NIRF imaging revealed significant differences in accumulation kinetics between the models. In conclusion, this technology can serve as an in vivo biomarker for antiangiogenesis treatment and angiogenesis phenotyping.

RGD-based strategies to target alpha(v) beta(3) integrin in cancer therapy and diagnosis.

The integrin α(v)ß(3) plays an important role in angiogenesis. It is expressed on tumoral endothelial cells as well as on some tumor cells. RGD peptides are well-known to bind preferentially to the α(v)ß(3) integrin. In this context, targeting tumor cells or tumor vasculature by RGD-based strategies is a promising approach for delivering anticancer drugs or contrast agents for cancer therapy and diagnosis. RGD-based strategies include antagonist drugs (peptidic or peptidomimetic) of the RGD sequence, RGD-conjugates, and the grafting of the RGD peptide or peptidomimetic, as targeting ligand, at the surface of nanocarriers. Although all strategies are overviewed, this review aims to particularly highlight the position of RGD-based nanoparticles in cancer therapy and imaging. This review is divided into three parts: the first one describes the context of angiogenesis, the role of the integrin α(v)ß(3), and the binding of the RGD peptide to this integrin; the second one focuses on RGD-based strategies in cancer therapy; while the third one focuses on RGD-based strategies in cancer diagnosis.

Transport of hepcidin, an iron-regulatory peptide hormone, into retinal pigment epithelial cells via oligopeptide transporters and its relevance to iron homeostasis.

Retinal pigment epithelial cells (RPE) express two transport systems (SOPT1 and SOPT2) for oligopeptides. Hepcidin is an iron-regulatory peptide hormone consisting of 25 amino acids. This hormone binds to ferroportin, an iron exporter expressed on the cell surface, and facilitates its degradation. Here we investigated if hepcidin is a substrate for SOPT1 and SOPT2 and if the hormone has any intracellular function in RPE. Hepcidin inhibited competitively the uptake of deltorphin II (a synthetic oligopeptide substrate for SOPT1) and DADLE (a synthetic oligopeptide substrate for SOPT2) with IC50 values in the range of 0.4-1.7 µM. FITC-hepcidin was taken up into RPE, and this uptake was inhibited by deltorphin II and DADLE. The entry of FITC-hepcidin into cells was confirmed by flow cytometry. Incubation of RPE with hepcidin decreased the levels of ferroportin mRNA. This effect was not a consequence of hepcidin-induced ferroportin degradation because excessive iron accumulation in RPE, which is expected to occur in these cells as a result of ferroportin degradation, did not decrease but instead increased the levels of ferroportin mRNA. This study reveals for the first time a novel intracellular function for hepcidin other than its established cell surface action on ferroportin.

The PI-3 kinase-Akt-MDM2-survivin signaling axis in high-risk neuroblastoma: a target for PI-3 kinase inhibitor intervention.

PURPOSE: Studies of SF1126, an RGDS targeted, water-soluble prodrug of LY294002, are currently nearing completion in two adult Phase I trials. Herein, we performed a preclinical evaluation of SF1126 as a PI-3K inhibitor for Phase I trials in the treatment of recurrent neuroblastoma (NB). METHODS: The effects of SF1126 on pAkt-MDM2 cell signaling, proliferation, apoptosis, and migration were determined using a panel of NB cell lines, and anti-tumor activity was determined using a xenograft model of NB. RESULTS: SF1126 blocks MDM2 activation, IGF-1 induced activation of Akt, and the upregulation of survivin induced by IGF-1. It also increases sensitivity to doxorubicin in vitro and was found to exhibit marked synergistic activity in combination with doxorubicin. Treatment disrupts the integrin αvß3/αvß5-mediated organization of the actin cytoskeleton as well as the α4ß1/α5ß1-mediated processes essential to metastasis. In vivo, SF1126 markedly inhibits tumor growth in NB xenografted mice (P < 0.05). CONCLUSIONS: A pan PI-3 kinase inhibitor has potent antitumor activity and induces apoptosis in multiple neuroblastoma cell lines. The observed effects of SF1126 on the p-Akt-MDM2-survivin axis suggest a patient selection paradigm in which NB tumors with increased pAkt-MDM2-survivin signaling may predict response to SF1126 alone or in combination with standard chemotherapy regimens that contain anthracyclines.

Inhibition of oligopeptide transporter suppress growth of human pancreatic cancer cells.

Oligopeptide transporters are abundantly expressed in various types of cancer cells. We here synthesized two novel dipeptides, l-phenylalanyl sarcosine (Phe-Sar) and 4-(4-methoxyphenyl)-l-phenylalanyl sarcosine (Bip(OMe)-Sar), and examined their effect on the growth of human pancreatic cancer AsPC-1 cells, which are known to highly express oligopeptide transporter PEPT1/SLC15A1. Growth of AsPC-1 cells was inhibited by these two peptides and a typical PEPT1/SLC15A1 substrate Gly-Sar. Growth inhibition by Gly-Sar, Phe-Sar and Bip(OMe)-Sar was concentration-dependent with half-maximal inhibitory concentration of 50, 0.91 and 0.55mM, respectively. These peptides also inhibited PEPT1-mediated [(3)H]Gly-Sar uptake with half-maximal inhibitory concentration of 2.6, 0.81 and 0.27mM, respectively. Thus, the rank order of the tumor cell growth inhibition by these three peptides was the same as that of PEPT1-inhibitory activity. Growth of AsPC-1 cells was also inhibited by 2-aminobicyclo(2,2,1)heptane-2-carboxylic acid (BCH), which is a typical inhibitor of amino acid transporter system L. The growth inhibition by BCH and Gly-Sar was additive, suggesting that these compounds act at distinct loci. Oligopeptide transporters thus appear to be a promising target for inhibition of pancreatic cancer progression. These results also proposed the idea that oligopeptide transporter is required for growth of AsPC-1 cells.

Critical roles of kisspeptins in female puberty and preovulatory gonadotropin surges as revealed by a novel antagonist.

Kisspeptins (Kp) have recently emerged as master regulators of the reproductive axis and among the most potent elicitors of GnRH-gonadotropin secretion. Despite their paramount importance in reproductive physiology and their potential therapeutic implications, development of Kp antagonists has remained elusive, and only recently has the first compound with the ability to block Kp actions in vitro and in vivo, namely p234, been reported. However, previous in vivo studies all used acute central injections, whereas characterization of the effects of the antagonist after continuous or systemic administration, which poses pharmacological challenges, is still pending. We report herein a comprehensive series of analyses on the impact of continuous intracerebroventricular infusion of p234 on puberty onset and the preovulatory surge of gonadotropins in the female rat. In addition, the effects of systemic (ip) administration of a tagged p234-penetratin, with a predicted higher permeability at the blood-brain barrier, on Kp-10 induced gonadotropin secretion were evaluated. Central infusion of p234 to pubertal females delayed vaginal opening and decreased uterine and ovarian weights at the expected time of puberty, without affecting body weight. Likewise, chronic intracerebroventricular administration of p234 for 4 d prevented the preovulatory surges of LH and FSH. In addition, systemic (ip) administration of p234-penetratin significantly attenuated acute LH and FSH responses to Kp-10, either after intracerebroventricular or ip injection of Kp. Our data document the validity of p234 for antagonizing Kp actions in vivo and provide direct experimental evidence for the important role of Kp signaling in the key events of female reproduction, such as puberty onset and the preovulatory surge of gonadotropins.

Kisspeptin signalling in the hypothalamic arcuate nucleus regulates GnRH pulse generator frequency in the rat.

BACKGROUND: Kisspeptin and its G protein-coupled receptor (GPR) 54 are essential for activation of the hypothalamo-pituitary-gonadal axis. In the rat, the kisspeptin neurons critical for gonadotropin secretion are located in the hypothalamic arcuate (ARC) and anteroventral periventricular (AVPV) nuclei. As the ARC is known to be the site of the gonadotropin-releasing hormone (GnRH) pulse generator we explored whether kisspeptin-GPR54 signalling in the ARC regulates GnRH pulses. METHODOLOGY/PRINCIPAL FINDINGS: We examined the effects of kisspeptin-10 or a selective kisspeptin antagonist administration intra-ARC or intra-medial preoptic area (mPOA), (which includes the AVPV), on pulsatile luteinizing hormone (LH) secretion in the rat. Ovariectomized rats with subcutaneous 17beta-estradiol capsules were chronically implanted with bilateral intra-ARC or intra-mPOA cannulae, or intra-cerebroventricular (icv) cannulae and intravenous catheters. Blood samples were collected every 5 min for 5-8 h for LH measurement. After 2 h of control blood sampling, kisspeptin-10 or kisspeptin antagonist was administered via pre-implanted cannulae. Intranuclear administration of kisspeptin-10 resulted in a dose-dependent increase in circulating levels of LH lasting approximately 1 h, before recovering to a normal pulsatile pattern of circulating LH. Both icv and intra-ARC administration of kisspeptin antagonist suppressed LH pulse frequency profoundly. However, intra-mPOA administration of kisspeptin antagonist did not affect pulsatile LH secretion. CONCLUSIONS/SIGNIFICANCE: These data are the first to identify the arcuate nucleus as a key site for kisspeptin modulation of LH pulse frequency, supporting the notion that kisspeptin-GPR54 signalling in this region of the mediobasal hypothalamus is a critical neural component of the hypothalamic GnRH pulse generator.

Mouse AMACO, a kidney and skin basement membrane associated molecule that mediates RGD-dependent cell attachment.

The VWA domain-containing extracellular matrix protein AMACO has not been extensively characterized and its function remains unknown. It has been proposed as a potential cancer marker and carries a rare O-glucosylation and O-fucosylation on its first EGF-like domain. AMACO is a basement membrane associated protein, however its exact localization has not been determined. Here we show by immunogold electron microscopy of mouse kidney and skin that AMACO does not occur within the basement membrane but rather subjacent to the basement membrane at its stromal surface. In skin, AMACO often colocalizes with triple-helical domains of collagen VII containing anchoring fibrils as they emerge from the basal lamina. However, the immunogold patterns for AMACO and the C-terminal end of collagen VII show discrete differences, indicating that AMACO and collagen VII do not colocalize at anchoring plaques. In contrast, the localization pattern of AMACO partially overlaps with that for collagen XVIII. In addition, mouse AMACO was shown to support beta1 integrin-mediated adhesion of a keratinocyte-like cell line, HaCaT, and a fibroblast cell line, Wi26, in an RGD-dependent manner, most likely using an RGD-motif near the C-terminus of AMACO. However, the loss of cell adhesion to the C-terminal part of the human AMACO, due to the unique absence of an RGD sequence in the human protein, suggests that cell adhesion is not AMACO's major function.