Eosinophil-derived TGFß1 controls the new bone formation in chronic rhinosinusitis with nasal polyps

BACKGROUND: Chronic rhinosinusitis with nasal polyps (CRSwNP) is characterized by chronic eosinophilic inflammation and new bone formation (NBF). These processes may be associated with each other in the pathogenesis and influence the severity and prognosis of the disease. However, it is still unclear how eosinophilic inflammation is involved in the NBF. METHODOLOGY: Sinus bone cells were isolated from ethmoid bone tissues of patients with CRSwNP and controls. Transforming growth factor beta 1 (TGFß1) and alkaline phosphatase (ALP) expression in sinus bone cells was determined using quantitative RT-PCR, immunoblotting, and immunohistochemistry. The co-localization of TGFß1 with eosinophils was assessed by immunofluorescence staining. Sinus bone cells were co-cultured with eosinophils (Eol-1 cell line), which were differentiated with butyrate, to measure the osteoblast differentiation activity of sinus bone cells. RESULTS: TGFß1 expression was increased in sinus bone tissues and correlated with CT scores in CRSwNP. TGFß1 was also increased in the submucosa of CRSwNP and co-localized predominantly with eosinophils compared with neutrophils Differentiated Eol-1 cells-derived TGFß1 increased ALP expression in sinus bone cells. Treatment with a TGFß inhibitor attenuated TGFß1-induced ALP expression and staining in sinus bone cells of CRSwNP, leading to loss of bone formation. CONCLUSIONS: Eosinophil-derived TGFß1 was enriched in the submucosa of CRSwNP, which induced ALP expression in sinus bone cells and NBF. Therefore, eosinophil-derived TGFß1 may mediate aberrant bone remodeling in CRSwNP.

Clinical significance of nutritional risk screening tool for hospitalised children with acute burn injuries: a cross-sectional study.

BACKGROUND: We assessed the nutritional risks among children hospitalised with acute burn injuries and their associated clinical outcomes using three nutritional risk screening (NRS) tools: Screening Tool for Risk of Impaired Nutritional Status and Growth (STRONGKIDS ), Pediatric Yorkhill Malnutrition Score (PYMS) and Screening Tool for the Assessment for Malnutrition in Pediatrics (STAMP). METHODS: This prospective cross-sectional study was conducted from October 2015 to November 2016, in a regional burn centre. Patients were screened by two independent observers, using the three NRS tools. RESULTS: A total of 100 children aged 3 months to 16.5 years were included. STRONGKIDS identified 16% of patients as having high risk, with being identified 45% by PYMS and 44% by STAMP. After adjustment for confounding factors in multivariate regression analysis, patients in the high-risk group had significantly longer median (SD) lengths of stay [medium versus high risk: STRONGKIDS , 9.5 (6.6) versus 15.0 (24.2) days; PYMS, 8.5 (4.4) versus 13.0 (16.1) days; STAMP, 9.0 (5.7) versus 11.0 (17.4) days] and greater median (SD) weight loss [medium versus high risk: STRONGKIDS, 0.15 (0.8) versus -0.35 (0.8) kg; STAMP, 0.5 (0.7) versus 0 (0.1) kg] than patients in the medium-risk group (P < 0.05). The strengths of agreement in the nutritional risk classification between the two observers were good (κ for STRONGKIDS = 0.61; PYMS = 0.79; STAMP = 0.75) (P < 0.01). CONCLUSIONS: The STRONGKIDS , PYMS and STAMP tools could be useful and practical for determining which hospitalised children with acute burn injuries will need additional nutritional intervention.

LSD1 demethylates HIF1α to inhibit hydroxylation and ubiquitin-mediated degradation in tumor angiogenesis.

Lysine-specific demethylase 1 (LSD1), which has been considered as a potential therapeutic target in human cancer, has been known to regulate many biological functions through its non-histone substrates. Although LSD1-induced hypoxia-inducible factor alpha (HIF1α) demethylation has recently been proposed, the effect of LSD1 on the relationship between HIF1α post-translational modifications (PTMs) and HIF1α-induced tumor angiogenesis remains to be elucidated. Here, we identify a new methylation site of the HIF1α protein antagonized by LSD1 and the interplay between HIF1α protein methylation and other PTMs in regulating tumor angiogenesis. LSD1 demethylates HIF1α at lysine (K) 391, which protects HIF1α against ubiquitin-mediated protein degradation. LSD1 also directly suppresses PHD2-induced HIF1α hydroxylation, which has a mutually dependent interplay with Set9-mediated HIF1α methylation. Moreover, the HIF1α acetylation that occurs in a HIF1α methylation-dependent manner is inhibited by the LSD1/NuRD complex. HIF1α stabilized by LSD1 cooperates with CBP and MTA1 to enhance vascular endothelial growth factor (VEGF)-induced tumor angiogenesis. Thus, LSD1 is a key regulator of HIF1α/VEGF-mediated tumor angiogenesis by antagonizing the crosstalk between PTMs involving HIF1α protein degradation.

Meal patterns across ten European countries - results from the European Prospective Investigation into Cancer and Nutrition (EPIC) calibration study.

OBJECTIVE: To characterize meal patterns across ten European countries participating in the European Prospective Investigation into Cancer and Nutrition (EPIC) calibration study. DESIGN: Cross-sectional study utilizing dietary data collected through a standardized 24 h diet recall during 1995-2000. Eleven predefined intake occasions across a 24 h period were assessed during the interview. In the present descriptive report, meal patterns were analysed in terms of daily number of intake occasions, the proportion reporting each intake occasion and the energy contributions from each intake occasion. SETTING: Twenty-seven centres across ten European countries. SUBJECTS: Women (64 %) and men (36 %) aged 35-74 years (n 36 020). RESULTS: Pronounced differences in meal patterns emerged both across centres within the same country and across different countries, with a trend for fewer intake occasions per day in Mediterranean countries compared with central and northern Europe. Differences were also found for daily energy intake provided by lunch, with 38-43 % for women and 41-45 % for men within Mediterranean countries compared with 16-27 % for women and 20-26 % for men in central and northern European countries. Likewise, a south-north gradient was found for daily energy intake from snacks, with 13-20 % (women) and 10-17 % (men) in Mediterranean countries compared with 24-34 % (women) and 23-35 % (men) in central/northern Europe. CONCLUSIONS: We found distinct differences in meal patterns with marked diversity for intake frequency and lunch and snack consumption between Mediterranean and central/northern European countries. Monitoring of meal patterns across various cultures and populations could provide critical context to the research efforts to characterize relationships between dietary intake and health.

Loss of the polycomb protein Mel-18 enhances the epithelial-mesenchymal transition by ZEB1 and ZEB2 expression through the downregulation of miR-205 in breast cancer.

The epithelial-mesenchymal transition (EMT) is the pivotal mechanism underlying the initiation of cancer invasion and metastasis. Although Mel-18 has been implicated in several biological processes in cancer, its function in the EMT of human cancers has not yet been studied. Here, we demonstrate that Mel-18 negatively regulates the EMT by epigenetically modulating miR-205. We identified miR-205 as a novel target of Mel-18 using a microRNA microarray analysis and found that Mel-18 increased miR-205 transcription by the inhibition of DNA methyltransferase-mediated DNA methylation of the miR-205 promoter, thereby downregulating its target genes, ZEB1 and ZEB2. Furthermore, the loss of Mel-18 promoted ZEB1- and ZEB2-mediated downregulation of E-cadherin transcription and also enhanced the expression of mesenchymal markers, leading to increased migration and invasion in MCF-7 cells. In MDA-MB-231 cells, Mel-18 overexpression restored E-cadherin expression, resulting in reduced migration and invasion. These effects were reversed by miR-205 overexpression or inhibition. A tumor xenograft with Mel-18 knockdown MCF-7 cells consistently showed increased ZEB1 and ZEB2 expression and decreased E-cadherin expression. Taken together, these results suggest that Mel-18 functions as a tumor suppressor by its novel negative control of the EMT, achieved through regulating the expression of miR-205 and its target genes, ZEB1 and ZEB2.

Serotonin induces melanogenesis via serotonin receptor 2A.

BACKGROUND: Serotonin (5-hydroxytryptamine, 5-HT) levels are increased by light exposure but the role and mechanism of 5-HT in the pigmentation of skin cells are unclear. OBJECTIVES: To clarify the effect of 5-HT on melanogenesis and to determine the 5-HT receptor subtype involved. METHODS: B16F10, SK-MEL-2 and Melan-A cells were cultured in Dulbecco's modified Eagle's medium with low fetal bovine serum. The three cell lines were treated with various concentrations of 5-HT, and 5-HT receptor agonists and antagonists. The involvement of the 5-HT receptor 2A (5-HTR2A) was examined by gene silencing and use of 5-HTR2A antagonists. RESULTS: 5-HT and the 5-HTR2A agonist, DOI, increased melanogenesis in the three cell lines. These increased events were suppressed by 5-HTR2A antagonists or gene silencing of the HTR2A gene. CONCLUSIONS: 5-HTR2A is involved in melanogenesis. These findings highlight the role of 5-HT and suggest new ways of controlling melanogenesis.

Modulation of firing activity by ATP in dopamine neurons of the rat substantia nigra pars compacta.

ATP acts as a neurotransmitter or co-neurotransmitter in many areas of the CNS and peripheral nervous systems; however, little is known about the expression and functional role of purinoceptors (P2) in midbrain dopaminergic neurons. Therefore, we investigated P2X receptor expression and regulation of spontaneous firing activity in dopaminergic neurons of the substantia nigra pars compacta (SNc) in rats using patch-clamp and Ca(2+)-imaging techniques. In most neurons, application of ATP (1 microM-1 mM) increased firing rate dose-dependently (EC(50)=1.26+/-0.26 microM, n=45). When the P2-receptor agonists such as 2-methylthio-adenosine 5'-triphosphate (2-MeSATP) or ATPgammaS were applied or pressure-applied to the neuron, the firing activity increased together with a rise in cytosolic Ca(2+) concentration ([Ca(2+)]c), but application of beta,gamma-methylene ATP (P2X(1, 3) agonist) or methylthio-adenosine 5'-diphosphate (P2Y(1) agonist) had no effect. In many neurons, the effect of ATP was abolished by the application of the P2-receptor antagonists, suramin or pyridoxal-phosphate-6-azophenyl-2',4'-disulfonic acid (PPADS). When ATP was applied in a Ca(2+)-free solution, there was no detectable change in [Ca(2+)]c, suggesting that ATP does not release Ca(2+) from intracellular stores. In the single-cell reverse transcription polymerase chain reaction (RT-PCR), we found that 65% of dopaminergic neurons expressed mRNAs for P2X receptors; positive amplifications of P2X(6) (57.1%), P2X(2/6) (25.0%), and P2X(4) mRNA (17.9%), respectively. From the above results, we could conclude that ATP modulates firing activities in the rat SNc dopaminergic neurons, possibly via P2X(2), P2X(2/6), and/or P2X(4) receptors.

Steroid-sparing effects of pentoxifylline in pulmonary sarcoidosis.

BACKGROUND: Agents that target pro-inflammatory cytokines may be useful in pulmonary sarcoidosis. OBJECTIVE: To determine effectiveness of a non-selective cyclic nucleotide phosphodiesterase (PDE) inhibitor, pentoxifylline (POF). DESIGN: Randomized, double-blind, placebo-controlled trial, SETTING: Clinical Research Center, National Institutes of Health. PATIENTS: 27 patients with biopsy-confirmed pulmonary sarcoidosis receiving prednisone. INTERVENTION: Placebo or POF (1200-2000 mg/day) for 10 months, as prednisone was tapered. MEASUREMENTS: Primary endpoints: sustained improvement in two or more pulmonary function parameters, or a combination of one pulmonary function parameter and dyspnea. RESULTS: Except for one patient, primary endpoints were not reached in POF-treated patients. Therefore, a post hoc analysis was performed. The observed relative risk reduction for flares associated with POF treatment was 54.9% (95% CI 0.21, 0.89) and the absolute risk reduction was 50.6% (95% CI 0.22, 0.80). Compared to placebo treatment, in the POF group, the mean prednisone dose was lower at 8 and 10 months (p = 0.007 and 0.01 respectively), and there was a trend towards less prednisone usage over the entire study period (p = 0.053), as determined by cumulative change analysis. CONCLUSIONS: Although our exploratory post hoc analysis suggested that POF reduced flares and had steroid-sparing effects, given the study limitations, definitive conclusions cannot be drawn regarding the efficacy of POF in pulmonary sarcoidosis. In addition, gastrointestinal side-effects, at the doses used, would seem to limit the use of POF in treating pulmonary sarcoidosis. Overall, however, this trial may provide a basis for using more specific, better-tolerated, PDE inhibitors in future clinical trials.

The clinical role of IL-23p19 in patients with rheumatoid arthritis.

OBJECTIVE: To determine the clinical implications of the over-expression of synovial and circulating interleukin (IL)-23p19 and the correlation between IL-23p19 and other cytokines such as IL-17, tumour necrosis factor (TNF)alpha, and IL-1beta in rheumatoid arthritis (RA). METHODS: Synovial fluid (SF) and sera of 22 patients with RA were obtained during knee arthrocentesis and stored at -20 degrees C. Tender/swollen joint counts, 100-mm visual analogue scale (VAS), erythrocyte sedimentation rate (ESR), C-reactive protein (CRP), rheumatoid factor (RF), and antibodies to cyclic citrullinated peptide (anti-CCP Ab) were measured. Bony erosions were determined by X-rays. Serum and SF IL-23p19, IL-17, TNFalpha, and IL-1beta concentrations were measured by sandwich enzyme-linked immunosorbent assay (ELISA). RESULTS: The concentration of IL-23p19 correlated with the concentration of IL-17 in SF and sera, and with the concentrations of TNFalpha and IL-1beta in sera. SF IL-23p19 concentration was higher in patients who had bony erosions than those who had not. However, there was no correlation between IL-23p19 concentrations and other clinical parameters of RA. CONCLUSION: Upregulated IL-23p19 in SF might be involved in joint destruction in RA through interplay with other cytokines such as IL-17, TNFalpha, and IL-1beta.

Terminal nerve gonadotrophin-releasing hormone (GnRH) neurones express multiple GnRH receptors in a teleost, the dwarf gourami (Colisa lalia).

Gonadotophin-releasing hormone (GnRH) peptide released from the terminal nerve (TN)-GnRH neurones of the dwarf gourami primarily modifies the electrical properties of various neurones, including the TN-GnRH neurones themselves. However, our knowledge on the expression of GnRH receptors (GnRHRs) in the TN-GnRH neurones is still limited. Here, we used the single-cell reverse transcriptase-polymerase chain reaction after whole-cell patch-clamp recording to study the distribution of various GnRHR types expressed in the individual TN-GnRH neurones. We found that TN-GnRH neurones express two of the three types of GnRHRs cloned in the dwarf gourami: GnRHR1-2 and -R2, but not -R1-1. Furthermore, in agreement with our previous findings, all TN-GnRH neurones contained mRNAs of salmon GnRH but not chicken GnRH-II.

Identification and molecular characterization of three GnRH ligands and five GnRH receptors in the spotted green pufferfish.

Gonadotropin-releasing hormone (GnRH) is thought to have diverse physiological functions. Understanding regulatory mechanisms of GnRH functions requires detailed knowledge of gene expressions of both GnRH ligands and receptors in a single species. This report concerns identification and molecular characterization of GnRH ligands and receptors in the spotted green pufferfish Tetraodon nigroviridis. It was identified that the pufferfish possessed three types of GnRH ligands and five types of GnRH receptors. All types of ligands and receptors showed different expression patterns, and were widely expressed both inside and outside the brain. Gonads expressed all the ligand and receptor subtypes. Two of five receptor subtypes could not be detected in the pituitary gland of reproductively active individuals, suggesting the existence of novel GnRH systems independent of hypothalamic-pituitary-gonadal axis. Alternative splicing was also observed for some receptor subtypes. The present results indicate that diversified gene expressions combined with molecular diversity contribute to the functional diversity of GnRH.

Gender specific effect of neonatal handling on stress reactivity of adolescent rats.

Early neonatal handling of rat pups produces dampened hypothalamic-pituitary-adrenal axis reactivity to stress in adult male offspring. However, less is known about whether there is a similar effect for females. Although, most studies of neonatal handling have examined subsequent effects during adulthood, adolescence is an important developmental stage for stress responsivity. To address these issues, the effect of neonatal handling on the endocrine stress response and brain activity of male and female rats was determined in response to acute restraint stress during adolescence. Consistent with previous findings in adult males, neonatal handling reduced restraint stress-induced hormone levels in adolescent males. However, in contrast, we found elevated plasma hormone concentrations in handled females. A gender-specific handling effect on brain activity was also evident, with significantly increased stress-induced activation of the posterior cingulate cortex of handled females, as measured by c-fos mRNA expression. The striking gender difference in the effect of early neonatal handling provides evidence that this must be considered as an important variable in subsequent stress responsivity induced by early manipulations.

Fenofibrate lowers abdominal and skeletal adiposity and improves insulin sensitivity in OLETF rats.

The effect of peroxisome proliferator-activated receptor (PPAR)-alpha activators on the liver is well established, but the other effects on muscle and adipose tissue about lipid metabolism and insulin sensitivity are not clear. We investigated whether PPAR-alpha activation affects adiposity of skeletal muscle as well as adipose tissue and improves insulin sensitivity in spontaneous type 2 diabetes model, Otsuka Long-Evans Tokushima Fatty (OLETF) rats. Thirty-three weeks of aged, 20 male OLETF rats were divided into two groups. Control group (n=10) was fed with chow and treatment group (n=10) with chow contained fenofibrate for 7 weeks. At the age of 40 weeks, all rats were examined with MRI, intravenous glucose tolerance test, and then sacrificed for measurement of fat mass and RNA analyses. The total fat (the sum of subcutaneous, mesenteric, epididymal, and retroperitoneal fat pads) measured by dissection was significantly reduced in treatment group. The signal intensity of muscular adiposity was significantly decreased in treatment group. The mRNA levels of FAT/CD36 and mitochondrial carnitine palmitoyltransferase I (M-CPT I) in liver were remarkably increased. Fasting plasma insulin and leptin levels, insulin response after intravenous glucose loading and homeostasis model assessment insulin resistance (HOMA(IR)) index were lowered in treatment group. Fenofibrate increase mitochondrial fatty acid beta-oxidation in liver but not in skeletal muscle and lower the plasma levels of triglyceride and free fatty acid. It might result in reduction of adiposity of truncal adipose tissue and skeletal muscle. We suggest that reduction of adiposity in trunk and skeletal muscle might improve insulin sensitivity.

Nicotinic acetylcholine receptor-mediated release of [3H]norepinephrine from developing and adult rat hippocampus: direct and indirect mechanisms.

The primary role of nicotinic acetylcholine receptors in adult and developing brain is to modulate neurotransmission. Using in vitro neurotransmitter release, we have examined mechanisms underlying nicotine-induced [(3)H]norepinephrine release from developing and adult rat hippocampus. At birth, nicotine significantly stimulated hippocampal [(3)H]norepinephrine release with a monotonic increase in maximal drug effect over the first ten postnatal days. No developmental changes in agonist or antagonist potency were observed. Comparison of synaptosomal and slice preparations, as well as examination of the effects of tetrodotoxin, indicated that at least two nicotinic acetylcholine receptor populations regulated [(3)H]norepinephrine release from neonatal and adult hippocampus; one localized on noradrenergic terminals, the other on adjacent cells. To further characterize the indirect mechanism of nicotine action in the adult, we examined the effects of pharmacological blockade of various neurotransmitter systems that provide excitatory input to hippocampal noradrenergic terminals. Whereas glutamate and muscarinic receptor blockade was ineffective, the GABA-A receptor antagonists, bicuculline and picrotoxin, inhibited the indirect component of nicotine-mediated [(3)H]norepinephrine release. Furthermore, pentobarbital, an allosteric effector at GABA-A receptors, potentiated the effect of submaximal concentrations of nicotine. These findings are consistent with the hypothesis that nicotine-induced GABA release serves as an additional stimulus for [(3)H]norepinephrine secretion within rat hippocampus.

GnRH agonist Buserelin affects colony-forming efficiency of HHUA and Jurkat cells.

In this study, cell proliferation was examined at low and high cell densities, using HHUA and Jurkat cell lines as experimental models for the antiproliferative and proliferation-enhancing effects of GnRH agonist, Buserelin, respectively. For efficient evaluation of Buserelin activity at low cell density, the colony-forming efficiency assay was adopted. Buserelin markedly affected colony-forming efficiency in a dose-dependent manner at low cell density; however, Buserelin had no effect at high cell density. The conditioned medium of HHUA cells inhibited the Buserelin action, whereas that of Jurkat cells mimicked it. These results suggest that each cell line secretes some substances which regulate cell proliferation, and that these substances can also change the effects of Buserelin. The measurement of colony-forming efficiency is a very effective way of eliminating autocrine and/or paracrine effects, and is a highly sensitive method for measuring GnRH activity.

Patterns of chemokine expression in models of Schistosoma mansoni inflammation and infection reveal relationships between type 1 and type 2 responses and chemokines in vivo.

To explore the roles of chemokines in type 1 and type 2 responses in vivo, we examined mRNA expression for a panel of up to 17 chemokines in experimental mouse models using Schistosoma mansoni. These studies revealed that Mig (monokine induced by gamma interferon), cytokine-responsive gene 2/10-kDa interferon-inducible protein, RANTES, lymphotactin, macrophage inflammatory protein 1beta (MIP-1beta), JE/monocyte chemoattractant protein 1, and MIP-2 are associated with type 1 egg-induced responses and that thymus-derived chemotactic agent 3 (TCA3), eotaxin, MIP-1alpha, and MIP-1gamma are associated with type 2 egg-induced responses. After cercarial infection, both type 1-associated and type 2-associated chemokines were elevated in the livers of infected mice presensitized with eggs and recombinant interleukin-12 (rIL-12), a regimen that diminishes pathology. Neutralization of IL-12 or gamma interferon during egg deposition reversed the effects of prior treatment with rIL-12, leading to a return to larger granulomas; persistently elevated expression of TCA3, eotaxin, and MIP-1alpha; and a marked reduction in the expression of type 1-associated chemokines despite the maintenance of a dominant type 1 cytokine response in the draining lymph nodes. Our findings suggest that there are patterns of coordinate chemokine expression characteristic of type 1 and type 2 responses in vivo; that the cells recruited by a given pattern of chemokines may differ, depending on the composition of peripheral populations; and that patterns of tissue expression of chemokines may determine the character of an inflammatory response independently of the dominant pattern of differentiation of antigen-specific T cells. Our data reveal new relationships between chemokines and polarized immune responses and suggest that end organ inflammation might be altered by chemokine blockade without necessitating reversal of the phenotype of the majority of differentiated T cells.

A novel class of inhibitors for steroid 5alpha-reductase: synthesis and evaluation of umbelliferone derivatives.

A series of umbelliferone derivatives was prepared and their 5alpha-reductase type 1 inhibitory activities were evaluated in cell culture systems. Our studies have identified a new series of potent 5alpha-reductase type 1 inhibitors and provided the basis for further development for the treatment of human endocrine disorders associated with overproduction of DHT by 5alpha-reductase type 1. The preliminary structure-activity relationship was described to elucidate the essential structural requirements.

Structure and biological activity of gonadotropin-releasing hormone isoforms isolated from rat and hamster brains.

Rat and hamster brain tissues were used to investigate the possible existence of a follicle stimulating hormone (FSH)-releasing factor with similar characteristics to the lamprey gonadotropin-releasing hormone III (lGnRH-III) form proposed in previous reports. The present studies involved isolation and purification of the molecule by high-performance liquid chromatography (HPLC), identification by radioimmunoassay, sequence analysis by automated Edman degradation, mass spectrometry and examination of biological activity. Hypothalamic extracts from both species contained an HPLC fraction that was immunoreactive to GnRH and coeluted with lGnRH-III and 9-hydroxyproline mGnRH ([Hyp(9)]GnRH). Determination of primary structure from purified total brain material demonstrated that the isolated molecule was [Hyp(9)]GnRH. This is the first report showing the presence of the posttranslationally modified form already known as [Hyp(9)]GnRH by primary sequence analysis. The biological activity of distinct GnRH peptides was also tested in vitro for gonadotropin release using rat pituitary primary cell cultures. The results showed that [Hyp(9)]GnRH stimulated both luteinizing hormone and FSH release, as already reported, whereas lGnRH-III had no action on the secretion of either gonadotropin.

Antimetastatic and antitumor effects of benzoquinonoid AC7-1 from Ardisia crispa.

An antimetastatic and cytostatic substance, termed AC7-1, was isolated from Ardisia crispa and identified as a benzoquinonoid compound, 2-methoxy-6-tridecyl-1,4-benzoquinone. It was originally characterized as the potent PAF (platelet-activating factor) receptor-binding antagonist with nonspecific antiplatelet effects on platelet aggregation induced by various agonists including PAF, ADP, thrombin and collagen. The nonspecific antiaggregatory properties of AC7-1 drew our interest given its possible relationship in integrin receptor-binding antagonistic activity. The integrin receptor plays an important role in metastasis and thrombosis as the cell surface transmembrane protein. Based on the aforementioned facts, the antimetastatic activities of AC7-1 were examined using various in vitro and in vivo metastasis assays. AC7-1 strongly blocked B16-F10 melanoma cell adhesion to extracellular matrix (ECM) and B16-F10 melanoma cell invasion. AC7-1 also remarkably inhibited pulmonary metastasis and tumor growth in vivo. AC7-1 inhibited B16-F10 melanoma cell adhesion to only specific synthetic peptides including RGDS. These findings suggest that antimetastatic activities of AC7-1 can be caused by blocking integrin-mediated adherence. We found AC7-1 to be a potential candidate for the development of a new antimetastatic drug.

Altered gabaa receptor subunit and splice variant expression in rats treated with chronic intermittent ethanol.

BACKGROUND: Intermittent chronic administration of ethanol to rats has been shown previously to produce a hyperexcitable, kindling-like state, accompanied by reduced inhibitory synaptic transmission in the hippocampus and changes in gamma-aminobutyric acid type A (GABAA) receptors. Further information is needed on the detailed changes in GABAA receptors and their time course and persistence, as is comparison to changes after chronic, continuous ethanol. METHODS: GABAA receptors were analyzed in the rat brain after chronic intermittent ethanol (CIE) by using radioligand binding, photoaffinity labeling of polypeptides, and estimates of messenger RNA (mRNA) levels of receptor subunits by reverse transcriptase-polymerase chain reaction (RT-PCR) and in situ hybridization. RESULTS: CIE rats were confirmed to have increased GABAA receptor binding of the benzodiazepine partial inverse agonist and ethanol antidote ligand Ro15-4513, due to increased expression of the alpha6 subunit polypeptide in the cerebellum, shown by photoaffinity labeling. Estimates of mRNA levels by use of RT-PCR did not reveal any significant increase in alpha6 or in several other receptor subunits in several brain regions, but a decrease in the ratio of the long and short splice variants (L/S) of the gamma2 subunit was detected in the hippocampus, especially the CA1 region. CONCLUSIONS: Changes in GABAA receptors were found in rats given CIE. Increased alpha6 subunit in the cerebellum was demonstrated by using both the binding to diazepam-insensitive sites for [3H]Ro15-4513 and increased levels of the 57-kDa alpha6 polypeptide after photoaffinity labeling with this ligand. This increase appeared after 30 doses of ethanol and decayed to normal 1 week after ethanol was discontinued. The transient change in cerebellar alpha6 subunit-containing receptors, also reportedly seen after chronic continuous ethanol, is thus unlikely to account for the persistently hyperexcitable, kindled, seizure-susceptible state seen in CIE. However, the significant decrease in gamma2 subunit L/S splice variant ratio in the hippocampus implies changes in GABAA receptor function, possibly involving protein phosphorylation by protein kinase C. Altered receptor trafficking and turnover associated with synaptic plasticity may contribute to the observed reduced inhibition in the hippocampus and other signs of alcohol dependence produced by CIE.