Estrogen receptors in medaka (Oryzias latipes) and estrogenic environmental contaminants: an in vitro-in vivo correlation

In many vertebrates, estrogens are necessary to promote the growth and differentiation of the female reproductive system during development, and have important reproductive roles in both males and females. Medaka (Oryzias latipes) has three estrogen receptor (ER) subtypes, ERα, ERβ1 and ERβ2. To evaluate the three medaka ER (mER)-ligand interactions, we applied the ERE-luciferase reporter assay system to characterize each ER subtype. In this transient transfection assay system using mammalian cells, the mER proteins displayed estrogen-dependent activation. 17β-Estradiol (E(2)) and op'-DDT showed high activation irrespective of ERs. Endosulfan also exhibited activation; with less/no transactivity measured using other pesticides, i.e., heptachlor, carbendazim, deltamethrin, acephate, dimethoate and amitraz. It was generally observed that ERβ2 had higher activation potential than ERα and ERβ1. To understand the molecular mechanism of estrogen action via ER, we also conducted E(2) treatment where we observed a trigger in ERβ2 expression upon E(2) exposure. The present data suggest that ERβ2 is essential for female gonad maintenance. The data were supported by induction of vitellogenin (VTG) mRNA in the liver and reduced VTG receptor mRNA expression in the gonad of both sexes. The present work will provide a basic tool allowing future studies to examine the receptor-ligand interactions and endocrine disrupting mechanisms, and also expands our knowledge of estrogen action on reproductive development in medaka.     

Synthesis, structure-activity relationships, and mechanism of action of anti-HIV-1 lamellarin α 20-sulfate analogues

Lamellarin α and six different types of lamellarin α 20-sulfate analogues were synthesized and their structure–activity relationships were investigated using a single round HIV-1 vector infection assay. All lamellarin sulfates having pentacyclic lamellarin core exhibited anti-HIV-1 activity at a 10 μM concentration range regardless of the number and position of the sulfate group. On the other hand, non-sulfated lamellarin α and ring-opened lamellarin sulfate analogues did not affect HIV-1 vector infection in similar concentrations. The lamellarin sulfates utilized in this study did not exhibit unfavorable cytotoxic effect under the concentrations tested (IC₅₀ > 100 μM). Confocal laser scanning microscopic analysis indicated that hydrophilic lamellarin sulfates were hardly incorporated in the cell. HIV-1 Env-mediated cell–cell fusion was suppressed by lamellarin sulfates. These results suggested that lamellarin sulfates have a novel anti-HIV-1 activity besides the previously reported integrase activity inhibition, possibly at a viral entry step of HIV-1 replication.     

Epidemiologic study of clinically amyopathic dermatomyositis and anti-melanoma differentiation-associated gene 5 antibodies in central Japan

Introduction

Several reports have found the onset or activity of inflammatory myopathies to show spatial clustering and seasonal association. We recently detected autoantibodies against melanoma differentiation-associated gene 5 (MDA-5) in more than 20% of patients with dermatomyositis. Anti-MDA-5 antibodies were associated with the presence of rapidly progressive interstitial lung disease in clinically amyopathic dermatomyositis (CADM). The present study aims to assess the growing prevalence of CADM and the geographical incidence of anti-MDA-5-positive patients.

Methods

We reviewed medical charts and examined the presence of anti-MDA-5 antibodies in 95 patients, including 36 CADM patients. Sera were obtained from 1994 through 2011. Statistical analyses were performed to assess whether CADM development and the presence of anti-MDA-5 antibodies were associated with various parameters, including age at disease onset, season of onset, annual positivity, and population of resident city.

Results

Tertiles based on the year when the sera were collected showed increasing tendencies of CADM and anti-MDA-5-positive patients among all of the dermatomyositis patients. From 1994 to 2010, the relative prevalence of CADM and anti-MDA-5 antibody-positive patients significantly increased. Interestingly, the presence of anti-MDA-5 antibodies in 26 patients was inversely associated with the population of their city of residence.

Conclusions

This is the first study to examine the distribution of anti-MDA-5-positive dermatomyositis phenotypes in Japan. Regional differences in the incidences of these phenotypes would suggest that environmental factors contribute to the production of antibodies against MDA-5, which triggers innate antiviral responses.     

Indigenous utilization of termite mounds and their sustainability in a rice growing village of the central plain of Laos

Background

This study was conducted to identify medicinal plants and spices used for medicine by the community of Beni-Sueif, Upper Egypt.

Methods

Ethnobotanical data from local people was collected using direct interviews and a semi-structured questionnaire.

Results

Forty-eight plant species belonging to twenty-seven families and forty-seven genera were encountered during the study. Their botanical and vernacular names, plant parts used and medicinal uses are given. Results of the study were analyzed using two quantitative tools. The factor informant consensus indicated the agreement in the use of plants and the fidelity level indicated the ratio between the number of informants who independently suggested the use of a species for the same major purpose and the total number of informants who mentioned the plant for any use. The results of the factor informant consensus showed that the cardiovascular category has the greatest agreement, followed by the immunological, gastrointestinal and respiratory categories. The most important species according to their fidelity are: Hibiscus sabdariffa L. for the cardiovascular category; Trigonella foenum-graecum L. for the immunological category; Mentha piperita L. for the gastrointestinal category and Pimpinella anisum L. for the respiratory category.

Conclusions

Medicinal plants are still used for treatment in Beni-Sueif community despite the availability of prescribed medications. Documentation of this ethnomedicinal knowledge is important. Evaluation of pharmacological activity for the promising medicinal plants is suggested.     

Differential analysis of site-specific glycans on plasma and cellular fibronectins: application of a hydrophilic affinity method for glycopeptide enrichment

Isolation of glycopeptides utilizing hydrogen bonding between glycopeptide glycans and a carbohydrate-gel matrix in the organic phase is useful for site-specific characterization of oligosaccharides of glycoproteins, when combined with mass spectrometry. In this study, recovery of glycopeptides was improved by including divalent cations or increasing the organic solvent in the binding solution, without losing specificity, whereas it was still less effective for those with a long peptide backbone exceeding 50 amino acid residues. The method was then applied to the analysis of glycan heterogeneities at seven N-glycosylation sites in each of the plasma and cellular fibronectins (FNs). There was a remarkable site-specific difference in fucosylation between these isoforms; Asn1244 selectively escaped the global fucosylation of cellular FN, whereas only Asn1007 and Asn2108 of the plasma isoform underwent modification. In addition, a new O-glycosylation site was identified at Thr279 in the connecting segment between the fibrin- and heparin-binding domain and the collagen-binding domain, and the glycopeptide was reactive to a peanut agglutinin lectin. Considering that another mucin-type O-glycosylation site lies within a different connecting segment, the O-glycosylation of FN was suggested to play a significant role in segregating the neighboring domains and thus maintaining the topology of FN and the domain functions. In addition, the method was applied to apolipoprotein B-100 (apoB100) whose N-glycan structures at 17 of 19 potential sites have been reported, and characterized the remaining sites. The results also demonstrated that the enriched glycopeptide provides resources for site-specific analysis of oligosaccharides in glycoproteomics.     

Transcriptional regulation of connective tissue growth factor by sphingosine 1-phosphate in rat cultured mesangial cells

Connective tissue growth factor (CTGF) is induced by transforming growth factor-beta (TGF-beta) via Smad activation in mesangial cells. We recently reported that sphingosine 1-phosphate (S1P) induces CTGF expression in rat cultured mesangial cells. However, the mechanism by which S1P induces CTGF expression is unknown. The present study revealed that S1P-induced CTGF expression is mediated via pertussis toxin-insensitive pathways, which are involved in the activation of small GTPases of the Rho family and protein kinase C. We also showed by luciferase reporter assays and chromatin immunoprecipitation that S1P induces CTGF expression via Smad activation as TGF-beta does.     

The N-terminal region of NTAK/neuregulin-2 isoforms has an inhibitory activity on angiogenesis

NTAK (neural- and thymus-derived activator for ErbB kinases), also known as neuregulin-2, is a member of the epidermal growth factor (EGF) family, which binds directly to ErbB3 and ErbB4 and transactivates ErbB2. Because ErbB signaling has been implicated in various angiogenic mechanisms, the effect of NTAK (which has at least nine isoforms due to alternative splicing) in angiogenesis is explored. One isoform, NTAKgamma, inhibited cell growth in terms of DNA synthesis and cell numbers in vascular endothelial cells specifically, whereas NTAKalpha and beta had no activity. On the other hand, NTAKgamma secreted by transfected MDA-MB-231 cells inhibited endothelial cell growth, and NTAKgamma expressed in endothelial cells by adenovirus infection suppressed cell growth in a dose-dependent manner. The EGF-like domain of NTAKgamma did not have this activity. The NTAKdelta isoform, which had the Ig-like domain but not the EGF-like domain, inhibited proliferation of endothelial cells. NTAKdelta prevented hyper-phosphorylation of the retinoblastoma tumor suppressor protein and caused G(1) arrest in endothelial cells. Both NTAKgamma and delta isoforms displayed anti-angiogenic activity in the chick embryo chorioallantoic membrane in vivo. These results suggest that the active site of NTAK is localized outside of the EGF-like domain but within the N-terminal region, including the Ig-like domain, of NTAK.     

Phosphoinositide 3-kinase in nitric oxide synthesis in macrophage: critical dimerization of inducible nitric-oxide synthase

Phosphoinositide 3-kinase (PI3K) has important functions in various biological systems, including immune response. Although the role of PI3K in signaling by antigen-specific receptors of the adaptive immune system has been extensively studied, less is known about the function of PI3K in innate immunity. In the present study, we demonstrate that macrophages deficient for PI3K (p85alpha regulatory subunit) are impaired in nitric oxide (NO) production upon lipopolysaccharide and interferon-gamma stimulation and thus vulnerable for intracellular bacterial infection such as Chlamydophila pneumoniae. Although expression of inducible nitric-oxide synthase (iNOS) is induced normally in PI3K-deficient macrophages, dimer formation of iNOS protein is significantly impaired. The amount of intracellular tetrahydrobiopterin, a critical stabilizing cofactor for iNOS dimerization, is decreased in the absence of PI3K. In addition, induction of GTP cyclohydrolase 1, a rate-limiting enzyme for biosynthesis of tetrahydrobiopterin, is greatly reduced. Our current results demonstrate a critical role of class IA type PI3K in the bactericidal activity of macrophages by regulating their NO production through GTP cyclohydrolase 1 induction.     

Phylogenetic relationship of the populations within and around Japan using 105 short tandem repeat polymorphic loci

We have analyzed 105 autosomal polymorphic short tandem repeat (STR) loci for nine East and South-eastern Asian populations (two Japanese, five Han Chinese, Thai, and Burmese populations) and a Caucasian population using a multiplex PCR typing system. All the STR loci are genomewide tetranucleotide repeat markers of which the total number of observed alleles and the observed heterozygosity were 756 and 0.743, respectively, for Japanese populations. Phylogenetic analysis for these allele frequency data suggested that the Japanese populations are more closely related with southern Chinese populations than central and/or northern ones. STRUCTURE program analysis revealed the almost clearly divided and accountable population structure at K=2–6, that the two Japanese populations always formed one group separated from the other populations and never belong to different groups at K≥3. Furthermore, our new allele frequency data for 91 loci were analyzed with those for 52 worldwide populations published by previous studies. Phylogenetic and multidimensional scaling (MDS) analyses indicated that Asian populations with large population size (six Han Chinese, three Japanese, two Southeast Asia) formed one distinct cluster and are closer to each other than other ethnic minorities in east and Southeast Asia. This pattern may be the caviar of comparing populations with greatly differing population sizes when STR loci were analyzed.