Hidetomo Iwano

Placental transfer of conjugated bisphenol A and subsequent reactivation in the rat fetus.

Background Bisphenol A (BPA), a well-known endocrine disruptor, is highly glucuronidated in the liver, and the resultant BPA-glucuronide (BPA-GA) is excreted primarily into bile. However, in rodents, prenatal exposure to low doses of BPA can adversely affect the fetus, despite the efficient drug-metabolizing systems of the dams. The transport mechanisms of BPA from mother to fetus are unknown. Objectives To test our hypothesis that BPA-GA—an inactive metabolite—is passed through the placenta to the fetus, where it affects the fetus after reactivation, we investigated the placental transfer of BPA-GA and reactivation to BPA in the fetus. Methods After performing uterine perfusion with BPA-GA in pregnant rats, we examined the expression and localization of the placental transporters for drug metabolites in the perfusate by reverse-transcriptase polymerase chain reaction and immunohistochemistry. We also investigated the deconjugation of BPA-GA in the fetus and examined uridine 5′-diphospho-glucuronosyltransferase (UGT) activity toward BPA and the expression of UGT isoforms in fetal liver. Results We detected BPA-GA and deconjugated BPA in the fetus and amniotic fluid after perfusion. In the trophoblast cells, organic anion-transporting polypeptide 4a1 (Oatp4a1) was localized on the apical membrane, and multidrug resistance-associated protein 1 (Mrp1) was localized to the basolateral membrane. We observed deconjugation of BPA-GA in the fetus; furthermore, we found the expression of UGT2B1, which metabolizes BPA, to be quite low in the fetus. Conclusions These results demonstrate that BPA-GA is transferred into the fetus and deconjugated in the fetus because of its vulnerable drug-metabolizing system.

Isolation from Sewage Influent and Characterization of Novel Staphylococcus aureus Bacteriophages with Wide Host Ranges and Potent Lytic Capabilities

ABSTRACT Staphylococcus aureus is a gram-positive pathogen that causes a variety of diseases, including bovine mastitis, which has severe economic consequences. Standard antibiotic treatment results in selection of resistant strains, leading to a need for alternative treatments, such as bacteriophage therapy. Forty-nine S. aureus isolates were obtained from the milk of mastitic cows for use in screening of staphylococcal phages. Fifteen isolates which were positive for both coagulase and hemolysin were assayed by PCR for variation in the X region and the immunoglobulin G-binding region of the protein A gene (spa) and in the carboxy terminus of the coagulase gene (coa) and for the presence of enterotoxin C, G, H, and I genes. The host ranges of 52 phages isolated from sewage influent were determined by performing spot tests with the 15 S. aureus isolates, and two phages were subsequently chosen for further analysis. ΦSA039 had the widest host range, producing clear plaques on 13 of the 15 isolates (87%), while ΦSA012 produced clear plaques on 8 isolates (53%) and was the only phage that produced a clear plaque on a nonmastitic S. aureus strain. Transmission electron microscopy revealed that the phages were similar sizes and belonged to the Myoviridae family. Measurement of optical densities during coculture with S. aureus isolates confirmed the breadth of the ΦSA039 host range and showed that ΦSA012 had potent lytic capability. ΦSA012-resistant bacteria did not appear for three of seven isolates tested (43%) after 65 h of incubation. These two phages are proposed as candidates for phage therapy of bovine mastitis.

Monocarboxylate transporter 1 (MCT1) plays a direct role in short-chain fatty acids absorption in caprine rumen

Despite the importance of short‐chain fatty acids (SCFA) in maintaining the ruminant physiology, the mechanism of SCFA absorption is still not fully studied. The goal of this study was to elucidate the possible involvement of monocarboxylate transporter 1 (MCT1) in the mechanism of SCFA transport in the caprine rumen, and to delineate the precise cellular localization and the level of MCT1 protein along the entire caprine gastrointestinal tract. RT‐PCR revealed the presence of mRNA encoding for MCT1 in all regions of the caprine gastrointestinal tract. Quantitative Western blot analysis showed that the level of MCT1 protein was in the order of rumen ≥ reticulum > omasum > caecum > proximal colon > distal colon > abomasum > small intestine. Immunohistochemistry and immunofluorescence confocal analyses revealed widespread immunoreactive positivities for MCT1 in the caprine stomach and large intestine. Amongst the stratified squamous epithelial cells of the forestomach, MCT1 was predominantly expressed on the cell boundaries of the stratum basale and stratum spinosum. Double‐immunofluorescence confocal laser‐scanning microscopy confirmed the co‐localization of MCT1 with its ancillary protein, CD147 in the caprine gastrointestinal tract. In vivo and in vitro functional studies, under the influence of the MCT1 inhibitors, p‐chloromercuribenzoate (pCMB) and p‐chloromercuribenzoic acid (pCMBA), demonstrated significant inhibitory effect on acetate and propionate transport in the rumen. This study provides evidence, for the first time in ruminants, that MCT1 has a direct role in the transepithelial transport and efflux of the SCFA across the stratum spinosum and stratum basale of the forestomach toward the blood side.

Histone H1 null vertebrate cells exhibit altered nucleosome architecture

In eukaryotic nuclei, DNA is wrapped around an octamer of core histones to form nucleosomes, and chromatin fibers are thought to be stabilized by linker histones of the H1 type. Higher eukaryotes express multiple variants of histone H1; chickens possess six H1 variants. Here, we generated and analyzed the phenotype of a complete deletion of histone H1 genes in chicken cells. The H1-null cells showed decreased global nucleosome spacing, expanded nuclear volumes, and increased chromosome aberration rates, although proper mitotic chromatin structure appeared to be maintained. Expression array analysis revealed that the transcription of multiple genes was affected and was mostly downregulated in histone H1-deficient cells. This report describes the first histone H1 complete knockout cells in vertebrates and suggests that linker histone H1, while not required for mitotic chromatin condensation, plays important roles in nucleosome spacing and interphase chromatin compaction and acts as a global transcription regulator.

UDP-GLUCURONOSYLTRANSFERASE ISOFORMS CATALYZING GLUCURONIDATION OF HYDROXY-POLYCHLORINATED BIPHENYLS IN RAT

Polychlorinated biphenyls (PCBs) are highly toxic environmental contaminants that can cause irreversible damage in humans and wildlife. The mechanism of toxicity is not clear, but biotransformation products such as hydroxy PCBs (OH-PCBs) are a major concern. Efforts to elucidate the metabolism of PCBs and their metabolites, however, have paid little attention to the structure of the compound to be eliminated. The objectives of this study were to clarify organ tissue distribution of the glucuronidation activities toward OH-PCBs and to determine the UDP-glucuronosyltranseferase (UGT) isoforms responsible for glucuronidation in relation to the OH-PCB structure. 2,4,6-Trichlorobiphenyl and 2,3,4,5-tetrachlorobiphenyl were incubated in primary culture of rat hepatocytes, and the metabolites were identified by HPLC. Organ tissue glucuronidation activities toward 10 OH-PCBs were investigated by reactions of microsomes prepared from brain, liver, small and large intestine, lung, kidney, and testis tissues. To determine substrate specificity of the isoforms toward the OH-PCBs, rat UGT isoforms UGT1A1, UGT1A3, UGT1A5, UGT1A6, UGT1A7, UGT2B1, UGT2B3, and UGT2B12 were expressed in yeast strain AH22. Glucuronidation of the PCBs was found to be contingent on their hydroxylation. The organ tissues had strong glucuronidation activities toward the OH-PCBs tested; and most OH-PCBs were glucuronidated by UGT1A1, UGT1A6, and UGT2B1, all of which were substrate-specific. In conclusion, glucuronidation activities of UGT1A1, UGT1A6, and UGT2B1 toward OH-PCBs is relative to expression of the isoforms in each tissue, and glucuronidation intensity of the isoforms is relative to the structure of the OH-PCB to be glucuronidated.

A new treatment for human malignant melanoma targeting L-type amino acid transporter 1 (LAT1): a pilot study in a canine model.

L-type amino acid transporter 1 (LAT1), an isoform of amino acid transport system L, transports branched or aromatic amino acids essential for fundamental cellular activities such as cellular growth, proliferation and maintenance. This amino acid transporter recently has received attention because of its preferential and up-regulated expression in a variety of human tumors in contrast to its limited distribution and low-level expression in normal tissues. In this study, we explored the feasibility of using LAT1 inhibitor as a new therapeutic agent for human malignant melanomas (MM) using canine spontaneous MM as a model for human MM. A comparative study of LAT expression was performed in 48 normal tissues, 25MM tissues and five cell lines established from MM. The study observed LAT1 mRNA levels from MM tissues and cell lines that were significantly (P<0.01) higher than in normal tissues. Additionally, MM with distant metastasis showed a higher expression than those without distant metastasis. Functional analysis of LAT1 was performed on one of the five cell lines, CMeC-1. [(3)H]l-Leucine uptake and cellular growth activities in CMeC-1 were inhibited in a dose-dependent manner by selective LAT1 inhibitors (2-amino-2-norbornane-carboxylic acid, BCH and melphalan, LPM). Inhibitory growth activities of various conventional anti-cancer drugs, including carboplatin, cyclophosphamide, dacarbazine, doxorubicin, mitoxantrone, nimustine, vinblastine and vincristine, were significantly (P<0.05) enhanced by combination use with BCH or LPM. These findings suggest that LAT1 could be a new therapeutic target for MM.

Smooth muscle layer-dependent distribution of 5-hydroxytryptamine7 receptor in the porcine myometrium

To analyse the mechanisms of muscle layer‐dependent inhibition of porcine myometrial contractility by 5‐hydroxytryptamine (5‐HT), the effects of 5‐HT, 5‐carboxamidotryptamine(5‐CT), 5‐methoxytryptamine (5‐MeOT), forskolin and cyclic adenosine 3′, 5′‐monophosphate (cyclic AMP) analogues on spontaneous and stimulant‐induced contractions were examined in longitudinal (LM) and circular muscles (CM). In addition, accumulation of cyclic AMP by 5‐HT and distribution of 5‐HT7 receptors in LM and CM layers were compared using biochemical and molecular approaches. 5‐HT receptor agonists inhibited the spontaneous contractions of LM and CM (5‐CT>5‐HT>5‐MeOT), but CM was more sensitive than was LM. The inhibition by the agonists was antagonized by methiothepin (100 nM). Carbachol‐, high‐K+‐, histamine‐ and Ca2+‐induced contractions were inhibited by 5‐HT with different responsivenesses (CM>LM). Even in the presence of 3‐isobutyl‐1‐methylxanthine (IBMX), the inhibition by 5‐HT in the CM was still more conspicuous than that in the LM. Compared with the CM, the inhibition of spontaneous contraction by forskolin, dibutyryl‐cyclic AMP and 8‐bromo‐cyclic AMP was marked in the LM. 5‐HT (1 nM–1 μM) increased the cyclic AMP in both muscle layers, but the increment in the CM was higher than that in the LM whether IBMX was present or not. LM and CM layers contained a single class of [3H]‐5‐CT binding sites with a similar Kd value (0.21–0.24 nM). However, Bmax (5‐HT7 receptor concentration) in the CM (120.6 fmol mg−1 protein) was higher than that in the LM (30.4 fmol mg−1 protein). The molecular study (reverse transcription polymerase chain reaction) demonstrated the expression of 5‐HT7 receptor mRNA in the CM was higher than that in the LM. These results suggest that the muscle layer‐dependent difference in inhibition by 5‐HT is not restricted to spontaneous contraction but applies to various contractions in the porcine myometrium. Different inhibition of the contractility by 5‐HT is caused by muscle layer‐related accumulation of cyclic AMP (CM>LM), due to smooth muscle‐layer dependent distribution (CM>LM) of 5‐HT7 receptors.

A simplified PCR assay for fast and easy mycoplasma mastitis screening in dairy cattle

A simplified polymerase chain reaction (PCR) assay was developed for fast and easy screening of mycoplasma mastitis in dairy cattle. Species of major mycoplasma strains [Mycoplasma (M.) bovis, M. arginini, M. bovigenitalium, M. californicum, M. bovirhinis, M. alkalescens and M. canadense] in cultured milk samples were detected by this simplified PCR-based method as well as a standard PCR technique. The minimum concentration limit for detecting mycoplasma by the simplified PCR was estimated to be about 2.5 × 103 cfu/mL and was similar to that of the standard PCR. We compared the specificity and sensitivity of the simplified PCR to those of a culture method. Out of 1,685 milk samples cultured in mycoplasma broth, the simplified PCR detected Mycoplasma DNA in 152 that were also positive according to the culture assay. The sensitivity and specificity of the simplified PCR were 98.7% and 99.7%, respectively, for detecting mycoplasma in those cultures. The results obtained by the simplified PCR were consistent with ones from standard PCR. This newly developed simplified PCR, which does not require DNA purification, can analyze about 300 cultured samples within 3 h. The results from our study suggest that the simplified PCR can be used for mycoplasma mastitis screening in large-scale dairy farms.

Antimicrobial susceptibilities of Mycoplasma isolated from bovine mastitis in Japan

Mycoplasma spp. are highly contagious pathogens and intramammary Mycoplasma infection is a serious issue for the dairy industry. As there is no effective vaccine for Mycoplasma infection, control depends on good husbandry and chemo-antibiotic therapy. In this study, antimicrobial susceptibility of Mycoplasma strains recently isolated from cases of bovine mastitis in Japan was evaluated by minimum inhibitory concentration (MIC). All Mycoplasma bovis strains were sensitive to pirlimycin, danofloxacin and enrofloxacin, but not kanamycin, oxytetracycline, tilmicosin or tylosin. M. californicum and M. bovigenitalium strains were sensitive to pirlimycin, danofloxacin, enrofloxacin, oxytetracycline, tilmicosin and tylosin, but not to kanamycin. This is the first report to describe the MIC of major antimicrobial agents for Mycoplasma species isolated from bovine mastitis in Japan.

Relationship between concentration of lingual antimicrobial peptide and somatic cell count in milk of dairy cows

Lingual antimicrobial peptide (LAP) belongs to the β-defensin family in cattle and is found in milk. LAP concentrations increase in milk from mastitic udders; however, the relationship between LAP concentrations and the somatic cell count (SCC) in milk remains to be elucidated in detail. Therefore, the present study was undertaken to investigate the relationship between LAP concentrations and the SCC in bovine milk to assess whether LAP may be used as an indicator of SCC. Milk was collected from 66 udders showing various SCCs. The SCC and LAP concentrations were measured in the milk. A significantly higher LAP concentration was observed in milk having 500-5000 × 10(3)cells/ml and >5000 × 10(3)cells/ml SCC groups than in lower SCC groups (<50 × 10(3)cells/ml and 50-500 × 10(3)cells/ml). A significantly positive correlation between LAP concentrations and SCCs in milk was observed (r=0.68). In milk samples with >26 nM of LAP, 92.0% of milk samples had high SCCs (>200 × 10(3)cells/ml). The concentration of LAP in milk infected with Staphylococcus aureus, Streptococcus bovis, Streptococcus dysgalactiae, and Escherichia coli was significantly higher than that in uninfected milk. These results suggest that the concentration of LAP can be a useful indicator of the SCC in dairy cows.