Molecular Classification of Enteroviruses Not Identified by Neutralization Tests

We isolated six viruses from patients diagnosed with aseptic meningitis or hand, foot, and mouth disease. The cytopathic effect of these viruses on cultured cells was like that of enteroviruses. However, viral neutralization tests against standard antisera were negative. Phylogenetic analysis with the complete VP4 nucleotide sequences of these 6 viruses and 29 serotypes of enteroviruses classified 3 of the viruses as serotype echovirus type 18 (EV18) and 3 as serotype human enterovirus 71 (HEV71). These results were confirmed by remicroneutralization tests with HEV-monospecific antisera or an additional phylogenetic analysis with the complete VP4 nucleotide sequences. Phylogenetic analysis with complete VP4 genes is more useful than neutralization tests with enterovirus serotype-specific antisera in identifying enterovirus serotypes

Study on magnetic thermal seeds coated with thermal-responsive molecularly imprinted polymers

We conceived a novel hybrid carrier of a thermal-responsive molecularly imprinted polymer (MIP) and a magnetic thermal seed (MTS) that showed a heat-generating ability under an alternate current (AC) magnetic field. Compared to our previous publications, we modify both the MIP and MTS to improve the feasibility for the hybrid carrier, briefly we have to achieve the accurate size control and narrower size distribution of MTS, and higher molecular recognition/release ability of MIP. Firstly, uniformly sized particles which are expected to show a large heat-generating ability under an AC magnetic field were successfully prepared by controlling the core creation. Then, an MIP targeted for selective adsorption of pemetrexed (PMX), a well-known anti-cancer drug, was prepared using N-carbobenzoxy-L-glutamic acid as a pseudo template. Finally, the preliminary hybridization of the MTS and the MIP-equivalent polymer coating was examined by introducing vinyl groups as methacrylic acid using a ligand exchanging method

STRUCTURE MODIFICATION OF ANDROGRAPHOLIDE TO IMPROVE ITS POTENCY AS ANTICANCER

Andrographolide, a diterpenoid lactone isolated from the herb of Andrographis paniculata and known to possess antitumor activity in breast cancer models was subjected to semisynthesis leading to the preparation of a number of derivatives. After protection of the two hydroxyl groups present at C-3 and C-19 to give 3,19-isopropylidene and 3,19-benzylidene andrographolides, the remaining hydroxyl group at C-14 of andrographolide was treated with acid anhydride or acid chloride under base condition. Unfortunately, the reactions gave only 14-dehydroandrographolide as well as unidentified diacyl compounds in replace of the target molecule 14-O-acyl andrographolide. An alternative procedure using neat acetic anhydride under reflux gave the acetyl derivatives. The resulted compounds exhibited cytotoxic activity against MCF-7 breast cancer cells with better growth inhibition than the parent compound andrographolide.   Keywords: andrographolide, acylation, anticancer, cytotoxic, breast cancer cells

Involvement of influx and efflux transport systems in gastrointestinal absorption of celiprolol

金沢大学医薬保健研究域薬学系Gastrointestinal absorption of several β-blockers is inhibited by citrus juices, although molecular mechanism(s) lying on their small intestinal absorption has not yet been identified. Here, we attempted to demonstrate involvement of both influx and efflux transporters in vivo in gastrointestinal absorption of celiprolol in mice. Plasma concentration of celiprolol (3 mg/kg) after oral administration was mostly under the limit of quantification in wild mice, whereas that in mdr1a/b knockout (mdr1a/b(-/-)) mice was much more obvious, indicating P-glycoprotein-mediated efflux. Then, the oral absorption of celiprolol in mdr1a/b(-/-) mice was further examined to investigate influx transport mechanism with avoiding effect of P-glycoprotein. Coadministration of bromosulfophthalein (BSP), an inhibitor of various influx transporters including organic anion transporting polypeptide (OATP) reduced plasma celiprolol concentration. Inhibition by BSP of celiprolol uptake from apical membranes was confirmed in Ussing-type chamber of small intestinal tissues. Uptake of celiprolol by human small intestinal transporter OATP-A/1A2 was also confirmed in Xenopus Laevis oocytes. Interestingly, OATP-A/1A2 accepts various b-blockers including acebutolol, atenolol and sotalol, oral absorption of which is inhibited by coadministration of citrus juice or telithromycin in human. Taken together, these findings have suggested fundamental role of influx transport system(s) in oral absorption of celiprolol. © 2008 Wiley-Liss, Inc. and the American Pharmacists Association

One autopsy case of cyanide-gas poisoning

A fishing-boat was smoked with cyanide-gas to rid vermin. A thirty-year old male was found dead in a cabin of the boat. Autopsy revealed fluid blood, and petechial haemorrhage in conjunctivas, thymus, heart and lungs. Lung, spleen, kidney and other organs were strongly congested. Bleedings in sternocleidomastoideus and sterunohyoideus muscles were found. The bloody foam and solution were also observed in trachea. From these autopsy findings, it was considered that he failed into severe dyspnea. Furthermore, postmortem lividity was bright pink color and the left cardiac blood was also bright pink, so there was markly different between the color of right and left cardiac blood. To make clear his cause of death, during the autopsy the screening test of cyanide, Schonbein-Pagenstecher method, was tried and then it was positive. Further toxicological analysis, quantitative measurement revealed 6.25 μg/ml of cyanide from his blood. From the results of autopsy and toxicological findings, his cause of death was diagnosed as the cyanide-gas poisoning

Immunohistochemical diagnosis and significance of forensic neuropathological changes

Immunohistochemistry is very useful when investigating the cause of death. Ischemic cell changes in the hippocampal neurons were not obvious in the brains damaged by hypoxic injury. However, it is suggested that even a moderate hypoxia, which may affect the neuronal proteins and metabolism, induced astrocytes is in the CA3 and CA4 regions, and that in patients with a history of hypoxic attacks neuronal damage may be severe even several hours after ischemic injury. Furthermore, hsp70 expression was found in the CA2, CA3 and CA4 regions of long-term survivors after severe hypoxic / ischemic injury. In forensic practice, detailed information about the duration and extent of a hypoxic / ischemic injury is often unavailable, so that immunohistochemical detection of hsp70 and glial cell staining can be of great value in diagnosing not only the hypoxic / ischemic injury during the process of death but also the victim’s past history of hypoxic attacks. In diffuse axonal injury, degeneration of axon and myelin, such as swelling and waving, were observed in survivors of more than 8 hours. Retraction balls appeared in survivors of more than 1 days. In longer term survivors, such as 3 or 5 months, breakdown of myelin and fat-granule cells were observed. In addition, retraction balls were also found. Immunohistochemical staining of 200 kD neurofilament was a very useful method to examine axonal changes, because antisera is specific for degenerative neurofilaments. In our study, all cases which had pathological findings of diffuse axonal injury (DAI)were associated with focal head injuries. From the immunohistochemical staining of neurons in the hippocampus, it was suggested that neurons in the hippocampus were injured by diffuse brain damage. Furthermore, repairing and protective mechanisms occurred especially from CA2 toCA4. It was considered that neuronal damage in diffuse brain injury was elucidated not only morphologically but also functionally. Therefore, in cases of suspected diffuse brain damage, it is recommended to examine the neuronal changes in addition to observing the findings of diffuse axonal injury. Immunohistochemical staining of the carotid body is potentially very useful for necropsy diagnosis, since it provides a method to detect evidence of mechanical asphyxia in suspected cases of manual and/or ligature strangulation

Impact of Cationic Amino Acid Transporter 1 on Blood- Retinal Barrier Transport of L-Ornithine

PURPOSE. To elucidate L-ornithine transport at the blood-retinal barrier (BRB). METHODS. Integration plot and retinal uptake index (RUI) were used to investigate the in vivo [ 3 H]L-ornithine transport across the BRB. In vitro transport studies of [ 3 H]L-ornithine were performed with TR-iBRB2 cells and RPE-J cells, the model cells of the inner and outer BRB, respectively. Immunohistochemistry was performed on cationic amino acid transporter 1 (CAT1/SLC7A1). RESULTS. The apparent influx permeability clearance of [ 3 H]L-ornithine was found to be 18. 7 lL/(minÁg retina), and the RUI of [ 3 H]L-ornithine was reduced by L-ornithine and L-arginine, suggesting the blood-to-retina transport of L-ornithine at the BRB. [ 3 H]L-Ornithine uptake by TR-iBRB2 cells showed a time-, temperature-and concentration-dependence with a MichaelisMenten constant (K m ) of 33.2 lM and a nonsaturable uptake rate (K d ) of 2.18 lL/(minÁmg protein). The uptake was Na þ -independent, and was inhibited by L-ornithine, L-arginine, and L-lysine, suggesting the involvement of CAT1 in L-ornithine transport at the inner BRB. Immunohistochemistry revealed the luminal and abluminal localization of CAT1 at the inner BRB, and at the basal localization at the outer BRB. Retinal pigment epithelium-J cells showed that the basal-to-cell (B-to-C) uptake of [ 3 H]L-ornithine was greater than that of the apical-tocell (A-to-C) uptake, and the B-to-C transport was inhibited by unlabeled L-ornithine, suggesting the involvement of CAT1 in the blood-to-cell transport of L-ornithine across the basal membrane at the outer BRB. CONCLUSIONS. These suggest the involvement of CAT1 in L-ornithine transport at the luminal and abluminal sides of the inner BRB and the basal side of the outer BRB

Involvement of Multidrug Resistance-Associated Protein 1 in Intestinal Toxicity of Methotrexate

金沢大学医薬保健研究域薬学系Purpose: Methotrexate (MTX) causes dose-limiting gastrointestinal toxicity due to exposure of intestinal tissues, and is a substrate of the multidrug resistance-associated protein (MRP) 1. Here we examine the involvement of MRP1, which is reported to be highly expressed in the proliferative crypt compartment of the small intestine, in the gastrointestinal toxicity of MTX. Methods: MTX was intraperitonealy administered to mrp1 gene knockout (mrp1(-/-)) and wild-type (mrp1(+/+)) mice. Body weight, food and water intake were monitored, intestinal histological studies and pharmacokinetics of MTX were examined. Results: mrp1(-/-) mice more severely decreased body weight, food and water intake than mrp1(+/+) mice. Almost complete loss of villi throughout the small intestine in mrp1(-/-) mice was observed, whereas the damage was only partial in mrp1(+/+) mice. Plasma concentration and biliary excretion profiles of MTX were similar in mrp1(-/-) and mrp1(+/+) mice, though accumulation of MTX in immature proliferative cells isolated from mrp1(-/-) mice was much higher compared to mrp1(+/+) mice. Immunostaining revealed localization of Mrp1 in plasma membrane of the intestinal crypt compartment in mrp1(+/+) mice, but not in mrp1(-/-) mice. Conclusion: Mrp1 determines the exposure of proliferative cells in the small intestine to MTX, followed by gastrointestinal toxicity. © 2009 Springer Science+Business Media, LLC