Biocommunication between Plants and Pollinating Insects through Fluorescence of Pollen and Anthers.

Flowering plants attract pollinators via various stimuli such as odor, color, and shape. Factors determining the foraging behavior of pollinators remain a major theme in ecological and evolutionary research, although the floral traits and cognitive ability of pollinators have been investigated for centuries. Here we show that the autofluorescence emitted from pollen and anthers under UV irradiation may act as another attractant for flower-visiting insects. We have identified fluorescent compounds from pollen and anthers of five plant species as hydroxycinnamoyl derivatives. The fluorescent compounds are also shown to quench UV energy and exhibit antioxidant activity, indicating a function as protectants of pollen genes from UV-induced damage. A two-choice assay using honeybees in the field demonstrated that they perceived the blue fluorescence emitted from the fluorescent compounds and were attracted to it. This result suggested that the fluorescence from pollen and anthers serves as a visual cue to attract pollinators under sunlight.

Reduction of endotoxin attenuates liver fibrosis through suppression of hepatic stellate cell activation and remission of intestinal permeability in a rat non-alcoholic steatohepatitis model.

Previous clinical studies have demonstrated that endotoxin/toll­like receptor 4 (TLR4) signaling is critical in the inflammatory pathways associated with non­alcoholic steatohepatitis (NASH). In human and animal studies, NASH was associated with portal lipopolysaccharide (LPS) and the plasma LPS level was hypothesized to be associated with small intestinal bacterial overgrowth, change in composition of the microbiota and increased intestinal permeability. The aim of the present study was to investigate the roles of endogenous endotoxin and TLR4 in the pathogenesis of NASH. The effects of antibiotics were assessed in vivo using a choline deficiency amino acid (CDAA)­induced experimental liver fibrosis model. Antibiotics, including polymyxins and neomycins, were orally administered in drinking water. Antibiotics attenuated hepatic stellate cell (HSC) activation and liver fibrosis via TGF­ß and collagen in an experimental hepatic fibrosis model. The mechanism by which antibiotics attenuated LPS­TLR4 signaling and liver fibrosis was assessed. Notably, TLR4 mRNA level in the liver was elevated in the CDAA group and the CDAA­induced increase was significantly reduced by antibiotics. However, no significant differences were observed in the intestine among all groups. Elevated mRNA levels of LPS binding protein, which was correlated with serum endotoxin levels, were recognized in the CDAA group and the CDAA­induced increase was significantly reduced by antibiotics. The intestinal permeability of the CDAA group was increased compared with the choline­supplemented amino acid group. The tight junction protein (TJP) in the intestine, determined by immunohistochemical analysis was inversely associated with intestinal permeability. Antibiotics improved the intestinal permeability and enhanced TJP expression. Inhibition of LPS­TLR4 signaling with antibiotics attenuated liver fibrosis development associated with NASH via the inhibition of HSC activation. These results indicated that reduction of LPS and restoration of intestinal TJP may be a novel therapeutic strategy for treatment of liver fibrosis development in NASH.

Combination of sorafenib and angiotensin-II receptor blocker attenuates preneoplastic lesion development in a non-diabetic rat model of steatohepatitis.

BACKGROUND: Given the well-documented adverse side effects of sorafenib, many sorafenib-treated patients may need the reduced initial dose of the compound, and an alternative sorafenib-based therapy, which exerts similar clinical benefit, is anticipated. An angiostatic therapy with sorafenib is considered one of the promising approaches for chemoprevention of hepatocellular carcinoma. The aim of the current study was to elucidate the combination effect of low dose of sorafenib and angiotensin-II receptor blocker (ARB) on hepatocarcinogenesis, especially in conjunction with angiogenesis. METHODS: The chemopreventive effect on the development of liver preneoplastic lesions, angiogenesis, and several indices was elucidated in rats. We also performed several sets of in vitro experiments to examine the mechanisms involved. RESULTS: Using a non-diabetic rat model of steatohepatitis with choline deficient L-amino acid-defined diet, sorafenib demonstrated marked inhibition of preneoplastic lesions in a dose dependent manner. Combined treatment with ARB (losartan) at a clinically comparable dose and half dose of sorafenib resulted in the inhibitory effect equivalent to that of common dose of sorafenib along with suppression of hepatic neovascularization and potent angiogenic factor, vascular endothelial growth factor. Furthermore, similar combined inhibitory outcomes were observed in several sets of in vitro studies. CONCLUSION: Since the combinatorial treatment using low doses of sorafenib and ARB could sufficiently induce inhibitory effect on the development of preneoplastic lesions at the magnitude similar to the conventional dose of sorafenib, this regimen may provide new strategy for patients intolerant of the usual dose of sorafenib in the future.

Influence of preparation method on polynucleotide conformation and pharmacological activity of lipoplex.

Conformations of polyinosinic acid [poly(I)] and polycytidylic acid [poly(C)] in liposomes (lipoplex) were investigated by both circular dichroism (CD) spectroscopy and fluorescence resonance energy transfer (FRET) measurements, and compared with those in aqueous solution. The results indicate that poly(I) and poly(C) take double-stranded structure in aqueous solution at pH 6.5-7.5 in the presence of NaCl at higher concentration than 50mM. Although lipoplex was prepared without NaCl to avoid aggregation of lipoplex particles, poly(I) and poly(C) were double-stranded in pre-mixed poly(I)/poly(C) lipoplex (pre-mixed LIC), prepared by adding a mixed solution of poly(I) and poly(C) to the cationic liposomes. However, poly(I) and poly(C) did not take double-stranded structure in separately mixed LIC, prepared by separate addition of poly(I) solution and poly(C) solution to the cationic liposomes. The physicochemical properties (particle diameter and zeta potential) of pre-mixed LIC and separately mixed LIC were not different, but the anti-proliferative effect of pre-mixed LIC on human epidermoid carcinoma A431 cells was about eight times greater than that of separately mixed LIC. Our results indicate that polynucleotide conformation in lipoplex is markedly influenced by the preparation method, and the polynucleotide conformation in lipoplex has a substantial effect on pharmacological activity.

Immunotherapy for nonalcoholic steatohepatitis using the multiple cytokine production modulator Y-40138.

AIM: To investigate the possible use of the multiple cytokine production modulator, Y-40138, as a novel immunotherapy in the rat nonalcoholic steatohepatitis (NASH) model. METHODS: We allocated 6-wk-old male F344 rats to choline-supplemented, L-amino acid-defined (CSAA) diet (control group), CSAA diet + Y-40138 (control + Y-40138 group), choline-deficient, L-amino acid-defined (CDAA) diet (NASH group), or CDAA diet + Y-40138 (NASH + Y-40138 group). In each group, we measured the plasma alanine aminotransferase (ALT) levels, and the plasma and liver levels of tumor necrosis factor-alpha (TNF-alpha), interferon-gamma (IFN-gamma), and interleukin-10 (IL-10). Tissue specimens of phosphate buffered saline-perfused liver were subjected to hematoxylin and eosin staining, Azan staining, Sirius red staining, and immunohistochemical staining (for Kupffer cells and TNF-alpha). We then extracted Kupffer cells from the collagenase-perfused livers using the Percoll gradient centrifugation method, and measured the TNF-alpha levels in the supernatant (in vitro TNF-alpha production by Kupffer cells) using an enzyme-linked immunosorbent assay kit. RESULTS: In comparison to the NASH group, serum ALT elevation was mild, production of serum and liver TNF-alpha and IFN-gamma was inhibited, and IL-10 production was increased in the NASH + Y-40138 group. Amelioration of liver histology was also noted in the NASH + Y-40138 group. Kupffer cell immunohistochemical staining revealed no differences between groups, whereas TNF-alpha immunohistochemical staining showed fewer stained cells in the NASH + Y-40138 group than in the NASH group. The TNF-alpha levels in the in-vitro Kupffer cell culture supernatant were lower in the NASH + Y-40138 group than in the NASH group. CONCLUSION: Administration of Y-40138 to NASH model rats reduced hepatic inflammation and suppressed fibrosis. These results indicate that the multiple cytokine production modulator, Y-40138, is promising as a novel treatment for NASH.

Drug-induced liver injury in Japan: An analysis of 1676 cases between 1997 and 2006.

At the 44th Annual Meeting of the Japan Society of Hepatology, 1674 cases of drug-induced liver injury (DILI), occurring between January 1997 and December 2006, were reviewed. Data were obtained by questionnaires completed by the 29 presenters of the special DILI session during the meeting. This article presents the review's findings, including the role of dietary supplements and Chinese herbal medicines in DILI.

Pneumatosis cystoides intestinalis following alpha-glucosidase inhibitor treatment: a case report and review of the literature.

A 69-year-old man was diagnosed as having myasthenia gravis (MG) in September 2004, and treated with thymectomy and prednisolone. He was then diagnosed as having steroid-induced diabetes mellitus, and received sulfonylurea (SU) therapy in May 2005. An alpha-glucosidase inhibitor (alphaGI) was added in March 2006, resulting in good glycemic control. He experienced symptoms of abdominal distention, increased flatus, and constipation in October 2007, and was admitted into our hospital in late November with hematochezia. Plain abdominal radiography revealed small linear radiolucent clusters in the wall of the colon. Computed tomography (CT) showed intramural air in the sigmoid colon. Colonoscopy revealed multiple smooth surfaced hemispherical protrusions in the sigmoid colon. The diagnosis of pneumatosis cystoides intestinalis (PCI) was made on the basis of these findings. As the alphaGI voglibose was suspected as the cause of this patient's PCI, treatment was conservative, ceasing voglibose, with fasting and fluid supplementation. The patient progressed well, and was discharged 2 wk later. Recently, several reports of PCI associated with alphaGI therapy have been published, predominantly in Japan where alphaGIs are commonly used. If the use of alphaGIs becomes more widespread, we can expect more reports of this condition on a global scale. The possibility of PCI should be considered in diabetic patients complaining of gastrointestinal symptoms, and the gastrointestinal tract should be thoroughly investigated in these patients.

Renin-angiotensin system inhibitors as therapeutic alternatives in the treatment of chronic liver diseases.

The renin-angiotensin system (RAS) is frequently activated in the patients with chronic liver diseases, and recent studies have shown that RAS plays a pivotal role in the progression of chronic liver diseases, i.e., liver fibrosis and hepatocellular carcinoma (HCC). Angiotensin-II (AT-II) reportedly stimulates contractility and proliferation of the activated hepatic stellate cells, and increases the transforming growth factor-beta (TGF-betabeta expression through angiotensin type-I receptors (AT1-R). Many studies have demonstrated that the clinically used angiotensin-converting enzyme inhibitors (ACE-I) and AT1-R blockers (ARB) significantly attenuated the liver fibrosis development in the experimental studies and clinical practice. AT-II also strongly promotes neovascularization, which plays a pivotal role in tumor development. AT-II induces a potent angiogenic factor; namely, the vascular endothelial growth factor (VEGF). It has been reported that ACE-I significantly attenuated the experimental HCC growth and hepatocarcinogenesis along with suppression of neovascularization. The VEGF expression in the tumor was suppressed by ACE-I, too. The combined treatment of ACE-I with other clinically used agents, such as interferon, imatinib mesylate, and vitamin K, shows more potent inhibitory effects on the development of liver fibrosis and HCC. Since RAS inhibitors are widely used in the clinical practice without serious side effects, they may represent a potential new therapeutic strategy against the progression of chronic liver diseases.

Onset of ischemic colitis following use of electrical muscle stimulation (EMS) exercise equipment.

Our patient was a 71-year-old man who presented with lower abdominal pain, and bloody and white mucosal stools. He purchased by mail-order an electrical muscle stimulation (EMS) device, which he strapped onto his lower abdomen, and for 2 consecutive days he underwent muscle stimulation comprising 600 contractions at 2.40 mA and 1.20 V over a 10 minute period. He experienced the onset of lower abdominal pain immediately following muscle stimulation on the second day, and then passed stools containing blood and white mucus. The cause was thought to be electrical and mechanical stimulation of the lower abdomen by the EMS equipment, either inducing colonic or vascular spasm, or dislodging thrombi associated with atrial fibrillation or atherosclerosis. This is the first known report of ischemic colitis associated with the use of EMS exercise equipment. We report this case in the belief that this condition is likely to become more common with increasing use of such devices.

Absorption and metabolism of bisphenol A, a possible endocrine disruptor, in the aquatic edible plant, water convolvulus (Ipomoea aquatica).

Water convolvulus, a vegetable, absorbed bisphenol A (BPA), an endocrine disruptor, from the medium. One week later, no BPA could be detected in the plant, indicating that BPA had been metabolized in the plant. BPA monoglucoside was detected as the BPA base at ca. 10% in the roots, some in the stems, but none in the leaves. (2)H-NMR analyses of MeOH extracts and hydrolyzates of the plant treated with BPA-d(16) showed the presence of metabolites (ca. 7% and 26%, respectively, as BPA equivalents) other than the glucoside. Over 50% of BPA might be polymerized and/or tightly bound in the plant residues.

Comparison of LNS-AmB, a novel low-dose formulation of amphotericin B with lipid nano-sphere (LNS), with commercial lipid-based formulations.

Three lipid-based delivery systems (AmBisome, Amphocil, and Abelcet) for amphotericin B (AmB) have been marketed to overcome the disadvantages associated with the clinical use of AmB. However, to show their efficacy, they need to be administered at higher doses than the conventional dosage form, Fungizone. In this study, we compared LNS-AmB, our new low-dose therapeutic system for AmB using lipid nano-sphere (LNS), with these commercial formulations in terms of their pharmacokinetics and efficacy. The plasma AmB levels yielded by LNS-AmB after intravenous administration to rats were much higher than those yielded by Amphocil or Abelcet, and similar to those yielded by AmBisome, but in dogs LNS-AmB yielded plasma AmB concentrations about three times higher than did AmBisome. In a carrageenin-induced pleurisy model in rats, LNS-AmB yielded AmB levels in the pleural exudate over 20 times those yielded by Amphocil or Abelcet, and similar to those yielded by AmBisome. From these pharmacokinetic results, it is clear that Amphocil and Abelcet are based on a quite distinct drug-delivery concept from LNS-AmB. In a rat model of localized candidiasis, LNS-AmB significantly inhibited the growth of Candida albicans in the pouch, whereas AmBisome did not, even though the AmB concentrations in the pouch were similar. This difference in antifungal activity between LNS-AmB and AmBisome was also found in vitro. That is, the antifungal activity of LNS-AmB against C. albicans was similar to that of Fungizone and dimethyl sulfoxide-solubilized AmB, while AmBisome showed weaker antifungal activity than did other formulations. Based on these results, the release of AmB from AmBisome was judged to be slow and slight. In a mouse model of systemic candidiasis, LNS-AmB (1.0mg/kg) was much more effective than AmBisome (8.0mg/kg) or Fungizone (1.0mg/kg). These results suggest that LNS-AmB maintained the potent activity of AmB against fungal cells even though the AmB was incorporated into LNS particles. We conclude that LNS-AmB may offer an improved therapeutic profile at lower doses than Fungizone and commercial lipid-based formulations.

Evaluation of the efficacy and toxicity of amphotericin B incorporated in lipid nano-sphere (LNS).

To develop a low-dose therapeutic system for amphotericin B (AmB), the efficacy and toxicity of lipid nano-sphere (LNS) incorporating AmB (LNS-AmB) were evaluated and compared with those of Fungizone, the conventional dosage form of AmB with sodium deoxycholate. LNS-AmB and Fungizone showed nearly equal activity against fungal cells both in vitro and in vivo. In contrast to Fungizone, however, LNS-AmB did not cause significant hemolysis. In addition, the vomiting toxicity of Fungizone was largely avoided by the use of LNS-AmB in dogs, in spite of the higher plasma AmB concentrations achieved by LNS-AmB. Therefore, LNS-AmB may be selective for fungal cells over mammalian cells. In a study of its toxicity and toxicokinetics in a regimen of daily 2-h intravenous infusions for 14 consecutive days, LNS-AmB showed less toxicity to the kidney than did Fungizone in spite of the higher plasma AmB concentrations achieved. LNS-AmB, therefore, allows the treatment of systemic fungal infections at low doses without the severe nephrotoxicity of Fungizone. Size-exclusion chromatography provided evidence that, when LNS-AmB was administered to rats, AmB was retained in the LNS particles in the blood circulation, but that when Fungizone was administered, AmB was transferred to high-density lipoproteins (HDL). AmB retained in LNS particles seemed to be less toxic to the kidney than was AmB associated with HDL. Consequently, LNS-AmB has the potential to become a low-dose therapeutic system for AmB, minimizing most of the severe side effects of AmB by decreasing the total dose required.

Extracellular matrix remodeling may predominate over hepatocyte injury in hepatocellular carcinoma development.

It has been suggested that both extracellular matrix (ECM) remodeling and persistent hepatocyte injury play important roles in liver carcinogenesis process. It is, however, still controversial which factor plays a predominant role. The aim of the present study was to examine the role of each factor in the liver enzyme-altered preneoplastic lesions, focusing on the relationship between the hepatocyte injury and fibrosis extension. The effects of two similar herbal medicines (Sho-saiko-to and Saiko-keishi-to: TJ-9 and TJ-10, respectively) were elucidated on the hepatocyte injury, fibrosis and preneoplastic lesions development using a choline-deficient-L-amino acid-defined (CDAA) diet rat liver carcinogenesis model. TJ-9 prevented fibrosis and glutathione S-transferase placental form (GST-P)-positive preneoplastic lesion development without reducing the hepatocyte injury as indicated by the serum markers. TJ-10 significantly protected against the hepatocyte injury. However, it did not exert any inhibitory effect on fibrosis and the development of preneoplastic lesions. Our in vitro study revealed that TJ-9 markedly suppressed the hepatic stellate cell activation whereas TJ-10 did not. These results suggested that the ECM remodeling plays a more important role than the persistent hepatocyte injury in the liver enzyme-altered preneoplastic lesion development in the rat.