Tacrolimus Inhibits the Revascularization of Isolated Pancreatic Islets

Aims

Immunosuppressive drugs could be crucial factors for a poor outcome after islet allotransplantation. Unlike rapamycin, the effects of tacrolimus, the current standard immunosuppressant used in islet transplantation, on graft revascularization remain unclear. We examined the effects of tacrolimus on islet revascularization using a highly sensitive imaging system, and analyzed the gene expression in transplanted islets by introducing laser microdissection techniques.

Methods

Islets isolated from C57BL/6-Tg (CAG-EGFP) mice were transplanted into the nonmetallic dorsal skinfold chamber on the recipients. Balb/c athymic mice were used as recipients and were divided into two groups: including a control group (n = 9) and tacrolimus-treated group (n = 7). The changes in the newly-formed vessels surrounding the islet grafts were imaged and semi-quantified using multi-photon laser-scanning microscopy and a Volocity system. Gene expression in transplanted islets was analyzed by the BioMark dynamic system.

Results

The revascularization process was completed within 14 days after pancreatic islet transplantation at subcutaneous sites. The newly-formed vascular volume surrounding the transplanted islets in the tacrolimus-treated group was significantly less than that in the control group (p<0.05). Although the expression of Vegfa (p<0.05) and Ccnd1 (p<0.05) was significantly upregulated in the tacrolimus-treated group compared with that of the control group, no differences were observed between the groups in terms of other types of gene expression.

Conclusions

The present study demonstrates that tacrolimus inhibits the revascularization of isolated pancreatic islets without affecting the characteristics of the transplanted grafts. Further refinements of this immunosuppressive regimen, especially regarding the revascularization of islet grafts, could improve the outcome of islet allotransplantation.     

Antiviral Activity of Glycyrrhizin against Hepatitis C Virus In Vitro

Glycyrrhizin (GL) has been used in Japan to treat patients with chronic viral hepatitis, as an anti-inflammatory drug to reduce serum alanine aminotransferase levels. GL is also known to exhibit various biological activities, including anti-viral effects, but the anti-hepatitis C virus (HCV) effect of GL remains to be clarified. In this study, we demonstrated that GL treatment of HCV-infected Huh7 cells caused a reduction of infectious HCV production using cell culture-produced HCV (HCVcc). To determine the target step in the HCV lifecycle of GL, we used HCV pseudoparticles (HCVpp), replicon, and HCVcc systems. Significant suppressions of viral entry and replication steps were not observed. Interestingly, extracellular infectivity was decreased, and intracellular infectivity was increased. By immunofluorescence and electron microscopic analysis of GL treated cells, HCV core antigens and electron-dense particles had accumulated on endoplasmic reticulum attached to lipid droplet (LD), respectively, which is thought to act as platforms for HCV assembly. Furthermore, the amount of HCV core antigen in LD fraction increased. Taken together, these results suggest that GL inhibits release of infectious HCV particles. GL is known to have an inhibitory effect on phospholipase A2 (PLA2). We found that group 1B PLA2 (PLA2G1B) inhibitor also decreased HCV release, suggesting that suppression of virus release by GL treatment may be due to its inhibitory effect on PLA2G1B. Finally, we demonstrated that combination treatment with GL augmented IFN-induced reduction of virus in the HCVcc system. GL is identified as a novel anti-HCV agent that targets infectious virus particle release.     

What Time Periods of the Day Are Concerning for Parents of Children with Attention Deficit Hyperactivity Disorder?

Background/Aim

The questionnaire-children with difficulties (QCD) is a parent-assessed questionnaire designed to evaluate a child’s difficulties in functioning during specific time periods of the day. In this study, the QCD was applied to determine the time periods of the day that are concerning for the parents of children with attention deficit hyperactivity disorder (ADHD). The results were compared with those for a community sample.

Methods

Elementary and junior high school students with ADHD (243 boys, 55 girls) and a community sample of children (518 boys, 618 girls) were enrolled in this study. Their behaviors were assessed by the QCD, the ADHD-rating scale (ADHD-RS), and the Oppositional Defiant Behavior Inventory (ODBI). The effects of gender (boy/girl) and diagnosis (ADHD/community sample) on the total QCD score were analyzed across each school grade (elementary/junior high school). Correlation coefficients between QCD and ADHD-RS/ODBI scores were analyzed.

Results

The QCD score for the ADHD group was significantly lower than that for the community sample (P < 0.001). There were significantly strong correlations between “evening” and ADHD-RS and ODBI scores for all children with ADHD (r > 0.41, P < 0.001) and between “night” and inattention and oppositional symptoms for the girls with ADHD (r > 0.40, P < 0.001).

Conclusions

Parents reported that children with ADHD faced greater difficulties in completing basic daily activities compared with the community controls, particularly in the evening. Furthermore, these difficulties were related to the severity of ADHD symptoms. The parents’ perceptions depended on the gender, ADHD and oppositional symptoms, and the time period of the day. This study determined that children with ADHD face greater difficulties in daily functioning compared with community sample children, that these difficulties are time-dependent, and that these difficulties were particularly experienced in the evening.     

Estimation of the Fraction of Cancer Cells in a Tumor DNA Sample Using DNA Methylation

Contamination of normal cells is almost always present in tumor samples and affects their molecular analyses. DNA methylation, a stable epigenetic modification, is cell type-dependent, and different between cancer and normal cells. Here, we aimed to demonstrate that DNA methylation can be used to estimate the fraction of cancer cells in a tumor DNA sample, using esophageal squamous cell carcinoma (ESCC) as an example. First, by an Infinium HumanMethylation450 BeadChip array, we isolated three genomic regions (TFAP2B, ARHGEF4, and RAPGEFL1) i) highly methylated in four ESCC cell lines, ii) hardly methylated in a pooled sample of non-cancerous mucosae, a pooled sample of normal esophageal mucosae, and peripheral leukocytes, and iii) frequently methylated in 28 ESCCs (TFAP2B, 24/28; ARHGEF4, 20/28; and RAPGEFL1, 19/28). Second, using eight pairs of cancer and non-cancer cell samples prepared by laser capture microdissection, we confirmed that at least one of the three regions was almost completely methylated in ESCC cells, and all the three regions were almost completely unmethylated in non-cancer cells. We also confirmed that DNA copy number alterations of the three regions in 15 ESCC samples were rare, and did not affect the estimation of the fraction of cancer cells. Then, the fraction of cancer cells in a tumor DNA sample was defined as the highest methylation level of the three regions, and we confirmed a high correlation between the fraction assessed by the DNA methylation fraction marker and the fraction assessed by a pathologist (r=0.85; p<0.001). Finally, we observed that, by correction of the cancer cell content, CpG islands in promoter regions of tumor-suppressor genes were almost completely methylated. These results demonstrate that DNA methylation can be used to estimate the fraction of cancer cells in a tumor DNA sample.     

Autophagy-mediated degradation is necessary for regression of cardiac hypertrophy during ventricular unloading

Cardiac hypertrophy occurs in response to a variety of stresses as a compensatory mechanism to maintain cardiac output and normalize wall stress. Prevention or regression of cardiac hypertrophy can be a major therapeutic target. Although regression of cardiac hypertrophy occurs after control of etiological factors, the molecular mechanisms remain to be clarified. In the present study, we investigated the role of autophagy in regression of cardiac hypertrophy. Wild-type mice showed cardiac hypertrophy after continuous infusion of angiotensin II for 14 days using osmotic minipumps, and regression of cardiac hypertrophy was observed 7 days after removal of the minipumps. Autophagy was induced during regression of cardiac hypertrophy, as evidenced by an increase in microtubule-associated protein 1 light chain 3 (LC3)-II protein level. Then, we subjected cardiac-specific Atg5-deficient (CKO) and control mice (CTL) to angiotensin II infusion for 14 days. CKO and CTL developed cardiac hypertrophy to a similar degree without contractile dysfunction. Seven days after removal of the minipumps, CKO showed significantly less regression of cardiac hypertrophy compared with CTL. Regression of pressure overload-induced cardiac hypertrophy after unloading was also attenuated in CKO. These results suggest that autophagy is necessary for regression of cardiac hypertrophy during unloading of neurohumoral and hemodynamic stress.     

Age- and size-based morphological comparison between the brown alga Sargassum macrocarpum (Heterokonta; Fucales) from different depths at an exposed coast in northern Kyoto, Japan

Although large perennial brown algae are known to show great morphological plasticity responding to specific environmental factors such as depth and wave exposure, there are few reports showing this morphological variability taking into account age or size composition. Here, we conducted age- and size-based morphological comparison between the perennial brown alga Sargassum macrocarpum C. Agardh from their upper depth limit, middle depth, and lower depth limit at an exposed coast in northern Kyoto, Japan. Model II regression was fitted for the relationships between age or stipe diameter (SD) and morphological variables including SD, holdfast weight (HW), number of main branches (MBN), total weight of main branches (MBW), thallus weight (TW), and thallus length (TL) of 30 specimens collected seasonally from each depth zone. The differences between depths in these regression equations were analyzed using SMATR. Although SD, HW, and MBN of the specimens did not differ between depths under both age- and SD-basis, there were significant differences between depths in MBW, TW, and TL, suggesting that the differences in TW between depths are resulted from differences in MBW. Whereas TW at the lower depth limit was lower than that at the upper depth limit or middle depth in many months, TL at the upper depth limit was shorter than that at the middle depth or lower depth limit in some months. These results suggest that S. macrocarpum at shallower depths tend to have short and bushy morphology, whereas those at deeper depths have long and less bushy morphology.     

Effects of heat treatment on oil-binding ability of rice flour

Heat-treated (120 °C for 120 min) rice flour showed high affinity to oil (oil-binding ability). This oil-binding ability could be observed by shaking the heat-treated rice flour (2.0 g), oil (4.0 mL), and water (20 mL) vigorously in a test tube, and the oil bound to the rice flour sank into the water. To examine the time-dependent levels of the oil-binding ability, rice flour was heat-treated at 120 °C for 10, 20, 40, 60, and 120 min, and the precipitated volume of oil/rice flour complex increased with an increase of the heating time. The oil-binding ability of the rice flour was not affected by the treatments with diethyl ether or boiled chloroform/methanol (2:1) solutions, which suggested no relationship to the oil in the rice flour, but was lost upon alkali (0.2% NaOH solution) or pepsin treatment, which suggested its relationship to the rice proteins.     

Synthesis and biological evaluation of C-glucosides with azulene rings as selective SGLT2 inhibitors for the treatment of type 2 diabetes mellitus: Discovery of YM543

Here, a series of C-glucosides with azulene rings in the aglycon moiety was synthesized and the inhibitory activities toward hSGLT1 and hSGLT2 were evaluated. Starting from the azulene derivative 7 which had relatively good SGLT2 inhibitory activity, compound 8a which has a 3-[(azulen-2-yl)methyl]phenyl group was identified as a lead compound for further optimization. Introduction of a phenolic hydroxyl group onto the central benzene ring afforded a potent and selective SGLT2 inhibitor 8e, which reduced blood glucose levels in a dose-dependent manner in rodent diabetic models. A mono choline salt of 8e (YM543) was selected as a clinical candidate for use in treating type 2 diabetes mellitus.     

Hepatitis C virus RNA replication in human stellate cells regulates gene expression of extracellular matrix-related molecules

Erlin1 and erlin2 are highly homologous, ∼40 kDa, endoplasmic reticulum membrane proteins that assemble into a ring-shaped complex with a mass of ∼2 MDa. How this complex is formed is not understood, but appears to involve multiple interactions, including a coiled-coil region that mediates lower-order erlin assembly, and a short hydrophobic region, termed the “assembly domain”, that mediates higher-order assembly into ∼2 MDa complexes. Here we have used molecular modeling, mutagenesis and cross-linking to examine the role of the assembly domain in higher-order assembly. We find (i) that the assembly domains of erlin1 and erlin2 are amphipathic helices, (ii) that erlin1 alone and erlin2 alone can assemble into ∼2 MDa complexes, (iii) that higher-order assembly is strongly inhibited by point mutations to the assembly domain, (iv) that three interacting hydrophobic residues in the assembly domain and aromaticity are essential for higher-order assembly, and (iv) that while erlins1 and 2 are equally capable of forming lower-order homo- and hetero-oligomers, hetero-oligomers are the most prevalent form when erlin1 and erlin2 are co-expressed. Overall, we conclude that the ∼2 MDa erlin1/2 complex is composed of an assemblage of lower-order hetero-oligomers, probably heterotrimers, linked together by assembly domain hydrophobic residues.