Studies on the Conjugated Lipids

Acid hydrolysis of cytosinine gave each one mole of cytosine, levulinic acid, ammonia and carbon dioxide. Reduction of cytosinine with PtO₂ afforded a mixture of dihydrocytosinine, 3-amino-tetrahydropyran-2-carboxylic acid and cytosine. Ozonolysis of N,N’-diacetylcytosinine methyl ester, followed by oxidation with hydrogen peroxide and acid hydrolysis gave erythro-d-β-hydroxyaspartic acid. These data permitted the assignment of structure (I) for cytosinine. Acid hydrolysis of uracinine gave uracil instead of cytosine, therefore, the structure (II) could be assigned to uracinine. Some stereochemical features and mechanism of levulinic acid formation were discussed.     

Frequency,Risk Factors and Survival Associated with an Intrasubsegmental Recurrence after Radiofrequency Ablation for Hepatocellular Carcinoma

Background

In the treatment of hepatocellular carcinoma (HCC), hepatic resection has the advantage over radiofrequency ablation (RFA) in terms of systematic removal of a hepatic segment.

Methods

We enrolled 303 consecutive patients of a single naïve HCC that had been treated by RFA at The University of Tokyo Hospital from 1999 to 2004. Recurrence was categorized as either intra- or extra-subsegmental as according to the Couinaud''s segment of the original nodule. To assess the relationship between the subsegments of the original and recurrent nodules, we calculated the kappa coefficient. We assessed the risk factors for intra- and extra-subsegmental recurrence independently using univariate and multivariate Cox proportional hazard regression. We also assessed the impact of the mode of recurrence on the survival outcome.

Results

During the follow-up period, 201 patients in our cohort showed tumor recurrence distributed in a total of 340 subsegments. Recurrence was categorized as exclusively intra-subsegmental, exclusively extra-subsegmental, and simultaneously intra- and extra-subsegmental in 40 (20%), 110 (55%), and 51 (25%) patients, respectively. The kappa coefficient was measured at 0.135 (95% CI, 0.079–0.190; P<0.001). Multivariate analysis revealed that of the tumor size, AFP value and platelet count were all risk factors for both intra- and extra-subsegmental recurrence. Of the patients in whom recurrent HCC was found to be exclusively intra-subsegmental, extra-subsegmental, and simultaneously intra- and extra-subsegmental, 37 (92.5%), 99 (90.8%) and 42 (82.3%), respectively, were treated using RFA. The survival outcomes after recurrence were similar between patients with an exclusively intra- or extra-subsegmental recurrence.

Conclusions

The effectiveness of systematic subsegmentectomy may be limited in the patients with both HCC and chronic liver disease who frequently undergo multi-focal tumor recurrence.     

Validity of ultrasound muscle thickness measurements for predicting leg skeletal muscle mass in healthy Japanese middle-aged and older individuals

Background

The skeletal muscle mass of the lower limb plays a role in its mobility during daily life. From the perspective of physical resources, leg muscle mass dominantly decreases after the end of the fifth decade. Therefore, an accurate estimate of the muscle mass is important for the middle-aged and older population. The present study aimed to clarify the validity of ultrasound muscle thickness (MT) measurements for predicting leg skeletal muscle mass (SM) in the healthy Japanese middle-aged and older population.

Findings

MTs at four sites of the lower limb and the bone-free lean tissue mass (LTM) of the right leg were determined using brightness-mode ultrasonography and dual-energy X-ray absorptiometry (DXA), respectively, in 44 women and 33 men, 52- to 78-years old. LTM was used as a representative variable of leg skeletal muscle mass. In the model-development group (30 women and 22 men), regression analysis produced an equation with R² and standard error of the estimate (SEE) of 0.958 and 0.3 kg, respectively: LTM (kg) = 0.01464 × (MT_SUM×L) (cm²) - 2.767, where MT_SUM is the sum of the product of MTs at four sites, and L is length of segment where MT is determined. The estimated LTM (7.0 ± 1.7 kg) did not significantly differ from the measured LTM (7.0 ± 1.7 kg), without a significant systematic error on a Bland-Altman plot. The application of this equation for the cross-validation group (14 women and 11 men) did not yield a significant difference between the measured (7.2 ± 1.6 kg) or estimated (7.2 ± 1.6 kg) LTM and systematic error.

Conclusion

The developed prediction equation may be useful for estimating the lean tissue mass of the lower extremity for the healthy Japanese middle-aged and older population.     

Sema6B,Sema6C,and Sema6D Expression and Function during Mammalian Retinal Development

In the vertebrate retina, the formation of neural circuits within discrete laminae is critical for the establishment of retinal visual function. Precise formation of retinal circuits requires the coordinated actions of adhesive and repulsive molecules, including repulsive transmembrane semaphorins (Sema6A, Sema5A, and Sema5B). These semaphorins signal through different Plexin A (PlexA) receptors, thereby regulating distinct aspects of retinal circuit assembly. Here, we investigate the physiological roles of three Class 6 transmembrane semaphorins (Sema6B, Sema6C, and Sema6D), previously identified as PlexA receptor ligands in non-retinal tissues, in mammalian retinal development. We performed expression analysis and also phenotypic analyses of mice that carry null mutations in each of genes encoding these proteins using a broad range of inner and outer retinal markers. We find that these Class 6 semaphorins are uniquely expressed throughout postnatal retinal development in specific domains and cell types of the developing retina. However, we do not observe defects in stereotypical lamina-specific neurite stratification of retinal neuron subtypes in Sema6B^−/− or Sema6C^−/−; Sema6D^−/− retinas. These findings indicate these Class 6 transmembrane semaphorins are unlikely to serve as major PlexA receptor ligands for the assembly of murine retinal circuit laminar organization.     

Structural Study of Cell Attachment Peptide Derived from Laminin by Molecular Dynamics Simulation

Peptides with cell attachment activity are beneficial component of biomaterials for tissue engineering. Conformational structure is one of the important factors for the biological activities. The EF1 peptide (DYATLQLQEGRLHFMFDLG) derived from laminin promotes cell spreading and cell attachment activity mediated by α2β1 integrin. Although the sequence of the EF2 peptide (DFATVQLRNGFPYFSYDLG) is homologous sequence to that of EF1, EF2 does not promote cell attachment activity. To determine whether there are structural differences between EF1 and EF2, we performed replica exchange molecular dynamics (REMD) simulations and conventional molecular dynamics (MD) simulations. We found that EF1 and EF2 had β-sheet structure as a secondary structure around the global minimum. However, EF2 had variety of structures around the global minimum compared with EF1 and has easily escaped from the bottom of free energy. The structural fluctuation of the EF1 is smaller than that of the EF2. The structural variation of EF2 is related to these differences in the structural fluctuation and the number of the hydrogen bonds (H-bonds). From the analysis of H-bonds in the β-sheet, the number of H-bonds in EF1 is larger than that in EF2 in the time scale of the conventional MD simulation, suggesting that the formation of H-bonds is related to the differences in the structural fluctuation between EF1 and EF2. From the analysis of other non-covalent interactions in the amino acid sequences of EF1 and EF2, EF1 has three pairs of residues with hydrophobic interaction, and EF2 has two pairs. These results indicate that several non-covalent interactions are important for structural stabilization. Consequently, the structure of EF1 is stabilized by H-bonds and pairs of hydrophobic amino acids in the terminals. Hence, we propose that non-covalent interactions around N-terminal and C-terminal of the peptides are crucial for maintaining the β-sheet structure of the peptides.     

EcoBalance 2018—Nexus of ideas: innovation by linking through life cycle thinking (9–12 October 2018, Tokyo,Japan)

The International Journal of Life Cycle Assessment -     

Biochemical characterization of the placeholder nucleosome for DNA hypomethylation maintenance

DNA methylation functions as a prominent epigenetic mark, and its patterns are transmitted to the genomes of offspring. The nucleosome containing the histone H2A.Z variant and histone H3K4 mono-methylation acts as a “placeholder” nucleosome for DNA hypomethylation maintenance in zebrafish embryonic cells. However, the mechanism by which DNA methylation is deterred by the placeholder nucleosome is poorly understood. In the present study, we reconstituted the placeholder nucleosome containing histones H2A.Z and H3 with the Lys4 mono-methylation. The thermal stability assay revealed that the placeholder nucleosome is less stable than the canonical nucleosome. Nuclease susceptibility assays suggested that the nucleosomal DNA ends of the placeholder nucleosome are more accessible than those of the canonical nucleosome. These characteristics of the placeholder nucleosome are quite similar to those of the H2A.Z nucleosome without H3K4 methylation. Importantly, the linker histone H1, which is reportedly involved in the recruitment of DNA methyltransferases, efficiently binds to all of the placeholder, H2A.Z, and canonical nucleosomes. Therefore, the characteristics of the H2A.Z nucleosome are conserved in the placeholder nucleosome without synergistic effects on the H3K4 mono-methylation.     

Freeze-dried platelets promote hepatocyte proliferation in mice

In recent years, platelets are reported to promote liver, as well as bone regeneration and dermal wound healing. Platelets are required not only for thrombocytopenia treating but also for regenerative therapy. Platelets cannot be stored beyond three days, therefore, shortage of fresh platelets sometimes occurs. To preserve platelets for a long duration without degrading growth factors, a freeze-dried technique is required. We report here that platelets can be preserved by freeze-drying, using a programmed freezing method to avoid intracellular ice crystal formation. Freeze-dried platelets kept their morphological countenance and response with the agonist of thrombin was well maintained. Freeze-dried platelets stored adenine nucleotides, PDGF, and IGF-1 the same as those of fresh platelets. Freeze dried platelets also preserved their proliferative effect on hepatocytes identical to that of fresh platelets. These results of our study suggest that freeze dried platelets will obviate the storage problem of fresh platelets.     

RhoG regulates anoikis through a phosphatidylinositol 3-kinase-dependent mechanism

In normal epithelial cells, cell-matrix interaction is required for cell survival and proliferation, whereas disruption of this interaction causes epithelial cells to undergo apoptosis called anoikis. Here we show that the small GTPase RhoG plays an important role in the regulation of anoikis. HeLa cells are capable of anchorage-independent cell growth and acquire resistance to anoikis. We found that RNA interference-mediated knockdown of RhoG promoted anoikis in HeLa cells. Previous studies have shown that RhoG activates Rac1 and induces several cellular functions including promotion of cell migration through its effector ELMO and the ELMO-binding protein Dock180 that function as a Rac-specific guanine nucleotide exchange factor. However, RhoG-induced suppression of anoikis was independent of the ELMO- and Dock180-mediated activation of Rac1. On the other hand, the regulation of anoikis by RhoG required phosphatidylinositol 3-kinase (PI3K) activity, and constitutively active RhoG bound to the PI3K regulatory subunit p85alpha and induced the PI3K-dependent phosphorylation of Akt. Taken together, these results suggest that RhoG protects cells from apoptosis caused by the loss of anchorage through a PI3K-dependent mechanism, independent of its activation of Rac1.     

Properties of blocking and non-blocking monoclonal antibodies specific for human macrophage galactose-type C-type lectin (MGL/ClecSF10A/CD301)

Monoclonal antibodies (mAbs) specific for the human macrophage galactose-type calcium-type lectin (MGL) were established. The recombinant extracellular domain of MGL was used to immunize a mouse, and 10 hybridoma clones were obtained. Binding of recombinant MGL to asialo-bovine submaxillary mucin was shown to be blocked by mAbs MLD-1, 4 and 6. Immunoprecipitation of MGL from lysates of COS-1 cells transfected with MGL cDNA (form 6A) was achieved with mAbs MLD-1, 4, 7, 8 and 16. Chimeric recombinant proteins between human MGL and mouse MGL1 were used to determine the location of the epitopes for these mAbs. mAbs MLD-8, 13, 15 and 16 interacted with the amino terminal side of the conserved WVDGTD sequence immediately upstream of QPD, whereas mAbs MLD-7, 12 and 17 interacted with the other side. mAbs MLD-1, 4, and 6 apparently required both sides of this boundary. mAbs MLD-15 and 16 were shown to recognize the protein products of alternatively spliced mRNA 6A/8A and 6C/8A, having deletions at the boundary of exons 7 and 8, in addition to full length and other spliced forms of MGL (6A, 6B and 6C), whereas the other mAbs bound only full length and forms 6A, 6B and 6C.