Isolation and Characterization of Antitumor Lipopolysaccharide from Proteus mirabilis

Systematic isolation of the cell constituents of Proteus mirabilis RMS–203 was performed to find out localization of antitumor principle only in the lipopolysaccharide (LPS) layer of the cell wall fraction.

LPS with strong antitumor activity was extracted from P. mirabilis RMS–203 by phenol-water method followed by purification on DEAE-Sephadex A–50 column chromatography.

The main components of purified LPS were galactose, hexosamine, 2-keto-deoxy-octonic acid (KDO), myristic acid, β-hydroxymyristic acid and α,ε-diaminopimelic acid.

The minimal effective dose of LPS against Ehrlich solid carcinoma in mice was 0.1~1.0 μg/mouse. LD₅₀ in mice and pyrogenicity in rabbits were 28 mg/kg and 10^?3–10^?5 μg/rabbit, respectively.     

Reducing Molecular Flexibility by Cyclization for Elucidation of Absolute Configuration by CD Calculations: Daurichromenic Acid

Circular dichroism (CD) calculations of flexible natural products have been difficult because of the large number of low‐energy conformers and ambiguous Boltzmann distributions. In this article, through electronic (ECD) and vibrational (VCD) studies on a natural product, (+)‐daurichromenic acid, we demonstrate that derivatization of a flexible molecule can dramatically reduce its flexibility. This work also shows the usefulness of derivatization for diminishing computational expenses required for optimization and CD calculations, and for increasing the reliability of the assignment of absolute configuration. Chirality 28:453–459, 2016. © 2016 Wiley Periodicals, Inc.     

Induced pluripotent stem cell‐derived melanocyte precursor cells undergoing differentiation into melanocytes

Induced pluripotent stem cell (iPSC) technology offers a novel approach for conversion of human primary fibroblasts into melanocytes. During attempts to explore various protocols for differentiation of iPSCs into melanocytes, we found a distinct and self‐renewing cell lineage that could differentiate into melanocytes, named as melanocyte precursor cells (MPCs). The MPCs exhibited a morphology distinctive from that of melanocytes, in lacking either the melanosomal structure or the melanocyte‐specific marker genes MITF, TYR, and SOX10. In addition, gene expression studies in the MPCs showed high‐level expression of WNT5A, ROR2, which are non‐canonical WNT pathway markers, and its related receptor TGFβR2. In contrast, MPC differentiation into melanocytes was achieved by activating the canonical WNT pathway using the GSK3β inhibitor. Our data demonstrated the distinct characteristic of MPCs' ability to differentiate into melanocytes, and the underlying mechanism of interfacing between canonical WNT signaling pathway and non‐canonical WNT signaling pathway.     

Role of phosphorylation on the structural dynamics and function of types III and IV intermediate filaments

Phosphorylation of types III and IV intermediate filaments (IFs) is known to regulate their organization and function. Phosphorylation of the amino-terminal head domain sites on types III and IV IF proteins plays a key role in the assembly/disassembly of IF subunits into 10 nm filaments, and influences the phosphorylation of sites on the carboxyl-terminal tail domain. These phosphorylation events are largely under the control of second messenger-dependent protein kinases and provide the cells a mechanism to reorganize the IFs in response to the changes in second messenger levels. In mitotic cells, Cdk1, Rho kinase, PAK1 and Aurora-B kinase are believed to regulate vimentin and glial fibrillary acidic protein phosphorylation in a spatio-temporal manner. In neurons, the carboxyl-terminal tail domains of the NF-M and NF-H subunits of heteropolymeric neurofilaments (NFs) are highly phosphorylated by proline-directed protein kinases. The phosphorylation of carboxyl-terminal tail domains of NFs has been suspected to play roles in forming cross-bridges between NFs and microtubules, slowing axonal transport and promoting their integration into cytoskeleton lattice and, in doing so, to control axonal caliber and stabilize the axon. The role of IF phosphorylation in disease pathobiology is discussed.     

Oncostatin M induces upregulation of claudin-2 in rodent hepatocytes coinciding with changes in morphology and function of tight junctions

In rodent livers, integral tight junction (TJ) proteins claudin-1, -2, -3, -5 and -14 are detected and play crucial roles in the barrier to keep bile in bile canaculi away from the blood circulation. Claudin-2 shows a lobular gradient increasing from periportal to pericentral hepatocytes, whereas claudin-1 and -3 are expressed in the whole liver lobule. Although claudin-2 expression induces cation-selective channels in tight junctions of epithelial cells, the physiological functions and regulation of claudin-2 in hepatocytes remain unclear. Oncostatin M (OSM) is a multifunctional cytokine implicated in the differentiation of hepatocytes that induces formation of E-cadherin-based adherens junctions in fetal hepatocytes. In this study, we examined whether OSM could induce expression and function of claudin-2 in rodent hepatocytes, immortalized mouse and primary cultured proliferative rat hepatocytes. In the immortalized mouse and primary cultured proliferative rat hepatocytes, treatment with OSM markedly increased mRNA and protein of claudin-2 together with formation of developed networks of TJ strands. The increase of claudin-2 enhanced the paracellular barrier function which depended on molecular size. The increase of claudin-2 expression induced by OSM in rodent hepatocytes was regulated through distinct signaling pathways including PKC. These results suggest that expression of claudin-2 in rodent hepatocytes may play a specific role as controlling the size of paracellular permeability in the barrier to keep bile in bile canaculi.     

In Vivo Metabolism of the Vitamin D Analog, 22-Oxacalcitriol: Evidence for Side-chain Truncation and 17-Hydroxylation

After intravenous administration of the vitamin D₃ analog, 22-oxacalcitriol (OCT), to normal rats plasma metabolites were investigated by HPLC, GC-MS and LC-MS. Five side-chain oxidation metabolites, 24R(OH)OCT, 24S(OH)OCT, (25R)-26(OH)OCT, (25S)-26(OH)OCT and 24oxoOCT, were identified by comparison with the corresponding synthetic compounds. These side-chain oxidation metabolites were similar to those of calcitriol [1,25(OH)₂ vitamin D₃] described previously. Besides these five metabolites, two unique side-chain cleavage metabolites, 20S(OH)-hexanor-OCT and 17,20S(OH)₂-hexanor-OCT, were identified as main metabolites in plasma by GC-MS and LC-MS using a specific chemical reaction. Our studies suggest that OCT is extensively metabolized and circulates in blood as a number of metabolites as well as unchanged OCT. This metabolism includes both unique pathways of C₂₃-O₂₂ cleavage and 17-hydroxylation, in addition to the side-chain oxidation metabolites similar to those of 1,25-(OH)₂D₃.