Biological characteristics of two lysines on human serum albumin in the high-affinity binding of 4Z,15Z-bilirubin-IXα revealed by phage display

4Z,15Z-bilirubin-IXα (4Z,15Z-BR), an endogenous compound that is sparingly soluble in water, binds human serum albumin (HSA) with high affinity in a flexible manner. A phage library displaying recombinant HSA domain II was constructed, after three rounds of panning against immobilized 4Z,15Z-BR, and eight clones with high affinity for the pigment were found to contain conserved basic residues, such as lysine or arginine, at positions 195 and 199. The wild type and two mutants, K195A and K199A, of whole HSA as well as stand-alone domain II were expressed in Pichia pastoris for ligand-binding studies. The binding of 4Z,15Z-BR to the K195A and K199A mutants was decreased in both whole HSA and the domain II proteins. The P-helicity conformer (P-form) of 4Z,15Z-BR was found to preferentially bind to the wild types and the K195A mutants, whereas the M-form bound to the K199A mutants. Photoconversion experiments showed that the P-form of 4Z,15Z-BR was transformed into highly water-soluble isomers at a much faster rate than the M-form. In addition, the M-form of 4Z,15Z-BR showed higher affinity for domain I than for domain II. The present findings suggest that, whereas both Lys195 and Lys199 in subdomain IIA are important for the high-affinity binding of 4Z,15Z-BR, Lys199 plays a more prominent role in the elimination of 4Z,15Z-BR.     

Hepatitis C virus infection suppresses the interferon response in the liver of the human hepatocyte chimeric mouse

Background and Aims

Recent studies indicate that hepatitis C virus (HCV) can modulate the expression of various genes including those involved in interferon signaling, and up-regulation of interferon-stimulated genes by HCV was reported to be strongly associated with treatment outcome. To expand our understanding of the molecular mechanism underlying treatment resistance, we analyzed the direct effects of interferon and/or HCV infection under immunodeficient conditions using cDNA microarray analysis of human hepatocyte chimeric mice.

Methods

Human serum containing HCV genotype 1b was injected into human hepatocyte chimeric mice. IFN-α was administered 8 weeks after inoculation, and 6 hours later human hepatocytes in the mouse livers were collected for microarray analysis.

Results

HCV infection induced a more than 3-fold change in the expression of 181 genes, especially genes related to Organismal Injury and Abnormalities, such as fibrosis or injury of the liver (P = 5.90E-16 ∼ 3.66E-03). IFN administration induced more than 3-fold up-regulation in the expression of 152 genes. Marked induction was observed in the anti-fibrotic chemokines such as CXCL9, suggesting that IFN treatment might lead not only to HCV eradication but also prevention and repair of liver fibrosis. HCV infection appeared to suppress interferon signaling via significant reduction in interferon-induced gene expression in several genes of the IFN signaling pathway, including Mx1, STAT1, and several members of the CXCL and IFI families (P = 6.0E-12). Genes associated with Antimicrobial Response and Inflammatory Response were also significantly repressed (P = 5.22×10⁻¹⁰ ∼ 1.95×10⁻²).

Conclusions

These results provide molecular insights into possible mechanisms used by HCV to evade innate immune responses, as well as novel therapeutic targets and a potential new indication for interferon therapy.     

Angulin-1 seals tricellular contacts independently of tricellulin and claudins

Tricellular tight junctions (tTJs) are specialized tight junctions (TJs) that seal the intercellular space at tricellular contacts (TCs), where the vertices of three epithelial cells meet. Tricellulin and angulin family membrane proteins are known constituents of tTJs, but the molecular mechanism of tTJ formation remains elusive. Here, we investigated the roles of angulin-1 and tricellulin in tTJ formation in MDCK II cells by genome editing. Angulin-1–deficient cells lost the plasma membrane contact at TCs with impaired epithelial barrier function. The C terminus of angulin-1 bound to the TJ scaffold protein ZO-1, and disruption of their interaction influenced the localization of claudins at TCs, but not the tricellular sealing. Strikingly, the plasma membrane contact at TCs was formed in tricellulin- or claudin-deficient cells. These findings demonstrate that angulin-1 is responsible for the plasma membrane seal at TCs independently of tricellulin and claudins.     

Tissue rinse liquid‐based cytology: a feasible tool for the intraoperative pathological evaluation of sentinel lymph nodes in breast cancer patients

K. Yamashiro, K. Taira, M. Nakajima, D. Okuyama, M. Azuma, H. Takeda, H. Suzuki, H. Jotoku, K. Watanabe, M. Takahashi, K. Taguchi and M. Tamura
Tissue rinse liquid‐based cytology: a feasible tool for the intraoperative pathological evaluation of sentinel lymph nodes in breast cancer patients Objectives: A unique diagnostic method was designed for the intraoperative pathological evaluation of sentinel lymph nodes (SLNs) in breast cancer patients, and the results were verified with 2 years of experience. Methods: Excised lymph nodes were cut into 2‐mm‐thick slices and rinsed thoroughly in CytoRich Red^®. The sliced tissues were embedded in a paraffin block. Three cytological glass slides of the cells exfoliated in CytoRich Red^® were prepared by the SurePath^® liquid‐based cytology (LBC) technique. Two slides were stained by the Papanicolaou method, and the remaining slide was immunostained with an anti‐keratin antibody. This process is called tissue rinse liquid‐based cytology (TRLBC). The results of TRLBC were compared with those of the final pathological diagnoses, including immunostaining with an anti‐keratin antibody on paraffin blocks (PB). Results: This study analysed 444 SLNs from 247 consecutive breast cancer patients. It required 35 minutes to complete the intraoperative diagnosis on a single node, and it took an additional 5 minutes per node if more than one node was submitted. When the results of PB were assumed to be the gold standard, the sensitivity and specificity of TRLBC were 81.9% and 96.1%, respectively. TRLBC detected all nodes with macrometastasis and 23 of 24 nodes with micrometastasis. Fifteen false‐negative TRLBC results were ‘isolated tumour cell clusters’ on PB, but there was one with micrometastasis histologically. Four of 14 false‐positive TRLBC results were proven to be true positive by supplementary examination using step sectioning of the paraffin blocks of the nodes. Conclusion: TRLBC is a feasible and promising intraoperative cytopathological tool showing a comparable efficacy to PB while still allowing the conventional postoperative histological examination.     

Orally available pyridinylpyrimidine derivatives as novel RANKL-induced osteoclastogenesis inhibitors

An HTS campaign led to the identification of 4-pyrroldino-2-(pyridin-2-yl)pyrimidine compound 1 as an RANKL-induced osteoclastogenesis inhibitor. The compound 1 showed high clearance values in microsomes, however. Modification of the pyrrolidino group to a benzylamino group improved human microsomal stability with a slight loss of in vitro activity. Substitution at the ortho position of the benzyl group ameliorated in vitro activity, and further fluorination of the benzyl group improved microsomal stability in rodents. Representative members of this series, compounds 20 and 23, exhibited efficacy in RANKL-induced osteopenic mice when administered orally at 0.3mg/kg.     

Soft coral Sarcophyton (Cnidaria: Anthozoa: Octocorallia) species diversity and chemotypes

Research on the soft coral genus Sarcophyton extends over a wide range of fields, including marine natural products and the isolation of a number of cembranoid diterpenes. However, it is still unknown how soft corals produce this diverse array of metabolites, and the relationship between soft coral diversity and cembranoid diterpene production is not clear. In order to understand this relationship, we examined Sarcophyton specimens from Okinawa, Japan, by utilizing three methods: morphological examination of sclerites, chemotype identification, and phylogenetic examination of both Sarcophyton (utilizing mitochondrial protein-coding genes MutS homolog: msh1) and their endosymbiotic Symbiodinium spp. (utilizing nuclear internal transcribed spacer of ribosomal DNA: ITS- rDNA). Chemotypes, molecular phylogenetic clades, and sclerites of Sarcophyton trocheliophorum specimens formed a clear and distinct group, but the relationships between chemotypes, molecular phylogenetic clade types and sclerites of the most common species, Sarcophyton glaucum, was not clear. S. glaucum was divided into four clades. A characteristic chemotype was observed within one phylogenetic clade of S. glaucum. Identities of symbiotic algae Symbiodinium spp. had no apparent relation to chemotypes of Sarcophyton spp. This study demonstrates that the complex results observed for S. glaucum are due to the incomplete and complex taxonomy of this species group. Our novel method of identification should help contribute to classification and taxonomic reassessment of this diverse soft coral genus.