Studies on the Chemical Synthesis of Potential Antimetabolites. 35.1 Synthesis of 2-Chloro-1-deazaadenosine and 2-Chloro-1-deazainosine as Candidate Inhibitors for S-Adenosylhomocysteinases and Methyltransferases

Abstract

The syntheses of 2-chloro-1-deazaadenosine (2) and 2-chloro-1-deazainosine (3) are described. Conversion of 7-ribosylated 6-chloro-1-deazapurine 3-oxide to the desired 2,6-disubstituted 9-ribosyl-1-deazapurines was effected by a series of reactions involving “deoxygenative chlorination” and transglycosylation in satisfactory yields.     

Linkage Mapping of Toll-Like Receptors (TLRs) in Japanese Flounder, <Emphasis Type="Italic">Paralichthys olivaceus</Emphasis>

Toll-like receptors (TLRs) are responsible for the recognition of specific pathogen-associated molecular patterns and consequently activate signal pathways leading to inflammatory and interferon responses. The region surrounding several TLRs was previously found to be associated with resistance to specific disease. Hence, we determined the location of 11 TLRs in Japanese flounder (Paralichthys olivaceus) using polymorphic microsatellite markers. TLR1 and TLR3 were located on linkage group (LG) 21 and 7, respectively. Membrane TLR5 and soluble TLR5 were mapped to LG22. TLR7 and TLR8 were mapped to LG3. TLR9 was found on LG1 and TLR14 and TLR21 were located on the same linkage group, LG10. TLR22 was found on LG8. Interestingly, TLR2 was mapped with the previously reported Poli9-8TUF microsatellite marker which is tightly associated with lymphocystis virus disease resistance. Therefore, TLR2 is a candidate gene for resistance to lymphocystis disease. These results imply that the location of a TLR associated with a particular disease may be valuable for the research on the relationship between host immune response and disease resistance.     

Spheroid formation and expression of liver-specific functions of human hepatocellular carcinoma-derived FLC-4 cells cultured in lactose-silk fibroin conjugate sponges

This study presents a hepatic tissue engineering application of three-dimensional (3D) porous sponges composed of lactose-silk fibroin (SF) conjugates (Lac-CY-SF) bearing β-galactose residues, hepatocyte-specific ligands. Lac-CY-SF sponges were prepared by freeze-drying, followed by immersion in a series of methanol aqueous solutions. Lac-CY-SF sponges showed heterogeneous pore structure with round pores about 100 μm in diameter and elongated pores 250-450 μm in length and 100-150 μm in breadth. To employ a 3D Lac-CY-SF culture system, human hepatocellular carcinoma-derived FLC-4 cells were seeded in Lac-CY-SF sponges and cultured up to 3 weeks. FLC-4 cell culture in collagen and SF sponges was also performed for comparison with the cell response to Lac-CY-SF sponges. Within 5 days of culture, FLC-4 cells cultured in Lac-CY-SF sponges, as well as the cells cultured in collagen sponges, formed multicellular spheroids with diameters from 30 to 100 μm more efficiently than did the cells cultured in SF sponges. After 3 weeks of culture, WST-1 viability assay revealed that shrinkage suppression of Lac-CY-SF sponges enabled the maintenance of viable FLC-4 cells for a long time, while the shrinkage and disintegration of collagen sponges prevented the maintenance of the cells. FLC-4 cells cultured in Lac-CY-SF sponges exhibited greater elevation of albumin secretion and sustained a higher albumin level compared with the cells cultured in collagen and SF sponges during the 3 week cultivation period. FLC-4 cells cultured in Lac-CY-SF sponges for 3 weeks expressed genes related to liver-specific functions such as transferrin and HNF-4α. On the other hand, the cells cultured in collagen and SF sponges for 3 weeks did not express these genes. These results indicated the very promising properties of Lac-CY-SF sponges as a scaffold for long-term culture of functional FLC-4 cells to study drug toxicity and hepatocyte metabolism in humans and develop a bioartificial liver model.     

Evaluating (13)C enrichment data of free amino acids for precise metabolic flux analysis

Metabolic flux analysis using (13)C enrichment data of intracellular free amino acids (FAAs) can improve the time resolution of flux estimation compared to analysis of proteinogenic amino acid data owing to the faster turnover times of FAAs. The nature of the (13)C enrichment dynamics of FAAs remains obscure, however, especially with regard to its dependence on culture conditions, even though an understanding of dynamic behavior is important for precise metabolic flux estimation. In this study, we analyzed the (13)C enrichment dynamics of free and proteinogenic amino acids in a series of continuous culture experiments with Escherichia coli. The results indicated that the effect of protein degradation on the (13)C enrichment of FAAs was negligible under cellular growth conditions. Furthermore, they showed that the time scale necessary for (13)C enrichment dynamics of FAAs to reach a steady state depends on culture conditions such as oxygen uptake rate, which was likely due to different pool sizes of intracellular metabolites. The results demonstrate the importance of analyzing (13)C enrichment dynamics for the precise estimation of metabolic fluxes using FAA data.     

A novel TK-NOG based humanized mouse model for the study of HBV and HCV infections

The immunodeficient mice transplanted with human hepatocytes are available for the study of the human hepatitis viruses. Recently, human hepatocytes were also successfully transplanted in herpes simplex virus type-1 thymidine kinase (TK)-NOG mice. In this study, we attempted to infect hepatitis virus in humanized TK-NOG mice and urokinase-type plasminogen activator-severe combined immunodeficiency (uPA–SCID) mice. TK-NOG mice were injected intraperitoneally with 6 mg/kg of ganciclovir (GCV), and transplanted with human hepatocytes. Humanized TK-NOG mice and uPA/SCID mice were injected with hepatitis B virus (HBV)- or hepatitis C virus (HCV)-positive human serum samples. Human hepatocyte repopulation index (RI) estimated from human serum albumin levels in TK-NOG mice correlated well with pre-transplantation serum ALT levels induced by ganciclovir treatment. All humanized TK-NOG and uPA–SCID mice injected with HBV infected serum developed viremia irrespective of lower replacement index. In contrast, establishment of HCV viremia was significantly more frequent in TK-NOG mice with low human hepatocyte RI (<70%) than uPA–SCID mice with similar RI. Frequency of mice spontaneously in early stage of viral infection experiment (8 weeks after injection) was similar in both TK-NOG mice and uPA–SCID mice. Effects of drug treatment with entecavir or interferon were similar in both mouse models. TK-NOG mice thus useful for study of hepatitis virus virology and evaluation of anti-viral drugs.