Surface plasmon resonance assay of inhibition by pharmaceuticals for thyroxine hormone binging to transport proteins

We developed a surface plasmon resonance (SPR) assay to estimate the competitive inhibition by pharmaceuticals for thyroxine (T4) binding to thyroid hormone transport proteins, transthyretin (TTR) and thyroxine binding globulin (TBG). In this SPR assay, the competitive inhibition of pharmaceuticals for introducing T4 into immobilized TTR or TBG on the sensor chip can be estimated using a running buffer containing pharmaceuticals. The SPR assay showed reproducible immobilization of TTR and TBG, and the kinetic binding parameters of T4 to TTR or TBG were estimated. The equilibrium dissociation constants of TTR or TBG measured by SPR did not clearly differ from data reported for other binding assays. To estimate the competitive inhibition of tetraiodothyroacetic acid, diclofenac, genistein, ibuprofen, carbamazepine, and furosemide, reported to be competitive or noncompetitive pharmaceuticals for T4 binding to TTR or TBG, their 50% inhibition concentrations (IC₅₀) (or 80% inhibition concentration, IC₈₀) were calculated from the change of T4 responses in sensorgrams obtained with various concentrations of the pharmaceuticals. Our SPR method should be a useful tool for predicting the potential of thyroid toxicity of pharmaceuticals by evaluating the competitive inhibition of T4 binding to thyroid hormone binding proteins, TTR and TBG.     

Helium‐based cold atmospheric plasma‐induced reactive oxygen species‐mediated apoptotic pathway attenuated by platinum nanoparticles

Plasma is generated by ionizing gas molecules. Helium (He)‐based cold atmospheric plasma (CAP) was generated using a high‐voltage power supply with low‐frequency excitation (60 Hz at 7 kV) and He flow at 2 l/min. Platinum nanoparticles (Pt‐NPs) are potent antioxidants due to their unique ability to scavenge superoxides and peroxides. These features make them useful for the protection against oxidative stress‐associated pathologies. Here, the effects of Pt‐NPs on He‐CAP‐induced apoptosis and the underlying mechanism were examined in human lymphoma U937 cells. Apoptosis was measured after cells were exposed to He‐CAP in the presence or absence of Pt‐NPs. The effects of combined treatment were determined by observing the changes in intracellular reactive oxygen species (ROS) and both mitochondrial and Fas dependent pathway. The results indicate that Pt‐NPs substantially scavenge He‐CAP‐induced superoxides and peroxides and inhibit all the pathways involved in apoptosis execution. This might be because of the SOD/catalase mimetic effects of Pt‐NPs. These results showed that the Pt‐NPs can induce He‐CAP desensitization in human lymphoma U937 cells.     

Formation of long‐lasting galls by overwintered nymphs in the Japanese aphid Quadrartus yoshinomiyai (Hemiptera: Aphididae: Hormaphidinae)

This paper examines the life history of a generation of galls created by the aphid Quadrartus yoshinomiyai (Hormaphidinae: Nipponaphidini) on its primary host plant, Distylium racemosum. First‐instar fundatrix nymphs of Q. yoshinomiyai initiated galls on stems of developing shoots in early April and incipient enclosed galls were found from later the same month. The galls lasted for up to 14 months, during which they grew to maturity, opened in early or mid‐April of the following year and dried up by the end of June. First‐instar fundatrix nymphs were found on winter buds, indicating that they hatched from eggs in autumn and overwintered as nymphs. These results suggest that Q. yoshinomiyai has a three‐year life cycle.     

Casein Kinase Iγ2 Down-Regulates Trafficking of Ceramide in the Synthesis of Sphingomyelin

Intracellullar trafficking of lipids is fundamental to membrane biogenesis. For the synthesis of sphingomyelin, ceramide is transported from the endoplasmic reticulum to the Golgi apparatus by the ceramide transfer protein CERT. CERT is phosphorylated by protein kinase D at S132 and subsequently multiple times in a serine-repeat motif, resulting in its inactivation. However, the kinase involved in the multiple phosphorylation remains unclear. Here, we identify the γ2 isoform of casein kinase I (CKIγ2) as a kinase whose overexpression confers sphingomyelin-directed toxin-resistance to Chinese hamster ovary cells. In a transformant stably expressing CKIγ2, CERT was hyperphosphorylated, and the intracellular trafficking of ceramide was retarded, thereby reducing de novo sphingomyelin synthesis. The reduction in the synthesis of sphingomyelin caused by CKIγ2 was reversed by the expression of CERT mutants that are not hyperphosphorylated. Furthermore, CKIγ2 directly phosphorylated CERT in vitro. Among three γ isoforms, only knockdown of γ2 isoform caused drastic changes in the ratio of hypo- to hyperphosphorylated form of CERT in HeLa cells. These results indicate that CKIγ2 hyperphosphorylates the serine-repeat motif of CERT, thereby inactivating CERT and down-regulating the synthesis of sphingomyelin.     

Enhanced target-specific accumulation of radiolabeled antibodies by conjugating arginine-rich peptides as anchoring molecules

We have devised and estimated a new strategy to prolong the residence time of radiolabeled antibodies in tumor in which an octaarginine peptide (R?) was used as an anchoring molecule to fix antibodies against CD20 (NuB2; IgG2a) on tumor cells. Conjugation of R? with antibodies was performed by maleimide-thiol chemistry using thiol groups generated by reducing the disulfide bonds of the antibody. The R?-conjugated NuB2 was then reacted with succinimidyl meta-[12?I]iodobenzoate to prepare [12?I]SIB-NuB2(I) (0.92 R?/NuB2) and [12?I]SIB-NuB2(III) (3.38 R?/NuB2). Both SIB-NuB2(I) and SIB-NuB2(III) exhibited size-exclusion HPLC elution profiles and immunoreactivity to CD20-positive cells similar to those of NuB2. NuB2(I) also possessed isoelectric focusing (IEF) profile similar to NuB2. However, NuB2(III) registered a broad IEF band toward higher pI. When incubated with CD20-positive cells, [12?I]SIB-NuB2(I) and [12?I]SIB-NuB2(III) exhibited 1.4 and 4.0 times higher cell-associated radioactivity than [12?I]SIB-NuB2. After the cells were washed and reincubated in a fresh medium for 3 h, [12?I]SIB-NuB2(I) and [12?I]SIB-NuB2(III) exhibited significantly higher cell-associated radioactivity than [12?I]SIB-NuB2. In biodistribution studies in normal mice, while both [12?I]SIB-NuB2(I) and [12?I]SIB-NuB2 exhibited similar biodistribution profiles, [12?I]SIB-NuB2(III) showed faster clearance from the blood and higher hepatic radioactivity levels than [12?I]SIB-NuB2. In SCID mice bearing CD20-positive xenografts, [131I]SIB-NuB2(I) exhibited significantly higher radioactivity in xenografts than those of [12?I]SIB-NuB2 with no significant increase being observed in other tissues. The findings indicate that appropriate R? modification of antibodies satisfies both specific targeting ability of antibody and strong cell-association property of R?, which was reflected in the increased radioactivity levels in tumor. These findings supported the applicability of this approach to enhance target-specific accumulation of radiolabeled antibodies.     

A quantitative structure-activity relationship study for structurally diverse HIV-1 protease inhibitors: Contribution of conformational flexibility to inhibitory activity

In this study, we investigated by linear regression model the SAR data of the 15 HIV-1 protease inhibitors possessing structurally diverse scaffolds. First, a regression model was developed only using the enzyme-inhibitor interaction energy as a term of the model, but did not provide a good correlation with the inhibitory activity (R2 = 0.580 and Q2 = 0.500). Then, we focused on the conformational flexibility of the inhibitors which may represent the diversity of the inhibitors, and added two conformational parameters into the model, respectively: the number of rotatable bonds of ligands (deltaSrot) and the distortion energy of ligands (deltaElig). The regression model by adding deltaElig successfully improved the quality of the model (R2 = 0.771 and Q2 = 0.713) while the model with deltaSrot was unsuccessful. The prediction for a training inhibitor by the deltaElig model also showed good agreement with experimental activity. These results suggest that the conformational flexibility of HIV-1 protease inhibitors directly contributes to the enzyme inhibition.     

Zinc is required for Fc epsilon RI-mediated mast cell activation

Zinc (Zn) is an essential nutrient, and its deficiency causes growth retardation, immunodeficiency, and neuronal degeneration. However, the precise roles and molecular mechanism(s) of Zn function in immune response have not been clarified. Mast cells (MCs) are granulated cells that play a pivotal role in allergic reactions and inflammation. The granules of MCs contain various chemical mediators and inflammatory cytokines that are released upon FcepsilonRI cross-linking. In this study, we report that Zn is essential for MC activation both in vitro and in vivo. We showed that a Zn chelator, N,N,N,N-tetrakis (2-pyridylmethyl) ethylenediamine, inhibited in vivo allergic reactions such as PCA and PSA. Consistent with this, N,N,N,N-tetrakis (2-pyridylmethyl) ethylenediamine significantly inhibited the FcepsilonRI-induced degranulation and cytokine production. We found that Zn was required for FcepsilonRI-induced translocation of granules to the plasma membrane, a process that we have shown to be important for MC degranulation. In addition, we showed that Zn was essential for plasma membrane translocation of protein kinase C and subsequent nuclear translocation of NF-kappaB, leading to cytokine production, such as IL-6 and TNF-alpha. These results revealed that Zn was involved in multiple steps of FcepsilonRI-induced MC activation and required for degranulation and cytokine production.     

Hepatitis C virus inhibits DNA damage repair through reactive oxygen and nitrogen species and by interfering with the ATM-NBS1/Mre11/Rad50 DNA repair pathway in monocytes and hepatocytes

Hepatitis C virus (HCV) infection is associated with the development of hepatocellular carcinoma and putatively also non-Hodgkin's B cell lymphoma. In this study, we demonstrated that PBMCs obtained from HCV-infected patients showed frequent chromosomal aberrations and that HCV infection of B cells in vitro induced enhanced chromosomal breaks and sister chromatid exchanges. HCV infection hypersensitized cells to ionizing radiation and bleomycin and inhibited nonhomologous end-joining repair. The viral core and nonstructural protein 3 proteins were shown to be responsible for the inhibition of DNA repair, mediated by NO and reactive oxygen species. Stable expression of core protein induced frequent chromosome translocations in cultured cells and in transgenic mice. HCV core protein binds to the NBS1 protein and inhibits the formation of the Mre11/NBS1/Rad50 complex, thereby affecting ATM activation and inhibiting DNA binding of repair enzymes. Taken together, these data indicate that HCV infection inhibits multiple DNA repair processes to potentiate chromosome instability in both monocytes and hepatocytes. These effects may explain the oncogenicity and immunological perturbation of HCV infection.     

Genetic Analysis of Bordetella pertussis Isolates from the 2008–2010 Pertussis Epidemic in Japan

A large pertussis epidemic occurred between 2008 and 2010 in Japan. To investigate epidemic strains, we analyzed 33 Bordetella pertussis isolates from the epidemic period by sequencing virulence-associated genes (fim3, ptxP, ptxA, and prn) and performing multilocus variable-number tandem repeat analysis (MLVA), and compared these results with those of 101 isolates from non-epidemic, earlier and later time periods. DNA sequencing of the fim3 allele revealed that the frequency of fim3B was 4.3%, 12.8%, 30.3%, and 5.1% within isolates in 2002–2004, 2005–2007, 2008–2010, and 2011–2012, respectively. The isolation rate of the fim3B strain therefore temporarily increased during the epidemic period 2008–2010. In contrast, the frequencies of the virulence-associated allelic variants, ptxP3, ptxA1, and prn2, increased with time during overall study period, indicating that these variants were not directly involved in the occurrence of the 2008–2010 epidemic. MLVA genotyping in combination with analysis of allele types showed that the prevalence of an MT27d strain temporarily increased in the epidemic period, and that this strain carried virulence-associated allelic variants (fim3B, ptxP3, ptxA1, and prn2) also identified in recent epidemic strains of Australia, Europe, and the US. Phenotypic analyses revealed that the serotype Fim3 strain was predominant (≥87%) during all the periods studied, and that the frequency of adhesion pertactin (Prn) non-expressing B. pertussis decreased by half in the epidemic period. All MT27d strains expressed Prn and Fim3 proteins, suggesting that B. pertussis MT27d strains expressing Prn and Fim3B have the potential to cause large epidemics worldwide.