Rational design,synthesis and in vitro evaluation of novel exo-methylene butyrolactone salicyloylamide as NF-κB inhibitor

(?)-Dehydroxymethylepoxyquinomicin ((?)-DHMEQ, 1) is a specific inhibitor of NF-κB. It binds to SH group in the specific cysteine residue of NF-κB components with its epoxide moiety to inhibit DNA binding. In the present research, we have designed and synthesized an epoxide-free analog called (S)-β-salicyloylamino-α-exo-methylene-?-butyrolactone (SEMBL, 3). SEMBL inhibited DNA binding of NF-κB component p65 in vitro. It inhibited LPS-induced NF-κB activation, iNOS expression, and inflammatory cytokine secretions. It also inhibited NF-κB and cellular invasion in ovarian carcinoma ES-2 cells. Moreover, its stability in aqueous solution was greatly enhanced compared with (?)-DHMEQ. Thus, SEMBL has a potential to be a candidate for a new anti-inflammatory and anticancer agent.     

The empirical Bayes estimators of fine‐scale population structure in high gene flow species

An empirical Bayes (EB) pairwise F_ST estimator was previously introduced and evaluated for its performance by numerical simulation. In this study, we conducted coalescent simulations and generated genetic population structure mechanistically, and compared the performance of the EBF_ST with Nei's G_ST, Nei and Chesser's bias‐corrected G_ST (G_{ST_NC}), Weir and Cockerham's θ (θ_WC) and θ with finite sample correction (θ_{WC_F}). We also introduced EB estimators for Hedrick’ G’_ST and Jost’ D. We applied these estimators to publicly available SNP genotypes of Atlantic herring. We also examined the power to detect the environmental factors causing the population structure. Our coalescent simulations revealed that the finite sample correction of θ_WC is necessary to assess population structure using pairwise F_ST values. For microsatellite markers, EBF_ST performed the best among the present estimators regarding both bias and precision under high gene flow scenarios (). For 300 SNPs, EBF_ST had the highest precision in all cases, but the bias was negative and greater than those for G_{ST_NC} and θ_{WC_F} in all cases. G_{ST_NC} and θ_{WC_F} performed very similarly at all levels of F_ST. As the number of loci increased up to 10 000, the precision of G_{ST_NC} and θ_{WC_F} became slightly better than for EBF_ST for cases with , even though the size of the bias remained constant. The EB estimators described the fine‐scale population structure of the herring and revealed that ~56% of the genetic differentiation was caused by sea surface temperature and salinity. The R package finepop for implementing all estimators used here is available on CRAN.     

Generation of membrane potential beyond the conceptual range of Donnan theory and Goldman-Hodgkin-Katz equation

Donnan theory and Goldman-Hodgkin-Katz equation (GHK eq.) state that the nonzero membrane potential is generated by the asymmetric ion distribution between two solutions separated by a semipermeable membrane and/or by the continuous ion transport across the semipermeable membrane. However, there have been a number of reports of the membrane potential generation behaviors in conflict with those theories. The authors of this paper performed the experimental and theoretical investigation of membrane potential and found that (1) Donnan theory is valid only when the macroscopic electroneutrality is sufficed and (2) Potential behavior across a certain type of membrane appears to be inexplicable on the concept of GHK eq. Consequently, the authors derived a conclusion that the existing theories have some limitations for predicting the membrane potential behavior and we need to find a theory to overcome those limitations. The authors suggest that the ion adsorption theory named Ling’s adsorption theory, which attributes the membrane potential generation to the mobile ion adsorption onto the adsorption sites, could overcome those problems.     

In vitro cytotoxicity assay to evaluate the toxicity of an electrophilic reactive metabolite using glutathione-depleted rat primary cultured hepatocytes

Glutathione plays an important role as not only a scavenger of reactive oxygen species but also in the conjugation or detoxification of electrophilic reactive metabolites, which has been thought to be one of the causes for idiosyncratic drug toxicity (IDT). Therefore, toxic responses to the reactive metabolites have been expected to be expressed more strongly in a glutathione-depleted condition. In the present study, we attempted to establish an in vitro cytotoxicity assay method to evaluate the toxicity of the reactive metabolite using rat primary cultured hepatocytes with cellular glutathione depletion by l-buthionine-S,R-sulfoximine. Also, we investigated whether the IDT risk is predictable by comparing the cytotoxic sensitivity between glutathione-depleted hepatocytes and untreated hepatocytes. Consequently, 10 drugs of 42 approved drugs, which were classified into 4 IDT categories (Withdrawn, Black box warning, Warning, and Safe), demonstrated higher cytotoxic sensitivity in the glutathione-depleted hepatocytes. Furthermore, a correlation was observed between the incidence of drugs with higher cytotoxic sensitivity in the glutathione-depleted hepatocytes and the IDT risk. The incidence was 50% in the Withdrawn category, 38% in the Black box warning category, 22% in the Warning category, and 8% in the Safe category. These results suggest that the IDT risk of some drugs may be predicted by comparing the cytotoxic sensitivity between them. Additionally, this method may be useful as a screening in the early stage of drug development where leads/candidates are optimized.     

Changes in Cerebral Blood Flow during Olfactory Stimulation in Patients with Multiple Chemical Sensitivity: A Multi-Channel Near-Infrared Spectroscopic Study

Multiple chemical sensitivity (MCS) is characterized by somatic distress upon exposure to odors. Patients with MCS process odors differently from controls. This odor-processing may be associated with activation in the prefrontal area connecting to the anterior cingulate cortex, which has been suggested as an area of odorant-related activation in MCS patients. In this study, activation was defined as a significant increase in regional cerebral blood flow (rCBF) because of odorant stimulation. Using the well-designed card-type olfactory test kit, changes in rCBF in the prefrontal cortex (PFC) were investigated after olfactory stimulation with several different odorants. Near-infrared spectroscopic (NIRS) imaging was performed in 12 MCS patients and 11 controls. The olfactory stimulation test was continuously repeated 10 times. The study also included subjective assessment of physical and psychological status and the perception of irritating and hedonic odors. Significant changes in rCBF were observed in the PFC of MCS patients on both the right and left sides, as distinct from the center of the PFC, compared with controls. MCS patients adequately distinguished the non-odorant in 10 odor repetitions during the early stage of the olfactory stimulation test, but not in the late stage. In comparison to controls, autonomic perception and negative affectivity were poorer in MCS patients. These results suggest that prefrontal information processing associated with odor-processing neuronal circuits and memory and cognition processes from past experience of chemical exposure play significant roles in the pathology of this disorder.     

Branched Chain Amino Acid Suppresses Hepatocellular Cancer Stem Cells through the Activation of Mammalian Target of Rapamycin

Differentiation of cancer stem cells (CSCs) into cancer cells causes increased sensitivity to chemotherapeutic agents. Although inhibition of mammalian target of rapamycin (mTOR) leads to CSC survival, the effect of branched chain amino acids (BCAAs), an mTOR complex 1 (mTORC1) activator remains unknown. In this study, we examined the effects of BCAA on hepatocellular carcinoma (HCC) cells expressing a hepatic CSC marker, EpCAM. We examined the effects of BCAA and/or 5-fluorouracil (FU) on expression of EpCAM and other CSC-related markers, as well as cell proliferation in HCC cells and in a xenograft mouse model. We also characterized CSC-related and mTOR signal-related molecule expression and tumorigenicity in HCC cells with knockdown of Rictor or Raptor, or overexpression of constitutively active rheb (caRheb). mTOR signal-related molecule expression was also examined in BCAA-treated HCC cells. In-vitro BCAA reduced the frequency of EpCAM-positive cells and improved sensitivity to the anti-proliferative effect of 5-FU. Combined 5-FU and BCAA provided better antitumor efficacy than 5-FU alone in the xenograft model. Stimulation with high doses of BCAA activated mTORC1. Knockdown and overexpression experiments revealed that inhibition of mTOR complex 2 (mTORC2) or activation of mTORC1 led to decreased EpCAM expression and little or no tumorigenicity. BCAA may enhance the sensitivity to chemotherapy by reducing the population of cscs via the mTOR pathway. This result suggests the utility of BCAA in liver cancer therapy.     

Controlled Chaos of Polymorphic Mucins in a Metazoan Parasite (Schistosoma mansoni) Interacting with Its Invertebrate Host (Biomphalaria glabrata)

Invertebrates were long thought to possess only a simple, effective and hence non-adaptive defence system against microbial and parasitic attacks. However, recent studies have shown that invertebrate immunity also relies on immune receptors that diversify (e.g. in echinoderms, insects and mollusks (Biomphalaria glabrata)). Apparently, individual or population-based polymorphism-generating mechanisms exists that permit the survival of invertebrate species exposed to parasites. Consequently, the generally accepted arms race hypothesis predicts that molecular diversity and polymorphism also exist in parasites of invertebrates. We investigated the diversity and polymorphism of parasite molecules (Schistosoma mansoni Polymorphic Mucins, SmPoMucs) that are key factors for the compatibility of schistosomes interacting with their host, the mollusc Biomphalaria glabrata. We have elucidated the complex cascade of mechanisms acting both at the genomic level and during expression that confer polymorphism to SmPoMuc. We show that SmPoMuc is coded by a multi-gene family whose members frequently recombine. We show that these genes are transcribed in an individual-specific manner, and that for each gene, multiple splice variants exist. Finally, we reveal the impact of this polymorphism on the SmPoMuc glycosylation status. Our data support the view that S. mansoni has evolved a complex hierarchical system that efficiently generates a high degree of polymorphism—a “controlled chaos”—based on a relatively low number of genes. This contrasts with protozoan parasites that generate antigenic variation from large sets of genes such as Trypanosoma cruzi, Trypanosoma brucei and Plasmodium falciparum. Our data support the view that the interaction between parasites and their invertebrate hosts are far more complex than previously thought. While most studies in this matter have focused on invertebrate host diversification, we clearly show that diversifying mechanisms also exist on the parasite side of the interaction. Our findings shed new light on how and why invertebrate immunity develops.     

Easy and Rapid Detection of Mumps Virus by Live Fluorescent Visualization of Virus-Infected Cells

Mumps viruses show diverse cytopathic effects (CPEs) of infected cells and viral plaque formation (no CPE or no plaque formation in some cases) depending on the viral strain, highlighting the difficulty in mumps laboratory studies. In our previous study, a new sialidase substrate, 2-(benzothiazol-2-yl)-4-bromophenyl 5-acetamido-3,5-dideoxy-α-D-glycero-D-galacto-2-nonulopyranosidonic acid (BTP3-Neu5Ac), was developed for visualization of sialidase activity. BTP3-Neu5Ac can easily and rapidly perform histochemical fluorescent visualization of influenza viruses and virus-infected cells without an antiviral antibody and cell fixation. In the present study, the potential utility of BTP3-Neu5Ac for rapid detection of mumps virus was demonstrated. BTP3-Neu5Ac could visualize dot-blotted mumps virus, virus-infected cells, and plaques (plaques should be called focuses due to staining of infected cells in this study), even if a CPE was not observed. Furthermore, virus cultivation was possible by direct pick-up from a fluorescent focus. In conventional methods, visible appearance of the CPE and focuses often requires more than 6 days after infection, but the new method with BTP3-Neu5Ac clearly visualized infected cells after 2 days and focuses after 4 days. The BTP3-Neu5Ac assay is a precise, easy, and rapid assay for confirmation and titration of mumps virus.