Preparation of Biodegradable Polymer Microcapsules Enclosing Anti-mite Agent

Many kinds of mite was caused by allergy and sick house syndrome, so it has become one of the social problems. Farnesyl acetone is one of the most potent anti-mite agent. However the vapor pressure of farnesyl acetone is high even at room temperature. This indicates the anti-mite effect has not been sustained even though the agent has strong anti-mite effect. In this study, we attempted to prepare polylactide microcapsule with farnesyl acetone for the purpose of sustainable dosage form of anti-mite agent. Microcapsule was prepared by solvent evaporation, which was a method of microencapsulation. The effects of preparation conditions on the enclosing efficiency, the release of microcapsule and anti-mite effect of microcapsule were investigated

Concerted action of activation-induced cytidine deaminase and uracil-DNA glycosylase reduces covalently closed circular DNA of duck hepatitis B virus

Covalently closed circular DNA (cccDNA) forms a template for the replication of hepatitis B virus (HBV) and duck HBV (DHBV). Recent studies suggest that activation-induced cytidine deaminase (AID) functions in innate immunity, although its molecular mechanism of action remains unclear, particularly regarding HBV restriction. Here we demonstrated that overexpression of chicken AID caused hypermutation and reduction of DHBV cccDNA levels. Inhibition of uracil-DNA glycosylase (UNG) by UNG inhibitor protein (UGI) abolished AID-induced cccDNA reduction, suggesting that the AID/UNG pathway triggers the degradation of cccDNA via cytosine deamination and uracil excision. © 2013 Federation of European Biochemical Societies

Dopaminergic neuroprotective effects of rotigotine via 5-HT1A receptors: Possibly involvement of metallothionein expression in astrocytes

Astrocytes exert neuroprotective effects through production of antioxidant molecules and neurotrophic factors. A recent study showed that stimulation of astrocyte serotonin 1A (5-HT1A) receptors promotes astrocyte proliferation and upregulation of the antioxidant molecules metallothionein (MT)-1,2, which protect dopaminergic neurons against oxidative stress. Rotigotine, an anti-parkinsonian drug, can bind to dopamine and 5-HT1A receptors. In this study, we examined neuroprotective effects of rotigotine in models of Parkinson's disease and involvement of astrocyte 5-HT1A receptors in neuroprotective effects of rotigotine against dopaminergic neurodegeneration. Rotigotine increased the number of astrocytes and MT-1,2 expression in cultured astrocytes. Pretreatment with conditioned media from rotigotine-treated astrocytes significantly inhibited 6-hydroxydopamine (6-OHDA)-induced dopaminergic neurotoxicity. These effects were completely blocked by a 5-HT1A antagonist or MT-1,2 specific antibody. Subcutaneous administration of rotigotine increased MT-1,2 expression in striatal astrocytes and prevented reduction of dopaminergic neurons in the substantia nigra of a 6-OHDA-lesioned mouse model of Parkinson's disease. These effects were blocked by co-administration with a 5-HT1A antagonist. These results suggest that rotigotine exerts neuroprotective effects through upregulation of MT expression in astrocytes by targeting 5-HT1A receptors. Our findings provide a possible therapeutic application of rotigotine to prevent dopaminergic neurodegeneration in Parkinson's disease

RNA editing of hepatitis B virus transcripts by activation-induced cytidine deaminase.

Activation-induced cytidine deaminase (AID) is essential for the somatic hypermutation (SHM) and class-switch recombination (CSR) of Ig genes. The mechanism by which AID triggers SHM and CSR has been explained by two distinct models. In the DNA deamination model, AID converts cytidine bases in DNA into uridine. The uridine is recognized by the DNA repair system, which produces DNA strand breakages and point mutations. In the alternative model, RNA edited by AID is responsible for triggering CSR and SHM. However, RNA deamination by AID has not been demonstrated. Here we found that C-to-T and G-to-A mutations accumulated in hepatitis B virus (HBV) nucleocapsid DNA when AID was expressed in HBV-replicating hepatic cell lines. AID expression caused C-to-T mutations in the nucleocapsid DNA of RNase H-defective HBV, which does not produce plus-strand viral DNA. Furthermore, the RT-PCR products of nucleocapsid viral RNA from AID-expressing cells exhibited significant C-to-T mutations, whereas viral RNAs outside the nucleocapsid did not accumulate C-to-U mutations. Moreover, AID was packaged within the nucleocapsid by forming a ribonucleoprotein complex with HBV RNA and the HBV polymerase protein. The encapsidation of the AID protein with viral RNA and DNA provides an efficient environment for evaluating AID's RNA and DNA deamination activities. A bona fide RNA-editing enzyme, apolipoprotein B mRNA editing catalytic polypeptide 1, induced a similar level of C-to-U mutations in nucleocapsid RNA as AID. Taken together, the results indicate that AID can deaminate the nucleocapsid RNA of HBV

Uracil DNA Glycosylase Counteracts APOBEC3G-Induced Hypermutation of Hepatitis B Viral Genomes: Excision Repair of Covalently Closed Circular DNA

The covalently closed circular DNA (cccDNA) of the hepatitis B virus (HBV) plays an essential role in chronic hepatitis. The cellular repair system is proposed to convert cytoplasmic nucleocapsid (NC) DNA (partially double-stranded DNA) into cccDNA in the nucleus. Recently, antiviral cytidine deaminases, AID/APOBEC proteins, were shown to generate uracil residues in the NC-DNA through deamination, resulting in cytidine-to-uracil (C-to-U) hypermutation of the viral genome. We investigated whether uracil residues in hepadnavirus DNA were excised by uracil-DNA glycosylase (UNG), a host factor for base excision repair (BER). When UNG activity was inhibited by the expression of the UNG inhibitory protein (UGI), hypermutation of NC-DNA induced by either APOBEC3G or interferon treatment was enhanced in a human hepatocyte cell line. To assess the effect of UNG on the cccDNA viral intermediate, we used the duck HBV (DHBV) replication model. Sequence analyses of DHBV DNAs showed that cccDNA accumulated G-to-A or C-to-T mutations in APOBEC3G-expressing cells, and this was extensively enhanced by UNG inhibition. The cccDNA hypermutation generated many premature stop codons in the P gene. UNG inhibition also enhanced the APOBEC3G-mediated suppression of viral replication, including reduction of NC-DNA, pre-C mRNA, and secreted viral particle-associated DNA in prolonged culture. Enhancement of APOBEC3G-mediated suppression by UNG inhibition was not observed when the catalytic site of APOBEC3G was mutated. Transfection experiments of recloned cccDNAs revealed that the combination of UNG inhibition and APOBEC3G expression reduced the replication ability of cccDNA. Taken together, these data indicate that UNG excises uracil residues from the viral genome during or after cccDNA formation in the nucleus and imply that BER pathway activities decrease the antiviral effect of APOBEC3-mediated hypermutation. © 2013 Kitamura et al

Structural studies of toxins and toxin-like proteins

Toxins are an ancient mechanism of interaction between cohabiting organisms: basal concentrations serve as an informal cue, enough as a warning signal; too much and the dialog is over. As such, the evolutionary race to arms led to the development of a vast trove of molecular unique biochemical mechanisms, from small molecules to protein toxins. The study of these mechanisms is not only essential for the treatment of toxin-related pathologies, but also as the potential source for novel therapeutic drugs. In this thesis, a series of studies of different toxins and toxin-like proteins are compiled. To further understand the biological function and relevance of each toxin, their detailed study and characterization were pursued. Here are described the advances made using a combination of different complementary biophysical and structural methods, chosen in each case to specifically target each molecule characteristics. In the first chapter, the general biological theme of this thesis is introduced: toxins, particularly protein toxins, their description, and classification, as well as the role of structural biology in the study of proteins in general. To set the theoretical background of the following chapters, are also described the general principles of two of the most prominent methods for the study of proteins in structural biology: nuclear magnetic resonance (NMR) spectroscopy, and X-ray diffraction. In the second chapter, the interaction between human FKBP12 chaperone protein and two similar bacterial small molecule toxins is detailed: rapamycin initially used as an anti-fungal before the discovery of its potent immunosuppressive properties as a mTOR inhibitor; and mycolactone, a bacterial toxin responsible for the disease Buruli ulcers in humans. In the third chapter, the cell-free protein expression system is introduced as a technique best suited for the expression of cytotoxic proteins and otherwise difficult targets, as explored further in the following chapters. In the fourth chapter, advancements towards the structural and conformational characterization of the membrane-inserted state of two similar pore-forming toxins are detailed: the bacterial Colicin Ia protein; and the human Bax protein, an apoptosis effector; using X-ray crystallography, solution NMR and solid-state NMR. Finally, in the fifth chapter, two FIC-domain bacterial toxins are investigated: the bacterial VbhTA toxin-antitoxin protein complex, and the structural determination with its cognate target, DNA GyraseB enzyme; and the auto-activation of the bacterial NmFIC protein; in both cases using a combination of X-ray crystallography and NMR spectroscopy, as well as other biophysical techniques

Reduced risk of recurrent myocardial infarction in homozygous carriers of the chromosome 9p21 rs1333049 c risk allele in the contemporary percutaneous coronary intervention era: A prospective observational study

Hara M, Sakata Y, Nakatani D on behalf of the OACIS Investigators, et al. Reduced risk of recurrent myocardial infarction in homozygous carriers of the chromosome 9p21 rs1333049 C risk allele in the contemporary percutaneous coronary intervention era: a prospective observational study. BMJ Open 2014;4:e005438. doi: 10.1136/bmjopen-2014-00543

APOBEC3 deaminases induce hypermutation in human papillomavirus 16 DNA upon beta interferon stimulation

Apolipoprotein B mRNA-editing catalytic polypeptide 3 (APOBEC3) proteins are interferon (IFN)-inducible antiviral factors that counteract various viruses such as hepatitis B virus (HBV) and human immunodeficiency virus type 1 (HIV-1) by inducing cytidine (C)-to-uracil (U) mutations in viral DNA and inhibiting reverse transcription. However, whether APOBEC3 proteins (A3s) can hypermutate human papillomavirus (HPV) viral DNA and exhibit antiviral activity in human keratinocyte remains unknown. Here we examined the involvement of A3s in the HPV life cycle using cervical keratinocyte W12 cells, which are derived from low-grade lesions and retain episomal HPV16 genomes in their nuclei. We focused on the viral E2 gene as a potential target for A3-mediated hypermutation because this gene is frequently found as a boundary sequence in integrated viral DNA. Treatment of W12 cells with beta interferon (IFN-ß) increased expression levels of A3s such as A3A, A3F, and A3G and induced C-to-U conversions in the E2 gene in a manner depending on inhibition of uracil DNA glycosylase. Exogenous expression of A3A and A3G also induced E2 hypermutation in W12 cells. IFN-ß-induced hypermutation was blocked by transfection of small interfering RNAs against A3G (and modestly by those against A3A). However, the HPV16 episome level was not affected by overexpression of A3A and A3G in W12 cells. This study demonstrates that endogenous A3s upregulated by IFN-ß induce E2 hypermutation of HPV16 in cervical keratinocytes, and a pathogenic consequence of E2 hypermutation is discussed. © 2014, American Society for Microbiology

RNA editing of hepatitis B virus transcripts by activation-induced cytidine deaminase

Activation-induced cytidine deaminase (AID) is essential for the somatic hypermutation (SHM) and class-switch recombination (CSR) of Ig genes. The mechanismby which AID triggers SHMand CSR has been explained by two distinct models. In the DNA deamination model, AID converts cytidine bases in DNA into uridine. The uridine is recognized by the DNA repair system, which produces DNA strand breakages and point mutations. In the alternative model, RNA edited by AID is responsible for triggering CSR and SHM. However, RNA deamination by AID has not been demonstrated. Here we found that C-to-T and G-to-A mutations accumulated in hepatitis B virus (HBV) nucleocapsid DNA when AID was expressed in HBV replicating hepatic cell lines. AID expression caused C-to-T mutations in the nucleocapsid DNA of RNase H-defective HBV, which does not produce plus-strand viral DNA. Furthermore, the RT-PCR products of nucleocapsid viral RNA from AID-expressing cells exhibited significant C-to-T mutations, whereas viral RNAs outside the nucleocapsid did not accumulate C-to-U mutations. Moreover, AID was packaged within the nucleocapsid by forming a ribonucleoprotein complex with HBV RNA and the HBV polymerase protein. The encapsidation of the AID protein with viral RNA and DNA provides an efficient environment for evaluating AID\u27s RNA and DNA deamination activities. A bona fide RNA-editing enzyme, apolipoprotein B mRNA editing catalytic polypeptide 1, induced a similar level of C-to-U mutations in nucleocapsid RNA as AID. Taken together, the results indicate that AID can deaminate the nucleocapsid RNA of HBV