Curcumin modulates the apolipoprotein B mRNA editing by coordinating the expression of cytidine deamination to uridine editosome components in primary mouse hepatocytes

Curcumin, an active ingredient of Curcuma longa L., can reduce the concentration of low-density lipoproteins in plasma, in different ways. We had first reported that curcumin exhibits hypocholesterolemic properties by improving the apolipoprotein B (apoB) mRNA editing in primary rat hepatocytes. However, the role of curcumin in the regulation of apoB mRNA editing is not clear. Thus, we investigated the effect of curcumin on the expression of multiple editing components of apoB mRNA cytidine deamination to uridine (C-to-U) editosome. Our results demonstrated that treatment with 50 µM curcumin markedly increased the amount of edited apoB mRNA in primary mouse hepatocytes from 5.13%–8.05% to 27.63%–35.61%, and significantly elevated the levels of the core components apoB editing catalytic polypeptide-1 (APOBEC-1), apobec-1 complementation factor (ACF), and RNA-binding-motif-protein-47 (RBM47), as well as suppressed the level of the inhibitory component glycine-arginine-tyrosine-rich RNA binding protein. Moreover, the increased apoB RNA editing by 50 µM curcumin was significantly reduced by siRNA-mediated APOBEC-1, ACF, and RBM47 knockdown. These findings suggest that curcumin modulates apoB mRNA editing by coordinating the multiple editing components of the editosome in primary hepatocytes. Our data provided evidence for curcumin to be used therapeutically to prevent atherosclerosis.

Novel fluoronucleoside analog NCC inhibits lamivudine-resistant hepatitis B virus in a hepatocyte model

ABSTRACT Antiviral drug resistance is the most important factor contributing to treatment failure using nucleos(t)ide analogs such as lamivudine for chronic infection with hepatitis B virus (HBV). Development of a system supporting efficient replication of clinically resistant HBV strains is imperative, and new antiviral drugs are needed urgently to prevent selection of drug-resistant HBV mutants. A novel fluorinated cytidine analog, NCC (N-cyclopropyl-4′-azido-2′-deoxy-2′-fluoro-β-d-cytidine), was recently shown to strongly inhibit human HBV in vitro and in vivo. This study was designed to evaluate the antiviral activity of NCC against lamivudine-resistant HBV. We generated a stable cell line encoding the major pattern of lamivudine-resistant mutations rtL180M/M204V and designated it "HepG2.RL1". Immuno-transmission electron microscopic examination and enzyme-linked immunosorbent assay were used to detect secretion of HBV-specific particles and antigens. Quantification of extracellular DNA and intracellular DNA of HepG2.RL1 cells by quantitative real-time polymerase chain reaction revealed >625-fold and >5556-fold increases in the 50% inhibitory concentration of lamivudine, respectively, compared with that for the wild-type virus. The results showed that NCC inhibited DNA replication and HBeAg production in wild-type or lamivudine-resistant HBV in a dose-dependent manner. In conclusion, screening for antiviral compounds active against lamivudine-resistant HBV can be carried out with relative ease using hepG2.RL1 cells. NCC is a potential antiviral agent against wild-type HBV and clinical lamivudine-resistant HBV and deserves evaluation for the treatment of HBV infection.

Establishment and identification of cell lines from type O blood Korean native pigs and their efficiency in supporting embryonic development via somatic cell nuclear transfer

Due to their similarities with humans in anatomy, physiology, and genetics miniature pigs are becoming an attractive model for biomedical research. We aim to establish and evaluate blood type O cells derived from Korean native pig (KNP), a typical miniature pig breed in Korea. Ten cell lines derived from 8 KNP piglets and one adult female KNP (kidney and ear tissues) were established. To confirm the presence of blood type O, genomic DNA, fucosyltransferase (FUT) expression, and immunofluorescence staining were examined. Additionally, fluorescence-activated cell sorting and somatic cell nuclear transfer were performed to investigate the normality of the cell lines and to evaluate their effectiveness in embryo development. We found no significant bands corresponding to specific blood group A, and no increase in FUT expression in cell lines derived from piglets No. 1, No. 4, No. 5, No. 8, and the adult female KNP; moreover, they showed normal levels of expression of α 1,3-galactosyltransferase and cytidine monophosphate-N-acetylneuraminic acid hydroxylase. There was no significant difference in embryo development between skin and kidney fibroblasts derived from the blood type O KNPs. In conclusion, we successfully established blood type O KNP cell lines, which may serve as a useful model in xenotransplantation research.

Cytidine-phosphate-guanosine oligodeoxynucleotides in combination with CD40 ligand decrease periodontal inflammation and alveolar bone loss in a TLR9-independent manner

Abstract Local administration of toll-like receptor 9 (TLR9), agonist cytidine-phosphate-guanosine oligodeoxynucleotide (CpG ODNs), and CD40 ligand (CD40L) can decrease ligature-induced periodontal inflammation and bone loss in wild type (WT) mouse. Objective: This study aimed to explore whether such effect is dependent on TLR9 signaling. Material and Methods: Purified spleen B cells isolated from WT C57BL/6J mice and TLR9 knockout (KO) mice were cultured for 48 hours under the following conditions: CD40L, CpG+CD40L, CpG at low, medium and high doses. We determined B cell numbers using a hemocytometer at 24 h and 48 h. Percentages of CD1dhiCD5+ B cells were detected by flow cytometry. Interleukin-10 (IL-10) mRNA expression and protein secretion were measured by quantitative real-time polymerase chain reaction (qRT-PCR) and by ELISA, respectively. The silk ligature was tied around the maxillary second molars for 14 days, during which the CpG+CD40L mixture or PBS was injected into palatal gingiva on days 3, 6, and 9. Results: For both WT and TLR9 KO mice, CpG significantly induced B cell proliferation, increased IL-10 mRNA expression and protein secretion of IL-10 but reduced CD1dhiCD5+ B cells population; local injection of CpG+CD40L mixture significantly decreased alveolar bone loss and the number of TRAP-positive cells adjacent to the alveolar bone surface, and significantly increased the gingival mRNA expression of IL-10 and decreased RANKL and IFN-γ mRNA expression. Conclusions: These results indicated that CpG plus CD40L decreased periodontal inflammation and alveolar bone loss in a TLR9-independent manner in ligature-induced experimental periodontitis.

Effective gene editing by high-fidelity base editor 2 in mouse zygotes

Targeted point mutagenesis through homologous recombination has been widely used in genetic studies and holds considerable promise for repairing disease-causing mutations in patients. However, problems such as mosaicism and low mutagenesis efficiency continue to pose challenges to clinical application of such approaches. Recently, a base editor (BE) system built on cytidine (C) deaminase and CRISPR/Cas9 technology was developed as an alternative method for targeted point mutagenesis in plant, yeast, and human cells. Base editors convert C in the deamination window to thymidine (T) efficiently, however, it remains unclear whether targeted base editing in mouse embryos is feasible. In this report, we generated a modified high-fidelity version of base editor 2 (HF2-BE2), and investigated its base editing efficacy in mouse embryos. We found that HF2-BE2 could convert C to T efficiently, with up to 100% biallelic mutation efficiency in mouse embryos. Unlike BE3, HF2-BE2 could convert C to T on both the target and non-target strand, expanding the editing scope of base editors. Surprisingly, we found HF2-BE2 could also deaminate C that was proximal to the gRNA-binding region. Taken together, our work demonstrates the feasibility of generating point mutations in mouse by base editing, and underscores the need to carefully optimize base editing systems in order to eliminate proximal-site deamination.

The Therapeutic Effect of Sauchinone on Inflammatory Arthritis in Mice

OBJECTIVE: Rheumatoid arthritis (RA) is a chronic inflammatory disease, mainly involving joints and bones. Sauchinone is an anti-inflammatory agent isolated from Saururus chinensis, which was used in oriental medicine. The aim of this study was to evaluate the therapeutic effect of sauchinone on inflammatory arthritis and underlying mechanism of anti-arthritic effect. METHODS: Mice with collagen induced arthritis (CIA) was intraperitoneally injected with sauchinone (20 mg/kg) or vehicle. The clinical and histological evaluations were performed with arthritis scoring and hematoxylin-eosin staining, respectively. CD4+ interleukin (IL) 17+ T cells were determined under Th17 skewing condition treated with sauchinone. To evaluate the effect of sauchinone on osteoclastogenesis, mice bone marrow macrophages (BMMs) and human peripheral blood mononuclear cells (PBMCs) were cultured with macrophage-colony stimulating factor and receptor activator of nuclear factor-κB ligand in the absence or presence of sauchinone. RESULTS: Sauchinone significantly attenuated the inflammatory arthritis in CIA mice both clinically and histologically. The proportion of Th17 cells were decreased with treatment with sauchinone in vivo and in vitro. The expressions of Th17 cell markers (IL-17 and retinoic acid receptor-related orphan receptor gamma t) and B cell markers (activation-induced cytidine deaminase) were downregulated in the presence of sauchinone. Sauchinone also suppressed the formation of tartrate-resistant acid phosphatase positive cells from mice BMMs and human PBMCs, and the expression of osteoclastogenic markers. CONCLUSION: Sauchinone alleviates inflammatory arthritis in mice through inhibition of Th17 differentiation and osteoclastogenesis. Sauchinone, one of traditional herbal medicine, could be a therapeutic candidate for the treatment of RA.

Activation-induced Cytidine Deaminase in B Cell Immunity and Cancers

Activation-induced cytidine deaminase (AID) is an enzyme that is predominantly expressed in germinal center B cells and plays a pivotal role in immunoglobulin class switch recombination and somatic hypermutation for antibody (Ab) maturation. These two genetic processes endow Abs with protective functions against a multitude of antigens (pathogens) during humoral immune responses. In B cells, AID expression is regulated at the level of either transcriptional activation on AID gene loci or post-transcriptional suppression of AID mRNA. Furthermore, AID stabilization and targeting are determined by post-translational modifications and interactions with other cellular/nuclear factors. On the other hand, aberrant expression of AID causes B cell leukemias and lymphomas, including Burkitt's lymphoma caused by c-myc/IgH translocation. AID is also ectopically expressed in T cells and non-immune cells, and triggers point mutations in relevant DNA loci, resulting in tumorigenesis. Here, I review the recent literatures on the function of AID, regulation of AID expression, stability and targeting in B cells, and AID-related tumor formation.

Activation-induced Cytidine Deaminase in B Cell Immunity and Cancers

Activation-induced cytidine deaminase (AID) is an enzyme that is predominantly expressed in germinal center B cells and plays a pivotal role in immunoglobulin class switch recombination and somatic hypermutation for antibody (Ab) maturation. These two genetic processes endow Abs with protective functions against a multitude of antigens (pathogens) during humoral immune responses. In B cells, AID expression is regulated at the level of either transcriptional activation on AID gene loci or post-transcriptional suppression of AID mRNA. Furthermore, AID stabilization and targeting are determined by post-translational modifications and interactions with other cellular/nuclear factors. On the other hand, aberrant expression of AID causes B cell leukemias and lymphomas, including Burkitt's lymphoma caused by c-myc/IgH translocation. AID is also ectopically expressed in T cells and non-immune cells, and triggers point mutations in relevant DNA loci, resulting in tumorigenesis. Here, I review the recent literatures on the function of AID, regulation of AID expression, stability and targeting in B cells, and AID-related tumor formation.

Neural compression-induced neuralgias: clinical evaluation of the effect of nucleotides associated with vitamin B12

The use of a combination of uridine triphosphate (UTP), cytidine monophosphate (CMP), and hydroxocobalamin was evaluated in a double-blind, randomized study in the treatment of neuralgia due to degenerative orthopedic alterations with neural compression. Following informed consent, 80 patients were randomized to a 30 day treatment period. The subjects received a thrice-daily oral treatment regimen of either the combination treatment (Group A: total daily dose of 9mg UTP, 15mg CMP, 6 mg hydroxocobalamin) or vitamin B12 alone (Group B: total daily dose of 6 mg hydroxocobalamin). Efficacy measures evaluated global patient condition from the perspective of the subject and the investigating physician pain ? measured by a visual-analog scale and functionality, using a patient-response questionnaire. The safety evaluation took into account physical evaluations and laboratory tests performed at each visit to the study center as well as the incidence and severity of adverse events. At the end of the 30-day treatment period, there were reductions in the pain scale scores in both groups, however there was a significantly larger reduction in the scores of the Group A patients. The Patient Global Evaluation scores improved in both groups but showed greater improvement in Group A, while the Physician Global Evaluation improved significantly only in Group A. A similar finding was observed in the scores of the Patient Functionality Questionnaire. Based on the findings of this clinical trial, we conclude that the combination of UTP, CMP, and vitamin B12 has a positive effect on pain and functionality improvement in the treatment of degenerative orthopedic alterations with neural compression, in the study population evaluated.

Determination of cytidine and adenosine in cordyceps by monolithiccapillary electrochromatography / 中国中药杂志

<p><b>OBJECTIVE</b>To develop a capillary electrochromatography method for determination of cytidine and adenosine in cordyceps with monolithic column.</p><p><b>METHOD</b>The total length of the home-made ploy-butyl methacrylate (PBMA) monolithic capillary electrochromatographic column was 34.5 cm with the effective length of 26.0 cm. The mobile phase was 20 mmol x L(-1) borax solution (adjusted pH to 3.5 using acetic acid); the operation voltage was 15 kV; sample injection pressure was 6 bar x 0.1 min; column temperature was 30 degrees C and the detection wavelength was set at 214 nm. The internal standard solution was 100 mg x L(-1) trimethoprim solution [ethanol-mobile phase (1 : 1) was used as the solvent].</p><p><b>RESULT</b>The results indicated that the concentrations of cytidine and adenosine within the range of 12.5-125 mg x L(-1) were linearly correlated with the relative peak areas, and the correlative coefficients (r) were 0.999 8 and 0.999 3, respectively. The LOD (S/N = 3) and LOQ (S/N = 10) of cytidine were 2.14 and 7.14 mg x L(-1), and those of adenosine were 1.88 and 6.25 mg x L(-1). The average recoveries of the two nucleosides were from 97.2% to 103.5% with relative standard deviation (RSD) within 0.9%-2.6% in three levels.</p><p><b>CONCLUSION</b>The method is effective and credible. It can be used to determine the contents of cytidine and adenosine in cordyceps.</p>

Induction of nucleoside phosphorylase in Enterobacter aerogenes and enzymatic synthesis of adenine arabinoside / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

Nucleoside phosphorylases (NPases) were found to be induced in Enterobacter aerogenes DGO-04, and cytidine and cytidine 5'-monophosphate (CMP) were the best inducers. Five mmol/L to fifteen mmol/L cytidine or CMP could distinctly increase the activities of purine nucleoside phosphorylase (PNPase), uridine phosphorylase (UPase) and thymidine phosphorylase (TPase) when they were added into medium from 0 to 8 h. In the process of enzymatic synthesis of adenine arabinoside from adenine and uracil arabinoside with wet cells of Enterobacter aerogenes DGO-04 induced by cytidine or CMP, the reaction time could be shortened from 36 to 6 h. After enzymatic reaction the activity of NPase in the cells induced remained higher than that in the cells uninduced.

ICAM-1 [469 T/C] gene polymorphism but not [241 G/A] is associated with Behcet's disease in the Lebanese population

To investigate the association of the 2 intracellular adhesion molecules-1 [ICAM-1] gene polymorphisms [thymidine/cytidine [T/C] 469 and guanosine/adenosine [G/A] 241] in Beh‡et's disease in Lebanon. We initiated the study in July 2003, and carried out the work in the research laboratory of Beirut Arab University, Beirut, Lebanon. We extracted the DNA by glass fiber matrix mini column. We amplified the ICAM gene by polymerase chain reaction [PCR] and tested the PCR products for the presence of the polymorphisms using a restriction enzyme specific for each polymorphism. We analyzed the results by agarose electrophoresis. We demonstrated the association of only one single nucleotide polymorphism [SNP] [K469] with Beh‡et's disease, while we could not detect the other SNP [G241A] in either controls or patients in the Lebanese population. The ICAM-1 gene polymorphism 469 T/C, but not 241 G/A, may encode risk for Beh‡et's disease in the Lebanese population

Antifungal Activity of the Extract from Paeonia japonica against Candida albicans / 대한의진균학회지

BACKGROUND: Candida albicans is one of the normal flora of the skin, mucous membranes and gastrointestinal tract. Vaginal candidiasis remains a significant problem in women of childbearing age, majority of the cases are caused by C. albicans and recurrence is common in spite of topical treatment. OBJECTIVE: The purpose of this study is to develop the antifungal agent from the medicinal herbs traditionally used in Korea. METHODS: In this study, extracts from roots of Paeonia japonica were examined for antifungal activities against C. albicans. Dried roots of Paeonia japonica were extracted with dichloromethane, methanol, water respectively and serially. Liquid column chromatography and thin layer chromatography were used to separate the fractions with antifungal activity, and mass spectrometric analysis was done to determine the mass. RESULTS: Dichloromethane extract showed the highest antifungal activity aginst C. albicans. Result of fractionation and mass spectrometric analysis revealed that there were six materials: propanal, cytidine, hexadecanoic acid, cholesterol, octadecanoic acid and a unidentified material. CONCLUSION: Dichloromethane extract from Paeonia japonica could be a candidate for a new antifungal agent against C. albicans.

Characterization of B Cells of Lymph Nodes and Peripheral Blood in a Patient with Hyper IgM Syndrome

PURPOSE: Hyper IgM syndrome(HIGM) is characterized by severe recurrent bacterial infections with decreased serum levels of IgG, IgA, and IgE but elevated IgM levels. Recently, it has been classified into three groups; HIGM1, HIGM2 and a rare form of HIGM. HIGM1 is a X-linked form of HIGM and has now been identified as a T-cell deficiency in which mutations occur in the gene that encodes the CD40 ligand molecule. HIGM2 is an autosomal recessive form of HIGM. Molecular studies have shown that the mutation of HIGM2 is in the gene that encodes activation-induced cytidine deaminase(AID). Recently, another rare form of X-linked HIGM syndrome associated with hypohydrotic ectodermal dysplasia has been identified. We encountered a patient with a varient form of HIGM2. To clarify the cause of this form of HIGM, we evaluated the peripheral B cells of this patient. METHODS: The lymphocytes of the patient were prepared from peripheral blood. B cells were immortalized with the infection of EBV. Cell cycle analysis was done with the immortalized B cells of the patient. Peripheral mononuclear cells were stained with monoclonal anti-CD40L antibody. Total RNA was extracted from the peripheral mononuclear cells. After RT-PCR, direct sequencing for CD40L gene and HuAID gene were done. Immunostainings of a lymph node for CD3, CD23, CD40, Fas-L, bcl-2, BAX were done. RESULTS: The peripheral B cells of this patient showed normal expression of CD40L molecule and normal sequencing of CD40L gene, and also normal sequencing of AID gene. Interestingly, the peripheral B cells of this patient showed a decreased population of G2/mitosis phase in cell cycles which recovered to normal with the stimulation of IL-4. CONCLUSION: We suspect that the cause of increased serum IgM in this patient may be from a decrease of G2/mitosis phase of the peripheral B cells, which may be from the decreased production or secretion of IL-4. Therefore, this may be a new form of HIGM.

Synthesis of some uridine and cytidine derivatives

Some modified nucleosides were prepared from 5,6-disubstituted uridine, 5-substituted cytidine and cytidine. Different nucleophilic substitution reactions were carried out in alkaline or neutral medium. Some of these reactions describe ring formation

Synthesis of some cytidine derivatives

Synthesis of some cytidine derivatives involving 4,5 cyclisation are performed by the individual reactions of both of 5-hydroxy, 5-mercapto and 5-amino cytidines with chloroethyl formate, thionyl chloride and thiophosgene successively. On the other hand, reactions of cytidine with chloroacetone or chloropropionaldehyde give 3,4-cyclized cytidine products

Leucemía mieloide aguda en niños del Hospital Universitario de Caracas (Período 1979-1989) / Acute myeloid leukemia in children at the University Hospital of Caracas (period 1979-1989)

En el Hospital Universitario de Caracas 52 nuevos casos de Laucemia Mieloblástica aguda (LMA) fueron tratados en dos series consecutivas entre 1979-1989. Concomitantemente se intentó correlacionar los hallazgos clínicos y de laboratorio al ingreso, y la respuesta al tratamiento, con los subtipos morfológicos (FAB) de LMA. Serie I (1979-1985): comprendió 37 niños que recibieron principalmente la terapia de inducción con Citosina Arabinosa 100 mg/m2/día, en una infusión continua por 7 días, además de Daunomicina 45 mg/m2/día, en los 3 primeros días del tratamiento. La serie II incluyó solo 15 pacientes tratados en igual forma, excepto por la Citosina Arabinosa que se administró durante 10 días. Los pacientes que alcanzaron remisión en ambas series recibieron luego 5 ciclos con el esquema de COAP como terapia de consolidación. Posteriormente, fueron a un tratamiento de mantenimiento semanal con 6-Thioguanima por 4 días más Citosina Arabinosa el 5to. día durante 24 meses en remisión contínua. el tratamineto de inducción se realizó en 44 niños, dado que 7 murieron durante la fase de inducción en aplasia y otro caso se descartó por Síndrome de Down. La remisión completa fue de 64 por ciento (21/33) en la Serie I y de 36 por ciento (4/11) en la Serie II; observandose en ambas series un 42 por ciento de LMA con un componente monocítico, la mayoría del tipo M4 y 80 por ciento (20/25) de los casos que alcanzaron remisión, presentaban diferenciación mieloide FAB M1-M4. La duración de la remisión fué corta, lo que sugiere fallas en el tratamiento citotócico recayendo el 60 por ciento en los primeros 6 meses. La sobrevida libre de enfermedad fue menor de 20 por ciento al año y de sólo 6 por ciento a los 30 meses, sin diferencias estadísticas significativas en las 2 Series. Se estima que la problemática asistencial crónica de nuestro hospital pudo haber influido en la alta tasa de mortalidad temprana. Esta es la primera casuística de niños con LMA reportada en Venezuela

Conformations of deoxydinucleoside phosphates containing pyrimidine-pyrimidine photodimers.

Conformational analysis of deoxydinucleoside monophosphates with the sequences TpT and CpC have been carried out with the incorporation of both cyclobutane type pyrimidine dimers and 6-4 photoadducts using the methods of molecular mechanics energy minimization. The effect of flexibility with respect to sugar geometries and glycosidic torsions have been studied and the relative energies of a large variety of structures have been compared. The salient features obtained from these calculations have been compared with the crystallographic and spectroscopic data on pyrimidine dimer incorporated deoxydinucleoside monophosphates. Effects of "inserting" the energetically favourable conformations of such structures into B-DNA helices have been discussed in terms of the distortions in helical structures.

Pyrimidine nucleoside catabolism in penicillium chrysoyenum

P. Chrysogenum utilized cytidine recognized as the sole source of nitrogen. This pyrimidine nucleoside supported the good growth of P. chrysogenum compared to that obtained with sodium nitrate. Dailyzed cell-free extracts of this organism hydrolyzed cytidine and uridine to the correspondings base and ribose by the pyrimidine nucleoside hydrolase. Pyrimidine nucleoside hydrolase was found to be constitutive. It was proved experimentally that this enzyme had hydrological activity and no phosphorylation occurred. Thymidine can not be cleaved under these experimental conditions. The pH optimum for cytidine and uridine hydrolysis was the same 6.2, and the optimum temperature was found to be 40C. It has been proved that pyrimidine nucleoside hydrolase differs from the purine nucleoside hydrolase in the same organism. The Km value of the enzyme was calculated and fond to be 13.5 x 10-3 M for cytidine and 16.6 x 10-3 M for uridine