Attenuation of a virulent swine acute diarrhea syndrome coronavirus strain via cell culture passage.

Swine acute diarrhea syndrome coronavirus (SADS-CoV) is a newly identified enteric alphacoronavirus that causes fatal diarrhea in newborn piglets in China. Here, we propagated a virulent strain SADS-CoV/CN/GDWT/2017 in Vero cells for up to 83 passages. Four strains of SADS-CoV/GDWT-P7, -P18, -P48 and -P83 were isolated and characterized. Sequence alignments showed that these four novel strains exhibited 16 nucleotide mutations and resultant 10 amino acid substitutions in open reading frame 1a/1b, spike, NS3a, envelope, membrane and nucleocapsid proteins. Furthermore, a 58-bp deletion in NS7a/7b was found in P48 and P83 strains, which led to the loss of NS7b and 38 amino acid changes of NS7a. Pig infection studies showed that the P7 strain caused typical watery diarrhea, while the P83 strain induced none-to-mild, delayed and transient diarrhea. This is the first report on cell adaption of a virulent SADS-CoV strain, which gives information on the potential virulence determinants of SADS-CoV.

12 novel atypical porcine pestivirus genomes from neonatal piglets with congenital tremors: A newly emerging branch and high prevalence in China.

Atypical porcine pestivirus (APPV), a newly discovered member of the genus Pestivirus, is considered to be associated with congenital tremors (CT) in piglets. From June 2016 to January 2018, 440 serum and tissue samples from CT-affected piglets in Anhui, Guangdong and Guangxi provinces were collected to detect APPV. The results showed a high level of 63.4% preference for APPV in 27 swine farms and complicated co-infection cases between APPV and other 12 swine viruses. Meanwhile, 12 novel APPV genomes were screened and identified. Results showed that complete genomes, Npro and NS5A genes of these novel 12 APPV sequences revealed 80.5%-99.8%, 78%-100% and 76.9%-99.8% nucleotide identities, respectively. Phylogenetic analyses based on sequences of full-length genomes, Npro and NS5A genes of APPV indicated three well-defined clades including a newly emerging branch in China. This study provides novel epidemiological information of APPV in China.

Detection and genome sequencing of porcine circovirus 3 in neonatal pigs with congenital tremors in South China.

Porcine circovirus 3 (PCV3) is a novel circovirus first discovered in the United States in piglets and sows with porcine dermatitis and nephropathy syndrome, reproductive failure, cardiac and multisystemic inflammation. Here, seven PCV3 strains were identified for the first time from neonatal pigs with clinical signs of congenital tremors (CT) in South China. The tissue tropism of PCV3 in CT-affected piglets was analysed by the real-time quantitative PCR, and the result showed that high loads of viral genomes were detected in the brains and hearts. The complete genomes of seven new PCV3 revealed 96.8%-99.6% nucleotide identities with eleven other PCV3 strains previously reported from the United States and China. Phylogenetic analysis based on the complete genome sequences showed that all PCV3 strains clustered together and were clearly separated from other circovirus species. This study reports on the first identification of PCV3 in CT-affected newborn piglets and provides the epidemiological information of neonatal piglets with CT in Guangdong and Guangxi Provinces of China.

Altered queuine modification of transfer RNA involved in the differentiation of human K562 erythroleukemia cells in the presence of distinct differentiation inducers.

Altered queuine modification of tRNA has been correlated to neoplasia and cell differentiation, but much of the existing evidence is only circumstantial. In the present study, we used several distinct differentiation inducers to measure changes in Q-family tRNA species during the erythroid differentiation of human K562 erythroleukemia cells. Treatment of K562 cells with 3.6 microM 1-beta-D-arabinofuranosylcytosine (ara-C), 1 mM sodium butyrate, 0.1 mM hemin, or 5 microM 5-azacytidine resulted in growth inhibition, erythroid differentiation, and changes in queuine content of tRNA. In the presence of the irreversible inducer ara-C, the queuine content of tRNA increased markedly when the cells differentiated into benzidine-positive erythroid cells, and cell growth was inhibited. The increase in the queuine content of tRNA in differentiated K562 cells was an irreversible event. In cells incubated with the reversible inducer sodium butyrate, an increase in the queuine content of tRNA was correlated with the increase in benzidine-positive erythroid cells throughout the culturing period. After removal of the drug at 48 h, the queuine content of tRNA decreased concomitant with a decrease in benzidine-positive cells. Treatment with another reversible inducer, hemin, caused only a transient increase in the queuine content of tRNA, which was not correlated with the steady increase in benzidine-positive erythroid cells. The agent 5-azacytidine slightly inhibited cell growth but did not significantly change the percentage of benzidine-positive cells and the queuine content of tRNA. We further clarified the changes in queuine content of tRNA by analyzing the Q-containing isoacceptors of Q-family tRNA species including tRNA(Tyr), tRNA(His), tRNA(Asp), and tRNA(Asn) by RPC-5 chromatography, and found that the change in queuine content of tRNA(Tyr) was greater than the other Q-family tRNA species during induction by ara-C, sodium butyrate, and hemin. Our results indicate that the change in queuine content of tRNA is an irreversible event of terminal differentiation in ara-C induction and is a transient event of reversible differentiation in hemin induction. Sodium butyrate induction might represent a status between irreversible and reversible differentiation. Q-containing isoacceptors of tRNA might potentially play an important biological role during K562 cell differentiation.

Relationship of the queuine content of transfer ribonucleic acids to histopathological grading and survival in human lung cancer.

To elucidate the significance of tRNA hypomodified with queuine to the grade of malignancies in human solid tumors, the amount of tRNA having guanosine in place of queuosine was determined in human lung cancer and normal lung tissue, by exchanging the unmodified guanosine residue for [3H]guanine. The reaction is catalyzed by guanine:queuine tRNA transglycosylase. Total tRNA was extracted from 23 different lung cancer specimens and the precursor of isoacceptor tRNA that contains guanine instead of queuine in the first or wobble position of the anticodon [(Q-)tRNA] content was determined. In 12 cases the (Q-)-tRNA was determined in normal lung tissues as well. In each individual, the (Q-)tRNA content in lung cancer tissue was higher than that of the normal lung tissue. The (Q-)tRNA content was not correlated to the surgicopathological staging of the patients but was highly correlated to the histopathological classification of the tumors. The amounts of (Q-)-tRNA were 1.75 +/- 0.67 (SD), 2.36 +/- 0.89, 3.77 +/- 1.39, 5.18 +/- 2.32, and 7.65 +/- 1.34 pmol/A260 in normal, well, moderately, moderately to poorly, and poorly differentiated tumors. The difference from normal to moderately differentiated or less differentiated groups was significant (P less than 0.05). In 10 patients with (Q-)tRNA higher than 3.5 pmol/A260, their cancers relapsed and only 2 were alive after 4 years. In 11 patients with (Q-)tRNA less than 3.5 pmol/A260 in their lung cancer tissues, 7 patients were still alive without any evidence of disease, 3 were dead, and 1 had recurrence of disease. These results, taken together with other previous studies, suggest that a decreased queuosine content of tRNA may be a general feature of neoplasms and may be useful for grading malignancy and perhaps also for the prediction of survival in human lung cancer.

Potentiation of cytotoxicity of 1-beta-D-arabinofuranosylcytosine for K562 human leukemic cells by cadeguomycin.

The treatment of K562 human myeloblastic leukemia cells and YAC-1 murine lymphoma cells with cadeguomycin at concentrations over 0.6 microM significantly enhanced the cytotoxicity of 1-beta-D-arabinofuranosylcytosine (ara-C). The degree of potentiation depended upon the antibiotic concentration. The treatment with 75 microM cadeguomycin for 18 h increased cellular uptake of [3H]ara-C into K562 cells and formation of ara-C nucleotides, as well as incorporation into nucleic acids. The level of the diphosphate of ara-C plus the triphosphate of ara-C was approximately 10 times higher in the cadeguomycin-treated cells than in the untreated cells by 30 min of incubation with [3H]ara-C. The extracts of 15 microM cadeguomycin-treated K562 cells showed increased activity of formation of ara-C nucleotides, resulting in 4- to 5-fold higher formation of the di- and triphosphates of ara-C than the control cell extracts. Cadeguomycin did not significantly change the level of ribonucleotide and deoxyribonucleotide pool in K562 cells. The mechanism of potentiation of ara-C by cadeguomycin was discussed.

Immunosuppressive effect of emodin, a free radical generator.

The possible mechanism of immunosuppressive effect of emodin (1,3,8-trihydroxy-6-methylanthraquinone) was investigated in this study. Human mononuclear cells (10(6) cells/ml) were stimulated with 0.25% phytohemagglutinin for 24, 48 and 72 h, and the proliferative response was determined by the uptake of tritiated thymidine. In the presence of emodin (10(-6) to 3 x 10(-5) M), the proliferative response was reduced in a dose-dependent manner. Emodin (3 x 10(-7) to 3 x 10(-5) M) also dose dependently reduced the proliferative response to mixed lymphocyte reaction. After 72 h exposure to emodin (10 microM), interleukin-1 (IL-1), interleukin-2 (IL-2) production and IL-2 receptor expression were all reduced. The structure-activity relationship of emodin and 10 other anthraquione derivatives indicates that the free hydroxyl group at the beta-position of the anthraquinone nucleus plays an important role in the immunosuppressive effect. The suppressive activity of emodin was significantly inhibited by catalase (a scavenger of hydrogen peroxide), but little affected by superoxide dismutase (a scavenger of superoxide radical) and mannitol (a scavenger of hydroxyl radical). Methylene blue and hemoglobin, guanylate cyclase inhibitors, did not significantly affect the suppressive activity of emodin. Nordihydroguaiaretic acid (a lipoxygenase inhibitor) significantly potentiated the suppressive activity whereas quinacrine (a phospholipase A2 inhibitor) and indomethacin (a cyclooxygenase inhibitor) did not significantly affect it. The results suggest that the immunosuppressive effect of emodin may be partly mediated through hydrogen peroxide generated from semiquinone and regulated by arachidonic acid metabolites or byproducts.

Gallotannins inhibit growth, water-insoluble glucan synthesis, and aggregation of mutans streptococci.

During screening for anti-plaque agents of plant origin, ethanolic extracts from Melaphis chinensis (Bell), the Chinese Nutgall, exhibited strong inhibition of glucosyltransferase (GTF), in vitro adherence and glucan-induced agglutination of Streptococcus mutans 3209 and S. sobrinus B13. More than 91% inhibition of water-insoluble glucan synthesis from sucrose by GTF was noted at a concentration as low as 7.8 micrograms/mL. Bactericidal effects on other mutans streptococci, S. salivarius, and Actinomyces viscosus were also evident. Through chemical fractionation and analyses, along with bioassays, the active components were identified as gallotannins.

Immunomodulating effects of the hydrolysis products of formosanin C and beta-ecdysone from Paris formosana Hayata.

Formosanin C (PF-3, I), a diosgenin glycoside with four sugars isolated from Paris formosana Hayata as main constituent, significantly showed immunomodulating effects on the proliferative response of mouse lymphocytes to concanavalin A (Con A). The partialy hydrolysis products of formosanin C, dioscin (II) and prosapogenin A of dioscin (III), also increased 3H-thymidine incorporation of Con A-stimulated lymphocytes maximally at 0.01 micrograms/ml, whereas formosanin C did so at 0.0001 micrograms/ml. However, trillin (IV) and diosgenin (V) obtained from the partial hydrolysis of formosanin C had no effects on these immune responses. Evidently, the immunomodulating activities increased in the order of increasing polarity. Probably the solubility in water was a factor. This demonstrated that the sugar moiety in the structure of formosanin C (I) displays a very important pattern for the effect on the proliferative response of mouse lymphocytes to Con A. On the other hand, these hydrolysis products at higher concentrations of 1 and 10 micrograms/ml reduced the cytotoxic effects on spleen cells as compared with formosanin C. The other constituent, beta-ecdysone (VI) isolated from the stems of Paris formosana Hayata also increased 3H-thymidine incorporation of Con A-stimulated splenocytes. At the concentration of 0.001 micrograms/ml, the stimulation index of beta-ecdysone (2) is higher than that of formosanin C (1.65), and at the concentration of 100 micrograms/ml, beta-ecdysone had no cytotoxicity for normal spleen cells whereas formosanin C at the lower concentration of 10 micrograms/ml showed cytotoxicity. Based on this study, beta-ecdysone (VI) is therefore a better immunomodulator than formosanin C.

Immunomodulators from Paris formosana Hayata.

Eight glycosides PF-1 to PF-8 were isolated from the leaves and stems of Paris formosana Hayata (Liliaceae), of which PF-3 (III) was the main compound. It was found that PF-1 to PF-3 (I-III) caused proliferative responses of mouse lymphocytes to concanavalin A and augmentation of mouse granulocyte/macrophage colony forming cells in mouse fibroblast cell L929 conditioned medium. PF-4(IV) and PF-8 showed significant immunomodulatory effects with very low toxicity: they not only caused the same immune responses as PF-1, 2 & 3 but also enhanced the proliferative response of human peripheral whole blood to phytohemagglutinin. PF-5 (V) also increased 3H-thymidine incorporation in ConA-stimulated lymphocytes and in PHA-stimulated human peripheral whole blood, and enhanced the proliferative response of granulocyte/macrophage colony stimulating activity. PF-6(VI) and PF-7(VII) augmented 3H-thymidine incorporation in granulocyte/macrophage colony-forming cells and mitogenic response of PHA-stimulated human peripheral whole blood cells from healthy adults. However, PF-1, 2 and 3 showed hemolytic action, while PF-4 to PF-8 had no hemolytic action at all. On the other hand, the hemolytic action of PF-3 derivatives was reduced as compared with PF-3 but their immune responses did not equal those of PF-3, only showing granulocyte/macrophage colony stimulating activity.

A controlled study of anxiety and morbid cognitions at initial screening for human immunodeficiency virus (HIV) in a cohort of people with haemophilia.

AIM: This study examines the relationship between anxiety, psychological state and Human Immunodeficiency Virus (HIV) stages as defined by the Centers for Disease Control at the time of initial screening for HIV in a cohort of people with haemophilia who were at risk of prior exposure to HIV transmission from blood products. METHOD: Psychological scores, immunological measures, and clinical data from case notes for 116 potentially HIV exposed people with haemophilia attending initial screening for HIV infection in 1984-1985, were used to examine the relationship between psychological variables, clinical state and their clinical classification under the Centres for Disease Control categorization. Psychometric test results were obtained for 63 HIV seronegative patients and 53 HIV seropositive patients. Planned comparisons, multiple and logistic regressions, were used to explain observed differences between seronegative and seropositive subjects. The potential confounders of sex, age, severity of haemophilia, haemophilia type and blood product usage were controlled. RESULTS: The major finding of this study was that higher levels of State Anxiety at the time of initial screening for HIV, were observed in those patients who lacked recognized symptoms of HIV infection and were seropositive, compared with seronegative subjects. The State Anxiety scores were predicted by HIV infection or alternatively CD4+ T-cell levels. CONCLUSION: The findings of this study suggest that HIV infection can produce psychological effects prior to any physical symptoms of infection being apparent.

Formation of mutagens by amino-carbonyl reactions.

The formation of mutagens by amino-carbonyl reactions of 20 kinds of amino acid and sugars after heating at 100 degrees C for 10 h was examined by the Ames test. The browned solutions of Gly, Ala, Val, Leu, Ile, Ser, Thr, Gln, Lys X HCl, Arg, Phe, Cys, Met and Pro with Glc caused mutation of Salmonella typhimurium TA100 and/or TA98 with or without S9 mix. The presence of S9 mix increased the mutagenic activity of the browned solutions of Cys and Phe with Glc on TA100 and of those of Gly, Ala, Val, Ile and Cys on TA98, but decreased the activity of other solutions. No revertants of Salmonella were induced by the browned solutions of Trp, Tyr, Asp, Asn, Glu and (Cys)2 with Glc. Among positive browned solutions, Cys, Lys, Arg and Phe had the stronger activity, but their activity was weak compared with that of pyrolysates or chemical mutagens such as Trp-P-1, Trp-P-2 and 4-nitroquinoline-N-oxide. The mutagenic activity of the browned solutions increased with prolongation of heating time and varied with the pH of the reaction mixture. Fru, Gal, Ara, Xyl, Man, Lac and Suc also had the ability to form mutagens in the browning reactions with amino acids.

Enhancement of pyrimidine nucleoside uptake into K562 and YAC-1 cells by cadeguomycin.

Cadeguomycin markedly stimulated the uptake of thymidine, deoxycytidine and uridine into the acid-insoluble fraction of K562 human leukemic cells, but did not significantly affect adenosine incorporation. The enhancement of pyrimidine nucleoside uptake was 6 approximately 17 fold over the control. Aspartate incorporation into nucleic acid was not significantly blocked by the antibiotic, suggesting that the stimulation of pyrimidine nucleoside incorporation is not due to the inhibition of de novo pyrimidine nucleotide synthesis. Net DNA and RNA syntheses, observed by [32P]phosphate uptake, were not significantly affected by cadeguomycin. The enzymatic activity of thymidine, deoxycytidine and uridine kinases was higher in cadeguomycin-treated cells than in untreated cells, suggesting that the enhancement of pyrimidine nucleoside uptake occurs in the phosphorylation process. The stimulatory activity of cadeguomycin of thymidine uptake was reversed by guanosine and deoxyguanosine, but not by adenosine and deoxyadenosine, suggesting that intracellular metabolism and/or action of cadeguomycin is related to that of guanosine and deoxyguanosine. The stimulation of pyrimidine nucleoside incorporation by cadeguomycin was also found with YAC-1 cells, but not with the other cell lines. The enhancement effect of the antibiotic seems to be not directly related to its cytotoxicity.

Biological activity of cadeguomycin. Inhibition of tumor growth and metastasis, immunostimulation, and potentiation of 1-beta-D-arabinofuranosylcytosine.

Cadeguomycin retarded growth of sc solid IMC carcinoma in CDF1 mice, and pulmonary metastasis of Lewis lung carcinoma in C57BL/6 mice. The antibiotic enhanced phagocytic activity of murine peritoneal macrophages and IL-1 production by P388D1 cells. Delayed type hypersensitivity was stimulated and interferon was induced by the drug. The results suggest that cadeguomycin inhibits tumor growth and metastasis in association with modification of the immune system. The cytotoxicity of arabinosylcytosine to K562 and YAC-1 cells was markedly enhanced by cadeguomycin in culture. The combined administration of arabinosylcytosine and cadeguomycin displayed potentiation in the inhibition of growth of ip-implanted P388 leukemia and metastasis of sc-implanted P388 leukemia to the regional lymph nodes. Cadeguomycin showed low toxicity for mice.

Cadeguomycin, a noval nucleoside analog antibiotic. I. The producing organism, production and isolation of cadeguomycin.

An actinomycete strain IM7912T was found to produce cadeguomycin, a new nucleoside analog antibiotic, together with tubercidin. Morphological, cultural and physiological studies showed that the organism belongs to the species Streptomyces hygroscopicus. Comparisons with S. hygroscopicus ISP 5578 revealed that both strains possess similar characteristics except for production of yellow or brownish yellow soluble pigment in some media. Cadeguomycin was isolated from the culture filtrate, separated from tubercidin, and purified.

Cadeguomycin, a novel nucleoside analog antibiotic. II. Improved purification, physicochemical properties and structure assessment.

Cadeguomycin, a new nucleoside analog antibiotic, has been purified as colorless needle crystals by recycling preparative HPLC. The antibiotic, C12H14O7N4, mp 231 approximately 239 degrees C (dec.), FD-MS: m/z 326 (M+); is a weakly acidic substance, showing UV gamma H2Omax (epsilon) 232 (19677), 272 (6881) and 298 nm (7607), and IR nu KBrmax 1650 (C = O) and 3420 (NH or OH) cm-1. The UV spectrum is similar to other pyrrolo[2,3-d]pyrimidines. The structure of cadeguomycin, 2-amino-3,4-dihydro-4-oxo-7-beta-D-ribofuranosyl-7H-pyrrolo[2,3-d]pyrimidine-5- carboxylic acid, has been elucidated by 1H NMR and 13C NMR in comparison with other pyrrolo[2,3-d]pyrimidines and their nucleosides.

Female genital tuberculosis: report of seven cases.

Seven cases of histologically proven genital tuberculosis (TB) encountered at the National Taiwan University Hospital in the past 16 years were reviewed. From a total of 54,576 gynecologic specimens submitted for pathologic examination, the occurrence of genital TB was 0.01%. Of these seven patients, one suffered from primary infertility and one from secondary infertility. The patient with primary infertility had been married for 29 years. The patient with secondary infertility underwent a tuboplasty due to bilateral hydrosalpinx. Genital TB was not diagnosed preoperatively in any of the seven cases. The preoperative diagnoses included: postmenopausal spotting, ovarian malignancy, cervicitis, ectopic pregnancy and hydrosalpinx with secondary infertility. This review suggests that genital TB is becoming rare in Taiwan. It is difficult to diagnose from clinical symptoms and is usually discovered on pathologic examination.

Role of autophagy in protection afforded by hypoxic preconditioning against MPP+-induced neurotoxicity in SH-SY5Y cells.

A sublethal preconditioning has been proposed as a neuroprotective strategy against several CNS neurodegenerative diseases. In this study, the involvement of autophagy in the protection provided by hypoxic preconditioning against 1-methyl-4-phenylpyridinium (MPP(+))-induced neurotoxicity was studied in SH-SY5Y neuroblastoma cells. In contrast to the cytotoxicity of 0.1% oxygen, 1% oxygen hypoxia for 24h did not cause significant cell death. A transient increase in LC3-II level, a biomarker of autophagy, was demonstrated during hypoxic treatment. At the same time, 8-h hypoxia increased fluorescence due to monodansylcadaverine, a specific dye for autophagosomes, in the treated cells. Co-incubation with bafilomycin A1 (10 nM) further increased hypoxia-induced LC3-II levels but 3-methyladenine (3-MA; 10 mM) reduced the elevation in LC3-II levels induced by 8-h hypoxia. Moreover, 8-h hypoxia increased free radical formation and nuclear HIF-1alpha level. Glutathione was found to diminish hypoxia-induced LC3-II elevation. In contrast to the elevated LC3-II level, 8-h hypoxia significantly decreased mitochondrial mass. Furthermore, a rebound elevation in mitochondrial mass was observed under 8-h hypoxia and subsequent 12-h normoxia. Prior hypoxia attenuated the MPP(+)-induced elevation in LC3-II levels and cell death. Moreover, hypoxic pretreatment inhibited MPP(+)-induced activation of caspase-3 and DNA fragmentation. Co-incubation with 3-MA during hypoxia prevented the protection afforded by hypoxic preconditioning against MPP(+)-induced increases in LC3-II levels and neurotoxicity. Taken together, our results suggest that sublethal hypoxia induces autophagy that is mediated by oxidative stress. Furthermore, autophagy may be involved in the protection provided by hypoxic preconditioning against MPP(+)-induced neurotoxicity, indicating a neuroprotective role of autophagy in hypoxic preconditioning.

Serum antibody levels to retinal S-antigen determined by ELISA in experimental and human uveitis.

Retinal S-antigen is thought to play an important role in the immunopathogenesis of uveitis. To investigate the role of S-antigen in human uveitis, an immunopotent bioactive S-antigen and a sensitive enzyme-linked immunosorbent assay (ELISA) were developed to determine the anti-S-antigen antibody contents in experimental autoimmune uveitis (EAU) and human uveitis. The bovine S-antigen was isolated by ammonium sulfate precipitation, Fractogel TSK HW-55 gel filtration and hydroxylapatite absorption chromatography. A test by SDS polyacrylamide electrophoresis showed that it was a highly purified protein. The purified S-antigen evoked EAU in 60% of eyes of guinea pigs even at the dose of 1 microgram. ELISA was developed to determine the contents of serum immunoglobulin G antibody against S-antigen. In the serum of S-antigen immunized rabbit, the antibody was measurable up to 1:160,000 in dilution. The antibody content appeared on the 10th day following inoculation and rose gradually until the 22nd day when the maximal antibody content was observed. Regarding human uveitis, the anti-S-antigen antibody was higher in 31 patients with Behçet's disease and 8 with Harada's disease than in normal controls although statistical significance existed only in the former (p less than 0.02). In this study, the method to purify the bovine retinal S-antigen proved relatively rapid and efficient. The ELISA developed herein appeared sensitive and well reproducible in the detection of anti-S-antigen antibody. The results showed that retinal autoimmunity may play a role in the pathogenesis in EAU and human uveitis.