Biochemical and Kinetic Characterization of Geranylgeraniol 18-Hydroxylase (CYP97C27) from Croton stellatopilosus Ohba

Geranylgeraniol 18-hydroxylase (EC 1.14.13.110) that exists solely in Croton stellatopilosus Ohba catalyses the last committed step of plaunotol biosynthetic pathways by conversion of geranylgeraniol (GGOH) to plaunotol. This enzyme and its gene are an attractive target for development of plaunotol production and its detailed biochemical properties need to be understood.   Recently, even though the gene (CYP97C27) coding for GGOH 18-hydroxylase has been identified, cloned, and expressed in Escherichia coli system, the enzyme activity has been detected mainly in the insoluble fraction (20,000 g). This means that biochemical and kinetic studies could not be undertaken. However, our previous study indicated that this enzyme activity was easily and specifically detected in the microsomal fraction (100,000 g) of a crude enzyme extract. Therefore, in this report we describe a comprehensive biochemical characterization of GGOH 18-hydroxylase activity in the microsomal fraction from C. stellatopilosus Ohba. The oxygen-dependent enzyme activity of GGOH 18-hydroxylase was inhibited by carbon monoxide and the inhibition was partially reversible upon illumination with white light. Kinetic studies of the GGOH 18-hydroxylase showed high affinity to GGOH and NADPH with apparent Km values of 0.8 and 53 µM, respectively. Furthermore, the enzyme activity was inhibited by P450 inhibitors, including ancymidol, metyrapone, miconazole, potassium cyanide and cytochrome c, with the IC50 values of 428, 65, 75, 66 and 8 µM, respectively. Based on the biochemical and kinetic characteristics, the GGOH 18-hydroxylase in the microsomal fraction is likely a P450 encoded by CYP97C27 gene as previously described.Keywords:  Plaunotol, Cytochrome P450, Enzyme Activity, Enzyme Inhibitor, MicrosomeAbbreviation: GGPP, Geranylgeranyl diphosphate; GGOH, geranylgeraniol; PMSF, phenylmethylsulfonyl fluoride; KCN, potasium cyanide; CO, carbon monoxid

Comparison of the diagnostic accuracy of commercial NS1-based diagnostic tests for early dengue infection

<p>Abstract</p> <p>Background</p> <p>We compared the diagnostic accuracy and reproducibility of commercially available NS1-based dengue tests and explored factors influencing their sensitivities.</p> <p>Methods</p> <p>Paired analysis of 310 samples previously characterized as positive (n = 218) and negative (n = 92) for viral isolation and/or RT-PCR and/or IgM seroconversion. Masked samples were tested by two observers with Platelia™ Dengue NS1 Ag, second generation Pan-E™ Dengue Early ELISA, SD Dengue NS1 Ag ELISA, Dengue NS1 Ag STRIP™, and SD BIOLINE™ Dengue Duo (NS1/IgM/IgG).</p> <p>Results</p> <p>SD BIOLINE™ NS1/IgM/IgG had the highest sensitivity (80.7% 95%CI 75-85.7) with likelihood ratios of 7.4 (95%CI 4.1-13.8) and 0.21 (95%CI 0.16-0.28). The ELISA-format tests showed comparable sensitivities; all below 75%. STRIP™ and SD NS1 had even lower sensitivities (<65%). The sensitivities significantly decreased in samples taken after 3 days of fever onset, in secondary infections, viral serotypes 2 and 4, and severe dengue. Adding IgM or IgG to SD NS1 increased its sensitivity in all these situations.</p> <p>Conclusions</p> <p>The simultaneous detection of NS1/IgM/IgG would be potentially useful for dengue diagnosis in both endemic and non endemic areas. A negative result does not rule out dengue. Further studies are required to assess the performance and impact of early laboratory diagnosis of dengue in the routine clinical setting.</p

Evidence and rationale for the World Health Organization recommended standards for Japanese encephalitis surveillance

<p>Abstract</p> <p>Background</p> <p>Japanese encephalitis (JE) is the most important form of viral encephalitis in Asia. Surveillance for the disease in many countries has been limited. To improve collection of accurate surveillance data in order to increase understanding of the full impact of JE and monitor control programs, World Health Organization (WHO) Recommended Standards for JE Surveillance have been developed. To aid acceptance of the Standards, we describe the process of development, provide the supporting evidence, and explain the rationale for the recommendations made in the document.</p> <p>Methods</p> <p>A JE Core Working Group was formed in 2002 and worked on development of JE surveillance standards. A series of questions on specific topics was initially developed. A literature review was undertaken and the findings were discussed and documented. The group then prepared a draft document, with emphasis placed on the feasibility of implementation in Asian countries. A field test version of the Standards was published by WHO in January 2006. Feedback was then sought from countries that piloted the Standards and from public health professionals in forums and individual meetings to modify the Standards accordingly.</p> <p>Results</p> <p>After revisions, a final version of the JE surveillance standards was published in August 2008. The supporting information is presented here together with explanations of the rationale and levels of evidence for specific recommendations.</p> <p>Conclusion</p> <p>Provision of the supporting evidence and rationale should help to facilitate successful implementation of the JE surveillance standards in JE-endemic countries which will in turn enable better understanding of disease burden and the impact of control programs.</p

Dengue: a continuing global threat.

Dengue fever and dengue haemorrhagic fever are important arthropod-borne viral diseases. Each year, there are ∼50 million dengue infections and ∼500,000 individuals are hospitalized with dengue haemorrhagic fever, mainly in Southeast Asia, the Pacific and the Americas. Illness is produced by any of the four dengue virus serotypes. A global strategy aimed at increasing the capacity for surveillance and outbreak response, changing behaviours and reducing the disease burden using integrated vector management in conjunction with early and accurate diagnosis has been advocated. Antiviral drugs and vaccines that are currently under development could also make an important contribution to dengue control in the future

Temporal evolution of human autoantibody response to cytoplasmic rods and rings structure during anti-HCV therapy with ribavirin and interferon-α

Autoantibodies to inosine monophosphate dehydrogenase-2 (IMPDH2), an enzyme involved in de novo biosynthesis of guanine nucleotides, are observed in a subset of hepatitis C virus (HCV) patients receiving interferon alpha (IFN-alpha) plus ribavirin. Anti-IMPDH2 antibodies display a peculiar cytoplasmic rod/ring (RR) pattern in IIF-HEp-2. We examined the dynamics of anti-RR autoimmune response with respect to immunoglobulin isotypes, titer, avidity, and protein targets in 80 sequential samples from 15 HCV patients (plus 12 randomly selected anti-RR-positive, totalizing 92 samples) collected over an 18-month period, including samples collected before, during, and after IFN-alpha + ribavirin treatment. Immunoprecipitation showed reactivity with the 55 kDa IMPDH2 protein in 12/15 patients (80 %) and 11/15 (73 %) reacted with IMPDH2 in a sandwich ELISA. During treatment, anti-IMPDH2 autoantibodies hit their highest levels after 6-12 months of treatment and decreased post-treatment, while anti-HCV antibodies levels were stable over time. Anti-IMPDH2 IgM levels increased up until the sixth month of treatment and remained stable thereafter, while IgG levels increased steadily up to the twelfth month. Both IgG and IgM decreased during the post-treatment period. IgG avidity increased steadily up to the twelfth month of treatment. in conclusion, this study showed that the temporal kinetics of IFN-alpha + ribavirin-induced humoral autoimmune response to IMPDH2 exhibited a considerably delayed pace of increase in antibody levels and avidity as well as in isotype class switch in comparison with a conventional humoral response to infectious agents. These unique findings uncover intriguing differences between the autoimmune response and the immune response to exogenous agents in humans.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Universidade Federal de São Paulo, Div Rheumatol, BR-04023062 São Paulo, BrazilUniv Florida, Dept Oral Biol, Gainesville, FL 32610 USAUniv Occupat & Environm Hlth, Sch Hlth Sci, Dept Clin Nursing, Yahata Nishi Ku, Kitakyushu, Fukuoka 8078555, JapanUniv Florida, Dept Med, Div Rheumatol & Clin Immunol, Gainesville, FL 32610 USAUniversidade Federal de São Paulo, Div Gastroenterol, BR-04023062 São Paulo, BrazilFleury Med & Hlth Labs, Div Immunol, BR-04102050 São Paulo, BrazilUniversidade Federal de São Paulo, Div Rheumatol, BR-04023062 São Paulo, BrazilUniversidade Federal de São Paulo, Div Gastroenterol, BR-04023062 São Paulo, BrazilFAPESP: 2010/50710-6FAPESP: 2011/12448-0CAPES: 9028-11-0CNPq: 305064/2011-8Web of Scienc