Biotin synthase mechanism: mutagenesis of the YNHNLD conserved motif

Biotin synthase, a member of the "radical SAM" family, catalyzes the final step of the biotin biosynthetic pathway, namely, the insertion of a sulfur atom into dethiobiotin (DTB). The active form of the enzyme contains two iron-sulfur clusters, a [4Fe-4S](2+) cluster liganded by Cys-53, Cys-57, and Cys-60 and the S-adenosylmethionine (AdoMet or SAM) cosubstrate and a [2Fe-2S](2+) cluster liganded by Cys-97, Cys-128, Cys-188, and Arg-260. Single-point mutation of each of these six conserved cysteines produced inactive variants. In this work, mutants of other highly conserved residues from the Y(150)NHNLD motif are described. They have properties similar to those of the wild-type enzyme with respect to their cluster content and characteristics. For all of them, the as-isolated form, which contains an air-stable [2Fe-2S](2+) center, can additionally accommodate an air-sensitive [4Fe-4S](2+) center which is generated by incubation under anaerobic conditions with Fe(2+) and S(2-). Their spectroscopic properties are similar to those of the wild type. However, they are inactive, except the mutant H152A that exhibits a weak activity. We show that the mutants, inactive in producing biotin, are also unable to cleave AdoMet and to produce the deoxyadenosyl radical (AdoCH(2)(*)). In the case of H152A, a value of 5.5 +/- 0.4 is found for the 5'-deoxyadenosine (AdoCH(3)):biotin ratio, much higher than the value of 2.8 +/- 0.3 usually observed with the wild type. This reveals a greater contribution of the abortive process in which the AdoCH(2)(*) radical is quenched by hydrogen atoms from the protein or from some components of the system. Thus, in this case, the coupling between the production of AdoCH(2)(*) and its reaction with the hydrogen at C-6 and C-9 of DTB is less efficient than that in the wild type, probably because of geometry's perturbation within the active site.     

PDCD6 additively cooperates with anti-cancer drugs through activation of NF-κB pathways

The expression of programmed cell death 6 (PDCD6) is known to be down-regulated in cancer cell lines and ovarian cancer tissues compared to normal cells and tissues. In the current study, we characterized the specific function of PDCD6 as a novel pro-apoptotic protein. To define the roles of PDCD6 and cisplatin in tumorigenesis, we either over-expressed PDCD6 or treated it with cisplatin in SKOV-3 ovarian cancer cells. Both PDCD6 and cisplatin respectively inhibited cancer cell proliferation in a dose-dependent manner. The combined treatment of PDCD6 and cisplatin was more effective at suppressing cell growth than with either drug treatment alone, but had no effect with the treatment of caspase-3 and caspase-9 inhibitors. Cleavages of caspase-3, -8, -9, and poly (ADP-ribose) polymerase (PARP) in PDCD6-overexpressing cells were significantly increased after cisplatin treatment. Cell cycle analysis highly correlated with down-regulation of cyclin D1 and CDK4, and the induction of p16 and p27 as a cyclin-dependent kinase inhibitor. Additionally, PDCD6 also suppressed the phosphorylation of signaling regulators downstream of PI3K, including PDK1 and Akt. PDCD6 promotes TNFα-dependent apoptosis through the activation of NF-κB signaling pathways, increasing Bax, p53, and p21 expression, while also down-regulating Bcl-2 and Bcl-xL expression. The p21 and p53 promoter luciferase activities were enhanced by PDCD6, while there was no affect in p53^−/− and p21^−/−. At the same time, p53 activity was confirmed by UV irradiation and siPDCD6. Taken together, these results provide evidence that PDCD6 can mediate the pro-apoptotic activity of cisplatin or TNFα through the down-regulation of NF-κB expression.     

Oral spirochetes implicated in dental diseases are widespread in normal human subjects and carry extremely diverse integron gene cassettes

The NIH Human Microbiome Project (HMP) has produced several hundred metagenomic data sets, allowing studies of the many functional elements in human-associated microbial communities. Here, we survey the distribution of oral spirochetes implicated in dental diseases in normal human individuals, using recombination sites associated with the chromosomal integron in Treponema genomes, taking advantage of the multiple copies of the integron recombination sites (repeats) in the genomes, and using a targeted assembly approach that we have developed. We find that integron-containing Treponema species are present in ～80% of the normal human subjects included in the HMP. Further, we are able to de novo assemble the integron gene cassettes using our constrained assembly approach, which employs a unique application of the de Bruijn graph assembly information; most of these cassette genes were not assembled in whole-metagenome assemblies and could not be identified by mapping sequencing reads onto the known reference Treponema genomes due to the dynamic nature of integron gene cassettes. Our study significantly enriches the gene pool known to be carried by Treponema chromosomal integrons, totaling 826 (598 97% nonredundant) genes. We characterize the functions of these gene cassettes: many of these genes have unknown functions. The integron gene cassette arrays found in the human microbiome are extraordinarily dynamic, with different microbial communities sharing only a small number of common genes.     

Accumulation of galloyl derivatives in a green alga, Spirogyra varians, in response to cold stress

The green alga Spirogyra varians accumulated antioxidative compounds in response to cold stress. When the algae were transferred from 20°C to 4°C, the amount of phenolic contents and flavonoids in the cell increased 17 times and 30 times, respectively, in 2 months. At this time, the radical scavenging activity of the methanolic extract of S. varians was 238 times higher than that of initial culture. To identify the responsible antioxidants, the methanolic extract was obtained from the algae grown at 4°C. HPLC analysis of the extract showed six compounds newly produced or increased over time. Four of the compounds were successfully purified, and the structures were identified using ¹H NMR spectroscopy. The compounds were galloyl derivatives—methyl gallate, 1-O-Galloyl-β-d-glucose, 1,2,3,6-tetra-O-Galloyl-β-d-glucose and 1,2,3,4,6-penta-O-Galloyl-β-d-glucose which are intermediates of the shikimate pathway.     

Direct fluorimetric sensing of UV-excited analytes in biological and environmental samples using molecularly imprinted polymer nanoparticles and fluorescence polarization

A rapid, robust, sensitive and economic sensing method, based on a molecularly imprinted polymer (MIP) synthetic antibody mimic, and fluorescence polarization analysis, for the direct detection of UV-excited fluorescent analytes in food and environmental samples was developed. Fluoroquinolone (FQ) antibiotics were used as fluorescent model analytes. Water-compatible MIP nanoparticles were synthesized with enrofloxacin (ENRO) as the imprinting template. Fluorescence polarization measurements then allow the direct determination of the amount of ENRO and other structurally related piperazine-based fluoroquinolones that bind to the MIP. No separation step was required since this technique distinguishes in situ analyte molecules bound to the MIP from the free analyte in solution. This assay was successfully applied for the first time to determine FQs in real samples, i.e. tap water and milk, without any prior concentration step, by simply adding a known amount of MIP. No interference by the sample components was observed even though the excitation was in the UV region. In tap water, a low limit of detection of 0.1 nM for ENRO was achieved with 5 μg mL(-1) of MIP. In milk, ENRO and danofloxacin, whose MRLs have been fixed at 0.28 μM and 0.08 μM, respectively, could be selectively measured and distinguished from other families of antibiotics. The procedure is very easy and practical as it consists of simply precipitating the milk proteins with acetonitrile and adding buffer and MIP to the supernatant before reading the polarization values with a spectrofluorimeter.     

Performance of ¿VIKIA Malaria Ag Pf/Pan¿ (IMACCESS®), a new malaria rapid diagnostic test for detection of symptomatic malaria infections

ABSTRACT: BACKGROUND: Recently, IMACCESS[REGISTERED SIGN] developed a new malaria test (VIKIA Malaria Ag Pf/Pan[TRADE MARK SIGN]), based on the detection of falciparum malaria (HRP-2) and non-falciparum malaria (aldolase). METHODS: The performance of this new malaria rapid diagnostic test (RDT) was assessed using 1,000 febrile patients seeking malaria treatment in four health centres in Cambodia from August to December 2011. The results of the VIKIA Malaria Ag Pf/Pan were compared with those obtained by microscopy, the CareStart Malaria[TRADE MARK SIGN] RDT (AccessBio[REGISTERED SIGN]) which is currently used in Cambodia, and real-time PCR (as "gold standard"). RESULTS: The best performances of the VIKIA Malaria Ag Pf/Pan[TRADE MARK SIGN] test for detection of both Plasmodium falciparum and non-P. falciparum were with 20--30 min reading times (sensitivity of 93.4% for P. falciparum and 82.8% for non-P. falciparum and specificity of 98.6% for P. falciparum and 98.9% for non-P. falciparum) and were similar to those for the CareStart Malaria[TRADE MARK SIGN] test. CONCLUSIONS: This new RDT performs similarly well as other commercially available tests (especially the CareStart Malaria[TRADE MARK SIGN] test, used as comparator), and conforms to the World Health Organization's recommendations for RDT performance. It is a good alternative tool for the diagnosis of malaria in endemic areas.     

Population characteristics of two seahorses, Hippocampus coronatus and Hippocampus mohnikei, around seagrass beds in the southern coastal waters of Korea

Population characteristics of two seahorse species, Hippocampus coronatus and Hippocampus mohnikei, in seagrass beds in the southern coastal waters of Korea were determined based on field observations. In Zostera beds in the Yeosu area, there was a mean density of 2.9 seahorses per 1,000?m² for H. coronatus and 1.4 seahorses per 1,000?m² for H. mohnikei. The greatest numbers of young individuals of both species were observed in July, with numbers decreasing through November. The male:female sex ratio was 1:1.6 for H. mohnikei and 1:1.7 for H. coronatus. Breeding males of H. coronatus and H. mohnikei were observed from July to November and from July to September, respectively. Within the study site, the populations of the two seahorse species exhibited low density and patchy distributions, with variation in temporal abundance being significantly correlated with water temperature. The findings of this study provide the basis for future research on the population status of H. coronatus and H. mohnikei in seagrass beds.     

sMEK1 enhances gemcitabine anti-cancer activity through inhibition of phosphorylation of Akt/mTOR

Recently, we reported that sMEK1 is down-regulated in cancer cells and tissues, and that it enhances the pro-proliferative effect as a novel pro-apoptotic protein. However, the biological mechanism of the sMEK1 tumor suppressor in the cellular signal pathway has not been well understood. In our current work, we examined whether sMEK1 could promote the cytotoxic activity of gemcitabine in the human ovarian carcinoma system. Initially, we attempted to use a treatment of gemcitabine traditional chemotherapeutic agent and over-expression of sMEK1 in OVCAR-3 cancer cells. The combined treatment of sMEK1 and gemcitabine was more effective at inhibiting cell proliferation than either chemotherapeutic agent treatment alone. In addition, sMEK1 actively contributes to cell migration through its ability to promote gemcitabine-inhibited cell migration in tumorigenesis. Cell cycle-related proteins are highly associated with the down-regulation of cyclin D1 and CDK4, and the promotion of p16 and p27 as a cyclin-dependent kinase inhibitor. At the same time, sMEK1 arrests cell cycle progression in the G(1)-G(0) phase, and activates p53 and p21 expression, whereas Bcl-2 and Bcl-xL protein expression is reduced. Additionally, sMEK1 and gemcitabine suppresses the phosphorylation of signaling modulators downstream of PI3K, such as PDK1 and Akt. The p53 and p21 promoter luciferase activities were promoted by either sMEK1 or gemcitabine, and sMEK1 and gemcitabine combined additively activated the promoter further. Furthermore, as expected, sMEK1 plus gemcitabine markedly reduced the phosphorylation of p70S6K and the phosphorylation of 4E-BP1, which is one of the best characterized targets of the mTOR complex cascade. Taken together, these results provide evidence that sMEK1 can effectively regulate the pro-apoptotic activity of gemcitabine through the up-regulation of p53 expression.     

Evaluation of the efficacy of a pre-pandemic H5N1 vaccine (MG1109) in mouse and ferret models

The threat of a highly pathogenic avian influenza (HPAI) H5N1 virus causing the next pandemic remains a major concern. In this study, we evaluated the immunogenicity and efficacy of an inactivated whole-virus H5N1 pre-pandemic vaccine (MG1109) formulated by Green Cross Co., Ltd containing the hemagglutinin (HA) and neuraminidase (NA) genes of the clade 1 A/Vietnam/1194/04 virus in the backbone of A/Puerto Rico/8/34 (RgVietNam/04xPR8/34). Administration of the MG1109 vaccine (2-doses) in mice and ferrets elicited high HI and SN titers in a dose-dependent manner against the homologous (RgVietNam/04xPR8/34) and various heterologous H5N1 strains, (RgKor/W149/06xPR8/34, RgCambodia/04xPR8/34, RgGuangxi/05xPR8/34), including a heterosubtypic H5N2 (A/Aquatic bird/orea/W81/05) virus. However, efficient cross-reactivity was not observed against heterosubtypic H9N2 (A/Ck/Korea/H0802/08) and H1N1 (PR/8/34) viruses. Mice immunized with 1.9 μg HA/dose of MG1109 were completely protected from lethal challenge with heterologous wild-type HPAI H5N1 A/EM/Korea/W149/06 (clade 2.2) and mouse-adapted H5N2 viruses. Furthermore, ferrets administered at least 3.8 μg HA/dose efficiently suppressed virus growth in the upper respiratory tract and lungs. Vaccinated mice and ferrets also demonstrated attenuation of clinical disease signs and limited virus spread to other organs. Thus, this vaccine provided immunogenic responses in mouse and ferret models even against challenge with heterologous HPAI H5N1 and H5N2 viruses. Since the specific strain of HPAI H5N1 virus that would potentially cause the next outbreak is unknown, pre-pandemic vaccine preparation that could provide cross-protection against various H5 strains could be a useful approach in the selection of promising candidate vaccines in the future.