Discovery of an Endosymbiotic Nitrogen-Fixing Cyanobacterium UCYN-A in Braarudosphaera bigelowii (Prymnesiophyceae)

Braarudosphaera bigelowii (Prymnesiophyceae) is a coastal coccolithophore with a long fossil record, extending back to the late Cretaceous (ca. 100 Ma). A recent study revealed close phylogenetic relationships between B. bigelowii, Chrysochromulina parkeae (Prymnesiophyceae), and a prymnesiophyte that forms a symbiotic association with the nitrogen-fixing cyanobacterium UCYN-A. In order to further examine these relationships, we conducted transmission electron microscopic and molecular phylogenetic studies of B. bigelowii. TEM studies showed that, in addition to organelles, such as the nucleus, chloroplasts and mitochondria, B. bigelowii contains one or two spheroid bodies with internal lamellae. In the 18S rDNA tree of the Prymnesiophyceae, C. parkeae fell within the B. bigelowii clade, and was close to B. bigelowii Genotype III (99.89% similarity). Plastid 16S rDNA sequences obtained from B. bigelowii were close to the unidentified sequences from the oligotrophic SE Pacific Ocean (e.g. {"type":"entrez-nucleotide","attrs":{"text":"HM133411","term_id":"311035799","term_text":"HM133411"}}HM133411) (99.86% similarity). Bacterial16S rDNA sequences obtained from B. bigelowii were identical to the UCYN-A sequence {"type":"entrez-nucleotide","attrs":{"text":"AY621693","term_id":"54125940","term_text":"AY621693"}}AY621693 from Arabian Sea, and fell in the UCYN-A clade. From these results, we suggest that; 1) C. parkeae is the alternate life cycle stage of B. bigelowii sensu stricto or that of a sibling species of B. bigelowii, and 2) the spheroid body of B. bigelowii originated from endosymbiosis of the nitrogen-fixing cyanobacterium UCYN-A.     

Circulating Levels of Fatty Acid-Binding Protein Family and Metabolic Phenotype in the General Population

Objective

Fatty acid-binding proteins (FABPs) are a family of 14-15-kDa proteins, and some FABPs have been to be used as biomarkers of tissue injury by leak from cells. However, recent studies have shown that FABPs can be secreted from cells into circulation. Here we examined determinants and roles of circulating FABPs in a general population.

Methods

From the database of the Tanno-Sobetsu Study, a study with a population-based cohort design, data in 2011 for 296 subjects on no medication were retrieved, and FABP1∼5 in their serum samples were assayed.

Results

Level of FABP4, but not the other isoforms, showed a gender difference, being higher in females than in males. Levels of all FABPs were negatively correlated with estimated glomerular filtration rate (eGFR), but a distinct pattern of correlation with other clinical parameters was observed for each FABP isoform; significant correlates were alanine aminotransferase (ALT), blood pressure (BP), and brain natriuretic peptide (BNP) for FABP1, none besides eGFR for FABP2, age, BP, and BNP for FABP3, age, waist circumference (WC), BP, BNP, lipid variables, high-sensitivity C-reactive protein (hsCRP), and HOMA-R for FABP4, and age, WC, BP, ALT, BNP, and HOMA-R for FABP5. FABP4 is the most strongly related to metabolic markers among FABPs. In a multivariate regression analysis, FABP4 level was an independent predictor of HOMA-R after adjustment of age, gender, WC, BP, HDL cholesterol, and hsCRP.

Conclusions

Each FABP isoform level showed a distinct pattern of correlation with clinical parameters, although levels of all FABPs were negatively determined by renal function. Circulating FABP4 appears to be a useful biomarker for detecting pre-clinical stage of metabolic syndrome, especially insulin resistance, in the general population.     

New Preclinical Antimalarial Drugs Potently Inhibit Hepatitis C Virus Genotype 1b RNA Replication

Background

Persistent hepatitis C virus (HCV) infection causes chronic liver diseases and is a global health problem. Although new triple therapy (pegylated-interferon, ribavirin, and telaprevir/boceprevir) has recently been started and is expected to achieve a sustained virologic response of more than 70% in HCV genotype 1 patients, there are several problems to be resolved, including skin rash/ageusia and advanced anemia. Thus a new type of anti-HCV drug is still needed.

Methodology/Principal Findings

Recently developed HCV drug assay systems using HCV-RNA-replicating cells (e.g., HuH-7-derived OR6 and Li23-derived ORL8) were used to evaluate the anti-HCV activity of drug candidates. During the course of the evaluation of anti-HCV candidates, we unexpectedly found that two preclinical antimalarial drugs (N-89 and its derivative N-251) showed potent anti-HCV activities at tens of nanomolar concentrations irrespective of the cell lines and HCV strains of genotype 1b. We confirmed that replication of authentic HCV-RNA was inhibited by these drugs. Interestingly, however, this anti-HCV activity did not work for JFH-1 strain of genotype 2a. We demonstrated that HCV-RNA-replicating cells were cured by treatment with only N-89. A comparative time course assay using N-89 and interferon-α demonstrated that N-89-treated ORL8 cells had more rapid anti-HCV kinetics than did interferon-α-treated cells. This anti-HCV activity was largely canceled by vitamin E. In combination with interferon-α and/or ribavirin, N-89 or N-251 exhibited a synergistic inhibitory effect.

Conclusions/Significance

We found that the preclinical antimalarial drugs N-89 and N-251 exhibited very fast and potent anti-HCV activities using cell-based HCV-RNA-replication assay systems. N-89 and N-251 may be useful as a new type of anti-HCV reagents when used singly or in combination with interferon and/or ribavirin.     

Liposome Vector Containing Biosurfactant-Complexed DNA as Herpes Simplex Virus Thymidine Kinase Gene Delivery System

For injectable-sized liposome complexed with DNA (lipoplexes) with high transfection efficiency of genes, we initially prepared small-sized liposomes by addition of biosurfactant. For selectivity of gene expression, the thymidine kinase (MK-tk) gene controlled by midkine was used for herpes simplex virus thymidine kinase (HSV-tk) gene therapy. Liposomes composed of 3([N-(N′,N′–dimethylaminoethane)-carbamoyl] cholesterol (DC-Chol), L-dioleoylphosphatidylethanolamine (DOPE), and a biosurfactant, such as β-sitosterol β-D-glucoside (Sit-G) for Sit-G-liposomes and mannosylerythrytol lipid A (MEL) for MEL-liposomes, produced about 300-nm-sized lipoplexes. Sit-G- and MEL-liposomes showed higher transfection efficiency of the luciferase marker gene and thymidine kinase activity in the presence of serum in the cells. The treatment with transfection of MK-tk gene by Sit-G-liposome and injection of ganciclovir significantly reduced tumor growth in a solid tumor model, compared with that by Sit-G-liposome alone. This finding suggested that Sit-G-liposome is a potential vector for HSV-tk gene therapy.     

Possible contribution of Bradyrhizobium on nitrogen fixation in sweet potatoes

Aims

Sweet potato (Ipomoea batatas) is known for its ability to grow under nitrogen-limited conditions. To clarify the possible contribution of biological nitrogen fixation, we tried to isolate and identify diazotrophic bacteria from sweet potatoes.

Methods

By using cultivation technique, we isolated putative endophytes, which possess nifH genes, from surface-sterilized sweet potatoes. Their nitrogen-fixing abilities were demonstrated by the acetylene reduction assay in a semi-solid malate medium and sweet potato extracts. We also examined the colonization of an isolated strain (AT1) in sweet potatoes and their influence on growth and nitrogen fixation in plants as assessed by an acetylene reduction assay and ¹⁵N-isotope dilution technique.

Results

The isolates were identified as strains of Bradyrhizobium sp. AT1, Paenibacillus sp. AS2 and Pseudomonas sp. T16 based on their 16S rRNA gene sequences. They showed acetylene reduction activity (ARA) in the semi-solid malate medium. Among them, B. sp. AT1 showed ARA in sweet potato extracts under micro-aerobic conditions whereas both P. sp. AS2 and P. sp. T16 showed no ARA. The inoculation of B. sp. AT1 to the sweet potatoes resulted in increases in the fresh weights and detection of ARA in the inoculated plants. Moreover, the reduction of ¹⁵N atom % was observed in the inoculated plants compared to uninoculated controls.

Conclusions

B. sp. AT1 actively expresses nitrogenase activity in sweet potatoes and may contribute to the nitrogen nutrition of host plants.     

ER stress response induced by the production of human IL-7 in rice endosperm cells

Rice seed has been used as a production platform for high value recombinant proteins. When mature human interleukin 7 (hIL-7) was expressed as a secretory protein in rice endosperm by ligating the N terminal glutelin signal peptide and the C terminal KDEL endoplasmic reticulum (ER) retention signal to the hIL-7 cytokine to improve production yield, this protein accumulated at levels visible by Coomassie Brilliant Blue staining. However, the production of this protein led not only to a severe reduction of endogenous seed storage proteins but also to a deterioration in grain quality. The appearance of aberrant grain phenotypes (such as floury and shrunken) was attributed to ER stress induced by the retention of highly aggregated unfolded hIL-7 in the ER lumen, and the expression levels of chaperones such as BiPs and PDIs were enhanced in parallel with the increase in hIL-7 levels. The activation of this ER stress response was shown to be mainly mediated by the OsIRE1-OsbZIP50 signal cascade, based on the appearance of unconventional splicing of OsbZIP50 mRNA and the induction of OsBiP4&5. Interestingly, the ER stress response could be induced by lower concentrations of hIL-7 versus other types of cytokines such as IL-1b, IL-4, IL-10, and IL-18. Furthermore, several ubiquitin 26S proteasome-related genes implicated in ER-associated degradation were upregulated by hIL-7 production. These results suggest that severe detrimental effects on grain properties were caused by proteo-toxicity induced by unfolded hIL-7 aggregates in the ER, resulting in the triggering of ER stress.     

Utilization of Amino Acids as a Sole Source of Nitrogen by Obligate Chemolithoautotroph Thiobacillus ferrooxidans

An obligate chemolithoautotroph, Thiobacillus ferrooxidans API 9–3, could utilize amino acids, other than glycine, methionine and phenylalanine, as a sole source of nitrogen. However, both the growth rate and growth yield were lower than those in Fe²⁺-NH4 -salts medium, suggesting that the ammonium ion was a superior nitrogen source for the strain compared to amino acids. Methionine and phenylalanine strongly inhibited the cell growth on Fe²⁺-NH4-salts medium at 10 mm. [¹⁴C]Glycine could not be taken up into the cells, and this meant the strain could not use glycine as a sole source of nitrogen. The uptake of [¹⁴C]leucine into the cells was dependent on the presence of Fe^{2 +}. When the strain was cultured on Fe^{2 +} - leucine (lOmm)-salts medium lacking an inorganic nitrogen source for 5 days at 30°C, 83.5% and 16.5% of the cellular carbon were derived from carbon dioxide and leucine, respectively, indicating that carbon dioxide was a superior carbon source for the bacterium compared to leucine. The ammonium ion did not inhibit the utilization of leucine for cellular carbon. Leucine uptake was markedly inhibited by inhibitors of protein synthesis, such as chloramphenicol (94.3% at 1 mm), streptomycin (57.2% at 5mm) and rifampin (77.2% at 0.1 mm), respectively. Carbon dioxide uptake was also completely inhibited by chloramphenicol at 4mm. These results suggest that the transport of both amino acids and carbon dioxide into the cells was dependent on protein synthesis.