Stable nuclear transformation of the diatom Chaetoceros sp.

A nuclear transformation system for the centric diatom Chaetoceros sp. has been established using two plasmids pTpfcp/nat and pTpNR/green fluorescent protein (GFP) that had been used for Thalassiosira pseudonana transformation. These contain the nourseothricin resistance gene (nat) with the fucoxanthin chlorophyll a/c binding protein (fcp) promoter/terminator from T. pseudonana and the enhanced green fluorescent protein gene (egfp), with the nitrate reductase (NR) promoter/terminator from T. pseudonana, respectively. Transformants were recovered in the presence of the antibiotic nourseothricin. One to four copies of both nat and egfp genes were integrated into genomic DNA of the transformants. Transformation efficiency was 1.5–6.0 transformants per 10⁸ cells. This work is the first report of stable genetic transformation of Chaetoceros, which is important as not only a constituent member of marine ecosystem but also feed for aquaculture.     

A protein aggregation based test for screening of the agents affecting thermostability of proteins

To search for agents affecting thermal stability of proteins, a test based on the registration of protein aggregation in the regime of heating with a constant rate was used. The initial parts of the dependences of the light scattering intensity (I) on temperature (T) were analyzed using the following empiric equation: I = K _agg(T−T ₀)², where K _agg is the parameter characterizing the initial rate of aggregation and T ₀ is a temperature at which the initial increase in the light scattering intensity is registered. The aggregation data are interpreted in the frame of the model assuming the formation of the start aggregates at the initial stages of the aggregation process. Parameter T ₀ corresponds to the moment of the origination of the start aggregates. The applicability of the proposed approach was demonstrated on the examples of thermal aggregation of glycogen phosphorylase b from rabbit skeletal muscles and bovine liver glutamate dehydrogenase studied in the presence of agents of different chemical nature. The elaborated approach to the study of protein aggregation may be used for rapid identification of small molecules that interact with protein targets.     

Sex- and age-related differences in morbidity rates of 2009 pandemic influenza A H1N1 virus of swine origin in Japan

Background

The objective of the present study was to determine whether the morbidity rates of the 2009 pandemic influenza A H1N1 virus (pdmH1N1) varied by age and/or sex.

Methods and Findings

Retrospective analysis of 2,024,367 cases of pdmH1N1 was performed using the national surveillance data from influenza sentinel points in Japan. The male-to-female morbidity ratios (M/F ratios) in nineteen age groups were estimated as the primary outcome. The M/F ratios for pdmH1N1 influenza were: >1 in age groups <20 years and ≥80 years (p<0.001); <1 in age groups 20–79 years (p<0.001). This data suggests that males <20 years of age may be more likely to suffer from pdmH1N1 influenza than females in the same age categories. When the infection pattern for pdmH1N1was compared with that of seasonal influenza outbreaks between 2000 and 2008, the M/F ratio for pdmH1N1 influenza was higher in ages 3–29 years and lower in ages 40–79 years. Because the present study was based on the national surveillance, it was impossible to estimate the morbidity rate for the Japanese population. It is also likely that the data did not capture asymptomatic or mild infections.

Conclusions

Although exposure to the pdmH1N1 virus is assumed to be similar in both boys and girls, M/F ratios were >1 in those younger than 20 years. The subsequent reversal of the M/F ratio in the adult generation could be due to several possibilities, including: greater immunity among adult males, more asymptomatic infections among males, less reporting of illness by males, or differences in exposure to the virus and probability of visiting a clinic. These results suggest that the infection and virulence patterns of pdmH1N1 are more complex than previously considered.     

Dimeric dUTPases, HisE, and MazG belong to a new superfamily of all-alpha NTP pyrophosphohydrolases with potential "house-cleaning" functions

Structure-guided analysis of the new dimeric dUTPase family revealed its sequence relationship to the phage T4 dCTPase, phosphoribosyl-ATP pyrophosphatase HisE, NTP pyrophosphatase MazG, and several uncharacterized protein families, including the human protein XTP3TPA (RS21-C6), which is overexpressed in embryonic and cancer cells. Comparison with the recently determined structure of a MazG-like protein from Sulfolobus solfataricus supported the unification of these enzymes in one superfamily of all-alpha NTP pyrophosphatases, suggesting that dimeric dUTPases evolved from a tetrameric MazG-like ancestor by gene duplication. Analysis of the structure of the Sulfolobus MazG points to 2-hydroxyadenosine (isoguanosine) triphosphate, a product of oxidative damage of ATP, as the most likely substrate. We predict that uncharacterized members of this superfamily perform "house-cleaning" functions by hydrolyzing abnormal NTPs and are functionally analogous to the structurally unrelated hydrolases of the Nudix superfamily. We outline probable tertiary and quaternary structures of the all-alpha NTP pyrophosphatase superfamily members.     

Alteration of substrate specificity of aspartase by directed evolution

Aspartase (l-aspartate ammonia-lyase, EC 4.3.1.1), which catalyzes the reversible deamination of l-aspartic acid to yield fumaric acid and ammonia, is highly selective towards l-aspartic acid. We screened for enzyme variants with altered substrate specificity by a directed evolution method. Random mutagenesis was performed on an Escherichia coli aspartase gene (aspA) by error-prone PCR to construct a mutant library. The mutant library was introduced to E. coli and the transformants were screened for production of fumaric acid-mono amide from l-aspartic acid-alpha-amide. Through the screening, one mutant, MA2100, catalyzing deamination of l-aspartic acid-alpha-amide was achieved. Gene analysis of the MA2100 mutant indicated that the mutated enzyme had a K327N mutation. The characteristics of the mutated enzyme were examined. The optimum pH values for the l-aspartic acid and l-aspartic acid-alpha-amide of the mutated enzyme were pH 8.5 and 6.0, respectively. The K(m) value and V(max) value for the l-aspartic acid of the mutated enzyme were 28.3 mM and 0.26 U/mg, respectively. The K(m) value and V(max) value for the l-aspartic acid-alpha-amide of the mutated enzyme were 1450 mM and 0.47 U/mg, respectively. This is the first report describing the alteration of the substrate specificity of aspartase, an industrially important enzyme.     

Amphidinolide H, a novel type of actin-stabilizing agent isolated from dinoflagellate

The effect of novel cytotoxic marine macrolide, amphidinolide H (Amp-H), on actin dynamics was investigated in vitro. Amp-H attenuated actin depolymerization induced by diluting F-actin. This effect remained after washing out of unbound Amp-H by filtration. In the presence of either Amp-H or phalloidin, lag phase, which is the rate-limiting step of actin polymerization, was shortened. Phalloidin decreased the polymerization-rate whereas Amp-H did not. Meanwhile, the effects of both compounds were the same when barbed end of actin was capped by cytochalasin D. Quartz crystal microbalance system revealed interaction of Amp-H with G-actin and F-actin. Amp-H also enhanced the binding of phalloidin to F-actin. We concluded that Amp-H stabilizes actin in a different manner from that of phalloidin and serves as a novel pharmacological tool for analyzing actin-mediated cell function.     

Identification and properties of complexes formed by myeloperoxidase with lipoproteins and ceruloplasmin

The first evidence of multi-component complexes formed by myeloperoxidase (MPO), ceruloplasmin (CP), and very low/low density lipoproteins (VLDL/LDL) obtained by electrophoresis, gel filtration, and photon-correlation spectroscopy (PCS) is presented in this paper. Complexes were observed when isolated MPO, CP, and VLDL/LDL were mixed and/or when MPO was added to the blood plasma. Complex LDL–MPO–CP was detected in 44 of 100 plasma samples taken from patients with atherosclerosis, and 33 of 44 samples also contained the VLDL–MPO–CP complex. MPO concentration in these patients’ plasma exceeded 800 ng/ml. Interaction of MPO with high density lipoproteins (HDL) was not revealed, as well as binding of CP to lipoproteins in the absence of MPO. Adding antibodies against apoB-100 to VLDL–MPO–CP and LDL–MPO–CP complexes results in release of lipoproteins. Using PCS the diameters of complexes under study were evaluated. By comparing concentrations of the components in complexes formed by MPO, CP, and lipoproteins their stoichiometry was assessed as 2VLDL:1MPO:2CP and 1LDL:1MPO:2CP. Lipoproteins affected the inhibition of MPO peroxidase activity by CP. The affinity of lipoproteins to MPO–CP complex was assessed using apparent dissociation constants determined as ～0.3 nM for VLDL and ～0.14 nM for LDL.     

Cranial vasculature in zebrafish forms by angioblast cluster-derived angiogenesis

Formation of embryonic vasculature involves vasculogenesis as endothelial cells differentiate and aggregate into vascular cords and angiogenesis which includes branching from the existing vessels. In the zebrafish which has emerged as an advantageous model to study vasculogenesis, cranial vasculature is thought to originate by a combination of vasculogenesis and angiogenesis, but how these processes are coordinated is not well understood. To determine how angioblasts assemble into cranial vasculature, we generated an etsrp:GFP transgenic line in which GFP reporter is expressed under the promoter control of an early regulator of vascular and myeloid development, etsrp/etv2. By utilizing time-lapse imaging we show that cranial vessels originate by angiogenesis from angioblast clusters, which themselves form by the mechanism of vasculogenesis. The two major pairs of bilateral clusters include the rostral organizing center (ROC) which gives rise to the most rostral cranial vessels and the midbrain organizing center (MOC) which gives rise to the posterior cranial vessels and to the myeloid and endocardial lineages. In Etsrp knockdown embryos initial cranial vasculogenesis proceeds normally but endothelial and myeloid progenitors fail to initiate differentiation, migration and angiogenesis. Such angioblast cluster-derived angiogenesis is likely to be involved during vasculature formation in other vertebrate systems as well.