Energetic approach to the folding of alpha/beta barrels

The folding of a polypeptide into a parallel (alpha/beta)8 barrel (which is also called a circularly permuted beta 8 alpha 8 barrel) has been investigated in terms of energy minimization. According to the arrangement of hydrogen bonds between two neighboring beta-strands of the central barrel therein, such an alpha/beta barrel structure can be folded into six different types: (1) left-tilted, left-handed crossover; (2) left-tilted, right-handed crossover; (3) nontilted, left-handed crossover; (4) nontilted, right-handed crossover; (5) right-tilted, left-handed crossover; and (6) right-tilted, right-handed crossover. Here "tilt" refers to the orientational relation of the beta-strands to the axis of the central beta-barrel, and "crossover" to the beta alpha beta folding connection feature of the parallel beta-barrel. It has been found that the right-tilted, right-handed crossover alpha/beta barrel possesses much lower energy than the other five types of alpha/beta barrels, elucidating why the observed alpha/beta barrels in proteins always assume the form of right tilt and right-handed crossover connection. As observed, the beta-strands in the energy-minimized right-tilted, right-handed crossover (alpha/beta)8-barrel are of strong right-handed twist. The value of root-mean-square fits also indicates that the central barrel contained in the lowest energy (alpha/beta)8 structure thus found coincides very well with the observed 8-stranded parallel beta-barrel in triose phosphate isomerase (TIM). Furthermore, an energetic analysis has been made demonstrating why the right-tilt, right-handed crossover barrel is the most stable structure. Our calculations and analysis support the principle that it is possible to account for the main features of frequently occurring folding patterns in proteins by means of conformational energy calculations even for very complicated structures such as (alpha/beta)8 barrels.     

FcγRIIIa expression on monocytes in rheumatoid arthritis: role in immune-complex stimulated TNF production and non-response to methotrexate therapy

Objective

The expression of FcγRIIIa/CD16 may render monocytes targets for activation by IgG-containing immune complexes (IC). We investigated whether FcγRIIIa/CD16 was upregulated in rheumatoid arthritis (RA), associated with TNF production in response to IC-stimulation, and if this predicted response to methotrexate therapy.

Methods

FcγRIIIa/CD16 expression on CD14^low and CD14⁺⁺ monocytes was measured by flow cytometry in healthy controls and RA patients (early and long-standing disease). Intracellular TNF-staining was carried out after in vitro LPS or heat-aggregated immunoglobulin (HAG) activation. FcγRIIIa/CD16 expression pre- and post-steroid/methotrexate treatment was examined.

Results

Increased FcγRIIIa/CD16 expression on CD14⁺⁺ monocytes in long-standing RA patients compared to controls was demonstrated (p = 0.002) with intermediate levels in early-RA patients. HAG-induced TNF-production in RA patients was correlated with the percentage of CD14⁺⁺ monocytes expressing FcγRIIIa/CD16 (p<0.001). The percentage of CD14⁺⁺ monocytes expressing FcγRIIIa/CD16 at baseline in early DMARD-naïve RA patients was negatively correlated with DAS28-ESR improvement 14-weeks post-methotrexate therapy (p = 0.003) and was significantly increased in EULAR non-responders compared to moderate (p = 0.01) or good responders (p = 0.003). FcγRIIIa/CD16 expression was not correlated with age, presence of systemic inflammation or autoantibody titers.

Conclusion

Increased FcγRIIIa/CD16 expression on CD14⁺⁺ monocytes in RA may result in a cell that has increased responsiveness to IC-stimulation. This monocyte subset may contribute to non-response to methotrexate therapy.     

Signaling pathways of LIGHT induced macrophage migration and vascular smooth muscle cell proliferation

The biological actions of LIGHT, a member of the tumor necrosis factor superfamily, are mediated by the interaction with lymphotoxin-beta receptor (LTbetaR) and/or herpes virus entry mediator (HVEM). Previous study demonstrated high-level expressions of LIGHT and HVEM receptors in atherosclerotic plaques. To investigate the role of LIGHT in the functioning of macrophages and vascular smooth muscle cells (VSMC) in relation to atherogenesis, we determined the effects of LIGHT on macrophage migration and VSMC proliferation. We found LIGHT through HVEM activation can induce both events. LIGHT-induced macrophage migration was associated with activation of signaling kinases, including MAPKs, PI3K/Akt, NF-kappaB, Src members, and FAK. Proliferation of VSMC was also shown relating to the activation of MAPKs, PI3K/Akt, and NF-kappaB, which consequently led to alter the expression of cell cycle regulatory molecules. Down-regulation of p21, p27, and p53, and inversely up-regulation of cyclin D and RB hyper-phosphorylation were demonstrated. In conclusion, LIGHT acts as a novel mediator for macrophage migration and VSMC proliferation, suggesting its involvement in the atherogenesis.     

Feeding and grazing impact by small marine heterotrophic dinoflagellates on heterotrophic bacteria

ABSTRACT. We investigated the feeding of the small heterotrophic dinoflagellates (HTDs) Oxyrrhis marina , Gyrodinium cf. guttula , Gyrodinium sp., Pfiesteria piscicida , and Protoperidinium bipes on marine heterotrophic bacteria. To investigate whether they are able to feed on bacteria, we observed the protoplasm of target heterotrophic dinoflagellate cells under an epifluorescence microscope and transmission electron microscope. In addition, we measured ingestion rates of the dominant heterotrophic dinoflagellate, Gyrodinium spp., on natural populations of marine bacteria (mostly heterotrophic bacteria) in Masan Bay, Korea in 2006–2007. Furthermore, we measured the ingestion rates of O. marina , G . cf. guttula , and P. piscicida on bacteria as a function of bacterial concentration under laboratory conditions. All HTDs tested were able to feed on a single bacterium. Oxyrrhis marina and Gyrodinium spp. intercepted and then ingested a single bacterial cell in feeding currents that were generated by the flagella of the predators. During the field experiments, the ingestion rates and grazing coefficients of Gyrodinium spp. on natural populations of bacteria were 14–61 bacteria/dinoflagellate/h and 0.003–0.972 day⁻¹, respectively. With increasing prey concentration, the ingestion rates of O. marina , G . cf. guttula , and P. piscicida on bacteria increased rapidly at prey concentrations of ca 0.7–2.2 × 10⁶ cells/ml, but increased only slowly or became saturated at higher prey concentrations. The maximum ingestion rate of O. marina on bacteria was much higher than those of G . cf. guttula and P. piscicida . Bacteria alone supported the growth of O. marina . The results of the present study suggest that some HTDs may sometimes have a considerable grazing impact on populations of marine bacteria, and that bacteria may be important prey.     

<Emphasis Type="Italic">Luteimonas aestuarii</Emphasis> sp. nov., isolated from tidal flat sediment

A novel bacterium B9^T was isolated from tidal flat sediment. Its morphology, physiology, biochemical features, and 16S rRNA gene sequence were characterized. Colonies of this strain are yellow and the cells are Gram-negative, rod-shaped, and do not require NaCl for growth. The 16S rRNA gene sequence similarity indicated that strain B9^T is associated with the genus Lysobacter (≤ 97.2%), Xanthomonas (≤ 96.8%), Pseudomonas (≤ 96.7%), and Luteimonas (≤ 96.0%). However, within the phylogenetic tree, this novel strain shares a branching point with the species Luteimonas composti CC-YY255^T (96.0%). The DNA-DNA hybridization experiments showed a DNA-DNA homology of 23.0% between strain B9^T and Luteimonas mephitis B1953/27.1^T. The G+C content of genomic DNA of the type strain is 64.7 mol% (SD, 1.1). The predominant fatty acids are iso-C_11:0, iso-C_15:0, iso-C_16:0, iso-C_17:0, iso-C_17:0 ω9c, and iso-C_11:0 3-OH. Combined analysis of the 16S rRNA gene sequences, fatty acid profile, and results from physiological and biochemical tests indicated that there is genotypic and phenotypic differentiation of the isolate from other Luteimonas species. For these reasons, strain B9^T was proposed as a novel species, named Luteimonas aestuarii. The type strain of the new species is B9^T (= KCTC 22048^T, DSM 19680^T).     

The motility and dynamic properties of intermediate filaments and their constituent proteins

Intermediate filament (IF) proteins exist in multiple structural forms within cells including mature IF, short filaments or 'squiggles', and non-filamentous precursors called particles. These forms are interconvertible and their relative abundance is IF type, cell type- and cell cycle stage-dependent. These structures are often associated with molecular motors, such as kinesin and dynein, and are therefore capable of translocating through the cytoplasm along microtubules. The assembly of mature IF from their precursor particles is also coupled to translation. These dynamic properties of IF provide mechanisms for regulating their reorganization and assembly in response to the functional requirements of cells. The recent findings that IF and their precursors are frequently associated with signaling molecules have revealed new functions for IF beyond their more traditional roles as mechanical integrators of cells and tissues.     

Characterization and application of oviductal epithelial cells in vitro in Gallus domesticus

Chicken oviductal epithelium produces large quantities of egg white protein in daily cycles. In this study, we cultured and characterized oviductal epithelial cells (OECs) from juvenile (10-wk-old) chickens and from actively laying (30-wk-old) hens. The juvenile OECs were maintained over passage 25 and were positive for toluidine blue, lectin-ConA, HPA, UEA-1, WFA, WGA, anti-OVA, anti-ESR1, and anti-PGR, whereas the adult OECs were cultured over passage 6 and were positive for toluidine blue, periodic acid-Schiff, lectin-ConA, WFA, WGA, anti-OVA, anti-ESR1, and anti-PGR. To investigate the optimal concentration of steroid hormones for inducing egg white protein genes in vitro, we examined the effects of estrogen, diethylstilbestrol, progesterone, and corticosterone on OECs. Results showed that oviduct-specific levels of avidin, ovalbumin, ovomucin, lysozyme, ESR1, and PGR gene expression were significantly elevated in steroid hormone-treated OECs compared with those of untreated cells (P < 0.05). Ovalbumin protein was also secreted into culture medium from hormone-treated OECs. In addition, to examine the application of OECs for avian transgenesis, we introduced human thrombopoietin (THPO)-expressing lentiviral vector controlled by a 3.5-kb ovalbumin promoter into cultured OECs, and THPO expression was significantly induced with diethylstilbestrol or progesterone in juvenile OECs (P < 0.05) and in adult OECs (P < 0.05). In conclusion, these data demonstrate the potential of cultured OECs as a model system for providing a better understanding of the regulation of gene expression and for the production of an avian transgenic bioreactor.     

Mixotrophy in the newly described phototrophic dinoflagellate Woloszynskia cincta from western Korean waters: feeding mechanism, prey species and effect of prey concentration

Woloszynskia species are dinoflagellates in the order Suessiales inhabiting marine or freshwater environments; their ecophysiology has not been well investigated, in particular, their trophic modes have yet to be elucidated. Previous studies have reported that all Woloszynskia species are photosynthetic, although their mixotrophic abilities have not been explored. We isolated a dinoflagellate from coastal waters in western Korea and established clonal cultures of this dinoflagellate. On the basis of morphology and analyses of the small/large subunit rRNA gene (GenBank accession number=FR690459), we identified this dinoflagellate as Woloszynskia cincta. We further established that this dinoflagellate is a mixotrophic species. We found that W. cincta fed on algal prey using a peduncle. Among the diverse prey provided, W. cincta ingested those algal species that had equivalent spherical diameters (ESDs) ≤12.6 μm, exceptions being the diatom Skeletonema costatum and the dinoflagellate Prorocentrum minimum. However, W. cincta did not feed on larger algal species that had ESDs≥15 μm. The specific growth rates for W. cincta increased continuously with increasing mean prey concentration before saturating at a concentration of ca. 134 ng C/ml (1,340 cells/ml) when Heterosigma akashiwo was used as food. The maximum specific growth rate (i.e. mixotrophic growth) of W. cincta feeding on H. akashiwo was 0.499 d(-1) at 20 °C under illumination of 20 μE/m(2) /s on a 14:10 h light-dark cycle, whereas its growth rate (i.e. phototrophic growth) under the same light conditions without added prey was 0.040 d(-1). The maximum ingestion and clearance rates of W. cincta feeding on H. akashiwo were 0.49 ng C/grazer/d (4.9 cells/grazer/d) and 1.9 μl/grazer/h, respectively. The calculated grazing coefficients for W. cincta on co-occurring H. akashiwo were up to 1.1 d(-1). The results of the present study suggest that grazing by W. cincta can have a potentially considerable impact on prey algal populations.     

Microsatellite analysis as a tool for discriminating an interfamily hybrid between olive flounder and starry flounder

An interspecific artificial hybrid was produced between two economically important aquaculture flatfish: olive flounder (Paralichthys olivaceus) and starry flounder (P. stellatus). This hybrid displays the rapid growth characteristic of the former and tolerance to low temperatures and low salinity of the latter, but the genetics of inheritance in this hybrid have not been elucidated. Polymorphic microsatellite markers developed for P. olivaceus and P. stellatus were tested to determine if these markers can be used for analysis of parentage and genetic inheritance. Multiplex PCR using two primer sets that were specific to each species produced PCR products of different sizes; these could be used for the identification of interspecific hybrids. Among the 192 primers derived from olive flounder, 25.5% of the primer sets successfully amplified genomic DNA from starry flounder, and 23% of the 56 primer sets originating from starry flounder amplified DNA from olive flounder. Analysis of genetic inheritance in the hybrid using seven of the 62 microsatellite markers common to both species demonstrated classic Mendelian inheritance of these markers in the hybrid progeny, with the exception of one locus identified as a null allele in the hybrid. These results demonstrate that cross-specific microsatellite markers can be used tools for parentage analysis of hybrid flatfish, for mapping quantitative trait loci, for marker-assisted selective breeding, and for studies of the evolution of fish.