Acanthoparyphium tyosenense (Digenea: Echinostomatidae): experimental confirmation of the cercaria and its complete life history in Korea

Cercaria yamagutii Ito, 1957, was found in the marine mesogastropods Lunatia fortuni and Glossaulax didyma from the tidelands of Simpo located at the estuary of the Mankyoung River, which runs to the western coast of Korea. Metacercariae were found in a marine bivalve Mactra veneriformis after being infected with C. yamagutii experimentally. When a sea gull, Larus crassiostris, was fed with the metacercariae collected from the infected M. veneriformis, adult worms were recovered 10 days later. It was confirmed that the parasites collected from L. crassiostris were Acanthoparyphium tyosenense Yamaguti, 1939. From the results of this life cycle study, it was determined that the first intermediate hosts of A. tyosenense are L. fortuni and G. didyma. The second intermediate and final hosts are M. veneriformis and L. crassiostris, respectively. Mactra veneriformis was experimentally infected with C. yamagutii isolated from L. fortuni and G. didyma by maintaining them in a water tank for 30 min at about 20 C. The cercariae entered M. veneriformis through their incurrent siphons. Five hours after infection, the cercariae tails began to separate from the bodies, and the cercariae formed cysts. Mature cysts were formed 340 hr (14 days) after infection and identified as the metacercariae of A. tyosenense. The prevalence of A. tyosenense metacercariae was 99.5% in naturally infected M. veneriformis. This is the first report of C. yamagutii as the cercaria of A. tyosenense, and the complete life cycle of A. tyosenense was established in Korea.     

The PDZ-binding motif of the avian NS1 protein affects transmission of the 2009 influenza A(H1N1) virus

By nature of their segmented RNA genome, influenza A viruses (IAVs) have the potential to generate variants through a reassortment process. The influenza nonstructural (NS) gene is critical for a virus to counteract the antiviral responses of the host. Therefore, a newly acquired NS segment potentially determines the replication efficiency of the reassortant virus in a range of different hosts. In addition, the C-terminal PDZ-binding motif (PBM) has been suggested as a pathogenic determinant of IAVs. To gauge the pandemic potential from human and avian IAV reassortment, we assessed the replication properties of NS-reassorted viruses in cultured cells and in the lungs of mice and determined their transmissibility in guinea pigs. Compared with the recombinant A/Korea/01/2009 virus (rK09; 2009 pandemic H1N1 strain), the rK09/VN:NS virus, in which the NS gene was adopted from the A/Vietnam/1203/2004 virus (a human isolate of the highly pathogenic avian influenza H5N1 virus strains), exhibited attenuated virulence and reduced transmissibility. However, the rK09/VN:NS-PBM virus, harboring the PBM in the C-terminus of the NS1 protein, recovered the attenuated virulence of the rK09/VN:NS virus. In a guinea pig model, the rK09/VN:NS-PBM virus showed even greater transmission efficiency than the rK/09 virus. These results suggest that the PBM in the NS1 protein may determine viral persistence in the human and avian IAV interface.     

The Validity of Quasi-Steady-State Approximations in Discrete Stochastic Simulations

In biochemical networks, reactions often occur on disparate timescales and can be characterized as either fast or slow. The quasi-steady-state approximation (QSSA) utilizes timescale separation to project models of biochemical networks onto lower-dimensional slow manifolds. As a result, fast elementary reactions are not modeled explicitly, and their effect is captured by nonelementary reaction-rate functions (e.g., Hill functions). The accuracy of the QSSA applied to deterministic systems depends on how well timescales are separated. Recently, it has been proposed to use the nonelementary rate functions obtained via the deterministic QSSA to define propensity functions in stochastic simulations of biochemical networks. In this approach, termed the stochastic QSSA, fast reactions that are part of nonelementary reactions are not simulated, greatly reducing computation time. However, it is unclear when the stochastic QSSA provides an accurate approximation of the original stochastic simulation. We show that, unlike the deterministic QSSA, the validity of the stochastic QSSA does not follow from timescale separation alone, but also depends on the sensitivity of the nonelementary reaction rate functions to changes in the slow species. The stochastic QSSA becomes more accurate when this sensitivity is small. Different types of QSSAs result in nonelementary functions with different sensitivities, and the total QSSA results in less sensitive functions than the standard or the prefactor QSSA. We prove that, as a result, the stochastic QSSA becomes more accurate when nonelementary reaction functions are obtained using the total QSSA. Our work provides an apparently novel condition for the validity of the QSSA in stochastic simulations of biochemical reaction networks with disparate timescales.     

Tensile properties of the medial patellofemoral ligament: The effect of specimen orientation

For recurrent patellar dislocation, reconstruction of the medial patellofemoral ligament (MPFL) with replacement autografts has often been performed but with only little data on the tensile properties of the MPFL to guide graft selection. With its complex anatomy and geometry, these properties are difficult to obtain. In this study, we showed how the orientation of the femur-MPFL-patella complex (FMPC) during uniaxial tensile testing can have a significant effect on its structural properties. Twenty two FMPCs were isolated from porcine stifle joints and randomly assigned to two groups of 11 each. For the first group, the specimens were loaded to failure with the patella oriented 30 degrees away from the direction of the applied load to mimic its orientation in situ, called natural orientation. In the second group, the patella was aligned in the direction of the tensile load, called non-natural orientation. The stiffness for the natural orientation group was 65±13 N/mm, 32% higher than that for the non-natural orientation group (50±17 N/mm; p<0.05). The ultimate loads were 438±128 N and 386±136 N, respectively (p>0.05). Ten out of 11 specimens in the natural orientation group failed at the femoral attachment (the narrowest portion of the MPFL) compared to 6 out of 11 in the non-natural orientation group. Our findings suggest that the specimen orientation that mimics the in-situ loading conditions of the MPFL should be used to obtain more representative data for the structural properties of the FMPC.     

Insulin stimulates production of nitric oxide via ERK in osteoblast cells

We explored to determine if iNOS could be induced by insulin in osteoblast-like UMR-106 cells. Insulin (100 nM) stimulated nitric oxide production by twofold and significantly increased iNOS mRNA and protein levels. Insulin also increased collagen synthesis, but had little effect on alkaline phosphatase activity. In contrast, IGF-1 had little effect on NO production below 10 nM and it stimulated NO production by only 57% at 100 nM. IGF-1 had little effect on collagen levels, whereas it inhibited alkaline phosphatase activities in a dose-dependent manner. When an MEK inhibitor was preincubated, insulin failed to stimulate NO production, whereas insulin dramatically increased NO production in the ERK1 overexpressed cells. Taken together, it is proposed that insulin increases iNOS mRNA, iNOS protein, and NO production, possibly via activation of ERK. These may play an important role in osteoblast functions such as collagen synthesis.     

Mechanism for antiplatelet effect of onion: AA release inhibition, thromboxane A(2)synthase inhibition and TXA(2)/PGH(2)receptor blockade

Antiplatelet actions of aqueous extract of onion were investigated in rat and human platelet. IC(50)values of onion extract for collagen-, thrombin-, arachidonic acid (AA)-induced aggregations and collagen-induced thromboxane A(2)(TXA(2)) formation were 0.17 +/- 0. 01, 0.23 + 0.03, 0.34 +/- 0.02 and 0.12 +/- 0.01 g/ml, respectively. [(3)H]-AA release induced by collagen (10 microg/ml) in rat platelet was decreased by onion compared to control (22.1 +/- 2.13 and 5.2 +/- 0.82% of total [(3)H]-AA incorporated, respectively). In fura-2 loaded platelets, the elevation of intracellular Ca(2+)concentration stimulated by collagen was inhibited by onion. Onion had no cytotoxic effect in platelet. Onion significantly inhibited TXA(2)synthase activity without influence on COX activity. Platelet aggregation induced by U46619, a stable TXA(2)mimetic, was inhibited by onion, indicating its antagonism for TXA(2)/PGH(2)receptor. These results suggest that the mechanism for antiplatelet effect of onion may, at least partly, involve AA release diminution, TXA(2)synthase inhibition and TXA(2)/PGH(2)receptor blockade.     

Development of cell line preservation method for research and industry producing useful metabolites by plant cell culture

The cell culture ofAngelica gigas Nakai producing decursin derivatives and immunostimulating polysaccharides was preserved in liquid nitrogen after pre-freezing in a deep freezer at −70°C for 480 min. The effects of the cryoprotectant and pretreatment before cooling were investigated to obtain the optimal procedure for cyropreservation. When compared to mannitol, sorbitol, or NaCl with a similar osmotic pressure, 0.7M sucrose was found to be the best osmoticum for the cryopreservation ofA. gigias cells. In the pre-culture medium, the cells in the exponential growth phase showed the best post-freezing survival after cryopre-servation. A mixture of sucrose, glycerol, and DMSO was found to be an effective cryoprotectant and a higher concentration of the cryoprotectant provided better cell viability. When compared with the vitrification, the optimum cryopreservation method proposed in this study would seem to be more effective for the long-term storage of suspension cells. The highest relative cell viability established with the optimal procedure was 89%.     

Interaction of heat shock protein 70 with membranes depends on the lipid environment

Heat shock proteins (hsp) are well recognized for their protein folding activity. Additionally, hsp expression is enhanced during stress conditions to preserve cellular homeostasis. Hsp are also detected outside cells, released by an active mechanism independent of cell death. Extracellular hsp appear to act as signaling molecules as part of a systemic response to stress. Extracellular hsp do not contain a consensus signal for their secretion via the classical ER-Golgi compartment. Therefore, they are likely exported by an alternative mechanism requiring translocation across the plasma membrane. Since Hsp70, the major inducible hsp, has been detected on surface of stressed cells, we propose that membrane interaction is the first step in the export process. The question that emerges is how does this charged cytosolic protein interact with lipid membranes? Prior studies have shown that Hsp70 formed ion conductance pathways within artificial lipid bilayers. These early observations have been extended herewith using a liposome insertion assay. We showed that Hsp70 selectively interacted with negatively charged phospholipids, particularly phosphatidyl serine (PS), within liposomes, which was followed by insertion into the lipid bilayer, forming high-molecular weight oligomers. Hsp70 displayed a preference for less fluid lipid environments and the region embedded into the lipid membrane was mapped toward the C-terminus end of the molecule. The results from our studies provide evidence of an unexpected ability of a large, charged protein to become inserted into a lipid membrane. This observation provides a new paradigm for the interaction of proteins with lipid environments. In addition, it may explain the export mechanism of an increasing number of proteins that lack the consensus secretory signals.