Frequency of D222G and Q223R hemagglutinin mutants of pandemic (H1N1) 2009 influenza virus in Japan between 2009 and 2010

Background

In April 2009, a novel swine-derived influenza A virus (H1N1pdm) emerged and rapidly spread around the world, including Japan. It has been suggested that the virus can bind to both 2,3- and 2,6-linked sialic acid receptors in infected mammals, in contrast to contemporary seasonal H1N1 viruses, which have a predilection for 2,6-linked sialic acid.

Methods/Results

To elucidate the existence and transmissibility of α2,3 sialic acid-specific viruses in H1N1pdm, amino acid substitutions within viral hemagglutinin molecules were investigated, especially D187E, D222G, and Q223R, which are related to a shift from human to avian receptor specificity. Samples from individuals infected during the first and second waves of the outbreak in Japan were examined using a high-throughput sequencing approach. In May 2009, three specimens from mild cases showed D222G and/or Q223R substitutions in a minor subpopulation of viruses infecting these individuals. However, the substitutions almost disappeared in the samples from five mild cases in December 2010. The D187E substitution was not widespread in specimens, even in May 2009.

Conclusions

These results suggest that α2,3 sialic acid-specific viruses, including G222 and R223, existed in humans as a minor population in the early phase of the pandemic, and that D222 and Q223 became more dominant through human-to-human transmission during the first and second waves of the epidemic. These results are consistent with the low substitution rates identified in seasonal H1N1 viruses in 2008.     

Eukaryotic translation initiation factor 4F architectural alterations accompany translation initiation factor redistribution in poxvirus-infected cells

Despite their self-sufficient ability to generate capped mRNAs from cytosolic DNA genomes, poxviruses must commandeer the critical eukaryotic translation initiation factor 4F (eIF4F) to recruit ribosomes. While eIF4F integrates signals to control translation, precisely how poxviruses manipulate the multisubunit eIF4F, composed of the cap-binding eIF4E and the RNA helicase eIF4A assembled onto an eIF4G platform, remains obscure. Here, we establish that the poxvirus infection of normal, primary human cells destroys the translational repressor eIF4E binding protein (4E-BP) and promotes eIF4E assembly into an active eIF4F complex bound to the cellular polyadenylate-binding protein (PABP). Stimulation of the eIF4G-associated kinase Mnk1 promotes eIF4E phosphorylation and enhances viral replication and protein synthesis. Remarkably, these eIF4F architectural alterations are accompanied by the concentration of eIF4E and eIF4G within cytosolic viral replication compartments surrounded by PABP. This demonstrates that poxvirus infection redistributes, assembles, and modifies core and associated components of eIF4F and concentrates them within discrete subcellular compartments. Furthermore, it suggests that the subcellular distribution of eIF4F components may potentiate the complex assembly.     

Jump exercise during remobilization restores integrity of the trabecular architecture after tail suspension in young rats.

Three-dimensional trabecular architecture was investigated in the femora of tail-suspended young growing rats, and the effects of jump exercise during remobilization were examined. Five-week-old male Wistar rats (n = 35) were randomly assigned to five body weight-matched groups: tail-suspended group (SUS; n = 7); sedentary control group for SUS (S(CON); n = 7); spontaneous recovery group after tail suspension (S+R(CON), n = 7); jump exercise group after tail suspension (S+R(JUM); n = 7); and age-matched control group for S+R(CON) and S+R(JUM) without tail suspension and exercise (S(CON)+R(CON); n = 7). Rats in SUS and S(CON) were killed immediately after tail suspension for 14 days. The jump exercise protocol consisted of 10 jumps/day, 5 days/wk, and jump height was 40 cm. Bone mineral density (BMD) of the femur and three-dimensional trabecular bone architecture at the distal femoral metaphysis were measured. Tail suspension induced a 13.6% decrease in total femoral BMD (P < 0.001) and marked deterioration of trabecular architecture. After 5 wk of free remobilization, femoral BMD, calf muscle weight, and body weight returned to age-matched control levels, but trabeculae remained thinner and less connected. On the other hand, S+R(JUM) rats showed significant increases in trabecular thickness, number, and connectivity compared with S+R(CON) rats (62.8, 31.6, and 24.7%, respectively; P < 0.05), and these parameters of trabecular architecture returned to the levels of S(CON)+R(CON). These results indicate that suspension-induced trabecular deterioration persists after remobilization, but jump exercise during remobilization can restore the integrity of trabecular architecture and bone mass in the femur in young growing rats.     

Nicotine suppresses tunicamycin-induced, but not thapsigargin-induced, expression of GRP78 during ER stress-mediated apoptosis in PC12 cells

We previously reported that nicotine protected against tunicamycin (Tm)-induced ER stress-mediated apoptosis, but not thapsigargin (Tg)-induced apoptosis in PC12 cells. In the present study, we report that the expression of glucose-regulated protein 78 (GRP78) was suppressed by nicotine in Tm-treated PC12 cells. Interestingly, the GRP78 expression was not changed by nicotine in Tg-treated cells. Moreover, nicotine reduced the activation of caspase-12 in Tm-treated cells, but not in Tg-treated cells. These results suggest that nicotine prevented Tm-induced ER stress-mediated apoptosis by attenuating an early stage of Tm-induced ER stress. It was possible that the suppression of GRP78 expression by nicotine was achieved through the suppression of the Ire1-XBP1 and/or ATF6 pathways. We observed that nicotine suppressed the Tm-induced, but not Tg-induced, splicing of XBP1 mRNA, and also suppressed the Tm-induced, but not Tg-induced, production of cleaved ATF6 in PC12 cells. These results indicate that the suppression of Ire1-XBP1 and ATF6 pathways contributes to the suppression of GRP78 expression by nicotine in Tm-treated PC12 cells, suggesting that nicotine suppresses a common step upstream of both the Ire1-XBP1 and ATF6 pathways which are required for the expression of GRP78 during Tm-induced ER stress.     

Regulation of arsenic trioxide-induced cellular responses by Mnk1 and Mnk2

Arsenic trioxide (As(2)O(3)) is a potent inducer of apoptosis of malignant cells in vitro and in vivo, but the precise mechanisms by which it mediates such effects are not well defined. We provide evidence that As(2)O(3) induces phosphorylation/activation of the MAPK signal-integrating kinases (Mnks) 1 and 2 in leukemia cell lines. Such activation is defective in cells with targeted disruption of the p38alpha MAPK gene, indicating that it requires upstream engagement of the p38 MAPK pathway. Studies using Mnk1(-/-) or Mnk2(-/-), or double Mnk1(-/-)Mnk2(-/-) knock-out cells, establish that activation of Mnk1 and Mnk2 by arsenic trioxide regulates downstream phosphorylation of the eukaryotic initiation factor 4E at Ser-209. Importantly, arsenic-induced apoptosis is enhanced in cells with targeted disruption of the Mnk1 and/or Mnk2 genes, suggesting that these kinases are activated in a negative-feedback regulatory manner, to control generation of arsenic trioxide responses. Consistent with this, pharmacological inhibition of Mnk activity enhances the suppressive effects of arsenic trioxide on primary leukemic progenitors from patients with acute leukemias. Taken together, these findings indicate an important role for Mnk kinases, acting as negative regulators for signals that control generation of arsenic trioxide-dependent apoptosis and antileukemic responses.     

Influence of tendon slack on electromechanical delay in the human medial gastrocnemius in vivo.

The purpose of this study was to clarify the influence of muscle-tendon complex stretch on electromechanical delay (EMD) in terms of the extent of tendon slack in the human medial gastrocnemius (MG). EMD and MG tendon length were measured at each of five ankle joint angles (-30, -20, -10, 0, and 5 degrees : positive values for dorsiflexion) using percutaneous electrical stimulation and ultrasonography, respectively. The extent of MG tendon slack was calculated as MG tendon length shortening, standardized with MG tendon slack length obtained at the joint angle (-16 degrees +/- 5 degrees ) where the passive ankle joint torque was zero. EMD at -30 degrees (19.2 +/-2.2 ms) and -20 degrees (17.2 +/- 1.3 ms) was significantly greater than that at -10 degrees (16.0 +/-2.3 ms), 0 degrees (15.0 +/-1.4 ms), and 5 degrees (14.8 +/-1.4 ms), and at 0 and 5 degrees, respectively. The relative EMD, normalized with the maximal EMD for each subject, decreased dependent on the extent of decrease in MG tendon slack. There were no significant differences in EMD among the joint angles (-10, 0, and 5 degrees ) where MG tendon slack was taken up. These results suggest that the extent of tendon slack is an important factor for determining EMD.     

Expression of highly sulfated keratan sulfate synthesized in human glioblastoma cells

Keratan sulfate (KS) proteoglycan is expressed in the extracellular matrix or cell surface in numerous tissues, predominantly in those of the cornea, cartilage, and brain. However, its structure, function, and regulation remain poorly understood. Our investigation of KS expression in glioblastoma cell lines using Western-blot and flow cytometry with anti-KS antibody (5D4) revealed that LN229 glioblastoma cell highly expresses KS on a cell surface. Real-time PCR analysis showed that LN229 expresses a high level of keratan sulfate Gal-6-sulfotransferase. Results of this study also demonstrate that recombinant 5D4-reactive aggrecan is produced in LN229. Taken together, these results suggest that LN229 produces 5D4-reactive highly sulfated KS and is useful to investigate the KS structure and function in glioblastoma.     

Effects of isometric training on the elasticity of human tendon structures in vivo.

The present study aimed to investigate the effect of isometric training on the elasticity of human tendon structures. Eight subjects completed 12 wk (4 days/wk) of isometric training that consisted of unilateral knee extension at 70% of maximal voluntary contraction (MVC) for 20 s per set (4 sets/day). Before and after training, the elongation of the tendon structures in the vastus lateralis muscle was directly measured using ultrasonography while the subjects performed ramp isometric knee extension up to MVC. The relationship between the estimated muscle force and tendon elongation (L) was fitted to a linear regression, the slope of which was defined as stiffness of the tendon structures. The training increased significantly the volume (7.6+/-4.3%) and MVC torque (33.9+/-14.4%) of quadriceps femoris muscle. The L values at force production levels beyond 550 N were significantly shorter after training. The stiffness increased significantly from 67.5+/-21.3 to 106.2+/-33.4 N/mm. Furthermore, the training significantly increased the rate of torque development (35.8 +/- 20.4%) and decreased electromechanical delay (-18.4+/-3.8%). Thus the present results indicate that isometric training increases the stiffness and Young's modulus of human tendon structures as well as muscle strength and size. This change in the tendon structures would be assumed to be an advantage for increasing the rate of torque development and shortening the electromechanical delay.     

Rac Regulates Vascular Endothelial Growth Factor Stimulated Motility

During angiogenesis endothelial cells migrate towards a chemotactic stimulus. Understanding the mechanism of endothelial cell migration is critical to the therapeutic manipulation of angiogenesis and ultimately cancer prevention. Vascular endothelial growth factor (VEGF) is a potent chemotactic stimulus of endothelial cells during angiogenesis. The endothelial cell signal transduction pathway of VEGF represents a potential target for cancer therapy, but the mechanisms of post-receptor signal transduction including the roles of rho family GTPases in regulating the cytoskeletal effects of VEGF in endothelial cells are not understood.

Here we analyze the mechanisms of cell migration in the mouse brain endothelial cell line (bEND3). Stable transfectants containing a tetracycline repressible expression vector were used to induce expression of Rac mutants. Endothelial cell haptotaxis was stimulated by constitutively active V12Rac on collagen and vitronectin coated supports, and chemotaxis was further stimulated by VEGF. Osteopontin coated supports were the most stimulatory to bEND3 haptotaxis, but VEGF was not effective in further increasing migration on osteopontin coated supports. Haptotaxis on support coated with collagen, vitronectin, and to a lesser degree osteopontin was inhibited by N17 Rac. N17 Rac expression blocked stimulation of endothelial cell chemotaxis by VEGF. As part of the chemotactic stimulation, VEGF caused a loss of actin organization at areas of cell-cell contact and increased stress fiber expression in endothelial cells which were directed towards pores in the transwell membrane. N17 Rac prevented the stimulation of cell-cell contact disruption and the stress fiber stimulation by VEGF.

These data demonstrate two pathways of regulating endothelial cell motility, one in which Rac is activated by matrix/integrin stimulation and is a crucial modulator of endothelial cell haptotaxis. The other pathway, in the presence of osteopontin, is Rac independent. VEGF stimulated chemotaxis, is critically dependent on Rac activation. Osteopontin was a potent matrix activator of motility, and perhaps one explanation for the absence of a VEGF plus osteopontin effect is that osteopontin stimulated motility was inhibitory to the Rac pathway.     

Photoswitching of peroxidase activity by position-specific incorporation of a photoisomerizable non-natural amino acid into horseradish peroxidase.

Horseradish peroxidase mutants containing L-p-phenylazophenylalanine (azoAla) at various positions were synthesized by using an Escherichia coli in vitro translation system. Among the 15 mutants examined, four mutants containing a single azoAla unit at the 6th, 68th, 142nd, and 179th positions, respectively, retained the peroxidase activity. The activity of the Phe68azoAla mutant was higher when the azobenzene group was in the cis form than in the trans form. On the contrary, the activity of the Phe179azoAla mutant disappeared when the azobenzene group was photoisomerized to the cis form, but recovered in the trans form. In the latter mutant, therefore, an on/off photoswitching of the peroxidase activity was attained.