The HA and NS Genes of Human H5N1 Influenza A Virus Contribute to High Virulence in Ferrets

Highly pathogenic H5N1 influenza A viruses have spread across Asia, Europe, and Africa. More than 500 cases of H5N1 virus infection in humans, with a high lethality rate, have been reported. To understand the molecular basis for the high virulence of H5N1 viruses in mammals, we tested the virulence in ferrets of several H5N1 viruses isolated from humans and found A/Vietnam/UT3062/04 (UT3062) to be the most virulent and A/Vietnam/UT3028/03 (UT3028) to be avirulent in this animal model. We then generated a series of reassortant viruses between the two viruses and assessed their virulence in ferrets. All of the viruses that possessed both the UT3062 hemagglutinin (HA) and nonstructural protein (NS) genes were highly virulent. By contrast, all those possessing the UT3028 HA or NS genes were attenuated in ferrets. These results demonstrate that the HA and NS genes are responsible for the difference in virulence in ferrets between the two viruses. Amino acid differences were identified at position 134 of HA, at positions 200 and 205 of NS1, and at positions 47 and 51 of NS2. We found that the residue at position 134 of HA alters the receptor-binding property of the virus, as measured by viral elution from erythrocytes. Further, both of the residues at positions 200 and 205 of NS1 contributed to enhanced type I interferon (IFN) antagonistic activity. These findings further our understanding of the determinants of pathogenicity of H5N1 viruses in mammals.     

Differentiation of Human Bone Marrow Mesenchymal Stem Cells to Chondrocytes for Construction of Three-dimensional Cartilage Tissue

A differentiation method of human bone marrow mesenchymal stem cells (MSCs) to chondrocytes was developed for the construction of a three-dimensional (3D) cartilage tissue. The adhesive cells, which were isolated from a human bone marrow aspirate were embedded in type I collagen in a poly-l-lactate-glycolic acid copolymer (PLGA) mesh and cultivated for 4 week together with growth factors. The degree of cellular differentiation was estimated by quantitative RT-PCR of aggrecan and type II collagen mRNAs and by staining with Safranin O. The 3D culture showed a higher degree of differentiation even without growth factors than the conventional pellet culture with growth factors, namely, dexamethasone and transforming growth factor (TGF)-β 3. The 3D culture for 2 week with the combined addition of dexamethasone, TGF-β 3, and insulin-like growth factor (IGF)-I reached a 30% expression of aggrecan mRNA compared with that in primary human chondrocytes, while the aggrecan mRNA expression in the conventional pellet culture was less than 2%. The sequential two-step differentiation cultivation, during which the cells were cultivated in 3D for 1 week after the conventional two-dimensional (2D) culture for 1 week, could markedly accelerate the expression of aggrecan mRNA compared with the 3D cultivation for 2 week.     

Role of positively charged amino acids in the M2D transmembrane helix of Ktr/Trk/HKT type cation transporters

Studies suggest that Ktr/Trk/HKT-type transporters have evolved from multiple gene fusions of simple K(+) channels of the KcsA type into proteins that span the membrane at least eight times. Several positively charged residues are present in the eighth transmembrane segment, M2(D), in the transporters but not K(+) channels. Some models of ion transporters require a barrier to prevent free diffusion of ions down their electrochemical gradient, and it is possible that the positively charged residues within the transporter pore may prevent transporters from being channels. Here we studied the functional role of these positive residues in three Ktr/Trk/HKT-type transporters (Synechocystis KtrB-mediated K(+) uniporter, Arabidopsis AtHKT1-mediated Na(+) uniporter and wheat TaHKT1-mediated K(+)/Na(+) symporter) by examining K(+) uptake rates in E. coli, electrophysiological measurements in oocytes and growth rates of E. coli and yeast. The conserved Arg near the middle of the M2(D) segment was essential for the K(+) transport activity of KtrB and plant HKTs. Combined replacement of several positive residues in TaHKT1 showed that the positive residue at the beginning of the M2(D), which is conserved in many K(+) channels, also contributed to cation transport activity. This positive residue and the conserved Arg both face towards the ion conducting pore side. We introduced an atomic-scale homology model for predicting amino acid interactions. Based on the experimental results and the model, we propose that a salt bridge(s) exists between positive residues in the M2(D) and conserved negative residues in the pore region to reduce electrostatic repulsion against cation permeation caused by the positive residue(s). This salt bridge may help stabilize the transporter configuration, and may also prevent the conformational change that occurs in channels.     

Flower form alteration by genetic transformation with the class B MADS-box genes of <Emphasis Type="Italic">Agapanthus</Emphasis><Emphasis Type="Italic">praecox</Emphasis> spp. <Emphasis Type="Italic">orientalis</Emphasis> in transgenic dicot and monocot plants

The class B genes, which belong to the MADS-box gene family, play important roles in regulating petal and stamen development in flowering plants. These genes exist in two different types termed DEF- and GLO-like genes, and the B-function is provided by heterodimers of a DEF- and a GLO-like gene product. In the present study, dicot (tobacco and lettuce) and monocot (Tricyrtis hirta) plants were transformed with the GLO-like gene of Agapanthus praecox ssp. orientalis ApGLO alone or in combination with the DEF-like gene of the same plant ApDEF. In two out of 10 transgenic tobacco plants containing ApGLO, sepals partially converted into petaloid organs. For lettuce, ray florets of four out of nine transgenic plants containing ApGLO also developed additional petaloid organs. In two out of five transgenic T. hirta plants containing both ApGLO and ApDEF, organs developed in whorl 4 showed noticeable morphological alteration: they were much longer compared with carpels of non-transgenic plants, and had purple spots overall on the surface as filaments of non-transgenic plants. No morphological alterations were observed in vegetative organs between transgenic and non-transgenic plants for all the three species. The results obtained in the present study indicate a possibility of molecular breeding for flower form alteration by genetic transformation with the class B MADS-box gene(s) of heterologous plant species.     

Growth characteristics of Sakhalin spruce (Picea glehnii) planted on the northern Japanese hillsides exposed to strong winds

Sakhalin spruce (Picea glehnii), a native species typically found in northern Japan, has been used in reforestation on hillsides exposed to strong winds. In the reforestation areas, there are south-facing (S-slope) and northwest-facing slopes (NW-slope). Climatic conditions are contrasting between the two slopes, with shallower snow cover on the S-slopes. We compared growth responses of the spruce to micro-environment between the S- and NW-slopes through soil nutrients, needle longevity, water status, photosynthesis, and nutrients in the needles. These parameters were measured in needles exposed above the snow in winter and in lower needles protected by snow cover. High-position needles suffered from drought stress, especially in winter, and needles were shed early in the year on both slopes. Low-position needles did not suffer from drought stress, and maintained a high photosynthetic rate on both slopes. However, needle longevity was reduced on the S-slope, and concentrations of nitrogen, phosphorus, and potassium in the needles decreased with needle age. Soil nutrient concentrations were low on the S-slope, which suggests that the needles on the S-slope may remobilize nutrients from aged needles to younger needles prior to shedding. This characteristic is probably an adaptation in Sakhalin spruce to poor soil conditions.     

Highly sensitive quantification of key regulatory oxysterols in biological samples by LC-ESI-MS/MS

We describe a highly sensitive and specific method for the quantification of key regulatory oxysterols in biological samples. This method is based upon a stable isotope dilution technique by liquid chromatography-tandem mass spectrometry (LC-MS/MS). After alkaline hydrolysis of human serum (5 microl) or rat liver microsomes (1 mg protein), oxysterols were extracted, derivatized into picolinyl esters, and analyzed by LC-MS/MS using the electrospray ionization mode. The detection limits of the picolinyl esters of 4beta-hydroxycholesterol, 7alpha-hydroxycholesterol, 22R-hydroxycholesterol, 24S-hydroxycholesterol, 25-hydroxycholesterol, 27-hydroxycholesterol, and 24S,25-epoxycholesterol were 2-10 fg (5-25 amol) on-column (signal-to-noise ratio = 3). Reproducibilities and recoveries of these oxysterols were validated according to one-way layout and polynomial equation, respectively. The variances between sample preparations and between measurements by this method were calculated to be 1.8% to 12.7% and 2.9% to 11.9%, respectively. The recovery experiments were performed using rat liver microsomes spiked with 0.05 ng to 12 ng of oxysterols, and recoveries of the oxysterols ranged from 86.7% to 107.3%, with a mean recovery of 100.6%. This method provides reproducible and reliable results for the quantification of oxysterols in small amounts of biological samples.