Characterization of a highly pathogenic H5N1 avian influenza A virus isolated from duck meat

Since the 1997 H5N1 influenza virus outbreak in humans and poultry in Hong Kong, the emergence of closely related viruses in poultry has raised concerns that additional zoonotic transmissions of influenza viruses from poultry to humans may occur. In May 2001, an avian H5N1 influenza A virus was isolated from duck meat that had been imported to South Korea from China. Phylogenetic analysis of the hemagglutinin (HA) gene of A/Duck/Anyang/AVL-1/01 showed that the virus clustered with the H5 Goose/Guandong/1/96 lineage and 1997 Hong Kong human isolates and possessed an HA cleavage site sequence identical to these isolates. Following intravenous or intranasal inoculation, this virus was highly pathogenic and replicated to high titers in chickens. The pathogenesis of DK/Anyang/AVL-1/01 virus in Pekin ducks was further characterized and compared with a recent H5N1 isolate, A/Chicken/Hong Kong/317.5/01, and an H5N1 1997 chicken isolate, A/Chicken/Hong Kong/220/97. Although no clinical signs of disease were observed in H5N1 virus-inoculated ducks, infectious virus could be detected in lung tissue, cloacal, and oropharyngeal swabs. The DK/Anyang/AVL-1/01 virus was unique among the H5N1 isolates in that infectious virus and viral antigen could also be detected in muscle and brain tissue of ducks. The pathogenesis of DK/Anyang/AVL-1/01 virus was characterized in BALB/c mice and compared with the other H5N1 isolates. All viruses replicated in mice, but in contrast to the highly lethal CK/HK/220/97 virus, DK/Anyang/AVL-1/01 and CK/HK/317.5/01 viruses remained localized to the respiratory tract. DK/Anyang/AVL-1/01 virus caused weight loss and resulted in 22 to 33% mortality, whereas CK/HK/317.5/01-infected mice exhibited no morbidity or mortality. The isolation of a highly pathogenic H5N1 influenza virus from poultry indicates that such viruses are still circulating in China and may present a risk for transmission of the virus to humans.     

Effect of phosphorylation on the interdomain interaction of the response regulator,NarL

DNA binding by the effector domain (NarLC) of the response regulator, NarL, is modulated by the phosphorylation state of the receiver domain (NarLN). The receiver domain appears to block the site of DNA binding in the nonphosphorylated state. Phosphorylation is proposed to disrupt this interaction, causing the effector domain to be released and free to bind DNA (Baikalov, I., Schroder, I., Kaczor-Grzeskowiak, M., Grzeskowiak, K., Gunsalus, R. P., and Dickerson, R. E. (1996) Biochemistry 35, 11053-61). To better understand this modulation, we analyzed the interaction between the two domains in the absence of a polypeptide linkage. Using multidimensional NMR, we mapped chemical shift changes that occurred during a titration between the two isolated domains. Specific residues in NarLC exhibit large chemical shift changes upon the addition of NarLN. These residues are primarily at the interface between the two domains as seen in the crystal structure. Using the residues with the largest chemical shift changes, we observed a dissociation constant of 88 +/- 7 microM. In the presence of 10 mM MgCl(2), the affinity is reduced 4-fold to about 350 microM. This work shows that the domains interact in trans and that this interaction, while fairly weak, provides a way to monitor the energetics of domain-domain interaction in this system. Phosphorylation of NarLN by a small-molecule phosphate donor, phosphoramidate, decreases this interaction about 25-fold from the nonphosphorylated sample. The results support the model that the mechanism of activation of NarL involves a disruption of the interdomain interface and suggests that the linker is not necessary for the transmission of signal across the domain interface. The linker does play a role in increasing the local concentration of the domains and therefore increasing the amount of closed conformation with respect to the open conformation. We estimate the levels of open conformation to be low (about 1%) in the nonphosphorylated state in the absence of magnesium ion and much higher in the phospho state (near 50%). This modulation of the open or active state via the interaction at the interface may also be applicable to other multidomain response regulator proteins.     

Constituents from the stems of Actinodaphne lancifolia

Two C(16)-lactonic compounds, actinolides A-B (1-2), were isolated from the stems of Actinodaphne lancifolia, together with five known lactones (3-7) and three known lignans (8-10). Their structures were determined spectroscopically, which included 2D NMR spectroscopic analysis.     

Cloning of gibberellin 3 beta-hydroxylase cDNA and analysis of endogenous gibberellins in the developing seeds in watermelon

We have isolated Cv3h, a cDNA clone from the developing seeds of watermelon, and have demonstrated significant amino acid homology with gibberellin (GA) 3 beta-hydroxylases. This cDNA clone was expressed in Escherichia coli as a fusion protein that oxidized GA(9) and GA(12) to GA(4) and GA(14), respectively. The Cv3h protein had the highest similarity with pumpkin GA 2 beta,3 beta-hydroxylase, but did not possess 2 beta-hydroxylation function. RNA blot analysis showed that the gene was expressed primarily in the inner parts of developing seeds, up to 10 d after pollination (DAP). In the parthenocarpic fruits induced by treatment with 1-(2-chloro-4-pyridyl)-3-phenylurea (CPPU), the embryo and endosperm of the seeds were undeveloped, whereas the integumental tissues, of maternal origin, showed nearly normal development. Cv3h mRNA was undetectable in the seeds of CPPU-treated fruits, indicating that the GA 3 beta-hydroxylase gene was expressed in zygotic cells. In our analysis of endogenous GAs from developing seeds, GA(9) and GA(4) were detected at high levels but those of GA(20) and GA(1) were very low. This demonstrates that GA biosynthesis in seeds prefers a non-13-hydroxylation pathway over an early 13-hydroxylation pathway. We also analyzed endogenous GAs from seeds of the parthenocarpic fruits. The level of bioactive GA(4 )was much lower there than in normal seeds, indicating that bioactive GAs, unconnected with Cv3h, exist in integumental tissues during early seed development.     

ProNGF induces p75-mediated death of oligodendrocytes following spinal cord injury

The neurotrophin receptor p75 is induced by various injuries to the nervous system, but its role after injury has remained unclear. Here, we report that p75 is required for the death of oligodendrocytes following spinal cord injury, and its action is mediated mainly by proNGF. Oligodendrocytes undergoing apoptosis expressed p75, and the absence of p75 resulted in a decrease in the number of apoptotic oligodendrocytes and increased survival of oligodendrocytes. ProNGF is likely responsible for activating p75 in vivo, since the proNGF from the injured spinal cord induced apoptosis among p75(+/+), but not among p75(-/-), oligodendrocytes in culture, and its action was blocked by proNGF-specific antibody. Together, these data suggest that the role of proNGF is to eliminate damaged cells by activating the apoptotic machinery of p75 after injury.     

Endophytic bacillus sp. isolated from the interior of balloon flower root

A bacterial strain, designated CY22, was isolated from the interior of balloon flower (Platycodon grandiflorum) root in the Republic of Korea. The isolate coproduced an iturin-like antifungal compound and a surfactin-like potent biosurfactant. Analysis of the 16S-rDNA of strain CY22 showed that the isolate was a member of Bacillus. High similarities were observed between strain CY22 and Bacillus sp. TKSP 24, and between strain CY22 and B. subtilis 168. Phylogenetic analysis based on 16S-rDNA sequences showed that strain CY22 was closely related to Bacillus sp. The main whole-cell fatty acids were anteiso-C15:0 (37%), C17:0 (5.1%), and iso-C15:0 (27.7%). DNA G+C content was 54 mol%. Based on phylogenetic inference, phenotypic and chemotaxonomic characteristics, this endophytic strain Bacillus sp. CY22 was assigned to the genus Bacillus.     

Effects of voluntary resistance exercise on heme biosynthesis in rats given glucocorticoid-injections

To identify the effects of a tower-climbing exercise on bone marrow heme biosynthesis and hematological status in rats given glucocorticoid-injections as a model of aging, 29 male Sprague-Dawley rats, 10 weeks of age, were assigned to three groups: a saline control (C, n = 9), a glucocorticoid-sedentary (GS, n = 10) group, and a glucocorticoid-exercise (GE, n = 10) group. The GS and GE groups were given 2 mg/kg prednisolone daily, and the C group was given 2 ml of saline daily, subcutaneously. Each group was meal-fed commercial rat chow isoenergetically and given free access to water for 8 weeks. The GE group were allowed to climb a 200-cm tower to drink water from a bottle set at the top of it. Weight gain during the 8-week experimental period was greater in the C group than in the GS and GE groups. The gastrocnemius and tibialis anterior muscles were heavier in the C group than in other groups. The hematological parameters were not influenced by glucocorticoid administration with or without climbing exercise. Bone marrow delta-aminolevulinic acid dehydratase activity was significantly higher in the GE group than in the C group. These results suggest that 8 weeks of climbing exercise increases heme biosynthesis without alteration of hematological status in rats given glucocorticoid-injections. Resistance exercise may be a preventive therapy for iron-deficiency anemia associated with aging.     

Proinflammatory cytokine profile in Vibrio vulnificus septicemic patients' sera

Vibrio vulnificus causes a fulminant and frequently fatal septicemia in susceptible hosts. The present study was designed to evaluate the proinflammatory cytokine profile in V. vulnificus septicemia patients' sera and the effect of doxycycline therapy on the levels of proinflammatory cytokines. Levels of proinflammatory cytokines, tumor necrosis factor (TNF)-alpha, interleukin (IL)-1beta and IL-6, were measured in the sera of V. vulnificus septicemic patients and normal healthy volunteers using colorimetric sandwich ELISA. The mean values of TNF-alpha, IL-1beta and IL-6 in the sera of V. vulnificus patients (n=33) increased by 210-, 232- and 40-fold in comparison with those of normal healthy volunteers (n=5), but only the IL-6 level showed a statistically significant difference (P<0.05) between the two groups. Sera from the cases for which doxycycline treatment histories were obvious were designated 'before-treatment' (TX). All the others were included in the after-TX group. In the before-TX group (n=5), the levels of TNF-alpha and IL-1beta significantly increased (P<0.05) in comparison with the after-TX group (n=5). IL-6 levels in the two groups showed no difference. In conclusion, the levels of the well known proinflammatory cytokines TNF-alpha, IL-1beta and IL-6 increased in the V. vulnificus septicemic patients' sera, and the levels of TNF-alpha and IL-1beta decreased significantly after doxycycline treatment. These data indicate that proinflammatory cytokines might play a critical role in V. vulnificus septicemia like in other endotoxemic shocks. The use of doxycycline as an effective bactericidal agent and as an effective modulator of proinflammatory cytokines is supported.     

Structure-antibacterial activity relationship of cecropin A derivatives

To investigate the effect of net positive charge, alpha-helicity and hydrophobicity of the peptides on antibacterial activity, we designed three kinds of cecropin A (CA) derivatives. Compared to CA, F3 with the highest net positive charge exhibited similar or slightly weaker antibacterial activity (MIC: 3.13 approximately 6.25 microM). F1 showed lower antibacterial activity than that of F3, even though it has higher hydrophobicity and a-helicity than F3. This result indicates that the net positive charge of cecropin A-like peptides appears to be a more important factor in antibacterial activity than alpha-helicity and hydrophobicity. The two peptides F1 and F2 possessed almost similar antibacterial activity, but F2 showed very lower activity in the membrane disruption than F1, suggesting other factors are involved in the antibacterial activity of the peptides as well as peptide-lipid interaction.     

Micromere lineages in the glossiphoniid leech Helobdella

In leech embryos, segmental mesoderm and ectoderm arise from teloblasts by lineages that are already relatively well characterized. Here, we present data concerning the early divisions and the definitive fate maps of the micromeres, a group of 25 small cells that arise during the modified spiral cleavage in leech (Helobdella robusta) and contribute to most of the nonsegmental tissues of the adult. Three noteworthy results of this work are as follows. (1) The c"' and dm' clones (3d and 3c in traditional nomenclature) give rise to a hitherto undescribed network of fibers that run from one end of the embryo to the other. (2) The clones of micromeres b" and b"' (2b and 3b in traditional nomenclature) die in normal development; the b" clone can be rescued to assume the normal c" fate if micromere c" or its clone are ablated in early development. (3) Two qualitative differences in micromere fates are seen between H. robusta (Sacramento) and another Helobdella sp. (Galt). First, in Helobdella sp. (Galt), the clone of micromere b" does not normally die, and contributes a subset of the cells arising exclusively from c" in H. robusta (Sacramento). Second, in Helobdella sp. (Galt), micromere c"' makes no definitive contribution, whereas micromere dm' gives rise to cells equivalent to those arising from c"' and dm' in H. robusta (Sacramento).