Influenza Infects Lung Microvascular Endothelium Leading to Microvascular Leak: Role of Apoptosis and Claudin-5

Severe influenza infections are complicated by acute lung injury, a syndrome of pulmonary microvascular leak. The pathogenesis of this complication is unclear. We hypothesized that human influenza could directly infect the lung microvascular endothelium, leading to loss of endothelial barrier function. We infected human lung microvascular endothelium with both clinical and laboratory strains of human influenza. Permeability of endothelial monolayers was assessed by spectrofluorimetry and by measurement of the transendothelial electrical resistance. We determined the molecular mechanisms of flu-induced endothelial permeability and developed a mouse model of severe influenza. We found that both clinical and laboratory strains of human influenza can infect and replicate in human pulmonary microvascular endothelium, leading to a marked increase in permeability. This was caused by apoptosis of the lung endothelium, since inhibition of caspases greatly attenuated influenza-induced endothelial leak. Remarkably, replication-deficient virus also caused a significant degree of endothelial permeability, despite displaying no cytotoxic effects to the endothelium. Instead, replication-deficient virus induced degradation of the tight junction protein claudin-5; the adherens junction protein VE-cadherin and the actin cytoskeleton were unaffected. Over-expression of claudin-5 was sufficient to prevent replication-deficient virus-induced permeability. The barrier-protective agent formoterol was able to markedly attenuate flu-induced leak in association with dose-dependent induction of claudin-5. Finally, mice infected with human influenza developed pulmonary edema that was abrogated by parenteral treatment with formoterol. Thus, we describe two distinct mechanisms by which human influenza can induce pulmonary microvascular leak. Our findings have implications for the pathogenesis and treatment of acute lung injury from severe influenza.     

Effect of sodium butyrate on the production, heterogeneity and biological activity of human thrombopoietin by recombinant Chinese hamster ovary cells

Human thrombopoietin (hTPO) is a heavily glycosylated protein with 6 and 24 potential N- and O-glycosylation sites, respectively. To determine the effect of sodium butyrate (NaBu) on the production and quality of hTPO in recombinant Chinese hamster ovary (rCHO) cells, NaBu (0-10 mM) was added to the cultures of exponentially growing cells. NaBu addition significantly increased both the specific and volumetric hTPO production, although it decreased the cell viability by apoptosis in a dose-dependent manner. The highest hTPO concentration of 82.2 +/- 5.6 microgml-1 was obtained in the culture with 3 mM NaBu addition. Compared with the culture without NaBu addition, the culture with 3 mM NaBu resulted in a 6.4-fold increase in qTPO and a 3.3-fold increase in the final hTPO concentration on day 7. However, NaBu deteriorated the quality of hTPO, resulting from increased heterogeneity, reduced acidic hTPO isoforms, reduced alpha(2 --> 3) sialylation, and decreased in vivo biological activity. We also found that the biological activity of hTPO in the culture with 3 mM NaBu addition collected on day 7 was 72% of that in the culture without NaBu addition. Taken together, the use of NaBu or its optimal concentration for high-level expression of a heavily glycosylated protein like hTPO should be determined by considering its detrimental effect on the quality of glycoprotein.     

Functional response of Harmonia axyridis (Pallas) (Coleoptera: Coccinellidae) to Aphis gossypii Glover (Homoptera: Aphididae) in the Laboratory

Stage specific functional response of Harmonia axyridis (Pallas) to varying densities of Aphis gossypii Glover was examined in a simplified cucumber leaf arena under laboratory conditions. All stages of H. axyridis were isolated individually for 24 h with different prey densities at 25 °C and a photoperiod of 16:8 (L:D) h. The number of prey consumed by the predator was checked at 3, 6, 12, and 24 h. All stages of H. axyridis showed a Type II functional response. Based on the random predator equation, estimated attack rates of H. axyridis at 24 h were 0.0037, 0.0442, 0.3590, 0.3228, and 0.1456, and estimated handling times were 4.1001, 2.4575, 0.7500, 0.2132, and 0.1708 h for the first, second, third, and fourth instars, and female adult, respectively.     

Denaturing gradient gel electrophoresis analysis of bacterial populations in 5-stage biological nutrient removal process with step feed system for wastewater treatment

Changes in the bacterial populations of a 5-stage biological nutrient removal (BNR) process, with a step feed system for wastewater treatment, were monitored by denaturing gradient gel electrophoresis (DGGE) of PCR-amplified 16S ribosomal DNA fragments. DGGE analysis indicated seasonal community changes were observed, however, community profiles of the total bacteria of each reactor showed only minor differences in the samples obtained from the same season. The number of major bands was higher in the summer samples, and decreased during the winter period, indicating that the microbial community structure became simpler at low temperatures. Since the nitrogen and phosphate removal efficiencies were highly maintained throughout the winter operation period, the bacteria which still remaining in the winter sample can be considered important, playing a key role in the present 5-stage BNR sludge. The prominent DGGE bands were excised, and sequenced to gain insight into the identities of the predominant bacterial populations present, and most were found to not be closely related to previously characterized bacteria. These data suggest the importance of culture-independent methods for the quality control of wastewater treatment.     

Alternatively spliced human genes by exon skipping--a database (ASHESdb)

Alternative splicing of mRNA allows many gene products with different functions to be produced from a single coding sequence. Exon skipping is the most commonly known alternative splicing mechanism. A comprehensive database of alternative splicing by exon skipping is made available for the human genome data. 1,229 human genes are identified to exhibit alternative splicing by exon skipping. Availability: http://sege.ntu.edu.sg/wester/ashes/.     

Backbone and side chain dynamics of mutant calmodulin-peptide complexes

The mechanism of long-range coupling of allosteric sites in calcium-saturated calmodulin (CaM) has been explored by characterizing structural and dynamics effects of mutants of calmodulin in complex with a peptide corresponding to the smooth muscle myosin light chain kinase calmodulin-binding domain (smMLCKp). Four CaM mutants were examined: D95N and D58N, located in Ca2+-binding loops; and M124L and E84K, located in the target domain-binding site of CaM. Three of these mutants have altered allosteric coupling either between Ca2+-binding sites (D58N and D95N) or between the target- and Ca2+-binding sites (E84K). The structure and dynamics of the mutant calmodulins in complex with smMLCKp were characterized using solution NMR. Analysis of chemical shift perturbations was employed to detect largely structural perturbations. 15N and 2H relaxation was employed to detect perturbations of the dynamics of the backbone and methyl-bearing side chains of calmodulin. The least median squares method was found to be robust in the detection of perturbed sites. The main chain dynamics of calmodulin are found to be largely unresponsive to the mutations. Three mutants show significantly perturbed dynamics of methyl-bearing side chains. Despite the pseudosymmetric location of Ca2+-binding loop mutations D58N and D95N, the dynamic response of CaM is asymmetric, producing long-range perturbation in D58N and almost none in D95N. The mutations located at the target domain-binding site have quite different effects. For M124L, a local perturbation of the methyl dynamics is observed, while the E84K mutation produces a long-range propagation of dynamic perturbations along the target domain-binding site.     

Evidence from biosynthetically incorporated strontium and FTIR difference spectroscopy that the C-terminus of the D1 polypeptide of photosystem II does not ligate calcium

Recent FTIR studies have provided evidence that the C-terminal alpha-COO(-) group of the D1 polypeptide at D1-Ala344 is a unidentate ligand of a Mn ion in photosystem II [Chu, H.-A., Hiller, W., and Debus, R. J. (2004) Biochemistry 43, 3152-3166; Kimura, Y., Mizusawa, N., Yamanari, T., Ishii, A., and Ono, T.-A. (2005) J. Biol. Chem. 280, 2078-2083]. However, the FTIR data could not exclude Ca ligation. Furthermore, the recent approximately 3.5 A X-ray crystallographic structural model positions the alpha-COO(-) group of D1-Ala344 near a Ca ion [Ferreira, K. N., Iverson, T. M., Maghlaoui, K., Barber, J., and Iwata, S. (2004) Science 303, 1831-1838]. Therefore, to conclusively establish whether the alpha-COO(-) group of D1-Ala344 ligates Mn or Ca, the symmetric carboxylate stretching mode of the alpha-COO(-) group of D1-Ala344 was identified in the S(2)-minus-S(1) FTIR difference spectrum of PSII particles having Sr substituted for Ca. Cells of the cyanobacterium Synechocystis sp. PCC 6803 were propagated in media having Sr substituted for Ca and containing either l-[1-(13)C]alanine or unlabeled ((12)C) alanine. The S(2)-minus-S(1) FTIR difference spectra of the purified PSII particles show that substituting Sr for Ca alters several carboxylate stretching modes, including some that may correspond to one or more metal ligands, but importantly does not alter the symmetric carboxylate stretching mode of the alpha-COO(-) group of D1-Ala344. In unlabeled PSII particles, this mode appears at approximately 1356 cm(-)(1) in the S(1) state and at either approximately 1337 or approximately 1320 cm(-)(1) in the S(2) state, irrespective of whether the PSII particles contain Ca or Sr. These data are inconsistent with Ca ligation and show, therefore, that the C-terminal alpha-COO(-) group of the D1 polypeptide ligates a Mn ion. These data also show that substituting Ca with the larger Sr ion perturbs other unidentified carboxylate groups, at least one of which may ligate the Mn(4) cluster.