Measuring the magnitude of internal motion in a complex hexasaccharide

For the development of a scheme for quantitative experimental estimation of internal motion in the complex human milk hexasaccharide lacto‐N‐di‐fuco hexose I (LNDFH I), we measured a large number of experimental residual dipolar couplings in liquid crystal orienting media. We present a total of 40 ¹³C? ¹H and ¹H? ¹H dipolar coupling values, each representing distinct directions of internuclear vectors. The NMR data were interpreted with established methods for analysis of rigid subdomains of the oligosaccharide as well as a novel method in which dipolar couplings were calculated over an ensemble of conformers from a solvent Molecular Dynamics trajectory using multiple linear regression analysis. The Lewis^b epitope region of LNDFH I assumed a single unique conformation with internal motion described by fluctuations of 5–10° in glycosidic dihedral angles consistent with previous studies. Greater flexibility was observed for the remaining GlcNAc1→3‐β‐D ‐Gal and β‐D ‐Gal1→4Glc linkages, with the former glycosidic linkage existing in a conformational exchange among three states. The results were also supported by similar results of calculations carried out with conformers obtained from a simple Monte Carlo simulation without explicit solvent. © 2010 Wiley Periodicals, Inc. Biopolymers 95: 39–50, 2011.     

Somatic cell nuclear transfer in the pig: control of pronuclear formation and integration with improved methods for activation and maintenance of pregnancy

To clone a pig from somatic cells, we first validated an electrical activation method for use on ovulated oocytes. We then evaluated delayed versus simultaneous activation (DA vs. SA) strategies, the use of 2 nuclear donor cells, and the use of cytoskeletal inhibitors during nuclear transfer. Using enucleated ovulated oocytes as cytoplasts for fetal fibroblast nuclei and transferring cloned embryos into a recipient within 2 h of activation, a 2-h delay between electrical fusion and activation yielded blastocysts more reliably and with a higher nuclear count than did SA. Comparable rates of development using DA were obtained following culture of embryos cloned from ovulated or in vitro-matured cytoplasts and fibroblast or cumulus nuclei. Treatment of cloned embryos with cytochalasin B (CB) postfusion and for 6 h after DA had no impact on blastocyst development as compared with CB treatment postfusion only. Inclusion of a microtubule inhibitor such as nocodozole with CB before and after DA improved nuclear retention and favored the formation of single pronuclei in experiments using a membrane dye to reliably monitor fusion. However, no improvement in blastocyst development was observed. Using fetal fibroblasts as nuclear donor cells, a live cloned piglet was produced in a pregnancy that was maintained by cotransfer of parthenogenetic embryos.     

Structure, Expression and Phylogenetic Analysis of the Gene Encoding Actin I in Pneumocystis Carinii

Actin is a major component of the cytoskeleton and one of the most abundant proteins found in eukaryotic cells. Comparative sequence analysis shows that this essential gene has been highly conserved throughout eukaryotic evolution making it useful for phylogenetic analysis. Complete cDNA clones for the actin-encoding gene were isolated and characterized from Pneumocystis carinii purified from immunosuppressed rat lungs. The nucleotide sequence encodes a protein of 376 amino acids. The predicted actin protein of P. carinii shares a high degree of conservation to other known actins. Only one major actin gene was found in P. carinii. The P. carinii actin sequence was compared with 30 other actin sequences. Gene phylogenies constructed using both neighbor-joining and protein parsimony methods places the P. carinii actin sequence closest to the majority of the fungi. Since the phylogenetic relationship of P. carinii to fungi and protists has been questioned, these data on the actin gene phylogeny support the grouping of P. carinii with the fungi.     

P blood group regulation of glycosphingolipid levels in human erythrocytes.

The neutral glycosphingolipid content of normal human erythrocytes was analyzed by a new method which utilizes high performance liquid chromatography. This rapid and accurate technique permits the quantitation of each of the major neutral glycolipids from individual blood samples. A correlation between the P blood group and the relative quantities of neutral glycosphingolipids is demonstrated. Erythrocytes from P1 individuals are shown to contain more globotriaosylceramide and less lactosylceramide than do erythrocytes from P2 individuals. The results of these experiments suggest the existence of a new phenotype in the P blood group system, and have further implications regarding the biosynthesis of the P blood group glycosphingolipids.     

Field Evaluation of Mulberry Root Structure with Regard to Phytoremediation

The root structure of a mature 12-year-old mulberry tree growing in a former waste disposal basin and the relationship of the tree's root system to the distribution of polycyclic aromatic hydrocarbons (PAHs) in soil/sludge beneath the tree were examined. Root excavation showed that large roots were restricted to the upper 60?cm of the root zone, whereas medium-sized (0.3 to 2.5?cm) and fine (<0.3?cm) roots were present throughout the root zone reaching depths of 110 cm. Comparison of the concentrations of PAHs in the root zone with those in the sludge beneath the root zone (>2.0?m) showed dramatic differences. In the upper 60?cm of the root zone, where roots have prevailed for potentially 12 years, the PAH concentrations were approximately 10 to 20% of those in the sludge. At lower rooting depths 60 to 100?cm, where fewer roots have been present for a shorter time period, the PAH concentrations have decreased but to a lesser degree. These field results provide evidence that the mulberry root system develops gradually over time in a vertical direction with fine roots serving as the leading edge of a moving front. The degradative properties of this biological front (root-microbe combination) toward toxic chemicals hold great importance for successful development of phytoremediation technology designed to degrade deep contaminants in former sludge basins.     

The maize lipoxygenase,ZmLOX10, mediates green leaf volatile,jasmonate and herbivore‐induced plant volatile production for defense against insect attack

Fatty acid derivatives are of central importance for plant immunity against insect herbivores; however, major regulatory genes and the signals that modulate these defense metabolites are vastly understudied, especially in important agro‐economic monocot species. Here we show that products and signals derived from a single Zea mays (maize) lipoxygenase (LOX), ZmLOX10, are critical for both direct and indirect defenses to herbivory. We provide genetic evidence that two 13‐LOXs, ZmLOX10 and ZmLOX8, specialize in providing substrate for the green leaf volatile (GLV) and jasmonate (JA) biosynthesis pathways, respectively. Supporting the specialization of these LOX isoforms, LOX8 and LOX10 are localized to two distinct cellular compartments, indicating that the JA and GLV biosynthesis pathways are physically separated in maize. Reduced expression of JA biosynthesis genes and diminished levels of JA in lox10 mutants indicate that LOX10‐derived signaling is required for LOX8‐mediated JA. The possible role of GLVs in JA signaling is supported by their ability to partially restore wound‐induced JA levels in lox10 mutants. The impaired ability of lox10 mutants to produce GLVs and JA led to dramatic reductions in herbivore‐induced plant volatiles (HIPVs) and attractiveness to parasitoid wasps. Because LOX10 is under circadian rhythm regulation, this study provides a mechanistic link to the diurnal regulation of GLVs and HIPVs. GLV‐, JA‐ and HIPV‐deficient lox10 mutants display compromised resistance to insect feeding, both under laboratory and field conditions, which is strong evidence that LOX10‐dependent metabolites confer immunity against insect attack. Hence, this comprehensive gene to agro‐ecosystem study reveals the broad implications of a single LOX isoform in herbivore defense.     

Interactions of Francisella tularensis with Alveolar Type II Epithelial Cells and the Murine Respiratory Epithelium

Francisella tularensis is classified as a Tier 1 select agent by the CDC due to its low infectious dose and the possibility that the organism can be used as a bioweapon. The low dose of infection suggests that Francisella is unusually efficient at evading host defenses. Although ~50 cfu are necessary to cause human respiratory infection, the early interactions of virulent Francisella with the lung environment are not well understood. To provide additional insights into these interactions during early Francisella infection of mice, we performed TEM analysis on mouse lungs infected with F. tularensis strains Schu S4, LVS and the O-antigen mutant Schu S4 waaY::TrgTn. For all three strains, the majority of the bacteria that we could detect were observed within alveolar type II epithelial cells at 16 hours post infection. Although there were no detectable differences in the amount of bacteria within an infected cell between the three strains, there was a significant increase in the amount of cellular debris observed in the air spaces of the lungs in the Schu S4 waaY::TrgTn mutant compared to either the Schu S4 or LVS strain. We also studied the interactions of Francisella strains with human AT-II cells in vitro by characterizing the ability of these three strains to invade and replicate within these cells. Gentamicin assay and confocal microscopy both confirmed that F. tularensis Schu S4 replicated robustly within these cells while F. tularensis LVS displayed significantly lower levels of growth over 24 hours, although the strain was able to enter these cells at about the same level as Schu S4 (1 organism per cell), as determined by confocal imaging. The Schu S4 waaY::TrgTn mutant that we have previously described as attenuated for growth in macrophages and mouse virulence displayed interesting properties as well. This mutant induced significant airway inflammation (cell debris) and had an attenuated growth phenotype in the human AT-II cells. These data extend our understanding of early Francisella infection by demonstrating that Francisella enter significant numbers of AT-II cells within the lung and that the capsule and LPS of wild type Schu S4 helps prevent murine lung damage during infection. Furthermore, our data identified that human AT-II cells allow growth of Schu S4, but these same cells supported poor growth of the attenuated LVS strain in vitro. Collectively, these data further our understanding of the role of AT-II cells in Francisella infections.     

Differences in mortality from acute myocardial infarction between coronary care unit and medical ward: treatment or bias?

To analyse the effectiveness of coronary care units in reducing mortality from myocardial infarction 18 hospitals ranging from large urban teaching hospitals to small country hospitals were stratified into four levels of care. Previous analysis had failed to show significant differences in the overall mortality in hospital among levels. There were significant differences in mortality, however, between those patients allocated to be cared for in the coronary care unit and those in the medical wards in the more advanced hospitals. The differences were largest in the hospitals with the most elaborate facilities (level 1) and non-existent in those with the least (level 4). Several analytical approaches to these observed differences indicated that they were: (a) reduced by adjustment for age and severity of infarction; (b) paralleled by differences in coexisting disease recorded on death certificates; (c) no longer significant at level 1 after allowing for differences in coexisting disease; and (d) not significant at any level after exclusion of patients first diagnosed at necropsy. These findings suggest that the observed differences in mortality between coronary care units and medical wards are largely due to bias in selection and diagnosis.     

Aroid scarabs in the genus Peltonotus Burmeister (Coleoptera,Scarabaeidae, Dynastinae): key to species and new distributional data

The southeast Asian scarab beetle genus Peltonotus Burmeister (Scarabaeidae, Dynastinae, Cyclocephalini) is reviewed. New country records for Peltonotus morio Burmeister (Myanmar and Vietnam), Peltonotus nasutus Arrow (southern China and Cambodia), and Peltonotus favonius Jameson and Wada (Myanmar) are reported, including a new record in the Palearctic/Sino-Japanese biogeographic region. The first female specimen of Peltonotus favonius is described. Biological associations with aroid inflorescences are reviewed, and human consumption of Peltonotus beetles is reported. A key to all species, paralectotype designations for Peltonotus nasutus, diagnoses, and distributions using dynamic mapping tools are included.