The cotton rat model of respiratory viral infections

Development of successful vaccines against human infectious diseases depends on using appropriate animal models for testing vaccine efficacy and safety. For some viral infections the task is further complicated by the frequently changing genetic make-up of the virus, as in the case of influenza, or by the existence of the little-understood phenomenon of vaccine-enhanced disease, as in the case of respiratory syncytial virus (RSV). The cotton rat Sigmodon hispidus has been used for years as an excellent small animal model of the RSV vaccine-enhanced disease. Recently, using cotton rats, we have demonstrated that vaccination against another paramyxovirus, human metapneumovirus (hMPV), can also lead to vaccine-enhanced disease. In addition to the study of paramyxoviruses, S. hispidus presents important advantages for the study of orthomyxoviruses such as influenza. The cotton rat is susceptible to infection with unadapted human influenza strains, and heterosubtypic immunity to influenza can be evoked in S. hispidus. The mechanisms of influenza, RSV, and hMPV pathogenesis and immunity can now be investigated in the cotton rat with the development of species-specific reagents for this animal model.     

White Vesicles in the Skin of Aplysia californicaCooper: A Proposed Excretory Function

White patches of skin that appear early in the development ofthe shell-less mollusc, Aplysia californica, are composed ofaggregations of vase-shaped vesicles, each a single, large cellwith an enlarged nucleus. Two layers of collagen, at 90°to each other, surround the vesicle membrane, together witha non-contiguous external layer of muscle. Energy dispersivespectroscopy and electron diffraction of the contents with scanningelectron microscopy (SEM) and transmission electron microscopy(TEM) failed to find a signal for halogens (involved with unpalatableskin and, therefore, defence against predation), but did recordprominent signals for calcium. Immature vesicles are filledwith an homogenous material that appeared to mature progressively,likely by nucleation, into numerous, small spherules, visibleby both SEM and TEM. Ultra-thin uranyl acetate stained sectionsof mature vesicles showed that the spherules had an intracrystallinematrix of radially arranged electron-dense material. The vesiclecell had none of the modifications characteristic of calciumor rhogocyte cell-types, which serve as calcium reservoirs forthe movement of this element to sites of shell formation andregeneration. These vesicles are likely involved with the excretionof excess calcium and not, as originally hypothesized, witheither defence against predators or calcium flux. In addition,the spherules that fill the vesicles are possibly composed ofthe less common polymorphs of calcium carbonate, vaterite andamorphous calcium carbonate. Excretion of the vesicle contentsappeared to be passive, since vesicles lacked an abundant contiguouslayer of muscle or a muscular release valve in the vesicle neck,but did have organized layers of collagen just outside the vesiclemembrane. We propose that release of vesicle contents probablyoccurs when the neck of the vesicle is broken. (Received 5 December 2005; accepted 28 April 2006)     

Contribution of the Human Parainfluenza Virus Type 3 HN-Receptor Interaction to Pathogenesis In Vivo

The envelope of human parainfluenza virus type 3 (HPF3) contains two viral glycoproteins, the hemagglutinin-neuraminidase (HN) protein and the fusion (F) protein. In a previous study, highly fusogenic variant HPF3 viruses were isolated, including two, C-0 and C-22, that exhibit increased avidity for sialic acid receptors due to single amino acid changes in the HN protein and one, C-28, that has decreased neuraminidase activity relative to that of the wild type (wt) and is delayed in the release of virus particles into the supernatant fluid. These variants form very large plaques and destroy a cell monolayer more rapidly than does wt HPF3 in cell culture. These variant viruses allowed us to formulate hypotheses about the roles of HN in pathogenesis. We investigated the behavior of wt HPF3 and the three variant viruses in the cotton rat model. In the cotton rat, there was no delayed clearance of any of the variant viruses compared to that of the wt. The variant plaque morphology was preserved in vivo, and there was no reversion to the wt phenotype in the infected animals. In spite of a slight advantage of wt virus in viral titer, there were no differences in the severities of peribronchiolitis between wt viruses and the variants. However, there were marked differences in severities in alveolitis and interstitial pneumonitis when each of the three variants was compared to the wt, with the variants causing enhanced disease. Thus, despite similar or lower viral titers and similar clearance rates, the variants caused more extensive disease in the lung. The results show that mutations in HN conferring altered fusion properties in cell culture also confer striking differences in the ability of HPF3 to cause extensive disease in the cotton rat lung and that this effect is dissociated from any effect on viral replication.     

NONCONSUMPTIVE PREDATOR‐DRIVEN MORTALITY CAUSES NATURAL SELECTION ON PREY

Predators frequently exert natural selection through differential consumption of their prey. However, predators may also cause prey mortality through nonconsumptive effects, which could cause selection if different prey phenotypes are differentially susceptible to this nonconsumptive mortality. Here we present an experimental test of this hypothesis, which reveals that nonconsumptive mortality imposed by predatory dragonflies causes selection on their damselfly prey favoring increased activity levels. These results are consistent with other studies of predator‐driven selection, however, they reveal that consumption alone is not the only mechanism by which predators can exert selection on prey. Uncovering this mechanism also suggests that prey defensive traits may represent adaptations to not only avoid being consumed, but also for dealing with other sources of mortality caused by predators. Demonstrating selection through both consumptive and nonconsumptive predator mortality provides us with insight into the diverse effects of predators as an evolutionary force.     

Infiltration of Alternatively Activated Macrophages in Cancer Tissue Is Associated with MDSC and Th2 Polarization in Patients with Esophageal Cancer

Myeloid derived suppressor cells (MDSCs) expand in cancer bearing hosts and contribute to tumor immune evasion. M2 macrophages constitute a major cellular component of cancer-related inflammation. However, the correlation between circulating MDSCs and infiltrating M2 macrophages in tumor tissues from patients with esophageal cancer (ECA), and its potential relationship with the polarization of Th2 cells remain unclear. In the present study, we showed the level of MDSCs in PBMC and Arg1 in plasma were significantly elevated in ECA patients, and the increased ratio of MDSC in PBMC was closely related to the expression of CD163 in cancer tissues. In addition, the ECA patients exhibited remarkable increases in the mRNA levels of IL-4 and GATA3, as well as the protein levels of IL-13 and IL-6, but IFN-γ and IL-12 in peripheral blood were decreased. Our data indicate that the increased Th2 cytokines are associated with MDSCs and M2 macrophages polarization, and foster the infiltration of CD163⁺M2 macrophages in cancer tissues, which promote the formation of immunosuppressive microenvironment in ECA patients.     

Purification and Characterization of an Extracellular,Thermo-Alkali-Stable,Metal Tolerant Laccase from Bacillus tequilensis SN4

A novel extracellular thermo-alkali-stable laccase from Bacillus tequilensis SN4 (SN4LAC) was purified to homogeneity. The laccase was a monomeric protein of molecular weight 32 KDa. UV-visible spectrum and peptide mass fingerprinting results showed that SN4LAC is a multicopper oxidase. Laccase was active in broad range of phenolic and non-phenolic substrates. Catalytic efficiency (k _cat/K _m) showed that 2, 6-dimethoxyphenol was most efficiently oxidized by the enzyme. The enzyme was inhibited by conventional inhibitors of laccase like sodium azide, cysteine, dithiothreitol and β-mercaptoethanol. SN4LAC was found to be highly thermostable, having temperature optimum at 85°C and could retain more than 80% activity at 70°C for 24 h. The optimum pH of activity for 2, 6-dimethoxyphenol, 2, 2′-azino bis[3-ethylbenzthiazoline-6-sulfonate], syringaldazine and guaiacol was 8.0, 5.5, 6.5 and 8.0 respectively. Enzyme was alkali-stable as it retained more than 75% activity at pH 9.0 for 24 h. Activity of the enzyme was significantly enhanced by Cu²⁺, Co²⁺, SDS and CTAB, while it was stable in the presence of halides, most of the other metal ions and surfactants. The extracellular nature and stability of SN4LAC in extreme conditions such as high temperature, pH, heavy metals, halides and detergents makes it a highly suitable candidate for biotechnological and industrial applications.     

Biophysical stratification of the Amazon basin

In field measurement programmes, stratified sampling can optimize sampling efficiency, but stratification is often undertaken subjectively, and is frequently based on a priori classification schemes such as those used for vegetation maps. In order to avoid the problems associated with a priori subjective schemes, we explore here an objective procedure, Regression Tree Analysis (RTA). RTA has previously been used in local-scale studies, but here we apply it to a very large study domain, namely the entire humid tropical zone of South America. The aim of the study was to develop an optimal sampling design in preparation for the Large Scale Biosphere-Atmosphere Experiment in Amazonia (LBA). Co-registered spatially continuous fields of rainfall, temperature, photosynthetically active radiation (PAR), the normalized difference index (NDVI), an index of surface moisture, and other independent variables were used to predict three dependent variables, annual net radiation (Rn), latent heat (LE) and net primary production (NPP). Rather than simply dividing the study area based on differing levels of the three dependent variables, empirical models were developed using RTA to indicate how the relationships between these and possible forcing variables vary across the study area. For each variable long-term seasonal indices such as annual average, monthly minimum and amplitude were used to exclude effects of temporal phase differences between the hemispheres. The resulting hierarchical models revealed variations in the interdependence of the forcing variables throughout the study area and therefore provided a basis for a stratified sampling and identifying the most important variables to be collected in LBA for the Amazon basin as a whole as well as optimizing the sampling scheme for scaling up findings from the field scale to larger areas.     

E.p.r.-spectroscopic studies on the molybdenum-iron site of nitrogenase from Clostridium pasteurianum.

The e.p.r. spectroscopy of the nitrogenase molybdenum-iron protein from Clostridium pasteurianum was re-investigated. The sharpness of the delta Ms = +/- 3 g'z peak from the +/- 3/2 Kramer's doublet enables the observation and quantification of incompletely resolved hyperfine splittings from the stable magnetic nuclei 95Mo and 57Fe in samples enriched in these isotopes. No couplings to 1H or 17O could be discerned by examination of spectra from samples exchanged into 2H2O and H2(17)O respectively. Simulation of the spectrum from 95Mo-enriched samples yields a hyperfine coupling of 2.9 MHz, and indicates that the earlier electron-nuclear-double-resonance-derived estimate of 8.1 +/- 0.2 MHz is substantially in error.