Bacterial contamination of tile drainage water and shallow groundwater under different application methods of liquid swine manure

A 2 year field experiment evaluated liquid manure application methods on the movement of manure-borne pathogens (Salmonella sp.) and indicator bacteria (Escherichia coli and Clostridium perfringens) to subsurface water. A combination of application methods including surface application, pre-application tillage, and post-application incorporation were applied in a randomized complete block design on an instrumented field site in spring 2007 and 2008. Tile and shallow groundwater were sampled immediately after manure application and after rainfall events. Bacterial enumeration from water samples showed that the surface-applied manure resulted in the highest concentration of E. coli in tile drainage water. Pre-tillage significantly (p < 0.05) reduced the movement of manure-based E. coli and C. perfringens to tile water and to shallow groundwater within 3 days after manure application (DAM) in 2008 and within 10 DAM in 2007. Pre-tillage also decreased the occurrence of Salmonella sp. in tile water samples. Indicator bacteria and pathogens reached nondetectable levels within 50 DAM. The results suggest that tillage before application of liquid swine manure can minimize the movement of bacteria to tile and groundwater, but is effective only for the drainage events immediately after manure application or initial rainfall-associated drainage flows. Furthermore, the study highlights the strong association between bacterial concentrations in subsurface waters and rainfall timing and volume after manure application.     

Ileal smooth muscle dysfunction and remodeling in cystic fibrosis

Patients with cystic fibrosis (CF) often suffer from gastrointestinal cramps and intestinal obstruction. The CF transmembrane conductance regulator (CFTR) channel has been shown to be expressed in vascular and airway smooth muscle (SM). We hypothesized that the absence of CFTR expression alters the gastrointestinal SM function and that these alterations may show strain-related differences in the mouse. The aim of this study was to measure the contractile properties of the ileal SM in two CF mouse models. CFTR(-/-) and CFTR(+/+) mice were studied on BALB/cJ and C57BL/6J backgrounds. Responsiveness of ileal strips to electrical field stimulation (EFS), methacholine (MCh), and isoproterenol was measured. The mass and the cell density of SM layers were measured morphometrically. Finally, the maximal velocity of shortening (Vmax) and the expression of the fast (+)insert myosin isoform were measured in the C57BL/6J ileum. Ileal hyperreactivity was observed in response to EFS and MCh in CFTR(-/-) compared with CFTR(+/+) mice in C57BL/6J background. This latter observation was not reproduced by acute inhibition of CFTR with CFTR(inh)172. BALB/cJ CFTR(-/-) mice exhibited a significant increase of SM mass with a lower density of cells compared with CFTR(+/+), whereas no difference was observed in the C57BL/6J background. In addition, in this latter strain, ileal strips from CFTR(-/-) exhibited a significant increase in Vmax compared with control and expressed a greater proportion of the fast (+)insert SM myosin isoform with respect to total myosin. BALB/cJ CFTR(-/-) ilium had a greater relaxation to isoproterenol than the CFTR(+/+) mice when precontracted with EFS, but no difference was observed in response to exogeneous MCh. In vivo, the lack of CFTR expression induces a different SM ileal phenotype in different mouse strains, supporting the importance of modifier genes in determining intestinal SM properties.     

A continuous-binding cross-linker model for passive airway smooth muscle

Although the active properties of airway smooth muscle (ASM) have garnered much modeling attention, the passive mechanical properties are not as well studied. In particular, there are important dynamic effects observed in passive ASM, particularly strain-induced fluidization, which have been observed both experimentally and in models; however, to date these models have left an incomplete picture of the biophysical, mechanistic basis for these behaviors. The well-known Huxley cross-bridge model has for many years successfully described many of the active behaviors of smooth muscle using sliding filament theory; here, we propose to extend this theory to passive biological soft tissue, particularly ASM, using as a basis the attachment and detachment of cross-linker proteins at a continuum of cross-linker binding sites. The resulting mathematical model exhibits strain-induced fluidization, as well as several types of force recovery, at the same time suggesting a new mechanistic basis for the behavior. The model is validated by comparison to new data from experimental preparations of rat tracheal airway smooth muscle. Furthermore, experiments in noncontractile tissue show qualitatively similar behavior, suggesting support for the protein-filament theory as a biomechanical basis for the behavior.     

The imbalanced supertree of flowering-plant phylogeny

Two contrasting approaches have been used to construct the overall tree of life from molecular data: one involves the analysis of single large datasets, while the other involves joining many independent smaller analyses into a supertree. A recent study uses the latter approach to produce the most complete phylogeny yet of flowering plant families.     

Establishment of a Genetically Marked Insect-Derived Symbiont in Multiple Host Plants

Alcaligenes xylosoxidans subsp. denitrificans, originally isolated from the cibarial region of the foregut of the glassy-winged sharpshooter (Homalodisca coagulata), was transformed using the Himar1 transposition system to express EGFP. Seedlings of six potential host plants were inoculated with transformed bacteria and 2 weeks later samples were taken 5 cm away and analyzed by quantitative real-time PCR using primers designed to amplify the gene insert. The largest colony of 3,591,427 cells/2 cm of A. xylosoxidans subsp. denitrificans was found in Citrus limon, with almost all plants testing positive in both trials. The amount of colonization decreased in the other plants tested in the following order: orange (Citrus sinensis sweet orange) > chrysanthemum (Chrysanthemum grandiflora cv. White Diamond) > periwinkle (Vinca rosea) > crepe myrtle (Lagerstroemia indica) > grapevine (Vitis vinifera cv. Chardonnay). The bacteriums preference for citrus paralleled the host insects preference for this same plant. Additional tests determined that A. xylosoxidans subsp. denitrificans thrives as a nonpathogenic, xylem-associated endophyte.     

(+)Insert smooth muscle myosin heavy chain (SM-B) isoform expression in human tissues

Two smooth muscle myosin heavy chain isoforms differ in their amino terminus by the presence [(+)insert] or absence [(–)insert] of a seven-amino acid insert. Animal studies show that the (+)insert isoform is predominantly expressed in rapidly contracting phasic muscle and the (–)insert isoform is mostly found in slowly contracting tonic muscle. The expression of the (+)insert isoform has never been demonstrated in human smooth muscle. We hypothesized that the (+)insert isoform is present in humans and that its expression is commensurate with the organ's functional requirements. We report, for the first time, the sequence of the human (+)insert isoform and quantification of its expression by real-time PCR and Western blot analysis in a panel of human organs. The (+)insert isoform mRNA and protein expression levels are significantly greater in small intestine compared with all organs studied except for trachea and are significantly greater in trachea compared with uterus and aorta. To assess the functional significance of this differential myosin isoform expression between organs, we measured the rate of actin filament movement (_max) when propelled by myosin purified from rat organs, because the rat and human inserts are identical and their remaining sequences show 93% identity. _max exhibits a rank correlation from the most tonic to the most phasic organ. The selective expression of the (+)insert isoform observed among human organs suggests that it is an important determinant of tissue shortening velocity. A differential expression of the (+)insert isoform could also account for altered contractile properties observed in human pathology. phasic and tonic smooth muscle; real-time polymerase chain reaction; in vitro motility assay     

Expiratory flow pattern and respiratory mechanics

During resting breathing, expiration is characterized by the narrowing of the vocal folds which, by increasing the expiratory resistance, raises mean lung volume and airway pressure. This is even more pronounced in the neonatal period, during which expirations with short complete airway closure are commonly occurring. We asked to which extent differences in expiratory flow pattern may modify the inspiratory impedance of the respiratory system. To this aim, newborn puppies, piglets, and adult rats were anesthetized, paralyzed, and ventilated with different expiratory patterns, (a) no expiratory load, (b) expiratory resistive load, and (c) end-inspiratory pause. The stroke volume of the ventilator and inspiratory and expiratory times were maintained constant, and the loads were adjusted in such a way that inflation always started from the resting volume of the respiratory system. After 1 min of each ventilatory pattern, mean inspiratory impedance and compliance of lung and respiratory system were measured. The values were unchanged or minimally altered by changing the type of ventilation. We conclude that the expiratory laryngeal loading is not primarily aimed to decrease the work of breathing. It is conceivable that the expiratory pattern is oriented to increase and control mean airway pressure in the regulation of pulmonary fluid reabsorption, distribution of ventilation, and diffusion of gases.     

Segregation of the NK-sensitive phenotype in human x mouse somatic cell hybrids reveals separate genetic control of recognition and postrecognition determinants

In an attempt to investigate the nature of tumor cell-derived membrane surface determinants involved in natural killer cell (NK) recognition or postrecognition events, we have constructed human X mouse interspecies somatic cell hybrids. Highly NK-sensitive (NKs) human tumor cells were fused with NK resistant (NKr) mouse fibroblasts (LMTK-) in polyethylene glycol and selected in hypoxanthine/aminopterin/thymidine medium and ouabain. Hybrids generated from NKs erythroleukemia cells (K-562) or NKs retinoblastoma cells (Y-79) with LMTK- displayed an intermediate NK-sensitive phenotype. One Y-79 X LMTK- hybrid (YL-22) retained a high level of susceptibility to NK binding and cytolysis, as determined by 51Cr release and in cold-target inhibition assays. On the other hand, human NKr RAJI cells generated NK-resistant hybrids when fused with LMTK- fibroblasts. Four hybrids (KL-12, YL-2, YL-22, and YL-43) displaying consistent NK sensitivity were subsequently cloned by limiting dilution. Various hybrid clones derived from the KL-12 hybrid (K-562 X LMTK-) demonstrated a range of NK-sensitive phenotypes. However, the uncloned KL-12 and most cloned lines derived from this hybrid competed against 51Cr-labeled K-562 targets as well as unlabeled K-562 parental cells, regardless of their NK-sensitive phenotype. These findings raise the possibility that chromosomal segregation may be affecting a postbinding step in this hybrid system. The NK-sensitive hybrids exhibited a limited number of human chromosomes as assessed by quinacrine banding. Furthermore, human transferrin receptor (TfR) expression, as monitored by flow cytometry using the B3/25 monoclonal antibody, demonstrated no clear correlation with NK sensitivity or competitive ability in either KL or YL hybrid clones, thus arguing against the involvement of the TfR in human NK recognition. These results suggest that the NK-sensitive phenotype in human tumor cells may be regulated by genes encoded by a limited number of human chromosomes.     

A morphological and immunohistochemical study of programmed cell death in Botryllus schlosseri (Tunicata,Ascidiacea)

The blastogenic cycle of the colonial ascidian Botryllus schlosseri concludes in a phase of selective cell and zooid death called takeover. Every week, all asexually derived parental zooids synchronously regress over a 30-h period and are replaced by a new generation. Here we document the sequential ultrastructural changes which accompany cell death during zooid degeneration. The principal mode of visceral cell death during takeover occurred by apoptosis, the majority of cells condensing and fragmenting into multiple membrane-bounded apoptotic bodies. Cytoplasmic organelles (mitochondria, basal bodies, striated rootlets) within apoptotic bodies retained ultrastructural integrity. Dying cells and fragments were then swiftly ingested by specialized blood macrophages or intraepithelial phagocytes and subsequently underwent secondary necrotic lysis. Certain organs (stomach, intestine) displayed a combination of necrotic and apoptotic changes. Lastly, the stomach, which demonstrated some of the earliest regressive changes, exhibited intense cytoplasmic immunostaining with a monoclonal antibody to ubiquitin at the onset of takeover. Affinity-purified rabbit antiserum against sodium dodecyl sulfate-denatured ubiquitin detected a characteristic 8.6-kDa mono-ubiquitin band by Western blot analysis. Collectively, these findings raise the possibility that cell death during takeover is a dynamic process which requires active participation of cells in their own destruction.     

Heteroduplex DNA Formation Is Associated with Replication and Recombination in Poxvirus-Infected Cells

Poxviruses are large DNA viruses that replicate in the cytoplasm of infected cells and recombine at high frequencies. Calcium phosphate precipitates were used to cotransfect Shope fibroma virus-infected cells with different DNA substrates and the recombinant products assayed by genetic and biochemical methods. We have shown previously that bacteriophage lambda DNAs can be used as substrates in these experiments and recombinants assayed on Escherichia coli following DNA recovery and in vitro packaging. Using this assay it was observed that 2-3% of the phage recovered from crosses between point mutants retained heteroduplex at at least one of the mutant sites. The reliability of this genetic analysis was confirmed using DNA substrates that permitted the direct detection of heteroduplex molecules by denaturant gel electrophoresis and Southern blotting. It was further noted that heteroduplex formation coincided with the onset of both replication and recombination suggesting that poxviruses, like certain bacteriophage, make no clear biochemical distinction between these three processes. The fraction of heteroduplex molecules peaked about 12-hr postinfection then declined later in the infection. This decline was probably due to DNA replication rather than mismatch repair because, while high levels of induced DNA polymerase persisted beyond the time of maximal heteroduplex recovery, we were unable to detect any type of mismatch repair activity in cytoplasmic extracts. These results suggest that, although heteroduplex molecules are formed during the progress of poxviral infection, gene conversion through mismatch repair probably does not produce most of the recombinants. The significance of these observations are discussed considering some of the unique properties of poxviral biology.