Effects of Cl2MDP-encapsulating liposomes in a murine model of Pseudomonas aeruginosa-induced sepsis

Pseudomonas aeruginosa is a pathogen that frequently causes acute lung injury, bacteremia and sepsis in critically ill patients. As tissue macrophages are a major producer of inflammatory mediators that contribute to septic physiology, and are essential for eliminating bacteria from the circulation, we investigated the role of tissue macrophages in the generation of both inflammatory and anti-inflammatory cytokines in septic shock by using our mouse model of P. aeruginosa pneumonia. To see the effects of tissue macrophage depletion, we intravenously injected dichloromethylene-diphosphonate (Cl2MDP)-encapsulating liposomes in mice. Two days after the liposome injection, we instilled cytotoxic P. aeruginosa (PA103) into the lung that disseminates and causes septic shock. After the infection, we collected blood and bronchoalveolar lavage fluids. The samples were then analyzed for TNF-alpha, MIP-2, and IL-10 concentration. We compared these results to control mice that received either liposomes without Cl2MDP or phosphate buffered saline alone. Plasma TNF-alpha, MIP-2, and IL-10 levels were significantly decreased in the tissue macrophage-depleted mice compared to the control groups of mice. Although depletion of tissue macrophages by Cl2MDP-liposome administration did not affect the severity of bacteremia or the survival of infected mice, these results imply that tissue macrophages have a major role in the production of both proinflammatory and anti-inflammatory cytokines in the circulation and in the causing septic physiology associated with P. aeruginosa pneumonia.     

Cross-correlation suppressed T1 and NOE experiments for protein side-chain 13CH2 groups

Relaxation measurements of side-chain ¹³CH₂-groups of uniformly ¹³C labeled human ubiquitin were performed at 600 MHz and 800 MHz magnetic field strength at 30°C. Dipole-dipole cross-correlated relaxation effects in T₁ experiments were suppressed by the combination of radio-frequency pulses and pulsed field gradients during the relaxation delay leading to monoexponential relaxation decays that allow a more accurate extraction of the ¹³C T₁ relaxation times. Heteronuclear ¹H-¹³C NOEs obtained by using different proton saturation schemes indicate that the influence of cross-correlation is small. The experimental T₁ and NOE data were interpreted in a model-free way in terms of a generalized order parameter and an internal correlation time.     

Evidence that acetyl phosphate functions as a global signal during biofilm development

We used DNA macroarray analysis to identify genes that respond to the status of the intracellular acetyl phosphate (acP) pool. Genes whose expression correlated negatively with the ability to synthesize acP (i.e. negatively regulated genes) function primarily in flagella biosynthesis, a result consistent with observations that we published previously (Prüss and Wolfe, 1994, Mol Microbiol 12: 973-984). In contrast, genes whose expression correlated positively with the ability to synthesize acP (i.e. positively regulated genes) include those for type 1 pilus assembly, colanic acid (capsule) biosynthesis and certain stress effectors. To our knowledge, this constitutes the first report that these genes may respond to the status of the intracellular acP pool. Previously, other researchers have implicated flagella, type 1 pili, capsule and diverse stress effectors in the formation of biofilms. We therefore tested whether cells altered in their ability to metabolize acP could construct normal biofilms, and found that they could not. Cells defective for the production of acP and cells defective for the degradation of acP could both form biofilms, but these biofilms exhibited characteristics substantially different from each other and from biofilms formed by their wild-type parent. We confirmed the role of individual cell surface structures, the expression of which appears to correlate with acP levels, in fim or fli mutants that cannot assemble type 1 pili or flagella respectively. Thus, the information gained by expression profiling of cells with altered acP metabolism indicates that acP may help to co-ordinate the expression of surface structures and cellular processes involved in the initial stages of wild-type biofilm development.     

Cryopreservation and long-term storage of pear germplasm

Summary Germplasm collections of vegetatively propagated crops are usually maintained as plants in fields or potted in greenhouses or screened enclosures. Safety duplication of these collections, as duplicate plants or separate collections, is costly and requires large amounts of space. Cryopreservation techniques which were recently developed for long-term storage of pear germalasm may offer an efficient alternative to conventional germplasm collection maintenance. Pear (Pyrus L.) germplasm may now be stored as seeds (species), dormant buds or pollen from field-grown trees, or shoot tips fromin vitro-grown plants (cultivars). Pear germplasm may now be cryopreserved and stored for long periods (> 100 yr) utilizing slow-freezing or vitrification ofin vitro-grown shoot-tips. Dormant bud freezing, pollen, and seed cryopreservation of other lines are being developed to complete the base collection forPyrus. This cryopreserved collection provides base (long-term) storage for the field-grown pear germplasm collection at the National Clonal Germplasm Repository, Corvallis, Oregon. Based on a presentation at the 1997 Congress on In Vitro Biology held in Washington, D.C., June 14–18, 1997.     

PDADMAC flocculation of Chinese hamster ovary cells: Enabling a centrifuge-less harvest process for monoclonal antibodies

High titer (>10 g/L) monoclonal antibody (mAb) cell culture processes are typically achieved by maintaining high viable cell densities over longer culture durations. A corresponding increase in the solids and sub-micron cellular debris particle levels are also observed. This higher burden of solids (≥15%) and sub-micron particles typically exceeds the capabilities of a continuous centrifuge to effectively remove the solids without a substantial loss of product and/or the capacity of the harvest filtration train (depth filter followed by membrane filter) used to clarify the centrate. We discuss here the use of a novel and simple two-polymer flocculation method used to harvest mAb from high cell mass cell culture processes. The addition of the polycationic polymer, poly diallyldimethylammonium chloride (PDADMAC) to the cell culture broth flocculates negatively-charged cells and cellular debris via an ionic interaction mechanism. Incorporation of a non-ionic polymer such as polyethylene glycol (PEG) into the PDADMAC flocculation results in larger flocculated particles with faster settling rate compared to PDADMAC-only flocculation. PDADMAC also flocculates the negatively-charged sub-micron particles to produce a feed stream with a significantly higher harvest filter train throughput compared to a typical centrifuged harvest feed stream. Cell culture process variability such as lactate production, cellular debris and cellular densities were investigated to determine the effect on flocculation. Since PDADMAC is cytotoxic, purification process clearance and toxicity assessment were performed.     

Long-distance dispersal helps germinating mahogany seedlings escape defoliation by a specialist caterpillar

Herbivores and pathogens with acute host specificity may promote high tree diversity in tropical forests by causing distance- and density-dependent mortality of seedlings, but evidence is scarce. Although Lepidoptera larvae are the most abundant and host-specific guild of herbivores in these forests, their impact upon seedling distributions remains largely unknown. A firm test of the mechanism underpinning the Janzen–Connell hypothesis is difficult, even for a single tree species, because it requires more than just manipulating seeds and seedlings and recording their fates. Experimental tests require: (1) an insect herbivore that is identified and highly specialised, (2) linkage to an in situ measure (or prevention) of herbivory, and (3) evaluation and confirmation among many conspecific adult trees across years. Here we present experimental evidence for a spatially explicit interaction between newly germinating seedlings of a Neotropical emergent tree, big-leaf mahogany (Swietenia macrophylla, Meliaceae), and caterpillars of a noctuid moth (Steniscadia poliophaea). In the understory of a southeastern Amazon forest, the proportion of attacks, leaf area lost, and seedling mortality due to this specialised herbivore peaked near Swietenia trees, but declined significantly with increasing distance from mature fruiting trees, as predicted by the Janzen–Connell hypothesis. We conclude that long-distance dispersal events (>50 m) provided an early survival advantage for Swietenia seedlings, and propose that the role of larval Lepidoptera as Janzen–Connell vectors may be underappreciated in tropical forests.     

Metal-catalyzed oxidation of the Werner syndrome protein causes loss of catalytic activities and impaired protein-protein interactions

Metal-catalyzed oxidation reactions target amino acids in the metal binding pocket of proteins. Such oxidation reactions generally result in either preferential degradation of the protein or accumulation of a catalytically inactive pool of protein with age. Consistently, levels of oxidized proteins have been shown to increase with age. The segmental, progeroid disorder Werner syndrome results from loss of the Werner syndrome protein (WRN). WRN is a member of the RecQ family of DNA helicases and possesses exonuclease and ATP-dependent helicase activities. Furthermore, each of the helicase and exonuclease domains of WRN contains a metal binding pocket. In this report we examined for metal-catalyzed oxidation of WRN in the presence of iron or copper. We found that WRN was oxidized in vitro by iron but not by copper. Iron-mediated oxidation resulted in the inhibition of both WRN helicase and exonuclease activities. Oxidation of WRN also inhibited binding to several known protein partners. In addition, we did not observe degradation of oxidized WRN by the 20 S proteasome in vitro. Finally, exposure of cells to hydrogen peroxide resulted in oxidation of WRN in vivo. Therefore, our results demonstrate that WRN undergoes metal-catalyzed oxidation in the presence of iron, and iron-mediated oxidation of WRN likely results in the accumulation of a catalytically inactive form of the protein, which may contribute to age-related phenotypes.     

Overexpression of MMP9 in macrophages attenuates pulmonary fibrosis induced by bleomycin

Pulmonary fibrosis is a common response to a variety of lung injuries, characterized by fibroblast/myofibroblast expansion and abnormal accumulation of extracellular matrix. An increased expression of matrix metalloprotease 9 (MMP9) in human and experimental lung fibrosis has been documented, but its role in the fibrotic response is unclear. We studied the effect of MMP9 overexpression in bleomycin-driven lung fibrosis using transgenic mice expressing human MMP9 in alveolar macrophages (hMMP9-TG). At 8 weeks post-bleomycin, the extent of fibrotic lesions and OH-proline content were significantly decreased in the TG mice compared to the WT mice. The decreased fibrosis in hMMP9-TG mice was preceded by a significant reduction of neutrophils and lymphocytes in bronchoalveolar lavage (BAL) at 1 and 4 weeks post-bleomycin, respectively, as well as by significantly less TIMP-1 than the WT mice. From a variety of cytokines/chemokines investigated, we found that BAL levels of insulin-like growth factor binding protein-3 (IGFBP3) as well as the immunoreactive protein in the lungs were significantly lower in hMMP9-TG mice compared with WT mice despite similar levels of gene expression. Using IGFBP-3 substrate zymography we found that BAL from TG mice at 1 week after bleomycin cleaved IGFBP-3. Further, we demonstrated that MMP9 degraded IGFBP-3 into lower molecular mass fragments. These findings suggest that increased activity of MMP9 secreted by alveolar macrophages in the lung microenvironment may have an antifibrotic effect and provide a potential mechanism involving IGFBP3 degradation.     

A meta‐analysis reveals a positive correlation between genetic diversity metrics and environmental status in the long‐lived seagrass Posidonia oceanica

The seagrass Posidonia oceanica is a key engineering species structuring coastal marine systems throughout much of the Mediterranean basin. Its decline is of concern, leading to the search for short‐ and long‐term indicators of seagrass health. Using ArcGIS maps from a recent, high‐resolution (1–4 km) modelling study of 18 disturbance factors affecting coastal marine systems across the Mediterranean (Micheli et al. 2013, http://globalmarine.nceas.ucsb.edu/mediterranean/ ), we tested for correlations with genetic diversity metrics (allelic diversity, genotypic/clonal diversity and heterozygosity) in a meta‐analysis of 56 meadows. Contrary to initial predictions, weak but significantly positive correlations were found for commercial shipping, organic pollution (pesticides) and cumulative impact. This counterintuitive finding suggests greater resistance and resilience of individuals with higher genetic and genotypic diversity under disturbance (at least for a time) and/or increased sexual reproduction under an intermediate disturbance model. We interpret the absence of low and medium levels of genetic variation at impacted locations as probable local extinctions of individuals that already exceeded their resistance capacity. Alternatively, high diversity at high‐impact sites is likely a temporal artefact, reflecting the mismatch with pre‐environmental impact conditions, especially because flowering and sexual recruitment are seldom observed. While genetic diversity metrics are a valuable tool for restoration and mitigation, caution must be exercised in the interpretation of correlative patterns as found in this study, because the exceptional longevity of individuals creates a temporal mismatch that may falsely suggest good meadow health status, while gradual deterioration of allelic diversity might go unnoticed.