Pulmonary collectins modulate strain-specific influenza a virus infection and host responses

Collectins are secreted collagen-like lectins that bind, agglutinate, and neutralize influenza A virus (IAV) in vitro. Surfactant proteins A and D (SP-A and SP-D) are collectins expressed in the airway and alveolar epithelium and could have a role in the regulation of IAV infection in vivo. Previous studies have shown that binding of SP-D to IAV is dependent on the glycosylation of specific sites on the HA1 domain of hemagglutinin on the surface of IAV, while the binding of SP-A to the HA1 domain is dependent on the glycosylation of the carbohydrate recognition domain of SP-A. Here, using SP-A and SP-D gene-targeted mice on a common C57BL6 background, we report that viral replication and the host response as measured by weight loss, neutrophil influx into the lung, and local cytokine release are regulated by SP-D but not SP-A when the IAV is glycosylated at a specific site (N165) on the HA1 domain. SP-D does not protect against IAV infection with a strain lacking glycosylation at N165. With the exception of a small difference on day 2 after infection with X-79, we did not find any significant difference in viral load in SP-A(-/-) mice with either IAV strain, although small differences in the cytokine responses to IAV were detected in SP-A(-/-) mice. Mice deficient in both SP-A and SP-D responded to IAV similarly to mice deficient in SP-D alone. Since most strains of IAV currently circulating are glycosylated at N165, SP-D may play a role in protection from IAV infection.     

Transient production of recombinant proteins by Chinese hamster ovary cells using polyethyleneimine/DNA complexes in combination with microtubule disrupting anti-mitotic agents

We have developed a simple and robust transient expression system utilizing the 25 kDa branched cationic polymer polyethylenimine (PEI) as a vehicle to deliver plasmid DNA into suspension-adapted Chinese hamster ovary cells synchronized in G2/M phase of the cell cycle by anti-mitotic microtubule disrupting agents. The PEI-mediated transfection process was optimized with respect to PEI nitrogen to DNA phosphate molar ratio and the plasmid DNA mass to cell ratio using a reporter construct encoding firefly luciferase. Optimal production of luciferase was observed at a PEI N to DNA P ratio of 10:1 and 5 mug DNA 10(6) cells(-1). To manipulate transgene expression at mitosis, we arrested cells in G2/M phase of the cell cycle using the microtubule depolymerizing agent nocodazole. Using secreted human alkaline phosphatase (SEAP) and enhanced green fluorescent protein (eGFP) as reporters we showed that continued inclusion of nocodazole in cell culture medium significantly increased both transfection efficiency and reporter protein production. In the presence of nocodazole, greater than 90% of cells were eGFP positive 24 h post-transfection and qSEAP was increased almost fivefold, doubling total SEAP production. Under optimal conditions for PEI-mediated transfection, transient production of a recombinant chimeric IgG4 encoded on a single vector was enhanced twofold by nocodazole, a final yield of approximately 5 microg mL(-1) achieved at an initial viable cell density of 1 x 10(6) cells mL(-1). The glycosylation of the recombinant antibody at Asn297 was not significantly affected by nocodazole during transient production by this method.     

Less is more: high pass filtering, to remove up to 99% of the surface EMG signal power, improves EMG-based biceps brachii muscle force estimates.

It is generally assumed that raw surface EMG (sEMG) should be high pass filtered with cutoffs of 10-30 Hz to remove motion artifact before subsequent processing to estimate muscle force. The purpose of the current study was to explore the benefits of filtering out much of the raw sEMG signal when attempting to estimate accurate muscle forces. Twenty-five subjects were studied as they performed rapid static, anisotonic contractions of the biceps brachii. Biceps force was estimated (as a percentage of maximum) based on forces recorded at the wrist. An iterative approach was used to process the sEMG from the biceps brachii, using progressively greater high pass cutoff frequencies (20-440 Hz in steps of 30 Hz) with first and sixth order filters, as well as signal whitening, to determine the effects on the accuracy of EMG-based biceps force estimates. The results indicate that removing up to 99% of the raw sEMG signal power resulted in significant and substantial improvements in biceps force estimates. These findings challenge previous assumptions that the raw sEMG signal power between about 20 and 500 Hz should used when estimating muscle force. For the purposes of force prediction, it appears that a much smaller, high band of sEMG frequencies may be associated with force and the remainder of the spectrum has little relevance for force estimation.     

Antimitotic antifungal compound benomyl inhibits brain microtubule polymerization and dynamics and cancer cell proliferation at mitosis, by binding to a novel site in tubulin

The antifungal agent benomyl [methyl-1-(butylcarbamoyl)-2-benzimidazolecarbamate] is used throughout the world against a wide range of agricultural fungal diseases. In this paper, we investigated the interaction of benomyl with mammalian brain tubulin and microtubules. Using the hydrophobic fluorescent probe 1-anilinonaphthalene-8-sulfonic acid, benomyl was found to bind to brain tubulin with a dissociation constant of 11.9 +/- 1.2 microM. Further, benomyl bound to at a novel site, distinct from the well-characterized colchicine and vinblastine binding sites. Benomyl altered the far-UV circular dichroism spectrum of tubulin and reduced the accessibility of its cysteine residues to modification by 5,5'-dithiobis-2-nitrobenzoic acid, indicating that benomyl binding to tubulin induces a conformational change in the tubulin. Benomyl inhibited the polymerization of brain tubulin into microtubules, with 50% inhibition occurring at a concentration of 70-75 microM. Furthermore, it strongly suppressed the dynamic instability behavior of individual brain microtubules in vitro as determined by video microscopy. It reduced the growing and shortening rates of the microtubules but did not alter the catastrophe or rescue frequencies. The unexpected potency of benomyl against mammalian microtubule polymerization and dynamics prompted us to investigate the effects of benomyl on HeLa cell proliferation and mitosis. Benomyl inhibited proliferation of the cells with an IC(50) of 5 microM, and it blocked mitotic spindle function by perturbing microtubule and chromosome organization. The greater than expected actions of benomyl on mammalian microtubules and mitosis together with its relatively low toxicity suggest that it might be useful as an adjuvant in cancer chemotherapy.     

Stability and Persistence of Alpine Stream Macroinvertebrate Communities and the Role of Physicochemical Habitat Variables

Macroinvertebrate communities in alpine streams have rarely been examined over more than two consecutive years or at sub-monthly temporal resolution during the summer melt season, in relation to a range of stream physicochemical habitat measurements. This paper addresses these research gaps by investigating the inter- (late melt season, 1996–2003) and intra-annual (bi-weekly; June–September, 2002–2003) community compositional stability and persistence of three alpine streams fed from different water sources (snow, glaciers and groundwater) in the Taillon–Gabiétous catchment, French Pyrénées. Inter-annual community stability and persistence decreased from 1996 to 2003; however, groundwater stream communities changed less than those in the main glacial stream. Intra-annual community stability varied spatially and temporally, particularly in relation to water quality variables (water temperature and suspended sediment concentration); water quantity (stream discharge) was less important perhaps due to taxa possessing adaptations to flow variability. The 15 most abundant taxa were consistently more stable and persistent than the entire stream community suggesting a common pool of taxa in these streams. Overall, the results support the view that streams originating from different alpine water sources are characterised by distinct benthic macroinvertebrate assemblages, and demonstrate the value of sampling at nested temporal scales (inter-annual to bi-weekly) for understanding how these stream ecosystems function.     

Response of Litter Decomposition to Simulated N Deposition in Disturbed, Rehabilitated and Mature Forests in Subtropical China

The response of decomposition of litter for the dominant tree species in disturbed (pine), rehabilitated (pine and broadleaf mixed) and mature (monsoon evergreen broadleaf) forests in subtropical China to simulated N deposition was studied to address the following hypothesis: (1) litter decomposition is faster in mature forest (high soil N availability) than in rehabilitated/disturbed forests (low soil N availability); (2) litter decomposition is stimulated by N addition in rehabilitated and disturbed forests due to their low soil N availability; (3) N addition has little effect on litter decomposition in mature forest due to its high soil N availability. The litterbag method (a total of 2880 litterbags) and N treatments: Control-no N addition, Low-N: −5 g N m⁻² y⁻¹, Medium-N: −10 g N m⁻² y⁻¹, and High-N: −15 g N m⁻² y⁻¹, were employed to evaluate decomposition_. Results indicated that mature forest, which has likely been N saturated due to both long-term high N deposition in the region and the age of the ecosystem, had the highest litter decomposition rate, and exhibited no significant positive and even some negative response to nitrogen additions. However, both disturbed and rehabilitated forests, which are still N limited due to previous land use history, exhibited slower litter decomposition rates with significant positive effects from nitrogen additions. These results suggest that litter decomposition and its responses to N addition in subtropical forests of China vary depending on the nitrogen status of the ecosystem.     

Carbonyldiphosphonate, a selective inhibitor of mammalian DNA polymerase delta

Twenty-three pyrophosphate analogues were screened as inhibitors of proliferating cell nuclear antigen independent DNA polymerase delta (pol delta) derived from calf thymus. Carbonyldiphosphonate (COMDP), also known as alpha-oxomethylenediphosphonate, inhibited pol delta with a potency (Ki = 1.8 microM) 20 times greater than that displayed for DNA polymerase alpha (pol alpha) derived from the same tissue. Characterization of the mechanism of inhibition of pol delta indicated that COMDP competed with the dNTP specified by the template and was not competitive with the template-primer. In the case of pol alpha, COMDP did not compete with either the dNTP or the polynucleotide substrate. COMDP inhibited the 3'----5' exonuclease activity of pol delta weakly, displaying an IC50 greater than 1 mM.     

Dectin-1: a signalling non-TLR pattern-recognition receptor

Dectin-1 is a natural killer (NK)-cell-receptor-like C-type lectin that is thought to be involved in innate immune responses to fungal pathogens. This transmembrane signalling receptor mediates various cellular functions, from fungal binding, uptake and killing, to inducing the production of cytokines and chemokines. These activities could influence the resultant immune response and can, in certain circumstances, lead to autoimmunity and disease. As I discuss here, understanding the molecular mechanisms behind these functions has revealed new concepts, including collaborative signalling with the Toll-like receptors (TLRs) and the use of spleen tyrosine kinase (SYK), that have implications for the role of other non-TLR pattern-recognition receptors in immunity.     

Effects of irradiance and temperature on photosynthesis in C3, C4 and C3/C4 Panicum species

Species in the Laxa and Grandia groups of the genus Panicum are adapted to low, wet areas of tropical and subtropical America. Panicum milioides is a species with C₃ photosynthesis and low apparent photorespiration and has been classified as a C₃/C₄ intermediate. Other species in the Laxa group are C₃ with normal photorespiration. Panicum prionitis is a C₄ species in the Grandia group. Since P. milioides has some leaf characteristics intermediate to C₃ and C₄ species, its photosynthetic response to irradiance and temperature was compared to the closely related C₃ species, P. laxum and P. boliviense and to P. prionitis. The response of apparent photosynthesis to irradiance and temperature was similar to that of P. laxum and P. boliviense, with saturation at a photosynthetic photo flux density of about 1 mmol m^-2 s^-1 at 30°C and temperature optimum near 30°C. In contrast, P. prionitis showed no light saturation up to 2 mmol m^-2 s^-1 and an optimum temperature near 40°C. P. milioides exhibited low CO₂ loss into CO₂-free air in the light and this loss was nearly insensitive to temperature. Loss of CO₂ in the light in the C₃ species, P. laxum and P. boliviense, was several-fold higher than in P. milioides and increased 2- to 5-fold with increases in temperature from 10 to 40°C. The level of dark respiration and its response to temperature were similar in all four Panicum species examined. It is concluded that the low apparent photorespiration in P. milioides does not influence its response of apparent photosynthesis to irradiance and temperature in comparison to closely related C₃ Panicum species.Abbreviations AP apparent photosynthesis - I CO₂ compensation point - gl leaf conductance; gm, mesophyll conductance - PPFD photosynthetic photon flux density - PR apparent photorespiration rate - RuBPC sibulose bisphosphate carboxylase     

Localization of a human gene homologous to the PrP gene on the p arm of chromosome 20 and detection of PrP-related antigens in normal human brain

Infectious fractions prepared from scrapie-infected hamster brains contain a protein, PrP 27-30, which shares antigenic determinants with polypeptides found in similarly prepared fractions from patients with Creutzfeldt-Jakob disease. cDNA sequences encoding the hamster PrP 27-30 identified homologous sequences in the human genome as well as in normal human brain mRNA preparations. Antibodies raised against the mouse PrP's identified antigenically related peptides in both normal hamster and human brain as well as in scrapie-infected hamster brain and CJD-affected human brain. By using in situ hybridization we localized the homologous human genomic sequences on the short arm of chromosome 20. Our results indicate that the reportedly unique proteins detected in human CJD preparations derive from normal human gene products.