Synergistic effects of food and predators on annual reproductive success in song sparrows

The behaviour literature is full of studies showing that animals in every taxon balance the probability of acquiring food with the risk of being preyed upon. While interactions between food and predators clearly operate at an individual scale, population-scale studies have tended to focus on only one factor at a time. Consequently, interactive (or 'synergistic') effects of food and predators on whole populations have only twice before been experimentally demonstrated in mammals. We conducted a 2 x 2 experiment to examine the joint effects of food supply and predator pressure on the annual reproductive success of song sparrows (Melospiza melodia). Our results show that these two factors do not operate in an additive way, but instead have a synergistic effect on reproduction. Relative to controls, sparrows reared 1.1 more young when food was added and 1.3 more when predator pressure was low. When these treatments were combined 4.0 extra young were produced, almost twice as many as expected from an additive model. These results are a cause for optimism for avian conservation because they demonstrate that remedial actions, aimed at simultaneously augmenting food and reducing predators, can produce dramatic increases in reproductive success.     

Expression of foreign LpxA acyltransferases in Neisseria meningitidis results in modified lipid A with reduced toxicity and retained adjuvant activity

A major problem in the development of vaccines against Gram-negative bacteria is the endotoxic -activity of lipopolysaccharide (LPS), which is determined by its lipid A moiety. Nevertheless, LPS would be an interesting vaccine component because of its immune-stimulating properties. In the present study, we have changed the fatty acid composition of Neisseria meningitidis LPS by replacing the lpxA gene of strain H44/76 with the Escherichia coli or Pseudomonas aeruginosa homologue. The majority of the O-linked 3-OH C12 in N. meningitidis lipid A was replaced by 3-OH C14 (strain HA01E) and 3-OH C10 (strain HA25P) respectively. Both strains, but most notably strain HA01E, had reduced amounts of LPS compared with the wild-type strain. In addition, growth was severely impaired for HA01E. The major outer membrane proteins were expressed normally. Outer membrane complexes of both strains normalized on their LPS content showed a 10-fold reduction in their ability to induce tumour necrosis factor (TNF)-alpha. Immunogenicity studies in BALB/c mice revealed that the adjuvant activity of the LPS was not affected. Thus, the replacement of the O-linked fatty acids in meningococcal lipid A results in immunogenic outer membranes with reduced endotoxic activity, more suitable for use in outer membrane vesicle vaccines.     

Evaluation of respiratory and cutaneous doses of chlorothalonil during re-entry in greenhouses

Five female workers were monitored for 5 consecutive days during re-entry into a greenhouse containing ornamental plants. Skin contamination (excluding hands) was evaluated with nine pads of filter paper placed on the skin. Hand contamination was assessed by washing with 95% ethanol. Respiratory exposure was evaluated by personal air sampling. The respiratory dose was based on a lung ventilation of 15 l/min. The doses absorbed were estimated assuming 10% skin absorption and 100% lung retention. Dislodgeable foliar residue was determined on days of re-entry to evaluate the decay of chlorothalonil. Chlorothalonil was analysed in the different matrices by GC-MS. Respiratory exposure was less than skin contamination, being 11.4+/-5.1% (mean+/-SD) of total exposure. The estimated total absorbed dose did not exceed the acceptable daily intake of 0.03 mg/kg body mass. The hands and unexposed skin of all workers were always found to be contaminated. Greater precautions are therefore needed to reduce skin exposure (clean gloves and suitable clean clothing every day).     

Conformation and self-assembly of a nystatin nitrobenzoxadiazole derivative in lipid membranes

Nystatin is a polyene (tetraene) macrolide antibiotic presenting antifungal activity that acts at the cellular membrane level. In the present study, we report the interaction of this antibiotic labelled at its amine group with 7-nitrobenz-2-oxa-1,3-diazole (NBD-Nys) with sterol-free and ergosterol- and cholesterol-containing 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) large unilamellar vesicles (LUV). The mean tetraene to NBD separating distance determined from fluorescence energy transfer measurements increased from 18 to 25.6 A upon antibiotic binding to the lipid vesicles, indicating that the monomeric labelled antibiotic adopts a more extended conformation in its lipid-bound state than in aqueous solution. The oligomeric state of membrane-bound NBD-Nys was also studied by resonance energy homotransfer between the NBD fluorophores. The decrease measured in its steady state fluorescence anisotropy upon increasing the surface concentration of the NBD-Nys is shown to be consistent with a random distribution of molecules on the surface of the liposomes. This data contradicts the sharp increase measured for nystatin mean fluorescence lifetime in the presence of 10 mol% ergosterol-containing POPC LUV within the same antibiotic concentration range and which is known to report nystatin oligomerization in the lipid vesicles. Therefore, we conclude that the amine group of nystatin is an essential requisite for the supramolecular organization/pore formation of this antibiotic.     

G-quadruplex DNA recognition by nucleophosmin: New insights from protein dissection

Background

Nucleophosmin (NPM1, B23) is a multifunctional protein that is involved in a variety of fundamental biological processes. NPM1/B23 deregulation is implicated in the pathogenesis of several human malignancies. This protein exerts its functions through the interaction with a multiplicity of biological partners. Very recently it is has been shown that NPM1/B23 specifically recognizes DNA G-quadruplexes through its C-terminal region.

Methods

Through a rational dissection approach of protein here we show that the intrinsically unfolded regions of NPM1/B23 significantly contribute to the binding of c-MYC G-quadruplex motif. Interestingly, the analysis of the ability of distinct NPM1/B23 fragments to bind this quadruplex led to the identifications of distinct NPM1/B23-based peptides that individually present a high affinity for this motif.

Results

These results suggest that the tight binding of NPM1/B23 to the G-quadruplex is achieved through the cooperation of both folded and unfolded regions that are individually able to bind it. The dissection of NPM1/B23 also unveils that its H1 helix is intrinsically endowed with an unusual thermal stability.

Conclusions

These findings have implications for the unfolding mechanism of NPM1/B23, for the G-quadruplex affinity of the different NPM1/B23 isoforms and for the design of peptide-based molecules able to interact with this DNA motif.

General observation

This study sheds new light in the molecular mechanism of the complex NPM1/G-quadruplex involved in acute myeloid leukemia (AML) disease.     

Morphological characterization of a human glioma cell l ine

A human malignant continuous cell line, named NG97, was recently established in our laboratory. This cell line has been serially subcultured over 100 times in standard culture media presenting no sign of cell senescence. The NG97 cell line has a doubling time of about 24 h. Immunocytochemical analysis of glial markers demonstrated that cells are positive for glial fibrillary acidic protein (GFAP) and S-100 protein, and negative for vimentin. Under phase-contrast microscope, cultures of NG97 showed cells with variable morphological features, such as small rounded cells, fusiform cells (fibroblastic-like cells), and dendritic-like cells. However, at confluence just small rounded and fusiform cells can be observed. At scanning electron microscopy (SEM) small rounded cells showed heterogeneous microextentions, including blebs and filopodia. Dendritic-like cells were flat and presented extensive prolongations, making several contacts with small rounded cells, while fusiform cells presented their surfaces dominated by microvilli.     

Dual roles for lipolysis and oxidation in peroxisome proliferation-activator receptor responses to electronegative low density lipoprotein

Low density lipoprotein (LDL) exists in various forms that possess unique characteristics, including particle content and metabolism. One circulating subfraction, electronegative LDL (LDL(-)), which is increased in familial hypercholesterolemia and diabetes, is implicated in accelerated atherosclerosis. Cellular responses to LDL(-) remain poorly described. Here we demonstrate that LDL(-) increases tumor necrosis factor alpha (TNFalpha)-induced inflammatory responses through NF kappa B and AP-1 activation with corresponding increases in vascular cell adhesion molecule-1 (VCAM1) expression. LDL receptor overexpression increased these effects. In contrast, exposing LDL(-) to the key lipolytic enzyme lipoprotein lipase (LPL) reversed these responses, inhibiting VCAM1 below levels seen with TNFalpha alone. LPL is known to act on lipoproteins to generate endogenous peroxisomal proliferator-activated receptor alpha (PPAR alpha) ligand, thus limiting inflammation. These responses varied according to the lipoprotein substrate triglyceride content (very low density lipoprotein > LDL > high density lipoprotein). The PPAR alpha activation seen with LDL, however, was disproportionately high. We show here that MUT LDL activates PPAR alpha to an extent proportional to its LDL(-) content. As compared with LDL(-) alone, LPL-treated LDL(-) increased PPAR alpha activation 20-fold in either cell-based transfection or radioligand displacement assays. LPL-treated LDL(-) suppressed NF kappa B and AP-1 activation, increasing expression of the PPAR alpha target gene I kappa B alpha, although only in the genetic presence of PPAR alpha and with intact LPL hydrolysis. Mass spectrometry reveals that LPL-treatment of either LDL or LDL(-) releases hydroxy-octadecadienoic acids (HODEs), potent PPAR alpha activators. These findings suggest LPL-mediated PPAR alpha activation as an alternative catabolic pathway that may limit inflammatory responses to LDL(-).     

Bacteriophage T4-Directed DNA Synthesis in Toluene-Treated Cells

DNA synthesis has been studied in T4-infected Escherichia coli cells made permeable to nucleotides by treatment with toluene. The rate of incorporation of labeled deoxyribonucleoside triphosphates into DNA at various times after infection is proportional to the in vivo rate. This in vitro incorporation is dependent on all four deoxyribonucleoside triphosphates (5-hydroxymethyldeoxy-cytidine triphosphate can substitute for dCTP) and Mg(2+). It is stimulated by rATP, partially inhibited by pancreatic DNase, and abolished by N-ethylmalei-mide and 1-beta-d-arabinofuranosylcytosine triphosphate. T4 amber DO (DNA negative) and temperature-sensitive DO mutants under nonpermissive conditions of infection fail to induce DNA synthesis in vitro. The synthesizing activity is intracellular and the DNA product is exclusively T4 DNA. The in vitro synthesis proceeds in a discontinuous manner involving synthesis and subsequent joining of small DNA fragments (about 10S in alkaline sucrose gradients) into larger molecules predominantly one-half the length of mature T4 DNA. No restriction of C-containing or nonglucosylated HMC-containing T4 DNA product is observed in this system.     

Phosphodiesterase-4 inhibition alters gene expression and improves isoniazid-mediated clearance of Mycobacterium tuberculosis in rabbit lungs

Tuberculosis (TB) treatment is hampered by the long duration of antibiotic therapy required to achieve cure. This indolent response has been partly attributed to the ability of subpopulations of less metabolically active Mycobacterium tuberculosis (Mtb) to withstand killing by current anti-TB drugs. We have used immune modulation with a phosphodiesterase-4 (PDE4) inhibitor, CC-3052, that reduces tumor necrosis factor alpha (TNF-α) production by increasing intracellular cAMP in macrophages, to examine the crosstalk between host and pathogen in rabbits with pulmonary TB during treatment with isoniazid (INH). Based on DNA microarray, changes in host gene expression during CC-3052 treatment of Mtb infected rabbits support a link between PDE4 inhibition and specific down-regulation of the innate immune response. The overall pattern of host gene expression in the lungs of infected rabbits treated with CC-3052, compared to untreated rabbits, was similar to that described in vitro in resting Mtb infected macrophages, suggesting suboptimal macrophage activation. These alterations in host immunity were associated with corresponding down-regulation of a number of Mtb genes that have been associated with a metabolic shift towards dormancy. Moreover, treatment with CC-3052 and INH resulted in reduced expression of those genes associated with the bacterial response to INH. Importantly, CC-3052 treatment of infected rabbits was associated with reduced ability of Mtb to withstand INH killing, shown by improved bacillary clearance, from the lungs of co-treated animals compared to rabbits treated with INH alone. The results of our study suggest that changes in Mtb gene expression, in response to changes in the host immune response, can alter the responsiveness of the bacteria to antimicrobial agents. These findings provide a basis for exploring the potential use of adjunctive immune modulation with PDE4 inhibitors to enhance the efficacy of existing anti-TB treatment.