Intracytoplasmic trapping of influenza virus by a lipophilic derivative of aglycoristocetin

We report on a new anti-influenza virus agent, SA-19, a lipophilic glycopeptide derivative consisting of aglycoristocetin coupled to a phenylbenzyl-substituted cyclobutenedione. In Madin-Darby canine kidney cells infected with influenza A/H1N1, A/H3N2, or B virus, SA-19 displayed a 50% antivirally effective concentration of 0.60 μM and a selectivity index (ratio of cytotoxic versus antiviral concentration) of 112. SA-19 was 11-fold more potent than unsubstituted aglycoristocetin and was active in human and nonhuman cell lines. Virus yield at 72 h p.i. was reduced by 3.6 logs at 0.8 μM SA-19. In contrast to amantadine and oseltamivir, SA-19 did not select for resistance upon prolonged virus exposure. SA-19 was shown to inhibit an early postbinding step in virus replication. The compound had no effect on hemagglutinin (HA)-mediated membrane fusion in an HA-polykaryon assay and did not inhibit the low-pH-induced refolding of the HA in a tryptic digestion assay. However, a marked inhibitory effect on the transduction exerted by retroviral pseudoparticles carrying an HA or vesicular stomatitis virus glycoprotein (VSV-G) fusion protein was noted, suggesting that SA-19 targets a cellular factor with a role in influenza virus and VSV entry. Using confocal microscopy with antinucleoprotein staining, SA-19 was proven to completely prevent the influenza virus nuclear entry. This virus arrest was characterized by the formation of cytoplasmic aggregates. SA-19 appeared to disturb the endocytic uptake and trap the influenza virus in vesicles distinct from early, late, or recycling endosomes. The aglycoristocetin derivative SA-19 represents a new class of potent and broad-acting influenza virus inhibitors with potential clinical relevance.     

A pharmacological study of NLP-12 neuropeptide signaling in free-living and parasitic nematodes

NLP-12a and b have been identified as cholecystokinin/sulfakinin-like neuropeptides in the free-living nematode Caenorhabditis elegans. They are suggested to play an important role in the regulation of digestive enzyme secretion and fat storage. This study reports on the identification and characterization of an NLP-12-like peptide precursor gene in the rat parasitic nematode Strongyloides ratti. The S. ratti NLP-12 peptides are able to activate both C. elegans CKR-2 receptor isoforms in a dose-dependent way with affinities in the same nanomolar range as the native C. elegans NLP-12 peptides. The C-terminal RPLQFamide sequence motif of the NLP-12 peptides is perfectly conserved between free-living and parasitic nematodes. Based on systemic amino acid replacements the Arg-, Leu- and Phe- residues appear to be critical for high-affinity receptor binding. Finally, a SAR analysis revealed the essential pharmacophore in C. elegans NLP-12b to be the pentapeptide RPLQFamide.     

Is the onset of senescence in leaf cells of intact plants due to low or high sugar levels?

This review examines the hypotheses that developmental programmed cell death in leaves is mediated (i) by sugar starvation in the leaf cells or (ii) by sugar accumulation in these cells. Experimental evidence for both hypotheses is critically discussed and found to be lacking. For example, some papers show that sugars prevent senescence of cut leaves placed in darkness, and prevent low sugar levels in the leaves. In these tests, the sugars seem to replace photosynthesis, hence the results have little relevance to leaf senescence in intact plants in the light. Low nitrogen nutrition and high light results in earlier senescence than the low nitrogen treatment alone. This is accompanied by high sugar levels in the leaves. The results have led to the idea that accumulation of sugars is the cause of the additional effect, or more generally, that sugar accumulation is always the direct cause of leaf senescence. Results from over-expressing, or knocking out, hexokinase genes tend to support the high sugar hypothesis, but pleiotropic effects confound this conclusion. In addition, several experiments show the effects of treatments on senescence without the increase in leaf sugar levels. Nonetheless, sugar levels are usually measured in whole leaves. Such an overall level does not reflect the differences in the onset of senescence between tissues and cells, and can therefore not be used as an argument for or against either of the two hypotheses. It is argued that future work should determine the time line of the concentrations of various sugars in various cells and cellular compartments, in relation to senescence processes in the same cells. Taken together, the data are not decisive. It is possible that neither of the two hypotheses is correct.     

Stress-relaxation of human patellar articular cartilage in unconfined compression: prediction of mechanical response by tissue composition and structure

Mechanical properties of articular cartilage are controlled by tissue composition and structure. Cartilage function is sensitively altered during tissue degeneration, in osteoarthritis (OA). However, mechanical properties of the tissue cannot be determined non-invasively. In the present study, we evaluate the feasibility to predict, without mechanical testing, the stress-relaxation response of human articular cartilage under unconfined compression. This is carried out by combining microscopic and biochemical analyses with composition-based mathematical modeling. Cartilage samples from five cadaver patellae were mechanically tested under unconfined compression. Depth-dependent collagen content and fibril orientation, as well as proteoglycan and water content were derived by combining Fourier transform infrared imaging, biochemical analyses and polarized light microscopy. Finite element models were constructed for each sample in unconfined compression geometry. First, composition-based fibril-reinforced poroviscoelastic swelling models, including composition and structure obtained from microscopical and biochemical analyses were fitted to experimental stress-relaxation responses of three samples. Subsequently, optimized values of model constants, as well as compositional and structural parameters were implemented in the models of two additional samples to validate the optimization. Theoretical stress-relaxation curves agreed with the experimental tests (R=0.95-0.99). Using the optimized values of mechanical parameters, as well as composition and structure of additional samples, we were able to predict their mechanical behavior in unconfined compression, without mechanical testing (R=0.98). Our results suggest that specific information on tissue composition and structure might enable assessment of cartilage mechanics without mechanical testing.     

Effects of nutrients and fish on periphyton and plant biomass across a European latitudinal gradient

Replicated, factorial mesocosm experiments were conducted across Europe to study the effects of nutrient enrichment and fish density on macrophytes and on periphyton chlorophyll a (chl-a) with regard to latitude. Periphyton chl-a densities and plant decline were significantly related to nutrient loading in all countries. Fish effects were significant in a few sites only, mostly because of their contribution to the nutrient pool. A saturation-response type curve in periphyton chl-a with nutrients was found, and northern lakes achieved higher densities than southern lakes. Nutrient concentration and phytoplankton chl-a necessary for a 50% plant reduction followed a latitudinal gradient. Total phosphorus values for 50% plant disappearance were similar from Sweden (0.27 mg L⁻¹) to northern Spain (0.35 mg L⁻¹), but with a sharp increase in southern Spain (0.9 mg L⁻¹). Planktonic chl-a values for 50% plant reduction increased monotonically from Sweden (30 μg L⁻¹) to València (150 μg L⁻¹). Longer plant growing-season, higher light intensities and temperature, and strong water-level fluctuations characteristic of southern latitudes can lead to greater persistence of macrophyte biomass at higher turbidities and nutrient concentration than in northern lakes. Results support the evidence that latitudinal differences in the functioning of shallow lakes should be considered in lake management and conservation policies.     

Powerful skin cancer protection by a CPD-photolyase transgene

BACKGROUND: The high and steadily increasing incidence of ultraviolet-B (UV-B)-induced skin cancer is a problem recognized worldwide. UV introduces different types of damage into the DNA, notably cyclobutane pyrimidine dimers (CPDs) and (6-4) photoproducts (6-4PPs). If unrepaired, these photolesions can give rise to cell death, mutation induction, and onset of carcinogenic events, but the relative contribution of CPDs and 6-4PPs to these biological consequences of UV exposure is hardly known. Because placental mammals have undergone an evolutionary loss of photolyases, repair enzymes that directly split CPDs and 6-4PPs into the respective monomers in a light-dependent and lesion-specific manner, they can only repair UV-induced DNA damage by the elaborate nucleotide excision repair pathway. RESULTS: To assess the relative contribution of CPDs and 6-4PPs to the detrimental effects of UV light, we generated transgenic mice that ubiquitously express CPD-photolyase, 6-4PP-photolyase, or both, thereby allowing rapid light-dependent repair of CPDs and/or 6-4PPs in the skin. We show that the vast majority of (semi)acute responses in the UV-exposed skin (i.e., sunburn, apoptosis, hyperplasia, and mutation induction) can be ascribed to CPDs. Moreover, CPD-photolyase mice, in contrast to 6-4PP-photolyase mice, exhibit superior resistance to sunlight-induced tumorigenesis. CONCLUSIONS: Our data unequivocally identify CPDs as the principal cause of nonmelanoma skin cancer and provide genetic evidence that CPD-photolyase enzymes can be employed as effective tools to combat skin cancer.