Hemagglutinin fusion peptide mutants in model membranes: structural properties, membrane physical properties, and PEG-mediated fusion

While the importance of viral fusion peptides (e.g., hemagglutinin (HA) and gp41) in virus-cell membrane fusion is established, it is unclear how these peptides enhance membrane fusion, especially at low peptide/lipid ratios for which the peptides are not lytic. We assayed wild-type HA fusion peptide and two mutants, G1E and G13L, for their effects on the bilayer structure of 1,2-dioleoyl-3-sn-phosphatidylcholine/1,2-dioleoyl-3-sn-phosphatidylethanolamine/Sphingomyelin/Cholesterol (35:30:15:20) membranes, their structures in the lipid bilayer, and their effects on membrane fusion. All peptides bound to highly curved vesicles, but fusion was triggered only in the presence of poly(ethylene glycol). At low (1:200) peptide/lipid ratios, wild-type peptide enhanced remarkably the extent of content mixing and leakage along with the rate constants for these processes, and significantly enhanced the bilayer interior packing and filled the membrane free volume. The mutants caused no change in contents mixing or interior packing. Circular dichroism, polarized-attenuated total-internal-reflection Fourier-transform infrared spectroscopy measurements, and membrane perturbation measurements all conform to the inverted-V model for the structure of wild-type HA peptide. Similar measurements suggest that the G13L mutant adopts a less helical conformation in which the N-terminus moves closer to the bilayer interface, thus disrupting the V-structure. The G1E peptide barely perturbs the bilayer and may locate slightly above the interface. Fusion measurements suggest that the wild-type peptide promotes conversion of the stalk to an expanded trans-membrane contact intermediate through its ability to occupy hydrophobic space in a trans-membrane contact structure. While wild-type peptide increases the rate of initial intermediate and final pore formation, our results do not speak to the mechanisms for these effects, but they do leave open the possibility that it stabilizes the transition states for these events.     

HOW AND WHEN DO LAMBS RECOGNIZE THE BLEATS OF THEIR MOTHERS?

ABSTRACT

In domestic sheep Ovis aries, the mother and the young display a preferential bond for each other that relies on multimodal inter-individual recognition. Lambs show a preference for their own dam shortly after birth, and this is important for their survival. The role of acoustic cues in this early preference for the mother is not clear. The aim of the present work was to analyze the timing of acoustic recognition of the mother and to identify the physical parameters used in the recognition of maternal bleats by the lamb.

In a first study, we investigated the ability of lambs to discriminate between the bleats of their own mother and an alien equivalent mother in a two-choice test. Both ewes were hidden behind a canvas sheet and lambs were not allowed to approach the dams closer than 1 m, thus preventing visual as well as olfactory perception. Tests were conducted 12 hr, 24 hr or 48 hr after birth (n=19 or 20/group). An indication of vocal discrimination was already present at 24 hr and at 48 hr lambs spent significantly more time near their mother than near the alien dam.

In a second step, we investigated which physical parameters of the bleats were important for recognition. For this, we conducted playback experiments with modified bleats at two weeks postpartum. Ours results show that lambs pay attention to a combination of various time, energy and frequency parameters: timbre (distribution of energy within the spectrum), amplitude and frequency modulations appear to be the most important parameters encoding the individual signature.

We conclude that vocal recognition between the ewe and her lamb plays an important role in the display of preferential mother-young bond from very early on. Our studies also demonstrate that the encoding of the individual signature is not limited to the frequency domain but rather involves a multiparametric encoding process.     

Determination of molecular order in supported lipid membranes by internal reflection Fourier transform infrared spectroscopy.

When polarized internal reflection infrared spectroscopy is used to determine molecular order in supported lipid membranes, the results are critically dependent on the accuracy of assumptions made about the evanescent electric field amplitudes in the membrane. In this work, we examine several expressions used for calculating evanescent electric field amplitudes in supported lipid monolayers and bilayers, and test their validity by measuring the infrared dichroism of poly-gamma-benzyl-L-glutamate and poly-beta-benzyl-L-aspartate under conditions in which their molecular order is known. Our results indicate that treating such systems as a simple single interface between two semi-infinite bulk phases is more accurate than the commonly employed thin-film approximation. This implies that earlier conclusions about molecular order in supported lipid membranes may require substantial revision.     

Nile perch and the transformation of Lake Victoria

The transformation of Lake Victoria that began in 1980 followed the population explosion of Nile perch Lates niloticus, causing the apparent extirpation of 500+ endemic haplochromine species and dramatic physico-chemical changes. Officially introduced in 1962–1963, but present earlier, the reasons for the long delay before its population exploded are discussed. The hypothesis that it occurred only after the haplochromine decline is evaluated, but haplochromines declined only after the Nile perch expansion began. The sudden eutrophication of the lake was attributed to Nile perch, but evidence of eutrophication from 1950 onwards led some researchers to conclude that it was the result of climatic changes. We conclude that the haplochromine destruction disrupted the complex food webs that existed prior to the upsurge of Nile perch. The depletion of fish biomass by Nile perch may have been the source of extra phosphorus responsible for the eutrophication of the lake. After the Nile perch explosion in 1980 the fish population came to be dominated by only three species, but fisheries productivity increased at least 10-fold. Fishing has caused demographic changes in Nile perch, which may have allowed some haplochromine species to recover. The condition of the lake appears to have stabilised since 2000, partly because the fish biomass has risen to at least 2 × 10⁶ t, replacing the ‘lost’ biomass and restoring some ecosystem functioning.     

Parameters of proteome evolution from histograms of amino-acid sequence identities of paralogous proteins

Background  

The evolution of the full repertoire of proteins encoded in a given genome is mostly driven by gene duplications, deletions, and sequence modifications of existing proteins. Indirect information about relative rates and other intrinsic parameters of these three basic processes is contained in the proteome-wide distribution of sequence identities of pairs of paralogous proteins.     

Deuterated docosahexaenoic acid protects against oxidative stress and geographic atrophy‐like retinal degeneration in a mouse model with iron overload

Oxidative stress plays a central role in age‐related macular degeneration (AMD). Iron, a potent generator of hydroxyl radicals through the Fenton reaction, has been implicated in AMD. One easily oxidized molecule is docosahexaenoic acid (DHA), the most abundant polyunsaturated fatty acid in photoreceptor membranes. Oxidation of DHA produces toxic oxidation products including carboxyethylpyrrole (CEP) adducts, which are increased in the retinas of AMD patients. In this study, we hypothesized that deuterium substitution on the bis‐allylic sites of DHA in photoreceptor membranes could prevent iron‐induced retinal degeneration by inhibiting oxidative stress and lipid peroxidation. Mice were fed with either DHA deuterated at the oxidation‐prone positions (D‐DHA) or control natural DHA and then given an intravitreal injection of iron or control saline. Orally administered D‐DHA caused a dose‐dependent increase in D‐DHA levels in the neural retina and retinal pigment epithelium (RPE) as measured by mass spectrometry. At 1 week after iron injection, D‐DHA provided nearly complete protection against iron‐induced retinal autofluorescence and retinal degeneration, as determined by in vivo imaging, electroretinography, and histology. Iron injection resulted in carboxyethylpyrrole conjugate immunoreactivity in photoreceptors and RPE in mice fed with natural DHA but not D‐DHA. Quantitative PCR results were consistent with iron‐induced oxidative stress, inflammation, and retinal cell death in mice fed with natural DHA but not D‐DHA. Taken together, our findings suggest that DHA oxidation is central to the pathogenesis of iron‐induced retinal degeneration. They also provide preclinical evidence that dosing with D‐DHA could be a viable therapeutic strategy for retinal diseases involving oxidative stress.     

Isolation and partial characterization of prochymosin and chymosin from cat

Extracts of cat gastric mucosa contain a zymogen that after activation shows partial immunochemical identity with chymosin (EC 3.4.23.4) from calf. Cat prochymosin has been purified by column chromatography and gel filtration, and cat chymosin was obtained after acid activation of the zymogen. The enzyme showed the optimum of general proteolytic activity at pH 2.5. The amino acid compositions of cat prochymosin and chymosin were similar to those of the corresponding proteins from calf. The first 27 residues of both cat prochymosin and chymosin have been sequenced. Among these 54 positions only 13 differences have been observed between the proteins from cat and calf. The results support the hypothesis that the chymosins form a group of neonatal gastric proteases with high milk-clotting activity, but with such weak general proteolytic activity that postnatal uptake of IgG is not hindered.     

A reassessment of decreased amino acid accumulation by ehrlich ascites tumor cells in the presence of metabolic inhibitors

This study was undertaken to examine the mechanism by which metabolic inhibition reduces amino acid active transport in ehrlich ascites tumor cells. At 37 degrees C the metabolic inhibitor combination 0.1 mM 2,4-dinitrophenol (DNP) + 10 mM 2- deoxy-D-glucose (DOG) reduced the cell ATP concentration to 0.10- 0.15 mM in less than 5 min. This inhibition was associated with a 20.6 percent +/- 6.4 percent (SD) decrease in the initial influx of α-aminoisobutyric acid (AIB), and a two- to fourfold increase in the unidirectional efflux. These effects could be dissociated from changes in cell Na(+) or K(+) concentrations. Cells incubated to the steady state in 1.0-1.5 mM AIB showed an increased steady-state flux in the presence of DNP + DOG. Steady- state fluxes were consistent with trans-inhibition of AIB influx and trans-stimulation of efflux in control cells, but trans- stimulation of both fluxes in inhibited cells. In spite of the reduction of the cell ATP concentration to less than 0.15 mM and greatly reduced transmembrane concentration gradients of Na(+) and K(+), cells incubated to the steady state in the presence of the inhibitors still established an AIB distribution ration 13.8 +/- 2.6. The results are interpreted to indicate that a component of the reduction of AIB transport produced by metabolic inhibition is attributable to other actions in addition to the reduction of cation concentration gradients. Reduction of cell ATP alone is not responsible for the effects of metabolic inhibition, and both the transmembrane voltage and direct coupling to substrate oxidation via plasma-membrane-bound enzymes must be considered as possible energy sources for amino acid active transport.