The HA2 subunit of influenza hemagglutinin inserts into the target membrane prior to fusion

The interaction between influenza virus and target membrane lipids during membrane fusion was studied with hydrophobic photoactivatable probes. Two probes, the newly synthesized bisphospholipid diphosphatidylethanolamine trifluoromethyl [3H]phenyl diazirine and the phospholipid analogue 1-palmitoyl-2(11-[4-[3-(trifluoromethyl)diazirinyl]phenyl]-[2-3H]- undecanoyl]-sn-glycero-3-phosphocholine (Harter, C., B?chi, T., Semenza, G., and Brunner , J. (1988) Biochemistry 27, 1856-1864), were used. Both labeled the HA2 subunit of the virus at low pH. By measuring virus-liposome interactions at 0 degrees C, it could be demonstrated that HA2 was inserted into the target membrane prior to fusion. As we have recently demonstrated, at this temperature, exposure of the fusion peptide of HA2 takes place within 15 s after acidification, but fusion does not start for 4 min (Stegmann, T., White, J. M., and Helenius, A. (1990) EMBO J. 9, 4231-4241). HA2 was labeled at least 2 min before fusion. No labeling of the HA1 subunit was seen. These data indicate that fusion is triggered by a direct interaction of the HA2 subunit of a kinetic intermediate form of HA with the lipids of the target membrane. Most likely, it is the fusion peptide of HA2 that is inserted into the target membrane. Just before fusion, HA is thus an integral membrane protein in both membranes. In contrast, the bromelain-derived ectodomain of HA was labeled by 1-palmitoyl-2(11-[4-[3-(trifluoromethyl)diazirinyl]phenyl]- [2-3H]undecanoyl)-sn-glycerol-3-phosphocholine at low pH but not by diphosphatidylethanolamine trifluoromethyl [3H]phenyl diazirine. This indicates that insertion of the fusion peptide of the bromelain-derived ectodomain of HA into a membrane differs from that of viral HA during fusion.     

The purine transferase from Trypanosoma cruzi as a potential target for bisphosphonate-based chemotherapeutic compounds

We identified and tested bisphosphonates as inhibitors of a protozoan molecular target. Computational modeling studies demonstrated that these compounds are mimics of the natural substrate of the enzyme. The most potent bisphosphonates in vitro are pamidronate and risedronate, which inhibit the purine transferase from Trypanosoma cruzi in the micromolar range.     

Temperature-induced conformational switch in intestinal fatty acid binding protein (IFABP) revealing an alternative mode for ligand binding

IFABP is a small beta-barrel protein with a short helix-turn-helix motif near the N-terminus that is thought to participate in the regulation of the uptake and delivery of fatty acids. In a previous work, we detected by near UV circular dichroism a reversible conformational transition of this protein occurring between 35 and 50 degrees C in the absence of fatty acids. The addition of the natural ligand oleic acid prevents this phenomenon. In both cases, the overall structure of the beta-barrel is maintained. This thermal transition is also detected by the fluorescent probe bis-anilino naphthalene sulfonic acid (bisANS) but not by its monomer ANS. In the present work, we studied in detail the interaction of each compound with IFABP as a function of temperature and in the absence or in the presence of oleic acid. A contrasting behavior was observed for these probes: (i) IFABP is able to bind two molecules of bisANS but only one molecule of ANS and (ii) oleic acid can fully displace ANS but only partially bisANS. Three independent lines of evidence, namely, fluorescence spectroscopy, circular dichroism, and limited proteolysis, indicate that there is an equilibrium among different conformations of IFABP, which differ in the extent of flexibility of the helical domain. This equilibrium can be shifted by raising temperature. bisANS is able to probe a population of IFABP in an altered state, which is more susceptible to cleavage by clostripain as compared to the apo-form, whereas the conformation of IFABP bound to oleic acid is characteristically more ordered. These results highlight the idea of an enhanced flexibility exhibited by IFABP that bears importance on its transport function, supporting the role of a dynamic entry portal region for the fatty acid ligand.     

Amphibians in Eurasian otter Lutra lutra diet: osteological identification unveils hidden prey richness and male‐biased predation on anurans

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First record of Latonia gigantea (Anura,Alytidae) from the Iberian Peninsula

The single extant species of the anuran genus Latonia lives in Israel, but in the fossil record the genus is known mainly from Europe, spanning from the Oligocene to the early Pleistocene. Here we describe new remains of Latonia from the early to late Miocene of the Vallès-Penedès Basin (NE Iberian Peninsula), coming from the following localities: Sant Mamet (MN4), Sant Quirze and Trinxera del Ferrocarril (MN7+8), and Castell de Barberà, Can Poncic 1 and Can Llobateres 1 (MN9). Fossils from the late Aragonian and early Vallesian are attributed to Latonia gigantea mainly because of the morphology of the ornamentation that covers the maxillae. In turn, an ilium from Sant Mamet is not diagnostic at the specific level and is assigned only to the genus Latonia. The newly reported remains represent the first record of L. gigantea in the Iberian Peninsula, where Latonia was previously known by a single report of Latonia cf. ragei from Navarrete del Río (MN2) and remains from other localities unassigned to species. Moreover, the Vallès-Penedès remains represent one of the southernmost records of the species known thus far. The presence of Latonia in these localities confirms the humid and warm environment suggested by the recorded mammal fauna.     

Cutaneous venom of Bombina variegata pachypus (Amphibia, Anura): effects on the growth of the human HL 60 cell line.

The effects of Bombina variegata cutaneous venom (Bvv) on eukaryotic cell growth has been assessed employing the human leukaemic cell line HL 60, by liquid and agar semisolid cultures and 51Cr release assay. HL 60 cells growth is impaired by Bvv in a dose-dependent fashion in both culture systems. The arrest of proliferation requires a contact time lower than 3 min and it is not reversed by washing and culturing the cells in a Bvv-free medium. Similarly, an extremely short exposure time is needed to determine maximum 51Cr release. Neither the agar medium nor the fetal calf serum interact with Bvv effects, which, according to the above findings, must be regarded as cytolytic in nature. In both liquid and the agar-semisolid culture Bvv cytolytic activity half life is about 8 hr. The cytolytic properties of Bvv are thought to be part of the chemical defence system of amphibian skin.     

Action of growing degree days on the morphogenesis and physiological responses of calla lily

The calla lily (Zantedeschia aethiopica) is an ornamental plant with growing acceptance in the market place that shows thermal constraints during planting. This study aimed to analyse its biological cycle in growing degree days (GDD) to determine the best time for planting and production of flower stalks. Rhizomes were planted in pots during all four seasons, and the pots were kept in a greenhouse. Growth analysis and gas exchange measurements were performed every 30 days. Rhizomes planted in the fall and summer showed greater shoot growth, a larger root system and increased production of flower stalks, and they remained in the vegetative phase longer before the onset of flowering, attaining a lower GDD value. Rhizomes planted in winter and spring matured early, which resulted in a higher GDD value and a decrease or lack of flower stalk production during this period. Calla lily rhizomes planted in the fall and summer showed higher water-use efficiency, light-use efficiency and net photosynthesis. It is possible to characterise the stages of development according to gas exchange. The calla lily must be cultivated with an irradiance of 250–400 µmol m^?2 s^?1 in a temperature range of 25–28 °C during the initial growth phase (up to 1,000 GDD). Thereafter, the temperature range should be reduced to between 13 and 15 °C until 3,500 GDD are reached, which was found to enable increased flower stalk production.     

Δ98Δ, a minimalist model of antiparallel β‐sheet proteins based on intestinal fatty acid binding protein

The design of β‐barrels has always been a formidable challenge for de novo protein design. For instance, a persistent problem is posed by the intrinsic tendency to associate given by free edges. From the opposite standpoint provided by the redesign of natural motifs, we believe that the intestinal fatty acid binding protein (IFABP) framework allows room for intervention, giving rise to abridged forms from which lessons on β‐barrel architecture and stability could be learned. In this context, Δ98Δ (encompassing residues 29–126 of IFABP) emerges as a monomeric variant that folds properly, retaining functional activity, despite lacking extensive stretches involved in the closure of the β‐barrel. Spectroscopic probes (fluorescence and circular dichroism) support the existence of a form preserving the essential determinants of the parent structure, albeit endowed with enhanced flexibility. Chemical and physical perturbants reveal cooperative unfolding transitions, with evidence of significant population of intermediate species in equilibrium, structurally akin to those transiently observed in IFABP. The recognition by the natural ligand oleic acid exerts a mild stabilizing effect, being of a greater magnitude than that found for IFABP. In summary, Δ98Δ adopts a monomeric state with a compact core and a loose periphery, thus pointing to the nonintuitive notion that the integrity of the β‐barrel can indeed be compromised with no consequence on the ability to attain a native‐like and functional fold.