Transcript profiling of papaya fruit reveals differentially expressed genes associated with fruit ripening

Papaya (Carica papaya L) fruit has a short shelf life due to fast ripening induced by ethylene, but little is known about the genetic control of ripening and attributes of fruit quality. Therefore, we identified ripening-related genes affected by ethylene using cDNA-AFLP (Amplified Fragment Length Polymorphism of cDNA). Transcript profiling of non-induced and ethylene-induced fruit samples was performed, and 71 differentially expressed genes were identified. Among those genes some involved in ethylene biosynthesis, regulation of transcription, and stress responses or plant defence were found (heat shock proteins, polygalacturonase-inhibiting protein, and acyl-CoA oxidases). Several transcription factors were isolated, and except for a 14-3-3 protein, an AP2 domain-containing factor, a salt-tolerant zinc finger protein, and a suppressor of PhyA-105 1, most of them were negatively affected by ethylene, including fragments of transcripts similar to VRN1, and ethylene responsive factors (ERF). With respect to fruit quality, genes related to cell wall structure or metabolism, volatiles or pigment precursors, and vitamin biosynthesis were also found. (C) 2010 Elsevier Ireland Ltd. All rights reserved.

A recombinant vaccine based on domain II of Plasmodium vivax Apical Membrane Antigen 1 induces high antibody titres in mice

The Apical Membrane Antigen 1 (AMA-1) is considered a promising candidate for development of a malaria vaccine against asexual stages of Plasmodium. We recently identified domain II (DII) of Plasmodium vivax AMA-1 (PvAMA-1) as a highly immunogenic region recognised by IgG antibodies present in many individuals during patent infection with P. vivax. The present study was designed to evaluate the immunogenic properties of a bacterial recombinant protein containing PvAMA-1 DII. To accomplish this, the recombinant protein was administered to mice in the presence of each of the following six adjuvants: Complete/Incomplete Freund`s Adjuvant (CFA/IFA), aluminium hydroxide (Alum), Quil A, QS21 saponin, CpG-ODN 1826 and TiterMax. We found that recombinant DII was highly immunogenic in BALB/c mice when administered in the presence of any of the tested adjuvants. Importantly, we show that DII-specific antibodies recognised the native AMA-1 protein expressed on the surface of P. vivax merozoites isolated from the blood of infected patients. These results demonstrate that a recombinant protein containing PvAMA-1 DII is immunogenic when administered in different adjuvant formulations, and indicate that this region of the AMA-1 protein should continue to be evaluated as part of a subunit vaccine against vivax malaria. (C) 2010 Elsevier Ltd. All rights reserved.

The anti-cancer agent guttiferone-A permeabilizes mitochondrial membrane: Ensuing energetic and oxidative stress implications

Guttiferone-A (GA) is a natural occurring polyisoprenylated benzophenone with cytotoxic action in vitro and anti-tumor action in rodent models. We addressed a potential involvement of mitochondria in GA toxicity (1-25 mu M) toward cancer cells by employing both hepatic carcinoma (HepG2) cells and succinate-energized mitochondria, isolated from rat liver. In HepG2 cells GA decreased viability, dissipated mitochondrial membrane potential, depleted ATP and increased reactive oxygen species (ROS) levels. In isolated rat-liver mitochondria GA promoted membrane fluidity increase, cyclosporine A/EGTA-insensitive membrane permeabilization, uncoupling (membrane potential dissipation/state 4 respiration rate increase), Ca(2+) efflux, ATP depletion, NAD(P)H depletion/oxidation and ROS levels increase. All effects in cells, except mitochondrial membrane potential dissipation, as well as NADPH depletion/oxidation and permeabilization in isolated mitochondria, were partly prevented by the a NAD(P)H regenerating substrate isocitrate. The results suggest the following sequence of events: 1) GA interaction with mitochondrial membrane promoting its permeabilization; 2) mitochondrial membrane potential dissipation; 3) NAD(P)H oxidation/depletion due to inability of membrane potential-sensitive NADP(+) transhydrogenase of sustaining its reduced state; 4) ROS accumulation inside mitochondria and cells; 5) additional mitochondrial membrane permeabilization due to ROS; and 6) ATP depletion. These GA actions are potentially implicated in the well-documented anti-cancer property of GA/structure related compounds. (C) 2011 Elsevier Inc. All rights reserved.

Snake venomics and antivenomics of Crotalus durissus subspecies from Brazil: Assessment of geographic variation and its implication on snakebite management

We report the comparative proteomic and antivenomic characterization of the venoms of subspecies cascavella and collilineatus of the Brazilian tropical rattlesnake Crotalus durissus. The venom proteomes of C. d. collilineatus and C. d. cascavella comprise proteins in the range of 4-115 kDa belonging to 9 and 8 toxin families, respectively. Collilineatus and cascavella venoms contain 20-25 main toxins belonging to the following protein families: disintegrin, PLA(2), serine proteinase, cysteine-rich secretory protein (CRISP), vascular endothelial growth factor-like (VEGF), L-amino acid oxidase, C-type lectin-like, and snake venom metalloproteinase (SVMP). As judged by reverse-phase HPLC and mass spectrometry, cascavella and collilineatus share about 90% of their venom proteome. However, the relative occurrence of the toxin families departs among the two C. durissus subspecies venoms. The most notable difference is the presence of the myotoxin crotamine in some C. d. collilineatus specimens (averaging 20.8% of the total proteins of pooled venom), which is absent in the venom of C. d. cascavella. On the other hand, the neurotoxic PLA2 crotoxin represents the most abundant protein in both C. durissus venoms, comprising 67.4% of the toxin proteome in C. d. collilineatus and 72.5% in C. d. cascavella. Myotoxic PLA(2)s are also present in the two venoms albeit in different relative concentrations (18.1% in C. d. cascavella vs. 4.6% in C. d. collilineatus). The venom composition accounts for the clinical manifestations caused by C. durissus envenomations: systemic neurotoxicity and myalgic symptoms and coagulation disturbances, frequently accompanied by myoglobinuria and acute renal failure. The overall compositions of C. d. subspecies cascavella and collilineatus venoms closely resemble that of C. d. terrificus, supporting the view that these taxa can be considered geographical variations of the same species. Pooled venom from adult C.d. cascavella and neonate C.d. terrificus lack crotamine, whereas this skeletal muscle cell membrane depolarizing inducing myotoxin accounts for similar to 20% of the total toxins of venom pooled from C.d. collilineatus and C.d. terrificus from Southern Brazil. The possible relevance of the observed venom variability among the tropical rattlesnake subspecies was assessed by antivenomics using anti-crotalic antivenoms produced at Instituto Butantan and Instituto Vital Brazil. The results revealed that both antivenoms exhibit impaired immunoreactivity towards crotamine and display restricted (similar to 60%) recognition of PLA(2) molecules (crotoxin and D49-myotoxins) from C. d. cascavella and C. d. terrificus venoms. This poor reactivity of the antivenoms may be due to a combination of factors: on the one hand, an inappropriate choice of the mixture of venoms for immunization and, on the other hand, the documented low immunogenicity of PLA(2) molecules. C. durissus causes most of the lethal snakebite accidents in Brazil. The implication of the geographic variation of venom composition for the treatment of bites by different C. durissus subspecies populations is discussed. (C) 2010 Elsevier B.V. All rights reserved.

Solution structure of amyloid beta-peptide(1-40) in a water-micelle environment. Is the membrane-spanning domain where we think it is?

The three-dimensional solution structure of the 40 residue amyloid beta-peptide, A beta(1-40), has been determined using NMR spectroscopy at pH 5.1, in aqueous sodium dodecyl sulfate (SDS) micelles, In this environment, which simulates to some extent a water-membrane medium, the peptide is unstructured between residues 1 and 14 which are mainly polar and likely solvated by water. However, the rest of the protein adopts an alpha-helical conformation between residues 15 and 36 with a kink or hinge at 25-27. This largely hydrophobic region is likely solvated by SDS. Based on the derived structures, evidence is provided in support of a possible new location for the transmembrane domain of A beta within the amyloid precursor protein (APP). Studies between pH 4.2 and 7.9 reveal a pH-dependent helix-coil conformational switch. At the lower pH values, where the carboxylate residues are protonated, the helix is uncharged, intact, and lipid-soluble. As the pH increases above 6.0, part of the helical region (15-24) becomes less structured, particularly near residues E22 and D23 where deprotonation appears to facilitate unwinding of the helix. This pH-dependent unfolding to a random coil conformation precedes any tendency of this peptide to aggregate to a beta-sheet as the pH increases. The structural biology described herein for A beta(1-40) suggests that (i) the C-terminal two-thirds of the peptide is an alpha-helix in membrane-like environments, (ii) deprotonation of two acidic amino acids in the helix promotes a helix-coil conformational transition that precedes aggregation, (iii) a mobile hinge exists in the helical region of A beta(1-40) and this may be relevant to its membrane-inserting properties and conformational rearrangements, and (iv) the location of the transmembrane domain of amyloid precursor proteins may be different from that accepted in the Literature. These results may provide new insight to the structural properties of amyloid beta-peptides of relevance to Alzheimer's disease.

Membrane-bounded nucleoids in microbial symbionts of marine sponges

In thin sections of resin-embedded samples of glutaraldehyde- and osmium tetroxide-fixed tissue from five genera of marine sponges, Stromatospongia, Astrosclera, Jaspis, Pseudoceratina and Axinyssa, cells of a bacteria-like symbiont microorganism which exhibit a membrane-bounded nuclear region encompassing the fibrillar nucleoid have been observed within the sponge mesohyl. The nuclear region in these cells is bounded by a single bilayer membrane, so that the cell cytoplasm is divided into two distinct regions. The cell wall consists of subunits analogous to those in walls of some Archaea. Cells of the sponge symbionts observed here are similar to those of the archaeal sponge symbiont Cenarchaeum symbiosum. (C) 1998 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.

In vitro human epidermal and polyethylene membrane penetration and retention of the sunscreen benzophenone-3 from a range of solvents

Purpose. To study epidermal and polyethylene membrane penetration and retention of the sunscreen benzophenone-3 (BP) from a range of single solvent vehicles and evaluate solvent effects on permeability parameters. Methods. The solubility of BP was measured in a number of solvents. Penetration of BP across human epidermis and high density polyethylene (HDPE) membranes was studied from 50% saturated solutions in each solvent. Results. Maximal BP fluxes from the solvents across the two membranes varied widely. Highest fluxes were observed from 90% ethanol (EtOH) for epidermis and from isopropyl myristate (IPM) and C12-15 benzoate alcohols (C12-15 BA) for HDPE membrane. Both the flux and estimated permeability coefficient and skin-vehicle partitioning of BP appeared to be related to the vehicle solubility parameter (delta(v)). The major effects of solvents on BP flux appear to be via changes in BP diffusivity through the membranes. Conclusions. Minimal penetration of sunscreens such as BP is best achieved by choosing vehicles with a delta(v) substantially different to the solubility parameter of the membrane.

Determination of chemical shielding tensor of an indole carbon and application of tryptophan orientation of a membrane peptide

Using tryptophan C-13-enriched at the C-4 (C epsilon(3)) of the indole, the orientation of the C epsilon(3) chemical shift tensor relative to the C epsilon(3)-H dipolar axis was determined from the C-13 chemical shift/C-13-H-1 dipolar 2D NMR powder pattern. The principal values obtained were 208, 137 and 15 ppm with sigma(33) perpendicular to the indole plane, and sigma(11) (least shielded direction) 5 degrees off the C epsilon(3)-H bond toward C xi(3). The side off the C epsilon(3)-H bond was determined by comparing the reduced chemical shift anisotropies obtained by solid-state NMR and from molecular dynamics calculations of [4-C-13] tryptophans in gramicidin A aligned in phospholipid membranes. (C) 1999 Elsevier Science B.V. All rights reserved.

The plasma membrane calcium pump, its role and regulation: New complexities and possibilities

Significant progress has been achieved in elucidating the role of the plasma membrane Ca2+-ATPase in cellular Ca2+ homeostasis and physiology since the enzyme was first purified and physiology since the enzyme was first purified and cloned a number of years ago. The simple notion that the PM Ca2+-ATPase controls resting levels of [Ca2+](CYT) has been challenged by the complexity arising from the finding of four major isoforms and splice variants of the Ca2+ pump, and the finding that these are differentially localized in various organs and subcellular regions. Furthermore, the isoforms exhibit differential sensitivities to Ca2+, calmodulin, ATP, and kinase-mediated phosphorylation. The latter pathways of regulation can give rise to activation or inhibition of the Ca2+ pump activity, depending on the kinase and the particular Ca2+ pump isoform. Significant progress is being made in elucidating subtle and more profound roles of the PM Ca2+-ATPase in the control of cellular function. Further understanding of these roles awaits new studies in both transfected cells and intact organelles, a process that will be greatly aided by the development of new and selective Ca2+ pump inhibitors. (C) 1999 Elsevier Science Inc.

Thylakoid membrane architecture

Confocal scanning laser microscopic observations were made on live chloroplasts in intact cells and on mechanically isolated, intact chloroplasts. Chlorophyll fluorescence was imaged to observe thylakoid membrane architecture. C-3 plant species studied included Spinacia oleracea L., Spathiphyllum sp. Schott, cv. 'Mauna Loa', and Pisum sativum L. C-4 plants were also investigated: Saccharum officinarum L., Sorghum bicolor L. Moench, Zea mays L. and Panicum miliaceum L. Some Spinacia chloroplasts were treated with 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) to enhance or sodium dithionite (SD) to reduce the photosystem II fluorescence signal. Confocal microscopy images of C-3 chloroplasts differed from electron microscopy pictures because they showed discrete spots of bright fluorescence with black regions between them. There was no evidence of fluorescence from stroma thylakoids. The thylakoid membrane system at times appeared to be string-like, with brightly fluorescing grana lined up like beads. C-4 bundle sheath chloroplasts were imaged from three different types of C-4 plants. Saccharum and Sorghum bundle sheath chloroplasts showed homogeneous fluorescence and were much dimmer than mesophyll chloroplasts. Zea had rudimentary grana, and dim, homogeneous intergrana fluorescence was visualised. Panicum contained thylakoids similar in appearance and string-like arrangement to mesophyll chloroplasts. Isolated Pisum chloroplasts, treated with a drop of 5 mM MgCl2 showed a thylakoid membrane system which appeared to be unravelling. Spongy mesophyll chloroplasts of Spinacia treated with 5 mM sodium dithionite showed a granal thylakoid system with distinct regions of no fluorescence. A time-series experiment provided evidence of dynamic membrane rearrangements over a period of half an hour.

Evolutionary conservation of the membrane fusion machine

Recent structural studies of proteins mediating membrane fusion reveal intriguing similarities between diverse viral and mammalian systems. Particularly striking is the close similarity between the transmembrane envelope glycoproteins from the retrovirus HTLV-1 and the filovirus Ebola. These similarities suggest similar mechanisms of membrane fusion. The model that fits most currently available data suggests fusion activation in viral systems is driven by a symmetrical conformational change triggered by an activation event such as receptor binding or a pH change. The mammalian vesicle fusion mediated by the SNARE protein complex most likely occurs by a similar mechanism but without symmetry constraints.

Interaction of RTD-1, a cyclic antimicrobial defensin from Rhesus macaque leukocytes, and its open chain analogue with membrane mimics

In contrast to other mammalian defensins, rhesus theta defensin-1 (RTD-1) is composed of just 18 amino acids with the backbone cyclized through peptide bonds. Antibacterial activities of both the native cyclic peptide and a linear form were examined, showing that the cyclic form was 3-fold more active than the open chain analogue, oRTD-1, although both peptides adopt very similar structures in water. It was suggested that the additional charges at the termini of oRTD-1 are the cause for its lower antimicrobial activity. Therefore, we studied the interaction of both peptides with membrane mimics composed of zwitterionic (PC) and negatively charged (PG) phospholipids, major lipid components of erythrocyte and bacterial cell membranes, respectively. Microcalorimetry showed that RTD-1 and oRTD-1 did not affect the phase behavior of PC liposomes, while in PG liposomes both peptides induced new phase transitions above the chain melting transition of the lipid. The shape and fraction differed between both peptides, depending also on their concentration, which will be discussed in terms of their antimicrobial activity.