Evaluation of bilayer disks as plant cell membrane models in partition studies

We have studied the partitioning of a set of phenolic compounds used as lignin precursor models into lipid bilayer disks and liposomes. The bilayer disks are open bilayer structures stabilized by polyethylene glycol-conjugated lipids. Our results indicate that disks generate more accurate partition data than do liposomes. Furthermore, we show that the partitioning into the membrane phase is reduced slightly if disks composed of 1,2-distearoyl-sn-glycero-3-phosphocholine and cholesterol are exchanged for disks with a lipid composition mimicking that of the root tissue of Zea mays L.     

Rac proteins and the control of axon development

Rac GTPases and their effectors control cellular morphogenesis in a wide range of developmental contexts by regulating the structure and dynamics of the actin cytoskeleton. Although much is known about the biochemistry of Racs and Rac regulators, less is known about how Racs control cellular morphogenesis, including axon development, in vivo. Recent loss-of-function genetic studies using model organisms have shown that Racs and their effectors are required for multiple aspects of axon development, including axon outgrowth, axon guidance and axon branching. Interestingly, these studies have also revealed that Rac activity is required to prune spurious axons and branches. Analyses of Racs and their upstream and downstream effectors suggest that Rac signaling is complex. Different neurons utilize distinct combinations of upstream Rac regulators during axon development, possibly reflecting responses to different axon path-finding signals, and Racs use distinct downstream effectors to mediate different aspects of axon development, possibly reflecting differential regulation of the lamellipodial and filopodial growth-cone actin-cytoskeleton domains underlying axon developmental events.     

The Rac GTP exchange factor TIAM-1 acts with CDC-42 and the guidance receptor UNC-40/DCC in neuronal protrusion and axon guidance

The mechanisms linking guidance receptors to cytoskeletal dynamics in the growth cone during axon extension remain mysterious. The Rho-family GTPases Rac and CDC-42 are key regulators of growth cone lamellipodia and filopodia formation, yet little is understood about how these molecules interact in growth cone outgrowth or how the activities of these molecules are regulated in distinct contexts. UNC-73/Trio is a well-characterized Rac GTP exchange factor in Caenorhabditis elegans axon pathfinding, yet UNC-73 does not control CED-10/Rac downstream of UNC-6/Netrin in attractive axon guidance. Here we show that C. elegans TIAM-1 is a Rac-specific GEF that links CDC-42 and Rac signaling in lamellipodia and filopodia formation downstream of UNC-40/DCC. We also show that TIAM-1 acts with UNC-40/DCC in axon guidance. Our results indicate that a CDC-42/TIAM-1/Rac GTPase signaling pathway drives lamellipodia and filopodia formation downstream of the UNC-40/DCC guidance receptor, a novel set of interactions between these molecules. Furthermore, we show that TIAM-1 acts with UNC-40/DCC in axon guidance, suggesting that TIAM-1 might regulate growth cone protrusion via Rac GTPases in response to UNC-40/DCC. Our results also suggest that Rac GTPase activity is controlled by different GEFs in distinct axon guidance contexts, explaining how Rac GTPases can specifically control multiple cellular functions.     

Prioritizing key resilience indicators to support coral reef management in a changing climate

Managing coral reefs for resilience to climate change is a popular concept but has been difficult to implement because the empirical scientific evidence has either not been evaluated or is sometimes unsupportive of theory, which leads to uncertainty when considering methods and identifying priority reefs. We asked experts and reviewed the scientific literature for guidance on the multiple physical and biological factors that affect the ability of coral reefs to resist and recover from climate disturbance. Eleven key factors to inform decisions based on scaling scientific evidence and the achievability of quantifying the factors were identified. Factors important to resistance and recovery, which are important components of resilience, were not strongly related, and should be assessed independently. The abundance of resistant (heat-tolerant) coral species and past temperature variability were perceived to provide the greatest resistance to climate change, while coral recruitment rates, and macroalgae abundance were most influential in the recovery process. Based on the 11 key factors, we tested an evidence-based framework for climate change resilience in an Indonesian marine protected area. The results suggest our evidence-weighted framework improved upon existing un-weighted methods in terms of characterizing resilience and distinguishing priority sites. The evaluation supports the concept that, despite high ecological complexity, relatively few strong variables can be important in influencing ecosystem dynamics. This is the first rigorous assessment of factors promoting coral reef resilience based on their perceived importance, empirical evidence, and feasibility of measurement. There were few differences between scientists' perceptions of factor importance and the scientific evidence found in journal publications but more before and after impact studies will be required to fully test the validity of all the factors. The methods here will increase the feasibility and defensibility of including key resilience metrics in evaluations of coral reefs, as well as reduce costs. Adaptation, marine protected areas, priority setting, resistance, recovery.     

Behavioural Responses of Dusky Dolphin Groups (Lagenorhynchus obscurus) to Tour Vessels off Kaikoura, New Zealand

Background

Commercial viewing and swimming with dusky dolphins (Lagenorhynchus obscurus) near Kaikoura, New Zealand began in the late 1980s and researchers have previously described changes in vocalisation, aerial behaviour, and group spacing in the presence of vessels. This study was conducted to assess the current effects that tourism has on the activity budget of dusky dolphins to provide wildlife managers with information for current decision-making and facilitate development of quantitative criteria for management of this industry in the future.

Methodology/Principal Findings

First-order time discrete Markov chain models were used to assess changes in the behavioural state of dusky dolphin pods targeted by tour vessels. Log-linear analysis was conducted on behavioural state transitions to determine whether the likelihood of dolphins moving from one behavioural state to another changed based on natural and anthropogenic factors. The best-fitting model determined by Akaike Information Criteria values included season, time of day, and vessel presence within 300 m. Interactions with vessels reduced the proportion of time dolphins spent resting in spring and summer and increased time spent milling in all seasons except autumn. Dolphins spent more time socialising in spring and summer, when conception occurs and calves are born, and the proportion of time spent resting was highest in summer. Resting decreased and traveling increased in the afternoon.

Conclusions/Significance

Responses to tour vessel traffic are similar to those described for dusky dolphins elsewhere. Disturbance linked to vessels may interrupt social interactions, carry energetic costs, or otherwise affect individual fitness. Research is needed to determine if increased milling is a result of acoustic masking of communication due to vessel noise, and to establish levels at which changes to behavioural budgets of dusky dolphins are likely to cause long-term harm. Threshold values from these studies would allow managers to set appropriate operational conditions based on quantifiable criteria.     

Melittin-Lipid interaction: A comparative study using liposomes, micelles and bilayerdisks

Comparison of melittin interaction with liposomes, bilayer disks and micelles showed that melittin binding to lipid aggregates is largely dictated by the amount of highly curved areas in the aggregates. The PEG-stabilised bilayer disks were characterised by a combination of small angle neutron scattering, cryo-transmission electron microscopy and dynamic light scattering. Importantly, the theoretically foreseen partial segregation of the lipid components, important for maintaining the structure of the bilayer disk, was confirmed. Steady state fluorescence spectroscopy indicated that melittin mainly resides at the rim of the bilayer disks. Results of the present study help increase the understanding of the mechanisms behind, and the physico-chemical factors affecting, melittin-lipid interaction. We suggest that bilayer disks, due to their stable structure, constitute interesting vehicles for transport of peptides that have high propensity to associate with lipid surfaces of high curvature.     

Structural basis for substrate selection by the translocation and assembly module of the β‐barrel assembly machinery

The assembly of proteins into bacterial outer membranes is a key cellular process that we are only beginning to understand, mediated by the β‐barrel assembly machinery (BAM). Two crucial elements of that machinery are the core BAM complex and the translocation and assembly module (TAM), with each containing a member of the Omp85 superfamily of proteins: BamA in the BAM complex, TamA in the TAM. Here, we used the substrate protein FimD as a model to assess the selectivity of substrate interactions for the TAM relative to those of the BAM complex. A peptide scan revealed that TamA and BamA bind the β‐strands of FimD, and do so selectively. Chemical cross‐linking and molecular dynamics are consistent with this interaction taking place between the first and last strand of the TamA barrel domain, providing the first experimental evidence of a lateral gate in TamA: a structural element implicated in membrane protein assembly. We suggest that the lateral gates in TamA and BamA provide different environments for substrates to engage, with the differences observed here beginning to address how the TAM can be more effective than the BAM complex in the folding of some substrate proteins.     

Nef stimulates human immunodeficiency virus type 1 replication in primary T cells by enhancing virion-associated gp120 levels: coreceptor-dependent requirement for Nef in viral replication

The Nef protein enhances human immunodeficiency virus type 1 (HIV-1) replication through an unknown mechanism. We and others have previously reported that efficient HIV-1 replication in activated primary CD4(+) T cells depends on the ability of Nef to downregulate CD4 from the cell surface. Here we demonstrate that Nef greatly enhances the infectivity of HIV-1 particles produced in primary T cells. Nef-defective HIV-1 particles contained significantly reduced quantities of gp120 on their surface; however, Nef did not affect the levels of virion-associated gp41, indicating that Nef indirectly stabilizes the association of gp120 with gp41. Surprisingly, Nef was not required for efficient replication of viruses that use CCR5 for entry, nor did Nef influence the infectivity or gp120 content of these virions. Nef also inhibited the incorporation of CD4 into HIV-1 particles released from primary T cells. We propose that Nef, by downregulating cell surface CD4, enhances HIV-1 replication by inhibiting CD4-induced dissociation of gp120 from gp41. The preferential requirement for Nef in the replication of X4-tropic HIV-1 suggests that the ability of Nef to downregulate CD4 may be most important at later stages of disease when X4-tropic viruses emerge.     

Primary metabolism in N2-fixing Alnus incana-Frankia symbiotic root nodules studied with 15N and 31P nuclear magnetic resonance spectroscopy

The primary nitrogen metabolism of the N₂-fixing root nodule symbiosis Alnus incana (L.)–Frankia was investigated by ³¹P and ¹⁵N nuclear magnetic resonance (NMR) spectroscopy. Perfusion of root nodules in a pulse–chase approach with ¹⁵N- or ¹⁴N-labeled NH₄⁺ revealed the presence of the amino acids alanine (Ala), -amino butyric acid, glutamine (Gln), glutamic acid (Glu), citrulline (Cit) and arginine (Arg). Labeling kinetics of the Gln amide-N and -amino acids suggested that the glutamine synthetase (GS; EC 6.3.1.2)–glutamate synthase (GOGAT; EC 1.4.1.13) pathway was active. Inhibition of the GS-catalyzed reaction by methionine sulphoximine abolished incorporation of ¹⁵N. Cit was labeled in all three N positions but most rapidly in the position, consistent with carbamoyl phosphate as the precursor to which Gln could be the amino donor catalyzed by carbamoyl phosphate synthase (CPS; EC 6.3.5.5). Ala biosynthesis occurred consistent with a flux of N in the sequence Gln–Glu–Ala. ³¹P NMR spectroscopy in vivo and of extracts revealed several metabolites and was used in connection with the ¹⁵N pulse–chase experiment to assess general metabolic status. Stable concentrations of ATP and UDP-glucose during extended perfusions showed that the overall root nodule metabolism appeared undisturbed throughout the experiments. The metabolic pathways suggested by the NMR results were confirmed by high activities of the enzymes GS, NADH-GOGAT and ornithine carbamoyltransferase (OCT; EC 2.1.3.3). We conclude that the primary pathway of NH₄⁺ assimilation in A. incana root nodules occurs through the GS–GOGAT pathway. Biosynthesis of Cit through GS–CPS–OCT is important and is a link between the first amino acid Gln and this final transport and storage form of nitrogen.Abbreviations AlaDH l-Alanine dehydrogenase - Cit Citrulline - CPS Carbamoyl phosphate synthase - GABA -Amino butyric acid - GOGAT Glutamate synthase - GS Glutamine synthetase - MDH Malate dehydrogenase - MSO Methionine sulphoximine - NMR Nuclear magnetic resonance - OCT Ornithine carbamoyltransferase - PEPC Phosphoenolpyruvate decarboxylase - UDPGlc Uridine 5-diphosphoglucose