Amide-resolved hydrogen—deuterium exchange measurements from membrane-reconstituted polypeptides using exchange trapping and semiselective two-dimensional NMR

Summary Amide-resolved, hydrogen-deuterium exchange from bee venom melittin reconstituted in fully hydrated vesicles suspended in D₂O buffer was measured using a technique involving (1) trapping samples throughout an exchange time course by rapid freezing and lyophilization; and (2) dissolving the dried peptide/lipid mixtures in deuteromethanol to record high-resolution spectra using semiselective excitation pulses to select peptide amide signals in the presence of large excess lipid signals. Two-dimensional, amide-selective GaussNOESY and fingerprint-selective off-diagonal PingCOSY spectra are shown to be suitable for rapid acquisition of amide-selective spectra, obtained throughout a time course of amide exchange in the membrane-bound state. Membrane-reconstituted melittin is shown to contain two sequences of exchange-stable amides, corresponding to helical regions on either side of the single proline residue.     

Divine powers and exchange with ‘others’ in Melanesia

Marshall Sahlins described divine king forms for a wide range of societies from Southeast Asia, Africa, and the Pacific, among others. In this article, I document a divine king form among the Fuyuge people of the Papuan highlands, revising my previous understanding of this powerful figure. At the same time, I argue there is an inextricable connection between Sahlins's theory of divine power and Marilyn Strathern's model of Melanesian gift exchange: both operate according to distinct ideas of otherness. The capacity to engage in transaction derives from cosmological sources while evidence of cosmological power is provided by the ability to engage in transactions with others in effective and powerful ways. More generally, I argue that conventional Melanesian figures of big-men, great men, and chiefs are all versions of the alterity of power in related political forms; each instantiates the mutual relations between cosmological and transactional otherness.     

Social exchange and reciprocity: confusion or a heuristic?

We propose that a “social exchange heuristic” is as important as the cheater detection mechanism for attaining mutual cooperation in social exchange. The social exchange heuristic prompts people to perceive a mixed-motive situation, such as the Prisoner's Dilemma (PD), as an Assurance Game (AG) situation in which cooperation is a personally better choice than defection insofar as the partner is cooperating as well. We demonstrate the operation of the social exchange heuristic through a comparison of the ordinary one-shot, simultaneous PD with the one-shot, sequential PD. Participants in the current experiments, involving a total of 261 volunteers, committed a logical error in the direction of favoring mutual cooperation as the situation involved more serious consequences. This result strongly suggests the operation of a domain specific “bias” that encourages pursuit of mutual cooperation in social exchange.     

Cl⁻/HCO₃⁻ exchange activity in fMLP-stimulated human neutrophils

It is well known that chemotactic agents active Na(+)/H(+) exchanger, increasing intracellular pH of neutrophils, but their effect on bicarbonate transporters have not been established yet. To study the effect of fMLP on the activity of Cl(-)/HCO(3)(-) exchange, the rate of pH recovery after acute Cl(-) readmission in cell subjected to an alkaline load by CO(2) washout in a Cl-free medium was measured. The activity of the exchanger was reduced to 72% of control when cells were pre-incubated for 5 min with 0.1 μM fMLP and reached 48% of control in steady state after acute exposure. After extracellular bicarbonate or TMA addition the rate recovery of intracellular pH was reduce at 72% and at 84%, respectively. The inhibitory effect on the intracellular pH recovery was not affected by blockers of Na(+)/H(+) exchange. We conclude from these studies that an increase of pH(i) produced for this chemotactic agent is facilitated by the simultaneous activation of Na(+)/H(+) exchange and inhibition of Cl(-)/HCO(3)(-) exchange in neutrophils.     

Activation segment exchange: a common mechanism of kinase autophosphorylation?

The crystal structure of the kinase domain from human checkpoint kinase 2 (Chk2) has shown, for the first time, the reciprocal exchange of activation segments between two adjacent molecules and provides the molecular basis for understanding the observed mode of Chk2 kinase activation via trans-autophosphorylation. With further examples of activation segment exchanged kinase domains now publicly available (i.e. Ste20-like kinase, Ser/Thr kinase 10 and Death-associated protein kinase 3), we suggest that this phenomenon represents a common mechanism of activation amongst a particular subset of protein kinases, that is, those that are dimeric (either transiently or constitutively), that undergo activation by autophosphorylation and that have activation segment amino acid sequences that do not resemble those of their substrate consensus sequence.     

Birth defects interstate data exchange: a battle worth fighting?

BACKGROUND: Regardless of where infants and children are delivered, diagnosed, or treated, an important aspect of population-based birth defects surveillance is ensuring the inclusion of children with birth defects in the catchment area. However, little is known as to how the lack of interstate birth defects data exchange affects program surveillance, monitoring, prevention, and referral activities. The study objectives were to determine the status of interstate birth defects data exchange agreements and to quantify statewide data on resident births occurring in nonresident states. METHODS: In 2004, surveys were distributed to all population-based birth defects programs in the United States to determine: 1) the types of interstate birth defects data exchange agreements that exist among birth defects programs, 2) perceived barriers in establishing exchange agreements, and 3) the extent to which out-of-state births affect a program's catchment area. The National Center for Health Statistics (NCHS) data for 2002 on live birth residency were used to determine the actual frequency of out-of-state live birth occurrence. RESULTS: Of the 52 states and territories that were surveyed, 65% (n = 34) responded. Approximately 21% (n = 7) of those that responded had an interstate data exchange agreement that allowed sharing of birth defects data with another state or a facility within another state. Approximately 53% (n = 18) of responding states indicated plans to develop an interstate birth defects data exchange agreement with other states, hospitals, or both. The NCHS data showed that the actual percentage of resident out-of-state live births ranged from 0.16 to 11.51. NCHS data also reveal that 78% of states would be able to capture >75% of their out-of-state births by sharing data on out-of-state births with the three neighboring states ranking highest in terms of such occurrences. CONCLUSIONS: Few states have interstate birth defects data exchange agreements, though all states have resident births occurring out of state. While suggestive, data beyond residency of live births are needed to quantify the degree to which the objectives of state-based birth defects programs are compromised. Resources exist to guide programs in establishing interstate data exchange agreements. Efforts to establish such agreements with only a few neighboring states could be a large step toward improving birth defects surveillance on a state, regional, and national level.     

Defective sarcoplasmic reticulum–mitochondria calcium exchange in aged mouse myocardium

Mitochondrial alterations are critically involved in increased vulnerability to disease during aging. We investigated the contribution of mitochondria–sarcoplasmic reticulum (SR) communication in cardiomyocyte functional alterations during aging. Heart function (echocardiography) and ATP/phosphocreatine (NMR spectroscopy) were preserved in hearts from old mice (>20 months) with respect to young mice (5–6 months). Mitochondrial membrane potential and resting O₂ consumption were similar in mitochondria from young and old hearts. However, maximal ADP-stimulated O₂ consumption was specifically reduced in interfibrillar mitochondria from aged hearts. Second generation proteomics disclosed an increased mitochondrial protein oxidation in advanced age. Because energy production and oxidative status are regulated by mitochondrial Ca²⁺, we investigated the effect of age on mitochondrial Ca²⁺ uptake. Although no age-dependent differences were found in Ca²⁺ uptake kinetics in isolated mitochondria, mitochondrial Ca²⁺ uptake secondary to SR Ca²⁺ release was significantly reduced in cardiomyocytes from old hearts, and this effect was associated with decreased NAD(P)H regeneration and increased mitochondrial ROS upon increased contractile activity. Immunofluorescence and proximity ligation assay identified the defective communication between mitochondrial voltage-dependent anion channel and SR ryanodine receptor (RyR) in cardiomyocytes from aged hearts associated with altered Ca²⁺ handling. Age-dependent alterations in SR Ca²⁺ transfer to mitochondria and in Ca²⁺ handling could be reproduced in cardiomyoctes from young hearts after interorganelle disruption with colchicine, at concentrations that had no effect in aged cardiomyocytes or isolated mitochondria. Thus, defective SR–mitochondria communication underlies inefficient interorganelle Ca²⁺ exchange that contributes to energy demand/supply mistmach and oxidative stress in the aged heart.Age is the main independent risk factor for cardiovascular morbidity and mortality.¹ It increases heart vulnerability to cardiac diseases as well as the severity of their clinical manifestations, and reduces the efficacy of cardioprotective interventions.² At the cellular level, some of the structural and functional age-dependent changes resemble those of failing cardiac myocytes.^{3, 4} Specifically, disturbed Ca²⁺ homeostasis and excitation–contraction coupling,⁵ as well as deficient mitochondrial energetics⁶ and excessive ROS production,⁷ have been consistently reported in senescent cardiomyocytes. These subcellular alterations likely contribute to the reduced adaptive capacity to stress (exercise, β-adrenergic stimulation) and increased vulnerability to disease of the aged hearts.In cardiac cells, electrochemical coupling and metabolic adaptations are based upon the coordination between sarcoplasmic reticulum (SR) and mitochondria tightly interconnected forming an interface to support local ionic exchange and signal transduction in a beat-to-beat basis.⁸ This privileged interorganelle communication facilitates mitochondrial ATP transport for SR Ca²⁺ cycling and ensures energy replenishment by reciprocal Ca²⁺ and ADP exchange. Ca²⁺ is taken up by mitochondria using a low-affinity uniporter whose activity is driven by the elevated Ca²⁺ concentration in the microenvironment present around ryanodine receptors (RyR).⁹ Indeed, the kinetics of mitochondrial Ca²⁺ uptake is more dependent on the concentration of Ca²⁺ at the SR–mitochondria contact points than on bulk cytosolic Ca²⁺ concentration.⁸ Mitochondrial Ca²⁺ uptake allows energy supply–demand matching through the activation of Krebs cycle dehydrogenases and electron transport chain activity, and at the same time it regulates the regeneration of Krebs-coupled antioxidative defenses (NAD(P)H).¹⁰Defective SR–mitochondria cross talk has been causally linked to the abnormal mitochondrial Ca²⁺ uptake in failing hearts and may underlie their increased oxidative stress.¹¹ Also, in diabetic cardiomyopathy, intracellular Ca²⁺ overload and depletion of energy stores appear to develop as a consequence of sequential SR–mitochondria dysfunction.¹² Atrial fibrillation has been associated with an increased fusion of mitochondria and a subsequent increased colocalization of giant mitochondria with SR, a subcellular remodeling process that contributes to the perpetuation of the arrhythmia.¹³ Because mitochondria are highly dynamic structures, some molecular links have been proposed to provide a stable physical interorganelle bridge^{14, 15} while others appear to facilitate direct tunneling of Ca²⁺ and other signaling mediators.¹⁶ In the present study, we hypothesized that aging may negatively impact on mitochondria–SR communication by mechanisms involving defective Ca²⁺ transmission, and we identified reduced physical interaction between RyR and mitochondrial voltage-dependent anion channel (VDAC) as the main responsible of this effect.     

Social exchange and solidarity: in-group love or out-group hate?

Men exhibit a stronger tendency to favor the in-group over the out-group compared to women. We examined whether this male-specific “coalitional psychology” represents in-group love or out-group hate. One hundred thirty-three college freshmen played a prisoner's dilemma game with a member of their own group and a member of another group. Both groups consisted of same-sex participants. An in-group bias (cooperation with the in-group at a level higher than cooperation with the out-group) based on expectations of cooperation from the in-group was observed for both men and women. When such expectations were experimentally eliminated, women did not show any in-group bias, whereas men still exhibited an in-group bias. This male-specific in-group bias was found to be a product of intragroup cooperation (in-group love) rather than a product of intergroup competition (out-group hate). These findings suggest that the male-specific coalitional psychology caters more toward the promotion of within-group solidarity than aggression against the out-group.     

Iron–sulfur cluster exchange reactions mediated by the human Nfu protein

Human Nfu is an iron–sulfur cluster protein that has recently been implicated in multiple mitochondrial dysfunctional syndrome (MMDS1). The Nfu family of proteins shares a highly homologous domain that contains a conserved active site consisting of a CXXC motif. There is less functional conservation between bacterial and human Nfu proteins, particularly concerning their Iron–sulfur cluster binding and transfer roles. Herein, we characterize the cluster exchange chemistry of human Nfu and its capacity to bind and transfer a [2Fe–2S] cluster. The mechanism of cluster uptake from a physiologically relevant [2Fe–2S](GS)₄ cluster complex, and extraction of the Nfu-bound iron–sulfur cluster by glutathione are described. Human holo Nfu shows a dimer-tetramer equilibrium with a protein to cluster ratio of 2:1, reflecting the Nfu-bridging [2Fe–2S] cluster. This cluster can be transferred to apo human ferredoxins at relatively fast rates, demonstrating a direct role for human Nfu in the process of [2Fe–2S] cluster trafficking and delivery.     

C-Terminal truncation affects subunit exchange of human αA-crystallin with αB-crystallin

In human lenses, C-terminal cleavage of αA-crystallin at residues 172,168, and 162 have been reported. The effect of C-terminal truncation of αA-crystallin on subunit exchange and heterooligomer formation with αB-crystallin and homooligomer formation with native αA-crystallin is not known. We have conducted fluorescence resonance energy transfer studies which have shown that the rates of subunit exchange of αA_1–172 and αA_1–168 with αB-wt were two-fold lower than for αA-wt interacting with αB-wt. The subunit exchange rate between αA_1–162 and αB-wt was six-fold lower. These data suggest that cleavage of the C-terminal residues could significantly affect heterooligomerization. On the other hand, the subunit exchange rates between αA-wt and the truncated αA-crystallins were either unchanged or only slightly decreased, which suggest that homooligomerization may not be significantly influenced by C-terminal truncation. The main conclusion from this study is that cleavage of C-terminal residues of αA-crystallin including the nine residues of the flexible tail is expected to significantly affect the formation of heteroaggregates. Reconstitution experiments showed that the presence of an intact C-terminus is essential for the formation of fully integrated heteroaggregates with equal proportion of αA and αB subunits.     

Plasma exchange in the long-term management of Waldenstr?m's macroglobulinemia.

Two patients with Waldenström''s macroglobulinemia (WM), which had become resistant to cytotoxic drugs, were treated for features of the hyperviscosity syndrome by repeated plasma exchange with the continuous-flow blood-cell separator over periods of 36 and 28 months, respectively. After four initial weekly plasma exchanges the procedure was carried out every 4 to 6 weeks and both patients tolerated it well. Relative viscosity of the serum was maintained within the normal range in one patient, and both patients remained free of symptoms of the hyperviscosity syndrome. The results suggest that treatment of WM by long-term "maintenance" plasma exchange alone should be considered in any patient with complications due to chemotherapy or whose disease fails to respond to chemotherapy.     

Ion exchange chromatographic conditions for obtaining individual subunits of soybean β-conglycinin

Soybean β-conglycinin is a complex protein possessing health-promoting properties. β-Conglycinin is a trimeric glycoprotein. Little information related to methods for separation of the individual chains forming β-conglycinin has been so far published and it is of great interest. As a consequence, less data on the bioactivities of α, α′ and β subunits of this glycoprotein have been published. The present research aimed to find out new alternative chromatographic conditions to obtain β-conglycinin subunits that are free of contaminating proteins. In the present short communication, we propose the use of a two-step ion exchange chromatographic protocol to achieve this goal. Firstly, β subunit was separated by means of anionic exchange fast protein liquid chromatography. Secondly, α and α′ chains were separated from each other by cationic exchange. Our data indicated the feasibility of proposed fractionation protocol to separate soybean β-conglycinin α and α′ subunits from other contaminating proteins and to obtain enough amounts of the three individual chains forming this glycoprotein for further characterization and application. The procedure may be easily up-scaled.     

Interspecies exchange of β 2-microglobulin and associated MHC and differentiation antigens

Radiolabeled human ₂-microglobulin (₂m) can bind to mouse histocompatibility (H-2) antigens on the cell surface or to partially purified H-2 antigens in solution. The complexes containing human ₂m and H-2 antigens from C3H (H-2^k) mice could be immunoprecipitated specifically with alloantisera, rabbit anti-H-2 xenoantisera, and with monoclonal H-2-specific antibodies. Specific association with H-2 antigens was also observed with other haplotypes. The only exception was B10.D2 (H-2 ^d ) from which complexes containing human ₂M could only be precipitated with anti-H-2 xenosera. Thus radiolabeled human ₂M can be used as a specific label for mouse H-2 antigens in precipitation and radioimmunoassays. The application of this finding extends to major histocompatibility complex antigens of other species, and to differentiation antigens with primary association with ₂m.Abbreviations used in this paper MHC major histocompatibility complex - ₂m ₂-microglobulin - LcH Lens culinaris hemagglutinin     

Radiation damping compensation of selective pulses in water–protein exchange spectroscopy

The observation of nuclear Overhauser effects (NOEs) between bound water and biological macromolecules such as proteins and nucleic acids can be improved by inverting the water resonance selectively while compensating for radiation damping effects. The efficiency of inversion, the offset profiles, and the appearance of 2D NOE-NOESY spectra can be improved in comparison with earlier methods.     

Conformational insight into multi-protein signaling assemblies by hydrogen–deuterium exchange mass spectrometry

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Retrotransfer in Escherichia coli conjugation: bidirectional exchange or de novo mating?

DNA can be transferred among eubacteria and to plants and fungi by related, plasmid-mediated processes collectively referred to as bacterial conjugation. Conjugation occurs between cells in contact with one another and results in the unidirectional delivery of DNA from a bacterial donor to a recipient. Recent experiments that have reexamined the directionality of DNA flow during conjugation have come to different conclusions, some suggesting that genetic material also flows from recipient cells into the donor and that this process, termed retrotransfer, is likewise directed by donor-encoded functions. Given that bacteria are perhaps united with all living creatures by conjugation, the possibility of gene flow into donor bacteria during conjugation raises interesting evolutionary and biocontainment issues. Here we report that plasmid transmission from bacterial recipients to donors is not a donor-mediated event. Movement of genetic material from recipients to donors was inhibited by streptomycin, which does not inhibit the conjugative donor, indicating that retrotransfer requires gene expression in recipients. Furthermore, retrotransfer was reduced in matings mediated by plasmids that encode strong entry exclusion, to a similar degree as matings between two donors. Therefore we suggest that retrotransfer is in fact newly initiated conjugation between transconjugants and donors.     

Discontinuous gas exchange in insects: is it all in their heads?

Some insects display an intermittent pattern of gas exchange while at rest, often going hours between breaths. These discontinuous gas exchange cycles (DGCs) are known to have evolved independently within five insect orders, but their possible adaptive benefit and evolutionary origin remain an enigma. Current research is primarily concerned with testing three adaptive hypotheses: that DGCs originally evolved or are currently maintained to (1) limit respiratory water loss, (2) enhance gas exchange in subterranean environments, or (3) limit oxidative damage. These adaptive explanations fail to unite a range of apparently contradictory observations regarding the insects that display DGCs and the conditions under which they occur. Here we argue that DGCs are explained by circadian, developmental, or artificially induced reductions in brain activity. We conclude that this pattern results from the thoracic and abdominal ganglia regulating ventilation in the absence of control from higher neural centers, and it is indicative of a sleeplike state.     

Estimating oxygen exchange across the air–water interface of a hypereutrophic lake

Patterns of estimates of oxygen flux (J) across the air–water interface of hypereutrophic Onondaga Lake, NY, U.S.A., are characterized for time scales ranging from diel to seasonal for an 8-month period. The analysis is supported by a high frequency (most often hourly) monitoring program, conducted with a robotic buoy, that included measurements of dissolved oxygen (DO), temperature, and fluorometric chlorophyll a in the lake's surface waters, vertical profiles of DO through the epilimnion, and wind speed and solar radiance. The magnitude and direction of J is demonstrated to vary dramatically at diel, day-to-day, and seasonal time scales. Thus, large errors in estimates of J may result from extrapolating flux calculations made from short-term data to longer time periods. The variations in J were driven by variations in metabolic activity and meteorology, and were mediated by departures from equilibrium DO concentrations and wind-driven turbulence. Extended periods of high J values are shown to coincide with intervals of large departures from equilibrium DO concentrations, but day-to-day differences are driven mostly by variations in wind. A distinct diel pattern of J estimates is manifested for average conditions, with substantially higher J values during daylight hours. This pattern reflects the common diel patterns of the drivers of both higher DO oversaturation and wind speed over those hours. It is demonstrated that the magnitude of J is substantial relative to net changes in the epilimnetic DO pool, and thus must be accommodated accurately in estimates of primary production and community respiration that are to be based on diel monitoring of DO in the water columns of productive lakes.