bluuebunny/crossref_metadata_2025 huggingface dataset:
In vivo bafilomycin-sensitive Na+ uptake in young freshwater fish
Authors: Fenwick J. C. , Bonga S. E. Wendelaar , Flik G.
Published: April 2021

In vivo treatment with external bafilomycin A1, a selective inhibitor of V-ATPase H+ pumps, reduced whole-body Na+ influx by up to 90 % in young tilapia and 70 % in young carp. The inhibition was rapidly reversible, with whole-body Na+ influx rebounding to 280 % of pre-treatment values within 20 min of removal from the bafilomycin. This rebound effect is consistent with the prior accumulation of protons during the period when the cells were exposed to bafilomycin. Bafilomycin also inhibited Cl− uptake, an effect that was still apparent 30 min after the removal of bafilomycin. These data provide circumstantial evidence for previous suggestions that Na+ uptake in freshwater fish is associated with a proton-motive force created by a proton pump and indirect evidence for the major significance of this mechanism in the branchial uptake of Na+ by freshwater fish.
Motor control of tongue movement during prey capture in plethodontid salamanders
Authors: Deban Stephen M. , Dicke Ursula
Published: April 2021

Four species of salamander of the family Plethodontidae were examined using electromyographic (EMG) recording during prey-capture behavior to test the hypotheses that the tongue retractor, tongue protractor and jaw depressor muscles are activated simultaneously and in a stereotyped pattern, as was found in other salamanders, and to determine whether species with different tongue morphologies and tongue protraction abilities exhibit different motor control strategies. The results show that sequential activation was observed far more frequently than simultaneous activation; the jaw depressor muscle was activated first, followed by the tongue protractor and then the tongue retractor. Species with short, attached tongues (Desmognathus quadramaculatus and Plethodon jordani) showed simultaneous activation more often than species with long, free tongues (Pseudotriton ruber and Hydromantes supramontis), which showed strongly non-simultaneous activation. Most EMG variables showed no effect of prey-capture success, suggesting that sensory feedback is not involved in modulating the motor pattern during the prey-capture strike. Hydromantes supramontis was examined for modulation of its motor pattern in response to prey distance, and several EMG variables were found to be positively correlated with tongue protraction distance. The motor pattern of strongly non-simultaneous activation of antagonistic tongue muscles has evolved along with the evolution of long, free tongues in plethodontids. The variable motor patterns observed provide further evidence that amphibian feeding in general is not as highly stereotyped as has been previously thought.
Feeding kinematics of phelsuma madagascariensis (Reptilia: gekkonidae): testing differences between iguania and scleroglossa
Authors: Delheusy Veronique , Bels Vincent L.
Published: April 2021

The kinematics of feeding in the gekkotan lizard Phelsuma madagascariensis (Scleroglossa) was investigated using high-speed cinematography (200–300 frames s−1 ) and X-ray films (64 frames s−1 ). Qualitative kinematic analysis of the head and jaw displacement of the prey to (capture) and within (reduction, transport, swallowing, licking) the buccal cavity are compared for two types of prey (crickets and mealworms) in 30 feeding sequences from four individuals. Maximal displacement of structures and timing of events are compared statistically to assess the differences among the phases and the prey using analysis of variance. P. madagascariensis uses its jaws only to capture the two types of prey item, and the capture jaw cycle is divided into fast-opening (FO), fast-closing (FC) and slow-closing (SC) stages only. As in iguanians and other scleroglossans, the reduction and transport cycles always involve a slow opening (SOI and SOII) stage before the FO stage, followed by FC and SC stages: this last stage was not easily identified in all feeding phase. Transport of the prey was followed by a large number of licking cycles. Our data show (i) that the capture profile in gekkotans is similar to that observed for other scleroglossans and different from that described for iguanians (e.g. the absence of an SO stage); (ii) that the kinematics of jaw and related hyo-lingual cycles of intraoral manipulation (reduction and transport) are similar in lizards with a very different hyo-lingual system (Iguania, Gekkota and Scincomorpha), suggesting a basic mechanism of feeding cycles in squamates, transformed in varanids and snakes; and (iii) that prey type affects the kinematics of capture and manipulation, although the high level of variation among lizards suggests a possible individual modulation of feeding mechanism. A principal components analysis was performed to compare capture and transport cycles in this study of P. madagascariensis (Gekkota) and a previous study of Oplurus cuvieri (Iguania). This analysis separated the capture cycle of each species, but the transport cycles were not completely separated. These results demonstrate the complexity of the modulation and evolution of feeding process in squamates.
Collagen-stimulated unidirectional translocation of cholesterol in human platelet membranes
Authors: Boesze-Battaglia Kathleen , Schimmel Richard J.
Published: April 2021

When human platelets are stimulated with collagen or thrombin, the asymmetric distribution of membrane lipids is disrupted as phosphatidylserine and phosphatidylethanolamine translocate from the inner monolayer to the outer monolayer. Coincident with the stimulus-dependent rearrangement of membrane phospholipids is a rapid redistribution of cholesterol from the outer to the inner membrane monolayer. This redistribution of cholesterol was observed when the stimulus was collagen or ADP. The data presented here show that epinephrine stimulation does not promote cholesterol translocation but does potentiate collagen-promoted movement of cholesterol. To investigate the process of cholesterol translocation, experiments were performed to determine whether collagen stimulated reverse cholesterol movement; i.e. from the inner to the outer monolayer. For this study, the fluorescent sterol cholestatrienol (C-3) was incorporated into platelet membranes by exchange from cholesterol-containing phosphatidylcholine small unilamellar vesicles. C-3 was then removed selectively from the outer monolayer by treatment of the platelets with bovine serum albumin (BSA). During the subsequent incubation of BSA-treated platelets, C-3 moved spontaneously into the outer from the inner monolayer. This translocation had an apparent half-time of approximately 25 min and was unaltered by the presence of collagen. These results suggest that collagen treatment of platelets selectively facilitates the inward movement of the sterol. We have hypothesized that cholesterol translocation may be thermodynamically driven as a result of an unfavorable entropy, resulting in cholesterol translocation out of an environment becoming enriched in phosphatidylethanolamine. The unidirectional nature of collagen-promoted cholesterol movement from the phosphatidylethanolamine-rich outer monolayer is consistent with this interpretation.
Sexual behaviour in Euplotes raikovi is accompanied by pheromone-induced modifications of ionic currents
Authors: Stock Christian , Krüppel Thomas , Key Gundula , Lueken Wolfgang
Published: April 2021

In the marine ciliate Euplotes raikovi, pheromone released by a complementary mating type (nonself pheromone) induces typical sexual behaviour, whereas self pheromone released by the same mating type generally has no effect. Nonself pheromone evokes a reduction of the mean walking speed by 66 %, a threefold increase in the frequency and duration of long-lasting rest phases and a doubling in the number of side-stepping reactions. Consequently, translocation is strongly reduced and the cells remain in a small area. This could increase the probability of finding a sexual partner for pair formation (conjugation). The usual pattern of rhythmic, spontaneous depolarizations controlling the walking rhythm is absent in nonself-pheromone-stimulated cells. The remaining depolarizations arise from a 4 mV hyperpolarized membrane potential and do not reach the usual amplitudes of 15–20 mV but only of 6–10 mV. In addition, the amplitudes of K+ currents are increased at depolarizations of more than 20 mV by at least 30 %. Hyperpolarization- and depolarization-activated Na+ current amplitudes are increased, whereas the Ca2+ current amplitude remains nearly unaffected.
Running, breathing and visceral motion in the domestic rabbit (Oryctolagus cuniculus): testing visceral displacement hypotheses
Authors: Simons Rachel S.
Published: April 2021

The relative motion of the visceral mass may be important to ventilation during running. A visceral piston hypothesis predicts that, during galloping, cranial motion of the liver during expiration and caudal motion of the liver during inspiration may characterize efficient quadrupedal mammalian locomotion. Although a theoretical model based on vibration mechanics casts doubt on this prediction, only limited direct measurements of visceral mass motion during galloping have been reported. In the present study, mechanical interactions between running, breathing and liver oscillations in the domestic rabbit are recorded using synchronized videographic, cineradiographic and pneumotachographic techniques. The analysis focuses on the variation in locomotor–respiratory coupling (LRC) and on the relative position of the liver. Results from running rabbits show (1) variation in phase angle between the locomotor and respiratory periods that is inconsistent with the 1:1 LRC ratio that has been reported for other galloping mammals; (2) a tendency towards a 1:1 LRC ratio at higher speeds and stride frequencies; and (3) that the relative motion of the liver is caudal during expiration and cranial during inspiration, which is inconsistent with the visceral piston hypothesis. The data presented here are generally consistent with the theoretical vibration mechanics model for liver motion and with a pneumatic stabilization hypothesis that the lungs serve an important role in the stabilization of the thorax during locomotion.
Nitric oxide stimulates the stress-activated protein kinase p38 in rat renal mesangial cells
Authors: Huwiler Andrea , Pfeilschifter Josef
Published: April 2021

Nitric oxide (NO) has gained increased attention as a diffusible universal messenger that plays a crucial role in the pathogenesis of inflammatory and autoimmune diseases. Recently, we reported that exogenous NO is able to activate the stress-activated protein kinase (SAPK) cascade in mesangial cells. Here, we demonstrate that exposure of glomerular mesangial cells to compounds releasing NO, including spermine-NO and (Z)-1-

{N-methyl-N-[6-(N-methylammoniohexyl)amino]diazen}-1-ium-1,2-diolate (MAHMA-NO), results in an activation of the stress-activated p38-mitogen-activated protein kinase (p38-MAPK) cascade as measured by the phosphorylation of the activator of transcription factor-2 (ATF2) in an immunocomplex kinase assay. Activation of the p38-MAPK cascade by a short stimulation (10 min) with the NO donor MAHMA-NO causes a large increase in ATF2 phosphorylation that is several times greater than that observed after stimulation with interleukin-1β, a well-known activator of the p38-MAPK pathway. Time course studies reveal that MAHMA-NO causes rapid and maximal activation of p38-MAPK after 10 min of stimulation and that activation declines to basal levels within 60 min. The longer-lived NO donor spermine-NO causes a comparable rapid activation of the p38-MAPK pathway; however, the increased activation state of p38-MAPK was maintained for several hours before control values were reattained after 24 h of stimulation. Furthermore, the NO donors also activated the classical extracellular signal-regulated kinase (ERK) p44-MAPK cascade as shown by phosphorylation of the specific substrate cytosolic phospholipase A2 in an immunocomplex kinase reaction. Both MAHMA-NO and spermine-NO cause a rapid activation of p44-MAPK after 10 min of stimulation. Interestingly, there is a second delayed peak of p44-MAPK activation after 4–24 h of stimulation with NO donors.

These results suggest that there is a differential activation pattern for stress-activated and mitogen-activated protein kinases by NO and that the integration of these signals may lead to specific cell responses.
Body bending during fast-starts in fish can be explained in terms of muscle torque and hydrodynamic resistance
Authors: Wakeling James M. , Johnston Ian A.
Published: April 2021

Fish fast-starts are rapid events caused by the simultaneous onset of muscle activity along one side of the body. Spine curvature and the strain and electromyographic activity in white muscle were measured for fast-starts in the common carp Cyprinus carpio. The first bend of the fast-start was powered by muscle on the concave side: muscle fibres on this side were activated and began shortening simultaneously between the length-specific longitudinal sites 0.3L and 0.56L, where L is total body length. However, there was an increasing delay in the timing of the first peak in body curvature and muscle strain along the length of the body. Modelling studies related the rate of body bending to the muscle torque and hydrodynamic resistance of the fish. The muscle torque produced on the spine was greatest in the central region of the trunk, and this acted against the moments of inertia of the fish mass and added mass of water. It was concluded that a wave of body bending can be generated as a result of the hydrodynamic resistance of the fish despite the initiation of that bending being simultaneous along the length of the body.
How polarization-sensitive interneurones of crickets see the polarization pattern of the sky: a field study with an opto-electronic model neurone
Authors: Labhart T.
Published: April 2021

Many insects gain directional information from the polarization pattern of the sky. Polarization vision is mediated by the specialized ommatidia of the dorsal rim area of the compound eye, which contains highly polarization-sensitive photoreceptors. In crickets Gryllus campestris, polarized light information conveyed by the dorsal rim ommatidia was found to be processed by polarization-opponent interneurones (POL-neurones). In this study, a field-proof opto-electronic model of a POL-neurone was constructed that implements the physiological properties of cricket POL-neurones as measured by previous electrophysiological experiments in the laboratory. Using this model neurone, both the strength of the celestial polarization signal and the directional information available to POL-neurones were assessed under a variety of meteorological conditions. We show that the polarization signal as experienced by cricket POL-neurones is very robust, both because of the special filtering properties of these neurones (polarization-antagonism, spatial low-pass, monochromacy) and because of the relatively stable e-vector pattern of the sky.
Optimizing Force and Velocity: Mandible Muscle Fibre Attachments in Ants
Authors: Paul Jürgen , Gronenberg Wulfila
Published: April 2021

To be able to perform swift and powerful movements, ant mandible closer muscles are composed of two subpopulations of muscle fibres: fast fibres for rapid actions and slow fibres for forceful biting. All these fibres attach to a sturdy and complex apodeme which conveys force into the mandible base. Fast muscle fibres attach directly to the apodeme. Slow fibres may attach directly or insert at individual thin filament processes of the apodeme which vary in length. Comparisons of different ant species suggest two basic principles underlying the design of mandible muscles. (1) Ants specialized for fast mandible movements generally feature long heads which contain long fast muscle fibres that attach to the apodeme at small angles. Their muscles comprise only a few filament-attached fibres and they maximize speed of action at the expense of force output. (2) Ants performing particularly forceful mandible movements, such as seed cracking, rely on many short parallel muscle fibres contained within a broad head capsule. Their slower muscles incorporate a large proportion of filament-attached fibres. Two simple models explain how the attachment angles are optimized with respect to force and velocity output and how filament-attached fibres help to generate the largest power output from the available head capsule volume.
Evolution of complexity in motor patterns and jaw musculature of tetraodontiform fishes
Authors: Friel John P. , Wainwright Peter C.
Published: April 2021

The prey-processing behavior and jaw-adducting musculature of tetraodontiform fishes provide a novel system for studying the evolution of muscles and their function. The history of this clade has involved a pattern of repeated ‘duplications’ of jaw muscles by physical subdivision of pre-existing muscles. As a result, the number of adductor mandibulae muscles in different taxa varies from as few as two to as many as eight. We used electromyography (EMG) to quantify motor-pattern variation of adductor mandibulae muscles in four tetraodontiform species during feeding events on prey items that varied in durability and elusiveness. Statistical analyses of variation in EMG variables revealed significant differences in motor patterns between duplicated muscles derived from a common ancestral muscle in seven of nine cases examined. Overall individual EMG timing variables (e.g. relative onset or duration of bursts) were slightly less likely to diverge functionally than amplitude variables (e.g. relative intensity of bursts). Functional divergence was found in significant overall differences between muscles and twice as frequently in significant muscle-by-prey interaction terms. Such interactions represent an underappreciated way in which motor patterns can evolve and diversify. Regional variation was documented in undivided muscles in two species, indicating that it is possible for functional subdivision to precede anatomical subdivision. This study shows that phylogenetic increases in the number of tetraodontiform jaw adductor muscles have been associated with increases in the functional complexity of the jaws at the level of muscle activation patterns.
CO2 excretion and postcapillary pH equilibration in blood-perfused turtle lungs
Authors: Stabenau Erich K. , Heming Thomas A.
Published: April 2021

Turtles possess a significant postcapillary CO2 partial pressure disequilibrium between arterial blood and alveolar gas. There are several possible explanations for this blood disequilibrium including a slow rate of erythrocyte physiological anion shift (Cl−/HCO3− exchange) or inaccessibility of plasma HCO3− to red blood cell or pulmonary carbonic anhydrase. The present study characterized the contribution of erythrocyte anion exchange and pulmonary and erythrocyte carbonic anhydrase to CO2 excretion and, hence, to postcapillary CO2–HCO3−–H+ equilibration in blood-perfused turtle (Pseudemys scripta) lungs. Turtle lungs perfused in situ with red cell suspensions containing inhibitors of erythrocyte anion exchange and/or pulmonary and red cell carbonic anhydrase produced significant postcapillary blood and pH disequilibria, while no disequilibria were measured when lungs were perfused with control red cell suspensions. Erythrocyte anion exchange and pulmonary intravascular carbonic anhydrase contributed 11 % and 9 %, respectively, to CO2 excretion during single-pass perfusion, whereas red cell and pulmonary carbonic anhydrase contributed 32 % to the measured CO2 excretion. The lack of a measurable disequilibrium during perfusion with control erythrocyte suspensions in this study suggests that alternative mechanisms may be responsible for the arterial–lung disequilibrium measured during breathing or diving episodes in turtles.
Heart rates and diving behavior of leatherback sea turtles in the eastern pacific ocean
Authors: Southwood Amanda L. , Andrews Russel D. , Lutcavage Molly E. , Paladino Frank V. , West Nigel H. , George Robert H. , Jones David R.
Published: April 2021

Heart rates and diving behavior of leatherback sea turtles (Dermochelys coriacea) were monitored at sea during the internesting interval. Instruments that recorded the electrocardiogram and the depth and duration of dives were deployed on six female leatherback turtles as they laid eggs at Playa Grande, Costa Rica. Turtles dived continually for the majority of the internesting interval and spent 57–68 % of the time at sea submerged. Mean dive depth was 19±1 m (mean ± S.D.) and the mean dive duration was 7.4±0.6 min. Heart rate declined immediately upon submergence and continued to fall during descent. All turtles showed an increase in heart rate before surfacing. The mean heart rate during dives of 17.4±0.9 beats min−1 (mean ± S.D.) was significantly lower than the mean heart rate at the surface of 24.9±1.3 beats min−1 (P<0.05). Instantaneous heart rates as low as 1.05 beats min−1 were recorded during a 34 min dive. The mean heart rate over the entire dive cycle (dive + succeeding surface interval; 19.4±1.3 beats min−1 ) was more similar to the heart rate during diving than to the heart rate at the surface. Although dive and surface heart rates were significantly different from each other, heart rates during diving were 70 % of heart rates at the surface, showing that leatherback turtles do not experience a dramatic bradycardia during routine diving.
Impulse conduction in a sponge
Authors: Leys S. P. , Mackie G. O. , Meech R. W.
Published: April 2021

All-or-none propagated electrical impulses were recorded from the hexactinellid sponge Rhabdocalyptus dawsoni using suction electrodes attached to lumps of aggregated sponge tissue grafted onto the surface of pieces of the same sponge. Impulses were normally evoked by means of externally applied electrical shocks. Recorded externally using an a.c.-coupled amplifier, the electrical event was triphasic and lasted approximately 30 s; integration gave a diphasic waveform. A further integration to give the form of the membrane action potential produced a monophasic signal. Impulses propagated at 0.27±0.1 cm s−1 with an absolute refractory period of 29 s and a relative refractory period of approximately 150 s. Concurrent thermistor flow meter recordings confirmed that water flow through the sponge was arrested following the passage of an impulse, presumably as result of the cessation of beating of the flagella in the flagellated chambers. Tactile stimuli also evoked impulses, as did addition of particulate material to the incoming water stream. Impulses continued to propagate through the sponge during arrests, indicating that the conduction and effector systems were independent. Sponges lack nerves, and a variety of evidence indicates that the conducting tissues are the syncytial trabecular reticulum and pinacoderm layers. Na+ -deficient solutions had little effect on the action potential, but propagation was blocked by 10 mmol l−1 Co2+, 1 mmol l−1 Mn2+ or 24 μmol l−1 nimodipine. Tetraethylammonium ions at 1–5 mmol l−1 also blocked propagation without prolonging the action potential. Impulse conduction in the sponge is discussed in relation to excitability and conduction in the protozoa and in plants and to non-nervous conduction in more advanced animals.
Luminal Acidification of Diverse Organelles by V-ATPase in Animal Cells
Authors: Futai Masamitsu , Oka Toshihiko , Sun-Wada Ge-Hong , Moriyama Yoshinori , Kanazawa Hiroshi , Wada Yoh
Published: April 2021

Eukaryotic cells contain organelles bounded by a single membrane in the cytoplasm. These organelles have differentiated to carry out various functions in the pathways of endocytosis and exocytosis. Their lumina are acidic, with pH ranging from 4.5 to 6.5. This article describes recent studies on these animal cell organelles focusing on (1) the primary proton pump (vacuolar-type H+-ATPase) and (2) the functions of the organelle luminal acidity. We also discuss similarities and differences between vacuolar-type H+-ATPase and F-type ATPase. Our own studies and interests are emphasized.
Structure and Regulation of Insect Plasma Membrane H+ V-ATPase
Authors: Wieczorek Helmut , Grüber Gerhard , Harvey William R. , Huss Markus , Merzendorfer Hans , Zeiske Wolfgang
Published: April 2021

H+ V-ATPases (V-ATPases) are found in two principal locations, in endomembranes and in plasma membranes. The plasma membrane V-ATPase from the midgut of larval Manduca sexta is the sole energizer of all transepithelial secondary transport processes. At least two properties make the lepidopteran midgut a model tissue for studies of general aspects of V-ATPases. First, it is a rich source for purification of the enzyme and therefore for structural studies: 20 larvae provide up to 0.5 mg of holoenzyme, and soluble, cytosolic V1 complexes can be obtained in even greater amounts of up to 2 mg.

Second, midgut ion-tranport processes are strictly controlled by the regulation of the V-ATPase, which is the sole energizer of all ion transport in this epithelium. Recent advances in our understanding the structure of the V1 and Vo complexes and of the regulation of the enzyme’s biosynthesis and ion-transport activity will be discussed.
H+V-ATPase-Dependent Luminal Acidification in the Kidney Collecting Duct and the Epididymis/Vas Deferens: Vesicle Recycling and Transcytotic Pathways
Authors: Brown Dennis , Breton Sylvie
Published: April 2021

Many vertebrate transporting epithelia contain characteristic ‘mitochondria-rich’ cells that express high levels of a vacuolar proton-pumping ATPase (H+V-ATPase) on their plasma membrane and on intracellular vesicles. In the kidney cortex, A-cells and B-cells are involved in proton secretion and bicarbonate secretion, respectively, in the distal nephron and collecting duct. A-cells have an H+V-ATPase on their apical plasma membrane and on intracellular vesicles, whereas the cellular location of the H+V-ATPase can be apical, basolateral, bipolar or diffuse in B-cells. The rat epididymis and vas deferens also contain a distinct population of H+V-ATPase-rich epithelial cells. These cells are involved in generating a low luminal pH, which is involved in sperm maturation and in maintaining sperm in an immotile state during their passage through the epididymis and vas deferens. In both kidney and reproductive tract, H+V-ATPase-rich cells have a high rate of apical membrane recycling. H+V-ATPase molecules are transported between the cell surface and the cytoplasm in vesicles that have a well-defined ‘coat’ structure formed of the peripheral V1 subunits of the H+V-ATPase. In addition, we propose that B-type intercalated cells have a transcytotic pathway that enables them to shuttle H+V-ATPase molecules from apical to basolateral plasma membrane domains. This hypothesis is supported by data showing that A-cells and B-cells have different intracellular trafficking pathways for LGP120, a lysosomal glycoprotein. LGP120 was found both on the basolateral plasma membrane and in lysosomes in B-cells, whereas no LGP120 was detectable in the plasma membrane of A-cells. We propose that the ‘polarity reversal’ of the H+V-ATPase in B-intercalated cells is mediated by a physiologically regulated transcytotic pathway that may be similar to that existing in some other cell types.
Structure and Function of the Fo Complex of the ATP Synthase from Escherichia Coli
Authors: Altendorf Karlheinz , Stalz Wolf-Dieter , Greie Jörg-Christian , Deckers-Hebestreit Gabriele
Published: April 2021

The membrane-bound ATP synthase (F1Fo) from mitochondria, chloroplasts and bacteria plays a crucial role in energy-transducing reactions. In the case of Escherichia coli, the reversible, proton-translocating ATPase complex consists of two different entities, F1 and Fo. The water-soluble F1 part carries the catalytic sites for ATP synthesis and hydrolysis. It is associated with the membrane-embedded Fo complex, which functions as a proton channel and consists of subunits a, b and c present in a stoichiometry of 1:2:12.

Subunit b was isolated by preparative gel electrophoresis, acetone-precipitated and renatured in a cholate-containing buffer. Reconstituted subunit b together with purified ac subcomplex is active in proton translocation and F1 binding, thereby demonstrating that subunit b had recovered its native conformation. Circular dichroism spectroscopy of subunit b reconstituted into liposomes revealed a rather high degree of α -helical conformation of 80 %.

After addition of a His6-tag to the N terminus of subunit a, a stable ab2 subcomplex was purified instead of a single subunit a, arguing in favour of a direct interaction between these subunits. After addition of subunit c and reconstitution into phospholipid vesicles, an Fo complex was obtained exhibiting rates of proton translocation and F1 binding comparable with those of wild-type Fo.

The epitopes of monoclonal antibodies against subunit c are located in the hydrophilic loop region (cL31–Q42) as mapped by enzyme-linked immunosorbent assay using overlapping synthetic heptapeptides. Binding studies revealed that all monoclonal antibodies (mAbs) bind to everted membrane vesicles irrespective of the presence or absence of F1. Although the hydrophilic region of subunit c, and especially the highly conserved residues cA40, cR41, cQ42 and cP43, are known to interact with subunits γ and ε of the F1 part, the mAb molecules have no effect on the function of Fo, either in proton translocation or in F1 binding. However, the F1 part and the mAb molecule(s) are bound simultaneously to the Fo complex, suggesting that not all c subunits are involved in the interaction with F1.
Cross-Linking and Electron Microscopy Studies of the Structure and Functioning of the Escherichia Coli ATP Synthase
Authors: Capaldi Roderick A. , Schulenberg Birte , Murray James , Aggeler Robert
Published: April 2021

ATP synthase, also called F1Fo-ATPase, catalyzes the synthesis of ATP during oxidative phosphorylation. The enzyme is reversible and is able to use ATP to drive a proton gradient for transport purposes. Our work has focused on the enzyme from Escherichia coli (ECF1Fo). We have used a combination of methods to study this enzyme, including electron microscopy and chemical cross-linking.

The utility of these two approaches in particular, and the important insights they give into the structure and mechanism of the ATP synthase, are reviewed.
Rate Acceleration of ATP Hydrolysis by F1Fo-ATP Synthase
Authors: Senior Alan E. , Nadanaciva Sashi , Weber Joachim
Published: April 2021

The rate acceleration of ATP hydrolysis by F1Fo-ATP synthase is of the order of 1011-fold. We present a cyclic enzyme mechanism for the reaction, relate it to known F1 X-ray structure and speculate on the linkage between enzyme reaction intermediates and subunit rotation. Next, we describe five factors known to be important in the Escherichia coli enzyme for the rate acceleration. First, the provision of substrate binding energy by residues lining the catalytic site is substantial; β-Lys155 and β-Arg182 are specific examples, both of which differentially support substrate MgATP versus product MgADP binding. Second, octahedral coordination of the Mg2+ in MgATP is crucial for both catalysis and catalytic site asymmetry. The residues involved are β-Thr156, β-Glu185 and β-Asp242. Third, there is stabilization of a pentacoordinate phosphorus catalytic transition state by residues β-Lys155, β-Arg182 and α-Arg376. Fourth, residue β-Glu181 binds the substrate water and stabilizes the catalytic transition state. Fifth, there is strong positive catalytic cooperativity, with binding of MgATP at all three sites yielding the maximum rate (Vmax); the molecular basis of this factor remains to be elucidated.
Steady-State and Pre-Steady-State Kinetics of the Mitochondrial F1Fo ATPase: Is Atp Synthase a Reversible Molecular Machine?
Authors: Vinogradov Andrei D.
Published: April 2021

H+-ATP synthase (F1Fo ATPase) catalyzes the synthesis and/or hydrolysis of ATP, and the reactions are strongly affected by all the substrates (products) in a way clearly distinct from that expected of a simple reversibly operating enzyme. Recent studies have revealed the structure of F1, which is ideally suited for the alternating binding change mechanism, with a rotating γ-subunit as the energy-driven coupling device. According to this mechanism ATP, ADP, inorganic phosphate (Pi) and Mg2+ participate in the forward and reverse overall reactions exclusively as the substrates and products. However, both F1 and F1Fo demonstrate non-trivial steady-state and pre-steady-state kinetics as a function of variable substrate (product) concentrations. Several effectors cause unidirectional inhibition or activation of the enzyme. When considered separately, the unidirectional effects of ADP, Pi, Mg2+ and energy supply on ATP synthesis or hydrolysis may possibly be explained by very complex kinetic schemes; taken together, the results suggest that different conformational states of the enzyme operate in the ATP hydrolase and ATP synthase reactions. A possible mechanism for an energy-dependent switch between the two states of F1Fo ATPase is proposed.
Crucial Role of the Membrane Potential for ATP Synthesis by F1Fo ATP Synthases
Authors: Dimroth Peter , Kaim Georg , Matthey Ulrich
Published: April 2021

ATP, the universal carrier of cell energy, is manufactured from ADP and phosphate by the enzyme ATP synthase using the free energy of an electrochemical gradient of protons (or Na+). The proton-motive force consists of two components, the transmembrane proton concentration gradient (ΔpH) and the membrane potential. The two components were considered to be not only thermodynamically but also kinetically equivalent, since the chloroplast ATP synthase appeared to operate on ΔpH only. Recent experiments demonstrate, however, that the chloroplast ATP synthase, like those of mitochondria and bacteria, requires a membrane potential for ATP synthesis. Hence, the membrane potential and proton gradient are not equivalent under normal operating conditions far from equilibrium. These conclusions are corroborated by the finding that only the membrane potential induces a rotary torque that drives the counter-rotation of the a and c subunits in the Fo motor of Propionigenium modestum ATP synthase.
Structure, Mechanism and Regulation of the Clathrin-Coated Vesicle and Yeast Vacuolar H+-ATPases
Authors: Forgac Michael
Published: April 2021

The vacuolar H+-ATPases (or V-ATPases) are a family of ATP-dependent proton pumps that carry out acidification of intracellular compartments in eukaryotic cells. This review is focused on our work on the V-ATPases of clathrin-coated vesicles and yeast vacuoles. The coated-vesicle V-ATPase undergoes trafficking to endosomes and synaptic vesicles, where it functions in receptor recycling and neurotransmitter uptake, respectively. The yeast V-ATPase functions to acidify the central vacuole and is necessary both for protein degradation and for coupled transport processes across the vacuolar membrane.

The V-ATPases are multisubunit complexes composed of two functional domains. The V1 domain is a 570 kDa peripheral complex composed of eight subunits of molecular mass 73–14 kDa (subunits A–H) that is responsible for ATP hydrolysis. The Vo domain is a 260 kDa integral complex composed of five subunits of molecular mass 100–17 kDa (subunits a, d, c, c′ and c′′) that is responsible for proton translocation. To explore the function of individual subunits in the V-ATPase complex as well as to identify residues important in proton transport and ATP hydrolysis, we have employed a combination of chemical modification, site-directed mutagenesis and in vitro reassembly.

A central question concerns the mechanism by which vacuolar acidification is controlled in eukaryotic cells. We have proposed that disulfide bond formation between conserved cysteine residues at the catalytic site of the V-ATPase plays an important role in regulating V-ATPase activity in vivo. Other regulatory mechanisms that are discussed include reversible dissociation and reassembly of the V-ATPase complex, changes in the tightness of coupling between proton transport and ATP hydrolysis, differential targeting of V-ATPases within the cell and control of the Cl− conductance that is necessary for vacuolar acidification.
Surface Oxidase and Oxidative Stress Propagation in Aging
Authors: Morré Dorothy M. , Lenaz Giorgio , Morré D. James
Published: April 2021

This report summarizes new evidence for a plasma-membrane-associated hydroquinone oxidase designated as CNOX (constitutive plasma membrane NADH oxidase) that functions as a terminal oxidase for a plasma membrane oxidoreductase (PMOR) electron transport chain to link the accumulation of lesions in mitochondrial DNA to cell-surface accumulations of reactive oxygen species. Previous considerations of plasma membrane redox changes during aging have lacked evidence for a specific terminal oxidase to catalyze a flow of electrons from cytosolic NADH to molecular oxygen (or to protein disulfides). Cells with functionally deficient mitochondria become characterized by an anaerobic metabolism. As a result, NADH accumulates from the glycolytic production of ATP. Elevated PMOR activity has been shown to be necessary to maintain the NAD+/NADH homeostasis essential for survival. Our findings demonstrate that the hyperactivity of the PMOR system results in an NADH oxidase (NOX) activity capable of generating reactive oxygen species at the cell surface. This would serve to propagate the aging cascade both to adjacent cells and to circulating blood components. The generation of superoxide by NOX forms associated with aging is inhibited by coenzyme Q and provides a rational basis for the anti-aging activity of circulating coenzyme Q.
Dynamic Mechanical Characterization of a Mutable Collagenous Tissue: Response of Sea Cucumber Dermis to Cell Lysis and Dermal Extracts
Authors: Szulgit Greg K. , Shadwick Robert E.
Published: April 2021

The dermis of the holothurian Cucumaria frondosa is a mutable collagenous tissue (MCT). In this study, the inner and outer regions of the dermis were separated and used to make two different tissue extracts. These extracts were applied to intact pieces of dermis, one invoking a stiff mechanical state and the other invoking a compliant state. The extracts were effective on tissues incubated in artificial sea water (ASW) and in those incubated in Ca2+-chelated ASW. Furthermore, the extracts were effective on both fresh tissues and tissues in which the cells had been lysed by freeze–thawing, indicating that the sites of action are in the extracellular matrix. Dynamic oscillatory shear tests and analyses were used to measure both the dynamic shear stiffness (G*) and the relative damping (tanδ) of the tissue. These two parameters proved to be inversely related to each other (i.e. when G* increased, tanδ decreased). A theoretical viscoelastic model is constructed to interpret the results of these tests. It is concluded that changes in the mechanical state of the tissue involve interactions between elastic elements within the tissue rather than an alteration of its viscous components.
Hagfish (Myxine Glutinosa) Red Cell Membrane Exhibits no Bicarbonate Permeability as Detected by 18O Exchange
Authors: Peters Thomas , Forster Robert E. , Gros Gerolf
Published: April 2021

The bicarbonate permeability of the plasma membrane of intact hagfish (Myxine glutinosa) red blood cells and the intracellular carbonic anhydrase activity of these cells were determined by applying the 18O exchange reaction using a special mass spectrometric technique. When the macromolecular carbonic anhydrase inhibitor Prontosil–Dextran was used to suppress any extracellular carbonic anhydrase activity, the mean intracellular acceleration of the CO2 hydration/HCO3− dehydration reaction over the uncatalyzed reaction (referred to as intracellular carbonic anhydrase activity Ai) was 21 320±3000 at 10 °C (mean ± S.D., N=9). The mean bicarbonate permeability of the red blood cell membrane was indistinguishable from zero. It can be concluded that CO2 transport within hagfish blood does not follow the classical scheme of CO2 transport in vertebrate blood. It is suggested that the combination of considerable intraerythrocytic carbonic anhydrase activity and low may serve to enhance O2 delivery to the tissue in the exceptionally hypoxia-tolerant hagfish.
Comb-Wax Discrimination by Honeybees Tested with the Proboscis Extension Reflex
Authors: Fröhlich Birgit , Riederer Markus , Tautz Jürgen
Published: April 2021

We used the proboscis extension reflex of honeybees to test their ability to discriminate between comb waxes of different ages (wax scales, 1-week-old wax, 2-to 3-year-old wax, 8-to 10-year-old wax). Such waxes differ in their chemical composition, and an ability to discriminate between them may aid the orientation of the bees in the nest.

To train the bees, we used whole extracts of waxes and four different fractions of the whole extract based on different elutions of solid-phase extractions (extract I, fraction A eluted with hexane and fraction B with diethylether; extract II, fraction B further subdivided into fraction C by elution with isopropylchloride and fraction D by elution with diethylether).

In a differential training regime (six learning and six test trials) with whole extracts or with the different fractions, we paired one type of wax with a reward and another with no reward. The bees learned to discriminate between all tested pairs of whole extracts. The two subfractions (fractions A and B) gave different results: the bees could discriminate between waxes of different ages when fraction B was used but not when fraction A was used. A further subdivision of fraction B into fractions C and D showed that only fraction D contained the elements that enabled bees to discriminate between old and new wax.

Fraction D makes up only 5–8 % of the total wax mass and contains hydroxy alkyl esters (5–6 % of the total wax mass), primary alcohols (0.3–0.5 % of the total wax mass) and acids (0.06–1.0 % of the total wax mass). Fractions A and C (together forming 62–64 % of the total wax mass), which consist of unbranched and branched aliphatic hydrocarbons and alkyl esters, could not be discriminated by the bees. The remaining wax mass (25–29 %) was eluted with a mixture of chloroform, methanol and water (13:5:1) as fraction E.
The Emetic Reflex in a Reptile (Crocodylus Porosus)
Authors: Andrews Paul L. R. , Axelsson Michael , Franklin Craig , Holmgren Susanne
Published: April 2021

The emetic (vomiting) reflex in a crocodilian, Crocodylus porosus, was characterised for the first time using the plant alkaloid veratrine (5 mg kg−1i.v. or i.p.) as an emetic stimulus. The latency to the onset of vomiting was 8.0±0.9 min (mean ± S.E.M., N=5 animals). Vomiting was preceded by a clearly defined set of prodromal behaviours including, in temporal sequence, rhythmic contraction of the pharynx, sneezing and jaw snapping. Expulsion of vomitus was not particularly forceful and was accompanied by lateral shaking of the head. Physiological studies revealed that vomiting was accompanied by oscillatory (9.1±0.7 oscillations over 29.7±3.6 s, N=9 episodes in three animals) increases in intraperitoneal pressure (7.0±0.9 kPa, cf. 0.7±0.1 kPa during respiration). The significance of these results is discussed in the context of the role(s) of vomiting as a protective reflex and as a mechanism for removal of indigestible food residues (e.g. fur, claws) from the gut.
Digital Motion Analysis As A Tool For Analysing The Shape And Performance of The Circulatory System in Transparent Animals
Authors: Schwerte Thorsten , Pelster Bernd
Published: April 2021

The analysis of perfusion parameters using the frame-to-frame technique and the observation of small blood vessels in transparent animals using video microscopy can be tedious and very difficult because of the poor contrast of the images. Injection of a fluorescent probe (fluorescein isothiocynate, FITC) bound to a high-molecular-mass dextran improved the visibility of blood vessels, but the gray-scale histogram showed blurring at the edges of the vessels. Furthermore, injection of the fluorescent probe into the ventricle of small zebrafish (Danio rerio) embryos (body mass approximately 1 mg) often resulted in reduced cardiac activity. Digital motion analysis, however, proved to be a very effective tool for analysing the shape and performance of the circulatory system in transparent animals and tissues. By subtracting the two fields of a video frame (the odd and the even frame), any movement that occurred within the 20 ms necessary for the acquisition of one field could be visualised. The length of the shifting vector generated by this subtraction, represented a direct measure of the velocity of a moving particle, i.e. an erythrocyte in the vascular system. By accumulating shifting vectors generated from several consecutive video frames, a complete trace of the routes over which erythrocytes moved could be obtained. Thus, a cast of the vascular system, except for those tiny vessels that are not entered by erythrocytes, could be obtained. Because the gray-scale value of any given pixel or any given group of pixels increased with the number of erythrocytes passing it, digital motion analysis could also be used to visualise the distribution of blood cells in transparent tissues. This method was used to describe the development of the peripheral vascular system in zebrafish larvae up to 8 days post-fertilisation. At this stage, food intake resulted in a clear redistribution of blood between muscle tissue and the gut, and α-adrenergic control of peripheral blood flow was established.
5-Hydroxytryptamine Modulates Pheromone-Evoked Local Field Potentials in The Macroglomerular Complex of The Sphinx Moth Manduca Sexta
Authors: Kloppenburg Peter , Heinbockel Thomas
Published: April 2021

Extra- and intracellular recordings from an intact brain preparation were used to study the effects of 5-hydroxytryptamine (5-HT or serotonin) on projection neurons in the sexually dimorphic macroglomerular complex (MGC) in the antennal lobe of the male moth Manduca sexta. The MGC is a group of three identified glomeruli specialized for synaptic processing of primary afferent information about the multi-component sex pheromone of the female. We investigated the modulatory effects of 5-HT on pheromone-evoked local field potentials in the MGC. The magnitude and duration of these potentials, which are thought to be generated by a population of pheromone-sensitive projection neurons of the MGC, were increased by 5-HT. Using intracellular recordings from the neurites of individual MGC projection neurons, we found that 5-HT increased the number of action potentials in response to pheromonal stimulation. These findings correlate well with earlier experiments that used other recording techniques. Our results are further evidence that 5-HT modulates a population of pheromone-sensitive MGC projection neurons that relay information about the pheromonal stimulus from the MGC to higher-order centers in the protocerebrum and are therefore pivotal for mate-finding and odor-guided behavior.