bluuebunny/crossref_metadata_2025 huggingface dataset:
Physiological optics in the hummingbird hawkmoth: a compound eye without ommatidia
Authors: Warrant Eric , Bartsch Klaus , Günther Claudia
Published: April 2021

The fast-flying day-active hawkmoth Macroglossum stellatarum (Lepidoptera: Sphingidae) has a remarkable refracting superposition eye that departs radically from the classical principles of Exnerian superposition optics. Unlike its classical counterparts, this superposition eye is highly aspherical and contains extensive gradients of resolution and sensitivity. While such features are well known in apposition eyes, they were thought to be impossible in superposition eyes because of the imaging principle inherent in this design. We provide the first account of a superposition eye where these gradients are not only possible, but also produce superposition eyes of unsurpassed quality. Using goniometry and ophthalmoscopy, we find that superposition images formed in the eye are close to the diffraction limit. Moreover, the photoreceptors of the superposition eyes of M. stellatarum are organised to form local acute zones, one of which is frontal and slightly ventral, and another of which provides improved resolution along the equator of the eye. This angular packing of rhabdoms bears no resemblance to the angular packing of the overlying corneal facets. In fact, this eye has many more rhabdoms than facets, with up to four rhabdoms per facet in the frontal eye, a situation which means that M. stellatarum does not possess ommatidia in the accepted sense. The size of the facets and the area of the superposition aperture are both maximal at the frontal retinal acute zone. By having larger facets, a wider aperture and denser rhabdom packing, the frontal acute zone of M. stellatarum provides the eye with its sharpest and brightest image and samples the image with the densest photoreceptor matrix. It is this eye region that M. stellatarum uses to fixate flower entrances during hovering and feeding. This radical departure from classical Exnerian principles has resulted in a superposition eye which has not only high sensitivity but also outstanding spatial resolution.
White muscle strain in the common carp and red to white muscle gearing ratios in fish
Authors: Wakeling James M. , Johnston Ian A.
Published: April 2021

White muscle strains were recorded using sonomicrometry techniques for 70 fast-starts in the common carp Cyprinus carpio L. High-speed ciné images were recorded simultaneously for 54 of these starts, and muscle strain was calculated independently from the digitized outlines of the fish. Sonomicrometry measurements of superficial muscle strain were not significantly different from the strain as calculated from the theory of simple bending of a homogeneous material: superficial muscle strain thus varied with chordwise distance from the spine. However, white muscle strain across a transverse section of the myotome shows less variation with chordwise position than would be expected from simple bending theory. Muscle strains measured using sonomicrometry thus do not necessarily represent the more uniform strain predicted for the whole section of the fish. White muscle strain can be accurately predicted from the spine curvatures as measured from the ciné images if the gearing ratio between the red and white muscle fibres is known. A model for calculating the gearing ratio from the helical muscle fibre geometry was re-evaluated using current data for the kinematics of fast-starting C. carpio. This model predicted a mean gearing ratio of 2.8 for these fast-starts. A quicker, alternative approach to estimating gearing ratio from the position of the centroid of white fibre area is proposed and results in ratios similar to those calculated from the model of helical geometry. White muscle strains in fish can thus be estimated from measurements of spine curvature and muscle distribution alone.
The development of microcracking and failure in bone depends on the loading mode to which it is adapted
Authors: Reilly Gwendolen C. , Currey John D.
Published: April 2021

During locomotion, the anterior cortex of the equine radius is loaded predominantly in tension, the posterior predominantly in compression. The anterior cortex is relatively strong in tension, the posterior in compression. We investigated the pattern of failure of specimens from the two cortices using laser scanning confocal microscopy.

All specimens were loaded in four-point bending to increasingly higher loads. We quantified the amount of diffuse microcracking on the tensile side of these specimens by observing the amount of light emitted under laser illumination. The amount of light emitted agreed well with subjective estimates of the amount of microcracking. Tensile microcracks first appeared at a strain of approximately 0.004, and all specimens showed considerable growth in microcrack density once the tensile strain had passed approximately 0.008.

In specimens from the posterior cortex, there was little compressive microcracking, and such cracks as were present were small and diffuse. These specimens failed on the tensile side first. In specimens from the anterior cortex, compression cracks were more numerous, longer and less diffuse, and specimens failed initially in compression.

The patterns of failure in the bone tissues of the two cortices are what would be expected assuming they were adapted to the mode of loading to which they are usually subjected.
Immune-mediated alterations in nociceptive sensory function in Aplysia californica
Authors: Clatworthy Andrea L. , Grose Eric
Published: April 2021

Nerve injury in Aplysia californica is accompanied by a profound long-lasting enhancement of the excitability of nociceptive sensory neurons that have axons in injured nerves. It is likely that a variety of signals are involved in triggering this injury-induced sensory plasticity. The objective of the present study was to determine whether cells of the cellular defense system (hemocytes) play a role in the modulation of sensory excitability following injury. In support of such an idea, we have shown previously that the induction of a cellular defense reaction close to sensory axons is accompanied by an increase in the excitability of sensory neurons with axons close to responding hemocytes. Furthermore, in the present study, we verified that, following axonal crush, numerous hemocytes accumulate at the injured site on the nerve. Using a hemocyte/nervous system co-culture preparation, we found that there were no significant differences in the expression of injury-induced sensory plasticity between sensory neurons incubated in the presence or absence of hemocytes. To overcome some potential limitations of our co-culture preparation, we used the endotoxin lipopolysaccharide (LPS) as a tool to activate the hemocytes. Sensory cells incubated in the presence of LPS and hemocytes were significantly more excitable than sensory cells incubated in the presence of LPS alone. We speculate that the addition of LPS to the incubation medium containing hemocytes enhanced the release of hemocyte-derived cytokine-like factors such as interleukin-1 and tumor necrosis factor. These cytokine-like factors may act as signals to modulate the expression of injury-induced sensory hyperexcitability.
Inducible NO synthase: role in cellular signalling
Authors: Beck Karl-Friedrich , Eberhardt Wolfgang , Frank Stefan , Huwiler Andrea , Meßmer Udo K. , Mühl Heiko , Pfeilschifter Josef
Published: April 2021

The discovery of endothelium-derived relaxing factor and its identification as nitric oxide (NO) was one of the most exciting discoveries of biomedical research in the 1980s. Besides its potent vasodilatory effects, NO was found under certain circumstances to be responsible for the killing of microorganisms and tumour cells by activated macrophages and to act as a novel, unconventional type of neurotransmitter. In 1992, Science picked NO as the ‘Molecule of the Year’, and over the past years NO has become established as a universal intercellular messenger that acutely affects important signalling pathways and, on a more long-term scale, modulates gene expression in target cells. These actions will form the focus of the present review.
Development of diving capacity in emperor penguins
Authors: Ponganis P. J. , Starke L. N. , Horning M. , Kooyman G. L.
Published: April 2021

To compare the diving capacities of juvenile and adult emperor penguins Aptenodytes forsteri, and to determine the physiological variables underlying the diving ability of juveniles, we monitored diving activity in juvenile penguins fitted with satellite-linked time/depth recorders and examined developmental changes in body mass (Mb), hemoglobin concentration, myoglobin (Mb) content and muscle citrate synthase and lactate dehydrogenase activities. Diving depth, diving duration and time-at-depth histograms were obtained from two fledged juveniles during the first 2.5 months after their depature from the Cape Washingon colony in the Ross Sea, Antarctica. During this period, values of all three diving variables increased progressively. After 8–10 weeks at sea, 24–41 % of transmitted maximum diving depths were between 80 and 200 m. Although most dives lasted less than 2 min during the 2 month period, 8–25 % of transmitted dives in the last 2 weeks lasted 2–4 min. These values are lower than those previously recorded in adults during foraging trips. Of the physiological variables examined during chick and juvenile development, only Mb and Mb content did not approach adult values. In both near-fledge chicks and juveniles, Mb was 50–60 % of adult values and Mb content was 24–31 % of adult values. This suggests that the increase in diving capacity of juveniles at sea will be most dependent on changes in these factors.
Brain blood flow and blood pressure during hypoxia in the epaulette shark Hemiscyllium ocellatum, a hypoxia-tolerant elasmobranch
Authors: Söderström Veronica , Renshaw Gillian M. C. , Nilsson Göran E.
Published: April 2021

The key to surviving hypoxia is to protect the brain from energy depletion. The epaulette shark (Hemiscyllium ocellatum) is an elasmobranch able to resist energy depletion and to survive hypoxia. Using epi-illumination microscopy in vivo to observe cerebral blood flow velocity on the brain surface, we show that cerebral blood flow in the epaulette shark is unaffected by 2 h of severe hypoxia (0.35 mg O2 l−1 in the respiratory water, 24 °C). Thus, the epaulette shark differs from other hypoxia- and anoxia-tolerant species studied: there is no adenosine-mediated increase in cerebral blood flow such as that occurring in freshwater turtles and cyprinid fish. However, blood pressure showed a 50 % decrease in the epaulette shark during hypoxia, indicating that a compensatory cerebral vasodilatation occurs to maintain cerebral blood flow. We observed an increase in cerebral blood flow velocity when superfusing the normoxic brain with adenosine (making sharks the oldest vertebrate group in which this mechanism has been found). The adenosine-induced increase in cerebral blood flow velocity was reduced by the adenosine receptor antagonist aminophylline. Aminophylline had no effect upon the maintenance of cerebral blood flow during hypoxia, however, indicating that adenosine is not involved in maintaining cerebral blood flow in the epaulette shark during hypoxic hypotension.
Water skating in the larvae of Dixella aestivalis (Diptera) and Hydrobius fuscipes (Coleoptera)
Authors: Brackenbury John
Published: April 2021

The kinematics of locomotion was investigated in the aquatic larvae of Dixella aestivalis and Hydrobius fuscipes with the aid of high-speed video recordings. Both insects are able to skate on the surface of the water using the dorso-apical tracheal gill as an adhesive organ or ‘foot’. Progress relies on the variable adhesion of the foot between ‘slide’ and ‘hold’ periods of the locomotory cycle. The flexural body movements underlying skating in D. aestivalis can be derived directly from the figure-of-eight swimming mechanism used in underwater swimming. The latter is shown to be similar to figure-of-eight swimming in chironomid larvae. This study shows how the deployment of a ‘foot’ enables simple side-to-side flexural movements of the body to be converted into effective locomotion at the air–water interface.
Vasodilation of swimbladder vessels in the european eel (Anguilla anguilla) induced by vasoactive intestinal polypeptide, nitric oxide, adenosine and protons
Authors: Schwerte Thorsten , Holmgren Susanne , Pelster Bernd
Published: April 2021

The effects of β-adrenergic stimulation, vasoactive intestinal polypeptide (VIP), adenosine, the nitric oxide (NO)-releasing agent sodium nitroprusside and of metabolic end-products of gas gland cell metabolism on swimbladder blood flow were investigated using saline- or blood-perfused swimbladder preparations of the freshwater European eel Anguilla anguilla. While β-adrenergic vasodilation was not detectable, a bolus injection of adenosine (100 μl, 10−7 mol l−1) and application of VIP (10−7 mol kg−1) caused a significant decrease in perfusion pressure in saline-perfused swimbladder preparations. Immunohistochemical analysis revealed the presence of VIP-immunoreactive nerve fibres in the swimbladder artery and in the swimbladder vein (seawater-adapted eels were used for immunohistochemical studies). Application of sodium nitroprusside also elicited a small, but significant, decrease in perfusion pressure in saline-perfused swimbladder preparations, while preincubation of swimbladder tissue with Nωnitro-L-arginine, a non-selective inhibitor of nitric oxide synthase, significantly enhanced the flow-induced increase in perfusion pressure. Lactate, the major metabolic end-product of gas gland cell metabolism, had no effect on perfusion pressure. In contrast, an increase in proton concentration in both saline- and blood-perfused preparations induced a vasodilation, as indicated by a significant decrease in perfusion pressure. The results demonstrate that VIP, NO, adenosine and protons may induce a vasodilation of swimbladder blood vessels. None of these effects, however, compares in time span with the previously described immediate, short-lasting vasodilation of swimbladder vessels elicited by pulse stimulation of the vagus nerve.
Secretion of primary urine by glomeruli of the hagfish kidney
Authors: Riegel J. A.
Published: April 2021

Adrenaline and noradrenaline increased the perfusion pressure (Pperf) and single glomerulus filtration rate (SGFR) of perfused hagfish glomeruli. Small amounts (0.1 % or 0.5 %) of bovine serum albumin (BSA) in perfusion fluids containing Ficoll 70 did not diminish the loss of colloid from hagfish glomerular capillaries as has been reported for other perfused capillaries. However, replacement of Ficoll 70 with an osmotically equivalent amount (3 %) of BSA appreciably reduced colloid loss. It was concluded that adrenaline and colloids enhanced flow through the urine-forming capillaries. Whereas adrenaline elevated the SGFR, colloid lowered the SGFR probably by a direct effect on the fluid permeability of the capillary walls.

The flow-enhancing effect of adrenaline was used to ensure the exposure of urine-forming capillaries to two inhibitors of active fluid transport, ouabain and 2,4-dinitrophenol (DNP). Both substances lowered the single glomerulus filtration fraction (SGFF), probably by affecting a fluid secretion mechanism. In addition, DNP diminished the flow-enhancing effect of adrenaline. This study provides relatively unequivocal evidence that fluid secretion underlies the formation of primary urine by the hagfish.
In vivo locomotor strain in the hindlimb bones of Alligator mississippiensis and Iguana iguana: implications for the evolution of limb bone safety factor and non-sprawling limb posture
Authors: Blob Richard W. , Biewener Andrew A.
Published: April 2021

Limb postures of terrestrial tetrapods span a continuum from sprawling to fully upright; however, most experimental investigations of locomotor mechanics have focused on mammals and ground-dwelling birds that employ parasagittal limb kinematics, leaving much of the diversity of tetrapod locomotor mechanics unexplored. This study reports measurements of in vivo locomotor strain from the limb bones of lizard (Iguana iguana) and crocodilian (Alligator mississippiensis) species, animals from previously unsampled phylogenetic lineages with non-parasagittal limb posture and kinematics. Principal strain orientations and shear strain magnitudes indicate that the limb bones of these species experience considerable torsion during locomotion. This contrasts with patterns commonly observed in mammals, but matches predictions from kinematic observations of axial rotation in lizard and crocodilian limbs. Comparisons of locomotor load magnitudes with the mechanical properties of limb bones in Alligator and Iguana indicate that limb bone safety factors in bending for these species range from 5.5 to 10.8, as much as twice as high as safety factors previously calculated for mammals and birds. Limb bone safety factors in shear (3.9–5.4) for Alligator and Iguana are also moderately higher than safety factors to yield in bending for birds and mammals. Finally, correlations between limb posture and strain magnitudes in Alligator show that at some recording locations limb bone strains can increase during upright locomotion, in contrast to expectations based on size-correlated changes in posture among mammals that limb bone strains should decrease with the use of an upright posture. These data suggest that, in some lineages, strain magnitudes may not have been maintained at constant levels through the evolution of a non-sprawling posture unless the postural change was accompanied by a shift to parasagittal kinematics or by an evolutionary decrease in body size.
Kinematics of intraoral transport and swallowing in the herbivorous lizard Uromastix acanthinurus
Authors: Herrel Anthony , Vree Frits de
Published: April 2021

The kinematics of intraoral transport and swallowing in lizards of the species Uromastix acanthinurus (Chamaeleonidae, Leiolepidinae) were investigated using cineradiography (50 frames s−1 ). Additional recordings were also made using high-speed (500 frames s−1 ) and conventional video systems (25 frames s−1 ). Small metal markers were inserted into different parts of the upper and lower jaw and the tongue. Cineradiographic images were digitised, and displacements of the body, head, upper and lower jaw and the tongue were quantified. Twenty additional variables depicting displacements and the timing of events were calculated. Multivariate analyses of variance indicated significant differences between feeding stages. Remarkably, only very few food-type-dependent differences were observed during intraoral transport, and no such differences could be demonstrated during swallowing. Using previously published data for the closely related insectivorous lizard Plocederma stellio, the effect of dietary specialisation in U. acanthinurus on the kinematic variables while eating locusts was examined. Species differed in a number of gape- and tongue-related variables. These differences may be related to differences in tongue structure between the species. Clearly, U. acanthinurus possesses a specialised gut and dental structure that allows them efficiently to cut pieces from whole leaves. However, a decrease in modulatory capacity seems to be a consequence of dietary specialisation in Uromastix acanthinurus.
Cardiovascular and gill microcirculatory effects of endothelin-1 in atlantic cod: evidence for pillar cell contraction
Authors: Stensløkken Kåre-Olav , Sundin Lena , Nilsson Göran E.
Published: April 2021

Endothelin-1 (ET-1) has been shown to cause a considerable increase in the vascular resistance of fish gills. In trout, recent evidence suggest that this is the result of pillar cell contraction in the gill lamellae. Using epi-illumination microscopy to observe the gill lamellae of anaesthetised Atlantic cod (Gadus morhua), we show that ET-1 (100 ng kg−1, injected into the ventral aorta) causes an increase in pillar cell diameter, consistent with pillar cell contraction, and a shift of intralamellar blood flow from the lamellar sheet to the outer marginal channels. Simultaneously, there was an increase in ventral aortic blood pressure, a reduction in cardiac output, an increase in gill vascular resistance and a reduction in the oxygen partial pressure of venous blood. All these effects were blocked by the ETA/ETB receptor antagonist bosentan (5 mg kg−1 ). Pillar cell contraction is likely to be a mechanism for matching the functional respiratory surface area with the instantaneous respiratory needs of the fish.
Crystallization, Structure and Dynamics of the Proton-Translocating P-Type ATPase
Authors: Scarborough Gene A.
Published: April 2021

Large single three-dimensional crystals of the dodecylmaltoside complex of the Neurospora crassa plasma membrane H+-ATPase (H+P-ATPase) can be grown in polyethylene-glycol-containing solutions optimized for moderate supersaturation of both the protein surfaces and detergent micellar region. Large two-dimensional H+ P-ATPase crystals also grow on the surface of such mixtures and on carbon films located at such surfaces. Electron crystallographic analysis of the two-dimensional crystals grown on carbon films has recently elucidated the structure of the H+ P-ATPase at a resolution of 0.8 nm in the membrane plane. The two-dimensional crystals comprise two offset layers of ring-shaped ATPase hexamers with their exocytoplasmic surfaces face to face. Side-to-side interactions between the cytoplasmic regions of the hexamers in each layer can be seen, and an interaction between identical exocytoplasmic loops in opposing hexamer layers holds the two layers together. Detergent rings around the membrane-embedded region of the hexamers are clearly visible, and detergent–detergent interactions between the rings are also apparent. The crystal packing forces thus comprise both protein–protein and detergent–detergent interactions, supporting the validity of the original crystallization strategy. Ten transmembrane helices in each ATPase monomer are well-defined in the structure map. They are all relatively straight, closely packed, moderately tilted at various angles with respect to a plane normal to the membrane surface and average approximately 3.5 nm in length. The transmembrane helix region is connected in at least three places to the larger cytoplasmic region, which comprises several discrete domains separated by relatively wide, deep clefts. Previous work has shown that the H+ P-ATPase undergoes substantial conformational changes during its catalytic cycle that are not changes in secondary structure. Importantly, the results of hydrogen/deuterium exchange experiments indicate that these conformational changes are probably rigid-body interdomain movements that lead to cleft closure. When interpreted within the framework of established principles of enzyme catalysis, this information on the structure and dynamics of the H+ P-ATPase molecule provides the basis of a rational model for the sequence of events that occurs as the ATPase proceeds through its transport cycle. The forces that drive the sequence can also be clearly stipulated. However, an understanding of the molecular mechanism of ion transport catalyzed by the H+ P-ATPase awaits an atomic resolution structure.
Analysis of the Membrane Domain of the Gastric H+/K+-ATPase
Authors: Munson Keith , Lambrecht Nils , Shin Jai Moo , Sachs George
Published: April 2021

A structure of the catalytic or alpha subunit of the H+/K+-ATPase, with ten transmembrane segments, and of the beta subunit, with a single such segment, was established using a combination of tryptic cleavage and peptide sequencing and in vitro translation. Sites at which covalent ligands bind to external surfaces were also defined by cleavage, separation and sequencing. Cys813 was found to be the common covalent binding site for all the substituted pyridyl methylsulfinyl benzimidazoles. The binding region of a K+-competitive reagent, the 1,2α-imidazo-pyridine SCH 28080, was defined by the kinetic effects of site-specific mutations. Amino acids substitutions in membrane-spanning segments M1, M3, M4 and M6 were found to influence the apparent inhibitor constant, Ki, to varying degrees, some having a large effect, some a moderate effect and some a slight effect, whereas some mutations had no effect. We interpret changes in Ki without effects on the apparent Michaelis constant, Km, as affecting SCH 28080 binding only. Mutation of Cys813 significantly affected the Ki for SCH 28080, explaining the prevention of benzimidazole inhibition by the imidazo-pyridine.

A model of the alpha subunit was constructed with a vestibule on the luminal surface of the pump bounded by M1–M6 and containing the SCH 28080 binding region. The cation binding site is suggested to be more towards the cytoplasmic face of the enzyme’s membrane domain. This model predicts the membrane peptide associations for the catalytic subunit. Biochemical and yeast two-hybrid methods place the beta subunit in association with M8, whereas similar methods place M5/6 in proximity to M9/10. These results, when combined with analysis of the two-dimensional crystals of the sarcoplasmic reticular Ca2+ and Neurospora crassa H+-ATPases, provide the basis for a tentative model of the arrangement of the six core segments of the gastric H+/K+-ATPase.
Composition and Assembly of the Yeast Vacuolar H+-ATPase Complex
Authors: Graham Laurie A. , Powell Ben , Stevens Tom H.
Published: April 2021

The proton-translocating ATPase (H+-ATPase) found on the membrane of the yeast vacuole is the best characterized member of the V-type ATPase family. Biochemical and genetic screens have led to the identification of 14 genes, the majority designated VMA (for vacuolar membrane ATPase) encoding subunits of the enzyme complex. At least eight genes encode for proteins comprising the peripherally associated catalytic V1 subcomplex, and six genes code for proteins forming the proton-translocating membrane Vo subcomplex. Several additional genes have been identified that encode proteins that are not part of the final V-ATPase complex yet are required for its assembly. These non-subunit Vma proteins function as dedicated V-ATPase assembly factors since their absence appears to inhibit assembly of the V-ATPase only. The assembly factors designated Vma12p, Vma21p and Vma22p have been localized to the membrane of the endoplasmic reticulum and aid the association of newly synthesized V-ATPase subunits translocated into the endoplasmic reticulum membrane. Two of these proteins, Vma12p and Vma22p, function together in an assembly complex that interacts directly with nascent V-ATPase subunits.
Coupling H+ Transport to Rotary Catalysis in F-Type ATP Synthases: Structure and Organization of the Transmembrane Rotary Motor
Authors: Fillingame R. H. , Jiang W. , Dmitriev O. Y.
Published: April 2021

H+-transporting F1Fo-type ATP synthases utilize a transmembrane H+ potential to drive ATP formation by a rotary catalytic mechanism. ATP is formed in alternating β subunits of the extramembranous F1 sector of the enzyme, synthesis being driven by rotation of the γ subunit in the center of the F1 molecule between the alternating catalytic sites. The H+ electrochemical potential is thought to drive γ subunit rotation by first coupling H+ transport to rotation of an oligomeric rotor of c subunits within the transmembrane Fo sector. The γ subunit is forced to turn with the c12 oligomeric rotor as a result of connections between subunit c and the γ and ε subunits of F1. In this essay, we will review recent studies on the Escherichia coli Fo sector. The monomeric structure of subunit c, determined by nuclear magnetic resonance (NMR), is discussed first and used as a basis for the rest of the review. A model for the structural organization of the c12 oligomer in Fo, deduced from extensive cross-linking studies and by molecular modeling, is then described. The interactions between the the a1b2 ‘stator’ subcomplex of Fo and the c12 oligomer are then considered. A functional interaction between transmembrane helix 4 of subunit a (aTMH-4) and transmembrane helix 2 of subunit c (cTMH-2) during the proton-release step from Asp61 on cTMH-2 is suggested. Current a–c cross-linking data can only be explained by helix–helix swiveling or rotation during the proton transfer steps. A model that mechanically links helix rotation within a single subunit c to the incremental 30 ° rotation of the c12 oligomer is proposed. In the final section, the structural interactions between the surface residues of the c12 oligomer and subunits ε and γ are considered. A molecular model for the binding of subunit ε between the exposed, polar surfaces of two subunits c in the oligomer is proposed on the basis of cross-linking data and the NMR structures of the individual subunits.
Cellular Role of the V-Atpase In Neurospora Crassa: Analysis of Mutants Resistant to Concanamycin or Lacking the Catalytic Subunit A
Authors: Bowman Emma Jean , Bowman Barry J.
Published: April 2021

Vacuolar ATPases (V-ATPases) are large complex enzymes that are structural and mechanistic relatives of F1Fo-ATPases. They hydrolyze ATP and pump protons across membranes to hyperpolarize membranes and, often, to acidify cellular compartments. The proton gradients generated are used to drive the movement of various compounds across membranes. V-ATPases are found in membranes of archaebacteria and some eubacteria, in various components of the endomembrane system of all eukaryotes and in the plasma membranes of many specialized eukaryotic cells. They have been implicated in a wide variety of cellular processes and are associated with several diseases. Bafilomycin and concanamycin, specific inhibitors of V-ATPases, have been instrumental in implicating the V-ATPase in many of these roles. To understand further the mechanism of inhibition by these antibiotics and the physiological role of the enzyme in the cell, we have isolated mutants of the filamentous fungus Neurospora crassa that are resistant to concanamycin. Concanamycin has a dramatic effect on hyphal morphology at acid pH and is lethal at basic pH. In the resistant mutants, the cells can germinate and grow, although abnormally, in basic medium. Thus far, none of the mutants we have characterized is mutated in a gene encoding a subunit of the V-ATPase. Instead, the largest class of mutants is mutated in the gene encoding the plasma-membrane H+-ATPase. Mutations in at least four uncharacterized genes can also confer resistance. Inactivation of the V-ATPase by disruption of vma-1, which encodes the catalytic subunit (A) of the enzyme, causes a much more severe phenotype than inhibition by concanamycin. A strain lacking vma-1 is seriously impaired in rate of growth, differentiation and capacity to produce viable spores. It is also completely resistant to concanamycin, indicating that the inhibitory effects of concanamycin in vivo are due to inhibition of the V-ATPase. How the multiplicity of ATPases within a cell is regulated and how their activity is integrated with other metabolic reactions is poorly understood. Mutant analysis should help unravel this puzzle.
Transport Properties of Cultured Branchial Epithelia from Freshwater Rainbow Trout: A Novel Preparation with Mitochondria-Rich Cells
Authors: Fletcher Mary , Kelly Scott P. , Pärt Peter , O’Donnell Michael J. , Wood Chris M.
Published: April 2021

A new double-seeded insert (DSI) technique is described for culture of branchial epithelial preparations from freshwater rainbow trout on filter supports. DSI epithelia contain both pavement cells and mitochondria-rich (MR) cells (15.7±2.5 % of total cell numbers). MR cells occur singly or in clusters, are voluminous, open apically to the ‘external environment’ and exhibit ultrastructural characteristics similar to those found in the ‘chloride cells’ of freshwater fish gills. After 6–9 days in culture with Leibovitz’s L-15 medium on both surfaces (symmetrical conditions), transepithelial resistance (TER) stabilized at values as high as 34 kΩ cm2, indicative of electrically ‘tight’ epithelia. The density of MR cells, the surface area of their clusters and transepithelial potential (TEP; up to +8 mV basolateral positive, mean +1.9±0.2 mV) were all positively correlated with TER. In contrast, preparations cultured using an earlier single-seeded insert (SSI) technique contained only pavement cells and exhibited a negligible TEP under symmetrical conditions. Na+/K+-ATPase activities of DSI preparations were comparable with those in gill filaments, but did not differ from those of SSI epithelia. Replacement of the apical medium with fresh water to mimic the in vivo situation (asymmetrical conditions) induced a negative TEP (−6 to −15 mV) and increased permeability to the paracellular marker PEG-4000. Under symmetrical conditions, unidirectional Na+ and Cl− fluxes were in balance, and there was no active transport by the Ussing flux ratio criterion. Under asymmetrical conditions, there were large effluxes, small influxes and evidence for active Cl− uptake and Na+ extrusion. Unidirectional Ca2+ fluxes were only 0.5–1.0 % of Na+ and Cl− fluxes; active net Ca2+ uptake occurred under symmetrical conditions and active net extrusion under asymmetrical conditions. Thus, DSI epithelia exhibit some of the features of the intact gill, but improvements in culture conditions are needed before the MR cells will function as true freshwater ‘chloride cells’.
Desensitisation of Chromaffin Cell Nicotinic Receptors Does Not Impede Catecholamine Secretion During Acute Hypoxia in Rainbow Trout (Oncorhynchus Mykiss)
Authors: Lapner Katherine N. , Montpetit Colin J. , Perry Steve F.
Published: April 2021

Experiments were performed on adult rainbow trout (Oncorhynchus mykiss) in vivo using chronically cannulated fish and in situ using a perfused posterior cardinal vein preparation (i) to characterise the desensitisation of chromaffin cell nicotinic receptors and (ii) to assess the ability of fish to secrete catecholamines during acute hypoxia with or without functional nicotinic receptors. Intra-arterial injection of nicotine (6.0×10−7 mol kg−1) caused a rapid increase in plasma adrenaline and noradrenaline levels; the magnitude of this response was unaffected by an injection of nicotine given 60 min earlier. Evidence for nicotinic receptor desensitisation, however, was provided during continuous intravenous infusion of nicotine (1.3×10−5 mol kg−1 h−1) in which plasma catecholamine levels increased initially but then returned to baseline levels. To ensure that the decline in circulating catecholamine concentrations during continuous nicotine infusion was not related to changes in storage levels or altered rates of degradation/clearance, in situ posterior cardinal vein preparations were derived from fish previously experiencing 60 min of saline or nicotine infusion. Confirmation of nicotinic receptor desensitisation was provided by demonstrating that the preparations derived from nicotine-infused fish were unresponsive to nicotine (10−5 mol l−1), yet remained responsive to angiotensin II (500 pmol kg−1). The in situ experiments demonstrated that desensitisation of the nicotinic receptor occurred within 5 min of receptor stimulation and that resensitisation was established 40 min later.

The ability to elevate plasma catecholamine levels during acute hypoxia (40–45 mmHg; 5.3–6.0 kPa) was not impaired in fish experiencing nicotinic receptor desensitisation. Indeed, peak plasma adrenaline levels were significantly higher in the desensitised fish during hypoxia than in controls (263±86 versus 69±26 nmol l−1; means ± S.E.M., N=6–9). Thus, the results of the present study demonstrate that activation of preganglionic sympathetic cholinergic nerve fibres and the resultant stimulation of nicotinic receptors is not the sole mechanism for eliciting catecholamine secretion during hypoxia.
A Tubular Network Associated with the Brush-Border Surface of the Aedes Aegypti Midgut: Implications for Pathogen Transmission by Mosquitoes
Authors: Zieler Helge , Garon Claude F. , Fischer Elizabeth R. , Shahabuddin Mohammed
Published: April 2021

The mosquito Aedes aegypti is capable of transmitting a variety of pathogens to man and to other vertebrates. The midgut of this insect has been well-studied both as the tissue where the first contact occurs between ingested pathogens and the insect host, and as a model system for blood meal digestion in blood-sucking insects. To understand better the nature of the midgut surface encountered by parasites or viruses, we used scanning electron microscopy to identify the most prominent structures and cell morphologies on the luminal midgut surface. The luminal side of the midgut is a complex and layered set of structures. The microvilli that are found on most, but not all, cells are covered by a network of fine strands that we have termed the microvilli-associated network (MN). The MN strands are membranous, as shown by a membrane bilayer visible in cross sections of MN strands at high magnification in transmission electron micrographs. The MN is found in blood-fed as well as unfed mosquitoes and is not affected by chitinase treatment, suggesting that it is not related to the chitinous peritrophic membrane that is formed only after blood feeding. The cells in the midgut epithelium have two distinct morphologies: the predominant cell type is densely covered with microvilli, while cells with fewer microvilli are found interspersed throughout the midgut. We used lectins to probe for the presence of carbohydrates on the midgut surface. A large number of lectins bind to the luminal midgut surface, suggesting that a variety of sugar linkages are present on the structures visualized by electron microscopy. Some of these lectins partially block attachment of malaria ookinetes to the midgut surface in vitro. Thus, the mosquito midgut epithelium, like the lining of mammalian intestines, is complex, composed of a variety of cell types and extensively covered with surface carbohydrate that may play a role in pathogen attachment.
The Scaling of Carbon Dioxide Release and Respiratory Water Loss in Flying Fruit Flies (Drosophila Spp.)
Authors: Lehmann Fritz-Olaf , Dickinson Michael H. , Staunton Jocelyn
Published: April 2021

By simultaneously measuring carbon dioxide release, water loss and flight force in several species of fruit flies in the genus Drosophila, we have investigated respiration and respiratory transpiration during elevated locomotor activity. We presented tethered flying flies with moving visual stimuli in a virtual flight arena, which induced them to vary both flight force and energetic output. In response to the visual motion, the flies altered their energetic output as measured by changes in carbon dioxide release and concomitant changes in respiratory water loss. We examined the effect of absolute body size on respiration and transpiration by studying four different-sized species of fruit flies. In resting flies, body-mass-specific CO2 release and water loss tend to decrease more rapidly with size than predicted according to simple allometric relationships. During flight, the mass-specific metabolic rate decreases with increasing body size with an allometric exponent of −0.22, which is slightly lower than the scaling exponents found in other flying insects. In contrast, the mass-specific rate of water loss appears to be proportionately greater in small animals than can be explained by a simple allometric model for spiracular transpiration. Because fractional water content does not change significantly with increasing body size, the smallest species face not only larger mass-specific energetic expenditures during flight but also a higher risk of desiccation than their larger relatives. Fruit flies lower their desiccation risk by replenishing up to 75 % of the lost bulk water by metabolic water production, which significantly lowers the risk of desiccation for animals flying under xeric environmental conditions.
Dorsal Unpaired Median Neurones in the Insect Central Nervous System: Towards a Better Understanding of the Ionic Mechanisms Underlying Spontaneous Electrical Activity
Authors: Grolleau Françoise , Lapied Bruno
Published: April 2021

The efferent dorsal unpaired median (DUM) neurones, which include octopaminergic neurones, are among the most intensively studied neurones in the insect central nervous system. They differ from other insect neurones in generating endogenous spontaneous overshooting action potentials. The second half of the 1980s is certain to be considered a turning point in the study of the ion channels underlying the electrical activity of DUM neurones. Recent advances made using the patch-clamp technique have stimulated an increasing interest in the understanding of the biophysical properties of both voltage-dependent and voltage-independent ion channels. Patch-clamp studies of DUM neurones in cell culture demonstrate that these neurones express a wide variety of ion channels. At least five different types of K+ channel have been identified: inward rectifier, delayed rectifier and A-like channels as well as Ca2+- and Na+-activated K+ channels. Moreover, besides voltage-dependent Na+ and Ca2+-sensitive Cl− channels, DUM neurones also express four types of Ca2+ channel distinguished on the basis of their kinetics, voltage range of activation and pharmacological profile. Finally, two distinct resting Ca2+ and Na+ channels have been shown to be involved in maintaining the membrane potential and in regulating the firing pattern. In this review, we have also attempted critically to evaluate these existing ion channels with regard to their specific functions in the generation of the different phases of the spontaneous electrical activity of the DUM neurone.
Object Fixation by The Blowfly During Tethered Flight in a Simulated Three-Dimensional Environment
Authors: Kimmerle Bernd , Eickermann Judith , Egelhaaf Martin
Published: April 2021

The ability of flies to detect and fixate objects moving relative to their background was investigated in a flight simulator during translational tethered flight. The fly experienced optic flow that depended on its own actions and reactions in a similar way as in free-flight (closed-loop) conditions. Fixation of an object required turning responses towards it. The simulated distances between the fly, object and background were varied systematically by changing the velocities with which the object and the background pattern moved from the frontal to the back part of the fly’s visual field. Fixation responses were only elicited when the object was simulated to be closer than the background. The fly’s fixation performance was better with close than with more distant objects. Since, under many stimulus conditions, fixation responses were either elicited or entirely failed to be elicited, it is concluded that object fixation behaviour is gated in the visuo-motor pathway.
Horizontal Flight of A Swallow (Hirundo Rustica) Observed In A Wind Tunnel, With A New Method for Directly Measuring Mechanical Power
Authors: Pennycuick C. J. , Hedenström Anders , Rosén Mikael
Published: April 2021

A swallow flying in the Lund wind tunnel was observed from the side and from behind, by two synchronised high-speed video cameras. The side-view camera provided a record of the vertical position of a white mark, applied to the feathers behind and below the eye, from which the vertical acceleration was obtained. The rear-view camera provided measurements of the mean angle of the left and right humeri above horizontal. From these data, the force acting on the body, the moment applied by each pectoralis muscle to the humerus and the rotation of the humerus were estimated and used to analyse the time course of a number of variables, including the work done by the muscles in each wing beat. The average mechanical power turned out to be more than that predicted on the basis of current estimates of body drag coefficient and profile power ratio, possibly because the bird was not flying steadily in a minimum-drag configuration. We hope to develop the method further by correlating the mechanical measurements with observations of the vortex wake and to apply it to birds that have been conditioned to hold a constant position in the test section.
The Macrophage in Acute Neural Injury: Changes in Cell Numbers Over time and Levels of Cytokine Production in Mammalian Central and Peripheral Nervous Systems
Authors: Leskovar Alenka , Moriarty Loren J. , Turek John J. , Schoenlein Ingrid A. , Borgens Richard B.
Published: April 2021

We evaluated the timing and density of ED-1-positive macrophage accumulation (ED 1 is the primary antibody for the macrophage) and measured cytokine production by macrophages in standardized compression injuries to the spinal cord and sciatic nerves of individual rats 3, 5, 10 and 21 days post-injury. The actual site of mechanical damage to the nervous tissue, and a more distant site where Wallerian degeneration had occurred, were evaluated in both the peripheral nervous system (PNS) and the central nervous system (CNS) at these time points. The initial accumulation of activated macrophages was similar at both the central and peripheral sites of damage. Subsequently, macrophage densities at all locations studied were statistically significantly higher in the spinal cord than in the sciatic nerve at every time point but one. The peak concentrations of three cytokines, tumor necrosis factor α (TNFα), interleukin-1 (IL-1) and interleukin-6 (IL-6), appeared earlier and were statistically significantly higher in injured spinal cord than in injured sciatic nerve. We discuss the meaning of these data relative to the known differences in the reparative responses of the PNS and CNS to injury.
Central Circuitry in the Jellyfish Aglantha Digitale: III. the Rootlet and Pacemaker Systems
Authors: Mackie G. O. , Meech R. W.
Published: April 2021

Tactile stimulation of the subumbrella of Aglantha digitale was found to evoke an escape swimming response similar to that evoked by stimulation of the outer surfaces of the margin but that does not involve the ring giant axon. Evidence is presented that conduction around the margin takes place via an interconnected system of rootlet interneurones. Confocal microscopy of carboxyfluorescein-filled axons showed that the rootlet neurones run out from the bases of the motor giant axons within the inner nerve ring and come into close contact with those of the neighbouring motor giant axons on either side. Transmission between the rootlet neurones has the properties of chemical synaptic transmission. A distinct type of fast excitatory postsynaptic potential (rootlet PSP) was recorded in motor giant axons following stimulation of nearby axons in 3–5 mmol l−1 Mn2+, which lowered the PSP below spike threshold. Immune labelling with anti-syntaxin 1 showed structures tentatively identified as synapses in the inner nerve ring, including some on the rootlet neurones. Neuromuscular junctions were not labelled.

A secondary consequence of stimulating motor giant axons was the triggering of events in the pacemaker system. Triggering was blocked in 105 mmol l−1 Mg2+, indicating a synaptic link. Activity in the pacemaker system led indirectly to tentacle contractions (as described in earlier papers in this series), but the contractions were not as sudden or as violent as those seen when escape swimming was mediated by the ring giant axon. Events triggered in the pacemaker system fed back into the motor giants, producing postsynaptic potentials that appeared as humps in the spike after-potential. The conduction velocity of events propagating in the relay system was increased when the rootlet pathway was simultaneously excited (piggyback effect).

With the addition of the rootlet pathway, the number of identified systems concerned with locomotion, feeding and tentacle contractions comes to fourteen, and the list is probably nearly complete.