Outer Entrance Research Articles

Mapping Potential Determinants of Peroxidative Activity in an Evolved Fungal Peroxygenase from Agrocybe aegerita.

Fungal unspecific peroxygenases (UPOs) are hybrid biocatalysts with peroxygenative activity that insert oxygen into non-activated compounds, while also possessing convergent peroxidative activity for one electron oxidation reactions. In several ligninolytic peroxidases, the site of peroxidative activity is associated with an oxidizable aromatic residue at the protein surface that connects to the buried heme domain through a long-range electron transfer (LRET) pathway. However, the peroxidative activity of these enzymes may also be initiated at the heme access channel. In this study, we examined the origin of the peroxidative activity of UPOs using an evolved secretion variant (PaDa-I mutant) from Agrocybe aegerita as our point of departure. After analyzing potential radical-forming aromatic residues at the PaDa-I surface by QM/MM, independent saturation mutagenesis libraries of Trp24, Tyr47, Tyr79, Tyr151, Tyr265, Tyr281, Tyr293 and Tyr325 were constructed and screened with both peroxidative and peroxygenative substrates. These mutant libraries were mostly inactive, with only a few functional clones detected, none of these showing marked differences in the peroxygenative and peroxidative activities. By contrast, when the flexible Gly314-Gly318 loop that is found at the outer entrance to the heme channel was subjected to combinatorial saturation mutagenesis and computational analysis, mutants with improved kinetics and a shift in the pH activity profile for peroxidative substrates were found, while they retained their kinetic values for peroxygenative substrates. This striking change was accompanied by a 4.5°C enhancement in kinetic thermostability despite the variants carried up to four consecutive mutations. Taken together, our study proves that the origin of the peroxidative activity in UPOs, unlike other ligninolytic peroxidases described to date, is not dependent on a LRET route from oxidizable residues at the protein surface, but rather it seems to be exclusively located at the heme access channel.

Derived muscle arrangements and their shared innervation patterns of external and internal cheek pouches in rodents.

Cheek pouches have evolved from the oral cavity in rodents and act as temporary food storage repositories. There are two types of opening, internal and external. Details about the complex cutaneous muscles controlling the pouches have still not been fully elucidated. To understand the shared and derived traits of the muscles surrounding the cheek pouch and their innervation, we carried out an evolutionary morphological study using two desert kangaroo rats (Dipodomys deserti) and three plains pocket gophers (Geomys bursarius) from each of the two families equipped with external cheek pouches, and four Syrian hamsters (Mesocricetus auratus) with internal cheek pouches. The most conspicuous derived trait of the muscles between the external and internal cheek pouches, the sphincter sacculi, surrounds almost all of the edge of the outer entrance of the pouch. It is present in both species with external pouches, but not in hamsters, which have internal pouches. Our neurological findings demonstrate that most pouch muscles are innervated by both the facial and the cervical nerves, regardless of the pouch type. In these dually innervated muscles, the ventromedial part of the muscles tends to be innervated dominantly or uniquely by the cervical nerves, which usually enter from the superficial or lateral aspect. As a trait shared with the cervical nerves innervating the propatagial muscles in aerodynamic mammals such as bats and flying squirrels, and panniculus carnosus in most mammals, our neurological evidence suggests that the cervical nerve has the potential to innervate derived cutaneous muscles in the cervicofacial region.

Unique nest entrance structure of Partamona helleri stingless bees leads to remarkable \u2018crash-landing\u2019 behaviour

Partamona helleri stingless bees construct a unique, funnel-shaped entrance that resolves an evolutionary conflict between foraging efficiency and defence. The large outer entrance allows many foragers to pass while the narrow inner entrance requires few guards to defend. This structure has given rise to a remarkable behaviour in returning foragers, which appear to approach the nest entrance at high speed and ‘crash’ head first into the entrance. We compared P. helleri landing behaviour with two related species with architecturally different entrances that land conventionally using their legs: Melipona scutellaris, whose narrow entrance allows only a single bee to pass, and Scaptotrigona depilis, which has a wide entrance tube. All three species initially decelerated on their approach to the nest entrance. However, 0.2 m from the entrance P. helleri began accelerating, whereas the other species continued to decelerate. Partamona helleri entered its nest at 1.14 ms−1, double the velocity of the other species. Despite its greater velocity, P. helleri made no fewer errors than the other species when attempting to enter its nest, probably by virtue of the large target provided by the outer entrance. We then used a bioassay that suggests that this behaviour is a defence against ambush predators at the nest entrance. Finally, we use a scaling argument to show that the crash impact should not cause any damage to a small animal such as a bee, such that no morphological adaptation is required.

Effect of Regulation of the Inlet Channel on Tides and Water Levels in Lake Hamana, Japan

Hamanako is an economically and culturally important tidal lake on the Pacific coast of Honshu, Japan. Between 1954 and 1973, entrance works were constructed to regulate the channel to the lake. In a previous paper, the tidal records from Maisaka just inside the lake, prior to regulation, were analyzed to explain the responses of the lake to the ocean tide and other water level changes. The present paper compares water level responses with those following regulation of the entrance. The principal differences are that at Maisaka the tidal range increased up to 1965, following the first stages of regulation, but it has been constant since then. In the inner part of the lake, regulation has increased the tidal range but reduced its variability and has reduced the mean water level in the lake. The southern sand shoal divides the lake into two tidal basins, with Maisaka lying in the smaller outer basin. Studies of the inner basin have shown that its tidal range continues to increase. A hydrodynamic model has been used to show that this increase is primarily due to scouring of the inner channel through the southern shoal. The model has also shown that the constancy of the Maisaka tidal amplitude since 1965 is not a static equilibrium, but is a balance between the effects of the outer entrance channel scouring and increasing the Maisaka tide, offset by the inner channel scouring and reducing the Maisaka tide.

Revisit on natural convection from vertical isothermal plate arrays--effects of extra plume buoyancy

In this study, natural convection from vertical isothermal parallel-plate arrays is examined using 2-D steady-state numerical analysis. Extended computation domains encompassing the single- or multi-channel plate arrays are adopted. Also investigated are the consequences of using computation domains without the inlet extension or/and the outlet extension. The plate height is fixed at 100 mm, the plate spacing is 7 mm, and the plate thickness is 1 mm. The Elenbaas Rayleigh number Ra’ is fixed at 62.8. The results show that setting the inlet boundary at the entrance of the channel would ignore the separation formed near the outer entrance corner and the associated flow resistance; while setting the outlet boundary at the exit of the channel would omit the extra buoyancy provided by the hot plume above the arrays. In multi-channel arrays, the average heat transfer coefficients in different individual channels (hs) are different from each other. The h is the highest in the central channel and the lowest in the edge channel. Except for the edge channels, the h values are higher than that of a single channel. The differences in h between the central and the edge channels increase with increasing number of channels. This phenomenon can be ascribed to (1) the stronger extra hot-plume buoyancy in the inner plume region and (2) the higher entrance-separation resistance in the outer channels. The overall convection heat transfer coefficients in the plate arrays increase with increasing number of plate channels. The conventional assumption that all the channels of a multi-plate array have similar heat transfer performance needs re-evaluation.

Crumbling of the masonry of ancient settlements on Santorini caused by the Hymenoptera Anthophora crinipes and Osmia latreillei

One of the most important archaeological findings of recent years has been the discovery of complete buildings of a Bronze Age settlement at Akrotiri on the island of Santorini (Thera). The settlement covers an area of over 200,000 square metres. The excavations conducted by Spyros Marinates brought to light a large number of buildings constructed of stones, mud and straw, with earth roofs. The walls were faced with clay. Almost every exposed mass of masonry in the settlement has been invaded by colonies of Hymenopterous insects consisting nests which are wreaking havoc on the walls, because they bore through the masonry to build their nests and then every time they go in and out of the tunnels, they wear away the dust of the walls which they have build for their own nests. Observations were carried out at Akrotiri in the spring and summer of 1985, 1986 and 1988. Glass collecting-tubes were placed at entry and exit points and at various places in the tunnels, to sellect the insects when the adults emerged in the spring, and insects also were collected with sweeping nets from the surrounding area and from light traps. Plants which were visited by adults of these species for pollen and nectar were also collected from round about. The insect species collected by these various methods were the following: Osmia (Chalcosinia) latredlei Spinola, 1806 (Hymenoptera: Megachilidae), Anthophora crinipes Smith, 1884 (Hymenoptera: Apidae), and Melecta albifrons albovaria Erichson 1840 ( Hymenoptera : Megachilidae). These three species were collected in all observation years in glass collectintubes. Of these A. crinipes and O. latreillei, which are solitary species, both build colonies with tunnels a short distance appart but not actually touching each other. The number of such colonies in the walls of the Akrotiri settlement is fairly large, and conspicuous through the holes of entrance and exit of the insects. M. albifrons albovaria is a predator on A. crinipes. It is known from the literature that all species of the genus Melecta prey on Anthophora species. The tunnels of both species are made by the females. At the end of each tunnel the female hollows out a number of cells and lays in supplies of pollen and nectar on which she deposits her eggs after mating. She then seals off each cell with a mixture of earth and saliva. Finally, when eggs have been deposited in all the cells, she closes the outer entrance of the tunnel. The larvae start developing once the tunnel has been sealed off. Thus after the hatching of the eggs, the insects spend more than thirty days as larvae, later on as prepupae. In this stage they overwinter and early in the spring enter the pupal stage. The first adults appeared from the last days of March. The females mate with the males and then start making tunnels and cells for their eggs. Adults of both sexes visit flowers to feed and also, in the case of the females, to collect pollen and nectar for the cells in which the larvae will develop. In the neighbourhood of Akrotiri we have observed adults of both species in question visiting flowers of the following species: Reichardia picroide(L.) Roth (Compositae), Echhon angustifolium Miller (Boraginaceae), Vicia vilosa Roth (Leguminosae), Euphorbia spp. (Euphorbiaceae), Salvia verticillata L. (Labiatae),Rosinarinuin officinalis L. (Labiatae), Morina persica L. (Dipsacaceae). Fahringer reported only Anthophora crinipes in Asia Minor and observed that adults visited the flowers of Salvia verticillata and Morina persica, but it is not clear if the insects collect pollen or nectar or both of these plants. The adults usually choose sunny days to fly out to the flowers, and during their flight a caracteristic buzzing is heard. The flying season be­gins in late March and goes on until the end of June, or some time later. The damage done by these insects to the fabric of the ancient settlement at Akrotiri is posing a serious problem for the preservation of the ruins in their present form. If it continues there is danger that parts of the masonry will be permanently deformed. Little heaps of fine pozzolana dust made by the insects going in and out of the tunnels is found on the ground below the entrance holes. In the last two years the team of archeaeologists has used light traps in and around the ruins, thereby apparently reducing the populations of these insects and limiting the damage done by them. On the basis of the insects collected in May 1988, the population of A. crinipes outnumbers that of O. larreillei (ratio 79:21).

Improved O2-tolerance in variants of a H2-evolving [NiFe]-hydrogenase from Klebsiella oxytoca HP1

In this study, we investigated the mechanism of O2 tolerance of Klebsiella oxytoca HP1 H2-evolving hydrogenase 3 (KHyd3) by mutational analysis and three-dimensional structure modeling. Results revealed that certain surface amino acid residues of KHyd3 large subunit, in particular those at the outer entrance of the gas channel, have a visible effect on its oxygen tolerance. Additionally, solution pH, immobilization and O2 partial pressure also affect KHyd3 O2-tolerance to some extent. We propose that the extent of KHyd3 O2-tolerance is determined by a balance between the rate of O2 access to the active center through gas channels and the deoxidation rate of the oxidized active center. Based on our findings, two higher O2-tolerant KHyd3 mutations G300E and G300M were developed.

Localizing the critical isthmus of postinfarct ventricular tachycardia: The value of pace-mapping during sinus rhythm

Most postinfarct ventricular tachycardias (VTs) are sustained by a reentrant mechanism. The "protected isthmus" of the reentrant circuit is critical for the maintenance of VTs and the target for catheter ablation. Various techniques based on conventional electrophysiology and/or detailed three-dimensional (3D) reconstruction of the VT circuit are used to unmask this isthmus. The purpose of this study was to assess pace-maps (PMs) to identify postinfarct VT isthmuses. We hypothesized that an abrupt change in paced QRS morphology may be used to identify a VT isthmus and be targeted for successful ablation. High-density 3D PMs were matched to the subsequent 3D endocardial reentrant VT activation mapping in 10 patients (8 men; age 70.7 ± 10.8 years) who underwent successful postinfarct VT ablation. At each pacing site in a given patient, the 12-lead ECG recorded during pacing was compared to that of VT, with the resulting matching percentage (up to 100% for perfect matches) allocated to this point to generate color-coded PMs. With respect to VT isthmuses, the best percentages of matching were found in the exit zones and isthmus exit part (89% ± 8% and 84% ± 7%, respectively) and the poorest adjacent to scar border in the outer entrance zones (23% ± 28%), in the entrance zones (39% ± 34%), and in the entrance part of the isthmus (32% ± 26%). The color-coded sequence (from the best to the poorest matching sites) on the PMs revealed figure-of-eight pictures matching the VT activation time maps and identifying VT isthmuses. Pace-mapping is useful for unmasking VT isthmuses in patients with well-tolerated postinfarct endocardial reentrant VTs.

High Ca2+ permeability of a peptide-gated DEG/ENaC from Hydra

Degenerin/epithelial Na+ channels (DEG/ENaCs) are Na+ channels that are blocked by the diuretic amiloride. In general, they are impermeable for Ca2+ or have a very low permeability for Ca2+. We describe here, however, that a DEG/ENaC from the cnidarian Hydra magnipapillata, the Hydra Na+ channel (HyNaC), is highly permeable for Ca2+ (PCa/PNa = 3.8). HyNaC is directly gated by Hydra neuropeptides, and in Xenopus laevis oocytes expressing HyNaCs, RFamides elicit currents with biphasic kinetics, with a fast transient component and a slower sustained component. Although it was previously reported that the sustained component is unselective for monovalent cations, the selectivity of the transient component had remained unknown. Here, we show that the transient current component arises from secondary activation of the Ca2+-activated Cl− channel (CaCC) of Xenopus oocytes. Inhibiting the activation of the CaCC leads to a simple on–off response of peptide-activated currents with no apparent desensitization. In addition, we identify a conserved ring of negative charges at the outer entrance of the HyNaC pore that is crucial for the high Ca2+ permeability, presumably by attracting divalent cations to the pore. At more positive membrane potentials, the binding of Ca2+ to the ring of negative charges increasingly blocks HyNaC currents. Thus, HyNaC is the first member of the DEG/ENaC gene family with a high Ca2+ permeability.

ATP hydrolysis-dependent asymmetry of the conformation of CFTR channel pore

Despite substantial efforts, the entire cystic fibrosis transmembrane conductance regulator (CFTR) protein proved to be difficult for structural analysis at high resolution, and little is still known about the actual dimensions of the anion-transporting pathway of CFTR channel. In the present study, we therefore gauged geometrical features of the CFTR Cl(-) channel pore by a nonelectrolyte exclusion technique. Polyethylene glycols with a hydrodynamic radius (R (h)) smaller than 0.95 nm (PEG 300-1,000) added from the intracellular side greatly suppressed the inward unitary anionic conductance, whereas only molecules with R (h) ≤ 0.62 nm (PEG 200-400) applied extracellularly were able to affect the outward unitary anionic currents. Larger molecules with R (h) = 1.16-1.84 nm (PEG 1,540-3,400) added from either side were completely excluded from the pore and had no significant effect on the single-channel conductance. The cut-off radius of the inner entrance of CFTR channel pore was assessed to be 1.19 ± 0.02 nm. The outer entrance was narrower with its cut-off radius of 0.70 ± 0.16 nm and was dilated to 0.93 ± 0.23 nm when a non-hydrolyzable ATP analog, 5'-adenylylimidodiphosphate (AMP-PNP), was added to the intracellular solution. Thus, it is concluded that the structure of CFTR channel pore is highly asymmetric with a narrower extracellular entrance and that a dilating conformational change of the extracellular entrance is associated with the channel transition to a non-hydrolytic, locked-open state.

The D-Diastereomer of ShK Toxin Selectively Blocks Voltage-gated K+ Channels and Inhibits T Lymphocyte Proliferation

The polypeptide toxin ShK is a potent blocker of Kv1.3 potassium channels, which are crucial in the activation of human effector memory T cells (T(EM)); selective blockers constitute valuable therapeutic leads for the treatment of autoimmune diseases mediated by T(EM) cells, such as multiple sclerosis, rheumatoid arthritis, and type-1 diabetes. The critical motif on the toxin for potassium channel blockade consists of neighboring lysine and tyrosine residues. Because this motif is sufficient for activity, an ShK analogue was designed based on D-amino acids. D-allo-ShK has a structure essentially identical with that of ShK and is resistant to proteolysis. It blocked Kv1.3 with K(d) 36 nm (2,800-fold lower affinity than ShK), was 2-fold selective for Kv1.3 over Kv1.1, and was inactive against other K(+) channels tested. D-allo-ShK inhibited human T(EM) cell proliferation at 100-fold higher concentration than ShK. Its circulating half-life was only slightly longer than that of ShK, implying that renal clearance is the major determinant of its plasma levels. D-allo-ShK did not bind to the closed state of the channel, unlike ShK. Models of D-allo-ShK bound to Kv1.3 show that it can block the pore as effectively as ShK but makes different interactions with the vestibule, some of which are less favorable than for native ShK. The finding that an all-D analogue of a polypeptide toxin retains biological activity and selectivity is highly unusual. Being resistant to proteolysis and nonantigenic, this analogue should be useful in K(+) channel studies; all-d analogues with improved Kv1.3 potency and specificity may have therapeutic advantages.

Comparative study in stingless bees (Meliponini) demonstrates that nest entrance size predicts traffic and defensivity

Stingless bees (Meliponini) construct their own species-specific nest entrance. The size of this entrance is under conflicting selective pressures. Smaller entrances are easier to defend; however, a larger entrance accommodates heavier forager traffic. Using a comparative approach with 26 species of stingless bees, we show that species with greater foraging traffic have significantly larger entrances. Such a strong correlation between relative entrance area and traffic across the different species strongly suggests a trade-off between traffic and security. Additionally, we report on a significant trend for higher forager traffic to be associated with more guards and for those guards to be more aggressive. Finally, we discuss the nest entrance of Partamona, known in Brazil as boca de sapo, or toad mouth, which has a wide outer entrance but a narrow inner entrance. This extraordinary design allows these bees to finesse the defensivity/traffic trade-off.

Excavations at Nineveh: The Halzi Gate

AbstractThe University of California Expedition to Nineveh excavated at the Halzi Gate in 1989 and 1990. The gate proved to be one of Nineveh's larger defensive gates and a scene of chaos and destruction. Tower 4 to the south of the gate was of irregular masonry, having been hastily rebuilt. The juxtaposition of a rough tower directly beside the fine ashlar masonry of the rest of the curtain wall of the gate appears to reflect a political instability that prevailed during the declining years of Assyrian rule. The late reconstruction of Tower 4 contrasts with the earlier attention to detail seen in building work of Sennacherib's reign (704–681 BC), when smoothly dressed orthostats lined the lower walls of the gate's large inner court and sophisticated waterproof membranes of bitumen-soaked reeds protected the mud-brick superstructures. Buried beneath the pavement in the corners of the court were apotropaic foundation figurines. Within the only partially excavated outer entrance passage at least twelve individuals died, perhaps while defending the city or trying to escape from it, as a stallion and a rider lay at the eastern limit of the gate passage. Within the reduced width of the gate passage were eight adult males, four adolescents and four children including one infant. Weaponry was found throughout the area and the scattered bodies lay where they had fallen. The associated small finds, which had not been looted, included silver jewelry, bronze personal items, stamp seals and a composite necklace.

Electrostatic Interaction between Charybdotoxin and a Tetrameric Mutant of Shaker K + Channels

The scorpion toxin, Charybdotoxin (CTX), blocks homotetrameric, voltage-gated K + channels by binding near the outer entrance to the pore in one of four indistinguishable orientations. We have determined the pH-dependence of CTX block of a tetrameric Shaker potassium channel with a single copy of a histidine replacing the wild-type phenylalanine at position 425. We compared this pH-dependence with that from homotetrameric channels with four copies of the mutation. We found that protonation of a single amino acid at position 425 had a large effect on the affinity of the channel for CTX—much larger than expected if only one of the four CTX binding orientations was disrupted. The pK a for the H +-ion induced protection from CTX block indicates that the electrostatic environment near position 425 is likely basic in nature, perhaps because of the proximity of lysine 427. We also examined the pH-dependence of block of channels with one and four copies of the histidine mutation by CTX containing neutralizing mutations of four basic residues on the active face of the toxin. The results suggested an orientation of CTX on the channel that places three of the positively charged CTX residues very near three of the four Shaker 425 positions.

Pore mutations alter closing and opening kinetics in Shaker K+ channels.

1. We have studied the effects of mutations of amino acids in the pore (positions 447 and 449) and the elevation of extracellular [K+] on the closing and opening kinetics of Shaker B K+ channels transiently expressed in Chinese hamster ovary (CHO) cells. 2. Mutant D447E had closing and C-type inactivation kinetics which were faster than the wild-type channel. These processes were slowed by increasing extracellular [K+] and in these conditions the channels exhibited linear instantaneous current-voltage relationships. Thus, the mutation seems to produce uniform decrease of occupancy by K+ in sites along the channel pore where the cation competes with closing and C-type inactivation. 3. In other mutants also showing K+-dependent fast C-type inactivation, closing was found to be slower than in the wild-type channel and insensitive to variations in external [K+]. These characteristics were particularly apparent in mutant T449K which even in high [K+] has a non-linear instantaneous current-voltage relationship with marked saturation of the inward current recorded at negative membrane potentials. Hence, in this channel type occupation by K+ of the pore appears to be non-uniform with low occupancy of sites near the outer entrance and saturation of the sites accessible from the internal solution. 4. The results show that channel closing is influenced by changes in the pore structure leading to alterations in the occupation of the channels by permeant cations. The differential effects of pore mutations and high external [K+] on closing and C-type inactivation indicate that the respective gates are associated with separate domains of the molecule. 5. Point mutations in the pore sequence can also lead to modifications in channel opening. In general, channels with fast C-type inactivation also show a fast rising phase of activation. However, these effects appear not to be due to primary modifications of the activation process but to arise from the coupling of activation and C-type inactivation. 6. These data, demonstrating that the pore structure influences most of the gating parameters of K+ channels, give further insight into the mechanisms underlying the modulation of K+ channel function by changes in the ionic composition in the extracellular milieu.

Calculated electrostatic gradients in recombinant human H-chain ferritin.

Calculations to determine the electrostatic potential of the iron storage protein ferritin, using the human H-chain homopolymer (HuHF), reveal novel aspects of the protein. Some of the charge density correlates well with regions previously identified as active sites in the protein. The three-fold channels, the putative ferroxidase sites, and the nucleation sites all show expectedly negative values of the electrostatic potential. However, the outer entrance to the three-fold channels are surrounded by regions of positive potential, creating an electrostatic field directed toward the interior cavity. This electrostatic gradient provides a guidance mechanism for cations entering the protein cavity, indicating the three-fold channel as the major entrance to the protein. Pathways from the three-fold channels, indicated by electrostatic gradients on the inner surface, lead to the ferroxidase center, the nucleation center and to the interior entrance to the four-fold channel. Six glutamic acid residues at the nucleation site give rise to a region of very negative potential, surrounding a small positively charged center due to the presence of two conserved arginine residues, R63, in close proximity (4.9 A), suggesting that electrostatic fields could also play a role in the nucleation process. A large gradient in the electrostatic potential at the 4-fold channel gives rise to a field directed outward from the internal cavity, indicating the possibility that this channel functions to expel cations from inside the protein. The 4-fold channel could therefore provide an exit pathway for protons during mineralization, or iron leaving the protein cavity during de-mineralization.

Two-sided action of protons on an inward rectifier K+ channel (IRK1).

A cloned inwardly rectifying potassium channel, IRK1, expressed in Xenopus oocytes was found to be sensitive to an extracellular acidic pH level of below 6, achieved by buffering with a membrane-impermeable buffer, phthalate. The voltage dependency of the suppressive effect of pH on the macroscopic current suggested that the location of the proton-sensitive site was at approximately 5% of the distance from the outer entrance to the pore. The single-channel conductance was reduced by protonation of the channel on the extracellular side. The external proton-binding site appears to consist of a single class of negatively charged groups with a pK of around 4.6. An intracellular acidic pH, buffered with membrane-permeable acetate, was found to inhibit, in a voltage-independent manner, the macroscopic IRK1 current with an approximate apparent pK of 5.6 and an approximate apparent Hill coefficient of 2.3. The single-channel activity was abolished by intracellular acidification down to pH 5.0.

Structural Model of the Outer Vestibule and Selectivity Filter of the Shaker Voltage-gated K+ Channel

A new generation of structural models were developed of the outer vestibule and ion-selective portion of the voltage-gated Shaker K+ channel. Some features of these models are similar to those that we have developed previously [Durrell and Guy (1992)Biophys. J. 62, 238–250; Guy (1990)In Monovalent Cations in Biological Systems (Pasternakk C.A., Ed.), pp. 31–58, CRC Press, Boca Raton, FL; Guy and Durell (1994)In Molecular Evolution of Physiological processes (Fambrough D., Ed.), pp. 197–212, The Rockefeller University Press, NY; Guy and Durell (1995)In Ion Channels and Genetic Diseases (Dawson D., Ed.), pp. 1–16, The Rockefeller University Press, NY] and other features were modified to make the models more consistent with recent experimental findings. The first part of the P segment is postulated, as always, to form a short α helix that spans only the outer portion of the membrane. The helix is tilted so that its C-terminal is nearer the pore than its N-terminal. The latter part of the P segment, P2, is postulated to have a relatively elongated conformation that is positioned approximately parallel to the axis of the pore. Four of the P2 segments assemble to form an ion-selective region that has two narrow regions; one formed by the Y445 side-chains at the outer entrance of the pore and one formed by the backbone of the T442 residues near the innermost part of the P segments. The S6 segment is postulated to form two α helices. The first S6 helix packs next to the P segments in our models. The NMR structures of two scorpion toxins, charybdotoxin and agitoxin 2, have been docked into the models of the outer vestibules. The shape of the outer vestibule has been modeled so that specific toxin-channel residue-residue interactions correspond to those that have been identified experimentally. Published by Elsevier Science Ltd

The Bordeaux Port Radar

At the end of 1963, to assist ships making use of the extensive facilities in the Gironde estuary, the port of Bordeaux installed surveillance radar covering the mouth of the Gironde. As it is still too early to assess its potentialities, this paper describes the technical arrangements and makes some general comments on the use of radar, suggested by the new installation.Ships approaching the Gironde estuary from the open sea follow the buoyed channel from the landfall buoy BXA, 18 kilometres west of Pointe de la Coubre. After picking up the pilot they enter the western or outer entrance passage, whose seaward termination lies 8 kilometres from the coast. This provides a dredged channel, 800 metres wide and 3500 metres long, running east–west. The depth of 12 metres at low water springs enables ships to cross the annular river bar separating the deep water on the seaward side from the mud banks at the mouth of the inner channel.

V.—Notes on the ‘Prison of Saint Catharine’ at Salamis in Cyprus

The ‘Prison of Saint Catharine’ is an ancient monument on the outskirts of the ruins of Salamis, on the east coast of Cyprus. It consists of two chambers, of which the inner, rectangular with gable roof east-to-west and door at one end, is cut out of a single block of limestone, and roofed with another, which projects slightly above the modern surface of the ground. The junction of the two blocks is about half-way up the gable roof. The outer chamber is much larger and lies transversely to the inner, with its long axis north and south, and the inner chamber door in the middle of its west wall. Nearly opposite in the east wall is the outer entrance, approached from ground-level by a descent of rough steps, between walls of large squared masonry, now much damaged. The walls of the outer chamber consist of enormous upright slabs, crowned by a massive cornice, of a wide cavetto between two fillets, of which the upper projects considerably beyond the lower. On this cornice rests a semicircular vault of very large stones, the largest of which are set on end and occupy as much as a third of the vault. Within, they are carefully dressed, like the wall surfaces, but outside they were left rough, and have suffered further damage from exposure. They were not, however, intended to be seen, for there are remains of an outer casing of massive squared masonry, consisting of a cornice below, of the same profile as that of the vaulted chamber; and over this a plinth of three courses, the upper and lower plain, the middle bearing a simple cyma moulding, convex above. One course of the wall face is traceable still above the plinth, about half-way up the vault. The ends of the vault above the cornice are filled with rough walling, mostly recent, but including a number of stones from the plinth. A breach in the north wall serves as an entrance now, with a modern flight of steps. The building is now buried up to the level of the cornice and the great roof-slab, in a low mound; but the natural ground-level is only about a metre lower.