Structural Comparison of Cephalopod Hemocyanins: Phylogenetic Significance

Hemocyanins, the respiratory molecules of cephalopod mollusks, are hollow cylinders with five internal arches. Three hemocyanins representative of three orders of cephalopods (Benthoctopus species, Octopoda; Vampyroteuthis infernalis, Vampyromorpha; Sepia officinalis, Sepioidea) were subjected to cryoelectron microscopy and three-dimensional (3D) reconstruction. The structure of Benthoctopus hemocyanin, solved at 26.4-A resolution, possesses arches comprising two identical functional units. The similarity between these functional units and the structure recently observed in X-ray crystallography for Octopus by Cuff et al. (J. Mol. Biol., 1998, 232, 522-529) allows the identification of their N- and C-terminal domains in the 3D reconstruction volume. Conversely, arches present in the 3D reconstruction volume of Sepia hemocyanin (21.8 A resolution) contain four functional units that are disposed differently. The strong resemblance between the reconstruction volumes of Vampyroteuthis (21.4-A resolution) and Benthoctopus hemocyanins suggests that Sepioidea diverged from a group containing Octopoda and Vampyromorpha.     

Sustainable Energy Source for Wearable Electronics Based on Multilayer Elastomeric Triboelectric Nanogenerators

Wearable electronics have attracted a wide range of attention with various functions due to the development of semiconductor industry and information technology. This work focuses on a triboelectric nanogenerator‐based self‐charging power system as a continuous energy source for wearable electronics. The triboelectric nanogenerator has a multilayer elastomeric structure with closely stacked arches as basic functional units. Owing to material and structural innovations, this triboelectric nanogenerator performs outstanding electric output with the maximum volume charge density ≈0.055 C m^?3 and practical properties for energy harvesting from body motions. Utilizing the triboelectric nanogenerator as outsole to harvest energy from walking or jogging, a pair of shoes is fabricated with the maximum equivalent charge current of each shoe being around 16.2 µA and specific fitness functions realized on each shoe separately without complex connections.     

Determination of volatile metabolites originating from mould growth on wall paper and synthetic media

Microbial volatile organic compounds (MVOCs) emitted from the mould species Penicillium expansum, P. chrysogenum, Aspergillus versicolor, A. fumigatus, A. niger and Cladosporium cladosporoides were analyzed by means of solid phase microextraction (SPME) and GCMS. The mould species were cultivated on the synthetic agar dichloran chloramphenicol (DG 18) and on wet wall paper. The production of MVOCs was monitored over several weeks to detect changes in the emission rates between the initial stage and later periods of growth. The cultivation on the synthetic agar resulted in MVOC patterns with a wide variety of signals. In contrast, the growth on wet wall paper led to changed MVOC patterns with less signals. The emission rates were drastically reduced. Components emitted by all six fungi species on wall paper were 2-pentanol and 2-pentanone. 1-Octen-3-ol was emitted by five fungi species. 2-Pentanol was only detected in considerable amounts during the first days of growth whereas 1-octen-3-ol had a more constant emission rate over the whole period of growth. On the basis of our studies some MVOCs could be proposed as specific for single fungi on wall paper, e.g. 1,3-dimethoxybenzene for A. versicolor and 2,4-pentandione for A. fumigatus.     

Augmin-dependent microtubule nucleation at microtubule walls in the spindle

The formation of a functional spindle requires microtubule (MT) nucleation from within the spindle, which depends on augmin. How augmin contributes to MT formation and organization is not known because augmin-dependent MTs have never been specifically visualized. In this paper, we identify augmin-dependent MTs and their connections to other MTs by electron tomography and 3D modeling. In metaphase spindles of human cells, the minus ends of MTs were located both around the centriole and in the body of the spindle. When augmin was knocked down, the latter population of MTs was significantly reduced. In control cells, we identified connections between the wall of one MT and the minus end of a neighboring MT. Interestingly, the connected MTs were nearly parallel, unlike other examples of end–wall connections between cytoskeletal polymers. Our observations support the concept of augmin-dependent MT nucleation at the walls of existing spindle MTs. Furthermore, they suggest a mechanism for maintaining polarized MT organization, even when noncentrosomal MT initiation is widespread.     

Electron microscopical structure analysis of yeast fatty-acid synthase at low resolution

From an electron microscopical tilt series of the multi-enzyme complex yeast fatty-acid synthase eight three-dimensional molecular structures were obtained by three-dimensional reconstruction of single molecules. The structures confirm present concepts showing a well defined central wall and a sequential arrangement of protein domains in the form of "arches". Additional structural details as ring-shaped parts in the central walls are recognizable. Because of the flattening and the irregular structural deterioration of the single molecules three-dimensional averaging was only partially successful; however, a satisfactory average from five molecules could be obtained. Attempts to find the symmetry of the subunit arrangement by applying correlation methods and by establishing a novel type of correlation analysis ("correlation tables") did not yield a clear proof. However, several strong indications of D3 symmetry were found.     

7种头足类动物墨汁的钠、钾含量分析

采用火焰光度计测定法,对虎斑乌贼(Sepia pharaonis)、金乌贼(Sepia esculenta)、拟目乌贼(Sepial ycidas)、日本无针乌贼(Sepiella japonica)、柏氏四盘耳乌贼(Euprymna berryi)(乌贼目)、剑尖枪乌贼(Uroteuthis edulis)(枪形目)和弯斑蛸(Octopus dollfusi)(八腕目)等7种头足类动物墨汁中的钠、钾含量进行了检测.结果显示:乌贼目墨汁的钠含量比八腕目和枪形目高.五种乌贼目动物中,金乌贼与拟目乌贼、日本无针乌贼、柏氏四盘耳乌贼,拟目乌贼与虎斑乌贼,虎斑乌贼与日本无针乌贼的墨汁钠含量存在显著的差异(P＜0.05),其余的组合无显著差异.不同目的动物墨汁的钾含量无明显的差异.     

Nuclear pores in the apoptotic cell

Summary During apoptosis, nuclear pores undergo strong modifications, which are described here in five different apoptotic models. Conventional electron microscopy, supported by freeze-fracture analysis, showed a constant migration of nuclear pores towards the diffuse chromatin areas. In contrast, dense chromatin areas appear pore-free and are frequently surrounded by strongly dilated cisternae. A possible functional significance of this pore behaviour during apoptosis is discussed.     

Electron microscope studies of oogonial wall and elemental composition of oogonia in Phytophthora

Scanning electron microscopy of oogonia of Phytophthora spp. showed that the oogonial wall was smooth in P. cactorum, P. citricola, P. heveae, and P. palmivora; finely granular in P. megasperma and P. megasperma var. sojae; and coarsely granular in P. parasitica. Transmission electron microscopy demonstrated that the oogonial wall in Phytophthora was composed of three layers with the middle layer being the least or the most electron dense. A coat of amorphous material was found on the entire outer surface of the oogonial wall. Elemental analysis of oogonia by means of a SEM electron probe microanalyzer revealed similar emission spectra among Phytophthora spp. with a characteristic peak for calcium.     

Electron flow to photosystem I from stromal reductants in vivo: the size of the pool of stromal reductants controls the rate of electron donation to both rapidly and slowly reducing photosystem I units

Electron donation from stromal reductants to photosystem I (PSI) was studied using the kinetics of P700(+) (the oxidized primary donor of PSI) reduction in the dark after irradiation of barley ( Hordeum vulgare L.) leaves. The leaves were treated with diuron and methyl viologen to abolish both the electron flow from PSII and PSI-driven cyclic electron transport. The redox state of P700 was monitored using the absorbance changes at 830 nm (Delta A(830)). Two exponentially decaying components with half-times of about 3 s (the slow component) and about 0.6 s (the fast one) were distinguished in the kinetic curves of Delta A(830) relaxation after a 1-s pulse of far-red light. The complex kinetics of P700(+) reduction thus manifested two types of PSI unit differing in the rate of electron input from stromal reductants. The rates of both kinetic components assayed after 1-s pulses were increased about 20-fold by a short (2-5 min) heat-pretreatment of leaves, indicating the accelerated input of electrons to both types of PSI unit. The increased rates of electron flow to P700(+) were even observed 1.5 h after the action of heat had been completed. Both kinetic components were dramatically slowed down upon irradiation of heat-treated leaves for 20-30 s. Their rates were restored after a short (20-30 s) period of darkness. A 5-min leaf exposure at 38 degrees C was sufficient to stimulate by severalfold the reduction of P700(+) pre-oxidized by a brief light pulse. In contrast, the acceleration of P700(+) reduction after a 1-min irradiation was observed only if leaves were subjected to temperatures above 40 degrees C. Neither heat treatment of leaves nor light-dark modulations in the rates of the fast and the slow components of P700(+) dark reduction influenced the relative magnitudes of the two kinetic components, providing strong additional evidence in favor of two distinct types of PSI existing per se in barley leaves. The key role in the control of the activity of electron donation to P700(+) in both rapidly and slowly reducing PSI units was attributed to the amount of stromal reductants available for P700(+) reduction. The latter was expected to be reduced under illumination in the presence of methyl viologen, while increased again in the dark. The regeneration of the pool of stromal reductants in the dark was likely provided by starch breakdown within the chloroplast stroma, but not by import of reducing equivalents from the cytosol. This was evidenced by much lower rates, compared with 1-h dark-adapted leaves, of dark reduction of both components of P700(+) in leaves stored for 24 h in the dark and thus depleted of starch but containing large amounts of glucose, the respiratory substrate.     

Probing the building blocks of eumelanins using scanning electron microscopy

Scanning electron microscopy (SEM) is used to examine the structure of natural and synthetic melanins. Eumelanin from Sepia officinalis and synthetic eumelanin are found to be structurally dissimilar. The natural sample has a significant structural order with subunits that have a lateral dimension of approximately 15 nm. The synthetic samples, on the other hand, appear to be amorphous solids. These results lend support for the existence of fundamental structural units proposed from the analyses of wide-angle X-ray diffraction measurements and previous mass-spectrometry results. These findings also provide insight into the disparate photophysical behavior of Sepia and synthetic eumelanin.     

Characterization of the performance of a 200-kV field emission gun for cryo-electron microscopy of biological molecules

The value of an electron microscope equipped with a field emission gun (FEG) was first revealed in materials science applications. More recently, the FEG has played a crucial role in breaking the 10A barrier in single-particle reconstructions of frozen hydrated biological molecules. The standard high-resolution performance tests for electron microscopes are made close to focus, at several hundreds of A underfocus at a magnification of 500,000x or more. While this is appropriate for materials science specimens, it is not suitable for observing frozen hydrated biological specimens with which the optimum underfocus is of the order of 1 micron or so and the magnification is limited by radiation damage to roughly 30,000 to 60,000x. Thus, in order to access the performance of a cryo-electron microscope for high-resolution 3D electron microscopy of biological molecules, additional tests are necessary. We present here resolution tests of a 200-kV FEG using frozen hydrated virus suspensions. The extent and amplitude of the contrast transfer function are used as a test of the performance. We propose that small spherical viruses close to 300A in diameter, such as the picornaviruses or phages, make good specimens for testing the performance of an electron microscope in cryo-mode.     

Dimer ribbons in the three-dimensional structure of sarcoplasmic reticulum

The three-dimensional structure of scallop sarcoplasmic reticulum membranes has been determined from electron micrographs of two classes of stain-filled tubules by helical reconstruction methods. These structures are characterized by dimer ribbons of Ca2+-ATPase molecules running diagonally around the tube wall. Deep right-handed grooves separate the ribbons. The elongated, curved units of the dimer (approximately 95 A long in the radial direction; 60 to 70 A axially, and about 30 A wide) are displaced axially by approximately 34 A and are connected at their outer ends by a bridge running nearly parallel to the tube axis. The monomers make a second contact at their inner ends. Adjacent units with the same orientation form a strong contact that is responsible for the ribbon appearance. Comparison of tubules of different diameter shows that one set of connections between the dimer ribbons is conserved: the inner ends of axially displaced dimers appear to make contact along a left-handed path almost perpendicular to the major grooves. The lipid bilayer cannot be clearly identified. The two-dimensional map obtained from flattened tubules is consistent with the three-dimensional reconstruction in showing dimer ribbons connected by a weak contact across the grooves, strongly resembling the inter-dimer bond observed in three dimensions. The two-dimensional map shows a 2-fold axis relating units of the dimer, but the three-dimensional tubes show a slight axial polarity that may arise from the presence of proteins other than the Ca2+-ATPase.     

Field emission scanning electron microscopy of microtubule arrays in higher plant cells

Summary Specimen preparation protocols that allow field emission scanning electron microscope imaging of microtubules in plant cells were developed, involving simultaneous permeabilization with saponin and stabilization of microtubules with taxol. All categories of microtubule array were observed in onion root tip cells and in tobacco BY-2 cells grown in suspension culture and synchronized to provide high frequencies of mitotic stages. Cortical arrays consist of overlapping microtubules with free ends; individual microtubules directly overlie individual microfibrils in the cell wall. Preprophase bands and spindle microtubule bundles were also imaged. Phragmoplasts revealed early stages of wall deposition in the included cell plates and features interpreted as relating to high rates of microtubule turnover at the growing margins. It was possible to combine high resolution three-dimensional imaging with immunogold labelling of microtubules. Individual gold particles were readily distinguished decorating microtubules in the preparations; the method should be vaulable for studying many features of plant cell microtubules and their associated macromolecules.Abbreviations FESEM field emission gun scanning electron microscope - MTSB microtubule stabilising buffer Dedicated to Professor Eldon H. Newcomb in recognition of his contributions to cell biology     

An analysis of interneuronal connections in the auditory cortex of awake cats]

A statistical analysis has been made of the interaction of the auditory cortex units in alert cats with chronically implanted electrodes. Three neurones with an amplitude ratio of 4:2:1 were singled out from the multineuronal activity. The dependence between the firing of two neurones was determined by the cross interval histograms. The relationships between 78 pairs of units were studied in 26 three units microsystems. About a third of the studied pairs functioned independently. The number of pairs with one-way and two-way connections was about equal (26 and 30 respectively). The neurones which generated spikes of high and medium amplitude, had the largest number of two-way connections. One-way connections were equally represented in all the three neurones, though with regard to direction they depended on the amplitude characteristics of the spikes. In neurones with large and medium spikes, output connections predominated, while in neurones with small spikes input connections predominated considerably. The connection could be of inhibitory, excitatory or mixed type. The inhibitory type of connections was the most frequent occurrence (57 out of 86). At prolonged recording (6 to 16 min) of spike activity, most of the functional connections persisted.     

Modulating the redox potential of the stable electron acceptor, Q(B), in mutagenized photosystem II reaction centers

One of the unique features of electron transfer processes in photosystem II (PSII) reaction centers (RC) is the exclusive transfer of electrons down only one of the two parallel cofactor branches. In contrast to the RC core polypeptides (psaA and psaB) of photosystem I (PSI), where electron transfer occurs down both parallel redox-active cofactor branches, there is greater protein-cofactor asymmetry between the PSII RC core polypeptides (D1 and D2). We have focused on the identification of protein-cofactor relationships that determine the branch along which primary charge separation occurs (P(680)(+)/pheophytin(-)(Pheo)). We have previously shown that mutagenesis of the strong hydrogen-bonding residue, D1-E130, to less polar residues (D1-E130Q,H,L) shifted the midpoint potential of the Pheo(D1)/Pheo(D1)(-) couple to more negative values, reducing the quantum yield of primary charge separation. We did not observe, however, electron transfer down the inactive branch in D1-E130 mutants. The protein residue corresponding to D1-E130 on the inactive branch is D2-Q129 which presumably has a reduced hydrogen-bonding interaction with Pheo(D2) relative to the D1-E130 residue with Pheo(D1). Analysis of the recent 2.9 ? cyanobacterial PSII crystal structure indicated, however, that the D2-Q129 residue was too distant from the Pheo(D2) headgroup to serve as a possible hydrogen bond donor and directly impact its midpoint potential as well as potentially determine the directionality of electron transfer. Our objective was to characterize the function of this highly conserved inactive branch residue by replacing it with a nonconservative leucine or a conservative histidine residue. Measurements of Chl fluorescence decay kinetics and thermoluminescence studies indicate that the mutagenesis of D2-Q129 decreases the redox gap between Q(A) and Q(B) due to a lowering of the redox potential of Q(B). The resulting increased yield of S(2)Q(B)(-) charge recombination in the D2-Q129 mutants leads to an increased susceptibility to photoinhibitory light presumably due to (3)P(680)-mediated oxidative damage. The results indicate that the D2-Q129 residue plays a critical role in stabilizing the charge-separated state in PSII and further documents the structural and functional asymmetry between the two cofactor branches in PSII.     

On the Similarity of Functional Connectivity between Neurons Estimated across Timescales

A central objective in neuroscience is to understand how neurons interact. Such functional interactions have been estimated using signals recorded with different techniques and, consequently, different temporal resolutions. For example, spike data often have sub-millisecond resolution while some imaging techniques may have a resolution of many seconds. Here we use multi-electrode spike recordings to ask how similar functional connectivity inferred from slower timescale signals is to the one inferred from fast timescale signals. We find that functional connectivity is relatively robust to low-pass filtering—dropping by about 10% when low pass filtering at 10 hz and about 50% when low pass filtering down to about 1 Hz—and that estimates are robust to high levels of additive noise. Moreover, there is a weak correlation for physiological filters such as hemodynamic or Ca²⁺ impulse responses and filters based on local field potentials. We address the origin of these correlations using simulation techniques and find evidence that the similarity between functional connectivity estimated across timescales is due to processes that do not depend on fast pair-wise interactions alone. Rather, it appears that connectivity on multiple timescales or common-input related to stimuli or movement drives the observed correlations. Despite this qualification, our results suggest that techniques with intermediate temporal resolution may yield good estimates of the functional connections between individual neurons.     

Morphology of reproductive accessory glands in female Sepia pharaonis (Cephalopoda: Sepiidae) sheds light on egg encapsulation

The pharaoh cuttlefish, Sepia pharaonis, is an important cephalopod fishery species in southeastern Asia, with understudied reproductive physiology. The present study aimed to investigate the cellular characteristics of epithelial cells found in the nidamental glands (NGs) and accessory NGs (ANGs), as well as the structural connections between these two glands in mature female S. pharaonis. A histological analysis revealed two types of epithelial cells in NGs: Alcian blue‐positive, PAS‐negative mucosubstance‐secreting cells and eosinophilic, PAS‐positive granule‐secreting cells. Using transmission electron microscopy, three types of epithelial cells were identified: cells with electron‐dense granules, cells with electron‐lucent granules, and cells with both cilia and microvilli in the apex. Mature ANGs contain an abundance of tubular units composed of epithelial cells resting on a thin layer of basal lamina. Innervated muscle cells are tightly adhered to the basal lamina. In addition, we observed epithelial canalization of ANG tubules penetrating through the connective tissue linking NGs and the walls of the tubules in ANGs, which allows the contents of the ANG tubules to be transported to the NGs. Our results suggest that ANGs participate in the encapsulation of the ova via the same pathway as NGs, which provides an important basis for future studies on the mechanism of protection provided by NGs and ANGs during embryonic development in S. pharaonis.     

A NEW BASIDIOMYCETOUS SEPTAL TYPE: THE MULTIPERFORATE SEPTUM IN KRIEGERIA ERIOPHORI

Septa from metabasidia and clamp connections of the heterobasidiomycetous sedge parasite Kriegeria eriophori were studied with light and electron microscopy. Septa from clamp connections subtending probasidia were reconstructed from serial sections. Septa at clamp connections are perforated by multiple, simple pores, while metabasidial septa possess single, central swellings which probably represent pores occluded by wall material. The occurrence of multiperforate septa in the fungi is reviewed. The septal morphology of K. eriophori is compared to that of simple-septate heterobasidiomycetes, and the systematic and functional implications are discussed.     

Similarities between G-proteins in visual cells of Sepia and cattle

In contrast to antisera against native transducin a polyclonal antiserum raised against heat-denatured bovine transducin crossreacts with the G-protein from Sepia visual cells. This antiserum recognizes a 44 kDa (G alpha) and a 36 kDa (G beta) protein band from Sepia photosensory membrane preparation. Furthermore we purified the antibody-binding G-protein from Sepia by binding it to light-activated rhodopsin of Sepia and GTP-induced extraction, similar to the purification of bovine transducin. This G-protein is probably involved in the phototransduction process. The purified Sepia G-protein did bind to vertebrate photosensoric membrane upon illumination, but was not eluted by GTP-containing buffer solution. After extensive bleaching, the G-protein became soluble.     

Exploring high-resolution cryo-ET and subtomogram averaging capabilities of contemporary DEDs

The potential of energy filtering and direct electron detection for cryo-electron microscopy (cryo-EM) has been well documented. Here, we assess the performance of recently introduced hardware for cryo-electron tomography (cryo-ET) and subtomogram averaging (STA), an increasingly popular structural determination method for complex 3D specimens. We acquired cryo-ET datasets of EIAV virus-like particles (VLPs) on two contemporary cryo-EM systems equipped with different energy filters and direct electron detectors (DED), specifically a Krios G4, equipped with a cold field emission gun (CFEG), Thermo Fisher Scientific Selectris X energy filter, and a Falcon 4 DED; and a Krios G3i, with a Schottky field emission gun (XFEG), a Gatan Bioquantum energy filter, and a K3 DED. We performed constrained cross-correlation-based STA on equally sized datasets acquired on the respective systems. The resulting EIAV CA hexamer reconstructions show that both systems perform comparably in the 4–6 Å resolution range based on Fourier-Shell correlation (FSC). In addition, by employing a recently introduced multiparticle refinement approach, we obtained a reconstruction of the EIAV CA hexamer at 2.9 Å. Our results demonstrate the potential of the new generation of energy filters and DEDs for STA, and the effects of using different processing pipelines on their STA outcomes.