Surface potential on the periplasmic side of the photosynthetic membrane of Rhodopseudomonas sphaeroides

Membrane surface potential on the periplasmic side of the photosynthetic membrane was estimated in cells, spheroplasts and chromatophores of Rhodopseudomonas sphaeroides. When the membrane potential (potential difference between bulk aqueous phases) was kept constant in the presence of carbonylcyanide m-chlorophenylhydrazone, addition of salt to a suspension of cells or spheroplasts induced a red shift in the carotenoid absorption spectrum which indicated a change in the intramembrane electrical field. The spectral shift is explained by a rise in electrical potential at the outside surface of the photosynthetic membrane due to a decrease in extent of the negative surface potential.The spectral shift occurred in the direction opposite to that in chromatophores, indicating that the sidedness of the membrane of cells or spheroplasts is opposite to that of chromatophores. The dependences of the extent of the potential change on concentration and valence of cations of salts agreed with the Gouy-Chapman relationship on the electrical diffuse double layer. The charge density on the periplasmic surface of the photosynthetic membrane was estimated to be ?2.9 · 10^?3 elementary charge per Ų, while that on the cytoplasmic side surface was calculated as ?1.9 · 10^?3 elementary charge per Ų (Matsuura, K., Masamoto, K., Itoh, S. and Nishimura, M. (1979) Biochim. Biophys. Acta 547, 91–102). Surface potential on the periplasmic side of the photosynthetic membrane was estimated to be about ?50 mV at pH 7.8 in the presence of 0.1 M monovalent salt.     

Interaction of membrane surface charges with the reconstituted ADP/ATP-carrier from mitochondria

Various modulating influences of negative and positive membrane charges on binding and transport properties of the reconstituted ADP/ATP carrier from mitochondria were investigated. The results are interpreted in terms of functional and structural asymmetries of the adenine nucleotide carrier embedded in the liposomal membrane. The surface potential of liposomes was measured directly either by potential-dependent adsorption of the fluorescent dye $\text{2-p-}\text{toluidinylnaphthalene}$ 6-sulfonate (TNS) or by the $\text{p}\text{K}$ shift of the lipophilic pH indicator pentadecylumbelliferone. These results were correlated with the following observations. (1) Negative surface potentials increase the apparent dissociation constant, $\text{K}{}_{\mathrm{<mtext>d</mtext>}}$, for binding of the negatively charged inhbitor carboxyatractylate to the reconstituted carrier protein. (2) Surface potentials modulate the apparent transport affinity, $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$, of the reconstituted adenine nucleotide carrier for ADP and ATP. The interaction of surface charges with the transport function was investigated with carrier proteins oriented both right-side-out and inside-out. Thus the influence of the surface potential on the function of the ADP/ATP carrier could be determined for the internal and external active sites of the translocator on the outer side of the membrane. Large discrepancies were observed not only between the potentials measured directly (fluorescent dyes) and those measured indirectly (binding and transport affinities), but also between the different surface potentials determined from the influence on the alternatively oriented carrier proteins. The effect of surface charges was rather weak on the cytosolic side of the translocator, whereas there was a strong influence of surface charges on the active site at the matrix side. The most obvious explanation, i.e., screening of negative membrane charges by positively charged amino acid residues at the protein surface, could be ruled out. Besides the modulation of binding affinities for substrates and inhibitors, an additional side-specific effect of surface charges on the transport velocity was observed. Again, the influence on the internal active site of the ADP/ATP carrier was found to be much higher than that on the cytosolic site. The observed effects can be explained by a definite structural asymmetry of the carrier embedded in the liposomal membrane. That site which is physiologically exposed to the cytosol is located at a considerable distance from the plane of the membrane, whereas the opposite site seems to be in close proximity to the membrane surface. Moreover, a spatial equivalence of carboxyatractylate binding site and nucleotide binding site at the external side of the carrier protein was concluded.     

Longitudinal organisation of the mollusc species in a theoretical French river

An analysis of mollusc communities collected at 272 stations was carried out, using multivariate statistical methods. The stations ranged from the sources to the lower reaches of large rivers. Nine malacotypes succeeded each other along this upstream-downstream gradient; comments are given regarding the composition of their communities. The organisation of the 52 mollusc species included corresponds to that of a theoretical river, representative of the French hydrographic system. The reduction of the abundance and of species richness in the lower reaches of large rivers is discussed. This revised version was published online in July 2006 with corrections to the Cover Date.     

Generation of daunomycin radicals on the outer side of the erythrocyte membrane

The generation of the daunomycin semiquinone was studied in intact red blood cells under CO atmosphere by ESR spectroscopy. The undialyzed hemolysates and the spin broadening agent chromium oxalate quenched the ESR signal, suggesting external location of the ESR-detectable radicals and their slow diffusion inside. A constant outward flow of O2- was detected by monitoring the approach to the steady state of the ESR signal of Cu,Zn superoxide dismutase externally added to red blood cells plus daunomycin in air. This suggests a reductase on the outer side of the erythrocyte membrane as the source of daunomycin radicals.     

Nucleosomal organization of chromatin in sperm nuclei of the bivalve mollusc Aulacomya ater

The sperm nuclei of Aulacomya ater, family Mitylidae, contain three proteins (X, Aa5 and Aa6) which are specific to this cell type coexisting with a set of five somatic-type histones. Information about the chromatin structure resulting from this kind of association is scarce. Therefore, we have probed the structure of this sperm chromatin through digestion with micrococcal nuclease in combination with salt fractionation. The data obtained have allowed us to propose a nucleosomal arrangement for this chromatin. However, two types of nucleosomes would be present in agreement with their protein components.     

Shoreward orientation involving geomagnetic cues in the nudibranch mollusc Tritonia diomedea

The marine nudibranch mollusc Tritonia diotnedea orients to the geomagnetic field in the laboratory, and has identifiable brain cells (Pd5, 6) which respond electrically when the ambient magnetic field is rotated artificially. Field studies reported here seek to determine if and why Tritonia diomedea uses geomagnetic cues to orient in the field. Animals were collected in their natural habitat using SCUBA, and placed on lines with magnetic headings parallel to the shore, at different locations with respect to their site of origin. Observations made at two or more tidal cycles later indicate that most animals move from the line in a direction corresponding to the original shoreward direction, regardless of the actual shoreward direction at the site of release, suggesting guidance by geomagnetic cues. Tritonia diomedea are close to neutrally buoyant, and subject to transport over great distances when dislodged by tidal currents or during escape swimming behavior. Since the natural distribution of food and mates is along the shoreline, shoreward orientation using geomagnetic cues, particularly when other cues are weak or ambiguous, may have adaptive value.     

Effects of different vertebrate growth factors on primary cultures of hemocytes from the gastropod mollusc,Haliotis tuberculata

Summary— A useful experimental system from primary cultures of hemocytes from Haliotis tuberculata has been established. Six days after initiation of the culture, the viability of hemocytes remained constant as measured by the MTT assay. In addition, hemocytes showed physiological responses as judged by protein and DNA syntheses in response to treatment with vertebrate growth factors. Porcine insulin and human epidermal growth factor (EGF) stimulated [³H]-leucine and [³H]-thymidine incorporation in hemocytes in a dose-dependent manner. No additive effect of insulin and EGF is observed either for [³H]-leucine or for [³H]-thymidine incorporation. The response of primary cultures of abalone hemocytes to vertebrate growth factors confirms their growth potential in vitro and provides a suitable model for further studies on regulation of the control of cellular processes such as cell growth, differentiation and migration in invertebrate cells.     

上川岛潮间带不同生境底栖软体动物物种多样性初步研究

目的:研究上川岛潮间带不同生境底栖软体动物物种多样性。方法:对上川岛潮间带(沙滩、泥沙滩、红树林泥滩、岩石滩) 4种不同生境的7个样方的底栖软体动物进行了生物多样性的初步调查。结果:共鉴定底栖软体动物14科30种,上川岛潮间带底栖软体动物物种丰富度指数为岩石滩(平均为1.784 ) >泥沙滩(平均为1.5 2 1) >红树林泥滩(平均为1.2 35 ) >沙滩(平均为0 .6 92 ) ;多样性指数岩石滩(平均为1.5 5 1) >红树林泥滩(平均为1.30 4 ) >泥沙滩(平均为1.16 2 ) >沙滩(平均为0 .4 0 3)。结论:底栖软体动物的种类及数量分布受底质、浪击等因素的影响。     

Ultrastructure of secretion in the atrial gland of a mollusc (Aplysia)

Extracts of the atrial gland of the sea hare Aplysia californiea (Mollusca) induce egg laying when injected into mature individuals. Since egg laying is controlled endogenously by a peptide secreted by neuroendocrine cells in the central nervous system, the relationship between the atrial gland and these central neurons has become an issue of interest. With the particular objective of examining secretory structures we undertook an ultrastructural study of the atrial gland and adjacent tissues. This study revealed that the atrial gland epithelium is composed of two major cell types: ‘goblet-like’ exocrine cells containing large electron-dense granules, and ciliated ‘capping cells’. A non-secretory, and possibly post-secretory, cell containing electron-lucent granules was noted. A region of the large hermaphroditic duct contiguous to the atrial gland, known as the red hemiduct, also displayed capping cells and secretory cells with large granules. The content of these granules is organized into crista-like condensations. The cell also contains iron-rich pigment inclusions.     

Anchorage of cyclodextrin glucanotransferase on the outer membrane of Escherichia coli

The gene encoding cyclodextrin glucanotransferase (CGTase) was successfully cloned from B. macerans by PCR. A recombinant plasmid pCS005 with a gene encoding the Lpp-OmpA-CGTase trifusion protein was constructed and transformed into E. coli for the surface display of CGTase. Results of immunoblotting analysis and protease accessibility on the fractionated cell membranes confirmed that the Lpp-OmpA-CGTase trifusion protein was successfully anchored on the outer membrane of E. coli. However, only 50% of the membrane-anchored trifusion proteins were displayed on the outer surface of E. coli with the remaining 50% un-translocated. The low efficiency of surface display is attributed to the large size of CGTase. Only a trace amount of CGTase activity was detected for both the whole cells and the cell debris fractions. Because the results of the protease accessibility study suggested that the trypsin-resistant conformation of CGTase was preserved in the membrane-anchored CGTase, we believe that the lack of enzyme activity is mainly due to the inaccessibility of the CGTase active site, near the N-terminus, for substrate molecules. It can be estimated that the critical size for surface display of protein in E. coli is approximately 70 kDa.     

Millimeter waves thermally alter the firing rate of the Lymnaea pacemaker neuron

The effects of millimeter waves (mm-waves, 75 GHz) and temperature elevation on the firing rate of the BP-4 pacemaker neuron of the pond snail Lymnaea stagnalis were studied by using microelectrode techniques. The open end of a rectangular waveguide covered with a thin Teflon film served as a radiator. Specific absorption rates (SARs), measured in physiological solution at the radiator outlet, ranged from 600 to 4200 W/kg, causing temperature rises from 0.3 to 2.2 °C, respectively. Irradiation at an SAR of 4200 W/kg caused a biphasic change in the firing rate, i.e., a transient decrease in the firing rate (69 ± 22% below control) followed by a gradual increase to a new level that was 68 ± 21% above control. The biphasic changes in the firing rate were reproduced by heating under the condition that the magnitude (2 °C) and the rate of temperature rise (0.96 °C/s) were equal to those produced by the irradiation (for an SAR of 4030 W/kg). The addition of 0.05 mM of ouabain caused the disappearance of transient responses of the neuron to the irradiation. It was shown that the rate of temperature rise played an important role in the development of a transient neuronal response. The threshold stimulus for a transient response of the BP-4 neuron found in warming experiments was a temperature rise of 0.0025 °C/s. Bioelectromagnetics 18:89–98, 1997. © 1997 Wiley-Liss, Inc.     

Morphology of wing mechanoreceptors involved in the wing retraction reflex in the pteropod mollusc Clione limacina

Tactile stimulation of the wings (parapodia) of actively swimming Clione limacina results in inhibition of swimming and retraction of the wings. Electrophysiological evidence suggests that wing mechanoreceptors have central cell bodies and wide innervation fields in the ipsilateral wing. Scanning electron microscopy of expanded wings reveals ciliary cone processes arranged in a pattern that is similar to the electrophysiologically‐determined innervation fields of wing mechanoreceptors. Transmission electron microscopy suggests that the ciliary cone structures are terminal processes of neuron‐like cells. Three‐dimensional reconstructions of serially‐sectioned terminal processes indicate that cell bodies are not found in the wing epithelium or immediately under the epithelium, further supporting the notion that the wing mechanoreceptors have central cell bodies.     

Expression, purification and the 1.8 angstroms resolution crystal structure of human neuron specific enolase

Human neuron-specific enolase (NSE) or isozyme gamma has been expressed with a C-terminal His-tag in Escherichia coli. The enzyme has been purified, crystallized and its crystal structure determined. In the crystals the enzyme forms the asymmetric complex NSE x Mg2 x SO4/NSE x Mg x Cl, where "/" separates the dimer subunits. The subunit that contains the sulfate (or phosphate) ion and two magnesium ions is in the closed conformation observed in enolase complexes with the substrate or its analogues; the other subunit is in the open conformation observed in enolase subunits without bound substrate or analogues. This indicates negative cooperativity for ligand binding between subunits. Electrostatic charge differences between isozymes alpha and gamma, -19 at physiological pH, are concentrated in the regions of the molecular surface that are negatively charged in alpha, i.e. surface areas negatively charged in alpha are more negatively charged in gamma, while areas that are neutral or positively charged tend to be charge-conserved.     

Charge clusters and the orientation of membrane proteins

Summary Although hydrophobic forces probably dominate in determining whether or not a protein will insert into a membrane, recent studies in our laboratory suggest that electrostatic forces may influence the final orientation of the inserted protein. A negatively charged hepatic receptor protein was found to respond totrans-positive membrane potentials as though electrophoresing into the bilayer. In the presence of ligand, the protein appeared to cross the membrane and expose binding sites on the opposite side. Similarly, a positively charged portion of the peptide melittin crosses a lipid membrane reversibly in response to atrans-negative potential. These findings, and others by Date and co-workers, have led us to postulate that transmembrane proteins would have hydrophobic transmembrane segments bracketed by positively charged residues on the cytoplasmic side and negatively charged residues on the extra-cytoplasmic side. In the thermodynamic sense, these asymmetrically placed charge clusters would create a compelling preference for correct orientation of the protein, given the inside-negative potential of most or all cells. This prediction is borne out by examination of the few transmembrane proteins (glycophorin, M13 coat protein, H-2K^b, HLA-A2, HLA-B7, and mouse Ig heavy chain) for which we have sufficient information on both sequence and orientation.In addition to the usual diffusion and pump potentials measurable with electrodes, the microscopic membrane potential reflects surface charge effects. Asymmetries in surface charge arising from either ionic or lipid asymmetries would be expected to enhance the bias for correct protein orientation, at least with respect to plasma membranes. We introduce a generalized form of Stern equation to assess surface charge and binding effects quantitatively. In the kinetic sense, dipole potentials within the membrane would tend to prevent positively charged residues from crossing the membrane to leave the cytoplasm. These considerations are consistent with the observed protein orientations. Finally, the electrostatic and hydrophobic factors noted here are combined in two hypothetical models of translocation, the first involving initial interaction of the presumptive transmembrane segment with the membrane; the second assuming initial interaction of a leader sequence.     

An identified cerebral interneuron initiates different elements of prey capture behavior in the pteropod mollusc,Clione limacina

The prey capture phase of feeding behavior in the pteropod molluscClione limacina consists of an explosive extrusion of buccal cones, specialized oral appendages which are used to catch the prey, and significant acceleration of swimming. Several groups of neurons which control different components of prey capture behavior inClione have been previously identified in the CNS. However, the question of their coordination in order to develop a normal behavioral reaction still remains open. We describe here a cerebral interneuron which has wide-spread excitatory and inhibitory effects on a number of neurons in the cerebral and pedal ganglia, directed toward the initiation of prey capture behavior inClione. This bilaterally symmetrical neuron, designated Cr-PC (Cerebral interneuron initiating Prey Capture), produced monosynaptic activation of Cr-A motoneurons, which control buccal cone extrusion, and inhibition of Cr-B and Cr-L motoneurons, whose spike activities maintain buccal cones in a withdrawn position inside the head in non-feeding animals. In addition, Cr-PC produced monosynaptic activation of a number of swim motoneurons and interneurons of the swim central pattern generator (CPG) in the pedal ganglia, pedal serotonergic Pd-SW neurons involved in a peripheral modulation of swimming and the serotonergic Heart Excitor neuron.     

A stochastic model for the membrane potential of a stimulated neuron

We present a simple model describing the transition between the prefiring, firing and postfiring phases of a single neuron in a large neural net. Using typical values for the physiological parameters that enter the model, we find average interspike times that are close to those reported in experimental measurements.     

Evidence for titratable gating charges controlling the voltage dependence of the outer mitochondrial membrane channel,VDAC

Summary A voltage-dependent anion-selective channel, VDAC, is found in outer mitochondrial membranes. VDAC's conductance is known to decrease as the transmembrane voltage is increased in either the positive or negative direction. Charged groups on the channel may be responsible for this voltage dependence by allowing the channel to respond to an applied electric field. If so, then neutralization of these charges would eliminate the voltage dependence. Channels in planar lipid bilayers which behaved normally at pH 6 lost much of their voltage dependence at high pH. Raising the pH reduced the steepness of the voltage dependence and raised the voltage needed to close half the channels. In contrast, the energy difference between the open and closed state in the absence of a field was changed very little by the elevated pH. The groups being titrated had an apparent pK of 10.6. From the pK and chemical modification, lysine epsilon amino groups are the most likely candidates responsible for VDAC's ability to respond to an applied electric field.     

Ancient DNA analysis identifies marine mollusc shells as new metagenomic archives of the past

Marine mollusc shells enclose a wealth of information on coastal organisms and their environment. Their life history traits as well as (palaeo‐) environmental conditions, including temperature, food availability, salinity and pollution, can be traced through the analysis of their shell (micro‐) structure and biogeochemical composition. Adding to this list, the DNA entrapped in shell carbonate biominerals potentially offers a novel and complementary proxy both for reconstructing palaeoenvironments and tracking mollusc evolutionary trajectories. Here, we assess this potential by applying DNA extraction, high‐throughput shotgun DNA sequencing and metagenomic analyses to marine mollusc shells spanning the last ~7,000 years. We report successful DNA extraction from shells, including a variety of ancient specimens, and find that DNA recovery is highly dependent on their biomineral structure, carbonate layer preservation and disease state. We demonstrate positive taxonomic identification of mollusc species using a combination of mitochondrial DNA genomes, barcodes, genome‐scale data and metagenomic approaches. We also find shell biominerals to contain a diversity of microbial DNA from the marine environment. Finally, we reconstruct genomic sequences of organisms closely related to the Vibrio tapetis bacteria from Manila clam shells previously diagnosed with Brown Ring Disease. Our results reveal marine mollusc shells as novel genetic archives of the past, which opens new perspectives in ancient DNA research, with the potential to reconstruct the evolutionary history of molluscs, microbial communities and pathogens in the face of environmental changes. Other future applications include conservation of endangered mollusc species and aquaculture management.