Light-activated single channel currents in Limulus ventral nerve photoreceptors

Light-activated single channel currents were measured in Limulus ventral photoreceptors in the cell-attached configuration at 14°C. The results show three channel types with conductances of 6.2, 10.4 and 28.7 pS. The most active channels have the 10 pS conductance; the open time histograms of these channels could be best fitted by the sum of two exponentials with time constants (and weights) of 0.58 ms (0.78) and 4.32 ms (0.22), suggesting two populations of channels or two open states. The mean open time was 1.38 ms. The open time histogram of the channels with the 29 pS conductance could be best fitted by a single exponential with a time constant of 3.35 ms. First latencies of the 10 pS channels were between 40 and 280 ms but those of the 29 pS conductance channels were 300 ms. These findings suggest that the two channel types are gated by two different intracellular transmitters or mechanisms. Offprint requests to: K. Nagy     

Modulation of inner mitochondrial membrane channel activity

Three classes of inner mitochondrial membrane (IMM) channel activities have been defined by direct measurement of conductance levels in membranes with patch clamp techniques in 150 mM K Cl. The 107 pS activity is slightly anion selective and voltage dependent (open with matrix positive potentials). Multiple conductance channel (MCC) activity includes several levels from about 40 to over 1000 pS and can be activated by voltage or Ca²⁺. MCC may be responsible for the Ca²⁺-induced permeability transition observed with mitochondrial suspensions. A low conductance channel (LCC) is activated by alkaline pH and inhibited by Mg²⁺. LCC has a unit conductance of about 15 pS and may correspond to the inner membrane anion channel, IMAC, which was proposed from results obtained from suspension studies. All of the IMM channels defined thus far appear to be highly regulated and have a low open probability under physiological conditions. A summary of what is known about IMM channel regulation and pharmacology is presented and possible physiological roles of these channels are discussed.     

Vorläufige Versuche über die Elastizität des Atraktoplasmas

Summary Spindles of nuclei mostly from metaphases of expiants in vitro, Set free according to Dan's method by means of solutions of Duponol (a sodium lauryl sulfonate), have been studied according to their elasticity. The isolated atractoplasm has shown a static modul of elasticityE0.8 to 1.6×10⁶ dynes×cm^–2, and, sometimes, a Poisson number of0.42, that is to say the atractoplasmic gel of the spindles of nuclei is certain elastically. The values of the modul of elongation agree satisfactory with those values recently ascertained by Crick and Hughes with cytoplasm oi explanted cells.     

Lumenal calcium modulates unitary conductance and gating of a plant vacuolar calcium release channel

The patch clamp technique has been used to investigate ion permeation and Ca²⁺-dependent gating of a voltage-sensitive Ca²⁺ release channel in the vacuolar membrane of sugar beet tap roots. Reversal potential measurements in bi-ionic conditions revealed a sequence for permeability ratios of Ca²⁺ Sr²⁺ Ba²⁺ > Mg²⁺ K⁺ which is inversely related to the size of the unitary conductances K⁺ Mg²⁺ Ba²⁺ > Sr²⁺ Ca²⁺, suggesting that ion movement is not independent. In the presence of Ca²⁺, the unitary K⁺ current is reduced in a concentration- and voltage-dependent manner by Ca²⁺ binding at a high affinity site (K _0.5 = 0.29 mm at 0 mV) which is located 9% along the electric field of the membrane from the vacuolar side. Comparison of reversal potentials obtained under strictly bi-ionic conditions with those obtained in the presence of mixtures of the two ions indicates that the channel forms a multi-ion pore. Lumenal Ca²⁺ also has an effect on voltage-dependent channel gating. Stepwise increases of vacuolar Ca²⁺ from micromolar to millimolar concentrations resulted in a dramatic increase in channel openings over the physiological voltage range via a shift in threshold for channel activation to less negative membrane potentials. The steepness of the concentration dependence of channel activation by Ca²⁺ at –41 mV predicts that two Ca²⁺ ions need to bind to open the gate. The implications of the results for ion permeation and channel gating are discussed.We thank Ian Jennings for writing and implementing some of the software used in this study and Anna J. Bate for technical assistance. The work was supported by grants from the Biotechnology and Biological Sciences Research Council to E.J. (PDF/14) and DS (PG87/529).     

Requirements for in vitro propagation of seven nitrogen-fixing Alnus species

Studies on the in vitro propagation of Alnus crispa, A. glutinosa, A. incana, A. japonica, A. rubra, A. sinuata and A. viridis indicated interspecific as well as intraspecific variations in their requirements for in vitro culture. The WPM and Blaydes media supported, respectively, growth of A. glutinosa and A. crispa but not that of both species, while the MS medium induced equal or significantly better growth than WPM and Blaydes media for both species. The optimum type and concentration of sugar to be used in the multiplication medium varied with species. Only A. glutinosa showed good growth on sucrose while glucose was optimum for all other species but at different concentrations. All species rooted in 3 weeks on half-strength MS medium including 1 M IBA. All clones of A. glutinosa and A. rubra rooted 100%, whereas easy-to-root and difficult-to-root clones were observed in the other species. In the rooting medium, glucose promoted rooting of the difficult-to-root clones better than sucrose. Survival following transfer to an artificial substrate was 100% for all species. Nodulation tests using pure cultures of two Frankia strains showed 100% nodulation on all Alnus clones.     

Ultrastructural localization of corticotropin, β-lipotropin,and αand β-endorphin in the porcine anterior pituitary

Summary The peroxidase-antiperoxidase immunocytochemical technique was used to identify the ACTH/endorphin cells in the porcine pituitary at the ultrastructural level and to determine the precise subcellular localization of the pro-ACTH/endorphin fragments. The cells display different aspects: 1) large, regular shapes with numerous and large secretory granules; 2) small, irregular and angular shapes with small granules aligned along the periphery of the cell; and 3) intermediate forms. The presence of and -endorphin not only in the same cells but also in the same secretory granules that contain ACTH and -LPH clearly indicates that both the precursor or its fragments and the abovementioned peptides are stored in the same granules and released simultaneously by the corticotropic cells. The presence of FSH in some corticotropic cells is also discussed.Abbreviations used in this Article ACTH corticotropin - -MSH -melanotropin (ACTH I–I3) - CLIP corticotropin-like intermediate lobe peptide (ACTH 18–39) - -LPH -lipotropin - -MSH -melanotropin (-LPH 41–58); -endorphin (-LPH 61–91); -endorphin (-LPH 61–76)     

Second-derivative Fourier transform infrared spectra of seaweed galactans

The Fourier transform infrared spectra of agar, agarose, -, -, and -carrageenan, and ofChondrus canaliculatus, Iridaea ciliata, I. membranacea, I. laminarioides andGracilaria chilensis polysaccharides were recorded in the 4000–400 cm^-1 region. The bands in the second derivative mode are sharper and more bands are resolved than in the normal spectra.Agar, agarose andG. chilensis phycocolloids exhibit diagnostic bands at 790 and 713 cm^-1. -, - and -carrageenans, and native carrageenan-type polysaccharides fromC. canaliculatus andIridaea species exhibit bands at around 1160, 1140, 1100, 1070, 1040, 1008, 610, and 580 cm^-1. Therefore, FT-IR spectroscopy in the second-derivative mode may be applied to differentiate between agar- and carrageenan-types seaweed galactans.     

Pattern and timing of evolutionary divergences among hominoids based on analyses of complete mtDNAs

We have examined and dated primate divergences by applying a newly established molecular/paleontological reference, the evolutionary separation between artiodactyls and cetaceans anchored at 60 million years before present (MYBP). Owing to the morphological transformations coinciding with the transition from terrestrial to aquatic (marine) life and the large body size of the animals (which makes their fossils easier to find), this reference can be defined, paleontologically, within much narrower time limits compared to any local primate calibration marker hitherto applied for dating hominoid divergences. Application of the artiodactyl/cetacean reference (A/C-60) suggests that hominoid divergences took place much earlier than has been concluded previously. According to a homogenous-rate model of sequence evolution, the primary hominoid divergence, i.e., that between the families Hylobatidae (gibbons) and Hominidae, was dated at 36 MYBP. The corresponding dating for the divergence betweenPongo (orangutan) andGorilla-Pan (chimpanzee)-Homo is 24.5 MYBP, that forGorilla vsHomo-Pan is 18 MYBP, and that forHomo vsPan 13.5 MYBP. The split between Sumatran and Bornean orangutans was dated at 10.5 MYBP and that between the common and pygmy chimpanzees at 7 MYBP. Analyses of a single gene (cytochromeb) suggest that the divergence within the Catarrhini, i.e., between Hominoidea and Old World monkeys (Cercopithecoidea), took place >40 MYBP; that within the Anthropoidea, i.e., between Catarrhini and Platyrrhini (New World monkeys), >60 MYBP; and that between Anthropoidea and Prosimii (lemur), 80 MYBP. These separation times are about two times more ancient than those applied previously as references for the dating of hominoid divergences. The present findings automatically imply a much slower evolution in hominoid DNA (both mitochondrial and nuclear) than commonly recognized.     

Structure of the fission yeast centromere cen3: Direct analysis of the reiterated inverted region

We determined the structure of the Schizosaccharomyces pombe centromere cen3 using direct genomic mapping and cosmid walking. The repetitive region of cen3 is approximately 110 kb, much longer than that of the previously determined cen1 and cen2 regions. The 30 kb long left and 60 kb right repetitive sequences are arranged with an inverted symmetry and flank the 1520 kb central domain. The repeat motifs in cen3, although they consist of the common centromeric repeat elements, are slightly different from those in cen1 and cen2. The cen3 repeat motifs appear to be reiterated four times in the left and nine times in the right side repetitive regions. We found that the central domain consists of the common 5 kb core sequence associated with the pair of innermost inverted sequences, most of which are reiterated only twice in the genome. Although their sizes differ significantly, the general features of cen1, cen2 and cen3 are similar, and a prototype, consensus structure for the fission yeast centromere may be deduced.by J.A. Huberman     

Spectrophotometric phytochrome measurements in light-grown Avena sativa L.

Phytochrome was studied spectrophotometrically in Avena sativa L. seedlings that had been grown for 6 d in continous white fluorescent light from lamps. Greening was prevented through the use of the herbicide San 9789. When placed in the light, phytochrome (P_tot) decreased with first order kinetics (_1/2 2 h) but reached a stable low level (2.5% of the dark level) after 36 h. This concentration of phytochrome remained constant in the light and during the initial hours of a subsequent dark period, but increased significantly after a prolonged dark period. Evidence suggests that the constant pool of phytochrome in the light is achieved through an equilibrium between synthesis of the red absorbing (P_r) and destruction of the far-red absorbing form (P_fr) of phytochrome. It is concluded that the phytochrome system in light-grown oat seedlings is qualitatively the same as that known from etiolated monocotyledonous seedlings, but different than that described for cauliflower florets.Abbreviations P_fr the far-red light absorbing form of phytochroma - P_r the red light absorbing form of phytochrome - P_tot P_r+P_fr - k_s rate constant of P_r synthesis - k_d rate constant of P_fr destruction - MOPS N-morpholino-3-propane-sulfonic acid - IRIS Tris (hydroxymethyl) amino methane - San 9789 4-chloro-5-(methyl amino)-2-(,,-trifluoro-m-tolyl)-3(2H)pyridazinone     

K-current fluctuations in inward-rectifying channels of frog skeletal muscle

Summary K currents and K-current fluctuations were recorded in inwardly rectifying K channels of frog skeletal muscle under voltage-clamp conditions. External application of 0.2 to 10mm Cs reduces the inward mean K current but produces a distinct increase of the spectral density of K-current fluctuations. The additional fluctuations arise from the random blocking by Cs ions. From the variance of current fluctuations, the steady-state current and the probability of the open unblocked channel an effective single-channel conductance ^* was calculated. ^* strongly depends on the external Cs concentration (7.8 pS at 0.2mm Cs, 2.1 pS at 10mm Cs). This dependence is interpreted in terms of a two-step blocking process: (1) a fast exchange of Cs ions between the external solution and a first binding site inside the channel which leads to the Cs-modulated effective single-channel conductance, and (2) a slow Cs binding to a second site deeper in the channel which produces the observed current fluctuations. With this hypothesis we obtained a real single-channel conductance of 10 pS and a real density ofn4 inwardly rectifying channels per m² of muscle surface area.     

Background concentration of 226Ra in terrestrial animals

Evidence that environmental levels of vanadium are increasing hasraised concern over the injection of vanadium into the environmentfrom anthropogenic sources. Two simple global mass balance models(simulating current and pre-industrial conditions) were developed todemonstrate the influence of anthropogenic vanadium on the globaldistribution of this trace metal. Current vanadium emissions owing toman's current industrial activities were estimated to comprise 30% oftotal atmosphere loading, 3% of total ocean loading, and 6% of totalland loading. These loadings were always considerably less than thoseresulting from non-anthropogenic sources or events. Differences notedbetween the pre-industrial and current models were not sufficientlygreat to suggest that injection of anthropogenic vanadium constitutes asignificant environmental threat on a global scale.     

Channel gating in the absence of agonist by a homooligomeric molluscan GABA receptor expressed inXenopus oocytes from a cloned cDNA

We have previously described the isolation of a complementary DNA (cDNA) from the freshwater molluscLymnaea stagnalis encoding a polypeptide that exhibits 50% identity to the ß-subunits of vertebrate -aminobutyric acid (GABA) type A (GABA_A) receptor. When expressed inXenopus laevis oocytes fromin vitro-transcribed RNA, the snail subunit forms functional homo-oligomeric receptors possessing chloride-selective ion channels. In recordings from voltage-clamped oocytes held at –60 mV, GABA induced an inward current, whereas application of the chloride-channel blocker picrotoxin (in the absence of agonist) elicited an apparent outward current. Single channel recordings obtained from cell-attached patches have revealed a single population of 20 pS channels, with an open probability greater than 90% (at a pipette potential of –100 mV) in the absence of GABA. The relationship between single channel current and pipette potential was linear over the studied range (–100 mV to +60 mV), but the open probability was less for hyperpolarizations than for depolarizations. The spontaneous channel openings were blocked by micromolar concentrations of picrotoxin. Functional hetero-oligomeric receptors were formed when the molluscan subunit was co-expressed in oocytes with the bovine GABA_A receptor 1-subunit, but the channels gated by these receptors did not open spontaneously.     

Kainate activated single channel cnrrents as revealed by domoic acid

We have studied the properties of kainic acid receptor-activated channels using domoic acid as an agonist. Similarities of the electrophysiological, pharmacological and noise properties of domoic acid and kainic acid-evoked currents confirm that domoate is a potent and specific agonist of the kainate receptor. Single-channel properties of domoic acid-evoked currents were directly determined from outside-out membrane patches for the first time, and results were compared with those obtained by fluctuation analysis of macroscopic currents. Small conductance cationic-selective channels of 4 pS and a mean open time of 2 to 3 ms were detected using both methods. Offprint requests to: O. Moran     

Effects of seed distribution and competitors on seed harvesting efficiency in heteromyid rodents

Summary The foraging strategies of four naturally co-existing heteromyid rodent species were investigated: Dipodomys deserti (100 g), D. merriami (38 g), Microdipodops pallidus (13 g), and Perognathus longimembris (7 g). In 208 over-night laboratory foraging trials animals were provided with millet seed distributed in clumped and scattered patterns. Net removal of seeds from the foraging arena and amounts of seeds in cheek pouches and in caches were determined. When alone in an areana none of these species specialized extensively on either clumped or scattered seeds, although each tended to take more clumped than scattered seeds. When placed together with other individuals, animals once again tended to cache more clumped than scattered seeds in all but one paired combination of species: P. longimembris cached more scattered than clumped seeds when opposed by D. deserti. This suggests that the smaller species obtained a less preferred distribution of seeds in the face of competition. The two smaller species showed a great reduction in general foraging success in the presence of either of the two larger species. In general, a species cached less seeds when faced by larger opponent species.     

Thermotropism and hydration properties of POPE and POPE-cholesterol systems as revealed by solid state2H and31P-NMR

The partial phase diagram and the hydration properties of the 1-palmitoyl-2-oleoyl-phosphatidylethanolamine (POPE)-water system, in the absence and presence of 30 mol% cholesterol, have been investigated by solid state phosphorus NMR of the lipid and deuterium NMR of heavy water. The POPE-D₂O phase diagram resembles other phosphatidylethanolamine (PE)-water systems: below water-to-lipid molar ratios (R_i) of 3 the lamellar gel (L or L_c)-to-hexagonal type II (H_II) phase sequence is observed on increasing the temperature. For R_i3 the thermotropic sequence (L or L_c)-L-H_II is detected. On increasing hydration from R_i=3, the H_II phase is detected from 40°C to 85°C whereas the gel phase is observed from 40°C to 30°C. The limiting hydrations of the gel, L and H_II phases are R_i 3, 17 and 20, respectively. The number of bound water molecules per lipid is ca. 8 in both the La and HII phases. The presence of cholesterol stabilizes the hexagonal phase 20°C below temperatures at which it is observed in its absence and reduces the limiting hydration of the fluid and hexagonal phases to Ri 9 and 14, respectively. The structure and/or dynamics of the water bound to the interface are markedly modified on going from the L to the H_II phase.Abbreviations NMR Nuclear magnetic resonance - DDPE 1,2-Didodecyl-rac-glycerol-3-phosphoethanol-amine - DHPE 1,2-Dihexadecyl-sn-glycerol-3-phosphoethanol-amine - DOPE 1,2-Dioleoyl-sn-glycerol-3-phosphoethanol-amine - POPE 1-Palmitoyl-2-oleoyl-sn-glycerol-3-phosphoetha-nolamine - DAPE 1,2-Diarachinoyl-sn-glycerol-3-phosphoethanol-amine - DMPC 1,2-Dimyristol-sn-glycerol-3-phosphocholine - DPPC 1,2-Dipalmitoyl-sn-glycerol-3-phosphocholine - T_c lamellar gel-to-lamellar fluid transition temperature - T_h lamellar fluid-to-hexagonal transition temperature     

Blue through UV polarization sensitivities in insects

A Signal-to-Noise optimization model has now been extended to explain the range of species-specific polarization sensitivities of insects. The different polarization sensitivities are shown to represent optimizations for the detection of plane polarized (Rayleigh-scattered) skylight over a range of atmospheric polarization conditions. Rhodopsin absorption spectra with peaks in the Blue (max450 nm) maximize detection efficiencies under conditions of high polarization. Rhodopsin absorption spectra peaking in the UV (max350 nm) maximize Signal-to-Noise Ratios for the detection of polarized skylight at the other extreme of low polarization.     

Sugar nucleotides dissipate ATP-generated transmembrane pH gradient in Golgi vesicles from suspension-cell protoplasts ofChenopodium rubrum L.

A microsomal vesicle fraction (GV) markedly enriched by the Golgi marker enzyme latent inosine diphosphatase (IDPase) has been isolated from photoautotrophic suspension-cell protoplasts ofChenopodium rubrum L. Addition of ATP creates a substantial pH gradient across the GV membrane as measured by accumulation of acridine orange. The GV showed a density of 1.14 g·cm^-3 by equilibrium density centrifugation on sucrose gradients. Coincidence of acridine-orange accumulation and IDPase activity was confirmed on Percoll gradients. Formation of the pH gradient half-saturates at 0.3 mM MgATP, peaks at pH 7, and is competitively inhibited by ADP (k _i0.1 mM), but not by P_i; it is hardly inhibited by orthovanadate, quickly dissipated by monensink ₂=18 nM), nigericin (k _1/2=25 nM), and sluggishly by N-ethylmaleimide (k _1/235 M). Inhibition by KNO₃ (k _1/26.7 mM) is incomplete (60%). Uridine 5-diphosphate (UDP)-glucose, UDP-galactose, but not UDP-mannose and the pertinent sugars, dissipate the ATP-generated pH gradient (k _1/210–20 mM UDP-glucose; optimum pH at 7.8). This UDP-glucose activity is accompanied by release of P_i, but not of glucose or sucrose. UDP-glucoseinduced P_i release from the GV saturates (k _1/2=1 mM UDP-glucose; optimum pH at 7) and is completely inhibited by the anion-channel blocker 4,4-diisothiocyano-2,2-stilbene disulfonic acid (DIDS;k _1/2=140 M). The GV incorporates UDP-[U-¹⁴C]glucose into an acid-labile, alkaline-stable macromolecular compound; this process is like-wise inhibited by DIDS. We propose a model including, inter alia, a UDP-glucose/uridine-5-monophosphate translocator and a phosphate-permeable anion channel to operate in Golgi vesicles ofChenopodium rubrum.Abbreviations AO acridine orange - DIDS 4,4-diisothiocyano-2,2-stilbene disulfonic acid - FCCP carbonyl cyanidep-trifluoromethyoxyphenyl hydrazone - GV Golgi-vesicle-enriched microsomal fraction - IDPase mosine diphosphatase     

Inward rectifier K channels in renal epithelioid cells (MDCK) activated by serotonin

Summary The present study has been performed to test for the effect of intracellular calcium and of serotonin on the channel activity in patches from subconfluent MDCK-cells. In inside-out patches, inwardly rectifying potassium-selective channels are observed with open probabilities of 0.01±0.01, 0.24±0.03 and 0.39±0.07, at 100 nmol/liter, 1 mol/liter or 10 mol/liter calcium activity, respectively. The single-channel slope conductance is 34±2 pS, if the potential difference across the patch (V ) is zero, and approaches 59±1 pS, ifV is –50 mV, cell negative. In the cell-attached mode, little channel activity is observed prior to application of serotonin (open probability=0.03±0.03). If 1 mol/liter serotonin is added to the bath perfusate, the open probability increases rapidly to a peak value of 0.34±0.04 within 8 sec. In continued presence of the hormone, the open probability declines to approach 0.06±0.02 within 30 sec. At zero potential difference between pipette and reference in the bath (i.e., the potential difference across the patch is equal to the potential difference across the cell membrane), the single-channel conductance is 59±4 pS. In conclusion, inwardly rectifying potassium channels have been identified in the cell membrane of subconfluent MDCK-cells, which are activated to a similar extent by increase of intracellular calcium activity to 1 mol/liter and by extracellular application of 1 mol/liter serotonin.     

Anion channels from rat brain synaptosomal membranes incorporated into planar bilayers

Summary Synaptic membranes from rat brain were incorporated into planar lipid bilayers, and the characteristics of two types of anion-selective channels (type I and type II) were investigated. In asymmetric BaCl₂ buffers (cis, 100mm/trans, 25mm), single channel conductances at –40 mV were 70 pS (type I) and 120 pS (type II). Permeability ratios (P _Na:P _Ba:P _Cl) calculated from the Goldman-Hodgkin-Katz current equation for type I and type II channels were 0.230.041 and 0.050.031, respectively. Both channels exhibited characteristic voltage-dependent bursting activities. Open probability for type I channels had a maximum of 0.7 at about 0 mV and decreased to zero at greater transmembrane potentials of either polarity. Type II channels were relatively voltage independent at negative voltages and were inactivated at highly positive voltages. Type I channels showed spontaneous irreversible inactivation often preceded by sudden transition to subconducting states. DIDS blocked type I channels only from thecis side, while it blocked type II channels from either side.