Showing papers by "Max Planck Society published in 1981"

Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches.

Abstract: 1. The extracellular patch clamp method, which first allowed the detection of single channel currents in biological membranes, has been further refined to enable higher current resolution, direct membrane patch potential control, and physical isolation of membrane patches. 2. A description of a convenient method for the fabrication of patch recording pipettes is given together with procedures followed to achieve giga-seals i.e. pipette-membrane seals with resistances of 10(9) - 10(11) omega. 3. The basic patch clamp recording circuit, and designs for improved frequency response are described along with the present limitations in recording the currents from single channels. 4. Procedures for preparation and recording from three representative cell types are given. Some properties of single acetylcholine-activated channels in muscle membrane are described to illustrate the improved current and time resolution achieved with giga-seals. 5. A description is given of the various ways that patches of membrane can be physically isolated from cells. This isolation enables the recording of single channel currents with well-defined solutions on both sides of the membrane. Two types of isolated cell-free patch configurations can be formed: an inside-out patch with its cytoplasmic membrane face exposed to the bath solution, and an outside-out patch with its extracellular membrane face exposed to the bath solution. 6. The application of the method for the recording of ionic currents and internal dialysis of small cells is considered. Single channel resolution can be achieved when recording from whole cells, if the cell diameter is small (less than 20 micrometer). 7. The wide range of cell types amenable to giga-seal formation is discussed.

A system for shotgun DNA sequencing.

Abstract: A multipurpose cloning site has been introduced into the gene for beta-galactosidase (beta-D-galactosidegalactohydrolase, EC 3.21.23) on the single-stranded DNA phage M13mp2 (Gronenborn, B. and Messing, J., (1978) Nature 272, 375-377) with the use of synthetic DNA. The site contributes 14 additional codons and does not affect the ability of the lac gene product to undergo intracistronic complementation. Two restriction endonuclease cleavage sites in the viral gene II were removed by single base-pair mutations. Using the new phage M13mp7, DNA fragments generated by cleavage with a variety of different restriction endonucleases can be cloned directly. The nucleotide sequences of the cloned DNAs can be determined rapidly by DNA synthesis using chain terminators and a synthetic oligonucleotide primer complementary to 15 bases preceeding the new array of restriction sites.

Crystallographic refinement and atomic models of a human Fc fragment and its complex with fragment B of protein A from Staphylococcus aureus at 2.9- and 2.8-A resolution.

Abstract: The model of human Fc fragment was refined at 2.9-A resolution. Two different automated procedures for crystallographic refinement were used [Deisenhofer, J., & Steigemann, W. (1975) Acta Crystallogr., Sec. B B31, 238; Jack, A., & Levitt, M. (1978) Acta Crystallogr., Sect. A A34, 931]. The final R value is 0.22. The dimer of CH3 domains closely resembles the CH1-CL aggregate in Fab fragments. There is no contact between CH2 domains. The contact between CH2 and CH3 domains has about one-third of the size of the CH3-CH3 contact. The carbohydrate, a branched chain of nine hexose units, covers parts of the C-contact face of the CH2 domain, shielding hydrophobic residues on this surface. Six atoms of the carbohydrate are within hydrogen-bonding distance of atoms in the CH2 domain. Crystallographic refinement of the complex between Fc fragment and fragment B of protein A from Staphylococcus aureus reduced the R value of the model is 0.24. A major part of the structure of fragment B consists of two alpha helics; the rest of the polypeptide chain is folded irregularly. In the crystal, fragment B forms two contacts with Fc fragment molecules. Contact 1 involves residues from both helices of fragment B, and residues from the CH2 and CH3 domains of FC, and is predominantly hydrophobic. Contact 2 is smaller than contact 1. Residues from the second helix and adjacent residues of fragment B and residues only from the CH3 domain of Fc contribute to contact 2. The nature of contact 2 is mainly polar and includes a sulfate ion. There are strong arguments that contact 1 is the fragment B-Fc contact formed in solution under physiological conditions, while contact 2 is a crystal contact.

Magnetic loop behind an interplanetary shock: Voyager, Helios and IMP-8 observations

Abstract: The flow behind an interplanetary shock was analyzed through the use of magnetic field and plasma data from five spacecraft, with emphasis on the magnetic cloud identified by a characteristic variation of the latitude angle of the magnetic field. The size of the cloud was found to be about 0.5 AU in radial extent and greater than 30 deg in azimuthal extent, with its front boundary almost normal to the radial direction. Because the field direction of the magnetic cloud as it moved past the spacecraft was observed to rotate nearly parallel to a plane, it is thought that the field configuration of the cloud was essentially two-dimensional. These results further suggest that the lines of force in the magnetic cloud formed loops, but it could not be determined whether these loops were open or closed.

The rapid isolation of clonable antigen‐specific T lymphocyte lines capable of mediating autoimmune encephalomyelitis

Abstract: The isolation and propagation of functional antigen-specific lines of T lymphoblasts is described. These lines were found to recognize foreign or self antigens in association with accessory cells of syngeneic major histocompatibility complex genotype. Intravenous inoculation of a T cell reactive only against myelin basic protein led to development of clinical paralysis in syngeneic rats. Thus, it is possible to study biological function as well as antigen specificity using T cell lines.

A Network Model for the Organization of Type IV Collagen Molecules in Basement Membranes

Abstract: Type IV collagen was solubilized from a tumor basement membrane either by acid extraction or by limited digestion with pepsin. The two forms were similar in composition and the size of the constituent chains but differed when examined by electron microscopy and in the fragment pattern produced by bacterial collagenase. The acid-soluble form showed after rotary shadowing strands mainly of a length of 320 nm which terminated in a globule, or two strands connected by a similar globule. The globule was identified as a non-collagenous domain (NC1) which under dissociating conditions could be separated into two peptides showing a monomer-dimer relationship. Higher aggregates of NC1 were visualized under non-dissociating conditions. Some of the acid-extracted molecules have retained the previously 7-S collagen domain. The pepsin-solubilized form lacked domain NC1 and consisted mainly of four triple-helical strands (length 356 nm) joined together at the 7-S domain (length 30 nm). Common to both forms of type IV collagen was a small collagenase-resistant domain NC2 which was composed of collagenous and non-collagenous elements and located between the 7-S domain and the major triple helix. These data indicate that the collagenous matrix of basement membranes consists of a regular network of type IV collagen molecules which is generated by two different interacting sites located at opposite ends of each molecule. The 7-S collagen domain connects four molecules while the NC1 domain connects two molecules. The maximal distance between identical cross-linking sites (7-S or NC1) was estimated to be about 800 nm comprising the length of two molecules.

Evidence for magnetic field reconnection at the Earth's magnetopause

Abstract: Eleven passes of the ISEE satellites through the frontside terrestrial magnetopause were identified, where the plasma velocity in the magnetopause and boundary layer was substantially larger than in the magnetosheath. The nature of the plasma flow, magnetic field, and energetic particle fluxes in these regions were examined, with a view to determining whether the velocity enhancements can be explained by magnetic field reconnection.

Inward current channels activated by intracellular Ca in cultured cardiac cells.

Abstract: Present concepts of excitable membrane function are based primarily on knowledge of two classes of channels: those activated by neurotransmitters1 and those activated by membrane potential2. Recent evidence suggests that these notions may have to be modified to include other channel types, such as special ion channels activated by membrane potential but regulated by ligands3–5. We report here studies on single channel currents recorded from heart muscle cells, in which we have found a channel, abundant in cardiac membrane, which does not seem to belong in any of the familiar categories. This channel shows little selectivity between cations, but excludes anions. It is activated by intracellular Ca ions but is not appreciably affected by membrane potential.

Ca-dependent K channels with large unitary conductance in chromaffin cell membranes

Abstract: Injection of Ca ions increases the membrane permeability of many types of cells (reviewed by Meech1), and in molluscan neurones, removal of Ca ions from the external solution suppresses a voltage-dependent K conductance2,3. These two phenomena may be related through a potential–dependent Ca influx2. The question then arises whether the voltage dependence of the corresponding K permeability system is an intrinsic property of this system or whether it is merely a consequence of the increase of Ca influx with depolarization. The resolution of this and similar problems has been hampered by technical difficulties, due to the presence of voltage–dependent Ca channels, of Ca–independent K conductances and of strong intra-cellular buffering for Ca ions. In the present study patches of membranes containing functional, Ca-dependent K channels have been isolated from chromaffin cells. It is shown that application of low Ca concentrations to the inner side of the membrane affects the properties of the channels, while Ca ions are ineffective on the outer side of the membrane. The method used here overcomes several of the limitations of the previous studies using cellular recording.

Molecular and functional analysis of the TOL plasmid pWWO from Pseudomonas putida and cloning of genes for the entire regulated aromatic ring meta cleavage pathway

Abstract: The genetic organization of the Pseudomonas putida plasmid pWWO-161, which encodes enzymes for the degradation of toluene and related aromatic hydrocarbons, has been investigated by transposition mutagenesis and gene cloning. Catabolic genes were localized to two clusters, one for upper pathway (hydrocarbon leads to carboxylic acid) enzymes and the other for lower pathway (carboxylic acid leads to tricarboxylic acid cycle) enzymes, that are separated by a 14-kilobase DNA segment. The physical organization of the catabolic genes thus reflects their functional organization into two regulatory blocks. The pWWO-161 DNA fragments Sst I fragment C and fragment D were cloned in a broad host range vector to produce plasmid pKT530. This hybrid encodes toluate oxygenase and all meta cleavage pathway enzymes, and it enables P. putida mt-2 and Escherichia coli K-12 cells to grow on m-toluate as sole carbon source. The pKT530 plasmid also carries xylS (a gene whose product has been postulated to regulate expression of the lower pathway genes) and the control sequences of the pathway that interact with this product, because catechol 2,3-oxygenase synthesis is specifically induced by m-toluate in both P. putida and E. coli. Evidence is presented that suggests the promoter operator of the meta pathway gene functions less effectively with the RNA polymerase or xylS product of E. coli than with the enzyme or product of P. putida.

Vaccination against autoimmune encephalomyelitis with T-lymphocite line cells reactive against myelin basic protein

Abstract: Despite differences in initiating events and pathophysiology, the aetiological agents of all autoimmune diseases are lymphocytes specifically reactive against normal constituents of the individual. Recently we have isolated and grown as a cell line rat T lymphocytes reactive against myelin basic protein (BP)1. This T-cell line originated from rats in which we had induced experimental autoimmune encephalomyelitis (EAE) by immunizing them against BP. Inoculation of syngeneic rats with the T-cell line led to the relatively rapid onset of EAE1. We report here that attenuation of this cell line provides an agent for establishing resistance to induction of active EAE. Intravenous (i.v.) inoculation of syngeneic rats with cells of the line attenuated by treatment with irradiation or mitomycin C augmented resistance to EAE caused by an encephalitogenic challenge with BP. Thus, aetiological agents of autoimmune disease, like those of microbial disease, when suitably attenuated can be used as effective vaccines.

Electron scattering by ionized impurities in semiconductors

Abstract: Theories of electron scattering by ionized impurities in semiconductors are reviewed. The early foundations based on the Born approximation and their subsequent refinements are discussed thoroughly. The phase-shift method which is not restricted to the Born approximation is also presented. The situation in heavily doped semiconductors is described. The theories are then compared critically with experiments. Finally, conclusions are drawn and some plausible lines of future work are outlined.

A model of extrusions and intrusions in fatigued metals I. Point-defect production and the growth of extrusions

Abstract: Based on recent experimental observations a realistic model of the evolution of the surface profile of persistent slip bands (PSBs) in fatigued metals is proposed. An essential feature of the model is the production of vacancies by the annihilation of edge dislocations which, combined with irreversible slip processes, leads to an elongation of the PSB parallel to the active slip vector. It is shown that a net extrusion forms rather rapidly. The growth processes and their dependence on temperature are discussed in detail. It is proposed that cracks initiate at the surface steps at the PSB–matrix interface and, at a later stage, also at the notch-peak profile from the random slip processes.

Predator evasion as an explanation of diurnal vertical migration by zooplankton.

Abstract: Diurnal vertical migrations of planktonic crustaceans are widespread phenomena both in freshwater lakes and in the sea. The adaptive significance of this behaviour is unclear. This movement from the warm surface layers, which provide abundant food, to colder, deeper water with its poor food supply must place the migrating animals at a disadvantage compared with individuals remaining near the surface. The reduced availability of food and the energy consumed in migration result in reduced growth and reproductive capacity of migrating individuals. Moreover, the development time of the eggs carried in the brood pouch or in egg sacs is prolonged in cold water and the birth rate is reduced. We have studied vertical migration in a deep lake and report here that two very similar Daphnia species have different strategies. Daphnia hyalina shows a pronounced diurnal migration whereas Daphnia galeata remains near the surface. Although the non-migrating D. galeata has a much higher birth rate than D. hyalina, the latter is numerically dominant, as D. galeata suffers a high mortality near the surface. These results support the hypothesis that predator avoidance is one of the most important factors in vertical migration.

Structure of the low‐latitude boundary layer

Abstract: Observations at high temporal resolution of the frontside magnetopause and plasma boundary layer, made with the LASL/MPE fast plasma analyzer onboard the ISEE 1 and 2 spacecraft, revealed a complex quasiperiodic structure of some of the observed boundary layers. A cool tailward streaming boundary layer plasma was seen intermittently, with intervening periods of hot tenuous plasma which has properties similar to the magnetospheric population. While individual encounters with the boundary layer plasma last only a few minutes, the total observation time may extend over one hour or more.

The acceleration of cosmic rays by shock waves

Abstract: The problem of the origin of galactic cosmic rays is a particularly difficult one despite the fact that rather detailed measurements of the properties of cosmic rays can be made, at least in the vicinity of the Sun. The current situation has been well reviewed by Lingenfelter (1) who points out that there are several linked problems to be solved, namely the question of sources and acceleration mechanisms, propagation within the galaxy, escape from the galaxy and of course solar modulation which affects the interpretation of the observations, especially below ~ 1 GeV/nuc. It is usually supposed that these problems can be treated separately, so that the sources (perhaps supernovae, pulsars, black holes, flare stars, etc.) simply provide cosmic rays with given elemental and isotopic abundances and given spectra, which then propagate independently by diffusion through the interstellar medium, producing secondaries and perhaps losing energy as they do so until they eventually leave the galaxy by some means, which is usually described in terms of a “free escape” boundary condition to the diffusion equations.

The dependence of onset and duration of sleep on the circadian rhythm of rectal temperature

Abstract: The sleep-wake cycle and the circadian rhythm of rectal temperature were recorded in subjects who lived singly in an isolation unit. In 10 subjects, the freerunning rhythms remained internally synchronized, 10 other subjects showed internal desynchronization. Times of onset and end of bedrest ("sleep") were determined in each cycle and referred to the phase of the temperature rhythm. In the synchronized subjects, onset of sleep occurred, on the average, 1.34 h before the minimum of temperature, and end of sleep 6.94 h thereafter, with narrow distributions. The desynchronized subjects had a broad bimodal distribution of sleep onsets (peaks 6.3 and 1.3 h before the minimum); the duration of sleep varied between more than 15 h when sleep began about 10 h before the temperature minimum, and less than 4 h when sleep began several hours after the minimum. The dependence of sleep duration on body temperature is interpreted as a continuing action of the coupling forces between the two rhythms after mutual synchronization is lost.

Fluctuations in the microsecond time range of the current through single acetylcholine receptor ion channels

Abstract: Acetylcholine-like drugs cause ion channels in the skeletal muscle endplate to open briefly1, producing, at random intervals, rectangular pulses of current with constant amplitude but random duration, that can be recorded by the patch clamp method2,3. However, even when the agonist concentration is so low that channel activations are very well separated, we have observed, with high resolution methods4, that openings may be interrupted by shut periods (gaps) so brief that they are very unlikely to arise from two independent channel activations. This sort of behaviour has been predicted on the basis that two or more openings might occur during the time for which the receptor remains occupied by agonist5,6. If this were correct, important new information about agonist activation of ion channels could be obtained from measurements of the gaps between openings. However, short gaps could arise in other ways: for example from brief blockage of the ion channel7, perhaps by the agonist itself. We now present results obtained with the acetylcholine-like agonist, suberyldicholine (SubCh, 20–100 nM), which suggest that the brief gaps do not result from ion channel block by the agonist itself, but which are consistent with a mechanism in which the channel opens and closes several times during a single agonist receptor occupancy. We have also observed that the number of short (<1 ms) current pulses is greater than we expected.

Constitution of terrestrial planets

Abstract: Reliable estimates of the bulk composition are so far restricted to the three planetary objects from which we have samples for laboratory investigation, i.e. the Earth, the Moon and the eucrite parent asteroid. The last, the parent body of the eucrite— diogenite family of meteorites, an object that like Earth and Moon underwent magmatic differentiations, seems to have an almost chondritic composition except for a considerable depletion of all moderately volatile (Na, K, Rb, F, etc.) and highly volatile (Cl, Br, Cd, Pb, etc.) elements. The Moon is also depleted in moderately volatile and volatile elements compared to carbonaceous chondrites of type 1 (Cl) and also compared to the Earth. Again normalized to Cl and Si the Earth’s mantle and the Moon are slightly enriched in refractory lithophile elements and in magnesium. It might be that this enrichment is fictitious and only due to the normalization to Si and that both Earth’s mantle and Moon are depleted in Si, which partly entered the Earth’s core in metallic form. The striking depletion of the Earth’s mantle for the elements V, Cr and Mn can also be explained by their partial removal into the core. The similar abundances of V, Cr and Mn in the Moon and in the Earth’s mantle indicate the strong genetic relationship of Earth and Moon. Apart from their contents of metallic iron, all siderophile elements, moderately volatile and volatile elements, Earth and Moon are chemically very similar. It might well be that, with these exceptions and that of a varying degree of oxidation, all the inner planets have a similar chemistry. The chemical composition of the Earth’s mantle, for which reliable and accurate data have recently been obtained from the study of ultramafic nodules, yields important information about the accretion history of the Earth and that of the inner planets. It seems that accretion started with highly reduced material, with all Fe as metal and even Si and Cr, V and Mn partly in reduced state, followed by the accretion of more and more oxidized matter.

Multiple conductance states of single acetylcholine receptor channels in embryonic muscle cells.

Abstract: Acetylcholine (ACh) activates in the synaptic membrane of skeletal muscle an inward current composed of many elementary currents1,2. High resolution current measurements in adult frog muscle have shown that the elementary current is a pulse-like event of unit amplitude, indicating that ACh opens ion channels which have only two conductance states, fully open or closed3. We now present evidence for a third conductance state. In the membrane of uninnervated embryonic rat muscle we observe that ACh activates two independent classes of currents of different amplitude and average duration, apparently arising from two populations of ACh receptor (AChR) channels. The currents from both classes show, at low incidence, transitions between a main level and a sublevel of lower amplitude. From this we conclude that AChR channels in embryonic muscle adopt, in addition to a ‘main’ conductance state, a ‘substate’ of lower conductance.

Symplectic Geometry of Radiative Modes and Conserved Quantities at Null Infinity

Abstract: The Hamiltonian description of massless spin zero- and one-fields in Minkowski space is first recast in a way that refers only to null infinity and fields thereon representing radiative modes With this framework as a guide, the phase space of the radiative degrees of freedom of the gravitational field (in exact general relativity) is introduced It has the structure of an infinite-dimensional affine manifold (modelled on a Frechet space) and is equipped with a continuous, weakly non-degenerate symplectic tensor field The action of the Bondi-Metzner-Sachs group on null infinity is shown to induce canonical transformations on this phase space The corresponding Hamiltonians - ie generating functions - are computed and interpreted as fluxes of supermomentum and angular momentum carried away by gravitational waves The discussion serves three purposes: it brings out, via symplectic methods, the universality of the interplay between symmetries and conserved quantities; it sheds new light on the issue of angular momentum of gravitational radiation; and, it suggests a new approach to the quantization of the 'true' degrees of freedom of the gravitational field