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Stress-induced physiological deficiencies in cells are reflected in structural, morphological and functional reactions of organelles. Although numerous investigations have focused on chloroplasts and mitochondria as main targets of different stressors in plant cells, there is insufficient information on the plant Golgi apparatus as stress sensor. By using the advantages of field emission scanning electron microscopy tomography in combination with classical ultrathin sectioning and transmission electron microscopic analyses, we provide structural evidence for common stress responses of the large and highly stable dictyosomes in the algal model system Micrasterias. Stress is induced by different metals such as manganese and lead, by starvation in 9 weeks of darkness or by inhibiting photosynthesis or glycolysis and by disturbing ionic homeostasis via KCl. For the first time a stress-induced degradation pathway of dictyosomes is described that does not follow "classical" autophagy but occurs by disintegration of cisternae into single membrane balls that seem to be finally absorbed by the endoplasmic reticulum (ER). Comparison of the morphological features that accompany dictyosomal degradation in Micrasterias to similar reactions observed during the same stress application in Nitella indicates an ubiquitous degradation process at least in algae. As the algae investigated belong to the closest relatives of higher land plants these results may also be relevant for understanding dictyosomal stress and degradation responses in the latter phylogenetic group. In addition, this study shows that two-dimensional transmission electron microscopy is insufficient for elucidating complex processes such as organelle degradation, and that information from three-dimensional reconstructions as provided by field emission scanning electron microscopy tomography is absolutely required for a comprehensive understanding of the phenomenon.

Magnetically-induced forces on the inertial masses on-board LISA Pathfinder are expected to be one of the dominant contributions to the mission noise budget, accounting for up to 40%. The origin of this disturbance is the coupling of the residual magnetization and susceptibility of the test masses with the environmental magnetic field. In order to fully understand this important part of the noise model, a set of coils and magnetometers are integrated as a part of the diagnostics subsystem. During operations a sequence of magnetic excitations will be applied to precisely determine the coupling of the magnetic environment to the test mass displacement using the on-board magnetometers. Since no direct measurement of the magnetic field in the test mass position will be available, an extrapolation of the magnetic measurements to the test mass position will be carried out as a part of the data analysis activities. In this paper we show the first results on the magnetic experiments during an end-to-end LISA Pathfinder simulation, and we describe the methods under development to map the magnetic field on-board. A post-publication change was made to this article on 26 Jun 2020 to add an author.

Cutaneous chlorellosis in a gazelle caused a chronic granulomatous inflammation in the subcutaneous tissue of the carpus. The diagnosis was based on the intense green color of the lesion and the presence of organisms identified as algae of the order Chlorellales. According to morphologic and ultrastructural features, the genus Bracteacoccus is most likely.

The main goal of LISA Pathfinder (LPF) mission is to estimate the acceleration noise models of the overall LISA Technology Package (LTP) experiment on-board. This will be of crucial importance for the future space-based Gravitational-Wave (GW) detectors, like eLISA. Here, we present the Bayesian analysis framework to process the planned system identification experiments designed for that purpose. In particular, we focus on the analysis strategies to predict the accuracy of the parameters that describe the system in all degrees of freedom. The data sets were generated during the latest operational simulations organised by the data analysis team and this work is part of the LTPDA Matlab toolbox. A post-publication change was made to this article on 26 Jun 2020 to add an author.

The tunic of Cystodytes dellechiajei (Poly- citoridae), a colony-forming species of the Ascidiacea that contains biologically active alkaloids, was investigated using light microscopy, laser-scanning microscopy and nuclear magnetic resonance techniques. The colonies contain numerous individual zooids, which are embedded in a common tunic. Each zooid is protected by a firm capsule of overlapping calcareous spicules. The colonies lack blood vessels in the tunic, but six morphologically different types of tunic cells were found: pigment cells, bladder cells, vacuolated filopodial cells, granular filopodial cells, morula cells and granular cells. Rod-like bacteria were found in the tunic matrix. Bladder cells and pigment cells could be identified as storage units for acid and pyridoacridine alkaloids, making the tunic inedible and repelling predators. Filopodial cells have long filopodia, which probably are connected to each other. They may be involved in transportation processes within the tunic tissue. The functions of the morula cells and the granular cells are unknown as yet. With its several specialised cells, the tunic of C. dellechiajei represents a dynamic living tissue containing biologically active compounds.

Mibefradil is a novel Ca2+ antagonist which blocks both high-voltage activated and low voltage-activated Ca2+ channels. Although L-type Ca2+ channel block was demonstrated in functional experiments its molecular interaction with the channel has not yet been studied. We therefore investigated the binding of [3H]-mibefradil and a series of mibefradil analogues to L-type Ca2+ channels in different tissues. [3H]-Mibefradil labelled a single class of high affinity sites on skeletal muscle L-type Ca2+ channels (KD of 2.5±0.4 nM, Bmax=56.4±2.3 pmol mg−1 of protein). Mibefradil (and a series of analogues) partially inhibited (+)-[3H]-isradipine binding to skeletal muscle membranes but stimulated binding to brain L-type Ca2+ channels and α1C-subunits expressed in tsA201 cells indicating a tissue-specific, non-competitive interaction between the dihydropyridine and mibefradil binding domain. [3H]-Mibefradil also labelled a heterogenous population of high affinity sites in rabbit brain which was inhibited by a series of nonspecific Ca2+ and Na+-channel blockers. Mibefradil and its analogue RO40-6040 had high affinity for neuronal voltage-gated Na+-channels as confirmed in binding (apparent Ki values of 17 and 1.0 nM, respectively) and functional experiments (40% use-dependent inhibition of Na+-channel current by 1 μM mibefradil in GH3 cells). Our data demonstrate that mibefradil binds to voltage-gated L-type Ca2+ channels with very high affinity and is also a potent blocker of voltage-gated neuronal Na+-channels. More lipophilic mibefradil analogues may possess neuroprotective properties like other nonselective Ca2+-/Na+-channel blockers. Keywords: Ca2+ channel blockers, sodium channel blockers, mibefradil Introduction The benzimidazolyl-substituted tetraline derivative mibefradil is a novel Ca2+ channel blocker with several interesting pharmacological properties. It blocks high-voltage-activated L-type Ca2+ channels in the cardiovascular system resulting in antihypertensive activity and slowing of heart rate without decreasing cardiac contractility (Clozel et al., 1997). Although mibefradil was withdrawn from the market due to pharmacokinetic interactions it remains an interesting pharmacological probe to investigate the contribution of individual ion channel subtypes for cardiovascular functions. In vascular and atrial myocytes it preferentially blocks low voltage-activated T-type Ca2+ channels suggesting that part of the bradycardic and vasodilating properties are mediated by block of these channels (Bezprozvanny & Tsien, 1995; Clozel et al., 1997). Heterologous expression of cloned Ca2+ channel α1 subunits confirmed block of both high (α1A, α1B, α1C, α1E; Bezprozvanny & Tsien, 1995) and low voltage-activated (α1G, α1H; Cribbs et al., 1998) Ca2+ channels by low micromolar concentrations of mibefradil. Block of L- (α1C) and non-L-type Ca2+ channels (α1A, α1B, α1E) occurred with similar IC50 values. It is therefore tempting to speculate that, if high affinity binding of tritiated mibefradil to L-type Ca2+ channels can be demonstrated, this radioligand should also be suitable for the reversible labelling of non-L-type Ca2+ channel complexes as well. So far the binding of mibefradil to L-type Ca2+ channels has not been quantified in much detail. In preliminary studies the affinity of mibefradil for cardiac and smooth muscle channels was derived from its potency to displace (−)-[3H]-devapamil from the phenylalkylamine (PAA) binding domain of the channel. IC50 estimates varied between 13 nM (Rutledge & Triggle, 1995) and 39 nM (Osterrieder & Holck, 1989). Direct labelling studies with [3H]-mibefradil were hampered by the presence of low affinity sites (Ratner et al., 1996). Mibefradil failed to modulate (+)-[3H]-isradipine binding to cardiac membranes and to polarized and depolarized cardiomyocytes (Rutledge & Triggle, 1995). This suggests that it binds to a site not coupled to the dihydropyridine (DHP) binding domain. In order to establish the usefulness of [3H]-mibefradil as a biochemical probe for voltage gated Ca2+ channels, we determined its binding properties in different excitable tissues using tritiated mibefradil and a series of unlabelled mibefradil analogues. We provide evidence that mibefradil labels a site on skeletal muscle and brain L-type Ca2+ channels which is tightly coupled to the DHP domain by a non-competitive binding mechanism. In addition, we found that mibefradil and its analogues also display high affinity for neuronal voltage-gated Na+-channels and potently block Na+-currents in GH3 cells.

A novel hyperthermophilic bacterium was isolated from pink filamentous streamers (pink filaments) occurring in the upper outflow channel (temperature, 82 to 88°C) of Octopus Spring in Yellowstone National Park, Wyo. The gram-negative cells grew at low salinity at temperatures up to 89°C in the neutral to alkaline pH range. Depending on the culture conditions, the organisms occurred as single motile rods, as aggregates, or as long filaments that formed streamer-like cell masses. The novel isolate grew chemolithoautotrophically with hydrogen, thiosulfate, and elemental sulfur as electron donors and oxygen as the electron acceptor. Alternatively, under aerobic conditions, formate and formamide served as sole energy and carbon sources. The novel isolate had a 16S rRNA sequence closely related to the 16S rRNA sequence obtained from uncultivated pink filaments. It represents a new genus in the order Aquificales , the type species of which we name Thermocrinis ruber (type strain, OC 1/4 [= DSM 12173]).

Shaker-related voltage-gated K+ (Kv) channels are assembled from ion-conducting K(v)alpha subunits, which are integral membrane proteins, and auxiliary K(v)beta subunits. This leads to the formation of highly diverse heteromultimeric Kv channels that mediate outward currents with a wide range of time courses for inactivation. Two principal inactivation mechanisms have been recognized: C-type inactivation correlated with carboxy-terminal K(v)alpha-subunit structures, and N-type inactivation conferred by 'ball' domains in the amino termini of certain K(v)alpha and K(v)beta subunits. Assembly of heteromultimers with one or more K(v)alpha- and/or K(v)beta ball domains appears to be an essential principle of the generation of A-type Kv channel diversity. Here we show that, unexpectedly, the presence of K(v)alpha- or K(v)beta-ball domains does not dominate the gating phenotype in heteromultimers containing Kv1.6alpha subunits. These heteromultimers mediate non-inactivating currents because of the dominant-negative activity of a new type of N-type inactivation-prevention (NIP) domain present in the Kv1.6 amino terminus. Mutations in the NIP domain lead to loss of function, and its transfer to another K(v)alpha subunit leads to gain of function. Our discovery of the NIP domain, which neutralizes the activity of K(v)alpha- and K(v)beta-inactivation gates, establishes a new determinant for the gating behaviour of heteromultimeric Kv channels.

Neurons require specific patterns of K+ channel subunit expression as well as the precise coassembly of channel subunits into heterotetrameric structures for proper integration and transmission of electrical signals. In vivo subunit coassembly was investigated by studying the pharmacological profile, distribution, and subunit composition of voltage-gated Shaker family K+ (Kv1) channels in rat cerebellum that are labeled by 125I-margatoxin (125I-MgTX; Kd, 0.08 pM). High-resolution receptor autoradiography showed spatial receptor expression mainly in basket cell terminals (52% of all cerebellar sites) and the molecular layer (39% of sites). Sequence-directed antibodies indicated overlapping expression of Kv1. 1 and Kv1.2 in basket cell terminals, whereas the molecular layer expressed Kv1.1, Kv1.2, Kv1.3, and Kv1.6 proteins. Immunoprecipitation experiments revealed that all 125I-MgTX receptors contain at least one Kv1.2 subunit and that 83% of these receptors are heterotetramers of Kv1.1 and Kv1.2 subunits. Moreover, 33% of these Kv1.1/Kv1.2-containing receptors possess either an additional Kv1.3 or Kv1.6 subunit. Only a minority of the 125I-MgTX receptors (

Voltage-gated potassium (K(V)) channels play key roles in setting the resting potential and in the activation cascade of human peripheral T lymphocytes. Margatoxin (MgTX), a 39-amino acid peptide from Centruroides margaritatus, is a potent inhibitor of lymphocyte K(V) channels. The binding of monoiodotyrosinyl margatoxin ([125I]MgTX) to plasma membranes prepared from either Jurkat cells, a human leukemic T cell line, or CHO cells stably transfected with the Shaker-type voltage-gated K+ channel, K(V)1.3, has been used to investigate the properties of lymphocyte K(V) channels. These data were compared with [125I]MgTX binding to heterotetrameric K(V) channels in rat brain synaptic plasma membranes [Knaus, H. G., et al. (1995) Biochemistry 34, 13627-13634]. The affinity for [125I]MgTX is 100-200 fM in either Jurkat or CHO/K(V)1.3 membranes, and the receptor density is 20-120 fmol/mg in Jurkat membranes or 1000 fmol/mg in CHO/K(V)1.3 membranes. In contrast to rat brain, [125I]MgTX binding to Jurkat and CHO/K(V)1.3 membranes exhibits an absolute requirement for K+, with no potentiation of binding by Na+. K(V)1.3 was the only K(V)1 series channel present in either CHO/K(V)1.3 or Jurkat plasma membranes as determined by immunoprecipitation of [125I]MgTX binding or by Western blot analyses using sequence-specific antibodies prepared against members of the K(V)1 family. The relative potencies of a series of peptidyl K(V) channel inhibitors was essentially the same for inhibition of [125I]MgTX binding to Jurkat, CHO, or rat brain membranes and for blocking 86Rb+ efflux from the CHO/K(V)1.3 cells, except that alpha-dendrotoxin was more potent at blocking binding to rat brain membranes than in the other assays. The characteristics of [125I]MgTX binding, the antibody profiles, and the effects of the peptidyl K(V) inhibitors all indicate that the [125I]MgTX receptor in Jurkat lymphocytes is comprised of a homomultimer of K(V)1.3, unlike the heteromultimeric arrangement of the receptor in rat brain.

Summary Plasmid DNA was encapsulated in liposomes to protect from degradation and to support the incorporation into sperm cells. Transgenic spermatozoa carry the foreign DNA to the target cell when fertilizing the egg. Several plasmid constructs were transferred into rabbits, cattle and chicken. The plasmid apparently can persist when provided with a murine amplification promoting sequence. The transgeneity of sperm cells as well as fetuses or offspring was demonstrated by dot blot, Southern blot analysis and PCR. Expression of the transgene was shown as enzymatic activities of the reporter genes SEAP or β-galactosidase and by gelelectrophoretic characterization of the expressed gene products. The transfer efficiency and the persistence of the gene constructs is significantly increased due to the use of sperm cells as gene carriers mediated by liposomes and the amplification element. Zusammenfassung Gentransfer mittels Liposomen und Spermien Plasmid-DNA wurde in Liposomen eingeschlossen, um sie vor enzymatischem Abbau zu schutzen und ihre Inkorporation in die Spermienzelle zu ermoglichen. Bei der Befruchtung bringt das Spermium die Fremd-DNA in die Eizelle. Es wurden verschiedene Genkonstrukte in Kaninchen, Rind und Huhn ubertragen. Das Plasmid kann offensichtlich persistieren, wenn es eine murine amplifikationsfordernde Sequenz enthalt. Die Transgenitat von Spermien, Feten und Nachkommen wurde mit Dot Blot, Southern Blot und PCR nachgewiesen, die Expression der Reportergene SEAP und β-Galaktosidase gelelektrophoretisch bzw. enzymatisch charakterisiert. Der Einsatz von Spermien und Liposomen zur Genubertragung sowie die Verwendung einer ‘amplification promoting sequence' erhohen die Transfereffizienz und die Persistenz der Genkonstrukte betrachtlich.

The HIV-1 Pr55gag precursors were previously shown to assemble and bud from a variety of different cell types as noninfectious virus-like particles (VLPs) resembling immature HIV virions. The use of these VLPs as an immunogenic and autologous carrier component may allow the presentation of defined epitopes deduced from reading frames other than gag to the immune system, thereby avoiding the induction of adverse immune responses. In order to identify domains within Pr55gag that can be replaced by immunologically relevant epitopes without affecting its capacity to assemble into VLPs, we deleted three domains of a predicted high surface probability. Deletion of amino acids 211-241 within p24CA and amino acids 436-471 within the p6LI portion of Pr55gag had no effect on the assembly, ultrastructure, biophysical properties, and yields of mutant VLPs when expressed via recombinant vaccinia viruses in mammalian cells. Deletion of amino acids 99-154 overlapping the p17MA/p24CA cleavage site completely abolished the capacity of the gag polyprotein to form VLPs and led to a reduction of immature Pr55 VLPs released into the cell-culture supernatants when coexpressed with wild-type Pr55gag. In contrast, assembly and budding of chimeric VLPs could be demonstrated after replacing amino acids 211-241 and 436-471 by immunologically relevant epitopes derived from reading frames other than Pr55gag (e.g., V3 loop; CD4-binding-domain; nef-CTL epitope) or after fusion of these sequences to the carboxy terminus of Pr55gag. The importance of these data for the development of novel HIV candidate vaccines is discussed., Erratum in: Virology 1994 Jun;201(2):424

This paper presents a quantitative assessment of the performance of the upcoming LISA Pathfinder geodesic explorer mission. The findings are based on the results of extensive ground testing and simulation campaigns using flight hardware and flight control and operations algorithms. The results show that, for the central experiment of measuring the stray differential acceleration between the LISA test masses, LISA Pathfinder will be able to verify the overall acceleration noise to within a factor two of the LISA requirement at 1 mHz and within a factor 6 at 0.1 mHz. We also discuss the key elements of the physical model of disturbances, coming from LISA Pathfinder and ground measurement, that will guarantee the LISA performance., Proceedings of the 8th LISA Symposium. To appear Class. Quantum Grav. 28 (2011)

'Candidatus Magnetobacterium bavaricum' is unusual among magnetotactic bacteria (MTB) in terms of cell size (8-10 µm long, 1.5-2 µm in diameter), cell architecture, magnetotactic behaviour and its distinct phylogenetic position in the deep-branching Nitrospira phylum. In the present study, improved magnetic enrichment techniques permitted high-resolution scanning electron microscopy and energy dispersive X-ray analysis, which revealed the intracellular organization of the magnetosome chains. Sulfur globule accumulation in the cytoplasm point towards a sulfur-oxidizing metabolism of 'Candidatus M. bavaricum'. Detailed analysis of 'Candidatus M. bavaricum' microhabitats revealed more complex distribution patterns than previously reported, with cells predominantly found in low oxygen concentration. No correlation to other geochemical parameters could be observed. In addition, the analysis of a metagenomic fosmid library revealed a 34 kb genomic fragment, which contains 33 genes, among them the complete rRNA gene operon of 'Candidatus M. bavaricum' as well as a gene encoding a putative type IV RubisCO large subunit.

Antigens in a particulate conformation were shown to be highly immunogenic in mammals. For this reason, the particle forming capacity of derivatives of the HIV-1 group specific core antigen p55 gag was assayed and compared dependent on various expression systems: recombinant bacteria, vaccinia- and baculoviruses were established encoding the entire core protein p55 either in its authentic sequence or lacking the myristylation consensus signal. Moreover, p55 gag was expressed in combination with the protease (p55-PR) or with the entire polymerase (p55-pol), respectively. Budding of 100-160 nm p55 core particles, resembling immature HIV-virions, was observed in the eucaryotic expression systems only. In comparison to the vaccinia virus driven expression of p55 in mammalian cells, considerably higher yields of particulate core antigen were obtained by infection of Spodoptera frugiperda (Sf9) insect cells with the recombinant Autographa californica nuclear polyhedrosis (AcMNPV) baculovirus. Mutation of the NH2-terminal myristylation signal sequence prevented budding of the immature core particles. Expression of the HIV p55-PR gene construct by recombinant baculovirus resulted in complete processing of the p55 gag precursor molecule in this system. The introduction of an artificial frameshift near the natural frameshift site resulted in constitutive expression of the viral protease and complete processing of p55, both in Escherichia coli and in vaccinia virus infected cells. Interestingly, significant processing of p55 resembling that of HIV infected H9 cells could also be achieved in the vaccinia system by fusing the entire pol gene to the gag gene. Moreover, processing was not found to be dependent on amino-terminal myristylation of the gag procursor molecule, which is in contrast to observations with type C and type D retrovirus. However, complete processing of p55 into p24, p17, p9 and p6 abolished particle formation. Purified immature HIV-virus like particles were highly immunogenic in rabbits, leading to a strong humoral immune response after immunization. Empty immature p55 gag particles represent a noninfectious and attractive candidate for a basic vaccine component.

The data analysis of the LISA Technology Package (LTP) will comprise a series of discrete experiments, each focusing on a particular noise measurement or characterization of the instrument in various operating modes. Each of these experiments must be analysed and planned in advance of the mission because the results of a given experiment will have an impact on those that follow. As such, a series of mock data challenges (MDCs) will be developed and carried out with the aim of preparing the analysis tools and optimizing the various planned analyses. The first of these MDCs (MDC1) is a simplified treatment of the dynamics along the axis joining the two test masses onboard LISA Pathfinder. The validation of the dynamical model by predicting the spectra of the interferometer output data is shown, a prediction for the data analysis is calculated and, finally, several simulated interferometer data sets are analysed and calibrated to equivalent out-of-loop test mass acceleration

Aerobic activated sludge granules are dense, spherical biofilms which can strongly improve purification efficiency and sludge settling in wastewater treatment processes. In this study, the structure and development of different granule types were analyzed. Biofilm samples originated from lab-scale sequencing batch reactors which were operated with malthouse, brewery, and artificial wastewater. Scanning electron microscopy, light microscopy, and confocal laser scanning microscopy together with fluorescence in situ hybridization (FISH) allowed insights into the structure of these biofilms. Microscopic observation revealed that granules consist of bacteria, extracellular polymeric substances (EPS), protozoa and, in some cases, fungi. The biofilm development, starting from an activated sludge floc up to a mature granule, follows three phases. During phase 1, stalked ciliated protozoa of the subclass Peritrichia, e.g., Epistylis spp., settle on activated sludge flocs and build tree-like colonies. The stalks are subsequently colonized by bacteria. During phase 2, the ciliates become completely overgrown by bacteria and die. Thereby, the cellular remnants of ciliates act like a backbone for granule formation. During phase 3, smooth, compact granules are formed which serve as a new substratum for unstalked ciliate swarmers settling on granule surfaces. These mature granules comprise a dense core zone containing bacterial cells and EPS and a loosely structured fringe zone consisting of either ciliates and bacteria or fungi and bacteria. Since granules can grow to a size of up to several millimeters in diameter, we developed and applied a modified FISH protocol for the study of cryosectioned biofilms. This protocol allows the simultaneous detection of bacteria, ciliates, and fungi in and on granules.

This chapter introduces the differential and integral calculus, the greatest inventions of all time in mathematics. We explain the ideas of Leibniz, the Bernoullis, and Euler. A rigorous treatment in the spirit of the 19th century will be the subject of Sections II1.5 and III.6.

Functions of several variables have their origin in geometry (e.g., curves depending on parameters (Leibniz 1694a)) and in physics. A famous problem throughout the 18th century was the calculation of the movement of a vibrating string (d’Alembert 1748, Fig. 0.1). The position of a string u(x, t) is actually a function of x, the space coordinate, and of t, the time. An important breakthrough for the systematic study of several variables, which occured around the middle of the 19th century, was the idea of denoting pairs (then n-tuples) $$ {\text{(}}{{\text{x}}_1}{\text{,}}{{\text{x}}_2}{\text{) = :x}}\quad {\text{(}}{{\text{x}}_1}{\text{,}}{{\text{x}}_2}{\text{,}} \ldots {\text{,}}{{\text{x}}_n}{\text{) = :x}}$$ by a single letter and of considering them as new mathematical objects. They were called “extensive Grosse” by Grassmann (1844, 1862), “complexes” by Peano (1888), and “vectors” by Hamilton (1853).

This chapter explains the origin of elementary functions and the impact of Descartes’s “Geometrie” on their calculation. The interpolation polynomial leads to Newton’s binomial theorem and to the infinite series for exponential, logarithmic, and trigonometric functions. The chapter ends with a discussion of complex numbers, infinite products, and continued fractions. The presentation follows the historical development of this subject, with the mathematical rigor of the period. The justification of dubious conclusions will be an additional motivation for the rigorous treatment of convergence in Chapter III.

Biochemical and genetic studies have suggested that a transmembrane tunnel structure penetrating the inner and outer membranes is formed during the lytic action of bacteriophage phi X174 protein E. In this study we directly visualized the lysis tunnel by using high-magnification scanning and transmission electron microscopy.

Bacteriophage PhiX174 encodes a single lysis gene, E, the function of which is necessary and sufficient to induce lysis of Escherichia coli. Here we present a novel model for E-lysis: physiological, genetic and biochemical data are presented which suggest that a transmembrane tunnel penetrating the inner and outer membrane is formed during the lytic action of protein E. Moreover, using high magnification scanning and transmission electron microscopy in this study, it was possible to visualize the transmembrane lysis structure directly.

High-resolution detection of phosphorylated histone H3 at serine 10 in mitotic barley chromosomes for scanning electron microscopy was shown using a novel application of indirect immunogold labeling with Nanogold. This method permits localization and quantification of signals in a three-dimensional context. Because the chromosome structure is well preserved, characterization of binding sites (chromomeres, parallel matrix fibers, solenoids), currently in the realm of nanometer decades, is possible. Quantification and three-dimensional localization of labels is possible with stereoscopic analysis. Limitations of the method pertain to the challenges in preservation of chromosome ultrastructure, accessibility of immunoreactants into the fixed chromatin and unspecific labeling. The differences between silver and gold enhancement and the current status of labeling efficiency are addressed.

Structural investigation and morphometry of meiotic chromosomes by scanning electron microscopy (in comparison to light microscopy) of all stages of condensation of meiosis I + II show remarkable differences during chromosome condensation in mitosis and meiosis I of rye (Secale cereale) with respect to initiation, mode and degree of condensation. Mitotic chromosomes condense in a linear fashion, shorten in length and increase moderately in diameter. In contrast, in meiosis I, condensation of chromosomes in length and diameter is a sigmoidal process with a retardation in zygotene and pachytene and an acceleration from diplotene to diakinesis. The basic structural components of mitotic chromosomes of rye are "parallel fibers" and "chromomeres" which become highly compacted in metaphase. Although chromosome architecture in early prophase of meiosis seems similar to mitosis in principle, there is no equivalent stage during transition to metaphase I when chromosomes condense to a much higher degree and show a characteristic "smooth" surface. No indication was found for helical winding of chromosomes either in mitosis or in meiosis. Based on measurements, we propose a mechanism for chromosome dynamics in mitosis and meiosis, which involves three individual processes: (i) aggregation of chromatin subdomains into a chromosome filament, (ii) condensation in length, which involves a progressive increase in diameter and (iii) separation of chromatids.

Scanning electron microscopy (SEM) proves to be an appropriate technique for imaging chromatin organization in meiosis I and II of rye (Secale cereale) down to a resolution of a few nanometers. It could be shown for the first time that organization of basic structural elements (coiled and parallel fibers, chromomeres) changes dramatically during the progression to metaphase I and II. Controlled loosening with proteinase K (after fixation with glutaraldehyde) provides an enhanced insight into chromosome architecture even of highly condensed stages of meiosis. By selective staining with platinum blue, DNA content and distribution can be visualized within compact chromosomes as well as in a complex arrangement of fibers. Chromatin interconnecting threads, which are typically observed in prophase I between homologous and non-homologous chromosomes, stain clearly for DNA. In zygotene transversion of chromatid strands to their homologous counterparts becomes evident. In pachytene segments of synapsed and non-synapsed homologs alternate. At synapsed regions pairing is so intimate that homologous chromosomes form one filament of structural entity. Chiasmata are characterized by chromatid strands which traverse from one homolog to its counterpart. Bivalents are characteristically fused at their telomeric regions. In metaphase I and II there is no structural evidence for primary and secondary constrictions.

K+ channels represent a large and diverse group of ion channels that play a fundamental role in controlling cell excitability. These channels regulate hormone release from endocrine cells, modulate the pattern of transmitter release from neurons, and set the level of contraction in arterial smooth muscle cells. At first glance, K+ channels can be subdivided into either voltage-gated K+ (Kv) channels or ligand-gated K+ channels, depending on the stimulus that triggers the conformational change that leads to channel opening. Within the wide family of K+ channels gated by voltage, the Kv channels are activated solely by membrane depolarization, whereas high-conductance Ca2+-activated K+ (BKCa) channels require both membrane depolarization and an increased level of cytosolic free Ca2+ ([Ca2+]i) to activate effectively. The use of molecular biology techniques has greatly extended our current understanding regarding the structure and existence of subfamilies of K+ channels. However, three major questions still are the subject of extensive investigation: (1) What is the molecular composition of the different types of K+ channels expressed in vivo, (2) what is the physiological function of these K+ channels, and (3) is the molecular composition and functional profile of K+ channels altered in pathophysiological states?