Proximity of excitatory synapses and astroglial gap junctions in layer IV of the mouse barrel cortex.

Neurons and astrocytes, the two major cell populations in the adult brain, are characterized by their own mode of intercellular communication--the synapses and the gap junctions (GJ), respectively. In addition, there is increasing evidence for dynamic and metabolic neuroglial interactions resulting in the modulation of synaptic transmission at the so-called "tripartite synapse". Based on this, we have investigated at the ultrastructural level how excitatory synapses (ES) and astroglial GJ are spatially distributed in layer IV of the barrel cortex of the adult mouse. We used specific antibodies for connexin (Cx) 30 and 43 to identify astroglial GJ, these two proteins are known to be present in the majority of astroglial GJ in the cerebral cortex. In electron-microscopic images, we measured the distance between two ES, between two GJ and between a GJ and its nearest ES. We found a ratio of two GJ per three ES in the hollow and septal areas. Taking into account the size of an astrocyte domain, the high density of GJ suggests the occurrence of reflexive type, i.e. GJ between processes of the same astrocyte. Interestingly, the distance between an ES and an astroglial GJ was found to be significantly lower than that between either two synapses or between two GJ. These observations indicate that the two modes of cell-to-cell communication are not randomly distributed in layer IV of the barrel cortex. Consequently, this feature may provide the morphological support for the recently reported functional interactions between neuronal circuits and astroglial networks.

Large Magnetoresistance in Fe/MgO/FeCo(001) Epitaxial Tunnel-Junctions on GaAs(001).

We present tunneling experiments on Fe~001!/MgO~20 Å!/FeCo~001! single-crystal epitaxial junctions of high quality grown by sputtering and laser ablation. Tunnel magnetoresistance measurements give 60% at 30 K, to be compared with 13% obtained recently on ~001!-oriented Fe/amorphous-Al2O3 /FeCo tunnel junctions. This difference demonstrates that the spin polarization of tunneling electrons is not directly related to the density of states of the free metal surface Fe~001! in this case but depends on the actual electronic structure of the entire electrode/barrier system.

Fast generation of spin-squeezed states in bosonic Josephson junctions

We describe methods for the fast production of highly coherent-spin-squeezed many-body states in bosonic Josephson junctions. We start from the known mapping of the two-site Bose-Hubbard (BH) Hamiltonian to that of a single effective particle evolving according to a Schrödinger-like equation in Fock space. Since, for repulsive interactions, the effective potential in Fock space is nearly parabolic, we extend recently derived protocols for shortcuts to adiabatic evolution in harmonic potentials to the many-body BH Hamiltonian. A comparison with current experiments shows that our methods allow for an important reduction in the preparation times of highly squeezed spin states.

Study of mesoscopic tunnel junctions between two normal metals

The main purpose of this work was to study different kinds of metal-based tunnel junctions at low temperatures. The problem which had to be solved was creating a junction with appropriate properties at these temperatures. The materials for junctions were found experimentally. The goal was to find an alloy material that can provide a high quality tunnel junction, which remains in the normal conductive state at low temperatures without applying magnetic field. The fabrication technology of such a device, based on an alloy of aluminium and manganese, is described in detail. In this thesis theoretical properties of tunnel junctions are considered and results of experiments with tunnel junctions are described and quantitative properties of the junctions are analyzed based on the experimental data.

Regulation of epidermal tight junctions by calcium ATPases and p38

The epidermis is the upper layer of the skin and keratinocytes are its most abundant cells. Tight junctions are cell junctions located in the granular layer of the epidermis. They maintain the polarity of the cells and regulate the movement of water-soluble molecules. Epidermal tight junctions may lose their integrity when there are defects in intercellular calcium regulation. Hailey-Hailey and Darier´s disease are dominantly inherited, blistering skin diseases. Hailey-Hailey disease is caused by mutations in the ATP2C1 gene encoding a calcium/manganese ATPase SPCA1 of the Golgi apparatus. Darier´s disease is caused by mutations in the ATP2A2 gene encoding a calcium ATPase SERCA2 of the endoplasmic reticulum. p38 regulates the differentiation of keratinocytes. The overall regulation of epidermal tight junctions is not well understood. The present study examined the regulation of tight junctions in the human epidermis with a focus on calcium ATPases and p38. Skin from Hailey-Hailey and Darier´s disease patients was studied by using immunofluorescence labeling which targeted intercellular junction proteins. Transepidermal water loss was also measured. ATP2C1 gene expression was silenced in cultured keratinocytes, by siRNA, which modeled Hailey-Hailey disease. Expression of intercellular junction proteins was studied at the mRNA and protein levels. Squamous cell carcinoma and normal human keratinocytes were used as a model for impaired and normal keratinocyte differentiation, and the role of p38 isoforms alpha and delta in the regulation of intercellular junction proteins was studied. Both p38 isoforms were silenced by adenovirus cell transduction, chemical inhibitors or siRNA and keratinocyte differentiation was assessed. The results of this thesis revealed that: i.) intercellular junction proteins are expressed normally in acantholytic skin areas of patients with Hailey-Hailey or Darier´s disease but the localization of ZO-1 expanded to the stratum spinosum; ii.) tight junction proteins, claudin-1 and -4, are regulated by ATP2C1 in non-differentiating keratinocytes; and iii.) p38 delta regulates the expression of tight junction protein ZO-1 in proliferating keratinocytes and in squamous cell carcinoma derived cells. ZO-1 silencing, however, did not affect the expression of other tight junction proteins, suggesting that they are differently regulated. This thesis introduces new mechanisms involved in the regulation of tight junctions revealing new interactions. It provides novel evidence linking intracellular calcium regulation and tight junctions.

Regulation of gap junctions by protein phosphorylation

Gap junctions are constituted by intercellular channels and provide a pathway for transfer of ions and small molecules between adjacent cells of most tissues. The degree of intercellular coupling mediated by gap junctions depends on the number of gap junction channels and their activity may be a function of the state of phosphorylation of connexins, the structural subunit of gap junction channels. Protein phosphorylation has been proposed to control intercellular gap junctional communication at several steps from gene expression to protein degradation, including translational and post-translational modification of connexins (i.e., phosphorylation of the assembled channel acting as a gating mechanism) and assembly into and removal from the plasma membrane. Several connexins contain sites for phosphorylation for more than one protein kinase. These consensus sites vary between connexins and have been preferentially identified in the C-terminus. Changes in intercellular communication mediated by protein phosphorylation are believed to control various physiological tissue and cell functions as well as to be altered under pathological conditions.

Gap junctions in the cardiovascular and immune systems

Gap junctions are clusters of intercellular channels directly connecting the cytoplasm of adjacent cells. These channels are formed by proteins named connexins and are present in all metazoan organisms where they serve diverse functions ranging from control of cell growth and differentiation to electric conduction in excitable tissues. In this overview we describe the presence of connexins in the cardiovascular and lympho-hematopoietic systems giving the reader a summary of the topics to be covered throughout this edition and a historical perspective of the discovery of gap junctions in the immune system.

Biophysical characteristics of gap junctions in vascular wall cells: implications for vascular biology and disease

The role gap junction channels play in the normal and abnormal functioning of the vascular wall is the subject of much research. The biophysical properties of gap junctions are an essential component in understanding how gap junctions function to allow coordinated relaxation and contraction of vascular smooth muscle. This study reviews the properties thus far elucidated and relates those properties to tissue function. We ask how biophysical and structural properties such as gating, permselectivity, subconductive states and channel type (heteromeric vs homotypic vs heterotypic) might affect vascular smooth muscle tone.

Gap junctions in isolated rat aorta: evidence for contractile responses that exhibit a differential dependence on intercellular communication

Connexin43 (Cx43) is a major gap junction protein present in the Fischer-344 rat aorta. Previous studies have identified conditions under which selective disruption of intercellular communication with heptanol caused a significant, readily reversible and time-dependent diminution in the magnitude of a1-adrenergic contractions in isolated rat aorta. These observations have indentified a significant role for gap junctions in modulating vascular smooth muscle tone. The goal of these steady-state studies was to utilize isolated rat aortic rings to further evaluate the contribution of intercellular junctions to contractions elicited by cellular activation in response to several other vascular spasmogens. The effects of heptanol were examined (0.2-2.0 mM) on equivalent submaximal (»75% of the phenylephrine maximum) aortic contractions elicited by 5-hydroxytryptamine (5-HT; 1-2 µM), prostaglandin F2a (PGF2a; 1 µM) and endothelin-1 (ET-1; 20 nM). Statistical analysis revealed that 200 µM and 500 µM heptanol diminished the maximal amplitude of the steady-state contractile responses for 5-HT from a control response of 75 ± 6% (N = 26 rings) to 57 ± 7% (N = 26 rings) and 34.9 ± 6% (N = 13 rings), respectively (P<0.05), and for PGF2a from a control response of 75 ± 10% (N = 16 rings) to 52 ± 8% (N = 19 rings) and 25.9 ± 6% (N = 18 rings), respectively (P<0.05). In contrast, 200 µM and 500 µM heptanol had no detectable effect on the magnitude of ET-1-induced contractile responses, which were 76 ± 5.0% for the control response (N = 38 rings), 59 ± 6.0% in the presence of 200 µM heptanol (N = 17 rings), and 70 ± 6.0% in the presence of 500 µM heptanol (N = 23 rings) (P<0.13). Increasing the heptanol concentration to 1 mM was associated with a significant decrease in the magnitude of the steady-state ET-1-induced contractile response to 32 ± 5% (21 rings; P<0.01); further increasing the heptanol concentration to 2 mM had no additional effect. In rat aorta then, junctional modulation of tissue contractility appears to be agonist-dependent.

Gap junctions in cells of the immune system: structure, regulation and possible functional roles

Gap junction channels are sites of cytoplasmic communication between contacting cells. In vertebrates, they consist of protein subunits denoted connexins (Cxs) which are encoded by a gene family. According to their Cx composition, gap junction channels show different gating and permeability properties that define which ions and small molecules permeate them. Differences in Cx primary sequences suggest that channels composed of different Cxs are regulated differentially by intracellular pathways under specific physiological conditions. Functional roles of gap junction channels could be defined by the relative importance of permeant substances, resulting in coordination of electrical and/or metabolic cellular responses. Cells of the native and specific immune systems establish transient homo- and heterocellular contacts at various steps of the immune response. Morphological and functional studies reported during the last three decades have revealed that many intercellular contacts between cells in the immune response present gap junctions or "gap junction-like" structures. Partial characterization of the molecular composition of some of these plasma membrane structures and regulatory mechanisms that control them have been published recently. Studies designed to elucidate their physiological roles suggest that they might permit coordination of cellular events which favor the effective and timely response of the immune system.

Tumor necrosis factor alpha increases epithelial barrier permeability by disrupting tight junctions in Caco-2 cells

The objectives of this study were to determine the effect of tumor necrosis factor alpha (TNF-α) on intestinal epithelial cell permeability and the expression of tight junction proteins. Caco-2 cells were plated onto Transwell® microporous filters and treated with TNF-α (10 or 100 ng/mL) for 0, 4, 8, 16, or 24 h. The transepithelial electrical resistance and the mucosal-to-serosal flux rates of the established paracellular marker Lucifer yellow were measured in filter-grown monolayers of Caco-2 intestinal cells. The localization and expression of the tight junction protein occludin were detected by immunofluorescence and Western blot analysis, respectively. SYBR-Green-based real-time PCR was used to measure the expression of occludin mRNA. TNF-α treatment produced concentration- and time-dependent decreases in Caco-2 transepithelial resistance and increases in transepithelial permeability to the paracellular marker Lucifer yellow. Western blot results indicated that TNF-α decreased the expression of phosphorylated occludin in detergent-insoluble fractions but did not affect the expression of non-phosphorylated occludin protein. Real-time RT-PCR data showed that TNF-α did not affect the expression of occludin mRNA. Taken together, our data demonstrate that TNF-α increases Caco-2 monolayer permeability, decreases occludin protein expression and disturbs intercellular junctions.

The role of cyclic nucleotides in modulation of crayfish neuromuscular junctions by a neuropeptide /

DF2, a heptapeptide, is a member of the family of FMRFamide-like peptides and has been shown to increase the amount of transmitter released at neuromuscular junctions of the crayfish, Procambarus clarkit Recent evidence has shown that protein kinase C (PKC), calcium/calmodulin-dependent protein kinase II (CaMKII) and the cAMPdependent protein kinase (PKA) play a role in the neuromodulatory pathway of DF2. The involvement of these kinases led to the prediction that a G-protein-coupled receptor (GPCR) is activated by DF2 due to the role that each kinase plays in traditional GPCR pathways seen in other organisms and in other cells. G-proteins can also act on an enzyme that generates cyclic guanosine monophosphate (cGMP) which mediates its effects through a cGMP-dependent protein kinase (PKG). This thesis addresses the question of whether or not DF2's effects on synaptic transmission in crayfish are mediated by the cyclic nucleotides cAMP and cGMP. The effects of DF2 on synaptic transmission were examined using deep abdominal extensor muscles of the crayfish Procambarus clarkii. An identified motor neuron was stimulated, and excitatory post-synaptic potentials (EPSPs) were recorded in abdominal extensor muscle LI . A number of activators and inhibitors were used to determine whether or not cAMP, PKA, cGMP and PKG mediate the effect of this peptide. Chemicals that are known to activate PKA (Sp-cAMPS) and/or PKG (8-pCPTcGMP) mimic and potentiate DF2's effect by increasing EPSP amplitude. Inhibitors of either PKA (Rp-cAMPS) or PKG (Rp-8-pCPT-cGMPS) block a portion of the increase in EPSP amplitude induced by the peptide. When both kinase inhibitors are applied simultaneously, the entire effect of DF2 on EPSPs is blocked. The PKG inhibitor blocks the effects of a PKG activator but does not alter the effect of a PKA activator on EPSP amplitude. Thus, the PKG inhibitor appears to be relatively specific for PKG. A trend in the data suggests that the PKA inhibitor blocks a portion of the response elicited by the PKG activator. Thus, the PKA inhibitor may be less specific for PKA. Phosphodiesterase inhibitors, which are known to inhibit the breakdown of cAMP (IBMX) and/or cGMP (mdBAMQ), potentiate the effect of the peptide. These results support the hypothesis that cAMP and cGMP, acting through their respective protein kinase enzymes, mediate the ability of DFi to increase transmitter output.

Studies on Fluxon Dynamics in Coupled Josephson Junctions

The thesis deals with detailed theoretical analysis of fluxon dynamics in single and in coupled Josephson junctions of different geometries under various internal and external conditions. The main objective of the present work is to investigate the properties of narrow Long Josephson junctions (LJJs) and to discuss the intriguing physics. In this thesis, Josephson junctions of three types of geometries, viz, rectangular, semiannular and quarter annular geometries in single and coupled format are studied to implement various fluxon based devices. Studies presented in this thesis reveal that mulistacked junctions are extremely useful in the fabrication of various super conducting electronic devices. The stability of the dynamical mode and therefore the operational stability of the proposed devices depend on parameters such as coupling strength, external magnetic fields, damping parameters etc. Stacked junctions offer a promising way to construct high-TC superconducting electronic components. Exploring the complex dynamics of fluxons in coupled junctions is a challenging and important task for the future experimental and theoretical investigations

Studies on Chaos and Synchronization in ac-driven Josephson junctions

The main goal of this thesis is to study the dynamics of Josephson junction system in the presence of an external rf-biasing.A system of two chaotically synchronized Josephson junction is studied.The change in the dynamics of the system in the presence of at phase difference between the applied fields is considered. Control of chaos is very important from an application point of view. The role Of phase difference in controlling chaos is discussed.An array of three Josephson junctions iS studied for the effect of phase difference on chaos and synchronization and the argument is extended for a system of N Josephson junctions. In the presence of a phase difference between the external fields, the system exhibits periodic behavior with a definite phase relationship between all the three junctions.Itdeals with an array of three Josephson junctions with a time delay in the coupling term. It is observed that only the outer systems synchronize while the middle system remain uncorrelated with t-he other two. The effect of phase difference between the applied fields and time-delay on system dynamics and synchronization is also studied. We study the influence of an applied ac biasing on a serniannular Josephson junction. It is found the magnetic field along with the biasing induces creation and annihilation of fluxons in the junction. The I-V characteristics of the junction is studied by considering the surface loss term also in the model equation. The system is found to exhibit chaotic behavior in the presence of ac biasing.

Dynamics of coupled Josephson junctions under the influence of applied fields

We investigate the effect of the phase difference of appliedfields on the dynamics of mutually coupledJosephsonjunctions. A phase difference between the appliedfields desynchronizes the system. It is found that though the amplitudes of the output voltage values are uncorrelated, a phase correlation is found to exist for small values of applied phase difference. The dynamics of the system is found to change from chaotic to periodic for certain values of phase difference. We report that by keeping the value of phase difference as π, the system continues to be in periodic motion for a wide range of values of system parameters. This result may find applications in devices like voltage standards, detectors, SQUIDS, etc., where chaos is least desired.