Effect of dopamine on synchronous neuronal oscillations in the globus pallidus-subthalamic nucleus network

Changes in the pattern of activity of neurones within the basal ganglia are relevant in the pathophysiology and symptoms of Parkinson’s disease. The globus pallidus (GP) – subthalamic nucleus (STN) network has been proposed to form a pacemaker driving regenerative synchronous bursting activity. In order to test whether this activity can be sustained in vitro a 20o parasagittal slice of mouse midbrain was developed which preserved functional connectivity between the STN and GP. Mouse STN and GP cells were characterised electrophysiologically by the presence or absence of a voltage sag in response to hyperpolarising current steps indicative of Ih and the presence of rebound depolarisations. The presence of evoked and spontaneous post-synaptic GABA and glutamatergic currents indicated functional connectivity between the STN and GP. In control slices, STN cells fired action potentials at a regular rate, activity which was unaffected by bath application of the GABAA receptor antagonist picrotoxin (50 μM) or the glutamate receptor antagonist CNQX (10 μM). Paired extracellular recordings of STN cells showed uncorrelated firing. Oscillatory burst activity was induced pharmacologically using the glutamate receptor agonist, NMDA (20 μM), in combination with the potassium channel blocker apamin (50 -100 nM). The burst activity was unaffected by bath application of picrotoxin or CNQX while paired STN recordings showed uncorrelated activity indicating that the activity is not produced by the neuronal network. Thus, no regenerative activity is evident in this mouse brain preparation, either in control slices or when bursting is pharmacologically induced, suggesting the requirement of other afferent inputs that are not present in the slice. Using single-unit extracellular recording, dopamine (30 μM) produced an excitation of STN cells. This excitation was independent of synaptic transmission and was mimicked by both the Dl-like receptor agonist SKF38393 (10 μM) and the D2-like receptor agonist quinpirole (10 μM). However, the excitation was partially reduced by the D1-like antagonist SCH23390 (2 μM) but not by the D2-like antagonists sulpiride (10 μM) and eticlopride (10 μM). Using whole-recordings, dopamine was shown to induce membrane depolarisation. This depolarisation was caused either by a D1-like receptor mediated increase in a conductance which reversed at -34 mV, consistent with a non-specific cation conductance, or a D2-like receptor mediated decrease in conductance which reversed around -100 mV, consistent with a potassium conductance. Bath application of dopamine altered the pattern of the burst-firing produced by NMDA an apamin towards a more regular pattern. This effect was associated with a decrease in amplitude and ll1crease in frequency of TTX-resistant plateau potentials which underlie the burst activity.

Activation of calcium-sensitive potassium channels in L6 skeletal myocytes by arginine vasopressin requires extracellular calcium

The effects of extracellular application of arginine vasopressin (AVP) upon membrane currents in L6 skeletal myocytes was investigated using the whole-cell configuration of the patch-clamp technique. At O mV AVP produced large amplitude, transient outward currents that reversed when the clamping potential was changed to -100 mV (negative to EK) The effects of alterations in the extracellular K+ concentration upon the current reversal potential suggested that the current elicited by AVP was carried mainly by K+ ions. Intracellular dialysis with 10 μM inositol 1,4,5-trisphosphate (InsP3) elicited similar currents but only in 6/14 cells. Inclusion of 5 mg ml-1 heparin in the intracellular solutions was ineffective at inhibiting the current responses to AVP. The AVP-induced current was totally abolished when the intracellular EGTA concentration was increased from 0.05 mM to 10 mM or Ca2+ was removed from the extracellular perfusing solution. These results suggest that AVP produces activation of a Ca2+-sensitive K+ conductance in L6 skeletal myocytes by a process dependent upon extracellular Ca2+ and not intracellular Ca2+ release. © 1995 Academic Press. All rights reserved.

Simplified recording of soft contact lens fit

Purpose: To determine the critical fitting characteristics of modern soft contact lens fits and from this to devise a simplified recording scheme. Methods: Ten subjects (aged 28.1 ± 7.4 years) wore eight different modern soft contact lenses. Video was captured and analysed of blink (central and up-gaze), excursion lag (up, down, right and left gaze) and push-up movement, centration and coverage. Results: Lens centration was on average close to the corneal centre. Movement on blink was significantly smaller in up-gaze than in primary-gaze (p<0.001). Lag was greatest in down-gaze and least in up-gaze (p<0.001). Push-up test recovery speed was 1.32±0.73mm/s. Overall lens movement was determined best by assessing horizontal lag, movement on blink in up-gaze and push-up recovery speed. Steeper lens base-curves did not have a significant effect on lens fit characteristics. Contact lens material did influence lens fit characteristics, particularly silicone-hydrogels which generally had lower centration and a faster push-up speed of recovery than HEMA lenses (p<0.05). Conclusion: Lag on vertical gaze, and movement on blink in primary gaze generally provide little extra information on overall lens movement compared to horizontal lag, movement on blink in up gaze and push-up recovery speed. They can therefore be excluded from a simplified recording scheme. A simplified and comprehensive soft contact lens fit recording system could consist of a cross-hairs indicating the centre of the cornea; a circle to indicate the lens centration; a mark on the relevant position of the circle to indicate any limbal incursion; a grade (‘B’) below for movement with blink in up-gaze, a grade (‘L’) to the side for horizontal lag and a grade above (‘P’) for the assessed push-up recovery speed.

An extracellular transglutaminase is required for apple pollen tube growth

An extracellular form of the calcium-dependent protein-cross-linking enzyme TGase (transglutaminase) was demonstrated to be involved in the apical growth of Malus domestica pollen tube. Apple pollen TGase and its substrates were co-localized within aggregates on the pollen tube surface, as determined by indirect immunofluorescence staining and the in situ cross-linking of fluorescently labelled substrates. TGase-specific inhibitors and an anti-TGase monoclonal antibody blocked pollen tube growth, whereas incorporation of a recombinant fluorescent mammalian TGase substrate (histidine-tagged green fluorescent protein: His6-Xpr-GFP) into the growing tube wall enhanced tube length and germination, consistent with a role of TGase as a modulator of cell wall building and strengthening. The secreted pollen TGase catalysed the cross-linking of both PAs (polyamines) into proteins (released by the pollen tube) and His6-Xpr-GFP into endogenous or exogenously added substrates. A similar distribution of TGase activity was observed in planta on pollen tubes germinating inside the style, consistent with a possible additional role for TGase in the interaction between the pollen tube and the style during fertilization.

Importance of tissue transglutaminase in repair of extracellular matrices and cell death of dermal fibroblasts after exposure to a solarium ultraviolet A source

Investigations were undertaken to study the role of the protein cross-linking enzyme tissue transglutaminase in changes associated with the extracellular matrix and in the cell death of human dermal fibroblasts following exposure to a solarium ultraviolet A source consisting of 98.8% ultraviolet A and 1.2% ultraviolet B. Exposure to nonlethal ultraviolet doses of 60 to 120 kJ per m2 resulted in increased tissue transglutaminase activity when measured either in cell homogenates, "in situ" by incorporation of fluorescein-cadaverine into the extracellular matrix or by changes in the epsilon(gamma-glutamyl) lysine cross-link. This increase in enzyme activity did not require de novo protein synthesis. Incorporation of fluorescein-cadaverine into matrix proteins was accompanied by the cross-linking of fibronectin and tissue transglutaminase into nonreducible high molecular weight polymers. Addition of exogenous tissue transglutaminase to cultured cells mimicking extensive cell leakage of the enzyme resulted in increased extracellular matrix deposition and a decreased rate of matrix turnover. Exposure of cells to 180 kJ per m2 resulted in 40% to 50% cell death with dying cells showing extensive tissue transglutaminase cross-linking of intracellular proteins and increased cross-linking of the surrounding extracellular matrix, the latter probably occurring as a result of cell leakage of tissue transglutaminase. These cells demonstrated negligible caspase activation and DNA fragmentation but maintained their cell morphology. In contrast, exposure of cells to 240 kJ per m2 resulted in increased cell death with caspase activation and some DNA fragmentation. These cells could be partially rescued from death by addition of caspase inhibitors. These data suggest that changes in cross-linking both in the intracellular and extracellular compartments elicited by tissue transglutaminase following exposure to ultraviolet provides a rapid tissue stabilization process following damage, but as such may be a contributory factor to the scarring process that results.

TG2, a novel extracellular protein with multiple functions

TG2 is multifunctional enzyme which can be secreted to the cell surface by an unknown mechanism where its Ca(2+)-dependent transamidase activity is implicated in a number of events important to cell behaviour. However, this activity may only be transient due to the oxidation of the enzyme in the extracellular environment including its reaction with NO probably accounting for its many other roles, which are transamidation independent. In this review, we discuss the novel roles of TG2 at the cell surface and in the ECM acting either as a transamidating enzyme or as an extracellular scaffold protein involved in cell adhesion. Such roles include its ability to act as an FN co-receptor for ß integrins or in a heterocomplex with FN interacting with the cell surface heparan sulphate proteoglycan syndecan-4 leading to activation of PKCa. These different properties of TG2 involve this protein in various physiological processes, which if not regulated appropriately can also lead to its involvement in a number of diseases. These include metastatic cancer, tissue fibrosis and coeliac disease, thus increasing its attractiveness as both a therapeutic target and diagnostic marker.

An investigation of thin film magnetic recording media

Mechanical, physical and chemical changes in the surface of commercial thin film metal evaporated magnetic recording media have been correlated to recording error and signal degradation measurements. Modified and adapted commercial Hi-8 video recorders have been used for sample generation whilst analytical techniques such as SXPS,IMS and SEM have been employed in the surface characterisation. The durability of the media was assessed through stop motion (still frame) and cycling tests, where error growth and signal degradation were measured as a function of running time. The tests were performed under ambient (22°C, 40% RH) and high humidity (22°C, 80% RH) conditions. Characterisation of the lubricant layer on each tape was performed through models based on XPS and angle resolved XPS. The lubricant thickness can significantly affect the durability and signal output level of a thin film tape and thus it is important that reliable quantification can be achieved. Various models were considered for determining the lubricant thickness although ultimately, the most suitable technique was deemed to be a model that assumed a uniform layer structure. In addition to thin film metal evaporated media, equivalent durability tests and surface analysis experiments were performed using a commercial metal particle tape in order that comparisons could be made between the two types of recording media. The signal performance of the thin film metal evaporated media was found to be quite different from that for the metal particle tape since dropout errors and signal degradation increased at a much earlier stage. Extensive surface analyses enabled the mechanisms responsible for media failure and error growth to be identified in the ME and MP tapes and these were found to result from cyclic stressing and fatigue on the immediate substrate of the media.

The tribology of laminated magnetic recording heads

This thesis investigates the mechanisms that lead to pole tip recession (PTR) in laminated magnetic recording heads (also known as "sandwich heads"). These heads provide a platform for the utilisation of advanced soft magnetic thin films in practical recording heads suitable for high frequency helical scan tape recording systems. PTR results from a differential wear of the magnetic pole piece from the tape-bearing surface of the head. It results in a spacing loss of the playback or read signal of 54.6dB per recording wavelength separation of the poles from the tape. PTR depends on the material combination used in the head, on the tape type and the climate - temperature and relative humidity (r.h.). Five head materials were studied: two non-magnetic substrate materials- sintered multi granular CaTi03 and composite CaTi03/ZrTi04/Ti02 and three soft magnetic materials- amorphous CoNbZr, and nanocrystalline FeNbSiN and FeTaN. Single material dummy heads were constructed and their wear rates measured when cycling them in a Hi-8 camcorder against commercially available metal particulate (MP) and metal evaporated (ME) tapes in three different climates: 25°C/20%r.h., 25°C/80%r.h. and 40°C/80%r.h. X-ray photoelectron spectroscopy (XPS) was used to examine changes the head surface chemistry. Atomic force microscopy (AFM) was used to examine changes in head and tape surface topography. PTR versus cycling time of laminated heads of CaTi03/ZrTiO4/Ti02 and FeTaN construction was measured using AFM. The principal wear mechanism observed for all head materials was microabrasion caused by the mating body - the tape surface. The variation in wear rate with climate and tape type was due to a variation in severity in this mechanism, except for tape cycling at 40°C in which gross damage was observed to be occurring to the head surface. Two subsidiary wear mechanisms were found: third body scratching (all materials) and grain pullout (both ceramics and FeNbSiN). No chemical wear was observed, though tribochemical reactions were observed on the metal head surfaces. PTR was found to be caused by two mechanisms - the first differential microabrasion of the metal and substrate materials and which was characterised by a low (~10nm) equilibrium value. The second was by deep ploughing by third body debris particles, thought mainly to be grain pullout particles. This level of PTR caused by this mechanism was often more severe, and of a non-equilibrium nature. It was observed more for ME tape, especially at 40°C/80%r.h. and 25°c/20%r.h. Two other phenomena on the laminated head pole piece were observed and commented upon: staining and ripple texturing.

The tribology of linear tape recording systems

This thesis is devoted to the tribology at the head~to~tape interface of linear tape recording systems, OnStream ADRTM system being used as an experimental platform, Combining experimental characterisation with computer modelling, a comprehensive picture of the mechanisms involved in a tape recording system is drawn. The work is designed to isolate the mechanisms responsible for the physical spacing between head and tape with the aim of minimising spacing losses and errors and optimising signal output. Standard heads-used in ADR current products-and prototype heads- DLC and SPL coated and dummy heads built from a AI203-TiC and alternative single-phase ceramics intended to constitute the head tape-bearing surface-are tested in controlled environment for up to 500 hours (exceptionally 1000 hours), Evidences of wear on the standard head are mainly observable as a preferential wear of the TiC phase of the AI203-TiC ceramic, The TiC grains are believed to delaminate due to a fatigue wear mechanism, a hypothesis further confirmed via modelling, locating the maximum von Mises equivalent stress at a depth equivalent to the TiC recession (20 to 30 nm). Debris of TiC delaminated residues is moreover found trapped within the pole-tip recession, assumed therefore to provide three~body abrasive particles, thus increasing the pole-tip recession. Iron rich stain is found over the cycled standard head surface (preferentially over the pole-tip and to a lesser extent over the TiC grains) at any environment condition except high temperature/humidity, where mainly organic stain was apparent, Temperature (locally or globally) affects staining rate and aspect; stain transfer is generally promoted at high temperature. Humidity affects transfer rate and quantity; low humidity produces, thinner stains at higher rate. Stain generally targets preferentially head materials with high electrical conductivity, i.e. Permalloy and TiC. Stains are found to decrease the friction at the head-to-tape interface, delay the TiC recession hollow-out and act as a protective soft coating reducing the pole-tip recession. This is obviously at the expense of an additional spacing at the head-to-tape interface of the order of 20 nm. Two kinds of wear resistant coating are tested: diamond like carbon (DLC) and superprotective layer (SPL), 10 nm and 20 to 40 nm thick, respectively. DLC coating disappears within 100 hours due possibly to abrasive and fatigue wear. SPL coatings are generally more resistant, particularly at high temperature and low humidity, possibly in relation with stain transfer. 20 nm coatings are found to rely on the substrate wear behaviour whereas 40 nm coatings are found to rely on the adhesive strength at the coating/substrate interface. These observations seem to locate the wear-driving forces 40 nm below the surface, hence indicate that for coatings in the 10 nm thickness range-· i,e. compatible with high-density recording-the substrate resistance must be taken into account. Single-phase ceramic as candidate for wear-resistant tape-bearing surface are tested in form of full-contour dummy-heads. The absence of a second phase eliminates the preferential wear observed at the AI203-TiC surface; very low wear rates and no evidence of brittle fracture are observed.

Mechanisms of transfer film formation on data recording heads

The increasing demand for high capacity data storage requires decreasing the head-to-tape gap and reducing the track width. A problem very often encountered is the development of adhesive debris on the heads at low humidity and high temperatures that can lead to an increase of space between the head and media, and thus a decrease in the playback signal. The influence of stains on the playback signal of reading heads is studied using RAW (Read After Write) tests and their influence on the wear of the heads by using indentation technique. The playback signal has been found to vary and the errors to increase as stains form a patchy pattern and grow in size to form a continuous layer. The indentation technique shows that stains reduce the wear rate of the heads. In addition, the wear tends to be more pronounced at the leading edge of the head compared to the trailing one. Chemical analysis of the stains using ferrite samples in conjunction with MP (metal particulate) tapes shows that stains contain iron particles and polymeric binder transferred from the MP tape. The chemical anchors in the binder used to grip the iron particles now react with the ferrite surface to create strong chemical bonds. At high humidity, a thin layer of iron oxyhydroxide forms on the surface of the ferrite. This soft material increases the wear rate and so reduces the amount of stain present on the heads. The stability of the binder under high humidity and under high temperature as well as the chemical reactions that might occur on the ferrite poles of the heads influences the dynamic behaviour of stains. A model of stain formation taking into account the channels of binder degradation and evolution upon different environmental conditions is proposed.

Modulation of tissue transglutaminase in tubular epithelial cells alters extracellular matrix levels: a potential mechanism of tissue scarring

The up-regulation and trafficking of tissue transglutaminase (TG2) by tubular epithelial cells (TEC) has been implicated in the development of kidney scarring. TG2 catalyses the crosslinking of proteins via the formation of highly stable e(?-glutamyl) lysine bonds. We have proposed that TG2 may contribute to kidney scarring by accelerating extracellular matrix (ECM) deposition and by stabilising the ECM against proteolytic decay. To investigate this, we have studied ECM metabolism in Opossum kidney (OK) TEC induced to over-express TG2 by stable transfection and in tubular cells isolated from TG2 knockout mice. Increasing the expression of TG2 led to increased extracellular TG2 activity (p < 0.05), elevated e(?-glutamyl) lysine crosslinking in the ECM and higher levels of ECM collagen per cell by 3H-proline labelling. Immunofluorescence demonstrated that this was attributable to increased collagen III and IV levels. Higher TG2 levels were associated with an accelerated collagen deposition rate and a reduced ECM breakdown by matrix metalloproteinases (MMPs). In contrast, a lack of TG2 was associated with reduced e(?-glutamyl) lysine crosslinking in the ECM, causing reduced ECM collagen levels and lower ECM per cell. We report that TG2 contributes to ECM accumulation primarily by accelerating collagen deposition, but also by altering the susceptibility of the tubular ECM to decay. These findings support a role for TG2 in the expansion of the ECM associated with kidney scarring.

Inhibition of transglutaminase activity reduces extracellular matrix accumulation induced by high glucose levels in proximal tubular epithelial cells

Diabetic nephropathy affects 30-40% of diabetics leading to end-stage kidney failure through progressive scarring and fibrosis. Previous evidence suggests that tissue transglutaminase (tTg) and its protein cross-link product epsilon(gamma-glutamyl)lysine contribute to the expanding renal tubulointerstitial and glomerular basement membranes in this disease. Using an in vitro cell culture model of renal proximal tubular epithelial cells we determined the link between elevated glucose levels with changes in expression and activity of tTg and then, by using a highly specific site directed inhibitor of tTg (1,3-dimethyl-2[(oxopropyl)thio]imidazolium), determined the contribution of tTg to glucose-induced matrix accumulation. Exposure of cells to 36 mm glucose over 96 h caused an mRNA-dependent increase in tTg activity with a 25% increase in extracellular matrix (ECM)-associated tTg and a 150% increase in ECM epsilon(gamma-glutamyl)lysine cross-linking. This was paralleled by an elevation in total deposited ECM resulting from higher levels of deposited collagen and fibronectin. These were associated with raised mRNA for collagens III, IV, and fibronectin. The specific site-directed inhibitor of tTg normalized both tTg activity and ECM-associated epsilon(gamma-glutamyl)lysine. Levels of ECM per cell returned to near control levels with non-transcriptional reductions in deposited collagen and fibronectin. No changes in transforming growth factor beta1 (expression or biological activity) occurred that could account for our observations, whereas incubation of tTg with collagen III indicated that cross-linking could directly increase the rate of collagen fibril/gel formation. We conclude that Tg inhibition reduces glucose-induced deposition of ECM proteins independently of changes in ECM and transforming growth factor beta1 synthesis thus opening up its possible application in the treatment other fibrotic and scarring diseases where tTg has been implicated.

Glycogen, the preferred energy source for extracellular protein formation and growth of Aeromonas salmonicida

A study was made of the effect of supplementing a rich 3% (w/v) tryptone soya broth (TSB) medium and a poorer 1.7% (w/v) tryptone-based medium with glucose, maltose and glycogen, as carbon sources, on growth and exoprotein formation by Aeromonas salmonicida. In TSB, glucose inhibited growth and repressed exoprotein formation whilst maltose and glycogen had little effect, up to 20 h, when compared with an unsupplemented control. By contrast, in the poorer medium, over a 24-h incubation period, growth was stimulated three-fold by glycogen, and whilst exoprotein formation was low in comparison with that observed in TSB, the greatest production was observed in the presence of glycogen. Extracellular alpha-amylase was measured in the tryptone medium in the presence of the three carbon sources and the highest level, produced in the presence of glycogen, was 1.6 times that with added maltose whilst none was detectable with glucose present. This pattern was repeated in the case of the maltose-inducible porin, LamB, of the outer membrane.

Eukaryotic translation initiation factor 3, subunit a, regulates the extracellular signal-regulated kinase pathway

The extracellular signal-regulated kinase (ERK) pathway participates in the control of numerous cellular processes, including cell proliferation. Since its activation kinetics are critical for to its biological effects, they are tightly regulated. We report that the protein translation factor, eukaryotic translation initiation factor 3, subunit a (eIF3a), binds to SHC and Raf-1, two components of the ERK pathway. The interaction of eIF3a with Raf-1 is increased by ß-arrestin2 expression and transiently decreased by epidermal growth factor (EGF) stimulation in a concentration-dependent manner. The EGF-induced decrease in Raf-1-eIF3a association kinetically correlates with the time course of ERK activation. eIF3a interferes with Raf-1 activation and eIF3a downregulation by small interfering RNA enhances ERK activation, early gene expression, DNA synthesis, expression of neuronal differentiation markers in PC12 cells, and Ras-induced focus formation in NIH 3T3 cells. Thus, eIF3a is a negative modulator of ERK pathway activation and its biological effects.