bluuebunny/crossref_metadata_2025 huggingface dataset:
Atomic force microscopy imaging of polycrystalline CuInSe2thin films
Authors: Alberts  , Hillie  , Demanet
Published: March 2003

In this study, atomic force microscopy (AFM) imaging has been used to study the structural properties of polycrystalline CuInSe2films, which are widely used as absorber materials in thin film solar cell devices. This technique demonstrated an excellent capability for the reproducible imaging of these rough polycrystalline materials. AFM imaging in combination with statistical analysis revealed distinct differences in the structural properties (i.e. grain width and height distributions, root‐mean‐square (RMS) and peak to valley (R(p–v)) roughness values) as a function of the specific growth technique used and the bulk composition of the films. In the case of Cu‐rich films, prepared by the H2Se/Ar treatment of Cu/In/Cu alloys, rough surface structures were in general observed. Statistical analysis revealed two distinct distribution of grains in these samples (1.0–2.5 μm and 3–5.5 μm) with large RMS andR(p–v)roughness values of 380 nm and 2.6 μm, respectively. In‐rich films were characterized by the presence of much smaller, roughly circular clusters with a significant reduction in both the width and height distributions as well as RMS andR(p–v)roughness values. The most successful growth techniques, in terms of producing homogeneous and dense films, were in the cases of H2Se/Ar treated metallic InSe/Cu/InSe alloys and the coevaporation of all materials to form CuInSe2. Both these techniques produced absorber films with very narrow grain width and height distributions as well as small roughness values. It was possible to establish that high efficiency devices are associated with the use of absorber films with narrow width distributions between 0.5 and 2 μm and small RMS (> 300 nm) roughness values. These values are used as a figure of merit in our laboratories to evaluate the structural properties of our CuInSe2thin films.
Polarization confocal microscopy and Congo Red fluorescence: a simple and rapid method to determine the mean cellulose fibril orientation in plants
Authors: Verbelen  , Kerstens
Published: March 2003

The mean or net preferential orientation of cellulose fibrils in plant cell walls is detected with polarization confocal laser scanning microscopy using the fluorescence dichroism of Congo Red. Single cells, arrays of cells in a tissue, or the epidermis of whole organs can be assayed in vivo. Aerial parts require an extra pectinase treatment because of the cuticle, which is impermeable to aqueous solutions. Peeling off the epidermis can be an elegant alternative, especially for leaves. With this method the net preferential fibril orientation can be related to the symmetry axis of the cell in quantitative terms. Data issuing from this approach are useful in current research on plant biomechanics.
The cytoskeleton in plant and fungal cell tip growth
Authors: Geitmann A. , Emons A. M. C.
Published: March 2003

Tip‐growing cells have a particular lifestyle that is characterized by the following features: (1) the cells grow in one direction, forming a cylindrical tube; (2) tip‐growing cells are able to penetrate their growth environment, thus having to withstand considerable external forces; (3) the growth velocity of tip‐growing cells is among the fastest in biological systems. Tip‐growing cells therefore appear to be a system well suited to investigating growth processes. The cytoskeleton plays an important role in cell growth in general, which is why tip‐growing cells provide an excellent model system for studying this aspect. The cytoskeletal system comprises structural elements, such as actin filaments and microtubules, as well as proteins that link these elements, control their configuration or are responsible for transport processes using the structural elements as tracks. Common aspects as well as differences in configuration and function of the cytoskeleton in various types of tip‐growing cells reveal the general principles that govern the relationship between the cytoskeleton and cell growth.
Stereological estimation of integral mixed curvature with application
Authors: Beneš V. , Hlawiczková M. , Voleník K.
Published: March 2003

This article addresses a bounded system of compact smooth surfaces in three‐dimensional space. Recently, an unbiased estimator of the integral mixed curvature of the given system based on a vertical sampling design has been proposed. The aims of the present paper are: (i) to show that the proposed estimator may have an infinite variance; (ii) to suggest a modification that has better statistical properties; (iii) to extend the point estimator to a function that monitors the curvature along the gradient microstructures; (iv) to present an application of the method to real microscopic images.
Orientation averaging of electron backscattered diffraction data
Authors: Humphreys F. J. , Bate P. S. , Hurley P. J.
Published: March 2003

The use of data averaging to improve the angular precision of electron backscattered diffraction (EBSD) maps is discussed. It is shown that orientations may be conveniently and rapidly averaged using the four Euler‐symmetric parameters which are coefficients of a quaternion representation. The processing of EBSD data requires the use of an edge preserving filter and a modified Kuwahara filter has been successfully implemented and tested. Three passes of such a filter have been shown to reduce orientation noise by a factor of ∼10. Application of the method to deformed and recovered aluminium alloys has shown that such data processing enables small subgrain misorientation (< 0.5°) to be detected reliably.
Far‐field emission of a tapered optical fibre tip: a theoretical analysis
Authors: Drezet A. , Woehl J. C. , Huant S.
Published: March 2003

The far‐field transmission pattern of a tapered optical tip with small aperture (radius ∼ < 40 nm) is modelled by solving Maxwell's equations in the radiation zone with boundary conditions appropriate to the conical geometry. The model is able to reproduce the large differences between the S and P polarizations observed previously in the emission profile of such a tip [Obermüller and Karrai. Appl. Phys. Lett. (1995) 67, 3408].
Time–frequency analysis: a tool to discriminate artefacts from near‐field optical data
Authors: Barchiesi D.
Published: March 2003

Near‐field optical data are non‐stationary, which means that their spectral content varies with the position of the tip, due to both the scanning‐probe recording process and the variations of the optical signal. Therefore time–frequency representations are potentially powerful tools for local characterization as they distribute the energy of the analysed signal over the time and frequency variables, and faithfully depict the signal local behaviour. In this paper, the time–frequency distributions are shown to be appropriate tools to analyse near‐field optical data by using it first on simulated data, and second on experimental near‐field optical images. Within this context, we observe that time–frequency analysis allows a possible separation of relevant optical signals from artefacts, especially in the usual case where the near‐field optical signal is lower band than the feedback data.
High efficiency beam splitter for multifocal multiphoton microscopy
Authors: Nielsen T. , Fricke M. , Hellweg D. , Andresen P.
Published: March 2003

In this article we present the development of a multibeam two‐photon laser scanning microscope. A new type of beam splitter to create the multitude of laser beams is described. This type of beam splitter has higher transmission and generates more uniform beams than can be achieved with the microlens approach used by other groups. No crosstalk exists between the different foci due to small temporal delays between the individual beams. The importance of dispersion compensation to obtain maximum efficiency of the microscope is discussed. With optimum compensation the fluorescence signal was raised by a factor of 14. Different modes of detecting the fluorescence signals and their effect on imaging speed and resolution are discussed.
Structural analyses in three‐dimensional atom probe: a Fourier transform approach
Authors: Vurpillot F. , Da Costa G. , Menand A. , Blavette D.
Published: March 2003

The three‐dimensional atom probe (3DAP) technique gives the elemental identities and the position of atoms within the small volume analysed (on the order of 10 × 10 × 100 nm3). The large number of atoms collected (up to two million) and the excellent spatial resolution of this instrument allows the observation of some crystallographic features of phases chemically identified. This paper shows that the application of a discrete Fourier transform algorithm to a 3DAP dataset provides information that is not easily accessible in real space. The derivation of the mean size of particles from Fourier intensities is an example. Using 3D ‘dark‐field’ imaging, single ordered grains were isolated from the disordered matrix of a ternary alloy. Moreover, the intrinsic spatial resolution of the instrument was evaluated by this method for pure metal; the resolution reaches 0.2 nm laterally and 0.06 nm in depth. This excellent resolution is shown to be sufficient to give access to the crystalline lattice. The use of image filtering in the reciprocal space enables for atomic columns to be imaged the first time from 3DAP data.
Fluorescence lifetime imaging in scanning microscopes: acquisition speed, photon economy and lifetime resolution
Authors: Gerritsen H. C. , Asselbergs M. A. H. , Agronskaia A. V. , Van Sark W. G. J. H. M.
Published: March 2003

In this paper a detailed discussion is presented of the factors that affect the fluorescence lifetime imaging performance of a scanning microscope equipped with a single photon counting based, two‐ to eight‐channel, time‐gated detection system. In particular we discuss the sensitivity, lifetime resolution, acquisition speed, and the shortest lifetimes that can be measured. Detection systems equipped with four to eight time‐gates are significantly more sensitive than the two time‐gate system. Only minor sensitivity differences were found between systems with four or more time‐gates. Experiments confirm that the lifetime resolution is dominated by photon statistics. The time response of the detector determines the shortest lifetimes that can be resolved; about 25 ps for fast MCP‐PMTs and 300–400 ps for other detectors. The maximum count rate of fast MCP‐PMTs, however, is 10–100 times lower than that of fast PMTs. Therefore, the acquisition speed with MCP‐PMT based systems is limited. With a fast PMT operated close to its maximum count rate we were able to record a fluorescence lifetime image of a beating myocyte in less than one second.
Freeze‐dried and resin‐embedded biological material is well suited for ultrastructure research
Authors: Edelmann L.
Published: March 2003

Transmission electron micrographs of different biological material, cryofixed, freeze‐dried and embedded in Spurr's resin, in Epon, or in Lowicryl, are presented. The structure preservation obtained either without or with application of chemical fixatives after drying showed that freeze‐dried embedded specimens are particularly well suited for new morphological, immunocytochemical and microanalytical studies aimed at detecting the life‐like subcellular distribution of mobile macromolecules and ions. The results also indicate that the removal of cell water by freeze‐drying from the areas of best cryofixation is relatively slow. Ultrathin sections of well cryofixed biological material embedded after freeze‐drying in Spurr's resin or Epon reveal cellular plasma phases with very fine granularities and well defined membranes in negative contrast. This may be due to the preservation of the original structure of cellular macromolecules with a considerable amount of their hydration water. Sublimation studies with differently hydrated and cryofixed macromolecules are suggested to settle this issue.
Optic fibre bundle contact imaging probe employing a laser scanning confocal microscope
Authors: Dubaj V. , Mazzolini A. , Wood A. , Harris M.
Published: March 2003

A small diameter (600 µm) fused optic fibre imaging bundle was used as a probe to compare fluorescent specimens by direct contact imaging using both a conventional fluorescence microscope and a laser scanning confocal microscope (LSCM) system. Green fluorescent polyester fibres placed on a green fluorescent cardboard background were used to model biological tissue. Axial displacement curves support the hypothesis that pinhole size in the LSCM system reduces the contribution of non‐focal plane light. Qualitative comparison showed that the LSCM system produced superior image quality and contrast over the conventional system. The results indicate that the new LSCM–probe combination is an improvement over conventional fluorescence–probe systems. This study shows the feasibility of employing such a small diameter probe in the investigation of biological function in difficult to access areas.
Transmitted light microscopy of a fibre reinforced metal
Authors: Moser B. , Rossoll A. , Weber L. , Beffort O. , Mortensen A.
Published: March 2003

A method is presented for studying fibre damage in continuous fibre reinforced composites. It is based on contrasting the transmission of light through intact translucent fibres with the light through fractured or dead‐ended fibres. The method is applied in order to detect processing‐induced fibre fractures in aluminium reinforced with continuous alumina fibres.
Prolonged Hormone Secretion from Neuroendocrine Cells of Aplysia is Indepentdent of Extracellular Calcium
Authors: Wayne Nancy L. , Kim Jiin , Lee Eugene
Published: March 2003

The role of Ca2+ from extracellular and intracellular sources in stimulating neurosecretion was investigated in four experiments using neuroendocrine bag cells of the marine mollusk Aplysia. (i) Bag cells were treated with either an extracellular calcium chelator (BAPTA) or Co2+‐substitution within 30 s after onset of an electrical afterdischarge to prevent influx of Ca2+ from extracellular fluid. These treatments shortened the duration of the afterdischarge, but did not significantly affect the overall pattern or total amount of egg laying hormone (ELH) secretion, suggesting that extracellular Ca2+ is not required for maintenance of ELH release. (ii) Substitution of Ba2+ for Ca2+ has previously been shown to support bag cell afterdischarges that trigger transient elevations in intracellular Ca2+. We showed that this treatment also stimulates ELH secretion, suggesting that Ca2+ release from intracellular stores can stimulate ELH secretion. (iii) To raise intracellular Ca2+ levels in the absence of an afterdischarge, the calcium ionophore X537A was used to transport Ca2+ across plasma and organelle membranes. When this treatment was combined with extracellular calcium chelators so that the only source of Ca2+ was from intracellular compartments, ELH secretion was stimulated. Taken together, these findings are consistent with the hypothesis that release of Ca2+ from intracellular stores is sufficient to stimulate ELH secretion.
Adrenomedullectomy Prevents the Suppression of Pulsatile Luteinising Hormone Release During Fasting in Female Rats
Authors: Cagampang  , Strutton  , Goubillon  , Coen
Published: March 2003

Fasting inhibits the pulsatile secretion of luteinising hormone (LH) in female rats, an effect which is potentiated by the presence of oestradiol (E2). We have previously described various pharmacological or surgical treatments that can rapidly restore the pulses in a fasting animal. Nevertheless, the central and peripheral mechanisms that mediate this suppression of the pulses remain unclear. We have recently shown that adrenomedullectomy prevents the suppression of LH pulses by insulin‐induced hypoglycaemia, a state which activates the sympathoadrenal axis. The present study was undertaken to establish whether this axis might contribute to the loss of the pulses that occurs in ovariectomised E2‐treated rats that have been fasted for 48 h. Following sham adrenomedullectomy LH pulses were observed in animals fed ad libitum; after 48 h of fasting the animals that had received this sham procedure showed a significant suppression of LH levels and LH pulse frequency. In contrast, adrenomedullectomy prevented the inhibition of the pulses by 48 h of fasting; it had no effect on the pulses in the absence of fasting. These results suggest that adrenomedullary activity plays a significant role in the fasting‐induced suppression of LH pulses in rats.
Evidence for Extensive Distribution of Oestrogen Receptor αImmunoreactivity in the Cerebral Cortex of Adult Rats
Authors: Butler  , Kalló  , Sjöberg  , Coen
Published: March 2003

The presence of oestrogen receptor alpha (ERα)‐immunoreactivity (IR) in the cerebral cortex of adult rats has been investigated. Previous studies have reported a negligible presence of ERα or ERα mRNA in this region of the adult rat brain. We have used immunoprecipitation and immunohistochemistry, with various antibodies and fixatives, to detect this protein in the cingulate cortex. When the tissue was fixed using paraformaldehyde alone only faint ERα–IR was observed at this site. In contrast, following fixation either with acrolein (with or without paraformaldehyde) or with a mixture of paraformaldehyde and glutaraldehyde there was extensive ERα–IR throughout layers II to VI; this was absent when the antibodies were preincubated with the peptide fragment used in their production. The presence of ERα–IR in the nonfixed cingulate cortex of adult rats was confirmed by immunoprecipitation.
Effect of Maternal Dexamethasone Treatment and Ambient Temperature on Prolactin Receptor Abundance in Brown Adipose and Hepatic Tissue in the Foetus and New‐born Lamb
Authors: Bispham  , Heasman  , Clarke  , Ingleton  , Stephenson  , Symonds
Published: March 2003

We investigated the influence of maternal dexamethasone treatment and ambient temperature on prolactin receptor (PRLR) abundance in brown adipose tissue (BAT) and hepatic tissue from foetuses and 6‐h‐old lambs delivered by caesarean section. Lambs were either delivered into a warm (30 °C; WD) or cool (15 °C; CD) ambient temperature at 140 days gestation, 2 days after dexamethasone treatment, or at 146 days gestation for controls. Uncoupling protein‐1 (UCP1) content of BAT was higher in dexamethasone‐treated groups compared to controls. A range of tissue‐specific PRLR isoforms was detected. For the long form of PRLR in BAT these isoforms had molecular weights of 66, 54, 34 and 19 kD compared with 88, 76, 66, 58, 54 and 48 kD in liver. In BAT, isoforms of the short form of PRLR had molecular weights of 66, 62, 54, 48, 33 and 31 kD compared with 82, 66, 56, 54, 48, 40 and 33 kD in liver. Dexamethasone treatment in CD lambs resulted in higher abundance of the 54 kD isoform of the short form of PRLR in liver, whilst in BAT dexamethasone resulted in a greater abundance of the 48 kD isoform of the short form, and lower abundance of the 66 kD isoform of the long form of PRLR, compared to controls. A negative correlation (r2=0.52) was observed between abundance of 66 kD isoform for the long form of PRLR and UCP1, compared with positive correlations (r2=0.58–0.60) for the abundance of the 54 and 48 kD isoforms for the short form of PRLR and UCP1. In conclusion, maternal dexamethasone treatment 1 week before term alters the abundance of PRLR isoforms in a tissue‐specific manner. This response is dependent on ambient temperature after birth and may provide a critical endocrine signal for maximising non‐shivering thermogenesis.
Prostaglandin E2 Inhibits Spontaneous Inhibitory Postsynaptic Currents in Rat Supraoptic Neurones via Presynaptic EP Receptors
Authors: Ibrahim  , Shibuya  , Kabashima  , Sutarmo  , Ueta  , Yamashita
Published: March 2003

Prostaglandin E2 (PGE2) has been implicated in the excitatory regulation of magnocellular neurones in the supraoptic nucleus (SON). We have recently reported that PGE2 excited SON neurones by directly activating postsynaptic PGE2 receptors (EP receptors) of a subclass other than EP1–3, but did not affect excitatory postsynaptic currents (EPSCs). In the present study, we examined presynaptic effects of PGE2 on rat SON neurones by measuring spontaneous inhibitory postsynaptic currents (IPSCs) by a slice patch‐clamp technique. PGE2 inhibited spontaneous IPSCs in a dose‐dependent and reversible manner. PGE2 selectively suppressed the frequency of IPSCs without affecting the amplitude of IPSCs in the presence of tetrodotoxin, a blocker of Na+ channels, indicating that the effects were presynaptic. The inhibitory effects of PGE2 on the frequency of IPSCs were mimicked by the EP1/EP3 agonists, 17PT‐PGE2 and sulprostone, and the EP2/EP3 agonist, misoprostol, whereas the EP2 agonist, butaprost, or the FP agonist, fluprostenol, had little effect. The effects of PGE2 on IPSCs were unaffected by the selective EP1 antagonist, SC‐51322. They were unaffected also by antagonists of GABAB and α2 adrenergic receptors, which are present at presynaptic terminals of GABA neurones in the SON and cause suppression of spontaneous IPSCs. The inhibitor of PG synthesis, indomethacin, had little effect on spontaneous IPSCs and on the inhibitory effects of PGE2 as well as of the GABAB agonist, baclofen, and noradrenaline. These results suggest that PGE2 inhibits release of GABA from the GABAergic terminals innervating SON neurones by activating presynaptic EP receptors, presumably of the EP3 subclass, and that such a presynaptic mechanism may play a role in the excitatory regulation of SON neurones by PGE2.
Expression of Proadrenomedullin Derived Peptides in the Mammalian Pituitary: Co‐Localization of Follicle Stimulating Hormone and Proadrenomedullin N‐20 Terminal Peptide‐Like Peptide in the Same Secretory Granules of the Gonadotropes
Authors: Montuenga  , Burrell  , Garayoa  , Llopiz  , Vos  , Moody  , García‐Ros  , Martínez  , Villaro  , Elsasser  , Cuttitta
Published: March 2003

Expression of proadrenomedullin‐derived peptides in the rat, cow and human pituitary was studied by a variety of techniques. Immunocytochemical detection showed a widespread expression of adrenomedullin peptide in the adenohypophysis and the neural lobe, with low expression in the intermediate pituitary. Proadrenomedullin N‐20 terminal peptide (PAMP)‐immunoreactivity was also present in the anterior pituitary but showed a more marked heterogeneous distribution, with cells going from very strong to negative immunostaining. Lower levels of PAMP were found in the neural lobe. Interestingly, the distribution of adrenomedullin and PAMP immunoreactivity in the anterior pituitary did not completely overlap. In the present study, we concentrated our efforts to determine which cell type of the adenohypophysis expresses PAMP. Paraffin and semithin serial sections immunostained for PAMP and the classical pituitary hormones revealed that a subpopulation of the gonadotropes expresses high levels of PAMP‐immunoreactive material. Ultrastructural analysis clearly showed PAMP‐immunoreactivity in the follicle stimulating hormone (FSH)‐containing large secretory granules of the gonadotropes, suggesting simultaneous secretion of PAMP and FSH by this cell type. Three mouse adenohypophysis‐derived cell lines (AtT20, GH3, and αT3‐1 derived from corticotropes, lacto/somatotropes and gonadotropes, respectively) were also analysed and showed expression of both proadrenomedullin‐derived peptides and their mRNA. Functional studies in these three cell lines showed that neither adrenomedullin nor PAMP was able to stimulate cAMP production in our experimental conditions. Taken together, our results support that proadrenomedullin derived peptides are expressed in the pituitary in cell‐specific and not overlapping patterns, that could be explained by differences in postranslational processing. Our data showing costorage of PAMP and FSH in the same secretory granules open a way by which PAMP could be involved in the control of reproductive physiology in a coordinated manner with FSH.
Neural Connections of Hypothalamic Neuroendocrine Nuclei in the Rat
Authors: Saeb‐Parsy  , Lombardelli  , Khan  , McDowall  , Au‐Yong  , Dyball
Published: March 2003

The secretion of many hormones, including oxytocin, vasopressin and growth hormone, is not constant but shows a day‐night rhythm. The suprachiasmatic nucleus (SCN) is thought to generate most mammalian biological rhythms and previous studies have reported suprachiasmatic efferents to the paraventricular nucleus (PVN) and the supraoptic nucleus (SON). We used in vivo extracellular electrophysiological techniques to show that the SCN also sends direct and indirect neural projections to the arcuate nucleus (ARC). This projection consisted of both excitatory and inhibitory components and may contribute to the entrainment of the rhythm in growth hormone secretion to the day–night cycle. Some SCN neurones appear to project to both the SON and the ARC. The SCN in turn receives excitatory and inhibitory inputs from the ARC and the peri‐nuclear zone of the SON (peri‐SON), which may provide feedback information, as well as allowing nonphotic entrainment of the SCN, for example, in response to feeding. Our data thus suggest extensive two‐way connections between the SCN and its target nuclei which may contribute to the generation of day‐night neuroendocrine rhythms. They also suggest the existence of indirect retinal projections to the ARC and PVN. We further investigated the retinal projection to the SCN. We were unable to demonstrate a significant difference in retinal input to those suprachiasmatic cells which had efferent projections to particular hypothalamic targets (SON and/or ARC), and those which did not.
Basic Fibroblast Growth Factor Priming Increases the Responsiveness of Immortalized Hypothalamic Luteinizing Hormone Releasing Hormone Neurones to Neurotrophic Factors
Authors: Gallo F. , Morale M. C. , Tirolo C. , Testa N. , Farinella Z. , Avola R. , Beaudet A. , Marchetti B.
Published: March 2003

The participation of growth factors (GFs) in the regulation of luteinizing hormone releasing hormone (LHRH) neuronal function has recently been proposed, but little is known about the role played by GFs during early LHRH neurone differentiation. In the present study, we have used combined biochemical and morphological approaches to study the ability of a number of GFs normally expressed during brain development, including basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), insulin and insulin‐like growth factor I (IGF‐I) to induce survival, differentiation, proliferation, and phenotypic expression of immortalized (GT1‐1) LHRH neurones in vitro, at early (3‐days in vitro, 3‐DIV) and late (8‐DIV) stages of neuronal differentiation. Comparison of GF‐treated vs untreated neurones grown in serum‐deprived (SD) medium demonstrated bFGF to be the most potent, and insulin the least active in promoting neuronal differentiation. Thus, at both 3‐DIV and 8‐DIV, but especially at 8‐DIV, bFGF induced the greatest increase in the total length and number of LHRH processes/cell and in growth cone surface area. bFGF was also the most active at 3‐DIV, and IGF‐I at 8‐DIV, in counteracting SD‐induced cell death, whereas EGF was the most potent in increasing [3H]thymidine incorporation. All GFs studied decreased the spontaneous release of LHRH from GT1‐1 cells when applied at 3‐DIV or 8‐DIV, except for insulin which was inactive at both time‐points and bFGF which was inactive at 8‐DIV. Pre‐treatment of GT1‐1 cells with a suboptimal (‘priming’) dose of bFGF for 12 h followed by application of the different GFs induced a sharp potentiation of the neurotrophic and proliferative effects of the latter and particularly of those of IGF‐I. Moreover, bFGF priming counteracted EGF‐induced decrease in LHRH release and significantly stimulated LHRH secretion following IGF‐I or insulin application, suggesting that bFGF may sensitize LHRH neurones to differentiating effects of specific GFs during development.
Corticotropin‐Releasing Hormone and Arginine Vasopressin: mRNA and Secretion are Differentially Regulated According to the Pattern of Exposure to Noradrenaline in Rat Hypothalamic Neurones
Authors: Haisenleder D. J.
Published: March 2003

Hypothalamic corticotropin‐releasing hormone (CRH) and arginine vasopressin (AVP) are secreted from the median eminence in a pulsatile manner and regulated by noradrenaline during stress. This study investigated the effect of pulsatile noraderanaline on CRH/AVP mRNAs and secretion. Foetal hypothalamic neurones were cultured on plastic coverslips, inserted into perifusion chambers and noraderanaline pulses given at various doses or pulse intervals for 24 h. CRH and AVP release rose in a dose dependant manner; however, maximal increases in mRNAs were seen with an intermediate noraderanaline pulse dose. The effect of noraderanaline pulse frequency was determined by giving noraderanaline pulses at intervals of 15–120 min vs continuous noraderanaline. Both pulsatile and continuous noraderanaline increased CRH and AVP release, but secretion was reduced after 22 h of treatment in the continuous noraderanaline and rapid pulse groups. CRH mRNA levels were maximally increased by medium interval pulses and AVP mRNA by rapid interval pulses. Neither CRH nor AVP mRNAs were stimulated by continuous noraderanaline. To determine noraderanaline specificity, pulses of veratridine (VER; 15–120 min intervals) vs continuous VER were examined. Only pulsatile VER increased CRH and AVP mRNAs, with maximal effects seen with the 60 min pulse interval for both. Thus, noraderanaline pulse pattern regulates CRH and AVP gene expression in both a coordinate and differential manner. Since noraderanaline plays an important role during stress, the pattern of noraderanaline signals may be critical to the observed changes in CRH and AVP expression.
Maternal Undernutrition in Early Gestation Alters Molecular Regulation of the Hypothalamic‐Pituitary‐Adrenal Axis in the Ovine Fetus
Authors: Hawkins P. , Hanson M. A. , Matthews S. G.
Published: March 2003

We have demonstrated previously that plasma adrenocorticotropin hormone and cortisol responses to exogenous and endogenous stimuli are reduced in fetuses of mildly undernourished ewes. In the present study, we examined the molecular regulation of fetal hypothalamic‐pituitary‐adrenal (HPA) axis function at 127–130 days gestation (dGA) following 15% reduction in maternal nutrition between 0 and 70 dGA. Usingin situhybridization, we found that corticotropin releasing hormone (CRH) mRNA expression in the hypothalamic paraventricular nucleus (PVN) was lower in fetuses from nutrient restricted ewes than in controls. Restricted fetuses also had greater levels of mRNA encoding preproenkephalin (PENK) and magnocellular arginine vasopressin (AVP) in the PVN. Expression of oxytocin mRNA and parvocellular AVP mRNA in the PVN and pro‐opiomelanocortin mRNA in the pituitary were unchanged. Glucocorticoid receptor mRNA expression was unaltered at the PVN, but was reduced (> 40%) in the anterior pituitary of restricted fetuses. Northern blot analysis demonstrated that levels of adrenal P450sccmRNA and P450C17mRNA were not different between the groups. We conclude that the reduction in HPA function reported previously is mediated, at least in part, by a decrease in expression of CRH mRNA and increase in PENK mRNA in the PVN.
Age‐Related Changes in Hypothalamic Gonadotropin‐Releasing Hormone and N‐Methyl‐d‐Aspartate Receptor Gene Expression, and their Regulation by Oestrogen, in the Female Rat
Authors: Gore A. C. , Oung T. , Woller M. J.
Published: March 2003

During reproductive ageing, the oestrous cycles of female rats become irregular and eventually cease. The mechanisms for reproductive senescence in rodents are believed to involve changes in hypothalamic neurones, including gonadotropin‐releasing hormone (GnRH) cells and their afferent inputs. In addition, effects of oestrogen on hypothalamic function may vary in animals of different ages. These issues were addressed using young (aged 4–5 months), middle‐aged (12–14 months) and old (24–26 months) female Sprague‐Dawley rats. Animals were ovariectomized and given oestrogen or vehicle replacement. They were killed and the preoptic area‐anterior hypothalamus (POA‐AH) and the medial basal hypothalamus‐median eminence (MBH‐ME) were dissected out, RNA extracted, and RNase protection assay used to quantify gene expression of several hypothalamic molecules. In the first experiment, GnRH RNA levels were measured in the POA‐AH. No effects of ageing or oestrogen were observed on GnRH gene expression. This finding suggests that ageing and oestrogen may affect GnRH release from neuroterminals independently of de novo biosynthesis, and that this may involve other neurones that affect GnRH neurosecretory function. In the second experiment, we investigated changes in N‐methyl‐d‐aspartate (NMDA) receptor subunit mRNA levels. These receptors play an important regulatory role in mediating effects of glutamate on GnRH function, and are themselves regulated by oestrogen and ageing. NMDA receptor subunit (NR) 1, 2a and 2b mRNA levels were quantified in the POA‐AH and MBH‐ME, the sites of GnRH perikarya and neuroterminals, respectively. In general, oestrogen had inhibitory effects on NR1 and NR2a, and differential effects on NR2b subunit mRNA levels. NMDA receptor subunit mRNA levels also changed during ageing: age‐related decreases in NR1 mRNA occurred in the MBH‐ME, and an age‐related increase in NR2b mRNA occurred in the POA‐AH. Taken together, these results demonstrate subunit‐ and region‐specific changes in hypothalamic NMDA receptor subunit gene expression with oestrogen and ageing. These alterations could have implications for the physiological effects of glutamate on its NMDA receptor, and impact the regulation of reproductive and other neuroendocrine and autonomic functions by hypothalamic glutamatergic inputs.
Metyrapone‐Induced Glucocorticoid Depletion Modulates Tyrosine Hydroxylase and Phenylethanolamine N‐Methyltransferase Gene Expression in the Rat Adrenal Gland by a Noncholinergic Transsynaptic Activation
Authors: Laborie C. , Van Camp G. , Bernet F. , Montel V. , Dupouy J. P.
Published: March 2003

The hypothalamic corticotropin‐releasing hormone system and the sympathetic nervous system are anatomically and functionally interconnected and hormones of the hypothalamic‐pituitary‐adrenocortical axis contribute to the regulation of catecholaminergic systems. To investigate the role of glucocorticoids on activity of the adrenal gland, we analysed plasma and adrenal catecholamines, tyrosine hydroxylase (TH) and phenylethanolamine N‐methyltransferase (PNMT) mRNA expression in rats injected with metyrapone or dexamethasone. Metyrapone‐treated rats had significantly lower epinephrine and higher norepinephrine production than control rats. Metyrapone increased TH protein synthesis and TH mRNA expression whereas its administration did not affect PNMT mRNA expression. Dexamethasone restored plasma and adrenal epinephrine concentrations and increased PNMT mRNA levels, which is consistent with an absolute requirement of glucocorticoids for PNMT expression. Adrenal denervation completely abolished the metyrapone‐induced TH mRNA expression. Blockage of cholinergic neurotransmission by nicotinic or muscarinic receptor antagonists did not prevent the metyrapone‐induced rise in TH mRNA. Finally, pituitary adenylate cyclase activating polypeptide (PACAP) adrenal content was not affected by metyrapone. These results provide evidence that metyrapone‐induced corticosterone depletion elicits transsynaptic TH activation, implying noncholinergic neurotransmission. This may involve neuropeptides other than PACAP.
Mechanisms of Cytosolic Ca2+ Suppression By Prostaglandin E2 Receptors in Rat Melanotrophs
Authors: Nagata T. , Harayama N. , Sasaki N. , Inoue M. , Tanaka K. , Toyohira Y. , Uezono Y. , Maruyama T. , Yanagihara N. , Ueta Y. , Shibuya I.
Published: March 2003

We have previously reported that voltage‐dependent Ca2+ (VDC) channels of rat melanotrophs are inhibited by prostaglandin E2 (PGE2). In this study, mechanisms involved in the inhibitory actions of PGE2 receptors of rat melanotrophs were analysed using reverse transcriptase‐polymerase chain reaction (RT‐PCR), Ca2+‐imaging and whole‐cell, patch‐clamp techniques with recently developed EP agonists, each of which is selective for the known four subclasses of EP receptors (EP1–4). PGE2 reversibly suppressed the cytosolic Ca2+ concentration ([Ca2+]i). The maximum reduction in [Ca2+]i by PGE2 was comparable to that by dopamine or to that by extracellular Ca2+ removal. RT‐PCR analysis of all four EP receptors revealed that EP3 and EP4 receptor mRNAs were expressed in the intermediate lobe. The effects of PGE2 to suppress [Ca2+]i were mimicked by the selective EP3 agonist, ONO‐AE‐248, whereas three other EP agonists, ONO‐DI‐004 (EP1), ONO‐AE1‐259 (EP2) and ONO‐AE1‐329 (EP4), had little or no effect on [Ca2+]i. All four G‐protein activated inward rectifying K+ (GIRK) channel mRNAs were identified in intermediate lobe tissues by RT‐PCR. Dopamine concentration‐dependently activated GIRK currents, whereas PGE2 did not activate GIRK currents, even at the concentration causing maximal inhibition of VDC channels. These results suggest that PGE2 acts on EP3 receptors to suppress Ca2+ entry of rat melanotrophs by selectively inhibiting VDC channels of these cells. We have compared the possible cellular and molecular mechanisms of inhibition by dopamine and PGE2.
The Anti‐Inflammatory Effect of Bee Venom Stimulation in a Mouse Air Pouch Model Is Mediated by Adrenal Medullary Activity
Authors: Kwon Y.‐B. , Kim H.‐W. , Ham T.‐W. , Yoon S.‐Y. , Roh D.‐H. , Han H.‐J. , Beitz A.‐J. , Yang I.‐S. , Lee J.‐H.
Published: March 2003

Cutaneous electrical or chemical stimulation can produce an anti‐inflammatory effect, which is dependent on adrenal medullary‐sympathetic activation. We have previously shown that peripheral injection of bee venom (BV) also produces a significant anti‐inflammatory effect that is neurally mediated. In the present study, we examined whether this anti‐inflammatory effect is also dependent on the adrenal gland using the mouse inflammatory air pouch model. Subcutaneous (s.c.) BV injection produced a marked suppression of leucocyte migration and tumour necrosis factor (TNF)‐α concentration induced by zymosan injection into the air pouch. The role of the adrenal gland in this suppression was evaluated in adrenalectomized mice. Adrenalectomy significantly reversed the suppression of leucocyte migration and TNF‐α elevation caused by BV. Serum concentrations of corticosteroid were increased in mice with zymosan‐induced air‐pouch inflammation and this increase was reduced by BV administration, suggesting that adrenal corticosteroid release is not involved in mediating the anti‐inflammatory effects of BV. To test this hypothesis, the corticosteroid receptor antagonist (RU486) was administered and found not to affect the BV‐induced inhibition of leucocyte migration. By contrast, pretreatment with the β‐adrenergic antagonist propranolol reversed the BV‐induced inhibitory effect on leucocyte migration. These results suggest that the anti‐inflammatory effect of s.c. BV administration is mediated in part by the release of catecholamines from the adrenal medulla.
In Vivo Gene Transfer Studies on the Regulation and Function of the Vasopressin and Oxytocin Genes
Authors: Murphy D. , Wells S.
Published: March 2003

Novel genes can be introduced into the germline of rats and mice by microinjecting fertilized one‐cell eggs with fragments of cloned DNA. A gene sequence can thus be studied within the physiological integrity of the resulting transgenic animals, without any prior knowledge of its regulation and function. These technologies have been used to elucidate the mechanisms by which the expression of the two genes in the locus that codes for the neuropeptides vasopressin and oxytocin is confined to, and regulated physiologically within, specific groups of neurones in the hypothalamus. A number of groups have described transgenes, derived from racine, murine and bovine sources, in both rat and mouse hosts, that mimic the appropriate expression of the endogenous vasopressin and genes in magnocellular neurones (MCNs) of the supraoptic and paraventricular nuclei. However, despite considerable effort, a full description of the cis‐acting sequences mediating the regulation of the vasopressin‐oxytocin locus remains elusive. Two general conclusions have nonetheless been reached. First, that the proximal promoters of both genes are unable to confer any cell‐specific regulatory controls. Second, that sequences downstream of the promoter, within the structural gene and/or the intergenic region that separates the two genes, are crucial for appropriate expression. Despite these limitations, sufficient knowledge has been garnered to specifically direct the expression of reporter genes to vasopressin and oxytocin MCNs. Further, it has been shown that reporter proteins can be directed to the regulated secretory pathway, from where they are subject to appropriate physiological release. The use of MCN expression vectors will thus enable the study of the physiology of these neurones through the targeted expression of biologically active molecules. However, the germline transgenic approach has a number of limitations involving the interpretation of phenotypes, as well as the large cost, labour and time demands. High‐throughput somatic gene transfer techniques, principally involving the stereotaxic injection of hypothalamic neuronal groups with replication‐deficient adenoviral vectors, are now being developed that obviate these difficulties, and which enable the robust, long‐lasting expression of biologically active proteins in vasopressin and oxytocin MCNs.
Termination of Neuroendocrine Refractoriness to Melatonin in Siberian Hamsters (Phodopus sungorus)
Authors: Kauffman A. S. , Freeman D. A. , Zucker I.
Published: March 2003

Siberian hamsters maintained from birth in a short day length (DL), unlike their long‐day counterparts, fail to undergo reproductive development by 5 weeks of age. Instead, reproductive maturation of short‐day males is delayed for approximately 20 weeks, at which point neuroendocrine refractoriness to the inhibitory effects of short DLs develops, resulting in growth of the gonads. To terminate refractoriness and re‐establish responsiveness to short photoperiods, 10–15 weeks of long‐day exposure is required. We assessed whether continuous exposure to long days is necessary to terminate refractoriness or whether the first few weeks of long days initiate a process that culminates several months later in the breaking of refractoriness. Male hamsters refractory to short DLs were transferred to a long‐day photoperiod, pinealectomized (PINx) after 0, 3, 6 or 15 weeks, and subsequently infused for 6 weeks with a short‐day melatonin signal. This melatonin treatment induces gonadal regression in photosensitive but not in photorefractory hamsters. Six percent of males PINx at week 0 and 88% of those PINx at week 15 underwent gonadal atrophy by the end of the melatonin infusion treatment initiated on week 15. Among hamsters PINx on week 6, 17% versus 76% underwent testicular involution in response to melatonin infusions initiated on week 6 and week 15, respectively. This finding indicates that a fraction of the long days that hamsters experience during spring and summer are sufficient to trigger the processes that restore responsiveness to short DLs. Additional groups of pineal‐intact photorefractory animals were given 3, 6 or 15 weeks of long‐day exposure and then returned to a short DL for several months; only those treated for 15 weeks terminated refractoriness. The breaking of refractoriness, once triggered by long‐day melatonin signals, proceeds to completion only in the absence of short‐day melatonin signals.
Cerana®– a new method for the restoration of teeth with prefabricated ceramic inlays
Authors: Ödman  , Nilsson  , Pietruszka
Published: March 2003

Cerana are all‐ceramic, prefabricated inlays which replace enamel and are produced in a lucite‐reinforced glass ceramic. To evaluate the clinical quality of Cerana inlay therapy, 38 patients with a total of 60 inlays were examined in a retrospective cross‐sectional study. The inlays were primarily of Class I type, but even Class II tunnel preparations and Class V were examined. The median age of the inlays was 1·5 years. A modified form of the California Dental Association criteria was used in the evaluation. The results show that all inlays were of excellent or acceptable character in terms of marginal fit, colour and surface roughness. None of the patients reported any post‐operative symptoms of pain. One restoration had secondary caries and a fractured marginal ridge. One restoration with poor anatomical form caused by a fractured filling and one restoration with deep marginal discolouration were discovered. Although the follow‐up time was short, the results show that Cerana can be considered an interesting alternative to other tooth‐coloured materials on the market.
A retrospective study of combined fixed–removable reconstructions with their analysis of failures
Authors: Studer S. P. , Mäder C. , Stahel W. , Schärer P. , O Department , Fixed F , Prosthodontics Removable , Materials Dental , O School , Medicine F Dental , University Zürich , Statistics And , O Swiss Federal Institute , F Technology  , Eth  , Zürich  , Switzerland
Published: March 2003

The survival rate and the reasons for failures of 130 combined fixed–removable reconstructions (CFR), incorporated in 112 patients, were assessed. Each CFR reconstruction was classified depending on its attachments: 76 reconstructions were attached with rigid, precise attachments, and constituted the rigid group; 54 reconstructions were attached with either semi‐precision or individual attachments and were defined as the semi‐rigid group. Of the 130 reconstructions, 41 were determined as complete successes, 39 as partial successes and 50 as failures, leading to 37 major repairs and to 13 new reconstructions. Three reconstructions failed due to technical reasons, 36 due to biological reasons and for 11 reconstructions, both categories of reasons were responsible for their failure. In total, technical reasons were counted 15 times in comparison to 73 biological reasons for those 50 failed reconstruction, with 29 fractured abutment teeth as the most common biological reason. Within the rigid group, 45 failed reconstructions were observed, whereas within the semi rigid group only 5 failures occurred, leading to an 8‐year survival estimate (± SD) of 30·1% (± 6·9%) for the rigid group and 93·1% (± 3·9%) for the semi rigid group. Beside the attachment type, the anatomy of the partially edentulous tooth arch in form of the free‐end situation and the dentate opposing jaw were identified as risk factors.
An investigation of the stress values on a tooth restored by amalgam
Authors: Toparli M. , Gökay N. , Aksoy T.
Published: March 2003

The study was carried out in two stages: (1) MOD amalgam cavities were prepared on maxillary second premolars and three strain gauges were attached to the palatal surface of each tooth. The teeth were filled using amalgam with and without base material (glass–ionomer). Stresses occurred during the hardening phase and also through mastication and were measured by strain‐gauge rosettes. (2) The stresses which occurred at the same points were calculated by the finite element method and compared with the values obtained experimentally. The highest stress values were observed when no base material was used.