bluuebunny/crossref_metadata_2025 huggingface dataset:
Bezafibrate‐induced improvement in glucose uptake and endothelial function in protease inhibitor‐associated insulin resistance
Authors: Nyström T. , Bratt G. , Sjöholm Å.
Published: March 2003

Abstract.  Nyström T, Bratt G, Sjöholm Å (Karolinska Institutet, Stockholm South Hospital, Stockholm, Sweden). Bezafibrate‐induced improvement in glucose uptake and endothelial function in protease inhibitor‐associated insulin resistance (Case Report). J Intern Med 2002; 252: 570–574.

Protease inhibitors used as therapy for HIV‐1 infection have been associated with metabolic side‐effects such as peripheral fat wasting, central adiposity, hyperlipidaemia and insulin resistance. This metabolic disorder is known as the lipodystrophy syndrome. This syndrome can be recognized by the early appearance of an increase in serum triglyceride, cholesterol and plasma‐free fatty acid levels. Here, we describe an HIV‐1 positive patient with the lipodystrophy syndrome in whom a decline in serum triglycerides was seen along with a significant improvement in glucose uptake following treatment with bezafibrate for 3 months. This improvement may be a consequence of the Randle effect, i.e. increased availability of plasma‐free fatty acids is negatively correlated to glucose uptake. We also noted a significant improvement in endothelial‐dependent flow‐mediated dilatation after treatment with bezafibrate. This effect could result from the increased glucose uptake observed and a decrease in insulin resistance secondary to the lowered triglyceride levels by bezafibrate. We conclude that bezafibrate may be of clinical utility in the lipodystrophy syndrome, through its beneficial effects on insulin resistance, glucose uptake and endothelial dysfunction.
Airway hyperresponsiveness in a large group of subjects with α1‐antitrypsin deficiency: a cross‐sectional controlled study
Authors: Malerba M. , Radaeli A. , Ceriani L. , Tantucci C. , Grassi V.
Published: March 2003

Abstract.  Malerba M, Radaeli A, Ceriani L, Tantucci C, Grassi V (University of Brescia, Brescia, Italy). Airway hyperresponsiveness in a large group of subjects with α1‐antitrypsin deficiency: a cross‐sectional controlled study. J Intern Med 2003; 253: 351–358.

Background.  It has been suggested that subjects with α1‐antitrypsin (AAT) deficiency, lacking a major antiprotease defence against airway inflammation, might be more susceptible of development of airway hyperresponsiveness (AHR). Moreover, lower AAT blood levels might also be able to influence the severity of AHR.

Objectives.  This study was aimed to investigate the prevalence of AHR in a large group of subjects with AAT deficiency included in the Italian Registry and to evaluate the relationship between AAT blood levels and the severity of AHR in this population.

Design.  Cross‐sectional controlled study.

Setting.  Regional Reference Centre for AAT deficiency in Brescia, Italy.

Methods.  A total of 114 subjects with AAT deficiency underwent pulmonary function tests. Eighty‐six were eligible to perform a bronchial provocation test with methacholine (MCh) (baseline FEV1 > 60% predicted) to assess the provocative dose producing a 20% fall of FEV1 (PD20FEV1). Similar measurements were performed in a control group of 27 age‐matched normal subjects.

Results.  The prevalence of AHR (PD20FEV1 < 2000 μg MCh) was not different between AAT deficiency subjects and controls (16.3% and 11.1%, respectively; P = 0.66), and also amongst two subgroups of AAT deficiency subjects divided according to different protease inhibitor (Pi) phenotypes (PiMZ‐MS, PiSZ‐ZZ). Hyperresponsive subjects with AAT deficiency, however, showed a positive correlation between AAT blood levels and PD20FEV1 values (r = 0.71, P < 0.01).

Conclusions.  These findings indicate that AAT deficiency subjects did not exhibit a greater prevalence of airway hyperresponsiveness as compared with control subjects, but suggest that, in the subset of AAT deficiency subjects hyperresponsive to MCh, lower levels of AAT are associated with a higher severity of AHR.
Association between vitamin D receptor gene polymorphisms and tubular citrate handling in calcium nephrolithiasis
Authors: Mossetti G. , Vuotto P. , Rendina D. , Numis F. G. , Viceconti R. , Giordano F. , Cioffi M. , Scopacasa F. , Nunziata V.
Published: March 2003

Abstract. Mossetti G, Vuotto P, Rendina D, Numis FG, Viceconti R, Giordano F, Cioffi M, Scopacasa F, Nunziata V (Federico II University Medical School, Naples, Italy). Association between vitamin D receptor gene polymorphisms and tubular citrate handling in calcium nephrolithiasis. J Intern Med 2003; 253: 194–200.

Objectives.  Hypocitraturia is a risk factor for calcium nephrolithiasis. 1,25(OH)2D3 influences renal citrate handling and enhances citraturia. The aim of this study was to evaluate the relationship between vitamin D receptor (VDR) allelic variant and urinary citrate excretion in recurrent stone formers (SF) patients.

Design.  Case–control study.

Subjects.  A total of 220 recurrent calcium oxalate SF patients and 114 healthy control (C) subjects were enrolled for this study. Subjects with urinary tract infections, hyperparathyroidism, cystinuria >70 μmol/24 h, gouty diathesis, renal tubular acidosis, renal failure, chronic diarrhoeal states, intake of thiazide diuretics, angiotensin‐converting enzyme (ACE)‐inhibitors, glucocorticoids or oestrogens were excluded.

A standard constant diet was given for 7 days. The 24‐h urinary citrate excretion and the active tubular reabsorption of filtered citrate (Rcit) were evaluated. Hypocitraturia was defined as a urinary citrate excretion lower than 1.7 mmol day−1. Stone formers patients and C were genotyped for BsmI and TaqI VDR alleles. Contingency table chi‐square tests were used to compare genotype frequencies in hypocitraturic SF patients, normocitraturic SF and C.

Results.  The prevalence of hypocitraturia in SF patients was 32.7% (72 of 200). Hypocitraturia in these patients resulted from excessive Rcit of a normal load of citrate. We found a different distribution (P < 0.05) of BsmI and TaqI VDR genotypes in hypocitraturic SF patients compared with normocitraturic SF and C. In particular, the prevalence of bb and TT VDR genotypes in hypocitraturic SF was significantly higher than in normocitraturic SF and C.

Conclusions.  These results point to a genetic association between BsmI and TaqI VDR polymorphisms and idiopathic hypocitraturia in calcium‐oxalate recurrent SF patients.
Changing sex ratio in acute coronary heart disease: data from Swedish national registers 1984–99
Authors: Rosengren A. , Thelle D. S. , Köster M. , Rosén M.
Published: March 2003

Abstract.  Rosengren A, Thelle DS, Köster M, Rosén M (Sahlgrenska University Hospital/Östra, Göteborg; and National Board of Health and Welfare, Stockholm; Sweden). Changing sex ratio in acute coronary heart disease: data from Swedish national registers 1984–99. J Intern Med 2003; 253: 301–310.

Objectives.  To examine trends in sex ratios for different manifestations of coronary disease.

Design.  National Swedish registers on hospital discharges and cause‐specific deaths were used to calculate age‐ and sex‐specific trends and sex ratios for coronary admissions and deaths.

Setting.  Nineteen Swedish counties, average population 4.8–5.1 million in the investigated age groups.

Subjects.  All patients aged 25–84 years with first hospital admissions or deaths as a result of coronary heart disease in 1984–99, in total 432 871 cases.

Main outcome measures.  Ratio men/women and rates (per 100 000) of acute myocardial infarction (AMI), acute admissions for angina and total of all acute admissions for any coronary disease.

Results.  Below age of 65 years AMI incidence decreased more for men than for women and rates of acute admissions for angina increased more in women than in men. In men and women above 65 years trends were almost identical. In 1984–87 the ratio men/women with respect to myocardial infarction was 5.6 at age 25–44 years, but decreased to 3.7 in 1996–99. Corresponding sex ratios for angina decreased from 3.2 to 1.8 and for total coronary heart disease from 4.7 to 2.8. Amongst men and women aged 45–54 years, the sex ratio with respect to myocardial infarction decreased from 5.6 to 4.1, for angina from 2.4 to 1.7 and for total acute coronary disease from 4.2 to 2.7. Ratios men/women decreased less at higher ages and remained unchanged throughout the period in the oldest age group.

Conclusions.  Overall, we found decreasing sex ratios at ages below 65, but above age 65 years trends in men and women were similar. These developments could be due to changing criteria for admission and diagnosis, but true differences in the clinical manifestation of coronary disease, possibly in response to secular trends in risk factor levels, cannot be excluded.
A silver impregnation utilizing only reagent‐grade chemicals for visualization of peripheral axons and fibroblasts
Authors: Novotny  , Mertens
Published: March 2003

A silver impregnation procedure is described that enables the representation of numerous tissue components. It especially visualizes nerves and fibroblasts, which may be clearly distinguished from other tissue elements. Since it can be performed on thick sections, three‐dimensional analysis of nerve terminations and fibroblasts in the tissues can be performed. The results are illustrated with the innervation of the rat snout and human labial sweat glands for nerves, and with bovine and pathological human material for fibroblasts. Axons are visualized as thin, sinuous black structures, sometimes, as in the case of autonomic efferents, with varicosities. Fibroblasts are revealed in their total extent by the darker staining of their nuclei and cytoplasm compared with that of the surrounding collagen. Cell processes can thus be followed for long distances, and may be seen to approach other cells.

Previously published methods for the visualization of nerves and fibroblasts depended upon the use of commercial formalin, which is subject to the manufacturers' modifications. The method presented here uses exclusively analytical‐grade reagents and distilled water. It is also less dependent than other methods on the fixation protocol.
Atomic force microscopy: influence of air drying and fixation on the morphology and viscoelasticity of cultured cells
Authors: Weyn  , Kalle  , Kumar‐Singh  , Van Marck  , Tanke  , Jacob
Published: March 2003

The influence of fixation, air‐drying and liquid‐imaging on the morphology as well as on the viscoelasticity of malignant mesothelioma cells was studied by atomic force microscopy. In this study, dehydrated cells were more easily scanned and offered faster data recording than hydrated cells. However, the influence of fixation strength was more noticeable. Strong fixation induced flattening of the cytoplasm and loss of nuclear structure, resulting in a clearly visible cytoskeleton which could be easily seen as fibres orientated in the direction of the cell growth. By contrast, the morphology of hydrated cells was influenced to a lesser degree on fixation and showed an overall ‘rounding’ of the surface with vague, ill‐defined structures. Nuclear areas of these samples were difficult to image.

Viscoelasticity measurements also exhibited large differences. Dehydrated cells were much harder and showed a uniform indentation profile over the whole cell that was independent of fixation. Indentation on hydrated cells was large and depended on the height of the measuring spot, the submembranous structure and, to a lesser extent, on fixation. To calculate an overall ‘cellular’ viscoelasticity, different methods were tested on these samples. Indentations of multiple, randomly chosen points, covering the whole cell, were measured and averaged to yield a mean indentation score. We avoided the thin and shadowed areas since it was shown that these regions were less suited for measuring. Using this design, large viscoelasticity differences were found, on which the influence of the external parameters could be shown. In another set‐up, layered imaging was tried. However, long data acquisition times caused cellular activation and rearrangement, making this scanning mode unsatisfactory.
Registration of serial sections of mouse liver cell nuclei
Authors: Baheerathan S. , Albregtsen F. , Danielsen H.E.
Published: March 2003

Image registration of biological tissue is essential for 3D reconstruction, which is important for visualizing and quantifying the 3D relationships between internal structures of an object. The biological role of DNA organization, which is an extremely complex 3D architecture within the cell nucleus, has come into focus since it has become clear that the chromatin structure in itself functions as a regulator of DNA. Thus, 3D reconstruction of cell nuclei based on consecutive series of high‐resolution ultrathin slices may provide new information about the chromatin structure and its organizational changes during carcinogenesis.

This work focuses mainly on the problem of registering successive serial transmission electron micrographs of ultrathin sections of mouse liver cell nuclei to analyse the 3D chromatin structure. A five‐step semiautomatic interactive registration method is proposed. The first two steps of the procedure correct the rotation and translation components by using the phase correlation. The third, fourth and fifth steps correct the global distortion, employing a point mapping method based on different ways of selecting the control points. In step three, the control points were automatically computed by phase correlating corresponding subimages of the reference and sensed image. A semiautomatic method is used in the fourth step to select the control points, i.e. an automated method for computing the centre of mass of manually identified anatomical structures in neighbouring slices. For the sections which could not be properly corrected by the four steps, a final step is introduced, where control points are manually selected in the reference and sensed images. An algorithm is proposed to examine the spatial distribution of selected control points.

Four sets of serial sections of mouse liver cell nuclei, each with approximately 100 sections, are registered by the proposed method and also registered manually for the comparison of registration accuracy. Artificial X–Z and Z–Y sections of registered series were visually compared for the smoothness of the nuclear membrane. To quantify the registration accuracy and the extent of registration, the correlation coefficient (C) and the overlap index (C0) were computed over the registered structure of interest. In addition to the visual comparison and the comparison of C and C0, the registered serial sets were compared by 3D GLCM‐based texture features in the Z direction. The results demonstrate that the proposed semiautomatic registration technique achieved accurate results comparable to the manual registration. The proposed registration method relies only on the operator for rough pinpointing of cellular structures. Therefore, it should provide better reproducibility, and allow the user to operate the system faster and in a more relaxed manner than in a manual registration.
Nanometric patterning of zinc by optical near‐field photochemical vapour deposition
Authors: Polonski V. V. , Yamamoto Y. , Kourogi M. , Fukuda H. , Ohtsu M.
Published: March 2003

A new technique, optical near‐field photochemical vapour deposition (NFO‐PCVD) enables maskless production of nanometric structures with controllable size, chemical composition and morphology. By placing a near‐field optical microscope inside the reaction chamber for photochemical vapour deposition we have deposited nanoscale metal patterns. We demonstrate for the first time, successfully deposited in the near‐field region, lines of metallic zinc with the observed stripe width of 20 nm.
Signal components in the environmental scanning electron microscope
Authors: Fletcher  , Thiel  , Donald
Published: March 2003

We demonstrate that the gas‐amplified secondary electron signal obtained in the environmental scanning electron microscope has both desired and spurious components. In order to isolate the contributions of backscattered and secondary electrons, two sets of samples were examined. One sample consisted of a pair of materials having similar secondary emission coefficients but different backscatter coefficients, while the other sample had a pair with similar backscatter but different secondary emission coefficients. Our results show how the contribution of the two electron signals varies according to the pressure of the amplifying gas. Backscatter contributions, as well as background due to gas ionization from the primary beam, become significant at higher pressure. Furthermore, we demonstrate that the relative amplification efficiencies of various electron signals are dependent upon the chemistry of the gas.
Adhesion artefacts in atomic force microscopy imaging
Authors: Paredes J. I. , Martínez‐Alonso A. , Tascón J. M. D.
Published: March 2003

Artefacts that affect contrast and arise from adhesion forces in atomic force microscopy images of aramid fibres (both fresh and plasma‐treated) are investigated. It is demonstrated that these stem not only from variations in the chemical composition of the surface but also from certain topographical features (which may appear hidden or enhanced in the images), resulting in changes in the lateral forces that are detected by the cantilever and are comparable to the vertical forces. It is also shown that both types of contribution to the forces can be uncoupled to yield images free from these artefacts, thus allowing more accurate quantitative measurements. These artefactual effects are also generally applicable to many other materials.
Near‐ and far‐field second‐harmonic imaging of quasi‐phase‐matching crystals
Authors: Vohnsen B. , Bozhevolnyi S. I.
Published: March 2003

Second‐harmonic scanning near‐ and far‐field optical microscopy of an electric‐field poled KTiOPO4 quasi‐phase‐matching crystal has been accomplished. This has been done in order to reveal the walls that form the intersections between inverted and non‐inverted crystal domains. The domain walls are seen clearly only in images recorded by means of second‐harmonic generation because of a large nonlinear contrast, and they appear as bright stripes when studied in a reflection geometry but they are dark when studied in transmission. The images show that the duty cycle of the quasi‐phase‐matching crystal differs from the ideal and that the walls are not completely smooth. These effects, in combination with the observed scattering from the domain walls, are expected to lower the output of the crystal when used for frequency doubling. We conclude that the wall thickness is no more than approximately 100 nm, which makes it a suitable test object for the resolution capabilities of scanning near‐field optical microscopes that are used for nonlinear imaging.
Combining AFM and FRET for high resolution fluorescence microscopy
Authors: Vickery S. A. , Dunn R. C.
Published: March 2003

Here we demonstrate a new microscopic method that combines atomic force microscopy (AFM) with fluorescence resonance energy transfer (FRET). This method takes advantage of the strong distance dependence in Förster energy transfer between dyes with the appropriate donor/acceptor properties to couple an optical dimension with conventional AFM. This is achieved by attaching an acceptor dye to the end of an AFM tip and exciting a sample bound donor dye through far‐field illumination. Energy transfer from the excited donor to the tip immobilized acceptor dye leads to emission in the red whenever there is sufficient overlap between the two dyes. Because of the highly exponential distance dependence in this process, only those dyes located at the apex of the AFM tip, nearest the sample, interact strongly. This limited and highly specific interaction provides a mechanism for obtaining fluorescence contrast with high spatial resolution. Initial results in which 400 nm resolution is obtained through this AFM/FRET imaging technique are reported. Future modifications in the probe design are discussed to further improve both the fluorescence resolution and imaging capabilities of this new technique.
In situ observation of the initial growth process of carbon nanotubes by time‐resolved high resolution transmission electron microscopy
Authors: Watanabe H. , Hisada Y. , Mukainakano S. , Tanaka N.
Published: March 2003

We succeeded in plan‐view dynamic observation of the initial formation process of carbon nanotubes from β‐SiC(1 1 1) surfaces by time‐resolved high resolution transmission electron microscopy. At 1360 °C, the flakes of graphite layers of a fibre orientation were formed on the SiC(1 1 1) surfaces. From the graphite layers, carbon nanotubes were formed perpendicular to the (1 1 1) plane of the SiC. A scanning tunnelling microscopy observation showed that the end of carbon nanotube was closed. These results indicate that the caps of the carbon nanotubes are formed by a lift of a part of the graphene along the [1 1 1] direction of the SiC through generation of pentagons and heptagons. Two types of carbon nanotube, single‐wall and double‐wall, were observed in plan‐view images. Different image intensity between an outer ring and an inner ring in double‐wall nanotubes suggests that the inner layers of multiwall nanotubes are formed after the outer ones.
Quantification of the graphical details of collagen fibrils in transmission electron micrographs
Authors: Xia Y. , Elder K.
Published: March 2003

A novel 2D image analysis technique is demonstrated. Using the digitized images of articular cartilage from transmission electron microscopy (TEM), this technique performs a localized ‘vector’ analysis at each region that is large enough to include several or tens of collagen fibrils but small enough to provide a fine resolution for the whole tissue. For each small and localized region, the morphology of the collagen fibrils can be characterized by three quantities essential to the nature of the tissue: the concentration of the fibrils, the overall orientation of the fibrils, and the anisotropy of the fibrils. This technique is capable of providing new insight to the existing technology by assigning quantitative attributes to the qualitative graphics. The assigned quantities are sensitive to the fine structure of the collagen matrix and meaningful in the architectural nature of the collagen matrix. These quantities could provide a critical linkage between the ultrastructure of the tissue and the macroscopic behaviours of the material. In addition, coarse‐graining the microscopic resolution of EM without compromising the essential features of the tissue's structure provides a direct view of the tissue's morphology and permits direct correlations and comparisons among interdisciplinary techniques.
Cylindrical diameters method for calibrating section thickness in serial electron microscopy
Authors: Fiala J. C. , Harris K. M.
Published: March 2003

An estimate of section thickness is required for measuring structures in serial section microscopy. Mean section thickness is estimated reliably by averaging the ratios of the diameters of cylindrical objects, such as mitochondria, to the number of sections they span. This cylindrical diameters method improves the accuracy of section thickness as inferred from the colour of sections floating in water. The cylindrical diameters method gives the same answer as that obtained by the minimal folds method. It is preferable because it can be done in a series that has no folds that can distort and obscure the objects that are being measured.
Radiation damage of water in environmental scanning electron microscopy
Authors: Royall C. P. , Thiel B. L. , Donald A. M.
Published: March 2003

Specimen damage from the electron beam poses a considerable problem with electron microscopy. This damage is particularly acute in environmental scanning electron microscopy (ESEM) for two reasons. Firstly, owing to its ability to stabilise insulating and hydrated specimens, ESEM lends itself to polymeric and biological materials that are typically highly beam‐sensitive. Secondly, water acts as a source of small, highly mobile free radicals, which accelerate specimen degradation.

By taking the results of single‐particle simulations of electron–water interactions, we determine the concentration of reactive species in a water specimen under ESEM conditions. We consider 12 species, which are produced in a Gaussian distribution, and annihilate according to a second‐order reaction scheme. Self‐diffusion along the concentration gradient is also modelled.

We find that the dominant reactive species is the hydroxyl (.OH) radical. Annihilation of this species is suppressed due to the lower concentration of reactants. The relatively stable hydrogen peroxide is also found at large concentrations. By comparing two beam energies, 5 and 25 keV, we find a drastic increase in the quantities of reactive species produced with beam energy. The longer range of 25 keV primary electrons spreads reactive species over a wider region, which then decay far more slowly.
Characterization of nitride thin films by electron backscatter diffraction
Authors: Trager‐Cowan C. , Sweeney F. , Hastie J. , Manson‐Smith S. K. , Cowan D. A. , McColl D. , Mohammed A. , O’Donnell K. P. , Zubia D. , Hersee S. D. , Foxon C. T. , Harrison I. , Novikov S. V.
Published: March 2003

Thin films incorporating GaN, InGaN and AlGaN are presently arousing considerable excitement because of their suitability for UV and visible light‐emitting diodes and laser diodes. However, because of the lattice mismatch between presently used substrates and epitaxial nitride thin films, the films are of variable quality.

In this paper we describe our preliminary studies of nitride thin films using electron backscattered diffraction (EBSD). We show that the EBSD technique may be used to reveal the relative orientation of an epitaxial thin film with respect to its substrate (a 90° rotation between a GaN epitaxial thin film and its sapphire substrate is observed) and to determine its tilt (a GaN thin film was found to be tilted by 13 ± 1° towards [1010]GaN), where the tilt is due to the inclination of the sapphire substrate (cut off‐axis by 10° from (0001)sapphire towards (1010)sapphire). We compare EBSD patterns obtained from As‐doped GaN films grown by plasma‐assisted molecular beam epitaxy (PA‐MBE) with low and high As4 flux, respectively. Higher As4 flux results in sharper, better defined patterns, this observation is consistent with the improved surface morphology observed in AFM studies. Finally, we show that more detail can be discerned in EBSD patterns from GaN thin films when samples are cooled.
Use of emission electron microscope for potential mapping in semiconductor microelectronics
Authors: Nepijko S. A. , Sedov N. N. , Schönhense G. , Escher M.
Published: March 2003

An emission electron microscope was used for visualization and measurement of the distribution of electric fields and potentials on the surface under study. The contrast of microfields is caused by the fact that slow‐moving electrons emitted from the object surface are deflected by these fields. The measurements were performed on a p‐n junction to which a voltage was applied. It is shown that the type of contrast from the p‐n junction can be reversed depending on the position of the contrast aperture restricting the electron beam. The same result was obtained by means of a computer simulation.
Video‐rate confocal endoscopy
Authors: Watson T. F. , Neil M. A. A. , Juškaitis R. , Cook R. J. , Wilson T.
Published: March 2003

Rigid endoscopes provide high quality optical images of reasonably accessible regions of the inner body, especially regions such as the aero‐digestive and genital tracts. In order to enhance the versatility of these instruments we describe a development that permits confocal endoscopic images to be obtained − along with traditional endoscopic images – in real‐time, from within the living patient. The system is based around a host lenslet‐array tandem scanning microscope, which is capable of producing images viewed directly by eye. These types of confocal microscope are configured for fluorescence imaging together with laser illumination. Hard and soft tissues in the mouth were imaged using this combined system.
Stereological estimation of tubular length
Authors: Gundersen H. J. G.
Published: March 2003

Very efficient and unbiased principles exist for estimating the total three‐dimensional (3D), two‐dimensional and zero‐dimensional amounts of arbitrary structure in 3D space. The total one‐dimensional length of real structure, in the ordinary sense, is an abstraction from the point of view of integral geometry. All stereological estimators of ‘tubular length’ are thus approximations. In addition, they are riddled by biases due to several types of artificial edges and other practical problems. This paper discusses several of these and proposes practical solutions of minimal biases.
Precision of circular systematic sampling
Authors: Cruz‐Orive L. M. , Gual‐Arnau X.
Published: March 2003

In design stereology, many estimators require isotropic orientation of a test probe relative to the object in order to attain unbiasedness. In such cases, systematic sampling of orientations becomes imperative on grounds of efficiency and practical applicability. For instance, the planar nucleator and the vertical rotator imply systematic sampling on the circle, whereas the Buffon–Steinhaus method to estimate curve length in the plane, or the vertical designs to estimate surface area and curve length, imply systematic sampling on the semicircle. This leads to the need for predicting the precision of systematic sampling on the circle and the semicircle from a single sample. There are two main prediction approaches, namely the classical one of G. Matheron for non‐necessarily periodic measurement functions, and a recent approach based on a global symmetric model of the covariogram, more specific for periodic measurement functions. The latter approach seems at least as satisfactory as the former for small sample sizes, and it is developed here incorporating local errors. Detailed examples illustrating common stereological tools are included.
Software techniques for EELS to realize about 0.3 eV energy resolution using 300 kV FEG‐TEM
Authors: Kimoto K. , Matsui Y.
Published: March 2003

In this short communication we describe some software techniques for electron energy‐loss spectrum measurement. We prepared DigitalMicrograph (Gatan) scripts for multiple spectrum acquisitions, quasi‐simultaneous acquisition of low‐loss and core‐loss spectra, energy drift correction, and other operations. Narrow zero‐loss spread of 0.27 eV is demonstrated using a 300 kV field‐emission transmission electron microscope (TEM) (Hitachi, HF‐3000) and a post‐column energy filter (Gatan, GIF2002). The core‐loss spectrum is acquired with an energy resolution of 0.36 eV with high reproducibility. The present software techniques effectively achieve the intrinsic energy resolution of electron sources. Sample scripts are provided in the Appendix.
A method of normalizing cathodoluminescence images of electron transparent foils for thickness contrast applied to InGaN quantum wells
Authors: Boyall N. M. , Durose K. , Watson I. M.
Published: March 2003

The uniformity of panchromatic cathodoluminescence (CL) from In0.09Ga0.91N/GaN quantum wells at 100 K was investigated using a combined transmission electron microscope–cathodoluminescence instrument. A technique for correcting CL images of electron‐transparent wedge specimens for thickness contrast artefacts is presented. The foil thickness was estimated using the dynamical formulation of the relationship between the thickness and the (experimentally observed) transmitted electron intensity. For a given thickness the CL intensity was calculated using the Everhart–Hoff depth‐dose function and also taking into account surface recombination losses. Experimental CL images were normalized by dividing by the calculated CL value at each point. The procedure was successful in calculating the underlying materials contrast in CL images of thin specimens of InGaN single quantum wells. Non‐uniformities in the CL emission on the scale of ∼0.7 µm were observed.
Serotonin and Carbachol Induced Suppression of Synaptic Excitability in Rat CA1 Hippocampal Area: Effects of Corticosteroid Receptor Activation
Authors: Hesen W. , Karten Y. J. G. , Van de witte S. V. , Jo M.
Published: March 2003

Serotonin (5HT) and the cholinergic analogue carbachol (CCh) act on neurons in the hippocampal CA1 area through pre‐ and post‐synaptic receptors. Previously, it was shown that post‐synaptic actions of 5HT and CCh are affected by corticosteroids: predominant activation of high affinity mineralocorticoid receptors resulted in small hyperpolarizing responses to 5HT and small depolarizing responses to CCh; additional activation of low affinity glucocorticoid receptors led to increased 5HT and CCh responses. In the present study, we examined the consequences of steroid modulation of these post‐synaptic membrane effects and/or possible pre‐synaptic effects by 5HT and CCh for the excitability in the CA1 area, using extracellular field potential or intracellular recordings from individual pyramidal neurons. Steroid treatment by itself did not affect the amplitude or paired pulse properties of synaptic responses. In slices from adrenally intact rats, both 5HT (3–30 μM) and CCh (1–10 μM) induced a dose‐dependent suppression of the synaptic field responses evoked in the CA1 area by stimulation of the Schaffer collaterals. No changes in these transmitter effects were observed after adrenalectomy. The 5HT induced suppression of the population spike amplitude was, however, reduced after selective occupation of mineralocorticoid receptors. Intracellularly, no significant steroid dependent modulation of (pre‐synaptic) 5HT evoked changes in synaptic responses was observed. These data suggest that the steroids modulate post‐synaptic but not pre‐synaptic 5HT effects and that this modulation is reflected in the excitability of the CA1 region. The CCh induced suppression of the population spike was not affected by corticosteroid receptor activation, indicating that the previously found steroid modulation of post‐synaptic CCh effects has no clear consequences for the CA1 excitability.
Multiple Hypothalamic Factors Regulate Pyroglutamyl Peptidase II in Cultures of Adenohypophyseal Cells: Role of the cAMP Pathway
Authors: Angel Vargas Miguel , Bourdais Julie , Sanchez Sonia , Uriostegui Bernardo , Moreno Eloy , Joseph‐Bravo Patricia , Charli Jean‐Louis
Published: March 2003

In the adenohypophysis, thyrotrophin‐releasing hormone (TRH) is inactivated by pyroglutamyl peptidase II (PPII), a TRH‐specific ectoenzyme localized in lactotrophs. TRH slowly downregulates surface PPII activity in adenohypophyseal cell cultures. Protein kinase C (PKC) activation mimics this effect. We tested the hypothesis that other hypothalamic factors controlling prolactin secretion could also regulate PPII activity in adenohypophyseal cell cultures. Incubation for 16 h with pituitary adenylate cyclase activator peptide 38 (PACAP; 10−6 M) decreased PPII activity. Bromocryptine (10−8 M), a D2 dopamine receptor agonist, or somatostatin (10−6 M) stimulated enzyme activity and blocked the inhibitory effect of [3‐Me‐His2]‐TRH, a TRH receptor agonist. Bromocryptine and somatostatin actions were suppressed by preincubation with pertussis toxin (400 ng ml−1). Because these hypophysiotropic factors transduce some of their effects using the cAMP pathway, we analysed its role on PPII regulation. Cholera toxin (400 ng ml−1) inhibited PPII activity. Forskolin (10−6 M) caused a time‐dependent decrease in PPII activity, with maximal inhibition at 12–16 h treatment; ED50 was 10−7 M. 3‐isobutyl‐1‐methylxanthine or dibutiryl cAMP, caused a dose‐dependent inhibition of PPII activity. These data suggest that increased cAMP down‐regulates PPII activity. The effect of PACAP was blocked by preincubation with H89 (10−6 M), a protein kinase A inhibitor, suggesting that the cAMP pathway mediates some of the effects of PACAP. Maximal effects of forskolin and 12‐O‐tetradecanoylphorbol 13‐acetate were additive. PPII activity, therefore, is independently regulated by the cAMP and PKC pathways. Because most treatments inhibited PPII mRNA levels similarly to PPII activity, an important level of control of PPII activity by these factors may be at the mRNA level. We suggest that PPII is subject to ‘homologous’ and ‘heterologous’ regulation by elements of the multifactorial system that controls prolactin secretion.
Electrochemical Stimulation of the Median Eminence Evokes FSH but not LH Release After LHRH Antagonist Treatment
Authors: Koppán Miklós , Kovács Magdolna , Mezö Imre , Flerkó Béla
Published: March 2003

Experimental data suggest that a follicle stimulating hormone‐releasing factor (FSH‐RF) distinct from luteinizing hormone‐releasing hormone (LHRH) exists. In the present study, we investigated, in short‐term ovariectomized (OVX) rats, whether FSH‐RF(s) can be released from nerve terminals by electrochemical stimulation (ECS) of the median eminence. To prevent the effect of LHRH liberated by ECS, 100 μg of a potent LHRH antagonist (MI‐1544) was administered to one group of OVX rats 60 min before ECS. Two groups of OVX rats were used as controls. One group was treated with the solvent of the LHRH antagonist 60 min before the ECS; the other group received sham‐ECS only. In‐vitro experiments using a hypothalamus‐pituitary coperifusion system were also performed to investigate the direct effect of ECS of the median eminence on LH and FSH release from pituitary cells. ECS in vivo induced 4.6‐fold (P<0.01) and 10.2‐fold (P<0.01) elevation of serum LH concentration, measured by RIA at 10 min and 60 min after ECS, respectively. Serum FSH concentrations increased 1.35‐fold at 10 min (P<0.01) and 1.50‐fold at 60 min (P<0.01) after ECS, compared with sham‐stimulated controls. Administration of LHRH antagonist attenuated the ECS‐induced release of LH by 44% at 10 min and prevented it entirely at 60 min after ECS. However, the ECS‐induced release of FSH was not modified by the antagonist at 10 min and was diminished by only 17% at 60 min after ECS, compared with solvent‐treated and stimulated controls. Immunohistological examination of the hypothalami showed that LHRH‐immunoreactivity was depleted in the region of ECS. In the study in vitro, substances released from the fragments of mediobasal hypothalami bearing ECS in the median eminence induced significant release of both LH and FSH, and the induced release of LH, but not FSH, was prevented by the LHRH antagonist. The present study suggests that FSH‐releasing factor(s) different from LHRH can be released from the median eminence and that a significant portion of FSH secretion is independent of the control of LHRH.
Ovarian Steroid Modulation of Neurokinin Contents in Hypothalamus, Pituitary, Trigeminal Nucleus, and Cervical Spinal Cord of the Ovariectomized Female Rat
Authors: Duval  , Lenoir  , Kerdelhue
Published: March 2003

Ovariectomized rats were treated with estradiol benzoate (EB) and progesterone in conditions known to negatively and positively regulate gonadotropin secretion. Injection with EB decreased the plasma concentration of substance P at the time of the positive feed‐back exerted by EB on gonadotropin secretion, while having no effect on the plasma concentration of neurokinin A. In the hypothalamus, EB injection enhanced the substance P and neurokinin A content, while progesterone reduced the substance P content. In the anterior pituitary, the substance P content was increased after progesterone, and this increase was blocked by EB. Conversely, in the posterior pituitary, the substance P content was reduced after progesterone, and this effect was enhanced by EB. In the trigeminal nucleus, the substance P content was increased after progesterone and EB, while only progesterone affected neurokinin A content. Finally, in the cervical spinal cord, the substance P and neurokinin A contents were reduced after EB. We conclude that neurokinin contents are controlled by ovarian steroids not only in the hypothalamo‐pituitary complex but also in the trigeminal nucleus and the cervical spinal cord.
A Study of the Gonadotropin Releasing Hormone Neuronal Network in the Median Eminence of the Rhesus Monkey (Macaca mulatta) Using a Post‐Embedding Immunolabelling Procedure
Authors: Durrant  , Plant
Published: March 2003

The purpose of this study was to describe the ultrastructural features of gonadotropin releasing hormone (GnRH) axonal processes in the median eminence of the monkey, using a post‐embedding immunogold labelling procedure. Evidence was also sought to evaluate the view that release of this peptide may be governed by direct inputs to GnRH axons in the median eminence. Plastic embedding was used to preserve ultrastructure, and a polyclonal rabbit anti‐GnRH was used as primary antibody. Immunogold labelling with 15‐nm particles was almost exclusively found overlying dense core vesicles (dcvs) and preabsorption of the primary antibody with synthetic GnRH eliminated this labelling. Morphometric analysis was performed on tissue from two monkeys. Four types of profiles containing GnRH immunoactive dcvs were observed. Type I profiles were morphologically unremarkable with a cross sectional area of approximately 0.6 μm2and probably represent intervaricose axon segments. Type II profiles, which were nominally larger than Type I structures, were characterized by a high density of round microvesicles, which were frequently concentrated along the neuronal membrane to form ‘synaptoid’ contacts with adjacent glia. Two additional and large GnRH profiles (>5 μm2) were observed. One (Type III) contained a high density of dcvs and mitochondria, and was considered analogous to an axonal swelling or Herring body in the magnocellular hypothalamo‐neurohypophysial system. The Type IV structure, which was considered not to be a Herring body because of the relative low density of mitochondria was innervated by a classical symmetrical synapse. The functional significance of these observations is discussed.
Effects of Chronic Stress on Hypothalamic lnterleukin‐1β, Interleukin‐2, and Gonadotrophin‐Releasing Hormone Gene Expression in Ovariectomized Rats
Authors: Tanebe  , Nishijo  , Muraguchi  , Ono
Published: March 2003

The influence of chronic stress on the expression of interleukin (IL)‐1β and IL‐2 mRNAs in ovariectomized rat brains, and the physiological consequences of the expression of these cytokines on hypothalamic‐pituitary‐gonadal (HPG) activity were investigated. Using polymerase chain reaction (PCR)‐assisted semiquantitative analysis, we demonstrated alterated expression of IL‐1β and IL‐2 mRNA during repeated cold stress; the expression of both IL‐β and IL‐2 mRNA increased in the medial preoptic area and ventromedial hypothalamus, and decreased in the lateral hypothalamic area. In the arcuate nucleus/median eminence, IL‐2 mRNA expression was dramatically decreased, in contrast to the increase in IL‐1β mRNA expression. Concomitant analysis of GnRH mRNA expression indicated significant suppression of GnRH synthesis in the chronic phase, and a strong negative correlation with cytokine expression in the medial preoptic area. Similar results were obtained in intact females exposed to this stress. These results, together with previous pharmacological studies, suggest that chronic stress may induce reproductive dysfunction through the effects of stress‐induced expression of endogenous cytokines.
Regulation of GABAA Receptor by Protein Tyrosine Kinases in Frog Pituitary Melanotrophs
Authors: Castel  , Louiset  , Anouar  , Le Foll  , Cazin  , Vaudry
Published: March 2003

The effects of protein tyrosine kinase (PTK) and PTK inhibitors on the GABAA receptor function were studied in cultured frog pituitary melanotrophs by using the patch‐clamp technique. Extracellular application of the PTK inhibitors genistein (10−9 to 10−5 M) or lavendustin A (10−12 to 10−7 M) provoked a bell‐shaped potentiation of the whole‐cell current induced by GABA (3×10−6 M). In contrast, at high concentrations, genistein (10−4 M) and lavendustin A (10−5 M) reversibly reduced the GABA‐evoked current. Daidzein and lavendustin B, the inactive analogs of genistein and lavendustin A, respectively, did not modify the current induced by GABA. In the inside‐out configuration, bath application of the recombinant PTK pp60c–src (75 U/ml) inhibited the GABA‐activated chloride current, and the inhibitory effect of pp60c–src was prevented by genistein (10−7 M). Immunoblotting revealed that genistein, at doses of 10−7 M or 10−4 M, markedly inhibited tyrosine phosphorylation of the β2/β3 subunits of the GABAA receptor. Extracellular application of the PKA activator Bt2cAMP (10−3 M), the PKA/PKC inhibitor H7 (10−5 M) and the Cam KII inhibitor W7 (10−5 M) reversibly diminished the whole‐cell GABA‐induced current. Internal application of H7 and W7 (10−4 M) did not modify the dose‐dependent effects of genistein. Internal application of sodium orthovanadate (10−4 M), a protein tyrosine phosphatase inhibitor, decreased the GABA‐evoked current and markedly reduced the potentiating effect of genistein. The present study provides the first evidence that, in frog pituitary melanotrophs, the GABAA receptor is phosphorylated at least on its β2/β3 subunits by an endogenous PTK. Our data also demonstrate that tyrosine phosphorylation exerts an inhibitory effect on GABAA receptor function.
In Vivo Modulation of Post‐Spike Excitability in Vasopressin Cells by κ‐Opioid Receptor Activation
Authors: Brown C. H. , Leng G.
Published: March 2003

An endogenous κ‐opioid agonist reduces the duration of phasic bursts in vasopressin cells. Non‐synaptic post‐spike depolarizing after‐potentials underlie activity during bursts by increasing post‐spike excitability and κ‐receptor activation reduces depolarizing after‐potential amplitude in vitro. To investigate the effects of κ‐opioids on post‐spike excitability in vivo, we analysed extracellular recordings of the spontaneous activity of identified supraoptic nucleus vasopressin cells in urethane‐anaesthetized rats infused with Ringer's solution (n = 17) or the κ‐agonist, U50,488H (2.5 µg/h at 0.5 µl/h; n = 23), into the supraoptic nucleus over 5 days. We plotted the mean hazard function for the interspike interval distributions as a measure of the post‐spike excitability of these cells. Following each spike, the probability of another spike firing in vasopressin cells recorded from U50,488H infused nuclei was markedly reduced compared to Ringer's treated vasopressin cells. To determine whether U50,488H could reduce post‐spike excitability in cells that displayed spontaneous phasic activity, we infused U50,488H (50 µg/h at 1 µl/h, i.c.v.), for 1–12 h while recording vasopressin cell activity. Nine of 10 vasopressin cells were silenced by i.c.v. U50,488H 15 ± 5 min into the infusion. Six cells exhibited spontaneous phasic activity before U50,488H infusion and recordings from three of these phasic cells were maintained until activity recovered; during U50,488H infusion, the activity of these three cells was irregular. Generation of the mean hazard function before and during U50,488H infusion revealed a reduction in post‐spike excitability during U50,488H infusion. Thus, κ‐receptor activation reduces post‐spike excitability in vivo; this may reflect inhibition of depolarizing after‐potentials and may thus underlie the reduction in burst duration of vasopressin cells caused by an endogenous κ‐agonist in vivo.