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At least 523 records · Page 29Linked to original sources
PubMed1996

Combined use of proacrosin immunocytochemistry and autosomal DNA in situ hybridisation for evaluation of human ejaculated germ cells.

The recently reported human pregnancies and births after fertilising oocytes with round spermatids recovered from the ejaculate of men with non-obstructive azoospermia have underscored the need for a more accurate evaluation of the nuclear and cytoplasmic maturation status of ejaculated germ cells. In this study we describe our first experience with a method combining the immunocytochemical visualisation of proacrosin with autosomal DNA fluorescence in situ hybridisation (FISH) to assess ejaculated germ cells from patients with a spermiogenesis defect. The proacrosin immunoreactivity, analysed with the use of the monoclonal antibody 4D4, has been detected in cells of round spermatid size presenting a haploid FISH figure as well as in larger cells whose ploidy corresponds to primary and secondary spermatocytes. These observations are in agreement with previously published results obtained, with the use of the same antibody, by immunocytochemical analysis of histological sections of testicular tissue. All the cells of round spermatid size possessing proacrosin immunoreactivity were found to be haploid by FISH. On the other hand, some of the haploid cells of round spermatid size did not possess proacrosin immunoreactivity. The structural pattern of proacrosin immunoreactivity was highly variable both in spermatids and in younger spermatogenic cells. These data show that cell size is the main criterion to be used for the identification of ejaculated round spermatids, whereas the presence of the developing acrosome represents only an auxiliary criterion. The scoring of acrosomal development in ejaculated spermatids may be useful as part of pre-treatment diagnosis before the inclusion of infertile couples in a spermatid conception programme.

Acrosin↗
PubMed1999

Stress induced changes in transmitter release from sympathetic varicosities of the mouse vas deferens.

Activation of the sympathetic nervous system is an important component of the response to stress, but the effects of prolonged stress on sympathetic neurotransmission have not been assessed. In the present study we have examined the effect of 3 to 10 days exposure to stress induced by frequent handling and sham injections on neurotransmitter release from sympathetic varicosities of the mouse vas deferens. DiOC2(5)-fluorescence was used to visualise the sympathetic varicosities so that extracellular electrodes could be placed over known numbers of varicosities to monitor transmitter release using electrophysiological techniques. The frequency of excitatory junction currents (EJCs) increased with increasing duration of exposure to stress. The mean and maximum EJC amplitude significantly increased by 107% and 43%, respectively after 10 days of exposure to stress. The density of sympathetic varicosities innervating smooth muscle of the mouse vas deferens was not changed throughout the duration of the exposure to stress. The findings from this study demonstrate that the efficacy of transmitter release from the sympathetic varicosities is altered by repeated exposure of mice to stressful stimuli, such as handling and sham injections. Since such procedures are routine in many pharmacological experiments, it is important that investigators are aware of these changes so that due consideration is given when interpreting the data obtained from animals treated in this way.

Animals↗
PubMed2003

Purinergic signalling in the medullary mechanisms of respiratory control in the rat: respiratory neurones express the P2X2 receptor subunit.

ATP is involved in central respiratory control and may mediate changes in the activity of medullary respiratory neurones during hypercapnia, thus playing an important role in central chemoreception. The main objective of this study was to explore further the role of ATP-mediated signalling in respiratory control and central chemoreception by characterising the profile of the P2X receptors expressed by physiologically identified respiratory neurones. In particular we determined whether respiratory neurones in the rostral ventrolateral medulla (VLM) express P2X2 receptor subunits of the ATP-gated ion channel, since ATP currents evoked at recombinant P2X2 receptors are potentiated by lowering extracellular pH. Experiments were performed on anaesthetised (pentobarbitone sodium 60 mg kg-1 I.P., then 10 mg kg-1 I.V. as required), gallamine-triethiodide-treated (10 mg kg-1 I.V., then 2-4 mg kg-1 h-1 I.V.) and artificially ventilated rats. The VLM respiratory neurones were classified according to the timing of their discharge pattern in relation to that of the phrenic nerve and by the exclusion of pump cells from the study population; these were labelled with Neurobiotin using the juxtacellular method, and visualised with fluorescence microscopy. It was found that a substantial proportion of the VLM respiratory neurones express the P2X2 receptor subunit. P2X2 receptor subunit immunoreactivity was detected in approximately 50 % (six out of 12) of expiratory neurones and in approximately 20 % (two out of 11) of neurones with inspiratory-related discharge (pre-inspiratory and inspiratory). In contrast, no Neurobiotin-labelled VLM respiratory neurones (n = 19) were detectably immunoreactive for the P2X1 receptor subunit. Microionophoretic application of ATP (0.2 M, 20-80 nA for 40 s) increased the activity of approximately 80 % (13 out of 16) of expiratory neurones and of approximately 30 % (five out of 18) of VLM neurones with inspiratory-related discharge. These effects were abolished by the P2 receptor blocker suramin (0.02 M, 80 nA), which also reduced the baseline firing in some expiratory neurones. These data indicate that modulation of P2X2 receptor function, such as that evoked by acidification of the extracellular environment during hypercapnia, may contribute to the changes in activity of the VLM respiratory neurones that express these receptors.

Adenosine Triphosphate↗
PubMed1992

Multiple mitochondrial DNA deletions in an elderly human individual.

We have used the polymerase chain reaction (PCR) to study deletions in the mitochondrial DNA (mtDNA) of an elderly human individual. An extended set of PCR primers has been utilised to identify 10 mitochondrial DNA deletions in a 69-year-old female subject with no known mitochondrial disease. The particular deletions visualised as PCR products depended on the primer pairs used, such that the more distantly separated PCR primers enabled visualisation of larger deletions. Some deletions were common to the heart, brain and skeletal muscle, whereas others were apparently specific to individual tissues. DNA sequencing analysis of PCR products showed that short direct repeat sequences (5 to 13 bp) flanked all deletion breakpoints; in most cases one copy of the repeat was deleted. It is proposed that the accumulation of such multiple deletions is a general phenomenon during the ageing process.

Aged↗
PubMed2002

Echo-planar magnetic resonance imaging of Gaviscon alginate rafts in-vivo.

Liquid Gaviscon and Gaviscon Advance are established reflux suppressant formulations. This study describes the use of echo-planar magnetic resonance imaging (EPI) to visualise non-invasively intragastric alginate rafts of Liquid Gaviscon and Gaviscon Advance in healthy subjects. Secondly, the feasibility of using relaxation rate (T2(-1)) measurements to monitor changes in the physicochemical properties of the rafts in-vivo is evaluated. Six subjects ingested 500 mL of a liquid meal and received a single dose of 20 mL Liquid Gaviscon or 10 mL Gaviscon Advance on 2 separate visits each and were imaged every 15 min. An alginate raft was observed in the stomach for all subjects and both treatments. The raft was observed to consist of a few large fragments on the majority of the scans for both products. At t = 60 min a raft was still present in all cases. Three-dimensional volume reconstructions showed, for the first time, the spatial distribution of the rafts within the gastric lumen. The T2(-1) data showed potential for assessment of dynamic changes in the physicochemical properties of the alginate rafts in-vivo. We conclude that EPI shows great potential in assessing alginate rafts formation in-vivo.

Adult↗
PubMed1992

Natural course of Crohn's disease after ileocolic resection: endoscopically visualised ileal ulcers preceding symptoms.

Forty two Crohn's disease patients were followed up after ileocolic resection with regard to symptoms and endoscopic appearance of the ileocolic anastomosis. Twenty eight patients resected because of colonic neoplasm served as controls. In all the Crohn's disease patients the ileal resection margin was disease free macroscopically at operation. In addition, intraoperative ileoscopy was performed in 13 and no sign of residual inflammation in the neoterminal ileum was seen. Endoscopy soon after surgery often showed preanastomotic ileal ulceration before symptoms appeared, whereas no anastomotic lesions were observed in the controls. Thus, 22 of 30 Crohn's disease patients examined had ulceration of the anastomotic area after three months, but only 10 had developed symptoms indicating relapse (73 v 33%). Corresponding figures in the 30 patients examined after one year were 93 v 37%, and in 14 patients after three years they were 100 and 86% respectively. The inflammatory lesions in all cases were preanastomotic, in the neoterminal ileum, and showed time related progression from aphthae to larger ulcers and stricture. The study suggests that endoscopically observed inflammatory lesions that appear soon after ileocolic resection for Crohn's disease signify new inflammation and not residual, persistent disease or incomplete anastomotic healing. The data further suggest that despite clinical remission after apparently radical intestinal resection, the bowel is permanently inflamed in Crohn's disease.

Adolescent↗
PubMed1999

CT data sets surface extraction for biomechanical modeling of long bones.

In modelling applications such as custom-made implants design is useful to have a surface representation of the anatomy of bones rather than the voxel-based representation generated by tomography systems. A voxel-to-surface conversion process is usually done by a 2D segmentation of the images stack. However, other methods allow a direct 3D segmentation of the CT or MRI data set. In the present work, two of these methods, namely the Standard Marching Cube (SMC) and the Discretized Marching Cube (DMC) algorithms, were compared in terms of local accuracy when used to reconstruct the geometry of a human femur. The SMC method was found to be more accurate than the DMC method. The SMC method was capable of reconstructing the inner and outer geometry of a human femur with a peak error lower than 0.9 mm and an average error comparable to the pixel size (0.3 mm). However, the large number of triangles generated by the algorithm may limit its adoption in many modelling applications. The peak error of the DMC algorithm was 1.6 mm but it produced approximately 70% less triangles than the SMC method. From the results of this study, it may be concluded that three dimensional segmentation algorithms are useful not only in visualisation applications but also in the creation of geometry models.

Algorithms↗
PubMed2003

ER quality control can lead to retrograde transport from the ER lumen to the cytosol and the nucleoplasm in plants.

Quality control in the secretory pathway is a fundamental step in preventing deleterious effects that may arise by the release of malfolded proteins into the cell or apoplast. Our aims were to visualise and analyse the disposal route followed by aberrant proteins within a plant cell in vivo using fluorescent protein technology. A green fluorescent protein (GFP) fusion was detected in the cytosol and the nucleoplasm in spite of the presence of an N-terminal secretory signal peptide. In contrast to secreted GFP, the fusion protein was retained in the cells where it was degraded slowly, albeit at a rate much higher than that of the endoplasmic reticulum (ER)-retained derivative GFP-HDEL. The fusion protein could not be stabilised by inhibitors of transport or the cytosolic proteasome. However, the protein is a strong lumenal binding protein (BiP) ligand. Complete signal peptide processing even after long-term expression in virus-infected leaves rules out the possibility that the documented accumulation in the cytosol and nucleoplasm is because of the bypassing of the translocation pores. The data are consistent with the hypothesis that the fusion protein is disposed off from the ER via a retrograde translocation back to the cytosol. Moreover, accumulation in the nucleoplasm was shown to be microtubule dependent unlike the well-documented diffusion of cytosolically expressed GFP into the nucleoplasm. The apparent active transport of the GFP fusion into the nucleoplasm may indicate an as yet undiscovered feature of the ER-associated degradation (ERAD) pathway and explain the insensitivity to degradation by proteasome inhibitors.

Biological Transport↗
PubMed1987

Cross sectional echocardiographic assessment of great artery diameters in infants and children.

The pulmonary trunk and aortic root were measured on cross sectional echocardiograms in 173 normal subjects aged from one day to 15 years. Fifteen neonates were reexamined 3-6 days later. The great vessels were visualised in the parasternal long axis and short axis views. All measurements were made in end diastole and end systole by the leading edge method. The internal diameter (inner surface to inner surface) of the pulmonary trunk was also measured. The diameters of the great vessels correlated best with the square root of body surface area. Individual variability in cardiac growth gave a wide scatter of normal values. This was controlled for by calculating the ratio of the pulmonary trunk to aortic root for each subject. This ratio showed little individual variability and, except for the neonatal period, was remarkably constant throughout infancy and childhood (1.06 (0.06)). In the first 24 hours of life the ratio of the pulmonary trunk to the aortic root was significantly larger (1.29 (0.12)) but within one week it decreased to the "normal" ratio found in the older age groups. These normal data should be useful in assessing patients with congenital heart disease, particularly those in whom pulmonary blood flow is abnormal.

Adolescent↗
PubMed1988

Dual-label autoradiographic analysis of human skin fibroblast and myoblast proteins by two-dimensional polyacrylamide gel electrophoresis using immobilised pH gradients in the first dimension.

Horizontal two-dimensional polyacrylamide gel electrophoresis with immobilised pH gradients in the first dimension has been applied to the analysis of human skin fibroblast and muscle myoblast total cell proteins. Excellent two-dimensional separations of skin fibroblast proteins were obtained using pH 4-10 immobilised pH gradient gels with a long interelectrode distance (16 cm), but resolution was degraded, particularly of the more acidic proteins, by the use of shorter (10 cm) gels. Improved resolution of acidic and basic proteins was obtained using separate pH 4-7 and pH 7-10 immobilised pH gradient gels respectively in the first dimension. Two-dimensional protein maps of skin fibroblast proteins were visualised both by silver staining and by autoradiography of samples labelled synthetically with [35S]methionine. Horizontal two-dimensional electrophoresis, using pH 4-7 and pH 7-10 immobilised pH gradient gels in the first dimension, was applied to the analysis of protein samples from skin fibroblasts and muscle myoblasts dual-labelled synthetically with [35S]methionine and [75Se]selenomethionine in an attempt to identify sets of proteins specific to each cell type. In addition, two-dimensional maps or protein samples derived from normal individuals and patients with Duchenne muscular dystrophy were compared to search for protein changes associated with the disease state. Although sets of qualitative protein spot differences were observed by visual inspection of the two-dimensional gels, more rigorous qualitative and quantitative analysis of the patterns using a computerised analysis system will be required to obtain the maximum amount of information from these data.

Autoradiography↗
PubMed1993

Specific atrial overexpression of G protein coupled human beta 1 adrenoceptors in transgenic mice.

OBJECTIVE: The aim was to develop a transgenic mouse model of atrial beta 1 adrenoceptor overexpression in order to create atrial alteration of the receptor transduction system. METHODS: Transgenic founders were generated after microinjection of the transgene construct into the pronucleus of fertilised mouse eggs. Heterozygous progeny were screened for RNA expression of the human beta 1 adrenoceptor gene under the control of a 0.56 kb proximal promoter of the human atrial natriuretic factor. One line, out of the three obtained, was selected and further characterised for overexpression of the human beta 1 adrenoceptor. Polymerase chain reaction was employed to detect beta 1 adrenoceptor mRNA, and 125I-cyanopindolol (ICYP) binding assays were used to quantify receptors in heart membranes. A quantitative autoradiographic ICYP binding technique was also used to visualise atrial and ventricular beta adrenoceptors in heart sections. RESULTS: The human beta 1 adrenoceptor was overexpressed specifically in the atria of transgenic mice. The level of the beta 1 adrenoceptor was 5-10-fold higher in transgenic mice compared to basal murine beta 1 adrenoceptors in non-transgenic control mice. Left and right atrial receptor overexpression was confirmed by in vitro autoradiography. The human receptors were able to couple to the murine stimulatory G proteins (Gs), as shown by high affinity binding site dosage using the beta adrenoceptor agonist isoprenaline. Isoprenaline displacement studies allowed the determination of two different affinity sites, one of high affinity (KH = 5.8 nM), and one of low affinity (KL = 520 nM). When expressed in terms of protein density (fmol.mg-1), atrial transgenic beta 1 adrenoceptors displayed a threefold increase in high affinity sites (KH) as compared to control mice. Preliminary electrocardiographic data showed supraventricular premature beats in 6/14 transgenic mice v 2/16 control mice. CONCLUSIONS: These transgenic mice may provide a useful pharmacological tool to investigate the pathophysiological consequences of the overactivation of atrial beta 1 adrenoceptor-adenylyl cyclase signalling system.

Animals↗
PubMed1988

The major protein of bull seminal plasma: biosynthesis and biological function.

We isolated the major protein of apparent Mr of 15,000-16,000 from seminal plasma as well as from seminal vesicle secretion of bull and proved by amino acid analysis and tryptic peptide mapping that the two proteins were identical. An antiserum against this major protein was employed to quantitate and identify the major protein in seminal plasma as well as seminal vesicle secretion. The antiserum did not cross-react with proteins from bovine or human plasma or follicular fluid respectively. Cell-free translation of poly(A)RNA from seminal vesicle tissue and immunoprecipitation yielded one major species with apparent Mr of 18,000. Using the anti-major protein antiserum, this major species was specifically immuno absorbed. Cloning and sequencing of a major protein-specific cDNA led to the identification of clone pMP17, encoding a precursor of the major protein of 128 amino acid residues. We proved that the major protein is identical to protein PDC 109 (Esch et al., Biochem. Biophys. Res. Comm. 113:861-867, 1983). The seminal vesicles synthesize major protein in an androgen-dependent fashion. In addition to intraluminal secretion of the vas deferens, ampullary spermatozoa revealed an intense immunoreaction which was restricted to the neck region of the sperm head and the middle piece, while the principal piece of the tail as well as the sperm head were devoid of immunoreactive material. Epididymal epithelium (as well as calf seminal vesicle epithelium) showed no immunoreactivity with major protein antiserum. Immunoelectron microscopy demonstrated that only spermatozoa devoid of a plasma membrane around the middle piece were able to bind the antiserum against major protein. After removal of the plasma membrane from epididymal spermatozoa, binding of major protein to subplasmalemmal binding sites was visualised using gold-labeled MP. Transblotting with gold-labeled MP demonstrated a protein of about 66 kDa which appears to represent the major protein-receptor. Binding of major protein to the receptor (after loss of the plasma membrane in the mid-piece region of the spermatozoa after contact with secretions from seminal vesicles) is interpreted as a physiological process presumably related to the onset of sperm motility.

Amino Acid Sequence↗
PubMed2000

Isolation and cloning of Streptomyces terminal fragments.

Streptomyces species have a linear chromosome of approximately 8 Mb in size. Many strains also carry linear plasmids. Most of these linear elements contain terminal proteins covalently bound to the 5' ends of the DNA. Using a method for the visualisation of terminal DNA fragments in agarose gels, it was possible to see three fragments in S. rimosus and five fragments in S. avermitilis. The method was also used to clone the 298 bp BamHI fragment carrying the left end of plasmid SLP2. Analysis of the sequence showed that the end resembled other Streptomyces chromosome and plasmid ends, but there were eight palindromes (instead of seven) and a tandem duplication of a 14 bp sequence.

Base Sequence↗
PubMed1995

Correlation between functional, radiological and anatomical abnormalities in upper airway obstruction (UAO) due to tracheal stenosis.

Few data are available on the relative sensitivity of different lung function tests in upper airway obstruction (UAO) and on the correlation of these tests with radiological estimations of tracheal stenosis. This paper is based on patients with a goiter or tracheal tumor and in healthy subjects breathing through rigid resistors, as well as on literature data. Lung function abnormalities present a specific pattern in UAO: this is characterised by a typical shape of the maximal flow-volume curve, a marked reduction in peakflow (PEF) and only minor changes in routine spirometry (e.g. FEV1, which thus tends to underestimate the stenosis). The clinical relevance of the lung function abnormalities in UAO can be estimated from their relationship with the exercise limitation: a moderate exercise limitation to about 60% pred is found if PEF is reduced to about 40% pred, and Raw is increased to about 600% pred (in which instance FEV1 is still 75% pred). This limitation grossly corresponds to a UAO of about 6 mm diameter. No good correlation is found between lung function abnormalities and the radiological stenosis (CT-scan or X-ray): CT-scan or X-ray are only moderately correlated with each other. Moreover, stenosis can be visualised through X-ray in only about 60% of the cases.

Adult↗
PubMed1994

Molecular dynamics simulations of oligonucleotides in solution: visualization of intrinsic curvature.

We have undertaken molecular dynamics simulations on the d(CGCAAAAAAGCG).d(CGCTTTTTTGCG) dodecamer in solution. In this study, we focus on aspects of conformation and dynamics, including the possibility of cross-strand hydrogen bonds. We compare our results with those from crystallography as well as infrared, Raman and NMR spectroscopy and cyclization kinetics. Our method of analysis allows us to visualise the curvature of the helix as a function of time during the simulation. We find that the major distortions of the helix axis path occur at the junctions between the (essentially straight) A-tract and the CG- and GC-tracts, although at one junction this is due to hyperflexibility (i.e., regions of high flexibility with no preferred direction of curvature), while at the other junction a static curvature is found (i.e., a preferred, sustained direction of curvature).

Base Sequence↗
PubMed2000

Real-time RT-PCR fluorescent detection of tomato spotted wilt virus.

A real-time reverse transcription-polymerase chain reaction assay based on TaqMan chemistry was developed for the detection and quantification of tomato spotted wilt virus (TSWV). This method enabled sensitive, reproducible and specific detection of TSWV in 'leaf soak' and total RNA extracts from infected plants. TaqMan reliably detected TSWV in as little as 500 fg total RNA. The assay was 10-fold more sensitive than visualisation of ethidium bromide-stained bands following agarose gel electrophoresis. TSWV isolates from various crops and locations were detected with a cycle threshold of 20-26 in 1 ng total RNA extracted from fresh or freeze-dried leaves. A dilution series of in vitro transcripts from a cloned 628 base pair fragment of TSWV S RNA served as standard for quantification of viral template in infected leaf samples. The TaqMan assay detected reproducibly 1000 molecules of the target transcript.

Base Sequence↗
PubMedDate not supplied

Investigating extended regulatory regions of genomic DNA sequences.

MOTIVATION: Despite the growing volume of data on primary nucleotide sequences, the regulatory regions remain a major puzzle with regard to their function. Numerous recognising programs considering a diversity of properties of regulatory regions have been developed. The system proposed here allows the specific contextual, conformational and physico-chemical properties to be revealed based on analysis of extended DNA regions. RESULTS: The Internet-accessible computer system RegScan, designed to analyse the extended regulatory regions of eukaryotic genes, has been developed. The computer system comprises the following software: (i) programs for classification dividing a set of promoters into TATA-containing and TATA-less promoters and promoters with and without CpG islands; (ii) programs for constructing (a) nucleotide frequency profiles, (b) sequence complexity profiles and (c) profiles of conformational and physico-chemical properties; (iii) the program for constructing the sets of degenerate oligonucleotide motifs of a specified length; and (iv) the program searching for and visualising repeats in nucleotide sequences. The system has allowed us to demonstrate the following characteristic patterns of vertebrate promoter regions: the TATA box region is flanked by regions with an increased G+C content and increased bending stiffness, the TATA box content is asymmetric and promoter regions are saturated with both direct and inverted repeats. AVAILABILITY: The computer system RegScan is available via the Internet at http://www.mgs.bionet.nsc. ru/Systems/RegScan, http://www.cbil.upenn.edu/mgs/systems/r egscan/.

Algorithms↗
PubMed2003

Depletion of the highly abundant protein albumin from human plasma using the Gradiflow.

Analysis of complex protein samples by two-dimensional electrophoresis (2-DE) is often more difficult in the presence of a few predominant proteins. In plasma, proteins such as albumin mask proteins of lower abundance, as well as significantly limiting the amount of protein that can be loaded onto the immobilized pH gradient strip. In this paper the Gradiflow, a preparative electrophoresis system, has been used to deplete human plasma of the highly abundant protein albumin under native and denatured conditions. A three step protocol incorporating a charge separation to collect proteins with an isoelectric point greater than albumin and two size separations to isolate proteins larger and smaller than albumin, was used. When the albumin depleted fractions were analysed on pH 3-10 2-DE gels, proteins that were masked by albumin were revealed and proteins not seen in the unfractionated plasma sample were visualised. Matrix-assisted laser desorption/ionisation-time of flight mass spectrometry analysis confirmed the identification of the protein that lies beneath albumin to be C4B-binding protein alpha chain. The liquid fractions from the Gradiflow separations were also analysed by liquid chromatography-tandem mass spectrometry to confirm the proteins were separated according to their size and charge mobility in an electric field.

Albumins↗