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PubMedDate not supplied

Direct diagnosis of carriers of Duchenne and Becker muscular dystrophy by amplification of lymphocyte RNA.

Rapid detection of deletion and duplication mutations that cause Duchenne and Becker muscular dystrophy was achieved in patients and carriers after amplification of small amounts of mRNA from peripheral blood lymphocytes. The entire coding region of the dystrophin mRNA was amplified in 10 sections by reverse transcription and nested polymerase chain reaction, and the products were directly visualised on acrylamide minigels with ethidium staining. Major structural gene mutations were identified by the appearance of a band of different size to that of the wild type. The altered band was readily detected in all patients and heterozygous relatives. This non-radioactive test of venous blood samples can be used for unambiguous and rapid identification of virtually all carriers of deletions or insertions within the dystrophin gene.

Base Sequence↗
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↗
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↗
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↗
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↗
PubMed1986

Regulatory peptides of the gastrointestinal and respiratory tracts.

The gastrointestinal and respiratory tracts contain numerous regulatory peptides produced by and released from specialised epithelial cells and the organ innervation. This complex system of endocrine cells and nerves is generally called "the diffuse neuroendocrine system". Markers are now available which permit the visualisation of the diffuse neuroendocrine system or its individual components. These include antibodies to neuron-specific enolase, chromogranin, neurofilament triplet proteins, the brain protein S100 and antibodies to a variety of regulatory peptides. Peptides present in the gut and lung innervation include: vasoactive intestinal polypeptide (VIP), peptide histidine isoleucine (PHI), galanin, substance P, calcitonin gene-related peptide (CGRP), neuropeptide tyrosine (NPY), somatostatin and cholecystokinin (the latter two are also localised to endocrine cells of the gut). Bombesin-immunoreactivity is found in nerves in the gut and in endocrine cells of the foetal/neonatal lung. Neuropeptides of the gut and lung originate either from local neurons (e.g. VIP, PHI, galanin) or extrinsic neurons localised in sensory ganglia (e.g. substance P and CGRP) or the sympathetic chain (e.g. NPY). Recent studies point to the involvement of regulatory peptides in diseases of the gut and lung. These, together with detailed distribution studies, provide supportive data on the putative role of the peptides in the control of normal bowel and respiratory functions. The gastrointestinal and respiratory tracts were within the systems investigated by Feyrter during his original observations on the existence of specialised epithelial cells with a putative regulatory function (Feyrter, 1938). These "endocrine/paracrine" cells were found to be scattered in epithelial organs throughout the body. In fact, endocrine cells of the respiratory tract are frequently referred to as "Feyrter's cells". The term "regulatory peptides" was introduced as a generic term (Polak and Bloom, 1983) after the finding that active peptides are produced both by cells of the diffuse endocrine or APUD (amine precursor uptake and decarboxylation) system (Pearse, 1983) and autonomic/sensory nerves. These peptides are released into the circulation from endocrine cells or locally from nerve terminals or paracrine cells. The concept of "gut/brain" peptides was dispelled after the findings that the respiratory tract was provided abundantly with numerous active peptides produced by and released from mucosal endocrine cells and/or the innervation.

Animals↗
PubMed1995

How the horse moves: 1. Significance of graphical representations of equine forelimb kinematics.

The kinematics of 24 two-year-old Dutch Warmblood horses were recorded at the trot (4 m/s) on a high-speed treadmill to study the coordination of joints within the equine forelimb. Joint angle-time, angle-angle, stick, and marker diagrams were used to show forelimb motion graphically. Because the kinematic data referred to the joint angles of the horse standing squarely and were time-standardised to the duration of the stride cycle, mean joint curves could be calculated for the total group. The motion of each segment in the equine forelimb during a complete stride is described and its function in intralimb coordination evaluated. It appeared that the rotation of the scapula and the cranio-caudal movement of the distal forelimb are synchronous and pendular. The carpal joint rapidly snaps into overextension at the beginning of the stance phase to enable the forelimb to work as a propulsive strut. The fetlock joint acts as an elastic spring, thereby conserving energy and, at the same time, absorbs oscillations generated by initial ground contact. Furthermore, the coordination between carpal and fetlock joints in the swing phase appears to be strongly influenced by inertia. Using the graphic tools evaluated in this paper, we were able to visualise the kinematics of the equine forelimb and relate these to specific functions of the forelimb in locomotion. This information can be used to select kinematic variables for clinical studies in which equine forelimb function has to be described and quantified.

Animals↗
PubMed1995

Multiplex PCR for identifying mycobacterial isolates.

AIMS: To develop a multiplex polymerase chain reaction (PCR) method to facilitate identification of mycobacterial isolates. METHODS: Type strains of 14 species of mycobacteria and 56 clinical isolates were lysed by boiling in TE Triton. The lysate (5 microliters) was used directly in a PCR reaction incorporating three pairs of PCR primers expected to amplify fragments from the genome of (a) all mycobacteria, (b) Mycobacterium tuberculosis complex only and (c) M avium only. PCR products were visualised by electrophoresis on agarose gels. RESULTS: Multiplex PCR applied to 14 type strains yielded patterns on electrophoresis which permitted identification of the mycobacterial isolates as M tuberculosis complex, M avium or as mycobacteria other than the former. The identification of 56 clinical isolates by multiplex PCR was consistent with other methods and was accomplished in less than one working day. CONCLUSIONS: This method may facilitate rapid and convenient identification of most clinical isolates of mycobacteria by PCR and gel electrophoresis. Further evaluation is warranted.

Base Sequence↗
PubMed1994

Consideration of time-dose-patterns in 3D treatment planning. An approach towards 4D treatment planning.

PURPOSE: The rendering of the 3D dose distribution together with anatomical information and the volumes of interest (VoI) is essential to get a visual impression of the treatment plan and to find modifications for the optimization of the dose distribution. The integration of biological effects into the 3D treatment planning is of interest for the assessment of different time-dose patterns. MATERIALS AND METHODS: One way of taking into account biological data is to relate the physical dose in critical structures to the corresponding tolerance dose. For that purpose the applied time-dose pattern has to be converted into the standard fractionation scheme being the basis of the tolerance dose. Generally any model can be used for these calculations. Here a modified incomplete repair model is used to calculate the relative biological dose distribution (RBD). The visualization of these biologically isoeffective dose distributions can be performed in the same manner as the physical dose so that the physical and biological dose distributions can by displayed side by side. As this is equivalent to introducing the time as a fourth dimension into 3D treatment planning this is called 4D treatment planning. RESULTS: From 3D dose matrices the biologically isoeffective dose distributions are calculated for the organs at risk. The changes introduced by different time-dose patterns are displayed using the same technique as for rendering 3D treatment plans. The visualisation of the three-dimensional biological dose distributions is shown by means of a patient with an oesophagus carcinoma. The RBD related to the tolerance dose of the organs at risk is displayed for different time-dose fractionations. CONCLUSION: The RBD distribution on a 3D treatment plan can be displayed in the same mode as the physical dose distribution. This offers additionally valuable information in a 3D treatment planning process about the dose to critical organs and the influence of different time-dose patterns.

Color↗
PubMed1992

Immunoperoxidase labelling of albumin at the endothelial cell surface of frog mesenteric microvessels.

Albumin was visualised at the endothelial cell surface of perfused frog mesenteric microvessels using immuno-peroxidase labelling. Vessels in the mesenteries of pithed frogs were washed free of blood and then perfused with frog Ringer solutions containing bovine serum albumin (BSA) at a concentration of 20 mg BSA ml-1, followed by a brief Ringer flush to remove excess albumin from the vessel lumen. The tissues were fixed in 1% glutaraldehyde and a double antibody labelling technique used to identify albumin within the tissue. A dense layer of peroxidase reaction product was seen, which extended 25-50 nm into the vessel lumen. It appeared as a continuous layer lining the luminal openings of interendothelial cell clefts and vesicles open to the luminal cell surface. In some vessels a more irregular layer of peroxidase labelled albumin was seen extending 150 to 200 nm into the vessel lumen, whilst in others clumps of peroxidase labelled albumin were also seen within the vessel lumen. These data offer direct evidence that BSA does interact with the endothelial cell surface of perfused frog mesenteric microvessels but suggest that a proportion is loosley or non-specifically bound to the cell surface and can be removed by a brief Ringer flush. The remainder appears more tightly bound and resistant to Ringer flush.

Animals↗
PubMed1995

[2-dimensional mapping and retinal and papillary microcirculation using scanning laser Doppler flowmetry].

PURPOSE: To present clinical applications of a new non-invasive method imaging in a high-definition the topography of perfused retinal vessels. METHOD: By a combination of a laser Doppler flowmeter with a scanning laser system the perfusion of the retina and the optic nerve head is visualized and quantified. The principles of measuring blood flow by Laser Doppler Flowmetry are based on the optical Doppler effect: laser light scattered by a moving particle is shifted in frequency by an amount delta f. Our data acquisition and evaluation system is a modified laser scanning tomograph. The technical data are: retinal area of measurement 2.7 mm x 0.7 mm, 10 degree-field with 256 points x 64 lines, measurement accuracy 10 microns, wavelength 670 nm and 790 nm, light power 100 microW, data acquisition time 2,048 s. Every line is scanned 128 times by a line-sampling rate of 4,000 Hz. By performing a discrete Fast Fourier Transformation over 128 intensities of each retinal point the laser Doppler-shift is calculated for each retinal point. With these data a 2-D map with 256 x 64 points of the retinal perfusion is created. The brightness of the picture-point is coded by the value of the Doppler shift. We estimated the reliability and the validity of the method. Perfusion-pictures of the superficial retinal layer and in the optic nerve head were presented. RESULTS: The reliability-coefficients r1 of "Flow", "Volume" and "Velocity" were 0.85, 0.83, and 0.85 respectively. The blood flow measurements by the presented method ("Scanning Laser Doppler Flowmetry") in an artificial capillary gave a linear relationship (r-value 0.973, p < 0.00001) between defined blood velocities and the measured blood flow. By the confocal technique, dependent on the focus, capillaries of the retinal superficial vasculature of the optic nerve head became visible with a high resolution. Off line the blood flow of areas of 110 microns x 110 microns were calculated in terms of laser Doppler flowmetry. CONCLUSION: "Scanning Laser Doppler Flowmetry" facilitates the visualisation of perfused retinal capillaries and vessels in high resolution. The representation of the function of the retinal circulation by SLDF leads to an image similar to the anatomical situation. The 2-dimensional mapping of local blood flow leads to a physiological picture of the retinal perfusion with visible vessels and capillaries.

Adult↗
PubMed1995

Theileria annulata sporozoite antigen fused to hepatitis B core antigen used in a vaccination trial.

A C terminal fragment (SR1) of SPAG-1, a sporozoite surface antigen of Theileria annulata, has been expressed as a fusion protein in the e1 loop of hepatitis B core antigen (HBcAg). This recombinant antigen (HBcAg-SR1) is produced in the form of self-assembling polyhedral particles which have been visualised under the electron microscope. Cattle immunised with HBcAg-SR1 produced high titres of neutralising antibodies. A significant T cell response to both the HBcAg and SR1 determinants was observed but evidence of a T suppressor determinant in SR1 was also revealed. Immunised cattle showed some evidence of protection to sporozoite challenge as assessed by severity of the disease. The significance of these findings for the development of a sub-unit vaccine against T. annulata is discussed.

Animals↗
PubMed1994

Visualisation by electron microscopy of the unique part of the cytoplasmic domain of a desmoglein, a cadherin-like protein of the desmosome type of cell junction.

Part of the cytoplasmic domain of a human desmoglein, Dsg1, a cadherin-like protein found in desmosomes of epithelial cells, has been visualised by electron microscopy. The cloned fragment contains five repeats of a 29 +/- 4 residue sequence unique to desmogleins, followed by a glycine-rich region. In rotary shadowed preparations the molecule consists of a globular head attached to a thin tail, the latter perhaps corresponding to the glycine-rich region. This portion of the molecule is thought to span the width of the inner dense plaque. The structure and dimensions concur well to the configuration deduced from the protein sequence.

Amino Acid Sequence↗
PubMedDate not supplied

Breast cancer imaging with radiolabelled peptide from complementarity-determining region of antitumour antibody.

Specific tumour imaging with radiolabelled monoclonal antibodies has been extensively investigated. Although some success has been reported, there are many limitations due to the slow kinetics, poor extravasation, catabolism by the reticuloendothelial system, and non-specific uptake of macromolecules such as antibodies. We have tried to overcome some of the problems associated with monoclonal antibodies while retaining their specificity by using an antibody-derived synthetic peptide. A synthetic pentadecapeptide (alpha M2) derived from the third heavy-chain complementarity-determining region (CDR-3H) of a tumour-associated monoclonal antibody was produced and shown to retain its specificity against the pan-carcinoma cell-surface antigen, polymorphic epithelial mucin, detected by the parent antibody. The peptide was radiolabelled with technetium-99m and injected intravenously to image malignant lesions in 26 women with primary, recurrent, or metastatic breast cancer. Visualisation of breast tumours and their metastases was obtained shortly after administration of alpha M2, and was optimum by 3 h. Overall, 57 (77%) of 74 sites were visualised. Successful imaging was achieved in 14 of 15 primary tumour sites and all of eight local recurrences. Five of six metastases in the opposite breast, eight of 15 metastatic axillary lymph nodes, and all of six metastatic supraclavicular lymph nodes were imaged. Metastatic sites in the lungs, mediastinum, chest wall, and liver were poorly visualised because of background cardiac blood pool. alpha M2 detected small lesions ( < 2 cm) as efficiently as larger ones. The peptide was rapidly (3 h) cleared from the circulation. No acute or chronic adverse reactions due to the alpha M2 were observed. Specific tumour targeting with the radiolabelled anticancer peptide alpha M2 offers new opportunities for breast cancer imaging and possibly therapy.

Adult↗