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Visualisation of biomedical datasets by use of growing cell structure networks: a novel diagnostic classification technique.

BACKGROUND: Medical research produces large multivariable datasets that are difficult to visualise and interpret intuitively. We describe a novel growing cell structure (GCS) technique that compresses multidimensional datasets into two dimensional maps with colour overlays that can be visually interpreted. METHODS: The two-dimensional map is self-discovered from the training set by distribution of cases to different nodes according to similarity between the cases at each node. Nodes are added to the map until there is no further significant reduction in error. The Parzen window method is used to estimate the probability distribution of the training cases, and this probability is converted to posterior class probabilities by use of Bayes' theorem. Classification performance can be assessed by means of receiver operating characteristic (ROC) curves. Colour maps of the values of each input variable at each node are constructed, which illustrate the relation between each input variable and the overall distribution of cases in the network map. FINDINGS: From a dataset of 11 input variables from 692 fine-needle aspirate samples from breast lesions, a 32-node network produced an area under the ROC curve of 0.96, which was not significantly different from that for logistic regression (0.98, z=1.09, p>0.05). Colour maps of the input variables showed that some variables had discrete distributions over exclusively benign or malignant areas of the network, and were thus discriminant, whereas others, such as foamy macrophages, covered both benign and malignant regions. INTERPRETATION: This technique produces dimensional compression that allows multidimensional data to be displayed as two-dimensional colour images. This envisioning of information allows the highly developed visuospatial abilities of human observers to perceive subtle inter-relations in the dataset.

Artificial Intelligence↗

Prefractionation of protein samples prior to two-dimensional electrophoresis.

Thousands of proteins may be visualised on a two-dimensional (2-D) gel, but only hundreds are present at levels sufficient for chemical analysis. Therefore, prefractionation of protein samples prior to 2-D polyacrylamide gel electrophoresis (PAGE) will be important for the investigation of proteins that are present at sub-picogram levels in physiological samples. We describe an approach to prefractionate protein samples prior to 2-D PAGE using the Gradiflow, which is a new (preparative) electrokinetic membrane apparatus designed to fractionate proteins in a number of different ways. We have fractionated human serum under nonreducing conditions using the 'reflux' mode, in which proteins are fractionated according to their relative mobility under controlled electrophoretic conditions, where the current is periodically reversed. We describe how fractionation occurs and present examples of enrichment of specific proteins.

Amino Acid Sequence↗

Blood pressure in the small-for-gestational age newborn.

AIM: The aim of the paper is to verify the existence of an inverse correlation between birth weight and blood pressure (BP) in neonates, infants and adolescents. METHODS: BP was measured at 7 days, 3, 6, 9, 12 months and 7-18 years in 432 subjects born at term at the Department of Pediatrics, Obstetrics and Reproductive Medicine, University of Siena; 228 of these subjects were small for gestational age (SGA) and 204 appropriate for gestational age (AGA). For small babies, BP was measured with a DYNAMAP oscillometer which provides digital visualisation of systolic, diastolic and mean arterial pressure and heart rate. In older children, a mercury sphygmomanometer was used. Statistical analysis was carried out with SPSS 8.01 software using the Kolmogorov-Smirnov test for normality of populations. RESULTS: Statistical analysis did not reveal any significant differences between SGA and AGA subjects in the various age classes of the first 12 months of life. Significant correlation was found between 7 and 18 years with differences in the various age classes for systolic pressure. Subjects with normal birthweight had lower systolic and diastolic BP. SGA males had higher risk of high systolic and diastolic pressure, whereas SGA females were only at higher risk for elevated diastolic pressure. CONCLUSIONS: SGA subjects should be monitored for BP and life-style between 7 and 18 years to risk of cardiovascular disease.

Adolescent↗

Robotic fundoplication in children.

Since January 2006, robotic assistance has been used for performing minimal invasive laparoscopic fundoplications in children. These patients were compared with those operated on with either the open surgical technique or the laparoscopic procedure. The first six children operated on with a fundoplication using the operation robot, da Vinci Surgical System from Intuitive Surgical, were included prospectively. As controls, data from the latest six children operated on using the open surgical procedure and the latest six children operated on using the minimal invasive laparoscopic technique were selected retrospectively. All the patients were operated on due to gastroesophageal reflux and were comparable in the De Meester score. The main outcome measures were the operating time, the use of postoperative analgesics, the duration of the postoperative hospital stay and the short-term outcome. There was no significant difference between the three groups concerning age, body weight and preoperative 24 h pH measurement. The mean operating time for the robotic group, 213 min, was the longer one, but the operating time for the latest four patients in the robotic group was similar to that for the laparoscopic group, 189 min. The postoperative hospital stay was shorter and a reduction in the use of analgesics postoperatively was noted. The reduction in the postoperative hospital stay and in the use of analgesics had been already noted with the introduction of the minimal invasive laparoscopic technique. There was no difference in short-term clinical outcome; the gastroesophageal reflux symptoms disappeared in all the patients. Robot-assisted laparoscopic fundoplication is comparable with the standard laparoscopic surgical procedure in terms of duration of operation, postoperative hospital stay, use of postoperative analgesics and short-term clinical outcome. The robotic surgery adds qualities to the surgical work when compared with open or laparoscopic surgery. These include better visualisation for the surgeon and greater precision in the movements of the instruments used.

Esophagoscopy↗

Human TPST1 transmembrane domain triggers enzyme dimerisation and localisation to the Golgi compartment.

TPST1 is a human tyrosylprotein sulfotransferase that uses 3'phosphoadenosine-5'phosphosulfate (PAPS) to transfer the sulfate moiety to proteins predominantly designated for secretion. To achieve a general understanding of the cellular role of human tyrosine-directed sulfotransferases, we investigated targeting, structure and posttranslational modification of TPST1. Golgi localisation of the enzyme in COS-7 and HeLa cells was visualised by fluorescence imaging techniques. PNGase treatment and mutational studies determined that TPST1 bears N-linked glycosyl residues exclusively at position Asn60 and Asn262. By alanine mutation of these asparagine residues, we could determine that the N-linked oligosaccharides do not have an influence on Golgi retention of TPST1. In concert with N and C-terminal flanking residues, the transmembrane domain of TPST1 was determined to act in targeting and retention of the enzyme to the trans-Golgi compartment. This domain exhibits a pronounced secondary structure in a lipid environment. Further in vivo FRET studies using the transmembrane domain suggest that the human tyrosylprotein sulfotransferase may be functional as homodimer/oligomer in the trans-Golgi compartment.

Amino Acid Sequence↗

Molecular modelling of glycoproteins by homology with non-glycosylated protein domains, computer simulated glycosylation and molecular dynamics.

OBJECTIVES: This study aims to visualise glycoproteins by computer graphics molecular modelling in order to research the dynamics of the oligosaccharide chains, determine their affects on protein conformation, antigenicity and function and to characterise oligosaccharide recognition determinants. With respect to the last, the modelling included the sialylpolylactosamine of thymocyte Thy-l and the sialyl Le(x)/Le(a) determinant present on brain Thy-l. METHODS: The following techniques were used: 1) database searching for homologies with non-glycosylated protein domains; 2) protein modelling on the basis of homology and secondary structure prediction techniques; 3) oligosaccharide construction using a simulated annealing approach utilising the AMBER forcefield with appropriate parameters in the Biosyn software environment; 4) creation of glycoprotein conjugates for further investigation by energy minimisation and molecular dynamics. RESULTS: This approach was successful in providing models of Thy-l and the carboxy terminus 27.5 kD of HIV-1 gp 120, by homology with immunoglobulin light chain folds and in one case (Thy-l) adding the oligosaccharide chains, phosphatidylinositol glycan anchor and lipid membrane and, in the other, adding additional highly glycosylated domains, and domains which folded by molecular dynamics. Significant affects on protein conformation were shown in the presence or absence of the lipid anchor and simulated membrane, but not by the N-linked oligosaccharide chains. CONCLUSIONS: The highly glycosylated molecules Thy-l and gp120, which are not expected to crystallise in their native state, were modelled by computer graphics simulated annealing or molecular dynamics from which interactions could be predicted which agree with experimental data on antibody binding and in vitro activity.

Carbohydrate Conformation↗

Prenatal diagnosis of beta thalassaemia based on restriction endonuclease analysis of amplified fetal DNA.

In the Mediterranean area, 50% of the beta thalassaemia mutations abolish or create a restriction endonuclease site in the beta globin gene. This study describes a new procedure for prenatal detection of these beta thalassaemia defects based on the direct visualisation, on an ethidium bromide stained polyacrylamide gel, of the discrete DNA fragments produced by restriction endonuclease digestion of fetal DNA, enzymatically amplified using the DNA polymerase from the thermophilus bacterium Thermus aquaticus. We applied this procedure to the Sardinian population to detect the nonsense mutation at codon 39 and the frameshift at codon 6 of the beta globin gene; these are the most frequent beta thalassaemia mutations in this population, accounting for 95% and 2.2% of the beta thalassaemia chromosomes. The main advantages of this procedure are simplicity (no radioactivity), sensitivity (0.2 microgram of DNA), and rapidity (12 hours). The very small amount of fetal material required makes amniotic fluid cell culture unnecessary and may decrease the fetal loss rate associated with trophoblast sampling. By circumventing the use of radioactive and non-radioactive probes, the spread of this technology to the high risk areas will be facilitated.

Amniocentesis↗

Robert Feulgen Prize Lecture 1995. Electronic light microscopy: present capabilities and future prospects.

Electronic light microscopy involves the combination of microscopic techniques with electronic imaging and digital image processing, resulting in dramatic improvements in image quality and ease of quantitative analysis. In this review, after a brief definition of digital images and a discussion of the sampling requirements for the accurate digital recording of optical images, I discuss the three most important imaging modalities in electronic light microscopy--video-enhanced contrast microscopy, digital fluorescence microscopy and confocal scanning microscopy--considering their capabilities, their applications, and recent developments that will increase their potential. Video-enhanced contrast microscopy permits the clear visualisation and real-time dynamic recording of minute objects such as microtubules, vesicles and colloidal gold particles, an order of magnitude smaller than the resolution limit of the light microscope. It has revolutionised the study of cellular motility, and permits the quantitative tracking of organelles and gold-labelled membrane bound proteins. In combination with the technique of optical trapping (optical tweezers), it permits exquisitely sensitive force and distance measurements to be made on motor proteins. Digital fluorescence microscopy enables low-light-level imaging of fluorescently labelled specimens. Recent progress has involved improvements in cameras, fluorescent probes and fluorescent filter sets, particularly multiple bandpass dichroic mirrors, and developments in multiparameter imaging, which is becoming particularly important for in situ hybridisation studies and automated image cytometry, fluorescence ratio imaging, and time-resolved fluorescence. As software improves and small computers become more powerful, computational techniques for out-of-focus blur deconvolution and image restoration are becoming increasingly important. Confocal microscopy permits convenient, high-resolution, non-invasive, blur-free optical sectioning and 3D image acquisition, but suffers from a number of limitations. I discuss advances in confocal techniques that address the problems of temporal resolution, spherical and chromatic aberration, wavelength flexibility and cross-talk between fluorescent channels, and describe new optics to enhance axial resolution and the use of two-photon excitation to reduce photobleaching. Finally, I consider the desirability of establishing a digital image database, the BioImage database, which would permit the archival storage of, and public Internet access to, multidimensional image data from all forms of biological microscopy. Submission of images to the BioImage database would be made in coordination with the scientific publication of research results based upon these data.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

Phosphatidic acid increases in response to noradrenaline and endothelin-1 in adult rabbit ventricular myocytes.

OBJECTIVE: The aim was to assess whether noradrenaline and endothelin-1 can stimulate endogenous production of phosphatidic acid in adult ventricular myocytes. METHODS: After stimulation of rabbit ventricular myocytes with noradrenaline and endothelin-1, total lipids were extracted using the Bligh and Dyer procedure and separated by thin layer chromatography, and phosphatidic acid was quantified using photodensitometric analysis of visualised lipids with CuSO4/H3PO4. RESULTS: Noradrenaline (10(-5) M) elicited a rapid increase in phosphatidic acid at 2 min, followed by a decrease at 5 min. A second delayed and sustained increase in phosphatidic acid occurred at 10 min. The response to noradrenaline (10(-9) to 10(-5) M) was concentration dependent with a half maximum response (EC50) of 3.1 x 10(-8) M and the maximum effect at 10(-6) M. The increase in phosphatidic acid production in response to noradrenaline was abolished by an alpha 1 adrenergic receptor blocking agent (2-[beta-(4-hydroxyphenyl)-ethylaminomethyl]tetralone) but unaffected by the beta adrenergic blocking agent L-propranolol. An increase in phosphatidic acid was also elicited in rabbit ventricular myocytes in response to endothelin-1. The response was time and concentration dependent with the maximal increase at 12 min, EC50 5.3 x 10(-9) M, and maximum effect at 10(-6) M. Both noradrenalin and endothelin-1 stimulated phosphatidylbutanol production in the presence of butanol (100 mM), indicating that both agonists activate phospholipase D. CONCLUSIONS: Noradrenaline at physiological concentrations elicits both a rapid and a delayed increase in phosphatidic acid in adult rabbit ventricular myocytes. Endothelial-1, at physiological concentrations, also stimulates an increase in the mass of phosphatidic acid in myocytes, but the increase induced by endothelin-1 is monophasic, in contrast to the biphasic response seen during stimulation with noradrenaline. Activation of phospholipase D contributes to the increase in phosphatidic acid seen during stimulation of myocytes with either noradrenaline or endothelin-1. These are the first data to characterise endogenous production of phosphatidic acid in isolated adult ventricular myocytes.

Adrenergic alpha-Antagonists↗

Detection by immunomagnetic PCR of Mycobacterium avium subsp. paratuberculosis in milk from dairy goats in Norway.

Milk samples from 340 individual goats in 34 dairy herds throughout Norway were examined for Mycobacterium avium subsp. paratuberculosis (M.a. paratuberculosis) by culture and immunomagnetic separation combined with PCR (IMS-PCR). The samples included three categories; (A) vaccinated dairy goats in herds with paratuberculosis; (B) vaccinated dairy goats in herds with no history of paratuberculosis; (C) unvaccinated goats in herds with no history of paratuberculosis. Viable M.a. paratuberculosis were not detected by culture in any sample, but 24 samples (7.1%) tested positive by IMS-PCR when the PCR products were visualised by dot blot hybridisation. PCR products from five milk samples originating from five different herds were sequenced; all showed 99% homology with the IS900 sequence from M.a. paratuberculosis.M.a. paratuberculosis were detected in all sampled categories. The percentage of IMS-PCR positive samples from herds where paratuberculosis had previously been reported was significantly lower than from herds where the infection had never been diagnosed (3.3 and 9.1%, respectively, P=0.048). Similar proportions of milk samples from vaccinated and non-vaccinated goats tested positive for the presence of M.a. paratuberculosis. Vaccinated goats older than 4 years tested positive more often than vaccinated animals less than 2 years old. Samples collected in May tested significantly more often positive than milk sampled during February-March (13.8 and 2.9%, respectively, P=0.001). This study showed that raw goats' milk in Norway might be contaminated with M.a. paratuberculosis.

Animals↗

The expression of different cytochemical markers in normal and axotomised dorsal root ganglion cells projecting to the nucleus gracilis in the adult rat.

Rat lumbar dorsal root ganglion neurones projecting to the nucleus gracilis in the brainstem were retrogradely labelled with Fluoro-Gold and analysed immunocytochemically for their expression of substance P-, calcitonin gene-related peptide-, galanin-, galanin message-associated peptide-, neuropeptide Y-, nitric oxide synthase- and carbonic anhydrase-like immunoreactivity as well as affinity to Griffonia (bandeiraea) simplicifolia lectin I--isolectin B4, RT97 and to choleragenoid. The analysis was made both in uninjured rats and in rats which had been subjected to unilateral sciatic nerve transection and partial resection 3 weeks earlier. The data showed that 6% of the L4 and L5 lumbar dorsal root ganglion cells that projected to the nucleus gracilis showed substance P-like immunoreactivity. Following nerve injury, none of the nucleus gracilis-projecting dorsal root ganglion cells showed substance P-like immunoreactivity. Nineteen per cent of the investigated cell population showed calcitonin gene-related peptide-like immunoreactivity in uninjured rats, but no nucleus gracilis-projecting calcitonin gene-related peptide-positive cells were found after nerve injury. Galanin- and galanin message-associated peptide-like immunoreactivity were found in 2% and 3%, respectively, of the Fluoro-Gold-labelled cell population normally and in 22% and 14%, respectively, after injury. No neuropeptide Y-positive cells were found in the Fluoro-Gold-labelled cell population normally, but after nerve injury, 96% of this population became neuropeptide Y-positive. Nitric oxide synthase-like immunoreactivity was found in 2% of the Fluoro-Gold-labelled cells normally and in 10% after injury. Two per cent of the Fluoro-Gold-labelled cells in the normal cases were stained by Griffonia (bandeiraea) simplicifolia lectin I--isolectin B4. After injury, however, no such double labelling was found. Thirty-four per cent of the Fluoro-Gold-labelled cell population was carbonic anhydrase positive normally, and 42% after injury. Seventy-five per cent of the Fluoro-Gold-labelled cells showed RT97 immunoreactivity normally and 12% after injury. Choleragenoid-like immunoreactivity was found in 99% of the Fluoro-Gold-labelled dorsal root ganglion cells normally and 81% after injury. Immunohistochemical visualisation of choleragenoid transganglionically transported from the injured sciatic nerve combined with neuropeptide Y immunocytochemistry showed that primary afferent fibres and terminals in the nucleus gracilis contain neuropeptide Y following peripheral nerve transection. Taken together, the results indicate that peripherally axotomised nucleus gracilis-projecting neurones undergo marked alterations in their cytochemical characteristics, which may be significant for the structural and functional plasticity of this system after injury.

Afferent Pathways↗

Use of polymerase chain reaction for early identification of Mycobacterium tuberculosis in positive cultures.

AIMS: To develop a readily applicable polymerase chain reaction (PCR) based technique which would permit the identification of Mycobacterium tuberculosis complex isolates from Bactec phials at an earlier stage than currently available methods. METHODS: Mycobacterial cells cultured in Bactec 12B medium were harvested by centrifugation. The cells were lysed by heating in distilled water. Oligonucleotide primers based on the sequence of the gene coding for the immunogenic protein MPB64 were then used to amplify a 240 base pair fragment of DNA directly from the crude cell lysate. The PCR product was visualised under ultraviolet light following electrophoresis of an aliquot in an agarose gel containing ethidium bromide. The sensitivity of the PCR was adjusted so that about 600 cfu of M tuberculosis gave a positive result. The lowest growth index at which this method of identification might be applied to Bactec phials was determined and a number of routine cultures giving a positive growth index examined. RESULTS: M tuberculosis was positively identified at the lowest growth index, as determined by the Bactec system. Of 45 routine cultures examined, with growth indexes ranging from 6 to 999, the 15 confirmed by conventional means to contain M tuberculosis were correctly identified from 1 ml of culture medium. CONCLUSIONS: The method described can be used to identify M tuberculosis isolates cultured in the Bactec system at the earliest detectable rise in growth index. It may therefore allow cultured mycobacteria to be identified at an earlier stage than conventional methods or the commercially available DNA probes adapted for use with the Bactec system.

Base Sequence↗

3-D video techniques in endoscopic surgery.

Three-dimensional visualisation of the operative field is an important requisite for precise and fast handling of open surgical operations. Up to now it has only been possible to display a two-dimensional image on the monitor during endoscopic procedures. The increasing complexity of minimal invasive interventions requires endoscopic suturing and ligatures of larger vessels which are difficult to perform without the impression of space. Three-dimensional vision therefore may decrease the operative risk, accelerate interventions and widen the operative spectrum. In April 1992 a 3-D video system developed at the Nuclear Research Center Karlsruhe, Germany (IAI Institute) was applied in various animal experimental procedures and clinically in laparoscopic cholecystectomy. The system works with a single monitor and active high-speed shutter glasses. Our first trials with this new 3-D imaging system clearly showed a facilitation of complex surgical manoeuvres like mobilisation of organs, preparation in the deep space and suture techniques. The 3-D-system introduced in this article will enter the market in 1993 (Opticon Co., Karlsruhe, Germany.

Animals↗

A combined radiolabelling and silver staining technique for improved visualisation, localisation, and identification of proteins separated by two-dimensional gel electrophoresis.

Two-dimensional gel electrophoresis (2-DE) remains the method of choice for the Separation of protein mixtures whilst mass spectrometry (MS) is rapidly becoming the premier tool for protein identification. When combined, 2-DE and MS form the current operating paradigm for classical proteomics. One of the key challenges of proteome research is that of detecting and identifying all of the elements (proteins) of a proteome. Silver staining and radiolabelling, e.g. with 35S-methionine ([35S]-met), represent two sensitive methods used to visualise many of the constitutive and synthesised elements of a proteome, respectively. The latter method allows a very low total protein loading on a two-dimensional (2-D) gel and challenges protein identification using current MS-based technology. Therefore, it is necessary to refer to and locate a radiolabelled spot's cognate on a preparatively loaded stained gel, or Western blot, and use that protein spot for identification. Unfortunately, the images of autoradiographs and preparative gels or blots, even of the same sample, often do not correspond making it difficult to accurately locate and select spots of interest by visual comparison. We have established a technique that permits the unambiguous localisation of radiolabelled proteins on the same silver stained 2-D gel. Protein identification of superimposed spots is described by peptide mass fingerprinting and database searching using matrix-assisted laser desorption/ionization-time of flight mass spectrometry and by peptide sequencing using tandem MS by hybrid quadrupole/orthogonal acceleration time of flight MS (Q-TOF).

Amino Acid Sequence↗

Cost analysis of the treatment of severe spinal spasticity with a continuous intrathecal baclofen infusion system.

OBJECTIVE: The purpose of our study was to analyse and evaluate the costs of continuous intrathecal baclofen administration as a modality in the treatment of severe spasticity in the Netherlands. DESIGN: A cost analysis was conducted as part of a prospective, multicentre, multidisciplinary, randomised and placebo-controlled clinical trial. The study covered the period from December 1991 to September 1995. The data on medical consumption and costs were collected over a 3-year period from different sources: administrative databases of health insurance companies, hospital registries and a patient survey. These data were structured by means of a flowchart analysis of the medical decision-making by specialists and general practitioners (GPs). They included data on in- and outpatient care, home care and care in nursing homes. The cost analysis was conducted using data from 18 patients included in the trial and from 15 so-called 'match' patients. The latter group are patients with comparable diseases leading to spasticity and living in comparable circumstances. Next to absolute costs (direct and indirect) of care and treatment for the 2 groups of patients, cost differences between the 2 groups were considered (differential cost analysis). SETTING: Per patient cost data, collected prospectively for 2 years during the phase of clinical evaluation, and retrospectively 1 year before implantation. The data were collected on patients from in- and outpatient care, home care and care in nursing home settings. PATIENTS AND PARTICIPANTS: The trial patients (8 men) had a mean age of 46 years; 11 patients had multiple sclerosis and 7 patients had spinal cord injuries. The match patients (7 men) had a mean age of 48 years; 9 patients had multiple sclerosis and 6 patients had spinal cord injuries. INTERVENTIONS: Trial patients were treated with a subcutaneously implanted programmable continuous infusion pump (SynchroMed, Medtronic), filled with baclofen (a muscle relaxant) to treat patients with chronic disabling spasticity who did not respond to a maximum dose of oral baclofen, dantrolene and tizanidine. MAIN OUTCOME MEASURES AND RESULTS: An analysis of hospital stay between both groups showed a significant difference during the implantation year. The average number of hospital days per patient in the year in the treated group was 31.5 days and in the match group was 18.7 days. Significant cost differences between both groups in the year that started with pump implantation and the following year can be attributed mostly to the costs of implantation of the pump and related hospitalisation days. The total costs of patient selection, testing, implanting the pump and follow-up amounted to $US28,473 for the first year. Savings must be taken into consideration as well. The savings of direct costs were due to withdrawal of oral medication (estimated annual total of between $US1950 and $US2800 per patient). Indirect savings on employment and nursing home costs, amounted annually to $US1047 and $US5814, respectively. Scenarios make it possible to consider policy consequences. The case of 'extending' the indications for this treatment to a larger population has been calculated and visualised. CONCLUSIONS: The costs of the therapy (continuous intrathecal infusion of baclofen) can be attributed mostly to implantation of the pump and related hospitalisation days. Savings originated from withdrawal of oral medication, job preservation and avoidance or delay of admission to a nursing home.

Adult↗

Neuropeptide B and W: neurotransmitters in an emerging G-protein-coupled receptor system.

Deorphanised G-protein-coupled receptors represent new and expanding targets for drug development. Neuropeptide B (NPB) and W (NPW) have recently been identified as the cognate endogenous ligands for the orphan receptor GPR7, now designated as NPBW(1). NPB and NPW also bound to a second related orphan receptor, GPR8, now designated as NPBW(2) that is present in humans but not rats or mice. In humans, high levels of NPW mRNA have been visualised in the substantia nigra, whereas moderate expression levels have been detected in the amygdala and hippocampus. In peripheral tissues, expression of NPW mRNA has been confirmed in the progenital system, comprising the kidney, testis, uterus, ovary and placenta, and also in stomach homogenates. Immunocytochemical, molecular biological and autoradiography techniques have revealed a discrete CNS distribution for NPBW(1) in human, mouse and rat. Highest expression of NPBW(1) mRNA and protein was identified in the amygdala and hypothalamic nuclei known to regulate feeding behaviour. [(125)I]-NPW bound with a single high affinity to rat amygdala, K(D)=0.44 nM and 150 fmol mg(-1) protein. Physiological studies demonstrate that intracerebroventricular infusion of NPBW(1) ligands modulates feeding behaviour, regulates the release of corticosterone, prolactin and growth hormone while also manipulating pain pathway. Mouse knockout models of the gene encoding either NPB or NPBW(1) have a gender-specific phenotype, with moderate obesity evident in males but not females. Further investigation is required to elucidate the precise physiological role of NPB and NPW as neurotransmitters.

Amino Acid Sequence↗

An inexpensive and rapid diagnostic method of Koi Herpesvirus (KHV) infection by loop-mediated isothermal amplification.

BACKGROUND: Koi Herpesvirus (KHV) affects both juvenile and adult common carp and koi, and is especially lethal to fry. The high mortalities caused by the disease have had a negative impact on the international koi trade. Different diagnostic techniques have been used to detect KHV, including: isolation of the virus in cell culture, electron microscopy, several PCR tests, ELISA and in situ hybridisation. All of these methods are time consuming, laborious and require specialised equipment. RESULTS: A rapid field diagnosis of KHV in common and koi carp was developed using loop-mediated isothermal amplification (LAMP). The LAMP reaction rapidly amplified nucleic acid with high specificity and efficiency under isothermal conditions using a simple water bath. Two methods of extracting DNA from host tissue were compared: extraction by boiling and by using a commercial extraction kit. A set of six primers--two inner primers, two outer primers and two loop primers--was designed from a KHV amplicon. The reaction conditions were optimised for detection of KHV in 60 min at 65 degrees C using Bst (Bacillus stearothermophilus) DNA polymerase. When visualised by gel electrophoresis, the products of the KHV LAMP assay appeared as a ladder pattern, with many bands of different sizes from 50 base-pairs (bp) up to the loading well. The KHV LAMP product could also be simply detected visually by adding SYBR Green I to the reaction tube and observing a colour change from orange to green. All samples positive for KHV by visual detection were confirmed positive by gel electrophoresis. The KHV LAMP had the same sensitivity as a standard PCR assay for the detection of KHV. CONCLUSION: This paper describes an accelerated LAMP assay for diagnosis of KHV. The entire procedure took only 90 minutes to produce a result: 15 minutes for DNA extraction; 60 min for the LAMP reaction; 2 min for visual detection using SYBR Green I. The test can be used under field conditions because the only equipment it requires is a water bath.

Animals↗

Immunohistochemical localisation of the 5-HT2C receptor protein in the rat CNS.

5-HT2C receptor mRNA has a widespread distribution in the human and rat CNS but the absence of a specific high affinity ligand has made autoradiographic localisation of the receptor difficult. By raising polyclonal antibodies against the rat 5-HT2C receptor protein this study reports the immunohistochemical distribution of this receptor in the rat CNS. A sephadex purified 5-HT2C antiserum visualised a single immunopositive band (54 kDa) in Western blots of membranes prepared from several rat brain regions and caused intense membrane immunofluorescence in HEK 293 cells transfected with h5-HT2C cDNA, which were both attenuated by incubation with the antigenic peptide sequence (200-300 microM). 5-HT2C-like immunoreactivity was located on neurones throughout the CNS. The most abundant 5-HT2C-like immunoreactive cell bodies were in the anterior olfactory nucleus, medial and intercalated amygdaloid nuclei, hippocampus layers CA1 to CA3, laterodorsal and lateral geniculate thalamic nuclei, caudate-putamen and several areas of the cortex (including piriform and frontal), consistent with this receptor being located postsynaptic to serotonergic neurones. Immunopositive neurones were also found in the dorsal raphé, suggesting that 5-HT2C receptors may be on some serotonergic neurones. The overall distribution of 5-HT2C-like immunoreactivity complements previous findings with conventional radioligands and agrees well with reported levels of 5-HT2C receptor mRNA.

Amino Acid Sequence↗