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Biomedical subjects

S M Nielsen

Publications and source records attributed to S M Nielsen.

At least 19 recordsLinked to original sources
PubMed2000

Constitutive activation of tethered-peptide/corticotropin-releasing factor receptor chimeras.

Constitutive activity, or ligand-independent activity, of mutant G protein-coupled receptors (GPCRs) has been described extensively and implicated in the pathology of many diseases. Using the corticotropin-releasing factor (CRF) receptor and the thrombin receptor as a model, we present a ligand-dependent constitutive activation of a GPCR. A chimera in which the N-terminal domain of the CRF receptor is replaced by the amino-terminal 16 residues of CRF displays significant levels of constitutive activation. The activity, as measured by intracellular levels of cAMP, is blocked in a dose-dependent manner by the nonpeptide antagonist antalarmin. These results support a propinquity effect in CRF receptor activation, in which the amino-terminal portion of the CRF peptide is presented to the body of the receptor in the proper proximity for activation. This form of ligand-dependent constitutive activation may be of general applicability for the creation of constitutively activated GPCRs that are regulated by peptide ligands such as CRF. These chimeras may prove useful in analyzing mechanisms of receptor regulation and in the structural analysis of ligand activated receptors.

Amino Acid Sequence↗
PubMed2000

Disulfide bridge engineering in the tachykinin NK1 receptor.

As in most other seven-transmembrane receptors, the central disulfide bridge from the extracellular end of TM-III to the middle of the second extracellular loop was essential for ligand binding in the NK1 receptor. However, introduction of "extra", single Cys residues in the second extracellular loop, at positions where disease-associated Cys substitutions impair receptor function in the vasopressin V2 receptor and in rhodopsin, did not cause mispairing with the Cys residues involved in this central disulfide bridge. Cys residues were introduced in the N-terminal extension and in the third extracellular loop, respectively, in such a way that disulfide bridge formation could be monitored by loss of substance P binding and breakage of the bridge could be monitored by gain of ligand binding. This disulfide bridge formed spontaneously in the whole population of receptors and could be titrated with low concentrations of reducing agent, dithiothreitol. Another putative disulfide bridge "switch" was constructed at the extracellular ends of TM-V and -VI, i.e., at positions where a high-affinity zinc site previously had been constructed with His substitutions. Disulfide bridge formation at this position, monitored by loss of binding of the nonpeptide antagonist [3H]LY303.870, occurred spontaneously only in a small fraction of the receptors. It is concluded that disulfide bridges form readily between Cys residues introduced appropriately in the N-terminal extension and the third extracellular loop, whereas they form with more difficulty between Cys residues placed at the extracellular ends of the transmembrane segments even at positions where high-affinity metal ion sites can be constructed with His residues.

Amino Acid Sequence↗
PubMed2000

Electroconvulsive stimuli enhance both neuropeptide Y receptor Y1 and Y2 messenger RNA expression and levels of binding in the rat hippocampus.

Repeated electroconvulsive stimulations and other seizure modalities produce an increase in neuropeptide Y synthesis and local release in the rat hippocampus, and perhaps as a consequence, a change in the concentration of neuropeptide Y binding sites in the same region. The aim of the present study was to determine possible changes in the expression of neuropeptide Y receptor subtypes affected by repeated stimulations in the hippocampus. Rats were exposed to 14 daily stimulations, and the brains were removed 24h after the last stimulation. For in vitro receptor autoradiography and in situ hybridisation histochemistry, the brains were frozen, sectioned, and levels of neuropeptide Y binding sites and messenger RNA expressions were determined quantitatively on sections from the same animals. In order to determine the contribution of different neuropeptide Y receptor subtypes, serial sections were incubated with either 125I-labelled peptide YY alone or the same radio-labelled peptide mixed with an excess of a number of displacing compounds with affinity for either neuropeptide Y receptor subtype Y1, Y2, or both. Binding studies revealed that the majority of peptide YY binding sites was represented by Y2, and that electroconvulsive stimulations reduced the binding capacity or the concentration of this receptor. A prominent reduction of Y1-preferring binding sites was determined in the dentate gyrus, and to a lesser extent in the CA1 and CA3 regions. Similarly, the treatment produced a significant reduction of Y2-preferring binding sites in the CA1 and CA3 region, but not in the granular cell layer of the dentate gyrus. Using semi-quantitative in situ hybridization, Y1 receptor messenger RNA level in the granular cell layer of the dentate increased by the stimulations. In the same region, Y2 receptor messenger RNA was expressed in low to undetectable amounts, but after the repeated stimulations, this transcript was found in moderate to high levels. These data suggest that the neuropeptide Yergic system in the dentate gyrus and the pyramidal cell layer are affected by the treatment, and that this includes both Y1 and Y2 receptor subtypes. Because levels of messenger RNA and binding are distinctly regulated, the turnover of both Y1 and Y2 molecules is strongly increased under electroconvulsive stimulations, suggesting that the intrahippocampal neuropeptide Yergic neurotransmission is also increased under the stimulations.

Animals↗
PubMed1998

[Arthritis as first symptom of leukemia in children].

Three children out of 30 (10%) referred for juvenile chronic arthritis had leukaemia. The patients had complained of intermittent musculoskeletal pain and painful joint swelling for three weeks, nine months and eighteen months prior to admission. On admission two of the patients had active arthritis with soft tissue swelling in one and three joints respectively. The third patient had only arthralgias and no joint swelling. All patients had slight anaemia, normal to slightly reduced thrombocyte count, slight neutropenia and absence of blasts in the peripheral blood. The correct diagnosis was made by bone marrow aspiration. Two children had acute lymphoblastic leukaemia and the third acute myeloblastic leukaemia. Leukaemia thus remains an important differential diagnosis in children presenting with musculoskeletal pain and/or arthritis.

Arthritis, Juvenile↗
PubMed1998

[Randomized comparison of intravenous immunoglobulin and methylprednisolone pulse therapy in children with newly diagnosed idiopathic thrombocytic purpura. The Danish ITP Study Group].

Forty three children with newly diagnosed idiopathic thrombocytopenic purpura (ITP), platelet count (pl.c.) below 20 x 10(9)/l, and either clinically significant bleeding or failure to show a spontaneous platelet rise within three days of admission were randomly allocated to treatment with intravenous infusions of either immunoglobulin (IVIG) 1 g/kg or methylprednisolone (MPPT) 30 mg/kg on two consecutive days. Prompt induction of partial remission with pl.c. > 50 x 10(9)/l after 72 hours was seen in 21/23 given IVIG versus 10/20 given MPPT (exact p = 0.003); mean pl.c.s after 72 hours were 188 versus 77 x 10(9)/l (2p < 0.001). Poor responders were then given the alternative infusions in addition. After six days, complete remission with pl.c. > 150 x 10(9)/l was achieved in 16/23 versus 10/20 (p = 0.16). During six months follow-up, there were no significant differences regarding relapse rates or chronic course. Eleven children with relapse were crossed over to the alternative treatment arm: the estimated treatment effect in pl.c. after 72 hours was 134 x 10(9)/l in favour of IVIG. These results indicate that IVIG infusions may be preferable to high-dose corticosteroids as initial treatment for children with ITP.

Anti-Inflammatory Agents↗
PubMed1998

Split-receptors in the tachykinin neurokinin-1 system--mutational analysis of intracellular loop 3.

Several membrane proteins have been functionally expressed from non-covalently coupled, contiguous segments especially with the split-site located between natural domains. Experiments using such 'split-proteins' were here performed in the tachykinin neurokinin-1 (NK1) receptor with co-expression of contiguous segments with split-sites positioned in various intracellular and extracellular loops. The construct where the split-site was located in intracellular loop 3 gave a reasonable expression level of substance-P-binding sites, i.e. 12% of wild-type expression. Of the other split-receptors tested, only the one with the split-site located just outside transmembrane (TM) segment-V gave any detectable substance P binding, which however only was 1% of the wild-type expression level. The construct with the split-site located in intracellular loop 3 bound all of the tested peptide agonists and non-peptide antagonists with normal affinity and was able to stimulate inositol phosphate turnover with a normal EC50 for substance P and an Emax according to the expression level. When intracellular loop 3 was either extended with 112 amino acid residues derived from the muscarine M2 receptor or, when major parts of the loop were deleted in the non-split NK1 receptor, the affinity for neither substance P nor for the prototype nonpeptide antagonist, CP96,345 was affected, yet an increase in EC50 for substance P was observed. Also in the split-receptor, most of intracellular loop 3 could be substituted or even deleted without affecting ligand affinity, although a decreased expression level was observed in constructs having major deletions. It is concluded, that the NK1 receptor is preferentially reconstituted by co-expression of a putative A-domain including TM-I-V and a B-domain including TM-VI and -VII. It is suggested that a number of rhodopsin-like 7TM receptors may function as two-domain structures based on the finding that a network of short loops has been highly conserved within each of the putative domains and, that these domains are separated by a relatively long and in respect of length poorly conserved loop, i.e. intracellular loop 3.

Amino Acid Sequence↗
PubMed1997

Engineering of metal-ion sites as distance constraints in structural and functional analysis of 7TM receptors.

G-protein-coupled receptors with their seven transmembrane (7TM) segments constitute the largest superfamily of proteins known. Unfortunately, still only relatively low resolution structures derived from electron cryo-microscopy analysis of 2D crystals are available for these proteins. We have used artificially designed Zn(II) metal-ion binding sites to probe 7TM receptors structurally and functionally and to define some basic distance constraints for molecular modeling. In this way, the relative helical rotation and vertical translocation of transmembrane helices TM-II, TM-III, TM-V, and TM-VI of the tachykinin NK-1 receptor have been restricted. Collectively, these zinc sites constitute a basic network of distance constraints that limit the degrees of freedom of the interhelical contact faces in molecular models of 7TM receptors. The construction of artificially designed metal-ion sites is discussed also in the context of probes for conformational changes occurring during receptor activation.

Binding Sites↗
PubMed1997

Renal CT after intravenous injection of nonionic dimeric or nonionic monomeric contrast media in healthy volunteers.

PURPOSE: To establish and compare time-density curves of the aorta, vena cava and renal cortex, medulla and pelvis after injection of the nonionic dimer iodixanol or the nonionic monomer iopromide contrast media. MATERIAL AND METHODS: In 36 healthy volunteers CT at the renal hilus was performed at regular intervals before and until 30 min after injection of 13.5 g I iodixanol or 15.0 g I iopromide. The density of the renal cortex, medulla, pelvis, aorta and inferior caval vein was measured. RESULTS: With injection of the 10% lower iodine dose in the iodixanol group, the profiles of the CT time-density curves were similar for all regions except for the renal pelvis. For the renal pelvis, significantly higher density values were obtained for the iodixanol group up to 10 min after injection. CONCLUSION: Although the present study shows that a lower iodine dose is needed for a dimeric compared to a monomeric nonionic contrast medium to obtain similar time-density curves, more studies are needed to establish the clinical importance of this for urography.

Adult↗
PubMed1997

Neuro-acanthocytosis--a rare cause of chorea.

Neuro-acanthocytosis is a rare neurological disorder characterised by stereotyped chorea, especially of the mouth, areflexia and acanthocytes seen in the peripheral blood. No cases have been described in the literature from South Africa. We report here a case of neuro-acanthocytosis seen in a black woman who presented to Johannesburg Hospital.

Acanthocytes↗
PubMed1996

Ultrasonographically guided fine-needle aspiration biopsy of intrathoracic tumors.

PURPOSE: Evaluation of diagnostic accuracy and complication frequency at ultrasonographically guided fine-needle aspiration biopsy of intrathoracic tumors. MATERIAL AND METHODS: A total of 134 ultrasonographically guided percutaneous fine-needle aspiration biopsies were performed on 128 patients with intrathoracic tumors abutting on the pleura. A cytologic diagnosis was obtained in 119 patients (93%). RESULTS: In 83 patients, cytologic malignancy was found; in 34, benignity; and in 2, cellular atypia. A total of 117 diagnoses were correct and 2 diagnoses were false-positive. A malignancy subtyping based on the cytologic aspirates was made in 80 patients. In 40 (93%) of 43 histologically proven subtypes, the subtyping was correct. The complication frequency in terms of pneumothorax was 3.7%. CONCLUSION: Ultrasonographically guided percutaneous fine-needle aspiration biopsy is a safe and reliable method of establishing the cytologic diagnosis of intrathoracic tumors.

Biopsy, Needle↗
PubMed1995

Conversion of antagonist-binding site to metal-ion site in the tachykinin NK-1 receptor.

Mutational analysis of the tachykinin NK-1 (refs 1-7), NK-2 (ref. 8) and angiotensin AT-1 (refs 9, 10) receptors indicates that non-peptide antagonists act through residues located between the seven transmembrane segments, whereas natural peptide agonists bind mainly to residues scattered in the exterior part of the receptor. The presumed contact points for the prototype NK-1 antagonist CP96,345 cluster on opposing faces of the outer portions of transmembrane helices V and VI (refs 1-5). Here we show that systematic introduction of histidyl residues at this antagonist-binding site in the human NK-1 receptor gradually converts it into a high-affinity metal-ion-binding site without affecting agonist binding. In a double mutant with histidine residues substituted at the top of transmembrane segments V and VI, respectively, Zn2+ inhibits binding of radiolabelled agonist peptide and efficiently blocks phosphoinositol turnover induced by substance P. We propose that Zn2+ and CP96,345 act as 'allosteric competitive' antagonists by stabilizing inactive conformations of the mutant and the wild-type receptor respectively. Introduction of metal-ion-binding sites could be used as a general tool in the structural and functional characterization of helix-helix interactions in G-protein-coupled receptors, as well as in other membrane proteins.

Allosteric Regulation↗
PubMed1995

Ultrasonographic in vitro examination of nonpalpable breast masses. A new method.

Specimen radiology is necessary for ensuring successful surgical excision of nonpalpable, clinically occult breast masses discovered by mammography. However, occasionally nonpalpable lesions are only detected by sonography. A simple method of ultrasonographic in vitro identification of nonpalpable lesions invisible on specimen radiographs is presented.

Adult↗
PubMed1995

Mediastinal staging of lung cancer. Is mediastinoscopy still essential?

The significance of computed tomography of the thorax and mediastinoscopy in pretherapeutic mediastinal assessment for the staging of lung cancer remains controversial. The presents study was designed to establish a standard approach to cervical mediastinoscopy for otolaryngologists, who in Denmark traditionally are involved in the staging of non-small cell lung cancer. Sixty-four potentially operable patients with non-small cell lung cancer underwent thoracic computed tomography prior to bronchoscopy and cervical mediastinoscopy. Thirty-six of the 43 mediastinoscopically negative patients additionally underwent thoracotomy, which in 32 cases was considered curative. Mediastinoscopy alone established the lung cancer diagnosis in 20% of the patients. In diagnosing lymph node metastases in the superior mediastinum, a criterion of 10 mm for abnormal enlargement resulted in an overall sensitivity and specificity of mediastinal computed tomography of 72% and 85%, respectively, and the overall false-negative and false-positive rates appeared to be 18% and 25%, respectively. No clinicopathological characteristics could be identified that influenced the occurrence of mediastinal metastases or the accuracy of computed tomography. It is concluded that mediastinoscopy remains essential in the evaluation of patients with presumed or verified non-small cell lung cancer. For otolaryngologists, the strategy of routine cervical mediastinoscopy, performed under general anesthesia in the same procedure as bronchoscopy, is advocated as a standard approach to preoperative mediastinal assessment for the staging of non-small cell lung cancer.

Adult↗
PubMed1993

Definition of inseparably fused ventricular myocardium in thoracopagus: fetal echocardiographic utility and pathologic refinement.

Correlative echocardiographic and pathological findings in a thoracopagus with conjoined hearts are reported. One twin had tricuspid atresia with discordant atrioventricular connections and concordant ventriculoarterial connections. The morphologic right ventricle was hypoplastic and there was a large muscular ventricular septal defect. The other twin had hypoplasia of the mitral valve anulus and left ventricle with double-outlet right ventricle and pulmonary valve atresia. The tricuspid valve was severely insufficient in part because of a large orifice and redundant, elongated leaflets with abnormal chordal attachments. The left ventricles of these two twins shared a perforated common "free wall" with at least two large defects allowing mixing of the circulations at that level. Not all anatomic details were established conclusively by fetal echocardiography; however, sufficient diagnostic information was obtained to support a decision not to aggressively resuscitate these twins after elective cesarean delivery at 31 weeks' gestation.

Adult↗