THE POTENTIAL TO USE TLD MEASUREMENTS TO VALIDATE THE OCCUPATIONAL RADIATION PROTECTION AT THE DEPARTMENT OF NUCLEAR MEDICINE.

Diagnostic examinations in nuclear medicine increase. This can cause a higher radiation burden to the personnel. The aim of this study was to create and apply a method to validate the occupational radiation protection at the Department of Nuclear Medicine using thermoluminescence dosemeters (TLD). Occupational doses in- and outside of the department and the dose rate distribution around the positron emission tomography/computed tomography (PET/CT) were validated in terms of Hp(10) using TLD-100. Backscatter correction factors were estimated to implement the TLD measurements on different materials. After correction for backscatter, the result indicates that doses in- and outside of the department are below recommended values and dose limits. The highest and the lowest dose rate were measured beside the 18F-autoinjector and the PET/CT gantry, respectively. The method of using TLD gives a good estimate of how the occupational radiation protection performs at the Department of Nuclear Medicine and can thus help in the optimization of radiation protection.

Multiple displacement amplification of isolated DNA from human gallstones: molecular identification of Helicobacter DNA by means of 16S rDNA-based pyrosequencing analysis.

BACKGROUND: Molecular typing of Helicobacter spp. in clinical biopsy specimens has become increasingly important. By means of nested polymerase chain reaction (PCR) amplification and Southern blot analysis of the PCR amplicons, we have shown that Helicobacter spp. DNA is present in human gallstones. In this study we have investigated the possibility of using multiple displacement amplification (MDA) of isolated gallstone DNA and pyrosequencing analysis for the molecular identification of Helicobacter spp. MATERIALS AND METHODS: DNA isolated from the nucleus of 33 human gallstones and one control strain were used in a MDA assay. Subsequently, pyrosequencing analysis was performed either directly on MDA-DNA using primers flanking the Helicobacter spp. 16S rDNA variable V3 region or on PCR amplicons derived from broad-range primers flanking the 16S rDNA variable V3, V4, and V9 regions. RESULTS: Pyrosequencing analysis of 16S rDNA derived from MDA-DNA revealed that Helicobacter spp.-like DNA was present in 25 of 33 (approximately 76%) gallstones. Using an H. pylori-specific Southern blot analysis, Helicobacter spp.-like DNA was present in 20 of 33 [approximately 61%] of the gallstones. Using MDA-DNA directly in pyrosequencing analysis, Helicobacter spp.-like DNA was present in 13 of 33 [approximately 39%] gallstones. CONCLUSIONS: We conclude that multiple displacement amplification combined with pyrosequencing enables a rapid and accurate molecular typing of Helicobacter spp. from small and precious biopsy specimens.

Multiple displacement amplification of DNA from human colon and rectum biopsies: bacterial profiling and identification of Helicobacter pylori-DNA by means of 16S rDNA-based TTGE and pyrosequencing analysis.

Amplifying bacterial DNA by PCR from human biopsy specimens has sometimes proved to be difficult, mainly due to the low amount of bacterial DNA present. Therefore, nested or semi-nested 16S rDNA PCR amplification has been the method of choice. In this study, we evaluate the potential use of whole genome amplification of total DNA isolated from human colon and rectum biopsy specimens, followed by 16S rDNA PCR amplification of multiple displacement amplified (MDA)-DNA. Subsequently, a H. pylori-specific 16S rDNA variable V3 region PCR assay was applied directly on MDA-DNA and, combined with pyrosequencing analysis; the presence of H. pylori in some biopsies from colon in patients with microscopic colitis was confirmed. Furthermore, temporal temperature gradient gel electrophoresis (TTGE) of 16S rDNA amplicons using primers flanking variable regions V3, V4, and V9, was used to establish bacterial profiles from individual biopsies. A variation of the bacterial profiles in the colonic mucosa in microscopic colitis and in normal rectal mucosa was observed. In conclusion we find the MDA technique to be a useful method to overcome the problem of insufficient bacterial DNA in human biopsy specimens.

Molecular cloning of a putative Ciona intestinalis cionin receptor, a new member of the CCK/gastrin receptor family.

Cionin, a peptide showing similarities with cholecystokinin and gastrin has been shown to be expressed in the gut and neural ganglion of the protochordate Ciona intestinalis. The present report describes the cloning of a putative cionin receptor (CioR), a new member of the CCK/gastrin family from the gastrointestinal tract of C. intestinalis. mRNA from the stomach of C. intestinalis was isolated using a modified RNA extraction procedure and, subsequently, reverse-transcribed into single-stranded cDNA by means of rapid amplification of 5'- and 3'-cDNA ends (RACE-PCR), followed by full-length PCR amplification. The cloned full-length PCR amplicons contained a short upstream open-reading frame (uORF) coding for a putative 16 amino acid long peptide, followed by a long open reading frame encoding a 526 amino acid putative CioR protein. At the amino acid level, the putative CioR protein shared 35-40% homology with cloned mammalian, chicken, and Xenopus laevis CCK receptors. Phylogenetic analysis revealed that the chicken and X. laevis CCK receptors are orthologues of the mammalian CCK2 receptors whereas CioR protein forms a clade with vertebrate cholecystokinin receptors. Moreover, we found that the CioR cDNA and deduced amino acid sequences were found to correspond to the annotated CCK/gastrin-like receptor gene on Scaffold 117 (C. intestinalis draft genome project, Joint Genome Institute database; http://www.jgi.doe.gov).

Molecular cloning of an unusual bicistronic cholecystokinin receptor mRNA expressed in chicken brain: a structural and functional expression study.

This report describes the molecular cloning and pharmacological characterization of a transiently expressed chicken brain cholecystokinin receptor (CCK-CHR) in COS-7 cells. A polymerase chain reaction (PCR)-based cloning strategy was applied using: (1) an initial PCR with deoxyinosine-containing primers designed to target conserved regions in CCK receptors, followed by (2) rapid amplification of cDNA ends (RACE), and (3) full-length PCR of the CCK-CHR cDNA. The full-length cloned bicistronic CCK-CHR cDNA contained a short upstream open reading frame (uORF) coding for a putative six-amino-acid-long peptide of unknown function, followed by a long open reading frame (lORF) encoding the 436-amino-acid-long CCK-CHR receptor protein. At the amino acid level, the CCK-CHR shared approximately 50% homology with mammalian and Xenopus laevis CCK receptors. The pharmacological profile of CCK-CHR resembled that of CCK-B receptors using agonists (CCK-8, CCK-4, gastrin-17), whereas CCK-CHR showed higher affinity for the CCK-A receptor antagonist, devazepide, than for the CCK-B receptor antagonist, L-365,260. To the best of our knowledge, this is the first description and functional expression study of a cloned chicken CCK receptor cDNA.

Pharmacological analysis of CCK(2) receptor ligands using COS-7 and SK-N-MC cells, expressing the human CCK(2) receptor.

A series of CCK(2) receptor ligands were analysed with respect to their interaction with binding sites in the membranes of COS-7 cells and SK-N-MC cells transiently expressing the human CCK(2) receptor (short isoform). The ligands were YF476, YM022, AG041R, L-740,093, JB93182, PD134308, and PD136450. Their binding was analysed by radioligand competition using [3H]L-365,260 as the labelled ligand. Saturation binding analysis indicated that [3H]L-365,260 interacted with a single class of binding sites. In competition binding experiments using COS-7-cell membranes, all seven ligands were incubated together with 2 nM [3H]L-365,260. The data for four of the compounds fitted a one-site model (pK(i) values: YM022: 9.2+/-0.02; YF476: 9.6+/-0.04; L-740,093: 9.2+/-0.01; and AG041R: 8.3+/-0.06), while the data for the three others fitted a two-site model (pK(i) values: JB93182: 8.8+/-0.04 and 6.0+/-0.15; PD134308: 9.0+/-0.04 and 6.1+/-0.15; and PD136450: 9.0+/-0.02 and 5.4+/-0.41). SK-N-MC cell membranes and 2 nM [3H]L-365,260 were incubated together with YM022, YF476, JB93182, and PD134308. The data for YM022 and YF476 fitted a one-site model (pK(i) values: YM022: 9.3+/-0.06; YF476: 9.4+/-0.02), while the data for JB93182 and PD134308 fitted a two-site model (pK(i) values: JB93182: 8.7+/-0.06 and 6.2+/-0.06; PD134308: 9.1+/-0.06 and 7.0+/-0.17). Competition binding experiments in the presence of the GTP-analogue guanylylimidodiphosphate, using either of the two cell types, produced similar binding data for PD134308 and JB93182 as in the absence of GTP-analogue. The human receptor seems to exist in a low and/or high affinity state. The shift from low to high affinity does not seem to reflect the degree of G protein coupling.