Ms. Ff. Mus. 1246 - Am Neu-Jahrs-Tag : Lobet den Herrn p ; C. A. T. B., 2 violini, viola, 2 oboe, 3 clarini, tympano, violoncello, organo

del S. Telemann

SADMFR guidelines for the use of Cone-Beam Computed Tomography/ Digital Volume Tomography

Cone-Beam Computed Tomography (CBCT) has been introduced in 1998. This radiological imaging procedure has been provided for dentistry and is comparable to computed tomography (CT) in medicine. It is expected that CBCT will have the same success in dental diagnostic imaging as computed tomography had in medicine. Just as CT is responsible for a significant rise in radiation dose to the population from medical X-ray diagnostics, CBCT studies will be accompanied by a significant increase of the dose to our patients by dentistry. Because of the growing concern for an uncritical and consequently rapidly increasing use of CBCT the Swiss Society of Dentomaxillofacial Radiology convened a first consensus conference in 2011 to formulate indications for CBCT, which can be used as guidelines. In this meeting, oral and maxillofacial surgery, orthodontics and temporomandibular joint disorders and diseases were treated and the most important and most experienced users of DVT in these areas were asked to participate. In general, a highly restrictive use of CBCT is required. Justifying main criterion for CBCT application is that additional, therapy-relevant information is expected that should lead to a significant benefit in patient care. All users of CBCT should have completed a structured, high-level training, just like that offered by the Swiss Society of Dentomaxillofacial Radiology.

N-terminal modifications improve the receptor affinity and pharmacokinetics of radiolabeled peptidic gastrin-releasing peptide receptor antagonists: examples of 68Ga- and 64Cu-labeled peptides for PET imaging

UNLABELLED Gastrin-releasing peptide receptors (GRPrs) are overexpressed on a variety of human cancers, providing the opportunity for peptide receptor targeting via radiolabeled bombesin-based peptides. As part of our ongoing investigations into the development of improved GRPr antagonists, this study aimed at verifying whether and how N-terminal modulations improve the affinity and pharmacokinetics of radiolabeled GRPr antagonists. METHODS The potent GRPr antagonist MJ9, Pip-d-Phe-Gln-Trp-Ala-Val-Gly-His-Sta-Leu-NH(2) (Pip, 4-amino-1-carboxymethyl-piperidine), was conjugated to 1,4,7-triazacyclononane, 1-glutaric acid-4,7 acetic acid (NODAGA), and 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA) and radiolabeled with (68)Ga and (64)Cu. The GRPr affinity of the corresponding metalloconjugates was determined using (125)I-Tyr(4)-BN as a radioligand. The labeling efficiency of (68)Ga(3+) was compared between NODAGA-MJ9 and NOTA-MJ9 in acetate buffer, at room temperature and at 95°C. The (68)Ga and (64)Cu conjugates were further evaluated in vivo in PC3 tumor xenografts by biodistribution and PET imaging studies. RESULTS The half maximum inhibitory concentrations of all the metalloconjugates are in the high picomolar-low nanomolar range, and these are the most affine-radiolabeled GRPr antagonists we have studied so far in our laboratory. NODAGA-MJ9 incorporates (68)Ga(3+) nearly quantitatively (>98%) at room temperature within 10 min and at much lower peptide concentrations (1.4 × 10(-6) M) than NOTA-MJ9, for which the labeling yield was approximately 45% under the same conditions and increased to 75% at 95°C for 5 min. Biodistribution studies showed high and specific tumor uptake, with a maximum of 23.3 ± 2.0 percentage injected activity per gram of tissue (%IA/g) for (68)Ga-NOTA-MJ9 and 16.7 ± 2.0 %IA/g for (68)Ga-NODAGA-MJ9 at 1 h after injection. The acquisition of PET images with the (64)Cu-MJ9 conjugates at later time points clearly showed the efficient clearance of the accumulated activity from the background already at 4 h after injection, whereas tumor uptake still remained high. The high pancreas uptake for all radiotracers at 1 h after injection was rapidly washed out, resulting in an increased tumor-to-pancreas ratio at later time points. CONCLUSION We have developed 2 GRPr antagonistic radioligands, which are improved in terms of binding affinity and overall biodistribution profile. Their promising in vivo pharmacokinetic performance may contribute to the improvement of the diagnostic imaging of tumors overexpressing GRPr.

Positron emission tomography (PET) imaging of prostate cancer with a gastrin releasing peptide receptor antagonist - from mice to men

UNLABELLED Ex vivo studies have shown that the gastrin releasing peptide receptor (GRPr) is overexpressed on almost all primary prostate cancers, making it a promising target for prostate cancer imaging and targeted radiotherapy. METHODS Biodistribution, dosimetry and tumor uptake of the GRPr antagonist ⁶⁴Cu-CB-TE2A-AR06 [(⁶⁴Cu-4,11-bis(carboxymethyl)-1,4,8,11-tetraazabicyclo(6.6.2)hexadecane)-PEG₄-D-Phe-Gln-Trp-Ala-Val-Gly-His-Sta-LeuNH₂] were studied by PET/CT in four patients with newly diagnosed prostate cancer (T1c-T2b, Gleason 6-7). RESULTS No adverse events were observed after injection of ⁶⁴Cu-CB-TE2A-AR06. Three of four tumors were visualized with high contrast [tumor-to-prostate ratio > 4 at 4 hours (h) post injection (p.i.)], one small tumor (T1c, < 5% tumor on biopsy specimens) showed moderate contrast (tumor-to-prostate ratio at 4 h: 1.9). Radioactivity was cleared by the kidneys and only the pancreas demonstrated significant accumulation of radioactivity, which rapidly decreased over time. CONCLUSION ⁶⁴Cu-CB-TE2A-AR06 shows very favorable characteristics for imaging prostate cancer. Future studies evaluating ⁶⁴Cu-CB-TE2A-AR06 PET/CT for prostate cancer detection, staging, active surveillance, and radiation treatment planning are necessary.

Screening, Identification and Preliminary Investigation of Target Transporters in Pregnancy Pathologies

Screening, Identification and Preliminary Investigation of Target Transporters in Pregnancy Pathologies. INTRODUCTION: Pre-eclampsia (PE), intrauterine growth restriction (IUGR) and gestational diabetes mellitus (GDM) are major sources of clinical morbidity and mortality in pregnant women worldwide. The mechanisms underlying these gestational diseases are complex and not yet fully understood, but one factor contributing to their development is impaired maternal-fetal nutrient transport. Therefore, we aimed to identify candidate membrane transporters involved in transplacental nutrient transfer associated with PE/IUGR or GDM. METHODS: Using in silico strategies, we analysed various gene expression data sets generated on different platforms focusing on solute carriers, ABC transporters and TRP channels in order to identify transporters that are differently expressed between patients and gestational age-matched controls. These bioinformatic analyses were combined with literature data to define a catalogue of target transporters that could be involved in the development of PE/IUGR or GDM. Transporters of interest were then analysed for gene expression using qRT-PCR in placental tissues of patients and controls. For validating the results on protein and functional level, we started to establish an in vitro assay using freshly isolated primary cytotrophoblast cells polarized on the Transwell® system. RESULTS: Using bioinformatics approaches, we initially identified 37 target membrane proteins which were mainly associated with the transport of amino acids, vitamins, and trace elements. At the current state of analysis, the amino acid transporters SLC7A7, SLC38A2, SLC38A5, and the thiamine transporter SLC19A3 showed significant differences in placental mRNA expression between controls and patients affected by PE and/or IUGR. Subsequent gene expression analysis in our in-house GDM placental tissue bank is still ongoing. CONCLUSIONS: Based on our in silico analyses, literature data and first follow-up in vitro validations, we were able to define potentially interesting candidate transporters implicated in PE/IUGR or GDM. To date, additional newly defined candidate targets are being analysed on mRNA level in PE/IUGR and GDM. Subsequent analyses on protein and functional level will reveal whether these targets could be of diagnostic or therapeutical interest in these pregnancy pathologies.

Mus Hs 1629 - Missae II : 1 ex db, 2 ex Gb : canto, alto, tenore, basso, 2 violini, alto viola, 2 corni, tympani et organo

Besetzung: Coro S, Coro A, Coro T, Coro B, Vl 1 2, Va, Trp 1 2, Timp, Org

Stereochemistry of Amino Acid Spacers Determines the Pharmacokinetics of (111)In-DOTA-Minigastrin Analogues for Targeting the CCK2/Gastrin Receptor.

The metabolic instability and high kidney retention of minigastrin (MG) analogues hamper their suitability for use in peptide-receptor radionuclide therapy of CCK2/gastrin receptor-expressing tumors. High kidney retention has been related to N-terminal glutamic acids and can be substantially reduced by coinjection of polyglutamic acids or gelofusine. The aim of the present study was to investigate the influence of the stereochemistry of the N-terminal amino acid spacer on the enzymatic stability and pharmacokinetics of (111)In-DOTA-(d-Glu)6-Ala-Tyr-Gly-Trp-Met-Asp-Phe-NH2 ((111)In-PP11-D) and (111)In-DOTA-(l-Glu)6-Ala-Tyr-Gly-Trp-Met-Asp-Phe-NH2 ((111)In-PP11-L). Using circular dichroism measurements, we demonstrate the important role of secondary structure on the pharmacokinetics of the two MG analogues. The higher in vitro serum stability together with the improved tumor-to-kidney ratio of the (d-Glu)6 congener indicates that this MG analogue might be a good candidate for further clinical study.

Evaluation of three different families of bombesin receptor radioantagonists for targeted imaging and therapy of gastrin releasing peptide receptor (GRP-R) positive tumors.

Two new classes of radiolabeled GRP receptor antagonists are studied and compared with the well-established statine-based receptor antagonist DOTA-4-amino-1-carboxymethylpiperidine-d-Phe-Gln-Trp-Ala-Val-Gly-His-Sta-Leu-NH2 (RM2, 1; DOTA:1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid; Sta:(3S,4S)-4-amino-3-hydroxy-6-methylheptanoic acid). The bombesin-based pseudopeptide DOTA-4-amino-1-carboxymethylpiperidine-d-Phe-Gln-Trp-Ala-Val-Gly-His-Leuψ(CHOH-CH2)-(CH2)2-CH3 (RM7, 2), and the methyl ester DOTA-4-amino-1-carboxymethylpiperidine-d-Phe-Gln-Trp-Ala-Val-Gly-His-Leu-OCH3 (ARBA05, 3) analogues are labeled with (111)In and evaluated in vitro in PC-3 cell line and in vivo in PC-3 tumor-bearing nude mice. Antagonist potency was assessed by immunofluorescence-based receptor internalization and Ca(2+) mobilization assays. The conjugates showed good binding affinity, the IC50 value of 2 (3.2 ± 1.8 nM) being 2 and 10 times lower than 1 and 3. Compared to (111)In-1, (111)In-2 showed higher uptake in target tissues such as pancreas (1.5 ± 0.5%IA/g and 39.8 ± 9.3%IA/g at 4 h, respectively), whereas the compounds had similar tumor uptake (11.5 ± 2.4%IA/g and 11.8 ± 3.9%IA/g at 4h, respectively). The displacement of the radioligand in vivo was different in different receptor positive organs and depended on the displacing peptide.

FUS/TLS contributes to replication-dependent histone gene expression by interaction with U7 snRNPs and histone-specific transcription factors.

Replication-dependent histone genes are up-regulated during the G1/S phase transition to meet the requirement for histones to package the newly synthesized DNA. In mammalian cells, this increment is achieved by enhanced transcription and 3' end processing. The non-polyadenylated histone mRNA 3' ends are generated by a unique mechanism involving the U7 small ribonucleoprotein (U7 snRNP). By using affinity purification methods to enrich U7 snRNA, we identified FUS/TLS as a novel U7 snRNP interacting protein. Both U7 snRNA and histone transcripts can be precipitated by FUS antibodies predominantly in the S phase of the cell cycle. Moreover, FUS depletion leads to decreased levels of correctly processed histone mRNAs and increased levels of extended transcripts. Interestingly, FUS antibodies also co-immunoprecipitate histone transcriptional activator NPAT and transcriptional repressor hnRNP UL1 in different phases of the cell cycle. We further show that FUS binds to histone genes in S phase, promotes the recruitment of RNA polymerase II and is important for the activity of histone gene promoters. Thus, FUS may serve as a linking factor that positively regulates histone gene transcription and 3' end processing by interacting with the U7 snRNP and other factors involved in replication-dependent histone gene expression.

Mus Hs 730 - Largo

[Carl Oestreich]

Dystrophic cardiomyopathy: role of TRPV2 channels in stretch-induced cell damage

Aims Duchenne muscular dystrophy (DMD), a degenerative pathology of skeletal muscle, also induces cardiac failure and arrhythmias due to a mutation leading to the lack of the protein dystrophin. In cardiac cells, the subsarcolemmal localization of dystrophin is thought to protect the membrane from mechanical stress. The absence of dystrophin results in an elevated stress-induced Ca2+ influx due to the inadequate functioning of several proteins, such as stretch-activated channels (SACs). Our aim was to investigate whether transient receptor potential vanilloid channels type 2 (TRPV2) form subunits of the dysregulated SACs in cardiac dystrophy. Methods and results We defined the role of TRPV2 channels in the abnormal Ca2+ influx of cardiomyocytes isolated from dystrophic mdx mice, an established animal model for DMD. In dystrophic cells, western blotting showed that TRPV2 was two-fold overexpressed. While normally localized intracellularly, in myocytes from mdx mice TRPV2 channels were translocated to the sarcolemma and were prominent along the T-tubules, as indicated by immunocytochemistry. Membrane localization was confirmed by biotinylation assays. Furthermore, in mdx myocytes pharmacological modulators suggested an abnormal activity of TRPV2, which has a unique pharmacological profile among TRP channels. Confocal imaging showed that these compounds protected the cells from stress-induced abnormal Ca2+ signals. The involvement of TRPV2 in these signals was confirmed by specific pore-blocking antibodies and by small-interfering RNA ablation of TRPV2. Conclusion Together, these results establish the involvement of TRPV2 in a stretch-activated calcium influx pathway in dystrophic cardiomyopathy, contributing to the defective cellular Ca2+ handling in this disease.

Copper-64 Labeled Macrobicyclic Sarcophagine Coupled to a GRP Receptor Antagonist Shows Great Promise for PET Imaging of Prostate Cancer

The gastrin-releasing peptide receptor (GRPr) is an important molecular target for the visualization and therapy of tumors and can be targeted with radiolabeled bombesin derivatives. The present study aims to develop statine-based bombesin receptor antagonists suitable for labeling with 64Cu for imaging by positron emission tomography (PET). The potent GRPr antagonist D-Phe-Gln-Trp-Ala-Val-Gly-His-Sta-Leu-NH2 was conjugated to the sarcophagine (3,6,10,13,16,19-hexaazabicyclo[6.6.6] icosane=Sar) derivative 5-(8-methyl-3,6,10,13,16,19-hexaaza-bicyclo[6.6.6]icosan-1-ylamino)-5-oxopentanoic acid (MeCOSar) via PEG4 (LE1) and PEG2 (LE2) spacers and radiolabeled with 64Cu2+ with >95% yield and specific activities of about 100 MBq/nmol. Both Cu(II) conjugates have high affinity for GRPr (IC50: natCu-LE1, 1.4±0.1 nM; natCu-LE2, 3.8±0.6 nM). The antagonistic properties of both conjugates were confirmed by Ca2+-flux measurements. Biodistribution studies of Cu-64-LE1 exhibited specific targeting of the tumor (19.6±4.7% IA/g at 1 h p.i.) and GRPr-positive organs. Biodistribution and PET images at 4 and 24 h postinjection showed increasing tumor-to-background ratios with time. This was illustrated by the acquisition of PET images showing high tumor-to-normal tissue contrast. This study demonstrates the high affinity of the MeCOSar-PEGx-bombesin conjugates to GRPr. The stability of 64Cu complexes of MeCOSar, the long half-life of 64Cu, and the suitable biodistribution profile of the 64Cu-labeled peptides lead to PET images of high contrast suitable for potential translation into the clinic.

Mus Hs 353 - Jubel-Cantate : zur Feÿer des 50jährigen Regierungsantritts Sr Maj: des Königs von Sachsen ; den 20: Sept. 1818 ; oder Erndte-Cantate ; op: 58

Musik von Carl Maria von Weber. Gedicht von Friedrich Kind. Nach Friedrich Kinds Jubel Cantate gedichtet von A. Wendt

GENETIC ORGANIZATION OF THE STRUCTURAL GENES FOR NITRATE REDUCTASE (TN5)

Nitrate reductase in Escherichia coli is a membrane-bound anaerobic enzyme that is repressed by oxygen and induced by nitrate. The genetic organization of the structural genes for the two larger subunits of nitrate reductase ((alpha) and (beta)) was determined by immunoprecipitation analysis of the formation of these proteins in nitrate reductase-deficient mutants resulting from transposon Tn5 mutagenesis. The results suggested that the genes encoding the (alpha) and (beta) subunits (narG and H) were arranged in an operon with transcription in the direction promoter(--->)(alpha)(--->)(beta). Segments of the chromosome containing the Tn5 inserts from several of the mutants were cloned into plasmid pBR322 and the positions of the transposons determined by restriction mapping. The Tn5 insertion sites were localized on two contiguous EcoRI fragments spanning about 6.6 kilobases of DNA. The narI gene (proposed to encode the (gamma) subunit) was positioned immediately downstream from the (beta)-gene (narH) by Southern analysis of Tn10 insertions into the narI locus. A Tn10 insertion into the narK locus, proposed to encode a nitrate-sensitive repressor of other anaerobic enzymes, was located about 1.5 kilobases upstream from the narGHI operon promoter. The narL locus, proposed to encode a nitrate-sensitive positive regulator of the narGHI operon and known to be genetically linked to the other nar genes, was demonstrated to lie outside a 19.3-kilobase region of the chromosome which encompasses the other nar genes. The physical limit of the narGHI promoter was defined by studying the effect of Tn5 insertions into a hybrid plasmid containing the functional operon. The points of origin of the coding regions for the (alpha) and (beta) genes were deduced by alignment of the chromosomal map of Tn5 insertion sites with the sizes of (alpha) and (beta) subunit fragments produced by plasmids carrying these Tn5 inserts in the nar operon. The coding region for the (alpha) subunit (143,000 daltons) begins about 250 nucleotides downstream from the deduced limit of the promoter region and includes about 4.0 kilobases of DNA; the region encoding (beta) (60,000 daltons) lies immediately downstream from the (alpha)-gene and is approximately 1.6 kilobases in length. The adjacent region encoding the (gamma) subunit (19,000 daltons) is approximately 0.5 kilobase in length. ^

Molecular regulatory mechanisms and embryonic tissue specification in sea urchin development

To understand how a eukaryote achieves differential transcription of genes in precise spatial patterns, the molecular details of tissue specific expression of the Strongylocentrotus purpuratus Spec2a gene were investigated by functional studies of the cis-regulatory components in the upstream enhancer. Regional activation of Spec2a in the aboral ectoderm is conferred by a combination of activators and repressors. The positive regulators include previously identified SpOtx and a trans-regulatory factor binding at the CCAAT site in the Spec2a enhancer. The nuclear protein binding to the CCAAT box was determined to be the heterotrimeric CCAAT binding factor (SpCBF). SpCBF also mediates general activation in the ectoderm. The negative regulators consist of an oral ectoderm repressor (OER), an endoderm repressor (ENR), and an S. Purpuratus goosecoid homologue (SpGsc). OER functions to prevent expression in the oral ectoderm, while ENR is required to repress endoderm expression. SpGsc antagonizes the SpOtx function by competing for binding at SpOtx target genes in oral ectoderm, where it functions as an active repressor. Thus, SpOtx and SpGsc perform collectively to establish and maintain the oral-aboral axis. Finally, purification of ENR and OER proteins from sea urchin blastula stage nuclear extracts was performed using site-specific DNA-affmity chromatography. ^