A new paradigm (Westphal-Paradigm) to study the neural correlates of panic disorder with agoraphobia.

Agoraphobia (with and without panic disorder) is a highly prevalent and disabling anxiety disorder. Its neural complexity can be characterized by specific cues in fMRI studies. Therefore, we developed a fMRI paradigm with agoraphobia-specific stimuli. Pictures of potential agoraphobic situations were generated. Twenty-six patients, suffering from panic disorder and agoraphobia, and 22 healthy controls rated the pictures with respect to arousal, valence, and agoraphobia-related anxiety. The 96 pictures, which discriminated best between groups were chosen, split into two parallel sets and supplemented with matched neutral pictures from the International Affective Picture System. Reliability, criterion, and construct validity of the picture set were determined in a second sample (44 patients, 28 controls). The resulting event-related "Westphal-Paradigm" with cued and uncued pictures was tested in a fMRI pilot study with 16 patients. Internal consistency of the sets was very high; parallelism was given. Positive correlations of picture ratings with Mobility Inventory and Hamilton anxiety scores support construct validity. FMRI data revealed activations in areas associated with the fear circuit including amygdala, insula, and hippocampal areas. Psychometric properties of the Westphal-Paradigm meet necessary quality requirements for further scientific use. The paradigm reliably produces behavioral and fMRI patterns in response to agoraphobia-specific stimuli. To our knowledge, it is the first fMRI paradigm with these properties. This paradigm can be used to further characterize the functional neuroanatomy of panic disorder and agoraphobia and might be useful to contribute data to the differentiation of panic disorder and agoraphobia as related, but conceptually different clinical disorders.

[Occlusive aortic disease as coral reef aorta--experience in 80 cases]. / Der stenosierende Aortenprozess als Coral Reef Aorta--Erfahrungen in 80 Patienten.

BACKGROUND: Coral reef aorta (CRA) is described as rock-hard calcifications in the visceral part of the aorta. These heavily calcified plaques grow into the lumen and can cause significant stenoses, leading to malperfusion of the lower limbs, visceral ischaemia or hypertension due to renal ischaemia. PATIENTS/METHODS: From 1/1984 to 11/2008, 80 patients (26 m, 54 f, mean age 61.6, range 14 to 86 years) underwent treatment in the Department of Vascular Surgery and Kidney Transplantation, Heinrich-Heine-University Hospital for CRA. The present study is based on a review of patient records and prospective follow-up in our outpatient clinic. RESULTS: The most frequent finding was renovascular hypertension (n=33, 41.3%) causing headache, vertigo and visual symptoms. Intermittent claudication due to peripheral arterial occlusive disease was found in 35 cases (43.8%). 15 patients (18.8%) presented with chronic visceral ischaemia causing diarrhoea, weight loss and abdominal pain. 79 patients (98.7%) underwent surgery; in 73 (93.7%) aortic reconstruction was achieved with thromboendarterectomy, on an isolated suprarenal segment in 7 (9.3%), an infrarenal segment in 21 (26.6%), and the supra- and infrarenal aorta in 45 cases (60%). Desobliteration of renal arteries was performed in 47 (one-sided n=8, 10.1%; both arteries n=39, 49.4%); the aortic bifurcation was desobliterated in 37 (46.8%), extension into iliac arteries was necessary in 29 cases (one-sided n=4, 5.1%; both arteries n=25, 31.6%). The coeliac trunk was desobliterated in 43% (n=34), the superior mesenteric artery in 44.3% (n=35) and the inferior mesenteric artery in 20.3% (n=16). In 15 cases additional revascularisation (bypass, transposition, graft interposition) was necessary. Surgical access was via a left-sided thoracoabdominal incision in 56.4% (n=45) and via laparotomy in 41.8% (n=33). The 30-day lethality was 8.7% (n=7). Postoperative complications requiring corrective surgery occurred in 11 patients (13.9%). Almost ⅓ of the patients (n=19, 27.5%) returned for follow-up after a mean of 52.6 months (range 3 to 215 months). Of these, there was significant clinical and diagnostic improvement in 16 (84.2%) and 3 (15.8%) were unchanged. Impairment was not observed. CONCLUSION: In spite of the existing and improving surgical techniques for the treatment of CRA, procedures are challenging and should be performed in centres with expertise.

The suitability of cytochrome-P4501A1 as a biomarker for PCB contamination in European eel (Anguilla anguilla).

European eel (Anguilla anguilla) elvers were intraperitoneally injected with different doses of 3,3',4,4'-tetrachlorobiyphenyl (PCB77) to examine and characterize the inductive effect of coplanar PCBs on CYP1A1 gene expression in liver and gills by using a semi-quantitative RT-PCR analysis. The influence of PCB77 injection on transcription activity of the housekeeping gene glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) was tested to determine its suitability as a reference gene for further quantitative gene expression analyses. Our results clearly indicate a significant dose-dependent increase in CYP1A1 gene expression in the gills of European eel, while in liver tissues a significant elevation in CYP1A1 gene expression was only detectable at highest contamination rates, indicating the potential of CYP1A1 differential gene expression analysis in gills as a biomarker for PCB contamination in eels. PCB77 contamination did not affect GAPDH transcription in gills but, at highest doses, resulted in a significant elevation in liver, speaking against GAPDH as a reference housekeeping gene after PCB exposure.

Identification of Hake species (Merluccius Genus) using sequencing and PCR-RFLP analysis of mitochondrial DNA control region sequences.

The use of DNA-based methodologies in identification of hake species belonging to the Merluccius genus was shown to be successful. A short fragment of the left hypervariable domain of the mitochondrial control region was amplified, sequenced, and digested from 11 hake species. The hake-specific PCR product, due to its limited size, was obtained in a variety of tissue samples with different levels of DNA concentration and degradation, including sterilized food products. On the basis of this phylogenetically informative 156-bp sequence were selected four restriction enzymes (ApoI, DdeI, DraIII, and MboII) that allow the hake species discrimination. Species identification by phylogenetic analysis of sequences or by PCR-RFLP methodologies is useful in a variety of scenarios including authentication of thermally processed food, detection of food components, and species determination of individuals whose morphological characters are removed.

Identification of flatfish (Pleuronectiforme) species using DNA-based techniques.

Identification of flatfish species using a DNA-based methodology was studied. The polymerase chain reaction was employed to obtain a 464 bp amplicon from mitochondrial cytochrome b gene. The sequences from this fragment belonging to 24 species were analyzed using a genetic distance method, and polymorphic sites were determined. The fragment was found to be highly polymorphic (231 sites), and this permitted the differentiation of most of the species. Phylogenetic tree construction was employed to allow the identification of flatfish species. As a result, each species was grouped in a well-differentiated clade, except for two pairs: Limanda ferruginea and L. limanda, and Solea impar and S. lascaris, which could not be differentiated. On the basis of the sequences obtained, restriction enzymes were selected to provide specific restriction profiles, which allow the differentiation of 21 species of flatfish in a faster and less expensive manner than sequencing. This polymerase chain reaction-restriction fragment length polymorphism methodology (PCR-RFLP) was tested using commercial samples.

Development of a DNA-based method aimed at identifying the fish species present in food products.

Analysis of restriction fragment length polymorphism (RFLP) profiles of a 464 bp amplicon obtained from the mitochondrial cytochrome b gene was used to differentiate between several different fish species. The method was tested by a collaborative study in which 12 European laboratories participated to ascertain whether the method was reproducible. Each laboratory was required to identify 10 unknown samples by comparison with RFLP profiles from authentic species. From a total of 120 tests performed, unknown samples were correctly identified in 96% of cases. Further work attempting to use the method to analyze mixed and processed fish samples was also performed. In all cases the species contained within mixed samples were correctly identified, indicating the efficacy of the method for detecting fraudulent substitution of fish species in food products.

Use of restriction fragment length polymorphism to distinguish between salmon species.

Identification of 10 salmon species using DNA-based methodology was investigated. Amplification of DNA was carried out using a primer set which amplified a region of the mitochondrial cytochrome b gene. Sequences of PCR-amplified DNA from the salmon species were used to select six restriction enzymes allowing species to be uniquely classified. RFLP patterns generated following analysis with each enzyme were resolved using polyacrylamide gel electrophoresis and visualized by silver staining. Results indicate that it is possible to differentiate between all 10 salmon species and that the technique could be easily adopted by the food industry for analysis of processed salmon products.

Identification of fish species after cooking by SDS-PAGE and urea IEF: a collaborative study.

A collaborative study, to validate the use of SDS-PAGE and urea IEF, for the identification of fish species after cooking has been performed by nine laboratories. By following optimized standard operation procedures, 10 commercially important species (Atlantic salmon, sea trout, rainbow trout, turbot, Alaska pollock, pollack, pink salmon, Arctic char, chum salmon, and New Zealand hake) had to be identified by comparison with 22 reference samples. Some differences in the recoveries of proteins from cooked fish flesh were noted between the urea and the SDS extraction procedures used. Generally, the urea extraction procedure appears to be less efficient than the SDS extraction for protein solubilization. Except for some species belonging to the Salmonidae family (Salmo, Oncorhynchus), both of the analytical techniques tested (urea IEF, SDS-PAGE) enabled identification of the species of the samples to be established. With urea IEF, two laboratories could not differentiate Salmo salar from Salmo trutta. The same difficulties were noted for differentiation between Oncorhynchus gorbuscha and Oncorhynchus keta samples. With SDS-PAGE, three laboratories had some difficulties in identifying the S. trutta samples. However, in the contrast with the previous technique, SDS-PAGE allows the characterization of most of the Oncorhynchus species tested. Only Oncorhynchus mykiss was not clearly recognized by one laboratory. Therefore, SDS-PAGE (Excel gel homogeneous 15%) appears to be better for the identification, after cooking, of fish such as the tuna and salmon species which are characterized by neutral and basic protein bands, and urea IEF (CleanGel) is better for the gadoid species, which are characterized by acid protein bands (parvalbumins). Nevertheless, in contentious cases it is preferable to use both analytical methods.

Fish muscle parvalbumins as marker proteins for native and urea isoelectric focusing.

An isoelectric point (pI) calibration kit containing fish muscle parvalbumins was prepared and tested for its suitability for isoelectric focusing (IEF) in the presence of 8 M urea. The pattern obtained by urea CleanGel IEF consisted of nine bands covering the pI range 4.96-5.64. This range is relevant for species identification of heated fish by urea IEF. The kit may also be used for native IEF in the low pH range, as demonstrated by running an extract made from the kit together with water-soluble fish muscle proteins on Servalyt Precotes 3-6.

A standardized method of identification of raw and heat-processed fish by urea isoelectric focusing: a collaborative study.

A urea-isoelectric focusing (urea-IEF) method of identifying fish species in processed fishery products was investigated as an interlaboratory collaborative study. The technique was optimized with respect to (i) protein extraction conditions, composition of the extraction solution (urea and SDS solutions), determination of protein concentrations of the fish extracts (five tested methods); (ii) nature of gel (with carrier ampholytes and Immobilines), conditions of rehydration of commercial dry gels, urea concentration; (iii) staining conditions, Coomassie blue and silver staining. The results of various experiments were compared to select the most appropriate methodology, with respect to the discrimination power of differentiating species with the minimal influence of heat processing, reproducibility, speed, and ease of application. The method recommended meets the requirements of food control and customs laboratories.

Visualization of the enzyme trimethylamine oxide demethylase in isoelectric focusing gels by an enzyme-specific staining method.

An enzyme-specific staining method for trimethylamine oxide demethylase (TMAO-ase) was developed. Direct visualization could be reached by coupling the reactions of the specific TMAO-ase assay with another reaction step generating as final product a dark-blue formazan. For these purposes 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) as tetrazolium salt and phenazine methosulfate (PMS) as electron transfer substance were used. Clear, dark-blue colored bands could be detected on 300 microm isoelectric focusing gels (IEF). Comparisons of enzyme-stained and protein-stained gels showed that diffusion could not be observed and that the band pattern of TMAO-ase could also be seen in the protein stain. The pI range where TMAO-ase was located was 5.6-6.6 for extracts and 6.2-6.6 for partially purified TMAO-ase. Specificity of stained TMAO-ase bands was assessed by the preparation of staining solution without the substrate trimethylamine oxide (TMAO) and by extraction of TMAO-ase from the gel and performance of the specific TMAO-ase assay.

Development of a sodium dodecyl sulfate-polyacrylamide gel electrophoresis reference method for the analysis and identification of fish species in raw and heat-processed samples: a collaborative study.

A collaborative study was carried out in seven European labs with the aim of achieving a sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) standard operation procedure to identify fish species in raw and cooked samples. Urea and SDS-containing solutions were evaluated as extractants. Several preelectrophoretic operations--such as treatment with RNase/DNase, ultrafiltration and desalting--and up to ten types of gels and three SDS-PAGE systems were considered. The SDS-containing solution allowed a higher protein extractability than urea. Unlike urea extraction, SDS extraction seemed not to be influenced so much by the state of the sample (raw, cooked at 60 degrees C, cooked at 85 degrees C). Desalting, ultrafiltration or treatment with RNase/DNase did not improve the discriminatory power of the protein patterns. Commercial homogeneous 15% ExcelGels, especially when they were silver stained, yielded good results and afforded higher reproducibility, thus allowing a better matching of results among the laboratories participating in this collaborative study. Under the optimized technical conditions described above, all the fish species tested, either raw and cooked, yielded reproducible and discriminant species-specific protein patterns.

Comparison of different methods to produce single-strand DNA for identification of canned tuna by single-strand conformation polymorphism analysis.

By using single-strand conformation polymorphism (SSCP) analysis of three amplicons of the cytochrome b gene obtained by the polymerase chain reaction (PCR) it was possible to differentiate between various species of tunas and bonitos processed as canned fish. Four different techniques were used to produce single-strand DNA (ssDNA): (i) Denaturation of double-strand DNA (dsDNA) by formamide and alkali, (ii) two-step asymmetrical PCR, (iii) one-step asymmetrical PCR, and (iv) exonuclease digestion of the phosphorylated strand of dsDNA. The technique rendering optimal results depended on the type of amplicon (i.e. the sequence).

Differentiation of scombroid fish species (tunas, bonitos and mackerels) by isoelectric focusing, titration curve analysis and native polyacrylamide gel electrophoresis of sarcoplasmic proteins.

Differentiation of scombroid fishes was possible by electrophoresis of sarcoplasmic proteins using either isoelectric focusing (IEF), titration curve analysis, or native polyacrylamide gel electrophoresis (PAGE). By IEF with Phast-Gels 3-9 species specific patterns, characterized by a few bands in the cathodal part of the gels, were obtained. This type of gel was also used for titration curve analysis. Here, too, the closely related species Thunnus thynnus and T. albacares gave different protein patterns. Native PAGE, native cathodal electrophoresis in Clean Gel 10% with buffer pH 5.5, proved to be a fast and simple method for differentiation of scombroid fishes. As most of the prominent sarcoplasmic proteins of these species have pIs in the neutral or alkaline pH range, they are positively charged at pH 5.5 and move to the cathode.

Performance and blood lactate on Gjessing and Concept II rowing ergometers.

The Gjessing (GE) and the wind resistance (Concept II, CII) rowing ergometers were compared in 11 trained subjects during incremental exercise. Maximum power was 255 (200-370) W on GE, but 294 (204-393) W in CII (median and range, p < 0.05). If power was directly measured by a strain gauge and a displacement transducer in the CII, a 5.1% (3.2%-7.8%) higher maximum performance was obtained (314 [223-413] W, p < 0.05). Maximum stroke rates were higher in GE (33 [27-37]/min) than in CII (29 [24-35]/min, NS). Blood lactate increased faster with work rate and lactic anaerobic threshold was therefore lower in GE. Blood lactate was higher for every heart rate for GE compared to CII. This suggests higher anaerobic effort in GE rowing.

Determination of the heating temperature of fishery products.

The German Fish Directive prescribes that products must be heated to the core temperature of +70 degrees C to kill existing larvae of nematodes. For subsequent determination of the heating temperature samples were extracted with water. The extracts were analysed for protein content, for protein patterns obtained by isoelectric focusing and by using the coagulation test. The suitability of these methods was investigated with heated extracts, heated minced fish flesh, and smoked herring and mackerel. Smoking was performed in the kiln of the institute at controlled temperatures. Analysis of commercial samples showed that the core temperature during smoking of herring and mackerel must have been clearly below 70 degrees C in several cases.

Fish species identification by peptide mapping of the myosin heavy chain.

Fish species of seafood made of washed fish flesh (e.g. imitation crab meat from surimi) were identified by peptide mapping of the myosin heavy chain (MHC). In the first step the MHC was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in 7.5% T gel, followed by a second SDS-PAGE in a 15% T gel with proteolytic digestion of the MHC in the gel. The resulting peptides gave species specific patterns.

Determination of the formaldehyde content in fishery products.

The influence of external factors, such as storage temperature and time on the content of free formaldehyde (FA) in fishery products is described. On the basis of the examination of several methods for the determination of free and bound FA, the following procedures are recommended: (1) for measuring free FA, the samples are extracted using 6% perchloric acid at room temperature and the FA content of the extracts is measured by the formation of 3,5-diacetyl-1,4-dihydrolutidine; (2) bound, acid-labile FA is released by steam distillation using 1% sulphuric acid, giving a pH value of about 1. The FA content of the distillates may be determined either by chromotropic acid assay or by the method described for the extracts.

Activities of chitinase and protease and concentration of fluoride in the digestive tract of Antarctic fishes feeding on krill (Euphausia superba Dana).

Studies on the digestion of krill by Notothenia rossii marmorata, Notothenia neglecta, Champsocephalus gunnari and Chaenocephalus aceratus showed that these Antarctic fish species are well equipped to feed on krill, as indicated by their high levels of chitinase and protease activity. Very high chitinolytic activities were determined in the stomach of the fish species. However, activities that were measured in intestine samples can be substantial, as well. Very strong protease activities were determined in samples of the stomach tissue and the intestinal contents. When krill were present in the guts, the concentrations of fluoride in the stomach and intestinal contents of N. rossii marmorata and Ch. gunnari were extremely high, while the tissues were practically devoid of fluoride.

Solubilization of fish muscle proteins with buffers containing sodium dodecyl sulfate.

The extraction of fish muscle protein using SDS containing solubilization buffers was studied varying the time and the temperature of solubilization, as well as pH and SDS concentration of the buffer. At pH less than 6 the myofibrillar proteins were incompletely solubilized; temperatures of 80-100 degrees C resulted in protein degradation observable in the SDS-PAGE. Samples of fish muscle containing high amounts of formaldehyde (50 mmoles FA/kg wet weight) could only be solubilised at 100 degrees C; on the other hand it was possible to solubilize cooked and/or canned products under mild conditions (2% SDS, 1% 2-ME, pH 8.9, shaking for 2 h at 60 degrees C).