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PubMed1993

A combined in situ hybridization and immunofluorescence procedure allowing visualisation of peptide mRNA and serotonin in single sections.

We describe a novel procedure for combining immunocytochemistry with in situ hybridisation. In contrast to previously published procedures, the technique involves immunofluorescence followed by in situ hybridization and is particularly suitable for antigens which are labile or sensitive to in situ hybridization processing. We have evaluated the technique using 5-hydroxytryptamine (5-HT, serotonin) immunofluorescence and neuropeptide in situ hybridization employing 35S-labelled oligonucleotide probes. Successful double labelling was obtained and showed that galanin messenger RNA (mRNA) is expressed by 5-HT immunoreactive cells in the dorsal raphe nucleus of the rat. In contrast, somatostatin mRNA in the same region is expressed by a separate non-serotonergic cell population. Double-labelled preparations produced using this technique can be conveniently viewed using epipolarised combined with epifluorescent illumination. Careful analysis of procedural variables revealed that it is not possible to carry out high-sensitivity 5-HT immunocytochemistry following in situ hybridization. The immunostaining is much poorer on slide-mounted sections than on free-floating sections, and 5-HT appears to be lost during the in situ hybridization steps of dehydration/delipidation and incubation in hybridization buffer. The procedure we describe avoids these problems but with a slight loss of in situ hybridization sensitivity.

Animals↗
PubMed1994

The effects of LPS and probucol on interleukin 1 (IL-1) and platelet-derived growth factor (PDGF) gene expression in the human monocytic cell line U-937.

We have investigated the effects of lipopolysaccharide (LPS) and probucol (a lipid soluble antioxidant) on the gene expression of interleukin 1 alpha and beta (IL-1 alpha and IL-1 beta), and platelet-derived growth factor A chain and B chain (PDGF-A and PDGF-B) in the human monocytic cell line U-937. Steady-state mRNA levels were measured quantitatively by the reverse transcription-polymerase chain reaction (RT-PCR) using a non-radioactive label. Cells were incubated with LPS, in the presence or absence of probucol for 20 h. The cells were harvested and RNA was then prepared, reverse-transcribed in the presence of an internal standard and subsequently amplified and labelled with digoxigenin-11-dUTP by the PCR reaction. The PCR products were subjected to agarose gel electrophoresis, blotted onto nylon membranes and visualised by immunological detection of digoxigenin followed by a chemiluminescent reaction. We found that: (1) LPS treatment of U-937 cells caused an up-regulation of gene expression of IL-1 beta and PDGF-A chain by approximately 250% and 100% respectively, although it did not stimulate the expression of IL-1 alpha nor PDGF-B chain mRNA. (2) Probucol treatment in vitro had no effect on the basal or LPS-stimulated mRNA levels of IL-1 alpha, IL-1 beta, PDGF-A and PDGF-B despite its reported activity in vivo. (3) PDGF-A and PDGF-B were expressed at a similar level in unstimulated U-937 cells approximately 10-50 copies/ng total RNA, whereas the expression of IL-1 beta mRNA was approximately 2-4 times higher than IL-1 alpha mRNA. (4) Finally, in U-937 monocytic cells the expression of IL-1 alpha and IL-1 beta, and PDGF-A and PDGF-B appear to be independently regulated.

Base Sequence↗
PubMedDate not supplied

Conventional ultrasonography versus Tissue Harmonic Imaging for the assessment of the common bile duct in cholecystectomized patients.

PURPOSE: The ultrasonographic study of the common bile duct in cholecystectomized patients is very often hampered by the presence of meteorism, scarring tissue, and internal adhesions. The aim of this study was to assess the usefulness of Tissue Harmonic Imaging (THI) in these clinical circumstances. In this technique, the image is formed by using not only the fundamental echo but also its harmonics, which improves the signal-to-noise ratio and considerably reduces the artefacts found in conventional US images. MATERIALS AND METHODS: Seventy-six consecutive cholecystecomized patients (53 F, 23 M, average age 56.9+/-14.5 SD, range 20-81) were assessed. Patients were examined on an empty stomach. The examinations were performed with conventional ultrasonography and with THI. Based on the capability to visualise the distal portion of the common bile duct and in particular to assess the intraluminal content, the study results were rated by three radiologists blinded to each other's evaluation as insufficient (1), sufficient (2), good (3) ad excellent (4). RESULTS: The average diameter of the common bile duct was 4.74+/-2.3 mm (range 1.6-13 mm); at conventional ultrasonography, the visualisation was insufficient in 30% of the cases, sufficient in 52%, good in 18%. With THI the visualisation was sufficient in 17% of the cases, good in 59% and excellent in 24%. Ten out of 76 patients (13%) had stones in the common bile duct. In these cases, conventional ultrasonography was insufficient for diagnosis in 5 patients. All three radiologist (A, B and C) rated THI better than fundamental ultrasonographic imaging (ETG) (p-value <0.0001, Wilcoxon signed rank test). The mean (SD) of the differences between THI and ETG rating were for radiologist A, B and C respectively: 1.25 (0.57), 1.22 (0.51), 1.09 (0.57). Differences for all the radiologists did not significantly vary between older (over 74 years) and younger patients (p-value = 0.35, Wilcoxon rank-sum test). With Cohen's Kw evaluation we obtained better inter-observer agreement (0.71 and 0.80 A and B; 0.7 and 0.79 A and C; 0.71 and 0.81 B and C by conventional ultrasonography and THI). CONCLUSIONS: THI proved to be superior to conventional ultrasonography in the study of the common bile duct in cholecystecomized patients and in particular in the identification of choledocholithiasis.

Adult↗
PubMed1999

Anti-proliferative effect of radiolabelled octreotide in a metastases model in rat liver.

Most neuroendocrine tumours and several other tumours, such as breast carcinoma and malignant lymphoma, express somatostatin receptors (SS-Rs). Lesions expressing these receptors can be visualised by receptor scintigraphy using a low radioactive dose of the radiolabelled SS analogue [111In-DTPA0]octreotide. This radioligand is internalised and transported to the lysosomes with a long residence time of 111In. The aim of this experimental study in rats was to investigate whether the same agent, given in a high radioactive dose, can be used for therapy of hepatic metastases of different tumour cell lines. The development of hepatic metastases was determined 21 days after direct injection of SS-R-positive or -negative tumour cells into the vena porta in rats. On day 1 and/or 8, animals were treated with 370 MBq (0.5 microg) [111In-DTPA0]octreotide. In one experiment, using SS-R-positive tumour cells, animals were pre-treated with a high dose of cold octreotide to block the SS-R by saturation. The number of SS-R-positive liver metastases was significantly decreased after treatment with [111In-DTPA0]octreotide. Blocking the SS-R by octreotide substantially decreased the efficacy of treatment with [111In-DTPA0]octreotide, suggesting that the presence of SS-R is mandatory. This was confirmed by the finding that the number of SS-R-negative liver metastases was not affected by treatment with [111In-DTPA0]octreotide. Therefore, we conclude that (i) high radioactive doses of [111In-DTPA0]octreotide for PRRT (peptide receptor radionuclide therapy) can inhibit the growth of SS-R-positive liver metastases in an animal model, (ii) PRRT is effective only if SS-Rs are present on the tumours, (iii) the effect of PRRT with [111In-DTPA0]octreotide can be reduced by pre-treatment with cold octreotide, which indicates that receptor binding is essential for PRRT. Our data suggest that PRRT with radiolabelled octreotide might be a new promising treatment modality for SS-R-positive tumours.

Animals↗
PubMed1995

Value of a new rapid non-radioactive sequencing method for analysis of the cytomegalovirus UL97 gene in ganciclovir-resistant strains.

Various DNA changes located within a restricted region of the UL97 open reading frame were shown to be associated with the resistance of cytomegalovirus strains to ganciclovir (GCV). In order to analyse this UL97 region in sensitive and GCV-resistant strains, a non-radioactive sequencing assay (Promega, Madison, WI, USA) which combines the dideoxy visualisation by silver-staining of the gel was used. Using this assay, polymerase chain reaction products from results were obtained within 1 day. Point mutations modifying the amino acid sequence of the putative UL97 catalytic site were detected in three isolates. These led to an alanine to valine substitution in residue 594 in one strain with reduced GCV sensitivity, and to a cysteine to glycine substitution in residue 592 in two GCV-resistant isolates. These mutations were different from the DNA changes previously mapped in GCV-resistant laboratory or field strains. No amino acid substitution in the UL97 catalytic site was found in GCV-sensitive isolates. Transfer marker experiments are in progress in order to test the significance of these DNA changes for GCV resistance. This rapid non-radioactive sequencing protocol could be a useful tool for analysing the UL97 region encoding the putative UL97 catalytic site of clinical isolates.

Amino Acid Sequence↗
PubMed1990

Characterisation and visualisation of [3H]dermorphin binding to mu opioid receptors in the rat brain. Combined high selectivity and affinity in a natural peptide agonist for the morphine (mu) receptor.

Dermorphin, Tyr-DAla-Phe-Gly-Tyr-Pro-Ser-NH2, a potent opioid peptide isolated from amphibian skin, is endowed with outstanding structural and biological features. It has no common structure with mammalian opioid peptides and is a unique example of a peptide, synthesized by an animal cell, which contains a D-amino acid in its native sequence. We have undertaken a complete evaluation of the receptor selectivity of dermorphin, together with the binding characteristics and receptor distribution of [3H]dermorphin in the rat brain. 1. Dermorphin was tested for its relative affinity to mu-, delta- and chi-opioid receptors by determining its potency in displacing the selective mu-receptor ligand [3H]Tyr-DAla-Gly-MePhe-Gly-ol (where Gly-ol = glycinol), the prototypic delta-receptor ligand [3H]Tyr-DPen-Gly-Phe-DPen (where DPen = beta, beta-dimethylcysteine) and the chi ligand [3H]ethylketocyclazocine from rat brain and/or guinea pig cerebellum membrane preparations. Inhibitory constant (Ki) values of dermorphin were 0.7 nM, 62 nM and greater than 5000 nM respectively for mu, delta and chi sites, indicating a selectivity ratio Ki(delta)/Ki(mu) = 88. Under similar conditions, Tyr-DAla-Gly-MePhe-Gly-ol, which is regarded as one of the most selective high-affinity mu-agonist available, exhibited a selectivity ratio of 84. 2. Specific binding properties of tritium-labeled dermorphin (52 Ci/mmol) were characterized in the rat brain. Equilibrium measurements performed over a large range of concentrations revealed a single homogeneous population of high-affinity binding sites (Kd = 0.46 nM; Bmax = 92 fmol/mg membrane protein). 3. Profound differences were observed in the potencies displayed by various selective opiates and opioids ligands in inhibiting the specific binding of [3H]dermorphin. The rank order of potency was in good agreement with that obtained with other mu-selective radiolabeled ligands. 4. Receptor autoradiography in vitro was used to visualize the distribution of [3H]dermorphin binding sites in rat brain. The labeling pattern paralleled that observed using other mu probes. Binding parameters and selectivity profile of [3H]dermorphin on slide-mounted sections were similar to those obtained with membrane homogenates. 5. Finally, intracerebroventricular administration of synthetic dermorphin into mice showed that this peptide is the most potent analgesic known to date, being up to 5 and 670 times more active than beta-endorphin and morphine, respectively. Higher doses induced catalepsy. The overall data collected demonstrate that dermorphin is the first among the naturally occurring peptides to be highly potent and nearly specific super-agonist towards the morphine (mu) receptor.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗
PubMed1993

Puffs and PCR: the in vivo dynamics of early gene expression during ecdysone responses in Drosophila.

The steroid hormone ecdysone orchestrates insect development by regulating gene networks. In Drosophila the most detailed description of ecdysone action is the sequential activation of early and late puffs in the polytene chromosomes of the late larval salivary gland. A number of these early puffs (2B5, 74EF and 75B) contain complex transcription units (Broad-Complex, E74 and E75 respectively) encoding families of regulatory proteins which are expressed in most if not all tissues. In vitro, transcripts of the different isoforms of these early genes as well as the ecdysone receptor (EcR) present varying dose response characteristics (Karim and Thummel, 1992, EMBO J. 11, 4083-4093). We have developed an in vivo approach using a reverse transcription-polymerase chain reaction assay (RT-PCR) so as to visualise these transcripts in the RNA extracted from a single salivary gland. Using one salivary gland lobe for developmental puff staging and the sister lobe for RT-PCR, we have obtained precise developmental profiles for these transcripts and have extended our study to other tissues and stages where puffing studies were not possible. In the salivary gland we have characterised three distinct ecdysone responses. For the mid and late third larval instar responses our results confirm and extend the conclusions of the in vitro studies concerning the temporal expression of the early gene isoforms. The relatively brief prepupal response contains elements in common with each of the larval responses and all three can be explained by the profiles of the respective ecdysone peaks. Interestingly EcR transcripts respond differently during each response. The analysis of different tissues of the same animal reveals subtle differences in the timing of the ecdysone response and isoform expression and suggests that this may reflect tissue differences in the ecdysone profiles. As these molecules have homologues in vertebrates, our analysis may have general implications for the organisation of hormonal responses in vivo.

Animals↗
PubMed2002

Effect of angiotensin converting enzyme inhibition on myocardial phosphoinositide metabolism visualised with 1-[1-11C]-butyryl-2-palmitoyl-rac-glycerol in myocardial infarction in the rat.

We recently reported that myocardial phosphoinositide (PI) metabolism can be visualised by 1-[1-11C]-butyryl-2-palmitoyl-rac-glycerol (11C-DAG) in rats with myocardial infarction (MI). Angiotensin II, the receptors for which are expressed predominantly in infarcted areas with active fibrogenesis rather than in non-infarcted regions, is involved in the upstream signalling systems of PI metabolism and plays an important role in the process of left ventricular (LV) remodelling after MI. We therefore hypothesised that the distribution of 11C-DAG after MI may be affected by the inhibition of angiotensin converting enzyme, which is one of the most important factors in the development of LV remodelling after MI. Rats were injected with 11C-DAG after 3 or 10 weeks of treatment with captopril or no treatment following coronary artery ligation, and quantitative autoradiography was performed. Cells occupying the infarcted region were identified by immunohistochemistry. Compared with untreated rats, treatment with captopril for 3 weeks after MI elicited a reduction in the 11C-DAG uptake in the infarcted region (P<0.05) but not in the non-infarcted region, and was associated with a 22% decrease in the heart weight/body weight ratio. The thallium-201 distribution in the infarcted area was similarly low in the rats with and rats without the 3-week captopril treatment after MI. Abundant macrophages and myofibroblasts occupied the infarcted area in both rats with and rats without the captopril treatment for 3 weeks after MI. The 11C-DAG radioactivity in the infarcted region in the untreated rats was lower 10 weeks after MI than 3 weeks after MI (P<0.01). This finding was in agreement with the results of immunohistochemistry demonstrating that the number and size of macrophages and myofibroblasts were remarkably reduced in rats 10 weeks after MI compared with 3 weeks after MI. Captopril treatment for 10 weeks after MI did not decrease the 11C-DAG radioactivity in the infarcted area further. These data suggest that 11C-DAG is useful for visually detecting regions with activated PI metabolism after MI, and that captopril reduces PI metabolism in the infarcted region in the relatively early phase of MI, which might contribute to the attenuation of ventricular remodelling.

Angiotensin-Converting Enzyme Inhibitors↗
PubMed2000

The replication origin of Azotobacter vinelandii.

The putative replication origin of Azotobacter vinelandii was cloned as an autonomously replicating fragment after ligation to an antibiotic resistance cartridge. The resulting plasmids could be isolated and labelled by Southern hybridisation with the antibiotic resistance cartridge as probe and also visualised by electron microscopy. These plasmids integrated into the chromosome after a few generations, even in the recA mutant of A. vinelandii. The integrated copy of the plasmid was re-isolated from the chromosome and the DNA and its subfragments were cloned in the plasmid vector pBR322. A 200-bp DNA fragment was sufficient to allow the replication of pBR322 in an Escherichia coli polA strain. Electron microscopic analysis of this plasmid showed that replication initiated mostly within the A. vinelandii DNA fragment. The nucleotide sequence of the putative replication origin and its flanking regions was determined. In the sequence of the 200-bp fragment many of the distinctive features found in other replication origins are lacking. A greater variation from the consensus DnaA binding sequence was observed in A. vinelandii. Direct sequencing of the relevant genomic fragment was also carried after amplifying it from A. vinelandii chromosomal DNA by PCR. This confirmed that no rearrangements had taken place while the cloned fragment was resident in E. coli. It was shown by hybridisation that the 200-bp chromosomal origin fragment of A. vinelandii was present in three other field strains of Azotobacter spp.

Azotobacter vinelandii↗
PubMed1996

Generation of three-dimensional medical thermograms.

To visualise non-invasively human organs in their true form and shape has intrigued mankind for centuries. Three-dimensional (3D) imaging is one recent development that has brought us closer to fulfilling the age-old quest of non-invasive visualisation so that diagnoses by doctors can be efficiently enhanced. Nowadays, 3D CT and MRI images have been very popular. Thermography is an important medical imaging technique that displays the temperature distribution on the surface of a human organ and it has been proved to be significant in offering a unique physiological reflection of pathology that may confirm or enhance the anatomic findings of other diagnostic imaging modalities. It is the only imaging modality that can evaluate pain whereas plain radiographs, CT and MRI, etc. can only depict structural anatomic abnormalities that may not always coincide with patients' clinical complaints. It is against this background that 3D thermograms have been developed. A set of comprehensive calibration procedures for the 3-camera system have been designed based on different models for the optical and infrared cameras. The accuracy of the results is high enough to produce 3D thermograms that can be used to correlate with the 3D images from other medical imaging modalities. One important achievement of the system is that the resultant 3D images are absolutely dimensioned and hence, it is particularly favourable for fully autonomous applications with robots. The system can also provide an overall picture of both the structural abnormalities and nervous responses of patients.

Algorithms↗
PubMed2006

Use of intra-cardiac ultrasound in the diagnosis of prosthetic valve malfunction.

A 46-year old lady was under regular follow up for aortic valve replacement done in 2002 for aortic stenosis. The valve was a Carbomedics 19 mm bi-leaflet aortic valve prosthesis for which she had adequate anticoagulation since implantation. She had a past history of end stage renal failure, type 2 diabetes, hypertension, cerebrovascular disease and systemic lupus erythematosus. She was asymptomatic and had a routine transthoracic echocardiogram performed which revealed that her aortic valve prosthesis was well seated with an elevated velocity across the valve; the aortic valve leaflets were poorly visualised but appeared to be mobile. A transoesophageal echocardiogram (TOE) confirmed the markedly increased forward velocity across the aortic valve (maximum velocity 4.6 m/s) with small aortic root raising the possibility of pressure recovery phenomenon. Once again the leaflets were not clearly seen but appeared mobile despite the use of deep trans-gastric view. Fluoroscopy was performed and revealed that one of the leaflets was not moving. The patient had an intra-cardiac ultrasound scan (ACUSON AcuNav; Siemens) with the probe of the scanner within the right atrium. A long-axis view demonstrated the prosthetic aortic valve leaflets clearly (Fig. 1). A short-axis view of the prosthetic valve revealed an echogenic area at the six o'clock position; this may be due to pannus formation (Fig. 2); colour flow across the valve during systole revealed absence of colour flow though one of the leaflets due to the leaflet being stuck (Fig. 3). The sewing cuff of the Carbomedics valve is coated with biolite carbon, which is an anti-thrombotic agent that prevents adhesion of thrombus or pannus on the sewing cuff. There are few reports of Carbomedics valve dysfunction by pannus formation in the mitral position but none in the aortic position. Fluoroscopy can be used to visualize mobility of valve leaflets but is unable to identify thrombus/pannus formation that may be causing the valve to stick. Intra-cardiac echocardiography (ICE) can provide additional information regarding potential causes of valve dysfunction such as pannus/thrombus formation. However, ICE does suffer from the same limitations as TOE in obtaining sufficiently adequate images of the aortic valve to allow appropriate assessment of leaflet motion and insight into flow channels; it is also more invasive and expensive compared with other imaging modalities. In our patient ICE provided better images than TOE, possibly due to the closer proximity of the probe to the aortic root compared with TOE. ICE is a novel tool in the assessment of prosthetic valve function, which can complement data obtained from transthoracic/transoesophageal echocardiography and fluoroscopy.

Aortic Valve Stenosis↗
PubMed1997

Antagonistic effect of synthetic peptides corresponding to the binding regions within fimbrial subunit protein from Porphyromonas gingivalis to human gingival fibroblasts.

Specific binding region within fimbrial subunit protein (fimbrilin) from Porphyromonas gingivalis strain 381 was studied in cultured human gingival fibroblasts. Fluorescent micrographs visualised FITC-labelled fimbriae of P. gingivalis specifically bound to normal human fibroblast cell line (Gin-1) along the cell surface. Flow cytometric analysis also revealed the binding of FITC-labelled fimbriae to Gin-1 cells. Synthetic peptides composed of residues 1-20 (AFGVGDDESKVAKLTVMVYN) of the fimbrilin from P. gingivalis, FP381 (1-20), FP381 (69-80; ALTTELTAENQE) and FP381 (171-181; DA-NYLTGSLTT) definitely inhibited P. gingivalis fimbria-binding to Gin-1 cells by enzyme-linked immunosorbent assay (ELISA). Furthermore, based on the Scatchard plot analysis of the binding of 125I-labelled P. gingivalis fimbriae to Gin-1 cells, the apparent dissociation constant (Kd) was calculated as 15.9 pM, and the number of binding sites (Rt) was estimated as 150 sites/cell. Binding studies of 125I-labelled FP381(171-181) also revealed the presence of a non-interacting, single class of affinity binding sites: the apparent Kd and Rt were 29.2 nM and 18440 sites/cell on Gin-1 cells, respectively. These results demonstrate that specific binding regions on P. gingivalis fimbriae to human gingival fibroblasts are present, and certain corresponding peptides clearly inhibited the binding of P. gingivalis fimbriae to human gingival fibroblasts.

Amino Acid Sequence↗
PubMed2003

[Sonografic evaluation of oxygenation in head and neck cancer].

BACKGROUND: Tumor oxygenation is an important aspect of radiosensitivity. The authors describe a new method for a non-invasive assessment of tumor oxygenation in head and neck cancer. PATIENTS AND METHODS: A group of 20 patients with neck metastases of squamous cell cancer of the head and neck region was surveyed. At first a pO (2)-polarography was performed in the metastatic cervical nodes to investigate the tissue oxygenation. In a second step, the vascularisation of these nodes was visualised by color duplex sonography. In order to evaluate the extent of vascularisation in these nodes, the density of color pixels was quantified by a custom-made software program. The color pixel density and the pO (2) values were correlated and the statistic significance was calculated by Pearson's test. RESULTS: The mean vascularisation as evaluated by the means of color duplex sonography was 7.78 % [95 % CI 6.04 - 9.51]. The interindividual pO (2) values in the stroma of metastatic lymph nodes ranged between 9.0 and 27.4 mmHg (16.6 [95 % CI 14.06 - 19.13]). The mean values of pO (2)-fractions < 2.5/< 5.0/10 mm Hg were 32.25 %, 44.25 % and 53.29 % respectively. The median value of the pO (2)-fraction was 10.49 % [95 % CI 7.13 - 13.85]. The vascularisation as evaluated by color pixel density showed a statistically significant correlation with the pO (2)-fractions < 5.0 and < 10 mmHg (p < 0.045 and < 0.0001) and with the mean (p < 0.002) and median values of tissue pO (2) values (p < 0.0001) in polarography. CONCLUSION: The results in a limited number of patients suggest, that the proposed sonographic method allows a reliable non-invasive evaluation of tissue oxygenation in cervical metastases of squamous cell head and neck cancers.

Adult↗
PubMed1996

Neuropeptide Y gene expression and receptor autoradiography in hypertensive and normotensive rat brain.

Neurones containing neuropeptide Y (NPY) may participate in central cardiovascular control by tonically influencing barosensitive neurones within the nucleus tractus solitarius. The present study has employed both in situ hybridisation histochemistry and receptor autoradiography, to visualise the expression of prepro-NPY mRNA in the forebrain and to determine the NPY receptor subtype(s) in the brainstem, respectively. Prepro-NPY gene expression was visualised in the hypothalamus, cortex, dentate gyrus and lateral reticular thalamus from age-matched spontaneously hypertensive rats (SHR) and normotensive Don Ryu rats (DRY) and Wistar Kyoto rats (WKY). Quantitative densitometry revealed an increase in the NPY transcript in the arcuate nucleus of SHR rats compared to their normotensive counterparts. Autoradiography using [125I]Bolton-Hunter-NPY (BH-NPY, 15 pM) demonstrated NPY binding sites in the area postrema, the commissural nucleus tractus solitarius (cNTS) and the inferior olivary complex. NPY (1 microM) and peptide YY (1 microM), but not [Leu31,Pro34]NPY (10-100 nM), fully inhibited the binding of [125I]BH-NPY. These results indicate that NPY receptors of the Y2 subtype predominate in the dorsal vagal complex. Unilateral nodose ganglionectomy resulted in a partial loss of NPY binding sites in the commissural NTS, but not the area postrema, suggesting that a proportion of binding sites (Y2 subtype) are present on central vagal terminals. While all three rat strains appear to have the same relative proportions of NPY receptor subtypes in the brainstem, the relevance of the differential NPY gene expression in the arcuate nucleus regarding central cardiovascular control mechanisms and/or the pathogenesis of hypertension remains to be elucidated.

Animals↗
PubMed1999

Observer variability in cancer detection during routine repeat (incident) mammographic screening in a study of two versus one view mammography.

OBJECTIVE: To examine the reasons for observer variability of cancer detection using one and two view mammography at incident (subsequent) screening and determine whether false negative results (non-recall of a cancer) are due to failure to detect the associated features(s) of the cancer on the mammogram, or misinterpretation of the observed feature(s) as not indicative of malignancy. SETTING: A random selection of cancers (invasive and in situ) seen as incident cases during the second screening round (January 1994-January 1997) in the South West London Breast Screening Service were used. This service uses two view mammography and double reading with arbitration by a third or further readers for all screens. METHODS: Mammograms of cases were mixed with those of controls in a 1:2 ratio in two test sets. Eleven experienced film readers, each reading both test sets, took part in the study. Initially the oblique view only was read, then, additionally, the craniocaudal view. Previous films were available to the readers. Data on abnormalities noted on the films and probability of recall were recorded and analysed. RESULTS: 387 valid readings of 36 cancers (30 invasive and six ductal carcinoma in situ) were made by 11 readers. The overall sensitivity increased from 79% with one view to 85% with two views. For invasive cancers < 10 mm the sensitivity was 71% with one view, but increased to 85% with two views. Recall of individual cancers by the readers varied substantially. With one view 15 (50%) of the 30 invasive cancers were recalled by all 11 readers, increasing to 18 (60%) with two views. Of the invasive cancers not recalled by all 11 readers, there was considerable disagreement, particularly for the smaller cancers. With one view 69% of invasive cancers < 10 mm were correctly marked on the proforma compared with 87% with two views. Invasive cancers > 10 mm were almost all marked on the proforma with one or two views. For invasive cancers, the misinterpreted feature that did not lead to recall was most commonly an asymmetry (42%), whereas for in situ cancers it was calcifications (67%). The finding of an irregular mass was the least misinterpreted feature. CONCLUSION: The study showed that of those invasive cancers detected at routine repeat screening by a programme using two view mammography and double reading with arbitration, at least 50% could be described as "difficult" (for example, "minimal" signs) to recall using the single reading of one view, even under "favourable" study conditions with two normal subjects per case. The finding that at least 87% of invasive cancers < 10 mm are detected (marked on the proforma) with two views, but only 69% with the one view, suggests that for single reading of mammograms with one view the detection of small invasive cancers is a major problem. This problem is helped by the second view. For invasive cancers > or = 10 mm, interpretation (benign or malignant) rather than detection (under these study conditions) was the major cause of recall failure. The most common signs to be misinterpreted were calcifications and asymmetries; once visualised an irregular mass was least likely to be misinterpreted. This study provides evidence that detection and interpretation of most invasive cancers is improved by increasing the number of views, and by increasing the number of readers.

Breast Neoplasms↗
PubMed2002

Homonucleotide stretches in chromosomal DNA of Campylobacter jejuni display high frequency polymorphism as detected by direct PCR analysis.

Homopolymeric nucleotide tracts have been previously identified in the genome sequence of Campylobacter jejuni 11168 [Parkhill et al., Nature 403 (2000) 665-668]. These tracts are believed to regulate contingency genes but as yet no phenotypic variation has been identified associated with many of these genes. To investigate homopolymeric tracts for genes for which there is no observable phenotype, a method was designed to visualise profiles of the various tract lengths directly at the genomic level by means of PCR and denatured polyacrylamide gel electrophoresis. Six of the seven contingency genes investigated displayed variation in the length of the respective homonucleotide tracts. Surprisingly, each contingency gene gave a typical peak profile that represented a conserved size distribution of polymorphic forms. For each gene studied, peak profiles were conserved between strains of C. jejuni. Duplicated genes, containing homonucleotide stretches, displayed locus-specific peak distributions for each gene copy. Contingency genes were polymorphic within single colonies, and the observed complex peak profiles suggested a frequency of slippage several orders of magnitude higher than reported for other organisms. No G7 (or C7) stretch was ever observed, and their absence from the complete genome suggests strong selection against their presence. In view of the predictable outcome of the process leading to these polymorphisms, it is hypothesised that the formation and/or selection of these tracts is not a random process, but is driven by as yet unknown mechanism(s). High-frequency polymorphism of these genes may be a mechanism by which C. jejuni survives selection bottlenecks between opportunities for growth within a host.

Animals↗
PubMed2004

Purification, identification and characterisation of seprase from bovine serum.

The study and identification for the first time of a soluble form of a seprase activity from bovine serum is presented. To date, this activity has only been reported to be an integral membrane protease but has been known to shed from its membrane. The activity was purified 30,197-fold to homogeneity, using a combination of column chromatographies, from bovine serum. Inhibition by DFP, resulting in an IC(50) of 100:nM confirms classification as a serine protease. The protease after separation and visualisation by native PAGE was subjected to tryptic digestion and the subsequent peptides sequenced. Each peptide sequenced was found to be present in the primary structure of seprase/fibroblast activation protein (FAP), a serine gelatinase specific for proline-containing peptides and macromolecules. Substrate specificity studies using kinetic, RP-HPLC and LC-MS analysis of synthetic peptides suggest that this peptidase has an extended substrate-binding region in addition to the primary specificity site S(1). This analysis revealed at least five subsites to be involved in enzyme-substrate binding, with the smallest peptide cleaved being a tetrapeptide. A proline residue in position P(1) was absolutely necessary therefore showing high primary substrate specificity for the Pro-X bond, while a preference for a hydrophobic residue at the C-terminal end of the scissile bond (P'(1)) was evident. The enzyme also showed complete insensitivity to the prolyl oligopeptidase specific inhibitors, JTP-4819, Fmoc-Ala-pyrrCN and Z-Phe-Pro-BT. To date, no physiological substrate has clearly been defined for this protease but its ability to effectively degrade gelatin suggests a candidate protein substrate in vivo and a possible role in extracellular matrix protein degradation.

Amino Acid Sequence↗
PubMed1994

Localisation of sulfakinin neuronal pathways in the blowfly Calliphora vomitoria.

The distribution of neurones immunoreactive to antisera raised against the undecapeptide C-terminal fragment of drosulfakinin II (DrmSKII), Asp-Gln-Phe-Asp-Asp-Tyr(SO3H)-Gly-His-Met-Arg-Phe-NH2, has been studied in the blowfly Calliphora vomitoria. Antisera were preabsorbed with combinations of the parent antigen, the tetrapeptide Phe-Met-Arg-Phe-NH2 and cholecystokinin, the vertebrate sulfated octapeptide (CCK-8), Asp-Tyr(SO3H)-Met-Gly-Trp-Met-Asp-Phe-NH2, in order to ensure specificity for the sulfakinin peptides of C. vomitoria (the nonapeptide callisulfakinin I is identical to drosulfakinin I and callisulfakinin II differs from DrmSK II only by the presence of -Glu3-Glu4- in place of -Asp3-Asp4-). Only four pairs of sulfakinin-immunoreactive neurons have been visualised in the entire nervous system. These occur in the brain: two pairs of cells situated medially in the caudo-dorsal region close to the roots of the ocellar nerve and two other pairs at the same level but positioned more laterally. Despite the small number of sulfakinin-immunoreactive cells, there are extensive projections to many areas of neuropile in the brain and the thoracic ganglion. The pathway of the medial sulfakinin cells extends into each of the three thoracic ganglia and a metameric arrangement of sulfakinin neuronal projections is also seen in the abdominal ganglia. Neither the dorsal neural sheath of the thoracic ganglion, nor the abdominal nerves contain sulfakinin-immunoreactive material. These observations suggest that the sulfakinins of the blowfly function as neurotransmitters or neuromodulators. They do not appear to have a direct role in gut physiology, as has been shown by in vitro bioassays for the sulfakinins of orthopterans and blattodeans. In addition to the neurones that display specific sulfakinin immunoreactivity, other cells within the brain and thoracic ganglion are immunoreactive to cholecystokinin/gastrin antisera. There are, therefore, at least two types of dipteran neuropeptides with amino acid sequences that are similar to the vertebrate molecules cholecystokinin and gastrin.

Amino Acid Sequence↗