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In situ hybridization and cytofluorometric analysis of cytokine mRNA during in vitro activation of human T cells.

We present an original method for in situ hybridization (ISH) using non isotopic probes and flow cytometry analysis that permits rapid detection of lymphokine transcripts at single cell level in an in vitro activated human Jurkat T cell line and in peripheral blood T cell subsets. After PMA and either ionomycin or ConA stimulation, cells were fixed and hybridized with digoxigenin (DIG)-labelled RNA antisense or sense probes specific for IL-2 and IFN-gamma. The level of cytokine gene expression in individual cells was visualised using FITC-conjugated anti-DIG antibody, and the resultant signal was analysed by flow cytometry. IL-2 mRNA was first detected in activated Jurkat T cells. Addition of cycloheximide 4 hours after the beginning of stimulation increased both the frequency of labelled cells and the amount of mRNA per cell, as determined by the mean fluorescence intensity. The specificity and sensitivity of IL-2 mRNA detection were tested by comparison with Northern blot analysis and in situ hybridization (ISH) with immuno-cytochemical staining. IL-2 and IFN-gamma mRNA were detectable in PBMC as early as 3 hours after in vitro stimulation with PMA and ionomycin. The frequency of positive cells and the amount of mRNA per cell peaked at 6-8 hours, when the percentages of IL-2 and IFN-gamma mRNA-containing cells reached 30-40% and 15-20%, respectively. The two lymphokines were expressed in both CD4+ and CD8+ T cells, but the frequency of IL-2 expressing cells and the amount of IL-2 mRNA per cell were higher in CD4+ (60%) than in CD8+ T cells (25%), whereas IFN-gamma were preferentially transcribed by CD8+ T cells (40%). The results obtained by this method were in accordance with the data obtained by Northern blot analysis, with cellular protein content estimated by immuno-fluorescence staining, and with IL-2 titration by bioassay. We compared the performance of this method with ISH using radioactive probes.

Animals↗

Dynamics of insulin-stimulated translocation of GLUT4 in single living cells visualised using green fluorescent protein.

Insulin increases glucose uptake by promoting the translocation of the GLUT4 isoform of glucose transporters to the plasma membrane. We have studied this process in living single cells by fusing green fluorescent protein (GFP) to the N-terminus (GFP-GLUT4) or C-terminus (GLUT4-GFP), of GLUT4. Both chimeras were expressed in a perinuclear compartment of CHO cells, and in a vesicular distribution through the cytosol. Insulin promoted an increase in plasma membrane fluorescence as a result of net translocation of the chimeras to the cell surface. GLUT4-GFP, but not GFP-GLUT4, was re-internalised upon the removal of insulin suggesting that a critical internalisation signal sequence exists in the N-terminus of GLUT4. The use of GFP thus allows an analysis of GLUT4 trafficking in single living cells.

Animals↗

Characterisation of Upd2, a Drosophila JAK/STAT pathway ligand.

The characterisation of ligands that activate the JAK/STAT pathway has the potential to throw light onto a comparatively poorly understood aspect of this important signal transduction cascade. Here, we describe our analysis of the only invertebrate JAK/STAT pathway ligands identified to date, the Drosophila unpaired-like family. We show that upd2 is expressed in a pattern essentially identical to that of upd and demonstrate that the proteins encoded by this region activate JAK/STAT pathway signalling. Mutational analysis demonstrates a mutual semi-redundancy that can be visualised in multiple tissues known to require JAK/STAT signalling. In order to better characterise the in vivo function of these ligands, we developed a reporter based on a natural JAK/STAT pathway responsive enhancer and show that ectopic upd2 expression can effectively activate the JAK/STAT pathway. While both Upd and Upd2 are secreted JAK/STAT pathway agonists, tissue culture assays show that the signal-sequences of Upd and Upd2 confer distinct properties, with Upd associated primarily with the extracellular matrix and Upd2 secreted into the media. The differing biophysical characteristics identified for Upd-like molecules have implications for their function in vivo and adds another aspect to our understanding of cytokine signalling in Drosophila.

Amino Acid Sequence↗

LALNVIEW: a graphical viewer for pairwise sequence alignments.

LALNVIEW is a graphical program for visualising local alignments between two sequences (protein or nucleic acids). Sequences are represented by coloured rectangles to give an overall picture of their similarities. LALNVIEW can display sequence features (exon, intron, active site, domain, propeptide, etc.) along with the alignment. When using LALNVIEW through our Web servers, sequence features are automatically extracted from database annotations (SWISS-PROT, GenBank, EMBL or HOVERGEN) and displayed with the alignment. LALNVIEW is a useful tool for analysing pairwise sequence alignments and for making the link between sequence homology and what is known about the structure or function of sequences. LALNVIEW executables for UNIX, Macintosh and PC computers are freely available from our server (http:// expasy.hcuge.ch/sprot/lalnview.html).

Acyltransferases↗

Ribosomes stalling on uORF1 in the Xenopus Cx41 5' UTR inhibit downstream translation initiation.

Translation of Xenopus laevis Connexin41 mRNA is strongly controlled by the three upstream open reading frames (uORFs) in its 5' untranslated region. Mutation of uAUG1 into AAG induced a 100-fold increase in translation of a green fluorescent protein (GFP) reporter ORF. The termination codon of uORF1 was mutated and the uORF was linked in-frame with the GFP ORF, enabling visualisation of initiation at uAUG1 by synthesis of an elongated GFP form. Unexpectedly, hardly any elongated GFP was made, suggesting that translation of uORF1 in wild-type mRNA causes constraining of the entry of 40S ribosomal subunits upstream of uORF1. A rare leucine codon, the third codon of uORF1, contributed to the slow translation and thus to slow scanning. Replacement of the rare leucine codon in uORF1 with a common leucine codon stimulated GFP translation. Remarkably, the rare leucine codon, the termination codon of uORF1, uAUG2 and uAUG3 all improved recognition of uAUG1. Apparently, the block formed by a stalled ribosome on any element in uORF1 prevented the landing of new ribosomal subunits next to the cap and therefore downregulated GFP translation.

5' Untranslated Regions↗

Endometrial destruction techniques for heavy menstrual bleeding.

BACKGROUND: Heavy menstrual bleeding (HMB) is a significant health problem in premenopausal women; it can reduce their quality of life and cause anaemia. First-line therapy has traditionally been medical therapy but this is frequently ineffective. On the other hand, hysterectomy is obviously 100% effective in stopping bleeding but is more costly and can cause severe complications. Endometrial ablation is less invasive and preserves the uterus, although long-term studies have found that the costs of ablative surgery approach the cost of hysterectomy due to the requirement for repeat procedures. A large number of techniques have been developed to 'ablate' (remove) the lining of the endometrium. The gold standard techniques (laser, transcervical resection of the endometrium and rollerball) require visualisation of the uterus with a hysteroscope and, although safe, require skilled surgeons. A number of newer techniques have recently been developed, most of which are less time consuming. However, hysteroscopy may still be required as part of the ablative techniques and some of them must be considered to be still under development, requiring refinement and investigation. OBJECTIVES: To compare the efficacy, safety and acceptability of methods used to destroy the endometrium to reduce HMB in premenopausal women. SEARCH STRATEGY: We searched the Cochrane Menstrual Disorders and Subfertility Group Specialised Register of controlled trials (April 2004). We also searched the Cochrane Central Register of Controlled Trials (CENTRAL) (The Cochrane Library Issue 4, 2004), MEDLINE (1966 to July 2004), EMBASE (1980 to July 2004), Current Contents (1993 to week 38, 2001), Biological Abstracts (1980 to June 2001), PsycLIT (1967 to August 2001), CINAHL (1982 to July 2004) and the metaregister of controlled trials and ISRCTN register (December 2004). We also searched reference lists of articles and contacted pharmaceutical companies and experts in the field. SELECTION CRITERIA: Randomised controlled trials comparing different endometrial ablation techniques in women with a complaint of heavy menstrual bleeding without uterine pathology. The outcomes included reduction of heavy menstrual bleeding, improvement in quality of life, operative outcomes, satisfaction with the outcome, complications and need for further surgery or hysterectomy. DATA COLLECTION AND ANALYSIS: The two review authors independently selected trials for inclusion, assessed trials for quality and extracted data. Attempts were made to contact authors for clarification of data in some trials. Adverse events were only assessed if they were separately measured in the included trials. MAIN RESULTS: In the comparison of the newer non-hysteroscopic techniques (second generation) with the gold standard hysteroscopic ablative techniques (first generation) overall, surgery was an average of 15 minutes shorter (weighted mean difference (WMD) 14.9, 95% CI 10.1 to 19.7), local anaesthesia was more likely to be employed (odds ratio (OR) 8.3, 95% CI 3.9 to 17.5) and equipment failure was more likely (OR 4.2, 95% CI 1.3 to 13.8) with second-generation ablation. Women undergoing newer ablative procedures were less likely to have fluid overload, uterine perforation, cervical lacerations and hematometra than women undergoing the more traditional type of ablation and resection techniques (OR 0.13, 95% CI 0.04 to 0.5; OR 0.21, 95% CI 0.07 to 0.7; OR 0.12, 95% CI 0.05 to 0.3 and OR 0.25, 95% CI 0.09 to 0.7, respectively). However, women were more likely to have nausea and vomiting and uterine cramping (OR 2.3, 95% CI 1.5 to 3.4 and OR 1.8, 95% CI 1.1 to 2.9, respectively). Some differences were also found in amenorrhoea rates and satisfaction rates, but there did not appear to be a trend over time so these results may be due to chance. AUTHORS' CONCLUSIONS: Endometrial ablation techniques continue to play an important role in the management of HMB. The rapid development of a number of new methods of endometrial destruction has made systematic comparisons between methods and with the 'gold standard' of transcervical resection of the endometrium (TCRE) difficult. Most of the newer techniques are technically easier than hysteroscopy-based methods to perform. However, uterine perforation, which is the major complication of endometrial ablation, cannot be excluded without hysteroscopy. Overall, the existing evidence suggests that success rates and complication profiles of newer techniques of ablation compare favourably with TCRE, although technical difficulties with new equipment need to be ironed out.

Adult↗

The application of statistical parametric mapping to 123I-FP-CIT SPECT in dementia with Lewy bodies, Alzheimer's disease and Parkinson's disease.

Dopaminergic loss can be visualised using (123)I-FP-CIT single photon emission computed tomography (SPECT) in several disorders including Parkinson's disease (PD) and dementia with Lewy bodies (DLB). Most previous SPECT studies have adopted region of interest (ROI) methods for analysis, which are subjective and operator-dependent. The purpose of this study was to investigate differences in striatal binding of (123)I-FP-CIT SPECT using the automated technique of statistical parametric mapping (SPM99) in subjects with DLB, Alzheimer's disease (AD), PD and healthy age-matched controls. This involved spatial normalisation of each subject's image to a customised template, followed by smoothing and intensity normalisation of each image to its corresponding mean occipital count per voxel. Group differences were assessed using a two-sample t test. Applying a height threshold of P <or= 0.05 corrected, the SPM[t] maps showed a significant bilateral reduced uptake in caudate, anterior and posterior putamen in DLB and PD subjects compared to AD subjects and controls. Significant reduction in binding was also observed bilaterally in the caudate nucleus in AD compared to controls. Striatal binding was indistinguishable between patients with DLB and PD. To investigate the usefulness of SPM as a decision aid in the evaluation of visually rated normal and abnormal patterns of uptake, receiver operator characteristic (ROC) curve analysis was performed using data from single-subject SPMs. The areas under the ROC curves were greater than 0.92, demonstrating comparable discriminatory power with visual rating. The automated voxel-based approach is a viable alternative to the subjective and often time-consuming method of ROI and, in addition, may have the potential to differentiate between normal and abnormal patterns of uptake in a manner similar to visual inspection.

Aged↗

Pre and post operative medical therapy for endometriosis surgery.

BACKGROUND: Endometriosis is a common gynaecological condition which affects approximately 10% of women of reproductive age (Askenazi 1997). There is a range of symptoms and most commonly women present with dysmenorrhoea, pelvic pain, infertility or a pelvic mass. Direct visualisation and biopsy during laparoscopy or laparotomy is the gold standard diagnostic test for this condition and enables the gynaecologist to identify the location, extent and severity of the disease. Surgical therapy can be performed concurrently with diagnostic surgery and may include removal (excision) or destruction (ablation) of endometriotic tissue, division of adhesions and removal of endometriotic cysts. Laparoscopic excision or ablation of endometriosis has been shown to be effective in the management of pain in mild-moderate endometriosis. Adjunctive medical treatment pre or post-operatively may prolong the symptom-free interval. OBJECTIVES: To determine the effectiveness of systemic medical therapies used for hormonal suppression before or after surgery for endometriosis, or before and after surgery for endometriosis in the eradication of endometriosis, improvement of symptoms, pregnancy rates and overall tolerability by comparing them with no treatment or placebo. SEARCH STRATEGY: We searched the Cochrane Menstrual Disorders and Subfertility group trials register (searched 10 September 2003), the Cochrane Central Register of Controlled Trials (CENTRAL) (The Cochrane Library Issue 3 2003), MEDLINE (January 1966 to September 2003), EMBASE (January 1985 to September 2003) and reference lists of articles. We also contacted researchers in the field. SELECTION CRITERIA: Trials were included if they were randomised controlled trials of the use of systemic medical therapies for hormonal suppression before or after, or before and after surgery for endometriosis. DATA COLLECTION AND ANALYSIS: Data extraction and quality assessment was performed independently by using relative risk or weighted mean difference and 95% confidence intervals. MAIN RESULTS: Eleven trials were included in the review. One study comparing pre-surgical medical therapy with surgery alone showed a significant improvement in AFS scores in the medical therapy group (WMD -9.60, 95% CI -11.42 to -7.78) but this may or may not be associated with better outcomes for the patients. Post surgical hormonal suppression of endometriosis compared to surgery alone (either no medical therapy or placebo) showed no benefit for the outcomes of pain or pregnancy rates but a significant improvement in disease recurrence (AFS scores (WMD -2.30, 95% CI -4.02 to -0.58)). There were no trials identified in the search that compared hormonal suppression of endometriosis before and after surgery with surgery alone. There is no significant difference between pre surgery hormonal suppression and post surgery hormonal suppression for the outcome of pain in the one trial identified (RR 1.01, 95% CI 0.49 to 2.07). Information concerning AFS scores and ease of surgery was reported only as a descriptive summary so any difference between the groups can not be quantified from the information in the report of this trial. REVIEWERS' CONCLUSIONS: There is insufficient evidence from the studies identified to conclude that hormonal suppression in association with surgery for endometriosis is associated with a significant benefit with regard to any of the outcomes identified. There may be a benefit of improvement in AFS scores with the pre-surgical use of medical therapy. The possible benefit should be weighed in the context of the adverse effects and costs of these therapies.

Chemotherapy, Adjuvant↗

Liposomes encapsulating polymeric chitosan based vesicles--a vesicle in vesicle system for drug delivery.

Drug delivery systems comprising vesicles prepared from one amphiphile encapsulating vesicles prepared from a second amphiphile have not been prepared previously due to a tendency of the bilayer components of the different vesicles to mix during preparation. Recently we have developed polymeric vesicles using the new polymer-palmitoyl glycol chitosan and cholesterol in a 2:1 weight ratio. These polymeric vesicles have now been encapsulated within egg phosphatidylcholine (egg PC), cholesterol (2:1 weight ratio) liposomes yielding a vesicle in vesicle system. The vesicle in vesicle system was visualised by freeze fracture electron microscopy. The mixing of the different bilayer components was studied by monitoring the excimer fluorescence of pyrene-labelled polymeric vesicles after their encapsulation within egg PC liposomes or hexadecyl diglycerol ether niosomes. A minimum degree of lipid mixing was observed with the polymeric vesicle-egg PC liposome system when compared to the polymeric vesicle-hexadecyl diglycerol ether niosome system. The polymeric vesicle-egg PC vesicle in vesicle system was shown to retard the release of encapsulated solutes. 28% of 5(6)-carboxyfluorescein (CF) encapsulated in the polymeric vesicle compartment of the vesicle in vesicle system was released after 4 h compared to the release of 62% of encapsulated CF from plain polymeric vesicles within the same time period.

Carbohydrate Sequence↗

FBSA: feature-based sequence alignment technique for very large sequences.

The ability to align pairs of very large molecular sequences is essential for a range of comparative genomic studies. However, given the complexity of genomic sequences, it has been difficult to devise a systematic method that can align - even within the same species - pairs of large sequences. Most existing approaches typically attempt to align nucleotide sequences while ignoring valuable features contained within them, eg they filter out low-complexity regions and retroelements before aligning the sequences. However, features are then added post-alignment for visualisation and analysis purposes. We argue that repetitive elements and other features (such as genes, exons and regulatory elements) should be part of the alignment process. A hierarchical approach that aligns the biologically relevant features before aligning the detailed nucleotide sequences has a number of interesting characteristics: (1) features define 'alignment anchor points' that can guide meaningful nucleotide alignment; (2) features can be weighted; (3) a hierarchical approach would identify only meaningful regions to be aligned; (4) nucleotide sequences can be described as sequences of features and non-features, providing a natural mechanism to divide the sequences for processing; and (5) computational speed is significantly faster than other approaches. In this paper, we describe and discuss a feature-based approach to aligning large genome sequences. We refer to this as 'feature-based sequence alignment'.

Algorithms↗

Amino acid uptake in the skeletal muscle measured using [11C]methylaminoisobutyrate (MEAIB) and PET.

An amino acid analogue, [(11)C]MeAIB, recently introduced for oncological positron emission tomography (PET) studies, is a highly selective substrate for insulin-sensitive amino acid transport system A. The aim of this study was to study the uptake kinetics of [(11)C]MeAIB in skeletal muscle in the fasting state and during insulin stimulation. Two dynamic PET studies were carried out in 11 healthy subjects, once in the fasting state and once during euglycaemic hyperinsulinaemia (serum insulin 67+/-12 mU l(-1)). Graphical analysis was used to calculate the fractional [(11)C]MeAIB uptake rate ( K(i)). Amino acid uptake was estimated by multiplying K(i) by the serum amino acid concentration. After tracer injection, rapid uptake in muscle tissue was detected both in the fasting state and during insulin stimulation and femoral muscles were clearly visualised in both studies. In the graphical analysis, the volume of distribution of [(11)C]MeAIB plotted against normalised plasma time yielded a linear curve (the slope of which = K(i)). The fractional [(11)C]MeAIB uptake rate ( K(i)) in the femoral muscle regions increased from 0.0070+/-0.0018 min(-1) (mean+/-SD) in the fasting state to 0.0079+/-0.0020 min(-1) ( P<0.05) during insulin stimulation. When compared with the fasting state, serum total amino acid concentration decreased from 2.49+/-0.22 to 2.16+/-0.18 mmol l(-1) ( P<0.0001) and the serum concentration of six amino acids typically using system A for their transport decreased from 0.72+/-0.1 to 0.63+/-0.07 mmol l(-1) ( P=0.0001) during hyperinsulinaemia. The calculated skeletal muscle total amino acid uptake and the uptake of the six amino acids typically using system A were similar in the fasting state and during insulin clamp (17.1+/-3.2 vs 17.7+/-3.7 micro mol kg(-1) min(-1), NS, and 5.0+/-1.3 vs 5.0+/-1.4 micro mol kg(-1) min(-1), NS, respectively). The uptake rates correlated with perfusion both in the fasting state and during hyperinsulinaemia ( P<0.05). [(11)C]MeAIB PET appears to be a feasible method for measurement of amino acid uptake in human skeletal muscle. As a tracer that is not metabolised in the tissues, [(11)C]MeAIB provides simple modelling and robust data analysis and thus provides a means to investigate amino acid uptake into muscle tissue in various disease conditions known to affect protein metabolism.

Amino Acids↗

A dot-matrix program with dynamic threshold control suited for genomic DNA and protein sequence analysis.

Graphical dot-matrix plots can provide the most complete and detailed comparison of two sequences. Presented here is DOTTER2, a dot-plot program for X-windows which can compare DNA or protein sequences, and also DNA versus protein. The main novel feature of DOTTER is that the user can vary the stringency cutoffs interactively, so that the dot-matrix only needs to be calculated once. This is possible thanks to a 'Greyramp tool' that was developed to change the displayed stringency of the matrix by dynamically changing the greyscale rendering of the dots. The Greyramp tool allows the user to interactively change the lower and upper score limit for the greyscale rendering. This allows exploration of the separation between signal and noise, and fine-grained visualisation of different score levels in the dot-matrix. Other useful features are dot-matrix compression, mouse-controlled zooming, sequence alignment display and saving/loading of dot-matrices. Since the matrix only has to be calculated once and since the algorithm is fast and linear in space, DOTTER is practical to use even for sequences as long as cosmids. DOTTER was integrated in the gene-modelling module of the genomic database system ACEDB3. This was done via the homology viewer BLIXEM in a way that also allows segments from the BLAST suite of searching programs to be superimposed on top of the full dot-matrix. This feature can also be used for very quick finding of the strongest matches. As examples, we analyse a Caenorhabditis elegans cosmid with several tandem repeat families, and illustrate how DOTTER can improve gene modelling.

Amino Acid Sequence↗

The Arabidopsis ABORTED MICROSPORES (AMS) gene encodes a MYC class transcription factor.

Visual screening of a T-DNA mutagenised population of Arabidopsis thaliana for an absence of silique elongation lead to the isolation of the aborted microspores (ams) mutant that shows a sporophytic recessive male sterile phenotype. Homozygous mutant plants are completely devoid of mature pollen. Pollen degeneration occurs shortly after release of the microspores from the tetrad, prior to pollen mitosis I. Premature tapetum and microspore degeneration are the primary defects caused by this lesion, while a secondary effect is visualised in the stamen filaments, which are reduced in length and lie beneath the receptive stigma at flower opening. The disrupted gene was isolated and revealed a T-DNA element to be inserted into the eighth exon of a basic helix-loop-helix (bHLH) gene located on chromosome II. This protein sequence contains a basic DNA binding domain and two alpha helices separated by a loop, typical of a transcription factor belonging to the MYC sub family of bHLH genes. Therefore, AMS plays a crucial role in tapetal cell development and the post-meiotic transcriptional regulation of microspore development within the developing anther.

Amino Acid Sequence↗

Diagnostic utility of tissue tracking echocardiography for the diagnosis of ischaemic heart disease.

INTRODUCTION: Both the resting electrocardiogram and standard echocardiography have limited value in detecting ischaemic heart disease (IHD) in patients with atypical symptoms or asymptomatic subjects. Tissue tracking (TT) is a novel method based on tissue Doppler echocardiography for the assessment of longitudinal apical myocardial motion. AIM: To assess diagnostic utility of TT mode in the diagnosis of IHD. METHODS: The study was performed in a group of 36 patients (aged 58+/-8 years, 15 males) with good acoustic window, sinus rhythm and normal left ventricular ejection fraction on standard echocardiography who were previously selected for coronary angiography. Systolic displacement of myocardium (TT) was assessed in all patients using apical views (4, 2, and 3-chamber) and 7-colour-coded visualisation expressing various apical displacements of the myocardium during systole. Group IHD(-) consisted of 16 patients with normal coronary angiography or insignificant lesions and group IHD(+) consisted of 20 patients with significant (>70%) coronary lesions. RESULTS: Despite similar prevalence of arterial hypertension and diabetes as well as similar pharmacological treatment patients from the IHD(+) group had a lower TT index (ratio of the sum of regional TT values to the number of analysed segments than the IHD(-) (patients 4.5+/-0.8 mm vs 5.9+/-0.9 mm respectively, p <0.001). CONCLUSIONS: Resting echocardiography with tissue tracking enables fast, non-invasive and semiquantitative evaluation of left ventricular function. This method of assessment of longitudinal layers of the left ventricle may be useful in the diagnosis of ischaemic heart disease.

Coronary Angiography↗

Distribution of cDNA for melanocortin receptor subtypes in human tissues.

Distribution of cDNA for five individual melanocortin receptor subtypes in 20 different human tissues was determined by PCR using subtype specific primers. PCR products were first visualised by agarose gel electrophoresis and ethidium bromide staining, and specific products were identified for melanocortin 1 receptor (MC1R) in pituitary and testis, for MC2R in adrenal gland, for MC3R in heart, for MC5R in adrenal gland, fat cells, kidney, leukocytes, lung, lymph node, mammary gland, ovary, pituitary, testis and uterus. The MC4R cDNA could not be detected by ethidium bromide staining. More tissues were revealed as positive when the DNA from PCR were hybridised with subtype specific radioactive probes. All the subtypes except MC4R could be detected in testis. MC4R could only be detected in pituitary. This is the first report describing the comprehensive distribution analysis of melanocortin receptor subtype cDNAs in human tissues, and provides a link between individual receptor subtype and diverse biological activities of melanocortic peptides in the respective target tissues.

Amino Acid Sequence↗

Vasoconstrictor endothelin receptors characterized in human renal artery and vein in vitro.

1. We have identified the endothelin receptors present in the media of human main stem renal artery and vein and characterized the subtypes mediating vasoconstriction in these blood vessels in vitro. 2. Messenger RNA encoding both ETA and ETB receptors was identified in the smooth muscle layer of human renal artery and vein by reverse transcriptase-polymerase chain reaction assay. In cryostat-cut cross-sections of both vessels autoradiographical visualisation suggested a majority of ETA receptors. Intense binding was obtained to the non-selective ligand [125I]-ET-1 and the ETA-selective [125I]-PD151242 but only weak labelling of sites by the ETB-selective [125I]-BQ3020. 3. ET-1 potently constricted renal artery and vein preparations with EC50 values of 4.06 nM and 1.00 nM, respectively. Sarafotoxin 6b was approximately ten times less potent than ET-1 with EC50 values of 36.3 nM and 13.8 nM respectively. In the renal artery, ET-3 and sarafotoxin 6c showed little or no activity up to 300 nM. Responses to these peptides were more variable in the renal vein. Preparations from three individuals did not respond to ET-3 but in three further cases, although ET-3 was much less potent than ET-1, full dose-response curves were obtained. S6c elicited dose-related contractions in vein preparations from 5/6 individuals and although more potent than ET-1, the maximum response was 30-60% of that obtained to ET-1. 4. ET-1-induced vasoconstriction of renal artery and vein was antagonized by the ETA-selective, BQ123 (3-10 microM). The dose-response curves to ET-1 were displaced in a parallel rightward fashion with no attenuation of the maximum responses. pA2 values were estimated to be 6.8 +/- 0.1 and 6.8 +/- 0.4 for artery and vein respectively.5. These data suggest that mRNA encoding both ETA and ETB receptors is present in the media of human main stem renal artery and vein. However, autoradiographical studies indicate that the majority of ET receptors expressed are of the ETA subtype. The relative potencies of ET-1 and ET-3 as vasoconstrictors of renal blood vessels in vitro is consistent with this being an ETA-mediated response,and therefore whilst responses to S6c indicate that constrictor ETB receptors may be present in renal veins from some individuals these are likely to be of less importance in these blood vessels.

Adult↗

Detection of Borrelia burgdorferi in cerebrospinal fluid by the polymerase chain reaction.

The polymerase chain reaction (PCR) was used to amplify specific DNA sequences from different clinical isolates of Borrelia burgdorferi and from cerebrospinal fluid (CSF) of two patients with Lyme disease of the central nervous system. The amplification products were separated by polyacrylamide gel electrophoresis and visualised by ethidium bromide staining. The definitive identification of amplified DNA as a part of the B. burgdorferi flagellin gene was achieved by hybridisation to a 40-base oligonucleotide probe complementary to a part of the spirochaetal gene but not to the primers. Attempts to cultivate borreliae from either patient were unsuccessful and one patient had no serological marker in serum or CSF to indicate borreliosis. Clinical symptoms of both patients regressed with antibiotic therapy. The PCR system is a powerful and rapid technique to amplify flagellin gene sequences from CSF of patients with neuroborreliosis. Only one-tenth of the time needed for cultivation was required from CSF sampling to diagnosis. Gene amplification might, for the first time, allow effective monitoring of therapy for patients with Lyme disease of the central nervous system.

Base Sequence↗

A novel strategy for the immunological tagging of cDNA constructs.

We describe the construction of pBact-myc, an expression vector that incorporates an immunological 'tag' into the produced polypeptide. When transfected into recipient cell lines, tagged protein fragments derived from any source can be visualised using a single monoclonal antibody (mAb). The neuronal-associated protein 2c (MAP2c) was tagged with a sequence encoding a peptide from the human c-myc gene. The preservation of normal function of the tagged protein was shown by transfecting it into cultured cell lines. No difference in binding ability to cellular microtubules could be observed between the myc-tagged MAP2c and the wild-type forms, and both produced the same characteristic changes in microtubule organisation. This approach is being used to study the biological function of selected fragments of MAP2c and other MAP-encoding genes. The pBact-myc expression vector represents a fast and convenient way to produce tagged polypeptides of selected sequences encoding whole proteins or fragments, for the analysis of their function in living cells.

Amino Acid Sequence↗