Trafficking and signalling of gonadotrophin-releasing hormone receptors: an automated imaging approach

Gonadotrophin-releasing hormone (GnRH) is a neuropeptide that mediates central control of reproduction by stimulating gonadotrophin secretion from the pituitary. It acts via 7 transmembrane region (7TM) receptors that lack C-terminal tails, regions that for many 7TM receptors, are necessary for agonist-induced phosphorylation and arrestin binding as well as arrestin-dependent desensitization, internalization and signalling. Recent work has revealed that human GnRH receptors (GnRHR) are poorly expressed at the cell surface. This apparently reflects inefficient exit from the endoplasmic reticulum, which is thought to be increased by pharmacological chaperones (non-peptide GnRHR antagonists that increase cell surface GnRHR expression) or reduced by point mutations that further impair GnRHR trafficking and thereby cause infertility. Here, we review recent work in this field, with emphasis on the use of semi-automated imaging to interrogate compartmentalization and trafficking of these unique 7TM receptors.This article is part of a themed section on Imaging in Pharmacology. To view the editorial for this themed section visit http://dx.doi.org/10.1111/j.1476-5381.2010.00685.x     

Analysis of VH genes used by neoplastic B cells in endemic Burkitt's lymphoma shows somatic hypermutation and intraclonal heterogeneity

Tumor cell lines from six typical cases of endemic Epstein-Barr virus (EBV) genome-positive Burkitt's lymphoma (BL) have been investigated for usage and mutational pattern of Ig VH genes. The neoplastic cells all had a t(8;14) (q24;q32) translocation involving the c-myc protooncogene. The VH genes were derived from VH1, VH3 and VH4, and both the IgM-positive (four cases) and IgG-positive (two cases) were extensively mutated from germline sequence. In two cases, early and late passage tumor cells were available, and the VH nucleotide sequences were identical, indicating that mutations had not accumulated in vitro. In a further case, there was evidence of sequence heterogeneity, which appeared to have been generated in vivo, indicating that the tumor cell VH gene was able to undergo posttranslocation somatic hypermutation. Analysis of the relatively nonpolymorphic VH4 genes for the pattern of replacement or silent mutations did not show a role for antigen selection in the expressed sequences.     

Intravascular local gene transfer mediated by protein- coated metallic stent

Objective To assess the feasibility, efficiency and selectivity of adenovirus- mediated ge ne transfer to local arterial wall by protein- coated metallic stent. Methods A replication- defective recombinant adenovirus carrying the Lac Z reporter gene for nuclear- specific β- galactosidase (Ad- βgal) was used in this study. Th e coating for metallic stent was made by immersing it in a gelatin solution cont aining crosslinker. The coated stents were mounted on a 4. 0 or3. 0mmpe rcutaneous transluminal coronary angioplasty (PTCA) balloon and submersed into a high- titer Ad- βgal viral stock (2×10(10)pfu/ml) for 3 min, and then im planted into the carotid arteries in 4 mini- swines and into the left anterior d escending branch of the coronary artery in 2 mini- swines via 8F large lumen gui ding catheters. The animals were sacrificed7 (n=4), 14 (n=1) and 21 (n=1) days after implantation, respectively. The β- galactosidase expression was as sessed by X- gal staining. Results The results showed that the expression of transgene was detected in all animal. In 1 of carotid artery with an intact intima, the β- gal expression was l imited to endothelial cells. In vessels with denuded endothelium, gene expressi on was found in the sub- intima, media and adventitia. The transfection efficie ncy of medial smooth muscle cells was 38. 6%. In 2 animals sacrificed 7 days af ter transfection, a microscopic examination of X- gal- stained samples did not s how evidence oftransfection in remote organs and arterial segments adjacent to the treated arterial site.Conclusions Adenovirus- mediated arterial gene transfer to endothelial, smooth muscle cells and adventitia by protein- coated metallic stent is feasible. The transfection efficiency is higher. The coated stent may act as a good carrier of adenovirus - mediated gene transfer and have a potential to prevent restenosis following PT CA.     

Antibodies to Cardiolipin in Stroke: Association with Mortality and Functional Recovery in Patients Without Systemic Lupus Erythematosus

Antibodies to cardiolipin were measured in 100 consecutive patientswith first ever stroke, on admission and at three and six monthsafter the acute event. One hundred healthy, age-and sex-matched,British elderly individuals were also screened for antibodiesto cardiolipin as a control group. Elevated levels of anticardiolipin antibody (i.e. 5SD abovethe laboratory control mean) were present in none of the controlgroup, but in 21 per cent of the patients with stroke. Thirteenof these 21 patients (62 per cent) died within three months,compared to 17 (21.5 per cent) of the seventy-nine patientswithout elevated levels of anticardiolipin antibodies (p<0.001).Six of the eight survivors with persistently elevated anticardiolipinantibodies had significant residual disability following stroke(Barthel score 0–9) compared to 11 of the 62 without (p<0.001).Two patients with initially raised anticardiolipin antibodieswho became independent at six months showed a progressive declinein the level of these antibodies to normal. The presence of high levels of anticardiolipin antibody didnot correlate with other recognized prognostic indices of stroke,except for incontinence. No correlation was noted between levelsof antibody to cardiolipin, antinuclear factor, antibody todouble-stranded DNA and C-reactive protein, either in the strokepatients or in the elderly control population. Hypertensionwas significantly more common in the patients with high anticardiolipinantibodies than in the rest of the patients in the stroke population(p=0.33). There was no correlation between levels of anticardiolipinantibody and age. Anticardiolipin antibody may be consideredas an independent prognostic marker for both mortality and clinicaloutcome after acute stroke.     

Liver Damage in Long-Term Anticonvulsant Therapy: A Serological and Histological Study

The prevalence of liver damage in patients receiving long-termanticonvulsant therapy was determined, using a new marker ofliver disease, the serum F protein concentration. Abnormal serumF protein concentrations were detected in 50 per cent of 34patients receiving anticonvulsant therapy. A retrospective analysisof post-mortem liver samples showed common histological abnormalitiesin three out of seven patients who had died whilst receivinganticonvulsant therapy. These changes were not seen in controlpatients. We suggest that chronic anticonvulsant therapy maycause significant hepatocellular damage.     

Pseudoaneurysms complicating pancreatitis: detection by CT

Complications of pancreatitis such as pseudocyst formation and abscess are well known to radiologists. Secondary formation of pseudoaneurysms has not been emphasized in the radiologic literature. The great morbidity and mortality associated with pseudoaneurysms emphasize the importance of early detection. Three patients are described whose angiographically proved pseudoaneurysms were demonstrated on contrast material-enhanced abdominal CT scans obtained for evaluation of pancreatitis. A homogeneously enhancing structure within or adjacent to a pancreatic pseudocyst or contiguous with a vascular structure should be considered highly suspicious for an associated pseudoaneurysm.