Identification and structure of the nerve growth factor binding site on TrkA.

Nerve growth factor (NGF) is involved in the development and maintenance of the nervous system and has been implicated as a possible therapeutic target molecule in a number of neurodegenerative diseases, especially Alzheimer's disease. NGF binds with high affinity to the extracellular region of a tyrosine kinase receptor, TrkA, which comprises three leucine-rich motifs (LRMs), flanked by two cysteine-rich clusters, followed by two immunoglobulin-like (Ig-like) domains. We have expressed the second Ig-like domain as a recombinant protein in E. coli and demonstrate that NGF binds to this domain with similar affinity to the native receptor. This domain (TrkAIg(2)) has the ability to sequester NGF in vitro, preventing NGF-induced neurite outgrowth, and in vivo, inhibiting NGF-induced plasma extravasation. We also present the three-dimensional structure of the TrkAIg(2) domain in a new crystal form, refined to 2.0 A resolution.

Recombinant human nerve growth factor for clinical trials: protein expression, purification, stability and characterisation of binding to infusion pumps.

Nerve growth factor (NGF) has been suggested to be of therapeutic benefit to patients with Alzheimer's disease. One of the early changes in this disease is a loss of cholinergic function within the brain, and NGF is able to rescue cholinergic neurons both in vitro and in vivo. We describe the production of recombinant human beta-NGF (rhNGF), using baculovirus infection of insect cells; its purification, formulation and subsequent stability for use in clinical trials. Tests were also carried out to monitor release of protein from infusion pumps and catheters for intracerebroventricular administration (icv). Initial problems with non-specific binding were overcome using a blocking formula.

Identification of syntaxin 1A as a novel binding protein for presenilin-1.

Mutations in the presenilin 1 gene have been shown to result in Alzheimer's disease. Presenilin 1 is a multi-transmembrane protein with a large hydrophilic loop near the C-terminus. This region is required for known functions of presenilin 1. We have constrained this loop within the active site of the bacterial protein, thioredoxin, to mimic its native conformational state. This hybrid protein was used as bait in a yeast two hybrid screen in an attempt to identify presenilin binding proteins. By this method syntaxin 1A, a synaptic plasma membrane protein, was identified as a novel binding protein for presenilin 1. In vitro experiments confirm the two-hybrid results suggesting that PS1 binds syntaxin under physiological conditions.

Effects of point-source PCB contamination on breeding performance and post-fledging survival in the dipper Cinclus cinclus.

Despite widespread information on the incidence and biochemical effects of polychlorinated biphenyls (PCBs) in birds, field studies of effects on population processes are still scarce. This is particularly so in passerines. We therefore assessed breeding performance and post-fledging survival in dippers, Cinclus cinclus, breeding along the Afon Mule, a Welsh river where previous work indicated locally elevated PCB concentrations. We hypothesised that marked toxicological effects, including endocrine disruption, would be detectable as altered clutch-size, hatching success, nest success, brood-size, post-fledging survival or recruitment to the breeding population. Congener-specific analysis of PCBs in failed and deserted dipper eggs during 1990-93 revealed that geometric mean concentrations were four to 20 times higher along the Mule than elsewhere in Wales, UK. PCB signatures also varied highly significantly from other locations, with congeners 138, 153 and 180 dominant at the contaminated site, but 118 or 170 more frequent in eggs elsewhere. Subsequent analysis of surface runoff confirmed a small breaker's yard as a point source. Neither laying dates, clutch-sizes, brood-sizes, nest failure nor hatching failure along the contaminated Mule (n=46 breeding attempts and 218 eggs) differed significantly from dippers along adjacent reference rivers (n=82 breeding attempts and 315 eggs) or Welsh rivers as a whole (n=332 breeding attempts and 1534 eggs). Subsequent recaptures of breeding dippers ringed initially as nestlings were nearly identical between the Mule (7.7% of 182 nestlings), the reference set (7.5% of 323 nestlings) and all other Welsh rivers (7.3% of 2821 nestlings). These data illustrate the value of congener-specific analysis of bird's eggs in indicating local PCB sources. The data show also that PCBs can occur in dipper eggs at total concentrations of 0.49 (geometric mean)-1.29 (upper quartile) microg g(-1) wet mass without effects on breeding performance and survival. These values are below those at which biochemical or reproductive effects on other passerines have been detected, but above current concentrations in any other population of European dippers for which PCB data are available. Population effects by PCBs on European dippers are therefore unlikely. Survivors from locally contaminated sites like that in our study might allow field assessments of second-generation effects on breeding performance in wild birds that have developed under PCB exposure.

A review of the likely causal pathways relating the reduced density of breeding dippers Cinclus cinclus to the acidification of upland streams.

Previous work has shown that the breeding density of a bird characteristic of upland streams, the dipper Cinclus cinclus, is markedly reduced at low pH in both Wales and Scotland. Populations also declined when streams became more acidic. Evidence of causal explanation for these relationships is that: (1) Food quantity is reduced in acidic streams, and important prey, including those rich in calcium, are scarce; (2) Blood chemistry in pre-breeding birds differs between acid and circumneutral streams, with plasma calcium reduced in those breeding at low pH. Skeletal sources of calcium are probably limited; (3) The time spent foraging by pre-breeding birds on acidic streams is markedly increased, even though overall energy costs on acidic and circumneutral streams are similar. Body condition is inferior to birds on circumneutral streams; (4) Egg laying is significantly delayed on acidic streams irrespective of an effect on laying of altitude, and clutch and brood sizes are significantly reduced; (5) Eggs are lighter and shells thinner at low pH; (6) Chick growth is reduced at low pH; (7) Contamination by heavy metals and persistent organochlorines is low in the populations on acidic streams and cannot explain the impaired breeding performance. None of these features can exclude the possibilities that acidic streams either hold populations of poor quality birds, which show the above features, or that acidity affects the breeding ecology of all dippers that attempt to breed at low pH. The qualitative outcome of these two alternatives is identical.

Patterns of contamination by organochlorines and mercury in the eggs of two river passerines in Britain and Ireland with reference to individual PCB congeners.

Unhatched eggs were collected in 1988 and 1990 from nests of the Eurasian Dipper Cinclus cinclus and the Grey Wagtail Motacilla cinerea in Wales, eastern Scotland and south-western Ireland. Mercury concentrations in Dipper eggs (geometric means 0.45-0.91 ppm (microg g(-1)) dry mass) were significantly lower in eastern Scotland than in either south-western Ireland or Wales, where the incidence of detectable residues increased markedly between 1988 (2% of eggs) and 1990 (69%). By contrast, DDE (geometric means 0.63-3.54 ppm in lipid), TDE (<0.01-1.80 ppm), DDT (<0.01-0.65 ppm), total PCBs (3.99-10.47 ppm), HEOD (0.39-0.61 ppm) and HCB (0.02-0.13 ppm) were all significantly higher in Scottish eggs than others. Around 33-46% of the total PCB burden in Dipper and Grey Wagtail eggs could be accounted for by six congeners (IUPAC numbers 118, 180, 101, 153, 138 and 170). Amongst these attributable PCBs, Dipper eggs from eastern Scotland were dominated by congener 153 (2,2',4,4',5,5'-hexachlorobiphenyl), whilst Welsh and Irish eggs were dominated by congener 118 (2,3',4,4',5-pentachlorobiphenyl). With the latter exception, all the individual congeners were found at significantly higher concentrations in Scottish eggs than others. Grey Wagtail eggs were dominated by congeners 118 and 101 (2,2,4,5,5'-pentachlorobiphenyl). In general, these congeners are common, widespread, and dominant components in the eggs of other wild birds for which data are available. Consistent with the low to medium levels of contaminants found in Dipper eggs, there was only slight evidence of any toxic effects. These included moderate shell thinning in relation to increasing DDE, and some evidence that contaminants had contributed to egg failure.

Loss of chromosome 11p alleles in cultured cells derived from Wilms' tumours.

Cell cultures have been produced from five Wilms' tumours. All cultures had a finite lifespan and a pattern of antigen expression which indicated that the cells were derived from the differentiated components of the tumours. No cells showed any of the expected characteristics of the putative Wilms' tumour stem cell. Nevertheless, in both cases where the original tumours showed a loss of heterozygosity at chromosome 11p alleles, the cultured cells also demonstrated a loss of heterozygosity. Thus these cell cultures definitely originated from Wilms' tumour tissue. The results demonstrate that cell cultures can be produced from the differentiated tissues present in Wilms' tumours and that these non-immortal cells show no 'transformed' phenotype, even though they possess the genetic changes present in the original tumour.

Long-term change in the suitability of welsh streams for dippers Cinclus cinclus as a result of acidification and recovery: a modelling study.

Using a recognized and widely used hydrochemical model, MAGIC, long-term changes in acidity were simulated at 104 sites in the acid sensitive region of upland Wales. Conditions were modelled in the future (2010) under different reductions in sulphate deposition from 0 to 90% of 1984 values. Chemical output from the model was used to simulate change in the chemical suitability of streams for a species of river bird, the Dipper Cinclus cinclus, known to be affected by acidification According to simulations, only reductions in sulphate deposition by over 50% of 1984 levels prevented decline in the number of streams chemically suitable for Dippers. Greater reductions in deposition in the model permitted some recovery except where conifer forestry occupied acid sensitive catchments. There are several uncertainties with the models in their present form.

SV40 transfection of human kidney epithelial cells and stability of chromosome 11.

Simian virus 40 (SV40) transformation has been used in a variety of mammalian cells and has been shown to extend their life span. We therefore decided to apply these results to normal kidney and tumoral cells derived from Wilms' patients. Wilms' tumour, a nephroblastoma which presents in early childhood, has been linked to deletions and rearrangements in chromosome 11. Analysis of gene structure in the 11p13 locus involved in the development of the tumour has been restricted by the very short life-span of the tumoral cells in vitro. We transfected normal kidney WT/NK, tumoral WT/T cells and human foetal kidney HFK cells with 2 SV40-derived plasmids SV3neo (pBR322-SV40-containing neomycin bacterial gene) and SVori- (pMK-origin mutated SV40). We isolated a high number of SV40-transfected cell lines. The efficiency of transfection appeared to be extremely low in WT/T cells compared with HFK and even WT/NK. The life span of the cell lines was increased in relation to their untransfected homologues. However, in all of the cell lines except 3, senescence occurred, after crisis step or not. We looked at different markers associated with SV40 transformation of mammalian cells and specifically with the presence of SV40 T antigen in the cells and its consequences: AIG, tumorigenicity, expression and insertion in genomic DNA of SV40 T antigen. Genetic studies involving karyotypic and restriction fragment length polymorphism (RFLP) analysis demonstrated that, despite a frequent pseudo-diploidy, the cell lines derived have conserved the 11p13 locus.

Egg mass and shell thickness in dippers Cinclus cinclus in relation to stream acidity in Wales and Scotland.

The shell thickness and mass of eggs of the dipper (Cinclus cinclus) collected on streams of different pH in Wales and Scotland were measured. The aluminium, phosphorus and calcium content of their invertebrate prey were also measured. In a regression analysis, significant at p<0.05, stream pH accounted for up to 7% of the variance in shell thickness, with shells decreasing by 2.5% of the overall mean with each unit of pH decline. In the Welsh sample, differences in shell thickness due to pH were small compared with differences between years across all sites. In data pooled between Scotland and Wales, pH accounted for 17% of the variance in egg mass, but a greater proportion (25%) in Scottish eggs alone. Aluminium concentrations in invertebrates showed no relationship with stream pH, but calcium levels in two insect orders increased significantly with pH. Calcium rich prey, such as Gammarus, were found only in circumneutral streams. The importance of calcium in the diet of dippers before and during egg formation is discussed. No evidence that aluminium in prey adversely affected dipper eggs was found.

Smoking and malignant disease: a general practice study.

This study, involving a practice population of 7,200 patients, compared the smoking habits of patients suffering from malignant disease (excluding skin cancer) with the smoking habits of controls matched for age and sex. Patients with malignancies were found to smoke more heavily than their controls; this difference was particularly marked between the younger patients and their controls. Data on the most prevalent malignancies (carcinomata of the breast, cervix, rectum, colon and bladder) revealed that this tendency to heavier smoking was more marked in patients with neoplasia of cervix or bladder than in patients with breast or colorectal neoplasms. The age of these patients at diagnosis was reviewed in relation to smoking habits. Although the numbers were small, it appeared that patients who smoke present at an earlier age than non-smokers, except in cases of neoplasia of the cervix.These results seem to justify further general practice studies into the links between smoking and malignant disease.