The STR system in the human phenylalanine hydroxylase gene: true fragment length obtained with fluorescent labelled PCR primers

We present a simple, fast, non-radioactive method for the analysis of the polymorphic short tandem repeat (STR) system in the human phenylalanine hydroxylase gene. Previously, sizing of the STR marker involved radiolabelling of PCR amplified fragments and resolution on denaturing polyacrylamide gels using M13 sequencing ladder as a standard. However, this method consistently gave sizes 2 bp longer than the known sequence. The fluorescent method presented here employs internal lane standards and enables accurate sizing of the fragments. To avoid confusion, we suggest that the true fragment lengths are used as reference values in the future. The analysis of STR alleles is valuable for population genetic studies and for targeted mutation screening in phenylketonuria (PKU). It can replace RFLP-based haplotype analysis for carrier detection, and we report its use for prenatal diagnosis in a Northern Irish family with PKU. The analysis of 250 Northern Irish chromosomes, including 128 PKU alleles, showed no significant difference between normal and PKU alleles, with fragment lengths of 238 and 242 bp most common in both groups.     

Molecular analysis of PKU in Ireland

Classical phenylketonuria (PKU: McKusick No. 261600) is caused by mutations occurring at the phenylalanine hydroxylase (PAH) locus on chromosome 12 and has a prevalence in Ireland of 1 in 4500. We examined 304 independent alleles from 350 patients for the presence of six mutations and have characterized VNTR alleles within the minisatellite region 3' to the PAH gene in patients carrying the most prevalent mutation. R408W was the most common mutation found, with a relative frequency of 42%. All other mutations had relative frequencies of <10%. VNTR analysis showed that the R408W mutation is associated with the VNTR-8 allele in the Irish population, indicating that R408W is associated with RFLP haplotype 1. This differs from that reported from eastern Europe where R408W is associated with RFLP haplotype 2/VNTR-3; an observation which has led several groups to propose a Balto-Slavic origin for this mutation. These results support the hypothesis of a second, independent founding event for the R408W mutation on an RFLP haplotype 1 VNTR-8 chromsome background in the Irish/Celtic population.     

Fructosamine and glycated haemoglobin in the assessment of long term glycaemic control in diabetes

Fructosamine and glycated haemoglobin were measured simultaneously in 147 children with diabetes. If glycated haemoglobin is considered as the 'gold standard' for long term glycaemic control, then fructosamine is a poor indicator of actual glycated haemoglobin values, with wide 95% confidence (fiducial) limits. This shows that it is impossible to accurately predict glycated haemoglobin concentrations and therefore, by implication, longer term glycaemic control, from measurements of fructosamine. As the major studies on the prevention of microvascular complications in diabetes have used glycated haemoglobin levels to assess glycaemic control, it is suggested that this measurement should be used in all children with diabetes in preference to the measurement of fructosamine.     

The binding of conformationally restricted antihistamines to histamine receptors

Some 1,1-diary 1–3-aminoprop-1-enes and 1,2-diaryl-4-amino-but-1-and ?2-enes, including isomers of triprolidine and pyrrobutamine, have been prepared, their geometrical configurations established by pmr spectroscopy, and their affinities for histamine receptors measured on the guinea-pig ileum. These isomers differed considerably in their affinities and a particularly large difference was observed with the isomers of triprolidine (1170:1). This is because the binding of 3-aminoprop-1-enes is enhanced when α-pyridyl and aminomethyl groups are trans to one another or when p-tolyl and aminomethyl groups are cis, whereas activity is reduced when these groups are in opposite configurations. There is also a considerable difference between the geometrical isomers of pyrrobutamine (ca 200:1) but the most active compounds all have the same configuration whether 3-aminoprop-1-enes or 4-aminobut-2-enes. For high activity it appears necessary to have a trans Ar.C: CH.CH₂.NC₄H₈ arrangement with the aromatic nucleus (α-pyridyl or phenyl) coplanar with the double bond, together with an aromatic function such as p-tolyl, benzyl or p-chlorobenzyl in a position cis to the aminomethyl group. All these compounds have restricted conformations so that the series serves as a useful model for the stereochemical requirements of the antihistamine receptor.     

Effects of diazepam and flumazenil on food competition behavior in high- and low-aggression pigeons

The food competition interaction test performed with food-restricted pigeons with previously consolidated dominance is a useful tool for the study of offensive and defensive social aggression. In the present study, we examined the effect of GABA-A-benzodiazepine (BZD) receptor manipulation on aggression, emotion, feeder control, and eating behavior in high- and low-aggression female pigeons maintained at 80% of their normal weight and exposed to food competition interactions. The pigeons were divided into pairs by previously ranked high-aggression females (total time spent in aggression over 60 s/5 min; n=6 pairs) and low-aggression females (time spent in aggression less than 10 s/5 min; n=6 pairs). In Experiment 1, a pigeon in each pair of high- and low-aggression subjects were treated daily with an oral dose of diazepam (DZP, 0.6 mg/kg/0.3 ml) for 8 days. The other animal received the vehicle. On Day 8, food competition trials (10 min) were performed 30 min after treatments. In Experiment 2, pigeons were injected subcutaneously with flumazenil (FZL, 0.1 mg/kg/1 ml) or saline and exposed to a food competition trial 30 min after injections. In Experiment 3, one animal in each pair received DZP for 8 days. The other animal received the vehicle. On Day 8, the DZP-treated subjects were injected subcutaneously with FZL (0.1 mg/ kg/1 ml) 30 min before the oral dose of DZP. Trials were performed 30 min after DZP or vehicle administration. In Experiment 1, it was found that the DZP group of high-aggression pigeons showed lower scores of aggression (P<.05) and emotional responses (P<.05) than controls. The other group-scored behaviors were not affected. The DZP low-aggressions, however, showed scores of aggression eightfold higher than their controls (P<.05) but the other scored behaviors were not changed. In Experiment 2, FZL injection did not induce intrinsic effects on aggression either in the high- or in the low-aggression group. Experiment 3 showed that the emotional and aggressive responses to DZP were neutralized by FZL. This shows that GABA-A-BZD receptor mechanisms are implicated in the DZP responses in high- and low-aggression pigeons.