Protein engineering and the study of structure--function relationships in receptors

Protein engineering is a powerful tool for studying relationships between receptor structure and function--providing that it is used and interpreted appropriately. Site-directed mutagenesis, deletion mutagenesis and construction of chimaeric proteins have all been used to characterize receptors. In this review, Walter Ward, David Timms and Alan Fersht describe the application of protein engineering, illustrating important concepts with experimental data. They explain that detailed study of function requires careful dissection of mechanistic steps. Care must also be taken when selecting replacement residues; mutation should not cause delocalized structural reorganization or else the true significance of functional change will remain unclear.     

Zinc deficiency and hyperprolactinaemia are not reversible causes of sexual dysfunction in uraemia

We selected a group of male dialysis patients complaining of sexual dysfunction in whom penile vascular insufficiency and drug-induced impotence had been excluded. Monitoring of nocturnal penile tumescence was used to confirm organic disturbance. Patients with normal serum prolactin concentrations (n = 18) had significantly lower serum zinc values than normal controls (P less than 0.001) and were entered in a 6-month double-blind study comparing oral zinc acetate with placebo. Patients with elevated prolactin concentrations (n = 8) were entered in a 3-month double-blind crossover study comparing oral pergolide mesylate with placebo. In the zinc study, serum zinc concentrations increased (P less than 0.05) in the zinc-treated but not the placebo-treated group. One of nine patients receiving zinc reported improved sexual function, as did two of nine patients receiving placebo. There were no significant changes in sperm counts, nocturnal penile tumescence, testosterone, sex hormone binding globulin or gonadotrophin concentrations in either treatment group. In the pergolide study, serum prolactin values decreased (P less than 0.01) in the pergolide but not in the placebo treatment period. One patient reported improved sexual function during the pergolide treatment period and two during the placebo period. There were no significant changes in sperm counts, nocturnal penile tumescence, testosterone, sex hormone binding globulin or gonadotrophin concentrations after pergolide. These studies show no benefit of zinc or pergolide compared with placebo in the treatment of uraemic impotence.     

Deletion of aldose reductase leads to protection against cerebral ischemic injury.

Previously, we reported that transgenic mice overexpressing endothelin-1 in astrocytes showed more severe neurological deficits and increased infarct after transient focal ischemia. In those studies, we also observed increased level of aldose reductase (AR), the first and rate-limiting enzyme of the polyol pathway, which has been implicated in osmotic and oxidative stress. To further understand the involvement of the polyol pathway, the mice with deletion of enzymes in the polyol pathway, AR, and sorbitol dehydrogenase (SD), which is the second enzyme in this pathway, were challenged with similar cerebral ischemic injury. Deletion of AR-protected animals from severe neurological deficits and large infarct, whereas similar protection was not observed in mice with SD deficiency. Most interestingly, AR(-/-) brains showed lowered expression of transferrin and transferrin receptor with less iron deposition and nitrotyrosine accumulation. The protection against oxidative stress in AR(-/-) brain was also associated with less poly(adenosine diphosphate-ribose) polymerase (PARP) and caspase-3 activation. Pharmacological inhibition of AR by Fidarestat also protected animals against cerebral ischemic injury. These findings are the first to show that AR contributes to iron- and transferrin-related oxidative stress associated with cerebral ischemic injury, suggesting that inhibition of AR but not SD may have therapeutic potential against cerebral ischemic injury.     

Atlantoaxial instability in Down's syndrome: clinical and radiological screening

One hundred and thirty children with Down's syndrome were screened for the presence of atlantoaxial instability, using both clinical examination and radiographs of the cervical spine taken in flexion and hyperextension views. Seven children were found to have radiological evidence of atlantoaxial instability, with an atlanto-dens interval greater than 5.0 mm in one or all positions. Although a full clinical history was obtained from the attending parent and each child underwent a complete neurological examination, there were no factors detected which differentiated between those with radiological evidence of atlantoaxial instability and those with a normal atlantodens distance. It is recommended that children with Down's syndrome be screened twice, at the ages of 5-10 years and at 15 years.     

Carrier diagnosis of Duchenne muscular dystrophy using restriction fragment length polymorphisms

Molecular probes that are tightly linked to and flank the Duchenne muscular dystrophy (DMD) locus, have been used to characterize DMD mutations and diagnose female carriers. Deletions within the Xp21 region were identified for 8 of 71 families studied. Using both DNA and CK studies, accurate (96 to 98%) carrier or noncarrier diagnoses were made for 51 of 75 females at risk in 24 families with a single affected male. DNA studies resulted in an alteration of predicted risk in 40% of the cases. Recombinant diagnostic methods are useful for carrier detection in families with one or more affected males.