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.     

Endoscopic stapling diverticulotomy: an audit of difficulties, outcome, and patient satisfaction.

OBJECTIVE: To evaluate the procedure in terms of technical difficulties, patient satisfaction, symptom relief, recurrence, and further management. STUDY DESIGN: Retrospective study including 41 patients who underwent endoscopic stapling diverticulotomy. Clinical notes were reviewed, and patients were sent a patient satisfaction questionnaire. Statistical analysis was done using the chi(2) square test. SETTING: Teaching hospital. RESULTS: Larger pouches had the best results; 81.578% were satisfied with the surgery, and 86.84% had improved swallowing. Complications presented in 6 patients (15%), including a perforated pouch and an esophageal perforation. Complications were more frequent in small pouches. CONCLUSIONS AND SIGNIFICANCE: Endoscopic stapling diverticulotomy offers rapid recovery in most patients, with early oral intake and short hospital stay. There is a high degree of symptoms relief, and the patient's satisfaction rate is high. The assessment of the pharyngeal pouch size is an important factor when deciding the best management for a patient with a pharyngeal pouch.     

Genetic analysis of the interleukin‐1 receptor antagonist and its homologue IL‐1L1 in alopecia areata: strong severity association and possible gene interaction‡

Alopecia areata is an inflammatory hair loss disease with a major genetic component. The presence of focal inflammatory lesions with perifollicular T‐cell infiltrates reflects the importance of local cytokine production in the pathogenesis. In addition to its fundamental pro‐inflammatory role, the interleukin‐1 (IL‐1) system has major effects on hair growth regulation in vitro, with the inhibitory actions of IL‐1α and IL‐1β being opposed by the receptor antagonist IL‐1ra. The novel interleukin‐1 like molecule 1 (IL‐1L1) which has greatest gene sequence homology with IL1RN, the gene encoding IL‐1ra, is another potential IL‐1 antagonist. In view of previous studies suggesting a significant role for IL1RN polymorphisms in the pathogenesis of autoimmune/inflammatory disease, we have analysed polymorphisms of IL‐1ra (IL1RN+2018) and its homologue IL‐1L1 (IL1L1+4734) in a case–control association study on 165 patients and a large number of matched controls. Homozygosity for the rare allele of IL1RN (IL1RN*2) was significantly associated with alopecia areata [odds ratio (OR) = 1.89, 95% CI (1.09, 3.28); P = 0.02], confirming our previous findings of significant association with the IL1RN variable number tandem repeat (VNTR). The results also revealed a novel association involving a polymorphism of the interleukin‐1 receptor antagonist homologue IL1L1 at position + 4734, IL1RN+2018, and alopecia areata. The effect of a genotype combining three copies of the rare alleles at the IL1RN and IL1L1 loci conferred a more than additive increase in the risk of disease compared to IL1RN+2018 or IL1L1+4734 alone [OR 3.37 (1.60, 7.06); P = 0.002], suggesting possible synergy between the IL1RN and IL1L1 genes. This effect was stronger in patients with severe disease (alopecia totalis/universalis) [OR 4.62 (1.87, 11.40), P = 0.0022], and in those with early age at onset (< 20 years) [OR = 6.38 (2.64, 15.42), P = 0.0002]. Our results suggest that these polymorphisms within IL1RN and IL1L1 themselves or a gene in linkage disequilibrium with IL1RN and IL1L1 predispose to the more severe forms of alopecia areata.     

cDNA clone encoding a high molecular weight antigen of Babesia bovis

An expression library was constructed by inserting cDNA copied from mRNA of the blood stages of Babesia bovis isolate KA into bacteriophage lambda gt11-amp3. An antigen-positive cDNA clone detected by screening the library with antibodies from cattle vaccinated with the KA isolate was shown to encode part of a high-molecular weight polypeptide antigen of B. bovis. This molecule was a dominant immunogen and was found by immunofluorescence to be within the parasite in infected erythrocytes.