Active amino acids of the Kell blood group protein and model of the ectodomain based on the structure of neutral endopeptidase 24.11

In addition to its importance in transfusion, Kell protein is a member of the M13 family of zinc endopeptidases and functions as an endothelin-3-converting enzyme. To obtain information on the structure of Kell protein we built a model based on the crystal structure of the ectodomain of neutral endopeptidase 24.11 (NEP). Similar to NEP, the Kell protein has 2 globular domains consisting mostly of alpha-helical segments. The domain situated closest to the membrane contains both the N- and C-terminal sequences and the enzyme-active site. The outer domain contains all of the amino acids whose substitutions lead to different Kell blood group phenotypes. In the model, the zinc peptidase inhibitor, phosphoramidon, was docked in the active site. Site-directed mutagenesis of amino acids in the active site was performed and the enzymatic activities of expressed mutant Kell proteins analyzed and compared with NEP. Our studies indicate that Kell and NEP use the same homologous amino acids in the coordination of zinc and in peptide hydrolysis. However, Kell uses different amino acids than NEP in substrate binding and appears to have more flexibility in the composition of amino acids allowed in the active site.     

Placental metastasis of maternal melanoma

Metastasis of maternal malignant tumor to the products of conception is a rare event. Melanoma is the most common maternal malignant tumor to metastasize to the placenta and the fetus. We report the case of a 28-year-old woman with melanoma during pregnancy. At parturition, histologic evaluation of the placenta revealed metastatic melanoma, and multiple organ metastasis developed. The infant was free of disease. Metastasis to products of conception portends a poor prognosis for the mother. To our knowledge, this report is the first of a patient with melanoma metastasis to the placenta to survive more than 7 months after parturition. As caretakers of patients with melanoma, dermatologists are in a position to coordinate and direct the care and follow-up treatment of affected patients.     

Comparative epidemiology of scrapie outbreaks in individual sheep flocks

Data recording the course of scrapie outbreaks in 4 sheep flocks (2 in Cheviot sheep and 2 in Suffolks) are compared. For each outbreak the data on scrapie incidence and sheep demography and pedigrees cover periods of years or decades. A key finding is that the incidence of clinical cases peaks in sheep 2-3 years old, despite very different forces-of-infection. This is consistent with age-specific susceptibility of sheep to scrapie, as has been reported for cattle to bovine spongiform encephalopathy and for humans to variant Creutzfeldt-Jakob disease. Scrapie incidence was higher in ewes than rams and at certain times of years, though these effects were not consistent between flocks. There was no evidence for high levels of vertical transmission.     

The management of childhood urinary tract infections

Urinary tract infections can result in significant morbidity and represent one of the most common urological conditions that the pediatrician and family practitioner encounter in the pediatric patient population. The prevalence of UTI in girls may be as high as 8.1%. UTIs also represent the most commonly identified serious bacterial infection in infants presenting with a febrile illness. Of febrile infants aged 2-3 months, 3-10% have a documented UTI. While the majority of the UTIs are not associated with any significant underlying conditions, the mere presence of a UTI is worrisome to most parents. An appropriate evaluation will determine which of these patients need referral. A brief summary is therefore presented to assist the primary care physician in the evaluation and management of childhood UTIs.     

Identification of the key amino acids of gliai cell line-derived neurotrophic factor family receptor alpha 1 involved in its biological function

Glial cell line-derived neurotrophic factor （GDNF） plays a critical role in neurodevelopment and survival of midbrain dopaminergic and spinal motor neurons in vitro and in vivo. The biological actions of GDNF are mediated by a two-receptor complex consisting of a glycosylphosphatidylinositol-linked cell surface molecule, the GDNF family receptor alpha 1 （GFR alpha 1）, and receptor protein tyrosine kinase Ret. Although structural analysis of GDNF has been extensively examined, less is known about the structural basis of GFR alpha 1 function. In this study, based on evolutionary trace method and relative solvent accessibility prediction of residues, a set of trace residues that are solvent-accessible was selected for site-directed mutagenesis. A series of GFR alpha 1 mutations was made, and PC12 cell lines stably expressing different GFR alpha 1 mutants were generated. According to the survival and differentiation responses of these stable PC12 cells upon GDNF stimulation and the GDNF- GFR alpha 1-Ret interaction assay, residues 152NN153, Arg259, and 316SNS318 in the GFR alpha 1 central region were found to be critical for GFR alpha 1 binding to GDNF and eliciting downstream signal transduction. The single mutation R259A in the GFR alpha 1 molecule simultaneously lost its binding ability to GDNF and Ret. However N152A/N153A or S316A/N317A/ S318A mutation in the GFR alpha 1 molecule still retained the ability to bind with Ret. These findings suggest that distinct structural elements in GFR alpha 1 may be involved in binding to GDNF and Ret.     

Practice MRI: Reducing the need for sedation and general anaesthesia in children undergoing MRI

The aim of this study was to evaluate the effectiveness of a practice magnetic resonance unit, in preparing children to undergo magnetic resonance procedures without general anaesthesia (GA) or sedation. The records of children who attended the practice MRI between February 2002 and April 2004 were retrospectively reviewed. Each record was assessed as to whether the child had passed or failed the practice MRI intervention. Those children who were considered to have passed and were proceeded to a clinical non‐GA MRI had the report of the clinical scan reviewed. If the scan had been reported as non‐diagnostic because of movement artefact it was classified as a failed scan, otherwise it was considered a pass. One hundred and thirty‐four children undertook a practice MRI (age range 4.1–16.1 years, median age 7.7 years, 47% boys) and 120/134 (90%) passed the practice session. In all, 117/120 (98%) subsequently had a clinical non‐GA MRI and 110/117 (94%) passed (median age 7.8 years, 47% boys). Preparation is a safe and effective method to reduce the need for sedation and GA in children undergoing a clinical MRI scan. It provides a positive medical experience for children, parents and staff, and results in cost savings for the hospital.     

Expression of inflammatory cytokines in placentas from women with preeclampsia

It is postulated that inadequate remodeling of the uterine spiral arteries in preeclampsia leads to focal ischemia and generation of inflammatory cytokines, such as tumor necrosis factor (TNF alpha) and interleukins (ILs), by the placenta. Our objective was to compare TNF alpha, IL-1 alpha, IL-1 beta, and IL-6 levels in placentas from patients with preeclampsia and normal term pregnancies. Because the placenta is a large heterogeneous organ, we analyzed multiple sites per placenta. On the average, there was a 3-fold variation in cytokine protein levels across the eight sites analyzed for each placenta. However, there were no significant overall differences among the normal term, preeclamptic, and preterm placentas from women without preeclampsia. There were also no significant differences in TNF alpha messenger ribonucleic acid between the normal term and preeclamptic placentas, although TNF alpha messenger ribonucleic acid levels were lower in placentas from preterm patients without diagnosis of preeclampsia than in the normal term placentas. In vitro, hypoxia stimulated the production of TNF alpha, IL-1 alpha and IL-1 beta, but not that of IL-6, by placental villous explants from both groups of patients, and this was not exaggerated in preeclampsia. Finally, although peripheral and uterine venous levels of TNF alpha were elevated in preeclamptic women compared with normal term patients, the ratio of uterine to peripheral venous TNF alpha levels was not significantly different from 1.0 for either patient group. Taken together, these results suggest that sources other than the placenta contribute to the elevated concentrations of TNF alpha and IL-6 found in the circulation of preeclamptic women.