A gating mutation at the internal mouth of the Kir6.2 pore is associated with DEND syndrome

Inwardly rectifying potassium (Kir) channels control cell membrane K+ fluxes and electrical signalling in diverse cell types. Heterozygous mutations in the human Kir6.2 gene (KCNJ11), the pore-forming subunit of the ATP-sensitive (K(ATP)) channel, cause permanent neonatal diabetes mellitus. However, the I296L mutation also results in developmental delay, muscle weakness and epilepsy. We investigated the functional effects of the I296L mutation by expressing wild-type or mutant Kir6.2/SUR1 channels in Xenopus oocytes. The mutation caused a marked increase in resting whole-cell K(ATP) currents by reducing channel inhibition by ATP, in both homomeric and simulated heterozygous states. Kinetic analysis showed that the mutation impaired ATP sensitivity indirectly, by stabilizing the open state of the channel and possibly also by means of an allosteric effect on ATP binding and/or transduction. The results implicate a new region in Kir-channel gating and suggest that disease severity is correlated with the extent of reduction in ATP sensitivity.     

Development of a tissue array for primary melanoma with long-term follow-up: discovering melanoma cell adhesion molecule as an important prognostic marker

Refining current prognostic capability is essential for improving the management of melanoma. This study was undertaken to develop a tumor array for the rapid assessment of novel prognostic markers in a series of specimens from melanoma patients with 7- to 10-year follow-up. A melanoma database of 120 patients with archival specimens was created after histopathological review of original specimens. A tissue array was developed allowing 480 biopsy samples from the 120 primary melanoma specimens to be embedded into a single paraffin block. This was sectioned and stained for the adhesion marker melanoma cell adhesion molecule (MCAM); after further review, 76 of the 120 specimens were suitable for further analysis. The slides were assessed by two independent observers without previous knowledge of the clinical outcome for staining positivity and stain intensity. Assessment of association between MCAM and clinicopathological features was carried out using chi-squared analysis, and univariate and Cox multivariate analyses were performed on the data. There was a high correlation between MCAM intensity and both Clark's level and Breslow thickness (Spearman correlation p < 0.001 for both). The data revealed that MACM was a highly specific prognostic marker for survival in univariate analysis (chi2 = 18, p < 0.0001). Subgroup analysis by stratification of the staining intensity revealed a sequentially worsening survival with increasing staining intensity (chi2 = 22.33, p < 0.0001). Multivariate analysis of survival showed MCAM to be an independent prognostic marker more accurate than all other clinicopathological parameters (p < 0.0001), including the Breslow depth. Further analysis within only intermediate-thickness tumors showed MCAM intensity added further refinement to outcome prediction (chi2 = 22.33, p < 0.0001). The tissue array provided a rapid method of analyzing up to 480 specimens within a single paraffin block. This will benefit many areas of plastic surgery research. The identification of adhesion markers revealed a valuable prognostic marker for predicting outcome and a potential target for therapeutic manipulation.     

Infectious disease and the decline of Steller sea lions (Eumetopias jubatus) in Alaska, USA: insights from serologic data

Serologic data were examined to determine whether infectious disease may have played a role in the decline of Steller sea lions (Eumetopias jubatus) in the Gulf of Alaska and Aleutian Islands, USA. Available published data, unpublished data, and recent collections (1997-2000) were compared and reviewed. Data were stratified by geography to compare the declining western Alaskan population in the Aleutian Islands through eastern Prince William Sound to the increasing population in southeastern Alaska. Prevalences of antibodies from the 1970s to the early 1990s were noted for Leptospira interrogans, Chlamydophila psittaci, Brucella spp., phocid herpesvirus-1, and calciviruses. Serum samples collected from 1997-2000 were tested for antibodies to these agents as well as to marine mammal morbilliviruses, canine parvovirus, and canine adenovirus-1 and -2. Conclusions could not be drawn about changes in antibody prevalence to these agents during the decline of Steller sea lions, however, because data were incomplete or not comparable as a result of inconsistencies in testing techniques. Despite these shortcomings, results provided no convincing evidence of significant exposure of Steller sea lions to morbilliviruses, Brucella spp., canine parvovirus, or L. interrogans. Steller sea lions have been exposed to phocid herpesviruses, caliciviruses, canine adenovirus, and C. psittaci or to cross-reactive organisms in regions of both increasing and decreasing sea lion abundance. Based on similar antibody prevalence estimates from the increasing and decreasing populations, these agents are unlikely to have been the primary cause of the population decline. They may have contributed to the decline or impeded population recovery, however, because of undetected mortality and morbidity or reductions of fecundity and body condition in animals under other stresses. Systematic monitoring for disease agents and their effects is needed to determine whether infectious disease currently plays a role in the decline and lack of recovery of Steller sea lions.     

Evidence for lectin activity of a plant receptor-like protein kinase by application of neoglycoproteins and bioinformatic algorithms

Detection of genes for putative receptor-like protein kinases, which contain an extracellular domain related to leguminous lectins, in plant genomes inspired the hypothesis that this part acts as sensor. Initial support for this concept came from proof for protein kinase activity. The next step, focusing on the protein of lombardy poplar (Populus nigra var. italica), is scrutiny for lectin activity. Consequently, we first pinpointed sets of high-scoring sequence pairs by extensive databank search. The calculations resulted in P-values in the range from 10(-14) to 10(-18) exclusively for leguminous lectins, the Pterocarpus angolensis agglutinin being front runner with P=3 x 10(-18) and thus most suitable template for modeling. The superimposition of the two folds gave notable similarity in the region responsible for binding carbohydrate and Ca(2+)/Mn(2+)-ions. Binding activity toward carbohydrates was detected by assaying a panel of (neo)glycoproteins as polyvalent probes, especially for alpha-l-rhamnose and glycans of asialofetuin. It was strictly dependent on Ca(2+)-ions, enhanced by Mn(2+)-ions and reached a K(D)-value of 34.3 nM for the neoglycoprotein with rhamnose as ligand. These results give further research direction to define physiological ligands, plant/bacterial rhamnose-containing saccharides and rhamnose-mimetic glycans or peptides being potential candidates.     

The catalytic copper of peptidylglycine alpha-hydroxylating monooxygenase also plays a critical structural role

Many bioactive peptides require amidation of their carboxy terminus to exhibit full biological activity. Peptidylglycine alpha-hydroxylating monooxygenase (PHM; EC 1.14.17.3), the enzyme that catalyzes the first of the two steps of this reaction, is composed of two domains, each of which binds one copper atom (CuH and CuM). The CuM site includes Met(314) and two His residues as ligands. Mutation of Met(314) to Ile inactivates PHM, but has only a minimal effect on the EXAFS spectrum of the oxidized enzyme, implying that it contributes only marginally to stabilization of the CuM site. To characterize the role of Met(314) as a CuM ligand, we determined the structure of the Met(314)Ile-PHM mutant. Since the mutant protein failed to crystallize in the conditions of the original wild-type protein, this structure determination required finding a new crystal form. The Met(314)Ile-PHM mutant structure confirms that the mutation does not abolish CuM binding to the enzyme, but causes other structural perturbations that affect the overall stability of the enzyme and the integrity of the CuH site. To eliminate possible effects of crystal contacts, we redetermined the structure of wt-PHM in the Met(314)Ile-PHM crystal form and showed that it does not differ from the structure of wild-type (wt)-PHM in the original crystals. Met(314)Ile-PHM was also shown to be less stable than wt-PHM by differential scanning calorimetry. Both structural and calorimetric studies point to a structural role for the CuM site, in addition to its established catalytic role.     

Stability and the evolvability of function in a model protein

Functional proteins must fold with some minimal stability to a structure that can perform a biochemical task. Here we use a simple model to investigate the relationship between the stability requirement and the capacity of a protein to evolve the function of binding to a ligand. Although our model contains no built-in tradeoff between stability and function, proteins evolved function more efficiently when the stability requirement was relaxed. Proteins with both high stability and high function evolved more efficiently when the stability requirement was gradually increased than when there was constant selection for high stability. These results show that in our model, the evolution of function is enhanced by allowing proteins to explore sequences corresponding to marginally stable structures, and that it is easier to improve stability while maintaining high function than to improve function while maintaining high stability. Our model also demonstrates that even in the absence of a fundamental biophysical tradeoff between stability and function, the speed with which function can evolve is limited by the stability requirement imposed on the protein.     

A multidimensional 1H NMR investigation of the conformation of methionine-enkephalin in fast-tumbling bicelles

Enkephalins are pentapeptides found in the central nervous system. It is believed that these neuropeptides interact with the nerve cell membrane to adopt a conformation suitable for their binding to an opiate receptor. In this work, we have determined the three-dimensional structure of methionine-enkephalin (Menk) in fast-tumbling bicelles using multidimensional (1)H NMR. Bicelles were selected as model membranes because both their bilayer organization and composition resemble those of natural biomembranes. The effect of the membrane composition on the peptide conformation was explored using both zwitterionic (PC bicelles) and negatively charged bicelles (Bic/PG). Pulsed field gradient experiments allowed the determination of the proportion of Menk bound to the model membranes. Approximately 60% of the water-soluble enkephalin was found to associate to the bicellar systems. Structure calculations from torsion angle and NOE-based distance constraints suggest the presence of both micro - and delta-selective conformers of Menk in each system and slightly different conformers in PC bicelles and Bic/PG. As opposed to previous studies of enkephalins in membrane mimetic systems, our results show that these opiate peptides could adopt several conformations in a membrane environment, which is consistent with the flexibility and poor selectivity of enkephalins.