Characterization of a novel mutation in the cardiac ryanodine receptor that results in catecholaminergic polymorphic ventricular tachycardia

Catecholaminergic polymorphic ventricular tachycardia (CPVT) is an arrhythmogenic disease that manifests as syncope or sudden death during high adrenergic tone in the absence of structural heart defects. It is primarily caused by mutations in the cardiac ryanodine receptor (RyR2). The mechanism by which these mutations cause arrhythmia remains controversial, with discrepant findings related to the role of the RyR2 binding protein FKBP12.6. The purpose of this study was to characterize a novel RyR2 mutation identified in a kindred with clinically diagnosed CPVT.Single-strand conformational polymorphism analysis and direct DNA sequencing were used to screen the RyR2 gene for mutations. Site-directed mutagenesis was employed to introduce the mutation into the mouse RyR2 cDNA. The impact of the mutation on the interaction between RyR2 and a 12.6 kDa FK506 binding protein (FKBP12.6) was determined by immunoprecipitation and immunoblotting and its effect on RyR2 function was characterized by single cell Ca²⁺ imaging and [³H]ryanodine binding.A novel CPVT mutation, E189D, was identified. The E189D mutation does not alter the affinity of the channel for FKBP12.6, but it increases the propensity for store-overload-induced Ca²⁺ release (SOICR). Furthermore, the E189D mutation enhances the basal channel activity of RyR2 and its sensitivity to activation by caffeine.The E189D RyR2 mutation is causative for CPVT and functionally increases the propensity for SOICR without altering the affinity for FKBP12.6. These observations strengthen the notion that enhanced SOICR, but not altered FKBP12.6 binding, is a common mechanism by which RyR2 mutations cause arrhythmias.Key words: arrhythmia, calcium, death sudden, genetics, ion channels     

Molecular systematics and paleobiogeography of the South American sigmodontine rodents [published erratum appears in Mol Biol Evol 1998 Feb;15(2):224]

The murid rodent subfamily Sigmodontinae contains 79 genera which are distributed throughout the New World. The time of arrival of the first sigmodontines in South America and the estimated divergence time(s) of the different lineages of South American sigmodontines have been controversial due to the lack of a good fossil record and the immense number of extant species. The "early-arrival hypothesis" states that the sigmodontines must have arrived in South America no later than the early Miocene, at least 20 MYA, in order to account for their vast present-day diversity, whereas the "late-arrival hypothesis" includes the sigmodontines as part of the Plio-Pleistocene Great American Interchange, which occurred approximately 3.5 MYA. The phylogenetic relationships among 33 of these genera were reconstructed using mitochondrial DNA (mtDNA) sequence data from the ND3, ND4L, arginine tRNA, and ND4 genes, which we show to be evolving at the same rate. A molecular clock was calibrated for these genes using published fossil dates, and the genetic distances were estimated from the DNA sequences in this study. The molecular clock was used to estimate the dates of the South American sigmodontine origin and the main sigmodontine radiation in order to evaluate the "early-" and "late-arrival" scenarios. We estimate the time of the sigmodontine invasion of South America as between approximately 5 and 9 MYA, supporting neither of the scenarios but suggesting two possible models in which the invading lineage was either (1) ancestral to the oryzomyines, akodonts, and phyllotines or (2) ancestral to the akodonts and phyllotines and accompanied by the oryzomyines. The sigmodontine invasion of South America provides an example of the advantage afforded to a lineage by the fortuitous invasion of a previously unexploited habitat, in this case an entire continent.      

Penam Sulfones and β-Lactamase Inhibition: SA2-13 and the Importance of the C2 Side Chain Length and Composition

β-Lactamases are the major reason β-lactam resistance is seen in Gram-negative bacteria. To combat this resistance mechanism, β-lactamase inhibitors are currently being developed. Presently, there are only three that are in clinical use (clavulanate, sulbactam and tazobactam). In order to address this important medical need, we explored a new inhibition strategy that takes advantage of a long-lived inhibitory trans-enamine intermediate. SA2-13 was previously synthesized and shown to have a lower k _react than tazobactam. We investigated here the importance of the carboxyl linker length and composition by synthesizing three analogs of SA2-13 (PSR-4-157, PSR-4-155, and PSR-3-226). All SA2-13 analogs yielded higher turnover numbers and k _react compared to SA2-13. We next demonstrated using protein crystallography that increasing the linker length by one carbon allowed for better capture of a trans-enamine intermediate; in contrast, this trans-enamine intermediate did not occur when the C2 linker length was decreased by one carbon. If the linker was altered by both shortening it and changing the carboxyl moiety into a neutral amide moiety, the stable trans-enamine intermediate in wt SHV-1 did not form; this intermediate could only be observed when a deacylation deficient E166A variant was studied. We subsequently studied SA2-13 against a relatively recently discovered inhibitor-resistant (IR) variant of SHV-1, SHV K234R. Despite the alteration in the mechanism of resistance due to the K→R change in this variant, SA2-13 was effective at inhibiting this IR enzyme and formed a trans-enamine inhibitory intermediate similar to the intermediate seen in the wt SHV-1 structure. Taken together, our data reveals that the C2 side chain linker length and composition profoundly affect the formation of the trans-enamine intermediate of penam sulfones. We also show that the design of SA2-13 derivatives offers promise against IR SHV β-lactamases that possess the K234R substitution.     

Molecular docking of thiamine reveals similarity in binding properties between the prion protein and other thiamine-binding proteins

Prion-induced diseases are a global health concern. The lack of effective therapy and 100 % mortality rates for such diseases have made the prion protein an important target for drug discovery. Previous NMR experimental work revealed that thiamine and its derivatives bind the prion protein in a pocket near the N-terminal loop of helix 1, and conserved intermolecular interactions were noted between thiamine and other thiamine-binding proteins. Furthermore, water-mediated interactions were observed in all of the X-ray crystallographic structures of thiamine-binding proteins, but were not observed in the thiamine–prion NMR study. To better understand the potential role of water in thiamine–prion binding, a docking study was employed using structural X-ray solvent. Before energy minimization, docked thiamine assumed a “V” shape similar to some of the known thiamine-dependent proteins. Following minimization with NMR-derived restraints, the “F” conformation was observed. Our findings confirmed that water is involved in ligand stabilization and phosphate group interaction. The resulting refined structure of thiamine bound to the prion protein allowed the 4-aminopyrimidine ring of thiamine to π-stack with Tyr150, and facilitated hydrogen bonding between Asp147 and the amino group of 4-aminopyrimidine. Investigation of the π-stacking interaction through mutation of the tyrosine residue further revealed its importance in ligand placement. The resulting refined structure is in good agreement with previous experimental restraints, and is consistent with the pharmacophore model of thiamine-binding proteins.     

Modifications of the C6-substituent of penicillin sulfones with the goal of improving inhibitor recognition and efficacy

In order to evaluate the importance of a hydrogen-bond donating substituent in the design of β-lactamase inhibitors, a series of C6-substituted penicillin sulfones, lacking a C2′ substituent, and having an sp³ hybridized C6, was prepared and evaluated against a representative classes A and C β-lactamases. It was found that a C6 hydrogen-bond donor is necessary for good inhibitory activity, but that this feature alone is not sufficient in this series of C6β-substituted penicillin sulfones. Other factors which may impact the potency of the inhibitor include the steric bulk of the C6 substituent (e.g., methicillin sulfone) which may hinder recognition in the class A β-lactamases, and also high similarity to the natural substrates (e.g., penicillin G sulfone) which may render the prospective inhibitor a good substrate of both classes of enzyme. The best inhibitors had non-directional hydrogen-bonding substituents, such as hydroxymethyl, which may allow sufficient conformational flexibility of the acyl-enzyme for abstraction of the C6 proton by E166 (class A), thus promoting isomerization to the β-aminoacrylate as a stabilized acyl-enzyme.     

Genome-wide identification,annotation and characterization of novel thermostable cytochrome P450 monooxygenases from the thermophilic biomass-degrading fungi Thielavia terrestris and Myceliophthora thermophila

Cytochrome P450 monooxygenases (P450s) are ubiquitous heme-thiolate proteins that have potential biotechnological application. Thermostable-P450s that can withstand hostile industrial conditions, such as high temperatures, extremes of pH and organic solvents, are needed for biotechnological usage. Here, for the first time, we report a large number of thermostable-P450s from two thermophilic biomass-degrading fungi, Myceliophthora thermophila and Thielavia terrestris. Genome-wide P450 analysis revealed the presence of 79 and 70 P450s (P450ome) in T. terrestris and M. thermophila. Authentic P450s containing both the P450 signature domains (EXXR and CXG) were classified as follows: T. terrestris (50 families and 56 subfamilies) and M. thermophila (49 families and 53 subfamilies). Bioinformatics analysis of P450omes suggested the presence of a large number of thermostable-P450s. Based on aliphatic index cut-off (>90), 14 and 11 P450s were determined to be thermostable in T. terrestris and M. thermophila. Among the thermostable P450s, six P450s from T. terrestris and three from M. thermophila had a melting temperature (Tm) of >65 °C, suggesting their hyperthermal tolerance. Analysis of the instability index of two ascomycete P450omes revealed the presence of 12 and 19 in vitro stable P450s in T. terrestris and M. thermophila. Overall, six P450s from T. terrestris and four from M. thermophila showed both thermal tolerance and in vitro stability. Thermophilic ascomycetes P450s are of potential interest from a structural, mechanistic and biotechnological point of view, as five P450s showed higher thermal tolerance and five showed higher in vitro stability compared to the well-characterized thermostable-P450s CYP175A1 (bacteria) and CYP119 (archaea).     

Sentinel node biopsy should be supplemented by axillary sampling in patients with small breast cancers

Axillary clearance provides important prognostic information but is associated with significant morbidity. Sentinel node biopsy can provide staging .141 patients with node negative early breast cancers-tumour size less than 1.5 cm measured clinically or by imaging had guided axillary sampling (sentinel lymph node biopsy in combination with axillary sampling). Four node axillary sampling improved the detection rate of axillary node metastases by 13.6% as compared to blue dye sentinel node biopsy alone. Positive sampled nodes strongly indicated the likelihood of further metastatic being revealed by axillary dissection (67%). Negative sampled nodes in combination with a positive sentinel node biopsy were associated with a much lower rate of further nodal involvement in the axillary clearance (8%).