Acyclic permutants of naturally occurring cyclic proteins - Characterization of cystine knot and beta-sheet formation in the macrocyclic polypeptide kalata B1

Kalata B1 is a prototypic member of the unique cyclotide family of macrocyclic polypeptides in which the major structural features are a circular peptide backbone, a triple stranded beta-sheet, and a cystine knot arrangement of three disulfide bonds. The cyclotides are the only naturally occurring family of circular proteins and have prompted us to explore the concept of acyclic permutation, i.e. opening the backbone of a cross-linked circular protein in topologically permuted ways. We have synthesized the complete suite of acyclic permutants of kalata B1 and examined the effect of acyclic permutation on structure and activity. Only two of six topologically distinct backbone loops are critical for folding into the native conformation, and these involve disruption of the embedded ring in the cystine knot. Surprisingly, it is possible to disrupt regions of the p-sheet and still allow folding into native-like structure, provided the cystine knot is intact. Kalata B1 has mild hemolytic activity, but despite the overall structure of the native peptide being retained in all but two cases, none of the acyclic permutants displayed hemolytic activity. This loss of activity is not localized to one particular region and suggests that cyclization is critical for hemolytic activity

Redox linked flavin sites in extracellular decaheme proteins involved in microbe-mineral electron transfer

Extracellular microbe-mineral electron transfer is a major driving force for the oxidation of organic carbon in many subsurface environments. Extracellular multi-heme cytochromes of the Shewenella genus play a major role in this process but the mechanism of electron exchange at the interface between cytochrome and acceptor is widely debated. The 1.8 Å x-ray crystal structure of the decaheme MtrC revealed a highly conserved CX8C disulfide that, when substituted for AX8A, severely compromised the ability of S. oneidensis to grow under aerobic conditions. Reductive cleavage of the disulfide in the presence of flavin mononucleotide (FMN) resulted in the reversible formation of a stable flavocytochrome. Similar results were also observed with other decaheme cytochromes, OmcA, MtrF and UndA. The data suggest that these decaheme cytochromes can transition between highly reactive flavocytochromes or less reactive cytochromes, and that this transition is controlled by a redox active disulfide that responds to the presence of oxygen

Paracetamol metabolism, hepatotoxicity, biomarkers and therapeutic interventions: a perspective

After over 60 years of therapeutic use in the UK, paracetamol (acetaminophen, N-acetyl-p-aminophenol, APAP) remains the subject of considerable research into both its mode of action and toxicity. The pharmacological properties of APAP are the focus of some activity, with the role of the metabolite N-arachidonoylaminophenol (AM404) still a topic of debate. However, that the hepatotoxicity of APAP results from the production of the reactive metabolite N-acetyl-p-benzoquinoneimine (NAPQI/NABQI) that can deplete glutathione, react with cellular macromolecules, and initiate cell death, is now beyond dispute. The disruption of cellular pathways that results from the production of NAPQI provides a source of potential biomarkers of the severity of the damage. Research in this area has provided new diagnostic markers such as the microRNA miR-122 as well as mechanistic biomarkers associated with apoptosis, mitochondrial dysfunction, inflammation and tissue regeneration. Additionally, biomarkers of, and systems biology models for, glutathione depletion have been developed. Furthermore, there have been significant advances in determining the role of both the innate immune system and genetic factors that might predispose individuals to APAP-mediated toxicity. This perspective highlights some of the progress in current APAP-related research

Stabilization of a-conotoxin AuIB: influences of disulfide connectivity and backbone cyclization

a-Conotoxins are peptides isolated from the venom ducts of cone snails that target nicotinic acetylcholine receptors (nAChRs). They are valuable pharmacological tools and have potential applications for treating a range of conditions in humans, including pain. However, like all peptides, conotoxins are susceptible to degradation, and to enhance their therapeutic potential it is important to elucidate the factors contributing to instability and to develop approaches for improving stability. AuIB is a unique member of the a-conotoxin family because the nonnative "ribbon" disulfide isomer exhibits enhanced activity at the nAChR in rat parasympathetic neurons compared with the native "globular" isomer. Here we show that the ribbon isomer of AuIB is also more resistant to disulfide scrambling, despite having a nonnative connectivity and flexible structure. This resistance to disulfide scrambling does not correlate with overall stability in serum because the ribbon isomer is degraded in human serum more rapidly than the globular isomer. Cyclization via the joining of the N- and C-termini with peptide linkers of four to seven amino acids prevented degradation of the ribbon isomer in serum and stabilized the globular isomers to disulfide scrambling. The linker length used for cyclization strongly affected the relative proportions of the disulfide isomers produced by oxidative folding. Overall, the results of this study provide important insights into factors influencing the stability and oxidative folding of a-conotoxin AuIB and might be valuable in the design of more stable antagonists of nAChRs

Dissecting the oxidative folding of circular cystine knot miniproteins

Cyclotides are plant proteins with exceptional stability owing to the presence of a cyclic backbone and three disulfide bonds arranged in a cystine knot motif. Accordingly, they have been proposed as templates to stabilize bioactive epitopes in drug-design applications. The two main subfamilies, referred to as the Möbius and bracelet cyclotides, require dramatically different in vitro folding conditions to achieve the native fold. To determine the underlying elements that influence cyclotide folding, we examined the in vitro folding of a suite of hybrid cyclotides based on combination of the Möbius cyclotide kalata B1 and the bracelet cyclotide cycloviolacin O1. The folding pathways of the two cyclotide subfamilies were found to be different and influenced by specific residues within intercysteine loops 2 and 6. Two changes in these loops, a substitution in loop 2 and an addition in loop 6, enabled the folding of a cycloviolacin O1 analogue under conditions in which folding does not occur in vitro for the native peptide. A key intermediate contains a native-like hairpin structure that appears to be a nucleation locus early in the folding process. Overall, these mechanistic findings on the folding of cyclotides are potentially valuable for the design of new drug leads. Copyright Mary Ann Liebert, Inc

Polyandrous females avoid costs of inbreeding

Why do females typically mate with more than one male? Female mating patterns have broad implications for sexual selection, speciation and conflicts of interest between the sexes, and yet they are poorly understood. Matings inevitably have costs, and for females, the benefits of taking more than one mate are rarely obvious. One possible explanation is that females gain benefits because they can avoid using sperm from genetically incompatible males, or invest less in the offspring of such males. It has been shown that mating with more than one male can increase offspring viability, but we present the first clear demonstration that this occurs because females with several mates avoid the negative effects of genetic incompatibility. We show that in crickets, the eggs of females that mate only with siblings have decreased hatching success. However, if females mate with both a sibling and a non-sibling they avoid altogether the low egg viability associated with sibling matings. If similar effects occur in other species, inbreeding avoidance may be important in understanding the prevalence of multiple mating

Association of surfactant protein A polymorphisms with otitis media in infants at risk for asthma

BACKGROUND: Otitis media is one of the most common infections of early childhood. Surfactant protein A functions as part of the innate immune response, which plays an important role in preventing infections early in life. This prospective study utilized a candidate gene approach to evaluate the association between polymorphisms in loci encoding SP-A and risk of otitis media during the first year of life among a cohort of infants at risk for developing asthma. METHODS: Between September 1996 and December 1998, women were invited to participate if they had at least one other child with physician-diagnosed asthma. Each mother was given a standardized questionnaire within 4 months of her infant's birth. Infant respiratory symptoms were collected during quarterly telephone interviews at 6, 9 and 12 months of age. Genotyping was done on 355 infants for whom whole blood and complete otitis media data were available. RESULTS: Polymorphisms at codons 19, 62, and 133 in SP-A1, and 223 in SP-A2 were associated with race/ethnicity. In logistic regression models incorporating estimates of uncertainty in haplotype assignment, the 6A(4)/1A(5)haplotype was protective for otitis media among white infants in our study population (OR 0.23; 95% CI 0.07,0.73). CONCLUSION: These results indicate that polymorphisms within SP-A loci may be associated with otitis media in white infants. Larger confirmatory studies in all ethnic groups are warranted

Evidence based medicine as science

Evidence based medicine has claimed to be science on a number of occasions but it is not clear that this status is deserved. Within philosophy of science four main theories about the nature of science are historically recognised: inductivism, falsificationism, Kuhnian paradigms and research programmes. If evidence based medicine is science knowledge claims should be derived using a process that corresponds to one of these theories. This paper analyses whether this is the case. In the first section, different theories about the nature of science are introduced. In the second section, the claim that evidence based medicine is science is reinterpreted as the claim that knowledge claims derived from randomised controlled trails and meta-analyses are science. In the third section the knowledge claims valued within evidence based medicine are considered from the perspective of inductivism, falsificationism, Kuhnian paradigms and research programmes. In the final section possible counter arguments are considered. It is argued that the knowledge claims valued by evidence based medicine are not justified using inductivism, falsificationism, Kuhnian paradigms or research programmes. If these are the main criteria for evaluating if something is science or not, evidence based medicine does not meet these criteria

Mitochondrial and nuclear genes suggest that stony corals are monophyletic but most families of stony corals are not (Order Scleractinia, Class Anthozoa, Phylum Cnidaria)

Modern hard corals (Class Hexacorallia; Order Scleractinia) are widely studied because of their fundamental role in reef building and their superb fossil record extending back to the Triassic. Nevertheless, interpretations of their evolutionary relationships have been in flux for over a decade. Recent analyses undermine the legitimacy of traditional suborders, families and genera, and suggest that a non-skeletal sister clade (Order Corallimorpharia) might be imbedded within the stony corals. However, these studies either sampled a relatively limited array of taxa or assembled trees from heterogeneous data sets. Here we provide a more comprehensive analysis of Scleractinia (127 species, 75 genera, 17 families) and various outgroups, based on two mitochondrial genes (cytochrome oxidase I, cytochrome b), with analyses of nuclear genes (ßtubulin, ribosomal DNA) of a subset of taxa to test unexpected relationships. Eleven of 16 families were found to be polyphyletic. Strikingly, over one third of all families as conventionally defined contain representatives from the highly divergent "robust" and "complex" clades. However, the recent suggestion that corallimorpharians are true corals that have lost their skeletons was not upheld. Relationships were supported not only by mitochondrial and nuclear genes, but also often by morphological characters which had been ignored or never noted previously. The concordance of molecular characters and more carefully examined morphological characters suggests a future of greater taxonomic stability, as well as the potential to trace the evolutionary history of this ecologically important group using fossils

Процесс анализа угроз, влияющих на экономическую устойчивость предприятия

На основании проведенного исследования были выявлены факторы возникновения угроз, их группировка по степени воздействию на экономическую устойчивость предприятий и рассмотрена формализация процесса анализа угроз экономической устойчивости предприятий. В условиях рыночной экономики невозможно управлять предприятием без учета влияния угроз, а для эффективного управления важно не только знать об их присутствии, а и правильно идентифицировать конкретную угрозу.На підставі проведеного дослідження були виявлені чинники виникнення загроз, їх угруповання по степені впливу на економічну стійкість підприємств і розглянута формалізація процесу аналізу загроз економічної стійкості підприємств. В умовах ринкової економіки неможливо керувати підприємством без вивчення впливу загроз, а для ефективного керування важливо не тільки знати про їх присутність, а і правильно ідентифікувати конкретну загрозу.On the basis of the conducted research the factors of origin of threats were exposed, their gourmet on a degree to influence on economic stability of enterprises and formalization of process of analysis of threats of economic stability of enterprises is considered. In the conditions of market economy it is impossible to manage an enterprise without taking into account influencing of threats, and for the effective management it is important not only to know about their presence, and to identify the concrete threat correctly