Insights into the Evolution and Emergence of a Novel Infectious Disease

Many zoonotic, novel infectious diseases in humans appear as sporadic infections with spatially and temporally restricted outbreaks, as seen with influenza A(H5N1). Adaptation is often a key factor for successfully establishing sustained human-to-human transmission. Here we use simple mathematical models to describe different adaptation scenarios with particular reference to spatial heterogeneity within the human population. We present analytical expressions for the probability of emergence per introduction, as well as the waiting time to a successful emergence event. Furthermore, we derive general analytical results for the statistical properties of emergence events, including the probability distribution of outbreak sizes. We compare our analytical results with a stochastic model, which has previously been studied computationally. Our results suggest that, for typical connection strengths between communities, spatial heterogeneity has only a weak effect on outbreak size distributions, and on the risk of emergence per introduction. For example, if or larger, any village connected to a large city by just ten commuters a day is, effectively, just a part of the city when considering the chances of emergence and the outbreak size distribution. We present empirical data on commuting patterns and show that the vast majority of communities for which such data are available are at least this well interconnected. For plausible parameter ranges, the effects of spatial heterogeneity are likely to be dominated by the evolutionary biology of host adaptation. We conclude by discussing implications for surveillance and control of emerging infections.     

Cyclin B2/cyclin-dependent kinase1 dissociation precedes CDK1 Thr-161 dephosphorylation upon M-phase promoting factor inactivation in Xenopus laevis cell-free extract

Cyclin-dependent kinase 1 (CDK1) is the enzymatic subunit of M-phase Promoting Factor (MPF). It is positively regulated by phosphorylation on Thr-161 and association with a cyclin B molecule. The role of Thr-161 dephosphorylation upon MPF inactivation remains unclear; nevertheless, degradation of cyclin B is thought to be a direct cause of MPF inactivation. However, MPF inactivation actually precedes cyclin B degradation in Xenopus cell-free extracts. Here we study in details the temporal relationship between histone H1 kinase (reflecting MPF activity) inactivation, Thr-161 dephosphorylation, CDK1-cyclin B2 dissociation and cyclin B2 proteolysis in such extracts. We show an asynchrony between inactivation of histone H1 kinase and degradation of cyclin B2. CDK1 dephosphorylation on Thr 161 is an even later event than cyclin B2 degradation, reinforcing the hypothesis that cyclin B dissociation from CDK1 is the key event inactivating MPF. Cyclins synthesized along with MPF inactivation could deliver shortly living active MPF molecules, potentially increasing the asynchrony between histone H1 kinase inactivation and cyclin B2 degradation. We confirm this by showing that in the absence of protein synthesis, such a tendency is lower, but nevertheless, still detectable. Finally, to characterise better CDK1/cyclin B dissociation, we show that CDK1 begins to dissociate from cyclin B2 before the very beginning of cyclin B2 degradation and that the diminution in CDK1-associated cyclin B2 is faster than the decline of its total pool. Thus, neither cyclin B2 degradation nor Thr-161 dephosphorylation participates directly in CDK1 inactivation as measured by histone H1 kinase decline upon the exit from mitotic M-phase in Xenopus embryo extract.     

Identification of a platyhelminth neuropeptide receptor

We report the characterisation of the first neuropeptide receptor from the phylum Platyhelminthes, an early-diverging phylum which includes a number of important human and veterinary parasites. The G protein-coupled receptor (GPCR) was identified from the model flatworm Girardia tigrina (Tricladida: Dugesiidae) based on the presence of motifs widely conserved amongst GPCRs. In two different assays utilising heterologous expression in Chinese hamster ovary cells, the Girardia GPCR was most potently activated by neuropeptides from the FMRFamide-like peptide class. The most potent platyhelminth neuropeptide in both assays was GYIRFamide, a FMRFamide-like peptide known to be present in G. tigrina. There was no activation by neuropeptide Fs, another class of flatworm neuropeptides. Also active were FMRFamide-like peptides derived from other phyla but not known to be present in any platyhelminth. Most potent among these were nematode neuropeptides encoded by the Caenorhabditis elegans flp-1 gene which share a PNFLRFamide carboxy terminal motif. The ability of nematode peptides to stimulate a platyhelminth receptor demonstrates a degree of structural conservation between FMRFamide-like peptide receptors from these two distinct, distant phyla which contain parasitic worms.     

Differences in regulation of the first two M-phases in Xenopus laevis embryo cell-free extracts

The first embryonic M-phase is special, being the time when paternal and maternal chromosomes mix together for the first time. Reports from a variety of species suggest that the regulation of first M-phase has many particularities; however, no systematic comparative study of the biochemical aspects of first and the following M-phases has been previously undertaken. Here, we ask whether the regulation of the first embryonic M-phase is modified, using Xenopus cell-free extracts. We developed new types of extract specific for the first and the second M-phase obtained either from parthenogenetic or from in vitro fertilized embryos. Analyses of these extracts confirmed that the amplitude of histone H1 kinase activity reflecting CDK1/cyclin B (or MPF for M-phase Promoting Factor) activity is higher and persists longer than during the second M-phase, and that levels of cyclins B1 and B2 are correspondingly higher during the first than the second embryonic M-phase. Inhibition of protein synthesis shortly before M-phase entry reduced mitotic histone H1 kinase amplitude, shortened the period of mitotic phosphorylation of chosen marker proteins, and reduced cyclin B1 and B2 levels, suggesting a role of B-type cyclins in regulating the duration of mitotic events. Moreover, addition of exogenous cyclin B to the extract prior the second mitosis brought forward the activation of mitotic histone H1 kinase but prolonged the duration of this activity. We also confirmed that the inhibitory phosphorylation of CDK1 on tyrosine 15 oscillates between the first two embryonic M-phases, but is clearly more pronounced before the first than the second mitosis, while the MAP kinase ERK2 tended to show greater activation during the first embryonic M-phase but with a similar duration of activation. We conclude that discrete differences exist between the first two M-phases in Xenopus embryo and that higher CDK1/cyclin B activity and B-type cyclin levels could account for the different characteristics of these M-phases.     

Structural and spectroscopic characterisation of the holmium double-decker partially oxidised and bi-radical phthalocyanine complexes with iodine

Two crystals of holmium(III) double-decker iodine doped phthalocyanines, HoPc₂I_5/3 (I) and HoPc₂I (II), were grown directly in the reaction of holmium chips with 1,2-dicyanobenzene under versatile quantity of iodine at 180-160 °C. The complex I crystallises in the P4/mcc space group of tetragonal system, while the complex II crystallises in the P2/c space group of monoclinic system. The space group of P4/mcc and z = 1 requires that the Ho(III) atom is statistically disordered in the HoPc₂I_5/3 structure. The iodine atoms form linear symmetrical triiodide ions in I, while the I⁻ ions in II. Assignment of iodine species as in the HoPc₂I_5/3 and I⁻ in HoPc₂I complexes point to the +5/9 and +1 oxidation state of the HoPc₂ unit in these complexes. Thus one Pc macrocycle of the double-decker HoPc₂ units has a non-integer oxidation state of −1.222 in I, while both Pc-rings are one-electron oxidised radical Pc⁻ in II. Magnetic susceptibilities of HoPc₂I_5/3 and HoPcI at room temperature are 4.56 × 10⁻² and 5.12 × 10⁻² emu/mol and the calculated magnetic moments are 10.46 and 11.08 μ_B, respectively. UV-Vis spectroscopic measurement of I and II in benzene solution were carried out and discussed.     

Characterization of an acetyltransferase that detoxifies aromatic chemicals in Legionella pneumophila

Legionella pneumophila is an opportunistic pathogen and the causative agent of Legionnaires' disease. Despite being exposed to many chemical compounds in its natural and man-made habitats (natural aquatic biotopes and man-made water systems), L. pneumophila is able to adapt and survive in these environments. The molecular mechanisms by which this bacterium detoxifies these chemicals remain poorly understood. In particular, the expression and functions of XMEs (xenobiotic-metabolizing enzymes) that could contribute to chemical detoxification in L. pneumophila have been poorly documented at the molecular and functional levels. In the present paper we report the identification and biochemical and functional characterization of a unique acetyltransferase that metabolizes aromatic amine chemicals in three characterized clinical strains of L. pneumophila (Paris, Lens and Philadelphia). Strain-specific sequence variations in this enzyme, an atypical member of the arylamine N-acetyltransferase family (EC 2.3.1.5), produce enzymatic variants with different structural and catalytic properties. Functional inactivation and complementation experiments showed that this acetyltransferase allows L. pneumophila to detoxify aromatic amine chemicals and grow in their presence. The present study provides a new enzymatic mechanism by which the opportunistic pathogen L. pneumophila biotransforms and detoxifies toxic aromatic chemicals. These data also emphasize the role of XMEs in the environmental adaptation of certain prokaryotes.     

On the transition from the meiotic to mitotic cell cycle during early mouse development

Here, we outline the mechanisms involved in the regulation of cell divisions during oocyte maturation and early cleavages of the mouse embryo. Our interest is focused on the regulation of meiotic M-phases and the first embryonic mitoses that are differently tuned and are characterized by specifically modified mechanisms, some of which have been recently identified. The transitions between the M-phases during this period of development, as well as associated changes in their regulation, are of key importance for both the meiotic maturation of oocytes and the further development of the mammalian embryo. The mouse is an excellent model for studies of the cell cycle during oogenesis and early development. Nevertheless, a number of molecular mechanisms described here were discovered or confirmed during the study of other species and apply also to other mammals including humans.     

On estimating similarity of artificial and real organisms

Summary In this paper, a dissimilarity measure for artificial organisms is proposed. The organisms are simulated in the Framsticks system [10]. Properties of agents are enumerated formally, and the heuristic algorithm for estimating overall phenetic dissimilarity of two agents is described. An example of performance is shown on two selected organisms. Two clustering experiments with interesting results are presented using the UPGMA method. The properties of the measure are then discussed. Computer simulations of complex systems and their characteristics are compared to biological systems, which may bring up ideas for further experiments related to biology.     

Combined structural and functional investigation of a C-3'-ketoreductase involved in the biosynthesis of dTDP-L-digitoxose

l-Digitoxose is an unusual dideoxysugar found attached to various pharmacologically active natural products, including the antitumor antibiotic tetrocarcin A and the antibiotics kijanimicin and jadomycin B. Six enzymes are required for its production starting from glucose 1-phosphate. Here we describe a combined structural and functional investigation of KijD10, an NADPH-dependent C-3'-ketoreductase that catalyzes the third step of l-digitoxose biosynthesis in the African soil-dwelling bacterium Actinomadura kijaniata. KijD10 belongs to the glucose-fructose oxidoreductase superfamily. For this investigation, both binary and ternary complexes of KijD10 were crystallized, and their structures were determined to 2.0 ? resolution or better. On the basis of these high-resolution structures, two potential active site acids were identified, Lys 102 and Tyr 186. These residues were individually mutated and the resultant proteins investigated both kinetically and structurally. The Y186F mutant protein demonstrated significant catalytic activity, and its structure was virtually identical to that of the wild-type enzyme except for the positioning of the nicotinamide ring. All lysine mutations, on the other hand, resulted in proteins with either abolished or drastically reduced catalytic activities. Structures for the K102A and K102E mutant proteins were determined and showed that the abrogation of catalytic activity was not a result of large conformational changes. Taken together, these data suggest that Lys 102 donates a proton to the C-3' keto group during the reaction and that Tyr 186 serves only an auxiliary role. This is in contrast to that proposed for glucose-fructose oxidoreductase and other family members in which the tyrosines, or in some cases similarly positioned histidines, are thought to play major catalytic roles.