Phosphorylation of the potyvirus capsid protein by protein kinase CK2 and its relevance for virus infection

We reported previously that the capsid protein (CP) of Potato virus A (PVA) is phosphorylated both in virus-infected plants and in vitro. In this study, an enzyme that phosphorylates PVA CP was identified as the protein kinase CK2. The alpha-catalytic subunit of CK2 (CK2alpha) was purified from tobacco and characterized using in-gel kinase assays and liquid chromatography-tandem mass spectrometry. The tobacco CK2alpha gene was cloned and expressed in bacterial cells. Specific antibodies were raised against the recombinant enzyme and used to demonstrate the colocalization of PVA CP and CK2alpha in infected tobacco protoplasts. A major site of CK2 phosphorylation in PVA CP was identified by a combination of mass spectrometric analysis, radioactive phosphopeptide sequencing, and mutagenesis as Thr-242 within a CK2 consensus sequence. Amino acid substitutions that affect the CK2 consensus sequence in CP were introduced into a full-length infectious cDNA clone of PVA tagged with green fluorescent protein. Analysis of the mutant viruses showed that they were defective in cell-to-cell and long-distance movement. Using in vitro assays, we demonstrated that CK2 phosphorylation inhibited the binding of PVA CP to RNA, suggesting a molecular mechanism of CK2 action. These results suggest that the phosphorylation of PVA CP by CK2 plays an important regulatory role in virus infection.     

Folate biosynthesis pseudogenes, PsifolP and PsifolK, and an O-sialoglycoprotein endopeptidase gene homolog in the phytoplasma genome

Phytoplasmas are wall-less phytopathogenic prokaryotes of small genome sizes that are obligate parasites of insect vectors and plant hosts. We have cloned a clover phyllody (CPh) phytoplasma DNA locus containing five potential coding sequences. Two were identified as pseudogenes (PsifolP and PsifolK) homologous to folP and folK genes, which encode dihydropteroate synthase (DHPS) and 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK), respectively, in other bacteria. Evolution of the phytoplasma presumably involved loss of functions through the formation of these and other pseudogenes during adaptation to obligate parasitism. The findings suggest that the phytoplasma lacks capacity for de novo folate biosynthesis and possesses a transport system for absorption of preformed folate from host cells. The PsifolP-PsifolK region was flanked by three open reading frames (ORFs) encoding a DegV family protein, a hypothetical protein with a P60-like lipoprotein domain homologous with the P60-like Mycoplasma hominis protein, and a glycoprotease (Gcp) protein that possibly functions as a host adaptation or virulence factor.     

Lessons learned from a regional strategy for resource allocation

Two qualitative case studies focus on the allocation of CDC funds distributed during 2002 for bioterrorism preparedness in two Texas public health regions (each as populous and complex as many states). Lessons learned are presented for public health officials and others who work to build essential public health services and security for our nation. The first lesson is that personal relationships are the cornerstone of preparedness. A major lesson is that a regional strategy to manage funds may be more effective than allocating funds on a per capita basis. One regional director required every local department to complete a strategic plan as a basis for proportional allocation of the funds. Control of communicable diseases was a central component of the planning. Some funds were kept at the regional level to provide epidemiology services, computer software, equipment, and training for the entire region. Confirmation of the value of this regional strategy was expressed by local public health and emergency management officials in a focus group 1 year after the strategy had been implemented. The group members also pointed out the need to streamline the planning process, provide up-to-date computer networks, and receive more than minimal communication. This regional strategy can be viewed from the perspective of adaptive leadership, defined as activities to bring about constructive change, which also can be used to analyze other difficult areas of preparedness.     

Analysis of effects of 2,2',5,5'-tetrachlorobiphenyl on the flux control in oxidative phosphorylation system in rat liver mitochondria

Modular kinetic analysis reveals that the environmental pollutant 2,2',5,5'-tetrachlorobiphenyl (2,2',5,5'-TCB) affects a large number of steps in oxidative phosphorylation in rat liver mitochondria. 2,2',5,5'-TCB increases membrane permeability to ions, and inhibits NADH dehydrogenase, cytochrome bc1, cytochrome oxidase (all in the respiratory chain) and ATP-synthase (in the phosphorylation subsystem). Surprisingly, flux control distribution does not change. A kinetic model for oxidative phosphorylation was used to simulate these findings, and it was found that combined large changes in the processes indicated indeed left the flux control largely unchanged. In addition, computational analysis with the model indicated that the adenine nucleotide translocator might be inhibited by 2,2',5,5'-TCB.     

Different paths – the same virulence: experimental study on avian single and co-infections with Plasmodium relictum and Plasmodium elongatum

Co-infections are prevalent worldwide, however, we are still struggling to understand interactions between different parasites and their impacts on host fitness. In the present experimental study we analysed the infection dynamics of two avian malarial parasites Plasmodium elongatum (genetic lineage pERIRUB01) and Plasmodium relictum (genetic lineage pSGS1) and their impacts on host health during single and co-infections. We reveal that P. elongatum intensity of parasitemia is enhanced by the presence of P. relictum during co-infection, while the parasitemia of P. relictum stays the same. This illustrates how development of a parasite (P. elongatum) which infects both mature and young (polychromatic) red blood cells (RBCs) is facilitated during co-infection with a parasite which specialises in adult RBCs only (P. relictum). The virulence of co-infections was similar to that of the more virulent parasite (P. elongatum). However, the profile of infection and the mechanisms that caused mortality were different. Birds infected with P. elongatum only start to die due to non-regenerative anaemia, when intensity of parasitemia is light and the number of polychromatic RBCs decrease dramatically. Meanwhile, co-infected birds start to die when the mean intensity of parasitemia reaches 10% and the number of polychromatic RBCs increases abnormally, reflecting regenerative anaemia. Our findings reveal that typically measured parameters of virulence (e.g., mortality rate, level of hematocrit) can be the same during single and co-infections, but the mechanisms responsible for the observed virulence can be different. This information serves a better understanding of the processes underpinning the interactions of co-infected parasite species.     

Sequence-variable mosaics: composites of recurrent transposition characterizing the genomes of phylogenetically diverse phytoplasmas

Phytoplasmas are cell wall-less prokaryotes characterized by small, AT-rich genomes that encode capabilities for obligate, transkingdom parasitism and pathogenicity in plants and insect vectors. Inability to isolate and characterize phytoplasmas in pure culture has led to adoption of the 'Candidatus species' convention to refer to distinct phytoplasma lineages. In this study, we provide evidence that multiple, sequence-variable mosaics (SVMs) of clustered genes and repetitive extragenic palindromes are characteristic features of phytoplasma genome architecture in phylogenetically diverse species. The findings suggest that the origin of SVMs was an ancient event in evolution of the phytoplasma clade, while current forms of SVMs are results of dramatic and more recent events. Sequence diversity of hypervariable regions indicated rapid evolution possibly involving capture of mobile elements recurrently targeted to SVMs. Multiple events of targeted mobile element attack, recombination, and rearrangement conceivably account for the composite structure of SVMs. Proteins encoded by the highly variable region included a lysophospholipase and other putatively secreted and/or transmembrane, cell surface-interacting proteins potentially significant in phytoplasma-host interactions.     

A 2-Hydroxypyridine Catabolism Pathway in Rhodococcus rhodochrous Strain PY11

Rhodococcus rhodochrous PY11 (DSM 101666) is able to use 2-hydroxypyridine as a sole source of carbon and energy. By investigating a gene cluster (hpo) from this bacterium, we were able to reconstruct the catabolic pathway of 2-hydroxypyridine degradation. Here, we report that in Rhodococcus rhodochrous PY11, the initial hydroxylation of 2-hydroxypyridine is catalyzed by a four-component dioxygenase (HpoBCDF). A product of the dioxygenase reaction (3,6-dihydroxy-1,2,3,6-tetrahydropyridin-2-one) is further oxidized by HpoE to 2,3,6-trihydroxypyridine, which spontaneously forms a blue pigment. In addition, we show that the subsequent 2,3,6-trihydroxypyridine ring opening is catalyzed by the hypothetical cyclase HpoH. The final products of 2-hydroxypyridine degradation in Rhodococcus rhodochrous PY11 are ammonium ion and α-ketoglutarate.     

The central core region of yeast ribosomal protein L11 is important for subunit joining and translational fidelity

Yeast ribosomal protein L11 is positioned at the intersubunit cleft of the large subunit central protuberance, forming an intersubunit bridge with the small subunit protein S18. Mutants were engineered in the central core region of L11 which interacts with Helix 84 of the 25S rRNA. Numerous mutants in this region conferred 60S subunit biogenesis defects. Specifically, many mutations of F96 and the A66D mutant promoted formation of halfmers as assayed by sucrose density ultracentrifugation. Halfmer formation was not due to deficiency in 60S subunit production, suggesting that the mutants affected subunit-joining. Chemical modification analyses indicated that the A66D mutant, but not the F96 mutants, promoted changes in 25S rRNA structure, suggesting at least two modalities for subunit joining defects. 25S rRNA structural changes were located both adjacent to A66D (in H84), and more distant (in H96-7). While none of the mutants significantly affected ribosome/tRNA binding constants, they did have strong effects on cellular growth at both high and low temperatures, in the presence of translational inhibitors, and promoted changes in translational fidelity. Two distinct mechanisms are proposed by which L11 mutants may affect subunit joining, and identification of the amino acids associated with each of these processes are presented. These findings may have implications for our understanding of multifaceted diseases such as Diamond–Blackfan anemia which have been linked in part with mutations in L11.     

Helminths of red foxes (Vulpes vulpes) and raccoon dogs (Nyctereutes procyonoides) in Lithuania

Red foxes and raccoon dogs are hosts for a wide range of parasites including important zoonotic helminths. The raccoon dog has recently invaded into Europe from the east. The contribution of this exotic species to the epidemiology of parasitic diseases, particularly parasitic zoonoses is unknown. The helminth fauna and the abundance of helminth infections were determined in 310 carcasses of hunted red foxes and 99 of raccoon dogs from Lithuania. Both species were highly infected with Alaria alata (94·8% and 96·5% respectively) and Trichinella spp. (46·6% and 29·3%). High and significantly different prevalences in foxes and raccoon dogs were found for Eucoleus aerophilus (97·1% and 30·2% respectively), Crenosoma vulpis (53·8% and 15·1%), Capillaria plica (93·3% and 11·3%), C. putorii (29·4% and 51·5%), Toxocara canis (40·5% and 17·6%) and Uncinaria stenocephala (76·9% and 98·8%). The prevalences of the rodent-transmitted cestodes Echinococcus multilocularis, Taenia polyacantha, T. crassiceps and Mesocestoides spp. were significantly higher in foxes than in raccoon dogs. The abundances of E. multilocularis, Mesocestoides, Taenia, C. plica and E. aerophilus were higher in foxes than those in raccoon dogs. A. alata, U. stenocephala, C. putorii and Echinostomatidae had higher abundances in raccoon dogs. The difference in prevalence and abundance of helminths in both animals may reflect differences in host ecology and susceptibility. The data are consistent with red foxes playing a more important role than raccoon dogs in the transmission of E. multilocularis in Lithuania.     

Chemical genetics define the roles of p38alpha and p38beta in acute and chronic inflammation

The p38 MAP kinase signal transduction pathway is an important regulator of proinflammatory cytokine production and inflammation. Defining the roles of the various p38 family members, specifically p38alpha and p38beta, in these processes has been difficult. Here we use a chemical genetics approach using knock-in mice in which either p38alpha or p38beta kinase has been rendered resistant to the effects of specific inhibitors along with p38beta knock-out mice to dissect the biological function of these specific kinase isoforms. Mice harboring a T106M mutation in p38alpha are resistant to pharmacological inhibition of LPS-induced TNF production and collagen antibody-induced arthritis, indicating that p38beta activity is not required for acute or chronic inflammatory responses. LPS-induced TNF production, however, is still completely sensitive to p38 inhibitors in mice with a T106M point mutation in p38beta. Similarly, p38beta knock-out mice respond normally to inflammatory stimuli. These results demonstrate conclusively that specific inhibition of the p38alpha isoform is necessary and sufficient for anti-inflammatory efficacy in vivo.