Rescue of foot-and-mouth disease viruses that are pathogenic for cattle from preserved viral RNA samples

Background

Foot and mouth disease is an economically important disease of cloven-hoofed animals including cattle, sheep and pigs. It is caused by a picornavirus, foot-and-mouth disease virus (FMDV), which has a positive sense RNA genome which, when introduced into cells, can initiate virus replication.

Principal Findings

A system has been developed to rescue infectious FMDV from RNA preparations generated from clinical samples obtained under experimental conditions and then applied to samples collected in the “field”. Clinical samples from suspect cases of foot-and-mouth disease (FMD) were obtained from within Pakistan and Afghanistan. The samples were treated to preserve the RNA and then transported to National Veterinary Institute, Lindholm, Denmark. Following RNA extraction, FMDV RNA was quantified by real-time RT-PCR and samples containing significant levels of FMDV RNA were introduced into susceptible cells using electroporation. Progeny viruses were amplified in primary bovine thyroid cells and characterized using antigen ELISA and also by RT-PCR plus sequencing. FMD viruses of three different serotypes and multiple lineages have been successfully rescued from the RNA samples. Two of the rescued viruses (of serotype O and Asia 1) were inoculated into bull calves under high containment conditions. Acute clinical disease was observed in each case which spread rapidly from the inoculated calves to in-contact animals. Thus the rescued viruses were highly pathogenic. The availability of the rescued viruses enabled serotyping by antigen ELISA and facilitated genome sequencing.

Conclusions

The procedure described here should improve the characterization of FMDVs circulating in countries where the disease is endemic and thus enhance disease control globally.     

Role of A1 adenosine receptors in regulation of vascular tone

The vascular response to adenosine and its analogs is mediated by four adenosine receptors (ARs), namely, A(1), A(2A), A(2B), and A(3). A(2A)ARs and/or A(2B)ARs are involved in adenosine-mediated vascular relaxation of coronary and aortic beds. However, the role of A(1)ARs in the regulation of vascular tone is less well substantiated. The aim of this study was to determine the role of A(1)ARs in adenosine-mediated regulation of vascular tone. A(1)AR-knockout [A(1)AR((-/-))] mice and available pharmacological tools were used to elucidate the function of A(1)ARs and the impact of these receptors on the regulation of vascular tone. Isolated aortic rings from A(1)AR((-/-)) and wild-type [A(1)AR((+/+))] mice were precontracted with phenylephrine, and concentration-response curves for adenosine and its analogs, 5'-N-ethyl-carboxamidoadenosine (NECA, nonselective), 2-chloro-N(6)-cyclopentyladenosine (CCPA, A(1)AR selective), 2-(2-carboxyethyl)phenethyl amino-5'-N-ethylcarboxamido-adenosine (CGS-21680, A(2A) selective), and 2-chloro-N(6)-3-iodobenzyladenosine-5'-N-methyluronamide (Cl-IBMECA, A(3) selective) were obtained to determine relaxation. Adenosine and NECA (0.1 microM) caused small contractions of 13.9 +/- 3.0 and 16.4 +/- 6.4%, respectively, and CCPA at 0.1 and 1.0 microM caused contractions of 30.8 +/- 4.3 and 28.1 +/- 3.9%, respectively, in A(1)AR((+/+)) rings. NECA- and CCPA-induced contractions were eliminated by 100 nM of 1,3-dipropyl-8-cyclopentylxanthine (DPCPX, selective A(1)AR antagonist). Adenosine, NECA, and CGS-21680 produced an increase in maximal relaxation in A(1)AR((-/-)) compared with A(1)AR((+/+)) rings, whereas Cl-IBMECA did not produce contraction in either A(1)AR((+/+)) or A(1)AR((-/-)) rings. CCPA-induced contraction at 1.0 microM was eliminated by the PLC inhibitor U-73122. These data suggest that activation of A(1)ARs causes contraction of vascular smooth muscle through PLC pathways and negatively modulates the vascular relaxation mediated by other adenosine receptor subtypes.     

Medicinal mushroom modulators of molecular targets as cancer therapeutics

Empirical approaches to discover anticancer drugs and cancer treatments have made limited progress in the past several decades in finding a cure for cancer. The expanded knowledge of the molecular basis of tumorigenesis and metastasis, together with the inherently vast structural diversity of natural compounds found in mushrooms, provided unique opportunities for discovering new drugs that rationally target the abnormal molecular and biochemical signals leading to cancer. This review focuses on mushroom low-molecular-weight secondary metabolites targeting processes such as apoptosis, angiogenesis, metastasis, cell cycle regulation, and signal transduction cascades. Also discussed in this review are high-molecular-weight polysaccharides or polysaccharide–protein complexes from mushrooms that appear to enhance innate and cell-mediated immune responses, exhibit antitumor activities in animals and humans, and demonstrate the anticancer properties of selenium compounds accumulated in mushrooms.     

U wave: an important noninvasive electrocardiographic diagnostic marker

Study of U waves exemplifies important clinical role of noninvasive electrocardiography in modern cardiology. Present article highlights significance of U waves with a clinical case and also summarizes in brief the history of the same.     

A kinetic model of the cytochrome bf complex. Evaluation of kinetic parameters

A kinetic model of the cytochrome bf complex was developed on the assumption that the Q-cycle operates. The bf complex was considered as a membrane enzyme catalyzing the electron transfer from plastoquinol to plastocyanine, which is coupled with proton translocation from the chloroplast stroma to the thylakoid lumen. The dependence of the electron transfer rates on the value of the transmembrane electric potential was taken into account. The model was applied to describe the experimental data on the flash-induced turnover of cytochromes b, plastocyanine, and the kinetics of proton deposition in the thylakoid lumen. The estimation of model parameters was performed.     

NADH oxidase activity of Bacillus subtilis nitroreductase NfrA1: Insight into its biological role

NfrA1 nitroreductase from the Gram-positive bacterium Bacillus subtilis is a member of the NAD(P)H/FMN oxidoreductase family. Here, we investigated the reactivity, the structure and kinetics of NfrA1, which could provide insight into the unclear biological role of this enzyme. We could show that NfrA1 possesses an NADH oxidase activity that leads to high concentrations of oxygen peroxide and an NAD⁺ degrading activity leading to free nicotinamide. Finally, we showed that NfrA1 is able to rapidly scavenge H₂O₂ produced during the oxidative process or added exogenously.

Structured summary

MINT-7990140: nfrA1 (uniprotkb:P39605) and nfrA1 (uniprotkb:P39605) bind (MI:0407) by X-ray crystallography (MI:0114)     

Isolation and characterization of coronary endothelial and smooth muscle cells from A1 adenosine receptor-knockout mice

Mice have been used widely in in vivo and in vitro cardiovascular research. The availability of knockout mice provides further clues to the physiological significance of specific receptor subtypes. Adenosine A(1) receptor (A(1)AR)-knockout (A(1)KO) mice and their wild-type (A(1)WT) controls were employed in this investigation. The heart and aortic arch were carefully removed and retroinfused with enzyme solution (1 mg/ml collagenase type I, 0.5 mg/ml soybean trypsin inhibitor, 3% BSA, and 2% antibiotics) through the aortic arch. The efflux was collected at 30-, 60-, and 90-min intervals. The cells were centrifuged, and the pellets were mixed with medium [medium 199-F-12 medium with 10% FBS and 2% antibiotics (for endothelial cells) and advanced DMEM with 10% FBS, 10% mouse serum, 2% GlutaMax, and 2% antibiotics (for smooth muscle cells)] and plated. Endothelial cells were characterized by a cobblestone appearance and positive staining with acetylated LDL labeled with 1,1'-dioctadecyl-3,3,3',3-tetramethylindocarbocyanine perchlorate. Smooth muscle cells were characterized by positive staining of smooth muscle alpha-actin and smooth muscle myosin heavy chain. Homogeneity of the smooth muscle cells was approximately 91%. Western blot analysis showed expression of smoothelin in the cells from passages 3, 7, and 11 in A(1)WT and A(1)KO mice. Furthermore, the A(1)AR was characterized by Western blot analysis using an A(1)AR-specific antibody. To our knowledge, this is the first isolation and successful characterization of smooth muscle cells from the mouse coronary system.     

Regulation of protein phosphatase type 1 and cell cycle progression by PfLRR1, a novel leucine-rich repeat protein of the human malaria parasite Plasmodium falciparum

The protein called 'suppressor of the dis2 mutant (sds22+)' is an essential regulator of cell division in fission and budding yeasts, where its deletion causes mitotic arrest. Its role in cell cycle control appears to be mediated through the activation of protein phosphatase type 1 (PP1) in Schizosaccharomyces pombe. We have identified the Plasmodium falciparum Sds22 orthologue, which we designated PfLRR1 as it belongs to the leucine-rich repeat protein family. We showed by glutathione-S-transferase pull-down assay that the PfLRR1 gene product interacts with PfPP1, that the PfLRR1-PfPP1 complex is present in parasite extracts and that PfLRR1 inhibits PfPP1 activity. Functional studies in Xenopus oocytes revealed that PfLRR1 interacted with endogenous PP1 and overcame the G2/M cell cycle checkpoint by promoting progression to germinal vesicle breakdown (GVBD). Confirmatory results showing the appearance of GVBD were observed when oocytes were treated with anti-PP1 antibodies or okadaic acid. Taken together, these observations suggest that PfLRR1 can regulate the cell cycle by binding to PP1 and regulating its activity.