Badnaviruses and banana genomes: a long association sheds light on Musa phylogeny and origin

Badnaviruses are double-stranded DNA pararetroviruses of the family Caulimoviridae. Badnaviral sequences found in banana are distributed over three main clades of the genus Badnavirus and exhibit wide genetic diversity. Interestingly, the nuclear genome of many plants, including banana, is invaded by numerous badnaviral sequences although badnaviruses do not require an integration step to replicate, unlike animal retroviruses. Here, we confirm that banana streak viruses (BSVs) are restricted to clades 1 and 3. We also show that only BSVs from clade 3 encompassing East African viral species are not integrated into Musa genomes, unlike BSVs from clade 1. Finally, we demonstrate that sequences from clade 2 are definitively integrated into Musa genomes with no evidence of episomal counterparts; all are phylogenetically distant from BSVs known to date. Using different molecular approaches, we dissected the coevolution between badnaviral sequences of clade 2 and banana by comparing badnavirus integration patterns across a banana sampling representing major Musa speciation events. Our data suggest that primary viral integrations occurred millions of years ago in banana genomes under different possible scenarios. Endogenous badnaviral sequences can be used as powerful markers to better characterize the Musa phylogeny, narrowing down the likely geographical origin of the Musa ancestor.     

The Angiotensin II Type 1 Receptor (AT1R) Closely Interacts with Large Conductance Voltage- and Ca2+-activated K+ (BK) Channels and Inhibits Their Activity Independent of G-protein Activation

Angiotensin II (ANG-II) and BK channels play important roles in the regulation of blood pressure. In arterial smooth muscle, ANG-II inhibits BK channels, but the underlying molecular mechanisms are unknown. Here, we first investigated whether ANG-II utilizes its type 1 receptor (AT1R) to modulate BK activity. Pharmacological, biochemical, and molecular evidence supports a role for AT1R. In renal arterial myocytes, the AT1R antagonist losartan (10 μm) abolished the ANG-II (1 μm)-induced reduction of whole cell BK currents, and BK channels and ANG-II receptors were found to co-localize at the cell periphery. We also found that BK inhibition via ANG-II-activated AT1R was independent of G-protein activation (assessed with 500 μm GDPβS). In BK-expressing HEK293T cells, ANG-II (1 μm) also induced a reduction of BK currents, which was contingent on AT1R expression. The molecular mechanisms of AT1R and BK channel coupling were investigated in co-transfected cells. Co-immunoprecipitation showed formation of a macromolecular complex, and live immunolabeling demonstrated that both proteins co-localized at the plasma membrane with high proximity indexes as in arterial myocytes. Consistent with a close association, we discovered that the sole AT1R expression could decrease BK channel voltage sensitivity. Truncated BK proteins revealed that the voltage-sensing conduction cassette is sufficient for BK-AT1R association. Finally, C-terminal yellow and cyan fluorescent fusion proteins, AT1R-YFP and BK-CFP, displayed robust co-localized Förster resonance energy transfer, demonstrating intermolecular interactions at their C termini. Overall, our results strongly suggest that AT1R regulates BK channels through a close protein-protein interaction involving multiple BK regions and independent of G-protein activation.     

Effect of inoculation method on the determination of decontamination efficacy against Bacillus spores

Decontamination studies investigating the effectiveness of products and processes for the inactivation of Bacillus species spores have traditionally utilized metering viable spores in a liquid suspension onto test materials (coupons). The current study addresses the representativeness of studies using this type of inoculation method compared to when coupons are dosed with a metered amount of aerosolized spores. The understanding of this comparability is important in order to assess the representativeness of such laboratory-based testing when deciding upon decontamination options for use against Bacillus anthracis spores. Temporal inactivation of B. anthracis surrogate (B. subtilis) spores on representative materials using fumigation with chlorine dioxide, spraying of a pH-adjusted bleach solution, or immersion in the solution was investigated as a function of inoculation method (liquid suspension or aerosol dosing). Results indicated that effectiveness, measured as log reduction, was statistically significantly lower when liquid inoculation was used for some material and decontaminant combinations. Differences were mostly noted for the materials observed to be more difficult to decontaminate (i.e., wood and carpet). Significant differences in measured effectiveness were also noted to be a function of the pH-adjusted bleach application method used in the testing (spray or immersion). Based upon this work and the cited literature, it is clear that inoculation method, decontaminant application method, and handling of non-detects (i.e., or detection limits) can have an impact on the sporicidal efficacy measurements.     

Polygalacturonase enzyme production from bacterial isolated from raw milk and green and black olives

Forty microbial strains isolated from raw milk samples and black and green olives were grown in MP5 (mineral pectin 5) medium containing 0.5% lemon pectin. All strains synthesized an extracellular polygalacturonase. Rhodotorula sp. ONRh9 (0.44 U x mL(-1)) and Leuconostoc sp. LLn1 (0.16 U x mL(-1)), which had a more active polygalacturonase in MP5 medium, were studied in MAPG5 medium containing polygalacturonic acid. Highest biomass and polygalacturonase production by these two strains were observed for polygalacturonic acid concentrations of 10 g x L(-1) (Rhodotorula sp. ONRh9) and 5 g x L(-1) (Leuconostoc sp. LLn1) and for initial pH values of 6 (Rhodotorula sp. ONRh9) and 5.5 (Leuconostoc sp. LLn1). The two strains grown in fermenters in MAPG5 medium generated the following results: with controlled initial pH, Rhodotorula sp. produced maximum biomass (DO) and polygalacturonase (PG) after 20 h (DO, 3.86; PG, 0.24 U x mL(-1)) of growth, and this level was sustained until the end of the culture; Leuconostoc sp. LLn1 synthesized more cells and polygalacturonase between 4 h (DO, 1.80; PG, 0.17 U x mL(-1)) and 24 h (DO, 3.90; PG, 0.27 U x mL(-1)) of culture. With uncontrolled initial pH, the cultures produced maximum biomass and polygalacturonase after 20 h (DO, 3.30; PG, 0.26 U x mL(-1)) for Rhodotorula sp. ONRh9 and 10 h (DO, 2.84; PG, 0.17 U x mL(-1)) for Leuconostoc sp. LLn1.     

Mediators of PGE2 synthesis and signalling downstream of COX-2 represent potential targets for the prevention/treatment of colorectal cancer

Colorectal cancer is a major cause of mortality and whilst up to 80% of sporadic colorectal tumours are considered preventable, trends toward increasing obesity suggest the potential for a further increase in its worldwide incidence. Novel methods of colorectal cancer prevention and therapy are therefore of considerable importance. Non-steroidal anti-inflammatory drugs (NSAIDs) are chemopreventive against colorectal cancer, mainly through their inhibitory effects on the cyclooxygenase isoform COX-2. COX enzymes represent the committed step in prostaglandin biosynthesis and it is predominantly increased COX-2-mediated prostaglandin-E₂ (PGE₂) production that has a strong association with colorectal neoplasia, by promoting cell survival, cell growth, migration, invasion and angiogenesis. COX-1 and COX-2 inhibition by traditional NSAIDs (for example, aspirin) although chemopreventive have some side effects due to the role of COX-1 in maintaining the integrity of the gastric mucosa. Interestingly, the use of COX-2 selective NSAIDs has also shown promise in the prevention/treatment of colorectal cancer while having a reduced impact on the gastric mucosa. However, the prolonged use of high dose COX-2 selective inhibitors is associated with a risk of cardiovascular side effects. Whilst COX-2 inhibitors may still represent viable adjuvants to current colorectal cancer therapy, there is an urgent need to further our understanding of the downstream mechanisms by which PGE₂ promotes tumorigenesis and hence identify safer, more effective strategies for the prevention of colorectal cancer. In particular, PGE₂ synthases and E-prostanoid receptors (EP1–4) have recently attracted considerable interest in this area. It is hoped that at the appropriate stage, selective (and possibly combinatorial) inhibition of the synthesis and signalling of those prostaglandins most highly associated with colorectal tumorigenesis, such as PGE₂, may have advantages over COX-2 selective inhibition and therefore represent more suitable targets for long-term chemoprevention. Furthermore, as COX-2 is found to be overexpressed in cancers such as breast, gastric, lung and pancreatic, these investigations may also have broad implications for the prevention/treatment of a number of other malignancies.     

Phenylpropanoids from Thapsia transtagana

Five phenylpropanoids have been isolated from the roots of Thapsia transtagana. Their structures have been elucidated by spectroscopic means.     

Draft Genome Sequence of Tsukamurella sp. Strain 1534

A draft genome sequence of Tsukamurella sp., an aerobic bacterium isolated from a human sputum specimen, is described here. A new virus or provirus, TPA4, was characterized.     

The β1-Subunit of the MaxiK Channel Associates with the Thromboxane A2 Receptor and Reduces Thromboxane A2 Functional Effects

The large conductance voltage- and Ca²⁺-activated K⁺ channel (MaxiK, BK_Ca, BK) is composed of four pore-forming α-subunits and can be associated with regulatory β-subunits. One of the functional roles of MaxiK is to regulate vascular tone. We recently found that the MaxiK channel from coronary smooth muscle is trans-inhibited by activation of the vasoconstricting thromboxane A₂ prostanoid receptor (TP), a mechanism supported by MaxiK α-subunit (MaxiKα)-TP physical interaction. Here, we examined the role of the MaxiK β1-subunit in TP-MaxiK association. We found that the β1-subunit can by itself interact with TP and that this association can occur independently of MaxiKα. Subcellular localization analysis revealed that β1 and TP are closely associated at the cell periphery. The molecular mechanism of β1-TP interaction involves predominantly the β1 extracellular loop. As reported previously, TP activation by the thromboxane A₂ analog U46619 caused inhibition of MaxiKα macroscopic conductance or fractional open probability (FP_o) as a function of voltage. However, the positive shift of the FP_o versus voltage curve by U46619 relative to the control was less prominent when β1 was coexpressed with TP and MaxiKα proteins (20 ± 6 mV, n = 7) than in cells expressing TP and MaxiKα alone (51 ± 7 mV, n = 7). Finally, β1 gene ablation reduced the EC₅₀ of the U46619 agonist in mediating aortic contraction from 18 ± 1 nm (n = 12) to 9 ± 1 nm (n = 12). The results indicate that the β1-subunit can form a tripartite complex with TP and MaxiKα, has the ability to associate with each protein independently, and diminishes U46619-induced MaxiK channel trans-inhibition as well as vasoconstriction.