Putative Calmodulin-Binding Domains in Aflatoxin Biosynthesis–Regulatory Proteins

The inhibition of aflatoxin production by trifluoperazine, an anticalmodulin (CaM) agent and the relevance of Ca²⁺/CaM-dependent phosphorylation and dephosphorylation during aflatoxin biosynthesis was previously reported. To identify proteins that may be regulated by CaM, an in silico analysis for putative CaM-binding domains (CaMBDs) in the aflatoxin-related proteins of Aspergillus parasiticus was performed using the CaM target database. Interestingly, the key regulators of aflatoxin biosynthesis such as AflR and AflJ contained predicted CaMBDs at their C-termini. Furthermore, potential phosphorylation sites for CaM-kinase II were present within these CaMBDs. In addition to other aflatoxin biosynthesis enzymes—such as Vbs, DmtA and OmtA, and the VeA protein (known to regulate the expression of AflJ and AflR)—also showed the presence of putative CaMBDs. Although the present report reaffirms earlier observations on CaM-mediated regulation of aflatoxin biosynthesis, it also opens new avenues for identifying the specific targets of CaM and elucidating the exact mechanism of initiation and regulation of aflatoxin biosynthesis.     

Fitness consequences of pheromone production and host selection strategies in a tree-killing bark beetle (Coleoptera: Curculionidae: Scolytinae)

Timing of arrival at a resource often determines an individual’s reproductive success. Tree-killing bark beetles can reproduce in healthy trees by attacking in adequate numbers to overcome host defences that could otherwise be lethal. This process is mediated by aggregation and antiaggregation pheromones. Beetles that arrive early in such a “mass attack” must contend with undiminished tree defences, and produce enough pheromones to attract more beetles, but have a head start on gallery construction and egg-laying. Beetles that arrive late may be impeded by competition and diminishing availability of phloem, but should experience fewer costs associated with pheromone production and battling tree defences. We investigated relationships between timing of arrival, body size, pheromone production and fitness in the southern pine beetle, Dendroctonus frontalis. In field experiments, we captured beetles that arrived early (pioneers) and late on slash pine trees, Pinus elliottii, and measured pheromone amounts in their hindguts. We marked gallery entrances of beetles as they landed on a tree and measured their reproductive success after the attack terminated. We found no difference in body size or pheromone amounts between early and late arrivers. Most beetles arrived at the middle of the attack sequence, and excavated longer galleries per day than early arrivers. The number of offspring produced per day by beetles that established galleries midway through mass attack was higher than those that arrived early or very late in the sequence. Our results suggest that beetles do not exhibit adaptive phenotypic plasticity in pre-landing pheromone production, depending on the extent of previous colonisation of a host. Rather, it appears that stabilising selection favours beetles that attack in the middle of the sequence, and contributes to attack synchrony. Synchronous attack on trees is essential before population booms characteristic of tree-killing bark beetles can occur in nature.     

Bactericidal and cyclooxygenase inhibitory diterpenes from Eremophila sturtii

Two serrulatane diterpenes, 3,8-dihydroxyserrulatic acid (1) and serrulatic acid (2), have been isolated from Eremophila sturtii through bioassay-guided fractionation. These compounds inhibit the inflammation pathway enzymes cyclooxygenase 1 and 2, and exhibit bactericidal activity against Staphylococcus aureus.     

Multiparameter assessments to determine the effects of sugars and antimicrobials on a polymicrobial oral biofilm

Clinical studies indicate relationships between dental plaque, a naturally formed biofilm, and oral diseases. The crucial role of nonmicrobial biofilm constituents in maintaining biofilm structure and biofilm-specific attributes, such as resistance to shear and viscoelasticity, is increasingly recognized. Concurrent analyses of the diverse nonmicrobial biofilm components for multiparameter assessments formed the focus of this investigation. Comparable numbers of Actinomyces viscosus, Streptococcus sanguinis, Streptococcus mutans, Neisseria subflava, and Actinobacillus actinomycetemcomitans cells were seeded into multiple wells of 96-well polystyrene plates for biofilm formation. Quantitative fluorescence and confocal laser scanning microscopy (CLSM) examined the influences of dietary sugars, incubation conditions, ingredients in oral hygiene formulations, and antibiotics on biofilm components. Biofilm extracellular polymeric substances (EPS) were examined with an optimized mixture of fluorescent lectins, with biofilm proteins, lipids, and nucleic acids detected with specific fluorescent stains. Anaerobic incubation of biofilms resulted in significantly more biofilm EPS and extractable carbohydrates than those formed under aerobic conditions (P < 0.05). Sucrose significantly enhanced biofilm EPS in comparison to fructose, galactose, glucose, and lactose (P < 0.05). CLSM demonstrated thicker biofilms under sucrose-replete conditions, along with significant increases in biofilm EPS, proteins, lipids, and nucleic acids, than under conditions of sucrose deficiency (P < 0.05). Agents in oral hygiene formulations (chlorhexidine, ethanol, and sodium lauryl sulfate), a mucolytic agent (N-acetyl-L-cysteine), and antibiotics with different modes of action (amoxicillin, doxycycline, erythromycin, metronidazole, and vancomycin) inhibited biofilm components (P < 0.05). Multiparameter analysis indicated a dose-dependent inhibition of biofilm EPS and protein by chlorhexidine and sodium lauryl sulfate, along with distinctive inhibitory patterns for subinhibitory concentrations of antibiotics. Collectively, these results highlight multiparameter assessments as a broad platform for simultaneous assessment of diverse biofilm components.     

Dynamic genetic interactions determine odor-guided behavior in Drosophila melanogaster

Understanding the genetic architecture of complex traits requires identification of the underlying genes and characterization of gene-by-gene and genotype-by-environment interactions. Behaviors that mediate interactions between organisms and their environment are complex traits expected to be especially sensitive to environmental conditions. Previous studies on the olfactory avoidance response of Drosophila melanogaster showed that the genetic architecture of this model behavior depends on epistatic networks of pleiotropic genes. We performed a screen of 1339 co-isogenic p[GT1]-element insertion lines to identify novel genes that contribute to odor-guided behavior and identified 55 candidate genes with known p[GT1]-element insertion sites. Characterization of the expression profiles of 10 p[GT1]-element insertion lines showed that the effects of the transposon insertions are often dependent on developmental stage and that hypomorphic mutations in developmental genes can elicit profound adult behavioral deficits. We assessed epistasis among these genes by constructing all possible double heterozygotes and measuring avoidance responses under two stimulus conditions. We observed enhancer and suppressor effects among subsets of these P-element-tagged genes, and surprisingly, epistatic interactions shifted with changes in the concentration of the olfactory stimulus. Our results show that the manifestation of epistatic networks dynamically changes with alterations in the environment.     

Design, synthesis, and biological activity of novel factor Xa inhibitors: 4-aryloxy substituents of 2,6-diphenoxypyridines

A novel series of triaryloxypyridines have been designed to inhibit factor Xa, a serine protease strategically located in the coagulation cascade. Inhibitor 5e has a K(I) against factor Xa of 0.12nM and is greater than 8000- and 2000-fold selective over two related serine proteases, thrombin and trypsin, respectively. The 4-position of the central pyridine has been identified as a site that tolerates various substitutions without deleterious effects on potency and selectivity. This suggests that the 4-position of the pyridine ring is an ideal site for chemical modifications to identify inhibitors with improved pharmacokinetic characteristics. This investigation has resulted in inhibitor 5d, which has an oral availability of 6% in dogs. The synthesis, in vitro activity, and in vivo profile of this class of inhibitors is outlined.     

Susceptibility of Tribolium castaneum life stages exposed to elevated temperatures during heat treatments of a pilot flour mill: influence of sanitation, temperatures attained among mills floors, and costs

The influence of sanitation on responses of life stages of the red flour beetle, Tribolium castaneum (Herbst) (Coleoptera: Tenebrionidae), was investigated in a pilot flour mill subjected to three, 24-h heat treatments by using forced-air gas heaters fueled by propane. Two sanitation levels, dusting of wheat flour and 2-cm-deep flour, were created in 25 plastic bioassay boxes, each holding eggs, young larvae, old larvae, pupae, and adults of T. castaneum plus two temperature sensors. Data loggers (48) were placed on the five mill floors to record air temperatures. The time required to reach 50 degrees C, time above 50 degrees C, and the maximum temperature among mill floors and in bioassay boxes were measured. The maximum temperature in bioassay boxes and in the mill was lower on the first floor than on other floors. This trend was apparent in time required to reach 50 degrees C and time above 50 degrees C, especially in compartments with 2-cm-deep flour. The mean +/- SE mortality of T. castaneum life stages on the first floor was 55.5 +/- 12.9-98.6 +/- 0.8%; it was 93.2 +/- 6.7-100 +/- 0.0% on other floors. Adults were the least susceptible stage. Mortality of T. castaneum stages in compartments with 2-cm-deep flour was generally lower than those with flour dust. Costs for the three heat treatments ranged from US$27,438 to $28,838. An effective heat treatment can be conducted within 24 h, provided temperatures on mill floors reach 50 degrees C in 8-12 h and are held above 50 degrees C for at least 10-14 h, with maximum temperatures held between 50 and 60 degrees C.     

Akt-dependent Skp2 mRNA translation is required for exiting contact inhibition, oncogenesis, and adipogenesis

The requirement of Akt for cell proliferation and oncogenesis is mammalian target of rapamycin complex 1 (mTORC1) dependent. SV40 large T expression in Akt-deficient cells restores cell proliferation rate, but is insufficient for exiting contact inhibition and oncogene-induced anchorage-independent growth, because of a failure to promote Skp2 mRNA translation. Skp2 mRNA and protein are induced upon exiting contact inhibition, which enables entry into mitosis. While Skp2 mRNA is induced in Akt-deficient cells, it is not translated, preventing entry into mitosis. Restoring Skp2 expression in Akt-deficient cells is sufficient to restore exit from contact inhibition and oncogenesis. Skp2 mRNA translation is dependent on mTORC1 and the eukaryotic translation initiation factor 4E (eIF4E). Thus, the requirement of Akt for exiting contact inhibition is mediated by the induction of Skp2 mRNA translation in eIF4E-dependent mechanism. These results provide a new insight into the role of the Akt/mTORC1/eIF4E axis in tumourigenesis. Akt-dependent Skp2 mRNA translation is also required for mitotic clonal expansion (MCE)--the earliest event in adipogenesis. Skp2 re-expression in Akt-deficient preadipocytes, which are impaired in adipogenesis, is sufficient to restore adipogenesis. These results uncover the mechanism by which Akt mediates adipogenesis.     

Protective effect of curcumin on pulmonary and cardiovascular effects induced by repeated exposure to diesel exhaust particles in mice

Particulate air pollution has been associated with increased risk of cardiopulmonary diseases. However, the underlying mechanisms are not fully understood. We have previously demonstrated that single dose exposure to diesel exhaust particle (DEP) causes lung inflammation and peripheral thrombotic events. Here, we exposed mice with repeated doses of DEP (15 μg/animal) every 2(nd) day for 6 days (a total of 4 exposures), and measured several cardiopulmonary endpoints 48 h after the end of the treatments. Moreover, the potential protective effect of curcumin (the yellow pigment isolated from turmeric) on DEP-induced cardiopulmonary toxicity was assessed. DEP exposure increased macrophage and neutrophil numbers, tumor necrosis factor α (TNF α) in the bronchoalveolar lavage (BAL) fluid, and enhanced airway resistance to methacoline measured invasively using Flexivent. DEP also significantly increased plasma C-reactive protein (CRP) and TNF α concentrations, systolic blood pressure (SBP) as well as the pial arteriolar thrombosis. It also significantly enhanced the plasma D-dimer and plasminogen activator inhibitor-1 (PAI-1). Pretreatment with curcumin by oral gavage (45 mg/kg) 1 h before exposure to DEP significantly prevented the influx of inflammatory cells and the increase of TNF α in BAL, and the increased airway resistance caused by DEP. Likewise, curcumin prevented the increase of SBP, CRP, TNF α, D-dimer and PAI-1. The thrombosis was partially but significantly mitigated. In conclusion, repeated exposure to DEP induced lung and systemic inflammation characterized by TNFα release, increased SBP, and accelerated coagulation. Our findings indicate that curcumin is a potent anti-inflammatory agent that prevents the release of TNFα and protects against the pulmonary and cardiovascular effects of DEP.