Genotoxic activity of <Emphasis Type="Italic">Eucalyptus globulus</Emphasis> essential oil in <Emphasis Type="Italic">Aspergillus nidulans</Emphasis> diploid cells

Eucalyptus globulus essential oil was evaluated for its genotoxic potential using a somatic segregation assay and a diploid strain of the fungus Aspergillus nidulans, heterozygous for nutritional and conidia color markers. The main compounds of the current essential oil sample were eucalyptol (49.0 %), α-pinene (8.9), β-pinene (1.5), globulol (6.9), α-eudesmol (1.12), spathulenol (1.42), γ-cadinene (1.45), trans-β-elemenone (1.23) and aromandendrene (2.3), totaling 74 % of oil. Oil at 0.12 and 0.25 μL/mL was found to increase the mitotic instability of the original diploid strain and the number of diploid mitotic recombinants of A. nidulans. The genotoxicity of the oil was associated with the induction of mitotic crossing-over or with oil-broken chromosomes.     

Molecular mechanisms of cell proliferation induced by low power laser irradiation

Low power laser irradiation (LPLI) promotes proliferation of multiple cells, which (especially red and near infrared light) is mainly through the activation of mitochondrial respiratory chain and the initiation of cellular signaling. Recently, the signaling proteins involved in LPLI-induced proliferation merit special attention, some of which are regulated by mitochondrial signaling. Hepatocyte growth factor receptor (c-Met), a member of tyrosine protein kinase receptors (TPKR), is phosphorylated during LPLI-induced proliferation, but tumor necrosis factor alpha (TNF-alpha) receptor has not been affected. Activated TPKR could activate its downstream signaling elements, like Ras/Raf/MEK/ERK, PI3K/Akt/eIF4E, PI3K/Akt/eNOS and PLC-gamma/PKC pathways. Other two pathways, ΔΨm/ATP/cAMP/JNK/AP-1 and ROS/Src, are also involved in LPLI-induced proliferation. LPLI-induced cell cycle progression can be regulated by the activation or elevated expressions of cell cycle-specific proteins. Furthermore, LPLI induces the synthesis or release of many molecules, like growth factors, interleukins, inflammatory cytokines and others, which are related to promotive effects of LPLI.     

Evaluation of a Stochastic Inactivation Model for Heat-Activated Spores of Bacillus spp.

Heat activates the dormant spores of certain Bacillus spp., which is reflected in the “activation shoulder” in their survival curves. At the same time, heat also inactivates the already active and just activated spores, as well as those still dormant. A stochastic model based on progressively changing probabilities of activation and inactivation can describe this phenomenon. The model is presented in a fully probabilistic discrete form for individual and small groups of spores and as a semicontinuous deterministic model for large spore populations. The same underlying algorithm applies to both isothermal and dynamic heat treatments. Its construction does not require the assumption of the activation and inactivation kinetics or knowledge of their biophysical and biochemical mechanisms. A simplified version of the semicontinuous model was used to simulate survival curves with the activation shoulder that are reminiscent of experimental curves reported in the literature. The model is not intended to replace current models to predict dynamic inactivation but only to offer a conceptual alternative to their interpretation. Nevertheless, by linking the survival curve''s shape to probabilities of events at the individual spore level, the model explains, and can be used to simulate, the irregular activation and survival patterns of individual and small groups of spores, which might be involved in food poisoning and spoilage.Heat inactivation kinetics of bacterial spores is a well-researched field. Much of the work on its relation to foods has focused on the heat-resistant spores of Clostridia, particularly those of Clostridium botulinum, which to this date serves as the reference organism in sterility calculations of low-acid foods (8, 32). The thermal resistance of Bacilli spores, although also extensively studied, has received less attention in the literature on food preservation. This is primarily because they are unlikely to germinate and produce cells that will survive and divide under the anaerobic conditions in a sterilized food container. Yet the mere possibility of viable Bacillus spores being present in processed foods has become an issue of food safety and a security concern. For this reason, there is a renewed interest in these spores'' heat resistance (2, 3, 6, 7, 16, 30). One of the peculiarities of certain Bacillus spores, like those of Bacillus sporothermodurans or Bacillus stearothermophilus, is that many of them can remain dormant unless activated by heat. The result is a survival curve that exhibits an “activation shoulder,” as shown schematically in Fig. ​Fig.11 and with published data in Fig. ​Fig.2.2. Thus, modeling this survival pattern, where the number of spores initially grows rather than declines, must account for the heat''s dual role of being a lethal agent and activator at the same time.Open in a separate windowFIG. 1.A schematic view of a survival curve having an activation shoulder. S(t) is the ratio between the number N(t) of viable spores at time t and the initial number N₀. Notice the discrepancy between the two ways to estimate the number of dormant spores, represented by the dashed and dotted gray lines.Open in a separate windowFIG. 2.Demonstration of the fit of equation 1 (solid line) and equation 2 (dashed line) to survival curves of B. stearothermophilus spores at two temperatures. Notice the postpeak concavity of the curves. In such cases, the estimated number of dormant spores reached by the tangent method will depend on the experiment duration. The original experimental data are from Sapru et al. (25).Traditionally, the thermal inactivation of both Clostridia and Bacilli spores has been thought to follow first-order kinetics (9, 12, 31), an assumption that has been frequently challenged in recent years (18, 21, 33, 35). The most publicized model of the simultaneous heat activation and inactivation of Bacillus spores in food is that proposed by Sapru et al. (24, 25), which is an improved version of models proposed earlier by Shull et al. (29) and Rodriguez et al. (23). All of these authors and others (1, 17) assumed that the activation of dormant spores follows first-order kinetics and so does their inactivation before and after activation. The temperature dependence of the corresponding exponential rate constants was assumed to follow the Arrhenius equation.Peleg (18, 20) and van Boekel (33, 35) have shown that none of the above assumptions was necessary and that the same survival data on Bacillus stearothermophilus reported by Sapru et al. (25) and other investigators (5) can be described by different kinds of alternative four-parameter empirical models, which have a slightly better fit. This was evident not only visually (Fig. ​(Fig.2)2) but also as judged by statistical criteria (34). Fig. ​Fig.22 shows the fit of the “double Weibullian” model proposed by van Boekel (33). It has the following form: (1) where S(t) = N(t)/N₀ is the survival/activation ratio, N₀ and N(t) are the initial and momentary number of countable spores, respectively, and b₁, b₂, n₁, and n₂ are adjustable temperature-dependent constants. Figure 2 also shows the fit of an ad hoc empirical model, a hybrid between the double Weibullian model and one previously proposed (20) that can be written in the following form: (2) or (3) where a₁, b₁, t_c2, and m₂ are adjustable temperature-dependent parameters. According to this model, a₁ is the asymptote of the first term on the right, b₁ is a time characteristic of the activation, t_c2 is a characteristic time of the inactivation, and m₂ is a parameter that represents the curve''s postpeak concavity. The structure of equation 2 or 3 dictates that the number of dormant spores must be finite and cannot exceed N₀ × 10^a1, if the logarithm is base 10, or N₀ × exp(a₁), if it is base e. (A demonstration that generates realistic-looking activation/inactivation curves using equation 3 as a model is available from Wolfram Research [http://demonstrations.wolfram.com/SurvivalCurvesOfBacilliSporesWithAnActivationShoulder/].)Corradini and Peleg (5) proposed a way to estimate the initial number of dormant spores from survival curves having an activation shoulder using a similar model, which was originally described in Peleg (20). They suggested that the intersection of a tangent to the survival curve drawn at its postpeak region with the time axis (Fig. ​(Fig.1)1) is not a recommended method to estimate the number of dormant spores and that it can render unrealistically high values if used. Also, where there is no evidence that the survival curve in the postpeak region ever becomes a straight line; the same survival curve will yield different estimates of the dormant spores'' initial number depending on the experiment''s duration. Moreover, if in the postpeak region the survival ratio drop rate progressively increases, as it most probably does (Fig. ​(Fig.2)2) (20, 33), then the number of dormant spores estimated by the tangent extrapolation method will grow indefinitely, despite the fact that it must be finite (1). Also, since the exponential inactivation rate can be a function of time as well as of temperature, the applicability of the Arrhenius equation as a secondary model might come into question. The same can also be said about the log-linearity of the D value''s temperature dependence if used instead of the Arrhenius equation.The question that arises in light of all the above is whether one can construct a conceptual population dynamic model of the activation/inactivation of spores without assuming any fixed kinetic order. The biochemical and biophysical mechanisms that govern bacterial spore germination, activation, and inactivation have been thoroughly investigated (11, 13, 14, 15, 22, 26-28). Still, it is not clear how processes within an individual spore can be translated into activation and survival patterns at the population level and how their manifestation can be expressed in a mathematical model. Whenever a system has inherent variability and knowledge of its working is incomplete or merely insufficient to develop a model from basic principles, one can, and sometimes must, resort to a probabilistic modeling approach. The general objective of this work has been to explore the merits and limitations of this option by developing a stochastic model of Bacilli spores'' heat activation and inactivation and examining its properties. The goal has not been to develop a new method to predict the spores'' survival under dynamic conditions—rate versions of the existing empirical models such as equation 1, 2, or 3 seem to be quite suitable for that—but to offer an alternative interpretation of the patterns reported and discussed in the literature.     

Purification, characterization, and mode of action of a rhamnogalacturonan hydrolase from Irpex lacteus, tolerant to an acetylated substrate

A novel rhamnogalacturonase (RGase) acting on an acetylated substrate was detected in the commercial preparation Driselase, an enzymatic mixture derived from the basidiomycete Irpex lacteus. The activity was isolated by hydrophobic interaction chromatography, gel filtration, and preparative isoelectric focusing, resulting in the isolation of five different rhamnogalacturonan hydrolases exhibiting various isoelectric points from 6.2 to 7.7. Sodium dodecyl sulfate polyacrylamide gel electrophoresis and mass spectrometry analyses after trypsin cleavage of the five fractions revealed that the five rhamnogalacturonases have a molar mass of 55 kDa without any divergences in the identified peptides. The RGase with a pI of 7.2 exhibited a pH optimum between 4.5 and 5 and a temperature optimum between 40°C and 50°C. Its mode of action was analyzed by mass spectrometry of the oligosaccharides produced after hydrolysis of acetylated and nonacetylated rhamnogalacturonan. Oligomers esterified by an acetyl group on the reducing galacturonic acid residue or fully acetylated were detected in the hydrolysate showing that the novel enzyme is able to bind acetylated galacturonic acid in its active site.     

Acidic and basic deprotection strategies of borane-protected phosphinothioesters for the traceless Staudinger ligation

The traceless Staudinger ligation has recently found various applications in the field of peptide synthesis and modification, including immobilization and cyclization strategies. In this report, we utilize the traceless Staudinger ligation in the formation of amide bonds, which allows the acquisition of acylated aminosugars and peptides as well as the cyclization of peptides. A key element in these synthetic procedures is the use of a borane-protected phosphinomethanethiol, which is demonstrated to be prone towards oxidation in its unprotected form, during the synthesis of phosphinothioesters. In combination with acidic and basic deprotection strategies for the borane-protected phosphinothioesters, amide bonds can be formed in the presence of azides in moderate to good overall yields.     

Molecular evolution of Legionella pneumophila dotA gene,the contribution of natural environmental strains

Given the role of DotA protein in establishing successful infections and the diversity of host cells interacting with Legionella pneumophila in nature, it is possible that this gene product is a target for adaptive evolution. We investigated the influence of L. pneumophila isolates from natural environments with the molecular evolution of this crucial virulence‐related gene. The population genetic structure of L. pneumophila was inferred from the partial sequences of rpoB and dotA of 303 worldwide strains. The topology of the two inferred trees was not congruent and in the inferred dotA tree the vast majority of the natural environmental isolates were clustered in a discrete group. The Ka/Ks ratio demonstrated that this group, contrary to all others, has been under strong diversifying selection. The alignment of all DotA sequences allowed the identification of several alleles and the amino acid variations were not randomly distributed. Moreover, from these results we can conclude that dotA from L. pneumophila clinical and man‐made environmental strains belong to a sub‐set of all genotypes existing in nature. A split graph analysis showed evidence of a network‐like organization and several intergenic recombination events were detected within L. pneumophila strains resulting in mosaic genes in which different gene segments exhibited different evolutionary histories. We have determined that the allelic diversity of dotA is predominantly found in L. pneumophila isolates from natural environments, suggesting that niche‐specific selection pressures have been operating on this gene. Indeed, the high level of dotA allelic diversity may reflect fitness variation in the persistence of those strains in distinct environmental niches and/or tropism to various protozoan hosts.     

Folding and Rescue of a Cystic Fibrosis Transmembrane Conductance Regulator Trafficking Mutant Identified Using Human-Murine Chimeric Proteins

Impairment of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl⁻ channel causes cystic fibrosis, a fatal genetic disease. Here, to gain insight into CFTR structure and function, we exploited interspecies differences between CFTR homologues using human (h)-murine (m) CFTR chimeras containing murine nucleotide-binding domains (NBDs) or regulatory domain on an hCFTR backbone. Among 15 hmCFTR chimeras analyzed, all but two were correctly processed, one containing part of mNBD1 and another containing part of mNBD2. Based on physicochemical distance analysis of divergent residues between human and murine CFTR in the two misprocessed hmCFTR chimeras, we generated point mutations for analysis of respective CFTR processing and functional properties. We identified one amino acid substitution (K584E-CFTR) that disrupts CFTR processing in NBD1. No single mutation was identified in NBD2 that disrupts protein processing. However, a number of NBD2 mutants altered channel function. Analysis of structural models of CFTR identified that although Lys⁵⁸⁴ interacts with residue Leu⁵⁸¹ in human CFTR Glu⁵⁸⁴ interacts with Phe⁵⁸¹ in mouse CFTR. Introduction of the murine residue (Phe⁵⁸¹) in cis with K584E in human CFTR rescued the processing and trafficking defects of K584E-CFTR. Our data demonstrate that human-murine CFTR chimeras may be used to validate structural models of full-length CFTR. We also conclude that hmCFTR chimeras are a valuable tool to elucidate interactions between different domains of CFTR.