Lipid Peroxidation Products Influence Calpain-1 Functionality In Vitro by Covalent Binding.

The objective of the current study was to evaluate the effects of lipid peroxidation products, malondialdehyde (MDA), hexenal, and 4-hydroxynonenal (HNE), on calpain-1 function, and liquid chromatography and tandem mass spectrometry (LC-MS/MS) identification of adducts on calpain-1. Calpain-1 activity slightly increased after incubation with 100 µM MDA but not with 500 and 1000 µM MDA. However, calpain-1 activity was lowered by hexenal and HNE at 100, 500, and 1000 µM. No difference in calpain-1 autolysis was observed between the control and 1000 µM MDA. However, 1000 µM hexenal and HNE treatments slowed the calpain-1 autolysis. Adducts of MDA were detected on glutamine, arginine, lysine, histidine, and asparagine residues via Schiff base formation, while HNE adducts were detected on histidine, lysine, glutamine, and asparagine residues via Michael addition. These results are the first to demonstrate that lipid peroxidation products can impact calpain-1 activity in a concentration-dependent manner and may impact the development of meat tenderness postmortem.

Expression profile and functional characterization of odorant binding proteins in a forest pest, Dioryctria abietella (Lepidoptera: Pyralidae).

In the forest ecosystem dominated by the Pinaceae plants, this boring pest Dioryctria abietella is subject to a variety of odorants derived from host and nonhost plants, in which olfactory-related proteins enriched in antennae are key behavioral modulators for the orientation of feeding and ovipositing hosts. Here, we addressed the odorant binding protein (OBP) gene family in D. abietella. Expression profiles revealed that the majority of OBPs were abundantly expressed in the antennae at a female-biased level. A male-antenna-biased DabiPBP1 was a strong candidate for detecting type I and type II pheromones of D. abitella female moths. Using a prokaryotic expression system combined with affinity chromatography, we harvested two antenna-dominant DabiOBPs. In the ligand-binding assays, the two DabiOBPs exhibited different odorant response spectra, as DabiOBP17 was tuned to most odorants with higher affinities compared to DabiOBP4. Of these, DabiOBP4 could strongly bind syringaldehyde and citral (dissociation constants (Ki) < 14 µM). A floral volatile, benzyl benzoate (Ki = 4.72 ± 0.20 µM), was the best ligand for DabiOBP17. Remarkably, several green leaf volatiles were found to strongly interact with DabiOBP17 (Ki < 8.5 µM), including Z3-hexenyl acetate, E2-hexenol, Z2-hexenal and E2-hexenal that may mediate a repellent response to D. abietella. Structural analyses of ligands revealed that the binding of the two DabiOBPs to odorants was associated with carbon-chain lengths and functional groups. Molecular simulations identified several key residues involved in the interactions of DabiOBPs and ligands, suggesting specific binding mechanisms. This study highlights olfactory roles of two antennal DabiOBPs in D. abietella, helping the identification of potentially behavioral compounds for the population control of this pest.

Microbial degradation of low-density polyethylene by Neopestalotiopsis phangngaensis.

Low-density polyethylene (LDPE) has been commercially used and accumulated as plastic solid waste. LDPE has also been found to be a non-degradable waste for decades and found as a pollution source in the environment. In this study, 65 fungi were screened for their biodegradation of LDPE. The fungi Neopestalotiopsis phangngaensis, Alternaria burnsii, Alternaria pseudoeichhorniae, and Arthrinium sacchari showed significant potential in LDPE biodegradation. These fungi were individually cultured with an LDPE sheet as a carbon source for 90 days. A maximum weight loss of the LDPE sheet was detected by the fungus N. phangngaensis (54.34%). This fungus also revealed the highest reduction rate of tensile strength of the LDPE sheet (0.33 MPa). The morphological surface of LDPE culturing with N. phangngaensis was crack, pit, and rough analyzed by scanning electron microscopy. The biodegradation of the LDPE sheet by N. phangngaensis was also confirmed by the Sturm test and analysis of enzymatic activities. The Sturm test showed the highest decomposition of the LDPE sheet by N. phangngaensis into CO2 with 2.14 g/L after incubation. Enzymatic activities of laccase, manganese peroxidase, and lignin peroxidase enzymes were found by N. phangngaensis during the LDPE degradation. The volatile organic compounds in culture supernatant of N. phangngaensis were also investigated. The major compounds were 3Z-diethyl acetal hexenal, 2E,4E-decadienol, and 2Z-diethyl acetal hexenal. This study reveals the utilization of the fungus N. phangngaensis as the carbon source at a considerable biodegradation rate without any prior treatment. Therefore, the fungus N. phangngaensis may be applied as an alternative degrader for LDPE degradation in the environment.

Mechanisms of Susceptibility and Resilience to PTSD: Role of Dopamine Metabolism and BDNF Expression in the Hippocampus.

Susceptibility and resilience to post-traumatic stress disorder (PTSD) are recognized, but their mechanisms are not understood. Here, the hexobarbital sleep test (HST) was used to elucidate mechanisms of PTSD resilience or susceptibility. A HST was performed in rats 30 days prior to further experimentation. Based on the HST, the rats were divided into groups: (1) fast metabolizers (FM; sleep duration < 15 min); (2) slow metabolizers (SM; sleep duration ≥ 15 min). Then the SM and FM groups were subdivided into stressed (10 days predator scent, 15 days rest) and unstressed subgroups. Among stressed animals, only SMs developed experimental PTSD, and had higher plasma corticosterone (CORT) than stressed FMs. Thus, resilience or susceptibility to PTSD was consistent with changes in glucocorticoid metabolism. Stressed SMs had a pronounced decrease in hippocampal dopamine associated with increased expressions of catecholamine-O-methyl-transferase and DA transporter. In stressed SMs, a decrease in monoaminoxidase (MAO) A was associated with increased expressions of hippocampal MAO-A and MAO-B. BDNF gene expression was increased in stressed FMs and decreased in stressed SMs. These results demonstrate relationships between the microsomal oxidation phenotype, CORT concentration, and anxiety, and they help further the understanding of the role of the liver−brain axis during PTSD.

Caffeoylputrescine-hexenal-mediated nonhost resistance against leafhoppers.

Despite its critical role in repelling damaging insects, our understanding of nonhost resistance against herbivores remains very limited. Recently, Bai et al. identified a novel caffeoylputrescine-green leaf volatile (GLV) compound in wild tobacco plants that confers nonhost resistance to Empoasca leafhoppers through high-throughput multi-omics analyses.

Hexobarbital Sleep Test for Predicting the Susceptibility or Resistance to Experimental Posttraumatic Stress Disorder.

Hexobarbital sleep test (HST) was performed in male Wistar rats (hexobarbital 60 mg/kg, i.p.) 30 days prior to stress exposure. Based on the duration of hexobarbital-induced sleep, rats were divided into two groups, animals with high intensity (fast metabolizers (FM), sleep duration <15 min) or low intensity of hexobarbital metabolism (slow metabolizers (SM), sleep duration ≥15 min). The SM and FM groups were then divided into two subgroups: unstressed and stressed groups. The stressed subgroups were exposed to predator scent stress for 10 days followed by 15 days of rest. SM and FM rats from the unstressed group exhibited different behavioral and endocrinological patterns. SM showed greater anxiety and higher corticosterone levels. In stressed animals, anxiety-like posttraumatic stress disorder (PTSD) behavior was aggravated only in SM. Corticosterone levels in the stressed FM, PTSD-resistant rats, were lower than in unstressed SM. Thus, HST was able to predict the susceptibility or resistance to experimental PTSD, which was consistent with the changes in glucocorticoid metabolism.

Identification of a Hexenal Reductase That Modulates the Composition of Green Leaf Volatiles.

Green leaf volatiles (GLVs), including six-carbon (C6) aldehydes, alcohols, and esters, are formed when plant tissues are damaged. GLVs play roles in direct plant defense at wound sites, indirect plant defense via the attraction of herbivore predators, and plant-plant communication. GLV components provoke distinctive responses in their target recipients; therefore, the control of GLV composition is important for plants to appropriately manage stress responses. The reduction of C6-aldehydes into C6-alcohols is a key step in the control of GLV composition and also is important to avoid a toxic buildup of C6-aldehydes. However, the molecular mechanisms behind C6-aldehyde reduction remain poorly understood. In this study, we purified an Arabidopsis (Arabidopsis thaliana) NADPH-dependent cinnamaldehyde and hexenal reductase encoded by At4g37980, named here CINNAMALDEHYDE AND HEXENAL REDUCTASE (CHR). CHR T-DNA knockout mutant plants displayed a normal growth phenotype; however, we observed significant suppression of C6-alcohol production following partial mechanical wounding or herbivore infestation. Our data also showed that the parasitic wasp Cotesia vestalis was more attracted to GLVs emitted from herbivore-infested wild-type plants compared with GLVs emitted from chr plants, which corresponded with reduced C6-alcohol levels in the mutant. Moreover, chr plants were more susceptible to exogenous high-dose exposure to (Z)-3-hexenal, as indicated by their markedly lowered photosystem II activity. Our study shows that reductases play significant roles in changing GLV composition and, thus, are important in avoiding toxicity from volatile carbonyls and in the attraction of herbivore predators.

Pedologic Factors Affecting Virgin Olive Oil Quality of "Chemlali" Olive Trees (Olea europaea L.).

The aim of this study examined the characterization of extra virgin olive oil samples from the main cultivar Chemlali, grown in five olive orchards with different soil type (Sandy, Clay, Stony, Brown, Limestone and Gypsum). Volatile compounds were studied using headspace-solid phase micro-extraction (HS-SPME) and gas chromatography-mass spectrometry (GC-MS) technics. Moreover, the sterol profile was established using gas chromatography-mass spectrometry. 35 different volatile compounds were identified: alcohols, esters, aldehydes, ketones and hydrocarbons. The chemical composition of the volatile fraction was characterized by the preeminence of 2-hexenal (32.75%) and 1-hexanol (31.88%). Three sterols were identified and characterized. For all olive oil samples, ß-sitosterol (302.25 mg/kg) was the most abundant sterol. Interestingly, our results showed significant qualitative and quantitative differences in the levels of the volatile compounds and sterols from oils obtained from olive trees grown in different soil type.

The constituents of essential oil in leaves of Karaj accession of Trigonella foenum graecum.

The chemical composition of the essential oils of Karaj accession of Trigonella foenum graecum leaves was detected by hydro-distillation and analysed by gas chromatography (GC-FID) and gas chromatography-mass spectroscopy (GC-MS) apparatuses for first time. Thirty-six compounds representing 95.3% of the total components were identified. The patterns of the main compounds were (2E)-Hexenal (26.61%), n-Hexadecanoic acid (10.14%) and (E)-b-Ionone (7.99%). Other notable constituents were Thymol (4.79%), 6,10,14-trimethyl-2-Pentadecanone (4.59%), Carvacrol (3.40%), (E)-Nerolidol (3.32%) and (2E,6Z)-Nonadienal (3.30%). (2E)-Hexenal was found as the most dominant component in this study.

Assessing the genotoxic effects of two lipid peroxidation products (4-oxo-2-nonenal and 4-hydroxy-hexenal) in haemocytes and midgut cells of Drosophila melanogaster larvae.

Lipid peroxidation products can induce tissue damage and are implicated in diverse pathological conditions, including aging, atherosclerosis, brain disorders, cancer, lung and various liver disorders. Since in vivo studies produce relevant information, we have selected Drosophila melanogaster as a suitable in vivo model to characterise the potential risks associated to two lipid peroxidation products namely 4-oxo-2-nonenal (4-ONE) and 4-hydroxy-hexenal (4-HHE). Toxicity, intracellular reactive oxygen species production, and genotoxicity were the end-points evaluated. Haemocytes and midgut cells were the evaluated targets. Results showed that both compounds penetrate the intestine of the larvae, affecting midgut cells, and reaching haemocytes. Significant genotoxic effects, as determined by the comet assay, were observed in both selected cell targets in a concentration/time dependent manner. This study highlights the importance of D. melanogaster as a model organism in the study of the different biological effects caused by lipid peroxidation products entering via ingestion. This is the first study reporting genotoxicity data in haemocytes and midgut cells of D. melanogaster larvae for the two selected compounds.

Predicting anxiety responses to halogenated glucocorticoid drugs using the hexobarbital sleep time test.

Glucocorticoids (GCs) are used to treat numerous diseases, but their use in limited by adverse side effects. One such effect is occasional increased anxiety. Since the intensity of hepatic microsomal oxidation has been shown to alter responses to GC, we examined the possibility that rats with lower rates of hepatic GC metabolism would have increased anxiety. We hypothesized that the resulting, excessive GC would stimulate brain monoamine oxidase A (MAO-A), which would reduce brain serotonin, and thereby increase anxiety. Hepatic microsomal oxidative intensity was evaluated by the hexobarbital sleep time (HST) test. Results showed that rats with lower rates of hepatic GC metabolism had elevated brain MAO-A activity, reduced brain serotonin, and more anxiety than rats with higher rates of hepatic GC metabolism. We suggest that the HST test, as an integrative test of microsomal oxidation status, should be useful for predicting individual sensitivity to GC and to other drugs metabolized by the hepatic microsomal oxidation system.

Duration of hexobarbital-induced sleep and monoamine oxidase activities in rat brain: Focus on the behavioral activity and on the free-radical oxidation.

The present study is focused on the relationship between monoamine oxidase (MAO) activity and hepatic content of cytochrome P450 (CYP), which reflects the status of microsomal oxidation. For vital integrative evaluation of hepatic microsomal oxidation in rats, the hexobarbital sleep test was used, and content of CYP was measured in hepatic microsomes. Rats with short hexobarbital sleep time (SHST) had higher content of microsomal CYP than rats with long hexobarbital sleep time (LHST). Whole brain MAO-A and MAO-B activities, serotonin and carbonylated protein levels were higher in SHST than in LHST rats. MAO-A and MAO-B activities were higher in brain cortex of SHST rats; MAO-A activity was higher only in hypothalamus and medulla of LHST. The same brain regions of LHST rats had higher concentrations of carbonylated proteins and lipid peroxidation products than in SHST rats. MAO activity was correlated with microsomal oxidation phenotype. Rats with higher hepatic content of CYP had higher activities of MAO-A and MAO-B in the brain and higher plasma serotonin levels than rats with lower microsomal oxidation. In conclusion, data obtained in this study showed a correlation between MAO activity and microsomal oxidation phenotype.

Using the Specialization Framework to Determine Degree of Dietary Specialization in a Herbivorous Woodrat.

To be considered a dietary specialist, mammalian herbivores must consume large quantities of a plant species considered "difficult" with respect to nutrient or toxin content, and possess specialized adaptations to deal with plant defensive compounds or low nutritional content. Populations of Neotoma lepida in the Great Basin consume Juniperus osteosperma, a plant heavily defended by terpenes, but a detailed dietary analysis of this population is lacking. Therefore, we investigated the extent of dietary specialization in this species in comparison with the better-studied specialist species, N. stephensi. Microhistological analysis of feces from N. lepida revealed that greater than 90% of their diet in nature was comprised of juniper. In laboratory tolerance trials, N. lepida tolerated a diet of 80% J. osteosperma, similar to that observed for N. stephensi. There was no difference in the abilities of N. lepida and N. stephensi to metabolize hexobarbital, a proxy compound for terpene metabolism. In preference tests of native and non-native juniper species, N. lepida did not exhibit a preference for its native or co-occurring juniper, J. osteosperma, over the non-native species, J. monosperma, whereas N. stephensi preferred its native or co-occurring juniper J. monosperma over non-native J. osteosperma. Behavioral and habitat differences between these woodrat species lead to the categorization of N. stephensi as an obligate juniper specialist with a small range that overlaps that of its preferred food, J. monosperma, and N. lepida as a facultative juniper specialist with a large range, and only a portion of its distribution containing populations that feed extensively on J. osteosperma.

Preparation, release and physicochemical characterisation of ethyl butyrate and hexanal inclusion complexes with ß- and γ-cyclodextrin.

Complexes of ethyl butyrate and hexanal encapsulated by ß-cyclodextrin (ß-CD) and γ-cyclodextrin (γ-CD) were prepared by coprecipitation, and gas chromatography was used to quantity the flavour compounds in the complexes. The ethyl butyrate-γ-CD complex had the highest inclusion ratio (12.20%) followed by the ethyl butyrate-ß-CD, hexanal-ß-CD and hexanal-γ-CD complexes (11.29, 4.41 and 3.33%, respectively). Release experiments were performed under different relative humidities (RH 93, 75 and 52%) and temperatures (4 and 25 °C). The flavour release behaviours of the complexes were described by the Avrami equation. The rate of flavour release was enhanced with both increasing temperature and RH, although the effect of RH was stronger. Physicochemical characterisation using FT-IR, XRD, DSC and SEM analyses demonstrated that crystalline complexes were formed. Both ß-CD and γ-CD were able to encapsulate ethyl butyrate and hexanal, and lower RH and temperature were more suitable for the storage of these complexes.

Lactic acid bacteria and natural antimicrobials to improve the safety and shelf-life of minimally processed sliced apples and lamb's lettuce.

Outbreaks of food-borne disease associated with the consumption of fresh and minimally processed fruits and vegetables have increased dramatically over the last few years. Traditional chemical sanitizers are unable to completely eradicate or kill the microorganisms on fresh produce. These conditions have stimulated research to alternative methods for increasing food safety. The use of protective cultures, particularly lactic acid bacteria (LAB), has been proposed for minimally processed products. However, the application of bioprotective cultures has been limited at the industrial level. From this perspective, the main aims of this study were to select LAB from minimally processed fruits and vegetables to be used as biocontrol agents and then to evaluate the effects of the selected strains, alone or in combination with natural antimicrobials (2-(E)-hexenal/hexanal, 2-(E)-hexenal/citral for apples and thyme for lamb's lettuce), on the shelf-life and safety characteristics of minimally processed apples and lamb's lettuce. The results indicated that applying the Lactobacillus plantarum strains CIT3 and V7B3 to apples and lettuce, respectively, increased both the safety and shelf-life. Moreover, combining the selected strains with natural antimicrobials produced a further increase in the shelf-life of these products without detrimental effects on the organoleptic qualities.

Characterization of polar organosulfates in secondary organic aerosol from the green leaf volatile 3-Z-hexenal.

Evidence is provided that the green leaf volatile 3-Z-hexenal serves as a precursor for biogenic secondary organic aerosol through the formation of polar organosulfates (OSs) with molecular weight (MW) 226. The MW 226 C6-OSs were chemically elucidated, along with structurally similar MW 212 C5-OSs, whose biogenic precursor is likely related to 3-Z-hexenal but still remains unknown. The MW 226 and 212 OSs have a substantial abundance in ambient fine aerosol from K-puszta, Hungary, which is comparable to that of the isoprene-related MW 216 OSs, known to be formed through sulfation of C5-epoxydiols, second-generation gas-phase photooxidation products of isoprene. Using detailed interpretation of negative-ion electrospray ionization mass spectral data, the MW 226 compounds are assigned to isomeric sulfate esters of 3,4-dihydroxyhex-5-enoic acid with the sulfate group located at the C-3 or C-4 position. Two MW 212 compounds present in ambient fine aerosol are attributed to isomeric sulfate esters of 2,3-dihydroxypent-4-enoic acid, of which two are sulfated at C-3 and one is sulfated at C-2. The formation of the MW 226 OSs is tentatively explained through photooxidation of 3-Z-hexenal in the gas phase, resulting in an alkoxy radical, followed by a rearrangement and subsequent sulfation of the epoxy group in the particle phase.

The historical significance of anaesthesia events at Pearl Harbor.

Up to the end of World War II, less than 10% of the general anaesthetics administered was with intravenous barbiturates. The remaining 90% of anaesthetics given in the USA were with diethyl ether. In the United Kingdom and elsewhere, chloroform was also popular. Diethyl ether administration was a relatively safe and simple procedure, often delegated to nurses or junior doctors with little or no specific training in anaesthesia. During the Japanese attack on the US bases at Pearl Harbor, with reduced stocks of diethyl ether available, intravenous Sodium Pentothal(®), a most 'sophisticated and complex' drug, was used with devastating effects in many of those hypovolaemic, anaemic and septic patients. The hazards of spinal anaesthesia too were realised very quickly. These effects were compounded by the dearth of trained anaesthetists. This paper presents the significance of the anaesthesia tragedies at Pearl Harbor, and the discovery in the next few years of many other superior drugs that caused medical and other health professionals to realise that anaesthesia needed to be a specialist medical discipline in its own right. Specialist recognition, aided by the foundation of the National Health Service in the UK, the establishment of Faculties of Anaesthesia and appropriate training in pharmacology, physiology and other sciences soon followed. Modern anaesthesiology, as we understand it today, was born and a century or more of ether anaesthesia finally ceased.

Identification of the female-produced sex pheromone of the plant bug Apolygus spinolae.

Apolygus spinolae (Meyer-Dür) (Heteroptera: Miridae) is an important pest of fruit and tea trees in Korea and Japan. Analyses of extracts of metathoracic scent glands revealed that those of female bugs contained hexyl butyrate, (E)-2-hexenyl butyrate, and (E)-4-oxo-2-hexenal in a ratio of 20:100:7. The glands of males contained the same three compounds, but the ratio of the components was quite different, with hexyl butyrate being the most abundant. Field trapping tests with various blends of the synthetic compounds dispensed from high-density polyethylene tubes showed that (E)-2-hexenyl butyrate and (E)-4-oxo-2-hexenal were essential for attraction of male A. spinolae, and catches with a wide range of ratios of these two compounds did not differ significantly. However, adding hexyl butyrate at 50 % or more of the (E)-2-hexenyl butyrate to the binary blend strongly inhibited attraction of males. Trap catches increased with increasing amounts of a 10:1 blend of (E)-2-hexenyl butyrate and (E)-4-oxo-2-hexenal from 0.011 to 11 mg loaded into the tube. Catches of males in traps baited with lures containing 1.1 mg of the binary blend were not significantly different from catches in traps baited with live virgin females.

Rabbit 3-hydroxyhexobarbital dehydrogenase is a NADPH-preferring reductase with broad substrate specificity for ketosteroids, prostaglandin D2, and other endogenous and xenobiotic carbonyl compounds.

3-Hydroxyhexobarbital dehydrogenase (3HBD) catalyzes NAD(P)⁺-linked oxidation of 3-hydroxyhexobarbital into 3-oxohexobarbital. The enzyme has been thought to act as a dehydrogenase for xenobiotic alcohols and some hydroxysteroids, but its physiological function remains unknown. We have purified rabbit 3HBD, isolated its cDNA, and examined its specificity for coenzymes and substrates, reaction directionality and tissue distribution. 3HBD is a member (AKR1C29) of the aldo-keto reductase (AKR) superfamily, and exhibited high preference for NADP(H) over NAD(H) at a physiological pH of 7.4. In the NADPH-linked reduction, 3HBD showed broad substrate specificity for a variety of quinones, ketones and aldehydes, including 3-, 17- and 20-ketosteroids and prostaglandin D2, which were converted to 3α-, 17ß- and 20α-hydroxysteroids and 9α,11ß-prostaglandin F2, respectively. Especially, α-diketones (such as isatin and diacetyl) and lipid peroxidation-derived aldehydes (such as 4-oxo- and 4-hydroxy-2-nonenals) were excellent substrates showing low K(m) values (0.1-5.9 µM). In 3HBD-overexpressed cells, 3-oxohexobarbital and 5ß-androstan-3α-ol-17-one were metabolized into 3-hydroxyhexobarbital and 5ß-androstane-3α,17ß-diol, respectively, but the reverse reactions did not proceed. The overexpression of the enzyme in the cells decreased the cytotoxicity of 4-oxo-2-nonenal. The mRNA for 3HBD was ubiquitously expressed in rabbit tissues. The results suggest that 3HBD is an NADPH-preferring reductase, and plays roles in the metabolisms of steroids, prostaglandin D2, carbohydrates and xenobiotics, as well as a defense system, protecting against reactive carbonyl compounds.