6-(7-Nitro-2,1,3-benzoxadiazol-4-ylthio) Hexanol Inhibits Proliferation and Induces Apoptosis of Endometriosis by Regulating Glutathione S-Transferase Mu Class 4.

Endometriosis is a chronic disease associated with a disrupted oxidative balance and chronic inflammation. In this study, we investigated the role of glutathione S-transferase Mu class 4 (GSTM4) in endometriosis and determined whether 6-(7-nitro-2,1,3-benzoxadiazol-4-ylthio) hexanol (NBDHEX) regulates GSTM4 expression to affect cellular functions and oxidative stress. GSTM4 expression was detected by immunohistochemistry in endometrium from 15 endometriosis patients and 15 healthy controls. Western blotting was used to detect the expression of GSTM4, proliferating cell nuclear antigen (PCNA), matrix metalloproteinase-9 (MMP-9), Survivin, B-cell lymphoma-extra-large (Bcl-XL), Bax, kelch-like ECH-associated protein 1 (Keap1), and nuclear factor-erythroid 2-related factor 2 (Nrf2) in primary endometrial stromal cells with endometriosis (EESC) and normal endometrial stromal cells (NESC). The effects of NBDHEX on cell proliferation, migration, and invasion were evaluated using Cell Counting Kit-8 (CCK8) and Transwell assays. Apoptosis was detected by flow cytometry. The expression of GSTM4 was significantly increased in endometrium from endometriosis patients. Upon NBDHEX treatment, ESC exhibited reduced proliferation, migration and invasion abilities, and increased apoptosis. NBDHEX decreased the expression of endometriosis prognostic markers (PCNA and MMP-9) and anti-apoptotic proteins (Survivin and Bcl-xl), while it increased the expression of the apoptotic protein Bax. It had no effect on Keap1 expression, and it decreased the expression of Nrf2. The effect of siRNA-mediated knockdown of GSTM4 was similar to that of suppressing GSTM4 expression with NBDHEX treatment. These results indicate that GSTM4 is highly expressed in endometriosis and its expression is inhibited by NBDHEX. Decreased expression of GSTM4 inhibits cell growth, migration, and invasion, and negatively regulates Nrf2 to affect oxidative stress-induced apoptosis. Our results suggest that GSTM4 may play a role in ameliorating the progression of endometriosis. NBDHEX may have therapeutic potential in the treatment of endometriosis.

Therapeutic Potential of TLR8 Agonist GS-9688 (Selgantolimod) in Chronic Hepatitis B: Remodeling of Antiviral and Regulatory Mediators.

BACKGROUND AND AIMS: GS-9688 (selgantolimod) is a toll-like receptor 8 agonist in clinical development for the treatment of chronic hepatitis B (CHB). Antiviral activity of GS-9688 has previously been evaluated in vitro in HBV-infected hepatocytes and in vivo in the woodchuck model of CHB. Here we evaluated the potential of GS-9688 to boost responses contributing to viral control and to modulate regulatory mediators. APPROACH AND RESULTS: We characterized the effect of GS-9688 on immune cell subsets in vitro in peripheral blood mononuclear cells of healthy controls and patients with CHB. GS-9688 activated dendritic cells and mononuclear phagocytes to produce IL-12 and other immunomodulatory mediators, inducing a comparable cytokine profile in healthy controls and patients with CHB. GS-9688 increased the frequency of activated natural killer (NK) cells, mucosal-associated invariant T cells, CD4+ follicular helper T cells, and, in about 50% of patients, HBV-specific CD8+ T cells expressing interferon-γ. Moreover, in vitro stimulation with GS-9688 induced NK-cell expression of interferon-γ and TNF-α, and promoted hepatocyte lysis. We also assessed whether GS-9688 inhibited immunosuppressive cell subsets that might enhance antiviral efficacy. Stimulation with GS-9688 reduced the frequency of CD4+ regulatory T cells and monocytic myeloid-derived suppressor cells (MDSCs). Residual MDSCs expressed higher levels of negative immune regulators, galectin-9 and programmed death-ligand 1. Conversely, GS-9688 induced an expansion of immunoregulatory TNF-related apoptosis-inducing ligand+ NK cells and degranulation of arginase-I+ polymorphonuclear MDSCs. CONCLUSIONS: GS-9688 induces cytokines in human peripheral blood mononuclear cells that are able to activate antiviral effector function by multiple immune mediators (HBV-specific CD8+ T cells, CD4+ follicular helper T cells, NK cells, and mucosal-associated invariant T cells). Although reducing the frequency of some immunoregulatory subsets, it enhances the immunosuppressive potential of others, highlighting potential biomarkers and immunotherapeutic targets to optimize the antiviral efficacy of GS-9688.

3,3-Dimethyl-1-butanol attenuates cardiac remodeling in pressure-overload-induced heart failure mice.

Trimethylamine N-oxide (TMAO) is closely related to cardiovascular diseases, particularly heart failure (HF). Recent studies shows that 3,3-dimethyl-1-butanol (DMB) can reduce plasma TMAO levels. However, the role of DMB in overload-induced HF is not well understood. In this research study, we explored the effects and the underlying mechanisms of DMB in overload-induced HF. Aortic banding (AB) surgery was performed in C57BL6/J mice to induce HF, and a subset group of mice underwent a sham operation. After surgery, the mice were fed with a normal diet and given water supplemented with or without 1% DMB for 6 weeks. Cardiac function, plasma TMAO level, cardiac hypertrophy and fibrosis, expression of inflammatory, electrophysiological studies and signaling pathway were analyzed at the sixth week after AB surgery. DMB reduced TMAO levels in overload-induced HF mice. Adverse cardiac structural remodeling, such as cardiac hypertrophy, fibrosis and inflammation, was elevated in overload-induced HF mice. Susceptibility to ventricular arrhythmia also significantly increased in overload-induced HF mice. However, these changes were prevented by DMB treatment. DMB attenuated all of these changes by reducing plasma TMAO levels, hence negatively inhibiting the p65 NF-κB signaling pathway and TGF-ß1/Smad3 signaling pathway. DMB plays an important role in attenuating the development of cardiac structural remodeling and electrical remodeling in overload-induced HF mice. This may be attributed to the p65 NF-κB signaling pathway and TGF-ß1/Smad3 signaling pathway inhibition.

Molecular basis for potentiation of Cx36 gap junction channel conductance by n-alcohols and general anesthetics.

In our recent study, we have demonstrated that short carbon chain n-alcohols (up to octanol) stimulated while long carbon chain n-alcohols inhibited the conductance of connexin (Cx) 36 (Cx36) gap junction (GJ) channels. In contrast, GJ channels composed of other types of Cxs all were inhibited by n-alcohols independent of their carbon chain length. To identify the putative structural domains of Cx36, responsible for the dual effect of n-alcohols, we performed structural modeling of Cx36 protein docking with hexanol and isoflurane that stimulated as well as nonanol and carbenoxolone that inhibited the conductance of Cx36 GJs and revealed their multiple common docking sites and a single pocket accessible only to hexanol and isoflurane. The pocket is located in the vicinity of three unique cysteine residues, namely C264 in the fourth, and C92 and C87 in the second transmembrane domain of the neighboring Cx36 subunits. To examine the hypothesis that disulphide bonding might be involved in the stimulatory effect of hexanol and isoflurane, we generated cysteine substitutions in Cx36 and demonstrated by a dual whole-cell patch-clamp technique that in HeLa (human cervix carcinoma cell line) and N2A (mouse neuroblastoma cell line) cells these mutations reversed the stimulatory effect of hexanol and isoflurane to inhibitory one, typical of other Cxs that lack respective cysteines and a specific docking pocket for these compounds. Our findings suggest that the stimulatory effect of hexanol and isoflurane on Cx36 GJ conductance could be achieved by re-shuffling of the inter-subunit disulphide bond between C264 and C92 to the intra-subunit one between C264 and C87.

A novel reverse micellar purification strategy for histidine tagged human interferon gamma (hIFN-γ) protein from Pichia pastoris.

In the present study, we have demonstrated the process development of human interferon gamma (hIFN-γ) (upstream to downstream). The codon optimized hIFN-γ gene was cloned in Pichia pastoris X-33 and the expression was evaluated in batch reactor study. The purification was carried out with modified nickel chelated reverse micellar system and compared with the existing Nickle- Nitrilotriacetic acid (NI-NTA) method. The parameter optimization for forward extraction demonstrated a significant enhancement of 72% in forward extraction efficiency (FEE). Furthermore, the factors governing back extraction efficiency (BEE) were also optimized with sequential optimization involving Taguchi orthogonal array and Artificial Neural Network linked Simulated Annealing Algorithm (ANN-SA). The optimization resulted in 91.2% back extraction efficiency of recombinant human interferon gamma (rhIFN-γ). The development of this purification system with optimized parameters led to an efficient recovery of 67.3% and improved purity of 79.54%. Alongside, the anti-proliferative activity in MCF-7 cell lines were also investigated and it demonstrated that at 60ngmL-1 concentration of rhIFN-γ more that 25%.

Spatial differences in (Z)-3-hexen-1-ol production preferentially reduces Spodoptera litura larva attack on the young leaves of Nicotiana benthamiana.

Plants synthesize specialized metabolites which possess extremely important ecological functions including direct defense, indirect defense, and signaling. The optimal defense theory (ODT) proposes that defensive metabolites are preferentially allocated to the tissues with high fitness value or in locations that are easily injured. In our present study, using the model plant Nicotiana benthamiana, we found that direct defense of N. benthamiana against Spodoptera litura (Fabricius) larvae showed spatial differences in the sites producing defensive chemicals. The upper leaves possessed significantly stronger direct defense ability than the middle and lower leaves. Interestingly, the strong defense ability of the upper leaves was not due to occurrences of well-known defensive metabolites such as nicotine and chlorogenic acid. After damage, the middle and lower leaves emitted higher amounts of (Z)-3-hexen-1-ol than the upper leaves, which could both attract larvae and significantly increase the amount of middle and lower leaf eaten by the larvae. The spatial difference in (Z)-3-hexen-1-ol emission may be due to spatial differences in expression of lipoxygenase (NbLOX2), which is responsible for the formation and emission of (Z)-3-hexen-1-ol. This study provided new insight into ODT, showing that plants effectively protect easily injured tissues through reduction in concentration of herbivore-feeding stimulant in the tissues.

Naturally Produced Defensive Alkenal Compounds Activate TRPA1.

(E)-2-alkenals are aldehydes containing an unsaturated bond between the alpha and beta carbons. 2-alkenals are produced by many organisms for defense against predators and secretions containing (E)-2-alkenals cause predators to stop attacking and allow the prey to escape. Chemical ecologists have described many alkenal compounds with 3-20 carbons common, having varied positions of double bonds and substitutions. How do these defensive alkenals act to deter predators? We have tested the effects of (E)-2-alkenals with 6-12 carbons on transient receptor potential channels (TRP) commonly found in sensory neurons. We find that (E)-2-alkenals activate transient receptor potential ankyrin subtype 1 (TRPA1) at low concentrations-EC50s 10-100 µM (in 0 added Ca(2+) external solutions). Other TRP channels were either weakly activated (TRPV1, TRPV3) or insensitive (TRPV2, TRPV4, TRPM8). (E)-2-alkenals may activate TRPA1 by modifying cysteine side chains. However, target cysteines include others beyond the 3 in the amino-terminus implicated in activation, as a channel with cysteines at 621, 641, 665 mutated to serine responded robustly. Related chemicals, including the aldehydes hexanal and decanal, and (E)-2-hexen-1-ol also activated TRPA1, but with weaker potency. Rat trigeminal nerve recordings and behavioral experiments showed (E)-2-hexenal was aversive. Our results suggest that TRPA1 is likely a major target of these commonly used defensive chemicals.

Primary alcohols activate human TRPA1 channel in a carbon chain length-dependent manner.

Transient receptor potential ankyrin 1 (TRPA1) is a calcium-permeable non-selective cation channel that is mainly expressed in primary nociceptive neurons. TRPA1 is activated by a variety of noxious stimuli, including cold temperatures, pungent compounds such as mustard oil and cinnamaldehyde, and intracellular alkalization. Here, we show that primary alcohols, which have been reported to cause skin, eye or nasal irritation, activate human TRPA1 (hTRPA1). We measured intracellular Ca(2+) changes in HEK293 cells expressing hTRPA1 induced by 1 mM primary alcohols. Higher alcohols (1-butanol to 1-octanol) showed Ca(2+) increases proportional to the carbon chain length. In whole-cell patch-clamp recordings, higher alcohols (1-hexanol to 1-octanol) activated hTRPA1 and the potency increased with the carbon chain length. Higher alcohols evoked single-channel opening of hTRPA1 in an inside-out configuration. In addition, cysteine at 665 in the N terminus and histidine at 983 in the C terminus were important for hTRPA1 activation by primary alcohols. Furthermore, straight-chain secondary alcohols increased intracellular Ca(2+) concentrations in HEK293 cells expressing hTRPA1, and both primary and secondary alcohols showed hTRPA1 activation activities that correlated highly with their octanol/water partition coefficients. On the other hand, mouse TRPA1 did not show a strong response to 1-hexanol or 1-octanol, nor did these alcohols evoke significant pain in mice. We conclude that primary and secondary alcohols activate hTRPA1 in a carbon chain length-dependent manner. TRPA1 could be a sensor of alcohols inducing skin, eye and nasal irritation in human.

Antimalarial activity of endoperoxide compound 6-(1,2,6,7-tetraoxaspiro[7.11]nonadec-4-yl)hexan-1-ol.

Plasmodium falciparum, the major causative parasite for the disease, has acquired resistance to most of the antimalarial drugs used today, presenting an immediate need for new antimalarial drugs. Here, we report the in vitro and in vivo antimalarial activities of 6-(1,2,6,7-tetraoxaspiro[7.11]nonadec-4-yl)hexan-1-ol (N-251) against P. falciparum and Plasmodium berghei parasites. The N-251 showed high antimalarial potencies both in the in vitro and the in vivo tests (EC(50) 2.3×10(-8) M; ED(50) 15 mg/kg (per oral)). The potencies were similar to that of artemisinin in vitro and greater than artemisinin's activity in vivo (p.o.). In addition, N-251 has little toxicity: a single oral administration at 2000 mg/kg to a rat gave no health problems to it. Administration of N-251 to mice bearing 1% of parasitemia (per oral 68 mg/kg, 3 times a day for 3 consecutive days) resulted in a dramatic decrease in the parasitemia: all the 5 mice given N-251 were cured without any recurrence, with no diarrhea or weight loss occurring in the 60 days of experiment. N-251 deserves more extensive clinical evaluation, desirably including future trials in the human.

Comparison of male and female emerald ash borer (Coleoptera: Buprestidae) responses to phoebe oil and (Z)-3-hexenol lures in light green prism traps.

We conducted trapping experiments for the emerald ash borer, Agrilus planipennis Fairmaire (Coleoptera: Buprestidae) in Michigan, U.S.A., and Ontario, Canada, to compare unbaited light green sticky prism traps with traps baited with phoebe oil, (Z)-3-hexenol (Z3-6:OH), or blends of other green leaf volatiles (GLVs) with Z3-6:OH. Traps were placed in the lower canopy of ash trees (Fraxinus spp.). Catches with Z3-6:OH-baited traps showed a significant male bias and these traps caught significantly more males than the unbaited controls at both sites. They were also superior to phoebe oil-baited traps and those baited with GLV blends. Catches with phoebe oil showed a significant female bias but there was no difference in the number of females captured between traps baited with phoebe oil or Z3-6:OH lures. Catches were analyzed at regular time intervals to examine the response of A. planipennis to the lures over the course of the flight season. Z3-6:OH-baited traps consistently caught more males than the controls at each interval throughout the flight season. Catches of females with Z3-6:OH and phoebe oil were significantly better than the controls early in the flight season but declined to control levels by midseason. Our results suggest that Z3-6:OH-baited green traps placed in the ash canopy would be a superior lure for detecting and monitoring A. planipennis throughout the flight season.

"Green odor" inhalation by stressed rat dams reduces behavioral and neuroendocrine signs of prenatal stress in the offspring.

Chronic maternal stress during pregnancy results in the "prenatally stressed" offspring displaying behavioral and neuroendocrine alterations that persist into adulthood. We investigated how inhalation of green odor (a mixture of equal amounts of trans-2-hexenal and cis-3-hexenol) by stressed dams might alter certain indices of prenatal stress in their offspring. These indices were depression-like behavior (increased immobility time in the forced-swim test) and acute restraint stress-induced changes in hypothalamo-pituitary-adrenocortical (HPA) axis activity [plasma corticosterone (CORT) and ACTH levels and the number of Fos-immunoreactive cells in the hypothalamic paraventricular nucleus (an index of neuronal activity)]. Pregnant rats were exposed to restraint stress for 60 min/day for 10 days (gestational days 10-19). The prenatally stressed offspring exhibited significant increases in depression-like behavior and in restraint stress-induced ACTH, CORT, and Fos responses, unless their dam had been exposed to green odor. The behavioral effect of the odor was also seen in offspring that were fostered by unstressed dams. The results obtained in the dams themselves were as follows. In vehicle-exposed stressed dams, but not in green odor-exposed ones, total body and adrenal weights were significantly decreased or increased, respectively. Depression-like behavior was not observed in the vehicle-exposed stressed dams themselves. Green odor inhalation prevented the impairment of maternal behavior induced by restraint stress. Thus, exposure of dams to stress may affect both the fetal brain and fetal HPA axis, and also maternal behavior, leading to altered behavioral and neuroendocrine responses in the offspring. Such effects may be prevented by the stressed dams inhaling green odor.

"Green odor" inhalation by rats down-regulates stress-induced increases in Fos expression in stress-related forebrain regions.

In the present study, on rats, a quantitative analysis of Fos protein immunohistochemistry was performed as a way of investigating the effects of inhalation of green odor (a mixture of equal amounts of trans-2-hexenal and cis-3-hexenol) on the neuronal activations in stress-related forebrain regions induced by acute and repeated stress. Rats were exposed to restraint stress for 90 min each day for 1, 2, 4, 7, or 11 consecutive days. The hypothalamic paraventricular nucleus (PVN), amygdala, hippocampus and paraventricular thalamic nucleus (PVT) were examined. Both acute and repeated restraint stress increased Fos-positive cells in the entire hypothalamic PVN, in the central and medial amygdala, and in PVT, although these responses declined upon repeated exposure to such stress. The stress-induced Fos responses were much weaker in rats that inhaled green odor during each day's restraint. No increases in Fos-positive cells were observed in the hippocampus in acutely stressed rats. The Fos-immunoreactive response to acute stress shown by the piriform cortex did not differ significantly between the vehicle+stress and green+stress groups. Green odor had inhibitory effects on the stress-induced corticosterone response, body-weight loss, and adrenal hypertrophy. These results suggest that in rats, green odor inhalation may, in an as yet unknown way, act on the brain to suppress activity in the neuronal networks involved in stress-related responses (such as activation of the hypothalamo-pituitary-adrenocortical axis and activation of the sympathetic nervous system, as well as stress-induced fear responses).

6-(7-Nitro-2,1,3-benzoxadiazol-4-ylthio)hexanol, a specific glutathione S-transferase inhibitor, overcomes the multidrug resistance (MDR)-associated protein 1-mediated MDR in small cell lung cancer.

In the present work, we have investigated the antitumor activity of 6-(7-nitro-2,1,3-benzoxadiazol-4-ylthio)hexanol (NBDHEX) on aggressive small cell lung cancer. NBDHEX not only is cytotoxic toward the parental small cell lung cancer H69 cell line (LC(50) of 2.3 +/- 0.6 micromol/L) but also overcomes the multidrug resistance of its variant, H69AR, which overexpresses the ATP-binding cassette transporter multidrug resistance-associated protein 1 (MRP1; LC(50) of 4.5 +/- 0.9 micromol/L). Drug efflux experiments, done in the presence of a specific inhibitor of MRP1, confirmed that NBDHEX is not a substrate for this export pump. Interestingly, NBDHEX triggers two different types of cell death: a caspase-dependent apoptosis in the H69AR cells and a necrotic phenotype in the parental H69 cells. The apoptotic pathway triggered by NBDHEX in H69AR cells is associated with c-Jun NH(2)-terminal kinase and c-Jun activation, whereas glutathione oxidation and activation of p38(MAPK) is observed in the NBDHEX-treated H69 cells. In contrast to the parental cells, the higher propensity to die through apoptosis of the H69AR cell line may be related to the lower expression of the antiapoptotic protein Bcl-2. Therefore, down-regulation of a factor crucial for cell survival makes H69AR cells more sensitive to the cytotoxic action of NBDHEX, which is not a MRP1 substrate. We have previously shown that NBDHEX is cytotoxic toward P-glycoprotein-overexpressing tumor cell lines. Therefore, NBDHEX seems a very promising compound in the search for new molecules able to overcome the ATP-binding cassette family of proteins, one of the major mechanisms of multidrug resistance in cancer cells.

Methyl salicylate, identified as primary odorant of a specific receptor neuron type, inhibits oviposition by the moth Mamestra brassicae L. (Lepidoptera, noctuidae).

The cabbage moth, Mamestra brassicae L. (Lepidoptera, Noctuidae), is a polyphagous species that is often choosing plants of Brassica as hosts for oviposition. In the search for biologically relevant odorants used by these moths, gas chromatography linked to electrophysiological recordings from single receptor neurons (RNs) has been employed, resulting in classification of distinct types of neurons. This study presents specific olfactory RNs responding to methyl salicylate (MeS) as primary odorant and showing a weak response to methyl benzoate, the 2 aromatic compounds occurring together in several plant species. In 2 cases, the neuron was colocated with another RN type responding to 6 green leaf volatiles: 1-hexanol, (3Z)-hexen-1-ol, (2E)-hexen-1-ol, (3Z)-hexenyl acetate, (2Z)-hexen-1-ol, and an unidentified compound. Whereas the specific RNs detected the minor amounts of MeS in some plants, the compound was not found by gas chromatography linked to mass spectrometry in intact plants, but it was found after herbivore attack. The behavioral effect of MeS was studied in outdoor test arenas with Brassica napus and artificial plants. These experiments indicated that mated M. brassicae females avoid plants with dispensers emitting MeS. As it is induced by caterpillar feeding, this compound may mediate a message to mated M. brassicae females that the plant is already occupied.

Aspergillus volatiles regulate aflatoxin synthesis and asexual sporulation in Aspergillus parasiticus.

Aspergillus parasiticus is one primary source of aflatoxin contamination in economically important crops. To prevent the potential health and economic impacts of aflatoxin contamination, our goal is to develop practical strategies to reduce aflatoxin synthesis on susceptible crops. One focus is to identify biological and environmental factors that regulate aflatoxin synthesis and to manipulate these factors to control aflatoxin biosynthesis in the field or during crop storage. In the current study, we analyzed the effects of aspergillus volatiles on growth, development, aflatoxin biosynthesis, and promoter activity in the filamentous fungus A. parasiticus. When colonies of Aspergillus nidulans and A. parasiticus were incubated in the same growth chamber, we observed a significant reduction in aflatoxin synthesis and asexual sporulation by A. parasiticus. Analysis of the headspace gases demonstrated that A. nidulans produced much larger quantities of 2-buten-1-ol (CA) and 2-ethyl-1-hexanol (EH) than A. parasiticus. In its pure form, EH inhibited growth and increased aflatoxin accumulation in A. parasiticus at all doses tested; EH also stimulated aflatoxin transcript accumulation. In contrast, CA exerted dose-dependent up-regulatory or down-regulatory effects on aflatoxin accumulation, conidiation, and aflatoxin transcript accumulation. Experiments with reporter strains carrying nor-1 promoter deletions and mutations suggested that the differential effects of CA were mediated through separate regulatory regions in the nor-1 promoter. The potential efficacy of CA as a tool for analysis of transcriptional regulation of aflatoxin biosynthesis is discussed. We also identify a novel, rapid, and reliable method to assess norsolorinic acid accumulation in solid culture using a Chroma Meter CR-300 apparatus.

Plant-plant signaling: ethylene synergizes volatile emission in Zea mays induced by exposure to (Z)-3-hexen-1-ol.

Leaf alcohol (Z)-3-hexen-1-ol (Z-3-ol) is emitted by green plants upon mechanical damage. Exposure of intact maize plants to Z-3-ol induces the emission of a volatile blend that is typically released after caterpillar feeding and attracts natural enemies of the herbivores [herbivore-induced volatile organic compounds (HI-VOC)]. Thus, it has been suggested that Z-3-ol might have a function in indirect plant defense mediating plant-plant signaling and intraplant information transfer. Here, we demonstrate that HI-VOC induction by Z-3-ol is synergized by the phytohormone ethylene. Exposure to Z-3-ol at doses of 100 and 250 nmol induced HI-VOC emission in intact maize plants. HI-VOC emissions increased by 2.5-fold when ethylene was added. The effect of ethylene was more pronounced (5.1- to 6.6-fold) when only total sesquiterpene release was considered. In contrast, ethylene alone had no inductive effect but rather decreased the emission of the constitutive maize volatile linalool. We suggest that ethylene plays a synergistic role in plant-plant signaling mediated by green leaf volatiles.

Functional and structural analysis of the GABAA receptor alpha 1 subunit during channel gating and alcohol modulation.

The substituted cysteine accessibility method has proven useful for investigating structural changes of the gamma-aminobutyric acid type A (GABA(A)) receptor during channel gating and allosteric modulation. In the present study, the surface accessibility and reaction rate of propyl- and hexyl-methanethiosulfonate to cysteine residues introduced into the third transmembrane segment of the GABA(A) receptor alpha(1) subunit were examined. GABA-induced currents in Xenopus oocytes expressing wild type and cysteine mutant GABA(A) receptors were recorded before and after application of methanethiosulfonate (MTS) reagents in the resting, GABA- or alcohol-bound (ethanol or hexanol) states. Our results indicate that a water-filled cavity exists around the Ala(291) and Tyr(294) residues of the third transmembrane segment, in agreement with previous results. Furthermore, our data indicate that a conformational change produced by alcohols (200 mM ethanol or 0.5 mM hexanol) exposure induces the water cavity around the A291C and Y294C residues to extend deeper, causing the A295C and F296C residues to become accessible to the MTS reagents. In addition, exposure of the A291C, Y294C, F296C, and V297C mutants to MTS reagents in the presence of GABA had significant effects on their GABA-induced currents, indicating that the water cavity around A291C and Y294C residues expanded to F296C and V297C by a structural movement caused by GABA binding. Our data show that GABA(A) receptor is a dynamic protein during alcohol modulation and channel gating.

Modulation of actomyosin motor function by 1-hexanol.

This study examines the effects of 1-hexanol as a perturbing agent on actomyosin ATPase and its related functions in the concentration range between 0 and 20 mM. In this range the denaturation of myosin subfragment 1 (S1), as measured by the inactivation rate of its K-EDTA-ATPase, and depolymerization of F-actin were insignificant. Major findings showed that hexanol had the following effects which were fully reversible, (a) a marked activation of S1 MgATPase (approximately 10-fold at 20 mM) without greatly affecting the enhancement of tryptophan fluorescence by formation of S1.ADP.Pi intermediate and the rate of ADP release from S1.ADP; (b) an inhibition of the maximum actin-activated ATPase activity; (c) an increase in the affinity of S1 for actin in the presence of ATP and a decrease in the presence of ADP or the absence of nucleotide; (d) a reduction in the sliding velocity of actin filaments in in vitro motility assays with myosin, and (e) a decrease in isometric tension of single skinned muscle fibers. Thus, the effects of hexanol on actomyosin interaction are distinct for the weak and strong binding states, consistent with a change in the hydrophobic interaction in the interface between myosin and actin accompanying the transition from the weak to the strong binding state. Hexanol also accelerates the Pi release from S1.ADP.Pi, which is the transition step from the weak to the strong binding state. The fact that hexanol accelerates Pi release suggests that this alcohol perturbs the S1.ADP.Pi conformation. We speculate that this intermediate-specific structural perturbation is related to the inhibition of the maximum actin-activated ATPase, in vitro motility, and isometric tension.

The effects of hexanol on Galpha(i) subunits of heterotrimeric G proteins.

UNLABELLED: Alcohols and other anesthetics interfere with the function of a variety of systems regulated by guanosine triphosphate (GTP)-binding proteins (G proteins). We examined the effect of hexanol on the activity of the alpha subunit (Galpha(i1)) of heterotrimeric G proteins. The GTP hydrolysis activity of recombinant Galpha(i1) was 0.029 mole Pi. mole Galpha(i1)(-1) x min(-1) and was inhibited by hexanol at concentrations larger than 10 mM, with a 50% inhibitory concentration of 22 mM. Circular dichroism spectroscopy revealed that hexanol decreased the denaturation temperature of Galpha(i1) from 47.2 degrees C to 42.5 degrees C without altering its secondary structure at 10 degrees C. Hexanol (30 mM) reduced the amount of monomeric Galpha(i1) in solution measured by size-exclusion chromatography, indicating that hexanol caused protein aggregation. However, the rate of GTPgammaS binding to Galpha(i) immunoprecipitated from airway smooth muscle membranes was not affected by 30 mM hexanol. Excluding the apparent inhibition of recombinant Galpha(i1) resulting from aggregation-induced artifact, we found no evidence that the hexanol-induced inhibition of receptor-activated Galpha(i)-coupled pathways in intact airway smooth muscle resulted from direct inhibition of the intrinsic rate of [(35)S]GTPgammaS binding to Galpha(i). IMPLICATIONS: Although the alpha subunit of heterotrimeric G proteins is a potential target of anesthetics, we found no evidence that hexanol affects the ability of the Galpha(i) subunit to bind or hydrolyze guanosine triphosphate, either in purified subunits or in subunits derived from smooth muscle cell membranes. This finding implies that this is not a mechanism by which hexanol interferes with receptor-G protein function.

Biochemical and structural changes in RBCs stored with different plasticizers: the role of hexanol.

BACKGROUND: PVC containers are plasticized with di(2-ethyl)hexylphthalate (DEHP) or a related phthalate. The toxicity of DEHP has been questioned. It has been proposed to use butyryltrihexylcitrate (BTHC) as the plasticizer. The purpose of this study was to determine if hexanol, a component of BTHC, plays a role in the preservation of RBCs stored in BTHC-plasticized PVC bags. STUDY DESIGN AND METHODS: WBC-reduced RBCs of ABO- and D-matched blood groups were prepared in 1-L polyolefin (PO) bags (PL732). Six 60-g aliquots were transferred to transfer packs made of PL146 (DEHP-plasticized) and PL2209 (BTHC-plasticized) and four PO (PL732) packs. To the PL146 and PL2209 packs, 30 mL of AS-1 was added. To three of the PO packs, 30 mL of AS-1 with sufficient DEHP, BTHC, or hexanol to achieve a final concentration of 3 mM was added, and to the final PO pack, 30 mL of AS-1 only was added (control). The units were stored for 6 weeks at 1 to 6 degrees C. RBC ATP, hemolysis, morphology, membrane lipids, deformability, and fluidity were measured. RESULTS: ATP levels were not significantly different in any of the systems after 6 weeks. Compared to the PO bags, hemolysis was lowest in the PL146 containers and was also significantly lower (p < 0.006) in the PO bags with added DEHP, BTHC, or hexanol. The accumulation of vesicles was significantly less in the units stored in the PL146 and PL2209 than in the PO plastic with or without added plasticizers or hexanol (p < or = 0.004). There was no significant difference in the formation of vesicles in any of the PO units (p > 0.05). There was no demonstrable change in the membrane fluidity of the RBCs during storage in any of the systems. The decrease in deformability was the same, and the losses of cholesterol and phospholipid during storage were similar in all the studies. CONCLUSIONS: The hexanol component of the BHTC plasticizer in a concentration of 144.6 microg per mL concentration suppresses hemolysis and vesiculation of RBCs during storage. The hexanol and DEHP that are slowly leached during storage have a greater effect in suppressing hemolysis and vesicle formation than when added extraneously to AS-1 in PO containers.