Selecting of a cytochrome P450cam SeSaM library with 3-chloroindole and endosulfan – Identification of mutants that dehalogenate 3-chloroindole

Cytochrome P450_cam (a camphor hydroxylase) from the soil bacterium Pseudomonas putida shows potential importance in environmental applications such as the degradation of chlorinated organic pollutants. Seven P450_cam mutants generated from Sequence Saturation Mutagenesis (SeSaM) and isolated by selection on minimal media with either 3-chloroindole or the insecticide endosulfan were studied for their ability to oxidize of 3-chloroindole to isatin. The wild-type enzyme did not accept 3-chloroindole as a substrate. Mutant (E156G/V247F/V253G/F256S) had the highest maximal velocity in the conversion of 3-chloroindole to isatin, whereas mutants (T56A/N116H/D297N) and (G60S/Y75H) had highest k_cat/K_M values. Six of the mutants had more than one mutation, and within this set, mutation of residues 297 and 179 was observed twice. Docking simulations were performed on models of the mutant enzymes; the wild-type did not accommodate 3-chloroindole in the active site, whereas all the mutants did. We propose two potential reaction pathways for dechlorination of 3-chloroindole. This article is part of a Special Issue entitled: Cytochrome P450 biodiversity and biotechnology, edited by Erika Plettner, Gianfranco Gilardi, Luet Wong, Vlada Urlacher, Jared Goldstone.     

Synthesis and antiviral evaluation of some novel tricyclic pyrazolo[3,4-b]indole nucleosides

Novel pyrazolo[3,4-b]indole nucleoside analogs were synthesized from the corresponding 3-formyl-2-chloroindole and 3-cyano-2-chloroindole nucleosides by treatment with hydrazine. Very few examples of pyrazolo[3,4-b]indole heterocycles have been published in the literature and this is the first synthesis of nucleoside analogs containing this heterocycle. These new pyrazolo[3,4-b]indole nucleosides were active against human cytomegalovirus and herpes simplex virus type 1, but this activity was not well separated from cytotoxicity.     

Synthesis and antioxidant activity evaluations of melatonin-based analogue indole-hydrazide/hydrazone derivatives

Melatonin (MLT) is a hormone synthesized from the pineal gland. It is a direct scavenger of free radicals, which is related to its capability to defend cells from oxidative stress. Recently MLT-related compounds are under investigation to establish which exhibit the maximum activity with the lowest side effects. In this study 5-chloroindole hydrazide/hydrazone derivatives were synthesized from 5-chloroindole-3-carboxaldehyde and phenyl hydrazine derivatives. All the compounds characterized and in vitro antioxidant activity was investigated against MLT and BHT. Most of the compounds showed strong inhibitory effect on the superoxide radical scavenging assay at 1?mM concentration (79 to 95%). Almost all the tested compounds possessed strong scavenging activity against the DPPH radical scavenging activity with IC(50) values (2 to 60 μM). Lastly, compound 1j revealed stronger inhibitory activity against MLT in the LP inhibitory assay at 0.1mM concentration (51%) while the rest of the compounds showed moderate inhibition.     

In vitro testing and the carcinogenic potential of several nitrosated indole compounds

4-chloro-methoxyindole is a naturally occurring compound in Vicia faba which can easily react with nitrite to form a N-nitroso compound. In this in vitro study, the potential genotoxic effects of nitrosated 4-chloro-6-methoxyindole and its structural analogue 4-chloroindole were evaluated for the first time by using both Salmonella and Chinese hamster V79 cells. Additionally, the inhibition of gap junctional intercellular communication in V79 cells by these compounds was determined; this is a validated parameter for tumor-promoting activity. Most assays were also performed with nitrosated indole-3-acetonitrile, a naturally occurring compound in brassicas. Both nitrosated chloroindoles were highly mutagenic to Salmonella typhimurium TA100 without the need of exogenous metabolic activation and were potent inducers of Sister Chromatid Exchanges. Nitrosated indole-3-acetonitrile generated the same effects, although at much higher concentrations. Equivocal results were obtained for the nitrosated chloroindoles in a forward mutation assay using the hypoxanthine guaninephosphoribosyltransferase locus. All nitrosated indole compounds significantly inhibited gap junctional intercellular communication. These results indicate that nitrosated chloroindoles and nitrosated indole-3-acetonitrile should be considered as mutagens and agents with potential tumor-promoting capacity.Abbreviations BrdU 5-bromo-2-deoxyuridine - 4Cl 4-chloroindole - 4C6MI 4-chloro-6-methoxy-indole - DMSO dimethyl sulfoxide - EBSS Earle's balanced salt solution - EMS ethyl methanesulfonate - GJIC gap junctional intercellular communication - HBSS Hanks balanced salt solution - HGPRT hypoxanthine guaninephosphoribosyl transferase - I₃A indole-3-acetonitrile - MNNG 1-methyl-1-nitroso-3-nitroguanidine - NOC N-nitroso compounds - NQO 4-nitroquinolone-N-oxide - SCE sister chromatid exchange - 6TG 6-thioguanine - TPA 12-O-tetradecanoylphorbol-13-acetate     

Detection of a new chloroperoxidase in Pseudomonas pyrrocinia

A new chloroperoxidase could be detected in Pseudomonas pyrrocinia ATCC 15,958, a bacterium that produces the antifungal antibiotic pyrrolnitrin. This enzyme was separated from a ferriprotoporphyrin IX containing bromoperoxidase which was also produced by this bacterium. The enzyme is capable of catalyzing the chorination of indole to 7-chloroindole. This procaryotic chloroperoxidase requires the presence of H2O2 and can also brominate monochlorodimedone, but cannot catalyze its chlorination. This enzyme is the first chloroperoxidase described from procaryotic sources.     

Comparison of Compositions of Odor Components of Natto and Cooked Soybeans

Natto, a traditional Japanese food product, was prepared from cooked soybeans by fermentation and its odor concentrate was obtained with a simultaneous distillation and extraction system. It was compared to those obtained from soybeans cooked for 0 ~ 3, 3 ~ 5.5 and 5.5 ~ 8 hr, respectively, by gas chromatography and gas chromatography-mass spectrometry. In the odor concentrates of the cooked soybeans, hexanal, (E)-2-hexenal and hexanol contributing to the green and grassy odor of soybeans disappeared or decreased while the cooking was in progress. 2-Pentylfuran and 1-octen-3-ol contributing to the beany odor remained even if the soybeans were cooked for 8 hr and were fermented into Natto. In the odor concentrate of Natto, pyrazines and sulfur containing compounds were important contributors to the characteristic odor of Natto. As the beany odor was not detected for Natto, it was concluded that the pyrazines and sulfur containing compounds cause the characteristic odor of Natto and mask the beany odor.     

Synthesis and antioxidant activity evaluations of melatonin-based analogue indole-hydrazide/hydrazone derivatives

Melatonin (MLT) is a hormone synthesized from the pineal gland. It is a direct scavenger of free radicals, which is related to its capability to defend cells from oxidative stress. Recently MLT-related compounds are under investigation to establish which exhibit the maximum activity with the lowest side effects. In this study 5-chloroindole hydrazide/hydrazone derivatives were synthesized from 5-chloroindole-3-carboxaldehyde and phenyl hydrazine derivatives. All the compounds characterized and in vitro antioxidant activity was investigated against MLT and BHT. Most of the compounds showed strong inhibitory effect on the superoxide radical scavenging assay at 1?mM concentration (79 to 95%). Almost all the tested compounds possessed strong scavenging activity against the DPPH radical scavenging activity with IC₅₀ values (2 to 60 µM). Lastly, compound 1j revealed stronger inhibitory activity against MLT in the LP inhibitory assay at 0.1mM concentration (51%) while the rest of the compounds showed moderate inhibition.     

Perceptual interactions between characteristic notes smelled above aqueous solutions of odorant mixtures

Twenty-two experienced panelists rated odor intensity of aqueous solutions of citral, octen-1-ol-3, and hexanal. The panel assessed unmixed components and mixtures (9 binary and 4 ternary). In sensory sessions dedicated to mixtures (n = 6), evaluation was focused on one target odor, presented at a fixed concentration. All components had lower odor intensity on mixed presentations. In many cases, information obtained from simpler systems was not extended to complex mixtures. In a mixture, the competition between odorant molecules on qualitative aspects (dominance/suppression) imbalanced components contribution, anticipated from the quantitative distribution. Hexanal appeared to be the potentially weaker odorant in paired combinations, whereas octen-1-ol-3 had a lower relative impact on ternary systems. Suppression of the odor of octen-1-ol-3 and a concomitant increase in the odor of hexanal was common to all ternary mixtures. Reciprocal inhibition of octen-1-ol-3 and citral odors through perceptual interactions was suspected. Mutual suppression is suspected to have eased the perception of hexanal intensity.     

Identification of new odoriferous compounds in human axillary sweat

3-Hydroxy-3-methylhexanoic acid (1) and the 3-sulfanylalkan-1-ols 2-5 were identified to contribute to the odor of human axillary sweat. Quantitative analyses of axillary sweat extracts from 50 healthy men showed an unambiguous correlation between the detected levels of 1 and the intensity of the axillary odor. Chiral-GC analyses revealed 1 to be a 72:28 mixture of the (S)/(R)-isomers. Optically pure (S)-1 (>97% ee) emanated a strong spicy note, which recalled typical axillary odors. 3-Methyl-3-sulfanylhexan-1-ol (2), the enantiomeric ratio of which equaled that of 1, was present in greater quantity than any of the other 3-sulfanylalkanols. Optically pure (S)-2 (>97% ee) had a strong meaty, fruity note, also reminiscent of axillary odor. The compounds identified, in particular (S)-1 and (S)-2, contribute significantly to the olfactory impression of human axillary odor.     

Biosynthetic studies on chlorinated indoles in Vicia faba

We identified L-4-chlorotriptophan (L-4-Cl-Trp) in two week old plants of Vicia faba. Deuterium labeled indole was incorporated to 4-chloroindole in these plants. Deuterium labeled L- and D-4-Cl-Trp were both synthesized and, when supplied to the plants only L-4-Cl-Trp was incorporated to 4-Cl-indoleacetic acid, although the incorporation ratio was very low.     

Characteristic vibration patterns of odor compounds from bread-baking volatiles upon protein binding: density functional and ONIOM study and principal component analysis

As the mechanism underlying the sense of smell is unclear, different models have been used to rationalize structure–odor relationships. To gain insight into odorant molecules from bread baking, binding energies and vibration spectra in the gas phase and in the protein environment [7-transmembrane helices (7TMHs) of rhodopsin] were calculated using density functional theory [B3LYP/6-311++G(d,p)] and ONIOM [B3LYP/6-311++G(d,p):PM3] methods. It was found that acetaldehyde (“acid” category) binds strongly in the large cavity inside the receptor, whereas 2-ethyl-3-methylpyrazine (“roasted”) binds weakly. Lys296, Tyr268, Thr118 and Ala117 were identified as key residues in the binding site. More emphasis was placed on how vibrational frequencies are shifted and intensities modified in the receptor protein environment. Principal component analysis (PCA) suggested that the frequency shifts of C–C stretching, CH₃ umbrella, C = O stretching and CH₃ stretching modes have a significant effect on odor quality. In fact, the frequency shifts of the C–C stretching and C = O stretching modes, as well as CH₃ umbrella and CH₃ symmetric stretching modes, exhibit different behaviors in the PCA loadings plot. A large frequency shift in the CH₃ symmetric stretching mode is associated with the sweet-roasted odor category and separates this from the acid odor category. A large frequency shift of the C–C stretching mode describes the roasted and oily-popcorn odor categories, and separates these from the buttery and acid odor categories.     

Flavor-Enhanced Modulation of Cerebral Blood Flow during Gum Chewing

Background

Flavor perception, the integration of taste and odor, is a critical factor in eating behavior. It remains unclear how such sensory signals influence the human brain systems that execute the eating behavior.

Methods

We tested cerebral blood flow (CBF) in the frontal lobes bilaterally while subjects chewed three types of gum with different combinations of taste and odor: no taste/no odor gum (C-gum), sweet taste/no odor gum (T-gum), and sweet taste/lemon odor gum (TO-gum). Simultaneous recordings of transcranial Doppler ultrasound (TCD) and near infrared spectrometer (NIRS) were used to measure CBF during gum chewing in 25 healthy volunteers. Bilateral masseter muscle activity was also monitored.

Results

We found that subjects could discriminate the type of gum without prior information. Subjects rated the TO-gum as the most flavorful gum and the C-gum as the least flavorful. Analysis of masseter muscle activity indicated that masticatory motor output during gum chewing was not affected by taste and odor. The TCD/NIRS measurements revealed significantly higher hemodynamic signals when subjects chewed the TO-gum compared to when they chewed the C-gum and T-gum.

Conclusions

These data suggest that taste and odor can influence brain activation during chewing in sensory, cognitive, and motivational processes rather than in motor control.     

Olfactory interference during inhibitory backward pairing in honey bees

Background

Restrained worker honey bees are a valuable model for studying the behavioral and neural bases of olfactory plasticity. The proboscis extension response (PER; the proboscis is the mouthpart of honey bees) is released in response to sucrose stimulation. If sucrose stimulation is preceded one or a few times by an odor (forward pairing), the bee will form a memory for this association, and subsequent presentations of the odor alone are sufficient to elicit the PER. However, backward pairing between the two stimuli (sucrose, then odor) has not been studied to any great extent in bees, although the vertebrate literature indicates that it elicits a form of inhibitory plasticity.

Methodology/Principal Findings

If hungry bees are fed with sucrose, they will release a long lasting PER; however, this PER can be interrupted if an odor is presented 15 seconds (but not 7 or 30 seconds) after the sucrose (backward pairing). We refer to this previously unreported process as olfactory interference. Bees receiving this 15 second backward pairing show reduced performance after a subsequent single forward pairing (excitatory conditioning) trial. Analysis of the results supported a relationship between olfactory interference and a form of backward pairing-induced inhibitory learning/memory. Injecting the drug cimetidine into the deutocerebrum impaired olfactory interference.

Conclusions/Significance

Olfactory interference depends on the associative link between odor and PER, rather than between odor and sucrose. Furthermore, pairing an odor with sucrose can lead either to association of this odor to PER or to the inhibition of PER by this odor. Olfactory interference may provide insight into processes that gate how excitatory and inhibitory memories for odor-PER associations are formed.     

Reductive dechlorination of polychlorinated biphenyls as affected by natural halogenated aromatic compounds

We investigated the effects of halogenated aromatic compounds (HACs) including naturally occurring ones (L-thyroxine, 3-chloro-L-tyrosine, 5-chloroindole, 2-chlorophenol, 4-chlorophenol and chlorobenzene) on polychlorinated biphenyl (PCB) dechlorination in sediment cultures. A PCB-dechlorinating enrichment culture of sediment microorganisms from the St. Lawrence River was used as an initial inoculum. When the culture was inoculated into Aroclor 1248 sediments amended with each of the six HACs, the extent of dechlorination was not enhanced by amendment with HACs. The dechlorination patterns in the HAC-amended sediments were nearly identical to that of the HAC-free sediments except the 3-chloro-L-tyrosine-amended ones where no dechlorination activity was observed. When these sediment cultures were transferred into fresh sediments with the same HACs, the dechlorination specificities remained the same as those of the initial inoculations. Thus, in the present study, the substrate range of the highly selected enrichment culture could not be broadened by the HACs. It appears that HACs affect PCB dechlorination mainly through population selection rather than enzyme induction of single population.     

雄性布氏田鼠对不同熟悉程度和动情状态下雌鼠气味的辨别

通过雄性布氏田鼠（Microtus brandti)对配偶和陌生雌鼠气味的辨别实验发现：（1）雌鼠动情状况对雄鼠气味行为反应的影响,当配偶雌鼠处于动情期时,被试雄鼠对动情陌生雌鼠巢垫物的溴闻和舔舐的持续时间都明显多于对非动情陌生雌鼠巢垫物的嗅闻和舔舐时间,而其他行为没有明显判别当配偶雌鼠处于非动情期时,雄鼠对动情的和非动情的陌生雌鼠的气味行为反应都没有明显判别当陌生雌鼠处于动情期时,被试雄鼠对动情配偶气味的嗅闻时间明显多于对非动情配偶的嗅闻时间,而其他行为反应没有明显差异;当陌生雌鼠处于非动情期时,被试雄鼠对动情配偶雌鼠巢垫物的嗅闻时间,挖掘频次,舔舐频次和时间都明显多于对非动情配偶气味的嗅闻,而且雄鼠在动情配偶气味周围搔扒体侧行为的发生频次和持续时间也多于在非动情配偶气味周围的搔扒行为;（2）熟悉性对雄鼠气味行为反应的影响,当配偶雌鼠和陌生雌鼠都处于动情期或都处于非动情期时,被试雄鼠对陌生雌鼠气味的探究行为（嗅问、挖掘和舔噬）和搔扒体侧行为明显多于对配偶雌鼠气味的探究;雄鼠访问陌生雌鼠气味源箱的频次和在其中的停留时间也都多于访问配偶雌鼠气味源箱的频次和停留时间;当配偶雌鼠处于动情期而陌生雌鼠处于非动情期时,被试雄鼠对配偶气味的嗅闻,挖掘和自身修饰行为明显多于对陌生雌鼠气味的行为反应,进入配偶气味源箱中的访问频次也多于进入陌生雌鼠气味源箱的访问频次。结果说明：雌鼠与被试雄鼠的熟悉程度和雌鼠的动情状况直接影响雄性布氏田鼠的社会探究和气味选择行为,即雄鼠偏好并选择动情雌鼠和陌生雌鼠,雄性布氏田鼠对雌鼠气味的行为反应也表现出田鼠属动物典型的多配制种类特征。     

Reshaping of bulbar odor response by nasal flow rate in the rat

Background

The impact of respiratory dynamics on odor response has been poorly studied at the olfactory bulb level. However, it has been shown that sniffing in the behaving rodent is highly dynamic and varies both in frequency and flow rate. Bulbar odor response could vary with these sniffing parameter variations. Consequently, it is necessary to understand how nasal airflow can modify and shape odor response at the olfactory bulb level.

Methodology and Principal Findings

To assess this question, we used a double cannulation and simulated nasal airflow protocol on anesthetized rats to uncouple nasal airflow from animal respiration. Both mitral/tufted cell extracellular unit activity and local field potentials (LFPs) were recorded. We found that airflow changes in the normal range were sufficient to substantially reorganize the response of the olfactory bulb. In particular, cellular odor-evoked activities, LFP oscillations and spike phase-locking to LFPs were strongly modified by nasal flow rate.

Conclusion

Our results indicate the importance of reconsidering the notion of odor coding as odor response at the bulbar level is ceaselessly modified by respiratory dynamics.     

Friends and foes from an ant brain's point of view--neuronal correlates of colony odors in a social insect

Background

Successful cooperation depends on reliable identification of friends and foes. Social insects discriminate colony members (nestmates/friends) from foreign workers (non-nestmates/foes) by colony-specific, multi-component colony odors. Traditionally, complex processing in the brain has been regarded as crucial for colony recognition. Odor information is represented as spatial patterns of activity and processed in the primary olfactory neuropile, the antennal lobe (AL) of insects, which is analogous to the vertebrate olfactory bulb. Correlative evidence indicates that the spatial activity patterns reflect odor-quality, i.e., how an odor is perceived. For colony odors, alternatively, a sensory filter in the peripheral nervous system was suggested, causing specific anosmia to nestmate colony odors. Here, we investigate neuronal correlates of colony odors in the brain of a social insect to directly test whether they are anosmic to nestmate colony odors and whether spatial activity patterns in the AL can predict how odor qualities like “friend” and “foe” are attributed to colony odors.

Methodology/Principal Findings

Using ant dummies that mimic natural conditions, we presented colony odors and investigated their neuronal representation in the ant Camponotus floridanus. Nestmate and non-nestmate colony odors elicited neuronal activity: In the periphery, we recorded sensory responses of olfactory receptor neurons (electroantennography), and in the brain, we measured colony odor specific spatial activity patterns in the AL (calcium imaging). Surprisingly, upon repeated stimulation with the same colony odor, spatial activity patterns were variable, and as variable as activity patterns elicited by different colony odors.

Conclusions

Ants are not anosmic to nestmate colony odors. However, spatial activity patterns in the AL alone do not provide sufficient information for colony odor discrimination and this finding challenges the current notion of how odor quality is coded. Our result illustrates the enormous challenge for the nervous system to classify multi-component odors and indicates that other neuronal parameters, e.g., precise timing of neuronal activity, are likely necessary for attribution of odor quality to multi-component odors.     

Innate attractiveness and associative learnability of odors can be dissociated in larval Drosophila

We investigate olfactory associative learning in larval Drosophila. A reciprocal training design is used, such that one group of animals receives a reward in the presence of odor X but not in the presence of odor Y (Train: X+ // Y), whereas another group is trained reciprocally (Train: X // Y+). After training, differences in odor preference between these reciprocally trained groups in a choice test (Test: X - Y) reflect associative learning. The current study, after showing which odor pairs can be used for such learning experiments, 1) introduces a one-odor version of such reciprocal paradigm that allows estimating the learnability of single odors. Regarding this reciprocal one-odor paradigm, we show that 2) paired presentations of an odor with a reward increase odor preference above baseline, whereas unpaired presentations of odor and reward decrease odor preference below baseline; this suggests that odors can become predictive either of reward or of reward absence. Furthermore, we show that 3) innate attractiveness and associative learnability can be dissociated. These data deepen our understanding of odor-reward learning in larval Drosophila on the behavioral level, and thus foster its neurogenetic analysis.     

Identification of amino acid residues involved in 4-chloroindole 3-hydroxylation by cytochrome P450 2A6 using screening of random libraries

Cytochrome P450 (P450) 2A6 is able to catalyze indole hydroxylation to form the blue dye indigo. The wild-type P450 2A6 enzyme was randomly mutated throughout the whole open reading frame and screened using 4-chloroindole hydroxylation, a substituted indole selected from 30 indole compounds for enhanced color development. Mutants with up to 5-fold increases of catalytic efficiency (k(cat)/K(m)) and 2-fold increases in k(cat) were selected after two rounds of screening. Important residues located both in (e.g., Thr305) and outside the active site (e.g., Ser224) were identified. The study utilized a better substrate for "indigo assay" to obtain new information on the structure-functional relationship of P450 2A6 that was not revealed by previous mutagenesis studies with this enzyme.     

The interruptive effect of electric shock on odor response requires mushroom bodies in Drosophila melanogaster

Nociceptive stimulus involuntarily interrupts concurrent activities. This interruptive effect is related to the protective function of nociception that is believed to be under stringent evolutionary pressure. To determine whether such interruptive effect is conserved in invertebrate and potentially uncover underlying neural circuits, we examined Drosophila melanogaster. Electric shock (ES) is a commonly used nociceptive stimulus for nociception related research in Drosophila. Here, we showed that background noxious ES dramatically interrupted odor response behaviors in a T‐maze, which is termed blocking odor response by electric shock (BOBE). The interruptive effect is not odor specific. ES could interrupt both odor avoidance and odor approach. To identify involved brain areas, we focused on the odor avoidance to 3‐OCT. By spatially abolishing neurotransmission with temperature sensitive Shibire^TS1, we found that mushroom bodies (MBs) are necessary for BOBE. Among the 3 major MB Kenyon cell (KCs) subtypes, α/β neurons and γ neurons but not α’/β’ neurons are required for normal BOBE. Specifically, abolishing the neurotransmission of either α/β surface (α/β_s), α/β core (α/β_c) or γ dorsal (γ_d) neurons alone is sufficient to abrogate BOBE. This pattern of MB subset requirement is distinct from that of aversive olfactory learning, indicating a specialized BOBE pathway. Consistent with this idea, BOBE was not diminished in several associative memory mutants and noxious ES interrupted both innate and learned odor avoidance. Overall, our results suggest that MB α/β and γ neurons are parts of a previously unappreciated central neural circuit that processes the interruptive effect of nociception.