Differential spectroscopic analysis of size-dependent phycobilisome from Spirulina maxima.

C-phycocyanin (C-Pc), a photosynthetic pigment for use as a fluorescent indicator or in pharmaceutical, food, and cosmetic products, exists in a phycobilisome complex with allophycocyanin (APC), phycoerythrin (PE), and linker polypeptides. This heterogeneity makes it difficult to quantify phycobilisome composition in an ultraviolet-visible (UV-vis) spectrum. In this study, derivative analysis of UV-vis spectra was successfully applied to display the distinct wavelengths at which C-Pc, APC, and PE have maximal peaks. In all samples, C-Pc of the largest portion had a "zero-crossing" first order, APC did not have a zero-crossing first order, and PE did not have first derivative for zero crossing or local minimum from the 500 and 700 nm, respectively. The results show that derivative analyses coupled with signal smoothing can be applied to elucidate the composition of phycobilisome under various conditions including purification and environment.

ERP study on the associations of peripheral oxytocin and prolactin with inhibitory processes involving emotional distraction.

BACKGROUND: Child maltreatment is a major health and social welfare problem, with serious and longstanding consequences. Impulse control ability plays an important role in reducing the risk of child maltreatment. The aim of this study was to investigate the associations of oxytocin (OXT) and prolactin (PRL) with behavior inhibition using children's facial expressions (angry or neutral) as emotional distractions. This may clarify a part of the neuroendocrinological mechanism that modulates impulse control ability in the context of child caregiving. METHODS: Participants were 16 females who had never been pregnant. Following venous blood sampling for OXT and PRL levels, participants performed an emotional Go/Nogo task during their follicular and luteal phases to test inhibitory control ability. Behavioral performance and event-related potentials (ERPs) during the task were measured. RESULTS: The results showed that there were significant fixed effects of OXT on behavioral performance, as measured by sensitivity (d-prime). This suggests that high peripheral OXT levels may be associated with better performance on the emotional Go/Nogo task, regardless of emotional distractors. PRL was associated with inhibitory processes as reflected by the Nogo-N2 and Nogo-P3. Particularly, high PRL levels were associated with the Nogo-N2 latency extension with the emotional distractors. CONCLUSIONS: Our findings suggest that OXT might be associated with improving behavioral performance regardless of emotional processes. It is suggested that processes related to PRL are related to premotor activities of behavioral inhibitions and emotions.

Enhanced Nogo-P3 amplitudes of mothers compared with non-mother women during an emotional Go/Nogo task.

BACKGROUND: It is known that emotion regulatory responses of humans are changed by the experiences they have, but in particular, they are changed by becoming a mother. A recent study has found how a woman's emotion regulatory response to a child's crying changes after becoming a mother. However, mothers' emotion regulatory responses other than those to children and the association between emotion regulatory response and parental stress are still unknown. METHODS: Eighteen healthy Japanese females (nine mothers and nine non-mothers) participated in the experiment. They performed an emotional Go/Nogo task, with facial expressions of others (angry, happy, and neutral faces) used as emotional stimuli. The percentage of correct responses, response time, and event-related potentials (ERPs) during the task was measured. RESULTS: This comparison revealed that the mother group had a larger P3 (Nogo-P3) amplitude than the non-mother group when Nogo trials were held. This indicates that in mothers, there was greater activation of the behavioral inhibition-related brain areas than in non-mother women when they inhibited inappropriate behavior following recognition of facial expressions of others. In addition, in the mother group, there was a negative correlation between parental stress levels and Nogo-P3 amplitudes evoked by angry faces. This suggests that there is a relation between the level of parental stress of mothers and their emotion regulatory responses to angry faces. CONCLUSIONS: Our results demonstrate that mothers' emotion regulatory processes may differ from those of non-mothers in response, not only to a child's crying but also to expressions of emotions by others, and also suggest that the inhibitory recognition activity of mothers can be affected by parental stress.

Effect of the Hand-Omitted Tool Motion on mu Rhythm Suppression.

In the present study, we investigated the effect of the image of hands on mu rhythm suppression invoked by the observation of a series of tool-based actions in a goal-directed activity. The participants were 11 university students. As a source of visual stimuli to be used in the test, a video animation of the porcelain making process for museums was used. In order to elucidate the effect of hand imagery, the image of hands was omitted from the original ("hand image included") version of the animation to prepare another ("hand image omitted") version. The present study has demonstrated that, an individual watching an instructive animation on the porcelain making process, the image of the porcelain maker's hands can activate the mirror neuron system (MNS). In observations of "tool included" clips, even the "hand image omitted" clip induced significant mu rhythm suppression in the right central area. These results suggest that the visual observation of a tool-based action may be able to activate the MNS even in the absence of hand imagery.

Engineering N-Glycosylation Pathway in Insect Cells: Suppression of ß-N-Acetylglucosaminidase and Expression of ß-1,4-Galactosyltransferase.

Most insect cells have a simple N-glycosylation process and consequently paucimannosidic or simple core glycans predominate. It has been proposed that ß-N-acetylglucosaminidase (GlcNAcase), a hexosaminidase in the Golgi membrane which removes a terminal N-acetylglucosamine (GlcNAc), might contribute to simple N-glycosylation profile in several insect cells including Drosophila S2. Here, we describe GlcNAcase suppression strategy using RNA interference (RNAi) to avoid the formation of paucimannosidic glycans in insect S2 cells. In addition, we describe coexpression of ß(1,4)-galactosyltransferase (GalT) as a strategy to improve N-glycosylation pattern and enable recombinant therapeutic proteins to be produced in S2 cells with more complex N-glycans.

Brain activation during processing of angry facial expressions in patients with alcohol dependency.

BACKGROUND: Alcoholism is associated with abnormal anger processing. The purpose of this study was to investigate brain regions involved in the evaluation of angry facial expressions in patients with alcohol dependency. METHODS: Brain blood-oxygenation-level-dependent (BOLD) responses to angry faces were measured and compared between patients with alcohol dependency and controls. RESULTS: During intensity ratings of angry faces, significant differences in BOLD were observed between patients with alcohol dependency and controls. That is, patients who were alcohol-dependent showed significantly greater activation in several brain regions, including the dorsal anterior cingulate cortex (dACC) and medial prefrontal cortex (MPFC). CONCLUSIONS: Following exposure to angry faces, abnormalities in dACC and MPFC activation in patients with alcohol dependency indicated possible inefficiencies or hypersensitivities in social cognitive processing.

Interaction between valence of empathy and familiarity: is it difficult to empathize with the positive events of a stranger?

BACKGROUND: Empathy in humans is thought to have evolved via social interactions caused by the formation of social groups. Considering the role of empathy within a social group, there might be a difference between emotional empathy for strangers and familiar others belonging to the same social group. In this study, we used the global field power (GFP) index to investigate empathic brain activity during observation of a cue indicating either a negative or positive image viewed by a stranger or close friend. METHODS: Sixteen healthy participants observed a partner performing an emotional gambling task displayed on a monitor. After the partner's choice-response, a frowning or smiling face symbol was simultaneously presented to the participant's monitor while a negative or positive emotional image was presented to the partner's monitor. All participants observed a control condition (CT) showing a computer trial, a stranger-observation condition (SO) showing the trial of a stranger, and a friend-observation condition (FO) to observe the trial of a close friend. During these observations, participants' event-related potentials (ERPs) were recorded to calculate GFP, and after the task, a subjective assessment of their feelings was measured. RESULTS: Positive emotion was significantly larger under the FO compared to the CT and the SO. Significantly larger negative emotion was found under the SO and FO compared to the CT. In response to a positive cue, significantly larger GFP during 300 to 600 ms was observed under the FO compared to the CT and SO. In response to a negative cue, significantly larger GFP was observed under the FO and SO compared to the CT. A significantly larger GFP under the SO was found in response to only a negative cue. Topographic map analysis suggested that these differences were related to frontal-occipital dynamics. GFP was significantly correlated with empathic trait. CONCLUSION: These results revealed that familiarity with another person has different effects depending on the valence of empathy. Negative empathy, including the danger perception function, might easily occur even among strangers, whereas positive empathy related to nursing and supporting an inner group does not happen easily with strangers.

Inter-individual relationships in empathic traits and feedback-related fronto-central brain activity: an event-related potential study.

BACKGROUND: Neuroimaging studies continue to indicate the major role the anterior cingulate cortex (ACC) plays in processing empathic responses. Error-related negativity (ERN), an event-related potential (ERP) thought to arise from the ACC, has been found to correlate with scores for individual empathic personality. This study investigated the relationship between empathic personality traits and the amplitude of feedback-related negativity (FRN), an ERP sourced from the ACC and similar to the ERN, using a task involving feedback of monetary gains or losses. METHODS: Sixteen healthy participants answered an empathy trait questionnaire and performed a gambling task to elicit FRN. Because FRN amplitude is thought to be associated with attention, motivation, emotional state, and anxiety trait, we performed a partial correlation analysis between the empathic trait score and FRN amplitude while controlling for variables. RESULTS: In partial correlation analysis, FRN amplitude was significantly inversely correlated with scores for personal distress and marginally correlated with scores for empathic concern and with total average score. DISCUSSION: The study revealed for the first time an association between FRN and emotional empathic traits, after controlling for variables that can affect FRN amplitude. However, we also found a reversed directional correlation contrary to our expectations. This fronto-central brain activity may be associated with empathic properties via dopaminergic neuronal function. Future study using these electric potentials as experimental tools is expected to help elucidate the neurological mechanism of empathy.

Expression and N-glycan analysis of human 90K glycoprotein in Drosophila S2 cells.

Human 90K (h90K; Mac-2-binding protein) glycoprotein is a potential pharmaceutical due to its inhibitory activity against cancer metastasis and expansion. Here, h90K glycoprotein was produced in insect Drosophila S2 cell system, and its N-glycan pattern was analyzed. A plasmid encoding h90K gene, fused with a hexahistidine tag under the control of Drosophila metallotionein promoter, was stably transfected into S2 cells. After copper sulfate induction, transfected S2 cells secreted recombinant h90K at a good expression level of 28mg/L in a 150-mL spinner flask culture. The purified recombinant h90K showed an apparent molecular weight of ∼78kDa which was much smaller than that (∼97kDa) of the natural h90K. Because de-N-glycosylated h90K appeared at ∼60kDa protein band, it was suggested that the recombinant h90K from S2 cells has small N-glycans with about half the molecular weight (∼18kDa) of N-glycans of the natural h90K. Through detail analyses using high-performance liquid chromatography and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, the S2-derived recombinant h90K was confirmed that it has simple paucimannosidic structures containing two or three mannose residues with core fucose as the major (∼79%) N-glycans.

Expression of ß-1,4-galactosyltransferase and suppression of ß-N-acetylglucosaminidase to aid synthesis of complex N-glycans in insect Drosophila S2 cells.

Previously, we have shown that simple paucimannosidic N-glycan structures in insect Drosophila S2 cells arise mainly because of ß-N-acetylglucosaminidase (GlcNAcase) action. Thus, in an earlier report, we suppressed GlcNAcase activity and clearly demonstrated that more complex N-glycans with two terminal N-acetylglucosamine (GlcNAc) residues were then synthesized. In the present work, we investigated the synergistic effects of ß-1,4-galactosyltransferase (GalT) expression and GlcNAcase suppression on N-glycan patterns. We found that the N-glycan pattern of human erythropoietin secreted by engineered S2 cells expressing GalT but not GlcNAcase was complete, even in small portion, except for sialylation; the N-glycan structures had two terminal galactose (Gal) residues. When GalT was expressed but GlcNAcase was not inhibited, N-glycan with GlcNAc and Gal at only one branch end was synthesized. Therefore, it will be possible to express a complete functional human glycoprotein in engineered Drosophila S2 cells by suppressing GlcNAcase and co-expressing additional glycosyltransferases of N-glycosylation pathway.

Suppression of beta-N-acetylglucosaminidase in the N-glycosylation pathway for complex glycoprotein formation in Drosophila S2 cells.

Most insect cells have a simple N-glycosylation process and consequently paucimannosidic or simple core glycans predominate. Previously, we have shown that paucimannosidic N-glycan structures are dominant in Drosophila S2 cells. It has been proposed that beta-N-acetylglucosaminidase (GlcNAcase), a hexosaminidase in the Golgi membrane which removes a terminal N-acetylglucosamine (GlcNAc), might contribute to simple N-glycosylation in several insects and insect-derived cells except S2 cells. In the present work, we investigated the substantial effects of GlcNAcase on N-glycan patterns in Drosophila S2 cells using two GlcNAcase suppression strategies: an mRNA-targeting approach using RNA interference (RNAi) and a protein-targeting approach using the specific chemical inhibitor 2-acetamido-1,2-dideoxynojirimycin (2-ADN). Using high-performance liquid chromatography (HPLC) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) analyses, we found that the N-glycosylation patterns of human erythropoietin (hEPO) secreted by stably transfected S2 cells were more complex following GlcNAcase suppression, which generated N-glycan structures with a terminal GlcNAc and/or galactose. These data demonstrate that GlcNAcase may be an important factor in the formation of paucimannosidic core N-glycans in Drosophila S2 cells and suggest that it may be possible to express complex glycoproteins in engineered Drosophila S2 cells by suppressing GlcNAcase in the N-glycosylation pathway.

Recombinant baculovirus-based multiple protein expression platform for Drosophila S2 cell culture.

A platform for selective and controllable expression of multiple foreign protein types was developed in insect cell culture. Based on the fact that baculovirus cannot replicate in nonpermissive Drosophila melanogaster Schneider line 2 (S2) cells, S2 cells that stably express human erythropoietin (hEPO) under the control of the S2-derived inducible metallothionein (MT) promoter were infected with three types of recombinant baculoviruses, each of which expressed a different fluorescent protein gene under the control of MT promoter. Addition of copper sulfate as an inducer to infected, stably transfected S2 cells resulted in simultaneous expression of hEPO and three fluorescent proteins. Expression profiles and levels of the three induced fluorescent proteins were similar in all single infected cells. Importantly, expression profiles and levels of hEPO were similar in both non-infected and infected cells, indicating that baculovirus expressed recombinant proteins do not adversely affect expression of host cell recombinant proteins. Expressions of the three fluorescent proteins were able to be selectively regulated by altering combination ratios of the three types of recombinant baculoviruses. Collectively, these data indicate that the baculovirus/stably transfected S2 cell system can be successfully used to express multiple foreign proteins in a controlled and selective manner without the burden of additional selection markers. Such a system would be expected to be attractive as a multiple protein expression platform for engineering metabolic or glycosylation pathways.

Statistical determination of optimal baculovirus infection condition for recombinant protein production in Drosophila S2 cells.

Insect Drosophila melanogaster S2 cell was developed as plasmid-based and, therefore, a nonlytic expression system for functional foreign proteins. To achieve multiple protein expressions, it was suggested that baculovirus be used on S2 cell system because baculovirus can infect S2 cells but cannot replicate inside the cells. Therefore, establishment of baculovirus infection conditions is the first important step and this should be properly optimized for production yield. We used statistical methodology to optimize the baculovirus infection conditions using green fluorescent protein (GFP) as a reporter protein. Consequently, we arrived at optimal infection conditions through a statistical regression method. The secreted GFP yield from vMT-GFP baculovirus-infected wild-type S2 cells under optimal infection conditions was >15-fold higher than that under nonoptimal conditions and comparable to that from stably transfected recombinant S2 cells.

Facile and rapid direct gold surface immobilization with controlled orientation for carbohydrates.

Effective surface immobilization is a prerequisite for numerous carbohydrate-related studies including carbohydrate-biomolecule interactions. In the present work, we report a simple and rapid modification technique for diverse carbohydrate types in which direct oriented immobilization onto a gold surface is accomplished by coupling the amine group of a thiol group-bearing aminophenyl disulfide as a new coupling reagent with an aldehyde group of the terminal reducing sugar in the carbohydrate. To demonstrate the generality of this proposed reductive amination method, we examined its use for three types of carbohydrates: glucose (monosaccharide), lactose (disaccharide), and GM1 pentasaccharide. Through successful mass identifications of the modified carbohydrates, direct binding assays on gold surface using surface plasmon resonance and electrochemical methods, and a terminal galactose-binding lectin assay using atomic force microscopy, we confirmed several advantages including direct and rapid one-step immobilization onto a gold surface and exposure of functional carbohydrate moieties through oriented modification of the terminal reducing sugar. Therefore, this facile modification and immobilization method can be successfully used for diverse biomimetic studies of carbohydrates, including carbohydrate-biomolecule interactions and carbohydrate sensor or array development for diagnosis and screening.

Recombinant mussel adhesive protein Mgfp-5 as cell adhesion biomaterial.

Mytilus galloprovincialis foot protein type-5 (Mgfp-5) is one of the mussel adhesive proteins that participate in adhesion with the substratum. We previously reported the production of recombinant Mgfp-5 in Escherichia coli and showed that the recombinant protein had superior adhesion abilities versus those of Cell-Tak, a commercially available mussel adhesive protein mixture. In the present work, we investigated the feasibility of using recombinant Mgfp-5 as a cell adhesion agent. Purified and tyrosinase-modified recombinant Mgfp-5 was used to adhere living anchorage-independent cells such as insect Drosophila S2 cells and human MOLT-4 cells onto glass slides. Our results revealed that these cell lines efficiently attached to recombinant Mgfp-5-coated glass surfaces, and that surface-immobilized S2 cells were viable and able to undergo cell division for up to 1 week. Cytochemical studies with 4',6-diamidino-2-phenylindole (DAPI) staining of nuclei and immunofluorescence for secreted foreign human erythropoietin (hEPO) from recombinant S2 cells and quantitative comparative analyses of S2 cell binding ability with Cell-Tak and poly-L-lysine, the main cell adhesion agent, were performed to demonstrate successful usage of recombinant Mgfp-5 for cell biological applications. Collectively, these results indicate that recombinant Mgfp-5 may be a useful new cell adhesion biomaterial for anchorage-independent cells.

Production and N-glycan analysis of secreted human erythropoietin glycoprotein in stably transfected Drosophila S2 cells.

Schneider 2 (S2) cells from Drosophila melanogaster have been used as a plasmid-based, non-lytic expression system for foreign proteins. Here, a plasmid encoding the human erythropoietin (hEPO) gene fused with a hexahistidine (His(6)) tag under the control of the Drosophila metallothionein (MT) promoter was stably transfected into Drosophila S2 cells. After copper sulfate induction, transfected S2 cells were found to secrete hEPO with a maximum expression level of 18 mg/L and a secretion efficiency near 98%. The secreted hEPO from Drosophila S2 had an apparent molecular weight of about 23-27 kDa which was significantly lower than a recombinant hEPO expressed in Chinese hamster ovary (CHO) cells (about 36 kDa). N-glycosidase F digestion almost completely eliminated the difference and resulted in the same molecular weight ( approximately 20 kDa) of de-N-glycosylated hEPO proteins. These data suggest that recombinant hEPO from S2 cells was modified with smaller N-glycans. Subsequently, the major N-glycans were identified following glycoamidase A digestion, labeling with 2-aminopyridine (PA), and two-dimensional high-performance liquid chromatography (HPLC) analysis in concert with exoglycosidase digestion. This analysis of N-glycans revealed that hEPO was modified to include paucimannosidic glycans containing two or three mannose residues with or without core fucose. A similar glycosylation pattern was observed on a recombinant human transferrin expressed in S2 cells. These results provide a detailed analysis of multiple N-glycan structures produced in a Drosophila cell line that will be useful in the subsequent application of these cells for the generation of heterologous glycoproteins.

Physiological responses induced by pleasant stimuli.

The specific physiological responses induced by pleasant stimuli were investigated in this study. Various physiological responses of the brain (encephaloelectrogram; EEG), autonomic nervous system (ANS), immune system and endocrine system were monitored when pleasant stimuli such as odors, emotional pictures and rakugo, a typical Japanese comical story-telling, were presented to subjects. The results revealed that (i) EEG activities of the left frontal brain region were enhanced by a pleasant odor; (ii) emotional pictures related to primitive element such as nudes and erotic couples elevated vasomotor sympathetic nervous activity; and (iii) an increase in secretory immunoglobulin A (s-IgA) and a decrease in salivary cortisol (s-cortisol) were induced by rakugo-derived linguistic pleasant emotion. Pleasant emotion is complicated state. However, by considering the evolutionary history of human being, it is possible to assess and evaluate pleasant emotion from certain physiological responses by appropriately summating various physiological parameters.

Expression of functional human transferrin in stably transfected Drosophila S2 cells.

Human transferrin (hTf) is a serum glycoprotein involved in Fe3+ transport. Here, a plasmid encoding the hTf gene fused with a hexahistidine (His6) epitope tag under Drosophila metallothionein promoter (pMT) was stably transfected into Drosophila melanogaster S2 cells as a nonlytic plasmid-based system. Following 3 days of copper sulfate induction, transfected S2 cells were found to secrete hTf into serum-free culture medium at a competitively high expression level of 40.8 microg/mL, producing 6.8 microg/mL/day in a 150-mL spinner flask culture. Purification of secreted recombinant hTf using immobilized metal affinity chromatography (IMAC) yielded 95.5% pure recombinant hTf with a recovery of 32%. According to MALDI-TOF mass spectrometry analysis, purified S2 cell-derived His6-tagged recombinant hTf had a molecular weight (76.4 kDa) smaller than that of native apo-hTf (78.0 kDa). 2-Dimensional gel electrophoresis patterns showed recombinant hTf had a simpler and less acidic profile compared to that of native hTf. These data suggest recombinant hTf was incompletely (noncomplex) glycosylated and lacked sialic acids on N-glycans. However, this difference in N-glycan structure compared to native hTf had no effect on the iron-binding activity of recombinant hTf. The present data show that a plasmid-based stable transfection S2 cell system can be successfully employed as an alternative for producing secreted functional recombinant hTf.

Co-expression of bacterial hemoglobin overrides high glucose-induced repression of foreign protein expression in Escherichia coli W3110.

Co-expression of Vitreoscilla hemoglobin (VHb) can enhance production of foreign proteins in several microorganisms, including Escherichia coli. Production of foreign proteins [green fluorescent protein (GFP) and organophosphorous hydrolase (OPH)] has been examined in two typical industrial E. coli strains, W3110 (a K12 derivative) and BL21 (a B derivative). In particular, we investigated the effects of VHb co-expression and media glucose concentration on target protein production. We employed the nar O(2)-dependent promoter for self-tuning of VHb expression based on the natural changes in dissolved O(2) levels over the duration of culture. Foreign protein production in strain BL21 was decreased by a high glucose concentration but co-expression of VHb had no effect on this. In contrast, co-expression of VHb in strain W3110 overrode the glucose-induced repression and resulted in steady expression of foreign proteins.

Characteristics of electroencephalographic responses induced by a pleasant and an unpleasant odor.

More than sensory stimuli, odorous stimuli were employed to facilitate the evocation of emotional responses in the present study. The odor-stimulated emotion was evaluated by investigating specific features of encephalographic (EEG) responses produced thereof. In this study, the concentrations of the same odor were altered; viz., the changes in odor-induced emotional level were compared with the concurrently monitored EEG response features. In addition, we performed the mental task to evoke the arousal state of the brain and investigated the resemblance of response characteristics of the resting state to the post-mental task resting state. Subjects having no abnormalities in the sense of smell included 12 male undergraduate and graduate students (age range: 22-26 years). Experiment I involved 2 types of odors that induced favorable odorous stimuli (pleasant induction); test-solutions were either diluted 150 (easily perceptible odorous sensation) or 500 (slightly perceptible odorous stimuli) times. Experiment II had 2 types of odors that evoked unfavorable odorous stimuli (unpleasant induction), and test-solutions with dilution rates similar to those of pleasant induction were prepared. Odorless distilled water was used as the control in both experiments. From results of rating the odorous stimuli of our compounds used, the candidates were respectively found to be appropriate in inducing the pleasant and unpleasant smell sensations. The analyses of EEG responses on inducing pleasant and unpleasant smell sensations revealed that the EEG activities of the left frontal region were enhanced. This finding may establish the hypothesis of a relationship prevailing between the positive approach-related emotion evoked by the visual sensation and the left hemisphere (Davidson, 1992; Tomarken et al., 1989). In other words, it can be interpreted that the negative withdrawal-related emotion may be associated with activities of the right hemisphere. However, this hypothesis may not be applicable to the unpleasant odors, as the unpleasant emotions are activated by the unpleasant odors not only in the bilateral frontal regions but also over an extensive area of the brain. As such, the pleasant emotions are evoked in the left frontal brain region while the unpleasant emotions are incited in the bilateral frontal and extensive regions in the brain with the odorous stimuli. Moreover, intrinsic EEG activities in response to the pleasant and unpleasant inputs were not observed after performing the mental tasks. In other words, EEG responses reflecting central nervous system activities elevated by loading of the mental tasks as a result of exposure to the pleasant and unpleasant odors may not apparently be observed.