Biomarkers for the evaluation of immunological properties during the shikoku walking pilgrimage.

It is important to determine the immunological properties for the maintenance of health. We chose the Shikoku Walking Pilgrimage to assess the proper biomarkers for the evaluation of immunological properties. We examined whether the Shikoku Walking Pilgrimage could have a positive effect on the mental and physical health of walking participants by using several biomarkers proposed by our laboratory. Twelve non-randomized healthy male volunteers including 3 twice attendees walked the Shikoku Walking Pilgrimage distance of 58.9 km over 3 days. Plasma, serum, urine, and saliva were collected from the volunteers during the pilgrimage and at 1 week before and after it. Immunological biomarkers, including lipid metabolism, oxidative stress, immune function, and catecholamines, were measured. Additionally, mood state scores, alertness, autonomic nervous system activity, and body motion levels during sleep were assessed. A significant decrease was observed in the subjective tension-anxiety levels and in the concentrations of serum low-density lipoprotein cholesterol, plasma hydroxyoctadecadienoic acid (HODE), and urine adrenaline during the pilgrimage as compared to the values of these parameters before the participants embarked on the pilgrimage. The serum levels of brain-derived neurotrophic factor (BDNF) were significantly increased 1 week after the pilgrimage relative to those assessed previously. No significant differences in subjective fatigue and the flicker perception threshold were observed. These results suggest that the Shikoku Walking Pilgrimage can exert a positive effect on mental and physical health as particularly shown in the reduction of tensionanxiety and oxidative stress without the accompaniment of fatigue. HODE correlated significantly with typical immunological marker natural killer cell activity and immunoglobulin G. This suggests that there are promising biomarkers such as HODE, NK activity, BDNF, LDL-c, and IgG for assessing the immunological properties.

Sphingosine kinase 1 expression is downregulated during differentiation of Friend cells due to decreased c-MYB.

Sphingosine kinase 1 (SPHK1) overexpression in malignant cells has been reported. Mouse Friend cells showed higher SPHK1 but not SPHK2 expression compared with other mouse cell lines. A Sphk1 promoter analysis demonstrated the region between -53bp and the first exon as the minimal promoter. Further promoter truncation revealed the importance of a MYB-binding site. EMSA using this region as the probe demonstrated one band containing c-MYB protein, and its intensity decreased during erythroid differentiation with hexamethylane bisacetamide (HMBA), a potent inducer of erythroid differentiation of Friend cells. ChIP assay also revealed in vivo binding of c-MYB. c-MYB overexpression and siRNA for c-Myb affected SPHK1 expression, confirming the important regulatory role of c-MYB in SPHK1 expression. HMBA reduced c-MYB expression rapidly. Induced differentiation by HMBA caused a marked and rapid reduction of SPHK1 mRNA, protein and enzyme activity leading to the rapid decrease of cellular sphingosine 1-phosphate level. Moreover, terminally differentiated cells did not resume SPHK1 expression. Compared with original Friend cells, stable overexpression of wild-type SPHK1 showed higher cell proliferation, resistance to cell death by serum depletion. Interestingly, HMBA-induced differentiation of these cells was delayed but not completely suppressed. In contrast, SPHK inhibitor and its siRNA inhibited cell growth and enhanced HMBA-induced differentiation significantly, suggesting that SPHK1 delayed HMBA-induced differentiation by its cell proliferation-promoting activity. Effects of pertussis toxin, a G-protein-coupled receptor inhibitor, and S1P receptor antagonist on Friend cell growth and differentiation were negligible, suggesting the importance of the intracellular SPHK1/S1P signaling in Friend cells.

Rejection of stimulus-related MEG artifacts using independent component analysis.

Electro-gustatory (EG) stimuli have been used to investigate the gustatory information processing in the human brain with precise temporal and spatial accuracy. But this technique was not widely applicable to magnetoencephalographic measurements due to the difficulty in eliminating huge stimulus-related artifact. In this study, we used independent component decomposition of the measured signal to reject the stimulus-related artifact from other brain activities based on information maximization (infomax) approach. Infomax ICA was applied to raw MEG data measured by 122 magnetometer channels producing 122 temporally independent components, and the trials for each component were averaged separately. Each independent component was visually inspected and the components obviously representing the EG stimulus-related artifacts were excluded from remixing and the signal projection onto the original MEG signal space to reconstruct the artifact-free MEG signals. Results showed that large EG stimulus-related artifacts were clearly removed from MEG waveforms. We were also able to separate blink-related components and magnetocardiographic components as well as the components representing alpha-band (8-13 Hz) spontaneous brain activity.

Effect of word familiarity on visually evoked magnetic fields.

This study investigated the effect of word familiarity of visual stimuli on the word recognizing function of the human brain. Word familiarity is an index of the relative ease of word perception, and is characterized by facilitation and accuracy on word recognition. We studied the effect of word familiarity, using "Hiragana" (phonetic characters in Japanese orthography) characters as visual stimuli, on the elicitation of visually evoked magnetic fields with a word-naming task. The words were selected from a database of lexical properties of Japanese. The four "Hiragana" characters used were grouped and presented in 4 classes of degree of familiarity. The three components were observed in averaged waveforms of the root mean square (RMS) value on latencies at about 100 ms, 150 ms and 220 ms. The RMS value of the 220 ms component showed a significant positive correlation (F=(3/36); 5.501; p=0.035) with the value of familiarity. ECDs of the 220 ms component were observed in the intraparietal sulcus (IPS). Increments in the RMS value of the 220 ms component, which might reflect ideographical word recognition, retrieving "as a whole" were enhanced with increments of the value of familiarity. The interaction of characters, which increased with the value of familiarity, might function "as a large symbol"; and enhance a "pop-out" function with an escaping character inhibiting other characters and enhancing the segmentation of the character (as a figure) from the ground.

A new dynamical approach to auditory evoked magnetic field by blind identification.

A new approach to understand neural dynamics underlying the generation of auditory evoked magnetic field is proposed. MEG time series data are temporally decorrelated by using a blind signal separation method. Two components are selected from their periodical property and a remixing matrix is applied to the two selected components to retrieve MEG signals of auditory evoked magnetic field. After principal component data for each sensor pairs are calculated, a minimum phase innovation model is identified from the viewpoint of statistical inverse problem. By using a blind identification method based on feedback system theory transfer functions can be evaluated to get a dynamical understanding of brain auditory functions. It is reported that all changes of their impulse responses between right and left hemisphere decay within about 40 ms, and that directional differences in transfer functions can be found.

Neural substrates involved in imitating finger configurations: an fMRI study.

Imitation plays a very important role in human cognition. Because previous neuroimaging studies on human imitation used rather simple actions as target stimuli, some aspects of imitation such as perceiving target actions or manipulating one's own mental image could not be studied. We used complicated non-symbolic (S-) and symbolic (S+) finger configurations as target stimuli in order to study the neural substrates involved in the perception of target actions and mental image manipulation during imitation. Bilateral supramarginal gyrus activation was detected when the S- condition was compared with the S+ condition. Our result suggests the involvement of the supramarginal gyrus especially for the imitation of novel actions.

Complete cysteine-scanning mutagenesis and site-directed chemical modification of the Tn10-encoded metal-tetracycline/H+ antiporter.

Bacterial Tn10-encoded metal-tetracycline/H(+) antiporter was the first found drug exporter and has been studied as a paradigm of antiporter-type major facilitator superfamily transporters. Here the 400 amino acid residues of this protein were individually replaced by cysteine except for the initial methionine. As a result, we could obtain a complete map of the functionally or structurally important residues. In addition, we could determine the precise boundaries of all the transmembrane segments on the basis of the reactivity with N-ethylmaleimide (NEM). The NEM binding results indicated the presence of a transmembrane water-filled channel in the transporter. The twelve transmembrane segments can be divided into three groups; four are totally embedded in the hydrophobic interior, four face a putative water-filled channel along their full length, and the remaining four face the channel for half their length, the other halves being embedded in the hydrophobic interior. These three types of transmembrane segments are mutually arranged with a 4-fold symmetry. The competitive binding of membrane-permeable and -impermeable SH reagents in intact cells indicates that the transmembrane water-filled channel has a thin barrier against hydrophilic molecules in the middle of the transmembrane region. Inhibition and stimulation of NEM binding in the presence of tetracycline reflects the substrate-induced protection or conformational change of the Tn10-encoded metal-tetracycline/H(+) antiporter. The mutations protected from NEM binding by tetracycline were mainly located around the permeability barrier in the N-terminal half, suggesting the location of the substrate binding site.

Safety and efficacy of water-in-oil-in-water emulsion vaccines containing Newcastle disease virus haemagglutinin-neuraminidase glycoprotein.

Subunit vaccines containing haemagglutinin-neuraminidase (HN) glycoprotein of Newcastle disease virus (NDV), formulated as water-in-oil-in-water (W/O/W) emulsions, were prepared. First, the suitable constituents of a W/O/W emulsion adjuvant were investigated with polyvalent vaccines using NDV, infectious bronchitis virus and Haemophilus paragallinarum. The W/O/W emulsion adjuvant, composed of the antigen in phosphate-buffered saline (PBS), liquid paraffin, squalene, diglyceryl monooleate, polysorbate 80 and PBS in a 30:25:10:5:2:28 ratio, induced a good antibody response with less adverse local reactions. HN protein of NDV was expressed by an improved baculovirus expression vector, a hybrid nucleopolyhedrovirus (HyNPV) between Autographa californica NPV and Bombyx mori NPV,and was prepared from silkworm pupae infected with the recombinant baculovirus, HyNPV-HN. Then, the W/O/W emulsion vaccine containing HN protein was prepared using the aforementioned constituents. Chickens showed 100, 100 and 80% protection against challenge exposure to virulent NDV at 4 weeks after vaccination with W/O/W emulsion vaccines containing 30, 6 and 3% of HyHPV-HN-infected pupae, respectively. The vaccines containing HN protein did not induce adverse local reactions at the site of injection. The subunit vaccine for NDV containing HN protein expressed in the recombinant baculovirus-infected pupae, formulated as a W/O/W emulsion vaccine composed of the antigen in PBS, liquid paraffin, squalene, diglyceryl monooleate, polysorbate 80 and PBS in a 30:25:10:5:2:28 ratio, was therefore found to be safe and effective.

Cysteine-scanning mutagenesis of transmembrane segments 4 and 5 of the Tn10-encoded metal-tetracycline/H+ antiporter reveals a permeability barrier in the middle of a transmembrane water-filled channel.

Cysteine-scanning mutants as to putative transmembrane segments 4 and 5 and the flanking regions of Tn10-encoded metal-tetracycline/H(+) antiporter (TetA(B)) were constructed. All mutants were normally expressed. Among the 57 mutants (L99C to I155C), nine conserved arginine-, aspartate-, and glycine-replaced ones exhibited greatly reduced tetracycline resistance and almost no transport activity, and five conserved glycine- and proline-replaced mutants exhibited greatly reduced tetracycline transport activity in inverted membrane vesicles despite their high or moderate drug resistance. All other cysteine-scanning mutants retained normal drug resistance and normal tetracycline transport activity except for the L142C and I143C mutants. The transmembrane (TM) regions TM4 and TM5 were determined to comprise 20 amino acid residues, Leu-99 to Ile-118, and 17 amino acid residues, Ala-136 to Ala-152, respectively, on the basis of N-[(14)C]ethylmaleimide ([(14)C]NEM) reactivity. The NEM reactivity patterns of the TM4 and TM5 mutants were quite different from each other. TM4 could be divided into two halves, that is, a NEM nonreactive periplasmic half and a periodically reactive cytoplasmic half, indicating that TM4 is tilted toward a water-filled transmembrane channel and that only its cytoplasmic half faces the channel. On the other hand, NEM-reactive mutations were observed periodically (every two residues) along the whole length of TM5. A permeability barrier for a membrane-impermeable sulfhydryl reagent, 4-acetamido-4'-maleimidylstilbene-2,2'-disulfonic acid, was present in the middle of TM5 between Leu-142 and Gly-145, whereas all the NEM-reactive mutants as to TM4 were not accessible to 4-acetamido-4'-maleimidylstilbene-2,2'-disulfonic acid, indicating that the channel-facing side of TM4 is located inside the permeability barrier. Tetracycline protected the G141C mutant from the NEM binding, whereas the other mutants in TM4 and TM5 were not protected by tetracycline.

Cysteine-scanning mutagenesis around transmembrane segments 1 and 11 and their flanking loop regions of Tn10-encoded metal-Tetracycline/H+ antiporter.

Putative transmembrane helices (TM) 1 and 11 in the metal-tetracycline/H(+) antiporter are predicted to be close to each other on the basis of disulfide cross-linking experiments of the double-cysteine mutants in the periplasmic loop regions (Kubo, Y., Konishi, S., Kawabe, T., Nada, S., and Yamaguchi, A. (2000) J. Biol. Chem. 275, 5270-5274). In this study, each amino acid from Asn-2 to Gly-44 in the putative TM1 and loop1-2 regions or that from Ser-328 to Gly-366 in TM11 and its flanking regions was individually replaced with cysteine. With respect to the TM1 region, 10 mutants, from T5C to L14C, were all not reactive with N-ethylmaleimide (NEM), and from D15C to I22C, NEM-reactive and non-reactive mutations periodically appeared every two residues. Three mutants, M23C to V25C, were all NEM-reactive, but the degree of the latter two mutants was very low. Seven mutants, from L26C to E32C, were all highly reactive with NEM. Therefore, the region of TM1 is composed of the 21 amino acid residues from Thr-5 to Val-25. It is a partially amphiphilic helix, that is, the N-terminal (cytoplasmic) half is embedded in the hydrophobic interior, and the C-terminal (periplasmic) half faces a water-filled channel. With respect to TM11, nine mutants, from S328C to G336C, and six mutants, from L361C to G366C, were all reactive with NEM. On the other hand, out of the 24 mutants, from L337C to S360C, 17 were not reactive with NEM, and the 7 NEM-reactive mutants were scattered, indicating that this region is a transmembrane segment. The 7 residues from Val-347 to Phe-353 including Pro-350 formed a central hydrophobic core, and the 7 NEM-reactive mutations were periodically distributed in its flanking regions, indicating that both ends of TM11 face a water-filled channel. Ala-354 is located at about 1/3 of the length from the periplasmic end of TM11. Disulfide cross-linking experiments on double-cysteine mutants having the combination of A354C and a cysteine-scanning mutation in the loop1-2 region indicated that loop1-2 is very flexible and close to the periplasmic end of TM11. Tetracycline prevented the cross-linking formation between the periplasmic ends of TM1 and TM11; however, it did not affect the cross-linking between loop1-2 and TM11, indicating that the substrate-induced conformational change involves a shift in the relative locations of TM1 and TM11.

Cysteine-scanning mutagenesis around transmembrane segment VI of Tn10-encoded metal-tetracycline/H(+) antiporter.

Each amino acid in putative transmembrane helix VI and its flanking regions, from Ser-156 to Thr-185, of a Cys-free mutant of the Tn10-encoded metal-tetracycline/H(+) antiporter (TetA(B)) was individually replaced by Cys. All of the cysteine-scanning mutants showed a normal level of tetracycline resistance except for the S156C mutant, which showed moderate resistance, indicating that there is no essential residue located in this region. All 20 mutants from S159C to W178C showed no reactivity with N-ethylmaleimide (NEM), whereas the mutants of the flanking regions from S156C to H158C and F179C to T185C were highly or moderately reactive with NEM. These results indicate that like transmembrane helices III and IX, the transmembrane helix VI comprising residues Ser-159-Trp-178 is totally embedded in the hydrophobic environment.

Dynamic cortical activation in mental image processing revealed by biomagnetic measurement.

The mental rotation task has been reported to activate the human parietal and extra-striate areas, based on the results of fMRI and PET analysis. In the present study, we investigated the dynamic properties of the distributed cortical activity related to mental rotation processes at high temporal resolution by means of brain magnetic field measurements and a linear inversion algorithm. Distributed neural activities during the mental rotation and control tasks were estimated for six subjects, and the differences in the activity distribution were analyzed. Statistically significant differences in the parietal and lateral posterior temporal region were detected 200-300 ms after the visual stimulus, indicating that the dorsal and ventral pathway were included in the mental image processing.

Site-directed chemical modification of cysteine-scanning mutants as to transmembrane segment II and its flanking regions of the Tn10-encoded metal-tetracycline/H+ antiporter reveals a transmembrane water-filled channel.

Cysteine-scanning mutants, E32C to G62C, of the metal-tetracycline/H+ antiporter were constructed in order to precisely determine the membrane topology around putative transmembrane segment II. None of the mutants lost the ability to confer tetracycline resistance, indicating that the cysteine mutation in each mutant did not alter the protein conformation. [14C]N-Ethylmaleimide (NEM) binding to these cysteine mutants in isolated membranes was then investigated. The peptide chain of this region passes through the membrane at least once because residues 36 and 65 are exposed on the outside and inside surfaces of the membrane, respectively (Kimura, T., Ohnuma, M., Sawai, T., and Yamaguchi, A. (1997) J. Biol. Chem. 272, 580-585). However, there was no continuous segment in which all of the introduced cysteine residues showed almost no reactivity with [14C]NEM. The proportion of the unbound positions in the second half downstream from position 45 was 55% (10/18), which was clearly higher than that in the first half (21%; 3/14), suggesting that the second half is a transmembrane segment. Positions reactive to NEM appear periodically in the second half. They are located on one side of the helical wheel, suggesting that this side of the transmembrane helix faces a water-filled channel. The cysteine mutants as to the reactive positions in the second half were severely inactivated by NEM except for the P59C mutant, whereas the A40C mutant was the only one inactivated by NEM in the first half. These results suggest that the water-filled channel along this helical region may be a substrate translocation pathway.

Different abilities of two types of Fc gamma receptor on guinea-pig macrophages to trigger the intracellular Ca2+ mobilization and O2- generation.

When exposed to hen ovalbumin (OA)-complexed IgG antibodies, guinea-pig macrophages undergo O2- generation and a rapid rise in the intracellular concentration of free Ca2+ ((Ca2+]i). These responses were found to depend on the IgG isotype of antibodies used; OA-complexed IgG2 antibody (OA gamma 2) induced these responses 3-5 times more intensively than did OA-complexed IgG1 antibody (OA gamma 1). The inhibitory effects of monoclonal antibody to Fc gamma receptor for IgG2 alone (Fc gamma 2R) and that to Fc gamma receptor for both IgG1 and IgG2 (Fc gamma 1/gamma 2R) showed that Fc gamma 2R triggered both an increase in [Ca2+]i and activation of the respiratory burst NADPH oxidase more effectively than did Fc gamma 1/gamma 2R. As the number of Fc gamma 2R molecules per macrophage is about one-half that of Fc gamma 1/gamma 2R molecules, the ability of Fc gamma 2R to trigger these responses may be much higher than that of Fc gamma 1/gamma 2R. This difference between their abilities was further demonstrated by measuring the responses induced by cross-linking of Fc gamma 2R or Fc gamma 1/gamma 2R molecules. In addition, the O2- generation with OA gamma 1 was found to be enhanced with cytochalasin B, and to be lowered by depletion of the intracellular Ca2+ of macrophages with Ionomycin and EGTA, though cytochalasin B and the Ca2+ depletion did not affect the O2- generation with OA gamma 2. These results suggest that the mechanisms of Fc gamma 2- and Fc gamma 1/gamma 2 R-mediated signal transmission leading to activation of the NADPH oxidase also differ from each other.

Identification of newborn rats by tattooing.

Pups were identified by toe clipping or tattooing the plantar surface of the paws on day 4 after delivery. Their growth, maturation, and reproductive capability were not affected by either identification method. In the toe clipping group, however, the duration until fall in the suspension test was significantly shortened, indicating that this identification method may not be suitable for some behavioural tests. The clipping also disturbs the skeletal investigation of toes and is not recommended from the view point of animal welfare. Palm tattooing, on the other hand, satisfies the fundamental requirements for long-term identification of rats.

Purification of a Brucella canis cell wall antigen by using immunosorbent columns and use of the antigen in enzyme-linked immunosorbent assay for specific diagnosis of canine brucellosis.

A cell wall antigen of Brucella canis was purified by immunosorbent columns. The antigen contained two proteins of 30 and 28 kilodaltons and a polysaccharide exhibiting a 12-kilodalton band upon 12.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Antibody to the purified antigen, which specifically reacted with the polysaccharide, was used as the first coating antibody in an enzyme-linked immunosorbent assay (ELISA) for serological diagnosis of canine brucellosis. Dogs inoculated orally with live B. canis were positive and dogs from B. canis-free colonies were negative in the ELISA. Of 199 dogs from a brucellosis-contaminated area, 116 with negative titers in the tube agglutination test (TAT), using heat-inactivated whole B. canis cells as the antigen, were also negative in the ELISA. Seventy-eight of the dogs with questionable titers in the TAT were divided into two groups: 20 dogs that were positive in the ELISA and 58 that were negative. Of five dogs with positive titers in the TAT, three were positive in the ELISA and the gel immunodiffusion test (GD) with crude B. canis extract as the antigen and were also culture positive for B. canis. One dog was positive in the ELISA and GD but gave a negative culture result. Serum from the remaining dog, which was positive with high titer in the TAT but negative in the ELISA and in culture for B. canis, formed a spur precipitate with a homologous precipitate in the GD. These results indicate that the ELISA is a specific serological test for B. canis infection in dogs.

Experiences with the identification of small rodents.

Own experiences with the identification of mice and rats using ear tags, ear punching, toe clipping (for newborns only) and ear tattooing are given and compared with methods presented in literature. By far, ear tattooing by the newly developed pincers is the most suitable method for marking adult rodents for life. Tattooing is easy to apply, easy to read and not harmful to the animals. For newborn pups on day 4 p. p., the s. c. injection of India ink into the palm of paw is recommended.