Inducing peripheral sympathetic nerve activity by therapeutic electrical stimulation.

PURPOSE: To examine whether the activity of peripheral sympathetic nerves in animals with spinal cord injury can be controlled using therapeutic electrical stimulation. METHODS: The spinal cords of 6 Wistar rats were severed at T12/T13 disk level and were given continuous therapeutic electrical stimulation. Microneurography was used to record sympathetic nerve activity at 24, 48, and 72 hours after severing the spinal cord. RESULTS: Integrated values of muscle sympathetic nerve activity after 72 hours of therapeutic electrical stimulation revealed significantly larger potentials on the stimulated side than the non-stimulated side. Skin sympathetic nerve activity showed no difference between the 2 sides. CONCLUSION: Therapeutic electrical stimulation was found to have a facilitatory effect on the muscle sympathetic nerve activity, whereas regulatory function was activated by the sympathetic nerves.

Molecular mapping of a fertility restorer gene for Owen cytoplasmic male sterility in sugar beet.

We report here the molecular mapping of a fertility restorer gene (named Rf1) for Owen cytoplasmic male sterility in sugar beet. Eight AFLP and two RAPD markers, tightly linked to the Rf1 locus, were identified using bulked segregant analysis. Three AFLP markers, mAFEM972, mAFEM976 and mAFEM985, were found to co-segregate with the Rf1 allele in our mapping populations. With the help of RFLP markers, previously mapped on the sugar beet genome, we showed that Rf1 is positioned in the terminal region of linkage group Kiel III/Koeln IV. This map location agrees well with that found for the restorer gene X, which suggests that the Rf1 locus corresponds to the X locus. The availability of the molecular markers will facilitate the selection of maintainer-pollinator lines in breeding program and provide the foundation for map-based cloning of the Rf1 gene.

The dependence of the auditory evoked N1m decrement on the bandwidth of preceding notch-filtered noise.

The auditory evoked response is known to be changed by a preceding sound. In this study we investigated by means of magnetoencephalography how a preceding notch-filtered noise (NFN) with different bandwidths influences the human auditory evoked response elicited by the following test stimulus. We prepared white noise (WN) and four NFNs which were derived from WN by suppressing frequency regions around 1 kHz with 1/8-, 1/4-, 1/2- and 1-octave bandwidths. Stimulation for 3 s with this set of noises resulted in differences in responsiveness to a 1-kHz test tone presented 500 ms after the offset of the noises. The N1m response to the 1-kHz test tone stimulus was at a minimum when the preceding NFN had 1/4-octave stop-band frequencies as compared with 1/8-, 1/2- and 1-octave NFN and WN. This N1m decrement is explained by the imbalanced neural activities caused by habituation and lateral inhibition in the auditory system. The results contribute to understanding of the inhibitory system in the human auditory cortex.

N1m recovery from decline after exposure to noise with strong spectral contrasts.

Comb-filtered noise (CFN, derived from white noise by suppressing regularly spaced frequency regions) was presented for 3 s followed by two types of test stimuli. One test stimulus (SB) was comprised of spectra centered in the stop-band regions of the CFN and the other test stimulus (PB) of spectra centered in the band pass regions of the CFN. Magnetoencephalographically recorded N1m responses evoked by SB stimuli were decreased relative to the N1m response evoked by PB stimuli. This effect was maximal when the interval between the CFN and test stimuli was short (0.5 s) but was detected at intervals up to 2 s. The results suggest lateral inhibition in the auditory cortex and point to a decay of inhibition lasting on the order of seconds.

The cytoplasmic male-sterile type and normal type mitochondrial genomes of sugar beet share the same complement of genes of known function but differ in the content of expressed ORFs.

The complete nucleotide sequence (501,020 bp) of the mitochondrial genome from cytoplasmic male-sterile (CMS) sugar beet was determined. This enabled us to compare the sequence with that previously published for the mitochondrial genome of normal, male-fertile sugar beet. The comparison revealed that the two genomes have the same complement of genes of known function. The rRNA and tRNA genes encoded in the CMS mitochondrial genome share 100% sequence identity with their respective counterparts in the normal genome. We found a total of 24 single nucleotide substitutions in 11 protein genes encoded by the CMS mitochondrial genome. However, none of these seems to be responsible for male sterility. In addition, several other ORFs were found to be actively transcribed in sugar beet mitochondria. Among these, Norf246 was observed to be present in the normal mitochondrial genome but absent from the CMS genome. However, it seems unlikely that the loss of Norf246 is causally related to the expression of CMS, because previous studies on mitochondrial translation products failed to detect the product of this ORF. Conversely, the CMS genome contains four transcribed ORFs (Satp6presequence, Scox2-2 , Sorf324 and Sorf119) which are missing from the normal genome. These ORFs, which are potential candidates for CMS genes, were shown to be generated by mitochondrial genome rearrangements.

Development of a real-time hand dose monitor for personnel in interventional radiology.

Medical procedures denoted as interventional radiology require operation near an X ray beam, which brings high dose exposures to the operators' hands. For the effectual control of their extremity doses, a prototype of a real-time wrist dosemeter has been developed, hand dose monitor (HDM), based on a single silicon detector. Experiments were performed to test its response to diagnostic X rays. The HDM was highly sensitive and showed a linear response down to doses of a few tens of microsieverts. Though dose rate, energy and angular dependence of the response were observed in some extreme conditions, the HDM was proved to be of practical use if it was appropriately calibrated. Since an HDM enables personnel to check their hand doses on a real-time basis, it would enable medical staff to control the exposure themselves.

Effect of thermosensitive liposomal doxorubicin with hyperthermia on primary tumor and lung metastases in hamster osteosarcoma.

We evaluated the effect of intravenous thermosensitive liposomal doxorubicin (TL-DOX) together with local hyperthermia on primary tumors in highly metastatic hamster osteosarcoma. This combination resulted in higher DOX concentrations in plasma, primary tumors and lungs than standard DOX under the same conditions. Tumor growth and lung metastasis were also inhibited more by TL-DOX and hyperthermia than by hyperthermia alone, DOX with or without hyperthermia, and TL-DOX without hyperthermia. In addition, gains in hamster body weight were not suppressed. These results suggest that the combination of TL-DOX and hyperthermia can control primary tumors and suppress lung metastasis in hamsters.

Activation of the Sarcophaga lectin gene promoter by (A + T)-stretch binding protein.

Previously, we purified and isolated a cDNA for (A + T)-stretch binding protein (ATBP) that binds to (A + T)-stretches in the 5' upstream region of the Sarcophaga lectin gene [Nakanishi-Matsui, M., Kubo, T. & Natori, S. (1995) Eur. J. Biochem. 230, 396-400]. Here, we used a luciferase reporter to examine the effect of ATBP on transcription of the Sarcophaga lectin gene. Deletion experiments revealed that ATBP activates the Sarcophaga lectin gene in a 5' upstream sequence-dependent manner, and that at least the N-terminal 25 residues, the three Zn-finger domains, an acidic domain and the third hydrophobic domain of ATBP are indispensable for its function. Furthermore, a synergistic effect was detected between ATBP and Dif, suggesting that ATBP is involved in the activation of insect immunity genes.

Identification of an inhibitor for interleukin 4-induced epsilon germline transcription and antigen-specific IgE production in vivo.

IgE plays a key role in the pathogenesis of allergic disease. Interleukin (IL) 4 is a potent and critical stimulator of immunoglobulin class switching from IgM to IgE in B cells. IL-4 induces the expression of epsilon germline transcript (epsilonGT), which is critical to initiate IgE production. While searching for molecules that inhibit epsilonGT expression induced by IL-4, we found that polyphenol strictinin, which was isolated from tea leaves, was able to inhibit the IL-4-induced epsilonGT expression in the human B cell line DND39. Strictinin also acted on human peripheral blood mononuclear cells obtained from healthy donors to inhibit IL-4-induced epsilonGT expression. Strictinin demonstrated similar inhibitory activity in peripheral blood mononuclear cells obtained from atopic donors. Interestingly, strictinin decreased ovalbumin-induced IgE production in mice, whereas the production of IgG and IgM was not affected. Furthermore, we found that the IL-4-induced STAT6 tyrosine phosphorylation, which is essential for IL-4-induced epsilonGT expression, was inhibited in DND39 cells upon treatment with strictinin. Taken together, these results suggest that strictinin can inhibit IgE production through the inhibition of IL-4-mediated signaling in B cells.

Cloning of complementary deoxyribonucleic acids encoding quail (Coturnix coturnix japonica) retinoic acid receptor ss isoforms and changes in their gene expression during gonadotropic growth.

Retinoids have important effects on the development of the reproductive system, where they act via their specific nuclear receptors: retinoic acid receptors (RARalpha, ss, gamma) and retinoid X receptors (RXRalpha, ss, gamma). The research reported here was conducted in an effort to clone quail RARbeta+ cDNA (qRARbeta) and to evaluate the expression of qRARbeta+ mRNAs in different tissues and during the development of gonadotropic organs. Two complete cDNAs of qRARbeta1 and qRARbeta2 were isolated by a combination of reverse transcription-polymerase chain reaction and 5'- and 3'-rapid amplification of cDNA ends techniques. An RNase protection assay revealed the widespread expression of qRARbeta1 and beta2 with large tissue-specific variations. The qRARbeta1 isoform was predominant in the testis, whereas qRARbeta2 was dominant in the other tissues examined with the exception of the brain, where both isoforms were almost equally expressed. In the developing testes, the qRARbeta1 mRNA level was high between 30 and 40 days of age, the period during which the testes grew rapidly. The level declined thereafter to its initial level. In contrast, qRARbeta2 mRNA did not exhibit obvious changes. In the developing oviducts, both qRARbeta1 and beta2 mRNAs reached their peak levels by 30 days of age, just before the rapid development of the oviduct occurred, and then decreased to almost undetectable levels when the oviduct developed to the laying stage (over 2.88 g in weight). Similar expression patterns of qRARbeta1 and beta2 were also observed in the developing follicles from the prehierarchical (<2-mm diameter) to the largest preovulatory follicle. In contrast, neither qRARbeta1 nor beta2 mRNA exhibited developmental changes in the brain. These results suggest that RARbeta+ may play an important role in the development of the reproductive systems of birds.

Regulation of hydroxyindole-O-methyltransferase gene expression in Japanese quail (Coturnix coturnix japonica).

Hydroxyindole-O-methyltrasferase (HIOMT) plays an important role as the final enzyme in the synthesis of melatonin. In this study, the expression of the HIOMT gene in Japanese quail was investigated with respect to tissue distribution and the effects of light and vitamin A deficiency. HIOMT mRNA in the pineal gland and eye had a clear daily rhythm with peak values in daytime. The testis also contained a detectable amount of HIOMT mRNA, which did not display a rhythmic change over a 24-h period. When birds were rendered vitamin A deficient through feeding with a vitamin A-free diet, the daily rhythm of the HIOMT gene almost disappeared in both the pineal gland and eye due to increases in the nighttime values. Our previous observations and these results suggest that vitamin A and a photo-signal are required to maintain the rhythmic expression of the HIOMT gene as well as the arylalkylamine N-acetyltransferase gene.

Cholinergic inputs to rostral ventrolateral medulla pressor neurons from hypothalamus.

The rostral ventrolateral medulla (RVLM) has cholinergic mechanisms responsible for pressor responses. Stimulation of the hypothalamic paraventricular nucleus (PVN) causes an increase of arterial pressure via activation of neurons in the RVLM. In this study, we examined whether PVN stimulation causes a pressor response via activation of cholinergic mechanisms in the RVLM. Male Wistar rats were used and they were anesthetized, paralyzed and artificially ventilated. Electrical stimulation of the PVN produced a pressor response. Microinjection of the muscarinic receptor antagonist scopolamine and the cholinesterase inhibitor physostigmine into the RVLM inhibited and potentiated, respectively, the pressor response induced by PVN stimulation. PVN stimulation also increased the firing rate of RVLM barosensitive neurons and the increase in the firing rate was inhibited and potentiated by scopolamine and physostigmine, respectively, iontophoretically applied on neurons. Microinjection of L-glutamate into the PVN produced a release of ACh in the RVLM. The inhibitory amino acid gamma-aminobutyric acid injected into the lateral parabrachial nucleus (LPBN) inhibited the pressor response induced by PVN stimulation. These results suggest that PVN stimulation causes an increase in arterial pressure via activation of cholinergic inputs in the RVLM. It appears that the pressor response is mediated, at least in part, via cholinergic inputs from the LPBN.

Concentrated expression of Ca2+/ calmodulin-dependent protein kinase II and protein kinase C in the mushroom bodies of the brain of the honeybee Apis mellifera L.

We have previously used the differential display method to identify a gene that is expressed preferentially in the mushroom bodies of worker honeybees and to show that it encodes a putative inositol 1,4,5-trisphosphate receptor (IP3R) homologue (Kamikouchi et al. [1998] Biochem. Biophys. Res. Commun. 242:181-186). In the present study, we examined whether the expression of some of the genes for proteins involved in the intracellular Ca2+ signal transduction is also concentrated in the mushroom bodies of the honeybee by isolating cDNA fragments that encode the Ca2+/calmodulin-dependent protein kinase II (CaMKII) and protein kinase C (PKC) homologues of the honeybee. In situ hybridization analysis revealed that the expression of these genes was also concentrated in the mushroom bodies of the honeybee brain: The CaMKII gene was expressed preferentially in the large-type Kenyon cells of the mushroom bodies, whereas that for PKC was expressed in both the large and small types of Kenyon cells. The expression of the genes for IP3R and CaMKII was concentrated in the mushroom bodies of the queen and drone as well as in those of the worker bee. Furthermore, the enzymatic activities of CaMKII and PKC were found to be higher in the mushroom bodies/central bodies than in the optic and antennal lobes of the worker bee brain. These results suggest that the function of the intracellular Ca2+ signal transduction is enhanced in Kenyon cells in comparison to other neuronal cell types in the honeybee brain.

A pitch glide activates an intermediate response between auditory N1 and mismatch negativity.

Pitch glides of a continuous tone elicit auditory N1-like responses. However, their characteristics have not well been investigated, and it remained unclear whether the response is an auditory true N1 or the mismatch negativity (MMN). We found here that a rapid pitch glide activates almost the same response as a true N1. On the contrary, as the rate of the pitch glide decreases, the response continuously varies the characteristics from true N1 to MMN. This suggests that there would exist intermediate responses between auditory N1 and MMN.

Regulation of the expression of serotonin N-acetyltransferase gene in Japanese quail (Coturnix japonica): II. Effect of vitamin A deficiency.

Levels of serotonin N-acetyltransferase [arylalkylamine N-acetyltransferase (AANAT); EC2.3.1.87] mRNA, AANAT activity, and melatonin display a rhythmic pattern in both the pineal gland and the retina. It has been shown that vitamin A is required to maintain the rhythm of melatonin synthesis in the pineal gland of Japanese quail. To understand the mechanism underlying the direct relationship among these factors, we developed an assay system sensitive enough to determine AANAT mRNA, AANAT activity, and melatonin content from a single pineal gland of Japanese quail. Positive direct relationships were found among these three parameters. We next deprived Japanese quail of vitamin A by feeding them a vitamin A-free diet supplemented with retinoic acid, and examined the effects of vitamin A deficiency on the expression of AANAT mRNA in the pineal gland and the retina. Vitamin A deficiency reduced both the expression of AANAT mRNA and melatonin content in the pineal gland. Retinal AANAT mRNA rhythm disappeared in vitamin A-deficient quails. Moreover, the responsiveness of the pineal gland and the retina to light was reduced by vitamin A deficiency when compared with the control group.

Histiocyte reaction in rabbit femurs to UHMWPE, metal, and ceramic particles in different sizes.

To assess the histologic reaction caused by biomaterial particles in different sizes around the bone-implant interface, we examined ultra-high molecular weight polyethylene (UHMWPE, average diameter of 11 microm), UHMWPE (99 microm), cobalt-chromium alloy (Co-Cr, 3.9 microm), stainless steel (SUS316L, 3.9 microm), alumina ceramics (3.9 microm), titanium alloy (Ti, 3.5 microm), Co-Cr (0.03 microm), and Ti (0.03 microm). After the longitudinal groove on a polymethylmethacrylate plug was filled with one type of the particles, the plug was inserted into the medullar canal of the distal end of rabbit femurs, and tissue block was resected 4 and 12 weeks after the insertion. Histiocytes were markedly accumulated around the particles of UHMWPE (11 microm), Co-Cr (3.9 microm), SUS316L (3.9 microm), Co-Cr (0.03 microm), and titanium alloy (0.03 microm). Around the UHMWPE particles (99 microm), a slight histiocytic reaction and bone formation were observed. Particles of alumina ceramics (3.9 microm) and titanium alloy (3.5 microm) which were in phagocytosable sizes also had few histiocytic reactions. Statistically, the material difference was more strongly related to the histiocyte reaction than to the particle size and calculated total surface area of particles. Our findings demonstrate that particles of different biomaterials and in different sizes induce different foreign-body histological reactions.

Pharmacological effects of SA96 (bucillamine) and its metabolites as immunomodulating drugs--the disulfide structure of SA-96 metabolites plays a critical role in the pharmacological action of the drug.

SA96 (generic name, bucillamine) is a disease-modifying anti-rheumatoid arthritis (RA) drug with immunological effects. This compounds has two sulfhydryl groups in its molecule, and the differences and similarities between this drug and D-penicillamine, which is also a sulfhydryl group-containing anti-rheumatic drug, have frequently been discussed. To clarify the pharmacological differences between these two drugs, we examined the concentrations of the compounds and its metabolites in serum and synovial fluid, paying special attention to the metabolites of SA96 produced in vivo. SA96 was metabolized in a very short time to SA981 which is a disulfide compound formed by intramolecular binding of two sulfhydryl groups, and transferred to synovial fluid. In addition SA981 had significant suppressive effects on IL-6 and IL-8 production by synovial cells in vitro. These results demonstrate that SA96, which has two sulfhydryl groups, exhibits anti-rheumatic effects via a pharmacological action clearly different from that of D-penicillamine.

Auditory evoked off-response: its source distribution is different from that of on-response.

Offset auditory responses were investigated by electroencephalography mainly in the 1970s, but since then no particular attention has been paid to them. Among the studies using magnetoencephalography (MEG) devices there are, to our knowledge, only three studies of the auditory off-response, and no significant variance has ever been observed between the source locations of on- and off-responses elicited from pure tones. We measured auditory evoked magnetic fields (AEFs) to various frequency pure tone stimulation in 5 healthy subjects with a 122-channel helmet-shaped magnetometer, and compared the distributions of the source locations of auditory N100m-Off (magnetic off-response around 100 ms) with those of N100m-On. Their spatial distributions were quite close to each other, and yet they were significantly different.

Vitamin A deficiency reduces the responsiveness of pineal gland to light in Japanese quail (Coturnix japonica).

Synthesis of melatonin in pineal gland is under the control of light environment. The recent finding of the presence of rhodopsin-like photopigment (pinopsin) and retinal in the avian pinealocytes has led to a hypothesis that vitamin A is involved in photoresponses of the pineal gland. We have thus analyzed the effect of vitamin A deficiency on the regulatory system of melatonin synthesis in the pineal gland of Japanese quail. Depletion of vitamin A from Japanese quails was attained by feeding them with a vitamin A-free diet supplemented with retinoic acid. In the vitamin A-deficient birds, diurnal rhythm in melatonin production persisted such that the phase of the wave was similar to that seen in the control birds. However, the amplitude of the nighttime surge of pineal melatonin was damped by vitamin A deficiency. When the control birds were briefly exposed to light at night, pineal melatonin dropped to the daytime level. In contrast, only slight decrease was observed in the vitamin A-deficient quails. The light responsiveness was restored after feeding the vitamin A-deficient quails with the control diet for 1 week. These results indicate that vitamin A plays essential roles in maintaining sufficient responsiveness of the avian pineal gland to photic input.

Histamine release from the hypothalamus induced by gravity change in rats and space motion sickness.

Freely moving rats were exposed to 2 g hypergravity in an animal centrifuge device to produce motion sickness. Histamine release from the anterior hypothalamus of the rats was measured in vivo with a microdialysis technique. After a 2-h load of 2 g hypergravity, rats ate kaolin. Because pica, eating a nonnutritive substance such as kaolin, is a behavioral index of motion sickness in rats, this finding indicates that the rats suffered from motion sickness. During 2 g hypergravity for 2-h, histamine release from the hypothalamus was transiently increased. In contrast, neither the transient increase of histamine release nor the kaolin consumption were induced by 2 g hypergravity in bilaterally labyrinthectomized rats. Pretreatment with alpha-fluoromethylhistidine, an inhibitor of histamine-synthesizing enzyme, decreased both the basal and hypergravity-induced releases of histamine from the hypothalamus and suppressed the kaolin consumption induced by hypergravity. Taken together, these findings suggest that the vestibular information of changes in gravity activate the histaminergic neuron system, resulting in the development of motion sickness. More prolonged stimulation, a 4-h load of 2 g hypergravity, induced significant increase of kaolin consumption on postdays 1-3, though rats ate kaolin on postdays 1-2 after 2 g hypergravity for 2 h. During 2 g hypergravity for 4 h, the initial transient increase of histamine release was followed by the gradual increase of histamine release after the end of centrifugation. It is suggested that rats adapted to the hypergravity environment after centrifugation for 4 h, but not 2 h, so that the change in gravity from 2 g to 1 g became a provocative stimulation. We, therefore, concluded that motion sickness in rats induced by a negative change in gravity can be used as a simulation of space motion sickness, which is induced by exposure to microgravity. Histaminergic activation in the development of motion sickness induced by negative change in gravity might be an underlying mechanism of space motion sickness.