Safety evaluation of arabinase (arabinan endo-1,5-α-L-arabinanase) from Aspergillus tubingensis.

Arabinase is an enzyme recognized for its ability to degrade arabinan, a plant cell wall constituent. It has been applied in the food industry most commonly for juice processing. One commercial source of arabinase is Aspergillus tubingensis (A. tubingensis), a black Aspergillus species. Given the intended use in food for human consumption, and noting its potential presence at trace levels in finished products, a series of safety studies including in vitro Ames and chromosome aberration assays, in vivo mammalian erythrocyte micronucleus and alkaline comet assays, and a 90-day rat oral toxicity study were conducted. No test article-related mutagenic activity was observed in the Ames assay. Although positive activity was observed in the chromosome aberration assay, this was not replicated in the in vivo genotoxicity assays including in preabsorptive cells. In the subchronic toxicity study, no test article-related adverse effects were observed following oral administration of arabinase at doses of 15.3, 153, or 1,530 mg total organic solids (TOS)/kg body weight/day to Sprague Dawley rats. The no-observed-adverse-effect level was considered to be the highest dose tested (1,530 mg TOS/kg body weight/day). The results of the genotoxicity studies and the subchronic toxicity study support the safe use of arabinase from A. tubingensis in food production.

Safety evaluation of nuclease P1 from Penicillium citrinum.

Nuclease P1 has been widely used in the food industry to enhance or create flavor. One commercial source of this enzyme is Penicillium citrinum, an anamorphic mesophilic fungus with a long history of safe use in Europe and Asia as a fermentation organism used in the production of ribonucleases. Given the intended use in food for human consumption, and noting its potential presence at trace levels in finished products, a series of safety studies including an in vitro Ames and chromosome aberration assay, an in vivo rat erythrocyte micronucleus assay and a 90-day oral toxicity study in rats were conducted. No mutagenic activity was observed in the Ames assay. Equivocal activity in the chromosome aberration assay was not replicated in the micronucleus assay at doses of up to 1007 mg total organic solids (TOS)/kg body weight (bw)/day. Following oral administration of nuclease P1 at dosages of 10.1, 101 or 1007 mg TOS/kg bw/day to Sprague-Dawley rats, no adverse effects on any study parameter were observed. The no-observed-adverse-effect level was considered to be 1007 mg TOS/kg bw/day. The results of the genotoxicity studies and subchronic rat study support the safe use in food production of nuclease P1 produced from P. citrinum.

Safety evaluation of AMP deaminase from Aspergillus oryzae.

Adenosine-5'-monophosphate (AMP) deaminase is an enzyme used to increase concentrations of 5'-inosine monophosphate in certain foods and beverages for flavoring purposes. One commercial source of this enzyme is Aspergillus oryzae, a filamentous fungus with a history of safe use in Asia as a fermentation organism used in the production of miso sauce and sake liquors. Noting the use of the enzyme in food intended for human consumption and potential presence at trace levels in finished goods, a series of safety studies including an in vitro Ames test and chromosome aberration assay with Chinese hamster lung fibroblasts were conducted along with a 90-day oral toxicity study in rats. AMP deaminase showed no evidence of genotoxicity in the in vitro tests. Following gavage administration of Sprague-Dawley rats at dosages of 19.8, 198.4, or 1984 mg total organic solids (TOS)/kg body weight (bw)/day for 90 days, no adverse effects on body weight gain, food consumption, hematology, clinical chemistry, urinalysis, ophthalmological and histopathological examinations were observed. The no-observed-adverse-effect level was considered to be 1984 mg TOS/kg bw/day, the highest dose tested. Results of the genotoxicity studies and subchronic rat study support the safe use of AMP deaminase produced from A. oryzae in food production.

Immunohistochemical evidence for the existence of novel mammalian neuropeptides related to the Hydra GLW-amide neuropeptide family.

The GLW-amide family is a neuropeptide family found in cnidarian species and is characterized by the C-terminal amino acid sequence -Gly-Leu-Trp-NH(2). To detect mammalian peptides structurally related to the GLW-amide family, we examined rat brain by immunohistochemistry with an anti-GLW-amide antibody. GLW-amide-like immunoreactivity (GLW-amide-LI) was observed in thin varicose fibers in some regions of the brain. Most neurons showing GLW-amide-LI were observed in the laterodorsal tegmental nucleus, pedunculopontine tegmental nucleus, and trigeminal/spinal ganglia. These results strongly suggest that the rat nervous system contains as yet unidentified GLW-amide-like peptides, and that GLW-amide-LI in the brain is a good marker for ascending projections from mesopontine cholinergic neurons.

Modification of AMPA receptor properties following environmental enrichment.

Environmental enrichment results in many modifications in the brain such as structural, behavioural, and biochemical changes. alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type receptors for excitatory amino acid glutamate are recently found to be involved in neuronal plasticity. In this study, we examined whether environmental enrichment modified the brain expression of mRNA for subunit composition of AMPA receptors in adult mice using the real-time quantitative PCR method and western blotting. Mice housed in enriched environments showed significantly higher levels of GluR2 and GluR4 subunits in the hippocampus compared to control mice. We concluded that environmental enrichment can change the expression of AMPA receptor subunits and thus might modify the potentials of brain plasticity.

Developmental regulation of activated ERK expression in the spinal cord and dorsal root ganglion of the chick embryo.

Mitogen-activated protein kinases (MAPKs) are involved in the intracellular pathways that respond to various extracellular signals. Extracellular signal-regulated kinase (ERK) is a member of MAPKs and has various functions in neural development. However, the in vivo distribution of the activated form of ERK (p-ERK) in the developing nervous system is not well understood. Here, we investigated the expression of p-ERK in the spinal cord and dorsal root ganglion (DRG) of chick embryos. In the spinal cord, p-ERK-positive cells appeared in the ventral ventricular zone on embryonic day 4 (E4). From E6 onward, they appeared in the gray matter and in the white matter, suggesting migration from the ventricular zone. A double labeling method revealed that these p-ERK-positive cells included oligodendrocyte precursors. In the dorsal horn, p-ERK-positive small cells appeared on E6. Subsequently, the positive cells in the dorsal horn increased transiently in number and then decreased markedly by E10. Motoneurons also expressed p-ERK transiently on E7. In the DRG, weak p-ERK immunoreaction appeared in the ventrolateral region on E5. From E6, the immunoreactivity became stronger and by E9 intense p-ERK-positive cells were observed throughout the DRG. These data provide a neuroanatomical framework to begin to examine the in vivo role of ERK in neural development.

Expression of serotonin2A receptors in Purkinje cells of the developing rat cerebellum.

Previous physiological and pharmacological studies have shown that the serotonin2A (5-HT2A) receptor is involved in cerebellar functions. However, the expression of 5-HT2A receptors in the developing cerebellum has not been elucidated to date. In the present immunohistochemical study, we examined developmental changes of the distribution of 5-HT2A receptors in Purkinje cells of the rat cerebellum from embryonic day 18 (E18) to postnatal day 21 (P21). The weak immunoreaction to 5-HT2A receptors was found in the deep cerebellar nuclei on E19. In the cerebellar cortex of the hemisphere and the posterior vermis, somata of Purkinje cells became weakly immunoreactive on P0. With the dendritic elongation and arborization, the immunoreaction appeared in the proximal parts of Purkinje cell dendrites. Distal parts of the dendrites became immunoreactive after P12, and were strongly immunolabeled by P21. The present study may provide a structural basis to investigate the roles of 5-HT2A receptors during the cerebellar development.

Lamina-selective changes in the density of synapses following perturbation of monoamines and acetylcholine in the rat medial prefrontal cortex.

The rat medial prefrontal cortex is known to have diverse brain functions such as learning and memory, attention, and behavioral flexibility. Although these functions are affected by monoamines (dopamine (DA), noradrenaline (NA) and serotonin (5-HT)) and acetylcholine (ACh), the detailed mechanisms remain unclear. These neuromodulators also have effects on synapse formation and maintenance, and regulate plasticity in the central nervous system (CNS). To clarify the effects of these neuromodulators on changes in the density of synapses in the rat medial prefrontal cortex, we separately administered a D1- or D2-antagonist, NA neurotoxin, 5-HT synthetic inhibitor, or muscarinic ACh antagonist for 1 week, and counted the number of synapses on electron microscopic photographs taken from the prelimbic area of the medial prefrontal cortex. The density of synapses in lamina I was regulated by DA via D1-like receptors, and that in laminae II/III was decreased by depletion of NA or ACh. However, 5-HT did not have a regulatory effect on the synaptic density throughout the layers in this brain region. The data in this study and our previous studies indicate that there are appreciable regional differences in the magnitude of biogenic amine-mediated synaptic plasticity in the rat CNS. These neuromodulators may have a trophic-like effect on the selected neuronal circuit to maintain synaptic contacts in the rat CNS. The synaptic density in the medial prefrontal cortex regulated by monoamines and ACh could be important not only for synaptic plasticity in this region but also for pharmacotherapeutic drug treatment.

Serum neurotrophin concentrations in autism and mental retardation: a pilot study.

To evaluate the availability of the serum neurotrophins for the diagnosis of the patients with neurodevelopmental disorder, we measured the serum concentration of brain-derived neurotrophic factor (BDNF) and neurotrophin-4 (NT-4) in the patients diagnosed with autism (n=18) and mental retardation (n=20), or healthy controls (n=16), using enzyme-linked immunosorbent assay. There tended to be a higher concentration of serum BDNF found in the autistic group ( P <0.05 by analysis of variance (ANOVA)) and the mental retardation group ( P <0.001 by ANOVA) compared to the control group. Serum NT-4 concentration tended to be increased in the mental retardation group (P <0.05 by ANOVA). We conclude that measuring the serum concentration of two neurotrophins, BDNF and NT-4, might be helpful to diagnose or classify disorders such as autism or mental retardation.

Developmental regulation of notochord-derived repulsion for dorsal root ganglion axons.

During the initial stages of development, the notochord provides repulsive signals for dorsal root ganglion (DRG) axons via semaphorin 3A/neuropilin-1, axonin-1/SC2, and other unknown repulsive molecules. The notochord is known to produce aggrecan, one of the chondroitin sulfate proteoglycans (CSPGs). We report here that adding aggrecan to the culture medium cannot only induce DRG growth cone collapse, but also inhibit DRG axonal growth. Using cocultures composed of tissues derived from chick embryos or neuropilin-1-deficient mice treated with chondroitinase ABC, we show the direct evidence that CSPGs are involved in notochord-derived repulsion for DRG axons. At later developmental stages, CSPGs are involved in perinotochordal sheath-derived axon repulsion, but not in notochord core-derived repulsion. We further demonstrate that TAG-1/axonin-1/SC2 is not involved in mediating repulsive activities by CSPGs, but is required for notochord core-derived axon repulsion. Thus, notochord-derived multiple axon repulsions act in a spatiotemporal-specific manner to shape the initial trajectories of DRG axons.

Regulation of dendrite formation of Purkinje cells by serotonin through serotonin1A and serotonin2A receptors in culture.

Serotonergic fibers and receptors appear in the rat cerebellum during early postnatal development. In the present study, we investigated the actions of serotonin (5-HT) and its receptors in the dendrite formation of Purkinje cells in organotypic cultures of anterior and posterior lobes of the cerebellum at postnatal day 7. In anterior lobes after 4 days in vitro (4DIV), the dendritic areas and branchings of Purkinje cells were increased by the treatment of 2 microM 5-HT, but decreased by 20 microM 5-HT. In posterior lobes after 4DIV, the dendritic areas of Purkinje cells were increased by 5-HT (2, 20 and 200 microM). In contrast, 5-HT treatment decreased dendritic areas of Purkinje cells in both anterior and posterior lobes after 7DIV. Next, we determined the actions of specific 5-HT receptors in mediating the effects of 5-HT by treatment with selective 5-HT receptor agonists. In anterior lobes after 4DIV, dendritic areas of Purkinje cells were increased by a 5-HT1A receptor agonist (8-OH-DPAT), whereas decreased by a 5-HT2A receptor agonist (DOI). The present study suggested that the dendrite formation of Purkinje cells is promoted by 5-HT through 5-HT1A receptors, but inhibited by 5-HT through 5-HT2A receptors.

P0 mRNA expression increases during gradual nerve elongation in adult rats.

Leg lengthening with nerve elongation is a common clinical treatment. We investigated morphological and molecular changes in peripheral nerves associated with femoral lengthening using animal models. Sciatic nerves of 13 week old male Wistar rats (n = 35) were elongated indirectly by leg lengthening for 14 days at 1 mm/day. At 3, 7, 14, 21, and 35 days following initiation of elongation, sciatic nerves on the elongated side and contralateral (control) side were excised at the midpoint of the femur. Internodal length was increased by 17%. Light and electron microscopic observation of transverse sections at 14 days showed elongated nerves appearing similar to control nerves with no degenerating axons and normal myelin thickness. We next examined changes of mRNA expression of a major myelin glycoprotein, P0, in elongated nerves using a quantitative reverse transcription-polymerase chain reaction and in situ hybridization. P0 mRNA expression in elongated nerves was increased during the first 3 weeks, with expression reaching 160% of control nerve expression at 14 days. Results of in situ hybridization were confirmatory. We concluded that myelin synthesis occurred during gradual nerve elongation. In adulthood, Schwann cells retain ability to synthesize myelin in response to nerve stretching.

Association between serotonin transporter gene polymorphism and chronic fatigue syndrome.

Interaction between the hypothalamo-pituitary-adrenal axis and the serotonergic system is thought to be disrupted in chronic fatigue syndrome (CFS) patients. We examined a serotonin transporter (5-HTT) gene promoter polymorphism, which affects the transcriptional efficiency of 5-HTT, in 78 CFS patients using PCR amplification of the blood genomic DNA. A significant increase of longer (L and XL) alleic variants was found in the CFS patients compared to the controls both by the genotype-wise and the allele-wise analyses (both p<0.05, by chi(2) test and Fisher's exact test). Attenuated concentration of extracellular serotonin due to longer variants may cause higher susceptibility to CFS.

Differential non-target-derived repulsive signals play a critical role in shaping initial axonal growth of dorsal root ganglion neurons.

Initial trajectories of dorsal root ganglion (DRG) axons are shaped by chemorepulsive signals from surrounding tissues. Although we have previously shown that axonin-1/SC2 expression on DRG axons is required to mediate a notochord-derived chemorepulsive signal, Dev. Biol. 224, 112-121), other molecules involved in the non-target-derived repulsive signals are largely unknown. Using coculture assays composed of tissues derived from the chick embryo or mutant mice treated with function-blocking antibodies and phosphatidylinositol-specific phospholipase C, we report here that the chemorepellent semaphorin 3A (Sema3A) and its receptor neuropilin-1 are required for mediating the dermamyotome- and notochord-derived, but not the ventral spinal cord-derived, chemorepulsive signal for DRG axons. The dermamyotome-derived chemorepulsion is exclusively dependent on Sema3A/neuropilin-1, whereas other molecules are also involved in the notochord-derived chemorepulsion. Chemorepulsion from the ventral spinal cord does not depend on Sema3A/neuropilin-1 but requires axonin-1/SC2 to repel DRG axons. Thus, differential chemorepulsive signals help shape the initial trajectories of DRG axons and are critical for the proper wiring of the nervous system.

Increased monoamine concentration in the brain and blood of fetal thalidomide- and valproic acid-exposed rat: putative animal models for autism.

Autism is defined as a congenital neurodevelopmental disorder in which serotonergic dysfunction may be involved in its pathogenesis. One of the characteristic laboratory findings in autistic patients is hyperserotonemia, although its mechanism has not been elucidated to date because of difficulties in studying human patients. Recent reports have demonstrated that thalidomide or valproic acid exposure during early embryonic days (first trimester) in humans causes higher incidence of autism. Morphologic abnormalities found in autism (e.g. cerebellar anomalies, reduced motor neuron numbers) have been reported in animals administered with these teratogens prenatally, suggesting the possibility of the use of these animals as an experimental autistic model. In this study, we evaluated monoamine levels in the brain and blood of rats exposed to teratogens prenatally. Of the groups exposed to thalidomide on embryonic day (E)2, E4, E7, E9, and E11, a significant increase of hippocampal serotonin was only observed in the group exposed on E9. Furthermore, E9 thalidomide and valproic acid exposure both resulted in an increase of hippocampal serotonin, frontal cortex dopamine, and hyperserotonemia. These results thus indicate that two potentially autism-inducing teratogens, thalidomide and valproic acid, have the same effect on early monoamine system development in the brain and the blood, which may explain the pathogenesis of autism.

Repeated cocaine administration differentially affects NMDA receptor subunit (NR1, NR2A-C) mRNAs in rat brain.

We investigated the effects of intermittent intraperitoneal (i.p.) injections of cocaine (20 mg/kg) on subunit mRNAs of N-methyl-D-aspartate (NMDA) receptors (NR1/NR2A-2C) in the rat brain by in situ hybridization using phosphor screen analysis. The level of NR1 subunit mRNA significantly increased in hippocampal complexes 1 h after a single i.p. injection of cocaine. After repeated cocaine injection, the mean scores of stereotyped behavior were increased with the number of injections. The level of NR1 subunit mRNA was obviously decreased in the striatum and cortices 24 h (early withdrawal) after a final injection following 14 days of subchronic administration. During the early withdrawal period, the amount of the NR1 subunit decreased in the nucleus accumbens, globus pallidus, and subiculum. In the dentate gyrus, the NR1 mRNA level significantly increased during early withdrawal in rats subchronically treated with cocaine. Levels of NR2B subunit mRNA were reduced in the cortices and striatum. During late withdrawal from cocaine, the level of NR2C subunit mRNA in the cerebellum was also reduced. These findings suggest that the disruption of NR1, NR2B, and NR2C subunits in the discrete brain regions occurs under the cocaine-related behavioral abnormalities and would be closely implicated in the initiation and expression of behavioral sensitization induced by repeated cocaine administration. Further studies on the changes in non-NMDA receptors are required to elucidate the biological significance of glutamate receptors for the mechanisms underlying the development of behavioral sensitization.

Runx3 controls the axonal projection of proprioceptive dorsal root ganglion neurons.

Dorsal root ganglion (DRG) neurons specifically project axons to central and peripheral targets according to their sensory modality. The Runt-related genes Runx1 and Runx3 are expressed in DRG neuronal subpopulations, suggesting that they may regulate the trajectories of specific axons. Here we report that Runx3-deficient (Runx3(-/-)) mice displayed severe motor uncoordination and that few DRG neurons synthesized the proprioceptive neuronal marker parvalbumin. Proprioceptive afferent axons failed to project to their targets in the spinal cord as well as those in the muscle. NT-3-responsive Runx3(-/-) DRG neurons showed less neurite outgrowth in vitro. However, we found no changes in the fate specification of Runx3(-/-) DRG neurons or in the number of DRG neurons that expressed trkC. Our data demonstrate that Runx3 is critical in regulating the axonal projections of a specific subpopulation of DRG neurons.

Repeated cocaine administration increases GABA(B(1)) subunit mRNA in rat brain.

The effects of a single and repeated administration of cocaine on GABA(B) receptor subunit mRNA was investigated in rat brain by in situ hybridization. Following a single administration of cocaine, no significant change was observed in any brain regions examined, neither 1 h nor 24 h after administration. During repeated administration of cocaine, behavioral sensitization with increased stereotyped behavior was observed. A significant increase in the level of GABA(B(1)) mRNA was observed in the nucleus accumbens (11.4%), CA1 field of the hippocampus (16.8%), and thalamus (16.5%) 1 day after repeated administrations of cocaine for 14 consecutive days. The level of mRNA returned to the basal level 1 week after the final injection of repeated cocaine treatment. The observed changes in the mRNA level after the repeated cocaine may imply changes of GABA(B(1)) subunit in molecular mechanisms which underlie development of behavioral sensitization.

An enriched environment increases noradrenaline concentration in the mouse brain.

Exposure to an enriched environment has been shown to have many positive effects on brain structure and function. In the present study, we examined the effects of environmental enrichment on monoaminergic neurons in the mouse brain. After being exposed to an enriched environment for 40 days, noradrenaline content was increased significantly in the parieto-temporo-occipital cortex, the cerebellum and the pons/medulla oblongata. In contrast, no changes were observed in serotonin or dopamine levels in these same regions.