Alteration of Neural Stem Cell Functions in Ataxia and Male Sterility Mice: A Possible Role of ß-Tubulin Glutamylation in Neurodegeneration.

Ataxia and Male Sterility (AMS) is a mutant mouse strain that contains a missense mutation in the coding region of Nna1, a gene that encodes a deglutamylase. AMS mice exhibit early cerebellar Purkinje cell degeneration and an ataxic phenotype in an autosomal recessive manner. To understand the underlying mechanism, we generated neuronal stem cell (NSC) lines from wild-type (NMW7), Nna1 mutation heterozygous (NME), and Nna1 mutation homozygous (NMO1) mouse brains. The NNA1 levels were decreased, and the glutamylated tubulin levels were increased in NMO1 cultures as well as in the cerebellum of AMS mice at both 15 and 30 days of age. However, total ß-tubulin protein levels were not altered in the AMS cerebellum. In NMO1 neurosphere cultures, ß-tubulin protein levels were increased without changes at the transcriptional level. NMO1 grew faster than other NSC lines, and some of the neurospheres were attached to the plate after 3 days. Immunostaining revealed that SOX2 and nestin levels were decreased in NMO1 neurospheres and that the neuronal differentiation potentials were reduced in NMO1 cells compared to NME or NMW7 cells. These results demonstrate that the AMS mutation decreased the NNA1 levels and increased glutamylation in the cerebellum of AMS mice. The observed changes in glutamylation might alter NSC properties and the neuron maturation process, leading to Purkinje cell death in AMS mice.

Effects of nicotine on odor-induced increases in regional blood flow in the olfactory bulb in rats.

The present study examined the effects of nicotinic acetylcholine receptor activation on the odor-induced blood flow response in the olfactory bulb. In urethane-anesthetized rats, odor stimulation (5% amyl acetate, 30 s) produced an increase in olfactory bulb blood flow (reaching 107% ± 3% of the pre-stimulus basal values), without changes in frontal cortical blood flow or mean arterial pressure. Intravenous injection of nicotine (30 µg/kg), a nicotinic acetylcholine receptor agonist, significantly augmented the odor-induced increase response of olfactory bulb blood flow, without changes in the basal blood flow level. The nicotine-induced augmentation of the olfactory bulb blood flow response to odor was negated by dihydro-ß-erythroidine, an α4ß2-preferring nicotinic acetylcholine receptor antagonist. Our results suggest that the activation of α4ß2-like neuronal nicotinic acetylcholine receptors in the brain potentiates an odor-induced blood flow response in the olfactory bulb.

Blood pressure-independent increase in the cortical cerebral blood flow induced by manual acupuncture of the auricular region in rats.

We aimed to determine whether acupuncture to the auricular region increases cortical regional cerebral blood flow (rCBF). The rCBF was measured using laser speckle contrast imaging in urethane-anesthetized rats. Acupuncture stimulation was performed manually at the auricular concha or abdomen. The former's stimulation significantly increased the rCBF of the bilateral cerebral cortex in the frontal, parietal, and occipital lobes without altering the systemic arterial pressure. In contrast, abdominal stimulation affected neither rCBF nor systemic arterial pressure. The increase in the rCBF was completely abolished by the severance of the somatic nerves that innervated the auricular region, comprising the trigeminal nerve, facial nerve, auricular branch of the vagal nerve, glossopharyngeal nerve, and great auricular nerve. Thus, application of acupuncture to the auricular region increases the rCBF without increasing arterial pressure.

Detection of vitellogenin incorporation into zebrafish oocytes by FITC fluorescence.

BACKGROUND: Large volumes of lymph can be collected from the eye-sacs of bubble-eye goldfish. We attempted to induce vitellogenin (Vtg) in the eye-sac lymph of bubble-eye goldfish and develop a method for visualizing Vtg incorporation by zebrafish oocytes using FITC-labeling. METHODS: Estrogen efficiently induced Vtg in the eye-sac lymph of goldfish. After FITC-labeled Vtg was prepared, it was injected into mature female zebrafish. RESULTS: Incorporation of FITC-labeled Vtg by zebrafish oocytes was detected in in vivo and in vitro experiments. The embryos obtained from zebrafish females injected with FITC-labeled Vtg emitted FITC fluorescence from the yolk sac and developed normally. CONCLUSION: This method for achieving Vtg incorporation by zebrafish oocytes could be useful in experiments related to the development and endocrinology of zebrafish oocytes.

Effects of 3,4-dichloroaniline on expression of ahr2 and cyp1a1 in zebrafish adults and embryos.

Arylhydrocarbon receptor (Ahr) and cytochrome P4501a1 (Cyp1a1) are members of the Ahr/Cyp1a1 pathway that oxygenates various toxic chemicals including aryl hydrocarbons. To elucidate Ahr/Cyp1a1 pathway responses in teleost fish tissues, we examined the effects of 3,4-dichloroaniline (3,4-DCA), a reference toxic compound known to activate the Ahr/Cyp1a1 pathway, on the expression of arh and cyp1a1 in zebrafish tissues and embryos by means of in situ hybridization (ISH). Our ISH analysis showed that cyp1a1 expression was markedly activated by 3,4-DCA in the gill and intestinal epithelia, skin epidermis, and liver parenchymal cells of adult zebrafish. Before differentiation of the gill, intestine, and liver, skin was the site of cyp1a1 activation in embryos. Unlike the cyp1a1 response, 3,4-DCA-mediated ahr activation was not marked in either adults or embryos, indicating a possibility that stable ahr transcripts persist in the cytoplasm of these cells to induce cyp1a1. Young oocytes (previtellogenic to early vitellogic stage) express ahr; however activation of cyp1a1 by 3,4-DCA was negligible in these oocytes, suggesting that ahr expression in oocytes is not directly linked to cyp1a1 activation. Based on our finding that skin epidermis up-regulates cyp1a1 in response to 3,4-DCA, we demonstrated that fin explants, which can be harvested without sacrificing fish, can be used as a standard for assaying cyp1a1 activation in addition to embryos that are now used.

BCR-ABL-independent and RAS / MAPK pathway-dependent form of imatinib resistance in Ph-positive acute lymphoblastic leukemia cell line with activation of EphB4.

OBJECTIVE: We investigated the mechanism responsible for imatinib (IM) resistance in Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph(+) ALL) cell lines. METHODS: We established cell lines from a patient with Ph(+) ALL at the time of first diagnosis and relapsed phase and designated as NPhA1 and NPhA2, respectively. We also derived IM-resistant cells, NPhA2/STIR, from NPhA2 under gradually increasing IM concentrations. RESULTS: NPhA1 was sensitive to IM (IC(50) 0.05 microm) and NPhA2 showed mild IM resistance (IC(50) 0.3 microm). NPhA2/STIR could be maintained in the presence of 10 microm IM. Phosphorylation of MEK and ERK was slightly elevated in NPhA2 and significantly elevated in NPhA2/STIR compared to NPhA1 cells. After treatment with IM, phosphorylation of MEK and ERK was not suppressed but rather increased in NPhA2 and NPhA2/STIR. Active RAS was also increased markedly in NPhA2/STIR after IM treatment. The expression of BCL-2 was increased in NPhA2 compared to NPhA1, but no further increase in NPhA2/STIR. Proliferation of NPhA2/STIR was significantly inhibited by a combination of MEK inhibitor and IM. Analysis of tyrosine phosphorylation status with a protein tyrosine kinase array showed increased phosphorylation of EphB4 in NPhA2/STIR after IM treatment. Although transcription of EphB4 was suppressed in NPhA1 and NPhA2 after IM treatment, it was not suppressed and its ligand, ephrinB2, was increased in NPhA2/STIR. Suppression of EphB4 transcripts by introducing short hairpin RNA into NPhA2/STIR partially restored their sensitivity to IM. CONCLUSIONS: These results suggest a new mechanism of IM resistance mediated by the activation of RAS/MAPK pathway and EphB4.

Motor activity and imagery modulate the body-selective region in the occipital-temporal area: a near-infrared spectroscopy study.

The extrastriate body area (EBA) lies in the occipital-temporal cortex and has been described as a "body-selective" region that responds when viewing other people's bodies. Recently, several studies have reported that EBA is also modulated when the subject moves or imagines moving their own body, even without visual feedback. The present study involved 3 experiments, wherein the first experiment was conducted to examine whether near-infrared spectroscopy (NIRS) could capture any activity in the EBA when viewing images of bodies. The second experiment was designed to elucidate whether this region also responds when the subjects move their own body, and the third to observe whether imagining carrying out a movement would activate EBA. Images of human bodies and chairs were used as the stimuli for the first experiment, simple hand movements carried out by the subject were used for the second and the act of imagining hand movements for the third. Our results confirmed that the region we defined as EBA was clearly activated when the subject viewed images of human bodies, carried out movements of their own body and imagined moving parts of their own body, thus demonstrating the usefulness of NIRS as a new brain imaging method. Moreover, we found a gender-based difference when imagining movement; male subjects showed a greater response than female subjects. This may reflect a gender difference in imagery skills; however, further research is needed to verify this hypothesis.

Conjugated bilirubin induces multidrug resistance-associated protein 2 mRNA expression and in vivo cisplatin resistance in rat hepatoma AH66 cells.

In vivo cisplatin resistance of rat ascites hepatoma AH66 cells is suggested to result from the induction of multidrug resistance-associated protein 2 (MRP2) expression by ascites fluid (ASF) in the peritoneal cavity. The in vitro cisplatin sensitivity of AH66 cells grown in assay medium containing 5% fetal bovine serum in Dulbecco's modified Eagle's medium (5% FBS DMEM) did not change when the cells were treated with probenecid, an inhibitor of anion transporters, while the decreased cisplatin sensitivity of AH66 cells cultured in an assay medium containing 5% ASF (5% ASF DMEM) was restored by probenecid. Furthermore, in an in vivo study, the survival span (%ILS) of AH66-bearing rats was markedly extended by combination therapy with cisplatin and probenecid, compared with either agent alone. The expression of MRP2 mRNA was increased when AH66 cells were cultured in medium containing 5% ASF or 5% bile for 24 h. The induction of MRP2 mRNA expression in AH66 cells was also observed in the presence of heat-denatured ASF. The bilirubin content in ASF was characteristically higher than that in FBS, normal rat serum or AH66-bearing rat serum. Unconjugated bilirubin did not change the expression of MRP2 mRNA, whereas conjugated bilirubin markedly increased it. The cisplatin uptake in AH66 cells after culture in 5% FBS DMEM containing conjugated bilirubin was about half that of the cells cultured in 5% FBS DMEM alone (p < 0.01). In addition, the cisplatin sensitivity of the cells was significantly lowered by the addition of conjugated bilirubin. The expression of MRP2 mRNA in rat normal hepatocytes was also increased after culture in medium containing 5% ASF or 5% bile. These results indicated that conjugated bilirubin, a component of ASF, induces the mRNA expression of MRP2, which is a determinant of the in vivo cisplatin resistance of AH66 cells.