Microcystin-LR-induced epithelial-mesenchymal transition-like cells acquire resistance to multi-toxins.

The protein phosphatase inhibitor microcystin-LR (MC-LR), a hepatocyte-selective cyanotoxin, induces phenotypic changes in HEK293 OATP1B3-expressing (HEK293-OATP1B3) cells, which include cytoskeletal reorganization (HEK293-OATP1B3-AD) and anoikis resistance (HEK293-OATP1B3-FL) transformed cells, respectively. These cells acquire resistance to MC-LR and partial epithelial-mesenchymal transition (EMT) characteristics. In cancer cells, EMT is generally involved in multi-drug resistance. Here, we focused on the multi-drug resistance of HEK293-OATP1B3-AD and HEK293-OATP1B3-FL cells. The MTT assay and immunoblotting were conducted to examine the responses of HEK293-OATP1B3, HEK293-OATP1B3-AD, and HEK293-OATP1B3-FL cells to multiple toxins and drugs that function as substrates for OATP1B3, including MC-LR, nodularin (Nod), okadaic acid (OA), and cisplatin (CDDP). HEK293-OATP1B3-AD and HEK293-OATP1B3-FL cells were more resistant to MC-LR, Nod, and OA than HEK293-OATP1B3 cells. Conversely, the three cell types were equivalently sensitive to CDDP. By using protein phosphatase assay, the reduction of the inhibitory effect of MC-LR and Nod on phosphatase activity might be one reason for the resistance to MC-LR and Nod in HEK293-OATP1B3-AD and HEK293-OATP1B3-FL cells. Furthermore, the parental HEK293-OATP1B3 cells showed enhanced p53 phosphorylation and stabilization after MC-LR exposure, while p53 phosphorylation was attenuated in HEK293-OATP1B3-AD and HEK293-OATP1B3-FL cells. Moreover, in HEK293-OATP1B3-AD and HEK293-OATP1B3-FL cells, AKT phosphorylation was higher than that of the parental HEK293-OATP1B3 cell line. These results suggest that the multi-toxin resistance observed in HEK293-OATP1B3-AD and HEK293-OATP1B3-FL cells is associated with AKT activation and p53 inactivation.

Effects of sleep hygiene education and behavioral therapy on sleep quality of white-collar workers: a randomized controlled trial.

Because poor sleep quality can reduce quality of life and increase prevalence of illness in workers, interventions are becoming increasingly important for businesses. To evaluate how sleep quality is affected by one-on-one behavioral modification when combined with group education, we conducted a randomized, controlled trial among day-shift white-collar employees working for an information-technology service company in Japan. Participants were randomly allocated to groups receiving either sleep hygiene group education (control group), or education combined with individual sleep modification training (one-on-one group). Occupational health professionals carried out both procedures, and sleep quality was assessed using the Pittsburgh Sleep Quality Index (PSQI). PSQI scores were obtained before and after the intervention period, and changes in scores were compared across groups after adjustments for age, gender, job title, smoking and drinking habits, body-mass index, and mental health as assessed using K6 scores. The average PSQI score for the control group decreased by 0.8, whereas that of the one-on-one group decreased by 1.8 (difference of 1), resulting in a significantly greater decrease in score for the one-on-one group (95% confidence interval: 0.02 to 2.0). These results show that, compared to sleep hygiene group education alone, the addition of individual behavioral training significantly improved the sleep quality of workers after only three months.

Randomized controlled trial on the effects of a combined sleep hygiene education and behavioral approach program on sleep quality in workers with insomnia.

To evaluate the effects of a combined sleep hygiene education and behavioral approach program on sleep quality in workers with insomnia, we conducted a randomized controlled trial at a design engineering unit in Japan. Employees evaluated for insomnia by the Athens Insomnia Scale (≥6 points) were divided into an intervention and control group. The intervention group received a short-term intervention (30 min) program that included sleep hygiene education and behavioral approaches (relaxation training, stimulus control, and sleep restriction) performed by occupational health professionals. We calculated differences in change in Pittsburgh Sleep Quality Index (PSQI) scores between the two groups from baseline to three months after the start of intervention after adjusting for gender, age, job title, job category, average number of hours of overtime during the study period, marital status, smoking habit, average number of days of alcohol consumption per week, exercise habits, K6 score, and baseline PSQI score. Results showed that the average PSQI score decreased by 1.0 in the intervention group but increased by 0.9 in the control group. Additionally, the difference in variation between the two groups was 1.9 (95% confidence interval: 0.6 to 3.4), which was significant. Taken together, these results indicate that the intervention program significantly improved the sleep quality of workers with insomnia.

Alterations of interneurons in the striatum and frontal cortex of mice during postnatal development.

We investigated the postnatal alterations of neuronal nuclei (NeuN)-positive neurons, parvalbumin (PV)-positive interneurons, neuronal nitric oxide synthase (nNOS)-positive interneurons, and neurotrophic factors in the mouse striatum and frontal cortex using immunohistochemistry. NeuN, PV, nNOS, nerve growth factor (NGF), and brain-derived neurotrophic factor (BDNF) immunoreactivity were measured in 1-, 2-, 4- and 8-week-old mice. Total number of NeuN-positive neurons was unchanged in the mouse striatum and frontal cortex from 1 up to 8 weeks of age. In contrast, a significant decrease in the number of PV-positive interneurons was observed in the striatum and frontal cortex of 1-, 2- and 4-week-old mice. Furthermore, a significant increase of nNOS-positive interneurons was found in the striatum and frontal cortex of 1- and/or 2-week-old mice. NGF-positive neurons were unchanged in the mouse striatum from 1 up to 8 weeks of age. In the frontal cortex, a significant increase in the number of NGF-positive neurons was observed only in 1-week-old mice. In contrast, a significant increase in the number of NGF-positive glia 1 cells was found in the striatum and frontal cortex of 4-week-old mice. Our double-labeled immunostaining showed that nNOS immunoreactivity was not found in PV-immunopositive interneurons. Furthermore, BDNF immunoreactivity was observed in both nNOS-positive and PV-positive interneurons in the striatum of 1- or 2-week-old mice. These results show that the maturation of nNOS-immunopositive interneurons precedes the maturation of PV-immunopositive interneurons in the striatum and frontal cortex during postnatal development. Furthermore, our results demonstrate that the expression of BDNF may play some role in the maturation of interneurons in the striatum and frontal cortex during postnatal development. Moreover, our findings suggest that the expression of NGF in glia cells may play some role in the maturation of glial cells and PV-positive interneurons in the striatum and frontal cortex during postnatal development.

Postnatal development of neurons, interneurons and glial cells in the substantia nigra of mice.

We investigated postnatal alterations of neurons, interneurons and glial cells in the mouse substantia nigra using immunohistochemistry. Tyrosine hydroxylase (TH), neuronal nuclei (NeuN), parvalbumin (PV), neuronal nitric oxide synthase (nNOS), glial fibrillary acidic protein (GFAP), ionized calcium-binding adaptor molecule 1 (Iba 1), CNPase (2',3'-cyclic nucleotide 3'-phosphodiesterase), brain-derived neurotrophic factor (BDNF) and glial cell-line-derived neurotrophic factor (GDNF) immunoreactivity were measured in 1-, 2-, 4- and 8-week-old mice. In the present study, the maturation of NeuN-immunopositive neurons preceded the production of TH in the substantia nigra during postnatal development in mice. Furthermore, the maturation of nNOS-immunopositive interneurons preceded the maturation of PV-immunopositive interneurons in the substantia nigra during postnatal development. Among astrocytes, microglia and oligodendrocytes, in contrast, the development process of oligodendrocytes is delayed in the substantia nigra. Our double-labeled immunohistochemical study suggests that the neurotrophic factors such as BDNF and GDNF secreted by GFAP-positive astrocytes may play some role in maturation of neurons, interneurons and glial cells of the substantia nigra during postnatal development in mice. Thus, our findings provide valuable information on the development processes of the substantia nigra.

Alterations of glial cells in the mouse hippocampus during postnatal development.

We investigated the postnatal alterations of neurons, astrocyte, oligodendrocyte, and microglia in the mouse hippocampal CA1 sector and dentate gyrus under the same conditions using immunohistochemistry. Neuronal nuclei (NeuN), Glial fibrillary acidic protein (GFAP), 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNPase), and ionized calcium binding adaptor molecule 1 (Iba 1) immunoreactivity were measured in 1-, 2-, 4-, and 8-week-old mice. Total number of NeuN-positive neurons was unchanged in the mouse hippocampal CA1 sector and dentate gyrus from 1 to 8 weeks of birth. In contrast, a significant increase in the number of GFAP-positive astrocytes was observed only in the hippocampal CA1 sector of 1-week-old mice when compared with 8-week-old animals. Thereafter, total number of GFAP-positive astrocytes was unchanged in the hippocampal CA1 sector and dentate gyrus from 2 to 8 weeks of birth. For microglia, a significant increase in the number of Iba 1-positive microglia was observed in the hippocampal CA1 sector and dentate gyrus of 1-, 2-, and 4-week-old mice as compared with 8-week-old animals. On the other hand, a significant decrease in the area of expression of CNPase-positive fibers was observed in the hippocampal CA1 sector of 1- and 2-week-old mice as compared with 8-week-old animals. In dentate gyrus, a significant decrease in the area of expression of CNPase-positive fibers was found in 1-, 2-, and 4-week-old mice. Furthermore, our double-labeled immunostaining showed that brain-derived neurotrophic factor (BDNF) immunoreactivity was observed in GFAP-positive astrocytes and Iba 1-positive microglia in the hippocampal CA1 sector and dentate gyrus of 1- and 2-week-old mice. These results show that glial cells may play some role in the maintenance and neuronal functions of hippocampal CA1 pyramidal neurons and granule cells of dentate gyrus during postnatal development. Furthermore, our results demonstrate that glial BDNF may play an important role in the maturation of oligodendrocyte in the hippocampal CA1 sector and dentate gyrus during postnatal development. Thus, our findings provide valuable information on the developmental processes.

Age-related changes of calcineurin and Akt1/protein kinase Balpha (Akt1/PKBalpha) immunoreactivity in the mouse hippocampal CA1 sector: an immunohistochemical study.

We investigated the age-related alterations of calcineurin and Akt1/protein kinase Balpha (Akt1/PKBalpha) immunoreactivity in the mouse hippocampal CA1 sector using immunohistochemistry. Calcineurin and Akt1/PKBalpha immunoreactivity was measured in 2-, 8-, 18-, 40-42- and 50-59-weeks-old animals. Diffuse calcineurin immunoreactivity was evident in pyramidal neurons of the hippocampal CA1 sector of 8-weeks-old mice. Densities of calcineurin immunoreactivity were lowered significantly in the hippocampal CA1 neurons of 2-weeks-old mice. In contrast, densities of calcineurin immunoreactivity were unchanged in the hippocampal CA1 neurons up to 40-42-weeks-old mice. However, densities of calcineurin immunoreactivity were increased significantly in the dendrites and plasma membranes of the hippocampal CA1 neurons of 50-59-weeks-old mice compared to 8-weeks old animals. Akt1/PKBalpha immunoreactivity was slightly detectable in the hippocampal CA1 sector of 8-weeks-old mice. A weak Akt1/PKBalpha immunoreactivity was found in cytoplasm of the hippocampal CA1 neurons and glial cells. Densities of Akt1/PKBalpha immunoreactivity were unchanged in the hippocampal CA1 neurons and glial cells of 2-weeks-old mice. In contrast, densities of Akt1/PKBalpha immunoreactivity were increased significantly in cytoplasm of neurons and glial cells of the hippocampal CA1 sector from 40-42 to 50-59 weeks after birth. The present study indicates that densities of calcineurin immunoreactivity and number of Akt1/PKBalpha immunoreactive cells were increased significantly in the hippocampal CA1 sector during aging processes. Our study also demonstrates that the activation of Akt1/PKBalpha signaling pathway may act defense mechanism against the neuronal dysfunction of the hippocampal CA1 sector caused by the activation of calcineurin signaling pathway during aging processes. These findings suggest that the calcineurin and Akt1/PKBalpha signaling pathway may be important targets for the development of novel therapeutic strategies for protection against age-related neurodegeneration.

Age-related changes of NGF, BDNF, parvalbumin and neuronal nitric oxide synthase immunoreactivity in the mouse hippocampal CA1 sector.

We investigated the age-related alterations in nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), parvalbumin and neuronal nitric oxide synthase (nNOS) immunoreactivity of the mouse hippocampal CA1 sector. NGF and BDNF immunoreactivity was unchanged in the hippocampal CA1 pyramidal neurons from 2 to 50-59 weeks of birth. In contrast, a significant increase in the NGF and BDNF immunoreactivity was observed in glial cells of the hippocampal CA1 sector from 40-42 to 50-59 weeks of birth. On the other hand, the number of parvalbumin- and nNOS-positive interneurons was unchanged in the hippocampal CA1 sector during aging processes, except for a significant decrease of nNOS-positive interneurons 2 weeks of birth. Our results indicate that NGF and BDNF immunoreactivity was unaltered in the hippocampal CA1 pyramidal neurons during aging processes. In contrast, a significant increase in the NGF and BDNF immunoreactivity was observed in glial cells of the hippocampal CA1 sector during aging processes. The present study also shows that the number of parvalbumin- and nNOS-positive interneurons was unchanged in the hippocampal CA1 sector during aging processes, except for a significant decrease of nNOS-positive interneurons 2 weeks of birth. These results demonstrate that the expression of glial NGF and BDNF may play a key role for helping survival and maintenance of pyramidal neurons and neuronal functions in the hippocampal CA1 sector during aging processes. Furthermore, our findings suggest that parvalbumin- and nNOS-positive interneurons in the hippocampal CA1 sector are resistant to aging processes. Moreover, our findings suggest that nitric oxide synthesized by the nNOS may play some role for neuronal growth during postnatal development.

Relationship between turnover and periodic health check-up data among Japanese hospital nurses: a three-year follow-up study.

The present study explored the relationship between turnover and periodic health check-up data among Japanese hospital nurses. The subjects were 379 registered nurses in a Japanese hospital and the duration of our study period was three years. By the proportional hazard model, we investigated the relative risks (RRs) of the turnover rate associated with the following: lifestyles (irregularity of meals, taking breakfast), work environment (working hours, work schedule), and health status (body mass index [BMI], serum total cholesterol [T-cho], taking medication, sleep disturbance). We obtained the data on turnover from October, 1997 to September, 2000. During the observed period we were able to follow up on 363 female nurses (96%) and the turnover total amongst them was 100. We divided the subjects into a younger (21-25-yr-old) and an older (>25-yr-old) group because of the interaction between age and turnover. In the younger group, we found that sleep disturbance influenced turnover significantly. On the other hand, in the older nurses, BMI and T-cho contributed to turnover significantly. Our results imply the possibility of a partial association between turnover and periodic health check-up data among the nurses.

[Comparison of Work Ability Index and cognitive function tests].

The purpose of this study was to evaluate the effect of aging with regard to scores for certain cognitive function tests and WAI (Work Ability Index), and to examine the relationship between cognitive function test scores and work ability as measured by WAI. The subjects were 139 male employees of a factory producing steel plate, and their average age was 48.1 yr (SD 16.4). The WAI and cognitive function tests were conducted and valid scores were obtained from 134 subjects as to WAI, and from 88 subjects as to cognitive function tests. The subjects were divided into two groups: young workers (under 45 yr) and middle-aged to elderly workers (45 yr and over). The WAI scores of the two groups were compared, but no significant differences were observed. Nevertheless, for two WAI items, WAI-2 and WAI-7, the scores of the middle-aged to elderly worker group were significantly higher than those of the young worker group. In contrast, the scores for WAI-3 of the middle-aged to elderly group were significantly lower than those of the young worker group. The cognitive function test scores for the two groups were also compared. The scores for Working Memory test, Tracking test, and Sentence-to-sentence Comparison test of the middle-aged to elderly worker group were significantly lower than those of the younger group. Moreover, for the middle-aged to elderly worker group, the average WAI-3 scores for those with good cognitive function test results and those with poor cognitive function test results were compared, but there were no significant differences. This result shows that deterioration of physical function caused by aging is not related to deterioration of cognitive function caused by aging in the subjects of this study. The reason for this may be that the subjects are blue-collar workers, and thus cognitive functions are less important for their work.