Lysophosphatidylethanolamine in Grifola frondosa as a neurotrophic activator via activation of MAPK

We found that Grifola frondosa extracts induced the activation of mitogen-activated protein kinase (MAPK) in cultured PC12 cells, a line of rat pheochromocytoma cells. The active substance was isolated by a few chromatographic steps, including high-performance liquid chromatography, and was identified to be lysophosphatidylethanolamine (LPE) from various structural analyses. LPE from G. frondosa (GLPE) was confirmed to induce the activation of MAPK of cultured PC12 cells and was found to suppress cell condensation and DNA ladder generation evoked by serum deprivation, suggesting that the GLPE had antiapoptotic effects. Moreover, GLPE caused morphological changes in and upregulation of neurofilament M expression of PC12 cells, demonstrating that the GLPE could induce neuronal differentiation of these cells. The activation of MAPK by GLPE was suppressed by AG1478, an antagonist of epidermal growth factor receptor (EGFR), and by U0126, an inhibitor of MAPK kinase (MEK1/2), but not by K252a, an inhibitor of TrkA, or by pertussis toxin. These results demonstrate that GLPE induced the MAPK cascade [EGFR-MEK1/2-extracellular signal-regulated protein kinases (ERK1/2)] of PC12 cells, the activation of which induced neuronal differentiation and suppressed serum deprivation-induced apoptosis. This study has clarified for the first time the involvement of the MAPK signal cascade in LPE actions.     

Purification by p-aminobenzoic acid (PABA)-affinity chromatography and the functional reconstitution of the nateglinide/H+ cotransport system in the rat intestinal brush-border membrane

(-)-N-(trans-4-isopropylcyclohexanecarbonyl)-D-phenylalanine (nateglinide) is a novel oral hypoglycemic agent possessing a peptide-type bond and a carboxyl group in its structure. Recently, we have shown that nateglinide transport occurs via the ceftibuten/H+ cotransport system, which is distinct from PepT1, and that the fluorescein/H+ cotransport system is involved in the uptake of nateglinide. The aim of this study was to characterize the functional properties of the intestinal nateglinide transporter. In the first part of this study, we demonstrated that the ceftibuten/H+ cotransport system is identical to the fluorescein/H+ cotransport system. We succeeded in purification of the nateglinide transporter from brush-border membranes of the rat small intestine using p-aminobenzoic acid (PABA)-affinity chromatography. We then investigated the functional properties of the nateglinide transporter using proteoliposomes prepared from the PABA-affinity chromatography elute. We demonstrated that nateglinide, ceftibuten, and fluorescein are transported by the same transporter in the intestine.     

Expression of the novel transcription factor OASIS, which belongs to the CREB/ATF family, in mouse embryo with special reference to bone development

The OASIS gene, which encodes a novel CREB/ATF family member, was isolated from long-term cultured astrocytes that were employed as an in vitro gliosis model. In the present study, we examined the expression pattern of the OASIS gene in the developing mouse embryo by in situ hybridization histochemistry and compared it with the expression of osteogenesis markers. OASIS mRNA expression was most strongly detected in preosteoblasts of the outer bony cortex of the ribs. Alveolar bone also showed strong signals for OASIS gene expression. OASIS mRNA was also localized to the preodontoblast of tooth buds. Expression began at embryonic day 12 (D12.5), peaked around D14.5-16.5, and continued to D18.5. The pattern of expression was very similar to that of hXBP-1 mRNA, which encodes another CREB/ATF family member. Spatiotemporal patterns of OASIS partly overlapped that of osteopontin, osteonectin, and alpha1 type I procollagen genes. Among these, the time course of OASIS mRNA expression was most similar to that of osteopontin mRNA expression, suggesting that the OASIS protein is involved in the late phase of osteoblast differentiation, as compared to the Cbfa1 that regulates early phases of osteoblast differentiation.     

Diacylglycerol kinase zeta is associated with chromatin, but dissociates from condensed chromatin during mitotic phase in NIH3T3 cells

Diacylglycerol kinase (DGK) converts diacylglycerol (DG) to phosphatidic acid, both of which act as second messengers to mediate a variety of cellular mechanisms. Therefore, DGK contributes to the regulation of these messengers in cellular signal transduction. Of DGK isozymes cloned, DGKzeta is characterized by a nuclear localization signal that overlaps with a sequence similar to the myristoylated alanine-rich C-kinase substrate. Previous studies showed that nuclear DG is differentially regulated from plasma membrane DG and that the nuclear DG levels fluctuate in correlation with cell cycle progression, suggesting the importance of nuclear DG in cell cycle control. In this connection, DGKzeta has been shown to localize to the nucleus in fully differentiated cells, such as neurons and lung cells, although it remains elusive how DGK behaves during the cell cycle in proliferating cells. Here we demonstrate that DGKzeta localizes to the nucleus during interphase including G1, S, and G2 phases and is associated with chromatin although it dissociates from condensed chromatin during mitotic phase in NIH3T3 cells. Furthermore, this localization pattern is also observed in proliferating spermatogonia in the testis. These results suggest a reversible association of DGKzeta with histone or its related proteins in cell cycle, plausibly dependent on their post-translational modifications.     

Connexin 43 contributes to differentiation of retinal pigment epithelial cells via cyclic AMP signaling

Retinal pigment epithelium (RPE) cells play important roles in the visual system that supports neurosensory retina homeostasis. Connexin (Cx) 43-mediated gap-junctional intercellular communication (GJIC) participates in the regulation of retinal organogenesis, but much of the function of Cx43 on the differentiation of RPE cells is unclear. Here, we report the involvement of Cx43 in RPE differentiation. Knockdown of Cx43 in RPE cells dramatically inhibited the differentiation, whereas Cx43-overexpression successfully induced RPE cell differentiation under de-differentiation conditions. From the experiments using GJIC inhibitors and C-terminus-truncated mutant of Cx43, it was clearly demonstrated that the regulation of RPE cell differentiation by Cx43 did not result from Cx43-mediated GJIC. The RPE cell differentiation induced by Cx43-overexpression was abolished by a cAMP antagonist. In contrast, the treatment with forskolin and phosphodiesterase inhibitor rolipram induced RPE cell differentiation under de-differentiation conditions. These findings indicate that Cx43 contributes to RPE differentiation via cAMP signaling.     

Kinetic analysis of the activation of photoactivated adenylyl cyclase (PAC), a blue-light receptor for photomovements of Euglena.

Photoactivated adenylyl cyclase (PAC) was first purified from a photosensing organelle (the paraflagellar body) of the unicellular flagellate Euglena gracilis, and is regarded as the photoreceptor for the step-up photophobic response. Here, we report the kinetic properties of photoactivation of PAC and a change in intracellular cAMP levels upon blue light irradiation. Activation of PAC was dependent both on photon fluence rate and duration of irradiation, between which reciprocity held well in the range of 2--50 micromol m(-2) s(-1)(total fluence of 1200 micromol m(-2)). Intermittent irradiation also caused activation of PAC in a photon fluence-dependent manner irrespective of cycle periods. Wavelength dependency of PAC activation showed prominent peaks in the UV-B/C, UV-A and blue regions of the spectrum. The time course of the changes in intracellular cAMP levels corresponded well with that of the step-up photophobic response. From this and the kinetic properties of PAC photoactivation, we concluded that an increase in intracellular cAMP levels evoked by photoactivation of PAC is a key event of the step-up photophobic response.     

SH2-B is required for both male and female reproduction

Many growth factors and hormones modulate the reproductive status in mammals. Among these, insulin and insulin-like growth factor I (IGF-I) regulate the development of gonadal tissues. SH2-B has been shown to interact with insulin and IGF-I receptors, although the role of SH2-B in these signals has not been clarified. To investigate the role of SH2-B, we generated mice with a targeted disruption of the SH2-B gene. Both male and female SH2-B(-/-) mice showed slight retardation in growth and impaired fertility. Female knockout mice possess small, anovulatory ovaries with reduced numbers of follicles and male SH2-B(-/-) mice have small testes with a reduced number of sperm. SH2-B(-/-) cumulus cells do not respond to either follicle-stimulating hormone or IGF-I. These data suggest that SH2-B plays a critical role in the IGF-I-mediated reproductive pathway in mice.     

Association between Combined Lifestyle Factors and Non-Restorative Sleep in Japan: A Cross-Sectional Study Based on a Japanese Health Database

Background

Although lifestyle factors such as cigarette smoking, excessive drinking, obesity, low or no exercise, and unhealthy dietary habits have each been associated with inadequate sleep, little is known about their combined effect. The aim of this study was to quantify the overall impact of lifestyle-related factors on non-restorative sleep in the general Japanese population.

Methods and Findings

A cross-sectional study of 243,767 participants (men, 39.8%) was performed using the Specific Health Check and Guidance System in Japan. A healthy lifestyle score was calculated by adding up the number of low-risk lifestyle factors for each participant. Low risk was defined as (1) not smoking, (2) body mass index<25 kg/m², (3) moderate or less alcohol consumption, (4) regular exercise, and (5) better eating patterns. Logistic regression analysis was used to examine the relationship between the score and the prevalence of non-restorative sleep, which was determined from questionnaire responses. Among 97,062 men (mean age, 63.9 years) and 146,705 women (mean age, 63.7 years), 18,678 (19.2%) and 38,539 (26.3%) reported non-restorative sleep, respectively. The prevalence of non-restorative sleep decreased with age for both sexes. Compared to participants with a healthy lifestyle score of 5 (most healthy), those with a score of 0 (least healthy) had a higher prevalence of non-restorative sleep (odds ratio, 1.59 [95% confidence interval, 1.29–1.97] for men and 2.88 [1.74–4.76] for women), independently of hypertension, hypercholesterolemia, diabetes, and chronic kidney disease. The main limitation of the study was the cross-sectional design, which limited causal inferences for the identified associations.

Conclusions

A combination of several unhealthy lifestyle factors was associated with non-restorative sleep among the general Japanese population. Further studies are needed to establish whether general lifestyle modification improves restorative sleep.     

Evaluation of varicella vaccine in childhood leukemia. Observation over 6 years

Since 1977, we have used a live attenuated varicella vaccine to immunize 10 children with acute leukemia. 8 patients had no adverse clinical reaction but 2 patients developed mild fever and papulovesicular rash after vaccination. All 9 tested children became seropositive after the vaccination. Also in all 3 children who were observed for more than 4 years, persistence of neutralizing antibody was detected. Most of the recipients were prevented from developing symptoms of varicella in spite of contact exposure. Two patients developed varicella when they were in severe immunosuppressive states but their symptoms were mild. None of the children developed herpes-zoster during the 6 year follow-up period. The results suggest that the varicella vaccine is effective in children with acute leukemia, and that long-term effectiveness can be expected.