Caffeine induces apoptosis by enhancement of autophagy via PI3K/Akt/mTOR/p70S6K inhibition

Caffeine is one of the most frequently ingested neuroactive compounds. All known mechanisms of apoptosis induced by caffeine act through cell cycle modulation or p53 induction. It is currently unknown whether caffeine-induced apoptosis is associated with other cell death mechanisms, such as autophagy. Herein we show that caffeine increases both the levels of microtubule-associated protein 1 light chain 3-II and the number of autophagosomes, through the use of western blotting, electron microscopy and immunocytochemistry techniques. Phosphorylated p70 ribosomal protein S6 kinase (Thr389), S6 ribosomal protein (Ser235/236), 4E-BP1 (Thr37/46) and Akt (Ser473) were significantly decreased by caffeine. In contrast, ERK1/2 (Thr202/204) was increased by caffeine, suggesting an inhibition of the Akt/mTOR/p70S6K pathway and activation of the ERK1/2 pathway. Although insulin treatment phosphorylated Akt (Ser473) and led to autophagy suppression, the effect of insulin treatment was completely abolished by caffeine addition. Caffeine-induced autophagy was not completely blocked by inhibition of ERK1/2 by U0126. Caffeine induced reduction of mitochondrial membrane potentials and apoptosis in a dose-dependent manner, which was further attenuated by the inhibition of autophagy with 3-methyladenine or Atg7 siRNA knockdown. Furthermore, there was a reduced number of early apoptotic cells (annexin V positive, propidium iodide negative) among autophagy-deficient mouse embryonic fibroblasts treated with caffeine than their wild-type counterparts. These results support previous studies on the use of caffeine in the treatment of human tumors and indicate a potential new target in the regulation of apoptosis.     

HMG domain containing SSRP1 is required for DNA demethylation and genomic imprinting in Arabidopsis

In Arabidopsis, DEMETER (DME) DNA demethylase contributes to reprogramming of the epigenetic state of the genome in the central cell. However, other aspects of the active DNA demethylation processes remain elusive. Here we show that Arabidopsis SSRP1, known as an HMG domain-containing component of FACT histone chaperone, is required for DNA demethylation and for activation and repression of many parentally imprinted genes in the central cell. Although loss of DNA methylation releases silencing of the imprinted FWA-GFP, double ssrp1-3;met1-3 mutants surprisingly showed limited activation of maternal FWA-GFP in the central cell, and only became fully active after several nuclear divisions in the endosperm. This behavior was in contrast to the dme-1;met1 double mutant in which hypomethylation of FWA-GFP by met1 suppressed the DNA demethylation defect of dme-1. We propose that active DNA demethylation by DME requires SSRP1 function through a distinctly different process from direct DNA methylation control.     

Rapid determination of free D-serine with chicken D-serine dehydratase

We have developed a simple, rapid, and inexpensive method of measuring the concentration of intrinsic free D-serine in tissue samples. This method uses chicken D-serine dehydratase in an enzymatic reaction to produce pyruvate, which is detected spectrophotometrically. Pyridoxal 5'-phosphate (PLP), a cofactor of D-serine dehydratase, increased pyruvate formation by 28%. The presence of Zn(2+) or ethylenediaminetetraacetic acid (EDTA) did not have any effect on pyruvate formation under the present assay conditions. In addition, this method was not affected by the presence of a large excess of L-serine, nor by the presence of tissue extracts, and accurately determined concentrations of 2-30 μM (200 pmol-3 nmol) of D-serine. The entire assay requires only 60 min. With this method, we determined the concentration of D-serine in various silkworm tissues. The results were in agreement with high performance liquid chromatography measurements. We found high concentrations of D-serine in silkworm larvae at day 3 of the fifth instar; specifically, 509 nmol g(-1) wet tissue in the midgut, 434 nmol g(-1) in the ovary, and 353 nmol g(-1) in the testis.     

The global DNA methylation surrogate LINE-1 methylation is correlated with MGMT promoter methylation and is a better prognostic factor for glioma

Gliomas are the most frequently occurring primary brain tumor in the central nervous system of adults. Glioblastoma multiformes (GBMs, WHO grade 4) have a dismal prognosis despite the use of the alkylating agent, temozolomide (TMZ), and even low grade gliomas (LGGs, WHO grade 2) eventually transform to malignant secondary GBMs. Although GBM patients benefit from promoter hypermethylation of the O(6)-methylguanine-DNA methyltransferase (MGMT) that is the main determinant of resistance to TMZ, recent studies suggested that MGMT promoter methylation is of prognostic as well as predictive significance for the efficacy of TMZ. Glioma-CpG island methylator phenotype (G-CIMP) in the global genome was shown to be a significant predictor of improved survival in patients with GBM. Collectively, we hypothesized that MGMT promoter methylation might reflect global DNA methylation. Additionally in LGGs, the significance of MGMT promoter methylation is still undetermined. In the current study, we aimed to determine the correlation between clinical, genetic, and epigenetic profiles including LINE-1 and different cancer-related genes and the clinical outcome in newly diagnosed 57 LGG and 54 GBM patients. Here, we demonstrated that (1) IDH1/2 mutation is closely correlated with MGMT promoter methylation and 1p/19q codeletion in LGGs, (2) LINE-1 methylation levels in primary and secondary GBMs are lower than those in LGGs and normal brain tissues, (3) LINE-1 methylation is proportional to MGMT promoter methylation in gliomas, and (4) higher LINE-1 methylation is a favorable prognostic factor in primary GBMs, even compared to MGMT promoter methylation. As a global DNA methylation marker, LINE-1 may be a promising marker in gliomas.     

Constitutive expression of insulin receptor substrate (IRS)-1 inhibits myogenic differentiation through nuclear exclusion of Foxo1 in L6 myoblasts

Insulin-like growth factors (IGFs) are well known to play essential roles in enhancement of myogenic differentiation. In this report we showed that initial IGF-I signal activation but long-term IGF-1 signal termination are required for myogenic differentiation. L6 myoblast stably transfected with myc-epitope tagged insulin receptor substrate-1, myc-IRS-1 (L6-mIRS1) was unable to differentiate into myotubes, indicating that IRS-1 constitutive expression inhibited myogenesis. To elucidate the molecular mechanisms underlying myogenic inhibition, IGF-I signaling was examined. IGF-I treatment of control L6 cells for 18 h resulted in a marked suppression of IGF-I stimulated IRS-1 association with the p85 PI 3-kinase and suppression of activation of Akt that correlated with a down regulation of IRS-1 protein. L6-mIRS1 cells, in contrast, had sustained high levels of IRS-1 protein following 18 h of IGF-I treatment with persistent p85 PI 3-kinase association with IRS-1, Akt phosphorylation and phosphorylation of the downstream Akt substrate, Foxo1. Consistent with Foxo1 phosphorylation, Foxo1 protein was excluded from the nuclei in L6-mIRS1 cells, whereas Foxo1 was localized in the nuclei in control L6 cells during induction of differentiation. In addition, L6 cells stably expressing a dominant-interfering form of Foxo1, Δ256Foxo1 (L6-Δ256Foxo1) were unable to differentiate into myotubes. Together, these data demonstrate that IGF-I regulation of Foxo1 nuclear localization is essential for the myogenic program in L6 cells but that persistent activation of IGF-1 signaling pathways results in a negative feedback to prevent myogenesis.     

Nuclear exportin receptor CAS regulates the NPI-1-mediated nuclear import of HIV-1 Vpr

Vpr, an accessory protein of human immunodeficiency virus type 1, is a multifunctional protein that plays an important role in viral replication. We have previously shown that the region between residues 17 and 74 of Vpr (Vpr(N17C74)) contained a bona fide nuclear localization signal and it is targeted Vpr(N17C74) to the nuclear envelope and then imported into the nucleus by importin α (Impα) alone. The interaction between Impα and Vpr is important not only for the nuclear import of Vpr but also for HIV-1 replication in macrophages; however, it was unclear whether full-length Vpr enters the nucleus in a manner similar to Vpr(N17C74). This study investigated the nuclear import of full-length Vpr using the three typical Impα isoforms, Rch1, Qip1 and NPI-1, and revealed that full-length Vpr is selectively imported by NPI-1, but not Rch1 and Qip1, after it makes contact with the perinuclear region in digitonin-permeabilized cells. A binding assay using the three Impα isoforms showed that Vpr bound preferentially to the ninth armadillo repeat (ARM) region (which is also essential for the binding of CAS, the export receptor for Impα) in all three isoforms. Comparison of biochemical binding affinities between Vpr and the Impα isoforms using surface plasmon resonance analysis demonstrated almost identical values for the binding of Vpr to the full-length isoforms and to their C-terminal domains. By contrast, the data showed that, in the presence of CAS, Vpr was released from the Vpr/NPI-1 complex but was not released from Rch1 or Qip1. Finally, the NPI-1-mediated nuclear import of Vpr was greatly reduced in semi-intact CAS knocked-down cells and was recovered by the addition of exogenous CAS. This report is the first to show the requirement for and the regulation of CAS in the functioning of the Vpr-Impα complex.     

Colocalization of 14-3-3 proteins with SOD1 in Lewy body-like hyaline inclusions in familial amyotrophic lateral sclerosis cases and the animal model

Background and Purpose

Cu/Zn superoxide dismutase (SOD1) is a major component of Lewy body-like hyaline inclusion (LBHI) found in the postmortem tissue of SOD1-linked familial amyotrophic lateral sclerosis (FALS) patients. In our recent studies, 14-3-3 proteins have been found in the ubiquitinated inclusions inside the anterior horn cells of spinal cords with sporadic amyotrophic lateral sclerosis (ALS). To further investigate the role of 14-3-3 proteins in ALS, we performed immunohistochemical analysis of 14-3-3 proteins and compared their distributions with those of SOD1 in FALS patients and SOD1-overexpressing mice.

Methods

We examined the postmortem brains and the spinal cords of three FALS cases (A4V SOD1 mutant). Transgenic mice expressing the G93A mutant human SOD1 (mutant SOD1-Tg mice), transgenic mice expressing the wild-type human SOD1 (wild-type SOD1-Tg mice), and non-Tg wild-type mice were also subjected to the immunohistochemical analysis.

Results

In all the FALS patients, LBHIs were observed in the cytoplasm of the anterior horn cells, and these inclusions were immunopositive intensely for pan 14-3-3, 14-3-3β, and 14-3-3γ. In the mutant SOD1-Tg mice, a high degree of immunoreactivity for misfolded SOD1 (C4F6) was observed in the cytoplasm, with an even greater degree of immunoreactivity present in the cytoplasmic aggregates of the anterior horn cells in the lumbar spinal cord. Furthermore, we have found increased 14-3-3β and 14-3-3γ immunoreactivities in the mutant SOD1-Tg mice. Double immunofluorescent staining showed that C4F6 and 14-3-3 proteins were partially co-localized in the spinal cord with FALS and the mutant SOD1-Tg mice. In comparison, the wild-type SOD1-Tg and non-Tg wild-type mice showed no or faint immunoreactivity for C4F6 and 14-3-3 proteins (pan 14-3-3, 14-3-3β, and 14-3-3γ) in any neuronal compartments.

Discussion

These results suggest that 14-3-3 proteins may be associated with the formation of SOD1-containing inclusions, in FALS patients and the mutant SOD1-Tg mice.     

Autism-associated gene expression in peripheral leucocytes commonly observed between subjects with autism and healthy women having autistic children

Autism spectrum disorder (ASD) is a severe neuropsychiatric disorder which has complex pathobiology with profound influences of genetic factors in its development. Although the numerous autism susceptible genes were identified, the etiology of autism is not fully explained. Using DNA microarray, we examined gene expression profiling in peripheral blood from 21 individuals in each of the four groups; young adults with ASD, age- and gender-matched healthy subjects (ASD control), healthy mothers having children with ASD (asdMO), and asdMO control. There was no blood relationship between ASD and asdMO. Comparing the ASD group with control, 19 genes were found to be significantly changed. These genes were mainly involved in cell morphology, cellular assembly and organization, and nerve system development and function. In addition, the asdMO group possessed a unique gene expression signature shown as significant alterations of protein synthesis despite of their nonautistic diagnostic status. Moreover, an ASD-associated gene expression signature was commonly observed in both individuals with ASD and asdMO. This unique gene expression profiling detected in peripheral leukocytes from affected subjects with ASD and unaffected mothers having ASD children suggest that a genetic predisposition to ASD may be detectable even in peripheral cells. Altered expression of several autism candidate genes such as FMR-1 and MECP2, could be detected in leukocytes. Taken together, these findings suggest that the ASD-associated genes identified in leukocytes are informative to explore the genetic, epigenetic, and environmental background of ASD and might become potential tools to assess the crucial factors related to the clinical onset of the disorder.     

Effects of living at two ambient temperatures on 24-h blood pressure and neuroendocrine function among obese and non-obese humans: a pilot study

The effects of environmental temperature on blood pressure and hormones in obese subjects in Japan were compared in two seasons: summer vs winter. Five obese (BMI, 32?±?5 kg/m²) and five non-obese (BMI, 23 ±3 kg/m²) men participated in this experiment at latitude 35°10′ N and longitude 136°57.9′ E. The average environmental temperature was 29?±?1 °C in summer and 3?±?1 °C in winter. Blood samples were analyzed for leptin, ghrelin, catecholamines, thyroid stimulating hormone (TSH), free thyroxine (fT₄), free triiodothyronine (fT₃), total cholesterol, triglycerides, insulin and glucose. Blood pressure was measured over the course of 24 h in summer and winter. A Japanese version of the Profile of Mood States (POMS) questionnaire was also administered each season. Systolic and diastolic blood pressures in obese men were significantly higher in winter (lower environmental temperatures) than in summer (higher environmental temperatures). Noradrenaline and dopamine concentrations were also significantly higher at lower environmental temperatures in obese subjects, but ghrelin, TSH, fT₃, fT₄, insulin and glucose were not significantly different in summer and winter between obese and non-obese subjects. Leptin, total cholesterol and triglyceride concentrations were significantly higher in winter in obese than non-obese men. Results from the POMS questionnaire showed a significant rise in Confusion at lower environmental temperatures (winter) in obese subjects. In this pilot study, increased blood pressure may have been due to increased secretion of noradrenaline in obese men in winter, and the results suggest that blood pressure control in obese men is particularly important in winter.