Bisleuconothine A Induces Autophagosome Formation by Interfering with AKT-mTOR Signaling Pathway.

We have previously reported that bisleuconothine A (Bis-A), a novel bisindole alkaloid isolated from Leuconotis griffithii, showed cytostatic activity in several cell lines. In this report, the mechanism of Bis-A-induced cytostatic activity was investigated in detail using A549 cells. Bis-A did not cause apoptosis, as indicated by analysis of annexin V and propidium iodide staining. Expression of all tested apoptosis-related proteins was also unaffected by Bis-A treatment. Bis-A was found to increase LC3 lipidation in MCF7 cells as well as A549 cells, suggesting that Bis-A cytostatic activity may be due to induction of autophagy. Subsequent investigation via Western blotting and immunofluorescence staining indicated that Bis-A induced formation but prevented degradation of autophagosomes. Mechanistic studies showed that Bis-A down-regulated phosphorylation of protein kinase B (AKT) and its downstream kinase, PRAS40, which is an mTOR repressor. Moreover, phosphorylation of p70S6K, an mTOR-dependent kinase, was also down-regulated. Down-regulation of these kinases suggests that the increase in LC3 lipidation may be due to mTOR deactivation. Thus, the cytostatic activity shown by Bis-A may be attributed to its induction of autophagosome formation. The Bis-A-induced autophagosome formation was suggested to be caused by its interference with the AKT-mTOR signaling pathway.

Sucutinirane-diterpene derivatives induce apoptosis via oxidative stress in HL-60 cells.

Previously, we reported the isolation of cassane-type diterpenes, sucutiniranes A-F, from the seeds of Bowdichia nitida. In this study, a series of sucutinirane derivatives was prepared, and their in vitro toxicity in the HL-60 cell line was evaluated. Then the action mechanism of a representative compound that induces cell death was investigated. Whereas C-6 or C-7 diol esters and ether decreased the activity against the HL-60 cell line, furan-oxidized derivatives 12 and 13 showed improvement or retention of the activity compared with those of the natural products sucutinirane A (11), E (1), and F (2). Treatment with sucutinirane derivative 13 elevated caspase 3/7 activity and also decreased expression of Bcl-2 family proteins, Mcl-1, and Bid. Derivative 13 generated reactive oxygen species in HL-60 cells, whose apoptotic effects were attenuated by the addition of an antioxidant, N-acetyl-L-cysteine. These results suggest that cassane butenolide 13 induces apoptosis in HL-60 via its oxidative effects.

Transport of PIP3 by GAKIN, a kinesin-3 family protein, regulates neuronal cell polarity.

Phosphatidylinositol-(3,4,5)-trisphosphate (PIP3), a product of phosphatidylinositol 3-kinase, is an important second messenger implicated in signal transduction and membrane transport. In hippocampal neurons, the accumulation of PIP3 at the tip of neurite initiates the axon specification and neuronal polarity formation. We show that guanylate kinase-associated kinesin (GAKIN), a kinesin-like motor protein, directly interacts with a PIP3-interacting protein, PIP3BP, and mediates the transport of PIP3-containing vesicles. Recombinant GAKIN and PIP3BP form a complex on synthetic liposomes containing PIP3 and support the motility of the liposomes along microtubules in vitro. In PC12 cells and cultured hippocampal neurons, transport activity of GAKIN contributes to the accumulation of PIP3 at the tip of neurites. In hippocampal neurons, altered accumulation of PIP3 by overexpression of GAKIN constructs led to the loss of the axonally differentiated neurites. Together, these results suggest that, in neurons, the GAKIN-PIP3BP complex transports PIP3 to the neurite ends and regulates neuronal polarity formation.

Development of action checkpoints for comfortable computer work.

We developed a manual including a checklist format for undertaking measures to prevent fatigue in visual display terminals (VDT) workers. With this manual, problems related to VDT work can be recognized by using checklists which allow for self-evaluation by the workers. The manual helps the workers to consider measures for improvement by themselves and will contribute to better occupational health education.

[Survey on visual and musculoskeletal symptoms in VDT workers].

With the spread of visual display terminals (VDT) in offices, the numbers of workers using VDT and the working hours at such equipment have increased rapidly in recent years. Also, preventive measures for fatigue have been proposed and the office-working environment has been improved. To examine the effects of the rapid changes in working conditions and environment on the health of VDT workers, we conducted a questionnaire survey in 2002. A self-reported questionnaire was distributed to 3,927 office workers; 2,374 (60.5%) responded. Subjects whose questionnaires had missing data were excluded from analysis. As a result, 1,406 (male: 1,069, female: 337) workers aged 20 to 59 were subjected to analysis. By a logistic regression model, we examined the association between VDT use and visual and musculoskeletal symptoms. Prevalence of eye strain and/or pain (72.1%) was the highest, followed by neck stiffness and/or pain (59.3%), low back stiffness and/or pain (30.0%) and hand or arm strain and/or pain (13.9%). Women consistently reported more discomfort than men. As a result of the logistic regression model, eye strain and/or pain was associated with dissatisfaction with airflow, but not with factors affecting visual symptoms as reported in previous studies, for example, reflection of light and blurred characters on the screen. It was thought that airflow appeared as a risk factor because the lighting environment had been improved in offices to prevent reflection of light on the screen. Neck stiffness and/or pain was associated with raising the shoulders during VDT work, the unsuitable shape of the computer mouse for a hand, and the inconvenient arrangement of the mouse in relation to the body. Hand or arm strain and/or pain were associated with the arrangement of the mouse and inappropriate height of the desk. Low back stiffness and/or pain were associated with dissatisfaction with the chair and using the keyboard without a wrist rest. Although measures to prevent fatigue had been implemented for VDT workers, risk factors for musculoskeletal symptoms would be the same as in previous studies.