Starvation-induced Hyperacetylation of Tubulin Is Required for the Stimulation of Autophagy by Nutrient Deprivation

The molecular mechanisms underlying microtubule participation in autophagy are not known. In this study, we show that starvation-induced autophagosome formation requires the most dynamic microtubule subset. Upon nutrient deprivation, labile microtubules specifically recruit markers of autophagosome formation like class III-phosphatidylinositol kinase, WIPI-1, the Atg12-Atg5 conjugate, and LC3-I, whereas mature autophagosomes may bind to stable microtubules. We further found that upon nutrient deprivation, tubulin acetylation increases both in labile and stable microtubules and is required to allow autophagy stimulation. Tubulin hyperacetylation on lysine 40 enhances kinesin-1 and JIP-1 recruitment on microtubules and allows JNK phosphorylation and activation. JNK, in turn, triggers the release of Beclin 1 from Bcl-2-Beclin 1 complexes and its recruitment on microtubules where it may initiate autophagosome formation. Finally, although kinesin-1 functions to carry autophagosomes in basal conditions, it is not involved in motoring autophagosomes after nutrient deprivation. Our results show that the dynamics of microtubules and tubulin post-translational modifications play a major role in the regulation of starvation-induced autophagy.     

Lactacystin stimulates arachidonic acid metabolism in rat liver cells: effects of cell density on arachidonic acid release, PGI2 production and cyclooxygenase activity

Lactacystin, an inhibitor of proteasome activity, amplifies prostaglandin I2 production by rat liver cells stimulated by 12-O-tetradecanoylphorbol-13-acetate, transforming growth factor-alpha or interleukin-1. Lactacystin also stimulates the cell's release of arachidonic acid (AA) and increases the cyclooxygenase activity in these cells. In serum deprived cells, the enhanced AA release is reduced, cyclooxygenase activity on exogenous AA is increased and endogenous production of prostaglandin I2 is unchanged. These findings suggest that, in vivo, the ratio of dividing to quiescent cells in a tissue may influence eicosanoid production. The increases in prostaglandin I2 production, AA release and cyclooxygenase activity on exogenous AA resulting from the combined lactacystin and 12-O-tetradecanoylphorbol-13-acetate treatment are inhibited by actinomycin or cycloheximide.     

Temporal and spatial variability in stable isotope compositions of a freshwater mussel: implications for biomonitoring and ecological studies

Stable isotopes can be used to elucidate ecological relationships in community and trophic studies. Findings are calibrated against baselines, e.g. from a producer or primary consumer, assumed to act as a reference to the isotopic context created by spatio-temporal attributes such as geography, climate, nutrient, and energy sources. The ability of an organism to accurately represent a community base depends on how, and over what time-scale, it assimilates ambient materials. Freshwater mussels have served as references for trophic studies of freshwater communities and as indicators of change in nutrient pollution load or source. Their suitability as reference animals has not yet been fully explored, however. We conducted a series of studies examining the suitability of freshwater mussels as isotopic baselines, using their ability to reflect variation in ambient nutrient loads as a case scenario. (1) We analyzed bivalve foot tissue δ¹⁵N and δ¹³C from 22 stream reaches in the Piedmont region of North Carolina, USA to show that compositions varied substantially among locations. Site mean bivalve δ¹³C values correlated with site ambient particulate organic matter (POM) δ¹³C values, and site mean bivalve δ¹⁵N values correlated with site ambient water dissolved δ¹⁵N-NO₃ values. (2) Similarity of results among sample types demonstrated that the minimally invasive hemolymph sample is a suitable substitute for foot tissue in δ¹⁵N analyses, and that small sample sizes generate means representative of a larger population. Both findings can help minimize the impact of sampling on imperiled freshwater mussel populations. (3) In a bivalve transplantation study we showed that hemolymph δ¹⁵N compositions responded to a shift in ambient dissolved δ¹⁵N-NO₃, although slowly. The tissue turnover time for bivalve hemolymph was 113 days. We conclude that bivalves serve best as biomonitors of chronic, rather than acute, fluctuations in stream nutrient loads, and provide initial evidence of their suitability as time-integrated isotopic baselines for community studies.     

The Photosynthetic Electron Transport Chain of Chlamydomonas reinhardi. VII. Photosynthetic Phosphorylation by a Mutant Strain of Chlamydomonas reinhardi Deficient in Active P700

Electron transport activity and absorbance changes associated with P700 were investigated in a mutant strain of Chlamydomonas reinhardi with impaired photosynthesis. This mutant strain, ac-8oa, cannot reduce NADP with electrons from either water or dye and ascorbate, but it has considerable Hill activity. The mutant strain shows none of the absorbance changes characteristic of P700. Although unable to carry out cyclic photosynthetic phosphorylation, ac-8oa is able to synthesize ATP when ferricyanide is provided as an electron acceptor.

These observations lead to the conclusion that a site for the coupling of photosynthetic phosphorylation with electron transport must exist between the 2 photochemical systems.

     

Neuronal influences on glial progenitor cell development

The role of cell-cell interactions in the development of bipotential glial progenitor cells in cultures of rat cerebellum and optic nerve was studied. In the cerebellar cultures, progenitor cells divide slowly and most of their progeny develop into additional progenitor cells. Progenitor cells isolated from postconfluent cultures of cerebellum, however, develop rapidly into oligodendrocytes when grown in a serum-free medium. Factors secreted or shed into the medium by young cerebellar interneurons stimulate optic nerve progenitor cells to divide and promote the survival of progenitor cells. These factors appear to alter the function of the internal clock that regulates the timing of oligodendrocyte differentiation. These results suggest that the neuronal microenvironment can influence the lineage decisions of multipotential glial progenitor cells.     

Phase I evaluation of intravenous ascorbic acid in combination with gemcitabine and erlotinib in patients with metastatic pancreatic cancer

Background

Preclinical data support further investigation of ascorbic acid in pancreatic cancer. There are currently insufficient safety data in human subjects, particularly when ascorbic acid is combined with chemotherapy.

Methods and Findings

14 subjects with metastatic stage IV pancreatic cancer were recruited to receive an eight week cycle of intravenous ascorbic acid (three infusions per week), using a dose escalation design, along with standard treatment of gemcitabine and erlotinib. Of 14 recruited subjects enrolled, nine completed the study (three in each dosage tier). There were fifteen non-serious adverse events and eight serious adverse events, all likely related to progression of disease or treatment with gemcitabine or erlotinib. Applying RECIST 1.0 criteria, seven of the nine subjects had stable disease while the other two had progressive disease.

Conclusions

These initial safety data do not reveal increased toxicity with the addition of ascorbic acid to gemcitabine and erlotinib in pancreatic cancer patients. This, combined with the observed response to treatment, suggests the need for a phase II study of longer duration.

Trial Registration

Clinicaltrials.gov {"type":"clinical-trial","attrs":{"text":"NCT00954525","term_id":"NCT00954525"}}NCT00954525     

The utility of epithelial-cell micronuclei in the assessment of intermittent exposures

Epithelial-cell micronuclei (MN) are potentially useful markers of occupational exposure to genotoxicants. With intermittent exposures, cells sampled either before or after a specific time interval, reflecting the time it takes for damaged cells to become available at the epithelial surface, are unlikely to be exposure-related. It may then be important to conduct an exposure-window analysis, with the goal of identifying the relevant exposures.We re-analysed individual exposure data from a previous study (Suruda et al. 1993) of MN formation in 22 male mortuary science students exposed to formaldehyde during a 90-day embalming class. We conducted an exposurewindow analysis and compared the results with those obtained with 90-day cumulative exposure. The window widths varied between 7 and 25 days, in 1 day increments, assuming a constant 7-day cell-cycle. We assessed the fit (likelihood-ratio test) of a linear regression model, regressing the change in buccal MN prevalence on formaldehyde exposure, using both asymptotic and non-asymptotic methods. Exposures defined from 7-15 to 7-18 days before specimen collection provided a slightly better fit than the 90-day cumulative exposure, with a doubling of the regression coefficient for the exposure effect (for the 7-16-days window LR = 5.32, p = 0.032, coefficient = 0.088 MN per 1000 cells per ppm-hr; 95% CI = 0.014, 0.16; for the 90-day cumulative exposure LR = 4.44, p = 0.048, coefficient = 0.045 MN per 1000 cells per ppm-hr, 95% CI = 0.0038, 0.086). Although hampered by the small number of subjects, these results reinforce the potential importance of exposure timing.     

Recognition of Apoptotic Cells by Epithelial Cells: CONSERVED VERSUS TISSUE-SPECIFIC SIGNALING RESPONSES*

During apoptosis, cells acquire new activities that enable them to modulate the fate and function of interacting phagocytes, particularly macrophages (mϕ). Although the best known of these activities is anti-inflammatory, apoptotic targets also influence mϕ survival and proliferation by modulating proximal signaling events, such as MAPK modules and Akt. We asked whether modulation of these same signaling events extends to epithelial cells, a minimally phagocytic cell type. We used BU.MPT cells, a mouse kidney epithelial cell line, as our primary model, but we also evaluated several epithelial cell lines of distinct tissue origins. Like mϕ, mouse kidney epithelial cells recognized apoptotic and necrotic targets through distinct non-competing receptors, albeit with lower binding capacity and markedly reduced phagocytosis. Also, modulation of inflammatory activity and MAPK-dependent signaling by apoptotic and necrotic targets was indistinguishable in kidney epithelial cells and mϕ. In contrast, modulation of Akt-dependent signaling differed dramatically between kidney epithelial cells and mϕ. In kidney epithelial cells, modulation of Akt was linked to target cell recognition, independently of phagocytosis, whereas in mϕ, modulation was linked to phagocytosis. Moreover, recognition of apoptotic and necrotic targets by kidney epithelial cells elicited opposite responses; apoptotic targets inhibited whereas necrotic targets stimulated Akt activity. These data confirm that nonprofessional phagocytes recognize and respond to dying cells, albeit in a manner partially distinct from mϕ. By acting as sentinels of environmental change, apoptotic and necrotic targets may permit neighboring viable cells, especially non-migratory epithelial cells, to monitor and adapt to local stresses.