Characterization of the signal that directs Bcl-xL, but not Bcl-2, to the mitochondrial outer membrane

It is assumed that the survival factors Bcl-2 and Bcl-xL are mainly functional on mitochondria and therefore must contain mitochondrial targeting sequences. Here we show, however, that only Bcl-xL is specifically targeted to the mitochondrial outer membrane (MOM) whereas Bcl-2 distributes on several intracellular membranes. Mitochondrial targeting of Bcl-xL requires the COOH-terminal transmembrane (TM) domain flanked at both ends by at least two basic amino acids. This sequence is a bona fide targeting signal for the MOM as it confers specific mitochondrial localization to soluble EGFP. The signal is present in numerous proteins known to be directed to the MOM. Bcl-2 lacks the signal and therefore localizes to several intracellular membranes. The COOH-terminal region of Bcl-2 can be converted into a targeting signal for the MOM by increasing the basicity surrounding its TM. These data define a new targeting sequence for the MOM and propose that Bcl-2 acts on several intracellular membranes whereas Bcl-xL specifically functions on the MOM

Regulatory feedback cycle of the insulin-degrading enzyme and the amyloid precursor protein intracellular domain: Implications for Alzheimer's disease

One of the major pathological hallmarks of Alzheimer´s disease (AD) is an accumulation of amyloid-β (Aβ) in brain tissue leading to formation of toxic oligomers and senile plaques. Under physiological conditions, a tightly balanced equilibrium between Aβ-production and -degradation is necessary to prevent pathological Aβ-accumulation. Here, we investigate the molecular mechanism how insulin-degrading enzyme (IDE), one of the major Aβ-degrading enzymes, is regulated and how amyloid precursor protein (APP) processing and Aβ-degradation is linked in a regulatory cycle to achieve this balance. In absence of Aβ-production caused by APP or Presenilin deficiency, IDE-mediated Aβ-degradation was decreased, accompanied by a decreased IDE activity, protein level, and expression. Similar results were obtained in cells only expressing a truncated APP, lacking the APP intracellular domain (AICD) suggesting that AICD promotes IDE expression. In return, APP overexpression mediated an increased IDE expression, comparable results were obtained with cells overexpressing C50, a truncated APP representing AICD. Beside these genetic approaches, also AICD peptide incubation and pharmacological inhibition of the γ-secretase preventing AICD production regulated IDE expression and promoter activity. By utilizing CRISPR/Cas9 APP and Presenilin knockout SH-SY5Y cells results were confirmed in a second cell line in addition to mouse embryonic fibroblasts. In vivo, IDE expression was decreased in mouse brains devoid of APP or AICD, which was in line with a significant correlation of APP expression level and IDE expression in human postmortem AD brains. Our results show a tight link between Aβ-production and Aβ-degradation forming a regulatory cycle in which AICD promotes Aβ-degradation via IDE and IDE itself limits its own production by degrading AICD

Using palynology to re-assess the Dead Sea laminated sediments – indeed varves?

Lacustrine laminated sediments are often varves representing annual rhythmic deposition. The Dead Sea high-stand laminated sections consist of mm-scale alternating detrital and authigenic aragonite laminae. Previous studies assumed these laminae were varves deposited seasonally. However, this assumption has never been robustly validated. Here we report an examination of the seasonal deposition of detrital-aragonite couplets from two well-known Late Holocene laminated sections at the Ze’elim fan-delta using palynology and grain-size distribution analyses. These analyses are complemented by the study of contemporary flash-flood samples and multivariate statistical analysis. Because transport affects the pollen preservation state, well–preserved (mostly) air-borne transported pollen was analysed separately from badly-preserved pollen and fungal spores, which are more indicative of water transport and reworking from soils. Our results indicate that (i) both detrital and aragonite laminae were deposited during the rainy season; (ii) aragonite laminae have significantly lower reworked and fungal spore concentrations than detrital and flash-flood samples; and (iii) detrital laminae are composed of recycling of local and distal sources, with coarser particles that were initially deposited in the Dead Sea watershed and later transported via run-off to the lake. This is in line with previous carbon balance studies that showed that aragonite precipitation occurs after the massive input of TCO2 associated with run-off episodes. Consequently, at least for the Holocene Ze’elim Formation, laminated sediments cannot be considered as varves. Older Quaternary laminated sequences should be re-evaluated

The induction of a neural-specific gene, SCG10, by nerve growth factor in PC12 cells is transcriptional, protein synthesis dependent, and glucocorticoid inhibitable

We describe features of the regulation of a neural-specific gene, SCG10, which is induced by nerve growth factor (NGF) during the neuronal differentiation of the rat pheochromocytoma cell line PC12. Induction of SCG10 mRNA occurs within 12–24 hr of exposure to NGF, is sustained in the continued presence of the neurotrophic factor, and involves a mechanism that is, at least in part, transcriptional. Unlike the rapid, transient transcriptional activations of genes such as c-fos, SCG10 induction requires ongoing protein synthesis, suggesting the participation of a de novo synthesized regulatory protein in mediating the effects of NGF on this gene. Although c-fos itself may play this role, its induction is clearly insufficient to cause an induction of SCG10. NGF, FGF, and, to a lesser extent, phorbol esters induced SCG10, whereas EGF and dibutyryl cAMP did not. In these characteristics, SCG10 induction appears to constitute a reliable molecular index of the transcription-dependent neuronal differentiation induced by NGF. Glucocorticoids, which inhibit NGF-induced neurite outgrowth from normal primary chromaffin cells, partially blocked SCG10 induction in PC12 cells. A reciprocal pattern of regulation by NGF and glucocorticoids was observed for tyrosine hydroxylase mRNA. These data suggest that environmental signals such as NGF may act on specific genes, both positively and negatively, to control the choice of alternative fates by developing neural crest cells

Controls on the Radiocarbon Reservoir Ages in the Modern Dead Sea Drainage System and in the Last Glacial Lake Lisan

From the 19th International Radiocarbon Conference held in Keble College, Oxford, England, April 3-7, 2006.Carbon isotopic and chemical compositions of freshwaters feeding the Dead Sea and the Sea of Galilee (i.e. perennial streams and floods along their stream profiles) were used to constrain the factors that dictate the reservoir ages (RA) of these lakes and the last glacial Lake Lisan. Runoff waters are characterized by high Ca2+, Mg2+, alkalinity, and radiocarbon contents (67-108 pMC), suggesting a major role for 14C atmospheric exchange reactions (carbonate rock dissolution alone will result in lower pMC values). These exchange processes were corroborated by dissolved inorganic carbon (DIC) and d13C trends throughout the flood profile. During the evolution from rain to incipient runoff, the 14CDIC of the water increases and is accompanied by a DIC increase and d13CDIC decrease, suggesting an addition of soil CO2, which is characterized by light d13C and high 14C content. When incipient runoffs evolve to floods, the opposite trends are observed. It appears that the Sea of Galilee, the Dead Sea, and its last glacial precursor, Lake Lisan, maintained uniform but specific RAs of 0.8 +/- 0.1, 2.3 +/- 0.1, and 1.6 +/- 0.3 kyr, respectively. However, applying the 14C contents of modern Dead Sea water sources to the water mass balance of Lake Lisan reveals that the RA of Lake Lisan is higher than that predicted by the mass balance. This discrepancy may reflect enhanced dissolution of carbonatic dust, changes in the amount of 14C exchanged in Judean Desert floods, or variations in the contribution of brine and saline springs. Furthermore, the small fluctuations in the Lisan RA (1.6 +/- 0.3 kyr) may reflect small, short-term changes in the relative contributions of these sources.The Radiocarbon archives are made available by Radiocarbon and the University of Arizona Libraries. Contact [email protected] for further information.Migrated from OJS platform February 202

Apoptosis Induced by Serum Deprivation of PC12 Cells Is Not Preceded by Growth Arrest and Can Occur at Each Phase of the Cell Cycle

Previous studies have shown that PC12 cells undergo apoptosis (programmed cell death) when deprived of serum. In the present study, we examined the relationship of this death process to the cell cycle. PC12 cell populations synchronized at different, specific phases of the cell cycle exhibit similar kinetics of cell death following deprivation of serum. Flow cytometry analysis was used to examine the levels of apoptotic death in these cell populations in relationship to their progression in the cell cycle during the course of serum deprivation. Such analysis revealed that the cells the during the G_o-G_1, S, and perhaps G_2-M phases and at the G_2 to G_1 transition. These results, therefore, suggest that the death of synchronized, serum-deprived PC12 cells occurs throughout the cell cycle and is not dependent on growth arrest. Flow cytometry methodology (acridine orange staining), which determines the RNA content of cells in relationship to their position in the cell cycle, was used to address these questions in nonsynchronized cells. These experiments revealed that, upon serum deprivation, an immediate loss of RNA occurred from cells in G_1, S, and G_2-M phases. This loss is accompanied by a slower appearance of cells with degraded DNA content. These results show that cells from all phases of the cell cycle are damaged upon serum deprivation and thus suggest that the apoptotic cell death of nonsynchronized PC12 cells may occur from each phase of the cell cycle

Apoptosis Induced by Serum Deprivation of PC12 Cells Is Not Preceded by Growth Arrest and Can Occur at Each Phase of the Cell Cycle

Previous studies have shown that PC12 cells undergo apoptosis (programmed cell death) when deprived of serum. In the present study, we examined the relationship of this death process to the cell cycle. PC12 cell populations synchronized at different, specific phases of the cell cycle exhibit similar kinetics of cell death following deprivation of serum. Flow cytometry analysis was used to examine the levels of apoptotic death in these cell populations in relationship to their progression in the cell cycle during the course of serum deprivation. Such analysis revealed that the cells the during the G_o-G_1, S, and perhaps G_2-M phases and at the G_2 to G_1 transition. These results, therefore, suggest that the death of synchronized, serum-deprived PC12 cells occurs throughout the cell cycle and is not dependent on growth arrest. Flow cytometry methodology (acridine orange staining), which determines the RNA content of cells in relationship to their position in the cell cycle, was used to address these questions in nonsynchronized cells. These experiments revealed that, upon serum deprivation, an immediate loss of RNA occurred from cells in G_1, S, and G_2-M phases. This loss is accompanied by a slower appearance of cells with degraded DNA content. These results show that cells from all phases of the cell cycle are damaged upon serum deprivation and thus suggest that the apoptotic cell death of nonsynchronized PC12 cells may occur from each phase of the cell cycle

Motor deficits and neurofibromatosis type 1 (NF1)-associated MRI impairments in a mouse model of NF1

Neurofibromatosis type 1 (NF1) is a genetic disorder characterized inter alia by cognitive and motor dysfunction and appearance of high-signal foci on T2-weighted images in the brain. Nf1+/− mice are useful models for studying aspects of NF1, including cognitive deficits. Here we assessed their motor performance and used quantitative transverse T2 relaxation MRI to identify structural abnormalities in their brains. Nf1+/− mice exhibited both enhanced and reduced T2 signals in distinct brain regions compared to wild-type mice, and their motor performance was impaired. As in NF1 patients, enhanced T2 signals in Nf1+/− mice were observed in the thalamus and basal ganglia. Reduced T2 signals were seen in motor-associated regions along the motor pathway, predominantly in the white matter of the cerebral peduncle and the optic tract. Correlation analysis between T2 signals and motor performance suggested that the motor deficits are associated with impairments in the cerebral peduncle and the amygdal

Non-canonical function of Bax in stress-induced nuclear protein redistribution

Bax and Bak (Bax/Bak) are essential pro-apoptotic proteins of the Bcl-2 family that trigger mitochondrial outer membrane permeabilization (MOMP) in a Bcl-2/Bcl-xL-inhibitable manner. We recently discovered a new stress-related function for Bax/Bak—regulation of nuclear protein redistribution (NPR) from the nucleus to cytoplasm. This effect was independent of Bax/Bak N-terminus exposure and not inhibited by Bcl-xL over-expression. Here, we studied the molecular mechanism governing this novel non-canonical response. Wild-type (WT) and mutant versions of Bax were re-expressed in Bax/Bak double-knockout mouse embryonic fibroblasts and their ability to promote NPR, apoptotic events, and changes in lamin A mobility was examined. Our results show that, in this system, Bax expression was sufficient to restore NPR such as in WT cells undergoing apoptosis. This activity of Bax was uncoupled from cytochrome c release from the mitochondria (indicative of MOMP) and required its membrane localization, α helices 5/6, and the Bcl-2 homology 3 (BH3) domain. Moreover, enrichment of Bax in the nuclear envelope by the so-called Klarsicht/ANC-1/Syne-1 homology domain effectively triggered NPR as in WT Bax, but without inducing MOMP or cell death. Bax-induced NPR was associated with impairment in lamin A mobility, implying a connection between these two nuclear envelope-associated events. Overall, the results indicate a new MOMP-independent, stress-induced Bax function on the nuclear envelope