Citations

The mechanism by which the proapoptotic Bcl-2 family members Bax and Bak release cytochrome c from mitochondria is incompletely understood. In this paper, we show that activator BH3-only proteins bind tightly but transiently to the Bak hydrophobic BH3-binding groove to induce Bak oligomerization, liposome permeabilization, mitochondrial cytochrome c release, and cell death. Analysis by surface plasmon resonance indicated that the initial binding of BH3-only proteins to Bak occurred with similar kinetics with or without detergent or mitochondrial lipids, but these reagents increase the strength of the Bak–BH3-only protein interaction. Point mutations in Bak and reciprocal mutations in the BH3-only proteins not only confirmed the identity of the interacting residues at the Bak–BH3-only protein interface but also demonstrated specificity of complex formation in vitro and in a cellular context. These observations indicate that transient protein–protein interactions involving the Bak BH3-binding groove initiate Bak oligomerization and activation.

Abstract The mechanism of cytotoxicity of farnesyltransferase inhibitors is incompletely understood and seems to vary depending on the cell type. To identify potential determinants of sensitivity or resistance for study in the accompanying clinical trial (Witzig et al, page 4882), we examined the mechanism of cytotoxicity of tipifarnib in human lymphoid cell lines. Based on initial experiments showing that Jurkat variants lacking Fas-associated death domain or procaspase-8 undergo tipifarnib-induced apoptosis, whereas cells lacking caspase-9 or overexpressing Bcl-2 do not, we examined changes in Bcl-2 family members. Tipifarnib caused dose-dependent up-regulation of Bim in lymphoid cell lines (Jurkat, Molt3, H9, DoHH2, and RL) that undergo tipifarnib-induced apoptosis but not in lines (SKW6.4 and Hs445) that resist tipifarnib-induced apoptosis. Further analysis demonstrated that increased Bim levels reflect inhibition of signaling from c-Raf to MEK1/2 and ERK1/2. Additional experiments showed that down-regulation of the Ras guanine nucleotide exchange factor RasGRP1 diminished tipifarnib sensitivity, suggesting that H-Ras or N-Ras is a critical farnesylation target upstream of c-Raf in lymphoid cells. These results not only trace a pathway through c-Raf to Bim that contributes to tipifarnib cytotoxicity in human lymphoid cells but also identify potential determinants of sensitivity to this agent.

BackgroundDiabetes is a risk factor for mild cognitive impairment (MCI) and dementia. However, the association between high normal fasting blood glucose (FBG) and dementia has not been studied.MethodsPolytomous logistic regression was used to assess the association of dementia and MCI with FBG in an age‐ and sex‐matched sample of 32 dementia patients, 27 amnestic MCI (aMCI) patients, and 31 normal controls (NC). Analyses were repeated for those with normal FBG. Correlations between FBG and cognitive test scores were obtained.ResultsControlling for age, gender, education, body mass index, Hachinski Ischemic Score, magnetic resonance imaging (MRI) stroke, and normalized brain, hippocampal, and white matter hyperintensity MRI volumes; higher FBG was associated with dementia versus aMCI status (OR = 3.13; 95% CI, 1.28–7.69). This association remained (OR = 7.75; 95% CI, 1.10–55.56) when analyses were restricted to subjects with normal FBG. When dementia patients were compared with NC adjusting for age, gender, and education, a significant association with FBG also was seen (OR = 1.83; 95% CI, 1.09–3.08), but it was lost when vascular covariates were added to the model. FBG was not associated with aMCI status versus NC. Higher FBG was correlated with poorer performance on the Trailmaking Test Part B (P = .003). The percentage of dementia patients with high normal FBG (90%) was significantly higher than that of aMCI patients with high normal FBG (32.9%) (χ2 = 13.9, P < .001).ConclusionsHigher FBG was associated with dementia (vs. aMCI) independent of vascular risk factors and MRI indicators of vascular disease, and remained a significant risk factor when analyses were restricted to subjects with normal FBG. The results of this cross‐sectional study suggest that a high normal level of FBG may be a risk factor for dementia.

Rationale: Activity of the large conductance Ca 2+ -activated K + (BK) channels is profoundly modulated by its β 1 subunit (BK-β 1 ). However, BK-β 1 expression is downregulated in diabetic vessels. The ubiquitin–proteasome system (UPS) is a major mechanism of intracellular protein degradation. Whether UPS participates in BK-β 1 downregulation in diabetic vessels is unknown. Objective: We hypothesize that UPS facilitates vascular BK-β 1 degradation in diabetes. Methods and Results: Using patch clamp and molecular biological approaches, we found that BK-β 1 –mediated channel activation and BK-β 1 protein expression were reduced in aortas of streptozotocin-induced diabetic rats and in human coronary arterial smooth muscle cells (CASMCs) cultured in high glucose. This was accompanied by upregulation of F-box only protein (FBXO)-9 and FBXO-32 (atrogin-1), the key components of the Skp1-Cullin-F-box (SCF) type ubiquitin ligase complex. BK-β 1 expression was suppressed by the FBXO activator doxorubicin but enhanced by FBXO-9 small interfering RNA or by the proteasome inhibitor MG-132. Cotransfection of atrogin-1 in HEK293 cells significantly reduced Flag-hSlo-β 1 expression by 2.16-fold, compared with expression of Flag-hSlo-β 1 V146A (a mutant without the PDZ-binding motif). After cotransfection with atrogin-1, the ubiquitination of Flag-hSlo-β 1 was increased by 1.91-fold, compared with that of hSlo-β 1 V146A, whereas cotransfection with atrogin-1ΔF (a nonfunctional mutant without the F-box motif) had no effect. Moreover, inhibition of Akt signaling attenuated the phosphorylation of forkhead box O transcription factor (FOXO)-3a and enhanced atrogin-1 expression, which in turn suppressed BK-β 1 protein levels in human CASMCs. Conclusions: Downregulation of vascular BK-β 1 expression in diabetes and in high-glucose culture conditions was associated with FOXO-3a/FBXO-dependent increase in BK-β 1 degradation.

Rationale : The large conductance Ca 2+ -activated K + (BK) channel, a key determinant of vascular tone, is regulated by angiotensin II (Ang II) type 1 receptor signaling. Upregulation of Ang II functions and downregulation of BK channel activities have been reported in diabetic vessels. However, the molecular mechanisms underlying Ang II-mediated BK channel modulation, especially in diabetes mellitus, have not been thoroughly examined. Objectives : The aim in this study was to determine whether caveolae-targeting facilitates BK channel dysfunction in diabetic vessels. Methods and Results : Using patch clamp techniques and molecular biological approaches, we found that BK channels, Ang II type 1 receptor, G αq/11 (G protein q/11 α subunit), nonphagocytic NAD(P)H oxidases (NOX-1), and c-Src kinases (c-Src) were colocalized in the caveolae of rat arterial smooth muscle cells and the integrity of caveolae in smooth muscle cells was critical for Ang II-mediated BK channel regulation. Most importantly, membrane microdomain targeting of these proteins was upregulated in the caveolae of streptozotocin-induced rat diabetic vessels, leading to enhanced Ang II-induced redox-mediated BK channel modification and causing BK channel and coronary dysfunction. The absence of caveolae abolished the effects of Ang II on vascular BK channel activity and preserved BK channel function in diabetes. Conclusions : These results identified a molecular scheme of receptor/enzyme/channel/caveolae microdomain complex that facilitates the development of vascular BK channel dysfunction in diabetes.

Objective: To determine apolipoprotein E (APOE)-ε4 and -ε2 frequencies and risk of mild cognitive impairment (MCI) and dementia in Shanghai, China. Methods: A total of 34 MCI and 34 dementia cases were recruited from an urban Memory Disorders Clinic and 32 controls were recruited from a residential community served by the clinic. Apolipoprotein E was genotyped using standard methods. Results: Among controls, frequencies were ε2, 0.11; ε3, 0.84; and ε4, 0.05; among MCI, 0.05, 0.77, and 0.18; and for dementia, 0.02, 0.84, and 0.15, respectively. In education-adjusted models, the odds ratio (OR) = 5.6 for dementia (95% CI = 1.09-29.3) and 4.7 for MCI (95% CI = 0.90-25.2) associated with any ε4 allele. The ε2 allele was inversely associated with dementia (OR = 0.12, 95% CI = 0.013-0.997) and MCI (OR = 0.38, 95% CI = 0.08-1.61). Conclusions: APOE-ε4 increases and -ε2 decreases the risk of dementia vs normal cognition. Similar trends were observed for amnestic mild cognitive impairment (aMCI).