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Cleavage of amyloid-β precursor protein (APP) at the Asp1 β-secretase site of the amyloid-β protein (Aβ) domain by β-site Aβ precursor protein-cleaving enzyme 1 (BACE1) is required for the generation of Aβ, a central component of neuritic plaques in the Alzheimer's disease (AD) brain. In this study, we found that Aβ Glu11 is the major β-secretase site for cleavage of APP by BACE1 to generate soluble secreted APP (sAPPβ)(606) and the C-terminal membrane-bound fragment (CTF)β product C89. Cleavage of C89 by γ-secretase resulted in truncated Aβ generation in a non-amyloidogenic pathway. A familial AD-associated Swedish APP mutation adjacent to Aβ Asp1 shifted the major APP β-secretase cleavage site from Aβ Glu11 to Asp1, resulting in significant increases in sAPPβ596 and CTFβ C99 generation and the C99/89 ratio, in turn leading to increased Aβ production in cultured cells in vitro and transgenic AD model mouse brains in vivo. Furthermore, increased BACE1 expression facilitated APP being processed by the β-secretase processing pathway rather than the α-secretase pathway, leading to more Aβ production. Our results suggest that potentiating BACE1 cleavage of APP at both the Asp1 and Glu11 sites, or shifting the cleavage from the Glu11 site to the Asp1 site, could result in increased Aβ production and facilitate neuritic plaque formation. Our study provides new insights into how alteration of BACE1 expression and β-secretase cleavage site selection could contribute to Alzheimer pathogenesis and the pharmaceutical potential of modulating BACE1 expression and its cleavage site selection.

Neuritic plaque is the pathological hallmark in Alzheimer's disease (AD). Amyloid-β protein (Aβ), the central component of neuritic plaques, is generated from amyloid-β precursor protein (APP) by β-site APP cleaving enzyme 1 (BACE1) and γ-secretase. β-site APP cleaving enzyme 2 (BACE2), a homolog of BACE1, functions differently from BACE1 in APP processing. BACE1 is the β-secretase essential for Aβ production, and BACE2, a θ-secretase, cleaves APP within the Aβ domain, preventing Aβ production. Elucidation of the mechanism underlying BACE2 degradation is important for defining its biological features and its potential role in Alzheimer's disease drug development. In this report we first showed that the half-life of BACE2 is approximately 20 h. Lysosomal inhibition increased BACE2 protein levels whereas proteasomal inhibition had no effect on BACE2 protein expression. Furthermore, we identified that macroautophagy mediated BACE2 degradation. Finally, we showed that lysosomal inhibition increased BACE2 cleavage of APP. Taken together, our in vitro study showed that BACE2 is degraded through the macrophagy-lysosome pathway and that lysosomal inhibition affects BACE2 processing of APP. Modulation of BACE2 degradation via the lysosomal pathway could be a new target for AD drug development.

A variety of amphiphilic helical peptides have been shown to exhibit a transition from adsorbing parallel to a membrane surface at low concentrations to inserting perpendicularly into the membrane at high concentrations. Furthermore, this transition has been correlated to the peptides' cytolytic activities. X-ray lamellar diffraction of diphytanoyl phosphatidylcholine-alamethicin mixtures revealed the changes of the bilayer structure with alamethicin concentration. In particular, the bilayer thickness decreases with increasing peptide concentration in proportion to the peptide-lipid molar ratio from as low as 1:150 to 1:47; the latter is near the threshold of the critical concentration for insertion. From the decreases of the bilayer thickness, one can calculate the cross sectional expansions of the lipid chains. For all of the peptide concentrations studied, the area expansion of the chain region for each adsorbed peptide is a constant 280 +/- 20 A2, which is approximately the cross sectional area of an adsorbed alamethicin. This implies that the peptide is adsorbed at the interface of the hydrocarbon region, separating the lipid headgroups laterally. Interestingly, the chain disorder caused by a peptide adsorption tends to spread over a large area, as much as 100 A in diameter. The theoretical basis of the long range nature of bilayer deformation is discussed.

We conducted a meta-analysis to summarize the evidence from prospective studies regarding the association between physical activity and breast cancer risk. A comprehensive search was conducted to identify eligible studies. The fixed or random effect model was used based on heterogeneity test. The dose–response relationship was assessed by restricted cubic spline model and multivariate random-effect meta-regression. Overall, 31 studies with 63,786 cases were included, and the combined relative risk (RR) with 95 % CI of breast cancer was 0.88 (0.85–0.91). In subgroup analysis by activity type, data from 27 studies including 37,568 cases for non-occupational activity (including recreational activity and household activity) and seven studies including 28,268 cases for occupational activity were used, and the RR (95 % CI) of breast cancer was 0.87 (0.83–0.91) and 0.90 (0.83–0.97), respectively. The inverse association was consistent among all subgroups analyses. Stronger association was found for subjects with BMI

Previous studies have shown that uric acid (UA) has strong anti-oxidant properties, and that high circulating levels of UA are prospectively associated with improved muscle function and cognitive performances in elderly Caucasians. We carried out a replication study in elderly Chinese using cross-sectional design.Data from 2006 individuals aged 50-74 years who participated in a population-based cross-sectional survey in Qingdao, China, were analyzed. Hand grip strength was measured in kilograms by using an electronic dynamometer. The sit-to-stand (STS) test time was used to represent lower limb strength. The Mini-Mental State Examination (MMSE) was used to estimate the participants' cognitive function. Lifestyle, comorbidities and laboratory measures were considered as potential confounders. Multiple linear regression models and binary logistic regression were fitted to find the association of UA with strength measures and cognitive performances.Participants were grouped according to UA tertiles (257.75 mmol/L, ≥ 257.75 and ≤ 359.00 mmol/L,359.00 mmol/L). Hand grip strength significantly increased across UA tertiles (26.4 ± 8.5 kg; 30.1 ± 10.5 kg; 35.0 ± 11.4 kg; P0.001), and prevalence of cognitive disorder declined across UA tertiles (7.9%, 4.9%, 3.1%; P=0.012). After adjusting for potential confounders, high UA level remained significantly associated with high grip strength (P=0.023) and decreased risk of cognitive disorder with an OR of 1.002 (95% CI 1.000-1.004; P=0.022). However, UA level was not significantly associated with STS time (P=0.780).Our findings suggested that notwithstanding the associated increased risk of cardiovascular disease, UA might play a protective role in aging-associated decline in muscle strength and cognitive function.

A meta-analysis was performed to assess the association between the methylenetetrahydrofolate reductase (MTHFR) A1298C genetic polymorphism and ischemic stroke. A comprehensive search was conducted to identify all case-control or cohort studies. The fixed or random effect pooled measure was selected based on the homogeneity between studies, as assessed by I(2). Meta-regression was used to explore the potential sources of between-study heterogeneity. Publication bias was estimated using Egger's linear regression test. Thirteen case-control studies corresponded to the inclusion criteria comprising 2133 patients and 2572 controls which were included in the present meta-analysis. After excluding articles that deviated from Hardy-Weinberg equilibrium in controls and the key contributors to between-study heterogeneity, significant associations between MTHFR A1298C genetic polymorphism and risk of ischemic stroke were observed in dominant (odds ratio [OR] 1.227, 95% confidence interval [CI] 1.062-1.416) and codominant (OR 1.138, 95% CI 1.007-1.286) inheritance models. Moreover, in the subgroup analysis based on region (Asia and Europe), significant associations were observed in most genetic models in Asia but not in Europe. This meta-analysis suggests that MTHFR A1298C genetic polymorphism is associated with increased risk of ischemic stroke, and the C allele may be an important risk factor for ischemic stroke.

Results from the recent meta-analysis suggested a favorable effect of green tea consumption and risk of lung cancer, while no significant association was found between black tea consumption and risk of lung cancer. Besides, a significantly positive association was found between coffee consumption and risk of lung cancer. However, the relationship of green tea and coffee consumption is unclear. Thus the dose-response relationship was assessed by restricted cubic spline model and multivariate random-effect meta-regression. Results suggested that a linear dose-response relationship exists between coffee consumption and risk of lung cancer, while the dose-response relationship is nonlinear between green tea consumption and risk of lung cancer.

To explore the synergistic effect between diabetes family history and abnormal waist-to-hip ratio (WHR) on the incidence of type 2 diabetes.Population-based case-control study was conducted. The interaction between diabetes family history and abnormal WHR was analysed by an additive model.After adjusting confounding factors by multiple logistic regression analysis, the synergy index was 5.63, the attributable risk was 1.49, and the attributable interaction percent was 52.9%.There was a synergistic effect between diabetes family history and abnormal WHR on the incidence of type 2 diabetes.

The Ca2+ release channel of skeletal muscle sarcoplasmic reticulum is modulated in a biphasic manner by the plant alkaloid ryanodine and there are two distinct binding sites on this channel for ryanodine. The Ca2+ release channel is a homotetramer with a subunit of 5037 amino acids. The ability of sarcoplasmic reticulum membranes to bind [3H]ryanodine to the high affinity site is lost upon proteolysis with trypsin. [3H]Ryanodine, however, bound before proteolysis remains bound after trypsin digestion. If the high affinity site is first occupied with [3H]ryanodine and then 100 microM ryanodine is added to occupy the low affinity sites, almost all of [3H]ryanodine bound to the high affinity site remains bound after proteolysis. Proteolysis causes the solubilized Ca2+ release channel containing bound [3H]ryanodine to undergo four discrete shifts in sedimentation (30 S-->28 S-->26 S-->19 S-->14 S). Polypeptides having apparent molecular masses of 76, 66, 56, 45, 37, and 27 kDa can be identified in the 14 S complex. The 76-, 56-, 45-, and 27-kDa polypeptides have been partially sequenced from the NH2 terminus. In addition, the 76-, 66-, and 27-kDa fragments are recognized by an antibody to the last 9 amino acids at the carboxyl terminus of the skeletal muscle ryanodine receptor and the 76-, 66-, and 37-kDa fragments are recognized by an antibody to a peptide matching the sequence 4670-4685. The 56-kDa and the 45-kDa fragments are not Ca2+ release channel fragments. Both high and low affinity ryanodine binding sites are found in the 14 S complex and are, therefore, most likely located between Arg-4475 and the carboxyl terminus.

Using oriented circular dichroism, we have found that magainin adopts an α-helical conformation with two distinct orientations when interacting with a lipid bilayer. At low concentrations, magainin is adsorbed parallel to the membrane surface. However, at high concentrations, magainin is inserted into the membrane. This transition occurs at roughly the same critical concentration required for cytolytic activity, implying that the membrane insertion is responsible for magainin's cell-lysing activity.

By focusing on four health variables, handgrip strength, near visual acuity, tooth loss and hearing level, this study examined the different patterns of age-related changes in these variables in Chinese aged from 50 to 74 years, as well as explored the relationship among the variables in a cross-sectional sample of 2006 individuals. The data exhibited high quality with a low missing rate of under 5% in any age groups for each variable. Effects of age and sex on the changes in the four health variables were assessed using multiple regression models with age and sex interactions included. Upon the highly significant effects of age on all four measurements, we observed substantially higher grip strength for men who, however, exhibited a faster age-related decline than for women. No sex difference or age–sex interaction was found in the number of teeth lost. Near visual acuity displayed a faster age-related decline in women than in men but neither the overall sex difference nor age–sex interaction reached statistical significance. For hearing function, while no sex difference was found at middle frequency, women had better sensitivity at high frequency and men were more sensitive at low frequency. Multivariate analysis did not support an age-related common mechanism underlying the four health variables.

A new technology of strengthening reinforced concrete (RC) structures using near-surface mounted (NSM) carbon fiber-reinforced polymer (CFRP) strips was used and a total of 6 T-shaped RC beams were investigated by a new modified beam-shear test program in order to study the two key factors influencing bond behavior of NSM-CFRP strips to concrete, including: bond length and loading mode. The bond failure modes and bond mechanisms were discussed, leading to a number of suggestions for engineering use.

Supply chain is not just a continuous or a discrete system, but a continuous-discrete combined system. Existing supply chain dynamics models cannot show the continuous-discrete combined features. In this paper, Agent-based Model and System Dynamics Model are used in AnyLogic to do modeling and simulation of supply chain, which contains factory, distributer, retailer and consumer. Bullship effect is showed in the simulation results.

The phase problem of membrane diffraction is usually solved by the swelling method; however, this method does not always resolve the phases unambiguously. An alternative method of phase determination using anomalous dispersion is illustrated by the multiple-wavelength diffraction of membranes containing gramicidin ion channels. The anomalously scattering atoms are thallium ions bound to the channel. The result determines the location of the ion-binding sites in the gramicidin channel and the electron-density profile of the membrane. The applicability and limitation of the anomalous-dispersion method are discussed.

We report the first X-ray diffraction on gramicidin in its membrane-active form by using uniformly aligned multilayer samples of membranes containing gramicidin and ions (Tl + , K + , Ba 2+ , Mg 2+ or without ions). Prom the difference electron density profiles, we found a pair of symmetrically located ion-binding sites for Tl + at 9.6(±0.3) A and for Ba 2+ at 13.0(±0.2) A from the midpoint of the gramicidin channel. The location of Ba 2+ -binding sites is near the ends of the channel, consistent with the experimental observation that divalent cations do not permeate but block the channel. The location of Tl + -binding sites is somewhat of a surprise. It was generally thought that monovalent cations bind to the first turn of the helix from the mouth of the channel. (It is now generally accepted that the gramicidin channel is a cylindrical pore formed by two monomers, each a single-stranded β 6.3 helix and hydrogen-bonded head-to-head at their N termini.) But our experiment shows that the Tl + -binding site is either near the bottom of or below the first helix turn.

XPF-ERCC1, a structure-specific endonuclease, is involved in nucleotide excision repair, crosslink repair and homologous recombination. XPF-ERCC1 is also found to interact with TRF2, a duplex telomeric DNA binding protein. We have previously shown that XPF-ERCC1 is required for TRF2-promoted telomere shortening. However, whether XPF-ERCC1 by itself has a role in telomere length maintenance has not been determined. Here we report that overexpression of XPF induces telomere shortening in XPF-proficient cells whereas XPF complementation suppresses telomere lengthening in XPF-deficient cells. These results suggest that XPF-ERCC1 can function as a negative mediator of telomere length maintenance. In addition, we find that introduction of wild type XPF into XPF-deficient cells leads to over 40% reduction in TRF2 association with telomeric DNA, indicating that XPF-ERCC1 negatively regulates TRF2 binding to telomeric DNA. Furthermore, we show that XPF carrying mutations in the conserved nuclease domain fails to control TRF2 association with telomeric DNA but it is competent for modulating telomere length maintenance. These results imply that XPF-ERCC1 controls TRF2 and telomere length maintenance through two distinctive mechanisms, with the former requiring its nuclease activity. Our results further imply that TRF2 association with telomeres may be deregulated in cells derived from XPF patients.

We present a new method for determining the orientation of alpha-helical sections of proteins or peptides in membrane. To apply this method, membranes containing proteins must be prepared in a multilayer array. Circular dichroism (CD) spectra of the multilayer sample are then measured at the normal as well as oblique incident angles with respect to the bilayer planes; we call such spectra oriented circular dichroism (OCD). The procedure of OCD measurement, particularly the ways to avoid the spectral artifacts due to the effects of dielectric interfaces, linear dichroism and birefringence, and the method of data analysis are described in detail. To illustrate the method, we analyze the OCD of alamethicin in diphytanoylphosphatidylcholine multilayers. We conclude unambiguously that the helical section of alamethicin is parallel to the membrane normal when the sample is in the full-hydration state, but the helical section rotates to the plane of membrane when the sample is in a low-hydration state. We also obtained the parallel and perpendicular CD spectra of alpha-helix, and found them to be in agreement with previous theoretical calculations based on the exciton theory. These spectra are useful for analyzing protein orientations in future experiments.

Human telomeres are associated with ATM and the protein complex consisting of MRE11, RAD50 and NBS1 (MRN), which are central to maintaining genomic stability. Here we show that when targeted to telomeres, wild-type RAD50 downregulates telomeric association of TRF1, a negative regulator of telomere maintenance. TRF1 binding to telomeres is upregulated in cells deficient in NBS1 or under ATM inhibition. The TRF1 association with telomeres induced by ATM inhibition is abrogated in cells lacking MRE11 or NBS1, suggesting that MRN and ATM function in the same pathway controlling TRF1 binding to telomeres. The ability of TRF1 to interact with telomeric DNA in vitro is impaired by ATM-mediated phosphorylation. We propose that MRN is required for TRF1 phosphorylation by ATM and that such phosphorylation results in the release of TRF1 from telomeres, promoting telomerase access to the ends of telomeres.

The skeletal muscle Ca 2+ release channel (RYR1) is a homotetramer with subunits of 565 kD. Although the channel part of this protein is probably formed by the C-terminal one fifth of the protein, most of the regulation of channel activity is likely to arise from intermolecular and intramolecular interactions of its large cytoplasmic domain. This cytoplasmic region of the protein is thought to contain binding sites for a variety of modulators, including the t-tubule voltage sensor, and mutations in some cases of malignant hyperthermia and central core disease. Regulation of channel activity must, therefore, involve long-distance allosteric modulation arising from changes in both intersubunit and intrasubunit interactions within the cytoplasmic domain of the RYR1 tetramer. Biochemical studies are beginning to elucidate some of the sites within the cytoplasmic domains important for modulting channel activity in the membrane-spanning domain. This review summarizes these findings and presents a working model for the regulation of the channel by the interactions of its cytoplasmic domains.

Down Syndrome (DS) patients develop characteristic Alzheimer's Disease (AD) neuropathology after their middle age. Prominent neuronal loss has been observed in the cortical regions of AD brains. However, the underlying mechanism leading to this neuronal loss in both DS and AD remains to be elucidated. Calcium overloading and oxidative stress have been implicated in AD pathogenesis. Two major isoforms of regulator of calcineurin 1 (RCAN1), RCAN1.1 and RCAN1.4, are detected in human brains. In this report we defined the transcriptional regulation of RCAN1.1 and RCAN1.4 by two alternative promoters. Calcium overloading upregulated RCAN1.4 expression by activating RCAN1.4 promoter through calcineurin-NFAT signaling pathway, thus forming a negative feedback loop in isoform 4 regulation. Furthermore, RCAN1.4 overexpression exacerbated calcium overloading-induced neuronal apoptosis, which was mediated by caspase-3 apoptotic pathway. Our results suggest that downregulating RCAN1.4 expression in neurons could be beneficial to AD patients.

This study presents evidence for a close relationship between the oxidation state of the skeletal muscle Ca2+release channel (RyR1) and its ability to bind calmodulin (CaM). CaM enhances the activity of RyR1 in low Ca2+and inhibits its activity in high Ca2+. Oxidation, which activates the channel, blocks the binding of125I-labeled CaM at both micromolar and nanomolar Ca2+concentrations. Conversely, bound CaM slows oxidation-induced cross-linking between subunits of the RyR1 tetramer. Alkylation of hyperreactive sulfhydryls (2+-free125I-CaM but not Ca2+/125I-CaM binding. These studies suggest that 1) the sites on RyR1 for binding apocalmodulin have features distinct from those of the Ca2+/CaM site, 2) oxidation may alter the activity of RyR1 in part by altering its interaction with CaM, and 3) CaM may protect RyR1 from oxidative modifications during periods of oxidative stress.

We demonstrate a technique for measuring x-ray (or neutron) scattering with the momentum transfer confined in the plane of membrane, for the purpose of studying lateral organization of proteins and peptides in membrane. Unlike freeze-fracture electron microscopy or atomic force microscopy which requires the membrane to be frozen or fixed, in-plane x-ray scattering can be performed with the membrane maintained in the liquid crystalline state. As an example, the controversial question of whether gramicidin forms aggregates in membrane was investigated. We used dilauroylphosphatidylcholine (DLPC) bilayers containing gramicidin in the molar ratio of 10:1. Very clear scattering curves reflecting gramicidin channel-channel correlation were obtained, even for the sample containing no heavy atoms. Thallium ions bound to gramicidin channels merely increase the magnitude of the scattering curve. Analysis of the data shows that the channels were randomly distributed in the membrane, similar to a computer simulation of freely moving disks in a plane. We suggest that oriented proteins may provide substantial x-ray contrast against the lipid background without requiring heavy-atom labeling. This should open up many possible new experiments.

Whereas the barrel-stave configuration is accepted by most investigators as a good description of the conducting state of alamethicin, there are conflicting interpretations on its nonconducting state; in the absence of an applied field, some found alamethicin molecules on the membrane surface, but others found them incorporated in the hydrophobic core of the membrane. This problem is resolved by the discovery of a phase-transitionlike behavior of alamethicin in the membrane. As a function of lipid/peptide ratio L/P and the chemical potential of water mu, alamethicin molecules were observed to switch between two states: in one, the majority of the peptide molecules bind parallel to the membrane surface; in another, the majority of the peptide molecules insert perpendicularly into the membrane. The state of alamethicin was monitored by the method of oriented circular dichroism (OCD; Wu, Y., H. W. Huang, and G. A. Olah, 1990, Biophys. J. 57:797-806) using aligned multilayer samples in the liquid crystalline L(alpha) phase. If L/P exceeds a critical value, most of the peptide molecules are on the membrane surface. If L/P is below the critical value, most of the peptide molecules are incorporated in the membrane when mu is high; when mu is low, most of them are again on the membrane surface. In a typical conduction experiment of voltage dependence, alamethicin molecules are in a partition equilibrium between the aqueous phase and the lipid phase before the application of voltage; in the lipid phase, the lipid/peptide ratio is such that most of alamethicin molecules are on the membrane surface. This is the nonconducting state of alamethicin. The OCD analysis showed that there is essentially no change in the secondary structure when alamethicin changes between the surface state and the inserted state. The voltage-gating mechanism can be explained if we assume that these surface peptide molecules probabilistically turn into the membrane core to form channels due to the dipole-electric field interactions. We speculate that the phase-transitionlike behavior is a manifestation of membrane-mediated intermolecular interactions between peptide molecules.