Alzheimer's disease: early alterations in brain DNA methylation at ANK1, BIN1, RHBDF2 and other loci.

We used a collection of 708 prospectively collected autopsied brains to assess the methylation state of the brain's DNA in relation to Alzheimer's disease (AD). We found that the level of methylation at 71 of the 415,848 interrogated CpGs was significantly associated with the burden of AD pathology, including CpGs in the ABCA7 and BIN1 regions, which harbor known AD susceptibility variants. We validated 11 of the differentially methylated regions in an independent set of 117 subjects. Furthermore, we functionally validated these CpG associations and identified the nearby genes whose RNA expression was altered in AD: ANK1, CDH23, DIP2A, RHBDF2, RPL13, SERPINF1 and SERPINF2. Our analyses suggest that these DNA methylation changes may have a role in the onset of AD given that we observed them in presymptomatic subjects and that six of the validated genes connect to a known AD susceptibility gene network.

Germline predictors of androgen deprivation therapy response in advanced prostate cancer.

OBJECTIVE: To evaluate whether germline variations in genes involved in sex steroid biosynthesis and metabolic pathways predict time to treatment failure for patients with advanced prostate cancer undergoing androgen deprivation therapy (ADT), because there are few known clinical predictors of response. PATIENTS AND METHODS: In a cohort of 304 patients with advanced prostate cancer undergoing ADT, we genotyped 746 single-nucleotide polymorphisms (SNPs) from 72 genes from germline DNA (680 tagSNPs from 58 genes and 66 candidate SNPs from 20 genes [6 genes common in both]). Association with the primary end point of time to ADT failure was assessed using proportional hazards regression models at the gene level (for genes with tagging SNPs) and at the SNP level. False discovery rates (FDRs) of 0.10 or less were considered noteworthy to account for multiple testing. RESULTS: At the gene level, TRMT11 showed the strongest association with time to ADT failure (P<.001; FDR=0.008). Two of 4 TRMT11 tagSNPs were associated with time to ADT failure. Median time to ADT failure for rs1268121 (A>G) was 3.05 years for the AA, 4.27 years for the AG, and 6.22 years for the GG genotypes (P=.002), and for rs6900796 (G>A), it was 2.42 years for the GG, 3.52 years for the AG, and 4.18 years for the AA genotypes (P<.001). No other gene level or SNP level tests had an FDR of 0.10 or less. CONCLUSION: Genetic variation in TRMT11 was associated with time to ADT failure. Confirmation of these preliminary findings in an independent cohort is needed.

Association of TNFSF8 polymorphisms with peripheral neutrophil count.

OBJECTIVE: To investigate the association between 347 single-nucleotide polymorphisms within candidate genes of the tumor necrosis factor, interleukin 1 and interleukin 6 families with neutrophil count. PATIENTS AND METHODS: Four hundred cases with heart failure after myocardial infarction (MI) were matched by age, sex, and date of incident MI to 694 controls (MI without post-MI heart failure). Both genotypes and neutrophil count at admission for incident MI were available in 314 cases and 515 controls. RESULTS: We found significant associations between the TNFSF8 poly morphisms rs927374 (P=5.1 x 10(-5)) and rs2295800 (P=1.3 x 10(-4)) and neutrophil count; these single-nucleotide polymorphisms are in high linkage disequilibrium (r(2)=0.97). Associations persisted after controlling for clinical characteristics and were unchanged after adjusting for case-control status. For rs927374, the neutrophil count of GG homozygotes (7.6±5.1) was 16% lower than that of CC homozygotes (9.0±5.2). CONCLUSION: The TNFSF8 polymorphisms rs927374 and rs2295800 were associated with neutrophil count. This finding suggests that post-MI inflammatory response is genetically modulated.

Efficacy of omega-3 fatty acids in mood disorders - a systematic review and metaanalysis.

OBJECTIVES: Existing efficacy trials of Omega-3 (omega-3) fatty acids in mood disorders have yielded inconsistent results. The current paper is an effort to provide a systematic review and meta-analysis to evaluate efficacy of omega-3 fatty acids in treatment of mood disorders. DESIGN: We searched Medline, Embase, PsychInfo, and the Cochrane Controlled Trials registry up to June 2008 for randomized trials investigating efficacy of omega-3 fatty acids in mood disorders.We conducted random effects meta-analyses.We used the I2 statistic to quantify between-study inconsistency, and conducted pre-specified subgroup analyses to explore potential explanations for inconsistency. OBSERVATIONS: We included 21 trials in our systematic review and found 13 trials to be eligible for meta-analysis. The pooled standardized mean difference in depressed mood states (n = 554 in 12 trials) was -0.47 (95% CI:-0.92,-0.02; I2 = 82.7; p = 0.07) and in manic mood states (n = 126 in 4 trials) was 0.22 (95% CI: -0.21, 0.65; I2 = 40.5; p = 0.31).We did not identify any treatment- subgroup interaction across forms of omega-3 fatty acids preparations (P = 0.99) or patient diagnosis (bipolar vs. unipolar depressive disorder; P = 0.96); there was a significant correlation between omega-3 fatty acids dose and treatment effect on depressive symptoms (r = 0.5, p = 0.04), but not on manic symptoms (P = 0.3). CONCLUSIONS: The available evidence suggests that omega-3 fatty acids are a potential treatment of depressive disorders, but not mania. The unexplained between-study inconsistency and imprecision of the pooled estimates mitigate this suggestion. Large randomized placebo-controlled trials are needed to better estimate the value of this intervention for patients with depression.

Tail-anchored protein biosynthesis at the endoplasmic reticulum: the same but different.

The post-translational integration of tail-anchored proteins at the endoplasmic reticulum represents a novel and distinct pathway for membrane protein synthesis. Studies of various precursors, exemplified by the synaptobrevins and cytochrome b5, indicate that multiple routes may facilitate their biosynthesis. There is clear evidence that both cytosolic factors and membrane components facilitate the efficient membrane insertion of at least some tail-anchored proteins. However, the nature of these mediators is currently unknown and their identification will be an essential step in defining the molecular basis of tail-anchored protein biogenesis.

Making membrane proteins at the mammalian endoplasmic reticulum.

Whereas protein biogenesis at the endoplasmic reticulum is well understood in the case of secretory proteins and simple membrane proteins, much less is known about the synthesis of multi-spanning integral membrane proteins. While it is clear that the multiple membrane-spanning domains of these proteins must be inserted into the lipid bilayer during biosynthesis, the mechanism by which their integration is achieved and their subsequent folding/assembly are poorly defined. In this review, we summarize our current understanding of protein synthesis at the endoplasmic reticulum and highlight specific features that are relevant to the biogenesis of multi-spanning membrane proteins.

The ABC's of staff retention.

The purpose of this article is to aid management in recognizing the key components to staff retention. Preparation for recruitment efforts, evaluating compensation, and establishing and maintaining good channels of communication are worthy undertakings for the purposes of reducing turnover. Combating turnover is really as easy as Appreciating your staff, rewarding the Behavior you want, and Continuing to ensure a work environment that is conducive to a place employees want to work. One of the key factors is listening. Do you hear what your staff is saying? Can you address their concerns? Do you communicate regularly with line staff? Are there ideas they have that can lead to improvements? Lead by example. If your staff sees your passion for the work, they will respect you and work hard to deliver what you have agreed are the goals on an individual and overall business objective level. Once you have established the aggregate levels that exist within your practice, you can move on to evaluating where each employee falls within the range.

Early events in glycosylphosphatidylinositol anchor addition. substrate proteins associate with the transamidase subunit gpi8p.

The addition of glycosylphosphatidylinositol (GPI) anchors to proteins occurs by a transamidase-catalyzed reaction mechanism soon after completion of polypeptide synthesis and translocation. We show that placental alkaline phosphatase becomes efficiently GPI-anchored when translated in the presence of semipermeabilized K562 cells but is not GPI-anchored in cell lines defective in the transamidase subunit hGpi8p. By studying the synthesis of placental alkaline phosphatase, we demonstrate that folding of the protein is not influenced by the addition of a GPI anchor and conversely that GPI anchor addition does not require protein folding. These results demonstrate that folding of the ectodomain and GPI addition are two distinct processes and can be mutually exclusive. When GPI addition is prevented, either by synthesis of the protein in the presence of cell lines defective in GPI addition or by mutation of the GPI carboxyl-terminal signal sequence cleavage site, the substrate forms a prolonged association with the transamidase subunit hGpi8p. The ability of the transamidase to recognize and associate with GPI anchor signal sequences provides an explanation for the retention of GPI-anchored protein within the ER in the absence of GPI anchor addition.

Glycoprotein folding in the endoplasmic reticulum: a tale of three chaperones?

The endoplasmic reticulum (ER) is a major site of protein synthesis and its inside, or lumen, is a major site of protein folding. The lumen of the ER contains many folding factors and molecular chaperones, which facilitate protein folding by increasing both the rate and the efficiency of this process. Amongst the many ER folding factors, there are three components that specifically modulate the folding glycoproteins bearing N-linked carbohydrate side chains. These components are calnexin, calreticulin and ERp57, and this review focuses on the molecular basis for their capacity to influence glycoprotein folding.

Disassembly of membrane-associated NSF 20S complexes is slow relative to vesicle fusion and is Ca(2+)-independent.

N-ethylmaleimide-sensitive fusion protein (NSF) and its co-factor soluble NSF attachment protein (alpha)-SNAP) are essential components of the synaptic vesicle fusion machinery and form part of a structurally-conserved 20S protein complex. However, their precise function, relative to fusion itself, is not clear. Using a UV-activated cross-linking approach, we have measured the rate at which a single round of NSF-driven ATP hydrolysis leads to 20S complex disassembly within synaptic membranes. Although this rate is substantially faster than previous estimates of NSF-dependent ATP hydrolysis, it remains much lower than published rates for fusion of synaptic vesicles. Furthermore, the stability of 20S complexes is unaffected by Ca(2+) at concentrations that elicit rapid membrane fusion. We conclude that the ATPase activity of NSF does not contribute directly to vesicle fusion, but more likely plays an earlier role in the synaptic vesicle cycle.

Protein targeting and translocation at the endoplasmic reticulum membrane--through the eye of a needle?

SRP-dependent and SRP-independent targeting routes deliver precursor proteins to the ER membrane translocon. These precursors are translocated into (for membrane proteins) and across (for secretory protein) the ER membrane via aqueous channels composed of oligomers of the Sec61 complex. Both ends of the ER translocon are 'gated' and the opening and closing of these gates are closely regulated. The lateral exit of hydrophobic polypeptide regions into the phospholipid bilayer also appears to be a carefully controlled process. Accessory components are transiently associated with active ER translocation sites and modify the nascent polypeptide as it appears on the luminal side of the membrane.

ERp57 functions as a subunit of specific complexes formed with the ER lectins calreticulin and calnexin.

ERp57 is a lumenal protein of the endoplasmic reticulum (ER) and a member of the protein disulfide isomerase (PDI) family. In contrast to archetypal PDI, ERp57 interacts specifically with newly synthesized glycoproteins. In this study we demonstrate that ERp57 forms discrete complexes with the ER lectins, calnexin and calreticulin. Specific ERp57/calreticulin complexes exist in canine pancreatic microsomes, as demonstrated by SDS-PAGE after cross-linking, and by native electrophoresis in the absence of cross-linking. After in vitro translation and import into microsomes, radiolabeled ERp57 can be cross-linked to endogenous calreticulin and calnexin while radiolabeled PDI cannot. Likewise, radiolabeled calreticulin is cross-linked to endogenous ERp57 but not PDI. Similar results were obtained in Lec23 cells, which lack the glucosidase I necessary to produce glycoprotein substrates capable of binding to calnexin and calreticulin. This observation indicates that ERp57 interacts with both of the ER lectins in the absence of their glycoprotein substrate. This result was confirmed by a specific interaction between in vitro synthesized calreticulin and ERp57 prepared in solution in the absence of other ER components. We conclude that ERp57 forms complexes with both calnexin and calreticulin and propose that it is these complexes that can specifically modulate glycoprotein folding within the ER lumen.

The Sec61 complex is located in both the ER and the ER-Golgi intermediate compartment.

The heteromeric Sec61 complex is composed of (alpha), beta and (gamma) subunits and forms the core of the mammalian ER translocon. Oligomers of the Sec61 complex form a transmembrane channel where proteins are translocated across and integrated into the ER membrane. We have studied the subcellular localisation of the Sec61 complex using both wild-type COS1 cells and cells transfected with GFP-tagged Sec61(alpha). By double labelling immunofluorescence microscopy the GFP-tagged Sec61(alpha) was found in both the ER and the ER-Golgi intermediate compartment (ERGIC) but not in the trans-Golgi network. Immunofluorescence studies of endogenous Sec61beta and Sec61(gamma) showed that these proteins are also located in both the ER and the ERGIC. Using the alternative strategy of subcellular fractionation, we have shown that wild-type Sec61(alpha), beta and (gamma), and GFP-tagged Sec61(alpha), are all present in both the ER and the ERGIC/Golgi fractions of the gradient. The presence of the Sec61 subunits in a post-ER compartment suggests that these proteins can escape the ER and be recycled back, despite the fact that none of them contain any known membrane protein retrieval signals such as cytosolic di-lysine or di-arginine motifs. We also found that another translocon component, the glycoprotein TRAM, was present in post-ER compartments as demonstrated by subcellular fractionation. Our data indicate that the core components of the mammalian ER translocon are not permanently resident in the ER, but rather that they are maintained in the ER by a specific retrieval mechanism.

The transient association of ERp57 with N-glycosylated proteins is regulated by glucose trimming.

The thiol-dependent reductase ERp57 has been shown to interact specifically with in vitro synthesised glycoproteins imported into canine pancreatic microsomes. On this basis, it was proposed that ERp57 forms part of a glycoprotein-specific folding 'machinery', present in the lumen of the endoplasmic reticulum (ER). In this study, we have investigated the interaction of ERp57 with newly synthesised proteins using semi-permeabilised mammalian cells (SP cells), in which the ER remains essentially intact and, hence, resembles that of a living cell. We demonstrate that ERp57 interacts preferentially with the glycosylated versions of soluble and membrane proteins, and that this interaction occurs in combination with calnexin and calreticulin. For the first time, we have performed a detailed analysis of the kinetics of ERp57 binding to newly synthesised glycoproteins. We find that ERp57 associates transiently with glycoproteins - a characteristic of molecular chaperones. Using mutant SP cells deficient in glucosidase I, we confirm that the binding of ERp57 to glycoproteins depends upon glucose trimming. We also demonstrate, for the first time, that the release of ERp57 from glycoprotein substrates is dependent upon glucose trimming. These data are combined to present a unified model for the role of ERp57/ER lectin complexes during glycoprotein folding in vivo.

Formation and turnover of NSF- and SNAP-containing "fusion" complexes occur on undocked, clathrin-coated vesicle-derived membranes.

Specificity of vesicular transport is determined by pair-wise interaction between receptors (SNAP receptors or SNAREs) associated with a transport vesicle and its target membrane. Two additional factors, N-ethylmaleimide-sensitive fusion protein (NSF) and soluble NSF attachment protein (SNAP) are ubiquitous components of vesicular transport pathways. However, the precise role they play is not known. On the basis that NSF and SNAP can be recruited to preformed SNARE complexes, it has been proposed that NSF- and SNAP-containing complexes are formed after SNARE-dependent docking of transport vesicles. This would enable ATPase-dependent complex disassembly to be coupled directly to membrane fusion. Alternatively, binding and release of NSF/SNAP may occur before vesicle docking, and perhaps be involved in the activation of SNAREs. To gain more information about the point at which so-called 20S complexes form during the transport vesicle cycle, we have examined NSF/SNAP/SNARE complex turnover on clathrin-coated vesicle-derived membranes in situ. This has been achieved under conditions in which the extent of membrane docking can be precisely monitored. We demonstrate by UV-dependent cross-linking experiments, coupled to laser light-scattering analysis of membranes, that complexes containing NSF, SNAP, and SNAREs will form and dissociate on the surface of undocked transport vesicles.

The Escherichia coli SRP and SecB targeting pathways converge at the translocon.

Two distinct protein targeting pathways can direct proteins to the Escherichia coli inner membrane. The Sec pathway involves the cytosolic chaperone SecB that binds to the mature region of pre-proteins. SecB targets the pre-protein to SecA that mediates pre-protein translocation through the SecYEG translocon. The SRP pathway is probably used primarily for the targeting and assembly of inner membrane proteins. It involves the signal recognition particle (SRP) that interacts with the hydrophobic targeting signal of nascent proteins. By using a protein cross-linking approach, we demonstrate here that the SRP pathway delivers nascent inner membrane proteins at the membrane. The SRP receptor FtsY, GTP and inner membranes are required for release of the nascent proteins from the SRP. Upon release of the SRP at the membrane, the targeted nascent proteins insert into a translocon that contains at least SecA, SecY and SecG. Hence, as appears to be the case for several other translocation systems, multiple targeting mechanisms deliver a variety of precursor proteins to a common membrane translocation complex of the E.coli inner membrane.

Membrane integration of Sec61alpha: a core component of the endoplasmic reticulum translocation complex.

The Sec61 complex is a central component of the endoplasmic reticulum (ER) translocation site. The complex consists of three subunits: Sec61alpha, Sec61beta and Sec61gamma, at least two of which (alpha and beta) are adjacent to nascent proteins during membrane insertion. Another component of the translocation machinery is the translocating chain-associating membrane (TRAM) protein, which is also adjacent to many nascent proteins during membrane insertion. Sec61alpha functions as the major component of a transmembrane channel formed by oligomers of the Sec61 complex. This channel is the site of secretory protein translocation and membrane protein integration at the ER membrane. Sec61alpha is a polytopic integral membrane protein, and we have studied its biosynthesis and membrane integration in vitro. Using a cross-linking approach to analyse the environment of a series of discrete Sec61alpha membrane-integration intermediates, we find: (i) newly synthesized Sec61alpha is adjacent to known components of the ER membrane-insertion site, namely Sec61alpha, Sec61beta and TRAM, and thus the integration of Sec61alpha appears to require a pre-existing Sec61 complex; (ii) a site-specific cross-linking analysis indicates that the first transmembrane domain of Sec61alpha remains adjacent to protein components of the ER-insertion site (specifically TRAM and Sec61beta) during the insertion of at least three subsequent transmembrane domains; and (iii) the membrane integration of Sec61alpha requires ER targeting by the signal-recognition particle.