Screening of soy and milk protein hydrolysates for their ability to activate the CCK1 receptor

The cholecystokinin receptor-type 1 (CCK1R) is a G-protein coupled receptor localized in the animal gastrointestinal tract. Receptor activation by the natural peptide ligand CCK leads to a feeling of satiety. In this study, hydrolysates from soy and milk proteins were evaluated for their potential to activate the CCK1R, assuming that bioactive peptides with a satiogenic effect can be used as an effective therapeutic strategy for obesity. Different protein hydrolysates were screened with a cell-based bioassay, which relies on the generation of a fluorescent signal upon receptor activation. Fluorescence was monitored using a fluorescence plate reader and confocal microscopy. Results from the fluorescence plate reader were biased by background autofluorescence of the protein hydrolysate matrices, which makes the fluorescence plate reader inappropriate for the evaluation of complex formulations. Measurements with the confocal microscope resulted in reliable and specific results. The latter approach showed that the gastrointestinal digested 7S fraction of soy protein demonstrates CCK1R activity.     

Expert System for Computer-assisted Annotation of MS/MS Spectra

An important step in mass spectrometry (MS)-based proteomics is the identification of peptides by their fragment spectra. Regardless of the identification score achieved, almost all tandem-MS (MS/MS) spectra contain remaining peaks that are not assigned by the search engine. These peaks may be explainable by human experts but the scale of modern proteomics experiments makes this impractical. In computer science, Expert Systems are a mature technology to implement a list of rules generated by interviews with practitioners. We here develop such an Expert System, making use of literature knowledge as well as a large body of high mass accuracy and pure fragmentation spectra. Interestingly, we find that even with high mass accuracy data, rule sets can quickly become too complex, leading to over-annotation. Therefore we establish a rigorous false discovery rate, calculated by random insertion of peaks from a large collection of other MS/MS spectra, and use it to develop an optimized knowledge base. This rule set correctly annotates almost all peaks of medium or high abundance. For high resolution HCD data, median intensity coverage of fragment peaks in MS/MS spectra increases from 58% by search engine annotation alone to 86%. The resulting annotation performance surpasses a human expert, especially on complex spectra such as those of larger phosphorylated peptides. Our system is also applicable to high resolution collision-induced dissociation data. It is available both as a part of MaxQuant and via a webserver that only requires an MS/MS spectrum and the corresponding peptides sequence, and which outputs publication quality, annotated MS/MS spectra (www.biochem.mpg.de/mann/tools/). It provides expert knowledge to beginners in the field of MS-based proteomics and helps advanced users to focus on unusual and possibly novel types of fragment ions.In MS-based proteomics, peptides are matched to peptide sequences in databases using search engines (1–3). Statistical criteria are established for accepted versus rejected peptide spectra matches based on the search engine score, and usually a 99% certainty is required for reported peptides. The search engines typically only take sequence specific backbone fragmentation into account (i.e. a, b, and y ions) and some of their neutral losses. However, tandem mass spectra—especially of larger peptides—can be quite complex and contain a number of medium or even high abundance peptide fragments that are not annotated by the search engine result. This can result in uncertainty for the user—especially if only relatively few peaks are annotated—because it may reflect an incorrect identification. However, the most common cause of unlabeled peaks is that another peptide was present in the precursor selection window and was cofragmented. This has variously been termed “chimeric spectra” (4–6), or the problem of low precursor ion fraction (PIF)¹ (7). Such spectra may still be identifiable with high confidence. The Andromeda search engine in MaxQuant, for instance, attempts to identify a second peptide in such cases (8, 9). However, even “pure” spectra (those with a high PIF) often still contain many unassigned peaks. These can be caused by different fragment types, such as internal ions, single or combined neutral losses as well as immonium and other ion types in the low mass region. A mass spectrometric expert can assign many or all of these peaks, based on expert knowledge of fragmentation and manual calculation of fragment masses, resulting in a higher degree of confidence for the identification. However, there are more and more practitioners of proteomics without in depth training or experience in annotating MS/MS spectra and such annotation would in any case be prohibitive for hundreds of thousands of spectra. Furthermore, even human experts may wrongly annotate a given peak—especially with low mass accuracy tandem mass spectra—or fail to consider every possibility that could have resulted in this fragment mass.Given the desirability of annotating fragment peaks to the highest degree possible, we turned to “Expert Systems,” a well-established technology in computer science. Expert Systems achieved prominence in the 1970s and 1980s and were meant to solve complex problems by reasoning about knowledge (10, 11). Interestingly, one of the first examples was developed by Nobel Prize winner Joshua Lederberg more than 40 years ago, and dealt with the interpretation of mass spectrometric data. The program''s name was Heuristic DENTRAL (12), and it was capable of interpreting the mass spectra of aliphatic ethers and their fragments. The hypotheses produced by the program described molecular structures that are plausible explanations of the data. To infer these explanations from the data, the program incorporated a theory of chemical stability that provided limiting constraints as well as heuristic rules.In general, the aim of an Expert System is to encode knowledge extracted from professionals in the field in question. This then powers a rule-based system that can be applied broadly and in an automated manner. A rule-based Expert System represents the information obtained from human specialists in the form of IF-THEN rules. These are used to perform operations on input data to reach appropriate conclusion. A generic Expert System is essentially a computer program that provides a framework for performing a large number of inferences in a predictable way, using forward or backward chains, backtracking, and other mechanisms (13). Therefore, in contrast to statistics based learning, the “expert program” does not know what it knows through the raw volume of facts in the computer''s memory. Instead, like a human expert, it relies on a reasoning-like process of applying an empirically derived set of rules to the data.Here we implemented an Expert System for the interpretation for high mass accuracy tandem mass spectrometry data of peptides. It was developed in an iterative manner together with human experts on peptide fragmentation, using the published literature on fragmentation pathways as well as large data sets of higher-energy collisional dissociation (HCD) (14) and collision-induced dissociation (CID) based peptide identifications. Our goal was to achieve an annotation performance similar or better than experienced mass spectrometrists (15), thus making comprehensively annotated peptide spectra available in large scale proteomics.     

Babies sleeping with parents: case-control study of factors influencing the risk of the sudden infant death syndrome. CESDI SUDI research group

ObjectiveTo investigate the risks of the sudden infant death syndrome and factors that may contribute to unsafe sleeping environments.DesignThree year, population based case-control study. Parental interviews were conducted for each sudden infant death and for four controls matched for age, locality, and time of sleep.SettingFive regions in England with a total population of over 17 million people.Subjects325 babies who died and 1300 control infants.ResultsIn the multivariate analysis infants who shared their parents'' bed and were then put back in their own cot had no increased risk (odds ratio 0.67; 95% confidence interval 0.22 to 2.00). There was an increased risk for infants who shared the bed for the whole sleep or were taken to and found in the parental bed (9.78; 4.02 to 23.83), infants who slept in a separate room from their parents (10.49; 4.26 to 25.81), and infants who shared a sofa (48.99; 5.04 to 475.60). The risk associated with being found in the parental bed was not significant for older infants (>14 weeks) or for infants of parents who did not smoke and became non-significant after adjustment for recent maternal alcohol consumption (>2 units), use of duvets (>4 togs), parental tiredness (infant slept ⩽4 hours for longest sleep in previous 24 hours), and overcrowded housing conditions (>2 people per room of the house).ConclusionsThere are certain circumstances when bed sharing should be avoided, particularly for infants under four months old. Parents sleeping on a sofa with infants should always be avoided. There is no evidence that bed sharing is hazardous for infants of parents who do not smoke.

Key messsages

• Cosleeping with an infant on a sofa was associated with a particularly high risk of sudden infant death syndrome
• Sharing a room with the parents was associated with a lower risk
• There was no increased risk associated with bed sharing when the infant was placed back in his or her cot
• Among parents who do not smoke or infants older than 14 weeks there was no association between infants being found in the parental bed and an increased risk of sudden infant death syndrome
• The risk linked with bed sharing among younger infants seems to be associated with recent parental consumption of alcohol, overcrowded housing conditions, extreme parental tiredness, and the infant being under a duvet

     

Catalytic site-directed gamma-secretase complex inhibitors do not discriminate pharmacologically between Notch S3 and beta-APP cleavages

The generation of gamma-secretase inhibitors which block the release of beta-amyloid peptide (Abeta) has long been an attractive therapeutic avenue for treatment or prevention of Alzheimer's disease (AD). Such inhibitors would reduce levels of Abeta available for aggregation into toxic assemblies that lead to the plaque pathology found in affected brain tissue. Cumulative evidence suggests that the S3 cleavage of Notch is also dependent on presenilins (PS) and is carried out by the multimeric PS-containing gamma-secretase complex. It is therefore possible that Notch function could be affected by gamma-secretase inhibitors. To assess the relationship between the cleavage of these substrates in the same system, Western blot cleavage assays have been established using a human cell line stably expressing both the beta-amyloid precursor protein (beta-APP) and the truncated Notch1 receptor fragment NotchDeltaE. Thus, a direct correlation may be made, following inhibitor treatment, of the decrease in the levels of the cleavage products, Abeta peptide and the Notch intracellular domain (NICD), as well as the increase in stabilized levels of both substrates. This analysis has been performed with a range of selected gamma-secretase inhibitors from six distinct structural classes. Changes in all four species usually occur in concert and with remarkably good agreement. A significant cleavage window is not clearly apparent in any case. Thus, these Notch and beta-APP cleavages cannot be dissected apart easily since they show the same pharmacological profile of inhibition. Whether this translates into proportionally reduced Notch signaling in vivo, however, remains to be seen.     

The regulative capacity of prespore amoebae as demonstrated by fluorescence-activated cell sorting and green fluorescent protein

The ability of prespore Dictyostelium discoideum amoebae to undergo redifferentiation so as to reestablish normal spore/stalk proportioning has been demonstrated in various ways over the years, beginning with the classic microdissection work of K. Raper. The discovery of anterior-like cells in the slug posterior, however, cast doubt on that ability, and more recent experiments using a cell-specific toxin suggested that prespore redifferentiation may not in fact occur. To reexamine this question, we performed fluorescence-activated cell sorting (FACS) upon amoebae expressing a mutated green fluorescent protein gene (S65T-GFP) under the control of a prespore-specific (PsA) promoter. FACS produced prespore cell populations with purities, measured by GFP expression, as high as 99. 5%. Sorted GFP(+) cells were developmentally competent and produced normally proportioned fruits, indistinguishable from those of "sham-sorted" (permissively gated, mixed GFP(+) and GFP(-)) amoebae. This result confirms the developmental totipotency of prespore amoebae.     

Partial uracil–DNA–glycosylase treatment for screening of ancient DNA

The challenge of sequencing ancient DNA has led to the development of specialized laboratory protocols that have focused on reducing contamination and maximizing the number of molecules that are extracted from ancient remains. Despite the fact that success in ancient DNA studies is typically obtained by screening many samples to identify a promising subset, ancient DNA protocols have not, in general, focused on reducing the time required to screen samples. We present an adaptation of a popular ancient library preparation method that makes screening more efficient. First, the DNA extract is treated using a protocol that causes characteristic ancient DNA damage to be restricted to the terminal nucleotides, while nearly eliminating it in the interior of the DNA molecules, allowing a single library to be used both to test for ancient DNA authenticity and to carry out population genetic analysis. Second, the DNA molecules are ligated to a unique pair of barcodes, which eliminates undetected cross-contamination from this step onwards. Third, the barcoded library molecules include incomplete adapters of short length that can increase the specificity of hybridization-based genomic target enrichment. The adapters are completed just before sequencing, so the same DNA library can be used in multiple experiments, and the sequences distinguished. We demonstrate this protocol on 60 ancient human samples.     

Effects of temozolomide (TMZ) on the expression and interaction of heat shock proteins (HSPs) and DNA repair proteins in human malignant glioma cells

We previously reported the association of HSPA1A and HSPB1 with high-grade astrocytomas, suggesting that these proteins might be involved in disease outcome and response to treatment. With the aim to better understand the resistance/susceptibility processes associated to temozolomide (TMZ) treatment, the current study was performed in three human malignant glioma cell lines by focusing on several levels: (a) apoptotic index and senescence, (b) DNA damage, and (c) interaction of HSPB1 with players of the DNA damage response. Three human glioma cell lines, Gli36, U87, and DBTRG, were treated with TMZ evaluating cell viability and survival, apoptosis, senescence, and comets (comet assay). The expression of HSPA (HSPA1A and HSPA8), HSPB1, O₆-methylguanine-DNA methyltransferase (MGMT), MLH1, and MSH2 was determined by immunocytochemistry, immunofluorescence, and Western blot. Immunoprecipitation was used to analyze protein interaction. The cell lines exhibited differences in viability, apoptosis, and senescence after TMZ administration. We then focused on Gli36 cells (relatively unstudied) which showed very low recovery capacity following TMZ treatment, and this was related to high DNA damage levels; however, the cells maintained their viability. In these cells, MGMT, MSH2, HSPA, and HSPB1 levels increased significantly after TMZ administration. In addition, MSH2 and HSPB1 proteins appeared co-localized by confocal microscopy. This co-localization increased after TMZ treatment, and in immunoprecipitation analysis, MSH2 and HSPB1 appeared interacting. In contrast, HSPB1 did not interact with MGMT. We show in glioma cells the biological effects of TMZ and how this drug affects the expression levels of heat shock proteins (HSPs), MGMT, MSH2, and MLH1. In Gli36 cells, the results suggest that interactions between HSPB1 and MSH2, including co-nuclear localization, may be important in determining cell sensitivity to TMZ.     

Selected non-steroidal anti-inflammatory drugs and their derivatives target gamma-secretase at a novel site. Evidence for an allosteric mechanism

Gamma-secretase is a multi-component enzyme complex that performs an intramembranous cleavage, releasing amyloid-beta (Abeta) peptides from processing intermediates of the beta-amyloid precursor protein. Because Abeta peptides are thought to be causative for Alzheimer's disease, inhibiting gamma-secretase represents a potential treatment for this neurodegenerative condition. Whereas inhibitors directed at the active center of gamma-secretase inhibit the cleavage of all its substrates, certain non-steroidal antiinflammatory drugs (NSAIDs) have been shown to selectively reduce the production of the more amyloidogenic Abeta(1-42) peptide without inhibiting alternative cleavages. In contrast to the majority of previous studies, however, we demonstrate that in cell-free systems the mode of action of selected NSAIDs and their derivatives, depending on the concentrations used, can either be classified as modulatory or inhibitory. At modulatory concentrations, a selective and, with respect to the substrate, noncompetitive inhibition of Abeta(1-42) production was observed. At inhibitory concentrations, on the other hand, biochemical readouts reminiscent of a nonselective gamma-secretase inhibition were obtained. When these compounds were analyzed for their ability to displace a radiolabeled, transition-state analog inhibitor from solubilized enzyme, noncompetitive antagonism was observed. The allosteric nature of radioligand displacement suggests that NSAID-like inhibitors change the conformation of the gamma-secretase enzyme complex by binding to a novel site, which is discrete from the binding site for transition-state analogs and therefore distinct from the catalytic center. Consequently, drug discovery efforts aimed at this site may identify novel allosteric inhibitors that could benefit from a wider window for inhibition of gamma (42)-cleavage over alternative cleavages in the beta-amyloid precursor protein and, more importantly, alternative substrates.