A walnut-containing meal had similar effects on early satiety, CCK, and PYY, but attenuated the postprandial GLP-1 and insulin response compared to a nut-free control meal.

Regular nut consumption is associated with lower adiposity and reduced weight gain in adulthood. Walnut feeding studies have observed minimal effect on body weight despite potential additional energy intake. Several mechanisms may explain why consuming nuts promotes weight control, including increased early phase satiety, possibly reflected in postprandial response of gastrointestinal and pancreatic peptides hypothesized to affect appetite. The purpose of this study was to compare postprandial insulin, glucagon and gastrointestinal peptide response and satiety following a meal with ∼54% of energy from walnuts or cream cheese, using a within-subject crossover study design in overweight/obese adults (N = 28). Sixty minutes after the walnut-containing meal, glucagon-like peptide-1 was lower than after the reference meal (p=0.0433), and peptide YY, cholecystokinin and ghrelin did not differ after the two meals. Sixty and 120 min after the walnut-containing meal, pancreatic polypeptide (p = 0.0014 and p = 0.0002) and glucose-dependent insulinotropic peptide (p < 0.0001 and p = 0.0079) were lower than after the reference meal, and 120 min after the walnut-containing meal, glucagon was higher (p=0.0069). Insulin and C-peptide increased at 60 min in response to both meals but were lower at 120 min after the walnut-containing meal (p=0.0349 and 0.0237, respectively). Satiety measures were similar after both meals. These findings fail to support the hypothesis that acute postprandial gastrointestinal peptide response to a walnut-containing meal contributes to increased satiety. However, inclusion of walnuts attenuated the postprandial insulin response, which may contribute to the more favorable lipid profile observed in association with regular walnut consumption

Competition between bound and free peptides in an ELISA-based procedure that assays peptides derived from protein digests

BACKGROUND: We describe an ELISA-based method that can be used to identify and quantitate proteins in biological samples. In this method, peptides in solution, derived from proteolytic digests of the sample, compete with substrate-attached synthetic peptides for antibodies, also in solution, generated against the chosen peptides. The peptides used for the ELISA are chosen on the basis of their being (i) products of the proteolytic (e.g. tryptic) digestion of the protein to be identified and (ii) unique to the target protein, as far as one can know from the published sequences. RESULTS: In this paper we describe the competition assay and we define the optimal conditions for the most effective assay. We have performed an analysis of the kinetics of interaction between the four components of the assay: the plastic substratum to which the peptide is bound, the bound peptide itself, the competing added peptide, and the antibody that is specific for the peptide and we compare the results of theoretical simulations to the actual data in some model systems. CONCLUSION: The data suggest that the peptides bind to the plastic substratum in more than one conformation and that, once bound, the peptide displays different affinities for the antibody, depending on how it has bound to the plat

An ELISA-based procedure for assaying proteins in digests of human leukocytes and cell lines, using specifically selected peptides and appropriate antibodies

BACKGROUND: We describe the application of an ELISA-based assay (the Peptidomatrix) that can be used to simultaneously identify and quantitate a number of proteins in biological samples. The biological sample (blood component, biopsy, culture or other) is first lysed to release all the proteins, without any additional separation. The denatured proteins in the sample are then digested in bulk with the desired proteolytic enzyme(s). The peptides in the digest are then assayed by appropriate antibodies, using a competition ELISA protocol. RESULTS: As an example of its use, the present paper applies the Peptidomatrix to the assay of four membrane proteins MDR1 (P-glycoprotein or ABCB1), MRP1 (ABCC1), BCRP/MXR (ABCG2) and the alpha subunit of the Na, K_ATPase (ATP1A1), present in a number of cell lines and in human lymphocytes. We show that we can detect and quantitate these proteins, using a series of peptide-antibody pairs, and that we can differentiate between cell lines or cell preparations that express the target proteins and those that do not. CONCLUSION: We have devised a simple, ELISA-based proteomics assay that enables the quantitation of designated proteins in a cell or tissue sample, and that can be used in any laboratory, with minimal specialized equipment

A transcription factor contributes to pathogenesis and virulence in streptococcus pneumoniae

To date, the role of transcription factors (TFs) in the progression of disease for many pathogens is yet to be studied in detail. This is probably due to transient, and generally low expression levels of TFs, which are the central components controlling the expression of many genes during the course of infection. However, a small change in the expression or specificity of a TF can radically alter gene expression. In this study, we combined a number of quality-based selection strategies including structural prediction of modulated genes, gene ontology and network analysis, to predict the regulatory mechanisms underlying pathogenesis of Streptococcus pneumoniae (the pneumococcus). We have identified two TFs (SP_0676 and SP_0927 [SmrC]) that might control tissue-specific gene expression during pneumococcal translocation from the nasopharynx to lungs, to blood and then to brain of mice. Targeted mutagenesis and mouse models of infection confirmed the role of SP_0927 in pathogenesis and virulence, and suggests that SP_0676 might be essential to pneumococcal viability. These findings provide fundamental new insights into virulence gene expression and regulation during pathogenesis.Layla K. Mahdi, Esmaeil Ebrahimie, David L. Adelson, James C. Paton, Abiodun D. Ogunniy

Effects of deletion of the Streptococcus pneumoniae lipoprotein diacylglyceryl transferase gene lgt on ABC transporter function and on growth in vivo

Lipoproteins are an important class of surface associated proteins that have diverse roles and frequently are involved in the virulence of bacterial pathogens. As prolipoproteins are attached to the cell membrane by a single enzyme, prolipoprotein diacylglyceryl transferase (Lgt), deletion of the corresponding gene potentially allows the characterisation of the overall importance of lipoproteins for specific bacterial functions. We have used a Δlgt mutant strain of Streptococcus pneumoniae to investigate the effects of loss of lipoprotein attachment on cation acquisition, growth in media containing specific carbon sources, and virulence in different infection models. Immunoblots of triton X-114 extracts, flow cytometry and immuno-fluorescence microscopy confirmed the Δlgt mutant had markedly reduced lipoprotein expression on the cell surface. The Δlgt mutant had reduced growth in cation depleted medium, increased sensitivity to oxidative stress, reduced zinc uptake, and reduced intracellular levels of several cations. Doubling time of the Δlgt mutant was also increased slightly when grown in medium with glucose, raffinose and maltotriose as sole carbon sources. These multiple defects in cation and sugar ABC transporter function for the Δlgt mutant were associated with only slightly delayed growth in complete medium. However the Δlgt mutant had significantly reduced growth in blood or bronchoalveolar lavage fluid and a marked impairment in virulence in mouse models of nasopharyngeal colonisation, sepsis and pneumonia. These data suggest that for S. pneumoniae loss of surface localisation of lipoproteins has widespread effects on ABC transporter functions that collectively prevent the Δlgt mutant from establishing invasive infection

Reduced ventricular proliferation in the foetal cortex following maternal inflammation in the mouse

It has been well established that maternal inflammation during pregnancy alters neurological function in the offspring, but its impact on cortical development and long-term consequences on the cytoarchitecture is largely unstudied. Here we report that lipopolysaccharide-induced systemic maternal inflammation in C57Bl/6 mice at embryonic Day 13.5 of pregnancy, as early as 8 h after challenge, caused a significant reduction in cell proliferation in the ventricular zone of the developing cerebral cortex, as revealed by quantification of anti-phospho-Histone H3 immunoreactivity and bromodeoxyuridine pulse labelling. The angle of mitotic cleavage, determined from analysis of haematoxylin and eosin staining, cyclin E1 gene expression and the pattern of β-catenin immunoreactivity were also altered by the challenge, which suggests a change from symmetric to asymmetric division in the radial progenitor cells. Modifications of cortical lamination and gene expression patterns were detected at post-natal Day 8 suggesting prolonged consequences of these alterations during embryonic development. Cellular uptake of proteins from the cerebrospinal fluid was observed in brains from lipopolysaccharide-treated animals in radial progenitor cells. However, the foetal blood–brain barrier to plasma proteins remained intact. Together, these results indicate that maternal inflammation can disrupt the ventricular surface and lead to decreased cellular proliferation. Changes in cell density in Layers IV and V at post-natal Day 8 show that these initial changes have prolonged effects on cortical organization. The possible shift in the fate of progeny and the resulting alterations in the relative cell numbers in the cerebral cortex following a maternal inflammatory response shown here will require further investigation to determine the long-term consequences of inflammation on the development of neuronal circuitry and behaviour

Dynamic Computational Model Suggests That Cellular Citizenship Is Fundamental for Selective Tumor Apoptosis

Computational models in the field of cancer research have focused primarily on estimates of biological events based on laboratory generated data. We introduce a novel in-silico technology that takes us to the next level of prediction models and facilitates innovative solutions through the mathematical system. The model's building blocks are cells defined phenotypically as normal or tumor, with biological processes translated into equations describing the life protocols of the cells in a quantitative and stochastic manner. The essentials of communication in a society composed of normal and tumor cells are explored to reveal “protocols” for selective tumor eradication. Results consistently identify “citizenship properties” among cells that are essential for the induction of healing processes in a healthy system invaded by cancer. These properties act via inter-cellular communication protocols that can be optimized to induce tumor eradication along with system recovery. Within the computational systems, the protocols universally succeed in removing a wide variety of tumors defined by proliferation rates, initial volumes, and apoptosis resistant phenotypes; they show high adaptability for biological details and allow incorporation of population heterogeneity. These protocols work as long as at least 32% of cells obey extra-cellular commands and at least 28% of cancer cells report their deaths. This low percentage implies that the protocols are resilient to the suboptimal situations often seen in biological systems. We conclude that our in-silico model is a powerful tool to investigate, to propose, and to exercise logical anti-cancer solutions. Functional results should be confirmed in a biological system and molecular findings should be loaded into the computational model for the next level of directed experiments

Association of CD40 Gene Polymorphisms with Sporadic Breast Cancer in Chinese Han Women of Northeast China

BACKGROUND: Breast cancer is a polygenetic disorder with a complex inheritance pattern. Single nucleotide polymorphisms (SNPs), the most common genetic variations, influence not only phenotypic traits, but also interindividual predisposition to disease, treatment outcomes with drugs and disease prognosis. The co-stimulatory molecule CD40 plays a prominent role in immune regulation and homeostasis. Accumulating evidence suggests that CD40 contributes to the pathogenesis of cancer. Here, we set out to test the association between polymorphisms in the CD40 gene and breast carcinogenesis and tumor pathology. METHODOLOGY AND PRINCIPAL FINDINGS: Four SNPs (rs1800686, rs1883832, rs4810485 and rs3765459) were genotyped by the polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) method in a case-control study including 591 breast cancer patients and 600 age-matched healthy controls. Differences in the genotypic distribution between breast cancer patients and healthy controls were analyzed by the Chi-square test for trends. Our preliminary data showed a statistically significant association between the four CD40 gene SNPs and sporadic breast cancer risk (additive P = 0.0223, 0.0012, 0.0013 and 0.0279, respectively). A strong association was also found using the dominant, recessive and homozygote comparison genetic models. In the clinical features analysis, significant associations were observed between CD40 SNPs and lymph node metastasis, human epidermal growth factor receptor 2 (C-erbB2), estrogen receptor (ER), progesterone receptor (PR) and tumor protein 53 (P53) statuses. In addition, our haplotype analysis indicated that the haplotype C(rs1883832)G(rs4810485), which was located within the only linkage disequilibrium (LD) block identified, was a protective haplotype for breast cancer, whereas T(rs1883832)T(rs4810485) increased the risk in the studied population, even after correcting the P value for multiple testing (P = 0.0337 and 0.0430, respectively). CONCLUSIONS AND SIGNIFICANCE: Our findings primarily show that CD40 gene polymorphisms contribute to sporadic breast cancer risk and have a significant association with clinicopathological features among Chinese Han women from the Heilongjiang Province

Evaluation of Cell Cycle Arrest in Estrogen Responsive MCF-7 Breast Cancer Cells: Pitfalls of the MTS Assay

Endocrine resistance is a major problem with anti-estrogen treatments and how to overcome resistance is a major concern in the clinic. Reliable measurement of cell viability, proliferation, growth inhibition and death is important in screening for drug treatment efficacy in vitro. This report describes and compares commonly used proliferation assays for induced estrogen-responsive MCF-7 breast cancer cell cycle arrest including: determination of cell number by direct counting of viable cells; or fluorescence SYBR®Green (SYBR) DNA labeling; determination of mitochondrial metabolic activity by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay; assessment of newly synthesized DNA using 5-ethynyl-2′-deoxyuridine (EdU) nucleoside analog binding and Alexa Fluor® azide visualization by fluorescence microscopy; cell-cycle phase measurement by flow cytometry. Treatment of MCF-7 cells with ICI 182780 (Faslodex), FTY720, serum deprivation or induction of the tumor suppressor p14ARF showed inhibition of cell proliferation determined by the Trypan Blue exclusion assay and SYBR DNA labeling assay. In contrast, the effects of treatment with ICI 182780 or p14ARF-induction were not confirmed using the MTS assay. Cell cycle inhibition by ICI 182780 and p14ARF-induction was further confirmed by flow cytometric analysis and EdU-DNA incorporation. To explore this discrepancy further, we showed that ICI 182780 and p14ARF-induction increased MCF-7 cell mitochondrial activity by MTS assay in individual cells compared to control cells thereby providing a misleading proliferation readout. Interrogation of p14ARF-induction on MCF-7 metabolic activity using TMRE assays and high content image analysis showed that increased mitochondrial activity was concomitant with increased mitochondrial biomass with no loss of mitochondrial membrane potential, or cell death. We conclude that, whilst p14ARF and ICI 182780 stop cell cycle progression, the cells are still viable and potential treatments utilizing these pathways may contribute to drug resistant cells. These experiments demonstrate how the combined measurement of metabolic activity and DNA labeling provides a more reliable interpretation of cancer cell response to treatment regimens