An investigation of pectin and pectic acid in dilute aqueous solution

Light-scattering measurements have been carried out on carefully clarified aqueous solutions of pectin and pectic acid. The strong dependence of the inverse excess scattering intensity on angle yields pectin chain dimensions that are inconsistent with the complete molecular dispersion of pectin molecules possessing the degrees of polymerization indicated by the measured osmotic molecular weights. Negligible or negative concertration dependences of the inverse scattering intensities coupled with the centrifugation, filtration, and gel permeation chromatography behavior of the samples likewise suggest nonequilibrium aggregation of the pectin molecules in solution. It is shown that the measured mean polymer chain dimensions can be rationalized in terms of the presence of a very small proportion of aggregated material.     

Silver staining DNA in polyacrylamide gels

This protocol describes a simple silver staining method used to visualize DNA fragments and other organic molecules with unsurpassed detail following traditional polyacrylamide gel electrophoresis (PAGE). Sensitivity rivals radioisotopic methods and DNA in the picogram range can be reliably detected. The described protocol is fast (approximately 1 h) and is implemented using readily available chemicals and materials. To achieve the sensitivity and visual clarity expected, quality reagents and clean handling are important. The updated protocol described here is based on the widely used method of Bassam et al. (1991), but provides improved image contrast and less risk of staining artefacts.     

Novel Molecular Insights into Classical and Alternative Activation States of Microglia as Revealed by Stable Isotope Labeling by Amino Acids in Cell Culture (SILAC)-based Proteomics*

Microglia, the resident immune cells of the brain, have been shown to display a complex spectrum of roles that span from neurotrophic to neurotoxic depending on their activation status. Microglia can be classified into four stages of activation, M1, which most closely matches the classical (pro-inflammatory) activation stage, and the alternative activation stages M2a, M2b, and M2c. The alternative activation stages have not yet been comprehensively analyzed through unbiased, global-scale protein expression profiling. In this study, BV2 mouse immortalized microglial cells were stimulated with agonists specific for each of the four stages and total protein expression for 4644 protein groups was quantified using SILAC-based proteomic analysis. After validating induction of the various stages through a targeted cytokine assay and Western blotting of activation states, the data revealed novel insights into the similarities and differences between the various states. The data identify several protein groups whose expression in the anti-inflammatory, pro-healing activation states are altered presumably to curtail inflammatory activation through differential protein expression, in the M2a state including CD74, LYN, SQST1, TLR2, and CD14. The differential expression of these proteins promotes healing, limits phagocytosis, and limits activation of reactive nitrogen species through toll-like receptor cascades. The M2c state appears to center around the down-regulation of a key member in the formation of actin-rich phagosomes, SLP-76. In addition, the proteomic data identified a novel activation marker, DAB2, which is involved in clathrin-mediated endocytosis and is significantly different between M2a and either M1 or M2b states. Western blot analysis of mouse primary microglia stimulated with the various agonists of the classical and alternative activation states revealed a similar trend of DAB2 expression compared with BV2 cells.Microglia, along with astrocytes, form the backbone of the immune response in the brain. Microglia, in particular, comprise 10–15% of the brain, varying by region and predominating in areas of the midbrain such as the hippocampus and substantia nigra (1). Separated from the systemic immune system by the blood-brain barrier, the brain''s immune response relies on the ability of microglia to act as a multifaceted immune cell; microglia are able to sense pathogens, toxins, injury, and cytokine levels, as well as respond in a neurotrophic or neurotoxic manner similar to the macrophage in the systemic immune system (2).Microglia can respond to insult and injury in a neurotoxic manner (3, 4) where activated microglia are able to induce pro-inflammatory cytokines to recruit other microglia and astrocytes in response to bacterial infection and produce a wide and varied array of factors including reactive oxygen species (ROS)¹, and reactive nitrogen species (RNS), cytokines and lipid mediators as well as remove cellular debris as a post-infection response through phagocytosis (5). As such, microglia protect themselves from their own toxic products through a series of antioxidant proteins regulated through the actions of nuclear factor, erythroid 2-like 2 protein (NFE2L2) (6). Microglia have been implicated in a growing number of CNS-associated diseases; classically activated microglia have been found in brain regions afflicted with Parkinson''s disease, Alzheimer''s disease, and AIDS-related dementia (7–9). Microglial activation has also been reported to play a role in brain injury because of chronic alcohol exposure (10–13).Raivich et al. described microglia response and phases as a linear set of stages that microglia pass through in response to injury, pathogens, or antibodies from the systemic immune system that have crossed the blood-brain barrier (14). The first stage is a quiescent resting state, followed by an alert stage characterized by increased expression of integrin-binding proteins, or cell adhesion molecules, such as CD11b. The homing stage of activation that follows is characterized by increased cell mobility and adhesion as microglia target sites of injury or invasion. The fourth stage is a phagocytic stage that is often termed the classical microglia response, characterized by production of neurotoxic factors such as ROS through a cell membrane-bound NADPH oxidase complex and RNS through the action of inducible nitric oxide synthase, iNOS, as well as phagocytosis of cellular debris. The final stage, known as the bystander activation stage, potentiates the microglia response by activating additional microglia through the production and release of pro-inflammatory cytokines such as tumor necrosis factor alpha (TNFα), interferon gamma (IFNγ), and interleukin-6 (IL-6).Our understanding of the role of microglia has broadened in recent years to include neurotrophic as well as neurotoxic features (15, 16). The presence of activated microglia does not always correlate to an inflammatory state in the local brain region, implying a noninflammatory or possibly neurotrophic role for these microglia. Microglia that display multiple activation states have been observed in the brains of Alzheimer''s patients (17). It has been suggested that microglia that enter an inflammatory neurotoxic state first change into a neurotrophic healing response prior to returning to their quiescent resting phase (1). As such, a new schema to describe microglia phenotype was required. M1 phase, which can be triggered in vivo and in vitro by lipopolysaccharide (LPS) and inflammatory cytokines, has been established to describe classically activated microglial cells that are similar to those found in the fourth and fifth stages of Raivich''s microglial hierarchy. Microglia do not return to a resting state without first receiving anti-inflammatory triggers that are released by other microglia. These additional stages have been classified as alternative activation and have multiple healing responses. Microglia can be induced into the first alternative activation stage, M2a, through treatment with interleukin-4 (IL-4), and/or interleukin-13 (IL-13). M2a is a healing phase typified by tissue repair and growth stimulation through the actions of various extracellular matrix factors. Most importantly, M2a microglia act as an anti-inflammatory counterpart to M1 phase microglia by competing for arginine, a nitrogen pool for the production of RNS during M1 phase; M2a phase microglia compete for this pool through the production of arginase-1 (ARG1) which converts arginine into ornithine (18). M2b phase is a mixed activation state that responds to viral infection and activated antibodies characterized by the production of the pro-inflammatory cytokines, TNFα and IL-6, in addition to reduction of IL-12 and increased production of IL-10 (19). M2b phase microglia can be reproduced, in vitro, by treating with IL-1β and LPS concurrently or activated IgA complexes, which bind to Fcγ receptors. M2c phase microglia can be induced through IL-10 exposure in vivo and in vitro, and the emergence of M2c microglia shuts down microglial immune response.In order to study microglia in a laboratory setting, enriched ex vivo microglia, primary microglia, or immortalized cell lines are required. BV2 immortalized mouse microglia have been described as producing 41% of the cytokines and chemokines produced by ex vivo cells as compared with 96% coverage by primary microglia. However, Wilcock et al. showed that BV2 cells were successful at producing the classical activators for all four microglia activation stages as measured by real-time polymerase chain reaction (17). In addition, proteomic analysis of pathway level changes may be able to smooth over the lack of full expression through high levels of accurate protein quantification.Because of their importance in immune response and possible role in multiple disease states, a thorough investigation of the differential proteomic expression in the various microglial activation states is required. Using SILAC-labeled immortalized BV2 microglial cells treated with activators of the various activation stages, a proteome profile that includes the major canonical microglial pathways across all four activation states, providing crucial information as to where in these pathways of various states diverge, was established. In addition, using the differential protein expression data, a novel marker of microglia activation, DAB2, was identified and confirmed in primary mouse microglia through Western blot analysis. The abundance of this protein, as well as other differentially expressed proteins identified in this study, may prove as novel indicators in differentiating and categorizing activated microglia in the brain.     

Posttranslational Modifications in Connexins and Pannexins

Posttranslational modification is a common cellular process that is used by cells to ensure a particular protein function. This can happen in a variety of ways, e.g., from the addition of phosphates or sugar residues to a particular amino acid, ensuring proper protein life cycle and function. In this review, we assess the evidence for ubiquitination, glycosylation, phosphorylation, S-nitrosylation as well as other modifications in connexins and pannexin proteins. Based on the literature, we find that posttranslational modifications are an important component of connexin and pannexin regulation.     

Chol Understandings of Suicide and Human Agency

According to ethnographic material collected since 2003, the Chol Mayan indigenous people in southern Mexico have different causal explanations for suicide. It can be attributed to witchcraft that forces victims to take their lives against their own will, to excessive drinking, or to fate determined by God. However, it can also be conceived of as a conscious decision made by a person overwhelmed by daily problems. Drawing from the theoretical framework developed by Laura M. Ahearn, inspired by practice theory, the paper contends that these different explanations operate within two different logics or understandings of human agency. The first logic attributes responsibility to supernatural causes such as witchcraft or divine destiny, and reflects Chol notions of personhood. The second logic accepts personal responsibility for suicide, and is related to processes of social change such as the introduction of wage labor, education and a market economy. The contemporary Chol resort to both logics to make sense of the human drama of suicide.     

Nuclear receptor binding protein 1 regulates intestinal progenitor cell homeostasis and tumour formation

Genetic screens in simple model organisms have identified many of the key components of the conserved signal transduction pathways that are oncogenic when misregulated. Here, we identify H37N21.1 as a gene that regulates vulval induction in let-60(n1046gf), a strain with a gain-of-function mutation in the Caenorhabditis elegans Ras orthologue, and show that somatic deletion of Nrbp1, the mouse orthologue of this gene, results in an intestinal progenitor cell phenotype that leads to profound changes in the proliferation and differentiation of all intestinal cell lineages. We show that Nrbp1 interacts with key components of the ubiquitination machinery and that loss of Nrbp1 in the intestine results in the accumulation of Sall4, a key mediator of stem cell fate, and of Tsc22d2. We also reveal that somatic loss of Nrbp1 results in tumourigenesis, with haematological and intestinal tumours predominating, and that nuclear receptor binding protein 1 (NRBP1) is downregulated in a range of human tumours, where low expression correlates with a poor prognosis. Thus NRBP1 is a conserved regulator of cell fate, that plays an important role in tumour suppression.     

Gestation age-related increase in 50-kDa rat uterine calcium-independent phospholipase A2 expression influences uterine sensitivity to polychlorinated biphenyl stimulation

Phospholipase A2 (PLA2) enzymes catalyze the rate-limiting step in eicosanoid production by liberating arachidonic acid from membrane phospholipids. There is limited information regarding the expression pattern and activity of uterine PLA2 enzymes during pregnancy. Polychlorinated biphenyls (PCBs) are a group of persistent environmental toxicants previously associated with decreased gestation length that are capable of activating PLA2. The purpose of the present study was to determine whether uterine sensitivity to PCB stimulation is dependent on PLA2 expression, comparing rat uterine PLA2 expression in Gestational Day (gd) 10 versus gd20. Western blot analysis revealed a significant increase in the expression of calcium-dependent PLA2G2A and a 50-kDa protein immunoreactive to calcium-independent PLA2G6 antibody in gd20 compared to gd10 rat uterine tissue. The increased expression of the 50-kDa PLA2G6 was associated with a gestational age-related increase in endometrial calcium-independent PLA2 activity that was sensitive to inhibition by bromoenol lactone (P < 0.05). Longitudinal uterine strips isolated from gd10 or gd20 rat were suspended in muscle baths to evaluate uterine contractions following exposure to the ortho substituted congener PCB 50. Exposure to 50 and 100 microM PCB 50 significantly increased the frequency of gd20, but not gd10, uteri compared to solvent (dimethyl sulfoxide) controls (P < 0.05). Pharmacologic inhibition of PLA2G6, but not PLA2G2A, attenuated PCB-induced stimulation of gd20 uterine contractions (P < 0.05). These data suggest that PCB 50 stimulates uterine contractions by activating endometrial PLA2G6. Furthermore, gestation age-related sensitivity to PCB is associated with an increase in the expression of a previously unidentified 50-kDa PLA2G6 in rat uterus.