A novel image cytometry-based Lactobacillus bacterial enumeration method for the production of kettle sour beer.

The demands for a variety of craft beer flavors have been increasing in the United States. To meet this rising demand, breweries have been experimenting with kettle sour beer that utilizes lactic acid-producing bacteria for fermentation. The current standard bacterial quantification method is insufficient for rigorous quality control, thus there is a need for a better method to monitor lactobacilli concentration in a kettle sour environment. In this work, an automated Lactobacillus counting method was developed using fluorescence-based image cytometry. Three commonly used species were cultured, the concentrations were measured using image cytometry and evaluated against the standard spread-plating method. This procedure was undertaken in vitro at different dilutions and the method was repeated with two species in a kettle sour environment at different time points. Both the in vitro and fermentation experiments were repeated three times. Results demonstrated that the new method was not significantly different when compared to the standard plating method in either controlled settings or within the kettle sour fermentation. The proposed method provides a rapid tool to monitor and control lactobacilli growth in kettle sour beer production, and allows for standardization of the products due to the availability of near instantaneous information for quality control.

Dual positive and negative regulation of GPCR signaling by GTP hydrolysis.

G protein-coupled receptors (GPCRs) regulate a variety of intracellular pathways through their ability to promote the binding of GTP to heterotrimeric G proteins. Regulator of G protein signaling (RGS) proteins increases the intrinsic GTPase activity of Galpha-subunits and are widely regarded as negative regulators of G protein signaling. Using yeast we demonstrate that GTP hydrolysis is not only required for desensitization, but is essential for achieving a high maximal (saturated level) response. Thus RGS-mediated GTP hydrolysis acts as both a negative (low stimulation) and positive (high stimulation) regulator of signaling. To account for this we generated a new kinetic model of the G protein cycle where Galpha(GTP) enters an inactive GTP-bound state following effector activation. Furthermore, in vivo and in silico experimentation demonstrates that maximum signaling output first increases and then decreases with RGS concentration. This unimodal, non-monotone dependence on RGS concentration is novel. Analysis of the kinetic model has revealed a dynamic network motif that shows precisely how inclusion of the inactive GTP-bound state for the Galpha produces this unimodal relationship.

Identification of caspase 3 motifs and critical aspartate residues in human phospholipase D1b and phospholipase D2a.

Stimulation of mammalian cells frequently initiates phospholipase D-catalyzed hydrolysis of phosphatidylcholine in the plasma membrane to yield phosphatidic acid (PA) a novel lipid messenger. PA plays a regulatory role in important cellular processes such as secretion, cellular shape change, and movement. A number of studies have highlighted that PLD-based signaling also plays a pro-mitogenic and pro-survival role in cells and therefore anti-apoptotic. We show that human PLD1b and PLD2a contain functional caspase 3 cleavage sites and identify the critical aspartate residues within PLD1b that affect its activation by phorbol esters and attenuate phosphatidylcholine hydrolysis during apoptosis.

E-cadherin and cell adhesion: a role in architecture and function in the pancreatic islet.

BACKGROUND/AIMS: The efficient secretion of insulin from beta-cells requires extensive intra-islet communication. The cell surface adhesion protein epithelial (E)-cadherin (ECAD) establishes and maintains epithelial tissues such as the islets of Langerhans. In this study, the role of ECAD in regulating insulin secretion from pseudoislets was investigated. METHODS: The effect of an immuno-neutralising ECAD on gross morphology, cytosolic calcium signalling, direct cell-to-cell communication and insulin secretion was assessed by fura-2 microfluorimetry, Lucifer Yellow dye injection and insulin ELISA in an insulin-secreting model system. RESULTS: Antibody blockade of ECAD reduces glucose-evoked changes in [Ca(2+)](i) and insulin secretion. Neutralisation of ECAD causes a breakdown in the glucose-stimulated synchronicity of calcium oscillations between discrete regions within the pseudoislet, and the transfer of dye from an individual cell within a cell cluster is attenuated in the absence of ECAD ligation, demonstrating that gap junction communication is disrupted. The functional consequence of neutralising ECAD is a significant reduction in insulin secretion. CONCLUSION: Cell adhesion via ECAD has distinct roles in the regulation of intercellular communication between beta-cells within islets, with potential repercussions for insulin secretion.

Dynamic interplay between the neutral glycosphingolipid CD77/Gb3 and the therapeutic antibody target CD20 within the lipid bilayer of model B lymphoma cells.

The centroblast-specific differentiation marker CD77 (Gb(3)), is the receptor for Shiga-like toxin (SLT). The dynamic relationship between Gb(3)/CD77 and key B-cell membrane proteins was studied in Burkitt's lymphoma cells with a focus on CD20. Engagement of Gb(3)/CD77 with SLT-B reduced the amount of CD20 and CXCR4 available, but levels of BCR, MHC Class II, CD21, CD27 and CD54 remained unchanged. Cholesterol depletion promoted a decrease in the number of sites accessed by CD20, CXCR4 and Gb(3)/CD77 antibodies. Constitutive localisation of Gb(3)/CD77 to lipid rafts was unperturbed by either SLT-B binding or cholesterol depletion, whereas the opposite was true for CD20. The effects were specific to SLT-B, highlighted by the inability of cholera toxin B-subunit to alter CD20 availability. Thus, the binding of Gb(3)/CD77 by its cognate ligand transmits information within the lipid bilayer of model lymphoma cells to impact the behaviour of selective proteins, most notably CD20, via a mechanism influenced by the level of cholesterol within the membrane.

The inhibitory region of troponin-I alters the ability of F-actin to interact with different segments of myosin.

Peptides corresponding to the N-terminus of skeletal myosin light chain 1 (rsMLC1 1-37) and the short loop of human cardiac beta-myosin (hcM398-414) have been shown to interact with skeletal F-actin by NMR and fluorescence measurements. Skeletal tropomyosin strengthens the binding of the myosin peptides to actin but does not interact with the peptides. The binding of peptides corresponding to the inhibitory region of cardiac troponin I (e.g. hcTnI128-153) to F-actin to form a 1 : 1 molar complex is also strengthened in the presence of tropomyosin. In the presence of inhibitory peptide at relatively lower concentrations the myosin peptides and a troponin I peptide C-terminal to the inhibitory region, rcTnI161-181, all dissociate from F-actin. Structural and fluorescence evidence indicate that the troponin I inhibitory region and the myosin peptides do not bind in an identical manner to F-actin. It is concluded that the binding of the inhibitory region of troponin I to F-actin produces a conformational change in the actin monomer with the result that interaction at different locations of F-actin is impeded. These observations are interpreted to indicate that a major conformational change occurs in actin on binding to troponin I that is fundamental to the regulatory process in muscle. The data are discussed in the context of tropomyosin's ability to stabilize the actin filament and facilitate the transmission of the conformational change to actin monomers not in direct contact with troponin I.

Breast cancer cell-derived EMMPRIN stimulates fibroblast MMP2 release through a phospholipase A(2) and 5-lipoxygenase catalyzed pathway.

Metalloproteinases (MMP) produced by both cancer and normal stromal fibroblast cells play a critical role in the metastatic spread of tumours, however little is known of the regulation of their release. In this report we demonstrate that breast cancer cells in culture release apparently full length soluble EMMPRIN that promotes the release of pro-MMP2 from fibroblasts. The generation of MMP2 is mediated by activation of phospholipase A(2) and 5-lipoxygenase. These results suggest that the production of soluble EMMPRIN, phospholipase A(2) and 5-lipoxygenase activities are sites for potential therapeutic intervention.

Antigen-stimulated activation of phospholipase D1b by Rac1, ARF6, and PKCalpha in RBL-2H3 cells.

Phospholipase D (PLD) activity can be detected in response to many agonists in most cell types; however, the pathway from receptor occupation to enzyme activation remains unclear. In vitro PLD1b activity is phosphatidylinositol 4,5-bisphosphate dependent via an N-terminal PH domain and is stimulated by Rho, ARF, and PKC family proteins, combinations of which cooperatively increase this activity. Here we provide the first evidence for the in vivo regulation of PLD1b at the molecular level. Antigen stimulation of RBL-2H3 cells induces the colocalization of PLD1b with Rac1, ARF6, and PKCalpha at the plasma membrane in actin-rich structures, simultaneously with cooperatively increasing PLD activity. Activation is both specific and direct because dominant negative mutants of Rac1 and ARF6 inhibit stimulated PLD activity, and surface plasmon resonance reveals that the regulatory proteins bind directly and independently to PLD1b. This also indicates that PLD1b can concurrently interact with a member from each regulator family. Our results show that in contrast to PLD1b's translocation to the plasma membrane, PLD activation is phosphatidylinositol 3-kinase dependent. Therefore, because inactive, dominant negative GTPases do not activate PLD1b, we propose that activation results from phosphatidylinositol 3-kinase-dependent stimulation of Rac1, ARF6, and PKCalpha.