Search

Increasing the operating temperature has been recommended for controlling opportunistic premise plumbing pathogen (OPPP) growth in buildings. However, tradeoffs with operation and both disinfectant residuals and disinfection byproducts (DBPs) are not well understood. Water heater storage units were operated to evaluate the impact of use pattern (high and low), operation temperature (∼45 °C and ∼60 °C) and disinfectant type (chlorine and chloramines) on disinfectant residual and DBP concentrations at the showerhead point of use (POU). Control studies with chlorinated water yielded a loss of residual by 72 hours at 22 °C and 3 hours at 48 °C and 60 °C. In the tank heater studies, free chlorine residuals decayed to below the limit of quantitation (LOQ) for all POU samples. The concentrations of total trihalomethanes (TTHMs) and five haloacetic acids (HAA5) at the POU and control sample locations increased by factors of 2.01 ± 0.21 and 1.46 ± 0.11, respectively, relative to the feed and peaked at the first point that the chlorine residual was below the LOQ, indicating a chlorine-limited reaction. The TTHM concentrations did not change thereafter, just as the HAA concentrations remained unchanged at 45 °C, but the HAA concentrations decreased at 60 °C, indicating thermal degradation. Strong decay patterns were found for four haloacetonitriles (HAN4) at elevated temperatures, and the decay was faster at 60 °C. In the chloramination system, total chlorine decreased passing through the tank, but a detectable residual was measured in all samples. No additional DBP formation relative to the feed was found. Under all scenarios, there was no indication of a tradeoff between controlling OPPPs and DBP formation when increasing the operating temperature to 60 °C.

The 14-3-3 proteins are a family of phosphoserine/phosphothreonine-binding molecules that control the function of a wide array of cellular proteins. We suggest that one function of 14-3-3 is to support cell survival. 14-3-3 proteins promote survival in part by antagonizing the activity of associated proapoptotic proteins, including Bad and apoptosis signal-regulating kinase 1 (ASK1). Indeed, expression of 14-3-3 inhibitor peptides in cells is sufficient to induce apoptosis. Interestingly, these 14-3-3 antagonist peptides can sensitize cells for effective killing by anticancer agents such as cisplatin. Thus, 14-3-3 may be part of the cellular machinery that maintains cell survival, and targeting 14-3-3-ligand interactions may be a useful strategy to enhance the efficacy of conventional anticancer agents.

14-3-3 proteins are a family of multifunctional phosphoserine binding molecules that can serve as effectors of survival signaling. Understanding the molecular basis for the prosurvival effect of 14-3-3 may lead to the development of agents useful in the treatment of disorders involving dysregulated apoptosis. One target of 14-3-3 is the proapoptotic Bcl-2 family member Bad. Serine phosphorylation of Bad is associated with 14-3-3 binding and inhibition of Bad-induced cell death, but the relative contributions of the three known phosphorylation sites to 14-3-3 binding have not been established. Here we demonstrate that S136 of Bad is vital for 14-3-3 interaction, but S112 seems to be dispensable. 14-3-3/Bad interaction was strictly dependent on the presence of phosphorylated S136 in vitro, in yeast, and in mammalian cells. However, mutation of S112 did not affect 14-3-3 binding. The death caused by wild-type and S112A Bad, but not that caused by S136A Bad, could be almost completely abrogated by 14-3-3. These data support a critical role for 14-3-3 in regulating Bad proapoptotic activity. The effect of 14-3-3 on Bad is controlled largely by phosphorylation of S136, whereas S112 may represent a 14-3-3-independent pathway.

The 14-3-3 proteins are a family of highly conserved eukaryotic regulatory molecules that play important roles in many biological processes including cell cycle control and regulation of cell death. They are able to carry out these effects through binding and modulating the activity of a host of signaling proteins. The ability of 14-3-3 to inhibit Bad and other proapoptotic proteins argues that 14-3-3 can support cell survival. To examine this issue in a global sense, a specific inhibitor of 14-3-3/ligand interactions, difopein, was used. Difopein expression led to induction of apoptosis. Studies using various components of survival and death signaling pathways were consistent with a vital role for 14-3-3/ligand interactions in signal transduction from upstream pro-survival kinases to the core apoptotic machinery. Because these kinases often become activated during oncogenesis, the effect of difopein on cell death induced by antineoplastic drugs was examined. It was found that difopein enhances the ability of cisplatin to kill cells. These data support the model that 14-3-3, through binding to Bad and other ligands, is critical for cell survival signaling. Inhibition of 14-3-3 may represent a useful therapeutic target for treatment of cancer and other diseases involving inappropriate cell survival.

The synthesis of a series of radiation-curable poly(urethane methacrylate) oligomers are reported, which, on exposure to electron-beam irradiation, result in adhesive films with pressure-sensitive properties. Variation of the chain terminator component of the formulation and the effect of this on adhesive properties were investigated. Adhesive systems utilizing Dowfax 9N6 nonylphenolethoxylate, 1-(2-hydroxyethyl)-2-pyrrolidinone, 2-hydroxyethyl salicylate, or 4-(2-hydroxyethyl)morpholine as alternative chain terminators performed in a manner that may make them suitable for medical applications. Terminators with higher ethoxylation levels (Brij 35, 58) conferred high hydrophilicity on the resulting adhesives. © 2001 John Wiley & Sons, Inc. J Appl Polym Sci 81: 3321–3326, 2001

CD40 is expressed on a diverse array of cell types from the hematopoeitic and non-hematopoeitic compartments. Within the hematopoeitic compartment, CD40 is found constitutively expressed on B cells, dendritic cells (DC) and macrophages. The function of CD40 in B cells has been documented as being essential in the control of humoral immunity. In DCs and macrophages, CD40 has been shown to be important in the induction of antigen-presenting cell (APC) maturation and effector function. CD40 is also expressed on non-hematopoeitic cells like keratinocytes, epithelial cells, and vascular endothelial cells and has been shown to be functionally important on these cell types.CD 154, the ligand for CD40, is a type II transmembrane glycoprotein of the TNF family. The human protein consists of a 22 aa cytoplasmic tail, a single 24 aa transmembrane domain and a 215 aa extracellular region (214 aa in the mouse), and human CD154 is 83% identical to murine CD154 at the nucleotide level. It is predominantly expressed on mature, activated CD4+ T cells, and can be inducibly expressed on THO, TH1 and TH2 cells. The expression of CD154 is tightly regulated, peaking 6-8 hr. postactivation in vitro and returning to resting levels by 24-48 hr. In addition to CD4+ T cells, CD 154 is reportedly found in a small population of activated CD8-t T cells, purified NK cells, monocytes, basophils, mast cells, activated eosinophils, and platelets. As one might expect due to the wide distribution of both the receptor and its ligand, especially on highly proliferative cells and antigen presenting cells, CD40-CD154 interactions have a very broad physiological role in the regulation of immune responses, as reviewed (1,2).

The efficacies of a series of six muscarinic cholinergic receptor agonists for stimulation of phosphoinositide breakdown and unidirectional efflux of 45Ca2+ in 1321N1 human astrocytoma cells were compared with the relative capacity of these agonists for formation of a GTP-sensitive high-affinity binding state in washed membranes. Carbachol and methacholine were ‘full’ agonists as regards phosphoinositide breakdown and Ca2+ mobilization, whereas bethanechol, arecoline and oxotremorine were ‘partial’ agonists for these two responses. Pilocarpine was the least efficacious of the six drugs tested. Except for pilocarpine, competition curves generated with the agonists and [3H]quinuclidinyl benzilate did not follow the Law of Mass Action for ligand interaction at a single site. Non-linear regression analyses of these data indicated that the data significantly better fit a two-, rather than a single-, site model with a high- and a low-affinity binding component. Competition curves generated in the presence of GTP were shifted to the right, and the extent of receptors in the high-affinity agonist-binding state was decreased. The relative efficacies of the six agonists for stimulation of phosphoinositide breakdown and Ca2+ mobilization were significantly correlated with the difference in affinities (KL/KH) between the two affinity states for each agonist. The relative efficacy of the agonists for stimulation of Ca2+ mobilization also was significantly correlated with the extent of receptors in the high-affinity state (%H) for each agonist. The results suggest that interaction with an as-yet unidentified guanine nucleotide regulatory protein is important in the mechanism whereby muscarinic receptors stimulate phosphoinositide breakdown in 1321N1 astrocytoma cells.

Phosphoinositide hydrolysis does not appear to desensitize in 1321N1 astrocytoma cells. The evidence for this is that 1) the rate of accumulation of [3H]inositol 1-phosphate is linear for up to 90 min in the presence of carbachol, 2) pretreatment of cells with 100 microM carbachol for 75 min does not diminish the subsequent ability of carbachol to increase [3H]inositol 1-phosphate accumulation, and 3) the production of all of the [3H]inositol phosphates including the polyphosphoinositide metabolites [3H]inositol bis- and trisphosphate continues for up to 75 min in the presence of carbachol and declines rapidly when the muscarinic receptor antagonist atropine is added. Only when cells are treated with carbachol for 2.5 hr or longer is there a reduction in carbachol-stimulated phosphoinositide hydrolysis, and this is associated with a decrease in muscarinic receptor number. There does appear to be desensitization of hormone-stimulated Ca2+ mobilization in 1321N1 cells, because treatment of these cells with carbachol for 75 min leads to loss of the subsequent ability of carbachol to stimulate unidirectional 45Ca2+ efflux. Histamine-stimulated 45Ca2+ efflux also is lost in cells pretreated with carbachol, indicating that the desensitization is heterologous. We conclude that desensitization of hormone-stimulated, unidirectional 45Ca2+ efflux cannot be accounted for by a loss of receptor-mediated phosphoinositide hydrolysis. If phosphoinositide hydrolysis or inositol triphosphate formation are signals for calcium mobilization, the site at which the calcium response desensitizes must be distal to the initial receptor-mediated activation of phospholipase C.

Activation of muscarinic receptors in human astrocytoma (1321N1) cells stimulates phosphoinositide metabolism and calcium mobilization. The muscarinic effect on phosphoinositide turnover is evidenced by increased formation of [3H]inositol 1-phosphate (Ins1P) and by increased [3H]inositol incorporation into PtdIns. The muscarinic effect on calcium mobilization is seen as a large increase in undirectional 45Ca2+ efflux from cells equilibrated with 45Ca2+ and a small increase in unidirectional 45Ca2+ influx. A series of muscarinic agonists was used to explore the relationship between phosphoinositide metabolism and unidirectional 45Ca2+ efflux. The maximal increases in [3H]Ins1P formation produced by carbachol and acetylcholine are similar and are much larger than those caused by oxotremorine and pilocarpine. The effects of these agonists on 45Ca2+ efflux are similar: carbachol and acetylcholine cause equivalent maximal increases in the rate of 45Ca2+ efflux whereas oxotremorine and pilocarpine cause submaximal 45Ca2+ efflux responses. The Kact values of carbachol and acetylcholine for stimulation of [3H]Ins1P formation are 40 microM and 1.5 microM, respectively. These values are only 2- to 3-fold higher than the respective Kact values for stimulating 45Ca2+ efflux. The finding that each of the muscarinic agonists tested has nearly identical efficacy and similar potency for stimulating [3H]Ins1P formation and 45Ca2+ efflux supports the idea that hormonal stimulation of phosphoinositide hydrolysis leads to calcium mobilization.

Muscarinic receptor activation stimulates phosphoinositide hydrolysis and inhibits cyclic AMP formation in dissociated embryonic chick heart cells. We used this preparation to examine the hypothesis that the putative M1 and M2 receptor subtypes are selectively coupled to these two responses. Atropine blocks the effects of carbachol on cyclic AMP formation and phosphoinositide breakdown with nearly identical KI values (1.9 and 0.8 nM); these values are close to the apparent KD (1.8 nM) of atropine competition for [3H]N-methylscopolamine binding. Pirenzepine blocks the effect of carbachol on cyclic AMP formation with a KI of 48 nM, a value similar to the apparent KD (23 nM) determined in radioligand-binding studies. In contrast, a higher concentration of pirenzepine is needed to inhibit carbachol-stimulated phosphoinositide hydrolysis (KI = 255 nM). Two selective agonists, McN-A343 and AHR 602, inhibit cyclic AMP formation but do not stimulate phosphoinositide hydrolysis in chick heart cells. Muscarinic receptor-mediated phosphoinositide hydrolysis in 1321N1 astrocytoma cells is also insensitive to McN-A343 or AHR 602 and is antagonized only by relatively high concentrations of pirenzepine. The M1 receptor, as previously defined, has high affinity for pirenzepine and is activated by McN-A343. We find that these ligands have greater activity at muscarinic receptors that inhibit cyclic AMP formation than at those that stimulate phosphoinositide hydrolysis. Thus, if different receptor subtypes are associated with these two responses, the M1 receptor regulates cyclic AMP rather than phosphoinositide metabolism. Our data also demonstrate that the chick heart has muscarinic receptors with high affinity for pirenzepine, and thus, in contrast to rat heart, appears to have predominantly M1 receptors.

14-3-3 proteins bind a variety of molecules involved in signal transduction, cell cycle regulation and apoptosis. 14-3-3 binds ligands such as Raf-1 kinase and Bad by recognizing the phosphorylated consensus motif, RSXpSXP, but must bind unphosphorylated ligands, such as glycoprotein Ib and Pseudomonas aeruginosa exoenzyme S, via a different motif. Here we report the crystal structures of the zeta isoform of 14-3-3 in complex with two peptide ligands: a Raf-derived phosphopeptide (pS-Raf-259, LSQRQRSTpSTPNVHMV) and an unphosphorylated peptide derived from phage display (R18, PHCVPRDLSWLDLEANMCLP) that inhibits binding of exoenzyme S and Raf-1. The two peptides bind within a conserved amphipathic groove on the surface of 14-3-3 at overlapping but distinct sites. The phosphoserine of pS-Raf-259 engages a cluster of basic residues (Lys49, Arg56, Arg60, and Arg127), whereas R18 binds via the amphipathic sequence, WLDLE, with its two acidic groups coordinating the same basic cluster. 14-3-3 is dimeric, and its two peptide-binding grooves are arranged in an antiparallel fashion, 30 A apart. The ability of each groove to bind different peptide motifs suggests how 14-3-3 can act in signal transduction by inducing either homodimer or heterodimer formation in its target proteins.

Polycystic ovaries (PCO) are detected using ultrasonography in a proportion of women who do not have clinical symptoms of the polycystic ovary syndrome (PCOS). The aim of this study was to compare the metabolic and endocrine differences between women with such ultrasound-detected PCO and women with PCOS, and to relate these changes to clinical presentation with particular reference to cycle irregularity. A group of 118 women showing PCO on vaginal ultrasound scan was divided into those who had no hyperandrogenaemia (n = 21) and those who had increased androgens and a clinical presentation normally associated with PCOS (n = 97). These were compared with a reference group of 26 normal subjects. Glucose tolerance, lipid concentrations and endocrine profiles were compared between groups. Apart from higher concentrations of androgens in the PCOS group, there were no significant differences between the PCO and PCOS groups in either fasting and stimulated insulin and glucose or in concentrations of sex hormone-binding globulin, gonadotrophins and blood lipids or in ovarian volume. Both PCO and PCOS subjects with cycle irregularity had significantly higher concentrations of serum fasting and stimulated insulin independent of androgens and body mass index than those with normal cycles. It was concluded that: (i) PCO and PCOS patients have equivalent disturbances in relation to insulin and glucose metabolism as well as lipid and lipoprotein disturbances compared to reference subjects; (ii) higher serum insulin values are associated with menstrual irregularity in both groups; (iii) ultrasound evidence for PCO predicts similar metabolic sequelae to PCOS and can therefore be used for studies of the genetics and long term risks for this condition.

Mutational replacements of specific residues in the GTP-binding pocket of the 21-kDa rasproteins (p21ras) reduce their GTPase activity. To test the possibility that the cognate regions of G protein α chains participate in GTP binding and hydrolysis, we compared signaling functions of normal and mutated α chains (termed αs) of Gs, the stimulatory regulator of adenylyl cyclase. αschains were expressed in an αs-deficient S49 mouse lymphoma cell line, cyc-. αsin which leucine replaces glutamine 227 (corresponding to glutamine 61 of p21ras) constitutively activates adenylyl cyclase and reduces the kcatfor GTP hydrolysis more than 100-fold. There is a smaller reduction in GTPase activity in another mutant in which valine replaces glycine 49 (corresponding to glycine 12 of p21ras). This mutant αsis a poor activator of adenylyl cyclase. Moreover, the glycine 49 protein, unlike normal αs, is not protected against tryptic cleavage by hydrolysis resistant GTP analogs; this finding suggests impairment of the mutant protein's ability to attain the active (GTP-bound) conformation. We conclude that αsresidues near glutamine 227 and glycine 49 participate in binding and hydrolysis of GTP, although the GTP binding regions of αsand p21rasare not identical.

Pertussis toxin was used to examine the role of the inhibitory guanine nucleotide regulatory protein, Ni, in muscarinic-receptor-mediated stimulation of phosphoinositide turnover and calcium mobilization. In cultured chick heart cells, pertussis-toxin treatment inhibited muscarinic-receptor-mediated attenuation of isoprenaline-stimulated cyclic AMP accumulation. This finding is consistent with the proposal that pertussis toxin blocks the capacity of Ni to couple muscarinic receptors to adenylate cyclase. In contrast, treatment of chick heart cells or 1321N1 human astrocytoma cells with pertussis toxin did not block muscarinic-receptor-mediated stimulation of phosphoinositide hydrolysis, as measured by [3H]inositol phosphate accumulation in the presence of Li+. Pertussis-toxin treatment also had little effect on basal and muscarinic-receptor-stimulated phosphatidylinositol synthesis, as measured by the incorporation of [3H]inositol into phosphatidylinositol. Activation of muscarinic receptors also enhances the rate of unidirectional 45Ca2+ efflux in 1321N1 cells; this response, like phosphoinositide hydrolysis, was not prevented by pertussis-toxin treatment. Our data suggest that muscarinic receptors are not coupled to phosphoinositide hydrolysis or calcium mobilization through Ni.

The interactions between CD40 on B cells and its ligand gp39 on activated T helper cells are known to be essential for the development of thymus-dependent humoral immunity. However, CD40 is also functionally expressed on thymic epithelial cells and dendritic cells, suggesting that gp39-CD40 interactions may also play a role in thymic education, the process by which self-reactive cells are deleted from the T cell repertoire. Six systems of negative selection were studied for their reliance on gp39-CD40 interactions to mediate negative selection. In all cases, when the antigen/superantigen was endogenously expressed (in contrast to exogenously administered), negative selection was blocked by loss of gp39 function. Specifically, blockade of gp39-CD40 interactions prevented the deletion of thymocytes expressing V beta 3, V beta 11, and V beta 12, specificities normally deleted in BALB/c mice because of the endogenous expression of minor lymphocyte-stimulating determinants. Independent verification of a role of gp39 in negative selection was provided by studies in gp39-deficient mice where alterations in T cell receptor (TCR) V beta expression were also observed. Studies were also performed in the AND TCR transgenic (Tg) mice, which bear the V alpha 11, V beta 3 TCR and recognize both pigeon cytochrome c (PCC)/IEk and H-2As. Neonatal administration of anti-gp39 to AND TCR Tg mice that endogenously express H-2As or endogenously produce PCC prevented the deletion of TCR Tg T cells. In contrast, deletion mediated by high-dose PCC peptide antigen (administered exogenously) in AND TCR mice was unaltered by administration of anti-gp39. In addition, deletion by Staphylococcus enterotoxin B in conventional mice was also unaffected by anti-gp39 administration. gp39 expression was induced on thymocytes by mitogens or by antigen on TCR Tg thymocytes. Immunohistochemical analysis of B7-2 expression in the thymus indicated that, in the absence of gp39, B7-2 expression was substantially reduced. Taken together, these data suggest that gp39 may influence negative selection through the regulation of costimulatory molecule expression. Moreover, the data support the hypothesis that, for negative selection to some endogenously produced antigens, negative selection may be dependent on TCR engagement and costimulation.

The G protein family of signal transducers includes five heterotrimers, which are most clearly distinguished by their different alpha chains. The family includes Gs and Gi, the stimulatory and inhibitory GTP-binding regulators of adenylate cyclase; Go, a protein of unknown function abundant in brain; and transducin 1 and transducin 2, proteins involved in retinal phototransduction. Using a bovine alpha t1 cDNA as a hybridization probe, we have isolated mouse cDNAs that encode alpha chains of two G proteins. One encodes a polypeptide of 377 amino acids (Mr 43,856), identified as alpha s because it specifically fails to hybridize with any transcript in an alpha s-deficient S49 mouse lymphoma mutant, cyc-; the other encodes a polypeptide of 355 amino acids (Mr 40,482), presumed to be alpha i. These alpha chains and those of the retinal transducins exhibit impressive sequence homology. Of the four, alpha t1 and alpha t2 are most alike (81% identical amino acid residues), whereas the presumptive alpha i is more similar than alpha s to alpha t1 (63% vs. 38% identical residues). Sequence homologies with p21ras and elongation factor Tu identify regions of the alpha chains that form the site for GTP binding and hydrolysis. Further comparison of the alpha-chain sequences suggests additional regions that may contribute to interactions with beta gamma subunits and the receptor and effector components of different signal transduction systems.