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Age-related macular degeneration is a progressive retinal disease that is associated with factors such as oxidative stress and inflammation. In this study, we evaluated the protective effects of SIG-1451, a non-steroidal anti-inflammatory compound developed for treating atopic dermatitis and known to inhibit Toll-like receptor 4, in light-induced photoreceptor degeneration. SIG-1451 was intraperitoneally injected into rats once per day before exposure to 1000 lx light for 24 h; one day later, optical coherence tomography showed a decrease in retinal thickness, and electroretinogram (ERG) amplitude was also found to have decreased 3 d after light exposure. Moreover, SIG-1451 partially protected against this decrease in retinal thickness and increase in ERG amplitude. One day after light exposure, upregulation of inflammatory response-related genes was observed, and SIG-1451 was found to inhibit this upregulation. Iba-1, a microglial marker, was suppressed in SIG-1451-injected rats. To investigate the molecular mechanism underlying these effects, we used lipopolysaccharide (LPS)-stimulated rat immortalised Müller cells. The upregulation of C-C motif chemokine 2 by LPS stimulation was significantly inhibited by SIG-1451 treatment, and Western blot analysis revealed a decrease in phosphorylated I-κB levels. These results indicate that SIG-1451 indirectly protects photoreceptor cells by attenuating light damage progression, by affecting the inflammatory responses. [ABSTRACT FROM AUTHOR]

Background: Chia seeds have gained importance as it is the highest known plant source of omega‐3 (ω3) polyunsaturated fatty acids. Specifically, chia seeds possess ω3 α‐linolenic acid (ALA) and ω6 linoleic acid (LA), together known as Vitamin F, which play an important role in maintaining skin function. Protein phosphatase 2A (PP2A) is a master regulatory protein that plays a critical role in skin barrier function and its activity is modulated by natural lipids. Aims: Obtain a chia seed extract (HYVIA™) with significant higher levels of Vitamin F, determine in vitro PP2A activity and skin hydration markers compared to other commercial chia seed extracts (CCSEs), and evaluate the potential skin hydration benefits clinically in human subjects. Methods: A PP2A demethylation assay was utilized to assess PP2A activity. In vitro studies utilizing normal human epidermal keratinocytes (NHEKs) were treated with HYVIA™ and gene expression of hydration markers (AQP3, HAS2) were measured by quantitative PCR (qPCR). A 16‐subject clinical trial was performed with 0.1% HYVIA™ formulated in a cream and applied topically to assess its skin moisturizing potential. Results: We demonstrate here that HYVIA™, ALA, and LA inhibit PP2A demethylation, boosting PP2A activity, while most other CCSEs do not. Unlike other CCSEs, HYVIA™ increases keratinocyte hydration factors aquaporin‐3 and hyaluronic acid synthase‐2 in vitro. Clinical assessment of 0.1% HYVIA™ cream shows that HYVIA™ improves skin hydration. Conclusions: HYVIA™ is a novel chia seed extract, enriched for Vitamin F, that modulates PP2A activity and clinically improves skin hydration and barrier function. [ABSTRACT FROM AUTHOR]

Utilizing a 3% SFC gel formulation (applied 2× daily), a majority of subjects had reduced average lesion counts, erythema, and 1-2 grade improvement on the IGA scale after 4 weeks. In the United States, rosacea has been estimated to affect at least 16 million, and worldwide incidence peaks as high as 18% in populations with "Celtic" heritage.[4] Rosacea is characterized by erythema, blushing, and inflammatory lesions. Pilot study demonstrates N-Succinyl-S-farnesyl-L-cysteine reduces erythema and inflammatory lesions in rosacea subjects. [Extracted from the article]

Background: SIG‐1273 is a novel cosmetic active that provides a broad spectrum of benefits to the skin. Considering the chronic skin exposure to pollution in urban areas, we sought to determine if SIG‐1273 could provide additional protection against skin aging by inhibiting pollutant‐induced cytotoxicity and inflammation. Objective: Determine if SIG‐1273 possesses antipollution properties in vitro and evaluate the potential anti‐aging benefits of Age IQ™ Night Cream clinically in human subjects. Methods: In vitro studies utilizing normal human epidermal keratinocytes (NHEKs), were co‐treated with urban dust (SRM 1649b) and SIG‐1273 (toxicity protection measured by MTS assay). A water‐soluble fraction of urban dust (UD‐WS) induces pro‐inflammatory cytokine release (IL‐8) from NHEKs (measured via ELISA). An 8‐week, 37‐subject clinical trial was performed with 0.05% SIG‐1273 formulated in Age IQ™ Night Cream and applied topically to assess its potential to reduce the appearance of aging. Results: In vitro studies using NHEKs demonstrate SIG‐1273 protects against urban dust‐induced cell toxicity, reducing cell death by 66% and concentration dependently inhibits UD‐WS‐induced IL‐8 production (IC50 = 20 nmol/L), outperforming niacinamide, ascorbic acid, and α‐tocopherol, commonly used actives in antipollution skin‐care products. Clinical assessment of Age IQ™ Night Cream shows it is effective in improving the appearance of facial skin aging including fine lines and wrinkles, skin texture, skin clarity/brightness, and firmness/elasticity. Conclusions: SIG‐1273, is demonstrated here for the first time to possess antipollution properties. Included as a key active ingredient in Age IQ™ Night Cream, this novel topical formulation provides benefits to individuals with aging skin. [ABSTRACT FROM AUTHOR]

Hyperphosphorylated α-synuclein in Lewy bodies and Lewy neurites is a characteristic neuropathological feature of Parkinson's disease (PD) and Dementia with Lewy bodies (DLB). The catalytic subunit of the specific phosphatase, protein phosphatase 2A (PP2A) that dephosphorylates α-synuclein, is hypomethylated in these brains, thereby impeding the assembly of the active trimeric holoenzyme and reducing phosphatase activity. This phosphatase deficiency contributes to the accumulation of hyperphosphorylated α-synuclein, which tends to fibrillize more than unmodified α-synuclein. Eicosanoyl-5- hydroxytryptamide (EHT), a fatty acid derivative of serotonin found in coffee, inhibits the PP2A methylesterase so as to maintain PP2A in a highly active methylated state and mitigates the phenotype of α-synuclein transgenic (SynTg) mice. Considering epidemiologic and experimental evidence suggesting protective effects of caffeine in PD, we sought, in the present study, to test whether there is synergy between EHT and caffeine in models of α-synucleinopathy. Coadministration of these two compounds orally for 6 mo at doses that were individually ineffective in SynTgmice and in a striatal α-synuclein preformed fibril inoculation model resulted in reduced accumulation of phosphorylated α-synuclein, preserved neuronal integrity and function, diminished neuroinflammation, and improved behavioral performance. These indices were associated with increased levels of methylated PP2A in brain tissue. A similar profile of greater PP2A methylation and cytoprotection was found in SHSY5Y cells cotreated with EHT and caffeine, but not with each compound alone. These findings suggest that these two components of coffee have synergistic effects in protecting the brain against α-synuclein-mediated toxicity through maintenance of PP2A in an active state. [ABSTRACT FROM AUTHOR]

Abstract: Cutibacterium (formerly Propionibacterium acnes) is a major contributor to the pathogenesis of acne. C. acnes initiates an innate immune response in keratinocytes via recognition and activation of toll‐like receptor‐2 (TLR2), a key step in comedogenesis. Tetramethyl‐hexadecenyl‐cysteine‐formylprolinate (SIG1459), a novel anti‐acne isoprenylcysteine (IPC) small molecule, is shown in this study to have direct antibacterial activity and inhibit TLR2 inflammatory signalling. In vitro antibacterial activity of SIG1459 against C. acnes was established demonstrating minimal inhibitory concentration (MIC = 8.5 μmol\L), minimal bactericidal concentration (MBC = 16.1 μmol\L) and minimal biofilm eradication concentration (MBEC = 12.5 μmol\L). To assess SIG1459's anti‐inflammatory activity, human keratinocytes were exposed to C. acnes and different TLR2 ligands (peptidoglycan, FSL‐1, Pam3CSK4) that induce pro‐inflammatory cytokine IL‐8 and IL‐1α production. Results demonstrate SIG1459 inhibits TLR2‐induced IL‐8 release from TLR2/TLR2 (IC50 = 0.086 μmol\L), TLR2/6 (IC50 = 0.209 μmol\L) and IL‐1α from TLR2/TLR2 (IC50 = 0.050 μmol\L). To assess the safety and in vivo anti‐acne activity of SIG1459, a vehicle controlled clinical study was conducted applying 1% SIG1459 topically (n = 35 subjects) in a head‐to‐head comparison against 3% BPO (n = 15 subjects). Utilizing the Investigator Global Assessment scale for acne as primary endpoint, results demonstrate 1% SIG1459 significantly outperformed 3% BPO over 8 weeks, resulting in 79% improvement as compared to 56% for BPO. Additionally, 1% SIG1459 was well tolerated. Thus, SIG1459 and phytyl IPC compounds represent a novel anti‐acne technology that provides a safe dual modulating benefit by killing C. acnes and reducing the inflammation it triggers via TLR2 signalling. [ABSTRACT FROM AUTHOR]

Soluble forms of oligomeric beta-amyloid (Aβ) are thought to play a central role in Alzheimer’s disease (AD). Transgenic manipulation of methylation of the serine/threonine protein phosphatase, PP2A, was recently shown to alter the sensitivity of mice to AD-related impairments resulting from acute exposure to elevated levels of Aβ. In addition, eicosanoyl-5-hydroxytryptamide (EHT), a naturally occurring component from coffee beans that modulates PP2A methylation, was shown to confer therapeutic benefits in rodent models of AD and Parkinson’s disease. Here, we tested the hypothesis that EHT protects animals from the pathological effects of exposure to elevated levels of soluble oligomeric Aβ. We treated mice with EHT-containing food at two different doses and assessed the sensitivity of these animals to Aβ-induced behavioral and electrophysiological impairments. We found that EHT administration protected animals from Aβ-induced cognitive impairments in both a radial-arm water maze and contextual fear conditioning task. We also found that both chronic and acute EHT administration prevented Aβ-induced impairments in long-term potentiation. These data add to the accumulating evidence suggesting that interventions with pharmacological agents, such as EHT, that target PP2A activity may be therapeutically beneficial for AD and other neurological conditions. [ABSTRACT FROM AUTHOR]

Objective Protein phosphatase 2A ( PP2A) is a heterotrimeric holoenzyme composed of a catalytic C subunit, a structural A subunit, and one of several regulatory B subunits that confer substrate specificity. The assembly and activity of PP2A are regulated by reversible methylation of the C subunit. α-Synuclein, which aggregates in Parkinson disease ( PD) and dementia with Lewy bodies ( DLB), is phosphorylated at Ser129, and PP2A containing a B55 α subunit is a major phospho-Ser129 phosphatase. The objective of this study was to investigate PP2A in α-synucleinopathies. Methods We compared the state of PP2A methylation, as well as the expression of its methylating enzyme, leucine carboxyl methyltransferase ( LCMT-1), and demethylating enzyme, protein phosphatase methylesterase ( PME-1), in postmortem brains from PD and DLB cases as well as age-matched Controls. Immunohistochemical studies and quantitative image analysis were employed. Results LCMT-1 was significantly reduced in the substantia nigra ( SN) and frontal cortex in both PD and DLB. PME-1, on the other hand, was elevated in the PD SN. In concert with these changes, the ratio of methylated PP2A to demethylated PP2A was markedly decreased in PD and DLB brains in both SN and frontal cortex. No changes in total PP2A or total B55 α subunit were detected. Interpretation These findings support the hypothesis that PP2A dysregulation in α-synucleinopathies may contribute to the accumulation of hyperphosphorylated α-synuclein and to the disease process, raising the possibility that pharmacological means to enhance PP2A phosphatase activity may be a useful disease-modifying therapeutic approach. [ABSTRACT FROM AUTHOR]

Background Isoprenylcysteine (IPC) small molecules were identified as a new class of anti-inflammatory compounds over 20 years ago. Since then, they have been developed as novel cosmetic functional ingredients (CFI) and topical drug candidates. SIG1273 is a second generation CFI that has previously been shown to provide a broad spectrum of benefits for the skin through its anti-inflammatory and antimicrobial properties. Objective To determine whether SIG1273 possesses anti-aging properties in vitro and evaluate the tolerability and activity of SIG1273 when applied topically to human subjects. Methods To model photoaging in vitro, human dermal fibroblasts (HDFs) were exposed in culture to UVA to induce collagenase (MMP-1) production. An in vitro wound-healing model was based on the activation of HDF migration into cell-free tissue culture surface. Hydrogen peroxide-induced oxidative stress was performed using HDFs to measure intracellular ROS activity. Radical scavenging capacity was determined using a colorimetric antioxidant assay kit (ABTS method). Lastly, a 4-week, 29-subject study was performed in which SIG1273 was applied topically as a cream to assess its tolerance and activity in reducing the appearance of aging. Results In vitro studies demonstrate SIG1273 inhibits UVA-induced MMP-1 production, hydrogen peroxide-induced oxidative stress and promotes wound healing. Moreover, SIG1273 was shown to be a radical scavenging antioxidant. Clinical assessment of SIG1273 cream (0.25%) showed it was well tolerated with significant improvement in the appearance of fine lines, coarse wrinkles, radiance/luminosity, pore size, texture/smoothness, hydration and increased firmness. Conclusions SIG1273 represents a novel CFI with antioxidant, anti-aging, and anti-inflammatory properties that when applied topically is well tolerated and provides benefits to individuals with aging skin. [ABSTRACT FROM AUTHOR]

Background Propionibacterium acnes is a major contributing factor to the inflammatory component of acne. The interaction of P. acnes with keratinocytes leads to an innate immune response via activation of toll-like receptors ( TLR2, TLR4) resulting in the production and secretion of pro-inflammatory mediators. SIG1273, an isoprenylcysteine small molecule modulates inflammatory signaling pathways and kills P. acnes. SIG1273 represents a novel cosmetic functional ingredient that provides relief from blemishes in acne prone skin. Objective To assess the keratinocyte response and microbial growth of SIG1273 in vitro and evaluate the tolerability of SIG1273 gel applied topically in acne prone subjects. Methods For in vitro studies, human keratinocytes were exposed in culture to live P. acnes and peptidoglycan ( PGN) to induce IL-8 production. P. acnes were cultured to determine minimal inhibitory concentration and minimal bactericidal concentration values. A total of 30 subjects were randomized in a double-blind controlled trial receiving 3% SIG1273 gel or vehicle for 6 weeks. Evaluation included inflammatory lesions, noninflammatory lesions, microcomedones, Sebutape scores, and P. acnes counts. Results In vitro studies demonstrate SIG1273 inhibits P. acnes-induced IL-8 production and inhibits P. acnes growth. SIG1273 gel was well tolerated with no signs of stinging, redness, or itching. Furthermore, improvement in some aspects of acne was observed in subjects applying SIG1273 gel, including inflammatory lesions, microcomedone counts and Sebutape scores. Facial scrubs taken to measure P. acnes colony-forming units showed those applying SIG1273 gel had ~1.0 Log 10 colony reduction over the length of the study, a statistically significantly improvement when compared with vehicle. No significant effects above vehicle were observed for noninflammatory lesions. Conclusions SIG1273 represents a novel cosmetic functional ingredient that provides a safe dual modulating benefit to individuals with acne prone skin by reducing P. acnes counts and reducing inflammation. [ABSTRACT FROM AUTHOR]

α-Synuclein (α-Syn) is a key protein that accumulates as hyperphosphorylated aggregates in pathologic hallmark features of Parkinson's disease (PD) and other neurodegenerative disorders. Phosphorylation of this protein at serine 129 is believed to promote its aggregation and neurotoxicity, suggesting that this post-translational modification could be a therapeutic target. Here, we demonstrate that phosphoprotein phosphatase 2A (PP2A) dephosphorylatesα-Syn at serine 129 and that this activity is greatly enhanced by carboxyl methylation of the catalytic C subunit of PP2A.α-Syn-transgenic mice raised on a diet supplemented with eicosanoyl-5-hydroxytryptamide, an agent that enhances PP2A methylation, dramatically reduced both α-Syn phosphorylation at Serine 129 and α-Syn aggregation in the brain. These biochemical changes were associated with enhanced neuronal activity, increased dendritic arborizations, and reduced astroglial and microglial activation, as well as improved motor performance. These findings support the notion that serine 129 phosphorylation ofα-Syn is of pathogenetic significance and that promoting PP2A activity is a viable disease-modifying therapeutic strategy for α-synucleinopathies such as PD. [ABSTRACT FROM AUTHOR]

Bacterial chemotaxis is mediated by two reversible protein modification chemistries: phosphorylation and carboxyl methylation. Attractants bind to membrane chemoreceptors that control the activity of a protein kinase which acts in turn to control flagellar motor activity. Coordinate changes in receptor carboxyl methylation provide a negative feedback mechanism that serves a memory function. Protein carboxyl methylation might play an analogous role in the nervous system. Two protein carboxyl methyltransferases serve to regulate signal transduction pathways in eukaryotic cells. One is highly expressed in the Purkinje layer of the cerebellum where it methyl esterifies prenylated cysteine residues at the carboxyl-termini of Ras-related and heterotrimeric G-proteins. The other is abundant throughout the brain where it methylates the carboxyl-terminus of protein phosphatase 2A. The phosphatase methyltransferase and the protein methylesterase that reverses phosphatase methylation are structurally related to the corresponding bacterial chemotaxis methylating and demethylating enzymes. Recent results indicate that deficiencies in phosphatase methylation play an important role in the etiology of Alzheimer's disease. [ABSTRACT FROM AUTHOR]

The small t antigen (ST) of DNA tumor virus SV40 facilitates cellular transformation by disrupting the functions of protein phosphatase 2A (PP2A) through a poorly defined mechanism. The crystal structure of the core domain of SV40 ST bound to the scaffolding subunit of human PP2A reveals that the ST core domain has a novel zinc-binding fold and interacts with the conserved ridge of HEAT repeats 3–6, which overlaps with the binding site for the B′ (also called PR61 or B56) regulatory subunit. ST has a lower binding affinity than B′ for the PP2A core enzyme. Consequently, ST does not efficiently displace B′ from PP2A holoenzymes in vitro. Notably, ST inhibits PP2A phosphatase activity through its N-terminal J domain. These findings suggest that ST may function mainly by inhibiting the phosphatase activity of the PP2A core enzyme, and to a lesser extent by modulating assembly of the PP2A holoenzymes. [ABSTRACT FROM AUTHOR]

Escherichia coil chemotaxis is mediated by membrane receptor/histidine kinase signaling complexes. Fusing the cytoplasmic domain of the aspartate receptor, Tar, to a leucine zipper dimerization domain produces a hybrid, IzTarC, that forms soluble complexes with CheA and CheW. The three-dimensional reconstruction of these complexes was different from that anticipated based solely on structures of the isolated components. We found that analogous complexes self-assembled with a monomeric cytoplasmic domain fragment of the serine receptor without the leucine zipper dimerization domain. These complexes have essentially the same size, composition, and architecture as those formed from IzTarC. Thus, the organization of these receptor/signaling complexes is determined by conserved interactions between the constituent chemotaxis proteins and may represent the active form in vivo. To understand this structure in its cellular context, we propose a model involving parallel membrane segments in receptor-mediated CheA activation in vivo. [ABSTRACT FROM AUTHOR]

The environmental topology of complex structures is used by Escherichia colito create traveling waves of high cell density, a prelude to quorum sensing. When cells are grown to a moderate density within a confining microenvironment, these traveling waves of cell density allow the cells to find and collapse into confining topologies, which are unstable to population fluctuations above a critical threshold. This was first observed in mazes designed to mimic complex environments, then more clearly in a simpler geometry consisting of a large open area surrounding a square (250 × 250 μm) with a narrow opening of 10-30 μm. Our results thus show that under nutrient- deprived conditions bacteria search out each other in a collective manner and that the bacteria can dynamically confine themselves to highly enclosed spaces. [ABSTRACT FROM AUTHOR]

Aspartate phosphorylation induces changes in protein conformation that are used to regulate processes ranging from gene expression and cell differentiation to cell motility and the generation of electrochemical gradients across membranes. We show here that dephosphorylation of the phosphoaspartate in the chemotaxis response regulator CheY can result in the loss of a water molecule that may be due to formation of a succinimide intermediate. [Copyright &y& Elsevier]

Response regulator proteins of two-component systems are usually activated by phosphorylation. The phosphorylated response regulator protein CheY∼P mediates the chemotaxis response in Escherichia coli. We performed random mutagenesis and selected CheY mutants that are constitutively active in the absence of phosphorylation. Although a single amino acid substitution can lead to constitutive activation, no single DNA base change can effect such a transition. Numerous different sets of mutations that activate in synergy were selected in several different combinations. These mutations were all located on the side of CheY defined by α4, β5, α5, and α1. Our findings argue against the two-state hypothesis for response regulator activation. We propose an alternative intermolecular mechanism that involves a dynamic interplay between response regulators and their effector targets. [ABSTRACT FROM AUTHOR]

Tau hyperphosphorylation is a central event in the development of Alzheimer’s Disease (AD). Protein phosphatase 2A (PP2A) heterotrimer formation is necessary for efficient dephosphorylation of the tau protein. S-Adenosylmethionine-dependent carboxyl methylation is essential for the assembly of PP2A heterotrimers. Epidemiological evidence indicates that elevated plasma homocysteine is an independent risk factor for AD. Homocysteine is a key intermediate in the methyl cycle and elevated plasma homocysteine results in a global decrease in cellular methylation. We propose that the PP2A methylation system is the link relating elevated plasma homocysteine to AD. [Copyright &y& Elsevier]

The phosphoprotein phosphatase 2A (PP2A) catalytic subunit contains a methyl ester on its C-terminus, which in mammalian cells is added by a specific carboxyl methyltransferase and removed by a specific carboxyl methylesterase. We have identified genes in yeast that show significant homology to human carboxyl methyltransferase and methylesterase. Extracts of wild-type yeast cells contain carboxyl methyltransferase activity, while extracts of strains deleted for one of the methyltransferase genes, PPM1, lack all activity. Mutation of PPM1 partially disrupts the PP2A holoenzyme in vivo and ppm1 mutations exhibit synthetic lethality with mutations in genes encoding the B or B′ regulatory subunit. Inactivation of PPM1 or overexpression of PPE1, the yeast gene homologous to bovine methylesteruse, yields phenotypes similar to those observed after inactivation of either regulatory subunit. These phenotypes can be reversed by overexpression of the B regulatory subunit. These results demonstrate that Ppm1 is the sole PP2A methyltransferase in yeast and that its activity is required for the integrity of the PP2A holoenzyme. [ABSTRACT FROM AUTHOR]

Phosphoprotein phosphatase 2A (PP2A) is a major phosphoserine/threonine protein phosphatase in all eukaryotes. It has been isolated as a heterotrimeric holoenzyme composed of a 65 kDa A subunit, which serves as a scaffold for the association of the 36 kDa catalytic C subunit, and a variety of B subunits that control phosphatase specificity. The C subunit is reversibly methyl esterified by specific methyltransferase and methylesterase enzymes at a completely conserved C-terminal leucine residue. Here we show that methylation plays an essential role in promoting PP2A holoenzyme assembly and that demethylation has an opposing effect. Changes in methylation indirectly regulate PP2A phosphatase activity by controlling the binding of regulatory B subunits to AC dimers. [ABSTRACT FROM AUTHOR]

Environmental stimuli attack the skin daily resulting in the generation of reactive oxygen species (ROS) and inflammation. One pathway that regulates oxidative stress in skin involves Protein Phosphatase 2A (PP2A), a phosphatase which has been previously linked to Alzheimer's Disease and aging. Oxidative stress decreases PP2A methylation in normal human dermal fibroblasts (NHDFs). Thus, we hypothesize agents that increase PP2A methylation and activity will promote skin health and combat aging. To discover novel inhibitors of PP2A demethylation activity, we screened a library of 32 natural botanical extracts. We discovered Grape Seed Extract (GSE), which has previously been reported to have several benefits for skin, to be the most potent PP2A demethylating extract. Via several fractionation and extraction steps we developed a novel grape seed extract called Activated Grape Seed Extract (AGSE), which is enriched for PP2A activating flavonoids that increase potency in preventing PP2A demethylation when compared to commercial GSE. We then determined that 1% AGSE and 1% commercial GSE exhibit distinct gene expression profiles when topically applied to a 3D human skin model. To begin to characterize AGSE's activity, we investigated its antioxidant potential and demonstrate it reduces ROS levels in NHDFs and cell-free assays equal to or better than Vitamin C and E. Moreover, AGSE shows anti-inflammatory properties, dose-dependently inhibiting UVA, UVB and chemical-induced inflammation. These results demonstrate AGSE is a novel, multi-functional extract that modulates methylation levels of PP2A and supports the hypothesis of PP2A as a master regulator for oxidative stress signaling and aging in skin. [ABSTRACT FROM AUTHOR]

The β-lactams are by far the most widely used and efficacious of all antibiotics. Over the past few decades, however, widespread resistance has evolved among most common pathogens. Streptococcus pneumoniae has become a paradigm for understanding the evolution of resistance mechanisms, the simplest of which, by far, is the production of β-lactamases. As these enzymes are frequently plasmid encoded, resistance can readily be transmitted between bacteria. Despite the fact that pneumococci are naturally transformable organisms, no β-lactamase-producing strain has yet been described. A much more complex resistance mechanism has evolved in S. pneumoniae that is mediated by a sophisticated restructuring of the targets of the β-lactams, the penicillin-binding proteins (PBPs); however, this may not be the whole story. Recently, a third level of resistance mechanisms has been identified in laboratory mutants, wherein non-PBP genes are mutated and resistance development is accompanied by deficiency in genetic transformation. Two such non-PBP genes have been described: a putative glycosyltransferase, CpoA, and a histidine protein kinase, CiaH. We propose that these non-PBP genes are involved in the biosynthesis of cell wall components at a step prior to the biosynthetic functions of PBPs, and that the mutations selected during β-lactam treatment counteract the effects caused by the inhibition of penicillin-binding proteins. [ABSTRACT FROM AUTHOR]

Chemotaxis responses in Escherichia coli are mediated by the phosphorylated response-regulator protein P-CheY. Biochemical and genetic studies have established the mechanisms by which the various components of the chemotaxis system, the membrane receptors and Che proteins function to modulate levels of CheY phosphorylation. Detailed models have been formulated to explain chemotaxis sensing in quantitative terms; however, the models cannot be adequately tested without knowledge of the quantitative relationship between P-CheY and bacterial swimming behavior. A computerized image analysis system was developed to collect extensive statistics on free-swimming and individual tethered cells. P-CheY levels were systematically varied by controlled expression of CheY in an E.coli strain lacking the CheY phosphatase, CheZ, and the receptor demethylating enzyme CheB. Tumbling frequency was found to vary with P-CheY concentration in a weakly sigmoidal fashion (apparent Hill coefficient ∼2.5). This indicates that the high sensitivity of the chemotaxis system is not derived from highly cooperative interactions between P-CheY and the flagellar motor, but rather depends on nonlinear effects within the chemotaxis signal transduction network. The complex relationship between single flagella rotation and free-swimming behavior was examined; our results indicate that there is an additional level of information processing associated with interactions between the individual flagella. An allosteric model of the motor switching process is proposed which gives a good fit to the observed switching induced by P-CheY. Thus the level of intracellular P-CheY can be estimated from behavior determinations: ∼30% of the intracellular pool of CheY appears to be phosphorylated in fully adapted wild-type cells. [ABSTRACT FROM AUTHOR]

Chemotaxis responses of Escherichia coli and Salmonella are mediated by type I membrane receptors with N-terminal extracytoplasmic sensing domains connected by transmembrane helices to C-terminal signaling domains in the cytoplasm. Receptor signaling involves regulation of an associated protein kinase, CheA. Here we show that kinase activation by a soluble signaling domain construct involves the formation of a large complex, with ∼14 receptor signaling domains per CheA dimer. Electron microscopic examination of these active complexes indicates a well defined bundle composed of numerous receptor filaments. Our findings suggest a mechanism for transmembrane signaling whereby stimulus-induced changes in lateral packing interactions within an array of receptor-sensing domains at the cell surface perturb an equilibrium between active and inactive receptor­kinase complexes within the cytoplasm. [ABSTRACT FROM AUTHOR]

We have previously shown that soluble fractions obtained from human HL-60 granulocytes contain a phospholipase C which is markedly stimulated by the stable GTP analogue guanosine 5'[3-O-thio]triphosphate (Camps, M., Hou, C., Jakobs, K. H. and Gierschik, P. (1990) Biochem. J. 271, 743-748]. To investigate whether this stimulation was due to a soluble α subunit of a heterotrimeric guanine-nucleotide-binding protein or a soluble low-molecular-mass GTP-binding protein, we have examined the effect of purified guanine-nucleotide-binding protein βγ dimers on the phospholipaseC-mediated formation of inositol phosphates by HL-60 cytosol. We found that βγ subunits, purified from bovine retinal transducin (βγt), markedly stimulated the hydrolysis of phosphatidylinositol 4,5bisphosphate by this phospholipase C preparation. The stimulation of phospholipase C by βγt was not secondary to a phospholipase-A2-mediated generation of arachidonic acid, was prevented by the GDP-liganded transducin α subunit and was additive to activation of phospholipase C by guanosine 5'-[3-O-thio]triphosphate. βγt also stimulated soluble phospholipase C from human and bovine peripheral neutrophils, as well as membrane-bound, detergent-solubilized phospholipase C from HL60 cells. Stimulation of soluble HL-60 phospholipase C was not restricted to βγt, but was also observed with highly purified βγ subunits from bovine brain. Fractionation of HL-60 cytosol by anion-exchange chromatography revealed the existence of at least two distinct forms of phospholipase C in HL-60 granulocytes. Only one of these forms was sensitive to stimulation by βγt, demonstrating that stimulation of phospholipase C by βγ subunits is isozyme specific. Taken together, our results suggest that guanine-nucleotide-binding protein βγ subunits may play an important and active role in mediating the stimulation of phospholipase C by heterotrimeric guanine-nucleotide-binding proteins. [ABSTRACT FROM AUTHOR]

In the Escherichia coli chemotaxis system, a family of chemoreceptors in the cytoplasmic membrane binds stimulatory ligands and regulates the activity of an associated histidine kinase CheA to modulate swimming behaviour and thereby cause a net migration towards attractants and away from repellents. The chemoreceptors themselves have been shown to be predominantly dimeric, but in the presence of the kinase CheA plus an adapter protein, CheW, much higher order structures have been observed. Recent results indicate that transmembrane signalling occurs within receptor clusters rather than through isolated dimers. We propose that the mechanism involves receptor arrays where binding of ligands at the outside surface of the membrane affects lateral packing interactions that cause perturbations in the organization of the signalling array at the opposing surface of the membrane. Results with receptor chimeras as well as findings with tyrosine kinase receptors suggest that this mechanism may represent a common theme in membrane receptor function. [ABSTRACT FROM AUTHOR]