In Salmonella enterica, the sirtuin-dependent protein acylation/deacylation system (SDPADS) maintains energy homeostasis during growth on low concentrations of acetate

ABSTRACT This report provides in vivo evidence for the posttranslational control of the acetyl coenzyme A (Ac-CoA) synthetase (AcsA) enzyme of Bacillus subtilis by the acuA and acuC gene products. In addition, both in vivo and in vitro data presented support the conclusion that the yhdZ gene of B. subtilis encodes a NAD+-dependent protein deacetylase homologous to the yeast Sir2 protein (also known as sirtuin). On the basis of this new information, a change in gene nomenclature, from yhdZ to srtN (for sirtuin), is proposed to reflect the activity associated with the YdhZ protein. In vivo control of B. subtilis AcsA function required the combined activities of AcuC and SrtN. Inactivation of acuC or srtN resulted in slower growth and cell yield under low-acetate conditions than those of the wild-type strain, and the acuC srtN strain grew under low-acetate conditions as poorly as the acsA strain. Our interpretation of the latter result was that both deacetylases (AcuC and SrtN) are needed to maintain AcsA as active (i.e., deacetylated) so the cell can grow with low concentrations of acetate. Growth of an acuA acuC srtN strain on acetate was improved over that of the acuA + acuC srtN strain, indicating that the AcuA acetyltransferase enzyme modifies (i.e., inactivates) AcsA in vivo, a result consistent with previously reported in vitro evidence that AcsA is a substrate of AcuA.

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Effect of Sodium Hypochlorite and Benzalkonium Chloride on the Structural Parameters of the Biofilms Formed by Ten Salmonella enterica Serotypes

Pathogens ◽

10.3390/pathogens8030154 ◽

2019 ◽

Vol 8 (3) ◽

pp. 154 ◽

Cited By ~ 3

Author(s):

Rosa Capita ◽

Silvia Fernández-Pérez ◽

Laura Buzón-Durán ◽

Carlos Alonso-Calleja

Keyword(s):

Sodium Hypochlorite ◽

Salmonella Enterica ◽

Benzalkonium Chloride ◽

Laser Scanning ◽

Surface Coverage ◽

Structural Parameters ◽

Laser Scanning Microscopy ◽

Confocal Laser ◽

Low Concentrations ◽

Practical Field

The influence of the strain on the ability of Salmonella enterica to form biofilms on polystyrene was investigated by confocal laser scanning microscopy. The effects of sodium hypochlorite with 10% active chlorine (SHY; 25,000, 50,000, or 100,000 ppm), and benzalkonium chloride (BZK; 1000, 5000, or 10,000 ppm) on twenty-four-hour-old biofilms was also determined. The biofilms of ten Salmonella enterica isolates from poultry (S. Agona, S. Anatum, S. Enteritidis, S. Hadar, S. Infantis, S. Kentucky, S. Thompson, S. Typhimurium, monophasic variant of S. Typhimurium 1,4,(5),12:i:-, and S. Virchow) were studied. Biofilms produced by S. Anatum, S. Hadar, S. Kentucky, and S. Typhimurium showed a trend to have the largest biovolume and the greatest surface coverage and thickness. The smallest biofilms (P < 0.01) in the observation field (14.2 × 103 µm2) were produced by S. Enteritidis and S. 1,4,(5),12:i:- (average 12.9 × 103 ± 9.3 × 103 µm3) compared to the rest of the serotypes (44.4 × 103 ± 24.7 × 103 µm3). Biovolume and surface coverage decreased after exposure for ten minutes to SHY at 50,000 or 100,000 ppm and to BZK at 5000 or 10,000 ppm. However, the lowest concentrations of disinfectants increased biovolume and surface coverage in biofilms of several strains (markedly so in the case of BZK). The results from this study suggest that the use of biocides at low concentrations could represent a public health risk. Further research studies under practical field conditions should be appropriate to confirm these findings.

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cAMP inhibits IP3-dependent Ca2+ release by preferential activation of cGMP-primed PKG

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.2001.281.5.g1238 ◽

2001 ◽

Vol 281 (5) ◽

pp. G1238-G1245 ◽

Cited By ~ 17

Author(s):

Karnam S. Murthy

Keyword(s):

Muscle Contraction ◽

Muscle Cells ◽

Dependent Protein Kinase ◽

Permeabilized Cells ◽

Phosphodiesterase 3 ◽

Low Concentrations ◽

Intact Muscle ◽

Dependent Protein ◽

Camp And Cgmp ◽

Permeabilized Muscle

The singular effects and interplay of cAMP- and cGMP-dependent protein kinase (PKA and PKG) on Ca2+ mobilization were examined in dispersed smooth muscle cells. In permeabilized muscle cells, exogenous cAMP and cGMP inhibited inositol 1,4,5-trisphosphate (IP3)-induced Ca2+ release and muscle contraction via PKA and PKG, respectively. A combination of cAMP and cGMP caused synergistic inhibition that was exclusively mediated by PKG and attenuated by PKA. In intact muscle cells, low concentrations (10 nM) of isoproterenol and sodium nitroprusside (SNP) inhibited agonist-induced, IP3-dependent Ca2+ release and muscle contraction via PKA and PKG, respectively. A combination of isoproterenol and SNP increased PKA and PKG activities: the increase in PKA activity reflected inhibition of phosphodiesterase 3 activity by cGMP, whereas the increase in PKG activity reflected activation of cGMP-primed PKG by cAMP. Inhibition of Ca2+ release and muscle contraction by the combination of isoproterenol and SNP was preferentially mediated by PKG. In light of studies showing that PKG phosphorylates the IP3 receptor in intact and permeabilized muscle cells, whereas PKA phosphorylates the receptor in permeabilized cells only, the results imply that inhibition of IP3-induced Ca2+ release is mediated exclusively by PKG. The effect of PKA on agonist-induced Ca2+ release probably reflects inhibition of IP3 formation.

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Effects of endothelin on rat renal proximal tubule Na(+)-Pi cotransport and Na+/H+ exchange

AJP Renal Physiology ◽

10.1152/ajprenal.1994.266.4.f658 ◽

1994 ◽

Vol 266 (4) ◽

pp. F658-F666 ◽

Cited By ~ 7

Author(s):

J. Guntupalli ◽

T. D. DuBose

Keyword(s):

Protein Kinase ◽

Proximal Tubule ◽

Collecting Duct ◽

Second Messengers ◽

Indirect Evidence ◽

Low Concentrations ◽

Proximal Tubular ◽

Dependent Protein ◽

Medullary Collecting Duct

Endothelin (ET), a powerful vasoconstrictive peptide, is distributed ubiquitously in various organs, including the vascular endothelium and tubules of the kidney. Although localized more abundantly to the glomerulus and inner medullary collecting duct, ET receptors have been identified in the proximal tubule. The possible effects of ET on proximal tubule transport and the potential role of second messengers in this process have not been described fully. To define the role of ET in proximal tubule transport, renal cortical slices were incubated for 3 min in the presence of various concentrations of ET. Incubation with low concentrations of ET-1 (1 x 10(-9) to 1 x 10(-11) M) within the physiological range stimulated both Na(+)-Pi cotransport and Na+/H+ exchange. Pretreatment with staurosporine (0.6 microM) for 25 min abolished completely the ET-induced effects on Na(+)-Pi cotransport and Na+/H+ exchange. Similarly, preincubation with phorbol ester 12-O-tetradecanoylphorbol-13-acetate (200 nM) also abolished the effects of ET on these transporters. Incubation with ET decreased significantly intracellular adenosine 3',5'-cyclic monophosphate (cAMP). Intravenous administration of pertussis toxin for 2 days prevented the ET-induced decrease in cAMP and abolished the stimulatory effects of ET on Na(+)-Pi cotransport and Na+/H+ exchange. These findings provide indirect evidence that ET participates in the regulation of proximal tubular Pi and bicarbonate homeostasis. These effects of ET are mediated by activation of protein kinase C and cAMP-dependent protein kinase A.

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Characterization of Ca2+-dependent endogenous phosphorylation of 160,000- and 150,000-Dalton proteins of sarcoplasmic reticulum

Biochemical Journal ◽

10.1042/bj2760089 ◽

1991 ◽

Vol 276 (1) ◽

pp. 89-96 ◽

Cited By ~ 9

Author(s):

I Orr ◽

Z Gechtman ◽

V Shoshan-Barmatz

Keyword(s):

Protein Kinase ◽

Sarcoplasmic Reticulum ◽

Atp Hydrolysis ◽

Binding Capacity ◽

Hill Coefficient ◽

Regulatory Function ◽

Phosphorylated Proteins ◽

Low Concentrations ◽

Phosphorylated Peptides ◽

Dependent Protein

The 160 and 150 kDa proteins of sarcoplasmic reticulum (SR) are phosphorylated endogenously. The phosphorylation of both proteins has a marked requirement for Ca2+. Half-maximal and maximal phosphorylation was obtained at about 1 nM- and 1 microM-Ca2+ respectively, and a Hill coefficient of about 0.5 was calculated. The phosphorylation is also dependent on NaF as an inhibitor of the SR phosphoprotein phosphatase. The phosphorylation of these proteins is very rapid, and maximal phosphorylation is achieved in less than 15 s. The phosphorylation of the 160 kDa and 150 kDa polypeptides is completely inhibited by 5 mM-MgCl2 and by 75 microM-LaCl3, by very low concentrations of different detergents, and by preincubation of the SR for 2 min at 60 degrees C. The inhibition by Mg2+ is due to stimulation of ATP hydrolysis, thereby decreasing ATP concentration. Different phosphorylated peptides were obtained by digestion with protease V8 of the 160 kDa and 150 kDa protein bands, suggesting that the 160 kDa and 150 kDa proteins are distinct. The two phosphorylated proteins are present in different fractions and preparations of SR, with or without [3H]PN200-110 binding capacity. These and other results suggest that the phosphorylated SR proteins are distinct from the alpha 1 and alpha 2 subunits of the voltage-gated Ca2+ channel of the T-system membranes. Different inhibitors and activators of protein kinase C and calmodulin-dependent protein kinase have no effect on the endogenous phosphorylation of both polypeptides, suggesting that the phosphorylation is regulated solely by Ca2+. A possible regulatory function for this phosphorylation system is described in the accompanying paper [Gechtman. Orr & Shoshan-Barmatz (1991) Biochem. J. 276.97-102].

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Enhanced Survival of Salmonella enterica in Vesicles Released by a Soilborne Tetrahymena Species

Applied and Environmental Microbiology ◽

10.1128/aem.71.3.1562-1569.2005 ◽

2005 ◽

Vol 71 (3) ◽

pp. 1562-1569 ◽

Cited By ~ 84

Author(s):

M. T. Brandl ◽

B. M. Rosenthal ◽

A. F. Haxo ◽

S. G. Berk

Keyword(s):

Public Health ◽

Salmonella Enterica ◽

Fluorescent Protein ◽

Survival Rates ◽

Human Pathogen ◽

Initial Ratio ◽

Calcium Hypochlorite ◽

Human Macrophages ◽

Low Concentrations ◽

Green Fluorescent

ABSTRACT Nondestructive ingestion by soilborne protozoa may enhance the environmental resiliency of important bacterial pathogens and may model how such bacteria evade destruction in human macrophages. Here, the interaction of Salmonella enterica serovar Thompson with a soilborne Tetrahymena sp. isolate was examined using serovar Thompson cells labeled with the green fluorescent protein. The bacteria were mixed in solution with cells of Tetrahymena at several ratios. During incubation with serovar Thompson, Tetrahymena cells released a large number of vesicles containing green fluorescent serovar Thompson cells. In comparison, grazing on Listeria monocytogenes cells resulted in their digestion and thus the infrequent release of this pathogen in vesicles. The number of serovar Thompson cells per vesicle increased significantly as the initial ratio of serovar Thompson to Tetrahymena cells increased from 500:1 to 5,000:1. The density of serovar Thompson was as high as 50 cells per vesicle. Staining with propidium iodide revealed that a significantly higher proportion of serovar Thompson cells remained viable when enclosed in vesicles than when free in solution. Enhanced survival rates were observed in vesicles that were secreted by both starved (F = 28.3, P < 0.001) and unstarved (F = 14.09, P < 0.005) Tetrahymena cells. Sequestration in vesicles also provided greater protection from low concentrations of calcium hypochlorite. Thus, the release of this human pathogen from Tetrahymena cells in high-density clusters enclosed in a membrane may have important implications for public health.

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A bioorthogonal chemical reporter for fatty acid synthase-dependent protein acylation

Journal of Biological Chemistry ◽

10.1016/j.jbc.2021.101272 ◽

2021 ◽

pp. 101272

Author(s):

Krithika P. Karthigeyan ◽

Lizhi Zhang ◽

David R. Loiselle ◽

Timothy A.J. Haystead ◽

Menakshi Bhat ◽

...

Keyword(s):

Fatty Acid ◽

Fatty Acid Synthase ◽

Chemical Reporter ◽

Dependent Protein ◽

Protein Acylation

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A bioorthogonal chemical reporter for fatty acid synthase-dependent protein acylation

10.1101/2021.05.07.443132 ◽

2021 ◽

Author(s):

Krithika P. Karthigeyan ◽

Lizhi Zhang ◽

David R. Loiselle ◽

Timothy A.J. Haystead ◽

Menakshi Bhat ◽

...

Keyword(s):

Fatty Acid ◽

Fatty Acid Synthase ◽

Matrix Protein ◽

De Novo ◽

Transmembrane Protein ◽

Cellular Protein ◽

Metabolic Labeling ◽

Dependent Manner ◽

Dependent Protein ◽

Protein Acylation

Cells acquire fatty acids from dietary sources or via de novo palmitate production by fatty acid synthase (FASN). Although most cells express FASN at low levels, it is upregulated in cancers and during replication of many viruses. The precise role of FASN in disease pathogenesis is poorly understood, and whether de novo fatty acid synthesis contributes to host or viral protein acylation has been traditionally difficult to study. We describe a cell permeable, click-chemistry compatible alkynyl-acetate analog (Alk-4) that functions as a reporter of FASN-dependent protein acylation. In a FASN-dependent manner, Alk-4 selectively labeled the cellular protein interferon-induced transmembrane protein 3 (IFITM3) at its palmitoylation sites, and the HIV-1 matrix protein at its myristoylation site. Alk-4 metabolic labeling also enabled biotin-based purification and identification of more than 200 FASN-dependent acylated cellular proteins. Thus, Alk-4 is a useful bioorthogonal tool to selectively probe FASN-mediated protein acylation in normal and diseased states.

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Improved ELISA for Selective Measurement of Adiponectin Multimers and Identification of Adiponectin in Human Cerebrospinal Fluid

Clinical Chemistry ◽

10.1373/clinchem.2007.085654 ◽

2007 ◽

Vol 53 (8) ◽

pp. 1541-1544 ◽

Cited By ~ 60

Author(s):

Hiroyuki Ebinuma ◽

Takashi Miida ◽

Toshimasa Yamauchi ◽

Yusuke Hada ◽

Kazuo Hara ◽

...

Keyword(s):

Molecular Weight ◽

Cerebrospinal Fluid ◽

Energy Homeostasis ◽

Serum Adiponectin ◽

Native Page ◽

Elisa System ◽

Low Concentrations ◽

Molecular Sizes ◽

The Mean ◽

Mean Concentration

Abstract Background: Human serum adiponectin exists in 3 multimer forms: high molecular weight (HMW), middle molecular weight, and low molecular weight (LMW), with some of the latter bound to albumin (Alb)-LMW. Some studies have suggested that adiponectin crosses the blood–brain barrier and plays a central role in energy homeostasis. Methods: To determine cerebrospinal fluid (CSF) adiponectin at extremely low concentrations, we modified the protocol of the ELISA system used to assay serum adiponectin. The 3 multimers of adiponectin were measured separately by pretreating CSF with 2 proteases. We measured the CSF adiponectin concentrations in anonymous human samples (n = 19). The molecular sizes of adiponectin in CSF pretreated with proteases or untreated were determined by use of native PAGE and immunoblotting. Results: The ELISA system measured adiponectin in the range of 1.0–167 μg/L. The between-assay imprecision estimates (CVs) were 6%–17% for the 3 forms. The mean total CSF adiponectin concentration (7.2 μg/L) was ∼1/1000 of the mean concentration in serum. Unlike serum adiponectin, the LMW and Alb-LMW forms predominated in all of the CSF samples. Immunoblotting analysis revealed that most LMW forms were bound to Alb, although the HMW form was detected in some samples. Conclusions: The modified ELISA system measures the 3 multimers separately and is sufficiently sensitive to measure adiponectin in CSF.

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The role of AMP-activated protein kinase in the androgenic potentiation of cannabinoid-induced changes in energy homeostasis

AJP Endocrinology and Metabolism ◽

10.1152/ajpendo.00421.2014 ◽

2015 ◽

Vol 308 (6) ◽

pp. E482-E495 ◽

Cited By ~ 15

Author(s):

Amanda Borgquist ◽

Cecilia Meza ◽

Edward J. Wagner

Keyword(s):

Protein Kinase ◽

Energy Intake ◽

Energy Homeostasis ◽

Cannabinoid Receptor ◽

Animal Experiments ◽

Subcutaneous Administration ◽

Postsynaptic Currents ◽

Compound C ◽

Electrophysiological Studies ◽

Dependent Protein

Orexigenic mediators can impact the hypothalamic feeding circuitry via the activation of AMP-dependent protein kinase (AMPK). Given that testosterone is an orexigenic hormone, we hypothesized that androgenic changes in energy balance are due to enhanced cannabinoid-induced inhibition of anorexigenic proopiomelanocortin (POMC) neurons via activation of AMPK. To this end, whole animal experiments were carried out in gonadectomized male guinea pigs treated subcutaneously with either testosterone propionate (TP; 400 μg) or its sesame oil vehicle (0.1 ml). TP-treated animals displayed increases in energy intake associated with increases in meal size. TP also increased several indices of energy expenditure as well as the p-AMPK/AMPK ratio in the arcuate nucleus (ARC) measured 2 and 24 h posttreatment. Subcutaneous administration of the CB1 receptor antagonist AM251 (3 mg/kg) rapidly blocked the hyperphagic effect of TP. This was mimicked largely upon third ventricular administration of AM251 (10 μg). Electrophysiological studies revealed that TP potentiated the ability of the cannabinoid receptor agonist WIN 55,212-2 to decrease the frequency of miniature excitatory postsynaptic currents in ARC neurons. TP also increased the basal frequency of miniature inhibitory postsynaptic currents. In addition, depolarization-induced suppression (DSE) is potentiated in cells from TP-treated animals and blocked by AM251. The AMPK inhibitor compound C attenuated DSE from TP-treated animals, whereas the AMPK activator metformin enhanced DSE from vehicle-treated animals. These effects occurred in a sizable number of identified POMC neurons. Collectively, these results indicate that the androgen-induced increases in energy intake are mediated via an AMPK-dependent augmentation in endocannabinoid tone onto POMC neurons.

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