Faculty Opinions recommendation of Endogenous protein S-Nitrosylation in E. coli: regulation by OxyR.

Mutagenesis in Escherichia coli, a subject of many years of study is considered to be related to DNA replication. DNA lesions nonrepaired by the error-free nucleotide excision repair (NER), base excision repair (BER) and recombination repair (RR), stop replication at the fork. Reinitiation needs translesion synthesis (TLS) by DNA polymerase V (UmuC), which in the presence of accessory proteins, UmuD', RecA and ssDNA-binding protein (SSB), has an ability to bypass the lesion with high mutagenicity. This enables reinitiation and extension of DNA replication by DNA polymerase III (Pol III). We studied UV- and MMS-induced mutagenesis of lambdaO(am)8 phage in E. coli 594 sup+ host, unable to replicate the phage DNA, as a possible model for mutagenesis induced in nondividing cells (e.g. somatic cells). We show that in E. coli 594 sup+ cells UV- and MMS-induced mutagenesis of lambdaO(am)8 phage may occur. This mutagenic process requires both the UmuD' and C proteins, albeit a high level of UmuD' and low level of UmuC seem to be necessary and sufficient. We compared UV-induced mutagenesis of lambdaO(am)8 in nonpermissive (594 sup+) and permissive (C600 supE) conditions for phage DNA replication. It appeared that while the mutagenesis of lambdaO(am)8 in 594 sup+ requires the UmuD' and C proteins, which can not be replaced by other SOS-inducible protein(s), in C600 supE their functions may be replaced by other inducible protein(s), possibly DNA polymerase IV (DinB). Mutations induced under nonpermissive conditions for phage DNA replication are resistant to mismatch repair (MMR), while among those induced under permissive conditions, only about 40% are resistant.

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Subcellular location and properties of bactericidal factors from human neutrophils.

Journal of Experimental Medicine ◽

10.1084/jem.164.5.1407 ◽

1986 ◽

Vol 164 (5) ◽

pp. 1407-1421 ◽

Cited By ~ 32

Author(s):

J E Gabay ◽

J M Heiple ◽

Z A Cohn ◽

C F Nathan

Keyword(s):

Protein S ◽

Subcellular Location ◽

Inhibitory Effect ◽

Human Neutrophils ◽

E Coli ◽

Fractionation Scheme ◽

Percoll Gradients ◽

Nonionic Detergent ◽

Almost All ◽

Triton X

We examined the subcellular location of bactericidal factors (BF) in human neutrophils, using an efficient fractionation scheme. Nitrogen bomb cavitates of DIFP-treated PMN were centrifuged through discontinuous Percoll gradients, each fraction extracted with 0.05 M glycine, pH 2.0, and tested for the killing of Escherichia coli. greater than 90% of BF coisolated with the azurophil granules. After lysis of azurophils, 98% of azurophil-derived BF (ADBF) sedimented with the membrane. ADBF activity was solubilized from azurophil membrane with either acid or nonionic detergent (Triton X-100, Triton X-114). Bactericidal activity was linear with respect to protein concentration over the range 0.3-30 micrograms/ml. 0.1-0.3 microgram/ml ADBF killed 10(5) E. coli within 30 min at 37 degrees C. At 1.4 micrograms/ml, 50% of 2 X 10(5) bacteria were killed within 5 min. ADBF was effective between pH 5-8, with peak activity at pH 5.5. Glucose (20 mM), EDTA (1-25 mM), and physiologic concentrations of NaCl or KCl had little or no inhibitory effect on ADBF. ADBF killed both Gram-positive and Gram-negative virulent clinical isolates, including listeria, staphylococci, beta-hemolytic streptococci, and Pseudomonas aeruginosa. Thus, under these conditions of cell disruption, fractionation, extraction, and assay, almost all BF in human PMN appeared to be localized to the membrane of azurophilic granules as a highly potent, broad-spectrum, rapidly acting protein(s) effective in physiologic medium. Some of these properties appear to distinguish ADBF from previously described PMN bactericidal proteins.

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C4b-binding protein exacerbates the host response to Escherichia coli

Blood ◽

10.1182/blood.v78.2.357.357 ◽

1991 ◽

Vol 78 (2) ◽

pp. 357-363 ◽

Cited By ~ 100

Author(s):

F Taylor ◽

A Chang ◽

G Ferrell ◽

T Mather ◽

R Catlett ◽

...

Keyword(s):

Escherichia Coli ◽

Acute Phase ◽

Protein C ◽

Activated Protein C ◽

Protein S ◽

Organ Damage ◽

Vascular Damage ◽

E Coli ◽

Infusion Group ◽

Sublethal Concentrations

Abstract Activated protein C is a plasma anticoagulant. For activated protein C to function as an anticoagulant, it must form a complex with protein S. Protein S anticoagulant activity is neutralized by formation of a reversible complex with C4b binding protein (C4bBP). C4bBP is an acute- phase plasma protein. When C4bBP levels increase, mass action forces the level of free protein S to decrease, giving rise to an acquired functional protein S deficiency. It has been proposed that these elevated C4bBP levels and the resultant acquired deficiency of protein S that occurs in inflammation could contribute to a hypercoagulable state. An experimental model to test this hypothesis was suggested by our previous studies that demonstrated that inhibition of protein C activation rendered baboons hypercoagulable in response to sublethal Escherichia coli infusion (J Clin Invest 79:918, 1987). We have extended these studies to examine the effect of inhibition of protein S activity with C4bBP in the host (baboon) response to infusion of sublethal concentrations of E coli organisms. Five sets of animals were studied: (1) those challenged with sublethal concentrations of E coli alone (0.4 x 10(10)/kg); (2) those supplemented only with C4bBP (20 mg/kg); (3) those challenged with the same level of E coli but supplemented with C4bBP (20 mg/kg); (4) those challenged with sublethal E coli and supplemented with C4bBP (20 mg/kg) and sufficient protein S (2.3 mg/kg) to fill the protein S binding sites on C4bBP; and (5) those challenged with lethal concentrations of E coli. Sublethal E coli infusion (group 1 animals) caused only an acute-phase response with no consumption of fibrinogen, detectable organ damage, or detectable tumor necrosis factor (TNF) in the plasma. C4bBP infusion (group 2 animals) resulted in no significant physiologic changes, no detectable plasma TNF, and little change in fibrinogen level. The group 3 animals, receiving both sublethal E coli and C4bBP, exhibited rapid consumption of fibrinogen, systemic organ damage, and detectable circulating TNF ultimately leading to death. The overall response of this group was very similar to the response of the group 5 animals receiving an LD100 dose of E coli. The group 4 animals, which were treated exactly as above except that C4bBP was supplemented with a slight excess of protein S, responded essentially like those that received sublethal E coli alone. These studies suggest that the elevation of C4bBP during an inflammatory response can contribute to fibrinogen consumption and vascular damage. This vascular damage may be associated with enhanced elaboration of cytokines like TNF.(ABSTRACT TRUNCATED AT 400 WORDS)

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Identification of a Reactivating Factor for Adenosylcobalamin-Dependent Ethanolamine Ammonia Lyase

Journal of Bacteriology ◽

10.1128/jb.186.20.6845-6854.2004 ◽

2004 ◽

Vol 186 (20) ◽

pp. 6845-6854 ◽

Cited By ~ 32

Author(s):

Koichi Mori ◽

Reiko Bando ◽

Naoki Hieda ◽

Tetsuo Toraya

Keyword(s):

Escherichia Coli ◽

Molecular Weight ◽

Protein S ◽

Amino Acid Residues ◽

Permeabilized Cells ◽

E Coli ◽

Cell Extracts ◽

Auxiliary Protein

ABSTRACT The holoenzyme of adenosylcobalamin-dependent ethanolamine ammonia lyase undergoes suicidal inactivation during catalysis as well as inactivation in the absence of substrate. The inactivation involves the irreversible cleavage of the Co-C bond of the coenzyme. We found that the inactivated holoenzyme undergoes rapid and continuous reactivation in the presence of ATP, Mg2+, and free adenosylcobalamin in permeabilized cells (in situ), homogenate, and cell extracts of Escherichia coli. The reactivation was observed in the permeabilized E. coli cells carrying a plasmid containing the E. coli eut operon as well. From coexpression experiments, it was demonstrated that the eutA gene, adjacent to the 5′ end of ethanolamine ammonia lyase genes (eutBC), is essential for reactivation. It encodes a polypeptide consisting of 467 amino acid residues with predicted molecular weight of 49,599. No evidence was obtained that shows the presence of the auxiliary protein(s) potentiating the reactivation or associating with EutA. It was demonstrated with purified recombinant EutA that both the suicidally inactivated and O2-inactivated holoethanolamine ammonia lyase underwent rapid reactivation in vitro by EutA in the presence of adenosylcobalamin, ATP, and Mg2+. The inactive enzyme-cyanocobalamin complex was also activated in situ and in vitro by EutA under the same conditions. Thus, it was concluded that EutA is the only component of the reactivating factor for ethanolamine ammonia lyase and that reactivation and activation occur through the exchange of modified coenzyme for free intact adenosylcobalamin.

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Postnatal changes in dolichol-pathway enzyme activities in cerebral cortex neurons

Biochemical Journal ◽

10.1042/bj2020087 ◽

1982 ◽

Vol 202 (1) ◽

pp. 87-95 ◽

Cited By ~ 13

Author(s):

V Idoyaga-Vargas ◽

H Carminatti

Keyword(s):

Cerebral Cortex ◽

Postnatal Development ◽

Enzyme Activities ◽

Synapse Formation ◽

Adult Stage ◽

Protein S ◽

Rat Cerebral Cortex ◽

Dolichyl Phosphate ◽

Endogenous Protein ◽

Dolichol Pathway

Neuronal perikarya were isolated from rat cerebral cortex at different stages of postnatal development. Membranes sedimenting at 100000 g were obtained from these neurons to study several glycosyltransferases of the dolichol pathway. Enzyme activities from stages before and during synapse formation were compared (days 5 and 15 respectively). Dolichyl diphosphate (Dol-P-P) N-acetylglucosamine, dolichyl phosphate mannose and dolichyl phosphate glucose synthases and the enzymes catalysing Dol-P-P-GlcNAc2Man9Glc3 formation were higher at day 15 of postnatal development. The glycosyl transfer of the latter compound to endogenous protein(s) as well as to a dinitrophenyl-heptapeptide was also measured. The activity was higher at day 15. Furthermore, the activity of dolichyl phosphate mannose synthase was also measured during the time when the number of synapses ceased to increase (day 36) and in the adult stage. The activity of dolichyl phosphate mannose synthase was higher at day 36 than at day 15, and declined in the adult stage. From these results it may be concluded that there is an increase in the glycosylation of asparagine-type glycoproteins during synapse formation in the neurons of the cerebral cortex.

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