scholarly journals Esterases in Serum-Containing Growth Media Counteract Chloramphenicol Acetyltransferase Activity In Vitro

1999 ◽  
Vol 43 (3) ◽  
pp. 655-660 ◽  
Author(s):  
Charles D. Sohaskey ◽  
Alan G. Barbour
Keyword(s):  
Human Serum ◽  
Horse Serum ◽  
Chloramphenicol Acetyltransferase ◽  
Rabbit Serum ◽  
Growth Media ◽  
Chloramphenicol Resistance ◽  
E Coli ◽  
Cell Extracts

ABSTRACT The spirochete Borrelia burgdorferi was unexpectedly found to be as susceptible to diacetyl chloramphenicol, the product of the enzyme chloramphenicol acetyltransferase, as it was to chloramphenicol itself. The susceptibilities of Escherichia coli and Bacillus subtilis, as well as that ofB. burgdorferi, to diacetyl chloramphenicol were then assayed in different media. All three species were susceptible to diacetyl chloramphenicol when growth media were supplemented with rabbit serum or, to a lesser extent, human serum. Susceptibility ofE. coli and B. subtilis to diacetyl chloramphenicol was not observed in the absence of serum, when horse serum was used, or when the rabbit or human serum was heated first. In the presence of 10% rabbit serum, a strain of E. colibearing the chloramphenicol acetyltransferase (cat) gene had a fourfold-lower resistance to chloramphenicol than in the absence of serum. A plate bioassay for chloramphenicol activity showed the conversion by rabbit, mouse, and human sera but not bacterial cell extracts or heated serum of diacetyl chloramphenicol to an inhibitory compound. Deacetylation of acetyl chloramphenicol by serum components was demonstrated by using fluorescent substrates and thin-layer chromatography. These studies indicate that esterases of serum can convert diacetyl chloramphenicol back to an active antibiotic, and thus, in vitro findings may not accurately reflect the level of chloramphenicol resistance by cat-bearing bacteria in vivo.

Comparison of Vibrio and Firefly Luciferases as Reporter Gene Systems for Use in Bacteria and Plants

1996 ◽  
Vol 23 (1) ◽  
pp. 75 ◽  
Author(s):  
SR Mudge ◽  
WR Lewis-Henderson ◽  
RG Birch
Keyword(s):  
Gene Expression ◽  
Reporter Gene ◽  
Ccd Camera ◽  
Reporter Gene Expression ◽  
In Vitro Assays ◽  
E Coli ◽  
Cell Extracts ◽  
Cooled Ccd

Luciferase genes from Vibrio harveyi (luxAB) and firefly (luc) were introduced into E. coli, Agrobacteriurn, Arabidopsis and tobacco. Transformed bacteria and plants were quantitatively assayed for luciferase activity using a range of in vitro and in vivo assay conditions. Both lux and luc proved efficient reporter genes in bacteria, although it is important to be aware that the sensitive assays may detect expression due to readthrough from distant promoters. LUX activity was undetectable by liquid nitrogen-cooled CCD camera assays on intact tissues of plants which showed strong luxAB expression by in vitro assays. The decanal substrate for the lux assay was toxic to many plant tissues, and caused chemiluminescence in untransformed Arabidopsis leaves. These are serious limitations to application of the lux system for sensitive, non-toxic assays of reporter gene expression in plants. In contrast, LUC activity was readily detectable in intact tissues of all plants with luc expression detectable by luminometer assays on cell extracts. Image intensities of luc-expressing leaves were commonly two to four orders of magnitude above controls under the CCD camera. Provided adequate penetration of the substrate luciferin is obtained, luc is suitable for applications requiring sensitive, non-toxic assays of reporter gene expression in plants.

scholarly journals The use of naturally occurring hybrid variants of chloramphenicol acetyltransferase to investigate subunit contacts

1981 ◽  
Vol 193 (2) ◽  
pp. 541-552 ◽  
Author(s):  
L C Packman ◽  
W V Shaw
Keyword(s):  
Chloramphenicol Acetyltransferase ◽  
Alpha Subunit ◽  
Type I ◽  
Chloramphenicol Resistance ◽  
Type Iii ◽  
Naturally Occurring ◽  
Alpha Beta ◽  
Beta 2

1. Hybrids of the tetrameric enzyme chloramphenicol acetyltransferase (EC 2.3.1.28) were formed in vivo in a strain of Escherichia coli which harbours two different plasmids, each of which normally confers chloramphenicol resistance and specifies an easily distinguished enzyme variant (type I or type III) which is composed of identical subunits. Cell-free extracts of the dual-plasmid strain were found to contain five species of active enzyme, two of which were the homomeric enzymes corresponding to the naturally occurring tetramers of the type-I (beta 4) and type-III (alpha 4) enzymes. The other three variants were judged to be the heteromeric hybrid variants (alpha 3 beta, alpha 2 beta 2, alpha beta 3). 2. The alpha 3 beta and alpha 2 beta 2 hybrids of chloramphenicol acetyltransferase were purified to homogeneity by combining the techniques of affinity and ion-exchange chromatography. The alpha beta 3 variant was not recovered and may be unstable in vitro. 3. The unique lysine residues that could not be modified with methyl acetimidate in each of the native homomeric enzymes were also investigated in the heteromeric tetramers. 4. Lysine-136 remains buried in each beta subunit of the parental (type I) enzyme and in each of the hybrid tetramers. Lysine-38 of each alpha subunit is similarly unreactive in the native type-III chloramphenicol acetyltransferase (alpha 4), but in the alpha 2 beta 2 hybird lysine-38 of each alpha subunit is fully exposed to solvent. Another lysine residue, fully reactive in the alpha 4 enzyme, was observed to be inaccessible to modification in the symmetrical hybrid. The results obtained for the alpha 3 beta enzyme suggest that lysine-38 in two subunits and a different lysine group (that identified in the alpha 2 beta 2 enzyme) in the third alpha subunit are buried. 5. A tentative model for the subunit interactions of chloramphenicol acetyltransferase is proposed on the basis of the results described.

scholarly journals Binding of FAD to 6-hydroxy-d-nicotine oxidase apoenzyme prevents degradation of the holoenzyme

1989 ◽  
Vol 258 (1) ◽  
pp. 187-192 ◽  
Author(s):  
R Brandsch ◽  
V Bichler ◽  
B Krauss
Keyword(s):  
Proteinase Inhibitors ◽  
Cysteine Proteinase ◽  
Cysteine Proteinase Inhibitor ◽  
Western Blots ◽  
E Coli ◽  
Cell Extracts ◽  
Arthrobacter Oxidans ◽  
Cloned Gene

Expression of the 6-hydroxy-D-nicotine oxidase (6-HDNO) gene from Arthrobacter oxidans cloned into Escherichia coli showed a marked temperature-dependence. Transformed E. coli cells grown at 30 degrees C exhibited a several-fold higher 6-HDNO activity than did cells grown at 37 degrees C. This effect did not depend on the promoter used for expression of the cloned gene in E. coli, nor was it an effect of 6-HDNO mRNA instability at 37 degrees C. Studies performed in vivo and in vitro revealed that an increased susceptibility of apo-6-HDNO to proteolytic attack at 37 degrees C was responsible for the observed phenomenon. Extracts from cells grown at 37 degrees C showed on Western blots a decrease in immunologically detectable 6-HDNO polypeptide when compared with extracts from cells grown at 30 degrees C. The 6-HDNO polypeptide is covalently modified by attachment of the cofactor FAD to a histidine residue. It could be shown that covalent flavinylation of the apoenzyme in vitro, i.e. formation of holoenzyme, by incubation of cell extracts with FAD and phosphoenolpyruvate protected the 6-HDNO polypeptide from degradation at 37 degrees C. Of a variety of proteinase inhibitors tested only the cysteine-proteinase inhibitor L-3-trans-carboxyoxiran-2-carbonyl-L-leucylagmatine (E64) prevented degradation, by up to 70%, of the apoenzyme.

PROSTACYCLIN (PGI2) FORMATION IN RELATION TO ANAPHYLATOXIN C5a GENERATION DURING RABBIT ENDOTOXIN SHOCK

1987 ◽  
Author(s):  
M Rampart ◽  
H Bult ◽  
A G Herman ◽  
P J Jose ◽  
T J Williams
Keyword(s):  
Blood Pressure ◽  
Arterial Blood Pressure ◽  
Endotoxin Shock ◽  
Rabbit Serum ◽  
Classical Pathway ◽  
E Coli ◽  
Arterial Blood ◽  
Anaphylatoxin C5a

Injection of endotoxin (LPS) in animals, a model for gram-negative septic shock, leads to intravascular activation of the complement system, and is one of the few conditions in which 6-oxo-PGF]CX and thromboxane (TX) B2 (non-enzymic metabolites of PGI2 and TXA2) can be detected in arterial blood. Previously we reported associations between complement activation, PGI2 biosynthesis and LPS-induced hypotension in rabbits. As C5a and C5adesArg trigger endothelial PGI2 formation in vitro, we have now measured the plasma levels of immunoreactive (ir) C5a in relation to generation of PGI2 and changes in arterial blood pressure in LPS shock. Pentobarbitone anaesthethized rabbits received LPS (E. coli 0111:B4, 0.5 mg/kg) or saline via the marginal ear vein. A catheter in the left carotid artery was used to collect blood and to monitor mean arterial blood pressure (MABP). Platelet and leukocyte numbers, haemolytic complement titre (CH50), and plasma ir6-oxo-PGFioc , irTXB2 and irC5a were measured 15 min before and at different times after saline or LPS injection. LPS caused a dose- and time-dependent formation of irC5a in rabbit serum in vitro, predominantly via the classical pathway. LPS also activated complement in vivo, as indicated by about 20 % reduction of CH50 titre (measured after 3h) and a marked increase of arterial irC5a (20-120 ng/ml) in the first 2 to 5 min. After 30 min, irC5a had returned to baseline levels (< 2-5 ng/ml) and remained so up to 3h after injection of LPS. This irC5a peak correlated with a shortlasting initiation of PGI2 release (from < 20 pg/ml up to 550 pg/ml) and a drop in MABP (from about 95 mmHg to 50 mmHg) 2-5 min after LPS. None of these changes occurred after saline injection.In conclusion, LPS activates complement in vivo with concomitant formation of C5a. This peptide may trigger -either directly or after phagocyte activation - endothelial PGI2 biosynthesis, leading to arterial hypotension. This is supported by the suppression of the initial rise of arterial ir6-oxo-PGF1α and hypotension in complement-depleted rabbits. Inhibition of C5a formation or activity may prove to be a meaningful approach to the treatment of septic circulatory shock.

scholarly journals The Function of Cytoplasmic Flavin Reductases in the Reduction of Azo Dyes by Bacteria

2000 ◽  
Vol 66 (4) ◽  
pp. 1429-1434 ◽  
Author(s):  
Rainer Russ ◽  
Jörg Rau ◽  
Andreas Stolz
Keyword(s):  
Azo Dyes ◽  
Reduction Rate ◽  
Constitutive Expression ◽  
Azo Compounds ◽  
Flavin Reductase ◽  
Whole Cells ◽  
E Coli ◽  
Cell Extracts

ABSTRACT A flavin reductase, which is naturally part of the ribonucleotide reductase complex of Escherichia coli, acted in cell extracts of recombinant E. coli strains under aerobic and anaerobic conditions as an “azo reductase.” The transfer of the recombinant plasmid, which resulted in the constitutive expression of high levels of activity of the flavin reductase, increased the reduction rate for different industrially relevant sulfonated azo dyes in vitro almost 100-fold. The flavin reductase gene (fre) was transferred to Sphingomonas sp. strain BN6, a bacterial strain able to degrade naphthalenesulfonates under aerobic conditions. The flavin reductase was also synthesized in significant amounts in theSphingomonas strain. The reduction rates for the sulfonated azo compound amaranth were compared for whole cells and cell extracts from both recombinant strains, E. coli, and wild-typeSphingomonas sp. strain BN6. The whole cells showed less than 2% of the specific activities found with cell extracts. These results suggested that the cytoplasmic anaerobic “azo reductases,” which have been described repeatedly in in vitro systems, are presumably flavin reductases and that in vivo they have insignificant importance in the reduction of sulfonated azo compounds.

scholarly journals Characterization of the Klebsiella aerogenes Urease Accessory Protein UreD in Fusion with the Maltose Binding Protein

2010 ◽  
Vol 192 (9) ◽  
pp. 2294-2304 ◽  
Author(s):  
Eric L. Carter ◽  
Robert P. Hausinger
Keyword(s):  
Binding Protein ◽  
Equilibrium Dialysis ◽  
Maltose Binding Protein ◽  
Accessory Protein ◽  
Klebsiella Aerogenes ◽  
E Coli ◽  
Cell Extracts ◽  
Recombinant Cells

ABSTRACT Assembly of the Klebsiella aerogenes urease metallocenter requires four accessory proteins, UreD, UreE, UreF, and UreG, to effectively deliver and incorporate two Ni2+ ions into the nascent active site of the urease apoprotein (UreABC). Each accessory protein has been purified and characterized with the exception of UreD due to its insolubility when it is overproduced in recombinant cells. In this study, a translational fusion was made between the maltose binding protein (MBP) and UreD, with the resulting MBP-UreD found to be soluble in Escherichia coli cell extracts and able to complement a ΔureD-urease cluster in this host microorganism. MBP-UreD was purified as a large multimer (>670 kDa) that bound approximately 2.5 Ni2+ ions (Kd of ∼50 μM, where Kd is the dissociation constant) per UreD protomer according to equilibrium dialysis measurements. Zn2+ directly competes with 10-fold higher affinity (∼4 Zn2+ ions per protomer; Kd of 5 μM) for the Ni2+ binding sites. MBP pulldown experiments demonstrated that the UreD domain of MBP-UreD formed in vivo complexes with UreF, UreG, UreF plus UreG, or UreABC when these proteins were overproduced in the same E. coli cells. In addition, a UreABC-(MBP-UreD)-UreFG complex was observed in cells producing all urease components. Comparative in vitro binding experiments with purified proteins demonstrated an approximate 1:1 binding ratio between the UreD domain of MBP-UreD and the UreF domain of the UreEF fusion, only weak or transient interaction between MBP-UreD and UreG, and no binding with UreABC. These studies are the first to describe the properties of purified UreD, and they extend our understanding of its binding partners both in vitro and in the cell.

scholarly journals In vitro and in vivo antibacterial efficacies of CFC-222, a new fluoroquinolone.

1997 ◽  
Vol 41 (10) ◽  
pp. 2209-2213 ◽  
Author(s):  
J H Kim ◽  
J A Kang ◽  
Y G Kim ◽  
J W Kim ◽  
J H Lee ◽  
...  
Keyword(s):  
Bacterial Infections ◽  
Protective Efficacy ◽  
Horse Serum ◽  
The Other ◽  
Infection Model ◽  
Gram Positive ◽  
Gram Negative ◽  
E Coli

CFC-222 is a novel fluoroquinolone containing a C-7 bicyclic amine moiety with potent antibacterial activities against gram-positive, gram-negative, and anaerobic organisms. We compared the in vitro and in vivo activities of CFC-222 with those of ciprofloxacin, ofloxacin, and lomefloxacin. CFC-222 was more active than the other fluoroquinolones tested against gram-positive bacteria. CFC-222 was particularly active against Streptococcus pneumoniae (MIC at which 90% of isolates are inhibited [MIC90], 0.2 microg/ml), Staphylococcus aureus (MIC90, 0.2 microg/ml for ciprofloxacin-susceptible strains), and Enterococcus faecalis (MIC90, 0.39 microg/ml). Against Escherichia coli and other members of the family Enterobacteriaceae, CFC-222 was slightly less active than ciprofloxacin (MIC90s for E. coli, 0.1 and 0.025 microg/ml, respectively). The in vitro activity of CFC-222 was not influenced by inoculum size, medium composition, or the presence of horse serum. However, its activity was decreased significantly by a change in the pH of the medium from 7.0 to 6.0, as was the case for the other quinolones tested. The in vivo protective efficacy of CFC-222 by oral administration was greater than those of the other quinolones tested in a mouse model of intraperitoneally inoculated systemic infection caused by S. aureus. CFC-222 exhibited efficacy comparable to that of ciprofloxacin in the same model of infection caused by gram-negative organisms, such as E. coli and Klebsiella pneumoniae. In this infection model, CFC-222 was slightly less active than ciprofloxacin against Pseudomonas aeruginosa. These results suggest that CFC-222 may be a promising therapeutic agent in various bacterial infections.

scholarly journals Bactericidal Monoclonal Antibodies Specific to the Lipopolysaccharide O Antigen from Multidrug-Resistant Escherichia coli Clone ST131-O25b:H4 Elicit Protection in Mice

2015 ◽  
Vol 59 (6) ◽  
pp. 3109-3116 ◽  
Author(s):  
Valéria Szijártó ◽  
Luis M. Guachalla ◽  
Zehra C. Visram ◽  
Katharina Hartl ◽  
Cecília Varga ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Monoclonal Antibodies ◽  
Human Serum ◽  
Significant Proportion ◽  
Multidrug Resistant ◽  
Bacterial Killing ◽  
Content Type ◽  
E Coli

ABSTRACTTheEscherichia colisequence type 131 (ST131)-O25b:H4 clone has spread worldwide and become responsible for a significant proportion of multidrug-resistant extraintestinal infections. We generated humanized monoclonal antibodies (MAbs) that target the lipopolysaccharide O25b antigen conserved within this lineage. These MAbs bound to the surface of live bacterial cells irrespective of the capsular type expressed. In a serum bactericidal assayin vitro, MAbs induced >95% bacterial killing in the presence of human serum as the complement source. Protective efficacy at low antibody doses was observed in a murine model of bacteremia. The mode of actionin vivowas investigated by using aglycosylated derivatives of the protective MAbs. The significant binding to liveE. colicells and thein vitroandin vivoefficacy were corroborated in assays using bacteria grown in human serum to mimic relevant clinical conditions. Given the dry pipeline of novel antibiotics against multidrug-resistant Gram-negative pathogens, passive immunization with bactericidal antibodies offers a therapeutic alternative to control infections caused byE. coliST131-O25b:H4.

Rapid, Refined, and Robust Method for Expression, Purification, and Characterization of Recombinant Human Amyloid-beta M1-42

2019 ◽  
Author(s):  
Priya Prakash ◽  
Travis Lantz ◽  
Krupal P. Jethava ◽  
Gaurav Chopra
Keyword(s):  
Amyloid Beta ◽  
Western Blots ◽  
Force Microscopy ◽  
E Coli ◽  
Atomic Force ◽  
Set Up ◽  
Short Time

Amyloid plaques found in the brains of Alzheimer’s disease (AD) patients primarily consists of amyloid beta 1-42 (Ab42). Commercially, Ab42 is synthetized using peptide synthesizers. We describe a robust methodology for expression of recombinant human Ab(M1-42) in Rosetta(DE3)pLysS and BL21(DE3)pLysS competent E. coli with refined and rapid analytical purification techniques. The peptide is isolated and purified from the transformed cells using an optimized set-up for reverse-phase HPLC protocol, using commonly available C18 columns, yielding high amounts of peptide (~15-20 mg per 1 L culture) in a short time. The recombinant Ab(M1-42) forms characteristic aggregates similar to synthetic Ab42 aggregates as verified by western blots and atomic force microscopy to warrant future biological use. Our rapid, refined, and robust technique to purify human Ab(M1-42) can be used to synthesize chemical probes for several downstream in vitro and in vivo assays to facilitate AD research.

scholarly journals A Peptide Found in Human Serum, Derived from the C-Terminus of Albumin, Shows Antifungal Activity In Vitro and In Vivo

2020 ◽  
Vol 8 (10) ◽  
pp. 1627
Author(s):  
Tecla Ciociola ◽  
Pier Paolo Zanello ◽  
Tiziana D’Adda ◽  
Serena Galati ◽  
Stefania Conti ◽  
...  
Keyword(s):  
Antifungal Activity ◽  
Human Serum ◽  
Fungicidal Activity ◽  
Galleria Mellonella ◽  
Serum Proteins ◽  
Morphological Alterations ◽  
C Terminus ◽  
Different Sources

The growing problem of antimicrobial resistance highlights the need for alternative strategies to combat infections. From this perspective, there is a considerable interest in natural molecules obtained from different sources, which are shown to be active against microorganisms, either alone or in association with conventional drugs. In this paper, peptides with the same sequence of fragments, found in human serum, derived from physiological proteins, were evaluated for their antifungal activity. A 13-residue peptide, representing the 597–609 fragment within the albumin C-terminus, was proved to exert a fungicidal activity in vitro against pathogenic yeasts and a therapeutic effect in vivo in the experimental model of candidal infection in Galleria mellonella. Studies by confocal microscopy and transmission and scanning electron microscopy demonstrated that the peptide penetrates and accumulates in Candida albicans cells, causing gross morphological alterations in cellular structure. These findings add albumin to the group of proteins, which already includes hemoglobin and antibodies, that could give rise to cryptic antimicrobial fragments, and could suggest their role in anti-infective homeostasis. The study of bioactive fragments from serum proteins could open interesting perspectives for the development of new antimicrobial molecules derived by natural sources.

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