A New Fluorogenic Substrate Method for Assay of Bacterial Endotoxins Using Limulus Hemocyte Lysate

1979 ◽  
Author(s):  
T Harada ◽  
M Ohki ◽  
M Niwa ◽  
S Iwanaga
Keyword(s):  
Antithrombin Iii ◽  
Assay Method ◽  
Sensitive Assay ◽  
Fluorogenic Substrate ◽  
Toxin Concentration ◽  
Amidase Activity ◽  
E Coli ◽  
Bacterial Endotoxins ◽  
Plasmin Inhibitor ◽  
Limulus Test

Limulus hemocyte lysate contains a proclotting enzyme, which is transformed to the active clotting enzyme in the presence of gram-negative bacterial endotoxins. The clotting enzyme coagulates a clottable protein, named coagulogen, contained also in the lysate. This gelation reaction of the lysate, named Limulus test, has been widely employed as a simple and very sensitive assay method for endotoxins. We developed a new fluorogenic substrate, Boc-Leu-Gly-Arg-4-methylcoumarin amide, for Limulus clotting enzyme and established an enzymatic assay method for endotoxins, using the substrate. Because the endotoxin mediates the activation of proclotting enzyme in the lysate, the measurement of amidase activity could be applicable for quantitation of the endotoxins. In fact, the amidase activity determined fluorometrically increased by increasing concentration of E. coli 0111: B4 endotoxin added to the lysate, and a linear relationship between the toxin concentration and the activity was observed in the range of 5X10-6to 5xl0-2 µg endotoxin. The method was a fifty times more sensitive than that of the Limulus test and was very reproducible. However, the method was not directly applicable for the assay of endotoxins in circulating blood, as the amidase activity was strongly inhibited by antithrombin III and α2-plasmin inhibitor. Thus, some pretreatment with heat or chloroform on plasma samples before the assay was required.

A New Fluorogenic Substrate Method for Assay of Bacterial Endotoxins Using Limulus Hemocyte Lysate

1979 ◽  
Author(s):  
T. Harada ◽  
M. Ohki ◽  
M. Niwa ◽  
S. Iwanaga
Keyword(s):  
Antithrombin Iii ◽  
Assay Method ◽  
Sensitive Assay ◽  
Fluorogenic Substrate ◽  
Toxin Concentration ◽  
Amidase Activity ◽  
E Coli ◽  
Bacterial Endotoxins ◽  
Plasmin Inhibitor ◽  
Limulus Test

Limulus hemocyte lysate contains a proclotting enzyme., which is transformed to the active clotting enzyme in the presence of gram-negative bacterial endotoxins. The clotting enzyme coagulates a clottable protein, named coagulogen, contained also in the lysate. This gelation reaction of the lysate, named Limulus test, has been widely employed as a simple and very sensitive assay method for endotoxins. We developed a new fluorogenic substrate, Boc-Leu-Cly-Arg-4-methyLcoumarin amide, for Limulus clotting enzyme and established an enzymatic assay method for endotoxins, using the substrate. Because the endotoxin mediates the activation of proclotting enzyme in the lysate, the measurement of amidasc activity could be applicable for quantitation of the endotoxins. In fact, the amidase activity determined fluorometrically increased by increasing concentration of E. coli 0111: B4 endotoxin added to the lysate, and a linear relationship between the toxin concentration and the activity was observed in the range of 5×10-6 to 5×10-2 ug endotoxin. The method was a fifty times more sensitive than that of the Limulus test and was very reproducible. However, the method was not directly applicable for the assay of endotoxins in circulating blood, as the amidase activity was strongly inhibited by antithrombin III and α2-plasmin inhibitor. Thus, some pretreatment with heat or chloroform on plasma samples before the assay was required.

PROTEINASE-INHIBITOR COMPLEXES (PIC) IN SEPTIC AND NON-SEPTIC SHOCK. COAGULATION; LEUKOCYTE AND BACTERIAL PROTEASE INHIBITION BY MEANS OF PLASMA-INHIBITOR REPLACEMENT

1987 ◽  
Author(s):  
R Egbring ◽  
R Seitz ◽  
M Wolf ◽  
L Lerch ◽  
T Menges
Keyword(s):  
Antithrombin Iii ◽  
Coagulation Factors ◽  
In Vitro Models ◽  
Protease Inhibition ◽  
E Coli ◽  
Bacterial Protease ◽  
Bacterial Endotoxins ◽  
At Iii ◽  
Cardiac Shock

In septic or cardiac shock antithrombin III-thrombin (AT III-Thr) and a1antitrypsin-elastase(a1AT-ELP) as well as a2antiplas-min-plasmin (a2AP-Pl) are found to be elevated to different extents. In cardiac shock AT III-Thr is predominantly increased, while in septic disorders a2AT-ELP as indicator of leukocyte stimulation is additionally found to be elevated. Stimuli for leukocyte activation are bacterial endotoxins, immune complexes, factor Xlla and others. The possible action of bacterial proteases during septic infections is only known in animal models. To stop hemorrhagic complications in disseminated intravascular coagulation (DIC) following septic (n=24) or non-septic (n=15) shock, we treated the patients with AT III concentrate and FFP in relatively high amounts containing a2macroglobulin (a2M), a1antitrypsin (a1AT) and others which are not available as concentrates. Subsequent to the procedure PIC's decreased, coagulation factors and inhibitors as well as thrombocyte counts increased. In in vitro models bacterial proteases have been shown to destroy a1AT, activate prothrombin and others. Only a2M may inhibit proteolytic activity of Staph aureus, N. meningitidis, P. aeroginosa and K1. pneumoniae and E. coli as our in vitro studies, using fibrin plates containing a2M, demonstrated. Not only bleeding or microthrombotic complications might be influenced by plasma derivative substitution, but also proteases released from bacteria

Effects of Human Antithrombin III on Mortality and Blood Coagulation Induced in Rabbits by Endotoxin

1984 ◽  
Vol 51 (02) ◽  
pp. 232-235 ◽  
Author(s):  
D C Triantaphyllopoulos
Keyword(s):  
Blood Coagulation ◽  
Bolus Dose ◽  
Antithrombin Iii ◽  
Coagulation Parameters ◽  
E Coli ◽  
At Iii ◽  
Baseline Values

SummaryTwenty-one rabbits were infused with 20μg/kg/hr of E. coli endotoxin for 6 hr. Eight of the animals were preinjected immediately before the infusion of endotoxin, with a bolus dose of human AT III calculated to increase the antithrombin content of the plasma by about 4 units/ml. All eight animals which were preinjected with AT III survived, while 5 of the 13 control rabbits infused with endotoxin alone died. The changes in coagulation parameters from the baseline values, between the 8 control rabbits which survived and the 8 animals which were preinjected with AT III were compared. The concentration of the preinjected human AT III declined significantly faster (P: <0.01) than that of the native rabbit AT III. AT III prevented the decline of F.XII throughout the infusion of the endotoxin. However, the decline in F.V, fibrinogen, prothrombin and platelets was not affected (P: >0.5) by the injection of AT III.

scholarly journals Live-Cell Imaging of Caspase Activation for High-Content Screening

2009 ◽  
Vol 14 (8) ◽  
pp. 956-969 ◽  
Author(s):  
Christophe Antczak ◽  
Toshimitsu Takagi ◽  
Christina N. Ramirez ◽  
Constantin Radu ◽  
Hakim Djaballah
Keyword(s):  
Cell Death ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Exposure Period ◽  
Live Cells ◽  
Assay Method ◽  
Caspase Activation ◽  
Fluorogenic Substrate ◽  
Automated Imaging ◽  
First Time

Caspases are central to the execution of programmed cell death, and their activation constitutes the biochemical hallmark of apoptosis. In this article, the authors report the successful adaptation of a high-content assay method using the DEVDNucView488™ fluorogenic substrate, and for the first time, they show caspase activation in live cells induced by either drugs or siRNA. The fluorogenic substrate was found to be nontoxic over an exposure period of several days, during which the authors demonstrate automated imaging and quantification of caspase activation of the same cell population as a function of time. Overexpression of the antiapoptotic protein Bcl-XL, alone or in combination with the inhibitor Z-VAD-FMK, attenuated caspase activation in HeLa cells exposed to doxorubicin, etoposide, or cell death siRNA. This method was further validated against 2 well-characterized NSCLC cell lines reported to be sensitive (H3255) or refractory (H2030) to erlotinib, where the authors show a differential time-dependent activation was observed for H3255 and no significant changes in H2030, consistent with their respective chemosensitivity profile. In summary, the results demonstrate the feasibility of using this newly adapted and validated high-content assay to screen chemical or RNAi libraries for the identification of previously uncovered enhancers and suppressors of the apoptotic machinery in live cells. ( Journal of Biomolecular Screening 2009:956-969)

Endotoxin-Like Activities of Mycoplasmal Lipopolysaccharides (Lipoglycans)

1980 ◽  
Vol 29 (3) ◽  
pp. 990-994
Author(s):  
Robert C. Seid ◽  
Paul F. Smith ◽  
Gabriel Guevarra ◽  
H. Donald Hochstein ◽  
Michael F. Barile
Keyword(s):  
Escherichia Coli ◽  
Synthetic Peptide ◽  
Active Role ◽  
Peptide Bond ◽  
Febrile Response ◽  
Thermoplasma Acidophilum ◽  
Amidase Activity ◽  
E Coli

Lipoglycans (previously designated lipopolysaccharides) from several species of Acholeplasma and from Thermoplasma acidophilum were examined for endotoxin-like activities as measured by the standard rabbit fever test and the Limulus amoebocyte lysate assay. The lipoglycans from Acholeplasma granularum, Achloplasma laidlawii, Acholeplasma modicum , and Acholeplasma oculi caused a febrile response at concentrations of 1 ng/ml per kg or greater, whereas with control Escherichia coli EC-2 lipopolysaccharides, 6.25 ng/ml per kg was required. Similar results were obtained in the Limulus amoebocyte lysate test. The minimum concentrations in nanograms per milliliter required to stimulate formation of a solid clot were: Acholeplasma axanthum , 0.22; A. granularum , 0.85; A. modicum , 0.51; A. laidlawii , 1.05; A. oculi , 0.74. Standard E. coli 1B lipopolysaccharide required a concentration of 0.125 ng/ml. Thermoplasma lipoglycan was least active, requiring 4.25 ng/ml. Clotting of the Limulus lysate proceeds by the activation by lipopolysaccharide plus Ca 2+ of a proenzyme which cleaves an arginine-lysine peptide bond of the coagulogen. The clotting and amidase activities are inactivated by deoxycholate and can be reactivated by addition of lipopolysaccharide and Ca 2+ . As with E. coli 1B lipopolysaccharide, acholeplasmal lipoglycans were shown to restore both clotting and amidase activities of the deoxycholate-inactivated Limulus clotting enzyme. The degree of restoration of amidase activity by mycoplasmal lipoglycans relative to E. coli lipopolysaccharide (1.00) were: A. axanthum , 1.71; A. modicum , 1.22; A. granularum , 0.61; and Thermoplasma , 0.37. The coagulating enzyme, restored with either E. coli lipopolysaccharide or mycoplasmal lipoglycans, was able to react with the synthetic peptide benzoyl-Ile-Glu-(γ-OCH 3 )-Gly-p-nitroaniline (an analog of the coagulogen) or with the purified coagulogen itself to form the clot. The mycoplasmal lipoglycans alone were incapable of promoting these reactions when incubated with the synthetic peptide or with the purified coagulogen, thereby ruling out the contamination of these lipoglycans with proteases capable of cleaving the same Arg-Lys peptide bond of the coagulogen. These results show that acholeplasmal lipoglycans possess endotoxin-like activities. Their passive or active role in disease remains to be established.

A New Sensitive Assay Method of Kallidrein-like Arginine esterases*

1971 ◽  
Vol 69 (4) ◽  
pp. 815-817 ◽  
Author(s):  
Hiroshi MORIYA ◽  
Noriko TODOKI ◽  
Chiake MORIWAKI ◽  
Yoshio HOJIMA
Keyword(s):  
Assay Method ◽  
Sensitive Assay

scholarly journals The Activity of Heparin Preparations Studied by Amidolytic and Clotting Methods

1977 ◽  
Author(s):  
A.N. Teien ◽  
U. Abildgaard ◽  
M. Höök ◽  
U. Lindahl
Keyword(s):  
Antithrombin Iii ◽  
Specific Activity ◽  
Factor Xa ◽  
Test System ◽  
Assay Method ◽  
Thrombin Time ◽  
Assay Methods ◽  
Specific Activities ◽  
The Difference ◽  
Heparin Preparation

Two heparin standards, heparin isolated from human mastocytoma tissue, four commercial heparins and two heparin preparations separated by affinity chromatography (“High affinity heparin”=HAH and “Low affinity heparin”=LAH) were assayed by the activated partial thromboplastin time method (APTT), the calcium thrombin time method (CaTT) and two amidolytic methods (measuring the accelerating effect of heparin on the inactivation of thrombin or factor Xa by antithrombin III), with and without plasma in the test system. The specific activities of the various heparins were expressed relative to that of the 3rd. Int. Standard (=100). Found specific activities ranged 3 - 198 (LAH and HAH, respectively). In all assay systems HAH had the highest specific activity, followed by one of the commercial preparations and the 3rd Int. Standard. LAH and human heparin had very low specific activities, except in the APTT test system, an assay method which in addition mirrors other anticoagulant effects of heparin than the acceleration of antithrombin III. Apart from the higher effect of LAH and human heparin on the APTT, the difference in specific activities found for each individual heparin preparation with these various assay methods was slight.In view of the reproducibility and simplicity of the amidolytic methods, it is suggested that they be adapted for heparin standardization.

scholarly journals Measuring serum oestradiol in women with Alzheimer's disease: the importance of the sensitivity of the assay method

2003 ◽  
pp. 67-72 ◽  
Author(s):  
E Hogervorst ◽  
J Williams ◽  
M Combrinck ◽  
A David Smith
Keyword(s):  
Alzheimer’S Disease ◽  
Alzheimer's Disease ◽  
Robust Regression ◽  
Meta Analysis ◽  
Hormone Replacement ◽  
Assay Method ◽  
Sensitive Assay ◽  
Significant Difference ◽  
Low Levels ◽  
The Difference

OBJECTIVE: Oestrogens could be protective against the development of Alzheimer's disease (AD) but reports on oestrogen levels in AD have been conflicting. DESIGN AND METHODS: A meta-analysis using robust regression was carried out to assess whether the sensitivity of the assays of past studies had affected the reported level of total oestradiol. We had also measured total oestradiol in women with AD (n=66) and controls (n=62) not using hormone replacement therapy. We used two assays for total oestradiol to assess the difference between sensitive (radioimmunoassay with a specific rabbit antibody: 3 pmol/l) and relatively insensitive (immunoassay: 37 pmol/l) assays. RESULTS: Meta-analysis using robust regression indicated that insensitive assays gave higher levels of total oestradiol when many samples fall below the level of sensitivity of the method. Earlier reports of low levels of total oestradiol in AD might be explained by this phenomenon, since total oestradiol levels (using the sensitive assay) in our controls were one third of those reported in the earlier studies. Using the sensitive assay we found that women with AD had significantly (P<0.01) higher levels (26+/-13 pmol/l) of total oestradiol than controls (21+/-13 pmol/l). Using the insensitive assay, there was no significant difference in the levels of total oestradiol. CONCLUSIONS: The sensitivity of the assay determines the reported value of the oestradiol levels. Studies using a sensitive assay do not report significantly lower levels of total oestradiol in women with AD. This weighs against the hypothesis that low levels of total oestradiol are a risk factor for AD.

A Sensitive Assay Method of Furosemide in Plasma and Urine by High-Performance Liquid Chromotography

1982 ◽  
Vol 4 (4) ◽  
pp. 381-384 ◽  
Author(s):  
Walter Snedden ◽  
Jagdish N. Sharma ◽  
Peter G. Fernandez
Keyword(s):  
High Performance ◽  
Assay Method ◽  
Sensitive Assay
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