scholarly journals Studies of the acetylcholinesterase from houseflies (Musca domestica L.) resistant and susceptible to organophosphorus insecticides

1975 ◽  
Vol 149 (2) ◽  
pp. 463-469 ◽  
Author(s):  
A L Devonshire
Keyword(s):  
Musca Domestica ◽  
Rate Constants ◽  
Gel Electrophoresis ◽  
Organophosphorus Compounds ◽  
Organophosphorus Insecticides ◽  
Triton X ◽  
Kinetics Of ◽  
Kinetics Of Inhibition ◽  
Single Enzyme

Acetylcholinesterase from the heads of insecticide-resistant and -susceptible houseflies (Musca domestica L.) was studied in vitro. The enzymes could not be distinguished electrophoretically, and their behaviour on polyacrylamide-disc-gel electrophoresis was influenced by the presence of Triton X-100 in both the homogenate and the gels. In the absence of detergent, the acetylcholinesterase was heterogeneous, but behaved as a single enzyme when it was present. By analogy with studies of acetylcholinesterase from other sources, these observations were attributed to aggregation of the enzyme when not bound by membranes. The enzyme from resistant flies was more slowly inhibited than the susceptible enzyme, bimolecular rate constants (ki) differing by approx. 4-20-fold for a range of organophosphorus compounds. The kinetics of inhibition of acetylcholinesterase were consistent with the results of electrophoresis, i.e. they corresponded to those of a single enzyme in the presence of Triton X-100, but a mixture of enzymes in its absence. The susceptibility of the more sensitive components in these mixtures was determined.

Platelet Microtubule Subunit Proteins

1979 ◽  
Vol 42 (05) ◽  
pp. 1630-1633 ◽  
Author(s):  
A G Castle ◽  
N Crawford
Keyword(s):  
Epithelial Cells ◽  
Gel Electrophoresis ◽  
Blood Platelets ◽  
Polyacrylamide Gel Electrophoresis ◽  
Polyacrylamide Gel ◽  
Lens Epithelial Cells ◽  
Molecular Weights ◽  
Kinetics Of ◽  
Microtubular Structures

SummaryBlood platelets contain microtubule proteins (tubulin and HMWs) which can be polymerised “in vitro” to form structures which resemble the microtubules seen in the intact platelet. Platelet tubulin is composed of two non-identical subunits a and p tubulin which have molecular weights around 55,000 but can be resolved in alkaline SDS-polyacrylamide gel electrophoresis. These subunits associate as dimers with sedimentation coefficients of about 5.7 S although it is not known whether the dimer protein is a homo- or hetero-dimer. The dimer tubulin binds the anti-mitotic drug colchicine and the kinetics of this binding are similar to those reported for neurotubulins. Platelet microtubules also contain two HMW proteins which appear to be essential and integral components of the fully assembled microtubule. These proteins have molecular weights greater than 200,000 daltons. Fluorescent labelled antibodies to platelet and brain tubulins stain long filamentous microtubular structures in bovine lens epithelial cells and this pattern of staining is prevented by exposing the cells to conditions known to cause depolymerisation of cell microtubules.

scholarly journals Initiation and regulation of fibrinolysis in human plasma at the plasminogen activator level

Blood ◽  
1987 ◽  
Vol 69 (5) ◽  
pp. 1354-1362 ◽  
Author(s):  
TC Wun ◽  
A Capuano
Keyword(s):  
Human Plasma ◽  
Plasminogen Activator ◽  
Gel Electrophoresis ◽  
Fibrinolytic Activity ◽  
High Rate ◽  
Bovine Aortic Endothelial Cell ◽  
Hep G2 ◽  
Triton X ◽  
Sepharose 4B

The initiation and regulation of fibrinolysis has been studied by reconstitution of fibrinolytic activity in human plasma in vitro. Depletion of tissue plasminogen activator (tPA) antigen by immunoadsorption of human plasma with anti-tPA Ig Sepharose 4B leads to total loss of spontaneous fibrinolytic activity determined by lysis of a thrombin-induced clot. Addition of physiological concentrations of purified tPA to tPA-depleted plasma restores fibrinolytic activity as a function of the length of time between tPA addition and clotting. Addition of free tPA to tPA-depleted plasma followed by immediate clotting results in a high rate of fibrinolysis. In contrast, when free tPA is allowed to incubate in plasma for 10 to 60 minutes prior to clot formation, the fibrinolytic activity of tPA is gradually lost. The loss of tPA-induced fibrinolytic activity in unclotted plasma is accompanied by decreased partitioning of tPA antigen into fibrin after clotting and is kinetically correlated with the formation of a 100 kilodalton (kDa) tPA complex as demonstrated by SDS-gel electrophoresis and fibrin-agar zymography. These results suggest that free tPA is susceptible to complexation by the plasma inhibitor in the absence of a clot. Fibrin formation renders tPA relatively inaccessible to inhibition. The tPA antigen isolated from stored plasma consists mainly of 100 kDa activity in SDS-gel electrophoresis and zymography, indicating that the tPA complex is resistant to dissociation by SDS. Upon rezymography of the sliced gel, only a 60 kDa tPA activity is found, suggesting that the activity at 100 kDa is at least partly due to free tPA dissociated from the complex during the first zymography. Conversion of tPA complex to enzymatically active free tPA also occurs with brief SDS exposure followed by incubation in the presence of excess Triton X-100 or by hydroxylamine treatment. These results reconcile the apparent discrepancy of the 100 kDA inhibitor-tPA complex manifesting plasminogen activation activity during zymography. The plasma tPA- inhibitor complex is precipitated strongly by antisera against plasminogen activator inhibitors (PAIs) of human Hep G2 hepatoma and HT- 1080 fibrosarcoma cells and weakly by antiserum against bovine aortic endothelial cell PAI but not by antiserum against a placental PAI (PAI- 2) suggesting that the plasma inhibitor is immunologically related to Hep G2, HT-1080 and possibly endothedial cell PAIs. Based on the above findings, a simple model for the initiation and regulation of plasma fibrinolysis at the PA level has been formulated.

scholarly journals In vitro antibacterial activity and in vivo efficacy of sulbactam-durlobactam against pathogenic Burkholderia species

2020 ◽  
pp. AAC.01930-20
Author(s):  
Krisztina M. Papp-Wallace ◽  
Adam B. Shapiro ◽  
Scott A. Becka ◽  
Elise T. Zeiser ◽  
John J. LiPuma ◽  
...  
Keyword(s):  
Human Pathogens ◽  
Minimal Inhibitory Concentrations ◽  
Class A ◽  
In Vitro Antibacterial Activity ◽  
Lung Infections ◽  
Kinetics Of ◽  
Kinetics Of Inhibition ◽  
Inhibitor Combination

The Gram-negative bacterial genus Burkholderia includes several hard-to-treat human pathogens: two biothreat species, B. mallei (causing glanders) and B. pseudomallei (causing melioidosis), and the B. cepacia complex (BCC) and B. gladioli, which cause chronic lung infections in persons with cystic fibrosis. All Burkholderia spp. possess an Ambler class A Pen β-lactamase, which confers resistance to β-lactams. The β-lactam-β-lactamase inhibitor combination sulbactam-durlobactam (SUL-DUR) is in clinical development for the treatment of Acinetobacter infections. Herein, we evaluated SUL-DUR for in vitro and in vivo activity against Burkholderia clinical isolates. We measured minimal inhibitory concentrations (MICs) of SUL-DUR against BCC and B. gladioli (N = 150), B. mallei (N = 30) and B. pseudomallei (N = 28); studied the kinetics of inhibition of the PenA1 β-lactamase from B. multivorans and the PenI β-lactamase from B. pseudomallei by durlobactam; tested for blaPenA1 induction by SUL-DUR; and evaluated in vivo efficacy in a mouse model of melioidosis. SUL-DUR inhibited growth of 87.3% of the BCC and B. gladioli strains and 100% of the B. mallei and B. pseudomallei strains at 4/4 μg/mL. Durlobactam potently inhibited PenA1 and PenI with k2/K values of 3.9 x 106 M−1s−1 and 2.6 x 103 M−1s−1 and Ki app of 15 nM and 241 nM, respectively, by forming highly stable covalent complexes. Neither sulbactam, durlobactam, nor SUL-DUR increased production of PenA1. SUL-DUR demonstrated activity in vivo in a murine melioidosis model. Taken together, these data suggest SUL-DUR may be useful as a treatment for Burkholderia infections.

scholarly journals Adenine nucleotides and magnesium ions in relation to control of mammalian cerebral-cortex hexokinase

1969 ◽  
Vol 112 (5) ◽  
pp. 579-586 ◽  
Author(s):  
H S Bachelard ◽  
P. S. G. Goldfarb
Keyword(s):  
Mixed Type ◽  
Competitive Inhibition ◽  
Adenine Nucleotides ◽  
Effective Control ◽  
Magnesium Ions ◽  
Soluble Enzyme ◽  
Kinetics Of ◽  
Kinetics Of Inhibition

1. The kinetics of inhibition of brain soluble cytoplasmic hexokinase by ADP were examined in relation to variations in the concentrations of Mg2+ and ATP. The type of inhibition observed was dependent on the Mg2+/ATP ratio. 2. ADP at Mg2+/ATP ratios 2:1 exhibited inhibition of the ‘mixed’ type; at Mg2+/ATP ratios 1:1 the inhibition appeared to be competitive with regard to ATP. 3. Inhibition by free ATP was observed when the Mg2+/ATP ratio was less than 1:1. The inhibition was also of the ‘mixed’ type with respect to MgATP2−. 4. The inhibitions due to ADP and to free ATP were not additive. The results suggested that there may be up to four sites in the soluble enzyme: for glucose, glucose 6-phosphate, ADP and MgATP2−. 5. The ‘free’ non-particulate intracellular Mg2+ concentration was measured and concluded to be about 1·5mm. 6. The concentrations in vivo of Mg2+ and ATP likely to be accessible to a cytoplasmic enzyme are suggested to be below those that yield maximum hexokinase rates in vitro. The enzymic rates were measured at relevant suboptimum concentrations of Mg2+ and ATP in the presence of ADP. Calculations that included non-competitive inhibition due to glucose 6-phosphate (56–65% at 0·25mm) resulted in net rates very similar to the measured rates for overall glycolysis. This system may therefore provide a basis for effective control of cerebral hexokinase.

scholarly journals Immune-modulating enzyme indoleamine 2,3-dioxygenase is effectively inhibited by targeting its apo-form

2018 ◽  
Vol 115 (13) ◽  
pp. 3249-3254 ◽  
Author(s):  
Micah T. Nelp ◽  
Patrick A. Kates ◽  
John T. Hunt ◽  
John A. Newitt ◽  
Aaron Balog ◽  
...  
Keyword(s):  
Cell Function ◽  
High Rate ◽  
Posttranslational Regulation ◽  
Indoleamine 2,3 Dioxygenase ◽  
Heme Enzyme ◽  
Enzyme Complexes ◽  
Kinetics Of ◽  
In Vitro Kinetics ◽  
Kinetics Of Inhibition

For cancer cells to survive and proliferate, they must escape normal immune destruction. One mechanism by which this is accomplished is through immune suppression effected by up-regulation of indoleamine 2,3-dioxygenase (IDO1), a heme enzyme that catalyzes the oxidation of tryptophan to N-formylkynurenine. On deformylation, kynurenine and downstream metabolites suppress T cell function. The importance of this immunosuppressive mechanism has spurred intense interest in the development of clinical IDO1 inhibitors. Herein, we describe the mechanism by which a class of compounds effectively and specifically inhibits IDO1 by targeting its apo-form. We show that the in vitro kinetics of inhibition coincide with an unusually high rate of intrinsic enzyme–heme dissociation, especially in the ferric form. X-ray crystal structures of the inhibitor–enzyme complexes show that heme is displaced from the enzyme and blocked from rebinding by these compounds. The results reveal that apo-IDO1 serves as a unique target for inhibition and that heme lability plays an important role in posttranslational regulation.

Kinetics of Inhibition of ADP-Induced Platelet Aggregation by Dihomo-β-Linolenic Acid (DHLA).

1979 ◽  
Author(s):  
M. Zuzel ◽  
A. Spencer
Keyword(s):  
Fatty Acids ◽  
Platelet Aggregation ◽  
Competitive Inhibition ◽  
Human Platelets ◽  
Inhibition Of Platelet Aggregation ◽  
Kinetics Of ◽  
Dose Dependent ◽  
Kinetics Of Inhibition ◽  
Primary Platelet

In the study of the effects on platelets of thromboxanes and prostaglandins (PG) large amounts of precursor fatty acids have frequently been added to platelet suspensions. In the case of DHLA, this results in a general inhibition of platelet reactions. We have employed kinetic analysis of the inhibition of ADP-induced primary platelet aggregation to estimate the potency, specificity and mode of action of DHLA on human platelets in citrated PRP. Platelet PG production was estimated from formation of malondialdehyde (MDA) and aspirin (ASA) was used to inhibit production-Inhibition of aggregation and MEA formation were dose-dependent and both were observed at DHLA concentrations of 0.1 mM and above. Inhibition of aggregation was of mixed type, consisting of an ASA-sensitive competitive component (K1 ≈ 0.2mM) and an ASA-insensitve component which was non-competitive and dominated inhibition at higher concentrations of DHLA. The KI (DHLA) of the non-competitive component varied from 0.4 to 1.5 mM with different batches of PRP. Other polyunsaturated fatty acids which are not PG precursors, did not cause competitive inhibition but were as potent as DHLA in the non-competitive inhibition of aggregation. The results show that a large part of the inhibition of platelet aggregation by DHLA in vitro is not due to its transformation into an inhibitor of platelet function in the PG production pathway.

scholarly journals Studies on Antimicrobial Activity and Kinetics of Inhibition by Plant Products in India (1990–2016)

2018 ◽  
Vol 101 (4) ◽  
pp. 948-955
Author(s):  
Ramesh Kumar Sharma ◽  
Bhupendra Kumar Rana
Keyword(s):  
Antimicrobial Activity ◽  
Antimicrobial Susceptibility ◽  
Antimicrobial Agents ◽  
Herbal Extracts ◽  
Analytical Approaches ◽  
Kinetics Of ◽  
Kinetics Of Inhibition ◽  
Regression Correlation

Abstract The antimicrobial activity of herbal extracts or plant isolates has usually been evaluated in India using different antimicrobial susceptibility testing methods generally based on diffusion and dilution. There are different analytical approaches for the reliable evaluation of antimicrobial activity ascribed to medicinal plants against selected pathogenic microorganisms. Obtained results may provide scientific bases for the selective use of these natural plants as healing drugs, crop-protecting pesticides, or shelf-life-extending solutions. In general, antimicrobial susceptibility methodologies involve in vivo and in vitro studies; at present, the in vitro evaluation of antibacterial activity appears more popular. Diffusion methods have some limitations, although they are extensively used to determine the susceptibility of organisms isolated from specimen samples to applied antimicrobials and vice versa. Dilution methods are preferred in the case of more precise antimicrobial activity estimation, in terms of minimum inhibitory concentration. With regard to the inherent antimicrobial nature of herbal compositions, herbs, and herbal extracts, Indian researchers have evaluated the reliability of these antimicrobial agents against selected pathogens and have shown them to be effective. Researchers have also tried to establish linear regression correlation analyses on the basis of available inhibition results. This research is still evolving, and interesting results may be expected in the future.

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