scholarly journals NADP-Isocitrate Dehydrogenase fromPseudomonas nautica: Kinetic Constant Determination and Carbon Limitation Effects on the Pool of Intracellular Substrates

1998 ◽  
Vol 64 (12) ◽  
pp. 4958-4964 ◽  
Author(s):  
Sylvie O. Roy ◽  
Ted T. Packard
Keyword(s):  
Isocitrate Dehydrogenase ◽  
Protein Concentration ◽  
Marine Bacterium ◽  
Culture Medium ◽  
Kinetic Constant ◽  
Exponential Phase ◽  
Carbon Limitation ◽  
Protein Levels ◽  
Constant Determination ◽  
Michaelis Constants

ABSTRACT Variations of intracellular concentrations of isocitrate and NADP+ were measured throughout all growth phases of the marine bacterium Pseudomonas nautica. The intracellular isocitrate concentration tracked the intracellular protein concentration throughout all phases of growth. It rapidly increased in early exponential phase to a maximum and fell to nearly zero in parallel with pyruvate exhaustion in the culture medium. The intracellular NADP+ and protein concentrations increased in parallel during the exponential phase but were poorly correlated. Even after carbon exhaustion, the intracellular NADP+concentration stayed high, as did protein levels. The results demonstrated that the intracellular isocitrate concentration, but not the intracellular NADP+ concentration, was affected by the carbon availability in the culture. They also suggest that, because of its variability, isocitrate, but not NADP+, plays the larger role in the control of the respiratory CO2production rate (R CO2 ). From initial rate studies, bisubstrate Michaelis constants and the dissociation constant were determined for NADP+-specific isocitrate dehydrogenase (IDH) from P. nautica. These studies support the hypothesis that the mechanism of IDH’s activity involves the ordered addition of the substrates,d-isocitrate and NADP+. Furthermore, the results support the use of a bisubstrate enzyme kinetic equation to model R CO2 in P. nautica.

Regulation of expression of the cyanide-insensitive terminal oxidase in Pseudomonas aeruginosa

Microbiology ◽  
2003 ◽  
Vol 149 (5) ◽  
pp. 1275-1284 ◽  
Author(s):  
Megan Cooper ◽  
Gholam Reza Tavankar ◽  
Huw D. Williams
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Stationary Phase ◽  
Culture Medium ◽  
Homoserine Lactone ◽  
Exponential Phase ◽  
Terminal Oxidase ◽  
Regulation Of Expression ◽  
Potent Inducer ◽  
Protein Levels ◽  
In The Wild

The regulation of the cyanide-insensitive oxidase (CIO) in Pseudomonas aeruginosa, a bacterium that can synthesize HCN, is reported. The expression of a cioA–lacZ transcriptional fusion, CioA protein levels and CIO activity were low in exponential phase but induced about fivefold upon entry into stationary phase. Varying the O2 transfer coefficient from 11·5 h−1 to 87·4 h−1 had no effect on CIO expression and no correlation was observed between CIO induction and the dissolved O2 levels in the growth medium. However, a mutant deleted for the O2-sensitive transcriptional regulator ANR derepressed CIO expression in an O2-sensitive manner, with the highest induction occurring under low-O2 conditions. Therefore, CIO expression can respond to a signal generated by low O2 levels, but this response is normally kept in check by ANR repression. ANR may play an important role in preventing overexpression of the CIO in relation to other terminal oxidases. A component present in spent culture medium was able to induce CIO expression. However, experiments with purified N-butanoyl-l-homoserine lactone or N-(3-oxododecanoyl)homoserine lactone ruled out a role for these quorum-sensing molecules in the control of CIO expression. Cyanide was a potent inducer of the CIO at physiologically relevant concentrations and experiments using spent culture medium from a ΔhcnB mutant, which is unable to synthesize cyanide, showed that cyanide was the inducing factor present in P. aeruginosa spent culture medium. However, the finding that in a ΔhcnB mutant cioA–lacZ expression was induced normally upon entry into stationary phase indicated that cyanide was not the endogenous inducer of the terminal oxidase. The authors suggest that the failure of O2 to have an effect on CIO expression in the wild-type can be explained either by the requirement for an additional, stationary-phase-specific inducing signal or by the loss of an exponential-phase-specific repressing signal.

scholarly journals The Effect of Rivaroxaban on CYP4F2 and Transcription Factors’ Activity in HUVECs

2021 ◽  
Vol 11 (22) ◽  
pp. 10851
Author(s):  
Ieva Ciapiene ◽  
Vacis Tatarunas ◽  
Agne Giedraitiene ◽  
Vaidotas Zvikas ◽  
Valdas Jakstas ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Transcription Factors ◽  
Protein Concentration ◽  
Culture Medium ◽  
Hepatic Metabolism ◽  
Esi Ms ◽  
Protein Levels ◽  
M 10 ◽  
The Impact

Interindividual variabilities between patients taking the anticoagulant rivaroxaban are a result of hepatic metabolism by CYP 450 enzymes. The objective of this study was to evaluate the impact of rivaroxaban on CYP4F2 and transcription factors’ activity in HUVECs. Rivaroxaban and its metabolites were detected by UPLC-ESI-MS and UPLC-QTOF-MS. CYP4F2, HNF4α, PXR and CAR expressions were determined in HUVECs by qPCR; CYP4F2 protein concentration was determined by ELISA. Rivaroxaban metabolites (M-1, M-2, M-5, M-8, M-10, M-11 and M-18) were detected in endothelial cells’ culture medium. Increasing concentrations of rivaroxaban determined lower 13-docosenamide concentrations. Rivaroxaban and dexamethasone reduced the expression of CYP4F2 when hsa-miR-24-3p—both CYP4F2 expression and CYP4F2 protein levels in HUVECs. The expression of the transcription factors HNF4α, PXR and CAR was not detected in HUVECs.

TEM study of a biofilm on copper corrosion

1996 ◽  
Vol 54 ◽  
pp. 220-221
Author(s):  
W. A. Chiou ◽  
N. Kohyama ◽  
B. Little ◽  
P. Wagner ◽  
M. Meshii
Keyword(s):  
Marine Bacterium ◽  
Culture Medium ◽  
Copper Alloys ◽  
Pure Copper ◽  
Localized Corrosion ◽  
Natural Seawater ◽  
Copper Corrosion ◽  
New Approach ◽  
The Past ◽  
Copper Tolerant

The corrosion of copper and copper alloys in a marine environment is of great concern because of their widespread use in heat exchangers and steam condensers in which natural seawater is the coolant. It has become increasingly evident that microorganisms play an important role in the corrosion of a number of metals and alloys under a variety of environments. For the past 15 years the use of SEM has proven to be useful in studying biofilms and spatial relationships between bacteria and localized corrosion of metals. Little information, however, has been obtained using TEM capitalizing on its higher spacial resolution and the transmission observation of interfaces. The research presented herein is the first step of this new approach in studying the corrosion with biological influence in pure copper.Commercially produced copper (Cu, 99%) foils of approximately 120 μm thick exposed to a copper-tolerant marine bacterium, Oceanospirillum, and an abiotic culture medium were subsampled (1 cm × 1 cm) for this study along with unexposed control samples.

scholarly journals Cerebrospinal fluid biomarkers of neuroinflammation in children with hydrocephalus and shunt malfunction

2021 ◽  
Vol 18 (1) ◽  
Author(s):  
Carolyn A. Harris ◽  
Diego M. Morales ◽  
Rooshan Arshad ◽  
James P. McAllister ◽  
David D. Limbrick
Keyword(s):  
Cerebrospinal Fluid ◽  
Inflammatory Cytokines ◽  
Protein Concentration ◽  
Shunt Revision ◽  
Csf Shunt ◽  
Shunt Failure ◽  
Revision Operation ◽  
Protein Levels ◽  
Anti Inflammatory ◽  
Pro Inflammatory Cytokines

Abstract Background Approximately 30% of cerebrospinal fluid (CSF) shunt systems for hydrocephalus fail within the first year and 98% of all patients will have shunt failure in their lifetime. Obstruction remains the most common reason for shunt failure. Previous evidence suggests elevated pro-inflammatory cytokines in CSF are associated with worsening clinical outcomes in neuroinflammatory diseases. The aim of this study was to determine whether cytokines and matrix metalloproteinases (MMPs) contribute towards shunt failure in hydrocephalus. Methods Using multiplex ELISA, this study examined shunt failure through the CSF protein concentration profiles of select pro-inflammatory and anti-inflammatory cytokines, as well as select MMPs. Interdependencies such as the past number of previous revisions, length of time implanted, patient age, and obstruction or non-obstruction revision were examined. The pro-inflammatory cytokines were IL-1β, IL-2, IL-5, IL-6, IL-8, IL-12, IL-17, TNF-α, GM-CSF, IFN-γ. The anti-inflammatory cytokines were IL-4 and IL-10, and the MMPs were MMP-2, MMP-3, MMP-7, MMP-9. Protein concentration is reported as pg/mL for each analyte. Results Patient CSF was obtained at the time of shunt revision operation; all pediatric (< 18), totaling n = 38. IL-10, IL-6, IL-8 and MMP-7 demonstrated significantly increased concentrations in patient CSF for the non-obstructed subgroup. Etiological examination revealed IL-6 was increased in both obstructed and non-obstructed cases for PHH and congenital hydrocephalic patients, while IL-8 was higher only in PHH patients. In terms of number of past revisions, IL-10, IL-6, IL-8, MMP-7 and MMP-9 progressively increased from zero to two past revisions and then remained low for subsequent revisions. This presentation was notably absent in the obstruction subgroup. Shunts implanted for three months or less showed significantly increased concentrations of IL-6, IL-8, and MMP-7 in the obstruction subgroup. Lastly, only patients aged six months or less presented with significantly increased concentration of IL-8 and MMP-7. Conclusion Non-obstructive cases are reported here to accompany significantly higher CSF cytokine and MMP protein levels compared to obstructive cases for IL-10, IL-6, IL-8, MMP-7 and MMP-9. A closer examination of the definition of obstruction and the role neuroinflammation plays in creating shunt obstruction in hydrocephalic patients is suggested.

scholarly journals Evaluation of airway inflammation in mechanically ventilated patients using cell count and protein concentration

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Rodopi Stamatiou ◽  
Vasiliki Tsolaki ◽  
Apostolia Hatziefthimiou ◽  
Epaminondas Zakynthinos ◽  
Demosthenes Makris
Keyword(s):  
Airway Inflammation ◽  
Protein Concentration ◽  
Cell Number ◽  
Mechanically Ventilated ◽  
Mechanically Ventilated Patients ◽  
Bronchial Secretions ◽  
Protein Levels ◽  
Tnf Α ◽  
Standardize Sampling ◽  
Ventilated Patients

AbstractMechanically ventilated (MV) patients may present airway inflammation and elevated secretion production. However, it is unknown whether cell and/or protein counts in bronchial samples may be useful to evaluate their clinical condition. Our aim was to standardize sampling and propose a new mechanical mucus dissolution in Tracheal-Bronchial secretions. In all patients, bronchial lining fluid aspiration (BLF), Bronchoalveolar lavage (BAL) and Bronchial Washings (BW40, BW5) were performed, while visible bronchial secretions were obtained via bronchoscopy (VBS) and blinded, via a common catheter for tracheobronchial aspiration (AC). Mucus was mechanically or DTT dissolved and cell number was count. Protein, albumin and TNF-α levels were measured, in mucus dissolved samples from control and MV patients. Cell number and protein levels were elevated in mucus dissolved compared to non-dissolved, or DTT dissolved. Cell number and TNF-α levels were elevated in MV patients compared to controls, while protein levels were lower in MV patients. Differences in cell and protein levels were observed in samples acquired using different sampling technics. Therefore, mechanical mucus dissolution provides a proper sample for evaluation, and the sampling technic used can influence the sample’s characteristics.

scholarly journals Carbon limitation drives GC content evolution of a marine bacterium in an individual-based genome-scale model

2018 ◽  
Vol 12 (5) ◽  
pp. 1180-1187 ◽  
Author(s):  
Ferdi L Hellweger ◽  
Yongjie Huang ◽  
Haiwei Luo
Keyword(s):  
Marine Bacterium ◽  
Gc Content ◽  
Scale Model ◽  
Carbon Limitation ◽  
Genome Scale ◽  
Genome Scale Model

scholarly journals FORMATION OF TRYPSIN FROM TRYPSINOGEN BY AN ENZYME PRODUCED BY A MOLD OF THE GENUS PENICILLIUM

1938 ◽  
Vol 21 (5) ◽  
pp. 601-620 ◽  
Author(s):  
M. Kunitz
Keyword(s):  
Temperature Coefficient ◽  
Acid Medium ◽  
Experimental Error ◽  
Protein Concentration ◽  
Liquid Culture ◽  
Culture Medium ◽  
Specific Activity ◽  
Crystalline Form ◽  
Liquid Culture Medium ◽  
Autocatalytic Activation

1. A powerful kinase which changes trypsinogen to trypsin was found to be present in the synthetic liquid culture medium of a mold of the genus Penicillium. 2. The concentration of kinase in the medium is increased gradually during the growth of the mold organism and continues to increase for some time even after the mold has ceased growing. 3. Mold kinase transforms trypsinogen to trypsin only in an acid medium. It differs thus from enterokinase and trypsin which activate trypsinogen best in a slightly alkaline medium. 4. The action of the mold kinase in the process of transformation of trypsinogen is that of a typical enzyme. The process follows the course of a catalytic unimolecular reaction, the rate of formation of a definite amount of trypsin being proportional to the concentration of kinase added. The ultimate amount of trypsin formed, however, is independent of the concentration of kinase used. 5. The formation of trypsin from trypsinogen by mold kinase is not accompanied by any measurable loss of protein. 6. The temperature coefficient of formation of trypsin from trypsinogen by mold kinase varies from Q5–15 = 1.70 to Q25–30 = 1.25 with a corresponding variation in the value of µ from 8100 to 4250. 7. Trypsin formed from trypsinogen by means of mold kinase is identical in crystalline form with the crystalline trypsin obtained by spontaneous autocatalytic activation of trypsinogen at pH 8.0. The two products have within the experimental error the same solubility and specific activity. A solution saturated with the crystals of either one of the trypsin preparations does not show any increase in protein concentration or activity when crystals of the other trypsin preparation are added. 8. The Penicillium mold kinase has a slight activating effect on chymo-trypsinogen the rate being only 1–2 per cent of that of trypsinogen. The activation, as in the case of trypsinogen, takes place only in an acid medium. 9. Mold kinase is rapidly destroyed when brought to pH 6.5 or higher, and also when heated to 70°C. In the temperature range of 50–60°C. the inactivation of kinase follows a unimolecular course with a temperature coefficient of Q10 = 12.1 and µ = 53,500. The molecular weight of mold kinase, as determined by diffusion, is 40,000.

scholarly journals Geranic Acid Formation, an Initial Reaction of Anaerobic Monoterpene Metabolism in DenitrifyingAlcaligenes defragrans

2000 ◽  
Vol 66 (7) ◽  
pp. 3004-3009 ◽  
Author(s):  
Udo Heyen ◽  
Jens Harder
Keyword(s):  
Culture Medium ◽  
Degradation Pathway ◽  
Unsaturated Hydrocarbon ◽  
Cell Suspensions ◽  
Carbon Limitation ◽  
Initial Reaction ◽  
Activation Reaction ◽  
Geranic Acid ◽  
Monoterpene Metabolism ◽  
In Cells

ABSTRACT Monoterpenes with an unsaturated hydrocarbon structure are mineralized anaerobically by the denitrifying β-proteobacteriumAlcaligenes defragrans. Organic acids occurring in cells ofA. defragrans and culture medium were characterized to identify potential products of the monoterpene activation reaction. Geranic acid (E,E-3,7-dimethyl-2,6-octadienoic acid) accumulated to 0.5 mM in cells grown on α-phellandrene under nitrate limitation. Cell suspensions of A. defragrans65Phen synthesized geranic acid in the presence of β-myrcene, α-phellandrene, limonene, or α-pinene. Myrcene yielded the highest transformation rates. The alicyclic acid was consumed by cell suspensions during carbon limitation. Heat-labile substances present in cytosolic extracts catalyzed the formation of geranic acid from myrcene. These results indicated that a novel monoterpene degradation pathway must be present in A. defragrans.

scholarly journals ENZYMATIC HYDROLYSIS KINETIC ANALYSIS OF THE VARIOUS SOURCE PROTEINS

2013 ◽  
Vol 17 (2) ◽  
pp. 15-20
Author(s):  
Aleksey Vinnov ◽  
Dmitro Prasol
Keyword(s):  
Chemical Composition ◽  
Enzymatic Hydrolysis ◽  
Kinetic Analysis ◽  
Protein Concentration ◽  
Proteolytic Enzymes ◽  
Sunflower Meal ◽  
Microbial Enzyme ◽  
Michaelis Constants ◽  
Plant Enzyme ◽  
Complex Dispersion

Abstract The chemical composition marble goby and sunflower meal is presented. The experimental results of enzymatic hydrolysis velocity in dependence of the protein concentration in substrate systems are set. Michaelis constants values for industrial proteolytic enzymes Corolase ® L10 and Corolase ® L7089 are calculated. The application experimental - theoretical kinetic analysis for protease / proteins congeniality determine in complex dispersion substrate systems expediency was confirmed. It is determined that microbial enzyme drag Corolase ® L7089 has a higher congeniality to proteins of all the tested substrates than plant enzyme drag Corolase ® L10

scholarly journals Evaluation of MMP Inhibitors Isolated from Ligustrum japonicum Fructus

Molecules ◽  
2019 ◽  
Vol 24 (3) ◽  
pp. 604
Author(s):  
Hojun Kim ◽  
Fatih Karadeniz ◽  
Chang-Suk Kong ◽  
Youngwan Seo
Keyword(s):  
Culture Medium ◽  
Mapk Pathway ◽  
Gelatin Zymography ◽  
Inhibitory Effect ◽  
Mmp Inhibitors ◽  
Protein Levels ◽  
Lead Compounds ◽  
Ligustrum Japonicum ◽  
Fibrosarcoma Cells ◽  
Human Fibrosarcoma Cells

The current study investigated the ability of two secoiridoids, GL-3 (1) and oleonuezhenide (2), isolated from the fruits of Ligustrum japonicum to inhibit MMP-2 and -9 activity in phorbol 12-myristate 13-acetate (PMA)-induced HT-1080 human fibrosarcoma cells. Both compounds 1 and 2 were able to exert lowered gelatin digestion activity for MMP-2 and -9 tested by gelatin zymography via suppressing the release of MMPs to culture medium according to ELISA results. Treatment with compounds was also able to suppress the expression of both mRNA and protein levels of MMP-2 and -9. Action mechanism behind the MMP inhibitory effect of the compounds was suggested to be via MAPK pathway indicated by decreased levels of phosphorylated p38, ERK and JNK proteins evaluated employing immunoblotting. Compound 1 was shown to be slightly more active to inhibit MMP-2 and -9, however, compound 2 showed more regular dose-dependency during inhibition. In conclusion, this study suggested that GL-3 and oleonuezhenide were notable natural origin potent MMP inhibitors and could serve as lead compounds for development of anti-invasive MMP inhibitors against tumor metastasis.

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