scholarly journals Purification and characterization of a calcium-independent acidic phospholipase A2 from rat lung

1994 ◽  
Vol 304 (1) ◽  
pp. 131-137 ◽  
Author(s):  
R Wang ◽  
C R Dodia ◽  
M K Jain ◽  
A B Fisher
Keyword(s):  
Phospholipase A2 ◽  
Dependent Manner ◽  
Rat Lung ◽  
Purification And Characterization ◽  
Ph Optimum ◽  
New Class ◽  
Terminal Amino ◽  
Dose Dependent

Several phospholipase A2 (PLA2) activities have been identified in rat lung homogenate and shown to be important in metabolism of lung phospholipids. One PLA2 activity is Ca(2+)-independent, active in vitro at pH 4, and inhibited by a substrate analogue, 1-hexadecyl-3-trifluoroethylglycero-sn-2-phosphomethanol (MJ33). Purification of this rat lung PLA2 by approx. 550-fold was carried out by sequential column chromatographies using DE-52, Sephacryl-100, heparin-Sepharose, and phenyl-Sepharose columns. The purified activity had an acidic pH optimum, was Ca(2+)-independent, was inhibited by MJ33 in a dose-dependent manner (50% inhibition at 3 mol%), was unaffected by treatment with p-bromophenacyl bromide or mercaptoethanol, and had a unique N-terminal amino acid sequence. The apparent molecular mass was 15 kDa on gel electrophoresis and activity was recovered in part by renaturation of protein from the gel. The properties of this enzyme suggest a new class of PLA2.

Production and utilization of monoclonal antibodies to human/rat corticotrophin-releasing factor-41

1990 ◽  
Vol 5 (2) ◽  
pp. 159-166 ◽  
Author(s):  
N. G. N. Milton ◽  
E. W. Hillhouse ◽  
S. A. Nicholson ◽  
C. H. Self ◽  
A. M. McGregor
Keyword(s):  
Monoclonal Antibodies ◽  
Enzyme Linked Immunosorbent Assay ◽  
Dependent Manner ◽  
Corticotrophin Releasing Factor ◽  
Tissue Extracts ◽  
Rat Pituitary ◽  
Hypothalamic Tissue ◽  
Dose Dependent

ABSTRACT Murine monoclonal antibodies against human/rat corticotrophin-releasing factor-41 (CRF-41) were produced and characterized for use in the immunological and biological characterization of CRF-41. Spleen cells from BALB/c mice immunized with CRF-41 conjugated to bovine γ-globulin were fused with a BALB/c-derived non-secretor X-63 myeloma line. Hybridomas were selected for CRF antibody production by enzyme-linked immunosorbent assay, and positive hybridomas cloned twice. Three monoclonal antibodies were obtained (KCHMB001, KCHMB002 and KCHMB003) and characterized as IgG1, IgG1 and IgG2a isotypes respectively, with affinity constants for rat CRF-41 of 30, 53 and 34 nmol/l respectively. All three monoclonal antibodies recognize an epitope contained between residues 34 and 41 of the human/rat sequence. The antibodies were able to neutralize the ACTH-releasing activity of rat CRF-41, applied to rat pituitary fragments in vitro, in a dose-dependent manner. Isoelectric focusing showed that KCHMB 003 detected bands of synthetic rat CRF-41 and rat [Met(O)21,38]-CRF-41 at pH 7·1 and 6·8 respectively. Use of KCHMB003 in a two-site enzyme-amplified immunoassay showed that this antibody recognizes both synthetic rat CRF-41 and immunoreactive CRF-41 in rat hypothalamic tissue extracts.

scholarly journals Characterization of RNA damage under oxidative stress in Escherichia coli

2012 ◽  
Vol 393 (3) ◽  
pp. 123-132 ◽  
Author(s):  
Min Liu ◽  
Xin Gong ◽  
Ravi Kumar Alluri ◽  
Jinhua Wu ◽  
Tene’ Sablo ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Escherichia Coli ◽  
Short Exposure ◽  
Dependent Manner ◽  
Ribosomal Rnas ◽  
Rna Oxidation ◽  
Folded Rna ◽  
Dose Dependent

Abstract We have examined the level of 8-hydroxyguanosine (8-oxo-G), an oxidized form of guanosine, in RNA in Escherichia coli under normal and oxidative stress conditions. The level of 8-oxo-G in RNA rises rapidly and remains high for hours in response to hydrogen peroxide (H2O2) challenge in a dose-dependent manner. H2O2 induced elevation of 8-oxo-G content is much higher in RNA than that of 8-hydroxydeoxyguanosine (8-oxo-dG) in DNA. Under normal conditions, the 8-oxo-G level is low in RNA isolated from the ribosome and it is nearly three times higher in non-ribosomal RNAs. In contrast, 8-oxo-G generated by a short exposure to H2O2 is almost equally distributed in various RNA species, suggesting that although ribosomal RNAs are normally less oxidized, they are not protected against exogenous H2O2. Interestingly, highly folded RNA is not protected from oxidation because 8-oxo-G generated by H2O2 treatment in vitro increases to approximately the same levels in tRNA and rRNA in both native and denatured forms. Lastly, increased RNA oxidation is closely associated with cell death by oxidative stress. Our data suggests that RNA is a primary target for reactive oxygen species and RNA oxidation is part of the paradox that cells have to deal with under oxidative stress.

scholarly journals Inhibitory action of polyamines on protein kinase C association to membranes

1987 ◽  
Vol 247 (1) ◽  
pp. 175-180 ◽  
Author(s):  
M Moruzzi ◽  
B Barbiroli ◽  
M G Monti ◽  
B Tadolini ◽  
G Hakim ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Activation Process ◽  
Dependent Manner ◽  
Kinase C ◽  
Association Reaction ◽  
Dose Dependent ◽  
Inside Out

Physiological activation of protein kinase C requires the interaction of this enzyme with cellular membranes [Nishizuka (1986) Science 233, 305-312]. In the present work a reconstituted system of protein kinase C and human inside-out erythrocyte vesicles was utilized to study the effect in vitro of naturally occurring polyamines on the activation process of protein kinase C. The active membrane-associated complex was conveniently determined by its ability to bind radioactive phorbol ester with an exact 1:1 stoichiometry. The association reaction of the enzyme to membrane was rapid, being complete within 1 min at 25 degrees C. The addition of polyamines, particularly spermine, greatly decreased in a dose-dependent manner the amount of protein kinase C bound to membranes (i.e. in the activated form). The effect observed was quite specific, since it was dependent on the chemical structure of the polyamine and it was manifest at micromolar concentrations of the polycation; the order of potency was spermine greater than spermidine greater than putrescine. A characterization of this effect is presented and possible physiological implications are discussed.

scholarly journals Characterization of an engineered live bacterial therapeutic for the treatment of phenylketonuria in a human gut-on-a-chip

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
M. Tyler Nelson ◽  
Mark R. Charbonneau ◽  
Heidi G. Coia ◽  
Mary J. Castillo ◽  
Corey Holt ◽  
...  
Keyword(s):  
Mechanistic Models ◽  
Human Gut ◽  
Primate Model ◽  
New Class ◽  
Testing Strategies ◽  
Dose Dependent ◽  
Engineered Strain

AbstractEngineered bacteria (synthetic biotics) represent a new class of therapeutics that leverage the tools of synthetic biology. Translational testing strategies are required to predict synthetic biotic function in the human body. Gut-on-a-chip microfluidics technology presents an opportunity to characterize strain function within a simulated human gastrointestinal tract. Here, we apply a human gut-chip model and a synthetic biotic designed for the treatment of phenylketonuria to demonstrate dose-dependent production of a strain-specific biomarker, to describe human tissue responses to the engineered strain, and to show reduced blood phenylalanine accumulation after administration of the engineered strain. Lastly, we show how in vitro gut-chip models can be used to construct mechanistic models of strain activity and recapitulate the behavior of the engineered strain in a non-human primate model. These data demonstrate that gut-chip models, together with mechanistic models, provide a framework to predict the function of candidate strains in vivo.

Oxygenation of 18-hydroxycorticosterone as the final reaction for aldosterone biosynthesis

1984 ◽  
Vol 107 (3) ◽  
pp. 395-400 ◽  
Author(s):  
Itaru Kojima ◽  
Etsuro Ogata ◽  
Hiroshi Inano ◽  
Bun-ichi Tamaoki
Keyword(s):  
Carbon Monoxide ◽  
Hydroxysteroid Dehydrogenase ◽  
Dependent Manner ◽  
In Vitro Production ◽  
Mitochondrial Preparation ◽  
Final Reaction ◽  
Vitro Production ◽  
Dose Dependent ◽  
Cytochrome P 450

Abstract. Incubation of 18-hydroxycorticosterone with the sonicated mitochondrial preparation of bovine adrenal glomerulosa tissue leads to the production of aldosterone, as measured by radioimmunoassay. The in vitro production of aldosterone from 18-hydroxycorticosterone requires both molecular oxygen and NADPH, and is inhibited by carbon monoxide. Cytochrome P-450 inhibitors such as metyrapone, SU 8000. SU 10603, SKF 525A, amphenone B and spironolactone decrease the biosynthesis of aldosterone from 18-hydroxycorticosterone. These results support the conclusion that the final reaction in aldosterone synthesis from 18-hydroxycorticosterone is catalyzed by an oxygenase, but not by 18-hydroxysteroid dehydrogenase. By the same preparation, the production of [3H]aldosterone but not [3H]18-hydroxycorticosterone from [1,2-3H ]corticosterone is decreased in a dose-dependent manner by addition of non-radioactive 18-hydroxycorticosterone.

Appraisal of Immunological Impacts of Melaleuca leucadendra Extract over Macrophage Performance in Vitro

2020 ◽  
Keyword(s):  
Nitric Oxide ◽  
Interleukin 2 ◽  
Dependent Manner ◽  
Reduced Pressure ◽  
Medical Plant ◽  
Dose Dependent ◽  
Level Production ◽  
Melaleuca Leucadendra ◽  
Phagocytic Killing

This trial research was performed to discuss the immune-influence of Melaleuca leucadendra ‘paper-bark tree’ dried leaves which is an important medical plant known in many regions in the world. The leaves were dissolved in a mixture of (ethanol + water) (3:1) mixture, then filtered, evaporated and dried under reduced pressure to obtain leaves extract. The macrophages of blood derived origin were provided from rats and mixed with three different leaves extracts doses in tissue culture plates and incubated then stained with fluorescent acridine orange and examined under fluorescent microscope to assess the phagocytic and killing potency. The wells contents were aspirated and assayed for nitric oxide and interleukin-2 levels. The results displayed an obvious increase in phagocytic, killing performance as well as nitric oxide and IL-2 level production than control in a dose dependent manner. The obtained results suggested the immune-stimulant impact of the paper-bark tree leaves.

Serotonin elicits long-lasting enhancement of rhythmic respiratory activity in turtle brain stems in vitro

2001 ◽  
Vol 91 (6) ◽  
pp. 2703-2712 ◽  
Author(s):  
Stephen M. Johnson ◽  
Julia E. R. Wilkerson ◽  
Daniel R. Henderson ◽  
Michael R. Wenninger ◽  
Gordon S. Mitchell
Keyword(s):  
Brain Stem ◽  
Respiratory Activity ◽  
Dependent Manner ◽  
Frequency Increase ◽  
Burst Frequency ◽  
Bath Application ◽  
Burst Amplitude ◽  
Dose Dependent ◽  
The Brain

Brain stem preparations from adult turtles were used to determine how bath-applied serotonin (5-HT) alters respiration-related hypoglossal activity in a mature vertebrate. 5-HT (5–20 μM) reversibly decreased integrated burst amplitude by ∼45% ( P < 0.05); burst frequency decreased in a dose-dependent manner with 20 μM abolishing bursts in 9 of 13 preparations ( P < 0.05). These 5-HT-dependent effects were mimicked by application of a 5-HT1A agonist, but not a 5-HT1B agonist, and were abolished by the broad-spectrum 5-HT antagonist, methiothepin. During 5-HT (20 μM) washout, frequency rebounded to levels above the original baseline for 40 min ( P < 0.05) and remained above baseline for 2 h. A 5-HT3 antagonist (tropesitron) blocked the post-5-HT rebound and persistent frequency increase. A 5-HT3 agonist (phenylbiguanide) increased frequency during and after bath application ( P < 0.05). When phenylbiguanide was applied to the brain stem of brain stem/spinal cord preparations, there was a persistent frequency increase ( P < 0.05), but neither spinal-expiratory nor -inspiratory burst amplitude were altered. The 5-HT3receptor-dependent persistent frequency increase represents a unique model of plasticity in vertebrate rhythm generation.

scholarly journals Antifungal activity of dendritic cell lysosomal proteins against Cryptococcus neoformans

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Benjamin N. Nelson ◽  
Savannah G. Beakley ◽  
Sierra Posey ◽  
Brittney Conn ◽  
Emma Maritz ◽  
...  
Keyword(s):  
Antifungal Activity ◽  
Cryptococcus Neoformans ◽  
Mammalian Cells ◽  
Cysteine Proteases ◽  
Dependent Manner ◽  
Life Threatening ◽  
Opportunistic Fungal Pathogen ◽  
Dose Dependent ◽  
Lysosomal Proteins

AbstractCryptococcal meningitis is a life-threatening disease among immune compromised individuals that is caused by the opportunistic fungal pathogen Cryptococcus neoformans. Previous studies have shown that the fungus is phagocytosed by dendritic cells (DCs) and trafficked to the lysosome where it is killed by both oxidative and non-oxidative mechanisms. While certain molecules from the lysosome are known to kill or inhibit the growth of C. neoformans, the lysosome is an organelle containing many different proteins and enzymes that are designed to degrade phagocytosed material. We hypothesized that multiple lysosomal components, including cysteine proteases and antimicrobial peptides, could inhibit the growth of C. neoformans. Our study identified the contents of the DC lysosome and examined the anti-cryptococcal properties of different proteins found within the lysosome. Results showed several DC lysosomal proteins affected the growth of C. neoformans in vitro. The proteins that killed or inhibited the fungus did so in a dose-dependent manner. Furthermore, the concentration of protein needed for cryptococcal inhibition was found to be non-cytotoxic to mammalian cells. These data show that many DC lysosomal proteins have antifungal activity and have potential as immune-based therapeutics.

scholarly journals Anti-tumor activity of a novel proteasome inhibitor D395 against multiple myeloma and its lower cardiotoxicity compared with carfilzomib

2021 ◽  
Vol 12 (5) ◽  
Author(s):  
Xuxing Shen ◽  
Chao Wu ◽  
Meng Lei ◽  
Qing Yan ◽  
Haoyang Zhang ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Proteasome Inhibitor ◽  
Osteoclast Differentiation ◽  
Dependent Manner ◽  
Serum Enzyme ◽  
Herg Channels ◽  
Anti Tumor Activity ◽  
Dose Dependent

AbstractCarfilzomib, a second-generation proteasome inhibitor, has significantly improved the survival rate of multiple myeloma (MM) patients, but its clinical application is still restricted by drug resistance and cardiotoxicity. Here, we identified a novel proteasome inhibitor, D395, and assessed its efficacy in treating MM as well as its cardiotoxicity at the preclinical level. The activities of purified and intracellular proteasomes were measured to determine the effect of D395 on the proteasome. CCK-8 and flow cytometry experiments were designed to evaluate the effects of D395 on cell growth and apoptosis. The effects of D395 and carfilzomib on serum enzyme activity, echocardiography features, cardiomyocyte morphology, and hERG channels were also compared. In our study, D395 was highly cytotoxic to MM cell lines and primary MM cells but not normal cells, and it was well tolerated in vivo. Similar to carfilzomib, D395 inhibited osteoclast differentiation in a dose-dependent manner. In particular, D395 exhibited lower cardiotoxicity than carfilzomib in all experiments. In conclusion, D395 is a novel irreversible proteasome inhibitor that has remarkable anti-MM activity and mild cardiotoxicity in vitro and in vivo.

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