scholarly journals Inactivation of Stress Induced 1-Aminocyclopropane Carboxylate Synthase in Vivo Differs from Substrate-Dependent Inactivation in Vitro

1990 ◽  
Vol 93 (4) ◽  
pp. 1482-1485 ◽  
Author(s):  
Pietro Spanu ◽  
Georg Felix ◽  
Thomas Boller
Keyword(s):  
Dependent Inactivation ◽  
Aminocyclopropane Carboxylate

L-Type CaV1.2 Calcium Channels: From In Vitro Findings to In Vivo Function

2014 ◽  
Vol 94 (1) ◽  
pp. 303-326 ◽  
Author(s):  
Franz Hofmann ◽  
Veit Flockerzi ◽  
Sabine Kahl ◽  
Jörg W. Wegener
Keyword(s):  
Calcium Channel ◽  
Splice Variants ◽  
Kinetic Properties ◽  
Expression Systems ◽  
Smooth Muscle Contractility ◽  
Dependent Inactivation ◽  
Heterologous Expression Systems ◽  
Channel Properties

The L-type Cav1.2 calcium channel is present throughout the animal kingdom and is essential for some aspects of CNS function, cardiac and smooth muscle contractility, neuroendocrine regulation, and multiple other processes. The L-type CaV1.2 channel is built by up to four subunits; all subunits exist in various splice variants that potentially affect the biophysical and biological functions of the channel. Many of the CaV1.2 channel properties have been analyzed in heterologous expression systems including regulation of the L-type CaV1.2 channel by Ca2+ itself and protein kinases. However, targeted mutations of the calcium channel genes confirmed only some of these in vitro findings. Substitution of the respective serines by alanine showed that β-adrenergic upregulation of the cardiac CaV1.2 channel did not depend on the phosphorylation of the in vitro specified amino acids. Moreover, well-established in vitro phosphorylation sites of the CaVβ2 subunit of the cardiac L-type CaV1.2 channel were found to be irrelevant for the in vivo regulation of the channel. However, the molecular basis of some kinetic properties, such as Ca2+-dependent inactivation and facilitation, has been approved by in vivo mutagenesis of the CaV1.2α1 gene. This article summarizes recent findings on the in vivo relevance of well-established in vitro results.

scholarly journals A structural mechanism for phosphorylation-dependent inactivation of the AP2 complex

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Edward A Partlow ◽  
Richard W Baker ◽  
Gwendolyn M Beacham ◽  
Joshua S Chappie ◽  
Andres E Leschziner ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Transmembrane Proteins ◽  
Active Conformation ◽  
Genetic Screens ◽  
Structural Mechanism ◽  
C Elegans ◽  
Dependent Inactivation ◽  
Clathrin Adaptor

Endocytosis of transmembrane proteins is orchestrated by the AP2 clathrin adaptor complex. AP2 dwells in a closed, inactive state in the cytosol, but adopts an open, active conformation on the plasma membrane. Membrane-activated complexes are also phosphorylated, but the significance of this mark is debated. We recently proposed that NECAP negatively regulates AP2 by binding open and phosphorylated complexes (Beacham et al., 2018). Here, we report high-resolution cryo-EM structures of NECAP bound to phosphorylated AP2. The site of AP2 phosphorylation is directly coordinated by residues of the NECAP PHear domain that are predicted from genetic screens in C. elegans. Using membrane mimetics to generate conformationally open AP2, we find that a second domain of NECAP binds these complexes and cryo-EM reveals both domains of NECAP engaging closed, inactive AP2. Assays in vitro and in vivo confirm these domains cooperate to inactivate AP2. We propose that phosphorylation marks adaptors for inactivation.

scholarly journals A structural mechanism for phosphorylation-dependent inactivation of the AP2 complex

2019 ◽  
Author(s):  
Edward A. Partlow ◽  
Richard W. Baker ◽  
Gwendolyn M. Beacham ◽  
Joshua S. Chappie ◽  
Andres E. Leschziner ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Transmembrane Proteins ◽  
Active Conformation ◽  
Genetic Screens ◽  
Structural Mechanism ◽  
C Elegans ◽  
Dependent Inactivation ◽  
Clathrin Adaptor

AbstractEndocytosis of transmembrane proteins is orchestrated by the AP2 clathrin adaptor complex. AP2 dwells in a closed, inactive state in the cytosol, but adopts an open, active conformation on the plasma membrane. Membrane-activated complexes are also phosphorylated, but the significance of this mark is debated. We recently proposed that NECAP negatively regulates AP2 by binding open and phosphorylated complexes (Beacham et al., 2018). Here, we report high-resolution cryo-EM structures of NECAP bound to phosphorylated AP2. The site of AP2 phosphorylation is directly coordinated by residues of the NECAP PHear domain that are predicted from genetic screens in C. elegans. Using membrane mimetics to generate conformationally open AP2, we find that a second domain of NECAP binds these complexes and cryo-EM reveals both domains of NECAP engaging closed, inactive AP2. Assays in vitro and in vivo confirm these domains cooperate to inactivate AP2. We propose that phosphorylation marks adaptors for inactivation.

scholarly journals Metabolic Drug-Drug Interaction Potential of Macrolactin A and 7-O-Succinyl Macrolactin A Assessed by Evaluating Cytochrome P450 Inhibition and Induction and UDP-Glucuronosyltransferase InhibitionIn Vitro

2014 ◽  
Vol 58 (9) ◽  
pp. 5036-5046 ◽  
Author(s):  
Soo Hyeon Bae ◽  
Min Jo Kwon ◽  
Jung Bae Park ◽  
Doyun Kim ◽  
Dong-Hee Kim ◽  
...  
Keyword(s):  
Cytochrome P450 ◽  
Drug Interactions ◽  
Antitumor Agents ◽  
Content Type ◽  
Dependent Inactivation ◽  
Competitive Inhibitors ◽  
Macrolactin A ◽  
Clinically Significant

ABSTRACTMacrolactin A (MA) and 7-O-succinyl macrolactin A (SMA), polyene macrolides containing a 24-membered lactone ring, show antibiotic effects superior to those of teicoplanin against vancomycin-resistant enterococci and methicillin-resistantStaphylococcus aureus. MA and SMA are currently being evaluated as antitumor agents in preclinical studies in Korea. We evaluated the potential of MA and SMA for the inhibition or induction of human liver cytochrome P450 (CYP) enzymes and UDP-glucuronosyltransferases (UGTs)in vitroto assess their safety as new molecular entities. We demonstrated that MA and SMA are potent competitive inhibitors of CYP2C9, withKivalues of 4.06 μM and 10.6 μM, respectively. MA and SMA also weakly inhibited UGT1A1 activity, withKivalues of 40.1 μM and 65.3 μM, respectively. However, these macrolactins showed no time-dependent inactivation of the nine CYPs studied. In addition, MA and SMA did not induce CYP1A2, CYP2B6, or CYP3A4/5. On the basis of anin vitro-in vivoextrapolation, our data strongly suggested that MA and SMA are unlikely to cause clinically significant drug-drug interactions mediated via inhibition or induction of most of the CYPs involved in drug metabolismin vivo, except for the inhibition of CYP2C9 by MA. Similarly, MA and SMA are unlikely to inhibit the activity of UGT1A1, UGT1A4, UGT1A6, UGT1A9, and UGT2B7 enzymesin vivo. Although further investigations will be required to clarify thein vivointeractions of MA with CYP2C9-targeted drugs, our findings offer a clearer understanding and prediction of drug-drug interactions for the safe use of MA and SMA in clinical practice.

Role of blue light in bactericidal effect against meat-borne pathogens and freshness maintaining of beef

2021 ◽  
Author(s):  
Shuanghua Luo ◽  
Xi Yang ◽  
Shuyan Wu ◽  
Minmin Liu ◽  
Xiujuan Zhang ◽  
...  
Keyword(s):  
Blue Light ◽  
Amino Acid Content ◽  
Bactericidal Effect ◽  
In Vivo Studies ◽  
Dependent Inactivation ◽  
A Minor ◽  
The Impact ◽  
First Time

Beef is rich in various nutrients while easily spoils due to contamination by pathogens, thus it is of great significance to develop a bactericidal method to inactivate meat-borne pathogens and meanwhile maintain the freshness of beef. For the first time, the present study investigated the bactericidal effect of blue light (BL) at 415 nm against four meat-borne pathogens (methicillin-resistant Staphylococcus aureus , Escherichia coli , Salmonella Typhimurium and Listeria monocytogenes ) in vitro and inoculated on the surface of fresh beef, respectively. When the non-illuminated beef was used as control, the population of the four pathogens did not change significantly ( P > 0.05), while BL-illuminated beef showed dose-dependent inactivation effect in both in vitro and in vivo studies. The experiments on beef cuts showed that 109.44 J/cm 2 of BL inactivated 90% of inoculated cells for the tested strains ( P < 0.05), and the impact of BL inactivation could be sustained in 7 days of cold storage. Notably, changes of lipid oxidation rate, water holding capacity and cooking loss value between the control and beef illuminated by 109.44 J/cm 2 at the same time were scarcely detected during the storage. BL had a minor but insignificant influence on surface color and free amino acid content. Moreover, the pH of illuminated beef increased slower ( P < 0.05) than that of non-illuminated beef. The present work demonstrated that BL could be a novel bactericidal and freshness-maintaining method for fresh beef.

Induction and Differentiation of Dental Epithelium in Vivo and in Vitro

1982 ◽  
Vol 40 ◽  
pp. 142-145
Author(s):  
E. J. Kollar
Keyword(s):  
Connective Tissue ◽  
Oral Mucosa ◽  
Basal Lamina ◽  
Functional Derivatives ◽  
Specific Source ◽  
Dental Epithelium ◽  
Connective Tissue Stroma

The differentiation and maintenance of many specialized epithelial structures are dependent on the underlying connective tissue stroma and on an intact basal lamina. These requirements are especially stringent in the development and maintenance of the skin and oral mucosa. The keratinization patterns of thin or thick cornified layers as well as the appearance of specialized functional derivatives such as hair and teeth can be correlated with the specific source of stroma which supports these differentiated expressions.

Ultrastructural Correlations between Clonal Human Tumor Colonies and in Vivo Neoplasms

1982 ◽  
Vol 40 ◽  
pp. 210-211
Author(s):  
M.J. Murphy ◽  
R.R. Price ◽  
J.C. Sloman
Keyword(s):  
Cultured Cells ◽  
Human Tumor ◽  
Cell Type ◽  
Tumor Mass ◽  
Initial Cell ◽  
Cloning Assay ◽  
Human Tumor Cloning ◽  
The Relationship

The in vitro human tumor cloning assay originally described by Salmon and Hamburger has been applied recently to the investigation of differential anti-tumor drug sensitivities over a broad range of human neoplasms. A major problem in the acceptance of this technique has been the question of the relationship between the cultured cells and the original patient tumor, i.e., whether the colonies that develop derive from the neoplasm or from some other cell type within the initial cell population. A study of the ultrastructural morphology of the cultured cells vs. patient tumor has therefore been undertaken to resolve this question. Direct correlation was assured by division of a common tumor mass at surgical resection, one biopsy being fixed for TEM studies, the second being rapidly transported to the laboratory for culture.

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