scholarly journals Viable “Haemophilus somnus” Induces Myosin Light-Chain Kinase-Dependent Decrease in Brain Endothelial Cell Monolayer Resistance

2007 ◽  
Vol 75 (9) ◽  
pp. 4572-4581 ◽  
Author(s):  
E. Behling-Kelly ◽  
David McClenahan ◽  
K. S. Kim ◽  
C. J. Czuprynski
Keyword(s):  
Endothelial Cell ◽  
Light Chain ◽  
Myosin Light Chain Kinase ◽  
Myosin Light Chain ◽  
Cell Monolayer ◽  
Brain Endothelial Cell ◽  
Endothelial Cell Monolayer ◽  
Necrosis Factor Alpha ◽  
Haemophilus Somnus

ABSTRACT “Haemophilus somnus” causes thrombotic meningoencephalitis in cattle. Our laboratory has previously reported that H. somnus has the ability to adhere to, but not invade, bovine brain endothelial cells (BBEC) in vitro. The goal of this study was to determine if H. somnus alters brain endothelial cell monolayer integrity in vitro, in a manner that would be expected to contribute to inflammation of the central nervous system (CNS). Monolayer integrity was monitored by measuring transendothelial electrical resistance (TEER) and albumin flux. BBEC incubated with H. somnus underwent rapid cytoskeletal rearrangement, significant increases in albumin flux, and reductions in TEER. Decreased monolayer TEER was preceded by phosphorylation of the myosin regulatory light chain and was partially dependent on tumor necrosis factor alpha and myosin light-chain kinase but not interleukin-1β. Neither heat-killed H. somnus, formalin-fixed H. somnus, nor purified lipooligosaccharide altered monolayer integrity within a 2-h incubation period, whereas conditioned medium from H. somnus-treated BBEC caused a modest reduction in TEER. The data from this study support the hypothesis that viable H. somnus alters integrity of the blood-brain barrier by promoting contraction of BBEC and increasing paracellular permeability of the CNS vasculature.

scholarly journals Inhibiting Myosin Light Chain Kinase Induces Apoptosis In Vitro and In Vivo

2005 ◽  
Vol 25 (14) ◽  
pp. 6259-6266 ◽  
Author(s):  
Fabeha Fazal ◽  
Lianzhi Gu ◽  
Ivanna Ihnatovych ◽  
YooJeong Han ◽  
WenYang Hu ◽  
...  
Keyword(s):  
Cell Death ◽  
Light Chain ◽  
Myosin Light Chain Kinase ◽  
Myosin Light Chain ◽  
Apoptotic Cells ◽  
Caspase Activation ◽  
Inhibitory Antibody ◽  
Necrosis Factor Alpha

ABSTRACT Previous short-term studies have correlated an increase in the phosphorylation of the 20-kDa light chain of myosin II (MLC20) with blebbing in apoptotic cells. We have found that this increase in MLC20 phosphorylation is rapidly followed by MLC20 dephosphorylation when cells are stimulated with various apoptotic agents. MLC20 dephosphorylation is not a consequence of apoptosis because MLC20 dephosphorylation precedes caspase activation when cells are stimulated with a proapoptotic agent or when myosin light chain kinase (MLCK) is inhibited pharmacologically or by microinjecting an inhibitory antibody to MLCK. Moreover, blocking caspase activation increased cell survival when MLCK is inhibited or when cells are treated with tumor necrosis factor alpha. Depolymerizing actin filaments or detaching cells, processes that destabilize the cytoskeleton, or inhibiting myosin ATPase activity also resulted in MLC20 dephosphorylation and cell death. In vivo experiments showed that inhibiting MLCK increased the number of apoptotic cells and retarded the growth of mammary cancer cells in mice. Thus, MLC20 dephosphorylation occurs during physiological cell death and prolonged MLC20 dephosphorylation can trigger apoptosis.

scholarly journals ATP increases the migration of microglia across the brain endothelial cell monolayer

2016 ◽  
Vol 36 (2) ◽  
Author(s):  
Tomoji Maeda ◽  
Manato Inagaki ◽  
Yu Fujita ◽  
Takehiro Kimoto ◽  
Chiaki Tanabe-Fujimura ◽  
...  
Keyword(s):  
Endothelial Cell ◽  
Matrix Metalloproteinases ◽  
Real Time ◽  
Culture System ◽  
Cell Monolayer ◽  
Brain Endothelial Cell ◽  
Endothelial Cell Monolayer ◽  
Microglial Migration ◽  
The Brain

To elucidate the mechanism of microglial migration across the blood–brain barrier (BBB), we developed an in vitro co-culture system and analysed real-time BBB integrity during transmigration. We show that ATP promotes microglia transmigration via a mechanism involving microglial matrix metalloproteinases (MMPs).

The Regulation of Myosin Light Chain Kinase by Ca++ and Calmodulin in Vitro and in Vivo

1982 ◽  
pp. 219-238 ◽  
Author(s):  
J. T. Stull ◽  
D. K. Blumenthal ◽  
B. R. Botterman ◽  
G. A. Klug ◽  
D. R. Manning ◽  
...  
Keyword(s):  
Light Chain ◽  
Myosin Light Chain Kinase ◽  
Myosin Light Chain

Is phosphorylation the main physiological action of myosin light chain kinase?

1994 ◽  
Vol 72 (11) ◽  
pp. 1377-1379 ◽  
Author(s):  
Setsuro Ebashi ◽  
Hideto Kuwayama
Keyword(s):  
Amino Acid ◽  
Light Chain ◽  
Myosin Light Chain Kinase ◽  
Myosin Light Chain ◽  
Actin Binding ◽  
In Vitro System ◽  
Beryllium Sulfate ◽  
Kinase Phosphorylation ◽  
Actin Binding Site

The 155-kDa component of bovine stomach, which exhibits a strong actomyosin (AM) activating activity and a relatively weak myosin light chain kinase (MLCK) activity, has a strong affinity for the actin filament and the actin-binding site is confined to an 80 amino acid residue on its N-terminal side. This affinity may play a crucial role in AM activation. Some reagents preferentially abolish either the AM-activating effect or MLCK activity. In conclusion, MLCK of the 155-kDa component does not play a fundamental role in activating the AM system as far as the in vitro system is concerned. The possible mechanism of AM activation by the component is discussed.Key words: myosin light chain kinase, phosphorylation of myosin light chain, leiotonin, wortmannin, beryllium sulfate.

scholarly journals Myosin light chain kinase (MYLK) coding polymorphisms modulate human lung endothelial cell barrier responses via altered tyrosine phosphorylation, spatial localization, and lamellipodial protrusions

2018 ◽  
Vol 8 (2) ◽  
pp. 204589401876417 ◽  
Author(s):  
Ting Wang ◽  
Mary E. Brown ◽  
Gabriel T. Kelly ◽  
Sara M. Camp ◽  
Joseph B. Mascarenhas ◽  
...  
Keyword(s):  
Endothelial Cell ◽  
Tyrosine Phosphorylation ◽  
Light Chain ◽  
Myosin Light Chain Kinase ◽  
Myosin Light Chain ◽  
Human Lung ◽  
Disease Risk ◽  
Spatial Localization ◽  
Cell Periphery ◽  
Nucleotide Polymorphisms

Sphingosine 1-phosphate (S1P) is a potent bioactive endogenous lipid that signals a rearrangement of the actin cytoskeleton via the regulation of non-muscle myosin light chain kinase isoform (nmMLCK). S1P induces critical nmMLCK Y464 and Y471 phosphorylation resulting in translocation of nmMLCK to the periphery where spatially-directed increases in myosin light chain (MLC) phosphorylation and tension result in lamellipodia protrusion, increased cell-cell adhesion, and enhanced vascular barrier integrity. MYLK, the gene encoding nmMLCK, is a known candidate gene in lung inflammatory diseases, with coding genetic variants (Pro21His, Ser147Pro, Val261Ala) that confer risk for inflammatory lung injury and influence disease severity. The functional mechanisms by which these MYLK coding single nucleotide polymorphisms (SNPs) affect biologic processes to increase disease risk and severity remain elusive. In the current study, we utilized quantifiable cell immunofluorescence assays to determine the influence of MYLK coding SNPs on S1P-mediated nmMLCK phosphorylation and translocation to the human lung endothelial cell (EC) periphery . These disease-associated MYLK variants result in reduced levels of S1P-induced Y464 phosphorylation, a key site for nmMLCK enzymatic regulation and activation. Reduced Y464 phosphorylation resulted in attenuated nmMLCK protein translocation to the cell periphery. We further conducted EC kymographic assays which confirmed that lamellipodial protrusion in response to S1P challenge was retarded by expression of a MYLK transgene harboring the three MYLK coding SNPs. These data suggest that ARDS/severe asthma-associated MYLK SNPs functionally influence vascular barrier-regulatory cytoskeletal responses via direct alterations in the levels of nmMLCK tyrosine phosphorylation, spatial localization, and lamellipodial protrusions.

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