An accelerometry-based, low cost and non-invasive respiration monitoring in anesthetized mice.

Respiratory rate monitoring is essential especially for anesthetized animals in veterinary and biomedical research. Current methods often rely on invasive or wearable devices, which can stress animals, especially smaller ones like rodents. Here we present a non-invasive, environmentally integrated device that detects subtle breathing movements through waveform analyzed data via a triaxial accelerometer under a flexible fabric sheet in a trampoline-like box.

Abemaciclib and Vacuolin-1 decrease aggregate-prone TDP-43 accumulation by accelerating autophagic flux.

(Macro)autophagy is a cellular degradation system for unnecessary materials, such as aggregate-prone TDP-43, a central molecule in neurodegenerative diseases including amyotrophic lateral sclerosis and frontotemporal lobar degeneration. Abemaciclib (Abe) and vacuolin-1 (Vac) treatments are known to induce vacuoles characterized by an autophagosome and a lysosome component, suggesting that they facilitate autophagosome-lysosome fusion. However, it remains unknown whether Abe and Vac suppress the accumulation of aggregate-prone TDP-43 by accelerating autophagic flux.

Dysregulation of the progranulin-driven autophagy-lysosomal pathway mediates secretion of the nuclear protein TDP-43.

The cytoplasmic accumulation of the nuclear protein transactive response DNA-binding protein 43 kDa (TDP-43) has been linked to the progression of amyotrophic lateral sclerosis and frontotemporal lobar degeneration. TDP-43 secreted into the extracellular space has been suggested to contribute to the cell-to-cell spread of the cytoplasmic accumulation of TDP-43 throughout the brain; however, the underlying mechanisms remain unknown. We herein demonstrated that the secretion of TDP-43 was stimulated by the inhibition of the autophagy-lysosomal pathway driven by progranulin (PGRN), a causal protein of frontotemporal lobar degeneration.

Pathologic HDAC1/c-Myc signaling axis is responsible for angiotensinogen transcription and hypertension induced by high-fat diet.

High-fat diet (HFD)-induced obesity is a cause of resistant hypertension. We have shown a possible link between histone deacetylases (HDACs) and renal angiotensinogen (Agt) upregulation in the HFD-induced hypertension, whereas the underlying mechanisms remain to be elucidated. Here, using a HDAC1/2 inhibitor romidepsin (FK228) and siRNAs, we determined roles of HDAC1 and HDAC2 in HFD-induced hypertension and found the pathologic signaling axis between HDAC1 and Agt transcription.

Abemaciclib and Vacuolin-1 induce vacuole-like autolysosome formation - A new tool to study autophagosome-lysosome fusion.

Macroautophagy (hereafter autophagy) is a conserved cellular degradation system, impairments in which have been implicated in the development of a wide range of diseases, including cancer and neurodegenerative diseases. Autophagy is mainly comprised of two processes: the formation of autophagosomes and autolysosomes. A detailed understanding of the formation of autophagosomes has been obtained in the past several decades.

MARK2 regulates directed cell migration through modulation of myosin II contractility and focal adhesion organization.

Cancer cell migration during metastasis is mediated by a highly polarized cytoskeleton. MARK2 and its invertebrate homolog Par1B are kinases that regulate the microtubule cytoskeleton to mediate polarization of neurons in mammals and embryos in invertebrates. However, the role of MARK2 in cancer cell migration is unclear.

Overexpression of progranulin increases pathological protein accumulation by suppressing autophagic flux.

Progranulin (PGRN) haploinsufficiency from autosomal dominant mutations in the PGRN gene causes frontotemporal lobar degeneration, which is characterized by cytoplasmic inclusions predominantly containing TDP-43 (FTLD-TDP). PGRN supplementation for patients with a PGRN gene mutation has recently been proposed as a therapeutic strategy to suppress FTLD-TDP. However, it currently remains unclear whether excessive amounts of PGRN are beneficial or harmful.

Possible roles of N- and C-terminal unstructured tails of CPI-17 in regulating Ca sensitization force of smooth muscle.

CPI-17 regulates the myosin phosphatase and mediates the agonist-induced contraction of smooth muscle. PKC and ROCK phosphorylate CPI-17 at Thr38 leading to a conformational change of the central inhibitory domain (PHIN domain). The N- and C-terminal tails of CPI-17 are predicted as unstructured loops and their sequences are conserved among mammals.

Kinase activity-tagged western blotting assay.

Determining cellular activities of protein kinases is a fundamental step for characterizing pathophysiological cell signaling pathways. Here, we optimized a nonradioactive method that detects protein kinases in tissues or cells after separation by SDS-PAGE and transfer onto polyvinylidene fluoride membranes. The method, kinase activity-tagged western blotting (KAT-WB), consists of five steps: electrophoresis of cell extracts that contain protein kinases, electroblotting proteins onto polyvinylidene fluoride membrane, denaturation-renaturation, phosphorylation, with or without an added substrate protein and immunodetection using anti-phospho-specific antibodies.

RSK2 contributes to myogenic vasoconstriction of resistance arteries by activating smooth muscle myosin and the Na/H exchanger.

Smooth muscle contraction is triggered when Ca/calmodulin-dependent myosin light chain kinase (MLCK) phosphorylates the regulatory light chain of myosin (RLC). However, blood vessels from -deficient mouse embryos retain the ability to contract, suggesting the existence of additional regulatory mechanisms. We showed that the p90 ribosomal S6 kinase 2 (RSK2) also phosphorylated RLC to promote smooth muscle contractility.

Correction to: Protein phosphatases 1 and 2A and their naturally occurring inhibitors: current topics in smooth muscle physiology and chemical biology.

The article Protein phosphatases 1 and 2A and their naturally occurring inhibitors: current topics in smooth muscle physiology and chemical biology, written by Akira Takai, Masumi Eto, Katsuya Hirano, Kosuke Takeya, Toshiyuki Wakimoto and Masaru Watanabe, was originally published electronically on the publisher's internet portal (currently SpringerLink) on 5th July 2017 without open access.

Protein phosphatases 1 and 2A and their naturally occurring inhibitors: current topics in smooth muscle physiology and chemical biology.

Protein phosphatases 1 and 2A (PP1 and PP2A) are the most ubiquitous and abundant serine/threonine phosphatases in eukaryotic cells. They play fundamental roles in the regulation of various cellular functions. This review focuses on recent advances in the functional studies of these enzymes in the field of smooth muscle physiology.

Unfair competition governs the interaction of pCPI-17 with myosin phosphatase (PP1-MYPT1).

The small phosphoprotein pCPI-17 inhibits myosin light-chain phosphatase (MLCP). Current models postulate that during muscle relaxation, phosphatases other than MLCP dephosphorylate and inactivate pCPI-17 to restore MLCP activity. We show here that such hypotheses are insufficient to account for the observed rapidity of pCPI-17 inactivation in mammalian smooth muscles.

Diversity and plasticity in signaling pathways that regulate smooth muscle responsiveness: Paradigms and paradoxes for the myosin phosphatase, the master regulator of smooth muscle contraction.

A hallmark of smooth muscle cells is their ability to adapt their functions to meet temporal and chronic fluctuations in their demands. These functions include force development and growth. Understanding the mechanisms underlying the functional plasticity of smooth muscles, the major constituent of organ walls, is fundamental to elucidating pathophysiological rationales of failures of organ functions.

Remodeling of the rat distal colon in diabetes: function and ultrastructure.

This study seeks to define and explain remodeling of the distal colon in the streptozotocin (STZ)-treated rat model of diabetes through analysis of resting and active length dependence of force production, chemical composition, and ultrastructure. Compared with untreated controls, the passive stiffness on extension of the diabetic muscle is high, and active force produced at short muscle lengths is amplified but is limited by an internal resistance to shortening. The latter are accounted for by a significant increase in collagen type 1, with no changes in types 3 and 4.

F-actin clustering and cell dysmotility induced by the pathological W148R missense mutation of filamin B at the actin-binding domain.

Filamin B (FLNB) is a dimeric actin-binding protein that orchestrates the reorganization of the actin cytoskeleton. Congenital mutations of FLNB at the actin-binding domain (ABD) are known to cause abnormalities of skeletal development, such as atelosteogenesis types I and III and Larsen's syndrome, although the underlying mechanisms are poorly understood. Here, using fluorescence microscopy, we characterized the reorganization of the actin cytoskeleton in cells expressing each of six pathological FLNB mutants that have been linked to skeletal abnormalities.

Reconstituted human myosin light chain phosphatase reveals distinct roles of two inhibitory phosphorylation sites of the regulatory subunit, MYPT1.

The myosin light chain phosphatase (MLCP) is a cytoskeleton-associated protein phosphatase-1 (PP1) holoenzyme and a RhoA/ROCK effector, regulating cytoskeletal reorganization. ROCK-induced phosphorylation of the MLCP regulatory subunit (MYPT1) at two sites, Thr696 and Thr853, suppresses the activity, although little is known about the difference in the role. Here, we developed a new method for the preparation of the recombinant human MLCP complex and determined the molecular and cellular basis of inhibitory phosphorylation.

Nuclear localization of CPI-17, a protein phosphatase-1 inhibitor protein, affects histone H3 phosphorylation and corresponds to proliferation of cancer and smooth muscle cells.

CPI-17 (C-kinase-activated protein phosphatase-1 (PP1) inhibitor, 17kDa) is a cytoplasmic protein predominantly expressed in mature smooth muscle (SM) that regulates the myosin-associated PP1 holoenzyme (MLCP). Here, we show CPI-17 expression in proliferating cells, such as pancreatic cancer and hyperplastic SM cells. Immunofluorescence showed that CPI-17 was concentrated in nuclei of human pancreatic cancer (Panc1) cells.

Endogenous inhibitor proteins that connect Ser/Thr kinases and phosphatases in cell signaling.

Protein phosphatase activity acts as a primary determinant of the extent and duration of phosphorylation of cellular proteins in response to physiological stimuli. Ser/Thr protein phosphatase-1 (PP1) belongs to the PPP superfamily, and is associated with regulatory subunits that confer substrate specificity, allosteric regulation, and subcellular compartmentalization. In addition, all eukaryotic cells contain multiple heat-stable proteins that originally were thought to inhibit phosphatase catalytic subunits released from the regulatory subunits, as a fail-safe mechanism.

Caffeine relaxes smooth muscle through actin depolymerization.

Caffeine is sometimes used in cell physiological studies to release internally stored Ca(2+). We obtained evidence that caffeine may also act through a different mechanism that has not been previously described and sought to examine this in greater detail. We ruled out a role for phosphodiesterase (PDE) inhibition, since the effect was 1) not reversed by inhibiting PKA or adenylate cyclase; 2) not exacerbated by inhibiting PDE4; and 3) not mimicked by submillimolar caffeine nor theophylline, both of which are sufficient to inhibit PDE.

Molecular mechanism of telokin-mediated disinhibition of myosin light chain phosphatase and cAMP/cGMP-induced relaxation of gastrointestinal smooth muscle.

Phospho-telokin is a target of elevated cyclic nucleotide concentrations that lead to relaxation of gastrointestinal and some vascular smooth muscles (SM). Here, we demonstrate that in telokin-null SM, both Ca(2+)-activated contraction and Ca(2+) sensitization of force induced by a GST-MYPT1(654-880) fragment inhibiting myosin light chain phosphatase were antagonized by the addition of recombinant S13D telokin, without changing the inhibitory phosphorylation status of endogenous MYPT1 (the regulatory subunit of myosin light chain phosphatase) at Thr-696/Thr-853 or activity of Rho kinase. Cyclic nucleotide-induced relaxation of force in telokin-null ileum muscle was reduced but not correlated with a change in MYPT1 phosphorylation.

Reciprocal regulation controlling the expression of CPI-17, a specific inhibitor protein for the myosin light chain phosphatase in vascular smooth muscle cells.

Cellular activity of the myosin light chain phosphatase (MLCP) determines agonist-induced force development of smooth muscle (SM). CPI-17 is an endogenous inhibitor protein for MLCP, responsible for mediating G-protein signaling into SM contraction. Fluctuations in CPI-17 expression occur in response to pathological stresses, altering excitation-contraction coupling in SM.