Methanol gel electrophoresis: Separation of human fast and slow myosin light chain 1 and other myofibrillar protein isoforms on a single gel format.

This report describes a novel sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) resolving gel format that consistently yields the electrophoretic separation of the fast and slow isoforms of human sarcomeric myosin light chain 1 (MLC1). The inclusion of methanol as a constituent of the resolving gel impacted the electrophoretic mobility of proteins across a broad range of molecular masses. There was greater separation of the fast and slow isoforms of human MLC1, as well as separation and high resolution of fast and slow isoforms of the three myosin heavy chain isoforms that are expressed in human skeletal muscle on the same gel format.

Targeting Troponin C with Small Molecules Containing Diphenyl Moieties: Calcium Sensitivity Effects on Striated Muscles and Structure-Activity Relationship.

Despite large investments from academia and industry, heart failure, which results from a disruption of the contractile apparatus, remains a leading cause of death. Cardiac muscle contraction is a calcium-dependent mechanism, which is regulated by the troponin protein complex (cTn) and specifically by the N-terminal domain of its calcium-binding subunit (cNTnC). There is an increasing need for the development of small molecules that increase calcium sensitivity without altering the systolic calcium concentration, thereby strengthening the cardiac function.

Targeting Troponin C with Small Molecules Containing Diphenyl Moieties: Calcium Sensitivity Effects on Striated Muscle and Structure Activity Relationship.

Despite large investments from academia and industry, heart failure, which results from a disruption of the contractile apparatus, remains a leading cause of death. Cardiac muscle contraction is a calcium-dependent mechanism, which is regulated by the troponin protein complex (cTn) and specifically by the N-terminal domain of its calcium binding subunit (cNTnC). There is an increasing need for the development of small molecules that increase calcium sensitivity without altering systolic calcium concentration, thereby strengthening cardiac function.

Small Molecule RPI-194 Stabilizes Activated Troponin to Increase the Calcium Sensitivity of Striated Muscle Contraction.

Small molecule cardiac troponin activators could potentially enhance cardiac muscle contraction in the treatment of systolic heart failure. We designed a small molecule, RPI-194, to bind cardiac/slow skeletal muscle troponin (Cardiac muscle and slow skeletal muscle share a common isoform of the troponin C subunit.) Using solution NMR and stopped flow fluorescence spectroscopy, we determined that RPI-194 binds to cardiac troponin with a dissociation constant K of 6-24 μM, stabilizing the activated complex between troponin C and the switch region of troponin I.

3-Chlorodiphenylamine activates cardiac troponin by a mechanism distinct from bepridil or TFP.

Despite extensive efforts spanning multiple decades, the development of highly effective Ca sensitizers for the heart remains an elusive goal. Existing Ca sensitizers have other targets in addition to cardiac troponin (cTn), which can lead to adverse side effects, such as hypotension or arrhythmias. Thus, there is a need to design Ca-sensitizing drugs with higher affinity and selectivity for cTn.

Desensitizing mouse cardiac troponin C to calcium converts slow muscle towards a fast muscle phenotype.

Key Points: The Ca -desensitizing D73N mutation in slow skeletal/cardiac troponin C caused dilatated cardiomyopathy in mice, but the consequences of this mutation in skeletal muscle were not known. The D73N mutation led to a rightward shift in the force versus pCa (-log [Ca]) relationship in slow-twitch mouse fibres. The D73N mutation led to a rightward shift in the force-stimulation frequency relationship and reduced fatigue resistance of mouse soleus muscle.

Euflammation attenuates peripheral inflammation-induced neuroinflammation and mitigates immune-to-brain signaling.

Peripheral inflammation can trigger a number of neuroinflammatory events in the CNS, such as activation of microglia and increases of proinflammatory cytokines. We have previously identified an interesting phenomenon, termed "euflammation", which can be induced by repeated subthreshold infectious challenges. Euflammation causes innate immune alterations without overt neuroimmune activation.

Interleukin 1 type 1 receptor restore: a genetic mouse model for studying interleukin 1 receptor-mediated effects in specific cell types.

Interleukin-1 (IL-1) mediates diverse neurophysiological and neuropathological effects in the CNS through type I IL-1 receptor (IL-1R1). However, identification of IL-1R1-expressing cell types and cell-type-specific functions of IL-1R1 remains challenging. In this study, we created a novel genetic mouse model in which IL-1R1 gene expression is disrupted by an intronic insertion of a loxP flanked disruptive sequence that can be deleted by Cre recombinase, resulting in restored IL-1R1 gene expression under its endogenous promoters.

Prostacyclin mediates endothelial COX-2-dependent neuroprotective effects during excitotoxic brain injury.

In a previous study, we found that intracerebral administration of excitotoxin (RS)-(tetrazole-5yl) glycine caused increased neural damage in the brain in an endothelial COX-2 deleted mouse line (Tie2Cre COX-2(flox/flox) ). In this study, we investigated whether prostacyclin might mediate this endothelial COX-2-dependent neuroprotection. Administration of excitotoxin into the striatum induced the production of prostacyclin (PGI2) in wild type, but not in endothelial COX-2 deleted mice.

Neuronal and nonneuronal COX-2 expression confers neurotoxic and neuroprotective phenotypes in response to excitotoxin challenge.

Treating acute brain injuries with COX-2 inhibitors can produce both neuroprotective and neurotoxic effects. This study investigated the role of COX-2 in modulating acute brain injury induced by excitotoxic neural damage. Intrastriatal injection of excitotoxin (RS)-(tetrazole-5yl) glycine elicited COX-2 expression in two distinct groups of cells.

Interleukin-1R3 mediates interleukin-1-induced potassium current increase through fast activation of Akt kinase.

Inflammatory cytokine interleukin-1 (IL-1) performs multiple functions in the central nervous system. The type 1 IL-1 receptor (IL-1R1) and the IL-1 receptor accessory protein (IL-1RAcP) form a functional IL-1 receptor complex that is thought to mediate most, if not all, IL-1-induced effects. Several recent studies, however, suggest the existence of a heretofore-unidentified receptor for IL-1.

Endothelial IL-1R1 is a critical mediator of EAE pathogenesis.

Interleukin-1 (IL-1) has been implicated in the disease progression of multiple sclerosis (MS). In the animal model of MS, experimental autoimmune encephalomyelitis (EAE), the induction of disease is significantly attenuated in mice lacking the type I IL-1 receptor (IL-1R1). In this study, we created a transgenic mouse (eIL-1R1 kd) in which IL-1R1 expression is knocked down specifically in endothelial cells.

Location-specific activation of the paraventricular nucleus of the hypothalamus by localized inflammation.

The existence of an immunological homunculus has been proposed, but evidence for location-specific response of the central nervous system to immunological stimulation is lacking. In this study, we show that inflammation induced by injection of casein into one of the causes c-fos expression in the paraventricular nucleus of the hypothalamus (PVN) in an asymmetrical manner: much stronger activation is always induced in the contralateral PVN. Unilateral sciatic nerve transection abolished the casein-induced PVN activation if casein was injected into the hindlimb with the nerve transection, but had no effect if casein was injected into the hindlimb with intact nerve innervation.

Endothelial-specific knockdown of interleukin-1 (IL-1) type 1 receptor differentially alters CNS responses to IL-1 depending on its route of administration.

Interleukin-1 (IL-1) has been implicated as a critical mediator of neuroimmune communication. In the brain, the functional receptor for IL-1, type 1 IL-1 receptor (IL-1R1), is localized primarily to the endothelial cells. In this study, we created an endothelial-specific IL-1R1 knockdown model to test the role of endothelial IL-1R1 in mediating the effects of IL-1.