During skeletal muscle excitation-contraction (EC) coupling, membrane depolarizations activate the sarcolemmal voltage-gated L-type Ca 2+ channel (Ca V 1.1). Ca V 1.1 in turn triggers opening of the sarcoplasmic Ca 2+ release channel (RyR1) via interchannel protein-protein interaction to release Ca 2+ for myofibril contraction. Simultaneously to this EC coupling process, a small and slowly activating Ca 2+ inward current through Ca V 1.1 is found in mammalian skeletal myotubes. The role of this Ca 2+ influx, which is not immediately required for EC coupling, is still enigmatic. Interestingly, whole-cell patch clamp experiments on freshly dissociated skeletal muscle myotubes from zebrafish larvae revealed the lack of such Ca 2+ currents. We identified two distinct isoforms of the pore-forming Ca V 1.1α 1S subunit in zebrafish that are differentially expressed in superficial slow and deep fast musculature. Both do not conduct Ca 2+ but merely act as voltage sensors to trigger opening of two likewise tissue-specific isoforms of RyR1. We further show that non-Ca 2+ conductivity of both Ca V 1.1α 1S isoforms is a common trait of all higher teleosts. This non-Ca 2+ conductivity of Ca V 1.1 positions teleosts at the most-derived position of an evolutionary trajectory. Though EC coupling in early chordate muscles is activated by the influx of extracellular Ca 2+ , it evolved toward Ca V 1.1-RyR1 protein-protein interaction with a relatively small and slow influx of external Ca 2+ in tetrapods. Finally, the Ca V 1.1 Ca 2+ influx was completely eliminated in higher teleost fishes.calcium conductivity | evolution | ion channels | slow and fast muscle | zebrafish

PurposeLeukotrienes and prostaglandins, products of arachidonic acid metabolism, sustain both systemic and lesion-localized inflammation. Tumor-associated Inflammation can also contribute to the pathogenesis of colon cancer. Patients with inflammatory bowel disease (IBD) have increased risk of developing colon cancer. The levels of 5-lipoxygenase (5-LO), the key enzyme for leukotrienes production, are increased in colon cancer specimens and colonic dysplastic lesions. Here we report that Zileuton, a specific 5-LO inhibitor, can prevent polyp formation by efficiently reducing the tumor-associated and systemic inflammation in APCΔ468 mice.Experimental DesignIn the current study, we inhibited 5-LO by dietary administration of Zileuton in the APCΔ468 mouse model of polyposis and analyzed the effect of in vivo 5-LO inhibition on tumor-associated and systemic inflammation.ResultsZileuton-fed mice developed fewer polyps and displayed marked reduction in systemic and polyp-associated inflammation. Pro-inflammatory cytokines and pro-inflammatory innate and adaptive immunity cells were reduced both in the lesions and systemically. As part of tumor-associated inflammation Leukotriene B4 (LTB4), product of 5-LO activity, is increased focally in human dysplastic lesions. The 5-LO enzymatic activity was reduced in the serum of Zileuton treated polyposis mice.ConclusionsThis study demonstrates that dietary administration of 5-LO specific inhibitor in the polyposis mouse model decreases polyp burden, and suggests that Zileuton may be a potential chemo-preventive agent in patients that are high-risk of developing colon cancer.

We determined the prevalence and antigenic specificity of autoantibodies against cytoskeletal proteins in patients affected with various autoimmune diseases. Sera collected from patients with rheumatoid arthritis, systemic lupus erythematosus or progressive systemic sclerosis, and normal volunteers, were examined for the presence of autoantibodies against cytoskeletal proteins by indirect immunofluorescence and enzyme-linked immunosorbent assay (ELISA). Patients with rheumatoid arthritis had the highest reactivity to cytoskeletal antigens on immunofluorescence assays using isolated muscle myofibrils (41/50) and L929 cells (37/50). Antigen-specific ELISA revealed significant immunoreactivity against actin (11/50) and myosin (15/50). In nine patients, immunoreactivity was seen against multiple cytoskeletal antigens. We concluded that the prevalence of IgG autoantibodies against cytoskeletal antigens, especially myofibrillar components actin and myosin, is elevated in patients with rheumatoid arthritis.

PurposeThe development of NIRF cathepsin activity probes offered the ability to visualize tumor associated tumor reaction and act as a surrogate marker to delineate the dysplastic lesions. One major type is a NIRF substrate of cathepsins (SBP), which is involved in catalytic way to produce high levels of fluorescence emission. The other major type (ABP) reacts with active cathepsins in stoichiometric manner since they bind covalently with their active center. Little is known about the sensitivity and the specificity of the NIRF probes to detect autochthonous developed dysplastic lesions. Dual laser NIRF endoscope provides a good tool to determine the efficiency of various NIRF probes in vivo in the same lesions.Experimental designIn the current study, we validated both types of NIRF probes by using the dual laser NIRF endoscope to detect lesions colon cancer mouse model (TS4Cre/cAPC +/lox).ResultsThe dual laser NIRF endoscope is emitting equal power with both lasers. It can detect with the same efficiency in 680 mode, as well as, 750 mode when NIFR probes of the same scaffold in vivo. When SBP and ABP were used, our results showed both probes are efficient enough to detect large polyps but small dysplastic lesions could not efficiently imaged with the ABP.ConclusionsThe dual laser NIRF endoscope is a powerful tool to validate probes. The probes that react catalytically with the active center of cathepsins are more efficient than the ones that react stoichiometrically in detecting small lesions.

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