High Expression of MYL9 Indicates Poor Clinical Prognosis of Epithelial Ovarian Cancer.

BACKGROUND: The prognosis of Epithelial Ovarian Cancer (EOC) is poor, but the prognostic biomarkers are neither sensitive nor specific. Therefore, it is very important to search novel prognostic biomarkers for EOC. OBJECTIVES: The present study aimed to investigate Myosin Light Chain 9(MYL9) expression in Epithelial Ovarian Cancer (EOC) tissues (including paraffin-embedded and fresh tissue samples) and its relationship with clinicopathological characteristics, as well as its potential prognostic value in patients with EOC. METHODS: Between March 2009 and December 2018, all of 184 paraffin-embedded cancer tissues from patients with EOC and 41 paratumor tissues, pathologically confirmed at the Memorial Hospital of Sun Yat-sen University and Integrated Hospital of Traditional Chinese Medicine, Southern Medical University, were collected for the present study and were assessed for MYL9 protein expression patterns using Immunohistochemistry (IHC). Furthermore, from August 2013 to November 2019, 16 fresh EOC tissues and their paired paratumor tissues, pathologically confirmed at the Integrated Hospital of Traditional Chinese Medicine, Southern Medical University were analyzed using Reverse-Transcription Quantitative PCR (RT-qPCR) to detect MYL9 mRNA expression levels. RESULTS: The results showed that MYL9 expression was higher in cancer tissues compared with that in paratumor tissues, and MYL9 overexpression was associated with shorter Recurrence Free Survival (RFS) and Overall Survival (OS) of EOC patients. Furthermore, multivariate Cox model analysis indicated that MYL9 overexpression was an independent poor survival prediction in patients with EOC. CONCLUSION: MYL9 is upregulated in EOC and may serve as a useful patent of prognostic biomarker in EOC, and it may demonstrate an important value for the clinical treatment and supervision of patients with EOC.

Atractylodes oil alleviates diarrhea-predominant irritable bowel syndrome by regulating intestinal inflammation and intestinal barrier via SCF/c-kit and MLCK/MLC2 pathways.

ETHNOPHARMACOLOGICAL RELEVANCE: Atractylodes lancea (Thunb.) DC. is a widely used traditional herb that is well known for treating spleen deficiency and diarrhea. According to traditional Chinese medicine (TCM) theory, diarrhea-predominant irritable bowel syndrome (IBS-D) is caused by cold and dampness, resulting in diarrhea and abdominal pain. Nevertheless, the effect and mechanism of Atractylodes on IBS-D are still unclear. AIM OF THE STUDY: This study was designed to confirm the therapeutic effect of Atractylodes lanceolata oil (AO) in a rat model of IBS-D, and to determine the mechanisms by which AO protects against the disease. MATERIALS AND METHODS: The chemical components in AO were determined using gas chromatography-mass spectrometry (GC-MS). The expression levels of 5-hydroxytryptamine (5-HT), vasoactive intestinal peptide (VIP), and surfactant protein (SP) in serum and colon tissue were measured using enzyme-linked immunosorbent assay (ELISA). Reverse transcription-polymerase chain reaction (RT-PCR), western blotting (WB), immunohistochemistry (IHC), and immunofluorescence (IF) were used to elucidate the mechanism of action of AO toward inflammation and the intestinal barrier in a rat model of IBS-D. RESULTS: The 15 chemical substances of the highest concentration in AO were identified using GC-MS. AO was effective against IBS-D in the rat model, in terms of increased body weight, diarrhea grade score, levels of interleukin-10 (IL-10), aquaporin 3 (AQP3), and aquaporin 8 (AQP8), and reduced fecal moisture content, levels of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), 5-HT, VIP, and SP, while also reducing intestinal injury, as observed using hematoxylin-eosin (HE) staining. In addition, the results indicated that AO increased the mRNA and protein expression levels of stem cell factor (SCF) and c-kit and enhanced the levels of zonula occludens-1 (ZO-1) and occludin, as well as decreased the levels of myosin light chain kinase (MLCK) and inhibited the phosphorylation of myosin light chain 2 (p-MLC2). CONCLUSIONS: AO was found to be efficacious in the rat model of IBS-D. AO inhibited the SCF/c-kit pathway, thereby reducing inflammation and protecting against intestinal barrier damage via the MLCK/MLC2 pathway.

The Effect of Orally Supplemented Melatonin on Larval Performance and Skeletal Deformities in Farmed Gilthead Seabream (Sparus aurata).

The gilthead seabream larval rearing in continuous light is common in most Mediterranean hatcheries to stimulate larval length growth and increase food consumption. Several studies have shown that continuous light affects larval development and increases the prevalence of skeletal deformities. Melatonin is a crucial pineal neurohormone that displays daily secretion patterns, stimulates cell proliferation and embryonic development in Atlantic salmon and zebrafish, and improves osseointegration in mice and humans. However, no studies have examined the effects of orally supplemented melatonin on skeletal deformities in Sparus aurata larvae. We administered exogenous melatonin to gilthead seabream larvae via enriched rotifers and nauplii of Artemia. Exogenous melatonin induced bone deformities and stimulated parathyroid hormone-related protein-coding gene (PTHrP) mRNA expression. In addition to the melatonin-induced PTHrP high expression level, the recorded non coordinated function of skeletal muscle and bone during growth can be the fountainhead of bone deformities. Both myosin light chain 2 (mlc2) and bone gamma-carboxyglutamate protein-coding gene (bglap) expression levels were significantly affected by melatonin administration in an inverse dose-response manner during the exogenous melatonin administration. This is the first study to report the effect of inducing melatonin bone deformities on Sparus aurata larvae reared under ordinary hatchery conditions.

The molecular effects of electrical stimulation on the muscle components of the urethra of female rats after trauma by vaginal distention.

AIMS: To evaluate the expression of genes and proteins related to the urethral muscles of female rats after trauma by vaginal distention (VD) and after electrical stimulation therapy (EST). METHODS: We compared the urethras of four groups of 20 animals each: control without trauma (C), 7 (recent-trauma) and 30 days (late-trauma) post-VD, and VD-treated with EST. We evaluated the expression of myogenic regulatory factors MYOD1 and myogenin (MYOG); skeletal muscle myosin heavy chain 1, 2, and 3 (MYH1, MYH2, and MYH3); smooth muscle MYH11; and myosin light chain 9 (MYL9). We used real-time quantitative polymerase chain reaction, Western blot analysis, and immunohistochemistry. RESULTS: MYOD1 and MYOG genes were overexpressed in the recent-trauma group compared with the other groups (P < .05). MYH1 and MYH3 genes were upregulated in the recent-trauma group compared with the control and EST groups (P < .05). The MYH2 gene was overexpressed in the late-trauma group (P < .05), while the MYH2 protein was significantly increased in the EST group compared with control, recent-trauma and late-trauma groups by 5-, 3-, and 2.7-fold change, respectively (P < .05). MYL9 and MYH11 messenger RNA were overexpressed in both trauma groups compared with control and EST groups (P < .05). MYH11 protein was not different among the study groups (P > .05). CONCLUSIONS: EST enhances the recovery of the damaged urethral tissue of rats mainly by acting on the striated-muscle components. The MYH2 pathway underlies the positive effects of EST in the external urethral sphincter.

Catharmus tinctorius volatile oil promote the migration of mesenchymal stem cells via ROCK2/Myosin light chain signaling.

MSC transplantation has been explored as a new clinical approach to stem cell-based therapies for bone diseases in regenerative medicine due to their osteogenic capability. However, only a small population of implanted MSC could successfully reach the injured areas. Therefore, enhancing MSC migration could be a beneficial strategy to improve the therapeutic potential of cell transplantation. Catharmus tinctorius volatile oil (CTVO) was found to facilitate MSC migration. Further exploration of the underlying molecular mechanism participating in the pro-migratory ability may provide a novel strategy to improve MSC transplantation efficacy. This study indicated that CTVO promotes MSC migration through enhancing ROCK2 mRNA and protein expressions. MSC migration induced by CTVO was blunted by ROCK2 inhibitor, which also decreased myosin light chain (MLC) phosphorylation. Meanwhile, the siRNA for ROCK2 inhibited the effect of CTVO on MSC migration ability and attenuated MLC phosphorylation, suggesting that CTVO may promote BMSC migration via the ROCK2/MLC signaling. Taken together, this study indicates that C. tinctorius volatile oil could enhance MSC migration via ROCK2/MLC signaling in vitro. C. tinctorius volatile oil-targeted therapy could be a beneficial strategy to improve the therapeutic potential of cell transplantation for bone diseases in regenerative medicine.

Anthocyanins inhibit tumor necrosis alpha-induced loss of Caco-2 cell barrier integrity.

An increased permeability of the intestinal barrier is proposed as a major event in the pathophysiology of conditions characterized by chronic gut inflammation. This study investigated the capacity of pure anthocyanins (AC), and berry and rice extracts containing different types and amounts of AC, to inhibit tumor necrosis alpha (TNFα)-induced permeabilization of Caco-2 cell monolayers. Caco-2 cells differentiated into intestinal epithelial cell monolayers were incubated in the absence/presence of TNFα, with or without the addition of AC or AC-rich plant extracts (ACRE). AC and ACRE inhibited TNFα-induced loss of monolayer permeability as assessed by changes in transepithelial electrical resistance (TEER) and paracellular transport of FITC-dextran. In the range of concentrations tested (0.25-1 µM), O-glucosides of cyanidin, and delphinidin, but not those of malvidin, peonidin and petunidin protected the monolayer from TNFα-induced decrease of TEER and increase of FITC-dextran permeability. Cyanidin and delphinidin acted by mitigating TNFα-triggered activation of transcription factor NF-κB, and downstream phosphorylation of myosin light chain (MLC). The protective actions of the ACRE on TNFα-induced TEER increase was positively correlated with the sum of cyanidins and delphinidins (r2 = 0.83) content in the ACRE. However, no correlation was observed between TEER and ACRE total AC, malvidin, or peonidin content. Results support a particular capacity of cyanidins and delphinidins in the protection of the intestinal barrier against inflammation-induced permeabilization, in part through the inhibition of the NF-κB pathway.

Inhibition of the invasion and migration of renal carcinoma 786­o­si3 cells in vitro and in vivo by Koelreuteria formosana extract.

Koelreuteria formosana ethanolic extract (KFEE) is obtained from natural plants that are endemic to Taiwan. In a previous study, it was demonstrated that KFEE inhibited low-density lipoprotein (LDL) and prevented oxidized LDL­induced apoptosis in endothelial cells. In the present study, KFEE was shown to inhibit the invasion and migration of 786­O­SI3 renal cell carcinoma (RCC) cells while not exhibiting any cytotoxic effects. 786­O­SI3 cells were treated with KFEE at numerous concentrations of ≤100 µg/ml for 24 h. In order to examine the effects of KFEE, cells were then subjected to a series of assays for cell viability (MTT), wound healing migration, cell invasion and migration, gelatin zymography, casein zymography and immunofluorescence, as well as western blot analysis. KFEE was shown to decrease levels of matrix metalloproteinase­2, phosphorylated (p­)focal adhesion kinase Try925, p­paxillin Ser178, p­mitogen­activated protein kinase kinase 1/2, p­myosin light chain and p­extracellular signal-regulated kinase 1/2 in 786-0-SI3 cells. Reduction of lung metastases was observed in KFEE-treated mice compared with vehicle­treated control mice. KFEE inhibited the invasion of RCC cells and may have the potential for use as a chemopreventive agent against RCC metastasis.

Atrial and ventricular specification of ADSCs is stimulated by different doses of BMP4.

To investigate the effect of BMP4 on cardiomyocyte differentiation of adipose tissue-derived stem cells (ADSCs), mouse ADSCs were treated with different concentrations of BMP4 in media containing fetal bovine serum (FBS) or Knockout™ Serum Replacement (KoSR). 3 weeks after cardiac induction, differentiated ADSCs expressed some cardiac-specific genes and proteins. BMP4 treatment upregulated the expression of cardiac transcription factors. In both FBS and KoSR-supplemented media, lower concentrations of BMP4 had a positive effect on the expression of MLC2A gene, while MLC2V was more expressed with higher concentrations of BMP4. BMP4 treatment in KoSR supplemented medium was more efficient for cardiac induction. Supplementation of culture media with insulin-transferrin-selenium improved the expression of MLC2A gene. The results of this study indicated that BMP4 is important for cardiac differentiation of the ADSCs. However, BMP4 was not enough for structural and functional maturation of the ADSC-derived cardiomyocytes.

Structure of the small Dictyostelium discoideum myosin light chain MlcB provides insights into MyoB IQ motif recognition.

Dictyostelium discoideum MyoB is a class I myosin involved in the formation and retraction of membrane projections, cortical tension generation, membrane recycling, and phagosome maturation. The MyoB-specific, single-lobe EF-hand light chain MlcB binds the sole IQ motif of MyoB with submicromolar affinity in the absence and presence of Ca(2+). However, the structural features of this novel myosin light chain and its interaction with its cognate IQ motif remain uncharacterized. Here, we describe the NMR-derived solution structure of apoMlcB, which displays a globular four-helix bundle. Helix 1 adopts a unique orientation when compared with the apo states of the EF-hand calcium-binding proteins calmodulin, S100B, and calbindin D9k. NMR-based chemical shift perturbation mapping identified a hydrophobic MyoB IQ binding surface that involves amino acid residues in helices I and IV and the functional N-terminal Ca(2+) binding loop, a site that appears to be maintained when MlcB adopts the holo state. Complementary mutagenesis and binding studies indicated that residues Ile-701, Phe-705, and Trp-708 of the MyoB IQ motif are critical for recognition of MlcB, which together allowed the generation of a structural model of the apoMlcB-MyoB IQ complex. We conclude that the mode of IQ motif recognition by the novel single-lobe MlcB differs considerably from that of stereotypical bilobal light chains such as calmodulin.

The effect of DA-9701 on 5-hydroxytryptamine-induced contraction of feline esophageal smooth muscle cells.

Serotonin, or 5-hydroxytryptamine (5-HT), is a monoamine neurotransmitter found in blood platelets, the gastrointestinal (GI) tract, and the central nervous system (CNS) of animals and humans. The signaling pathways of 5-hydroxytryptamine (5-HT)-induced contractions in cat esophageal smooth muscle cell (ESMC)s have been identified, but the downstream components of the 5-HT signaling pathway remain unclear. DA-9701 is the standardized extract of the Pharbitis nil Choisy seed (Pharbitidis Semen, Convolvulaceae) and the root of Corydalis yahusuo W.T. Wang (Corydalis Tuber, Papaveraceae). DA-9701 is known to have strong gastroprokinetic effects and a good safety profile. In this study, we investigated the 5-HT signaling pathway at the G-protein level, and we explored the mechanisms by which DA-9701 induces smooth muscle contraction. Freshly isolated smooth muscle cells were harvested from the feline esophagus, and cells were permeabilized to measure their length. 5-HT produced esophageal smooth muscle contractions in a dose-dependent manner. Furthermore, 5-HT produced a relatively long-acting contraction. 5-HT binds to the 5-HT2, 5-HT3 and 5-HT4 receptors to induce smooth muscle contraction in feline ESMCs. These receptors, which are located in esophageal smooth muscle, are coupled to Gαq, Gαo and Gαs. These G proteins activate PLC, which leads to Ca2+/calmodulin-dependent MLCK activation, resulting in MLC20 phosphorylation and cell contraction. Conversely, DA-9701 inhibits 5-HT-induced contraction by inhibiting MLC20 phosphorylation.

Areca nut-induced buccal mucosa fibroblast contraction and its signaling: a potential role in oral submucous fibrosis--a precancer condition.

Betel quid (BQ) chewing is an oral habit that increases the risk of oral cancer and oral submucous fibrosis (OSF), a precancerous condition showing epithelial atrophy and tissue fibrosis. Persistent fibroblast contraction may induce the fibrotic contracture of tissue. In this study, we found that areca nut extract (ANE) (200-1200 µg/ml) stimulated buccal mucosa fibroblast (OMF)-populated collagen gel contraction. Arecoline but not arecaidine-two areca alkaloids, slightly induced the OMF contraction. Exogenous addition of carboxylesterase (2U/ml) prevented the arecoline- but not ANE-induced OMF contraction. OMF expressed inositol triphosphate (IP3) receptors. ANE-induced OMF (800 µg/ml) contraction was inhibited by U73122 [phospholipase C (PLC) inhibitor] and 2-aminoethoxydiphenyl borate (IP3 receptor antagonist), respectively. Ethylene glycol tetraacetic acid and verapamil, two calcium mobilization modulators, also suppressed the ANE-induced OMF contraction. ANE induced calcium/calmodulin kinase II and myosin light chain (MLC) phosphorylation in OMF. Moreover, W7 (a Ca(2+)/calmodulin inhibitor), HA1077 (Rho kinase inhibitor), ML-7 (MLC kinase inhibitor) and cytochalasin B (actin filament polymerization inhibitor) inhibited the ANE-induced OMF contraction. Although ANE elevated reactive oxygen species (ROS) level in OMF, catalase, superoxide dismutase and N-acetyl-L-cysteine showed no obvious effect on ANE-elicited OMF contraction. These results indicate that BQ chewing may affect the wound healing and fibrotic processes in OSF via inducing OMF contraction by ANE and areca alkaloids. AN components-induced OMF contraction was related to PLC/IP3/Ca(2+)/calmodulin and Rho signaling pathway as well as actin filament polymerization, but not solely due to ROS production.

Molecular and functional characterization of an Entamoeba histolytica protein (EhMLCI) with features of a myosin essential light chain.

Entamoeba histolytica, a protozoan parasite of humans, relays on its striking motility to survive and invade host tissues. Characterization of the molecular components involved in motile processes is crucial to understand its pathogenicity. Although protein components of myosin II hexamers have been predicted from E. histolytica genome data, only a heavy chain of myosin, EhmhcA, has been characterized so far. We have cloned an E. histolytica cDNA sequence that best matched Dictyostelium discoideum myosin essential light chain and found that the cloned sequence is transcribed as an mRNA of 0.445 kb which could encode a protein of 16.88 kDa, within the predicted range for a myosin light chain. In silico analyses revealed that the protein sequence, named EhMLCI, shows two consensus domains for binding MHC, but lacks the N-terminal sequence for actin binding, as in A2 type myosin essential light chains. A single EF-hand calcium-binding domain was identified in the C-terminus and several high score predictability sites for serine and tyrosine phosphorylation. Antibodies to recombinant EhMLCI identified two proteins of approximately 17 and 15 kDa in trophozoite extracts, the latter phophorylated in tyrosines. Serine phosphorylation was not detected. Immunomicroscopy revealed EhMLCI cortical and cytoplasmic distribution in trophozoites and true colocalization with EhmhcA determined by PCC. Co-immunoprecipitation corroborated EhMLCI interaction with EhmhcA. EhMLCI was also localized in actomyosin-containing complexes. Differential partition of phospho-tyrosinated EhMLCI into cell fractions containing the soluble form of EhmhcA and its lack of serine phosphorylation suggest its possible participation in a novel down regulatory mechanism of myosin II activity in E. histolytica.

Kinetic analysis reveals differences in the binding mechanism of calmodulin and calmodulin-like protein to the IQ motifs of myosin-10.

Myo10 is an unconventional myosin with important functions in filopodial motility, cell migration, and cell adhesion. The neck region of Myo10 contains three IQ motifs that bind calmodulin (CaM) or the tissue-restricted calmodulin-like protein (CLP) as light chains. However, little is known about the mechanism of light chain binding to the IQ motifs in Myo10. Binding of CaM and CLP to each IQ motif was assessed by nondenaturing gel electrophoresis and by stopped-flow experiments using fluorescence-labeled CaM and CLP. Although the binding kinetics are different in each case, there are similarities in the mechanism of binding of CaM and CLP to IQ1 and IQ2: for both IQ motifs Ca(2+) increased the binding affinity, mainly by increasing the rate of the forward steps. The general kinetic mechanism comprises a two-step process, which in some cases may involve the binding of a second IQ motif with lower affinity. For IQ3, however, the kinetics of CaM binding is very different from that of CLP. In both cases, binding in the absence of Ca(2+) is poor, and addition of Ca(2+) decreases the K(d) to below 10 nM. However, while the CaM binding kinetics are complex and best fitted by a multistep model, binding of CLP is fitted by a relatively simple two-step model. The results show that, in keeping with growing structural evidence, complexes between CaM or CaM-like myosin light chains and IQ motifs are highly diverse and depend on the specific sequence of the particular IQ motif as well as the light chain.

A plant-derived remedy for repair of infarcted heart.

BACKGROUND: Myocardial infarction (MI) due to coronary artery disease remains one of the leading causes of premature death. Replacement of infarcted heart tissue with regenerating myocardium from endogenous progenitor pools or exogenously introduced stem cells remains a therapeutic ideal. Their impracticality mainly lies in their low efficiency in cardiogenic differentiation (CD). Our recent studies with an acute MI animal model have already demonstrated the therapeutic effect of the MeOH extract of Geum japonicum (EGJ), providing clear evidence of myocardial regeneration. METHODS AND FINDINGS: The present study further isolated the active component contained in EGJ using bioassay-guided isolation and investigated its efficacy in the treatment of infarcted heart in animal MI models. We demonstrated that substantial repair of infarcted heart in animal MI models by EGJ can be mimicked by the isolated candidate compound (cardiogenin) in MI animal models. Clear evidence of newly regenerated endogenous mesenchymal stem cells (MSCs) derived cardiomyocytes was observed throughout the infarct zone, accompanied by significantly improved functional performance of the heart. Transplantation of MSCs pretreated with EGJ or cardiogenin into a MI animal model also resulted in substantial regeneration of functional myocardium, implying that the activated MSCs carry all the necessary blueprints for myocardial regeneration. Signaling pathways specific to cell survival, CD identified in embryonic heart induction and angiogenesis were activated in both cardiogenin-treated MSCs and cardiogenin-induced regenerating myocardium. CONCLUSIONS: This study has demonstrated the therapeutic effects of cardiogenin in infarcted heart repair, and identified the associated signalling pathways for effective cardiogenic differentiation of MSCs, cell survival and angiogenesis. These findings should enable new treatment strategies for MI to be developed immediately.

Icariin-mediated expression of cardiac genes and modulation of nitric oxide signaling pathway during differentiation of mouse embryonic stem cells into cardiomyocytes in vitro.

AIM: To investigate effects of icariin on cardiac gene expression and the modulation of nitric oxide (NO) signal transduction during the differentiation of embryonic stem (ES) cells into cardiomyocytes in vitro. METHODS: The expression levels of cardiac developmental-dependent genes were measured using reverse transcription-polymerase chain reaction (RT-PCR). The chronotropic responses of cardiomyocytes to b-adrenoceptor stimulation were determined. The levels of cAMP and cGMP in ES cells were measured using radioimmunoassay. Endogenous NO levels were measured by using the Griess reaction. Aminoguanidine (AG) was used to confirm the influence of icariin on the endogenous NO signal pathway. RESULTS: Icariin significantly elevated mRNA levels of cardiac transcription factors GATA4 and Nkx2.5, and cardiac-specific alpha-MHC, MLC-2v and beta-AR genes in a concentration- and time-dependent manner (P<0.05). Cardiomyocytes derived from embryoid body (EB) treated with icariin were more sensitive to isoprenaline (P<0.01). Treatment of ES cells with icariin resulted in a continued elevation in the cAMP/cGMP ratio before a shift to the cardiomyocyte phenotype (P<0.05). AG decreased the NO level, and delayed and decreased the incidence of contracting EB to only approximately 35% on d 5+11, an effect that could be rescued by icariin. When cells were cocultured with icariin and AG, the percentage of beating EB reached a peak level of 73% on d 5+11 (P<0.05). CONCLUSION: The inducible effects of icariin were partly related to increase in the expression of cardiac developmental-dependent genes, and elevation of the cAMP/cGMP ratio in ES cells, as well as upregulation of endogenous NO generation during the early stages of cardiac development.

Identification of a differential gene HUMMLC2B between F1 hybrids Landrace x Yorkshire and their female parents Yorkshire.

In order to investigate heterosis on a molecular basis, suppression subtractive hybridization was used to analyze the differences in gene expression between porcine F1 hybrids Landrace x Yorkshire and their female parents Yorkshire. From two specific subtractive cDNA libraries, the clones screened out by reverse Northern high-density blots screening were chosen to clone full-length cDNA by RACE. An expression-upregulated gene for Yorkshire skeletal muscle, designated as HUMMLC2B, was identified. Porcine HUMMLC2B contains an open reading frame (ORF) encoding 169 amino acids residues with 59 and 115 nucleotides in the 5' and 3' untranslated regions (UTRs), respectively. In the porcine genome, it contains seven exons separated by six introns. High allelic variations and four SINEs were detected in it. Comparison of derived amino acid sequence of HUMMLC2B with database sequences revealed highly conserved 12 amino acid residues in a putative calcium-binding region. RT-PCR analysis showed a tissue-specific pattern of expression in skeletal muscle and a similar level of expression during skeletal muscle development. The possible role of HUMMLC2B and its relation to porcine heterosis are discussed.

Inducible effects of icariin, icaritin, and desmethylicaritin on directional differentiation of embryonic stem cells into cardiomyocytes in vitro.

AIM: To investigate the possible inducible effects of icariin, icaritin, and desmethylicaritin on the directional differentiation of embryonic stem (ES) cells into cardiomyocytes in vitro. METHODS: ES cells were cultivated as embryoid bodies (EBs) in hanging drops with icariin, icaritin, or desmethylicaritin. ES cells treated with retinoic acid and with solvent were used as positive and negative controls, respectively. The cardiomyocytes derived from the ES cells were verified using immunocytochemistry. The expression of cardiac developmental-dependent genes was detected using the reverse transcription-polymerase chain reaction (RT-PCR) method. Cell cycle distribution and apoptosis were analyzed using flow cytometry to determine the partly inducible effect mechanisms involved. RESULTS: The total percentage of beating EBs treated with 10(-7) mol/L icariin, icaritin, or desmethylicaritin was 87% (P<0.01), 59% (P<0.01), and 49%, respectively. All the beating cardiomyocytes derived from the ES cells expressed cardiac-specific proteins for a-actinin and troponin T. Among them, 10(-7) mol/L icariin treatment resulted in a significantly advanced and increased mRNA level of a-cardiac major histocompatibility complex (MHC) and myosin light chain 2v (MLC-2v) in EBs in the early cardiac developmental stage. Before shifting to the cardiomyocyte phenotype, icariin could evoke the accumulation of ES cells in G0/G1 and accelerate apoptosis of the cell population (P<0.05). CONCLUSION: Icariin facilitated the directional differentiation of ES cells into cardiomyocytes at a concentration of 10(-7) mol/L. The promoting effect of icariin on cardiac differentiation was related to increasing and accelerating gene expression of a-cardiac MHC and MLC-2v, as well as regulating the cell cycles and inducing apoptosis.

Human fast skeletal myosin light chain 2 cDNA: isolation, tissue specific expression of the single copy gene, comparative sequence analysis of isoforms and evolutionary relationships.

A cDNA clone encoding human fast skeletal myosin regulatory light chain (HSRLC) has been isolated and characterized from a fetal muscle cDNA library. The cDNA contains the coding sequence of 170 amino acids (aa) and 58 and 91 nucleotides in the 5' and 3' untranslated regions (UTRs), respectively. HSRLC is encoded by a single copy gene in the human genome and shows a tissue-specific pattern of expression in skeletal muscle. Comparison of derived amino acid sequence of HSRLC with database sequences reveals highly conserved 12 amino acid residues in a putative calcium-binding region. HSRLC is unique among all RLC sequences in having three consecutive potential phosphorylatable serine residues. The Cys-129 of HSRLC corresponds to the critical Gly-117 of scallop RLC that is essential for its regulatory function. The clusters of hydrophobic residues that are believed to stabilize the binding of NH2-terminal of RLC with myosin heavy chain show high sequence conservation in RLCs. Besides identifying specific targets for functional studies of HSRLC by mutagenesis, the results support the concept of an ancestral gene from which the RLC genes have evolved.

Familial hypertrophic cardiomyopathy mutations in the regulatory light chains of myosin affect their structure, Ca2+ binding, and phosphorylation.

The effect of the familial hypertrophic cardiomyopathy mutations, A13T, F18L, E22K, R58Q, and P95A, found in the regulatory light chains of human cardiac myosin has been investigated. The results demonstrate that E22K and R58Q, located in the immediate extension of the helices flanking the regulatory light chain Ca(2+) binding site, had dramatically altered Ca(2+) binding properties. The K(Ca) value for E22K was decreased by approximately 17-fold compared with the wild-type light chain, and the R58Q mutant did not bind Ca(2+). Interestingly, Ca(2+) binding to the R58Q mutant was restored upon phosphorylation, whereas the E22K mutant could not be phosphorylated. In addition, the alpha-helical content of phosphorylated R58Q greatly increased with Ca(2+) binding. The A13T mutation, located near the phosphorylation site (Ser-15) of the human cardiac regulatory light chain, had 3-fold lower K(Ca) than wild-type light chain, whereas phosphorylation of this mutant increased the Ca(2+) affinity 6-fold. Whereas phosphorylation of wild-type light chain decreased its Ca(2+) affinity, the opposite was true for A13T. The alpha-helical content of the A13T mutant returned to the level of wild-type light chain upon phosphorylation. The phosphorylation and Ca(2+) binding properties of the regulatory light chain of human cardiac myosin are important for physiological function, and alteration any of these could contribute to the development of hypertrophic cardiomyopathy.

Myosin light chain replacement in the heart.

Myosin-actin cross-bridge kinetics are an important determinant for cardiac systolic and diastolic function. We compared the effects of myosin light chain substitutions on the ability of the fibers to contract in response to calcium and in their ability to produce power. Transgenesis was used to effect essentially complete replacement of the target contractile protein isoform specifically in the heart. Atrial and ventricular fibers derived from the various transgenic (TG) lines were skinned, and the force-velocity relationships, unloaded shortening velocities, and Ca(2+)-stimulated Mg(2+)-ATPase activities were determined. Replacement with an ectopic isoform resulted in significant changes in cross-bridge cycling kinetics but without any overt effects on morbidity or mortality. To confirm that this result was not light chain specific, a modified alpha-myosin heavy chain isoform that resulted in significant changes in force development was also engineered. The animals appeared healthy and have normal lifespans, and the changes in force development did not result in significant remodeling or overt hypertrophy. We conclude that myosin light chains can control aspects of cross-bridge cycling and alter force development. The myosin heavy chain data also show that changes in the kinetics of force development and power output do not necessarily lead to activation of the hypertrophic response or significant cardiac remodeling.