AIMTo detect effect of the different frequency of chronic electrical stimulation (CES) on myofibrillar isoform, myosin heavy chain (MHC) and metabolic enzyme activities.

METHODSThe histochemical method and SDS-polyacrylamide gel electrophoresis were respectively employed.

RESULTS(1)There were a significant increase in I myo-fibrillar isoform and I MHC isoform and decrease in II B myofibrillar isoform and II B MHC isoforms in the chronic low frequency electrical stimulation (CLFES) 10 Hz and 20 Hz groups, but opposite results were found in the chronic high frequency electrical stimulation (CHFES) 50 Hz and 100 Hz groups. (2) There were a significant increase in the aerobic-oxidative enzyme activities and capacity, and a concomitant significant drop in glycolysis enzyme activities in CLFES groups, but opposite results were found in CHFES 50 Hz and 100 Hz groups.

CONCLUSIONIt was suggested that there was a significant dependent relation between chronic electrical stimulation frequency and myofibrilla isoforms, myosin heavy chain (MHC) and metabolic enzyme activities.

Adaptation, Physiological ; Animals ; Diaphragm ; enzymology ; metabolism ; physiology ; Electric Stimulation ; Muscle Contraction ; Myosin Heavy Chains ; metabolism ; Nonmuscle Myosin Type IIB ; metabolism ; Protein Isoforms ; Rabbits

Myosin light chain 9 (MYL9) is a regulatory light chain of myosin, which plays an important role in various biological processes including cell contraction, proliferation and invasion. MYL9 expresses abnormally in several malignancies including lung cancer, breast cancer, prostate cancer, malignant melanoma and others, which is closely related to the poor prognosis, but the clinical significance for its expression varies with different types of cancer tissues. Further elucidating the molecular mechanism of MYL9 in various types of malignant tumor metastasis is of great significance for cancer prevention and treatment. At the same time, as a molecular marker and potential target, MYL9 may have great clinical value in the early diagnosis, prognosis prediction, and targeted treatment of malignant tumors.
Biomarkers ; Humans ; Lung Neoplasms ; Male ; Myosin Light Chains/metabolism* ; Prognosis ; Prostatic Neoplasms

OBJECTIVETo study the role of myosin light chain kinase (MLCK) in intestinal epithelial barrier dysfunction after hypoxia.

METHODSThe Caco-2 monolayers developed with Transwell inserts were exposed to hypoxia for 0 h (NC group), 2, 6, 8, 12 and 24 h (H group), and 6 h hypoxic specimens were treated with 100 mol/L ML-9 (T group). The transepithelial electrical resistance (TER) of monolayers was measured with an ohmmeter. The tight junction protein ZO-1 of monolayers was analyzed by immunofluorescence assay. The protein expressions of phosphorylated myosin light chain (p-MLC) and MLCK were detected by Western blotting.

RESULTSThe TER of monolayers in H group at 6, 8, 12 and 24 h was 422 +/- 17, 427 +/- 27, 403 +/- 40 and 426 +/- 22 ohms respectively, which was significantly lower than that of NC group (451 +/- 27 ohms, P < 0.05). The TER of monolayers in T group was 558 +/- 110 ohms, which was significantly higher than that in H group at each time point ( P < 0.01). The ZO-1 of monolayers in H group at 6 h was irregular in arrangement, with interruptions and rugae, and sawtooth. These abnormalities were ameliorated in T group (regular in arrangement, with little or without ruga and sawtooth). The protein expressions of p-MLC and MLCK in H group at each time point were higher than those in NC group.

CONCLUSIONSIntestinal epithelial barrier dysfunction after hypoxia can be mediated by MLCK.

Caco-2 Cells ; Epithelium ; metabolism ; physiopathology ; Humans ; Hypoxia ; metabolism ; physiopathology ; Intestinal Absorption ; Intestinal Mucosa ; metabolism ; physiopathology ; Intestines ; cytology ; metabolism ; physiopathology ; Myosin Light Chains ; metabolism ; Myosin-Light-Chain Kinase ; metabolism

BackgroundOutflow tract (OFT) septation defects are a common cause of congenital heart disease. Numerous studies have focused on the septation mechanism of the OFT, but have reported inconsistent conclusions. This study, therefore, aimed to investigate the septation of the aortic sac and the OFT in the early embryonic human heart.

MethodsSerial sections of 27 human embryonic hearts from Carnegie stage (CS) 10 to CS19 were immunohistochemically stained with antibodies against α-smooth muscle actin (α-SMA) and myosin heavy chain.

ResultsAt CS10-CS11, the OFT wall was an exclusively myocardial structure that was continuous with the aortic sac at the margin of the pericardial cavity. From CS13 onward, the OFT was divided into nonmyocardial and myocardial portions. The cushion formed gradually, and its distal border with the OFT myocardium was consistently maintained. The aortic sac between the fourth and sixth aortic arch arteries was degenerated. At CS16, the α-SMA-positive aortopulmonary septum formed and fused with the two OFT cushions, thus septating the nonmyocardial portion of the OFT into two arteries. At this stage, the cushions were not fused. At CS19, the bilateral cushions were fused to septate the myocardial portion of the OFT.

ConclusionsData suggest that the OFT cushion is formed before the aortopulmonary septum is formed. Thus, the OFT cushion is not derived from the aortopulmonary septum. In addition, the nonmyocardial part of the OFT is septated into the aorta and pulmonary trunk by the aortopulmonary septum, while the main part of the cushion fuses and septates the myocardial portion of the OFT.

Actins ; metabolism ; Alkaline Phosphatase ; metabolism ; Aorta ; embryology ; Heart ; embryology ; Heart Valves ; embryology ; Humans ; Immunohistochemistry ; Myosin Heavy Chains ; metabolism

The signal transduction pathways within corporal smooth muscle cells are the intracellular molecular mechanisms of corporal smooth muscle tone regulation. Various neurotransmitters activate the membrane receptor proteins or intracellular enzyme pathways and result in the production of extracellular chemical signals. Second messenger molecules and ions transmit and amplify the signals, and subsequently induce the relaxation of smooth muscle cells and penile erection. Therefore, the study of signal transduction in cavernous smooth muscle play an important role in understanding the physiology of erection and the pathophysiology of erectile dysfunction as well as in developing new selective drugs for the treatment of erectile dysfunction.
Calcium ; metabolism ; Humans ; Male ; Muscle, Smooth ; metabolism ; Myosin-Light-Chain Kinase ; physiology ; Penis ; metabolism ; Signal Transduction ; physiology

OBJECTIVETo compare the effect of high frequency oscillatory ventilation (HFOV) and conventional mandatory ventilation (CMV) on the myocardial function of rabbits with inhalation injury.

METHODSSteam inhalation injury model was reproduced in 16 New Zealand albino rabbits. They were randomly divided into CMV group (n = 8) and HFOV group (n = 8) by drawing lots, and they received ventilation in metered volume and HFOV treatment respectively. Heart blood was drawn from rabbits before they were sacrificed 4 hours after treatment to determine the plasma activity of lactate dehydrogenase 1 (LDH1) and creatine phosphorylated kinase (CPK-MB). Myocardial tissue from left ventricle was harvested and homogenized to determine the concentration of TNF-α and IL-8, the activity of caspase-1, and the activity of myosin-light-chain kinase (MLCK) and the ATPase of myosin light chain (MLC-ATPase) by enzyme-linked immunosorbent assay, spectrophotometry, and the nuclide liquid scintillation technique respectively. Part of the myocardial tissue sample was examined pathologically. Data were processed with analysis of variance.

RESULTS(1) The activities of LDH1 and CPK-MB in plasma were obviously higher in CMV group than in HFOV group [(643 ± 108), (342 ± 48) U vs. (233 ± 92), (186 ± 36) U, with F value respectively 10.326 and 9.846, P values all below 0.01]. (2) The contents of TNF-α, IL-8 and the activity of caspase-1 in myocardial tissue homogenate were obviously higher in CMV group than in HFOV group [(181 ± 35), (89 ± 19) pg/g, and (0.56 ± 0.27) g/g protein vs. (94 ± 21), (43 ± 11) pg/g, and (0.24 ± 0.12) g/g protein, with F value respectively 8.239, 7.826, 5.716, P values all below 0.01]. (3) The activities of MLC-ATPase and MLCK were lower in CMV group than in HFOV group [(0.24 ± 0.12) µmol×mg(-1)×min(-1), (3.3 ± 1.1) mmol×mg(-1)×min(-1) vs. (0.48 ± 0.16) µmol×mg(-1)×min(-1), (7.7 ± 1.7) mmol×mg(-1)×min(-1), with F value respectively 4.125, 4.766, P values all below 0.01]. (4) No obvious necrosis, degeneration or inflammatory cell infiltration was observed in myocardial tissue of rabbits in 2 groups under light microscope; but the myocardial fiber was slightly swollen, and it was less marked in the HFOV group.

CONCLUSIONSThe influence of HFOV on myocardial myosin phosphorylation system of rabbits with inhalation injury is less than that of CMV.

Animals ; Burns, Inhalation ; metabolism ; physiopathology ; therapy ; High-Frequency Ventilation ; Myocardium ; metabolism ; Myosin-Light-Chain Kinase ; metabolism ; Rabbits ; Respiration, Artificial

The study was purposed to investigate the expression and function of non-muscle myosin heavy chain-IIA (NMMHC-IIA) in Fechtner syndrome in order to explore the pathologic changes of kindy disease and the mechanism of granulocyte inclusion body formation. NMMHC-IIA levels in granulocytes were analyzed by Western-blot, the expressions of NMMHC-IIA, IIB in HEK-293 cells were detected by RT-PCR and were analyzed by co-immunoprecipitation. The results indicated that the IIA/beta-actin ratio for Fechtner syndrome granulocytes was (0.35 +/- 0.12), and obviously decreased as compared with that of normal control (0.87 +/- 0.18) (p < 0.01). The IIA and IIB expressed higher in HEK-293 cells. The interaction of IIA and IIB was confirmed by co-immunoprecipitation in HEK-293 cells. It is concluded that dominant-negative effect of NMMHC-IIA is involved in the formation of inclusion bodies. IIA and IIB show obvious interaction, IIB partly compensates the IIA defect derived from MYH9 mutations, and may delay or prevent the development of clinically relevant abnormalities.
Blood Platelet Disorders ; genetics ; metabolism ; pathology ; Cell Line ; Granulocytes ; pathology ; Humans ; Inclusion Bodies ; pathology ; Kidney ; cytology ; embryology ; metabolism ; Mutation ; Nonmuscle Myosin Type IIA ; genetics ; metabolism ; physiology ; Nonmuscle Myosin Type IIB ; genetics ; metabolism ; physiology ; Syndrome ; Thrombocytopenia ; genetics ; metabolism ; pathology

After a series of studies, we found that the intestinal permeability was increased, tight junction protein (zonula occluden-1) obviously decreased and redistributed, accompanied by an increase in expression of myosin light chain (MLC) phosphorylation in severely burned rats. After using inhibitor of MLC kinase (ML-9 2 mg/kg) or of Rho-associated kinase (Y-27632 2 mg/kg), above-mentioned changes could be alleviated. Therefore, to regulate the MLC phosphorylation of tight junction protein and perijunctional actin-myosin ring may be one of the key links to lessen the intestinal epithelium permeability after burn injury.
Animals ; Burns ; metabolism ; Intestinal Mucosa ; metabolism ; Intestines ; metabolism ; Membrane Proteins ; metabolism ; Myosin Light Chains ; metabolism ; Permeability ; Phosphoproteins ; metabolism ; Phosphorylation ; Rats ; Zonula Occludens-1 Protein ; rho-Associated Kinases ; metabolism

OBJECTIVETo investigate the effect of combination of interferon-gamma (IFN-γ) and tumor necrosis factor-alpha (TNF-α) on intestinal epithelial barrier function.

METHODSThe Caco-2 monolayers were cultured in DMEM nutrient solution, and then they were inoculated in 24-well or 6-well plate with Transwell inserts.They were divided into control group (ordinary treatment), IFN-γ group (with addition of 10 ng/mL IFN-γ), TNF-α group (with addition of 10 ng/mL TNF-α), and IFN-γ plus TNF-α group (with addition of 10 ng/mL TNF-α and 10 ng/mL IFN-γ). Monolayers inoculated in 24-well plate were collected for determination of transepithelial electrical resistance (TER) with an ohmmeter at post treatment hour (PTH) 0, 6, 12, 24, 36, and 48, the permeability of monolayers with fluorescein isothiocyanate-labeled dextran (FITC-dextran) tracer method at PTH 48, the distribution and morphological change of tight junction occludin with immunofluorescence assay at PTH 48. Monolayers inoculated in 6-well plate were collected for determination of protein expression of occludin, myosin light chain kinase (MLCK), and phosphorylated MLC (pMLC) with Western blot at PTH 24. Data were processed with one-way analysis of variance and t test.

RESULTS(1) There was no obvious difference in TER in control group at each time point (F = 0.86, P > 0.05). TER in IFN-γ group and TNF-α group were gradually decreased during PTH 6-48, but showed no statistical difference as compared with that at PTH 0 (with F value respectively 1.69, 2.47, P values all above 0.05). TER in IFN-γ plus TNF-α group was significantly decreased from PTH 24 as compared with that at PTH 0 (t = 4.97, P < 0.05) and that in each of the other three groups (F = 11.54, P < 0.05). (2) The permeability of monolayers in IFN-γ plus TNF-α group [(1197 ± 215)pmol] was significantly higher than that in control group, IFN-γ group, and TNF-α group [(303 ± 93), (328 ± 76), (797 ± 177) pmol, with t value respectively 4.8, 5.0, 6.9, P values all below 0.01]. (3) There was no statistical difference in occludin expression at PTH 24 among four groups (F = 0.26, P > 0.05). The occludin in control group at PTH 48 was regular in arrangement, while that in IFN-γ and TNF-α groups was irregular in arrangement. The arrangement of occludin in IFN-γ plus TNF-α group at PTH 48 was interrupted, with obvious redistribution in cytoplasm. (4) The protein expression of pMLC in IFN-γ plus TNF-α group (0.95 ± 0.05) was significantly higher than that in control group, IFN-γ group, or TNF-α group (0.57 ± 0.12, 0.56 ± 0.07, 0.59 ± 0.10, respectively, F = 17.97, P < 0.01). The protein expression of MLCK in IFN-γ plus TNF-α group (1.57 ± 0.36) was also significantly higher than that in control, IFN-γ, TNF-α groups (0.85 ± 0.18, 1.04 ± 0.23, 1.00 ± 0.07, respectively, F = 9.05, P < 0.05).

CONCLUSIONSCombination of IFN-γ and TNF-α can induce intestinal epithelial barrier dysfunction by up-regulating MLCK protein expression and promoting MLC phosphorylation.

Caco-2 Cells ; Epithelial Cells ; drug effects ; metabolism ; Humans ; Interferon-gamma ; pharmacology ; Intestinal Mucosa ; cytology ; physiopathology ; Membrane Proteins ; metabolism ; Myosin Light Chains ; metabolism ; Myosin-Light-Chain Kinase ; metabolism ; Occludin ; Tumor Necrosis Factor-alpha ; pharmacology

OBJECTIVE: To investigate the expression of myosin heavy chain (MHC) in normal laryngeal muscle and the difference between the adductor and abductor. METHOD: Seven patients with total laryngectomy were enrolled in this study. The adductor muscles were acquired from the lateral cricoarytenoid (LCA) muscle and the abductor muscles were acquired from the posterior cricoarytenoid (PCA) muscle. The expression of myosin heavy chain were detected with fluorescent quantitative reverse transcription polymerase chain reaction (RT-PCR) and immunofluorescence staining respectively. RESULT: (1) MHC-II b was expressed in laryngeal muscles at mRNA levels, and not expressed at the protein level; (2) At both mRNA level and protein level, the expression of MHC-I was higher in the PCA muscles than in the LCA muscles while MHC-II level was higher in the LCA muscles than in the PCA muscles. CONCLUSION (1) MHC-II b protein was not expressed in human laryngeal muscles; (2) Phenotypic differences were significant in laryngeal adductor and abductor muscles based on their different functions. PCA contained larger percentage of MHC-I fibers, while LCA contained more MHC-II fibers.
Aged ; Female ; Humans ; Laryngeal Muscles ; chemistry ; metabolism ; Male ; Middle Aged ; Myosin Heavy Chains ; genetics ; metabolism ; Phenotype ; Protein Isoforms