Cyclic ADP-ribose (cADPR) releases Ca²⁺ from ryanodine receptor (RyR)-sensitive calcium pools in various cell types. In cardiac myocytes, the physiological levels of cADPR transiently increase the amplitude and frequency of Ca²⁺ (that is, a rapid increase and decrease of calcium within one second) during the cardiac action potential. In this study, we demonstrated that cADPR levels higher than physiological levels induce a slow and gradual increase in the resting intracellular Ca²⁺ ([Ca²⁺](i)) level over 10 min by inhibiting the sarcoendoplasmic reticulum Ca²⁺ ATPase (SERCA). Higher cADPR levels mediate the tyrosine-dephosphorylation of α-actin by protein tyrosine phosphatase 1B (PTP1B) present in the endoplasmic reticulum. The tyrosine dephosphorylation of α-actin dissociates phospholamban, the key regulator of SERCA, from α-actin and results in SERCA inhibition. The disruption of the integrity of α-actin by cytochalasin B and the inhibition of α-actin tyrosine dephosphorylation by a PTP1B inhibitor block cADPR-mediated Ca²⁺ increase. Our results suggest that levels of cADPR that are relatively higher than normal physiological levels modify calcium homeostasis through the dephosphorylation of α-actin by PTB1B and the subsequent inhibition of SERCA in cardiac myocytes.
Action Potentials ; Adenosine Diphosphate* ; Adenosine Triphosphatases ; Calcium ; Cyclic ADP-Ribose ; Cytochalasin B ; Endoplasmic Reticulum ; Homeostasis ; Muscle Cells* ; Myocytes, Cardiac ; Protein Tyrosine Phosphatase, Non-Receptor Type 1* ; Protein Tyrosine Phosphatases* ; Reticulum ; Ryanodine Receptor Calcium Release Channel ; Tyrosine

OBJECTIVETo investigate the clinical, pathological and genetic characteristics in a family with autosomal dominant Emery-Dreifuss muscular dystrophy (AD-EDMD).

METHODSClinical data and skeletal muscle specimens were collected from two patients (the proband and her daughter) for pathological analysis. DNA samples of the proband and her family members (7 persons from 3 generations) were obtained for PCR amplification and direct DNA sequencing of the lamin A/C (LMNA) gene. Haplotype analysis was performed after the identification of mutation.

RESULTSThe proband had typical clinical manifestation of EDMD: joint contracture, progressive muscle weakness and atrophy and cardiac conduction dysfunction. Muscular pathology revealed myopathic changes combined with slight neuropathic changes. A heterozygous missense mutation 1583 (C to G)(T528R) was identified in exon 9 of the LMNA gene in the two patients, but not in other family members. Haplotype analysis indicated that the proband and her daughter shared the same causative haplotype.

CONCLUSIONThis is the first report of the phenotype and genotype of AD-EDMD in Chinese.

Adult ; Asian Continental Ancestry Group ; genetics ; Base Sequence ; Child ; DNA Mutational Analysis ; Female ; Haplotypes ; genetics ; Heterozygote ; Humans ; Immunohistochemistry ; Male ; Muscular Dystrophy, Emery-Dreifuss ; genetics ; metabolism ; pathology ; physiopathology ; Mutation ; Pedigree ; Phenotype

The identification of five marine-derived shell traditional Chinese medicine (TCM) recorded in the Chinese Pharmacopoeia were studied. Using near infrared technology (NIR) combined with principal component analysis (PCA) methods, Ostreae Concha, Haliotidis Concha, and Margaritifera Concha could be efficiently distinguished from Meretricis Concha together with Arcae Concha. In the first principal components, Ostreae Concha exhibited obvious differences with high loadings in 4 236, 5 263, 7 142 cm(-1) concerning to the contents of CaCO3 and H2O in the samples. Arcae Concha and Meretricis Concha displayed significant differences with others in the second principal components, which can be illustrated by high loadings in 5 000 -4 430 cm(-1) areas. It is indicated that the second principal components might be related to organics which contained NH and CH groups, for example proteins. Meanwhile, our data showed a correlation between the function of these shell TCM and their distribution in the PCA plot. These results suggested that organic components in marine-derived shell TCM could not be neglected for their quality control.
Animal Shells ; chemistry ; Animals ; Calcium Carbonate ; analysis ; Medicine, Chinese Traditional ; methods ; Mollusca ; chemistry ; classification ; Principal Component Analysis ; Seawater ; Species Specificity ; Spectroscopy, Near-Infrared ; methods

OBJECTIVEThe effect of vascular endothelial growth factor(165) (VEGF(165)) on intracellular free magnesium ([Mg(2+)](i)) and the relationship between Mg(2+) and angiogenesis in human umbilical vein endothelial cells (HUVECs) were investigated in this study.

METHODS[Mg(2+)](i) in HUVECs loaded with fluorescent magnesium indicator mag-fura-2 were quantitatively detected with the use of intracellular cation measurement system. HUVECs were obtained from normal fetus and cultured in M199 with 0.2 fetal bovine serum. The angiogenesis effects of VEGF(165) were observed in presence of 0 mmol/L, 1 mmol/L or 2 mmol/L of extracellular Mg(2+).

RESULTSVEGF(165) significantly increased [Mg(2+)](i) in a dose-dependent manner independent of extracellular Mg(2+), Na(+) and Ca(2+) and this effect could be blocked by pretreatment with VEGF(165) receptor-2 (KDR) inhibitor (SU1498). The angiogenesis induced by VEGF(165) was significantly inhibited cells with 0 mmol/L extracellular Mg(2+), the angiogenesis effects of VEGF(165) were similar in cells with 1 mmol/L and 2 mmol/L extracellular Mg(2+) and these effects could be blocked by SU1498.

CONCLUSIONSThese results suggest that the [Mg(2+)](i) increase induced by VEGF(165) originates from intracellular Mg(2+) pools and promotes angiogenesis via KDR-dependent signaling pathways.

Cations, Divalent ; Cells, Cultured ; Endothelial Cells ; metabolism ; Humans ; Magnesium ; metabolism ; Neovascularization, Physiologic ; Signal Transduction ; Vascular Endothelial Growth Factor A ; metabolism ; Vascular Endothelial Growth Factor Receptor-2 ; metabolism

Objective To investigate the clinical efficacy of electrothermal acupuncture plus joint mobilization in treating knee osteoarthritis.Methods One hundred patients with knee osteoarthritis were randomized to treatment and control groups, 50 cases each. The control group received Maitland's technique and the treatment group, electrothermal acupuncture in addition. The Osteoarthritis Index score was recorded and cytokines (IL-1, IL-6 and TNF-α) contents were measured in the two groups before and after four weeks of treatment. The clinical therapeutic effects were compared between the two groups.Results The total efficacy rate was 91.5％ in the treatment group and 80.0％ in the control group; there was a statistically significant difference between the two groups (P<0.05). There were statistically significant pre-/post-treatment differences in the WOMAC score and cytokines in the two groups (P<0.01,P<0.05). There were statistically significant post-treatment differences in the WOMAC score and cytokines between the treatment and control groups (P<0.05).Conclusion Electrothermal acupuncture plus joint mobilization is an effective way to treat knee osteoarthritis.

AIMThe mechanism of vascular endothelial growth factor165 (VEGF165) on intracellular free magnesium ([Mg2+]i) in human umbilical vein endothelial cells (HUVECs) was investigated.

METHODS[Mg2+]i in HUVECs loaded with fluorescent magnesium indicator mag-fura-2 were quantitatively detected the use of intracellular cation measurement system.

RESULTSVEGF165 significantly increased [Mg2+]i in the extracellular Mg2+ and this effect could be blocked by pretreatment with tyrosine kinase inhibitors (tyrphostin A23 and genistein), phosphatidylinositol 3-kinase (PI3K) inhibitors (wortmannin and LY294002) and phospholipase Cgamma (PLCgamma) inhibitor (U73122). In contrast, phospholipase Cgamma (PLCgamma) inhibitor analog (U73343), mitogen-activated protein kinase inhibitors (SB202190 and PD98059) had no effect on the VEGF165-induced [Mg2+]i increase.

CONCLUSIONThe increase of [Mg2+]i by VEGF165 originates from intracellular Mg2+ pool through tyrosine kinase/ PI3K/PLCgamma-dependent signaling pathways.

Cells, Cultured ; Human Umbilical Vein Endothelial Cells ; cytology ; metabolism ; physiology ; Humans ; Magnesium ; metabolism ; Neovascularization, Physiologic ; drug effects ; Phosphatidylinositol 3-Kinases ; metabolism ; Phospholipase C gamma ; metabolism ; Protein-Tyrosine Kinases ; metabolism ; Signal Transduction ; Vascular Endothelial Growth Factor A ; physiology

In this report, we study the effects of over-expression of Lin28a and Lin28b on let-7 family activity in HeLaS3. Firstly, we constructed pAAV2neo-Lin28a and pAAV2neo-Lin28b to express Lin28a and Lin28b, respectively. Then, pAAV2neo-Lin28a and pAAV2neo-Lin28b were transfected into HeLaS3, selected with G418 and obtained cell lines, HeLaS3/pAAV2neo-Lin28a and HeLaS3/pAAV2neo-Lin28b, to express Lin28a and Lin28b stably. Thereafter, we constructed eight plasmid vectors for detection of let-7 family activity based on pAAV2neo-Gluc-(Fluc). These vectors were further packaged into recombinant adeno-associated viral vectors (rAAV) which were used as sensors, nominated as Asensors, to detect inhibition activity of miRNA at post-transcriptional level. Subsequently, with HeLaS3 as a control, we assayed expression levels of Lin28a and Lin28b by Western blot, detected expression levels of let-7 family by QRT-PCR, and tested let-7 family activity by Asensors in HeLaS3/pAAV2neo-Lin28a and HeLaS3/pAAV2neo-Lin28b. Results demonstrated that both HeLaS3/pAAV2neo-Lin28a and HeLaS3/pAAV2neo-Lin28b could express Lin28a and Lin28b effectively. Compared with HeLaS3, the expression level of let-7 family except let-7e declined in HeLaS3/pAAV2neo-Lin28a. But declining extent among members of let-7 family was different. The let-7 family activity also decreased while the decreasing extent varied among members. Furthermore, the activity level was not consistent with its expression level for the same member in let-7 family. Compared with HeLaS3, both expression level and activity level of let-7 family in HeLaS3/ pAAV2neo-Lin28b were decreased. However, the decreasing extent of let-7 family expression changes was larger than that of HeLaS3/pAAV2neo-Lin28a while the decreasing extent of activity changes was similar. In this study, we established a method to detect and compare post-transcriptional inhibition level mediated by miRNA complementary targets. We firstly clarified the effect of Lin28a and Lin28b on let-7 family activity profile and found that this effect was not the same as that at expression level of let-7 family, suggesting that it was more comprehensive to understand miRNA regulation roles to detect both miRNA expression and activity. This paves a way for further research on mechanism of regulation of let-7 family.
Cell Line, Tumor ; DNA-Binding Proteins ; genetics ; metabolism ; HeLa Cells ; Humans ; MicroRNAs ; genetics ; metabolism ; Protein Processing, Post-Translational ; genetics ; RNA-Binding Proteins

OBJECTIVETo analyze the spatial distribution of hemorrhagic fever with renal syndrome (HFRS) in Haidian district, Beijing and to explore the geographical characteristics of HFRS in highly endemic areas.

METHODSAdministration boundary layer was established under the background of 1:100,000 map in the ArcInfo 8.1 software. The HFRS cases from 1997 to 2002 were positioned on the map. Highly endemic areas were identified by spatial cluster analysis using SaTScan 3.0 software. Distribution of HFRS cases was shown in different colors and contours by spatial analysis of geographic information system (GIS).

RESULTSSpatial Cluster Analysis of confirmed cases of HFRS identified in 1997 - 2002 in Haidian district showed that HFRS patients were not randomly distributed. The highly endemic areas were founded in Sujiatuo township, Yongfeng township, Shangzhuang township, Wenquan town and Bei'anhe township (relative risk = 4.43, P = 0.001). A thematic map of HFRS in haidian district was set up.

CONCLUSIONHFRS infections were not randomly distributed, since the distribution was related to geographic-environmental factors.

China ; epidemiology ; Hemorrhagic Fever with Renal Syndrome ; epidemiology ; Humans ; Urban Health ; statistics & numerical data ; Urbanization

Gap junction protein, connexin, is expressed in endothelial cells of vessels, glomerulus, and renin secreting cells of the kidney. The purpose of this study was to investigate the role of gap junction in renin secretion and its underlying mechanisms using As 4.1 cell line, a renin-expressing clonal cell line. Renin release was increased proportionately to incubation time. The specific gap junction inhibitor, 18-beta glycyrrhetinic acid (GA) increased renin release in dose-dependent and time- dependent manners. Heptanol and octanol, gap junction blockers, also increased renin release, which were less potent than GA. GA-stimulated renin release was attenuated by pretreatment of the cells with amiloride, nifedipine, ryanodine, and thapsigargin. GA dose-dependently increased intracellular Ca2+ concentration, which was attenuated by nifedipine, nimodipine, ryanodine, and thapsigargin. However, RP-cAMP, chelerythrine, tyrphostin A23, or phenylarsine oxide did not induced any significant change in GA-stimulated increase of intracellular Ca2+ concentration. These results suggest that gap junction plays an important role on the regulation of renin release and intracellular Ca2+ concentration in As 4.1 cells.
Amiloride ; Calcium* ; Cell Line* ; Connexins ; Endothelial Cells ; Gap Junctions* ; Glycyrrhetinic Acid ; Heptanol ; Kidney ; Nifedipine ; Nimodipine ; Renin* ; Ryanodine ; Thapsigargin

BACKGROUND: Increasing evidence has shown that visit-to-visit variability (VVV) of blood pressure (BP) is associated with an increased risk of cardiovascular disease (CVD). The objective of this study was to evaluate the impact of VVV of systolic blood pressure (SBP) and diastolic blood pressure (DBP) on the risk of CVD among patients with type 2 diabetes mellitus (T2DM) in China. METHODS: We conducted a retrospective cohort study of 10,163 T2DM patients who were not previously diagnosed with CVD from January 2008 to December 2012 in Ningbo, China. The VVV of BP was calculated using five metrics, including standard deviation (SD), coefficient of variation (CV), variation independent of mean, average real variability, and successive variability (SV) of measurements, obtained over a 24-month measurement period. Hazard ratios and 95% confidence intervals (CIs) were estimated by Cox proportional hazards regression models for the associations of variability in BP with risk of CVD. RESULTS: A total of 894 CVD events were observed during a median follow-up of 49.5 months. The hazard ratio in the highest quintile of SD of SBP was 1.24 (95% CI, 1.01 to 1.52) compared with patients in the lowest quintile. The association between higher VVV of DBP and risk of CVD was not consistent across different metrics and sensitivity analyses. CONCLUSION: Higher VVV of SBP was associated with an increased risk of CVD, irrespective of the mean SBP level. Future studies are needed to confirm these findings.
Blood Pressure ; Cardiovascular Diseases ; China ; Cohort Studies ; Diabetes Mellitus, Type 2 ; Follow-Up Studies ; Humans ; Retrospective Studies