[Analysis and Modeling of Risk Factors for Children with Intestinal Acute Graft-Versus-Host Disease after Allogeneic Hematopoietic Stem Cell Transplantation].

OBJECTIVE: To analyze factors associated with intestinal acute graft-versus-host disease (aGVHD) after allogeneic hematopoietic stem cell transplantation(allo-HSCT) in children and to develop a prediction model for intestinal aGVHD after allo-HSCT in children. METHODS: The clinical data of 62 children who underwent allo-HSCT at the Department of Hematology of the People's Hospital of Xinjiang Uygur Autonomous Region from February 2018 to September 2021 were retrospectively analyzed. Intestinal aGVHD was evaluated according to the Mount Sinai Acute GVHD International Consortium (MAGIC) grading criteria, the variables were screened by LASSO (least absolute shrinkage and selection operator) regression analysis with 10-fold cross-validation, and developed a model for predicting intestinal aGVHD after allo-HSCT in children. RESULTS: A total of 33 (53.2%) of the 62 children developed intestinal aGVHD, of which 25 were degree II and 8 were degree III-IV. The results of screening variables by 10-fold cross-validated LASSO regression showed that the significant variables included ethnic minorities (OR =7.229; 95%CI: 2.337-22.354), platelet (PLT) (OR =0.971; 95%CI: 0.932-0.993), uric acid (UA) (OR =0.971; 95%CI: 0.935-0.988), C-reactive protein (CRP) (OR =1.217; 95%CI: 1.053-1.545), and viral infection (OR =10; 95%CI: 3.021-32.668), and these variables were independently associated with intestinal aGVHD in children (all P <0.05). A prediction model was constructed based on above variables. The area under the receiver operating characteristic (ROC) curve (AUC) of the model was calculated, and the AUC value was 0.985 (0.966-1), the Brier score was 0.055. The evaluation showed that the model has a high degree of discrimination and calibration. CONCLUSION: Ethnic minorities, low PLT, low UA, high CRP, and viral infections are independently associated with intestinal aGVHD in children, and early attention should be paid to these high-risk children.

Precise Differentiation of Wobble-Type Allele via Ratiometric Design of a Ligase Chain Reaction-Based Electrochemical Biosensor for CYP2C19*2 Genotyping of Clinical Samples.

Due to the comparable stability between the perfect-base pair and the wobble-base pair, a precise differentiation of the wobble-type allele has remained a challenge, often leading to false results. Herein, we proposed a ligase chain reaction (LCR)-based ratiometric electrochemical DNA sensor, namely, R-eLCR, for a precise typing of the wobble-type allele, in which the traditionally recognized "negative" signal of wobble-base pair-mediated amplification was fully utilized as a "positive" one and a ratiometric readout mode was employed to ameliorated the underlying potential external influence and improved its detection accuracy in the typing of the wobble-type allele. The results showed that the ratio between current of methylene blue (IMB) and current of ferrocene (IFc) was partitioned in three regions and three types of wobble-type allele were thus precisely differentiated (AA homozygote: IMB/IFc > 2; GG homozygote: IMB/IFc < 1; GA heterozygote: 1 < IMB/IFc < 2); the proposed R-eLCR successfully discriminated the three types of CYP2C19*2 allele in nine cases of human whole blood samples, which was consistent with those of the sequencing method. These results evidence that the proposed R-eLCR can serve as an accurate and robust alternative for the identification of wobble-type allele, which lays a solid foundation and holds great potential for precision medicine.

Nucleic Acid Amplification by Template-Dominated Click Chemistry for Ultrasensitive DNA/RNA Detection on an Electrochemical Readout Platform.

As an enzyme-free exponential nucleic acid amplification method, the click chemistry-mediated ligation chain reaction (ccLCR) has shown great prospects in the molecular diagnosis. However, the current optics-based ccLCR is challenged by remarkable nonspecific amplification, severely hindering its future application. This study demonstrated that the severe nonspecific amplification was generated probably due to high random collision in the high DNA probe concentration (µM level). To solve this hurdle, a nucleic acid template-dominated ccLCR was constructed using nM-level DNA probes and read on an electrochemical platform (cc-eLCR). Under the optimal conditions, the proposed cc-eLCR detected a low-level nucleic acid target (1 fM) with a single-base resolution. Furthermore, this assay was applied to detect the target of interest in cell extracts with a satisfactory result. The proposed cc-eLCR offers huge possibility for click chemistry-mediated enzyme-free exponential nucleic acid amplification in the application of medical diagnosis and biomedical research.

Gender differences in acute toxicity, toxicokinetic and tissue distribution of amphotericin B liposomes in rats.

Amphotericin B (AmB), an effective polyene drug with broad spectrum antifungal activity, is used for serious fungal infections. Liposomal amphotericin B (LAmB) is a lipid dosage form, which has a significantly improved toxicity profile compared with conventional amphotericin B deoxycholate (DAmB). This study focused on verifying the gender differences in the acute toxicity of LAmB and further exploring its causes. Acute toxicity study of LAmB and DAmB were performed in rats, and toxicity responses and mortality of different sexes were observed and recorded. Concentrations of AmB in rat plasma and tissues were determined by a fully validated UPLC-MS/MS assay. The results demonstrated that LAmB showed significant gender differences in acute toxicity, with more severe toxic symptoms and higher mortality for female rats at different doses, but the same differences were not observed for DAmB under the same condition. To explore the cause of differences, toxicokinetic and tissue distribution studies were performed and the results showed that female animals had higher drug exposure, longer half-life and lower plasma clearance compared to male rats, and the drug was mostly distributed in the liver and kidneys, in which female rats displayed a significant higher concentration than that of male rats.

RIP1/RIP3/MLKL mediates dopaminergic neuron necroptosis in a mouse model of Parkinson disease.

Parkinson's disease (PD) is the second most common neurodegenerative disorder and is characterized by severe neuronal loss. Necroptosis, or programmed cell necrosis, is mediated by the receptor interacting protein kinase-1 and -3/mixed lineage kinase domain-like protein (RIP1/RIP3/MLKL) pathway, and is involved in several neurodegenerative diseases. Here we aimed to explore the involvement of necroptosis in 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine hydrochloride (MPTP)-induced PD and determine the potential mechanisms. We found that the protein levels of RIP1, RIP3, and MLKL increased significantly in a MPTP-induced mouse PD model. High expression of RIP1/RIP3/MLKL was associated with severe loss of dopaminergic neurons. Pretreatment with necrostatin-1 or the knockout of the RIP3/MLKL gene to block necroptosis pathway dramatically ameliorated PD by increasing dopamine levels and rescuing the loss of dopaminergic neurons, independent of the apoptotic pathway. Moreover, upregulation of inflammatory cytokines in MPTP-treated mice was partially inhibited by deletion of RIP3 or MLKL gene, indicating that a positive feedback loop exists between these genes and inflammatory cytokines. Our data indicate that RIP1/RIP3/MLKL-mediated necroptosis is involved in the pathogenesis of MPTP-induced PD. Downregulating the expression of RIP1, RIP3, or MLKL can significantly attenuate MPTP-induced PD. Future therapy targeting necroptosis may be a promising new option.

Kimura's Disease Presenting As Scrotal Mass: A Difficult Diagnosis.

Herein we report a case of Kimura's disease with unusual manifestations. A 46-year- old Chinese man presented with mass in scrotum which gradually increased in size for approximately 7 years. A computerized tomographic scan of abdomen revealed a soft-tissue density shadow in scrotum and enlargement of lymph nodes in groin and retroperitoneum. The scrotal mass was excised and the biopsy specimen revealed angiolymphoid hyperplasia with infiltration of eosinophils. In conclusion, clinical doctors should pay attention that the patient with eosinophilia and scrotal mass could be indicative for Kimura's disease.

A new material for tissue engineered vagina reconstruction: Acellular porcine vagina matrix.

Acellular matrix materials have been widely used to repair various tissues and organs. According to the plastic principle, when a part of the body is lost, it should be replaced with a similar material. Therefore, the use of a homologous organ-specific acellular vaginal tissue in vagina reconstruction repair surgery may show good results. However, the acellular vagina matrix (AVM) form large vertebrates is difficult to isolate. In this study, we described a multistep method to prepare porcine AVM and evaluated the efficacy of acellularization. We also investigated the biomechanical properties, biological activity elements, and biocompatibility of the porcine AVM. We then used this material to reconstruct a rat vagina and performed further morphologic and functional analyses. Small intestinal submucosa (SIS), which is a commonly used acellular matrix material, was used in a control group. Histological examination, DNA content analysis, and agarose gel electrophoresis revealed that the decellularization procedure was effective. The AVM had acceptable biomechanical properties and sufficient growth factor production (VEGF, FGF, TGF-ß1, and PDGF-BB) compared with that of the SIS. Subcutaneous transplantation in rats showed that the AVM had good biocompatibility. The tissue-engineered vagina using the AVM more resembled normal-appearing tissue than did that using SIS following morphologic and functional analyses. The AVM has great potential for application in vaginal reconstructive surgery. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 1949-1959, 2017.

Inhibitory effect of compound NS5806 on cardiac transient outward potassium channel dependents on interaction between auxiliary subunits / 中国药理学与毒理学杂志

OBJECTIVE Transient outward potassium current (Ito) plays a crucial role in cardiac phase 1 repolarization and the channels are assembled by pore-forming α-subunits (Kv4.2 or Kv4.3) and auxiliary subunits (KChIP2 and DPP6). Previous studies have found that the compound NS5806 increases Ito in canine ventricular cardiomyocytes through slowing current decay. Here, we reported that NS5806 produced an acute inhibitory action on Ito in mouse ventricular cardiomyocytes and human induced pluripotent stem cell-derived cardiomyocytes (hiPS-CM). METHODS Whole-cell patch-clamp was used to record Ito in native myocytes and in HEK cells expressing cloned Kv4.x/KChIP2/DPP6 channels; Western-blot detected the channel protein expression. RESULTS In isolated mouse ventricular cardiomyocytes, NS5806 (0.1-30 μmol·L-1) inhibited Ito in a concentration-dependent manner with IC50 of 6.6±1.9 μmol·L-1. The current decay was significantly accelerated with a time constant from 53.8±5.5 to 41.8±3.0 ms at +60 mV (P<0.01). Similarly, NS5806 concentration-dependently reduced the Ito peak current amplitude with an acceleration of current decay. In addition, NS5806 increased IKv4.2/KChIP2 and delayed current decay, but decreased IKv4.2/KChIP2/DPP6 with the acceleration of current decay. The inhibitory action on the current was more potent if DPP6 expression level was increased from Kv4.2/KChIP2/DPP61:1:1 to 1:1:3. Western-blot showed a higher expression of DPP6 protein in mouse heart and in hiPS- CM compared to canine heart. Moreover, specific knock- down DPP6 expression by siRNA antagonized the inhibitory action of NS5806 in hiPS-CM. Our results pointed to an important role of DPP6 subunit in the regulation of NS5806 on the channel. By using molecular docking simulation, five interaction sites with high possibility between KChIP2 and DPP6 were identified. Mutations of those sites changed the inhibitory action of NS58056 into excitatory effect on the current with the delay of current decay. CONCLUSION NS5806 significantly inhibits Ito by accelerating current decay in mouse cardiomyocytes and hiPS-CM. The effect depends on the interaction between DPP6 and KChIP2 subunits.

[The effect of lidocaine on hERG (+) channels].

We studied the effects of the lidocaine on the hERG K(+) channels with a focus on the electrophysiology of the heart. The hERG current was recorded using the conventional whole-cell patch clamp technique and the channel protein expression level was measured with Western blot in HEK 293 cells stablely expressed hERG K(+) channels. The langendorff perfusion system was used to record the ECG from isolated rabbit heart. Lidocaine inhibited hERG current in a concentration-dependent manner at 0.3-1 000 µmol·L(-1), the IC(50) value was 88.63 ±7.99 µmol·L(-1). The inhibitory action was enhanced by positive votalge without changing the votalge-dependent activation of the channel. However, lidocaine inhibited hERG current in a frequency-dependent manner. In addition, chronic incubation of lidocaine did not change the hERG K(+) channel protein expression. ECG recordings in the isolated perfused rabbit heart demonstrated that lidocaine( > 100 µmol·L(-1)) did not affected QTc interval, but decreased the heart rate and prolonged the PR interval and QRS duration. Our results demonstrate that lidocaine potentially inhibits the hERG K(+) current at a high concentration, but does not prolonged the QTc of ECG.

[Role of calcineurin in down-regulation of left ventricular transmural voltage- dependent K(+) currents in mice with heart failure].

The aim of the present study was to investigate the role of calcineurin in the down-regulation of left ventricular transmural voltage-dependent K(+) currents in heart failure. Transverse aorta was banded by using microsurgical techniques to create mouse heart failure model. Sham-operated (Sham) or aorta banded (Band) mice were randomized to receive calcineurin inhibitor cyclosporine A (CsA) or vehicle. The densities and kinetic properties of voltage-dependent K(+) currents, as well as action potential (AP), of left ventricular subendocardial (Endo) and subepicardial (Epi) myocytes were determined by using whole-cell patch-clamp technique. The results showed that calcineurin activity was significant higher in Endo myocytes than that in Epi ones in all the groups. Compared with Sham group, Band mice showed significantly increased calcineurin activity both in Endo and Epi myocytes. CsA significantly reduced calcineurin activity in Band mice. CsA treatment in Band mice partially reversed the down-regulation of Ito density, completely reversed the down-regulation of IK,slow density both in Endo and Epi myocytes, and Iss density in Endo myocytes. In addition, CsA treatment in Band mice partially antagonized the prolongation of action potential duration (APD), and APD at 50% (APD50) and 90% repolarization (APD90) were significantly reduced. Because of non-parallel shortening of APD in Endo and Epi myocytes, the ratio of Endo/Epi APD90 was reduced from 4.8:1 in Band mice to 2.6:1 in CsA-treated mice, which was close to that in Sham mice. The results suggest that non-parallel activation of calcineurin in Endo and Epi myocytes contributes to the down-regulation of transmural voltage-dependent K(+) currents and the amplification of transmural dispersion of repolarization (TDR) in left ventricular failure hearts. Inhibition of calcineurin may be a potential new therapeutic strategy to prevent and cure arrhythmias and sudden death in heart failure.

Yishen Jiangzhuo Granules affect tubulointerstitial fibrosis via a mitochondrion-mediated apoptotic pathway.

OBJECTIVE: To investigate the effect of Yishen Jiangzhuo Granules, YSJZG) on mitochondrial injury and regeneration and renal tubular epithelial cell apoptosis in chronic renal failure (CRF) rats and explore its mechanism from molecular pathology, gene, protein levels, and relative pathway. METHODS: The CRF rat model was established using 5/6 nephrectomy. Sixty rats were randomly divided into six groups: sham-operation group, model (CRF) group, Niaoduqing Granules-treated group [5 g/(kg.day)], low-, moderate-, and high-dose [L-YSJZG, M-YSJZG, H-YSJZG at 3, 6, and 9 g/(kg day)] YSJZG-treated group (n=10 each). The levels of serum creatinine (Scr), blood urea nitrogen (BUN), and 24-h urine protein were assessed after 10 weeks of treatment. The tubulointerstitial injury and collagen deposition were evaluated using periodic acid-schiff stain and Masson staining. Renal tubular epithelial cell apoptosis was assessed using the terminal deoxynucleotidyl transferase dUTP nick end labeling assay, mitochondrial injury was observed using an electron microscope, and superoxide dismutase (SOD), glutathione (GSH) and malondialdehyde (MDA) levels were assessed using chromometry. Transforming growth factor-ß1 (TGF-ß1) expression was assessed using immunohistochemistry. The expressions of Bax, Bcl-2, peroxisome proliferator-activated receptor γ coactivator- 1α (PGC-1α), mitochondrial transcription factor A (Tfam), mitogen-activated protein kinases (MAPK) phosphorylation were evaluated by Western blot. RESULTS: YSJZG decreased the 24-h urine protein, BUN, Scr, remnant kidney weight-to-body weight ratio, renal tubular injury, deposition of collagen, and the apoptosis of renal tubular epithelial cells in a dose-dependent manner. YSJZG dose-dependently restored the number and structure of mitochondria and the expression of Tfam and PCG-1α, up-regulated the expression of Bcl-2, and inhibited the expression of Bax. YSJZG also dose-dependently inhibited TGF-ß1 expression, increased SOD and GSH activity, decreased the MDA level, and inhibited p38MAPK and pERK1/2 phosphorylation (all P<0.01). CONCLUSION: YSJZG improved the renal function in rats with CRF and inhibited the progression of tubulointerstitial fibrosis by dose-dependently alleviating mitochondrial injury, restoring the expression of Tfam and PCG-1α, and inhibiting renal tubular epithelial cell apoptosis through inhibiting activation of reactive oxygen species-MAPK signaling.

Biomechanical testing of a unique built-in expandable anterior spinal internal fixation system.

BACKGROUND: Expandable screws have greater pullout strength than conventional screws. The purpose of this study was to compare the biomechanical stability provided by a new built-in expandable anterior spinal fixation system with that of 2 commonly used anterior fixation systems, the Z-Plate and the Kaneda, in a porcine partial vertebral corpectomy model. METHODS: Eighteen porcine thoracolumbar spine specimens were randomly divided into 3 groups of 6 each. A vertebral wedge osteotomy was performed by removing the anterior 2/3 of the L1 vertebral body and the T15/L1 disc. Vertebrae were fixed with the Z-Plate, Kaneda, or expandable fixation system. The 3-dimensional spinal range of motion (ROM) of specimens in the intact state (prior to osteotomy), injured state (after osteotomy), and after internal fixation were recorded. The pullout strength and maximum torque of common anterior screws, the expandable anterior fixation screw unexpanded, and the expandable anterior fixation screw expanded was tested. RESULTS: After internal fixation, the expandable device and Z-plate system exhibited higher left bending motion than the Kaneda system (5.50° and 5.37° vs. 5.04, p = 0.001 and 0.008, respectively), and the Z-plate and Kaneda groups had significantly higher left axial and right axial rotation ROM as compared to the expandable device group (left axial rotation: 5.23° and 5.02° vs. 4.53°; right axial rotation: 5.23° and 5.08° vs. 4.49°). The maximum insertion torque of the expandable device was significantly greater than of a common screw (5.10 vs. 3.75 Ns). The maximum pullout force of the expandable device expanded was significantly higher than that of the common screw and the expandable device unexpanded (3,035.48 N vs. 1,827.38 N and 2,333.49 N). CONCLUSIONS: The built-in anterior fixation system provides better axial rotational stability as compared to the other 2 systems, and greater maximum torque and pullout strength than a common fixation screw.

[Colorimetric characterization of LCD based on wavelength partition spectral model].

To establish a colorimetrical characterization model of LCDs, an experiment with EIZO CG19, IBM 19, DELL 19 and HP 19 LCDs was designed and carried out to test the interaction between RGB channels, and then to test the spectral additive property of LCDs. The RGB digital values of single channel and two channels were given and the corresponding tristimulus values were measured, then a chart was plotted and calculations were made to test the independency of RGB channels. The results showed that the interaction between channels was reasonably weak and spectral additivity property was held well. We also found that the relations between radiations and digital values at different wavelengths varied, that is, they were the functions of wavelength. A new calculation method based on piecewise spectral model, in which the relation between radiations and digital values was fitted by a cubic polynomial in each piece of wavelength with measured spectral radiation curves, was proposed and tested. The spectral radiation curves of RGB primaries with any digital values can be found out with only a few measurements and fitted cubic polynomial in this way and then any displayed color can be turned out by the spectral additivity property of primaries at given digital values. The algorithm of this method was discussed in detail in this paper. The computations showed that the proposed method was simple and the number of measurements needed was reduced greatly while keeping a very high computation precision. This method can be used as a colorimetrical characterization model.

[Alisol B inhibited complement 3a-induced human renal tubular epithelial to mesenchymal transition].

OBJECTIVE: To study whether alisol B could inhibit complement 3a (C3a) induced renal tubular epithelial-mesenchymal transition (EMT). METHODS: The in vitro cultured human renal tubular epithelial HK-2 cells were intervened with 5 ng/mL transforming growth factor-beta (TGF-beta), 0.1 micromol C3a, and 0.1 micromol C3a + 10 micromol alisol B, respectively. The mRNA and protein expressions of alpha-SMA, E-cadherin, and C3 were detected using RT-PCR, Western blot, and immunofluorescence, respectively. RESULTS: The mRNA and protein expressions of C3 in HK-2 cells were up-regulated after intervention of C3a (P < 0.01), the mRNA and protein expressions of alpha-SMA in HK-2 cells were obviously enhanced (P < 0.01), the mRNA and protein expressions of E-cadherin obviously decreased (P < 0.01). When compared with the group intervened by exogenous C3a, after intervention of alisol B, the mRNA and protein expressions of alpha-SMA in HK-2 cells were obviously reduced (P < 0.01), the mRNA and protein expressions of E-cadherin obviously increased (P < 0.05). CONCLUSIONS: Exogenous C3a could induce renal tubular EMT. Alisol B was capable of suppressing C3a induced EMT.

Adenovirus-vectored shRNAs targeted to the highly conserved regions of VP1 and 2B in tandem inhibits replication of foot-and-mouth disease virus both in vitro and in vivo.

Foot-and-mouth disease is a highly contagious and economically important disease of cloven-hoofed animals. RNA interference (RNAi) can be used as a rapid and specific antiviral approach. It was shown that treatment with recombinant adenovirus (Ad(VP1-2B)) carrying shRNAs targeted to the VP1 and 2B genes of FMDV expressed in tandem had marked antiviral effects against FMDV both in IBRS-2 cells and guinea pigs. Treatment with Ad(VP1-2B) both before and after FMDV infection was most effective in IBRS-2 cells, as the FMDV RNA transcripts could not be detected within 48 h post-challenge (hpc), and the viral RNA copy number at 72 hpc was only 0.02% of that in the positive control group. Delivery of Ad(VP1-2B) reduced significantly the susceptibility of guinea pigs to FMDV infection. All guinea pigs were protected within 3 days post challenge (dpc) when they were injected twice with the same dose of Ad(VP1-2B), and a third treatment with the same dose of Ad(VP1-2B) at 3 dpc was necessary to confer longer lasting protection (up to 6 dpc). In conclusion, application of such a adenovirus vector to inhibit more than one viral gene may be an advantageous method for prevention and therapy of FMDV infection.

Angiotensin II decreases spontaneous firing rate of guinea-pig sino-atrial node cells.

Angiotensin II (Ang II) plays an important role in the regulation of cardiac function, but its electrophysiological effects on sino-atrial (SA) node are not well understood. In this study, the immediate effect of Ang II on action potentials and ionic currents were investigated by using whole-cell patch-clamps in single guinea-pig SA node pacemaker cells. We demonstrated that Ang II exerted a negative effect on spontaneous firing rate, with a concomitant reduction in the slope of diastolic depolarization. The inhibitory effect of Ang II on spontaneous activity displayed a concentration-dependent manner in the range of 1-1000 nM, with IC50 of 8.34 nM. Ang II type 1 (AT1) receptor antagonist valsartan (1 µM) abolished the inhibitory effect. In contrast, Ang II type 2 (AT2) receptor antagonist, PD123319 (1 µM) didn't affect the action of Ang II. Ang II had no significant effect on hyperpolarization-activated current (If) in SA node cells. However, it significantly slowed the deactivation of the slowly activated delayed rectifier K+ current (Iks) and increased the tail current density. Furthermore, Ang II decreased the current density of L-type Ca2+ current in SA node cells. Our data demonstrate that Ang II reduces the auto rhythm of SA node cells via enhancing Iks and reducing ICaL. The result suggests a potential mechanism by which elevated levels of Ang II may be involved in the occurrence of SA node dysfunction in cardiac pathophysiology.

[Effects of bone morphogenic protein-7 on transdifferentiation and the expression of connective tissue growth factor of human renal tubular epithelial cells induced by transforming growth factor-beta1].

OBJECTIVE: To investigate the effects of bone morphogenic protein (BMP)-7 upon epithelial-to-mesenchymal transition (EMT) and the expression of connective tissue growth factor (CTGF) in human renal proximal tubular epithelial cells (HK-2) induced by transforming growth factor-beta1, (TGF-beta1) and to explore the possible mechanisms of BMP-7 for the inhibition and reversal of renal interstitial fibrosis. METHODS: HK-2 cells were treated with TGF-beta1 or a combination of TGF-beta1 and BMP-7. RT-PCR and Western blot were used to determine the mRNA and protein expression of alpha-SMA, E-cadherin and CTGF. For EMT reversal experiments, when TGF-beta1-induced EMT occurred, then the medium was removed and replaced with medium containing 200 ng/ml BMP-7. After 48 h, the morphological changes and the expression of E-cadherin were assessed by phase contrast microscopy or immunofluorescent microscopy. RESULTS: The control cells displayed typical cobblestone morphology of epithelial cells. 3 ng/ml TGF-beta1 induced profound morphologic changes after 48 h, with cells becoming elongated in shape, but addition of 200 ng/ml BMP-7 for 48 h restored the epithelial morphology of HK-2 cells. Indirect immunofluorescence showed that treatment of 3 ng/ml TGF-beta1 resulted in a distinct loss of E-cadherin staining in the plasma membrane of HK-2 cells but subsequent treatment with 200 ng/ml BMP-7 largely restored the E-cadherin protein staining. 3 ng/ml TGF-beta1 significantly up-regulated the mRNA and protein expression of alpha-SMA, reduced the expression of E-cadherin and increased the expression of CTGF after 48 h (vs. control, P < 0.01). BMP-7 dramatically suppressed the mRNA and protein expression of alpha-SMA, restored the expression of E-cadherin and prevented the expression of CTGF in a dose-dependent manner after co-incubation with TGF-beta1 for 48 h (400 ng/ml BMP-7 + TGF-beta1 vs. TGF-beta1, alone, P < 0.01). CONCLUSIONS: BMP-7 exerts its antifibrotic effect partially through blocking and reversing TGF-beta1-induced EMT and downregulating the expression of CTGF in human renal tubular epithelial cells.

Effects of strophanthidin on intracellular calcium concentration in ventricular myocytes of guinea pig.

Effect of strophanthidin (Str) on intracellular calcium concentration ([Ca2+]i) was investigated on isolated ventricular myocytes of guinea pig. Single ventricular myocytes were obtained by enzymatic dissociation technique. Fluorescent signal of [Ca2+]i was detected with confocal microscopy after incubation of cardiomycytes in Tyrode' s solution with Fluo3-AM. The result showed that Str increased [Ca2+]i in a concentration-dependent manner. The ventricular myocytes began to round-up into a contracture state once the peak level of [Ca2+]i was achieved in the presence of Str (10 micromol L(- 1)), but remained no change in the presence of Str (1 and 100 nmol L(-1)). Tetrodotoxin (TTX), nisodipine, and high concentration of extracellular Ca2+ changed the response of cardiomycytes to Str (1 and 100 nmol L(-1)) , but had no obvious effects on the action of Str (10 micromol L(-1)). The elevation of [Ca2+]i caused by Str at all of the detected concentrations was partially antagonized by rynodine (10 micromol L(-1)) or the removal of Ca2+ from Tyrode's solution. In Na+, K+ -free Tyrode' s solution, the response of cardiomycytes in [Ca2+]i elevation to Str (10 micromol L(-1)) was attenuated, while remained no change to Str (1 and 100 nmol L(-1)). TTX, nisodipine, and high concentration of extracellular Ca2+ changed the response of cardiomycytes to Str at all of the detected concentrations in Na+, K+ -free Tyrode's solution. The study suggests that the elevation of [Ca2+]i by Str at the low (nomomolar) concentrations is partially mediated by the extracellular calcium influx through Ca2+ channel or a "slip mode conductance" of TTX sensitive Na+ channel. While the effect of Str at high (micromolar) concentrations was mainly due to the inhibition of Na+, K+ -ATPase. Directly triggering the release of intracellular Ca2+ from sarcoplasmic reticulum (SR) by Str may be also involved in the mechanism of [Ca2+]i elevation.

[Transmural L-type calcium current in a pressure-overloaded mouse model with heart failure].

Transmural electrical heterogeneity plays an important role in the normal dispersion of repolarizaion and propagation of excitation in the heart. The amplification of transmural electrical heterogeneity contributes to the genesis of arrhythmias in cardiac hypertrophy and failure. We established a mouse model with cardiac failure by aortic banding and investigated the possible contribution of L-type calcium current (I(Ca-L)) to transmural electrical heterogeneity in both normal and failing hearts. Single myocytes were enzymatically isolated from subendocardial and subepicardial myocardium of the free left ventricle wall. The recordings of action potential and I(Ca-L) were performed using the conventional whole-cell patch-clamp technique. The results showed that: (1) The action potential duration at 90% repolarization (APD(90)) of the subendocardial myocytes in normal control mice was (38.2 +/- 6.44) ms, which was significantly longer than that of the subepicardial myocytes [(15.672 +/- 5.31) ms]. The ratio of APD(90) for subendocardial/subepicardial myocytes was about 2.5:1. The peak I(Ca-L) density in subendocardial myocytes was (-2.7 +/- 0.49) pA/pF, which was not different from that in subepicardial myocytes [(-2.54 +/- 0.53) pA/pF]. (2) In failing hearts, both action potential duration at 50% repolarization (APD(50)) and APD(90) were remarkably prolonged either in subendocardial or subepicardial myocytes compared to that in sham hearts. The subendocardial myocytes had much longer APD. The ratio of APD(90) for subendocardial/subepicardial myocytes changed to about 4.2:1. (3) I(Ca-L) density in subendocardial myocytes was significantly decreased in failing hearts compared with that in sham hearts. At four test potentials from +10 mV to +40 mV, the density of I(Ca-L) from subendocardial myocytes in failing hearts was decreased by 20.2%, 21.4%, 21.6% and 25.7%, respectively (P<0.01). However, no significant difference was observed in I(Ca-L) density from subepicardial myocytes in failing hearts. There was no significant difference in the kinetic properties of I(Ca-L) in subendocardial and subepicardial myocytes between the band and sham groups. We conclude that I(Ca-L) may not contribute to the physiological transmural electrical heterogeneity in mouse hearts. The electrical heterogeneity is exaggerated and the density of I(Ca-L) is decreased in the subendocardial myocytes, but not in the subepicardial myocytes in failing hearts. The results obtained suggest that the decreased density of I(Ca-L) in subendocardial myocytes is possibly an adaptive response to the prolongation of action potential due to delayed depolarization and may reduce the transmural dispersion of repolarization in heart failure.