OBJECTIVETo study the changes of p38MAPK expressions, the frequency of apoptosis and the distribution of cell cycle of hunan Glioma U251 cells after HCMV infection.

METHODSThe expression of total p38 (both phosphorylated and nonphosphorylated p38) and phosphorylated p38 in U251 cells were detected by Western blotting at 15 min, 30 min, 1 h, 6 h, 10 h, 16 h, 24 h, 36 h and 48 h after HCMV infection. The apoptosis percentage and the cell cycle distribution of U251 cells at 2 d, 5 d and 7 d after HCMV infection were detected by flow cytometry (FCM).

RESULTSThe results of Western blotting demonstrated that a strong increase in phosphorylated p38 was detected from 6 h to 10 h after HCMV infection, with mean gray scales 186.33 +/- 7.51 (t = 5.37, P < 0.01) and 188.00 +/- 7.02 (t = 5.26, P < 0.01 for all) at 6 h and 10 h, respectively, and p38 phosphorylation decreased to the basic level at 16 h after HCMV infection. But the overall levels of p38 protein were not significantly altered during the course of infection. FCM analysis showed that HCMV could significantly increase the apoptotic rates of U251 cells compared with controls (t = 10.84, P < 0.01), and the apoptotic percentages of the cells reached to peak [(10.18 +/- 1.24)%] at 5 d after HCMV infection. The data of FCM showed that HCMV could decrease the number of U251 cells in G1 phase and arrest the cells in S and G2 phase. The numbers of G1 phase U251 cells were significantly lowered to (56.50 +/- 2.57)% (t = 26.45, P < 0.01), (62.33 +/- 2.64)% (t = 21.20, P < 0.01) and (67.45 +/- 4.44)% (t = 10.61, P < 0.01), respectively at 2 d, 5 d and 7 d after infection.

CONCLUSIONHCMV could activate p38MAPK pathway and trigger apoptosis and interfere cell cycle in U251 cells.

Apoptosis ; Blotting, Western ; Cell Cycle ; Cell Line, Tumor ; Cytomegalovirus ; isolation & purification ; Cytomegalovirus Infections ; metabolism ; physiopathology ; Flow Cytometry ; Glioma ; metabolism ; microbiology ; pathology ; Humans ; MAP Kinase Signaling System ; Phosphorylation ; p38 Mitogen-Activated Protein Kinases ; metabolism

OBJECTIVETo evaluate the effects of W-7, a calmodulin inhibitor, on transmural dispersion of repolarization (TDR), early after depolarization (EAD) and torsade de pointes (TdP) induction after administration of d-sotalol in isolated rabbit heart.

METHODSTdP was induced by d-sotalol (30 micromol/L), bradycardia, and hypokalemic (1.5 mmol/L)/hypomagnesaemic (0.35 mmol/L) solution in isolated female rabbit hearts. Thirty six rabbit hearts were divided into 4 groups (n = 9 each): d-sotalol alone, d-sotalol + W-7 (20 micromol/L), d-sotalol + W-7 (50 micromol/L), and d-sotalol + W-7 (100 micromol/L). Monophasic action potentials (MAPs) of the left ventricular epimyocardium (Epi), midmyocardium (M), and endomyocardium (Endo) were recorded simultaneously with ECG. The incidence of EAD and TdP were observed as well.

RESULTSTreatment with d-sotalol alone prolonged ventricular MAP duration and QT interval, increased TDR, and evoked high incidence of EAD (9/9) and spontaneous TdP (7/9) in hypokalemic/hypomagnesaemic solution in female rabbit heart. W-7 concentration-dependently decreased incidence of TdP (4/9 in 20 micromol/L; 2/9 in 50 micromol/L; 1/9 in 100 micromol/L). This effect of W-7 coincided with the decreased incidence of EAD (5/9 in 20 micromol/L; 4/9 in 50 micromol/L; 1/9 in 100 micromol/L). However, the d-sotalol-induced prolongation of QT interval and TDR was not significantly altered by W-7 at the three concentration used.

CONCLUSIONSIn isolated female rabbit hearts, calmodulin antagonist W-7 suppresses d-sotalol-induced TdP without altering TDR but does suppress EAD. The effects observed with W-7 also suggest a possible important role for calmodulin-activated enzymes in the induction of TdP.

Animals ; Calmodulin ; antagonists & inhibitors ; Enzyme Inhibitors ; therapeutic use ; Female ; In Vitro Techniques ; Rabbits ; Sotalol ; adverse effects ; Sulfonamides ; therapeutic use ; Torsades de Pointes ; chemically induced ; prevention & control

BACKGROUND: Calcium calmodulin-dependent kinase Ⅱ (CaMKⅡ) can be more active in patients with left ventricular hypertrophy (LVH), which in turn causes phosphorylation of ryanodine receptors, resulting in inactivation and the instability of intracellular calcium homeostasis. The present study aimed to determine the effect of CaMKⅡ–ryanodine receptor pathway signaling in rabbits with left ventricular hypertrophy and triggered ventricular arrhythmia. METHODS: Forty New Zealand rabbits were randomized into four groups (10 per group): sham group, LVH group, KN-93 group (LVH+KN-93), and ryanodine group (LVH+ryanodine). Rabbits in the LVH, KN-93, and ryanodine groups were used to establish a left ventricular hypertrophy model by the coarctation of the abdominal aorta, while those in the sham group did not undergo the coarctation. After eight weeks, action potentials (APs) were recorded simultaneously in the endocardium and epicardium, and a transmural electrocardiogram (ECG) was also recorded in the rabbit left ventricular wedge model. Drugs were administered to the animals in the KN-93 and ryanodine groups, and the frequency of triggered APs and ventricular tachycardia was recorded after the rabbits were given isoprenaline (1 mol/L) and high-frequency stimulation. RESULTS: The frequency (animals/group) of triggered APs was 0/10 in the sham group, 10/10 in the LVH group, 4/10 in the KN-93 group, and 1/10 in the ryanodine group. The frequencies of ventricular tachycardia were 0/10, 9/10, 3/10, and 1/10, respectively. The frequencies of polymorphic ventricular tachycardia or ventricular fibrillation were 0/10, 7/10, 2/10, and 1/10, respectively. The frequencies of triggered ventricular arrhythmias in the KN-93 and ryanodine groups were much lower than those in the LVH group (P<0.05). CONCLUSIONS: KN-93 and ryanodine can effectively reduce the occurrence of triggered ventricular arrhythmia in rabbits with LVH. The CaMKⅡ–ryanodine signaling pathway can be used as a new means of treating ventricular arrhythmia.

The cDNA encoding the precursor of cobrotoxin was cloned from the venom gland of the Chinese continental cobra ( Naja naja atra ) by RT-PCR. Its deduced amino acid sequence analysis showed that the mature protein was identical to that identified from the Taiwan cobra ( Naja naja atra ) by protein sequencing technique. The cDNA encoding the mature protein was then subcloned into the expression vector pMAL-P2. The gene of CMl 1, which was formed by ligation of the fragments of the synthetic oligonucleotides, was also cloned into the expression vector pMAL-P2. After induction of IPTG, both of the neurotoxins were overexpressed as soluble fusion proteins which were confirmed by SDS-PAGE and western blotting. The expressed fusion proteins was purified by sepharose 6B-amylose affinity chromatography and DEAE-sepharose FF chromatography. Both of the recombinant proteins achieved after digestion by factor Xa showed the in vivo toxicity.

Objective The aim of this study is to observe the effect of combined amiodarone and antiarrhythmic peptide (AAP10) use on the incidence of induced ventricular arrhythmias in healed myocardial infarction (MI) rabbits. Methods Twenty Japanese rabbits underwent thoracotomy without coronary artery ligation( Sham, group A) ,the middle left circumflex branch were ligated to induce MI in 180 Japanese rabbits. Eight weeks after operation, 124 rabbits survived MI operation and were divided into four groups: control group (group B, n =31 ), amiodarone group (group C, n =31 ), AAP10 group (group D,n =31 ) and amiodarone plus AAP10 group ( group E, n =31 ). The A and B and D groups were treated with saline 2 ml/d, the C and E groups were treated with 2 ml saline containing amiodarone 100 mg· kg-1·d-1. All rabbits were examined by echocardiogram at 12 weeks after operation, then anesthetized by sodium barbital, the left wedge ventricular preparations were cannulated and artery perfused by Tyrode's solution in vitro in the absence (Group A, B and C) and presence of AAP10 (500 nmol/L, Group D and E). The volume electrocardiogram, QT Interval, QRS interval, effective refractory period (ERP), the T-peak to Tend interval ( Tp-e ), and ventricular tachycardia episodes induced by programmed stimulation were recorded.The Tp-e/QT ratio was calculated. After perfusion, gap junctions protein connexin 43 (Cx43) expression was detected by Western blot and immunofluorescence. Results The incidence of induced ventricular tachycardia episodes of group A, B, C, D, E was 0, 62. 5%, 26. 9%, 40.0%, 22. 2% respectively. The incidence of induced ventricular tachycardia episodes of group E was less than group B. The Tp-e/QT ratio in group B, C, D were greater than in group A. The Tp-e/QT ratio of group E was less than group B. The myocardial Cx43 in the group B was down-regulated and disorganized compared to group A, up-regulated in group C and E compared to group B, up-regulated in group E compared to group D. The Cx43 in the heart of group D and E were well organized than in group B and C. Conclusions The artery perfused rabbits wedge preparations with healed myocardial infarction with high incidence of induced ventricular tachycardia episodes are good platform for ventricular arrhythmias research. Combined amiodarone and AAP10 use could decrease the Tp-e/QT ratio and the incidence of induced ventricular tachycardia episodes. Amiodarone and AAP10have synergistic effects on upregulating Cx43 and preventing ventricular arrhythmias in this rabbit model of healed myocardial infarction.

OBJECTIVETo determine current density of voltage-gated potassium channels and Kv1.3 express in T-lymphocyte derived from patients with acute coronary syndrome (ACS).

METHODSPeripheral blood mononuclear cells were collected from 12 patients with ACS and 10 control donors. Whole-cell patch clamp technique was used to record the outward K(+) currents (IK) and western blots technique was used to detect the express of Kv1.3 protein in lymphocyte.

RESULTS(1) The current density of voltage-gated potassium channel was significantly higher in ACS patients [(269 +/- 94) pA/pF] than in controls [(191 +/- 64) pA/pF, P < 0.01] while membrane capacitance was similar between the two groups. (2) Kv1.3 protein expression was also significantly increased in ACS patients than in controls (P < 0.01).

CONCLUSIONThe lymphocyte voltage-gated potassium channel is upregulated in patients with ACS suggesting a role of Kv activation in the pathophysiology of ACS.

Acute Coronary Syndrome ; metabolism ; Adult ; Aged ; Female ; Humans ; Kv1.3 Potassium Channel ; metabolism ; Male ; Middle Aged ; Patch-Clamp Techniques ; T-Lymphocytes ; metabolism

Objective The purpose of our study is to observe the voltage-gated potassium channel Kv1.3 expression on CD4+CD28null T cells from the peripheral blood of ACS patients by the patch clamp technique.Methods Kv1.3 potassium channels expression from 17 patients with ACS and 11 healthy agematched normal controls was detected in single cell ( CD4+ CD28null T cells and CD4 + CD28 + T cells) by fluorescence microscopy and patch clamp.Results The percent of CD4+ CD28null T cells are higher in the ACS(6.97% ± 2.05% ) than that in the controls ( 1.38% ± 0.84%, P < 0.05 ).The concentration of hsCRP is directly correlated with the number of the CD4+CD28nullT cells in the ACS ( r = 0.52, P < 0.05). The conductance[ (6.89 ± 1.17) nS vs.(3.36 ± 0.66) nS ], dens [ ( 1.95 ± 0.80) n/μm2 vs.( 1.13 ± 0.57) n/μm2] and numbers[ (574.5±97.6) n/cell vs.(280.3±55.3) n/cell] of the Kv1.3 channels on the CD4+ CD28nullT cells are significantly higher than those on the CD4 + CD28 + T cells ( all P < 0.01 ) in ACS patients, but were similar on CD4 + CD28 + T cells between ACS patients and controls.conclusion The CD4+ CD28nullT cells in the ACS and the numbers of Kv1.3 channels on the CD4 + CD28nullT cells from the ACS patients are significantly upregulated and might contribute to the pathogenesis of ACS.

OBJECTIVETo explore the value of (18)F-FDG PET/CT in the diagnosis and treatment evaluation in patients with pretreatment or recurrent extranodular natural killer/T-cell lymphoma nasal type (ENTCL).

METHODS(18)F-FDG PET/CT images and clinical records of 35 cases (67 scans) of pathologically confirmed ENTCL treated in our hospital within the last 9 years were analyzed. The imaging characteristics of the upper aerodigestive tract (UAT) and the non-aerodigestive tract (NUAT) lesions were analyzed. Lesion distribution, clinical stages, SUVmax and patient survival data were compared between pretreatment and recurrent cases.

RESULTSs All the ENTCL lesions were hypermetabolic. The UAT lesions involved mainly the nasal cavity and pharynx, while the NUAT lesions may involve the lymph nodes and all the organs. UAT lesions were more common in pretreatment cases while NUAT lesions tended to increase in recurrent cases. The SUVmax of pretreatment and recurrent lesions were 10.4∓4.4 and 9.6∓5.2, and showed no significant difference among patients with different lesion distribution patterns, clinical stages, or treatment history. The tumor remission rate evaluated by PET/CT were higher in cases with an initial diagnosis than in those with recurrence [(89.5% (17/19) vs 33.3% (5/15), P<0.005)]. Cox regression analysis revealed no significant differences in the survival rates among patients with different treatment history, clinical stages, lesion distribution patterns, or SUVmax levels (P>0.05).

CONCLUSION(18)F-FDG PET/CT can sensitively detect the pretreatment or recurrent lesions in ENTCL patients and helps in accurate tumor staging and curative effect evaluation.

Fluorodeoxyglucose F18 ; Humans ; Lymphoma, Extranodal NK-T-Cell ; diagnostic imaging ; Neoplasm Recurrence, Local ; Neoplasm Staging ; Positron Emission Tomography Computed Tomography

OBJECTIVETo evaluate the effects of antiarrhythmic peptide (AAP10) on ventricular arrhythmias in rabbits with healed myocardial infarction (OMI).

METHODSThirty rabbits were randomly divided into three groups (n = 10 each): Sham group, left thoracotomy was performed without coronary ligation; OMI group and OMI + AAP10 group, the circumflex coronaries were ligated. Three months post operation, the electrophysiological and antiarrhythmic effects of AAP10 were assessed in the arterially perfused rabbit left ventricular wedge preparation. Sham and OMI group were perfused with Tyrode's solution and OMI + AAP10 group was perfused with Tyrode's solution + AAP10 (80 nmol/L). Transmembrane action potentials were recorded simultaneously from endocardium and epicardium together with a transmural ECG by use of 2 separate intracellular floating microelectrodes. The stimulus-response-interval (SRI) of the epicardium and the incidence of ventricular tachycardia (VT) were observed. Whole heart and left ventricular weights, the left ventricular thickness at infarct border zone were measured.

RESULTSWhole heart and left ventricular weights as well as the left ventricular thickness at the infarct border zone significantly increased post infarction. VT was induced in 8 out of 10 rabbits in OMI group and in 2 out of 10 rabbits in OMI + AAP10 group (P < 0.05). SRI was also significantly shortened in OMI + AAP10 group compared to OMI group [SRI-1: (20.59 +/- 0.79) ms vs. (28.71 +/- 0.55) ms; SRI-2: (30.42 +/- 0.74) ms vs. (38.67 +/- 0.49) ms, all P < 0.01]. However, the action potential morphology and duration were similar between OMI and OMI + AAP10 groups.

CONCLUSIONThe antiarrhythmic peptide (AAP10) can increase gap junctional intercellular conductance without affecting the action potential morphology and duration and decrease the incidence of inducible ventricular tachycardia.

Animals ; Arrhythmias, Cardiac ; etiology ; prevention & control ; Male ; Myocardial Infarction ; complications ; physiopathology ; Oligopeptides ; pharmacology ; Rabbits ; Random Allocation

OBJECTIVETo investigate the effect of KN-93, a calmodulin kinase II inhibitor, on ventricular arrhythmias in rabbits with cardiac hypertrophy.

METHODSFemale New Zealand white rabbits were randomly divided into four groups (n = 10 each): Sham; LVH; LVH + KN-92 and LVH + KN-93 group. LVH was induced by partially constricting the abdominal aorta. In Sham group, the abdominal aorta was exposed without constriction. Eight weeks later, the arterially perfused left ventricular wedge preparations were made and transmembrane action potentials (TAP) from epicardium and endocardium and transmural ECG were simultaneously recorded. Incidence of early after depolarization (EAD) and torsade de pointes (Tdp), QT interval, action potential duration (APD) and transmural depolarization dispersion (TDR) at different cycle lengths were observed under slow stimulation (2000 - 4000 ms), hypokalemic (2 mmol/L) and hypomagnesaemic (0.25 mmol/L) Tyrode's solution perfusion.

RESULTSLeft ventricular hypertrophy was detected in LVH group by echocardiography and not affected by KN-92 and KN-93. Perfused with hypokalemic, hypomagnesaemic Tyrode's solution and under slow stimulation (2000 - 4000 ms), the incidences of EAD and Tdp in Sham group, LVH group, LVH + KN-92 group (0.5 micromol/L) and LVH + KN-93 group (0.5 micromol/L) were 0/10, 10/10, 9/10, 5/10 and 0/10, 5/10, 4/10, 1/10, respectively. With 1 micromol/L KN-92 and KN-93, the incidences of EAD and Tdp in LVH + KN-92 and LVH + KN-93 group were 9/10, 3/10 and 4/10, 1/10 respectively. The QT interval, APD and TDR were not affected by KN-93.

CONCLUSIONThe calmodulin kinase II inhibitor KN-93 can effectively suppress ventricular arrhythmias in rabbits with cardiac hypertrophy by decreasing EAD.

Animals ; Arrhythmias, Cardiac ; complications ; drug therapy ; Benzylamines ; pharmacology ; Calcium-Calmodulin-Dependent Protein Kinase Type 2 ; antagonists & inhibitors ; Cardiomegaly ; complications ; drug therapy ; Disease Models, Animal ; Male ; Protein Kinase Inhibitors ; pharmacology ; Rabbits ; Sulfonamides ; pharmacology