[Effects of Rosa roxburghii Pomace Biochar on Yield and Quality of Chinese Cabbage and Soil Properties].

In order to explore the effect of Rosa roxburghii pomace biochar on the yield and quality of Chinese cabbage and soil properties and realize the resource utilization of R. roxburghii pomace, a pot experiment was conducted to study the effect of R. roxburghii pomace biochar on the yield and quality of Chinese cabbage and soil properties by setting five biochar application rates of 0 % (CK), 1 % (T1), 3 % (T2), 5 % (T3), and 7 % (T4). The results showed that:① The application of R. roxburghii pomace biochar could significantly improve the yield and quality of Chinese cabbage, and the effect was the best at a 5 % biochar application rate. The yield, soluble solids, soluble sugar, vitamin C, total nitrogen, total phosphorus, and total potassium content of Chinese cabbage increased by 71.51 %, 40.14 %, 33.65 %, 38.08 %, 9.03 %, 28.85 %, and 35.38 %, respectively, compared with those in CK. ② The application of biochar from R. roxburghii pomace could significantly improve soil properties and increase soil nutrient content and availability. The effect was better at a 5 % biochar application rate. The soil pH, organic matter, total nitrogen, alkali-hydrolyzable nitrogen, available phosphorus, and available potassium content increased by 41.06 %, 134.84 %, 157.48 %, 140.79 %, 341.75 %, and 627.13 %, respectively, compared with those in CK. The contents of available Fe, Mn, Cu, and Zn and exchangeable Ca and Mg increased by 37.68 %, 61.69 %, 400.00 %, 4 648.84 %, 617.17 %, and 351.42 %, respectively, compared with those in CK. ③ The application of biochar from R. roxburghii pomace could significantly enhance soil enzyme activity. Compared with those in the CK treatment, soil urease, acid phosphatase, catalase, and sucrase increased by 51.43 %-362.86 %, 90.63 %-134.14 %, 21.40 %-85.12 %, and 82.92 %-218.43 %, respectively. ④ Redundancy analysis showed that soil AK; exchangeable Ca, SOM, and AP; and available Zn were the main factors affecting the yield and quality of Chinese cabbage, and there was a significant positive correlation between them. In summary, the application of R. roxburghii pomace biochar can significantly increase the yield and quality of Chinese cabbage and improve soil properties. The preparation of R. roxburghii pomace into biochar can provide a theoretical reference for the rational utilization of R. roxburghii pomace resources.

Insect group II chitinase OfChtII promotes chitin degradation during larva-pupa molting.

The insect group II chitinase (ChtII, also known as Cht10) is a unique chitinase with multiple catalytic and chitin-binding domains. It has been proven genetically to be an essential chitinase for molting. However, ChtII's role in chitin degradation during insect development remains poorly understood. Obtaining this knowledge is the key to fully understanding the chitin degradation system in insects. Here, we investigated the role of OfChtII during the molting of Ostrinia furnacalis, a model lepidopteran pest insect. OfChtII was expressed earlier than OfChtI (OfCht5) and OfChi-h, at both the gene and protein levels during larva-pupa molting as evidenced by quantitative polymerase chain reaction and western blot analyses. A truncated OfChtII, OfChtII-B4C1, was recombinantly expressed in Pichia pastoris cells and purified to homogeneity. The recombinant OfChtII-B4C1 loosened compacted chitin particles and produced holes in the cuticle surface as evidenced by scanning electron microscopy. It synergized with OfChtI and OfChi-h when hydrolyzing insoluble α-chitin. These findings suggested an important role for ChtII during insect molting and also provided a strategy for the coordinated degradation of cuticular chitin during insect molting by ChtII, ChtI and Chi-h.

Downregulation of miR-3568 Protects Against Ischemia/Reperfusion-Induced Cardiac Dysfunction in Rats and Apoptosis in H9C2 Cardiomyocytes Through Targeting TRIM62.

microRNA-3568 (miR-3568) has been reported to be associated with atherosclerosis. Only few data describe the expression and underlying mechanism of miR-3568 in regulating cardiac ischemia-reperfusion (I/R) injury such as apoptosis. In this study, we therefore sought to investigate the potential function of miR-3568 in simulated I/R-induced apoptosis in H9C2 cardiomyocytes and related signaling pathways involved. Flow cytometry was performed to examine the cell apoptosis. The expression of miR-3568, Survivin, Bcl-2, ERK, JNK, p38, AKT, and STAT3 was measured by western blot and quantitative real-time PCR. The correlation between TRIM62 and p-STAT3 was measured by co-immunoprecipitation and ubiquitination. We found that miR-3568 expression in simulated I/R-induced H9C2 cardiomyocytes was increased in a time-dependent manner. miR-3568 mimic transfection in H9C2 cardiomyocytes significantly enhanced cell apoptosis, decreased the expression of Bcl-2 and Survivin, and activated STAT3 signaling, which were reversed by miR-3568 inhibitor. The direct interaction between miR-3568 and the 3'-untranslated region (UTR) of TRIM62 mRNA was confirmed by dual-luciferase reporter assay. TRIM62 overexpression or AG490, a selective inhibitor of JAK2/STAT3 significantly, significantly inhibited I/R and miR-3568 mimic induced cell apoptosis and STAT3 activation. TRIM62 was found to interact with and induce ubiquitination of p-STAT3. The facilitating role of miR-3568 in I/R injury was also observed in our in vivo rat models. In conclusion, our study suggests that miR-3568 promotes simulated I/R-induced apoptosis in H9C2 cardiomyocytes through targeting TRIM62.

[Observation on therapeutic effect of "Tiaoren Tongdu acupuncture" on premature ovarian insufficiency of kidney deficiency].

OBJECTIVE: To observe the clinical efficacy of "Tiaoren Tongdu acupuncture" and oral estradiol and dydrogesterone tablets (femoston) on premature ovarian insufficiency of kidney deficiency. METHODS: A total of 50 patients with premature ovarian insufficiency of kidney deficiency were randomized into an observation group and a control group, 25 cases in each one.In the observation group, "Tiaoren Tongdu acupuncture" was applied at Baihui (GV 20), Zhongwan (CV 12), Guanyuan (CV 4), Qihai (CV 6), Zhongji (CV 3), Yaoyangguan (GV 3), Yaoshu (GV 2), Mingmen (GV 4), etc. once every 2 days, 1 month as a course. In the control group, femoston was prescribed for oral administration, one tablet per time, once a day, 1 month as a course. Both of the two groups were given consecutive treatment for 3 courses. Before and after treatment, the clinical symptoms, menstrual improvement as well as the changes of estradiol (E2), luteotrophic hormone (LH) and follicle-stimulating hormone (FSH) in serum were observed in the two groups. RESULTS: After treatment, the clinical symptoms and menstrual conditions were improved (P<0.01), the levels of FSH and LH were significantly reduced (P<0.01), and the levels of E2 were significantly increased in the two groups (P<0.01). There were no significant difference in menstrual improvement rate and menstrual improvement time between the observation group and the control group (P<0.05), the recurrence rate of menopause and clinical symptom score improvement in the observation group were superior to the control group (P<0.05). In the observation group, the level of E2 in serum was lower and the levels of FSH and LH in serum were significantly lower than those in the control group (P<0.05, P<0.01). In the observation group, the rate of adverse reaction was 4.0% (1/25), which was lower than 36.0% (9/25) in the control group (P<0.05). CONCLUSION: "Tiaoren Tongdu acupuncture" has better therapeutic effect for premature ovarian insufficiency of kidney deficiency. It is superior to femoston in improving clinical symptoms and recurrence rate of menopause as well as reducing the levels of FSH and LH.

The pattern-recognition molecule mindin binds integrin Mac-1 to promote macrophage phagocytosis via Syk activation and NF-κB p65 translocation.

Mindin has a broad spectrum of roles in the innate immune system, including in macrophage migration, antigen phagocytosis and cytokine production. Mindin functions as a pattern-recognition molecule for microbial pathogens. However, the underlying mechanisms of mindin-mediated phagocytosis and its exact membrane receptors are not well established. Herein, we generated mindin-deficient mice using the CRISPR-Cas9 system and show that peritoneal macrophages from mindin-deficient mice were severely defective in their ability to phagocytize E  coli. Phagocytosis was enhanced when E  coli or fluorescent particles were pre-incubated with mindin, indicating that mindin binds directly to bacteria or non-pathogen particles and promotes phagocytosis. We defined that 131 I-labelled mindin binds with integrin Mac-1 (CD11b/CD18), the F-spondin (FS)-fragment of mindin binds with the αM -I domain of Mac-1 and that mindin serves as a novel ligand of Mac-1. Blockade of the αM -I domain of Mac-1 using either a neutralizing antibody or si-Mac-1 efficiently blocked mindin-induced phagocytosis. Furthermore, mindin activated the Syk and MAPK signalling pathways and promoted NF-κB entry into the nucleus. Our data indicate that mindin binds with the integrin Mac-1 to promote macrophage phagocytosis through Syk activation and NF-κB p65 translocation, suggesting that the mindin/Mac-1 axis plays a critical role during innate immune responses.

Therapeutic targeting of liver cancer with a recombinant DNA vaccine containing the hemagglutinin-neuraminidase gene of Newcastle disease virus via apoptotic-dependent pathways.

A total of ~38.6 million mortalities occur due to liver cancer annually, worldwide. Although a variety of therapeutic methods are available, the efficacy of treatment at present is extremely limited due to an increased risk of malignancy and inherently poor prognosis of liver cancer. Gene therapy is considered a promising option, and has shown notable potential for the comprehensive therapy of liver cancer, in keeping with advances that have been made in the development of cancer molecular biology. The present study aimed to investigate the synergistic effects of the abilities of the hemagglutinin neuraminidase protein of Newcastle disease virus (NDV), the pro-apoptotic factor apoptin from chicken anaemia virus, and the interferon-γ inducer interleukin-18 (IL-18) in antagonizing liver cancer. Therefore, a recombinant DNA plasmid expressing the three exogenous genes, VP3, IL-18 and hemagglutinin neuraminidase (HN), was constructed. Flow cytometry, acridine orange/ethidium bromide staining and analysis of caspase-3 activity were performed in H22 cell lines transfected with the recombinant DNA plasmid. In addition, 6-week-old C57BL/6 mice were used to establish a H22 hepatoma-bearing mouse model. Mice tumor tissue was analyzed by immunohistochemistry and scanning electron microscopy. The results of the present study revealed that the recombinant DNA vaccine containing the VP3, IL-18 and HN genes inhibited cell proliferation and induced autophagy via the mitochondrial pathway in vivo and in vitro.

Dragon (RGMb) induces oxaliplatin resistance in colon cancer cells.

Colorectal cancer (CRC) is one of the most commonly diagnosed cancers and a major cause of cancer mortality. Chemotherapy resistance remains a major challenge for treating advanced CRC. Therefore, the identification of targets that induce drug resistance is a priority for the development of novel agents to overcome resistance. Dragon (also known as RGMb) is a member of the repulsive guidance molecule (RGM) family. We previously showed that Dragon expression increases with CRC progression in human patients. In the present study, we found that Dragon inhibited apoptosis and increased viability of CMT93 and HCT116 cells in the presence of oxaliplatin. Dragon induced resistance of xenograft tumor to oxaliplatinin treatment in mice. Mechanistically, Dragon inhibited oxaliplatin-induced JNK and p38 MAPK activation, and caspase-3 and PARP cleavages. Our results indicate that Dragon may be a novel target that induces drug resistance in CRC.

Immunoglobulin E induces colon cancer cell apoptosis via enhancing cyp27b1 expression.

The pathogenesis of colon cancer (Cca) is to be further investigated. Vitamin D deficiency is associated with cancer growth; the underlying mechanism is unclear. Published data indicate that Cca cells express CD23. This study tests a hypothesis that exposure to IgE induces Cca cell apoptosis. In this study, the effect of ligation of CD23 by IgE on the expression of cyp27b1 was performed with Cca cells. The induction of apoptosis of Cca cells by IgE was assessed in a cell culture model. We observed that Cca cells express CD23; ligation of CD23 with IgE on Cca cells increased the expression of cyp27b1 in Cca, which promoted the conversion of VD3 to calcitriol, the latter increased the expression of FasL by Cca cells, and induced apoptosis of Cca cells. In conclusion, IgE is capable of inducing the cancer cell apoptosis via ligating CD23 and converting VD3 to calcitriol. The results suggest that IgE may have therapeutic potential in the treatment of Cca.

Exome sequencing identifies novel compound heterozygous IFNA4 and IFNA10 mutations as a cause of impaired function in Crohn's disease patients.

Previous studies have highlighted the role of genetic predispositions in disease, and several genes had been identified as important in Crohn's disease (CD). However, many of these genes are likely rare and not associated with susceptibility in Chinese CD patients. We found 294 shared identical variants in the CD patients of which 26 were validated by Sanger sequencing. Two heterozygous IFN variants (IFNA10 c.60 T > A; IFNA4 c.60 A > T) were identified as significantly associated with CD susceptibility. The single-nucleotide changes alter a cysteine situated before the signal peptide cleavage site to a stop code (TGA) in IFNA10 result in the serum levels of IFNA10 were significantly decreased in the CD patients compared to the controls. Furthermore, the IFNA10 and IFNA4 mutants resulted in an impairment of the suppression of HCV RNA replication in HuH7 cells, and the administration of the recombinant IFN subtypes restored DSS-induced colonic inflammation through the upregulation of CD4(+) Treg cells. We identified heterozygous IFNA10 and IFNA4 variants as a cause of impaired function and CD susceptibility genes in Chinese patients from multiple center based study. These findings might provide clues in the understanding of the genetic heterogeneity of CD and lead to better screening and improved treatment.

Depressive neurosis treated by acupuncture for regulating the liver--a report of 176 cases.

OBJECTIVE: To observe therapeutic effect of acupuncture for regulating the liver on depressive neurosis. METHODS: In a multi-center randomized controlled trial, 440 patients were divided into 3 groups: Acupuncture group for regulating the liver (Acup., 176 cases) was treated by acupuncture at Siguan Points, i.e., bilateral Hegu (LI 4) and Taichong (LR 3), Baihui (GV 20) and Yintang (EX-HN3) plus ear-acupuncture, Prozac group (P., 176 cases) by oral administration of Prozac, and Non-acupoint needling group (NAN, 88 cases) by acupuncture at non-acupoints as acupuncture placebo. Self-rating Depression Scale (SDS) was examined before treatment, and one month, two and three months after treatment respectively to evaluate therapeutic effect, and Rating Scale for Side Effects (SERS) was used to evaluate the safety. RESULTS: After one month of treatment, SDS scores in Acup. Group were significantly lower than that in P. Group (P < 0.05) and than that in NAN Group (P < 0.01), and SDS scores in P. Group were lower than that in NAN Group (P < 0.05), showing the SDS scores in Acup. Group < P. Group < NAN Group. After 2 months of treatment, SDS scores in Acup. Group were also significantly lower than that in P. Group (P < 0.01) and than that in NAN Group (P < 0.01), and SDS scores in P. Group were also lower than that in NAN Group (P < 0.05), showing the SDS scores in Acup. Group 0.05), showing the SERS scores in Acup. Group < NAN Group < P. Group. No side effect was found in Acup. and NAN groups. CONCLUSION: The therapeutic effect of acupuncture on depressive neurosis is better than or similar to that of Prozac but with less side effect.

Differentiation of Wharton's jelly primitive stromal cells into insulin-producing cells in comparison with bone marrow mesenchymal stem cells.

Primitive stromal cells can be isolated from umbilical cord Wharton's jelly (UC-PSCs). Umbilical cord can be easily obtained without causing pain to donors, and the procedure avoids ethical and technical issues. UC-PSCs are more primitive than mesenchymal stem cells (MSCs) isolated from some other tissue sources. In this study, UC-PSCs were induced to differentiate into insulin-producing cells, and compared with bone marrow-derived MSCs (BM-MSCs) for their pancreatic differentiation potential. UC-PSCs showed significantly higher proliferation than BM-MSCs. During pancreatic induction, UC-PSCs formed larger islet-like cell clusters than BM-MSCs. Immunocytochemical analysis showed that higher expression of the pancreatic-specific transcription factor PDX-1 was detected in differentiated UC-PSCs than in differentiated BM-MSCs. Flow cytometry analysis demonstrated that the percentage of differentiated UC-PSCs expressing pancreatic-specific marker C-peptide was 72% higher than differentiated BM-MSCs. Radioimmunoassay revealed that differentiated UC-PSCs secreted significantly more insulin than differentiated BM-MSCs. These results demonstrated that UC-PSCs had higher pancreatic differentiation potential than BM-MSCs. Therefore, UC-PSCs are more suitable for pancreatic tissue engineering in the treatment of type I diabetes than BM-MSCs.

Biocompatibility of poly(3-hydroxybutyrate-co-3-hydroxyvalerate-co-3-hydroxyhexanoate) with bone marrow mesenchymal stem cells.

As a new member of the polyhydroxyalkanoate (PHA) family, poly(3-hydroxybutyrate-co-3-hydroxyvalerate-co-3-hydroxyhexanoate) (PHBVHHx) was produced by recombinant Aeromonas hydrophila 4AK4. PHBVHHx showed a rougher surface and had higher hydrophobicity than the well-studied polymers poly(L-lactic acid) (PLA) and poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx). Human bone marrow mesenchymal stem cells (MSCs) adhered better on PHBVHHx film than on tissue culture plates (TCPs), PLA film and PHBHHx film. The cell number on the PHBVHHx film was 115% higher than that on the TCPs, 66% higher than on the PHBHHx film and 263% higher than on the PLA film (p<0.01). PHBVHHx also supported the osteogenic differentiation of MSCs. Previous studies have shown that all PHA polymers tested were either poorer than or equal to TCPs for supporting cell growth. PHBVHHx is the only PHA polymer to significantly increase cell numbers compared with TCPs. These data demonstrate that PHBVHHx could be a promising biomaterial for bone tissue engineering.

Promoting effects of serotonin on hematopoiesis: ex vivo expansion of cord blood CD34+ stem/progenitor cells, proliferation of bone marrow stromal cells, and antiapoptosis.

Serotonin is a monoamine neurotransmitter that has multiple extraneuronal functions. We previously reported that serotonin exerted mitogenic stimulation on megakaryocytopoiesis mediated by 5-hydroxytryptamine (5-HT)2 receptors. In this study, we investigated effects of serotonin on ex vivo expansion of human cord blood CD34+ cells, bone marrow (BM) stromal cell colony-forming unit-fibroblast (CFU-F) formation, and antiapoptosis of megakaryoblastic M-07e cells. Our results showed that serotonin at 200 nM significantly enhanced the expansion of CD34+ cells to early stem/progenitors (CD34+ cells, colony-forming unit-mixed [CFU-GEMM]) and multilineage committed progenitors (burst-forming unit/colony-forming unit-erythroid [BFU/CFU-E], colony-forming unit-granulocyte macrophage, colony-forming unit-megakaryocyte, CD61+ CD41+ cells). Serotonin also increased nonobese diabetic/severe combined immunodeficient repopulating cells in the expansion culture in terms of human CD45+, CD33+, CD14+ cells, BFU/CFU-E, and CFU-GEMM engraftment in BM of animals 6 weeks post-transplantation. Serotonin alone or in addition to fibroblast growth factor, platelet-derived growth factor, or vascular endothelial growth factor stimulated BM CFU-F formation. In M-07e cells, serotonin exerted antiapoptotic effects (annexin V, caspase-3, and propidium iodide staining) and reduced mitochondria membrane potential damage. The addition of ketanserin, a competitive antagonist of 5-HT2 receptor, nullified the antiapoptotic effects of serotonin. Our data suggest the involvement of serotonin in promoting hematopoietic stem cells and the BM microenvironment. Serotonin could be developed for clinical ex vivo expansion of hematopoietic stem cells for transplantation. Disclosure of potential conflicts of interest is found at the end of this article.

Inositol-1,4,5-trisphosphate and ryanodine-dependent Ca2+ signaling in a chronic dog model of atrial fibrillation.

Ca2+ signaling regulation plays an important role in triggering and/or maintaining atrial fibrillation (AF). Little is known about the relationship of the inositol-1,4,5-triphosphate receptors (InsP3Rs) and ryanodine receptors (RyRs) in left atrium to chronic AF. In this study, we investigated the expression and function of InsP3R1, InsP3R2 and RyR2 in a chronic dog model of AF. AF was induced in 6 dogs by rapid right atrial pacing for 24 weeks, and a sham procedure was performed in 5 dogs (control group). The intact left atrial myocytes were used to examine the expression and function of InsP3Rs, RyRs by BODIPY(O,R) TR-X ryanodine, heparin-fluorescein conjugate, and were stimulated by caffeine, ATP to release Ca2+ through RyRs, InsP3Rs separately. We also assessed the molecular components of left atrial tissue underlying the amount of RyR2, InsP3R1 and InsP3R2 determined by RT-PCR, immunohistochemistry and Western blot analysis. In the chronic AF group, the Ca2+ released through RyRs is not altered, but the Ca2+ released through InsP3Rs increased significantly. RyR2 distributed in cytosol of myocytes, cellular membrane; its expression significantly decreased in AF group compared to controls. InsP3R1 distributed in cytosol, InsP3R2 distributed not only in cytosol, cellular membrane, but also in nuclear envelope and intercalated discs. The InsP3R1 and InsP3R2 expression significantly increased in chronic AF group compared to controls. These results indicated that in a chronic dog model of AF, the expression and function of RyR2 down-regulated; on the contrary, the expression and function of InsP3R1, InsP3R2 up-regulated, and InsP3R2 may be the major InsP3Rs, which regulate intracellular or even intercellular Ca2+ signal transmission.

[Effect of 5-hydroxtryptamine on megakaryocytopoiesis--review].

5-hydroxtryptamine (5-HT, serotonin) has been recognized not only as a neurotransmitter and vasoactive agent, but also as a growth factor. 5-HT mainly binds to 5-HT(2) receptors or 5-HT(1) receptors on cell surface to stimulate cell proliferation through Ras or MAPK pathway in many cell types. It has been reported that 5-HT stimulates megakaryocytopoiesis via 5-HT receptors. The possible mechanism of 5-HT on the proliferation and differentiation of megakaryocytes (MK) has been discussed in this review article. In early stage of megakaryocytopoiesis, 5-HT may bind to 5-HT(2B) receptor on megakaryocytes, and promotes their proliferation and differentiation. In the late stage, 5-HT may involve in the platelet release procedure by inducing nitric oxide (NO) synthesis via 5-HT(2A) receptors. 5-HT can also antagonize the apoptotic effect induced by thrombospondin-1 (TSP-1) which is a platelet alpha granule protein and has synergic effect with platelet-derived growth factor (PDGF) to enhance megakaryocytes proliferation. Therefore, 5-HT is likely to be an important substance in the feedback regulation of thrombopoiesis. In this review the 5-HT and its receptors, 5-HT as cell growth factor, pathway of 5-HT stimulating cell proliferation and influance of 5-HT on MK-progenitor cells were summarized.

The effect of 5-hydroxtryptamine on the regulation of megakaryocytopoiesis.

5-Hydroxtryptamine (5-HT, serotonin) has been recognized not only as a neurotransmitter and vasoactive agent, but also as a growth factor. 5-HT mainly binds to 5-HT2 receptors or 5-HT1 receptors on cell surfaces to stimulate cell proliferation through Ras or MAPK (mitogen-activated protein kinase) pathways in many cell types. It has been reported that 5-HT stimulates megakaryocytopoiesis via 5-HT receptors (5-HTR). The possible mechanism by which 5-HT regulates the proliferation and differentiation of megakaryocytes (MK) is discussed in this review article. In early stages of megakaryocytopoiesis, 5-HT may bind to 5-HT2B receptors on MK to promote their proliferation and differentiation. In the late stages, 5-HT may be involved in platelet release by inducing nitric oxide (NO) synthesis via 5-HT2A receptors. 5-HT can also antagonize the apoptotic effect induced by thrombospondin-1 (TSP-1) which is a platelet alpha-granule protein and has synergic effects with platelet-derived growth factor (PDGF) to enhance MK proliferation. Therefore, 5-HT is likely to be an important substance in the feedback regulation of thrombopoiesis.

[Expression and function changes of ryanodine receptors and inositol 1,4,5-triphosphate receptors of atrial myocytes during atrial fibrillation].

OBJECTIVE: To investigate the expression and function changes of inositol 1,4,5-triphosphate receptor (IP3R) and ryanodine receptor (RyR) in the atrial myocytes during atrial fibrillation. METHODS: Ten adult mongrel dogs were randomly divided into 2 groups: 5 dogs underwent continuous rapid atrial pacing (500 beats/min) for twenty-four weeks to create persistent atrial fibrillation, and the other 5 size-matched dogs without pacemaker implantation were used as controls. Twenty-four weeks after the dogs' hearts were taken out and the canine atrial myocytes were isolated by enzymatic dissociation: fluorescent indicator Fluo-3/AM was added into the buffer to load the myocytes and then the Ca(2+) concentration was determined by confocal microscopy. BODIPY TR-X ryanodine was added into the buffer to stain the myocytes. Caffeine and ATP were added separately to stimulate the release of Ca(2+) from RyR. RESULTS: (1) The expression of RyR in the sarcoplasmic reticulum of the atrial myocytes of the control group was (2.70 +/- 0.23), significantly higher than that of the atrial fibrillation group (0.25 +/- 0.14, P < 0.05). RyR was expressed mostly around the nucleus and only expressed in a small amount in the nucleus in the atrial fibrillation group. However, it was not expressed in the nucleus of the control group. The expression of IP3R in the atrial fibrillation group was significantly higher than that of the control group (P < 0.05). (2) After caffeine stimulation, the concentration in the atrial myocytes of the control group was (1.74 +/- 0.16), significantly higher than that of the fibrillation group (1.26 +/- 0.06, P < 0.05). (3) After ATP stimulation the Ca(2+) concentration in the atrial myocytes of the control group was (1.23 +/- 0.23), not significantly increased in comparison with that before ATP stimulation; however, the Ca(2+) concentration in the atrial myocytes of the fibrillation group after ATP stimulation was (2.29 +/- 0.65), significantly increased in comparison with that before ATP stimulation (P < 0.05). CONCLUSIONS: (1) The expression of RyR is down-regulated, the function of RyR is decreased, and it is expressed in the nucleus during atrial fibrillation which shows that RyR is possibly translocated into the nucleus. (2) The expression of IP3R is up-regulated and the function of IP3R is increased during atrial fibrillation, which may be one of the major mechanisms of intracellular Ca(2+)-overload during atrial fibrillation.