N6-methyldeoxyadenosine modification difference contributes to homocysteine-induced mitochondrial perturbation in rat hippocampal primary neurons and PC12 cells.

Elevated homocysteine (Hcy) levels are detrimental to neuronal cells and contribute to cognitive dysfunction in rats. Mitochondria plays a crucial role in cellular energy metabolism. Interestingly, the damaging effects of Hcy in vivo and in vitro conditions exhibit distinct results. Herein, we aimed to investigate the effects of Hcy on mitochondrial function in primary neurons and PC12 cells and explore the underlying mechanisms involved. The metabolic intermediates of Hcy act as methyl donors and play important epigenetic regulatory roles. N6-methyldeoxyadenosine (6 mA) modification, which is enriched in mitochondrial DNA (mtDNA), can be mediated by methylase METTL4. Our study suggested that mitochondrial perturbation caused by Hcy in primary neurons and PC12 cells may be attributable to mtDNA 6 mA modification difference. Hcy could activate the expression of METTL4 within mitochondria to facilitate mtDNA 6 mA status, and repress mtDNA transcription, then result in mitochondrial dysfunction.

GLUT1-mediated microglial proinflammatory activation contributes to the development of stress-induced spatial learning and memory dysfunction in mice.

BACKGROUND: Stress is a recognized risk factor for cognitive decline, which triggers neuroinflammation involving microglial activation. However, the specific mechanism for microglial activation under stress and affects learning and memory remains unclear. METHODS: The chronic stress mouse model was utilized to explore the relationship between microglial activation and spatial memory impairment. The effect of hippocampal hyperglycemia on microglial activation was evaluated through hippocampal glucose-infusion and the incubation of BV2 cells with high glucose. The gain-and loss-of-function experiments were conducted to investigate the role of GLUT1 in microglial proinflammatory activation. An adeno-associated virus (AAV) was employed to specifically knockdown of GLUT1 in hippocampal microglia to assess its impact on stressed-mice. RESULTS: Herein, we found that chronic stress induced remarkable hippocampal microglial proinflammatory activation and neuroinflammation, which were involved in the development of stress-related spatial learning and memory impairment. Mechanistically, elevated hippocampal glucose level post-stress was revealed to be a key regulator of proinflammatory microglial activation via specifically increasing the expression of microglial GLUT1. GLUT1 overexpression promoted microglial proinflammatory phenotype while inhibiting GLUT1 function mitigated this effect under high glucose. Furthermore, specific downregulation of hippocampal microglial GLUT1 in stressed-mice relieved microglial proinflammatory activation, neuroinflammation, and spatial learning and memory injury. Finally, the NF-κB signaling pathway was demonstrated to be involved in the regulatory effect of GLUT1 on microglia. CONCLUSIONS: We demonstrate that elevated glucose and GLUT1 expression induce microglia proinflammatory activation, contributing to stress-associated spatial memory dysfunction. These findings highlight significant interplay between metabolism and inflammation, presenting a possible therapeutic target for stress-related cognitive disorders.

Blood-brain barrier dysfunction mediated by the EZH2-Claudin-5 axis drives stress-induced TNF-α infiltration and depression-like behaviors.

Growing evidence suggests that neurovascular dysfunction characterized by blood-brain barrier (BBB) breakdown underlies the development of psychiatric disorders, such as major depressive disorder (MDD). Tight junction (TJ) proteins are critical modulators of homeostasis and BBB integrity. TJ protein Claudin-5 is the most dominant BBB component and is downregulated in numerous depression models; however, the underlying mechanisms remain elusive. Here, we demonstrate a molecular basis of BBB breakdown that links stress and depression. We implemented an animal model of depression, chronic unpredictable mild stress (CUMS) in male C57BL/6 mice, and showed that hippocampal BBB breakdown was closely associated with stress vulnerability. Concomitantly, we found that dysregulated Cldn5 level coupled with repression of the histone methylation signature at its promoter contributed to stress-induced BBB dysfunction and depression. Moreover, histone methyltransferase enhancer of zeste homolog 2 (EZH2) knockdown improved Cldn5 expression and alleviated depression-like behaviors by suppressing the tri-methylation of lysine 27 on histone 3 (H3K27me3) in chronically stressed mice. Furthermore, the stress-induced excessive transfer of peripheral cytokine tumor necrosis factor-α (TNF-α) into the hippocampus was prevented by Claudin-5 overexpression and EZH2 knockdown. Interestingly, antidepressant treatment could inhibit H3K27me3 deposition at the Cldn5 promoter, reversing the loss of the encoded protein and BBB damage. Considered together, these findings reveal the importance of the hippocampal EZH2-Claudin-5 axis in regulating neurovascular function and MDD development, providing potential therapeutic targets for this psychiatric illness.

Norepinephrine promotes glioma cell migration through up-regulating the expression of Twist1.

BACKGROUND: Glioma cells are characterized by high migration ability, resulting in aggressive growth of the tumors and poor prognosis of patients. It has been reported that the stress-induced hormone norepinephrine (NE) contributes to tumor progression through mediating a number of important biological processes in various cancers. However, the role of NE in the regulation of glioma migration is still unclear. Epithelial-to-mesenchymal transition (EMT) is one of the most important steps for tumor migration and metastasis. Twist1, as a key regulator of EMT, has been found to be elevated during glioma migration. But it is still unknown whether Twist1 is involved in the effect of NE on the migration of glioma cells. METHODS: Wound healing assay and transwell assay were conducted to evaluate the migration of glioma cells upon different treatments. The mesenchymal-like phenotype and the expression of Twist1 after NE treatment were assessed by cell diameters, real-time PCR, western blot and immunofluorescence staining. The gain-and loss-of-function experiments were carried out to investigate the biological function of Twist1 in the migration induced by NE. Finally, the clinical significance of Twist1 was explored among three public glioma datasets. RESULTS: In this study, our finding revealed a facilitative effect of NE on glioma cell migration in a ß-adrenergic receptor (ADRB)-dependent way. Mechanistically, NE induced mesenchymal-like phenotype and the expression of Twist1. Twist1 overexpression promoted glioma cells migration, while knockdown of Twist1 abolished the discrepancy in the migration ability between NE treated glioma cells and control cells. In addition, the clinical analysis demonstrated that Twist1 was up-regulated in malignant gliomas and recurrent gliomas, and predicted a poor prognosis of glioma patients. CONCLUSIONS: NE enhanced the migration ability of glioma cells through elevating the expression of Twist1. Our finding may provide potential therapeutic target for protecting patients with glioma from the detrimental effects of stress biology on the tumor progression.

Chronic stress promotes glioma cell proliferation via the PI3K/Akt signaling pathway.

High malignancy and high mortality of glioma render it urgent to elucidate the underlying mechanisms of glioma carcinogenesis and explore novel targets for therapy. Epidemiologic and clinical studies have revealed that chronic stress promotes the progression of various solid tumors and is correlated with poor prognosis; however, findings reporting the involvement of chronic stress in glioma are rare. In the present study, a chronic restraint animal model and a chronic stress cell model were established to explore the effects of chronic stress on glioma and its molecular mechanisms. The results revealed that chronic stress promoted glioma growth in vivo, and the serum levels of the stress hormones glucocorticoid (GC) and noradrenaline (NE) were significantly increased. In addition, GC and NE were verified to accelerate the proliferation of glioma cells in vitro. Mechanistically, the phosphatidylinositol 3­kinase (PI3K)/Akt signaling pathway was revealed to be activated under stress conditions, and inhibition of the expression of p­Akt could restrain the stress hormone­induced glioma cell proliferation. In addition, our data indicated that the GC receptor (GR) and ß­adrenergic receptors (ADRBs) were both required for the biological functions of GC and NE in glioma cells. In conclusion, these results indicated that chronic stress and the stress hormones GC and NE activated PI3K/Akt signaling through binding to GR and ADRBs, thereby promoting glioma cell growth. Our findings may provide potential therapeutic targets and pave the way for the development of new strategies to protect patients with glioma from the detrimental effects of stress on tumor progression.

Mesenchymal stem cells modified by FGF21 and GLP1 ameliorate lipid metabolism while reducing blood glucose in type 2 diabetic mice.

OBJECTIVE: The purpose of this study was to investigate the therapeutic effects of genetically modified mesenchymal stem cells (MSCs) in the treatment of type 2 diabetes mellitus (T2DM) in order to identify a new method for treating diabetes that differs from traditional medicine and to provide a new means by which to fundamentally improve or treat diabetes. METHODS: MSCs derived from adipose tissue were modified to overexpress FGF21 and GLP1, which was achieved through lentiviral particle transduction. The cells were transplanted into BKS.Cg-Dock7m+/+Leprdb/Nju mice (T2DM mouse model). Injections of physiological saline (0.1 mL) and liraglutide (0.5 mg/kg) were used as negative and positive controls, respectively. ELISA or Western blotting was used for protein analysis, and quantitative real-time PCR was used for gene expression analysis. RESULTS: Genetic modification had no effects on the morphology, differentiation ability, or immunophenotype of MSCs. Moreover, MSC-FGF21+GLP1 cells exhibited significantly increased secretion of FGF21 and GLP1. In the T2DM mouse model, the transplantation of MSC-FGF21+GLP1 cells ameliorated the changes in blood glucose and weight, promoted the secretion of insulin, enhanced the recovery of liver structures, and improved the profiles of lipids. Moreover, FGF21 and GLP1 exerted synergistic effects in the regulation of glucolipid metabolism by controlling the expression of insulin, srebp1, and srebp2. CONCLUSION: Stem cell treatment based on MSCs modified to overexpress the FGF21 and GLP1 genes is an effective approach for the treatment of T2DM.

BNIP3L promotes cardiac fibrosis in cardiac fibroblasts through [Ca2+]i-TGF-ß-Smad2/3 pathway.

Fibrosis is an important, structurally damaging event that occurs in pathological cardiac remodeling, leading to cardiac dysfunction. BNIP3L is up-regulated in pressure overload-induced heart failure and has been reported to play an important role in cardiomyocyte apoptosis; however, its involvement in cardiac fibroblasts (CFs) remains unknown. We prove for the first time that the expression of BNIP3L is significantly increased in the CFs of rats undergoing pressure overload-induced heart failure. Furthermore, this increased BNIP3L expression was confirmed in cultured neonatal rat CFs undergoing proliferation and extracellular matrix (ECM) protein over-expression that was induced by norepinephrine (NE). The overexpression or suppression of BNIP3L promoted or inhibited NE-induced proliferation and ECM expression in CFs, respectively. In addition, [Ca2+]i, transforming growth factor beta (TGF-ß) and the nuclear accumulation of Smad2/3 were successively increased when BNIP3L was overexpressed and reduced when BNIP3L was inhibited. Furthermore, the down-regulation of TGF-ß by TGF-ß-siRNA attenuated the increase of BNIP3L-induced fibronectin expression. We also demonstrated that the increase of BNIP3L in CFs was regulated by NE-AR-PKC pathway in vitro and in vivo. These results reveal that BNIP3L is a novel mediator of pressure overload-induced cardiac fibrosis through the [Ca2+]i-TGF-ß-Smad2/3 pathway in CFs.

Diet-induced elevation of circulating HSP70 may trigger cell adhesion and promote the development of atherosclerosis in rats.

Although accumulating evidence indicates that heat shock protein 70 (HSP70) could be secreted into plasma and its levels have been found to have an ambiguous association with atherosclerosis, our knowledge for the exact role of circulating HSP70 in the development of atherosclerosis is still limited. In the present study, we report an adhesion-promoting effect of exogenous HSP70 and evaluate the potential involvement of elevated circulating HSP70 in the development of atherosclerosis. Time-dependent elevation of plasma HSP70 was found in diet-induced atherosclerotic rats, whose effect was investigated through further in vitro experiments. In rat aortic endothelial cell (RAEC) cultures, exogenous HSP70 incubation neither produced cell injuries by itself nor had protective effects on cell injuries caused by Ox-LDL or homocysteine. However, exogenous HSP70 administration could lead to a higher adhesion rate between rat peripheral blood monocytes (PBMCs) and RAECs. This adhesion-promoting effect appeared only when PBMCs, rather than RAECs, were pretreated with HSP70 incubation. PBMCs in an HSP70 environment released more IL-6 to supernatant, which subsequently up-regulated the expression of ICAM-1 in RAECs. These results indicate that the diet-induced elevation of circulating HSP70 could trigger cell adhesion with the help of IL-6 as a mediator, which provides a novel possible mechanism for understanding the role of circulating HSP70 in the pathogenesis of atherosclerosis.

[Detection of endotoxin activity in water environment and analysis of influence factors for TAL assay].

Endotoxins, derived from cell walls of most Gram-negative bacteria and some cyanobacteria, are common pyrogen and highly immunogenic molecules, and related to many diseases. In this paper, a detection method for endotoxin activity in water environment using kinetic-turbid assay of Tachypleus Amebocyte Lysate (TAL) was established, the influence of pH and salts on TAL assay was investigated. Results showed that it was favorable for TAL assay in the pH range of 6.0-8.4, at low pHs, inhibition results were observed and opposite results were obtained at high pHs. The pH should be adjusted by Tris-HCl (pH = 7.4) buffer before the endotoxin detection. No significant interference was shown in the detection of water containing NaCl, Na2SO4, CaCl2, MgCl2 and KCl with a concentration of less than 50 mg x L(-1), however, the inhibition occurred at the concentration up to 1000-10,000 mg x L(-1). Only 2. 5 mg x L(-1) of FeCl, Fe2(SO4)3, AlCl3 and Al2 (SO4)3 caused significant inhibition. Endotoxin activities of ultrapure water, tap water and recreational water were detected by TAL assay, and their endotoxin activities were < 0.06 EU x mL(-1), 0.46 EU x mL(-1) and 432. 68 EU x mL(-1), respectively.

Inhibition of cystathionine ß-synthase is associated with glucocorticoids over-secretion in psychological stress-induced hyperhomocystinemia rat liver.

Hyperhomocysteinemia (HHcy), a pathological condition characterized by an increase in plasma concentration of total homocysteine (Hcy), is recognized as a risk factor for several diseases. The transsulfuration pathway is the main metabolic fate of Hcy utilization, which requires the activity of cystathionine ß-synthase (CBS). Our results showed the development of HHcy induced by psychological stress was mainly derived from a reduction of CBS activity in the liver, which was accompanied by a significant decrease in its mRNA level. It suggested that the hepatic CBS enzyme regulated by stress at the level of transcription would have a profound effect on circulating Hcy levels. The expression of Sp3, a negative factor for cbs transcription, obviously increased in hepatocytes nuclei of stressed rats, but Sp1 was not altered. It indicated that Sp3 was the key point of variations in cbs transcription caused by stress. Meanwhile, we detected that augmented plasma Hcy concentrations correlated with glucocordicoids (GCs) over-secretion in response to stress, and CBS mRNA levels were markedly lowered in GCs-treated rat hepatocytes. Further results found that glucocorticoids receptor (GR) expression in hepatocyte nuclei of stress rats and GR nuclear translocation ratio was increased, and the same results were proved by experiments in vitro, i.e., GR nuclear translocation and Sp3 expression was remarkably increased in GCs-treated hepatocytes. Moreover, results from ChIP suggested GCs enhanced the binding of GR to the regulatory region of the Sp3 promoter. These results indicated that GCs inhibit CBS transcription by up-regulating Sp3 in psychological stress-induced HHcy.

Prohibitin protects against oxidative stress-induced cell injury in cultured neonatal cardiomyocyte.

Oxidative stress is one of the main causes of myocardial injury, which is associated with cardiomyocyte death. Mitochondria play a key role in triggering the necrosis and apoptosis pathway of cardiomyocytes under oxidative stress. Although prohibitin (PHB) has been acknowledged as a mitochondrial chaperone, its functions in cardiomyocytes are poorly characterized. The present research was designed to investigate the cardioprotective role of PHB in mitochondria. Oxidative stress can increase the PHB content in mitochondria in a time-dependent manner. Overexpression of PHB in cultured cardiomyocytes by transfection of recombinant adenovirus vector containing PHB sense cDNA resulted in an increase of PHB in mitochondria. Compared with the non-transfection cardiomyocytes, PHB overexpression could protect the mitochondria from oxidative stress-induced injury. The mitochondria-mediated apoptosis pathway was consistently suppressed in PHB-overexpressed cardiomyocytes after hydrogen peroxide (H(2)O(2)) treatment, including a reduced change in mitochondrial membrane permeability transition and an inhibited release of cytochrome c from mitochondria to cytoplasma. As a result, the oxidative stress-induced cardiomyocyte apoptosis was suppressed. These data indicated that PHB protected the cardiomyocytes from oxidative stress-induced damage, and that increasing PHB content in mitochondria constituted a new therapeutic target for myocardium injury.

Hsp70 may protect cardiomyocytes from stress-induced injury by inhibiting Fas-mediated apoptosis.

Expression of Hsp70 is an endogenous mechanism by which living cells adapt to stress and the protection of Hsp70 may interfere with the apoptotic machinery in a variety of ways. Here, we observed the change of Hsp70 expression in rat myocardium under stress and explored the protective effect of Hsp70 on the Fas-mediated pathway to cardiomyocyte apoptosis. The results showed that restraint stress led to cardiac dysfunction and structural damage of the myocardium, as well as activation of the Fas pathway. A similar increase in the Fas expression level, caspase-8/3 activity, and the apoptotic rate of the cardiomyocyte also were found, which indicated that Fas-mediated apoptosis of cardiomyocytes might be one of the mechanisms of cardiomyocyte injury induced by stress. Changes in Hsp70 levels and distribution occurred during the stress process, which correlated with the severity of myocardium injury. Heat preconditioning induced the upregulation of Hsp70 synthesis, which in turn may have mitigated subsequent restraint stress-induced damage, including electrocardiography (ECG) abnormality, myocardium damage, and cell death. Moreover, Hsp70 overexpression induced by heat preconditioning had no effect on Fas expression in the cardiomyocyte, but could inhibit activation of caspase-8/3 induced by the Fas signaling pathway and, as a result, prevent cell apoptosis. These results suggest that Hsp70 is capable of protecting the cardiomyocyte from stress-induced injury by inhibiting Fas-mediated apoptosis, and Hsp70 could be considered a target in future drugs to prevent cardiovascular injury caused by stress.

Dedifferentiation of adult human myoblasts induced by ciliary neurotrophic factor in vitro.

Ciliary neurotrophic factor (CNTF) is primarily known for its important cellular effects within the nervous system. However, recent studies indicate that its receptor can be highly expressed in denervated skeletal muscle. Here, we investigated the direct effect of CNTF on skeletal myoblasts of adult human. Surprisingly, we found that CNTF induced the myogenic lineage-committed myoblasts at a clonal level to dedifferentiate into multipotent progenitor cells--they not only could proliferate for over 20 passages with the expression absence of myogenic specific factors Myf5 and MyoD, but they were also capable of differentiating into new phenotypes, mainly neurons, glial cells, smooth muscle cells, and adipocytes. These "progenitor cells" retained their myogenic memory and were capable of redifferentiating into myotubes. Furthermore, CNTF could activate the p44/p42 MAPK and down-regulate the expression of myogenic regulatory factors (MRFs). Finally, PD98059, a specific inhibitor of p44/p42 MAPK pathway, was able to abolish the effects of CNTF on both myoblast fate and MRF expression. Our results demonstrate the myogenic lineage-committed human myoblasts can dedifferentiate at a clonal level and CNTF is a novel regulator of skeletal myoblast dedifferentiation via p44/p42 MAPK pathway.

Mitochondrial mechanism of heat stress-induced injury in rat cardiomyocyte.

Heat stress results in cardiac dysfunction and even cardiac failure. To elucidate the cellular and molecular mechanism of cardiomyocyte injury induced by heat stress, the changes of structure and function in cardiac mitochondria of heat-exposed Wistar rats and its role in cardiomyocyte injury were investigated. Heat stress induced apoptosis and necrosis of cardiomyocytes in a time- and dose-dependent fashion. In the mitochondria of heat-stressed cardiomyocytes, the respiratory control rate and oxidative phosphorylation efficiency (P:O) were decreased gradually with the rise of rectal temperature. The Ca2+ -adenosine triphosphatase activity and Ca2+ content were also reduced. Exposing isolated mitochondria to the heat stress induced special internal environmental states including Ca2+ overload, oxidative stress, and altered mitochondrial membrane permeability transition (MPT). In vivo, the heat stress-induced mitochondrial MPT alteration was also found. The changes of mitochondrial MPT resulted in the release of cytochrome c from mitochondria into the cytosol, and in turn, caspase-3 was activated. Transfection of bcl-2 caused Bcl-2 overexpression in cardiomyocyte, which protected the mitochondria and reduced the heat stress-induced cardiomyocyte injury. In conclusion, it appears that the destruction of mitochondrial structure and function not only resulted in the impairment of physiological function of cardiomyocytes under heat stress but may also further lead to severe cellular injury and even cell death. These findings underline the contribution of mitochondria to the injury process in cardiomyocytes under heat stress.

Proteomic analysis of mitochondrial proteins in cardiomyocytes from chronic stressed rat.

Chronic restraint stress induces cardiac dysfunction as well as cardiomyocyte injury including severe ultrastructural alteration and cell death, but its mechanism and molecular basis remain unclear. Mitochondria play a key role in regulating cell life. For exploring mitochondrial proteins which correlate with stress-induced injury, two-dimensional electrophoresis and matrix-assisted laser desorption/ionization mass spectrometry (MALDI-TOF MS) were applied. After comparing the protein profiles of myocardial mitochondria between a chronic restraint stress group and a control group, 11 protein spots were found altered, seven of which were identified by MALDI-TOF MS. Among the seven proteins, five proteins involved in the Krebs cycle and lipid metabolism in mitochondria decreased after chronic restraint stress. They were identified as carnitine palmitoyltransferase 2, mitochondrial acyl-CoA thioesterase 1, isocitrate dehydrogenase 3 (NAD+) alpha, fumarate hydratase 1 and pyruvate dehydrogenase beta. The last two proteins, creatine kinase and prohibitin, increased after chronic restraint stress. Biochemical tests for energy metabolism in mitochondria also supported the proteomic results. These findings provide clues for understanding the mechanism of dysfunction or injury in cardiomyocytes induced by chronic stress.

[Negative regulation of homocysteine metabolism by stress in rats].

To investigate the effect of stress on homocysteine metabolism in the rat and explore the mechanism as well as the key regulatory link of stress-induced hyperhomocysteinemia, male Wistar rats were treated with restraint stress while control rats received routine treatment. By HPLC-fluorometry, the homocysteine level in rat plasma was determined. Cystathionine beta-synthase (CBS) activity in blood, heart, liver and kidney was measured by radioisotope assay using [(14)C]-serine as the labeled substrate. Total RNA was isolated from rat liver after restraint stress. RT-PCR and Northern blot were used to estimate the level of CBS mRNA. The results showed that hyperhomocysteinemia was induced by restraint stress. The highest CBS enzyme activity was seen in rat livers. A decrease in hepatic activities of CBS was found in restraint stress rats. The 29.4% +/-2.5% reduction in the activity of CBS was accompanied by a 44.1% +/-3.4% decrease in its mRNA level. CBS enzyme activity was slightly elevated in the kidney of stressed rats while it was almost undeterminable in the cardiovascular system. The study suggests that stress leads to an inhibition of the transsulfuration pathway in homocysteine metabolism. The hepatic CBS influenced by stress at the level of transcription exerts a profound effect on the circulating levels of homocysteine. The liver is the key organ where stress affects homocysteine metabolism.

Prokaryotic expression, polyclonal antibody preparation, and sub-cellular localization analysis of Na+, K+-ATPase beta2 subunit.

Na+, K+-ATPase beta2 subunit (NKA1b2) is not only a regulator of Na+, K+-ATPase, but also functions in the interaction between neuron and glia cells as a Ca2+-dependent adhesion molecule. To further study the function of NKA1b2, the anti-NKA1b2 polyclonal antibody was prepared to recognize the outer-membrane carboxyl portion segment of NKA1b2. The coding region for amino acids 190-290 at the carboxyl portion of NKA1b2 (NKA1b2-CP) was sub-cloned into the vector pGEX-4T-2 and introduced into the Escherichia coli BL21(DE3) cell for efficient soluble expression. The amino acid sequence of expressed protein was determined using mass spectrometry following Mascot analysis. After purification, GST-NKA-beta2-CP was used to immunize the adult rabbits following standard protocols. The produced antiserum could detect the NKA1b2 protein expressed not only in the prokaryotic cells (E. coli) but also in the eukaryotic cells (COS7) transfected with NKA1b2 expression vector (pEGFP-NKA1b2). Furthermore, the antiserum was used for determining the localization of NKA1b2 in primary culture of neonatal rat neurons using immunohistochemical technique. Results demonstrated that NKA1b2 was localized both in the cytoplasm and cellular membrane. The preparation of anti-NKA-beta2-CP polyclonal antibody will facilitate further functional study on NKA1b2.

[Identification of interaction and interaction domains between neuroglobin and Na(+), K(+)-ATPase beta2 subunit].

The pre-transformed human fetal brain cDNA library was used to screen the protein interacting with neuroglobin by using yeast two hybrid system III from ClonTech Inc. The protein encoded by one of the clones interacting with neuroglobin (NGB) was confirmed to be the C terminus of the Na(+), K(+)-ATPase beta2 subunit (NKA1b2) based on amino acid sequences. Then the full-length coding region cDNA sequence of NKA1b2 was obtained from human fetal brain cDNA library by PCR. A set of experiments were designed to test the interaction between NGB and NKA1b2. Interaction between NGB and NKA1b2 was confirmed by binding assay in vitro. Furthermore, the interaction was also proved by co-immunoprecipitation test in vivo. Moreover, the structure integrity of neuroglobin was found to be essential for the interaction between NGB and NKA1b2 by yeast two hybrid method with a series of neuroglobin truncated mutants.

[Mechanisms of protection effect of bcl-2 gene transfection on heat-stressed cardiomyocytes].

AIM: To study the mechanisms of protection of bcl-2 gene transfection against heat-stressed cardiomyocytes. METHODS: Cardiomyocytes were isolated and cultured. bcl-2 was transfected into cardiomyocytes with Lipofectamine transfection methods. The cardiomyocytes were stressed by heat. The change of H+ -ATPase synthesis activity of cardiomyocytes mitochondria caused by bcl-2 transfection was measured by chemical radiation method. The changes of Caspase 3 activity of cardiomyocytes caused by bcl-2 transfection was measured by fluorometric analysis. RESULTS: bcl-2 transfection could increase the H+ -ATPase synthesis activity of cardiomyocytes mitochondria under heat stress at 41 degrees C and 43 degrees C and could decrease the Caspase 3 activity of cardiomyocytes under heat stress at 41 degrees C and 43 degrees C. CONCLUSION: The protection effect of bcl-2 transfection on heat-stressed cardiomyocytes may be associated with preserved H+ ATPase synthesis activity of cardiomyocytes mitochondria and the activity of Caspase 3 of cardiomyocytes.