Apoptosis ; Calcineurin ; metabolism ; Chymotrypsin ; metabolism ; Humans ; Lysosomes ; enzymology ; Mitochondria ; metabolism ; pathology ; Mitochondrial Dynamics ; Neuroblastoma ; metabolism ; pathology

Calcineurin ; metabolism ; Cardiovascular System ; growth & development ; metabolism ; Humans ; NFATC Transcription Factors ; metabolism ; Signal Transduction

OBJECTIVETo discuss the significance of calcineurin (CaN) and nuclear factor of active T cells 1 (NFATc1) in the damage mechanism of the testis of rats with chronic fluorosis.

METHODSEighteen clear class SD male rats, aging 6 week-old, were randomly divided into 3 groups, 6 rats in each. The rats of control group were fed with tap water (NaF < 1 mg/L) and the experimental rats were exposed to NaF (lower group: 5 mg/L, higher group: 50 mg/L) to established the chronic fluorosis model. After 8 months, we observed the occurrence of dental fluorosis among rats in different groups, and the contents of urine fluoride were detected by fluorine ion selective electrode method. The body of the rats were weighted as well as their testis. The testis tissues were stained with hematoxylin-eosin and observed under light microscope to find the morphological changes. The expression of CaN and NFATc1's protein and mRNA in testis were detected by Immunocytochemistry (IHC) and In-situ hybridization (ISH).

RESULTSThe number of rats which was found dental fluorosis were separately 0, 4 and 5 in control group, low dose group and high dose group (χ(2) = 10.60, P < 0.05). The contents of urine fluoride were gradually increased in control group, low group and high group, which were (1.26 ± 0.17), (2.06 ± 0.64) and (7.69 ± 1.96)mg/L, respectively (F = 36.57, P < 0.05). The body weight were significantly different in all three groups(629.00 ± 16.00), (585.17 ± 17.27), (560.50 ± 16.07)g, F = 26.67, P < 0.05) and the testis weight were without statistical difference ((2.58 ± 0.17), (2.43 ± 0.31), (2.35 ± 0.38)g, F = 0.91, P > 0.05). Compared with the control group, the testicular structures were damaged in the experimental groups and especially significant in high dose group. The expression of CaN (59.10 ± 5.62, 77.93 ± 4.16, 101.69 ± 6.31, F = 74.18, P < 0.05) and NFATc1's (76.11 ± 4.41, 93.42 ± 3.85, 120.42 ± 9.31, F = 92.4, P < 0.05) protein in testis tissues were increased by the fluorine concentration. The mRNA expression of CaN and NFATc1 were separately (CaN: 58.76 ± 7.70, 82.01 ± 6.88, 99.47 ± 8.33, F = 42.65, P < 0.05 and NFATc1: 59.39 ± 4.74, 90.02 ± 5.37, 121.15 ± 7.69, F = 155.47, P < 0.05). There were positive correlation between the expression of CaN and NFATc1's protein and mRNA expression (r = 0.899, r = 0.908).

CONCLUSIONThe changes in the signaling pathway of expression of CaN may be involved in the injury mechanism of testis tissues of rats with chronic fluorosis.

Animals ; Calcineurin ; metabolism ; Fluoride Poisoning ; metabolism ; Male ; Rats ; Rats, Sprague-Dawley ; Signal Transduction ; Testis ; metabolism ; Transcription Factors ; metabolism

Amyotrophic lateral sclerosis (ALS) is the most common motor neuron disease. At present, no definite ALS biomarkers are available. In this study, exosomes from the plasma of patients with ALS and healthy controls were extracted, and differentially expressed exosomal proteins were compared. Among them, the expression of exosomal coronin-1a (CORO1A) was 5.3-fold higher than that in the controls. CORO1A increased with disease progression at a certain proportion in the plasma of patients with ALS and in the spinal cord of ALS mice. CORO1A was also overexpressed in NSC-34 motor neuron-like cells, and apoptosis, oxidative stress, and autophagic protein expression were evaluated. CORO1A overexpression resulted in increased apoptosis and oxidative stress, overactivated autophagy, and hindered the formation of autolysosomes. Moreover, CORO1A activated Ca²⁺-dependent phosphatase calcineurin, thereby blocking the fusion of autophagosomes and lysosomes. The inhibition of calcineurin activation by cyclosporin A reversed the damaged autolysosomes. In conclusion, the role of CORO1A in ALS pathogenesis was discovered, potentially affecting the disease onset and progression by blocking autophagic flux. Therefore, CORO1A might be a potential biomarker and therapeutic target for ALS.
Mice ; Animals ; Amyotrophic Lateral Sclerosis/pathology* ; Calcineurin/metabolism* ; Motor Neurons/pathology* ; Microfilament Proteins/metabolism* ; Cytoskeletal Proteins/metabolism*

AIMTo evaluate whether protein phosphorylation and dephosphorylation in nuclei play roles in the development of myocardial hypertrophy, distribution of protein kinases and phosphatases in cell fractions were determined.

METHODSThe model of hypertensive rat was established by abdominal aortic constriction. Velocity and isopyknic gradient centrifugation was employed to fractionate rat myocardium to membrane, cytosol and nuclei. Enzymatic methods were employed to determine kinases and phosphatases.

RESULTSCompare with control group, the activity of CaMK increased by 101.1% (P < 0.01) and 40.16% (P < 0.01) respectively in nuclear and membranous fractions, changed without significance in cytosolic fraction; the activity of calcineurin in nuclei increased by 43.57%, (P < 0.05), lightly changed without significance in membranous and cytosolic fractions.

CONCLUSIONNuclear translocation of CaMK and calcineurin, might play important roles on overload-induced cardiac hypertrophy.

Animals ; Calcineurin ; metabolism ; Calcium ; metabolism ; Calcium-Calmodulin-Dependent Protein Kinases ; metabolism ; Cardiomyopathy, Hypertrophic ; metabolism ; pathology ; physiopathology ; Cell Nucleus ; metabolism ; Male ; Rats ; Rats, Wistar

OBJECTIVETo investigate the effects of polybrominated diphenyl ether-153 (BDE-153) exposure during lactation period on the calcium ion (Ca(2+)) concentration and calcium-activated enzyme levels in cerebral cortical cells among adult rats and to provide a scientific basis for the study on the developmental neurotoxicity of BDE-153.

METHODSForty newborn male rats were randomly and equally divided into four groups according to their body weights and litters: 1, 5, and 10 mg/kg BDE-153 groups and olive oil solvent control group. On postnatal day 10 (PND 10), the BDE-153 groups were administrated BDE-153 (0.1 ml/10 g body weight) by intraperitoneal injection, while the olive oil solvent control group was given an equal volume of olive oil. Two months later, these rats were decapitated, and the cerebral cortex was separated quickly on an ice-cold dish. The Ca(2+) concentration in cerebral cortical cells was measured by flow cytometry. The activities of calcineurin (CaN) and Ca(2+)-Mg(2+)-ATP enzyme were determined by colorimetric method. The mRNA and protein expression of calpain-1 and calpain-2 was measured by real-time quantitative PCR and Western blot.

RESULTSThe mean fluorescence intensities of intracellular Ca(2+) in control group and 1, 5, and 10 mg/kg BDE-153 groups were 10.83, 1.48, 1.93, and 0.62, respectively; the 1, 5, and 10 mg/kg BDE-153 groups had significantly lower intercellular Ca(2+) concentrations than the control group (P < 0.05). The activities of CaN and Ca(2+)-Mg(2+)-ATP enzyme and mRNA and protein expression of calpain-1 showed no significant differences between the 1, 5, and 10 mg/kg BDE-153 groups and control group (P > 0.05). The protein expression of calpain-2 increased as the dose of BDE-153 rose. Compared with the control group (mRNA: 0.81±0.26; protein: 0.15±0.07), the 5 and 10 mg/kg BDE-153 groups had significantly higher mRNA expression of calpain-2 (5 mg/kg BDE-153 group: 1.16±0.52; 10 mg/kg BDE-153 group: 1.32±0.23) and significantly higher protein expression of calpain-2 (5 mg/kg BDE-153 group: 0.31±0.07; 10 mg/kg BDE-153 group: 0.37±0.06) (P < 0.05). The 10 mg/kg BDE-153 group had significantly higher protein expression of calpain-2 than the 1 mg/kg BDE-153 group (0.37±0.06 vs 0.22±0.07, P < 0.05).

CONCLUSIONCa(2+-) mediated calpain-2 activation may be one of the main mechanisms of BDE-153 neurotoxicity.

Animals ; Animals, Newborn ; Ca(2+) Mg(2+)-ATPase ; metabolism ; Calcineurin ; metabolism ; Calcium ; metabolism ; Calpain ; metabolism ; Cerebral Cortex ; metabolism ; Male ; Polybrominated Biphenyls ; toxicity ; Rats ; Rats, Sprague-Dawley

OBJECTIVETo investigate the regulation of calcineurin (CaN) by endoplasmic reticulum stress (ERS) in podocytes in vitro and in vivo at the stage microalbuminuria in diabetic nehropathy (DN).

METHODSThe urinary albumin excretions of C57BLKS/J (Lepr) db/db and db/m mice at the ages of 6, 9, and 12 weeks were measured. The expressions of CaN and synaptopodin of these mice were observed. In immortalized mouse podocytes, the expression of podocyte CaN incubated with different concentrations of paltimate was quantitatively determined by real-time PCR. The changes of CaN incubated with paltimate with or without ursodeoxy-cholic acid (UDCA) were analyzed by confocal microscopy and Western blotting.

RESULTSAs urine protein increased, the expression of CaN was enhanced and the expression of synaptopodin was reduced in early stage DN db/db mice potocytes. In immortalized mouse podocytes, as the concentrations of palmitate increased, CaN mRNA increased. By confocal microscopy, the fluorescence intensity of CaN increased in palmitate treatment group. After co-incubation with palmitate and UDCA, the fluorescence intensity decreased. The similar results were shown by Western blotting.

CONCLUSIONAt the stage of microalbuminuria in DN, ERS in podocytes up-regulates the expression of CaN.

Animals ; Calcineurin ; metabolism ; Cells, Cultured ; Diabetes Mellitus, Experimental ; metabolism ; Diabetic Nephropathies ; metabolism ; Endoplasmic Reticulum Stress ; Male ; Mice ; Mice, Inbred C57BL ; Microfilament Proteins ; metabolism ; Podocytes ; metabolism

OBJECTIVETo observe the effect of total ginsenosides (TG) on monocrotaline (MCT) induced right ventricular hypertrophy rats, and to explore its correlation with calcineurin (CaN) pathway.

METHODSFifty male Sprague Dawley rats were randomly divided into the normal control group, the MCT model group, and the low, middle, high dose TG treatment groups, 10 in each group. All medication was performed by peritoneal injection for 18 days. Right ventricular peak systolic pressure (RVSP), right ventricular hypertrophy index (RVHI), and right ventricular weight/body weight (RVW/BW) were measured. Intracellular free calcium concentrations were measured by Ca2+ fluorescence indicator Fura2/AM. The atrial natriuretic factor (ANF) and CaN mRNA expression of the myocardial tissue were quantitatively analyzed by Real-time PCR. The protein expression of CaN was detected by Western blot.

RESULTSCompared with the MCT model group, preventive treatment of TG at the 3 doses could significantly reduce RVSP, RVHI, RVW/BW, and ANF mRNA expression, and decrease Ca2+ concentration in myocardial cells, CaN mRNA and protein expression in the myocardial tissue.

CONCLUSIONTG could obviously improve MCT-induced right ventricular hypertrophy, which was possibly achieved through suppressing MCT-activated CaN signal transduction.

Animals ; Atrial Natriuretic Factor ; Calcineurin ; metabolism ; Calcineurin Inhibitors ; therapeutic use ; Ginsenosides ; therapeutic use ; Heart Ventricles ; Hypertrophy, Right Ventricular ; drug therapy ; metabolism ; Male ; Monocrotaline ; Myocardium ; Myocytes, Cardiac ; RNA, Messenger ; Rats ; Rats, Sprague-Dawley ; Signal Transduction

AIMTo investigate the significance of the calcineurin (CaN) activation in active lupus nephritis patient.

METHODSPeripheral blood mononuclear cells (PBMCs) were separated from twenty-one active LN patients and 12 healthy controls. Phosphatase activity of CaN was determined using the CaN assay kit by measuring the content of released PO4. Reverse transcription-PCR was used to detect the expression of CD40L mRNA. Flow cytometry analysis was used to detect the expression of CD40L in LN PBMC.

RESULTS(1) Increased activation of CaN in spontaneous cultured PBMC in active LN group was found as compared with control group (46.08 +/- 5.58 vs 8.81 +/- 3.61, P < 0.01). In stimulated by PMA/Ionomycin , activity of CaN in active LN group was also higher than that of control (69.34 +/- 12.59 vs 37.12 +/- 11.57, P < 0.01). (2) Relative content of CD40L in PBMC in active LN groups increased significantly as compared with the control groups under spontaneous and PMA/Ionomycin-induced culture, respectively (P < 0.01). (3) FK506 reduced significantly production of CD40L in spontaneous and PMA/Ionomycin-induced PBMC of LN.

CONCLUSIONElevated activation of CaN in active LN may participate in regulation overexpression of CD40L in PBMC of LN. Through inhibiting CaN activity, FK506 may prevent abnormal activation of CD40-CD40L costimulatory pathway in lupus nephritis.

Adolescent ; Adult ; CD40 Ligand ; metabolism ; Calcineurin ; metabolism ; Case-Control Studies ; Cells, Cultured ; Female ; Humans ; Leukocytes, Mononuclear ; metabolism ; Lupus Nephritis ; blood ; metabolism ; Male ; Middle Aged ; Tacrolimus ; pharmacology ; Young Adult

Calreticulin (CRT) is an essential Ca(2+)-binding chaperone existing in endoplasmic reticulum (ER) or sarcoplasmic reticulum (SR), and is involved in intracellular Ca(2+) homeostasis and protein folding. Ischemic postconditioning (I-postC), a newly discovered endogenous protective phenomenon, induces CRT up-regulation. The present study aimed to investigate the cardioprotective mechanism of CRT up-regulation induced by hypoxic postconditioning (H-postC). Primary cultured neonatal rat cardiomyocytes were exposed to 2 h of hypoxia followed by 24 h of reoxygenation. Postconditioning was carried out by two cycles of 10 min of reoxygenation and 20 min of rehypoxia after 2 h of hypoxia. Antisense oligodeoxynucleotides (AS-ODNs) were used to inhibit CRT expression 36 h before hypoxia. Cardiomyocytes were randomly divided into 6 groups as follows (n=4): control, hypoxia/reoxygenation (H/R), H-postC, AS, AS + H/R, and AS + H-postC. Morphological studies, lactate dehydrogenase (LDH) activity assay in culture medium, and flow cytometry were used to detect cardiomyocyte necrosis and apoptosis. Intracellular Ca(2+) concentration was detected by fluorescent Fluo-3/AM staining through laser confocal microscope, and p-nitrophenyl phosphate (PNPP) was used as substrate to measure calcineurin (CaN) activity. The expression of CRT, CaN, nuclear factor kappa B (NFκB) and apoptosis-related proteins, such as Bcl-2, Bax and C/EBP homologous protein (CHOP) were detected by Western blot. The results were as follows. (1) H-postC protected neonatal cardiomyocytes from H/R injury. Compared with H/R group, cell survival rate increased by 17.1%, apoptotic rate and LDH leakage decreased by 6.67% and 27.9% in H-postC group, respectively (P<0.05). (2) H-postC induced mild up-regulation of CRT expression. Inhibition of CRT by AS-ODNs attenuated the cardioprotection of H-postC partly. Compared with H-postC group, cell survival rate decreased by 8.98%, and apoptotic rate and LDH leakage increased by 1.74% and 13.6% in AS + H-postC group, respectively (P<0.05), but intracellular Ca(2+) concentration, CaN activity, and expression of CaN and NFκB did not change significantly (P>0.05), suggesting that CRT participates in endogenous protection, not through Ca(2+)-CaN pathway. (3) H-postC inhibited the expression of pro-apoptosis proteins such as Bax and CHOP, but induced up-regulation of anti-apoptosis protein Bcl-2. Inhibition of CRT by AS-ODNs partly inhibited the changes in apoptosis-related proteins expression induced by H-postC, suggesting that CRT participates in the anti-apoptosis effect of H-postC through regulating expression of apoptosis-related proteins. These results indicate that CRT up-regulation induced by H-postC is involved in the cardioprotection through regulating expression of apoptosis-related proteins, not through Ca(2+)-CaN pathway in neonatal cardiomyocytes.
Animals ; Apoptosis ; Calcineurin ; metabolism ; Calreticulin ; metabolism ; Cell Hypoxia ; Cell Survival ; Cells, Cultured ; Ischemic Postconditioning ; Myocytes, Cardiac ; metabolism ; Oxygen ; metabolism ; Rats ; Up-Regulation