The purpose of this study was to study the role of neurofilament (NF) mRNA and calpain in NF reduction of acrylamide (ACR) neuropathy. Male Wistar adult rats were injected i.p. every other day with ACR (20 mg/kg·bW or 40 mg/kg·bW) for 8 weeks. NF mRNA expression was detected using RT-PCR and the calpain concentration was determined using western blot analysis. The NF mRNA expression significantly decreased while the level of m-calpain and μ-calpain significantly increased in two ACR-treated rats groups regardless of the ACR dose. The light NF (NF-L) protein expression was significantly correlated with NF-L mRNA expression. Combined with previous data, the concentrations of three NF subunits were negatively correlated with the calpain levels. These findings suggest that NF-L mRNA and calpain mediated the reduction in NF of ACR neuropathy.
Acrylamide ; toxicity ; Animals ; Calpain ; metabolism ; Gene Expression Regulation ; drug effects ; Intermediate Filaments ; genetics ; Male ; Peripheral Nervous System Diseases ; chemically induced ; genetics ; metabolism ; RNA, Messenger ; genetics ; metabolism ; Rats

OBJECTIVETo investigate the splice variants of the calpain 3 gene existing in human skeletal muscle tissue and white blood cells, and to explore the feasibility of gene diagnosis using CAPN3 mRNA extracted from peripheral leukocytes.

METHODSTotal RNA was extracted from peripheral blood and skeletal muscle tissue in healthy individuals. CAPN3 cDNAs were determined by reverse transcriptase polymerase chain reaction and DNA sequencing. CAPN3 cDNAs from peripheral leukocytes were compared with sequences obtained from skeletal muscle tissue.

RESULTSRT-PCR and DNA sequencing showed that the CAPN3 cDNAs comprised 24 exons in human skeletal muscle tissue, while the number of exons was 23 in white blood cells. Exon 15 was spliced out in human white blood cells.

CONCLUSIONSplice variants exist in human skeletal muscle tissue and white blood cells. Gene diagnosis may omit the mutations of exon 15 using mRNA extracted from peripheral leukocytes. These findings suggest that mutation analysis of the CAPN3 cDNA should use skeletal muscle tissue as materials instead of peripheral blood.

Calpain ; genetics ; DNA Mutational Analysis ; DNA, Complementary ; genetics ; Exons ; genetics ; Humans ; Leukocytes ; metabolism ; Muscle Proteins ; genetics ; Muscle, Skeletal ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction

Primary neuronal culture is a powerful tool to study neuronal development, aging, and degeneration. However, cultured neurons show signs of cell death after 2 or 3 weeks. Although the mechanism underlying this phenomenon has not been elucidated, several preventive methods have been identified. Here we show that the neuronal loss in primary cortical culture involves calpain activation and subsequent neuronal cell death. Neuronal loss during cultivation showed destruction of neurites and synapses, and a decrease in neuron numbers. micro-Calpain and micro-calpain were initially activated and accumulated by increased RNA expression. This neuronal death exhibited neurodegenerative features, such as conversion of p35 to p25, which is important in the developmental process and in the pathogenesis of Alzheimer's disease. But, postnatal and aged rat cortex did not show calpain activation and prolonged processing of p35 to p25, in contrast to the long-term culture of cortical neurons. In addition, the inhibition of calpains by ALLM or ALLN blocked the conversion of p35 to p25, indicating that the calpain activity is essential for the neurodegenerative features of cell death. Taken together, this study shows that the neuronal loss in primary cortical cultures involves neurodegeneration-like cell death through the activation of calpains and the subsequent processing of p35 to p25, but not developmental apoptosis or aging. Our results suggest that the long term primary culture of cortical neurons represent a valuable model of neurodegeneration, such as Alzheimer's disease.
Transcription, Genetic/genetics ; Time Factors ; Rats ; Phosphotransferases/*metabolism ; Neurons/*cytology/*metabolism ; Cells, Cultured ; Cell Shape ; Caspases/antagonists & inhibitors/metabolism ; Calpain/antagonists & inhibitors/genetics/*metabolism ; *Apoptosis ; Animals

Entamoeba histolytica is an enteric tissue-invading protozoan parasite that can cause amebic colitis and liver abscess in humans. E. histolytica has the capability to kill colon epithelial cells in vitro; however, information regarding the role of calpain in colon cell death induced by ameba is limited. In this study, we investigated whether calpains are involved in the E. histolytica-induced cell death of HT-29 colonic epithelial cells. When HT-29 cells were co-incubated with E. histolytica, the propidium iodide stained dead cells markedly increased compared to that in HT-29 cells incubated with medium alone. This pro-death effect induced by ameba was effectively blocked by pretreatment of HT-29 cells with the calpain inhibitor, calpeptin. Moreover, knockdown of m- and micro-calpain by siRNA significantly reduced E. histolytica-induced HT-29 cell death. These results suggest that m- and micro-calpain may be involved in colon epithelial cell death induced by E. histolytica.
Calpain/antagonists & inhibitors/genetics/*metabolism ; *Cell Death ; Cell Line ; Cell Survival/drug effects ; Dipeptides/metabolism ; Entamoeba histolytica/*pathogenicity ; Epithelial Cells/*parasitology ; Gene Knockdown Techniques ; Humans

After invasion of red blood cells, malaria matures within the cell by degrading hemoglobin avidly. For enormous protein breakdown in trophozoite stage, many efficient and ordered proteolysis networks have been postulated and exploited. In this study, a potential interaction of a 60-kDa Plasmodium falciparum (Pf)-heat shock protein (Hsp60) and Pf-calpain, a cysteine protease, was explored. Pf-infected RBC was isolated and the endogenous Pf-Hsp60 and Pf-calpain were determined by western blot analysis and similar antigenicity of GroEL and Pf-Hsp60 was determined with anti-Pf-Hsp60. Potential interaction of Pf-calpain and Pf-Hsp60 was determined by immunoprecipitation and immunofluorescence assay. Mizoribine, a well-known inhibitor of Hsp60, attenuated both Pf-calpain enzyme activity as well as P. falciparum growth. The presented data suggest that the Pf-Hsp60 may function on Pf-calpain in a part of networks during malaria growth.
Amino Acid Sequence ; Calpain/genetics/*metabolism ; Chaperonin 60/chemistry/genetics/*metabolism ; Erythrocytes/parasitology ; Humans ; Malaria, Falciparum/parasitology ; Molecular Sequence Data ; Plasmodium falciparum/chemistry/enzymology/genetics/*metabolism ; Protein Binding ; Protozoan Proteins/chemistry/genetics/*metabolism ; Sequence Alignment

To explore the apoptotic effect of simvastatin on K562 cells through endoplasmic reticulum stress, morphological change of apoptotic cells was observed by Hoechst33258 fluorescent staining under fluorescent microscope. Apoptosis rate of cells was determined with annexinV-FITC/PI double staining by flow cytometry; Intracellular calcium concentration ([Ca2+]i) was measured by laser scanning confocal microscope (LSCM); The expression levels of glucose regulated protein 78 (GRP78) and calpain gene mRNA were determined by RT-PCR; The expression levels of caspase-3, -6, -7, -9, -12, calpain and GRP78 proteins were evaluated by Western blotting. In this study, K562 cells treated with simvastatin for 72 h exhibited typical morphological change of apoptosis cells. After 72 h exposed to 10, 20, 30 micromol x L(-1) simvastatin, the apoptotic rates of K562 cells were 12.41%, 19.08% and 23.41%, respectively. Simvastatin induced the increase of [Ca2+]i in K562 cells, fluorescent intensities were 43, 54, and 64, respectively. The expression levels of GRP78 and calpain gene mRNA were up-regulated. The cleavage and activation of caspase-3, -6, -7, -9, -12 and upregulation of GRP78 expression were determined by Western blotting. These findings suggest that endoplasmic reticulum is an important pathway of apoptosis in cells and participates simvastatin-induced apoptosis in K562 cells. It is implied that simvastatin may be suitable for clinical usage in the treatment of myeloma patients.
Apoptosis ; drug effects ; Calcium ; metabolism ; Calpain ; genetics ; metabolism ; Caspases ; metabolism ; Endoplasmic Reticulum ; drug effects ; metabolism ; Enzyme Inhibitors ; pharmacology ; Heat-Shock Proteins ; genetics ; metabolism ; Humans ; K562 Cells ; RNA, Messenger ; metabolism ; Simvastatin ; pharmacology ; Thapsigargin ; pharmacology

The purpose of this study was to investigate the effect of inhibition of calpain on retinal ganglion cell-5 (RGC-5) necroptosis following oxygen glucose deprivation (OGD). RGC-5 cells were cultured in Dulbecco's-modified essential medium and necroptosis was induced by 8-h OGD. PI staining and flow cytometry were performed to detect RGC-5 necrosis. The calpain expression was detected by Western blotting and immunofluorescence staining. The calpain activity was tested by activity detection kit. Flow cytometry was used to detect the effect of calpain on RGC-5 necroptosis following OGD with or without N-acetyl-leucyl-leucyl-norleucinal (ALLN) pre-treatment. Western blot was used to detect the protein level of truncated apoptosis inducing factor (tAIF) in RGC-5 cells following OGD. The results showed that there was an up-regulation of the calpain expression and activity following OGD. Upon adding ALLN, the calpain activity was inhibited and tAIF was reduced following OGD along with the decreased number of RGC-5 necroptosis. In conclusion, calpain was involved in OGD-induced RGC-5 necroptosis with the increased expression of its downstream molecule tAIF.
Animals ; Apoptosis Inducing Factor ; biosynthesis ; genetics ; Calpain ; biosynthesis ; genetics ; Gene Expression Regulation ; drug effects ; Glucose ; metabolism ; Humans ; Leupeptins ; administration & dosage ; Mice ; Oxygen ; metabolism ; Retinal Ganglion Cells ; metabolism ; pathology ; Retinal Necrosis Syndrome, Acute ; genetics ; pathology

OBJECTIVETo investigate the uppressive effects of par-4 antisense oligodeoxynucleotide on the up-regulation of intracellular calcium concentration in PC12 cell induced by glutamate and its anti-apoptosis effects.

METHODSCationic lipid-mediated par-4 antisense oligodeoxynucleotide (par-4-AS-ODN) was transfected into PC12 cells and followed by glutamate for treatment. Mismatch oligodeoxy-nucleotide (MS-ODN) was used as the control. Morphological assessment and evaluation of the anti-apoptosis effects of par-4-AS-ODN on PC12 cells were performed by laser scanning confocal microscopy by double staining of the cells with Hoechest 33258/propidium iodide (Hoe/PI) and flow cytometry respectively. The mRNA and protein levels of calpain 10 and par-4 were measured by RT-PCR and Western blot. Intracellular calcium concentration was determined by using laser scanning confocal microscope with Fura-2/AM as the fluorescent dye.

RESULTSPar-4-AS-ODN repress the increase of par-4 protein in PC12 cell (52.3 +/- 5.0 vs 90.0 +/- 3.2, < 0.01). Par-4-AS-ODN significantly inhibited the apoptosis of PC12 cells induced by glutamate (53% vs 31%, < 0.01). Par-4-AS-ODN significantly suppress the up-regulation of intracellular calcium concentration in PC12 cells induced by glutamate (Rate of fluorescent density: 167.9 +/- 32.4 vs 228.8 +/- 36.8, < 0.01). Par-4-AS-ODN inhibited the increase of calpain 10 mRNA in PC12 cells induced by glutamate (46.3 +/- 3.7 vs 34.8 +/- 2.1, < 0.01).

CONCLUSIONSpar-4-AS-ODN enables to inhibit apoptosis of PC12 cells induced by glutamate. The mechanism of the inhibition may be closely related to suppression of the up-regulation of intracellular calcium concentration and calpain transcription expression.

Animals ; Apoptosis ; drug effects ; Apoptosis Regulatory Proteins ; biosynthesis ; genetics ; Calcium ; metabolism ; Calpain ; biosynthesis ; genetics ; Glutamic Acid ; pharmacology ; Oligodeoxyribonucleotides, Antisense ; pharmacology ; PC12 Cells ; RNA, Messenger ; biosynthesis ; genetics ; Rats ; Transfection ; Up-Regulation

Entamoeba histolytica is a tissue-invasive protozoan parasite causing dysentery in humans. During infection of colonic tissues, amoebic trophozoites are able to kill host cells via apoptosis or necrosis, both of which trigger IL-8-mediated acute inflammatory responses. However, the signaling pathways involved in host cell death induced by E. histolytica have not yet been fully defined. In this study, we examined whether calpain plays a role in the cleavage of pro-survival transcription factors during cell death of colonic epithelial cells, induced by live E. histolytica trophozoites. Incubation with amoebic trophozoites induced activation of m-calpain in a time- and dose-dependent manner. Moreover, incubation with amoebae resulted in marked degradation of STAT proteins (STAT3 and STAT5) and NF-kappaB (p65) in Caco-2 cells. However, IkappaB, an inhibitor of NF-kappaB, was not cleaved in Caco-2 cells following adherence of E. histolytica. Entamoeba-induced cleavage of STAT proteins and NF-kappaB was partially inhibited by pretreatment of cells with a cell-permeable calpain inhibitor, calpeptin. In contrast, E. histolytica did not induce cleavage of caspase-3 in Caco-2 cells. Furthermore, pretreatment of Caco-2 cells with a calpain inhibitor, calpeptin (but not the pan-caspase inhibitor, z-VAD-fmk) or m-calpain siRNA partially reduced Entamoeba-induced DNA fragmentation in Caco-2 cells. These results suggest that calpain plays an important role in E. histolytica-induced degradation of NF-kappaB and STATs in colonic epithelial cells, which ultimately accelerates cell death.
Caco-2 Cells ; Calcium-Binding Proteins ; Calpain/genetics/metabolism ; Caspase 3/genetics/metabolism ; Caspases ; *Cell Death ; Colon/cytology ; Entamoeba histolytica/*physiology ; Epithelial Cells/cytology/parasitology ; Humans ; I-kappa B Proteins/metabolism ; Intestinal Mucosa/cytology ; NF-kappa B/genetics/*metabolism ; RNA Interference ; RNA, Small Interfering ; STAT3 Transcription Factor/genetics/*metabolism ; STAT5 Transcription Factor/genetics/*metabolism ; Signal Transduction

Familial amyotrophic lateral sclerosis (fALS) is caused by mutations in Cu/Zn-superoxide dismutase (SOD1), and SOD1 aggregation and calcium toxicity are involved in neuronal death. However, the effect of altered calcium homeostasis on the SOD1 aggregation is unknown. To investigate whether calcium triggers mutant SOD1 aggregation in vitro, human mutant SOD1 (G93A) was transfected into motor neuronal cell line (VSC 4.1 cells). These cells were then treated with calcium ionophore A23187 or agents that induce intracellular calcium release like cyclic ADP ribose, ryanodine or thapsigargin. A23187 was found to increase mutant SOD1 aggregation and neuronal nitric oxide synthase (nNOS) expression. Moreover, the NOS inhibitor (L-NAME) and a NO-dependent cyclic GMP cascade inhibitor (ODQ) reduced SOD1 aggregation, whereas an exogenous NO donor (GSNO) increased mutant SOD1 aggregation, which was also prevented by NOS or cGMP cascade inhibitor. Our data demonstrate that calcium-influx increases SOD1 aggregation by upregulating NO in cultured motor neuronal cells.
Amyotrophic Lateral Sclerosis/genetics/metabolism ; Animals ; Calcimycin/pharmacology ; Calcium/*metabolism ; Calpain/metabolism ; Caspase 3/metabolism ; Cell Line ; Humans ; Ionophores/pharmacology ; Motor Neurons/*metabolism ; Multiprotein Complexes ; Mutation ; Nitric Oxide/*metabolism ; Rats ; Recombinant Proteins/chemistry/genetics/metabolism ; Superoxide Dismutase/chemistry/genetics/*metabolism ; Transfection