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Calpain, a Ca2+-dependent cysteine protease, has been implicated in neuronal apoptosis following spinal cord injury. In this study, activation of calpain was investigated in motor neurons of adult spinal cord slices from the mouse, using a cell-permeable fluorogenic calpain substrate and Western blotting. Calpain was rapidly activated in the motor neurons of excised spinal cord slices and calpain activity was observed both in the cytoplasm and the nuclei. In these neurons, nuclear and chromatin condensation were pronounced. Both calpain inhibitor VI and EGTA (ethyleneglycol-bis(beta-aminoethyl ether) N' ,N' ,N' ,N' -tetraacetic acid) inhibited calpain activation and subsequent appearance of apoptotic nuclei. In contrast, the general caspase inhibitor Z-VAD.fmk had no effect. Calpain activation was also observed in the slices by Western blotting using an antibody to 150-kD calpain-cleaved alpha-fodrin fragment. These results show that calpain is rapidly activated in injured motor neurons and imply that this activation could be responsible for execution of caspase-independent apoptosis in injured adult motor neurons.

Background: Lymphocytes are a group of white blood cells with a variety of roles their integrity is crucial for the body's immune responses. Cadmium, a heavy metal and environmental pollutant, is known as a toxicant to exert its adverse effects on some sort of cells including blood cells. Research Design: In this study, human lymphocytes were divided into 3 groups: (1) lymphocytes at 0-h, (2) lymphocytes at 24 h (control), (3) lymphocytes treated with cadmium chloride (15 μM). Lymphocyte viability and plasma membrane integrity were assessed in these groups. In addition, the occurrence of apoptosis was investigated by assessment of nucleus diameter and flow cytometry. Activation of caspase-3 was also detected by immunocytochemistry. Results: Result showed that lymphocyte's viability and plasma membrane integrity decreased in lymphocytes treated with cadmium as compared with the control group. Decreased nucleus diameter and result of flow cytometry demonstrated cadmium-induced apoptosis in human lymphocytes. Furthermore, lymphocytes treated with cadmium displayed intensely activated caspase-3 immunoreactivity in their cytoplasm. Conclusion: In conclusion, cadmium not only negatively effect on viability and plasma membrane, but also induces caspase-dependent apoptosis in human lymphocytes. [ABSTRACT FROM AUTHOR]

Oxidative stress‐induced apoptosis in spermatozoa may lead to male infertility. Environmental pollutants and heavy metals such as cadmium cause harmful effects on the reproductive system and sperm parameters through the induction of oxidative stress. Silymarin, as a potent antioxidant, is able to inhibit oxidative stress. This study was performed to investigate the protective effects of silymarin on cadmium‐induced toxicity in human spermatozoa. Sperm samples were divided into the following five groups: (a) spermatozoa at 0 min, (b) spermatozoa in the control group, (c) spermatozoa treated with cadmium chloride (20 μM), (d) spermatozoa treated with silymarin (2 μM)+ cadmium chloride (20 μM) and (e) spermatozoa treated with silymarin (2 μM). Sperm parameters related to apoptosis, such as DNA fragmentation, nucleus diameter, mitochondrial membrane potential (MMP) and expression of caspase‐3, were evaluated in all groups. After 180 min, spermatozoa treated with cadmium chloride showed a significant decrease in nucleus diameter and MMP but a significant increase in DNA fragmentation; however, caspase‐3 expression remained unchanged. At this time point, silymarin in the silymarin + cadmium chloride group could significantly reverse the adverse effects of cadmium chloride on these parameters.Silymarn could partly compensate for the caspase‐independent apoptosis in the spermatozoa. Therefore, oxidative stress could be a consequence for cadmium toxicity. [ABSTRACT FROM AUTHOR]

Introduction: Since Curcuma longa extract and curcumin have been shown to be potent antioxidant agents, they were used in cultured adult mouse spinal cord slices to investigate whether they can inhibit apoptosis in motor neurons. Methods: Slices from the thoracic region of adult mice spinal cord were divided into four groups: 1. Freshlyprepared slices (time 0); 2. Control; 3. Slices treated by curcumin (20 μM); and 4. Slices treated by Curcuma longa extract (1000 ppm). The control and the treated slices were cultured for 6 hours. MTT [3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide] assay was used to evaluate slice viability in the four groups. Morphological and biochemical features of apoptosis were studied using fluorescent staining (propidium iodide and Hoechst 33342) and TUNEL method, respectively. Data were analyzed using one-way ANOVA. Results: The viability of slices cultured for 6 hours (control) was considerably decreased compared to freshly-prepared slices. Motor neurons from slices cultured for 6 hours showed morphological and biochemical features of apoptosis. At this time point, the application of either curcumin or Curcuma longa extract not only increased the viability of cultured slices compared to the control, but also they could inhibit morphological and biochemical features of apoptosis in the motor neurons. Conclusion: Oxidative stress might be a possible mechanism for the induction of apoptosis of motor neurons in cultured spinal cord slices. [ABSTRACT FROM AUTHOR]

Objective: The apoptosis of motor neurons is a critical phenomenon in spinal cord injuries. Adult spinal cord slices were used to investigate whether voltage sensitive calcium channels and Na+/Ca2+ exchangers play a role in the apoptosis of motor neurons. Materials and Methods: In this experimental research, the thoracic region of the adult mouse spinal cord was sliced using a tissue chopper and the slices were incubated in a culture medium in the presence or absence of N/L type voltage sensitive calcium channels blocker (loperamide, 100 μM) or Na+/Ca2+ exchangers inhibitor(bepridil, 20 μM) for 6 hours. 3-(4, 5-dimethylthiazol-2-yl)-2, 5 diphenyl tetrazolium (MTT) staining was used to assess slice viability while morphological features of apoptosis in motor neurons were studied using fluorescent staining. Results: After 6 hours in culture, loperamideand bepridil not only increased slice viability, but also prevented motor neuron apoptosis and significantly increased the percentage of viable motor neurons in the ventral horns of the spinal cord. Conclusion: The results of this study suggest that voltage sensitive calcium channels and Na+/Ca2+ exchanger might be involved in the apoptosis of motor neurons in adult spinal cord slices. [ABSTRACT FROM AUTHOR]

Apoptosis, a form of programmed cell death that occurs under physiological as well as pathological conditions, is characterized by morphological and biochemical features. While the importance of caspases in apoptosis is established, several noncaspase proteases (Ca2+-dependent proteases) such as calpain may play a role in the execution of apoptosis. The calpain family consists of two major isoforms, calpain I and calpain II which require μM and mM Ca2+ concentrations to initiate their activity. An increase in intracellular Ca2+ level is thought to trigger a cascade of biochemical processes including calpain activa- tion. Once activated, calpains degrade membrane, cytoplasmic and nuclear substrates, leading to the breakdown of cellular architecture and finally apoptosis. The activation of calpain has been implicated in neuronal apoptosis following spinal cord injuries and neurodegenerative diseases. This review focuses on calpain with an emphasis on its key role in the proteolysis of cellular protein substrates following apoptosis. [ABSTRACT FROM AUTHOR]

Objective: The aim of this study was to investigate the preventive effect of the polyamine component N, N, N′, N′-tetrakis(2-aminoethyl)2,2-dimethylproparie-1,3-diamine (tdmtn) on apoptotic motor neurons in adult mouse spinal cord slices. Materials and Methods: Thoracic region of adult mouse spinal cords was sliced by a tissue chopper into 400 urn slices and cultured in medium in the presence or absence of tdmtn for 6 hours. Morphological a features of apoptosis were evaluated using fluorescent staining with propidium iodide and Hoechst 33342. The appearance of nucleosomal DNA fragmentation was studied using agarose gel electrophoresis. Our results were analyzed using the one-way ANOVA and Tukey's tests. Results: After 6 hours in culture, motor neurons displayed morphological sings of apoptosis including cell shrinkage, as well as nuclear and chromatin condensation. DNA extracted from slices cultured for 24 hours revealed nucleosomal DNA fragmentation on agarose gel electrophoresis. Tdmtn reduced the occurrence of apoptosis in motor neurons and significantly (p<0.01) increased the viability of these neurons after 6 hours of culturing. Conclusion: Tdmtn, as a polyamine component, could probably prevent the occurrence of motor neuron apoptosis through calcium chelation. [ABSTRACT FROM AUTHOR]