ABSTRACT
Blockade of hypoxia-caused nonmyocytes apoptosis helps improve survival and mitigate ventricular remodeling and dysfunction during the chronic stage of myocardial infarction. But tools affecting nonmyocyte apoptosis are very rare. Sphingosylphosphorylcholine (SPC), a naturally occurring bioactive sphingolipid in plasma, was proved to protect cardiomyocyte against apoptosis in an ischemic model in our previous study. Here, we showed that SPC also inhibited hypoxia-induced apoptosis in myofibroblasts, an important type of nonmyocytes in the heart. Calmodulin (CaM) is an identified receptor of SPC. We clarified that SPC inhibited myofibroblast apoptosis through CaM as evidenced by decreased cleaved caspase 3, PARP1 and condensed nucleus. Furthermore, the employment of inhibitor and agonist of p38 and STAT3 suggests that SPC inhibits myofibroblast apoptosis by regulating the phosphorylation of p38 and STAT3, and they act as downstream of CaM. The present work may provide new evidence on the regulation of myofibroblasts apoptosis by SPC and a novel target for heart remodeling after hypoxia.
Subject(s)
Apoptosis/drug effects , MAP Kinase Signaling System/drug effects , Myofibroblasts/drug effects , Phosphorylcholine/analogs & derivatives , Sphingosine/analogs & derivatives , Animals , Calmodulin/metabolism , Calmodulin/physiology , Cell Hypoxia , Fibrosis , Mice, Inbred C57BL , Myocardial Infarction/drug therapy , Myocardial Infarction/pathology , Myocardium/cytology , Myofibroblasts/enzymology , Myofibroblasts/metabolism , Phosphorylcholine/pharmacology , Phosphorylcholine/therapeutic use , Rats, Wistar , STAT3 Transcription Factor/metabolism , STAT3 Transcription Factor/physiology , Sphingosine/pharmacology , Sphingosine/therapeutic use , p38 Mitogen-Activated Protein Kinases/metabolism , p38 Mitogen-Activated Protein Kinases/physiologyABSTRACT
p28, a protein derived from toad (Bufo bufo gargarizans) oocytes, has a high level of sequence homology to mouse UCH L1. We report here that it is a toad ubiquitin carboxyl-terminal hydrolase (UCH), termed tUCH for its ability to hydrolyze the UCH substrate ubiquitin ethyl ester (UboEt). The similarities of secondary structures between tUCH and UCH L1 highlight that they might have common functions. The total extracted proteins both from immature and matured oocytes contain 2% tUCH. The enzyme kinetic constants (Km and Kc) both of the tUCH and UCH L1 reveal that they possess similar catalytic properties on their common substrate Ub-AMC. Anti-tUCH monoclonal antibody (tUCH mAb) can recognize tUCH and dominant-negative tUCH (tUCH C(90)S), but not mouse UCH L1, suggesting that it does not target on the conservative UCH active sites. Furthermore, anti-tUCH mAb when injected into oocytes blocked the progesterone-induced GVBD but anti-tUCH mAb could not inhibit the tUCH catalytic hydrolysis of Ub-AMC, revealing that the implication of tUCH in the oocyte maturation regulation is not dependent on its UCH activity.
Subject(s)
Bufo bufo/physiology , Oocytes/physiology , Ubiquitin Thiolesterase/physiology , Amino Acid Sequence , Animals , Antibodies, Monoclonal/immunology , Molecular Sequence Data , Progesterone , Sequence Homology, Amino Acid , Ubiquitin Thiolesterase/immunologyABSTRACT
By means of in situ hybridization and immunohistochemistry, the protein localization and gene expression of cyclin B1 in spermatogenic cells were characterized during the spermatogenesis of rabbits. The results showed that the cyclin B1 mRNA in rabbit spermatogenic epithelium was expressed dominantly in primary spermatocytes. The expression was observed in round spermatids with a gradual decline in the process of metamorphosis of the spermatids, but not in elongated spermatids and sperms. Cyclin B1 protein was expressed in mitotic spermatogonia and meiotic spermatocytes and was observed predominantly in round and elongated spermatids. These results indicate that the expression of cyclin B1 mRNA and localization of cyclin B1 protein are dependent on the developmental stages of spermatogenic cells.
Subject(s)
Cyclin B1/genetics , Spermatocytes/metabolism , Spermatogenesis/genetics , Animals , Cyclin B1/metabolism , Gene Expression Regulation, Developmental , Male , RNA, Messenger/genetics , Rabbits , Spermatids/metabolism , Spermatocytes/cytology , Spermatogonia/metabolismABSTRACT
p28, a 28kD protein from toad (Bufo bufo gargarizans) oocytes, was identified by using p13(suc1)-agarose affinity chromatography. Sequence homology analysis of the full-length cDNA of p28 (Gene Bank accession number: AF 314091) indicated that it encodes a protein containing 224 amino-acids with about 55% identities and more than 70% positives to human, rat or mouse UCH-L1, and contains homological functional domains of UCH family. Anti-p28 monoclonal antibody, on injecting into the oocytes, could inhibit the progesterone-induced resumption of meiotic division in a dose-dependent manner. The recombinant protein p28 showed similar SDS/PAGE behaviors to the native one, and promoted ubiquitin ethyl ester hydrolysis, a classical catalytic reaction for ubiquitin carboxyl terminal hydrolases (UCHs). The results in this paper reveal that a novel protein, p28, exists in the toad oocytes, is a UCH L1 homolog, was engaged in the process of progesterone-induced oocyte maturation possibly through an involvement in protein turnover and degradation.
