Somatic cell nuclear transfer (SCNT) has various applications in research, as well as in the medical field and animal husbandry. However, the efficiency of SCNT is low and the accurate mechanism of SCNT in murine embryo development is unreported. In general, the developmental rate of SCNT murine embryos is lower than in vivo counterparts. In previous studies, polo-like kinase 1 (Plk1) was reported to be a crucial element in cell division including centrosome maturation, cytokinesis, and spindle formation. In an initial series of experiments in this study, BI2536, a Plk1 inhibitor, was treated to in vivo-fertilized embryos and the embryos failed to develop beyond the 2-cell stage. This confirmed previous findings that Plk1 is crucial for the first mitotic division of murine embryos. Next, we investigated Plk1's localization and intensity by immunofluorescence analysis. In contrast to normally developed embryos, SCNT murine embryos that failed to develop exhibited two types of Plk1 expressions; a low Plk1 expression pattern and ectopic expression of Plk1. The results show that Plk1 has a critical role in SCNT murine embryos. In conclusion, this study demonstrated that the SCNT murine embryos fail to develop beyond the 2-cell stage, and the embryos show abnormal Plk1 expression patterns, which may one of the main causes of developmental failure of early SCNT murine embryos.
Animal Husbandry ; Cell Division ; Centrosome ; Cytokinesis ; Ectopic Gene Expression ; Embryonic Development ; Embryonic Structures ; Female ; Fluorescent Antibody Technique ; Nuclear Transfer Techniques ; Phosphotransferases ; Pregnancy

Previously, we have reported that CKAP2 is involved in the maintenance of centrosome integrity, thus allowing for proper mitosis in primary hepatocytes. To understand this biological process, we identified the mitosis-specific phosphorylation sites in mouse CKAP2 and investigated CKAP’s possible role in cell cycle progression. Because we observed mouse CKAP2 depletion in amplified centrosomes and aberrant chromosomal segregation, which was rescued by ectopic expression of wild-type CKAP2, we focused on the centrosome duplication process among the various aspects of the cell cycle. Among the identified phosphorylation sites, T603 and possibly S608 were phosphorylated by CDK1–cyclin B1 during mitosis, and the ectopic expression of both T603A and S608A mutants was unable to restore the centrosomal abnormalities in CKAP2-depleted cells. These results indicated that the phosphorylation status of CKAP2 during mitosis is critical for controlling both centrosome biogenesis and bipolar spindle formation.
Animals ; Biological Processes ; Cell Cycle ; Centrosome* ; Ectopic Gene Expression ; Hepatocytes ; Mice ; Mitosis ; Phosphorylation*

Oligoasthenozoospermia, teratozoospermia or low sperm motility is the main cause of male infertility. Low sperm motility can be induced by abnormalities of the sperm tail structure and sperm function. The outer dense fiber protein 2 (ODF2) is a protein fiber maintaining cytoskeleton, as a major component of the mammalian sperm tail and centrosome, and its abnormality is closely related to asthenospermia. Recent studies indicate that ODF2 includes many proteins of the same name and homologous splices located in the sperm centrosomes and spindles of cleaved-embryos, necessary for animal ciliogenesis and associated with sperm capacitation. The features of ODF2 indicate that it is not a single-structural protein. This paper reviews the known functions of ODF2, paving a ground for further studies of the relationship between the ODF2 protein and fertilization.
Animals ; Asthenozoospermia ; complications ; Azoospermia ; complications ; Centrosome ; chemistry ; Cytoskeleton ; chemistry ; Heat-Shock Proteins ; physiology ; Humans ; Infertility, Male ; etiology ; Male ; Sperm Motility ; physiology ; Sperm Tail ; Spermatozoa ; physiology

BACKGROUND: Mutations in centrosomal protein genes have been identified in a number of genetic diseases in brain development, including microcephaly. Centrosomal P4.1-associated protein (CPAP) is one of the causal genes implicated in primary microcephaly. We previously proposed that CPAP is essential for mother centriole maturation during mitosis. METHODS: We immunostained CPAP-depleted cells using the ninein antibody, which selectively detects subdistal appendages in mature mother centrioles. RESULTS: Ninein signals were significantly impaired in CPAP-depleted cells. CONCLUSION: The results suggest that CPAP is required for mother centriole maturation in mammalian cells. The selective absence of centriolar appendages in young mother centrioles may be responsible for asymmetric spindle pole formation in CPAP-depleted cells.
Brain ; Cell Cycle ; Centrioles* ; Centrosome ; Humans ; Microcephaly ; Mitosis ; Mothers* ; Spindle Poles

BACKGROUND: Aneuploidy has been suggested as one of the major causes of cancer from the time of Boveri. In support of this notion, many studies have shown that cancer cells exhibit aneuploidy. However, there are evidences that do not support the aneuploidy hypothesis. We have previously reported that the spindle assembly checkpoint protein BubR1 is acetylated in mitosis and that the acetylation of BubR1 is crucial for checkpoint maintenance and chromosome-spindle attachment. Mice heterozygous for acetylation-deficient BubR1 (K243R/+) spontaneously develop cancer with chromosome instability. As K243R/+ mice develop hepatocellular carcinoma, we set out to test if chromosome mis-segregation was the cause of their liver cancer. METHODS: Primary hepatocytes in the regenerating liver after partial hepatectomy (PH) were analyzed and compared for various mitotic parameters. RESULTS: Primary hepatocytes isolated from K243R/+ mice after PH displayed a marked increase of chromosome misalignment, accompanied by an increase of micronuclei. In comparison, the number of nuclei per cell and the centrosome numbers were not different between wild-type and K243R/+ mice. Taken together, chromosome mis-segregation provokes tumorigenesis in mouse liver. CONCLUSION: Our results corroborate that PH provides a reliable tool for assessing mitotic infidelity and cancer in mice.
Acetylation ; Aneuploidy ; Animals ; Carcinogenesis* ; Carcinoma, Hepatocellular ; Centrosome ; Chromosomal Instability ; Hepatectomy* ; Hepatocytes ; Hydrogen-Ion Concentration ; Liver ; Liver Neoplasms ; M Phase Cell Cycle Checkpoints ; Mice* ; Mitosis

In humans and most mammals, the sperm centrosome is primarily responsible for nucleating and organizing the sperm astar, which pushes the sperm head toward the oocyte center and guides the migration of the female pronucleus, completing the fertilization process. There are about 200 kinds of protein in the human sperm centrosome. Currently, most of the researches focus on the centrin protein. Further studies on the functions of different human sperm centrosomal proteins may contribute to the understanding of the causes of the failures in assisted reproductive technology (ART). And in ART, morphological observation of the sperm neck integrity is the only way for primary evaluation of the function of the sperm centrosome.
Calcium-Binding Proteins ; physiology ; Centrosome ; physiology ; Chromosomal Proteins, Non-Histone ; physiology ; Humans ; Male ; Reproductive Techniques, Assisted ; Spermatozoa ; cytology

OBJECTIVETo analyze the centrosome abnormalities in the malignant transformation of human bronchial epithelial cells (BEAS-2B) induced by coal tar pitch smoke extracts and to investigate the role and action mechanism of centrosome in the lung cancer induced by coal tar pitch.

METHODSMedium-temperature coal tar pitch smoke extracts were used to treat immortalized human bronchial epithelial cells (BEAS-2B) and establish a malignant transformation model. The treated BEAS-2B cells were used as exposure group, and solvent control group and normal control group were also set for passage culture. The changes of centrosome in BEAS-2B cells seeded on coverslips were evaluated by indirect immunofluorescence assay. The mRNA expression of p53, p21, and cyclin E in BEAS-2B cells was measured by real-time quantitative RT-PCR, and their protein levels in BEAS-2B cells seeded on coverslips were measured by semiquantitative immunohistochemical analysis.

RESULTSThe overall rate of centrosome abnormalities in BEAS-2B cells at passage 20 was 6.56±1.01% in the exposure group, significantly higher than those in the normal control group (3.40±0.86%) and solvent control group (3.14±0.59%) (P < 0.05). In addition, the exposure group had a significantly higher overall rate of centrosome abnormalities in BEAS-2B cells at passage 30 compared with the normal control group and solvent control group (22.39±9.5% vs 4.34±1.04%, P < 0.05; 22.39±9.5% vs 4.33±1.20%, P < 0.05). Compared with the normal control group and solvent control group, the exposure group had significantly decreased mRNA and protein expression of p53 and significantly increased mRNA and protein expression of cyclin E in BEAS-2B cells at passages 20 and 30 (P < 0.05).

CONCLUSIONCentrosome abnormalities occur before the malignant transformation in BEAS-2B cells treated with coal tar pitch smoke extracts, and they may be mediated by the p53/p21/cyclin E signaling pathway.

Cell Line ; Cell Transformation, Neoplastic ; metabolism ; pathology ; Centrosome ; metabolism ; pathology ; Coal Tar ; Cyclin E ; metabolism ; Epithelial Cells ; cytology ; metabolism ; Humans ; Signal Transduction ; Smoke ; Tumor Suppressor Protein p53 ; metabolism

OBJECTIVETo construct a mammalian expression plasmid of the BC022687 gene and investigate the expression and localization of the fusion protein in Chinese hamster ovary (CHO) cells.

METHODSThe BC022687 coding sequence was amplified by polymerase chain reaction (PCR) and subcloned into the pEGFP-C1 vector carrying the gene of green fluorescence protein (GFP). After the target region was sequenced, the recombinant plasmid was transfected into CHO cells, and its expression in the CHO cells was determined by Western blot. The localization of GFP-tagged BC022687 in the CHO cells was observed by laser scanning confocal microscopy.

RESULTSBC022687 was successfully cloned into the mammalian expression vector pEGFP-C1, with the restriction fragment length of 950 bp. The expression of the fusion protein was confirmed, with the relative molecular weight of 64 000. The GFP-tagged BC022687 protein was mainly localized in the cytoplasm, and also presented in the centrioles in the transfected CHO cells.

CONCLUSIONThe successful construction of the plasmid expressing BC022687 in CHO cells has laid a foundation for further studies on the role of this protein in ciliogenesis.

Animals ; CHO Cells ; Centrosome ; metabolism ; Cilia ; metabolism ; Cricetinae ; Cricetulus ; DNA, Complementary ; Genetic Vectors ; Male ; Mice ; Plasmids ; Recombinant Fusion Proteins ; genetics ; Transfection

Antineoplastic Agents ; therapeutic use ; BRCA2 Protein ; metabolism ; Breast Neoplasms ; drug therapy ; metabolism ; pathology ; Cell Cycle Proteins ; metabolism ; physiology ; Centrosome ; metabolism ; Drug Screening Assays, Antitumor ; Female ; Humans ; Microtubule-Associated Proteins ; metabolism ; Neoplasm Invasiveness ; Nuclear Proteins ; metabolism ; Phosphorylation ; Prognosis ; Protein-Serine-Threonine Kinases ; metabolism ; physiology ; Proto-Oncogene Proteins ; metabolism ; physiology ; RNA, Small Interfering ; pharmacology ; Tumor Suppressor Protein p53 ; metabolism

BACKGROUND: Although the pericentric inversion of chromosome 9, inv(9)(p11q13), is generally considered a normal variation, it is also associated with solid tumors and several hematologic malignancies such as biphenotypic acute leukemia, ALL, AML, and myeloproliferative neoplasms. However, to the best of our knowledge, there have been no reports that suggest an association between CML and constitutional pericentric inversion of chromosome 9. The purpose of this retrospective study was to investigate the frequency and clinical features of CML patients with concomitant inv(9) and t(9;22)(q34;q11.2) variation at our institution. METHODS: We reviewed the bone marrow chromosome database entries between October 2006 and December 2008 to identify patients with concomitant inv(9) and t(9;22) variations. Laboratory and clinical data of the patients were obtained from the electronic medical record system. RESULTS: Among the 51 CML patients, 4 (7.8%) had concomitant inv(9) and t(9;22) variations. CONCLUSIONS: Although the association between inv(9) variation and CML is still controversial, we believe that hematologists should consider the role of constitutional inv(9) variation in CML patients to avoid overlooking the impaired engraftment potential of hematopoietic stem cells harboring inv(9). Therefore, we suggest that more effort should be invested to develop cytogenetic tests for detecting constitutional inv(9) variation in CML patients.
Adult ; Asian Continental Ancestry Group/*genetics ; Centrosome ; *Chromosome Inversion ; *Chromosomes, Human, Pair 9 ; Female ; Humans ; Karyotyping ; Leukemia, Myeloid, Acute/diagnosis/*genetics ; Male ; Middle Aged ; Republic of Korea ; Retrospective Studies ; Translocation, Genetic