Ken Hayashi

Cyclin-dependent kinase 2 (Cdk2) is required for centrosome duplication in mammalian cells.

Centrosome duplication is indispensable for the formation of the bipolar mitotic spindle. Surprisingly, even if DNA replication or mitosis is inhibited, centrosome duplication can still occur [1] [2] [3] [4] [5]. Thus, it remains unknown how centrosome duplication is coordinated with the cell cycle. Here, we show that centrosome duplication requires cyclin-dependent kinase 2 (Cdk2) in mammalian cells. We have found that in Chinese hamster ovary (CHO) cells, whereas centrosome duplication is not inhibited by hydroxyurea (HU) treatment, which arrests the cells in S phase, it is inhibited by mimosine treatment, which arrests the cells in late G1 phase. Cdk2 activity was higher in HU-treated cells than in mimosine-treated cells. Remarkably, inhibition of the Cdk2 activity in HU-treated cells with butyrolactone I or roscovitine [6], or by expression of the Cdk inhibitor p21(Waf1/Cip1), blocked the continued centrosome duplication. Moreover, overexpression of Cdk2 reversed the inhibition of centrosome duplication by mimosine treatment. These results indicate a requirement of Cdk2 activity for centrosome duplication and therefore suggest an underlying mechanism for the coordination of centrosome duplication with the cell cycle.

Analysis of translesion replication across an abasic site by DNA polymerase IV of Escherichia coli.

Unrepaired replication-blocking DNA lesions are bypassed by specialized DNA polymerases, members of the Y super-family. In Escherichia coli the major lesion bypass DNA polymerase is pol V, whereas the function of its homologue, pol IV, is not fully understood. In vivo analysis showed that pol V has a major role in bypass across an abasic site analog, with little or no involvement of pol IV. This can result from the inability of pol IV to bypass the abasic site, or from in vivo regulation of its activity. In vitro analysis revealed that purified pol IV, in the presence of the beta subunit DNA sliding clamp, and the gamma complex clamp loader, bypassed a synthetic abasic site with very high efficiency, reaching 73% in 2 min. Bypass was observed also in the absence of the processivity proteins, albeit at a 10- to 20-fold lower rate. DNA sequence analysis revealed that pol IV skips over the abasic site, producing primarily small deletions. The RecA protein inhibited bypass by pol IV, but this inhibition was alleviated by single-strand binding protein (SSB). The fact that the in vitro bypass ability of pol IV is not manifested under in vivo conditions suggests the presence of a regulatory factor, which might be involved in controlling the access of the bypass polymerases to the damaged site in DNA.

Effects of platelet activating factor on mouse embryo implantation in vitro

PurposeOur purpose was to assess the role of platelet activating factor (PAF) in embryo implantation, we examined the effects of PAF and a PAF antagonist on the in vitro implantation of mouse embryos, using an in vitro embryo culture system in the absence of the endometrium.MethodsBDF1 mouse pronuclear-stage embryos were cultured until they developed to the two-cell, the four- to eight-cell, or the morula stage in the absence of PAF or its antagonist CV6209. The medium was then changed and supplemented with PAF or CV6209 at various concentrations. We also examined the reversible effects of PAF addition to the media containing the PAF antagonist.ResultsThe addition of PAF to the culture from the two-cell stage significantly (P <0.05) increased the rates of embryo implantation in vitro (control, 69.8%; 10−10 MPAF, 90.1%; 10−9 MPAF, 95.5%). Similarly, the addition of PAF to the cultures from the four- to eight-cell and morula stage also significantly (P <0.05) increased their rates of implantation in vitro. In contrast, the addition of CV6209 to the culture significantly (P <0.01) decreased the rates of embryo implantation in vitro. CV6209 also decreased the rate of blastocyst formation. The degree of inhibition by CV6209 decreased with the advancing stage of embryos. The addition of PAF to media containing CV6209 reversed the inhibition and restored the implantation rate in vitro.ConclusionsThese results suggest that PAF may act directly on the mouse embryo and favor its implantation like an autocrine activating factor, irrespective of the presence or absence of the endometrium.

Cytogenetic Survey of Subfertile Males in Japan

Chromosomal analysis of 554 consecutive Japanese male patients attending a subfertility clinic revealed 18 (3.2%) major chromosomal aberrations. The incidence of sex chromosomal anomalies and autosomal anomalies is 1.3 and 2.0%, respectively, which is significantly higher than that for the general population. All 7 sex chromosomal anomalies were found among the azoo- or oligozoospermic patients, while the incidence of autosomal anomalies for the normozoospermic patients was statistically similar to that for the azoo- or oligozoospermic patients. Therefore, cytogenetic analysis is recommended not only for patients with reduced sperm density but also for subfertile patients with normozoospermia.

Azoospermic Male with a Balanced Y-Autosome Translocation

Structural chromosomal aberrations, such as translocations or inversions, may cause spermatogenic failure. We report a rare chromosomal anomaly 46,X,t(Y;3)(q12;p21) in an azoospermic male with normal phenotype. Twenty-two cases of a balanced Y-autosome translocation have been reported in the literature and the majority of them have been detected among azoospermic males at male infertility clinics. This structural chromosomal anomaly brings spermatogenic arrest at the primary or secondary spermatocyte.

de novo Reciprocal Translocation t(6;14)(q27;q13.3) in a Child with Infantile Spasms

Summary: A boy with infantile spasms was found to have a balanced de novo translocation, 6q;14q. The karyotype was 46, XY, t(6;14)(q27;q13.3). He had mental retardation and microcephaly but no dysmorphic features. Whether or not there is a relationship between the translocation in our patient and his infantile spasms is unknown. If there is a connection, a gene or genes on chromosome 14 may be implicated in the seizure disorders that are common in patients with ring chromosome 14 syndrome or proximal partial trisomy 14 syndrome.

The cloning of size-heterogeneous, Y-specific repetitive DNAs and their clinical application

SummaryFour Y-specific DNAs of different sizes were isolated by screening a human Y-chromosome gene-library. After determining their structural characteristics, the possibility for their clinical application was examined. The results are as follows:1.The 4 cloned DNAs had inserts of 3.3, 2.0, 1.9 and 1.4 kb; they were subcloned into plasmid pUC19, and designated pKY-2, pKY-3, pKY-4 and pKY-5, respectively.2.All four clones hybridized specifically with the Y-specific 3.5 kb DNA but not with female DNA. However, they did not cross-hybridize with the 2.1 kb fragment known to be another Y-specific repetitive DNA of human genomes.3.The newly cloned probes were applied to the detection of Y-specific DNA in abnormal Y-chromosomes [Turner Syndrome (45, X/46, X, −X, + marker), 46, XY-female, mosaicism (46, XY/47, XYY), 46, XX-male and super male (47, XY4.Since the newly cloned DNAs did not hybridize with other eukaryotic genome DNAs such as monkey, rat, mouse, chicken, frog, or fish, their human gene specificities were confirmed.

Glioblastoma Multiforme with a Peculiar Chromosomal Aberration—Double Minute Chromosomes

The chromosomal aberration of double minute chromosomes (DMS) is a peculiar structural anomaly characterized by the occurrence of strongly varying numbers of often very small, paired chromatin bodies, in addition to the ordinary chromosomes. A glioblastoma multiforme with DMS was found in a 73-year-old female patient, who had received neither radiation nor chemotherapy before the tissue was explanted. She received computerized tomography of the brain because of severe headache, and a brain tumor was found in the right parieto-occipital lobe. The brain tumor was extirpated subtotally and the operative specimen indicated glioblastoma multiforme. In the cytogenetic study, nine cells containing DMS in this tumor tissue showed the hyperdiploid picture, with the total number of chromosomes varying from 50 to 57. Monosomy was found on chromosomes #10, 12, D group, #17, F group, and #21, trisomy on chromosomes A group, #6, 7, 8, 9, 16, and X, nullisomy on chromosome #22, in the karyotype analysis. All of the nine cells containing DMS showed karyotypic abnormalities, while DMS were not seen in the cells with normal karyotypes. Marker chromosomes ranging from 12 to 16, other than DMS, were seen in all of the nine cells. The number of DMS varied considerably among the cells, ranging from 17 to 133. The number of DMS correlated neither with the total number of the chromosomes nor with the number of marker chromosomes. There was no chromosomal abnormality indicating homogeneously staining regions in all of the cells observed in this tumor tissue. In the course of in vitro culture, both the cells with DMS and DMS within the cells gradually diminished in number.

DNA polymerase activities in human placenta

SummaryActivities of DNA polymerase α and β were assayed in crude extracts prepared from human placenta. Polymerase α activity was high in early pregnancy but low during the 2nd trimester. Polymerase β activity did not change significantly with gestational weeks. The increase in polymerase α activity in early pregnancy may be closely related to mitosis at the time of placental formation; there might also be some type of accelerating factor for polymerase in early pregnancy.