Tatsuo Yagura

Immunolocalization of Ku-proteins (p80/p70): Localization of p70 to nucleoli and periphery of both interphase nuclei and metaphase chromosomes☆

Distribution on both nuclei and metaphase chromosomes of Ku-proteins, recognized by autoantibodies from a patient with systemic lupus erythematosus, has been studied using a specific monoclonal antibody (mAbH6) that recognizes p70, one Ku-protein. Observation with either a conventional fluorescent microscope or a confocal laser scanning microscope revealed mAbH6-stained p70 antigen localized on both nuclear periphery and nucleoli of human interphase cells. The specific staining of nucleoli with mAbH6 has been confirmed using isolated nucleoli from rat liver in which the staining was seen as fine granules surrounding nucleolar DNA. During mitosis p70 antigen moved away from association with the nuclear envelope region to localization on the periphery of condensed chromosomes with no apparent staining of chromosome interior. The p70 antigen was copurified with DNA fragments by immunoaffinity column chromatography using mAbH6. The mAbH6 staining of both nuclear periphery and nucleoli was lost upon digestion with DNase I at low concentrations. These results suggest that p70 antigen is connected with these nuclear structures through DNA.

Antibacterial Properties of Some Cyclic Organobismuth(III) Compounds

ABSTRACT Bismuth compounds are known for their low levels of toxicity in mammals, and various types of bismuth salts have been used to treat medical disorders. As part of our program to probe this aspect of bismuth chemistry, cyclic organobismuth compounds 1 to 8 bearing a nitrogen or sulfur atom as an additional ring member have been synthesized, and their antimicrobial activities against five standard strains of gram-negative and gram-positive bacteria were assessed. The eight-membered-ring compounds, compounds 1 to 3, exhibited MICs of less than 0.5 μg/ml against Staphylococcus aureus and were more active than the six-membered ones, compounds 5 to 8 (MICs, 4.0 to 16 μg/ml). The gram-positive bacteria (Staphylococcus aureus, Bacillus subtilis, and Enterococcus faecalis) were more susceptible to both types of ring compounds than the gram-negative ones (Escherichia coli and Pseudomonas aeruginosa). Treatment with polymyxin B nonapeptide increased the susceptibility of E. coli to cyclic organobismuth compounds, indicating the low permeability of the outer membrane of gram-negative bacteria to the compounds. Compound 1 also had activity against methicillin-resistant S. aureus, which had an MIC for 90% of the hospital stock strains of 1.25 μg/ml. The killing curves for S. aureus treated with compound 1 or 3 revealed a static effect at a low dose (2× the MIC). However, when S. aureus was treated with 10× the MIC of compound 1 or 3, there was an approximately 3-log reduction in the viable cell number after 48 h of treatment. Electron microscopic inspection demonstrated a considerable increase in the size of S. aureus and the proportion of cells undergoing cell division after treatment with compound 1 at 0.5× the MIC.

Heterocyclic organobismuth(III) induces apoptosis of human promyelocytic leukemic cells through activation of caspases and mitochondrial perturbation

We have synthesized novel heterocyclic organobismuth compounds that have potent antibacterial properties. In this study, we examined their anticancer activity and addressed the cellular mechanisms involved. Heterocyclic organobismuth compounds showed anticancer activities in various human cancer cell lines. These compounds have particularly potent anticancer activities against leukemia cell lines. One of them, bi-chlorodibenzo [c,f][1,5] thiabismocine (compound 3), inhibited the growth of the human promyelocytic leukemia cell line HL-60 at a concentration of 0.22 microM. Low concentrations of compound 3 (0.22-0.44 microM) induced apoptosis, whereas at a higher concentration (>1.1 microM) it causes acute necrosis. During the apoptosis, caspase-3, -8, and -9 were activated but caspase-12 was not. A broad caspase inhibitor (z-VAD-fmk), and caspase-3 (z-DEVD-fmk) and caspase-9 (z-LEHD-fmk) inhibitors suppressed the compound 3-induced apoptosis, but a caspase-8 inhibitor (z-IETD-fmk) was less effective, suggesting that the caspase-8 activity only partially participates in the apoptosis. In the apoptotic cells, cytochrome c was released from mitochondria to cytosol and a loss of mitochondrial transmembrane potential (DeltaPsi(m)) was detected. Compound 3-induced apoptosis was associated with enhanced generation of intracellular reactive oxygen species (ROS). Pretreatment of the cells with N-acetyl-L-cysteine or catalase suppressed the apoptosis. On the other hand, buthionine sulfoximine enhanced the compound 3-induced collapse of DeltaPsi(m) and apoptosis. Taken together, these results indicate that compound 3 is a potent inducer of apoptosis, triggering a caspase-3-mediated mechanism via the generation of ROS and release of cytochrome c from mitochondria, suggesting a potential mechanism for the anticancer activity of compound 3.

Aphidicolin induces alterations in Golgi complex and disorganization of microtubules of HeLa cells upon long-term administration.

Treatment of HeLa cells with aphidicolin at 5 or 0.5 μg/ml induced cell cycle arrest at G1/S or G2/M phase, respectively, and was accompanied by unbalanced cell growth. Long‐term administration of aphidicolin (more than 48 h) resulted in noticeable loss of reproductive capacity though cells were viable at the time of treatment. Immunofluorescence with anti‐Golgi membrane protein monoclonal antibody (mAbG3A5) showed disfigurement of the characteristic mesh‐like configuration when cells were treated for more than 48 h. Interestingly, we found that the fragmented Golgi complex formed a ring around the nucleus in more than 20% of the cells. Immunoelectron microscopy using mAbG3A5 antibody demonstrated that the stack structure of the fragmented Golgi complex in aphidicolin‐arrested cells appeared partially broken up and seemed to have converted to a vesicle‐like structure. Analysis using an antibody to tubulin and anticentrosome human autoimmune serum showed that alterations in the Golgi complex were induced even by the lower 0.5 μg/ml dose. These alterations were accompanied by both changes in the distribution of microtubules and an increase in the number of centrosomes. These cells lost their distinct perinuclear microtubule organiz‐ing center (MTOC). On the other hand, treatment with aphidicolin at 5 μg/ml did not induce multiplication of the centrosome although the loss of distinct MTOC was still evident. No changes took place in the Golgi complex, microtubule, or centrosome of cells treated with 0.5 μg/ml aphidicolin when cycloheximide was added simultaneously to the culture. J. Cell. Physiol. 176:602–611, 1998.

A novel organobismuth compound, 1-[(2-di-p-tolylbismuthanophenyl)diazenyl]pyrrolidine, induces apoptosis in the human acute promyelocytic leukemia cell line NB4 via reactive oxygen species

A novel organobismuth compound, 1-[(2-di-p-tolylbismuthanophenyl)diazenyl]pyrrolidine (4), which has 1-(phenyldiazenyl)pyrrolidine (1) substituent in a benzene ring of tri(p-tolyl)bismuthane (2), was synthesized and tested for biological activity toward human tumor cell lines. 4 had a potent anti-proliferative effect on human cancer cell lines, although both 1 and 2 exhibited only weak activity. The sensitivity of leukemic cell lines to 4 was relatively high; IC(50) values for the human leukemia cell line NB4 and cervical cancer cell line HeLa were 0.88 μM and 5.36 μM, respectively. Treatment of NB4 cells with 4 induced apoptosis, loss of mitochondrial membrane potential (ΔΨ(mt)) and the generation of cellular reactive oxygen species (ROS). 1 and 2 did not induce apoptosis and had only a marginal effect on ΔΨ(mt) and the generation of ROS. N-acetyl cysteine (NAC) reduced the generation of ROS and conferred protection against 4-induced apoptosis, indicating a role for oxidative stress. 4 did not inhibit the polymerization of tubulin in vitro. 1-[2-(di-p-tolylstibanophenyl)diazenyl]pyrrolidine (3), which has the same chemical structure as 4 but contains antimony in place of bismuth, did not show any cytotoxic activity. The results suggest that the conjugated structure of the diazenylpyrrolidine moiety and bismuth center are key to the bioactivity of 4.

A protein homologous to human Ku p70-protein is required for reconstitution ofXenopus sperm pronuclei

The monoclonal antibody mAbH6, which recognizes one human Ku-protein (p70), cross-reacted with the counterpart protein fromXenopus eggs. TheXenopus antigen purified with a mAbH6-Sepharose column was a complex of 88 kDa and 72 kDa proteins. The role of Ku protein in nuclear structure formation was studied using a cell-free nuclear assembly extract derived fromXenopus interphase eggs. The protein was distributed on the surface of demembranated sperm chromatin and in the membrane vesicle fraction of the nuclear assembly extract. Addition of mAbH6 to the assembly extracts prevented the completion of reconstitution of pronuclei from demembranated sperm chromatins, although partial decondensation mediated by nucleoplasmin was not impeded. As a result, the sperm chromatin remained in partially swollen structures or formed round but small anomalous nuclei. Nuclear membranes were formed on the nuclei in mAbH6-inhibited extract systems, but DNA synthesis was largely decreased, suggesting the incomplete reconstitution of pronuclei. The incorporation of lamin to the nuclei was inhibited by mAbH6. It is suggested that theXenopus Ku-homologous protein has a role in the formation of the lamin layer after nuclear membrane reconstitution.

Size difference in catalytic polypeptides of two active forms of mouse DNA polymerase α and separation of the primase subunit from one form, DNA replicase

There are two active forms of DNA polymerase alpha in mouse cells. One form (DNA replicase) is a DNA polymerase associated with primase activity and the other form (7.3 S polymerase) has no primase activity (Yaugar, T., Kozu, T. and Seno, T. (1982) J. Biol. Chem. 257, 11121-11127). The primase activity was dissociated from partially purified DNA replicase by hydroxyapatite column chromatography in buffer containing dimethyl sulfoxide and ethylene glycol. Nearly homogeneous primase, consisting of a 58 kDa polypeptide was obtained by glycerol gradient sedimentation and DEAE-cellulose column chromatography. Experiments on the effect of proteinase treatment and measurement of the molecular weight of the catalytic polypeptide of DNA replicase after its dissociation from the primase polypeptide indicated that the primase is not part of the DNA polymerase molecule, but an independent protein associated with DNA polymerase alpha, and that the latter is a 115 kDa catalytic polypeptide. The other form of DNA polymerase alpha, 7.3 S polymerase, consists of a 72 kDa catalytic polypeptide. Thus, the two forms of mouse DNA polymerase alpha have partially, if not completely, different catalytic polypeptide structures, suggesting that the 7.3 S polymerase is not simply formed from DNA replicase by dissociation of the primase subunit.

Hyperleptinemia elicited by the 5-HT precursor, 5-hydroxytryptophan in mice: involvement of insulin

Mechanisms for hyperleptinemia elicited by a serotonin (5-hydroxytryptamine, 5-HT) precursor, 5-hydroxytryptophan (5-HTP), were investigated. 5-HTP elicited apparent increases in serum leptin levels of mice. Administration of 5-HTP did not alter expression of leptin mRNA in white adipose tissues. Furthermore, neither 5-HTP nor 5-HT increased leptin secretion from isolated fat pads of mice. Since insulin is known to enhance leptin release, involvement of insulin in 5-HTP-induced hyperleptinemia was examined. 5-HTP significantly elevated serum insulin levels. In mice treated with streptozotocin, which depletes insulin, 5-HTP did not increase serum leptin levels. These results suggest that hyperinsulinemia participates the elevation of serum leptin levels elicited by 5-HTP.

Inhibition of a novel subspecies of DNA polymerase α by 2′-deoxy-2′-azidoadenosine 5′-triphosphate

Abstract DNA polymerase α1, a subspecies of DNA polymerase α of Ehrlich ascites tumor cells, was associated with a novel RNA polymerase activity and utilized poly(dT) and single-stranded circular fd DNA as a template without added primer in the presence of ribonucleoside triphosphates and a specific stimulating factor. DNA synthesis in the above system was inhibited by the ATP analogue, 2′-deoxy-2′-azidoadenosine 5′-triphosphate more than the DNA synthesis with poly(dT)·oligo(rA) by DNA polymerase α1 and RNA synthesis by mouse RNA polymerases I and II. Kinetic analysis showed that the analogue inhibited DNA polymerase α1 activity on poly(dT) competitively with respect to ATP, suggesting that the analogue inhibited RNA synthesis by the associated RNA polymerase activity.

MOLECULAR CLONING AND SEQUENCING OF CDNAS ENCODING HOMOLOGUES OF HUMAN KU70 AND KU80 AUTOANTIGEN FROM XENOPUS AND THEIR EXPRESSION IN VARIOUS XENOPUS TISSUES

We isolated cDNA clones encoding Ku70 and Ku80 homologues of Xenopus laevis from a cDNA library prepared from Xenopus oocytes. The nucleotide sequences of these Ku70 and Ku80 homologues have coding sequences of 1833 bp and a 611 aa protein, and 2178 bp and a 726 aa protein, respectively. The amino acid sequences deduced from the open reading frame of the Ku70 and Ku80 cDNA clones were highly homologous to those from Ku genes previously isolated, such as human (ca. 65% and ca. 62% identity, respectively) and mouse (ca. 65% and ca. 60%), and show a certain degree of homology to Drosophila (ca. 27% with Ku70), Caenorhabditis elegans (ca. 20% with Ku80) and Saccharomyces cerevisiae (ca. 23% and ca. 19%). Our detailed comparison of the predicted amino acid sequences among these species revealed the highly conserved octa-peptide LPFXXDIR common to both Xenopus Ku70 and Ku80 homologues in the region showing the high homology throughout the species tested. A Northern analysis using specific cDNA probes showed that Ku poly(A)+ mRNAs are expressed at high levels in Xenopus adult oocyte and testis.