Esperanto for histones: CENP-A, not CenH3, is the centromeric histone H3 variant.

The first centromeric protein identified in any species was CENP-A, a divergent member of the histone H3 family that was recognised by autoantibodies from patients with scleroderma-spectrum disease. It has recently been suggested to rename this protein CenH3. Here, we argue that the original name should be maintained both because it is the basis of a long established nomenclature for centromere proteins and because it avoids confusion due to the presence of canonical histone H3 at centromeres.

A human centromere protein, CENP-B, has a DNA binding domain containing four potential alpha helices at the NH2 terminus, which is separable from dimerizing activity.

The alphoid DNA-CENP-B (centromere protein B) complex is the first sequence-specific DNA/protein complex detected in the centromeric region of human chromosomes. In the reaction, CENP-B recognizes a 17-bp sequence (CENP-B box) and assembles two alphoid DNA molecules into a complex, which is designated complex A (Muro, Y., H. Masumoto, K. Yoda, N. Nozaki, M. Ohashi, and T. Okazaki. 1992. J. Cell Biol. 116:585-596). Since CENP-B gene is conserved in mammalian species and CENP-B boxes are found also in mouse centromere satellite DNA (minor satellite), this sequence-specific DNA-protein interaction may be important for some kind of common centromere function. In this study we have characterized the structure of CENP-B and CENP-B-alphoid DNA complex. We have shown by chemical cross-linking that CENP-B formed a dimer, and have estimated by molecular weight determination the composition of complex A to be a CENP-B dimer and two molecules of alphoid DNA. The DNA binding domain has been delimited within the NH2-terminal 125-amino acid region containing four potential alpha-helices using truncated CENP-B made in Escherichia coli cells. We have shown that CENP-B had sites highly sensitive to proteases and that the DNA binding domain was separable from the dimerizing activity by the proteolytic cleavage at 20 kD from the COOH terminus of the molecule. Thus, CENP-B may organize a higher order structure in the centromere by juxtaposing two CENP-B boxes in the alphoid DNA repeat through both the DNA-protein and protein-protein interactions.

A human centromere antigen (CENP-B) interacts with a short specific sequence in alphoid DNA, a human centromeric satellite.

We report the interaction between a human centromere antigen and an alphoid DNA, a human centromeric satellite DNA, which consists of 170-bp repeating units. A cloned alphoid DNA fragment incubated with a HeLa cell nuclear extract is selectively immunoprecipitated by the anticentromere sera from scleroderma patients. Immunoprecipitation of the DNA made by primer extension defines the 17-bp segment on the alphoid DNA that is required for formation of DNA-antigen complex. On the other hand, when proteins bound to the biotinylated alphoid DNA carrying the 17-bp motif are recovered by streptavidin agarose and immunoblotted, the 80-kD centromere antigen (CENP-B) is detected. DNA binding experiments for proteins immunoprecipitated with anticentromere serum, separated by gel electrophoresis, and transferred to a membrane strongly suggest that the 80-kD antigen specifically binds to the DNA fragment with the 17-bp motif. The 17-bp motif is termed the "CENP-B box." Alphoid monomers with the CENP-B box are found in all the known alphoid subclasses, with varying frequencies, except the one derived from the Y chromosome so far cloned. These results imply that the interaction of the 80-kD centromere antigen with the CENP-B box in the alphoid repeats may play some crucial role in the formation of specified structure and/or function of human centromere.

Centromere protein B assembles human centromeric alpha-satellite DNA at the 17-bp sequence, CENP-B box.

We purified 15,000-fold from HeLa cell nuclear extract the centromere antigen that reacts specifically with the 17-bp sequence, designated previously as CENP-B box, in human centromeric alpha-satellite (alphoid) DNA by a two-step procedure including an oligonucleotide affinity column. The purified protein was identified as the centromere protein B (CENP-B) by its mobility on SDS-PAGE (80 kD), and reactivities to a monoclonal antibody raised to CENP-B (bacterial fusion protein) and to anticentromere sera from patients with autoimmune diseases. Direct binding by CENP-B of the CENP-B box sequence in the alphoid DNA has been proved using the purified CENP-B by DNA mobility-shift assay, Southwestern blotting, and DNase I protection analysis. The binding constant of the antigen to the CENP-B box sequence is 6 x 10(8) M-1. DNA mobility-shift assays indicated that the major complex formed between the CENP-B and the DNA contains two DNA molecules, suggesting the importance of the CENP-B/CENP-B box interaction in organization of higher ordered chromatin structures in the centromere and/or kinetochore. Location of DNA binding and dimerization domains in CENP-B was discussed based on the DNA mobility-shift assays performed with a protein fraction containing intact and partial cleavage products of CENP-B.

Giant Faraday Rotation in Metal-Fluoride Nanogranular Films.

Magneto-optical Faraday effect is widely applied in optical devices and is indispensable for optical communications and advanced information technology. However, the bismuth garnet Bi-YIG is only the Faraday material since 1972. Here we introduce (Fe, FeCo)-(Al-,Y-fluoride) nanogranular films exhibiting giant Faraday effect, 40 times larger than Bi-YIG. These films have a nanocomposite structure, in which nanometer-sized Fe, FeCo ferromagnetic granules are dispersed in a Al,Y-fluoride matrix.

Dpb11 controls the association between DNA polymerases alpha and epsilon and the autonomously replicating sequence region of budding yeast.

Dpb11 is required for chromosomal DNA replication and the S-phase checkpoint in Saccharomyces cerevisiae. Here, we report detection of a physical complex containing Dpb11 and DNA polymerase epsilon (Dpb11-Polepsilon complex). During the S phase of the cell cycle, Dpb11 associated preferentially with DNA fragments containing autonomously replicating sequences (ARSs), at the same time as Polepsilon associated with these fragments. Association of Dpb11 and Polepsilon with these fragments was mutually dependent, suggesting that the Dpb11-Polepsilon complex associates with the ARS. Moreover, Dpb11 was required for the association of Polalpha-primase with the fragments. Thus, it seems likely that association of the Dpb11-Polepsilon complex with the ARS fragments is required for the association of the Polalpha-primase complex. Hydroxyurea inhibits late-origin firing in S. cerevisiae, and the checkpoint genes, RAD53 and MEC1, are involved in this inhibition. In the presence of hydroxyurea at temperatures permissive for cell growth, Polepsilon in dpb11-1 cells associated with early- and late-origin fragments. In wild-type cells, however, it associated only with early-origin fragments. This indicates that Dpb11 may also be involved in the regulation of late-origin firing. Overall, these results suggest that Dpb11 controls the association between DNA polymerases alpha and epsilon and the ARS.

Analysis of protein-DNA and protein-protein interactions of centromere protein B (CENP-B) and properties of the DNA-CENP-B complex in the cell cycle.

We previously reported that centromere protein B (CENP-B) forms a stable complex (designated complex A) containing two alphoid DNAs in vitro. Domains in the CENP-B polypeptide involved in the formation of complex A were determined in the present study with truncated derivatives expressed in Escherichia coli and in rabbit reticulocyte lysates. It was revealed by gel mobility shift analyses that polypeptides containing the NH2-terminal DNA-binding domain bind a DNA molecule as a monomer, while dimerizing at a novel hydrophobic domain in the COOH-terminal region of 59 amino acid residues. This polypeptide dimerization activity at the COOH-terminal region was also confirmed with the two-hybrid system in Saccharomyces cerevisiae cells. The results thus proved that CENP-B polypeptides form a homodimer at the COOH-terminal hydrophobic domain, each binding a DNA strand at their NH2-terminal domains. The dimerization and DNA-binding domains fall into two of the three completely conserved sequences found in human and mouse CENP-B, and complex A-forming activity was also detected in nuclear extracts of mouse cells. Metaphase-specific phosphorylation of CENP-B was also detected, but this had no effect on its complex A-forming activity. On the basis of the present results, we propose that CENP-B plays an important role in the assembly of specific centromere structures by forming unique DNA-protein complexes at the sites of CENP-B boxes on the centromeric repetitive DNA both in interphase nuclei and on mitotic chromosomes.

Sld2, which interacts with Dpb11 in Saccharomyces cerevisiae, is required for chromosomal DNA replication.

The DPB11 gene, which genetically interacts with DNA polymerase II (epsilon), one of three replicative DNA polymerases, is required for DNA replication and the S phase checkpoint in Saccharomyces cerevisiae. To identify factors interacting with Dbp11, we have isolated sld (synthetically lethal with dpb11-1) mutations which fall into six complementation groups (sld1 to -6). In this study, we characterized SLD2, encoding an essential 52-kDa protein. High-copy SLD2 suppressed the thermosensitive growth defect caused by dpb11-1. Conversely, high-copy DPB11 suppressed the temperature-sensitive growth defect caused by sld2-6. The interaction between Sld2 and Dpb11 was detected in a two-hybrid assay. This interaction was evident at 25 degreesC but not at 34 degreesC when Sld2-6 or Dpb11-1 replaced its wild-type protein. No interaction between Sld2-6 and Dpb11-1 could be detected even at 25 degreesC. Immunoprecipitation experiments confirmed that Dpb11 physically interacts with Sld2. sld2-6 cells were defective in DNA replication at the restrictive temperature, as were dpb11-1 cells. Further, in dpb11-1 and sld2-6 cells, the bubble-shaped replication intermediates formed in the region of the autonomously replicating sequence reduced quickly after a temperature shift. These results strongly suggest the involvement of the Dpb11-Sld2 complex in a step close to the initiation of DNA replication.

CENP-B binds a novel centromeric sequence in the Asian mouse Mus caroli.

Minor satellite DNA, found at Mus musculus centromeres, is not present in the genome of the Asian mouse Mus caroli. This repetitive sequence family is speculated to have a role in centromere function by providing an array of binding sites for the centromere-associated protein CENP-B. The apparent absence of CENP-B binding sites in the M. caroli genome poses a major challenge to this hypothesis. Here we describe two abundant satellite DNA sequences present at M. caroli centromeres. These satellites are organized as tandem repeat arrays, over 1 Mb in size, of either 60- or 79-bp monomers. All autosomes carry both satellites and small amounts of a sequence related to the M. musculus major satellite. The Y chromosome contains small amounts of both major satellite and the 60-bp satellite, whereas the X chromosome carries only major satellite sequences. M. caroli chromosomes segregate in M. caroli x M. musculus interspecific hybrid cell lines, indicating that the two sets of chromosomes can interact with the same mitotic spindle. Using a polyclonal CENP-B antiserum, we demonstrate that M. caroli centromeres can bind murine CENP-B in such an interspecific cell line, despite the absence of canonical 17-bp CENP-B binding sites in the M. caroli genome. Sequence analysis of the 79-bp M. caroli satellite reveals a 17-bp motif that contains all nine bases previously shown to be necessary for in vitro binding of CENP-B. This M. caroli motif binds CENP-B from HeLa cell nuclear extract in vitro, as indicated by gel mobility shift analysis. We therefore suggest that this motif also causes CENP-B to associate with M. caroli centromeres in vivo. Despite the sequence differences, M. caroli presents a third, novel mammalian centromeric sequence producing an array of binding sites for CENP-B.

Centromere protein B of African green monkey cells: gene structure, cellular expression, and centromeric localization.

Centromere protein B (CENP-B) is a centromeric DNA-binding protein which recognizes a 17-bp sequence (CENP-B box) in human and mouse centromeric satellite DNA. The African green monkey (AGM) is phylogenetically closer to humans than mice and is known to contain large amounts of alpha-satellite DNA, but there has been no report of CENP-B boxes or CENP-B in the centromere domains of its chromosomes. To elucidate the AGM CENP-B-CENP-B box interaction, we have analyzed the gene structure, expression, biochemical properties, and centromeric localization of its CENP-B. The amino acid sequence deduced from the cloned AGM CENP-B gene was established to be highly homologous to that of human and mouse CENP-B. In particular, the DNA binding and homodimer formation domains demonstrated 100% identity to their human and mouse counterparts. Immunoblotting and DNA mobility shift analyses revealed CENP-B to be expressed in AGM cell lines. As predicted from the gene structure, the AGM CENP-B in the cell extracts exhibited the same DNA binding specificity and homodimer forming activity as human CENP-B. By indirect immunofluorescent staining of AGM mitotic cells with anti-CENP-B antibodies, a centromere-specific localization of AGM CENP-B could be demonstrated. We also isolated AGM alpha-satellite DNA with a CENP-B box-like sequence with CENP-B affinity. These results not only prove that CENP-B functionally persists in AGM cells but also suggest that the AGM genome contains the recognition sequences for CENP-B (CENP-B boxes with the core recognition sequence or CENP-B box variants) in centromeric satellite DNA.

Replication slippage between distant short repeats in Saccharomyces cerevisiae depends on the direction of replication and the RAD50 and RAD52 genes.

Small direct repeats, which are frequent in all genomes, are a potential source of genome instability. To study the occurrence and genetic control of repeat-associated deletions, we developed a system in the yeast Saccharomyces cerevisiae that was based on small direct repeats separated by either random sequences or inverted repeats. Deletions were examined in the LYS2 gene, using a set of 31- to 156-bp inserts that included inserts with no apparent potential for secondary structure as well as two quasipalindromes. All inserts were flanked by 6- to 9-bp direct repeats of LYS2 sequence, providing an opportunity for Lys+ reversion via precise excision. Reversions could arise by extended deletions involving either direct repeats or random sequences and by -1-or +2-bp frameshift mutations. The deletion breakpoints were always associated with short (3- to 9-bp) perfect or imperfect direct repeats. Compared with the POL+ strain, deletions between small direct repeats were increased as much as 100-fold, and the spectrum was changed in a temperature-sensitive DNA polymerase delta pol3-t mutant, suggesting a role for replication. The type of deletion depended on orientation relative to the origin of replication. On the basis of these results, we propose (i) that extended deletions between small repeats arise by replication slippage and (ii) that the deletions occur primarily in either the leading or lagging strand. The RAD50 and RAD52 genes, which are required for the recombinational repair of many kinds of DNA double-strand breaks, appeared to be required also for the production of up to 90% of the deletions arising between separated repeats in the pol3-t mutant, suggesting a newly identified role for these genes in genome stability and possibly replication.

Construction of YAC-based mammalian artificial chromosomes.

To construct a mammalian artificial chromosome (MAC), telomere repeats and selectable markers were introduced into a 100 kb yeast artificial chromosome (YAC) containing human centromeric DNA. This YAC, which has a regular repeat structure of alpha-satellite DNA and centromere protein B (CENP-B) boxes, efficiently formed MACs that segregated accurately and bound CENP-B, CENP-C, and CENP-E. The MACs appear to be about 1-5 Mb in size and contain YAC multimers. Structural analyses suggest that the MACs have not acquired host sequences and were formed by a de novo mechanism. The accurate segregation of the MACs suggests they have potential as vectors for introducing genes into mammals.

[Tricuspid valve plasty associated with removal of an infected pacemaker lead: report of a case].

We performed tricuspid valve plasty in a 72-year-old woman with pacemaker lead infection and septicemia. All the infected pacemaker system was removed under cardiopulmonary bypass. Because of advanced adhesion and infection, we needed partial resection and plasty of the tricuspid valve. Postoperative echocardiography revealed only mild tricuspid regurgitation and the recurrence of infection has been avoided. Our technique of valve plasty was useful in a patient with advanced infection of both pacemaker leads and tricuspid valve leaflets.

[The third repair for pseudoaneurysm following Bentall procedure: report of a case].

A 77-year-old male, who had undergone the Bentall procedure 27 years ago, was admitted to our hospital for the repair of postoperative pseudoaneurysm. This was the 3rd repair, and the pseudoaneurysm was close to the sternum. Total extracorporeal circulation was established with femorofemoral cannulation and sternotomy was performed under deep hypothermia. During sternotomy, we encountered massive hemorrhage due to injury of the aortic graft. We coped effectively with the situation utilizing temporary circulatory arrest. Aortic graft reimplantation was performed under continuous retrograde cerebral perfusion. Collapse of the suture line of the left coronary orifice was recognized and was reconstructed. The patient was discharged uneventfully on the 26th postoperative day.

[Treatment strategy for acute type A aortic dissection; how to manage mesenteric ischemia].

Mesenteric ischemia is a dreaded complication of acute type A aortic dissection. From January 1994 to December 2004, 134 patients with acute type A aortic dissection were operated. Eleven patients showed postoperative mesenteric ischemia. Mortality of such patients was much higher than that without mesenteric ischemia (81.8 vs. 10.6% , p < 0.0001). Preoperative mesenteric and/or lower extremity ischemia were revealed to be the risk factors of postoperative mesenteric ischemia. Our strategy to manage these patients is as follows; patients who are suffering mesenteric and/or lower extremity ischemia preoperatively, or those whose computed tomography (CT) shows stenosis, obstruction, or dissection of the superior mesenteric artery, should be recognized as high-risk patients of postoperative mesenteric ischemia. Their mesenteric circulation should be examined directly with laparotomy after the central repair. If the mesenteric circulation seems to be suboptimal, iliac artery-superior mesenteric artery bypass should be performed.

[Clinical outcome of aortic valve replacement with 19-mm prosthetic valve in elderly patients].

From 1979 to June 2005, 90 patients aged 65 or older underwent aortic valve replacement with 19-mm prosthetic valve. They were 84 women and 6 men, with a mean age of 74. The mean body surface area was 1.35 m2. Bioprosthetic valves were implanted in 77 patients (85.6%). In-hospital mortality was 2.2% (2 of 90). There were 13 late deaths. New York Heart Association (NYHA) functional class improved to class I in most of survivors. Survival rates for 5 and 10 years were 84.9 and 71.2%, respectively. The outcome of aortic valve replacement with 19-mm prosthetic valve in elderly patients was excellent.

Molecular cloning and expression of aminopeptidase A isoforms from rat hippocampus.

The full-length cDNA encoding aminopeptidase A (APAL) was cloned from a rat hippocampus cDNA library. A short variant aminopeptidase A (APAS), produced by deletion, was also cloned. In the case of APAL, the longest open reading frame encodes 945 amino acid residues with a calculated molecular mass of 108 kDa, and the deduced amino acid sequence shows 76, 86 and 78% identity with its human, murine and porcine counterparts, respectively. Rat aminopeptidase A mRNAs were detected in the kidney, liver, heart and brain by Northern blot analysis. When overexpressed in COS-1 cells, APAL shows apparent aminopeptidase A activity, whereas APAS does not.

Purification of a human centromere antigen (CENP-B) and application of DNA immunoprecipitation to quantitative assay for anti-CENP-B antibodies.

Centromere protein-B (CENP-B) was purified from HeLa nuclear extract by using a combination of Q-sepharose ion change and oligonucleotide sepharose column chromatography. CENP-B was purified more than 10,000 times and was analyzed by immunoblotting and DNA immunoprecipitation. Purified CENP-B was used as an antigen to develop DNA immunoprecipitation for rapid and specific detection of anti-CENP-B antibody in human sera. In this analysis, none of the tested sera immunoprecipitated DNA alone. All of the 40 anticentromere antibody (ACA)-positive sera immunoprecipitated CENP-B-alphoid DNA fragment complex, whereas 10 healthy control sera and 10 other autoantibody positive sera did not. Five of the ACA-positive sera, which did not show reactivity of CENP-B in immunoblotting analysis, immunoprecipitated CENP-B-DNA complex. In some sera antigenicity to CENP-B may be weakened by denaturation.

Kinetic study of the reaction of ebselen with peroxynitrite.

The second-order rate constant for the reaction of ebselen with peroxynitrite (ONOO-) is (2.0+/-0.1) X 10(6) M(-1) s(-1) at pH > or = 8 and 25 degrees C, 3-4 orders of magnitude higher than the rate constants observed for cysteine, ascorbate, or methionine. The activation energy is relatively low, 12.8 kJ/mol. This is the fastest reaction of peroxynitrite observed so far. It may allow Se-containing compounds to play a novel role in the defense against peroxynitrite, one of the important reactive species generated during inflammatory processes.

Dissociation of m-calpain subunits occurs after autolysis of the N-terminus of the catalytic subunit, and is not required for activation.

Calpain is a heterodimeric, intracellular Ca(2+)-dependent, "bio-modulator" that alters the properties of substrates through site-specific proteolysis. It has been proposed that calpains are activated by autolysis of the N-terminus of the large subunit and/or its dissociation into the subunits. It is, however, unclear whether the dissociation into subunits is required for the expression of protease activity and/or for in vivo function. Recently, the crystal structure of m-calpain in the absence of Ca(2+) has been resolved. The 3D structure clearly shows that the N-terminus of the m-calpain large subunit (mCL) makes contact with the 30K subunit, suggesting that autolysis of the N-terminus of mCL changes the interaction of both subunits. To examine the relationship between autolysis, dissociation, and activation, we made and analysed a series of N-terminal mutants of mCL that mimic the autolysed forms or have substituted amino acid residue(s) interacting with 30K. As a result, the mutant m-calpains, which are incapable of autolysis, did not dissociate into subunits, whereas those lacking the N-terminal 19 residues (Delta 19), but not those lacking only nine residues (Delta 9), dissociated into subunits even in the absence of Ca(2+). Moreover, both Delta 9 and Delta 19 mutants showed an equivalent reduced Ca(2+) requirement for protease activity. These results indicate that autolysis is necessary for the dissociation of the m-calpain subunits, and that the dissociation occurs after, but is not necessary for, activation.