Lamin B2 prevents chromosome instability by ensuring proper mitotic chromosome segregation.

The majority of human cancer shows chromosomal instability (CIN). Although the precise mechanism remains largely uncertain, proper progression of mitosis is crucial. B-type lamins were suggested to be components of the spindle matrix of mitotic cells and to be involved in mitotic spindle assembly; thus, B-type lamins may contribute to the maintenance of chromosome integrity. Here, using a proteomic approach, we identified lamin B2 as a novel protein involved in CIN. Lamin B2 expression decreased in colorectal cancer cell lines exhibiting CIN, as compared with colorectal cancer cell lines exhibiting microsatellite instability (MIN), which is mutually exclusive to CIN. Importantly, lamin B2 knockdown in MIN-type colorectal cancer cells induced CIN phenotypes such as aneuploidy, chromosome mis-segregation and aberrant spindle assembly, whereas ectopic expression of lamin B2 in CIN-type colorectal cancer cells prevented their CIN phenotypes. Additionally, immunohistochemical analysis showed a lower expression of lamin B2 in cancer tissues extracted from patients with sporadic colorectal cancer (CIN-type) than that from patients with hereditary non-polyposis colorectal cancer (HNPCC; MIN type). Intriguingly, mitotic lamin B2 in MIN cancer cells was localized outside the spindle poles and mitotic lamin B2 localization was diminished in CIN cancer cells, suggesting an important role of lamin B2 in proper mitotic spindle formation. The obtained results suggest that lamin B2 maintains chromosome integrity by ensuring proper spindle assembly and that its downregulation causes CIN in colorectal cancer.

Successful tocilizumab and tacrolimus treatment in a patient with rheumatoid arthritis complicated by systemic lupus erythematosus.

We report a 37-year-old female of intractable rheumatoid arthritis (RA) complicated by systemic lupus erythematosus (SLE), who was successfully treated with a combination of tocilizumab (TCZ) and tacrolimus. She was diagnosed with RA when she was 21 years old, and was administered oral prednisolone, injectable gold and salazosulfapyridine, but deformity of her hands gradually developed. She developed high fever and thrombocytopenia when she was 35 years old. Renal involvement, pericarditis, positive antinuclear antibody and high level of anti-double-stranded DNA antibody were found and the patient was diagnosed with SLE. Polyarthritis and immunological abnormalities developed despite aggressive immunosuppressive therapy including high-dose corticosteroids and intravenously administered cyclophosphamide. Tacrolimus (TAC) therapy gave only partial improvement of joint symptoms. After the initiation of combination therapy with TCZ, not only was a complete remission of RA obtained, but also the serum levels of SLE markers dramatically decreased. Our report suggests the possibility that this combination therapy is effective in treating SLE as well as RA.

In vitro and in vivo analysis of a JAK inhibitor in rheumatoid arthritis.

Multiple cytokines play a pivotal role in the pathogenesis of rheumatoid arthritis (RA). The appropriate intracellular signalling pathways must be activated via cytokine receptors on the cell surface, and the tyrosine kinases transduce the first 'outside to in' signals to be phosphorylated after receptor binding to its ligand. Among them, members of the Janus kinase (JAK) family are essential for the signalling pathways of various cytokines and are implicated in the pathogenesis of RA. The in vitro, ex vivo and in vivo effects of a JAK inhibitor CP-690,550 (tofacitinib) for the treatment of RA are reported. In vitro experiments indicated that the effects of tofacitinib were mediated through suppression of interleukin 17 (IL-17) and interferon γ production and proliferation of CD4 T cells, presumably Th1 and Th17. A treatment study was conducted in the severe combined immunodeficiency (SCID)-HuRAg mice, an RA animal model using SCID mice implanted with synovium and cartilage from patients. Tofacitinib reduced serum levels of human IL-6 and IL-8 in the mice and also reduced synovial inflammation and invasion into the implanted cartilage. A phase 2 double-blind study using tofacitinib was carried out in Japanese patients with active RA and inadequate response to methotrexate (MTX). A total of 140 patients were randomised to tofacitinib 1, 3, 5, 10 mg or placebo twice daily and the American College of Rheumatology 20% improvement criteria (ACR20) response rate at week 12, a primary end point, was significant for all tofacitinib treatment groups. Thus, an orally available tofacitinib in combination with MTX was efficacious and had a manageable safety profile. Tofacitinib at 5 and 10 mg twice a day appears suitable for further evaluation to optimise the treatment of RA.

New insight into the mitotic chromosome structure: irregular folding of nucleosome fibers without 30-nm chromatin structure.

Mitotic chromosomes are essential structures for the faithful transmission of replicated genomic DNA into two daughter cells during cell division. A long strand of DNA is wrapped around a core histone and forms a nucleosome. The nucleosome has long been assumed to be folded into 30-nm chromatin fibers. However, how the nucleosome or 30-nm chromatin fiber is organized into mitotic chromosomes remains unclear, although condensins and topoisomerase IIα are implicated in the condensation process. In fact, what do mitotic chromosomes look like in living cells? When frozen hydrated human mitotic cells were observed using cryo-electron microscopy (cryo-EM), higher-order structures including 30-nm chromatin fibers were not found. We thus propose that the nucleosome fibers in the bulk of mitotic chromosomes do not form 30-nm chromatin fibers but instead exist in a highly irregular state that is locally similar to a polymer melt. We provide new insight into mitotic chromosome structure.

Heme arginate pretreatment attenuates pulmonary NF-kappaB and AP-1 activation induced by hemorrhagic shock via heme oxygenase-1 induction.

Hemorrhagic shock followed by resuscitation (HSR) induces oxidative stress that leads to acute lung injury. Heme oxygenase-1 (HO-1), the rate-limiting enzyme in heme catabolism, is induced by oxidative stress and is thought to play an important role in the protection from oxidative tissue injuries. We previously demonstrated that HO-1 induction by heme arginate (HA), a strong inducer of HO-1, ameliorated HSR-induced lung injury and inflammation. Cellular redox state is known to modulate the DNA biding activity of the transcription factors; nuclear factor-kappaB (NF-kappaB) and activator protein-1 (AP-1). In the present study, we treated rats with HA (30 mg/kg of hemin) 18 h prior to HSR and examined its effect on the DNA binding activity of NF-kappaB and AP-1 at 1.5 h after HSR. HSR significantly increased the DNA binding activity of NF-kappaB as well as AP-1, while HA pretreatment markedly attenuated the activities of these transcription factors. In contrast, administration of tin mesoporphyrin, a specific competitive inhibitor of HO activity, to HA-pretreated animals abolished the suppressive effect of HA on the activities of NF-kappaB and AP-1, and increased these activities to almost the same level as those in HSR animals. Our findings indicate that HA pretreatment can significantly suppress the increased activity of NF-kappaB and AP-1 induced by HSR by virtue of its ability to induce HO-1. Our findings also suggest that HO-1 induced by HA pretreatment ameliorates HSR-induced lung injury at least in part mediated through the suppression of the activities of these transcription factors.

Specific targeting of insect and vertebrate telomeres with pyrrole and imidazole polyamides.

DNA minor groove-binding compounds (polyamides) that target insect and vertebrate telomeric repeats with high specificity were synthesized. Base pair recognition of these polyamides is based on the presence of the heterocyclic amino acids pyrrole and imidazole. One compound (TH52B) interacts uniquely and with excellent specificity (K(d) = 0.12 nM) with two consecutive insect-type telomeric repeats (TTAGG). A related compound, TH59, displays high specificity (K(d) = 0.5 nM) for tandem vertebrate (TTAGGG) and insect telomeric repeats. The high affinity and specificity of these compounds were achieved by bidentate binding of two flexibly linked DNA-binding moieties. Epifluorescence microscopy studies show that fluorescent derivatives of TH52B and TH59 stain insect or vertebrate telomeres of chromosomes and nuclei sharply. Importantly, the telomere-specific polyamide signals of HeLa chromosomes co-localize with the immunofluorescence signals of the telomere-binding protein TRF1. Our results demonstrate that telomere-specific compounds allow rapid estimation of relative telomere length. The insect-specific compound TH52 was shown to be incorporated rapidly into growing Sf9 cells, underlining the potential of these compounds for telomere biology and possibly human medicine.

Decline in taste and odor discrimination abilities with age, and relationship between gustation and olfaction.

It is important to learn about changes in both taste and odor perceptions with increasing age, because the taste of foods we encounter in our daily life is strongly affected by their smell. This study discusses the difference in qualitative taste and odor discrimination between the elderly and the young. Tastants and odorants used in this study were presented not as single stimuli but as a taste mixture (sucrose and tartaric acid) and an odor mixture (beta-phenylethyl alcohol and gamma-undecalactone). The results showed that quality discrimination abilities of the elderly subjects for both taste and odor were significantly lower than those of the young subjects, indicating a decline in quality discrimination abilities related to age. Also, a moderate but significant correlation was observed between the taste discrimination ability and the odor discrimination ability. We measured thresholds for single-taste and odor components in mixtures and compared them between the elderly and the young to investigate the cause for these findings.

Nucleotide dependent structural and kinetic changes in Xenopus rad51.1-DNA complex stimulating the strand exchange reaction: destacking of DNA bases and restriction of their local motion.

Rad51 is a eukaryotic homologue of RecA and it catalyzes the DNA strand exchange reaction in homologous recombination. This protein, like RecA, requires ATP as a cofactor for activity. We investigated the mechanism of activation of this protein by the nucleotide cofactor by studying the effect of various nucleotides, particularly ATP, ADP and the non-hydrolyzable analog of ATP, adenosine-5'-O-(3-thiotriphosphate) (ATPgammaS) on the DNA binding of a Xenopus Rad51 protein (XRad51.1). DNA binding was studied in solution by monitoring the fluorescence changes of etheno-modified fluorescent poly(dA) or fluorescein-labeled oligo(dT) and by filter binding assay. Active nucleotides (ATP, dATP) changed the DNA binding mode of XRad51.1. In the active complex, the DNA bases were destacked and their motion was highly restricted. Dissociation of XRad51.1 from DNA was accelerated by ATP and dATP, as was dissociation of RecA from DNA. In contrast to these similarities with RecA, the XRad51.1-DNA complex was dissociated by the non-hydrolyzable analog of ATP (ATPgammaS) and this dissociation was not significantly accelerated by ADP. The effect of ATP hydrolysis on the XRad51.1-DNA complex differs from that on the RecA-DNA complex. ATP hydrolysis may not be essential for the strand exchange reaction whereas the changes in the DNA structure by ATP are important.

[Comparison of the amnesic effects of propofol and isoflurane anesthesia].

Sixty adult patients were assigned to one of 3 groups according to the anesthetic technique chosen: isolfurane inhalation, propofol infusion at a higher rate, and propofol infusion at a lower rate supplemented with epidural anesthesia. When the patients regained consciousness and orientation after surgery, they were shown a digit and asked to confirm it verbally. Memory of the digit was tested on the following day. The percentages of the patients who were able to recall the digit were significantly different among the 3 groups, 5%, 40% and 80%, respectively. We concluded that the amnesic effect of propofol was weaker than that of isoflurane when compared immediately after anesthesia. This tendency was more conspicuous when the dose of propofol was less with epidural anesthesia.

Nucleotide cofactor-dependent structural change of Xenopus laevis Rad51 protein filament detected by small-angle neutron scattering measurements in solution.

Rad51 protein, a eukaryotic homologue of RecA protein, forms a filamentous complex with DNA and catalyzes homologous recombination. We have analyzed the structure of Xenopus Rad51 protein (XRad51.1) in solution by small-angle neutron scattering (SANS). The measurements showed that XRad51.1 forms a helical filament independently of DNA. The sizes of the cross-sectional and helical pitch of the filament could be determined, respectively, from a Guinier plot and the position of the subsidiary maximum of SANS data. We observed that the helical structure is modified by nucleotide binding as in the case of RecA. Upon ATP binding under high-salt conditions (600 mM NaCl), the helical pitch of XRad51.1 filament was increased from 8 to 10 nm and the cross-sectional diameter decreased from 7 to 6 nm. The pitch sizes of XRad51.1 are similar to, though slightly larger than, those of RecA filament under corresponding conditions. A similar helical pitch size was observed by electron microscopy for budding yeast Rad51 [Ogawa, T., et al. (1993) Science 259, 1896-1899]. In contrast to the RecA filament, the structure of XRad51.1 filament with ADP is not significantly different from that with ATP. Thus, the hydrolysis of ATP to ADP does not modify the helical filament of XRad51.1. Together with our recent observation that ADP does not weaken the XRad51.1/DNA interaction, the effect of ATP hydrolysis on XRad51.1 nucleofilament should be very different from that on RecA.

Purification and characterization of XRad51.1 protein, Xenopus RAD51 homologue: recombinant XRad51.1 promotes strand exchange reaction.

BACKGROUND: The RAD51 gene of Saccharomyces cerevisiae is homologous to the Escherichia coli recA gene and plays a key role in genetic recombination and DNA double-strand break repair. To construct an improved experimental system of homologous recombination in higher eukaryotes, we have chosen the South African clawed frog, Xenopus laevis, whose egg extracts might be useful for the in vitro studies. We identified and characterized a Xenopus homologue of RAD51 gene, the XRAD51.1. RESULTS: Recombinant XRad51.1 was expressed in E. coli. The purified XRad51.1 protein showed ssDNA-dependent ATPase activity and promoted the DNA strand exchange reaction between two 55-mer oligonucleotides. The binding stoichiometry of XRad51.1 to ssDNA was determined by fluorescence of poly(d epsilonA), a chemically modified poly(dA), and was found to be about six bases/XRad51.1 monomer in a nucleoprotein filament, a similar value to E. coli RecA protein. The kinetics of the fluorescence change of poly(d epsilonA) after XRad51.1 binding in the presence of ATP was significantly different from that observed with RecA protein. The affinity of XRad51.1 to ssDNA in the presence of ATP was higher than that of RecA protein, and the dissociation of the XRad51.1-ssDNA complex was slower than the RecA-ssDNA complex. CONCLUSIONS: Purified recombinant XRad51.1 protein promoted the strand exchange between short DNA molecules. While the binding stoichiometry of XRad51.1 protein to ssDNA was identical to that of the RecA protein, XRad51.1 has a significantly higher affinity and binding stability to ssDNA than did the RecA protein in the presence of ATP.

RAD51 homologues in Xenopus laevis: two distinct genes are highly expressed in ovary and testis.

The RAD51 gene is a eukaryotic counterpart of the Escherichia coli recA gene which is involved in genetic recombination. Two distinct Xenopus laevis RAD51 cDNA clones (XRAD51.1 and XRAD51.2) were isolated from an oocyte cDNA library using the human RAD51 cDNA (HsRAD51) as a probe. Sequence analysis revealed that 98.2% of the amino-acid residues were identical between XRAD51.1 and XRAD51.2, and that both were 95% identical to HsRAD51. Both of the XRAD51 genes were expressed at a higher level in ovary and testis than in other somatic tissues, suggesting their involvement in meiotic recombination. The expression of XRAD51.1 was about eightfold in excess of that of XRAD51.2 in all of the tissues examined. Analysis of the rates of synonymous substitution in the coding sequences of the two XRAD51 suggests that these two genes diverged about 50 million years ago. The structural similarities of the XRAD51 proteins to RecA in E. coli and Rad51 in yeasts or vertebrates are discussed.

Analysis of biological function of poly(ADP-ribosyl)ation in Drosophila melanogaster.

To understand the biological function of poly(ADP-ribosyl)ation of proteins, we have isolated and characterized the gene for poly(ADP-ribose) polymerase from Drosophila melanogaster. Two approaches were taken to analyze the function of the poly(ADP-ribosyl)ation reaction. The first is analysis of the homology of the amino acid sequences of poly(ADP-ribose) polymerase from phylogenetically different eukaryotes, namely human, mouse, bovine, chicken, Xenopus laevis and Drosophila melanogaster and elucidation of the conserved amino acid sequences that appear to be important for the function of poly(ADP-ribose) polymerase. Analysis of the recombinant poly(ADP-ribose) polymerase which had truncated or mutated motifs expressed in E coli would confirm the importance of the conserved amino acid sequence. The interaction of poly(ADP-ribose) polymerase with other proteins involved in DNA repair, replication, recombination and transcription will clarify the function of poly(ADP-ribosyl)ation. The second approach is to get the mutants which have disruption in the poly(ADP-ribose) polymerase gene and to analyse the phenotypes of these mutants. The characterization of these mutants will be discussed.

Absolute configuration of L-methionine sulfoximine as a toxic principle in Cnestis palala (Lour.) Merr.

An unusual amino acid, L-methionine sulfoximine (1), has been isolated from the fresh seeds of Cnestis palala (Lour.) Merr. [Connaraceae]. The absolute configuration of the natural sulfoximine (1) was confirmed to be 2(S)-methionine S(S)-sulfoximine [(2S,SS)-2-amino-4-(S-methylsulfonimidoyl)-n-butanoic acid] by comparison of the [alpha]D value and IR spectrum with those of authentic samples obtained through the optical resolution of synthetic materials. Acute toxicity of the seeds of C. palala in a beagle dog was also studied.