Critical-sized marginal defects around implants in the rabbit mandible.

BACKGROUND: The mandible of the rabbit is considered a reliable model to be used to study bone regeneration in defects. The aim of the present study was to evaluate the formation of new bone around implants installed in defects of either 5 or 10 mm in the mandible of rabbits. MATERIALS AND METHODS: In 12 rabbits, 3 mm deep circumferential defect, either 5 or 10 mm in diameter, were prepared bilaterally and an implant was placed in the center. A collagen membrane was placed to close the entrance. After 10 weeks, biopsies were taken, histological slides were prepared, and different regions of the defects were analyzed. RESULTS: Similar amounts of new bone were found in both defects. However, most of the 5 mm defects were filled with new bone. New bone was observed closing the entrance of the defect and laid onto the implant surface. Only in a few cases the healing was incomplete. Despite a similar percentage of new bone found within the 10 mm defects, the healing was incomplete in most of the cases, presenting a low rate of bone formation onto the implant surface within the defect. Only one case presented the closure of the entrance. CONCLUSIONS: The dimensions of the defect strongly influenced the healing so that a circumferential marginal defect of 10 mm around an implant in the mandible body should be considered a critical-sized defect. The presence of the implant and of residues of teeth might have strongly influenced the healing.

Influence on Implant Bone Healing of a Collagen Membrane Placed Subjacent the Sinus Mucosa-A Randomized Clinical Trial on Sinus Floor Elevation.

BACKGROUND: Perforation of the sinus mucosa is quite a frequent complication that might occur during sinus floor elevation. The perforation is often protected with a collagen membrane to avoid the extrusion of graft particles within the sinus. However, this procedure might hinder the innate osteogenic potential of the sinus mucosa. Hence, the aim of the study was to evaluate the influence of a placement of a collagen membrane subjacent the Schneiderian membrane during sinus floor elevation on implant bone healing. METHODS: Twenty volunteers took part in the trial. Ten were randomly included in the group that received a collagen membrane subjacent the sinus mucosa (Mb group), and ten did not receive the membrane (non-Mb group). A collagenated corticocancellous porcine bone was used to fill the elevated space. Six 6 months after the sinus floor elevation, a mini implant was placed transcrestally and retrieved after a further 3 months. Histological analyses were then performed on the full body of the mini implant as well as on its coronal and apical portions. RESULTS: The new bone apposition proportion onto the implant surface was similar in the Mb and non-Mb groups, both in the apical and coronal portions of the mini implants. A lesser amount of graft was found in contact with the surface. New bone density around the mini implants were similar both in the apical and coronal portions. However, a statistically higher proportion of graft particles was found in the Mb group compared to the non-membrane group. CONCLUSIONS: The placement of a collagen membrane subjacent the sinus mucosa did not affect bone healing at implants and bone density.

MAJIN Links Telomeric DNA to the Nuclear Membrane by Exchanging Telomere Cap.

In meiosis, telomeres attach to the inner nuclear membrane (INM) and drive the chromosome movement required for homolog pairing and recombination. Here, we address the question of how telomeres are structurally adapted for the meiotic task. We identify a multi-subunit meiotic telomere-complex, TERB1/2-MAJIN, which takes over telomeric DNA from the shelterin complex in mouse germ cells. TERB1/2-MAJIN initially assembles on the INM sequestered by its putative transmembrane subunit MAJIN. In early meiosis, telomere attachment is achieved by the formation of a chimeric complex of TERB1/2-MAJIN and shelterin. The chimeric complex matures during prophase into DNA-bound TERB1/2-MAJIN by releasing shelterin, forming a direct link between telomeric DNA and the INM. These hierarchical processes, termed "telomere cap exchange," are regulated by CDK-dependent phosphorylation and the DNA-binding activity of MAJIN. Further, we uncover a positive feedback between telomere attachment and chromosome movement, revealing a comprehensive regulatory network underlying meiosis-specific telomere function in mammals.

[Postoperative Analgesia in Peritonectomy for Patients with Peritoneal Surface Malignancy].

BACKGROUND: We investigated the efficacy of postoperative analgesia in peritonectomy for patients with peritoneal surface malignancy, by comparing peripheral nerve block (PNB) with intravenous patient controlled analgesia (iv-PCA) group to patient controlled epidural analgesia (PCEA) group. METHODS: Forty one patients of PNB+iv-PCA group received ultrasound guided rectus sheath block (using 0.25% levobupivacaine 20 ml bilaterally) and posterior transversus abdominis plane block (30 ml bilaterally) after induction of anesthesia. Then, iv-PCA with fentanyl was commenced before skin incision and 10 mg morphine was given 1 hr before the end of surgery. Fifty eight patients of PCEA group received 5-6 mg morphine epidural block at T6-7 or T7-8 before induction of anesthesia, and PCEA of 4-5 mg morphine and 200 ml normal saline was commenced before skin incision. When PCA showed inadequate effect for post- operative pain, flurbiprofen was used for all patients. RESULTS: The rate of administration of flurbiprofen during the first 24 hr after surgery was significantly lower in PNB+iv-PCA group. The duration of the first administration of flurbiprofen after extubation was significantly longer in PNB+iv-PCA group. Levobupivacaine (0.25%) 100 ml for PNB was safe to use without complications. CONCLUSIONS: PNB+iv-PCA decreased the rate of administration of flurbiprofen as compared to PCEA for postoperative pain after peritonectomy.

The dissection of meiotic chromosome movement in mice using an in vivo electroporation technique.

During meiosis, the rapid movement of telomeres along the nuclear envelope (NE) facilitates pairing/synapsis of homologous chromosomes. In mammals, the mechanical properties of chromosome movement and the cytoskeletal structures responsible for it remain poorly understood. Here, applying an in vivo electroporation (EP) technique in live mouse testis, we achieved the quick visualization of telomere, chromosome axis and microtubule organizing center (MTOC) movements. For the first time, we defined prophase sub-stages of live spermatocytes morphologically according to GFP-TRF1 and GFP-SCP3 signals. We show that rapid telomere movement and subsequent nuclear rotation persist from leptotene/zygotene to pachytene, and then decline in diplotene stage concomitant with the liberation of SUN1 from telomeres. Further, during bouquet stage, telomeres are constrained near the MTOC, resulting in the transient suppression of telomere mobility and nuclear rotation. MTs are responsible for these movements by forming cable-like structures on the NE, and, probably, by facilitating the rail-tacking movements of telomeres on the MT cables. In contrast, actin regulates the oscillatory changes in nuclear shape. Our data provide the mechanical scheme for meiotic chromosome movement throughout prophase I in mammals.

RBMX: a regulator for maintenance and centromeric protection of sister chromatid cohesion.

Cohesion is essential for the identification of sister chromatids and for the biorientation of chromosomes until their segregation. Here, we have demonstrated that an RNA-binding motif protein encoded on the X chromosome (RBMX) plays an essential role in chromosome morphogenesis through its association with chromatin, but not with RNA. Depletion of RBMX by RNA interference (RNAi) causes the loss of cohesin from the centromeric regions before anaphase, resulting in premature chromatid separation accompanied by delocalization of the shugoshin complex and outer kinetochore proteins. Cohesion defects caused by RBMX depletion can be detected as early as the G2 phase. Moreover, RBMX associates with the cohesin subunits, Scc1 and Smc3, and with the cohesion regulator, Wapl. RBMX is required for cohesion only in the presence of Wapl, suggesting that RBMX is an inhibitor of Wapl. We propose that RBMX is a cohesion regulator that maintains the proper cohesion of sister chromatids.

A conserved KASH domain protein associates with telomeres, SUN1, and dynactin during mammalian meiosis.

In yeasts and worms, KASH (Klarsicht/ANC-1/Syne/homology) domain and SUN (Sad-1/UNC-84) domain nuclear envelope (NE) proteins play a crucial role in meiotic chromosome movement and homologue pairing. However, although the vertebrate SUN domain protein SUN1 is involved in these processes, its partner has remained identified. Based on subcellular localization screening in mouse spermatocytes, we identified a novel germ cell-specific protein, KASH5, that localized exclusively at telomeres from the leptotene to diplotene stages in both spermatocytes and oocytes. KASH5 possesses hitherto unknown KASH-related sequences that directly interacted with SUN1 and mediated telomere localization. Thus, KASH5 is a mammalian meiosis-specific KASH domain protein. We show that meiotic chromosome movement depended on microtubules and that KASH5 interacted with the microtubule-associated dynein-dynactin complex. These results suggest that KASH5 connects the telomere-associated SUN1 protein to the cytoplasmic force-generating mechanism involved in meiotic chromosome movement. Our study strongly suggests that the meiotic homologue-pairing mechanism mediated by the SUN-KASH NE bridge is highly conserved among eukaryotes.

[Influence of remifentanil on postoperative pain with intravenous patient-controlled analgesia following total knee arthroplasty].

BACKGROUND: There is a controversy about the development of acute opioid tolerance during remifentanil infusion for postoperative pain. We investigated whether intraoperative remifentanil infusion leads to rapid development of tolerance. We also investigated whether adjuvant analgesic is effective or not. METHODS: One hundred and thirty patients scheduled for TKA were divided into 4 groups; Non-remifentanil infusion group (30 patients; group A). This group was given a bolus of fentanyl 0.2 mg at the induction of anesthesia. In remifentanil anesthesia group (100 patients), remifentanil was given 0.2-0.3 microg x kg(-1) min(-1) and divided into three groups; 23 patients group B given a bolus of 0.2 mg fentanyl at induction of anesthesia, 38 patients group C given a bolus of fentanyl 0.2 mg at skin closure, and 39 patients group D given a bolus of fentanyl 0.2 mg at skin closure and administrated diclofenac sodium suppository rectally at induction of anesthesia. Intravenous patient controlled analgesia (PCA) of fentanyl was commenced before skin incision for all patients. Pain was scored at rest and during movement for 24 postoperative hours. RESULTS: Numerical rating scale (NRS) scores during movement after 24 hours in group B were significantly higher than in group A. And, the NRS scores in group D tended to be lower than the other groups, but there were no significant differences. CONCLUSIONS: Intraoperative remifentanil increased postoperative pain during movement. Remifentanil seems to cause acute torelance. NSAIDs given before operation may be useful for postoperative analgesia.

Assembly states of the nucleosome assembly protein 1 (NAP-1) revealed by sedimentation velocity and non-denaturing MS.

Proteins often exist as ensembles of interconverting states in solution which are often difficult to quantify. In the present manuscript we show that the combination of MS under nondenaturing conditions and AUC-SV (analytical ultracentrifugation sedimentation velocity) unambiguously clarifies a distribution of states and hydrodynamic shapes of assembled oligomers for the NAP-1 (nucleosome assembly protein 1). MS established the number of associated units, which was utilized as input for the numerical analysis of AUC-SV profiles. The AUC-SV analysis revealed that less than 1% of NAP-1 monomer exists at the micromolar concentration range and that the basic assembly unit consists of dimers of yeast or human NAP-1. These dimers interact non-covalently to form even-numbered higher-assembly states, such as tetramers, hexamers, octamers and decamers. MS and AUC-SV consistently showed that the formation of the higher oligomers was suppressed with increasing ionic strength, implicating electrostatic interactions in the formation of higher oligomers. The hydrodynamic shapes of the NAP-1 tetramer estimated from AUC-SV agreed with the previously proposed assembly models built using the known three-dimensional structure of yeast NAP-1. Those of the hexamer and octamer could be represented by new models shown in the present study. Additionally, MS was used to measure the stoichiometry of the interaction between the human NAP-1 dimer and the histone H2A-H2B dimer or H3-H4 tetramer. The present study illustrates a rigorous procedure for the analysis of protein assembly and protein-protein interactions in solution.

The nuclear scaffold protein SAF-A is required for kinetochore-microtubule attachment and contributes to the targeting of Aurora-A to mitotic spindles.

Segregation of chromosomes during cell division requires correct formation of mitotic spindles. Here, we show that a scaffold attachment factor A (SAF-A), also known as heterogeneous nuclear ribonucleoprotein-U, contributes to the attachment of spindle microtubules (MTs) to kinetochores and spindle organization. During mitosis, SAF-A was localized at the spindles, spindle midzone and cytoplasmic bridge. Depletion of SAF-A by RNA interference induced mitotic delay and defects in chromosome alignment and spindle assembly. We found that SAF-A specifically co-immunoprecipitated with the chromosome peripheral protein nucleolin and the spindle regulators Aurora-A and TPX2, indicating that SAF-A is associated with nucleolin and the Aurora-A-TPX2 complex. SAF-A was colocalized with TPX2 and Aurora-A in spindle poles and MTs. Elimination of TPX2 or Aurora-A from cells abolished the association of SAF-A with the mitotic spindle. Interestingly, SAF-A can bind to MTs and contributes to the targeting of Aurora-A to mitotic spindle MTs. Our finding indicates that SAF-A is a novel spindle regulator that plays an essential role in kinetochore-MT attachment and mitotic spindle organization.

Visualization of mitotic HeLa cells by advanced polarized light microscopy.

The conventional polarized light imaging system can observe sub-microscopic molecular order non-destructively, quantitatively and without labeling or staining. Recently, a more sophisticated version, Abrio imaging system, than the conventional polarized light imaging system, was developed. This advanced polarized light imaging system has simplified the process of birefringent imaging, higher sensitivity and accuracy irrespective of sample orientation. By performing time-lapse observations using the advanced polarized light microscopy, we visualized mitotic spindles, especially kinetochore microtubules, of HeLa cells. Moreover, we successfully visualized the detailed structure of several filament bundles, which possibly are components of the contractile ring. Here, we report the potential of advanced polarized light imaging systems for imaging mitotic HeLa cells and the new insights obtained during this microscopic study.

H1.X with different properties from other linker histones is required for mitotic progression.

We report here the characterization of H1.X, a human histone H1 subtype. We demonstrate that H1.X accumulates in the nucleolus during interphase and is distributed at the chromosome periphery during mitosis. In addition, the results of fluorescence recovery after photobleaching indicate that the exchange of H1.X on and off chromatin is faster than that of the other H1 subtypes. Furthermore, RNA interference experiments reveal that H1.X is required for chromosome alignment and segregation. Our results suggest that H1.X has important functions in mitotic progression, which are different from those of the other H1 subtypes.

PHB2 protects sister-chromatid cohesion in mitosis.

Cohesion between sister chromatids is essential for proper chromosome segregation in mitosis. In vertebrate mitotic cells, most cohesin is removed from the chromosome arms [1-4], but centromeric cohesin is protected by shugoshin until the onset of anaphase [5]. However, the mechanism of this protection of centromeric cohesion is not well understood. Here, we demonstrate that prohibitin 2 (PHB2) is involved in the regulation of sister-chromatid cohesion during mitosis in HeLa cells. PHB2 is an evolutionarily conserved protein in eukaryotes and has multiple functions, such as transcriptional regulation and cell viability and development [6-8]. However, its functions in mitosis have not yet been determined. We show that depletion of PHB2 by RNA interference (RNAi) causes premature sister-chromatid separation and defects in chromosome congression accompanied by mitotic arrest by spindle-checkpoint activation. In the absence of PHB2, cohesin is dissociated from centromeres during early mitosis, although the centromeric localization of shugoshin is preserved. Thus, our findings suggest that, in addition to the shugoshin, PHB2 is also required to protect the centromeric cohesion from phosphorylation by Plk1 during early mitosis and that its function is essential for proper mitotic progression.

Histone H2A mobility is regulated by its tails and acetylation of core histone tails.

Histone tail domains play important roles in cellular processes, such as replication, transcription, and chromosome condensation. Histone H2A has one central and two tail domains, and their functions have mainly been studied from a biochemical perspective. In addition, analyses based on visualization have been employed for functional analysis of some chromatin proteins. In this study, we analyzed histone H2A mobility in vivo by two-photon FRAP, and elucidated that the histone H2A N- and C-terminal tails regulate its mobility. We found that histone H2A mobility was increased following treatment of host cells with a histone deacetylase inhibitor. Our results support a model in which core histone tails directly regulate transcription by interacting with nucleosome DNA via electrostatic interactions.