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Purpose: In the revised Regulation on Prevention of Ionizing Radiation Hazards (April 2020), the equivalent dose limit for the lens of the eye was lowered to "100 mSv in 5 years and 50 mSv in 1 year." It is necessary to reduce occupational exposure in the healthcare sector. The purpose of this study was to facilitate comparison with the equivalent dose limit in an endoscopic retrograde cholangiopancreatography (ERCP) examination by measuring the scattered dose in an X-ray room as an individual dose equivalent., Methods: The scattered dose was measured by dosimeters positioned in a grid pattern in the X-ray room. The dose rate was measured with and without the use of a protective cloth (0.25 mm Pb equivalent) suspended around the X-ray tube extending to the patient. The dose reduction rate of the material was evaluated during the simulated ERCP., Results: The maximum dose rate at the position of the surgeon performing an ERCP inspection in the standing position was 2.9 mSv/h (3 mm dose equivalent rate) at 150 cm from the ground. The number of ERCP examinations to comply with the equivalent dose limit of the equivalent dose of the lens of the eye without the protective cloth was 28 per year., Conclusion: The results of this study suggest that individual dose equivalent measurements in the X-ray clinic may facilitate comparison with the equivalent dose limit.

Background: Collecting lymphatics have lymph-drainage function with contraction of smooth muscle cells. Patients with edema have lost this drainage function due to degeneration of smooth muscle cells. Lymphaticovenular (LV) anastomosis salvages smooth muscle cells from reversible degeneration (mild edema), but muscle cells cannot be recovered from irreversible degeneration (severe edema). Therefore, in severe edema, LV anastomoses cannot reestablish the drainage function of the lymphatic system.To overcome this weakness of LV bypass methods for severe edema, new methods were instituted for repair of this missing drainage function using a lymphadiposal flap from the contralateral foot for hemilateral edema, or transfer of lateral thoracic lymph nodes for bilateral edema., Methods: A total of 13 cases were repaired with lymphadiposal flaps and additional LV anastomoses. These cases have frequent phlegmon or cellulitis or resisted to previous LV anastomoses and/or compression therapy. The ages ranged from 15 to 75 years. There were four cases of primary edema and nine cases of secondary edema., Results: Regarding the lymphadiposal flap (n = 8), three cases showed an excellent response (37.5%; no need for compression therapy), four cases had a good response (50%; improvement with compression), one case showed no change (12.5%; no improvement), and there were no cases of deterioration. Regarding the lateral thoracic lymph nodes transfer (n = 5), two cases had a good response (40%), three showed no improvement (60%), and there were no cases of deterioration., Conclusion: It is concluded that lymphadiposal flap or lymph nodes transfer is suitable for severe edema having frequent cellulitis in unilateral or bilateral lower extremities resisting previous LV anastomoses and/or compression therapy., (Thieme Medical Publishers 333 Seventh Avenue, New York, NY 10001, USA.)

Vascular smooth muscle cell polyploidization occurs during normal development and is enhanced under physiologic stress, but the mechanism of this cell cycle has not been explored. We show via time-lapse video imaging and immunofluorescence analyses that primary vascular smooth muscle cells (VSMC) undergo an endomitotic-type cell cycle, including a normal progression through part of mitosis. Mononuclear polyploid cells are generated by defects in sister chromatid separation and/or segregation, and cellular binucleation occurs by reversal of cytokinesis. To obtain further leads to regulators involved, we examined the chromosomal passenger proteins, Aurora B, inner centromere protein and Survivin, and concluded that Aurora B and inner centromere protein are normally colocalized in centromeres, the midzone, and the midbody during mitosis. Survivin, however, is dim and diffused; it does not colocalize with either Aurora B or inner centromere protein in VSMC, which could account for defects in sister chromatid separation and/or segregation and reversal of cytokinesis. In accordance with the reported dependency of Aurora B activity on Survivin, the Aurora B substrate, vimentin, is not phosphorylated during cytokinesis. Finally, the data show that ectopically expressed Survivin inhibits polyploidization in vascular smooth muscle cells. Hence, aberrant chromosome passenger protein activity and endomitosis are associated with VSMC polyploidization.

MAIL (molecule-possessing ankyrin repeats induced by lipopolysaccharide) is a nuclear IkappaB protein that is also termed interleukin-1-inducible nuclear ankyrin repeat protein or inhibitor of nuclear factor kappaB (IkappaB) zeta. In this study, we generated Mail-/- mice to investigate the roles of MAIL in whole organisms. Mail-/- mice grew normally until 4-8 weeks after birth, when they began to develop lesions in the skin of the periocular region, face, and neck. MAIL mRNA and protein were constitutively expressed in the skin of wild type controls, especially in the keratinocytes. Serum IgE was higher in Mail-/- mice than in normal. Histopathological analysis indicated that the Mail-/- skin lesions appeared to be atopic dermatitis (AD) eczema with inflammatory cell infiltration. In addition, markedly elevated expression of some chemokines such as thymus and activation-regulated chemokine was detected in the Mail-/- skin lesions, similar to that observed in the skin of patients with AD. In Mail-/- mice, MAIL-deficient keratinocytes might be activated to produce chemokines and induce intraepidermal filtration of inflammatory cells, resulting in the onset of the AD-like disease. These findings suggest that MAIL is an essential molecule for homeostatic regulation of skin immunity. The Mail-/- mouse is a valuable new animal model for research on AD.

A number of proteins accumulate in the anaphase spindle midzone, but the interaction and precise role of these proteins in midzone organization remain obscure. Here, we found that the microtubule-bundling protein PRC1 bound separately to the three motor proteins, KIF4, MKLP1 and CENP-E, but not to the chromosomal passenger proteins. In KIF4-deficient cells, the central spindle was disorganized, and all midzone-associated proteins including PRC1 failed to concentrate at the midline, instead being dispersed along the loosened microtubule bundles of the central spindle. This suggests that KIF4 is essential for the organization of central spindles and for midzone formation. In PRC1-deficient cells, no midzone was formed, KIF4 and CENP-E did not localize to the disconnected half-spindle, and MKLP1 and chromosomal passenger proteins localized to discrete subdomains near microtubule plus ends in the half-spindle. Thus, PRC1 is required for interaction of the two half-spindles and for localization of KIF4 and CENP-E. These results suggest that KIF4 and its binding partner PRC1 play essential roles in the organization of central spindles and midzone formation.

Megakaryocytes skip late anaphase and cytokinesis during endomitosis. We found normal expression and localization of a fundamental regulator of mitosis, Aurora-B/AIM-1, during prophase in polyploidizing mouse bone marrow megakaryocytes. At late anaphase, however, Aurora-B/AIM-1 is absent or mislocalized. Megakaryocytes treated with a proteasome inhibitor display Aurora-B/AIM-1 properly expressed and localized to the midzone, suggesting that protein degradation contributes to this atypical appearance. In contrast, survivin, an Aurora-B/AIM-1 coregulator of mitosis, is not detected at any stage of the endomitotic cell cycle, and in most megakaryocytes proteasome inhibition does not rescue this phenotype. To further explore the importance of reduced Aurora-B/AIM-1 for polyploidization, it was overexpressed in megakaryocytes of transgenic mice. The phenotype includes increased transgenic mRNA, but not protein, in polyploidy megakaryocytes, further suggesting that Aurora-B/AIM-1 is regulated at the protein level. Aurora-B/AIM-1 protein is, however, elevated in diploid transgenic megakaryocytes. Transgenic mice also exhibit enhanced numbers of megakaryocytes with increased proliferative potential, and some mice exhibit mild decreases in ploidy level. Hence, the molecular programming involved in endomitosis is characterized by the mislocalization or absence of at least 2 critical mitotic regulators, Aurora-B/AIM-1 and survivin. Future studies will examine the impact of survivin restoration on mouse megakaryocyte polyploidization.

Mammalian spermatogenesis involves drastic morphological changes leading to the development of the mature sperm. Sperm development includes formation of the acrosome and flagellum, translocation of nucleus-acrosome to the cell surface, and condensation and elongation of the nucleus. In addition, spermatogenic cell progenies differentiate as cohorts of units interconnected by intercellular bridges. Little is known about the structural components involved in the establishment of conjoined spermatogenic cells and the mechanism of nuclear shaping of the male gamete. We identified two isoforms of delta-tubulin and found that the long isoform is predominantly expressed in testis, while the short isoform is expressed in all tissues examined. We also found that delta-tubulin forms intercellular bridges conjoining sister spermatogenic cells. In addition, delta-tubulin is a component of the perinuclear ring of the manchette, which acts on translocation and elongation of the nucleus. Furthermore, small rings clearly distinct from the intercellular bridges, which might mature to perinuclear ring of the manchette in later stages of spermatogenesis, were detected on the cell surface of round spermatids. These results suggest that delta-tubulin is a component of two types of ring, the intercellular bridges and the perinuclear rings, which may be involved in morphological changes of spermatid to mature sperm.

A matured megakaryocyte releases thousands of platelets through a drastic morphological change, proplatelet formation (PPF). The megakaryocyte/erythrocyte-specific transcription factor, p45 NF-E2, is essential for initiating PPF, but the factor regulating PPF has not been identified. Here we report that estradiol synthesized in megakaryocytes triggers PPF. We demonstrate that a key enzyme for steroid hormone biosynthesis, 3beta-hydroxysteroid dehydrogenase (3beta-HSD), is a target of p45 NF-E2, and rescues PPF of p45 NF-E2-deficient megakaryocytes. We also show that estradiol is synthesized within megakaryocytes, and that extracellular estradiol stimulates PPF, inhibition of 3beta-HSD activity blocks PPF, and estrogen receptor antagonists inhibit platelet production in vivo. We conclude that autocrine estradiol action regulates platelet production by triggering PPF.

Recent data in yeast and Drosophila suggest a domain-like centromere structure with a modified chromatin core and flanking regions of heterochromatin. We have analyzed a functional human centromere and defined a region of increased chromosome scaffold/matrix attachment that overlaps three other distinct and nonoverlapping domains for constitutive centromere proteins CENP-A and CENP-H, and heterochromatin protein HP1. Transcriptional competency is intact throughout the S/MAR-enriched region and within the CENP-A- and CENP-H-associated chromatin. These results provide insights into the relationship between centromeric chromatin and transcriptional competency in vivo, highlighting the permissibility of transcription within the constitutively modified, nonheterochromatic chromatin of a functional eukaryotic centromere.

The crooked neck (crn) gene of Drosophila melanogaster encodes a scaffold protein carrying multiple tetratricopeptide repeat (TPR) motifs, and its mutation results in a reduction in the number of neuroblasts and lethality during larval stages. Here, we isolated two structurally related genes from a rat embryonic brain cDNA library. One gene is the rat orthologue of crn, which encodes 690 amino acids including 16 copies of TPR. The other gene, ATH55, encodes an 855 amino acid protein including 21 TPR motifs, which presumably represents a rat crn homologue and an orthologue of human XAB2. Both genes are highly expressed in embryonic brain but their expressions decrease during development. ATH55-like immunoreactivity is present in the ventricular zone and newly formed cortical plate, while CRN-like immunoreactivity is more abundant in a younger ventricular zone. In agreement, both proteins were found to be enriched in cultured neural stem cells and to decrease in response to cell differentiation signals. As indicated for the yeast CRN-like protein, ATH55 and CRN immunoreactivities were both recovered in the nuclear fraction and detected in the splicing complex carrying pre-mRNA. These findings suggest that both TPR-motif-containing proteins are involved in RNA processing of mammalian neural stem cells and their immediate descendants.

Background: We previously reported that the expressions of two CXC chemokines, neutrophil activating peptide-2 (NAP-2) and platelet factor-4 (PF-4), were induced by megakaryocyte-specific cytokine thrombopoietin (TPO) in mouse bone marrow megakaryocytes. The roles of these chemokines on megakaryocyte maturation/differentiation processes, including polyploidization and proplatelet formation (PPF) remain unresolved., Results: NAP-2 and PF-4 suppressed the PPF of mature megakaryocytes freshly prepared from mouse bone marrow as well as that of the megakaryocyte progenitors, c-Kit+CD41+ cells, isolated from mouse bone marrow and cultured with TPO. NAP-2 and PF-4 inhibited polyploidization of c-Kit+CD41+ cells in the presence of TPO, and also inhibited the proliferation of c-Kit+CD41+ cells., Conclusions: NAP-2 and PF-4 produced by TPO stimulation in megakaryocytes suppress megakaryocyte maturation and proliferation as a feedback control.

It was reported that human hematopoietic stem cells in bone marrow were restricted to the CD34(+)KDR(+) cell fraction. We found that expression levels of Flk-1, a mouse homologue of KDR, were low or undetectable in mouse Lin(-)c-Kit(+)Sca-1(+)CD34(low/-) cells as well as Hoechst33342(-) cells (side population), which have long-term reconstitution capacity. Furthermore, neither Flk-1(+)CD34(low/-) cells nor Flk-1(+)CD34(+) cells had long-term reconstitution capacity in mouse. Taken together with other observations using Flk-1-deficient mice, these results indicate that Flk-1 is essential for the development of hematopoietic stem cells in embryo but not for the function of hematopoietic stem cells in adult mouse bone marrow.

CENP-H has recently been discovered as a constitutive component of the centromere that co-localizes with CENP-A and CENP-C throughout the cell cycle. The precise function, however, remains poorly understood. We examined the role of CENP-H in centromere function and assembly by generating a conditional loss-of-function mutant in the chicken DT40 cell line. In the absence of CENP-H, cell cycle arrest at metaphase, consistent with loss of centromere function, was observed. Immunocytochemical analysis of the CENP-H-deficient cells demonstrated that CENP-H is necessary for CENP-C, but not CENP-A, localization to the centromere. These findings indicate that centromere assembly in vertebrate cells proceeds in a hierarchical manner in which localization of the centromere-specific histone CENP-A is an early event that occurs independently of CENP-C and CENP-H.

The regulator of G-protein signaling (RGS) negatively regulates the alpha subunit of G proteins by accelerating their intrinsic guanosine triphosphatase (GTPase) activity. Here are reported the isolation and characterization of a novel mouse RGS, termed RGS18, which is a new member of RGS subfamily B. Northern blot analysis showed that RGS18 messenger RNA was detected predominantly in spleen and hematopoietic cells, and immunohistochemical studies demonstrated that RGS18 was expressed in megakaryocytes, platelets, granulocytes/monocytes, and, weakly, in hematopoietic stem cells, but not in lymphocytes or erythrocytes. Although various subcellular localizations of RGS have been reported, RGS18 was found to be localized in cytoplasm in megakaryocytes. In vitro binding assays of RGS18 with megakaryocyte cell lysates with or without AlF(4)(-) treatment demonstrated that RGS18 specifically binds to 2 alpha subunits of the G protein, Galphai and Galphaq. Furthermore, RGS18 clearly exhibited GTPase-activating protein (GAP) activity for Galphai and Galphaq but not for Galphas or Galpha12. In addition, chemokine stromal-derived factor 1 (SDF-1), which has been reported to stimulate megakaryocyte colony formation in the presence of thrombopoietin, affected the binding of RGS18 to Galphai but not to Galphaq. Therefore, the newly isolated RGS18 turned out to be a new member of the RGS family bearing GAP activity for Galphai, which might be stimulated by SDF-1 in megakaryocytes, as well as for Galphaq. Thus, RGS18 may play an important role in proliferation, differentiation, and/or migration of megakaryocytes.

We isolated a novel gene termed interleukin (IL)-1-inducible nuclear ankyrin-repeat protein (INAP), of which expression was specifically induced by IL-1 in OP9 stromal cells. The INAP has ankyrin-repeat motifs and shares weak amino acid sequence homology with Bcl-3 and other IkappaB family members. The human genomic INAP gene found in the NCBI data base is located at chromosome 3q3.11. Northern blot analyses revealed that INAP was not expressed in any examined tissues without stimulation, but INAP expression was rapidly and transiently induced by IL-1 although not by tumor necrosis factor alpha nor by phorbol 12-myristate 13-acetate in OP9 cells. Immunoblots with anti-INAP-specific antibody demonstrated that INAP was rapidly and specifically produced by IL-1 stimulation and was predominantly localized in the nucleus. Immunofluorescence stainings showed that the INAP newly synthesized by IL-1 stimulation was promptly translocated into the nucleus, and FLAG-tagged INAP forcibly expressed in NIH/3T3 cells was also specifically localized in the nucleus. The possible interaction of INAP with RelA/p65, NF-kappaB1/p50, NF-kappaB2/p52, C/EBPbeta, and retinoid X receptor was examined, but we could detect none of these interactions in the nuclear extracts of IL-1-stimulated cells. Unlike Bcl-3 and other IkappaB family members, INAP may play a unique role in IL-1-induced specific gene expression and/or signal transduction in the nucleus.

Centromere and kinetochore proteins have a pivotal role in centromere structure, kinetochore formation and sister chromatid separation. However, the molecular architecture and the precise dynamic function of the centromere-kinetochore complex during mitosis remain poorly understood. Here we report the isolation and characterization of human CENP-H. Confocal microscopic analyses of HeLa cells with anti-human CENP-H-specific antibody demonstrated that CENP-H colocalizes with inner kinetochore plate proteins CENP-A and CENP-C in both interphase and metaphase. CENP-H was present outside centromeric heterochromatin, where CENP-B is localized, and inside the kinetochore corona, where CENP-E is localized during prometaphase. Furthermore, CENP-H was detected at neocentromeres, but not at inactive centromeres in stable dicentric chromosomes. In vitro binding assays of human CENP-H with centromere-kinetochore proteins suggest that the CENP-H binds to itself and MCAK, but not to CENP-A, CENP-B or CENP-C. CENP-H multimers were observed in cells in which both FLAG-tagged CENP-H and hemagglutinin-tagged CENP-H were expressed. These results suggest that CENP-H multimers localize constitutively to the inner kinetochore plate and play an important fundamental role in organization and function of the active human centromere-kinetochore complex.

Previously we have shown that the V(H) and V(L) fragments of an anti-hen egg lysozyme (HEL) antibody HyHEL-10 are weakly associated but can be driven together by antigen. By joining these antibody variable domains to the cytoplasmic portion of the murine erythropoietin receptor, we created a chimeric growth factor receptor that could be activated by HEL. After co-transfection with two plasmids encoding the respective chimeric receptors in IL-3 dependent murine pro-B Ba/F3 cells, a portion of the cells survived under antigen dependent stimulation without IL-3. These surviving cells all showed coexpression of the two chimeric receptor chains and demonstrated HEL dose-dependent growth stimulation without IL-3. When another IL-3 dependent cell line 32D was transfected with a variant of such chimeric receptor with a linker peptide (Gly-Ser-Gly) inserted between V(H)/V(L) and EpoR domains, an improved growth response was attained. These observations suggest the utility of heterodimeric Fv chimeric receptors in creating cells that respond to monomeric antigen.

Background: Activation of the cyclin-dependent kinase cdc2-cyclin B1 at the G2/M transition of the cell cycle requires dephosphorylation of threonine-14 and tyrosine-15 in cdc2, which in higher eukaryotes is brought about by the Cdc25C phosphatase. In Xenopus, there is evidence that a kinase cascade comprised of xPlkk1 and Plx1, the Xenopus polo-like kinase 1, plays a key role in the activation of Cdc25C during oocyte maturation. In the mammalian somatic cell cycle, a polo-like kinase homologue (Plk1) also functions during mitosis, but a kinase upstream of Plk is still unknown., Results: We show here that human Ste20-like kinase (SLK), which is a ubiquitously expressed mammalian protein related to xPlkk1, can phosphorylate and activate murine Plk1. During progression through the G2 phase of the mammalian cell cycle, the activity of endogenous SLK is increased. The amount of SLK protein is decreased in quiescent and differentiating cells. Treatment with okadaic acid induces a phosphorylation-dependent enhancement of SLK activity., Conclusions: We propose that SLK has a role in the regulation of Plk1 activity in actively dividing cells during the somatic cell cycle. SLK itself is suggested to be regulated by phosphorylation.

Macromolecular centromere-kinetochore complex plays a critical role in sister chromatid separation, but its complete protein composition as well as its precise dynamic function during mitosis has not yet been clearly determined. Here we report the isolation of a novel mouse kinetochore protein, CENP-H. The CENP-H, with an apparent molecular mass of 33 kDa, was found to contain a coiled-coil structure and a nuclear localization signal. The CENP-H transcripts were relatively scarce but were detectable in most tissues and embryos at various stages of development. Immunofluorescence stainings of mouse fibroblast cells with anti-CENP-H-specific antibody demonstrated that the CENP-H is specifically and constitutively localized in kinetochores throughout the cell cycle; this was also confirmed by stainings with anti-centromere-specific antibody. Thus the newly isolated CENP-H may play a role in kinetochore organization and function throughout the cell cycle.

Anaphase-promoting complex or cyclosome (APC) is a ubiquitin ligase which specifically targets mitotic regulatory factors such as Pds1/Cut2 and cyclin B. Identification of the subunits of multiprotein complex APC in several species revealed the highly conserved composition of APC from yeast to human. It has been reported, however, that vertebrate APC is composed of at least eight subunits, APC1 to APC8, while budding yeast APC is constituted of at least 12 components, Apc1 to Apc13. It has not yet been clearly understood whether additional components found in budding yeast, Apc9 to Apc13, are actually composed of mammalian APC. Here we isolated and characterized human APC10/Doc1, and found that APC10/Doc1 binds to APC core subunits throughout the cell cycle. Further, it was found that APC10/Doc1 is localized in centrosomes and mitotic spindles throughout mitosis, while it is also localized in kinetochores from prophase to anaphase and in midbody in telophase and cytokinesis. These results strongly support the notion that human APC10/Doc1 may be one of the APC core subunits rather than the transiently associated regulatory factor.