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Monoalkyl oxalates are among the most important building blocks being applied to the synthesis of a variety of significant classes of compounds or applied to various cutting-edge reactions. However, their commercial availability is limited. Their synthetic methods are also limited because of the difficulty to synthesize them, and those hitherto reported are carried out in organic solvents often with the use of toxic reagents with mostly low to modest yields. Here we have developed practical synthesis of monoalkyl oxalates in aqueous media by applying the highly efficient selective monohydrolysis reactions of symmetric diesters which we reported previously. The best conditions apply an aqueous NaOH solution with relatively nontoxic THF or acetonitrile as a co-solvent at around 0-5 °C. The procedures are simple and environmentally friendly without requiring toxic or expensive reagents, yet yielding the corresponding half-esters in high yields with high purities. All the half-esters prepared here are stable over a long period of time. Therefore, our studies are expected to offer practical green methods for the synthesis of monoalkyl oxalates.

Treatment with systemic corticosteroids is often used for acute liver failure (ALF), but this has increased the number of profoundly immunocompromised patients and cases of opportunistic infection.Between January 2007 and December 2012, all patients (n = 51) referred to the Chiba University Hospital for treatment of ALF were studied. Patients with prothrombin activity of 40 % or less of the standardized values were defined as having ALF. Patient age, sex, cause of ALF, alanine aminotransferase and total bilirubin levels, prothrombin activity and total amount of corticosteroid were analyzed to determine the factors associated with the occurrence of opportunistic infection.Opportunistic infections occurred in 21.6 % (n = 11) of ALF patients. Thirty-five patients underwent systemic corticosteroid therapy, and 31.4 % of those patients showed opportunistic infections. Cytomegalovirus (n = 9, 81.8 %) and Pneumocystis jiroveci (n = 6, 54.5 %) were the microorganisms frequently suspected as the causes of opportunistic infection. In 7 (63.6 %) of the 11 cases of opportunistic infection, 2 or more species of microorganism were detected. Seven patients (63.6 %) with opportunistic infection were cured by treatment. Cox regression analysis for the patients who underwent systemic corticosteroid therapy steroid treatment revealed that age over 52 years (compared to younger patients: odds ratio = 9.62, 95 % confidence interval = 1.22-76.9) was only the predictive factor for the occurrence of opportunistic infection.Opportunistic infections are not rare in ALF patients, and the appropriate diagnosis and treatment of these infections are critical during ALF treatment.

Pandemic influenza A (H1N1) virus (AH1pdm) emerged in April 2009. An inactivated, split-virus, unadjuvanted AH1pdm vaccine was manufactured in Japan, and vaccination was initiated with top priority for healthcare workers (HCWs) on October 19, 2009. A retrospective cohort study was conducted to evaluate the effectiveness of a single-dose vaccine for HCWs in a hospital in Japan. A total of 1,567 (84.5%) of 1,854 HCWs were vaccinated. Thirty-seven were infected with AH1pdm before the vaccine became available, and were excluded. The other 250 were not vaccinated for personal reasons. We analyzed the influenza infection rate with or without vaccination and related adverse events. Among the 1,817 HCWs without previous infection, 37 were infected with AH1pdm; 13 (5.2%) of 250 unvaccinated HCWs became infected, which was a significantly higher rate than the 24 (1.5%) of 1,567 vaccinated HCWs (P=0.001). Multivariate analysis revealed that age of 20-29 years was a risk factor for infection (adjusted odds ratio [aOR], 3.7; P0.001), and that vaccination was a preventive factor (aOR, 0.20; P0.001). Adverse events occurred in 549 of 1,060 HCWs, but most were mild. Although vaccination was carried out during AH1pdm epidemic expansion, the single-dose AH1pdm vaccine proved effective in HCWs, and severe adverse events were rare.

Background Definitive diagnosis is crucial in reducing morbidity and mortality from pandemic influenza A H1N1 2009 (A/H1N1/2009), especially in high-risk populations. We recently developed a rapid diagnosis kit (RDK) capable of specifically detecting A/H1N1/2009. Objectives To evaluate the diagnostic capability of the RDK in a multicenter, prospective trial. Study design Samples were obtained by nasal swab from patients with suspected influenza. The diagnostic capability of the RDK was compared with that of the standard, real-time reverse transcription-polymerase chain reaction (RT-PCR) method. Results Of 266 patients who met the criteria, 122 and 92 were positive for A/H1N1/2009 influenza by PCR and by the newly developed RDK, respectively. The sensitivity, specificity and positive and negative predictive values of the RDK were 73.0%, 97.9%, 96.7% and 81.0%, respectively. A/H1N1/2009 detection rates by the RDK were significantly lower in samples obtained from patients more than 3 days after onset than in samples obtained between 1 and 2 days. Conclusions The A/H1N1/2009-specific RDK is a reliable test that can be used easily at a patient's bedside for rapid diagnosis of A/H1N1/2009. This test will be of key importance in the control of A/H1N1/2009.

Water outlets for washing hands and medical equipment are essential for preventing hospital infection. The present study clarified the effects of water flow volume on the identification and quantitative evaluation of bacteria found around spouts in the 17 hand-washing stations. Pseu-domonas aeruginosa was detected from 4 sta-tions before adjustment and 2 after adjustment. Although no significant difference was identified in the detection rate of P. aeruginosa (p = 0.368), when combining P. aeruginosa and glucose non-fermentative Gram-negative bacilli (NFB), the number of stations with P. aeruginosa and/or NFB decreased significantly from 15 before adjustment to 9 after adjustment (p = 0.023). Before adjust-ment, quantity of bacteria was “2+” for 3 stations and “1+” for 7 stations, but was “1+” for 3 stations and “2+” for 0 stations after adjustment. These results show that quantity of bacteria could be reduced from spouts by adjusting flow volume. These results were also supported by experiments for cleanliness using Adenosine 5’-triphosphate bioluminescence me- thod.

We report three cases of severe opportunistic infection in patients with acute liver failure (ALF). These cases included a 56-year-old man infected with Cryptococcus neoformans, cytomegarovirus (CMV), Candida albicans and Aspergillus fumigates, as well as a 62-year-old man and a 54-year-old woman infected with pneumocystis pneumonia, CMV and Aspergillus fumigatus. One patient died as a result of the infection rather than liver failure. We stress the importance of opportunistic infection as a consideration in the use of immunosuppressive agents for the treatment of ALF.

BACKGROUND AND PURPOSE: In reviewing MR images of patients with spontaneous intracranial hypotension (SIH), we found an accentuated decrease in the subcortical white matter on fluid-attenuated inversion recovery (FLAIR) images. Our aim was to determine whether the signal intensity of the subcortical white matter decreases on FLAIR and T2-weighted images in SIH. MATERIALS AND METHODS: We retrospectively examined pretreatment MR images including 7 FLAIR and 10 T2-weighted images obtained from 10 patients with SIH and follow-up images (5 FLAIR and 7 T2-weighted images). Two observers measured the signal intensities in the subcortical white matter on MR images at the level of the centrum semiovale and, to calculate the signal intensity ratios, measured those of the adjacent cortex and corpus callosum. Furthermore, 4 observers performed visual evaluation for accentuated signal intensity decreases for receiver operating characteristic (ROC) analysis. RESULTS: The intensity ratios of the subcortical white matter, both to the adjacent cortex and corpus callosum, were significantly different between the control and pretreatment images in SIH and between pretreatment and follow-up images in SIH on FLAIR images, whereas these showed no significant differences between the control and follow-up images in SIH. On visual inspection, an accentuated decrease in signal intensity in the subcortical white matter was shown on pretreatment FLAIR images, which returned to the control level on follow-up images. However, on the T2-weighted images we could hardly recognize the decrease in the signal intensity. CONCLUSIONS: Awareness of the decreased signal intensity of the subcortical white matter on FLAIR images could help in the diagnosis of SIH.

The track records of the use of anti-methicillin-resistant Staphylococcus aureus agents (anti-MRSA agents) in a 5-year period (2001.4-2006.3) were collected, and cases in which anti-MRSA agents were used for >4 days were selected. In each case, the results of laboratory data and bacterial examination before and after administering the anti-MRSA agents were investigated retrospectively. In addition, it was also investigated in each case whether therapeutic drug monitoring (TDM) was carried out. It was observed that the number of patients treated with anti-MRSA agents and the total dose of anti-MRSA agents used tended to increase over time, except for arbekacin sulfate. It was, however, shown that treatment with anti-MRSA agents resulted in significant decreases in body temperature, C-reactive protein, and white blood cell counts. Bacterial examination was conducted in 75.6% of the patients treated with anti-MRSA agents, with MRSA being detected in 72.4% of the cases examined. On the other hand, TDM was also conducted in 60% of the cases, but this was at a lower percentage than that of the other examinations. Quantitative bacterial examination after treatment with anti-MRSA agents indicates that TDM can be considered important for the appropriate use of anti-MRSA agents.

Phorbol-12-myristate-13-acetate (PMA) significantly elevated c-mpl promoter activity and the protein kinase C (PKC) inhibitors GF 109203, H7 and calphostin C conspicuously reduced the steady level of the activity. Destruction of the -107Sp1 and the -57Sp1 sites in the c-mpl promoter enhancer region resulted in decrease of the promoter activity by 49.6% and 48.2%, respectively, and destruction of -69Ets and -28Ets elements dramatically decreased the activity by 93.4% and 82.6%, respectively, while mutation of -77GATA moderately reduced the activity by 28.6%. We conclude that the expression of the c-mpl gene is modulated by transcription through a PKC-dependent pathway and that Ets elements at -69 and -28 nucleotides in front of the transcription start site are critical that Sp1(-107) and Sp1(-57) are also important and that GATA(-77) is less involved as a positive regulatory element in c-mpl gene expression induced by PMA in CMK cells.

The immunogenicity of pandemic influenza A H1N1 virus (A/H1pdm) vaccine might be modified by prior seasonal trivalent influenza vaccine (sTIV) administration. We conducted a retrospective analysis of immunogenicity of 243 health care workers (number of sTIV-positive [sTIV+] subjects, 216; number of sTIV−subjects, 27) by hemagglutination inhibition. There was no significant difference in the ratios of antibody titers of ≥40 (41.2% versus 48.1%;P= 0.49) and fold increases in geometric mean titer (3.8 versus 4.5;P= 0.37). sTIV injected 7 to 10 days prior to A/H1pdm vaccine administration did not interfere with the immunogenicity of the latter.

Expression levels of Gb3/CD77 synthase together with Gb3/CD77 antigen were analyzed using human hematopoietic tumor cell lines and normal cells. Among about 40 kinds of cells, Burkitt lymphoma cells showed the highest gene expression concomitant with the expression levels of Gb3/CD77. Unexpectedly, megakaryoblastic leukemia lines also expressed fairly high levels of mRNA of Gb3/CD77 synthase and its product. A megakaryoblastic leukemia line, MEG-01 was sensitive to verotoxins from Escherichia coli O157 and apoptosis was induced via the caspase pathway. We also demonstrated that the cell surface Gb3/CD77 expression was reduced on differentiated MEG-01 although the mRNA level of the α1,4Gal-T gene increased. In this case, the localization of Gb3/CD77 was changed from the cell surface to the cytoplasm as stained with a granular pattern, co-localizing with platelet GPIIb-IIIa, indicating that some of them were platelet precursors. Small particles outside of cells also showed similar staining patterns. These results agreed with the previous report that platelets produced in mature megakaryoblasts abundantly contained Gb3/CD77 antigen. Here, we propose the possibility that verotoxins bind immature megakaryoblasts and induce their apoptosis, leading to the arrest of platelet generation in the bone marrow. This may be one of the causes of thrombocytopenia in patients with hemolytic uremic syndrome.

Sixty-two infants with MLL gene-rearrangement-positive acute lymphoblastic leukemia (MLL-r ALL) were treated with the MLL03 protocol of the Japanese Pediatric Leukemia/Lymphoma Study Group: short-course intensive chemotherapy followed by early allogeneic hematopoietic stem cell transplantation (HSCT) within 4 months of the initial induction. The 4-year event-free survival and overall survival rates were 43.2% (95% confidence interval (CI)=30.7-55.1%) and 67.2% (53.8-77.4%), respectively. A univariate analysis showed younger age (

The circumstances of medium speed diesel engine have been severe to keep up with the environmental problem such as global warming or IMO regulation. To adapt the market trend, the authors developed a series of“Neo”medium speed diesel engines, model 22HLX, 28HLX and 34HLX. This paper describes design features, development processes and principal inspection results of these engines. On the development, extensive cost analysis of existing engines was made to realize extremely low cost/output rate. Performance simulation, FEM analysis, 3D module planning and component tests were also carried out. As the result, the engines having high reliability and friendly handling with advantageous performance were developed. These engines achieved high output with increasing power rate (24.5MPa⋅m/s) as the product of high brake mean effective pressure (2.45MPa) and increased mean piston speed (10m/s) . High thermal efficiency was achieved by prolonged piston stroke, soft-Miller-cycle and high performance radial turbocharger. Lightweight mono-block piston and compound oil-groove big-end bearing, for example, were the key components to realize such high output engines. Concurrent engineering and virtual factory production were applied by means of unity administration of allied factories. First productions were successfully launched from 1998 to 1999, and the longest running hours reached over 18000 hours in October of 2001. The FOC, LOC and exhaust gas temperature was at low level as expected. At the first principal inspection, the piston top land and piston ring grooves were far clean rather than previous engines. Piston and cylinder liner condition was perfect.

Intravenous ribavirin was given to nine patients who had developed severe adenovirus-induced hemorrhagic cystitis (AD-HC) which was resistant to conventional therapy or where there was involvement of other organs after allogeneic BMT. Three patients recovered completely from AD-HC, two of whom had been resistant to vidarabine. All three had received sibling BMTs (2 HLA matched, 1 HLA mismatched). Five patients who received BMTs from related (2 HLA mismatched) or unrelated (1 HLA matched, 2 HLA mismatched) showed an improvement in symptoms but had recurrent AD-HC after discontinuation of ribavirin. Improvement in clinical symptoms and termination of virus excretion were well correlated. The last patient who received a mismatched unrelated BMT died during ribavirin therapy. Ribavirin was notably more effective among patients receiving BMTs from siblings in contrast to patients receiving BMTs from alternative donors (

Hematopoietic stem cell transplantation for X-linked thrombocytopenia from a mild symptomatic carrier

There exists limited information about the usefulness of hemopoietic stem cell transplantation (HSCT) for the treatment of patients with refractory Langerhans cell histiocytosis (LCH). We report here four Japanese pediatric patients with multisystem LCH disease who underwent HSCT between 1994 and 1997. Two of the four patients are doing well without any relapse. However, neither of them shows improved sequelae 3 to 4 years after allogeneic HSCT, although the graft was rejected in one of the cases. The remaining two patients died of septic shock. A review of the literature of 11 patients revealed four fatalities after the use of HSCT in the treatment of LCH. Three of these were due to active LCH and three deaths occurred within 2 months after HSCT. To establish the usefulness of HSCT for refractory LCH, further studies are required.

To search for the existence of a tumour-suppressor gene (TSG) associated with oral squamous cell carcinoma (SCC), PCR analysis of microsatellite polymorphisms corresponding to 14 loci which map to chromosome 7q21.3-qter was performed to screen 35 patients with oral SCC for loss of heterozygosity (LOH). LOH was observed in at least one of the loci in 19 of 34 (55.9%) informative cases. Among the loci tested, frequent LOH was restricted at D7S522 on chromosome 7q31.1, which was measured within 1 cM. Furthermore, we detected microsatellite instability (MI) in 11 of 35 (31.4%) cases tested. Our observations indicate that alterations of chromosome 7q are associated with oral SCC tumorigenesis and that 7q31.1 might harbour at least one putative TSG.

Thrombopoietin (TPO, c-Mpl ligand) is considered to play an important role in the regulation of megakaryocytopoiesis and platelet production by activating the cytokine receptor c-Mpl. We have examined the binding of 125I-TPO to the human megakaryocytic cell line, CMK, and to primary human megakaryocytes. Scatchard analysis of TPO binding to its cognate receptor in megakaryocytic cells suggested the existence of a single class of c-Mpl receptors. CMK cells exhibited 1223 receptors per cell with a dissociation constant (Kd) of Kd = 223 pM, whereas primary human megakaryocytes exhibited 12140 receptors per cell and a dissociation constant of Kd = 749 pM. The pretreatment of CMK cells and primary bone marrow megakaryocytes with TPO resulted in a decreased binding of TPO to the c-Mpl receptors. This down-regulation was observed within 3 h and was not inhibited by cycloheximide. Phorbol ester, an activator of protein kinase C, also inhibited TPO binding to the c-Mpl receptors by reducing the number of these receptors. The pretreatment of CMK cells with IL-3, IL-6 and DMSO, all of which induced the differentiation of CMK cells, did not affect the binding of TPO to the c-Mpl receptors. These results suggest an additional mechanism, where protein kinase C may help to regulate the binding of TPO to these cells.

In infantile leukemias and therapy-related leukemias, the MLL gene is frequently found to be disrupted and fused to various translocation partner genes, such as AF4/FEL, LTG9/AF9 and LTG19/ENL as a result of 11q23 translocations. We previously showed that the N-terminal portion common to various chimeric MLL products, as well as to MLL-LTG9 and MLL-LTG19, localizes in the nuclei, and therefore suggested that it might play an important role in leukemogenesis. In the present study, MLL-AF6 chimeric products found in the t(6;11) (q27;q23) translocation were analysed since AF6, a Ras-binding protein, exhibits a different subcellular localization from that of LTG9/AF9 and LTG19/ENL. Immunofluorescence staining data and cell fractionation analyses demonstrated that MLL-AF6 chimeric products localize in the nuclei despite the fact that AF6 itself localizes in the cytoplasm, confirming the importance of the nuclear localization of chimeric MLL products. The region in the N-terminal portion of MLL responsible for this nuclear localization was examined and found to be a region containing AT-hook motifs.

It is well known that interleukin-3 (IL-3) stimulates the proliferation of megakaryocyte colonies. On the other hand, it remains unclear whether IL-3 also promotes the maturation of megakaryocytes. In this report, we investigated the effects of IL-3 on megakaryocyte maturation in vitro using the human megakaryocytic cell line, CMK, which excludes the influence of bone marrow accessory cells. CMK cells were incubated both with and without 2-50 U/ml of recombinant human IL-3 (rhIL-3) and then analyzed for the effects on proliferation, cell size, DNA ploidy and the expression of platelet glycoproteins. We initially confirmed that CMK cells express IL-3 receptors (IL-3R) on the surface, and rhIL-3 stimulates the proliferation of CMK cells, showing their IL-3R function normally. With addition of rhIL-3 to the CMK cell culture system, increments in cell size, a significant shift toward high ploidy classes, and an increased expression of platelet glycoproteins CD29, CD41, CD42a, CD42b and CDw49f were newly observed. These findings suggest that rhIL-3 acts directly on human megakaryocytes and has the ability to directly promote both proliferation and maturation of megakaryocytes, without the help of accessory cells. Furthermore, rhIL-3 induced/enhanced the expression of mRNA for granulocyte-macrophage colony stimulating factor (GM-CSF) and leukemia inhibitory factor (LIF) in CMK cells. Therefore, the effects of rhIL-3 on the maturation of megakaryocytic cell lines demonstrated in this study may be partly mediated by the action of cytokines such as LIF and GM-CSF, which are induced by IL-3 in the megakaryocytic cell line.

A novel human leukaemic cell line, designated CTS, was established from the peripheral blood of a 13-year-old girl suffering from acute myeloblastic leukaemia (AML) in relapse. CTS cells expressed CD7, CD13, CD33, CD34 and HLA-DR antigens, and showed ultrastructural myeloperoxidase activity. In addition, CTS cells showed DNA rearrangements of the immunoglobulin heavy chain gene and the light kappa chain gene, and deletions of the T-cell receptor delta 1 gene. Cytogenetic analysis revealed a human female diploid karyotype with a t(6;11)(q27;q23) chromosomal translocation. Molecular studies demonstrated a DNA rearrangement of the MLL gene, the expression of a truncated 11.0 kb MLL mRNA and the detection of the MLL/AF-6 fusion transcript in CTS cells. To our knowledge, this cell line is the first report of a human leukaemic cell line with a t(6;11) chromosomal translocation. CTS cells showed no significant proliferative response to the cytokines, IL-2, IL-3, IL-6, IL-11, GM-CSF, G-CSF, EPO, SCF, but were induced to differentiate to the T-cell, B-cell, erythroid or megakaryocytic lineage in the presence of particular cytokines. This CTS cell line may provide a useful tool in the study of the oncogenesis of mixed lineage leukaemia with 11q23 abnormalities and for the analysis of growth and differentiation of pluripotent stem cells.

Multidrug resistance is a major clinical problem in chemotherapy of malignant disease. Acute megakaryoblastic leukemia (AMKL) is a rare form of childhood leukemia, and is often more resistant to many anticancer chemotherapeutic drugs compared to other types of childhood leukemia. There have been reports of the increased expression in hematologic malignancy of multidrug resistant (mdr-1) gene, which encodes for a transmembrane glycoprotein P-glycoprotein that acts as an efflux pump for structurally unrelated chemotherapeutic drugs. We investigated the malignant cells of 15 newly diagnosed childhood AMKL patients by immunocytochemical analysis and found P-glycoprotein expression in all samples from these patients. RNA prepared from five patients at the time of presentation confirmed the expression of mdr-1 specific message in all cases by Northern blot analysis. These results imply that malignant cells from all childhood AMKL might express the mdr-1/P-glycoprotein.

Multidrug resistance is a severe clinical problem in the chemotherapy of malignant disease. Acute megakaryoblastic leukemia (AMKL) is a rare form of childhood leukemia, and is often resistant to many anti-cancer chemotherapeutic drugs. Here we report the expression of the mdr-1/P-glycoprotein in a cell line, CMK, established from a patient with AMKL. Expression of mdr-1 mRNA in CMK11-5 cells, a well differentiated subline, was higher than in CMK6 cells, a poorly differentiated subline. The level of P-glycoprotein was also higher in CMK11-5 cells. The cytokines interferon-gamma (IFN-gamma), GM-CSF and IL-3, which were shown to induce megakaryocytic differentiation of CMK cells, enhanced the expression of the mdr-1 mRNA and levels of P-glycoprotein. These results imply that differentiated megakaryocytic cells may have higher levels of the P-glycoprotein expression, suggesting a possible normal physiological function of P-glycoprotein in mature megakaryocytes.

The 26 S proteasome complex catalyzing ATP-dependent breakdown of ubiquitin-ligated proteins was purified from spinach leaves to near homogeneity by chromatography on DEAE-cellulose, gel filtration on Biogel A-1.5, and glycerol density gradient centrifugation. The purified enzyme was shown to degrade multi-ubiquitinated, but not unmodified, lysozymes in an ATP-dependent fashion coupled with ATPase activity supplying energy for proteolysis and isopeptidase activity to generate free ubiquitin. By nondenaturing electrophoresis, the purified enzyme was separated into two distinct forms of the 26 S complex, named 26 S alpha and 26 S beta proteasomes, with different electrophoretic mobilities. The 26 S proteasome was found to consist of multiple polypeptides with molecular masses of 23-35 and 39-115 kDa, which were thought to be those of a 20 S proteasome with multicatalytic proteinase activity and an associated regulatory part with ATPase and deubiquitinating activities, respectively. The subunit multiplicity of the spinach 26 S proteasome closely resembled that of rat liver with minor differences in certain components. No sulfhydryl bond was involved in the assembly of this multicomponent polypeptide complex. Electron microscopy showed that the 26 S proteasome complex had a "caterpillar"-like shape, consisting of four central protein layers, assumed to be the 20 S proteasome, with asymmetric V-shaped layers at each end. These structural and functional characteristics of the spinach 26 S proteasome showed marked similarity to those of the mammalian 26 S proteasomes reported recently, suggesting that the 26 S proteasome is widely distributed in eukaryotic cells and is of general importance for catalyzing the soluble energy- and ubiquitin-dependent proteolytic pathway.

In Japan, we encountered a pandemic expansion of novel influenza A(H1N1) in September 2009, but the impact on patients with underlying disease remained unclear. The Tokyo Children Cancer Study Group (TCCSG) established a "novel influenza information-sharing system" to share real time information on how the novel influenza affects pediatric patients with cancer or other hematologic disorders. To facilitate reporting, we limited the items to only the basic data (underlying disease, age, sex), influenza-associated data (diagnostic method, therapy and outcome) and allowed space for free comments. We could share the information promptly, and found that this system worked well. One hundred and fifteen patients were reported between September 2009 and February 2010. Although eight patients needed to be hospitalized, none of the patients died, were admitted to intensive care units or demonstrated sequelae. The novel influenza A(H1N1) did not have a strong impact on pediatric patients with cancer or hematologic disorder at least during the 2009-2010 season.

To characterize the CMK11-5 subclone of a human megakaryocytic leukemia cell line (CMK), an ultrastructural study on adhesion was performed. The CMK11-5 cells showed irreversible attachment to rabbit aortic subendothelium accompanied by several morphological changes such as flattening and the spreading out of pseudopodia. These morphological changes are similar to those observed in normal platelets during attachment. The attachment of CMK11-5 cells to rabbit aortic subendothelium indicates that human megakaryocytes may possess an adhesion function like that of platelets. The CMK cell line and its CMK11-5 subclone with megakaryocytic properties appear to be useful for studying the function of megakaryocytes.

To study the involvement of cyclins in cell-cycle progression, changes of mRNA levels for three G1 cyclins (cyclin C, D1 and E) and cyclin A were studied in a leukemia cell line, HEL cells, before and after incubation with 12-O-tetradecanoylphorbol-13-acetate (TPA). Unexpectedly, the cyclin D1 mRNA level markedly increased in the HEL cells when the cells were growth-arrested by TPA, while the amounts of cyclin E and A mRNAs decreased to an almost undetectable level in HEL cells after incubation with TPA. The similar marked increment of the cyclin D1 mRNA level was observed in other leukemia cell lines, CMK and HL-60 cells, after incubation with TPA. The cyclin C mRNA was not detected in HEL cells before and after incubation with TPA.

The expressions of platelet-specific glycoprotein(GP)s Ib, IIb and IIIa were analyzed in 2 megakaryoblastic cell lines: CMK and UT-7. Phorbol-12 myristate 13-acetate (PMA) treatment of CMK induced expressions of GPs IIb and IIIa that peaked on the 4th day post-treatment, while treated UT-7 cells showed maximal levels of these GPs during the 6-8th days. Antibody staining to detect the formation of GPIb alpha after PMA induction showed the presence of the intact GP in UT-7 and the degraded form in CMK. In UT-7, synthesis of GPIIb mRNA increased on days 4-6 after PMA treatment, in parallel with the increase of GPIIb. PMA increased the cytoskeletal protein (actin and myosin) contents in both lines, but in contrast to the two GPs, the increase in these proteins started immediately after PMA addition to the cells. Cell surface proteins of CMK and UT-7 cells were rapidly modified after PMA induction. Especially notable were the degradations of 93-kDa and 140-kDa proteins that occurred on days 1-2 after PMA treatment. These studies demonstrate that the expression of platelet-specific proteins shows a different time dependency than the increment of cytoskeletal proteins, indicating that the syntheses of these two classes of proteins are most likely induced through different mechanisms.

A phase III observational study evaluating a single-dose of an inactivated, split-virus, unadjuvanted AH1pdm vaccine in HCW was conducted. A safe and effective vaccine was needed after the emergence of AH1pdm in April 2009. We analyzed the immunogenicity and safety of the vaccine. A total of 409 subjects were enrolled and given 15 μg hemagglutinin antigen by s.c. injection. Antibody titers were measured using hemagglutination-inhibition antibody assays before vaccination and 28 days after. The co-primary immunogenicity end-points were the proportion of subjects with antibody titers of 1:40 or more, the proportion of subjects with either seroconversion or a significant increase in antibody titer, and the factor increase in geometric mean titer. We collected 389 pair samples. Antibody titers of 1:40 or more were observed in 148 of 389 subjects (38.0%, 95% CI: 33.2-42.9). The immunogenicity was also confirmed in other end-points, but was not sufficient and was lower than in previous reports. A total of 96 of adverse events was reported: 51 local events and 57 systemic events. There were 12 subjects with both local and systemic events. Nearly all events were mild to moderate except in four subjects. A single 15-μg dose of AH1pdm vaccine did not induce sufficient immunogenicity in HCW, with mild-to-moderate vaccine-associated adverse events. We need to consider further improvement of the AH1pdm vaccine program in HCW for the prevention of nosocomial infection, as well as for the benefit of HCW.

Infection control committee as the central decision-making and infection control team (ICT) for carrying out all aspects of infection control are important for hospital infection control. Standard precautions are used for all patients and apply to all body fluids except for sweat, whether or not they contain visible blood. Transmission-based precautions involve airborne, droplet, contact vector-borne and common vehicle precautions. Airborne precautions are for tuberculosis, varicella and measles. Small particles (5 microm) can be dispersed widely by air currents. Droplet precautions are for influenza, rubella, pertussis, and so on. Transmission via large droplet (5 microm) requires close contact within 1 to 2 m. Contact transmissions are for blood-borne pathogens, such as hepatitis B and C virus and HIV, bacterial infections via percutaneous and mucocutaneous exposure, and so on. Vaccinations for measles, rubella, varicella, mumps, influenza and hepatitis B virus to health-care workers are important. Influenza virus is mainly transmitted via droplet and contact, but also via airborne transmission in the aerosol-generating procedures such as intubation and suctioning. WHO declared a public health emergency in April 2009. A novel influenza has spread rapidly across the globe, and it will be necessary to prepare for the outbreak in this autumn and winter.

The proteasome (multicatalytic protease complex), a high molecular weight protein complex, has been purified from spinach leaves by successive chromatography on DEAE-cellulose, Bio-Gel A-1.5m, DEAE-TOYOPEARL 650C, and DEAE-5PW. The molecular mass was estimated to be 850 kDa by gel filtration. Polyacrylamide gel electrophoresis of the proteasome gave a single protein band under nondenaturing conditions and at least 10 bands in the range of 21-32 kDa in the presence of sodium dodecyl sulfate. By electron microscopy after negative staining with uranyl acetate, the proteasome from spinach appeared as symmetrical ring-shaped particles. The substrate specificity of proteasomes indicates that they contain at least three types of activity, namely, chymotrypsin-like, Staphylococcus aureus V8 protease-like, and trypsin-like activities. The former two activities were enhanced by poly-L-lysine or sodium dodecyl sulfate. Moreover, we examined the immunological reactivities of proteasomes from various eukaryotes. As a result, cross-immunoreactivities of some subunits were observed. These properties of the proteasome are similar to those of proteasomes isolated from various other eukaryotic sources.

The CMK cell line is an acute megakaryoblastic leukemia cell line established from a patient with Down's syndrome, and is known to possess characteristics of normal megakaryocytes. Several cytokines with the ability to stimulate megakaryopoiesis, such as interleukin-3 (IL-3), interleukin-6 (IL-6) and granulocyte-macrophage colony-stimulating factor (GM-CSF), stimulated colony formation by CMK cells. The present study revealed that tumor necrosis factor-alpha (TNF-alpha) stimulated colony formation by CMK cells; the potency was almost equal to that of IL-3, IL-6 or GM-CSF. Scatchard plot analysis revealed that CMK cells possess two types of specific binding sites for TNF-alpha. The high-affinity binding sites had an affinity constant of 0.18 nM, and numbered 5,000. The low-affinity binding sites had an affinity constant of 1.8 nM and numbered 19,000. These results raise the possibility that TNF-alpha can act as a growth-stimulating agent on megakaryocyte-lineage cell line.

The Tokyo Children's Cancer Study Group (TCCSG) has performed prospective randomized controlled studies since 1984 for childhood acute lymphoblastic leukemia (ALL). Four hundred and ninety-eight newly diagnosed patients were treated with 5 different regimens designated as S1, S2 for a standard risk group (SRG), H1 and H2 for a high risk group (HRG) and HEX for an extremely high risk group of patients. We added intermediate-dose methotrexate as early intensification therapy in the S2 and H2 groups to reduce extramedullary and medullary relapses. Event-free survival of all patients at 4 years 6 months from the start of this regimen (median follow-up period 32 months) was 67.5%. The CNS relapse rate was significantly decreased to 2.2% compared to previously reported studies and our own experience. There were some unexpected complications of the CNS such as myelopathy and/or leukoencephalopathy. For the SRG early ID-MTX seemed to be useful to improve the prognosis, but triple intrathecal injections (TIT) should be limited in number. The 24Gy cranial irradiation (CRX) was effective but possibly excessive for the HRG. As evidenced by the success of the HEX group regimen, more intensive multi-drug consolidation in the early post-remission phase might be necessary to improve further the prognosis and to make it possible to shorten the treatment periods.

The human immunodeficiency virus (HIV) is capable of infecting certain cells of hematopoietic lineage, particularly monocyte-macrophages and T lymphocytes. Recently, the possibility that cells of megakaryocytic lineage are susceptible to HIV infection has been raised. We have characterized infection of the permanent megakaryocytic cell line CMK by HIV in vitro. CMK cells were easily infected by HIV type 2 (HIV-2), producing significant amounts of virus in culture. Infection appeared to be mediated by the CD4 surface antigen on CMK cells. Three different strains of HIV-1 were able to minimally infect CMK cells, suggesting there may be isolates of HIV tropic for megakaryocytes. Infection of CMK cells led to downregulation of the CD4 surface antigen but no discernable change in expression of megakaryocyte-associated proteins glycoprotein Ib and glycoprotein IIb/IIIa. These observations support the likelihood that megakaryocytes are susceptible to HIV infection, and cell lines of megakaryocytic origin may provide a useful model to study effects of the retrovirus on megakaryocyte function.

Forty-eight Japanese infants with acute lymphoid leukemia (ALL) (n = 24) and acute nonlymphoid leukemia (ANLL) (n = 24) were analyzed on the basis of clinical and laboratory data. Morphologically, 20 of the 24 infants with ALL were of the FAB L1 type, and 20 of the 24 infants with ANLL were of the M4 or M5 type. Markedly enlarged liver and spleen, and hyperleukocytosis (more than 50,000/microliters) were seen in 9, 12, and 14 infants with ALL and 10, 11, and 10 infants with ANLL, respectively. Initial CNS leukemia was evident in 2 infants. Chromosome studies of the leukemic cells showed abnormal karyotypes in 9 of the 21 infants with ALL and 19 of the 22 infants with ANLL, consisting mainly of translocation 11, 12, and inversion 16. By surface marker analysis, only 7 of the 22 infants with ALL (32%) were diagnosed as having common ALL (HLA-DR+, CD19+, CD10+). Of the 15 infants with ANLL, 12 and 5 infants also showed reactivity to HLA-DR and CD19, respectively. All of the 5 ANLL infants with lymphoid markers showed different leukemic cell features at the time of relapse. Twelve of the 19 infants with ALL (63%) who achieved a complete remission relapsed within the first 2 years; 8 of the 21 with ANLL (38%) relapsed within the first year. Analysis of event-free survival shows that the ALL infants with hyperleukocytosis have a poorer prognosis than those without hyperleukocytosis (p less than 0.05). Infant leukemia originates in a multipotent cell with lymphoid and myeloid features, and intensive multiagent chemotherapy is necessary for the treatment of infants with acute leukemia.

CMK is a human cell line derived from a megakaryoblastic leukaemia. It has characteristics of the megakaryocytic lineage, such as the presence of platelet peroxidase, membrane glycoproteins (GP)Ib and GPIIb/IIIa, alpha-granules, and demarcation membranes. The cell line proliferates autonomously in serum-containing medium. Here we report that the cell line expresses the gene for IL-6 and releases small quantities of the cytokine into the medium. Addition of exogenous IL-6 to cultures seeded into medium was found to promote growth of the cells. Conversely, addition of a neutralizing anti-IL-6 antibody inhibited cell growth. These data support the notion that autocrine IL-6 is one of the factors accounting for autonomous growth of the cell line.

A patient with severe diarrhea was successfully diagnosed as having acute intestinal GVHD on stool smear through detection of detached intestinal epithelial cells with apoptosis. Since a stool smear can be easily obtained non-invasively, it is a possible tool for the diagnosis of acute intestinal GVHD.

The track records of the use of anti-methicillin-resistant Staphylococcus aureus agents (anti-MRSA agents) in a 5-year period (2001.4-2006.3) were collected, and cases in which anti-MRSA agents were used for4 days were selected. In each case, the results of laboratory data and bacterial examination before and after administering the anti-MRSA agents were investigated retrospectively. In addition, it was also investigated in each case whether therapeutic drug monitoring (TDM) was carried out. It was observed that the number of patients treated with anti-MRSA agents and the total dose of anti-MRSA agents used tended to increase over time, except for arbekacin sulfate. It was, however, shown that treatment with anti-MRSA agents resulted in significant decreases in body temperature, C-reactive protein, and white blood cell counts. Bacterial examination was conducted in 75.6% of the patients treated with anti-MRSA agents, with MRSA being detected in 72.4% of the cases examined. On the other hand, TDM was also conducted in 60% of the cases, but this was at a lower percentage than that of the other examinations. Quantitative bacterial examination after treatment with anti-MRSA agents indicates that TDM can be considered important for the appropriate use of anti-MRSA agents.

The objectives of this study were to develop a population pharmacokinetic model and to determine the covariates affecting the pharmacokinetics of busulfan in Japanese pediatric patients who received high-dose oral busulfan as a conditioning regimen before hematopoietic stem cell transplantation. Population analysis was performed using retrospective therapeutic drug monitoring data (including test dose data) from 103 children. Their ages ranged from 2 months to 11 years old (mean age, 30 months; median age, 18 months). The plasma concentration of busulfan in all 1028 samples was measured with the same high-performance liquid chromatography method. Maximum likelihood estimates were sought for pharmacokinetic parameters with the NONMEM program. The best structural covariate-free model for busulfan was a one-compartment model with an exponential error model to account for intersubject variability and a proportional error model to account for intrasubject variability. The apparent oral clearance was found to be correlated with age, aspartate transaminase, and type of disease (malignant disease or other). The apparent volume of distribution was related to body weight. The busulfan formulation (1% powder form or crystal form) and dose (milligrams per kilogram) influenced the absorption rate constant. It was estimated that oral clearance expressed per kilogram of body weight is low at early infancy, then increases to a maximum at approximately 2 years of age and, thereafter, decreases. In conclusion, we have developed a population pharmacokinetic model of oral busulfan in children, particularly for those younger than 4 years old, that takes into consideration not only body size, but also several other covariates.

We analyzed the outcomes of 44 children with hepatitis associated aplastic anemia (HAA) who received immunosuppressive therapy (IST) with antithymocyte globulin (ATG) and cyclosporine (CsA). Fourteen (31.8%) patients achieved complete response and 17 (38.6%) achieved partial response, for an overall response rate of 70.4% after 6 months. Seven non-responders received bone marrow transplantation from an HLA-matched unrelated donor and 6 out of 7 are alive. The probability of overall survival at 10 years was 88.3+/-4.9%, which supports the role of IST with ATG and CsA as treatment of choice for children with HAA without an HLA identical sibling donor.

We have analyzed the effect of stress caused by the exposure of BALB/c mice to microgravity change on the susceptibility to herpes simplex virus type 1 (HSV-1) infection. Mice exposed to microgravity for 10 seconds showed a decreased rate of survival from HSV-1 infection. Murine interferon (MuIFN) administration to mice after exposure to microgravity partly diminished the decrease in survival. After repeated exposure to microgravity, mice showed tolerance to the stress. These results indicate that microgravity-stress leads to a down regulation of resistance to virus infection in relation to the modulation of the central nerve system in mice. Responsibility of the immune system dependent on cytokines, including IFN, for down regulation of the resistance is also suggested. Tail-suspended mice were also less resistant to virus infection.

Forty-four infants with acute lymphoblastic leukemia (ALL) characterized by MLL gene rearrangements were treated on a protocol of intensive chemotherapy followed by hematopoietic stem cell transplantation (HSCT) between November 1998 and June 2002. The remission induction rate was 91.0%, and the 3-year overall survival and event-free survival (EFS) rates, with 95% confidence intervals, were 58.2% (43.5%-72.9%) and 43.6% (28.5%-58.7%), respectively. Univariate analysis of EFS by presenting features indicated a poorer outcome in patients younger than 6 months of age with high white blood cell counts (/= 100 x 10(9)/L; EFS rate, 9.4% versus 55.1% for all others, P = .0036) and in those with central nervous system invasion (EFS rate, 10.0% versus 56.9% for all others, P = .0073). The 3-year posttransplantation EFS rate for the 29 patients who underwent HSCT in first remission was 64.4% (46.4%-82.4%). In this subgroup, only the timing of HSCT (first remission versus others) was a significant risk factor by multivariate analysis (P.0001). These results suggest that early introduction of HSCT, possibly with a less toxic conditioning regimen, may improve the prognosis for infants with MLL(+) ALL. Identification of subgroups or patients who respond well to intensified chemotherapy alone should have a high priority in future investigations.

Low-temperature germination is one of the major determinants for stable stand establishment in the direct seeding method in temperate regions, and at high altitudes of tropical regions. Quantitative trait loci (QTLs) controlling low-temperature germinability in rice were identified using 122 backcross inbred lines (BILs) derived from a cross between temperate japonica varieties, Italica Livorno and Hayamasari. The germination rate at 15 degrees C was measured to represent low-temperature germination and used for QTL analysis. The germination rate at 15 degrees C for 7 days of Italica Livorno and Hayamasari was 98.7 and 26.8%, respectively, and that of BILs ranged from 0 to 83.3%. Using restriction fragment length polymorphism (RFLP) and simple sequence repeat (SSR) markers, we constructed a linkage map which corresponded to about 90% of the rice genome. Three putative QTLs associated with low-temperature germination were detected. The most effective QTL, qLTG-3-1 on chromosome 3, accounted for 35.0% of the total phenotypic variation for low-temperature germinability. Two additional QTLs, qLTG-3-2 on chromosome 3 and qLTG-4 on chromosome 4, were detected and accounted for 17.4 and 5.5% of the total phenotypic variation, respectively. The Italica Livorno alleles in all detected QTLs increased the low-temperature germination rate.

Genes expressed with day/night rhythms in the mouse liver were searched for by microarray analysis using an in-house array harboring mouse liver cDNAs. The rhythmic expression with a single peak and trough level was confirmed by RNA blot analysis for 3beta-Hsd and Gabarapl1 genes exhibiting a peak in the light phase and Spot14, Hspa8, Hspa5, and Hsp84-1 genes showing a peak in the dark phase. On the other hand, mRNA levels for all of the three fibrinogen subunits, Aalpha, Bbeta and gamma, exhibited two peaks each in the light and dark phases in a synchronized manner. This two-peaked rhythmic pattern of fibrinogen genes as well as the single peak-trough pattern of other genes was diminished or almost completely lost in the liver of Clock mutant mice, suggesting that the two-peaked expression is also under the control of oscillation-generating genes. In constant darkness, the first peak of the expression rhythm of fibrinogen genes was almost intact, but the second peak disappeared. Therefore, although the first peak in the subjective day is a component of the innate circadian rhythm, the second peak seems to require light stimuli. Fasting in constant darkness caused shifts of time phases of the circadian rhythms. Protein levels of the fibrinogen subunits in whole blood also exhibited circadian rhythms. In the mouse and human loci of the fibrinogen gene cluster, a number of sequence elements resembling circadian transcription factor-binding sites were found. The fibrinogen gene locus provides a unique system for the study of two-peaked day/night rhythms of gene expression in a synchronized form.

It is reported that the stay in the space develop anemia, throbocytopenia, and altered function and structure of red blood cell. The mechanism of these abnormalities was not clarified yet. TPO has been shown to stimulate both megakaryocyte colony growth from marrow progenitor cells and the maturation of immature megakaryocyte to form functional platelet. This process include massive cytoskeletal rearrangement, such as proplatelet formation and fragmentation of proplatelet. Our previous reports (Fuse and Sato, 2001, Fuse et al, 2001) showed an inverse relationship between decreased platelet count and increased TPO concentrations in peripheral blood of mouse was induced by parabolic flight (PF). We have studied which gravity change during PF involved this phenomenon.

Expression levels of Gb3/CD77 synthase together with Gb3/CD77 antigen were analyzed using human hematopoietic tumor cell lines and normal cells. Among about 40 kinds of cells, Burkitt lymphoma cells showed the highest gene expression concomitant with the expression levels of Gb3/CD77. Unexpectedly, megakaryoblastic leukemia lines also expressed fairly high levels of mRNA of Gb3/CD77 synthase and its product. A megakaryoblastic leukemia line, MEG-01 was sensitive to verotoxins from Escherichia coli O157 and apoptosis was induced via the caspase pathway. We also demonstrated that the cell surface Gb3/CD77 expression was reduced on differentiated MEG-01 although the mRNA level of the alpha1,4Gal-T gene increased. In this case, the localization of Gb3/CD77 was changed from the cell surface to the cytoplasm as stained with a granular pattern, co-localizing with platelet GPIIb-IIIa, indicating that some of them were platelet precursors. Small particles outside of cells also showed similar staining patterns. These results agreed with the previous report that platelets produced in mature megakaryoblasts abundantly contained Gb3/CD77 antigen. Here, we propose the possibility that verotoxins bind immature megakaryoblasts and induce their apoptosis, leading to the arrest of platelet generation in the bone marrow. This may be one of the causes of thrombocytopenia in patients with hemolytic uremic syndrome.