Haplotype-based, case-control study of the receptor (calcitonin) activity-modifying protein (RAMP) 1 gene in essential hypertension.

The adrenomedullin receptor is a complex molecule that comprises the calcitonin-receptor-like receptor (CRLR) and the receptor-activity-modifying protein (RAMP). RAMP1 is a vasodilation factor, and RAMP1-deficient mice (RAMP1(-/-)) exhibit inflammatory responses with a significant transient increase in serum calcitonin-gene-related peptide levels and proinflammatory cytokines when compared with RAMP1(+/+) mice. The purpose of the present study was to investigate the relationships between essential hypertension (EH) and RAMP1 gene single-nucleotide polymorphisms (SNPs) or haplotypes in a Japanese population via a case-control study. Based on a database search of the National Center of Biotechnology Information website and the HapMap project, we chose six RAMP1 gene SNPs and performed an association study involving 263 patients with EH and 267 age-matched normotensive (NT) subjects. There was no significant difference between the EH and NT groups with regard to overall distribution of genotypes or SNP alleles. However, the haplotype-based case-control analysis revealed that there was a significant difference between the EH and NT groups with regard to overall distribution of the allele combinations at three SNPs-rs3754701-rs3769048-rs10199956-(P=0.002). The T-A-T haplotype was significantly more common in the EH group (10.3%) than in the NT control group (6.1%) (P=0.047). These results suggested that this T-A-T RAMP1 gene haplotype might have utility as a genetic marker for EH and that the RAMP1 gene or a neighbouring gene may be associated with increased susceptibility to EH.

The genetic basis of speciation in the Giliopsis lineage of Ipomopsis (Polemoniaceae).

One of the most powerful drivers of speciation in plants is pollinator-mediated disruptive selection, which leads to the divergence of floral traits adapted to the morphology and behavior of different pollinators. Despite the widespread importance of this speciation mechanism, its genetic basis has been explored in only a few groups. Here, we characterize the genetic basis of pollinator-mediated divergence of two species in genus Ipomopsis, I. guttata and I. tenuifolia, using quantitative trait locus (QTL) analyses of floral traits and other variable phenotypes. We detected one to six QTLs per trait, with each QTL generally explaining small to modest amounts of the phenotypic variance of a backcross hybrid population. In contrast, flowering time and anthocyanin abundance (a metric of color variation) were controlled by a few QTLs of relatively large effect. QTLs were strongly clustered within linkage groups, with 26 of 37 QTLs localized to six marker-interval 'hotspots,' all of which harbored pleiotropic QTLs. In contrast to other studies that have examined the genetic basis of pollinator shifts, our results indicate that, in general, mutations of small to modest effect on phenotype were involved. Thus, the evolutionary transition between the distinct pollination modes of I. guttata and I. tenuifolia likely proceeded incrementally, rather than saltationally.

Down-scattered neutron imaging detector for areal density measurement of inertial confinement fusion.

A custom developed (6)Li glass scintillator (APLF80+3Pr) for down-scattered neutron diagnostics in inertial confinement fusion experiments is presented. (6)Li provides an enhanced sensitivity for down-scattered neutrons in DD fusion and its experimentally observed 5-6 ns response time fulfills the requirement for down-scattered neutron detectors. A time-of-flight detector operating in the current mode using the APLF80+3Pr was designed and its feasibility observing down-scattered neutrons was demonstrated. Furthermore, a prototype design for a down-scattered neutron imaging detector was also demonstrated. This material promises viability as a future down-scattered neutron detector for the National Ignition Facility.

Haplotype-based case-control study of receptor (calcitonin) activity-modifying protein-1 gene in cerebral infarction.

Calcitonin gene-related peptide (CGRP) receptor is a complex molecule that consists of calcitonin receptor-like receptor and receptor activity-modifying protein-1 (RAMP1). It was recently reported that RAMP1-deficient mice (RAMP1(-/-)) showed inflammatory responses with a transiently significant increase in serum CGRP levels and proinflammatory cytokines when compared with RAMP1(+/+) mice. The aim of this study was to investigate the relationship between the human RAMP1 gene and cerebral infarction (CI) using single-nucleotide polymorphisms (SNPs) in a Japanese population. We selected six SNPs in the human RAMP1 gene (rs3754701, rs3769048, rs7557078, rs1584243, rs10199956 and rs7590387) and performed a case-control study using each SNP and haplotype in 171 CI patients and 234 controls. There were no significant differences in overall distribution of genotype and allele frequencies of the SNPs between the CI and control groups. However, there was a significant difference in overall distribution between the CI and control groups (P<0.001) in the haplotype-based case-control study with the combinations of rs3754701-rs3769048-rs7590387. The T-A-C susceptibility haplotype for CI was significantly more frequent than in the control group (P=0.0024). The results suggest that the T-A-C haplotype is a genetic marker for CI, and that RAMP1 or neighbouring genes are associated with increased susceptibility to CI.

Primary central nervous system post-transplant lymphoproliferative disorder presenting as cerebral hemorrhage after unrelated bone marrow transplantation.

We present a rare case of cerebral hemorrhage due to Epstein-Barr virus (EBV)-associated post-transplant lymphoproliferative disorder (PTLD). A 58-year-old man with myelodysplastic syndrome received allogeneic hematopoietic stem cell transplantation from an unrelated donor after being conditioned with fludarabine, melphalan, and total body irradiation. Tacrolimus and methotrexate were given for graft-versus-host disease (GVHD) prophylaxis. On day 23, he developed acute GVHD, which was successfully treated with prednisolone (PSL). The tapering of PSL failed because of extensive chronic GVHD involving the liver and lungs, and mycophenolate mofetil was added on day 244. On day 340, the patient suddenly complained of severe headache. Computed tomography confirmed subcortical hemorrhage, and he died on day 348. The autopsy revealed atypical lymphocytes infiltrating the brain and meninges, which were positive for B-cell-associated antigens and EBV-encoded RNA, and thus EBV-associated PTLD was diagnosed.

Interindividual variation of maximal blood levels of tacrolimus after its oral administration in hematopoietic cell transplant recipients.

We investigated the pharmacokinetics of oral tacrolimus in 31 hematopoietic cell transplant recipients, identifying 2 subgroups based on the differences between C(0) (trough) and C(max) (maximal) levels: group A (n = 21; 68%) with a C(max)-C(0) value of <10 ng/mL, and group B (n = 10; 32%) with a C(max)-C(0) value of >or=10 ng/mL. Although the C(0) and C(12) values were not significantly different between the 2 groups, the mean area under the concentration curve for 12 hours (AUC(0-12)) was significantly greater in group B than group A (200.9 +/- 36.3 vs 155.1 +/- 43.1 ng.h/mL; P < .05), and the mean half-life was significantly shorter in group B than group A (13.55 +/- 6.70 vs 18.17 +/- 6.30 hours; P < .05). Thus after the oral administration of tacrolimus, we observed a notably high AUC due to high peak level, which we were unable to predict simply by measuring the trough level. A pharmacokinetic analysis of each patient was essential to optimize the oral tacrolimus dose.

Tacrolimus and methotrexate for the prophylaxis of graft-versus-host disease after unrelated donor cord blood transplantation for adult patients with hematologic malignancies.

Eighteen patients with hematologic malignancies underwent cord blood transplantation (CBT) from unrelated donors after being conditioned with myeloablative or reduced-intensity regimens, and received tacrolimus and methotrexate (15 mg/m(2) on day 1, 10 mg/m(2) on days 3 and 6) as graft-versus-host disease (GVHD) prophylaxis. The median number of nucleated cells in infused cord blood was 2.66 x 10(7)/kg (range 1.90 to 4.15 x 10(7)/kg). Engraftment was achieved in 16 of 18 patients. The median time to absolute neutrophil count >0.5 x 10(9)/L was 21.5 days (range 17 to 32), and the median time to platelet count >2.0 x 10(9)/L was 36 days (range 26 to 57). Of the 16 evaluable patients, five and eight had grades I and II acute GVHD, respectively, and none had grades III/IV acute GVHD. The cumulative incidence of grade II acute GVHD was 44.4%. Chronic GVHD occurred in 7 of 15 evaluable patients: limited type in three patients, extensive type in four patients. Of the 18 patients, 14 were alive and disease-free between 173 and 1514 days after CBT (median 746 days). The probability of disease-free survival at 2 years was 79.1%. These results, although in a retrospective study, suggested that tacrolimus and short-term methotrexate effectively prevented the occurrence of severe acute GVHD after unrelated CBT, and may contribute to a high survival rate.

Sperm protein 17 influences the tissue-specific malignancy of clear cell adenocarcinoma in human epithelial ovarian cancer.

Clear cell adenocarcinoma of the ovary has a poor prognosis due to chemoresistance and early metastasis to the lymph nodes. It also can result in endometriosis and is the second most frequent type of ovarian cancer in Japan. Serous adenocarcinoma of the ovary is another common epithelial cancer tissue subtype in Japan, and it is highly sensitive to chemotherapy. In the current study, we examined the differential expression of genes in these types of ovarian cancer and tried to analyze their functions, especially as they relate to chemoresistance. We used differential display to compare clear cell carcinoma and serous adenocarcinoma of the ovary. We identified sperm protein 17 (SP17) as a candidate gene related to the chemoresistance of clear cell carcinoma. Its differential expression was confirmed by real-time polymerase chain reaction. Because the function of the SP17 gene in ovarian cancer is not known, we examined the effect of small interfering RNA targeting the SP17 gene on the chemoresistance and proliferation of ES-2 ovarian cancer cells to paclitaxel, currently the most effective treatment for ovarian cancer. We found that this treatment decreased the chemoresistance of these cells to paclitaxel. Our results strongly suggest that SP17 plays a role in the resistance of clear cell carcinoma to chemotherapy without influencing their ability to proliferate.

Total body irradiation and granulocyte colony-stimulating factor-combined high-dose cytarabine as a conditioning regimen in allogeneic hematopoietic stem cell transplantation for advanced myelodysplastic syndrome: a single-institute experience.

In this study, we retrospectively evaluated the efficacy and safety of total body irradiation (TBI) and granulocyte colony-stimulating factor (G-CSF)-combined high-dose cytarabine as a conditioning regimen for allogeneic hematopoietic stem cell transplantation (HSCT) in patients with advanced myelodysplastic syndrome (MDS). We evaluated 22 patients with advanced MDS, including refractory anemia with excess blasts (RAEB; n=10), RAEB in transformation (n=2), acute myelogenous leukemia transformed from MDS (n=6) and chronic myelomonocytic leukemia (n=4). The conditioning regimen consisted of 12 Gy of TBI and high-dose cytarabine (3 g/m(2)) every 12 h for 4 days, and the cytarabine was combined with continuous administration of G-CSF. The stem cell sources were bone marrow or peripheral blood stem cells from human leukocyte antigen (HLA)-identical siblings (n=12) and bone marrow from HLA serologically matched unrelated donors (n=10). Three patients experienced disease relapse, two of whom died of disease progression. Of 22 patients, 16 are currently alive and disease-free. The 5-year estimated overall survival, disease-free survival, relapse and non-relapse mortality rates are 76.7, 72.2, 16.6 and 14.1%, respectively. These results suggest that G-CSF-combined high-dose cytarabine could be a promising component of the conditioning regimen of allogeneic HSCT for advanced MDS, providing a low incidence of both relapse and treatment-related mortality.

Inhibition of the mammalian target of rapamycin (mTOR) by rapamycin increases chemosensitivity of CaSki cells to paclitaxel.

Paclitaxel, a potent anti-neoplastic agent, has been found to be effective against several tumours, including cervical cancer. However, the exact mechanism underlying the cytotoxic effects of pacitaxel, especially in the survival-signalling pathway, is poorly understood. The aim of this study was to investigate the molecular pathway of the cytotoxic effect of paclitaxel in human cervical cancer cell lines. Four human cervical cancer cell lines were treated for 24 h with various concentration of paclitaxel, and the sensitivity was analysed by an MTT assay. The cell cycle progression and sub-G1 population were analysed by flow cytometry. Apoptosis was further measured by DNA fragmentation and microscope examination. The protein expression was determined by Western blot analysis. Our results showed that HeLa cells demonstrated the highest sensitivity to paclitaxel, whereas CaSki cells showed the lowest. In cervical cancer cells, paclitaxel induced apoptosis through an intrinsic pathway with prior G2/M arrest. In addition, we showed that paclitaxel downregulated the phosphorylation of Akt in both HeLa and CaSki cells. Interestingly, in CaSki cells, which were more suggestive of a resistant phenotype, paclitaxel induced the activation of mTOR as a downstream target of Akt. Pre-treatment with rapamycin inhibited activation of mTOR signalling and significantly enhanced the sensitivity of CaSki cells to paclitaxel by increasing apoptotic cell death. This effect was mediated, at least partly, through caspase activation. Overall, paclitaxel exerts its anti-tumour effects on cervical cancer cells by inducing apoptosis through intrinsic pathway, and rapamycin targeted to mTOR can sensitise paclitaxel-resistant cervical cancer cells.

Dose-adjusted preemptive therapy for cytomegalovirus disease based on real-time polymerase chain reaction after allogeneic hematopoietic stem cell transplantation.

We have prospectively evaluated the efficacy of real-time PCR-guided preemptive therapy for CMV diseases in allogeneic hematopoietic stem cell transplant recipients with grades II-IV acute GVHD. The dose of ganciclovir was adjusted according to the viral load determined by real-time polymerase chain reaction (PCR). On detecting CMV reactivation in the plasma, ganciclovir was initiated at a dose of 5 mg/kg body weight once daily, and the dose was increased to twice daily if viral load continued to increase after initiating ganciclovir. In 39 evaluable patients, CMV reactivation assessed by real-time PCR became positive in 30 (77%). One developed CMV gastroenteritis before PCR became positive. Thus the remaining 29 patients were treated preemptively with ganciclovir. The dose of ganciclovir was increased in 12 patients (41%) of preemptively treated patients for increasing viral load. CMV diseases were diagnosed in two patients (one gastroenteritis and one retinitis), and late CMV disease was diagnosed in one patient (gastritis). The treatment was generally well-tolerated, but three patients (10%) developed neutropenia (neutrophil count less than 1.0 x 10(9)/l). In conclusion, real-time PCR-guided preemptive therapy with decreased dose of ganciclovir is feasible and does not increase the frequency of CMV diseases if the dose is adjusted according to the viral load.

Expression and sequence analyses of p33(ING1) gene in myeloid leukemia.

p33(ING1) is a novel candidate tumor suppressor gene which is involved in the regulation of apoptosis. p33(ING1) interacts with p53 signaling pathway and regulates cellular growth. It has reported that the expression of p33(ING1) mRNA was decreased in lymphoid malignancies. We thus investigated the potential involvement of p33(ING1) abnormalities in myeloid leukemias. However, the levels of p33(ING1) transcript were almost equal in 3 AML cell lines and 10 fresh AML samples. In addition, neither point mutations nor deletions in p33(ING1) gene were found in myeloid leukemias. These results suggest that p33(ING1) may not be a major candidate tumor suppressor gene in myeloid leukemias.

A quantitative extraction method for the determination of trace amounts of both butyl- and phenyltin compounds in sediments by gas chromatography-inductively coupled plasma mass spectrometry.

A simple and reliable extraction method was developed for quantitative determination of both butyl- and phenyltin compounds in sediments by capillary gas chromatography combined with inductively coupled plasma mass spectrometry (GC-ICP-MS). Both types of organotin compounds were extracted quantitatively from sediment by mechanical shaking into tropolone-toluene and HCl-methanol. After phase separation and pH adjustment, these organotins were ethylated with sodium tetraethylborate. The method was evaluated by analyzing PACS-2 and NIES No. 12 sediment certified reference materials. The dibutyltin (DBT; 1.14 +/- 0.02 micrograms g-1) and tributyltin (TBT; 1.01 +/- 0.04 micrograms g-1) values observed in PACS-2 sediment closely matched the certified values (DBT, 1.09 +/- 0.15; TBT, 0.98 +/- 0.13 microgram g-1 as tin). The monobutyltin (MBT) value was higher (0.62 +/- 0.02 microgram g-1) by more than two fold over the reference value (0.3 microgram g-1 as tin). The concentrations of TBT (0.18 +/- 0.04 microgram g-1) and triphenyltin (TPhT; 0.0099 +/- 0.002 microgram g-1) in the NIES No. 12 sediment were also in good agreement with the certified and reference values of TBT (0.19 +/- 0.03 microgram g-1 as compound) and TPhT (0.008 microgram g-1 as compound), respectively. Recoveries of TBT, tripentyltin (TPeT) and TPhT from spiked sediments were satisfactory (TBT, 102 +/- 3.4%; TPrT, 96 +/- 3.4%; TPhT, 99 +/- 8.5%). The detection limits as tin were in the range 0.23-0.48 ng g-1 for a 0.5 g sample size. It is also noteworthy that clean-up of the extract is not necessary because of the superior selectivity of ICP-MS detection. The present method was successfully applied to marine sediment samples.

Tin speciation in the femtogram range in open ocean seawater by gas chromatography/inductively coupled plasma mass spectrometry using a shield torch at normal plasma conditions.

A sensitive method for the determination of ultratrace organotin species in seawater is described. The merits and demerits of derivatization methods using Grignard reagent or sodium tetraethylborate (NaBEt4) were evaluated in terms of derivatization efficiency, applicability to the programmed temperature vaporization (PTV) method, and procedural blanks. The sensitivity of the gas chromatography/inductively coupled plasma mass spectrometry (GC/ICPMS) was improved by more than 100-fold by operating the shield torch at normal plasma conditions, compared with that obtained without using it. The absolute detection limit as tin reached subfemtogram (fg) levels. Furthermore, the detection limit in terms of relative concentration was improved 100-fold by using the PTV method, which enabled the injection of a large sample volume of as much as 100 microL without loss of analyte. When the organotin species in seawater were extracted into hexane with a preconcentration factor of 1000 after ethylation with NaBEt4 and a 100 microL aliquot of the extract was injected into the GC, the instrumental detection limit in relative concentration reached 0.01 pg/L in original seawater. Sources of contamination of organotin species during the sample preparation were examined, and a purification method of NaBEt4 was developed. Finally, the method was successfully applied to open ocean seawater samples containing organotin species at the level of 1-100 pg/L.

High-speed voltammetry: dual measurement of dopamine and serotonin.

A high-speed voltammetric system was designed and tested for dual measurement of dopamine (DA) and serotonin (5-HT) at 250-ms intervals. First, an anodic-cathodic square-wave pulse was delivered to activate the electrode (carbon fiber, 7 microm in diameter), then DA and 5-HT oxidation currents (current intensity) were measured when potentials were stepped from 100 to 250 mV and 300 to 450 mV, respectively. To isolate DA and 5-HT current intensities, the current observed at 100 mV was subtracted from that at 250 mV, and the current observed at 300 mV was subtracted from that at 450 mV, respectively. Measurements were performed every 250 ms. In vitro, DA and 5-HT current intensities increased with increasing concentrations of DA and 5-HT, respectively. The DA current intensity was not affected by the addition of the DA metabolite 3,4-dihydroxyphenylacetic acid (10(-6) M) or ascorbic acid (10(-5) M), but the 5-HT current intensity was affected by the addition of the 5-HT metabolite 5-hydroxyindoleacetic acid (10(-6) M) or uric acid (10(-5) M). Electrodes were used for several months without any change in sensitivity. In vivo, following intraperitoneal injection of L-DOPA to rats, an increase in striatal DA release was observed but there was no increase in release of 5-HT. Following intraperitoneal injection of 5-hydroxytryptamine there was an increase in 5-HT release but not DA release. This high-speed system was capable of obtaining stable, long-term dual measurements of DA and 5-HT in vitro and in vivo.

Pneumococcal psoas abscess.

A 47-year-old woman was admitted to our hospital because of severe low back pain. A computed tomography (CT) scan revealed a left sided psoas muscle abscess. On the first hospital day, US-guided drainage was performed. Streptococcus pneumoniae was isolated from the pus. Thereafter, the open drainage of the abscess and antibiotic treatment were given with subsequent clinical improvement. Only 10 cases of pneumococcal psoas abscess have been previously reported in the world literature.

Effect of polishing on cyclic fatigue strength of CAD/CAM ceramics.

The biaxial flexural strength and cyclic fatigue biaxial flexural strength of CAD/CAM ceramics polished with #220, 400, 600 and 1000 diamond pads were measured in an effort to determine the effect of surface roughness on fatigue behavior of dental ceramics. The surface roughness was improved after polishing with a smaller diamond grain pad. The flexural strengths of the specimens polished with #220, 400, 600 and 1000 diamond pad were 75.2, 76.6, 82.2, and 83.3 MPa, respectively; the fatigue flexural strength of those with #220, 400, 600 and 1000 were 53.0, 58.1, 60.0, and 61.5 MPa, respectively. Both the flexural and fatigue flexural strengths increased with improvement of surface profile. These results suggest the importance of polishing of dental ceramics for not only the static strength but also the cyclic fatigue strength.

Differential time courses of exogenous 1-methyl-4-phenyl-1,2,3, 6-tetrahydropyridine and its metabolite MPP+ in the rat striatum and nucleus accumbens measured using in vivo voltammetry.

The dopaminergic neurotoxin 1-methyl-4-phenyl-1,2,3, 6-tetrahydropyridine (MPTP) has been shown to affect nigrostriatal projection neurons to a greater extent than substantia nigra neurons that project to the nucleus accumbens. To investigate this preferential vulnerability, the intracerebral pharmacokinetics of locally-applied MPTP was investigated using in vivo voltammetry. First, we examined whether MPTP and MPP+ were measurable in vitro. At the most efficient oxidation potential for MPTP (850 mV), its metabolite MPP+ was also partly oxidized, whereas at that for MPP+ (650 mV), MPTP was not oxidized. Then, in vivo measurements were taken less than 1 mm from the site of infusion of MPTP. MPTP and endogenously produced MPP+ peaked later and took longer to return to baseline in the nucleus accumbens than in the striatum. Systemic monoamine oxidase-B inhibitor pargyline delayed the peak and return to baseline of endogenously produced MPP+ in the nucleus accumbens. Exogenously applied MPP+ also took longer to peak and return to baseline in the nucleus accumbens. These results indicate that the difference in the pharmacokinetics of exogenously applied MPTP in the striatum and nucleus accumbens may be due to a difference in uptake in these regions, and that the difference in pharmacokinetics of endogenously produced MPP+ may be due to differences in both uptake and monoamine oxidase-B activity.