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.

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.

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.

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.

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.

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.

Locus coeruleus neurons projecting to the forebrain and the spinal cord in the cat.

The intranuclear organization of the cat locus coeruleus neurons was investigated anatomo-physiologically. The locus coeruleus neurons project to the forebrain through the dorsal noradrenergic bundle and to the spinal cord. Horseradish peroxidase, a retrograde tracer, was pressure-injected into either the dorsal noradrenergic bundle or the ventrolateral funiculus of the high cervical cord (C1-C2). The cats (n = 12) were killed after a 2- or 3-day survival period. The frontal sections (100 micron) throughout the locus coeruleus were observed by light microscope after carrying out the diaminobenzidine reaction. The labeled locus coeruleus neurons were located predominantly in the rostral locus coeruleus proper and locus coeruleus alpha when horseradish peroxidase was injected into the dorsal noradrenergic bundle, whereas they were predominantly located in the caudal locus coeruleus alpha and subcoeruleus when horseradish peroxidase was injected into the spinal cord. In the electrophysiological experiments, cats (n = 30) were anesthetized with alpha-chloralose and two stimulating electrodes were placed stereotaxically in the dorsal noradrenergic bundle and the ipsilateral ventrolateral funiculus of the high cervical cord (C1-C2), respectively. Monophasic square-wave pulses (2.5 Hz, 100 microsecond duration, 800 microA) were delivered. A recording glass electrode, filled with 2 M NaCl saturated with Fast Green, was placed in the locus coeruleus. Neurons with different conduction velocities, which were evoked by the antidromic stimulation of the dorsal noradrenergic bundle and spinal cord, were verified in the locus coeruleus and the adjacent areas. The slow conductive neurons with a conduction velocity of less than 1 m/s had a slow firing rate (1.6 +/- 0.9/s). They were located predominantly in the rostral locus coeruleus proper and locus coeruleus alpha by the dorsal noradrenergic bundle stimulation. From the anatomical and electrophysiological experimental results, it was concluded that the conduction velocities of the horseradish peroxidase-labeled neurons observed in locus coeruleus proper and locus coeruleus alpha were mostly slow and less than 1 m/s. Most of the slow conductive neurons were considered to be noradrenergic. Neurons evoked antidromically by both the dorsal noradrenergic bundle and spinal cord stimulation were not observed.

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.

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.

A triangular conditioning voltage wave does not influence spontaneous neuronal activity in the rat striatum.

A triangular potential wave applied shortly before each measuring triple pulse has been used in in vivo voltammetric experiments to ensure the maintenance of a high level of stability and sensitivity in the carbon fiber electrode. To investigate whether a triangular wave (0- +/- 1500 mV, 10 V/s slope) and a consecutive triple pulse affect spontaneous neuronal firing, extracellular recordings were made in the rat striatum at a distance of about 200-300 microns from the carbon fiber electrode while these potentials were being delivered. No significant change in the rate of spontaneous firing was found. A triangular pulse has proven to be very effective in lengthening the measurement lifetime of the carbon fiber electrode without interfering with the spontaneous activity of striatal neurons.

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.

Effect of exogenous L-dopa on behavior in the rat: an in vivo voltammetric study.

L-DOPA was administered intraperitoneally (i.p.) or intraventricularly (i.v.t.) to freely moving rats to investigate the effects of exogenous L-DOPA itself on behavior. Striatal dopamine (DA) in the extracellular fluid was examined with microcomputer-controlled in vivo voltammetry, and behavioral change was observed. When L-DOPA was administered (i.p.) after pretreatment with benserazide, a peripheral DOPA decarboxylase inhibitor, behavioral change was elicited before the elevation in DA and suppressed before its reduction. After pretreatment with NSD-1015, a central DOPA decarboxylase inhibitor, behavioral change was also elicited, although DA was still not increased. When L-DOPA was injected (i.v.t.), the behavioral effect was manifested at once; DA was still unchanged at this time, but it increased after behavioral activity reached the maximum level. L-DOPA was also injected (i.v.t.) into rats with striatal lesions induced by 6-hydroxydopamine (i.v.t.). Behavioral change was manifested promptly after the injection. When the dose-response curves to different dosages of L-DOPA were examined in normal rats without striatal lesions, it was found to exhibit a steeper rise than that of DA. Finally, when rats were injected (i.p. or i.v.t.) with 3-O-methyl-DOPA (3-methoxytyrosine), a major metabolite of L-DOPA, no behavioral change was elicited, and no increase in DA was recognized. These experimental results indicated that L-DOPA is related directly to the manifestation of behavioral change.

Duration of catalepsy correlates with increased intrastriatal sulpiride.

To investigate the mechanism underlying sulpiride-induced catalepsy, we simultaneously examined cataleptic behavior and the kinetics of the dopamine receptor antagonist, sulpiride of dopamine, and the dopamine metabolite 3,4-dihydroxyphenylacetic acid (DOPAC), using in vivo voltammetry. After intrastriatal administration of sulpiride to freely moving rats, the levels increased, peaked at 20 min, and remained elevated for more than 3 h. Sulpiride-induced cataleptic behavior also continued for 3 h. Levels of DOPAC peaked 180 min after the injection and did not return to baseline within the experimental period. Thus, the time-course of cataleptic behavior correlated better with elevated extracellular levels of sulpiride than with that of DOPAC. These findings suggest that sulpiride induces catalepsy via a direct action.

The dopamine D2 receptor antagonist sulpiride causes long-lasting serotonin release.

The effects of the dopamine D2 receptor antagonist sulpiride on extracellular levels of serotonin (5-hydroxytryptamine, 5-HT) and the 5-HT metabolite 5-hydroxyindoleacetic acid (5-HIAA) were examined by using in vivo voltammetry. Sulpiride (1 or 3 mM, 2 microl over 24 min) was administered to freely moving rats via a cannula implanted in the striatum and 5-hydroxyindole levels were measured by using a carbon fiber voltammetry electrode implanted in the ipsilateral striatum. Six to 8 h after injection, 5-hydroxyindole levels increased 3-fold, peaked 1 to 2 days post-injection, and returned to normal levels within 2 to 4 days. These effects were suppressed by pretreatment with p-chlorophenylalanine. Two days after sulpiride injection, high-performance liquid chromatography of striatal homogenates revealed that although the 5-HT concentration was unchanged, the 5-HIAA concentration was increased significantly. These results suggest that the long-lasting elevation of 5-hydroxyindole concentrations was primarily due to increased 5-HT release.

Cervical radiculomyelopathy due to calcification of the ligamenta flava.

Three cases of cervical radiculomyelopathy caused by calcification of the ligamenta flava of the cervical spine are reported. A review of the literature yielded 13 cases of calcification of the cervical ligamenta flava with findings similar to those in our cases. Specifically, we observed that 1) all patients were more than 50 years of age; 2) most were female; and 3) the calcifications appeared consistently as oval nodules, located symmetrically and paramedially in and around the region of the fifth cervical vertebra. Such characteristic similarities in both clinical and radiologic features are important in considering the etiology of calcification of the cervical ligamenta flawa.

Malignant hypertension in a patient with primary aldosteronism with elevated active renin concentration.

A 40-year-old male, with a past history of hypertension but receiving no medical treatment, was referred. He manifested malignant hypertension (190/130 mmHg; Keith-Wagener III), renal dysfunction (serum creatinine, 3.8 mg/dl), and elevated plasma aldosterone (450 pg/ml) and active renin concentration (ARC, 104 pg/ml). His blood pressure was controlled with multiple antihypertensive agents and ARC thus decreased (4.3 pg/ml), but aldosterone remained elevated. Abdominal magnetic resonance imaging (MRI) revealed a right adrenal adenoma, and aldosterone-producing adenoma was confirmed by adrenal venous sampling. Primary aldosteronism very rarely develops to malignant hypertension, and even in that case ARC is suppressed. Therefore this is a rare case of primary aldosteronism complicated with malignant hypertension and high ARC.

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.

Artificial G-load and chemical changes of saliva.

Effectiveness of artificial G load is expected when it is used as a countermeasure against various bio-medical mishaps which are taking place in the micro-gravity environment, and probably more importantly, to prepare re-entry to the earth 1-G environment. Regarding the salivary chemical analysis, it has been known that, in case of (experimental) motion sickness, symptomatic outputs are delivered through the autonomic nervous system, and changes in salivary chemical components occur. In this study, accordingly, quantitative analyses of salivary potassium, sodium, chloride and total protein were performed before and after various levels of G loads which were produced by our short-radius human centrifuge. When the results were compared, the subjects who failed 60 minutes ride exhibited significantly higher post-load levels of potassium and sodium than those of successful 60 minutes riders. However, the differences between two groups were less clear in chloride and total protein. The result of present study indicates that salivary potassium and sodium are good indicators to represent autonomic nervous system function under the stressful condition produced by an artificial G load.

[Results of annual health examination for the aged provided by the law that are predictive of increased mortality risk].

Possible risk factors associated with mortality were studied in a community using data derived from annual mass health examinations for the aged mandated by law. A total of 1,804 adults (685 men and 1,119 women) aged 40 or older in A-town, located on Tsushima Island, Nagasaki Prefecture, Japan who had participated in annual health examinations at least once between 1984 and 1990, were followed for a mean period of 4.9 years. After adjustment for age using Cox proportional hazards models, in men liver dysfunction (aspartate aminotransferase > 40 U/l or alanine aminotransferase > 35 U/l), fasting blood glucose > or = 110 mg/dl and glucosuria, and in women serum creatinine > or = 1.2 mg/dl, fasting blood glucose > or = 110 mg/dl and proteinuria were found to be associated with a significantly increased risk of total mortality. In multivariate analysis using all independent variables that were significantly associated with mortality in age-adjusted bivariate analysis, in men liver dysfunction and hyperglycemia, and in women hypercreatininemia and hyperglycemia, were significant predictors of mortality. These independent variables remained significant or marginally significant predictors of total mortality even after excluding the effects of 3 pancreatic cancer cases with liver dysfunction or hyperglycemia or 12 deaths within the first year of follow-up, being associated with at least two-fold increased hazard rate ratios. From these results, it is recommended that persons with these risk factors be followed intensively and counseled by public health personnel to modify risk factors.