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Conventional plant gene editing requires laborious tissue-culture-mediated transformation, which restricts the range of applicable plant species. In this study, we developed a heritable and tissue-culture-free gene editing method in Nicotiana benthamiana using tobacco ringspot virus (TRSV) as a vector for in planta delivery of Cas9 and single-guide RNA (sgRNA) to shoot apical meristems. Agrobacterium-mediated inoculation of the TRSV vector induced systemic and heritable gene editing in Nicotiana benthamiana PHYTOENE DESATURASE. Transient downregulation of RNA silencing enhanced gene editing efficiency, resulting in an order of magnitude increase (0.8-13.2%) in the frequency of transgenerational gene editing. While the TRSV system had a preference for certain sgRNA sequences, co-inoculation of a TRSV vector carrying only Cas9 and a tobacco rattle virus vector carrying sgRNA successfully introduced systemic mutations with all five tested sgRNAs. Extensively gene-edited lateral shoots occasionally grew from plants inoculated with the virus vectors, the transgenerational gene editing frequency of which ranged up to 100%. This virus-mediated heritable gene editing method makes plant gene editing easy, requiring only the inoculation of non-transgenic plants with a virus vector(s) to obtain gene-edited individuals., (© The Author(s) 2024. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists.)

Background: The study aims to explore MRI phenotypes that predict glioblastoma's (GBM) methylation status of the promoter region of MGMT gene (pMGMT) by qualitatively assessing contrast-enhanced T1-weighted intensity images., Methods: A total of 193 histologically and molecularly confirmed GBMs at the Kansai Network for Molecular Diagnosis of Central Nervous Tumors (KANSAI) were used as an exploratory cohort. From the Cancer Imaging Archive/Cancer Genome Atlas (TCGA) 93 patients were used as validation cohorts. "Thickened structure" was defined as the solid tumor component presenting circumferential extension or occupying >50% of the tumor volume. "Methylated contrast phenotype" was defined as indistinct enhancing circumferential border, heterogenous enhancement, or nodular enhancement. Inter-rater agreement was assessed, followed by an investigation of the relationship between radiological findings and pMGMT methylation status., Results: Fleiss's Kappa coefficient for "Thickened structure" was 0.68 for the exploratory and 0.55 for the validation cohort, and for "Methylated contrast phenotype," 0.30 and 0.39, respectively. The imaging feature, the presence of "Thickened structure" and absence of "Methylated contrast phenotype," was significantly predictive of pMGMT unmethylation both for the exploratory ( p  = .015, odds ratio = 2.44) and for the validation cohort ( p  = .006, odds ratio = 7.83). The sensitivities and specificities of the imaging feature, the presence of "Thickened structure," and the absence of "Methylated contrast phenotype" for predicting pMGMT unmethylation were 0.29 and 0.86 for the exploratory and 0.25 and 0.96 for the validation cohort., Conclusions: The present study showed that qualitative assessment of contrast-enhanced T1-weighted intensity images helps predict GBM's pMGMT methylation status., Competing Interests: None declared., (© The Author(s) 2024. Published by Oxford University Press, the Society for Neuro-Oncology and the European Association of Neuro-Oncology.)

Since the Fukushima Dai-ichi Nuclear Power Plant (F1-NPP) accident in 2011, radiation effects on wildlife in the contaminated areas have been a major concern. The outskirts of the F1-NPP are mainly rural areas, where many rice fields, streams and reservoirs are located. We searched for wild medaka (small aquarium fish) around the F1-NPP and found two wild medaka habitats (S1 and S2). S1 is a stream located 4 km from the F1-NPP, where the ambient dose equivalent rate was 0.4-0.9 μSv/h (2013-14), and S2 is a reservoir located 7.5 km from the F1-NPP, where the ambient dose equivalent rate was 9.8-22 μSv/h (2013-14 and 2017-18). Dosimeters were placed for one day at the locations where the medaka were captured, and the absorbed dose rates were estimated. Radiation effects on wild medaka were examined using micronucleus assay between 2013 and 2018. No significant difference in frequency of micronucleated gill cells was observed among the wild medaka from S1, S2 and our cultivated medaka that were used as a control., (© The Author(s) 2020. Published by Oxford University Press on behalf of The Japanese Radiation Research Society and Japanese Society for Radiation Oncology.)

Genome editing technology is important for plant science and crop breeding. Genome-edited plants prepared using general CRISPR-Cas9 methods usually contain foreign DNA, which is problematic for the production of genome-edited transgene-free plants for vegetative propagation or highly heterozygous hybrid cultivars. Here, we describe a method for highly efficient targeted mutagenesis in Nicotiana benthamiana through the expression of Cas9 and single-guide (sg)RNA using a potato virus X (PVX) vector. Following Agrobacterium-mediated introduction of virus vector cDNA, >60% of shoots regenerated without antibiotic selection carried targeted mutations, while ≤18% of shoots contained T-DNA. The PVX vector was also used to express a base editor consisting of modified Cas9 fused with cytidine deaminase to introduce targeted nucleotide substitution in regenerated shoots. We also report exogenous DNA-free genome editing by mechanical inoculation of virions comprising the PVX vector expressing Cas9. This simple and efficient virus vector-mediated delivery of CRISPR-Cas9 could facilitate transgene-free gene editing in plants., (© The Author(s) 2020. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists.)

Objectives: Using a murine model of systemic Kawasaki disease (KD)-like vasculitis induced by Candida albicans cell-wall-derived mannan · β-glucan · protein complexes, the objective was to elucidate the relationships of β-glucan receptor dectin-1 (D1) and α-mannan receptor dectin-2 (D2) to the onset of that vasculitis. Methods: The incidence and histological severity of vasculitis were compared among mice lacking the genes for D1 or D2 (i.e. D1 -/- and D2 -/- ) and wild-type (WT) mice. Results: The incidences of vasculitis in the three animal groups were 100% (18/18) in the WT group, 100% (18/18) in the D1 -/- group, and 0% (0/18) in the D2 -/- group. In the WT and D1 -/- mice, severe inflammatory cell infiltration, consisting mainly of neutrophils and macrophages, was seen in the aortic root and the coronary arteries. On the other hand, in the D2 -/- mice, not even mild vascular lesions such as endoarteritis were seen. Conclusion: Recognition of α-mannan by D2 played an important role in the onset of vasculitis in the studied murine model.

Amborella trichopoda is placed close to the base of the angiosperm lineage (basal angiosperm). By genome-wide RNA sequencing, we identified 184C-to-U RNA editing sites in the plastid genome of Amborella. This number is much higher than that observed in other angiosperms including maize (44 sites), rice (39 sites) and grape (115 sites). Despite the high frequency of RNA editing, the biased distribution of RNA editing sites in the genome, target codon preference and nucleotide preference adjacent to the edited cytidine are similar to that in other angiosperms, suggesting a common editing machinery. Consistent with this idea, the Amborella nuclear genome encodes 2-3 times more of the E- and DYW-subclass members of pentatricopeptide repeat proteins responsible for RNA editing site recognition in plant organelles. Among 165 editing sites in plastid protein coding sequences in Amborella, 100 sites were conserved at least in one out of 38 species selected to represent key branching points of the angiosperm phylogenetic tree. We assume these 100 sites represent at least a subset of the sites in the plastid editotype of ancestral angiosperms. We then mapped the loss and gain of editing sites on the phylogenetic tree of angiosperms. Our results support the idea that the evolution of angiosperms has led to the loss of RNA editing sites in plastids., (� The Author(s) 2019. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

We conducted a detailed study of lymphangiogenesis and subsequent lymph node metastasis in early-stage esophageal squamous cell carcinoma (ESCC) using immunostaining for D2-40 and vascular endothelial growth factor (VEGF)-C and D. The study materials included 13 samples of normal squamous epithelium, 6 samples of low-grade intraepithelial neoplasia (LGIN), and 60 samples of superficial ESCC (M1 and M2 cancer 24; M3 or deeper cancer 36). We assessed lymphatic vessel density (LVD) using D2-40 and immunoreactivity for VEGF-C and D in relation to histological type, lymphatic invasion, and lymph node metastasis. LVD in M1 and M2 lesions and M3 or deeper lesions was significantly higher than in normal squamous epithelium (P < 0.001). High expression of VEGF-C and D was observed in M1 and M2 cancer and in M3 or deeper cancer, but not in normal squamous epithelium or LGIN. LVD in VEGF-C- and D-positive cases was significantly higher than in negative cases (P < 0.001). In M3 or deeper cancer, the correlation between VEGF-C or D status and lymphatic invasion or lymph node metastasis was not significant. LVD in cases with positive lymphatic invasion and those with lymph node metastasis was significantly higher than in cases lacking either (P = 0.02 and 0.03, respectively). ESCC cells produce VEGF-C and D from the very early stage of progression. VEGF-C and D activate lymphangiogenesis, and this increase of lymphatic vessels leads to lymphatic invasion and subsequent lymph node metastasis.

Aims: Beta-blockers (BBs) and calcium antagonists (CAs) are reported to aggravate ST-segment elevation in some patients with Brugada syndrome (BrS). The feasibility of their long-term use in BrS still remains unknown. We investigated the safety of long-term use of BB and CA in BrS patients., Methods and Results: Of the 360 consecutive BrS patients, 29 [5: a history of ventricular fibrillation (VF), 17: syncope, 7: asymptomatic] took BB and/or CA (BB: 22, CA: 8) for more than 1 year for the treatment of co-morbidities such as atrial tachyarrhythmia, vasospastic angina, and neurally mediated syncope. The electrocardiographic changes and clinical outcome after the treatment were evaluated. Eleven patients showed type 1 electrocardiogram (ECG) at baseline. BBs and CAs were used within normal dosage range in all patients. After starting a BB and/or CA, type 1 ECG was still observed in 9 patients. There were no significant differences in the ECG parameters such as the amplitude of J-point, QRS duration, and corrected QT intervals before and after starting BB and/or CA. During follow-up of 89 ± 65 months after initiation of the drugs, 1 patient experienced a VF recurrence without significant changes of ECG parameters 2 years after BB therapy was started., Conclusion: Long-term intake of BB or CA within normal dosage range was not associated with the aggravation of ECG parameters and clinical outcome in patients with BrS. The use of BBs and CAs is acceptable under careful observation.

Aims: This study was undertaken to assess the association between plaque features at culprit lesions assessed by frequency-domain optical coherence tomography (FD-OCT) and impaired microvascular perfusion estimated by intracoronary electrocardiogram (IcECG) after elective percutaneous coronary intervention (PCI). Furthermore, we investigated whether IcECG could predict future cardiac events., Methods and Results: This study consisted of 84 patients who underwent both FD-OCT and IcECG during PCI. Patients were classified into two groups based on ST-segment elevation (ST-E) on IcECG after the procedure; ST-E (-) group (n = 53) and ST-E (+) group (n = 31). Minimum fibrous cap thickness was significantly thinner in the ST-E (+) group than in the ST-E (-) group (240 μm [IQR 180 to 310] vs. 100 μm [IQR 60 to 120], P < 0.001). Plaque rupture (7.5% vs. 35.5%, P = 0.001), lipid-rich plaque (75.5% vs. 100%, P < 0.001), the thin cap fibroatheroma (0% vs. 25.8%, P < 0.001) on pre-FD-OCT, protrusion (18.9% vs. 56.7%, P < 0.001), and intra-stent dissection (15.1% vs. 50.0%, P < 0.001) on post-FD-OCT were significantly more frequently found in the ST-E (+) group than in the ST-E (-) group. The incidence of MACE (cardiac death, myocardial infarction, revascularization, hospitalization for heart failure) during 1-year was significantly higher in the ST-E (+) group than in the ST-E (-) group (5.7% vs. 19.4%, P < 0.05)., Conclusion: Plaque features assessed by FD-OCT might be associated with impaired microvascular perfusion and ST-segment elevation on IcECG after the procedure could predict 1-year cardiac events after elective PCI., (Published on behalf of the European Society of Cardiology. All rights reserved. © The Author 2017. For permissions, please email: journals.permissions@oup.com.)

Split-protein methods-where a protein is split into two inactive fragments that must re-assemble to form an active protein-can be used to regulate the activity of a given protein and reduce the size of gene transcription units. Here, we show that a Staphylococcus aureus Cas9 (SaCas9) can be split, and that split-SaCas9 expressed from Agrobacterium can induce targeted mutagenesis in Nicotiana benthamiana. Since SaCas9 is smaller than the more commonly used Cas9 derived from Streptococcus pyogenes, the split-SaCas9 provides the smallest tool yet for clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) plant genome editing. Both sets of split-SaCas9 (&#95;430N/431C and &#95;739N/740C) exhibited genome-editing activity, and the activity of split-SaCas9&#95;739N/740C was almost the same as that of full-length SaCas9. This result indicates that split-SaCas9&#95;739N/740C is suitable for use in targeted mutagenesis. We also show that the split-SaCas9 fragment expressed from Tomato mosaic virus could induce targeted mutagenesis together with another fragment expressed from Agrobacterium, suggesting that a split-SaCas9 system using a plant virus vector is a promising tool for integration-free plant genome editing. Split-SaCas9 has the potential to regulate CRISPR/Cas9-mediated genome editing activity in plant cells both temporally and spatially., (© The Author 2017. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists.)

Objective: We investigated the prevalence of Lynch syndrome and Lynch-like syndrome among Japanese colorectal cancer patients, as there have been no credible data from Japan., Methods: Immunohistochemical analyses for mismatch repair proteins (MLH1, MSH2, MSH6 and PMS2) were carried out in surgically resected, formalin-fixed paraffin-embedded specimens obtained from 1,234 newly diagnosed colorectal cancer patients between March 2005 and April 2014. The presence/absence of the BRAF V600E mutation and hypermethylation of the MLH1 promoter was analyzed where necessary. Genetic testing was finally undertaken in patients suspected as having Lynch syndrome., Results: By the universal screening approach with immunohistochemical analysis for mismatch repair proteins followed by analyses for the BRAF V600E mutation and MLH1 promoter methylation status, 11 (0.9%) of the 1,234 patients were identified as candidates for genetic testing. Out of the 11 patients, 9 (0.7%) were finally diagnosed as having Lynch syndrome; the responsible genes included MLH1 (n = 1), MSH2 (n = 4), EPCAM (n = 1) and MSH6 (n = 3). The remaining two patients (0.2%) were regarded as having Lynch-like syndrome, since biallelic somatic deletion of the relevant mismatch repair genes was detected in the absence of germline mismatch repair alterations. None of the cases was identified as having germline MLH1 epimutation., Conclusions: The prevalence of Lynch syndrome among all newly diagnosed cases of colorectal cancer in Japan is in the same range as that recently reported by studies in Western population. The prevalence of Lynch-like syndrome seems to be extremely low., (© The Author 2016. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com)

Background: Recent studies have shown that arterial stiffness is reduced after meal intake. We evaluated the acute response of central hemodynamics to glucose loading and the variation in their responses among normal glucose tolerance (NGT), impaired glucose tolerance (IGT), and diabetes mellitus (DM)., Methods: The study enrolled 85 patients with known or suspected coronary artery disease who underwent a 75-g oral glucose tolerance test. Central hemodynamic measurements were assessed using radial applanation tonometry at fasting, 60, and 120 minutes after glucose loading., Results: Glucose loading decreased the augmentation index normalized to a heart rate of 75 bpm (AIx@75) (81.6±13.9 to 74.5±14.1%, P < 0.01) and central systolic blood pressure (SBP) (115±22 to 109±21mm Hg, P < 0.01) at 120 minutes without a significant change in brachial SBP (126±25 to 125±25mm Hg, P = 0.93). Glucose loading decreased central SBP in NGT and IGT groups but did not affect the DM group. Change in AIx@75 at 120 minutes after glucose loading was blunted in IGT and DM groups compared with the NGT group (-5.7±4.4 vs. -3.6±4.1 vs. -9.3±6.2%, P < 0.01). Multivariate logistic regression analysis identified DM as an independent factor associated with the presence of blunted response of AIx to glucose loading., Conclusions: Oral glucose loading decreased central SBP and AIx@75 without a significant change in brachial SBP, and these central hemodynamic responses were blunted in patients with DM., (© American Journal of Hypertension, Ltd 2015. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Aims: Published reports regarding inferolateral early repolarization (ER) syndrome (ERS) before 2013 possibly included patients with Brugada-pattern electrocardiogram (BrP-ECG) recorded only in the high intercostal spaces (HICS). We investigated the significance of HICS ECG recording in ERS patients., Methods and Results: Fifty-six patients showing inferolateral ER in the standard ECG and spontaneous ventricular fibrillation (VF) not linked to structural heart disease underwent drug provocation tests by sodium channel blockade with right precordial ECG (V1-V3) recording in the 2nd-4th intercostal spaces. The prevalence and long-term outcome of ERS patients with and without BrP-ECG in HICS were investigated. After 18 patients showing type 1 BrP-ECG in the standard ECG were excluded, 38 patients (34 males, mean age; 40.4 ± 13.6 years) were classified into four groups [group A (n = 6;16%):patients with ER and type 1 BrP-ECG only in HICS, group B (n = 5;13%):ERS with non-type 1 BrP-ECG only in HICS, group C (n = 8;21%):ERS with non-type 1 BrP-ECG in the standard ECG, and group D (n = 19;50%):ERS only, spontaneously or after drug provocation test]. During follow-up of 110.0 ± 55.4 months, the rate of VF recurrence including electrical storm was significantly higher in groups A (4/6:67%), B (4/5:80%), and C (4/8:50%) compared with D (2/19:11%) (A, B, and C vs. D, P < 0.05)., Conclusions: Approximately 30% of the patients with ERS who had been diagnosed with the previous criteria showed BrP-ECG only in HICS. Ventricular fibrillation mostly recurred in patients showing BrP-ECG in any precordial lead including HICS; these comprised 50% of the ERS cohort., (Published on behalf of the European Society of Cardiology. All rights reserved. © The Author 2015. For permissions please email: journals.permissions@oup.com.)

Endocytoscopy (ECS) is a novel endoscopic technique that allows detailed diagnostic examination of the gastrointestinal tract at the cellular level. We previously reported that use of ECS at ×380 magnification (GIF-Y0002) allowed a pathologist to diagnose esophageal squamous cell carcinoma (ESCC) with high sensitivity (94.9%) but considerably low specificity (46.7%) because this low magnification did not reveal information about nuclear abnormality. In the present study, we used the same magnifying endoscope to observe various esophageal lesions, but employed digital 1.6-fold magnification to achieve an effective magnification of ×600, and evaluated whether this improved the diagnostic accuracy in distinguishing neoplastic from non-neoplastic lesions.We examined the morphology of surface cells using vital staining with toluidine blue and compared the histological features of 40 cases, including 19 case of ESCC and 21 non-neoplastic esophageal lesions (18 cases of esophagitis, 1 case of glycogenic acanthosis, 1 case of leiomyoma, and 1 case of normal squamous epithelium). One endoscopist classified the lesions using the type classification, and we consulted one pathologist for judgment of the ECS images as 'neoplastic', 'borderline', or 'non-neoplastic'. At ×600 magnification, the pathologist confirmed that nuclear abnormality became evident, in addition to the information about nuclear density provided by observation at ×380. The overall sensitivity and specificity with which the endoscopist was able to predict neoplastic lesions using the type classification was 100% (19/19) and 90.5% (19/21), respectively, in comparison with values of 94.7% (18/19 cases) and 76.2% (16/21), respectively, for the pathologist using a magnification of ×600. The pathologist diagnosed two non-neoplastic lesions and one case of ESCC showing an apparent increase of nuclear density with weak nuclear abnormality as 'borderline'. Among the 21 non-cancerous lesions, two cases of esophagitis that were misdiagnosed by the endoscopist were also misinterpreted as 'neoplastic' by the pathologist. We have shown, by consultation with a pathologist, that an ECS magnification of ×600 (on a 19-inch monitor) is adequate for recognition of nuclear abnormality. We consider that it is feasible to diagnose esophageal neoplasms on the basis of ECS images, and that biopsy histology can be omitted if a combination of increased nuclear density and nuclear abnormality is observed., (© 2014 International Society for Diseases of the Esophagus.)

Background: Individuals with substance use disorders typically exhibit a predilection toward instant gratification with apparent disregard for the future consequences of their actions. Indirect evidence suggests that low dopamine D2-type receptor availability in the striatum contributes to the propensity of these individuals to sacrifice long-term goals for short-term gain; however, this possibility has not been tested directly. We investigated whether striatal D2/D3 receptor availability is negatively correlated with the preference for smaller, more immediate rewards over larger, delayed alternatives among research participants who met DSM-IV criteria for methamphetamine (MA) dependence., Methods: Fifty-four adults (n = 27 each: MA-dependent, non-user controls) completed the Kirby Monetary Choice Questionnaire, and underwent positron emission tomography scanning with [(18)F]fallypride., Results: MA users displayed steeper temporal discounting (p = 0.030) and lower striatal D2/D3 receptor availability (p < 0.0005) than controls. Discount rate was negatively correlated with striatal D2/D3 receptor availability, with the relationship reaching statistical significance in the combined sample (r = -0.291, p = 0.016) and among MA users alone (r = -0.342, p = 0.041), but not among controls alone (r = -0.179, p = 0.185); the slopes did not differ significantly between MA users and controls (p = 0.5)., Conclusions: These results provide the first direct evidence of a link between deficient D2/D3 receptor availability and steep temporal discounting. This finding fits with reports that low striatal D2/D3 receptor availability is associated with a higher risk of relapse among stimulant users, and may help to explain why some individuals choose to continue using drugs despite knowledge of their eventual negative consequences. Future research directions and therapeutic implications are discussed., (© The Author 2015. Published by Oxford University Press on behalf of CINP.)

Objective: This study was performed to clarify whether size criteria could be applied to the prediction of metastasis to the mesorectal, internal iliac and obturator lymph nodes in patients with lower rectal cancer., Methods: A total of 915 lymph nodes (416 mesorectal, 199 internal iliac and 300 obturator) from 53 patients with lower rectal cancer who underwent a curative resection were examined; 83 lymph nodes were positive. The sizes of the lymph nodes immediately after removal and those of paraffin-embedded sections were compared for 175 lymph nodes. Then, size criteria for discriminating the status of metastasis were evaluated in 915 paraffin-embedded lymph nodes., Results: Regardless of the metastatic status and the site of the lymph nodes, a positive relationship was observed between the sizes of the lymph nodes immediately after removal and those of paraffin-embedded sections (P < 0.01). The area under the curve generated by a receiver-operating characteristics curve showed no significant differences between the short-axis diameter and the long-axis diameter, regardless of the lymph node location. Specifically, the optimal cutoff value of the short-axis diameter for discriminating the status of metastasis in mesorectal, internal iliac and obturator lymph nodes extrapolated to the living body was set at 6.2, 5.0 and 4.8 mm, with accuracies of 72.4, 63.8 and 59.3% and with positive predictive values of 29.9, 16.3 and 7.1%, respectively., Conclusions: Size criteria were applicable for discriminating the metastatic status of lymph nodes from three different locations, although the positive predictive values of the size criteria for the internal iliac and obturator lymph nodes were lower than that for the mesorectal lymph nodes., (© The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Decision-making involves frontolimbic and dopaminergic brain regions, but how prior choice outcomes, dopamine neurotransmission, and frontostriatal activity are integrated to affect choices is unclear. We tested 60 healthy volunteers using the Balloon Analogue Risk Task (BART) during functional magnetic resonance imaging. In the BART, participants can pump virtual balloons to increase potential monetary reward or cash out to receive accumulated reward; each pump presents greater risk and potential reward (represented by the pump number). In a separate session, we measured striatal D2/D3 dopamine receptor binding potential (BPND) with positron emission tomography in 13 of the participants. Losses were followed by fewer risky choices than wins; and during risk-taking after loss, amygdala and hippocampal activation exhibited greater modulation by pump number than after a cash-out event. Striatal D2/D3 BPND was positively related to the modulation of ventral striatal activation when participants decided to cash out and negatively to the number of pumps in the subsequent trial; but negatively related to the modulation of prefrontal cortical activation by pump number when participants took risk, and to overall earnings. These findings provide in vivo evidence for a potential mechanism by which dopaminergic neurotransmission may modulate risk-taking behavior through an interactive system of frontal and striatal activity., (© The Author 2013. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

Kawasaki disease (KD) is an acute vasculitis syndrome of unknown aetiology in children. The administration of Candida cell wall antigens induced KD-like coronary vasculitis in mice. However, the responses of KD patients to Candida cell wall antigen are unknown. In this study, we examined the response of KD patients to β-glucan (BG), one of the major fungal cell wall antigens, by measuring the anti-BG titre. In KD patients, the anti-C. albicans cell wall BG titre was higher than that in normal children. The anti-BG titre was also higher in KD patients compared to children who served as control subjects. The efficacy of intravenous immunoglobulin (IVIG) therapy in KD is well established. We categorized the KD patients into three groups according to the therapeutic efficacy of intravenous immunoglobulin (IVIG) and compared the anti-BG titre among these groups. Anti-BG titres were similar in the control group and the non-responsive group. In the fully responsive group, the anti-BG titre showed higher values than those in the normal children. This study demonstrated clinically that KD patients have high antibody titres to Candida cell wall BG, and suggested the involvement of Candida cell wall BG in the pathogenesis of KD. The relationship between IVIG therapy and anti-BG titre was also shown. These results provide valuable insights into the therapy and diagnosis of KD., (© 2014 British Society for Immunology.)

Objective: The esophagus is the only organ where changes in the superficial microvasculature from normal squamous epithelium to invasive cancer are evident by magnifying endoscopy. We investigated in detail the features of angiogenesis in early-stage esophageal cancer using CD34 and CD105 immunostaining, and also the correlation between angiogenesis and mononuclear cell infiltration., Materials and Methods: Using 10 samples of normal squamous epithelium, 7 samples of low-grade intraepithelial neoplasia, and 45 samples of superficial esophageal cancer, we determined the microvessel density at hot spots showing positive staining for CD34 and CD105. We observed the histological features of CD34- and CD105-positive microvessels that corresponded to observations made by magnifying endoscopy. We then investigated the correlation between microvessel density and each histological situation or the grade of mononuclear cell infiltration., Results: The histological features of CD34- and CD105-positive microvessels were able to explain the morphological changes in the microvasculature during cancer progression observed by magnifying endoscopy. The microvessel density for CD34 or CD105 was significantly correlated with each of the histological types (P < 0.001, rS = 0.51 and 0.76, respectively). Mononuclear cell infiltration at CD105 hot spots was most frequent in M1 and M2 cancer (94.7%). The correlation between the degree of mononuclear cell infiltration and microvessel density for CD105 staining was also significant (P < 0.001, rS = 0.49)., Conclusions: The microvessel density based on CD34 and CD105 immunostaining can be used to corroborate observations of superficial esophageal squamous cell carcinoma made by magnifying endoscopy. Mononuclear cell infiltration may play an important role in angiogenesis at the early stage of cancer progression., (© The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Aims: Coronary flow velocity reserve (CFVR) measurement in three major coronary arteries by transthoracic echocardiography is a promising and non-invasive method for detecting myocardial ischaemia. Its value when compared with fractional flow reserve (FFR) is unknown. Our aim was to determine the diagnostic accuracy of CFVR in three major coronary arteries for detecting ischaemia compared with FFR., Methods: This is a prospective study in 172 vessels of 140 patients with at least one ≥50% stenosis in a major epicardial artery as determined by visual assessment on computed tomography coronary angiography. We performed CFVR measurement by transthoracic echocardiography within 48 h before coronary angiography and FFR measurement. The cut-off value of CFVR was estimated by the receiver operating characteristic (ROC) curve based on that of FFR ≤0.75., Results: The CFVR was 1.86 ± 0.36 in coronary arteries with FFR ≤0.75 (n = 79) and 2.54 ± 0.48 in those with FFR >0.75 (n = 93, P < 0.0001). CFVR with cut-off of 2.2, determined by the ROC curve, was 85% sensitive and 79% specific in predicting the stenotic condition of the coronary artery with FFR ≤0.75 in three major vessels. In each vessel, the sensitivity and specificity were 85 and 78% (left anterior descending coronary artery), 94 and 83% (right coronary artery), and 88 and 88% (left circumflex coronary artery). CFVR was indirect proportional to FFR (r = 0.56, P < 0.0001) and to per cent diameter stenosis (r = 0.26, P = 0.0008)., Conclusions: The non-invasive CFVR measurement could be a reliable stenosis-specific method for determining the haemodynamic significance of three major coronary arteries.

Oxidative stress is recognized as one of the pathogenic mechanisms involved in neurodegenerative disease. However, recent evidence has suggested that regulation of cellular fate in response to oxidative stress appears to be dependent on the stress levels. In this study, using HT22 cells, we attempted to understand how an alteration in the oxidative stress levels would influence neuronal cell fate. HT22 cell viability was reduced with exposure to high levels of oxidative stress, whereas, low levels of oxidative stress promoted cell survival. Erk1/2 activation induced by a low level of oxidative stress played a role in this cell protective effect. Intriguingly, subtoxic level of H2O2 induced expression of a growth factor, progranulin (PGRN), and exogenous PGRN pretreatment attenuated HT22 cell death induced by high concentrations of H2O2 in Erk1/2-dependent manner. Together, our study indicates that two different cell protection mechanisms are activated by differing levels of oxidative stress in HT22 cells.

Jejunal carcinoma in patients with familial adenomatous polyposis has been rarely reported, and little is known about its association with genetic alterations of the APC gene. A 52-year-old woman with familial adenomatous polyposis underwent palliative resection of the proximal jejunum because of two circumferential tumors associated with peritoneal carcinomatosis. A histological examination revealed that one tumor was a poorly differentiated adenocarcinoma, and that the other was a moderately differentiated adenocarcinoma with adenomatous components. The patient did not respond to standard chemotherapy and died of disseminated disease 8 months after surgery. A genetic analysis of the APC gene identified somatic mutations in each tumor (c.4450delAG and p.R1450X) in addition to the germline mutation (c.3984del5), all of which form stop codons, resulting in truncated APC products. This report is the first description of how a second hit to the APC gene can be involved in carcinogenesis of the jejunum in familial adenomatous polyposis.

Aims: The importance of oxidized low-density lipoprotein (oxLDL) has been implicated in the process of plaque rupture. However, few previous studies demonstrated the relationship between plaque morphology and oxLDL. We evaluated the relationship between coronary plaque vulnerability assessed by optical coherence tomography (OCT) and circulating malondialdehyde-modified low-density lipoprotein (MDA-LDL)., Methods and Results: OCT was used to determine plaque vulnerability in 102 patients with acute coronary syndrome (ACS; n = 53) and stable angina pectoris (SAP; n = 49). Circulating levels of MDA-LDL were measured by using enzyme-linked immunosorbent assay. Thin-cap fibroatheromas (TCFAs; defined as lipid-rich with plaque cap thickness <65 μm) were detected more frequently in ACS than in SAP (83% vs. 16%, P < 0.001). The circulating levels of MDA-LDL were significantly higher in patients with ACS compared with SAP (P = 0.008). The levels of MDA-LDL were significantly higher in SAP patients with TCFA than those with non-TCFA (P < 0.001). Although the levels of MDA-LDL were not significant between ACS patients with TCFA and those with non-TCFA, patients with ruptured TCFA had higher levels of MDA-LDL compared with those with morphologically intact TCFA (P = 0.023). MDA-LDL levels were associated with the presence of TCFA (odds ratio, 1.45 per 10-unit increment of MDA-LDL; 95% CI, 1.24-1.68; P< 0.001) in multivariable logistic regression analysis., Conclusion: Circulating MDA-LDL levels might be associated with the presence of TCFA in the culprit lesion.

Objective: In the surgical treatment for lower rectal cancer, preoperative selection of patients at high risk for lateral lymph node metastasis is important, since lateral lymph node dissection might impair genitourinary functions. We examined whether the status of lateral lymph node metastasis can be predicted from the lymph node size., Methods: The subjects were 533 (35 positive and 498 negative) lateral lymph nodes from 47 patients with lower rectal cancer who underwent curative resection with lateral lymph node dissection. The sizes of the lateral lymph nodes immediately after removal and those in paraffin-embedded sections were compared for 108 lateral lymph nodes from 13 patients. In addition, receiver-operating characteristic curves were generated for the 533 paraffin-embedded lateral lymph nodes from the 47 patients to determine the optimal cut-off size for discriminating between positive and negative lateral lymph nodes., Results: Irrespective of the presence/absence of metastasis and the long-/short-axis diameter, a positive relationship was noted between the sizes of the lateral lymph nodes measured immediately after removal and those measured on paraffin-embedded sections (P< 0.01). The area under the curve for the short-axis diameter differed little from that for the long-axis diameter (0.77 vs. 0.76, P =0.80). The optimal cut-off values of the short- and long-axis diameter extrapolated to the living body were 5.4 and 8.4 mm, respectively, with an accuracy of 72.8% for the short-axis diameter and 71.9% for the long-axis diameter., Conclusions: Prediction of the status of lateral lymph node metastasis from the lymph node size (long-/short-axis diameter) may be a simple and reliable method. The optimal cut-off diameter should be validated in prospective imaging studies.

A transition edge sensor (TES) microcalorimeter has been developed for use as an energy dispersive X-ray spectrometer. The TES microcalorimeter is a thermal detector that enables one to determine the energy of an incident photon by measuring the resultant increase in temperature. In this work, a Ti/Au TES microcalorimeter was developed to measure LX rays emitted by transuranium elements. The phase transition temperature was set at ~200 mK by using a bilayer structure composed of a 110-nm-thick Au layer and a 40-nm-thick Ti layer. An Au of 5 µm thickness was deposited on the Ti/Au bilayer to achieve an absorption efficiency of 35-80 % for the energy range of LX rays (10-25 keV). The developed TES microcalorimeter was irradiated with LX rays emitted by an (241)Am source at an operating temperature of 140 mK. An energy resolution of ~80 eV (full width at the half maximum) was obtained for L(β1)X ray of 17.75 keV.

We describe a 74-year-old male who underwent open stent repair for an infrarenal abdominal aortic aneurysm with a severely calcified aortic neck. The stent graft was constructed by covering a 50-mm long Gianturco Z stent (diameter: 20 mm) with a Dacron prosthesis (diameter: 20 mm). The stented Dacron graft was inserted into the calcified aortic neck, was then sutured to the trimmed aneurysmal wall, and was anastomosed to a bifurcated prosthesis. The distal ends of the bifurcated prosthesis were anastomosed to both common femoral arteries, and the terminal aorta was closed. The patient had an uneventful postoperative course. This procedure may be a feasible and safe way to repair infrarenal abdominal aortic aneurysm with a severely calcified aortic neck.

Aims: Although some recent guidelines recommend an early invasive strategy for non-ST-segment elevation acute coronary syndrome (NSTEACS), several studies have failed to identify any benefit for very early intervention for NSTEACS. The no-reflow phenomenon may inhibit the expected benefit from very early recanalization for NSTEACS subjects. The aim of this study was to investigate whether optical coherence tomography (OCT) could predict no-reflow in patients with NSTEACS., Methods and Results: This study comprised 83 consecutive patients with NSTEACS who underwent OCT and successful emergent primary stenting. On the basis of post-stent TIMI flow, patients were divided into two groups: no-reflow group (n = 14) and reflow group (n = 69). Thin-cap fibroatheroma (TCFA) was defined as a plaque presenting lipid content for >90 degrees , and with thinnest part of the fibrous cap measuring <70 microm. Thin-cap fibroatheroma were more frequently observed in the no-reflow group than in the reflow group (50% vs. 16%, P = 0.005). The frequency of the no-reflow phenomenon increases according to the size of the lipid arc in the culprit plaque. Final TIMI blush grade also deteriorated according to the increase in the lipid arc. A multivariable logistic regression model revealed that lipid arc alone was an independent predictor of no-reflow (odds ratio 1.018; CI 1.004-1.033; P = 0.01)., Conclusion: Optical coherence tomography can predict no-reflow after percutaneous coronary intervention (PCI) in NSTEACS. The lipid contents of a culprit plaque may play a key role in damage to the microcirculation after PCI for NSTEACS. From our results, it is found that OCT is useful tool for stratifying risk for PCI for NSTEACS.

We recently determined the structure of a unique type of 1,3-beta-D-glucan obtained from Aureobasidium pullulans (AP-FBG) and found that it reacted with the antibodies in human sera. The reactivity of AP-FBG to the antibodies was stronger than that of 1,3-beta-D-glucan obtained Grifola frondosa (GRN) but weaker than that of 1,3-beta-D-glucan from Candida albicans (CSBG). Here, we demonstrated that AP-FBG reacted to IgG antibodies, especially those of the subclasses IgG2, IgG1, and IgG3, in human sera. Moreover, the results of competitive enzyme-linked immunosorbent assays (ELISAs) using various glucan competitors showed that these IgGs recognized branched chains at position 6. This is the first study to report that the branched chains at position 6 of beta-D-glucan strongly contribute to its recognition by antibodies in human sera. This high reactivity of AP-FBG to human IgG could be advantageous for the use of this glucan in medicine, e.g., as an immunostimulatory agent.

Objective: Vascular endothelial growth factor (VEGF)-C is known to be associated with angiogenesis and lymphangiogenesis in various cancers. However, little is known about the clinical significance of determining the blood level of VEGF-C in patients with colorectal cancer., Methods: Plasma levels of VEGF-C in patients with colorectal cancer (n = 127) and normal healthy volunteers (n = 23) were determined by the sandwich enzyme-linked immunosorbent assay., Results: The plasma VEGF-C concentration did not significantly differ between patients with colorectal cancer and healthy controls (P = 0.53). However, subgroup analysis showed that deeper tumor invasion (P = 0.04), more severe lymphatic invasion (P = 0.03) and venous invasion (P < 0.01) were correlated with an elevated level of plasma VEGF-C. Among the patients (n = 109) who underwent potentially curative surgery, the plasma level of VEGF-C was higher in patients who developed recurrence (n = 35) than in those who did not (n = 74) (P = 0.04). In addition, disease-free (P = 0.02) and overall survival times (P = 0.02) were shorter in patients with a high level (>1840 pg/ml) of plasma VEGF-C than in those with a low level (< or =1840 pg/ml) when the cut-off value was determined on the basis of the median value in colorectal cancer patients. Multivariate analysis with the Cox proportional hazard model demonstrated that the plasma VEGF-C level along with Dukes' stage was an independent factor affecting overall survival (P = 0.03)., Conclusion: These results suggest that determining the plasma level of VEGF-C would be useful for predicting lymphatic invasion, venous invasion and poor outcome of patients with colorectal cancer.

This study examined the effects of odors on sustained attention during a vigilance task. Two essential oils (lavender and eucalyptus) and two materials (l-menthol and linalyl acetate) were compared with a control. The increase in reaction time was significantly lower with lavender than with the control. The results suggest that the administration of lavender helped to maintain sustained attention during the long-term task.

Background: Cytomegalovirus (CMV) is the most important pathogen affecting the outcome of renal transplantation. The combination of CMV-seronegative transplant recipients with CMV-seropositive transplant donors places recipients at the highest risk of CMV disease. In cases of congenital CMV infection, existing immunity only partially protected mothers from reinfection with a different genotypic strain. The effect of differences in infected CMV strains between CMV-seropositive transplant donors and CMV seropositive transplant recipients on the outcome of transplantation remains unclear., Methods: In this prospective multicenter study, the presence of antibodies against strain-specific glycoprotein H epitopes in 84 CMV-seropositive transplant donor/CMV-seropositive transplant recipient renal transplantation cases were determined, and their relationships to acute transplant rejection, CMV infection, degree of antigenemia, and CMV disease were evaluated., Results: Among the 84 donor/recipient pairs, 45 and 32 had matched and mismatched strain-specific glycoprotein H antibodies, respectively. Acute transplant rejection in the mismatched group was more frequent than it was in the matched group (63% vs. 22%; P=.005). CMV disease was also more frequently observed in the mismatched group (28% vs. 9%; P=.026). The mismatched group had a higher level of antigenemia (P=.019)., Conclusions: Our results illustrate more adverse events in the cases with a CMV-seropositive transplant donor and a CMV-seropositive transplant recipient in which the glycoprotein H antibodies are mismatched, suggesting that reinfection with a different CMV strain results in more complications.

Objectives: We review cases of pseudoaneurysm formation of the graft anastomosis sites following repair of type A acute aortic dissection by our original leak-proof technique for dissected aortic wall reinforcement with xenopericardium and gelatin-resorcinol-formalin (GRF) glue., Cases: A 47-year-old male presented inferior acute myocardial infarction with bradycardia and cardiogenic shock 34 months after the initial total arch replacement for acute aortic dissection. The patient underwent reoperation with total arch replacement and coronary artery bypass grafting to the right coronary artery. There was rupture of the proximal anastomosis with clotted pseudoaneurysm formation extending over the right ventricle. The right coronary artery was compressed by the pseudoaneurysm. The distal anastomosis also ruptured with localized pseudoaneurysm formation. Twenty-two patients with type A acute aortic dissection underwent aortic repair by our original leak-proof technique for dissected aortic wall reinforcement with xenopericardium and gelatin-resorcinol-formalin glue between 1997-2003. Four patients developed redissection of the anastomosis sites, which required reoperation, including the current case., Discussion: The cause of redissection was unclear, however, use of GRF glue itself might develop tissue damage and redissection of the aorta, and also glued xenopericardium strip reinforcement in our original technique might accelerate damage to the aortic wall., Conclusion: Follow-up examination is mandatory for the patient of aortic repair with the use of GRF glue.

Background: Sensorineural hearing loss (SNHL) is the most common congenital disease. Longitudinal studies of infants with congenital cytomegalovirus (CMV) infection have demonstrated an association between CMV and SNHL. However, because of the lack of suitable neonatally collected specimens, the proportion of CMV-associated SNHL has not been defined, nor has the relationship between CMV and the major genetic causes of SNHL, such as mutations in the GJB2 gene., Methods: Sixty-seven children with severe SNHL were analyzed for CMV and human herpesvirus 6 (HHV-6) infections and for GJB2 mutations. DNA specimens were prepared from dried umbilical cords, which are available for everyone born in Japan. Four children with typical symptomatic infection at birth served as positive control subjects., Results: Congenital CMV infection and GJB2 mutations were identified in 15% and 24% of the patients, respectively. HHV-6 was not detected. All children with CMV-associated cases developed SNHL before they were 2 years old. Most children with CMV-associated SNHL had no obvious clinical abnormality at birth, and their viral loads were lower than those of symptomatic children., Conclusions: Congenital CMV infection is an important cause of severe SNHL, and it has an incidence comparable to that of GJB2-associated SNHL.

Our previous data indicated that a Myxococcus xanthus sensor-type adenylyl cyclase (CyaA) functions in signal transduction during osmotic stress. However, the cAMP-mediated signal transduction pathway in this bacterium was unknown. Here, we isolated a clone from a M. xanthus genomic DNA library using oligonucleotide probes designed based on the conserved cAMP-binding domains of the cAMP-dependent protein kinase (PKA) regulatory subunits. The clone contained two open-reading frames (ORFs), cbpA and cbpB, encoding hydrophilic proteins with one and two cAMP-binding domains, respectively. The CbpB exhibited partial primary structural similarity to PKA regulatory subunits. cbpA and cbpB mutants, generated by gene disruption, showed normal growth, development and spore germination. However, the cbpB mutant cultured under high- or low-temperature conditions exhibited a marked reduction in growth. cbpB mutant cells were also more sensitive to osmotic stress than wild-type cells. The cbpA mutant possessed normal resistance to such stress. The phenotype of cbpB mutant was similar to those of PKA regulatory subunit mutants of some eukaryotic microorganisms.

2'-O-Me RNA/ENA chimera oligonucleotides complementary to exon 45 and 46 of the dystrophin gene induced exon 45 and 46 skipping of the dystrophin pre-mRNA, respectively. The induction of exon skipping by the most effective 2'-O-Me RNA/ENA chimeras led to the expression of dystrophin in dystrophin-deficient myocytes by correcting the translational reading frames. Also, in the process of 2'-O-Me RNA/ENA chimera optimization to induce exon skipping in several exons, it was found that the optimized target sequences of the chimeras included guanosine- or adenosine-rich sequences that might function as spiking enhancer sequences (SES).

Grifolan, GRN, is a fungal antitumor beta-glucan isolated from Grifola frondosa. Various studies suggested that the underlying mechanism of the antitumor activity of GRN is strongly related to immune modulation. In the previous publication (Adachi et al., 1994; Okazaki et al., 1995), we have shown that GRN activates macrophages to produce tumor necrosis factor (TNF) in vitro. In this study, the structural unit essential to produce TNF was examined by chemical modifications of GRN. GRN suspended in distilled water was treated at 150 degrees C for up to 3 h. Addition of the resulting turbid solution to the RAW 264.7 macrophage-like cell line produced TNF, and the relative activity was diminished in relation to the heat treatment period. The fractions with a heating period longer than 15 min did not show any activity. After centrifugation of the resulting solution, significant activity was shown by precipitate fractions, suggesting that the insoluble form of GRN is important for TNF production. Interestingly, the precipitate fraction obtained from 9 min of treatment also had significant activity. In addition, admixing the soluble fraction with the particles significantly inhibited the TNF production. In contrast to these observations, the high-molecular-mass subfraction of the soluble fraction prepared by ultrafiltration produced significant amounts of TNF. Similar phenomena were shown with sodium hydroxide treatment and dimethylsulfoxide treatment. These facts strongly suggested that insoluble as well as a high molecular mass soluble form of GRN are required for TNF production by macrophages.

We have recently shown that pretreatment with endothelin-1 (ET-1) for 20 min stimulates GLUT4 translocation in a PI3-kinase-dependent manner in 3T3-L1 adipocytes (Imamura, T. et al., J Biol Chem 274:33691-33695). This study presents another pathway by which ET-1 potentiates glucose transport in 3T3-L1 adipocytes. ET-1 treatment (10 nM) leads to approximately 2.5-fold stimulation of 2-deoxyglucose (2-DOG) uptake within 20 min, reaching a maximal effect of approximately 4-fold at approximately 6 h, and recovering almost to basal levels after 24 h. Insulin treatment (3 ng/ml) results in an approximately 5-fold increase in 2-DOG uptake at 1 h, and recovering to basal levels after 24 h. The ETA receptor antagonist, BQ 610, inhibited ET-1 induced glucose uptake both at 20 min and 6 h, whereas the ETB receptor antagonist, BQ 788, was without effect. Interestingly, ET-1 stimulated 2-DOG uptake at 6 h, not at 20 min, was almost completely blocked by the protein-synthesis inhibitor, cycloheximide and the RNA-synthesis inhibitor, actinomycin D, suggesting that the short-term (20 min) and long-term (6 h) effects of ET-1 involve distinct mechanisms. GLUT4 translocation assay showed that 20 min, but not 6 h, exposure to ET-1 led to GLUT4 translocation to the plasma membrane. In contrast, 6 h, but not 20 min, exposure to ET-1 increased expression of the GLUT1 protein, without affecting expression of GLUT4 protein. ET-1 induced 2-DOG uptake and GLUT1 expression at 6 h were completely inhibited by the MEK inhibitor, PD 98059, and partially inhibited by the PI3-kinase inhibitor, LY 294002, and the G alpha i inhibitor, pertussis toxin. The PLC inhibitor, U 73122, was without effect. These findings suggest that ET-1 induced GLUT1 protein expression is primarily mediated via MAPK, and partially via PI3K in 3T3-L1 adipocytes.

Fluid and protein secretion by the submandibular glands of 25-day-old rats were examined and compared in response to three cholinergic and four peptidergic sialogogues at various doses. All cholinergic and peptidergic agonists used were potent sialogogues for the submandibular glands of the weanling rats over the wide range of doses used. The cholinergic agonists, bethanechol and methacholine and the peptidergic agonists, substance P, substance PTyr8 and eledoisin-related peptide used intravenously, acted similarly to each other on the submandibular glands of the rats, late in the natural weaning period, but carbachol and physalaemin had slightly different effects. Of the peptidergic agonists, physalaemin was the most potent sialogogue among four tachykinins tested at the low dose. The types of protein secreted by the submandibular glands of the weanling rats in response to all sialogogues used here were typical of the beta-type. These results indicate that all agonists used could mainly stimulate the acinar cells of the submandibular glands of the weanling rats which have already fully developed functionally at this time.

We performed a randomized, crossover study comparing the effects of nicardipine and trichlormethiazide on insulin sensitivity in 8 untreated essential hypertensive patients. They were treated either with nicardipine or trichlormethiazide for 12 weeks, after which they were treated with the alternative drug for a further 12 weeks. Insulin sensitivity was determined by the hyperinsulinemic euglycemic clamp technique. Nicardipine had no adverse effect on insulin sensitivity. Trichlormethiazide decreased insulin sensitivity index by 12%. As nicardipine does not have an adverse effect on insulin sensitivity, it should be preferred in patients who have a proven metabolic abnormality.

We obtained rabbit antibodies by injecting immunogenic conjugates which were prepared by combining covalently 1-O-(15'-carboxypentadecyl)-2-O-acetyl-sn-glycero-3- phosphocholine(acetyl-CPGPC), 1-O-(15'-carboxypentadecyl)-2-O-N,N-dimethylcarbamoyl-sn-glycero-3 - phosphocholine (dimethylcarbamoyl-CPGPC), or 1-O-(15'-carboxypentadecyl)-2-O-N-butyl-carbamoyl-sn-glycero-3-pho sphocholine (butylcarbamoyl-CPGPC) with protein (BSA or KLH), respectively, and examined the specificity of the resulting antibodies by comparison with inhibition of the binding of iodolabeled CPGPC derivatives to the antibodies by corresponding or related phospholipids. Acetyl-CPGPC and dimethylcarbamoyl-CPGPC possessed haptenic activity causing production of antibodies reactive with PAF. Changes of the substituents at sn-2 in the antigens affected the specificity of the resulting antibodies. The affinity of the substituents to the antibodies decreased in the following order: acetyl much greater than dimethylcarbamoyl and butylcarbamoyl for antibodies to acetyl-CPGPC-KLH; dimethylcarbamoyl greater than acetyl much greater than butylcarbamoyl for antibodies to dimethylcarbamoyl-CPGPC-BSA; and butylcarbamoyl greater than dimethylcarbamoyl greater than acetyl for antibodies to butylcarbamoyl-CPGPC-BSA. Naturally occurring phospholipids, including lysoPAF, phosphatidylcholine, lysophosphatidylcholine, and sphingomyelin, revealed no cross-reactivities with these antibodies. Anti-dimethylcarbamoyl-CPGPC-BSA IgG and anti-acetyl-CPGPC-KLH IgG inhibited a PAF-induced aggregation of washed rabbit platelets in a dose-dependent manner. In contrast, anti-butylcarbamoyl-CPGPC-BSA IgG did not affect a PAF-induced platelet aggregation, nor did preimmune IgG.

An enzyme-labelled immunoassay for plasma cortisol was developed. For this alkaline-phosphatase was conjugated through 21-hemisuccinate of cortisol using water-soluble carbodiimide. An antibody was raised in rabbits against corisol-21-hemisuccinate bovine serum albumin. Before assay 10 mul samples of plasma were mixed with glutamate buffer, pH 3.3, and boiled to denature endogenous cortisol-binding globulin and alkaline-phosphatase. Separation of "bound and free" cortisol was done by the double antibody method. A linear relationship was obtained between the volume of plasma and the amount of cortisol. The minimal detectable level of plasma cortisol was 1 mug/dl and the coefficients of variation were 2.7%-4.4% (within assays) and 4.7%-6.0% (between assays). Cortisol values determined by the present method correlated well with those determined by radioimmunoassay. The present method of enzyme-labelled immunoassay is suitable for routine clinical analysis of plasma cortisol.

beta-Alanyltyrosine derivative of 2',5'-tetraadenylate 5'-triphosphate, pppA2'p5'A2'-p5'A2'p5'A-beta-Ala-Tyr was prepared by coupling of periodate-oxidized pppA2'p5'-A2'p5'A2'p5'A with beta-alanyltyrosine methyl ester, followed by reduction with sodium cyanoborohydride. Its stability to 2',5'-phosphodiesterase and phosphatase was investigated in mouse L cell extract. The 5'-triphosphate of the compound was cleaved gradually to form the 5'-dephosphorylated derivative, A2'p5'A2'p5'A2'p5'A-beta-Ala-Tyr, followed by slow degradation of the 2',5'-phosphodiester bond. On the other hand, pppA2'p5'A2'p5'A2'p5'A was hydrolyzed very quickly under the same conditions. The tetramer derivative bound tightly to the 2',5'-oligoadenylate-dependent endoribonuclease in rabbit reticulocyte lysate or mouse L cell extract and inhibited protein synthesis of mouse L cells more effectively than the unmodified 2',5'-tetraadenylate 5'-triphosphate. The corresponding trimer derivative had slightly weaker activities than the unmodified trimer for binding to the endoribonuclease and for inhibition of protein synthesis. The compound, pppA2'p5'A2'p5'-A2'p5'A-beta-Ala-Tyr, was iodinated easily at the tyrosine residue with 125I, giving a high-specific-radioactivity derivative which was used as a radio-labeled probe in a radiobinding assay for 2',5'-oligoadenylate.

A novel 125I-labeled derivative of 2',5'-triadenylate 5'-triphosphate, pppA2'p5'A2'p5'A, with high specific radioactivity was synthesized by coupling of periodate-oxidized pA2'p5'A2'p5'A with beta-alanyltyrosine methyl ester followed by 5'-triphosphorylation and iodination with 125I. Antisera toward 2',5'-oligoadenylate 5'-triphosphate were produced in rabbits by immunization with the conjugate of pppA2'p5'A2'p5'A2'p5'A with bovine serum albumin, and an antiserum with high specificity and high sensitivity for 2',5'-oligoadenylates was selected and tested extensively. Radioimmuno assaying of 2',5'-oligoadenylates was carried out by a competitive double antibody method in which the amount of the antibody bound to the 125I-labeled probe was measured after precipitation with goat anti-rabbit IgG. The concentration of pppA2'p5'A2'p5'A required for 50% inhibition of the binding between the antiserum and the probe was 0.6 nM. The cross reactivity of the antiserum with the 3',5'-triadenylate was more than 10,000 times weaker compared to in the case of 2',5'-oligoadenylates. Very low or no cross reaction was observed with ATP, AMP, and adenosine. The radioimmuno assay using the 125I-labeled compound and the antiserum allows the direct analysis of 2',5'-oligoadenylates in the range of 4 fmol to 1 pmol (0.04-10 nM in a 100 microliter sample). This assay was applied to the measurement of the activity of 2',5'-oligoadenylate synthetase in cells stimulated by interferon. The properties of the 125I-labeled derivative of pppA2'p5'A2'p5'A are described.

We describe a sensitive, simple, and rapid two-site immunoenzymometric assay for thyrotropin in dried blood samples on filter paper, for use in screening for neonatal primary hypothyroidism. In this method, two dried-blood spots of 3 mm diameter (equivalent to about 5.4 microliter of blood) are incubated overnight with anti-thyrotropin-beta-D-galactosidase complex in an anti-thyrotropin coated tube. Then the enzyme activity in the washed tube is determined fluorophotometrically. The range of thyrotropin measurable is 10 to 160 milli-int. units/L blood. Values for thyrotropin in dried blood samples determined by this method and those determined by radioimmunoassay correlated highly (r = 0.96).

We have developed a highly sensitive radioimmunoassay of thyroxine and thyrotropin for mass screening for neonatal hypothyroidism. This assay involves a single disc (3 mm diameter) of dried blood on filter paper. The minimum detectable concentrations are 15 pg/tube (10 microgram/L) for thyroxine and 15 nano-int. units/tube (10 milli-int. units/L) for thyrotropin; intra- and interassay CV's are less than 15% in both assays. The high sensitivity of this method is due to use of labeled thyroxine with high specific activity (3 kCi/g) and of an anti-thyrotropin serum with high affinity (Keq = 7.8 x 10(11) L/mol). With this method, 11337 newborns were screened; a follow-up study revealed that only newborns with both high thyrotropin and low thyroxine concentrations had permanent hypothyroidism. We conclude that this method is sensitive, simple, and reliable and that the recall rate with this method is much lower than that of tests for measuring thyroxine or thyrotropin alone.