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Backgroud Aims: Regenerative therapies employing cell therapy products (CTPs) have attracted considerable attention. Biodistribution (BD) evaluation of CTPs is mainly performed to clarify the cell survival time, engraftment, and distribution site. This evaluation is crucial for predicting the efficacy and safety profiles of clinical studies based on non-clinical BD study outcomes. However, no internationally unified method has been established for assessing cell BD after administration. Here, we aimed to standardize the BD assay method used for CTPs, conducting the following evaluations using the same protocol across multiple study facilities: (1) in vitro validation of quantitative polymerase chain reaction (qPCR) and droplet digital PCR (ddPCR) analyses using the primate-specific Alu gene, and (2) in vivo BD studies after the intravenous administration of human mesenchymal stem cells (hMSCs) to immunodeficient mice, commonly used in non-clinical tumorigenicity studies., Methods: Quality control samples were prepared and analyzed by adding a fixed number of human-derived cells to several mouse tissues. The respective quantitative performances of the qPCR and ddPCR methods were compared for accuracy and precision. hMSCs were intravenously administered to immunodeficient mice, and tissues were collected at 1, 4, and 24 h after administration., Results: Both methods demonstrated an accuracy (relative error) generally within ±50% and a precision (coefficient of variation) generally less than 50%. While differences in calibration curve ranges were observed between qPCR and ddPCR, no significant differences in quantification were found among the assay facilities. The BD of hMSCs in mice was evaluated at seven facilities (qPCR at three facilities; ddPCR at four facilities), revealing similar tissue distribution profiles in all facilities, with the lungs showing the highest cell distribution among the tissues tested., Conclusions: Quantitative evaluation of qPCR and ddPCR using Alu sequences was conducted, demonstrating that the test method can be adapted for BD evaluation., Competing Interests: Declarations of competing interest The authors have no commercial, proprietary or financial interest in the products or companies described in this article except for YY, who is an employee of Bio-Rad Laboratories., (Copyright © 2024 International Society for Cell & Gene Therapy. Published by Elsevier Inc. All rights reserved.)

The DHCR7 enzyme converts 7-DHC into cholesterol. Mutations in DHCR7 can block cholesterol production, leading to abnormal accumulation of 7-DHC and causing Smith-Lemli-Opitz syndrome (SLOS). SLOS is an autosomal recessive disorder characterized by multiple malformations, including microcephaly, intellectual disability, behavior reminiscent of autism, sleep disturbances, and attention-deficit/hyperactivity disorder (ADHD)-like hyperactivity. Although 7-DHC affects neuronal differentiation in ex vivo experiments, the precise mechanism of SLOS remains unclear. We generated Dhcr7 deficient (dhcr7 -/- ) zebrafish that exhibited key features of SLOS, including microcephaly, decreased neural stem cell pools, and behavioral phenotypes similar to those of ADHD-like hyperactivity. These zebrafish demonstrated compromised myelination, synaptic anomalies, and neurotransmitter imbalances. The axons of the dhcr7 -/- zebrafish showed increased lysosomes and attenuated autophagy, suggesting that autophagy-related neuronal homeostasis is disrupted., Competing Interests: Declaration of competing interest All authors declare that they have no conflicts of interest regarding the contents of this article., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

V-ATPase is an ATP hydrolysis-driven proton pump involved in the acidification of intracellular organelles and systemic acid-base homeostasis through H + secretion in the renal collecting ducts. V-ATPase dysfunction is associated with hereditary distal renal tubular acidosis (dRTA). ATP6V1B1 encodes the B1 subunit of V-ATPase that is integral to ATP hydrolysis and subsequent H + transport. Patients with pathogenic ATP6V1B1 mutations often exhibit an early onset of sensorineural hearing loss. However, the mechanisms underlying this association remain unclear. We employed morpholino oligonucleotide-mediated knockdown and CRISPR/Cas9 gene editing to generate Atp6v1ba-deficient (atp6v1ba -/- ) zebrafish as an ortholog model for ATP6V1B1. The atp6v1ba -/- zebrafish exhibited systemic acidosis and significantly smaller otoliths compared to wild-type siblings. Moreover, deficiency in Atp6v1ba led to degeneration of inner ear hair cells, with ultrastructural changes indicative of autophagy. Our findings indicate a critical role of ATP6V1B1 in regulating lysosomal pH and autophagy in hair cells, and the results provide insights into the pathophysiology of sensorineural hearing loss in dRTA. Furthermore, this study demonstrates that the atp6v1ba -/- zebrafish model is a valuable tool for further investigation into disease mechanisms and potential therapies for acidosis-related hearing impairment., Competing Interests: Declaration of competing interest All authors declare that they have no conflicts of interest regarding the contents of this article., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Vaccinia-related kinase 1 (VRK1) is a serine/threonine kinase, for which mutations have been reported cause to neurodegenerative diseases, including spinal muscular atrophy, characterized by microcephaly, motor dysfunction, and impaired cognitive function, in humans. Partial Vrk1 knockdown in mice has been associated with microcephaly and impaired motor function. However, the pathophysiological relationship between VRK1 and neurodegenerative disorders and the precise mechanism of VRK1-related microcephaly and motor function deficits have not been fully investigated. To address this, in this study, we established vrk1-deficient (vrk1 -/- ) zebrafish and found that they show mild microcephaly and impaired motor function with a low brain dopamine content. Furthermore, vrk1 -/- zebrafish exhibited decreased cell proliferation, defects in nuclear envelope formation, and heterochromatin formation in the brain. To our knowledge, this is the first report demonstrating the important role of VRK1 in microcephaly and motor dysfunction in vivo using vrk1 -/- zebrafish. These findings contribute to elucidating the pathophysiological mechanisms underlying VRK1-mediated neurodegenerative diseases associated with microcephaly., Competing Interests: Declaration of competing interest The authors declare no conflict of interests., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Background: Gamma knife (GK) thalamotomy has been used as a treatment option for essential tremor (ET). Numerous studies on GK use in ET treatment have reported more varied responses and complication rates., Methods: Data from 27 patients with ET who underwent GK thalamotomy were retrospectively analyzed. The Fahn-Tolosa-Marin Clinical Rating Scale for Tremor, handwriting, and spiral drawing were evaluated. Postoperative adverse events and magnetic resonance imaging findings were also evaluated., Results: The mean age at GK thalamotomy was 78.1 ± 4.2 years. The mean follow-up period was 32.5 ± 19.4 months. The preoperative postural tremor, handwriting, and spiral drawing scores were 3.4 ± 0.6, 3.3 ± 1.0, and 3.2 ± 0.8, respectively, all of which showed significant improvements to 1.5 ± 1.2 (55.9% improvement, P < 0.001), 1.4 ± 1.1 (57.6% improvement, P < 0.001), and 1.6 ± 1.3 (50% improvement, P < 0.001), respectively, at the available final follow-up evaluations. Three patients presented with no improvement in tremor. Six patients presented with adverse effects, including complete hemiparesis, foot weakness, dysarthria, dysphagia, lip numbness, and finger numbness, at the final follow-up period. Two patients presented with serious complications, including complete hemiparesis due to massive widespread edema and chronic encapsulated expanding hematoma. One patient died of aspiration pneumonia following severe dysphagia secondary to chronic encapsulated expanding hematoma., Conclusions: GK thalamotomy is an efficient procedure for treating ET. Careful treatment planning is necessary to reduce complication rates. The prediction of radiation complications will increase the safety and effectiveness of GK treatment., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Study Objective: Hysteroscopic surgery criteria for patients with cesarean scar defect (CSD) are unclear. Therefore, this study aimed to explore the indication of hysteroscopic surgery for secondary infertility owing to CSD., Design: Retrospective cohort study., Setting: Single university hospital., Patients: Seventy patients with secondary infertility owing to symptomatic CSD who underwent hysteroscopic surgery under laparoscopy between July 2014 and February 2022 were included., Interventions: Clinical data, including basic patient information, preoperative residual myometrial thickness (RMT), and postoperative pregnancy status, were collected from medical records. Patients were divided into postoperative pregnancy and nonpregnancy groups. A receiver operating characteristic curve was drawn, and the optimal cutoff value was calculated based on the area under the curve to predict pregnancy after hysteroscopic surgery., Measurements and Main Results: No complications were observed in any cases. Among the 70 patients, 49 patients (70%) became pregnant after hysteroscopic surgery. There was no significant difference in patient characteristics between the pregnancy and nonpregnancy groups. In the receiver operating characteristic curve analysis for patients aged <38 years, the value of the area under the curve was 0.77 (sensitivity, 0.83; specificity, 0.78) when optimal cutoff of RMT was 2.2 mm. There was a significant difference in preoperative RMT between the pregnancy and nonpregnancy groups (3.3 mm and 1.7 mm, respectively) in patients aged <38 years., Conclusion: For RMT ≥2.2 mm, hysteroscopic surgery was reasonable for secondary infertility owing to symptomatic CSD, particularly in patients aged <38 years., (Copyright © 2023 AAGL. Published by Elsevier Inc. All rights reserved.)

Autosomal recessive primary microcephaly (MCPH) is a rare congenital disorder characterized by a below average brain volume at birth and is associated with neurodevelopmental disorders such as growth retardation and intellectual disability. Mutations in ANKLE2 have been identified as one of the causes of MCPH (MCPH16). ANKLE2 is a target molecule of the Zika virus NS4a protein that interferes with ANKLE2 function, resulting in severe microcephaly. ANKLE2 is essential for organizing the nuclear envelope and chromatin structures during the mitotic-end process via barrier to autointegration factor (BAF) dephosphorylation. However, the precise mechanism by which the loss of ANKLE2 function causes the pathogenesis of microcephaly remains unclear. In this study, we generated Ankle2-deficient zebrafish (ankle2 -/- ) with a significant reduction in brain size compared with that of their control siblings. The ankle2 -/- brain showed a significant decrease in the number of radial glial progenitor cells, suggesting that Ankle2 deficiency in zebrafish causes neurogenesis defects. Furthermore, ankle2 -/- male zebrafish showed infertility owing to defects in spermatogenesis. Notably, microcephaly was overcome by vrk1 morpholino knockdown or vrk1 heterozygous deletion. In addition, spermatogenesis in ankle2 -/- zebrafish males was partially restored by the vrk1 heterozygous deletion, although infertility was not resolved. These results indicate that ANKLE2 and VRK1 coordinate with each other for BAF phosphorylation to maintain normal mitosis during neurogenesis and spermatogenesis., Competing Interests: Declaration of competing interest The authors declare no conflicts of interest., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Cleavage factor polyribonucleotide kinase subunit 1 (CLP1), an RNA kinase, plays essential roles in protein complexes involved in the 3'-end formation and polyadenylation of mRNA and the tRNA splicing endonuclease complex, which is involved in precursor tRNA splicing. The mutation R140H in human CLP1 causes pontocerebellar hypoplasia type 10 (PCH10), which is characterized by microcephaly and axonal peripheral neuropathy. Previously, we reported that RNA fragments derived from isoleucine pre-tRNA introns (Ile-introns) accumulate in fibroblasts of patients with PCH10. Therefore, it has been suggested that this intronic RNA fragment accumulation may trigger PCH10 onset. However, the molecular mechanism underlying PCH10 pathogenesis remains elusive. Thus, we generated knock-in mutant mice that harbored a CLP1 mutation consistent with R140H. As expected, these mice showed progressive loss of the upper motor neurons, resulting in impaired locomotor activity, although the phenotype was milder than that of the human variant. Mechanistically, we found that the R140H mutation causes intracellular accumulation of Ile-introns derived from isoleucine pre-tRNAs and 5' tRNA fragments derived from tyrosine pre-tRNAs, suggesting that these two types of RNA fragments were cooperatively or independently involved in the onset and progression of the disease. Taken together, the CLP1-R140H mouse model provided new insights into the pathogenesis of neurodegenerative diseases, such as PCH10, caused by genetic mutations in tRNA metabolism-related molecules., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Exosc2 is one of the components of the exosome complex involved in RNA 3' end processing and degradation of various RNAs. Recently, EXOSC2 mutation has been reported in German families presenting short stature, hearing loss, retinitis pigmentosa, and premature aging. However, the in vivo function of EXOSC2 has been elusive. Herein, we generated Exosc2 knockout (exosc2 -/- ) zebrafish that showed larval lethality 13 days post fertilization, with microcephaly, loss of spinal motor neurons, myelin deficiency, and retinitis pigmentosa. Mechanistically, Exosc2 deficiency caused impaired mRNA turnover, resulting in a nucleotide pool imbalance. Rapamycin, which modulated mRNA turnover by inhibiting the mTOR pathway, improved nucleotide pool imbalance in exosc2 -/- zebrafish, resulting in prolonged survival and partial rescue of neuronal defects. Taken together, our findings offer new insights into the disease pathogenesis caused by Exosc2 deficiency, and might help explain fundamental molecular mechanisms in neuronal diseases, such as Alzheimer's disease, amyotrophic lateral sclerosis, and spinal muscular atrophy., Competing Interests: Declaration of competing interest The authors declare no conflicts of interest., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Background: The endoscopic endonasal approach (EEA) has been accepted as an alternative option for diseases at the craniovertebral junction. However, the inferior destination through the endoscopic endonasal approach is anatomically higher than that of the transoral approach. Therefore, preoperative assessment of accessibility is mandatory for appropriate selection of indication. Using a navigation system, we examined the inferior limit through the endonasal route and evaluated the relationships between surrounding anatomicl structures and the lowest point., Methods: This study included patients who underwent endoscopic transsphenoidal surgery for intrasellar lesions at our hospital (N = 23). At the start of surgery, the lowest point (target point [TP]) was marked with a straight probe under guidance of the navigation system. We measured 4 parameters on preoperative computed tomography: nasal length, hard palate length, anterior-posterior diameter of the nasopharynx, and nasopalatine angle. Patients were classified into groups depending on whether the TP was at or above (group A) or below (group B) the hard palatine line., Results: TPs were above the hard palatine line in 15 patients (group A) and below the hard palatine line in 8 patients (group B). No TPs reached the nasopalatine line. Nasal length (P = 0.03) and nasopalatine angle (P = 0.01) were larger in group B than in group A. There were no significant differences in anterior-posterior diameter of the nasopharynx or hard palate length., Conclusions: The hard palatine line is a reliable parameter for assessing the inferior limit of the endoscopic endonasal approach. Nostril size affects accessibility with surgical instruments., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Fragments of transfer RNA (tRNA), derived either from pre-tRNA or mature tRNA, have been discovered to play an essential role in the pathogenesis of various disorders such as neurodegenerative disease. CLP1 is an RNA kinase involved in tRNA biogenesis, and mutations in its encoding gene are responsible for pontocerebellar hypoplasia type-10. Mutation of the CLP1 gene results in the accumulation of tRNA fragments of several different kinds. These tRNA fragments are expected to be associated with the disease pathogenesis. However, it is still unclear which of the tRNA fragments arising from the CLP1 gene mutation has the greatest impact on the onset of neuronal disease. We found that 5' tRNA fragments derived from tyrosine pre-tRNA (5' Tyr-tRF) caused p53-dependent neuronal cell death predominantly more than other types of tRNA fragment. We also showed that 5' Tyr-tRF bound directly to pyruvate kinase M2 (PKM2). Injection of zebrafish embryos with PKM2 mRNA ameliorated the neuronal defects induced in zebrafish embryos by 5' Tyr-tRF. Our findings partially uncovered a mechanistic link between 5' Tyr-tRF and neuronal cell death that is regulated by PKM2., Competing Interests: Declaration of competing interest The authors declare no conflict of interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)

CLP1 plays an essential role in the protein complex involved in mRNA 3'-end formation and polyadenylation as well as in the tRNA splicing endonuclease (TSEN) complex involved in the splicing of precursor tRNAs. NOL9 localizes in the nucleolus of cells and plays an essential role in ribosomal RNA maturation. Both CLP1 and NOL9 are RNA kinases that phosphorylate the 5' end of RNAs. From the evidence that phosphorylation of the 5' end of a siRNA is essential for its efficient RNA cleavage, it was expected that CLP1 and NOL9 would be corresponding molecules. However, there had been no direct evidence that this is the case. In this study, murine NOL9 showed no apparent RNA kinase activity in cells or even in an RNA kinase assay using recombinant murine NOL9 protein. Although siRNA efficiency was decreased in CLP1 kinase-dead (Clp1 K/K ) cells, it was not influenced by NOL9 overexpression. These findings indicate that in mouse cells it is CLP1 that mainly acts to phosphorylate the 5' end of RNAs in the siRNA pathway, with no apparent involvement of NOL9., Competing Interests: Declaration of competing interest The authors declare no conflict of interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)

KIF13B, a kinesin-3 family motor, was originally identified as GAKIN due to its biochemical interaction with human homolog of Drosophila discs-large tumor suppressor (hDLG1). Unlike its homolog KIF13A, KIF13B contains a carboxyl-terminal CAP-Gly domain. To investigate the function of the CAP-Gly domain, we developed a mouse model that expresses a truncated form of KIF13B protein lacking its CAP-Gly domain (KIF13BΔCG), whereas a second mouse model lacks the full-length KIF13A. Here we show that the KIF13BΔCG mice exhibit relatively higher serum cholesterol consistent with the reduced uptake of [ 3 H]CO-LDL in KIF13BΔCG mouse embryo fibroblasts. The plasma level of factor VIII was not significantly elevated in the KIF13BΔCG mice, suggesting that the CAP-Gly domain region of KIF13B selectively regulates LRP1-mediated lipoprotein endocytosis. No elevation of either serum cholesterol or plasma factor VIII was observed in the full length KIF13A null mouse model. The deletion of the CAP-Gly domain region caused subcellular mislocalization of truncated KIF13B concomitant with the mislocalization of LRP1. Mechanistically, the cytoplasmic domain of LRP1 interacts specifically with the alternatively spliced I 3 domain of DLG1, which complexes with KIF13B via their GUK-MBS domains, respectively. Importantly, double mutant mice generated by crossing KIF13A null and KIF13BΔCG mice suffer from perinatal lethality showing potential craniofacial defects. Together, this study provides first evidence that the carboxyl-terminal region of KIF13B containing the CAP-Gly domain is important for the LRP1-DLG1-KIF13B complex formation with implications in the regulation of metabolism, cell polarity, and development., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Malaria and babesiosis are bloodborne protozoan infections for which the emergence of drug-resistant strains poses a threat. Our previous phage display cDNA screens established the essentiality of Plasmodium falciparum signal peptide peptidase (SPP) in asexual development at the blood stage of malaria infection. Given the structural similarities between SPP inhibitors and HIV protease inhibitors, we screened ten HIV protease inhibitors and selected Lopinavir and Atazanavir for their ability to inhibit PfSPP activity. Using a transcription-based assay, we observed that Lopinavir inhibits both parasite-and host-derived SPP activities whereas Atazanavir inhibited only parasite derived SPP activity. Consistent with their inhibitory effect on Plasmodium growth, both Lopinavir and Atazanavir strongly inhibited intraerythrocytic Babesia microti growth ex vivo. Moreover, Lopinavir prevented the steep rise in Babesia microti parasitemia typically observed in rag1-deficient mice. Our data provide first evidence that inhibition of parasite-derived SPPs by HIV protease inhibitors offers a promising therapeutic avenue for the treatment of severe babesiosis and infections caused by other Apicomplexa parasites., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Malaria remains a major global threat to human health and economic development. Microvascular lesions caused by Plasmodium falciparum -infected human erythrocytes/red blood cells are hallmarks of severe pathogenesis contributing to high mortality, particularly in children from sub-Saharan Africa. In this study, we used a phage display complementary DNA library screening strategy to identify P falciparum glutamic acid-rich protein (PfGARP) as a secreted ligand that recognizes an ectodomain of human erythrocyte anion-exchanger, band 3/AE1, as a host receptor. Domain mapping of PfGARP revealed distinct nonoverlapping repeats encoding the immune response epitopes and core erythrocyte-binding activity. Synthetic peptides derived from the erythrocyte-binding repeats of PfGARP induced erythrocyte aggregation reminiscent of the rosetting phenomenon. Using peptides derived from the immunogenic repeats, a quantitative immunoassay was developed to detect a selective immune response against PfGARP in human plasma samples obtained from patients in rural Mali, suggesting the feasibility of PfGARP as a potential biomarker of disease progression. Collectively, our results suggest that PfGARP may play a functional role in enhancing the adhesive properties of human erythrocytes by engaging band 3 as a host receptor. We propose that immunological and pharmacological inhibition of PfGARP may unveil new therapeutic options for mitigating lesions in cerebral and pregnancy-associated malaria., (© 2019 by The American Society of Hematology.)

Dematin is a relatively low abundance actin binding and bundling protein associated with the spectrin-actin junctions of mature erythrocytes. Primary structure of dematin includes a loosely folded core domain and a compact headpiece domain that was originally identified in villin. Dematin's actin binding properties are regulated by phosphorylation of its headpiece domain by cyclic adenosine monophosphate-dependent protein kinase. Here, we used a novel gene disruption strategy to generate the whole body dematin gene knockout mouse model (FLKO). FLKO mice, while born at a normal Mendelian ratio, developed severe anemia and exhibited profound aberrations of erythrocyte morphology and membrane stability. Having no apparent effect on primitive erythropoiesis, FLKO mice show significant enhancement of erythroblast enucleation during definitive erythropoiesis. Using membrane protein analysis, domain mapping, electron microscopy, and dynamic deformability measurements, we investigated the mechanism of membrane instability in FLKO erythrocytes. Although many membrane and cytoskeletal proteins remained at their normal levels, the major peripheral membrane proteins spectrin, adducin, and actin were greatly reduced in FLKO erythrocytes. Our results demonstrate that dematin plays a critical role in maintaining the fundamental properties of the membrane cytoskeleton complex., (© 2016 by The American Society of Hematology.)

Plasmodium falciparum invasion of human red blood cells (RBCs) is an intricate process requiring a number of distinct ligand-receptor interactions at the merozoite-erythrocyte interface. Merozoite surface protein 1 (MSP1), a highly abundant ligand coating the merozoite surface in all species of malaria parasites, is essential for RBC invasion and considered a leading candidate for inclusion in a multiple-subunit vaccine against malaria. Our previous studies identified an interaction between the carboxyl-terminus of MSP1 and RBC band 3. Here, by employing phage display technology, we report a novel interaction between the amino-terminus of MSP1 and RBC glycophorin A (GPA). Mapping of the binding domains established a direct interaction between malaria MSP1 and human GPA within a region of MSP1 known to potently inhibit P falciparum invasion of human RBCs. Furthermore, a genetically modified mouse model lacking the GPA- band 3 complex in RBCs is completely resistant to malaria infection in vivo. These findings suggest an essential role of the MSP1-GPA-band 3 complex during the initial adhesion phase of malaria parasite invasion of RBCs., (© 2015 by The American Society of Hematology.)

Introduction: The purpose of this study was to investigate the ability of swept-source optical coherence tomographic (SS-OCT) imaging to detect a second mesiobuccal canal (MB2) in maxillary molars compared with visual inspection (VI) and dental operating microscopy (DOM)., Methods: Forty extracted human maxillary molars were examined. After the removal of half the crown and access cavity preparation, the existence of MB2 canals was confirmed based on cross-sectional images of each tooth obtained by micro-computed tomographic scanning as the reference standard. Then, the pulp chamber floor was scanned by SS-OCT imaging. Three dentists independently evaluated the SS-OCT images and the pulp chamber floor under VI and DOM for the presence of MB2 canals. The detection rate of MB2 canals for SS-OCT imaging, VI, and DOM was calculated, and statistical analysis was performed., Results: MB2 canals existed in 19 of 40 teeth (47.5%) using micro-CT imaging. Sensitivity of DOM (0.947) was significantly higher than that of SS-OCT imaging (0.632). Specificity of SS-OCT imaging (0.714) was significantly higher than that of DOM (0.333). No statistically significant differences were found for accuracy among the 3 methods. Kappa values of SS-OCT, VI, and DOM were 0.526, 0.417, and 0.326, respectively., Conclusions: SS-OCT imaging is noninvasive, involves no ionizing radiation, and is accurate for the detection of MB2 canals., (Copyright © 2014 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.)

Purpose: This study explored the pharmacodynamic and pharmacokinetic effects of combining perampanel (PER) with commonly co-administered AEDs., Method: A strong stimulus intensity (three-fold higher than after-discharge threshold) was used to elicit drug-resistant seizures in a rat amygdala kindling model. Vehicle, low-dose PER (0.75 mg/kg), or high-dose PER (1.5mg/kg), in combination with vehicle, levetiracetam (LEV) 50mg/kg, lamotrigine (LAM) 20mg/kg, carbamazepine (CBZ) 20mg/kg, or valproic acid (VPA) 200mg/kg, were administered intraperitoneally to groups of 6-13 rats. Seizure score, electroencephalography (EEG) seizure duration, and motor seizure duration were evaluated, with pharmacodynamic interactions determined by two-way analysis of variance (ANOVA). Motor impairment was evaluated by rotarod test and two-way ANOVA., Results: High-dose PER, but not low-dose PER, LEV, LAM, CBZ, or VPA, reduced EEG seizure duration, motor seizure duration, and seizure score compared with vehicle alone. However, when low-dose PER was administered in combination with LEV, LAM, CBZ, or VPA, seizure severity parameters were reduced compared with the concomitant AEDs alone. These pharmacodynamic interactions were statistically significant in some cases, but the same AED combinations were not associated with statistically significant neurotoxic interactions. Efficacy may have been slightly affected by changes in PER plasma concentrations in the presence of other AEDs:PER plasma concentrations increased with LEV or LAM co-administration, and decreased with CBZ or VPA co-administration., Conclusion: Overall, these data support published Phase III data demonstrating the efficacy of PER as adjunctive therapy for the treatment of refractory partial-onset seizures in patients aged ≥ 12 years., (Copyright © 2014 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Central post-stroke pain (CPSP) including thalamic pain is one of the most troublesome sequelae that can occur after a cerebrovascular accident. Although the prevalence of CPSP among stroke patients is relatively low, the persistent, often treatment-refractory, painful sensations can be a major problem and decrease the affected patient's quality of life. To better understand of the pathophysiological basis of CPSP, we developed and characterized a new mouse model of thalamic CPSP. This model is based on a hemorrhagic stroke lesion with collagenase in the ventral posterolateral nucleus of the thalamus. Histopathological analysis indicated that the thalamic hemorrhage produced a relatively confined lesion that destroys the tissue within the initial bleed, and also showed the presence of activated microglia adjacent to the core of hemorrhagic lesions. Behavioral analysis demonstrated that the animals displayed diclofenac-, morphine- or pregabalin-resistant mechanical allodynia and thermal hyperalgesia of the hind paw contralateral to the lesion for over 112 days. However, we found that minocycline, a microglial inhibitor, significantly ameliorated mechanical allodynia and thermal hyperalgesia. These results suggest that this model might be proved as a useful animal model for studying the neuropathology of thalamic syndrome, and developing improved therapeutics for CPSP., (Copyright © 2013 Elsevier Ireland Ltd and the Japan Neuroscience Society. All rights reserved.)

Two cases of spontaneous epidural emphysema that occurred during asthmatic attacks in a 13-year-old and a 15-year-old are reported here. Epidural emphysema was diagnosed in both cases by using computed tomography (CT), and in 1 case by using magnetic resonance imaging (MRI). Neither patient had neurological findings. Both patients were discharged with no respiratory difficulties. It is generally believed that a diagnosis of epidural emphysema can only be made on CT. In this report, MRI was used to make the diagnosis of subdural emphysema, and it demonstrated that the air was localized within the epidural fat., (2012 The Japanese Respiratory Society. Published by Elsevier B.V. All rights reserved)

The effect of aging on the number, size, conduction velocity, and vasoconstrictive function of unmyelinated fibers in ovarian nerve accompanying the ovarian artery was studied in adult (4-7mo) and aged (28-31mo) rats. Morphological observation by electron microscopy showed that the ovarian nerve contains mainly unmyelinated fibers with only a small percentage (less than 4%) of myelinated fibers in either age group. The number of unmyelinated fibers tended to decrease in aged rats (717±59) compared to adult rats (801±48), especially in fibers of smaller diameter, although this difference was not statistically significant. The maximum conduction velocity of unmyelinated fibers within the ovarian nerve was similar when compared between adult (1.05±0.04m/s) and aged (1.02±0.05m/s) rats. Under anesthesia, electrical stimulation of the distal portion of a severed ovarian nerve reduced ovarian blood flow, as measured by laser Doppler flowmetry, when the stimulus intensity was above the threshold for unmyelinated C fibers. Stimulation of the ovarian nerve with supra-maximum intensity (10V) at 2-20Hz frequencies produced frequency-dependent reductions in ovarian blood flow in both adult and aged rats. There were no significant differences in magnitude of the reduction in ovarian blood flow with comparable frequencies of electrical stimulation of the ovarian nerve between adult and aged rats. Collectively, these data indicate that unmyelinated C fibers in ovarian nerve are maintained in number, size, conduction ability, and vasoconstrictor function in aged rats., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Monodispersed molecularly imprinted polymer particles selective for cholesterol were prepared by the copolymerization of styrene and divinylbenzene in the presence of template silica gel particles (particle size: 5 μm; pore size: 10 nm) functionalized with cholesterol on the surface, followed by dissolution of the cholesterol-bonded silica gel with a NaOH aqueous solution. Transmission and scanning electron micrographs of the molecularly imprinted polymer (MIP) particles revealed good monodispersity and porous structure. The MIP particles were packed into a high performance liquid chromatographic column, and its recognition ability of cholesterol was evaluated using cholesterol, cholesterol esters and fatty acid methyl esters by comparison with the non-imprinted polymer (NIP) particles prepared from styrene and divinylbenzene without cholesterol. The MIP particles showed a high affinity for cholesterol and cholesterol esters (K(MIP)'/K(NIP)' > 5.7)., (Copyright © 2010 Elsevier B.V. All rights reserved.)

For the separation of aromatic amines, two types of monodispersed porous polymer resins were prepared by the copolymerization of 2-vinylpyridine and 4-vinylpyridine with divinylbenzene in the presence of template silica gel particles (particle size 5 microm), followed by dissolution of the template silica gel in an alkaline solution. The transmission electron micrographs and the scanning electron micrograph revealed that these templated polymer resins have a spherical morphology with a good monodispersity and porous structure. Using these monodispersed polymer resins, the high-performance liquid chromatographic separation of aromatic amines in the mobile phases of pHs 2.0, 2.9, 4.1, 7.2 and 11.7 were carried out. The 2-vinylpyridine-divinylbenzene copolymer resins showed slightly stronger retentions for aromatic amines than the 4-vinylpyridine-divinylbenzene copolymer resins. Under acidic conditions (around pH 2.0), aniline and the toluidines showed no retention on these copolymer resins due to the repulsion between the cationic forms of these amines and pyridinium cations in the stationary phase, whereas less basic aromatic amines or non-basic acetanilide showed slight retentions. Above pH 4.1, the separation of aromatic amines with these polymer resins showed a typical reversed-phase mode separation. Therefore, the separation patterns of aromatic amines are effectively tunable by changing the pH value of the mobile phases. A good separation of eight aromatic amines was achieved at pH 2.9 using the 2-vinylpyridine-divinylbenzene copolymer resins.

Using an HPLC column packed with monodispersed vinylbenzeneboronic acid-divinylbenzene (V-D) copolymer resins, the elution behaviors of the mono- and disaccharides were studied under different pH mobile phases. The monodispersed V-D copolymer resins were prepared by the copolymerization of 4-vinylbenzeneboronic acid and divinylbenzene in the presence of template silica gel particles (particle size: 5 microm; pore size: 10 nm), followed by dissolution of the template silica gel using a NaOH solution. Similarly, styrene-divinylbenzene (S-D) copolymer resins as the control resins were also synthesized. The transmission electron micrographs of these polymer resins revealed a good monodispersity. The complexation behavior of the saccharides was evaluated by comparison of the peak area eluted through the V-D column for that through the S-D column. Four aldopentoses (D-ribose, D-arabinose, D-xylose, and D-lyxose) and four aldohexoses (D-glucose, D-mannose, D-galactose, and D-talose) were retained completely at pH 11.9. Especially, ribose and talose were totally retained even under acidic and neutral conditions. For the disaccharides, unlike sucrose and maltose, palatinose was completely retained in basic mobile phases.

Introduction: The murine local lymph node assay (LLNA) is a well-established alternative to the guinea pig maximization test (GPMT) or Buehler test (BT) for the assessment of the skin sensitizing ability of drugs and chemicals. Daicel Chemical Industries Ltd. has developed a modified LLNA based on the adenosine triphosphate (ATP) content (LLNA-DA). We conducted 2 interlaboratory validation studies to evaluate the reliability and relevance of LLNA-DA., Methods: The experiment involved 17 laboratories, wherein 14 chemicals were examined under blinded conditions. In the first study, 3 chemicals were examined in 10 laboratories and the remaining 9 were examined in 3 laboratories. In the second study, 1 chemical was examined in 7 laboratories and the remaining 4 chemicals were examined in 4 laboratories. The data were expressed as the ATP content for each chemical-treated group, and the stimulation index (SI) for each chemical-treated group was determined as the increase in the ATP content relative to the concurrent vehicle control group. An SI of 3 was set as the cut-off value for exhibiting skin sensitization activity., Results: The results of the first study obtained in the experiments conducted for the 3 chemicals that were examined in all the 10 laboratories and for 5 of the remaining 9 chemicals were sufficiently consistent with small variations in their SI values. The sensitivity, specificity, and accuracy of LLNA-DA against those of GPMT/BT were 7/8 (87.5%), 3/3 (100%), and 10/11 (90.9%), respectively. In the second study, all the 5 chemicals studied demonstrated acceptably small interlaboratory variations., Discussion: In the first study, a large variation was observed for 2 chemicals; in the second study, this variation was small. It was attributed to the application of dimethylsulfoxide as the solvent for the metallic salts. In conclusion, these 2 studies provide good evidence for the reliability of the LLNA-DA.

Objective: We have previously reported that intravesical oxybutynin chloride with hydroxypropylcellulose (modified intravesical oxybutynin) is an effective therapeutic agent for patients with detrusor overactivity. In this study, we report on the efficacy, safety and side effects of modified intravesical oxybutynin administration in children with neurogenic bladder., Patients: Modified intravesical oxybutynin (1.25mg/5 mL, twice a day) was administered to four children (three males and one female) with neurogenic bladder (detrusor overactivity and/or low compliance bladder), who were previously unresponsive to or experienced intolerable side effects from oral medications. A cystometrogram was obtained before, 1 week after, and 1 year after the first intravesical instillation of modified oxybutynin. We also carefully observed anticholinergic side effects, occurrence of urinary tract infection and degree of incontinence during this treatment., Results: After 1 week, both cystometric bladder capacity and compliance were improved in all patients, and detrusor overactivity was undetectable in three of four patients. At 1 year, there was further improvement in bladder compliance in three patients, and detrusor overactivity was not observed in two patients. Significant improvement in the degree of incontinence was achieved. No systemic anticholinergic side effects were observed in any of the patients. One patient with vesicoureteral reflux discontinued the therapy after 2 months due to upper urinary tract infections., Conclusion: Modified intravesical oxybutynin is an effective and relatively safe therapeutic option for children with neurogenic bladders.

We found reduced locomotor activity (LA) under fasting in systemic carnitine-deficient juvenile visceral steatosis (jvs(-/-)) mice. When food was withdrawn at 8:00 a.m. (lights-off at 7:00 p.m., 12h/cycle), the nocturnal LA of jvs(-/-) mice was much less than the control (jvs(+/+) and jvs(+/-)) mice. LA recovered under carnitine or sucrose administration, but not under medium-chain triglyceride. In addition, fasted jvs(-/-) mice, without any energy supply, were activated by modafinil, a stimulator of the dopamine pathway. These results suggest that the reduced LA is not adequately explained by energy deficit. As the fasted jvs(-/-) mice showed lower body core temperature (BT), we examined the central nervous system regulating LA and BT. We found lower percentage of c-Fos positive orexin neurons in the lateral hypothalamus and reduced orexin-A concentration in the cerebrospinal fluid of fasted jvs(-/-) mice. Sleep analysis revealed that fasted jvs(-/-) mice had disruption of prolonged wakefulness, with a higher frequency of brief episodes of non-REM sleep during the dark period than fasted jvs(+/+) mice. These results strongly suggest that the reduced LA in fasted jvs(-/-) mice is related to the inhibition of orexin neuronal activity.

Plasma von Willebrand factor (VWF) has been identified as an indispensable factor for platelet adhesion and thrombus formation on a collagen surface under flow conditions. VWF binds to collagen and then tethers platelets to the collagen surface through interaction with platelet glycoprotein Ib and also contributes to the thrombus formation on the collagen surface. In the present study, we demonstrated that the addition of VWF/factor VIII complex or purified VWF (> 2 ristocetin cofactor activity units/mL) increased platelet adhesion to the collagen surface in platelet-reduced blood ( approximately 5 x 10(4) platelets/microL) to the normal level. VWF had no stimulatory effect when it was allowed to bind to the collagen surface before blood flow was initiated. Addition of an excess of FITC (fluorescein-5-isothiocyanate)-labeled VWF to platelet-reduced blood under these flow conditions demonstrated that the VWF was mainly incorporated into the platelet aggregates. These results indicated that the supplemented VWF stimulates the platelet adhesion onto the collagen surface by enhancing platelet aggregation in the platelet-reduced condition. This also suggests a possibility that supplementation of VWF to individuals with thrombocytopenia might be effective for increasing their hemostatic potential.

The mammalian mitochondrial (mt) ribosome (mitoribosome) is a bacterial-type ribosome but has a highly protein-rich composition. Almost half of the rRNA contained in the bacterial ribosome is replaced with proteins in the mitoribosome. Escherichia coli elongation factor G (EF-G Ec) has no translocase activity on the mitoribosome but EF-G mt is functional on the E.coli ribosome. To investigate the functional equivalency of the mt and E.coli ribosomes, we prepared hybrid mt and E.coli ribosomes. The hybrid mitoribosome containing E.coli L7/12 (L7/12 Ec) instead of L7/12 mt clearly activated the GTPase of EF-G Ec and efficiently promoted its translocase activity in an in vitro translation system. Thus, the mitoribosome is functionally equivalent to the E.coli ribosome despite their distinct compositions. The mt EF-Tu-dependent translation activity of the E.coli ribosome was also clearly enhanced by replacing the C-terminal domain (CTD) of L7/12 Ec with the mt counterpart (the hybrid E.coli ribosome). This strongly indicates that the CTD of L7/12 is responsible for EF-Tu function. These results demonstrate that functional compatibility between elongation factors and the L7/12 protein in the ribosome governs its translational specificity.

Inflammation is progressed by the action of pro-inflammatory cytokines, including IL-1, TNF, IFN-gamma and IL-6, and is resolved by anti-inflammatory cytokines, such as IL-4, IL-10, IL-13, IFN-alpha and transforming growth factor-beta (TGF-beta). Intracellular signal transduction pathways initiated by these cytokines have been studied extensively; these pathways ultimately activate transcription factors, such as NF-kappa B, Smad and STATs. Recently, negative feedback regulation of these pathways, mediated by molecules such as A-20, Smad7 and CIS/SOCS family proteins, has been identified. Negative regulation of cytokine signaling is quite important for inflammation.

Neuromedin U (NMU), a hypothalamic peptide, has been known to be involved in feeding behavior as a catabolic signaling molecule. However, little is known about the participation of NMU in the neuronal network. One NMU receptor, NMU2R, is abundantly expressed in the hypothalamic paraventricular nucleus, where corticotrophin-releasing hormone (CRH) is synthesized. The functions of CRH, regulation of stress response and feeding behavior, are comparable with those of NMU. Here, we have investigated the functional relationships between NMU and CRH using CRH knockout (KO) mice. Intracerebroventricular administration of NMU suppressed dark-phase food intake and fasting-induced feeding in wild-type mice. In contrast, these suppressions were not observed in CRH KO mice. NMU-induced increases in oxygen consumption and body temperature were attenuated in CRH KO mice. These results suggest that NMU plays a role in feeding behavior and catabolic functions via CRH. This study demonstrates a novel hypothalamic pathway that links NMU and CRH in the regulation of feeding behavior and energy homeostasis.

Intracellular transglutaminases (protein-glutamine: amine gamma-glutamyltransferase, EC 2.3.2.13) are calcium-dependent thiol enzymes that catalyze the covalent cross-linking of proteins, including those in the erythrocyte membrane. Several studies suggest that the activation of some transglutaminases is positively regulated by the calcium-dependent cysteine protease, mu-calpain. Using mu-calpain null (Capn1(-/-)) mouse erythrocytes, we demonstrate that the activation of soluble as well as membrane-bound forms of transglutaminase (TG2) in mouse erythrocytes was independent of mu-calpain. Also, the absence of mu-calpain or any detectable cysteine protease did not affect the transglutaminase activity in the erythrocyte lysate. Our studies also identify physiological substrates of mu-calpain in the erythrocyte membrane and show that their cleavage has no discernible effect on the transglutaminase mediated cross-linking of membrane proteins. Taken together, these data suggest the existence of a calpain-independent mechanism for the activation of transglutaminase 2 by calcium ions in the mouse erythrocytes and presumably also in non-erythroid cells.

The suppressor of cytokine signaling/cytokine-inducible SH2 containing proteins are cytokine inducible and are negative regulators of the signal transducers and activators of the transcription signaling pathway. We investigated the mechanism regulating signal transducers and activators of transcription and the suppressor of cytokine signaling/cytokine-inducible SH2 containing protein family in keratinocytes, one of the major target cells for cytokines. Suppressor of cytokine signaling 1 mRNA was upregulated 3 h post-interferon gamma, and a 8.1-fold increase in the suppressor of cytokine signaling 1 mRNA occurred 48 h post-interferon gamma. The suppressor of cytokine signaling 3 mRNA was also upregulated from 1 h post-interferon gamma, and a 6.7-fold increase in the suppressor of cytokine signaling 3/cytokine-inducible SH2 containing protein 3 mRNA occurred between 6 and 12 h post-interferon gamma. Interleukin-6 exposure for 1 h enhanced the expression of the suppressor of cytokine signaling 3/cytokine-inducible SH2 containing protein 3 mRNA, but the suppressor of cytokine signaling 1/JAB mRNA was not induced by interleukin-6. Interleukin-4 upregulated the suppressor of cytokine signaling 1/JAB and cytokine-inducible SH2 containing protein 1 mRNA, with 3.4-fold and 5.1-fold increases in mRNA observed at 1 h post-interleukin-4, respectively. In contrast, epidermal growth factor, which phosphorylates signal transducers and activators of transcription 3, did not influence the level of the suppressor of cytokine signaling/cytokine-inducible SH2 containing protein family mRNA expression. Transfection of an adenovirus vector expressing the suppressor of cytokine signaling 1/JAB completely inhibited interferon gamma-dependent signal transducers and activators of transcription 1 phosphorylation and interleukin-4-dependent signal transducers and activators of transcription 6 phosphorylation. Transfection of adenovirus vector expressing the suppressor of cytokine signaling 1/JAB did not inhibit interleukin-6-dependent signal transducers and activators of transcription 3 phosphorylation-several reports show that the suppressor of cytokine signaling 1/JAB is a potent inhibitor of signal transducers and activators of transcription 3 signaling in the myeloid leukemia M1 cell. Transfection of the adenovirus vector expressing suppressor of cytokine signaling 3/cytokine-inducible SH2 containing protein 3 completely inhibited interleukin-6-dependent signal transducers and activators of transcription 3 phosphorylation and partially inhibited interferon gamma-dependent signal transducers and activators of transcription 1 phosphorylation. Transfection of the adenovirus vector expressing suppressor of cytokine signaling 3/cytokine-inducible SH2 containing protein 3, however, did not inhibit interleukin-4-dependent signal transducers and activators of transcription 6 phosphorylation. Transfection of the adenovirus vector expressing cytokine-inducible SH2 containing protein 1 had no effect on signal transducers and activators of transcription 1, 3, and 6 signaling in normal keratinocytes. Therefore, the relationship between signal transducers and activators of transcription and suppressor of cytokine signaling is unique in the keratinocytes, and the suppressor of cytokine signaling regulates cytokine signals in these cells.

Ghrelin is a novel brain-gut peptide that stimulates food intake and body weight gain. We studied the anabolic effect of ghrelin in a cancer cachexia mouse model. SEKI, a human melanoma cell line, was inoculated into nude mice to examine the effects of ghrelin on food intake and body weight. The intraperitoneal administration of ghrelin twice a day (6 nmol/mice/day) for 6 days suppressed weight loss in SEKI-inoculated mice and increased the rate of weight gain in vehicle-treated nude mice. Ghrelin administration also increased food intake in both SEKI- and vehicle-treated mice. Both the weight of white adipose tissue and the plasma leptin concentration were reduced in tumor-inoculated mice compared with vehicle-treated mice; these factors increased following ghrelin administration. The levels of both ghrelin peptide and mRNA in the stomach were upregulated in tumor-inoculated mice. The anabolic effect of ghrelin efficiently reverses the cachexia in mice bearing SEKI human melanoma. Ghrelin therefore may have a therapeutic ability to ameliorate cancer cachexia.

Neuromedin U (NMU) is a hypothalamic peptide that has been recently found to reduce food intake, but few is known about its other functions in the central nervous system. We here studied behavioral activities induced by an intracerebroventricular (ICV) administration of NMU in rats and mice. NMU increased gross locomotor activity, face washing behavior, and grooming. NMU-induced stress response was significantly abolished by pretreatment with an antagonist of corticotropin-releasing hormone (CRH), alpha-helical CRH (9-41) (alpha-hCRH), or anti-CRH IgG. NMU did not induce locomotor activity in CRH knockout mice. NMU that interacts anatomically and/or functionally with the CRH system is a novel physiological regulator of stress response., (Copyright 2001 Academic Press.)

The effect of shear stress on the release of prostacyclin (PGI2) from cultured endocardial endothelial cells (EECs) was investigated. EECs were harvested from the right ventricle (RV) and the left ventricle (LV) of porcine heart. Confluent EECs were incubated under various degrees of shear stress (0.2, 1, 4 and 6 dyne/cm2) and PGI2 release from each cell was measured. PGI2 release from LV-EECs and RV-EECs was enhanced by the elevation of shear stress in a shear-dependent manner with a rapid increase at the onset of flow; however, there was no significant difference in PGI2 production between RV-EECs and LV-EECs. production of PGI2 was significantly inhibited from cells exposed to 8-(dimetilamino) octyl 3,4,5-trymethoxybenzoate hydrochloride (10 and 100 microM: an inhibitor of intracellular calcium mobilization) or cyclopiazonic acid (10 microM: an endoplasmic reticulum Ca2+-ATPase inhibitor). These results indicate that shear stress enhances PGI2 release from cultured EECs and that mechanotransduction of shear stress depends on calcium mobilization in EECs.

Glycophorin A is the major transmembrane sialoglycoprotein of red blood cells. It has been shown to contribute to the expression of the MN and Wright blood group antigens, to act as a receptor for the malaria parasite Plasmodium falciparum and Sendai virus, and along with the anion transporter, band 3, may contribute to the mechanical properties of the red blood cell membrane. Several lines of evidence suggest a close interaction between glycophorin A and band 3 during their biosynthesis. Recently, we have generated mice where the band 3 expression was completely eliminated by selective inactivation of the AE1 anion exchanger gene, thus allowing us to study the effect of band 3 on the expression of red blood cell membrane proteins. In this report, we show that the band 3 -/- red blood cells contain protein 4.1, adducin, dematin, p55, and glycophorin C. In contrast, the band 3 -/- red blood cells are completely devoid of glycophorin A (GPA), as assessed by Western blot and immunocytochemistry techniques, whereas the polymerase chain reaction (PCR) confirmed the presence of GPA mRNA. Pulse-label and pulse-chase experiments show that GPA is not incorporated in the membrane and is rapidly degraded in the cytoplasm. Based on these findings and other published evidence, we propose that band 3 plays a chaperone-like role, which is necessary for the recruitment of GPA to the red blood cell plasma membrane.

Background: It has been reported that the thromboexclusion operation is a good method for managing certain difficult thoracic aortic aneurysms., Methods: Forty-four patients underwent graft replacement (group 1) and 14, the thromboexclusion operation (group 2). We reviewed the long-term results of the thromboexclusion operation and compared them with those of graft replacement in our institutions., Results: The hospital mortality rate in groups 1 and 2 was 29.5% (13 patients) and 35.7% (4 patients), respectively. In group 1, the one late death (2.3%) was due to heart failure and in group 2, three of the four late deaths (28.6%) were due to rupture of the excluded thoracic aorta, and one late death was due to heart failure. Long-term follow-up was possible for 23 patients in group 1 and 5 patients in group 2. Survival 3 years after operation was significantly better in group 1 than in group 2 (p < 0.05). Long-term follow-up with blood pressure measurements, chest roentgenograms, electrocardiograms, and echocardiograms showed no significant differences between the preoperative and postoperative findings. However, in group 2, left ventricular hypertrophy and hypertension, which had not been present preoperatively, were found in all of the patients. Also, 1 patient has had persistent hemoptysis., Conclusions: The thromboexclusion operation has introduced unanticipated problems that were recognized at long-term follow-up.

We have found that a 15 kDa protein (p15) produced by a fungus of Helicosporium genus induced characteristic morphological changes on neurites extending from rat cerebral cortex neurons in culture. In the presence of p15, neurite elongation from cortical neurons was markedly inhibited, and they extended short, slender, and less branched neurites. Laminin-induced promotion of neurite outgrowth was also dramatically suppressed. In contrast, p15 promoted nerve growth factor (NGF)-induced neurite outgrowth from PC12 cells, although treatment of cells with p15 alone had little effect. Partial amino acid sequence analysis of p15 revealed that it had limited homology to plant lectins. These results suggest that the mechanism of neurite outgrowth was considerably different between cortical neurons and PC12 cells and that p15 modulated neurite outgrowth in cell type-specific manners.

A sensitive staining method of horseradish peroxidase-labeled immunoglobulins on nitrocellulose membrane was established by employing a reaction chain leading to formazan formation with phenol as a substrate of peroxidase and NADH as a hydrogen donor to reduce nitro blue tetrazolium. Higher concentrations of NADH relative to phenol were necessary to increase the intensity of staining and to ensure a wide dose-response range of color production with respect to the applied enzyme activities. By an optimized tetrazolium method in combination with antibody-affinity blotting, as low as 4 ng/ml alpha-fetoprotein was detected and 3-4-fold greater color intensities in a working assay range as compared with those of existing methods were obtained. The present technique of peroxidase staining may prove to have a wide application for the enzyme immunoassay using blotting modalities.