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During the fertilization of fish eggs, the hardening of the egg envelope is mediated by transglutaminase (hTGase). After fertilization, TGase undergoes processing. We isolated hTGase from extracts of unfertilized and water-activated rainbow trout eggs. Rainbow trout hTGase (Rt-hTGase) appeared as an 80 kDa protein, and its processed form was 55 kDa. Their N-terminal amino acid sequences were nearly identical, suggesting processing in the C-terminal region. The specific activities were not significantly different, indicating that C-terminal processing does not activate the enzyme itself. We cloned the cDNA by reverse transcription polymerase chain reaction (RT-PCR) using degenerate primers followed by RACE-PCR. The deduced amino acid sequence of the cDNA was similar to that of factor XIII subunit A (FXIIIA). Molecular phylogenetic and gene syntenic analyses clearly showed that hTGase was produced by duplication of FXIIIA during the evolution to Teleostei. The 55 kDa processed form of Rt-hTGase is predominantly composed of an enzyme domain predicted from the amino acid sequence of the cDNA. It is hypothesized that the C-terminal domain of Rt-hTGase binds to egg envelope proteins, and that processing allows the enzyme to move freely within the egg envelope, increasing substrate-enzyme interaction and thereby accelerating hardening., (© The Author(s) 2024. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Amyotrophic lateral sclerosis (ALS) is a severe motor neuron disease with uncertain genetic predisposition in most sporadic cases. The spatial architecture of cell types and gene expression are the basis of cell-cell interactions, biological function and disease pathology, but are not well investigated in the human motor cortex, a key ALS-relevant brain region. Recent studies indicated single nucleus transcriptomic features of motor neuron vulnerability in ALS motor cortex. However, the brain regional vulnerability of ALS-associated genes and the genetic link between region-specific genes and ALS risk remain largely unclear. Here, we developed an entropy-weighted differential gene expression matrix-based tool (SpatialE) to identify the spatial enrichment of gene sets in spatial transcriptomics. We benchmarked SpatialE against another enrichment tool (multimodal intersection analysis) using spatial transcriptomics data from both human and mouse brain tissues. To investigate regional vulnerability, we analysed three human motor cortex and two dorsolateral prefrontal cortex tissues for spatial enrichment of ALS-associated genes. We also used Cell2location to estimate the abundance of cell types in ALS-related cortex layers. To dissect the link of regionally expressed genes and ALS risk, we performed burden analyses of rare loss-of-function variants detected by whole-genome sequencing in ALS patients and controls, then analysed differential gene expression in the TargetALS RNA-sequencing dataset. SpatialE showed more accurate and specific spatial enrichment of regional cell type markers than multimodal intersection analysis in both mouse brain and human dorsolateral prefrontal cortex. Spatial transcriptomic analyses of human motor cortex showed heterogeneous cell types and spatial gene expression profiles. We found that 260 manually curated ALS-associated genes are significantly enriched in layer 5 of the motor cortex, with abundant expression of upper motor neurons and layer 5 excitatory neurons. Burden analyses of rare loss-of-function variants in Layer 5-associated genes nominated NOMO1 as a novel ALS-associated gene in a combined sample set of 6814 ALS patients and 3324 controls (P = 0.029). Gene expression analyses in CNS tissues revealed downregulation of NOMO1 in ALS, which is consistent with a loss-of-function disease mechanism. In conclusion, our integrated spatial transcriptomics and genomic analyses identified regional brain vulnerability in ALS and the association of a layer 5 gene (NOMO1) with ALS risk., (© The Author(s) 2024. Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

Multiple system atrophy is a neurodegenerative disease with α-synuclein pathology predominating in the striatonigral and olivopontocerebellar systems. Mixed pathologies are considered to be of low frequency and mostly comprise primary age-related tauopathy or low levels of Alzheimer's disease-related neuropathologic change. Therefore, the concomitant presence of different misfolded proteins in the same brain region is less likely in multiple system atrophy. During the neuropathological evaluation of 21 consecutive multiple system atrophy cases, we identified four cases exhibiting an unusual discrepancy between high Thal amyloid-β phase and low transentorhinal Braak neurofibrillary tangle stage. We mapped α-synuclein pathology, measured the size and number of glial cytoplasmic inclusions and compared the amyloid-β peptides between multiple system atrophy and Alzheimer's disease. In addition, we performed α-synuclein seeding assay from the affected putamen samples. We performed genetic testing for APOE , MAPT , PSEN1 , PSEN2 and APP . We refer to the four multiple system atrophy cases with discrepancy between amyloid-β and tau pathology as 'amyloid-β-predominant Alzheimer's disease neuropathologic change-multiple system atrophy' to distinguish these from multiple system atrophy with primary age-related tauopathy or multiple system atrophy with typical Alzheimer's disease neuropathologic change. As most multiple system atrophy cases with mixed pathologies reported in the literature, these cases did not show a peculiar clinical or MRI profile. Three amyloid-β-predominant Alzheimer's disease neuropathologic change-multiple system atrophy cases were available for genetic testing, and all carried the APOE ɛ4 allele. The extent and severity of neuronal loss and α-synuclein pathology were not different compared with typical multiple system atrophy cases. Analysis of amyloid-β peptides revealed more premature amyloid-β plaques in amyloid-β-predominant Alzheimer's disease neuropathologic change-multiple system atrophy compared with Alzheimer's disease. α-Synuclein seeding amplification assay showed differences in the kinetics in two cases. This study highlights a rare mixed pathology variant of multiple system atrophy in which there is an anatomical meeting point of amyloid-β and α-synuclein, i.e. the striatum or cerebellum. Since biomarkers are entering clinical practice, these cases will be recognized, and the clinicians have to be informed that the prognosis is not necessarily different than in pure multiple system atrophy cases but that the effect of potential α-synuclein-based therapies might be influenced by the co-presence of amyloid-β in regions where α-synuclein also aggregates. We propose that mixed pathologies should be interpreted not only based on differences in the clinical phenotype but also on whether protein depositions regionally overlap, potentially leading to a different response to α-synuclein-targeted therapies., Competing Interests: G.G.K. holds a shared patent for the 5G4 antibody and received royalty for 5G4 synuclein antibody. Other authors report no competing interests in relation to the work described., (© The Author(s) 2024. Published by Oxford University Press on behalf of the Guarantors of Brain.)

Ascomycete lectins may play an important role in their life cycle. In this report, we mined a ricin B-type lectin, named CmRlec, from the Cordyceps militaris genome by homology search. Furthermore, we succeeded in the soluble expression of CmRlec using β-glucuronidase as a solubilization tag and demonstrated that this lectin is a novel chitin-recognizing lectin., (© The Author(s) 2023. Published by Oxford University Press on behalf of Japan Society for Bioscience, Biotechnology, and Agrochemistry.)

Our study aimed to compare the treatment outcomes between endoscopic submucosal dissection (ESD) with an insulated-tip knife (ESD-IT) and a needle-type knife (ESD-N) for large superficial esophageal neoplasms, as no study of this kind has been previously reported. We used the dataset of a multicenter, randomized controlled trial that compared conventional ESD (C-ESD) and traction-assisted ESD (TA-ESD) for superficial esophageal neoplasms. We compared the procedural outcomes between ESD-IT and ESD-N in a post hoc analysis and conducted sub-analyses based on traction assistance and electrical knife type. We included 223 (EST-IT, n = 169; ESD-N, n = 54) patients with no significant differences in baseline characteristics. The operator handover rate due to ESD difficulties was significantly higher in ESD-N (ESD-IT = 0.6% vs. ESD-N = 13.0%, P = 0.001), while the injection volume was significantly higher in ESD-IT than in ESD-N (40.0 vs. 20.5 mL, P < 0.001). Other outcomes were comparable between both groups (procedural time: 51.0 vs. 49.5 minute, P = 0.89; complete resection: 90.5% vs. 90.7%, P > 0.99; and complication rate: 1.8% vs. 3.7%, P = 0.60 for ESD-IT and ESD-N, respectively). In the sub-analyses, the handover rate was significantly lower with TA-ESD than with C-ESD for ESD-N (3.2% vs. 26.1%, P = 0.034), and a significantly smaller injection volume was used in TA-ESD than in C-ESD for ESD-IT (31.5 vs. 47.0 mL, P < 0.01). ESD with either endoscopic device achieved favorable treatment outcomes with low complication rates. The handover rate in ESD-N and the injection volume in ESD-IT improved with the traction method., (© The Author(s) 2022. Published by Oxford University Press on behalf of International Society for Diseases of the Esophagus. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Mango (Mangifera indica L.) kernels are usually discarded as waste, but they contain many pharmacological properties and bioactivities. In this study, we isolated antiobesity agents from mango kernels that inhibit intracellular lipid formation in 3T3-L1 adipocytes. Two phenolic acids, ethyl gallate and ethyl digallate, and 2 tannin acids, 1,2,3,4,6-penta-O-galloyl-β-d-glucose (PGG) and 3-O-digalloyl-1,2,4,6-tetra-O-β-d-glucose (HGG), were identified from mango kernels and were found to be suppressed lipid accumulation as evidenced by Oil Red O staining. Furthermore, ethyl digallate, PGG, and HGG significantly downregulated the mRNA expression of adipogenic transcription factors such as C/EBPα and PPARγ. However, ethyl gallate did not affect the expression of these transcription factors. Our findings reveal the presence of antiobesity compounds in mango kernels, implying its therapeutic role against obesity., (© The Author(s) 2022. Published by Oxford University Press on behalf of Japan Society for Bioscience, Biotechnology, and Agrochemistry.)

Background: Implementation of personalized medicine requires the accessibility of tumor molecular profiling in order to allow prioritization of appropriate targeted therapies for individual patients. Our aim was to study the role of comprehensive genomic profiling assays that may inform treatment recommendations for patients with solid tumors., Materials and Methods: We performed a prospective study to evaluate the feasibility of application of the FoundationOne CDx panel-which detects substitutions, insertions and deletions, and copy number alterations in 324 genes, select gene rearrangements, and genomic signatures including microsatellite instability and tumor mutation burden (TMB)-to patients with advanced or recurrent solid tumors before its approval in Japan., Results: A total of 181 samples were processed for genomic testing between September 2018 and June 2019, with data being successfully obtained for 175 of these samples, yielding a success rate of 96.7%. The median turnaround time was 41 days (range, 21-126 days). The most common known or likely pathogenic variants were TP53 mutations (n = 113), PIK3CA mutations (n = 33), APC mutations (n = 32), and KRAS mutations (n = 29). Among the 153 patients assessed for TMB, the median TMB was 4 mutations/Mb, and tumors with a high TMB (≥10 mutations/Mb) were more prevalent for lung cancer (11/32) than for other solid tumor types (9/121, Fisher's exact test p < .01). No clear trend toward increased efficacy for immune checkpoint inhibitor (ICI) monotherapy or ICI combination chemotherapy in patients with a high programmed cell death-ligand 1 tumor proportion score or a high TMB was apparent. Among the 174 patients found to harbor known or likely pathogenic actionable alterations, 24 individuals (14%) received matched targeted therapy., Conclusion: The FoundationOne CDx assay was performed with formalin-fixed, paraffin-embedded tumor specimens with a success rate of >95%. Such testing may inform the matching of patients with cancer with investigational or approved targeted drugs., Implications for Practice: This prospective cohort study was initiated to investigate the feasibility and utility of clinical application of FoundationOne CDx. A total of 181 samples were processed for genomic testing between September 2018 and June 2019, with data being successfully obtained for 175 of these samples, yielding a success rate of 96.7%, and 24 individuals (14%) received matched targeted therapy., (© 2020 The Authors. The Oncologist published by Wiley Periodicals LLC on behalf of AlphaMed Press.)

Tau is a microtubule associated protein in the brain that aggregates in Alzheimer's disease to form pathological tangles and neurites. Insoluble tau aggregates composed of the microtubule binding region (MTBR) of tau are highly associated with the cognitive and clinical symptoms of Alzheimer's disease. In contrast, levels of soluble forms of tau, such as CSF total tau and phosphorylated tau-181 and tau-217, increase prior to tau aggregation in Alzheimer's disease, but these biomarkers do not measure the MTBR of tau. Thus, how CSF MTBR-tau is altered in Alzheimer's disease remains unclear. In this study, we used sequential immunoprecipitation and chemical extraction methods followed by mass spectrometry to analyse MTBR-tau species in Alzheimer's disease and control CSF. We quantified MTBR-tau-specific regions in the CSF and identified that species containing the region beginning at residue 243 were the most highly correlated with tau PET and cognitive measures. This finding suggests that CSF level of tau species containing the upstream region of MTBR may reflect changes in tau pathology that occur in Alzheimer's disease and could serve as biomarkers to stage Alzheimer's disease and track the development of tau-directed therapeutics., (© The Author(s) (2020). Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Sialic acids (Sias) are an outermost-situated sugar of glycoproteins and glycolipids to play important roles in various biological phenomena. They are often modified by additional substituents, such as O -acetyl group, to display more than 50 different structures in nature. Of those modified Sia, nothing is known about the occurrence and biological functions of sulfated Sias (SiaSs) in mammals. To elucidate the significance of sialic acid sulfation, we investigated various mammalian-cultured cell lines for the expression of SiaS using the specific antibody 3G9. First, SiaS is expressed in a cell line-dependent and a cell density-dependent manner. Second, in CHO cells, the expression of SiaS is reversibly induced by treatment with the antibiotic G418. Taken together, the expression of SiaS is changed by intrinsic and extrinsic factors in mammalian cells. This is the first demonstration of regulated expression of SiaS.

Background: We assessed the safety and effectiveness of baloxavir marboxil administration in Japanese children with influenza., Methods: This open-label study administered 1 weight-adjusted dose of baloxavir to 107 children aged 1-11 years with laboratory-confirmed, febrile influenza virus infection of ≤48 hours duration., Results: Adverse events (AEs) were reported in 34.6% of patients, most commonly vomiting (7.5%); no serious AEs or AEs causing discontinuation occurred. The median time to alleviation of influenza illness was 44.6 hours (95% confidence interval, 38.9-62.5 hours), to resolution of fever was 21.4 hours, and to sustained cessation of infectious viral shedding was 24.0 hours. However, viruses with amino acid substitutions in the viral polymerase acidic protein at position I38 (PA/I38T/M) emerged in 18 of 77 (23.4%) patients. Emergence was associated with longer infectious virus detectability (median time, 180.0 hours) and time to illness alleviation (median, 79.6 vs 42.8 hours in patients without PA/I38T/M-substituted viruses). Among patients with PA/I38T/M-substituted virus emergence, those with baseline hemagglutinin inhibition (HAI) antibody titer <40 experienced delay in time to illness alleviation (median, 85.4 vs 56.0 hours in patients with higher baseline HAI antibody titer)., Conclusions: A single, oral dose of baloxavir marboxil was well tolerated and rapidly reduced viral titers, but the common emergence of PA/I38T/M-substituted viruses warrants consideration of alternative dosing regimens in young children., Clinical Trials Registration: Japan Pharmaceutical Information Center Clinical Trials Information (Japic CTI-163417)., (© The Author(s) 2019. Published by Oxford University Press for the Infectious Diseases Society of America.)

Age at onset of Alzheimer's disease is highly variable, and its modifiers (genetic or environmental) could act through epigenetic changes, such as DNA methylation at CpG sites. DNA methylation is also linked to ageing-the strongest Alzheimer's disease risk factor. DNA methylation age can be calculated using age-related CpGs and might reflect biological ageing. We conducted a clinical, genetic and epigenetic investigation of a unique Ashkenazi Jewish family with monozygotic triplets, two of whom developed Alzheimer's disease at ages 73 and 76, while the third at age 85 has no cognitive complaints or deficits in daily activities. One of their offspring developed Alzheimer's disease at age 50. Targeted sequencing of 80 genes associated with neurodegeneration revealed that the triplets and the affected offspring are heterozygous carriers of the risk APOE ε4 allele, as well as rare substitutions in APP (p.S198P), NOTCH3 (p.H1235L) and SORL1 (p.W1563C). In addition, we catalogued 52 possibly damaging rare variants detected by NeuroX array in affected individuals. Analysis of family members on a genome-wide DNA methylation chip revealed that the DNA methylation age of the triplets was 6-10 years younger than chronological age, while it was 9 years older in the offspring with early-onset Alzheimer's disease, suggesting accelerated ageing., (© The Author(s) (2019). Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

The G4C2-repeat expansion in C9orf72 is the most common known cause of amyotrophic lateral sclerosis and frontotemporal dementia. The high phenotypic heterogeneity of C9orf72 patients includes a wide range in age of onset, modifiers of which are largely unknown. Age of onset could be influenced by environmental and genetic factors both of which may trigger DNA methylation changes at CpG sites. We tested the hypothesis that age of onset in C9orf72 patients is associated with some common single nucleotide polymorphisms causing a gain or loss of CpG sites and thus resulting in DNA methylation alterations. Combined analyses of epigenetic and genetic data have the advantage of detecting functional variants with reduced likelihood of false negative results due to excessive correction for multiple testing in genome-wide association studies. First, we estimated the association between age of onset in C9orf72 patients (n = 46) and the DNA methylation levels at all 7603 CpG sites available on the 450 k BeadChip that are mapped to common single nucleotide polymorphisms. This was followed by a genetic association study of the discovery (n = 144) and replication (n = 187) C9orf72 cohorts. We found that age of onset was reproducibly associated with polymorphisms within a 124.7 kb linkage disequilibrium block tagged by top-significant variation, rs9357140, and containing two overlapping genes (LOC101929163 and C6orf10). A meta-analysis of all 331 C9orf72 carriers revealed that every A-allele of rs9357140 reduced hazard by 30% (P = 0.0002); and the median age of onset in AA-carriers was 6 years later than GG-carriers. In addition, we investigated a cohort of C9orf72 negative patients (n = 2634) affected by frontotemporal dementia and/or amyotrophic lateral sclerosis; and also found that the AA-genotype of rs9357140 was associated with a later age of onset (adjusted P = 0.007 for recessive model). Phenotype analyses detected significant association only in the largest subgroup of patients with frontotemporal dementia (n = 2142, adjusted P = 0.01 for recessive model). Gene expression studies of frontal cortex tissues from 25 autopsy cases affected by amyotrophic lateral sclerosis revealed that the G-allele of rs9357140 is associated with increased brain expression of LOC101929163 (a non-coding RNA) and HLA-DRB1 (involved in initiating immune responses), while the A-allele is associated with their reduced expression. Our findings suggest that carriers of the rs9357140 GG-genotype (linked to an earlier age of onset) might be more prone to be in a pro-inflammatory state (e.g. by microglia) than AA-carriers. Further, investigating the functional links within the C6orf10/LOC101929163/HLA-DRB1 pathway will be critical to better define age-dependent pathogenesis of frontotemporal dementia and amyotrophic lateral sclerosis.

Background/objectives: The peri-oral muscles-including orbicularis oris-are critical in maintaining equilibrium in tooth position. Lip incompetence (LI) can thus be a factor in malocclusion. We therefore aimed to validate a technique to evaluate not only muscle activity via electromyography (EMG) but also muscle endurance and fatigue via blood flow (BF) for LI., Subjects/methods: Subjects were classified into increased muscle tension/lip incompetent (experimental) and normal muscle tension/lip competent (control) groups. Each subject then exerted force on a custom-made traction plate connected to a tension gauge. Using laser speckle imaging and electromyographic measurements, we characterized muscle activity and corresponding BF rates in these subjects in various states of resting, loading, and recovery., Results: Results showed a significant difference between the experimental and control groups, notably in the rate of change in BF to the inferior orbicularis oris muscle under conditions of increasing load (graded exertion). Furthermore, the data suggested that the muscles in the control group undergo a more prolonged (and therefore presumably more complete) recovery than muscles in the experimental group. These factors of reduced BF and short recovery may combine to accelerate muscle fatigue and produce LI., Limitations: The sample used here was controlled for malocclusion (including open bite) to eliminate this type of confounding effect., Conclusions/implications: From these findings, we conclude that reduced BF and inadequate recovery in the orbicularis oris muscles may be more significant than EMG activity in the assessment of LI.

Objective: Monosodium urate (MSU) has been shown to promote interleukin-1β (IL-1β) secretion in human monocytes, but the priming signals for NOD-like receptor family pyrin domain containing 3 (NLRP3) inflammasome pathway remains elusive. In this study, we investigated the role of Tumor necrosis factor-alpha (TNF-α) on MSU-mediated IL-1β induction in human neutrophils., Methods: Human neutrophils were stimulated with MSU, in the presence or absence of TNF-α priming. The cellular supernatants were analyzed for IL-1β, IL-18, and caspase-1 by enzyme-linked immunosorbent assay (ELISA) methods. Pro-IL-1β mRNA expressions in human neutrophils were analyzed by real-time PCR method., Results: TNF-α stimulation induced pro-IL-1β mRNA expression; however, MSU stimulation did not induce pro-IL-1β mRNA expression in human neutrophils. TNF-α alone or MSU stimulation did not result in efficient IL-1β secretion in human neutrophils, whereas in TNF-α-primed neutrophils, MSU stimulation resulted in a marked IL-1β and IL-18 secretion. TNF-α-primed neutrophils secreted cleaved caspase-1 (p20), in response to MSU stimulation., Conclusion: Our data demonstrate that priming of human neutrophils with TNF-α promotes uric acid-mediated IL-1β secretion in the absence of microbial stimulation. These findings provide insights into the neutrophils-mediated inflammatory processes in gouty arthritis.

Lactoferrin (LF) is produced by exocrine glands including salivary gland, and has various functions including infection defense. However, the transfer of LF from peripheral organs into the brain remains unclear. To clarify the kinetics of salivary LF (sLF), we investigated the consequences of sialoadenectomy and bovine LF (bLF) sublingual administration in rats. The salivary glands were removed from male Wistar rats, and we measured rat LF levels in the blood and brain at 1 week post-surgery. We also examined the transfer of LF into the organs of the rats after sublingual administration of bLF. Rat LF levels in the blood and brain were significantly reduced by sialoadenectomy. Sublingual bLF administration significantly increased bLF levels in the brain, which then decreased over time. These results indicate that LF is transferred from the sublingual mucosa to the brain, in which favorable effects of sLF on brain will be expected via the sublingual mucosa.

Aims: The Mason-Likar modified electrocardiogram (ML-ECG) can be interchanged with standard 12 lead ECG electrode positions (standard ECG) without affecting the diagnostic interpretation during sinus rhythm, but the morphological differences during ventricular arrhythmias have not been sufficiently evaluated. This study aimed to elucidate the morphological changes in the ML-ECG precordial leads., Methods and Results: In 53 consecutive patients with premature ventricular contractions predicted to originate from the outflow tract (OT-PVCs), the arrhythmias were analysed by those two ECG methods. The OT-PVC origin sites, which were predicted by currently published criteria with the respective ECG methods prior to catheter ablation, were compared with the successful ablation sites. Compared with the standard-ECG, S-waves in the ML-ECG became shallower in leads V1-4 (P < 0.05 in lead V1; P < 0.001 in leads V2-4), and pseudo-R-waves in lead V1 appeared in seven patients. The precordial leads transition zone shifted counter-clockwise in 18 patients in the ML-ECG. In leads I and aVL, the negative deflection amplitudes of the ML-ECG were greater than those of the standard ECG (P < 0.001), and polarity reversals in lead I appeared in 18 patients. The R-wave amplitudes in all ML-ECG inferior leads were greater than those in the standard-ECG leads (all for P < 0.001). Those changes had an effect on the diagnostic indexes for the localization, and the specificity of the criteria for the ML-ECG was poorer than that for the standard-ECG., Conclusion: Great differences were found between those two ECG methods. Predicting OT-PVC origins by diagnostic criteria with the ML-ECG might result in a misdiagnosis and inefficient ablation., (Published on behalf of the European Society of Cardiology. All rights reserved. © The Author 2015. For permissions please email: journals.permissions@oup.com.)

Proteins play important roles in cell functions such as enzymes, cell trafficking, neurotransmission, muscle contraction and hormone secretion. However, some proteins are very difficult to be crystallized and their structures are undetermined. Several techniques have been developed to elucidate the structure of macromolecules; X-ray or electron crystallography, nuclear magnetic resonance spectroscopy, and high-resolution electron microscopy. Among them, electron microscopy based single particle reconstruction (SPA) technique is a computer-aided structure determination method. This method reconstructs the 3D structure from projection images of dispersed protein. A large number of two-dimensional particle images are picked up from EM films, aligned and classified to generate 2D averages, and used to reconstruct the 3D structure by assigning the Euler angle of each 2D average. Due to the necessity of elaborate collaboration between the classical biology and the innovative information technology including parallel computing, scientists needed to break unseen barriers to get a start of this analysis. However, recent progresses in electron microscopes, mathematical algorithms, and computational abilities greatly reduced the height of barriers and expanded targets that are considered to be primarily addressable using single particle analysis. Membrane proteins are one of these targets to which the single particle analysis is successfully applied for the understanding of their 3D structures. For this purpose, we have developed various SPA methods [1-5] and applied them to different proteins [6-8].Here, we introduce reconstructed proteins, and discuss the availability of this technique. The intramembrane-cleaving proteases (I-CLiPs) that sever the transmembrane domains of their substrates have been identified in a range of organisms and play a variety of roles in biological conditions. I-CLiPs have been classified into three groups: serine-, aspartyl- and metalloprotease-type. Signal peptide peptidase (SPP) is an atypical aspartic protease that hydrolyzes peptide bonds within the transmembrane domain of substrates and is implicated in several biological and pathological functions. The structure of human SPP was determined by SPA at a resolution of 22 Å [8]. SPP forms a slender, bullet-shaped homotetramer with dimensions of 85 x 85 x 130 Å. The SPP complex has four concaves on the rhombus-like sides, connected to a large chamber inside the molecule. For the tetrameric assembly, the N-terminal region of SPP was found to be sufficient. Moreover, when N-terminal region was overexpressed, the formation of the endogenous SPP tetramer was inhibited, which suppressed the proteolytic activity within cells. From these data, the N-terminal region is considered to work as the structural scaffold.Transmembrane (TM) translocation of newly synthesized secretion proteins and membrane proteins are carried out by a Sec translocon protein complex. The polypeptide-conducting pore is formed by the SecYEG-SecA complex in bacteria, and the membrane protein SecDF is necessary for the efficient transport of proteins. However the molecular mechanism how SecDF realized efficient transport is not clear. A previous X-ray structural study of the whole protein and subdomain suggest that SecDF has at least two conformational variants, which could reflect molecular dynamics of this protein. To confirm this hypothesis, we analyzed the 3D structure of SecDF using dark field STEM electron tomography and single particle reconstruction. We determined two different whole SecDF protein structures which well explains the X-ray data. From these data, we would like to propose the possible molecular mechanism of SecDF during polypeptide translocation., (© The Author 2014. Published by Oxford University Press on behalf of The Japanese Society of Microscopy. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

High-throughput immuno-electron microscopy is required to capture the protein-protein interactions realizing physiological functions. Atmospheric scanning electron microscopy (ASEM) allows in situ correlative light and electron microscopy of samples in liquid in an open atmospheric environment. Cells are cultured in a few milliliters of medium directly in the ASEM dish, which can be coated and transferred to an incubator as required. Here, cells were imaged by optical or fluorescence microscopy, and at high resolution by gold-labeled immuno-ASEM, sometimes with additional metal staining. Axonal partitioning of neurons was correlated with specific cytoskeletal structures, including microtubules, using primary-culture neurons from wild type Drosophila, and the involvement of ankyrin in the formation of the intra-axonal segmentation boundary was studied using neurons from an ankyrin-deficient mutant. Rubella virus replication producing anti-double-stranded RNA was captured at the host cell's plasma membrane. Fas receptosome formation was associated with clathrin internalization near the surface of primitive endoderm cells. Positively charged Nanogold clearly revealed the cell outlines of primitive endoderm cells, and the cell division of lactic acid bacteria. Based on these experiments, ASEM promises to allow the study of protein interactions in various complexes in a natural environment of aqueous liquid in the near future.

Sialic acids (Sia) are involved in many biological activities and frequently exist as monosialyl residues at the non-reducing terminal end of glycoconjugates. Occasionally, polymerized structures in the form of disialic acid (diSia), oligosialic acid (oligoSia) and polysialic acid (polySia) are also found in glycoconjugates. In particular, polySia, which is an evolutionarily conserved epitope from sea urchin to humans, is one of the most biologically important glycotopes in vertebrates. The biological functions of polySia, especially on neural cell adhesion molecules, have been well studied and an in-depth body of knowledge concerning polySia has been accumulated. However, considerably less attention has been paid to glycoproteins containing di- and oligoSia groups. However, advances in analytical methods for detecting oligo/polymerized structures have allowed the identification and characterization of an increasing number of glycoproteins containing di/oligo/polySia chains in nature. In addition, sophisticated genetic techniques have also helped to elucidate the underlying mechanisms of polySia-mediated activities. In this review, recent advances in the study of the chemical properties, distribution and functions of di-, oligo- and polySia residues on glycoproteins are described.

Data mining from noisy data/images is one of the most important themes in modern science and technology. Statistical image processing is a promising technique for analysing such data. Automation of particle pickup from noisy electron micrographs is essential, especially when improvement of the resolution of single particle analysis requires a huge number of particle images. For such a purpose, reference-based matching using primary three-dimensional (3D) model projections is mainly adopted. In the matching, however, the highest peaks of the correlation may not accurately indicate particles when the image is very noisy. In contrast, the density and the heights of the peaks should reflect the probability distribution of the particles. To statistically determine the particle positions from the peak distributions, we have developed a density-based peak search followed by a peak selection based on average peak height, using multi-reference alignment (MRA). Its extension, using multi-reference multiple alignment (MRMA), was found to enable particle pickup at higher accuracy even from extremely noisy images with a signal-to-noise ratio of 0.001. We refer to these new methods as stochastic pickup with MRA (MRA-StoPICK) or with MRMA (MRMA-StoPICK). MRMA-StoPICK has a higher pickup accuracy and furthermore, is almost independent of parameter settings. They were successfully applied to cryo-electron micrographs of Rice dwarf virus. Because current computational resources and parallel data processing environments allow somewhat CPU-intensive MRA-StoPICK and MRMA-StoPICK to be performed in a short period, these methods are expected to allow high-resolution analysis of the 3D structure of particles.

Three-dimensional (3D) protein structures facilitate the understanding of their biological functions and provide valuable information for developing medicines. Single-particle analysis (SPA) from electron microscopy (EM) is a structure determination method suitable for macromolecules. To achieve a high resolution using combinations of several SPA software packages, 'workflow' optimization and comparative evaluation by scoring results are essential. Two-dimensional (2D) averaging is a key step for 3D reconstruction. The integrated convergence-evaluation oriented system (IC-EOS) proposed here provides an effective tool for customizing 2D averaging. This assesses the behavior and characteristics of workflows and evaluates the convergence of iteration steps without human intervention. We chose five base measurements for quantifying convergence: resolution, variance, similarity, shift-distance and rotation-angle. Curve fitting to history graphs scored their stability. We call this score 'fluctuation'. The number of particle images discarded from the library and the number of classification groups were examined to see their effects on optimization levels and fluctuation of measurements, allowing the IC-EOS to select the most appropriate workflow for the target. A case study using a bacterial sodium channel and a simulation study using GroEL showed that resolution of 2D averaging improved with relatively stricter particle selection. With fewer groups, resolutions of class averages improved, but similarities between class-averages and their constituent particle images degraded. Fluctuation was useful for selecting adequate conditions, even when achieved values alone were not conclusive. The vote method, using fluctuation, was robust against noise and enabled a decision without exhaustive search trials. Thus, the IC-EOS is a step toward full automation of SPA.

Recently genome-wide association studies have identified significant association between Alzheimer's disease (AD) and variations in CLU, PICALM, BIN1, CR1, MS4A4/MS4A6E, CD2AP, CD33, EPHA1, and ABCA7. However, the pathogenic variants in these loci have not yet been found. We conducted a genome-wide scan for large copy number variation (CNV) in a dataset of Caribbean Hispanic origin (554 controls and 559 AD cases that were previously investigated in a SNP-based genome-wide association study using Illumina HumanHap 650Y platform). We ran four CNV calling algorithms to obtain high-confidence calls for large CNVs (>100 kb) that were detected by at least two algorithms. Global burden analyses did not reveal significant differences between cases and controls in CNV rate, distribution of deletions or duplications, total or average CNV size; or number of genes affected by CNVs. However, we observed a nominal association between AD and a ∼470 kb duplication on chromosome 15q11.2 (P = 0.037). This duplication, encompassing up to five genes (TUBGCP5, CYFIP1, NIPA2, NIPA1, and WHAMML1) was present in 10 cases (2.6%) and 3 controls (0.8%). The dosage increase of CYFIP1 and NIPA1 genes was further confirmed by quantitative PCR. The current study did not detect CNVs that affect novel AD loci identified by recent genome-wide association studies. However, because the array technology used in our study has limitations in detecting small CNVs, future studies must carefully assess novel AD genes for the presence of disease-related CNVs.

Determining the mobile signal used by plants to defend against biotic and abiotic stresses has proved elusive, but jasmonic acid (JA) and its derivatives appear to be involved. Using deuterium-labeled analogs, we investigated the distal transport of JA and jasmonoyl-isoleucine (JA-Ile) in response to leaf wounding in tobacco (Nicotiana tabacum) and tomato (Solanum lycopersicum) plants. We recovered [(2)H(2)-2]JA ([(2)H(2)]JA) and [(2)H(3)-12]JA-Ile ([(2)H(3)]JA-Ile) in distal leaves of N. tabacum and S. lycopersicum after treating wounded leaves with [(2)H(2)]JA or [(2)H(3)]JA-Ile. We found that JA-Ile had a greater mobility than JA, despite its lower polarity, and that application of exogenous JA-Ile to wounded leaves of N. tabacum led to a higher accumulation of JA and JA-Ile in distal leaves compared with wounded control plants. We also found that exudates from the stem of S. lycopersicum plants with damaged leaflets contained JA and JA-Ile at higher levels than in an undamaged plant, and a significant difference in the levels of JA-Ile was observed 30 min after wounding. Based on these results, it was found that JA-Ile is a transportable compound, which suggests that JA-Ile is a signaling cue involved in the resistance to biotic and abiotic stresses in plants., (© The Author 2011. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists.)

Treating the leaves of sweet pepper (Capsicum annuum L. var. grossum) with an aqueous solution of cellulase resulted in a four-fold increase in the salicylic acid level compared to a control plant. The level of endogenous azelaic acid was also elevated by the cellulase treatment. Azelaic acid has recently been reported to act as a mobile "priming" agent to arm plants against pathogenic attack. Our results are consistent with this and that the cellulase treatment enhanced the ability of sweet pepper to withstand viral attack.

Glycosaminoglycans (GAGs), large anionic glycopolymers, are the glycan portion of proteoglycans and are important components of the extracellular matrix. Recently we reported that polysialic acid, a polyanionic glycopolymer specific to the brain, binds neurotrophic factors to form a large complex. It is not clear whether GAGs also bind neurotrophic factors to form a large complex. In the present study, we demonstrate that a brain-derived neurotrophic factor (BDNF) dimer directly binds GAGs other than chondroitin and hyaluronic acid to form a large complex. Neurotrophin-3 showed similar GAG binding properties. Furthermore, BDNF, after forming a large complex with GAG, bound to the BDNF receptors tropomyosin-related kinase (Trk) B and p75 neurotrophin receptor (NTR). These findings suggest that GAGs function to produce a reservoir of BDNF and other neurotrophic factors, and may serve to regulate their local concentrations on the cell surface.

In plants, the mobile signal needed for wound-induced systemic acquired resistance (WSR) has been elusive. The signal compound involved in WSR is supposed to be JA or its derivatives. On the basis of kinetic study of the accumulation of JA or its derivatives, it was discovered that JA, JA-Ile, tuberonic acid (TA, 12-OH epi-JA), and tuberonic acid glucoside (TAG) accumulated in systemic tissues in response to mechanical wounding stress in the tobacco plant (Nicotiana tabacum). Attempts to recover deuterium-labeled JA in systemic leaves after feeding the wounded leaves with deuterium-labeled JA were successfully done. It was also found that the translocated deuterium-labeled JA was metabolized to TA in systemic leaves under feeding of deuterium-labeled JA to the wounding leaves.

A PCR method to detect porcine DNA was developed for verifying the allergen labeling of foods and for identifying hidden pork ingredients in processed foods. The primer pair, F2/R1, was designed to detect the gene encoding porcine cytochrome b for the specific detection of pork with high sensitivity. The amplified DNA fragment (130 bp) was specifically detected from porcine DNA, while no amplification occurred with other species such as cattle, chicken, sheep, and horse. When the developed PCR method was used for investigating commercial food products, porcine DNA was clearly detected in those containing pork in the list of ingredients. In addition, 100 ppb of pork in heated gyoza (pork and vegetable dumpling) could be detected by this method. This method is rapid, specific and sensitive, making it applicable for detecting trace amounts of pork in processed foods.

Acute-phase proteins are an important marker of inflammation and sometimes have a role in the general defense response towards tissue injury. In the present study, we identified a 32-kDa protein that was immunoreactive with monoclonal antibody 2-4B (mAb.2-4B), which is specific to di/oligoNeu5Gc structures, and that behaved as an acute-phase protein following stimulation with either turpentine oil or lipopolysaccharides. The 32-kDa protein was identified as carbonic anhydrase II (CA-II), based on matrix-assisted laser desorption/ionization time-of-flight mass spectrometry analyses of the purified protein. Mouse and human CA-II was immunoreactive and immunoprecipitated with mAb.2-4B, but contained no sialic acid. In addition to mAb.2-4B, the mAb. S2-566 an antibody specific for diNeu5Ac-containing glycans, recognized the CA-II, whereas an anti-oligo/polysialic acid antibody did not. These results indicate that a part of the CA-II protein structure mimics the disialic acid structure recognized by the monoclonal antibodies. This is the first report that CA-II circulates in the serum following inflammation.

The egg envelope, referred to as zona pellucida (ZP) in mammalian eggs, is a fibrous and noncollagenous extracellular matrix surrounding vertebrate eggs, and composed of three to four homologous glycoproteins with a common ZP domain. In birds, a liver-derived ZP glycoprotein (ZP1/ZPB1) is transported through the bloodstream to ovarian follicles and joins the egg-envelope matrix construction together with the other ZP glycoproteins, such as ZPC and ZPD/ZPX2, both secreted from follicular granulosa cells. We report here that, through its ZP domain, ZPB1 specifically associates with ZPC, which might lead to the construction of egg-envelope matrix. The ZPB1 in laying hen's serum specifically bound to ZPC, but not to ZPX2, separated by SDS-PAGE and blotted on a membrane. Hemagglutinin (HA)-tagged ZPC expressed in a mammalian cell line (COS-7) cells was processed and secreted as a mature-form into the culture medium. From the culture supernatant of ZPC-expressing transfectants cultured in the presence of ZPB1, both ZPB1 and ZPC were recovered as heterocomplexes by immunoprecipitation using either anti-HA or anti-ZPB1 antibody. Interestingly, a monoclonal antibody, 8E1, which immunoprecipitated free ZPB1, did not immunoprecipitate the ZPB1-ZPC heterocomplexes. An 8E1 epitope was mapped on a C-terminal region of the ZP domain in a ZPB1 molecule by identifying an 8E1-positive peptide using mass spectroscopy. Furthermore, by laser scanning confocal microscopy, ZPB1 and ZPC were observed to colocalize on the surface of ZPC-expressing transfectants cultured in the presence of ZPB1, whereas almost no ZPC was detected on the surface of the transfectants cultured in the absence of ZPB1. Taken together, these results suggest that ZPB1 transported into ovarian follicles encounters and associates with ZPC secreted from granulosa cells, resulting in the formation of heterocomplexes around an oocyte. In addition, it appears that such ZPB1-ZPC complexes accumulated on the oocyte surface act as a scaffold for subsequent matrix construction events including ZPX2 association.

The syndrome of apparent mineralocorticoid excess (AME) is characterized by persistent hypertension and hypokalemia, which is caused by impaired inactivation of cortisol (F) to cortisone (E). The thyroid hormone has been known to influence the F to E conversion leading to efficacious inactivation of F into E. However, there have been no reports regarding the clinical manifestation of secondary AME due to hypothyroidism. Here we report an elderly patient who manifested AME, showing persistent hypertension with hypokalemia induced by primary hypothyroidism. Maintenance of euthyroid conditions ameliorated the concurrent AME and restored adrenal secretion of aldosterone after the recovery of the F to E shuttle. This case report would broaden our clinical recognition regarding acquired AME in relation to thyroid dysfunction.

Corticobasal syndrome (CBS) is a rare cognitive and movement disorder characterized by asymmetric rigidity, apraxia, alien-limb phenomenon, cortical sensory loss, myoclonus, focal dystonia, and dementia. It occurs along the clinical spectrum of frontotemporal lobar degeneration (FTLD), which has recently been shown to segregate with truncating mutations in progranulin (PGRN), a multifunctional growth factor thought to promote neuronal survival. This study identifies a novel splice donor site mutation in the PGRN gene (IVS7+1G-->A) that segregates with CBS in a Canadian family of Chinese origin. We confirmed the absence of the mutant PGRN allele in the RT-PCR product which supports the model of haploinsufficiency for PGRN-linked disease. This report of mutation in the PGRN gene in CBS extends the evidence for genetic and phenotypic heterogeneity in FTLD spectrum disorders.

Polysialoglycoprotein (PSGP) in salmonid fish egg is a unique glycoprotein bearing alpha2,8-linked polysialic acid (polySia) on its O-linked glycans. Biosynthesis of the polySia chains is developmentally regulated and only occurs at later stage of oogenesis. Two alpha2,8-polysialyltransferases (alpha2,8-polySTs), PST (ST8Sia IV) and STX (ST8Sia II), responsible for the biosynthesis of polySia on N-glycans of glycoproteins, are known in mammals. However, nothing has been known about which alpha2,8-polySTs are involved in the biosynthesis of polySia on O-linked glycans in any glycoproteins. We thus sought to identify cDNA encoding the alpha2,8-polyST involved in polysialylation of PSGP. A clone for PST orthologue, rtPST, and two clones for the STX orthologue, rtSTX-ov and rtSTX-em, were identified in rainbow trout. The deduced amino acid sequence of rtPST shows a high identity (72-77%) to other vertebrate PSTs, while that of rtSTX-ov shows 92% identity with rtSTX-em and a significant identity (63-76%) to other vertebrate STXs. The rtPST exhibited the in vivo alpha2,8-polyST activity, although its in vitro activity was low. However, the rtSTXs showed no in vivo and very low in vitro activities. Interestingly, co-existence of rtPST and rSTX-ov in the reaction mixture synergistically enhanced the alpha2,8-polyST activity. During oogenesis, rtPST was constantly expressed, while the expression of rtSTX-ov was not increased until polySia chain is abundantly biosynthesized in the later stage. rtSTX-em was not expressed in ovary. These results suggest that the enhanced expression of rtSTX-ov under the co-expression with rtPST may be important for the biosynthesis of polySia on O-linked glycans of PSGP.

Mammalian genes subject to genomic imprinting often form clusters and are regulated by long-range mechanisms. The distal imprinted domain of mouse chromosome 7 is orthologous to the Beckwith-Wiedemann syndrome domain in human chromosome 11p15.5 and contains at least 13 imprinted genes. This domain consists of two subdomains, which are respectively regulated by an imprinting center. We here report the finished-quality sequence of a 0.6-Mb region encompassing the more centromeric subdomain. The sequence contains four imprinted genes (Ascl2/Mash2, Ins2, Igf2 and H19) and reveals previously unidentified CpG islands and tandem repeats, which may be features of imprinted genes. Most interestingly, a unique 210-kb segment consisting almost exclusively of tandem repeats and retroelements is identified. This segment, located between Th and Ins2, has features of heterochromatin-forming DNA and is highly methylated at CpG sites. The segment exhibits asynchronous replication on the parental chromosomes, a feature of the imprinted domains. We propose that this repeat segment could serve either as a boundary between the two subdomains or as a target for epigenetic chromatin modifications that regulate imprinting.

Polysialoglycoprotein (PSGP) is a major cortical alveolus glycoprotein of rainbow trout eggs that is characterized by the attachment of a polysialic acid structure to its O-glycan chains. It has been demonstrated that the polysialic acid structure is synthesized by at least three sialyltransferases mostly localized in cortical alveoli. Here we have cloned a cDNA encoding the sialyltransferase, designated rtST6GalNAc, responsible for the transfer of the first sialic acid residue onto the O-glycan chain of PSGP. This enzyme belongs to the vertebrate ST6GalNAc II family, and is strongly expressed in ovaries. Of those O-glycoproteins tested as substrates, asialo-PSGP is the best substrate. These results indicate that rtST6GalNAc is the enzyme responsible for the sialylation of PSGP during oogenesis. Furthermore, the rtST6GalNAc mRNA is expressed throughout oogenesis, is down-regulated at the late yolk vesicle stage (May), and then up-regulated during vitellogenesis (until August). This developmental profile is highly similar to that of STL2, a cortical alveolus lectin, while it is quite different from that of PSGP, which is extensively expressed at the yolk vesicle stage and down-regulated at later stages. Thus, not all cortical alveolus components are transcribed concomitantly. This is the first description of a developmental change in the transcription of a glycosyltransferase during oogenesis.

To analyze the influence of hepatitis C virus nonstructural protein 5A (NS5A) on apoptosis, we established Huh7 cells that stably express NS5A, and induced apoptosis using tumor necrosis factor (TNF)-alpha. The viability of control Huh7 cells was reduced to 40%, compared with untreated cells, after TNF-alpha treatment, whereas that of Huh7-NS5A cells was reduced only to 80%. DNA fragmentation also decreased to <50% in Huh7-NS5A compared with control cells. Nuclear factor-kappaB activation was the same in both cell types, whereas caspase-8, -9, and -3 activity was decreased in Huh7-NS5A cells, compared with control cells, which indicates that the inhibition of caspase-8 activation is responsible for the antiapoptotic effect of the NS5A protein. The coexpression of NS5A did not inhibit apoptosis induced by caspase-8 or Fas-associating death domain protein expression. These findings suggest that the NS5A protein inhibits the apoptotic effect of TNF-alpha upstream of caspase-8 in the apoptosis cascade.

Human umbilical cord blood (UCB) cells have many advantages as grafts for cell transplantation because of the immaturity of newborn cells compared with adult cells. In contrast to their hematopoietic and mesenchymal potential, it remains unclear whether UCB cells have endodermal competence. Here, with a view to utilize UCB cells for cell transplantation into injured liver, we investigated the hepatic potential of UCB cells both in vitro and in vivo. We determined the most efficient conditions leading UCB cells to produce albumin (ALB). In a novel primary culture system supplemented with a combination of growth/differentiation factors, about 50% of UCB cells in 21-day cultures expressed ALB, and the ALB(+) cells coexpressed hepatocyte lineage markers. The ALB-expressing cells were able to proliferate in the culture system. Moreover, in the cell-transplantation model into liver-injured severe combined immunodeficient mice, inoculated UCB cells developed into functional hepatocytes in the liver, which released human ALB into the sera of the recipient mice. In conclusion, this study demonstrates that human UCB is a source of transplantable hepatic progenitor cells. Our findings may have relevance to clinical application of UCB-derived cell transplantation as a novel therapeutic option for liver failure.

A 64-year-old man who underwent hepatectomy for hepatocellular carcinoma (HCC) 4 years ago followed by transarterial chemoembolization for recurrent foci 1 year later had a thrombus in the inferior vena cava (IVC). Tumor thrombus derived from HCC was suspected owing to the increase in size and hepatic arteriography was performed. Common hepatic arteriography demonstrated a small stain suggesting a recurrent HCC in the remnant liver. However, CT during hepatic arteriography revealed that the stain localized not in the liver but within the thrombus in the IVC; there was no recurrent HCC in the liver. The dense stain associated with thin neovasculature developed through the thrombus was recognized. The thrombus spontaneously regressed 8 months later. It was speculated that the neovasculature played an important role in a process of absorption of the thrombus. One should note that the stain shown on angiography does not always suggest that the lesion is localized in the liver and is malignant.

Background: This study was conducted to investigate the recommended dose of paclitaxel for use in combination with a fixed dose of carboplatin and to evaluate the toxicity and efficacy of carboplatin-paclitaxel combination chemotherapy in patients with epithelial ovarian cancer., Methods: One hundred and ten patients were enrolled in the Phase I/II study and 97 patients were evaluated for further analysis, excluding 13 ineligible patients or patients with infringement of protocol: 15 patients for the Phase I and 82 for the Phase II study. In the Phase I trial, we studied dose escalation using a carboplatin dose of AUC 5 and paclitaxel levels of 150, 175 and 200 mg/m(2). The grades of toxicity of the regimen of all patients enrolled in the Phase II study (n = 82), the progression-free survival time (PFS) of optimal-debulked patients and complete responders (n = 62) and the response rate of suboptimal-debulked patients (n = 39) were investigated., Results: After observing grade 4 neutropenia in four of six patients in the paclitaxel 200 mg/m(2) administration group, we chose 175 mg/m(2) as the recommended dose of paclitaxel in this regimen. At this dose, the median of PFS and response rate were 432 days (range, 19-907 days) and 66.7%, respectively., Conclusion: Combination chemotherapy using paclitaxel 175 mg/m(2) and carboplatin AUC 5 is very well tolerated and highly effective for the treatment of ovarian cancer.

To explore the relationship between responses to interferon (IFN) and the mutation patterns in the IFN sensitivity-determining region (ISDR; amino acid positions 2209-2248) in the NS5A gene of hepatitis C virus genotype 1b, a cohort of 334 patients was analyzed. The number of mutations in the ISDR was higher in patients with sustained response (SR) than in patients with transient or no response (P<.001). Patients with viruses mutated at positions 2209 (P=.02), 2216 (P=.01), or 2227 (P=.02) more frequently experienced SR than did those without these mutations. Mutation occurred most frequently at position 2218, where the presence of cysteine was significantly associated with SR. Thus, the mutation pattern in the ISDR affects the virologic response to IFN and reflects different influences on the function of the NS5A protein. ISDR sequence analysis would allow the prediction of clinical IFN efficacy in individual patients.

Merocyanine 540 (MC540) is a widely used dye probe for membranous environments. However, fundamental knowledge of the spectral features of this dye in aqueous and hydrophobic environments is still lacking. Such knowledge is important because biomembranes involve a hydrophobic environment surrounded by a hydrophilic environment. Because many investigations so far have been performed based on indistinct spectral estimations, the interpretation of the data obtained using this dye as a fluorescent transmembrane probe remains controversial. In order to determine the exact spectra in both aqueous and hydrophobic environments, we adopted principal factor analysis (PFA), a method of multivariate analysis. The PFA method can also determine the number of molecular species present in the reaction mixture, which is three in pure water and two in phospholipid suspension. Two of the species in both water and phospholipid suspension were the monomer and dimer. The third species in water was the trimer, but its amount was so small at 10 microM MC540 solution that the spectral data in water can be approximated neglecting this molecular species. The monomer spectrum changed its form markedly with a bathochromic shift when transferred from the water to phospholipid environment, whereas the dimer remained similar in its shape except for a remarkable red shift. In water, the dissociation constants, K(1) and K(2), for the assumed stacking-model reactions, M+M <--> M(2) and M+M(2) <--> M(3), were 3.1 x 10(-4) M and 5.7 x 10(-4) M, respectively. In the phospholipid environment, the dissociation constant K* for the assumed stacking-model reaction, M(*)+M(*) <--> *M(2), was 1.9x10(-5)M. The fluorescent intensities of MC540 were also measured in both water and phospholipid environments. A comparison based on the absorption and fluorescence spectra suggested that the temporal increase in the amount of the monomer on the excitable membrane contributes to the fluorescent intensity change observed in the transmembrane potential change.

The extracellular domain of human EGF receptor (sEGFR) produced by CHO cells has been used in various biophysical studies to elucidate the molecular mechanism of EGF-induced receptor activation. We have found that the CHO sEGFR contains one oligosaccharide chain attached to an atypical N-glycosylation consensus sequence, Asn(32 )-X( 33 )-Cys(34 ). The oligosaccharide structure at Asn(32 ) is a mixture of the monosialo and asialo forms of a core fucosylated biantennary complex-type oligosaccharide. Deletion of this atypical glycosylation site by replacement of Asn(32 ) with lysine changed neither the expression nor function of the full length EGFR in CHO cells. The glycosylation at Asn(32 ) in CHO sEGFR was incomplete: 20% of Asn(32 ) remained unmodified. Thus, CHO sEGFR itself is heterogeneous with respect to the glycosylation at Asn(32 ), which may cause problems in biophysical studies. An attempt to remove the oligosaccharide at Asn(32 ) enzymatically did not succeed under nondenaturing conditions. Therefore, sEGFR with the mutation of Asn(32) -> Lys(32 )is useful for biophysical and biochemical studies, and, particularly, for X-ray crystallography.

The prevalence and clinical significance of naturally occurring mutations in the full-length surface and overlapping polymerase genes of hepatitis B virus (HBV) were analyzed in 42 patients with chronic hepatitis. Mutations were observed in 10 patients (24%) in the a determinant region, which is the neutralizing epitope within the major hydrophilic region of the surface gene. A high proportion of these mutations (17/18; 94%) occurred in the first loop, unlike mutations induced by immunization. The presence of serum antibody to hepatitis B surface antigen was significantly associated with these mutations. No other region of the surface gene contained any cluster of mutations. These results suggest that escape mutations commonly contribute to persistency in the natural course of HBV infection. In contrast, mutations affecting the major catalytic domains of the polymerase gene, which could alter susceptibility to antiviral nucleoside analogues, were not detected at all.

A close correlation was reported between amino acid mutations in the nonstructural protein 5A (interferon [IFN] sensitivity-determining region [ISDR]) of hepatitis C virus (HCV)-1b and the response to IFN therapy. The dynamic change of ISDR quasispecies during IFN treatment was investigated in 22 patients. In 18 nonresponders, the number of ISDR mutations in major quasispecies decreased after therapy (P=.039). In each nonresponder, the percentage of wild-type (no mutations in the ISDR) quasispecies increased after treatment (P=.008), whereas the percentages of intermediate- (1-3 mutations) and mutant-type (>/=4 mutations) quasispecies decreased (P=.037 and P=.043, respectively). No mutant-type quasispecies were detected after therapy. Four complete responders had only quasispecies with >/=3 mutations before therapy. Thus, HCVs have fewer mutations in the ISDR after IFN therapy than those before therapy. These IFN-resistant HCVs were already present before therapy as minor quasispecies and were selected by IFN in nonresponders.

To assess the interaction of alcohol and HCV infection in hepatocarcinogenesis, we prospectively studied 447 patients with liver cirrhosis (LC) who presented to our out-patient clinics in a month; 163 patients with habitual drinking (AL-LC) who had taken more than 72 g alcohol per day (HCV positive 79 cases: HCV+AL; HCV negative 84 cases: AL); 176 with HCV infection but without alcohol intake; 39 with HB infection; and 82 with liver disease from other etiologies such as primary biliary cirrhosis (PBC). In the HCV group, HCC developed in 15 patients in the first year and 10 in the second year; the cumulative appearance rate was 11% and 16%, respectively. There was no difference in the HCC appearance rate between the two groups. In the AL group, the cumulative HCC occurrence rate was only 2% in the first year, and 2% in the second year. The appearance rate was significantly lower in the AL group compared with the HCV and the HCV+AL groups. One-hundred and fourteen patients (94 with HCV, 20 with HCV+AL) who had a history of blood transfusion more than 10 years ago were selected. A year-adjusted disease occurrence rate calculated by the Kaplan-Meier method showed that the HCV+AL group had a significantly higher disease occurrence rate than the HCV group. Theses results suggest that although alcohol alone does not become an independent risk factor for HCC from LC, it may accelerate the development of HCC caused by HCV, at least in the group with a history of blood transfusion.

To determine risk factors for hepatocellular carcinoma (HCC) in Japan, we studied 575 patients with liver cirrhosis (LC) who presented to our out-patient clinics in a month; 217 patients with habitual drinking with or without viral infections (AL-LC), 235 with hepatitis C virus (HCV) infection, 40 with hepatitis B virus (HB) infection, and 82 with liver diseases from other aetiologies such as primary biliary cirrhosis (PBC). HCC was found in 28% of AL-LC, 27% of HCV-LC, and 18% of HB-LC. HCV antibody examined by the second-generation HCV antibody assay (HCV II) was positive in 49% of AL-LC without HCC, and 84% of AL-LC with HCC. HCV was significantly associated with the development of HCC with an odds ratio of 1.90 by multivariate analysis, whereas alcohol alone did not become a risk factor, with an odds ratio of 0.65. When both variables were combined (HCV+AL), however, the odds ratio was increased twice to 3.65. One-hundred and thirteen patients who had a history of blood transfusion more than 10 years ago were selected to assess the interaction between alcohol and HCV in hepatocarcinogenesis. A year-adjusted disease occurrence rate calculated by the Kaplan-Meyer method showed that HCV+AL had a significantly higher disease occurrence rate than HCV alone. These results suggest that although alcohol alone may not become a risk factor for HCC, it may potentiate the development of HCC caused by HCV. HCV+AL is considered to be the highest risk group; strict abstinence is necessary for these patients.

A dot blot hybridization procedure was developed to detect human blood meals in engorged mosquitoes. A biotinylated DNA probe allowed the detection of 10-100 ng of human DNA, discriminated well between human and nonhuman sources of blood, and cross-reacted only with monkey DNA. Results showed that this method was a specific and sensitive technique for the identification of blood meals up to 100 h after ingestion. The nonisotopic label offers easy handling without the problems inherent in the use of radioisotopes, and it can be adapted for use in routine field tests.