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We developed phosphorylation reagents with a nitrobenzyl hydrophobic tag and used them for 5'-phosphorylation of chemically or transcriptionally synthesized RNA. The capability of hydrophobic tags to synthesize 5'-monophosphorylated RNA was evaluated based on the yield of the desired oligonucleotides, stability of protecting groups during cleavage/deprotection, separation ability in reverse-phase HPLC (RP-HPLC), and deprotection efficiency after RP-HPLC purification. The results showed that a nitrobenzyl derivative with a tert-butyl group at the benzyl position was most suitable for RNA 5'-phosphorylation. Using the developed phosphorylation reagent, we chemically synthesized 5'-phosphorylated RNA and confirmed that it could be purified by RP-HPLC and the following deprotection. In addition, we demonstrated complete chemical synthesis of minimal mRNA by chemical capping of 5'-monophosphorylated RNA. Ribonucleoside 5'-monophosphates with hydrophobic protecting groups have also been developed and used as substrates to transcriptionally synthesize 5'-phosphorylated RNA with >1000 bases. From the mixture of the by-products and the desired RNA, only 5'-monophosphorylated RNA could be effectively isolated by RP-HPLC. Furthermore, monophosphorylated RNA can be converted into circular mRNA via RNA ligase-mediated cyclization. Circular mRNA expression of nanoluciferase in cultured cells and mice. These techniques are important for the production of chemically synthesized mRNA and circular mRNA., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Objective: The Japanese Psycho-Oncology Society and the Japanese Association of Supportive Care in Cancer have recently revised the clinical practice guidelines for delirium in adult cancer patients. This article reports the process of developing the revised guidelines and summarizes the recommendations made., Methods: The guidelines were developed in accordance with the Medical Information Network Distribution Service creation procedures. The guideline development group, consisting of multi-disciplinary members, created three new clinical questions: non-pharmacological intervention and antipsychotics for the prevention of delirium and trazodone for the management of delirium. In addition, systematic reviews of nine existing clinical questions have been updated. Two independent reviewers reviewed the proposed articles. The certainty of evidence and the strength of the recommendations were graded using the grading system developed by the Medical Information Network Distribution Service, following the concept of The Grading of Recommendations Assessment, Development, and Evaluation system. The modified Delphi method was used to validate the recommended statements., Results: This article provides a compendium of the recommendations along with their rationales, as well as a short summary., Conclusions: These revised guidelines will be useful for the prevention, assessment and management of delirium in adult cancer patients in Japan., (© The Author(s) 2023. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permission@oup.com.)

A subcortical pathway through the superior colliculus and pulvinar has been proposed to provide the amygdala with rapid but coarse visual information about emotional faces. However, evidence for short-latency, facial expression-discriminating responses from individual amygdala neurons is lacking; even if such a response exists, how it might contribute to stimulus detection is unclear. Also, no definitive anatomical evidence is available for the assumed pathway. Here we showed that ensemble responses of amygdala neurons in monkeys carried robust information about open-mouthed, presumably threatening, faces within 50 ms after stimulus onset. This short-latency signal was not found in the visual cortex, suggesting a subcortical origin. Temporal analysis revealed that the early response contained excitatory and suppressive components. The excitatory component may be useful for sending rapid signals downstream, while the sharpening of the rising phase of later-arriving inputs (presumably from the cortex) by the suppressive component might improve the processing of facial expressions over time. Injection of a retrograde trans-synaptic tracer into the amygdala revealed presumed monosynaptic labeling in the pulvinar and disynaptic labeling in the superior colliculus, including the retinorecipient layers. We suggest that the early amygdala responses originating from the colliculo-pulvino-amygdalar pathway play dual roles in threat detection., (© The Author(s) 2022. Published by Oxford University Press.)

Objectives: We aimed to clarify the clinical implication of ultrasound (US)-detected foot joint inflammation in tightly controlled patients with rheumatoid arthritis (RA)., Methods: We evaluated bilateral foot joints (second to fifth metatarsophalangeal joints of forefoot; tarsometatarsal, cuneonavicular and midtarsal joints of midfoot) of 430 RA patients for synovitis using Power Doppler (PD) imaging by US. We made a cross-sectional and a 3-year longitudinal analysis about the associations of US-detected synovitis with clinical, laboratory and radiographic data as well as foot-specific outcomes using a self-administered foot evaluation questionnaire (SAFE-Q)., Results: The US-detected foot synovitis was seen in 28% of patients. The US-detected synovitis was closely related to 28 joint-disease activity score (DAS28) more in the forefoot than in the midfoot, while related to joint destruction in both. Multiple regression analyses showed significant associations between midfoot PD positivity and SAFE-Q in the remission group. SAFE-Q was worsened after the 3-year interval, but PD positivity at baseline did not contribute to the changes. On the other hand, destruction of the joints with US-detected synovitis significantly progressed in 3 years than with not., Conclusions: US-detected synovitis on foot joints were related to systemic inflammation, clinical symptoms, and future joint destruction with region specificity., (© 2021 Japan College of Rheumatology.)

Objective: Although recent clinical trials showed that ultrasound (US) remission is not required to achieve good outcomes at the group level, it currently remains unclear whether the prognosis of individual patients in clinical remission, but not US remission, i.e. those with subclinical sonographic synovitis (SSS), is favorable. However, it is no longer acceptable to perform US on all patients in order to identify those with SSS. Therefore, the present study was initiated to elucidate the conditions under which SSS is frequently detected., Methods: In total, 563 consecutive RA patients were recruited. Bilateral 2-5 MCP, wrist, ankle, and 2-5 MTP joints were scanned by US, and Gray scale and Power Doppler (PD) images were scored semi-quantitatively. Clinical data were obtained by physicians who were blind to US results. Changes in the modified Total Sharp Score (mTSS) of tocilizumab (TCZ) users were calculated., Results: A total of 402 patients were included. SSS was more frequently detected in patients with more severe joint deformity, even if they were in remission. In contrast, a high Patient Global Assessment of Disease (PtGA) did not reflect SSS. Furthermore, the relationship between PtGA and PD scores was weak. Although the frequency of SSS was high in TCZ user, the presence of SSS in TCZ users not always results in the progression of mTSS., Conclusions: While remission is overestimated in patients with severe joint deformity, underestimations may occur in those who do not fulfill remission criteria because of a high PtGA.

Background: The Japanese Psycho-Oncology Society and Japanese Association of Supportive Care in Cancer recently launched the clinical practice guidelines for delirium in adult cancer patients. The aim of the guidelines was to provide evidence-based recommendations for the clinical assessment and management of delirium in cancer patients. This article reports the process of developing the guideline and summarizes the recommendations made., Methods: The guidelines were developed in accordance with the Medical Information Network Distribution Service creation procedures. The guideline development group, consisting of multidisciplinary members, formulated nine clinical questions. A systematic literature search was conducted to identify relevant articles published prior to through 31 May 2016. Each article was reviewed by two independent reviewers. The level of evidence and the strength of the recommendations were graded using the grading system developed by the Medical Information Network Distribution Service, following the concept of The Grading of Recommendations Assessment, Development and Evaluation system. The modified Delphi method was used to validate the recommendation statements., Results: This article provides a summary of the recommendations with rationales for each, as well as a short summary., Conclusions: These guidelines will support the clinical assessment and management of delirium in cancer patients. However, additional clinical studies are warranted to further improve the management of delirium., (© The Author(s) 2020. Published by Oxford University Press.)

In the history of viral research, one of the important biological features of bacteriophage Mu is the ability to expand its host range. For extending the host range, the Mu phage encodes two alternate tail fibre genes. Classical amber mutation experiments and genome sequence analysis of Mu phage suggested that gene products (gp) of geneS (gpS = gp49) and gene S' (gpS' = gp52) are tail fibres and that gene products of geneU (gpU = gp50) and geneU' (gpU' = gp51) work for tail fibre assembly or tail fibre chaperones. Depending on the gene orientation, a pair of genes 49-50 or 52-51 is expressed for producing different tail fibres that enable Mu phage to recognize different host cell surface. Since several fibrous proteins including some phage tail fibres employ their specific chaperone to facilitate folding and prevent aggregation, we expected that gp50 or gp51 would be a specific chaperone for gp49 and gp52, respectively. However, heterologous overexpression results for gp49 or gp52 (tail fibre subunit) together with gp51 and gp50, respectively, were also effective in producing soluble Mu tail fibres. Moreover, we successfully purified non-native gp49-gp51 and gp52-gp50 complexes. These facts showed that gp50 and gp51 were fungible and functional for both gp49 and gp52 each other., (© The Author(s) 2019. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.)

Aberrant wound healing presents as inappropriate or insufficient tissue formation. Using a model of musculoskeletal injury, we demonstrate that loss of transforming growth factor-β activated kinase 1 (TAK1) signaling reduces inappropriate tissue formation (heterotopic ossification) through reduced cellular differentiation. Upon identifying increased proliferation with loss of TAK1 signaling, we considered a regenerative approach to address insufficient tissue production through coordinated inactivation of TAK1 to promote cellular proliferation, followed by reactivation to elicit differentiation and extracellular matrix production. Although the current regenerative medicine paradigm is centered on the effects of drug treatment ("drug on"), the impact of drug withdrawal ("drug off") implicit in these regimens is unknown. Because current TAK1 inhibitors are unable to phenocopy genetic Tak1 loss, we introduce the dual-inducible COmbinational Sequential Inversion ENgineering (COSIEN) mouse model. The COSIEN mouse model, which allows us to study the response to targeted drug treatment ("drug on") and subsequent withdrawal ("drug off") through genetic modification, was used here to inactivate and reactivate Tak1 with the purpose of augmenting tissue regeneration in a calvarial defect model. Our study reveals the importance of both the "drug on" (Cre-mediated inactivation) and "drug off" (Flp-mediated reactivation) states during regenerative therapy using a mouse model with broad utility to study targeted therapies for disease. Stem Cells 2019;37:766-778., (©AlphaMed Press 2019.)

Objective: It is known that depression and anxiety occur more frequently in pancreatic cancer patients than in those with other malignancies. However, few studies have assessed depression and anxiety using reliable psychiatric diagnostic tools. The purpose of this study was to determine the prevalence of depression and anxiety among pancreatic cancer patients before and 1 month after the start of anticancer treatment using reliable psychiatric diagnostic tools, and to identify factors that predict their occurrence., Methods: Pancreatic cancer patients were consecutively recruited. Structured clinical interviews were used to determine the presence of affective disorders, anxiety disorders and adjustment disorders. Baseline interviews were performed prior to initiation of anticancer treatment, while follow-up interviews were performed 1 month after treatment was started. Medical, demographic and psychosocial backgrounds were also assessed as predictive factors., Results: One hundred and ten patients participated in the baseline interview and 91 in the follow-up interview. Depression and anxiety were observed in 15 patients (13.6%) at the baseline, and 15 patients (16.5%) at the follow-up. Lack of confidants was associated with depression and anxiety at the baseline. At the baseline, sadness, lower Karnofsky Performance Status and prior experience with the death of a family member due to cancer predicted newly diagnosed depression and anxiety at the follow-up., Conclusion: A considerable percentage of pancreatic cancer patients experienced depression and anxiety. Multidimensional psychosocial predictive factors were found and optimal psychological care should incorporate early detection of sadness., (© The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Because of unique linguistic characteristics, the prevalence rate of developmental dyslexia is relatively low in the Japanese language. Paradoxically, Japanese children have serious difficulty analysing phonological processes when they have dyslexia. Neurobiological deficits in Japanese dyslexia remain unclear and need to be identified, and may lead to better understanding of the commonality and diversity in the disorder among different linguistic systems. The present study investigated brain activity that underlies deficits in phonological awareness in Japanese dyslexic children using functional magnetic resonance imaging. We developed and conducted a phonological manipulation task to extract phonological processing skills and to minimize the influence of auditory working memory on healthy adults, typically developing children, and dyslexic children. Current experiments revealed that several brain regions participated in manipulating the phonological information including left inferior and middle frontal gyrus, left superior temporal gyrus, and bilateral basal ganglia. Moreover, dyslexic children showed altered activity in two brain regions. They showed hyperactivity in the basal ganglia compared with the two other groups, which reflects inefficient phonological processing. Hypoactivity in the left superior temporal gyrus was also found, suggesting difficulty in composing and processing phonological information. The altered brain activity shares similarity with those of dyslexic children in countries speaking alphabetical languages, but disparity also occurs between these two populations. These are initial findings concerning the neurobiological impairments in dyslexic Japanese children.

Background: There have been no longitudinal studies in Japan examining national-level data for suicide risk by marital and employment status. We examined the age-adjusted relative suicide risk (RR) by marital and employment status from national data acquired for all suicides in Japan occurring in the past 25 years., Methods: All deaths identified as suicides according to ICD-9 and ICD-10 were extracted from vital statistics data of Japan for the years 1980, 1985, 1990, 1995, 2000 and 2005. Population statistics for Japanese residents aged ≥15 years were obtained from the census., Results: Suicide rates for almost all categories analyzed decreased in both genders between 1985 and 1990 and increased between 1995 and 2000, especially among men. Unemployed and divorced men had a consistently higher RR in each year analyzed. Unemployed and divorced women had a higher risk than those in other categories, especially in 2000 and 2005. In women, particularly in 1980, 1985 and 1990, those who were unemployed and never married had a similar RR to those who were unemployed and divorced., Conclusions: Unemployed and divorced people were at a high risk of suicide over the past 25 years, particularly in 2000 and 2005. Our findings suggest that the effects of divorce and unemployment on suicide risk are synergistic.

Colostrum is a complex mixture of bioactives that promotes neonate growth. Recently, we have found by in vivo study that skimmed, sterilized, and concentrated bovine late colostrum (SCBLC), obtained from a Holstein herd on days 6-7 after parturition, had an ability to maintain intestinal integrity. In the present study we investigated effects of SCBLC on rat intestinal IEC-6 cell proliferation in vitro. A fraction containing αs1-casein was found to have a robust stimulation effect as compared to other protein fractions from SCBLC and even the αs1-casein fraction from milk from other Holstein herds. Furthermore, the SCBLC αs1-casein molecule demonstrated not only slightly slower mobility on both SDS- and native-PAGE than other bovine milk αs1-caseins, but also a peculiar conformation reminiscent of moltenglobule in the circular dichroism spectrum. These findings may be of relevant to the competence of SCBLC to preserve intestinal integrity.

Native alpha-lactalbumins (α-LA) from equine, bovine, and human milk were not cytotoxic. However, after treatment with trifluoroethanol (TFE), all three α-LAs exhibited cytotoxicity. Toxic potencies were distinctly different among them. Equine α-LA was the most robust, bovine α-LA was moderate, and human α-LA was weak. There were no significant structural changes as between the native and the TFE-treated α-LAs.

Context: Neuropilin-2 (Nrp2) is a coreceptor for vascular endothelial growth factor-D (VEGF-D) that is expressed on the surface of endothelial cells. Recently, Nrp2 was shown to play a role in lymph node metastasis and promotion of cancer cell migration. VEGF-D also promotes lymphangiogenesis, which in turn promotes tumor metastasis., Objective: The aim was to study the role of neuropilin-2 in lymph node metastasis in human papillary thyroid carcinoma (PTC)., Design: Expression of Nrp2 was studied by immunohistochemistry and the relationship between Nrp2 expression and lymph node metastasis, VEGF-D expression and other established clinicopathological variables were analyzed in PTC. The effects of neutralizing anti-Nrp2 antibody on VEGF-D-induced invasion and migration were assessed in PTC cell lines., Results: Nrp2 expression was observed in 64.3% (36 of 56) of the PTC patients. Nrp2 expression was significantly correlated with lymph node metastasis (P = 0.0216) and VEGF-D expression (P = 0.0034). VEGF-D was shown to promote filopodia formation and cancer cell migration and invasion by K1 and B-CPAP cells. These responses were significantly blocked by neutralizing anti-Nrp2 antibody., Conclusion: Nrp2 expression was correlated with lymph node metastasis and VEGF-D expression in PTC. Our data also showed a role for Nrp2 in regulating VEGF-D-induced invasion and migration in vitro.

Previous studies have reported the existence of an association between brain-derived neurotrophic factor and major depression. However, the possible role of brain-derived neurotrophic factor in the pathophysiology of major depression after cancer diagnosis has not yet been investigated. Subjects were collected using the Lung Cancer Database project. Using the cut-off scores on the depression subscale of the Hospital Anxiety and Depression Scale (HADS-D), 81 subjects with depression (HADS-D > 10) and 81 subjects without depression (HADS-D < 5) were selected. The two groups were matched for age, sex, clinical stage and performance status. The serum brain-derived neurotrophic factor levels were measured using an enzyme-linked immunosorbent assay method. The serum brain-derived neurotrophic factor levels were not statistically different between the subjects in the depression group [29.1 (13.6) ng/ml; mean (SD)] and the non-depression group [31.4 (10.6) ng/ml] (P = 0.22). In a stratified analysis by gender, however, the mean serum brain-derived neurotrophic factor level in the depression group tended to be lower than that in the non-depression group among women (n = 24 pairs, P = 0.06). Major depression after cancer diagnosis is not associated with serum brain-derived neurotrophic factor levels.

We purified two isozymes of coniferyl alcohol dehydrogenase (CADH I and II) to homogeneity from cell-free extracts of Streptomyces sp. NL15-2K. The apparent molecular masses of CADH I and II were determined to be 143 kDa and 151 kDa respectively by gel filtration, whereas their subunit molecular masses were determined to be 35,782.2 Da and 37,597.7 Da respectively by matrix-assisted laser-desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS). Thus, it is probable that both isozymes are tetramers. The optimum pH and temperature for coniferyl alcohol dehydrogenase activity were pH 9.5 and 45 °C for CADH I and pH 8.5 and 40 °C for CADH II. CADH I oxidized various aromatic alcohols and allyl alcohol, and was most efficient on cinnamyl alcohol, whereas CADH II exhibited high substrate specificity for coniferyl alcohol, and showed no activity as to the other alcohols, except for cinnamyl alcohol and 3-(4-hydroxy-3-methoxyphenyl)-1-propanol. In the presence of NADH, CADH I and II reduced cinnamaldehyde and coniferyl aldehyde respectively to the corresponding alcohols.

Previous rotavirus infection studies used the focus reduction assay extensively to evaluate cellular responses to viral infection, but this technique has a number of limitations. In this study, we developed a simplified, accurate rotavirus infection assay to evaluate the effects of inhibitory substances on rotavirus infection in vitro by measurement of the fluorescence intensities of stained cells.

We investigated the effects of vitamin C administration on vitamin C-specific transporters in ODS/ShiJcl-od/od rat livers. The vitamin C-specific transporter levels increased in the livers of the rats not administered vitamin C and decreased in the livers of those administered vitamin C at 100 mg/d, indicating that these transporter levels can be influenced by the amount of vitamin C administered.

Clostridial cellulosomes are cellulolytic complexes that are formed by highly specific interactions between one of the repeated cohesin modules present in the scaffolding protein and a dockerin module of the catalytic components. Although Clostridium thermocellum Xyn11A dockerin has a typical C. thermocellum dockerin sequence, in which two amino acid residues are species specifically conserved within the two segments of the dockerin modules, it can recognize Clostridium josui cohesin modules in a non-species-specific manner. The importance of these two conserved amino acids, which are part of the recognition site of the cohesin and dockerin interaction, was investigated by introducing mutations into the first and/or the second segments of the Xyn11A dockerin. Mutations in the first segment did not affect the interactions between dockerin and C. thermocellum and C. josui cohesins. However, mutations in the second segment prevented binding to cohesin proteins. A second round of mutations within the first segment re-established the affinity for both the C. thermocellum and the C. josui cohesins. However, this was not observed for a 'conventional' dockerin, Xyn10C. These results suggest that the combination of the first and second dockerin segments is important for the target recognition., (© 2010 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.)

We found that skimmed and concentrated bovine late colostrum (SCBLC) obtained from normal cows at 6-7 d after parturition exhibited high potency in inhibiting replication of human rotavirus (HRV) in vitro. Furthermore, prophylactic oral administration of SCBLC once before inoculation of HRV prevented the development of diarrhea in suckling mice in vivo. SCBLC from normal cows might be useful in the prevention of HRV-induced severe gastroenteritis in immunocompromised hosts.

Lactophorin is a heat-stable phosphoglycoprotein, also known as milk glycosylation-dependent cell adhesion molecule 1 (GlyCAM-1). Bovine 18 kDa lactophorin was purified by heparin affinity chromatography from cow's milk whey. Its N-glycans were obtained by proteomic techniques, including two-dimensional polyacrylamide gel electrophoresis (2D-PAGE), followed by in-gel digestion with peptide-N(4)-(N-acetyl-beta-glucosaminyl)-asparagine amidase (PNGase F). The released N-glycans were derivatized with 2-aminopryridine (PA) and analyzed by matrix-assisted laser desorption ionization quadruple ion trap time of flight mass spectrometry (MALDI-QIT-TOF MS). Among the MS analyzed peaks, 15 peaks were found to be N-glycan molecules as detected by MS(2) analysis. These glycans consisted of mono-sialylated bi-, tri-, and tetra-antennary complex-type N-glycans carrying Gal-GlcNAc (LacNAc) or GalNAc-GlcNAc (LacdiNAc) with and without core-fucose.

Rotaviruses are the leading cause of severe dehydrating diarrhea in children worldwide. We have found that high-M(r) glycoprotein fraction (F1) from cow's milk whey has potent inhibitory activity against human rotavirus (HRV) in cell culture. The present study was undertaken to identify and characterize the components responsible for this inhibitory activity. F1 was initially heated at 95 degrees C for 30 min, rendering milk antibodies inert, subjected to ammonium sulfate fractionation, and then resolved by two-dimensional polyacrylamide gel electrophoresis. After electroelution, we found that a heat-stable milk protein lactophorin C-terminal fragment (LP16) and bovine milk fat globule membrane protein PAS6/7 strongly inhibited the replication of HRV MO strains in MA104 cells. Furthermore, we found that prophylactic oral administration of F1 once before inoculation of the HRV MO strain obviously prevented the development of diarrhea in vivo. These non-immunoglobulin components are a promising candidate for a prophylactic food additive against HRV infection.

Cellulosomes are cellulolytic complexes produced by anaerobic bacteria, and are composed of a scaffolding protein and several catalytic components. The complexes are formed by highly specific interactions of one of the reiterated cohesin modules of the scaffolding protein with a dockerin module of the catalytic components. The affinities of a dockerin module of Clostridium thermocellum CelJ (Cel9D-Cel44A) for several cohesin modules from C. thermocellum and Clostridium josui scaffolding proteins were quantitatively measured by surface plasmon resonance analysis. The recombinant CelJ dockerin-containing protein interacted with three recombinant C. josui cohesin proteins as well as recombinant C. thermocellum cohesin proteins beyond the so-called 'species specificity' of the dockerin and cohesin interactions. However, this protein did not recognize a second cohesin module from the C. josui scaffolding protein, suggesting that the catalytic components are not necessarily arranged randomly on a scaffolding protein in native cellulosomes.

Objective: This study was conducted to evaluate the efficacy and safety of pegylated liposomal doxorubicin (PLD) in Japanese patients with Müllerian carcinoma having a therapeutic history of platinum-based chemotherapy., Methods: Patients who were diagnosed with Müllerian carcinoma (epithelial ovarian carcinoma, primary carcinoma of fallopian tube and peritoneal carcinoma) by histological examination and had received the initial platinum-based chemotherapy were included in the study. The study drug was administered to the patients at 50 mg/m(2) every 4 weeks., Results: Seventy-four patients were enrolled in the study. All patients had received platinum-based chemotherapy as first-line regimen and more than 90% of patients had also received taxanes. The overall response rate was 21.9% (95% confidence interval, 13.1-33.1%) and 38.4% of patients had stable disease. The median time to progression was 166 days. The major non-haematological toxicities were hand-foot syndrome (Grade 3; 16.2%) and stomatitis (Grade 3; 8.1%). Myelosuppression such as leukopenia (Grade 3; 52.7%, Grade 4; 6.8%), neutropenia (Grade 3; 31.1%, Grade 4; 36.5%) and decreased haemoglobin (Grade 3; 14.9%, Grade 4; 2.7%) were the most common haematological toxicities., Conclusion: We confirmed that a 50 mg/m(2) every 4 weeks regimen of PLD was active in Japanese patients with Müllerian carcinoma having a therapeutic history of platinum-based chemotherapy and toxicity was manageable by dose modification of PLD or supportive care.

The use of bovine brain has been prohibited in many countries because of the world-wide prevalence of mad cow disease, and thus porcine brain is expected to be a new source for the preparation of gangliosides. Here, we report the presence of a ganglioside in porcine brain which is strongly resistant to hydrolysis by endoglycoceramidase, an enzyme capable of cleaving the glycosidic linkage between oligosaccharides and ceramides of various glycosphingolipids. Five major gangliosides (designated PBG-1, 2, 3, 4, 5) were extracted from porcine brain by Folch's partition, followed by mild alkaline hydrolysis and PBA column chromatography. We found that PBG-2, but not the others, was strongly resistant to hydrolysis by the enzyme. After the purification of PBG-2 with Q-Sepharose, Silica gel 60 and Prosep-PB chromatographies, the structure of PBG-2 was determined by GC, GC-MS, FAB-MS and NMR spectroscopy as Fucalpha1-2Galbeta1-3GalNAcbeta1-4(NeuAcalpha2-3)Galbeta1-4Glcbeta1-1'Cer (fucosyl-GM1a). The ceramide was mainly composed of C18:0 and C20:0 fatty acids and d18:1 and d20:1 sphingoid bases. The apparent kcat/Km for fucosyl-GM1a was found to be 30 times lower than that for GM1a, indicating that terminal fucosylation makes GM1a resistant to hydrolysis by the enzyme. This report indicates the usefulness of endoglycoceramidase to prepare fucosyl-GM1a from porcine brain.

Docosahexaenoic acid (DHA, 22:6n-3)-containing phospholipids are a ubiquitous component of the central nervous system and retina, however their physiological and pharmacological functions have not been fully elucidated. Here, we report a novel DHA-containing phosphatidylcholine (PC) in a marine single cell eukaryote, Schizochytrium sp. F26-b. Interestingly, 31.8% of all the fatty acid in F26-b is DHA, which is incorporated into triacylglycerols and various phospholipids. In phospholipids, DHA was found to make up about 50% of total fatty acid. To identify phospholipid species containing DHA, the fraction of phospholipids from strain F26-b was subjected to normal phase high-performance liquid chromatography (HPLC). It was found that DHA was incorporated into PC, lyso-PC, phosphatidylethanolamine, and phosphatidylinositol. The major DHA-containing phospholipid was PC in which 32.5% of the fatty acid was DHA. The structure of PC was analyzed further by phospholipase A2 treatment, fast atom bombardment mass spectrometry, and 1H- and 13C-NMR after purification of the PC with reverse phase HPLC. Collectively, it was clarified that the major PC contains pentadecanoic acid (C15:0) at sn-1 and DHA at sn-2; the systematic name of this novel PC is therefore "1-pentadecanoyl-2-docosahexaenoyl-sn-glycero-3-phosphocholine."

Background: The lung cancer database project was established in 1999 at the National Cancer Center Hospital East, Japan, as an ongoing project to integrate data on various factors in lung cancer patients. The aim of the project was to construct a large-scale cancer registry for lung cancer that would contribute to basic research and clinical research in the future., Methods: Between July 1999 and July 2004, consecutive lung cancer patients were recruited into this project. The baseline survey consisted of self-administered questionnaires concerning various demographic data, health habits and psychological factors. Medical information was obtained from the patients' medical charts. Urine specimens and blood samples were collected, and DNA was extracted from blood lymphocytes., Results: Out of the 2506 patients who were asked to participate in the project, 2036 (81%) patients with newly diagnosed, untreated primary lung cancer were enrolled. The final analytic cohort consisted of 1995 patients. Virtually all of the 1995 patients (corresponding rate, 99%) completed the questionnaires on demographic data and health habits. The corresponding rates for the questionnaires on psychological factors and dietary habits were 99 and 94%, respectively. In a follow-up survey conducted to determine vital status as of December 2004, a total of 1051 patients (53%) had died and 44 patients (2%) were lost to follow-up., Conclusions: This paper overviews the rationale for initiating the lung cancer database project, Japan. This database should prove useful for researchers examining the pathogenesis of lung cancer and may contribute to the formulation of a framework for cancer treatment.

Objective: The purpose of the present study was to obtain preliminary findings regarding psychiatric disorders and background characteristics among Japanese family members of cancer patients., Methods: We investigated the psychiatric diagnosis and background factors of family members of cancer patients by analyzing the consultation data of patients referred to the Psychiatry Division, National Cancer Centre Hospital East, Japan., Results: Of a total of 1469 psychiatric consultation, 47 (3.2%) family members were referred, and 85% of them were spouses. The most common patient cancer site was the lung. Approximately one-half of the referred family encountered patients' end-of-life issues. The most common psychiatric diagnoses were adjustment disorders, followed by major depression., Conclusions: These preliminary findings suggest that psychosocial support for family members is not fully delivered and development of a comprehensive support system for caregivers of cancer patients is an urgent issue in the clinical oncology setting in Japan.

The contribution of the lipid part of lipopolysaccharide (LPS) to recognition by minor spike H protein of bacteriophage phiX174 was investigated by comparing the interactions of H protein with LPS and its deacylated derivatives. The fluorescence and circular dichroism (CD) spectra of H protein increased upon binding to intact LPS and a partially deacylated derivative. In contrast, completely deacylated derivatives showed lower affinities and almost no fluorescence or CD changes of H protein. These results demonstrate that the lipid part of LPS is responsible for the conformational change of minor spike H protein, which would function as a trigger for phage DNA ejection for infection of the host cell.

The interaction between the type-II dockerin domain of the scaffoldin protein CipA and the type-II cohesin domain of the outer layer protein SdbA is the fundamental mechanism for anchoring the cellulosome to the cell surface of Clostridium thermocellum. We constructed and purified a dockerin polypeptide and a cohesin polypeptide, and determined affinity constants of the interaction between them by the surface plasmon resonance method. The dissociation constant (K(D)) value was 1.8 x 10(-9) M, which is a little larger than that for the combination of a type-I dockerin and a type-I cohesin.

The binding of spike H and G proteins of bacteriophage phiX174 with lipopolysaccharides (LPSs) were evaluated by a competitive enzyme-linked plate assay using the biotin-labeled LPS of Escherichia coli C, one of a host strain, and the non-labeled LPSs having different R-core polysaccharide lengths. H protein promptly decreased its affinity when some saccharide residues were truncated from the outer R-core. However, G protein showed significant affinity to the LPSs lacking all the residues of the outer R-core and some of the inner R-core. Thus, G protein rather than H protein well recognized the residues of the inner R-core of LPS.

A histidine-tagged G protein of bacteriophage phi X174 (HisG) bound strongly with lipopolysaccharide (LPS) of Escherichia coli C, one of a phi X174-sensitive Ra strain. The dissociation constant, Kd, was measured to be 0.16 +/- 0.04 microM by fluorometric titration. HisG showed slightly less affinity to LPSs of the insensitive Rc and Rd 2 strains having shorter R-core polysaccharide sequences than that of the sensitive Ra strains. The difference between the two types of LPS was demonstrated by CD spectra; LPSs of the sensitive strains increased the signal intensity for beta-sheet, while the insensitive strains decreased it. The chemically degraded LPS derivatives lacking a hydrophobic lipid region showed much less affinity to HisG, indicating the importance of the lipid region of LPS for strong binding with HisG. On the other hand, since the degraded derivatives increased the intensity of CD spectra, the polysaccharide region is thought to contribute to the conformation change of the protein.

Background: The amount of dehydroascorbic acid contained within total ascorbic acid (oxidized as well as non-oxidized forms) in plasma, hereafter referred to as the dehydroascorbic acid fraction, may be a measure of oxidative stress during haemodialysis. In the present study, we determined this fraction in chronic haemodialysis patients., Methods: Using high performance liquid chromatography, dehydroascorbic acid and total ascorbic acid levels were measured in 80 maintenance haemodialysis patients for a period of > 2 years as well as in 49 controls, to examine a possible association of these compounds with clinical parameters and/or drugs taken by the patients., Results: Dialysis patients who had an increased plasma urate level (P < 0.05) and had been taking allopurinol (P < 0.05) or NSAID (non-steroid anti-inflammatory drugs) (P < 0.01), and dialysis patients who were younger (< or = 55 years), as compared with older dialysis patients (P < 0.01), were found to have a lower dehydroascorbic acid fraction by multivariate analysis. Mean plasma dehydroascorbic acid levels and dehydroascorbic acid fractions were significantly lower in the younger haemodialysis patients (4.8 +/- 0.7 micromol/l and 28.4 +/- 3.9%) than in healthy younger controls (13.3 +/- 1.1 micromol/l and 41.1 +/- 1.8%) (P < 0.0001 and P < 0.01, respectively). Moreover, a correlation was found between plasma dehydroascorbic acid fraction and plasma lipid peroxide (r = 0.66, P < 0.01) in patients who had not been taking allopurinol and/or NSAID., Conclusion: We found that dehydroascorbic acid fraction was related to patients' age, plasma urate level and to taking allopurinol or NSAID. Dehydroascorbic acid fraction may be another indirect index of oxidative stress.

Background: Serum creatine kinase-MB isoenzyme (CK-MB) is widely used as a marker of myocardial injury. We prepared recombinant human CK (r-hCK) MB isoenzyme and examined its potential for use as a control material for assay of CK-MB in serum., Methods: cDNAs encoding CK-M and CK-B subunits were inserted into the same plasmid vector, followed by transformation of Escherichia coli. The resulting three types of CK isoenzymes were purified by conventional chromatography., Results: The ratio of MB to MM to BB was 50:40:10 on the basis of CK activity. Highly purified CK-MB with a specific activity of 533 U/mg was produced in a yield of 5.7 mg/g of packed cells. Purified r-hCK-MB had the isoelectric point (pI 5.3) and molecular size (46 kDa for the subunit) of native CK-MB. Its immunoreactivity in an ELISA using antibody against native heart enzyme was similar to that of cardiac CK-MB. The r-hCK-MB retained >90% activity for at least 4 months at 11 degrees C in a delipidated serum matrix in a liquid form at a concentration of 118 U/L., Conclusions: r-hCK-MB shows key properties of the native cardiac isoenzyme and may be useful as a control and calibrator for serum assays of CK-MB.

The spike G protein of bacteriophage phiX174 was prepared as a hexa histidine-tagged G protein (HisG). In the enzyme-linked plate assay, HisG bound specifically to lipopolysaccharides (LPSs) of the phiX174-sensitive strains, and did not bind to LPSs of the phiX174-insensitive strains. The truncated G protein obtained after trypsin digestion of HisG had the similar affinity to the LPSs to HisG, indicating that eight amino acid residues from the N-terminus are not essential to the binding with the LPSs.

The spike H protein of bacteriophage phiX174 was prepared as a hexa histidine-tagged fusion (HisH). On enzyme-linked plate assaying, HisH was found to bind specifically to the lipopolysaccharides (LPSs) of phiX174-sensitive strains, Escherichia coli C and Salmonella typhimurium Ra chemotype, having the complete oligosaccharide sequence of the R-core on the LPSs. In sharp contrast, HisH bound weakly to the LPSs of phiX174-insensitive strains, i.e. E. coli F583 (Rd(2)) lacking some terminal saccharides and E. coli O111: B4 (smooth strain) having additional O-repeats on the R-core. The fluorescence spectra of HisH changed dose-dependently in the case of the LPS of E. coli C, the intensity increasing and the emission peak shifting to the shorter wavelength side, which was attributable to the hydrophobic interaction of HisH with the LPS. The binding equilibrium was analyzed by fluorometric titration to determine the dissociation constant K(d), 7.02 +/- 0.37 microM, and the Gibbs free energy change DeltaG(0), -29.1 kJ mol(-1) (at 22 degrees C, pH 7.4). Based on the temperature dependence of (K)d in a van't Hoff plot, the standard enthalpy change DeltaH(0) and the entropy change DeltaS(0) were calculated to be +23.7 kJ mol(-1) and 179 J mol(-1) K(-1) at 22 degrees C, respectively, and this binding was thereby concluded to be an entropy-driven reaction.

As the aqueous sphere has been proposed to be an important source medium for the virus infection of land animals, the glycolipids of some aquatic organisms were examined for human influenza A virus-binding activity. Active compounds were not found among the eight echinoderm gangliosides, but two active non-sialylated glycoglycerolipids were isolated from an aquatic bacterium, Corynebacterium aquaticum. The structural formula of one of them, H632A, was elucidated to be 1-14-methyl-hexadecanoyl-3-alpha-D-galactopyranosyl-(1-->3)-6-(12-met hyl-tetradecanoyl)-1-alpha-D-mannopyranosyl]-sn-glycerol. The latter together with reported one elsewhere, S365A, 1-14-methyl-hexadecanoyl-3-[alpha-D-mannopyranosyl-(1-->3)-6-(12-meth yl-tetradecanoyl)-1-alpha-D-mannopyranosyl]-sn-glycerol, apparently bound to three human influenza viruses, A/PR/8/34 (H1N1), A/Aichi/2/68 (H3N2), and A/Memphis/1/71 (H3N2), exhibiting 7-12% (H632A) and 10-22% (S365A) of the activities of the control substances (Neu5Acalpha2-3-paragloboside and Neu5Acalpha2-6- paragloboside). Additionally, these glycolipids were assumed to have virus-neutralizing activities for the following two reasons: (i) The hemagglutination and hemolysis activities of the viruses were inhibited by the glycolipid. (ii) The leakage of a cytosolic enzyme (lactate dehydrogenase) from Madin-Darby canine kidney cells on virus infection was prevented by the glycolipids to nearly the same extent as by fetuin. This is the first evidence of the binding- and neutralizing-abilities of native glycoglycerolipids as to influenza viruses.

Protein phosphorylation is a key regulatory mechanism of the organization and dynamics of the actin cytoskeleton during cell motility, differentiation, and cytokinesis. The level of protein phosphorylation is dependent on the relative activities of both protein kinases and protein phosphatases. In this paper, we examined the effect of phoslactomycins (PLMs) on the regulation of the cytoskeleton of NIH/3T3 fibroblasts. Treatment of cells with PLM-F (10 microM) induced actin filament depolymerization after 4 h. This effect was reversible and actin filaments were reformed 1 h after removal of the inhibitors. As PLM-F had no effect at all on polymerization of purified actin in vitro, it is thought that PLMs induce actin depolymerization through an indirect mechanism. An in vitro assay showed PLMs inhibited protein phosphatase 2A at lower concentrations (IC50 4.7 microM) than protein phosphatase 1. An in situ phosphorylation assay also revealed that PLM-F treatment stimulated the phosphorylation of intracellular vimentin. These results suggest that phoslactomycins are protein phosphatase 2A-specific inhibitors and that protein phosphatase 2A is involved in regulation of the organization of the actin cytoskeleton.

Aims: Brugada syndrome is considered to be a distinctive subgroup of idiopathic ventricular fibrillation. Identification of the circadian pattern of ventricular fibrillation would contribute to the elucidation of its underlying pathophysiology, but this pattern remains unknown in patients with Brugada syndrome., Methods: and Results A total of 12 consecutive Brugada syndrome patients (46+/-14 years, all male) who underwent implantation of an implantable cardioverter defibrillator were studied. The distribution of the time of ventricular fibrillation detection was examined and classified into four 6-hour time periods of the day. The mean follow-up period following implantation was 777+/-535 days. In six out of the 12 patients, ventricular fibrillation occurred during follow-up. The data logs revealed that ventricular fibrillation was detected 30 times (range, 3-9). Ventricular fibrillation was observed more frequently at night ( 1800 h to 0600 h) than in the day (0600 h to 1800 h) (93.3% [28/30] vs 6.7%[2/30], P<0.001), and during sleep than while awake (86.7% [26/30] vs 13.3%[4/30], P<0.001). Ventricular fibrillation occurred most frequently between midnight and 0600 h in patients with ventricular fibrillation episodes during sleep (76.9% [20/26] vs 23.1%[6/26], P<0.01)., Conclusion: These results suggest that increased nocturnal vagal activity and withdrawal sympathetic activity may play an important role in the arrhythmogenesis of the Brugada syndrome.

We report a patient with nasopharyngeal cancer with long-term follow-up of more than 16 years after the first course of radiotherapy in 1981. He developed a lung metastasis in 1996 after having a second course of radiotherapy for neck recurrence in 1989. The patient was a 42-year-old man with a nasopharyngeal tumor and a fixed upper neck metastasis (T1N1M0), which was treated with definitive radiotherapy. He manifested regional recurrence, at the margin of the radiation portal, with an 8 year disease-free interval, which was treated successfully by definitive re-irradiation. He developed a solitary lung metastasis, which was treated by video-assisted thoracoscopic lung resection, 7 years disease-free after the second course of radiotherapy. For 20 months after the removal of the lung metastasis he has been generally well without any signs of recurrence of sequelae. This case indicates the efficacy of definitive re-irradiation for regional recurrence and the necessity for long-term observation after radiation therapy for nasopharyngeal cancer.

In response to stimulation of B-cells through cell surface IgM, the activity of the serine/threonine protein phosphatase PP1, but not PP2A, was transiently decreased and reached a minimum 10-20 min after the stimulation. The decrease was more profound in the immature B-cell line WEHI-231, than in the mature B-cell line BAL-17. Under these conditions, PP1alpha, an isoform of PP1, showed unique alterations in the patterns of several spots with distinct isoelectic points in the Western blot after two-dimensional electrophoresis, whereas another isoform, PP1delta, did not show any alteration. PP1gamma1 and PP1gamma2 were not detected in B-cells. Similar alterations in these spots were observed in B-cells stimulated by PMA. When partially purified PP1 consisting of PP1alpha and PP1delta was incubated with [gamma-32P]ATP and PKC, radioactive spots of PP1alpha could be detected, but no spot of PP1delta was detected. Because differences in sequence among PP1 isoforms are mostly restricted to their C-terminals, phosphorylation rates of the C-terminal peptides containing the PKC-phosphorylation motif were compared. The C-terminal peptide of PP1alpha is a better substrate for PKC than those of PP1gamma1 and PP1gamma2, and is phosphorylated at the serine residue corresponding to Ser-325 of PP1alpha. The corresponding C-terminal region of PP1delta does not contain the phosphorylation site. On the other hand, there was a large difference in subcellular distribution of PP1delta, but not PP1alpha, between immature and mature B-cells. From these results, it was strongly suggested that PP1alpha is involved, via phosphorylation by PKC, in the regulation of signal transduction in response to the stimulation of B-cells through cell surface IgM.

To examine structural features of proline which are essential for the proline-directed phosphorylation by cdc2 kinase or cdk5, we prepared the peptide representing the cdc2 kinase phosphorylation site at Ser-55 in vimentin [Ser-Leu-Tyr-Ser-Ser-Ser55-Pro56-Gly-Gly58-Ala-Tyr-NH2], the peptide containing arginine in place of Gly-58, and their derivatives containing various N-methylamino acids or proline homologs in place of Pro-56, and tested them as substrates for the kinases. While substitution of the proline by proline homologs (L-pipecolic acid or L-azetidine-2-carboxylic acid) increased the K(m) value 2- to 4-fold at utmost, substitution by N-methylamino acids (sarcosine, L-N-methylalanine, L-N-methylvaline, or L-N-methylleucine) increased the K(m) value 7- to 40-fold for cdc2 kinase. For cdk5, these substitutions led to parallel effects on the K(m) value to those found for cdc2 kinase; cdk5 recognized the peptides with a proline specificity similar to that for cdc2 kinase. These results suggest that the pyrrolidine ring of proline is important for substrate recognition by cdc2 kinase or cdk5. Molecular dynamics and molecular mechanics simulations indicated that the pyrrolidine ring of proline is optimal to stabilize a beta-turn at the phosphorylation site and that the K(m) values of the peptides for the enzymes might be related to the probability of the turn structure. The results obtained here also suggest that the pyrrolidine ring of proline is required to maintain a high V(max) value for cdc2 kinase or especially for cdk5. These will aid in designing specific substrates or inhibitors for cdc2 kinase or cdk5.

Site-specific phosphorylation of intermediate filament (IF) proteins on serine and threonine residues leads to dynamic alterations in filament structure. Site- and phosphorylation state-specific antibodies for IF proteins can visualize spatial and temporal distribution of site-specific IF protein phosphorylations in the cell. These antibodies are also useful to identity IF kinases involved in cellular events, including cell signaling and cell cycle.

The thermotropic effect of ganglioside GD1a on the phosphatidylcholine membrane immobilized on the porous cellulose acetate filter was investigated by ESR spin-labeling and DSC. The spin-labeled GD1a having 12-DOXYL-stearic acid instead of the long acyl chain of the ceramide portion (GD1a*) was incorporated into the model membrane. An ESR examination of the membrane showed that GD1a* undergoes an anisotropic rotation in the wide range of temperature -30-70 degrees C. By monitoring the overall splitting (2T parallel) of spin-labeled phosphatidylcholine (PC*), the model membranes were found to show decreased fluidity in accordance with the GD1a content. The phase transition temperature (Tm) of distealoylphosphatidylcholine (DSPC) model membranes could be estimated by the measurement of ESR and DSC. The effects of GD1a were found to be more significant in broadening the phase transition rather than in elevating the Tm of DSPC model membranes.

We identified the phosphorylation sites of glial fibrillary acidic protein (GFAP) for cdc2 kinase and Ca(2+)-calmodulin (CaM)-dependent protein kinase II. GFAP was phosphorylated to approximately 0.2 mol of phosphate/mol of GFAP by cdc2 kinase, and this phosphorylation did not induce disassembly of the filament structure. On the other hand, GFAP was phosphorylated to approximately 1.9 mol of phosphate/mol of GFAP by Ca(2+)-CaM-dependent protein kinase II, and this phosphorylation did induce disassembly of the filament. Sequential analysis of the purified phosphopeptides revealed that only Ser8 on GFAP was phosphorylated by cdc2 kinase, whereas Ser13, Ser17, Ser34, and Ser389 on GFAP were phosphorylated by Ca(2+)-CaM-dependent protein kinase II.

Using a new cannulation method, which completely prevented interruption of circulation to brain tissue during surgical operations, we could prepare a functionally intact hemoglobin-free isolated perfused rat head in situ. The frequency distribution (8-25 Hz) and amplitude of a spontaneous electroencephalogram, EEG (50 microV), were kept within the normal ranges for up to 4 h after the perfusion was started. Our preparation gave characteristic flash-evoked EEG responses through the eyes. The administration of bicuculline elicited an epileptic seizure similar to that in normal rats. Near-infrared difference spectra of the rat head obtained on an aerobic to anaerobic transition were similar to those of cytochrome oxidase in isolated mitochondria as well as in the purified state. Under the aerobic perfusion conditions, both heme a+a3 and copper in cytochrome oxidase were in the fully oxidized states, showing that normoxia was maintained in the brain tissue. The redox behaviors of these two chromophores in the brain were identical to those observed in isolated mitochondria. The usefulness of brain perfusion for bridging in vitro and in vivo studies is well documented.

A case is reported of prolonged survival after radical hysterectomy for poorly differentiated adenocarcinoma of the endometrium and resection of metastatic carcinoma of the brain followed by radiation therapy. The 43-year-old patient has survived for seven years after hysterectomy and six years 10 months after excision of the brain metastasis. Our results show that the surgical excision of a single metastatic lesion of the brain with postoperative irradiation offers hope of prolonged survival in patients with a solitary brain metastasis and no evident systemic disease.