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Immune reconstitution after human leukocyte antigen (HLA)-mismatched (haploidentical) hematopoietic stem cell transplantation (haplo-HCT) can significantly influence long-term outcomes. The three possible HLA haplotypes after transplantation are: one carried by both the patient and the donor (shared HLA), one by donor only (donor-specific HLA), and one by patient only (host-specific HLA), and the donor T cells remain restricted to one of these three haplotypes. Understanding the presence of donor T cells restricted to each haplotype may provide more detailed insights into post-transplant immune response and potentially provide valuable information for the development of chimeric antigen receptor T cell or T cell receptor T cell constructs. In this study, patients or donors with HLA-A24 or HLA-A2 were tested with HLA-A*24:02- and A*02:01-restricted cytomegalovirus (CMV)-specific tetramers for detecting the respective HLA-restricted T cells. Sixty-four samples from 40 patients were assayed. More than half of the patients at day 90 and all patients by day 900 had shared HLA-restricted T cells. After day 90, half of the patients had donor-specific HLA-restricted T cells, but no host-specific HLA-restricted T cells were found. In the comparative analysis of the transplant types, shared HLA-restricted T cells were positive in all three categories: haplo-HCT (50%), 2-haplo-mis-HCT (75%), and spousal HCT (67%). Furthermore, donor-specific HLA-restricted T cells demonstrated positivity in haplo-HCT at 57% and in 2-haplo-mis-HCT at 60%, with a threshold of 0.01%. Donor-specific HLA-restricted T cells for spousal HCT were not examined due to the lack of an appropriate HLA combination for the tetramers. The presence of shared HLA-restricted T cells explains the host defense after HLA-haploidentical transplantation, while the presence of donor-specific HLA-restricted T cells may account for host defense against hematotropic viruses, such as CMV. However, this study failed to detect host-specific HLA-restricted T cells, leaving the host defense against epitheliotropic viruses unresolved, thus requiring further investigation., Competing Interests: Declaration of competing interest There are no conflicts of interest to report., (Copyright © 2024. Published by Elsevier B.V.)

Anaerobic microbial acetogenesis is ubiquitous on Earth, and thus plays an important role in the global carbon cycle. The mechanism of carbon fixation in acetogens has attracted great interest from various studies for combatting climate change, and even for studying ancient metabolic pathways. Here, we developed a new, simple method for investigating carbon flows in the metabolic reaction of acetogen by conveniently and accurately determining the relative abundance of individual acetate- and/or formate-isotopomers formed in 13 C labeling experiments. We measured the underivatized analyte by gas chromatography-mass spectrometry (GC-MS) coupled with a direct aqueous sample injection technique. The individual abundance of analyte isotopomers was calculated by the mass spectrum analysis using the least-squares approach. The validity of the method was demonstrated by determining known mixtures of unlabeled and 13 C-labeled analytes. The developed method was applied to study the carbon fixation mechanism of the well-known acetogen Acetobacterium woodii grown on methanol and bicarbonate. We provided a quantitative reaction model for methanol metabolism of A. woodii, which indicated that methanol was not the sole carbon precursor of the acetate methyl group and that 20-22% of the methyl group was formed from CO 2 . In contrast, the carboxyl group of acetate appeared to form exclusively by CO 2 fixation. Thus, our simple method, without laborious analytical procedures, has broad utility for the study of biochemical and chemical processes related to acetogenesis on Earth., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Konomi Suda reports financial support was provided by Japan Society for the Promotion of Science. Hideyuki Tamaki reports financial support was provided by Japan Society for the Promotion of Science., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Autophagy is regulated by phosphoinositides. We have previously shown that phosphatidylinositol 4-phosphate (PtdIns(4)P) is localized in the autophagosomal membrane. Additionally, in yeast cells, phosphatidylinositol 4-kinases Pik1p and Stt4p play important roles in the formation of the autophagosome and its fusion with the vacuole, respectively. In this study, we analyzed the primary role of PtdIns(4)P phosphatases in yeast autophagy. The PtdIns(4)P labeling densities in the membranes of the vacuoles, mitochondria, nucleus, endoplasmic reticulum, and plasma membrane dramatically increased in the phosphatase deletion mutants sac1∆ and sjl3∆, and the temperature-sensitive mutant sac1 ts /sjl3∆ at the restrictive temperature. GFP-Atg8 processing assay indicated defective autophagy in the sac1∆ and sac1 ts /sjl3∆ mutants. In contrast to the localization of PtdIns(4)P in the luminal leaflet of autophagosomal membranes in the wild-type yeast, PtdIns(4)P was localized in both the luminal and cytoplasmic leaflets of the autophagosomal membranes in the sac1∆ strain. In addition, the number of autophagic bodies in the vacuole significantly decreased in the sac1∆ strain, although autophagosomes were present in the cytoplasm. In the sac1 ts /sjl3∆ strain, the number of autophagosomes in the cytoplasm dramatically decreased at the restrictive temperature. Considering that the numbers of autophagosomes and autophagic bodies in the sjl3∆ strain were comparable to those in the wild-type yeast, we found that the autophagosome could not be formed when PtdIns(4)P phosphatase activities of both Sac1p and Sjl3p were diminished. Together, these results indicate that the turnover of PtdIns(4)P by phosphatases is essential for autophagosome biogenesis., (Copyright © 2022 Elsevier B.V. All rights reserved.)

To date, the only methanoarchaea isolated directly from methane hydrate bearing sediments were Methanoculleus submarinus Nankai-1 T and Methanoculleus sp. MH98A. Here, we provide the genome of Methanoculleus taiwanensis CYW4 T isolated from the deep-sea subseafloor sediment at the Deformation Front offshore southwestern Taiwan, where methane hydrate deposits are likely located. Through comparative genomics analyses of nine Methanoculleus strains from various habitats, 2-3 coding genes for trehalose synthases were found in all nine Methanoculleus genomes, which were not detected in other methanogens and are therefore suggested as a signature of genus Methanoculleus among methane-producing archaea. In addition, the structural genes adjacent to trehalose synthase genes are comprised of the signaling module of Per-Arnt-Sim (PAS) domain-containing proteins, Hsp20 family proteins, arabinose efflux permeases and multiple surface proteins with fasciclin-like (FAS) repeat. This indicates that trehalose synthase gene clusters in Methanoculleus might play roles in the response to various stresses and regulate carbon storage and modification of surface proteins through accumulation of trehalose. The non-gas hydrate-associated Methanoculleus strains harbor carbon-monoxide dehydrogenase (cooS/acsA) genes, which are important for the conversion of acetate to methane at the step of CO oxidation/CO 2 reduction in acetoclastic methanogens and further implies that these strains may be able to utilize CO for methanogenesis in their natural habitats. In addition, both genomes of M. bourgensis strains MS2 T and MAB1 harbor highly abundant transposase genes, which may be disseminated from microbial communities in their habitats, sewage treatment plants and biogas reactors, which are breeding grounds for antibiotic resistance. Through comparative genomic analyses, we gained insight into understanding the life of strictly anaerobic methane-producing archaea in various habitats, especially in methane-based deep-sea ecosystems., (Copyright © 2019. Published by Elsevier B.V.)

Autophagy is a degradative cellular pathway that protects eukaryotic cells from starvation/stress. Phosphatidylinositol 4-kinases, Pik1p and Stt4p, are indispensable for autophagy in budding yeast, but participation of PtdIns-4 kinases and their product, phosphatidylinositol 4-phosphate [PtdIns(4)P], is not understood. Nanoscale membrane lipid distribution analysis showed PtdIns(4)P is more abundant in yeast autophagosomes in the luminal leaflet than the cytoplasmic leaflet. PtdIns(4)P is confined to the cytoplasmic leaflet of autophagosomal inner and outer membranes in mammalian cells. Using temperature-conditional single PIK1 or STT4 PtdIns 4-kinase mutants, autophagic bodies in the vacuole of PIK1 and STT4 mutant cells dramatically decreased at restrictive temperatures, and the number of autophagosomes in the cytosol of PIK1 mutants cells was also decreased, whereas autophagosome levels of STT4 mutant cells were comparable to that of wild-type and STT4 mutant cells at permissive temperatures. Localization of PtdIns(4)P in the luminal leaflet in the biological membrane is a novel finding, and differences in PtdIns(4)P distribution suggest substantial differences between yeast and mammals. We also demonstrate in this study that Pik1p and Stt4p play essential roles in autophagosome formation and autophagosome-vacuole fusion in yeast cells, respectively., (Copyright © 2019 Elsevier B.V. All rights reserved.)

In budding yeast Saccharomyces cerevisiae, PtdIns(4)P serves as an essential signalling molecule in the Golgi complex, endosomal system, and plasma membrane, where it is involved in the control of multiple cellular functions via direct interactions with PtdIns(4)P-binding proteins. To analyse the distribution of PtdIns(4)P in yeast cells at a nanoscale level, we employed an electron microscopy technique that specifically labels PtdIns(4)P on the freeze-fracture replica of the yeast membrane. This method minimizes the possibility of artificial perturbation, because molecules in the membrane are physically immobilised in situ. We observed that PtdIns(4)P is localised on the cytoplasmic leaflet, but not the exoplasmic leaflet, of the plasma membrane, Golgi body, vacuole, and vesicular structure membranes. PtdIns(4)P labelling was not observed in the membrane of the endoplasmic reticulum, and in the outer and inner membranes of the nuclear envelope or mitochondria. PtdIns(4)P forms clusters of <100 nm in diameter in the plasma membrane and vacuolar membrane according to point pattern analysis of immunogold labelling. There are three kinds of compartments in the cytoplasmic leaflet of the plasma membrane. In the present study, we showed that PtdIns(4)P is specifically localised in the flat undifferentiated plasma membrane compartment. In the vacuolar membrane, PtdIns(4)P was concentrated in intramembrane particle (IMP)-deficient raft-like domains, which are tightly bound to lipid droplets, but not surrounding IMP-rich non-raft domains in geometrical IMP-distributed patterns in the stationary phase. This is the first report showing microdomain formations of PtdIns(4)P in the plasma membrane and vacuolar membrane of budding yeast cells at a nanoscale level, which will illuminate the functionality of PtdIns(4)P in each membrane., (Copyright © 2018 Elsevier GmbH. All rights reserved.)

Objectives: The purpose of this study was to investigate the effect of anodal transcranial direct-current stimulation (tDCS) applied over the primary motor (M1) or the primary somatosensory (S1) cortices on somatosensory evoked magnetic fields (SEFs) following median nerve stimulation., Methods: Anodal tDCS was applied for 15min on the left motor or somatosensory cortices at 1mA. SEFs were recorded following right median nerve stimulation using a magnetoencephalography (MEG) system before and after the application of tDCS. SEFs was measured and compared before and after tDCS was applied over M1 or S1., Results: The source strengths for the P35m and P60m increased after tDCS was applied over M1 and that for the P60m increased after tDCS was applied over S1. The mean equivalent current dipole (ECD) location for the P35m was located significantly anterior to that of the N20m, but only during post 1 (10-20min after tDCS was applied over M1)., Conclusion: Our results indicated that the anodal tDCS applied over M1 affected the P35m and P60m sources on SEF components, while that applied over S1 influenced the P60m source., Significance: We demonstrated anodal tDCS applied over M1 or S1 can modulate somatosensory processing and components of SEFs, confirming the hypothesis for locally distinct generators of the P35m and P60m sources., (Copyright © 2014 International Federation of Clinical Neurophysiology. Published by Elsevier Ireland Ltd. All rights reserved.)

Background: The motor cortex in the human brain can be modulated by the application of transcranial static magnetic field stimulation (tSMS) through the scalp. However, the effect of tSMS on the excitability of the primary somatosensory cortex (S1) in humans has never been examined., Objective: This study was performed to investigate the possibility of non-invasive modulation of S1 excitability by the application of tSMS in healthy humans., Methods: tSMS and sham stimulation over the sensorimotor cortex were applied to 10 subjects for periods of 10 and 15 min. Somatosensory evoked potentials (SEPs) following right median nerve stimulation were recorded before and immediately after, 5 min after, and 10 min after tSMS from sites C3' and F3 of the international 10-20 system of electrode placement. In another session, SEPs were recorded from 6 of the 10 subjects every 3 min during 15 min of tSMS., Results: Amplitudes of the N20 component of SEPs at C3' significantly decreased immediately after 10 and 15 min of tSMS by up to 20%, returning to baseline by 10 min after intervention. tSMS applied while recording SEPs every 3 min and sham stimulation had no effect on SEP., Conclusions: tSMS is able to modulate cortical somatosensory processing in humans, and thus might be a useful tool for inducing plasticity in cortical somatosensory processing. Lack of change in the amplitude of SEPs with tSMS implies that use of peripheral nerve stimulation to cause SEPs antagonizes alteration of the function of membrane ion channels during exposure to static magnetic fields., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Objective: Symptoms consistent with neurological decompression sickness (DCS) in commercial breath-hold (Ama) divers has been reported from a few districts of Japan. The aim of this study was to detect circulating intravascular bubbles after repetitive breath-hold diving in a local area where DCS has been reported in Ama divers., Methods: The participants were 12 partially assisted (descent using weights) male Ama divers. The equipment (AQUALAB system) consisted of continuous-wave Doppler with a 5-MHz frequency, and the Doppler probe was placed in the precordial site with the ultrasonic wave directed into the pulmonary infundibulum. We carried out continuous monitoring for 10 minutes at the end of the series of repetitive dives, and the recordings were made on numerical tracks and graded in a blind manner by 2 experienced investigators, according to the Spencer Doppler code., Results: Depths and number of dives were 8 to 20 m and 75 to 131 times. Mean diving duration and surface interval were 64 ± 12 seconds and 48 ± 8 seconds, respectively (mean ± SD). We detected the lowest grade of intravascular bubbles (Spencer's grade I) in an Ama diver whose mean surface interval was only 35.2 ± 6.2 seconds. His mean descending, bottom, and ascending times were 10.4 ± 1.6 seconds, 39.2 ± 8 seconds, and 18.2 ± 3.0 seconds, respectively, over the course of 99 dives., Conclusions: Intravascular bubbles may be formed after repetitive breath-hold dives with short surface intervals or after a long breath-holding session in Ama divers. Symptoms consistent with neurological accidents in repetitive breath-hold diving may be caused in part by the intravascular presence of bubbles, indicating the need for safety procedures., (Copyright © 2014 Wilderness Medical Society. Published by Elsevier Inc. All rights reserved.)

Posttranslational protein modifications (PTMs) are necessary for cells to function properly. The role of PTMs in regulating immune responses, specifically those mediated by dendritic cells (DCs), which are critical for both innate and adaptive immunity, is not well understood. Utilizing multiple but complementary approaches, we determined the role of an important but less understood type of PTM, namely, neddylation, in regulating DC functions. Inhibition of neddylation suppressed the release of proinflammatory cytokines by DCs in response to Toll-like receptor, nucleotide oligomerization domain-like receptor, and noninfectious CD40L stimulation. These effects were more profound than those mediated by the proteasome inhibitor bortezomib or a commonly used antiinflammatory agent, dexamethasone. Targeting neddylation also suppressed the ability of DCs to stimulate murine allogeneic T cells in vitro and in vivo and human allogeneic T-cell responses in vitro. Mechanistic studies demonstrated that inhibition of neddylation reduced both canonical and noncanonical nuclear factor-κB (NF-κB) activity. Neddylation inhibition prevented the degradation of inhibitor-κB and thus reduced the translocation and activation of NF-κB, but without perturbation of the mitogen-activated protein kinase/extracellular signal-regulated kinase pathway. Thus, blocking neddylation could be a novel strategy for mitigating immune-mediated disease processes.

Hydroxyapatite chromatography is a very important step in the purification of voltage-dependent anion channels (VDACs) and several members of solute carrier family 25 (Slc25) from isolated mitochondria. In the presence of Triton X-100, VDACs and Slc25 members present a peculiar property, i.e., a lack of interaction with hydroxyapatite, resulting in their presence in the flow-through fraction of hydroxyapatite chromatography. This property has allowed selective isolation of VDACs and Slc25 members from a mixture of total mitochondrial proteins. However, the reason why only these few proteins are selectively obtained in the presence of Triton X-100 from the flow-though fraction of hydroxyapatite chromatography has not yet been adequately understood. In this study, when we examined the protein species in the flow-through fractions by proteomic analysis, VDAC isoforms, Slc25 members, and some other membrane proteins were identified. All the mitochondrial proteins had in common high hydrophobicity over their entire protein sequences. When the proteins were fused to soluble proteins, the fused proteins showed affinity for hydroxyapatite even in the presence of Triton X-100. Based on these results, we discussed the molecular basis of the interactions between proteins and hydroxyapatite in the presence of Triton X-100., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Nursing- and healthcare-associated pneumonia (NHCAP) has been proposed by the Japanese Respiratory Society as a new category of pneumonia considering the characteristics of the Japanese medical care environment. It is necessary to ascertain the epidemiology and clinical outcomes of NHCAP. A prospective study was conducted of patients with pneumonia who were hospitalized at our hospital from August 2011 to July 2012. We compared 192 cases of NHCAP with 114 cases of community-acquired pneumonia (CAP). Compared with CAP, NHCAP had a higher disease severity, higher 30-day mortality rate (10.9 vs. 3.5 %, P = 0.022), and longer length of hospital stay (median, 12 vs. 8 days, P < 0.001). Streptococcus pneumoniae was the most frequent causative pathogen in both NHCAP and CAP (33.9 vs. 34.8 %, P = 0.896). The incidence of atypical pathogens in NHCAP was low (1.7 %). Multidrug-resistant (MDR) pathogens were isolated more frequently in NHCAP than in CAP, but there was no significant difference (11.0 vs. 4.5 %, P = 0.135). Among 192 NHCAP patients, 122 (63.5 %) were aspiration pneumonia. Aspiration pneumonia was associated with poor outcomes and was considered a major characteristic of NHCAP. Our study suggested that many patients with NHCAP do not need broad-spectrum antibiotic therapy targeting MDR pathogens. Excess mortality in NHCAP patients is the result of patient backgrounds or disease severity rather than the presence of MDR pathogens.

Homeodomain-only protein X (HOPX)-β promoter methylation was recently shown to be frequent in human cancers and was suggested as tumor suppressor gene in esophageal and gastric cancer. The aim of this study was to investigate the mechanistic roles of HOPX-β promoter methylation and its clinical relevance in colorectal cancer (CRC). HOPX-β promoter methylation was assessed in human CRC cell lines and 294 CRC tissues. HOPX mRNA and protein levels were measured in relation to HOPX-β promoter methylation. The effects of forced HOPX expression on tumorigenesis were studied using in vitro and in vivo assays. The association between HOPX-β promoter methylation and clinical relevance of CRC patients was determined. HOPX-β promoter methylation is cancer-specific and frequently found in CRC cell lines and tissues, resulting in the down-regulation of HOPX mRNA and protein levels. In CRC cell lines, forced expression of HOPX suppressed proliferation, invasion, and anchorage-independent growth. DNA microarray analyses suggested critical downstream genes that are associated with cancer cell proliferation, invasion or angiogenesis. In a mouse xenograft model, HOPX inhibited tumorigenesis and angiogenesis. Finally, HOPX-β promoter methylation was associated with worse prognosis of stage III CRC patients (hazard ratio= 1.40, P = .035) and also with poor differentiation (P = .014). In conclusion, HOPX-β promoter methylation is a frequent and cancer-specific event in CRC progression. This epigenetic alteration may have clinical ramifications in the diagnosis and treatment of CRC patients.

The interindividual variation in the rate of drug metabolism and disposition has been known for many years. Pharmacogenomics dealing with heredity and response to drugs is a part of science that attempts to explain variability of drug responses and to search for the genetic basis of such variations or differences. Genetic polymorphisms of drug metabolizing enzymes and drug transporters have been found to play a significant role in the patients' responses to medication. Accumulating evidence demonstrates that certain nonsynonymous polymorphisms have great impacts on the protein stability and degradation, as well as the function of drug metabolizing enzymes and transporters. The aim of this review article is to address a new aspect of protein quality control in the endoplasmic reticulum and to present examples regarding the impact of nonsynonymous single-nucleotide polymorphisms on the protein stability of thiopurine S-methyltransferase as well as ATP-binding cassette (ABC) transporters including ABCC4, cystic fibrosis transmembrane conductance regulator (CFTR, ABCC7), ABCC11, and ABCG2. Furthermore, we will discuss the molecular mechanisms underlying posttranslational modifications (intramolecular and intermolecular disulfide bond formation and N-linked glycosylation) and ubiquitin-mediated proteasomal degradation of ABCG2, one of the major drug transporter proteins in humans., (Copyright © 2011 Wiley-Liss, Inc.)

ADP-ribosylation factor 6 (ARF6) is a small GTPase that regulates neuronal morphogenesis processes such as axonal, dendritic, and spine formation possibly through the actin cytoskeleton and membrane trafficking. In an attempt to define the molecular mechanisms that regulate neuronal morphogenesis by ARF6, we identified vezatin as a novel binding partner of active GTP-bound ARF6 using yeast two-hybrid screening. Vezatin was able to bind specifically to GTP-ARF6 among the ARF family. In the adult mouse brain, vezatin exhibited widespread gene expression with high levels in the hippocampus and medial habenular nucleus. In hippocampal neurons, vezatin was localized at dendrites as well as cell bodies. Knockdown of endogenous vezatin significantly reduced total dendritic length and arborization of cultured hippocampal neurons, while overexpression of vezatin increased dendritic length. Our present study suggests that vezatin may regulate dendritic formation as a downstream effector of ARF6.

Bone marrow (BM)-derived hematopoietic cells, which are major components of tumor stroma, determine the tumor microenvironment and regulate tumor phenotypes. Cyclooxygenase (COX)-2 and endogenous prostaglandins are important determinants for tumor growth and tumor-associated angiogenesis; however, their contributions to stromal formation and angiogenesis remain unclear. In this study, we observed that Lewis lung carcinoma cells implanted in wild-type mice formed a tumor mass with extensive stromal formation that was markedly suppressed by COX-2 inhibition, which reduced the recruitment of BM cells. Notably, COX-2 inhibition attenuated CXCL12/CXCR4 expression as well as expression of several other chemokines. Indeed, in a Matrigel model, prostaglandin (PG) E2 enhanced stromal formation and CXCL12/CXCR4 expression. In addition, a COX-2 inhibitor suppressed stromal formation and reduced expression of CXCL12/CXCR4 and a fibroblast marker (S100A4) in a micropore chamber model. Moreover, stromal formation after tumor implantation was suppressed in EP3-/- mice and EP4-/- mice, in which stromal expression of CXCL12/CXCR4 and S100A4 was reduced. The EP3 or EP4 knockout suppressed S100A4+ fibroblasts, CXCL12+, and/or CXCR4+ stromal cells as well. Immunofluorescent analyses revealed that CXCL12+CXCR4+S100A4+ fibroblasts mainly comprised stromal cells and most of these were recruited from the BM. Additionally, either EP3- or EP4-specific agonists stimulated CXCL12 expression by fibroblasts in vitro. The present results address the novel activities of COX-2/PGE2-EP3/EP4 signaling that modulate tumor biology and show that CXCL12/CXCR4 axis may play a crucial role in tumor stromal formation and angiogenesis under the control of prostaglandins.

The inhibition of intestinal breast cancer resistance protein (BCRP), which restricts the absorption of xenobiotics, may increase the systemic availability of its substrates. The aim of this study was to evaluate the inhibitory effects of herbal extracts and their constituents on BCRP-mediated transport. The inhibitory effects of 9 herbal extracts and 23 isoflavonoids, including soybean-derived isoflavones, on BCRP-mediated methotrexate (MTX) transport were evaluated using BCRP-expressing membrane vesicles. The structure-inhibitory potency relationship was investigated by multiple factor analysis. Extracts of soybean, Gymnema sylvestre, black cohosh and passion flower and rutin strongly inhibited BCRP-mediated transport of MTX at 1 mg/ml, while inhibition by chlorella, milk thistle and Siberian ginseng extracts was weak. Among the 23 isoflavonoids examined, all of which inhibited BCRP-mediated transport, coumestrol showed the most potent inhibition (IC(50)=63 nM). The inhibitory potencies of 6 isoflavonoid glucosides were 10- to 100-fold lower than those of the corresponding aglycones. The addition of a 5-hydroxyl or 6-methoxyl moiety tended to potentiate the inhibition. The inhibitory potency of daidzein was decreased 100-fold by 7-glucuronidation, but was virtually unaffected by 4'-sulfation. Thus, some herbal and dietary supplements and isoflavonoids may increase the systemic availability of BCRP substrates when concomitantly given orally.

Fucoidan from Cladosiphon okamuranus and its sulfate derivatives were prepared. Sulfate contents of native and oversulfated fucoidan were estimated to be 13.5% and 32.8%, respectively. The results of (1)H NMR suggest that 2,4-di-O-sulfo-, 2-mono-O-sulfo- and 4-mono-O-sulfo-l-fucopyranose were involved in oversulfated fucoidan and 4-mono-O-sulfo-l-fucopyranose was involved in native fucoidan. The oversulfated fucoidan reduced the proliferation of U937 cells in a dose-dependent manner, but the activity of native fucoidan was weak. The sulfate content and substituting position of sulfate group might be important factors of anti-proliferative activity in U937 cells. To examine whether the anti-proliferative activity of oversulfated fucoidan was caused by induction of apoptosis, apoptosis assay, caspase-3 activity assay and Western blotting analysis were performed. These results indicated that the oversulfated fucoidan induced apoptosis via caspase-3 and -7 activation-dependent pathway.

To clarify the clinical impact of influenza A on the development of febrile seizures (FS), consecutive FS patients brought to our hospital between October 2003 and September 2004 were prospectively surveyed. Patients infected with influenza A (influenza A patients) and those uninfected with influenza (non-influenza patients) were compared with regard to clinical characteristics of FS. Influenza infection was determined by rapid antigen test and/or serologically. Associations of influenza A with atypical findings of FS, including partial seizures, prolonged seizures, multiple seizures during the same illness, and 30-min or longer prolonged postictal impairment of consciousness (PPIC), were analyzed by multiple logistic regression. A total of 215 patients (47 influenza A and 168 non-influenza patients) were enrolled in the study. Age was significantly higher in the influenza A group (39.85+/-22.16 months vs. 27.51+/-17.14 months, P<0.001). Of 42 patients aged 48 months or older, which corresponded to the 80th percentile for age, 15 (35.7%) were influenza A patients, with a significantly higher incidence of such patients than in the subgroup of patients aged 47 months or younger (32/173, 18.5%) (P=0.015). On multiple logistic regression analysis, influenza A was independently associated with PPIC (odds ratio: 4.44, 95% confidence interval: 1.52-12.95, P=0.006), but not with other atypical findings. The positive association of influenza A with PPIC suggests that influenza may affect state of consciousness at the same time that it induces seizures with fever.

Expression of CYP1A1 mRNA in mouse hepatocytes in primary culture was investigated. The expression was obvious on day 3 of culture without addition of any known ligands of the aryl hydrocarbon receptor and increased with culture period. Removal of insulin from and addition of hydrogen peroxide to the medium enhanced and suppressed the expression, respectively. The CYP1A1 mRNA expression was also enhanced in the presence of anti-oxidant, t-butylhydroquinone, in the medium. Several kinds of kinase inhibitors markedly increased the CYP1A1 mRNA expression. In contrast, the inhibitory expression was prolonged in the presence of okadaic acid, a potent inhibitor of serine/threonine phosphatase PP1 and PP2. These observations suggest that there might be a repressive pathway in the regulation of CYP1A1 mRNA expression and that the presently observed expression pathway differs at several points from those previously reported, such as ligand-activated aryl hydrocarbon receptor- or omeprazole-mediated expression. Modulation of CYP1A2 mRNA expression after exposing hepatocytes to agents affecting phosphorylation pathways differed from that of CYP1A1 mRNA. This implies that regulatory pathways for CYP1A1 and CYP1A2 expression may differ.

In acute-type leukemia, no method for the prediction of relapse following allogeneic stem cell transplantation based on minimal residual disease (MRD) levels is established yet. In the present study, MRD in 72 cases of allogeneic transplantation for acute myeloid leukemia, acute lymphoid leukemia, and chronic myeloid leukemia (accelerated phase or blast crisis) was monitored frequently by quantitating the transcript of WT1 gene, a "panleukemic MRD marker," using reverse transcriptase-polymerase chain reaction. Based on the negativity of expression of chimeric genes, the background level of WT1 transcripts in bone marrow following allogeneic transplantation was significantly decreased compared with the level in healthy volunteers. The probability of relapse occurring within 40 days significantly increased step-by-step according to the increase in WT1 expression level (100% for 1.0 x 10(-2)-5.0 x 10(-2), 44.4% for 4.0 x 10(-3)-1.0 x 10(-2), 10.2% for 4.0 x 10(-4)-4.0 x 10(-3), and 0.8% for < 4.0 x 10(-4)) when WT1 level in K562 was defined as 1.0). WT1 levels in patients having relapse increased exponentially with a constant doubling time. The doubling time of the WT1 level in patients for whom the discontinuation of immunosuppressive agents or donor leukocyte infusion was effective was significantly longer than that for patients in whom it was not (P <.05). No patients with a short doubling time of WT1 transcripts (< 13 days) responded to these immunomodulation therapies. These findings strongly suggest that the WT1 assay is very useful for the prediction and management of relapse following allogeneic stem cell transplantation regardless of the presence of chimeric gene markers.

Survival of transplanted Zostera marina L. (eelgrass) and environmental conditions (water quality, bottom sediments, sedimentation on leaves and flow regime) were studied concurrently in the center, edge, and at the outside of a eelgrass meadow located in a eutrophic coastal zone in northern Hiroshima Bay, Seto Inland Sea, Japan. Eelgrass transplants at the outside of the meadow declined significantly, whereas those at the center were consistently well established. Silt content in the bottom sediments at the outside was higher than that at the center. The sediment was oxic from the surface to 2 cm deep at the center, whereas those at the edge and the outside were reductive almost from the surface. The sediment characteristics typical in eutrophic water seemed to be a factor responsible for the deterioration of eelgrass meadows. Although suspended solid concentrations in the water columns were almost the same, the amount of sediments deposited on leaves of eelgrass at the outside was higher than that at the center of the meadow. The amount of the deposition at the outside seems to be enough to inhibit photosynthesis; i.e. photosynthetic photon flux density (PPFD) available for eelgrass was only 36% of that without any deposition. The deposition in the center, however, was small enough to allow 84% of the original PPFD. Flow rates, determined at 30 cm above the bottom, a half height of average eelgrass, suggested that the rate at the outside was not enough to remove deposited sediments from the surface of eelgrass leaves. Thus, the large amount of sediment deposition caused by water pollution and/or eutrophication seemed to be another factor to inhibit the survival of eelgrass at the outside edge of the meadow.

Wilms tumor gene WT1 is expressed at high levels in hematopoietic malignancies, such as leukemias and myelodysplastic syndromes (MDS), and in various kinds of solid tumors, including lung cancer, and it exerts an oncogenic function in these malignancies. IgM and IgG WT1 antibodies were measured by means of dot blot assay in 73 patients with hematopoietic malignancies (16 acute myeloid leukemia [AML], 11 acute lymphoid leukemia [ALL], 13 chronic myeloid leukemia [CML], and 33 MDS) and 43 healthy volunteers. Immunoglobulin IgM, IgG, and IgM+IgG WT1 antibodies were detected in 40 (54.8%), 40 (54.8%), and 24 (32.8%), respectively, of the 73 patients with hematopoietic malignancies, whereas 7 (16.2%), 2 (4.7%), and none of the 43 healthy volunteers had IgM, IgG, or IgM+IgG WT1 antibodies, respectively. Furthermore, immunoglobulin isotype class switching of WT1 antibodies from IgM to IgG occurred in conjunction with disease progression from refractory anemia (RA) to RA with excess of blasts (RAEB), and further to RAEB in transformation (RAEB-t) in MDS patients. These results showed that humoral immune responses against the WT1 protein could be elicited in patients with WT1-expressing hematopoietic malignancies, and they suggested that the helper T-cell responses needed to induce humoral immune responses and immunoglobulin isotype class switching from IgM to IgG were also generated in these patients. Our findings may provide new insight into the rationale for elicitation of cytotoxic T-cell responses against the WT1 protein in cancer immunotherapy using the WT1 vaccine.

Alpha-N-acetylgalactosaminidase (alpha-GalNAc-ase; EC 3.2.1.49) is an exoglycosidase specific for the hydrolysis of terminal alpha-linked N-acetylgalactosamine in various sugar chains. The cDNA, nagA, encoding alpha-GalNAc-ase from Acremonium sp. was cloned, sequenced, and expressed in yeast Saccharomyces cerevisiae. The nagA contains an open reading frame which encodes for 547 amino acid residues including 21 residues of a signal peptide in its N-terminal. The calculated molecular mass of mature protein from the deduced amino acid sequence of nagA is 57260 Da, which corresponds to the value obtained from SDS-PAGE of native and recombinant enzymes treated with endo-beta-N-acetylglucosaminidase H. The amino acid sequence of NagA showed significant similarity to those of eukaryotic alpha-GalNAc-ases and alpha-galactosidases (alpha-Gal-ases), particularly alpha-Gal-ase A (AglA) from Aspergillus niger. Phylogenetic analysis revealed that NagA does not belong to the cluster of vertebrate alpha-GalNAc-ase and alpha-Gal-ase but forms another cluster with AglA and yeast alpha-Gal-ases. Thus, the evolutionary origin of the fungal alpha-GalNAc-ase is suggested to be different from that of vertebrate alpha-GalNAc-ase. This is the first report of a microbial alpha-GalNAc-ase gene.

The crystal structure of galactinol dihydrate has been determined by X-ray diffraction. The crystal belongs to the orthorhombic system, space group P2(1)2(1)2, a = 15.898(6), b = 19.357(5), c = 5.104(4) A, and Z = 4. The structure was refined to R = 0.044 for 1818 observed structure amplitudes. The primary hydroxyl group exhibits twofold orientational disorder. The linkage conformation is close to those of alpha-(1 --> 4) linkages in methyl alpha-maltotrioside tetrahydrate and erlose trihydrate. Although there is no interring hydrogen bond in galactinol, an indirect interring hydrogen bond including a water molecule is present. The observed conformation is additionally stabilized by the indirect interring hydrogen bond. The global minimum in the relaxed-residue energy map based on the MM3(92) force-field is close to the observed conformation in the crystal structure. All hydroxyl, ring and water oxygen atoms are involved in a complex three-dimensional hydrogen-bonding network.

Cell growth and differentiation are regulated by nutrient availability in the yeast Saccharomyces cerevisiae. Under conditions of nitrogen limitation, diploid cells of S. cerevisiae differentiate to a filamentous growth known as a pseudohyphal growth, while haploid cells produce invasive filaments which penetrate the agar in nutrient-rich medium. We have found that GPR1, which encodes a putative G-protein-coupled receptor, is required for both pseudohyphal and invasive growth. Pseudohyphal growth was defective in Deltagpr1/Deltagpr1 mutant strain and this defect was reversed by addition of cAMP. Also, haploid Deltagpr1 mutant strain was defective in invasive growth. Northern blot analysis revealed that the transcriptional level of FLO11, which encodes a recently identified cell surface flocculin required for pseudohyphal growth, was reduced in Deltagpr1 mutant strain. These results indicate that GPR1 regulates both pseudohyphal and invasive growth by a cAMP-dependent mechanism., (Copyright 2000 Academic Press.)

How cells monitor the availability of nutrition and transduce signals is a fundamental, unanswered question. We have found that Gpr1p, a recently identified G-protein (Gpa2p) coupled receptor in yeast Saccharomyces cerevisiae, regulate the cellular cAMP level in response to glucose. The glucose-induced higher cAMP level found in the strain with GPA2 in multicopy plasmid decreased by deletion of GPR1 gene. A transient increase of cAMP in response to glucose was not observed in a Deltagpr1 mutant strain and this defect was complemented and restored by introducing GPR1 gene with YCp vector. Gpr1p was also required for the increase of cAMP in response to other fermentable sugars. Both membrane proximal regions o the third cytosolic loop in Gpr1p, which has been shown to be important for coupling to G-proteins, were also required for glucose-induced transient increase of cAMP. Our findings suggest that Gpr1p is part of the nutrition sensing machinery most likely acting as a receptor to monitor glucose as well as other fermentable sugars and regulate cellular cAMP levels., (Copyright 1998 Academic Press.)

The WT1 gene is a tumor-suppressor gene that was isolated as a gene responsible for Wilms' tumor, a childhood kidney neoplasm. We have previously reported that the WT1 gene is strongly expressed in leukemia cells with an increase in its expression levels at relapse and an inverse correlation between its expression levels and prognosis, thus making it a novel tumor marker for leukemic blast cells. Furthermore, WT1 antisense oligomers have been found to inhibit the growth of leukemic cells. These results strongly suggested the involvement of the WT1 gene in human leukemogenesis. The present study was performed to prove our hypothesis that the WT1 gene plays a key role in leukemogenesis and performs an oncogenic function in hematopoietic progenitor cells, rather than a tumor-suppressor gene function. 32D cl3, an interleukin-3-dependent myeloid progenitor cell line, differentiates into mature neutrophils in response to granulocyte colony-stimulating factor (G-CSF). However, when transfected wild-type WT1 gene was constitutively expressed in 32D cl3, the cells stopped differentiating and continued to proliferate in response to G-CSF. As for signal transduction mediated by G-CSF receptor (G-CSFR), Stat3alpha was constitutively activated in wild-type WT1-infected 32D cl3 in response to G-CSF, whereas, in WT1-uninfected 32D cl3, activation of Stat3alpha was only transient. However, most interesting was the fact that G-CSF stimulation resulted in constitutive activation of Stat3beta only in wild-type WT1-infected 32D cl3, but not in WT1-uninfected 32D cl3. Thus, WT1 expression constitutively activated both Stat3alpha and Stat3beta. A transient activation of Stat1 was detected in both wild-type WT1-infected and uninfected 32D cl3 after G-CSF stimulation, but no difference in its activation was found. No activation of MAP kinase was detected in both wild-type WT1-infected and uninfected 32D cl3 after G-CSF stimulation. These results demonstrated that WT1 expression competed with the differentiation-inducing signal mediated by G-CSFR and constitutively activated Stat3, resulting in the blocking of differentiation and subsequent proliferation. Therefore, the data presented here support our hypothesis that the WT1 gene plays an essential role in leukemogenesis and performs an oncogenic function in hematopoietic progenitor cells and represent the first demonstration of an important role of the WT1 gene in signal transduction in hematopoietic progenitor cells.

The Saccharomyces cerevisiae GPR1 (G-protein coupled receptor) gene was isolated using two-hybrid system with a heterotrimeric GTP binding protein alpha subunit Gpa2p as a bait. The GPR1 gene encodes 961 amino acids with predicted seven transmembrane segments and two large cytosolic regions as third cytosolic loop with 350 amino acids where asparagine-rich region was found and the C-terminal region with 283 amino acids. The Gpr1p interacted with Gpa2p at C-terminal region with 131 amino acid residues as well as third cytosolic loop. Disruption of the GPR1 gene was not lethal and did not affect to the cell growth. The Gpr1p-GFP fusion protein localized at the cell surface. These results suggest that Gpr1p is a G-protein coupled receptor which localized at plasma membrane. It is likely that a Gpr1p monitors the extracellular signal such as nutrition and transduce it via Gpa2p a possible positive regulator of cAMP level.

To clarify whether the expression of the WT1 gene in leukemic cells is aberrant or merely reflects that in normal counterparts, the expression levels of the WT1 gene were quantitated for normal hematopoietic progenitor cells. Bone marrow (BM) and umbilical cord blood (CB) cells were fluorescence-activated cell sorting (FACS)-sorted into CD34+ and CD34- cell populations, and the CD34+ cells into nine subsets (CD34+ CD33-, CD34+ CD33+, CD34+ CD38-, CD34+ CD38+, CD34+ HLA-DR-, CD34+ HLA-DR+, CD34+ c-kit(high), CD34+ c-kit(low), and CD34+ c-kit-) according to the expression levels of CD34, CD33, CD38, HLA-DR, and c-kit. Moreover, acute myeloid leukemic cells were also FACS-sorted into four populations (CD34+ CD33-, CD34+ CD33+, CD34- CD33+, and CD34- CD33-). FACS-sorted normal hematopoietic progenitor and leukemic cells and FACS-unsorted leukemic cells were examined for the WT1 expression by quantitative reverse transcriptase-polymerase chain reaction. The WT1 expression in the CD34+ and CD34- cell populations and in the nine CD34+ subsets of BM and CB was at either very low (1.0 to 2.4 x 10(-2)) or undetectable (< 10(-2)) levels (the WT1 expression level of K562 cells was defined as 1.0), whereas the average levels of WT1 expression in FACS-sorted and -unsorted leukemic cells were 2.4 to 9.3 x 10(-1). Thus, the WT1 expression levels in normal hematopoietic progenitor cells were at least 10 times less than those in leukemic cells. Therefore, we could not find any normal counterparts of BM or CB that expressed the WT1 at levels comparable with those in leukemic cells. These results indicate an aberrant overexpression of the WT1 gene in leukemic cells and imply the involvement of this gene in human leukemogenesis.

Thirty-one patients (27 with acute myeloid leukemia [AML], 2 with acute lymphocytic leukemia [ALL], and 2 with acute mixed lineage leukemia [AMLL]) treated with conventional chemotherapy (CHT) and 23 patients (13 AML, 5 ALL, and 5 with chronic myeloid leukemia [CML]) treated with allogeneic bone marrow transplantation (BMT) were monitored for WT1 expression levels in BM and peripheral blood (PB) by reverse transcriptase-polymerase chain reaction over a long-term period (mean, 29 months for CHT and 24 months for BMT). Sixteen of the patients in the CHT group and 3 in the BMT group who had achieved complete remission suffered clinical relapse. In 10 of these patients, WT1 expression that had returned to normal BM levels (< 10(-3); the WT1 expression level of K562 cells was defined as 1.0) after complete remission (CR) either gradually or rapidly increased again to abnormal levels 1 to 18 months (mean, 7 months) before clinical relapse became apparent. In another 9 patients, WT1 expression never returned to normal BM levels even after CR and the subsequent relapse was accompanied by a rapid increase in WT1 expression to levels higher than 10(-2) (10(-3) levels in PB). On the other hand, the remaining 35 patients (15 CHT and 20 BMT) maintained their CR. In 29 of these patients (11 CHT and 18 BMT), WT1 expression either gradually or rapidly decreased to normal BM levels, whereas in the other 6 (4 CHT and 2 BMT), low or very low levels of WT1 mRNAs (10(-3) to 10(-2) in BM and 10(-5) to 10(-3) in PB) remain detectable, but without any clinical signs of relapse. A clear correlation was found to exist between the minimal residual disease (MRD) detected in the paired BM and PB samples for all types of leukemias (AML, ALL, and CML), with MRD in PB being approximately one-tenth of that in BM. WT1 quantitation of 168 paired BM and PB samples showed that PB samples were superior to BM samples for the detection of MRD. We conclude that monitoring of WT1 expression levels in BM and PB makes it possible to rapidly assess the effectiveness of individual treatment and diagnose clinical relapse in the early stage for all leukemia patients regardless of the presence or absence of tumor-specific DNA markers.

Analyses of epitopes and biological activity were made on two preparations of recombinant human thyrotropin produced in Chinese hamster ovary cells. Seven monoclonal antibodies recognizing four epitopes on alpha subunit of hTSH (hTSH alpha) and three on beta subunit (hTSH beta) were used for the analysis. Binding activities of the two rhTSH with each antibody were almost identical with that of a pituitary-derived reference hTSH, except at one epitope on hTSH alpha. Their immunoreactivity measured by four commercial immunoassay kits and their bioactivity by thyrotropin dependent FRTL-5 cell system, however, agreed closely with those of the reference hTSH. From these results and its constant availability, rhTSH will be a good candidate for a future standard material in assays for hTSH.

Glutathione S-transferase Y-2 mRNA synthesis was induced in yeast Issatchenkia orientalis approximately 37-fold by cultivation with o-dinitrobenzene. A cDNA library complementary to poly (A)+RNA of I. orientalis grown with o-dinitrobenzene was screened by colony hybridization. Twenty positive clones were obtained from 6,000 clones and seven of twenty positive clones expressed glutathione S-transferase activity in E. coli. One of the expressing clones harboring plasmid pHT108 had 28 times more glutathione S-transferase activity induced by Isopropyl-beta-D-thio-galactopyranoside than a strain harboring plasmid pUC118. Expressed glutathione S-transferase Y-2 protein comigrated with yeast glutathione S-transferase Y-2 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis as detected by immunoblot analysis.