BACKGROUND: The effectiveness of adjuvant transcatheter arterial chemo- or/and chemoembolization therapy after curative hepatectomy of initial hepatocellular carcinoma (HCC) is controversial. This study aimed to evaluate whether hepatectomy combined with adjuvant transcatheter arterial infusion therapy (TAI) for initial HCC has better long-term survival outcomes than hepatectomy alone. METHODS: From January 2012 to December 2014, a prospective randomized controlled trial of patients with initial HCC was conducted. Then, 114 initial HCC patients were recruited to undergo hepatectomy with adjuvant TAI (TAI group, n=55) or hepatectomy alone (control group, n=59) at our institution. The TAI therapy was performed twice, at 3 and 6 months after curative hepatectomy (UMIN 000011900). RESULTS: The patients treated with TAI had no serious side effects, and operative outcomes did not differ between the two groups. No significant differences were found in the pattern of intrahepatic recurrence or time until recurrence between the two groups. Moreover, no significant differences were found in the relapse-free survival or overall survival. Low cholinesterase level (<200) had been identified as a risk factor affecting relapse-free survival. Furthermore, compared with surgery alone, adjuvant TAI with hepatectomy improved the overall survival for lower-cholinesterase patients. CONCLUSIONS: Adjuvant TAI is safe and feasible, but it cannot reduce the incidence of postoperative recurrence or prolong survival for patients who underwent curative hepatectomy for initial HCC.
Title: Application of two newly identified and characterized feruloyl esterases from Streptomyces sp. in the enzymatic production of ferulic acid from agricultural biomass Uraji M, Arima J, Inoue Y, Harazono K, Hatanaka T Ref: PLoS ONE, 9:e104584, 2014 : PubMed
Ferulic acid (FA), a component of hemicellulose in plant cell walls, is a phenolic acid with several potential applications based on its antioxidant properties. Recent studies have shown that feruloyl esterase (FAE) is a key bacterial enzyme involved in FA production from agricultural biomass. In this study, we screened a library of 43 esterases from Streptomyces species and identified two enzymes, R18 and R43, that have FAE activity toward ethyl ferulate. In addition, we characterized their enzyme properties in detail. R18 and R43 showed esterase activity toward other hydroxycinnamic acid esters as well, such as methyl p-coumarate, methyl caffeate, and methyl sinapinate. The amino acid sequences of R18 and R43 were neither similar to each other, nor to other FAEs. We found that R18 and R43 individually showed the ability to produce FA from corn bran; however, combination with other Streptomyces enzymes, namely xylanase and alpha-l-arabinofuranosidase, increased FA production from biomass such as corn bran, defatted rice bran, and wheat bran. These results suggest that R18 and R43 are effective FAEs for the enzymatic production of FA from biomass.
BACKGROUND/PURPOSE: Human carboxylesterase 2 (CES2) is the key enzyme for metabolic activation of irinotecan (CPT-11). The aim was to evaluate the clinical implications of CES2 RNA expression in neuroblastoma cells. METHODS: CES2 RNA expression was determined by real-time reverse transcription-polymerase chain reaction in five neuroblastoma cell lines and 42 clinical samples of untreated neuroblastoma. Sensitivity to CPT-11 was assessed by WST-8 colorimetric assays. Induction of apoptosis was evaluated by flow cytometry after CPT-11 exposure. Protein expression of CES2 was evaluated by Western blotting analysis. CES2 RNA expression in clinical samples was investigated for its associations with the clinicopathological characteristics. RESULTS: CES2 RNA expression was observed in neuroblastoma cells, and its expression in neuroblastoma cell lines was positively correlated with sensitivity to CPT-11 and apoptosis after CPT-11 exposure in vitro. CES2 RNA expression was correlated with the protein levels of CES2 in vitro. CES2 RNA expression was significantly higher in patients with a characteristic related to advanced disease. CONCLUSIONS: Our results suggest the potential of clinical application of CPT-11 in neuroblastoma treatment for patients with advanced disease.
Ferulic acid (FA), which is present in the cell walls of some plants, is best known for its antioxidant property. By combining a commercial enzyme that shows FA esterase activity with several Streptomyces carbohydrate-hydrolyzing enzymes, we succeeded in enhancing the enzymatic production of FA from defatted rice bran. In particular, the combination of three xylanases, an alpha-L-arabinofuranosidase, and an acetyl xylan esterase from Streptomyces spp. produced the highest increase in the amount of released FAs among all the enzymes in the Streptomyces enzymes library. This enzyme combination also had an effect on FA production from other biomasses, such as raw rice bran, wheat bran, and corncob.
The term 'sake yeast' is generally used to indicate the Saccharomyces cerevisiae strains that possess characteristics distinct from others including the laboratory strain S288C and are well suited for sake brewery. Here, we report the draft whole-genome shotgun sequence of a commonly used diploid sake yeast strain, Kyokai no. 7 (K7). The assembled sequence of K7 was nearly identical to that of the S288C, except for several subtelomeric polymorphisms and two large inversions in K7. A survey of heterozygous bases between the homologous chromosomes revealed the presence of mosaic-like uneven distribution of heterozygosity in K7. The distribution patterns appeared to have resulted from repeated losses of heterozygosity in the ancestral lineage of K7. Analysis of genes revealed the presence of both K7-acquired and K7-lost genes, in addition to numerous others with segmentations and terminal discrepancies in comparison with those of S288C. The distribution of Ty element also largely differed in the two strains. Interestingly, two regions in chromosomes I and VII of S288C have apparently been replaced by Ty elements in K7. Sequence comparisons suggest that these gene conversions were caused by cDNA-mediated recombination of Ty elements. The present study advances our understanding of the functional and evolutionary genomics of the sake yeast.
Familial lecithin-cholesterol acyltransferase (LCAT) deficiency (FLD) is a rare genetic disease characterized by corneal opacities, normocytic anemia, dyslipidemia, and proteinuria progressing to chronic renal failure. In all FLD cases, a mutation has been found in the coding sequence of the LCAT gene. FLD is clinically distinguished from an acquired form of LCAT deficiency by the presence of corneal opacities. Here we describe a 36-year-old woman presenting with clinical, pathological, and laboratory data compatible with FLD. Her mother and elder sister had corneal opacities. However, genetic analysis revealed there were no mutations in the LCAT coding sequences and no alterations in LCAT mRNA expression. Furthermore, we were unable to find any underlying conditions that may lead to LCAT deficiency. The present case therefore demonstrates that LCAT deficiency may be caused by factors other than mutations in the coding sequence and we suggest that a translational or posttranslational mechanism may be involved.
The Carica papaya lipase-catalyzed acylation of benzylcarbinols with vinyl hexanoate proceeded smoothly and enantiospecifically (E > 200), affording the R-esters and leaving the S-alcohols intact. Thus, this plant lipase proved to be a promising biocatalyst for the resolution of alcohols as well as for that of carboxylic acids reported earlier.
AIMS: Immunosuppressive acidic protein (IAP) is a potent biological marker for immunological surveillance in patients with malignant tumors. This study aimed to investigate the significance of serum IAP as an index of disease status, clinicopathological findings and prognosis in colorectal cancer. METHODS: A total of 101 patients with colorectal cancer and 80 normal volunteers were included in this retrospective trial. Preoperative serum IAP was assayed using a commercially available enzyme-linked immunosorbent assay kit. RESULTS: The serum IAP level in the patients, which was not associated with clinicopathological features except for tumor size, was significantly higher than that in controls. The serum IAP level was closely correlated with percent body weight loss, serum albumin and cholinesterase, and percentage of circulating lymphocytes reflecting the host's nutritional and immunological conditions. Interestingly, these parameters were not associated with factors reflecting disease progression except for tumor size. The prognosis of patients with higher IAP levels was significantly worse than that of patients with lower IAP levels. Furthermore, an elevated serum IAP level was an independent prognostic marker in all patients. CONCLUSION: The preoperative serum IAP level may reflect the general condition of colorectal cancer patients, and thus may predict long-term survival independently of stage progression.
BACKGROUND: Dementia of Alzheimer type (DAT) has been diagnosed objectively by using single photon emission tomography (SPECT). Donepezil hydrochloride (donepezil) is available for the symptomatic treatment of DAT. In a quantitative evaluation of therapeutic response in DAT, to compare with regional cerebral blood flows (rCBF) of various lesions before and after treatment, uptake in some sorts of cerebral regions of interests (ROIs) were used to be measured. But ROI analysis has problems such as poor reproducibility and lack of objectivity. The aim of this study was to investigate the evaluation of therapeutic response by three-dimensional stereotaxic ROI template (3DSRT), fully automated ROI analysis software, which can objectively estimate rCBF. METHODS: SPECT studies and Alzheimer's Disease Assessment Scale Japan cognitive Subscale function test ADAS-Jcog, as recognitive function test were performed for 22 patients (16 females, 6 males mean age = 73.6 years) who were diagnosed as DAT. On 3DSRT, we compared ratios of the rCBF values of the parietal lobes, temporo-occipital lobes, hippocampus, corpus callosum and the frontal lobes/cerebellar hemispheres before and after medical treatment. To determine a cut-off number of areas exhibiting improved blood flow optimal as an indicator of improvement in cognitive function in response to treatment, receiver operating characteristics (ROC) analysis of number of areas improved blood flow was performed. RESULTS: The number of cases exhibiting changes in cognitive function was greatest when the cut-off number of areas exhibiting improved blood flow was set at 5. CONCLUSIONS: The possibility of evaluation of therapeutic response to Donepezil in patients with DAT using 3DSRT was thus demonstrated by our study.
BACKGROUND: Our aim was to clarify the significance of widely accepted irinotecan (CPT-11)/5-fluorouracil (5-FU) combinations in colon cancer by investigating their sequential effect. METHODS: The sequential effect of CPT-11/5-FU in two colon cancer cell lines, LoVo and SW480, was evaluated by WST-8 colorimetric assay. The cell cycle distributions of each drug were analyzed by flow cytometry, and then the chemoresistant mechanisms and expression of a drug transporter (MDR1), the bcl-2 apoptotic pathway, metabolizing enzymes [carboxylesterase (CE), dihydropyrimidine dehydrogenase], and target enzymes (topoisomerase I, thymidine synthase) associated with sequence-dependent cytotoxicity were examined. RESULTS: The cytotoxicity of 5-FU (10, 100, 1000 microM) followed by CPT-11 (1 microM) was significantly greater than that of CPT-11 (1 microM) followed by 5-FU (10, 100, 1000 microM) (P < 0.05). Reverse transcription-polymerase chain reaction analysis revealed that exposure to 5-FU downregulated both MDR1 and bcl-2 mRNA and simultaneously upregulated CE2 mRNA expression, suggesting enhancement of subsequent CPT-11 cytotoxicity. CONCLUSIONS: The cytotoxic effects of the CPT-11/5-FU combinations were shown to be schedule-dependent in human colon cancer cells. The findings suggest that 5-FU followed by CPT-11 administration might be the optimal sequence for CPT-11/5-FU treatment of advanced colon cancer.
Title: Genome sequence of Xanthomonas oryzae pv. oryzae suggests contribution of large numbers of effector genes and insertion sequences to its race diversity Inoue Y, Takeya M, Sasaki A, Kaki H Ref: , 39:275, 2005 : PubMed
The plant-pathogenic prokaryote Xanthomonas oryzae pv. oryzae (Xoo) causes bacterial blight, one of the most important diseases of rice. The bacterium is a model organism for the analysis of plant-pathogen interaction, because more than 30 races differing in virulence and 25 resistance genes in rice have been reported to date. We present here the complete genome sequence of Xoo strain MAFF 311018. The size of the genome was 4,940,217 bp, in a single circular chromosome. The genome structure of Xoo MAFF 311018 was characterized by large numbers of effector (avr) genes of the avrBs3/pth family and insertion sequences (ISs). RFLP analysis of diverse strains using ISXo1 as a probe suggests that the prevalence of mobile elements in this species, which can bring about genome inversions and rearrangement, may have played a major role in generating the high degree of genetic diversity and race differentiation characteristic of this pathogen. The Xoo MAFF 311018 sequence was also highly similar to those of X. axonopodis pv. citri and X. campestris pv. campestris with the exception of the large number of effectors and IS elements, and numerous inversions and rearrangements.
We encountered three cases of organophosphate poisoning treated with pralidoxime iodide (PAM) and whole-bowel irrigation without atropine sulfate. All patients recovered without persistence or recurrence of toxic symptoms and without any somatic after effects. In case 1, a 48-year-old woman ingested approximately 5 g of ethylthiometon in a suicide attempt. She was transferred to the hospital because of cardiopulmonary arrest. After resuscitation, she was transferred to our center. She was placed on a ventilator and received i.v. PAM and polyethylene glycol-electrolyte through a nasojejunal tube for whole-bowel irrigation. Six days later, serum ChE was improved. In case 2, a 51-year-old man ingested approximately 30 g of malathion in a suicide attempt and was transferred to our center because of dyspnea. He was treated with PAM and whole-bowel irrigation, but did not require a respirator. Serum ChE already showed improvement the following day. In case 3, a 31-year-old man ingested approximately 50 g of DEP in a suicide attempt and was transferred to our center because of unconsciousness. He was treated with a respirator, PAM and whole-bowel irrigation. Serum ChE improved within two days. These cases suggest the possibility that preferential whole-bowel irrigation without atropine sulfate prevents the persistence or recurrence of the toxic effects of organophosphate.
NO-1886 is a lipoprotein lipase (LPL) activator. Administration of NO-1886 results in an increase in plasma high-density lipoprotein cholesterol (HDL-C) and a decrease in plasma triglyceride (TG) levels. The aim of this study was to ascertain whether NO-1886 improves fatty liver caused by high-fat feeding in streptozotocin (STZ)-induced diabetic rats. Administration of NO-1886 resulted in increased plasma HDL-C levels and decreased TG levels without affecting total cholesterol and glucose levels in the diabetic rats. NO-1886 dose-dependently decreased liver TG contents and cholesterol contents, resulting in improvement of fatty liver. NO-1886 also reduced plasma aspartate aminotransferase (AST) and alanine aminotransferase (ALT) that accompany fatty liver. The liver cholesterol contents were inversely correlated with plasma HDL-C levels (r = -0.5862, P <.001) and were positively correlated with plasma TG levels (r = 0.4083, P <.003). The liver TG contents were inversely correlated with plasma HDL-C levels (r = -0.6195, P <.001) and were positively correlated with plasma TG levels (r = 0.5837, P <.001). There was no correlation between plasma cholesterol levels, and cholesterol and TG contents in liver. These results indicate that reducing plasma TG levels and elevating in HDL-C levels may result in improving fatty liver.
We encountered a case of mixed poisoning by organophosphate and methanol. Each poisoning was comparatively frequent but we reviewed no cases of mixed poisoning of them. A 49-year-old woman was transferred to our hospital because of oral ingestion of organophosphorous compound (about 14 g of fenitrothion) and glass cleaner (about 40 g of methanol) for suicidal purpose. She underwent general antioverdose treatment including gastric lavage, activated charcoal and cathartics. For fenitrothion poisoning, she received atropine and pralidoxime. For methanol poisoning, she was treated with hemodialysis. Three days later, she moved to psychiatric ward from emergency ward without aftereffects and was given a diagnosis of depression.
Title: The glutamate receptor agonist, AMPA, induces acetylcholine release in guinea pig cochlea; a microdialysis study Hoya N, Ogawa K, Inoue Y, Takiguchi Y, Kanzaki J Ref: Neuroscience Letters, 311:206, 2001 : PubMed
Acetylcholine (Ach) has been considered a major neurotransmitter in the inner ear efferent nerve endings. A bioassay analysis has shown that the electrical stimulation of the crossed olivocochlear bundle increased the Ach-like activity in the perilymph. Applying in vivo microdialysis techniques and high-performance liquid chromatography to the perilymph, the change of Ach level was thus measured before and after alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA), a glutamate receptor agonist, was added to the perfusate. Ach was only detectable when the perfusate contained an acetylcholinesterase inhibitor. The level of Ach increased 2-3-fold immediately after AMPA was administered. Our data suggest that the afferent stimulation, such as the administration of AMPA, may therefore induce the release of Ach from the efferent nerve endings.
Platelet-activating factor (PAF) is a potent mediator of inflammatory injury in renal diseases. PAF is degraded to inactive products by PAF acetylhydrolase. Recently, a point mutation (G to T transversion) of the PAF acetylhydrolase gene was observed at position 994, and this mutation was found to contribute to the variability in plasma PAF levels, with undetectable plasma PAF acetylhydrolase activity occurring in homozygous patients (TT genotype) and reduced levels of activity in heterozygous patients (GT genotype). Therefore, we investigated the effect of the PAF acetylhydrolase gene mutation on the pathogenesis and progression of immunoglobulin A (IgA) nephropathy. Genomic DNA was obtained from 89 children with IgA nephropathy and 100 controls. We identified the PAF acetylhydrolase gene mutation (G994T) by polymerase chain reaction. There was no significant difference in genotypic frequency between patients and controls. However, urinary protein excretion at the time of biopsy was significantly greater in patients with the GT/TT genotypes than in those with the GG genotype. The percentage of glomeruli with mesangial cell proliferation was significantly greater in patients with the GT/TT genotypes than in those with the GG genotype. These results indicate the PAF acetylhydrolase gene mutation may influence the degree of proteinuria and the extent of mesangial proliferation in the early stage of childhood IgA nephropathy.
Title: [Evaluation of transcatheter hepatic segmental or subsegmental infusion of SMANCS for treatment of hepatocellular carcinoma] Inoue Y, Tomoda K, Oi H, Nakamura H Ref: Japanese Journal of Cancer & Chemotherapy, 1:56, 1998 : PubMed
A total of seventeen patients with hepatocellular carcinoma (HCC), nineteen HCCs, who underwent as an initial treatment transcatheter hepatic segmental or subsegmental arterial administration of SMANCS alone for hepatocellular carcinoma (HCC), were studied to evaluate the efficacy and complication of that treatment. The initial treatments provided CR in eight patients (47%), and repeat administrations of SMANCS achieved CR in an additional four patients (24%). The initial treatment provided a dense deposit of Lipiodol in the twelve tumors (63%), in five of which Lipiodol was thereafter washed out in some portions of the tumor. Complete necrosis was obtained in nine (75%) of fourteen hypervascular tumors, and in two (40%) of five intermediately vascular or hypovascular tumors. Segmental or subsegmental administration of SMANCS was well tolerated with self-controlled abdominal pain or fever well responding to medication. Ascites was seen in three cases, and atrophy of the segment infused occurred in five patients. Cholinesterase significantly reduced at one week and one month, then recovered to baseline two to three months after initial treatment. The cumulative survival rates were 77% at 1 year, 66% at 2 years, and 53% at 5 years in the whole patients. The survival rate was 100% at 5 years in the Child A group. In the patients who obtained CR using SMANCS alone, the survival rates were 89% at 1 year, 74% at 2 years and 56% at 5 years. Although this method may transiently deteriorate hepatic function, segmental or subsegmental administration of SMANCS may be an excellent therapeutic method for treatment of HCC and promising for use in properly selected patients.
In a previous study, we demonstrated that Platelet-activating Factor (PAF) acetylhydrolase purified from bovine brain cortical cytosol consists of two mutually homologous catalytic subunits (alpha1 and alpha2) and one putative regulatory beta subunit. The latter is a product of the LIS1 gene, which is defective in the Miller-Dieker syndrome, a form of lissencephaly. In this study, we examined the expression patterns of these three subunits in the developing rat brain. All three subunits were expressed in embryonic brain, whereas only alpha2 and beta subunit were detected in the adult brain by Western blotting. Biochemical analyses revealed that the alpha1/alpha2 heterodimer and alpha2/alpha2 homodimer are major catalytic units of embryonic and adult brain PAF acetylhydrolases, respectively. The alpha1 transcript and protein were detected predominantly in embryonic and postnatal neural tissues, such as the brain and spinal cord. Furthermore, we found using primary cultured cells isolated from neonatal rat brain that alpha1 protein were expressed only in neurons but not in glial cells and fibroblasts. In contrast, alpha2 and beta transcripts and proteins were detected both in neural and non-neural tissues, and their expression level was almost constant from fetal stages through adulthood. These results indicate that alpha1 expression is restricted to actively migrating neurons in rats and that switching of catalytic subunits from the alpha1/alpha2 heterodimer to the alpha2/alpha2 homodimer occurred in these cells during brain development, suggesting that PAF acetylhydrolase plays a role(s) in neuronal migration.
BACKGROUND: Platelet-activating factor (PAF) may be involved in the pathogenesis of steroid-responsive nephrotic syndrome (SRNS). PAF is degraded to inactive products by PAF acetylhydrolase. We have investigated whether PAF acetylhydrolase gene mutation is involved in SRNS in Japanese children. METHODS: We identified a point mutation in the PAF acetylhydrolase gene (G994T) using the polymerase chain reaction in 101 Japanese children with SRNS and 100 healthy Japanese. RESULTS: There was no difference in the genotype and allele frequencies between patients with SRNS and normal controls. The mean number of relapses during the first year after onset was significantly higher in the 26 patients who were heterozygous for the mutant allele (GT) than in 75 wild-type homozygotes (GG) (2.61 +/- 1.98 vs. 1.33 +/- 1.35; P = 0.0019). CONCLUSIONS: We conclude that analysis of the PAF acetylhydrolase gene mutation at position 994 in Japanese children with SRNS allows the identification of patients who are more likely to have a disease relapse.
Title: Biochemical and molecular characterization of the polyhydroxybutyrate depolymerase of Comamonas acidovorans YM1609, isolated from freshwater Kasuya K, Inoue Y, Tanaka T, Akehata T, Iwata T, Fukui T, Doi Y Ref: Applied Environmental Microbiology, 63:4844, 1997 : PubMed
Comamonas acidovorans YM1609 secreted a polyhydroxybutyrate (PHB) depolymerase into the culture supernatant when it was cultivated on poly(3-hydroxybutyrate) [P(3HB)] or poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [P(3HB-co-3HV)] as the sole carbon source. The PHB depolymerase was purified from culture supernatant of C. acidovorans by two chromatographic methods, and its molecular mass was determined as 45,000 Da by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The enzyme was stable at temperatures below 37 degrees C and at pH values of 6 to 10, and its activity was inhibited by diisopropyl fluorophosphonate. The liquid chromatography analysis of water-soluble products revealed that the primary product of enzymatic hydrolysis of P(3HB) was a dimer of 3-hydroxybutyric acid. Kinetics of enzymatic hydrolysis of P(3HB) film were studied. In addition, a gene encoding the PHB depolymerase was cloned from the C. acidovorans genomic library. The nucleotide sequence of this gene was found to encode a protein of 494 amino acids (M(r), 51,018 Da). Furthermore, by analysis of the N-terminal amino acid sequence of the purified enzyme, the molecular mass of the mature enzyme was calculated to be 48,628 Da. Analysis of the deduced amino acid sequence suggested a domain structure of the protein containing a catalytic domain, fibronectin type III module as linker, and a putative substrate-binding domain. Electron microscopic visualization of the mixture of P(3HB) single crystals and a fusion protein of putative substrate-binding domain with glutathione S-transferase demonstrated that the fusion protein adsorbed strongly and homogeneously to the surfaces of P(3HB) single crystals.
Acetylcholinesterase (AChE) activity was topographically investigated in the presumptive cardiac conduction tissue regions visualized by HNK-1 immunoreactivity in rat embryos, and AChE-positive cells were examined with the electron microscope. On embryonic day (ED) 14.5, when HNK-1 was most intensely visualized, AChE activity could not be detected enzyme-histochemically in the conduction tissue regions, except in the ventricular trabeculae and part of the AV node. On ED 16.5, however, the AChE activity was clearly demonstrated in some parts of the developing conduction tissue. One exception was the AV node region, where an AChE-positive area was in close proximity to an area showing HNK-1 immunoreactivity but did not overlap. Furthermore, AChE activity was demonstrated predominantly in the ventricular trabeculae, including cardiac myocytes, but was rather weak in the atrium. With the electron microscope, AChE reaction products were observed predominantly intracellularly in both developing conduction tissue cells and developing ordinary myocytes, and no reactivity was found in neuronal components. From ED 18.5 until birth, both AChE activity and HNK-1 immunoreactivity faded away in the conduction tissue. Thus, transient AChE activity in the embryonic heart seems to be different from the developing adult form and may be related to a morphogenetic function in embryonic tissues, as proposed by other authors.
