Title: Preparation of Chiral Triacylglycerols, sn-POO and sn-OOP, via Lipase-mediated Acidolysis Reaction Yamamoto Y, Yoshida H, Nagai T, Hara S Ref: J Oleo Sci, 67:207, 2018 : PubMed
It is well known that lipases are useful tools for preparing various structured triacylglycerols (TAGs). However, the lipase-mediated preparation of chiral TAGs has never been reported. This study aimed to prepare chiral TAGs (viz., 1-palmitoyl-2,3-dioleoyl-sn-glycerol (sn-POO) or 1,2-dioleoyl-3-palmitoyl-sn-glycerol (sn-OOP)) via lipase mediated acidolysis, using triolein (TO) and palmitic acid (P) as substrates. Three commercially available lipases (viz., Lipozyme RM-IM(), Lipozyme TL-IM(), and Lipase OF()) were used. Lipozyme RM-IM() resulted in an increase 1P-2O (sn-POO + sn-OOP + 1,3-dioleoyl-2-palmitoyl-sn-glycerol) content with reaction time, which plateaued at 2~24 h (max. yield 47.1% at 4 h). The highest sn-POO/sn-OOP ratio of ca. 9 was obtained at 0.25 h, and the rate got close to 1 with reaction time (sn-POO/sn-OOP = 1.3 at 24 h). Lipozyme TL-IM() resulted in a lower 1P-2O synthesis rate than Lipozyme RM-IM(), where its highest sn-POO/sn-OOP ratio of ca. 2 was obtained at 0.25 h and did not vary much further with reaction time. In the case of Lipase OF(), its reaction rate for 1P-2O synthesis was lower than that of the other two lipases, and the highest sn-POO/sn-OOP ratio of ca. 1.4 was obtained at 0.5 h, reaching closer to 1 with a longer reaction time. Reaction solvents (viz., hexane, acetone, and benzene) also affected the 1P-2O preparation, where the highest 1P-2O content was obtained with the solvent-free system. Furthermore, the solvent-free system showed a higher reaction rate for 1P-2O synthesis than did the hexane system, with no effect on chiral specificity of the lipase for the TAG molecules. These results suggested that among three types of commercial lipase, Lipozyme RM-IM() is the most useful for the preparation of chiral TAGs by acidolysis reaction.
The plant gibberellin (GA) receptor GID1 shows sequence similarity to carboxylesterase (CXE). Here, we report the molecular evolution of GID1 from establishment to functionally diverse forms in eudicots. By introducing 18 mutagenized rice GID1s into a rice gid1 null mutant, we identified the amino acids crucial for GID1 activity in planta. We focused on two amino acids facing the C2/C3 positions of ent-gibberellane, not shared by lycophytes and euphyllophytes, and found that adjustment of these residues resulted in increased GID1 affinity toward GA4, new acceptance of GA1 and GA3 carrying C13-OH as bioactive ligands, and elimination of inactive GAs. These residues rendered the GA perception system more sophisticated. We conducted phylogenetic analysis of 169 GID1s from 66 plant species and found that, unlike other taxa, nearly all eudicots contain two types of GID1, named A- and B-type. Certain B-type GID1s showed a unique evolutionary characteristic of significantly higher nonsynonymous-to-synonymous divergence in the region determining GA4 affinity. Furthermore, these B-type GID1s were preferentially expressed in the roots of Arabidopsis, soybean, and lettuce and might be involved in root elongation without shoot elongation for adaptive growth under low-temperature stress. Based on these observations, we discuss the establishment and adaption of GID1s during plant evolution.
Title: Cloning and sequencing of cDNA and genomic DNA encoding serine carboxypeptidase of Fusarium moniliforme that was copurified with phosphatase Yoshida H, Iizuka M, Norioka N, Norioka S Ref: J Biosci Bioeng, 103:521, 2007 : PubMed
A previous study [Yoshida, H. et al., J. Biochem., 140, 813-823 (2006)] revealed that a protein of unknown nature was copurified with PDM phosphatase of Fusarium moniliforme. In this study, the identity of this protein was investigated. The results of homology search for the tryptic peptides derived from the purified preparation of PDM phosphatase strongly suggested that it might be serine carboxypeptidase. In fact, carboxypeptidase activity was demonstrated in the preparation and partial separation of carboxypeptidase from PDM phosphatase was achieved by gel filtration high-performance liquid chromatography. Cloning and sequencing of the full-length cDNA encoding the carboxypeptidase was successfully conducted. The cDNA possessed an open reading frame for a protein of 575 amino acid residues with a molecular mass of 64,650 Da, which was highly homologous to certain fungal serine carboxypeptidases. Comparison of the deduced amino acid sequence with the N-terminal sequence of the separated carboxypeptidase revealed that the mature enzyme starts at serine 56 of the precursor and has a molecular mass of 58,487 Da. Cloning and sequencing of the genomic DNA corresponding to the cDNA demonstrated that the gene of carboxypeptidase consists of four exons. A limited number of close homologs of F. moniliforme carboxypeptidase were detected among fungi by homology search and their evolutionary relationship was discussed.
We succeeded in purifying the major glycolipid fraction in the class of sulfoquinovosyl diacylglycerol, monogalactosyl diacylglycerol and digalactosyl diacylglycerol (DGDG) from a green vegetable, spinach (Spinacia oleracea L.). This glycolipid fraction was an inhibitor of DNA polymerases and a growth inhibitor of NUGC-3 human gastric cancer cells, and, interestingly, the activities were much stronger when the fraction was hydrolyzed by lipase. Glycolipids in the hydrolyzed fraction consisted of sulfoquinovosyl monoacylglycerol (SQMG), monogalactosyl monoacylglycerol (MGMG) and DGDG. In the in vivo antitumor assay using Greene's melanoma, the fraction containing SQMG, MGMG and DGDG showed to be a promising suppressor of solid tumors. Spinach glycolipid fraction might be a potent antitumor compound if directly injected into a tumor-carrying body, and this fraction may be a healthy food material that has antitumor activity.
Title: Three-dimensional structures of enzymes useful for beta-lactam antibiotic production Barends TR, Yoshida H, Dijkstra BW Ref: Curr Opin Biotechnol, 15:356, 2004 : PubMed
Significant advances have been made in the structure-based engineering of enzymes useful for beta-lactam antibiotic production. Structure-based engineering of penicillin G acylase and cephalosporin acylase has resulted in improved enzymes for use in enzymatic production processes. The structures of many other enzymes that could be used in the production of beta-lactam antibiotics, such as enzymes from the beta-lactam biosynthetic pathway and beta-lactam antibiotic-converting enzymes, have been determined. The interest in these structures suggests that the future may see an even more extensive use of rationally engineered biocatalysts in antibiotic production than today.
Donepezil is a selective acetylcholinesterase inhibitor approved for the symptomatic treatment of mild to moderate Alzheimer's disease (AD). Since behavioral symptoms severely affect quality of life for AD patients and their caregivers, predicting behavioral responses to donepezil will be useful in managing patients with AD. In this study, we analyzed 70 consecutive cases with mild to moderate AD. Caregivers were interviewed with the Neuropsychiatric Inventory for behavioral assessment and 4-point improvement at week 12 was accepted as a treatment response. Twenty-one (30.0%) patients showed a behavioral response, while 42 (60.0%) showed no behavioral change and 7 (10.0%) worsened. Dysphoria, anxiety and apathy significantly improved after treatment among the responder group. The baseline profile including age, sex, Mini-Mental State Examination (MMSE), the Alzheimer's Disease Assessment Scale (ADAS-cog) and the Geriatric Depression Scale did not differ significantly among the three groups. Statistical Parametric Mapping analysis of single photon emission computed tomography (SPECT) images at baseline showed that cerebral blood flow in the premotor and parietotemporal cortices was significantly higher in the responder group than in the worse group. The present study suggested usefulness of SPECT imaging in the prediction of behavioral response to donepezil among AD patients even with similar psychiatric symptoms and cognitive functions.
Title: A new method of functional scintiphotosplenoportography using technetium-99m-DTPA-galactosylated human serum albumin Kaneko M, Onda M, Tajiri T, Yoshida H, Kumita S, Kumazaki T Ref: Hepato-Gastroenterology, 47:1636, 2000 : PubMed
BACKGROUND/AIMS: We investigated whether a new method for scintiphotosplenoportography using technetium-99m-DTPA-galactosylated human serum albumin could visualize the portal venous system and simultaneously assess hepatic function. METHODOLOGY: Thirty-two patients underwent scintiphotosplenoportography, including 4 with metastatic tumor in an otherwise normal liver, 5 with chronic hepatitis, 9 with cirrhosis, and 14 with both hepatocellular carcinoma and cirrhosis. The tracer (3 mg, 185 MBq) was injected into the spleen under ultrasonic guidance. Hepatic function was evaluated using the receptor index and the index of blood clearance, both calculated by regions of interest for liver and for heart. Spleen-to-liver times were calculated by subtracting peak activity times over the spleen from those over the portal vein. RESULTS: Correlations of blood clearance and receptor indices with plasma retention rate of indocyanine green at 15 min (respectively, r = 0.616, P < 0.0005 and r = -0.669, P < 0.0001), prothrombin time (r = -0.605, P < 0.0005 and r = 0.710, P < 0.0001), the hepaplastin test (r = -0.526, P < 0.005 and r = 0.605, P < 0.0005), serum albumin (r = -0.488, P < 0.005 and r = 0.640, P < 0.0001), and serum cholinesterase (r = -0.600, P < 0.0005 and r = 0.671, P < 0.0001) were significant. Blood clearance and receptor indices also were significantly related to clinical stage, underlying liver disease, features of radioisotopic images, and presence of esophagogastric varices (P < 0.01). Additionally spleen-to-liver time reflected progressive hepatic damage. CONCLUSIONS: Our new method of scintiphotosplenoportography using technetium-99m-DTPA-galactosylated human serum albumin can assess hepatic function and portal hemodynamics simultaneously. Results correlate with accepted, conventional diagnostic techniques. This method represents a potent new method for evaluating the hepatic system.
Title: Correlations between plasma platelet-activating factor acetylhydrolase (PAF-AH) activity and PAF-AH genotype, age, and atherosclerosis in a Japanese population Yamada Y, Yoshida H, Ichihara S, Imaizumi T, Satoh K, Yokota M Ref: Atherosclerosis, 150:209, 2000 : PubMed
Platelet-activating factor acetylhydrolase (PAF-AH), a plasma enzyme that hydrolyzes PAF and oxidized phospholipids, is thought to be involved in protecting cells against oxidative stress. A G(994) (M allele)-->T (m allele) mutation in the plasma PAF-AH gene, which results in a Val(279)-->Phe substitution in the mature protein, leads to a loss of catalytic activity. To elucidate the relationships among PAF-AH enzyme activity, genotype, age, and atherosclerosis, we assayed these parameters in a large Japanese population (n=3932) that consisted of three groups; a control group (healthy individuals; n=1684), a risk-factor group (individuals having at least one conventional risk factor for atherosclerosis; n=1398), and a diseased group (patients who had suffered a myocardial infarction or stroke; n=850). We observed a significantly increased frequency of the m allele in the diseased group as compared with the control or risk-factor groups. Plasma PAF-AH activity increased significantly with age in women in the control group with the MM and Mm genotypes, and in men in the control group with the MM genotype, but not in men with the Mm genotype. In both the risk-factor and diseased groups, however, no correlation was observed between plasma PAF-AH activity and age in subjects with either genotype. These results suggest that in individuals with the MM genotype, plasma PAF-AH activity may be increased in response to stresses induced by PAF and/or oxidized phospholipids that might accumulate with age, but that this response is not evident or reduced in healthy individuals with the m allele, or in subjects with atherosclerotic disease, or having risk factors. Together with our previous findings, the G(994)-->T mutation in the PAF-AH gene may be one of the genetic determinants for atherosclerotic disease in the Japanese population.
Platelet-activating factor (PAF) acetylhydrolase is an enzyme that inactivates PAF. Deficiency of this enzyme is caused by a missense mutation in the gene. We previously found a higher prevalence of this mutation in patients with ischemic stroke. This fact suggests that the mutation might enhance the risk for stroke through its association with hypertension. We have addressed this hypothesis by analyzing the prevalence of the mutation in hypertension. We studied 138 patients with essential hypertension, 99 patients with brain hemorrhage, and 270 healthy controls. Genomic DNA was analyzed for the mutant allele by the polymerase-chain reaction. The prevalence of the mutation was 29.3% (27.4% heterozygotes and 1.9% homozygotes) in controls and 36.2% in hypertensives and the difference was not significant. The prevalence in patients with brain hemorrhage was significantly higher than the control: 32.6% heterozygotes and 6.1% homozygotes (p <0.05). PAF acetylhydrolase deficiency may be a genetic risk factor for vascular diseases.
Title: A mutation in plasma platelet-activating factor acetylhydrolase (Val279-->Phe) is a genetic risk factor for stroke Hiramoto M, Yoshida H, Imaizumi T, Yoshimizu N, Satoh K Ref: Stroke, 28:2417, 1997 : PubMed
BACKGROUND AND PURPOSE: Platelet-activating factor (PAF) is a phospholipid with multiple actions that include thrombosis and inflammation. It is inactivated by a plasma enzyme, PAF acetylhydrolase. Deficiency of this enzyme in plasma is caused by a missense mutation in the gene (Val279-->Phe). We have studied a possible association of this mutation with the risk of stroke. SUBJECTS AND METHODS: We studied 120 consecutive patients with cerebral thrombosis. The control group consisted of 134 patients matched for age and sex with minor complaints but without stroke. Genomic DNA was analyzed for the mutant allele by a specific polymerase-chain reaction. Plasma PAF acetylhydrolase activity was determined by the method of Stafforini et al. RESULTS: The prevalence of the mutant gene was 43.4% in stroke patients (39.2% heterozygotes and 4.2% homozygotes), which was significantly higher than the 25.4% in control subjects (22.4% heterozygotes and 3.0% homozygotes) (chi 2 = 9.22, P < .01). The prevalence was slightly higher in stroke patients without hypertension than those with hypertension, but the difference was not significant. The patients with family histories of stroke had a slightly higher but not a significant prevalence of the mutant gene as compared with those without family histories of stroke. Plasma PAF acetylhydrolase activity was higher in patients than in control subjects, in normal subjects, or patients with a heterozygous genotype. CONCLUSIONS: These results suggest that plasma PAF acetylhydrolase deficiency may be a risk factor for stroke. This may explain the relatively high prevalence of stroke in Japan, as the mutation is more common among Japanese than Caucasians.
Deficiency of plasma platelet-activating factor (PAF) acetylhydrolase is an autosomal recessive syndrome that has been associated with severe asthma in Japanese children. Acquired deficiency has been described in several human diseases usually associated with severe inflammation. PAF acetylhydrolase catalyzes the degradation of PAF and related phospholipids, which have proinflammatory, allergic, and prothrombotic properties. Thus, a deficiency in the degradation of these lipids should increase the susceptibility to inflammatory and allergic disorders. Miwa et al. reported that PAF acetylhydrolase activity is absent in 4% of the Japanese population, which suggests that it could be a common factor in such disorders, but the molecular basis of the defect is unknown. We show that inherited deficiency of PAF acetylhydrolase is the result of a point mutation in exon 9 and that this mutation completely abolishes enzymatic activity. This mutation is the cause of the lack of enzymatic activity as expression in E. coli of a construct harboring the mutation results in an inactive protein. This mutation as a heterozygous trait is present in 27% in the Japanese population. This finding will allow rapid identification of subjects predisposed to severe asthma and other PAF-mediated disorders.
Title: Genetic lipid storage disease with lysosomal acid lipase deficiency in rats. Yoshida H, Kuriyama M Ref: Lab Anim Sci, 40:486, 1990 : PubMed
Title: Poster: Blockade of cholinergic receptors by PrBCM in the rat cerebral cortex causes deficits in passive avoidance learning Yoshida H, Fukuchi I, Nakahiro M, Uchida S Ref: Trends in Pharmacological Sciences, Suppl:95, 1986 : PubMed
Title: Studies on soluble proteins released from the synaptic vesicles of rat brain cortex Matsuda T, Saito K, Katsuki S, Hata F, Yoshida H Ref: Journal of Neurochemistry, 18:713, 1971 : PubMed
Title: Effects of metabolic inhibitors on potassium- and acetylcholine-stimulated incorporation of phosphate into phospholipids of rat-brain cortex slices Yoshida H, Quastel JH Ref: Biochimica & Biophysica Acta, 57:67, 1962 : PubMed
