Confinement of Metalloenzymes in PEGylated Liposomes to Formulate Colloidal Catalysts for Antioxidant Cascade.

Antioxidant cascade reactions detoxifying reactive oxygen species are of significance to control oxidative stresses-triggered diseases. In the present work, the antioxidant catalysts were prepared through the confinement of dual metalloenzymes in liposomes. The amino groups of superoxide dismutase (SOD) were conjugated to the carboxyl groups-bearing liposomes encapsulated with the catalase (CAT) to formulate a spatially organized antioxidant reaction network. The activity of SOD and CAT in the liposomal system was evaluated in detail on the basis of the prolonged xanthine oxidase/xanthine reaction producing superoxide anion radicals (O2̇-) and hydrogen peroxide (H2O2) coupled with redox reactions of cytochrome c. The liposome-confined SOD and CAT molecules were clearly demonstrated to catalyze the sequential disproportionation of O2̇- and H2O2 at 25 °C in a potassium phosphate buffer solution (pH = 7.8) under moderate transfer resistance with respect to the intermediate product (H2O2) within the liposomes. Furthermore, the liposomal catalysts were modified with the poly(ethylene glycol) (PEG)-conjugated lipids with the molecular mass of the PEG moiety of about 5000 through the post-PEGylation approach. The mean hydrodynamic diameter of the PEGylated liposomal catalysts was 140-150 nm. The dual enzyme activity in liposomes and the thermal stability of the encapsulated CAT were practically unaffected by the PEGylation. The above liposome-based antioxidant catalysts are highly biocompatible, PEG-modifiable, and reactive, thereby making the catalysts potentially applicable to therapeutic materials exhibiting functionality similar to cellular peroxisomes.

Rapid leakage from PEGylated liposomes triggered by bubbles.

Liposomes are applicable to fabrication of colloidal carriers of drugs and proteins. Physicochemical stimuli-triggered leakage from liposomes offers a wide variety of applications in biochemical and biomedical fields. In this work, effects of bubbles on the characteristics of PEGylated liposomes encapsulating 5(6)-carboxyfluorescein were examined. The liposomes were composed of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and 1-10 mol% 1,2-distearoyl-sn-glycero-3-phosphoethanolamine conjugated with poly(ethylene glycol) (DSPE-PEG). The mean molecular mass Mr,PEG of the PEG moiety was 550 or 5000. A bubble column was used for generating air bubbles at a superficial gas velocity of 0.58-0.88 cm s-1. Leakage from the PEGylated liposomes was remarkably accelerated at 25 or 40 °C by introducing air to a liposome suspension at pH 7.4, whereas the dye molecules practically remained encapsulated in the liposomes being suspended in static liquid. The apparent rate constant for the dye release from the liposomes composed of DOPC and 1 mol% DSPE-PEG (Mr,PEG = 5000) being suspended in the gas-liquid flow was 168 times larger than that obtained with respect to the same liposomes in static liquid. Leakage from non-PEGylated liposomes was not pronounced even in the gas-liquid flow. Furthermore, the release rate of the dye from the PEGylated liposomes in liquid shear flow (no bubble) was clearly smaller than that in the gas-liquid flow, meaning that the interaction between bubbles and the liposomes was responsible for the observed rapid leakage. Adsorption of the PEGylated lipids to bubbles was indicated to induce leaky lipid bilayers, which was discussed on the basis of the conformational state of the PEG moiety.

Immobilized carbonic anhydrase: preparation, characteristics and biotechnological applications.

Carbonic anhydrase (CA) is an essential metalloenzyme in living systems for accelerating the hydration and dehydration of carbon dioxide. CA-catalyzed reactions can be applied in vitro for capturing industrially emitted gaseous carbon dioxide in aqueous solutions. To facilitate this type of practical application, the immobilization of CA on or inside solid or soft support materials is of great importance because the immobilization of enzymes in general offers the opportunity for enzyme recycling or long-term use in bioreactors. Moreover, the thermal/storage stability and reactivity of immobilized CA can be modulated through the physicochemical nature and structural characteristics of the support material used. This review focuses on (i) immobilization methods which have been applied so far, (ii) some of the characteristic features of immobilized forms of CA, and (iii) biotechnological applications of immobilized CA. The applications described not only include the CA-assisted capturing and sequestration of carbon dioxide, but also the CA-supported bioelectrochemical conversion of CO2 into organic molecules, and the detection of clinically important CA inhibitors. Furthermore, immobilized CA can be used in biomimetic materials synthesis involving cascade reactions, e.g. for bone regeneration based on calcium carbonate formation from urea with two consecutive reactions catalyzed by urease and CA.

Enhanced Heat Stability of α-Chymotrypsin through Single-Enzyme Confinement in Attoliter Liposomes.

The entrapment of α-chymotrypsin (α-CT) within 70-140 nm liposomes formed from POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) leads to an unexpected and remarkable increase in the thermal stability of the enzyme. This finding is based on the observation that heating aqueous suspensions of α-CT-containing POPC liposomes to 80 °C for 30 minutes resulted in partial enzyme inactivation, whereas the same treatment of aqueous solutions of free α-CT inactivated the enzyme completely. The stabilizing effect of enzyme confinement in the attoliter volumes of the liposomes was found to increase with decreasing numbers of α-CT molecules per liposome. Single-enzyme confinement was particularly effective, as intermolecular interactions between heat-denatured α-CT molecules (causing irreversible inactivation) are not possible.

Liposomes as chaperone mimics with controllable affinity toward heat-denatured formate dehydrogenase from Candida boidinii.

Chaperone machinery in living systems can catch denatured enzymes and induce their reactivation. Chaperone mimics are beneficial for applying enzymatic reactions in vitro. In this work, the affinity between liposomes and thermally denatured enzymes was controlled to stabilize the enzyme activity. The model enzyme is formate dehydrogenase from Candida boidinii (CbFDH) which is a homodimer and negatively charged in the phosphate buffer solution (pH 7.2) used. The activity of free CbFDH readily decreased at 58 °C following the first-order kinetics with the half-life t1/2 of 27 min. The turbidity measurements showed that the denatured enzyme molecules formed aggregates. The liposomes composed of zwitterionic phosphatidylcholines (PCs) stabilized the CbFDH activity at 58 °C, as revealed with six different PCs. The PC liposomes were indicated to bind to the aggregate-prone enzyme molecules, allowing reactivation at 25 °C. The cofactor ß-reduced nicotinamide adenine dinucleotide (NADH) also stabilized the enzyme activity. The affinity between liposomes and denatured CbFDH could be modulated by incorporating cationic 1,2-dioleoyloxy-3-trimethylammonium propane chloride (DOTAP) in PC membranes. The t1/2 values significantly increased in the presence of liposomes ([lipid] = 1.5 mM) composed of PC and DOTAP at the mole fraction f(D) of 0.1. On the other hand, the DOTAP-rich liposomes (f(D) ≥ 0.7) showed strong affinity toward denatured CbFDH, accelerating its deactivation. The liposomes with low charge density function as chaperone mimics that can efficiently catch the denatured enzymes without interfering with their intramolecular interaction for reactivation.

Selective oxidation of D-amino acids catalyzed by oligolamellar liposomes intercalated with D-amino acid oxidase.

D-Amino acid oxidase (DAO) is structurally unstable and exhibits broad specificity to D-amino acids. In this work, we fabricated a stable liposomal DAO system with high apparent substrate specificity. Permeability of the membrane composed of POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) was highly selective between the d-forms of alanine (Ala) and serine (Ser). The permeability coefficient of d-Ala and d-Ser at 25 °C was 3.59 and 0.27 pm/s, respectively, as determined with the dialysis method. On the other hand, the chiral environment of POPC membrane showed no clear selectivity between the enantiomers of Ala or Ser. POPC liposomes encapsulating DAO from porcine kidney selectively catalyzed the oxidation of hydrophobic D-phenylalanine (D-Phe) over D-Ala and D-Ser because of their intrinsic membrane permeability. As a different type of liposomal DAO, the enzyme molecules were conjugated to the surface of activated lipids-bearing liposomes. The activity of liposome-conjugated DAO showed significantly higher stability at 50 °C than free DAO at low enzyme concentrations ranging from 2.5 to 10 mg/L. Then, the DAO-conjugated liposomes were coated with POPC bilayers to give the oligolamellar structure intercalated with the DAO molecules. The additional bilayers allowed to induce the permeability resistance-based substrate specificity and strengthened the stabilizing effect on the DAO activity. The oligolamellar liposomes fabricated can be a colloidal platform for integrating the functions of lipid membrane to stabilize DAO and to modulate its substrate specificity.

Role of proteoglycan synthesis genes in osteosarcoma stem cells.

Osteosarcoma stem cells (OSCs) contribute to the pathogenesis of osteosarcoma (OS), which is the most common malignant primary bone tumor. The significance and underlying mechanisms of action of proteoglycans (PGs) and glycosaminoglycans (GAGs) in OSC phenotypes and OS malignancy are largely unknown. This study aimed to investigate the role of PG/GAG biosynthesis and the corresponding candidate genes in OSCs and poor clinical outcomes in OS using scRNA-seq and bulk RNA-seq datasets of clinical OS specimens, accompanied by biological validation by in vitro genetic and pharmacological analyses. The expression of ß-1,3-glucuronyltransferase 3 (B3GAT3), one of the genes responsible for the biosynthesis of the common core tetrasaccharide linker region of PGs, was significantly upregulated in both OSC populations and OS tissues and was associated with poor survival in patients with OS with high stem cell properties. Moreover, the genetic inactivation of B3GAT3 by RNA interference and pharmacological inhibition of PG biosynthesis abrogated the self-renewal potential of OSCs. Collectively, these findings suggest a pivotal role for B3GAT3 and PG/GAG biosynthesis in the regulation of OSC phenotypes and OS malignancy, thereby providing a potential target for OSC-directed therapy.

Effects of Cooking Methods on Caffeoylquinic Acids and Radical Scavenging Activity of Sweet Potato.

The effects of cooking methods, including steaming, deep-frying, and baking, on the phenolic content, 1,1-diphenyl-2-picrylhydrazyl radical scavenging activity, and isomerization of caffeoylquinic acids in sweet potato were investigated. A high correlation was observed between antioxidant capacity and total phenolic content. Deep-frying treatment resulted in higher antioxidant capacity with increasing heating time. The major phenolic components of raw sweet potat were 5-caffeoylquinic acid (CQA) and 3,5-dicaffeoylquinic acid (diCQA), which were reduced by heat treatment due to the isomerization of 5-CAQ to 3- and 4-CQA, and 3,5-diCQA to 3,4- and 4,5-diCQA. Moreover, 5-CQA was more stable than 3,5-diCQA even at 100 °C. Our results demonstrated that by controlling the cooking temperature and time, new bioactive compounds such as mono- and diCQA derivatives can be produced from sweet potato. These data indicate a potential approach for the development of new functional foods from sweet potato by controlling cooking temperature and time.

Liposome-Papain Conjugates for Catalytic Digestion of Antibody Producing Fab Fragments.

Papain is useful for the enzymatic digestion of various proteins to produce functional peptides or protein fragments. Immobilized papain being reactive toward proteins and easily removable from a reaction mixture is worth developed. In the present work, liposomes were applied as colloidal carriers of papain for the catalytic digestion of polyclonal immunoglobulin G (IgG). Papain was covalently conjugated at pH = 7.0 via tris-succinimidyl aminotriacetate (TSAT) to liposomes incorporated with 5 mol % poly(ethylene glycol)-tethered lipid with a reactive amino group. The papain-conjugated liposome (liposome-papain) catalyzed the hydrolysis of Nα-benzoyl-l-arginine 4-nitroanilide hydrochloride (BAPNA) at pH = 5.0-7.0. The activity of liposome-papain significantly increased with increasing temperature from 25 to 50 °C. The Michaelis constant Km was determined with respect to the liposome-papain- and free papain-catalyzed reactions with BAPNA at 37 °C as Km = 1.11 ± 0.13 and 11.6 ± 2.9 mM, respectively. Liposome-papain was applied to the catalytic digestion of 10 mg·mL-1 IgG at 37 °C for 24 h at pH = 5.0-7.0. The reaction mixture could be analyzed without pretreatment by using the affinity columns immobilized with the protein A or protein L ligand because colloidal liposome-papain quickly flowed through the chromatographic stationary phase, exhibiting little proteolytic effect on the proteinaceous ligands. The analysis clearly demonstrated the catalytic production of antigen-binding fragments (Fab) from IgG in an enzyme concentration- and pH-dependent manner. Liposome-papain with 15 or 50 mol % anionic lipids also catalyzed the formation of Fab from IgG. The above results demonstrated that liposome-papain was useful to digest IgG and to purify Fab formed with the affinity chromatography.

Causality Assessment Between Drugs and Fatal Cerebral Haemorrhage Using Electronic Medical Records: Comparative Evaluation of Disease-Specific and Conventional Methods.

INTRODUCTION: A new algorithm for causality assessment of drugs and fatal cerebral haemorrhage (ACAD-FCH) was published in 2021. However, its use in clinical practice has not been verified. OBJECTIVES: This study aimed to explore the practical value of the ACAD-FCH when applying information available in clinical practice. METHODS: The medical records of patients who died at the University of Tokyo Hospital in 2020 were reviewed, and cases with intracranial haemorrhage were selected. Two evaluators independently assessed these cases using three methods (the ACAD-FCH, Naranjo algorithm, and WHO-UMC scale). The number of 'Yes', 'No', and 'No information/Do not know' responses to each question by both evaluators were summed and compared. Inter-rater reliability was evaluated for each method using agreement rates and kappa coefficients with 95% confidence intervals (CI). RESULTS: Among 316 deaths, 24 cases with intracranial haemorrhage were evaluated. The proportion of ?No information/Do not know' responses for each question was 35.6% (95% CI 31.4-40.6%) for the ACAD-FCH and 66.9% (95% CI 62.5-71.1%) for the Naranjo algorithm. The respective agreement rates and kappa coefficients were 0.917 (0.798-1.00) and 0.867 (0.675-1.00) for the ACAD-FCH, 0.708 (0.512-0.904) and 0.139 (-0.236 to 0.513) for the Naranjo algorithm, and 0.50 (0.284-0.716) and 0.326 (0.110-0.541) for the WHO-UMC scale, respectively. CONCLUSION: Our findings suggest the utility of the ACAD-FCH when assessing death cases with intracranial haemorrhage. However, larger studies including intra-rater assessments are warranted for further validation of this algorithm.

Preparation and Catalytic Properties of Carbonic Anhydrase Conjugated to Liposomes through a Bis-Aryl Hydrazone Bond.

Liposomes (lipid vesicles) with sizes of about 100-200 nm carrying surface-bound (immobilized) water-soluble enzymes are functionalized molecular compartment systems for possible applications, for example, as therapeutic materials or as catalytic reaction units for running reactions in aqueous media in vitro. One way of covalently attaching enzyme molecules under mild conditions in a controlled way to the surface of preformed liposomes is to apply the spectrophotometrically traceable bis-aryl hydrazone (BAH) bond between the liposome and the enzyme molecules of interest. Using bovine carbonic anhydrase (BCA), an aqueous dispersion of liposome-BAH-BCA - conjugates of defined composition was prepared. The liposomes used consisted of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), N-(methylpolyoxyethylene oxycarbonyl)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE-PEG), and N-(aminopropylpolyoxyethylene oxycarbonyl)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE-PEG-NH2). The amino group of some of the DSPE-PEG-NH2 molecules present in the liposomes were converted into an aromatic aldehyde, which (after purification) reacted with (purified) BCA molecules that had on their surface on average one acetone protected aromatic hydrazine. After purification of the liposome-BAH-BCA conjugate dispersion obtained, it was characterized in terms of (i) BCA activity, (ii) overall BCA structure, and (iii) storage stability. For an average liposome of 138 nm diameter, about 1200 BCA molecules were attached to the outer liposome surface. Liposomally bound BCA was found to exhibit (i) similar catalytic activity at 25 °C and (ii) similar storage stability when stored in a dispersed state in aqueous solution at 4 °C as free BCA. Measurements at 5 °C clearly showed that liposome-BAH-BCA is able to catalyze the hydration of carbon dioxide to hydrogen carbonate.

Bioinformatic analysis reveals potential relationship between chondrocyte senescence and protein glycosylation in osteoarthritis pathogenesis.

Osteoarthritis (OA) is the most common degenerative and progressive joint disease. Cellular senescence is an irreversible cell cycle arrest progressive with age, while protein glycosylation is the most abundant post-translational modification, regulating various cellular and biological pathways. The implication of either chondrocyte senescence or protein glycosylation in the OA pathogenesis has been extensively and individually studied. In this study, we aimed to investigate the possible relationship between chondrocyte senescence and protein glycosylation on the pathogenesis of OA using single-cell RNA sequencing datasets of clinical OA specimens deposited in the Gene Expression Omnibus database with a different cohort. We demonstrated that both cellular senescence signal and protein glycosylation pathways in chondrocytes are validly associated with OA pathogenesis. In addition, the cellular senescence signal is well-connected to the O-linked glycosylation pathway in OA chondrocyte and vice-versa. The expression levels of the polypeptide N-acetylgalactosaminyltransferase (GALNT) family, which is essential for the biosynthesis of O-Glycans at the early stage, are highly upregulated in OA chondrocytes. Moreover, the expression levels of the GALNT family are prominently associated with chondrocyte senescence as well as pathological features of OA. Collectively, these findings uncover a crucial relationship between chondrocyte senescence and O-linked glycosylation on the OA pathophysiology, thereby revealing a potential target for OA.

Ameliorative effect of the oil components derived from sweet potato shochu on impaired short-term memory in mice after amyloid ß25-35 injection.

Sweet potato shochu oil is a by-product of shochu production and usually discarded although some physiological functions are considered. In this study, we investigated the effects of shochu oil on short-term memory using a murine model of spontaneous alternating behavior induced by the intracerebroventricular (ICV) administration of amyloid ß25-35 (Aß25-35). Mice were orally administered shochu oil for 15 days. Experiments with a Y-maze model revealed that the Aß25-35 caused a significant decrease in spontaneous alternation behavior, and supplementation with shochu oil significantly improved this behavior. DNA microarray analysis revealed that the administration of shochu oil downregulated the expression of S100a9 and Ptgs2, which reportedly exacerbate amyloid ß deposition in Alzheimer's disease. The administration of shochu oil upregulated the expression of Dnaja1 and PP2A, which is typically downregulated in Alzheimer's disease. These data suggest that shochu oil possible ameliorates on impaired short-term memory in mice after amyloid ß25-35 injection, as indicated by its effects on improving spontaneous alternation behavior and modulating the expressions of related genes.

Efficient Entrapment of Carbonic Anhydrase in Alginate Hydrogels Using Liposomes for Continuous-Flow Catalytic Reactions.

A versatile approach to entrap relatively small enzymes in hydrogels allows their diverse biotechnological applications. In the present work, bovine carbonic anhydrase (BCA) was efficiently entrapped in calcium alginate beads with the help of liposomes. A mixture of sodium alginate (3 wt %) and carbonic anhydrase-liposome conjugates (BCALs) was dripped into a Tris-HCl buffer solution (pH = 7.5) containing 0.4 M CaCl2 to induce the gelation and curing of the dispersed alginate-rich droplets. The entrapment efficiency of BCALs, which was defined as the amount of catalysts entrapped in alginate beads relative to that initially charged, was 98.7 ± 0.2% as determined through quantifying BCALs in the filtrate being separated from the beads. When free BCA was employed, on the other hand, a significantly lower entrapment efficiency of 27.2 ± 4.1% was obtained because free BCA could pass through alginate matrices. Because the volume of a cured alginate bead (10 µL) entrapped with BCALs was about 2.5 times smaller than that of an original droplet, BCALs were densely present in the beads to give the concentrations of lipids and BCA of 4.6-8.3 mM and 1.1-1.8 mg/mL, respectively. Alginate beads entrapped with BCALs were used to catalyze the hydrolysis of 1.0 mM p-nitrophenyl acetate (p-NA) at pH = 7.5 using the wells of a microplate or 10 mL glass beakers as batch reactors. Furthermore, the beads were confined in a column for continuous-flow hydrolysis of 1.0 mM p-NA for 1 h at a mean residence time of 8.5 or 4.3 min. The results obtained demonstrate that the conjugation of BCA to liposomes gave an opportunity to achieve efficient and stable entrapment of BCA in alginate hydrogels for applying to catalytic reactions in bioreactors.

Hair growth-promoting activity of components derived from sweet potato shochu.

Sweet potato shochu oil is one of the by-products of sweet potato shochu production. We investigated the functionality and industrial use of shochu oil as a food-derived raw material. Because of the increased incidence of self-consciousness in people owing to thinning hair, in this study, we examined the hair growth-inducing effects of shochu oil. Minoxidil, the only topical medication approved for hair growth treatment in Japan, was used as a control for the evaluation of hair growth-promoting activity of shochu oil. Human follicle dermal papilla cells treated with shochu oil showed upregulated expression of vascular endothelial growth factor in a concentration-dependent manner, indicating that shochu oil induced the activation of the hair growth cycle. In vivo, epidermal treatment with shochu oil also promoted hair growth in C3H mice. More than 35 components were detected in shochu oil via gas chromatography-mass spectrometry. The main components, accounting for 98.5% of shochu oil, were as follows, in order of decreasing concentration: ethyl palmitate, ethyl linoleate, ethyl oleate, ethyl stearate, ethyl caprate, ethyl laurate, ethyl myristate, and ethyl α-linolenate. Among these, ethyl palmitate, ethyl linoleate, and ethyl α-linolenate promoted hair growth in C3H mice. These results indicate that shochu oil can be used as a hair restorer. To the best of our knowledge, this study is the first to demonstrate the hair growth-promoting activity of shochu oil.

A two-enzyme cascade reaction consisting of two reaction pathways. Studies in bulk solution for understanding the performance of a flow-through device with immobilised enzymes.

Enzyme-catalysed cascade reactions in flow-through systems with immobilised enzymes currently are of great interest for exploring their potential for biosynthetic and bioanalytical applications. Basic studies in this field often aim at understanding the stability of the immobilised enzymes and their catalytic performance, for example, in terms of yield of a desired reaction product, analyte detection limit, enzyme stability or reaction reproducibility. In the work presented, a cascade reaction involving the two enzymes bovine carbonic anhydrase (BCA) and horseradish peroxidase (HRP) - with hydrogen peroxide (H2O2) as HRP "activator" - was first investigated in great detail in bulk solution at pH = 7.2. The reaction studied is the hydrolysis and oxidation of 2',7'-dichlorodihydrofluorescein diacetate (DCFH2-DA) to 2',7'-dichlorofluorescein (DCF), which was found to proceed along two reaction pathways. This two-enzyme cascade reaction was then applied for analysing the performance of BCA and HRP immobilised in glass fiber filters which were placed inside a filter holder device through which a DCFH2-DA/H2O2 substrate solution was pumped. Comparison was made between (i) co-immobilised and (ii) sequentially immobilised enzymes (BCA first, HRP second). Significant differences for the two arrangements in terms of measured product yield (DCF) could be explained based on quantitative UV/vis absorption measurements carried out in bulk solution. We found that the lower DCF yield observed for sequentially immobilised enzymes originates from a change in one of the two possible reaction pathways due to enzyme separation, which was not the case for enzymes that were co-immobilised (or simultaneously present in the bulk solution experiments). The higher DCF yield observed for co-immobilised enzymes did not originate from a molecular proximity effect (no increased oxidation compared to sequential immobilisation).

High Permeability of Polyunsaturated Lipid Bilayers As Applied to Attoliter Enzyme Reactors.

Phospholipid vesicles encapsulated with enzymes have potential applications for artificial organelles. A critical problem associated with the compartmentalized enzymes is their low reactivity because of the permeability resistance of lipid bilayers to substrates. In the present work, the polyunsaturated bilayers of 1,2-dilinolenoyl-sn-glycero-3-phosphocholine (18:3-PC) were elucidated to be highly permeable to 5(6)-carboxyfluorescein at high temperatures up to 60 °C and applied to fabricate vesicle-based reactive enzyme reactors. d-Amino acid oxidase (DAO) from porcine kidney was encapsulated in 18:3-PC vesicles with each aqueous volume of 3.4 × 10-21 m3 (=3.4 aL). The DAO-containing vesicles were highly reactive at 40 °C toward d-alanine being added to bulk solution at pH 9.0 and stably catalyzed following two types of reactions. One is the DAO-catalyzed continuous production of H2O2 in the vesicles for 30 min being detected by the free peroxidase-catalyzed oxidation of o-dianisidine in bulk solution. The other is the cascade reaction in the vesicles coencapsulating DAO and catalase being followed for 5 h on the basis of the concentration of unreacted d-alanine. In the latter reaction, the intermediate product H2O2 was decomposed by catalase producing oxygen allowing its cyclical use for the DAO-catalyzed oxidation. Furthermore, thanks to the highly temperature-dependent permeability of 18:3-PC bilayers, on/off-like switching in the activity could be induced with respect to the vesicle-confined enzyme by shifting the reaction temperature between 20 and 40 °C. The above reactive vesicles can offer the opportunity of the H2O2-based reliable detection of d-amino acids and the continuous optical resolution of racemic mixtures of amino acids.

Aggregation of chlorophyll a induced in self-assembled membranes composed of DMPC and DHPC.

The J-aggregate of chlorophyll a (Chla) functions as a light-harvesting antenna in natural systems. In this study, we employed the phospholipid membranes composed of longer-chain 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and shorter-chain 1,2-dihexanoyl-sn-glycero-3-phosphocholine (DHPC), as a platform to induce Chla aggregates. The DMPC/DHPC assembly at the mixing ratio (q) = 1.5 induced J-aggregates of Chla at 20 °C with a total lipid concentration of 20 mM. While, Chla aggregates were not observed in the membranes at q = ∞ (DMPC vesicles) and q = 0 (DHPC micelles). The surroundings Chla molecules in DMPC/DHPC at q = 1.5 were estimated to comprise a less polar environment, based on the deconvolution analysis of Soret band spectrum (400-440 nm). The photo-reduction activity of Chla J-aggregates was investigated in lower lipid concentration conditions.

Dopamine receptor agonists reverse behavioral abnormalities of alpha-synuclein transgenic mouse, a new model of Parkinson's disease.

Parkinson's disease (PD) is characterized by loss of nigral dopaminergic (DAergic) neurons and presence of Lewy bodies, whose major component is alpha-synuclein. We had previously generated transgenic mice termed Syn130m that express truncated human alpha-synuclein (amino acid residues 1-130) in DAergic neurons. Syn130m mice showed significant loss of DAergic neurons in the substantia nigra pars compacta. Subsequently, the striatal DA level and spontaneous locomotor activity of the mice were decreased significantly. In the present study, we investigated behavioral responses of Syn130m mice to L-DOPA and DA receptor agonists. Administration of L-DOPA dose dependently ameliorated the reduction of spontaneous locomotor activity of Syn130m mice. Similarly, D(2) agonists, quinpirole and talipexole, and a D1/D2 agonist, pergolide, were effective against the reduction. Syn130m mice also showed significant reduction in exploratory behavior compared with non-Tg littermates when they were placed in a novel environment, but this abnormality was ameliorated by treatment with pergolide. These results strongly suggest that the behavioral abnormalities of Syn130m mice were caused by low striatal DA content. On the other hand, the expression of postsynaptic D(2)-like receptors (DRD2) in the striatum was not increased in Syn130m mice, although the low striatal DA level is known to induce compensatory expression of DRD2. Because the abnormalities could be rectified by treatment with DA receptor agonists, it is likely that Syn130m mice provide a useful tool to explore therapeutic possibilities for PD as a new animal model of the disease.

Common behavioral influences of the ErbB1 ligands transforming growth factor alpha and epiregulin administered to mouse neonates.

Ligands for epidermal growth factor (EGF) receptor (ErbB1), such as EGF, transforming growth factor alpha (TGFalpha), and epiregulin, are enriched in body fluids and blood and regulate development of various peripheral organs. It remains however how such circulating polypeptide growth factors influence brain development and function. Here, we performed peripheral injections of TGFalpha and epiregulin to mouse neonates and evaluated immediate physical and neurochemical development and later behavioral consequences. Subcutaneous administration of TGFalpha and epiregulin increased phosphorylation of brain ErbB1, suggesting their effects on brain development. Repeated their injections similarly enhanced physical development of eyelid opening and tooth eruption during early postnatal stage and resulted in abnormal behavioral traits in the adult stage. Acoustic startle responses of mice treated with these growth factors as neonates were enhanced and prepulse inhibition was decreased without an apparent correlation between prepulse inhibition level and startle intensity. Locomotor activity and fear-learning performance with tone and context cues were not altered, however. These results suggest that circulating ErbB1 ligands in the periphery of neonates have some common influences on later behavioral traits. Abnormal ErbB1 ligand production at neonatal and potentially prenatal stages might therefore associate with neurodevelopmental disorders such as schizophrenia.