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Background Staphylococcus aureus (S. aureus) is frequently detected in the skin of patients with atopic dermatitis (AD), and involved in the flare of AD. There are some evidence-specific strains of S. aureus affect the severity of AD. However, the mechanism of predominant colonization and the aggravation of dermatitis by certain strains of S. aureus in the patients with AD are still unknown. Objective To reveal the characteristics of S. aureus from patients with AD (S. aureus-AD), we analyzed the interaction of S. aureus-AD and keratinocytes in comparison with those of S. aureus laboratory strains (S. aureus-stand.). Methods We stimulated HaCaT cells, keratinocyte cell line, and human epidermal keratinocytes by heat-killed S. aureus strains, then evaluated immune response of keratinocytes by ELISA, immunofluorescence staining, and flow cytometry. Results Upon incubation with keratinocytes, three out of four strains of heat-killed S. aureus-AD were strongly agglutinated inside the cytoplasm. In the cells, they are located in lysosomes and promoted the secretion of interleukin-1α (IL-1α). These reactions were not observed by any of four strains of S. aureus-stand. and S. epidermidis and were abolished by the treatment of S. aureus with proteinase K. Moreover, the IL-1α secretion was diminished by the inhibition of Toll-like receptor 9 (TLR9). Conclusion S. aureus-AD accumulates in lysosome of keratinocytes by means of bacterial cell wall proteins and induces IL-1α via TLR9.

The complete hydrolysis of konjac glucomannan (KGM) with an acid or enzyme generally takes a long time. To accelerate KGM hydrolysis without diminishing the conventional quality, a diluted acid hydrolysis of KGM with sulfuric acid was conducted using a microwave digestion system. The optimum conditions of microwave-assisted acid hydrolysis for KGM were: 10 mL of 0.25 M sulfuric acid, hydrolysis temperature of 135°C (microwave power of 600 W), and total microwave-irradiation time of 45 min. The yields of the component sugars, mannose and glucose, from two konjac powders were similar to those by conventional acid hydrolysis with 1 M sulfuric acid in a boiling water bath for 5 h. Furthermore, a pretest for microwave-assisted acid hydrolysis using mixtures of konjac powder and starch at different ratios proved that their konjac content can be calculated by determining the mannose generated by the new rapid hydrolysis method, if the raw materials are provided.

Teranishi T, Kondo I, Tanino G, Miyasaka H, Sakurai H, Kaga J, Suzuki Y, Matsushima A, Kawakita M, Sonoda S. An analysis of falls occurring in a convalescence rehabilitation ward — a decision tree classification of fall cases for the management of basic movements —. Jpn J Compr Rehabil Sci 2013; 4: 7-13. Purpose: A simple and discriminative method for evaluating the ability to maintain balance, the Standing test for Imbalance and DisEquilibrium (SIDE), was developed for fall risk management. As a preliminary step toward clinical application of the SIDE, this study aimed to determine a method of assessing the expected fall risk by analyzing fall cases using a decision tree classification for the management of basic movements. Subjects and method: The subjects of this study were 513 patients who had been discharged from a convalescence rehabilitation ward over a one-year period (from January 1, 2010 to December 31, 2010). In cases when a fall occurred, we investigated the period from hospitalization and the classification of the patient, which was made with a decision tree describing the permitted activities of the patient and the management of the activities. The frequency of fall occurrence was determined by considering the number of patients who fell during every 15-day period during hospitalization. Results: One hundred twenty patients fell during hospitalization. In total, 163 falls occurred. The incidence of falls was 4.65‰. Thirty patients suffered multiple falls. The falls occurred significantly more frequently during the first 15 days after admission. From classification by the decision tree, 62 falls occurred while the patients were being managed using physical restraints and/or sensors, 55 falls occurred during unpredictable actions of patients when the patients were not using physical restraints or sensors, and 26 falls occurred during permitted activities of patients. Conclusion: Simple assessments of balance and adherence are necessary to reduce the occurrence of patient falls during the early period after admission.

Both growth and thermoregulation are energetically costly, and many studies implicate an energetic tradeoff between them. Moreover, fur is known to ameliorate thermoregulatory costs in adult mammals, but its role in maintaining energy balance during growth is unclear. This study tested for an energetic tradeoff between growth and thermoregulation in juvenile Siberian hamsters (Phodopus sungorus) and the effect of an insulative pelage on intrinsic growth rate. Hamsters weaned at 18 days of age and left fully furred or deprived of all dorsal fur by shaving at 20 days of age, were housed at 10 degrees C or 23 degrees C. Body mass, length, and food consumption were measured until hamsters were 35 days old. Thermal challenge, whether by low ambient temperature or shaving, resulted in increased food intake and decreased efficiency at converting food into body mass. Body mass and length were not affected by the thermal challenges. These results suggest that there is no mandatory tradeoff between growth and thermoregulation in this species, particularly when food is in abundant supply. Although fur was not necessary for normal growth to proceed, it ameliorated energetic costs associated with thermoregulation, and may play a role in maintaining energy balance under conditions of limited food availability.

Treatment of lithium ester enolates with magnesium alkylidene carbenoids, generated from 1-chlorovinyl p-tolyl sulfoxides with isopropylmagnesium chloride via the sulfoxide–magnesium exchange reaction, gave β,γ-unsaturated esters in moderate to good yields. When this reaction was conducted with the lithium ester enolates of α-chlorocarboxylic acid esters, allenic esters were obtained. This procedure provides an unprecedented way for the synthesis of β,γ-unsaturated esters and allenic esters from ketones with the construction of a carbon–carbon bond between α- and β-positions.

An arginine specific protease, Sp-protease, was purified by column chromatography from freeze-dried Spirulina platensis using a five-step process. Purified Sp-protease has a molecular weight of 80 kDa. It hydrolyzed the synthetic substrates containing arginine residue in the P1 position but did not hydrolyze synthetic substrates containing other amino acid residues, including lysine residue in the P1 position. Among the synthetic substrates tested, a substrate of plasminogen activator (Pyr-Gly-Arg-MCA) was hydrolyzed most effectively with the enzyme (Km = 5.5 x 10(-6) M), and fibrin gel was solubilized via activation of intrinsic plasminogen to plasmin with the enzyme. Activity was inhibited completely with camostat mesilate (Ki = 1.1 x 10(-8) M) and leupeptin (Ki = 3.9 x 10(-8) M) but was not inhibited with Nalpha-tosyl-L-lysine chloromethyl ketone (TLCK). The optimum pH of the enzyme has a range of pH 9.0 to pH 11.0. The optimum temperature was 50 degrees C; the enzyme was stable at 0-50 degrees C.

Biliverdin, the precursor of bilirubin, was adsorbed onto the synthetic clay mineral, smectite, in a mixed solution of benzene and methyl alcohol (7:3 v/v) to form a biliverdin–smectite conjugate. The adsorption of biliverdin to smectite followed the Freundlich equation with an adsorption coefficient of 1.0. The water-soluble conjugate had a green color with the absorbance maxima at 679 and 378 nm. The biliverdin–smectite conjugate became photostable in water to irradiation with UV, although the biliverdin in benzyl alcohol was rapidly decolorized.

Phycocyanin with blue color, phycocyanobilin bound to protein, with the absorption maximum at 614 nm is quite unstable against urea-treatment because of the denaturation of the protein conformation. To prevent the bleaching of phycocyanin by urea, amino groups of lysine residues in the phycocyanin molecule were modified with DSP [dithiobis(succinimidyl propionate)] to cross-link amino groups in the protein molecule in order to maintain the high order structure of phycocyanin. The color development of DSP-modified phycocyanin became stable, but unmodified phycocyanin was markedly decreased to 20% of the original absorbance at 614 nm against urea-treatment. Splitting of the S–S linkage of DSP-modified phycocyanin molecule by 2-mercaptoethanol caused the reduction of color development of phycocyanin under urea-treatment. The color development of phycocyanin is closely associated with its high order structure of protein.

Hemin (Fe(3+)) was adsorbed onto synthetic smectite (clay mineral) intercalated with a quaternary alkenylammonium compound, dioleyldimethylammonium chloride (DOA), to form a hemin-smectite-DOA conjugate. The hemin-smectite-DOA conjugate was soluble in organic solvents such as benzene and toluene to form a transparent colloidal solution with a light yellow color. Its absorption spectrum in benzene showed two bands, 600 and 568 nm, in the visible region and a sharp Soret band at 400 nm with the molar extinction coefficient of 7.5 x 10(4) M(-1) cm(-1). The formation of the conjugate of smectite and DOA was confirmed by X-ray diffraction analysis: the basal spacing, d(001), of hemin-smectite-DOA conjugate was 19 A which is an expansion of the interlayer space by 5 A based upon the basal spacing of smectite of 14 A. Hemin-smectite-DOA conjugate catalyzed the peroxidase-like reaction in organic solvents using benzoyl peroxide as the hydrogen acceptor and leucocrystal violet as the hydrogen donor. The temperature-dependent peroxidase-like activity of the conjugate was compared with peroxidase activity of horseradish peroxidase. The hemin-smectite-DOA conjugate exhibited higher activity as the temperature was increased from 30 to 70 degrees C, while horseradish peroxidase activity was reduced as the temperature was increased.

Hemin (Fe(3+)) was adsorbed onto synthetic smectite (clay mineral) in acetone to form a hemin-smectite conjugate. The hemin-smectite conjugate became soluble in water to form a transparent colloidal solution with a dark brown color. Its absorption spectrum in water showed a sharp Soret band at 398 nm with the molar extinction coefficient as epsilon(398nm) = 11.6 x 10(4) M(-1) cm(-1), which is in good agreement with epsilon(398nm) = (12.2 +/- 3) x 10(4) M(-1) cm(-1) of monomeric hematin (1). Hemin (Fe(3+))-smectite conjugate had a peroxidase-like activity in the presence of hydrogen peroxide (a hydrogen acceptor) and guaiacol (a hydrogen donor) in aqueous solution and its activity was higher than that of hematin. Hemin (Fe(3+))-smectite conjugate in water was reduced by adding sodium dithionite to form a heme (Fe(2+))-smectite conjugate which is also a transparent colloidal solution in water. Its absorption spectrum in aqueous solution was surprisingly in close agreement with that of oxyhemoglobin. Its peak positions of alpha, beta, and Soret bands were located in only a 9--3 nm shift to shorter wavelengths in comparison with those of oxyhemoglobin. Therefore, heme (Fe(2+))-smectite conjugate was bound to O(2) to form O(2)-heme (Fe(2+))-smectite conjugate. The addition of carbon monoxide, CO, to O(2)-heme (Fe(2+))-smectite conjugate caused the formation of CO-heme (Fe(2+))-smectite conjugate with a similar absorption spectrum of carboxyhemoglobin (HbCO) accompanied by shifting 8--10 nm to shorter wavelength. Therefore, the transformation of O(2)-heme (Fe(2+))-smectite conjugate to CO-heme (Fe(2+))-smectite conjugate was accompanied by shifting of 7, 4, and 3 nm to shorter wavelengths in the alpha, beta, and Soret bands respectively, which are similar to the spectral change from oxyhemoglobin to carboxyhemoglobin. Also the ratio (1:1.6) of the molar extinction coefficient of Soret band of O(2)-heme (Fe(2+))-smectite conjugate and CO-heme (Fe(2+))-smectite conjugate was surprisingly agreement with ratio (1:1.5) of oxyhemoglobin and carboxyhemoglobin. The phenomenon shown above was unexpectedly found during the course of study of bioconjugate of a bioactive substance, hemin (Fe(3+)) or heme (Fe(2+)), and a clay mineral, smectite, in place of the protein of globin in hemoglobin.

Polyethylene glycol (PEG) conjugates of protein antigens induce antigen-specific immune tolerance of helper T (Th)-cells. However, the mechanism of this Th-cell tolerance has remained unelucidated. Using transgenic mice with ovalbumin (OVA)-specific T-cell receptor (TCR) genes, we examined the response of OVA-specific Th-cells towards tolerogenic PEG-conjugate of OVA in vitro and in vivo. When stimulated with PEG--OVA in vitro, transgenic OVA-specific Th-cells proliferated and produced interleukin 2, the levels of which were comparable to those induced by unmodified OVA. In contrast, PEG--OVA administered into the circulation of transgenic mice induced unresponsiveness in peripheral OVA-specific Th-cells. Moreover, in the thymus of these transgenic mice, the frequency of immature CD4+CD8+ (double positive) thymocytes was reduced. A similar phenomenon was not observed in transgenic mice treated with unmodified OVA. As autoreactive T-cells are known to be clonally deleted at the immature double positive stage in the thymus. Th-cell tolerance induced by PEG--protein antigens is at least in part mediated by central tolerance in the thymus, and is likely caused by the markedly enhanced stability of PEG--protein conjugates in the circulatory system.

Chlorophyllin a was conjugated with alpha-(3-aminopropyl)-omega-methoxypoly(oxyethylene), PEG-NH(2), to form the PEG-chlorophyllin conjugate through acid-amide bonds. The PEG-chlorophyllin conjugate was stable toward light illumination under anaerobic condition in comparison with chlorophyllin a. The conjugate catalyzed the reduction of methyl viologen in the presence of 2-mercaptoethanol and the evolution of hydrogen gas in the presence of methyl viologen (an electron carrier), 2-mercaptoethanol (an electron donor) and hydrogenase (Scheme 1). Furthermore, the PEG-chlorophyllin conjugate catalyzed the photoreduction of NADP(+) or NAD(+) in the presence of ascorbate as an electron donor and ferredoxin-NADP(+) reductase as the coupling enzyme. Utilizing the reducing power of NADPH generated by the PEG-chlorophyllin conjugate under the illumination, CO(2) fixation was accomplished by the synthesis of malate (C(4)) from pyruvate (C(3)) and CO(2) in the presence of malic enzyme (Scheme 2). These reactions mentioned above did never proceed in dark or without each enzyme.

Chlorophyll a was adsorbed to a synthetic smectite intercalated by poly(vinylpyrrolidone) (PVP) to form the chlorophyll-PVP-smectite conjugate (Chl-PVP-SME) having an absorption maximum at 677 nm. The conjugate was found to be stable toward light illumination in comparison with chlorophyll-smectite, chlorophyll-PVP, and free chlorophyll a. Chl-PVP-SME had a photoinduced activity for catalyzing the reduction of methyl viologen. Furthermore, the evolution of hydrogen gas was observed when an aqueous suspension containing Chl-PVP-SME, methyl viologen (an electron carrier), 2-mercaptoethanol (an electron donor), and hydrogenase was illuminated by visible light.

Lipase modified with polyethylene glycol became soluble and active in organic solvents, and catalyzed regioselective deacetylation of peracetylated monosaccharide derivatives in 1,1,1-trichloroethane. The deacetylation occurred only at the positions of C-4 and C-6 of the glycopyranoside ring. Especially, peracetylated methyl β-D-xylopyranoside and peracetylated L-serine-β-D-xylopyranoside were hydrolyzed only at the position of C-4. Subsequently, one of the resulting products, that is L-serine-2,3-di-O-acetyl-β-D-xylopyranoside, was coupled with galactose residue to obtain L-serine-4-O-(β-D-galactopyranosyl)-β-D-xylopyranoside, a model compound of the carbohydrate-protein linkage region of proteoglycans.

Apoptotic cell death of murine leukemia cells induced by E. coli L-asparaginase was studied. Deprivation of L-asparagine from the culture of L5178Y cells by L-asparaginase caused the fragmentation of chromosomal DNA of the leukemia cells within 24 h. Prior to the degradation of DNA, cell cycles of L5178Y cells were found to be arrested in G1 phase, and evidence of the DNA strand breaks was initially observed in G1 phase cells as early as 8 h after the asparaginase treatment. Therefore, apoptosis of leukemia cells induced by L-asparaginase is an event that is associated with the cell cycle arrest in G1 phase.

Chlorophyll a was adsorbed onto smectite to form chlorophyll-smectite conjugate, which became photostable against light illumination. The chlorophyll-smectite conjugate caused the photoreduction of nitro blue tetrazolium, NBT, through the formation of superoxide anion, and the conjugate retained the photosensitive activity even after 20 hour illumination. Furthermore, the conjugate deposited on SnO2 electrode caused electron transfer under light illumination. The anodic photocurrent rose at −200 mV versus Ag/AgCl electrode (KCl saturated) and reached a maximum level at approximately +100 mV. The photocurrent spectrum was in good agreement with the absorption spectrum of chlorophyll-smectite conjugate in an aqueous solution and the quantum efficiency was approximately 1% at 670 nm.

Chlorophyll a was adsorbed onto freeze-dried bentonite to form chlorophyll-bentonite conjugate, which was photostable against light illumination [1]. Physiological function of chlorophyll-bentonite conjugate is explored; increasing the amount of chlorophyll adsorbed onto bentonite leads to enhancement of the photostability accompanied with shifting the absorbance maximum to longer wavelength. Furthermore, the conjugate exhibits a high photoreduction ability of nitro blue tetrazolium, NBT, through the formation of superoxide anion without any loss of photosensitive activity even after 50 hr illumination.

Ovalbumin (OVA) was chemically modified with poly(ethylene glycol) (PEG) derivatives: activated PEG2, 2,4-bis[O-methoxypoly(ethylene glycol)]-6-chloro-s-triazine and activated PM13, copolymer of poly(oxyethylene) allyl methyl diether and maleic anhydride. Pretreatment of BALB/c mice with PEG2- or PM13-OVA suppressed the production of both IgG- and IgE-class anti-OVA antibodies induced by subsequent immunizations with unmodified OVA. PEG2-OVA had higher potency to suppress OVA-specific immune response than PM13-OVA. Although extensive modification (62%) of amino groups in the OVA molecule with activated PEG2 was required to diminish most of its immunoreactivity towards anti-OVA antibodies, only a low degree of modification (27%) was sufficient to induce tolerogenicity to OVA. Thus, the loss of immunoreactivity of PEG2-OVA was not prerequisite for its tolerogenic capacity. Moreover, the use of completely denatured PEG2-OVA immunogen lead to the loss of its ability to induce immune tolerance to native OVA. These observations are discussed in relation to the regulatory mechanism of immune response.

Chemical modification of enzymes and other bioactive molecules with polyethylene glycol derivatives, activated PEG and PM, can eliminate some of the drawbacks of the biomolecules and/or give them new functions in biotechnological processes. PEG- or PM-lipase becomes soluble and active in organic solvents so that the reverse reactions of hydrolysis proceed effectively, not only in organic media but also in straight substrates without any solvent. These include ester synthesis and ester exchange reactions including lactone synthesis and optical resolution Enzymes such as lipase and asparaginase modified with activated PMs gain stabilization towards heat and urea denaturation and, for asparaginase in vivo, prolongation of clearance time. Photostabilization of natural pigments, magnetization of enzymes and effective affinity partitioning are archieved by modification with PEG derivatives.

Chlorophyllin a prepared by the saponification of chlorophyll a was conjugated with α-(3-aminopropyl)-ω-methoxypoly(oxyethylene), PEG-NH2, to form PEG-chlorophyllin through acid-amide bonds. PEG-chlorophyllin was readily soluble not only in aqueous solution but also in organic solvents. The absorption maxima of the conjugate were located at 657 nm and 418 nm in aqueous solution, and at 656 nm and 422 nm in benzene. The conjugate seems to be more stable toward light-illumination than uncoupled chlorophyllin. Furthermore, PEG-chlorophyllin possessed a potent photosensitizing activity to produce superoxide anion during the illumination, although chlorophyllin immediately lost its activity by light-illumination.

Among the various plant lectins, pokeweed mitoge (PWM) is most effective in enhancing the cytotoxicity of human lymphokine-activated killer (LAK) cells. However, the use of PWM in adoptive immunotherapy has been limited due to the strong immune response against the protein of plant origin. Amino groups in PWM was modified with 2,4-bis[O-methoxypoly(ethylene glycol)]-6-chioro-s-triazine, activated PEG2, to form PEG-PWM conjugates. Its immunoreactivity towards anti-PWM antibodies was reduced by increasing the degree of modification of amino groups in PWM. PEG-PWM, in which 54% of amino groups in PWM was modified with activated PEG2, had a nearly complete reduction of immunoreactivity. Intraperitoneal administration of PEG-PWM to mice did not produce substantial levels of anti-PWM antibodies. Nevertheless, PEG-PWM retained the ability to induce the maximum levels of cytotoxicity of human LAK cells in vitro.

Lipase from Pseudomonas cepacia was modified with 2,4-bis[O-methoxypoly(ethylene glycol)]-6-chloro-s-triazine(activated PEG2) to form PEG-lipase. The PEG-lipase, soluble and active in organic solvents, catalyzes asymmetric alcoholysis of racemic δ-decalactone in alcohols to form (R)-5-hydroxydecanoic acid alkyl esters. The yield was 69% with 83% enantiomeric excess after 3 hr-reaction in n-decanol at 50°C. The advantage of this reaction is that the alcoholysis proceeds efficiently in straight hydrophobic substrates without any organic solvents.

Almost all natural pigments exhibit defects when the pigments are extracted from living bodies; they are much less stable to light than synthetic dyes, and this accounts for why these pigments have not been widely utilized industrially. Another reason is often the poor solubility of natural pigments both in aqueous media and in organic solvents. to overcome these drawbacks, chlorophyll a, β-carotene and astaxanthin were adsorbed on the silk protein, fibroin, to form pigment-fibroin conjugates. These conjugates exhibit high stability to visible and UV light. Similarly, melanin, an insoluble dermal pigment, was coupled with polyethylene glycol (PEG). The PEG-melanin conjugate is soluble not only in aqueous media but also in organic solvents, and exhibits a photoprotective effect to UV radiation on the growth of Escherichia coli cells.

Chlorophyll a was adsorbed onto a synthetic smectite, hectorite, in benzene to form a chlorophyll-smectite conjugate. The adsorption proceeded in two steps, involving monolayer and then multilayer adsorption of chlorophyll a onto smectite. The chlorophyll-smectite conjugate became a transparent colloidal solution with a green color on the addition of water. The absorption maximum of chlorophyll a in the red region shifted to longer wavelength by increasing the amount of chlorophyll a adsorbed onto smectite, accompanied by an effective photostabilization.

L-Asparaginase from Escherichia coli was coupled with two types of comb-shaped copolymer of poly-(ethylene glycol) derivative and maleic anhydride (activated PM), having molecular weights of 13,000 and 100,000 (activated PM13 and PM100, respectively) with multivalent reaction sites. After single intravenous injections of PM100-asparaginase and nonmodified asparaginase into rats, the enzymic activity of PM100-asparaginase in serum was well retained for at least 11 days, and the serum L-asparagine concentration remained undetectable for 27 days. The half-lives of PM100-asparaginase and nonmodified asparaginase were 50 and 1.5 h, respectively. Stabilization of L-asparaginase toward heat, urea, and acidity was caused by modifying the enzyme with activated PM13 and PM100. Especially, PM100-asparaginase retained high enzymic activity toward heat and urea, compared with PM13-asparaginase. It was suggested that these modifiers with a comb-shaped form and with multivalent reactive sites cover the whole surface of the asparaginase molecule and stabilize its conformation possibly through multiple covalent bindings and through various noncovalent interactions.

Lipases from various microorganisms were chemically modified with polyethylene glycol derivatives: 2,4-bis[O-methoxypoly(ethylene glycol)]-6-chloro-s-triazine (activated PEG2, a chain-shaped polymer) and copolymer of polyoxyethylene allyl methyl diether and maleic anhydride (activated PM, a comb-shaped polymer). Because each polymer is amphipathic, the modified lipases become soluble not only in aqueous solution but also in hydrophobic media. They exhibit potent catalytic actions for ester synthesis and ester exchange reactions, the reverse reaction of hydrolysis, in transparent organic solvents and also in oily substrates without organic solvents. With PEG2-lipases, macrocyclic lactone and gefarnate (geranyl farnesylacetate) were synthesized in high yields from 16-hydroxy-hexadecanoic acid ethyl ester and from farnesylacetic acid and geraniol in organic solvents, respectively. The modified lipase catalyzed the esterification preferentially with the (R)-isomer of secondary alcohols. Because the ester synthesis reactions with modified lipase proceeded in the transparent benzene system, the kinetic parameters (Michaelis constant and maximum velocity) were obtained by reciprocal plotting according to the Michaelis equation. With comb-shaped polymer as modifier, PM-lipase catalyzed effectively the reverse reaction of hydrolysis in organic solvents. The properties of each modified lipase are discussed in relation to those of the nonmodified lipase.

A protease (Df-protease) from house dust mites (Dermatophagoides farinae), which is closely related to mite-induced allergic diseases, catalyzes the activation of kallikrein-kinin system in plasma (3, 6, 12). Df-protease was competitively inhibited by guanidine and amidine derivatives with low-molecular weight. The kinetic study revealed that the Ki values of 1-[p-(p-amidinophenoxycarbonyl)benzoyl]-D-proline benzyl ester acetate and N-[p-(2-methoxycarbonyl-4-amidinophenoxycarbonyl)benzoyl]-N-phenylglycine ethyl ester acetate were 5×10 -9 M and 6×10 -9 M, respectively

Bovine serum albumin (BSA) was chemically modified with two types of comb-shaped copolymers of polyethylene glycol derivative and maleic anhydride, one with the molecular weight of 13,000 (activated PM13) and the other with 100,000 (activated PM100), to form PM13- and PM100-BSA. The immunoreactivity of BSA was markedly reduced by coupling with each modifier and was completely lost when 30% or 20% of amino groups in BSA were modified with activated PM13 or PM100, respectively. The esterase activity of PM13- and PM100-BSA without immunoreactivity were retained 63% and 93% of non-modified one, respectively. These results were discussed with those of modified-asparaginases(1).

Treatment of lithium ester enolates with magnesium alkylidene carbenoids, generated from 1-chlorovinyl p-tolyl sulfoxides with isopropylmagnesium chloride via the sulfoxide–magnesium exchange reaction, gave β,γ-unsaturated esters in moderate to good yields. When this reaction was conducted with the lithium ester enolates of α-chlorocarboxylic acid esters, allenic esters were obtained. This procedure provides an unprecedented way for the synthesis of β,γ-unsaturated esters and allenic esters from ketones with the construction of a carbon–carbon bond between α- and β-positions.

Lipase fromPseudomonas fluorescens was coupled with a copolymer of polyoxyethylene allyl methyl diether and maleic anhydride, activated PM. The PM-lipase became soluble and active in organic solvents, and also heat stable. It catalyzed the ester synthesis in benzene and ester hydrolysis in an aqueous system with high enzymic activity.

A serine protease was earlier purified to homogeneity from a culture extract of mites and its possible involvement in the allergic reaction was proposed [K. Takahashi et al. (1990): Int. Arch. Allergy Appl. Immun., 91, 80-85]. The serine protease thus obtained was found in the present study to be trypsin-like protease and had high substrate specificity towards the synthetic substrate for blood coagulation factor XIIa. The enzyme gave rise to rhythmic contraction of rat uterine horns in factor XII-deficient human plasma, but not in prekallikrein-deficient plasma. These findings lead us to conclude that this enzyme activates the kallikrein-kinin system in plasma through hydrolysis of prekallikrein in the plasma.

A new approach in biotechnological processes is to use lipase modified with polyethylene glycol(PEG) which has both hydrophilic and hydrophobic properties. The PEG-lipase is soluble in organic solvents such as benzene and chlorinated hydrocarbons and exhibits high enzymic activity in organic solvents. The PEG-lipase catalyses the reverse reaction of hydrolysis in organic solvents; ester synthesis and ester exchange reactions. The PEG-lipase can also be conjugated to magnetite (Fe3O4). The magnetic lipase catalyses ester synthesis in organic solvents and can be readily recovered by magnetic force without loss of enzymic activity.

An aqueous extract from a mite culture, of Dermatophagoides farinae, activated prekallikrein to kallikrein in normal plasma. Crude protein preparation, obtained by ammonium sulfate precipitation (95 % saturation) from the extract, exhibited high activity (0.81 units/mg protein) towards a synthetic substrate of coagulation factor XIIa, Boc-Gln-Gly-Arg-MCA, and had also activity to form kallikrein in human plasma deficient in coagulation factor XII. Treatment of the protein preparation with phenylmethylsulfonyl fluoride (PMSF), an inhibitor of serine enzyme, gave rise to inactivation of both activities. Thus, the serine protease specific for Boc-Gln-Gly-Arg-MCA in mite cultures of D.farinae was purified by ammonium sulfate precipitation and chromatographies on p-aminobenzamidine-sepharose CL-4B, DEAE-Toyopearl 650M, Sephadex G-75 superfine and Sephacryl S-200. The purified protease was homogeneous electrophoretically, and its molecular weight was estimated to be 30,000. The optimum pH and temperature were around 7.5 and 50 °C, respectively. The specific activity was 36 units/mg protein at pH 7.4 and 37 °C. The activity was completely inhibited by PMSF. The serine protease had the activity to activate the kallikrein-kinin system in normal human plasma.

A peptidase from Japanese cedar pollen, Jc-peptidase, was clarified to preferentially hydrolyze an MCA substrate of Phe-MCA (L-phenylalanyl-4-methylcoumaryl-7-amide). This study examined substrate specificities of Jc-peptidase using oligopeptides. Jc-peptidase hydrolyzed Phe-Phe and Tyr-Phe effectively and hydrolyzed Leu-Phe, Met-Phe, and Arg-Phe moderately. Other substrates such as Ala-Phe, Asp-Phe, and Pro-Phe were not hydrolyzed with the peptidase. Results obtained with a series of aminoacyl-Phe peptides were compatible with the facts obtained for MCA substrates except for Arg-MCA. Effects of amino acid residues in the P1' position were also examined using Phe-amino acids. An N-terminal phenylalanine residue was actually released from bioactive peptides such as molluscan cardioexcitatory neuropeptide (FMRF-NH(2)). Because the activity was inhibited with Zn(2+) and EDTA, Jc-peptidase was inferred to belong to the metalloproteases. The N-terminal amino acid sequence was determined to be APIGVQLEIEENYVHMYNGF and an internal sequence to be EIFAATFNVDEETEA, but no homology with other proteins was found.

Phenylketonuria (PKU) is an autosomal recessive genetic disease caused by the defects in the phenylalanine hydroxylase (PAH) gene. Individuals homozygous for defective PAH alleles show elevated levels of systemic phenylalanine and should be under strict dietary control to reduce the risk of neuronal damage associated with high levels of plasma phenylalanine. Researchers predict that plant phenylalanine ammonia-lyase (PAL), which converts phenylalanine to nontoxic t-cinnamic acid, will be an effective therapeutic enzyme for the treatment of PKU. The problems of this potential enzyme therapy have been the low stability in the circulation and the antigenicity of the plant enzyme. Recombinant PAL originated from parsley (Petroselinum crispum) chemically conjugated with activated PEG2 [2,4-bis(O-methoxypolyethyleneglycol)-6-chloro-s-triazine] showed greatly enhanced stability in the circulation and was effective in reducing the plasma concentration of phenylalanine in the circulation of mice. PEG-PAL conjugate will be an effective therapeutic enzyme for the treatment of PKU.

An aminopeptidase, Jc-peptidase, was purified from Japanese cedar pollen by seven steps, including precipitation with ammonium sulfate, ion-exchange chromatography, gel filtration, hydrophobic interaction chromatography on phenyl-agarose, and high-performance liquid chromatography. Purified Jc-peptidease has a molecular weight of 42 kDa and hydrolyzes the synthetic substrates of L-phenylalanyl-4-methylcoumaryl-7-amide (Phe-MCA) with Km = 5 x 10(-5) M, Tyr-MCA with Km = 7 x 10(-4) M, Leu-MCA with Km = 1 x 10(-3) M, and Met-MCA with Km = 1 x 10(-3) M. Other MCA analogues such as Arg-MCA or Glu-MCA failed to serve as its substrates. The activity was inhibited in the presence of phebestin, [(2S,3R)-3-amino-2-hydroxy-4-phenylbutanoyl-L-valyl]-L-phenylalanine, with Ki = 4.7 x 10(-5) M, or bestatin, [(2S,3R)-3-amino-2-hydroxy-4-phenylbutanoyl]-L-leucine, with Ki = 1.1 x 10(-4) M. According to amino acid sequence analysis, the N-terminal amino group seems to be blocked. The physiological function of the aminopeptidase (Jc-peptidase) has not been clarified in vivo.

Chlorophyllide a was coupled with α-(3-aminopropyl)-ω-methoxypoly(oxyethylene) (PEG–NH2) to form a PEG–chlorophyllide conjugate through an acid–amide bond. The conjugate catalyzed the reduction of methylviologen in the presence of 2-mercaptoethanol. It also catalyzed the photoreduction of NADP+ or NAD+ in the presence of ascorbate as an electron donor and ferredoxin-NADP+ reductase as the coupling enzyme. Utilizing the reducing power of NADPH generated by PEG-chlorophyllide conjugate under illumination, glutamate was synthesized from 2-oxoglutarate and NH4+ in the presence of glutamate dehydrogenase. PEG-chlorophyllide conjugate was quite stable toward light illumination compared with chlorophyll a. The increase in the molecular weight of PEG in the PEG-chlorophyllide conjugates was accompanied by the enhancement of photostability of the conjugate and also by the increased solubility in the aqueous solution. © 2001 John Wiley & Sons, Inc. Biotechnol Bioeng 76: 86–90, 2001.

It was reported that Df-protease from house dust mite (Dermatophagoides farinae) catalyzes the activation of the kallikrein−kinin system in human plasma and is closely associated with mite-induced allergy. Therefore, to prevent the release of kinin by Df-protease, the inhibitory activity of polyphenols including catechins and flavonols was tested in vitro and in vivo. Among them, myricetin and epigallocatechin gallate (EGCg) effectively inhibited the amidase activity of Df-protease with Ki values of 1 × 10-8 and 6 × 10-4 M, respectively. The kinin release in human plasma was extensively inhibited by the addition of EGCg in comparison with myricetin. Enhancement of vascular permeability in guinea pigs caused by Df-protease was markedly suppressed by EGCg. Keywords: Mite protease; kallikrein−kinin system; catechin; allergic disease; protease inhibitor; epigallocatechin gallate

A fluorescent substance was isolated from the cyanobacterium with a yield of 4.5 mg per 10 g of dried Spirulina (Arthrospira) platensis cells by gentle extraction and ethanol fractionation followed by column chromatography. The fluorescent substance, which has absorption maxima at 256 nm and 362 nm (pH 8.4), was identified as biopterin-alpha-glucoside by spectrophotometry and nuclear magnetic resonance spectroscopy. Biopterin-alpha-glucoside prevented decolorization of the photosynthetic pigments, chlorophyll a, phycocyanin, and carotenoids in photosynthetic vesicles of Spirulina platensis cells, by ultraviolet irradiation.

L-Asparaginase modified with comb-shaped polyethylene glycol derivative, activated PM, markedly prolonged the clearance time in vivo and enhanced the stability against heat (65°C), urea (4.0 M) and acidity (pH 4.0).

Chemical modification of proteins and other bioactive molecules with polyethylene glycol (PEG) or its derivatives (PM) can be used to tailor molecular properties to particular applications, eliminating disadvantageous properties or conferring new molecular functions. Complexes of therapeutic proteins and PEG or PM show reduced immunoreactivity, prolonged clearance times and improved biostability. Modification with PEG can also increase the solubility and activity of enzymes in organic solvents, thus extending their potential for application in organic syntheses and biotransformation processes.