Analysis of the Epitopes Recognized by Mouse Monoclonal Antibodies Directed to Yersinia enterocolitica Heat-Shock Protein 60

To determine amino acid sequences of the epitopes recognized by monoclonal antibodies (mAbs) 3C8 and 5C3 directed against Yersinia enterocolitica heat-shock protein (HSP60), a dot blot analysis was perfomed using synthesized peptides of Y. enterocolitica HSP60 such as peptides p316-342, p327-359, p340-366, p316-326, p316-321, p319-323, and p321-326 which represent positions of amino acids in Y. enterocolitica HSP60. The dot blot analysis revealed that 5C3 mAb reacted with p316-342, p316-326 and p321-326, and 3C8 mAb p316-342 and p316-326. These results indicate that the epitopes recognized by the mAbs were associated with eleven amino acids, Asp Leu Gly Gln Ala Lys Arg Val Val Ile Asn, of p316-326. The sequence homology between p316-326 of Y. enterocolitica HSP60 and the rest of the HSP60 family suggests that the five amino acids of Lys, Arg, Val, Ile and Asn, which are highly conserved in the HSP60 family, might be related with the epitope recognized by 3C8. In contrast, it was also demonstrated that three amino acids of Leu, Gly and Val, which are not well conserved in the HSP60 family, might be related to the epitope recognized by 5C3.     

Immobilization ofβ-fructofuranosidase fromAureobasidium on DEAE-cellulose

Summary -Fructofuranosidase, which produces fructo-oligosaccharides (1-kestose and nystose) from sucrose, was purified fromAureobasidium and immobilized on DEAE-cellulose at especially high efficiency (95%). The enzymatic profiles of the immobilized enzyme were almost identical to those of the native form except that the stability was slightly improved. The immobilized enzyme was stable during long-term continuous reaction for up to 360 h.     

Embryonic Development and Postnatal Changes in Free d-Aspartate and d-Serine in the Human Prefrontal Cortex

Abstract: We have analyzed free chiral amino acids (aspartate and serine) in the human frontal cortex at different ontogenic stages (from 14 weeks of gestation to 101 years of age) by HPLC with fluorometric detection after derivatization with N-tert -butyl-oxycarbonyl- l -cysteine and o -phthaldialdehyde. Exceptionally high levels of free d -aspartate and d -serine were demonstrated in the fetal cortex at gestational week 14. The ratios of d -aspartate and of d -serine to the total corresponding amino acids were also high, at 0.63 and 0.27, respectively. The concentration of d -aspartate dramatically decreased to a trace level by gestational week 41 and then remained very low during all postnatal stages. In contrast, the frontal tip contained persistently high levels of d -serine throughout embryonic and postnatal life, whereas the d -amino acid content in adolescents and aged individuals was about half of that in the fetuses. Because d -aspartate and d -serine are known to have selective actions at the NMDA-type excitatory amino acid receptor, the present data suggest that these d -amino acids might play a pivotal role in cerebral development and functions that are related to the NMDA receptor.     

Enantioselective pharmacokinetics of homochlorcyclizine: III. Simultaneous determination of (+)- and (−)- homochlorcyclizine in human urine by high-performance liquid chromatography

A method is described for the simultaneous determination of (+)- and (−)-homochlorcyclizine (HCZ) in human urine by high-performance liquid chromatography on a chiral stationary phase of ovomucoid-bonded silica. The pH of the buffer and organic modifier in the mobile phase markedly affected the chromatographic separation. A mobile phase of methanol—0.02 M acetate buffer (pH 4.7) (25:75, v/v) at a flow-rate of 1.0 ml/min was used for the urine assays. The ultraviolet absorption was monitored at 240 nm, and diphenhydramine was employed as the internal standard for the quantitation. (+)-HCZ, (−)-HCZ and the internal standard were eluted at retention times of 15, 25 and 8 min, respectively. The limit of determination for HCZ enantiomers was ca. 50 ng/ml of urine. One of the metabolites in human urine, which was a quaternary ammonium-linked glucuronide, could also be determined in a manner similar to unchanged HCZ after β-glucuronidase hydrolysis. A pharmacokinetic study was conducted with three healthy volunteers, who each received a single oral dose of racemic HCZ (20 mg). Distinct differences were found between the two enantiomers, particularly in the metabolic process, that is, the urinary excretion as (−)-HCZ-glucuronide within 48 h was ca. four times higher than that of the (+)-isomer. This method should be very useful for enantioselective pharmacokinetic studies of HCZ.     

Purification and properties of extracellularβ-fructofuranosidases fromAureobasidium sp. ATCC 20524

Summary Two extracellular -fructofuranosidases (E-1 andE-2) fromAureobasidium sp. ATCC 20524, producing 1-kestose (1^F--fructofuranosyl-sucrose) from sucrose, were purified to homogeneity. Molecular weights of the enzymes were estimated to be about 304000 (E-1) and 315000 (E-2) Da by gel filtration. The enzymes contained 33% (w/w) (E-1) and 27% (w/w) (E-2) carbohydrate. TheK _m values for sucrose ofE-1 andE-2 andE-2 were 0.34 and 0.28 M, respectively. were 0.34 and 0.28 M, respectively. The enzymatic profiles of these enzymes were almost identical to intracellular enzymesP-1 andP-2 except for the differences in carbohydrate content andK _m values ofE-2 andP-2.     

Preparation of Optically Pure cis-2,4-Dimethyl-l-cyclohexanones,the Key Intermediates in Cycloheximide Synthesis,Using Microbial Resolution

Asymmetric hydrolysis of acetate (10) of (±)-t-2,t-4-dimethyl-r-l-cyclohexanol with Bacillus subtilis var. niger gave (?)-(lS,2S,4S)-2,4-dimethyl-l-cyclohexanol (6a) and (+)-(1R,2R,4R)-acetate (10b) with high optical purities. Optically pure (?) and (+)-alcohols (6a and 6b) were prepared via corresponding 3,5-dinitrobenzoates. Oxidation of alcohols (6a and 6b) with chromic acid gave optically pure (?)-(2S,4S) and (+)-(2R,4R)-2,4-dimethyl-l-cyclohexanones (2a and 2b), respectively.     

Synthesis of Streptovitacin-C2 Isomers

(2R*,4S*,6S*,αS*)- and (2R,4R,6R,αS)-Streptovitacin-C₂ (STV-C₂) (1a and 1b) were synthesized by an aldol condensation of (2R*,4S*)- or (2R,4R)-2,4-dimethyl-2-trimethylsiloxy-1-cyclohexanone (15a or 15b) with 4-(2-oxoethyl)-2,6-piperidinedione (16), which was followed by desilylation of the products. The stereochemistry of the synthesized STV-C₂ isomers (1a and 1b) was elucidated by NMR. STV-C₂ isomers (1a and 1b) did not show strong antimicrobial activity against Saccharomyces cerevisiae and Pyricularia oryzae.     

Solid State 13C-NMR Analysis of Cell Wall Components of Fusarium oxysporum

An abdominal fat accumulation complicated by high blood triglycerides is regarded as a risk factor of metabolic syndrome. Feeding powdered nacre, mother of pearl, from Pinctada maxima, resulted in reduced body weight, visceral fat amount, and blood triglyceride level without influencing the food intake, body length, or amount of muscular tissue, suggesting that nacre powder specifically could decrease visceral fat.     

Dynamic light scattering and circular dichroism studies on heat-induced gelation of hard-keratin protein aqueous solutions

Animal hairs consist of aggregates of dead cells filled with keratin protein gel. We succeeded in preparing water-soluble hard-keratin proteins and reconstructing the keratin gels by heat-induced disulfide linkages in vitro. Here, the roles of intermolecular hydrophobic interaction and disulfide bonding between the proteins in the gel were discussed. Water-soluble keratin proteins consisting of mixtures of type I ( approximately 48 kDa) and type II ( approximately 61 kDa) were prepared from wool fibers as S-carboxymethyl alanyl disulfide keratin (CMADK). The gelation was achieved by heating an aqueous solution containing at least 0.8 wt % CMADK at 100 degrees C. CMADK solutions with different urea or N-ethylmaleimide concentrations or pH were exposed to dynamic light scattering (DLS) and circular dichroism (CD). DLS clarified the gelation point of CMADK solutions and provided information on the changes in keratin cluster size. DLS suggested two types of gelation mechanism. One was the regenerated chemical disulfide bonding between keratins from CMAD parts of chains. After the gel formed, this bond became important to maintain the gel structure. The other was the physical assembly due to hydrophobic interaction between alpha-helix parts of keratin chains. This hydrophobic assembly also played an important role during gelation. CD confirmed a conformational change in the keratin protein, resulting heat-induced gelation. CD clarified the relationship between keratin protein conformation and gelation, i.e., a rodlike conformation with many alpha-helix structures was necessary to associate keratin chains and form a gel network.