Rapid process for purification of an extracellular beta-xylosidase by aqueous two-phase extraction.

A rapid process for purification of an extracellular beta-xylosidase with high purity was developed. The manipulation involved the precipitation of protein from culture medium and the extraction of enzyme from the resuspended crude protein solution by an aqueous-two phase separation. A linear random copolymer, PE62, with 20% ethylene oxide and 80% propylene oxide was employed in both stages of the purification. The enzyme was precipitated effectively by using 10% (w/v) PE62 and 5% (w/v) Na2HPO4. The aqueous two-phase extraction was performed with PE62 (10%)-NaH2PO4 (15%) as phase-forming reagent. SDS-PAGE analysis revealed that the purified enzyme is near homogeneity. The yield is about 100% with a purification factor of 8.8-fold. The whole process can be completed within an hour without any column chromatography.

Effective extraction and purification of beta-xylosidase from Trichoderma koningii fermentation culture by aqueous two-phase partitioning.

Effective extraction of protein from bulk medium is an important technique in bioresearch. In the present study, we describe an extracellular beta-xylosidase from the fermentation supernatant of Trichoderma koningii G-39 that was successfully extracted and purified simultaneously in a single step by using an aqueous two-phase partitioning method. This two-phase system was prepared by dissolving suitable amount of poly(ethylene glycol) (PEG) and sodium dihydrogenphosphate (NaH(2)PO(4)) in aqueous solution. beta-Xylosidase was recovered with high yield and high concentration in the bottom salt-rich phase when 25% (w/v) PEG 1500 and 20-25% (w/v) NaH(2)PO(4) were applied. Based on a 1-liter scale extraction, the purity of the enzyme was enhanced at least 33-fold. The total activity increased 422% in comparison with that in the untreated filtrate. The effectiveness and simplicity may make this technique potentially useful in various applications. The transxylosylation activity of the enzyme purified by this technique was also investigated.

Mechanistic study of beta-xylosidase from Trichoderma koningii G-39.

The catalytic mechanism of the beta-xylosidase purified from the culture filtrate of Trichoderma koningii G-39 was investigated. By NMR spectroscopy, the stereochemistry of the enzyme catalyzing the hydrolysis of 2,4-dinitrophenyl and p-nitrophenyl-beta-D-xylosides was found unequivocally to involve retention of the anomeric configuration. Based on the k(cat) values of a series of arylxylosides with leaving group pK(a)s in the range of 4-10, an extended Bronsted plot was constructed with a slope (beta(lg)) near zero. Enzymatic hydrolysis of aryl-beta-D-xylosides in acetate buffer (pH 4.0) containing 3 or 5% methanol showed a constant product ratio (methylxyloside/xylose), indicating the presence of a common intermediate, probably the xylosyl-enzyme intermediate. In the presence of DTT, the k(cat) values of p-cyanophenyl-beta-D-xylopyranoside and p-nitrophenyl-beta-D-xylopyranoside increased greatly. A two-step mechanism involving the formation and breakdown of the xylosyl-enzyme intermediate was therefore proposed. The rate-limiting step is the breakdown of the intermediate. The secondary deuterium kinetic isotope effect (k(H)/k(D)) measured for 2,4-dinitrophenyl-beta-D-xyloside was 1.02+/-0.01, suggesting that the transition state for breakdown of the xylosyl-enzyme intermediate is S(N)2-like.

Biphasic effects of pu-chung-i-chi-tang on sedation and excitation.

The biphasic effects of Pu-Chung-I-Chi-Tang (PCT) on sedation and excitation in acute treatment or after one-week consecutive treatment were studied. The results indicated that PCT produces sedation in acute treatment and excitation after one-week consecutive treatment. The sedative mechanism of PCT in acute treatment might be due to an increase in serotonergic activity and a decrease in dopaminergic activity. However, the excitatory mechanism of PCT after one-week consecutive treatment might involve the increase in dopaminergic activity and the decrease in serotonergic activity.

mtDNA polymorphism in East Asian Populations, with special reference to the peopling of Japan.

Nucleotide sequences of the major noncoding (D-loop) region of human mtDNA from five East Asian populations including mainland Japanese, Ainu, Ryukyuans, Koreans, and Chinese were analyzed. On the basis of a comparison of 482-bp sequences in 293 East Asians, 207 different sequence types were observed. Of these, 189 were unique to their respective populations, whereas 18 were shared between two or three populations. Among the shared types, eight were found in common between the mainland Japanese and Koreans, which is the largest number in the comparison. The intergenic COII/tRNA(Lys) 9-bp deletion was observed in every East Asian population with varying frequencies. The D-loop sequence variation suggests that the deletion event occurred only once in the ancestry of East Asians. Phylogenetic analysis revealed that East Asian lineages were classified into at least 18 monophyletic clusters, though lineages from the five populations were completely intermingled in the phylogenetic tree. However, we assigned 14 of the 18 clusters for their specificity on the basis of the population from which the maximum number of individuals in each cluster was derived. Of note is the finding that 50% of the mainland Japanese had continental specificity in which Chinese or Koreans were dominant, while < 20% of either Ryukyuans or Ainu possessed continental specificity. Phylogenetic analysis of the entire human population revealed the closest genetic affinity between the mainland Japanese and Koreans. Thus, the results of this study are compatible with the hybridization model on the origin of modern Japanese. It is suggested that approximately 65% of the gene pool in mainland Japanese was derived from the continental gene flow after the Yayoi Age.

Orosomucoid phenotyping with monoclonal antibodies:polymorphic occurrence of ORM1*Q0 in aboriginal Taiwanese populations.

Three monoclonal antibodies (OR35, OR40 and OR48) against orosomucoid (ORM) were prepared for the phenotyping of the human ORM system. The OR35 and OR48 antibodies recognized ORM1 and ORM2 products, respectively. OR40 reacted strongly to the products of ORM1 but poorly to those of ORM2. With the help of these monoclonal antibodies, ORM phenotyping was performed on 658 individuals from nine subpopulations of aboriginal Taiwanese, with close attention to two individuals with an ORM1 Q0 homozygous phenotype. The ORM1*Q0 allele was found to be at a polymorphic frequency in eight of the nine subpopulations.

Polymorphisms of complement component I and C1R subcomponent of C1 in nine aboriginal Taiwanese populations.

Complement component I (IF) and C1R subcomponent of C1 (C1R) types were determined by isoelectric focusing and subsequent immunoblotting techniques for 658 individuals from nine aboriginal Taiwanese populations. The frequency of the IF*A allele ranges from 0.075 (Bunun) to 0.430 (Saisiat), and a new variant allele IF*B2 was found to have polymorphic frequency in the Atayal. The frequency of the C1R*1 allele ranges from 0.410 (Yami) to 0.650 (Atayal), and the frequency of the C1R*2 allele ranges from 0.265 (Atayal) to 0.586 (Saisiat). The C1R*5 allele was found in five populations (Atayal, Bunun, Ami, Puyuma, Yami), and the C1R*9 allele was found in two populations (Tsou, Puyuma). The results indicate a remarkable degree of genetic variability among these populations. The variability may reflect long-term genetic and geographic isolation of each population.

Seroepidemiological survey of human T-lymphotropic retrovirus among indigenous populations in Taiwan.

The prevalence of human T-lymphotropic retrovirus (HTLV) was examined in Taiwan's indigenous populations. In all, 797 healthy subjects in Taiwan including Han Chinese and nine indigenous populations (Ami, Atayal, Bunun, Saisiat, Paiwan, Puyuma, Rukai, Tsuo, and Yami) were examined for the presence of antibodies to HTLV by particle agglutination, indirect immunofluorescence and Western blot test. Two seropositive cases were found in this screening. One Saisiat male and a Han Chinese female were seropositive for HTLV. The Western blot profile indicated the virus was type-1 HTLV.

Seroepidemiology of adult T-cell leukemia virus infection and analysis of sero-positive cases in Taiwan.

For a seroepidemiologic study of adult T-cell leukemia virus (ATLV) infection in Taiwan, the gelatin particle agglutination technique and the indirect immunofluorescence method were used for anti-ATLV titration. Sporadic sero-positive cases were found all over the Taiwan districts except among the aborigines (0/947). Sero-positive rates ranged from 0 to 5.6% (except ATL family) and a total of 48 cases were found in 3682 Han-Chinese. Among them 9 cases were newly found in family surveys, and 39 cases were observed in random samples. As an average positive rate was 1.0%, by calculation about 80,000 sero-positive cases are supposed to be present in Taiwan. A most remarkable feature of the sero-positive cases was the high rate in couples. Various patterns of sero-positive cases existed in pedigrees. Anti-ATLV positive sera of Chinese living in Taiwan and Japanese were compared by immunoprecipitation and there was no difference between them. The possible infection route from Japan to Taiwan is discussed.

Relative sensitivity of cell culture systems in the detection of herpes simplex viruses.

Guinea pig embryonic fibroblasts were more sensitive and McCoy and Hep-2 cells were less sensitive than human foreskin fibroblasts in parallel titrations of herpes simplex virus. No difference in sensitivity was found for five lines of human fetal lung fibroblasts (including WI-38 cells), two lines of human embryo fibroblasts, one line of human foreskin fibroblasts, cells from a human fetal kidney, amnion cells from a human placenta, the Chang liver cell, the HeLa cell, and a line of mink cells. The cell doubling level of human or guinea pig fibroblast lines did not affect their sensitivity. One hundred ninety-five clinical specimens submitted for herpes simplex virus isolations were tested in parallel in primary rabbit kidney, guinea pig embryo, and human fetal lung fibroblast cultures. The percentages of positive or false-negative cultures were essentially the same for the three types of cells.

Seroepidemiology of adult T-cell leukemia virus in Taiwan.

A survey of carriers of adult T-cell leukemia virus (ATLV) detected as anti-ATLV associated antigens was made in Taiwan. Among 2545 adults aged 30 years or more examined, seropositive donors amounted to 0.9% in the Han Chinese but none in the aborigines.

Analysis of hydroxyzine hydrochloride, meperidine hydrochloride and atropine sulfate in glass and plastic syringes.

The apparent stability of combinations of hydroxyzine hydrochloride and meperidine hydrochloride (50 mg/2 ml each) and of these two drugs (50 mg/2.5 ml each) and atropine sulfate (0.4 mg/2.5 ml) in prefilled glass and plastic syringes was studied. Syringes (3 ml) containing the combinations were stored at 25 C and 3 C for 10 days and analyzed at specific time intervals. Absorption spectra, chromatographic characteristics and pH were determined in addition to visual inspection. Results of these qualitative tests indicated that the mixtures apparently were stable for 10 days at room temperature or when refrigerated. No differences were found between solutions stored in glass and those stored in plastic syringes. Degradation of the syringe contents or appearance of additional constituents was not detected in any of the admixtures, and they were considered to be chemically compatible within the limitations of the study. The study suggests that storage of these combinations in syringes is feasible but the results cannot be extrapolated to drug solutions or syringes other than those studied.