Determination of free D-proline and D-leucine in the brains of mutant mice lacking D-amino acid oxidase activity.

A new procedure to accurately measure a trace amount of d-proline in biological samples has been developed. This D-amino acid was derivatized with 4-fluoro-7-nitro-2,1,3-benzoxadiazole and was determined by a column-switching HPLC system, a combination of a micro-ODS column and a chiral column. The detection limit for D-proline spiked in a mouse cerebrum sample is 1 fmol (injection amount, S/N = 3). Within-day precision and day-to-day precision obtained for spiked d-proline (10 fmol) are 2.14 and 5.35% (RSD), respectively. Using the new method, the amount of free D-proline in eight brain regions and sera of mutant ddY/DAO- mice, lacking D-amino acid oxidase activity, and control ddY/DAO+ mice was determined. The amount of free D-leucine was also investigated. The amount and distribution of D-proline in the brains of ddY/DAO+ mice and ddY/DAO- mice are almost the same, and relatively high amounts of D-proline have been observed in the pituitary gland and in the pineal gland. On the other hand, the amount of D-leucine is different between the two strains. In the brains of ddY/DAO+ mice, a relatively high amount of D-leucine has been observed in the pineal gland compared with other regions. In the brains of ddY/DAO- mice, D-leucine amounts are approximately 10 times higher than those obtained in ddY/DAO+ mice and regional difference has not been observed, while the amounts of L-proline and L-leucine are not significantly different between the two strains. In the serum, the amounts of both free D-proline and d-leucine are significantly higher in the ddY/DAO- mice than those obtained in ddY/DAO+ mice.     

Characteristics of β-galactosidase purified from cell suspension cultures of carrot

Five glycosidase activities from cell homogenate of carrot ( Daucus carota L. cv. Kintoki) cell cultures were assayed after extraction successively by phosphate buffer (pH 7.0) and the buffer plus 2 M NaCl. A β-galactosidase (EC 3.2.1.23) was isolated in a highly purified state from the buffer-soluble protein fraction by ammonium sulfate fractionation and chromatography on CM-Sephadex C-50, DEAE-Sephadex A-50 and Sephadex G-200. The molecular weight of this enzyme was ca 104 000 and the isoelectric point was pH 7.8. The optimal activity occurred at pH 4.4 with McIlvaine buffer. The K_m and V_max values were 1.67 m M and 201 units (mg protein)⁻¹, respectively, for p -nitrophenyl β- d -galactopyranoside. The enzyme activity was strongly inhibited by Zn²⁺, Cu²⁺, Hg²⁺ and d -galactono-1,4-lactone. The enzyme acted on the β-1,4-linked galactan prepared from citrus pectin in an exo-fashion. Furthermore, the enzyme was slightly involved in the hydrolysis of the pectic polymer and cell walls purified from carrot cell cultures.     

Expression of the selectable marker gene bsrm in BALB/MK cells induces apoptosis by overproduction of hydrogen peroxide.

Transduction of the retroviral vector LBmSN, which expresses the blasticidin S resistance gene bsrm in the murine keratinocyte cell line BALB/MK, induces death in these cells. Cell death is caused by a factor called DOKEB (death factor obtained from keratinocytes expressing bsrm), which is released before the cells' death. In this report we describe and discuss the purification and characterization of DOKEB. Our results were as follows. (i) The 5-day-old medium from the modified BALB/MK cells with LBmSN was used for purification and characterization by filtration and chromatography: DOKEB was a stable and highly hydrophilic compound, with a molecular mass less than that of 1 amino acid. (ii) The conditioned medium containing DOKEB was reactive against thiobarbituric acid and dichlorofluorescein diacetate. (iii) DOKEB activity was neutralized by the incubation of the conditioned medium with catalase. Therefore, our conclusion is that the BALB/MK cells expressing bsrm produce a large amount of hydrogen peroxide, which catalyzes the process of apoptosis of those cells.     

Alteration of pectic polysaccharides in cell walls, extracellular polysaccharides, and glycan-hydrolytic enzymes of growth-restricted carrot cells under calcium deficiency

Suspension-cultured carrot ( Daucus carota L. cv. Kintoki) cells were grown in calcium (Ca²⁺)-deficient and normal liquid media. Cell growth was limited by the Ca²⁺ deficiency. Similar amounts of pectic fractions were extracted from the walls of control and Ca²⁺-deprived cells, but the fractions from the walls of Ca²⁺-deprived cells showed a substantial decrease in galacturonic acid content. However, after 15 days of culture, Ca²⁺-deprived cells released galacturonic acid-rich extracellular polysaccharides at twice the rate of control cells. The polysaccharides consisted of a mixture of several polymers containing predominantly arabinose, galactose and galacturonic acid. Ca²⁺-deprived cells also secreted three times more extracellular proteins, containing many glycan-hydrolytic enzymes, into the medium than did normal cells. SDS-PAGE analysis revealed several distinct changes in the polypeptide pattern in the medium of control and Ca²⁺-deprived cells. Activities of α -galactosidase, β -glucosidase and exo- polygalacturonase increased considerably during Ca²⁺ deficiency, whereas α - l -arabinofuranosidase and β -galactosidase activities were much reduced.     

PCR-RFLP analysis of cytomegalovirus infections associated with bone marrow transplantation in Japanese children.

In order to investigate the longitudinal molecular epidemiology of cytomegalovirus (CMV) infections associated with bone marrow transplantation (BMT) in Japanese children, we analyzed 36 CMV strains from 11 cases. Three regions (DNA polymerase, glycoprotein H, and immediate-early regions) of CMV DNA were amplified by polymerase chain reaction (PCR), and amplified products were each digested with two restriction enzymes, followed by electrophoresis. These restriction fragment length polymorphism (RFLP) analyses allowed the differentiation of 36 strains into 13 genotypes. Each patient excreted his or her own CMV with distinct genotype over the study period of up to one year. CMVs of two different genotypes were recovered during a one-month study from one recipient, who received a peripheral blood stem cell transplantation. Although the majority of patients and donors were CMV-seropositive before BMT, multiple CMV infections might not be common and the reactivation of latently infected CMV might be prominent in Japanese children receiving transplants.     

The interaction of sodium alginate with univalent cations

The M/G ratio, dyad and triad frequencies in the sodium alginate chain, were determined from ¹³C-nmr spectra. The interactions of sodium alginate in solution with the univalent cations K⁺ ion and Na⁺ ion have been investigated by viscometry and membrane osmometry. The dependencies of intrinsic viscosity, Huggins constant, and second virial coefficient on ionic strength were observed, and the maximums in reduced viscosity were obtained in low KCl and NaCl concentrations, respectively. These show that the electroviscous effects play an important role in polyelectrolyte solution, and the effect of the Na⁺ ion on aqueous solution of sodium alginate is greater than the K⁺ ion. The experimental observations are interpreted in terms of ion-pair formation with carboxyl groups of mannuronate and isolated guluronate residues and cooperation “egg-box” binding between polyguluronate chain sequence. The difference of interaction between univalent cations and alginate chains in solution is attributed to the ability of their binding with the polyion, which depends on the properties of ions itself. © 1998 John Wiley & Sons, Inc. Biopoly 46: 395–402, 1998