Purification and properties of a novel enzyme,N-acylamino acid racemase,from Streptomyces atratus Y-53

A novel enzyme, N-acylamino acid racemase, was purified to homogeneity from Streptomyces atratus Y-53 and characterized. This enzyme catalyzes the interconversion of optically active N-acylamino acids. The relative molecular mass (M_r) of the enzyme was estimated to be about 41 000 and 244 000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration, respectively, indicating that the enzyme is composed of six subunits with an equal M_r. The enzyme showed a broad substrate specificity toward N-acylamino acids, such as N-acetylmethionine, N-chloroacetylphenylalanine and N-chloroacetylvaline. The apparent Michaelis constant (K_m) values for N-acetyl-l-methionine and N-acetyl-d-methionine were calculated to be 15.2 and 5.6 mm, respectively. Enzyme activity was markedly enhanced by divalent metal ions, such as Co²⁺, Mg²⁺ and Mn²⁺, and was inhibited by metal-chelating reagent, indicating that the enzyme is a metalloenzyme. We propose to name the enzyme N-acylamino acid racemase (acylamino acid racemase). Correspondence to: S. Tokuyama     

Expression of Regeneration-Associated Antigens in Normal and Retinoid-Treated Regenerating Limbs of Ambystoma mexicanum

The expression of two regeneration-associated antigens in the blastemas of normal and retinoid-treated regenerating limbs of axolotl ( Ambystoma mexicanum ) was examined.
One antigen, 55C12, which was similar to tenascin in expression pattern and molecular weight profile, was weakly expressed in the perichondrium and tendon of normal limbs. In the regenerating limbs, the amount of 55C12 antigen increased near the amputation site within 7 days and almost all cells of the blastema mesenchyme came to be positive to the antigen at 20 days, although those of epidermis and most stump tissues were negative. When the regenerating limbs were treated with Am80, a synthetic retinoid, which induced proximo-distal duplication, the expression of 55C12 antigen in the blastema became weak temporarily and was reactivated in the anterior region of the blastema. This expression pattern suggests that the duplicated limb is formed by the preferential growth of this 55C12-positive anterior blastema region.
The other antigen, 117C1, was faintly expressed in the epidermis, dermis, muscle, perichondrium and cartilage of normal limbs, and intensely expressed in the blastema mesenchyme and wound epidermis. The Am80 treatment, however, induced no changes in the expression pattern of 117C1.
These results suggest that these antigens may distinguish two different regions of the blastema in normal regeneration and retinoid-induced duplication.     

Rhodium-catalyzed synthesis of 2(5H)-furanones from terminal alkynes and non-substituted alkynes under water-gas shift reaction conditions

Rhodium-catalyzed synthesis of 2(5H)-furanones from alkynes under water-gas shift reaction conditions was studied. By improving the reaction conditions for internal alkynes reported previously, the reaction could be extended to terminal alkynes. Terminal alkynes are selectively converted into 3- and 4-substituted 2(5H)-furanones (2 and 3). When acetylene itself is used, 2(5H)-furanone (2n) is obtained in a good yield. Examination of reaction solutions by IR spectroscopy and some other experimental findings suggest that the active species would be an alkyne-coordinated monomeric rhodium anion. A new reaction path is proposed.     

Analysis of effector T cells against the murine syngeneic tumor MethA in mice orally administered antitumor polysaccharide SPR-901

The growth of MethA tumor was significantly inhibited by oral administration of the -glucan SPR-901 in BALB/c (+/+) mice but not in nude mice. Mice treated orally with SPR-901 exhibited an augmentation of antigen-specific resistance against rechallenge with the tumor cells. The tumor-neutralizing activity of regional lymph node cells from MethA-bearing mice against the tumor was augmented by oral administration of SPR-901. The tumor-neutralizing activity of lymph node cells from SPR-901-treated mice mainly appeared in Lyt2+cells. Furthermore, lymphokine-activated killer activity of these cells was enhanced by administration of SPR-901. The antitumor effect of SPR-901 was abrogated in mice depleted of either L3T4+ or Lyt2+ cells, and in cyclosporin-A-treated mice. These results suggest that Lyt2+ cells are important effector cells in MethA-bearing mice orally adminstered SPR-901 and that functional exertion of both Lyt2+ and L3T4+T cells is necessary for the antitumor effect of orally administered SPR-901 in vivo.     

The effect of methyl palmoxirate on incorporation of [U-14C]palmitate into rat brain

We examined the dose response, time course and reversibility of the effect of methyl 2-tetradecylglycidate (McN-3716, methyl palmoxirate or MEP), an inhibitor of -oxidation of fatty acids, on incorporation of radiolabeled palmitic acid ([U-¹⁴C]PA) from plasma into brain lipids of awake rats. MEP (0.1, 1 and 10 mg/kg) or vehicle was administered intravenously from 10 min to 72 hr prior to infusion of [U-¹⁴C]PA. Two hr pretreatment with MEP (0.1 to 10 mg/kg) increased brain organic radioactivity 1.2 to 1.8 fold and decreased brain aqueous radioactivity by 1.2 to 3.0 fold when compared to control values. At 10 mg/kg, MEP significantly increased brain organic fraction from 40% in controls to 85%, 30 min to 6 hr pretreatment, and resulted in a redistribution of the radiolabeled fatty acid toward triacylglycerol. MEP changed the lipid/aqueous brain ratio of incorporated [U-¹⁴C]PA from 0.67 to 5.7. The incorporation rate coefficient, k^*, was significantly increased by MEP (10 mg/kg) at 2 hr (31%), 4 hr (59%) and 6 hr (34%). All effects were reversed by 72 hr, consistent with a half-life of 2 days for carnitine palmitoyl transferase I. These results indicate that intravenous MEP may be used with [1-¹¹C]palmitic acid for studying brain lipid metabolism in vivo by positron emission tomography, as it significantly reduces the large unincorporated aqueous fraction that would result in high background radioactivity.     

IRE-Bubble PCR: A Rapid Method for Efficient and Representative Amplification of Human Genomic DNA Sequences from Complex Sources

A significant issue in the analysis of any genomic DNA segment is the generation of a unique set of short single-copy sequences that are representative of that region. In this report we describe a novel technique, IRE-bubble PCR, which was designed to amplify the human DNA content of somatic cell hybrids, YACs, cosmids, and λ phage and result in greater complexity and representation than standard inter-IRE, PCR. Here we demonstrate that IRE-bubble PCR is species specific and that it results in the generation of a product that is at least 10-fold more complex and representative than that produced by standard inter-IRE PCR. In addition, we have addressed the factors that contribute to the representation of the IRE-bubble PCR product and show how they may be used to further increase the complexity of this reaction. Finally, we have illustrated how the complexity and distribution of products generated by IRE-bubble PCR can be exploited and applied to FISH mapping and "chromosome painting" as well as to the generation of STSs targeted to specific chromosomal or subchromosomal regions.     

cDNA Cloning and Expression of the Plastidic Copper/Zinc-Superoxide Dismutase from Spinach (Spinacia oleracea L.) Leaves

A cDNA clone for copper/zinc-superoxide dismutase (Cu/Zn-SOD)was isolated from spinach (Spinacia oleracea L.) leaves. Itsnucleotide sequence showed that it codes for a precursor polypeptideof 222 amino acids, including the NH₂-terminal 68-residue extensionwhich corresponds to a plastidic transit peptide. Northern hybridization,using plastidic and cytosolic Cu/Zn-SOD cDNAs as the probes,revealed that these two genes are differentially expressed inthe roots and leaves of spinach. ¹Present address: Department of Biochemistry and Microbiology,Cook College, Rutgers University New Brunswick, NJ 08903-0231,U.S.A.     

Characterization of peroxidases in luminol chemiluminescence coupled with copper-catalysed oxidation of cysteamine

Hydrogen peroxide formed during the course of the copper(II)-catalysed oxidation of cysteamine with oxygen was continuously determined by a peroxidase (POD)-catalysed luminol chemiluminescence (CL) method. Horseradish peroxidase (HRP), lactoperoxidase (LPO) and Arthromyces ramosus peroxidase (ARP) were used as a CL catalyst. The respective PODs gave specific CL intensity-time profiles. HRP caused a CL delay, and ARP gave a time-response curve which followed the production rate of H₂O₂. LPO gave only a weak CL flash which decayed promptly. These differences of CL response curves could be explained in terms of the different reactivities of PODs for superoxide anion and the different formation rate of luminol radicals in the peroxidation of luminol catalysed by POD.     

Movement of actin away from the center of reconstituted rabbit myosin filament is slower than in the opposite direction.

By decreasing ionic strength slowly, thick filaments of several micrometers in length were obtained from purified rabbit skeletal muscle myosin. Dark-field observation showed these filaments with their center scattering light extensively. Active movement of actin filaments complexed with tetramethyl rhodamine-phalloidin along the reconstituted myosin filaments was observed. Actin filaments moved towards the center of myosin filaments at a speed of 3.9 +/- 1.6 microns s-1 (mean +/- SD, n = 40) and often continued to move beyond the center towards the tip of the opposite side at a lower speed. The speed of the movement away from the center was 1.0 +/- 0.6 microns s-1 (n = 59). Thus, the functional bipolarity in terms of the movement speed which was first found in native thick filaments of molluscan smooth muscle is also seen in reconstituted filaments from purified rabbit skeletal muscle myosin. The difference of the speed between the two directions is considered to be due to properties of myosin molecules themselves.     

Cell Sorting and Chondrogenic Aggregate Formation in Limb Bud Recombinants and in Culture

The cartilage pattern of the developing chick limb changes along the proximal-distal (PD) axis. It is assumed that these spatial changes are brought about by differences in the cellular properties of distal mesoderm, the progress zone (PZ). To examine whether these differences are actually maintained in the individual cells composing the PZ, we dissociated early (stage 20) and late (stage 25) PZ tissues into single cells, then mixed and recombined them with ectodermal jackets. The recombinants were grafted to limb bud stumps and allowed to develop into limb-like structures. Early PZ cells were distributed within whole cartilage elements along the PD axis of the limb-like structures, while cells from late PZ participated only in the formation of distal cartilage elements.
A difference in distribution pattern between the cells of early and late PZ in mixed culture was also observed. Cells of early PZ aggregated rapidly in patches and formed cartilage nodules, while the cells of late PZ distributed in regions surrounding these cell aggregates and gradually differentiated to cartilage cells. These results suggest that the cellular properties in the PZ concerning the rate of chondrogenic aggregate formation change during limb bud development, and that this change may relate to the cartilage pattern formation along the PD axis.