Enzymatic preparation of [1, 3-13C]dihydroxyacetone phosphate from [13C]methanol and hydroxypyruvate using the methanol-assimilating system of methylotrophic yeasts

Dihydroxyacetoone synthase (EC 2.2.1.3), which is a key enzyme of the C₁-compound-assimilating pathway in yeasts, catalyzes transketolation between formaldehyde and hydroxypyruvate, leading to the formation of dihydroxyacetone and CO₂. When [¹³C]formaldehyde was used as a substrate with dihydroxyacytone synthase from Candida boidinii 2201, ¹³C was confirmed to be incorporated to the C-1 and C-3 positions of dihydroxyacetone, and the ¹³C content of each carbon (atoms/100 atoms) was estimated to be 50%. [¹³C]Methanol was also useful for the enrichment of dihydroxyacetone with ¹³C, when alcohol oxidase from a methylotrophic yeast was added for the conversion of methanol to formaldehyde. A fed-batch reaction with periodic addition of the substrates was required for the accumalation of ¹³C-labelled dihydroxyacetone at a higher concentration, because the enzyme system was relatively susceptible to the C donor, formaldehyde or methanol. The optimum conditions for the production gave 160mM (14.4 mg/ml) dihydroxyacetone for 180 min; the molar yield relative to methanol added was 80%. Diyhdroxyacetone kinase (EC 2.7.1.29) from methanol-grown Hansenula polymorpha CBS 4732 was a suitable enzyme for the phosphorylation of dihydroxyacytone. The phosphorylation system, comprising of dihydroxyacetone kinase, adenylate kinase, and ATP, could be coupled with the system for dihydroxyacetone production. A fed-batch reaction afforded 185 mM [1, 3-¹³C]dihydroxyacetone phosphate from [¹³C]methanol; the molar yield of the ester relative to methanol added was 92.5%     

A heterogeneous electrophysiological profile of bone marrow-derived mast cells

Electrophysiological properties of mouse bone marrow-derived mast cells (BMMC) were studied under the whole-cell clamp configuration. About one third of the cells were quiescent, but others expressed either inward or outward currents. Inwardly rectifying (IR) currents were predominant in 14% of the cells, and outwardly rectifying (OR) currents in 24%. The rest (22%) of the cells exhibited both inward and outward currents. The IR currents were eliminated by 1 mm Ba²⁺, and were partially inhibited by 100 μm quinidine. The reversal potential was dependent on extracellular K⁺, thereby indicating that K⁺ mediated the IR currents. The negative conductance region was seen at potentials positive to E _K. The OR currents did not apparently depend on the extracellular K⁺ concentration, but were reduced by lowering the extracellular Cl^? concentration. The OR currents were partially blocked by 1 mm Ba²⁺, and were further blocked by a Cl^? channel blocker, 4,4′-diisothiocyano-2, 2′-stilbenedisulfonate (DIDS). In addition, the reversal potential of the OR currents was positively shifted by decreasing the ratio of external and internal Cl^? concentrations, suggesting that Cl^? was a major ion carrier. In cells exhibiting IR currents, the membrane potential varied among cells and tended to depolarize by elevating the external K⁺ concentration. In cells with OR currents, the resting potential was hyperpolarized in association with an increase in conductance. These results suggest that BMMC have a heterogeneous electrophysiological profile that may underlie a variety of ion channels expressed in different phenotypes of mast cells. Activities of both the inwardly rectifying K⁺ channel and the outwardly rectifying Cl^? channel seem to contribute to the regulation of the membrane potential.     

Changes in chorion proteins induced by the exudate released from the egg cortex at the time of fertilization in the teleost, Oryzias latipes

The chorion of unfertilized medaka Oryzias latipes eggs consists of two major proteins (77–73 and 49 kDa) and a minor 150 kDa protein. Upon fertilization, these major chorion proteins are polymerized to insoluble high molecular weight proteins via the temporary formation of several new proteins (132, 114, 62 and 61 kDa). Increasing chorion toughness is closely related to the formation of high molecular weight proteins and the increasing insolubility of the chorion proteins. The changes in chorion proteins and hardening could be induced in vitro in isolated chorions by an egg exudate, which includes cortical alveolar contents. The effects of temperature and pH on the egg exudate-induced changes in chorion proteins were examined in the present study. The major proteins could be digested by proteolytic enzymes. The 49 kDa protein was PAS-positive. Analysis with polyclonal antibodies against the major proteins demonstrated that the temporarily formed 62 and 61 kDa proteins were derived from the 77–73 kDa protein and that higher molecular weight proteins, newly formed in the process of chorion hardening, contained the same epitopes as did the 77–73 and 49 kDa proteins. The results suggest that the changes in chorion proteins of the medaka egg at the time of fertilization can be induced by an enzyme(s) released from the egg cortex into the perivitelline space.     

Molecular Evolution of the Duplicated Amy Locus in the Drosophila Melanogaster Species Subgroup: Concerted Evolution Only in the Coding Region and an Excess of Nonsynonymous Substitutions in Speciation

From the analysis of restriction maps of the Amy region in eight sibling species belonging to the Drosophila melanogaster species subgroup, we herein show that the patterns of duplication of the Amy gene are almost the same in all species. This indicates that duplication occurred before speciation within this species subgroup. From the nucleotide sequence data, we show a strong within-species similarity between the duplicated loci in the Amy coding region. This is in contrast to a strong similarity in the 5' and 3' flanking regions within each locus (proximal or distal) throughout the species subgroup. This means that concerted evolution occurred only in the Amy coding region and that differentiated evolution between the duplication occurred in the flanking regions. Moreover, when comparing the species, we also found a significant excess of nonsynonymous substitutions. In particular, all the fixed substitutions specific to D. erecta were found to be nonsynonymous. We thus conclude that adaptive protein evolution occurred in the lineage of D. erecta that is a ``specialist' species for host plants and probably also occurs in the process of speciation in general.     

Comparison of the DNA content of megakaryocytes identified immunologically with that identified morphologically

 We devised a new microfluorometric method for determining the ploidy of megakaryocytes identified immunologically in bone marrow smears. The smears were immunostained by incubation with mouse monoclonal anti-glycoproteins (GP) IIb antibodies, followed by fluorescein isothiocyanate-conjugated goat anti-mouse IgG antibodies. They were then stained with 4′,6-diamidino-2-phenylindole (DAPI). Megakaryocytes were identified by their GPIIb immunofluorescence using a microfluorometer and, after the filters were changed, their DNA content was assayed by measuring the intensity of DAPI fluorescence. This intensity was shown to be proportional to the DNA content when the aperture of the objective lens was reduced. We compared these results with those obtained when megakaryocytes were identified morphologically, using DAPI staining after Wright-Giemsa destaining. In all 12 normal controls, the ploidy peaks were shown to be 16N by both methods, and the mean ploidy detected by the immunological method was only reduced 0.961 times relative to the estimate from the morphological method. In contrast, in eight myelodysplastic syndrome (MDS) patients, the ploidy peaks were either 8N or 4N and the mean was reduced by 0.906 times (P=0.018). Thus we could immunologically identify small megakaryocytes which we could not identify morphologically. Therefore, this method is useful for measuring megakaryocytic ploidy, especially in the pathological megakaryocytes of MDS patients. Accepted: 29 April 1997     

Isolation and characterization of Enterobacter cloacae mutants which are defective in chemotaxis toward inorganic phosphate.

Enterobacter cloacae IFO3320 is attracted to Pi when cells are starved for Pi. Two Tn1737KH-induced mutants, which were constitutive for alkaline phosphatase, failed to exhibit Pi taxis even under conditions of Pi limitation. Both of the mutant strains exhibited normal chemotactic responses to peptone, suggesting that they are specifically defective in Pi taxis. Cloning and sequence analysis showed that the TN1737KH insertions were located in either the pstA or pstB genes which encode the channel-forming proteins of the Pi-specific transport (Pst) system in E. cloacae. These results suggest that the E. cloacae Pst system is required for Pi chemoreception.     

A Novel Type of Substrate Specificity of Rice BAPAase (Benzoyl-L-arginine p-nitroanilide Hydrolase) with Mixed Endopeptidase and Carboxypeptidase

The substrate specificity of rice embryo benzoyl-L-argininep-nitroanilide hydrolase (BAPAase) was examined. No endopeptidaseactivity toward protein substrates was detectable. Small peptides(less than 8 residues) and amide, ester substrates, however,were hydrolyzed very well at the carboxyl side of the lysineor arginine residue. No other peptide bond was hydrolyzed. TheN-terminal arginine of the substrates was released very slowly.Peptides with lysine or arginine penultimate to the C-terminalposition were hydrolyzed well and released an amino acid. Theoxidized insulin B chain (30 residues) was cleaved very slowlyat the C-terminal Lys-Ala bond, whereas an Arg-Gly bond at aninner position was not cleaved. The hydrolytic rate increasedafter the chain length was shortened by chymotryptic digestion.These results show that the rice embryo BAPAase is a novel enzymewhich has mixed endopeptidase-carboxypeptidase activity towardthe Arg-X and Lys-X bonds of small peptides, a characteristicintermediate between trypsin and serine carboxypeptidase. Thisenzyme may act in the breakdown of small peptides that havephysiological functions. (Received May 26, 1984; Accepted August 29, 1984)     

Purification and Characterization of Rice Embryo BAPAase (Benzoyl-L-arginine p-nitroanilide Hydrolase)

Benzoyl-L-arginine p-nitroanilide hydrolase (BAPAase), whichhas both endopeptidase and carboxypeptidase activity towardthe Arg-X or Lys-X bond of small peptides [Shibata and Doi (1984)Plant & Cell Physiol. 25: 1421], was purified from riceembryos by ammonium sulfate and polymin fractionations and byion exchange, gel exclusion and hydrophobic chromatographies.The purified enzyme was homogeneous when analyzed by polyacrylamidegel electrophoresis. It was unstable in the absence of surface-activereagents such as Triton X-100. Maximum activity for benzoyl-Largininep-nitroanilide (L-BAPA) or carboxypeptidase activity towardbutoxycarbonyl-Gly-Lys-Leu was obtained at pH 9.0. L-BAPA athigh concentrations inhibited the enzyme's activity. Di-isopropylphosphofluoridate, N-tosyl-L-lysine chloromethyl ketone, leupeptinand antipain, which are specific inhibitors of trypsin, inhibitedBAPAase activity, but soybean and rice bran trypsin inhibitorhad no effect on it. Sulfhydryl reagents strongly inhibitedthe BAPAase activity. (Received May 26, 1984; Accepted August 29, 1984)     

Relation of mode of administration of testosterone to evocation of male sex behavior in frogs

In Rana pipiens, mating behavior could be induced readily in intact males by several pituitary implantations, but never in castrates. Systemic testosterone injection (1 mg daily), with or without pituitary implantation, failed to restore mating behavior in castrated frogs. On the other hand, intracranial implantation of testosterone (approximately 60-μg pellets in which testosterone is mixed with cholesterol 1:1) in castrates evoked mating behavior, including mating calls and clasping. The most effective implantation site was the rostral part of the preoptic nucleus. Thus, the rostral part of the preoptic nucleus is the androgen-sensitive site which governs sexual behavior in this species. The relative ineffectiveness of systemic injection of testosterone is discussed.     

Rat alpha1-acid glycoprotein. Purification and immunological estimation of its serum concentration.

A rapid method is described for the preparation of serum alpha1-acid glycoprotein from rats with inflammation induced with turpentine oil injection. The protein obtained by two purification steps, batchwise adsorption with DEAE-cellulose followed by chromatography on CM-cellulose, was proved to be native alpha1-acid glycoprotein in a high degree of purity by electrophoretical, immunological, ultracentrifugal and carbohydrate analysis. The monospecific and potent antiserum to this protein was prepared by immunizing rabbits with the desialyzed material emulsified with Freund's incomplete adjuvant. Using purified alpha1-acid glycoprotein and its specific antiserum, the concentration of alpha1-acid glycoprotein in rat serum was determined by single radial immunodiffusion. Abnormally high levels of its concentration (5-6 times higher than the control) were observed in inflammatory and tumor bearing rats.