The Isolation and Properties of a Factor in Taro Tuber that Agglutinates Spores of Ceratocystis fimbriata, Black Rot Fungus

A factor which agglutinates spores of Ceratocystis fimbriatain the presence of Ca²⁺ was isolated from taro tuber (Corocasiaesculenta Schott, cv. Shiro). The elemental composition of theisolated factor was as found by analysis: C (33.27%), H (4.27%),O (61.90%), N (0.56%); as calculated for C₆₉H₁₀₆O₉₇N₁: C (33.13%),H (4.27%), O (62.04%), N (0.56%). This factor is composed mainlyof galacturonic acid (85% of its dry weight) and contains arabinose,fucose and an unidentified component as its minor components. The differential spore-agglutinating activity of this factordepends on the pH of the assay medium, differential agglutinatingactivity being present at pH 6.5 toward germinated spores ofvarious strains of C. fimbriata. The differential agglutinationof the spores of these strains changed with the growth stage:Ungerminated spores and hyphae of the strains tested were agglutinatedto the same extent, whereas the germinated spores of these strainswere agglutinated differently. When ungerminated and germinated spores of the strains weretreated with pronase, Macerozyme or phospholipase D, their reactivityto the factor changed. Sonication also caused changes in thereactivity of the spores to the factor; germinated spores ofthe sweet potato strain became highly sensitive to it. Insensitivityto the factor was restored in sonicated spores incubated witha substance released from the spores during sonication. Theseresults are discussed in relation to host-parasite specificity. (Received May 19, 1982; Accepted November 9, 1982)     

Alteration of nuclear proto-oncogene expression by erythropoietin (Epo) in Epo-responsive murine cell lines

The signal transduction system of erythropoietin (Epo) and the accompanying molecular control mechanism of proliferation and differentiation of erythroid progenitors remains largely unknown. In this study, the effect of Epo on the expression of nuclear oncogenes was investigated in two murine cell lines which respond to the hormone in different ways: ELM-I-1 cells proliferate independently of Epo, but differentiate in response to the hormone, while the growth of DA-1ER cells is absolutely dependent on Epo or interleukin (IL) 3. The cell lines were stimulated with Epo or IL-3, and total RNA was extracted. Then expression of nuclear proto-oncogenes (c-myc, c-fos and c-myb) was analyzed by northern blotting. The change in c-fos expression observed during the first two h following stimulation with either stimulant were common to both cell lines; a rapid and temporary increment. Before stimulation, c-myc and c-myb were strongly expressed in both lines. No apparent change in c-myc expression was observed during the first two h of stimulation, while c-myb expression in ELM-I-1 cells was slightly reduced 1 h after stimulation with Epo but not with IL-3. Three days after stimulation with Epo, but not with IL-3, only ELM-I-1 produced hemoglobin and expressed a lower amount of c-myb mRNA. These data suggest the importance of c-fos in the early signaling system of Epo, and the involvement of c-myb in erythroid differentiation but not in proliferation.     

Analysis of the binding sites of Gd3+ on Ca(2+)-transporting ATPase of the sarcoplasmic reticulum through its effects on fluorescence of tryptophan residues and a covalently attached fluorescent probe N-(1-anilinonaphthyl-4)maleimide.

Interaction of Ca2+ and Gd3+ ions with Ca(2+)-transporting ATPase of the sarcoplasmic reticulum (SR-ATPase) was analyzed. Binding of Ca2+ to the transport site caused an enhancement of intrinsic fluorescence of SR-ATPase. Gd3+ also induced fluorescence enhancement. However, the effects of Ca2+ and Gd3+ were additive rather than competitive, indicating that the Gd(3+)-binding site responsible for this enhancement is distinct from the Ca(2+)-transport site. Gd3+ ions at concentrations higher than 10 microM caused a marked fluorescence quenching, indicating an additional interaction at low-affinity binding sites. Interaction of Ca2+ with the transport site led to a quenching of fluorescence of N-(1-anilinonaphthyl-4)maleimide (ANM) covalently attached at SHN [as defined in Yasuoka-Yabe, K. & Kawakita, M. (1983) J. Biochem. 94, 665-675]. In this case the effects of Ca2+ and Gd3+ were mutually exclusive, indicating that Ca2+ and Gd3+ were competing for the same binding site (i.e. the transport site) to affect ANM fluorescence. Competition between Ca2+ and Gd3+ for the Ca(2+)-transport site was also demonstrated by direct measurement of Ca(2+)-binding using nitrocellulose membrane filters. Affinity of Gd3+ for the Ca(2+)-transport site was a little lower than that of Ca2+. Based on these results it was concluded that Gd3+ has at least three kinds of binding sites on SR-ATPase, namely the Ca(2+)-transport site, the Gd(3+)-specific high-affinity site, and a number of low-affinity sites.(ABSTRACT TRUNCATED AT 250 WORDS)     

Alteration of cell cycle progression in human leukemia cell line (KOPM-28) induced by 12-o-tetradecanoylphorbol-13-acetate

Terminal cell differentiation usually results in an irreversible arrest in the G1 phase of the cell cycle and loss of cell renewal ability. Human promyelocytic leukemia HL-60 cells induced with 12-o-tetradecanoylphorbol-13-acetate (TPA) differentiate into monocytes/macrophages and accumulate in G1. We determined the effect of TPA on the growth kinetics of a human leukemia cell line (KOPM-28), which developed several of the characteristics of megakaryocytes in response to TPA, such as the surface antigen complex IIb/IIIa, platelet peroxidase and polyploidy. Cell growth was immediately and completely inhibited by TPA. Flow cytometric analysis of cellular DNA content revealed a gradual decrease in cells in G1 and an accumulation of cells in G2. These data suggest that TPA prolonged G1 and rapidly arrested the cells in G2. Synchronized cells were utilized to further analyze the rapid G2 arrest. Cells arrested with aphidicolin at the G1/S interphase were released, and the effects of TPA (added at different intervals) on cell cycle progression were examined 14 h after release. The results showed that TPA added at the end of the S phase, as well as at the G1/S interphase incompletely but distinctly arrested cells in G2. Moreover, G2 arrest was observed when TPA was added to cells released from a colcemid-induced G2/M block, suggesting that cells already in G2 were inhibited by TPA from moving through M to G1. Since some cells became multi-nucleated in the course of incubation with TPA, this G2 accumulation may have resulted at least in part from a prolongation of the phase or a transient G2 block. These changes in cell cycle progression induced by TPA may be characteristic of and/or related to megakaryocytic differentiation of hemopoietic precursor cells.     

Affinity labeling of ras oncogene product p21 with guanosine diphospho- and triphosphopyridoxals

Purified v-rasH p21 overproduced in Escherichia coli was treated with guanosine diphospho- and triphosphopyridoxals (GP2- and GP3-PL), affinity labeling reagents specific to a lysyl residue located in the guanine nucleotide binding site. GP2-PL and GP3-PL inhibited [3H]GDP binding to p21 competitively. Incubation of p21 with GP2-PL and GP3-PL followed by reduction with NaBH4 resulted in 40 and 50% loss of [3H]GDP binding activity, respectively, whereas the addition of excess GDP completely protected p21 from the inactivation. The tryptic digest of p21 which was modified with GP2-PL or GP3-PL in the presence or absence of protective GDP and subsequently reduced by NaBH4 was analyzed by reverse phase high performance liquid chromatography. The profile of the effluent monitored by the fluorescence from the pyridoxyl moiety showed the existence of peptides which were specifically labeled only in the absence of GDP. Structural analyses of these peptides allowed us to identify the labeled residue as Lys-16. These results suggest that Lys-16 is located in the guanine nucleotide binding site, close to the beta- or gamma-phosphate group of the nucleotide.     

Selenium Compound Protects Corneal Epithelium against Oxidative Stress

The ocular surface is strongly affected by oxidative stress, and anti-oxidative systems are maintained in corneal epithelial cells and tear fluid. Dry eye is recognized as an oxidative stress-induced disease. Selenium compound eye drops are expected to be a candidate for the treatment of dry eye. We estimated the efficacy of several selenium compounds in the treatment of dry eye using a dry eye rat model. All of the studied selenium compounds were uptaken into corneal epithelial cells in vitro. However, when the selenium compounds were administered as eye drops in the dry eye rat model, most of the selenium compounds did not show effectiveness except for Se-lactoferrin. Se-lactoferrin is a lactoferrin that we prepared that binds selenium instead of iron. Se-lactoferrin eye drops suppressed the up-regulated expression of heme oxygenase-1, cyclooxygenase-2, matrix metallopeptidase-9, and interleukin-6 and also suppressed 8-OHdG production in the cornea induced by surgical removal of the lacrimal glands. Compared with Se-lactoferrin, apolactoferrin eye drops weakly improved dry eye in high dose. The effect of Se-lactoferrin eye drops on dry eye is possibly due to the effect of selenium and also the effect of apolactoferrin. Se-lactoferrin is a candidate for the treatment of dry eye via regulation of oxidative stress in the corneal epithelium.     

Molecular cloning and functional expression of the human Golgi UDP-N-acetylglucosamine transporter.

We have cloned the human UDP-N-acetylglucosamine (UDP-GlcNAc) transporter cDNA, which was recognized through a homology search in the expressed sequence tags database (dbEST) based on its similarity to the human UDP-galactose transporter. The chromosomal location of the UDP-GlcNAc transporter gene was assigned to chromosome 1p21 by fluorescence in situ hybridization (FISH). The transporter was expressed ubiquitously in every tissue so far examined. Expression of the transporter cDNA in CHO-K1 cells in its native and in a C-terminally HA-tagged form indicated that the human UDP-GlcNAc transporter was localized in the Golgi apparatus. The membrane vesicles prepared from yeast cells expressing the cDNA product exhibited UDP-GlcNAc-specific transporting activity. Comparison among UDP-galactose, CMP-sialic acid, and UDP-GlcNAc transporters from several organisms enabled us to identify residues highly conserved among the transporters and residues specific for each group of transporters.     

CHANGES OF THYMIDINE KINASE IN THE DEVELOPING RAT BRAIN

Abstract— Thymidine kinase (ATP: thymidine-5'-phosphotransferase EC 2.7.1.21) of the supernatant fraction from 6-day-old rat brain possessed a pH optimum of 8.0 and required the presence of 5mM-ATP and 2.5 mM-MgCl₂ for maximum activity. The activity was completely inhibited by addition of 1.8 mM-TTP. The enzyme activity was lost if the same supernatant fraction was refrozen and thawed. K_m was 2.8 × 10⁻⁶ M for [6-³H]thymidine.
Following subcellular fractionation of rat brain, the greatest proportion and highest specific activity of thymidine kinase was found in the supernatant fraction. Thymidine kinase activities reached a maximum at 6 days of age and then dropped sharply during maturation. Comparative studies of thymidine kinase activities of cerebrum, cerebellum and the remainder of the brain during growth indicated that the activity in the cerebellum was usually higher than those in the cerebrum and the remainder, and the biggest differences obtained at 6 days after birth corresponded with the peak in cerebellar activity.     

THYMIDINE METABOLISM AND DEOXYRIBONUCLEIC ACID SYNTHESIS IN THE DEVELOPING RAT BRAIN

—In growing rat brain, the specific activity of DNA at 12 h after the subcutaneous injection of [³H]thymidine underwent a sharp rise during the first 6 days of life, dropping just as precipitously by 15 days, thereafter continuing to decrease with increasing age. When [³H]thymidine was given to 6-day-old rats, a considerable amount was taken up immediately into the brain. Thymidine taken up into the acid-soluble fraction was readily phosphorylated to its nucleotides, thymidine mono-, di-, and triphosphate (TMP, TDP and TTP) within only 30 min following injection. The highest specific activity was found in TTP. The incorporation of of [3H]thymidine into DNA took place over a longer period of time after injection.