Biochemical Characterization of Secreted Proteases During Growth in Tetrahymena pyriformis WH-14: Comparison of Extracellular with Intracellular Proteases

Tetrahymena pyriformis strain WH-14 secreted large quantities of intracellular proteases into its culture medium during growth. Extracellular enzymes were purified to homogeneity from cell-free medium by ammonium sulfate precipitation, CM-Sephadex column chromatography, gel filtration, and DEAE-cellulose column chromatography. The DEAE-cellulose eluates were separated into four peaks (P-I, P-II, P-III, and P-IV), each of which exhibited a different specific activity toward azocasein and α-N-benzoyl-DL-arginine-ρ-nitroanilide (Bz-Arg-Nan). These four forms of the protease showed similarity in amino acid composition, molecular weight (21,000–24,000), and antigenic reactivity. They had pH optima at neutral range. P-I showed the highest specificity to azocasein whereas P-IV was most effective toward the synthetic substrates. The Km values for hydrolysis of Bz-Arg-Nan were 2.4, 1.6, 1.3, and 1.4 mM for P-I, P-II. P-III, and P-IV, respectively, and the corresponding Kcat/Km values were 5.0, 9.4, 28.5, and 114.3 S^-1.M^-1. These properties of secreted proteases were compared with those of intracellular proteases purified by the same procedure except for the initial Triton X-100 extraction. There were similarities in specific activity toward two substrates, molecular weight, Km, pH optima, and antigenic reactivity between the proteases from two sources, providing evidence that the intracellular proteases may be secreted into the extracellular medium without modification.     

Histological Detection of Chicken δ-Crystallin DNA Sequences Introduced into Mouse Teratocarcinoma Cell Lines

In situ hybridization techniques to detect specific DNA sequences in histological sections were developed for the purpose of analyzing experimental chimeras produced by combination of mouse teratocarcinoma (TCC) cells stably carrying chicken δ-crystallin DNA sequences and normal mouse embryos. Various hybridization conditions for detection of exogenous DNA sequences were compared in samples of solid tumors of TCC lines. Of the conditions examined, denaturation of DNA in alkali and hybridization at 68°C in 6x SSC in the presence of dextran sulphate was the best for detecting δ-crystallin DNA sequences. With ³H-labelled probe under these conditions, virtually all nuclei containing more than 100 copies of chicken δ-crystallin sequences were labelled sufficiently to be distinguishable from nuclei without chicken sequences. This technique could be applied to other experimental chimeras in which specific DNA sequences can be used as markers of certain cell lineages.     

K-Casein Suppresses Melanogenesis in Cultured Pigment Cells

The effects of bovine milk proteins on melanogenesis in B16 cells were examined. Both whey protein isolate and casein exhibited depigmenting properties. Among the major protein components of milk—including β-lactoglobulin, α-lactalbumin, α-, β-, and k-casein—only K-casein exhibited the depigmenting effect. However, the carboxyl terminal peptide of K-casein, glycomacropeptide, did not show this activity. Also, K-casein promoted the proliferation of the cells and inhibited the activity of tyrosinase in the cells. These results indicate that K-casein acts as a melanogenesis-suppressing modulator.     

Persistent Nucleoli in Early Postimplantation of Rat Embryos

Ultrastructural changes of the nucleolus in mitotic embryonic ectodermal cells of 7 1/2-day and 7 2/3-day rat embryos were examined. It was found that the nucleolus was broken down into small fragments during late prophase and metaphase, and that some of these fragments persisted in the cytoplasm of telophase cell (persistent nucleoli). No interphase embryonic ectodermal cells contained persistent nucleoli. Persistent nucleoli were also found in telophase cells of extraembryonic ectoderm, extraembryonic visceral endoderm and parietal endoderm of the embryos, but they disappeared in interphase cells. Persistent nucleoli in telophase cells tended to decrease in size with embryonic age, and they had almost completely disappeared in neuroectodermal cells of the telencephalon in 14 1/2-day embryos. They were concluded to be remnants of disappearing nucleoli in embryonic cells that were cycling too rapidly to permit their nucleoli to disappear completely.     

RAPID ALDOSTERONE-INDUCED CELL VOLUME INCREASE OF ENDOTHELIAL CELLS MEASURED BY THE ATOMIC FORCE MICROSCOPE

Atomic force microscopy (AFM) is a useful technique for imaging the surface of living cells in three dimensions. The authors applied AFM to obtain morphological information of individual cultured endothelial cells of bovine aorta under stationary and strain conditions and to simultaneously measure changes in cell volume in response to aldosterone. This mineralocorticoid hormone is known to have acute, non-genomic effects on intracellular pH, intracellular electrolytes and inositol-1,4,5-triphosphate production. In this study whether endothelial cells under tension change their volume in response to aldosterone was tested. Such changes were already shown in human leukocytes measured by Coulter counter. In contrast to leukocytes that are more or less spherical and live in suspension, endothelial cells exhibit a complex morphology and adhere to a substrate. Thus, measurements of discrete cell volume changes in endothelial cells under physiological condition is only feasible with more sophisticated techniques. By using AFM we could precisely measure the absolute cell volume of individual living endothelial cells. Before the addition of aldosterone the cell volume of mechanically stressed endothelial cells mimicking arterial blood pressure was 1827±172fl. Cell volume was found to increase by 28% 5min after hormone exposure. Twenty-five minutes later cell volume was back to normal despite the continuous presence of aldosterone in the medium. Amiloride, a blocker of the plasma membrane Na⁺/H⁺exchanger prevented the initial aldosterone-induced volume increase. Taken together, AFM disclosed a transient swelling of endothelial cells induced by the activation of an aldosterone sensitive plasma membrane Na⁺/H⁺exchanger.     

Purification and Characterization of Phospholipase C Preferentially Hydrolysing Phosphatidylcholine in Tetrahymena Membranes

A phospholipase C (PLC) activity that preferentially hydrolyses phosphatidylcholine to diacylglycerol and phosphorylcholine was found to be present in Tetrahymena pyriformis, strain W and most of its activity was recovered in the membrane fraction. This enzyme was extracted with 1% Triton X-100 from the membrane fraction and purified to apparent homogeneity by sequential chromatographies on Fast Q-Sepharose, hydroxyapatite HCA-100S, Mono Q and Superose 12 gel filtration columns. The purified enzyme had specific activity of 2083 nmol of diacylglycerol released/mg of protein/min for dipalmitoylphosphatidylcholine hydrolysis. Its apparent molecular mass was 128 kDa as determined by SDS-polyacrylamide gel electrophoresis and was 127 kDa by gel filtration chromatography, indicating that the enzyme is present in a monomeric form. The enzyme exhibited an optimum pH 7.0 and the apparent K_m value was determined to be 166 μM for dipalmitoylphosphatidylcholine. A marked increase was observed in phosphatidylcholine hydrolytic activity in the presence of 0.05% (1.2 mM) deoxycholate. Ca²⁺ but not Mg²⁺ enhanced the activity at a concentration of 2 mM. This purified phospholipase C exhibited a preferential hydrolytic activity for phosphatidylcholine but much less activity was observed for phosphatidylinositol (～ 9%) and phosphatidylethanolamine (～ 2%).     

ULTRASTRUCTURAL CHANGES IN EMBRYONIC ECTODERMAL CELLS OF THE NEURULATING RAT EMBRYO

Embryonic ectodermal cells of rat embryos were examined by light and electron microscopy during the early stage of neurulation. Before the onset of neurulation (day 9–6 hr embryos), the cells underwent certain characteristic ultrastructural changes; that is, apical cytoplasmic protrusions and free spherules appeared, numerous vacuoles were formed in the cytoplasm, mitochondria showed ballooning, and the endoplasmic reticulum became dilated. The amniotic cells derived from the embryonic ectoderm exhibited the same ultrastructural changes, but those from the extraembryonic mesoderm did not. Embryonic mesodermal cells and neuroectodermal cells also did not show these changes. In the middle stage of neurulation (day 9–12 hr embryos), the embryonic ectodermal cells and the amniotic cells derived from the embryonic ectoderm assumed a flat squamous shape. None of the ultrastructural changes observed in day 9–6 hr embryos were noted in these cells. The functional significance of the production of apical cytoplasmic protrusions and free spherules in the embryonic ectodermal cells and amniotic cells is discussed in relation to similar phenomena reported to occur in other cell types.     

A Thermostable Acid α-Glucosidase from Tetrahymena thermophila: Purification and Characterization

An acid α-glucosidase (EC 3.2.1.20) was purified to homogeneity from the culture medium of Tetrahymena thermophila CU 399. Its general molecular, catalytic and immunological properties were compared to those of the T. pyriformis W enzyme. The enzyme from T. thermophila was a 105-kD monomer and the N-terminus (25 amino acid residues) displayed some homology with that of T. pyriformis enzyme. The purified enzyme was most active at 56° C and showed resistance to thermal inactivation. The acid α-glucosidase appears to have α-1,6-glucosidase as well as α-1,4-glucosidase activity. The Km values determined with p-nitrophenyl-α-glucopyranoside, maltose, isomaltose and glycogen were 0.7 mM, 2.5 mM, 28.5 mM and 18.5 mg/ml, respectively. The enzyme was antigenically distinct from T. pyriformis acid α-glucosidase.     

Oviposition site selection by a lepidopteran leafminer in response to heterogeneity of leaf surface conditions: structural traits and microclimates

1. Leafminer larvae are sedentary and make feeding tracks called mines. Their spatial distribution in trees affects their growth and survival through interaction with the heterogeneity of environments, such as leaf traits and microclimate. Lepidopteran leafminers that mine lower leaf surfaces have shown evolutionary radiation, suggesting that lower surfaces improve leafminer performance. 2. The lepidopteran multivoltine leafminer Phyllocnistis sp. Zeller (Gracirallidae: Phyllocnistinae) uses the Japanese privet Ligustrum japonicum Thunb. (Oleaceae). It mines only the lower‐surface epidermal layer of primary shoot leaves early in the occurrence season, but once lammas shoots appear, which happens in seasons other than spring, it preferentially uses the lower surface, but also uses the upper surface of the leaves. This study examined whether selection of oviposition sites was associated with the structural traits and microclimate of the leaf surface. 3. The shift of oviposition site from primary to lammas shoot leaves followed increasing hardness and epidermal cell wall thickness of primary shoot leaves during leaf development, and mine initiation rates decreased below 20% after oviposition on mature primary shoot leaves. Preference for the lower surface was related to the thinner cuticle. However, the thinner cuticle of the upper surface on lammas shoot leaves allowed Phyllocnistis sp. to expand its mining sites to both surfaces with no decrease in mine initiation and emergence rates. 4. Microclimates (leaf surface and mine temperatures) did not differ between upper and lower surfaces, suggesting that microclimate did not affect oviposition site selection by Phyllocnistis sp. These results suggest that the adaptive radiation of lower‐surface mining may have been influenced by the leaf surface characteristics.     

Presence of two glycolytic gene clusters in a severe pathogenic line of Candidatus Phytoplasma asteris

Phytoplasmas are plant-pathogenic bacteria that are associated with numerous plant diseases. We have previously reported the complete genomic sequence of Candidatus Phytoplasma asteris, OY strain, OY-M line, which causes mild symptoms. The phytoplasma genome lacks several important metabolic genes, implying that the consumption of metabolites by phytoplasmas in plants may cause disease symptoms. Here we show that the approximately 30-kb region including the glycolytic genes was tandemly duplicated in the genome of OY-W phytoplasma, which causes severe symptoms. Almost duplicated genes became pseudogenes by frameshift and stop-codon mutations, probably because of their functional redundancy. However, five kinds of genes, including two glycolytic genes, remained full-length ORFs, suggesting that it is advantageous for the phytoplasma to retain these genes in its lifestyle. In particular, 6-phosphofructokinase is known as a rate-limiting enzyme of glycolysis, implying that the different number of glycolytic genes between OY-W and OY-M may influence their respective glycolysis activities. We previously reported that the phytoplasma population of OY-W was higher than that of OY-M in their infected plants. Taking this result into account, the higher consumption of the carbon source may affect the growth rate of phytoplasmas and also may directly or indirectly cause more severe symptoms.