Suppression of Inhibitory Effects of Copper on Enzymatic Activities by Copper-Binding Substances from Athyrium yokoscense Assayed in vitro

A metal-tolerant fern, Athyrium yokoscense, is capable of growingin highly copper-contaminated soil, but cupric chloride inhibitedthe activities of some enzymes extracted from the fern. Thefunction in the detoxification of copper of two copper-bindingsubstances was investigated by examination of their effectson various enzymes assayed in vitro, i.e. acid phosphatase (orthophosphoric-monoesterphosphohydrolase [acid optimum], EC 3.1.3.2 [EC] ), glucose-6-phosphatedehydrogenase (D-glucose-6-phosphate: NADP⁺ 1-oxidoreductase,EC 1.1.1.49 [EC] ) and isocitrate dehydrogenase (threo-Ds-isocitrate:NADP⁺ oxidoreductase [decarboxylating], EC 1.1.1.42 [EC] ). The twocopper-binding substances, whose apparent molecular weightsare 9.5 kDa and 2 kDa, were previously obtained from the solublecytoplasmic fraction of the fern root. The 9.5-kDa substance,which is a cysteine-rich peptide induced as a result of exposureof the fern to copper, was found to suppress almost entirelythe inhibitory effects of the metal on the enzymes. The suppressoractivity of the peptide was nearly as effective as that of ethylenediaminetetraaceticacid. The 2-kDa substance, which is also found in fern thathas not been exposed to copper, had a more modest suppressoractivity. These results indicate that the 9.5-kDa substancemay contribute to the copper-tolerance of the fern growing incopper-contaminated soil. (Received August 26, 1988; Accepted March 17, 1989)     

Twenty-four-hour variations in subcellular structures of rat type II alveolar epithelial cells

Summary Subcellular structures of type II alveolar epithelial cells in the rat lung were analyzed at six evenly spaced times over 24 h (light period: 06.00 h–18.00 h), using a morphometric technique. The cell volumes were maximal at 16.00 h and minimal at 08.00 h. The volume and surface densities of rough endoplasmic reticulum and mitochondria were low during the light period, and high during the dark period. Morphometric parameters of multivesicular bodies did not significantly fluctuate over 24 h, but they increased from 04.00 h to 08.00 h. The volume densities of lamellar bodies increased from 16.00 h to 20.00 h, and decreased from 00.00 h to 08.00 h. The change in numerical densities of lamellar bodies was inversely correlated to that in the volume densities. As shown by electron microscopy, small lamellar bodies predominated at 08.00 h, larger lamellar bodies increasing at 16.00h. Composite bodies often appeared at 08.00 h and 12.00 h. Type II cells thus appear to fluctuate, showing three phases over 24 h: formation, accumulation and secretion of lamellar bodies. In particular, it is noteworthy that the accumulation stage occurs during the resting phase of the rat, whereas the secretion stage occurs during its body-active phase.     

Fertilization Potential of the Medaka Egg

Fertilization of the medaka egg in 10% Ringer's solution generates a depolarization of 4 mV just before the appearance of a characteristically longer hyperpolarization (25). The depolarization appears to be the result of a nonspecific leak triggered by sperm stimulation and the amplitude of the depolarization is thought to be independent of [Ca²⁺]_o (25). We have investigated the ionic dependence of this depolarization. An initial small depolarization (3–4 mV; duration, 5–8 sec) is followed by a rising phase of a spike-like depolarization in the range of 10–60 mV. The amplitude of this spike-like depolarization is propodional to log [Ca²⁺], ranging from 0.33–18 mM. Calcium antagonists, e.g. 10 mM cobalt or 10 μg/ml verapamil in 10% Ringer do not block the depolarization in the presence of 1.1 mM CaCl₂.     

Reverse Changes in Plasma Membrane Properties upon Deacclimation of Mulberry Trees (Morus bombysis Koidz.)

To gain more insight into the relation between plasma membranechanges and cold hardiness in mulberry trees (Morus bombysisKoidz. cv Goroji), biochemical and biophysical changes in theplasma membrane were studied during cold deacclimation in spring.The majority of the changes in the plasma membranes that occurredduring the cold acclimation process in the fall/winter werereversed following deacclimation in the spring. Significantdecreases in phospholipid content, degree of unsaturation inphospholipid fatty acids, and membrane fluidity were observedin the plasma membranes during cold deacclimation. The sterolto phospholipid ratio increased with decreasing cold hardiness.Reverse changes were also detected in the majority of proteinand glycoprotein components. These reversible changes in theplasma membranes are considered to be involved in the mechanismof cold hardiness of plants. ¹Contribution No. 2766 from the Institute of Low TemperatureScience. (Received July 10, 1985; Accepted October 25, 1985)     

Structural characterization of a novel mono-sulfated gangliotriaosylceramide containing a 3-O-sulfatedN-acetylgalactosamine from rat kidney

A novel mono-sulfated glycosphingolipid based on the gangliotriaose core structure was isolated from rat kidney. The isolation procedure involved extraction of lipids with chloroform/methanol, mild alkaline methanolysis, column chromatographies with anion exchangers and silica beads. The structure was characterized by compositional analysis, FTIR spectroscopy, methylation analysis,¹H-NMR spectroscopy and negative-ion liquid secondary ion mass spectrometry (LSIMS) using the intact glycolipid and its desulfation product. The two dimensional chemical shift correlated spectroscopy provided information on the sugar sequence as well as anomeric configurations, and indicated the presence of a 3-O-sulfatedN-acetylgalactosamine within the molecule. Negative-ion LSIMS with high- and low-energy collision-induced dissociation defined the sugar sequence and ceramide composition, confirming the presence of a sulfatedN-acetylgalactosamine at the non-reducing terminus. From these results, the complete structure was proposed to be HSO₃-3GalNAc1-4Gal1-4Glc1-1Cer (Gg₃Cer III³-sulfate, SM2b). Abbreviations: Abbreviations for sulfated glycolipids [17] follow the modifications of the nomenclature system of Svennerholm for gangliosides [37], and the designation of the other glycosphingolipids follows the IUPAC-IUB recommendations [38]. Cer, ceramide; LacCer, lactosylceramide, Gal1-4Glc1-1Cer; Gg₃Cer, gangliotriaosylceramide, GalNAc1-4Gal1-4Glc1-1Cer; Gg₄Cer, gangliotetraosylceramide, Gal1-3GalNAc1-4Gal1-4Glc1-1Cer; iGb₄Cer, isoglobotetraosylceramide, GalNAc1-3Gal1-3Gal1-4Glc1-1Cer; Gb₄Cer, globotetraosylceramide, GalNAc1-3Gal1-4Gal1-4Glc1-1Cer; SM4s, galactosylceramide sulfate, GalCer I³-sulfate; SM3, lactosylceramide sulfate, LacCer II³-sulfate; SM2a, Gg₃Cer II³-sulfate; SM2b, Gg₃Cer III³-sulfate; SB2, Gg₃Cer II³,III³-bis-sulfate; SM1a, Gg₄Cer II³-sulfate; SM1b, Gg₄Cer IV³-sulfate; SB1a, Gg₄Cer II³,IV³-bissulfate; GLC, gas-liquid chromatography; GC-MS, gas chromatography-mass spectrometry; DQF, double quantum filtered; COSY, chemical-shift-correlated spectroscopy; LSIMS, liquid secondary ion mass spectrometry; CID, collision-induced dissociation; MS/MS, tandem mass spectrometry.     

Expression and localization of Lewisx glycolipids and GD1a ganglioside in human glioma cells

We analysed the glycolipid composition of glioma cells (N-370 FG cells), which are derived from a culture of transformed human fetal glial cells. The neutral and acidic glycolipid fractions were isolated by column chromatography on DEAE-Sephadex and analysed by high-performance thin-layer chromatography (HPTLC). The neutral glycolipid fraction contained 1.6 µg of lipid-bound glucose/galactose per mg protein and consisted of GlcCer (11.4% of total neutral glycolipids), GalCer (21.5%), LacCer (21.4%), Gb4 (21.1%), and three unknown neutral glycolipids (23%). These unknown glycolipids were characterized as Lewis^x (fucosylneolactonorpentaosyl ceramide; Le^x), difucosylneolactonorhexaosyl ceramide (dimeric Le^x), and neolactonorhexaosyl ceramide (nLc6) by an HPTLC-overlay method for glycolipids using specific mouse anti-glycolipid antibodies against glycolipid and/or liquid-secondary ion (LSI) mass spectrometry. The ganglioside fraction contained 0.6 µg of lipid-bound sialic acid per mg protein with GD1a as the predominant ganglioside species (83% of the total gangliosides) and GM3, GM2, and GM1 as minor components. Trace amounts of sialyl-Le^x and the complex type of sialyl-Le^x derivatives were also present. Immunocytochemical studies revealed that GD1a and GalCer were primarily localized on the surface of cell bodies. Interestingly, Le^x glycolipids and sialyl-Le^x were localized not only on the cell bodies but also on short cell processes. Especially, sialyl-Le^x glycolipid was located on the tip of fine cellular processes. The unique localization of the Le^x glycolipids suggests that they may be involved in cellular differentiation and initiation of cellular growth in this cell line.     

Biochemical systematics of Japanese fireflies of the subfamily Luciolinae and their flash communication systems

Japanese fireflies of the subfamily Luciolinae are biochemically analyzed using 13 allozymes, and the phylogenetic relationships obtained from this analysis are compared with their flash communication systems. As a result, the Japanese Luciolinae can be divided into three groups.Hotaria parvula andH. tsushimana together withLuciola yayeyamana andL. kuroiwae from the first group, and they use the same communication system.L. lateralis, Curtos okinawana, andC. costipennis make up the second group, and their communication systems are also the same.L. cruciata makes up the last one, and its communication system is different from the other fireflies of Luciolinae. Therefore, their taxonomical arrangement and communication systems are not congruent. However, the genetic similarity deduced by allozymic analysis of the members of the Japanese Luciolinae is highly consistent with their flash communication systems.     

Effects of ligation on ethanol-induced Balbiani ring puffing in salivary glands of Chironomus

Treatment of Chironomus larvae with dilute (0.5%–1.0%) ethanol results in puffing changes similar to those obtained with galactose in the Balbiani rings (BRs) of the salivary gland chromosomes. A shift in the relative size of BR1 and BR2 in chromosome 4 of C. pallidivittatus or C. tentans was observed within 1–2 days after ethanol treatment. The exceptional Balbiani ring, BR6 in chromosome 3, began to appear within 1 day after ethanol treatment of C. pallidivittatus and was fully developed after 3–4 days. Prepupae appeared to be refractory to the treatment. To localize possible controls of BR puffing in Chironomus, ligatures were made at various positions along the thorax and the anterior abdominal segments of the ethanoltreated larvae. In surviving larvae, ligated anterior to the brain or posterior to the salivary glands, induction of BR6 could be detected. In contrast, little or no BR6 puff induction was found in animals ligated in the middle of the second segment approximately between the brain and the salivary glands. No shift in the BR1/BR2 relation occurred with any of the ligations combined with ethanol treatment.     

A retarded rate of DNA replication and normal level of DNA repair in Werner's syndrome fibroblasts in culture

We investigated the cloning efficiency, DNA repair, and the rate of DNA replication in the skin fibroblasts from patients with Werner's syndrome (WS) of an autosomal recessive premature aging disease. Five WS strains exhibited normal levels of sensitivity toward X-ray and UV killings and repair of X-ray induced single strand breaks of DNA (rejoining) and UV damage to DNA (unscheduled DNA synthesis). The sedimentation of newly synthesizing DNA in alkaline sucrose gradients demonstrated a characteristic feature that only the elongation rate of DNA chains, estimated by the molecular weight increase, was significantly slower during early passages in WS cells than in normal Hayflick Phase II fibroblasts. In addition, plating efficiencies as well as the replicative potentials of five WS strains were more limited than those of normal cells under the identical culture conditions. It seems therefore that at least in the WS cells tested, the slow rate of DNA replication may be more related to the shortened lifespan and enhanced cell death, as manifestation of premature senescence at the cellular level, than be the DNA repair ability.