A simple method for preparing physiologically active mitochondria from plant leaves rich in oils and phenolics

Amberlite XAD-7, a nonionic polyacrylate adsorbent, was found to be a very effective protectant for isolating mitochondria from tissues rich in oils and phenolics. Physiologically active, well-coupled mitochondria were successfully prepared from young green leaf tissues of citrus, apple, pear and tobacco.Abbreviations DNP 2,4-dinitrophenol - CCCP carbonyl cyanide m-chlorophenylhydrazone - BSA bovine serum albumin - PVP polyvinylpyrrolidone     

Spatial relationship among individuals of the Japanese lacertidTakydromus tachydromoides (Sauria,Lacertidae)

The home range ofTakydromus tachydromoides was studied in a grassland area from April 1977 to November 1978. The mean size of home range did not differ markedly between sexes; 136.5 m² for males and 130.8 m² for females. Home ranges of adults overlapped greatly in each sex, and the lizard was considered to be non-territorial. Individuals showed return movement to a definite area (sleeping site) within the home range, and the home range did not shift within a year or between years. Characteristics of the home range of this grassland-inhabiting lizard were discussed in relation to resource abundance and predation pressure.     

Detection of carcinogen-induced DNA breaks by nick translation in permeable cells

A nick-translation reaction with $\text{E. coli}$ DNA polymerase I (pol. I) was used to detect $\text{in situ}$ DNA breaks produced by chemical carcinogens. Normal human fibroblasts treated with N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) in various doses were permeabilized with lysolecithin, and were nick translated in the presence of [³H]dCTP and pol. I. The radioactivity incorporated increased with MNNG concentration, and was directly proportional to the poly(ADP-ribose) synthetase activity. Other DNA-damaging agents such as bleomycin or 4-nitroquinoline 1-oxide also caused the nick translation rate to increase. When MNNG-treated cells were cultured in fresh medium containing no MNNG, the increase in the rate of nick translation in permeable cells became less and this decrease was abolished by addition of aphidicolin or cytosine arabinoside. The nick translation method described here may be a useful means for estimating intrinsic DNA breaks in cells treated with carcinogens.     

Diacylglycerol kinase from pig brain. Purification and phospholipid dependencies

Diacylglycerol kinase (EC 2.7.1.-) was purified 1,650-fold from pig brain cytosol. The purified enzyme showed a single protein band on polyacrylamide gel electrophoresis in the presence and absence of sodium dodecyl sulfate. The molecular weight of the kinase was estimated to be 78,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A similar value (76,000) was obtained by Sephadex G-150 gel filtration. The activity of the purified enzyme was markedly enhanced by either deoxycholate or phospholipids. The extent of activation by phospholipids was in the order of phosphatidylcholine greater than lysophosphatidylcholine greater than phosphatidylethanolamine approximately equal to phosphatidylserine greater than sphingomyelin. Other phospholipids and unsaturated fatty acids were ineffective. Phosphatidylcholines from egg yolk and pig brain, and dioleoyl phosphatidylcholine were similarly effective. Saturated phosphatidylcholines with acyl chain lengths shorter than palmitate also gave a considerable activation. The activity with phosphatidylcholine was from 1.5- to 2.5-fold higher than that measured with deoxycholate. A very small amount of phosphatidylinositol or phosphatidylglycerol potently inhibited the phosphatidylcholine-dependent (but not deoxycholate-dependent) kinase activity. The inhibition by phosphatidylinositol was varied according to its molar ratio to phosphatidylcholine. As little as about 2.5 mol per cent of phosphatidylinositol resulted in 50% inhibition of the phosphatidylcholine-dependent kinase activity. The deoxycholate- and phosphatidylcholine-dependent kinase activities showed almost the same Km values for the substrates. In both cases, the apparent Km values for ATP and diacylglycerol were 300 microM and about 60 microM, respectively. The kinase required Mg2+ for its activity. When compared to deoxycholate, phosphatidylcholine was more effective at higher Mg2+ concentrations. The deoxycholate-dependent activity showed a broad pH optimum at around 8.0, whereas the phosphatidylcholine-dependent activity formed a clear peak at pH 7.4.     

Histochemical application of mild alkaline hydrolysis for selective elimination of O-glycosidically linked glycoproteins

A new technique to eliminate O-glycosidically linked glycoprotein (mucin-type glycoprotein) selectively has been developed. Composite paraffin sections were collodionized before and after alkaline treatment with 0.5 M NaOH in 70% ethanol; the effect of this procedure on mucosubstances was examined using the periodic acid-Schiff reaction. Exposure to alkaline hydrolysis for 72 to 144 hours at 4 C led to a complete loss of periodic acid-Schiff reactivity of epithelial mucins in rat sublingual gland, stomach and small intestine, but that of fuzzy coat, thyroid colloid, collagen fibers and tracheal cartilage was well preserved. These results agreed fairly well with biochemical findings. The present study also revealed that materials prepared by freeze-substitution provided the most satisfactory results.     

Ferredoxins in Developing Spinach Cotyledons: The Presence of Two Molecular Species

An antibody for ferredoxin was used to investigate the developmentof ferredoxin during the greening of spinach cotyledons. Ferredoxinwas present in 8-day-old etiolated cotyledons and increasedwith illumination, which means that the synthesis of ferredoxinwas both light dependent and independent. The ferredoxin purified from etiolated cotyledons, greeningcotyledons, and mature leaves was a mixture of two chemicallydistinct molecular species; ferredoxin I and II. The relativecontents of these two species varied with the stage of developmentand the conditions used. Ferredoxin I was identical with that isolated previously asvalidated by its amino acid sequence [Matsubara and Sasaki (1968)J. Biol. Chem. 243: 1732]. The complete amino acid sequenceof the second component, ferredoxin II, was determined as well.It was composed of 97 amino acid residues and differed fromferredoxin I by 25 residues. (Received October 16, 1982; Accepted December 14, 1982)     

Benzyladenine-Enhanced Growth of Attached Young Bean Leaves. Studies with Inhibitors of Nucleic Acid and Protein Synthesis

Benzyladenine (BA) stimulated division but not expansion ofmesophyll cells and repressed chlorophyll accumulation in attachedyoung bean leaves. Even in the presence of fluorodeoxyuridine(FUdR) or mitomycin C which causes complete suppression of BA-inducedincrease in DNA content, BA increased RNA and protein contentsand fresh weight, but decreased chlorophyll accumulation. Moreover,BA n the presence of FUdR induced marked cell expansion. Inthe presence of a-amanitin (AM), BA did not produce any changein DNA content, fresh weight or cell size. All of the BA effectswere observed even in the presence of fluorouracil (FU) plusthymidine (TdR). AM and cycloheximide added 0–12 h effectively inhibitedBA-stimulated cell division but showed no effect if added at18 h. FU plus TdR added 0–18 h had almostno effect onthe cell number at 24 h. These results indicate that BA stimulates the mRNA synthesisnecessary for induction of cell division, and that the synthesisof cytoplasmic rRNA is not always necessary for BA-stimulatedcell division, and moreover, that BA stimulates expansion growthof cells in which DNA synthesis is suppressed. (Received August 16, 1982; Accepted March 31, 1983)     

Separation of the Effects of Photoperiod and Hormones on Sex Expression in Cucumber

The effects of photoperiod on the sex expression of cucumberflowers were examined in relation to the endogenous GA and ethyleneproduction with short day (SD)- and long day (LD)-sensitivecultivars under controlled environmental conditions. Growth of plants was greatly reduced under LD in both typesof cultivars. Reflecting this, GA activity was greater and ethyleneevolution less in plants grown under SD than in those underLD. However, the number of pistillate flowers increased underSD and LD in SD- and LD-sensitive cultivar, respectively, indicatingthat a change in sex expression did not coincide with hormonalchanges in the SD-sensitive cultivar. Thus, the effect of photoperiodon the sex expression was found to be separate, at least inpart, from the effect of GA or ethylene in cucumber. (Received September 16, 1982; Accepted December 7, 1982)     

An inducible lactone hydrolase yielding 2,5-diphenyl-3-hydroxy-4-oxo-2-hexendioic acid from pulvinic acid

The metabolism of vulpinic acid by an unclassified soil micro-organism was studied. A new compound, 2,5-diphenyl-3-hydroxy-4-oxo-2-hexendioic acid (DHOHA) was isolated from the reaction mixture of a cell-free preparation and pulvinic acid. The existence of a hydrolase which catalyses the conversion of vulpinic acid to pulvinic acid was detected in cell-free preparation, and an inducible lactone hydrolase capable of converting pulvinic acid to DHOHA was purified 130-fold and characterized. This enzyme had a MW of ca 34 000, a K_m for pulvinic acid at pH optimum (pH 7.0) less than 10 ^{? 6} M, pI = 5.0, and was inhibited by p-chloromercuriphenylsulfonate and diethylpyrocarbonate. The enzyme was highly specific for pulvinic acid. The initial degradative steps proposed for this organism are vulpinic acid → pulvinic acid → DHOHA.     

Expression of human immunoglobulin E epsilon chain cDNA in E. coli.

Using the cDNA of human epsilon chain, three expression plasmids that code directly the constant portion of the epsilon chain (C epsilon 1-C epsilon 4, C epsilon 2-C epsilon 4 and C epsilon 3-C epsilon 4 domains) were constructed. These epsilon chain peptides were synthesized in E. coli under the control of the trp promoter-operator. The bacterially produced peptides have the antigenicity of human epsilon chain and gave the molecular weights equal to the values calculated from the amino acid sequence of the constructed plasmids.