Small circular DNA complexes in eucaryotic cells

A small number of eucaryotic cells (100 to 1000 cells) were pressed by mica sheet; then the extruded contents were adsorbed on mica and processed for electron microscopy. In the absence of divalent cation, small polydisperse circular DNA molecules bound to proteins or membrane material were preferentially adsorbed. The small circular DNA complexes have been found in every eucaryotic cell, primary lymphoid tissue cells of bursa and thymus, primary cell lines of retina and liver, and established cultured cell lines of embryonal teratocarcinoma, F9 and PCC3, HeLa and 3T6. Size distribution of these DNA complexes varies, depending on the cell source. The circles less than 1 μm in contour length predominate in cultured cell lines and the larger ones in primary cell lines and cells in situ. Polydisperse covalently closed circular DNAs were recovered from thymus lymphocytes by the conventional dye-CsCl buoyant density method. Their size distribution was similar to that of the small circular DNA complexes detected by the mica-press-adsorption method. They are present in several tens to hundreds of copies per cell representing, at a maximum, 0.02% of the total cellular DNA. The possibility that small circular DNA complexes may result from gene rearrangement as well as from replicon “misfiring” (A. Varshavsky, 1981, Proc. Nat. Acad. Sci. USA 78, 3673–3677) are discussed.     

The concentrations and thermodynamic activities of cations in intact Bryopsis chloroplasts

The internal cation levels of chloroplasts isolated from a green sea alga, Bryopsis maxima, were studied. Atomic absorption spectroscopy, combined with the determination of the sorbitol-impermeable and water-permeable spaces, revealed that chloroplasts contain an extremely high concentration of K⁺ and high levels of Na⁺, Mg²⁺ and Ca²⁺. A method was developed to estimate the thermodynamic activities of monovalent and divalent cations present in chloroplasts. pH changes induced by the addition of an ionophore (plus an H⁺ carrier), which makes the outer limiting membranes of chloroplasts permeable to both a cation and H⁺, were determined. Provided that the external pH was set equal to the internal pH, the internal concentration of the cation was estimated by determining the external cation concentration which gave rise to no electrochemical potential difference of the cation and hence no pH change on addition of the ionophore. The internal pH was determined by measuring distributions of radioactive methylamine and 5,5-dimethyloxazolidine-2,4-dione between the chloroplast and medium (Heldt, H.W., Werdan, K., Milovancev, M. and Geller, G. (1973) Biochim. Biophys. Acta 314, 224–241). The internal pH was also estimated by measuring pH changes caused by the disruption of the outer limiting membrane with Triton X-100. The results indicate that a significant part of the monovalent cations and most of the divalent cations are attracted into a diffuse layer adjacent to the negatively charged surfaces of membranes and proteins, or form complexes with organic and inorganic compounds present in the intact chloroplasts.     

Synthesis of 2,2,2-trichloroethyl 3,6-di-O-benzyl-2-deoxy-2-phthalimido-β-d-glucopyranoside,and its reaction with glycosyl halides.

2,2,2-trichloroethyl 3,6-di-O-benzyl-2-deoxy-2-phthalimido-β-d-glucopyranoside (9) was synthesized in 6 steps from the readily available 1,3,4,6-tetra-O-acetyl-2-deoxy-2-phthalimido-β-d-glucopyranose in 25% overall yield by employing the stannyl method for the regioselective activation of hydroxyl groups. Dibenzyl ether 9 was then glycosylated with appropriate glycosyl donors to afford lactosamine and chitobiose derivatives in good yield.     

Synthesis of 3,6-di-O-acetyl-2-deoxy-2-phthalimido-4-O-(2,3,4,6-tetra-O-acetyl-β-d-galactopyranosyl)-β-d-glucopyranosyl chloride

A lactosaminyl donor, 3,6-di-O-acetyl-2-deoxy-2-phthalimido-4-O-(2,3,4,6-tetra-O-acetyl-β-d-galactopyranosyl)-β-d- glucopyranosyl chloride, was synthesized in 10 steps, starting from 1,3,4,6-tetra-O-acetyl-2-deoxy-2-phthalimido-β-d-glucopyranose. Benzyl 3,6-di-O-benzyl-2-deoxy-2-phthalimido-β-d-glucopyranoside was prepared by regioselective benzylation at the primary hydroxyl group by the stannyl method, and was used as a key intermediate.

     

Disappearance of calcium-induced phase separation in phosphatidylserine-phosphatidylcholine membranes caused by protonation and by electric current

Disappearance of Ca²⁺-induced phase separation in phosphatidylserine-phosphatidylcholine membranes has been studied under several conditions by monitoring electron spin resonance spectrum of spin-labeled phosphatidylcholine. The membranes were prepared in Millipore filters. Electron micrographs of the preparations showed formation of multilayered structures lined on the pore surface. The phase separation was disappeared when the membrane was soaked in non-buffered salt solution (100 ml KCl, pH 5.5). It was markedly contrasting that when the bathing salt solution was buffered no disappearance was observed. Disappearance of the phase separation was also observed when the Ca²⁺-treated membrane was transferred to acidic salt solutions ($\text{?}\text{pH 2.5}$) or to low ionic strength media ($\text{? 10}\text{mM}$) buffered at pH 5.5, and then to the buffered salt solution (100 mM KCl, pH 5.5). These are due to replacement of Ca²⁺ by proton, proton-induced separation, followed by disappearance of the phase separation inthe buffered salt solution. Biological significance of the competition between Ca²⁺ and proton for the phase separation or domain formation in the membranes was emphasized.     

Concanavalin A-peroxidase-diaminobenzidine (Con A-PO-DAB)-alcian blue (AB)

Summary A method has been established for the dual staining of complex carbohydrates in light microscopy. It is a combined concanavalin A-peroxidase-diaminobenzidine (Con A-PO-DAB)-alcian blue (AB) (pH 2.5) method, and with this method it is possible to color -D-glucosyl and -D-mannosyl residues and acidic groupings of complex carbohydrates in tissues brown and blue respectively. Histochemical experiments using histological sections with reactive complex carbohydrates and casein films containing carbohydrates of known chemical structure have substantiated the validity of the above significance of the dual staining. Thus, the present dual staining method is a reliable one and a new addition to a series of dual staining techniques hitherto employed in the light microscopic histochemistry of complex carbohydrates.This investigation was supported in part by a Grant-in-Aid from the Japanese Education Ministry (1975)     

Metabolism of PG in man: Effect of furosemide on the excretion of the main metabolite of PG F2α

The excretion rates of main urinary metabolite of PG F₂α were measured radioimmunologically in 4 healthy persons and in 13 essential hypertensives. The resting values were 9.3±0.73 in the former and 10.4±2.17 ng/min in the latter. There was no significant differences between them. The excretion of the metabolite decresed prominently after the administration of furosemide. The percent decrease was 57% in healthy persons and 70% in essential hypertension. The percent result supports that furosemide inhibit the catabolism of PG F₂α.     

Effect of light on nonphotosynthetic microorganisms. II. Photochromogenicity in genus Streptomyces.

The effect of light on the pigmentation of various strains belonging to the genus Streptomyces was investigated. It was revealed that six species of streptomycetes, S. massaporeus, S. phaeopurpureus, S. chibaensis, S. salmonicida, S. fluvissimus and S. longispororuber, were photochromogenic, i.e., these strains can be photoinduced to synthesize pigments. On the basis of these results, pigmentation in Streptomyces is discussed.     

Studies on the circadian rhythm of phosphoenolpyruvate carboxykinase. III. Circadian rhythm in the kidney.

Phosphoenolypyruvate carboxykinase [EC 4.1.1.21] activity in rat kidney shows a circadian rhythm with the highest activity between 0200 h and 0800 h and the lowest activity between 1400 h and 2000 h. The rhythm was observed in both sexes and throughout the year. Actinomycin D and cycloheximide effectively blocked the circadian increase in enzyme activity. These findings suggest that the circadian increase in phosphoenolypyruvate carboxykinase activity is due to net synthesis of enzyme protein through newly synthesized mRNA. In experiments with kidney cortex slices, gluconeogenesis from the radioactive precursor, [14C]malic acid, was considerably higher at 0200 h than at 1400 h, varying in parallel with the change in the enzyme activity.