Sequence-specific modification of DNA by 6-hydroxybenzo[a]pyrene

6-Hydroxybenzo[a]pyrene cleaved phi X174 supercoiled DNA to open circular DNA in the presence of heavy metal ions. It induced an alkali-labile modification in DNA via an oxygen-radical-mediated reaction; the most frequent alkali-labile sites were on the 3' side of the pyrimidine residues of the pyrimidine cluster.     

Eimeria tenella, E. necatrix, E. acervulina, E. maxima, and E. brunetti: potent anticoccidial activity of an uridine analog, 1-(beta-D-ribofuranosyl)-2(1H)-pyrazinone 4-oxide

The anticoccidial activity of an uridine analog, 1-(beta-D-ribofuranosyl)-2(1H)-pyrazinone 4-oxide (emimycin riboside), against five species of chicken Eimeria was tested individually in battery experiments. With 16 ppm of the compound in feed, marked anticoccidial activity was obtained against Eimeria tenella, E. necatrix, E. acervulina, E. maxima, and E. brunetti. The last named species was more drug-sensitive than the others--dietary levels of at least 8 ppm of the drug exhibited good protection and eliminated practically all clinical signs. The battery tests with delayed and restricted medications showed that emimycin riboside affected the development of parasites in first and second generation schizogony of the life cycle of E. tenella.     

Changes in Lipoxygenase Components of Rice Seedlings during Germination

Changes in lipoxygenase (LOX) activity were followed duringthe germination of rice seeds. The enzyme activity of 3-day-oldseedlings was 20 times higher than that of ungerminated seeds.Sixty per cent of the increased activity was found in shoots.The increase in LOX activity was mainly due to an increase inlipoxygenase-2 (LOX-2), a minor component in ungerminated seeds;this increase was inhibited by cycloheximide. LOX-2 was isolatedfrom the 3-day-old seedlings and compared for its enzymologicalproperties with rice lipoxygenase-3 (LOX-3), a major componentin ungerminated seeds. Both LOX-2 and LOX-3 were stable at pH5 to 8, but LOX-2 was more heatstable than LOX-3. Apparent K_mvalues of LOX-2 and LOX-3 for linoleic acid were 170 and 59µM, and those for linolenic acid were 5,300 and 88 µM,respectively. Both LOXs were inhibited by some metal ions andantioxidants. (Received February 5, 1986; Accepted May 9, 1986)     

Synergistic effects of the myc and ras oncogenes on ganglioside synthesis by BALB/c 3T3 fibroblasts

The effects of oncogene activation on glycosphingolipid (GSL) synthesis by a mouse fibroblast clonal cell line were studied. A transfectant that expressed the activated ras gene showed a definite change in the composition of acidic GSLs, probably an increase in polysialoganglioside, while one that expressed the myc gene showed only a slight change. Neither transfectant grew in soft agar. However, another transfectant, which expressed both the myc and ras genes, and grew in soft agar, showed a more dramatic increase in the acidic GSL component. Thus, activations of the myc and ras oncogenes have a synergistic effect on GSL synthesis during transformation.     

Conformational changes of papain induced on interaction with thiol proteinase inhibitors from newborn rat epidermis

The conformational changes of the papain molecular on interaction with two thiol proteinase inhibitors (TPI(1) and TPI(2] from newborn rat epidermis were studied by measuring circular dichroism (CD), the difference absorption spectrum, and the fluorescence spectrum due to tryptophan residues in papain. The far-ultraviolet CD band of papain between 210 and 230 nm was distinctly reduced on interaction with both inhibitors. Also, the near-ultraviolet CD spectrum of TPI(1)-bound papain changed between 285 and 320 nm as well as that of the TPI(2)-bound enzyme. The difference absorption spectrum for TPI(1)-bound papain exhibited two distinct peaks at 276.5 and 282 nm, indicating perturbation of aromatic amino acid residues. The fluorescence intensity of papain was significantly decreased on interaction with both inhibitors, which showed pH-dependency on an ionizable group, with pK values of 8.5 and 7.9 for TPI(1) and TPI(2), respectively. The complex formation of papain with both inhibitors caused a reduction of the susceptibility of a tryptophan residue, probably tryptophan-177, to chemical modification with N-bromosuccinimide. These results suggest that the active site involving histidine-159 in the papain molecule was much influenced by the alteration of the microenvironment of tryptophan-177 as a part of the interaction site for these two thiol proteinase inhibitors.     

Daphnia curvirostris Eylmann in Japanese high mountain waters (Cladocera: Daphniidae)

A species of Daphnia, Daphnia curvirostris Eylmann, found in high mountain lakes and ponds in central Japan is described. Although there were some differences in the shape of the male rostrum and the chromosome number between European populations as described by Johnson (1952) and Trentini (1980), and Japanese ones collected from high mountain waters, Japanese specimens had many characteristics similar to the taxon D. curvirostris of Europe.     

Light-induced potential and current across a large bacteriorhodopsin-asolectin planar membrane stabilized on a polyacrylamide gel surface

A phospholipid bilayer membrane was spread from an organic solvent solution between a polyacrylamide gel surface and an aqueous buffer solution. The membrane was quite similar to the conventional black lipid membrane, but was of a large size and was stable since it was supported on the gel surface. Bacteriorhodopsin, impregnated into the membrane, generated membrane potential and current upon illumination. The induced current was large, and this was attributed to the large area of the present membrane. Remarkable responses of the light-induced potential and current were also observed with a thick layer of organic solvent containing phospholipids. The effects of applied membrane potential, carbonylcyanide-m-chlorophenyl hydrazone (CCCP) and gramicidin were examined on these photoresponses. Steady-state current, which is due to protons flowing through the membrane, was enormously enhanced by applying membrane potential opposite to the photopotential or by adding gramicidin to the membrane-forming solution.     

Subaxolemmal cytoskeleton in squid giant axon. II. Morphological identification of microtubule- and microfilament-associated domains of axolemma

In the preceding paper (Kobayashi, T., S. Tsukita, S. Tsukita, Y. Yamamoto, and G. Matsumoto, 1986, J. Cell Biol., 102:1710-1725), we demonstrated biochemically that the subaxolemmal cytoskeleton of the squid giant axon was highly specialized and mainly composed of tubulin, actin, axolinin, and a 255-kD protein. In this paper, we analyzed morphologically the molecular organization of the subaxolemmal cytoskeleton in situ. For thin section electron microscopy, the subaxolemmal cytoskeleton was chemically fixed by the intraaxonal perfusion of the fixative containing tannic acid. With this fixation method, the ultrastructural integrity was well preserved. For freeze-etch replica electron microscopy, the intraaxonally perfused axon was opened and rapidly frozen by touching its inner surface against a cooled copper block (4 degrees K), thus permitting the direct stereoscopic observation of the cytoplasmic surface of the axolemma. Using these techniques, it became clear that the major constituents of the subaxolemmal cytoskeleton were microfilaments and microtubules. The microfilaments were observed to be associated with the axolemma through a specialized meshwork of thin strands, forming spot-like clusters just beneath the axolemma. These filaments were decorated with heavy meromyosin showing a characteristic arrowhead appearance. The microtubules were seen to run parallel to the axolemma and embedded in the fine three-dimensional meshwork of thin strands. In vitro observations of the aggregates of axolinin and immunoelectron microscopic analysis showed that this fine meshwork around microtubules mainly consisted of axolinin. Some microtubules grazed along the axolemma and associated laterally with it through slender strands. Therefore, we were led to conclude that the axolemma of the squid giant axon was specialized into two domains (microtubule- and microfilament-associated domains) by its underlying cytoskeletons.     

Streptococcal cytoplasmic pH is regulated by changes in amount and activity of a proton-translocating ATPase

The Streptococcus faecalis H+-ATPase (F1 X F0 complex) level was elevated when the cytoplasmic pH was shifted below 7.5. The elevated level was attained by the increase in functional unit (F1 X F0 complex) in membranes, but not by the activation of the enzyme. Our data strongly suggested that the increase in enzyme arises from stimulation of enzyme biosynthesis. When calls growing at pH 7.6 were transferred to an acid medium with a pH below 7, the amount of H+-ATPase increased. The amount of H+-ATPase decreased to the basal level when the medium was alkalized again. Cytoplasmic pH was not controlled normally in cells where a change in the amount of H+-ATPase was inhibited. Based on these findings and previous data (Kobayashi, H. (1985) J. Biol. Chem. 260, 72-76), we propose a model for the regulatory mechanism of streptococcal cytoplasmic pH: the pH is regulated by changes in amount and activity of the H+-ATPase, which are dependent on the cytoplasmic pH.     

Mouse lymphoma L1210 cells acquire a new cystine transport activity upon adaptation in vitro

Summary Mouse lymphoma L1210 cells maintained in vitro at a high cell density for a certain time period adapted themselves to the in vitro environment and were able to grow indefinitely. From these adapted cells, more than 30 clones were isolated. They all had much higher activity to take up cystine than the original L1210 cells, supporting a previous view that the deficiency of the cystine uptake limits the survival and growth of L1210 cells in vitro. The cystine uptake of one cloned cell line was characterized. The enhanced uptake of cystine in these cells was mainly mediated by a Na⁺-independent, saturable system and was potently inhibited by glutamate and some other anionic amino acids, but less by aspartate. Such activity of cystine uptake was not observed in the original L1210 cells. The results suggest that, upon adaptation in vitro, L1210 cells acquire a new cystine transport activity necessary for survival and growth in vitro.