Use of ryanodine for functional removal of the calcium store in smooth muscle cells of the guinea-pig

Calcium store of the skinned fibers of the guinea-pig portal vein, pulmonary artery and taenia caeci consisted of two classes: one with both Ca-induced Ca release (CICR) and inositol 1,4,5-trisphosphate (IP3)-induced Ca release (IICR) mechanisms (S alpha) and the other only with IICR mechanisms (S beta). Ryanodine, applied during the CICR was activated, locked the CICR channels open, but the drug had practically no effect on the IICR mechanism. Thus, after the ryanodine treatment the Ca store with the CICR (S alpha) lost its capacity to hold Ca. Changes in the agonist-evoked contraction of intact muscle due to the ryanodine treatment suggest that agonists release Ca from S alpha which produces the initial phase of contractures.     

Branching Photocycle of Sensory Rhodopsin in Halobacterium Halobium

Temperature effect of the photocyle of sensory rhodopsin (sR) was studied by nanosecond spectroscopy. Though the formation yield of sR_M (sR₃₇₀) was sharply decreased with temperature, those of sR_K (sR₆₈₀) and sR_L were insensitive to temperature changes. These results show the existence of the branching process back to sR from sR_L. The absorption maxima for sR_K and sR_L were 595 ± 5 and 555 ± 15 nm, respectively.     

Scattered light intensity fluctuation in the canine ventricular myocardium: correlation with inotropic drug effect

Scattered light intensity fluctuation (SLIF) of coherent light by a strip of ventricular muscle during diastole is believed to be due to asynchronous cellular motion within the myocyte as a result of spontaneous release of Ca from the sacoplamic reticulum. Previous studies have shown a correlation between inotropic agents, such as ouabain and elevated extracellular Ca or decreased extracellular Na, and SLIF. The purpose of this study was to see if this correlation could be extended to other inotropic agents. The digitalis genin, ouabagenin, produces inotropy by increasing intracellular free Ca. In toxic concentrations the drug produces abnormal aftercontractions by spontaneous Ca release from the sarcoplasmic reticulum. On the other hand, the Ca channel agonist BAY k 8644 is also positively inotropic, but its effect is associated with a decrease in Ca release from the sarcoplasmic reticulum, manifested by conversion of "rest potentiation" to "rest depression." The effects of these inotropic agents on the power spectra of SLIF were dissimilar. Both frequency and amplitude of SLIF were increased after ouabagenin (1 microM), but these changes were most marked after the onset of toxicity, at which time contractility was decreased, rather than during the positive inotropic response. In contrast, BAY k 8644 (1 microM) decreased SLIF at all levels of inotropic response. The beta-adrenoceptor stimulant drug, dobutamine, and the adenylate cyclase activator, forskolin, produced minimal increase in SLIF at inotropic concentrations but caused a large increase in SLIF only after the onset of toxicity. These results suggest that SLIF is a better indicator of intracellular Ca overload and toxic oscillatory contractions in the presence of an inotrope and not of increased inotropy, per se.     

Metabolism of galactosylceramide in the twitcher mouse, an animal model of human globoid cell leukodystrophy

The metabolism of galactosylceramide was investigated in normal and twitcher mice, an animal model for human globoid cell leukodystrophy. The findings were compared with data obtained on human tissues. In vitro studies demonstrated that there were two genetically distinct enzymes that hydrolyze galactosylceramide: galactosylceramidase I and II. The former was deficient in the twitcher, while the latter was intact. beta-Galactosidase preparations purified from normal mouse liver possessed the activity to hydrolyze galactosylceramide when the assay conditions for galactosylceramidase II was used. Therefore, galactosylceramidase II was considered to be identical to GM1 ganglioside beta-galactosidase. In contrast to the human enzyme, the murine beta-galactosidase had a relatively high Km value toward galactosylceramide. The galactosylceramide-loading test demonstrated that the twitcher fibroblasts hydrolyzed the lipid at lower rates than seen in cases of human globoid cell leukodystrophy fibroblasts. These differences in galactosylceramidase II between murine and human tissues suggest that galactosylceramide accumulates in twitcher mice but not in humans with globoid cell leukodystrophy, even though galactosylceramidase I is genetically deficient in both human and this mouse model.     

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.     

Time-resolved x-ray study of effect of sinusoidal length change on tetanized frog muscle.

Time-resolved x-ray diffraction studies were done on frog skeletal muscles with synchrotron radiation by applying sinusoidal length changes of frequency 10 Hz and amplitude approximately 1% to isometrically contracting muscles at approximately 17 degrees C. Distinct periodic intensity changes were observed in the 14.3-nm myosin meridional reflection and the equatorial 1,0 and 1,1 reflections. Response of the 14.3-nm reflection to the sinusoidal length change was nonlinear, as evidenced by a large second harmonic in its oscillatory intensity change, whereas the response of the equatorial 1,1 reflection was closely linear, as evidenced by almost sinusoidal intensity change. Intensity change of the 1,0 reflection was nearly antiphase to that of the 1,1 reflection. Integral widths of the 14.3-nm meridional reflection measured along the meridian and of the equatorial 1,1 reflection remained almost constant during tension development, while that of the 1,0 reflection tended to decrease. The widths of the 14.3-nm meridional reflection perpendicular to the meridian and of the equatorial 1,0 reflection appeared to undergo oscillatory changes in response to the sinusoidal length changes.     

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.     

Isolation and Sequencing of a Genomic Clone for Mouse Brain Specific Small RNA

We isolated a mouse genomic clone that hybridized with small RNA present in the cytoplasm of the brain. The RNA was about 150 nucleotides long. This RNA seemed to be specific to the brain, since it was not found in the liver or kidney. The clone DNA contained a sequence homologous to 82-nucleotide "identifier" core sequence of cDNA clones of rat. The sequence contained a split promoter for RNA polymerase III and was flanked by a 12-nucleotide direct repeat (ATAAATAATTTA).     

Effects of norharman on the mutagenicity of chlorophenylhydroxylamine and its metabolism with rat liver S9

o,p-Chlorophenylhydroxylamines (CPHAs) (10468-16-3, 823-86-9) only demonstrated mutagenicity in the presence of S9 mix and norharman (NOH) (244-63-3), as well as chloronitrobenzenes. The mutagenic activity of o-CPHA was 30 times higher than that of p-CPHA. When o-CPHA was preincubated with S9 mix without NOH, the mutagenic activity disappeared rapidly. The decrease in activity during the preincubation was suppressed by addition of NOH. HPLC analysis revealed that o-CPHA was metabolized to o-chloroaniline (o-CA) (95-51-2) and that the metabolic reduction was inhibited by NOH. When microsomes containing NADPH were used instead of S9 mix, o-CPHA exhibited only very weak mutagenicity. The activity in the microsome system, however, was greatly enhanced by adding cytosol or ascorbic acid (50-81-7). These phenomena were only observed in the conventional plate incorporation method. In the case of the liquid incubation assay, in which test compound metabolism and tester strain mutation only occur in the liquid incubation medium, the mutagenic activity of o-CPHA in the microsome system with NOH was comparable to that in the S9 system, indicating that o-CPHA was activated by an enzyme in microsomes in the presence of NOH. Consequently, it was concluded that NOH not only affects the metabolic inactivation of o-CPHA to o-CA by S9, but also the metabolic activation of o-CPHA by microsomes. No appreciable enhancing effects of cytosol and ascorbic acid were observed in the liquid incubation assay, suggesting that these factors affect the stability of CPHA or an active metabolite. The microsome activation of o-CPHA was dependent on NADPH and oxygen; SKF-525A (62-68-0), metyrapone (54-36-4) and alpha-naphthoflavone (604-59-1) inhibited the mutagenic activity by about 50%, suggesting that cytochrome P-450 was involved in the metabolic activation.     

Behavior of Chloroplast Nucleus during Chloroplast Development and Degeneration in Chlamydomonas reinhardii

Changes in morphology of chloroplast nuclei (cp-nuclei), totalcp-DNA content, number of cp-nuclei, oxygen-evolution activityand chlorophyll (a and b) content were examined during the degenerationand development of chloroplasts, using Chlamydomonas reinhardiicells which had been incubated on solid medium for various periods. Under 4'-6-diamidino-2-phenylindole (DAPI) epifluorescence microscopy,each cell that had been incubated for 7 days had one cell nucleus,one cup-shaped chloroplast and about 10 small, dispersed cp-nucleiin the chloroplast. One day after incubation of these cellson fresh medium, the cell volume and cp-nuclei increased insize 2-3 fold, but rapidly decreased in size after cell division.After about 7 days of incubation, cells ceased to divide andcp-nuclei began to associate with each other. At about 20 daysthey formed a ring-shaped structure surrounding the pyrenoid,followed by condensation into one cp-nuclear particle near thepyrenoid. When 41-day-old cells, having only one cp-nucleus,were reinoculated on fresh solid medium, the cp-nucleus increasedin size 2–3 fold, divided into several cp-nuclear particlesand then dispersed into the chloroplast, forming a bead-likestructure, before cell division. From microscopic fluorometry,a 4-fold increase in total cp-DNA content per chloroplast, withoutan increase in the number of cp-nuclear particles per chloroplast,occurred one day after the start of the experiment and one dayafter reinoculation of 41-day-old cells onto fresh medium. Theprocess of condensation of dispersed cp-nuclear particles intoone cp-nucleus during degeneration of the chloroplast was notaccompanied by any change in total cp-DNA content per chloroplast.A large peak of oxygen-evolution (0.6–0.9 pmoles/cell/hour)was seen one day after inoculation and reinoculation of thecells. The chlorophyll content (a+b) was high (1.2–2.2pg/cell) during the first week of incubation, after which itgradually decreased. (Received December 18, 1985; Accepted April 2, 1986)