Incorporation of Uracil by Synchronous Chlorella

Light-dark synchronized Chlorella fusca was used to study the incorporation of radioactive uracil into the trichloroacetic acid insoluble fraction. In autospores the incorporation measured in darkened cells usually started immediately after uraci addition, and the time course showed three distinct linear phases with abrupt transitions between them. Chromatographic analyses of the radioactive pool components showed that the total amounts of monophosphate, diphosphate and triphosphate nucleotides of uracil did not limit the incorporation and thus did not cause the abrupt rate shifts. In autospores, 5, 9 and 14 h old cells, the initial incorporation rate increased with increasing initial uracil concentration to reach a constant value above 0.5 μ.M. Addition of glucose to the cells increased the incorporation rate over the whole uracil concentration range tested with autospores, but did not influence the rates of the other cells. The incorporation rate varied during the synchronous cycle in a manner which closely resembled the pattern for uptake and degradation rates, with the most pronounced similarity from 9 h onwards.     

Major factors affecting isocitrate lyase activity in <Emphasis Type="Italic">Rhodobacter capsulatus</Emphasis> B10 under phototrophic conditions

External factors affecting the activity of isocitrate lyase (ICL) in Rhodobacter capsulatus B10 grown under controlled photoheterotrophic anaerobic conditions were investigated. The activity of this enzyme was found to depend on the history of the inoculum and on the growth phase on acetate medium. Intracellular degradation of ICL under unfavorable conditions was shown. However, transition of the growing culture from acetate to lactate did not result in active degradation of the enzyme. When transferred to acetate, Rba. capsulatus could grow without the lag phase and did not exhibit ICL activity, suggesting another anaplerotic pathway in Rba. capsulatus cells. Since emergence of the ICL activity in the cells grown on acetate results in an increase in its growth rate, the glyoxylate bypass plays an important role in acetate metabolism of Rba. Capsulatus.     

Isolation and biochemical properties of two types of microbody from Neurospora crassa cells.

Cells of the Neurospora crassa slime mutant grown in sucrose medium exhibited low activities of glyoxysomal marker enzymes isocitrate lyase (ICL), malate synthetase (MS), and malate dehydrogenase. Transfer of the cells to a medium containing acetate as sole carbon source ("acetate medium") induced a strong increase in the activities of these enzymes in both the soluble and the crude particulate cell fraction. Soluble isocitrate lyase activity increased rapidly after a lag phase of about 45 minutes. Addition of 0.1 mM cycloheximide to the acetate medium 3 hours after transfer of the cells halted the rise of isocitrate lyase activity in either cell fraction, but the inhibition of the incorporation of ICL activity into the particulate cell fraction was delayed by 1 hour. Addition of 20 g/l glucose resulted in the immediate decrease of both soluble and particulate ICL activities. Transfer to acetate medium induced no change in the activities of other microbody marker enzymes such as catalase, uricase or D-amino acid oxidase. Resolution of crude homogenates of "slime" cells by sucrose density gradient centrifugation yielded two major protein bands: A mitochondrial band at a density of 1.180 kg/l showing maximum activites of fumarase, isocitrate dehydrogenase and cytochrome c oxidase, and a microbody-rich band which obviously consisted of two types of organelles with different biochemical properties. Maximum activities of ICL and MS sedimented at a density of 1.21 kg/l while the peaks of particulate uricase and catalase activities were recovered at 1.24 kg/l.     

Regulation of the Synthesis of Isocitrate Lyase and the Corresponding mRNA in the Green Alga Chlorogonium

In the unicellular green alga Chlorogonium elongatum the level of isocitrate lyase (ICL), the rate of its synthesis and the level of ICL-mRNA measured by in vitro translation are considerably increased after addition of acetate to the culture medium of autotrophically precultured cells. Almost identical increases are obtained independently of whether the cells are cultured after the addition of acetate in the dark (heterotrophically) or in the light (mixotrophically). Transfer of heterotrophic cells to autotrophic conditions results in a fast decrease of ICL-mRNA and ICL protein, while a transfer to mixotrophic conditions causes no alterations in both molecular species. Therefore the concentration of ICL and its translatable mRNA is controlled only by acetate and is unaffected by light.     

Effect of culture conditions on the isocitrate dehydrogenase and isocitrate lyase activities in Chlamydomonas reinhardtii

In the green alga Chlamydomonas reinhardtii , nitrogen staravation induced a reversible increase (2-fold) in NAD-isocitrate dehydrogenase (NAD-IDH; EC 1.1.1.41) and NADP-isocitrate dehydrogenase (NADP-IDH; EC 1.1.1.42) activities. Both enzymes were not affected by the concentration of CO₂, the dark or the nature of the nitrogen source (nitrate, nitrite, or ammonium). When cells growing autotrophically were transferred to heterotrophic conditions, a 40% reduction of the NAD-IDH activity was detected, a 2-fold increase of NADP-IDH was observed and isocitrate lyase (ICL; EC 4.1.3.1) activity was induced. The replacement of autotrophic conditions led to the initial activity levels. NAD- and NADP-IDH activities showed markedly different patterns of increase in synchronous cultures of this alga obtained by 12 h light/12 h dark transitions. While NAD-IDH increased in the last 4 h of the dark period, NADP-IDH increased during the last 4 h of the light period, remaining constant for the rest of the cycle.     

Regulation of isocitrate lyase inRhodobacter capsulatus E1F1

InRhodobacter capsulatus E1F1, isocitrate lyase (ICL) (EC 4.5.3.1) is a regulatory enzyme whose levels are increased in the presence of acetate as the sole carbon source. Acetate activated isocitrate lyase in a process dependent on energy supply and de novo protein synthesis. In contrast to isocitrate lyase, isocitrate dehydrogenase (ICDH) activity was independent of the carbon source used for growth and significantly increased in darkened cells. Pyruvate or yeast extract prevented in vivo activation of isocitrate lyase in cells growing on acetate. The enzyme was reversibly inactivated to a great extent in vitro by pyruvate and other oxoacids presumably involved in acetate metabolism. These results suggest that, inR. capsulatus E1F1, isocitrate lyase is regulated by both enzyme synthesis and oxoacid inactivation.     

Induction of Chlorosis,ROS Generation and Cell Death by a Toxin Isolated from Pyricularia oryzae

The ethyl acetate extract of the conidia germination fluid from an Avena isolate (Br58) of Pyricularia oryzae had chlorosis-inducing activity on oat leaf segments. The same activity was also present in the acetone extract of an oatmeal agar culture of Br58. Fungal cultures were used for a large-scale preparation. A series of acetone and ethyl acetate extraction monitored by chromatography was used to isolate an active fraction. The active principle was purified by HPLC. We show by NMR and LC/MS that the toxin was an oxidized C18 unsaturated fatty acid named Mag-toxin. Mag-toxin induced chlorosis on oat leaf segments incubated in the light but not in the dark. Reactive oxygen species (ROS) and cell death were induced by Mag-toxin in oat cells. The sub-cellular localization of ROS generation induced by the toxin treatment was correlated with the location of mitochondria. Interestingly, the induction of ROS generation and cell death by Mag-toxin was light-independent.     

Regulation of glyoxylate cycle enzymes in Saccharomycopsis lipolytica. I. Effect of the carbon source on isocitrate lyase and malate synthase activity

Comparative studies on the activities of isocitrate lyase (ICL) and malate synthase (MS) were carried out with Saccharomycopsis lipolytica incubating the yeast on media with different carbon sources. When cells were incubated in minimal medium with glucose, the activities of both enzymes were very low. In contrast, in minimal medium with acetate enhanced enzyme activities could be demonstrated. It is probably that the synthesis of ICL is repressed in presence of glucose. Furthermore the activity of ICL was inhibited by tricarboxylic acid cycle intermediates like succinic acid and oxalacetic acid. It was concluded that the syntheses of enzymes are derepressed. When cells of Sm. lipolytica were incubated in minimal medium with acetate, a high enzyme activity is evident. Synthesis of ICL on acetate was inhibited by cycloheximide and actinomycin D. The results were discussed comparing them with data obtained from other organisms.     

Lipoprotein lipase activity of rat cardiac muscle. Changes in the enzyme activity during incubations of isolated cardiac-muscle cells in vitro.

1. Isolated cardiac-muscle cells from the hearts of adult rats were shown to retain a high amount of viability during 4 h of incubation when viability was assessed by Trypan Bue stain exclusion and intracellular enzyme leakage. 2. The cells also retained their ability to take up O2 and utilize added substrates over the period of incubation at both 25 and 30 degrees C. 3. When cells from the hearts of fed rats were incubated in a buffered-salts solution at pH 7.4 in the presence of amino acids and heparin, lipoprotein lipase activity in the medium increased progressively. 4. During these incubations the intracellular activity of the enzyme remained constant and the total activity of lipoprotein lipase in the system (cells plus medium) increased by 80% over the 4 h of incubation at 25 degrees C. 5. In the absence of heparin only low amounts of enzyme activity were detectable in the medium and the total lipoprotein lipase activity in the system remained constant. 6. The measurement of lipoprotein lipase activity in either fresh homogenates of the cells or in homogenates of acetone/diethyl ether-dried powders of the cells had no effect on the overall pattern of activity change during the incubations, although as reported previously the total activity detected with acetone/diethyl either-dried preparations was approx. 3-fold higher than with fresh cell homogenates. 7. The observations were compared with published data on lipoprotein lipase activity changes in neonatal heart cell cultures maintained in vitro.     

Enzyme profiles of Thiobacillus versutus after aerobic and denitrifying growth: Regulation of isocitrate lyase

The specific activities of the tricarboxylic acid (TCA) cycle enzymes in Thiobacillus versutus were invariably lower after aerobic growth as compared to denitrifying growth in acetate- or succinate-limited chemostat cultures. Of the glyoxylate cycle enzymes, isocitrate lyase (ICL) activity was nil during aerobic and 76 nmol·min^-1·mg^-1 protein during denitrifying growth on acetate whereas malate synthase (MS) did not change. In succinate-grown cells ICL was always near nil. The change in ICL and MS was followed after pulse additions of acetate and nitrate to an aerobic acetate-limited chemostat culture made anaerobic prior to the first pulse. ICL remained nil during denitrifying growth after the first pulse but increased to 47 and 81 nmol ·min^-1·mg^-1 protein after the second and third pulse, respectively. MS remained unaltered. The appearance of ICL was dependent upon de novo protein synthesis. During transition in a steady state culture on acetate from oxygen to nitrate as terminal electron acceptor, denitrifying growth started after 0.6 volume replacements. The resumption of growth was concomitant with an increase in TCA cycle enzyme activities. ICL was observed only after two volume replacements. During the reverse transition, ICL disappeared at a rate twice the dilution rate. SDS polyacrylamide gelectrophoresis of cell-free extracts containing ICL showed a major protein band with a R_f value identical to purified ICL and a mol·wt. of 60,000. ICL from T. versutus was inhibited by 1.5 mM itaconate but not by 10 mM phosphoenolpyruvate. Its activity was dependent upon the presence of Mg²⁺ and cysteine.Abbreviations TCA Tricarboxylic acid - ICL isocitrate lyase - MS malate synthase - FPLC fast protein liquid chromatography - maximum specific oxygen consumption rate     

Pool Control of Uracil Uptake by Synchronous Chlorella

Experiments with autospores of Chlorella fusca showed that the uptake rate of uracil was reduced as much as 40 % as the result of formation of soluble pools of uracil and its metabolites from exogenous uracil. Using synchronous cultures similar results were obtained with cells at other developmental stages.     

The influence of organic substrates and inhibitors on the induction of a respiratory oscillation in the cyanobacterium Anacystis nidulans

The rate of respiratory oxygen uptake of the cyanobacterium (blue-green alga) Anacystis nidulans oscillated under certain physiological conditions after light pulses or after addition of sodium acetate. The oscillation started either by the photosynthetic inhibition of respiration or by the stimulation of oxygen uptake caused by sodium acetate. The photosynthetic inhibition of respiration decreased the rate of oxygen uptake to about 20% of the rate in the dark.Starved cells (48 h dark) had lost the inducibility of the oscillation. In starved as well as in non-starved cells oscillations were inducible in the presence of fructose or glucose. Well developed oscillations were not promoted further. All other substances tested as substrates did not restore the inducibility of the oscillation in starved cells. The induction of the oscillation was inhibited by iodoacetamide (0.1 mM), p-hydroxymercuribenzoate (0.1 mM) and sodium fluoride (100 mM). It is suggested that a flow from glyceraldehyde-3-phosphate to the incomplete tricarboxylic acid cycle is a prerequisite for the oscillation.     

Reconsideration of the taxonomy of ellipsoidal species of Chlorella (Trebouxiophyceae, Chlorophyta), with establishment of Watanabea sen. nov.

Subcultures of SAG 211–9b and 1AM C-211, ultimately derived from CCAP 211/9b, a strain isolated by Pringsheim in 1939 and identified as Chlorella sac-charophila (Kruger) Migula were observed using light and electron microscopy. Their morphology proved to be basically identical. Both have two forms of cells, one (E-form) narrowly to broadly ellipsoidal, the other (S-form) ovoid to spheroidal. The cell wall of both forms is composed of a single smooth layer. The chloroplast of young cells is trough-like or saucer-shaped with a smooth margin, while that of mature cells is band- or cup-shaped with deep incisions. The thylakoid lamellae are loosely stacked and neither form has a pyrenoid. Both types of cells are capable of producing autospores: eight to 16 in E-form cells, two to four in S-form cells. These morphological features are different from those of C. saccharophila, which has a pyrenoid and produces only one form of autospores. In the absence of any existing genus that includes Chlorella-like algae with a simple cell wall, no pyrenoid, and two forms of mature cells and autospores, a new genus, Watanabea, is proposed with the type species W. reniformis.     

Genetics and function of isocitrate lyase in Coprinus

Summary Thirteen chromosomal loci have been identified which affect acetate metabolism in Coprinus. Mutants at only two loci, acu-1 and acu-7, are deficient in isocitrate lyase (ICL) (EC4.1.3.1) activity, acu-1 mutants are unable to induce ICL because they lack acetyl-CoA synthetase which is required to convert acetate to the metabolic inducer of ICL. acu-7 is the structural gene for ICL. This was shown by selecting temperature sensitive acu ⁺ revertants resulting from a second mutation within the acu-7 gene. One such severtant was shown to produce an ICL protein which was more thermolabile than the wild type enzyme. Other workers have postulated that ICL activity is important during asexual morphogenesis in fungi. No evidence was found for this in Coprinus. The morphological mutant oidial, which produces abundant asexual spores even in submerged culture, had the same low uninduced level of ICL activity as the wild type. Moreover, an acu-7 mutation had no effect on the expression of the oidial phenotype.     

Lipoprotn lipase content and triglyceride fatty acid uptake in adipose tissue of rats of differing body weights

Increasing body weight appears to alter lipid metabolism in adipose tissue. We have measured the content of lipoprotein lipase and the uptake of chylomicron triglyceride fatty acids in epididymal fat pads of rats of different weights. In order that the results might be expressed in terms of cell numbers, the relationship between the weights of fat pads and the numbers and volumes of fat cells isolated from them was determined. Highly significant correlations were found between fat pad weight and both the number and the volume of the individual adipocytes. In rats weighing from 140 to 350 g, the increase in the size of fat pads was attributable almost equally to increases in cell size and in cell number. Lipoprotein lipase activity was measured in acetone powders of whole fat pads and of isolated fat cell preparations. With both, lipoprotein lipase activity per cell diminished significantly as the weight of fat tissue increased, i.e., larger fat cells contained less enzyme per cell than smaller cells. The uptake of triglyceride fatty acid radioactivity was measured after incubation of fat pads with radiolabeled rat lymph chylomicrons in flasks containing either buffer alone or with added glucose or glucose plus insulin. The addition of glucose and insulin led to a mean increase of 70% in the uptake of radioactivity, but larger adipocytes were stimulated less than smaller cells. This resulted in a significant negative correlation between the weights of fat pads and the uptake of radioactivity. Enlargement of fat cells also led to a diminution in their capacity to esterify fatty acids.     

The genetics of aryl hydrocarbon hydroxylase induction in mice: A single gene difference between C57BL/6J and DBA/2J

Twenty-one inbred strains of mice were surveyed for inducibility of hepatic aryl hydrocarbon hydroxylase (AHH) activity by the carcinogen 3-methylcholanthrene (MC). In 11 strains given MC, AHH activity increased 1.3- to 5-fold (inducible), whereas ten strains responded with a less than 0.5-fold increase (noninducible). Neither the inducible nor the noninducible class was homogeneous, and in each considerable variation was found in both the basal activity of AHH and the response to MC. Strains DBA/2J and C57BL/6J were chosen to represent the noninducible and inducible classes, respectively. In the crosses (C57BL/6 × DBA/2)F₁ × DBA/2 and (C57BL/6 × DBA/2)F₂, inducibility segregated as a single autosomal dominant gene. The gene symbols Ahh ⁱ and Ahh ⁿ are proposed for the alleles present in C57BL/6J and DBA/2J, respectively. No genetic linkage was found between the Ahh locus and the following loci: b, d, Es-1, Es-3, Gpd-1, Hbb, Id-1, Pgm-1, and sex. Some implications of this work in the study of mammalian enzyme induction and chemically induced carcinogenesis are discussed. There is a positive correlation between AHH inducibility and the development of an inflammatory response to the topical application of the carcinogen 7,12-dimethylbenzanthracene.     

O6-methylguanine methyltransferase increases before S phase in normal human cells but does not increase in hypermutable Bloom's syndrome cells

The regulation of the O6-methylguanine methyltransferase was examined during cell proliferation in hypermutable Bloom's syndrome fibroblasts and normal human skin fibroblasts. During synchronous growth following serum stimulation normal human cells enhanced methyltransferase activity 2.4-fold in the absence of exogenous damage as a normal regulatory event during the cell cycle. Methyltransferase activity was increased prior to the induction of DNA replication and of DNA polymerase and was diminished when each replicative activity was maximal. In contrast, although methyltransferase levels in quiescent cells are equivalent, hypermutable Bloom's syndrome cells did not increase methyltransferase at any interval in the cell cycle.