The regulation of synthesis of mitochondrial enzymes in regreening and division-synchronized Euglena cultures

The effect of light and carbon nutrition on the synthesis of citrate synthase (EC 4.1.3.7) and malate dehydrogenase (EC 1.1.1.37) in dark-grown resting (carbon deficient) and in phototrophic division-synchronized cultures of Euglena gracilis Klebs strain z were investigated. Exposure of dark-grown Euglena to white or red light produced a transient increase in the specific activities of citrate synthase and malate dehydrogenase but blue light (of equal energy) was ineffective. Citrate-synthase activity increased at the end of the light phase and in early dark phase in phototrophic cultures division-synchronized by a regime of 14 h light-10 h dark. The addition of ethanol or malate produced a twofold increase in citrate-synthase activity compared with phototrophic cultures. White and blue light, but not red light, produced a transient repression of the metabolite-induced increase in citrate-synthase activity in division-synchronized cultures. Since only red light could effect a transient increase in the specific activity of mitochondrial enzymes, and the blue-red plastid receptor should respond to both blue and red light, the synthesis of mitochondrial enzymes in regreening cultures may be under the control of a new photoreceptor responding only to red light. In division-synchronized phototrophic cells the primary effector of synthesis of mitochondrial enzymes is not light but carbon nutrition.     

Photosynthetic adaptation of pea plants grown at different light intensities: State 1 - State 2 transitions and associated chlorophyll fluorescence changes

A study of pea plants grown at different light intensities has been made. Using a leaf oxygen electrode, it was shown that plants grown under low light intensities had lower saturated rates of photosynthesis than high-light-grown plants however, at low light intensities the photosynthetic rates were similar for both types of plants. State 1- State 2 transitions have been monitored with attached leaves using a modulated fluorescence technique. It is shown that peas grown under low light intensities (20 W m^-2) had a faster State 1 to State 2 transition when compared with medium-(50 W m^-2) and high-(70 W m^-2) light-grown plants. Measurement of fast-fluorescence-induction curves in the absence of 3-(3′,4′-dichlorophenyl)-1,1-dimethylurea (DCMU) have shown that low-light plants are, when in State 1, more effective at using Photosystem-two (PSII) light to reduce their plastoquinone pool than high-light plants. Transition from State 1 to State 2 for all plants led to a decrease in the reduction level of the plastoquinone pool inidcating that the transition had increased electron flow through Photosystem one (PSI) relative to PSII. Analyses of fast fluorescence induction in the presence of DCMU indicate that low-light-grown plants have a higher PSII-α/PSII-β ratio than high-light-grown plants. Such a difference is in line with the increase in the PSII/PSI ratio of low-light plants and is reflected in their high chlorophyll b/chlorophyll a ratio and their larger appressed to non-appressed thylakoid-membrane areas. It is suggested that these two latter factors give rise to the faster State 1 - State 2 transitions in low-light plants.     

Role of the tumor suppressor IQGAP2 in metabolic homeostasis: possible link between diabetes and cancer

Deficiency of IQGAP2, a scaffolding protein expressed primarily in liver leads to rearrangements of hepatic protein compartmentalization and altered regulation of enzyme functions predisposing development of hepatocellular carcinoma and diabetes. Employing a systems approach with proteomics, metabolomics and fluxes characterizations, we examined the effects of IQGAP2 deficient proteomic changes on cellular metabolism and the overall metabolic phenotype. Iqgap2 ^?/?mice demonstrated metabolic inflexibility, fasting hyperglycemia and obesity. Such phenotypic characteristics were associated with aberrant hepatic regulations of glycolysis/gluconeogenesis, glycogenolysis, lipid homeostasis and futile cycling corroborated with corresponding proteomic changes in cytosolic and mitochondrial compartments. IQGAP2 deficiency also led to truncated TCA-cycle, increased anaplerosis, increased supply of acetyl-CoA for de novo lipogenesis, and increased mitochondrial methyl-donor metabolism necessary for nucleotides synthesis. Our results suggest that changes in metabolic networks in IQGAP2 deficiency create a hepatic environment of a ‘pre-diabetic’ phenotype and a predisposition to non-alcoholic fatty liver disease which has been linked to the development of hepatocellular carcinoma.     

Effect of Growth Irradiance on Photosynthesis and Transpiration inPhaseolus vulgaris L.

In comparison with primary leaves of French bean plants grown under a photon flux density of 100 μeinstein m-2 s-1 (LP), leaves grown under 400 μeinstein m-2 s-1 (HP) were thicker (contained 82 to 104% more dry matter per blade area), had 44 to 48% higher stomatal frequency, 18 to 26% more chlorophyll (a + b) per leaf area unit and 31 to 42% less chlorophyll (a + b) per dry matter unit, 41% higher photosynthetic and 38% higher transpiration rates at light saturation, 33% higher stomatal conductance and 40% higher Photosystem 2 (H2O → K3[Fe(CN)6]) activity of isolated chloroplasts. There were no significant differences in the Photosystem 1 (TMPD/Ascorbate → MV) activity per unit amount of chlorophyll. Higher growth irradiance increased the ratio of frequencies of stomata in the upper/lower epidermes.     

Knockdown of LYRM1 Rescues Insulin Resistance and Mitochondrial Dysfunction Induced by FCCP in 3T3-L1 Adipocytes

LYR motif-containing 1 (LYRM1) was recently discovered to be involved in adipose tissue homeostasis and obesity-associated insulin resistance. We previously demonstrated that LYRM1 overexpression might contribute to insulin resistance and mitochondrial dysfunction. Additionally, knockdown of LYRM1 enhanced insulin sensitivity and mitochondrial function in 3T3-L1 adipocytes. We investigated whether knockdown of LYRM1 in 3T3-L1 adipocytes could rescue insulin resistance and mitochondrial dysfunction induced by the cyanide p-trifluoromethoxyphenyl-hydrazone (FCCP), a mitochondrion uncoupler, to further ascertain the mechanism by which LYRM1 is involved in obesity-associated insulin resistance. Incubation of 3T3-L1 adipocytes with 1 µM FCCP for 12 h decreased insulin-stimulated glucose uptake, reduced intracellular ATP synthesis, increased intracellular reactive oxygen species (ROS) production, impaired insulin-stimulated Glucose transporter type 4 (GLUT4) translocation, and diminished insulin-stimulated tyrosine phosphorylation of Insulin receptor substrate-1 (IRS-1) and serine phosphorylation of Protein Kinase B (Akt). Knockdown of LYRM1 restored insulin-stimulated glucose uptake, rescued intracellular ATP synthesis, reduced intracellular ROS production, restored insulin-stimulated GLUT4 translocation, and rescued insulin-stimulated tyrosine phosphorylation of IRS-1 and serine phosphorylation of Akt in FCCP-treated 3T3-L1 adipocytes. This study indicates that FCCP-induced mitochondrial dysfunction and insulin resistance are ameliorated by knockdown of LYRM1.     

Cytoplasmic and outer membranes separation in Rhodopseudomonas sphaeroides

A cell envelope fraction has been prepared after mechanical disruption of lysozyme-EDTA spheroplasts from depigmented Rhodopseudomonas sphaeroides (aerobically grown in the light). On linear sucrose gradients this fraction can be separated in a cytoplasmic membrane fraction and an outer membrane fraction. The cytoplasmic fraction (buoyant density: 1.18 g/cm³) has been characterized by its succinic dehydrogenase activity and by its composition. The outer membrane fraction (buoyant density: 1.21 g/cm³) does not contain any respiratory activity nor hemoproteins. The same fractionation has been done on cells repigmented in the dark by lowering the O₂ pressure. In that case the same two fractions have been detected in addition to the chromatophore fraction (buoyant density: 1.14 g/cm³). However both, and specially the outer membrane fraction, were contaminated by chromatophore material.     

The mitochondrial localization of hexokinase in pea leaves

Up to 80% of total cellular hexokinase (EC 2.1.7.4) activity in pea (Pisum sativum L.) leaves was found to be associated with particulate fractions. Fractionation on sucrose density gradients showed this particulate activity to be associated exclusively with mitochondria. In the presence of glucose and ATP, the bound mitochondrial hexokinase could support rates of O₂ uptake of up to 30% of normal ADP-stimulated rates. This stimulation of O₂ uptake by hexokinase was completely sensitive to oligomycin, indicating that it resulted from an increase in the supply of ADP for mitochondrial oxidative phosphorylation. Spectrophotometric measurements of the mitochondrial hexokinase activity showed that ADP could support rapid rates of activity provided oxidizable substrates were also present to support the conversion of ADP to ATP in oxidative phosphorylation. Carboxyatractyloside, an inhibitor of adenine-nucleotide uptake by mitochondria, inhibited this ADP-supported activity, but had no effect on hexokinase activity in the presence of added ATP, demonstrating that the hexokinase enzyme was located external to the inner mitochondrial membrane. Oligomycin also inhibited ADP-supported activity but had no effect on ATP-supported hexokinase activity. Glucose (K_m 53 μM) was the preferred substrate of pea-leaf mitochondrial hexokinase compared with fructose (K_m 5.1 mM). Hexokinase was not solubilised in the presence of glucose-6-phosphate.     

Glutamine synthetase,glutamate synthase and glutamate dehydrogenase in Rhizobium japonicum strains grown in cultures and in bacteroids from root nodules of Glycine max

The growth yields of three strains of Rhizobium japonicum (CB 1809, CC 723, CC 705) in culture solutions containing L-glutamate were about twice those grown with ammonium. The activities of glutamine synthetase (GS; EC 6.3.1.2) and glutamate dehydrogenase (GDH; EC 1.4.1.4) were dependent on the nitrogen source in the medium and also varied with growth. Both NADPH-and NADH-dependent glutamate synthase (GOGAT; EC 1.4.1.13) and NADPH-dependent GDH were found in strains grown with either glutamate or ammonium but NADH-linked GDH was only detected in glutamate-grown cells. Glutamine synthetase was adenylylated in cells grown with NH ₄ ⁺ (90%) and to lesser extent in those grown with L-glutamate (50%). In root nodules produced by the three strains in Glycine max (L.) Merr., the bulk of GS was located in the nodule cytosol (60–85%). The enzyme was adenylylated in bacteroids (43–75%) and in the nodule tissues (52–68%). The enzyme in cell-free extracts of Rh. japonicum (CC 705) grown in culture solutions containing glutamate and in bacteroids (CC 705) was deadenylylated by snake-venom phosphodiesterase. L-methionine-DL-sulfoximine restricted the incoporation of ¹⁵N-labelled (NH₄)₂SO₄ into cells of strains CB 1809 and CC 705, as well as in bacteroids of strain CC 705. It is noteworthy that appreciable activities for GDH were found in the free-living rhizobia grown on glutamate. Thus the presence of an enzyme does not necessarily imply that a particular pathway is operative in assimilating ammonium into cell nitrogen. Based on ¹⁵N studies, the GS-GOGAT pathway of rhizobia (strains CB 1809 and CC 705) is important when grown in culture solutions as well as in bacteroids from root nodules of G. max.     

Relationships between circadian rhythm of chilling resistance and acclimation to chilling in cotton seedlings

Cotton (Gossypium hirsutum L. cv. Deltapine 50) seedlings grown under light-dark cycles of 12:12h at 35°C showed rhythmic daily changes in chilling resistance. Chilling treatment (5°C, 48h) started at the beginning or middle of the daily light period resulted in a substantial growth inhibition of the seedlings upon return to 35°C whereas when chilling was started at the beginning or middle of the dark period the subsequent growth of the seedlings was much less inhibited. This rhythm in chilling resistance persisted under continuous light for three 24-h periods, indicating that it is of an endogenous nature. Seedlings grown under continuous light from germination showed no daily changes in resistance, but a rhythm was initiated by introduction of a dark period of 6h or longer. In 24-h cycles with different light and dark periods, maximal resistance was reached just before the start of dark period. Seedlings grown at 35°C could be acclimated to chilling by exposure to low, non-damaging temperatures (25–15°C). A short-term (6h) exposure to 25°C started at the resistant phase resulted in a large increase in resistance during the following otherwise sensitive phase. The resistance induced by the low temperature matched or slightly exceeded the maximal resistance reached during the resistant phase of the daily rhythm of chilling. The low-temperature-induced resistance and the daily rhythmic increase in resistance were not additive, indicating a common mechanism for the two kinds of resistances. An adaptive advantage of a combination of a rapid temperature-induced acclimation and the daily rhythmic increase in resistance is suggested.     

Rhythmic changes in ribonuclease activity in relation to nucleic acid synthesis in cotyledons ofChenopodium rubrum L. and to floral induction of the plants

Ribonuclease (RNAse) activity was investigated in cotyledons ofChenopodium rubrum plants subjected to various conditions of illumination (photoperiodic induction, continuous light, induction cancelled by interrupting the dark period by a light-break). At the end of the dark period of the single inductive cycles RNAse activity of induced plants was inferior to that of plants grown in continuous light. At the end of the first two cycles the activity was lowest after the interruption of the dark period by light. The investigation of the enzyme in 6h intervals showed rhythmic changes in activity to occur in induced plants. Enzyme activity followed a pattern opposed to this of nucleic acid (NA) synthesis in the cotyledons. In plants from continuous light the enzyme activity did not show any rhythm and in plants having obtained a light-break during the inductive period the rhythm was less distinct than in the induced ones. The period length of the endogenous rhythm of NA synthesis in the cotyledons is about half as long as this of flowering and the peaks of flowering coincide with the throughs of NA synthesis.     

The blue-light reaction in plasmodia of Physarum polycephalum is coupled to respiration

The influence of inhibitors of energy metabolism (2-deoxy-D-glucose, monoiodoacetate, KCN) as well as various substrates for respiration (sodium acetate, glycine, glutamine, α-ketoglutarate, pyruvate) were investigated with respect to the effect of blue light (450 nm) on contractile behaviour of plasmodial strands of Physarum polycephalum. When the energy metabolism is not experimentally modified, blue light induces a prolongation of the period of the contraction-relaxation cycle. This effect appears within 2–3 min and seems to represent the primary reaction of this organism to blue light. Inhibition of respiration by KCN completely abolished this response to blue-light irradiation. In contrast, an impediment of glycolysis enhanced the effect. This indicates that the reaction to blue light is related to respiration, i.e., to the function of mitochondria. Among different substrates for respiration only α-ketoglutarate combined with pyruvate and applied in the presence of inhibitors of glycolysis showed an enhancement of the photoresponse, i.e., a prolongation of the period and an increase of the amplitude of the force oscillations. This indicates that the pyruvate and α-ketoglutarate-dehydrogenase complexes functioning in mitochondrial respiration are involved in the primary blue-light reaction of plasmodia of Physarum polycephalum.     

Raphanusanin-mediated resistance to pathogens is light dependent in radish and Arabidopsis thaliana

Raphanusanin (Ra) is a light-induced inhibitor of hypocotyl growth that responds to unilateral blue light illumination in radish seedlings. We have previously shown that Ra regulates genes that are involved in common defense mechanisms. Many genes that are induced by Ra are also positively regulated by early blue light. To extend the understanding of the role of Ra in pathogen defense, we evaluated the effects of Ra on radish and Arabidopsis thaliana (A. thaliana) infected with the necrotrophic pathogen Botrytis cinerea (B. cinerea) and biotrophic pathogen Pseudomonas syringae (P. syringae). Radish and A. thaliana were found to be resistant to both pathogens when treated with Ra, depending on the concentration used. Interestingly, Ra-mediated resistance to P. syringae is dependent on light because Ra-treated seedlings exhibited enhanced susceptibility to P. syringae infection when grown in the dark. In addition to regulating the biotic defense response, Ra inhibited seed germination and root elongation and enhanced the growth of root hairs in the presence of light in radish and A. thaliana. Our data suggest that Ra regulates the expression of a set of genes involved in defense signaling pathways and plays a role in pathogen defense and plant development. Our results show that light may be generally required not only for the accumulation of Ra but also for its activation during the pathogen defense response.     

Preliminary studies of the effects of an induced phosphate deficiency on carbohydrate-nitrogen ratios inAlternanthera philoxeroides [Centrospermae: Amaranthacea] and on the feeding ofAgasicles hygrophila [Col.: Chrysomelidae]

In no-choice tests but not in choice tests, alligatorweed flea beetles,Agasicles hygrophila Selman & Vogt (Col.: Chrysomelidae) exposed to alligatorweed plants grown with two levels of mineral nutrition fed significantly more on those grown in full mineral nutrient than those grown with deficient phosphate. Chemical analysis showed that the full nutrient plants had more ethanol-soluble nitrogen compounds but less total carbohydrate than the phosphate-deficient plants. The response of beetles to phosphate-deficient alligatorweed may thus result from a change in the carbohydrate-nitrogen composition of host plants, though further investigation is needed for confirmation. Flea beetle response was identical to terminal and mature leaf tissue of full nutrient plants.     

Submitochondrial localization and characteristics of thymidine kinase molecular forms in parental and kinase-deficient hela cells

Although HeLa (BU25) cells are deficient in cytosol dT kinase activity, they contain two mitochondrial dT kinases with disc PAGE mobilities (R _m) of 0.4 and 0.6 and isoelectric points (pI) of 8.4 and 5.6, respectively. Mitochondrial extracts of parental HeLa S3 contain the two HeLa (BU25) activities, but also a cytosol-like enzyme (0.25 R _m, pI 9.8). The 0.6-R _m (pI 5.6) mitochondrial activity utilizes ribonucleoside 5′-triphosphates other than ATP (dATP) as phosphate donors and is sensitive to dCTP inhibition. The predominant HeLa S3 cytosol (0.25 R _m) enzyme and the 0.4 R _m mitochondrial enzymeefficiently utilize only ATP as a phosphate donor and are relatively insensitive to dCTP inhibition. Submitochondrial fractionation studies have shown that (1) 74–98% of the mitochondrial dT kinase is located in the matrix plus inner membrane fractions; (2) the matrix fraction has the highest specific activity, contains all the 0.6-R _m activity, most of the HeLa S3 0.25-R _m activity, and some 0.4-R _m activity; (3) the inner membrane fraction is the major site of the 0.4-R _m activity but the outer membrane fraction also contains the 0.4 R _m activity; and (4) all HeLa S3 submitochondrial fractions contain the 0.25-R _m dT kinase activity.     

Identification of three proteins involved in fertilization and parthenogenetic development of a brown alga,Scytosiphon lomentaria

Metabolic pathways of cell organelles may influence the expression of nuclear genes involved in fertilization and subsequent zygote development through a retrograde regulation. In Scytosiphon lomentaria, inheritance of chloroplast is biparental but mitochondria are maternally inherited. Male and female gametes underwent different parthenogenetic outcomes. Most (>99 %) male gametes did not differentiate rhizoid cells or survived beyond four-cell stage, while over 95 % of female gametes grew into mature asexual plants. Proteomic analysis showed that the protein contents of male and female gametes differed by approximately 1.7 %, 12 sex-specific proteins out of 700 detected proteins. Three sex-specific proteins were isolated and identified using CAF-MALDI mass spectrometry and RACE-PCR. Among them, a male gamete-specific homoaconitate hydratase (HACN) and a female gamete-specific succinate semialdehyde dehydrogenase (SSADH) were predicted to be the genes involved in mitochondrial metabolic pathways. The expression level of both mitochondrial genes was dramatically changed at the fertilization event. During parthenogenetic development the male-specific HACN and GTP-binding protein were gradually down-regulated but SSADH stayed up-regulated up to 48 h. To observe the effect of chemicals on the expression of these genes, male and female gametes were treated with γ-aminobutyric acid (GABA), hydrogen peroxide and l-ascorbic acid. Among them GABA treatment significantly reduced SSADH gene expression in female gamete but the same treatment induced high upregulation of the gene in male gamete. GABA treatment affected the behavior of gametes and their parthenogenetic development. Both gametes showed prolonged motile stage, retarded settlement and subsequent parthenogenetic development. Our results suggest that male and female gametes regulate mitochondrial metabolic pathways differentially during fertilization, which may be the reason for their physiological and behavioral differences.