Mechanism of the elevation in cardiolipin during HeLa cell entry into the S-phase of the human cell cycle

CL (cardiolipin) is a key phospholipid involved in ATP generation. Since progression through the cell cycle requires ATP we examined regulation of CL synthesis during S-phase in human cells and investigated whether CL or CL synthesis was required to support nucleotide synthesis in S-phase. HeLa cells were made quiescent by serum depletion for 24 h. Serum addition resulted in substantial stimulation of [methyl-(3)H]thymidine incorporation into cells compared with serum-starved cells by 8 h, confirming entry into the S-phase. CL mass was unaltered at 8 h, but increased 2-fold by 16 h post-serum addition compared with serum-starved cells. The reason for the increase in CL mass upon entry into S-phase was an increase in activity and expression of CL de novo biosynthetic and remodelling enzymes and this paralleled the increase in mitochondrial mass. CL de novo biosynthesis from D-[U-(14)C]glucose was elevated, and from [1,3-(3)H]glycerol reduced, upon serum addition to quiescent cells compared with controls and this was a result of differences in the selection of precursor pools at the level of uptake. Triascin C treatment inhibited CL synthesis from [1-(14)C]oleate but did not affect [methyl-(3)H]thymidine incorporation into HeLa cells upon serum addition to serum-starved cells. Barth Syndrome lymphoblasts, which exhibit reduced CL, showed similar [methyl-(3)H]thymidine incorporation into cells upon serum addition to serum-starved cells compared with cells from normal aged-matched controls. The results indicate that CL de novo biosynthesis is up-regulated via elevated activity and expression of CL biosynthetic genes and this accounted for the doubling of CL seen during S-phase; however, normal de novo CL biosynthesis or CL itself is not essential to support nucleotide synthesis during entry into S-phase of the human cell cycle.     

Heart Mitochondrial TTP Synthesis and the Compartmentalization of TMP

The primary pathway of TTP synthesis in the heart requires thymidine salvage by mitochondrial thymidine kinase 2 (TK2). However, the compartmentalization of this pathway and the transport of thymidine nucleotides are not well understood. We investigated the metabolism of [³H]thymidine or [³H]TMP as precursors of [³H]TTP in isolated intact or broken mitochondria from the rat heart. The results demonstrated that [³H]thymidine was readily metabolized by the mitochondrial salvage enzymes to TTP in intact mitochondria. The equivalent addition of [³H]TMP produced far less [³H]TTP than the amount observed with [³H]thymidine as the precursor. Using zidovudine to inhibit TK2, the synthesis of [³H]TTP from [³H]TMP was effectively blocked, demonstrating that synthesis of [³H]TTP from [³H]TMP arose solely from the dephosphorysynthase pathway that includes deoxyuridine triphosphatelation of [³H]TMP to [³H]thymidine. To determine the role of the membrane in TMP metabolism, mitochondrial membranes were disrupted by freezing and thawing. In broken mitochondria, [³H]thymidine was readily converted to [³H]TMP, but further phosphorylation was prevented even though the energy charge was well maintained by addition of oligomycin A, phosphocreatine, and creatine phosphokinase. The failure to synthesize TTP in broken mitochondria was not related to a loss of membrane potential or inhibition of the electron transport chain, as confirmed by addition of carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone and potassium cyanide, respectively, in intact mitochondria. In summary, these data, taken together, suggest that the thymidine salvage pathway is compartmentalized so that TMP kinase prefers TMP synthesized by TK2 over medium TMP and that this is disrupted in broken mitochondria.     

Evaluation of radiation dose resulting from the ingestion of [3H]- and [14C]thymidine in the rat

Average doses to rat tissues from the ingestion of 2-[14C]thymidine were compared with those from methyl-[3H]thymidine or 6-[3H]thymidine. Among the three precursors, [14C]thymidine gave the highest dose to spleen and small intestine. The doses to other tissues from [14C]thymidine were almost the same or lower as compared with those from [3H]thymidine, irrespective of the 9 times higher beta-ray energy of 14C than that of 3H. In the case of [14C]thymidine, most of the dose was given by radioactivity incorporated into the organic tissue constituents (non-volatile radioactivity). In the case of [3H]thymidine, however, the dose contributions by non-volatile radioactivity were very small and the major contributions were rather from volatile radioactivity (3HHO), formed by degradation of [3H]thymidine. No significant difference in their total doses was found between the two [3H]precursors, but the dose from non-volatile radioactivity alone was 2-3 times higher with methyl-[3H]thymidine than with 6-[3H]thymidine. Estimates of the dose to cell nuclei in various tissues after the ingestion of [3H]thymidine were also made in order to predict more precisely possible radiation hazards.     

Herpes Simplex Virus Type 2 Functions Expressed During Stimulation of Human Cell DNA Synthesis

Experiments were designed to identify herpes simplex virus type 2 (HSV-2)-specific functions expressed during stimulation of human embryo fibroblast DNA synthesis. Cultures were partially arrested in DNA synthesis by pretreatment with 5-fluorouracil and maintenance in low-serum (0.2%) medium during virus infection. Results showed that continuous [methyl-(3)H]thymidine uptake into cellular DNA was ninefold greater in HSV-2-infected than in mock-infected cultures measured after 24 h of incubation at 42 degrees C. Shifting mock-infected cultures from low- to high-serum (10%) medium also caused some stimulation, but [methyl-(3)H]thymidine uptake was only twofold greater than in cells maintained with low serum. Plating efficiencies of both HSV-2-infected and mock-infected cells at 42 degrees C were essentially the same and ranged from 37 to 76% between zero time and 72 h of incubation. De novo RNA and protein syntheses were continuously required for HSV-2 stimulation of cellular DNA synthesis. HSV-2 infection markedly enhanced transport, phosphorylation, and rate of incorporation of [methyl-(3)H]thymidine into cellular DNA, starting at 3 h and reaching a maximum by 12 h; after 12 h, these processes gradually declined to low levels. In mock-infected cells these processes remained at low levels throughout the observation period. Pretreatment of cells with interferon or addition of arabinofuranosylthymine at the time of virus infection inhibited stimulation caused by HSV-2. 5-Bromodeoxyuridine density-labeled experiments revealed that HSV-2 stimulates predominantly semiconservative DNA replication and some DNA repair. Stimulation of [methyl-(3)H]thymidine into cellular DNA correlated with detection of virus-specific thymidine kinase activity. In conclusion, HSV-2 stimulation of cellular DNA synthesis appeared to involve at least four virus-specific functions: induction of thymidine transport, HSV-2 thymidine kinase activity, semiconservative replication, and repair of cellular DNA.     

The tritium isotope effect of sn-glycerol 3-phosphate oxidase and the effects of clofenapate and N-(2-benzoyloxyethyl)norfenfluramine on the esterification of glycerol phosphate and dihydroxyacetone phosphate by rat liver mitochondria

1. Owing to a (3)H isotope effect, the mitochondrial sn-glycerol 3-phosphate oxidase (EC 1.1.99.5) had a mean activity which was 8.4 times less with sn-[2-(3)H]-rather than with sn-[1-(14)C]glycerol 3-phosphate as a substrate. 2. A method for measuring the simultaneous synthesis of lipid from glycerol phosphate and dihydroxyacetone phosphate in rat liver mitochondria is described. 3. The lipid synthesized by rat liver mitochondria from sn-[1-(14)C]glycerol 3-phosphate was mainly phosphatidate and lysophosphatidate, whereas that synthesized from dihydroxy[1-(14)C]acetone phosphate was mainly acyldihydroxyacetone phosphate. 4. Additions of NADPH facilitated the conversion of acyldihydroxyacetone phosphate into lysophosphatidate and phosphatidate. 5. Hydrazine (1.4mm) or KCN (1.4mm) inhibited the synthesis of lipids from dihydroxyacetone phosphate but not from glycerol phosphate. 6. Clofenapate (1-2.5mm) inhibited the synthesis of lipids from dihydroxyacetone phosphate but slightly stimulated synthesis from glycerol phosphate. 7. The methanesulphonate of N-(2-benzoyloxyethyl)norfenfluramine, at 0.25-0.75mm, inhibited lipid synthesis from both glycerol phosphate and dihydroxyacetone phosphate.     

The subcellular location in rat kidney of the peripheral benzodiazepine acceptor

In rat kidney high-affinity binding sites for [3H]Ro-5-4864 and [3H]PK-11195 with the properties of the peripheral-type acceptor were found enriched in mitochondrial (M) and light-mitochondrial-lysosomal (L) fractions on differential centrifugation. When the combined M and L fractions were subjected to sucrose density gradient centrifugation, these binding sites were found enriched at a density of 1.155 g/ml coincident with a population of light mitochondria, whereas a population of heavier mitochondria (rho = 1.175 g/ml) had few or no binding sites. Transmission electron microscopy showed that whereas the heavier mitochondria appeared highly pure and intact, the lighter mitochondria appeared less intact and to be contaminated with vesicular structures. After fractionation of the light mitochondria and vesicles by centrifugation, both fractions showed the same ratio of [3H]Ro-4864 binding sites to monoamine oxidase activity consistent with the vesicles being of mitochondrial outer-membrane origin. Digitonin pre-treatment had no effect on the density of acceptor-rich fractions on sucrose density gradient centrifugation. However, pretreatment with succinate/iodophenylnitrophenylphenyltetrazolium (INT) perturbed equally the density of acceptor-rich fractions and mitochondrial marker enzymes. When mitochondrial fractions were subjected to sonication prior to density gradient centrifugation the binding sites were now found highly enriched in a much lighter fraction coincident with the monoamine oxidase activity and thus consistent with being outer-membrane vesicles. When a mitochondrial fraction was subjected to hypotonic treatment before assay no evidence for activation/unmasking of binding sites was found. The hypotonic treatment did not release any inhibitor of the binding sites. These results are consistent with the peripheral benzodiazepine acceptor having an outer-membrane location on a sub-population of rat kidney mitochondria. Those mitochondria showing high levels of the acceptor are either light mitochondria or appear more susceptible to osmotic damage than those mitochondria in which the acceptor is absent or at low levels.     

3H]thymidine incorporation into whole liver as an alternative to [3H]thymidine incorporation into DNA as a parameter of cell proliferation in regenerating liver tissue in rats

OBJECTIVE: To monitor liver regeneration following partial hepatectomy, liver cell proliferation can be measured by assaying in vivo [3H]thymidine incorporation into liver cell DNA. We hypothesized that [3H]thymidine incorporation into whole liver tissue parallels [3H]thymidine incorporation into liver cell DNA, both in high proliferating and low proliferating liver. STUDY DESIGN: Liver cell proliferation in rats after partial hepatectomy or a sham operation was studied by measuring incorporation of [3H]thymidine into various fractions of liver tissue on days 1, 2, 3, 4 and 10 after surgery. RESULTS: [3H]thymidine incorporation into whole liver tissue and in the protein fraction correlated well with DNA-specific [3H]thymidine incorporation into regenerating (r > .80, P < .0001) and nonregenerating liver (r > .69, P < .005). [3H]thymidine incorporation into DNA was < 5% of the total amount of administered [3H]thymidine in both sham-operated and hepatectomized rats. Significant differences in [3H]thymidine incorporation into partially hepatectomized livers as compared to sham-operated rat livers were found on days 1 and 2 (whole liver tissue and protein fraction) or day 1 (DNA) after surgery. CONCLUSION: [3H]thymidine incorporation into whole liver tissue is a simple technique that can be used for the study of liver cell proliferation after partial hepatectomy in rats.     

Enzymes of deoxythymidine triphosphate biosynthesis in Neurospora crassa mitochondria.

Intact mitochondria of Neurospora crassa incorporate deoxythymidine 5'-monophosphate (dTMP) into deoxyribonucleic acid but not the label from (methyl-3H) deoxythymidine. Mitochondrial homogenates contain deoxythymidylate kinase (EC 2.7.4.9), deoxycytidylate aminohydrolase (dCMP deaminase) (EC 3.5.4.12), and thymidylate synthetase (EC 2.1.1b), but not thymidine kinase (EC 2.7.1.21) activity. dTMP kinase is loosely bound to the mitochondrial membrane and is solubilized by 0.4 M KCl in mitochondrial homogenates, the dCMP aminohydrolase deaminase) is bound to the inner membrane and is not solubilized by 0.4 M KCl. dTMP synthetase activity is found in the 2,000 times g particulate fractions by homogenization of mitochondria in 0.4 M KCl. The dCMP deaminase activity found in the particulate fraction of the inner membrane is efficiently regulated by the products of the pathway: deoxycytidine 5'-triphosphate activates whereas deoxythymidine 5'-triphosphate inhibits, as found for the soluble enzyme from other sources. These data indicate that mitochondria of N. crassa contain specific enzymes for the biosynthesis of deoxythymidine triphosphate.     

Insulin-like growth factor I (IGF-I) production and the presence of IGF-I receptors in rat medullary thyroid carcinoma cell line 6-23 (clone 6)

To clarify whether insulin-like growth factor I (IGF-I) is an autocrine growth factor of rat medullary thyroid carcinoma (MTC) cell line, 6-23 (clone 6), IGF-I binding to MTC cell membranes, IGF-I levels in the conditioned culture medium of MTC cells and the effects of IGF-I on methyl-[3H]thymidine incorporation to MTC cells were examined. Scatchard analysis of saturation binding studies revealed the association constant and the maximal binding capacity were 1.0 x 10(9) M-1 and 199 fmol/mg of membrane protein, respectively. The binding of [125I]IGF-I to MTC cell membranes was inhibited by unlabeled IGF-I, IGF-II and insulin; the relative potencies were IGF-I greater than IGF-II much greater than insulin, suggesting the presence of type I IGF receptors in MTC cells. IGF-I levels in the conditioned culture medium of MTC cells were 120 +/- 3 pM (mean + SE). IGF-I (10(-10) to 10(-8) M) dose-dependently stimulated methyl-[3H]thymidine incorporation to MTC cells. These findings suggest a possible role of IGF-I as an autocrine growth factor for MTC cells.     

Biogenesis of mammalian mitochondria in the renoprival kidney. II. Aspects of mitochondrial DNA synthesis

Mitochondrial DNA (m-DNA) content and factors which might control its concentration were investigated in the renoprival kidney at various times after unilateral nephrectomy. On the basis of mitochondrial protein, m-DNA increased 30% in the renoprival kidney at 24 hr and returned to normal by 48 hr. The total tissue content of m-DNA was also increased at 24 hr. The specific activity of [³H]thymidine incorporated into m-DNA in vivo was decreased markedly at 24 hr after mononephrectomy; at the same time there was a threefold increase of [³H]thymidine incorporation into total cellular DNA. The incorporation into m-DNA was above normal at 48 hr. The mitochondrial specific DNase was decreased 60% at 24 and 36 hr post-mononephrectomy. There was no significant difference in the total radioactivity or total optical density at 260 nm of the acid soluble extract from mitochondria isolated at various times after mononephrectomy. The incorporation of [³H]thymidine into TMP and TDP in the renoprival kidney was not different from normal but there was a decrease in the incorporation into TTP. It is suggested that the increase in mitochondrial DNA could be due to a decrease in the rate of degradation rather than an increase in synthesis.     

Alterations of MCF-7 human breast cancer cell after prostaglandins PGA1 and PGF2 alpha treatment

Treatment of monolayer cultures of MCF-7 cells with prostaglandins PGA1 and PGF2 alpha inhibited cell proliferation, reduced the rate of labeled precursor incorporation into DNA, RNA, and protein, and induced morphological changes in a dose-dependent manner. The rate of [3H]thymidine incorporation was increased by PGA1 at 10(-10)-10(-8) M, while a sharp decrease was observed at 10(-6)-10(-4) M (p less than 0.05 and p less than 0.005). PGF2 alpha inhibited [3H]thymidine incorporation at all concentrations tested. Similar results were obtained for [3H]uridine incorporation with both PGs. PGA1 inhibited [3H]leucine incorporation at 10(-4) M, but increased incorporation at 10(-10)-10(-6) M. At the ultrastructural level, neither PG induced morphological alterations at 10(-12)-10(-8) M. However, at 10(-6)-10(-4) M both PGA1 and PGF2 alpha diminished the number and size of cell surface projections; some cells appeared to completely lack microvilli. Disorganization of mitochondrial cristae and increased electron density of the matrix were also evident.     

Identification of the protein responsible for pyruvate transport into rat liver and heart mitochondria by specific labelling with [3H]N-phenylmaleimide.

1. N-Phenylmaleimide irreversibly inhibits pyruvate transport into rat heart and liver mitochondria to a much greater extent than does N-ethylmaleimide, iodoacetate or bromopyruvate. alpha-Cyanocinnamate protects the pyruvate transporter from attack by this thiol-blocking reagent. 2. In both heart and liver mitochondria alpha-cyanocinnamate diminishes labelling by [3H]N-phenylmaleimide of a membrane protein of subunit mol.wt. 15000 on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. 3. Exposure of mitochondrial to unlabelled N-phenylmaleimide in the presence of alpha-cyanocinnamate, followed by removal of alpha-cyanocinnamate and exposure to [3H]N-phenylmaleimide, produced specific labelling of the same protein. 4. Both labelling and kinetic experiments with inhibitors gave values for the approximate amount of carrier present in liver and heart mitochondria of 100 and 450 pmol/mg of mitochondrial protein respectively. 5. The turnover numbers for net pyruvate transport and pyruvate exchange at 0 degrees C were 6 and 200 min-1 respectively.     

Selective incorporation of thymidine in the organelle DNA of Polytoma obtusum

Polytoma obtusum was found to selectively incorporate exogenous thymidine into its leukoplast DNA. The nuclear DNA was unable to incorporate [3H]thymidine, although both DNA species could be labeled with radioactive adenine with similar efficiencies. The mitochondrial DNA (mitDNA), which had a buoyant density of 1.714 g/ml and was banded slightly on the heavier side of the nuclear DNA peak, was also found to incorporate a small amount of [3H]thymidine. These observations suggest that P. obtusum lack cytoplasmic thymidine kinase, whereas the enzyme is present in both leukoplast and mitochondria.     

Excitatory Amino Acids Stimulate Inositol Phospholipid Hydrolysis and Reduce Proliferation in Cultured Astrocytes

Excitatory amino acids stimulated inositol phospholipid hydrolysis in primary cultures of astrocytes, as reflected by an increased formation of [3H]inositol monophosphate [( 3H]InsP) in the presence of 10 mM Li+. Quisqualate was the most potent activator of inositol phospholipid hydrolysis, followed by glutamate and ibotenate. Kainate exhibited low activity, whereas N-methyl-D-aspartate (NMDA) and alpha-amino-3-hydroxy-5-methylisoxazolepropionate (AMPA) were inactive. The increase in [3H]InsP formation induced by glutamate was potentiated after 12-h exposure to the proliferative agent epidermal growth factor (EGF), suggesting that activation of the mitotic cycle leads to an enhanced coupling of glutamate recognition sites with phospholipase C. To study how glutamate receptors are involved in regulating cell proliferation, we have measured [methyl-3H]thymidine incorporation in cultured astrocytes. Excitatory amino acids reduced thymidine incorporation with a pharmacological profile similar to that observed for the stimulation of inositol phospholipid hydrolysis. Quisqualate acted as a potent antiproliferative agent, both under basal conditions and in cells stimulated to proliferate by addition of EGF or phorbol 12-tetradecanoate 13-acetate. Glutamate and ibotenate reduced [methyl-3H]thymidine incorporation at high concentrations, whereas kainate, AMPA, and NMDA were virtually inactive. The action of quisqualate on both inositol phospholipid hydrolysis and thymidine incorporation was attenuated by 2-amino-4-phosphonobutyrate, which acted as a weak agonist/competitive antagonist. Other excitatory amino acid receptor antagonists were not effective.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mitochondrial DNA is synthesized during meiosis and spermiogenesis in the mouse

Following intratesticular injection of [3H]thymidine, spermatozoa were isolated from the caput and cauda epididymis of CD-1 mice. Quantitation of the amount of radiolabelled DNA in whole spermatozoa, sperm heads (nuclei) and sperm midpiece-tails (mitochondria) at time intervals 3-36 days postinjection demonstrated mitochondrial DNA (mtDNA) is synthesized during meiosis and early spermiogenesis. mtDNA synthesis terminates in mid-spermiogenesis suggesting that the mitochondrial genome in spermatozoa has a half-life of at least 15 days.     

ORAL CHEMORECEPTOR ORGANS OF BULLFROG TADPOLES DURING METAMORPHOSIS*

Degeneration of the premetamorphic papillae and development of the fungiform papillae during metamorphosis of bullfrog tadpoles were investigated by electrophysiological and scanning electron microscopic methods. Premetamorphic papillae were observed during the early metamorphic stages, and these degenerated rapidly at about metamorphic stage 20. The anlage of the tongue appeared at about metamorphic stage 10, but the anlage of the fungiform papillae appeared at about metamorphic stage 18. The microvilli at the apex of the fungiform papillae were observed at about metamorphic stage 21. At metamorphic stage 24 the fungiform papillae had a similar structure to that of adult frogs. Taste responses were recorded from the glossopharyngeal nerve of the tadpole. The responses to 1 M sucrose and 0.01 M quinine hydrochloride could be observed at metamorphic stage 6 or later, though during stage 20 the responses were very weak. The response to 0.02 M ammonium chloride appeared at metamorphic stage 6, but disappeared at stage 20 and did not reappear later. These results indicate that the fungiform papillae become functional as chemo-receptor organs at about metamorphic stage 21 and that, before the fungiform papillae function, the premetamorphic papillae serve as chemoreceptor organs in the tadpole.     

Modification of the biosynthesis and composition of polyglycerophosphatides in outer and inner mitochondrial membranes by cytidine liponucleotides

The biosynthesis of [3H]polyglycerophosphatides ([3H]phosphatidylglycerophosphate and [3H]phosphatidylglycerol) in mitochondrial and submitochondrial (outer and inner) membranes isolated from guinea pig liver was examined. Experimental results have established that the amount of biosynthesized [3H]polyglycerophosphatides and the relative amounts of biosynthesized [3H]phosphatidylglycerol and [3H]phosphatidylglycerolphosphate can be influenced by varying the composition of fatty acids in CDP-diglycerides and by altering the incubation time of the mixture containing CDP-diglycerides (obligatory precursor), sn-[2-3H]glycerol-3-phosphate and mitochondria or submitochondrial membranes. The changes thus obtained in respect to the amount and composition of biosynthesized [3H]polyglycerophosphatides are different in mitochondria and submitochondrial membranes. The highest amount of biosynthesized [3H]polyglycerophosphatides was obtained with CDP-didecanoin and inner mitochondrial membranes. The greatest accumulation of [3H]phosphatidylglycerol with CDP-didecanoin was obtained in mitochondria and outer mitochondrial membranes, while in inner mitochondrial membranes the amounts of [3H]phosphatidylglycerol and [3H]phosphatidylglycerolphosphate accumulated were approximately the same. In general, prolongation of the incubation time decreased the relative amounts of [3H]phosphatidylglycerolphosphate and increased the amount of accumulated [3H]phosphatidylglycerol, but the absolute amounts of these [3H]polyglycerophosphatides were more dependent on fatty acid composition of CDP-diglycerides tested. The following cytidine liponucleotides were tested: CDP-didecanoin, CDP-dipalmitin, CDP-diolein, and CDP-diglycerides containing saturated and unsaturated fatty acids similar to those in egg yolk lecithin. The formation of [3H]cardiolipin from [3H]phosphatidylglycerol in the presence of CDP-didecanoin and Mn2+ was found in both the outer and inner mitochondrial membranes.     

Measurement of the formation of betaine aldehyde and betaine in rat liver mitochondria by a high pressure liquid chromatography-radioenzymatic assay.

A new assay procedure for measurement of rat liver mitochondrial choline dehydrogenase was developed. Oxidation of [methyl-14C]choline to [methyl-14C]betaine aldehyde and [methyl-14C]betaine was measured after isolating these compounds using HPLC. We observed that NAD+ was required for conversion of betaine aldehyde to betaine in rat liver mitochondria. In the absence of this cofactor, oxidation of choline led to the accumulation of betaine aldehyde. The apparent Km of the mitochondrial choline dehydrogenase for choline was 0.14-0.27 mM, which is significantly lower than previously reported. A partially purified preparation of choline dehydrogenase catalyzed betaine aldehyde formation only in the presence of exogenous electron acceptors (e.g., phenazine methosulfate). This preparation failed to catalyze the formation of betaine even in the presence of NAD+, indicating that betaine aldehyde dehydrogenase may be a separate enzyme from choline dehydrogenase.     

Control of catabolism of mitochondria in oncogenesis. I. Increase of hepatic mitochondrial population during feeding inactive or marginally active amino azo dyes: absence of de novo biosynthesis

The mechanism of the well established phenomenon that the number of liver mitochondria increases during administration of 2-methyl-4-dimethylaminoazobenzene (2-Me-DAB) has been investigated. Fed to rats, both 2-Me-DAB (0.06%) and 4-diethylaminoazobenzene (4-DEAB) (0.0635 %) increase the amount of liver mitochondria by 47% and 31%, respectively. It was established that this is not due to de novo mitochondriogenesis. The increase in the amount of mitochondria correlates with an ~ 10% decrease of total liver protein per g of tissue. Mitochondrial ATP synthesis, which is a prerequisite of any anabolic situation, is drastically impaired following feeding of 2-Me-DAB beyond 1 week as indicated by a very substantial decrease of State 3 respiration, the respiratory control index, and the $\text{ADP}\text{O}$ ratio. Determination of the polysome profile and polysome/monosome ratio at intervals during 2-Me-DAB administration showed no change, despite the fact that mitochondrial components are coded for in both nuclear and mitochondrial DNA. During 4-DEAB administration there was, however, a small but definite rise of the polysome/monosome ratio. Administration of 2-Me-DAB up to 42 days brought about drastic inhibition of the incorporation of [³H]thymidine into both DNA's (approx. 59% with mitochondrial DNA and approx. 77% with nuclear DNA), indicating that these templates could not possibly be involved in the substantial increase of the mitochondrial population. The data suggest that the increase results from a steady accumulation due to increase of the half-life of mitochondria, owing possibly to an inhibition of lysosomal catabolic enzymes.     

Acclimation,Hydrogen Peroxide,and Abscisic Acid Protect Mitochondria against Irreversible Chilling Injury in Maize Seedlings

Our previous results indicated that 3-d-old dark-grown chilling-sensitive maize (Zea mays L.) seedlings did not survive 7 d of 4[deg]C chilling stress, but 69% of them survived similar stress when the seedlings were either preexposed to 14[deg]C for 3 d or pretreated with 0.1 mM H2O2 for 4 h at 27[deg]C (T.K. Prasad, M.D. Anderson, B.A. Martin, C.R. Stewart [1994] Plant Cell 6: 65-74) or 1 mM abscisic acid (ABA) for 24 h at 27[deg]C (M.D. Anderson, T.K. Prasad, B.A. Martin, C.R. Stewart [1994] Plant Physiol 105: 331-339). We discovered that chilling imposed oxidative stress on the seedlings. Since H2O2 accumulated during the periods of both acclimation and nonacclimation, we concluded that H2O2 had dual effects at low temperature: (a) During acclimation, its early transient accumulation signals the induction of antioxidant enzymes such as catalase 3 and peroxidase to scavenge H2O2; and (b) at 4[deg]C in nonacclimated seedlings, it accumulates to damaging levels in the tissues because of low levels of these and perhaps other antioxidant enzymes. Three-day-old seedlings pretreated with H2O2 (a mild oxidative stress) or ABA showed induced chilling tolerance. In the present study, we investigated whether mitochondria are a target for chilling-induced oxidative stress and, if so, what differences do acclimation, H2O2, or ABA make to protect mitochondria from irreversible chilling injury. The results indicated that chilling, in general, impairs respiratory activity, the cytochrome pathway of electron transport, and ATPase activity regardless of the treatment. In pretreated seedlings, the activities of catalase 3 and peroxidase in the mitochondria increased severalfold compared with control and nonacclimated seedlings. The increases in these antioxidant enzymes imply that mitochondria are under oxidative stress and such increases could initiate a protective mechanism in the mitochondria. Mitochondrial respiration is partially cyanide resistant during chilling stress and also after the 1st d of recovery. Upon further recovery over 3 d, in contrast to nonacclimated seedlings, the mitochondria of acclimation-, H2O2-, and ABA-treated seedlings showed the following recovery features. (a) The mitochondrial respiration changed from a cyanide-resistant to a cyanide-sensitive cytochrome pathway, (b) cytochrome oxidase activity recovered to control levels, (c) the ability of mitochondria to generate ATP was regained, and (d) the antioxidant enzyme activities remained at or above control levels. Based on these results, we conclude that chilling impairs mitochondrial function and that chilling-induced oxidative stress seems to be a factor, at least in part, for causing possible irreversible damage to the mitochondrial membrance components. Acclimation, H2O2, and ABA provide a protective mechanism by inducing antioxidant enzymes to protect mitochondria from irreversible oxidative damage that is absent in nonacclimated seedlings. Therefore, we conclude that the ability of the seedlings to recover from chilling injury is, at least in part, due to the ability of the mitochondria to resume normal function.