EXPRESSION OF THE MITOCHONDRIAL GENOME IN HELA CELLS : XV. Effect of Inhibition of Mitochondrial Protein Synthesis on Mitochondrial Formation

The effect of selective inhibition of mitochondrial protein synthesis by chloramphenicol at 40 or 200 µg/ml on the formation of mitochondria in HeLa cells was investigated. HeLa cells, under the conditions used in the present work, grow at a decreasing rate for at least four cell generations in the presence of 40 µg/ml chloramphenicol, and for two generations in the presence of 200 µg/ml chloramphenicol. The progressive cell growth inhibition which begins after 2 days of exposure of the cells to 40 µg/ml chloramphenicol is immediately or gradually reversible, upon removal of the drug, for periods up to at least 8 days of treatment, though there is a progressive loss of cloning efficiency. In cells which have been treated for 6–7 days with 40 or 200 µg/ml of chloramphenicol, mitochondrial protein synthesis occurs at a normal or near-normal rate 1 h after removal of the drug. Mitochondria increase normally in number and show a normal size and amount of cristae in the presence of either concentration of drug. However, in 4–5% of the mitochondrial profiles the cristae appear to be arranged in unusual, circular, looped or whorled configuration.     

ESTABLISHMENT AND CHARACTERIZATION OF ETHIDIUM BROMIDE RESISTANCE IN SIMIAN VIRUS 40-TRANSFORMED HAMSTER CELLS : Effects on Mitochondria In Vivo

This study describes the isolation and subsequent characterization of four mammalian cell lines resistant to ethidium bromide (EB). Treatment of the simian virus 40- (SV40) transformed hamster cell line F5-1 first led to the establishment of the F2 cell line, which is resistant to 2 µg EB/ml. At this concentration cytochromes c and b are present in almost normal or only slightly diminished amounts, whereas cytochromes a + a₃ show an obvious decrease. The mitochondria of the F2 cell show a normal ultrastructure, not distinct from the parental cell line F5-1, and contain closed circular DNA. The sensitive parental F5-1 cells, however, when exposed to the same dye concentration exhibit the typical EB-induced ultrastructural changes in the mitochondria, and no more component I mitochondrial DNA can be demonstrated. 1 yr after establishment we derived from the F2 cell three more cell lines, resistant against 4, 8, and 16 µg of EB/ml. These cell lines, termed F4, F8, and F16, respectively, also revealed relatively intact-appearing mitochondria, although distinguishable from F5-1 and F2 mitochondria by a more condensed or unorthodox cristae conformation. F4, F8, and F16 cell lines contained closed circular mitochondrial DNA in the same position as that of the parental F5-1 cells, when analyzed in an isopycnic CsCl-EB gradient. A small shoulder at the lower density side of the DNA I peaks was observed. The newly acquired drug resistance of the F cells is hereditarily transmitted to the progeny cells and retained even after a period of growth in EB-free medium.     

Expression of blaA Underlies Unexpected Ampicillin-Induced Cell Lysis of Shewanella oneidensis

Shewanella oneidensis is a facultative anaerobic γ-proteobacterium possessing remarkably diverse respiratory capacities for reducing various organic and inorganic substrates. As a veteran research model for investigating redox transformations of environmental contaminants the bacterium is well known to be a naturally ampicillin-resistant microorganism. However, in this study we discovered that ampicillin has a significant impact on growth of S. oneidensis. Particularly, cell lysis occurred only with ampicillin at levels ranging from 0.49 to 6.25 µg/ml but not at 50 µg/ml. This phenotype is attributable to insufficient expression of the β-lactamase BlaA. The subsequent analysis revealed that the blaA gene is strongly induced by ampicillin at high (50 µg/ml), but not at low levels (2.5 µg/ml). In addition, we demonstrated that penicillin binding protein 5 (PBP5), the most abundant low molecular weight PBP (LMW PBP), is the only one relevant to β-lactam resistance under the tested conditions. This nonessential PBP, largely resembling its Escherichia coli counterpart in functionality, mediates expression of the blaA gene.     

Some features of mitochondria and fluffy layer in regenerating rat liver

1. Mitochondria and fluffy layer were prepared from control and regenerating rat liver. Differential and density-gradient centrifugation were used to fractionate the preparations, which were examined for protein content, density and the activity of cytochrome c oxidase, succinate dehydrogenase, NAD–isocitrate dehydrogenase and NADP–isocitrate dehydrogenase. 2. During regeneration the mitochondrial protein content of the liver fell by 18% from the control value of 18·4mg. of protein/g. of liver (wet wt.) and by 3 weeks had risen to 130% of the control value. It then declined slowly. 3. The fluffy-layer protein content (4·7mg./g. of liver) varied inversely as the mitochondrial content and increased by 70% in the early stages (10 days) of liver regeneration. The results suggest that fluffy layer may partially represent both partly formed and broken-down mitochondria. 4. NAD– and NADP–isocitrate dehydrogenases differed in their behaviour during liver regeneration. 5. The succinate-dehydrogenase and NADP–isocitrate-dehydrogenase activity of fluffy layer was high and rose during the early stages of liver regeneration (1 week). Succinate dehydrogenase and cytochrome c oxidase were concentrated in the lighter fluffy-layer particles 10 days to 3 weeks after partial hepatectomy. The significance of this with respect to mitochondrial formation is discussed. 6. Mitochondrial fractions possessed a certain degree of heterogeneity in enzymic activity when separated according to size and density. The mean density of heavy mitochondria was 1·198, light mitochondria 1·193. Fluffy layer was nearly homogeneous in control liver, but during regeneration considerable heterogeneity became evident. The significance of the heterogeneity is discussed.     

ROLE OF THE MITOCHONDRIAL GENOME DURING EARLY DEVELOPMENT IN MICE : Effects of Ethidium Bromide and Chloramphenicol

The role of the mitochondrial genome in early development and differentiation was studied in mouse embryos cultured in vitro from the two to four cell stage to the blastocyst (about 100 cells). During this period the mitochondria undergo morphological differentiation: progressive enlargement followed by an increase in matrix density, in number of cristae, and in number of mitochondrial ribosomes. Mitochondrial ribosomal and transfer RNA synthesis occurs from the 8 to 16 cell stage on and contributes to the establishment of a mitochondrial protein-synthesizing system. Inhibition of mitochondrial RNA- and protein-synthesis by 0.1 µg/ml of ethidium bromide or 31.2 µg/ml of chloramphenicol permits essentially normal embryo development and cellular differentiation. Mitochondrial morphogenesis is also nearly normal except for the appearance of dilated and vesicular cristae in blastocyst mitochondria. Such blastocysts are capable of normal postimplantation development when transplanted into the uteri of foster mothers. Higher concentrations of these inhibitors have general toxic effects and arrest embryo development. It is concluded that mitochondrial differentiation in the early mouse embryo occurs through the progressive transformation of the preexisting mitochondria and is largely controlled by the nucleocytoplasmic system. Mitochondrial protein synthesis is required for the normal structural organization of the cristae in blastocyst mitochondria. Embryo development and cellular differentiation up to the blastocyst stage are not dependent on mitochondrial genetic activity.     

DIRECT COUNTING AND SIZING OF MITOCHONDRIA IN SOLUTION

Resistive particle counting has been developed for the accurate sizing and counting of mitochondria in solution. The normal detection limit with a 30 µ aperture is 0.48 µ diameter, or 0.056 µ³ particle volume The mean volume of rat liver mitochondria was 0.42 µ³ or 0.93 µ in diameter. The average value for numbers of particles per milligram of mitochondrial protein was 4.3 x 10³, and per gram of rat liver was about 11 x 10¹⁰. These values compare satisfactorily with those derived by light microscopy and electron microscopy. The mean volume for mitochondria from rat heart was 0 60 µ³ and from rat kidney cortex, 0.23 µ³. These values agree within 15% of those determined by electron microscopy of whole tissue. Mitochondrial fragility and contaminating subcellular organelles were shown to have little influence on the experimentally determined size distributions The technique may be applied to rapid swelling studies, as well as to estimations of the number and size of mitochondria from animals under different conditions such as liver regeneration and hormonal, pathological, or drug-induced states Mitochondrial DNA, RNA, cytochrome c-oxidase, cytochrome (a ÷ a₃), and iron were nearly constant per particle over large differences in particle size. Such data may be particularly valuable for biogenesis studies and support the hypothesis that the net amount per particle of certain mitochondrial constituents remains constant during mitochondrial growth and enlargement     

Toxicity of Tioxazafen to Meloidogyne Incognita and Rotylenchulus Reniformis

Tioxazafen is a seed-applied nematicide used in row crops. Currently, there are no data on nematode toxicity, nematode recovery, or effects of low concentrations of tioxazafen on nematode infection of a host root for Meloidogyne incognita or Rotylenchulus reniformis. Nematode toxicity and recovery experiments were conducted in water solutions of tioxazafen, while root infection assays were conducted on tomato. Nematode paralysis was observed after 24 hr of exposure at 27.0 µg/ml tioxazafen for both the nematode species. Based on an assay of nematode motility, 24-hr EC₅₀ values of 57.69 µg/ml and 59.64 µg/ml tioxazafen were calculated for M. incognita and R. reniformis, respectively. Tioxazafen rates of 2.7 µg/ml and 27.0 µg/ml reduced the nematode hatch after 3 d of exposure for both the nematode species. There was no recovery in nematode motility after the 24-hr exposure of M. incognita and R. reniformis to their corresponding 48-hr EC₅₀ values of 47.15 µg/ml and 47.25 µg/ml tioxazafen, respectively. Mortality of M. incognita continued to increase after 24 hr exposure, whereas R. reniformis mortality remain unchanged after nematodes were rinsed and removed for 48 hr from the tioxazafen solution. A 24-hr exposure to low concentrations of 0.38 to 47.15 µg/ml for M. incognita and 47.25 µg/ml for R. reniformis reduced the infectivity of each nematode species on tomato roots. The toxicity of tioxazafen was similar between nematode species; however, a greater rate of tioxazafen was needed to suppress R. reniformis infection of tomato than for M. incognita.     

The alpha-subunit of the maize F(1)-ATPase is synthesised in the mitochondrion

The F₁-ATPase complex has been purified from maize (Zea mays L.) mitochondria and shown to consist of five subunits with mol. wts. of 58 000 (α), 56 000 (β), 35 000 (γ), 22 000 (δ) and 8000 (ε). The α-subunit co-migrates on one- and two- dimensional isoelectric focussing-SDS polyacrylamide gels with the major polypeptide synthesised by isolated mitochondria. One-dimensional proteolytic peptide mapping and immunoprecipitation confirms that the α-subunit is a mitochondrial translation product and therefore presumably encoded in mitochondrial DNA. This contrasts with the situation in animal and fungal cells where all five subunits of the F₁-ATPase are encoded by the nuclear genome and synthesised on cytosolic ribosomes.     

Respiration and Alternative Oxidase in Corn Seedling Tissues during Germination at Different Temperatures

Respiration rates of Zea mays L. seedling tissues grown at 30 and 14°C were measured at 25°C at different stages of seedling growth. Accumulation of heat units was used to define the developmental stages to compare respiration between the two temperatures. At both temperatures, respiration rates of most tissues were highest at the youngest stages, then declined with age. Respiration rates of mesocotyl tissue were the most responsive to temperature, being nearly twofold higher when grown at 14 compared to 30°C. Alternative pathway respiration increased concomitantly with respiration and was higher in mesocotyls grown in the cold. When seedlings were started at 30 then transferred to 14°C, the increase in alternative pathway respiration due to cold was not observed unless the seedlings were transferred before 2 days of growth. Seedlings transferred to 14°C after growth at 30°C for 2 days had the same alternative oxidase capacity as seedlings grown at 30°C. Seedlings grown at 14°C for 10 to 12 days, then transferred to 30°C, lost alternative pathway respiratory capacity over a period of 2 to 3 days. Western blots of mitochondrial proteins indicated that this loss of capacity was due to a loss of the alternative oxidase protein. Some in vitro characteristics of mitochondria were determined. The temperature optimum for measurement of alternative oxidase capacity was 15 to 20°C. At 41°C, very little alternative oxidase was measured, i.e., the mitochondrial oxygen uptake was almost completely sensitive to cyanide. This inactivation at 41°C was reversible. After incubation at 41°C, the alternative oxidase capacity measured at 25°C was the similar to when it was measured at that temperature directly. Isolated mitochondria lost alternative oxidase capacity at the same rate when incubated at 41°C as they did when incubated at 25°C. Increasing the supply of electrons to isolated mitochondria increased the degree of engagement of the alternative pathway, whereas lower temperature decreased the degree of engagement. Lower temperatures did not increase the degree of engagement of the pathway in intact tissues. We interpret these observations to indicate that the greater capacity of alternative oxidase in cold-grown seedlings is a consequence of development at these low temperatures which results in elevated respiration rates. Low temperature itself does not cause greater capacity or engagement of the alternative oxidase in mitochondria that have developed under warm temperatures. Our hypothesis would be that the low growth temperatures require the seedlings to have a higher respiration rate for some reason, e.g., to prevent the accumulation of a toxic metabolite, and that the alternative pathway functions in that respiration.     

Temperature Effects on Mitochondrial Respiration in Phaseolus acutifolius A. Gray and Phaseolus vulgaris L

Electron transport, using succinate as a substrate, was measured polarographically in mitochondria isolated from Phaseolus vulgaris and P. acutifolius plants at 25°C and 32°C. Mitochondria isolated from P. vulgaris plants grown at 32°C had reduced electron transport and were substantially uncoupled. Growth at 32°C had no effect on electron transport or oxidative phosphorylation in P. acutifolius compared to 25°C grown plants. Mitochondria isolated from 25°C grown P. vulgaris plants measured at 42°C were completely uncoupled. Similarly treated P. acutifolius mitochondria remained coupled. The uncoupling of P. vulgaris was due to increased proton permeability of inner mitochondrial membrane. The alternative pathway was more sensitive to heat than the regular cytochrome pathway. At 42°C, no alternative pathway activity was detected. The substantially greater heat tolerance of P. acutifollus compared to P. vulgaris mitochondrial electron transport suggests that mitochondrial sensitivity to elevated temperatures is a major limitation to growth of P. vulgaris at high temperatures and is an important characteristic conveying tolerance in P. acutifolius.     

Quantification of Campylobacter spp. in Chicken Rinse Samples by Using Flotation prior to Real-Time PCR

Real-time PCR is fast, sensitive, specific, and can deliver quantitative data; however, two disadvantages are that this technology is sensitive to inhibition by food and that it does not distinguish between DNA originating from viable, viable nonculturable (VNC), and dead cells. For this reason, real-time PCR has been combined with a novel discontinuous buoyant density gradient method, called flotation, in order to allow detection of only viable and VNC cells of thermotolerant campylobacters in chicken rinse samples. Studying the buoyant densities of different Campylobacter spp. showed that densities changed at different time points during growth; however, all varied between 1.065 and 1.109 g/ml. These data were then used to develop a flotation assay. Results showed that after flotation and real-time PCR, cell concentrations as low as 8.6 × 10² CFU/ml could be detected without culture enrichment and amounts as low as 2.6 × 10³ CFU/ml could be quantified. Furthermore, subjecting viable cells and dead cells to flotation showed that viable cells were recovered after flotation treatment but that dead cells and/or their DNA was not detected. Also, when samples containing VNC cells mixed with dead cells were treated with flotation after storage at 4 or 20°C for 21 days, a similar percentage resembling the VNC cell fraction was detected using real-time PCR and 5-cyano-2,3-ditolyl tetrazolium chloride-4′,6′-diamidino-2-phenylindole staining (20% ± 9% and 23% ± 4%, respectively, at 4°C; 11% ± 4% and 10% ± 2%, respectively, at 20°C). This indicated that viable and VNC Campylobacter cells could be positively selected and quantified using the flotation method.     

Mitochondria Are the Target Organelle of Differentiation-Inducing Factor-3, an Anti-Tumor Agent Isolated from Dictyostelium Discoideum

Differentiation-inducing factor-3 (DIF-3), found in the cellular slime mold Dictyostelium discoideum, and its derivatives such as butoxy-DIF-3 (Bu-DIF-3) are potent anti-tumor agents. However, the precise mechanisms underlying the actions of DIF-3 remain to be elucidated. In this study, we synthesized a green fluorescent derivative of DIF-3, BODIPY-DIF-3, and a control fluorescent compound, Bu-BODIPY (butyl-BODIPY), and investigated how DIF-like molecules behave in human cervical cancer HeLa cells by using both fluorescence and electron microscopy. BODIPY-DIF-3 at 5–20 µ M suppressed cell growth in a dose-dependent manner, whereas Bu-BODIPY had minimal effect on cell growth. When cells were incubated with BODIPY-DIF-3 at 20 µM, it penetrated cell membranes within 0.5 h and localized mainly in mitochondria, while Bu-BODIPY did not stain the cells. Exposure of cells for 1–3 days to DIF-3, Bu-DIF-3, BODIPY-DIF-3, or CCCP (a mitochondrial uncoupler) induced substantial mitochondrial swelling, suppressing cell growth. When added to isolated mitochondria, DIF-3, Bu-DIF-3, and BOIDPY-DIF-3, like CCCP, dose-dependently promoted the rate of oxygen consumption, but Bu-BODIPY did not. Our results suggest that these bioactive DIF-like molecules suppress cell growth, at least in part, by disturbing mitochondrial activity. This is the first report showing the cellular localization and behavior of DIF-like molecules in mammalian tumor cells.     

Metabolism of propionate by sheep liver. Stimulation of the mitochondrial rate by factors from the cell sap

1. The rate of metabolism of propionate by aged sheep-liver mitochondria in the presence of oxygen + carbon dioxide (95:5) was 5·0 (± s.e.m. 0·8) μmoles/mg. of mitochondrial N/hr. 2. When aged in the presence of the mitochondrial supernatant the rate was increased. Mitochondria from 0·33g. of liver, when combined with the corresponding mitochondrial supernatant from 0·08g. of liver, metabolized propionate at a rate of 11·4 (± s.e.m. 1·2) μmoles/mg. of mitochondrial N/hr. This rate is comparable with rates previously obtained with aged nuclear-free homogenates. 3. Two factors in the mitochondrial supernatant were detected, which when combined reproduced the effect of the fresh supernatant and prevented loss of activity on aging. One of these was non-diffusible and was recovered by fractionation of the dialysed mitochondrial supernatant with ammonium sulphate. The second factor was present in an ultrafiltrate of fresh mitochondrial supernatant and in boiled mitochondrial supernatant; it was isolated and identified as l(+)-glutamate. 4. The effect of the non-diffusible factor was due to protection of the mitochondria from the aging process, whereas glutamate served both in this capacity and as a direct stimulant of propionate metabolism at low concentration.     

STUDIES ON MITOCHONDRIA : II. Mitochondrial DNA of Thoracic Muscles of Schistocerca gregaria

The buoyant densities of the nuclear and mitochondrial DNA from the thoracic muscles of Schistocerca gregaria were found to be 1 702 and 1.689 g/cm³, respectively, corresponding to guanine plus cytosine (G + C) content of 42.2 and 30% A preliminary treatment of the mitochondrial pellet with DNase (25°C, 20 min) is necessary to eliminate the contaminating nuclear DNA. The mitochondrial DNA renatures readily after heat denaturation and incubation at 65°C. The DNA released from the mitochondrial pellet by osmotic shock consists of circular open and closed molecules with a contour length around 5 µ The instability of insect mitochondria in in vitro preparations is discussed.     

Proinflammatory Cytokines Stimulate Mitochondrial Superoxide Flashes in Articular Chondrocytes In Vitro and In Situ

Objective

Mitochondria play important roles in many types of cells. However, little is known about mitochondrial function in chondrocytes. This study was undertaken to explore possible role of mitochondrial oxidative stress in inflammatory response in articular chondrocytes.

Methods

Chondrocytes and cartilage explants were isolated from wild type or transgenic mice expressing the mitochondrial superoxide biosensor - circularly permuted yellow fluorescent protein (cpYFP). Cultured chondrocytes or cartilage explants were incubated in media containing interleukin-1β (10 ng/ml) or tumor necrosis factor-α (10 ng/ml) to stimulate an inflammatory response. Mitochondrial imaging was carried out by confocal and two-photon microscopy. Mitochondrial oxidative status was evaluated by “superoxide flash” activity recorded with time lapse scanning.

Results

Cultured chondrocytes contain abundant mitochondria that show active motility and dynamic morphological changes. In intact cartilage, mitochondrial abundance as well as chondrocyte density declines with distance from the surface. Importantly, sudden, bursting superoxide-producing events or “superoxide flashes” occur at single-mitochondrion level, accompanied by transient mitochondrial swelling and membrane depolarization. The superoxide flash incidence in quiescent chondrocytes was ∼4.5 and ∼0.5 events/1000 µm²*100 s in vitro and in situ, respectively. Interleukin-1β or tumor necrosis factor-α stimulated mitochondrial superoxide flash activity by 2-fold in vitro and 5-fold in situ, without altering individual flash properties except for reduction in spatial size due to mitochondrial fragmentation.

Conclusions

The superoxide flash response to proinflammatory cytokine stimulation in vitro and in situ suggests that chondrocyte mitochondria are a significant source of cellular oxidants and are an important previously under-appreciated mediator in inflammatory cartilage diseases.     

Protective Effect of Boerhaavia diffusa L. against Mitochondrial Dysfunction in Angiotensin II Induced Hypertrophy in H9c2 Cardiomyoblast Cells

Mitochondrial dysfunction plays a critical role in the development of cardiac hypertrophy and heart failure. So mitochondria are emerging as one of the important druggable targets in the management of cardiac hypertrophy and other associated complications. In the present study, effects of ethanolic extract of Boerhaavia diffusa (BDE), a green leafy vegetable against mitochondrial dysfunction in angiotensin II (Ang II) induced hypertrophy in H9c2 cardiomyoblasts was evaluated. H9c2 cells challenged with Ang II exhibited pathological hypertrophic responses and mitochondrial dysfunction which was evident from increment in cell volume (49.09±1.13%), protein content (55.17±1.19%), LDH leakage (58.74±1.87%), increased intracellular ROS production (26.25±0.91%), mitochondrial superoxide generation (65.06±2.27%), alteration in mitochondrial transmembrane potential (ΔΨm), opening of mitochondrial permeability transition pore (mPTP) and mitochondrial swelling. In addition, activities of mitochondrial respiratory chain complexes (I-IV), aconitase, NADPH oxidase, thioredoxin reductase, oxygen consumption rate and calcium homeostasis were evaluated. Treatment with BDE significantly prevented the generation of intracellular ROS and mitochondrial superoxide radicals and protected the mitochondria by preventing dissipation of ΔΨm, opening of mPTP, mitochondrial swelling and enhanced the activities of respiratory chain complexes and oxygen consumption rate in H9c2 cells. Activities of aconitase and thioredoxin reductase which was lowered (33.77±0.68% & 45.81±0.71% respectively) due to hypertrophy, were increased in BDE treated cells (P≤0.05). Moreover, BDE also reduced the intracellular calcium overload in Ang II treated cells. Overall results revealed the protective effects of B. diffusa against mitochondrial dysfunction in hypertrophy in H9c2 cells and the present findings may shed new light on the therapeutic potential of B. diffusa in addition to its nutraceutical potentials.     

Further observations on the production of amino acids by rat-liver mitochondria and other subcellular fractions

1. Rat-liver mitochondria showed a decrease in amino acid production after preparation in 0·25m-sucrose containing EDTA (1mm), but an increase in water content. When EDTA was replaced by Mn²⁺ (1mm) or succinate (1mm), both amino acid production and water content were lowered, whereas preparation in 0·9% potassium chloride caused an increase in both. 2. Amino acid production by rat-liver homogenates prepared in 0·9% potassium chloride or 0·25m-sucrose was similar (q_{amino acid} 0·047 and 0·042 respectively aerobically). After freezing-and-thawing q_{amino acid} values were approximately doubled, and approached that of a homogenate prepared in water. 3. All cations tested inhibited amino acid production by mitochondria, Hg²⁺ and Zn²⁺ being the most effective in tris–hydrochloric acid buffer. In phosphate buffer Mg²⁺ and Mn²⁺ had no effect. Of the anions tested only pyrophosphate and arsenate had any inhibitory effect at final concn. 1mm. 4. Iodosobenzoate (1mm) and p-chloromercuribenzenesulphonate (1mm) inhibited mitochondrial amino acid production by 70–80%, whereas soya-bean trypsin inhibitor, EDTA and di-isopropyl phosphorofluoridate inhibited by a maximum of 30%. Respiratory inhibitors had no effect. 5. Rat-liver homogenate and subcellular fractions each showed an individual pattern of inhibition when a series of inhibitors was tested. 6. Amino acid production by mitochondria was decreased by up to 50% in the presence of oxidizable substrate, apart from α-glycerophosphate and palmitate, which had no effect. CoA stimulated amino acid production in tris–hydrochloric acid but not in phosphate buffer, α-oxoglutarate abolishing the stimulation. 7. Cysteine and glutathione stimulated amino acid production by whole mitochondria by 30%, but only reduced glutathione stimulated production in broken mitochondria. 8. Adrenocorticotrophic hormone and growth hormone stimulated mitochondrial amino acid production by 21–24%, whereas insulin inhibited production by 25%. 9. Coupled oxidative phosphorylation increased amino acid production by up to 154% at 25° and 40°. The increase was abolished by 2,4-dinitrophenol. 10. Amino acid incorporation in mitochondria was accompanied by an increase in amino acid production, both being decreased by chloramphenicol. 11. Mitochondrial production of ninhydrin-positive material was increased in the presence of albumin. The biggest increase was noted for the soluble fraction of broken mitochondria. No increase was found in the presence of ¹⁴C-labelled algal protein or denatured mitochondrial protein.     

CHOLINE METABOLISM AND MEMBRANE FORMATION IN RAT HEPATOMA CELLS GROWN IN SUSPENSION CULTURE : II. Phosphatidylcholine Synthesis during Growth Cycle and Fluctuation of Mitochondrial Density

The incorporation of methyl-labeled choline into phosphorylcholine and phosphatidylcholine of cellular membranes by Novikoff rat hepatoma cells (line N1S1-67) during growth in suspension culture was investigated. Upon initiation of a fresh culture at 10⁵ cells/ml, the rate of synthesis of phosphorylcholine by the cells was four to five times greater than that of the synthesis of phosphatidylcholine. While the rate of synthesis of the latter remained relatively constant, the rate of phosphorylation of choline decreased progressively during the course of the growth cycle of the culture to 10–20% of the initial rate when the culture reached stationary phase at 3 x 10⁶ cells/ml. The decrease in phosphorylcholine synthesis during the growth cycle was not due to depletion of choline in the medium or a decrease in its concentration, but was correlated with a decrease in choline kinase activity of the cells as measured in cell-free extracts. Newly synthesized phosphatidylcholine was detectable in cells only as an integral part of cellular membranes. Its distribution among various cytoplasmic membrane structures separated by isopycnic centrifugation in sucrose density gradients remained relatively constant during the growth cycle. About 50% was associated with the mitochondria, and the remainder with plasma membrane fragments and other membranous structures with mean densities of about 1.15 and 1.13 g/cm³, respectively. However, the density of the mitochondria increased from about 1.167 g/cm³ in early exponential phase cells to about 1.190 g/cm³ in stationary phase cells. The finding that the density of the entire propulation of mitochondria changed simultaneously and progressively is in agreement with the view that mitochondria grow by addition of phospholipids and structural proteins and increase in number by division.     

Finding of the Low Molecular Weight Inhibitors of Resuscitation Promoting Factor Enzymatic and Resuscitation Activity

Background

Resuscitation promoting factors (RPF) are secreted proteins involved in reactivation of dormant actinobacteria, including Mycobacterium tuberculosis. They have been considered as prospective targets for the development of new anti-tuberculosis drugs preventing reactivation of dormant tubercle bacilli, generally associated with latent tuberculosis. However, no inhibitors of Rpf activity have been reported so far. The goal of this study was to find low molecular weight compounds inhibiting the enzymatic and biological activities of Rpfs.

Methodology/Principal Findings

Here we describe a novel class of 2-nitrophenylthiocyanates (NPT) compounds that inhibit muralytic activity of Rpfs with IC₅₀ 1–7 µg/ml. Fluorescence studies revealed interaction of active NPTs with the internal regions of the Rpf molecule. Candidate inhibitors of Rpf enzymatic activity showed a bacteriostatic effect on growth of Micrococcus luteus (in which Rpf is essential for growth protein) at concentrations close to IC₅₀. The candidate compounds suppressed resuscitation of dormant (“non-culturable”) cells of M. smegmatis at 1 µg/ml or delayed resuscitation of dormant M. tuberculosis obtained in laboratory conditions at 10 µg/ml. However, they did not inhibit growth of active mycobacteria under these concentrations.

Conclusions/Significance

NPT are the first example of low molecular weight compounds that inhibit the enzymatic and biological activities of Rpf proteins.     

Optimization and Validation of FePro Cell Labeling Method

Current method to magnetically label cells using ferumoxides (Fe)-protamine (Pro) sulfate (FePro) is based on generating FePro complexes in a serum free media that are then incubated overnight with cells for the efficient labeling. However, this labeling technique requires long (>12–16 hours) incubation time and uses relatively high dose of Pro (5–6 µg/ml) that makes large extracellular FePro complexes. These complexes can be difficult to clean with simple cell washes and may create low signal intensity on T2* weighted MRI that is not desirable. The purpose of this study was to revise the current labeling method by using low dose of Pro and adding Fe and Pro directly to the cells before generating any FePro complexes. Human tumor glioma (U251) and human monocytic leukemia cell (THP-1) lines were used as model systems for attached and suspension cell types, respectively and dose dependent (Fe 25 to 100 µg/ml and Pro 0.75 to 3 µg/ml) and time dependent (2 to 48 h) labeling experiments were performed. Labeling efficiency and cell viability of these cells were assessed. Prussian blue staining revealed that more than 95% of cells were labeled. Intracellular iron concentration in U251 cells reached ∼30–35 pg-iron/cell at 24 h when labeled with 100 µg/ml of Fe and 3 µg/ml of Pro. However, comparable labeling was observed after 4 h across the described FePro concentrations. Similarly, THP-1 cells achieved ∼10 pg-iron/cell at 48 h when labeled with 100 µg/ml of Fe and 3 µg/ml of Pro. Again, comparable labeling was observed after 4 h for the described FePro concentrations. FePro labeling did not significantly affect cell viability. There was almost no extracellular FePro complexes observed after simple cell washes. To validate and to determine the effectiveness of the revised technique, human T-cells, human hematopoietic stem cells (hHSC), human bone marrow stromal cells (hMSC) and mouse neuronal stem cells (mNSC C17.2) were labeled. Labeling for 4 hours using 100 µg/ml of Fe and 3 µg/ml of Pro resulted in very efficient labeling of these cells, without impairing their viability and functional capability. The new technique with short incubation time using 100 µg/ml of Fe and 3 µg/ml of Pro is effective in labeling cells for cellular MRI.