Ornithine decarboxylase prevents tumor necrosis factor alpha-induced apoptosis by decreasing intracellular reactive oxygen species

Ornithine decarboxylase (ODC) plays an essential role in various biological functions, including cell proliferation, differentiation and cell death. However, how it prevents the cell apoptotic mechanism is still unclear. Previous studies have demonstrated that decreasing the activity of ODC by difluoromethylornithine (DFMO), an irreversible inhibitor of ODC, causes the accumulation of intracellular reactive oxygen species (ROS) and cell arrest, thus inducing cell death. These findings might indicate how ODC exerts anti-oxidative and anti-apoptotic effects. In our study, tumor necrosis factor alpha (TNF-) induced apoptosis in HL-60 and Jurkat T cells. The kinetic studies revealed that the TNF- -induced apoptotic process included intracellular ROS generation (as early as 1 h after treatment), the activation of caspase 8 (3 h), the cleavage of Bid (3 h) and the disruption of mitochondrial membrane potential ( _m) (6 h). Furthermore, ROS scavengers, such as glutathione (GSH) and catalase, maintained _m and prevented apoptosis upon treatment. Putrescine and overexpression of ODC had similar effects as ROS scavengers in decreasing intracellular ROS and preventing the disruption of _m and apoptosis. Inhibition of ODC by DFMO in HL-60 cells only could increase ROS generation, but did not disrupt _m or induce apoptosis. However, DFMO enhanced the accumulation of ROS, disruption of _m and apoptosis when cells were treated with TNF- . ODC overexpression avoided the decline of Bcl-2, prevented cytochrome c release from mitochondria and inhibited the activation of caspase 8, 9 and 3. Overexpression of Bcl-2 maintained _m and prevented apoptosis, but could not reduce ROS until four hours after TNF- treatment. According to these data, we suggest that TNF- induces apoptosis mainly by a ROS-dependent, mitochondria-mediated pathway. Furthermore, ODC prevents TNF- -induced apoptosis by decreasing intracellular ROS to avoid Bcl-2 decline, maintain _m, prevent cytochrome c release and deactivate the caspase cascade pathway.     

Die optischen Übertragungseigenschaften der Komplexaugen von Drosophila

Summary The transfer properties of the optical system in the arthropod compound eye are determined by the interommatidial angle , influencing the resolving power, and by the width of the visual fields of single ommatidia , influencing the response at high spatial frequencies of brightness distributions in the object space. The energy transfer/ receptor is proportional to ( )² and decreases with in-inreasing approximation of the perfect-imaging condition: gD 0; 0. However, a value > 0 has to be maintained in order to overcome the threshold of nervous excitation at a certain minimum-brightness level. Theoretical treatment yields /=0.62 to 0.88 as the corresponding optimum-imaging relation. The actual ratio can be derived from measurements of the optomotor reactions to the movement of periodic brightness patterns. The approximate value 0.76 is obtained from the fruitfly Drosophila with normal and mutant eye pigmentation. As a result, the parameters of this imaging system are found to be established in a way that enables optimum performance at sufficient illumination. An dieser Stelle möchte ich Dr. W. Reichardt für sein eingehendes Interesse und manche anregende Diskussion über die Sehvorgänge im Komplexauge meinen Dank sagen. Dr. K. Kirschfeld verdanke ich ebenfalls wertvolle Hinweise. Herrn E. Freiberg bin ich für die Anfertigung der Abbildungen sehr verbunden.     

Preparation of a series of sulfated tetrasaccharides from shark cartilage chondroitin sulfate D using testicular hyaluronidase and structure determination by 500 MHz1H NMR spectroscopy

Six tetrasaccharide fractions were isolated from shark cartilage chondroitin sulfate D by gel filtration chromatography followed by HPLC on an amine-bound silica column after exhaustive digestion with testicular hyaluronidase. Their structures were determined unambiguously by one- and two-dimensional 500 MHz¹H NMR spectroscopy in conjunction with HPLC analysis of chondroitinase AC-II digests of the tetrasaccharides. One fraction was found to contain two tetrasaccharide components. All the seven tetrasaccharides shared the common core structure GlcA1-3GalNAc1-4GlcA1-3GalNAc with various sulfation profiles. Four were disulfated comprising of two monosulfated disaccharide units GlcA1-3GalNAc(4-sulfate) and/or GlcA1-3GalNAc(6-sulfate), whereas the other three were hitherto unreported trisulfated tetrasaccharides containing a disulfated disaccharide unit GlcA(2-sulfate)1-3GalNAc(6-sulfate) and a monosulfated disaccharide unit GlcA1-3GalNAc(4-or 6-sulfate). These sulfated tetrasaccharides were demonstrated to serve as appropriate acceptor substrates for serum -N-acetylgalactosaminyltransferase, indicating their usefulness as authentic oligosaccharide substrates or probes for the glycobiology of sulfated glycosaminoglycans.Abbreviations NFU National formulary unit - COSY correlation spectroscopy - HOHAHA homonuclear Hartmann-Hahn - 1D or 2D one- or two-dimensional - IdoA l-iduronic acid - GlcA d-gluco-4-enepyranosyluronic acid - Di-0S GlcA1-3GalNAc - Di-4S GlcA1-3GalNAc(4-sulfate) - Di-4S GlcA1-3GalNAc(4-sulfate) - Di-6S GlcA1-3GalNAc(6-sulfate) - Di-6S GlcA1-3GalNAc(6-sulfate) - Di-diS _d GlcA(2-sulfate)1-3GalNAc(6-sulfate) - Di-diS_E GlcA1-3GalNAc(4, 6-disulfate) - U G, U, 2S, 4S, and 6S represent GlcA, GalNAc, GlcA, 2-O-sulfate, 4-O-sulfate, and 6-O-sulfate, respectively     

Protocol-dependence of equivalent circuit parameters of toad urinary bladder

Summary Determinations of current-voltage relationships are widely employed in the characterization of epithelial sodium transport. In order to determine the protocol dependence of transport parameters in the toad urinary bladder, studies were carried out in the presence and absence of amiloride, an inhibitor of active sodium transport. With symmetric positive and negative perturbations of the transepithelial electrical potential difference (0±100 mV) for 30 sec, the amiloride-sensitive current-voltage (i ^a -) relationship was near linear over the range –75+100 mV, indicating constancy of the conductance ^a and the apparent electromotive force E _Na, lumped parameters of the standard electrical equivalent circuit model of the active transport system. With a reverse protocol (±1000 mV) or 15 min perturbations thei ^a - relationships were highly nonlinear. Nonlinearity reflected voltage dependence of parameters: perturbations that increased active transport decreased E _Na and increased ^a, as evaluated from 10 sec perturbations of ; slowing of active transport produced the converse changes. These effects are usefully analyzed in both quasi-steady states and true steady states by means of a detailed equivalent circuit incorporating the significant ionic currents across each plasma membrane. Precise understanding of the significance of ^a and E _Na will require characterization of the partial ionic conductances on perturbation of .     

Theoretical stability diagram of solvent-containing black lipid films

Recently, we have developed an analytical, semi-microscopic theory for the macroscopic behavior of a solvent-containing black lipid film subjected to an electric cross film voltage, . Here we employ the theoretical expressions derived for the disjoining pressure, _D, the film elasticity, _F, and the film tension, _F, to construct the stability diagram of the film, in the _D-. Depending on its state (_D, ), the film is stable or is prone to squeezing or bending deformations. For a monooleate film we show how the destruction of the plane film due to a periodic thickness fluctuation (squeezing) is facilitated by two mechanisms: i) lowering of _D at fixed ; ii) lowering of at fixed _D, provided that the film is in a stable state characterized by _D<–7.03×10³ dyne/cm² and >0 mV. Bending of a low tension film (single interface tension _s 0.025 dyne/cm¹) can be achieved only for >170 mV and _D > –8.7 × 10⁴ dyne/cm². Finally, we demonstrate the existence of a marginal state ( _D ⁰ , ⁰) where the film is predicted to exhibit strong fluctuations both in the squeezing and in the bending mode.     

A dominant mutation for high oleic acid content in sunflower (Helianthus annuus L.) seed oil is genetically linked to a single oleate-desaturase RFLP locus

Pervenets is a sunflower mutant with a seed oil oleic acid content greater than 65%. It was obtained after mutagenesis treatment on VNIIMK 8931. Several commercial varieties derived from Pervenets and breeding materials with a high oleic acid content have been marketed. However, the genetics of this trait are still not fully understood by breeders. To characterize the Pervenets mutation, we studied RFLP in relation to high oleic acid content. We performed diversity analyses on 239 genotypes with cDNA sequences coding for 9- and 12-desaturases as probes. The 12 RFLPs enabled us to identify at least two independent loci. One 12 RFLP allele (12HOS) was strictly correlated to high oleic acid content, whereas no correlation was found between 9-desaturase polymorphism and high oleic acid content. These results enabled to us estimate the genetic distance between the marker and the Pervenets mutation loci. An F₂ segregating population of 107 plants confirmed the correlation between high oleic acid content and 12HOS, indicating tight genetic linkage. The nature of the Pervenets dominant mutation and the complexity of the high oleic acid content trait are discussed.     

ATP-induced ΔpH formation in chloroplast ATP synthase proteoliposomes

Summary A procedure to reconstitute CF₀CF₁ proteoliposomes by gel filtration through a Sephadex-column pre-equilibrated with valinomycin and potassium is described. Proteoliposomes reconstituted by this procedure catalyze an ATP-induced pH of 2.5 to 3.5 units. pH was measured with either 9-aminoacridine or with the pH indicator pyranine trapped inside the proteoliposomes. CF₀CF₁ proteoliposomes prepared by conventional techniques catalyzed an ATP-induced formation, but were unable to catalyze an ATP-induced pH even in the presence of valinomycin.The ATP-induced pH was sensitive to uncouplers and energy transfer inhibitors and was increased at low temperatures. It is suggested that ATP-induced pH was observed in these proteoliposomes due to the efficient removal of intravesicular ammonium introduced with the CF₀CF₁ preparation. The ammonium acted as an internal buffer, and thus prevented an observable pH formation.     

Steroid synthesizing cellular sites in the ovary of the domestic pigeon Columba livia (Gmelin): a histochemical study.

Synopsis The ovary of the domestic pigeon,Columba livia, has been assayed histochemically for the localization of ⁵-3-hydroxysteroid dehydrogenase (⁵-3-HSDH), 17-hydroxysteroid dehydrogenase (17-HSDH), 11-hydroxysteroid dehydrogenase (11-HSDH), glucose-6-phosphate dehydrogenase (G6P-DH) and NADH-diaphorase activities during different periods of the reproductive cycle. ⁵-3-HSDH, 17-HSDH, 11-HSDH, G6P-DH and NADH-diaphorase activity was found in the theca interna of growing, atretic and postovulatory follicles, the granulosa of ovulatory, atretic and postovulatory follicles, and interstitial gland cells during the pre-incubation and the laying periods. During the incubation and squab feeding periods only ⁵-3-HSDH, G6P-DH and NADH-diaphorase activities were observed in the above mentioned cells. The steroidogenic potential of atretic follicles depends upon the type of atresia a follicle undergoes.     

Variation in leaf carbon isotope discrimination in Encelia farinosa: implications for growth,competition, and drought survival

Population-level variation in the leaf carbon isotope discrimination () values was examined in Encelia farinosa, a common Sonoran Desert shrub. There was approximately a 2 range in values among different plants. These differences in values among neighboring plants were maintained through time, both under conditions when neighbors were present and after neighbors had been removed. Individuals with high values were found to have an accelerated growth rate when these plants were released from competition for water. Individuals with low values were better able to persist through long-term drought. These data suggest possible tradeoffs between conditions favoring high- and low--value plants within a natural population. Given the temporal variability in precipitation between years and spatial variability in microhabitat quality in the Sonoran Desert, variation in values among E. farinosa plants will be maintained within a population.     

Interaction of cytochrome c L with methanol dehydrogenase from Methylophaga marina 42:

The reaction of methanol dehydrogenase with cytochrome c _L from Methylophaga marina and the reactions of the non-physiological substrates, Wurster's blue and ascorbic acid, with both proteins were studied as a function of temperature (4–32 °C), pressure (1–2000 bar) and ionic strength using the optical high pressure stopped-flow method. The thermodynamic parameters H, S and V were determined for all reactions where electron transfers are involved. These data allowed the determination of the Maxwell relationships which proved the internal thermodynamic consistency of the system under study. A conformational change on the cytochrome c _L level was deduced from both breaks in the Arrhenius plots and the variation of the V with temperature.Abbreviations MOPS 4-morpholinepropanesulfonic acid - CHES 2-(cyclohexylamino)ethanesulfonic acid - MDH methanol dehydrogenase - EDTA ethylenedinitrilotetraacetic acid disodium salt - BTB bromothymol blue (3,3-dibromothymolsulfoneph-thalein) - PQQ 2,7,9-tricarboxy-lH-pyrrolo-[2,3f]quinoline-4,5-dione - cytochrome c _HH mammalian horse heart cytochrome c     

Energetic basis of cadmium toxicity in Staphylococcus aureus

In washed cells of cadmium-sensitive Staphylococcus aureus 17810S oxidizing glutamate, initial Cd²⁺⁺⁺ influx via the Mn²⁺ porter down membrane potential () was fast due to involvement of energy generated by two proton pumps—the respiratory chain and the ATP synthetase complex working in the hydrolytic direction. Such an unusual energy drain for rapid initial Cd²⁺ influx is suggested to be due to a series of toxic events elicited by Cd²⁺ accumulation down generated via the redox proton pump: (i) strong inhibition of glutamate oxidation accompanied by a decrease of electrochemical proton gradient ( _H ⁺) formation via the respiratory chain, (ii) automatic reversal of ATP synthetase from biosynthetic to hydrolytic mode, which was monitored by a decrease of _H ⁺-dependent ATP synthesis, (iii) acceleration of the initial Cd²⁺ influx down generated the reversed ATP synthetase, the alternative proton pump hydrolyzing endogenous ATP. The primary, cadmium-sensitive targets in strain 17810S seem to be dithiols located in the cytoplasmic glutamate oxidizing system, prior to the membrane-embedded NADH oxidation system. Inhibition by Cd²⁺ of _H ⁺-dependent ATP synthesis and of pH gradient (pH)-linked [¹⁴C]glutamate transport is a secondary effect due to cadmium-mediated inhibition of _H ⁺ generation at the cytoplasmic level. In washed cells of cadmium-resistant S. aureus 17810R oxidizing glutamate, Cd²⁺ accumulation was prevented due to activity of the plasmid-coded Cd²⁺ efflux system. Consequently, _H ⁺-producing and -requiring processes were not affected by Cd²⁺.     

Average phase difference theory and 1∶1 phase entrainment in interlimb coordination

The dynamics of coupled biological oscillators can be modeled by averaging the effects of coupling over each oscillatory cycle so that the coupling depends on the phase difference between the two oscillators and not on their specific states. Average phase difference theory claims that mode locking phenomena can be predicted by the average effects of the coupling influences. As a starting point for both empirical and theoretical investigations, Rand et al. (1988) have proposed d/dt= — K sin ), with phase-locked solutions =arcsin( /K), where is the difference between the uncoupled frequencies and K is the coupling strength. Phase-locking was evaluated in three experiments using an interlimb coordination paradigm in which a person oscillates hand-held pendulums. was controlled through length differences in the left and right pendulums. The coupled frequency _c was varied by a metronome, and scaled to the eigenfrequency _v of the coupled system K was assumed to vary inversely with _c. The results indicate that: (1) and K contribute multiplicatively to (2) =0 or = regardless of K when =0; (3) 0 or regardless of when K is large (relative to ); (4) results (1) to (3) hold identically for both in phase and antiphase coordination. The results also indicate that the relevant frequency is _c/_v rather than _c. Discussion high-lighted the significance of confirming =arcsin(/K) for more general treatments of phase-locking, such as circle map dynamics, and for the 11 phase-entrainment which characterizes biological movement systems.     

Modelling based method for pharmacokinetic hypotheses test

The aim of this work is to propose methods to test mechanism of synergy of toxic agents in bees. A synergy between prochloraz, an imidazole fungicide, and deltamethrin, a pyrethroid insecticide, was demonstrated experimentally. The hypothesis is that prochloraz modifies the penetration or the metabolism of deltamethrin. This hypothesis is tested using a pharmacokinetic box model. A previous experimental work showed that bee instantaneous mortalities were higher, from the time t ₁ to the time t ₂ after spraying, in groups sprayed with deltamethrin at dose D ₀ in the presence of prochloraz (+P) than in those sprayed with deltamethrin alone at a dose time as high (). We postulate that accrued mortality is proportional to the cumulated internal deltamethrin (ID ₂). ID ₂ of treatment (+P) had to be greater than ID ₂ of treatment () during the period from t ₁ to t ₂ so that the hypothesis would be consistent with the experimental data. The limit, for which the hypothesis is conceivable, is the ID ₂₍₎ = ID _2(+P ) curve. We study, in particular, the asymptotic behaviour of the limit curve when different parameters of the kinetic model tend to 0 or . These limits allow to verify quickly and easily whether a mechanism is conceivable or not As the limits are calculated with algebraic values, the test can be used for other synergies.     

The relation of proton motive force,adenylate energy charge and phosphorylation potential to the specific growth rate and efficiency of energy transduction inBacillus licheniformis under aerobic growth conditions

The magnitude of the proton motive force (p) and its constituents, the electrical () and chemical potential (-ZpH), were established for chemostat cultures of a protease-producing, relaxed (rel ^–) variant and a not protease-producing, stringent (rel ⁺) variant of an industrial strain ofBacillus licheniformis (respectively referred to as the A- and the B-type). For both types, an inverse relation of p with the specific growth rate was found. The calculated intracellular pH (pH_in) was not constant but inversely related to . This change in pH_in might be related to regulatory functions of metabolism but a regulatory role for pH_in itself could not be envisaged. Measurement of the adenylate energy charge (EC) showed a direct relation with for glucose-limited chemostat cultures; in nitrogen-limited chemostat cultures, the EC showed an approximately constant value at low and an increased value at higher . For both limitations, the ATP/ADP ratio was directly related to .The phosphorylation potential (G'p) was invariant with . From the values for G'p and p, a variable H⁺/ATP-stoichiometry was inferred: H⁺/ATP=1.83+0.52µ, so that at a given H⁺/O-ratio of four (4), the apparent P/O-ratio (inferred from regression analysis) showed a decline of 2.16 to 1.87 for =0 to _max (we discuss how more than half of this decline will be independent of any change in internal cell-volume). We propose that the constancy of G'p and the decrease in the efficiency of energy-conservation (P/O-value) with increasing are a way in which the cells try to cope with an apparent less than perfect coordination between anabolism and catabolism to keep up the highest possible with a minimum loss of growth-efficiency. Protease production in nitrogen-limited cultures as compared to glucose-limited cultures, and the difference between the A- and B-type, could not be explained by a different energy-status of the cells.Abbreviations CCCP carbonylcyanide-p-trichloromethoxyphenylhydrazone - DW dry weight of biomass - F Faraday's constant, 96.6 J/(mV × mol) - Fo chemostat outflow-rate (ml/h) - FCCP carbonylcyanide-p-trifluoromethoxyphenylhydrazone - G'p phosphorylation potential, the Gibbs energy change for ATP-synthesis from ADP and P_i - G'⁰p standard Gibbs energy change at specified conditions - H⁺/ATP number of protons translocated through - ATP synthase in synthesis of one ATP - H⁺/O protons translocated during transfer of 2 electrons from substrate to oxygen - specific growth rate (1/h) - _H+ transmembrane electrochemical proton potential, J/mol - M_b molar weight (147.6 g/mol) of bacteria with general cell formula C_6.0H_10.8O_3.0N_1.2 - pH_out,in extracellular, intracellular pH - P_i (intracellular) inorganic phosphate - p proton motive force, mV - pH transmembrane pH-difference - transmembrane electrical potential, mV - P/O number of ADP phosphorylated to ATP upon reduction of one O^2– to H₂O by two electrons transferred through the electron transfer chain - P/O (H⁺/O) × (H⁺/ATP)^–1 - P/O_F, P/O_N P/O with the two electrons donated by resp. (NADH + H⁺) and FADH - q specific rate of consumption or production (mol/g DW × h) - rel ⁺,rel ^– stringent, relaxed genotype - R universal gas constant, 8.36 J/(mol × degree) - T absolute temperature - TPMP⁺ triphenylmethylphosphonium ion - TPP⁺ tetraphenyl phosphonium ion - Y growth yield, g DW/mol - Z conversion constant=61.8 mV for 310 K (37 °C) - ZpH transmembrane proton potential or chemical potential, mV     

A Mössbauer spectroscopy study of cellular acquisition of iron from pyoverdine byPseudomonas aeruginosa

Summary Mössbauer spectroscopy was used to investigate the cellular acquisition of iron byPseudomonas aeruginosa which had been incubated with ferripyoverdine for 20, 40, 60, 120 or 360 min. Studies revealed that no ferripyoverdine accumulated in the cells at any of these times and that the amounts and kinds of iron complexes produced by cellular metabolism vary with time. At 20 and 40 min a ferric species, with isomer shift =0.38–0.42 mm/s and quadrupole splitting E _Q=0.94–0.92 mm/s, was the major iron metabolite comprising approximately 80% of the iron. At later times at least three other ferric species appeared with =0.54 0.72, E _Q = 0.84 1.07 mm/s. Ferrous species, =1.43 1.77 mm/s and E _Q = 2.69 1.82 mm/s, were also seen at times as early as 20 min and comprised as much as 17% of the total iron at 20 and 40 min. The parameters of all these species identify them as being six-coordinated high-spin complexes. In addition a low-spin species, =0.19 mm/s E _Q=0.67 0.91 mm/s, never before reported in cells, appeared at 60, 120, and 360 min as one of the major iron metabolites (50% or more). All isomer shifts are measured with respect to natural iron.     

The ability of acidic pH,growth inhibitors,and glucose to increase the proton motive force and energy spilling of amino acid-fermenting <Emphasis Type="Italic">Clostridium sporogenes</Emphasis> MD1 cultures

Clostridium sporogenes MD1 grew rapidly with peptides and amino acids as an energy source at pH 6.7. However, the proton motive force (p) was only –25 mV, and protonophores did not inhibit growth. When extracellular pH was decreased with HCl, the chemical gradient of protons (ZpH) and the electrical membrane potential () increased. The p was –125 mV at pH 4.7, even though growth was not observed. At pH 6.7, glucose addition did not cause an increase in growth rate, but increased to –70 mV. Protein synthesis inhibitors also significantly increased . Non-growing, arginine-energized cells had a of –80 mV at pH 6.7 or pH 4.7, but was not detected if the F₁F₀ ATPase was inhibited. Arginine-energized cells initiated growth if other amino acids were added at pH 6.7, and and ATP declined. At pH 4.7, ATP production remained high. However, growth could not be initiated, and neither nor the intracellular ATP concentration declined. Based on these results, it appears that C. sporogenes MD1 does not need a large p to grow, and p appears to serve as a mechanism of ATP dissipation or energy spilling.Mandatory disclaimer: Proprietary or brand names are necessary to report factually on available data; however, the USDA neither guarantees nor warrants the standard of the product, and the use of the name by the USDA implies no approval of the product, and exclusion of others that may be suitable.     

A suitable method for construction and cloning hybrid plasmids containing EcoRI-fragments of E. coli genome.

Summary A convenient procedure for the isolation of specificEcoRI-fragments ofE. coli genome and their amplification on Km-resistance plasmid vector CK 11 is described. The hybrid molecules were constructedin vitro usingEcoRI-digestion, followed by ligation. Then appropriatedE. coli strain was transformed with ligated DNA mixture and hybrid plasmids CK 11-arg ⁺, CK 11-his ⁺, CK 11-thr ⁺ and CK 11-leu ⁺ containing loci ofE. coli genome were selected by molecular cloning. The hybrid plasmids obtained consisted of oneEcoRI-fragment of initial plasmid CK 11 and one respective specific portion ofE. coli genome.     

Reverse genetic analysis of the glutathione metabolic pathway suggests a novel role of<Emphasis Type="Italic"> PHGPX</Emphasis> and<Emphasis Type="Italic"> URE2</Emphasis> genes in aluminum resistance in<Emphasis Type="Italic"> Saccharomyces cerevisiae</Emphasis>

We have taken a systematic genetic approach to study the potential role of glutathione metabolism in aluminum (Al) toxicity and resistance, using disruption mutants available in Saccharomyces cerevisiae. Yeast disruption mutants defective in phospholipid hydroperoxide glutathione peroxidases (PHGPX; phgpx1 , phgpx2 , and phgpx3), were tested for their sensitivity to Al. The triple mutant, phgpx1 /2/3, was more sensitive to Al (55% reduction in growth at 300 M Al) than any single phgpx mutant, indicating that the PHGPX genes may collectively contribute to Al resistance. The hypersensitivity of phgpx3 to Al was overcome by complementation with PHGPX3, and all PHGPX genes showed increased expression in response to Al in the wild-type strain (YPH250), with maximum induction of approximately 2.5-fold for PHGPX3. Both phgpx3 and phgpx1/2/3 mutants were sensitive to oxidative stress (exposure to H₂O₂ or diamide). Lipid peroxidation was also increased in the phgpx1/2/3 mutant compared to the parental strain. Disruption mutants defective in genes for glutathione S-transferases (GSTs) (gtt1 and gtt2), glutathione biosynthesis (gsh1 and gsh2), glutathione reductase (glr1) and a glutathione transporter (opt1) did not show hypersensitivity to Al relative to the parental strain BY4741. Interestingly, a strain deleted for URE2, a gene which encodes a prion precursor with homology to GSTs, also showed hypersensitivity to Al. The hypersensitivity of the ure2 mutant could be overcome by complementation with URE2. Expression of URE2 in the parental strain increased approximately 2-fold in response to exposure to 100 M Al. Intracellular oxidation levels in the ure2 mutant showed a 2-fold (non-stressed) and 3-fold (when exposed-to 2 mM H₂O₂) increase compared to BY4741; however, the ure2 mutant showed no change in lipid peroxidation compared to the control. The phgpx1/2/3 and ure2 mutants both showed increased accumulation of Al. These findings suggest the involvement of PHGPX genes and a novel role of URE2 in Al toxicity/resistance in S. cerevisiae.Communicated by D.Y. Thomas     

Effect of different carbon sources on the biosynthesis of polyunsaturated fatty acids ofα-linolenic acid family in culture of minimal deviation hepatoma 7288 c cells

Summary The effect of three different carbon sources on the biosynthesis of polyunsaturated fatty acids of the-linolenic acid series was investigated in hepatoma tissue culture (HTC) cells. Alpha linolenic acid was converted to higher homologs by a desaturating route that synthetized mainly 18:4 (6, 9, 12, 15), 20:4 (8, 11, 14, 17) and 20:5 (5, 8, 11, 14, 17) and an elongating route that produced 20:3 (11, 14, 17) and 20:4 (5, 11, 14, 17) acids. Fasting decreased both biosynthetic routes whereas glucose reactivated only the elongating pathway. Lactalbumin hydrolysate enhanced significantly only the desaturating route whereas glycerol was inactive. Glucose and aminoacids increased similarly the incorporation of labeled linolenic acid in the cells. The results are independent of hormonal effects.Members of the Carrera del Investigador Científico of the Consejo Nacional de Investigaciones Científicas y Técnicas, Argentina.     

Steady state changes in mitochondrial electrical potential and proton gradient in perfused liver from rats fed a high fat diet

In this work the protonmotive force (p), as well as the subcellular distribution of malate, ATP, and ADP were determined in perfused liver from rats fed a low fat or high fat diet, using density gradient fractionation in non acqueous solvents.Rats fed a high fat diet, despite an enhanced hepatic oxygen consumption, exhibit similar p to that found in rats fed a low fat diet, but when we consider the two components of p, we find a significant decrease in mitochondrial/cytosolic pH difference (pH_m) and a significant increase in mitochondrial membrane potential (_m) in rats fed a high fat diet compared to rats fed a low fat diet, which tend to compensate each other. In rats fed a high fat diet the concentration ratio of malate and ATP/ADP does not reflect the changes in pH_m and _m, which represent the respective driving force for their transport.The findings are in line with an increase in substrate supply to the respiratory chain which is, however, accompanied by a higher energy turnover in livers from HFD rats. By this way the liver could contribute to the lack of weight gain from the high caloric intake in HFD rats.