The mitochondrial membrane potential (deltapsi(m)) in apoptosis; an update

Mitochondrial dysfunction has been shown to participate in the induction of apoptosis and has even been suggested to be central to the apoptotic pathway. Indeed, opening of the mitochondrial permeability transition pore has been demonstrated to induce depolarization of the transmembrane potential (_m), release of apoptogenic factors and loss of oxidative phosphorylation. In some apoptotic systems, loss of _m may be an early event in the apoptotic process. However, there are emerging data suggesting that, depending on the model of apoptosis, the loss of _m may not be an early requirement for apoptosis, but on the contrary may be a consequence of the apoptotic-signaling pathway. Furthermore, to add to these conflicting data, loss of _m has been demonstrated to not be required for cytochrome c release, whereas release of apoptosis inducing factor AIF is dependent upon disruption of _m early in the apoptotic pathway. Together, the existing literature suggests that depending on the cell system under investigation and the apoptotic stimuli used, dissipation of _m may or may not be an early event in the apoptotic pathway. Discrepancies in this area of apoptosis research may be attributed to the fluorochromes used to detect _m. Differential degrees of sensitivity of these fluorochromes exist, and there are also important factors that contribute to their ability to accurately discriminate changes in _m.     

Anaerobic biosynthesis of unsaturated fatty acids in the cyanobacterium,Oscillatoria limnetica

The mechanism for synthesis of monounsaturated fatty acids under aerobic and anaerobic conditions was studied in the facultative anaerobic cyanobacterium, Oscillatoria limnetica. The hexadecenoic acid (C161) of aerobically grown O. limnetica was shown to contain both the 7 (79%) and 9 (21%) isomers, while the octadecenoic (C181) acid was entirely the 9 acid. Incorporation of [2-¹⁴C] acetate into the fatty acids under aerobic conditions resulted in synthesis of the 7 and 9 C161 and the 9 C181. Synthesis of unsaturated fatty acids in the presence of DCMU required sulfide. Anaerobic incubations in the presence of DCMU and sulfide (less than 0.003% atmospheric oxygen) resulted in a two-fold increase in monounsaturated fatty acids of both 7 and 9 C161 and 9 and 11 C181. The synthesis of these isomers is characteristic of a bacterialtype, anaerobic pathway.Abbreviations DCMU 3(3,4-dichlorophenyl)-1,1-dimethylurea - MFA monounsaturated fatty acid     

SCM2, a tryptophan permease in Saccharomyces cerevisiae, is important for cell growth

SCM2, a novel gene encoding a yeast tryptophan permease, was cloned as a high-copy-number suppressor of cse2-1. The cse2-1 mutation causes cold sensitivity, temperature sensitivity and chromosome missegregation. However, only the cold-sensitive phenotype of cse2-1 cells is suppressed by SCM2 at high copy. SCM2 is located on the left arm of yeast chromosome XV, adjacent to SUP3 and encodes a 65 kDa protein that is highly homologous to known amino acid permeases. Four out of five disrupted scm2 alleles (scm21-4) cause slow growth, whereas one disrupted allele (scm25) is lethal. Cells with both the scm21 and trp1-101 mutations exhibit a synthetic cold-sensitive phenotype and grow much more slowly at the permissive temperature than cells with a single scm21 or trp1-101 mutation. A region of the predicted SCM2 protein is identical to the partial sequence recently reported for the yeast tryptophan permease TAP2, indicating that SCM2 and TAP2 probably encode the same protein.     

Kinetics of the flash-induced electrochromic absorbance change in the presence of background illumination. Turnover rate of the electron transport. I. Isolated intact chloroplasts

We investigated the flash-induced electrochromic absorbance change, A ₅₁₅, of isolated intact chloroplasts in continuous monochromatic background light of different intensities and wavelengths. From the variation of the amplitude of A ₅₁₅ in background illumination the steady-state turnover time of electron transport was found to be around 100 msec and the slowest process could be assigned to a photosystem 1 driven cycle. The change of pH induced by nigericin, ATP, or ADP did not modify substantially the turnover time.In contrast to earlier observations the slow rise (10 msec) of A ₅₁₅ in untreated chloroplasts persists also at high-intensity background illumination exciting both photosystems. The proportion of the slow rise of A ₅₁₅ in nigericin-treated chloroplasts increases in the presence of background light. This result on the slow rise is discussed in terms of two different models existing in the literature.     

Protonmotive force in Brevibacterium flavum: the lack of intracellular pH homeostasis

Brevibacterium flavum 22LD-P cells were shown to maintain a transmembrane pH gradient (pH) from 0.6 to 1.8–2 units and a transmembrane electric potential difference () from 0 to 200 mV depending on the pH and ionic composition of the incubation medium, grwoth substrate and concentration of cells. decreased from 120–140 mV to 0 when medium pH was lowered from neutral to 5.0–5.5 and increased to 180–200 mV when medium pH was raised to 8–9 in cells utilizing acetate or endogenous substrate. Cells growing on sucrose, kept around 100–120 mV at neutral as well as acidic medium pH. Intracellular pH in the acetate utilizing or endogenously respiring cells was maintained with the range of 8.9 to 5.5 at medium pH ranging from 9.1 to 4.0, respectively. Sucrose grown cells were able to maintain a more stable intracellular pH. Endogenously respiring cells in potassium phosphate buffer at high biomass concentrations maintained larger pH and relatively smaller , than the same cells in diluted suspensions. Cells in sodium phosphate buffer possessed larger and almost no pH, but was still dependent on biomass concentration.The lack of intracellular pH homeostasis and the collapse of at acid medium pH are discussed in the context of cell membrane proton permeability.     

A study on Na+-coupled oxidative phosphorylation: ATP formation supported by artificially imposed ΔpNa and ΔpK inVibrio alginolyticus cells

Addition of Na⁺ to the K⁺-loadedVibrio alginolyticus cells, creating a 250-fold Na⁺ gradient, is shown to induce a transient increase in the intracellular ATP concentration, which is abolished by the Na⁺/H⁺ antiporter, monensin. The pNa-supported ATP synthesis requires an additional driving force supplied by endogenous respiration or, alternatively, by a K⁺ gradient (high [K⁺] inside). In the former case, ATP formation is resistant to the protonophorous uncoupler. Dicyclohexylcarbodiimide and diethylstilbestrol, but not vanadate, completely inhibit Na⁺ pulse-induced ATP formation. The data agree with the assumption that Na⁺-ATP-synthase is involved in oxidative phosphorylation inV. alginolyticus. Interrelation of H⁺ and Na⁺ cycles in bacteria is discussed.Abbreviations and electrochemical gradients of H⁺ and Na⁺, respectively - transmembrane electric potential difference - pH, pNa, and pK concentration gradients of H⁺, Na⁺, and K⁺, respectively - CCCP carbonyl cyanidem-chlorophenylhydrazone - DCCD N,N-dicyclohexylcarbodiimide - DES diesthylstilbestrol - HQNO 2-heptyl-4-hydroxyquinolineN-oxide - Tricine N[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine     

Protonmotive force driven 6-deoxyglucose uptake by the oral pathogen,Streptococcus mutans Ingbritt

Streptococcus mutans Ingbritt was grown in glucose-excess continuous culture to repress the glucose phosphoenolpyruvate phosphotransferase system (PTS) and allow investigation of the alternative glucose process using the non-PTS substrate, (³H) 6-deoxyglucose. After correcting for non-specific adsorption to inactivated cells, the radiolabelled glucose analogue was found to be concentrated approximately 4.3-fold intracellularly by bacteria incubated in 100 mM Tris-citrate buffer, pH 7.0. Mercaptoethanol or KCl enhanced 6-deoxyglucose uptake, enabling it to be concentrated internally by at least 8-fold, but NaCl was inhibitory to its transport. Initial uptake was antagonised by glucose but not 2-deoxyglucose. Evidence that 6-deoxyglucose transport was driven by protonmotive force (p) was obtained by inhibiting its uptake with the protonophores, 2,4-dinitrophenol, carbonylcyanide m-chlorophenylhydrazine, gramicidin and nigericin, and the electrical potential difference () dissipator, KSCN. The membrane ATPase inhibitor, N,N¹-dicyclohexyl carbodiimide, also reduced 6-deoxyglucose uptake as did 100 mM lactate. In combination, these two inhibitors completely abolished 6-deoxyglucose transport. This suggests that the driving force for 6-deoxyglucose uptake is electrogenic, involving both the transmembrane pH gradient (pH) and . ATP hydrolysis, catalysed by the ATPase, and lactate excretion might be important contributors to pH.Abbreviations DNP 2,4-dinitrophenol - CCCP carbonylcyanide m-chlorophenylhydrazone - DCCD N,N¹-dicyclohyxyl carbodiimide - p protonmotive force - pH transmembrane pH gradient - transmembrane electrical potential difference     

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.     

Epimerization of Hydroxyl Group in Lupan Series Triterpenoids

Two methods of obtaining 3-betulinic acid and related compounds from their 3-epimers were studied: the reaction of bimolecular substitution and the stereoselective reduction of 3-ketoderivatives. The substitution of acyloxy by formyloxy group in 3--tosyllupeol or of the belulin hydroxyl by benzoyloxy group resulted only in ^{2, 3}-elimination products, with none of the expected products of bimolecular substitution being found. The catalytic hydrogenation of betulonic acid over Raney nickel resulted only in reduction of the isopropenyl double bond, whereas the use of 5% Ru/C gave a 60 : 40 mixture of epimers of dihydrobetulinic acid. Practically the same mixture of betulinic acid epimers was obtained when reducing betulonic acid with L-Selectride. The cytotoxic activity of 3-betulinic acid increased toward the Bro melanoma cells and decreased toward the MS melanoma cells.     

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.     

Lactate conversion to acetate,CO2 and H2 in cell suspensions of Desulfovibrio vulgaris (Marburg): indications for the involvement of an energy driven reaction

Cell suspensions of Desulfovibrio vulgaris were found to catalyze, in the absence of sulfate, the complete conversion of 1 lactate to 1 acetate, 1 CO₂, and 2 H₂ (G₀=-8.8 kJ/mol) and of 1 pyruvate to 1 acetate, 1 CO₂, and 1 H₂ (G₀=-52 kJ/mol). Protonophores, the proton translocating ATPase inhibitor N,N-dicyclohexylcarbodiimide, and arsenate specifically inhibited H₂ formation from lactate but not from pyruvate. The results suggest that lactate oxidation to pyruvate and H₂ (G ₀=+43.2 kJ/mol) is energy driven.     

Positional specificity in the incorporation of isomeric cis- and trans-octadecenoic acids into glycerolipids of cultured soya cells

Heterotrophically grown cell suspension cultures of soya (Glycine max L.) were incubated with two different mixed substrates consisting of positional isomers of either cis-[1-¹⁴C]octadecenoic acids (8 to 15) or trans-[1-¹⁴C]octadecenoic acids (8 to 16), each with known composition. With both substrates, about one-fourth of the radioactivity supplied was incorporated into the diacylglycerophosphocholines, while another one-fourth of the radioactivity was almost equally distributed between diacylglycerophos-phoethanolamines and triacylglycerols. All the positional isomers of cis-and trans-octadecenoic acids supplied to the cells were readily incorporated into various classes of glycerolipids. None of the octadecenoic acids was isomerized, elongated or desaturated during incubation. From the cis-octadecenoic acids, only the naturally occurring 9-isomer (oleic acid) was preferentially incorporated into position 2 of diacylglycerophosphocholines, diacylglycerophospho-ethanolamines, and triacyglycerols; all the other isomers exhibited a strong affinity for position 1 of the glycerophospholipids and positions 1 and 3 of the triacylglycerols. From the trans-octadecenoic acids, only the 9-isomer (elaidic acid) was preferentially incorporated into position 2 of diacylglycerophospho-cholines and triacylglycerols; all the other isomers preferred position 1 and positions 1 and 3, respectively, of these lipids. In diacylglycerophospho-ethanolamines, however, each of the trans-octadecenoic acids, including the 9-isomer, exhibited a strong affinity for position 1. Apparently, the enzymes involved in the incorporation of exogenous monounsaturated fatty acids into membrane lipids of plant cells can recognize the preferred substrate in a mixture of closely related isomers.     

Estimation of Membrane Potential Δψ in Reconstituted Plasma Membrane Vesicles Using a Numerical Model of Oxonol VI Distribution

A model of membrane potential-dependent distribution of oxonol VI to estimate the electrical potential difference across Schizosaccharomyces pombe plasma membrane vesicles (PMV) has been developed. was generated by the H⁺-ATPase reconstituted in the PMV. The model treatment was necessary since the usual calibration of the dye fluorescence changes by diffusion potentials (K⁺ + valinomycin) failed. The model allows for fitting of fluorescence changes at different vesicle and dye concentrations, yielding in ATP-energized PMV of 80 mV. The described model treatment to estimate may be applicable for other reconstituted membrane systems.     

A novel eye morphology induced by a P element in somatic tissue of Drosophila melanogaster.

Summary We found a specific eye morphology designated as Square, which is induced when some Drosophila melanogaster strains harboring P elements are crossed with the 2–3 strain carrying a modified P element, P[ry ⁺, 2–3], which produces transposase in somatic tissue. This phenotype was dominant and also induced in the reciprocal crosses. Square was induced when the 2–3 strain was crossed with Q and M strains such as the sn^w (M) strain carrying three small P elements but not with P strains. Inheritance of Square was also tested and its phenotype was not transmitted to the next generation. These results suggest that Square is caused by the transposition of P elements in somatic cells.     

Molecular mapping of a new gene Rrs4 CI 11549 for resistance to barley scald (Rhynchosporium secalis)

Doubled haploid (DH) progeny from a cross between the scald susceptible barley (Hordeum vulgare L.) cultivar Ingrid and the resistant accession CI 11549 (Nigrinudum) was evaluated for resistance in the pathogen Rhynchosporium secalis (Oudem) J.J. Davis. Two linked and incompletely dominant loci confer resistance CI 11549 against isolate 4004. One is an allele at the complex Rrs1 locus on chromosome 3H close to the centromere; the other is located 22 cM distally on the long arm. The latter locus is designated Rrs4. In BC₃-lines into Ingrid from CI 2222 (another Nigrinudum) resistance seems governed by one locus close to the telomeric region of chromosome 7H, probably allelic to Rrs2. In neither case did we find any trace of the recessive gene rh8 reported to be present in Nigrinudum. Various resistance donors of Ethiopian origin designated as Nigrinudum, Jet or Abyssinian were identical to a great extent with respect to markers, but differed in resistance to different isolates of scald or in barley yellow dwarf virus (BYDV) resistance. The implications for their use as differentials in scald tests and screening of germplasm collections are discussed.     

Cyclosporin A-sensitive decrease in the transmembrane potential across the inner membrane of liver mitochondria induced by low concentrations of fatty acids and Ca2+

At low Ca²⁺ concentrations the pore of the inner mitochondrial membrane can open in substates with lower permeability (Hunter, D. R., and Haworth, R. A. (1979) Arch. Biochem. Biophys., 195, 468-477). Recently, we showed that Ca²⁺ loading of mitochondria augments the cyclosporin A-dependent decrease in transmembrane potential () across the inner mitochondrial membrane caused by 10 M myristic acid but does not affect the stimulation of respiration by this fatty acid. We have proposed that in our experiments the pore opened in a substate with lower permeability rather than in the classic state (Bodrova, M. E., et al. (2000) IUBMB Life, 50, 189-194). Here we show that under conditions lowering the probability of classic pore opening in Ca²⁺-loaded mitochondria myristic acid induces the cyclosporin A-sensitive decrease and mitochondrial swelling more effectively than uncoupler SF6847 does, though their protonophoric activities are equal. In the absence of P_i and presence of succinate and rotenone (with or without glutamate) cyclosporin A either reversed or only stopped decrease induced by 5 M myristic acid and 5 M Ca²⁺. In the last case nigericin, when added after cyclosporin A, reversed the decrease, and the following addition of EGTA produced only a weak (if any) increase. In P_i-containing medium (in the presence of glutamate and malate) cyclosporin A reversed the decrease. These data show that the cyclosporin A-sensitive decrease in by low concentrations of fatty acids and Ca²⁺ cannot be explained by specific uncoupling effect of fatty acid. We propose that: 1) low concentrations of Ca²⁺ and fatty acid induce the pore opening in a substate with a selective cation permeability, and the cyclosporin A-sensitive decrease results from a conversion of to pH gradient due to the electrogenic cation transport in mitochondria; 2) the ADP/ATP-antiporter is involved in this process; 3) higher efficiency of fatty acid compared to SF6847 in the Ca²⁺-dependent pore opening seems to be due to its interaction with the nucleotide-binding site of the ADP/ATP-antiporter and higher affinity of fatty acids to cations.     

Sodium ions are necessary for growth and energy transduction in the marine cyanobacterium Oscillatoria brevis

The maximal growth rate of the marine cyanobacterium Oscillatoria brevis was reached at 200–400 mM NaCl and pH 9.0–9.6. NaCl was found (i) to stimulate the rate of the light-supported generation across the cytoplasmic membrane of the cells and (ii) to decrease the sensitivity of level and motility of the O. brevis trichomes to protonophorous uncouplers. The Na⁺/H⁺ antiporter, monensin, increased both and the uncoupler sensitivity of the cells. The data obtained agree with the assumption that O. brevis possesses a primary Na⁺ pump in its cytoplasmic membrane.Abbreviations ATP adenosine-5-triphosphate - TTFB tetrachlortrifluoromethylimidazol - CCCP carbonyl cyanide m-chlorophenylhydrazone - Na⁺ transmembrane electrochemical potential differences of Na⁺ - transmembrane electric potential difference - pNa transmembrane pNa difference     

A survey of the response of Rhododendron to in vitro culture

The responses of 7 genotypes of Rhodendron to culture conditions and their establishment as shoot cultures are described. The genotypes represent a broad genetic diversity in the genus. After sterilization and an acclimation period of 3 to 12 months, all the selections were established as shoot cultures on Woody Plant Medium (WPM) supplemented with N⁶(-²-isopenteny) adenine (2iP). Plants with strong episodic growth cycles required the longer acclimation periods. Utilizing shoots from these cultures, the response to a cytokinin series of 0 to 32 M 2iP or BAP (6-Benzylaminopurine) was analyzed. BAP proved toxic to all but the elipidote and lepidote rhododendrons (R. mucronulatum, R. x Boule de Neige, and R. x PJM); however, even with these selections, 2iP stimulated greater shoot multiplication rates. The optimum 2iP level for shoot multiplication varied little with the genotype and levels of 4 to 16 M generally proved optimal, depending on the specific selection. Adventitious shoot production was observed in 3 selections (R. canadense, R. x Boule de Neige and R. x PJM), but only at 2iP levels above 8 M. Shoot multiplication rates of 7 to 21 times were observed, depending on the selection. Using an average utilizable shoot production rate of 40 shoots per culture per 6 week subculture period, some 75,000 shoots can be generated per square meter of culture space per year. The harvested shoots (microcuttings) rooted readily out-of-culture and the resultant plants grew like seedlings.     

Biochemical composition of three algal species proposed as food for captive freshwater mussels

To identify potential diets for rearing captive freshwater mussels, the protein, carbohydrate (CHO), and lipid contents of two green algae, Neochloris oleoabundans, Bracteacoccus grandis, and one diatom, Phaeodactylum tricornutum, were compared at different growth stages. The fatty acid and sterol composition were also identified. Protein was greatest (55–70%) for all species at late log growth stage (LL), and declined in late stationary (LS) growth. CHO was greatest at LS stage for all species (33.9–56.4% dry wt). No significant change in lipid levels occurred with growth stage, but tended to increase in N. oleoabundans. Mean lipid content differed significantly in the order: N. oleoabundans > P. tricornutum > B. grandis. Total fatty acids (TFA) were higher at LS stage compared to other stages in the two green algae, and stationary stage in the diatom. Mean unsaturated fatty acids (UFA) as %TFA was significantly higher in N. oleoabundans than the other species. The green algae contained high percentages of C-18 polyunsaturated fatty acids (PUFAs), while the diatom was abundant in C-16 saturated and mono-unsaturated fatty acids and C-20 PUFA fatty acids. Growth stage had no effect on sterol concentration of any species. B. grandis showed significantly higher sterol levels than the other species except P. tricornutum at S stage. B. grandis was characterized by predominantly ⁵, C-29 sterols, while N. oleoabundans synthesized ^5,7, ^5,7,22 , and ⁷, C-28 sterols. P. tricornutum produced primarily a ^5,22, C-28 sterol, and a small amount of a ^7,22, C-28 sterol.     

ATP is required for K+ active transport in the archaebacterium Haloferax volcanii

The Archaebacterium Haloferax volcanii concentrates K⁺ up to 3.6 M. This creates a very large K⁺ ion gradient of between 500- to 1,000-fold across the cell membrane. H. volcanii cells can be partially depleted of their internal K⁺ but the residual K⁺ concentration cannot be lowered below 1.5 M. In these conditions, the cells retain the ability to take up potassium from the medium and to restore a high internal K⁺ concentration (3 to 3.2 M) via an energy dependent, active transport mechanism with a K _m of between 1 to 2 mM. The driving force for K⁺ transport has been explored. Internal K⁺ concentration is not in equilibrium with m suggesting that K⁺ transport cannot be accounted for by a passive uniport process. A requirement for ATP has been found. Indeed, the depletion of the ATP pool by arsenate or the inhibition of ATP synthesis by N,N-dicyclohexylcarbodiimide inhibits by 100% K⁺ transport even though membrane potential m is maintained under these conditions. By contrast, the necessity of a m for K⁺ accumulation has not yet been clearly demonstrated. K⁺ transport in H. volcanii can be compared with K⁺ transport via the Trk system in Escherichia coli.Abbreviations CCCP Carbonylcyanide m-chlorophenyl-hydrazone - DCCD N,N-dicyclohexylcarbodiimide - MES 2-[N-morpholino] ethane sulfonic acid - MOPS 3-[N-morpholino] propane sulfonic acid - TRIS Tris (hydroxymethyl) aminomethane - TPP tetraphenyl phosphonium