Addition Of Dinitrophenol Research Articles

Mo1959 Basolateral IKCA Channel Inhibition by Octreotide Prevents the Increase in Mucosal Permeability Induced by Chemical Hypoxia in Human Colon: Implications for Preventing Sepsis After Major Abdominal Surgery

Background: Intestinal ischemia occurs frequently during major abdominal surgery, causing increasedmucosal permeability and bacterial translocation, thus increasing the risk of postoperative sepsis.We previously showed that chemical hypoxia stimulated basolateral, intermediate conductance, Ca2+-sensitive K+ (IKCa) channels and doubled paracellular shunt conductance (GS) (which reflects mucosal permeability) in human colon in vitro, changes which were completely inhibited by the serosal addition of IKCa channel blockers TRAM-34 and clotrimazole (Am J Physiol Gastrointest Liver Physiol 300:G146-G153,2011). Aim: To determine whether inhibition of basolateral IKCa channels by somatostatin (SOM) and its synthetic analog octreotide (OCT) (Am J Physiol Gastrointest Liver Physiol 277:G967-G975,1999) prevents the increase in GS induced by chemical hypoxia in human colon. Methods: With informed consent, sheets of muscle-stripped normal human distal colon (from cancer patients) were mounted in an Ussing chamber, and bathed with high-K+ gluconate solution (mucosal side) and high-Na+ gluconate solution (serosal side) at 37oC. While passing 2-sec rectangular current pulses (I; 50 μA), transepithelial voltage (VT), total tissue conductance (GT; = I/ΔVT) and calculated short-circuit current (Isc; = VT.GT) were monitored until constant values were obtained. Tissues acting as ‘controls' were gassed throughout with 95%O2/5%CO2. Other tissues gassed with 95%O2/5%CO2 were switched to 95%N2/5%CO2 when 100 μM dinitrophenol (DNP) was added mucosally and serosally to induce chemical hypoxia over 5-min. 2 μM SOM was added to hypoxic tissues either 10-min before or immediately after addition of DNP. Nystatin (500 U/ml) was then added mucosally to permeabilize the apical membrane, and VT, GT and Isc monitored at 10-sec intervals to steady-state. Similar experiments in hypoxic tissues established the dose response to OCT. GS was estimated from the y-axis intercept of the linear relationship between GT and Isc (J Membr Biol 45:81108,1979). Results: GS was significantly greater in hypoxic tissues (5.52±1.22 mS/cm2; n= 10) than in controls (3.65±1.34 mS/cm2; n=11, P<0.005). GS was similar in hypoxic tissues irrespective of whether SOM was added before (3.28±1.56 mS/cm2; n=6) or after (3.48±1.21 mS/cm2; n=6) the application of DNP. GS in both SOM groups was significantly lower than in tissues treated with DNP alone (P<0.05), but similar to GS in controls. Dose response data indicated that 0.2 μM OCT and 2 μM OCT/SOM had the same protective effect on GS. Conclusion: Our results suggest that perioperative administration of OCT (a long-acting synthetic analog of SOM) during major abdominal surgery may prevent the increase in intestinal mucosal permeability associated with intestinal ischemia, thereby decreasing the likelihood of bacterial translocation and post-operative sepsis.

Acute Mitochondrial Actions of Glitazones on the Liver: a Crucial Parameter for their Antidiabetic Properties

Background/aims: Glitazones are synthetic insulin-sensitizing drugs which act as agonists of peroxisome proliferator-activated receptor gamma (PPARγ). However, TZDs action does not exclude independent PPARγ-activation effects. Remarkably, direct mitochondrial action of these agents has not been fully studied yet. Methods: Oxygen consumption rates (JO₂) were measured using a Clark-type oxygen electrode in intact hepatocytes and isolated liver mitochondria. Mitochondrial reactive oxygen species (ROS) production was quantified by fluorescence assay. Moreover, activities of mitochondrial respiratory chain complex I, II and III were spectrometrically determined. Results: Pioglitazone and rosiglitazone inhibited JO₂ in liver cells and mitochondria. This inhibition affected the state 3 of respiration (in the presence of ADP) and the uncoupled state (after addition of dinitrophenol). Moreover, these agents dramatically reduced mitochondrial ROS production in all situations tested. We also demonstrated that both glitazones specifically inhibited the activities of complex I and complex III, by 50% and 35% respectively. Additionally, they do not modify neither the oxidative phosphorylation yield nor the permeability transition pore opening. Conclusions: Pioglitazone and rosiglitazone reduce both respiration intensity and ROS production, acutely and by a probable PPARγ-independent way, through inhibition of complex I and III activities. This new finding could positively contribute to their anti-diabetic properties.

Differential inhibition of nitrification by three metabolic inhibitors

In a soil slurry the effects of two uncouplers of oxidative phosphorylation, DNP (2,4 dinitrophenol) and CCCP (carbonyl cyanide m‐chlorophenyl hydrazone), and one inhibitor of terminal oxidase (KCN) on biological oxidation (nitrification) of NH4 + and NO2 ‐ to NO3 ‐ were studied. DNP did not affect, KCN reduced, and CCCP fully inhibited the oxidation of both NH4 + and NO2 ‐ to NO3 ‐Addition of DNP to soil slurry resulted in formation of excess NO3 ‐ (over that of the control) which was stoichiometrically close to the amount of nitrogen in the added DNP. It is suggested that the ineffectiveness of DNP as nitrification inhibitor was partly due to its rapid hydrolysis and formation of excess NO2 ‐which was in turn nitrified to NO3 ‐ It is concluded that in comparison to DNP, CCCP and KCN introduce no artefacts into the experimental cultures and are thus better suited for studies involving inhibition of biological nitrification than DNP.

Evidence for active transport of 3H-androgens across the epididymal epithelium in the rat.

The effect of metabolic inhibitors on transepithelial movement of 3H-androgens was investigated by in vivo perifusion and subsequent micropuncture of caput and cauda epididymal tubules. Epididymal tissue (adenosine triphosphate (ATP) concentrations were determined at 1 h after exposure of perifusion fluid with metabolic inhibitor. To determine whether or not metabolic inhibitor alters intraluminal androgen-binding protein concentration or androgen binding to interstitial proteins in the caput epididymis, 3H-dihydrotestosterone (DHT) binding to interaluminal androgen-binding protein and bound vs free androgen ratio in the interstitial fluid after 1 h perifusion with fluid containing metabolic inhibitor around caput epididymal tubules were examined. Proluminal movement of 3H-androgens and tissue ATP concentrations in the caput and cauda epididymis were significantly decreased by addition of dinitrophenol (DNP) or potassium cyanide to the perifusion fluid. Relative intraluminal androgen-binding protein protein concentration and bound vs free 3H-androgen ratio in the interstitial fluid were not altered when DNP or potassium cyanide was added to the perifusion fluid. These results demonstrate for the first time that an energy-dependent mechanism may be involved in the epididymal androgen uptake.

Involvement of the CDC25 gene product in the signal transmission pathway of the glucose-induced RAS-mediated cAMP signal in the yeast Saccharomyces cerevisiae

Addition of glucose or related fermentable sugars to derepressed cells of the yeast Saccharomyces cerevisiae triggers a RAS-protein-mediated cAMP signal, which induces a protein phosphorylation cascade. Yeast strains without a functional CDC25 gene were deficient in basal cAMP synthesis and in the glucose-induced cAMP signal. Addition of dinitrophenol, which in wild-type strains strongly stimulates in vivo cAMP synthesis by lowering intracellular pH, did not enhance the cAMP level. cdc25 disruption mutants, in which the basal cAMP level was restored by the RAS2val19 oncogene or by disruption of the gene (PDE2) coding for the high-affinity phosphodiesterase, were still deficient in the glucose- and acidification-induced cAMP responses. These results indicate that the CDC25 gene product is required not only for basal cAMP synthesis in yeast but also for specific activation of cAMP synthesis by the signal transmission pathway leading from glucose to adenyl cyclase. They also show that intracellular acidification stimulates the pathway at or upstream of the CDC25 protein. When shifted to the restrictive temperature, cells with the temperature sensitive cdc25-5 mutation lost their cAMP content within a few minutes. After prolonged incubation at the restrictive temperature, cells with this mutation, and also those with the temperature sensitive cdc25-1 mutation, arrested at the 'start' point (in G1) of the cell cycle, and subsequently accumulated in the resting state G0. In contrast with cdc25-5 cells, however, the cAMP level did not decrease and normal glucose- and acidification-induced cAMP responses were observed when cdc25-1 cells were shifted to the restrictive temperature.(ABSTRACT TRUNCATED AT 250 WORDS)

Regulation of Activity of an ATP-Dependent Protease, Clp, by the Amount of a Subunit, ClpA, in the Growth of Escherichia coli Cells1

The activity of an ATP-dependent protease, Clp, was examined in Escherichia coli SG1110 (lon-) in various growth phases. The ATP-dependent proteolytic activity (Clp activity) in a crude extract of the cells changed with the growth phase. Cells in the early exponential growth phase showed the lowest activity, but then the activity increased dramatically with cell growth. The highest Clp activity was found in the cells in the late exponential and early stationary phases, however, the activity returned to the original level on prolonged culturing. These changes in Clp activity were closely correlated to the amount of one of the components of Clp, Clp A, which was quantitated immunochemically with antibodies against the Clp A protein. However, the amount of the other component of Clp, Clp P, did not change with the growth phase. These results suggest that the activity of Clp in the cells is regulated by the amount of Clp A in various growth phases. We next examined the effect of the cellular ATP level on Clp activity, because ATP is a cofactor for Clp protease in vitro. The addition of dinitrophenol (DNP) and sodium azide reduced the intracellular concentration of ATP, but had no effect on the Clp activity or the level of the Clp A protein when these drugs were added to the culture at the stationary phase. On the other hand, these drugs elevated both the Clp activity and the Clp A amount in exponentially growing cells, whose cellular ATP level was also reduced.(ABSTRACT TRUNCATED AT 250 WORDS)

Requirement of one functional RAS gene and inability of an oncogenic ras variant to mediate the glucose-induced cyclic AMP signal in the yeast Saccharomyces cerevisiae.

Addition of glucose to Saccharomyces cerevisiae cells grown on a nonfermentable carbon source triggers a cyclic AMP (cAMP) signal, which induces a protein phosphorylation cascade. In a yeast strain lacking functional RAS1 and RAS2 genes and containing a bcy mutation to suppress the lethality of RAS deficiency, the cAMP signal was absent. Addition of dinitrophenol, which stimulates in vivo cAMP synthesis by lowering intracellular pH, also did not enhance the cAMP level. A bcy control strain, with functional RAS genes present, showed cAMP responses similar to those of a wild-type strain. In disruption mutants containing either a functional RAS1 gene or a functional RAS2 gene, the cAMP signal was not significantly different from the one in wild-type cells, indicating that RAS function cannot be a limiting factor for cAMP synthesis during induction of the signal. Compared with wild-type cells, the cAMP signal decreased in intensity with increasing temperature in a ras2 disruption mutant. When the mutant RAS2Val-19, which carries the equivalent of the human H-rasVal-12 oncogene, was grown under conditions in which RAS1 expression is repressed, the cAMP signal was absent. The oncogene product is known to be deficient in GTPase activity. However, the amino acid change at position 19 (or 12 in the corresponding human oncogene product) might also have other effects, such as abolishing receptor interaction. Such an additional effect probably provides a better explanation for the lack of signal transmission than the impaired GTPase activity. When the RAS2Val-19 mutant was grown under conditions in which RAS1 is expressed, the cAMP signal was present but significantly delayed compared with the signal in wild-type cells. This indicates that oncogenic RAS proteins inhibit normal functioning of wild-type RAS proteins in vivo and also that in spite of the presence of the RAS2(Val-19) oncogene, adenyl cyclase is not maximally stimulated in vivo. Expression of only the RAS(Val-19) gene product also prevented most of the stimulation of cAMP synthesis by dinitrophenol, indicating that lowered intracellular pH does not act directly on adenyl cyclase but on a step earlier in the activation pathway of the enzyme. The results obtained with the control bcy strain, the RAS2(Val-19) strain under conditions in which RAS1 is expressed, and with dinitrophenol show that the inability of the oncogene product to mediate the cAMP signal is not due to feedback inhibition by the high protein kinase activity in strains containing the RAS2(Val-19) oncogene. Hence, the present results show that the RAS protein in S. cerevisiae are involved in the transmission of the glucose-induced cAMP signal and that the oncogenic RAS protein is unable to act as a signal transducer. The RAS protein in S. cerevisiae apparently act similarly to the Gs proteins of mammalian adenyl cyclase, but instead of being involved in hormone signal transmission, they function in a nutrient-induced signal transmission pathway.

Requirement of One Functional RAS Gene and Inability of an Oncogenic ras Variant to Mediate the Glucose-Induced Cyclic AMP Signal in the Yeast Saccharomyces cerevisiae

Addition of glucose to Saccharomyces cerevisiae cells grown on a nonfermentable carbon source triggers a cyclic AMP (cAMP) signal, which induces a protein phosphorylation cascade. In a yeast strain lacking functional RAS1 and RAS2 genes and containing a bcy mutation to suppress the lethality of RAS deficiency, the cAMP signal was absent. Addition of dinitrophenol, which stimulates in vivo cAMP synthesis by lowering intracellular pH, also did not enhance the cAMP level. A bcy control strain, with functional RAS genes present, showed cAMP responses similar to those of a wild-type strain. In disruption mutants containing either a functional RAS1 gene or a functional RAS2 gene, the cAMP signal was not significantly different from the one in wild-type cells, indicating that RAS function cannot be a limiting factor for cAMP synthesis during induction of the signal. Compared with wild-type cells, the cAMP signal decreased in intensity with increasing temperature in a ras2 disruption mutant. When the mutant RAS2Val-19, which carries the equivalent of the human H-rasVal-12 oncogene, was grown under conditions in which RAS1 expression is repressed, the cAMP signal was absent. The oncogene product is known to be deficient in GTPase activity. However, the amino acid change at position 19 (or 12 in the corresponding human oncogene product) might also have other effects, such as abolishing receptor interaction. Such an additional effect probably provides a better explanation for the lack of signal transmission than the impaired GTPase activity. When the RAS2Val-19 mutant was grown under conditions in which RAS1 is expressed, the cAMP signal was present but significantly delayed compared with the signal in wild-type cells. This indicates that oncogenic RAS proteins inhibit normal functioning of wild-type RAS proteins in vivo and also that in spite of the presence of the RAS2(Val-19) oncogene, adenyl cyclase is not maximally stimulated in vivo. Expression of only the RAS(Val-19) gene product also prevented most of the stimulation of cAMP synthesis by dinitrophenol, indicating that lowered intracellular pH does not act directly on adenyl cyclase but on a step earlier in the activation pathway of the enzyme. The results obtained with the control bcy strain, the RAS2(Val-19) strain under conditions in which RAS1 is expressed, and with dinitrophenol show that the inability of the oncogene product to mediate the cAMP signal is not due to feedback inhibition by the high protein kinase activity in strains containing the RAS2(Val-19) oncogene. Hence, the present results show that the RAS protein in S. cerevisiae are involved in the transmission of the glucose-induced cAMP signal and that the oncogenic RAS protein is unable to act as a signal transducer. The RAS protein in S. cerevisiae apparently act similarly to the Gs proteins of mammalian adenyl cyclase, but instead of being involved in hormone signal transmission, they function in a nutrient-induced signal transmission pathway.

After-hyperpolarizations produced in frog motoneurons by excitatory amino acid analogues

After-hyperpolarizations (AHPs) produced in frog motoneurons by applications of the excitatory amino acid analogues quisqualate (QUIS), N-methyl- d-aspartate (NMDA), and kainate (KA) were studied in the isolated hemisected frog spinal cord using sucrose gap techniques. AHPs were present following 98% of QUIS-induced depolarizations, but were seen in only 35% and 15% of NMDA- and KA-evoked responses respectively. AHPs produced by QUIS are produced both by direct effects of QUIS on motoneuron membranes and by indirect effects mediated through a synaptic process involving interneurons. Thus, applications of Mg 2+, Mn 2+, or tetrodotoxin (TTX) in concentrations sufficient to block synaptic transmission and interneuronal firing, reduced, but did not abolish the AHPs produced by QUIS. In contrast, NMDA- and KA-AHPs appear to be entirely mediated by indirect means as block of synaptic transmission and interneuronal firing eliminated AHPs produced by these substances. Exposure of the cord to Mn 2+ after addition of TTX did not affect the size of QUIS-AHPs. In the presence of TTX, QUIS-AHPs were reduced or completely blocked by addition of dinitrophenol (DNP) and sodium cyanide, by dihydro-ouabain, by removal of K + from the superfusate, by cooling, and by replacement of 50% of the external Na + with Li +. The results suggest that the QUIS-AHPs are largely the result of the direct effect of the excitatory amino acid agonist on motoneuron membranes and is caused by activation of an electrogenic Na + pump. AHPs following depolarizations evoked by NMDA and KA are presumably the result of indirect actions of these latter analogues on interneurons.

The effect of dinitrophenol on magnesium transport across an isolated preparation of sheep rumen epithelium.

Mg transport across an isolated preparation of sheep rumen epithelium was studied in vitro. The efflux (flux from the mucosal side to serosal side) and the influx (flux with serosal to mucosal side) were estimated with 28Mg. Under physiological conditions a significant net efflux of Mg was observed, in which the unidirectional efflux was seven to eight times that of the influx. In experiments where ATP synthesis was blocked by the addition of dinitrophenol, Mg efflux was reduced to that of the influx. These observations support the conclusion that Mg absorption across the rumen wall of the sheep is an active process.

Prolonged post-tetanic depolarization of frog dorsal root fibers

In the isolated frog spinal cord, tetanic stimulation of a dorsal root resulted in a sustained negative potential recorded from that root. This negative potential was followed by a large (5–12 mV) and slow (5–60 s) negative after-potential which persisted after the train of impulses and which was succeeded by a long-lasting positive potential. The negative after-potential was postulated to be largely a consequence of excess extracellular K + accumulated during the tetanus, because the negative after-potential and the extracellular [K +] measured with K +-sensitive microelectrodes were a function of the stimulating current, the stimulation frequency and the duration of the tetanus. Both measures were augmented by procedures which interfered with the clearance of K + from the extracellular space (addition of dinitrophenol or ouabain, partial substitution of Li + for Na +, elimination of K +from the superfusate and cooling). The negative after-potential and the liberation of much of the excess extracellular K + were reduced by increasing the superfusing divalent ion concentration with 4mM Ca 2+or 50–100 μM Mn 2+. Both measures were augmented in low Ca 2+-containing Ringer's solution. The results with low concentrations of Ca 2+, Na + and K + and with high concentrations of Ca 2+ indicate that the negative after-potential was not caused by a conductance increase for these cations. Reduction of extracellular [Cl −] did not change the negative after-potential making it unlikely that this anion is a factor in its production. The negative after-potential was not mediated by γ-aminobutyrate because its amplitude and duration were increased by the γ-aminobutyrate antagonist picrotoxin, and were reduced by pentobarbital which enhances the actions of γ-aminobutyrate. Application of the excitatory amino acid antagonists dl-α-aminoadipic acid and glutamic acid diethylester eliminated the negative after-potential and markedly reduced the amount of K + liberated by a tetanus. These results suggest that the K + release that produces the negative after-potential may result from the action of an excitatory amino acid such as l-glutamate or l-aspartate either synaptically released or released from nonsynaptic regions of afferent fibers.

Functioning of Mitochondria-Bound Hexokinase in Rat Brain in Accordance with Generation of ATP inside the Organelle

The function of mitochondria-bound hexokinase, the enzymatic form peculiar to the brain, in utilization of ATP generated inside the organelles, was examined by incubating rat brain mitochondrial fraction with [14C]glucose under various conditions. Addition of succinate and ADP to the incubation medium increased glucose 6-phosphate formation by the mitochondrial hexokinase and caused a smaller increase in ATP concentration in the mitochondria. The glucose phosphorylation was markedly inhibited by the addition of dinitrophenol, potassium cyanide, and oligomycin, and the ATP concentration was decreased. On the other hand, addition of atractyloside suppressed the glucose phosphorylation without affecting the mitochondrial hexokinase activity, whereas addition of antiserum against the mitochondrial hexokinase inhibited both glucose 6-phosphate formation and hexokinase activity. A part of both the glucose phosphorylation and hexokinase activities, however, remained even in the presence of the maximum dose of the anti-hexokinase serum and atractyloside. These results indicate the active utilization of intrinsically generated ATP by the mitochondria-bound hexokinase, a part of which may be located away from the surface of the mitochondrial membrane.

Ammonium uptake and metabolism by mitrogen fixing bacteria. II. Klebsiella pneumoniae.

The primary steps of N2, ammonia and nitrate metabolism in Klebsiella pneumoniae grown in a continuous culture are regulated by the kind and supply of the nitrogenous compound. Cultures growing on N2 as the only nitrogen source have high activities of nitrogenase, unadenylated glutamine synthetase and glutamate synthase and low levels of glutamate dehydrogenase. If small amounts of ammonium salts are added continuously, initially only part of it is absorbed by the organisms. After 2-3 h complete absorption of ammonia against an ammonium gradient coinciding with an increased growth rate of the bacteria is observed. The change in the extracellular ammonium level is paralleled by the intracellular glutamine concentration which in turn regulates the glutamine synthesis and an induction of glutamate dehydrogenase synthesis. Upon deadenylation these events are reversed.--Addition of dinitrophenol causes transient leakage of intracellular ammonium into the medium.

Regulation of pyruvate dehydrogenase in isolated rat liver mitochondria. Effects of octanoate, oxidation-reduction state, and adenosine triphosphate to adenosine diphosphate ratio.

Factors which influence the distribution of pyruvate dehydrogenase between its active, unphosphorylated form (PDHa) and its inactive, phosphorylated form (PDHb) have been examined in isolated rat liver mitochondria. A rapid freezing method was developed for the extraction of pyruvate dehydrogenase from incubated mitochondria which prevented interconversions between PHDa and PDHb which normally occur when mitochondria are collected by centrifugal methods. The intramitochondrial ATP:ADP ration was varied over a 100-fold range by the addition of dinitrophenol, oligomycin, or both substances to mitochondria oxidizing 2-oxoglutarate. PDHa activity was found to be inversely proportional to the intramitochondrial ATP:ADP ratio but was not closely correlated with the extramitochondrial adenine nucleotide levels. When mitochondria were incubated in State 4 with succinate and rotenone, the addition of pyruvate increased PDHa activity more than 10-fold without appreciably altering the mitochondrial ATP:ADP ratio. These observations are most readily explained by the known inhibitory effects of pyruvate and ADP on PDHa kinase. PDHa activity could be maintained at a high level by incubating mitochondria in a condition resembling State 3 by the addition of succinate, glucose, and hexokinase. The further addition of octanoate reduced PDHa activity by 60% without appreciably altering the ATP:ADP ratio. Rotenone had a sililar effect. When added in the presence of octanoate, rotenone further decreased PDHa activity whereas 4-pentenoate led to an increase in activity. The effects of octanoate on PDHa activity were not seen when mitochondria were incubated in the presence of high levels of pyruvate, though pyruvate oxidation was till diminished by over 50%. The data suggest that octanoate addition favors the PDHa kinase reaction leading to inactivation of PDHa, and in addition causes the accumulation of NADH and acetyl-CoA which are recognized competitive inhibitors of pyruvate dehydrogenase.

ENHANCED RATE OF14C-SOLUTE RELEASE TO THE FREE SPACE BY THE PHLOEM OF VICIA FABA STEMS PARASITISED BY CUSCUTA

SUMMARY By washing out 14C-labelled assimilates from the free space of stem segments of Vicia faba L., considerably more label could be removed from a stem parasitised by Cuscuta species than from a normal one. This could mean that the moving of assimilates from the host phloem to the parasite at least partly occurs through the apoplast. The assumed unloading of the host phloem is apparently under metabolic control since it appeared to be inhibited at 0°C and after addition of dinitrophenol.

Biochemical effects of cuprizone on mouse liver and heart mitochondria

Mitochondria isolated from livers of mice fed the copper-chelating agent, cuprizone, showed a decrease in state 3 respiration, respiratory control ratios and ADP/O or P/O ratios. This inhibition of state 3 respiration was not relieved by the addition of dinitrophenol (DNP). These inhibitory effects apparently involved all three sites of mitochondrial-coupled oxidative phosphorylation as well as possibly having an effect on substrate-level oxidative phosphorylation. Cuprizone produced a greater degree of inhibition of phosphorylation than of oxidation and therefore has the characteristics of an inhibitor uncoupler. Liver mitochondria isolated from control mice or from normal rats were unaffected by treatment in vitro with cuprizone. In contrast to liver mitochondria, mitochondria isolated from the hearts of mice fed cuprizone show no defects in oxidative phosphorylation.

Stimulation and inhibition of membrane-dependent ATP synthesis in chloroplasts by artificially induced K+ gradients.

The imposition of a potassium ion gradient simultaneous with a pH transition of Valinomycin treated spinach chloroplasts results in a modulation of ATP synthesis elicited by the acid-base transition. A gradient from the outside to the interior of the granum results in a stimulation of ATP synthesis. Under these conditions the synthesis of ATP is sensitive to the addition of dinitrophenol. A gradient of the opposite sense results in an inhibition of ATP synthesis and the yield is not further reduced by the addition of dinitrophenol. The data support the concept that ion imbalance caused by Valinomycin facilitated K+ movement across the energy conserving membrane of the chloroplast can lead to membrane potential differences of sufficient magnitude to influence the yield of ATP in a membrane-dependent energy conserving system.

Studies on the maintenance of development in tetrahymena pyriformis GL-C. I. An analysis of the mechanism of resorption of developing oral structures.

AbstractResorption of differentiating oral structures of Tetrahymena pyriformis GL‐C can be brought about by exposures to high and to low temperatures, to antagonists of RNA and of protein synthesis, and to inhibitors of energy metabolism. The resorption process is accompanied by a blockage (or reversal) of cell division furrowing. At a late developmental stage, oral primordium development and the cell division process become simultaneously stabilized, so that both processes can be completed (although often at a reduced rate) even in the presence of the agent. The stage at which stabilization takes place is independent of the agent employed.The sequence of events observed during the resorption process was generally the same regardless of the agent which brought the process about. However, the rate of resorption depended on the agent and also on the concentration at which it was employed. At a sublethal high temperature (34°C) and in the presence of antagonists of RNA and protein synthesis, resorption was rapid, being completed in an hour or less. On the other hand, in the presence of 2,4‐dinitrophenol (at a concentration just above the threshold for blocking cell division) resorption was extremely slow and incomplete. At low temperatures, resorption was also slow, the rate depending on the temperature; a linear relationship was obtained if the logarithm of the resorption rate was plotted against the reciprocal of the absolute temperature (Arrhenius plot).Following the addition of dinitrophenol or the onset of low temperature treatment, there was an initial interval during which oral development was blocked or considerably retarded, but oral primordium resorption had not yet commenced. If cells were returned to optimal growth conditions (by washing out the DNP or returning samples from the cold to 28°C) late in this interval, they underwent immediate and rapid resorption of oral primordia in which membranelle differentiation had begun. This resorption process always went to completion. Resorption is thus an irreversible process, which is in some way pre‐determined or triggered by dinitrophenol, low temperature, and probably the other agents also.The time required to bring about the triggering of resorption was ascertained by exposing synchronized cultures to DNP or low temperature treatments of differing durations. Resorption was triggered quite rapidly by DNP, and more slowly by low temperature. Furthermore, the time required to trigger resorption at 1°C was much longer than the time required at 9°C. Hence the triggering of resorption probably involves some metabolic activity, but not a substantial energy requirement. It is suggested, in the discussion, that the resorption process involves the localized enzymatic degradation of the developing oral structures and that the triggering of this process probably corresponds to the activation and/or mobilization of the degradative enzymes. Alternative hypotheses of the nature of this triggering mechanism are presented and discussed.

Transport Systems for 1,4-Diaminobutane, Spermidine, and Spermine in Escherichia coli

Uptake processes for 1,4-diaminobutane, spermidine, and spermine have been studied in Escherichia coli. The uptake systems can be divided into two components. One is a rapid adsorption that is not dependent on active metabolic processes, and can be observed at 0° as well as at 37°. The adsorbed amine can be largely removed by washing the bacteria with the nonradioactive amine or with salt-containing media. The amount of the adsorbed amine was less than 1% of the added amine for 1,4-diaminobutane, 3 to 5% for spermidine, and 7 to 15% for spermine. The second is a larger, time-dependent component which depends on metabolic processes, does not occur at 0°, and is markedly decreased by the omission of glucose or by the addition of dinitrophenol. The accumulated amines cannot be removed by washing the bacteria with solutions of the nonradioactive amines; they are released, however, by treatment of the bacteria with toluene or butanol-1. Studies are also reported on the effect of pH, concentration, and inhibitors on these systems.

Studies on controlled and released respiration in animal and vegetal halves of the embryo of the sea urchin, Paracentrotus lividus

Summary Eggs of the sea urchin Paracentrotus lividus were separated in the 16–32 cell stage in animal and vegetal halves. These were allowed to develop, and the respiration of the halves was measured in the microrespirometer of Linderstrom-Lang and Holter. The measurements referred to advanced cleavage stages (≥128 cells) and to the mesenchyme blastula stage or beginning gastrula stage. The medium was either pure sea water or sea water within the addition of 5×10 −5 M 2,4-dinitrophenol. In the first medium the “controlled” in the latter the “released” respiration was measured. A survey of the results is given in Figs. 1 and 2. In both stages which were subject to examination, the respiratory control ratios (released respiration over controlled respiration) in animal halves were high, about 2.9, a value corresponding to that found immediately after fertilization in whole eggs suspended in normal sea water [14]. In advanced cleavage stage, the respiratory rate seemed to be higher in vegetal than in animal halves. Addition of dinitrophenol caused a certain decrease in the respiratory rate (Fig. 1). Vegetal halves in the mesenchyme blastula state (Fig. 2), on the other hand, showed a respiratory rate not significantly different from that in animal halves. Upon addition of dinitrophenol only a rather low increase in the respiratory rate occurred (Fig. 2). Controls were made in order to ascertain that the changes in respiratory rate were not due to damage caused by the process of separating the halves. As the released respiration expresses the maximal electron transport capacity [24], it may also be considered an approximate measure of the amount of respiratory enzyme chains present in the embryos. Thus, a considerable synthesis of these enzymes must take place in the isolated animal halves. This is in agreement with the increasing incorporation of amino acids in isolated animal halves, demonstrated by Markman [21]. The results with isolated vegetal halves at mesenchyme blastula stage indicate, on the other hand, that the amount of respiratory enzyme chains is lower than in animal halves, which again corresponds to studies on incorporation of labeled amino acids [21]. The different values of the respiratory control ratio in animal and vegetal halves indicate differences in metabolic state. A high value of the ratio is in keeping with an accumulation of energy-rich compounds, whereas lower values indicate that a certain steady state has been approached. The interactions between animal and vegetal regions which display themselves in the normal development are discussed in the light of the results obtained. Financial support was given from the Swedish Natural Sciences Research Council, the Swedish Cancer Society and the Consiglio Nazionale delle Recerche (Rome). These grants are most gratefully acknowledged.