The effects of 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone on isolated pea chloroplasts

The effects of a quinone analogue, 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone(DBMIB), on the photochemical activities of isolated pea chloroplastshave been investigated. DBMIB completely inhibits pseudo-cyclicflow with methyl viologen (MV) and partially inhibits non-cyclicflow with ferricyanide (FeCN) in both coupled and uncoupledsystems. It is also shown that, under some circumstances, DBMIBacts like an uncoupler in that it stimulates electron flow andinhibits cyclic photophosphorylation. ¹ Present address: 32(1), 6 1/4 Mile, Prome Road, Kamayut P.O., Rangoon, Burma. (Received May 21, 1973; )     

Comparison of normalisation methods for surface-enhanced laser desorption and ionisation (SELDI) time-of-flight (TOF) mass spectrometry data

Background  

Mass spectrometry for biological data analysis is an active field of research, providing an efficient way of high-throughput proteome screening. A popular variant of mass spectrometry is SELDI, which is often used to measure sample populations with the goal of developing (clinical) classifiers. Unfortunately, not only is the data resulting from such measurements quite noisy, variance between replicate measurements of the same sample can be high as well. Normalisation of spectra can greatly reduce the effect of this technical variance and further improve the quality and interpretability of the data. However, it is unclear which normalisation method yields the most informative result.     

Uptake of ATP analogs by isolated pea chloroplasts and their effect on CO2 fixation and electron transport.

1. The ATP analog, adenylyl-imidodiphosphate rapidly inhibited CO2-dependent oxygen evolution by isolated pea chloroplasts. Both alpha, beta- and beta, gamma-methylene adenosine triphosphate also inhibited oxygen evolution. The inhibition was relieved by ATP but only partially relieved by 3-phosphoglycerate. Oxygen evolution with 3-phosphoglycerate as substrate was inhibited by adenylyl-imidodiphosphate to a lesser extent than CO2-dependent oxygen evolution. The concentration of adenylylimidodiphosphate required for 50% inhibition of CO2-dependent oxygen evolution was 50 micronM. 2. Although non-cyclic photophosphorylation by broken chloroplasts was not significantly affected by adenylyl-imidodiphosphate, electron transport in the absence of ADP was inhibited by adenylyl-imidodiphosphate to the same extent as by ATP, suggesting binding of the ATP analog to the coupling factor of phosphorylation. 3. The endogenous adenine nucleotides of a chloroplast suspension were labelled by incubation with [14C]ATP and subsequent washing. Addition of adenylyl-imidodiphosphate to the labelled chloroplasts resulted in a rapid efflux of adenine nucleotides suggesting that the ATP analog was transported into the chloroplasts via the adenine nucleotide translocator. 4. It was concluded that uptake of ATP analogs in exchange for endogenous adenine nucleotides decreased the internal ATP concentration and thus inhibited CO2 fixation. Oxygen evolution was inhibited to a lesser extent in spinach chloroplasts which apparently have lower rates of adenine nucleotide transport than pea chloroplasts.     

Over-reduction of cultured tobacco cells mediates changes in respiratory activities

Regulation of respiration in Nicotiana tabacum suspension cultures was studied using the respiratory inhibitor myxothiazol and the oxidative phosphorylation uncoupler carbonylcyanide p -(trifluoromethoxy)phenylhydrazone (FCCP). Myxothiazol (2 μ M ) or FCCP (2 μ M ) almost completely inhibited cell growth for about 24 h, after which the cultures could resume a growth rate similar to that of the untreated culture. During the first 18 h of myxothiazol treatment, rates of cellular respiration were similar to control cultures. The capacity of the alternative pathway was significantly higher than control 2–3 h after myxothiazol treatment and increased further after 18 h. The capacity of the alternative pathway in FCCP-treated tobacco cells also increased but to a lesser extent. ATP/ADP ratios decreased for at least 9 h after either myxothiazol or FCCP treatments, which triggered an increase in glycolytic flux and an over-reduction of cultured tobacco cells, as demonstrated by increases in levels of reactive oxygen species and cellular ethanol. The generation of reactive oxygen species served as a signal for an induction of alternative oxidase, which in turn relieved the aerobic fermentation and over-reduction of the cells.     

HAT: Hypergeometric Analysis of Tiling-arrays with application to promoter-GeneChip data

Background  

Tiling-arrays are applicable to multiple types of biological research questions. Due to its advantages (high sensitivity, resolution, unbiased), the technology is often employed in genome-wide investigations. A major challenge in the analysis of tiling-array data is to define regions-of-interest, i.e., contiguous probes with increased signal intensity (as a result of hybridization of labeled DNA) in a region. Currently, no standard criteria are available to define these regions-of-interest as there is no single probe intensity cut-off level, different regions-of-interest can contain various numbers of probes, and can vary in genomic width. Furthermore, the chromosomal distance between neighboring probes can vary across the genome among different arrays.     

Respiratory activities in chloramphenicol-treated tobacco cells

Chloramphenicol (CAP) inhibited tobacco cell growth as shown by a reduction (34%) of cell mass 4 days after treatment. The rates of cell respiration were slightly higher than control under coupled conditions. However, CAP-treated cells showed a decreased maximal capacity of the cytochrome pathway (48%) and an increased maximal capacity of alternative path (56%) 4 days after treatment. In purified mitochondria, the rates of NADH or malate oxidation under state 4 conditions were not significantly changed by CAP treatment. However, the state 3 rates were 34–40% lower in CAP-treated than in control mitochondria. Succinate oxidation decreased by 31–46% under both state 4 and state 3 conditions after CAP treatment. The activities of complexes I, III, and IV, which contain mitochondrially encoded subunits, decreased by about 50% in CAP-treated mitochondria. There was also a decrease in the contents of mitochondrial cytochromes. Unexpectedly, the activities of complex II and the matrix-facing rotenone-insensitive NADH dehydrogenase, which are thought to be nuclear-encoded, also declined. The activities of external NADH dehydrogenase, NAD-linked malic enzyme, and fumarase remained unchanged after CAP treatment. There was a slight increase in the activity and protein level of alternative oxidase. An electrochemical gradient across the mitochondrial membranes was observed by Rhodamine 123 staining in CAP-treated cells. However, the morphology of most of the mitochondria changed from spherical to vermicular. A method for purifying a high yield of intact mitochondria from tobacco cell suspension cultures is described.     

Statistical modelling of mitochondrial power supply

By experiment and theory, formulae are derived to calculate the response of mitochondrial power supply, in flux and potential, to an ATP consuming enzyme load, incorporating effects of varying amounts of (i) enzyme, (ii) total circulating adenylate, and (iii) inhibition of the ATP/ADP translocase. The formulae, which apply between about 20% and 80% of maximum respiration, are the same as for the current and voltage of an electrical circuit in which a battery with potential, linear in the logarithm of the total adenylate, charges another battery whose opposing potential is also linear in the same logarithm, through three resistances. These resistances produce loss of potential due to dis-equilibrium of (i) intramitochondrial oxidative phosphorylation, (ii) the ATP/ADP translocase, and (iii) the ATP-consuming enzyme load. The model is represented geometrically by the following configuration: when potential is plotted against flux, the points lie on two pencils of lines each concurrent at zero respiration, the two pencils describing the respective characteristics of the mitochondrion and enzyme. Control coefficients and elasticities are calculated from the formulae.