A pitfall in the computer-aided quantitation of autoradiograms

Swillens, Stéphane; Cochaux, Pascale; Lecocoq, Raymond

doi:10.1016/0968-0004(89)90097-2

Cited by 22 publications

(15 citation statements)

References 5 publications

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“…with increasing proton concentration, although even at pH 5 the label bound was only twice that for LHCIIc, despite the much higher levels of CF, subunit TI1 (sidATP synthase); (cHxN),C binding to the two LHCIIa bands, in contrast, showed a sharp increase at pH 7-8, reaching a similar binding stoichiometry to that shown by LHCIIc (we ascribe the apparent discrepancy of this data with the band intensity on autoradiographs, see Fig. 2, to the problems inherent in quantitation via densitometry ; Swillens et al, 1989). This increase in binding with increasing pH shows a degree of qualitative correlation with the increase in the capacity of broken chloroplasts to show (cHxN),C-sensitive, rapidly relaxing, DCMU-reversible chlorophyll fluorescence quenching.…”

Section: Effect Of Ph On Labelling Of Thylakoid Proteinsmentioning

confidence: 82%

“…2). Since densitometry of autoradiographs (particularly when using film which has not been preflashed) is unreliable as a quantitative measure of radioactivity (Swillens et al, 1989), these results were quantified by solubilising slices of gel carrying the Lhcb4 and LhcbS proteins (and other proteins comigrating with them) and assaying the bound (cHxN),C by scintillation counting ; the band at the dye-front, including labelled CF, subunit 111, was also assayed in this way. with increasing proton concentration, although even at pH 5 the label bound was only twice that for LHCIIc, despite the much higher levels of CF, subunit TI1 (sidATP synthase); (cHxN),C binding to the two LHCIIa bands, in contrast, showed a sharp increase at pH 7-8, reaching a similar binding stoichiometry to that shown by LHCIIc (we ascribe the apparent discrepancy of this data with the band intensity on autoradiographs, see Fig.…”

Section: Effect Of Ph On Labelling Of Thylakoid Proteinsmentioning

confidence: 99%

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Higher Plant Light‐Harvesting Complexes LHCIIa and LHCIIc are Bound by Dicyclohexylcarbodiimide During Inhibition of Energy Dissipation

Walters

Ruban

Horton

1994

European Journal of Biochemistry

126

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We have investigated the binding to proteins of the photosynthetic apparatus of the carboxymodifying agent dicyclohexylcarbodiimide, (cHxN),C ; this inhibits the protective dissipation of excess absorbed light energy (qE) by the light-harvesting apparatus of photosystem I1 (LHCII), suggesting that carboxyl amino-acid side chains within hydrophobic protein domains may be involved in qE. (cHxN),I4C was used to label thylakoids and photosystem I1 particles, so as to identify proteins which may be involved in the detection of lumen pH during qE induction. Of six thylakoid proteins labelled with (cHxN),C under conditions where qE is efficiently induced, three are associated with photosystem I, and none with the bulk LHCII. PSII-associated label is bound to three minor components of LHCII, identified as LHCIIa (two species) and LHCIIc, as shown by protein sequencing of tryptic fragments of purified complexes. pH titration of qE formation and protein labelling in coupled thylakoids showed that both qE and labelling of LHCIIa increased at pH 7-8.In higher plants, chlorophyll and carotenoid molecules are bound to specific proteins to form light-harvesting complexes (LHCI, LHCII), which efficiently capturc and deliver excitation energy to photosystem I (PSI) and photosystem I1 (PSII) reaction centres. Four different types of LHCII (LHCIIa-d), which differ in their polypeptide and pigment content, have been identified (Peter and Thornber, 1991). The polypeptides comprising LHCII are encoded by members of the Lhc gene family; Lhcbl, Lhcb2 and Lhcb3 encode 28-, 27-and 25-kDa polypeptides, respectively, comprising LHCIIb; Lhcb4 encodes the 30-31-kDa protein of LHCIIa; Lhcb.5 encodes the LHCIIc 26-kDa protein; the LHCIId 24-kDa protein is encoded by Lhcbd . LHCIIb binds up to 65% of PSII chlorophyll and is often referred to as the bulk light-harvesting complex; LHCIIa, c and d are minor complexes, each accounting for 3-5% (Peter and Thornber, 1991 ;Dainese and Bassi, 1991 ;.When the light absorbed by LHCII is greater than that capable of being used in photosynthesis, the photosynthetic apparatus is protected from the damaging effects of overexcitation by the rapid induction of a process of non-radiative energy dissipation known as qE Demmig-Adams, 1990;Horton and Ruban, 1992). Measurement of radiationless dissipation of energy via non-photochemical

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Section: Effect Of Ph On Labelling Of Thylakoid Proteinsmentioning

confidence: 82%

Section: Effect Of Ph On Labelling Of Thylakoid Proteinsmentioning

confidence: 99%

Higher Plant Light‐Harvesting Complexes LHCIIa and LHCIIc are Bound by Dicyclohexylcarbodiimide During Inhibition of Energy Dissipation

Walters

Ruban

Horton

1994

European Journal of Biochemistry

126

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“…To determine the relative radioactivity per cell incorporated into the plasmids, the intensity must be divided by the cell number in each membrane-elution fraction (c). (d) The relative amounts of radioactivity per cell incorporated into the F'lac plasmid 20-kb band (-) and the minichromosome pAL49 band (O) were determined by dividing the intensity in panel b by the cell number in panel c. The first peak in the F'lac plasmid occurs prior to membrane-elution fraction 9 (54 min), and the second peak occurs in fraction 20 (120 min). The first peak in the minichromosome occurs in membraneelution fraction 15 (90 min), and the second peak occurs around fraction 26 (156 min).…”

Section: Resultsmentioning

confidence: 99%

Cell-cycle-specific F plasmid replication: regulation by cell size control of initiation

1991

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F plasmid replication during the Escherichia coli division cycle was investigated by using the membraneelution technique to produce cells labeled at different times during the division cycle and scintillation counting for quantitative analysis of radioactive plasmid DNA. The F plasmid replicated, like the minichromosome, during a restricted portion of the bacterial division cycle; i.e., F plasmid replication is cell-cycle specffic. The F plasmid replicated at a different time during the division cycle than a minichromosome present in the same cell. F plasmid replication coincided with doubling in the rate of enzyme synthesis from a plasmid-encoded gene. When the cell cycle age of replication of the F plasmid was determined over a range of growth rates, the cell size at which the F plasmid replicated followed the same rules as did replication of the bacterial chromosome-initiation occurred when a constant mass per origin was achieved-except that the initiation mass per origin for the F plasmid was different from that for the chromosome origin. In contrast, the high-copy mini-R6K plasmid replicated throughout the division cycle.Plasmids are stably inherited, independently replicating circles of DNA. As they are stably maintained in a culture, their replication must be attuned to the rate of cell growth and division. For low-copy plasmids, the F plasmid being an example, there must be at least one round of replication per division cycle. This requirement, coupled with a specific segregation mechanism, would enable low-copy plasmids to be stably maintained with few plasmid-free segregants. One way of coupling plasmid replication to the rate of cell growth and division would be to have replication of all plasmids occur at one specific time during the division cycle. The minichromosome, as well as the bacterial chromosome, replicates in a cell cycle-specific manner. Even though there are many copies of the minichromosome per cell, the minichromosomes replicate at the same time as the chromosomal origin initiates replication (13).The most recent results obtained by using the membraneelution method and quantitative autoradiography to measure F plasmid replication indicate that the F plasmid replicates throughout the division cycle (14); i.e., F plasmid replication is cell-cycle independent. Mini-F plasmids containing only one of several F plasmid replication origins (oriS) were also shown to replicate throughout the division cycle. These results are in sharp contrast to the large body of earlier work that had consistently shown a doubling in the ability of a cell to synthesize plasmid-encoded ,-galactosidase at a specific time during the division cycle (3,6,8,9,17,22). These enzyme experiments indicated that the F plasmid replicates in a cell-cycle-specific manner. How can the random replication of the F plasmid determined by autoradiography be reconciled with cell-cycle-specific replication determined by enzyme induction?We reinvestigated the problem of F plasmid replication during the division cycle by using the membr...

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“…After incubation for 20 min at ambient temperature, samples were loaded onto a 5% native polyacrylamide gel and resolved at 4 o C for 4 h at 450 V. Immediately afterward, the gel was dried under vacuum at 80 o C for 60 min, and subjected to autoradiography or was analyzed quantitatively on a GS-525 Molecular Analyzer phosphorimager with the supplied Molecular Analyst software (BioRad, Hercules CA). Phosphorimaging screens were erased immediately before use to increase the sensitivity of detection by lowering the nonspecific background phosphorescence [68]. SigmaPlot software (Systat) was used to analyze the quantified intensities.…”

Section: Electrophoretic Mobility Shift Assays (Emsa)mentioning

confidence: 99%

Modulation of the activation of Stat1 by the interferon-γ receptor complex

Krause¹,

He²,

Pestka

2006

Cell Res

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The activation of Stat1 by the interferon-gamma (IFN-γ) receptor complex is responsible for the transcription of a significant portion of IFN-γ induced genes. Many of these genes are responsible for the induction of an apoptotic state in response to IFN-γ. In the absence of Stat1 activation, IFN-γ instead induces a proliferative response. Modifying Stat1 activation by IFN-γ may have pharmacological benefits. We report that the rate of activation of Stat1 can be altered in HeLa cells by overexpressing either the IFN-γR1 chain or the IFN-γR2 chain. These alterations occur in hematopoietic cell lines: Raji cells and monocytic cell lines, which have average and above-average IFN-γR2 surface expression, activate Stat1 similarly to HeLa cells and HeLa cells overexpressing IFNγR2, respectively. The rapid Stat1 activation seen in HeLa cells can be inhibited by overexpressing a chimeric IFN-γR2 chain that does not bind Jak2 or (when high concentrations of IFN-γ are used) by overexpressing IFN-γR1. These data are consistent with a model in which the recruitment of additional Jak2 activity to a signaling complex accelerates the rate of Stat1 activation. We conclude that the rate of activation of Stat1 in cells by IFN-γ can be modified by regulating either receptor chain and speculate that pharmacological agents which modify receptor chain expression may alter IFN-γ receptor signal transduction.

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A pitfall in the computer-aided quantitation of autoradiograms

Cited by 22 publications

References 5 publications

Higher Plant Light‐Harvesting Complexes LHCIIa and LHCIIc are Bound by Dicyclohexylcarbodiimide During Inhibition of Energy Dissipation

Higher Plant Light‐Harvesting Complexes LHCIIa and LHCIIc are Bound by Dicyclohexylcarbodiimide During Inhibition of Energy Dissipation

Cell-cycle-specific F plasmid replication: regulation by cell size control of initiation

Modulation of the activation of Stat1 by the interferon-γ receptor complex

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