DNA Segments Sensitive to Single-Strand-Specific Nucleases Are Present in Chromatin of Mitotic Cells

It was observed before that DNAin situin chromatin of mitotic cells is more sensitive to denaturation than DNA in chromatin of interphase cells. DNA sensitivity to denaturation, in these studies, was analyzed by exposing cells to heat or acid and using acridine orange (AO), the metachromatic fluorochrome which can differentially stain double-stranded (ds) vs single-stranded (ss) nucleic acids, as a marker of the degree of DNA denaturation. However, without prior cell treatment with heat or acid no presence of single-stranded DNA in either mitotic or interphase cells was detected by this assay. In the present experiments we demonstrate that DNAin situin mitotic cells, without any prior treatment that can induce DNA denaturation, is sensitive to ss-specific S1 and mung bean nucleases. Incubation of permeabilized human T cell leukemic MOLT-4, promyelocytic HL-60, histiomonocytic lymphoma U937 cells, or normal PHA-stimulated lymphocytes with S1 or mung bean nucleases generated extensive DNA breakage in mitotic cells. DNA strand breaks were detected using fluorochrome-labeled triphosphonucleotides in the reaction catalyzed by exogenous terminal deoxynucleotidyl transferase. Under identical conditions of the cells’ exposure to ss-specific nucleases, DNA breakage in interphase cells was of an order of magnitude less extensive compared to mitotic cells. The data indicate that segments of DNA in mitotic chromosomes, in contrast to interphase cells, may be in a conformation which is sensitive to ss nucleases. This may be a reflection of the differences in the torsional stress of DNA loops between interphase and mitotic chromatin. Namely, greater stress in mitotic loops may lead to formation of the hairpin-loop structures by inverted repeats; such structures are sensitive to ss nucleases. The present method of detection of such segments appears to be more sensitive than the use of AO. The identification of mitotic cells based on sensitivity of their DNA to ss nucleases provides an additional method for their quantification by flow cytometry.     

Effect of Protease Inhibitors on Early Events of Apoptosis

Proteolysis is an early event of apoptosis which appears to be associated with activation of the endonuclease which is responsible for internucleosomal DNA cleavage. The present study was designed to reveal the possible role of proteolysis in other early events, such as chromatin condensation, nuclear breakdown, and destabilization ofin situDNA double-stranded structure. Apoptosis of human leukemic HL-60 cells and rat thymocytes was induced by different agents, including DNA topoisomerase inhibitors, an RNA antimetabolite, and the glucocorticosteroid, prednisolone. DNA degradation was evaluated by pulsed field and conventional gel electrophoresis and by the presence ofin situDNA strand breaks. DNA stability was estimated by the measure of its sensitivityin situto denaturation. Chromatin condensation, nuclear breakdown, and other morphological changes were monitored by interference contrast and UV microscopy following cell staining with the DNA-specific fluorochrome 4′,6-diamidino-2-phenylindole. Several irreversible or reversible serine protease inhibitors prevented internucleosomal DNA degradation, nuclear breakdown, and destabilization of DNA double-stranded structure. The effective inhibitors, however, did not prevent the onset of chromatin condensation, nor the loss of the fine structural framework, nor the initial step of DNA cleavage generating DNA fragments of ≥50 kb in size. The data indicate that in both cell systems the activity of proteases sensitive to the inhibitors tested is needed for internucleosomal DNA cleavage to occur. The data also suggest that these proteases may be involved in dissolution of the nuclear envelope. Because nuclear matrix proteins and histones stabilize DNAin situ,and the decrease in DNA stability which occurs during apoptosis is precluded by the inhibitors, it is likely that serine proteases may degrade DNA stabilizing proteins. The activity of these proteases, however, appears needed neither for DNA cleavage to ≥50-kb fragments nor for the onset of chromatin condensation which is associated with dissolution of the structural framework of the nucleus.     

SurA assists the folding of Escherichia coli outer membrane proteins.

Many proteins require enzymatic assistance in order to achieve a functional conformation. One rate-limiting step in protein folding is the cis-trans isomerization of prolyl residues, a reaction catalyzed by prolyl isomerases. SurA, a periplasmic protein of Escherichia coli, has sequence similarity with the prolyl isomerase parvulin. We tested whether SurA was involved in folding periplasmic and outer membrane proteins by using trypsin sensitivity as an assay for protein conformation. We determined that the efficient folding of three outer membrane proteins (OmpA, OmpF, and LamB) requires SurA in vivo, while the folding of four periplasmic proteins was independent of SurA. We conclude that SurA assists in the folding of certain secreted proteins.     

Cyclic electron flow around Photosystem II in vivo

The oxygen flash yield (Y_O2) and photochemical yield of PS II (_{PS II}) were simultaneously detected in intact Chlorella cells on a bare platinum oxygen rate electrode. The two yields were measured as a function of background irradiance in the steady-state and following a transition from light to darkness. During steady-state illumination at moderate irradiance levels, Y_O2 and _{PS II} followed each other, suggesting a close coupling between the oxidation of water and Q_A reduction (Falkowski et al. (1988) Biochim. Biophys. Acta 933: 432–443). Following a light-to-dark transition, however, the relationship between Q_A reduction and the fraction of PS II reaction centers capable of evolving O₂ became temporarily uncoupled. _{PS II} recovered to the preillumination levels within 5–10 s, while the Y_O2 required up to 60 s to recover under aerobic conditions. The recovery of Y_O2 was independent of the redox state of Q_A, but was accompanied by a 30% increase in the functional absorption cross-section of PS II (_{PS II}). The hysteresis between Y_O2 and the reduction of Q_A during the light-to-dark transition was dependent upon the reduction level of the plastoquinone pool and does not appear to be due to a direct radiative charge back-reaction, but rather is a consequence of a transient cyclic electron flow around PS II. The cycle is engaged in vivo only when the plastoquinone pool is reduced. Hence, the plastoquinone pool can act as a clutch that disconnects the oxygen evolution from photochemical charge separation in PS II.Abbreviations ADRY acceleration of the deactivation reactions of the water-splitting enzyme (agents) - Chl chlorophyll - cyt cytochrome - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - F_O minimum fluorescence yield in the dark-adapted state - F_I minimum fluorescence yield under ambient irradiance or during transition from the light-adapted state - F_M maximum fluorescence yield in the dark-adapted state - F_M maximum fluorescence yield under ambient irradiance or during transition from light-adapted state - F_V, F_V variable fluorescence (F_V=F_M–F_O ; F_V=F_M–F_I) - FRR fast repetition rate (fluorometer) - _{PS II} quantum yield of Q_A reduction (_{PS II}=(F_M – F_O)/F_M or _{PS II})=(F_M= – F_I=)/F_M=) - LHCII Chl a/b light harvesting complexes of Photosystem II - OEC oxygen evolving complex of PS II - P680 reaction center chlorophyll of PS II - PQ plastoquinone - POH₂ plastoquinol - PS I Photosystem I - PS II Photosystem II - RC II reaction centers of Photosystem II - PS II the effective absorption cross-section of PHotosystem II - TL thermoluminescence - Y_O2 oxygen flash yield The US Government right to retain a non-exclusive, royalty free licence in and to any copyright is acknowledged.     

Non-photochemical fluorescence quenching and the diadinoxanthin cycle in a marine diatom

The diadinoxanthin cycle (DD-cycle) in chromophyte algae involves the interconversion of two carotenoids, diadinoxanthin (DD) and diatoxanthin (DT). We investigated the kinetics of light-induced DD-cycling in the marine diatom Phaeodactylum tricornutum and its role in dissipating excess excitation energy in PS II. Within 15 min following an increase in irradiance, DT increased and was accompanied by a stoichiometric decrease in DD. This reaction was completely blocked by dithiothreitol (DTT). A second, time-dependent, increase in DT was detected 20 min after the light shift without a concomitant decrease in DD. DT accumulation from both processes was correlated with increases in non-photochemical quenching of chlorophyll fluorescence. Stern-Volmer analyses suggests that changes in non-photochemical quenching resulted from changes in thermal dissipation in the PS II antenna and in the reaction center. The increase in non-photochemical quenching was correlated with a small decrease in the effective absorption cross section of PS II. Model calculations suggest however that the changes in cross section are not sufficiently large to significantly reduce multiple excitation of the reaction center within the turnover time of steady-state photosynthetic electron transport at light saturation. In DTT poisoned cells, the change in non-photochemical quenching appears to result from energy dissipation in the reaction center and was associated with decreased photochemical efficiency. D1 protein degradation was slightly higher in samples poisoned with DTT than in control samples. These results suggest that while DD-cycling may dynamically alter the photosynthesis-irradiance response curve, it offers limited protection against photodamage of PS II reaction centers at irradiance levels sufficient to saturate steady-state photosynthesis.Abbreviations CAP chloramphenicol - D1 PS II reaction center protein - DD diadinoxanthin - DD cycle-diadinoxanthin cycle - DT diatoxanthin - DTT dithiothreitol - FCP fucoxanthin chlorophyll a-c protein - F_m maximum fluorescence yield in the dark-adapted state - F_o minimum fluorescence yield in the dark-adapted state - F_m and F_o maximum and minimum fluorescence yields respectively in some light adapted state - F_v maximum variable fluorescence yield in the dark-adapted state - I_k Irradiance at the intercept of the initial slope of the photosynthesis-irradiance curve and the maximum photosynthetic rate - k_D first order rate constant for nonradiative de-excitation of excitions in the PS II antenna - k_d first order rate constant for non-radiative de-excitation of excitons in the PS II reaction center - k_F first order rate constant for fluorescence - k_T first order rate constant for exciton transfer to the reaction center - k_t first order rate constant for exciton transfer from the reaction center to the antenna - Rubisco ribulose bisphosphate carboxylase - SV_m Stern-Volmer quenching coefficient of the maximum fluorescence yield - SV_o Stern-Volmer quenching coefficient of the miniximum fluorescence yield - _{PS II} apparent absorption cross-section of PS II - _arr average interval between exciton arrival to the PS II reaction center (ms) - _rem average interval between electron turnover during photosynthesis in the PS II reaction center (ms) - _d the probability that an exciton is non-radiatively dissipated in the reaction center - _T the probability that an exciton in the antenna is transferred to the reaction center - _t the probability that an exciton is transferred back from the reaction center to the antenna     

cDNA approaches to isolation of the mouse mutant weaver gene

The mouse autosomal recessive mutant gene weaver (wv) results in abnormalities in cerebellum, substantia nigra and testis. Although a subtracted cDNA library prepared by removing P31 (wv/wv) sequences from a P1 (wv/+) library should contain mainly nonrepetitive neonatal sequences, unfortunately, repetitive sequences still appear during screening. Two clones, one repetitive, the other not, are used to illustrate the problems encountered in attempting to isolate the weaver gene from a subtracted cDNA library.Special issue dedicated to Dr. Sidney Ochs.     

Effect of linolenic acid on release of the 43 kDa polypeptide and manganese from photosystem II membranes depleted of the 33 kDa extrinsic polypeptide

The effect of linolenic acid (18:3) on release of the 43 kDa polypeptide and manganese from photosystem II ( PS II ) membranes depleted of extrinsic polypeptides was studied. In both control and NaCl-washed particles which were depleted of the extrinsic 23 and 16 kDa polypeptides, the 18:3 treatment caused a 20% release of the 33 and 43 kDa polypeptides. In CaCl₂, (or urea + NaCl)-washed particles, which were depleted of the 33 kDa polypeptide in addition to the 23 and 16 kDa polypeptides, the release of the 43 kDa polypeptide increased to 70%, whereas only 25% of the 47 kDa polypeptide was removed. These findings suggest (i) that the 33 and the 43 kDa polypeptides are neighbows in the photosynthetic membrane and (ii) that the 33 kDa polypeptide shields the 43 kDa polypeptide against the action of 18:3. Incubation of CaCl₂, or (urea + NaCI)-treated PSII particles in the presence or absence of 18:3 resulted in the loss of only 2 of the 4 Mn atoms present per reaction center. this indicates that the 2 Mn atoms more firmly associated with PSII are not affected by the removal of the extrinsic 16, 23 and 33 kDa polypeptides, and the intrinsic 43 kDa polypeptide. nor by the treatment with linolenic acid.     

De novo design of the hydrophobic core of ubiquitin.

We have previously reported the development and evaluation of a computational program to assist in the design of hydrophobic cores of proteins. In an effort to investigate the role of core packing in protein structure, we have used this program, referred to as Repacking of Cores (ROC), to design several variants of the protein ubiquitin. Nine ubiquitin variants containing from three to eight hydrophobic core mutations were constructed, purified, and characterized in terms of their stability and their ability to adopt a uniquely folded native-like conformation. In general, designed ubiquitin variants are more stable than control variants in which the hydrophobic core was chosen randomly. However, in contrast to previous results with 434 cro, all designs are destabilized relative to the wild-type (WT) protein. This raises the possibility that beta-sheet structures have more stringent packing requirements than alpha-helical proteins. A more striking observation is that all variants, including random controls, adopt fairly well-defined conformations, regardless of their stability. This result supports conclusions from the cro studies that non-core residues contribute significantly to the conformational uniqueness of these proteins while core packing largely affects protein stability and has less impact on the nature or uniqueness of the fold. Concurrent with the above work, we used stability data on the nine ubiquitin variants to evaluate and improve the predictive ability of our core packing algorithm. Additional versions of the program were generated that differ in potential function parameters and sampling of side chain conformers. Reasonable correlations between experimental and predicted stabilities suggest the program will be useful in future studies to design variants with stabilities closer to that of the native protein. Taken together, the present study provides further clarification of the role of specific packing interactions in protein structure and stability, and demonstrates the benefit of using systematic computational methods to predict core packing arrangements for the design of proteins.     

Apoptosis in anititumor strategies: Modulation of cell cycle or differentiation

There is a strong evidence that administration of antitumor drugs triggers apoptotic death of target cells. A characteristic feature of appotosis is active participation of the affected cell in its demise. Attempts have been made, therefore, to potentiate the cytotoxicity of a variety of agents by modulating the propensity of cells to respond by apoptosis. Several strategies to enhance apoptosis that involve modulation of the cell cycle or differentiation are discussed. Loss of control of the G₁ checkpoint in tumor cells allows one to design treatments that arrest normal cells at the checkpoint and attempt to selectively kill tumor cells with S phase specific drugs. The possibility of a restoration of the apoptosis triggering function of the tumor suppressor gene p53 when the G₁ checkpoint function is abolished is expected to increase tumor cells' sensitivity to S phase poisons. Because induction of apoptosis by many antitumor drugs is cell cycle phase specific, drug combinations that preferentially trigger apoptosis at different phases of the cycle, or recruitment of cells to the sensitive phase, offer another antitumor strategy. There is also evidence that apoptosis is potentiated when cell differentiation is triggered follwing DNA damage. This observation suggests that strategies which combine DNA damaging and differentiating drugs, under conditions where the latter are administered following DNA damage caused by the former, may be successful.     

Identification of a wheat (Triticum aestivum) cell line lacking a specific divalent cation requirement

A wheat (Triticum aestivum L.) cell line, derived from anther culture of an F₁ hybrid, has exogenous Ca²⁺, to that of calcium-dependent cells grown on complete medium. The calcium-independent cell line has been grown in the absence of Ca²⁺ for more than 1.5 years. The cell line grew at a rate similar to that on complete medium for up to 12 weeks, if supplied with any one of the divalent cations, Ca²⁺, Mg²⁺, Mn²⁺, Zn²⁺, Cu²⁺ or Co²⁺, but declined and appeared necrotic when all 6 of these were removed from the medium. The calcium-independence trait, while identified in tissue culture, was also observed in germinated immature embryos of the same hybrid and one of its parental inbred lines.