Search

Human mitochondrial transcription factor A (TFAM) distorts DNA into a U-turn, as shown by crystallographic studies. The relevance of this U-turn is associated with transcription initiation at the mitochondrial light strand promoter (LSP). However, it has not been yet discerned whether a tight U-turn or an alternative conformation, such as a V-shape, is formed in solution. Here, single-molecule FRET experiments on freely diffusing TFAM/LSP complexes containing different DNA lengths show that a DNA U-turn is induced by progressive and cooperative binding of the two TFAM HMG-box domains and the linker between them. SAXS studies further show compaction of the protein upon complex formation. Finally, molecular dynamics simulations reveal that TFAM/LSP complexes are dynamic entities, and the HMG boxes induce the U-turn against the tendency of the DNA to adopt a straighter conformation. This tension is resolved by reversible unfolding of the linker, which is a singular mechanism that allows a flexible protein to stabilize a tight bending of DNA., This work was supported by the Ministry of Economy and Competitiveness (MINECO) (BFU2012-33516 and BFU2015-70645-R to M.S., and BIO2012-32868 and BFU2014-61670-EXP to M.O.); Generalitat de Catalunya (SGR2009-1366 and 2014-SGR-997 to M.S., and SGR2009- 1348, 2014 SGR-134 to M.O.); the Instituto Nacional de Bioinforma´tica; the European Union (FP7-HEALTH-2010-261460, FP7-PEOPLE-2011- 290246, and FP7-HEALTH-2012-306029-2 to M.S., and H2020-EINFRA-2015-1-675728 and H2020-EINFRA-2015-676556 to M.O.); and the European Research Council (ERC-2011-ADG_20110209-291433 to M.O.). A.R.-C. was awarded with a ‘‘Junta para la Ampliacio´n de Estudios’’ (Programa JAE) fellowship from Consejo Superior de Investigaciones Cientı´ficas (CSIC). The Structural Biology Unit at IBMB-CSIC is a ‘‘Maria de Maeztu’’ Unit of Excellence awarded by the Ministry of Economy and Competitiveness (MINECO) under MDM-2014-0435. IRB Barcelona is the recipient of a Severo Ochoa Award of Excellence from the Ministry of Economy and Competitiveness (MINECO). The CBS is a member of the French Infrastructure for Integrated Structural Biology (FRISBI), a national infrastructure supported by the French National Research Agency (ANR-10-INBS-05).

Nucleosomes play a dual role in compacting the genome and regulating the access to DNA. To unravel the underlying mechanism, we study fluorescently labeled mononucleosomes by multi-parameter FRET measurements and characterize their structural and dynamic heterogeneity upon NaCl-induced destabilization. Species-selective fluorescence lifetime analysis and dynamic photon distribution analysis reveal intermediates during nucleosome opening and lead to a coherent structural and kinetic model. In dynamic octasomes and hexasomes the interface between the H2A-H2B dimers and the (H3-H4)2 tetramer opens asymmetrically by an angle of ≈20° on a 50 and 15 µs time scale, respectively. This is followed by a slower stepwise release of the dimers coupled with DNA unwrapping. A mutation (H2A-R81A) at the interface between H2A and H3 facilitates initial opening, confirming the central role of the dimer:tetramer interface for nucleosome stability. Partially opened states such as those described here might serve as convenient nucleation sites for DNA-recognizing proteins., Nucleosomes compact the genome and regulate access to specific DNA sequences. Here the authors employ single-molecule FRET studies to characterize nucleosome dynamics at different salt concentrations and dissect nucleosome disassembly into elementary steps.

Nucleosomes are important for chromatin compaction and gene regulation; their integrity depends crucially on the structural properties of the histone tails. Recent all-atom molecular dynamics simulations revealed that removal of the N-terminal tails of histone H3, known to destabilize nucleosomes, causes a rearrangement of two arginines of histone H2A, namely R81 and R88 by altering the electrostatic environment of the H2A α3 domain. Whether this rearrangement is the cause or the effect of decreased stability, is unclear. Here, we emulate the altered electrostatic environment that was found after H3 tail clipping through charge-modifying mutations to decouple its impact on intranucleosomal interactions from that of the histone tails. Förster resonance energy transfer experiments on recombinant nucleosomes and all-atom molecular dynamics simulations reveal a compensatory role of those amino acids in nucleosome stability. The simulations indicate a weakened interface between H2A-H2B dimers and the (H3-H4)2 tetramer, as well as between dimers and DNA. These findings agree with the experimental observations of position and charge dependent decreased nucleosome stability induced by the introduced mutations. This work highlights the importance of the H2A α3 domain and suggests allosteric effects between this domain and the outer DNA gyre as well as the H3 N-terminal tail.

Opening of the nucleosome structure is essential for accessing genomic DNA. To study the mechanism of this process, we monitor the distance between various fluorescently labeled positions on mononucleosomes by single-molecule Forster resonance energy transfer (FRET). Here, we compare nucleosomes reconstituted from recombinant mouse, Xenopus, and yeast histones. As DNA sequences we compared, the effect of 5S rDNA, MMTV-B sequence, and Widom 601 DNA. The stability, as measured by the salt concentration at the opening transition midpoint, is lowest for yeast, followed by Xenopus and mouse. The 601 DNA sequence builds much more stable nucleosomes and the distribution of FRET efficiencies is narrower than for those reconstituted on 5S rDNA or MMTV-B sequences. The opening pathway through an intermediate state, as found for Xenopus histones, could be verified for the mouse and yeast systems and for the different DNA sequences, suggesting a general mechanism for accessing nucleosomal DNA. © 2013 International Society for Advancement of Cytometry

Background:: A consensus has not been established for the standard treatment of hyperkalemia in the neonatal population. Most treatment regimens include a dextrose/insulin infusion. Additional agents used include calcium, sodium bicarbonate, polystyrene sulfonate, and albuterol. This study assessed the safety and efficacy of a potassium cocktail (k-cocktail) containing dextrose, insulin, calcium gluconate, and sodium lactate for treatment of neonatal hyperkalemia. Objective: To determine whether modifications to a potassium cocktail formulation, based on a prior quality improvement project, resulted in a decrease in the incidence of hyperglycemia and acidosis associated with its use, and to evaluate the effectiveness of the k-cocktail in lowering serum potassium levels and the incidence of adverse effects. Methods: We conducted a retrospective cohort study of neonates with hyperkalemia who received 2 k-cocktail formulations (group 1 [n = 13], original formulation, dextrose:insulin 5:1; group 2 [n = 26], modified formulation, dextrose: insulin 3.3:1). Group 2 subjects were matched 2:1 by gestational age and birth weight with those in group 1. Variables related to safety and effectiveness of therapy were assessed by medical record review. The following tests were used to assess group differences: χ2, Fisher exact, 2-tailed t-tests, and mixed linear models. Results: The incidence of hyperglycemia during the modified k-cocktail infusion in group 2 decreased from 76.9% to 217% (p = 0.001). Serum blood glucose concentrations increased during the infusion, on average, for group 1 infants and were unchanged during the infusion for those in group 2. The incidence of acidosis during the infusion was similar between groups (group 1 [76.9%] vs group 2 [68.2%]; p = 0.58). No significant adverse events were observed. Serum potassium concentrations decreased similarly in both groups. Conclusions: An intravenous infusion including a dextrose:insulin ratio of 3.3:1. compared with a higher ratio, results in less hyperglycemia and appears to be as effective in decreasing potassium concentrations in newborns.

Nucleosomes are multi-component macromolecular assemblies which present a formidable obstacle to enzymatic activities that require access to the DNA, e.g. DNA and RNA polymerases. The mechanism and pathway(s) by which nucleosomes disassemble to allow DNA access are not well understood. Here we present evidence from single molecule FRET experiments for a previously uncharacterized intermediate structural state before H2A-H2B dimer release, which is characterized by an increased distance between H2B and the nucleosomal dyad. This suggests that the first step in nucleosome disassembly is the opening of the (H3-H4)(2) tetramer/(H2A-H2B) dimer interface, followed by H2A-H2B dimer release from the DNA and, lastly, (H3-H4)(2) tetramer removal. We estimate that the open intermediate state is populated at 0.2-3% under physiological conditions. This finding could have significant in vivo implications for factor-mediated histone removal and exchange, as well as for regulating DNA accessibility to the transcription and replication machinery.

The nucleosome has a central role in the compaction of genomic DNA and the control of DNA accessibility for transcription and replication. To help understanding the mechanism of nucleosome opening and closing in these processes, we studied the disassembly of mononucleosomes by quantitative single-molecule FRET with high spatial resolution, using the SELEX-generated “Widom 601” positioning sequence labeled with donor and acceptor fluorophores. Reversible dissociation was induced by increasing NaCl concentration. At least 3 species with different FRET were identified and assigned to structures: ( i ) the most stable high-FRET species corresponding to the intact nucleosome, ( ii ) a less stable mid-FRET species that we attribute to a first intermediate with a partially unwrapped DNA and less histones, and ( iii ) a low-FRET species characterized by a very broad FRET distribution, representing highly unwrapped structures and free DNA formed at the expense of the other 2 species. Selective FCS analysis indicates that even in the low-FRET state, some histones are still bound to the DNA. The interdye distance of 54.0 Å measured for the high-FRET species corresponds to a compact conformation close to the known crystallographic structure. The coexistence and interconversion of these species is first demonstrated under non-invasive conditions. A geometric model of the DNA unwinding predicts the presence of the observed FRET species. The different structures of these species in the disassembly pathway map the energy landscape indicating major barriers for 10-bp and minor ones for 5-bp DNA unwinding steps.

DNA accessibility in the cell nucleus depends on its packaging. The stability of the basic compaction unit, the nucleosome, is regulated through the interactions between DNA and histones. Natural sequence variation along the DNA strongly modulates nucleosome stability. Histone variations, mutations and posttranslational modifications alter intra- and internucleosomal interactions. We are investigating interactions which hold nucleosomes together by measuring distances in reconstituted samples using single molecule Forster energy transfer.

Nucleosomes compact the genome and regulate access by wrapping and unwrapping DNA. Here we characterized NaCl-induced opening of mononucleosomes by single-molecule FRET, using donor/acceptor labels on the DNA and various histones. By species-selective fluorescence lifetime and photon distribution analysis we identified new nucleosome opening intermediates and developed a kinetic model [1]. Opening proceeds through a weakening of the H2A-H2B dimer/(H3-H4)2 tetramer interface on a 0.1 ms time scale, then by a slower two-step release of the dimers coupled to DNA unwrapping, extending from several ms to minutes. Nucleosome opening and detachment of histone dimers proceed asymmetrically depending on the DNA sequence.Mutations at the H2A/H3 interface (H2A R81A, R88A, R81A/R88A, R81E/R88E) facilitate the initial opening, confirming the importance of the dimer:tetramer interface for nucleosome stability. This is also supported by molecular dynamics (MD) simulations that show enhanced DNA fluctuations for the mutants. Partially opened states such as described here might be a convenient nucleation point for DNA-recognizing proteins.For characterizing the role of histone tails in nucleosome interaction and stability, we performed MD simulations in explicit and implicit solvent on the H4 tail interacting with the acidic patch of a neighboring nucleosome. The simulations show that H4K16 acetylation decreases the interactions between H4 tail and the acidic patch of the neighboring nucleosome, a process that is central to the regulation of chromatin compaction [2,3].References[1] A. Gansen, A. Valeri, F. Hauger, S. Felekyan, S. Kalinin, K. Toth, J. Langowski, and C. A. M. Seidel. Nucleosome disassembly intermediates characterized by single-molecule FRET. Proc Natl Acad Sci U S A, 106(36):15308-13, Sep 2009.[2] J. Erler, R. Zhang, L. Petridis, X. Cheng, J. C. Smith, and J. Langowski. The role of histone tails in the nucleosome: A computational study. Biophys. J., 107(12):2911-2922, 2014.[3] R. Zhang, J. Erler, J. Langowski. How does histone acetylation regulate chromatin accessibility? A computational study of the role of H4K16 in inter-nucleosome interaction. Biophys. J., under revision.

Quantification of mRNA in single cells provides direct insight into how intercellular heterogeneity plays a role in disease progression and outcomes. Quantitative polymerase chain reaction (qPCR), the current gold standard for evaluating gene expression, is insufficient for providing absolute measurement of single-cell mRNA transcript abundance. Challenges include difficulties in handling small sample volumes and the high variability in measurements. Microfluidic digital PCR provides far better sensitivity for minute quantities of genetic material, but the typical format of this assay does not allow for counting of the absolute number of mRNA transcripts samples taken from single cells. Furthermore, a large fraction of the sample is often lost during sample handling in microfluidic digital PCR. Here, we report the absolute quantification of single-cell mRNA transcripts by digital, one-step reverse transcription PCR in a simple microfluidic array device called the self-digitization (SD) chip. By performing the reverse transcription step in digitized volumes, we find that the assay exhibits a linear signal across a wide range of total RNA concentrations and agrees well with standard curve qPCR. The SD chip is found to digitize a high percentage (86.7%) of the sample for single-cell experiments. Moreover, quantification of transferrin receptor mRNA in single cells agrees well with single-molecule fluorescence in situ hybridization experiments. The SD platform for absolute quantification of single-cell mRNA can be optimized for other genes and may be useful as an independent control method for the validation of mRNA quantification techniques.

Using Forster resonance energy transfer (FRET) measurements in bulk and on single molecules, we assessed the structural role of histone acetylation and of some point mutations in nucleosomes reconstituted on the 170bp long Widom 601 DNA sequence. By measuring distances between fluorescently labeled parts of the nucleosome we could follow the salt-induced nucleosome disassembly, using donor-acceptor pairs on the ends or in the internal part of the nucleosomal DNA, and on histones. The single molecule observations helped to characterize the intermediate states as well as the distributions of the populations [1,2].

The metabolic pathways of methane formation vary with environmental conditions, but whether this can also be linked to changes in the active archaeal community structure remains uncertain. Here, we show that the suppression of aceticlastic methanogenesis by methyl fluoride (CH(3)F) caused surprisingly little differences in community composition of active methanogenic archaea from a rice field soil. By measuring the natural abundances of carbon isotopes we found that the effective dose for a 90% inhibition of aceticlastic methanogenesis in anoxic paddy soil incubations was

By using a circularly polarized pulse that is tuned well below resonance, we create a virtual population of spin-polarized coherent excitons in a GaAs/AlGaAs multiple quantum well, and we study its effects on the polarization state of a weak signal pulse using a combination of time-resolved and spectrally resolved ellipsometric techniques. From these data the polarization state contributions to an all-optical polarization switch, which exhibits a ~415 fs switching time and large contrast ratios, are identified and the performance of the switch is demonstrated.

Nucleosomes, as the basic packing unit of chromatin, regulate DNA accessibility and have significant influence on gene expression. Two copies of each histone protein (H2A, H2B, H3, H4) build up the protein octamer, around which approximately 150 bp of DNA are wrapped.The N-terminal tails of the histones protrude from the nucleosome; they are important for inter- and intranucleosomal interactions. Nucleosome disassembly may be forced in vitro by increasing salt concentration and may be followed by Forster resonance energy transfer (FRET) between fluorophores. We use reconstituted Xenopus laevis nucleosomes, fluorescently labeled on different positions[2] to compare the disassembly of nucleosomes containing mutated histones or modifications to the wild type.Here we focus on the dynamics of the N-tail of H3. Mutated versions of H3 labeled with Alexa488 were used to examine whether there are interactions between the labeled H3 tail and the labeled DNA or other labeled histones. First results show an interaction of the H3 tail and the DNA in vicinity of the dyad axis.The second part is about the functional relevance of a particular region of H2A. Molecular dynamic simulations on ‘tailless’ variants of H3 and H2A have suggested conformational changes affecting two arginines of H2A. Furthermore a conformational change of the protruding part of the DNA was described, which is due to internal conformational changes of H2A[3].From those observations the question arises whether these amino acids are necessary for stable octamers and/or nucleosomes, and how they influence DNA breathing, unwrapping, and stability of entire nucleosomes. To address these questions we generated recombinant H2A proteins incorporating site-specific mutations (R81A, R88A, R81AR88A, R81E, R88E, R81ER88E). Our results show a decreasing stability associated with position (Wt > R88 > R81 > R81R88) and charge (RA > RE) of the amino acids.

Nucleosome structure and stability affect genetic accessibility by altering the local chromatin morphology. Recent FRET experiments on nucleosomes have given valuable insight into the structural transformations they can adopt. Yet, even if performed under seemingly identical conditions, experiments performed in bulk and at the single molecule level have given mixed answers due to the limitations of each technique. To compare such experiments, however, they must be performed under identical conditions. Here we develop an experimental framework that overcomes the conventional limitations of each method: single molecule FRET experiments are carried out at bulk concentrations by adding unlabeled nucleosomes, while bulk FRET experiments are performed in microplates at concentrations near those used for single molecule detection. Additionally, the microplate can probe many conditions simultaneously before expending valuable instrument time for single molecule experiments. We highlight this experimental strategy by exploring the role of selective acetylation of histone H3 on nucleosome structure and stability; in bulk, H3-acetylated nucleosomes were significantly less stable than non-acetylated nucleosomes. Single molecule FRET analysis further revealed that acetylation of histone H3 promoted the formation of an additional conformational state, which is suppressed at higher nucleosome concentrations and which could be an important structural intermediate in nucleosome regulation.

Nucleosomes were reconstituted from 170 bp long fragments of 5S rDNA and an optimal positioning sequence, the Selex 601, with recombinant histones. In free-solution single pair Förster resonance energy transfer (spFRET) measurements of the distance between fluorescently labeled bases in the nucleosomal DNA, the samples exhibited structural diversity. The structural heterogeneity correlated with the stability of the complexes and depended on the DNA sequence and histone acetylation. The stability of the nucleosomes was assessed via dilution-driven disruption: histone acetylation decreased nucleosome stability. The spFRET experiments used a new approach for data acquisition and analysis that we term "deliberately detuned detection" (D3). This permits the separation of subpopulations in the samples even for the low-FRET regime characteristic for the linker-DNA labeled nucleosomes. Thus, it became possible to study in more detail histone acetylation- and salt-dependent structural variations using either end- or internally labeled DNAs on the nucleosome. We found that the distance distribution of the fluorophore pairs on the linker DNA ends was much more sensitive to histone acetylation or sequence variation than that of labels on the internal part of the DNA, which was more tightly associated with the histone core. spFRET on freely diffusing nucleosomes allows us therefore to localize the influence of histone modifications and DNA sequence variations on the nucleosome structure and dynamics.

We demonstrate photon-number discrimination using a novel semiconductor detector that utilizes a layer of self-assembled InGaAs quantum dots (QDs) as an optically addressable floating gate in a GaAs/AlGaAs δ-doped field-effect transistor. When the QDOGFET (quantum dot, optically gated, field-effect transistor) is illuminated, the internal gate field directs the holes generated in the dedicated absorption layer of the structure to the QDs, where they are trapped. The positively charged holes are confined to the dots and screen the internal gate field, causing a persistent change in the channel current that is proportional to the total number of holes trapped in the QD ensemble. We use highly attenuated laser pulses to characterize the response of the QDOGFET cooled to 4 K. We demonstrate that different photon-number states produce well resolved changes in the channel current, where the responses of the detector reflect the Poisson statistics of the laser light. For a mean photon number of 1.1, we show that decision regions can be defined such that the QDOGFET determines the number (0, 1, 2, or ≥3) of detected photons with a probability of accuracy ≥83 % in a single-shot measurement.

We applied fluorescence detection methods on the single-molecule level to study structural variations and dynamic processes occurring within nucleosomes. Four fluorescent nucleosome constructs were made by attaching donor and acceptor fluorophores to different positions of two nucleosome positioning sequences and reconstituting nucleosomes by salt dialysis. The photochemical and biochemical stability of nucleosomes under single-molecule conditions was optimized by adding inert protein and free radical capturing additives, allowing us to define the best experimental conditions for single-molecule spectroscopy on highly diluted solutions of nucleosome complexes. We could demonstrate for the first time the resolution of conformational subpopulations of nucleosomes by single-pair fluorescence resonance energy transfer in a freely diffusing system and could show the effect of thermally induced nucleosome repositioning.

Summary The effects of 3 different conditioning programmes on muscle glycogen concentration in horses were examined. Speed of exercise was selected according to the blood lactate values for each horse derived from a standardised exercise test before beginning a conditioning programme. Six 2-year-old Haflinger stallions were assigned randomly to one of 3 conditioning programmes according to a 6 × 3 latin square design: 45 min at their individual v1.5 or v2.5 and 25 min at v4. Each conditioning programme lasted 6 weeks (21 exercise sessions), followed by 5 weeks without conditioning (resting period). All exercise was carried out on a treadmill inclined at 17%. Muscle biopsies were taken 5 times from the gluteus medius muscle at 2 cm and 6 cm depth: before the start and in the middle of the conditioning period, then at Days 2, 9 and 35 after the last exercise session. It was found that glycogen concentration was not affected by conditioning until 9 days after finishing conditioning at v1.5 and v2.5 for 45 min (P

A novel temporally-gated technique and standard differential detection techniques are used to study quantum beats involving the light and heavy hole excitons in GaAs quantum wells. The contributions of intravalence band coherence are separated from those of conduction-to-valence band coherence and the contributions of dynamic exciton-exciton correlations are identified by taking advantage of the polarization selection rules for the excitonic transitions.

In order to analyse the immunological changes in patients with endocrine orbitopathy (EO) the antigenic character of orbital connective tissue was studied. Counter-stimulation assays of patients' lymphocytes with autologous retrobulbar fibroblasts resulted in a markedly increased lymphocyte proliferation in comparison to incubation with retrobulbar control fibroblasts. Proliferation tests of retrobulbar T cell lines showed significant responses to autologous retro-orbital connective tissue proteins, with molecular weights of 6-10 kD and 19-26 kD. Phenotypic analysis of orbital T cell lines indicates that they consisted predominantly of CD4+ cells. Hyaluronic acid production of orbital fibro blasts following co-cultivation with lymphocytes of EO patients or controls revealed a threefold increased synthesis in patients with EO. Furthermore, distribution pattern of orbital extracellular matrix glycosaminoglycans (GAG) differs in EO patients in comparison to controls. The results suggest the presence of autoreactive T cells directed against antigens of orbital fibroblasts, whose stimulation results in an augmented GAG synthesis in patients with EO.

Near-room-temperature operation of an all-optical polarization switch based on the virtual excitation of spin-polarized carriers in semiconductor quantum wells is demonstrated. The device is shown to exhibit a pulse-width-limited switching time, a contrast ratio of >18dB, an optical bandwidth of ∼3THz, and an energy throughput of >0.1% using a thin (40 wells) GaAs∕AlGaAs sample. The results of differential transmission measurements are used to identify the dominant switching mechanisms and to monitor the spin and temporal dynamics of the carriers excited during the operation of the switch.

Measurements of density of an isobutyric acid (COOD)/D2O mixture of critical composition (upper critical point; critical composition: xDA,c = 0.113; xDA: mole fraction of acid; critical temperature: Tc = 45.738°C) as function of temperature in the one fluid phase region are reported. The function p = P1 + P2t + P3t(1 - α) is fitted to the data (p: density; P1, P2, P3: constants; t: reduced temperature difference (T–Tc)/Tc; α: critical exponent, α = 0.11). P3 has a positive value. Consequently, the coefficient dTc/dP (pressure dependence of critical temperature) has to be negative for thermodynamic reasons. This is confirmed by direct measurements. The experimentally determined values of P1, P3 and dTc/dP together with the value of the correlation length of local concentration fluctuations taken from the literature are consistent with the predictions of the amplitude relation of two-scale factor universality. — For isobutyric acid/H2O, D2O mixtures of critical composition there exists a linear relation between the coefficient dTc/dP and the equivalent fraction of exchangeable protons Y (=nH/(nH + nD)). dTc/dP decreases from dTc/dP = −40.9 mK bar−1 for DA/D2O (Y=0) to dTc/dP = −55.0 mK bar−1 for HA/H2O (Y=1).

Thermodynamic considerations for determining the number of components of mixtures with a miscibility gap in the presence of isotope exchange reactions are presented using the system isobutyric acid (COOH)/D2O as an example. — Systems of that kind are thermodynamically two component systems as long as one liquid phase exists. If two liquid phases coexist each phase separately is a three component system. The consequences of this analysis are studied by investigating the liquid/liquid coexistence curves of the systems isobutyric acid (COOD)/D2O and n-butyric acid (COOH)/D2O. Thermodynamische Uberlegungen zur Bestimmung der Zahl der Komponenten in Mischungen mit einer Mischungslucke, in denen Isotopenaustauschreaktionen stattfinden konnen, werden am Beispiel des Systems Isobuttersaure (COOH)/D2O dargestellt. Derartige Systeme sind thermodynamisch gesehen zweikomponentige Systeme, solange eine flussige Phase existiert. — Koexistieren zwei flussige Phasen, dann ist jede dieser Phasen getrennt betrachtet ein dreikomponentiges System. Die Schlusfolgerungen, die sich aus dieser Analyse ergeben, werden erlautert anhand von Untersuchungen der flussig/flussig Koexistenzkurven der Systeme Isobuttersaure (COOD)/D2O und n-Buttersaure (COOD)/D2O.

The coexistence surface of the ternary system isobutyric acid/H/sub 2/O, D/sub 2/O in which two independent isotope-exchange reactions take place is determined by chemical analysis of the two fluid phases coexisting at temperatures below the upper critical solution temperature. The acid is generated by hydrolysis of isobutyric acid anhydride in H/sub 2/O, D/sub 2/O mixtures of known composition. The form of the coexistence surface practically does not change with the equivalent fraction Y of exchangeable protons. The critical composition determined on the basis of the equal volume criterion decreases slightly with increasing values of Y (about 3%). The critical temperature is a linear function of Y. It increases by about 20 K. In the systems isobutyric acid (COOH)/D/sub 2/O, confirming prediction of Knobler and Scott. The properties of the coexistence surface of the system isobutyric acid/H/sub 2/O, D/sub 2/O, appear to be typical for critical systems in which isotope-exchange reactions take place. This is shown by studies of the temperature of phase separation vs. composition of the systems phenol (OH)/D/sub 2/O and 2-butoxyethanol (OH)/D/sub 2/O near the upper and lower critical point, respectively.

Actin distribution in serially passaged embryonic mouse fibroblasts has been visualized by the anti-actin-PAP method; the organization of the microfilaments has been observed by electron microscopy (SEM and TEM). Four successive actin patterns have been identified: early (few well-organized bundles of microfilaments), middle-aged (many well-organized bundles and patches around the nucleus), late (numerous ill-organized filamentous structures and diffuse perinuclear-actin) and "senescent" (heavy packs of short microfilaments around the nucleus). All the observed actin-positive filaments were disrupted by cytochalasin B treatment. The cytoplasmic actin complex was cell-age and not cell-size-dependent; it behaved differently from the cytoplasmic microtubular complex to serially subcultivated fibroblasts. Measurements of the cell-protein content (Lowry's method) and SDS-polyacrylamide gel electrophoresis (Laemmli's method) have been performed in the successive population doubling levels (PDL) of the primary cultures. Triton-insoluble actin increased in parallel with total protein and reached about 4% of the total proteins in all the PDLs. Triton-soluble actin also increase at the beginning of the middle-aged period (generally 6 PDL) and another in declining cultures (generally 10 PDL). Total actin amounted to about 8% of the total proteins in early fibroblasts, to about 16% at the beginning of the middle-aged period and to about 20% in the declining terminal cultures. Taking into account all the known characteristics of subcultivated primary cultures, we tentatively consider the evolution of the fibroblasts as an in vitro differentiation followed by true in vitro senescence in the declining cultures. Regarding the cytoplasmic actin-complex, senescence would be characterized by a sharp increase in soluble actin, an unbalanced ratio between soluble and insoluble actin and an impairment of the ability of the microfilaments to form well-organized bundles.

The 3H-actinomycin binding sites in mature ovarian oocytes of Xenopus laevis labelled after fixation were studied by electron microscopic autoradiography. Two different methods were compared: (a) labelling of floating ultrathin sections, (b) labelling of fixed oocytes before embedding. In each case, the oocytes were fixed either with 6% glutaraldehyde in Millonig buffer or with acetified methanol. Two different emulsions, Ilford L4 and Gevaert Scientia Nuc 307, were applied to the sections. Some autoradiograms were quantitatively analysed. The first method (a) failed to give satisfactory results as it proved impossible to eliminate the high nonspecific background. Method (b) on the contrary produced highly reproducible autoradiograms with identical distribution of radioactivity: strong labelling of the nuclei of follicular cell, significant labelling of the yolk platelets and very weak cytoplasmic background (table 1). Moreover it has been shown that the superficial layer of the yolk platelets contains at least twice as many grains as their crystalline core (table 2).

Aging, aphidicolin, serum deprivation and cytochalasin B induce a decrease in the rate of DNA synthesis, an increase in cell flattening (cell surface increase) and an extension of the cytoplasmic microtubular complex (CMTC). Age and experimental conditions affect the protein content of the cell, but there is no relationship between cell morphology and cell protein content. Serum deprivation, aphidicolin and cytochalasin B are more effective on DNA synthesis and cytoplasmic actin complex (CAC) of late than of early fibroblasts. Despite these facts, the cell morphology of late cells is fairly stable and is not affected by experimental conditions, which exert an "aging effect" upon the cell morphology in earlier cultures. Colchicine acts upon the CMTC, cell morphology and DNA synthesis at all ages of the cultures. It also induces disruption of the CAC, the intensity of the disruption depending on both the length of the treatment and the age of the culture: the sensitivity of the actin-microfilaments to colchicine increases with the mitotic age of the cells. We suggest that the microtubular integrity is needed, but not sufficient, to preserve the organization of the CAC into microfilaments. We propose a logical model comprising feedback loops between the number of the mitotic cycles, the rate of DNA synthesis, the extention rate of the plasma membranes and CMTC in normal fibroblasts. CMTC is associated, in this model, with the expression of negative or positive controls, depending on the grade of its extension (Fig. 9).

The coexistence surface of the ternary system isobutyric acid/H/sub 2/O, D/sub 2/O in which two independent isotope-exchange reactions take place is determined by chemical analysis of the two fluid phases coexisting at temperatures below the upper critical solution temperature. The acid is generated by hydrolysis of isobutyric acid anhydride in H/sub 2/O, D/sub 2/O mixtures of known composition. The form of the coexistence surface practically does not change with the equivalent fraction Y of exchangeable protons. The critical composition determined on the basis of the equal volume criterion decreases slightly with increasing values of Y (about 3%). The critical temperature is a linear function of Y. It increases by about 20 K. In the systems isobutyric acid (COOH)/D/sub 2/O, confirming prediction of Knobler and Scott. The properties of the coexistence surface of the system isobutyric acid/H/sub 2/O, D/sub 2/O, appear to be typical for critical systems in which isotope-exchange reactions take place. This is shown by studies of the temperature of phase separation vs. composition of the systems phenol (OH)/D/sub 2/O and 2-butoxyethanol (OH)/D/sub 2/O near the upper and lower critical point, respectively.

Populations of embryonic mouse fibroblasts undergo 10 +/- 2 doublings in vitro prior to cessation of growth. We have studied various properties of the DNA of such cells as they undergo this process of in vitro ageing. Following a 1-h pulse with [3H]thymidine the labelling of growing cell populations decreases progressively with serial subcultivation. At early passage levels, the decrease in labelling between passages is rapid, but after about five passages the decline is much slower. Following a long pulse with [3H]thymidine (up to 4 days), up to 50% of the cells in the final passage become labelled. The binding of [3H]actinomycin to nuclei decreases progressively during serial subcultivation. Under conditions of quiescence, induced by serum deprivation, the cells withdraw from the S-phase. Feulgen cytophotometry reveals a wide spectrum of cellular DNA contents in terminal cultures, with 38% of cells possessing more DNA than equivalent early passage cells. Under these conditions, 40% of the fibroblasts bind less actinomycin than any early passage cells. Neither variations in DNA amount nor differences in cell-cycle phases can explain these alterations, which are thought to be related to age-dependent changes in chromatin condensation.

The nucleoli of the premeiotic, pachytene and young diplotene (A, B, C, up to 100µm diameter) oocytes are heavily labelled after 7 h of [3H]uridine incorporation, which is in good agreement with the hypothesis that nucleolar multiplication occurs throughout all these early stages of oogenesis. The total labelling of the nucleolar, nuclear and cytoplasmic volumes has been calculated from the uridine high-resolution autoradiograms and the estimated amounts are discussed. The chromatin of several premeiotic oocytes and the cap of all the pachytene oocytes are strongly labelled after 7 h of [3H]thymidine incorporation. We did not find any thymidine incorporation in the young A, B or C diplotene oocytes.

Changes undergone during the in vitro maturation of oocytes from 8 different amphibian species have been followed by cytochemical methods (Unna staining, Feulgen reaction, PAS reaction). Critical stages of maturation in Xenopus laevis have been studied by electron microscopy. Attention has been mainly focused on two types of structures: 1. 1. Large basophilic bodies, rich in RNA and glycogen, which form or accumulate at the basal end of the nuclear membrane under hormonal stimulation. They probably correspond to polysomal aggregates and they seem directly involved in the rupture of the germinal vesicle. When the basal nuclear membrane begins to open, the nuclear sap becomes invaded by ribosomes and glycogen and later on by mitochondria and small yolk platelets. 2. 2. Feulgen positive bodies, which correspond to the nucleolar organizers: they appear at the basal end of the nuclear membrane when the latter begins to break down. Later, they move toward the egg cortex, where they ultimately vanish. The effects of actinomycin, puromycin, cycloheximide, and hydroxyurea on the formation of these two kinds of nucleic acid-containing bodies have been studied. The significance of these observations is discussed. © 1970., Journal Article, SCOPUS: ar.j, info:eu-repo/semantics/published

THE cuticle of the earthworm's gizzard is generally thought to be chitinous ; but there was no experimental evidence for this view, until the observation by Rudall1, who obtained an X-ray diffraction diagram similar to that found in Aphrodite bristles previously described by Lotmar and Picken2 as a modified chitin (β-chitin). Although after purification he obtained a different diagram, he nevertheless concluded that the gizzard membrane was probably chitinous in nature. This identification is much less convincing in view of the fact that the structural proteins are always found together with a certain amount of polysaccharides3.