Ulrich-Walter Grummt

Demystifying chromosome preparation and the implications for the concept of chromosome condensation during mitosis

The processes taking place during routine chromosome preparation are not well understood. In this study, the morphological changes in amniotic fluid cells, blood lymphocytes, and bone marrow cells in the metaphase stage were examined under an inverted microscope during chromosome preparation. The putative processes that occur during chromosome preparation were simulated in suspension, and the cells were treated with different mixtures of hypotonic solution, fixative, methanol, acetic acid, and water. Evaporation of the fixative was performed under normal atmospheric conditions and under vacuum at different levels of humidity. Freeze fracture electron microscopy was used to analyze the effects of fixative on the cell membrane. Confocal microscopic analysis was used to investigate three-dimensionally the effects of hypotonic treatment on the positions of chromosomes in fixed mitotic lymphocytes. Chromosome preparation-induced changes in the lengths of single chromosomes were also investigated. The results show that chromosome spreading involves significant water-induced swelling of mitotic cells during evaporation of the fixative from the slide, which is a prerequisite for chromosomal elongation, the production of metaphase spreads for chromosome analysis, and the appearance of Giemsa banding patterns. Hypotonic treatment is essential for well-spread metaphase chromosomes because it moves the chromosomes from a central to a more peripheral position in the cell, where they can be stretched more effectively during mitotic swelling. Like mitotic cells, isolated single chromosomes also have their own potential to swell and lengthen during chromosome preparation. We hypothesize that chromosome preparation leads to a genome-wide chromosomal region–specific opening of chromatin structures as GTG-light bands and sub-bands. Living cells may possess a similar mechanism, which is used only to open single chromatin structures to facilitate transcription. We propose the concept of chromosomal region–specific protein swelling.

Torsional barriers in biphenyl, 2,2′-bipyridine and 2-phenylpyridine

Abstract The rotational barriers for biphenyl, 2,2′-bipyridine and 2-phenylpyridine were calculated with HF, B3LYP, BPW91, and MP2 and double-zeta polarized basis sets. The methods differ significantly in the near-planar region, and are in close agreement otherwise, with DFT results similar to experiment over the whole range (for biphenyl). Steric repulsions, stabilizing C–H–N′ interactions, π-conjugation and electron correlation give rise to DFT-calculated dihedrals for biphenyl of about 40° and similar 0° and 90° barriers, dihedrals of 23° and an almost non-existent 0° barrier for 2-phenylpyridine and two minima for 2,2′-bipyridine, which is strongly stabilized by two C–H–N′ interactions in the coplanar conformation with anti-standing nitrogens.

The DNA-Based Structure of Human Chromosome 5 in Interphase

In contrast to those of metaphase chromosomes, the shape, length, and architecture of human interphase chromosomes are not well understood. This is mainly due to technical problems in the visualization of interphase chromosomes in total and of their substructures. We analyzed the structure of chromosomes in interphase nuclei through use of high-resolution multicolor banding (MCB), which paints the total shape of chromosomes and creates a DNA-mediated, chromosome-region-specific, pseudocolored banding pattern at high resolution. A microdissection-derived human chromosome 5-specific MCB probe mixture was hybridized to human lymphocyte interphase nuclei harvested for routine chromosome analysis, as well as to interphase nuclei from HeLa cells arrested at different phases of the cell cycle. The length of the axis of interphase chromosome 5 was determined, and the shape and MCB pattern were compared with those of metaphase chromosomes. We show that, in lymphocytes, the length of the axis of interphase chromosome 5 is comparable to that of a metaphase chromosome at 600-band resolution. Consequently, the concept of chromosome condensation during mitosis has to be reassessed. In addition, chromosome 5 in interphase is not as straight as metaphase chromosomes, being bent and/or folded. The shape and banding pattern of interphase chromosome 5 of lymphocytes and HeLa cells are similar to those of the corresponding metaphase chromosomes at all stages of the cell cycle. The MCB pattern also allows the detection and characterization of chromosome aberrations. This may be of fundamental importance in establishing chromosome analyses in nondividing cells.

Conjugated polymers with 2,2′-bipyridine and diethinylenebenzene units: absorption and luminescence properties

Alternating oligomers and polymers consisting of 2,2′-bipyridine and diethinylenebenzene units and corresponding model compounds were synthesized and investigated in dilute solutions by absorption spectroscopy and by stationary and time-resolved emission spectroscopy. The strictly linear (rod-like) π-chain oligomers/polymers were compared with the angularly linked oligomers/polymers and with related model compounds. The model compounds which already show the essential spectroscopic properties of the oligomers/polymers consist of three (hetero)aromatics linearly connected by two diethenylene groups. These models exhibit fluorescence quantum yields close to unity and short fluorescence decay times around 1 ns. Fluorescence anisotropy and rotational relaxation times are consistent with the Stokes–Einstein equation and the Perrin equation. The absorption and emission spectra of the polymers and their radiative rate constants determined by fluorescence quantum yield and lifetime and according to the Strickler/Berg equation show a conjugation length of one to two repetition units. The conjugation along the chain is stronger in linear than in angular polymers and stronger in alkoxy-substituted than in unsubstituted polymers. In angular polymers at least two different emitting segments were found. The shortened mean lifetimes and the reduced fluorescence quantum yields and anisotropies of the oligomers/polymers indicate an additional radiationless deactivation channel which is opened by energy migration along the chain. Rates of energy transfer calculated for linear and angular polymers correlate with rates of radiationless deactivation. Copyright

Detailed energy decomposition of the rotational barrier in 2,2′-bipyridine: a density functional study

Abstract Detailed analysis of the torsional profile for 2,2′-bipyridine with different model chemistries and basis sets shows a strong stabilization of the coplanar nitrogen anti conformation and a second minimum for the cis conformation with a dihedral of about 40°. The entropic contributions are unfavourable near the minima, contrary to the enthalpy curve. The electronic energy barrier is comprised of small geometry relaxation effects, and mostly dominated by contributions from exchange and electron correlation. The barrier for a dihedral φ of 0° is mostly due to steric repulsion of the hydrogens, and to a lesser extent due to Coulomb repulsion of the nitrogen lone pairs.

Limitations of the sum-over-states approach to second-order hyperpolarizabilities: para-nitroaniline and all-trans-1,3,5,7-octatetraene

Abstract A systematic investigation of the effects of various configuration interaction (CI) approximations, input frequencies, and geometry variations on the sum-over-states calculated second-order hyperpolarizabilities is performed using AM1 semiempirical NDDO theory (NDDO/SOS/CI). Para-nitroaniline and 1,3,5,7-all-trans-octatetraene are chosen as test compounds. It turns out that it is necessary to include at least single and double excitations (CISD) and to have active windows containing the complete scheme of π- and n-type orbitals to get converged CI solutions. These approximations are thus limited to molecules, which are too small to be of interest for application in non-linear optical organic materials. For non-suitable low-level CI-approximations, artificial effects on calculated γ are obtained. Information about the hyperpolarizability resonant regions and the excitation pathways for the compounds can be obtained in a consistent manner by the use of the above-mentioned methodologically stable CI-approximation. The geometry dependence of γ is visualized by hyperpolarizability torsional profiles and hypersurfaces and by comparison of the values for molecule builder-, semiempirical- and DFT-derived structures. From these findings, we emphasize the use of DFT-derived structures as starting points for semiempirical SOS calculations of hyperpolarizabilities.

CHEMICAL DEPOSITION OF SILVER NANOCLUSTERS ON SELF-ASSEMBLED ORGANIC MONOLAYERS. A STRATEGY TO CONTACT INDIVIDUAL MOLECULES

Abstract Self-assembled monolayers (SAMs) on gold(111) surfaces were prepared consisting of a matrix of n -dodecane thiol and a mercaptan as a host which is terminated by a four-electron reducing agent. Upon treatment with ammoniated silver nitrate solutions silver nanoclusters are generated on the SAM surface. Physical development allows enlargement of the clusters.

Implementation of an NDDO/CI/SOS approach for second‐order hyperpolarizabilities

We report the implementation of the sum-over-states (SOS) formalism for neglect of diatomic differential overlap (NDDO) Hamiltonians Austin model 1 (AM1), parametric method 3 (PM3), and modified neglect of differential overlap (MNDO) for calculation of third-order nonlinear optical properties into the program package VAMP and its application to third harmonic generation (THG). Extensive comparisons between THG experimental data and published MOPAC/FF to VAMP/PECI/SOS and AMPAC/FF calculated values were carried out in gas phase and in solvent for a data set of 236 compounds of the general type DπA and conjugated π systems. Great care was taken to derive the global minimum conformers, yielding significant deviations of the geometries derived by the three Hamiltonians. The data set therefore gives an overview of the shortcomings and strengths of the semiempirical methods. Here, the implementations of solvent effects in both semiempirical packages are especially problematic in the case of elongated molecules, so a threshold for molecular globularity had to be defined to eliminate erroneous data. The correlation statistics presented for γ are in acceptable agreement for the whole data set as for all experimentally well-defined substance classes with scalable correlation slopes smaller than unity. The data become more reliable for large γ, probably due to more precise experimental values. Inclusion of solvent effects raises the polarizabilities of the molecules consistently. These results enable us to qualitatively predict trends for small as well as large second-order polarizabilities, to derive scaling functions for quantitative predictions, and to calculate experimentally nonaccessible tensor elements of γ. The SOS formalism even allows us to obtain insights into the frequency dependence of second-order hyperpolarizability effects beyond THG.

Deactivation of excited 4′-dialkylamino-9-styrylacridines

Abstract The absorption and emission spectra and deactivation of excited styrylacridines are determined by the flexibility and charge transfer properties of the donor-acceptor chromophore system. The fluorescence and excitation spectra, as well as the kinetics, vary with the excitation and emission wavelength respectively. Principal component analysis (PCA) and global analysis of the spectra and kinetics of the E isomers reveal at least two absorbing and two emitting components in fluid solution at room temperature as well as in a frozen matrix. At 77 K, the fluorescence of the Z isomer is also observed. The Sn ← S1 transition of the Z isomer is the only transient detectable from picosecond time-resolved absorption experiments in solution. The main competing deactivation channels of the S1 state are E → Z isomerization, charge transfer-induced internal conversion and fluorescence.

Photochromism and thermochromism of dithizone in solution

Abstract Dithizone behaves as a photochromic system (green⇌←red) in pure hexane; in acetone and acetonitrile a second transient is observed. As proved by the kinetic deuterium isotope effect and base catalysis of the thermal reverse reaction the photochromism is interpreted in terms of tautomeric and geometric transformations. A proposal is presented for a possible structural assignment of the phototransients.