Improvement of protein secondary structure prediction by combination of statistical algorithms and circular dichroism.

Three different approaches (propensity curve shifting, hydropathy index evaluation, and iterative attribution/cancellation of secondary structure) to the use of secondary structure percentages derived from circular dichroism measurements to improve the success rate of a protein secondary structure prediction method, without using decision constants, are described and compared. Propensity-curve shifting appears to be the best-performing approach, bearing an increase of 5.3% in the success rate of single-residue structural prediction when exact information on the secondary structure, obtained by X-ray crystallography, is employed; with information of an accuracy comparable to that obtainable by circular dichroism, the improvement stays between 3.5 and 4.9%, for a three-state prediction. Although developed with circular dichroism in mind, the method can use percentages of secondary structure obtained by any other experimental methodology from which they can be inferred, for instance Raman spectroscopy and infrared spectroscopy.

Computer acquisition and analysis of thermal profiles from differential scanning calorimeter.

In this paper we describe a computerized system for data recovery from differential scanning calorimetry of mammalian cells and their biopolymers. The 'in-house'-designed preamplifier, digital acquisition, control card and its real-time software provide us with a powerful workstation to acquire and analyze large quantities of data on-line and off-line. The final data are obtained after eliminating noise interference using both hardware and software filters. Fourier analysis is also performed for a more refined thermodynamic characterization. All software is written in Basic and Fortran 77 under the DOS 3.10 operating system on a personal computer.

Circular intensity differential scattering and chromatin-DNA structure. A combined theoretical approach.

Circular intensity differential scattering (CIDS) has been proven a powerful method in determining the higher-order structure of large biopolymers, such as chromatin. Theoretical predictions of the expected differential light scattering of circularly polarized light have previously been made for chromatin, either within the Born approximation, treating nucleosomes as noninteracting, oblate ellipsoids, or within a multiple dipole approximation, treating nucleosomes as interacting spheres. In order to conduct a meaningful interpretation of the CIDS signal in terms of given geometric parameters of the chiral structure, we have in this paper combined the two approaches considering the mutual interactions of ellipsoidal nucleosomes. In the process we have also found a confirmation for the validity of the Born approximation itself.

Cell versus nuclear morphometry of serum-stimulated fibroblasts: nuclear changes precede cell changes.

Changes in the cell and nuclear morphology of sparsely plated WI38 fibroblasts were followed as a function of time after increasing the serum concentration from 0.3% to 10%. Quantitative measurements were carried out in parallel on Feulgen-stained nuclei and Wright-stained cells using the Quantimet 720-D image analysis system. We report a rapid, significant change in nuclear morphology indicative of nuclear rounding taking place within 30 min after increasing the serum concentration. In contrast, cell morphology showed only a slight change within the first 30 min but showed a significant change, also indicative of cell rounding, between 30 min and 3 h after increasing the concentration. Thus our results indicate a coupling between cell and nuclear morphology, but one in which nuclear changes precede cellular changes. As variations in both cell and nuclear morphology have been linked to the control of cell growth and transformation we also discuss briefly the implications of our results in relation to the regulation of cell growth and transformation.

Biophysical characterization of normal T-lymphocytes and Sézary cells.

Blood lymphocytes from 18 patients with cutaneous T-cell lymphoma (Sézary syndrome and mycosis fungoides) were characterized using multiparameter laser flow microfluorimetry (FMF) and automated image analysis (AIA) and the results correlated with routine blood smears, cytogenetic studies and observations made on PHA-stimulated normal T-lymphocytes in vitro. Specimens from all 9 patients with Sézary syndrome and 5 of 9 patients with mycosis fungoides contained one or more discrete subpopulations of neoplastic (Sézary) lymphocytes that were detected by FMF. Studies with AIA demonstrated that neoplastic T-lymphocytes are distinguished from normal quiescent (G0) lymphocytes not only by alterations in DNA content (aneuploidy) but also by chromatin structuring (increased chromatin dispersion), which may be a more sensitive index of neoplastic transformation than ploidy levels. In several patients, small and large Sézary cells were present with DNA-chromatin properties quite similar to normal cycling G1 and G2 lymphocytes respectively, but their presence was not explained by an increase in proliferative activity in the blood. These findings indicate that Sézary syndrome consists of a heterogeneous group of related disorders differing in terms of the Sézary cell population. The response to treatment and prognosis may differ accordingly.

Coupling of nuclear morphometry to cell geometry and growth in human fibroblasts.

The nuclear morphology of WI-38 cells plated on increasing thicknesses of the polymer, poly-2-hydroxyethylmethacrylate, was studied. Changes in nuclear morphometry were found to parallel changes in cellular morphometry: namely a transition to a more rounded and compact conformation, with increasing thicknesses of the polymer. Such changes in nuclear morphometry were opposite to those already observed after stimulation of proliferation by serum addition and similar to those observed with increasing confluency. These results suggest that coupling between cell and nuclear geometry may represent a mechanism for the control of proliferation by cell shape in nontransformed cells.

Multiparameter geometric and densitometric analysis of the G0-G1 transition of WI-38 cells.

Automated image analyses were performed using Feulgen stained smears of WI-38 cells that were either confluent, or that had received a nutritional stimulus to proliferate 3 hr before collection. These experiments show that it is possible to observe changes in morphometric and densitometric parameters of nuclei that correlate with structural and functional differences in isolated chromatins from quiescent G0 and proliferation G1 cells that have been demonstrated by other means. Scatter plot analyses of the data indicated the presence of nuclear images from the stimulated G1 population that had the same deoxyribonucleic acid content as the confluent G0 cells, but had greater areas, perimeters and horizontal projections and smaller mean free paths, form factors, and average optical densities. Multiparameter cluster analysis permits, even minimally, an objective, model-independent identification of G0 from G1 cells that present an increased nuclear dispersion (i.e., lower average optical density) systematically accompanied by increased nuclear convolution (i.e., lower form factor), both compatible with the reported increase in available binding sites with respect to G0 cells.