ALBERT

Notes: Summary Optical Fourier analysis was applied for evaluation of the differences between normal and pathologically changed bone tissue. Collagen fibers were used as markers of bone structure. To prove the usefulness of this technique for objective mathematical analysis of these differences the spatial distribution of collagen fiber bundles was evaluated in normal and osteopetrotic bone. The variation in the spatial distribution of collagen fiber bundles in cross sections of femur diaphyses was evaluated quantitatively by optical diffraction in three groups of Fatty Orl-op strain rats, i.e. phenotypically normal animals, osteopetrotic (op/op) mutants and op/op-mutants cured by transplantation of normal syngenic bone marrow. The histological sections of decalcified bone were stained with Sirus-Red and then photographed under polarizing microscope. The Sirus-Red staining was used to enhance the natural birefringency of collagen fibers. Diffractograms obtained from microphotographs of selected bone section areas, i.e. outer and inner circumferential lamellae and haversian bone of normal and cured op/op animals as well as whole cortical bone and woven bone filling the medullary cavities in op/op mutants were analysed separately. Diffractograms contain summarized information on the size and relative position of histological structures. The radial distribution of light energy informs on the size and/or distances between the structures, while angular distribution gives the relative position of these structures in histological sections. The radial and angular distribution of light energy were evaluated for each diffractogram with an electronic detector. The obtained distributions were described by several sets of parameters concerning the position, level and shape of local maxima and minima. Out of these parameters five with the highest discriminant power were chosen for further mathematical analysis. This analysis was based on the calculation of the position of centroids in the multidimensional space described by the linear functions of the chosen parameters for each of the evaluated bone section areas. The centroids (mean values of discriminant scores of each group) represent the centers of gravity of the analysed groups, while the separation of the centroids tested by the F-test illustrates the differences between the respective groups of selected bone section areas. A high level of separation of centroids was found when osteopetrotic bone was compared with normal one, what means that the spatial distribution, size and interstructural distances between the collagen fiber bundles in bone tissue in these two groups of animals differ markedly. A similar situation was observed when osteopetrotic bone was compared with bone tissue obtained from op/op mutants cured by transplantation of normal syngenic bone marrow. On the other hand, the level of separation of centroids was low when bone tissue of cured op/op mutants was compared with the control one, a finding which corresponds to the less pronounced histological differences between these two groups of animals. Computer-aided classification on single microphotographs of selected bone section areas to the known a priori type of bone tissue was performed. The percentage of cases correctly classified to one of the eight groups of selected bone section areas equals 47. The probability of reaching such a result by chance was estimated as less than 10−4. The percentage of cases classified correctly to one of any two statistically different groups was higher than 90. These observations demonstrate that optical diffractometry allows to describe in numerical terms the differences between normal and pathologically changed bone tissue, and therefore might be used for automatic evaluation of histopathological sections and interlaboratory comparative studies.

Notes: Summary Arterial smooth muscle cells in contractile and synthetic state were analyzed by optical diffractometry. Cell sections (80–90 nm) were photographed in an electron microscope and diffraction patterns of the plates (negatives) were produced using a helium-neon laser. Radial and angular distributions of light intensity in the diffractograms were measured and digitized using an electronic detector plate consisting of ring- and wedge-shaped photosensitive elements; radial distributions provide information about size of structures and distances between them and angular distributions about spatial orientation of structures in the images. Micrographs of nuclei and cytoplasm were analyzed separately (40–50 plates in each group). Computerized statistical analysis of radial distributions of light intensity showed that the nuclear chromatin pattern differed between cells in contractile and synthetic state. The probability that the observed difference could have arisen purely by chance was less than 10−5. Computer-aided classification to the a priori known cell group was correct in 96.5% of the cases. Analysis of radial distributions of light intensity similarly showed marked differences in cytoplasmic structure between cells in contractile state (dominated by bundles of myofilaments) and synthetic state (dominated by cisternae of rough endoplasmic reticulum). The probability that the observed difference could have arisen purely by chance was less than 10−5. Computer-aided classification to the a priori known cell group was correct in 92.0% of the cases. In contrast, analysis of angular distributions of light intensity did not indicate any statistically significant differences between contractile and synthetic state cells. A likely reason is that both myofilaments and cisternae of rough endoplasmic reticulum were arranged in parallel. The results demonstrate that optical diffractometry is a useful method for image analysis in studies of cell fine structure. It provides information about size and orientation of structures with poorly defined shape and is particularly well suited for studies on cell differentiation and effects of pharmacological and other experimental treatments on cell fine structure. It represents an alternative and a complement to stereology for quantitative and objective evaluation of morphological data.

Notes: Summary The effect of 1-hydroxyethylidene-1,1-bisphosphonate (HEBP) and dichloromethylidene-bisphosphonate (Cl2MBP) on the structure of the organic matrix of heterotopically induced bone in guinea pig was studied. Heterotopic bone formation was induced by transplantation of allogenic urinary bladder epithelium. Starting from the day of transplantation the animals were treated subcutaneously with HEBP and Cl2MBP with a dose of 12.5 mg P/kg/day during 35 days. The control group was injected with 0.9% NaCl solution. The advantage of heterotopic bone induction as an experimental model is the fact that the applied drugs act on de novo bone formation. Collagen fibers were treated as markers of bone because their size and spatial arrangement reflect the structure and maturity of organic matrix of this tissue. Decalcified histological sections of induced bone, taken 35 days after implantation of inductor, were stained by the picrosirius method. This staining enhances the natural birefringency of collagen fibers and allows for better and specific visualization of collagen fibers bundles under polarizing microscope. In this way the amount of information in the analysed image is increased. Thirty five microphotographs were analysed from each of the investigated groups with the use of optical diffractometry. The radial distribution of light intensity in diffraction patterns was analysed what allowed to evaluate spatial frequencies connected with the width of collagen bundles in induced bone tissue. Since the spatial arrangement of collagen fibers in newly formed bone is random, analysis of angular distribution of light intensity in diffractograms was not performed. Using discriminant analysis the significant differences between all three studied groups of animals were found. The widest differences were found between the Cl2MBP and HEBP treated animals. They were larger than those between each of these two groups and the control one. In control as well as in HEBP treated animals thicker bundles of collagen fibers were more frequently observed than in the Cl2MBP treated group, while in the latter thin bundles, nondetected in the former two groups were found. Generally, the radial distribution of light intensity in diffraction patterns of the HEBP treated animals resembles more that in the control group than in the Cl2MBP treated one. The different effects of the two analysed bisphosphonates (BPs) on the organic bone matrix of heterotopically induced bone is interpreted as differences in their influence on osteogenic cells and/or as differences in their direct influence on extracellular collagen fiber bundles formation.

Notes: Summary The aim of this paper is to show the possibility of objective mathematical description of changes occurring in the shape of cells in the process of transformation. The evaluation of the changes in cell shape of the chosen cell lines differing in transformation grade was performed by the use of Fourier analysis of the shape. Any two-dimensional contour can be described with specific accuracy in a mathematical manner using the closed form Fourier series of cosines. The components forming the analysed shape, called harmonics, are independent and uncorrelated measures of their contribution to the total shape. The shape of each cell can be represented by the spectrum of harmonic amplitudes. To quote the paper by Healy-Williams and Williams (1981): “The observed shape is partitioned into series, where gross shape, as elongation or triangularity, is measured by the harmonic amplitudes of the lower harmonic order and increasingly fine scaled surface sculpture is measured at higher orders”. The statistically evaluated results allow the objective comparison of the cell shapes of several compared cell lines differing in transformation grades. Malignant transformation is supposed to be a multistep process. The different grades of transformation could be defined by several parameters as changes in the morphology of the cells, their ability to compete with fibroblasts, their life span, their angiogenic potency, their invasiveness in vitro and their tumorigenicity in nude mice. In this paper several human urothelial cell lines of normal and tumor origin differing in their transformation grade (TGr I–III) were compared by the use of Fourier analysis of their shape. TGr I cultures have finite life span but do not need intermittent collagenase treatment to prevent fibroblast overgrowth. TGr II cultures acquire infinite growth potential, here defined as capacity to survive at least 70 passages. They are neither tumorigenic nor invasive. TGr III cultures show infinite growth transformation, increased angiogenicity and ability to invade normal host tissue in vitro. They produce progressively growing tumors in nude mice. The following human uroepithelial cell lines differing in the degree of transformation were studied and compared by statistical evaluation of the harmonic amplitudes deseribing mathematically the cell shape: Two cell lines derived from human transitional cell carcinoma (TCC): 1. Hu 1703S classified as TGr I, 2. Hu 1703He classified as TGr III. It was found that these two cell lines differ in all harmonics. Two cell lines derived from morphologically normal human bladder epithelium: 3. HCV-29 classified as TGr II. The confluent (HCV-29 confl) and peripherial (HCV-29 periph) parts of the HCV-29 cultures were studied separately. 4. HCV-29T “spontaneously” transformed in culture, subline of HCV-29 classified as TGr III. 5. Normal human fibroblast line was used for comparison and control. — It was shown that the confluent part of the epithelial HCV-29 line (HCV-29 confl) which is not tumorigenic was identical in shape to the cells of HCV-29T tumorigenic cultures. The parameters describing the shape of cells in the periphery of HCV-29 cultures (HCV-29 periph) were nearly identical to those of human fibroblasts. Both HCV-29 lines are statistically different from the Hu 1703He line. The applied technique allows for the numerical description of cell shape — the one of many changeable parameters in the process of transformation. These numerical data could be analysed by statistical methods, which allow to define the similarities or differences between the compared cell lines in a quantitative way.

Notes: Abstract Pectin methylesterase (PME) is responsible for the demethylation of pectin prior to pectin's degradation by the combined activities of polygalacturonase and pectate lyase. We have differentially screened a maize pollen cDNA library to detect cDNA clones whose genes are specifically expressed in pollen. One group of clones resulting from this screen showed similarity (between 18% and 41% identity) with plant and fungal PMEs. The full-length clone from this group, ZmC5, identifies a small gene family (at least 2 members) when used as a probe on genomic Southern blots. Northern analysis reveals that the ZmC5 transcript is expressed specifically in late pollen development. This tissue-specific gene expression programme is further confirmed in transgenic tobacco plants harbouring ZmC5 promoter/GUS chimeric gene constructs.