The three-dimensional ultrastructure of interphasic and metaphasic nucleolar argyrophilic components studied with high-voltage electron microscopy in thick sections

The three-dimensional structure of the nucleolar argyrophilic components was studied by recording stereo-pairs of tilted thick sections--0.5-2 microns thick--observed with 200 and 300 kV high-voltage electron microscopy (HVEM). Using a very specific silver staining method, the argyrophilic components were stained with a high contrast relatively to the unstained background, thus allowing their study with a high resolution within thick sections. This study was performed on compact nucleoli (of HL60 and K562 cells), on reticulated nucleoli (of human breast cancerous cells) and on metaphasic nucleolar organizer regions (NORs). In compact nucleoli argyrophilic components show a 'knotted rope-like' structure in which knots are constituted of one central fibrillar centre surrounded at some distance by loops of the dense fibrillar component and in which the rope is constituted of dense fibrillar component. In reticulated nucleoli silver deposits are confined to the surface of the nucleolonema as several strands twisted at the periphery of the fibrillar component. During metaphase some NORs get a characteristic crescent-shaped structure disposed at the periphery of some chromosomes.     

The Three-dimensional Study of Chromosomes and Upstream Binding Factor-immunolabeled Nucleolar Organizer Regions Demonstrates Their Nonrandom Spatial Arrangement during Mitosis

The volumic rearrangement of both chromosomes and immunolabeled upstream binding factor in entire well-preserved mitotic cells was studied by confocal microscopy. By using high-quality three-dimensional visualization and tomography, it was possible to investigate interactively the volumic organization of chromosome sets and to focus on their internal characteristics. More particularly, this study demonstrates the nonrandom positioning of metaphase chromosomes bearing nucleolar organizer regions as revealed by their positive upstream binding factor immunolabeling. During the complex morphogenesis of the progeny nuclei from anaphase to late telophase, the equal partitioning of the nucleolar organizer regions is demonstrated by quantification, and their typical nonrandom central positioning within the chromosome sets is revealed.     

Adriamycin resistance is characterized by ultrastructural changes in human leukaemic K562 cells in vitro.

Ultrastructural changes associated with adriamycin (ADM) resistance have been investigated in the human K562 leukaemic cell line: sensitive K562 cells, a resistant subline cultured in the continuous presence of ADM and resistant cells without ADM Transmission electron microscopy (TEM) study revealed that K562-resistant cells displayed ultrastructural modifications of the cell surface, chromatin and nucleolus conformation. Alterations were not directly related to the presence of adriamycin as deprivated cells exhibited modificated characters through a slow progressive recovery phenomenon.     

Growth and characterization of isolated bovine tracheal gland cells in culture. Influence of a reconstituted basement membrane matrix

We describe a method for establishing the culture of bovine tracheal submucosal gland (BTG) cells, in which we have also examined the influence of a reconstituted basement membrane matrix derived from the Engelbreth-Holm-Swarm tumor (EHS) on the growth and morphological differentiation of these cells. BTG cells have been isolated by tissue enzymatic digestion using trypsin, deoxyribonuclease I, elastase, hyaluronidase and EGTA for 1 hr at 37 degrees C. Afterwards, cells and tissue were collected by centrifugation and were incubated for 15 min with 15% newborn calf serum to inactivate the proteolytic enzymes. Enzymatic digestion using only trypsin, centrifugation and inactivation steps were repeated three times. Using this protocol, we obtained 15 +/- 4 (X 10(6] cells per g of tracheal submucosa with 72 +/- 2% (n = 5) cell viability. On microscopic observation, isolated cells were mainly composed of serous type glandular cells. Cells were cultured in a 1:1 medium of Dulbecco's Modified Eagle's/Ham's F12 supplemented with 10% fetal calf serum and subcultured in either plastic flasks or flasks coated with EHS matrix. On the plastic, the BTG cells exhibited at confluency an epithelioid appearance. They stained positively with the immunofluorescent anticytokeratin antibody and contained PAS-staining granules. By electron microscopy, lactoferrin, a protein marker specific to the serous cells, was demonstrated immunocytochemically in small secretory vesicles. BTG cells cultured on EHS matrix revealed a significantly increased growth in comparison to those cultured on plastic. In post-confluent culture of BTG cells on EHS matrix, we observed numerous dome-like structures formed by differentiated cells which were joined together around luminal spaces.     

Allometric equations for integrating remote sensing imagery into forest monitoring programmes

Remote sensing is revolutionizing the way we study forests, and recent technological advances mean we are now able – for the first time – to identify and measure the crown dimensions of individual trees from airborne imagery. Yet to make full use of these data for quantifying forest carbon stocks and dynamics, a new generation of allometric tools which have tree height and crown size at their centre are needed. Here, we compile a global database of 108753 trees for which stem diameter, height and crown diameter have all been measured, including 2395 trees harvested to measure aboveground biomass. Using this database, we develop general allometric models for estimating both the diameter and aboveground biomass of trees from attributes which can be remotely sensed – specifically height and crown diameter. We show that tree height and crown diameter jointly quantify the aboveground biomass of individual trees and find that a single equation predicts stem diameter from these two variables across the world's forests. These new allometric models provide an intuitive way of integrating remote sensing imagery into large‐scale forest monitoring programmes and will be of key importance for parameterizing the next generation of dynamic vegetation models.     

Evaluation of automated pipelines for tree and plot metric estimation from TLS data in tropical forest areas

Background and AimsTerrestrial LiDAR scanning (TLS) data are of great interest in forest ecology and management because they provide detailed 3-D information on tree structure. Automated pipelines are increasingly used to process TLS data and extract various tree- and plot-level metrics. With these developments comes the risk of unknown reliability due to an absence of systematic output control. In the present study, we evaluated the estimation errors of various metrics, such as wood volume, at tree and plot levels for four automated pipelines.MethodsWe used TLS data collected from a 1-ha plot of tropical forest, from which 391 trees >10 cm in diameter were fully processed using human assistance to obtain control data for tree- and plot-level metrics.Key ResultsOur results showed that fully automated pipelines led to median relative errors in the quantitative structural model (QSM) volume ranging from 39 to 115 % at the tree level and 10 to 134 % at the 1-ha plot level. For tree-level metrics, the median error for the crown-projected area ranged from 46 to 59 % and that for the crown-hull volume varied from 72 to 88 %. This result suggests that the tree isolation step is the weak link in automated pipeline methods. We further analysed how human assistance with automated pipelines can help reduce the error in the final QSM volume. At the tree scale, we found that isolating trees using human assistance reduced the error in wood volume by a factor of 10. At the 1-ha plot scale, locating trees with human assistance reduced the error by a factor of 3.ConclusionsOur results suggest that in complex tropical forests, fully automated pipelines may provide relatively unreliable metrics at the tree and plot levels, but limited human assistance inputs can significantly reduce errors.     

Semiautomatic quantification of silver-stained nucleolar organizer regions in tissue sections and cellular smears.

Silver staining of nucleoli reveals argyrophilic proteins associated with nucleolar organizer region (Ag-NOR) proteins. Argyrophilic components appear as dots about 1 micron in diameter dispersed throughout the nucleolus (Ag-NOR dots). The count of Ag-NOR dots is a useful index for improving the cancer diagnosis and determination of prognosis. Here we describe software developed on a medium-cost image analyzer in order to evaluate the mean area of NORs and their number relative to an internal reference, the number and areas of clusters of NORs and the area of the nucleus. Statistical analysis of the data was performed during counting. The first application concerned counting NOR dots during mitosis in cell imprints; those counts were 2.3, 15.3 and 55.56 for the metaphase, telophase and interphase, respectively (relative to unitary dots of metaphase cells). In the second application we demonstrated a significant difference in NOR numbers between two groups of prostatic cancers with good and poor prognoses (6.05 +/- 2.79 SD and 7.96 +/- 3.01, respectively; with Student's t test, = 1.999; P = .05).