Are physicians prepared for whole genome sequencing? a qualitative analysis.

Although the integration of whole genome sequencing (WGS) into standard medical practice is rapidly becoming feasible, physicians may be unprepared to use it. Primary care physicians (PCPs) and cardiologists enrolled in a randomized clinical trial of WGS received genomics education before completing semi-structured interviews. Themes about preparedness were identified in transcripts through team-based consensus-coding. Data from 11 PCPs and 9 cardiologists suggested that physicians enrolled in the trial primarily to prepare themselves for widespread use of WGS in the future. PCPs were concerned about their general genomic knowledge, while cardiologists were concerned about how to interpret specific types of results and secondary findings. Both cohorts anticipated preparing extensively before disclosing results to patients by using educational resources with which they were already familiar, and both cohorts anticipated making referrals to genetics specialists as needed. A lack of laboratory guidance, time pressures, and a lack of standards contributed to feeling unprepared. Physicians had specialty-specific concerns about their preparedness to use WGS. Findings identify specific policy changes that could help physicians feel more prepared, and highlight how providers of all types will need to become familiar with interpreting WGS results.

A genotype risk score predicts type 2 diabetes from young adulthood: the CARDIA study.

AIMS/HYPOTHESIS: Genotype does not change over the life course and may thus facilitate earlier identification of individuals at high risk for type 2 diabetes. We hypothesised that a genotype score predicts incident type 2 diabetes from young adulthood and improves diabetes prediction models based on clinical risk factors alone. METHODS: The Coronary Artery Risk Development in Young Adults (CARDIA) study followed young adults (aged 18-30 years, mean age 25) serially into middle adulthood. We used Cox regression to build nested prediction models for incident type 2 diabetes based on clinical risk factors assessed in young adulthood (age, sex, race, parental history of diabetes, BMI, mean arterial pressure, fasting glucose, HDL-cholesterol and triacylglyercol), without and with a 38-variant genotype score. Models were compared with C statistics and continuous net reclassification improvement indices (NRI). RESULTS: Of 2,439 participants, 830 (34%) were black and 249 (10%) had a BMI ≥ 30 kg/m(2) at baseline. Over a mean 23.9 years of follow-up, 215 (8.8%) participants developed type 2 diabetes. The genotype score significantly predicted incident diabetes in all models, with an HR of 1.08 per risk allele (95% CI 1.04, 1.13) in the full model. The addition of the score to the full model modestly improved reclassification (continuous NRI 0.285; 95% CI 0.126, 0.433) but not discrimination (C statistics 0.824 and 0.829 in full models with and without score). Race-stratified analyses were similar. CONCLUSIONS/INTERPRETATION: Knowledge of genotype predicts type 2 diabetes over 25 years in white and black young adults but may not improve prediction over routine clinical measurements.

The Indian type 2 diabetes risk score also helps identify those at risk of macrovasvular disease and neuropathy (CURES-77).

AIMS: To see whether the diabetic individuals identified by the Indian Diabetes Risk Score (IDRS) also have a higher prevalence of diabetes related complications. METHODS: Type 2 diabetic subjects were selected from the Chennai Urban Rural Epidemiology Study in south India. Four field stereo retinal colour photography was done and diabetic retinopathy [DR] was classified according to Early Treatment Diabetic Retinopathy Study grading system. Coronary artery disease was diagnosed using Minnesota coding of 12-lead electrocardiograms. Diabetic peripheral neuropathy (DPN) was diagnosed if vibratory perception threshold [VPT] of the right great toe measured by biothesiometry was > or =20. The criterion for diagnosis of peripheral vascular disease (PVD) was an ankle-brachial index < 0.9. Macroalbuminuria was diagnosed if urinary albumin excretion was > or =300 microg/mg creatinine. A total of 1476 individuals who had information on all test parameters were included for analysis. RESULTS: Subjects with IDRS score > or =60 had significantly higher prevalence of coronary artery disease (CAD) [9.2% vs. 5.4%, p = 0.043], DPN [29.2% vs. 8.8%, p < 0.001] and PVD [4.8% vs. 1.9%, p = 0.038] compared to subjects with IDRS score <60. However, the prevalence of DR and macroalbuminuria did not differ between the two IDRS subgroups. Age explained much of the observed differences in prevalence of CAD, PVD and DPN between the two IDRS subgroups. CONCLUSIONS: This study further extends the clinical usefulness of IDRS to predicting diabetic complications like CAD, PVD and DPN as well.

Protective effect of superoxide dismutase against hair graying in a mouse model.

Oxygen free radicals play a role in the aging process, and the protective effect of various antioxidants has been intensively studied, in particular for cutaneous aging. Besides hereditary factors, free radical-mediated damage to melanocytes of the hair follicle has been considered as a mechanism for aging of the hair. It was the aim of this study to evaluate the role of photosensitization reactions for hair graying and to demonstrate potential protective effects of superoxide dismutase (SOD). Mice with black hair were depilated with the fingertips on a surface of 6 x 2.5 cm on both sides of the dorsum. The right side received five applications of a SOD-containing gel before exposure to psoralen (concentration 0.5 mg/mL) plus UV-A (365 nm, 4 J/cm2). The left side was pretreated in the same way with a gel free of SOD. When the hair started growing again, the SOD-protected side was covered with black hair, whereas the hair on the vehicle-treated side was gray or white in 27 of the 30 animals studied. The 0.01% SOD concentration was as protective as the 0.1% concentration. Heat-inactivated SOD, applied in another five animals, was not protective. Using fluorescent labeling of the SOD with fluorescein isothiocyanate, epifluorescence microscopy and digital imaging processing, we show that SOD applied to the skin surface penetrates through the follicular appendages, as well as through the unbroken stratum corneum. Our findings suggest that superoxide radicals, generated by interaction of UV-A light with the sensitizer, initiated the formation of secondary products with well-known DNA-damaging effects, such as lipid peroxidation products and tumor necrosis factor alpha. SOD prevented the damage to melanocyte DNA by dismutating superoxide. Photosensitization may be another mechanism for hair graying, which can be influenced by antioxidants. Given the large number of exogenous and endogenous sensitizers, this mechanism deserves further study for human hair graying.

Shear stress modulates tumour cell adhesion to the endothelium.

The adhesion of breast adenocarcinoma cells (MDA-MB-231) to human umbilical vein endothelial cells (HUVEC) was studied in whole blood and under varying flow conditions. This study was done on HUVEC either kept under static conditions or pre-conditioned in flow for 2 hours at a shear stress of 5 or 13 dyn/cm(2). Coverslips coated by HUVEC were placed in a parallel plate perfusion chamber and perfused at a shear rate of 300 or 1500 sec(-1) with heparin-anticoagulated blood containing 111In labelled MDA-MB-231 cells. We report here the optimal conditions for studying the adhesion of MDA-MB-231 to endothelial cells under shear constraints corresponding to those observed into small and medium sized arteries.

Weightlessness acts on human breast cancer cell line MCF-7.

Because cells are sensitive to mechanical forces, weightlessness might act on stress-dependent cell changes. Human breast cancer cells MCF-7, flown in space in a Photon capsule, were fixed after 1.5, 22 and 48 h in orbit. Cells subjected to weightlessness were compared to 1 g in-flight and ground controls. Post-flight, fluorescent labeling was performed to visualize cell proliferation (Ki-67), three cytoskeleton components and chromatin structure. Confocal microscopy and image analysis were used to quantify cycling cells and mitosis, modifications of the cytokeratin network and chromatin structure. Several main phenomena were observed in weightlessness: The perinuclear cytokeratin network and chromatin structure were looser; More cells were cycling and mitosis was prolonged. Finally, cell proliferation was reduced as a consequence of a cell-cycle blockade; Microtubules were altered in many cells. The results reported in the first point are in agreement with basic predictions of cellular tensegrity. The prolongation of mitosis can be explained by an alteration of microtubules. We discuss here the different mechanisms involved in weightlessness alteration of microtubules: i) alteration of their self-organization by reaction-diffusion processes, and a mathematical model is proposed, ii) activation or deactivation of microtubules stabilizing proteins, acting on both microtubule and microfilament networks in cell cortex.

Mitochondria transfection by oligonucleotides containing a signal peptide and vectorized by cationic liposomes.

The progress of research in gene therapy allows hope for treatment of mitochondrial genetic disorders provided that efficient methods for gene transfer into mitochondria can be found. In this work, we have used an oligonucleotide coupled covalently to a mitochondria-targeted peptide at one end and a cationic liposome prepared from trimethyl aminoethane carbamoyl cholesterol iodide (TMAEC-Chol) to carry it in living cells. With a fluorescent probe to label the oligonucleotide at the other end and by means of confocal microscopy, we show that such modified oligonucleotides complexed to liposomes enter into the cytoplasm of human fibroblasts in primary culture, and then, after dissociation from the complexes, they penetrate into the mitochondria. The fluorescence was still observed after 8 days, suggesting the continued presence of oligonucleotides. At the concentrations used for this study, the cationic liposomes have practically no effect on cell growth, as revealed by the MTT assay.

Remodeling of vimentin cytoskeleton correlates with enhanced motility of promyelocytic leukemia cells during differentiation induced by retinoic acid.

The intermediate filament (IFs) cytoskeleton is one of the major determinants for the mechanical properties of cytoplasm. Vimentin is the major IFs protein in peripheral blood neutrophils. We investigated its expression and function during neutrophil differentiation using the promyelocytic leukemia cell line NB4. The differentiation of NB4 cells along the neutrophil lineage and the monocytic pathway was induced by all-trans retinoic acid (ATRA) and phorbol esters (PMA), respectively. We demonstrated a down-regulation of vimentin after ATRA treatment of NB4 cells by immunoblotting and immunofluorescence. The architecture of the vimentin cytoskeleton in differentiated NB4 cells resembled that observed in mature neutrophils. In contrast, we showed a slight increase of vimentin content in phorbol ester (PMA)-treated NB4 cells. The structural features of the vimentin cytoskeleton obtained by image analysis showed significant differences in network density and directionality between ATRA-treated NB4 cells and controls. The functional consequence of the cytoskeletal remodeling for the mechanical properties of NB4 cells was assessed in migration assays. After ATRA treatment, we found a 4-fold increased migration of NB4 cells across transwell membranes with a 8 microm pore size without any cell size modification. No significant differences between PMA-treated NB4 cells and control cells could be observed using similar tests. These results indicate that both vimentin expression and network architecture are tightly controlled during neutrophil differentiation to regulate the mechanical properties of these cells.

Delivery and pathway in MCF7 cells of DNA vectorized by cationic liposomes derived from cholesterol.

We have investigated the delivery and the pathway in tumoral MCF7 cells of DNA carried by liposomes prepared from (trimethyl aminoethane carbamoyl cholesterol iodide (TMAE-Chol), a cholesterol-based cationic lipid with a quaternary ammonium on the polar head. The structure of DNA-liposome complexes depends on the length of DNA and on the lipid-DNA charge ratio X. Spherical beads constitute fine structures of the observed complexes even when they appear as aggregates. For oligonucleotide transfer, dissociation from liposomes after transfection, penetration of the oligonucleotides into nuclei, and a long resident time were observed. For plasmid transfer, a correlation between the variation in the transfection level and the ultrastructure of complexes was demonstrated. The results showed a cellular route of lipid/plasmid complexes from the beginning by endocytosis, entrapped into endosomes, released by the latter until entry in the perinuclear area, and then penetration of plasmids inside the nuclei resulting in the observed expression of the beta-galactosidase gene.

Immunolocalization of actin in root statocytes of Lens culinaris L.

Lentil root statocytes show a strict structural polarity of their organelles with respect to the g vector. These cells are involved in the perception of gravity and are responsible for the orientation of the root. Actin filaments take part in the positioning of their organelles and could also be involved in the transduction of the gravitropic signal. A pre-embedding immunogold silver technique was carried out with a monoclonal antibody in order to study the distribution of actin cytoskeleton in the statocytes at the electron microscopic level. Some areas were never labelled (cell wall, vacuole, nucleoplasm, mitochondria, starch grains of the amyloplasts) or very slightly labelled (stroma of the amyloplasts). The labelling was scattered in the cytoplasm always close to, or on the nuclear and amyloplast envelopes and the tonoplast. Associations of 2 to 6 dots in file were observed, but these short files were not oriented in one preferential direction. They corresponded to a maximum distance of 0.9 micron. This work demonstrated that each statocyte organelle was enmeshed in an actin web of short filaments arranged in different ways. The images obtained by rhodaminephalloidin staining were in accordance with those of immunogold labelling. The diffuse fluorescence of the cytoplasm could be explained by the fact that the meshes of the web should be narrow. The vicinity of actin and of the amyloplasts envelope could account for the movement of these organelles that was observed in spatial microgravity.

Cellular penetration of fluorescently labeled superoxide dismutases of various origins.

BACKGROUND: Using fluorescently labeled superoxide dismutase (SOD) and flow cytometry, we have shown previously that the enzyme CuZn SOD (EC 1.15.1.1) from bovine erythrocytes binds rapidly to the cell surface with slow uptake into the cell during the following hours. The degree of labeling was most important for monocytes in comparison to other blood cells (erythrocytes, lymphocytes, and neutrophils) and fibroblasts. In agreement with the flow-cytometric findings, the inhibition of superoxide production was more important for SOD-pretreated monocytes than for neutrophils, as demonstrated with the cytochrome c reduction assay. It was thus of interest to confirm the observed differences between monocytes and neutrophils with confocal laser microscopy, study in greater detail the kinetics of binding, penetration, and intracellular localization of the enzyme, and compare the results obtained with bovine CuZn SOD with those from SODs of other origins and carrying different active sites. MATERIALS AND METHODS: Recombinant human (rh), bovine, and equine CuZn SODs, as well as rh and E. coli Mn SODs, were studied before use with respect to specific activity and purity (HPLC, SDS-PAGE electrophoresis). Fluorescein isothiocyanate was covalently conjugated to the various SODs for study with high-resolution confocal scanning laser microscopy. Superoxide production by monocytes and neutrophils was measured with the cytochrome c assay. RESULTS: As expected from our experiments with flow cytometry, only rare neutrophils were labeled with FITC-SOD, even with the longest incubation time of 3 hr and the highest dose of 1500 units/ml. In addition, they showed a localized fluorescence pattern that was quite different from the diffuse punctate fluorescence pattern of monocytes. Lymphocytes were not labeled at all. The rapid binding to the cellular surface of monocytes was confirmed, and even after 5 min of preincubation, FITC-SOD was found on a small percentage of monocytes. This was correlated with a reduction in superoxide release after phorbolmyristate acetate (PMA) stimulation by 40%. An interesting finding was the perinuclear accumulation of the penetrated SOD after the longest pretreatment of 3 hr, suggesting a barrier against further progression. Indeed, through confocal microscopy we were able to exclude any fluorescence at the nuclear level. While the fluorescence labeling patterns and the kinetics of penetration were quite similar for bovine, equine, and rh CuZn SOD, the Mn SODs showed poor labeling, correlated with a weak inhibitory effect on cytochrome c reduction, which was not statistically significant. CONCLUSIONS: The rapid binding of native CuZn SODs on the surface of monocytes, leading to reduced superoxide release by these cells, explains the observation that beneficial effects of injected SOD lasted for months despite rapid clearance of the enzyme from the bloodstream, according to pharmacodynamic studies. The preferential binding to monocytes, in contrast to neutrophils, may play a role in chronic inflammatory diseases in which the monocytes are in an activated state. The differences in binding capacity between CuZn SODs and Mn SODs, correlated with different inhibitory effects of superoxide production by monocytes, may also have therapeutic significance.

Subcellular localization of RhoA and ezrin at membrane ruffles of human endothelial cells: differential role of collagen and fibronectin.

Endothelial cells and the regulation of their migration are of prime importance in many physiological and pathological processes such as angiogenesis. RhoA, an important Rho family member known to trigger actin reorganization, has been shown to mediate the formation of focal adhesions and stress fibers in quiescent fibroblasts. However, recent studies have emphasized its functional diversity and its implication in migration or metastatic processes in different cell types other than fibroblasts. Its role in endothelial cells is little known. In this study, we were interested by analyzing in human endothelial cells the subcellular redistribution of endogenous RhoA and the reorganization of cytoskeletal actin induced by two important extracellular matrix proteins, collagen and fibronectin. This paper shows a translocation of RhoA and its association with cortical actin in focal contact domains at membrane ruffles and at lamellipodia of spread or migrating endothelial cells, in the absence of any soluble mitogen stimulation. Furthermore, RhoA was found colocalized with ezrin, a member of the ERM family proteins newly described as important membrane-actin cytoskeleton linkers, at early membrane ruffles of endothelial cells spread on collagen but not on fibronectin. The present study points out that extracellular matrix, depending on the nature of its components, may promote distinct assemblies of focal contact constitutive proteins and strongly suggests that endothelial RhoA, like Rac1, may be an important mediator of matrix signaling pathway regulating endothelial cell adhesiveness and motility, independently of growth factor stimulation.

Adhesion-induced intracellular signalling in endothelial cells depends on the nature of the matrix.

The adhesion of a human microvascular endothelial cell line to its own matrix was studied in comparison with adhesion of the same cells to fibronectin or thrombospondin-1. These endothelial cells adhered preferentially to their matrix whereas an equal cell number was attached to fibronectin or thrombospondin-1. The adhesion of cells to thrombospondin-1 was mediated by the N-terminal heparin binding domain of thrombospondin-1 as shown by the use of a recombinant fragment, N18. Cells adhering to their matrix displayed a morphology and a cytoskeleton organization very similar to that observed in vivo with an apical immunostaining for actin stress fibers and a fine basal labeling for vinculin. Cells on fibronectin were extensively spread and rapidly assembled stress fibers and focal contacts. Cells adherent to thrombospondin-1 presented large lamellae rich in actin but devoid of vinculin and only few actin fibers were observed. Depending on the substratum used, adhering endothelial cells displayed also different tyrosine phosphorylation patterns on electrophoresis. Our observations indicate that endothelial cells adhering to their matrix present an activation state intermediate between that induced by a "hyperadhesive" protein like fibronectin and that generated by a moderate, indeed anti-adhesive, protein like thrombospondin-1.

Transforming growth factor-beta 1 increases the adhesion of MDA-MB-231 mammary adenocarcinoma cells to the microvascular subendothelium.

The increase of tumor cell adhesion to the subendothelium in the presence of TGF-beta 1 is thought to be mediated by two major events: an enrichment of extracellular matrix proteins secreted by endothelial cells and an increase of the integrins on the surface of tumor cells. In this study, we analyzed the effect of TGF-beta 1 on the adhesion of a mammary adenocarcinoma cell line (MDA-MB-231) to the matrix of human microvascular endothelial cells (HMEC-1). The adhesion of TGF-beta 1-treated tumor cells to a non-treated matrix or to purified matrix proteins was enhanced, while no increase was observed when non-treated tumor cells were let to adhere to a matrix secreted by HMEC-1 in the presence of the cytokine. Thus, the increase of cell adhesion was due to the effect of TGF-beta 1 on tumor cells and not to the matrix enrichment induced by this cytokine. The hyper-adhesion was inhibited by the RGD peptide and EDTA indicating that integrins were involved. Integrin subunits concentrations (alpha 5, alpha v and beta 1) on the surface of TGF-beta 1-treated tumor cells were not modified, while confocal microscopy showed a reorganization of beta 1 integrin subunits on the cell surface and in the cytoplasm resulting in actin fibers reorganization in the cytoskeleton. This indicates that the enhanced adhesion of TGF-beta 1-treated MDA-MB-231 cells to the subendothelium is due to a qualitative change of integrins.

Quantitative analysis of cytokeratin network topology in the MCF7 cell line.

BACKGROUND: In the MCF7 human breast cancer cell line, several patterns of cytokeratin networks are observed, depending on the intracellular localization. Our hypothesis is that architectural variations of cytokeratin networks depend on local tensions or forces appearing spontaneously in the cytoplasm. The aim of this work was to discriminate between the different patterns and to quantitate these variations. MATERIALS AND METHODS: Image analysis procedures were developed to extract cytokeratin filament networks visualized by immunofluorescence and confocal microscopy. Two methods were used to segment sets of curvilinear objects. The first, the "mesh-approach," based on classical methods of mathematical morphology, takes into account global network topology. The second, the "filament-approach" (novel), is meant to account for individual element morphology. These methods and their combination allow the computation of several features at two levels of geometry: global (network topology) and local (filament morphology). RESULTS: Variations in cytokeratin networks are characterized by their connectivity, density, mesh structure, and filament shape. The connectivity and the density of a network describe its location in a local "stress-force" zone or in a "relaxed" zone. The mesh structure characterizes the intracellular localization of the network. Moreover, the filament shape reflects the intracellular localization and the occurrence of a "stress-force" zone. CONCLUSIONS: These features permitted the quantitation of differences within the network patterns and within the specific filament shapes according to the intracellular localization. Further experiments on cells submitted to external forces will test the hypothesis that the architectural variations of intermediate filaments reflect intracytoplasmic tensions.

Cytoskeletal association of the A and B nucleoside diphosphate kinases of interphasic but not mitotic human carcinoma cell lines: specific nuclear localization of the B subunit.

The human A and B subunits of nucleoside diphosphate kinase (NDP kinase), encoded by the nm23-H1 and nm23-H2 genes, respectively, associate as homo- or heterohexamers to be catalytically active for the synthesis of nucleoside triphosphates. Despite 88% identity, they appear to possess specific functions. The nm23-H1 gene is implicated in tumor progression and metastasis, and the nm23-H2 gene product is a transcription factor for c-myc. To determine if these distinct functions reflect different subcellular localizations, the distribution of the A and B NDP kinases was analyzed by immunocytofluorescence microscopy in human breast cancer cell lines (MCF-7 and MDA-MB-231) using highly specific polyclonal and monoclonal antibodies. Interphasic cells exhibited a granular and filamentous cytoplasmic staining, particularly intense around nuclei, with both anti-NDP kinase A and B antibodies. The filamentous component observed with either anti-A or anti-B antibodies was altered in parallel to tubulin labeling with compounds interacting with microtubules, such as taxol and colchicine. Confirming published biochemical data, a partial colocalization with the vimentin network was observed in the MDA-231 cell line. A nuclear and nucleolar localization of NDP kinase B was shown by confocal microscopy which was not observed with the A enzyme. In dividing cells, NDP kinase labeling was punctiform and was not colocalized with the mitotic spindle. In conclusion, the A and B NDP kinases are similarly distributed in cytosol, associated partly to microtubules supporting a role in nucleotide channeling. Only the B enzyme is present in nuclei in accord with its role as a DNA binding protein. Their altered localization in dividing cells suggests colocalization with yet unidentified structures which are not intermediate filament aggregates.

Low-molecular-weight dextran derivatives (f-CMDB) enter the nucleus and are better cell-growth inhibitors compared with parent CMDB polymers.

Carboxymethyldextrans-benzylamide (CMDB) are dextran derivatives that are statistically substituted with carboxymethyl and benzylamide groups. These molecules display a variety of biological effects, one of which is their inhibitory activity against mammary tumor cell growth, both in vitro and in vivo. We and others have previously shown that the effects of CMDB on cell growth are related to their ability to interact with the growth factor FGF-2. The binding modifies the conformation of FGF-2, leading to the suppression of its mitogenic activity. Here, the method previously reported to fragment natural polysaccharide fucans has been applied to CMDB (80,000 g/mol). f-CMDB (fragmented CMDB) of molecular weights from 6000 to 20,000 g/mol were found to be more potent inhibitors of MCF7 mammary tumor cell growth than high-molecular-weight CMDB. Confocal microscopy experiments using CMDB and f-CMDB labeled with the fluorophore DTAF (5-([4,6-dichlorotriazine-2-yl]amino) fluorescein) indicate that only low-molecular-weight f-CMDB penetrate into the nucleus of MCF7 cells. It is thus assumed that the better inhibitory properties demonstrated by f-CMDB, compared with CMDB, are related to their better ability to penetrate the nucleus and interact with nuclear targets, including topoisomerase II. The DNA relaxation properties of the latter are inhibited in vitro by both CMDB and f-CMDB. These findings could help us to develop models of low-molecular-weight oligosaccharide derivatives exhibiting better antiproliferative and antitumor properties.

Application of confocal scanning laser microscopy to 3-D DNA image cytometry of prostatic lesions.

OBJECTIVE: To explore the potential of three-dimensional (3-D) image cytometry for the measurement of DNA content in prostatic specimens using confocal scanning laser microscopy (CSLM) and 3-D image analysis. STUDY DESIGN: Thick tissue slices (100 microns), stained for DNA with chromomycin A3, from four patients with benign hyperplasia, prostatic intraepithelial neoplasia (PIN), well- and poorly differentiated adenocarcinoma of the prostate, were studied. Two different blocks from the same slice were studied for each case. Cell nuclei were segmented automatically. DNA content and nuclear volume were measured. RESULTS: DNA histograms showed a single peak in the diploid range for the hyperplasia and PIN cases. For the case of well-differentiated carcinoma, two peaks were observed, one in the diploid range and one in the tetraploid range. The two peaks were observed on two different blocks of the same tissue slice. Poorly differentiated carcinoma was characterized by an aneuploid distribution. For the cases of PIN and carcinoma, we observed a considerable variation in nuclear volume. CONCLUSION: The results indicate the potential of 3-D image cytometry for the measurement of DNA content in prostatic specimens while preserving tissue architecture.

Fluorescent and radiolabeling of polysaccharides: binding and internalization experiments on vascular cells.

Glycosaminoglycans (GAGs) such as heparan sulfates are complex carbohydrate polymers. These structural components of the extracellular matrix are essential for the adhesion, migration, and regulation of cellular growth. To understand the physiological role of GAGs and GAG analogues, a practical approach consists of labeling and detecting them in cell extracts, or analyzing binding domains and their distributions into the cells. We propose a convenient and reliable method for preparing and labeling amino-enriched, polysaccharides with the fluorescent derivative 5-[(4,6-dichlorotriazine-2-yl)amino]-fluorescein (DTAF). Radioiodination is then performed on the DTAF moiety. This method was applied to polysaccharides known to inhibit vascular smooth-muscle cell (SMC) proliferation such as functionalized dextrans derived from poly(alpha 1-6 glucose) and fucan, poly(L-fucose 4-sulfate) extracted from brown seaweed. Using autoradiography and confocal microscopy, we observed the fixation and internalization of labeled antiproliferative products in SMCs from rat aorta. These probes can be useful for the understanding of polysaccharide-cell interactions. In addition, the method presented here can be applied to various synthetic or natural biomedical materials.