High resolution morphological analysis of in situ human chromosomes.

The purpose of this study was to analyze the inner structure of chromosomes in cells arrested, fixed and cryosectioned in metaphase. The chromosomes in metaphase maps prepared using standard cytogenetic protocols, are usually covered by cellular debris, which obscures the structural details on the surface and limits analysis by techniques when using nanometric resolution. By using cryosectioning, the debris is removed and it is possible to analyze the internal structure of the chromosomes. We described the ultrastructure of chromosome sections fixed with either acetic acid, methanol or glutaraldehyde, evaluating the effect and the influence of the fixative on the morphology. Furthermore, we subjected those cells previously fixed with glutaraldehyde to osmic maceration in order to better visualize the intracellular structure. All samples were examined with a Field Emission In Lens Scanning Electron Microscope (FEISEM), which allows high-resolution analysis of biological samples without any metal coating. The results showed a package morphology in samples fixed with glutaraldehyde, mainly due to the high capacity of the fixative to strongly crosslink the proteins. In contrast, the fibrillar structure seen in cryosections fixed with acetic acid/methanol is due to the propensity of the fixatives to extract and remove proteins. We propose that in situ chromosomes fixed with glutaraldehyde and then osmicated are a good model for studying the inner structure of chromosomes by using high resolution scanning electron microscopy.

Characterization and cell cycle kinetics of hepatocytes during rat liver regeneration: in vivo BrdUrd incorporation analysed by flow cytometry and electron microscopy.

The incorporation of bromodeoxyuridine (BrdUrd) into newly synthesized DNA has been analysed during hepatocellular regeneration induced by partial hepatectomy in young rats. The kinetic state of the liver has been studied by flow cytometric analysis of the incorporated BrdUrd, while the fine localization of DNA replication sites through the cell cycle has been investigated at the ultrastructural level by the immunogold technique. Eighteen hours after partial hepatectomy flow cytometry revealed an early S phase distribution which corresponded to a specific staining of the interchromatin domains of the hepatocyte nucleus. Thirty-four hours after hepatectomy, on the other hand, when most cells were in late S, a specific staining of heterochromatin domains was observed. The effect of the BrdUrd technique on nuclear aggregation has also been analysed and discussed. The results demonstrate that specific patterns of DNA replication can be recognized during the cell cycle and that flow cytometry and electron microscopy appear to be complementary in the kinetic study of liver regeneration.

High-resolution FEISEM detection of DNA centromeric probes in HeLa metaphase chromosomes.

HeLa metaphase chromosome spreads were hybridized with centromeric biotinylated DNA probes and detected with gold-conjugated anti-biotin antibodies. Chromosomes were observed by an in-lens field emission scanning electron microscope (FEISEM), which permits detection of biological samples without any coating. DNA probes were well localized in the centromeric region of chromosomes and there was clear discrimination between 10 nm fibers that hybridized to DNA probes and those that did not hybridize. This approach shows that in situ hybridization can be directly visualized at the FEISEM level by evaluating only secondary electron emission, which allows physical localization of the hybridized probe with high resolution so that backscatter detection represents only a control. Because chromosomes maintain the 10-nm fiber organization after in situ hybridization procedures, our data suggest that this fiber represents the lowest order of chromatin arrangement that permits transitory DNA denaturation.

Ultrastructural aspects of the DNA polymerase alpha distribution during the cell cycle.

We studied the nuclear topography of the replicating enzyme DNA polymerase alpha in HeLa cells by transmission electron microscopy and field emission in lens scanning electron microscopy. Cells were synchronized at the G1/S-phase boundary and samples of the different phases of the cell cycle were labeled with an anti-DNA polymerase alpha antibody detected by an immunogold reaction. DNA synthesis was detected by immunogold labeling after bromodeoxyuridine administration. The typical labeling pattern of DNA polymerase alpha observed in G1- and S-phase cells was represented by circular structures 80-100 nm in diameter surrounding an electron-dense area. In double labeled samples these circular structures were associated with bromodeoxyuridine-containing DNA replication sites, forming rosette-like structures. Field emission scanning electron microscopy performed on ultrathin cryosections revealed the chromatin fibers underlying DNA polymerase alpha complexes and showed that the size of the rosette-like structures corresponded to the diameter of chromatin foldings. G2- and M-phase cells showed a spread distribution of DNA polymerase alpha. The evidence of DNA polymerase alpha circular arrangement exclusively in G1- and S-phase cells, obtained by such different approaches, allowed us to consider the three-dimensional structures as DNA replication areas.

High-resolution detection of newly synthesized DNA by anti-bromodeoxyuridine antibodies identifies specific chromatin domains.

We analyzed the incorporation of bromodeoxyuridine (BrdUrd) into DNA in exponentially growing murine erythroleukemia cells (FLC-745), using fluorescent anti-BrdUrd antibodies with light microscopy and flow cytometry. The fine localization of the DNA replicating sites was investigated at the ultrastructural level by using a second antibody conjugated with colloidal gold. The latter approach, which does not require acidic denaturation of the DNA, enables preservation of good morphology and obtains a better resolution power than that of electron microscopic autoradiography, the percentage of labeled cells obtained with the two techniques being comparable. After short BrdUrd pulses, characteristic distribution of the labeling can be identified in the heterochromatin, in interchromatin domains, or at the boundary between the dispersed and the condensed chromatin. Similar patterns are also observable in the nuclear structures which condense after acid denaturation, suggesting that DNA replication takes place at fixed sites associated with the nuclear matrix.

Immunocytochemical detection of phosphatidylinositol 4,5-bisphosphate localization sites within the nucleus.

Phosphatidylinositol 4,5-bisphosphate (PIP2) is a key element of signal transduction, being the preferential substrate of specific phospholipases that produce second messengers such as inositol trisphosphate (IP3) and diacylglycerol (DG). Because PIP2 has been cytochemically identified by monoclonal antibodies not only in the cytoplasmic membranes but also in the nuclear envelope and within the nucleus, we performed a study by immunoblotting and by confocal and electron microscopic immunocytochemistry to identify the nuclear sites of PIP2 localization and to exclude any cross-reactivity of the antibody with other nuclear molecules. The results confirm the specificity of the immunolabeling and indicate that PIP2 is localized at precise intranuclear sites both in in situ and in isolated nuclei. They also show that a significant amount of the phospholipid is retained by the cytoskeleton and by the inner nuclear matrix in in situ matrix preparations. Moreover the sensitivity of the immunocytochemical reaction is capable of detecting quantitative variations of PIP2 nuclear content induced by agonists that modulate the signal transduction system at the nuclear level.

Immunocytochemical discrimination between early and late S phase: a new approach to the study of gingival epithelium proliferation in rats.

The renewal of the free gingival margin epithelium in rats was studied evaluating 5-bromodeoxyuridine (BrdU) incorporation in proliferating cells by means of an immunocytochemical method. We found a close correspondence between light and electron microscopy patterns of BrdU incorporation at a nuclear level. BrdU was localized in the inner interchromatin regions in cells starting DNA synthesis, while it was localized in the peripheral heterochromatin domains in cells terminating the S phase. This possibility of discriminating cells in early S phase from cells in late S is able to provide far more information as to the time at which a labeled cell starts proliferation than that obtainable with 3H-thymidine autoradiography. This, in turn, permits detection of cells that start proliferation in a wide period of time by means of a single BrdU administration. Rats treated at 7 a.m. demonstrated higher proliferation than rats treated at 7 p.m., supporting the existence of circadian variations in the epithelial renewal. Proliferative events take place by consecutive activation of at least three replication waves, producing clusters of labeled cells which could be observed in rats sacrificed at 10 a.m. In rats treated once with BrdU at 7 a.m., the clusters were localized in both the basal and suprabasal layer of the epithelium; in rats further injected with BrdU at the same time, the clusters increased in size, progressively extending throughout the epithelium. In this way, the renewal of the free gingival margin epithelium does not proceed randomly, but by consecutive activation of discrete units or clusters of basal cells, which then extend to the upper layers. This can be followed at a morphological level as a progression of labeled cells, which move from the basal layer to the epithelium surface in approximately 82-85 hours.

[Microscopic research on arachnoid villi in various mammals]. / Ricerca microscopica sui villi aracnoidali in alcuni mammiferi.

The sagittal sinus in the man, dog, cat and rabbit was studied; we have noted remarkable histological differences within the zoological succession in the system of discharge of the cerebrospinal fluid in the venous system. The differences, probably, are chargeable to the different metabolic encephalic exigences which in the man are exalted and which justify the presence of the more complicated structures.

Uptake of tritiated phosphatidylcholine by isolated rat liver nuclei studied by electron microscope autoradiography in albumin embedded specimens.

The transfer of phosphatidylcholine from multilamellar vesicles to isolated rat liver nuclei was studied by means of electron microscope autoradiography. To avoid the possible loss or the artifactual redistribution of the phospholipid occurring during dehydration with organic solvents and plastic embedding, the fixed specimens were embedded in aqueous albumin, which was then hardened by glutaraldehyde and dehydrated physically. The quantitative analyses of the autoradiograms demonstrated that part of the labelled phosphatidylcholine was taken up by the isolated nuclei and was transferred inside the nucleoplasm. The uptake corresponded to the loss of the vesicular arrangement, probably owing to the formation of a lipoprotein complex with the nuclear proteins. The results provide evidence that the lipid-induced changes of transcriptional activity occur upon the actual interaction of the exogenous phospholipid with the inner nuclear components.

Changes of chromatin organization induced by phospholipids.

Isolated nuclei represent a suitable model for studying the influence of exogenous phospholipids, normally found as minor chromatin components, on the nuclear structure, which, in turn, could be related to the observed modifications of DNA and RNA synthesis. The morphological modifications induced on chromatin RNP granules and nuclear matrix have been analyzed both with conventional thin sectioning and with an original method based on image analysis of freeze-fractured and replicated nuclear samples. The results obtained support the hypothesis that anionic phospholipids, by removing histone H1, induce a transition of the chromatin from solenoid to nucleosome conformation and favour the RNA polymerizing activity which results in an increased release of RNP particles, while neutral phospholipids, probably affecting the matrix structure, partly impare the RNP maturation and transport, with consequent increase of chromatin condensation.

Electron microscopy microsampling of isolated nuclei sorted by flow cytometry.

This paper describes an efficient method to concentrate for electron microscopic examination minute quantities of subcellular particles obtained by cytofluorimetric sorting. The advantages of this micromethod, based on diafiltration on Millipore filters under constant positive nitrogen pressure, are discussed.

Changes in ribonucleoprotein particle and chromatin organization induced by liposomes in isolated nuclei.

Nuclei isolated from rat liver, incubated in the presence of liposomes of different phospholipids, undergo typical modifications: chromatin dispersion and reduction of the interchromatin granules in nuclei incubated with negatively charged liposomes and increase of the chromatin density and of the number and size of the interchromatin granules in nuclei incubated with neutral liposomes. The possibility that the observed modifications are caused by an impairment of the transport and translocation of ribonucleoproteins belonging to the inner nuclear matrix, is suggested by the results obtained by radiotracer techniques on the release of RNA from liposome-incubated nuclei.

Chromatin fibers organization within the nucleus.

In order to evaluate at the ultrastructural level the three dimensional arrangement of the dispersed chromatin during the intephase, the immunogold detection of Bromodeoxyuridine (BrdU), of the DNA polymerase alpha and of the proliferating cell nuclear antigen (PCNA) was performed on human HL60 leukemia cells and nuclear matrices extracted from the same cellular model. The Field Emission In lens Scanning Electron Microscopy analysis of the ultrathin cryosectioned cells revealed the presence of a chromatin three dimensional network where the different constituents appeared repetitively assembled. Also the nuclear matrix showed a repetitive structure, on which the deprivation of the DNA corresponded to the selective loss of particular class sized fibers. The single or multiple combined immunolocalization of different structures involved in the DNA replication, where BrdU, DNA polymerase alpha and PCNA represent, respectively, the substratum, the polymerizing enzyme and a regulator of the reaction, allowed the understanding of its reciprocal spatial relationship on the dispersed interphasic chromatin and the role of the nuclear matrix in the replicative process.

Chromatin organization in isolated nuclei: flow cytometric characterization employing forward and perpendicular light scatter.

Flow cytometric perpendicular and forward light scatters have been employed to evaluate whether the changes in chromatin organization due to ionic strength, Mg++ concentration and pH, visible in electron microscopy, can be monitored by flow cytometry. The average intensity of the perpendicular light scatter signal increased as nuclear chromatin became decondensed by lowering the ionic strength or releasing H1 histone at low pH values. These results indicate that flow cytometry signals and in particular the perpendicular light scatter allow the detection of the conformational transitions in chromatin and may therefore be useful for studying cell cycle associated morphological changes in isolated nuclei.

Cytochemical localization of DNA loop attachment sites to the nuclear lamina and to the inner nuclear matrix.

The rat liver nuclear matrix, obtained by endogenous nuclease digestion and extraction with low and high ionic strength media, contains residual DNA fragments that are considered to represent the attachment sites of the chromatin domains to the nucleoskeleton. These sites, protected against nuclease digestion by their binding with the nucleoskeleton proteins, should be either mainly linked to the peripheral lamina or to the inner nuclear matrix. The DNA fragment distribution at the level of the different components of the nuclear matrix has been evaluated in samples embedded in Epon and in hydrophilic resins by means of the DNase-gold technique. The labeling obtained suggests that the chromatin loops are prevailingly associated with the interior of the matrix; in fact about twice of the label is present in the inner matrix with respect to the peripheral lamina area. These results confirm the hypothesis that in interphase the chromatin maintains an organization similar to that of chromosomes, with loops radiating from a central scaffold, instead of being mainly attached to the lamina as otherwise suggested.

Improved bromodeoxyuridine/DNA analysis by anti-BudR monoclonal antibody versus right angle light scatter.

Dynamic cell cycle analysis is based on the incorporation of labelled precursors into DNA. Although antibodies to BrdU are very useful for analysing in flow cells which synthesize DNA, this approach has two main limitations. First, the detection of low incorporating cells is often difficult; second, four parameter flow cytometry is not able to correlate cell cycle to any other cellular marker. We have developed a methodology that, employing an IgGH + L as a second antibody and side scatter instead of propidium iodide fluorescence, allows a better discrimination of BudR+ cells. This approach allows the collection of an extra-fluorescent signal, and the analysis of specific cellular markers within the cell cycle.

The impairment of natural killer function in the healthy aged is due to a postbinding deficient mechanism.

In order to study the fine mechanisms that underlie the impairment of non-MHC-restricted cytolytic activity which occurs during human aging, we examined by multiparametric flow cytometry the binding and lytic activities of human natural killer cells. The flow analysis revealed a striking increase of the CD16+8- subset, together with a significant decrease of CD8bright cells and total T cells (CD3+). Aging had no influence on the CD8dim subset. The total lytic activity expressed by PBL as well as their binding efficiency to K562 targets were moderately but not significantly increased in the elderly. In contrast, the cytotoxicity of the single target-bound natural killer cell (i.e., lytic efficiency) was deeply impaired in aged subjects, suggesting that the NK functional impairment observed in aging is located at postbinding level.

Proliferative response of human marginal gingiva to phlogistic process and titanium implant.

In order to detect proliferative processes in human marginal gingiva in pathological conditions and after externalization of titanium implants, we have attempted BrdU incorporation after "in vitro" incubation of tissue fragments. In comparison with healthy controls, immunocytochemical detection of samples from patients affected by hypertrophic gengivitis shows a good number of proliferating cells in the basal layer of the epithelium, while only in one case can positiveness be detected after externalization of titanium implants. Since after reduction of inflammation by hygienic treatment a low number of proliferating cells can be observed only in the regions where pathological alterations are also present, we suggest that the increase in tissue proliferation may be closely dependent on the intensity of the inflammatory process. All these data demonstrate that in vitro BrdU incubation of tissue fragments represents a suitable method to evaluate cell proliferation in human tissue.

[Characterization of the convoluted seminiferous tubule wall. II. Ultrastructural alteration after testis retention in the abdominal cavity]. / Caratterizzazione della parete del tubulo seminifero contorto. II) Modificazioni ultrastrutturali dopo ritenzione del testicolo in cavita' addominale.

An ultrastructural analysis has been carried out on the seminiferous tubules after different periods of stay of the rat testis in the abdominal cavity. With respect to control rats, no changes are detectable after 5 days, while after 15 and 30 days considerable modifications take place. A number of cellular features are modified, such as the nuclear shape and the perinuclear vesicles, while the cytoplasmic filaments show a less ordered appearance. In addition, the extracellular amorphous matrix undergoes a large increase, due either to an enhanced production or to a reduced turnover related with a lowered cell activity.

[Characterization of the convoluted seminiferous tubule wall. I. Ultrastructural aspects after the ligation of the vas deferens and the efferent tubules]. / Caratterizzazione della parete del tubulo seminifero contorto. I) Aspetti ultrastrutturali dopo legatura del condotto deferente e dei tubuli efferenti.

The structure of the wall of the rat seminiferous tubules has been studied by electron microscopy. No significant modifications have been revealed after the ligature of the vas deferens while on the contrary the ligature of the efferent ductules produces a reduction of the contractile filaments and of the pinocytosis vesicles, as well as an increase of the amount of collagen fibers in the basal membrane.