Probing DNA structure and function with a multiwavelength fluorescence confocal laser microscope

Three levels of organization in DNA structure in the interphase cell nucleus are assessed by confocal laser scanning microscopy: (i) the conformational state of the double helix; (ii) the distribution of eu- and heterochromatin; and (iii) the localization of replication complexes throughout S phase. Multi-parameter measurements were carried out in each optical section using two laser sources and combined stereoscopic reconstructions were used to assess the co-localization of nuclear components. DNA is highly polymorphic and can adopt a variety of different helical conformations as well as unusual structures (curved, cruciform, multi-stranded). We have assessed by laser scanning microscopy the presence of left-handed Z-DNA in polytene chromosomes of Diptera as well as the spatio-temporal distribution of Z-DNA binding proteins in whole-mount Drosophila embryos and ovaries. We have determined the 3-D distribution of replication sites relative to heterochromatin regions, nucleoli and nuclear membrane by using short pulses of BrdU incorporation in synchronized mouse and human fibroblasts. Replication sites were visualized with a monoclonal anti-BrdU antibody combined with DNA fluorescent staining and antibody labelling of nuclear lamin. The implications of dynamic DNA movement and structural rearrangement to the organization of the nucleus in domains are discussed.     

Histochemical and ultrahistochemical localization of heavy metals in calf organs

Cadmium, zinc, selenium, and copper were administered, singly or in combination, orally or subcutaneously. Experiment I included 32 calves of both sexes; six received Cd (two groups), Zn, Cd, and Zn, and Cd and Se (two groups) and one group was a control. In Experiment II (21 bulls), three were given Cd, Cd, and Cu, and Cd and Zn, respectively, and one group was a control. For light microscopy, in Experiment I the highest amounts of silver granules were present in the samples of liver, small intestine, and vesicular gland of all the exposed groups; in Experiment II the most affected organs were liver, kidney, and small intestine. For electron microscopy, in Experiment I, after administration of Cd and Zn, the highest amounts of granules were seen in the cytoplasm of hepatocytes and cells of the proximal and distal renal tubules and the lowest amounts were found in glandular cells of the pancreas. Administration of Cd and Se resulted in the presence of large numbers of granules in the nuclei and nucleoli of spermatogonies. In Experiment II, ingestion of Cd and Zn in feed led to the appearance of highest amounts of granules in the nucleoli, nuclei, and cytoplasm of cells in testes, kidneys, and pancreas. Following Cd intake, the highest accumulation of granules was observed in the nucleoli of hepatocytes and cells of the proximal and distal renal tubules. Combined Cd and Cu produced the highest number of granules in cells of the proximal and distal renal tubules and in the nucleoli and nuclei of germinal epithelium.     

Observations on the nucleolar staining by ethanolic phosphotungstic acid.

In addition to the already known reactivity of heterochromatin masses and synaptonemal complexes for ethanolic phosphotungstic acid, nucleoli from Sertoli cells show a preferential electron microscopic staining of the pars fibrosa. This ultrastructural pattern can be correlated with intranucleolar differentiations observed in light microscopy after staining of semithin sections with Unna's polychrome blue.     

Ultrastructural comparison of ion beam and radiofrequency plasma etching effects on biological tissue sections

Three dry etching techniques (Ar+ ion beam, O₂+ ion beam, O₂ radiofrequency electrodeless discharge) were compared with respect to preferential etching and damage to the ultrastructure of glutaraldehyde-fixed Epon-embedded frog skeletal muscle sections. SEM and TEM studies were performed on both unstained and stained (osmium tetroxide, uranyl acetate) sections. Etching effects were observed to differ for the various ion beam or plasma etching techniques. Whereas selective retention of electron dense structures (e.g. Z lines, nuclear heterochromatin) was observed for oxygen plasma etching, preferential etching of these components was observed using O₂+ ion beam bombardment. Selectively etched Z lines and etch-resistant nucleoli were observed for both reactive (O₂+) and inert (Ar+) ion beam sputtering after sufficiently high ion doses. The above suggest that selective etching under keV ion beam irradiation is related more to physical sputtering processes (momentum transfer) than to the chemical reactivity of the incident ion. Heavy metal post-fixation and staining had no qualitative effect on the nature of the selective etching phenomena. The above findings are significant in that they potentially influence both electron and ion microprobe measurements of etched biological specimens.     

Intravital dual-colored visualization of colorectal liver metastasis in living mice using two photon laser scanning microscopy

A major challenge of cancer biology is to visualize the dynamics of the metastatic process in secondary organs at high optical resolution in vivo real-time. Here, we presented intravital, dual-colored imaging of liver metastasis formation from a single cancer cell to metastatic colonies in the living liver of living mice using two photon laser scanning microscopy (TPLSM). Red fluorescent protein expressing murine (SL4) or human (HT29) colorectal cancer cell lines were inoculated to the spleen of green fluorescent protein expressing mice. Intravital TPLSM was performed by exteriorizing and fixing the liver lobe of living mice. This was repeated several times for the long-term imaging of the same mouse. Viable cancer cells in the living liver of living mice were visualized intravitally at a magnification of over 600×. Single cancer cells were arrested within hepatic sinusoids 2 h after injection. Platelet aggregation surrounding a cancer cell was observed, indicating a phenomenon of tumor-cell induced platelet aggregation. Cancer cells were extravasated from hepatic sinusoids to the space of Disse. Protrusions of Kupffer cells surrounding a cancer cell were observed, indicating that Kupffer cells appear to phagocytose cancer cells. SL4 cells formed liver metastatic colonies with extensive stromal reaction. Liver metastases by HT29 cells were observed as a cluster of micrometastatic nodules. High-resolution, dual-colored, real-time visualization of cancer metastasis using intravital TLPSM can help to understand spatiotemporal tumor-host interactions during metastatic processes in the living organs of living animals.     

Cytotoxic effects of permethrin on mouse liver and spleen cells

This study analyzed the histopathological and histochemical effects of different dosages of permethrin on liver and spleen cells of mice, in order to evaluate the toxic potential of this substance and the possible impairments that this chemical causes in different tissues of nontarget organisms (laboratorial conditions). The results showed that permethrin caused severe alterations in the liver cells, reducing the size of the nuclei and causing hydropic degeneration of the hepatocytes, in addition to stimulating the proliferation of Kupffer cells, altered the amount of proteins, polysaccharides, lipids, and vacuoles in the cytoplasm of the hepatocytes and congested the hepatic capillaries. As for the spleen of the treated mice, no alterations were observed in the morphology in relation to the control group, what would suggest that the spleen would continue performing its functions, without suffering morphological alterations even in the presence of the toxic agent.     

Effects of metals cadmium and chromium alone and in combination on the liver and kidney tissue of male Spraque‐Dawley rats: An ultrastructural and electron‐energy‐loss spectroscopy investigation

Heavy metal pollution has increased in the last decades. Water sources are contaminated and human exposure is often long term exposure to variable amounts of different metals. In this study, male Sprague‐Dawley rats were exposed via oral gavage for 28 days to cadmium (Cd) and chromium (Cr), alone and in combination at concentrations 1000 times the human World Health Organization's acceptable water limits. Rat equivalent dosages were used. Blood markers of liver and kidney function were measured, changes to cellular morphology was determined with transmission electron microscopy and the intracellular metal localisation was determined with the electron energy‐loss spectroscopy and energy filtered transmission electron microscopy analysis. Both Cd and Cr caused changes to the nuclear and mitochondrial membranes and irregular chromatin condensation of hepatocytes. Cr exposure caused dilation of the rough endoplasmic reticulum (rER). The combination caused nuclear and mitochondrial membrane damage as well as irregular chromatin condensation. In the kidney tissue, Cd caused irregular chromatin condensation in the cells of the proximal convoluted tubule (PCT). Cr caused changes to the outer nuclear and mitochondrial membrane and chromatin structure. The combination group caused membrane damage, irregular chromatin condensation and rER changes in the PCT. All the metal groups showed damage to the endothelial cells and pedicles, but not to the mesangial cells. Cd and Cr bio‐accumulation was observed in the nucleus, mitochondria and rER of the liver and kidney and therefore are responsible for the cellular observed damage that can cause functional changes to the tissues and organs.     

Characterization of ovarian nuclei with the parameter of power ratio

The nucleoli and chromatin clumps of ovarian cells contain important features in discriminating malignant cells from normal ones. In geometric properties, the ovarian nucleoli and chromatin clumps appear as irregularly shaped dark spots in the nuclear images from specimens immunohistochemically stained with antibody to Mib-1. Malignant cells often have more active and larger nucleoli and chromatin clumps. However, estimating the size of the nucleoli or chromatin clumps is a difficult task since it is not easy to recognize and accurately separate the regions of nucleoli and chromatin clumps from the rest of the nuclei that are highly irregular and variant in contents and intensities. In this paper, we develop a method to derive a parameter called power ratio that is proportionally related to the size of nucleoli and chromatin clumps based on an ideal nuclear model without the region segmentation of nucleoli or chromatin clumps. Results of characterization of the parameter and comparison between malignant and normal cells are provided.     

Determination of the number of cells with multiple nucleoli in histological sections (author's transl)]

A method has been described for correction of a bias, when cells with more than one nucleolus are counted by counting their nucleoli. The true numbers of cells and nucleoli per cell can be estimated by a system of linear equations, with coefficients depending on the thickness of the histological specimen, the average nuclear diameter and the probabilities of nuclear slices, which one gets by cutting a cell nucleus with a certain number of nucleoli. The presuppositions for this method have been discussed with respect to different practical situations. Uncorrected and corrected numbers of motoneurons in the nucl. n. oculomotorii of Tupaia belangeri have been compared.     

Intestinal and liver changes after chronic ketamine and ketamine plus alcohol treatment

The effects of long‐term chronic ketamine treatment on the intestine and the liver were studied in the ICR mice which had daily intraperitoneal injection of ketamine at 30mg/kg per day for 7 months. The intestine showed no significant pathology after treatment but had a decrease of the positive sites of proliferative cell nuclear antigen in the mucosae of the intestines after ketamine and ketamine plus alcohol (added in the last month) treatment. No significant apoptosis (via TUNEL) nor necrosis (via lactic acid dehydrogenase) was detected in the intestines of all control and ketamine‐treated groups, with the exception of an increase of lactic acid dehydrogenase in the mucosae of the intestines of the ketamine plus alcohol group. In the liver, loss of glycogen was observed in animals after ketamine and ketamine plus alcohol treatment, in addition to the pathology reported in a previous work. The decrease in quantity of glycogen in the liver reflected either a failure of glycogen synthesis from glucose or an increase of glycogenolysis in the liver. Microsc. Res. Tech. 75:1170–1175, 2012. © 2012 Wiley Periodicals, Inc.     

Intranuclear paracrystals observed in striated muscle specific LIM protein-deficient mouse cardiomyocytes

A paracrystalline structure was observed within left ventricular cardiomyocyte nuclei of MLP(-/-) mice. The paracrystal possessed cross lines, approximately 8.0 micro m long and 0.3 micro m wide, with a slender spindle shape and a periodicity of 13 nm. Paracrystals were best observed along the longitudinal orientation of myofibrils and were detected in less than 10% of the nuclei observed. One dimension of the protein unit forming the paracrystal was 8.5 nm long. The electron density of the paracrystal appeared to be slightly higher than that of heterochromatin, suggesting that RNA-associated proteins are constituents of the paracrystal. This is the first report of intranuclear paracrystals in cardiomyocytes, which appear to be unique to MLP(-/-) mice.     

Biology of transforming growth factor beta in hepatocarcinogenesis

TGF-beta is an important factor in the regulation of liver growth. It is an inhibitor of hepatocyte DNA synthesis and may induce active cell death, e.g., to remove excessive tissue mass. Studies using transgenic mice suggest that expression in the resting liver has to be well balanced; either under- or overexpression appear to cause an increased turnover of hepatocytes and to predispose to hepatocarcinogenesis. TGF-beta overexpression is frequently observed in human hepatocellular carcinomas, probably as a late event in tumor development. In men and mice, TGF-beta overexpression appears to be associated with loss of TGF-beta responsiveness often by disruption of TGF-beta signaling. However, mechanisms as mutations in TGF-beta receptor II or Smad2 and 4 genes, frequently observed in other human cancers, have only rarely been observed in hepatocellular carcinomas. Further studies may clarify the mechanisms by which hepatocellular tumors escape TGF-beta growth control, as well as analyze possible roles of TGF-beta overexpression in immunosuppression and angiogenesis.     

1, 2 di‐substituted idopyranose from Vitex negundo l. Protects against streptozotocin‐induced diabetes by inhibiting nuclear factor‐kappa B and inducible nitric oxide synthase expression

The aim of this study is to investigate the mechanism of an action of compound isolated from Vitex negundo in streptozotocin‐induced diabetic mice. Light microscopic examination of liver, kidney and pancreatic sections of streptozotocin‐induced diabetic mice showed changes like coarsening of acinar cells of endoplasmic reticulum, destruction of β‐cells, and alteration in their secretory function were observed in the pancreas. Changes like dilation of vein, unusual concentric arrangement of hepatocytes, and liver fibrosis were observed in the liver. Thickening of tubules and expansion of glomerulus were observed in kidneys. All these altered parameters were reversed close to normal condition upon treatment using idopyranose. The results show the antidiabetic potential of idopyranose. Interestingly, liver, kidney, and pancreatic sections of diabetic mice fed with the isolated 1, 2 di‐substituted idopyranose showed regeneration of hepatocytes, nephrocytes, as well as β‐cells and acinar region appeared normal with increased numbers of β‐cells. To understand the probable mechanism of action of 1, 2 di‐substituted idopyranose, we analyzed proinflammatory inducible nitric oxide synthase (iNOS) and nuclear factor‐kappa B (NF‐κB) expression by immunohistochemistry and the results showed an increased iNOS and NF‐κB levels in streptozotocin‐induced diabetic liver, kidney and pancreas. Such high iNOS and NF‐κB levels were inhibited in 1, 2 di‐substituted idopyranose treated mice. The results suggest that 1, 2 di‐substituted idopyranose helps in the protection of hepatocytes, nephrocytes and pancreatic β‐cells probably by its action against NF‐κB and iNOS mediated inflammation in streptozotocin‐induced diabetes. Microsc. Res. Tech., 2011. © 2010 Wiley‐Liss, Inc.     

Pronounced loss of cell nuclei and anisotropic deformation of thick sections

The local deformation and variations in section thickness are studied in 100-μm thick vibratome sections of well-fixed human brain tissue. During processing, including drying on glass slides, the section thickness is reduced to less than half, but close to the edges there is less shrinkage of the section thickness. Close to both surfaces there is a pronounced reduction in the number of neuronal nucleoli. At the scale of the original section, the upper 15 μm and the lower 10 μm are depleted. The loss is most pronounced at the upper surface, which is unprotected during processing. In the central 70% of the section height, where one would ordinarily use an optical disector for sampling, there is no indication of non-uniform shrinkage. The simplest explanation for the observed loss of nucleoli is that all cells opened by the knife may lose their nuclei across an unprotected section surface. The observations do not generalize to other tissues and other preparation techniques, but illustrate the magnitude of some of the problems for uniform sampling and unbiased estimation in very thick sections. The uniform optical disector sampling of nucleoli in thick sections, as opposed to that of cell nuclei, raises a special problem, which is discussed briefly.     

Morphological changes in vero cells postinfection with dengue virus type‐2

Although no specific antiviral tablets or injections that can kill the dengue virus are currently available, adequate care and treatment could control its morbidity. Interaction of dengue virus to target cells could be an important feature for virus propagation. Ultrastructural analysis of this interaction was studied with vero cells. Vero cells were treated with Dengue virus type‐2 at different time intervals at multiplicity of infection (m.o.i) < 10, m.o.i > 10, and m.o.i = 100. It was found that m.o.i < 10 is best to study morphological changes. At an m.o.i > 10 apoptosis occurs and at m.o.i. = 100, cell necrosis occurs. While studying morphological changes, it was found that at 30 min postinfection cells have morphology very similar to that of the control cells although some have irregular outline and show cytoplasmic projections and intense cytoplasmic vacuolization. After 1–12 hours postinfection (h.p.i), the nuclei ran from normal looking to diffuse. Nuclear membrane begins to disintegrate. Some nucleoli are difficult to be seen. The cytoplasm appears as a mottled, lumps diffuse mass distributed throughout the cytosol, with dense lysosomes and myelin figures, also in the mitochondria. In later hours (24 h.p.i), the intranuclear euchromatin is dispersed and heterochromatin forms peripheral clumps. The cytoplasmic processes are short and few in numbers. A proportion of damaged mitochondria with disrupted cristae appear, suggesting that dengue virus may induce mitochondrial dysfunction and nucleus and mitochondria may be the primary organelles helping in dissemination of virus. Microsc. Res. Tech. 2011. © 2010 Wiley‐Liss, Inc.     

Cytotoxicity of fipronil on mice liver cells

In the present study, mice livers were examined following exposure to different doses of fipronil (15, 25, and 50 mg/kg). Histological and histochemical techniques were used to determine the cytotoxic potential of this compound and to assess the damage it caused to livers. Mice were divided into four groups: control group and groups I, II, and III were exposed to 15, 25, and 50 mg/kg fipronil, respectively. Our findings revealed cytological, morphohistological, and histochemical alterations in liver cells of animals from groups I, II, and III compared to group control animals. These changes included Kupffer-cell proliferation, hepatocyte hypertrophy, accumulation and distribution of proteins, polysaccharides, lipids, and vacuoles in the cytoplasm of hepatocytes, and congestion of blood vessels. These phenotypes mainly characterize the following: (a) autophagic processes, (b) steatosis, and (c) cell death by necrosis, which demonstrate the damage caused by fipronil on nontarget organisms in artificial conditions.     

Action of the chemical agent fipronil (active ingredient of acaricide Frontline®) on the liver of mice: an ultrastructural analysis

Fipronil, active ingredient of the acaricide Frontiline®, is a phenyl‐pyrazolic derivative, and its efficacy in the elimination of several plagues, even in low concentrations, has already been demonstrated; however, its effect on nontarget organisms has not been thoroughly explained. In this sense, the objective of this study was to evaluate the effects of different dosages of fipronil on the liver of mice in artificial conditions. Results showed that the animals exposed to fipronil present significant ultrastrucutural changes in hepatic cells with evident cellular and cytoplasm disorganization in hepatocytes characterized by an increase in the number of organelles, mainly mitochondria and rough endoplasmic reticulum, organelles that, in the case of the exposed animals, were probably responsible for the enzymes' synthesis that have the function of inactivating the toxic metabolites. A fat accumulation in the hepatocytes' cytoplasm (steatosis) was observed, in addition to extended vacuolated areas, mainly in regions next to the cell nucleus. Alterations observed in the nuclei of the hepatocytes pointed out cell death processes. Moreover, Kupffer cells increased in number (hyperplasia) suggesting an increase in the phagocytic activity of the liver in the exposed animals. Microsc. Res. Tech., 2011. © 2011 Wiley Periodicals, Inc.     

Ultrastructural and developmental toxicity of potato and tomato leaf extracts to beet armyworm,Spodoptera exigua (lepidoptera: noctuidae)

Beet Armyworm, Spodoptera exigua is a herbivorous moth and a serious pest of many economically important plants, which are used as food sources. Because of rigorous standards of food quality, usage of synthetic insecticides in crop protection, against pests, is limited. Solanaceae plant extracts may be a relatively cheap source of efficient natural insecticides that can limit usage of synthetic substances. Their biological activity is not fully known. In particular, ultrastructural studies, using transmission electron microscopy, are not usual. In the present article we describe the effects of sublethal concentrations of tomato and potato leaf extracts against S. exigua. Acute lethal effects were not observed. Both extracts exerted similar effects within midgut and fat body cells. Midgut cells were not significantly altered while fat body cells showed prominent swelling of nuclear envelope and endoplasmic reticulum, vacuolization of mitochondria and fusion of fat droplets. These changes were much more intensive within groups exposed to potato than tomato extracts at highest concentration at least. Light microscopy was used to observe and document developmental alterations of S. exigua exposed to potato and tomato leaf extracts. Potato leaf extracts significantly decreased hatching success and caused morphological malformations of imagoes. Among them, malformations of wings were the most prominent. Interestingly, these effects were not observed within populations exposed to tomato extracts at highest concentration at least.     

Stevenel's Blue, an excellent stain for optical microscopical study of plastic embedded tissues

Stevenel's Blue is a reliable, rapid, and clean, one-step polychromatic stain for 1 micron thick epoxy sections. The staining solution, originally used by L. Stevenel (1918) to stain human parasites, is made by adding diluted potassium permanganate (2%) to an aqueous solution of the methylene blue (1.3%) and redissolving the precipitate thoroughly, by boiling in water bath and filtering. Staining is carried out in a Coplin jar at 60 degrees C for approximately 10 minutes for tissues embedded in Epon 812 or Poly 812, or 20 minutes for tissues embedded in Spurr's medium. The sections are rinsed, air dried, and mouted in Permount. The staining solution is very stable, and does not tend to form precipitates on the tissue. The stain brings excellent histological differentiation to nuclear, cytoplasmic, and extracellular components. Incorporation of the stain by elements within each tissue varies from intense to light with a subtle gradation of intermediate shades of purple and blue tones. For most cell structures the density of the stain parallels the electron density of that structure as seen under the electron microscope. For example, nucleoli and heterochromatin stain in dark purple while euterochromatin appear in a light blue shade. In all cases, the embedding media remains unstained. The bond between Stevenel's Blue and the tissues is stable, remaining unaltered by the mounting medium. It is also resistant to time-fading.