Morphologic changes in the pineal gland of rats exposed to continuous darkness

We examined the pineal structure of rats exposed to constant darkness (DD) at light microscopic level. Two groups of rats were exposed to 12:12 light/dark cycle (LD) or DD from their prenatal ontogenesis and then for 3 months after birth. The gland structure of DD rats was observed to have an active appearance. Some of the observed pinealocytes with light nuclei from DD rats were determined to contain double nucleoli. Nuclear area and perimeter of both dark and light types were greater in rats kept in DD than in LD. Rats exposed to DD had more cells with light nuclei and lesser cells with dark ones than rats kept in LD. No significant differences in nuclear characteristics of intermediate type were found between rats kept in LD and those kept in DD. The activity of mammalian pineal can be altered by light conditions to which the animal is exposed.     

Quantitative DNS-Bestimmungen von Ameisenhirnzellen

Brains of workers and males of the ant species Lasius fuliginosus were Feulgen stained. The amount of Feulgen dye of diploid nuclei of the “Perikaryenschicht” were measured microspectrophotometrically. When the slides were kept in the dark at 4° C the colour intensity did not decrease during half a year. Hydrolysis in Schiffs reagent with 0,09 N HCl did not influence the colour significantly. When the same method was used the results were more or less reproducible; 6 of 8 series gave identical values. — Feulgen dye and nuclear volume of diploid cells of ants from a natural habitat decrease from spring to autumn by ca 30 % and 10% respectively. — Ants which were kept in the laboratory for nearly two years did not show such a regular change in the Feulgen dye in their brain cells. These nuclei never reached the colour intensity of those ants from a natural habitat. — No difference in the Feulgen intensity has been found between ants kept in 4° C and at room temperature, respectively. The staining maximum was reached after 1 h by animals from the natural habitat, after 2 h by those from the laboratory. As expected the amount of Feulgen dye of haploid male cells was half of that of diploid worker cells, but the nuclear volume of haploid male cells was larger than half of that of worker cells (Zusammenfassung see p. 93).     

123 Permanent Fluorescent Nuclear Staining of Binucleate Dinoflagellates

Typically, fluorescent microscopy of dinoflagellate nuclei is of poor resolution, due mainly to visual obstruction of the nuclei by plastids, pigment granules, and thecal plates. Moreover, the usual slide mounts using buffered glycerol are temporary, and fade after a week or so. We have developed a procedure to clear pigments from dinoflagellates, followed by fluorescent staining of the nuclei. The cells are then prepared as permanent mounts using an ultraviolet light-catalyzed resin to produce stained samples which may be kept for at least three years with little loss of fluorescence. This procedure can also be used to prepare plastic embedded dinoflagellate cells which can then be sectioned at 1–2 nm, fluorescent stained, and permanently mounted. Suitable nuclear stains are DAPI, Hoechst 33258, ethidium bromide and acridine orange. The dinoflagellate (dinokaryotic), and endosymbiont (eukaryotic) nuclei are clearly visualized, revealing individual chromosomes in the dinoflagellate nucleus, and a highly lobed morphology of the endosymbiont nucleus.     

Preliminary study on the effect of temperature on the transport of thyroxine (T4) into the body tissues and sub-cellular fractions of liver and muscle of carp, Cyprinus carpio

In order to determine if the inability of thyroxine to induce cellular effects at low temperature is mediated through a temperature-sensitive system for the translocation of T4 into the nucleus, the effect of temperature on the uptake of T4 by body tissues and sub-cellular fractions of carp liver and muscle was studied in vivo. A single injection of 125I-T4 (1 micro C/10 g body weight) was given intraperitoneally to juvenile carp maintained at 15 and 25 C. Uptake from the peritoneal cavity was rapid. All the tissues exhibited maximum radioactivity at 2 hour after the injection. Fish kept at 25 C showed another peak at 8 hour and those at 15 C at 48 hour after the single injection. Transfer of T4 from the cytoplasm to nuclei was not blocked at lower temperatures. For example, in liver at 8 hour, nuclei from fish tissues kept at lower or higher temperatures had equal amounts of radioactivity. Muscle nuclei had 15% more radioactivity than liver nuclei when expressed as radioactivity/g tissue. Since there are comparable amounts of activity in the nuclei at both temperatures, some other mechanism/s than a simple block in transport from cytoplasm to nuclei is operating. There are some indications that nutritional status of fish may be playing some role in this respect.     

Circadiane änderungen im endokrinen system der Honigbiene, Apis mellifera, effekt von haft und Ocellenblendung

By PAF-staining it can be shown how the amount of neurosecretion in the pars intercerebralis (PI) and in the corpora cardiaca (CC) changes during the course of the day. Simultaneously, the density of nuclei in the corpora allata (CA) was measured. The secretion in the PI and CC is minimal at midday. With this the relatively small changes in the density of nuclei in the CA are negatively correlated. When normal animals are kept in a daylight cage, the content of secretion in the PI and CC does not decrease during the morning as it does in free flying foragers. It one places bees with black varnished ocelli in this cage, the amount of secretion increases very much, and some animals show an extremely high density of nuclei in the CA. With the help of a simple model, the results are discussed as an ocellary controlling influence on humoral regulation of motor activity.     

A selective block of nuclear actin export stabilizes the giant nuclei of Xenopus oocytes

Actin is a major cytoskeletal element and is normally kept cytoplasmic by exportin 6 (Exp6)-driven nuclear export. Here, we show that Exp6 recognizes actin features that are conserved from yeast to human. Surprisingly however, microinjected actin was not exported from Xenopus laevis oocyte nuclei, unless Exp6 was co-injected, indicating that the pathway is inactive in this cell type. Indeed, Exp6 is undetectable in oocytes, but is synthesized from meiotic maturation onwards, which explains how actin export resumes later in embryogenesis. Exp6 thus represents the first example of a strictly developmentally regulated nuclear transport pathway. We asked why Xenopus oocytes lack Exp6 and observed that ectopic application of Exp6 renders the giant oocyte nuclei extremely fragile. This effect correlates with the selective disappearance of a sponge-like intranuclear scaffold of F-actin. These nuclei have a normal G2-phase DNA content in a volume 100,000 times larger than nuclei of somatic cells. Apparently, their mechanical integrity cannot be maintained by chromatin and the associated nuclear matrix, but instead requires an intranuclear actin-scaffold.     

Culture of Pollen Tubes for Chromosomal Analysis at the Pollen Tube Division

It is possible to grow pollen tubes routinely for cytological analysis of nuclei at the pollen tube division. Pollen has been grown successfully after temperature, pressure, gas, moisture, and radiation treatments. The technic for growing Tradescantia pollen is described, but any method is satisfactory which ensures that: (a) the pollen is kept dry before sowing, (b) a minimum of time elapses between sowing pollen on culture medium and placing in a moist growing box, and (c) the growing pollen tubes are not allowed to dry out. Pollen of other species can be grown by the same methods.     

Diurnal rhythmicity of nuclear and cytoplasmic polyadenylylated ribonucleic acids and or polyadenylate-dependent polyadenylate polymerase in rat liver

The hepatic concentration of polyadenylylated RNA was measured in rats kept under LD 12:12. In the nuclei, the concentration was maximal during the dark phase, whereas in the cytoplasm, the highest values were measured during the light phase. The activity of nuclear polyadenylate-dependent polyadenylate polymerase showed a rhythm of low amplitude with a maximum at light-off.     

Automatic segmentation of cell nuclei in Feulgen-stained histological sections of prostate cancer and quantitative evaluation of segmentation results

Digital image analysis of cell nuclei is useful to obtain quantitative information for the diagnosis and prognosis of cancer. However, the lack of a reliable automatic nuclear segmentation is a limiting factor for high-throughput nuclear image analysis. We have developed a method for automatic segmentation of nuclei in Feulgen-stained histological sections of prostate cancer. A local adaptive thresholding with an object perimeter gradient verification step detected the nuclei and was combined with an active contour model that featured an optimized initialization and worked within a restricted region to improve convergence of the segmentation of each nucleus. The method was tested on 30 randomly selected image frames from three cases, comparing the results from the automatic algorithm to a manual delineation of 924 nuclei. The automatic method segmented a few more nuclei compared to the manual method, and about 73% of the manually segmented nuclei were also segmented by the automatic method. For each nucleus segmented both manually and automatically, the accuracy (i.e., agreement with manual delineation) was estimated. The mean segmentation sensitivity/specificity were 95%/96%. The results from the automatic method were not significantly different from the ground truth provided by manual segmentation. This opens the possibility for large-scale nuclear analysis based on automatic segmentation of nuclei in Feulgen-stained histological sections.     

A Novel Dictionary Based Computer Vision Method for the Detection of Cell Nuclei

Cell nuclei detection in fluorescent microscopic images is an important and time consuming task in a wide range of biological applications. Blur, clutter, bleed through and partial occlusion of nuclei make individual nuclei detection a challenging task for automated image analysis. This paper proposes a novel and robust detection method based on the active contour framework. Improvement over conventional approaches is achieved by exploiting prior knowledge of the nucleus shape in order to better detect individual nuclei. This prior knowledge is defined using a dictionary based approach which can be formulated as the optimization of a convex energy function. The proposed method shows accurate detection results for dense clusters of nuclei, for example, an F-measure (a measure for detection accuracy) of 0.96 for the detection of cell nuclei in peripheral blood mononuclear cells, compared to an F-measure of 0.90 achieved by state-of-the-art nuclei detection methods.     

Annulate lamellae in hamster pineal gland

Pineal glands from young adult hamsters (Mesocricetus auratus Water-house) kept on a 14-10 light-dark photoperiod contain annulate lamellae. These annulate lamellae are apparently limited to the light pinealocytes. They are found in close proximity to nuclei, the Golgi apparatus, and agranular endoplasmic reticulum. There is also an interesting association or microtubules with the annulate lamellae. It seems reasonable that the presence of annulate lainellae in the pinealocytes may have some correlation with the physiological function or functions of these cells.     

ChIC and ChEC; genomic mapping of chromatin proteins

To map the genomic interaction sites of chromatin proteins, two related methods were developed and experimentally explored in Saccharomyces cerevisiae. The ChIC method (chromatin immunocleavage) consists of tethering a fusion protein (pA-MN) consisting of micrococcal nuclease (MN) and staphylococcal protein A to specifically bound antibodies. The nuclease is kept inactive during the tethering process (no Ca2+). The ChEC method (chromatin endogenous cleavage) consists of expressing fusion proteins in vivo, where MN is C-terminally fused to the proteins of interest. The specifically tethered nucleases are activated with Ca2+ ions to locally introduce double-stranded DNA breaks. We demonstrate that ChIC and ChEC map proteins with a 100-200 bp resolution and excellent specificity. One version of the method is applicable to formaldehyde-fixed nuclei, another to native cells with comparable results. Among various model experiments, these methods were used to address the conformation of yeast telomeres.     

Behavior of HEp-2 cell line cells, their nuclei and nucleoli during cultivation and under the effect of Na-ds-RNA

The behavior of human larynx cancer cells (HEp-2) and of their nuclei and nucleoli during the cultivation without the influence of Na-ds-RNA and after its introduction into the medium was investigated by methods of cytomorphometry and cytophotometry. The density of monolayer (the number of cells on the area unit), percentage of two-nuclear cells, the number of nucleoli in the nuclei, mitotic coefficient, volume and total surface of nuclei and nucleoli have been measured. In addition, the mass of DNA in the nuclei and that of the total RNA and DNA in the nuclei and in each nucleolus was measured. Cells in the culture, not subjected to the influence of Na-ds-RNA, were weakly differentiated, kept active proliferation, and their population contained a small number of two-nuclear elements and a high share of multi-nuclear cell. During cultivation, these indices became even more pronounced, which is typical for the increase in cell malignancy. Under the influence of Na-ds-RNA, the proliferate activity decreases, the number of double-nuclear cells increases, while that of multi-nucleolar cell decreases; also, the share of cells with one- and two-nucleolar nuclei increases. The authors conclude that Na-ds-RNA may have antineoplastic activities, clearly evidenced from its influence on the culture of transformed HEp-2 cells.     

An efficient procedure for isolation of nuclei from plant protoplasts

Summary The present communication describes an easy, efficient and rapid method for isolation of nuclei from plant protoplasts. Release of nuclei is accomplished by disruption of protoplasts in an appropriate buffer containing a very low concentration (0.01%) of the detergent Triton X-100. The pH of the nuclei isolation buffer (5.3) played a critical role in the recovery of stable nuclei in large numbers. Supplementation of buffer (10 mM MES) with spermine (0.1 mM), dithiothreitol (2.5 mM), ethylenediaminetetraacetic acid (2.5 mM) and Nad and KCl (10 mM each) improved nuclear yield and quality. With the method developed it is possible to routinely recover 95% nuclei from the protoplasts within 30 minutes. The nuclear preparations are of high purity with little detectable cytoplasmic contamination and no clumping of the nuclei. The structural integrity of the nuclei has been assessed and confirmed by Nomarski differential interference contrast optics and ultrastructural observations.     

The polymerization of actin. A study of the nucleation reaction.

We compared the properties of the nuclei that accumulate in 7.5 mM-KCl in ATP-G-actin solutions and of the oligomers that are formed by sonication of either G-actin or F-actin. We found that the ability of the above species to prime the polymerization of actin decays with different rates. The nuclei are stable in 7.5 mM-KCl (they decay with a rate constant of 1.5 X 10(-3) s -1 at pH 7.8 at 22 degrees C in the absence of KCl). The oligomers formed by sonication of either G-actin or F-actin, once the sonication is stopped, revert to simpler structures or evolve into F-actin, depending on the KCl concentration in which they are kept. In 10.5 mM-KCl at pH 7.8 at 22 degrees C their priming ability decays with a rate constant of 6 X 10(-3) s -1. We propose that the nuclei that form spontaneously in 7.5 mM-KCl are not directly susceptible to elongation. They must first be converted into activated nuclei, which exist in very low concentration at the steady state. The activated nuclei are directly susceptible to elongation, they have a short life and they decay rapidly into the ground state unless the elongation reaction occurs. Sonication displaces the steady-state concentration in favour of the activated state.     

Comparative studies on sex chromosomes in different species

InLocusta migratoria (XO),Mus musculus, Rattus norvegicus, Mesocricetus auratus, Cricetulus griseus andHomo sapiens typical sex vesicle structures are visible in early meiotic prophase stages up to pachynema. The structures include whole sex chromosomes or parts thereof. The heterologous parts and the solitary X chromosome ofLocusta pass diplonema, diakinesis and first metaphase nearly in mitotic shape. Entirely heterologous sex chromosomes are kept together by a unilateral and achiasmatic end connection. The sex vesicle is interpreted as a special structure of allocyclic sex chromosomes or parts of them, corresponding in early meiotic stages to the chromocenters of mitotic interphase nuclei. The formation of the sex vesicle is independent of the orthoploidy of nuclei and of the DNA ratio between autosomes and sex chromosomes. Heteropycnotic behaviour of sex chromosomes in spermatids is interpreted as a condition capable of blocking genetic activity, like in the Barr bodies of female somatic nuclei, giving equal chances of fertilization to both types of gametes.Based on a paper read at the XI International Congress of Genetics, of which an abstract has appeared in the congress proceedings, Genetics Today, Vol. 1, p. 299 (1963).     

The protein composition of Friend cell nuclear matrix stabilized by various treatments. Different recovery of nucleolar proteins B23 and C23 and nuclear lamins

Summary— Using two-dimensional polyacrylamide gels stained with Coomassie blue we have studied the protein composition of the nuclear matrix obtained from mouse erythroleukemic nuclei kept at O°C throughout the isolation procedure to prepare the high ionic strength resistant fraction (control matrix) or stabilized in vitro or in vivo by different procedures prior to subfractionation (ie 37°C incubation of isolated nuclei; sodium tetrathionate exposure of purified nuclei; heat shock of intact cells). When the matrix obtained from 37°C incubated nuclei was compared with the control matrix, striking differences in the polypeptide pattern were seen if the protein was obtained in both cases from an equivalent number of nuclei. On the other hand, if the same amount of protein for both the samples was applied to the gels the differences were less evident. Sodium tetrathionate stabilization of isolated nuclei and heat shock of intact cells produced a matrix protein pattern that was very similar and differed from that of the in vitro heat-exposed matrix. Using specific polyclonal antisera, we demonstrate that nucleolar proteins B23/numatrin and C23/nucleolin were very abundant in the matrix obtained from chemically-treated nuclei or in vivo heat-stabilized nuclei but were recovered in very small amounts (B23) or completely absent (C23) in the matrix prepared from nuclei heated to 37°C in vitro. Differences were seen also in the recovery of nuclear lamins, and especially lamin B, that was poorly represented in the sodium tetrathionate-stabilized matrix. The results demonstrate that in mouse erythroleukemia cells the increased recovery of nuclear matrix protein that is seen after in vitro heating of isolated nuclei is predominantly due to an additional recovery of the same types of polypeptides that are detected also in the absence of such a treatment. The data also indicate that in vivo heat shock of intact cells produces a nuclear matrix protein pattern that is more similar to the pattern seen after stabilization of purified nuclei with sodium tetrathionate and differs significantly from that obtained by exposing nuclei to 37°C in vitro, unlike to that what previous reports have indicated.     

Immunocytochemical localization of bovine serum albumin (BSA) in the liver and testis of rats injected with testosterone-BSA, hydrocortisone-BSA or corticosterone-BSA

Through observations of colloidal gold with silver enhancement, we have demonstrated that 2-nm colloidal gold labeled-testosterone-bovine serum albumin (BSA) conjugate or hydrocortisone-BSA conjugate injected intravenously enters the hormone-target cell nuclei of rats (Nishimura and Ichihara, 1997; Nishimura and Nakano, 1997, 1999). To confirm immunocytochemically whether the nature of BSA in the steroid hormone-BSA conjugates (steroid-BSAs) remains intact in the hormone-target cell nuclei, testosterone-BSA, hydrocortisone-BSA or corticosterone-BSA was injected into the vascular system of rats, then the liver and testes of rats killed 2 h postinjection were reacted with FITC-conjugated anti-BSA antibody, and examined under fluorescence microscopy and confocal laser scanning microscopy. In the liver of rat injected with testosterone-BSA, the fluorescence was observed in the nuclei of endothelial cells, but not in the nuclei of hepatocytes, hepatic stellate cells and Kupffer cells. In the liver of rat injected with hydrocortisone-BSA, intense fluorescence was seen in the nuclei of hepatic stellate cells, but did not seem to be present in the nuclei of the other three kinds of cells. In the liver of rat injected with corticosterone-BSA, the fluorescence seemed to be in a few nuclei of hepatic stellate cells, and appeared as speckles in a few nuclei of the hepatocytes and Kupffer cells. In some seminiferous tubules of rat injected with testosterone-BSA, fluorescence was observed in the nuclei of spermatocytes and spermatids. These results suggest that BSA conjugated with steroid hormone can enter the hormone-target cell nuclei with its antigenicity kept intact, and that the fate of steroid-BSAs is decided at the cell membrane level.     

Possible role of SV40 T antigen in cell transformation]

The effect of purified SV40 T antigen on DNA synthesis in isolated nuclei from the confluent culture of CV-1 cells was studied. In the presence of T antigen the incorporation of [3H]TTP into DNA was found to be 2 to 3 times as high as in the control nuclei. The resulting labelled DNA was subjected to alkaline sucrose gradient centrifugation, which revealed the presence of 4S DNA species, corresponding to Okazaki fragments of animal cells. The latter finding suggests a replicative mode of DNA synthesis induced by T antigen. T antigen isolated from the cells infected with SV40 tsA-mutant and kept at a nonpermissive (41 degrees) temperature fails to stimulate DNA synthesis in isolated nuclei from resting cells. On storage at 4 degrees SV40 T antigen gradually loses its ability to stimulate DNA synthesis and by the 8th day even suppresses it when tested on isolated nuclei from a growing cell culture. No effect of T antigen on the endonuclease-induced reparative synthesis of DNA could be observed. The data described suggest that T antigen is directly involved in the control of DNA synthesis in the cells infected or transformed with SV40.     

Novel features of hamster pinealocyte ultrastructure

Pineal glands from young adult hamsters (Mesocricetus auratus Water-house) kept on a 12 : 12 light : dark photoperiod were examined by electron microscopy. Ultrastructural features of nuclei, mitochondria, and centrioles that have not been reported previously are presented in this paper. Of particular interest is the presence of modified centrioles in normal hamster pinealocytes.