A cytochemical and radioautographic study of the ultrastructural organization of puff-like fibrillar structures in plant interphase nuclei (Allium porrum)

1979 ◽  
Vol 39 (1) ◽  
pp. 13-27
Author(s):  
J.G. Lafontaine ◽  
B.T. Luck ◽  
D. Dontigny
Keyword(s):  
Phosphotungstic Acid ◽  
Proteinase K ◽  
Ultrastructural Organization ◽  
Allium Porrum ◽  
Interphase Nuclei ◽  
Dna And Rna ◽  
Spherical Structures ◽  
Nuclear Structures ◽  
Additional Support ◽  
Chromosome Segments

Loose, fibrillar, spherical structures have been observed during recent years in interphase nuclei of both animal and plant cells. These nuclear formations have been referred to as karyosomes, fibrillar bodies, micropuffs and centromeres. In order to gain further information on the nature of these structures, a cytochemical and radioautographic investigation was undertaken using plant meristematic cells (Allium porrum). For that purpose roots were fixed with either formaldehyde or glutaraldehyde in order to carry out cytochemical tests for DNA, RNA and proteins. Certain of the preparations were also first digested with DNase, RNase or proteinase K and then stained according to different procedures. Other specimens were labelled with thymidine for high-resolution radioautographic observations. Staining with diaminobenzidine (DAB) revealed that these nuclear puff-like formations consisted partly of a loose fibrillar meshwork containing nucleic acids. Part of this fine fibrillar reticulum persisted whether the preparations were digested with DNase or RNase before staining with DAB, thus indicating that these nuclear structures contained both DNA and RNA. The fact that these formations incorporate thymidine furnished additional support for the view that they correspond to specific chromosome segments. Staining with ethanolic phosphotungstic acid or digestion of specimens with proteinase K showed that these loose fibrillar structures also consisted of proteins. Judging from their ultrastructure, their association with the chromatin reticulum as well as from their cytochemical characteristics, these nuclear formations most likely correspond to centromeres. In view of the presence of DNA within these structures, it is possible to distinguish them from other equally spherical nuclear formations, observed in certain plant species, that have generally been referred to as karyosomes or micronucleoli and that appear to consist of ribonucleoproteins.

An ultracytochemical study of the nucleoplasm in meristematic plant cell (Allium porrum) interphase nuclei

1983 ◽  
Vol 61 (10) ◽  
pp. 2554-2565 ◽  
Author(s):  
B. T. Luck ◽  
J. G. Lafontaine
Keyword(s):  
Plant Cell ◽  
Osmium Tetroxide ◽  
Phosphotungstic Acid ◽  
Large Population ◽  
Allium Porrum ◽  
Structural Elements ◽  
Interphase Nuclei ◽  
En Bloc ◽  
Fibrillar Material ◽  
Ultrastructural Characteristics

In conventionally processed specimens, the nucleoplasm of plant reticulate interphase nuclei consists of a mixture of fibrils and granules of varying sizes. The degree of granularity of the nucleoplasm varies, however, with the fixative used. The finest texture of the nucleoplasm is provided by osmium tetroxide fixation, but judging by its greater transparency, material is undoubtedly extracted. In specimens fixed with glutaraldehyde only evidence of clumping of the nucleoplasm is observed. The nucleoplasm exhibits a finer, less granular texture following postfixation with osmium tetroxide. Although extraction of nucleoplasmic material is noted in samples fixed in formaldehyde, a much improved preservation is obtained when this fixative is used with osmium tetroxide. Following preservation with these two fixatives and staining en bloc with bismuth the ultrastructural characteristics of the nucleoplasm are particularly well revealed. Apart from fine fibrillar material permeating the nucleoplasmic zones, a large population of granules of different sizes is also observed. Part of the material may be shown, using phosphotungstic acid staining, to consist of diffuse chromatin. The fact, however, that the nucleoplasm also contains much RNA-containing structural elements is revealed by the persistence of a coarse interchromatin reticulum after extraction of specimens with deoxyribonuclease and staining with 3,3′-diaminobenzidine.

An Ultrastructural and Radio-Autographic Study of the Evolution of the Interphase Nucleus in Plant Meristematic Cells (Allium Porrum)

1974 ◽  
Vol 14 (2) ◽  
pp. 263-287
Author(s):  
J. G. LAFONTAINE ◽  
A. LORD
Keyword(s):  
Interphase Nucleus ◽  
Structural Changes ◽  
Tritiated Thymidine ◽  
Light And Electron Microscopy ◽  
Allium Porrum ◽  
Meristematic Cells ◽  
Fibrillar Material ◽  
Nuclear Structures ◽  
S Period ◽  
Dense Chromatin

Radioautography under both light and electron microscopy was exploited to investigate the structural changes of the chromatin reticulum which characterizes the interphase nucleus of a number of plants. Allium porrum meristematic plant cells were used for this purpose. In this species, the telophase chromosomes uncoil into dense strands which, during the G1 period, gradually give rise to a coarse reticulum. There then follows an extensive unravelling of portions of these strands, and high-resolution radioautography reveals that labelling with tritiated thymidine predominantly occurs over zones of the nucleus consisting of diffuse fine fibrillar material. As the S-period progresses, a chromatin reticulum reappears throughout the nuclear cavity, the tortuous strands being approximately 0.25 µm in diameter. Most of the radioautographic grains still remain over the light nucleoplasmic areas but a number of these are now located on the outermost portion of the dense chromatin profiles. By the end of the S-period, the chromatin strands are slightly thicker (ca. 0.3 µm) and form a looser reticulum. Labelling has decreased noticeably in nuclei of that period, the radioautographic grains being grouped into clusters resting over more or less spherical regions of the chromatin reticulum. Judging from their localization at the surface of the nucleolus or close to the nuclear envelope, these structures correspond to chromocentres. The additional interesting finding that such nuclear structures appear much less compactly organized strongly suggests that chromocentres undergo important conformational modifications during duplication of their DNA.

In Vitro Biological Activity of Adriamycin

1970 ◽  
Vol 56 (3) ◽  
pp. 137-148 ◽  
Author(s):  
Rosella Silvestrini ◽  
Carmela Gambarucci ◽  
Teresa Dasdia
Keyword(s):  
Biological Activity ◽  
Cellular Proliferation ◽  
Therapeutic Index ◽  
Dna And Rna ◽  
Total Inhibition ◽  
A Cell ◽  
Nuclear Structures ◽  
Higher Doses

Adriamycin is an antibiotic, isolated from cultures of a mutant of Streptomyces peucetius, var. caesius, with a chemical structure very similar to daunomycin but with a higher therapeutic index in experimental tumors. The biological activity of this antibiotic has been studied in vitro on the HeLa cell strain. Adriamycin quickly penetrates into the cells and fixes to the nuclear structures with a marked localization at the level of the perinucleolar chromatin. It causes a marked and immediate disturbance of the mitotic process, viz. pre-prophasic inhibition at the low doses and mitotic block at the higher doses. Even the synthesis of DNA and RNA, evaluated autoradiographically as incorporation of 3H-thymidine and 3H-uridine, appear markedly inhibited. The viability of the cells, tested both as regards capacity to give rise to colonies and as regards proliferative activity of a cell population, was seriously reduced, in a degree proportional to the period of treatment and to the concentration of the antibiotic, until total inhibition. In comparison with daunomycin, adriamycin exerts an immediate antimitotic and anti-metabolic effect which, at equivalent doses, is slightly lower than that of daunomycin. The long-term antiproliferative activity on cellular proliferation is however, identical for the two antibiotics.

scholarly journals PHYSICAL CHEMICAL STUDIES ON THE SPECIFIC INTERACTION OF AN ACRIFLAVINE-PHOSPHOTUNGSTIC ACID COMPLEX WITH DOUBLE-STRANDED NUCLEIC ACIDS

1970 ◽  
Vol 47 (2) ◽  
pp. 500-511 ◽  
Author(s):  
Charles T. Ladoulis ◽  
Thomas J. Gill
Keyword(s):  
Organic Cation ◽  
Phosphotungstic Acid ◽  
Physiological Activity ◽  
Specific Interaction ◽  
Genetic Material ◽  
Acid Complex ◽  
Dna And Rna ◽  
Physical Chemical ◽  
Chemical Studies ◽  
Definition Of

The detailed definition of the structure of DNA in chromosomes and in interphase chromatin is important for correlating the structure of the genetic material with various states of physiological activity. A general approach to developing specific reagents for a variety of such studies in solution and in tissues is to combine a chemically specific organic cation with the electron-opaque phosphotungstic acid (PTA) molecule. The reagent described in this paper was made from the interaction of acriflavine and phosphotungstic acid. The acriflavine-PTA complex (a) displays some unique absorption and fluorescence properties, (b) binds specifically to DNA and RNA by intercalation of the acriflavine moiety, and (c) is electron opaque. In addition, it binds to double-stranded synthetic polynucleotides, but not to a variety of proteins, nucleoproteins, or polysaccharides.

scholarly journals Methicillin-ResistantStaphylococcus aureusBiofilms and Their Influence on Bacterial Adhesion and Cohesion

2016 ◽  
Vol 2016 ◽  
pp. 1-14 ◽  
Author(s):  
Khulood Hamid Dakheel ◽  
Raha Abdul Rahim ◽  
Vasantha Kumari Neela ◽  
Jameel R. Al-Obaidi ◽  
Tan Geok Hun ◽  
...  
Keyword(s):  
Biofilm Formation ◽  
Protein A ◽  
Extracellular Dna ◽  
Proteinase K ◽  
Staphylococcal Protein A ◽  
Sodium Metaperiodate ◽  
Predominant Genotype ◽  
Dna And Rna ◽  
Proteinase K Treatment ◽  
Main Components

Twenty-five methicillin-resistantStaphylococcus aureus(MRSA) isolates were characterized by staphylococcal protein A gene typing and the ability to form biofilms. The presence of exopolysaccharides, proteins, and extracellular DNA and RNA in biofilms was assessed by a dispersal assay. In addition, cell adhesion to surfaces and cell cohesion were evaluated using the packed-bead method and mechanical disruption, respectively. The predominant genotype wasspatype t127 (22 out of 25 isolates); the majority of isolates were categorized as moderate biofilm producers. Twelve isolates displayed PIA-independent biofilm formation, while the remaining 13 isolates were PIA-dependent. Both groups showed strong dispersal in response to RNase and DNase digestion followed by proteinase K treatment. PIA-dependent biofilms showed variable dispersal after sodium metaperiodate treatment, whereas PIA-independent biofilms showed enhanced biofilm formation. There was no correlation between the extent of biofilm formation or biofilm components and the adhesion or cohesion abilities of the bacteria, but the efficiency of adherence to glass beads increased after biofilm depletion. In conclusion, nucleic acids and proteins formed the main components of the MRSA clone t127 biofilm matrix, and there seems to be an association between adhesion and cohesion in the biofilms tested.

scholarly journals Effects of water soluble oncostatic fraction from Rheum officinale Baill. rhizomes on Allium cepa root meristem. I. The mitotic activity

2015 ◽  
Vol 45 (4) ◽  
pp. 429-443
Author(s):  
A. Dawidowicz-Grzegorzewska
Keyword(s):  
Allium Cepa ◽  
Root Meristem ◽  
Soluble Fraction ◽  
Water Soluble ◽  
Total Water ◽  
Lower Intensity ◽  
Interphase Nuclei ◽  
Meristematic Cells ◽  
Dna And Rna ◽  
Inhibition Of Dna Synthesis

The effects of the oncostatic extracts from <i>Rheum officinale</i> rhizomes on the activity of meristematic cells from <i>Allium cepa</i> roots were investigated. A statistically significant decrease of the IM value was noted as well as of the total number of mitoses during incubation. The disturbances in the course of mitosis and cytokinesis are described and discussed. The kind of disturbances during postincubation points to damage of the S and G2 phases of the interphase nuclei. Cytochemical and autoradiographic studies demonstrated a diminished intensity of staining of DNA and RNA and inhibition of DNA synthesis during incubation, this leading in tum to a lower intensity of protein staining in postincubation. Disturbances in mitosis and cytokinesis after treatment wth 2,6-dihydroxyantraquinone, supposed to be the antimitotically active compound of the extract, are the same as those produced by the total water soluble fraction.

Electron Microscopic Radioautography: The Sites of Nuclear RNA Synthesis in Early Embryonic Cells

1968 ◽  
Vol 26 ◽  
pp. 4-5
Author(s):  
Shuichi Karasaki
Keyword(s):  
Rna Synthesis ◽  
Sea Water ◽  
Ultrastructural Organization ◽  
Embryonic Cells ◽  
Electron Microscopic ◽  
Interphase Nuclei ◽  
Thin Sections ◽  
Arbacia Punctulata ◽  
The Relationship ◽  
Physical Developer

The relationship between RNA synthesis and ultrastructural organization of chromatin fibrils was examined in the interphase nuclei of the sea urchin embryos (Arbacia punctulata). The embryos at various stages of development were treated for 1 or 3 hours in sea water with 50 μC/ml of 3H-5-uridine. The samples were fixed in OSO4 or glutaraldehyde (GTA) and embedded in Epon or glycol methacrylate (GMA). Radioautographs were made by applying a thin film of Ilford L4 emulsion directly from an expandable wire loop to thin sections mounted on grids. Following exposure of 10 to 20 weeks, they were developed in Microdol-X or physical developer.In the cleavage and early blastula embryos, each interphase nucleus contains diffusely distributed chromatin and shows only very low incorporation of 3H-uridine into RNA (Fig.1). During these periods, many nucleolus-like bodies occur within the nucleus although they are not labeled with the isotope.

An ultracytochemical study of nucleolar organization in meristematic plant cells (Allium porrum)

1980 ◽  
Vol 43 (1) ◽  
pp. 37-58
Author(s):  
B.T. Luck ◽  
J.G. Lafontaine
Keyword(s):  
Nucleic Acids ◽  
Outer Region ◽  
Staining Technique ◽  
Subsequent Treatment ◽  
Proteinase K ◽  
Allium Porrum ◽  
High Concentration ◽  
Nucleolar Material ◽  
Protein Rich ◽  
Nucleolar Components

The interphase nucleolus in Allium porrum, as in many of the plant species studied so far, is highly heterogeneous in ultrastructure owing to the presence of coarse, contorted, thread-like structures, or nucleolonemata. Each nucleolonema appears to be sharply twisted and to give rise to a skein within the nucleolar mass. In order to characterize further these nucleolar components, a variety of cytochemical techniques were exploited. For that purpose, specimens were mostly fixed in 4% formaldehyde and stained in the block according to procedures known to reveal the presence of nucleic acids or proteins. Certain specimens were also digested with deoxyribonuclease, ribonuclease or proteinase K before staining. By staining with phosphotungstic acid or bismuth oxynitrate, the presence of a high concentration of proteins can be demonstrated within thin (0.15 micrometer), filamentous structures which are believed to correspond to the outer region of the nucleolonema. Such convoluted formations disappear upon sufficiently long extraction with proteinase K. Using Bernhard's regressive staining technique for chromatin, the distribution of this substance throughout the nucleolar mass was found to match closely that of the nucleolonemata as revealed by several other procedures. As a last test for investigating the cytochemical make-up of the nucleolus, blocks of tissues were stained with 3,3′-diaminobenzidine, a substance known to react specifically with nucleic acids. When such specimens are digested with ribonuclease for 1 h, there persist within the nucleolus, fibrillogranular zones the localization of which is highly reminiscent of that of the nucleolonemata. Combination of ribonuclease hydrolysis with subsequent treatment with proteinase K (30 min) induces the extraction of a large proportion of the nucleolar material, the persisting loose and rather evenly distributed fibrils exhibiting a diamter of 3–5 nm. The possibility is considered that these units may correspond to chromatin fibrils although they have most likely been displaced from their original localization during the extraction procedures. Our cytochemical data suggest that, in Allium porrum, the nucleolonema is approximately 0.3 micrometer in diameter and may consist of a central axis from which chromatin loops project radially. A possible interpretation for the presence of protein-rich, 0.1 micrometer-thick, annular structures throughout the nucleolonemal skein is that the newly synthesized RNP products are accumulated transiently at the extremities of these loops before migrating to the immediately adjacent granular nucleolar zones.

scholarly journals Proteinase K-sensitive disease-associated ovine prion protein revealed by conformation-dependent immunoassay

2006 ◽  
Vol 401 (2) ◽  
pp. 475-483 ◽  
Author(s):  
Alana M. Thackray ◽  
Lee Hopkins ◽  
Raymond Bujdoso
Keyword(s):  
Prion Protein ◽  
Phosphotungstic Acid ◽  
Limited Proteolysis ◽  
Proteinase K ◽  
Genotypic Differences ◽  
Time Resolved Fluorescence ◽  
Time Resolved ◽  
Natural Scrapie ◽  
Proteinase K Treatment ◽  
Sheep Scrapie

PrPSc [abnormal disease-specific conformation of PrP (prion-related protein)] accumulates in prion-affected individuals in the form of amorphous aggregates. Limited proteolysis of PrPSc results in a protease-resistant core of PrPSc of molecular mass of 27–30 kDa (PrP27–30). Aggregated forms of PrP co-purify with prion infectivity, although infectivity does not always correlate with the presence of PrP27–30. This suggests that discrimination between PrPC (normal cellular PrP) and PrPSc by proteolysis may underestimate the repertoire and quantity of PrPSc subtypes. We have developed a CDI (conformation-dependent immunoassay) utilizing time-resolved fluorescence to study the conformers of disease-associated PrP in natural cases of sheep scrapie, without using PK (proteinase K) treatment to discriminate between PrPC and PrPSc. The capture-detector CDI utilizes N-terminal- and C-terminal-specific anti-PrP monoclonal antibodies that recognize regions of the prion protein differentially buried or exposed depending on the extent of denaturation of the molecule. PrPSc was precipitated from scrapie-infected brain stem and cerebellum tissue following sarkosyl extraction, with or without the use of sodium phosphotungstic acid, and native and denatured PrPSc detected by CDI. PrPSc was detectable in brain tissue from homozygous VRQ (V136 R154 Q171) and ARQ (A136 R154 Q171) scrapie-infected sheep brains. The highest levels of PrPSc were found in homozygous VRQ scrapie-infected brains. The quantity of PrPSc was significantly reduced, up to 90% in some cases, when samples were treated with PK prior to the CDI. Collectively, our results show that the level of PrPSc in brain samples from cases of natural scrapie display genotypic differences and that a significant amount of this material is PK-sensitive.

Nuclear division in the trophic stages of Iodamoeba bütschlii (Prowazek, 1912) Dobell, 1919

Parasitology ◽  
1959 ◽  
Vol 49 (3-4) ◽  
pp. 543-551 ◽  
Author(s):  
Chia-Tung Pan
Keyword(s):  
Nuclear Membrane ◽  
Interphase Nucleus ◽  
Daughter Cell ◽  
Nuclear Division ◽  
Early Prophase ◽  
Nuclear Morphology ◽  
Type I ◽  
Interphase Nuclei ◽  
Nuclear Structures

1. Study of the nuclear morphology of a large number of haematoxylin-stained specimens of Iodamoeba bütschlii has permitted description of the process of nuclear division in this organism.2. The interphase nuclei of the trophozoites were classified into four types (types I to IV) depending on the distribution of the periendosomal granules. The development of the four types of interphase nuclei appears to follow the process of nuclear division.3. During the early prophase stages the periendosomal granules are incorporated into the endosome. More than twenty small granules are formed from the organizing endosome.4. In late metaphase stages more than ten, and probably twelve, granules can usually be counted per set of the daughter chromosomes.5. The centrodesmus may extend beyond the nuclear boundary during the anaphase stages, while the nuclear membrane appears intact.6. The periendosomal granules reappear by the end of telophase stages when the two daughter nuclei are nearly completely separated.7. In the freshly separated daughter cell the nucleus may still retain the trailing tail and appear tadpole-shaped, but the periendosomal granules are usually arranged like those in the type I interphase nucleus.8. The validity of the genus Iodamoeba is re-emphasized on the basis of the characteristic nuclear structures and the unique process of nuclear division as here described for I. bütschlii.

Sign in / Sign up

Export Citation Format

Share Document