Nucleolar Organizer Regions In Pituitary Gland Adenomas

Nucleolar organizer regions are DNA segments concerned with the synthesis of ribosomal RNA (rRNA). Some specific proteins related to NORs can be demonstrated by a silver technique and are then referred to as Ag-NORs. The mean number of Ag-NORs per cell has been used as a marker of cell proliferation in recent years and there is some evidence to suggest that Ag-NOR counts can be used to differentiate between normal and neoplastic cells. Paraffin block sections of 42 pituitary gland adenomas and 24 normal adult pituitary glands have been examined using the Ag-NOR staining technique. Touch imprints of nine adenomas and 12 normal adult pituitary glands were also examined using the same Ag-NOR staining technique. Using light microscopy at x2000 magnification, the Ag-NORs were located as black dots in the cell nuclei and the mean number of Ag-NORs per cell was determined. No significant difference was observed between blocked sections and imprints of adenomas or between imprints and paraffin sections of normal glands. However, a significant difference was observed between the mean number of Ag-NORs in pituitary gland adenomas and normal glands in paraffin block sections and touch preparations (P less than 0.001). The modified Ag-NOR technique can be a useful aid in the differential diagnosis between normal pituitary gland tissue and pituitary adenomas, especially during surgical operations.     

Silver Binding Nucleolar Organizer Regions In Adrenocortical Neoplasia

The possible contribution of the silver colloid technique for staining nucleolar organizer regions in the distinction between benign and malignant adrenocortical neoplasms was investigated. Nine cases of adenoma, eight cases of carcinoma, nine cases of hyperplasia and four normal adrenal cortex specimens were examined. The mean silver binding nucleolar organizer region (Ag-NOR) value in adenoma was 4.29, and in carcinoma 7.16 (P less than 0.001). Adenomas with diameters greater than 3 cm had significantly higher Ag-NOR counts than smaller adenomas. For normal cortex, the mean Ag-NOR value was 2.05 and in hyperplasia, 3.62. The results indicate that the Ag-NOR technique can help in differential diagnosis between benign and malignant adrenocortical lesions and thus may have a prognostic value.     

睾酮对大鼠再生肝细胞核仁组织区影响的初步研究

以银染核仁组织区(argymphil nucleolar organiting regions,AgNORs)阳性颗粒数为指标,结合放射免疫技术,研究了大鼠部分肝切除(partial hepatectomy,PH)后,睾酮对余留肝细胞核仁组织区的影响。结果显示,pH后0～24h,各种方式(假手术、皮下注射芝麻油或睾酮)处理组,AgNORs数都下降,随后持续升高。在PH后48h和72h,低剂量(0．5ms／kg体重)睾酮对AgNORs数影响最显著,都明显高于其它两个睾酮处理组;中等剂量(2．5ms／kg体重)和高剂量睾酮(5ms／kg体重)处理对AgNORs数的影响与芝麻油处理组相比都无显著差异,但均高于假手术组;假手术后72h的AgNORs恢复到术前水平。血清睾酮含量测定显示,芝麻油处理组PH后0～48h睾酮持续下降,48h后不再下降,48h和72h时的睾酮浓度显著低于假手术组;低剂量处理,血清睾酮浓度在PH后0～24h下降,然后持续上升,在48h、72h达到假手术组的2～3倍;中等剂量和高剂量处理组,血清睾酮在PH后0～72h持续升高,是假手术组的6～7倍。以上结果初步表明,睾酮对PH后肝细胞中AgNORs的影响有两种情况：(1)在血清中的浓度是生理水平的2～3倍时,起促进作用;(2)6～7倍于生理水平时,基本无作用。     

Study of the Nucleolar Organizer Regions in Palinurus elephas (Crustacea: Decapoda)

Using fluorescence in situ hybridization (FISH), chromomycin (CMA₃) staining and silver staining, we studied the nucleolar organizer regions in the spiny lobster Palinurus elephas in order to extend our knowledge on the karyology of this commercially important species. Multiple NORs have been detected by FISH, and CMA₃ showed a good correspondence between the localization of GC-rich heterochromatin and the ribosomal genes mapped by FISH. In contrast, the number of Ag-positive regions was higher than the number of FISH and CMA₃ signals, which may be explained by silver staining of the kinetochores. A variability in the number of FISH and CMA₃ signals has been detected in metaphases I and II which is probably due to the occurrence of rDNA cistrons on B chromosomes.     

Gold Toning Improves the Visualization of Nucleolar Organizer Regions in Paraffin Embedded Tissues

A modification of the silver colloid technique for staining nucleolar organizer regions in paraffin embedded tissues is described. This modification involves the application of a gold toning step with subsequent gold reduction, if necessary, following incubation of sections in the standard silver colloid solution. Silver stained nucleolar organizer regions (AgNORs) in toned sections are more sharply delineated when compared to untoned controls. in high grade tumors the addition of the toning step results in significantly higher AgNOR counts due to the ability to discriminate more easily individual AgNORs in argyrophilic aggregates within the nucleus. It is recommended, because of enhanced visualization, that this modification of the silver colloid technique be used in studies involving quantification of AgNORs in tissue sections.     

Gold Toning Improves the Visualization of Nucleolar Organizer Regions in Paraffin Embedded Tissues

A modification of the silver colloid technique for staining nucleolar organizer regions in paraffin embedded tissues is described. This modification involves the application of a gold toning step with subsequent gold reduction, if necessary, following incubation of sections in the standard silver colloid solution. Silver stained nucleolar organizer regions (AgNORs) in toned sections are more sharply delineated when compared to untoned controls. in high grade tumors the addition of the toning step results in significantly higher AgNOR counts due to the ability to discriminate more easily individual AgNORs in argyrophilic aggregates within the nucleus. It is recommended, because of enhanced visualization, that this modification of the silver colloid technique be used in studies involving quantification of AgNORs in tissue sections.     

Silver Staining of the Nucleolar Organizer Regions (NORS) in Semithin Lowicryl Sections

The one-step silver technique was applied to semithin Lowicryl sections of root meristem cells of Allium cepa and a human tumor cell line (TG cells). In vegetal cells, after 5 min of staining reaction, the Ag-NOR proteins formed ring-shaped structures peripherally within the nucleolus. In animal cells silver granules were distributed over the entire nucleolus. The specificity of the staining reaction was increased by incubation of the sections in NH₄Cl and Schiff's reagent prior to Ag-NOR silver staining.     

癌症患者淋巴细胞核仁形成区活性变异的研究

应用银染-G带复合显示方法研究了肺癌、胃癌、肠癌、乳腺癌患者的外周淋巴细胞核仁形成区(NOR)活性。与正常对照相比,肺癌第15号染色体的Ag-NOR频率及Ag-NOR总频率增加,乳腺癌第14号染色体的Ag-NOR频率减少,胃癌第14号染色体Ag-NOR频率减少而第22号染色体的Ag-NOR频率增加。肠癌未见明显变异。结果提示不同部位的肿瘤具有不同的优势银染型,rRNA基因的表达可能存在肿瘤部位的特异性。     

A Technique for the Simultaneous Staining of Both Nucleolar Organizer Regions and Kinetochores of Human Chromosomes with Silver

Pretreatment of human metaphase chromosomes with NaOH at a pH of 8.5, followed by staining with silver nitrate, differentially stains both the nucleolar organism regions on the 10 acrocentric chromosomes as well as the kinetochore centers on all 46 chromosomes.     

人类核仁形成区结构性变异与随体联合的研究

本文以硝酸银染色法研究了200对正常夫妇及200对唐氏综合征(DS)患者双亲淋巴细胞染色体核仁形成区(NOR)。结果发现双核仁形成区(dNOR)的检出率在两组人群中均为0.5％;随体联合(SA)的结果表明,负有dNOR染色体的SA频率并不随其核糖体RNA(rRNA)基因含量的明显增加而上升,提示除rRNA基因含量及其活性外,尚有其他因素影响SA的形成。dNOR携带者与DS的发生亦无明显关系。     

The Three-dimensional Study of Chromosomes and Upstream Binding Factor-immunolabeled Nucleolar Organizer Regions Demonstrates Their Nonrandom Spatial Arrangement during Mitosis

The volumic rearrangement of both chromosomes and immunolabeled upstream binding factor in entire well-preserved mitotic cells was studied by confocal microscopy. By using high-quality three-dimensional visualization and tomography, it was possible to investigate interactively the volumic organization of chromosome sets and to focus on their internal characteristics. More particularly, this study demonstrates the nonrandom positioning of metaphase chromosomes bearing nucleolar organizer regions as revealed by their positive upstream binding factor immunolabeling. During the complex morphogenesis of the progeny nuclei from anaphase to late telophase, the equal partitioning of the nucleolar organizer regions is demonstrated by quantification, and their typical nonrandom central positioning within the chromosome sets is revealed.     

Silver Staining for Nucleolar Organizing Regions of Vertebrate Chromosomes

Refinements to a simple, one-step silver staining technique for nucleolar organizing regions are described. These include fixation of silver stained material with sodium thiosulfate and standardization of silver development conditions for different groups of vertebrates. The central advantages to the method are that it is rapid, reliable, simple, and inexpensive. Additional benefits include (i) consistent and uniform silver staining of nucleolar organizing regions, (ii) few reduced silver deposits elsewhere on the chromosomes or on the slides, (iii) generally unaltered chromosome morphology after silver treatment, and (iv) relative permanence of Permounted preparations. The method works equally well on chromosomes made from cell cultures and from solid tissues of live specimens.     

Organization of Organelles within Hyphae of Ashbya gossypii Revealed by Electron Tomography

Ashbya gossypii grows as multinucleated and constantly elongating hyphae. Nuclei are in continuous forward and backward motion, also move during mitosis, and frequently bypass each other. Whereas these nuclear movements are well documented, comparatively little is known about the density and morphology of organelles which very likely influence these movements. To understand the three-dimensional subcellular organization of hyphae at high resolution, we performed large-scale electron tomography of the tip regions in A. gossypii. Here, we present a comprehensive space-filling model in which most membrane-limited organelles including nuclei, mitochondria, endosomes, multivesicular bodies, vacuoles, autophagosomes, peroxisomes, and vesicles are modeled. Nuclei revealed different morphologies and protrusions filled by the nucleolus. Mitochondria are very abundant and form a tubular network with a polarized spherical fraction. The organelles of the degradative pathways show a clustered organization. By analyzing vesicle-like bodies, we identified three size classes of electron-dense vesicles (∼200, ∼150, and ∼100 nm) homogeneously distributed in the cytoplasm which most likely represent peroxisomes. Finally, coated and uncoated vesicles with approximately 40-nm diameters show a polarized distribution toward the hyphal tip with the coated vesicles preferentially localizing at the hyphal periphery.     

Electron Tomography of Neuronal Mitochondria: Three-Dimensional Structure and Organization of Cristae and Membrane Contacts

The structure of neuronal mitochondria from chick and rat was examined using electron microscope tomography of chemically fixed tissue embedded in plastic and sliced in ≈500-nm-thick sections. Three-dimensional reconstructions of representative mitochondria were made from single-axis tilt series acquired with an intermediate voltage electron microscope (400 kV). The tilt increment was either 1° or 2° ranging from −60° to +60°. The mitochondrial ultrastructure was similar across species and neuronal regions. The outer and inner membranes were each ≈7 nm thick. The inner boundary membrane was found to lie close to the outer membrane, with a total thickness across both membranes of ≈22 nm. We discovered that the inner membrane invaginates to form cristae only through narrow, tubular openings, which we call crista junctions. Sometimes the cristae remain tubular throughout their length, but often multiple tubular cristae merge to form lamellar compartments. Punctate regions, ≈14 nm in diameter, were observed in which the inner and outer membranes appeared in contact (total thickness of both membranes ≈14 nm). These contact sites are known to a play a key role in the transport of proteins into the mitochondrion. It has been hypothesized that contact sites may be proximal to crista junctions to facilitate transport of proteins destined for the cristae. However, our statistical analyses indicated that contact sites are randomly located with respect to these junctions. In addition, a close association was observed between endoplasmic reticulum membranes and the outer mitochondrial membrane, consistent with the reported mechanism of transport of certain lipids into the mitochondrion.     

Composition and Behavior of the Nucleolar Organizer of the Nucleolar Variant of Axolotls; Review and Comparison to Other Systems

SYNOPSIS. Studies on the nudeohis in various species of Amphibia,including the Mexican axolotl, have contributed to an understandingof the function of the nucleolar organizing region. In the firstpart of this paper we have reviewed some of the research onnucleolar variants in the axolotl. These variants were recognizedin different strains of laboratory reared animals. The phenotypesinclude diminished nucleolar size and possible alterations inthe levels of rDNA. The expression of several of these variantnucleolar phenotypes does not, however, reflect a simple deletionof rRNA genes. Evidence from analyses of several mutant andapparently normal strains indicate that some animals have relativelyhigh levels of rRNA redundancy, while others, including someanimals which display variant nucleolar sizes, display "normal"levels. Data on the inheritance of rRNA genome sizes in the axolotlis reviewed. The observation that intermediate levels of redundancywere detected in some crosses is discussed in terms of inheritancedata from other organisms. In the second part of this reviewa variety of information, obtained from studies of various organisms,on nucleolar expression is discussed.     

Evidence for a Role of CLIP-170 in the Establishment of Metaphase Chromosome Alignment

CLIPs (cytoplasmic linker proteins) are a class of proteins believed to mediate the initial, static interaction of organelles with microtubules. CLIP-170, the CLIP best characterized to date, is required for in vitro binding of endocytic transport vesicles to microtubules. We report here that CLIP-170 transiently associates with prometaphase chromosome kinetochores and codistributes with dynein and dynactin at kinetochores, but not polar regions, during mitosis. Like dynein and dynactin, a fraction of the total CLIP-170 pool can be detected on kinetochores of unattached chromosomes but not on those that have become aligned at the metaphase plate. The COOH-terminal domain of CLIP-170, when transiently overexpressed, localizes to kinetochores and causes endogenous full-length CLIP-170 to be lost from the kinetochores, resulting in a delay in prometaphase. Overexpression of the dynactin subunit, dynamitin, strongly reduces the amount of CLIP-170 at kinetochores suggesting that CLIP-170 targeting may involve the dynein/dynactin complex. Thus, CLIP-170 may be a linker for cargo in mitosis as well as interphase. However, dynein and dynactin staining at kinetochores are unaffected by this treatment and further overexpression studies indicate that neither CLIP-170 nor dynein and dynactin are required for the formation of kinetochore fibers. Nevertheless, these results strongly suggest that CLIP-170 contributes in some way to kinetochore function in vivo.Microtubules (MTs)¹ in vertebrate somatic cells are involved in intracellular transport and distribution of membranous organelles. Fundamental to this role are their tightly controlled, polarized organization, and unusual dynamic properties (Hirokawa, 1994) and their interaction with a complex set of MT-based motor proteins (Hirokawa, 1996; Sheetz, 1996; Goodson et al., 1997). During mitosis, they contribute to the motility of centrosomes, the construction of spindle poles (Karsenti et al., 1996; Merdes and Cleveland, 1997), and the dynamic movements of kinetochores (Rieder and Salmon, 1994) and chromosome arms (Barton and Goldstein, 1996; Vernos and Karsenti, 1996). The motor protein cytoplasmic dynein, drives the transport toward MT minus-ends of a variety of subcellular organelles (Schnapp and Reese, 1989; Schroer et al., 1989; Holzbaur and Vallee, 1994). Dynactin is a molecular complex originally identified as being essential for dynein-mediated movement of salt-washed vesicles in vitro (reviewed in Schroer, 1996; Schroer and Sheetz, 1991). Genetic studies in fungi, yeast, and flies have shown that the two complexes function together to drive nuclear migration, spindle and nuclear positioning and to permit proper neuronal development (Eshel et al., 1993; Clark and Meyer, 1994; Muhua et al., 1994; Plamann et al., 1994; McGrail et al., 1995; Karsenti et al., 1996). Biochemical studies suggest a direct interaction between certain subunits of dynein and dynactin (Karki and Holzbaur, 1995; Vaughan and Vallee, 1995). In vivo, the two molecules may bind one another transiently, since they have not been isolated as a stable complex.There is good evidence indicating that the dynein/dynactin complex, together with other motors (Eg5, and a minus-end oriented kinesin-related protein) and a structural protein (NuMa), drive the focusing of free microtubule ends into mitotic spindle poles (Merdes and Cleveland, 1997; Waters and Salmon, 1997)_. A trimolecular complex composed of NuMa and dynein/dynactin may be crucial in this process in both acentriolar (Merdes et al., 1996), and centriolar spindles (Gaglio et al., 1997). A number of findings also indicate that the combined actions of dynein and dynactin at the kinetochore contribute to chromosome alignment in vertebrate somatic cells. First, the initial interaction between polar spindle MTs and kinetochores seems to involve a tangential capture event (Merdes and De Mey, 1990; Rieder and Alexander, 1990) which is followed by a poleward gliding along the surface lattice of the MT (Hayden et al., 1990). Both in vivo and in vitro (Hyman and Mitchison, 1991) this gliding movement appears similar to the dynein-mediated retrograde transport of vesicular organelles along MTs. Consistent with this is the finding that both dynein (Pfarr et al., 1990; Steuer et al., 1990) and its activator, dynactin (Echeverri et al., 1996), are present at prometaphase kinetochores. Overexpression of dynamitin, a 50-kD subunit of the dynactin complex, results in the partial disruption of the dynactin complex and in the loss, from kinetochores, of dynein, as well as dynactin. Therefore, it has been proposed that dynactin mediates the association of dynein with kinetochores. Abnormal spindles with poorly focused poles are observed and the cells become arrested in pseudoprometaphase (Echeverri et al., 1996). Despite these findings, rigorous proof for a role of the dynein motor complex in kinetochore motility is still lacking, and its role may differ between lower and higher eucaryotes, and between mitosis and meiosis.CLIP-170 (Rickard and Kreis, 1996) is needed for in vitro binding of endocytic transport vesicles to MTs (Pierre et al., 1992). It is a nonmotor MT-binding protein that accumulates preferentially in the vicinity of MT plus ends and on early endosomes and endocytic transport vesicles in nondividing cells (Rickard and Kreis, 1990; Pierre et al., 1992). Like many MT-binding proteins, CLIP-170 is a homodimer whose NH₂-terminal head domains and COOH-terminal tail domains flank a central α-helical coiled-coil domain. The binding of CLIP-170 to MTs involves a 57–amino acid sequence present twice in the head domain (Pierre et al., 1992) and is regulated by phosphorylation (Rickard and Kreis, 1991). The COOH-terminal domain has been proposed to participate in targeting to endocytic membranes (Pierre et al., 1994). The fact that the latter move predominantly toward microtubule minus ends in a process most likely mediated by cytoplasmic dynein and dynactin (Aniento and Gruenberg, 1995), suggests that CLIP-170 may act in concert with this motor complex, and may be subject to regulated interactions with one or more dynactin or dynein subunits at the vesicle membrane.Here we report that during mitosis, CLIP-170 codistributes with dynein and dynactin at kinetochores, but not spindle poles. Evidence is presented that the COOH-terminal domain of CLIP-170 is responsible for its kinetochore targeting, and that this may be mediated by the complex of dynein and dynactin. The effects on mitotic progression of overexpression of wild type and several deletion mutants of CLIP-170 provide evidence for the involvement of CLIP-170 in kinetochore function early in mitosis. We also present in vivo evidence that neither CLIP-170 nor the complex of dynein and dynactin are required for formation of kinetochore fibers.