Populational polymorphisms in silver staining of nucleolus organizer regions (NORs) in human acrocentric chromosomes

Summary The Ag stainability of the nucleolus organizer region (NOR) was studied in the acrocentric chromosomes identified by Q banding of cultured lymphocytes in 41 karyotypically normal persons (33 males and 8 females) originating from southeast Estonia. The data obtained are compared with those established earlier for a combined Vienna-Ulm population of 51 karyotypically normal persons (see Mikelsaar et al., 1977a). Significant differences between the two populations in the frequency and patterns of Ag-positive NORs were found. The following findings were most striking: the frequency of Ag-positive NORs in chromosome 14 and in the totals was significantly lower in the Estonian population than in the Vienna-Ulm population (P<0.01). The average modal number of Ag-positive NORs per individual was 7.8 in the Estonian population and 8.7 in the Vienna-Ulm sample (P<0.01). If the data of the two populations were combined the frequency of positive NORs was significantly (P<0.05) lower in chromosome 22 than in 13,15, and 21, but not 14.     

Development of silver stained structures during spermatogenesis in different genera of Formicidae

Development of silver stained structures during the spermatogenesis of Tapinoma nigerrlum, Pheidole pallidula, Tetramorium caespitum, Tetramorium semilaeve, Lasius niger and Plagiolepis schmitzii are studied. Nucleolar masses are only observed in early prophase. Obvious NORs are present in metaphase in all genera and species studied. However, there are no nucleolar Ag precipitates after metaphase. A resumption of silver stainability occurs in round spermatids. The majority of these genera present differential activity between the existing NORs. In T. nigerrimum there is primary or secondary NOR activity in all chromosomes of the complement, although there are interpopulation differences in relation to the NOR activity. In the remaining genera only certain chromosomes present NOR activity. Interpopulation genetic differences and environmental factors can cause differential activity of secondary NORs as observed in Tapinoma nigerrimum.     

Chromosomal Characteristics of rDNA in European Grayling <Emphasis Type="Italic">Thymallus thymallus</Emphasis> (Salmonidae)

The chromosomal characteristics, locations and variations of two classes of ribosomal DNA (5S and 18S) were studied in European grayling karyotype (Thymallus thymallus, Salmonidae). Major rDNA sites as revealed by sequential CMA₃/Ag staining and confirmed by in situ hybridization with a 18S rDNA probe were situated in two loci and were found to be polymorphic in size and displaying several distinct forms. The 5S rDNA was located by PRINS on three pairs of subtelocentric chromosomes, additional minor signal was present at the centromere of one metacentric element. 5S sites were not associated with NORs. The dosage compensation mechanism was proposed as an explanation of high frequency of lethal rDNA-deleted forms of the NOR-bearing chromosomes. Double variable pattern in the number and location of NORs supported the bi-directional evolution of salmonid rDNA loci.     

The ultrastructure and argentophilic properties of the nucleolus organizer and centromeric regions of the chromosomes in early mouse embryogenesis

The Ag-staining of metaphase chromosomes in one-cell mouse embryos shows that the nucleolus organizer regions (NORs) are Ag-negative, whereas centromeric regions (CRs) are Ag-positive. Starting from 8-16-cell embryos, NORs stained by AgNO3 constantly, CRs remaining argentophobic. On the ultrathin sections of multicell embryos, Ag(+)-NORs differ from the chromosomal arms: they consist of loosely filaments about 6-8 nm in diameter, characterized by a low electron density. On the contrary, at one-cell stage Ag(-)-NORs are not morphologically identified: chromosomal bodies consist of uniform DNP-fibrils about 20 nm in diameter. These data permit to suppose that extended rDNA may form supranucleosomal and nucleosomal DNP-fibrils in the absence of Ag-proteins. The Ag(+)- and Ag(-)-CRs contain 10-20 nm DNP-fibrils mainly, although their density at multicell stages is higher than in one-cell mouse embryos.     

Regional differences in immunostainability of isolated metaphase chromosomes of Indian muntjac with anti-Z-DNA antibody

The distribution of Z-form DNA along the length of metaphase chromosomes of Indian muntjac was studied by indirect immunofluorescence procedures using an antibody specific to the Z-DNA conformation. Several fixation conditions were compared for reproducible detection of Z-DNA in isolated metaphase chromosomes. Fixation of chromosomes with 45% acetic acid alone gave reproducible reactivity with the antibody. When fixation was done either with Carnoy's solution (3:1 methanol:acetic acid) or with 75% alcohol alone, the antibody binding was at background level. Acetic acid-fixed chromosomes exhibited intense fluorescence both at C-band heterochromatin and at nucleolus organizer regions (NORs). The euchromatic regions had weakly, but clearly, stained bands, which were quite similar to the chromomycin A₃ R-bands. After treatment with topoisomerase I, the immunofluorescence at NORs and R-bands disappeared, but only a slight decrease in immunofluorescence intensity was observed at C-band regions. We suggest that this difference in the immunoreactivity of NORs and R-bands from C-bands reflects a difference in gene activity among these regions. Possible molecular mechanisms involved in Z-DNA immunoreactivity are discussed, based on SDS-polyacrylamide gel electrophoretic analysis of chromosomal proteins after extraction of metaphase chromosomes with different fixative solutions.Abbreviations PI propidium iodide - NOR(s) nucleolus organizer region(s) - SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis Deceased, April 23, 1988     

FISH-mapping of 18S ribosomal RNA genes and telomeric sequences in the Japanese bitterlings Rhodeus ocellatus kurumeus and Tanakia limbata (Pisces,Cyprinidae) reveals significant cytogenetic differences in morphologically similar karyotypes

The Japanese rose bitterling, Rhodeus ocellatus kurumeus, and the oily bitterling, Tanakia limbata, were cytogenetically studied by silver (Ag)- and chromomycin A₃ (CMA₃)-staining, by C-banding and by mapping of the 18S ribosomal genes and of the (TTAGGG) _n telomeric sequence. These two representative species of related genera of the subfamily Acheilognathinae show very similar chromosome complements. Nevertheless, significant differences in the chromosomal distribution of nucleolus organizer regions (NORs) and interstitial telomeric sequences were observed. Whereas R. ocellatus kurumeus shows a single NOR-bearing chromosome pair, T. limbata is characterized by a higher number of variable NORs. Multiple telomeric sequence sites were found at the pericentromeric regions of several chromosomes in the rose bitterling. No telomeric sequence sites were detected near centromeres, but they were found to be scattered along the NORs in the oily bitterling. Two karyoevolutive trends might have been identified in the subfamily.     

Disposition of ribosomal DNAs in the chromosomes of perennial oats (Poaceae: Aveneae)

The chromosomal loci of 5S and 45S ribosomal DNAs (rDNAs) and the activity of nucleolar‐organizing regions (NORs) were analysed in perennial oats of the genera Ammophila, Amphibromus, Arrhenatherum, Avena, Deschampsia, and Helictotrichon s.l. (Poaceae: Aveneae) using fluorescence in situ hybridization, staining with chromomycin/4′,6‐diamidino‐2‐phenylindole (DAPI), and silver impregnation. All chromosomes with a secondary constriction were nucleolar active. In chromosomes without a secondary constriction, NORs corresponded exclusively to broad bands of 45S rDNA with chromomycin‐positive, DAPI‐negative, and silver‐positive stainability. Additional minor bands of 45S rDNA showed no nucleolar activity. 5S rDNA was localized mostly in loci different from the nucleolar‐active 45S rDNA. If both rDNAs occurred within the same chromosome, they were at largely corresponding distances from the centromere, irrespective of their particular localization in either the same chromosome arm or in opposite arms. In the latter case, 5S rDNA was never more distal to the centromere than 45S rDNA. A new model was devised to explain this non‐random distribution of both rDNAs in nucleolar‐organizing chromosomes, which identified the Rabl orientation of chromosomes as ensuring a spatial proximity of 5S to 45S rDNA in interphase nuclei, even if they were localized in opposite arms. The possible role of the Rabl orientation in determining the spread and accumulation of 5S rDNA sequences in further chromosomes of the genome was discussed. B chromosomes were devoid of 5S rDNA, but most contained 45S rDNA and were nucleolar active. In some large groups of species, the number and arrangement of 5S and 45S rDNA sites in the chromosomes were remarkably uniform, especially in Helictotrichon subgenus Helictotrichon and Helictotrichon subgenus Pratavenastrum. Such distribution patterns have survived many speciation processes and have also remained widely unchanged in polyploids. © 2007 The Linnean Society of London, Botanical Journal of the Linnean Society, 2007, 155 , 193–210.     

Long-term influence of adjuvant therapy on natural killer cell activity in breast cancer

Summary Unstimulated IFN- and IL2-stimulated (NK) cell activities were investigated in patients with breast cancer who had received either local radiotherapy alone or adjuvant chemotherapeutic treatment with CMF combined with radiotherapy 12 to 18 months previously. When tested against the primarily NK-sensitive K562 cell line, patients who had received adjuvant chemotherapeutic treatment with CMF were shown to have a significantly decreased unstimulated and IFN-stimulated NK cell activity, as compared to both patients after radiotherapy only (P<0.002) and P<0.005, respectively) and healthy control persons (P<0.05). The former group of patients also had a significantly decreased IFN-stimulated NK cell activity, when tested against the primarily NK-insensitive Chang hepatoma cell line, as compared to patients after radiotherapy only (P<0.005) and healthy controls (P<0.05). Moreover, patients after radiotherapy only proved to have a significantly increased unstimulated (P<0.01) and IFN-stimulated NK cell activity (K562: P<0.05; Chang hepatoma cell line: P<0.05), as compared to healthy control individuals. In contrast, no difference in IL2-stimulated NK cell activity was detected. The investigation for the expression of CD3 and/or Leu 19 antigens as phenotypic markers of cells with non-MHC restricted cytotoxicity showed a significantly lower percentage of cells with the CD3+ phenotype in patients with breast cancer, irrespective of the chosen post-operative treatment, as compared to healthy controls (P<0.01). Finally, patients with breast cancer who had received radiotherapy only had a significant trend towards an increased percentage of CD³+/Leu 19+ PMNC, as compared to both patients after CMF treatment (P<0.05) and healthy controls (P<0.025). We conclude that patients with breast cancer vary on a long-term basis in their NK activity and in the phenotype of their PMNC depending on their post-operative adjuvant management.Abbreviations NK natural killer cells - IFN interferon - IL 2 interleukin 2 - CMF cyclophosphamide, methotrexate, fluorouracil - ER oestrogen receptor - PMNC peripheral blood mononuclear cells - MHC major histocompatibility complex - CTL cytotoxic T lymphocytes     

Serum TNFRII: A promising biomarker for predicting the risk of subcentimetre lung adenocarcinoma

Early diagnosis of lung adenocarcinoma requires effective risk predictors. TNFRII was reported to be related to tumorigenesis, but remained unclear in lung cancer. This research set out to investigate the relationship between the sTNFRII (serum TNFRII) level and the risk of lung adenocarcinoma less than 1 cm in diameter. Seventy-one pairs of subcentimetre lung adenocarcinoma patients and healthy controls were analysed through multiplex bead-based Luminex assay and found a significantly lower expression of sTNFRII in patients with subcentimetre lung adenocarcinoma than that in the healthy controls (P < .001), which was further verified through ONCOMINE database analysis. Increased levels of sTNFRII reduced the risk of subcentimetre lung adenocarcinoma by 89% (P < .001). Patients with a higher level of BLC had a 2.70-fold (P < .01) higher risk of subcentimetre adenocarcinoma. Furthermore, a higher BLC/TNFRII ratio was related to a 35-fold higher risk of subcentimetre adenocarcinoma. TNFRII showed good specificity, sensitivity and accuracy (0.72, 0.75 and 0.73, respectively), with an AUC of 0.73 (P < .001). In conclusion, the present study assessed the value of sTNFRII as a potential biomarker to predict the risk of subcentimetre lung adenocarcinoma and provided evidence for the further use of TNFRII as an auxiliary marker in the diagnosis of subcentimetre lung adenocarcinoma.     

microRNA-21 表达水平对新疆地区食管癌患者早期诊断及预后评估的临床价值

目的:探讨microRNA-21表达水平对新疆地区食管癌早期诊断及预后评估的临床价值。方法:收集我院收治的100例食管癌患者,其中哈萨克族患者50例,汉族患者50例,采用RT-PCR以及RT-qPCR的方法对患者血清microRNA-21表达水平进行检测并比较。结果:食管癌患者癌组织中microRNA-21表达水平均高于癌旁组织,差异具有统计学意义(P0.05);有淋巴结转移、ⅡB+Ⅲ期、低分化癌组织中的miRNA-21相对表达量高于无淋巴结转移、I+ⅡA期以及高分化癌组织患者,差异具有统计学意义(P0.05);miRNA-21在不同民族、性别、年龄分组中表达无明显差异(P0.05)。结论:microRNA-21表达水平对新疆地区食管癌患者癌组织中表达水平较高,在有淋巴转移、ⅡB+Ⅲ期以及低分化癌组织中表达水平较高,民族、性别以及年龄对microRNA-21水平的影响较小,因此microRNA-21检测对食管癌的早期诊断及预后判断具有指导意义。     

Comparative chromosomal studies on two minnow fish, Phoxinus phoxinus (Linnaeus, 1758) and Eupallasella perenurus (Pallas, 1814); an associated cytogenetic-taxonomic considerations

The present work provides new data on the banding pattern of two cyprinid fish species Phoxinus phoxinus and Eupallasella perenurus from Poland. C-banding, silver-staining (Ag), and fluorescent staining with chromomycin A₃ techniques were used to describe the karyotypes. Both of the species karyotypes of 2n=50 were characterised by one pair of acrocentric chromosomes, the largest in the set, and by two pairs of NOR-bearing chromosomes. In the chromosome set of Ph. phoxinus Ag-stained NORs were located on telomeres of two metacentric and two submetacentric chromosomes, but in most metaphases only one of the two homologous was observed. The karyotype of E. perenurus was characterised by Ag-NOR regions at a telomeric position on the shorter arm of two submetacentric chromosome pairs. In most metaphases only three NOR-bearing chromosomes were observed. In both investigated species the location of the A₃ positive signals corresponded with the location of Ag-stained NORs and these sites were associated with heterochromatin shown as C-bands. The results of cytogenetical studies on other related, mainly the North American phoxinins, species are compared and discussed.     

Chromosome banding and FISH with rDNA probe in the diploid and tetraploid loach Misgurnus anguillicaudatus

The chromosomes of the diploid and tetraploid loach Misgurnus anguillicaudatus were analyzed by staining with Ag, chromomycin A₃ (CMA₃)/distamycin A (DA), and DA/4′,6-diamidino-2-phenylindole (DAPI), and using fluorescence in situ hybridization (FISH) with 5.8S + 28S rDNA as a probe. Nucleolus organizer regions (NORs) were mapped to the telomeric region of the short arms of the largest (first) metacentric chromosome pair in the diploid loach with 2n = 50 and the homologous quartet in the tetraploid loach with 4n = 100. The NORs were positive at the same region of the first metacentric chromosome for Ag and CMA₃/DA stainings, but negative for DA/DAPI staining. Four signals at the homologs within the same quartet suggest the duplication of the entire genome from diploid to tetraploid status. However, a size difference was detected between the rDNA signals by FISH and CMA₃ banding.     

Karyotype analysis of Eucinostomus argenteus, E. gula, E. harengulus, and Eugerres plumieri (Teleostei, Gerreidae) from Florida and Puerto Rico

The karyotypes of four gerreids of the western Atlantic Ocean are documented. A diploid chromosome complement of 48 telocentric chromosomes was found in the four species (2N=48t, fundamental number FN=48). No differences were detected either in the number of chromosomes of the standard karyotype, in their karyotype size, or between the karyotypes derived from male or female specimens of any of the species. Chromosome length decreased progressively and slightly from pair 1 to pair 24. The Ag–NOR karyotypes of E. argenteus and E. harengulus were characterized by the position of the nucleolar organizer regions next to the centromere in chromosome pair 1, whereas in E. gula and E. plumieri Ag–NORs were detected in pair 4. The other 46 chromosomes showed a light staining of the centromere with no terminal or intermediate heterochromatic blocks. All Eucinostomus species showed Ag–NORs of similar size, while Eugerres plumieri showed Ag–NORs 10–20% larger than Eucinostomus species. A combination of size and position of the Ag–NORs identified E. gula, while size alone identified E. plumieri. However, the ancestral state for size and position of Ag–NORs could not be established. There was no differential staining of the chromosomes by G-banding. The karyotype of the gerreids appears similar to the hypothetical ancestral karyotype of fish. The phylogenetic relationships among these species could not be established because of the lack of chromosome G-bands. Most likely this indicates a homogeneous distribution of GC nucleotides in the chromosomes.     

Polymorphism of major ribosomal gene chromosomal sites (NOR-phenotypes) in the hybridogenetic fish <Emphasis Type="Italic">Squalius alburnoides</Emphasis> complex (Cyprinidae) assessed through crossing experiments

Chromosomal locations of major ribosomal sites, i.e. NOR-phenotypes, were assigned in Squalius alburnoides complex using sequential chromomycin A₃ (CMA₃)- and silver (Ag)-staining. This hybridogenetic Iberian minnow comprises diploid, triploid and tetraploid forms that arose by interspecific hybridisation between S. pyrenaicus and an unknown species. Inheritance of NOR patterns was studied by means of crossing experiments involving most diploid–polyploid forms of the S. alburnoidescomplex with identified specific genotype constitution. In all the specimens studied, the NORs were localised in the short arms of submetacentric chromosomes. Although S. pyrenaicus presented only one pair of NOR-bearing chromosomes, the data from experimental crosses evidenced that S. alburnoides complex was characterised by a multiple NOR phenotype composed of one chromosome pair with stable NORs and two chromosome pairs with NOR site polymorphism of presence/absence type. These data suggest that the karyotype of the unknown parental species of the S. alburnoidescomplex should have a multiple NOR pattern and emphasised the role of the all-male diploid linage in the dynamics and evolutionary potential of the S.alburnoidescomplex allowing the preservation of the missing ancestor genome. Cross-analyses evidenced that in spite of the high polymorphic nature of NORs in this fish complex, we have no reason to reject the hypothesis that their inheritance patterns were in accordance with Mendelian segregation.     

Intercellular NOR-AG variability in man

Summary A new modification of the Ag I technique has been developed using human cultured blood lymphocytes, which involves ultra-violet irradiation of chromosome preparations during incubation in AgNO₃. This technique enables detection in a short incubation time all NORs capable of being stained with silver. A peculiar morphological change in Ag-stainable NORs during the incubation is described, which can be used as a criterion of the completion of Ag staining. With the refined Ag-staining procedure, acrocentric marker chromosomes were studied which showed one or two satellite stalks within the same individual. Ag staining was highly coincident with this variability.     

Banded karyotype of spined loach Cobitis taenia and triploid Cobitis from Poland

The present work provides new data on the banding pattern of diploid Cobitis taenia and its triploid hybrid females, which belong to the diploid–polyploid complex in the Vistula River tributary. C-banding, silver-staining (Ag), and fluorescent staining with chromomycin A₃ techniques were used to describe the diploid and triploid karyotype. The karyotype of Cobitis taenia of 2n=48 was characterised by one pair of NOR-bearing subtelocentric chromosomes and at least four chromosomes with CMA₃-positive sites. The C-positive heterochromatin was present in the centromeres of almost all chromosomes and the pericentromeric regions of several metacentric and submetacentric chromosomes. The triploid females of 3n=74 had two pairs of chromosomes with active NORs. The NORs-sites were located terminally on two biarmed and two uniarmed chromosomes. The CMA₃-staining revealed at least six A₃-positive sites. The C-banded and A₃-stained triploid karyotype was composed of haploid set of Cobitis taenia and diploid set of unidentified species, so heterochromatin pattern confirmed the possibility of their hybrid origin. The characteristics of banded diploid and triploid karyotype, and the hypothetical karyotype of an unknown species of 2n=50 is discussed. This revised version was published online in July 2006 with corrections to the Cover Date.     

CD4(+)CD25high regulatory T cells increase with tumor stage in patients with gastric and esophageal cancers

Purpose: Regulatory T cells (T regs) can inhibit immune responses mediated by T cells. It has been shown that there is an increased proportion of T regs in several different human malignancies, although the actual mechanism remains unclear. In the present study, we evaluated the prevalence of CD4(+)CD25^high T regs in PBMCs from patients with gastric and esophageal cancers in relation to the clinical outcome. Methods: PBMCs in 72 patients with gastric cancer and 42 patients with esophageal cancer were evaluated for the proportion of CD4(+)CD25^high T cells, as a percentage of the total CD4(+) cells, by flow cytometric analysis with triple-color staining. Actuarial overall survival rates of the patients were analyzed by the Kaplan–Meier method. Results: The percentages of CD4(+)CD25^high T cells for cases of gastric cancer (4.9±1.2%) and esophageal cancer (5.2±2.1%) were significantly higher than those for healthy donors (1.9±1.1%, P<0.01). There were significant differences in the prevalence of CD4(+)CD25^high T cells between the early and advanced disease stages, both in gastric cancer (stage I vs. III, P<0.05; stage I vs. IV, P<0.05) and esophageal cancer (stage I vs. IV, P<0.05). The patients with a high proportion of CD4(+)CD25^high T cells showed poorer survival rates in comparison to those with a low proportion, in both gastric and esophageal cancers. After patients received curative resections of gastric cancers (n=57), the increased proportions of CD4(+)CD25^high T cells were significantly reduced, and the levels were almost equal to those in normal healthy donors. In addition, studies of gastric cancer patients with postoperative recurrent tumors (n=6) revealed that the prevalence of CD4(+)CD25^high T cells individually increased compared to 2 months after the operations. CD4(+)CD25^high T cells expressed FOXP3 mRNA and had abundant CD45RO and intracellular CTLA-4 molecules. Conclusions: These results strongly suggest that tumor-related factors induce and expand CD4(+)CD25^high T regs.     

非小细胞肺癌患者血清CYFRA21-1、VEGF及CEA的表达及与临床病理特征的相关性研究

目的:研究非小细胞肺癌(NSCLC)患者血清细胞角蛋白19片段(CYFRA21-1)、血管内皮生长因子(VEGF)、癌胚抗原(CEA)的表达及与临床病理特征的相关性。方法:选取2015年12月至2016年4月在我院接受治疗的NSCLC患者120例作为观察组,另选取同期在我院接受治疗的肺部良性病变患者50例作为良性对照组,比较两组患者血清中CYFRA21-1、VEGF及CEA的表达,分析观察组患者血清中CYFRA21-1、VEGF及CEA的表达与临床病理特征之间的关系,采用Pearson相关系数分析观察组患者血清中CYFRA21-1、VEGF、CEA的相关性。结果:观察组患者血清中的CYFRA21-1、VEGF及CEA水平均高于良性对照组(P0.05)。鳞状细胞癌患者血清中CYFRA21-1水平高于腺癌患者,CEA水平低于腺癌患者(P0.05),鳞状细胞癌患者和腺癌患者血清中VEGF水平比较无统计学差异(P0.05)。TNM分期为III-IV期的观察组患者血清中CYFRA21-1、VEGF及CEA水平均明显高于I-II期患者,有统计学差异(P0.05)。经Pearson相关系数分析显示,观察组患者血清中CYFRA21-1与VEGF、CEA呈正相关(r=0.512,0.423,P=0.000,0.000),VEGF与CEA呈正相关(r=0.452,P=0.000)。结论:NSCLC患者血清中CYFRA21-1、VEGF及CEA呈高表达,且CYFRA21-1、CEA与病理类型和TNM分期有关,VEGF与TNM分期有关,且三种指标存在一定的相关性。