A karyological study of swamp and dry valley populations of Siberian fir (<Emphasis Type="Italic">Abies sibirica</Emphasis> Ledeb.)

The karyological data on populations of Siberian fir (Abies sibirica Ledeb.) growing in lowland swamp and dry valleys are given. The diploid set of both populations included 24 chromosomes (2n = 24). Polykaryogram analysis revealed seven pairs of metacentric and five pairs of submetacentric chromosomes. The revealed differences between the populations included the absolute length of chromosomes, number of nucleolar organizer regions, and number of nucleoli. Changed chromosome numbers (mixoploidy and aneuploidy) as well as chromosomal aberrations were recorded. For the first time, mitosis was studied in this species and anaphase/telophase aberrations were revealed. The population of Siberian fir growing under extreme conditions of lowland swamp featured the widest range of mutations.Translated from Izvestiya Akademii Nauk, Seriya Biologicheskaya, No. 1, 2005, pp. 23–29.Original Russian Text Copyright © 2005 by Sedelnikova, Pimenov.     

Analysis of nuclear proteins in primary spermatocytes of Drosophila hydei: the correlation of nuclear proteins with the function of the Y chromosomal loops

The protein content of spermatocyte nuclei from X/Y males and mutants of D. hydei which lack different Y chromosomal loop forming sites, was compared with that of X/0 males in ¹⁴C/³H double labelling experiments. Proteins of 45,000, 52,000, 54,000, 66,000, 80,000, 84,000, and 170,000 Dalton are found to be enriched in nuclei containing two or more active Y chromosomal loop forming sites. These proteins are also present in the nuclei of X0 males. In the complete absence of the Y-chromosomal loops proteins of 35,000, 46,000, 58,000 and 110,000 Dalton become enriched in the spermatocyte nuclei. — Analysis of the nuclear RNP of spermatocytes led to the isolation of an hnRNP-containing fraction with an S-value of >900S (RNP-PP). — In the RNP-PP of XY males labelled protein material associated with hnRNA is enriched by a factor of 3 in respect to the X0 genotype. The nuclear RNP has a heterogenous buoyant density in CsCl of p = 1.33 to 1.43 g/cm³. RNase T₁ treatment of the crude nuclear RNP from XY males prior to sucrose gradient analysis shows that the 66,000 Dalton protein which is also strongly enriched in the nuclei in the presence of active Y chromosomal loop forming sites, is the main protein associated with protected RNA-sequences of 80–120 and 200–300 nucleotides in length. Competitive nitrocellulose filter binding assays reveal that the 66,000 Dalton protein predominantly forms in 2 M NaCl stable RNA/protein complexes with the poly A ⁺hnRNA of the RNP-PP. These RNP complexes have a buoyant density of p = 1.43 g/cm³ in CsCl. The results are discussed in relation to the nuclear structure and the function of the Y chromosomal loops during spermatogenesis in Drosophila hydei.     

Nuclear division in isolated protoplasts from cells of higher plants grown in vitro

Summary Protoplasts were isolated from cell suspensions of Haplopappus gracilis. The cell walls were degraded by the cellulase preparation Onozuka P 1500 at a concentration of 5%. Sorbitol was found to work well as osmotic stabilizer in concentrations of 0.4–0.6 M. The protoplasts were cultured in growth medium after isolation; 3–5% went through nuclear division once and less than 1% also for a second time. No nuclear fusion was observed.     

Aktomyosinartige Filamente im Teilungsmakronukleus des Ciliaten Ichthyophthirius Multifiliis

Just before nuclear division, the chromosomal elements within the large, highly polyploid macronucleus of I. multifiliis carry out rotational movements. Electron micrographs of cells fixed during the rotational movements show islets filled with microfilaments in various states of aggregation. Both thick (80–200 Å) and thin (30–80 Å) filaments occur, either as a highly dense network or as straight, in part parallel, filaments embedded in a filamentous network of lower density. Other islets of the macronucleus contain large and dense aggregates of filaments, sometimes with globular particles measuring 50–60 Å arranged along the thick filaments, occasionally forming cross-bridges with the thinner ones. — After incubation of the cells before fixation in a contractionsolution containing 0.002 M ATP, all nuclear islets show a nearly uniform appear ance of filamentous aggregates: numerous long and thick filaments are arranged in parallel with thin filaments with which they are in some parts connected by bridges. The probable myosinoid and actinoid nature of thick and thin filaments is discussed. It is suggested that the pre-divisional intranuclear rotational movement is a mechanism to avoid aneuploidy by producing a random arrangement of replicated hereditary units prior to division.     

Establishment of oil palm cell suspensions and plant regeneration

Primary globular callus from immature zygotic embryos and friable embryogenic tissue derived from mature zygotic embryos were used to establish suspension cultures. Callus cultures were established either on modified Y3 or MS medium containing 475–500 M 2,4-D or 250 M picloram and 0.3% (w/v) activated charcoal. Suspension cultures of both cell lines were established in modified Y3 medium containing 10 M 2,4-D. The establishment of cell suspensions from friable embryogenic tissue took only 2 months, in contrast with suspensions from primary globular callus which took 3–5 months to establish. Embryo differentiation was observed only in cell suspensions derived from the friable embryogenic tissue after plating aliquots on regeneration medium. Germinated embryos were recovered and plantlets were successfully established under greenhouse conditions.Abbreviations CET compact embryogenic tissue - FET friable embryogenic tissue - CIM callus induction medium - PGC primary globular callus - 2,3-D 2,4-dichlorphenoxyacetic acid Y3-Eeuwens' medium - MS Murashige & Skoog medium - PVP-40 polyvinylpyrrolidone - KM Kao & Michayluk vitamins - ABA abscisic acid     

The effects of various soils,different provenances and air pollution on root tip chromosomes in Norway spruce

The classification of chromosomal aberrations was used to characterize different factors affecting chromosomes in the root meristem of Norway spruce [Picea abies (L.) Karst.] trees. It is important to know the most significant factor affecting the chromosomes in the root meristem of plants at natural sites. The results suggest that an intensive site effect is more significant than the soil or the provenance of the individual. This cytogenetic plant test system was also used to investigate 5-year-old spruce trees exposed in environmental chambers to elevated concentrations of carbon dioxide (750 cm³m^–3) and ozone (0.08 cm³m^–3) as single variables or in combination, and then transferred to a field for observation of a memory effect. The fumigated variants showed an increased number of chromosomal aberrations compared to the controls, which carried on as a memory effect in the root meristems far beyond the fumigation period.     

Cytogenetic studies in motile sperm from normal men

Summary Most studies on human sperm chromosomes from normal men involve the heterologous fertilization of zona free hamster eggs by unselected human sperm. In this work, we have performed cytogenetic studies of highly motile sperm, selected by a swim-up method. A total of 505 motile human sperm complements from three normal donors was analysed. The total frequency of sperm with chromosomal abnormalities (10.9%; 6.9% structural aberrations and 4.0% aneuploidy) and the sex ratio (50.4% X49.6% Y) were similar to those obtained from whole semen samples. Our results indicate that the selection of motile sperm does not imply chromosomal selection.     

Quantitative autoradiography of α1 adrenoceptors with [3H]tamsulosin in human hypertrophied prostate using computerized image analysis

Summary Fourteen specimens of human hypertrophied prostate were evaluated for the distribution of ₁ adrenoceptors using autoradiography with a computerized image analysis system. The hypertrophied prostatic specimens, obtained at open prostatectomy, were dissected vertically to the urethra, and sectioned at 10 m. They were immersed in 1 nM of specific ₁ ligand, [³H]tamsulosin chloride ([³H]tamsulosin) and exposed to autoradiographic film. The images were analysed by a computerized image analysis system. The total binding of [³H]tamsulosin in the whole section (n = 14) was 0.82 ± 0.21 (mean ± se) nCi mg^–1. The autographic data were correlated with data obtained in a membrane-binding assay. The prostatic tissue studied was divided into urethral, glandular and stromal zones, the latter two zones being further divided into the inner and outer areas. The total binding of [³H]tamsulosin in the urethral zone (n = 7) was 0.65 ± 0.32 nCi mg^–1. The glandular zone contained significantly more abundant ₁ adrenoceptors than the stromal zone and their densities (glandular vs stromal) were 1.15 ± 0.19 nCi mg^–1 (n = 14) vs 0.72 ± 0.15 nCi mg^–1 (n = 14), respectively (p < 0.05). The data from the whole section were not affected by prostatic weight. This method described enabled the distribution of the receptors in different sites to be evaluated both morphologically and quantitatively.     

Regeneration from leaf-base explants of <Emphasis Type="Italic">Lolium perenne</Emphasis> L. and <Emphasis Type="Italic">Lolium multiflorum</Emphasis> L.

The regenerability of Lolium perenne and Lolium multiflorum was investigated using leaf-base sections excised from plantlets grown in vitro. Young leaf bases were cut into three sections each 2 mm in length, the lowest 0–2 mm section adjacent to, but not including, the apical meristem. Callus development followed by embryogenesis occurred only in the lowest 2 mm leaf-base sections of L. perenne cv. Limes, whereas L. multiflorum leaf bases showed an embryogenic response along the entire 6 mm section. Rooted, green plantlets were obtained from 100% of the 0–2 mm sections of L. perenne cv. Limes and from 91%, 65% and 26% of leaf-base sections at 0–2, 2–4 and 4–6 mm respectively from L. multiflorum. Microprojectile bombardment of leaf-base explants resulted in transient expression of the -glucuronidase gene.     

Centromeric association of a microchromosome

Summary A supernumerary microchromosome measuring 0.5–1 m found in over half of the metaphases of a CREST scleroderma patient and his daughter has been characterized by various cytogenetic techniques. The microchromosome consisted of constitutive heterochromatin and contained nuclear antigens reacting with specific anti-kinetochore antibodies. The most remarkable property of the microchromosome was its non-random position: it was closely associated with the centromere of any of the normal chromosomes in the majority of the metaphases. Furthermore, an inordinately high rate of Y chromosome aneuploidy was found in the CREST scleroderma patient. The origin and structure of the microchromosome, its possible connection with the CREST variant of scleroderma, and the phenomenon of centromeric association are discussed.     

Search for chromosomal variations among gas-exposed persons in Bhopal

Summary A chromosomal survey using standard lymphocyte cultures employing different media and G-banding techniques was initiated in 1984. This study became particularly important following the tragic gaseous exposure of the population in Bhopal at midnight on 2 December 1984. We have been able to formulate a chromosomal profile for each person whom we have studied; during 1986–1988, 154 persons were examined twice. Among seemingly normal individuals, as many as 20% might possess some chromosomal abnormality; of these, 50% may develop, at a later date, some kind of pathological complication (such as tumours, recurrent abortion or transmission of defects to their offspring). The people exposed to methyl isocyanate have repeatedly shown Robertsonian translocations, mostly in acrocentric chromosomes 13 and 21. Other types of translocations have been studied among all exposed (53) and normal (101) persons; the involvement of chromosomes 5, 9, 11, 14 and 16 is statistically significant (P= <0.001). One of the major clinical symptoms is dyspnoea; we have estimated that almost all seriously dyspnoeic patients have developed at least two categories of chromosomal aberrations, one of which is Robertsonian translocation, in at least 10% metaphases. Our chromosomal survey will be of significance because we are able to identify people with chromosomal aberrations that might be correlated with future pathological consequences of the accident. The chromosomal load that can be sustained with an apparently normal phenotype can also be measured.     

Biogeneration of ethyl valerate by whole cells of Pseudomonas fragi CRDA 037 in aqueous medium

Summary The production of ethyl valerate by aqueous suspensions of whole cells of Pseudomonas fragi was followed over 8 h after the addition of ethanol and valeric acid. Initial quantity of esters formed ranged from 10 to 160 moles/100 ml under the following conditions: pH 8–9, 8–12°C, 5% (w/v) cell concentration, 0.01–0.05M valeric acid and 0.02–0.1M ethanol.     

Nuclear DNA changes,genome differentiation and evolution inNicotiana (Solanaceae)

A survey of 51 species fromNicotiana subgg.Tabacum, Rustica andPetunioides has shown that evolution was accompanied by a five-fold variation in nuclear DNA amounts. This variation, however, was not directly correlated with the changes in chromosome number. Drastic rearrangement of karyotypes is characteristic for the evolution ofNicotiana spp. Significant gain or loss in nuclear DNA has often accompanied such changes, but DNA variation has also occurred without significant changes in karyotype arrangements.—The distribution of nuclear DNA is discontinuous inNicotiana, species cluster into DNA groups with consistently regular increments in the mean DNA amounts. The discontinuities are viewed as steady states in terms of genomic balance and biological fitness.—Changes in the amount of nuclear DNA and in the heterochromatin are compared with the morphological, chromosomal and adaptive changes which accompanied speciation in 14 subgeneric sections. The evolutionary significance of DNA variation is discussed.     

Reproductive success of Coregonus species in areas loaded by effluents from paper mills

The annual reproductive success of Coregonus albula and C. lavaretus in some polluted and clean areas of the central parts of L. Päijänne was estimated in 1981–90 on the basis of occurrence of larvae in shore seine and seine net samples after the ice melted. In polluted areas (0–5 km from Kaipola paper mill) larvae of coregonids were found only occasionally. In semipolluted areas (5–15 km from the paper mill) the densities of larvae were usually less than 0.1 ind m^–2. In clean areas the mean densities of larvae ranged from 0.45 to 5.34 inds m^–2. A relatively high reproductive success of vendace was observed in 1984–85, moderate success in 1982–83 and low success in 1986–90. The larvae of whitefish were scarce in both polluted and semipolluted areas and their density ranged from 0.01 to 0.15 ind m^–2 in clean shores. The highest density was observed in 1989. In spite of the improvement of the quality of water in the area in the 1980s, the reproductive success of coregonids is still low in a great part of the basin which may depend on adverse oxygen conditions of the hypolimnion and sediment and the toxic effects of the effluents.     

Aurea Mediocritas: The Importance of a Balanced Genome

Aneuploidy, defined as an abnormal number of chromosomes, is a hallmark of cancer. Paradoxically, aneuploidy generally has a negative impact on cell growth and fitness in nontransformed cells. In this work, we review recent progress in identifying how aneuploidy leads to genomic and chromosomal instability, how cells can adapt to the deleterious effects of aneuploidy, and how aneuploidy contributes to tumorigenesis in different genetic contexts. Finally, we also discuss how aneuploidy might be a target for anticancer therapies.As Horace famously wrote in his Odes, the “golden mean” is the secret to a happy, balanced life. Recent work, reviewed here, emphasizes the importance of this kind of balance for the genetics of human cells.Maintaining a stable genome is critical for the preservation of genetic information during the life span of an organism. Despite mechanisms designed to ensure a diploid karyotype, errors can and do occur during chromosome segregation that result in the gain and loss of whole chromosomes. In vitro estimates suggest that normal, diploid cells missegregate a chromosome once every 100 cell divisions (Thompson and Compton 2008). The in vivo rate of chromosome missegregation is unknown, but could vary between different cell types. Even if the rate is low, an abnormal number of chromosomes, or aneuploidy, could have a significant impact on normal cell physiology, as well as tumorigenesis.Aneuploidy, at the level of the organism, is detrimental and generally incompatible with life. In humans, only three aneuploidies—trisomy 13, 18, and 21—are viable, and only trisomy 21 is compatible with a life span beyond infancy (Hassold et al. 2007). Despite the deleterious consequences of aneuploidy in normal physiological contexts, an abnormal number of chromosomes is one of the hallmarks of cancer cells. Aneuploidy is found in ∼90% of solid tumors and >50% of blood cancers (Beroukhim et al. 2010; Mitelman et al. 2013). Whether aneuploidy is a cause or consequence of cell transformation is a frequent topic of debate. The challenge for establishing a causal relationship stems from the complexity of cancer cells, in which numerical chromosome abnormalities are rarely found in isolation but are usually accompanied by other genomic alterations, such as point mutations, translocations, and microsatellite instability. This complexity makes it difficult to define the initiating event(s) in tumorigenesis.This review focuses on whole-chromosome aneuploidy, although it has been shown that the gain and loss of chromosome arms is also a common occurrence in cancer cells (Beroukhim et al. 2010; Mitelman et al. 2013). We review the molecular pathways leading to aneuploidy, the effects of aneuploidy on cellular physiology, and the links between aneuploidy and tumorigenesis. Finally, we also explore the exciting concept of targeting aneuploidy as a novel therapeutic approach in treating cancer.     

Genotoxic effects of the pesticides Rubigan, Omite and Rovral in root-meristem cells of Crepis capillaris L.

Three pesticides have been studied for their genotoxicity by the use of assays in the plant Crepis capillaris, aimed at measuring chromosomal aberrations, micronuclei and sister chromosome exchange (SCE). The fungicides Rubigan 12 EC (fenarimol) and Rovral 25 Flo (iprodione) and the insecticide Omite 57 E (propargite) are all widely used nowadays. The aim of our study was to evaluate the genotoxic effects of these pesticides at concentrations corresponding to those applied in agricultural practice. In preliminary experiments we found that these concentrations do not influence cell proliferation and do not inhibit the growth of root meristems. In all experiments formulated commercial products were used. From the results we conclude that the three pesticides did not induce chromosomal aberrations as estimated by metaphase and anaphase analyses. They were also not capable to induce SCE. Rubigan did not induce micronucleus formation even at the highest concentration tested, but Omite and Rovral markedly increased micronucleus formation. The MN response depended on the sampling time and the concentration used, which showed a significant dose–response correlation (r = 0.978, P < 0.01 and r = 0.941, P < 0.01, respectively). A greater increase in micronucleus frequency was observed after Rovral treatment, where the highest concentration gave a response 8–10-fold above the negative control. Both pesticides induced high frequencies of lagging chromosomes, even after exposure to the lower test concentrations. The presence of lagging chromosomes is an indication of anti-microtubule activity of the pesticides tested. This effect was more strongly expressed after exposure to the two higher concentrations of Omite and Rovral. In this case a complete destruction of the mitotic spindle was observed, resulting in C-mitoses as well as in numerical aberrations—polyploidy and aneuploidy. The present findings suggest that Omite and Rovral at concentrations comparable to those used in practice can be regarded as potential aneugens.     

Counting viableAzotobacter chroococcum in vertisols

Methods for preparing soil suspensions for countingAzotobacter chroococcum in vertisols by soil dilution and plating were investigated. Mechanical methods to promote disaggregation of soil and Azotobacter microcolonies by shaking soil suspensions with glass beads (10% w/v) or coarse sand (1–2 mm fraction) increased soil dispersion and Azotobacter colony counts. Chemical methods for disaggregation were unsatis-factory. The non-ionic detergent Agral (0.004, 0.02, 0.1, 0.5 and 2.5% w/v) had no significant effect on soil dispersion and Azotobacter count. Both sodium pyrophosphate (0.03, 0.1, 0.3 and 0.9% w/v) and sodium metaphosphate as Calgon (0.022, 0.066, 0.2, 0.6 and 1.8% w/v) increased soil dispersion but were toxic to Azotobacter. Increasing time of shaking soil: distilled water suspensions increased deflocculation of the clay and Azotobacter counts to a maximum after 6–23 hours shaking. Comparable results were obtained within 30–60 minutes of shaking with coarse sand, but shaking with coarse sand beyond 2 hours reduced counts through mechanical damage to cells. Counts from suspensions in physiological saline (0.75% NaCl) and in distilled water were similar. Counts from suspensions in Jensen's mineral base shaken for <3 hrs were lower than from distilled water due to flocculation fo the soil by^Ca2+ ions, but were higher on extended shaking up to 23 hours due to better cell protection. Shaking soil suspensions in distilled water with 10% w/v coarse sand for 30 minutes is recommended when counting Azotobacter in vertisols.     

Semen Abnormalities,Sperm DNA Damage and Global Hypermethylation in Health Workers Occupationally Exposed to Ionizing Radiation

Background

Cytogenetic studies have demonstrated that low levels of chronic radiation exposure can potentially increase the frequency of chromosomal aberrations and aneuploidy in somatic cells. Epidemiological studies have shown that health workers occupationally exposed to ionizing radiation bear an increased risk of hematological malignancies.

Objectives

To find the influence of occupational radiation exposure on semen characteristics, including genetic and epigenetic integrity of spermatozoa in a chronically exposed population.

Methods

This cross sectional study included 134 male volunteers of which 83 were occupationally exposed to ionizing radiation and 51 were non-exposed control subjects. Semen characteristics, sperm DNA fragmentation, aneuploidy and incidence of global hypermethylation in the spermatozoa were determined and compared between the non-exposed and the exposed group.

Results

Direct comparison of the semen characteristics between the non-exposed and the exposed population revealed significant differences in motility characteristics, viability, and morphological abnormalities (P<0.05–0.0001). Although, the level of sperm DNA fragmentation was significantly higher in the exposed group as compared to the non-exposed group (P<0.05–0.0001), the incidence of sperm aneuploidy was not statistically different between the two groups. However, a significant number of hypermethylated spermatozoa were observed in the exposed group in comparison to non-exposed group (P<0.05).

Conclusions

We provide the first evidence on the detrimental effects of occupational radiation exposure on functional, genetic and epigenetic integrity of sperm in health workers. However, further studies are required to confirm the potential detrimental effects of ionizing radiation in these subjects.     

Sequence-specific interactions of wound-inducible nuclear factors with mannopine synthase 2′ promoter wound-responsive elements

A 318 bp mannopine synthase 2 (mas2) promoter element from the T-DNA of Agrobacterium tumefaciens can direct wound-inducible and root-preferential expression of a linked uidA gene in transgenic tobacco plants. Wound inducibility is further enhanced by sucrose in the medium. Promoter deletion analysis indicated that the sucrose enhancement is conferred by a region extending from –318 to –213. DNase I footprinting indicated that an A/T-rich DNA sequence in this region is protected by tobacco nuclear factors. Regions extending from –103 to +66 and from –213 to –138 directed wound-inducibile expression of a linked uidA gene when placed downstream of a CaMV 35S enhancer or upstream of a truncated (–209) CaMV 35S promoter, respectively. DNase I footprinting analyses indicated that proteins from wounded tobacco leaves specifically bound to three contiguous motifs downstream of the mas2 TATA box. In addition to a common retarded band formed by the upstream wound-responsive element complexed with proteins from either wounded or unwounded tobacco leaves, two unique retarded bands were observed when this element was incubated with protein from wounded leaves. Methylation interference analysis additionally identified an unique motif composed of promoter elements and nuclear factors derived specifically from wounded tobacco leaves. We propose a model to describe the involvement of nuclear factors with mas2 promoter elements in wound-inducible gene expression.     

The Degree of Segmental Aneuploidy Measured by Total Copy Number Abnormalities Predicts Survival and Recurrence in Superficial Gastroesophageal Adenocarcinoma

Background

Prognostic biomarkers are needed for superficial gastroesophageal adenocarcinoma (EAC) to predict clinical outcomes and select therapy. Although recurrent mutations have been characterized in EAC, little is known about their clinical and prognostic significance. Aneuploidy is predictive of clinical outcome in many malignancies but has not been evaluated in superficial EAC.

Methods

We quantified copy number changes in 41 superficial EAC using Affymetrix SNP 6.0 arrays. We identified recurrent chromosomal gains and losses and calculated the total copy number abnormality (CNA) count for each tumor as a measure of aneuploidy. We correlated CNA count with overall survival and time to first recurrence in univariate and multivariate analyses.

Results

Recurrent segmental gains and losses involved multiple genes, including: HER2, EGFR, MET, CDK6, KRAS (recurrent gains); and FHIT, WWOX, CDKN2A/B, SMAD4, RUNX1 (recurrent losses). There was a 40-fold variation in CNA count across all cases. Tumors with the lowest and highest quartile CNA count had significantly better overall survival (p = 0.032) and time to first recurrence (p = 0.010) compared to those with intermediate CNA counts. These associations persisted when controlling for other prognostic variables.

Significance

SNP arrays facilitate the assessment of recurrent chromosomal gain and loss and allow high resolution, quantitative assessment of segmental aneuploidy (total CNA count). The non-monotonic association of segmental aneuploidy with survival has been described in other tumors. The degree of aneuploidy is a promising prognostic biomarker in a potentially curable form of EAC.