Effect of local UV irradiation of the generative nuclei of Paramecium caudatum on the sexual functions of the cells]

A study has been made of the functional pattern of the micronucleus (Mi) during the sexual process in ciliates Paramecium caudatum which are descendants of those individuals whose Mi was locally irradiated with UV in dose of 3060 erg/mm2. It was demonstrated that these descendants (cells of UV-clones) were able to enter into the sexual process whatever morphological type of their Mi might be. In 4 of 14 tested clones, the initial mating type got changed for the opposite one, which may be associated with the Mi genome alteration following UV irradiation. The nuclear reorganization during the sexual process was studied in three UV irradiated clones. The local UV irradiated of the Mi is followed by the number of abnormalities in the derivates of Mi during the metagamic phase of the sexual process in clones whose Mi underwent serious changes. Exconjugants obtained from UV-clones with such changes in Mi and normal test-clones demonstrated a lower viability.     

A Cytochemical Study of ACPase and ALPase Activity in the Laminar Hair Cells of Bresenia shreberi

The ultrastructural distribution of acid phosphatase (ACPase) alkaline phosphatase (ALPase) and the changes of their activities in the laminar hair cells of Bresenia shreberi Geml. were studied by means of lead phosphate preciptation method during their development. The ACPase activity became detactable at the stage of mucilage secretion of the head cell, which was located at endoplasmic reticulum (Er),cuticle (Cu) and tonoplast (Tp) of the head cell, and nuclear membrane (NM), plasmodesmata (P1) and wall ingrowths (WI) of the stalk cell, and WI and plasmolemma of basal cell. ALPase activity showed its first appearance at the mitochondria(Mi) of the basal cell at the stage of head cell formation. At the stage of head cell secretion, ALPase activity was located at Er, Cu, Mi of head cell, and NM, Go, Er, WI and P1 of stalk cell. ALPase activity at Mi of basal cell was most intensive. The results indicated that ACPase and ALPase possessed the function of promoting cell differentiation and material transfer in the process of hair cell development and that the material for synthesizing mucilage in head cell was provided by the basal cells and the parenchyma cells nearby.     

Evolution of amitosis of the ciliate macronucleus: gain of the capacity to divide

Ciliates exhibit nuclear dimorphism, i.e. they have a germline micronucleus and a somatic macronucleus. Macronuclei are differentiated from mitotic sisters of micronuclei. The macronuclei of "higher ciliates" are polyploid and divide acentromerically ("amitotically"); they differentiate once per life cycle. By contrast, Karyorelict (KR) ciliate macronuclei are nearly diploid and cannot divide; they must differentiate at every cell cycle. Diverse lines of evidence are presented to support the hypothesis that ancestral ciliate macronuclei were incapable of division (as in living karyorelict ciliates) and that higher ciliates gained, perhaps independently more than once, the ability to divide the macronucleus. Selective pressures that could have driven the evolution and macronuclear division and two plausible step-wise pathways for the evolution of macronuclear division are proposed. These hypotheses are relevant to our understanding of amitosis mechanisms, evolution of nuclear dimorphism, and phylogenetic classification of ciliates.     

Evolution of Amitosis of the Ciliate Macronucleus: Gain of the Capacity to Divide

Ciliates exhibit nuclear dimorphism, i.e. they have a germline micronucleus and a somatic macronucleus. Macronuclei are differentiated from mitotic sisters of micronuclei. The macronuclei of "higher ciliates" are polyploid and divide acentromerically ("amitotically"); they differentiate once per life cycle. By contrast, Karyorelict (KR) ciliate macronuclei are nearly diploid and cannot divide; they must differentiate at every cell cycle. Diverse lines of evidence are presented to support the hypothesis that ancestral ciliate macronuclei were incapable of division (as in living karyorelict ciliates) and that higher ciliates gained, perhaps independently more than once, the ability to divide the macronucleus. Selective pressures that could have driven the evolution and macronuclear division and two plausible step-wise pathways for the evolution of macronuclear division are proposed. These hypotheses are relevant to our understanding of amitosis mechanisms, evolution of nuclear dimorphism, and phylogenetic classification of ciliates.     

Clinical and kinetic characteristics of a temporarily nonproliferating subpopulation of bone marrow blast cells from acute leukemia patients (based on data on DNA autoradiography and cytophotometry

The morphology of Paramecium bursaria cells, both originally possessing 2 micronuclei (MI), or one MI, has been studied after a local UV irradiation of the germ nuclei. Elimination of one of the MI in bimicronuclear cells usually did not lead to general cytological damage in the UV progeny. Irradiation of MI in the unimicronuclear cells resulted in the following: producing of amicronuclear (MI-) UV subclones, 86% of which appeared to be non-viable. The MI- cells are characterized by the appearance of somatic deciliation and the loss of mobility, disorganization structures of the oral apparatus, the feeding process and the cell division. It is remarkable that the macronucleus (MA) of cells from those cultures displays fragmentation and aberrant division. The structure of MA was changed. Dimensions of the MI- ciliates decreased (by 1.7--1.8 times in average); sometimes these cells had 3--5 contractile vacuoles with one pore. These data suggest that the MI has important role during the vegetative phase of the cell life. Possible asexual cellular functions of the MI and the role of the break of nuclear interactions in the appearance of the morphological damage are discussed.     

Mitogenic activation of B cells in vitro: the properties of adherent accessory cells as revealed by partition analysis

The requirement of B cells activated by mitogen (dextran sulfate plus lipopolysaccharide) for accessory cells was studied by partition analysis. Small numbers of splenic B cells were activated to clonal growth, as determined by visual inspection, and to immunoglobulin (Ig) synthesis, as determined by release of Ig into the culture fluid. By placing irradiated adherent cells in the periphery of the microculture wells and forcing responding cells to different areas of the well (slant experiments), it was observed that no cell contact was necessary for B cell activation, and that "promoted" contact ("Rock and Roll" experiments) does not increase the efficiency of activation. Sequential microcultures suggest that only some irradiated adherent cells act as accessory cells, but they can perform this function to more than one B cell. Attempts to perform limiting dilution analysis by varying irradiated adherent cell input showed non-single-hit behavior. When the data were rearranged, taking into account the distribution of irradiated adherent cells, then single-hit behavior with about 1 to 5% of irradiated adherent cells acting as an accessory cells for B cell clonal activation was observed. The evidence suggests that an uncommon irradiated adherent cell releases a soluble factor necessary for B cell activation and/or clonal proliferation.     

Expansion of the Candida albicans cell envelope in different morphological forms of the fungus.

Modes of cell envelope expansion were monitored in developing cells of Candida albicans 73/055 to which polystyrene beads were attached. Eight different conditions of culture medium, pH and temperature were used to promote growth in a variety of morphological forms. The cells were observed microscopically during growth in Sykes-Moore perfusion chambers, and sequential measurements of distances between the bead and the parent cell, and the bead and the apical tip were used to distinguish apical envelope expansion from general envelope expansion. Morphology index (Mi) was determined at each time point as an estimate of each cell's morphology. Calculations based on the measurements showed that general envelope expansion was inversely proportional to Mi, but that general expansion greater than 20% occurred only in cells with a final Mi less than 2.0, indicating that regulation of apical and general envelope expansion alone may be insufficient to determine the different morphologies seen in cells with higher Mi. The rate of expansion of the perimeter of cells was linearly proportional to the final Mi. This observation suggests that commitment to morphological development in C. albicans may in part involve commitment to a rate of envelope expansion, which itself helps determine the final morphology of a cell.     

The microelectrophoresis of the DNA in individual intact and gamma-irradiated thymocytes

The in vitro gamma-irradiated mouse thymocytes were embedded in low melting agarose at 37 degrees C. After getting at 4 degrees C, the cells were lysed in neutral detergent solution containing proteinase K and ethidium bromide. Microscopic visualization of single lysed and stained cells showed the presence of the central "core" (nuclear matrix) surrounded with "halo" (relaxed nuclear DNA). During electrophoresis (2-5 V/sm, 5 min) this "halo" migrated towards the anode forming a "tail". The use of microdensitometric system provided measuring the size of the tail (L) and quantity of migrated DNA (S) for individual cells as well as obtaining the distribution of these parameters among the cells. The latter may be characteristic of heterogeneity of the cell population. It was shown that L and S increased linearly with the dose irradiation at least between 0.2 and and 5.0 Gy. In irradiated thymocyte (3 Gy) the DNA repair occurred within 10-20 min, but residual DNA damage could be observed even after 60 min of incubation. These damages may initiate the degradation of DNA in irradiated thymocytes that was observed after the repair of DNA.     

肽链释放因子和核糖体蛋白L11在八肋游仆虫细胞中的定位

原生动物纤毛虫是一类单细胞真核生物,其蛋白质合成终止过程中密码子使用的特殊性使其成为研究蛋白质合成终止机制的一个经典模型。为了能够有效地分析生物大分子在该细胞中的功能作用位点,本研究根据该生物染色体结构的特征,构建了含有红色荧光蛋白基因的大核人工染色体EoMAC_R,并与之前构建的含绿色荧光蛋白基因的大核染色体EoMAC_G一起,对蛋白质合成终止有关的3个重要因子核糖体大亚基蛋白L11、多肽链释放因子eRF1和eRF3在八肋游仆虫细胞中进行了荧光共定位分析。结果显示,在八肋游仆虫细胞中,蛋白质翻译过程主要位于"C"形大核内侧区域。构建的人工染色体能够作为一种有效的工具,对目的蛋白质在八肋游仆虫细胞中进行定位分析。     

Mass entrapment and lysis of Mesodinium rubrum cells in mucus threads observed in cultures with Dinophysis

The entrapment and death of the ciliate Mesodinium rubrum in the mucus threads in cultures with Dinophysis is described and quantified. Feeding experiments with different concentrations and predator–prey ratios of Dinophysis acuta, Dinophysis acuminata and M. rubrum to study the motility loss and aggregate formation of the ciliates and the feeding behaviour of Dinophysis were carried out. In cultures of either Dinophysis species, the ciliates became entrapped in the mucus, which led to the formation of immobile aggregates of M. rubrum and subsequent cell lysis. The proportion of entrapped ciliates was influenced by the concentration of Dinophysis and the ratio of predator and prey in the cultures. At high cell concentrations of prey (136 cells mL⁻¹) and predator (100 cells mL⁻¹), a maximum of 17% of M. rubrum cells became immobile and went through cell lysis. Ciliates were observed trapped in the mucus even when a single D. acuminata cell was present in a 3.4 mL growth medium. Both Dinophysis species were able to detect immobile or partly immobile ciliates at a distance and circled around the prey prior to the capture with a stretched out peduncle. Relatively high entrapment and lysis of M. rubrum cells in the mucus threads indicates that under certain conditions Dinophysis might have a considerable impact on the population of M. rubrum.     

Activation of lymphocytes alters Fc receptor-beta 2-microglobulin interrelationship on the lymphocyte surface

The effect of anti-beta 2-microglobulin (anti-B2Mi) on the expression of Fc receptors (FcR) of human lymphocytes was compared on resting and activated cells. Previously we reported that anti-B2Mi induces a "co-shedding" of FcR with the beta 2-microglobulin (B2Mi)-anti-B2Mi complexes when used under the conditions where the redistribution of membrane molecules is allowed (Sármay et al., Cell. Immunol. 56, 452, 1980; Sármay et al. Immunology 36, 339, 1979). Furthermore our group also described two types of FcR-bearing cells, one which shed their FcR during a temperature shift from 4 to 37 degrees C (FcRI+ cells) and the other which has an immobile type FcR under the same circumstances (FcRI+ cells) (Sándor et al., Immunology 38, 553, 1979; Sármay et al., Immunology 34, 315, 1978). In this work we have characterized the FcR released from the membrane as a consequence of anti-B2Mi treatment. We have found that they are the mobile, FcRI type. It was proved that the shedding of this FcRI is a consequence of the anti-B2MI-induced transformation of FcRII into the FcRI form on the membrane of the antibody-treated lymphocytes. On the activated T cells, however, anti-B2Mi is incapable of inducing the same phenomenon in the early phase of activation. In contrast, FcR expression is blocked by anti-B2Mi treatment similarly to that on resting lymphocytes, on the surface of activated B cells, or on activated T cells in the later phases of activation.     

Degeneration of interstitial gland cells of normal and irradiated dog ovary

The interstitial gland cells of the ovary were studied by electron microscopy in seven Beagle dogs during advanced metestrus. Additionally, the findings of three dogs with an increased activation of the interstitial gland cells after 12 Gy whole-body X-irradiation were also presented. Three groups of interstitial gland cells were distinguished: the immature, the mature and the degenerating cells. The steroid-specific cell organelles of mature interstitial cells were highly developed after irradiation-induced stimulation. At the onset of generation, a circular arrangement of dilated endoplasmic vesicles (demarcation channel) characterized the cells of non-irradiated and of irradiated ovaries. During the early stage of degeneration, different degrees of nuclear shrinkage and of vacuolation of the cytoplasm were seen. A lace-like vacuolation was common in the irradiated ovaries. During the advanced stage of degeneration, the cysts ruptured with resultant shedding of the cytoplasmic organelles into the intercellular space. A new cellular membrane was simultaneously reconstituted and a fibrocyte-like cell appeared. The process is referred as "secretory degeneration" and is presented as a hypothesis.     

Insights into the Evolution of Nuclear Dualism in the Ciliates Revealed by Phylogenetic Analysis of rRNA Sequences

The small subunit rRNA gene sequences of the karyorelictean ciliates, Loxodes striatus and Protocruzia sp., and the heterotrichian ciliates, Climacostomum virens and Eufolliculina uhligi , were used to test the evolution of nuclear dualism in the Phylum Ciliophora. Phylogenies derived using a least squares distance method, neighbour joining, and maximum parsimony demonstrate that the karyorelictean ciliates sensu Small and Lynn, 1985 do not form a monophyletic group. However, Loxodes and the heterotrich ciliates form the first branch in the ciliate lineage, and Protocruzia branches, in distance methods, basal to the spirotrich lineage. It is proposed that Protocruzia be removed from the Class Karyorelictea, and placed in closer taxonomic association with the spirotrich lineage. The distribution of nuclear division types along the phylogenetic tree is consistent with the notion that macronuclei incapable of division represent a derived rather than a primitive or "karyorelictid" character trait.     

两种纤毛虫营养细胞和休眠细胞蛋白组成的比较分析

应用生化抽提和SDS-PAGE方法显示,膜状急纤虫(Tachysoma pellionella)的营养细胞含38条蛋白谱带,休眠细胞含29条蛋白谱带,两者共有谱带为26条,特有谱带各为12条和3条,相似度为77.6%;休眠细胞的包囊壁含22条蛋白谱带,细胞脱包囊后残留的包囊壁含15条蛋白谱带,两者共有谱带为14条,特有谱带各为8条和1条,相似度为76%。包囊游仆虫(Euplotes encysticus)的营养细胞和休眠细胞均含23条蛋白谱带,两者共有谱带为19条,特有谱带各为4条,相似度为82.6%;休眠细胞的包囊壁和细胞脱包囊后残留的包囊壁均含20条蛋白谱带,两者共有谱带为19条,特有谱带均为1条,相似度为95%。结果表明,两种纤毛虫的营养细胞向休眠细胞转化过程中,细胞结构的主要蛋白质成分发生了明显变化,这些变化与细胞在不同生理状态下结构的分化及其生命活动特征相关。形成“毛基体吸收型包囊”的急纤虫与形成“毛基体非吸收型包囊”的游仆虫相比较,前者营养期和休眠期细胞蛋白组成有更明显的差异,这可能与其细胞结构更大程度的脱分化有关;根据纤毛虫休眠细胞的包囊壁和细胞脱包囊后残留的包囊壁两者蛋白组成的差异推测,前者包囊壁可能含有与休眠细胞生命活动相联系的“活性”成分。     

Intestinal ciliate fauna of the Asian elephant from Gaziantep,Turkey and the description of Brevitentaculum antebum n. g., n. sp.

The aims of this investigation were to identify and quantify ciliates inhabiting the large intestine of Asian elephants living in Gaziantep Zoo, Turkey, and to describe a new suctorian genus and species. Species composition and distribution of intestinal ciliates voided in the feces of two Asian elephants were examined. Fifteen species of intestinal ciliates, representing 7 genera, were identified. One new suctorian genus and species, Brevitentaculum antebum n. g., n. sp., was described. This new species has two short truncated terminal projections, with two longitudinally lined bands located between the two projections, near the convex surface of the cell. Short clavate-like tentacles are in two fascicles near each base of terminal projections on the flattened surface of the body. Ciliate densities in the two fecal samples were 4.5 × 10⁴ mL⁻¹ and 10 × 10⁴ mL⁻¹.     

Downregulation of Wnt signaling by sonic hedgehog activation promotes repopulation of human tumor cell lines

Tumor repopulation after radiotherapy is a big obstacle for clinical cancer therapy. The molecular mechanisms of tumor cell repopulation after radiotherapy remain unclear. This study investigated the role of sonic hedgehog (SHH) and Wnt signaling pathways in tumor repopulation after radiotherapy in an in vitro repopulation model. In this model, irradiated dying tumor cells functioned as feeder cells, whereas luciferase-labeled living tumor cells acted as reporter cells. Proliferation of reporter cells was measured by bioluminescence imaging. Results showed that irradiated dying HT29 and Panc1 tumor cells significantly stimulated the repopulation of living cells in their respective cultures. In HT29 and Panc1 cells, radiation significantly inhibited Wnt activity. In the irradiated dying HT29 and Panc1 cells, the level of the activated nuclear β-catenin was significantly decreased. Treatment with the Wnt agonist 68166 significantly decreased, whereas treatment with Wnt antagonist significantly increased, repopulation in HT29 and Panc1 tumor cells in a dose-dependent manner. β-catenin short-hairpin RNA (shRNA) also significantly promoted tumor cell repopulation. The level of secreted frizzled related protein-1 (SFRP1), hedgehog and Gli1 were increased in irradiated cells. Our results highlight the interaction between Wnt and SHH signaling pathways in dying tumor cells and suggest that downregulation of Wnt signaling after SHH activation is negatively associated with tumor repopulation.KEY WORDS: Colon cancer, Pancreatic cancer, Radiotherapy, Repopulation, Wnt signaling     

Nuclear Roles in the Post-Conjugant Development of the Ciliate Euplotes aediculatus II. Experimentally Induced Regeneration of Old Macronuclear Fragments1

Following conjugation in ciliates, the usual fate of the old pre-conjugant macronucleus is resorption. In some species, however, old macronuclei, or their fragments, have the ability to reform functional vegetative macronuclei when new macronuclear anlagen are defective. The present work on Euplotes shows that if anlagen are allowed to carry out their essential roles in early exconjugant development, including influence on cortical reorganization such that feeding can resume, they can then be permanently damaged by UV-microbeam irradiation and regeneration of old macronuclear fragments can occur. E. aediculatus exconjugants were anlage-irradiated at 40–60 hr of development and the irradiated cells cultured individually and fed. Squashes revealed enlargement and anteriorward migration of the persistent (posterior) macronuclear fragments. The first post-conjugant fission of such cells was delayed (times ranged 6–43 days) and did not seem to involve the damaged anlagen, which remained rudimentary, did not divide along with the cells, and were subsequently resorbed. It appeared that cell fission was supported by the fragments of the old macronuclei, which either divided or partitioned themselves between the two daughter cells. Mating tests performed on early clones derived from irradiated exconjugants revealed ample conjugation competence; intraclonal conjugation in such clones was also apparent. The absence of the immature period seen in normal exconjugants provides further evidence that the clones arose from cells with regenerated macronuclei.     

The quest for the ancestor of the Ciliophora: a brief review of the continuing problem

Although the assumption has long been made that the Ciliophora arose from flagellates, limited progress has been made in determining exactly what extant group - flagellate or other - may best resemble the ancestral "preciliate" assemblage. The distinctiveness of the ciliates and of the many protist phyla containing flagellated stages in their life cycles makes the recognition of potentially homologous features difficult. The suggestion that there may be a phylogenetic relationship between dinoflagellates and ciliates, while seeming unlikely from consideration of a number of specialized characters, is attracting increased attention. This is because some basic similarities (possibly shared derived characters) are characteristic of many species from both groups: cortical alveoli, tubular mitochondrial cristae, kinetidal systems, acentric mitoses with persisting nuclear envelope, functional cytostome, extrusomes (mucocytsts and explosive trichocysts), locomotory organelles, and small subunit rRNAs (close structural similarity values). But many questions remain unanalyzed or unanswered, and caution is still advisable in drawing any firm conclusions about the evolutionary closeness of ciliates and dinoflagellates.     

Degradation of the nuclear matrix is a common element during radiation-induced apoptosis and necrosis

Human promyelocytic leukemia (HL60) cells were irradiated with 10 or 50 Gy of X rays and studied for up to 72 h postirradiation to determine the mode of death and assess changes in the nuclear matrix. After 50 Gy irradiation, cells were found to die early, primarily by apoptosis, while cells irradiated with 10 Gy died predominantly by necrosis. Disassembly of the nuclear lamina and degradation of the nuclear matrix protein lamin B occurred in cells undergoing radiation-induced apoptosis or necrosis. However, using Western blotting and a recently developed flow cytometry assay to detect changes in nuclear matrix protein content, we found that the kinetics and mechanisms of disassembly of the nuclear lamina are different for each mode of cell death. During radiation-induced apoptosis, cleavage and degradation of lamin B to a approximately 28-kDa fragment was detected in most cells within 4-12 h after irradiation. Measurements of dual-labeled apoptotic cells revealed that nonrandom DNA fragmentation was evident prior to or concomitant with breakdown of the nuclear lamina. Disassembly of the nuclear lamina during radiation-induced necrosis occurred much later (between 30-60 h after irradiation), and a different cleavage pattern of lamin B was observed. Degradation of the nuclear lamina was also inhibited in apoptosis-resistant BCL2-overexpressing HL60 cells exposed to 50 Gy until approximately 48 h after irradiation. These data indicate that breakdown of the nuclear matrix may be a common element in radiation-induced apoptosis and necrosis, but that the mechanisms and temporal patterns of breakdown of the nuclear lamina during apoptosis are distinct from those of necrosis.     

Experimental set up for the irradiation of biological samples and nuclear track detectors with UV C

Aim

In this work we present a methodology to produce an “imprint” of cells cultivated on a polycarbonate detector by exposure of the detector to UV C radiation.

Background

The distribution and concentration of ¹⁰B atoms in tissue samples coming from BNCT (Boron Neutron Capture Therapy) protocols can be determined through the quantification and analysis of the tracks forming its autoradiography image on a nuclear track detector. The location of boron atoms in the cell structure could be known more accurately by the simultaneous observation of the nuclear tracks and the sample image on the detector.

Materials and Methods

A UV C irradiator was constructed. The irradiance was measured along the lamp direction and at different distances. Melanoma cells were cultured on polycarbonate foils, incubated with borophenylalanine, irradiated with thermal neutrons and exposed to UV C radiation. The samples were chemically attacked with a KOH solution.

Results

A uniform irradiation field was established to expose the detector foils to UV C light. Cells could be seeded on the polycarbonate surface. Both imprints from cells and nuclear tracks were obtained after chemical etching.

Conclusions

It is possible to yield cellular imprints in polycarbonate. The nuclear tracks were mostly present inside the cells, indicating a preferential boron uptake.