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Mild cognitive impairment (MCI) is considered a nosological entity or a translational state between normal aging and sporadic Alzheimer's disease (AD). From brain tissue to peripheral blood samples, it is evident that the early markers of metabolic dysfunction observed in AD have also been found in MCI subjects. These observations obtained from MCI and AD subjects leave open the possibility that mitochondrial dysfunction-induced oxidative damage happening a priori of symptom onset, may trigger other pathological hallmarks, namely Aβ oligomerization. In this study, we used a citoplasmic hybrid (cybrid) model created by the repopulation of human teratocarcinoma (NT2) cells depleted of endogenous mitochondrial DNA (mtDNA) with platelets from age-matched controls, MCI and AD subjects. We found mitochondrial deficits in MCI and AD cybrids as compared with controls, such as a decrease in cytochrome c oxidase (COX) activity, a decrease in mitochondrial membrane potential and in mitochondrial cytochrome c content. Consequently, we analyzed parameters of oxidative damage and found that AD and MCI cybrids exhibit an increase in lipid peroxides, higher production of superoxide radicals, and higher content in protein carbonyls. Since our data clearly show alterations in mitochondrial-mediated oxidative damage in MCI cybrids we propose that mitochondrial dysfunction is an early event in idiopathic AD. Moreover, we found that mitochondrial Aβ oligomeric content increases in AD, which may exacerbate initial mitochondrial damage. Altogether, our data strongly supports a key role for mitochondria/ mtDNA in aged-driven AD pathology.

Background: Lewy bodies (LB) are a neuropathological hallmark of Parkinson's disease (PD) and other synucleinopathies. The role their formation plays in disease pathogenesis is not well understood, in part because studies of LB have been limited to examination of post-mortem tissue. LB formation may be detrimental to neuronal survival or merely an adaptive response to other ongoing pathological processes. In a human cytoplasmic hybrid (cybrid) neural cell model that expresses mitochondrial DNA from PD patients, we observed spontaneous formation of intracellular protein aggregates ("cybrid LB" or CLB) that replicate morphological and biochemical properties of native, cortical LB. We studied mitochondrial morphology, bioenergetics and biogenesis signaling by creating stable sub-clones of three PD cybrid cell lines derived from cells expressing CLB., Results: Cloning based on CLB expression had a differential effect on mitochondrial morphology, movement and oxygen utilization in each of three sub-cloned lines, but no long-term change in CLB expression. In one line (PD63(CLB)), mitochondrial function declined compared to the original PD cybrid line (PD63(Orig)) due to low levels of mtDNA in nucleoids. In another cell line (PD61(Orig)), the reverse was true, and cellular and mitochondrial function improved after sub-cloning for CLB expression (PD61(CLB)). In the third cell line (PD67(Orig)), there was no change in function after selection for CLB expression (PD67(CLB))., Conclusions: Expression of mitochondrial DNA derived from PD patients in cybrid cell lines induced the spontaneous formation of CLB. The creation of three sub-cloned cybrid lines from cells expressing CLB resulted in differential phenotypic changes in mitochondrial and cellular function. These changes were driven by the expression of patient derived mitochondrial DNA in nucleoids, rather than by the presence of CLB. Our studies suggest that mitochondrial DNA plays an important role in cellular and mitochondrial dysfunction in PD. Additional studies will be needed to assess the direct effect of CLB expression on cellular and mitochondrial function.

Embryogenic callus formation in different larch species from Siberia (Larix sibirica, L. gmelinii, and L. sukaczewii) was carried out on MSGm medium supplemented with growth regulators (2.4-D and BAP) and followed one and the same scheme: elongation of somatic cells and their asymmetric division with formation of initial and tube cells. The cells of embryo initial underwent sequential divisions and formed embryonic globules which caused the formation of somatic embryos. Somatic embryos became mature and germinated by addition of ABA and PEG into the medium. Long-term proliferating cell lines and regenerant plants were obtained in Sukachev larch and its hybrid with Siberian larch. The success of somatic embryogenesis depended on the genotype of the donor tree.

Previously we reported that endoplasmic reticulum (ER)-mitochondria crosstalk is involved in amyloid-β (Aβ)-induced apoptosis. Now we show that mitochondrial dysfunction affects the ER stress response triggered by Aβ using cybrids that recreate the defect in mitochondrial cytochrome c oxidase (COX) activity detected in platelets from Alzheimer's disease (AD) patients. AD and control cybrids were treated with Aβ or classical ER stressors and the ER stress-mediated apoptotic cell death pathway was accessed. Upon treatment, we found increased glucose-regulated protein 78 (GRP78) levels and caspase-4 activation (ER stress markers) which were more pronounced in AD cybrids. Treated AD cybrids also exhibited decreased cell survival as well as increased caspase-3-like activity, poli-ADP-ribose-polymerase (PARP) levels and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)-positive apoptotic cells. Finally, we showed that Aβ-induced caspase-3 activation in both cybrid cell lines was prevented by dantrolene, thus implicating ER Ca(2+) release in ER stress-mediated apoptosis. Our results demonstrate that mitochondrial dysfunction occurring in AD patients due to COX inhibition potentiates cell susceptibility to Aβ-induced ER stress. This study further supports the close communication between ER and mitochondria during apoptosis in AD., (Copyright © 2012 Elsevier Inc. All rights reserved.)

The bone morphogenetic protein (BMP) signaling pathway regulates survival, proliferation, and differentiation of several cell types in multiple tissues, including the thymus. Previous reports have shown that BMP signaling negatively regulates T-cell development. Here, we study the subpopulation of early human intrathymic progenitors expressing the type IA BMP receptor (BMPRIA) and provide evidence that CD34(+)CD1a(-)BMPRIA(+) precursor cells mostly express surface cell markers and transcription factors typically associated with NK cell lineage. These CD34(+) cells mostly differentiate into functional CD56(+) natural killer (NK) cells when they are cocultured with thymic stromal cells in chimeric human-mouse fetal thymic organ cultures and also in the presence of SCF and IL-15. Moreover, autocrine BMP signaling can promote the differentiation of thymic NK cells by regulating the expression of key transcription factors required for NK cell lineage (eg, Id3 and Nfil3) as well as one of the components of IL-15 receptor, CD122. Subsequently, the resulting population of IL-15-responsive NK cell precursors can be expanded by IL-15, whose action is mediated by BMP signaling during the last steps of thymic NK cell differentiation. Our results strongly suggest that BMPRIA expression identifies human thymic NK cell precursors and that BMP signaling is relevant for NK cell differentiation in the human thymus.

In this research that was performed on isolated neurons of mollusk Lymnaea stagnalis, using neuron enucleation, the cytoplast was obtained which was then fused with another neuron resulting in cybrid formation. The experiments performed have shown that the isolated neurons are able to fuse with each other, forming binuclear neurons; also, like all other cells, they could be enucleated with the formation of cyto- and karyoplasts and, after fusion, they can form cell body-cytoplast, cytoplasts-karyoplast, and other complexes. This is associated with the appearance of all doubtless indicators of fusion described for fusion of nerve cell bodies. This work demonstrates the possibility to artificially fuse the amputated neuroplasm fragment with neuronal cell body--the metabolic center of another cell. Theoretically, this means that in vivo amputated neuronal process also can be fused with a novel cell.

Background: Direct damage to DNA is generally accepted as the main initiator of mutation and cancer induced by environmental carcinogens or ionising radiation. However, there is accumulating evidence suggesting that extracellular/extranuclear targets may also have a key role in mediating the genotoxic effects of ionising radiation. As the possibility of a particle traversal through the cytoplasm is much higher than through the nuclei in environmental radiation exposure, the contribution to genotoxic damage from cytoplasmic irradiation should not be ignored in radiation risk estimation. Although targeted cytoplasmic irradiation has been shown to induce mutations in mammalian cells, the precise mechanism(s) underlying the mutagenic process is largely unknown., Methods: A microbeam that can target the cytoplasm of cells with high precision was used to study mechanisms involved in mediating the genotoxic effects in irradiated human-hamster hybrid (A(L)) cells., Results: Targeted cytoplasmic irradiation induces oxidative DNA damages and reactive nitrogen species (RNS) in A(L) cells. Lipid peroxidation, as determined by the induction of 4-hydroxynonenal was enhanced in irradiated cells, which could be suppressed by butylated hydroxyl toluene treatment. Moreover, cytoplasmic irradiation of A(L) cells increased expression of cyclooxygenase-2 (COX-2) and activation of extracellular signal-related kinase (ERK) pathway., Conclusion: We herein proposed a possible signalling pathway involving reactive oxygen/nitrogen species and COX-2 in the cytoplasmic irradiation-induced genotoxicity effect.

Two dozen hybrid clones were produced by fusion of diploid embryonic stem (ES) cells positive for green fluorescent protein (GFP) with tetraploid fibroblasts derived from DD/c and C57BL-I(I)1RK mice. Cytogenetic analysis demonstrated that most cells from these hybrid clones contained near-hexaploid chromosome sets. Additionally, the presence of chromosomes derived from both parental cells was confirmed by polymerase chain reaction (PCR) analysis of polymorphic microsatellites. All hybrid cells were positive for GFP and demonstrated growth characteristics and fibroblast-like morphology. In addition, most hybrid cells were positive for collagen type I, fibronectin, and lamin A/C but were negative for Oct4 and Nanog proteins. Methylation status of the Oct4 and Nanog gene promoters was evaluated by bisulfite genomic sequencing analysis. The methylation sites (CpG-sites) of the Oct4 and Nanog gene promoters were highly methylated in hybrid cells, whereas the CpG-sites were unmethylated in the parental ES cells. Thus, the fibroblast genome dominated the ES genome in the diploid ES cell/tetraploid fibroblast hybrid cells. Immunofluorescent analysis of the pluripotent and fibroblast markers demonstrated that establishment of the fibroblast phenotype occurred shortly after fusion and that the fibroblast phenotype was further maintained in the hybrid cells. Fusion of karyoplasts and cytoplast derived from tetraploid fibroblasts with whole ES cells demonstrated that karyoplasts were able to establish the fibroblast phenotype of the reconstructed cells but not fibroblast cytoplasts. Thus, these data suggest that the dominance of parental genomes in hybrid cells of ES cell/somatic cell type depends on the ploidy of the somatic partner.

The intermediate filament (IF) proteins have been recently found as dynamic structures that influence several aspects of cell homeostasis. Here, two alternative approaches to study the dynamics of IF proteins are described: the formation of cell hybrids by the fusion of different parental cells, and the transfection of keratin genes in cultured cells. In the first case, the selection of parental cell lines and the use of specific antibodies allow us to study how IF proteins recombine and copolymerize to form the heterokaryon cytoskeleton by immunofluorescence. In the second approach, some modifications of conventional transfection protocols allow the synchronized expression conditions, making it suitable for the analysis of the incorporation of a newly synthesized IF protein into the preexisting IF cytoskeleton of transfected cells.

In the present work we examined the status of nucleolus organizing regions of mitotic chromosomes (NOR) in hybrid cells obtained by fusion of the mouse teratocarcinoma cells PCC4aza1 and adult mouse spleenocytes upon cultivation of hybrid cells under different conditions. We have shown that extended cultivation of hybrid cells in medium supplemented with HAT (hypoxanthine, aminopterin, thymidine) promotes the maintenance of NO-chromosomes, whereas under nonselective conditions elimination of NO-chromosome occurs. In nonselective medium the number of active, i. e. Ag-positive, NORs has been augmented comparatively to that observed under selective conditions. This observation directly indicates that reprogramming of the parental cell genomes in hybrid cells includes changes in the status of chromosomal NORs. The number of active NORs depends on conditions of hybrid cells culturing and may be changed by either of the two major ways--by elimination of NO-chromosomes (under nonselective conditions) or by inactivation of some NORs, when the general number of NO-chromosomes remains unaltered (under selective conditions).

When hybrid cells are created, not only nuclear genomes of parental cells unite but their cytoplasm as well. Mitochondrial DNA (mtDNA) is a convenient marker of cytoplasm allowing one to gain insight into the organization of hybrid cell cytoplasm. We analyzed the parental mtDNAs in hybrid cells resulting from fusion of Mus musculus embryonic stem (ES) cells with splenocytes and fetal fibroblasts of DD/c mice or with splenocytes of M. caroli. Identification of the parental mtDNAs in hybrid cells was based on polymorphism among the parental mtDNAs for certain restrictases. We found that intra- and inter-specific ES cell-splenocyte hybrid cells lost entirely or partially mtDNA derived from the somatic partner, whereas ES cell-fibroblast hybrids retained mtDNAs from both parents in similar ratios with a slight bias. The lost of the "somatic" mitochondria by Es-splenocyte hybrids implies non-random segregation of the parental mitochondria as supported by a computer simulation of genetic drift. In contrast, ES cell-fibroblast hybrids show bilateral random segregation of the parental mitochondria judging from analysis of mtDNA in single cells. Preferential segregation of "somatic" mitochondria does not depend on the differences in sequences of the parental mtDNAs but depends on replicative state of the parental cells.

Diesel exhaust particle (DEP) has been identified as a class 2A human carcinogen and closely related to the increased incidence of respiratory allergy, cardiopulmonary morbidity and mortality, and risk of lung cancer. However, the molecular mechanisms of DEP mutagenicity/carcinogenicity are still largely unknown. In the present study, we focused on the mutagenicity of DEPs in human-hamster hybrid (A(L)) cells and evaluated the role of cell-particle interaction in mediating mutagenic process. We found that DEPs formed micron-sized aggregates in the medium and located mainly in large cytoplasmic vacuoles of cells by 24h treatment. The cellular granularity was increased by DEP treatment in a dose-dependent manner. DEPs resulted in a dose-dependent increase of mutation yield at CD59 locus in A(L) cells, while inflicting minimal cytotoxicity. There was a more than two-fold increase of mutation yield at CD59 locus in A(L) cells exposed to DEPs at a dose of 50mug/ml. Such induction was significantly reduced by concurrent treatment with phagocytosis inhibitors, cytochalasin B and ammonium chloride (p<0.05). These results provided direct evidence that DEPs was mutagenic in mammalian cells and that cell-particle interaction played an essential role in the process.

Reactive nitrogen and oxygen species (O2*-, H2O2, NO* and ONOO-) have been strongly implicated in the pathophysiology of neurodegenerative and mitochondrial diseases. In the present study, we examined the effects of nitrosative and/or nitrative stress generated by DETA-NO {(Z)-1-[2-aminoethyl-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate}, SIN-1 (3-morpholinosydnonimine hydrochloride) and SNP (sodium nitroprusside) on U87MG glioblastoma cybrids carrying wt (wild-type) and mutant [A3243G (Ala3243-->Gly)] mtDNA (mitochondrial genome) from a patient suffering from MELAS (mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-like episodes). The mutant cybrids had reduced activity of cytochrome c oxidase, significantly lower ATP level and decreased mitochondrial membrane potential. However, endogenous levels of reactive oxygen species were very similar in all cybrids regardless of whether they carried the mtDNA defects or not. Furthermore, the cybrids were insensitive to the nitrosative and/or nitrative stress produced by either DETA-NO or SIN-1 alone. Cytotoxicity, however, was observed in response to SNP treatment and a combination of SIN-1 and glucose-deprivation. The mutant cybrids were significantly more sensitive to these insults compared with the wt controls. Ultrastructural examination of dying cells revealed several characteristic features of autophagic cell death. We concluded that nitrosative and/or nitrative stress alone were insufficient to trigger cytotoxicity in these cells, but cell death was observed with a combination of metabolic and nitrative stress. The vulnerability of the cybrids to these types of injury correlated with the cellular energy status, which were compromised by the MELAS mutation.

The multicolor-banding (mcb) technique is a fluorescence in situ hybridization (FISH)-banding approach, which is based on region-specific microdissection libraries producing changing fluorescence intensity ratios along the chromosomes. The latter are used to assign different pseudocolors to specific chromosomal regions. Here we present the first three available mcb-probe sets for the Mus musculus chromosomes 3, 6, and 18. In the present work, the creation of the microdissection libraries was done for the first time on mouse/human somatic cell hybrids. During creation of the mcb-probes, the latter enabled an unambiguous identification of the, otherwise in GTG-banding, hardly distinguishable murine chromosomes.

Chromosome segregation of the parental chromosomes was studied in 20 interspecific hybrid clones obtained by fusion of Mus musculus embryonic stem cells with Mus caroli splenocytes. FISH analysis with labeled species specific probes and microsatellite markers was used for identification of the parental chromosomes. Cytogenetic analysis has shown significant intra- and interclonal variability in chromosome numbers and ratios of the parental chromosomes in the hybrid cells: six clones contained all M. caroli chromosomes, nine clones showed moderate segregation of M. caroli chromosomes (from 1 to 7), and five clones showed extensive loss of M. caroli chromosomes (from 12 to complete loss of all M. caroli autosomes). Both methods demonstrated "cryptic" segregation of the somatic partner chromosomes. For instance, five clones with near-tetraploid chromosome sets contained only few M. caroli chromosomes (from 1 to 8). The data obtained suggest that the tetraploid chromosome set per se is not a sufficient criterion for conclusion on the absence of chromosome loss in the hybrid cells. Note that "cryptic" chromosome segregation occurred at a high frequency in the examined hybrid clones. Thus, "cryptic" segregation should be borne in mind for assessing pluripotency and genome reprogramming of embryonic stem hybrid cells.

In the mid-1960s the #620 cell passage line of a murine lymphoma-leukemia was developed at the Section of Clinical Tumor Virology and Immunology, Department of Medicine, The University of Texas M.D. Anderson Hospital in Houston, TX. The diploid lymphoma cells released a retrovirus and were antigenic in young adult Swiss (YAS) mice. Small lymphoma cell inocula were rejected with immunity acquired against large inocula of lymphoma cells. Tissue sections revealed the "starry sky" configurations. In one of the tissue cultures set up from lymphoma #620, a cell line consisting of large round poly- or tetraploid cells arose and was referred to as lymphoma cell line #818. The #818 cells grew in suspension cultures and in the form of large, lethal ascitic tumors in YAS mice. Diploid #620 lymphoma cells stained for retroviral antigens; #818 cells stained both for retroviral antigens and immunoglobulins. Fluids withdrawn from #818 cultures neutralized the leukemia virus in spleen focus assays. Immunoglobulin precipitated from #818 suspension culture fluids strongly and specifically neutralized the leukemia virus. The growth of #620 or #818 cells in YAS mice treated with rabbit anti-lymphoma cell immune sera was strongly inhibited but culture fluids of #818 cells showed weak and insignificant inhibition against leukemia-lymphoma #620 (in one experiment, unpublished). In two experiments #620 lymphoma cells were co-inoculated with immune spleen cells into the peritoneal cavities of YAS mice. The immune spleen cells derived from mice that rejected #620 cell inocula or were actively immunized with a photodynamically inactivated mouse leukemia virus vaccine. In the peritoneal cavities of mice co-inoculated with #620 cells and immune spleen cells, clones of large round cells emerged with tetra- or polyploid chromosomal modes. These cells stained for leukemia viral antigens and immunoglobulins. When passaged in YAS mice these cells induced lethal ascites tumors. It was concluded as early as in 1968-69 that an immune plasma cell can fuse with a lymphoma cell, if the lymphoma cell expresses retroviral antigens against which the plasma cell is producing a specific antibody. Some human lymphoma-leukemia cells express retroviral antigens and/or budding retroviral particles, whether due to the acquisition of new env sequences by incomplete resident endogenous retroviral genomes or due to the entry of exogenous retroviruses into lymphopoietic stem cells. In the Discussion illustrations are provided for the human cell line #778 established from a patient with "lymphosarcoma cell leukemia" in 1966. The malignant cells released unidentified retrovirus-like particles and fused with one another and with reactive lymphoid cells of the host. It should be investigated further if human lymphoma-leukemia cells could fuse with an immune plasma cell of the host and thus alter the clinical course of the disease.

Background: Adult human peripheral blood CD34-positive (CD34+) cells appear to transform into cardiomyocytes in the injured hearts of severe combined immunodeficient mice. It remains unclear, however, whether the apparent transformation is the result of transdifferentiation of the donor stem cells or of fusion of the donor cell with the cardiomyocyte of the recipients., Methods and Results: We performed flow cytometry analyses of cells isolated from the hearts of mice that received human CD34+ cells. Human HLA-ABC antigen and cardiac troponin T or Nkx2.5 were used as markers for cardiomyocytes derived from human CD34+ cells, and HLA-ABC and VE-cadherin were used to identify the transformed endothelial cells. The double-positive cells were collected and interphase fluorescence in situ hybridization was used to detect the expression of human and mouse X chromosomes in these cells. We found that 73.3% of nuclei derived from HLA+ and troponin T+ or Nkx2.5+ cardiomyocytes contain both human and mouse X chromosomes and 23.7% contain only human X chromosome. In contrast, the nuclei of HLA-, troponin T+ cells contain only mouse X chromosomes. Furthermore, 97.3% of endothelial cells derived from CD34+ cells contained human X chromosome only., Conclusions: Thus, both cell fusion and transdifferentiation may account for the transformation of peripheral blood CD34+ cells into cardiomyocytes in vivo.

Following hybridization with embryonic stem (ES) cells, somatic genomes are epigenetically reprogrammed and acquire pluripotency. This results in the transcription of somatic genome-derived tissue-specific genes upon differentiation. During nuclear reprogramming, it is expected that DNA and chromatin modifications, believed to function in cell-type-specific epigenotype memory, should be significantly modified. Indeed, current evidence indicates that acetylation and methylation of histone H3 and H4 amino termini play a major role in the regulation of gene activity through the modulation of chromatin conformation. Here, we show that the reprogrammed somatic genome of ES hybrid cells becomes hyperacetylated at H3 and H4, while lysine 4 (K4) of H3 becomes globally hyper-di- and -tri-methylated. In the Oct4 promoter region, histones H3 and H4 are acetylated and H3-K4 is highly tri-methylated on both the ES and reprogrammed somatic genomes, which correlates with gene activation and DNA demethylation. However, H3-K4 is also di- and tri-methylated in the promoter regions of Neurofilament-M (Nfm), Nfl, and Thy-1, which are all silent in both ES and hybrid cells. Thus, H3-K4 di- and tri-methylation of reprogrammed somatic genomes is independent of gene activity and represents one of the major events that occurs during somatic genome reprogramming towards a transcriptional activation-permissive state.

We created and studied new cybrid cell lines from sporadic Alzheimer's disease (SAD) or control (CTL) subjects to assess mitochondrial abnormalities just after metabolic selection ("early passage") and again six passages later ("late passage"). Cytochrome oxidase (CO) activities in early passage SAD cybrids created independently from the same platelet samples were highly correlated. Early passage SAD and CTL cybrids showed equivalent mitochondrial morphologies. Late passage SAD cybrids showed increased mitochondrial number, reduced mitochondrial size, and an approximately eightfold increase in morphologically abnormal mitochondria. Deficiency of SAD cybrid mitochondrial membrane potentials (DeltaPsi(M)) increased with passage. Mitochondrial bromodeoxyuridine (BrdU) uptake to estimate mitochondrial DNA (mtDNA) synthesis did not change with passage in CTL but increased in SAD cybrids. With time in culture, SAD mtDNA appears to replicate faster in cybrids, yielding cells with relative worsening of bioenergetic function. Metabolically deleterious SAD mitochondrial genes, like those in yeast, may have a replicative advantage over nondeleterious mitochondrial genes that assume dominance in CTL cybrids.

The human-Chinese hamster hybrid cell line XR-C1#8, containing human chromosome 8, was used as a model system to investigate the relative importance of cellular enzymatic environment and chromosomal structure for modulating the efficiency of repair of UV-induced DNA damage. The hybrid cells were irradiated with UVC light and the extent of cytogenetic damage, detected as frequencies of sister chromatid exchanges (SCEs), was compared between the human and the hamster chromosomes. The combination of immunofluorescent staining for SCEs and chromosome painting with fluorescence in situ hybridization allowed the simultaneous analysis of SCEs in the human and hamster chromosomes. The aim of the present study was to determine if the differences in biological response to comparable UV treatments observed between human and hamster cells were maintained in the hybrid cells in which human and hamster chromosomes are exposed in the same cellular environment. The analysis of replication time of human chromosome 8 indicated the active status of this chromosome in XR-C1#8 hybrid cells. The frequencies of SCEs for human chromosome 8 and a hamster chromosome of comparable size were 0.35 +/- 0.52, 0.80 +/- 0.73, 1.24 +/- 2.24 and 0.36 +/- 0.12, 0.71 +/- 0.2, 0.97 +/- 0.27, respectively, after irradiation with 0, 5, and 10 J/m2. The persistence of UV-induced SCEs after three cell cycles was also analyzed, both for the human and hamster chromosomes. The observed frequencies of SCEs were 0.40 +/- 0.57, 0.62 +/- 1.05, 0.58 +/- 0.83 and 0.26 +/- 0.08, 0.67 +/- 0.18, 0.69 +/- 0.24, in human and hamster chromosomes respectively, after treatment with 0, 10, and 20 J/m2 of UVC light. No significant differences could be observed between the human and hamster chromosomes. These results suggest that the enzymatic environment of human and hamster cells has the main role, in comparison to the structural organization of human and hamster chromosomes, for determining the different level of repair of UV-induced DNA damage observed in these two species., (Copyright 2003 S. Karger AG, Basel)

The detrimental effects associated with exposure to ionizing radiation have long been thought to result from the direct targeting of the nucleus leading to DNA damage; however, the emergence of concepts such as radiation-induced genomic instability and bystander effects have challenged this dogma. After cellular exposure to ionizing radiation, we have isolated a number of clones of Chinese hamster-human hybrid GM10115 cells that demonstrate genomic instability as measured by chromosomal destabilization. These clones show dynamic and persistent generation of chromosomal rearrangements multiple generations after the original insult. We hypothesize that these unstable clones maintain this delayed instability phenotype by secreting factors into the culture medium. To test this hypothesis we transferred filtered medium from unstable cells to unirradiated GM10115 cells. No GM10115 cells were able to survive this medium. This phenomenon by which GM10115 cells die when cultured in medium from chromosomally unstable GM10115 clones is the death-inducing effect. Medium transfer experiments indicate that a factor or factors is/are secreted by unstable cells within 8 h of growth in fresh medium and result in cell killing within 24 h. These factors are stable at ambient temperature but do not survive heating or freezing, and are biologically active when diluted with fresh medium. We present the initial description and characterization of the death-inducing effect. This novel epigenetic effect of radiation has implications for radiation risk assessment and for health risks associated with radiation exposure.

Transplantation of allogenic bone marrow from HLA-identical sibs to patients with acute and chronic leukemia receiving immunosuppressive therapy is associated with the appearance of erythrocytes simultaneously carrying donor and recipient antigenic markers: AB0 system, rhesus factor and its subtypes, M and N antigens. Integration of genes responsible for each antigen is realized independently presumably at the level of stem cell, which ensures long-term (>3 years) repopulation of these erythrocytes. Experiments on inbred mice showed that transplantation of allogenic bone marrow is associated with an increase of chromosome number in 39% bone marrow cells 4 days after transplantation, which indicate the possibility of integration of whole chromosomes.

Control over neuronal growth is a fundamental objective in neuroscience, cell biology, developmental biology, biophysics, and biomedicine and is particularly important for the formation of neural circuits in vitro, as well as nerve regeneration in vivo [Zeck, G. & Fromherz, P. (2001) Proc. Natl. Acad. Sci. USA 98, 10457-10462]. We have shown experimentally that we can use weak optical forces to guide the direction taken by the leading edge, or growth cone, of a nerve cell. In actively extending growth cones, a laser spot is placed in front of a specific area of the nerve's leading edge, enhancing growth into the beam focus and resulting in guided neuronal turns as well as enhanced growth. The power of our laser is chosen so that the resulting gradient forces are sufficiently powerful to bias the actin polymerization-driven lamellipodia extension, but too weak to hold and move the growth cone. We are therefore using light to control a natural biological process, in sharp contrast to the established technique of optical tweezers [Ashkin, A. (1970) Phys. Rev. Lett. 24, 156-159; Ashkin, A. & Dziedzic, J. M. (1987) Science 235, 1517-1520], which uses large optical forces to manipulate entire structures. Our results therefore open an avenue to controlling neuronal growth in vitro and in vivo with a simple, noncontact technique.

The alpha-synuclein gene is implicated in the pathogenesis of Parkinson's disease. Although alpha-synuclein function is uncertain, the protein has homology to the chaperone molecule 14-3-3. In addition, alpha-synuclein can bind to 14-3-3, and both alpha-synuclein and 14-3-3 bind to many of the same proteins. Because 14-3-3 binds to and activates tyrosine hydroxylase, the rate-limiting enzyme in dopamine (DA) biosynthesis, we explored whether alpha-synuclein also bound to tyrosine hydroxylase and influenced its activity. Immunoprecipitation revealed an interaction between alpha-synuclein and tyrosine hydroxylase in brain homogenates and MN9D dopaminergic cells. Colocalization of alpha-synuclein with tyrosine hydroxylase was confirmed by immunoelectron microscopy. To explore the consequences of the interaction, we measured the effect of recombinant alpha-synuclein on tyrosine hydroxylase activity in a cell-free system and observed a dose-dependent inhibition of tyrosine hydroxylase by alpha-synuclein. To measure the impact of alpha-synuclein on tyrosine hydroxylase in dopaminergic cells, we stably transfected MN9D cells with wild-type or A53T mutant alpha-synuclein. Overexpression of wild-type or A53T mutant alpha-synuclein did not significantly alter tyrosine hydroxylase protein levels in our stably transfected cells. However, overexpressing cell lines had significantly reduced tyrosine hydroxylase activity and a corresponding reduction in dopamine synthesis. The reduction in cellular dopamine levels was not caused by increased dopamine catabolism or dopamine efflux. These data suggest that alpha-synuclein plays a role in the regulation of dopamine biosynthesis, acting to reduce the activity of tyrosine hydroxylase. If so, a loss of soluble alpha-synuclein, by reduced expression or aggregation, could increase dopamine synthesis with an accompanying increase in reactive dopamine metabolites.

We present evidence of hybridization between Meth A sarcoma cells and syngeneic as well as semigeneic peritoneal macrophages. The resultant hybrids are characterized by morphology, membrane markers, ploidy, chromosomal content and functional features. Briefly, after a few days of coculture, cells appeared with morphology intermediate between the 2 original cell types. Typical macrophage surface molecules appeared in the hybrids. Meth A cells were labeled with red fluorescence and macrophages with green fluorescence. After 4 days in vitro, hybrids with yellow fluorescence appeared. Macrophages from BALB.K mice (H-2 K(k)) were cocultivated with Meth A cells from BALB/c mice (H-2 K(d)). The semigeneic hybrids displayed both specificities, as demonstrated by flow cytometry. The hybrids appeared moderately phagocytic, less so than the macrophages and markedly more so than the essentially nonphagocytic Meth A cells. The hybrids had a mean number of 76 chromosomes, as opposed to 53 in the Meth A cells and 40 in the macrophages. The macrophage DNA index was set at 1; Meth A cells were found to have an index of 1.6 in G1 phase, and the hybrids had a 2.6 index. The hybrids grew more slowly in vitro than Meth A cells, but grew faster in vivo., (Copyright 2002 Wiley-Liss, Inc.)

To understand the relationship between the location of organelles and cellular function, we examined the dynamic state of cytoplasmic organelles and cytoskeleton in polynuclear Ehrlich ascites tumor (EAT) cells fused with hemagglutinating virus of Japan (HVJ; Sendai virus) by confocal laser scanning microscopy. Irregular fused cells gradually became spherical during culture, and nuclei and mitochondria were redistributed in the fused cell; nuclei formed a cluster surrounded by mitochondria. F-actin, vimentin, and microtubules were also reorganized with the redistribution of cell organelles. Further, when the morphological change was inhibited by L4-1, a chlorophyll-like substance derived from silkworm faeces, or pyropheophorbide-a, the arrangement of organelles and cytoskeleton remained disturbed, suggesting that the movement of the cytoskeleton is closely associated with cell shape and the distribution of cytoplasmic organelles., (Copyright 2002 Elsevier Science Ltd. All rights reserved.)

Rat brain presynaptic 5-HT3 serotonin receptors, members of the ligand-gated ion channel superfamily, induce changes in nerve terminal [Ca2+]i in a manner distinct from that found for somatic 5-HT3 receptors. Here, we assessed the role of postsynaptic target in regulating the nature of presynaptic receptor-induced responses, using the hybrid neuroblastoma cell line NG108-15 as a model neuronal system that expresses 5-HT3 receptors. Using immunocytochemistry, 5-HT3 receptors were found to be present on the presynaptic-like varicosities of differentiated NG108-15 cells, indicating that these receptors possess an inherent ability to localize to potential presynaptic sites. In the absence of postsynaptic target, 5-HT3 receptors localized to the varicosities induce rapid but transient changes in [Ca2+]i that were initiated by voltage-gated Ca2+ channels, as assessed using Ca2+ channel blockers, these properties being typical of those found for somatic 5-HT3 receptors. In co-cultures containing rat myotubes, with which NG108-15 cells form functional cholinergic synapses, the 5-HT3 receptor-induced changes in [Ca2+]i in the axonal varicosities shifted over time (three to 10 days) to that found for brain nerve endings: sustained responses that were insensitive to blockade by antagonists of voltage-gated Ca2+ channels. The effect of co-culturing myotubes with the NG108-15 cells was mimicked by conditioned media from myotube cultures. These results indicate that regulatory molecules from the target postsynaptic cell dictate the functional responses elicited by presynaptic 5-HT3 receptors. Because the target-induced changes required several days before they were evident, we hypothesize that changes in protein expression, perhaps the consequence of altered gene regulation, underlie the changes in the responses to 5-HT3 receptor activation in the axonal varicosities of this neuronal cell line.

The effects of several K(+)-selective neutral ionophores on membrane electrical characteristics of differentiated NG108-15 (neuroblastoma X glioma hybrid) cells were examined. Specifically, alterations in membrane resting potential (V(m)), input resistance (R(in)) and electrically-induced action potential generation were determined upon bath application of enniatin (0.1-10 microg/ml), nonactin (0. 1-10 microM) and valinomycin (0.1-10 microM). Although some cells exhibited a slight hyperpolarization and/or reduced R(in), i.e. membrane electrical correlates of enhanced K(+) loss, neither V(m) nor R(in) were significantly altered by any of the ionophores. However, valinomycin and especially nonactin affected action potentials induced by electrical stimulation. This was apparent in the ablation of action potentials in some cells and in the occurrence of degenerative changes in action potential shape in others. The simultaneous administration of the neutral ionophores and the protonophore CCCP or the superfusion of enniatin, nonactin or valinomycin in high (50 mM) glucose-containing physiological solution did not yield more extensive alterations in V(m) or R(in). These data suggest that the neutral ionophores are unable to materially enhance K(+) flux above the relatively high resting level in NG108-15 cells. Thus, alterations in action potentials appear to be unrelated to K(+) transport activity.

Radiation hybrid (RH) mapping has proven to be an extremely powerful approach to constructing high density maps of human chromosomes and is experiencing increased use in other animals, including cattle. A 5000 rad bovine whole-genome radiation hybrid panel was recently constructed in order to integrate existing cattle linkage maps with evolutionarily conserved genes and provide high resolution comparative maps relative to humans and mice. We utilized this panel to construct a 19 marker framework map of bovine chromosome 1 (BTA1), which included 8 Type I loci and 11 Type II loci ordered with at least 1000:1 odds. A 35 marker comprehensive map including 15 Type I loci and 20 Type II loci was also produced. Of the 15 Type I loci ordered on the comprehensive map, three are ordered on HSA3 and five are ordered in three blocks on HSA21 on the human cytogenetic maps.

Electron transport chain (ETC) dysfunction may arise from mitochondrial genetic, nuclear genetic, or toxic etiologies. Cytoplasmic hybrid (cybrid) systems can help distinguish between these possibilities by facilitating expression of suspect mitochondrial DNA (mtDNA) within a nuclear and environmentally controlled context. Perpetuation of ETC dysfunction in cybrids is consistent with an mtDNA pathogenesis while defect correction is not. We previously used cybrids to screen sporadic Parkinson's disease, Alzheimer's disease, and amyotrophic lateral sclerosis patients for mtDNA mutation with positive results. To further address the fidelity of these experiments, we created and characterized cybrids expressing mtDNA from persons with Huntington's disease (HD), an autosomal dominant, nuclear DNA-determined disorder in which mitochondrial ETC functioning is abnormal. On ETC, oxidative stress, and calcium homeostasis assays HD cybrid lines were indistinguishable from control cybrid lines. These data support the use of the cybrid technique for mtDNA mutation screening in candidate diseases., (Copyright 1999 Academic Press.)

The present study was aimed to facilitate karyotyping of human blastomeres using the metaphase-inducing factors present in unfertilized eggs. A rapid technique for karyotyping would have wide application in the field of preimplantation genetic diagnosis. When cryopreserved in-vitro matured bovine oocytes were fused with human blastomeres, the transferred human nuclei were forced into metaphase within a few hours. Eighty-seven human blastomeres from abnormal or arrested embryos were fused with bovine oocytes in a preclinical study. Fusion efficiency was 100%. In 21 of the hybrid cells, no trace of human chromatin was found. Of the remaining 66, 64 (97%) yielded chromosomes suitable for analysis. The method was used to karyotype embryos from two patients with maternal translocations. One embryo which was judged to be karyotypically normal was replaced in the first patient, resulting in one pregnancy with a normal fetus. None of the second patient's embryos was diagnosed as normal, and hence none was transferred. The results of the present study demonstrated that the ooplasmic factors which induce and maintain metaphase in bovine oocytes can force transferred human blastomere nuclei into premature metaphase, providing the basis for a rapid method of karyotyping blastomeres from preimplantation embryos and, by implication, cells from other sources.

Fusion of the highly tumorigenic Burkitt's lymphoma (BL) cell line BL60-P7 with the nontumorigenic autologous EBV-immortalized lymphoblastoid cell line (LCL) IARC 277 results in suppression of the tumorigenic phenotype of the parental cell line BL60-P7 after s.c. inoculation into T cell-deficient nude mice. We analyzed whether, after long-term cultivation of these lymphoma hybrid cells, expression of tumorigenicity could be observed and correlated to the loss of particular chromosomes or chromosomal fragments, akin to numerous nonlymphoid hybrid cell models described previously. Two years after fusion, in vitro proliferation of some BL x LCL hybrid cells accelerated, and they partially lost LCL-typical aggregation. However, no major changes in the expression pattern of B cell-associated surface antigens and the EBV latent membrane protein LMP 1 were observed. Cytogenetic evaluation of these cells revealed spontaneous loss of chromosomes. Karyotyping of long-term cultivated hybrid cells demonstrated the occurrence of disomy for each chromosome in at least one metaphase analyzed. Therefore, if suppression of tumorigenicity in these hybrid cells would have been the result of the presence of a single LCL-derived chromosome, there should have been a high probability of its loss, leading to tumorigenic segregants. Surprisingly, the tumorigenic phenotype remained suppressed in nude mice. To induce chromosomal breakage and maldistribution, in addition to spontaneous chromosomal loss, the hybrid cell lines were irradiated at various doses. Again, none of the hybrid cell clones treated in this manner became tumorigenic in nude mice. Immunohistological analysis of the regressing hybrid cell tumors revealed that the hybrid cells had retained their LCL-like differentiation phenotype in vivo. In addition, infiltration with mononuclear cells of murine origin was observed in these regressing hybrid grafts. We conclude that suppression of the tumorigenic Burkitt's lymphoma phenotype in these hybrid cells cannot be attributed to a function encoded by a distinct chromosome or chromosomal fragment. Rather, the unexpected stable nontumorigenic phenotype reflects a LCL-specific activated B-cell phenotype of these hybrids, most probably induced by the expression of numerous copies of episomal latent EBV proteins.

The assembly and function of respiratory-competent mitochondria in eukaryotic cells depends on collaboration between the nuclear and mitochondrial genomes, but the molecular mechanisms underlying such cross-talk are poorly understood. Microcell-mediated chromosome transfer has been used to transfer intact chromosomes from one mammalian cell to another, helping to map loci implicated in different diseases and in the senescence process. In the present work, we show that microcells have a significant number of mitochondria which can be transferred to another cell simultaneously with a limited number of chromosomes. By fusing microcells from a colon carcinoma cell line with a mitochondrial DNA (mtDNA)-less osteosarcoma cell line, we were able to isolate transmitochondrial hybrids containing only one of three selectable chromosomes and mtDNA from the donor cell. The proportion of transmitochondrial hybrids containing one chromosomal marker with respect to the total transmitochondrial hybrids and cybrids was approximately 1% and no hybrids were isolated containing more than one nuclear marker. The genetic data correlated well with the composition and structure of the microcell preparations, which showed the presence of cytoplast-like structures and microcells containing mitochondria surrounding the micronuclei. Microcell-mediated mtDNA and chromosome transfer can be used to identify nuclear factors implicated in mtDNA maintenance and gene expression, as well as to investigate nuclear factors which modulate clinical phenotypes in mitochondrial disorders.

A combination of microscopy (video-enhanced contrast microscopy) and spectrophotometry (which analyses online the spectra from the microscopic picture) is presented. This set-up allows the in vivo microscopic observation of cellular and subcellular structures as well as simultaneous online determination of cellular chromophores. To test our equipment we measured spectra of isolated red blood cells and NG 108-15 cells and correlated these spectra with the corresponding video-enhanced microscopic picture. By labelling specific cell organelles of astrocytes with in vivo fluorescent dyes, we visualized mitochondria and captured the corresponding spectra.

We have assembled a first-generation anchor map of the mouse genome using a panel of 94 whole-genome-radiation hybrids (WG-RHs) and 271 sequence-tagged sites (STSs). This is the first genome-wide RH anchor map of a model organism. All of the STSs have been previously localized on the genetic map and are located 8.8 Mb apart on average. This mouse WG-RH panel, known as T31, has an average retention frequency of 27.6% and an estimated potential resolution of 145 kb, making it a powerful resource for efficient large-scale expressed sequence tag mapping. [All of the mapping data for the maps presented here have been deposited at the Research Genetics, Inc., web site and can be freely accessed and downloaded at http://www.resgen.com/.]

We have previously found that human chromosome 3 was fragmented in the course of in vivo tumor growth of monochromosomal human/mouse (A9 fibrosarcoma parent) microcell hybrids in SCID mice. Marker analysis of tumor cell lines has identified a regularly eliminated 7 cM segment on 3p21.3 referred to as the common eliminated region (CER). The same region is frequently affected by LOH in a variety of human carcinomas. The present study is a comparative chromosome painting, reverse painting, and PCR marker analysis of microcell hybrids (MCHs) that originally contained an intact chromosome 3 from two alternative donors, during and after four passages in SCID mice. We found regular elimination of 3p in parallel with preferential retention of 3q. In addition to CER on 3p, we can now define a common retained region (CRR) on 3q. It includes eight markers between D3S1282 (3q25-q26) and D3S1265 (3q27-qter) and spans approximately 43 cM. These observations are concordant with the frequent loss of corresponding 3p regions and the frequent retention, with occasional amplification, of 3q in several types of human tumors.

The role of adhesion molecule L1 in synapse formation was examined by transient transfection of L1 cDNA in neuroblastoma x glioma hybrid NG108-15 cells. L1 overexpression was found in approximately 50% of the transfected NG108-15 cell population. Neurite outgrowth induced by 0.25 mM dibutyryl cyclic AMP (cAMP) was much greater in L1-transfected NG108-15 cells than that in nontransfected and mock-transfected cells. The proportion of cells with neurites and the number of neurites per cells were increased in L1-transfected cells after 2 days of dibutyryl cAMP treatment. The proportion of cells with branched neurites and the average length of neurites were higher at day 4. A significantly higher rate of synapse formation with myotubes was apparent in the late phase of coculture (days 4-7) in L1-transfected cells than in control cells. The miniature end-plate potential frequency in myotubes was the same for the three types of NG108-15 cells. These results show that overexpression of L1 in NG108-15 cells facilitates synaptic connections by enhancing branching and elongation of neurites induced with dibutyryl cAMP, rather than by increasing probability of acetylcholine release.

To study the dynamics of keratin intermediate filaments, we fused two different types of epithelial cells (PtK2 and BMGE+H) and studied how the keratins from the parental cells recombine and copolymerize to form the heterokaryon cytoskeleton. The behaviour of the keratins during this process was followed by immunofluorescence using specific antibodies. After fusion, the parental cytoskeletons undergo a depolymerization process most apparent in the region adjacent to the fusion area. The depolymerized subunits spread throughout the heterokaryon and copolymerize into a new hybrid cytoskeleton. The complete process is very rapid, occurring in 3-4 hours, thus demonstrating the highly dynamic nature of the keratin cytoskeleton. Although newly synthesised subunits contribute to the formation of the hybrid cytoskeleton, the process takes place with similar kinetics in the absence of protein synthesis, showing the dynamic nature of the keratins from pre-existing cytoskeletons. During this process, specific keratins behave differently. Keratins K8, K18, K5 and K10 are mobilised from the parental cytoskeletons and reassemble rapidly into the hybrid cytoskeleton (3-6 hours), whereas K14 requires a substantially longer period (9-24 hours). Thus, different keratins, even when they form part of the same heterodimeric/tetrameric complexes, as is the case for K5 and K14, exhibit different dynamics. This suggests that individual polypeptides or homopolymeric complexes rather than exclusively heterodimeric/ tetrameric subunits, as is currently thought, can also take part in keratin intermediate filament assembly and dynamics. Biochemical analysis performed in the absence of protein synthesis revealed greater amounts of K5 than of K14 in the soluble pool of BMGE+H cells. Crosslinking and immunoprecipitation experiments indicated an excess of monomeric K5, as well as of K5/K14 heterodimers and K5 homodimers in the soluble pool. These results are in agreement with the different dynamic behaviour of these keratins observed in immunofluorescence. On the contrary, the phosphorylation levels of K5 and K14 are similar in both the soluble pool and the polymerized fraction, suggesting that phosphorylation does not play an important role in the different dynamics displayed by these two proteins. In summary, our results demonstrate that, following fusion, the keratin intermediate filament network reshapes rather rapidly and that keratins are highly dynamic proteins, although this mobility depends on each particular polypeptide.

We have previously shown that four markers spanning the 3p24-p21.3 region, THRB, AP20R, D3S1029, and D3S32, were regularly eliminated from three human chromosome 3 (chr3)/mouse microcell hybrids (MCHs) during tumor growth in SCID mice. In an attempt to narrow down the eliminated region, we have studied 22 new SCID mouse tumors derived from 5 MCH lines carrying human chr3. They were analyzed by fluorescence in situ hybridization (FISH), Southern blotting, and polymerase chain reaction (PCR). MCHs that carried human chr1, chr8, chr13, and chr17 were examined as controls. We could identify a common eliminated region (CER) at 3p21.3, bordered distally by D3S1260 and proximally by D3S643/D3F15S2. Eight of 53 chr3-specific PCR markers, AP20R, D3S966, D3S3559, D3S1029, WI-7947, D3S2354, AFMb362wb9, and D3S32. were localized within the CER. This finding is consistent with the notion that a tumor suppressor gene may be located in this area, as suggested by frequent loss of heterozygosity (LOH) within this region observed in several types of solid tumors.

NG108-15 neuroblastoma cells differentiated with 0.1 M of all-trans retinoic acid (RA) were processed for immunohistochemical analysis using polyclonal antisera against the delta opioid receptor (DOR) and the N-Methyl-D-Aspartate receptor (NMDAR1) to determine the cellular sites for possible functional associations between DOR and NMDAR1 receptors. In this study, 6 days of RA treatment resulted in prominent morphological differentiation characterized by the appearance of numerous axon- and dendrite-like processes and formation of networks between the cell clusters. An immunocytochemical approach allowed the demonstration of antibody concentration-dependent differences, not evident in ligand binding studies, in the distribution of DOR and NMDA receptor protein between cell soma and processes. RA-differentiated cultures showed positive DOR-like immunostaining (DOR-LI) throughout the cell bodies as well as on the newly acquired processes. In contrast, NMDAR1-like immunoreactivity (NMDAR1-LI) in the RA-treated cells was detected in the cell soma and processes only with the higher concentration of the antiserum. With the lower concentration of the antibody the NMDAR1-LI was not detected in the processes and was limited to a punctuate subcellular distribution in the soma. The DOR-LI pattern of distribution in NG108-15 cells differentiated with RA appeared to be consistent with the DOR-LI detected in the CNS. The NMDAR1-LI distribution in these cells is similar to brain tissue with respect to its presence on the newly acquired processes. However, it differed from brain in that a much higher abundance of NMDAR1 receptors was observed in the cell soma. This differential distribution of DOR and NMDAR1 receptors in the RA-treated NG108-15 cells could provide a basis for future studies of drug-induced changes in these two receptors.

Phospholipases A2 (PLA2) form an extended class of enzymes that play an important role in signal transduction. Phospholipase A2-like (PLA2L) belongs to the secreted forms of phospholipases A2, but constitutes a new subgroup. We have assigned the gene for this enzyme to human chromosome 8q24-qter using fluorescence in situ hybridization and radiation hybrid mapping techniques.

The metaphase I and anaphase I stages of meiosis of wheat x rye hybrids carrying the ph1b mutation were analyzed by genomic in situ hybridization. This technique allows distinction between three different types of wheat-rye associations in metaphase I configurations as well as detection of wheat-rye recombinant chromosomes in anaphase I cells. The frequency of associations between wheat and rye chromosomes greatly exceeded the level of wheat-rye recombination found in the three hybrids examined. Extremely distal associations, which account for about 50% of the total wheat-rye metaphase I chromosomal pairing, can explain such a discrepancy between metaphase I and anaphase I data. It is further discussed whether these associations reflect very distally located chiasmata or nonchiasmatic pairing. The sizes of the segments exchanged in wheat-rye recombinant chromosomes provide cytological evidence that wheat-rye recombination is restricted to the distal chromosomal regions.

Chromosomes formed de novo which originated from the centromeric region of mouse chromosome 7, have been analysed. These new chromosomes were formed by apparently similar large-scale amplification processes, and are organized into amplicons of approximately 30 Mb. Centromeric satellite DNA was found to be the constant component of all amplicons. Satellite DNA sequences either bordered the large euchromatic amplicons (E-type amplification), or made up the bulk of the constitutive heterochromatic amplicons (H-type amplification). Detailed analysis of a heterochromatic megachromosome formed de novo by an H-type amplification revealed that it is composed of a tandem array of 10-12 large (approximately 30 Mb) amplicons each marked with integrated "foreign' DNA sequences at both ends. Each amplicon is a giant palindrome, consisting of two inverted doublets of approximately 7.5-Mb blocks of satellite DNA. Our results indicate that the building units of the pericentric heterochromatin of mouse chromosomes are approximately 7.5-Mb blocks of satellite DNA flanked by non-satellite sequences. We suggest that the formation de novo of various chromosome segments and chromosomes seen in different cell lines may be the result of large-scale E- and H-type amplification initiated in the pericentric region of chromosomes.

We have analysed the replication of the heterochromatic megachromosome that was formed de novo by a large-scale amplification process initiated in the centromeric region of mouse chromosome 7. The megachromosome is organized into amplicons approximately 30 Mb in size, and each amplicon consists of two large inverted repeats delimited by a primary replication initiation site. Our results suggest that these segments represent a higher order replication unit (megareplicon) of the centromeric region of mouse chromosomes. Analysis of the replication of the megareplicons indicates that the pericentric heterochromatin and the centromere of mouse chromosomes begin to replicate early, and that their replication continues through approximately three-quarters of the S-phase. We suggest that a replication-directed mechanism may account for the initiation of large-scale amplification in the centromeric regions of mouse chromosomes, and may also explain the formation of new, stable chromosome segments and chromosomes.

The response of acute promyelocytic leukemia (APL) peripheral blood and bone marrow cells to trans-retinoic acid (RA) was cytogenetically characterized during RA treatment using the techniques of premature chromosome condensation (PCC) and fluorescence in situ hybridization (FISH). Before treatment, the predominant immature bone marrow cells were found to have t(15;17), whereas the residual mature granulocytes were diploid and lacked evidence of the translocation. In response to RA treatment, an increase in the leukocyte count was noted. The majority of these cells exhibited a t(15;17). Subsequently (eg, between days 6 and 23), 32% to 91% of the maturing myeloid cells still exhibited t(15;17). The appearance of t(15;17) in gradually maturing elements suggests that RA contributed to a release of the maturation block of the leukemic elements. As responding patients obtained complete remission, diploid elements without evidence of the translocation prevailed in the blood and bone marrow. In 16 patients studied after 1 month in complete remission, all but 2 showed all diploid cells. The residual t(15;17) cells disappeared 18 days later in 1 patient, whereas the second patient exhibited clinical evidence of relapse 20 days later. These results suggest that response of patients with APL to RA is associated with maturation, subsequent loss of the mature leukemic elements, and preferential regeneration of normal diploid hematopoietic elements.

The activity of cell proliferation in human chorionic villi (CV) obtained from early spontaneous abortions (SAB) and from elective abortions (EAB) was determined by means of premature chromosome condensation (PCC), and by Northern blot analysis of expression of proliferation antigens Ki67 and PCNA. The rate of chromosome aberrations among the SABs was 57%, while the EABs, taken as controls, were shown to be chromosomally normal. PCCs were induced by fusion of the chorionic interphase cells with mitotic Chinese hamster ovary (CHO) cells. To analyze the proliferation stages of the chorionic G1-interphases, the potential proliferation index (PPI) was ascertained. The PPI values found in SABs ranged from 36% to 97% as described for intensely proliferating tissues. They did not differ from the values found in the controls (34-90%), indicating maintenance of proliferation activity of CV cells even after death of the embryo in missed abortions. No significant PPI differences were observed between the abortion groups with different cytogenetic results. The expression of proliferation associated antigens Ki67 and PCNA showed no significant differences between mean values of the total of SABs and of the controls. However, the comparison of mean values of chromosomally abnormal with chromosomally normal SABs revealed a significantly reduced Ki67 activity in the SABs with chromosome aberrations. A similar results was obtained when comparing mean values of Ki67 and PCNA expression from trisomic SABs with those of the controls. By further subdivision of chromosome aberrations a decrease for the expression of both antigens in chorionic villi from early lethal trisomies, and a significantly increased Ki67- and PCNA-expression in triploidies became evident.

Chicken blastodermal cells (stage X) were fused to mouse enucleated oocytes with either no or high maturation promoting factor (MPF) activity. High MPF levels always induced premature chromosome condensation (PCC) irrespective of the number of nuclei fused to a single oocyte. When a single blastodermal cell was fused to a single oocyte without MPF activity the nucleus remained intact for up to 3 h and thereafter underwent PCC. A quite different situation was observed after multiple fusion of several blastodermal cells to a single oocyte without MPF activity. Here, the transplanted nuclei remained intact even after prolonged culture but underwent extensive swelling. DNA synthesis was detected in almost all unfused blastodermal cells. However, after the fusion of several blastodermal cells to a single oocyte no DNA synthesis could be detected. These results provide further evidence that MPF is the universal cell-cycle regulator in the animal kingdom. Its activity is blocked (or neutralised) after fusion to several S-phase cells. Interestingly, our results further suggest that DNA synthesis is suppressed in meiotic cytoplasm even in the presence of an intact nuclear envelope.

One-cell parthenogenetic haploid embryos and blastomeres of the 2- and 4-cell diploid mouse embryos were observed in vitro for the occurrence of two cytoplasmic activities: the cortical activity and the chromatin condensation activity. For this purpose anucleated halves (AHs) and nucleated halves (NHs) were produced by bisection of one-cell embryos and of blastomeres. The cortical activity (manifested by surface deformations) was observed only during the first cleavage cycle. In AHs the surface activity began at the same time as in NHs and disappeared before the time of the cleavage division of nucleated halves. Anucleate fragments of blastomeres from 2- and 4-cell embryos did not exhibit any cortical activity. In the absence of the native nucleus the chromatin condensation activity (assayed by premature chromatin condensation of interphase thymocyte nuclei introduced into cytoplasts by cell fusion) could also have been detected only in the first cleavage cycle. In AHs this activity appeared at the time when NHs started to cleave and disappeared after the NHs finished the first cleavage division. AHs obtained from 2-cell and 4-cell stage blastomeres did not reveal condensation activity.