Cell nucleus

Long-term success of distraction osteogenesis depends on the ability of the surrounding soft tissues to tolerate distraction forces and to adapt to the resulting increase in skeletal length and volume. The hypothesis tested in this study was that mandibular elongation by distraction induces myocyte proliferation. Unilateral mandibular angle osteotomies were performed in 16 Yucatan minipigs. The hemimandibles were lengthened using semiburied distraction devices (Synthes Maxillofacial, Paoli, PA) with 0-day latency, 1, 2, or 4 mm/d distraction rates, and a neutral fixation period of twice the gap size in days. In 2 additional animals, the dissection and osteotomies were performed, and distraction devices were placed without activation (sham control). At the end of neutral fixation, tissues were taken from masseter muscle overlying the osteotomy and the equivalent region on the unoperated side (contralateral control). Proliferation of myocytes was estimated using immunohistochemical localization with antibodies against proliferating cell nuclear antigen (PCNA). Muscle overlying the distracted mandible showed 6-fold more PCNA-positive myocytes (16.8% +/- 11.3%) than the contralateral control side (2.8% +/- 1.1%, P < .0001). In the 2 sham-control animals, there was a low index of PCNA-positive myocytes on both the osteotomy (2%, 5%) and the contralateral sides (1%, 2%). The results of this study suggest that distraction of the porcine mandible by the protocol described induces myocyte proliferation in the masseter muscle. A proliferative response may contribute to improved long-term stability of mandibular expansion by distraction osteogenesis.

Mitogen- and stress-activated kinases (MSKs) are important substrates of the mitogen-activated protein kinase (MAPK)-activated protein kinase family. MSK1 and MSK2 are both nuclear serine/threonine protein kinases, with MSK1 being suggested to potentially play a role in breast cancer cell proliferation, cell cycle progression, cell migration, invasion and tumour growth. The aim of the current study was to assess MSK1 protein expression in breast cancer tumour specimens, evaluating its prognostic significance. A large cohort of 1902 early stage invasive breast cancer patients was used to explore the expression of MSK1. Protein expression was examined using standard immunohistochemistry on tissue microarrays. Low MSK1 protein expression was associated with younger age (P = 0.004), higher tumour grade (P < 0.001), higher Nottingham Prognostic Index scores (P = 0.007), negative ER (P < 0.001) and PR (P < 0.001) status, and with triple-negative (P < 0.001) and basal-like (P &...

To obtain a deeper insight into the genes and gene networks involved in the development of placentopathies, we have assessed global gene expression in three different models of placental hyperplasia caused by interspecies hybridization (IHPD), cloning by nuclear transfer, and mutation of the Esx1 gene, respectively. Comparison of gene expression profiles of approximately 13,000 expressed sequence tags (ESTs) identified specific subsets of genes with changed expression levels in IHPD, cloned, and Esx1 mutant placentas. Of interest, only one gene of known function and one EST of unknown function were found common to all three placentopathies; however, a significant number of ESTs were common to IHPD and cloned placentas. In contrast, only one gene was shared between IHPD and Esx1 mutant, and cloned and Esx1 mutant placentas, respectively. These genes common to different abnormal placental growth genotypes are likely to be important in the occurrence of placentopathy. Developmental Dynamics 230: 149-164, 2004.

The purpose of this paper was to study the reaction of rat subcutaneous connective tissue to the implantation of dentin tubes filled with white mineral trioxide aggregate (MTA), a material that will be marketed. The tubes were implanted into rat subcutaneous tissue and the animals were sacrificed after 7 and 30 days. The undecalcified pieces were prepared for histological analysis with polarized light and von Kossa technique for mineralized tissues. Granulations birefringent to polarized light and an irregular structure like a bridge were observed next to the material; both were von Kossa positive. Also, in the dentin wall tubules a layer of birefringent granulations was observed. The results were similar to those reported for gray MTA, indicating that the mechanisms of action of the white and gray MTA are similar.

The aggregation of R-synuclein is believed to play an important role in the pathogenesis of Parkinson's disease as well as other neurodegenerative disorders ("synucleinopathies"). However, the function of R-synuclein under physiologic and pathological conditions is unknown, and the mechanism of R-synuclein aggregation is not well understood. Here we show that R-synuclein forms a tight 2:1 complex with histones and that the fibrillation rate of R-synuclein is dramatically accelerated in the presence of histones in vitro. We also describe the presence of R-synuclein and its co-localization with histones in the nuclei of nigral neurons from mice exposed to a toxic insult (i.e., injections of the herbicide paraquat). These observations indicate that translocation into the nucleus and binding with histones represent potential mechanisms underlying R-synuclein pathophysiology.

Obtaining karyotypes from human spermatozoa after microinjection into Syrian golden hamster oocytes is difficult and the hitherto reported results are unsatisfactory. This may be related to the injection and culture technique or to the high susceptibility of the hamster oocytes to undergo parthenogenetic activation or both. Therefore, we investigated the hamster oocyte-human sperm microinjection model using the following two approaches: (i) application of contemporary techniques for injection (touching the sperm tail) and culture (hamster embryo culture medium, HECM-3, 10% CO 2 ) and (ii) omission of Ca 2⍣ from the injection medium. Thus, in the first series of experiments, 252 hamster oocytes were injected with human spermatozoa. Among the 219 (87%) oocytes that survived the injection procedure, the mean percentages of male pronucleus formation [two pronuclei (2PN), two polar bodies (PB)], mitotic metaphase entry and sperm chromosome spreads were 41.4, 27.8 and 18.2% respectively. Analysis of the oocytes which failed to develop the male pronucleus following injection revealed that most of them had developed only the hamster female PN while the sperm nuclei were either intact or swollen (partially decondensed), indicating that failure of oocyte activation was not the likely reason for the failure of male PN formation in these oocytes. In the next series of experiments, sibling oocytes were alternately injected with spermatozoa suspended either in the regular (1.9 mM Ca 2⍣ ) or Ca 2⍣ -free injection medium (experiment set 2, n ⍧ 278). A significant improvement was noted in the mean percentages of oocytes with 2PN, 2PB, metaphase entry and sperm chromosome spreads in the Ca 2⍣ -free group versus the regular group (2PN, 2PB: 51 versus 36.6%, metaphase entry: 36.3 versus 26.9% and sperm chromosome spreads: 28 versus 20.4%; all P < 0.04). Thus, parthenogenetic activation appears to be one of the contributing factors for the failure of male PN formation after heterospecific hamster ICSI. From these experiments it can be concluded that application of the advanced injection and culture techniques and omission of Ca 2⍣ from the injection medium are promising for the routine application of the hamster oocyte microinjection for karyotyping of human spermatozoa with poor fertilizing capacity.

Our recent study has shown that beetroot juice protects against N-nitrosodimethylamine (NDEA)-induced liver injury and increases the activity of phase II enzymes, suggesting the activation of the nuclear factor erythroid-2-related factor 2 (Nrf2)–antioxidant response element (ARE) pathway. The aim of the present study was to further explore the mechanism of the activity of beetroot by evaluating the cytoprotective effects of its major component. The influence of betanin (BET) on the activation of Nrf2 and the expression of GSTA, GSTP, GSTM, GSTT, NQO1 and HO-1 was assessed in two hepatic cell lines: non-tumour THLE-2 and hepatoma-derived HepG2 cell lines. The level of the tumour suppressor p53 in both cell lines and the methylation of GSTP in HepG2 cells were also evaluated. Treatment of both cell lines with 2, 10 and 20 μm of BET resulted in the translocation of Nrf2 from the cytosol to the nucleus. The mRNA and nuclear protein levels of Nrf2 and the binding of Nrf2 to ARE sequence...

Unfertilized ovaries isolated from immature female flowers of coconut (Cocos nucifera L.) were tested as a source of explants for callogenesis and somatic embryogenesis. The correct developmental stage of ovary explants and suitable in vitro culture conditions for consistent callus production were identified. The concentration of 2,4-dichlorophenoxyacetic acid (2,4-D) and activated charcoal was found to be critical for callogenesis. When cultured in a medium containing 100 µM 2,4-D and 0.1% activated charcoal, ovary explants gave rise to 41% callusing. Embryogenic calli were sub-cultured into somatic embryogenesis induction medium containing 5 µM abscisic acid, followed by plant regeneration medium (with 5 µM 6-benzylaminopurine). Many of the somatic embryos formed were complete with shoot and root poles and upon Communicated by P. Debergh

This work examines the cellular localization of holocarboxylase synthetase (HCS) and its association to chromatin during different stages of development of Drosophila melanogaster. While HCS is well known for its role in the attachment of biotin to biotin-dependent carboxylase, it also regulates the transcription of HCS and carboxylases genes by triggering a cGMP-dependent signal transduction cascade. Further, its presence in the nucleus of cells suggests additional regulatory roles, but the mechanism involved has remained elusive. In this study, we show in D. melanogaster that HCS migrates to the nucleus at the gastrulation stage. In polytene chromosomes, it is associated to heterochromatin bands where it co-localizes with histone 3 trimethylated at lysine 9 (H3K9met3) but not with the euchromatin mark histone 3 acetylated at lysine 9 (H3K9ac). Further, we demonstrate the association of HCS with the hsp70 promoter by immunofluorescence and chromatin immuno-precipitation (ChIP) of associated DNA sequences. We demonstrate the occupancy of HCS to the core promoter region of the transcriptionally inactive hsp70 gene. On heat-shock activation of the hsp70 promoter, HCS is displaced and the promoter region becomes enriched with the TFIIH subunits XPD and XPB and elongating RNA pol II, the latter also demonstrated using ChIP assays. We suggest that HCS may have a role in the repression of gene expression through a mechanism involving its trafficking to the nucleus and interaction with heterochromatic sites coincident with H3K9met3.

A thorough understanding of the mechanisms of R-, C-and G-banding will come only from studies of the binding of Giemsa dyes to isolated and characterized preparations of heterochromatin and euchromatin. Since such studies require an exact knowledge of the optical characteristics of Giemsa, the spectral adsorption curves and extinction coefficients of Giemsa and its component dyes at various concentrations in the presence and absence of DNA were determined. — Although Giemsa is a complex mixture of thiazin dyes plus eosin; methylene blue, and azure A, B or C alone gave good banding. Thionin, with no methyl groups, gave poor or no banding. Eosin was not a necessary component for banding. — The most striking characteristic of the thiazin dyes is that they are strongly metachromatic, i.e., their adsorption spectra and extinction coefficients change as the concentration of the dye increases or as they bind to positively charged compounds (chromotropes). These changes, especially for methylene blue, are described in detail and allow a distinction between concentration dependent binding to DNA by intercalation and binding by side stacking.

Cytosine methylation is attracting new attention for regulatory roles in gene expression and there is an increasing interest in detecting, at a single-base resolution, any 5-methylcytosine in genes from complex genomes. Differential base modification by chemicals followed by PCR-based genomic sequencing procedures can provide the resolution, sensitivity, and specificity required for such a goal. The various methods available are not devoid of artifacts but if used carefully and in combination, very reliable information can be obtained. We compare the methods using bisulfite and conventional PCR with those using either hydrazine or potassium permanganate and ligation-mediated PCR and provide a step-by-step description of the corresponding procedures.

Transient receptor potential channels (TRPC) are plasma membrane, non-selective cationic channels and have been proposed as candidates involved in the regulation of cellular Ca2+ influx. TRPC are involved in metabotropic glutamate receptor (mGluR)-mediated excitatory post-synaptic currents (EPSCs) in the dopaminergic neurons of the substantia nigra. We previously observed several subtypes of TRPC to be expressed at an mRNA level in

is associated with more severe liver damage and an increased risk of fulminant disease. HDV is a single-stranded RNA virus that encodes a single protein, the delta antigen, which is expressed in two forms, small (S-HDAg) and large (L-HDAg). Here we show that although HDV ribonucleoproteins are mainly detected in the nucleus, they are also present in the cytoplasm of cells infected with HDV or transfected with HDV cDNA. Making use of an heterokaryon assay, we demonstrate that HDV ribonucleoproteins shuttle continuously between the nucleus and the cytoplasm. In the absence of HDV RNA, both forms of the delta antigen are retained in the nucleus, whereas in the absence of the delta antigen, HDV RNA is predominantly detected in the cytoplasm. Coexpression of HDV RNA and S-HDAg (which binds to the viral RNA and contains a nuclear localization signal) results in nuclear accumulation of the viral RNA. This suggests that HDV RNA mediates export of viral particles to the cytoplasm whereas the delta antigen triggers their reimport into the nucleus.

Previously we described a method to estimate the average number of virus genomes expressed in an infected cell. By analyzing the color spectrum of cells infected with a mixture of isogenic pseudorabies virus (PRV) recombinants expressing three fluorophores, we estimated that fewer than seven incoming genomes are expressed, replicated, and packaged into progeny per cell. In this report, we expand this work and describe experiments demonstrating the generality of the method, as well as providing more insight into herpesvirus replication. We used three isogenic PRV recombinants, each expressing a fluorescently tagged VP26 fusion protein (VP26 is a capsid protein) under the viral VP26 late promoter. We calculated a similar finite limit on the number of expressed viral genomes, indicating that this method is independent of the promoter used to transcribe the fluorophore genes, the time of expression of the fluorophore (early versus late), and the insertion site of the fluorophore gene in the PRV genome (UL versus US). Importantly, these VP26 fusion proteins are distributed equally in punctate virion assembly structures in each nucleus, which improves the signal-to-noise ratio when determining the color spectrum of each cell. To understand how the small number of genomes are distributed among the replication compartments, we used a two-color fluorescent in situ hybridization assay. Most viral replication compartments in the nucleus occupy unique nuclear territories, implying that they arose from single genomes. Our experiments suggest a correlation between the small number of expressed viral genomes and the limited number of replication compartments.

Analyses of genome orthologs in cancer on the background of tumor heterogeneity, coupled with the recent identification that the tumor propagating capacity resides within a very small fraction of cells (the tumor stem cells-TSCs), has not been achieved. Here, we describe a strategy to explore genetic drift in the mitochondrial genome accompanying varying stem cell dynamics in epithelial ovarian cancer. A major and novel outcome is the identification of a specific mutant mitochondrial DNA profile associated with the TSC lineage that is drastically different from the germ line profile. This profile, however, is often camouflaged in the primary tumor, and sometimes may not be detected even after metastases, questioning the validity of whole tumor profiling towards determining individual prognosis. Continuing mutagenesis in subsets with a mutant mitochondrial genome could result in transformation through a cooperative effect with nuclear genes - a representative example in our study is a tumor suppressor gene viz. cAMP responsive element binding binding protein. This specific profile could be a critical predisposing step undertaken by a normal stem cell to overcome a tightly regulated mutation rate and DNA repair in its evolution towards tumorigenesis. Our findings suggest that varying stem cell dynamics and mutagenesis define TSC progression that may clinically translate into increasing tumor aggression with serious implications for prognosis.

In over 90% of cervical cancers and cancer-derived cell lines, the p53 tumor suppressor pathway is disrupted by human papillomavirus (HPV). The HPV E6 protein promotes the degradation of p53 and thus inhibits the stabilization and activation of p53 that would normally occur in response to HPV E7 oncogene expression. Restoration of p53 function in these cells by blocking this pathway should promote a selective therapeutic affect. Here we show that treatment with the small molecule nuclear export inhibitor, leptomycin B, and actinomycin D leads to the accumulation of transcriptionally active p53 in the nucleus of HeLa, CaSki, and SiHa cells. Northern blot analyses showed that both actinomycin D and leptomycin B reduced the amount of HPV E6-E7 mRNA whereas combined treatment with the drugs showed almost complete disappearance of the viral mRNA. The combined treatment activated p53-dependant transcription, and increases in both p21 WAF1͞CIP1 and Hdm2 mRNA were seen. The combined treatment resulted in apoptotic death in the cells, as evidenced by nuclear fragmentation and PARP-cleavage indicative of caspase 3 activity. These effects were greatly reduced by expressing a dominant negative p53 protein. The present study shows that small molecules can reactivate p53 in cervical carcinoma cells, and this reactivation is associated with an extensive biological response, including the induction of the apoptotic death of the cells.

Parental (genomic) imprinting is the process by which the differential expression of maternal and paternal alleles at certain genetic loci in mammalian embryos occurs. Such loci are implicated in the control of fetal, placental and neonatal growth, and, more generally, in diverse aspects of fetal nutrient acquisition and maternal-fetal interactions. Not surprisingly, the aberrant expression of imprinted genes is implicated in a range of embryonic and fetal abnormalities. We outline how an evolutionary theory, based on classic parent-offspring conflict theory, relates to certain fetal growth abnormalities. In particular, we suggest that growth abnormalities resulting from the manipulation of preimplantation mammalian embryos in vitro (for example large calf syndrome) may reflect the occurrence of genetic conflict over the fetal growth programme in the early preimplantation period.

Store-operated calcium entry (SOCE) is the predominant Ca2+ entry mechanism in nonexcitable cells and controls a variety of physiological and pathological processes. Although significant progress has been made in identifying the components required for SOCE, the molecular mechanisms underlying it are elusive. The present study provides evidence for a direct involvement of kinase suppressor of Ras 2 (KSR2) in SOCE. Using lymphocytes and fibroblasts from ksr2−/− mice and shKSR2-depleted cells, we find that KSR2 is critical for the elevation of cytosolic Ca2+ concentration. Specifically, our results show that although it is dispensable for Ca2+-store depletion, KSR2 is required for optimal calcium entry. We observe that KSR2 deficiency affects stromal interaction molecule 1 (STIM1)/ORAI1 puncta formation, which is correlated with cytoskeleton disorganization. Of interest, we find that KSR2-associated calcineurin is crucial for SOCE. Blocking calcineurin activity impairs STIM1/ORAI1 pun...

There have been 2 putative prostatic cancer precursors, prostatic intraepithelial neoplasia (PIN) and atypical adenomatous hyperplasia (adenosis), but PIN remains as a well-known precancerous condition. To describe recent advances in knowledge of PIN and to better define the diagnostic criteria and differential diagnosis of PIN. Review of the pertinent literature and our experience. The presence of ductal/acinar epithelial changes including nuclear enlargement, prominent nucleoli, chromatin alterations, and luminal complexity is an easy way to identify the disorder. Four main patterns of high-grade PIN (HGPIN) have been described: tufting, micropapillary, cribriform, and flat. In addition, variants of HGPIN have also been described. Both HGPIN and prostatic carcinoma share an increased incidence and severity with advancing age and with high rates of occurrence in the peripheral zone of the prostate. Furthermore, HGPIN and prostate cancer share genetic and molecular markers as well, ...

After invading host cells, Toxoplasma gondii multiplies within a parasitophorous vacuole (PV) that is maintained by parasite proteins secreted from organelles called dense granules. Most dense granule proteins remain within the PV, and few are known to access the host cell cytosol. We identify GRA16 as a dense granule protein that is exported through the PV membrane and reaches the host cell nucleus, where it positively modulates genes involved in cellcycle progression and the p53 tumor suppressor pathway. GRA16 binds two host enzymes, the deubiquitinase HAUSP and PP2A phosphatase, which exert several functions, including regulation of p53 and the cell cycle. GRA16 alters p53 levels in a HAUSP-dependent manner and induces nuclear translocation of the PP2A holoenzyme. Additionally, certain GRA16-deficient strains exhibit attenuated virulence, indicating the importance of these host alterations in pathogenesis. Therefore, GRA16 represents a potentially emerging subfamily of exported dense granule proteins that modulate host function.

Coiled bodies are nuclear organelles that contain components of at least three RNA-processing pathways: pre-mRNA splicing, histone mRNA 3′- maturation, and pre-rRNA processing. Their function remains unknown. However, it has been speculated that coiled bodies may be sites of splicing factor assembly and/or recycling, play a role in histone mRNA 3′-processing, or act as nuclear transport or sorting structures. To study the dynamics of coiled bodies in living cells, we have stably expressed a U2B&quot;–green fluorescent protein fusion in tobacco BY-2 cells and in Arabidopsis plants. Time-lapse confocal microscopy has shown that coiled bodies are mobile organelles in plant cells. We have observed movements of coiled bodies in the nucleolus, in the nucleoplasm, and from the periphery of the nucleus into the nucleolus, which suggests a transport function for coiled bodies. Furthermore, we have observed coalescence of coiled bodies, which suggests a mechanism for the decrease in coiled bo...

This study compared the effects of ad libitum (AL) overfeeding and moderate or marked dietary restriction (DR) on the pathogenesis of aged-related pituitary gland changes in Sprague-Dawley (SD) rats. SD rats were fed Purina Certified Rodent Diet AL (group 1), DR at 72-79% of AL (group 2), DR at 68-72% of AL (group 3) or DR at 47-48% of AL (group 4) for 106 weeks. Interim necropsies were performed at 13, 26, and 53 weeks, after a 7-day 5-bromo-2-deoxyuridine (BrdU)-filled minipump implantation. Body weights, organ weights and insulin-like growth factor 1 (IGF-1) serum levels were measured at interim and final necropsies. Serum levels of prolactin (PRL), progesterone, estradiol, luteinizing hormone (LH) and follicle stimulating hormone (FSH) were measured at 53 and/or 106 weeks. In addition to the routine histopathologic examination, determination of 7 stereologic parameters after pituitary immunohistochemistry of PRL, growth hormone (GH) and BrdU was done in both sexes at 13, 26, and 53 weeks. Body and pituitary weights were proportional to the food intake. In AL-fed rats, hyperplastic and neoplastic changes developed early and progressed with age, affecting almost all animals by 106 weeks. These changes were associated with high PRL serum levels. Pituitary adenomas were the most common cause of death in both sexes. In DR rats, a delayed onset and a decreased incidence of pituitary tumors were observed in association with decreased serum IGF-1, PRL, estradiol, and LH levels. The results of the stereological analysis demonstrated that, compared to AL-fed rats, pituitary glands from DR rats contained lower PRL and GH secreting cell volumes, and a lower epithelial cell BrdU labeling index, which correlated with a lower incidence of pituitary tumors at study termination. Moderate and marked degrees of DR delayed the onset of pituitary tumors in a temporaland dose-related manner. In contrast to marked DR, which dramatically reduced the incidence of hyperplastic and neoplastic pituitary gland changes, moderate DR delayed the onset but did not prevent the development of pituitary tumors.

Mitochondrial dysfunction is a hallmark of cancer cells. Consistent with this phenotype mutations in mitochondrial genome have been reported in all cancers examined to date. However, it is not clear whether mitochondrial genomic status in human cells affects nuclear genome stability and whether proteins involved in inter-genomic cross talk are involved in tumorigenesis. Using cell culture model and cybrid cell technology, we provide evidence that mitochondrial genetic status impacts nuclear genome stability in human cells. In particular our studies demonstrate 1) that depletion of mitochondrial genome (rho 0 ) leads to chromosomal instability (CIN) reported to be present in variety of human tumors and 2) rho 0 cells show transformed phenotype. Our study also demonstrates that mitochondrial genetic status plays a key role in regulation of a multifunctional protein APE1 (also known as Ref1 or HAP1) involved in transcription and DNA repair in the nucleus and the mitochondria. Interestingly we found that altered expression of APE1 in rho 0 cells and tumorigenic phenotype can be reversed by exogenous transfer of wild type mitochondria in rho 0 cells. Furthermore, we demonstrate that APE1 expression is altered in variety of primary tumors. Taken together, these studies suggest that inter-genomic cross talk between mitochondria and the nucleus plays an important role in tumorigenesis and that APE1 mediates this process. D

Growing evidence suggests that oxidative stress is involved in the neuronal degeneration and can promote the aggregation of α-synuclein. However, the role of α-synuclein under physiological and pathological conditions remains poorly understood. In the present study, we examined the possible interaction between the α-synuclein and oxidative stress. In a dopaminergic cell line MES23.5, we have found that the 200μM H2O2

The Tradescantia-Micronucleus (Trad-MCN) test is a simple short-term bioassay for various gaseous and liquid forms of chemical agents, and physical agents such as radiation. 140 agents, directly or indirectly related to human health, were screened for their mutagenicity. Plant cuttings of young inflorescences, in which the pollen mother cells undergo various stages of meiosis, were maintained in nutrient solution for experimentation. Treatments were made either by absorption of the soluble agents through the stem, by diffusion of gaseous agents through the leaves and buds, by exposure to internal/external radiation or by in situ exposure to air pollutants. Micronuclei formed from damaged chromosomes served as the indicators of mutagenicity. Results of 140 agents tested are listed in 9 different categories. (1) Carcinogens/mutagens, (2) common beverages, (3) common chemicals, (4) drugs, (5) pesticides, (6) common household chemicals, (7) radiation and radioisotopes, (8) in situ monitoring, (9) complex environmental mixtures. Out of 140 agents tested, 52 showed positive, 20 showed borderline positive responses and 5 showed strong toxicity. Test results of 41 agents in the present study showed 67% congruity with Ames test results found in the literature.

Cisplatin is a broad-spectrum anticancer drug that is also widely used in experimental studies on DNA damage-induced apoptosis. Induction of apoptosis within 24-48 hr requires cisplatin concentrations that are at least one order of magnitude higher than the IC 50 . Here, we show that such high, apoptosis-inducing cisplatin concentrations induce cellular superoxide formation and that apoptosis is inhibited by superoxide scavengers. The same concentration limit and the requirement for superoxide are also true for induction of caspase activation in enucleated cells (cytoplasts), showing that cisplatin-induced apoptosis occurs independently of nuclear DNA damage. In contrast, lower cisplatin concentrations, which do not induce acute apoptosis, are sufficient for induction of DNA damage signaling. We propose that the antiproliferative effects of cisplatin at IC 50 doses involve premature senescence and secondary, nonstress-induced apoptosis. The higher doses currently used in in vitro studies lead to acute, stress-induced apoptosis that involves induction of superoxide but is largely DNA damage-independent. ' 2006 Wiley-Liss, Inc.

Euphrasia includes perennial or annual green parasitic plants, and has a disjunct bipolar distribution except for one transtropical connection across the high mountains of Oceania. The disjunction is coupled with strikingly contrasting patterns of morphological diversity between the southern and northern hemispheres, making it an exciting model to study processes of evolutionary diversification which shaped present floras. We inferred the relationships among 51 species representing 14 of the 15 sections of the genus based on nrDNA ITS and cpDNA trnL intron, trnL-trnF and atpB-rbcL intergenic spacers. Maximum parsimony and Bayesian inference support monophyly of the genus and of several intrageneric groups characterized by morphology, ploidy level, and geographic range. Molecular phylogenetic dating using Bayesian ''relaxed" clock methods suggests that the earliest Euphrasia radiations occurred minimum 11-8 Mya with bipolarity being achieved 7-5 Mya. Biogeographic analyses using explicit modelbased approach inferred Eurasia as an ancestral area for the genus. The most parsimonious reconstruction found by a dispersal-vicariance analysis requires 17 dispersals to account for the current biogeographic pattern and supports Eurasian origin for Euphrasia. Both long-distance dispersal and across land vicariance can be invoked to explain the diversification in the genus, which experienced rapid radiations driven by new ecological opportunities of the late Pliocene and Pleistocene but also retained a set of local endemic or relict species of an earlier origin.

Subunit B of diphtheria toxin (DT), which consists of two domains: R (receptor-binding) and T (transmembrane), plays an important role in toxin-receptor binding on the cell-targets and in transportation of catalytic subunit A to the cell cytosol. Recombinant analogues of the subunit B are promising representatives in the unique class of transporting proteins, able to deliver different types of biologically active molecules to cell cytosol. In the development of these protein constructs understanding of the role of each of the DT fragments in determination of transporting pathways of endocytosed complex toxin-receptor is urgently required. We have studied in this work the T-domain effect on intracellular transport of recombinant fragments of DT. We have compared intracellular transport of the R-domain and the subunit B, the last one was consisted of both R-domain and T-domain. Recombinant fragments of DT used in this work were labeled with fluorescent proteins, which allowed applying...

The DJ-1 gene encodes a ubiquitous, highly conserved protein. Here, we show that DJ-1 mutations are associated with PARK7, a monogenic form of human parkinsonism. The function of the DJ-1 protein remains unknown, but evidence suggests its involvement in the oxidative stress response. Our findings indicate that loss of DJ-1 function leads to neurodegeneration. Elucidating the physiological role of DJ-1 protein may promote understanding of the mechanisms of brain neuronal maintenance and pathogenesis of Parkinson's disease.

Oculodermal melanocytosis (nevus of Ota) is a benign, congenital, melanocytic pigmentary disorder, most commonly involving the area innervated by the first and second divisions of the trigeminal nerve. We describe a case of cutaneous melanoma that developed in a nevus of Ota.

The objectives of this study were to assess cytokinesis-block micronucleus cytome (CBMN Cyt) assay parameters and also oxidative DNA damage in patients with active acromegaly and controls and to assess the relationship between age, serum insulin-like growth factor 1 (IGF-1) levels, pituitary adenoma diameters, 8-hydroxy-2′-deoxyguanosine (8-OHdG) levels and CBMN Cyt assay parameters in patients with active acromegaly. Design: The study population included 30 patients with active acromegaly and 30 age-and sex-matched healthy controls. CBMN Cyt assay parameters in peripheral blood lymphocytes of patients with active acromegaly and controls were evaluated and plasma 8-OHdG levels were measured. Results: Frequencies of micronucleus (MN), nucleoplasmic bridges (NPBs) and nuclear buds (NBUDs) in lymphocytes of patients with acromegaly were found to be significantly higher than those in controls (p b 0.001, p b 0.001, p b 0.001, respectively). The frequencies of apoptotic and necrotic cells in lymphocytes of patients with acromegaly were found to be significantly higher than those in controls (p b 0.001 and p b 0.001 respectively). No statistically significant differences in the number of cells in metaphase, the number of bi-nucleated cells (M2), the number of tri-nucleated cells (M3), the number of tetra-nucleated cells (M4) and nuclear division index (NDI) values were observed between patients and controls (p N 0.05). Plasma 8-OHdG (ng/ml) levels in patients with acromegaly were found to be significantly higher than those in controls (p b 0.005). MN frequency in the lymphocytes of patients with acromegaly increased with elevated serum IGF-1 levels (p b 0.05), whereas the number of NPBs and the frequency of apoptotic cells decreased with elevated serum IGF-1 levels (p b 0.01 and p b 0.05 respectively). Conclusions: Both the increase in chromosomal/oxidative DNA damage and the positive association between MN frequency and serum IGF-1 levels may predict an increased risk of malignancy in acromegalic patients. Long-term follow-up of patients with acromegaly will be necessary to establish the degree of cancer risk in this population.

The highly conserved Wnt signalling pathway plays an important role in embryonic development and disease pathogenesis, most notably cancer. The 'canonical' or β-catenin-dependent Wnt signal initiates at the cell plasma membrane with the binding of Wnt proteins to Frizzled:LRP5/LRP6 receptor complexes and is mediated by the translocation of the transcription co-activator protein, β-catenin, into the nucleus. β-Catenin then forms a complex with T-cell factor (TCF)/lymphoid enhancer binding factor (LEF) transcription factors to regulate multiple gene programmes. These programmes play roles in cell proliferation, migration, vasculogenesis, survival and metabolism. Mutations in Wnt signalling pathway components lead to constitutively active Wnt signalling that drives aberrant expression of these programmes and development of cancer. It has been a longstanding and challenging goal to develop therapies that can interfere with the TCF/LEF-β-catenin transcriptional complex. This review will focus on the (i) structural considerations for targeting the TCF/LEF-β-catenin and co-regulatory complexes in the nucleus, (ii) current molecules that directly target TCF/LEF-β-catenin activity and (iii) ideas for targeting newly discovered components of the TCF/LEF-β-catenin complex and/or downstream gene programmes regulated by these complexes.

In previous studies from our laboratory, the presence in highly purified liver nuclei of metabolic pathways for processing ethanol (EtOH), N -nitrosodimethylamine (NDMA), carbon tetrachloride and chloroform was reported. All these chemicals are known to be metabolized in liver microsomes, via cytochrome P450 2E1 (CYP2E1)-mediated processes. In the present work we checked whether rat liver nuclei from rats chronically drinking an alcohol-containing liquid diet exhibited an enhanced ability to metabolize chemicals known to require CYP2E1 participation for given metabolic transformations. The nicotinamide adenosine dinucleotide phosphate (NADPH)-requiring metabolism of p -nitrophenol to p-nitrocathecol; the activation of carbon tetrachloride to trichloromethyl radicals, covalently binding to proteins; and the ring hydroxylation of aniline and o -toluidine were studied. Comparison of the obtained nuclear activities against the one present in the microsomal counterpart, and their respective response to the EtOH inductive effect after repetitive exposure to it, was studied. The obtained results showed that rat liver nuclei exhibited less pnitrophenol hydroxylase activity than microsomes, but it was inducible by repetitive alcohol drinking to equivalent levels of those of microsomes from control animals. Nuclei exhibited the ability to activate CCl 4 , which was significantly enhanced by alcohol drinking. Aniline was ring hydroxylated in liver microsomes but not in nuclei from either control or EtOH-treated animals. In contrast, nuclei and microsomes metabolized o-toluidine to ring hydroxylated products. They are considered less toxic in nature but other authors reported a genotoxic effect for one of them. The production of the ring hydroxylated metabolites was enhanced by repetitive EtOH drinking. Results suggest that nuclear metabolism of xenobiotics might be relevant for either activations or detoxications mediated by CYP2E1 and that repetitive exposure to EtOH might significantly modulate those processes. Toxicology and Industrial Health 2006; 22: 367 Á374.

STAT6, NF-κB (p50) and C/EBPβ transcription factors (TF) were examined with respect to CD23 regulation. Electrophoretic mobility shift assay (EMSA), competition and supershift analysis demonstrated that STAT6 binds the CD23a promoter but with a lower affinity than the consensus site. STAT6 -/mice were analyzed for CD23 levels and showed reduced expression after CD40 ligand trimer (CD40LT) stimulation. However, normal CD23 expression and even some IgE production was induced in STAT6 -/mice with CD40LT/IL-4. EMSA analysis indicated that the CD23a STAT site was bound by a protein in nuclear extracts from CD40 ⍨ IL-4-stimulated STAT6 -/-B cells. Western blot analysis of these nuclear extracts demonstrated the presence of STAT3 and STAT5, suggesting that these STATs can induce CD23 in this situation. Further supporting evidence was obtained by showing that IL-2 and IL-4 both synergize with CD40 in an identical manner for CD23 induction on STAT6 -/-B cells. EMSA analysis of the two putative NF-κB sites confirmed binding to both, although one site bound with a higher affinity than the second. Analysis of p50 -/mice indicated that this subunit was not necessary for CD23 induction or CD40/IL-4-induced IgE production. Finally, no role for C/EBP was observed in CD23 induction by EMSA or by CD23 induction analysis in C/EBPβ -/mice, whereas the absence of C/EBPβ did have an effect on IgE production and lipopolysaccharide-induced B cell proliferation. Based on these data, a model is presented which suggests that CD23 superinduction results from STAT and NF-κB interaction.

The pineal of viscacha (Lagostomus maximus maximus) is formed by two main cellular populations of pinealocytes and interstitial cells. A connective capsule surrounds the gland sending numerous septa containing blood vessels and nerves. In the specimens collected during summer the pinealocyte displays a defined contour and a clear cytoplasm with abundant ribosomes and numerous cisternae of endoplasmic reticulum. The pinealocytes observed during winter have cytological characteristics of active cells with dense and elongated mitocondria, dense bodies, and a developed Golgi complex. These seasonal glandular changes could be related to the environmental condition and mainly to the summer photoperiod. The presence of abundant nerve terminals in the perivascular spaces suggests a neural control on the blood vessels.