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Clonal Lines (clonal + line)
Selected AbstractsOestrogenic activity of isobutylparaben in vitro and in vivoJOURNAL OF APPLIED TOXICOLOGY, Issue 4 2002P. D. Darbre Abstract The alkyl esters of p -hydroxybenzoic acid (parabens) are used widely as preservatives in foods, pharmaceuticals and cosmetics to which the human population is exposed. Recent studies have reported that methylparaben, ethylparaben, n -propylparaben and n -butylparaben all possess oestrogenic activity in several in vitro assays and in animal models in vivo. This study reports on the oestrogenic activity of isobutylparaben in a panel of assays in vitro and in vivo. Isobutylparaben was able to displace [3H]oestradiol from cytosolic oestrogen receptor , of MCF7 human breast cancer cells by 81% at 100 000-fold molar excess. Using a clonal line of MCF7 cells containing a stably transfected oestrogen-responsive ERE-CAT reporter gene, CAT gene expression could be increased by isobutylparaben such that the magnitude of the response was the same at 10,5 M isobutylparaben as with 10,8 M 17,-oestradiol. Isobutylparaben could also increase expression of the endogenous oestrogen-responsive pS2 gene in MCF7 cells and maximal expression at 10,5 M isobutylparaben could be inhibited with the anti-oestrogen ICI 182 780. The proliferation of two oestrogen-dependent human breast cancer cell lines MCF7 and ZR-75-1 could be increased with isobutylparaben such that at concentrations of 10,5 M the proliferation response was of the same magnitude as with 10,8 M 17,-oestradiol. Evidence for oestrogen receptor mediation of proliferation effects was provided by the inability of isobutylparaben to influence the growth of oestrogen-unresponsive MDA-MB-231 human breast cancer cells and by the ability of the anti-oestrogen ICI 182 780 to inhibit the isobutylparaben effects on MCF7 cell growth. The proliferation response to 10,10 M 17,-oestradiol was not antagonized with isobutylparaben at any concentration from 10,9 M to 10,4 M in either MCF7 or ZR-75-1 cells. Finally, subcutaneous administration of isobutylparaben was able to increase the uterine weight in the immature mouse after three daily doses of 1.2 or 12.0 mg per mouse. Previous work using linear-alkyl-chain parabens has shown that oestrogenic activity increases with alkyl chain length from methylparaben to n -butylparaben. The results here show that branching of the alkyl chain to isobutylparaben increases oestrogenic activity beyond that of the equivalent length linear alkyl chain in n -butylparaben. Copyright © 2002 John Wiley & Sons, Ltd. [source] Recombination is suppressed over a large region of the rainbow trout Y chromosomeANIMAL GENETICS, Issue 6 2009R. B. Phillips Summary The previous genetic mapping data have suggested that most of the rainbow trout sex chromosome pair is pseudoautosomal, with very small X-specific and Y-specific regions. We have prepared an updated genetic and cytogenetic map of the male rainbow trout sex linkage group. Selected sex-linked markers spanning the X chromosome of the female genetic map have been mapped cytogenetically in normal males and genetically in crosses between the OSU female clonal line and four different male clonal lines as well as in outcrosses involving outbred OSU and hybrids between the OSU line and the male clonal lines. The cytogenetic maps of the X and Y chromosomes were very similar to the female genetic map for the X chromosome. Five markers on the male maps are genetically very close to the sex determination locus (SEX), but more widely spaced on the female genetic map and on the cytogenetic map, indicating a large region of suppressed recombination on the Y chromosome surrounding the SEX locus. The male map is greatly extended at the telomere. A BAC clone containing the SCAR (sequence characterized amplified region) Omy - 163 marker, which maps close to SEX, was subjected to shotgun sequencing. Two carbonyl reductase genes and a gene homologous to the vertebrate skeletal ryanodine receptor were identified. Carbonyl reductase is a key enzyme involved in production of trout ovarian maturation hormone. This brings the number of type I genes mapped to the sex chromosome to six and has allowed us to identify a region on zebrafish chromosome 10 and medaka chromosome 13 which may be homologous to the distal portion of the long arm of the rainbow trout Y chromosome. [source] Fluorescent Plasmodium berghei sporozoites and pre-erythrocytic stages: a new tool to study mosquito and mammalian host interactions with malaria parasitesCELLULAR MICROBIOLOGY, Issue 6 2001Ramya Natarajan To track malaria parasites for biological studies within the mosquito and mammalian hosts, we constructed a stably transformed clonal line of Plasmodium berghei, PbFluspo, in which sporogonic and pre-erythrocytic liver-stage parasites are autonomously fluorescent. A cassette containing the structural gene for the FACS-adapted green fluorescent protein mutant 2 (GFPmut2), expressed from the 5, and 3, flanking sequences of the circumsporozoite (CS) protein gene, was integrated and expressed at the endogenous CS locus. Recombinant parasites, which bear a wild-type copy of CS, generated highly fluorescent oocysts and sporozoites that invaded mosquito salivary glands and were transmitted normally to rodent hosts. The parasites infected cultured hepatocytes in vitro, where they developed into fluorescent pre-erythrocytic forms. Mammalian cells infected by these parasites can be separated from non-infected cells by fluorescence activated cell sorter (FACS) analysis. These fluorescent insect and mammalian stages of P. berghei should be useful for phenotypic studies in their respective hosts, as well as for identification of new genes expressed in these parasite stages. [source] Investigation of the genetic diversity among isolates of Salmonella enterica serovar Dublin from animals and humans from England, Wales and IrelandJOURNAL OF APPLIED MICROBIOLOGY, Issue 5 2002E. Liebana Aims: To assess the degree of genetic diversity among animal Salmonella Dublin UK isolates, and to compare it with the genetic diversity found among human isolates from the same time period. Methods and Results: One hundred isolates (50 human and 50 animal) were typed using plasmid profiling, XbaI-pulsed field gel electrophoresis (PFGE) and PstI- SphI ribotyping. Antimicrobial resistance data to 16 antibiotics was presented, and the presence of class-I integrons was investigated by real-time PCR. Seven different plasmid profiles, 19 ribotypes and 21 PFGE types were detected. A combination of the three methods allowed clear differentiation of 43 clones or strains. Eighteen isolates were resistant to at least one antimicrobial; five of them were multi-resistant and of these, only three presented class I integrons. Conclusions: Ribotyping data suggest the existence of at least three very different clonal lines; the same distribution in well-defined groups was not evident from the PFGE data. The existence of a variety of clones in both animals and humans has been demonstrated. A few prevalent clones seem to be widely disseminated among different animal species and show a diverse geographical and temporal distribution. The same clones were found in animals and humans, which may infer that both farm and pet animals may act as potential vehicles of infection for humans. Some other clones seem to be less widely distributed. Clustering analysis of genomic fingerprints of Salmonella Dublin and Salm. Enteritidis isolates confirms the existence of a close phylogenetic relationship between both serotypes. Significance and Impact of the Study: This paper describes the utility of a multiple genetic typing approach for Salm. Dublin. It gives useful information on clonal diversity among human and animal isolates. [source] CLONAL HERBAL EXTRACTS AS ELICITORS OF PHENOLIC SYNTHESIS IN DARK-GERMINATED MUNGBEANS FOR IMPROVING NUTRITIONAL VALUE WITH IMPLICATIONS FOR FOOD SAFETYJOURNAL OF FOOD BIOCHEMISTRY, Issue 3 2002PATRICK McCUE ABSTRACT Plant phenolics are secondary metabolites that confer beneficial properties to the plants that produce them. Extracts made from plants that produce these phytochemicals are increasingly being recognized for their antimicrobial properties. In this study, we investigated extracts made from high-phenolics-producing clonal lines of oregano and thyme for potential as elicitors of phenolic antioxidant production in dark-germinated mungbean (Vigna radiata,). Mungbean was dark-germinated under the rationale that any energy stored in the bean seed in the form of starch may potentially be utilized for enhanced phenolics production, since without a light source the dark-germinated seedling may not stimulate the development of photosynthetic components. Wafer-based herb extracts showed the greatest ability to stimulate phenolic content in dark-germinated mungbeans. Three of the oregano extracts were investigated further and showed an ability to stimulate glucose-6-phosphate dehydrogenase (G6PDH), guaiacol peroxidase (GPX), and antioxidant activity. These results suggest that the extracts contain an active elicitor that stimulates phenolic antioxidant content, as well as activity of the pentose-phosphate pathway. In addition, the results of this study suggest that extracts of high-phenolics-producing clonal plants may have potential in the food and agriculture industry as seed treatments for preventing bacterial infection in germinating sprouts by stimulating phenolic antioxidant-producing pathways, as well as for increasing the nutritional value of sprouts for human consumption. [source] A NEW LARVAL FISH BIOASSAY FOR TESTING THE PATHOGENICITY OF PFIESTERIA SPP. (DINOPHYCEAE),JOURNAL OF PHYCOLOGY, Issue 3 2003Vincent J. Lovko Water quality, microbial contamination, prior fish health, and variable results have been major impediments to identifying the cause and mechanism of fish mortality in standard aquarium-format Pfiesteria bioassays. Therefore, we developed a sensitive 96-h larval fish bioassay for assessing Pfiesteria spp. pathogenicity using six-well tissue culture plates and 7-day-old larval cyprinodontid fish. We used the assay to test pathogenicity of several clonal lines of Pfiesteria piscicida Steidinger and Burkholder and P. shumwayae Glasgow and Burkholder that had been cultured with algal prey for 2 to 36 months. The P. shumwayae cultures exhibited 80%,100% cumulative mortality in less than 96 h at initial zoospore densities of approximately 1000 cells·mL,1. No fish mortalities occurred with P. piscicida at identical densities or in controls. In a dose-response assay, we demonstrated a strong positive correlation between dinospore density and fish mortality in a highly pathogenic culture of P. shumwayae, generating a 96-h LD50 of 108 zoospores·mL,1. Additionally, we applied the assay to evaluate a 38-L P. shumwayae bioassay that was actively killing fish and compared results with those from exposures of juvenile tilapia (Oreochromis niloticus) in a 500-mL assay system. Water from the fish-killing 38-L assay was filtered and centrifuged to produce fractions dominated by dinoflagellates, bacteria, or presumed ichthyotoxin (cell-free fraction). After 96 h, the larval fish assay exhibited 50%,100% cumulative mortality only in fractions containing dinoflagellates, with no mortalities occurring in the other fractions. The 500-mL bioassay with tilapia produced inconsistent results and demonstrated no clear correlation between mortality and treatment. The new larval fish bioassay was demonstrated as a highly effective method to verify and evaluate dinoflagellate pathogenicity. [source] Immunodetection and Characterization of Antigens Expressed by Uncinula necatorJOURNAL OF PHYTOPATHOLOGY, Issue 11-12 2002V. L. Markovic Abstract Conidia from four genetically distinct isolates of Uncinula necator (Schw.) Burr. were used to raise a polyclonal antiserum. Immunofluorescent detection of the fungus on Vitis vinifera (cv. Chardonnay) indicated that the antiserum bound specifically to fungal antigens present on both conidia and hyphae, with no detection of underlying berry tissues. The antibody reacted with three antigens present on the conidia (Mr 21, 29 and > 250 kDa). Immunoreactivity with the 21 kDa antigen was dependent upon the preservation of at least one disulphide bond linkage. Evidence from immunoblot staining and enzyme immunoassay indicated that only a small proportion of the recognized epitopes contained carbohydrate moieties. The antibody detected homologous U. necator conidial antigens in a plate trapped antigen-enzyme-linked immunoabsorbent assay (PTA-ELISA), with a linear range of detection extending from 1000 to 9000 conidia/ml at a 1/5000 dilution of serum. Under these conditions the immunoassay also detected antigens from pooled heterologous U. necator isolates. The antiserum exhibited cross-reactivity with antigens present on Aspergillus, Pithomyces and Sporobolomyces species co-isolated from powdery mildew-infected grapes, which could not be removed by fractionation of the antiserum on antigen affinity columns. Monoclonal antibodies were subsequently produced to avoid the problems of cross-reactivity associated with the polyclonal antibody. Antibodies produced from two clonal lines exhibited specificity for U. necator and were shown to detect a 21 kDa conidial antigen. Use of either of these antibodies enabled the differentiation of grapes grouped on the basis of powdery mildew disease levels. [source] Human neural stem cells genetically modified for brain repair in neurological disordersNEUROPATHOLOGY, Issue 3 2004Seung U. Kim Existence of multipotent neural stem cells (NSC) has been known in developing or adult mammalian CNS, including humans. NSC have the capacity to grow indefinitely and have multipotent potential to differentiate into three major cell types of CNS, neurons, astrocytes and oligodendrocytes. Stable clonal lines of human NSC have recently been generated from the human fetal telencephalon using a retroviral vector encoding v-myc. One of the NSC lines, HB1.F3, carries normal human karyotype of 46XX and has the ability to self-renew, differentiate into cells of neuronal and glial lineages, and integrate into the damaged CNS loci upon transplantation into the brain of animal models of Parkinson disease, HD, stroke and mucopolysaccharidosis. F3 human NSC were genetically engineered to produce L-dihydroxyphenylalanine (L-DOPA) by double transfection with cDNA for tyrosine hydroxylase and guanosine triphosphate cylohydrolase-1, and transplantation of these cells in the brain of Parkinson disease model rats led to L-DOPA production and functional recovery. Proactively transplanted F3 human NSC in rat striatum, supported the survival of host striatal neurons against neuronal injury caused by 3-nitropro-pionic acid in rat model of HD. Intravenously introduced through the tail vein, F3 human NSC were found to migrate into ischemic lesion sites, differentiate into neurons and glial cells, and improve functional deficits in rat stroke models. These results indicate that human NSC should be an ideal vehicle for cell replacement and gene transfer therapy for patients with neurological diseases. In addition to immortalized human NSC, immortalized human bone marrow mesenchymal stem cell lines have been generated from human embryonic bone marrow tissues with retroviral vectors encording v-myc or teromerase gene. These immortalized cell lines of human bone marrow mesenchymal stem cells differentiated into neurons/glial cells, bone, cartilage and adipose tissue when they were grown in selective inducing media. There is further need for investigation into the neurogenic potential of the human bone marrow stem cell lines and their utility in animal models of neurological diseases. [source] Recombination is suppressed over a large region of the rainbow trout Y chromosomeANIMAL GENETICS, Issue 6 2009R. B. Phillips Summary The previous genetic mapping data have suggested that most of the rainbow trout sex chromosome pair is pseudoautosomal, with very small X-specific and Y-specific regions. We have prepared an updated genetic and cytogenetic map of the male rainbow trout sex linkage group. Selected sex-linked markers spanning the X chromosome of the female genetic map have been mapped cytogenetically in normal males and genetically in crosses between the OSU female clonal line and four different male clonal lines as well as in outcrosses involving outbred OSU and hybrids between the OSU line and the male clonal lines. The cytogenetic maps of the X and Y chromosomes were very similar to the female genetic map for the X chromosome. Five markers on the male maps are genetically very close to the sex determination locus (SEX), but more widely spaced on the female genetic map and on the cytogenetic map, indicating a large region of suppressed recombination on the Y chromosome surrounding the SEX locus. The male map is greatly extended at the telomere. A BAC clone containing the SCAR (sequence characterized amplified region) Omy - 163 marker, which maps close to SEX, was subjected to shotgun sequencing. Two carbonyl reductase genes and a gene homologous to the vertebrate skeletal ryanodine receptor were identified. Carbonyl reductase is a key enzyme involved in production of trout ovarian maturation hormone. This brings the number of type I genes mapped to the sex chromosome to six and has allowed us to identify a region on zebrafish chromosome 10 and medaka chromosome 13 which may be homologous to the distal portion of the long arm of the rainbow trout Y chromosome. [source] Rapid changes in clonal lines: the death of a ,sacred cow'BIOLOGICAL JOURNAL OF THE LINNEAN SOCIETY, Issue 1 2003HUGH D. LOXDALE It is well established that asexually reproducing viruses and prokaryotes mutate rapidly. In contrast, the eukaryotic clone is often still treated as if it is genetically homogeneous within and between populations, i.e. that it is assumed to show genetic fidelity. However, such fidelity has rarely been tested empirically using the range of high-resolution molecular markers now available, culminating with direct sequencing of the DNA. If such a biological entity as a ,clone' really did exist, it would be a fantastic entity, differing from everything else known in biology, i.e. it would possess a population mean but no variance for any particular trait. It would not be amenable to selection and adaptive variation and would thus be unchanging in time and space. In this paper, we argue that the general acceptance of clonal fidelity is a scientific convenience, since the rate of asexual reproduction of eukaryotes is not as fast as that of bacteria and hence it is easier to accept fidelity as a ,fact' rather than test for it. We propose that part of the acceptance of fidelity may have a cultural basis and thereby is a kind of ,pre-Darwinian relic'. Instead, a clonal genotype is perhaps largely a function of marker resolution, i.e. dependent on the number and type of markers employed. If this is so and were enough of the genome explored, perhaps each individual within a clone would be found to differ genetically at particular regions of the chromosomes. The question of what constitutes a clone is not just a semantic one and impacts directly on recent attempts to understand and produce ,artificial' clones, especially of mammals. New research is already confirming that mutations and epigenetic influences play a crucial role in the success of cloning attempts. © 2003 The Linnean Society of London. Biological Journal of the Linnean Society, 2003, 79, 3,16. [source] Cell volume and rate of proliferation, but not protein expression pattern, distinguish pup/intimal smooth muscle cells from subcultured adult smooth muscle cellsCELL PROLIFERATION, Issue 5 2001E. McKilligin Smooth muscle cells from neonatal rats and from injured blood vessels grow with a characteristic cobblestone morphology that distinguishes them from adult smooth muscle cells. This has led to the proposition that there are two distinct types of smooth muscle cells with different proliferative capacity. Here we systematically compare the properties of subcultured adult smooth muscle cells in culture and clonal lines of cobblestone smooth muscle cells from both neonatal rats and injured vessels. The cobblestone smooth muscle cells have a significantly smaller average cell volume, estimated using two different flow cytometry measurements. However, the two types of smooth muscle cells have indistinguishable protein expression patterns when the levels of more than 20 different proteins (including cytoskeletal proteins, matrix proteins, cytokines, cytokine receptors, adhesion molecules and enzymes) are measured by quantitative immunofluorescence. Furthermore, in contrast to previous observations, we demonstrate that both types of smooth muscle cells secrete a powerful mitogenic activity. The higher cell density achieved by the cobblestone smooth muscle cells in culture was responsible for the earlier reports that this mitogenic activity was secreted only by cobblestone smooth muscle cells. We conclude that many of the differences seen between cobblestone smooth muscle cells and adult smooth muscle cells in vitro (proliferation rate, morphology, protein expression pattern, secretion of mitogenic activity) could be attributable to a stable difference in the median cell volume of the cultures. [source] | |||||||||||||